Renal ciliopathies are the leading cause of inherited kidney failure. In autosomal dominant polycystic kidney disease (ADPKD), mutations in the ciliary gene PKD1 lead to the induction of CCL2, which promotes macrophage infiltration in the kidney. Whether or not mutations in genes involved in other renal ciliopathies also lead to immune cells recruitment is controversial. Through the parallel analysis of patients' derived material and murine models, we investigated the inflammatory components of nephronophthisis (NPH), a rare renal ciliopathy affecting children and adults. Our results show that NPH mutations lead to kidney infiltration by neutrophils, macrophages and T cells. Contrary to ADPKD, this immune cell recruitment does not rely on the induction of CCL2 in mutated cells, which is dispensable for disease progression. Through an unbiased approach, we identified a set of inflammatory cytokines that are upregulated precociously and independently of CCL2 in murine models of NPH. The majority of these transcripts is also upregulated in NPH patient renal cells at a level exceeding those found in common non-immune chronic kidney diseases. This study reveals that inflammation is a central aspect in NPH and delineates a specific set of inflammatory mediators that likely regulates immune cell recruitment in response to NPH genes mutations.

Novel protein kinase C (nPKC) family member, protein kinase C epsilon (PKCε) is an AGC kinase superfamily member. It is associated with neurological and metabolic diseases as well as human cancers. No study so far has been conducted to identify genetic variations and their effect on PKCε folding and functioning. The present study aimed to identify mutational hotspots in PKCε and disease-causing non-synonymous variants (nsSNPs) along with the investigation of nsSNP impact on protein dynamics. Twenty-nine in silico tools were applied to determine nsSNP deleteriousness, their impact on protein dynamics and disease association, along with the prediction of PKCε post-translational modification (PTM) sites. The present study's outcomes indicated that most nsSNPs were concentrated in the PKCε hinge region and C-terminal tail. Most pathogenic variants mapped to the kinase domain. Regulatory domain variants influenced PKCε interaction with molecular players whereas kinase domain variants were predicted to impact its phosphorylation pattern and protein-protein interactions. Most PTM sites were mapped to the hinge region. PKCε nsSNPs have an association with oncogenicity and its expression dysregulation is responsible for poor overall survival. Understanding nsSNP structural impact is a primary step necessary for delineating the relationship of genetic level differences with protein phenotype. The obtained knowledge can eventually help in disease diagnosis and therapy design.

Multiple sclerosis (MS) is a complex and demyelinating disease of the central nervous system. One of the challenges of the post-genome-wide association studies (GWAS) era is to understand the molecular basis of statistical associations to reveal gene networks and potential therapeutic targets. The L3MBTL3 locus has been associated with MS risk by GWAS. To identify the causal variant of the locus, we performed fine mapping in a cohort of 3440 MS patients and 1688 healthy controls. The variant that best explained the association was rs6569648 (P = 4.13E-10, odds ratio = 0.71, 95% confidence interval (CI) = 0.64-0.79), which tagged rs7740107, located in intron 7 of L3MBTL3. The rs7740107 (A/T) variant has been reported to be the best expression and splice quantitative trait locus (eQTL and sQTL) of the region in up to 35 human genotype-tissue expression (GTEx) tissues. By sequencing RNA from blood of 17 MS patients and quantification by digital qPCR, we determined that this eQTL/sQTL originated from the expression of a novel short transcript starting in intron 7 near rs7740107. The short transcript was translated into three proteins starting at different translation initiation codons. These N-terminal truncated proteins lacked the region where L3MBTL3 interacts with the transcriptional regulator Recombination Signal Binding Protein for Immunoglobulin Kappa J Region which, in turn, regulates the Notch signalling pathway. Our data and other functional studies suggest that the genetic mechanism underlying the MS association of rs7740107 affects not only the expression of L3MBTL3 isoforms, but might also involve the Notch signalling pathway.

Noise-induced hearing loss (NIHL) is a multifactorial disease caused by environmental, genetic and epigenetic variables. SUMOylation is a post-translational modification that regulates biological processes. The objective of this study was to determine the link between genetic variation in the chromobox 4 (CBX4) and the risk of NIHL. This study applied a case-control design with 588 cases and 582 controls, and the sample was predominantly male (93.76%). The T allele of CBX4 rs1285250 was found to be significantly linked with NIHL (P = 0.002) and showed strong associations in both the codominant and recessive models (TT versus CC, P = 0.005; TT/TC versus CC, P = 0.009). By constructing a mouse model of hearing loss because of noise exposure, changes in hearing thresholds were observed in noise-exposed mice, along with a decrease in the number of cochlear hair cells. Furthermore, noise promotes cochlear hair cell apoptosis by inducing SP1/CBX4 pathway activation. Further functional studies demonstrated that SP1 has an influence on the promoter activity of the CBX4 rs1285250 intron, with the promoter activity of the T allele being higher than that of the C allele. Knockdown of transcription factor SP1 reduced the expression of CBX4 expression and simultaneously reduced apoptosis in HEI-OC1 cells. Together, our findings have shown that CBX4 genetic polymorphism rs1285250 T-allele was associated with increased risk of NIHL and might be used as biomarkers for male workers exposed to noise. Furthermore, we speculate that the CBX4 of rs1285250 T-allele leads to a stronger potential enhancer activity from a predicted gain of stronger SP1 binding.

