High prevalence of multilocus pathogenic variation in neurodevelopmental disorders in the Turkish population.

Neurodevelopmental disorders (NDDs) are clinically and genetically heterogenous; many such disorders are secondary to perturbation in brain development and/or function. The prevalence of NDDs is > 3%, resulting in significant sociocultural and economic challenges to society. With recent advances in family-based genomics, rare-variant analyses, and further exploration of the Clan Genomics hypothesis, there has been a logarithmic explosion in neurogenetic "disease-associated genes" molecular etiology and biology of NDDs; however, the majority of NDDs remain molecularly undiagnosed. We applied genome-wide screening technologies, including exome sequencing (ES) and whole-genome sequencing (WGS), to identify the molecular etiology of 234 newly enrolled subjects and 20 previously unsolved Turkish NDD families. In 176 of the 234 studied families (75.2%), a plausible and genetically parsimonious molecular etiology was identified. Out of 176 solved families, deleterious variants were identified in 218 distinct genes, further documenting the enormous genetic heterogeneity and diverse perturbations in human biology underlying NDDs. We propose 86 candidate disease-trait-associated genes for an NDD phenotype. Importantly, on the basis of objective and internally established variant prioritization criteria, we identified 51 families (51/176 = 28.9%) with multilocus pathogenic variation (MPV), mostly driven by runs of homozygosity (ROHs) - reflecting genomic segments/haplotypes that are identical-by-descent. Furthermore, with the use of additional bioinformatic tools and expansion of ES to additional family members, we established a molecular diagnosis in 5 out of 20 families (25%) who remained undiagnosed in our previously studied NDD cohort emanating from Turkey.

Phenotypic heterogeneity associated with KIF21A: Two new cases and review of the literature.

Our understanding of genetic and phenotypic heterogeneity associated with the clinical spectrum of rare diseases continues to expand. Thorough phenotypic descriptions and model organism functional studies are valuable tools in dissecting the biology of the disease process. Kinesin genes are well known to be associated with specific disease phenotypes and a subset of kinesin genes, including KIF21A, have been associated with more than one disease. Here we report two patients with KIF21A variants identified by exome sequencing; one with biallelic variants, supporting a novel KIF21A related syndrome with recessive inheritance and the second report of this condition, and another with a heterozygous de novo variant allele representing a phenotypic expansion of the condition described to date. We provide detailed phenotypic information on both families, including a novel neuropathology finding of neuroaxonal dystrophy associated with biallelic variants in KIF21A. Additionally, we studied the dominant variant in Saccharomyces cerevisiae to assess variant pathogenicity and found that this variant appears to impair protein function. KIF21A associated disease has mounting evidence for phenotypic heterogeneity; further patients and study of an allelic series are required to define the phenotypic spectrum and further explore the molecular etiology for each of these conditions.

A biallelic frameshift indel in PPP1R35 as a cause of primary microcephaly.

Protein phosphatase 1 regulatory subunit 35 (PPP1R35) encodes a centrosomal protein required for recruiting microtubule-binding elongation machinery. Several proteins in this centriole biogenesis pathway correspond to established primary microcephaly (MCPH) genes, and multiple model organism studies hypothesize PPP1R35 as a candidate MCPH gene. Here, using exome sequencing (ES) and family-based rare variant analyses, we report a homozygous, frameshifting indel deleting the canonical stop codon in the last exon of PPP1R35 [Chr7: c.753_*3delGGAAGCGTAGACCinsCG (p.Trp251Cysfs*22)]; the variant allele maps in a 3.7 Mb block of absence of heterozygosity (AOH) in a proband with severe MCPH (-4.3 SD at birth, -6.1 SD by 42 months), pachygyria, and global developmental delay from a consanguineous Turkish kindred. Droplet digital PCR (ddPCR) confirmed mutant mRNA expression in fibroblasts. In silico prediction of the translation of mutant PPP1R35 is expected to be elongated by 18 amino acids before encountering a downstream stop codon. This complex indel allele is absent in public databases (ClinVar, gnomAD, ARIC, 1000 genomes) and our in-house database of 14,000+ exomes including 1800+ Turkish exomes supporting predicted pathogenicity. Comprehensive literature searches for PPP1R35 variants yielded two probands affected with severe microcephaly (-15 SD and -12 SD) with the same homozygous indel from a single, consanguineous, Iranian family from a cohort of 404 predominantly Iranian families. The lack of heterozygous cases in two large cohorts representative of the genetic background of these two families decreased our suspicion of a founder allele and supports the contention of a recurrent mutation. We propose two potential secondary structure mutagenesis models for the origin of this variant allele mediated by hairpin formation between complementary GC rich segments flanking the stop codon via secondary structure mutagenesis.

