Human Molecular Genetics

It is desirable to quantify the prediction accuracy of polygenic scores (PGS) for disease on the scale of liability and adjusted for case-control ascertainment in the test sample, because that allows comparison across prevalence and ascertainment. Previous expressions have focused in their derivation and implementation on linear regression on the observed 0-1 scale followed by a transformation of the coefficient of determination (R2) to the scale of liability, adjusted for ascertainment. Yet most statistical analyses with empirical data use logistic regression. The differences in scale have led to confusion and incorrect transformations in the literature. Here we provide a new derivation and simple equation, validated by simulation, that allows a direct transformation from the empirical results from logistic regression to the scale of liability.

Hypokalemic periodic paralysis (HypoPP) is an inherited skeletal muscle ion channelopathy of CACNA1S or SCN4A characterized by recurrent episodes of weakness, often triggered by rest after exercise or by reduced K+ (carbohydrate ingestion, stress). Here, we describe a multigenerational family in whom a KCNA7 missense variant [c.834A>C (p.Arg278Ser)] co-segregated with susceptibility to recurrent attacks of weakness in association with hypokalemia as low as 1.3 mEq/L and a 40% decrement of the compound muscle action potential after exercise, suggesting an additional HypoPP gene. Arg278 is the outermost positively charged residue (R1) within the S4 transmembrane segment of the voltage-sensor domain of KV1.7, orthologous to the canonical patten of arginine missense mutations at R1 or R2 in S4 segments for HypoPP-mutant CaV1.1 and NaV1.4 channels. To determine the functional consequences of Arg278Ser, we expressed wild-type and mutant KV1.7 channels in Xenopus oocytes and HEK293 cells. Arg278Ser, but not wild-type KV1.7, generated an anomalous inwardly rectifying current at hyperpolarized membrane potentials that was nonselective Na+ or K+, consistent with the anomalous gating pore conductance that causes susceptibility to HypoPP. Other pathogenic variants of KCNA7 have previously been implicated only in inherited cardiac arrhythmias. Collectively, the genetic, structural and electrophysiological findings support KCNA7 Arg278Ser as a pathogenic variant underlying HypoPP and implicate KCNA7 as a disease gene for periodic paralysis, extending the established gating-pore mechanism of HypoPP to KV1.7.

Pathogenic variants in MAPK8IP3 (JIP3) cause Neurodevelopmental Disorder with or without variable Brain Abnormalities (NEDBA), characterized by cognitive impairment, global developmental delay, motor dysfunction, abnormal muscle tone, behavioral dysregulation including autism and attention deficit hyperactivity disorder (ADHD), seizures, and structural brain abnormalities. While more than 30 largely de novo MAPK8IP3 variants have been reported, the functional impact of variants across JIP3 protein structural domains is poorly defined. To address this knowledge gap, we compare clinical features of individuals with a truncating (p.E27X) or one of two missense (p.R578C and p.R1146C) variants from distinct JIP3 functional protein domains to corresponding knock-in mouse models. Our findings showed that all individuals, regardless of variant type, exhibited delays in language and gross motor function, but individual variants were associated with distinct motor, cognitive, and psychiatric symptoms. Corresponding homozygous p.E27X and p.R1147C variant mice resulted in embryonic lethality, consistent with essential roles for JIP3 in early neurodevelopment. Behavioral characterization of viable heterozygous mice revealed variant-specific locomotor, motor coordination, and hindlimb clasping defects that closely recapitulate clinical observations. Heterozygous p.R579C mice exhibited reduced locomotion, impaired motor performance, and hypertonia-like clasping, mirroring human motor deficits. In contrast, p.R1147C mice displayed hyperactivity, hindlimb clasping, and decreased brain weight, paralleling human clinical features. Together, our findings demonstrate that while distinct MAPK8IP3 variants lead to some shared phenotypes, they are also associated with distinct phenotypes that could reveal domain-specific aspects of JIP3 function. This work establishes the first domain-resolved in vivo rodent models of NEDBA and provides a validated translational platform for mechanistic investigation and preclinical therapeutic testing.