Manganese (Mn) is an essential mineral, but excess exposure can cause dopaminergic neurotoxicity. Restless legs syndrome (RLS) is a common neurological disorder, but the etiology and pathology remain largely unknown. The purpose of this study was to identify the role of Mn in the regulation of an RLS genetic risk factor BTBD9, characterize the function of BTBD9 in Mn-induced oxidative stress and dopaminergic neuronal dysfunction. We found that human subjects with high blood Mn levels were associated with decreased BTBD9 mRNA levels, when compared with subjects with low blood Mn levels. In A549 cells, Mn exposure decreased BTBD9 protein levels. In Caenorhabditis elegans, loss of hpo-9 (BTBD9 homolog) resulted in more susceptibility to Mn-induced oxidative stress and mitochondrial dysfunction, as well as decreased dopamine levels and alternations of dopaminergic neuronal morphology and behavior. Overexpression of hpo-9 in mutant animals restored these defects and the protection was eliminated by mutation of the forkhead box O (FOXO). In addition, expression of hpo-9 upregulated FOXO protein levels and decreased protein kinase B levels. These results suggest that elevated Mn exposure might be an environmental risk factor for RLS. Furthermore, BTBD9 functions to alleviate Mn-induced oxidative stress and neurotoxicity via regulation of insulin/insulin-like growth factor signaling pathway.

Recessive mutations in the CTNS gene encoding the lysosomal transporter cystinosin cause cystinosis, a lysosomal storage disease leading to kidney failure and multisystem manifestations. A Ctns knockout mouse model recapitulates features of cystinosis, but the delayed onset of kidney manifestations, phenotype variability and strain effects limit its use for mechanistic and drug development studies. To provide a better model for cystinosis, we generated a Ctns knockout rat model using CRISPR/Cas9 technology. The Ctns-/- rats display progressive cystine accumulation and crystal formation in multiple tissues including kidney, liver and thyroid. They show an early onset and progressive loss of urinary solutes, indicating generalized proximal tubule dysfunction, with development of typical swan-neck lesions, tubulointerstitial fibrosis and kidney failure, and decreased survival. The Ctns-/- rats also present crystals in the cornea, and bone and liver defects, as observed in patients. Mechanistically, the loss of cystinosin induces a phenotype switch associating abnormal proliferation and dedifferentiation, loss of apical receptors and transporters, and defective lysosomal activity and autophagy in the cells. Primary cultures of proximal tubule cells derived from the Ctns-/- rat kidneys confirmed the key changes caused by cystine overload, including reduced endocytic uptake, increased proliferation and defective lysosomal dynamics and autophagy. The novel Ctns-/- rat model and derived proximal tubule cell system provide invaluable tools to investigate the pathogenesis of cystinosis and to accelerate drug discovery.

Bloom syndrome (BS) is an autosomal recessive disease clinically characterized by primary microcephaly, growth deficiency, immunodeficiency and predisposition to cancer. It is mainly caused by biallelic loss-of-function mutations in the BLM gene, which encodes the BLM helicase, acting in DNA replication and repair processes. Here, we describe the gene expression profiles of three BS fibroblast cell lines harboring causative, biallelic truncating mutations obtained by single-cell (sc) transcriptome analysis. We compared the scRNA transcription profiles from three BS patient cell lines to two age-matched wild-type controls and observed specific deregulation of gene sets related to the molecular processes characteristically affected in BS, such as mitosis, chromosome segregation, cell cycle regulation and genomic instability. We also found specific upregulation of genes of the Fanconi anemia pathway, in particular FANCM, FANCD2 and FANCI, which encode known interaction partners of BLM. The significant deregulation of genes associated with inherited forms of primary microcephaly observed in our study might explain in part the molecular pathogenesis of microcephaly in BS, one of the main clinical characteristics in patients. Finally, our data provide first evidence of a novel link between BLM dysfunction and transcriptional changes in condensin complex I and II genes. Overall, our study provides novel insights into gene expression profiles in BS on an sc level, linking specific genes and pathways to BLM dysfunction.

