BACKGROUND: It is unclear whether gut microbiota (GM) affects the risk of optic neuritis (ON) through the "gut-brain" axis and the "gut-retina" axis. To examine the causal relationship between GM and ON, we conducted Mendelian randomization (MR) study. METHODS: Up to 18,340 samples of 24 population-based cohorts were included in genome-wide association study (GWAS) of 196 GM taxa. ON outcomes were selected from the FinnGen GWAS (951 ON cases and 307,092 controls). In addition, the GWAS based on UK Biobank (UKB) (105 ON cases and 456,243 controls) was used for further exploration. Inverse variance weighted (IVW) was carried out to estimate their effects on ON risk and the MR assumptions were evaluated in sensitivity analyses. RESULTS: Among the 196 GM taxa, the IVW results confirmed that Family -Peptococcaceae (P = 2.17 × 10-3), Genus- Hungatella (P = 4.57 × 10-3) and genus-Eubacterium_rectale_group (P = 0.02) were correlated with the risk of ON based on Finngen GWAS. Based on data from UKB, Genus- Eubacterium_hallii_group (P = 1.50 × 10-3) and Genus- Ruminococcaceae_UCG_002 (P = 0.02) were correlated with the risk of ON. At the phylum, class and order levels, no GM taxa were causally related to ON (P > 0.05). Heterogeneity (P > 0.05) and pleiotropy (P > 0.05) analysis confirmed the robustness of the MR results. CONCLUSION: Our MR findings support the causal effect of specific GM taxa on ON. GM may affect the risk of ON through the "gut-brain" axis and the "gut-retina" axis. However, further research is needed to confirm the relevant mechanism of the relationship between GM and ON.

Advancing age is a major risk factor of Alzheimer's disease (AD). The worldwide prevalence of AD is approximately 50 million people, and this number is projected to increase substantially. The molecular mechanisms underlying the aging-associated susceptibility to cognitive impairment in AD are largely unknown. As a hallmark of aging, cellular senescence is a significant contributor to aging and age-related diseases including AD. Senescent neurons and glial cells have been detected to accumulate in the brains of AD patients and mouse models. Importantly, selective elimination of senescent cells ameliorates amyloid beta and tau pathologies and improves cognition in AD mouse models, indicating a critical role of cellular senescence in AD pathogenesis. Nonetheless, the mechanisms underlying when and how cellular senescence contributes to AD pathogenesis remain unclear. This review provides an overview of cellular senescence and discusses recent advances in the understanding of the impact of cellular senescence on AD pathogenesis, with brief discussions of the possible role of cellular senescence in other neurodegenerative diseases including Down syndrome, Parkinson's disease, multiple sclerosis, and amyotrophic lateral sclerosis.

Inflammatory bowel disease (IBD), with Crohn's disease and ulcerative colitis as main subtypes, is a prototypical multifactorial disease with both genetic and environmental factors involved. Genetically, IBD covers a wide spectrum from monogenic to polygenic forms. In polygenic disease, many genetic variants each contribute a small amount to disease risk. With the advent of genome-wide association studies (GWAS), it became possible to find these variants and corresponding genes, leading so far to the discovery of ca 240 loci associated with IBD. Together, these however explain only 20-25% of the heritability of IBD, leaving a large portion unaccounted for. This missing heritability might be hidden in common variants with even lower effect than the ones currently found through GWAS, but also in rare variants which can be found through large-scale sequencing studies or potentially in multiplex families. In this review, we will give an overview of the current knowledge about the genetics of non-monogenic IBD and how it differs from the monogenic form(s), and future perspectives. The history of IBD genetic studies from twin studies over linkage studies to GWAS, and finally large-scale sequencing studies and the revisiting of multiplex families will be discussed.

Pain often occurs in parallel with neuropsychiatric disorders. However, the underlying mechanisms and potential causality have not been well studied. We collected the genome-wide association study (GWAS) summary statistics of 26 common pain and neuropsychiatric disorders with sample size ranging from 17,310 to 482,730 in European population. The genetic correlation between pair of pain and neuropsychiatric disorders, as well as the relevant cell types were investigated by linkage disequilibrium (LD) score regression analyses. Then, transcriptome-wide association study (TWAS) was applied to identify the potential shared genes by integrating the gene expression information and GWAS. In addition, Mendelian randomization (MR) analyses were conducted to infer the potential causality between pain and neuropsychiatric disorders. Among the 169 pairwise pain and neuropsychiatric disorders, 55 pairs showed positive correlations (median rg = 0.43) and 9 pairs showed negative correlations (median rg =  -0.31). Using MR analyses, 26 likely causal associations were identified, including that neuroticism and insomnia were risk factors for most of short-term pain, and multisite chronic pain was risk factor for neuroticism, insomnia, major depressive disorder and attention deficit/hyperactivity disorder, and vice versa. The signals of pain and neuropsychiatric disorders tended to be enriched in the functional regions of cell types from central nervous system (CNS). A total of 19 genes shared in at least one pain and neuropsychiatric disorder pair were identified by TWAS, including AMT, NCOA6, and UNC45A, which involved in glycine degradation, insulin secretion, and cell proliferation, respectively. Our findings provided the evidence of shared genetic structure, causality and potential shared pathogenic mechanisms between pain and neuropsychiatric disorders, and enhanced our understanding of the comorbidities of pain and neuropsychiatric disorders.

Exocytosis is the process by which secretory vesicles fuse with the plasma membrane to deliver materials to the cell surface or to release cargoes to the extracellular space. The exocyst-an evolutionarily conserved octameric protein complex-mediates spatiotemporal control of SNARE complex assembly for vesicle fusion and tethering the secretory vesicles to the plasma membrane. The exocyst participates in diverse cellular functions, including protein trafficking to the plasma membrane, membrane extension, cell polarity, neurite outgrowth, ciliogenesis, cytokinesis, cell migration, autophagy, host defense, and tumorigenesis. Exocyst subunits are essential for cell viability; and mutations or variants in several exocyst subunits have been implicated in human diseases, mostly neurodevelopmental disorders and ciliopathies. These conditions often share common features such as developmental delay, intellectual disability, and brain abnormalities. In this review, we summarize the mutations and variants in exocyst subunits that have been linked to disease and discuss the implications of exocyst dysfunction in other disorders.

Regulatory elements are the genomic regions that interact with transcription factors to control cell-type-specific gene expression in different cellular environments. A precise and complete catalog of functional elements encoded by the human genome is key to understanding mammalian gene regulation. Here, we review the current state of regulatory element annotation. We first provide an overview of assays for characterizing functional elements, including genome, epigenome, transcriptome, three-dimensional chromatin interaction, and functional validation assays. We then discuss computational methods for defining regulatory elements, including peak-calling and other statistical modeling methods. Finally, we introduce several high-quality lists of regulatory element annotations and suggest potential future directions.

Monogenic intestinal epithelial disorders, also known as congenital diarrheas and enteropathies (CoDEs), are a group of rare diseases that result from mutations in genes that primarily affect intestinal epithelial cell function. Patients with CoDE disorders generally present with infantile-onset diarrhea and poor growth, and often require intensive fluid and nutritional management. CoDE disorders can be classified into several categories that relate to broad areas of epithelial function, structure, and development. The advent of accessible and low-cost genetic sequencing has accelerated discovery in the field with over 45 different genes now associated with CoDE disorders. Despite this increasing knowledge in the causal genetics of disease, the underlying cellular pathophysiology remains incompletely understood for many disorders. Consequently, clinical management options for CoDE disorders are currently limited and there is an urgent need for new and disorder-specific therapies. In this review, we provide a general overview of CoDE disorders, including a historical perspective of the field and relationship to other monogenic disorders of the intestine. We describe the genetics, clinical presentation, and known pathophysiology for specific disorders. Lastly, we describe the major challenges relating to CoDE disorders, briefly outline key areas that need further study, and provide a perspective on the future genetic and therapeutic landscape.

Although COVID-19 is mostly a pulmonary disease, it is now well accepted that it can cause a much broader spectrum of signs and symptoms and affect many other organs and tissue. From mild anosmia to severe ischemic stroke, the impact of SARS-CoV-2 on the central nervous system is still a great challenge to scientists and health care practitioners. Besides the acute and severe neurological problems described, as encephalopathies, leptomeningitis, and stroke, after 2 years of pandemic, the chronic impact observed during long-COVID or the post-acute sequelae of COVID-19 (PASC) greatly intrigues scientists worldwide. Strikingly, even asymptomatic, and mild diseased patients may evolve with important neurological and psychiatric symptoms, as confusion, memory loss, cognitive decline, chronic fatigue, associated or not with anxiety and depression. Thus, the knowledge on the correlation between COVID-19 and the central nervous system is of great relevance. In this sense, here we discuss some important mechanisms obtained from in vitro and in vivo investigation regarding how SARS-CoV-2 impacts the brain and its cells and function.

Diarrhoeal disorders in childhood extend beyond the inflammatory bowel diseases. Persistent and severe forms of diarrhoea can occur from birth and are associated with significant morbidity and mortality. These disorders can affect not only the gastrointestinal tract but frequently have extraintestinal manifestations, immunodeficiencies and endocrinopathies. Genomic analysis has advanced our understanding of these conditions and has revealed precision-based treatment options such as potentially curative haematopoietic stem cell transplant. Although many new mutations have been discovered, there is frequently no clear genotype-phenotype correlation. The functional effects of gene mutations can be studied in model systems such as patient-derived organoids. This allows us to further characterise these disorders and advance our understanding of the pathophysiology of the intestinal mucosa. In this review, we will provide an up to date overview of genes involved in diarrhoeal disorders of early onset, particularly focussing on the more recently described gene defects associated with protein loosing enteropathy.

Aging is a progressive multifaceted functional decline of a biological system. Chronic age-related conditions such as neurodegenerative diseases are leading causes of death worldwide, and they are becoming a pressing problem for our society. To address this global challenge, there is a need for novel, safe, and effective rejuvenation therapies aimed at reversing age-related phenotypes and improving human health. With gene expression being a key determinant of cell identity and function, and in light of recent studies reporting rejuvenation effects through genetic perturbations, we propose an age reversal strategy focused on reprogramming the cell transcriptome to a youthful state. To this end, we suggest using transcriptomic data from primary human cells to predict rejuvenation targets and develop high-throughput aging assays, which can be used in large perturbation screens. We propose neural cells as particularly relevant targets for rejuvenation due to substantial impact of neurodegeneration on human frailty. Of all cell types in the brain, we argue that glutamatergic neurons, neuronal stem cells, and oligodendrocytes represent the most impactful and tractable targets. Lastly, we provide experimental designs for anti-aging reprogramming screens that will likely enable the development of neuronal age reversal therapies, which hold promise for dramatically improving human health.

For several years, experts have warned about the lack of diversity in genetic research databases, and researchers have devoted time and resources to recruiting subjects from underrepresented subgroups. In this study, we review published reports in academic journals of genetic studies of Alzheimer's disease to note whether demographic diversity was indicated in the reports and, if so, the extent of representation of non-European subjects over the period from 1997 to 2022. We use multivariate regression analysis to analyze changes over time and to explain variation across studies. Our analysis indicates that reported diversity has not changed over time. Rather, it appears to have remained relatively constant, since Genome-Wide Association Studies (GWASs) were first used in the 1990s. We find most variation to be across journals rather than within journals, suggesting that characteristics of journals are an important influence on the dissemination of research with diverse samples. Lack of racial diversity in genetic databases used to develop clinical applications could lead to disparities in the effectiveness of those applications for underrepresented groups.