Age-related macular degeneration (AMD) is the leading cause of irreversible central vision loss in older adults. The highest genetic risk for AMD maps to Chromosome 10q26. However, due to strong linkage disequilibrium between two genes, ARMS2 and HTRA1 located in this region, it has been difficult to determine whether one or both genes contribute to the AMD-disease risk. Functional studies of ARMS2 have been hampered due to the lack of animal models, as orthologs are only found in the higher primates and the gene is absent in rodents. To model ARMS2 biology in vivo, we used CRISPR-Cas9 to insert the complete human ARMS2 gene carrying the A69S risk variant (rs10490924), into a region of homology between humans and mice, on mouse Chromosome 7, 3.5 kb upstream of Htra1, preserving its native promoter, spacing, and orientation. We also generated a mouse model bearing its wild-type counterpart ARMS2A69 (ARMS2WT). RNA analysis revealed tissue- and cell-type-specific pattern of ARMS2 expression enriched in neural tissues, testis, and eye, and within the eye, in retinal horizontal cells and the retinal pigment epithelium (RPE). ARMS2 expression was consistent with human expression data and unaltered by the A69S mutation. By 12 months, ARMS2A69S mice develop AMD-relevant phenotypes including increased fundus spots, A69S-specific thinning of the outer nuclear layer, and reduced scotopic ERG amplitudes. Transcriptomic profiling of postnatal day 14 retina and RPE-enriched fractions revealed reduced mitochondrial oxidative-phosphorylation genes, corroborated by reduced retinal mtDNA copy number and lower maximal respiration, alongside preliminary results that suggest sphingolipid remodeling in the RPE. This humanized model links the ARMS2 A69S variant to mitochondrial dysfunction and provides an in vivo platform for defining AMD mechanisms and testing therapies.

Huntington disease is a fatal neurodegenerative disorder caused by CAG repeat expansion encoding polyglutamine in the HTT gene. Recent studies have shown that loss of CAA/CCA interruptions within polyglutamine-coding CAG tracts and adjacent polyproline-coding region are linked to earlier disease onset. It has been hypothesized that somatic repeat instability, influenced by these interrupted CAG tracts, may mediate this effect. Here we demonstrate that HTT CAA/CCA-loss variant linked to early disease onset exacerbates mutant HTT toxicity in cellular models through mechanisms independent of somatic repeat instability. The CAA/CCA-loss variant exhibits significantly higher toxicity than canonical HTT sequences in both transient expression and stable cell line models. Notably, this enhanced toxicity persists in knockout cells of the mismatch repair gene MSH3 where somatic instability is blunted, with a consistent toxicity hierarchy (CAA/CCA-loss > CCA-loss > CAA-loss > canonical HTT) in both wildtype and MSH3 knockout cells. Furthermore, the CAA/CCA-loss variant generates elevated levels of repeat-associated non-AUG (RAN) translation products. In HEK293-based cellular models, these results suggest that the disease-accelerating effects of HTT CAA/CCA-loss variants involve intrinsic properties of the altered sequence context, highlighting the importance of understanding sequence-specific mechanisms in HD pathogenesis beyond polyglutamine length and somatic instability.

Nail-patella syndrome (NPS) is a multisystem disorder caused by pathogenic variants in LMX1B and is characterized by dysplasia of the nails and patellae as well as extraskeletal complications such as progressive nephropathy and glaucoma. We generated a CRISPR/Cas9 knock-in mouse carrying the R252Q substitution, corresponding to a human LMX1B variant associated with renal-predominant disease. Phenotypic analysis revealed that homozygous mice were viable, but they displayed marked growth retardation and severe bilateral ocular opacity. Interestingly, while this model exhibited clear skeletal and ocular defects, the renal phenotype was relatively mild, although increased urinary albumin excretion, focal glomerular basement membrane abnormalities, and subtle changes in renal gene expression were detected. Beyond the classical NPS hallmarks, mutant mice also displayed midbrain morphological abnormalities, suggesting broader developmental consequences of this LMX1B variant. This patient-derived variant model not only recapitulates the pleiotropic features of NPS but also demonstrates organ-specific susceptibility to the R252Q substitution, providing a foundation for elucidating the complex molecular mechanisms underlying multisystem disease.

