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Truncating variants in exons 33 and 34 of the SNF2-related CREBBP activator protein (SRCAP) gene cause the neurodevelopmental disorder (NDD) Floating-Harbor syndrome (FLHS), characterized by short stature, speech delay, and facial dysmorphism. Here, we present a cohort of 33 individuals with clinical features distinct from FLHS and truncating (mostly de novo) SRCAP variants either proximal (n = 28) or distal (n = 5) to the FLHS locus. Detailed clinical characterization of the proximal SRCAP individuals identified shared characteristics: developmental delay with or without intellectual disability, behavioral and psychiatric problems, non-specific facial features, musculoskeletal issues, and hypotonia. Because FLHS is known to be associated with a unique set of DNA methylation (DNAm) changes in blood, a DNAm signature, we investigated whether there was a distinct signature associated with our affected individuals. A machine-learning model, based on the FLHS DNAm signature, negatively classified all our tested subjects. Comparing proximal variants with typically developing controls, we identified a DNAm signature distinct from the FLHS signature. Based on the DNAm and clinical data, we refer to the condition as "non-FLHS SRCAP-related NDD." All five distal variants classified negatively using the FLHS DNAm model while two classified positively using the proximal model. This suggests divergent pathogenicity of these variants, though clinically the distal group presented with NDD, similar to the proximal SRCAP group. In summary, for SRCAP, there is a clear relationship between variant location, DNAm profile, and clinical phenotype. These results highlight the power of combined epigenetic, molecular, and clinical studies to identify and characterize genotype-epigenotype-phenotype correlations.

Whole-exome sequencing (WES) has increasingly enabled new pathogenic gene variant identification for undiagnosed neurodevelopmental disorders and provided insights into both gene function and disease biology. Here, we describe seven children with a neurodevelopmental disorder characterized by microcephaly, profound developmental delays and/or intellectual disability, cataracts, severe epilepsy including infantile spasms, irritability, failure to thrive, and stereotypic hand movements. Brain imaging in these individuals reveals delay in myelination and cerebral atrophy. We observe an identical recurrent de novo heterozygous c.892C>T (p.Arg298Trp) variant in the nucleus accumbens associated 1 (NACC1) gene in seven affected individuals. One of the seven individuals is mosaic for this variant. NACC1 encodes a transcriptional repressor implicated in gene expression and has not previously been associated with germline disorders. The probability of finding the same missense NACC1 variant by chance in 7 out of 17,228 individuals who underwent WES for diagnoses of neurodevelopmental phenotypes is extremely small and achieves genome-wide significance (p = 1.25 × 10-14). Selective constraint against missense variants in NACC1 makes this excess of an identical missense variant in all seven individuals more remarkable. Our findings are consistent with a germline recurrent mutational hotspot associated with an allele-specific neurodevelopmental phenotype in NACC1.

Congenital muscular dystrophy (CMD) is a clinically and genetically heterogeneous group of inherited muscle disorders. In patients, muscle weakness is usually present at or shortly after birth and is progressive in nature. Merosin deficient congenital muscular dystrophy (MDC1A) is a form of CMD caused by a defect in the laminin-α2 gene (LAMA2). Laminin-α2 is an extracellular matrix protein that interacts with the dystrophin-dystroglycan (DGC) complex in membranes providing stability to muscle fibers. In an N-ethyl- N-nitrosourea mutagenesis screen to develop zebrafish models of neuromuscular diseases, we identified a mutant fish that exhibits severe muscular dystrophy early in development. Genetic mapping identified a splice site mutation in the lama2 gene. This splice site is highly conserved in humans and this mutation results in mis-splicing of RNA and a loss of protein function. Homozygous lama2 mutant zebrafish, designated lama2d501/cl501, exhibited reduced motor function and progressive degeneration of skeletal muscles and died at 8-15 days post fertilization. The skeletal muscles exhibited damaged myosepta and detachment of myofibers in the affected fish. Laminin-α2 deficiency also resulted in growth defects in the brain and eye of the mutant fish. This laminin-α2 deficient mutant fish represents a novel disease model to develop therapies for modulating splicing defects in congenital muscular dystrophies and to restore the muscle function in human patients with CMD. [ABSTRACT FROM AUTHOR]