The clinical and molecular spectrum of QRICH1 associated neurodevelopmental disorder.

De novo variants in QRICH1 (Glutamine-rich protein 1) has recently been reported in 11 individuals with intellectual disability (ID). The function of QRICH1 is largely unknown but it is likely to play a key role in the unfolded response of endoplasmic reticulum stress through transcriptional control of proteostasis. In this study, we present 27 additional individuals and delineate the clinical and molecular spectrum of the individuals (n = 38) with QRICH1 variants. The main clinical features were mild to moderate developmental delay/ID (71%), nonspecific facial dysmorphism (92%) and hypotonia (39%). Additional findings included poor weight gain (29%), short stature (29%), autism spectrum disorder (29%), seizures (24%) and scoliosis (18%). Minor structural brain abnormalities were reported in 52% of the individuals with brain imaging. Truncating or splice variants were found in 28 individuals and 10 had missense variants. Four variants were inherited from mildly affected parents. This study confirms that heterozygous QRICH1 variants cause a neurodevelopmental disorder including short stature and expands the phenotypic spectrum to include poor weight gain, scoliosis, hypotonia, minor structural brain anomalies, and seizures. Inherited variants from mildly affected parents are reported for the first time, suggesting variable expressivity.

Bi-allelic Pathogenic Variants in TUBGCP2 Cause Microcephaly and Lissencephaly Spectrum Disorders.

Lissencephaly comprises a spectrum of malformations of cortical development. This spectrum includes agyria, pachygyria, and subcortical band heterotopia; each represents anatomical malformations of brain cortical development caused by neuronal migration defects. The molecular etiologies of neuronal migration anomalies are highly enriched for genes encoding microtubules and microtubule-associated proteins, and this enrichment highlights the critical role for these genes in cortical growth and gyrification. Using exome sequencing and family based rare variant analyses, we identified a homozygous variant (c.997C>T [p.Arg333Cys]) in TUBGCP2, encoding gamma-tubulin complex protein 2 (GCP2), in two individuals from a consanguineous family; both individuals presented with microcephaly and developmental delay. GCP2 forms the multiprotein γ-tubulin ring complex (γ-TuRC) together with γ-tubulin and other GCPs to regulate the assembly of microtubules. By querying clinical exome sequencing cases and through GeneMatcher-facilitated collaborations, we found three additional families with bi-allelic variation and similarly affected phenotypes including a homozygous variant (c.1843G>C [p.Ala615Pro]) in two families and compound heterozygous variants consisting of one missense variant (c.889C>T [p.Arg297Cys]) and one splice variant (c.2025-2A>G) in another family. Brain imaging from all five affected individuals revealed varying degrees of cortical malformations including pachygyria and subcortical band heterotopia, presumably caused by disruption of neuronal migration. Our data demonstrate that pathogenic variants in TUBGCP2 cause an autosomal recessive neurodevelopmental trait consisting of a neuronal migration disorder, and our data implicate GCP2 as a core component of γ-TuRC in neuronal migrating cells.

Homozygous Missense Variants in NTNG2, Encoding a Presynaptic Netrin-G2 Adhesion Protein, Lead to a Distinct Neurodevelopmental Disorder.

NTNG2 encodes netrin-G2, a membrane-anchored protein implicated in the molecular organization of neuronal circuitry and synaptic organization and diversification in vertebrates. In this study, through a combination of exome sequencing and autozygosity mapping, we have identified 16 individuals (from seven unrelated families) with ultra-rare homozygous missense variants in NTNG2; these individuals present with shared features of a neurodevelopmental disorder consisting of global developmental delay, severe to profound intellectual disability, muscle weakness and abnormal tone, autistic features, behavioral abnormalities, and variable dysmorphisms. The variants disrupt highly conserved residues across the protein. Functional experiments, including in silico analysis of the protein structure, in vitro assessment of cell surface expression, and in vitro knockdown, revealed potential mechanisms of pathogenicity of the variants, including loss of protein function and decreased neurite outgrowth. Our data indicate that appropriate expression of NTNG2 plays an important role in neurotypical development.

The Genomics of Arthrogryposis, a Complex Trait: Candidate Genes and Further Evidence for Oligogenic Inheritance.