Spinocerebellar ataxia type 3 (SCA3) is an inherited, fatal neurodegenerative disease caused by a pathological CAG repeat expansion in the ATXN3 gene, resulting in the selective degeneration of vulnerable neuronal populations. Recent work has identified impairments in oligodendrocyte maturation as a novel and robust feature of SCA3 pathogenesis. Oligodendrocytes synthesize myelin structural components through the endoplasmic reticulum (ER), rendering this organelle essential for white matter integrity. Despite this, the role of ER function in SCA3 remains unclear. In this study, we show that loss of FICD-mediated AMPylation, a post-translational modification regulating the ER-resident HSP70 chaperone, BiP, rescues motor impairments in a transgenic SCA3 mouse model. Ficd-/- SCA3 mice exhibit significantly reduced levels of nuclear ATXN3 in vulnerable brain regions, while Ficd+/+ littermates show an increased burden of AMPylated BiP in the spinal cord, identifying aberrant AMPylation as a novel contributor of SCA3 pathology. Using unbiased proteomics, we demonstrate that Ficd deletion mitigates the pathological decrease in myelin structural proteins and oligodendrocyte maturation factors, restoring levels of mature, myelinating oligodendrocytes. In parallel, we show that Ficd activates SREBP2-dependent cholesterol biosynthesis to support myelination. Taken as a whole, these findings posit ER homeostasis as a critical driver of oligodendrocyte pathology and identify FICD as a novel target for alleviating non-neuronal toxicity in SCA3.

Background: Genome-wide association studies (GWASs) have identified thousands of genomic regions in which common variants affect blood pressure, but have yielded limited mechanistic insight. This study used a novel method of genetic analysis based on identifying core genes on which the _trans_-effects of common variants coalesce to influence blood pressure. Methods: We undertook GWASs of mean arterial pressure (MAP) and body mass index (BMI) in 373,882 individuals aged less than 60 years in the Our Future Health study. We used summary statistics from GWASs of proteins on the SomaScan and Olink platforms to compute genome-wide aggregated trans-effects (GATE) scores for each protein, and tested for association of MAP with these scores. Results: The strongest GATE score association with MAP was for lipoprotein lipase (LpL, encoded by LPL_. Genetic up-regulation of circulating levels of LpL was associated with lower MAP but higher BMI. Associations of MAP and BMI with GATE scores for circulating levels of proteins encoded by three other genes involved in lipid handling -- CD300LG, ADIPOQ, TIMP4 -- were similar to those with LpL. GATE scores for all four of these proteins were inversely associated with measurements by NMR spectroscopy of plasma triglyceride in chylomicrons and extremely large VLDL particles. Conclusions: These results point to a key role in hypertension for proteins that regulate post-prandial clearance of triglyceride-rich lipoproteins, independently of adiposity. This is consistent with experimental studies showing that accumulation of lipids in endothelial cells impairs endothelial function.

Amyotrophic lateral sclerosis (ALS) shows well-established sex differences in incidence, age at onset and progression, but their biological basis is unclear. We hypothesised that the DNA methylation changes associated with ALS differ between the sexes, which would point to sex-specific disease processes. To test this, we performed a blood-based epigenome-wide association meta-analysis of ALS cases and controls from Project MinE, an international ALS genomics consortium, and an independent Australian cohort, comprising 8,946 individuals after quality control (5,129 males and 3,817 females; 8,942 with complete covariates in the primary models). We analysed 781,648 autosomal CpGs using complementary sex-stratified case-control models and an ALS-by-sex interaction model that directly tested whether ALS-associated methylation effects differed by sex. Sex-stratified analyses identified no genome-wide significant differences between male and female ALS effects. In contrast, interaction testing identified seven robust sex-dependent ALS-associated differentially methylated positions (P < 9 x 10-8), mapping to KCNQ1, ADPRH, HLA-F, GHRLOS, IRF5, RAD51B and one intergenic region. The strongest signal was at KCNQ1 (P = 3.1 x 10-9). Six of the seven remained genome-wide significant in a four-stratum sensitivity analysis, and none was detected in previous conventional ALS or population sex-associated methylation analyses. Together, these findings suggest a distinct sex-conditional component of the ALS blood methylome, implicating immune, metabolic and ion-channel loci and providing new candidates for investigating biological heterogeneity between males and females with ALS.

