The interocular distance, or orbital telorism, is a distinctive craniofacial trait that also serves as a clinically informative measure. While its extremes, hypo- and hypertelorism, have been linked to monogenic disorders and are often syndromic, little is known about the genetic determinants of interocular distance within the general population. We derived orbital telorism measures from cranial magnetic resonance imaging by calculating the distance between the eyeballs' centre of gravity, which showed a good reproducibility with an intraclass correlation coefficient of 0.991 (95% confidence interval 0.985-0.994). Heritability estimates were 76% (standard error = 12%) with a family-based method (N = 364) and 39% (standard error = 2.4%) with a single nucleotide polymorphism-based method (N = 34 130) and were unaffected by adjustment for height (model II) and intracranial volume (model III) or head width (model IV). Genome-wide association studies in 34 130 European individuals identified 56 significantly associated genomic loci (P < 5 × 10-8) across four different models of which 46 were novel for facial morphology, and overall these findings replicated in an independent sample (N = 10 115) with telorism-related horizontal facial distance measures. Genes located nearby these 56 identified genetic loci were 4.9-fold enriched for Mendelian hypotelorism and hypertelorism genes, underlining their biological relevance. This study provides novel insights into the genetic architecture underlying interocular distance in particular, and the face in general, and explores its potential for applications in a clinical setting.

The most frequent genetic cause of focal epilepsies is variations in the GAP activity toward RAGs 1 complex genes DEP domain containing 5 (DEPDC5), nitrogen permease regulator 2-like protein (NPRL2) and nitrogen permease regulator 3-like protein (NPRL3). Because these variations are frequent and associated with a broad spectrum of focal epilepsies, a unique pathology categorized as GATORopathy can be conceptualized. Animal models recapitulating the clinical features of patients are essential to decipher GATORopathy. Although several genetically modified animal models recapitulate DEPDC5-related epilepsy, no models have been reported for NPRL2- or NPRL3-related epilepsies. Here, we conditionally deleted Nprl2 and Nprl3 from the dorsal telencephalon in mice [Emx1cre/+; Nprl2f/f (Nprl2-cKO) and Emx1cre/+; Nprl3f/f (Nprl3-cKO)] and compared their phenotypes with Nprl2+/-, Nprl3+/- and Emx1cre/+; Depdc5f/f (Depdc5-cKO) mice. Nprl2-cKO and Nprl3-cKO mice recapitulated the major abnormal features of patients-spontaneous seizures, and dysmorphic enlarged neuronal cells with increased mechanistic target of rapamycin complex 1 signaling-similar to Depdc5-cKO mice. Chronic postnatal rapamycin administration dramatically prolonged the survival period and inhibited seizure occurrence but not enlarged neuronal cells in Nprl2-cKO and Nprl3-cKO mice. However, the benefit of rapamycin after withdrawal was less durable in Nprl2- and Nprl3-cKO mice compared with Depdc5-cKO mice. Further studies using these conditional knockout mice will be useful for understanding GATORopathy and for the identification of novel therapeutic targets.

Mutations in NMNAT1, a key enzyme involved in the synthesis of NAD+ in the nucleus, lead to an early onset severe inherited retinal degeneration (IRD). We aimed to understand the role of nuclear NAD+ in the retina and to identify the molecular mechanisms underlying NMNAT1-associated disease, using a mouse model that harbors the p.V9M mutation in Nmnat1 (Nmnat1V9M/V9M). We identified temporal transcriptional reprogramming in the retinas of Nmnat1V9M/V9M mice prior to retinal degeneration, which begins at 4 weeks of age, with no significant alterations in gene expression at 2 weeks of age and over 2600 differentially expressed genes by 3 weeks of age. Expression of the primary consumer of NAD+ in the nucleus, PARP1, an enzyme involved in DNA damage repair and transcriptional regulation, as well as 7 other PARP family enzymes, was elevated in the retinas of Nmnat1V9M/V9M. This was associated with elevated levels of DNA damage, PARP-mediated NAD+ consumption and migration of Iba1+/CD45+ microglia/macrophages to the subretinal space in the retinas of Nmnat1V9M/V9M mice. These findings suggest that photoreceptor cells are especially sensitive to perturbation of genome homeostasis, and that PARP-mediated cell death may play a role in other genetic forms of IRDs, and potentially other forms of neurodegeneration.