Domestic dogs display a wide array of heritable behaviors that have intermediate genetic complexity thanks to a long history of human-influenced selection. Comparative genetics in dogs could address the scarcity of non-human neurogenetic systems relevant to human neuropsychiatric disorders, which are characterized by mental, emotional, and behavioral symptoms and involve vastly complex genetic and non-genetic risk factors. Our review describes the diverse behavioral "phenome" of domestic dogs, past and ongoing sources of behavioral selection, and the state of canine behavioral genetics. We highlight two naturally disordered behavioral domains that illustrate how dogs may prove useful as a comparative, forward neurogenetic system: canine age-related cognitive dysfunction, which can be examined more rapidly given the attenuated lifespan of dogs, and compulsive disorders, which may have genetic roots in purpose-bred behaviors. Growing community science initiatives aimed at the companion dog population will be well suited to investigating such complex behavioral phenotypes and offer a comparative resource that parallels human genomic initiatives in scale and dimensionality.

Contactin-associated protein-like 2 (CNTNAP2) gene encodes for CASPR2, a presynaptic type 1 transmembrane protein, involved in cell-cell adhesion and synaptic interactions. Biallelic CNTNAP2 loss has been associated with "Pitt-Hopkins-like syndrome-1" (MIM#610042), while the pathogenic role of heterozygous variants remains controversial. We report 22 novel patients harboring mono- (n = 2) and bi-allelic (n = 20) CNTNAP2 variants and carried out a literature review to characterize the genotype-phenotype correlation. Patients (M:F 14:8) were aged between 3 and 19 years and affected by global developmental delay (GDD) (n = 21), moderate to profound intellectual disability (n = 17) and epilepsy (n = 21). Seizures mainly started in the first two years of life (median 22.5 months). Antiseizure medications were successful in controlling the seizures in about two-thirds of the patients. Autism spectrum disorder (ASD) and/or other neuropsychiatric comorbidities were present in nine patients (40.9%). Nonspecific midline brain anomalies were noted in most patients while focal signal abnormalities in the temporal lobes were noted in three subjects. Genotype-phenotype correlation was performed by also including 50 previously published patients (15 mono- and 35 bi-allelic variants). Overall, GDD (p < 0.0001), epilepsy (p < 0.0001), hyporeflexia (p = 0.012), ASD (p = 0.009), language impairment (p = 0.020) and severe cognitive impairment (p = 0.031) were significantly associated with the presence of biallelic versus monoallelic variants. We have defined the main features associated with biallelic CNTNAP2 variants, as severe cognitive impairment, epilepsy and behavioral abnormalities. We propose CASPR2-deficiency neurodevelopmental disorder as an exclusively recessive disease while the contribution of heterozygous variants is less likely to follow an autosomal dominant inheritance pattern.

Asthenozoospermia is one of the main factors leading to male infertility, but the genetic mechanisms have not been fully elucidated. Variants in the androglobin (ADGB) gene were identified in an infertile male characterized by asthenozoospermia. The variants disrupted the binding of ADGB to calmodulin. Adgb-/- male mice were infertile due to reduced sperm concentration (< 1 × 106 /mL) and motility. Spermatogenesis was also abnormal, with malformation of both elongating and elongated spermatids, and there was an approximately twofold increase in apoptotic cells in the cauda epididymis. These exacerbated the decline in sperm motility. It is surprising that ICSI with testicular spermatids allows fertilization and eventually develops into blastocyst. Through mass spectrometry, we identified 42 candidate proteins that are involved in sperm assembly, flagella formation, and sperm motility interacting with ADGB. In particular, CFAP69 and SPEF2 were confirmed to bind to ADGB. Collectively, our study suggests the potential important role of ADGB in human fertility, revealing its relevance to spermatogenesis and infertility. This expands our knowledge of the genetic causes of asthenozoospermia and provides a theoretical basis for using ADGB as an underlying genetic marker for infertile males.

Genomics research related to Indigenous people has been at worst exploitative and at best, retrospectively on a journey to improve effective engagement of Indigenous individuals and communities. Genomics can positively impact all stages of clinical management, and to improve genomic effectiveness researchers aggregate genomic data from diverse global sub-populations, such as shared ancestry groupings, as people within these groupings will have a greater proportion of shared DNA traits. While genomics is already being used worldwide to improve lives, its utility and effectiveness has not been maximized for individuals with Indigenous ancestry. Several large datasets of human genetic variation have been made publicly available, of which the most widely used is the Genome Aggregation Database (gnomAD), but none of these databases currently contain any population-specific data for Indigenous populations. There are many reasons why Indigenous people have been largely left out of genomics research and, because of this, miss out on the benefits offered. It is also clear that if research is to be effective, it needs to be done 'with' and not 'on' Indigenous communities. This systematic review of the literature regarding Indigenous peoples (in high income countries) and genomics aims to review the existing literature and identify areas of strength and weakness in study design and conduct, focusing on the effectiveness of Indigenous community engagement.

TMEM151A, located at 11q13.2 and encoding transmembrane protein 151A, was recently reported as causative for autosomal dominant paroxysmal kinesigenic dyskinesia (PKD). Here, through comprehensive analysis of sporadic and familial cases, we expand the clinical and mutation spectrum of PKD. In doing so, we clarify the clinical and genetic features of Chinese PKD patients harboring TMEM151A variants and further explore the relationship between TMEM151A mutations and PKD. Whole exome sequencing was performed on 26 sporadic PKD patients and nine familial PKD pedigrees without PRRT2 variants. Quantitative real-time PCR was used to assess the gene expression of frameshift mutant TMEM151A in a PKD patient. TMEM151A variants reported to date were reviewed. Four TMEM151A variants were detected in four unrelated families with 12 individuals, including a frameshift mutation [c.606_607insA (p.Val203fs)], two missense mutations [c.166G > A (p.Gly56Arg) and c.791T > C (p.Val264Ala)], and a non-pathogenic variant [c.994G > A (p.Gly332Arg)]. The monoallelic frameshift mutation [c.606_607insA (p.Val203fs)] may cause TMEM151A mRNA decay, suggesting a potential pathogenic mechanism of haploinsufficiency. Patients with TMEM151A variants had short-duration attacks and presented with dystonia. Our study provides a detailed clinical description of PKD patients with TMEM151A mutations and reports a new disease-causing mutation, expanding the known phenotypes caused by TMEM151A mutations and providing further detail about the pathoetiology of PKD.

Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) gene-editing system (CRISPR-Cas) is a valuable tool for fundamental and applied research applications. Significant improvements in editing efficacy have advanced genome editing strategies into phase 3 human clinical trials. However, recent studies suggest that our understanding of editing outcomes has lagged behind the developments made in generating the edits themselves. While many researchers have analyzed on- and off-target events through the lens of small insertions or deletions at predicted sites, screens for larger structural variants (SVs) and chromosomal abnormalities are not routinely performed. Full and comprehensive validation of on- and off-target effects is required to ensure reproducibility and to accurately assess the safety of future editing applications. Here we review SVs associated with CRISPR-editing in cells of human origin and highlight the methods used to detect and avoid them.

Human disorders of the enteric nervous system (ENS), e.g., Hirschsprung's disease, are rarely associated with major central nervous system involvement. We describe two families each segregating a different homozygous truncating variant in KIF26A with a unique constellation of severe megacolon that resembles Hirschsprung's disease but lacks aganglionosis as well as brain malformations that range from severe to mild. The intestinal phenotype bears a striking resemblance to that observed in Kif26a-/- mice where KIF26A deficiency was found to cause abnormal GDNF-Ret signaling resulting in failure to establish normal neuronal networks despite myenteric neuronal hyperplasia. Very recently, a range of brain developmental phenotypes were described in patients and mice with KIF26A deficiency and were found to result from abnormal radial migration and increased apoptosis. Our report, therefore, reveals a recognizable autosomal-recessive human KIF26A deficiency phenotype characterized by severe ENS dysfunction and a range of brain malformations.

Fetal hyperechogenic kidneys (HEK) is etiologically a heterogeneous disorder. The aim of this study was to identify the genetic causes of HEK using prenatal chromosomal microarray analysis (CMA) and exome sequencing (ES). From June 2014 to September 2022, we identified 92 HEK fetuses detected by ultrasound. We reviewed and documented other ultrasound anomalies, microscopic and submicroscopic chromosomal abnormalities, and single gene disorders. We also analyzed the diagnostic yield of CMA and ES and the clinical impact the diagnosis had on pregnancy management. In our cohort, CMA detected 27 pathogenic copy number variations (CNVs) in 25 (25/92, 27.2%) fetuses, with the most common CNV being 17q12 microdeletion syndrome. Among the 26 fetuses who underwent further ES testing, we identified 7 pathogenic/likely pathogenic variants and 8 variants of uncertain significance in 9 genes in 12 fetuses. Four novel variants were first reported herein, expanding the mutational spectra for HEK-related genes. Following counseling, 52 families chose to continue the pregnancy, and in 23 of them, postnatal ultrasound showed no detectable renal abnormalities. Of these 23 cases, 15 had isolated HEK on prenatal ultrasound. Taken together, our study showed a high rate of detectable genetic etiologies in cases with fetal HEK at the levels of chromosomal (aneuploidy), sub-chromosomal (microdeletions/microduplications), and single gene (point mutations). Therefore, we speculate that combined CMA and ES testing for fetal HEK is feasible and has good clinical utility. When no genetic abnormalities are identified, the findings can be transient, especially in the isolated HEK group.

Dravet syndrome is a devastating epileptic syndrome characterized by intractable epilepsy with an early age of onset, regression of developmental milestones, ataxia, and motor deficits. Loss-of-function pathogenic variants in the SCN1A gene are found in the majority of patients with Dravet syndrome; however, a significant number of patients remain undiagnosed even after comprehensive genetic testing. Previously, it was shown that intronic elements in the SCN1A gene called poison exons can incorporate into SCN1A mRNA, leading to haploinsufficiency and potentially causing Dravet syndrome. Here, we developed a splicing reporter assay for all described poison exons of the SCN1A gene and validated it using previously reported and artificially introduced variants. Overall, we tested 18 deep-intronic single nucleotide variants and one complex allele in the SCN1A gene. Our approach is capable of evaluating the effect of both variants affecting cis-regulatory sequences and splice-site variants, with the potential to functionally annotate every possible variant within these elements. Moreover, using antisense-modified uridine-rich U7 small nuclear RNAs, we were able to block poison exon incorporation in mutant constructs, an approach that could be used as a promising therapeutic intervention in Dravet syndrome patients with deep-intronic variants.

Ichthyosis is a genetically heterogeneous genodermatosis characterized by severely rough, dry and scaly skin. We report two consanguineous families with congenital ichthyosis. Combined positional mapping and exome sequencing of the two families revealed novel homozygous likely deleterious variants in PRSS8 (encoding prostasin) within a linkage locus on chromosome 16. One variant involved a canonical splice site and was associated with reduced abundance of the normal transcript, while the other was a missense variant that altered a highly conserved residue. The phenotype of Prss8 knockout mouse bears a striking resemblance to the one we describe in human patients, including the skin histopathology. Our data suggest a novel PRSS8-related ichthyosis disorder.

DBR1 encodes the only known human lariat debranching enzyme and its deficiency has been found to cause an autosomal recessive inborn error of immunity characterized by pediatric brainstem viral-induced encephalitis (MIM 619441). We describe a distinct allelic disorder caused by a founder recessive DBR1 variant in four families (DBR1(NM_016216.4):c.200A > G (p.Tyr67Cys)). Consistent features include prematurity, severe intrauterine growth deficiency, congenital ichthyosis-like presentation (collodion membrane, severe skin peeling and xerosis), and death before the first year of life. Patient-derived fibroblasts displayed the characteristic accumulation of intron lariats in their RNA as revealed by targeted and untargeted analysis, in addition to a marked reduction of DBR1 on immunoblot analysis. We propose a novel DBR1-related developmental disorder that is distinct from DBR1-related encephalitis susceptibility and highlight the apparent lack of correlation with the degree of DBR1 deficiency.