Genetic ancestry composition has been used extensively as a predictor of clinical outcomes, multiomic biomarkers, and endophenotypes, but the models have most often used untransformed ancestry fractions as explanatory variables. Under a simplex constraint results from this type of model can be uninterpretable and misleading. We demonstrate the use of multiple transformed log ratio coordinates of ancestral proportions as predictors in a study of excreted myeloperoxidase (MPO), a neutrophilic enzyme used as a fecal biomarker to measure intestinal inflammation and enteropathy. We show how to convert these difficult-to-interpret effects into association results for shifts between two ancestries under an isoancestry constraint (holding an ancestry fraction constant) using post-estimation average marginal effects and explain their geometric interpretation. We found that increasing proportion of African ancestry was associated with increased levels of excreted MPO in low-income household infants in three Northeastern Brazil studies, with a smaller negative secondary association of European ancestry, all under a tri-continental ancestry composition (African, European, Amerindian). While the maximum log(MPO) outcome variance explained by differences in genetic ancestry was modest at a few percent, we show evidence that these differences in heritability were context-dependent on measurement of MPO under active enteric infection. The results were attenuated by socio-economic status, but a signal remained. Overall, we found that African genomes carry additional alleles that enable a more intensive neutrophil response to enteric infection relative to European and Amerindian genomes, illuminating a possible host genetic background role in modulating immune response to enteric diseases and pathogens.

Neuroblastoma amplified sequence gene (NBAS) variants are associated with short stature, optic atrophy, and Pelger-Huet anomaly (SOPH) syndrome. We previously identified compound heterozygous variants in NBAS to cause atypical Osteogenesis Imperfecta (OI), with these patients presenting with short stature, developmental delay and recurrent long-bone fractures. However, skeletal disease progression due to these variants and the disease mechanisms underlying NBAS-associated OI remain poorly understood. Here, we provide a clinical update on previously identified patients and investigate the role of NBAS during skeletal development using zebrafish knockout and patient-specific missense variant zebrafish models. Homozygous knockout larvae exhibited delayed operculum development, reduced bone ossification, and defects in Meckels cartilage morphology and its underlying cellular structure. Homozygous missense larvae displayed milder cartilage defects without any major defects to early skeletal structures. Seemingly opposing phenotypes were observed in compound heterozygous zebrafish carrying the knockout and missense alleles in trans, with no obvious phenotypes seen in the Meckels cartilage and accelerated operculum development observed. Together, our results demonstrate that different nbas variants differentially affect skeletal development, suggesting complex spectrums of phenotypic and pathogenic mechanisms in NBAS-associated atypical OI.

Nemaline myopathy is a severe inherited skeletal muscle disorder characterized by profound muscle weakness, impaired mobility, and early mortality in severe cases. Despite the devastating clinical burden, no disease-modifying therapies are currently available. Nemaline myopathy-causing genes either encode sarcomeric structural proteins or regulators of sarcomeric protein stability, resulting in primary defects in sarcomere structure and function. Recessive mutations in KLHL40 cause one of the most severe forms of nemaline myopathy, frequently resulting in perinatal lethality. Given its loss-of-function mechanism, KLHL40-associated nemaline myopathy is an attractive candidate for gene replacement therapy. We therefore evaluated the therapeutic potential of systemic AAV9-mediated KLHL40 gene replacement in a mouse model of severe nemaline myopathy. A single systemic administration of an AAV9 vector encoding human KLHL40 during early disease produced durable rescue of survival, skeletal muscle pathology, motor performance, and contractile function in KLHL40-deficient mice. Therapeutic benefit was accompanied by restoration of sarcomere organization and normalization of myofiber size without detectable long-term histopathological abnormalities in major organs. Collectively, our findings provide a strong preclinical foundation for developing AAV9-mediated KLHL40 gene replacement as a treatment for severe KLHL40-associated nemaline myopathy.