Arthrogryposis is a clinical finding that is present either as a feature of a neuromuscular condition or as part of a systemic disease in over 400 Mendelian conditions. The underlying molecular etiology remains largely unknown because of genetic and phenotypic heterogeneity. We applied exome sequencing (ES) in a cohort of 89 families with the clinical sign of arthrogryposis. Additional molecular techniques including array comparative genomic hybridization (aCGH) and Droplet Digital PCR (ddPCR) were performed on individuals who were found to have pathogenic copy number variants (CNVs) and mosaicism, respectively. A molecular diagnosis was established in 65.2% (58/89) of families. Eleven out of 58 families (19.0%) showed evidence for potential involvement of pathogenic variation at more than one locus, probably driven by absence of heterozygosity (AOH) burden due to identity-by-descent (IBD). RYR3, MYOM2, ERGIC1, SPTBN4, and ABCA7 represent genes, identified in two or more families, for which mutations are probably causative for arthrogryposis. We also provide evidence for the involvement of CNVs in the etiology of arthrogryposis and for the idea that both mono-allelic and bi-allelic variants in the same gene cause either similar or distinct syndromes. We were able to identify the molecular etiology in nine out of 20 families who underwent reanalysis. In summary, our data from family-based ES further delineate the molecular etiology of arthrogryposis, yielded several candidate disease-associated genes, and provide evidence for mutational burden in a biological pathway or network. Our study also highlights the importance of reanalysis of individuals with unsolved diagnoses in conjunction with sequencing extended family members.

Identifying Genes Whose Mutant Transcripts Cause Dominant Disease Traits by Potential Gain-of-Function Alleles.

Premature termination codon (PTC)-bearing transcripts are often degraded by nonsense-mediated decay (NMD) resulting in loss-of-function (LoF) alleles. However, not all PTCs result in LoF mutations, i.e., some such transcripts escape NMD and are translated to truncated peptide products that result in disease due to gain-of-function (GoF) effects. Since the location of the PTC is a major factor determining transcript fate, we hypothesized that depletion of protein-truncating variants (PTVs) within the gene region predicted to escape NMD in control databases could provide a rank for genic susceptibility for disease through GoF versus LoF. We developed an NMD escape intolerance score to rank genes based on the depletion of PTVs that would render them able to escape NMD using the Atherosclerosis Risk in Communities Study (ARIC) and the Exome Aggregation Consortium (ExAC) control databases, which was further used to screen the Baylor-Center for Mendelian Genomics disease database. This analysis revealed 1,996 genes significantly depleted for PTVs that are predicted to escape from NMD, i.e., PTVesc; further studies provided evidence that revealed a subset as candidate genes underlying Mendelian phenotypes. Importantly, these genes have characteristically low pLI scores, which can cause them to be overlooked as candidates for dominant diseases. Collectively, we demonstrate that this NMD escape intolerance score is an effective and efficient tool for gene discovery in Mendelian diseases due to production of truncated or altered proteins. More importantly, we provide a complementary analytical tool to aid identification of genes associated with dominant traits through a mechanism distinct from LoF.

Biallelic and monoallelic variants in PLXNA1 are implicated in a novel neurodevelopmental disorder with variable cerebral and eye anomalies.

PURPOSE: To investigate the effect of PLXNA1 variants on the phenotype of patients with autosomal dominant and recessive inheritance patterns and to functionally characterize the zebrafish homologs plxna1a and plxna1b during development. METHODS: We assembled ten patients from seven families with biallelic or de novo PLXNA1 variants. We describe genotype-phenotype correlations, investigated the variants by structural modeling, and used Morpholino knockdown experiments in zebrafish to characterize the embryonic role of plxna1a and plxna1b. RESULTS: Shared phenotypic features among patients include global developmental delay (9/10), brain anomalies (6/10), and eye anomalies (7/10). Notably, seizures were predominantly reported in patients with monoallelic variants. Structural modeling of missense variants in PLXNA1 suggests distortion in the native protein. Our zebrafish studies enforce an embryonic role of plxna1a and plxna1b in the development of the central nervous system and the eye. CONCLUSION: We propose that different biallelic and monoallelic variants in PLXNA1 result in a novel neurodevelopmental syndrome mainly comprising developmental delay, brain, and eye anomalies. We hypothesize that biallelic variants in the extracellular Plexin-A1 domains lead to impaired dimerization or lack of receptor molecules, whereas monoallelic variants in the intracellular Plexin-A1 domains might impair downstream signaling through a dominant-negative effect.