OBJECTIVE: This retrospective study aimed to investigate the value of whole exome sequencing (WES) for clubfoot (CF) fetuses with or without other structural abnormalities and to further explore the genetic causes of fetal CF. METHODS: this study included 83 singleton pregnancies diagnosed with fetal CF referred to our center between January 2016 and March 2022; cases were divided into two groups: isolated CF and non-isolated CF. After excluding cases with positive karyotyping and chromosomal microarray analysis results, WES was performed for the eligible fetuses and parents. Monogenic variants detected by WES and perinatal outcomes were recorded and evaluated at postnatal follow-up. RESULTS: overall, clinically significant variations were identified in 12.0% (10/83) of fetuses, and the detection rate was significantly higher in the non-isolated than in the isolated CF group (8/36, 22.2% vs. 2/47, 4.3%, p = 0.031). We additionally detected eight (9.6%) fetuses harboring variants of unknown significance. We identified 11 clinically significant variations correlating with clinical phenotypes in nine genes from ten fetuses, with KLHL40 being the most frequent (n = 2). Furthermore, we observed a significant difference in termination and survival rates between isolated and non-isolated CF cases (27.6 vs. 77.8% and 59.6 vs. 19.4%, p < 0.001 for both). CONCLUSION: our data indicate that WES has a high additional diagnostic yield for the molecular diagnosis of fetal CF, markedly enhancing existing prenatal diagnostic capabilities and expanding our understanding of intrauterine genetic disorders, thus assisting us to better interpret fetal phenotype in the future.

The tissue-specific incidence of cancers and their genetic basis are poorly understood. Although prior studies have shown global correlation across tissues for cancer risk single-nucleotide polymorphisms (SNPs) identified through genome-wide association studies (GWAS), any shared functional regulation of gene expression on a per SNP basis has not been well characterized. We set to quantify cis-mediated gene regulation and tissue sharing for SNPs associated with eight common cancers. We identify significant tissue sharing for individual SNPs and global enrichment for breast, colorectal, and Hodgkin lymphoma cancer risk SNPs in multiple tissues. In addition, we observe increasing tissue sharing for cancer risk SNPs overlapping with super-enhancers for breast cancer and Hodgkin lymphoma providing further evidence of tissue specificity. Finally, for genes under cis-regulation by breast cancer SNPs, we identify a phenotype characterized by low expression of tumor suppressors and negative regulators of the WNT pathway associated with worse freedom from progression and overall survival in patients who eventually develop breast cancer. Our results introduce a paradigm for functionally annotating individual cancer risk SNPs and will inform the design of future translational studies aimed to personalize assessment of inherited cancer risk across tissues.

Currently, routine genetic investigation for male infertility includes karyotyping analysis and PCR for Y chromosomal microdeletions to provide prognostic information such as sperm retrieval success rate. However, over 85% of male infertility remain idiopathic. We assessed 101 male patients with primary infertility in a retrospective cohort analysis who have previously received negative results from standard-of-care tests. Mate-pair genome sequencing (large-insert size library), an alternative long-DNA sequencing method, was performed to detect clinically significant structural variants (SVs) and copy-number neutral absence of heterozygosity (AOH). Candidate SVs were filtered against our in-house cohort of 1077 fertile men. Genes disrupted by potentially clinically significant variants were correlated with single-cell gene expression profiles of human fetal and postnatal testicular developmental lineages and adult germ cells. Follow-up studies were conducted for each patient with clinically relevant finding(s). Molecular diagnoses were made in 11.1% (7/63) of patients with non-obstructive azoospermia and 13.2% (5/38) of patients with severe oligozoospermia. Among them, 12 clinically significant SVs were identified in 12 cases, including five known syndromes, one inversion, and six SVs with direct disruption of genes by intragenic rearrangements or complex insertions. Importantly, a genetic defect related to intracytoplasmic sperm injection (ICSI) failure was identified in a patient with non-obstructive azoospermia, illustrating the additional value of an etiologic diagnosis in addition to determining sperm retrieval rate. Our study reveals a landscape of various genomic variants in 101 males with idiopathic infertility, not only advancing understanding of the underlying mechanisms of male infertility, but also impacting clinical management.

Immune dysregulation has been consistently reported in psychiatric disorders, however, the causes and mechanisms underlying immune dysregulation in psychiatric disorders remain largely unclear. Here we conduct a Mendelian randomization study by integrating plasma proteome and GWASs of schizophrenia, bipolar disorder and depression. The primate-specific immune-related protein BTN3A3 showed the most significant associations with all three psychiatric disorders. In addition, other immune-related proteins, including AIF1, FOXO3, IRF3, CFHR4, IGLON5, FKBP2, and PI3, also showed significant associations with psychiatric disorders. Our study showed that a proportion of psychiatric risk variants may contribute to disease risk by regulating immune-related plasma proteins, providing direct evidence that connect the genetic risk of psychiatric disorders to immune system.

Constitutional complex chromosomal rearrangements (CCRs) are rare cytogenetic aberrations arising in the germline via an unknown mechanism. Here we analyzed the breakpoint junctions of microscopically three-way or more complex translocations using comprehensive genomic and epigenomic analyses. All of these translocation junctions showed submicroscopic genomic complexity reminiscent of chromothripsis. The breakpoints were clustered within small genomic domains with junctions showing microhomology or microinsertions. Notably, all of the de novo cases were of paternal origin. The breakpoint distributions corresponded specifically to the ATAC-seq (assay for transposase-accessible chromatin with sequencing) read data peak of mature sperm and not to other chromatin markers or tissues. We propose that DNA breaks in CCRs may develop in an accessible region of densely packaged chromatin during post-meiotic spermiogenesis.

Arthrogryposis multiplex congenita forms a broad group of clinically and etiologically heterogeneous disorders characterized by congenital joint contractures that involve at least two different parts of the body. Neurological and muscular disorders are commonly underlying arthrogryposis. Here, we report five affected individuals from three independent families sharing an overlapping phenotype with congenital contractures affecting shoulder, elbow, hand, hip, knee and foot as well as scoliosis, reduced palmar and plantar skin folds, microcephaly and facial dysmorphism. Using exome sequencing, we identified homozygous truncating variants in FILIP1 in all patients. FILIP1 is a regulator of filamin homeostasis required for the initiation of cortical cell migration in the developing neocortex and essential for the differentiation process of cross-striated muscle cells during myogenesis. In summary, our data indicate that bi-allelic truncating variants in FILIP1 are causative of a novel autosomal recessive disorder and expand the spectrum of genetic factors causative of arthrogryposis multiplex congenita.

Age-related macular degeneration (AMD), cataract, and glaucoma are leading causes of blindness worldwide. Previous genome-wide association studies (GWASs) have revealed a variety of susceptible loci associated with age-related ocular disorders, yet the genetic pleiotropy and causal genes across these diseases remain poorly understood. By leveraging large-scale genetic and observational data from ocular disease GWASs and UK Biobank (UKBB), we found significant pairwise genetic correlations and consistent epidemiological associations among these ocular disorders. Cross-disease meta-analysis uncovered seven pleiotropic loci, three of which were replicated in an additional cohort. Integration of variants in pleiotropic loci and multiple single-cell omics data identified that Müller cells and astrocytes were likely trait-related cell types underlying ocular comorbidity. In addition, we comprehensively integrated eye-specific gene expression quantitative loci (eQTLs), epigenomic profiling, and 3D genome data to prioritize causal pleiotropic genes. We found that pleiotropic genes were essential in nerve development and eye pigmentation, and targetable by aflibercept and pilocarpine for the treatment of AMD and glaucoma. These findings will not only facilitate the mechanistic research of ocular comorbidities but also benefit the therapeutic optimization of age-related ocular diseases.

CHAMP1 disorder is a genetic neurodevelopmental condition caused by mutations in the CHAMP1 gene that result in premature termination codons. The disorder is associated with intellectual disability, medical comorbidities, and dysmorphic features. Deletions of the CHAMP1 gene, as part of 13q34 deletion syndrome, have been briefly described with the suggestion of a milder clinical phenotype. To date, no studies have directly assessed differences between individuals with mutations in CHAMP1 to those with deletions of the gene. We completed prospective clinical evaluations of 16 individuals with mutations and eight with deletions in CHAMP1. Analyses revealed significantly lower adaptive functioning across all domains assessed (i.e., communication, daily living skills, socialization, and motor skills) in the mutation group. Developmental milestones and medical features further showed difference between groups. The phenotypes associated with mutations, as compared to deletions, indicate likely difference in pathogenesis between groups, where deletions are acting through CHAMP1 haploinsufficiency and mutations are acting through dominant negative or gain of function mechanisms, leading to a more severe clinical phenotype. Understanding this pathogenesis is important to the future of novel therapies for CHAMP1 disorder and illustrates that mechanistic understanding of mutations must be carefully considered prior to treatment development.

Complex congenital chromosome abnormalities are rare but often cause severe symptoms. However, the structures and biological impacts of such abnormalities have seldomly been analyzed at the molecular level. Previously, we reported a Japanese female patient with severe developmental defects. The patient had an extra dicentric chromosome 21 (chr21) consisting of two partial chr21 copies fused together within their long arms along with two centromeres and many copy number changes. In this study, we performed whole-genome, transcriptional, and DNA methylation analyses, coupled with novel bioinformatic approaches, to reveal the complex structure of the extra chromosome and its transcriptional and epigenetic changes. Long-read sequencing accurately identified the structures of junctions related to the copy number changes in extra chr21 and suggested the mechanism of the structural changes. Our transcriptome analysis showed the overexpression of genes in extra chr21. Additionally, an allele-specific DNA methylation analysis of the long-read sequencing data suggested that the centromeric region of extra chr21 was hypermethylated, a property associated with the inactivation of one centromere in the extra chromosome. Our comprehensive analysis provides insights into the molecular mechanism underlying the generation of the extra chromosome and its pathogenic roles.

Turner syndrome (TS) is a chromosomal disorder caused by complete or partial loss of the second sex chromosome and exhibits phenotypic heterogeneity, even after accounting for mosaicism and karyotypic variation. Congenital heart defects (CHD) are found in up to 45 percent of girls with TS and span a phenotypic continuum of obstructive left-sided lesions, with bicuspid aortic valve (BAV) being the most common. Several recent studies have demonstrated a genome-wide impact of X chromosome haploinsufficiency, including global hypomethylation and altered RNA expression. The presence of such broad changes to the TS epigenome and transcriptome led others to hypothesize that X chromosome haploinsufficiency sensitizes the TS genome, and several studies have demonstrated that a second genetic hit can modify disease susceptibility in TS. The objective of this study was to determine whether genetic variants in known heart developmental pathways act synergistically in this setting to increase the risk for CHD, specifically BAV, in TS. We analyzed 208 whole exomes from girls and women with TS and performed gene-based variant enrichment analysis and rare-variant association testing to identify variants associated with BAV in TS. Notably, rare variants in CRELD1 were significantly enriched in individuals with TS who had BAV compared to those with structurally normal hearts. CRELD1 is a protein that functions as a regulator of calcineurin/NFAT signaling, and rare variants in CRELD1 have been associated with both syndromic and non-syndromic CHD. This observation supports the hypothesis that genetic modifiers outside the X chromosome that lie in known heart development pathways may influence CHD risk in TS.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a condition caused by mutations in NOTCH3 and results in a phenotype characterised by recurrent strokes, vascular dementia and migraines. Whilst a genetic basis for the disease is known, the molecular mechanisms underpinning the pathology of CADASIL are still yet to be determined. Studies conducted at the Genomics Research Centre (GRC) have also identified that only 15-23% of individuals clinically suspected of CADASIL have mutations in NOTCH3. Based on this, whole exome sequencing was used to identify novel genetic variants for CADASIL-like cerebral small-vessel disease (CSVD). Analysis of functionally important variants in 50 individuals was investigated using overrepresentation tests in Gene ontology software to identify biological processes that are potentially affected in this group of patients. Further investigation of the genes in these processes was completed using the TRAPD software to identify if there is an increased number (burden) of mutations that are associated with CADASIL-like pathology. Results from this study identified that cell-cell adhesion genes were positively overrepresented in the PANTHER GO-slim database. TRAPD burden testing identified n = 15 genes that had a higher number of rare (MAF < 0.001) and predicted functionally relevant (SIFT < 0.05, PolyPhen > 0.8) mutations compared to the gnomAD v2.1.1 exome control dataset. Furthermore, these results identified ARVCF, GPR17, PTPRS, and CELSR1 as novel candidate genes in CADASIL-related pathology. This study identified a novel process that may be playing a role in the vascular damage related to CADASIL-related CSVD and implicated n = 15 genes in playing a role in the disease.