A pair of paralogs, Chchd3 and Chchd6, two components of mitochondrial contact site and cristae organizing system (MICOS), have been identified to be candidate pathogenetic genes in congenital heart disease (CHD). Previous research found that knockdown (KD) of the single Chchd3/6 (Chchd3) gene and other MICOS components in Drosophila impaired heart function, likely due to a deficit in mitochondrial organization, ATP production, actomyosin levels, and thus severely diminished contractility. However, the underlying mechanisms of how MICOS deficiency leads to these defects are not clear. Here, we performed genetic manipulations in the Drosophila heart to probe for possible interactions between MICOS-compromised mitochondria and other organelles and processes. We found that moderate reduction in Pink1/parkin-mediated mitophagy synergistically aggravated cardiac Chchd3 KD phenotypes, indicating a major interaction. Further, Chchd3 KD increased the level of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. Interestingly, KD of catalase (CAT) also elevated cardiac ROS levels, but surprisingly did not compromise contractility either by itself or in combination with Chchd3 KD to aggravate the cardiac phenotype. However, CAT overexpression (OE) in Chchd3 KD hearts restored contractility, but only partially, even though elevated ROS due to Chchd3 KD was fully normalized. Similarly, counteracting ER stress by overexpressing Xbp1 (or spliced mouse Xbp1) also partially rescued the heart function defects induced by Chchd3 KD. Overall, these data indicate a critical role of mitophagy and ER/oxidative stress in cardiac homeostasis involving Chchd3, which suggests that deficiency of MICOS function contributes to heart dysfunction via multiple stress responsive pathways.

BackgroundOverweight and obese individuals often exhibit lower mortality rates or better prognoses than lean or normal-weight individuals with stroke and other diseases, a phenomenon called the "obesity paradox". Carotid atherosclerosis is the primary cause of ischemic stroke, and body weight serves as a reliable surrogate for adiposity in mice. MethodsPhenotypic and genetic connections of carotid atherosclerosis with body weight were evaluated in 299 F2 mice derived from BALB/cJ and LP/J Apoe knockout (Apoe-/-) mice. F2 mice were fed a Western diet for 12 weeks. Atherosclerotic lesion sizes in left carotid arteries, body weight, coat color, plasma lipids, glucose, small dense LDL ApoB, and malondialdehyde were measured, and 11,000 single nucleotide polymorphism (SNP) markers were genotyped. ResultsCarotid lesion sizes inversely correlated with body weight in both sexes. Genome-wide scans identified two significant quantitative trait loci (QTLs) for carotid atherosclerosis on chromosomes (Chr) 6 and 15 in an additive sex model, and five QTLs on Chr 6, 7, 12, 13, and 15 in an interactive sex model. Adjusting for body weight variation downgraded Chr 15 QTL (Cath5) in both models, whereas other QTLs upgraded in the additive sex model and downgraded in the interactive sex model. Human syntenic region of Cath5 associated with carotid intima-medial thickness (cIMT) and waist-to-hip ratio (WHR). ConclusionsThese findings indicate that the obesity paradox in carotid atherosclerosis is partially driven by shared genetic components that exert opposing effects on adiposity and plaque development and act through sex-dependent mechanisms.