Laryngeal squamous cell cancer (LSCC) is the second most prevalent malignancy occurring in the head and neck with a high incidence and mortality rate. Immunotherapy has recently become an emerging treatment for cancer. It is therefore essential to explore the role of tumour immunity in laryngeal cancer. Our study first delineated and evaluated the comprehensive immune infiltration landscapes of the tumour microenvironment in LSCC. A hierarchical clustering method was applied to classify the LSCC samples into two groups (high- and low-infiltration groups). We found that individuals with low immune infiltration characteristics had significantly better survival than those in the high-infiltration group, possibly because of the elevated infiltration of immune suppressive cells, such as regulatory T cells and myeloid-derived suppressor cells, in the high-infiltration group. Differentially expressed genes between two groups were involved in some immune-related terms, such as antigen processing and presentation. A univariate Cox analysis and least absolute shrinkage and selection operator analysis were performed to identify an immune gene-set-based prognostic signature (IBPS) to assess the risk of LSCC. The prognostic model comprising six IBPSs was successfully verified to be robust in different cohorts. The expression of the six IBPSs was detected by immunohistochemistry in 110 cases of LSCC. In addition, different inflammatory profiles and immune checkpoint landscape of LSCC were found between two groups. Hence, our model could serve as a candidate immunotherapeutic biomarker and potential therapeutic target for laryngeal cancer.

Despite the high morbidity and mortality rates associated with colorectal cancer (CRC), the aberrant genes and mechanisms driving CRC pathogenesis remain poorly understood. Chromosome instability (CIN), or ongoing changes in chromosome numbers, is a predominant form of genome instability associated with ~85% of CRCs, suggesting it may be a key mechanism driving CRC oncogenesis. CIN enables the acquisition of copy number alterations conferring selective growth, proliferation and survival advantages that promote cellular transformation. Despite these associations, the aberrant genes underlying CIN remain largely unknown. Candidate CIN gene FBXO7 encodes an F-box protein, a subunit of the SKP1-CUL1-FBOX (SCF) complex that confers substrate specificity to the complex and targets proteins for subsequent degradation by the 26S proteasome. Recently, the genes encoding the three core SCF complex members were identified as CIN genes; however, it is unknown whether F-box proteins exhibit similar integral roles in maintaining chromosome stability. Using short- small interfering RNA (siRNA) and long- (CRISPR/Cas9) term approaches, we show that reduced FBXO7 expression induces CIN in various colonic epithelial cell contexts, whereas FBXO7 knockout clones also exhibit hallmarks associated with cellular transformation, namely increased clonogenic and anchorage-independent growth. Collectively, these data demonstrate that FBXO7 is required to maintain genome stability identifying FBXO7 a novel CIN gene whose reduced expression may contribute to CRC development and progression.

Mutations in the mitochondrial protein CHCHD2 cause autosomal dominant Parkinson's disease characterized by the preferential loss of substantia nigra dopamine (DA) neurons. Therefore, understanding the function of CHCHD2 in neurons may provide vital insights into how mitochondrial dysfunction contributes to neurodegeneration in PD. To investigate the normal requirement and function of CHCHD2 in neurons, we first examined CHCHD2 levels and showed that DA neurons have higher CHCHD2 levels than other neuron types, both in vivo and in co-culture. We then generated mice with either a targeted deletion of CHCHD2 in DA neurons or a deletion in the brain or total body. All three models were viable, and loss of CHCHD2 in the brain did not cause degeneration of DA neurons. Mice lacking CHCHD2 in DA neurons did display sex-specific changes to locomotor activity, but we did not observe differences in assays of muscle strength, exercise endurance or motor coordination. Furthermore, mitochondria derived from mice lacking CHCHD2 did not display abnormalities in OXPHOS function. Lastly, resilience to CHCHD2 deletion could not be explained by functional complementation by its paralog CHCHD10, as deletion of both CHCHD10 and CHCHD2 did not cause degeneration of DA neurons in the midbrain. These findings support the hypothesis that pathogenic CHCHD2 mutations cause PD through a toxic gain-of-function, rather than loss-of-function mechanism.