Enlargement of the endolymphatic sac, duct, and vestibular aqueduct (EVA) is the most common inner ear malformation identified in patients with sensorineural hearing loss. EVA is associated with pathogenic variants in SLC26A4. However, in European-Caucasian populations, about 50% of patients with EVA carry no pathogenic alleles of SLC26A4. We tested for the presence of variants in CHD7, a gene known to be associated with CHARGE syndrome, Kallmann syndrome, and hypogonadotropic hypogonadism, in a cohort of 34 families with EVA subjects without pathogenic alleles of SLC26A4. In two families, NM_017780.4: c.3553A > G [p.(Met1185Val)] and c.5390G > C [p.(Gly1797Ala)] were detected as monoallelic CHD7 variants in patients with EVA. At least one subject from each family had additional signs or potential signs of CHARGE syndrome but did not meet diagnostic criteria for CHARGE. In silico modeling of these two missense substitutions predicted detrimental effects upon CHD7 protein structure. Consistent with a role of CHD7 in this tissue, Chd7 transcript and protein were detected in all epithelial cells of the endolymphatic duct and sac of the developing mouse inner ear. These results suggest that some CHD7 variants can cause nonsyndromic hearing loss and EVA. CHD7 should be included in DNA sequence analyses to detect pathogenic variants in EVA patients. Chd7 expression and mutant phenotype data in mice suggest that CHD7 contributes to the formation or function of the endolymphatic sac and duct.

Hypertension is a polygenic disease that affects over 1.2 billion adults aged 30-79 worldwide. It is a major risk factor for renal, cerebrovascular, and cardiovascular diseases. The heritability of hypertension is estimated to be high; nevertheless, our understanding of its underlying mechanisms remains scarce and incomplete. This study covered the entries from European ancestry from the UK-Biobank (UKB), with 74,090 cases diagnosed with essential (primary) hypertension and 200,734 controls. We compared the findings from large-scale genome-wide association studies (GWAS) to the gene-based method of proteome-wide association studies (PWAS). We focused on 70 statistically significant associated genes, most of which failed to reach significance in variant-based GWAS. A total of 30% of the PWAS-associated genes were validated against independent cohorts, including the Finnish Biobank. Furthermore, gene-based analyses that were performed on both sexes revealed sex-dependent genetics with a stronger genetic component associated with females. Analysis of systolic and diastolic blood pressure measurements confirms a strong genetic effect associated with females. We demonstrated that gene-based approaches provide insight into the underlying biology of hypertension. Specifically, the expression profiles of the identified genes exposed the enrichment of endothelial cells from multiple organs. Furthermore, females' top-ranked significant genes are involved in cellular immunity. We conclude that studying hypertension and blood pressure via gene-based association methods improves interpretability and exposes sex-dependent genetic effects, which enhances clinical utility.

GWAS has identified thousands of loci associated with disease, yet the causal genes within these loci remain largely unknown. Identifying these causal genes would enable deeper understanding of the disease and assist in genetics-based drug development. Exome-wide association studies (ExWAS) are more expensive but can pinpoint causal genes offering high-yield drug targets, yet suffer from a high false-negative rate. Several algorithms have been developed to prioritize genes at GWAS loci, such as the Effector Index (Ei), Locus-2-Gene (L2G), Polygenic Prioritization score (PoPs), and Activity-by-Contact score (ABC) and it is not known if these algorithms can predict ExWAS findings from GWAS data. However, if this were the case, thousands of associated GWAS loci could potentially be resolved to causal genes. Here, we quantified the performance of these algorithms by evaluating their ability to identify ExWAS significant genes for nine traits. We found that Ei, L2G, and PoPs can identify ExWAS significant genes with high areas under the precision recall curve (Ei: 0.52, L2G: 0.37, PoPs: 0.18, ABC: 0.14). Furthermore, we found that for every unit increase in the normalized scores, there was an associated 1.3-4.6-fold increase in the odds of a gene reaching exome-wide significance (Ei: 4.6, L2G: 2.5, PoPs: 2.1, ABC: 1.3). Overall, we found that Ei, L2G, and PoPs can anticipate ExWAS findings from widely available GWAS results. These techniques are therefore promising when well-powered ExWAS data are not readily available and can be used to anticipate ExWAS findings, allowing for prioritization of genes at GWAS loci.

Chemoradiation-induced hearing loss (CRIHL) is one of the most devasting side effects for nasopharyngeal carcinoma (NPC) patients, which seriously affects survivors' long-term quality of life. However, few studies have comprehensively characterized the risk factors for CRIHL. In this study, we found that age at diagnosis, tumor stage, and concurrent cisplatin dose were positively associated with chemoradiation-induced hearing loss. We performed a genome-wide association study (GWAS) in 777 NPC patients and identified rs1050851 (within the exon 2 of NFKBIA), a variant with a high deleteriousness score, to be significantly associated with hearing loss risk (HR = 5.46, 95% CI 2.93-10.18, P = 9.51 × 10-08). The risk genotype of rs1050851 was associated with higher NFKBIA expression, which was correlated with lower cellular tolerance to cisplatin. According to permutation-based enrichment analysis, the variants mapping to 149 hereditary deafness genes were significantly enriched among GWAS top signals, which indicated the genetic similarity between hereditary deafness and CRIHL. Pathway analysis suggested that synaptic signaling was involved in the development of CRIHL. Additionally, the risk score integrating genetic and clinical factors can predict the risk of hearing loss with a relatively good performance in the test set. Collectively, this study shed new light on the etiology of chemoradiation-induced hearing loss, which facilitates high-risk individuals' identification for personalized prevention and treatment.

Leveraging genome-wide association statistics generated from a large study of amyotrophic lateral sclerosis (ALS; 29,612 cases and 122,656 controls) and UK Biobank (UKB; 4,024 phenotypes, up to 361,194 participants), we conducted a phenome-wide analysis of ALS genetic liability and identified 46 genetically correlated traits, such as fluid intelligence score (rg = - 0.21, p = 1.74 × 10-6), "spending time in pub or social club" (rg = 0.24, p = 2.77 × 10-6), non-work related walking (rg = - 0.25, p = 1.95 × 10-6), college education (rg = - 0.15, p = 7.08 × 10-5), "ever diagnosed with panic attacks (rg = 0.39, p = 4.24 × 10-5), and "self-reported other gastritis including duodenitis" (rg = 0.28, p = 1.4 × 10-3). To assess the putative directionality of these genetic correlations, we conducted a latent causal variable analysis, identifying significant genetic causality proportions (gĉp) linking ALS genetic liability to seven traits. While the genetic component of "self-reported other gastritis including duodenitis" showed a causal effect on ALS (gĉp = 0.50, p = 1.26 × 10-29), the genetic liability to ALS is potentially causal for multiple traits, also including an effect on "ever being diagnosed with panic attacks" (gĉp = 0.79, p = 5.011 × 10-15) and inverse effects on "other leisure/social group activities" (gĉp = 0.66, p = 1 × 10-4) and prospective memory result (gĉp = 0.35, p = 0.005). Our subsequent Mendelian randomization analysis indicated that some of these associations may be due to bidirectional effects. In conclusion, this phenome-wide investigation of ALS polygenic architecture highlights the widespread pleiotropy linking this disorder with several health domains.

We provide the first whole genome Copy Number Variant (CNV) study addressing Roma, along with reference populations from South Asia, the Middle East and Europe. Using CNV calling software for short-read sequence data, we identified 3171 deletions and 489 duplications. Taking into account the known population history of the Roma, as inferred from whole genome nucleotide variation, we could discern how this history has shaped CNV variation. As expected, patterns of deletion variation, but not duplication, in the Roma followed those obtained from single nucleotide polymorphisms (SNPs). Reduced effective population size resulting in slightly relaxed natural selection may explain our observation of an increase in intronic (but not exonic) deletions within Loss of Function (LoF)-intolerant genes. Over-representation analysis for LoF-intolerant gene sets hosting intronic deletions highlights a substantial accumulation of shared biological processes in Roma, intriguingly related to signaling, nervous system and development features, which may be related to the known profile of private disease in the population. Finally, we show the link between deletions and known trait-related SNPs reported in the genome-wide association study (GWAS) catalog, which exhibited even frequency distributions among the studied populations. This suggests that, in general human populations, the strong association between deletions and SNPs associated to biomedical conditions and traits could be widespread across continental populations, reflecting a common background of potentially disease/trait-related CNVs.

Migraine-a painful, throbbing headache disorder-is the most common complex brain disorder, yet its molecular mechanisms remain unclear. Genome-wide association studies (GWAS) have proven successful in identifying migraine risk loci; however, much work remains to identify the causal variants and genes. In this paper, we compared three transcriptome-wide association study (TWAS) imputation models-MASHR, elastic net, and SMultiXcan-to characterise established genome-wide significant (GWS) migraine GWAS risk loci, and to identify putative novel migraine risk gene loci. We compared the standard TWAS approach of analysing 49 GTEx tissues with Bonferroni correction for testing all genes present across all tissues (Bonferroni), to TWAS in five tissues estimated to be relevant to migraine, and TWAS with Bonferroni correction that took into account the correlation between eQTLs within each tissue (Bonferroni-matSpD). Elastic net models performed in all 49 GTEx tissues using Bonferroni-matSpD characterised the highest number of established migraine GWAS risk loci (n = 20) with GWS TWAS genes having colocalisation (PP4 > 0.5) with an eQTL. SMultiXcan in all 49 GTEx tissues identified the highest number of putative novel migraine risk genes (n = 28) with GWS differential expression at 20 non-GWS GWAS loci. Nine of these putative novel migraine risk genes were later found to be at and in linkage disequilibrium with true (GWS) migraine risk loci in a recent, more powerful migraine GWAS. Across all TWAS approaches, a total of 62 putative novel migraine risk genes were identified at 32 independent genomic loci. Of these 32 loci, 21 were true risk loci in the recent, more powerful migraine GWAS. Our results provide important guidance on the selection, use, and utility of imputation-based TWAS approaches to characterise established GWAS risk loci and identify novel risk gene loci.

Waardenburg syndrome (WS) is a rare inherited autosomal dominant disorder caused by SOX10, PAX3, MITF, EDNRB, EDN3, and SNAI2. A large burden of pathogenic de novo variants is present in patients with WS, which may be derived from parental mosaicism. Previously, we retrospectively analyzed 90 WS probands with family information. And the frequency of de novo events and parental mosaicism was preliminary investigated in our previous study. In this study, we further explored the occurrence of low-level parental mosaicism in 33 WS families with de novo variants and introduced our procedure of quantifying low-level mosaicism. Mosaic single nucleotide polymorphisms (SNPs) were validated by amplicon-based next-generation sequencing (NGS); copy-number variants (CNVs) were validated by droplet-digital polymerase chain reaction (ddPCR). Molecular validation of low-level mosaicism of WS-causing variants was performed in four families (12.1%, 4/33). These four mosaic variants, comprising three SNVs and one CNV, were identified in SOX10. The rate of parental mosaicism was 25% (4/16) in WS families with de novo SOX10 variants. The lowest allele ratio of a mosaic variant was 2.0% in parental saliva. These de novo WS cases were explained by parental mosaicism conferring an elevated recurrence risk in subsequent pregnancies of parents. Considering its importance in genetic counseling, low-level parental mosaicism should be systematically investigated by personalized sensitive testing. Amplicon-based NGS and ddPCR are recommended to detect and precisely quantify the mosaicism for SNPs and CNVs.