Inguinal hernias are some of the most frequently diagnosed conditions in clinical practice and inguinal hernia repair is the most common procedure performed by general surgeons. Studies of inguinal hernias in non-European populations are lacking, though it is expected that such studies could identify novel loci. Further, the cumulative lifetime incidence of inguinal hernia is nine times greater in men than women, however, it is not clear why this difference exists. We conducted a genome-wide association meta-analysis of inguinal hernia risk across 513 120 individuals (35 774 cases and 477 346 controls) of Hispanic/Latino, African, Asian and European descent, with replication in 728 418 participants (33 491 cases and 694 927 controls) from the 23andMe, Inc dataset. We identified 63 genome-wide significant loci (P < 5 × 10-8), including 41 novel. Ancestry-specific analyses identified two loci (LYPLAL1-AS1/SLC30A10 and STXBP6-NOVA1) in African ancestry individuals. Sex-stratified analyses identified two loci (MYO1D and ZBTB7C) that are specific to women, and four (EBF2, EMX2/RAB11FIP2, VCL and FAM9A/FAM9B) that are specific to men. Functional experiments demonstrated that several of the associated regions (EFEMP1 and LYPLAL1-SLC30A10) function as enhancers and show differential activity between risk and reference alleles. Our study highlights the importance of large-scale genomic studies in ancestrally diverse populations for identifying ancestry-specific inguinal hernia susceptibility loci and provides novel biological insights into inguinal hernia etiology.

The genetic regulation of ovarian development remains largely unclear. Indeed, in most cases of impaired ovarian development-such as 46,XX disorders of sex development (DSD) without SRY, and premature ovarian insufficiency (POI)-the genetic causes have not been identified, and the vast majority of disease-associated sequence variants could lie within non-coding regulatory sequences. In this study, we aimed to identify enhancers of five ovarian genes known to play key roles in early ovarian development, basing our analysis on the expression of enhancer derived transcripts (eRNAs), which are considered to characterize active enhancers. Temporal expression profile changes in mouse WT1-positive ovarian cells were obtained from cap analysis of gene expression at E13.5, E16.5 and P0. We compared the chronological expression profiles of ovarian-specific eRNA with expression profiles for each of the ovarian-specific genes, yielding two candidate sequences for enhancers of Wnt4 and Rspo1. Both sequences are conserved between mouse and human, and we confirmed their enhancer activities using transient expression assays in murine granulosa cells. Furthermore, by sequencing the region in patients with impaired ovarian development in 24 patients, such as POI, gonadal dysgenesis and 46,XX DSD, we identified rare single nucleotide variants in both sequences. Our results demonstrate that combined analysis of the temporal expression profiles of eRNA and mRNA of target genes presents a powerful tool for locating cis-element enhancers, and a means of identifying disease-associated sequence variants that lie within non-coding regulatory sequences, thus advancing an important unmet need in forward human genetics.

Retinal diseases exhibit extensive genetic heterogeneity and complex etiology with varying onset and severity. Mutations in over 200 genes can lead to photoreceptor dysfunction and/or cell death in retinal neurodegeneration. To deduce molecular pathways that initiate and/or drive cell death, we adopted a temporal multiomics approach and examined molecular and cellular events in newborn and developing photoreceptors before the onset of degeneration in a widely-used Pde6brd1/rd1 (rd1) mouse, a model of autosomal recessive retinitis pigmentosa caused by PDE6B mutations. Transcriptome profiling of neonatal and developing rods from the rd1 retina revealed early downregulation of genes associated with anabolic pathways and energy metabolism. Quantitative proteomics of rd1 retina showed early changes in calcium signaling and oxidative phosphorylation, with specific partial bypass of complex I electron transfer, which precede the onset of cell death. Concurrently, we detected alterations in central carbon metabolism, including dysregulation of components associated with glycolysis, pentose phosphate and purine biosynthesis. Ex vivo assays of oxygen consumption and transmission electron microscopy validated early and progressive mitochondrial stress and abnormalities in mitochondrial structure and function of rd1 rods. These data uncover mitochondrial overactivation and related metabolic alterations as determinants of early pathology and implicate aberrant calcium signaling as an initiator of higher mitochondrial stress. Our studies thus provide a mechanistic framework with mitochondrial damage and metabolic disruptions as early drivers of photoreceptor cell death in retinal degeneration.