Anomalous pulmonary venous return (APVR) is a potentially lethal congenital heart disease. Elucidating the genetic etiology is crucial for understanding its pathogenesis and improving clinical practice, whereas its genetic basis remains largely unknown because of complex genetic etiology. We thus performed whole-exome sequencing for 144 APVR patients and 1636 healthy controls and report a comprehensive atlas of APVR-related rare genetic variants. Novel singleton, loss-of-function and deleterious missense variants (DVars) were enriched in patients, particularly for genes highly expressed in the developing human heart at the critical time point for pulmonary veins draining into the left atrium. Notably, PLXND1, encoding a receptor for semaphorins, represents a strong candidate gene of APVR (adjusted P = 1.1e-03, odds ratio: 10.9-69.3), accounting for 4.17% of APVR. We further validated this finding in an independent cohort consisting of 82 case-control pairs. In these two cohorts, eight DVars were identified in different patients, which convergently disrupt the GTPase-activating protein-related domain of PLXND1. All variant carriers displayed strikingly similar clinical features, in that all anomalous drainage of pulmonary vein(s) occurred on the right side and incorrectly connected to the right atrium, which may represent a novel subtype of APVR for molecular diagnosis. Studies in Plxnd1 knockout mice further revealed the effects of PLXND1 deficiency on severe heart and lung defects and cellular abnormalities related to APVR such as abnormal migration and vascular formation of vascular endothelial cells. These findings indicate the important role of PLXND1 in APVR pathogenesis, providing novel insights into the genetic etiology and molecular subtyping for APVR.

Nephronophthisis-related ciliopathies (NPHP-RC) comprises a group of inherited kidney diseases, caused by mutations in genes encoding proteins localizing to primary cilia. NPHP-RC represents one of the most frequent monogenic causes of renal failure within the first three decades of life, but its molecular disease mechanisms remain unclear. Here, we identified biallelic ANKS6 mutations in two affected siblings with late-onset chronic kidney disease by whole-exome sequencing. We employed patient-derived fibroblasts generating an in vitro model to study the precise biological impact of distinct human ANKS6 mutations, completed by immunohistochemistry studies on renal biopsy samples. Functional studies using patient-derived cells showed an impaired integrity of the ciliary inversin compartment with reduced cilia length. Further analyses demonstrated that ANKS6 deficiency leads to a dysregulation of Hippo-signaling through nuclear yes-associated protein (YAP) imbalance and disrupted ciliary localization of YAP. In addition, an altered transcriptional activity of canonical Wnt target genes and altered expression of non-phosphorylated (active) β-catenin and phosphorylated glycogen synthase kinase 3β were observed. Upon ciliation, ANKS6 deficiency revealed a deranged subcellular localization and expression of components of the endocytic recycling compartment. Our results demonstrate that ANKS6 plays a key role in regulating the Hippo pathway, and ANKS6 deficiency is linked to dysregulation of signaling pathways. Our study provides molecular clues in understanding pathophysiological mechanisms of NPHP-RC and may offer new therapeutic targets.

Spinal muscular atrophy (SMA) is characterized by the loss of alpha motor neurons in the spinal cord and a progressive muscle weakness and atrophy. SMA is caused by loss-of-function mutations and/or deletions in the survival of motor neuron (SMN) gene. The role of SMN in motor neurons has been extensively studied, but its function and the consequences of its loss in muscle have also emerged as a key aspect of SMA pathology. In this study, we explore the molecular mechanisms involved in muscle defects in SMA. First, we show in C2C12 myoblasts, that arginine methylation by CARM1 controls myogenic differentiation. More specifically, the methylation of HuR on K217 regulates HuR levels and subcellular localization during myogenic differentiation, and the formation of myotubes. Furthermore, we demonstrate that SMN and HuR interact in C2C12 myoblasts. Interestingly, the SMA-causing E134K point mutation within the SMN Tudor domain, and CARM1 depletion, modulate the SMN-HuR interaction. In addition, using the Smn2B/- mouse model, we report that CARM1 levels are markedly increased in SMA muscles and that HuR fails to properly respond to muscle denervation, thereby affecting the regulation of its mRNA targets. Altogether, our results show a novel CARM1-HuR axis in the regulation of muscle differentiation and plasticity as well as in the aberrant regulation of this axis caused by the absence of SMN in SMA muscle. With the recent developments of therapeutics targeting motor neurons, this study further indicates the need for more global therapeutic approaches for SMA.

This study aimed to identify the genes and small RNAs (sRNAs) expressed by the human endogenous retrovirus K (HERV-K) HML2 and their associations with the immune process of systemic lupus erythematosus (SLE). RNA-Seq data including 99 SLE patients and 18 controls (GSE72420) was obtained from the Gene Expression Omnibus. Differentially expressed genes (DEGs) as well as HML2-DEGs between SLE patients and normal controls were identified. Five HML2-DEGs involved in immune-regulating function were identified using weighted gene co-expression network analysis. The associations between these genes and the proportions of immune cells were determined by CIBERSORT. Ten candidate HML2-encoded sRNAs were identified based on specific criteria, and three of them were further validated in SLE patients by qRT-PCR. The diagnostic values of these three sRNAs were evaluated in SLE and lupus nephritis (LN). This study suggested that HML2 genes and their encoded sRNAs might be involved in the immune regulation and progress of SLE. These potential sRNAs might function as regulatory molecules and diagnostic biomarkers of SLE and LN.