Mutations in TDP2, encoding tyrosyl-DNA phosphodiesterase 2, have been associated with a syndromal form of autosomal recessive spinocerebellar ataxia, type 23 (SCAR23). This is a very rare and progressive neurodegenerative disorder described in only nine patients to date, and caused by splice site or nonsense mutations that result in greatly reduced or absent TDP2 protein. TDP2 is required for the rapid repair of DNA double-strand breaks induced by abortive DNA topoisomerase II (TOP2) activity, important for genetic stability in post-mitotic cells such as neurons. Here, we describe a sibship that is homozygous for the first TDP2 missense mutation (p.Glu152Lys) and which presents with clinical features overlapping both SCAR23 and Fanconi anemia (FA). We show that in contrast to previously reported SCAR23 patients, fibroblasts derived from the current patient retain significant levels of TDP2 protein. However, this protein is catalytically inactive, resulting in reduced rates of repair of TOP2-induced DNA double-strand breaks and cellular hypersensitivity to the TOP2 poison, etoposide. The TDP2-mutated patient-derived fibroblasts do not display increased chromosome breakage following treatment with DNA crosslinking agents, but both TDP2-mutated and FA cells exhibit increased chromosome breakage in response to etoposide. This suggests that the FA pathway is required in response to TOP2-induced DNA lesions, providing a possible explanation for the clinical overlap between FA and the current TDP2-mutated patients. When reviewing the relatively small number of patients with SCAR23 that have been reported, it is clear that the phenotype of such patients can extend beyond neurological features, indicating that the TDP2 protein influences not only neural homeostasis but also other tissues as well.

Risk prediction models hold great promise to reduce the impact of cancer in society through advanced warning of risk and improved preventative modalities. These models are evolving and becoming more complex, increasingly integrating genetic screening data and polygenic risk scores as well as calculating risk for multiple types of a disease. However, unclear regulatory compliance requirements applicable to these models raise significant legal uncertainty and new questions about the regulation of medical devices. This paper aims to address these novel regulatory questions by presenting an initial assessment of the legal status likely applicable to risk prediction models in Canada, using the CanRisk tool for breast and ovarian cancer as an exemplar. Legal analysis is supplemented with qualitative perspectives from expert stakeholders regarding the accessibility and compliance challenges of the Canadian regulatory framework. While the paper focuses on the Canadian context, it also refers to European and U.S. regulations in this domain to contrast them. Legal analysis and stakeholder perspectives highlight the need to clarify and update the Canadian regulatory framework for Software as a Medical Device as it applies to risk prediction models. Findings demonstrate how normative guidance perceived as convoluted, contradictory or overly burdensome can discourage innovation, compliance, and ultimately, implementation. This contribution aims to initiate discussion about a more optimal legal framework for risk prediction models as they continue to evolve and are increasingly integrated into landscape for public health.

Self-domestication could contribute to shaping the biology of human brain and consequently the predisposition to neurodevelopmental disorders. Leveraging genome-wide data from the Psychiatric Genomics Consortium, we tested the enrichment of self-domestication and neural crest function loci with respect to the heritability of autism spectrum disorder, schizophrenia (SCZ in East Asian and European ancestries, EAS and EUR, respectively), attention-deficit/hyperactivity disorder, obsessive-compulsive disorder, and Tourette's syndrome (TS). Considering only self-domestication and neural-crest-function annotations in the linkage disequilibrium score regression (LDSC) model, our partitioned heritability analysis revealed statistically significant enrichments across all disorders investigated. The estimates of the heritability enrichments for self-domestication loci were similar across neurodevelopmental disorders, ranging from 0.902 (EAS SCZ, p = 4.55 × 10-20) to 1.577 (TS, p = 5.85 × 10-5). Conversely, a wider spectrum of heritability enrichment estimates was present for neural crest function with the highest enrichment observed for TS (enrichment = 3.453, p = 2.88 × 10-3) and the lowest for EAS SCZ (enrichment = 1.971, p = 3.81 × 10-3). Although these estimates appear to be strong, the enrichments for self-domestication and neural crest function were null once we included additional annotations related to different genomic features. This indicates that the effect of self-domestication on the polygenic architecture of neurodevelopmental disorders is not independent of other functions of human genome.

Hereditary spastic paraplegias (HSPs) are a group of neurodegenerative disorders characterized by progressive dysfunction of corticospinal motor neurons. Mutations in Atlastin1/Spg3, a small GTPase required for membrane fusion in the endoplasmic reticulum, are responsible for 10% of HSPs. Patients with the same Atlastin1/Spg3 mutation present high variability in age at onset and severity, suggesting a fundamental role of the environment and genetic background. Here, we used a Drosophila model of HSPs to identify genetic modifiers of decreased locomotion associated with atlastin knockdown in motor neurons. First, we screened for genomic regions that modify the climbing performance or viability of flies expressing atl RNAi in motor neurons. We tested 364 deficiencies spanning chromosomes two and three and found 35 enhancer and four suppressor regions of the climbing phenotype. We found that candidate genomic regions can also rescue atlastin effects at synapse morphology, suggesting a role in developing or maintaining the neuromuscular junction. Motor neuron-specific knockdown of 84 genes spanning candidate regions of the second chromosome identified 48 genes required for climbing behavior in motor neurons and 7 for viability, mapping to 11 modifier regions. We found that atl interacts genetically with Su(z)2, a component of the Polycomb repressive complex 1, suggesting that epigenetic regulation plays a role in the variability of HSP-like phenotypes caused by atl alleles. Our results identify new candidate genes and epigenetic regulation as a mechanism modifying neuronal atl pathogenic phenotypes, providing new targets for clinical studies.

CLEC16A is a membrane-associated C-type lectin protein that functions as a E3-ubiquitin ligase. CLEC16A regulates autophagy and mitophagy, and reportedly localizes to late endosomes. GWAS studies have associated CLEC16A SNPs to various auto-immune and neurological disorders, including multiple sclerosis and Parkinson disease. Studies in mouse models imply a role for CLEC16A in neurodegeneration. We identified bi-allelic CLEC16A truncating variants in siblings from unrelated families presenting with a severe neurodevelopmental disorder including microcephaly, brain atrophy, corpus callosum dysgenesis, and growth retardation. To understand the function of CLEC16A in neurodevelopment we used in vitro models and zebrafish embryos. We observed CLEC16A localization to early endosomes in HEK293T cells. Mass spectrometry of human CLEC16A showed interaction with endosomal retromer complex subunits and the endosomal ubiquitin ligase TRIM27. Expression of the human variant leading to C-terminal truncated CLEC16A, abolishes both its endosomal localization and interaction with TRIM27, suggesting a loss-of-function effect. CLEC16A knockdown increased TRIM27 adhesion to early endosomes and abnormal accumulation of endosomal F-actin, a sign of disrupted vesicle sorting. Mutagenesis of clec16a by CRISPR-Cas9 in zebrafish embryos resulted in accumulated acidic/phagolysosome compartments, in neurons and microglia, and dysregulated mitophagy. The autophagocytic phenotype was rescued by wild-type human CLEC16A but not the C-terminal truncated CLEC16A. Our results demonstrate that CLEC16A closely interacts with retromer components and regulates endosomal fate by fine-tuning levels of TRIM27 and polymerized F-actin on the endosome surface. Dysregulation of CLEC16A-mediated endosomal sorting is associated with neurodegeneration, but it also causes accumulation of autophagosomes and unhealthy mitochondria during brain development.

Hereditary hyperekplexia is a rare neuronal disorder characterized by an exaggerated startle response to sudden tactile or acoustic stimuli. In this study, we present a Miniature Australian Shepherd family showing clinical signs, which have genetic and phenotypic similarities with human hereditary hyperekplexia: episodes of muscle stiffness that could occasionally be triggered by acoustic stimuli. Whole genome sequence data analysis of two affected dogs revealed a 36-bp deletion spanning the exon-intron boundary in the glycine receptor alpha 1 (GLRA1) gene. Further validation in pedigree samples and an additional cohort of 127 Miniature Australian Shepherds, 45 Miniature American Shepherds and 74 Australian Shepherds demonstrated complete segregation of the variant with the disease, according to an autosomal recessive inheritance pattern. The protein encoded by GLRA1 is a subunit of the glycine receptor, which mediates postsynaptic inhibition in the brain stem and spinal cord. The canine GLRA1 deletion is located in the signal peptide and is predicted to cause exon skipping and subsequent premature stop codon resulting in a significant defect in glycine signaling. Variants in GLRA1 are known to cause hereditary hyperekplexia in humans; however, this is the first study to associate a variant in canine GLRA1 with the disorder, establishing a spontaneous large animal disease model for the human condition.

Premature ovarian insufficiency (POI) is a common cause of infertility in women, characterised by amenorrhea and elevated FSH under the age of 40 years. In some cases, POI is syndromic in association with other features such as sensorineural hearing loss in Perrault syndrome. POI is a heterogeneous disease with over 80 causative genes known so far; however, these explain only a minority of cases. Using whole-exome sequencing (WES), we identified a MRPL50 homozygous missense variant (c.335T > A; p.Val112Asp) shared by twin sisters presenting with POI, bilateral high-frequency sensorineural hearing loss, kidney and heart dysfunction. MRPL50 encodes a component of the large subunit of the mitochondrial ribosome. Using quantitative proteomics and western blot analysis on patient fibroblasts, we demonstrated a loss of MRPL50 protein and an associated destabilisation of the large subunit of the mitochondrial ribosome whilst the small subunit was preserved. The mitochondrial ribosome is responsible for the translation of subunits of the mitochondrial oxidative phosphorylation machinery, and we found patient fibroblasts have a mild but significant decrease in the abundance of mitochondrial complex I. These data support a biochemical phenotype associated with MRPL50 variants. We validated the association of MRPL50 with the clinical phenotype by knockdown/knockout of mRpL50 in Drosophila, which resulted abnormal ovarian development. In conclusion, we have shown that a MRPL50 missense variant destabilises the mitochondrial ribosome, leading to oxidative phosphorylation deficiency and syndromic POI, highlighting the importance of mitochondrial support in ovarian development and function.

Biallelic SHQ1 variant-related neurodevelopmental disorder is extremely rare. To date, only six affected individuals, from four families, have been reported. Here, we report eight individuals, from seven unrelated families, who exhibited neurodevelopmental disorder and/or dystonia, received whole-genome sequencing, and had inherited biallelic SHQ1 variants. The median age at disease onset was 3.5 months old. All eight individuals exhibited normal eye contact, profound hypotonia, paroxysmal dystonia, and brisk deep tendon reflexes at the first visit. Varying degrees of autonomic dysfunction were observed. One individual had cerebellar atrophy at the initial neuroimaging study, however, three individuals showed cerebellar atrophy at follow-up. Seven individuals who underwent cerebral spinal fluid analysis all had a low level of homovanillic acid in neurotransmitter metabolites. Four individuals who received 99mTc-TRODAT-1 scan had moderate to severe decreased uptake of dopamine in the striatum. Four novel SHQ1 variants in 16 alleles were identified: 9 alleles (56%) were c.997C > G (p.L333V); 4 (25%) were c.195T > A (p.Y65X); 2 (13%) were c.812T > A (p.V271E); and 1 (6%) was c.146T > C (p.L49S). The four novel SHQ1 variants transfected into human SH-SY5Y neuronal cells resulted in a retardation in neuronal migration, suggestive of SHQ1 variant correlated with neurodevelopmental disorders. During the follow-up period, five individuals still exhibited hypotonia and paroxysmal dystonia; two showed dystonia; and one had hypotonia only. The complex interactions among movement disorders, dopaminergic pathways, and the neuroanatomic circuit needs further study to clarify the roles of the SHQ1 gene and protein in neurodevelopment.