Sotos syndrome (SS), the most common overgrowth with intellectual disability (OGID) disorder, is caused by inactivating germline mutations of NSD1, which encodes a histone H3 lysine 36 methyltransferase. To understand how NSD1 inactivation deregulates transcription and DNA methylation (DNAm), and to explore how these abnormalities affect human development, we profiled transcription and DNAm in SS patients and healthy control individuals. We identified a transcriptional signature that distinguishes individuals with SS from controls and was also deregulated in NSD1-mutated cancers. Most abnormally expressed genes displayed reduced expression in SS; these downregulated genes consisted mostly of bivalent genes and were enriched for regulators of development and neural synapse function. DNA hypomethylation was strongly enriched within promoters of transcriptionally deregulated genes: overexpressed genes displayed hypomethylation at their transcription start sites while underexpressed genes featured hypomethylation at polycomb binding sites within their promoter CpG island shores. SS patients featured accelerated molecular aging at the levels of both transcription and DNAm. Overall, these findings indicate that NSD1-deposited H3K36 methylation regulates transcription by directing promoter DNA methylation, partially by repressing polycomb repressive complex 2 (PRC2) activity. These findings could explain the phenotypic similarity of SS to OGID disorders that are caused by mutations in PRC2 complex-encoding genes.

Age-related macular degeneration (AMD) and central serous chorioretinopathy (CSC) are common diseases that can cause vision loss in older and younger populations. These diseases share pathophysiological conditions derived from retinal pigment epithelium (RPE) dysfunction. Tumor necrosis factor receptor superfamily 10A (TNFRSF10A)-LOC389641 with the same lead single-nucleotide polymorphism (SNP) (rs13278062) is the only overlapped susceptibility locus found in both AMD and CSC through genome-wide association studies. This lead SNP has been reported to alter the transcriptional activity of TNFRSF10A. This study aimed to elucidate the function of TNFRSF10A in RPE degeneration using human primary RPE cells and Tnfrsf10 knockout (Tnfrsf10-/-) mice. TNFRSF10A was found to be localized in human RPE. In vitro assays revealed that a T allele of rs13278062, the risk allele for AMD and CSC, downregulated TNFRSF10A transcription in RPE, leading to decreased cell viability and increased apoptosis through protein kinase C-α (PKCA) downregulation. Treatment with phorbol 12-myristate 13-acetate, a PKC activator, rescued the cell viability. Morphological RPE abnormality was found in the retina of Tnfrsf10-/- mice. Our data suggest that downregulation of TNFRSF10A expression inactivates PKCA signaling and causes cellular vulnerability of the RPE, which may contribute to the pathogenesis of AMD and CSC.

Childhood socioeconomic position (SEP) is a major determinant of health and well-being across the entire life course. To effectively prevent and reduce health risks related to SEP, it is critical to better understand when and under what circumstances socioeconomic adversity shapes biological processes. DNA methylation (DNAm) is one such mechanism for how early life adversity 'gets under the skin'. In this study, we evaluated the dynamic relationship between SEP and DNAm across childhood using data from 946 mother-child pairs in the Avon Longitudinal Study of Parents and Children (ALSPAC). We assessed six SEP indicators spanning financial, occupational, and residential domains during very-early childhood (ages 0-2), early childhood (ages 3-5), and middle childhood (ages 6-7). Epigenome-wide DNAm were measured at 412956 CpGs from peripheral blood at age 7. Using an innovative two-stage structured life course modeling approach, we tested three life-course hypotheses for how SEP shapes DNAm profiles-accumulation, sensitive period, and mobility. We showed that changes in the socioeconomic environment were associated with the greatest differences in DNAm, and that middle childhood may be a potential sensitive period when socioeconomic instability is especially important in shaping DNAm. Top SEP-related DNAm CpGs were overrepresented in genes involved in pathways important for neural development, immune function, and metabolic processes. Our findings highlight the importance of socioeconomic stability during childhood and if replicated, may emphasize the need for public programs to help children and families experiencing socioeconomic instability and other forms of socioeconomic adversity.

The human choroid is a heterogeneous, highly vascular connective tissue that dysfunctions in age-related macular degeneration (AMD). In this study, we performed single-cell RNA sequencing on 21 human choroids, 11 of which were derived from donors with early atrophic or neovascular AMD. Using this large donor cohort, we identified new gene expression signatures and immunohistochemically characterized discrete populations of resident macrophages, monocytes/inflammatory macrophages and dendritic cells. These three immune populations demonstrated unique expression patterns for AMD genetic risk factors, with dendritic cells possessing the highest expression of the neovascular AMD-associated MMP9 gene. Additionally, we performed trajectory analysis to model transcriptomic changes across the choroidal vasculature, and we identified expression signatures for endothelial cells from choroidal arterioles and venules. Finally, we performed differential expression analysis between control, early atrophic AMD, and neovascular AMD samples, and we observed that early atrophic AMD samples had high expression of SPARCL1, a gene that has been shown to increase in response to endothelial damage. Likewise, neovascular endothelial cells harbored gene expression changes consistent with endothelial cell damage and demonstrated increased expression of the sialomucins CD34 and ENCM, which were also observed at the protein level within neovascular membranes. Overall, this study characterizes the molecular features of new populations of choroidal endothelial cells and mononuclear phagocytes in a large cohort of AMD and control human donors.