Rahman syndrome (RMNS) is a rare genetic disorder characterized by mild to severe intellectual disability, hypotonia, anxiety, autism spectrum disorder, vision problems, bone abnormalities and dysmorphic facies. RMNS is caused by de novo heterozygous mutations in the histone linker gene H1-4; however, mechanisms underlying impaired neurodevelopment in RMNS are not understood. All reported mutations associated with RMNS in H1-4 are small insertions or deletions that create a shared frameshift, resulting in a H1.4 protein that is both truncated and possessing an abnormal C-terminus frameshifted tail (H1.4 CFT). To expand understanding of mutations and phenotypes associated with mutant H1-4, we identified new variants at both the C- and N-terminus of H1.4. The clinical features of mutations identified at the C-terminus are consistent with other reports and strengthen the support of pathogenicity of H1.4 CFT. To understand how H1.4 CFT may disrupt brain function, we exogenously expressed wild-type or H1.4 CFT protein in rat hippocampal neurons and assessed neuronal structure and function. Genome-wide transcriptome analysis revealed ~ 400 genes altered in the presence of H1.4 CFT. Neuronal genes downregulated by H1.4 CFT were enriched for functional categories involved in synaptic communication and neuropeptide signaling. Neurons expressing H1.4 CFT also showed reduced neuronal activity on multielectrode arrays. These data are the first to characterize the transcriptional and functional consequence of H1.4 CFT in neurons. Our data provide insight into causes of neurodevelopmental impairments associated with frameshift mutations in the C-terminus of H1.4 and highlight the need for future studies on the function of histone H1.4 in neurons.

The common null polymorphism (R577X) in the ACTN3 gene is present in over 1.5 billion people worldwide and results in the absence of the protein α-actinin-3 from the Z-discs of fast-twitch skeletal muscle fibres. We have previously reported that this polymorphism is a modifier of dystrophin-deficient Duchenne Muscular Dystrophy. To investigate the mechanism underlying this, we use a double knockout (dk)Actn3KO/mdx (dKO) mouse model, which lacks both dystrophin and sarcomere α-actinin-3. We used dKO mice and mdx dystrophic mice at 12 months (aged) to investigate the correlation between morphological changes to the fast-twitch dKO EDL and the reduction in force deficit produced by an in vitro eccentric contraction protocol. In the aged dKO mouse, we found a marked reduction in fibre branching complexity that correlated with protection from eccentric contraction induced force deficit. Complex branches in the aged dKO EDL fibres (28%) were substantially reduced compared to aged mdx EDL fibres (68%), and this correlates with a graded force loss over three eccentric contractions for dKO muscles (~36% after first contraction, ~66% overall) compared to an abrupt drop in mdx upon the first eccentric contraction (~75% after first contraction, ~89% after three contractions). In dKO, protection from eccentric contraction damage was linked with a doubling of SERCA1 pump density the EDL. We propose that the increased oxidative metabolism of fast-twitch glycolytic fibres characteristic of the null polymorphism (R577X) and increase in SR Ca2+ pump proteins reduces muscle fibre branching and decreases susceptibility to eccentric injury in the dystrophinopathies.

Autism spectrum disorder (ASD) and intellectual disability (ID) often exist together in patients. The RAB39B gene has been reported to be mutated in ID patients with additional clinical features ranging from ASD, macrocephaly, seizures and/or early-onset parkinsonism. Here, we describe a novel RAB39B nonstop mutation [Xq28; c.640 T > C; p.(*214Glnext*21)] in a family with ASD, severe ID and poor motor coordination, and we assessed the pathogenicity of the mutation. A heterologous cell system and a Rab39b knockdown (KD) murine model, which mimic the nonstop mutation, were used to validate the deleterious effect of the RAB39B mutation. The mutation led to RAB39B protein instability, resulting in its increased degradation and consequent downregulation. Using a Rab39b KD mouse model, we demonstrated that the downregulation of RAB39B led to increased GluA2 lacking Ca2+-permeable AMPAR composition at the hippocampal neuronal surface and increased dendritic spine density that remained in an immature filopodia-like state. These phenotypes affected behavioural performance in a disease-specific manner. Rab39b KD mice revealed impaired social behaviour but intact social recognition. They also showed normal anxiety-like, exploratory and motivational behaviours but impaired working and associative memories. In conclusion, we found a novel RAB39B nonstop variant that segregated in a family with a clinical phenotype including ID, ASD and poor motor coordination. The pathogenicity of mutations causing the downregulation of RAB39B proteins, impacting AMPAR trafficking and dendritic spine morphogenesis, reinforced the idea that AMPAR modulation and dendritic spine assets could be considered hallmarks of neurodevelopmental disorders.