The complement system regulator CD55 was initially found to carry the Cromer blood group system antigens, and its complete loss of function was subsequently revealed to cause a severe monogenic gastrointestinal syndrome characterized by protein-losing enteropathy and susceptibility to venous thrombosis. Here we present homozygosity to the CD55 c.596C>T; p.Ser199Leu variant, which was previously described as the Cromer Dr(a-) genotype, in two Bukharan Jewish CD55-deficiency patients with variable disease severity. We confirm that this missense variant causes aberrant splicing and deletion of 44 bp in exon 5, leading to premature termination and low expression of the CD55 protein. Furthermore, Patient 1 exhibited a mildly abnormal B cell immunophenotyping profile. By population screening we established that this variant is highly prevalent in the Bukharan Jewish population, with a carrier frequency of 1:17, suggesting that many similar patients are un- or mis-diagnosed. The phenotypic variability, ranging from abdominal pain when eating a high-fat diet to the full CD55-deficiency phenotype, is likely related to modifiers affecting the proportion of the variant that is able to escape aberrant splicing. Establishing the diagnosis of CD55-deficiency in a timely manner, even in patients with milder symptoms, may have a critical effect on their management and quality-of-life since treatment with the complement inhibitor eculizumab is highly effective in ameliorating disease manifestations. Awareness of founder mutations within certain populations can further guide genetic testing and prevent a diagnostic odyssey, by placing this CD55 variant high on the differential diagnosis.

Type 2 diabetes (T2D) is a critical risk factor for peripheral artery disease (PAD). However, the sex differences in genetic basis, causality, and underlying mechanisms of the two diseases are still unclear. Using sex-stratified and ethnic-based GWAS summary, we explored the genetic correlation and causal relationship between T2D and PAD in both ethnicities and sexes by linkage disequilibrium score regression, LAVA and six Mendelian Randomization approaches. We observed stronger genetic correlations between T2D and PAD in females than males in East Asians and Europeans. East Asian females exhibit higher causal effects of T2D on PAD than males. The gene-level analysis found KCNJ11 and ANK1 genes associated with the cross-trait of T2D and PAD in both sexes. Our study provides genetic evidence for the sex difference of genetic correlations and causal relationships between PAD and T2D, indicating the importance of using sex-specific strategies for monitoring PAD in T2D patients.

One of the most severe forms of infertility in humans, caused by gametogenic failure, is non-obstructive azoospermia (NOA). Approximately, 20-30% of men with NOA may have single-gene mutations or other genetic variables that cause this disease. While a range of single-gene mutations associated with infertility has been identified in prior whole-exome sequencing (WES) studies, current insight into the precise genetic etiology of impaired human gametogenesis remains limited. In this paper, we described a proband with NOA who experienced hereditary infertility. WES analyses identified a homozygous variant in the SUN1 (Sad1 and UNC84 domain containing 1) gene [c. 663C > A: p.Tyr221X] that segregated with infertility. SUN1 encodes a LINC complex component essential for telomeric attachment and chromosomal movement. Spermatocytes with the observed mutations were incapable of repairing double-strand DNA breaks or undergoing meiosis. This loss of SUN1 functionality contributes to significant reductions in KASH5 levels within impaired chromosomal telomere attachment to the inner nuclear membrane. Overall, our results identify a potential genetic driver of NOA pathogenesis and provide fresh insight into the role of the SUN1 protein as a regulator of prophase I progression in the context of human meiosis.

Loss-of-function variants in AP3D1 have been linked to Hermansky-Pudlak syndrome (HPS) 10, a severe multisystem disorder characterized by oculocutaneous albinism, immunodeficiency, neurodevelopmental delay, hearing loss (HL), and neurological abnormalities, fatal in early childhood. Here, we report a consanguineous family who presented with presumably isolated autosomal recessive (AR) HL. Whole-exome sequencing was performed on all core family members, and selected patients were screened using array-based copy-number analysis and karyotyping. Candidate variants were validated by Sanger sequencing and assessed in silico. A homozygous, likely pathogenic p.V711I missense variant in AP3D1 segregated with the HL. The family was characterized by thorough medical and laboratory examination. The HL was consistent across patients and accompanied by neurological manifestations in two brothers. The sole female patient was diagnosed with premature ovarian failure. Further findings, including mild neutropenia and reduced NK-cell cytotoxicity in some as well as brain alterations in all homozygous patients, were reminiscent of HPS10, though milder and lacking the characteristic albinism. Previously unrecognized, milder, isolated HL was identified in all heterozygous carriers. A protein model indicates that the variant interferes with protein-protein interactions. These results suggest that a missense variant alters inner-ear-specific functions leading to HL with mild HPS10-like symptoms of variable penetrance. Milder HL in heterozygous carriers may point towards semi-dominant inheritance of this trait. Since all previously reported HPS10 cases were pediatric, it is unknown whether the observed primary ovarian insufficiency recapitulates the subfertility in Ap3d1-deficient mice.

The minichromosome maintenance (MCM) complex acts as a DNA helicase during DNA replication, and thereby regulates cell cycle progression and proliferation. In addition, MCM-complex components localize to centrosomes and play an independent role in ciliogenesis. Pathogenic variants in genes coding for MCM components and other DNA replication factors have been linked to growth and developmental disorders as Meier-Gorlin syndrome and Seckel syndrome. Trio exome/genome sequencing identified the same de novo MCM6 missense variant p.(Cys158Tyr) in two unrelated individuals that presented with overlapping phenotypes consisting of intra-uterine growth retardation, short stature, congenital microcephaly, endocrine features, developmental delay and urogenital anomalies. The identified variant affects a zinc binding cysteine in the MCM6 zinc finger signature. This domain, and specifically cysteine residues, are essential for MCM-complex dimerization and the induction of helicase activity, suggesting a deleterious effect of this variant on DNA replication. Fibroblasts derived from the two affected individuals showed defects both in ciliogenesis and cell proliferation. We additionally traced three unrelated individuals with de novo MCM6 variants in the oligonucleotide binding (OB)-fold domain, presenting with variable (neuro)developmental features including autism spectrum disorder, developmental delay, and epilepsy. Taken together, our findings implicate de novo MCM6 variants in neurodevelopmental disorders. The clinical features and functional defects related to the zinc binding residue resemble those observed in syndromes related to other MCM components and DNA replication factors, while de novo OB-fold domain missense variants may be associated with more variable neurodevelopmental phenotypes. These data encourage consideration of MCM6 variants in the diagnostic arsenal of NDD.

Fatty acid elongase ELOVL5 is part of a protein family of multipass transmembrane proteins that reside in the endoplasmic reticulum where they regulate long-chain fatty acid elongation. A missense variant (c.689G>T p.Gly230Val) in ELOVL5 causes Spinocerebellar Ataxia subtype 38 (SCA38), a neurodegenerative disorder characterized by autosomal dominant inheritance, cerebellar Purkinje cell demise and adult-onset ataxia. Having previously showed aberrant accumulation of p.G230V in the Golgi complex, here we further investigated the pathogenic mechanisms triggered by p.G230V, integrating functional studies with bioinformatic analyses of protein sequence and structure. Biochemical analysis showed that p.G230V enzymatic activity was normal. In contrast, SCA38-derived fibroblasts showed reduced expression of ELOVL5, Golgi complex enlargement and increased proteasomal degradation with respect to controls. By heterologous overexpression, p.G230V was significantly more active than wild-type ELOVL5 in triggering the unfolded protein response and in decreasing viability in mouse cortical neurons. By homology modelling, we generated native and p.G230V protein structures whose superposition revealed a shift in Loop 6 in p.G230V that altered a highly conserved intramolecular disulphide bond. The conformation of this bond, connecting Loop 2 and Loop 6, appears to be elongase-specific. Alteration of this intramolecular interaction was also observed when comparing wild-type ELOVL4 and the p.W246G variant which causes SCA34. We demonstrate by sequence and structure analyses that ELOVL5 p.G230V and ELOVL4 p.W246G are position-equivalent missense variants. We conclude that SCA38 is a conformational disease and propose combined loss of function by mislocalization and gain of toxic function by ER/Golgi stress as early events in SCA38 pathogenesis.

The molecular basis of Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome remains largely unknown. Pathogenic variants in WNT4 and HNF1B have been confirmed in a small percent of individuals. A variety of copy number variants have been reported, but causal gene(s) remain to be identified. We hypothesized that rare structural variants (SVs) would be present in some individuals with MRKH, which could explain the genetic basis of the syndrome. Large molecular weight DNA was extracted from lymphoblastoid cells from 87 individuals with MRKH and available parents. Optical genome mapping (OGM) was performed to identify SVs, which were confirmed by another method (quantitative PCR, chromosomal microarray, karyotype, or fluorescent in situ hybridization) when possible. Thirty-four SVs that overlapped coding regions of genes with potential involvement in MRKH were identified, 14 of which were confirmed by a second method. These 14 SVs were present in 17/87 (19.5%) of probands with MRKH and included seven deletions, three duplications, one new translocation in 5/50 cells-t(7;14)(q32;q32), confirmation of a previously identified translocation-t(3;16)(p22.3;p13.3), and two aneuploidies. Of interest, three cases of mosaicism (3.4% of probands) were identified-25% mosaicism for trisomy 12, 45,X(75%)/46,XX (25%), and 10% mosaicism for a 7;14 translocation. Our study constitutes the first systematic investigation of SVs by OGM in individuals with MRKH. We propose that OGM is a promising method that enables a comprehensive investigation of a variety of SVs in a single assay including cryptic translocations and mosaic aneuploidies. These observations suggest that mosaicism could play a role in the genesis of MRKH.

A recent genome-wide association study on dyslexia in 51,800 affected European adults and 1,087,070 controls detected 42 genome-wide significant single nucleotide variants (SNPs). The association between rs2624839 in SEMA3F and reading fluency was replicated in a Chinese cohort. This study explores the genetic overlap between Chinese and English word reading, vocabulary knowledge and spelling, and aims at replicating the association in a unique cohort of bilingual (Chinese-English) Hong Kong Chinese twins. Our result showed an almost complete genetic overlap in vocabulary knowledge (r2 = 0.995), and some genetic overlaps in word reading and spelling (r2 = 0.846, 0.687) across the languages. To investigate the region near rs2624839, we tested proxy SNPs (rs1005678, rs12632110 and rs12494414) at the population level (n = 305-308) and the within-twin level (n = 342-344 [171-172 twin pairs]). All the three SNPs showed significant associations with quantitative Chinese and English vocabulary knowledge (p < 0.05). The strongest association after multiple testing correction was between rs12494414 and English vocabulary knowledge at the within-twin level (p = 0.004). There was a trend of associations with word reading and spelling in English but not in Chinese. Our result suggested that the region near rs2624839 is one of the common genetic factors across English and Chinese vocabulary knowledge and unique factors of English word reading and English spelling in bilingual Chinese twins. A larger sample size is required to validate our findings. Further studies on the relationship between variable expression of SEMA3F, which is important to neurodevelopment, and language and literacy are encouraged.