Previous genome-wide association studies (GWAS) of hematological traits have identified over 10 000 distinct trait-specific risk loci. However, at these loci, the underlying causal mechanisms remain incompletely characterized. To elucidate novel biology and better understand causal mechanisms at known loci, we performed a transcriptome-wide association study (TWAS) of 29 hematological traits in 399 835 UK Biobank (UKB) participants of European ancestry using gene expression prediction models trained from whole blood RNA-seq data in 922 individuals. We discovered 557 gene-trait associations for hematological traits distinct from previously reported GWAS variants in European populations. Among the 557 associations, 301 were available for replication in a cohort of 141 286 participants of European ancestry from the Million Veteran Program. Of these 301 associations, 108 replicated at a strict Bonferroni adjusted threshold ($\alpha$= 0.05/301). Using our TWAS results, we systematically assigned 4261 out of 16 900 previously identified hematological trait GWAS variants to putative target genes. Compared to coloc, our TWAS results show reduced specificity and increased sensitivity in external datasets to assign variants to target genes.

Hypothalamic hamartoma with gelastic seizures is a well-established cause of drug-resistant epilepsy in early life. The development of novel surgical techniques has permitted the genomic interrogation of hypothalamic hamartoma tissue. This has revealed causative mosaic variants within GLI3, OFD1 and other key regulators of the sonic-hedgehog pathway in a minority of cases. Sonic-hedgehog signalling proteins localize to the cellular organelle primary cilia. We therefore explored the hypothesis that cilia gene variants may underlie hitherto unsolved cases of sporadic hypothalamic hamartoma. We performed high-depth exome sequencing and chromosomal microarray on surgically resected hypothalamic hamartoma tissue and paired leukocyte-derived DNA from 27 patients. We searched for both germline and somatic variants under both dominant and bi-allelic genetic models. In hamartoma-derived DNA of seven patients we identified bi-allelic (one germline, one somatic) variants within one of four cilia genes-DYNC2I1, DYNC2H1, IFT140 or SMO. In eight patients, we identified single somatic variants in the previously established hypothalamic hamartoma disease genes GLI3 or OFD1. Overall, we established a plausible molecular cause for 15/27 (56%) patients. Here, we expand the genetic architecture beyond single variants within dominant disease genes that cause sporadic hypothalamic hamartoma to bi-allelic (one germline/one somatic) variants, implicate three novel cilia genes and reconceptualize the disorder as a ciliopathy.

Juvenile dermatomyositis (JDM) is a rare, severe autoimmune disease and the most common idiopathic inflammatory myopathy of children. JDM and adult-onset dermatomyositis (DM) have similar clinical, biological and serological features, although these features differ in prevalence between childhood-onset and adult-onset disease, suggesting that age of disease onset may influence pathogenesis. Therefore, a JDM-focused genetic analysis was performed using the largest collection of JDM samples to date. Caucasian JDM samples (n = 952) obtained via international collaboration were genotyped using the Illumina HumanCoreExome chip. Additional non-assayed human leukocyte antigen (HLA) loci and genome-wide single-nucleotide polymorphisms (SNPs) were imputed. HLA-DRB1*03:01 was confirmed as the classical HLA allele most strongly associated with JDM [odds ratio (OR) 1.66; 95% confidence interval (CI) 1.46, 1.89; P = 1.4 × 10-14], with an independent association at HLA-C*02:02 (OR = 1.74; 95% CI 1.42, 2.13, P = 7.13 × 10-8). Analyses of amino acid positions within HLA-DRB1 indicated that the strongest association was at position 37 (omnibus P = 3.3 × 10-19), with suggestive evidence this association was independent of position 74 (omnibus P = 5.1 × 10-5), the position most strongly associated with adult-onset DM. Conditional analyses also suggested that the association at position 37 of HLA-DRB1 was independent of some alleles of the Caucasian HLA 8.1 ancestral haplotype (AH8.1) such as HLA-DQB1*02:01 (OR = 1.62; 95% CI 1.36, 1.93; P = 8.70 × 10-8), but not HLA-DRB1*03:01 (OR = 1.49; 95% CR 1.24, 1.80; P = 2.24 × 10-5). No associations outside the HLA region were identified. Our findings confirm previous associations with AH8.1 and HLA-DRB1*03:01, HLA-C*02:02 and identify a novel association with amino acid position 37 within HLA-DRB1, which may distinguish JDM from adult DM.