RNA modifications affect many aspects of RNA metabolism and are involved in the regulation of many different biological processes. Mono-methylation of adenosine in the N1 position, N1-methyladensoine (m1A), is a reversible modification that is known to target rRNAs and tRNAs. m1A has been shown to increase tRNA structural stability and induce correct tRNA folding. Recent studies have begun to associate the dysregulation of epitranscriptomic control with age-related disorders such as Alzheimer's disease. Here, we applied the newly developed m1A-quant-seq approach to map the brain abundant m1A RNA modification in the cortex of an Alzheimer's disease mouse model, 5XFAD. We observed hypomethylation in both mitochondrial and cytosolic tRNAs in 5XFAD mice compared with wild type. Furthermore, the main enzymes responsible for the addition of m1A in mitochondrial (TRMT10C, HSD17B10) and cytosolic tRNAs (TRMT61A) displayed decreased expression in 5XFAD compared with wild-type mice. Knockdown of these enzymes results in a more severe phenotype in a Drosophila tau model, and differential m1A methylation is correlated with differences in mature mitochondrial tRNA expression. Collectively, this work suggests that hypo m1A modification in tRNAs may play a role in Alzheimer's disease pathogenesis.

The 5-year overall survival (OS) of pancreatic ductal adenocarcinoma (PDAC) is only 10%, partly owing to the lack of reliable diagnostic and prognostic biomarkers. The raw gene-cell matrix for single-cell RNA-seq (scRNA-seq) analysis was downloaded from the GSA database. We drew cell atlas for PDAC and normal pancreatic tissues. The inferCNV analysis was used to distinguish tumor cells from normal ductal cells. We identified differential expression genes (DEGs) by comparing tumor cells and normal ductal cells. The common DEGs were used to conduct prognostic and diagnostic model using univariate and multivariate Cox or logistic regression analysis. Four genes, MET, KLK10, PSMB9 and ITGB6, were utilized to create risk score formula to predict OS and to establish diagnostic model for PDAC. Finally, we drew an easy-to-use nomogram to predict 2-year and 3-year OSs. In conclusion, we developed and validated the prognostic and diagnostic model for PDAC based on scRNA-seq and bulk-seq datasets.

The IFT-B complex mediates ciliary anterograde protein trafficking and membrane protein export together with the BBSome. Bardet-Biedl syndrome (BBS) is caused by mutations in not only all BBSome subunits but also in some IFT-B subunits, including IFT74/BBS22 and IFT27/BBS19, which form heterodimers with IFT81 and IFT25, respectively. We found that the IFT25-IFT27 dimer binds the C-terminal region of the IFT74-IFT81 dimer and that the IFT25-IFT27-binding region encompasses the region deleted in the BBS variants of IFT74. In addition, we found that the missense BBS variants of IFT27 are impaired in IFT74-IFT81 binding and are unable to rescue the BBS-like phenotypes of IFT27-knockout (KO) cells. Furthermore, the BBS variants of IFT74 rescued the ciliogenesis defect of IFT74-KO cells, but the rescued cells demonstrated BBS-like abnormal phenotypes. Taken together, we conclude that the impaired interaction between IFT74-IFT81 and IFT25-IFT27 causes the BBS-associated ciliary defects.

Cornelia de Lange syndrome (CdLS) is a rare multiorgan developmental disorder caused by pathogenic variants in cohesin genes. It is a genetically and clinically heterogeneous dominant (both autosomal and X-linked) rare disease. Increasing experimental evidence indicates that CdLS is caused by a combination of factors, such as gene expression dysregulation, accumulation of cellular damage and cellular aging, which collectively contribute to the CdLS phenotype. The CdLS phenotype overlaps with a number of related diagnoses such as KBG syndrome and Rubinstein-Taybi syndrome both caused by variants in chromatin-associated factors other than cohesin. The molecular basis underlying these overlapping phenotypes is not clearly defined. Here, we found that cells from individuals with CdLS and CdLS-related diagnoses are characterized by global transcription disturbance and share common dysregulated pathways. Intriguingly, c-MYC (subsequently referred to as MYC) is downregulated in all cell lines and represents a convergent hub lying at the center of dysregulated pathways. Subsequent treatment with estradiol restores MYC expression by modulating cohesin occupancy at its promoter region. In addition, MYC activation leads to modification in expression in hundreds of genes, which in turn reduce the oxidative stress level and genome instability. Together, these results show that MYC plays a pivotal role in the etiopathogenesis of CdLS and CdLS-related diagnoses and represents a potential therapeutic target for these conditions.