Several congenital muscular dystrophies caused by defects in known or putative glycosyltransferases are commonly associated with hypoglycosylation of a-dystroglycan (a-DG) and a marked reduction of its receptor function. We have investigated changes in the processing and function of a-DG resulting from genetic manipulation of LARGE, the putative glycosyltransferase mutated both in Largemyd mice and in humans with congenital muscular dystrophy 1D (MDC1D). Here we show that overexpression of LARGE ameliorates the dystrophic phenotype of Largemyd mice and induces the synthesis of glycan-enriched a-DG with high affinity for extracellular ligands. Notably, LARGE circumvents the a-DG glycosylation defect in cells from individuals with genetically distinct types of congenital muscular dystrophy. Gene transfer of LARGE into the cells of individuals with congenital muscular dystrophies restores a-DG receptor function, whereby glycan-enriched a-DG coordinates the organization of laminin on the cell surface. Our findings indicate that modulation of LARGE expression or activity is a viable therapeutic strategy for glycosyltransferase-deficient congenital muscular dystrophies. [ABSTRACT FROM AUTHOR]

RAC1 is a widely studied Rho GTPase, a class of molecules that modulate numerous cellular functions essential for normal development. RAC1 is highly conserved across species and is under strict mutational constraint. We report seven individuals with distinct de novo missense RAC1 mutations and varying degrees of developmental delay, brain malformations, and additional phenotypes. Four individuals, each harboring one of c.53G>A (p.Cys18Tyr), c.116A>G (p.Asn39Ser), c.218C>T (p.Pro73Leu), and c.470G>A (p.Cys157Tyr) variants, were microcephalic, with head circumferences between −2.5 to −5 SD. In contrast, two individuals with c.151G>A (p.Val51Met) and c.151G>C (p.Val51Leu) alleles were macrocephalic with head circumferences of +4.16 and +4.5 SD. One individual harboring a c.190T>G (p.Tyr64Asp) allele had head circumference in the normal range. Collectively, we observed an extraordinary spread of ∼10 SD of head circumferences orchestrated by distinct mutations in the same gene. In silico modeling, mouse fibroblasts spreading assays, and in vivo overexpression assays using zebrafish as a surrogate model demonstrated that the p.Cys18Tyr and p.Asn39Ser RAC1 variants function as dominant-negative alleles and result in microcephaly, reduced neuronal proliferation, and cerebellar abnormalities in vivo . Conversely, the p.Tyr64Asp substitution is constitutively active. The remaining mutations are probably weakly dominant negative or their effects are context dependent. These findings highlight the importance of RAC1 in neuronal development. Along with TRIO and HACE1, a sub-category of rare developmental disorders is emerging with RAC1 as the central player. We show that ultra-rare disorders caused by private, non-recurrent missense mutations that result in varying phenotypes are challenging to dissect, but can be delineated through focused international collaboration. [ABSTRACT FROM AUTHOR]

Summary Background: In a systematic analysis of inherited forms of cardiomyopathy, we previously identified a family with X-linked dilated cardiomyopathy characterised by a mutation in the rod region of dystrophin. We have now attempted to eludicate the genetic mechanism involved in this disease, as well as the role of dystrophin-associated glycoproteins.Methods: The affected dystrophin epitope, which lacks binding to the dys-1 antibody, was analysed by single-strand conformation polymorphism analysis, reverse-transcription PCR, and DNA sequencing. Effects on dystrophin-associated glycoproteins were studied by immunohistochemistry and western blotting.Findings: A translation-termination mutation (C4148T) in exon 29 of the dystrophin gene was found in all affected family members. Alternative splicing rescued the reading frame and led to the expression of a dystrophin molecule lacking 50 aminoacids both in cardiac and skeletal muscle. Immunohistochemical analysis of the dystrophin-associated proteins revealed a reduction of beta-sarcoglycan and [DELTA]-sarcoglycan in the sarcolemma of cardiac muscle but not skeletal muscle tissue. However, western blotting revealed similar amounts of sarcoglycan subunits in both tissues.Interpretation: The molecular mechanism of this subtype of X-linked cardiomyopathy may be explained by a conformational change in exon-29-deleted dystrophin, resulting in disruption of the sarcoglycan assembly in heart muscle but not skeletal muscle. [ABSTRACT FROM AUTHOR]