Background: Both fetal and maternal genomes contribute to birthweight, but the timing and relative magnitude of their effects during gestation remain unclear. Polygenic risk scores (PRSs), constructed from genome-wide association studies (GWAS), capture the cumulative influence of common birthweight-associated variants to quantify genetic predisposition to fetal growth. We constructed fetal and maternal PRSs for birthweight in two UK birth cohorts, the Pregnancy Outcome Prediction Study (POPS) and Born in Bradford (BiB), to evaluate their associations with estimated fetal weight (EFW) throughout pregnancy and with birthweight at delivery. We also investigated whether PRSs could improve risk stratification of infants born below the 10th centile by distinguishing those with pathological intrauterine growth restriction (IUGR) from those who are constitutionally small-for-gestational-age (SGA). Methods: In POPS and BiB, we used LDpred2-auto to estimate PRSs based on the largest available maternal and fetal GWAS for birthweight. We longitudinally assessed EFW associations with PRSs using linear mixed effects models in POPS and BiB. We evaluated birthweight associations with PRSs using linear models, where birthweight was adjusted for gestational age and fetal sex. We estimated R2 and conducted variance decomposition analyses for all models. Genetically-predicted birthweights were estimated from joint models of fetal and maternal PRSs plus ten genetic principal components (PCs). BiB analyses were conducted separately for genetically-inferred European (EUR) and South Asian (SAS) ancestry. Results: While PRSfetus was significantly associated with EFW at baseline ([~]22 weeks gestation) across cohorts, PRSmother was not. Both the interaction of PRSfetus and PRSmother with gestational age (GA) were significantly associated with EFW. The effect of PRSmother on EFW increased more quickly across gestation than the effect of PRSfetus in POPS, but this finding was not replicated in BiB. Jointly, fetal and maternal polygenic risk explained 4% of variance in EFW in POPS and 1% in BiB-EUR. In POPS, fetal and maternal PRSs showed independent contributions to EFW variance of 3% and 0.5%, respectively. Joint and univariable linear models showed significant positive associations of PRSfetus and PRSmother with observed birthweight. In POPS, PRSfetus and PRSmother accounted for [~]10% and [~]4% of variance in birthweight over covariates alone, respectively, which was replicated in BiB-EUR. Maternal and fetal genetic effects jointly explained 11% of birthweight variance in POPS, similar to BiB-EUR, at 12%; however, the variance explained was reduced by half in BiB-SAS, at 6%. Among POPS babies born <10th birthweight centile, the magnitude of the difference between observed and genetically-predicted birthweight was greater in the group judged clinically to be IUGR vs. SGA (p=0.008; point estimate=0.29, 95%CI: 0.12, 0.47). Conclusions: Maternal and fetal PRSs jointly account for [~]11-12% of birthweight variance in European ancestry groups, but much less in a South Asian cohort. In POPS, the effect of birthweight-associated SNPs on estimated fetal weight increases more rapidly across pregnancy for maternal than for fetal genetic effects. Deviation from genetically-predicted birthweight may be a useful metric to help distinguish growth-restricted from constitutionally small babies.

Ancestry-related genomic variation may contribute to phenotypic diversity. However, its cellular consequences remain difficult to evaluate. To address this, we developed a human induced pluripotent stem cell (hiPSC)-based framework for investigating such phenotypes. We used the Japanese population as a model of Jomon-related and continental East Asian ancestry admixture. Jomon ancestry, estimated using the Jomon allele score (JAS), was associated with clinical measures, including renal-related traits and serum triglycerides in 34,152 Tohoku Medical Megabank participants. Peripheral blood mononuclear cell transcriptomics of donors from the upper and lower extremes of the JAS distribution (n = 10) suggested group-level differences in immune-related gene expression. To test whether the serum triglyceride association coincided with hepatic cellular phenotypes, we differentiated hiPSCs from these donors into hepatocyte-like cells. Neither intracellular triglycerides nor lipid-related gene expression differed between groups. These findings highlight the value of a genetically characterized hiPSC-based framework for investigating ancestry-associated phenotypes across cellular contexts.