Better methods are required to interpret the pathogenicity of disease-associated variants of uncertain significance (VUS), which cannot be actioned clinically. In this study, we explore the use of an animal model (Caenorhabditis elegans) for in vivo interpretation of missense VUS alleles of TMEM67, a cilia gene associated with ciliopathies. CRISPR/Cas9 gene editing was used to generate homozygous knock-in C. elegans worm strains carrying TMEM67 patient variants engineered into the orthologous gene (mks-3). Quantitative phenotypic assays of sensory cilia structure and function (neuronal dye filling, roaming and chemotaxis assays) measured how the variants impacted mks-3 gene function. Effects of the variants on mks-3 function were further investigated by looking at MKS-3::GFP localization and cilia ultrastructure. The quantitative assays in C. elegans accurately distinguished between known benign (Asp359Glu, Thr360Ala) and known pathogenic (Glu361Ter, Gln376Pro) variants. Analysis of eight missense VUS generated evidence that three are benign (Cys173Arg, Thr176Ile and Gly979Arg) and five are pathogenic (Cys170Tyr, His782Arg, Gly786Glu, His790Arg and Ser961Tyr). Results from worms were validated by a genetic complementation assay in a human TMEM67 knock-out hTERT-RPE1 cell line that tests a TMEM67 signalling function. We conclude that efficient genome editing and quantitative functional assays in C. elegans make it a tractable in vivo animal model for rapid, cost-effective interpretation of ciliopathy-associated missense VUS alleles.

Mis-expression of DUX4 in skeletal muscle causes facioscapulohumeral muscular dystrophy (FSHD). Human DUX4 and mouse Dux are retrogenes derived from retrotransposition of the mRNA from the parental DUXC gene. Primates and rodents have lost the parental DUXC gene, and it is unknown whether DUXC had a similar role in driving an early pluripotent transcriptional program. Dogs and other Laurasiatherians have retained DUXC, providing an opportunity to determine the functional similarity to the retrotransposed DUX4 and Dux. Here, we identify the expression of two isoforms of DUXC mRNA in canine testis tissues: one encoding the canonical double homeodomain protein (DUXC), similar to DUX4/Dux, and a second that includes an in-frame alternative exon that disrupts the conserved amino acid sequence of the first homeodomain (DUXC-ALT). The expression of DUXC in canine cells induces a pluripotent program similar to DUX4 and Dux and induces the expression of a similar set of retrotransposons of the ERV/MaLR and LINE-1 families, as well as pericentromeric satellite repeats; whereas DUXC-ALT did not robustly activate gene expression in these assays. Important for preclinical models of FSHD, human DUX4 and canine DUXC show higher conservation of their homeodomains and corresponding binding motifs compared with the conservation between human DUX4 and mouse Dux, and human DUX4 activates a highly similar transcriptional program in canine cells. Together, these findings show that retrotransposition resulted in the loss of an alternatively spliced isoform and that DUXC containing mammals might be good candidates for certain preclinical models ofFSHD.

Skin deficiency of kinesin family member 3A causes disrupted skin barrier function and promotes development of atopic dermatitis (AD). It is not known how well Kif3aK14∆/∆ mice approximate the human AD transcriptome. To determine the skin transcriptomic profile of Kif3aK14∆/∆ mice and compare it with other murine AD models and human AD, we performed RNA-seq of full-thickness skin and epidermis from 3- and 8-week-old Kif3aK14∆/∆ mice and compared the differentially expressed genes (DEGs) with transcriptomic datasets from mite-induced NC/Nga, flaky tail (Tmem79ma/ma Flgft/ft), and filaggrin-mutant (Flgft/ft) mice, as well as human AD transcriptome datasets including meta-analysis derived atopic dermatitis [MADAD] and the pediatric atopic dermatitis [PAD]. We then interrogated the Kif3aK14∆/∆ skin DEGs using the LINCS-L1000 database to identify potential novel drug targets for AD treatment. We identified 471 and 901 DEGs at 3 and 8 weeks of age, respectively, in the absence of Kif3a. Kif3aK14∆/∆ mice had 3.5-4.5 times more DEGs that overlapped with human AD DEGs compared to the flaky tail and Flgft/ft mice. Further, 55%, 85% and 75% of 8-week Kif3aK14∆/∆ DEGs overlapped with the MADAD and PAD non-lesional and lesional gene lists, respectively. Kif3aK14∆/∆ mice spontaneously develop a human AD-like gene signature, which better represents pediatric non-lesional skin compared to other mouse models including flaky tail, Flgft/ft and NC/Nga. Thus, Kif3aK14∆/∆ mice may model pediatric skin that is a precursor to the development of lesions and inflammation, and hence may be a useful model to study AD pathogenesis.