Clonal hematopoiesis because of somatic mutations in hematopoietic stem/progenitor cells is an age-related phenomenon and commonly observed when sequencing blood DNA in elderly individuals. Several genes that are implicated in clonal hematopoiesis are also associated with Mendelian disorders when mutated in the germline, potentially leading to variant misinterpretation. We performed a literature search to identify genes associated with age-related clonal hematopoiesis followed by an OMIM query to identify the subset of genes in which germline variants are associated with Mendelian disorders. We retrospectively screened for diagnostic cases in which the presence of age-related clonal hematopoiesis confounded exome sequencing data interpretation. We found 58 genes in which somatic mutations are implicated in clonal hematopoiesis, while germline variants in the same genes are associated with Mendelian (mostly neurodevelopmental) disorders. Using five selected cases of individuals with suspected monogenic disorders, we illustrate how clonal hematopoiesis in either variant databases or exome sequencing datasets poses a pitfall, potentially leading to variant misclassification and erroneous conclusions regarding gene-disease associations.

Cognitive impairment is a common non-motor complication of Parkinson's disease (PD). Glucocerebrosidase gene (GBA1) variants are found in 10-15% of PD cases and are numerically the most important risk factor for PD and dementia with Lewy bodies. Accumulation of α-synuclein and tau pathology is thought to underlie cognitive impairment in PD and likely involves cholinergic as well as dopaminergic neurons. Neural crest stem cells were isolated from both PD patients with the common heterozygous N370S GBA1 mutation and normal subjects without GBA1 mutations. The stem cells were used to generate a cholinergic neuronal cell model. The effects of the GBA1 variant on glucocerebrosidase (GCase) protein and activity, and cathepsin D, tau and α-synuclein protein levels in cholinergic neurons were examined. Ambroxol, a GCase chaperone, was used to investigate whether GCase enhancement was able to reverse the effects of the GBA1 variant on cholinergic neurons. Significant reductions in GCase protein and activity, as well as in cathepsin D levels, were found in GBA1 mutant (N370S/WT) cholinergic neurons. Both tau and α-synuclein levels were significantly increased in GBA1 mutant (N370S/WT) cholinergic neurons. Ambroxol significantly enhanced GCase activity and decreased both tau and α-synuclein levels in cholinergic neurons. GBA1 mutations interfere with the metabolism of α-synuclein and tau proteins and induce higher levels of α-synuclein and tau proteins in cholinergic neurons. The GCase pathway provides a potential therapeutic target for neurodegenerative disorders related to pathological α-synuclein or tau accumulation.

Repeat associated non-AUG (RAN) translation of CGG repeats in the 5'UTR of FMR1 produces toxic proteins that contribute to fragile X-associated tremor/ataxia syndrome (FXTAS) pathogenesis. The most abundant RAN product, FMRpolyG, initiates predominantly at an ACG upstream of the repeat. Accurate FMRpolyG measurements in FXTAS patients are lacking. We used data-dependent acquisition and parallel reaction monitoring (PRM) mass spectrometry coupled with stable isotope labeled standard peptides to identify signature FMRpolyG fragments in patient samples. Following immunoprecipitation, PRM detected FMRpolyG signature peptides in transfected cells, and FXTAS tissues and cells, but not in controls. We identified two amino-terminal peptides: an ACG-initiated Ac-MEAPLPGGVR and a GUG-initiated Ac-TEAPLPGGVR, as well as evidence for RAN translation initiation within the CGG repeat itself in two reading frames. Initiation at all sites increased following cellular stress, decreased following eIF1 overexpression and was eIF4A and M7G cap-dependent. These data demonstrate that FMRpolyG is quantifiable in human samples and FMR1 RAN translation initiates via similar mechanisms for near-cognate codons and within the repeat through processes dependent on available initiation factors and cellular environment.