Birk-Barel syndrome (BBS), also known as KCNK9 imprinting syndrome, is a neurodevelopmental disorder caused by mutations in the maternally expressed paternally imprinted KCNK9 gene that encodes the TASK3 potassium channel. The first mutation identified in patients with BBS was a loss-of-function variant that reduces the potassium current mediated by TASK3. Subsequent studies have uncovered additional pathogenic variants, including gain-of-function mutations, that increase TASK3 activity. Here, we show that both mutations resulted in the expression of the hyperactive Task3M159I variant or the hypoactive Task3G236R variant, which caused early postnatal lethality in mice when maternally inherited, confirming the monogenic nature of BBS. These results indicate that the pathogenesis of BBS involves mechanisms other than alterations in channel activity. Consequently, the proposed therapeutic strategies, such as pharmacological modulation of the TASK3 channel function or epigenetic reactivation of the paternal KCNK9 allele, do not seem to be viable options. These findings also demonstrate that TASK3 knockout mice are not suitable models for studying KCNK9 imprinting syndrome or for developing therapies for affected patients. Therefore, there is an urgent need for animal models that allow conditional expression of BBS-associated mutations.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common dominant muscular dystrophies and remains without an approved disease modifying therapy. Caused by the aberrant expression of the cytotoxic gene DUX4, FSHD is typically diagnosed in adulthood, however clinical onset in children (<18 years of age) is often associated with a more severe and rapid disease. While clinical trials are underway, a lack of human-specific pre-clinical models limit effective testing of potential therapies, particularly in children. To fill this gap, we describe here the development of induced pluripotent stem cell-derived 2-and 3-dimensional skeletal muscle models of children with clinically defined mild, moderate, and severe FSHD. These iPSC-derived muscle models replicate key features of FSHD, including aberrant DUX4 mRNA expression, muscle atrophy, and weakness, which correlate with the individuals specific disease severity. Next, we assessed the efficacy of adenine base editing (ABE) as a potential gene therapy approach to treat FSHD. DUX4-targeted ABE reduced DUX4 mRNA expression, improved muscle area and force generation in the most severe individual. Together this work supports the use of iPSC-derived skeletal muscle models as a less invasive method to study childhood-onset FSHD and establishes targeted DUX4 gene editing therapies as a potential treatment approach.

Craniofacial malformations account for one third of all congenital anomalies. Genetic factors play a vital role, yet the list of causal genes and their mechanisms are far from complete. As part of a larger effort to sequence patients with micrognathia and Pierre-Robin sequence, we identified two candidate pathogenic missense variants in C-terminal binding protein 1 (CTBP1) along with a heterozygous early stop missense variant in alkaline phosphatase (ALPL) in a proband with mandibular hypoplasia. Ctbp1 has been shown to regulate Wnt/{beta}-Catenin signaling but it has not yet been implicated in craniofacial development. Here we generated two orthologous variants of Ctbp1 mimicking the patient variants using genome editing in mice and explored the micrognathia phenotype in combination with a previously reported Alpl null allele. Ctbp1Q148H/G238S; Alplnull/Wt complex heterozygous mutants have smaller mandibles recapitulating the human mandibular hypoplasia. We identified that a reduction in cell proliferation and active {beta}-Catenin levels could possibly account for the micrognathia phenotype in the Ctbp1; Alpl complex heterozygous. These data uncover a novel role for Ctbp1 in craniofacial development and highlight the complex genetic and molecular signaling in the pathogenesis of craniofacial malformations.

IRX4 is a TALE- homeodomain transcription factor which is essential for cardiac development. In murine models, Irx4 deficiency leads to impaired ventricular function and results in cardiomyopathy. To elucidate the role of IRX4 in human congenital heart disease (CHD), Sanger sequencing of the IRX4 gene was performed in 205 individuals with non-syndromic CHD, 24 Down syndrome (DS) cases with CHD, 27 DS cases without CHD, and 150 healthy control individuals. Two novel (p.Ser24Asn and p.Thr217Iso) and one reported variant (rs2232376) were identified in non-syndromic CHD. Concurrently, rs2232376 was also detected in DS with CHD. The first novel (p.Ser24Asn) and reported (rs2232376) variants lie in the N-terminal region while the second novel (Thr217Iso) variant lies within the TALE homeodomain. In silico structural modelling suggested that both the novel variants (p.Ser24Asn and Thr217Iso) induce conformational changes in the IRX4 protein, potentially altering its DNA-binding affinity. A significant reduced expression of IRX4 muteins was noted in Western blotting by both variants (p.Ser24Asn and Thr217Iso). Furthermore, luciferase reporter assays demonstrated decline in the activity of Nanog promoter and HEY2 enhancer in response to both the variants which was further corroborated by decrease mRNA expression in qRT-PCR. Additional downstream targets, including Nfyc, Nppa, and Bmp10, also exhibited anomalous expression due to both the variants (p.Ser24Asn and Thr217Iso). Altogether, the aberrant expression of muteins as well as downstream target genes along with compromised activities of promoters substantiate the pathogenic potential of the identified IRX4 variants and underscore the critical role of IRX4 in regulating multiple stages of cardiogenesis.