Erratum in Hum Mol Genet. 2022 Apr 21;:

Monogenic early onset osteoporosis (EOOP) is a rare disease defined by low bone mineral density (BMD) that results in increased risk of fracture in children and young adults. Although several causative genes have been identified, some of the EOOP causation remains unresolved. Whole-exome sequencing revealed a de novo heterozygous loss-of-function mutation in Wnt family member 11 (WNT11) (NM_004626.2:c.677_678dup p.Leu227Glyfs*22) in a 4-year-old boy with low BMD and fractures. We identified two heterozygous WNT11 missense variants (NM_004626.2:c.217G > A p.Ala73Thr) and (NM_004626.2:c.865G > A p.Val289Met) in a 51-year-old woman and in a 61-year-old woman, respectively, both with bone fragility. U2OS cells with heterozygous WNT11 mutation (NM_004626.2:c.690_721delfs*40) generated by CRISPR-Cas9 showed reduced cell proliferation (30%) and osteoblast differentiation (80%) as compared with wild-type U2OS cells. The expression of genes in the Wnt canonical and non-canonical pathways was inhibited in these mutant cells, but recombinant WNT11 treatment rescued the expression of Wnt pathway target genes. Furthermore, the expression of RSPO2, a WNT11 target involved in bone cell differentiation, and its receptor leucine-rich repeat containing G protein-coupled receptor 5 (LGR5), was decreased in WNT11 mutant cells. Treatment with WNT5A and WNT11 recombinant proteins reversed LGR5 expression, but Wnt family member 3A (WNT3A) recombinant protein treatment had no effect on LGR5 expression in mutant cells. Moreover, treatment with recombinant RSPO2 but not WNT11 or WNT3A activated the canonical pathway in mutant cells. In conclusion, we have identified WNT11 as a new gene responsible for EOOP, with loss-of-function variant inhibiting bone formation via Wnt canonical and non-canonical pathways. WNT11 may activate Wnt signaling by inducing the RSPO2-LGR5 complex via the non-canonical Wnt pathway.

Changes in the N-glycosylation of immunoglobulin G (IgG) are often observed in pathological states, such as autoimmune, inflammatory, neurodegenerative, cardiovascular diseases and some types of cancer. However, in most cases, it is not clear if the disease onset causes these changes, or if the changes in IgG N-glycosylation are among the risk factors for the diseases. The aim of this study was to investigate the casual relationships between IgG N-glycosylation traits and 12 diseases, in which the alterations of IgG N-glycome were previously reported, using two sample Mendelian randomization (MR) approach. We have performed two sample MR using publicly available summary statistics of genome-wide association studies of IgG N-glycosylation and disease risks. Our results indicate positive causal effect of systemic lupus erythematosus (SLE) on the abundance of N-glycans with bisecting N-acetylglucosamine in the total IgG N-glycome. Therefore, we suggest regarding this IgG glycosylation trait as a biomarker of SLE. We also emphasize the need for more powerful GWAS studies of IgG N-glycosylation to further elucidate the causal effect of IgG N-glycome on the diseases.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease caused by homozygous deletions or mutations in survival motor neuron gene 1 (SMN1). Currently, the primary therapeutic strategy for SMA is to increase the level of SMN via correcting SMN2 splicing (nusinersen and risdiplam). However, some patients with SMA do not respond to such treatments, thereby warranting a need to develop new therapeutic strategies. We have previously reported that SMN2 expression is epigenetically regulated by DNA methylation levels of the SMN2 promoter region. In the present study, we determined that methyl-CpG-binding protein 2 (MeCP2) may bind to this critical promoter region (nt-167 to 43). Antisense oligonucleotides (ASO-P1 and ASO-P2) were designed to target the key methylation sites in the SMN2 promoter region, which enhanced the overall transcription and functional protein expression levels in the SMA cell lines. These results were similar to those observed in nusinersen-treated SMA cells. Moreover, a combined treatment of ASO-P1 and ASO-NUS in SMA cell lines further increases fl-SMN2 transcript and SMN protein levels. The delivery of ASO-P1 to the central nervous system of severe SMA mice corrected the molecular, pathological, and functional phenotypes of this disease and increased survival rates. Our findings suggest that the key methylation regions in the SMN2 promoter region may be a novel therapeutic target for SMA.