Bi-allelic mutations in the gene coding for human trans-membrane anterior-posterior transformation protein 1 (TAPT1) result in a broad phenotypic spectrum, ranging from syndromic disease with severe skeletal and congenital abnormalities to isolated early-onset cataract. We present here the first patient with a frameshift mutation in the TAPT1 gene, resulting in both bilateral early-onset cataract and skeletal abnormalities, in addition to several dysmorphic features, in this way further expanding the phenotypic spectrum associated with TAPT1 mutations. A tapt1a/tapt1b double knock-out (KO) zebrafish model generated by CRISPR/Cas9 gene editing revealed an early larval phenotype with eye malformations, loss of vision, increased photokinetics and hyperpigmentation, without visible skeletal involvement. Ultrastructural analysis of the eyes showed a smaller condensed lens, loss of integrity of the lens capsule with formation of a secondary lens and hyperplasia of the cells in the ganglion and inner plexiform layers of the retina. Transcriptomic analysis pointed to an impaired lens development with aberrant expression of many of the crystallin and other lens-specific genes. Furthermore, the phototransduction and visual perception pathways were found to be significantly disturbed. Differences in light perception are likely the cause of the increased dark photokinetics and generalized hyperpigmentation observed in this zebrafish model. In conclusion, this study validates TAPT1 as a new gene for early-onset cataract and sheds light on its ultrastructural and molecular characteristics.

CLCN2 encodes a two-pore homodimeric chloride channel protein (CLC-2) that is widely expressed in human tissues. The association between Clcn2 and the retina is well-established in mice, as loss-of-function of CLC-2 can cause retinopathy in mice; however, the ocular phenotypes caused by CLCN2 mutations in humans and the underlying mechanisms remain unclear. The present study aimed to define the ocular features and reveal the pathogenic mechanisms of CLCN2 variants associated with retinal degeneration in humans using an in vitro overexpression system, as well as patient-induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) cells and retinal organoids (ROs). A patient carrying the homozygous c.2257C > T (p.R753X) nonsense CLCN2 mutation was followed up for > 6 years. Ocular features were comprehensively characterized with multimodality imaging and functional examination. The patient presented with severe bilateral retinal degeneration with loss of photoreceptor and RPE. In vitro, mutant CLC-2 maintained the correct subcellular localization, but with reduced channel function compared to wild-type CLC-2 in HEK293T cells. Additionally, patient iPSC-derived RPE cells carrying the CLCN2 mutation exhibited dysfunctional ClC-2 chloride channels and outer segment phagocytosis. Notably, these functions were rescued following the repair of the CLCN2 mutation using the CRISPR-Cas9 system. However, this variant did not cause significant photoreceptor degeneration in patient-derived ROs, indicating that dysfunctional RPE is likely the primary cause of biallelic CLCN2 variant-mediated retinopathy. This study is the first to establish the confirmatory ocular features of human CLCN2-related retinal degeneration, and reveal a pathogenic mechanism associated with biallelic CLCN2 variants, providing new insights into the cause of inherited retinal dystrophies.

Identifying causal genes at GWAS loci can help pinpoint targets for therapeutic interventions. Expression studies can disentangle such loci but signals from expression quantitative trait loci (eQTLs) often fail to colocalize-which means that the genetic control of measured expression is not shared with the genetic control of disease risk. This may be because gene expression is measured in the wrong cell type, physiological state, or organ. We tested whether Mendelian randomization (MR) could identify genes at loci influencing COVID-19 outcomes and whether the colocalization of genetic control of expression and COVID-19 outcomes was influenced by cell type, cell stimulation, and organ. We conducted MR of cis-eQTLs from single cell (scRNA-seq) and bulk RNA sequencing. We then tested variables that could influence colocalization, including cell type, cell stimulation, RNA sequencing modality, organ, symptoms of COVID-19, and SARS-CoV-2 status among individuals with symptoms of COVID-19. The outcomes used to test colocalization were COVID-19 severity and susceptibility as assessed in the Host Genetics Initiative release 7. Most transcripts identified using MR did not colocalize when tested across cell types, cell state and in different organs. Most that did colocalize likely represented false positives due to linkage disequilibrium. In general, colocalization was highly variable and at times inconsistent for the same transcript across cell type, cell stimulation and organ. While we identified factors that influenced colocalization for select transcripts, identifying 33 that mediate COVID-19 outcomes, our study suggests that colocalization of expression with COVID-19 outcomes is partially due to noisy signals even after following quality control and sensitivity testing. These findings illustrate the present difficulty of linking expression transcripts to disease outcomes and the need for skepticism when observing eQTL MR results, even accounting for cell types, stimulation state and different organs.

Cerebral organoids are comprised of diverse cell types found in the developing human brain, and can be leveraged in the identification of critical cell types perturbed by genetic risk variants in common, neuropsychiatric disorders. There is great interest in developing high-throughput technologies to associate genetic variants with cell types. Here, we describe a high-throughput, quantitative approach (oFlowSeq) by utilizing CRISPR-Cas9, FACS sorting, and next-generation sequencing. Using oFlowSeq, we found that deleterious mutations in autism-associated gene KCTD13 resulted in increased proportions of Nestin+ cells and decreased proportions of TRA-1-60+ cells within mosaic cerebral organoids. We further identified that a locus-wide CRISPR-Cas9 survey of another 18 genes in the 16p11.2 locus resulted in most genes with > 2% maximum editing efficiencies for short and long indels, suggesting a high feasibility for an unbiased, locus-wide experiment using oFlowSeq. Our approach presents a novel method to identify genotype-to-cell type imbalances in an unbiased, high-throughput, quantitative manner.

Constitutional mismatch repair deficiency (CMMRD) is an aggressive and highly penetrant cancer predisposition syndrome. Because of its variable clinical presentation and phenotypical overlap with neurofibromatosis, timely diagnosis remains challenging, especially in countries with limited resources. Since current tests are either difficult to implement or interpret or both we used a novel and relatively inexpensive functional genomic assay (LOGIC) which has been recently reported to have high sensitivity and specificity in diagnosing CMMRD. Here we report the clinical and molecular characteristics of nine patients diagnosed with cancer and suspected to have CMMRD and highlight the challenges with variant interpretation and immunohistochemical analysis that led to an uncertain interpretation of genetic findings in 6 of the 9 patients. Using LOGIC, we were able to confirm the diagnosis of CMMRD in 7 and likely exclude it in 2 patients, resolving ambiguous result interpretation. LOGIC also enabled predictive testing of asymptomatic siblings for early diagnosis and implementation of surveillance. This study highlights the varied manifestations and practical limitations of current diagnostic criteria for CMMRD, and the importance of international collaboration for implementing robust and low-cost functional assays for resolving diagnostic challenges.

The application of next-generation sequencing (NGS) to clinical practice is still hampered by the ability to interpret the clinical relevance of novel variants and the difficulty of evaluating their effect in specific tissues. Here, we applied integrated genomic approaches for interrogating blood samples of two unrelated individuals with neurodevelopmental disorders and identified a novel neuro-pathogenic role for the Mitogen-Activated Protein Kinase 4 gene (MAP4K4). In particular, we identified two novel frameshift variants in coding exons expressed in the blood and neuronal isoforms. Both variants were predicted to generate non-sense-mediated decay. By transcriptome analysis, we simultaneously demonstrated the deleterious effect of the identified variants on the splicing activity and stability of MAP4K4 mRNA. Therefore, we propose MAP4K4 as a novel causative gene for non-syndromic and syndromic neurodevelopmental disorders. Altogether, we prove the efficacy of an integrated approach of exome and transcriptome sequencing in the resolution of undiagnosed cases by leveraging the analysis of variants in genes expressed in peripheral blood.

Phosphomannomutase 2 (PMM2) deficiency causes Congenital Disorder of Glycosylation (PMM2-CDG), but does not have a recognised association with Inflammatory Bowel Disease (IBD). A distinct clinical syndrome of hyperinsulinism and autosomal recessive polycystic kidney disease (HIPKD) arises in the context of a specific variant in the PMM2 promotor, either in homozygosity, or compound heterozygous with a deleterious PMM2 variant. Here, we describe the development of IBD in three patients with PMM2-HIPKD, with onset of IBD at 0, 6, and 10 years of age. In each case, intestinal inflammation coincided with the unusual finding of gastric antral foveolar hyperplasia. IBD disease was of variable severity at onset but well controlled with conventional and first-line biologic treatment approaches. The organ-level pattern of disease manifestations in PMM2-HIPKD-IBD may reflect a loss of cis-acting regulatory control by hepatocyte nuclear factor 4 alpha (HNF4A). Analysis of published transcriptomic data suggests that IBD most likely arises due to an impact on epithelial cellular function. We identify a specific pattern of variation in PMM2 as a novel association of early-onset IBD with distinctive gastric pathology.

Exome sequencing (ES) has become the method of choice for diagnosing rare diseases, while the availability of short-read genome sequencing (SR-GS) in a medical setting is increasing. In addition, new sequencing technologies, such as long-read genome sequencing (LR-GS) and transcriptome sequencing, are being increasingly used. However, the contribution of these techniques compared to widely used ES is not well established, particularly in regards to the analysis of non-coding regions. In a pilot study of five probands affected by an undiagnosed neurodevelopmental disorder, we performed trio-based short-read GS and long-read GS as well as case-only peripheral blood transcriptome sequencing. We identified three new genetic diagnoses, none of which affected the coding regions. More specifically, LR-GS identified a balanced inversion in NSD1, highlighting a rare mechanism of Sotos syndrome. SR-GS identified a homozygous deep intronic variant of KLHL7 resulting in a neoexon inclusion, and a de novo mosaic intronic 22-bp deletion in KMT2D, leading to the diagnosis of Perching and Kabuki syndromes, respectively. All three variants had a significant effect on the transcriptome, which showed decreased gene expression, mono-allelic expression and splicing defects, respectively, further validating the effect of these variants. Overall, in undiagnosed patients, the combination of short and long read GS allowed the detection of cryptic variations not or barely detectable by ES, making it a highly sensitive method at the cost of more complex bioinformatics approaches. Transcriptome sequencing is a valuable complement for the functional validation of variations, particularly in the non-coding genome.

Neurodevelopmental disorders (NDDs) and congenital anomalies (CAs) are rare disorders with complex etiology. In this study, we investigated the less understood genomic overlap of copy number variants (CNVs) in two large cohorts of NDD and CA patients to identify de novo CNVs and candidate genes associated with both phenotypes. We analyzed clinical microarray CNV data from 10,620 NDD and 3176 CA cases annotated using Horizon platform of GenomeArc Analytics and applied rigorous downstream analysis to evaluate overlapping genes from NDD and CA CNVs. Out of 13,796 patients, only 195 cases contained 218 validated de novo CNVs. Eighteen percent (31/170) de novo CNVs in NDD cases and 40% (19/48) de novo CNVs in CA cases contained genomic overlaps impacting developmentally constraint genes. Seventy-nine constraint genes (10.1% non-OMIM entries) were found to have significantly enriched genomic overlap within rare de novo pathogenic deletions (P value = 0.01, OR = 1.58) and 45 constraint genes (13.3% non-OMIM entries) within rare de novo pathogenic duplications (P value = 0.01, OR = 1.97). Analysis of spatiotemporal transcriptome demonstrated both pathogenic deletion and duplication genes to be highly expressed during the prenatal stage in human developmental brain (P value = 4.95 X 10-6). From the list of overlapping genes, EHMT1, an interesting known NDD gene encompassed pathogenic deletion CNVs from both NDD and CA patients, whereas FAM189A1, and FSTL5 are new candidate genes from non-OMIM entries. In summary, we have identified constraint overlapping genes from CNVs (including de novo) in NDD and CA patients that have the potential to play a vital role in common disease etiology.