Retinitis pigmentosa (RP) is caused by one of many possible gene mutations. The National Institutes of Health recommends high daily doses of vitamin A palmitate for RP patients. There is a critical knowledge gap surrounding the therapeutic applicability of vitamin A to patients with the different subtypes of the disease. Here, we present a case report of a patient with RP caused by a p.D190N mutation in Rhodopsin (RHO) associated with abnormally high quantitative autofluorescence values after long-term vitamin A supplementation. We investigated the effects of vitamin A treatment strategy on RP caused by the p.D190N mutation in RHO by exposing Rhodopsin p.D190N (RhoD190N/+) and wild-type (WT) mice to experimental vitamin A-supplemented and standard control diets. The patient's case suggests that the vitamin A treatment strategy should be further studied to determine its effect on RP caused by p.D190N mutation in RHO and other mutations. Our mouse experiments revealed that RhoD190N/+ mice on the vitamin A diet exhibited higher levels of autofluorescence and lipofuscin metabolites compared to WT mice on the same diet and isogenic controls on the standard control diet. Vitamin A supplementation diminished photoreceptor function in RhoD190N/+ mice while preserving cone response in WT mice. Our findings highlight the importance of more investigations into the efficacy of clinical treatments like vitamin A for patients with certain genetic subtypes of disease and of genotyping in the precision care of inherited retinal degenerations.

Transcriptome-wide association studies (TWAS) integrate genome-wide association study (GWAS) data with gene expression (GE) data to identify (putative) causal genes for complex traits. There are two stages in TWAS: in Stage 1, a model is built to impute gene expression from genotypes, and in Stage 2, gene-trait association is tested using imputed gene expression. Despite many successes with TWAS, in the current practice, one only assumes a linear relationship between GE and the trait, which however may not hold, leading to loss of power. In this study, we extend the standard TWAS by considering a quadratic effect of GE, in addition to the usual linear effect. We train imputation models for both linear and quadratic gene expression levels in Stage 1, then include both the imputed linear and quadratic expression levels in Stage 2. We applied both the standard TWAS and our approach first to the ADNI gene expression data and the IGAP Alzheimer's disease GWAS summary data, then to the GTEx (V8) gene expression data and the UK Biobank individual-level GWAS data for lipids, followed by validation with different GWAS data, suitable model checking and more robust TWAS methods. In all these applications, the new TWAS approach was able to identify additional genes associated with Alzheimer's disease, LDL and HDL cholesterol levels, suggesting its likely power gains and thus the need to account for potentially nonlinear effects of gene expression on complex traits.

Erratum in Hum Mol Genet. 2023 Jan 6;32(2):351.

Mutations in genes that lead to dysfunctional cilia can cause a broad spectrum of human disease phenotypes referred to as ciliopathies. Many ciliopathy-associated proteins are localized to the evolutionary conserved ciliary transition zone (TZ) subdomain. We identified biallelic missense and nonsense mutations in the gene encoding the transmembrane protein TMEM218 in unrelated patients with features related to Bardet-Biedl, Joubert and Meckel-Gruber syndrome (MKS) and characterized TMEM218 as a major component of the ciliary TZ module. Co-immunoprecipitation assays resulted in the physical interaction of TMEM218 with the MKS module member TMEM67/Meckelin that was significantly reduced by the TMEM218 missense change harboured by one of our patients. We could further validate its pathogenicity by functional in vivo analysis in zebrafish (Danio rerio) as a well-established vertebrate model for ciliopathies. Notably, ciliopathy-related phenotypes were most prominent by genetic interactions with the NPHP module component Nphp4. Conclusively, we describe TMEM218 as a new disease gene for patients with a wide spectrum of syndromic ciliopathy phenotypes and provide evidence for a synergistic interaction of TMEM218 and the NPHP module crucial for proper ciliary function.

Variants in multiple lysosomal enzymes increase Parkinson's disease (PD) risk, including the genes encoding glucocerebrosidase (GCase), acid sphingomyelinase (ASMase) and galactosylceramidase. Each of these enzymes generates ceramide by hydrolysis of sphingolipids in lysosomes, but the role of this common pathway in PD pathogenesis has not yet been explored. Variations in GBA1, the gene encoding GCase, are the most common genetic risk factor for PD. The lysosomal enzyme cathepsin B has recently been implicated as an important genetic modifier of disease penetrance in individuals harboring GBA1 variants, suggesting a mechanistic link between these enzymes. Here, we found that ceramide activates cathepsin B, and identified a novel role for cathepsin B in mediating prosaposin cleavage to form saposin C, the lysosomal coactivator of GCase. Interestingly, this pathway was disrupted in Parkin-linked PD models, and upon treatment with inhibitor of ASMase which resulted in decreased ceramide production. Conversely, increasing ceramide production by inhibiting acid ceramidase activity was sufficient to upregulate cathepsin B- and saposin C-mediated activation of GCase. These results highlight a mechanistic link between ceramide and cathepsin B in regulating GCase activity and suggest that targeting lysosomal ceramide or cathepsin B represents an important therapeutic strategy for activating GCase in PD and related disorders.