Huntington's disease (HD) is an incurable neurodegenerative disorder caused by a CAG expansion in the huntingtin gene (HTT). Post-translational modifications of huntingtin protein (HTT), such as phosphorylation, acetylation and ubiquitination, have been implicated in HD pathogenesis. Arginine methylation/dimethylation is an important modification with an emerging role in neurodegeneration; however, arginine methylation of HTT remains largely unexplored. Here we report nearly two dozen novel arginine methylation/dimethylation sites on the endogenous HTT from human and mouse brain and human cells suggested by mass spectrometry with data-dependent acquisition. Targeted quantitative mass spectrometry identified differential arginine methylation at specific sites in HD patient-derived striatal precursor cell lines compared to normal controls. We found that HTT can interact with several type I protein arginine methyltransferases (PRMTs) via its N-terminal domain. Using a combination of in vitro methylation and cell-based experiments, we identified PRMT4 (CARM1) and PRMT6 as major enzymes methylating HTT at specific arginines. Alterations of these methylation sites had a profound effect on biochemical properties of HTT rendering it less soluble in cells and affected its liquid-liquid phase separation and phase transition patterns in vitro. We found that expanded HTT 1-586 fragment can form liquid-like assemblies, which converted into solid-like assemblies when the R200/205 methylation sites were altered. Methyl-null alterations increased HTT toxicity to neuronal cells, while overexpression of PRMT 4 and 6 was beneficial for neuronal survival. Thus, arginine methylation pathways that involve specific HTT-modifying PRMT enzymes and modulate HTT biochemical and toxic properties could provide targets for HD-modifying therapies.

Although previous studies identified numerous single nucleotide polymorphisms (SNPs) and their target genes predisposed to prostate cancer (PrCa) risks, SNP-related splicing associations are rarely reported. In this study, we applied distance-based sQTL analysis (sQTLseekeR) using RNA-seq and SNP genotype data from benign prostate tissue (n = 467) and identified significant associations in 3344 SNP-transcript pairs (P ≤ 0.05) at PrCa risk loci. We characterized a common SNP (rs7247241) and its target gene (PPP1R14A) located in chr19q13, an sQTL with risk allele T associated with upregulation of long isoform (P = 9.99E-7). We confirmed the associations in both TCGA (P = 2.42E-24) and GTEX prostate cohorts (P = 9.08E-78). To functionally characterize this SNP, we performed chromatin immunoprecipitation qPCR and confirmed stronger CTCF and PLAGL2 binding in rs7247241 C than T allele. We found that CTCF binding enrichment was negatively associated with methylation level at the SNP site in human cell lines (r = -0.58). Bisulfite sequencing showed consistent association of rs7247241-T allele with nearby sequence CpG hypermethylation in prostate cell lines and tissues. Moreover, the methylation level at CpG sites nearest to the CTCF binding and first exon splice-in (ψ) of PPP1R14A was significantly associated with aggressive phenotype in the TCGA PrCa cohort. Meanwhile, the long isoform of the gene also promoted cell proliferation. Taken together, with the most updated gene annotations, we reported a set of sQTL associated with multiple traits related to human prostate diseases and revealed a unique role of PrCa risk SNP rs7247241 on PPP1R14A isoform transition.

Mitochondria have a complex communication network with the surrounding cell and can alter nuclear DNA methylation (DNAm). Variation in the mitochondrial DNA (mtDNA) has also been linked to differential DNAm. Genome-wide association studies have identified numerous DNAm quantitative trait loci, but these studies have not examined the mitochondrial genome. Herein, we quantified nuclear DNAm from blood and conducted a mitochondrial genome-wide association study of DNAm, with an additional emphasis on sex- and prediabetes-specific heterogeneity. We used the Young Finns Study (n = 926) with sequenced mtDNA genotypes as a discovery sample and sought replication in the Ludwigshafen Risk and Cardiovascular Health study (n = 2317). We identified numerous significant associations in the discovery phase (P < 10-9), but they were not replicated when accounting for multiple testing. In total, 27 associations were nominally replicated with a P < 0.05. The replication analysis presented no evidence of sex- or prediabetes-specific heterogeneity. The 27 associations were included in a joint meta-analysis of the two cohorts, and 19 DNAm sites associated with mtDNA variants, while four other sites showed haplogroup associations. An expression quantitative trait methylation analysis was performed for the identified DNAm sites, pinpointing two statistically significant associations. This study provides evidence of a mitochondrial genetic control of nuclear DNAm with little evidence found for sex- and prediabetes-specific effects. The lack of a comparable mtDNA data set for replication is a limitation in our study and further studies are needed to validate our results.