Inadequate representation of non-European ancestry populations in genome-wide association studies (GWAS) has limited opportunities to isolate functional variants. Fine-mapping in multi-ancestry populations should improve the efficiency of prioritizing variants for functional interrogation. To evaluate this hypothesis, we leveraged ancestry architecture to perform comparative GWAS and fine-mapping of obesity-related phenotypes in European ancestry populations from the UK Biobank (UKBB) and multi-ancestry samples from the Population Architecture for Genetic Epidemiology (PAGE) consortium with comparable sample sizes. In the investigated regions with genome-wide significant associations for obesity-related traits, fine-mapping in our ancestrally diverse sample led to 95% and 99% credible sets (CS) with fewer variants than in the European ancestry sample. Lead fine-mapped variants in PAGE regions had higher average coding scores, and higher average posterior probabilities for causality compared to UKBB. Importantly, 99% CS in PAGE loci contained strong expression quantitative trait loci (eQTLs) in adipose tissues or harbored more variants in tighter linkage disequilibrium (LD) with eQTLs. Leveraging ancestrally diverse populations with heterogeneous ancestry architectures, coupled with functional annotation, increased fine-mapping efficiency and performance, and reduced the set of candidate variants for consideration for future functional studies. Significant overlap in genetic causal variants across populations suggests generalizability of genetic mechanisms underpinning obesity-related traits across populations.

Comorbid conditions can be driven by underlying pleiotropic and causal mechanisms that can provide insights into shared molecular and biological processes contributing to disease risk. Endometriosis is a chronic condition affecting one in nine women of reproductive age and poses many challenges including lengthy diagnostic delays and limited treatment efficacy owing to poor understanding of disease aetiology. To shed light on the underlying biological mechanisms and to identify potential risk factors, we examine the epidemiological and genomic relationship between endometriosis and its comorbidities. In the UK Biobank 292 ICD10 codes were epidemiologically correlated with endometriosis diagnosis, including gynaecological, immune, infection, pain, psychiatric, cancer, gastrointestinal, urinary, bone and cardiovascular traits. A subset of the identified comorbidities (n = 76) underwent follow-up genetic analysis. Whilst Mendelian randomisation suggested causality was not responsible for most comorbid relationships, 22 traits were genetically correlated with endometriosis, including pain, gynaecological and gastrointestinal traits, suggestive of a shared genetic background. Pleiotropic genetic variants and genes were identified using gene-based and colocalisation analysis. Shared genetic risk factors and potential target genes suggest a diverse collection of biological systems are involved in these comorbid relationships including coagulation factors, development of the female reproductive tract and cell proliferation. These findings highlight the diversity of traits with epidemiological and genomic overlap with endometriosis and implicate a key role for pleiotropy in the comorbid relationships.

Neuronal ceroid lipofuscinoses (NCLs) are neurodegenerative lysosomal storage diseases which are considered among the most frequent causes of dementia in childhood worldwide This study aimed to identify the gene variants, molecular etiologies, and clinical features in 23 unrelated Iranian families with NCL. In total, 29 patients with neuronal ceroid lipofuscinoses (NCLs), diagnosed based on clinical manifestations, MRI neuroimaging, and electroencephalography (EEG), were recruited for this study. Through whole-exome sequencing (WES), functional prediction, Sanger sequencing, and segregation analysis, we found that 12 patients (41.3%) with mutations in the CLN6 gene, 7 patients (24%) with the TPP1 (CLN2) gene variants, and 4 patients (13.7%) with mutations in the MFSD8 (CLN7) gene. Also, mutations in each of the CLN3 and CLN5 genes were detected in 2 cases and mutations of each PPT1 (CLN1) and CLN8 gene were observed in only 1 separate patient. We identified 18 different mutations, 11 (61%) of which are novel, never have been reported before, and the others have been previously described. The gene variants identified in this study expand the number of published clinical cases and the variant frequency spectrum of the neuronal ceroid lipofuscinoses (NCLs) genes; moreover, the identification of these variants supplies foundational clues for future NCL diagnosis and therapy.

The co-occurrence of migraine and glycemic traits has long been reported in observational epidemiological studies, but it has remained unknown how they are linked genetically. We used large-scale GWAS summary statistics on migraine, headache, and nine glycemic traits in European populations to perform cross-trait analyses to estimate genetic correlation, identify shared genomic regions, loci, genes, and pathways, and test for causal relationships. Out of the nine glycemic traits, significant genetic correlation was observed for fasting insulin (FI) and glycated haemoglobin (HbA1c) with both migraine and headache, while 2-h glucose was genetically correlated only with migraine. Among 1703 linkage disequilibrium (LD) independent regions of the genome, we found pleiotropic regions between migraine and FI, fasting glucose (FG), and HbA1c, and pleiotropic regions between headache and glucose, FI, HbA1c, and fasting proinsulin. Cross-trait GWAS meta-analysis with glycemic traits, identified six novel genome-wide significant lead SNPs with migraine, and six novel lead SNPs with headache (Pmeta < 5.0 × 10-8 and Psingle-trait < 1 × 10-4), all of which were LD-independent. Genes with a nominal gene-based association (Pgene ≤ 0.05) were significantly enriched (overlapping) across the migraine, headache, and glycemic traits. Mendelian randomisation analyses produced intriguing, but inconsistent, evidence for a causal relationship between migraine and headache with multiple glycemic traits; and consistent evidence suggesting increased fasting proinsulin levels may causally decrease the risk of headache. Our findings indicate that migraine, headache, and glycemic traits share a common genetic etiology and provide genetic insights into the molecular mechanisms contributing to their comorbid relationship.

Biological samples containing DNA that is attributed to deceased relatives, can now undergo genetic testing at a reasonable cost due to revolutionary improvements in sampling, sequencing, and analytical techniques. This artifact DNA testing, or 'artDNA', includes genetic analysis of hair locks, stamps, envelopes with saliva traces or teeth. ArtDNA can reveal valuable information about a deceased relative or one's genetic background, but it also presents novel ethical dilemmas and legal uncertainties for genetic researchers and commercial testing services. In this paper, we provide an analysis of some of the unique ethical and legal risks of such testing and provide needed recommendations for practitioners of private family artDNA testing. ArtDNA testing generates ethical and legal risks regarding the privacy and autonomy of deceased individuals, the rights of living relatives over their ancestor's genetic information, and the rights of living persons to control their own genetic information. To mitigate these risks, practitioners can conduct certain preliminary testing to ascertain the identity of a DNA donor and estimate the time that has elapsed postmortem. Generally, the ethical and legal concerns will be higher when a shorter period has passed between the death of the DNA donor and the time of artifact DNA testing. Regardless, all artDNA testing present some risks, and practitioners should exercise professional judgement as necessary.

Deleterious variants in collagen genes are the most common cause of hereditary connective tissue disorders (HCTD). Adaptations of the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) criteria are still lacking. A multidisciplinary team was set up for developing specifications of the ACMG/AMP criteria for COL1A1, COL1A2, COL2A1, COL3A1, COL5A1, COL5A2, COL11A1, COL11A2 and COL12A1, associated with various forms of HCTD featuring joint hypermobility, which is becoming one of the most common reasons of referral for molecular testing in this field. Such specifications were validated against 209 variants, and resulted effective for classifying as pathogenic and likely pathogenic null alleles without downgrading of the PVS1 level of strength and recurrent Glycine substitutions. Adaptations of selected criteria reduced uncertainties on private Glycine substitutions, intronic variants predicted to affect the splicing, and null alleles with a downgraded PVS1 level of strength. Segregation and multigene panel sequencing data mitigated uncertainties on non-Glycine substitutions by the attribution of one or more benignity criteria. These specifications may improve the clinical utility of molecular testing in HCTD by reducing the number of variants with neutral/conflicting interpretations. Close interactions between laboratory and clinicians are crucial to estimate the a priori utility of molecular test and to improve medical reports.

Congenital diarrheas and enteropathies (CODEs) constitute a heterogeneous group of individually rare disorders manifesting with infantile-onset chronic diarrhea. Genomic deletions in chromosome 16, encompassing a sequence termed the 'intestine-critical region (ICR)', were recently identified as the cause of an autosomal recessive congenital enteropathy. The regulatory sequence within the ICR is flanked by an unannotated open reading frame termed PERCC1, which plays a role in enteroendocrine cell (EEC) function. We investigated two unrelated children with idiopathic congenital diarrhea requiring home parenteral nutrition attending the Irish Intestinal Failure Program. Currently 12 and 19-years old, these Irish male patients presented with watery diarrhea and hypernatremic dehydration in infancy. Probands were phenotyped by comprehensive clinical investigations, including endoscopic biopsies and serum gastrin level measurements. Following negative exome sequencing, PCR and Sanger sequencing of the entire coding region and intron boundaries of PERCC1 were performed for each proband and their parents. In both patients, serum gastrin levels were low and failed to increase following a meal challenge. While no deletions involving the ICR were detected, targeted sequencing of the PERCC1 gene revealed a shared homozygous c.390C > G stop gain variant. We report clinical and molecular findings in two unrelated patients harboring a shared homozygous variant in PERCC1, comprising the first description of a point mutation in this gene in association with CODE. That both parenteral nutrition dependent children with unexplained diarrhea at our institution harbored a PERCC1 mutation underscores the importance of its inclusion in exome sequencing interpretation.

Neurofibromatosis type 1 results from loss-of-function NF1 pathogenic variants (PVs). Up to 30% of all NF1 PVs disrupt mRNA splicing, including deep intronic variants. Here, we retrospectively investigated the spectrum of NF1 deep intronic PVs in a cohort of 8,090 unrelated individuals from the University of Alabama at Birmingham (UAB) dataset with a molecularly confirmed neurofibromatosis type 1. All variants were identified through their effect on the NF1 transcript, followed by variant characterization at the DNA-level. A total of 68 distinct variants, which were ≥ 20 nucleotides away from the closest exon-intron junction, were identified in 2.5% unrelated individuals with NF1 (200/8,090). Nine different pathogenic splice variants, identified in 20 probands, led to exonization of different parts of intron 30 [23.2] or 31 [23a]. The two major NF1 transcript isoforms, distinguished by the absence (type I) or presence (type II) of the alternatively spliced cassette exon 31 [23a], are equally expressed in blood in control individuals without NF1 or NF1-affected individuals carrying their PV not in the introns flanking exon 31 [23a]. By fragment and cloning analysis we demonstrated that the exonization of intron 31 [23a] sequences due to deep intronic PV predominantly affects the NF1 isoform II. Seven additional (likely) pathogenic NF1 deep intronic variants not observed in the UAB dataset were found by classification of 36 variants identified by a literature search. Hence, the unique list of these 75 deep intronic (likely) PVs should be included in any comprehensive NF1 testing strategy.

Single-nucleotide variants (SNVs) and copy number variations (CNVs) are the most common genomic variations that cause phenotypic diversity and genetic disorders. MALDI-TOF-MS is a rapid and cost-effective technique for multi-variant genotyping, but it is challenging to efficiently detect CNVs and clustered SNVs, especially to simultaneously detect CNVs and SNVs in one reaction. Herein, a novel strategy termed Target-Allele-Specific Probe Single-Base Extension (TASP-SBE) was devised to efficiently detect CNVs and clustered SNVs with MALDI-TOF-MS. By comprehensive use of traditional SBE and TASP-SBE strategies, a MALDI-TOF-MS assay was also developed to simultaneously detect 28 α-/β-thalassemia mutations in a single reaction system, including 4 α-thalassemia deletions, 3 HBA and 21 HBB SNVs. The results showed that all 28 mutations were sensitively identified, and the CNVs of HBA/HBB genes were also accurately analyzed based on the ratio of peak height (RPH) between the target allele and reference gene. The double-blind evaluation results of 989 thalassemia carrier samples showed a 100% concordance of this assay with other methods. In conclusion, a one-tube MALDI-TOF-MS assay was developed to simultaneously genotype 28 thalassemia mutations. This novel TASP-SBE was also verified a practicable strategy for the detection of CNVs and clustered SNVs, providing a feasible approach for multi-variants analysis with MALDI-TOF-MS technique.