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is a common, severe craniofacial malformation that imposes significant medical, psychosocial and financial burdens. NSCL/P is a multifactorial disorder with genetic and environmental factors playing etiologic roles. Currently, only 25% of the genetic variation underlying NSCL/P has been identified by linkage, candidate gene and genome-wide association studies. In this study, whole-genome sequencing and genome-wide genotyping followed by polygenic risk score (PRS) and linkage analyses were used to identify the genetic etiology of NSCL/P in a large three-generation family. We identified a rare missense variant in PDGFRA (c.C2740T; p.R914W) as potentially etiologic in a gene-based association test using pVAAST (P = 1.78 × 10-4) and showed decreased penetrance. PRS analysis suggested that variant penetrance was likely modified by common NSCL/P risk variants, with lower scores found among unaffected carriers. Linkage analysis provided additional support for PRS-modified penetrance, with a 7.4-fold increase in likelihood after conditioning on PRS. Functional characterization experiments showed that the putatively causal variant was null for signaling activity in vitro; further, perturbation of pdgfra in zebrafish embryos resulted in unilateral orofacial clefting. Our findings show that a rare PDGFRA variant, modified by additional common NSCL/P risk variants, have a profound effect on NSCL/P risk. These data provide compelling evidence for multifactorial inheritance long postulated to underlie NSCL/P and may explain some unusual familial patterns.

α-syntrophin (α-syn) and α-dystrobrevin (α-dbn), two components of the dystrophin-glycoprotein complex, are essential for the maturation and maintenance of the neuromuscular junction (NMJ) and mice deficient in either α-syn or α-dbn exhibit similar synaptic defects. However, the functional link between these two proteins and whether they exert distinct or redundant functions in the postsynaptic organization of the NMJ remain largely unknown. We generated and analyzed the synaptic phenotype of double heterozygote (α-dbn+/-, α-syn+/-), and double homozygote knockout (α-dbn-/-; α-syn-/-) mice and examined the ability of individual molecules to restore their defects in the synaptic phenotype. We showed that in double heterozygote mice, NMJs have normal synaptic phenotypes and no signs of muscular dystrophy. However, in double knockout mice (α-dbn-/-; α-syn-/-), the synaptic phenotype (the density, the turnover and the distribution of AChRs within synaptic branches) is more severely impaired than in single α-dbn-/- or α-syn-/- mutants. Furthermore, double mutant and single α-dbn-/- mutant mice showed more severe exercise-induced fatigue and more significant reductions in grip strength than single α-syn-/- mutant and wild-type. Finally, we showed that the overexpression of the transgene α-syn-GFP in muscles of double mutant restores primarily the abnormal extensions of membrane containing AChRs that extend beyond synaptic gutters and lack synaptic folds, whereas the overexpression of α-dbn essentially restores the abnormal dispersion of patchy AChR aggregates in the crests of synaptic folds. Altogether, these data suggest that α-syn and α-dbn act in parallel pathways and exert distinct functions on the postsynaptic structural organization of NMJs.

Duchenne muscular dystrophy (DMD) is a fatal X-linked genetic disorder affecting approximately 1 in 5000 male births worldwide. DMD is caused by mutations in the dystrophin gene. Dystrophin is essential for maintaining muscle cell membrane integrity and stability by linking the cytoskeleton to the extracellular matrix, which protects myofibers from contraction-induced damage. Loss of dystrophin leads to mechanically induced skeletal and cardiac muscle damage. Although the disease is not evident in DMD patients at birth, muscular dystrophy rapidly progresses and results in respiratory and cardiac muscle failure as early as the teenage years. Premature death in DMD patients is due to cardiac arrhythmias and left ventricular dysfunction. Currently, there is no effective treatment for DMD-related cardiac failure. Recently, we have shown that a Food and Drug Administration-approved small molecule, sunitinib, a multi-targeted tyrosine kinase inhibitor can mitigate skeletal muscle disease through an increase in myogenic capacity, cell membrane integrity, and improvement of skeletal muscle function via regulation of STAT3-related signaling pathway. Chronic activation of STAT3 has been shown to promote cardiac hypertrophy and failure. In this study, we examined the effects of long-term sunitinib treatment on cardiac pathology and function. Our results showed sunitinib treatment reduced STAT3 phosphorylation in the heart muscle of mdx mice, improved cardiac electrical function, increased cardiac output and stroke volume, decreased ventricular hypertrophy, reduced cardiomyocytes membrane damage, fibrotic tissue deposition and slightly decreased cardiac inflammation. Together, our studies support the idea that sunitinib could serve as a novel treatment to slow cardiomyopathy progression in DMD. One Sentence Summary In this study, we determined if sunitinib, a Food and Drug Administration-approved drug, could reduce the pathology and improve cardiac function in an animal model for DMD.