Deficiencies in vesicular transport mediated by TRAPPC4 are associated with severe syndromic intellectual disability.

The conserved transport protein particle (TRAPP) complexes regulate key trafficking events and are required for autophagy. TRAPPC4, like its yeast Trs23 orthologue, is a core component of the TRAPP complexes and one of the essential subunits for guanine nucleotide exchange factor activity for Rab1 GTPase. Pathogenic variants in specific TRAPP subunits are associated with neurological disorders. We undertook exome sequencing in three unrelated families of Caucasian, Turkish and French-Canadian ethnicities with seven affected children that showed features of early-onset seizures, developmental delay, microcephaly, sensorineural deafness, spastic quadriparesis and progressive cortical and cerebellar atrophy in an effort to determine the genetic aetiology underlying neurodevelopmental disorders. All seven affected subjects shared the same identical rare, homozygous, potentially pathogenic variant in a non-canonical, well-conserved splice site within TRAPPC4 (hg19:chr11:g.118890966A>G; TRAPPC4: NM_016146.5; c.454+3A>G). Single nucleotide polymorphism array analysis revealed there was no haplotype shared between the tested Turkish and Caucasian families suggestive of a variant hotspot region rather than a founder effect. In silico analysis predicted the variant to cause aberrant splicing. Consistent with this, experimental evidence showed both a reduction in full-length transcript levels and an increase in levels of a shorter transcript missing exon 3, suggestive of an incompletely penetrant splice defect. TRAPPC4 protein levels were significantly reduced whilst levels of other TRAPP complex subunits remained unaffected. Native polyacrylamide gel electrophoresis and size exclusion chromatography demonstrated a defect in TRAPP complex assembly and/or stability. Intracellular trafficking through the Golgi using the marker protein VSVG-GFP-ts045 demonstrated significantly delayed entry into and exit from the Golgi in fibroblasts derived from one of the affected subjects. Lentiviral expression of wild-type TRAPPC4 in these fibroblasts restored trafficking, suggesting that the trafficking defect was due to reduced TRAPPC4 levels. Consistent with the recent association of the TRAPP complex with autophagy, we found that the fibroblasts had a basal autophagy defect and a delay in autophagic flux, possibly due to unsealed autophagosomes. These results were validated using a yeast trs23 temperature sensitive variant that exhibits constitutive and stress-induced autophagic defects at permissive temperature and a secretory defect at restrictive temperature. In summary we provide strong evidence for pathogenicity of this variant in a member of the core TRAPP subunit, TRAPPC4 that associates with vesicular trafficking and autophagy defects. This is the first report of a TRAPPC4 variant, and our findings add to the growing number of TRAPP-associated neurological disorders.

The role of FREM2 and FRAS1 in the development of congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) has been reported twice in individuals with a clinical diagnosis of Fraser syndrome, a genetic disorder that can be caused by recessive mutations affecting FREM2 and FRAS1. In the extracellular matrix, FREM2 and FRAS1 form a self-stabilizing complex with FREM1, a protein whose deficiency causes sac CDH in humans and mice. By sequencing FREM2 and FRAS1 in a CDH cohort, and searching online databases, we identified five individuals who carried recessive or double heterozygous, putatively deleterious variants in these genes which may represent susceptibility alleles. Three of these alleles were significantly enriched in our CDH cohort compared with ethnically matched controls. We subsequently demonstrated that 8% of Frem2ne/ne and 1% of Fras1Q1263*/Q1263* mice develop the same type of anterior sac CDH seen in FREM1-deficient mice. We went on to show that development of sac hernias in FREM1-deficient mice is preceded by failure of anterior mesothelial fold progression resulting in the persistence of an amuscular, poorly vascularized anterior diaphragm that is abnormally adherent to the underlying liver. Herniation occurs in the perinatal period when the expanding liver protrudes through this amuscular region of the anterior diaphragm that is juxtaposed to areas of muscular diaphragm. Based on these data, we conclude that deficiency of FREM2, and possibly FRAS1, are associated with an increased risk of developing CDH and that loss of the FREM1/FREM2/FRAS1 complex, or its function, leads to anterior sac CDH development through its effects on mesothelial fold progression.

Wolff-Parkinson-White syndrome: De novo variants and evidence for mutational burden in genes associated with atrial fibrillation.

BACKGROUND: Wolff-Parkinson-White (WPW) syndrome is a relatively common arrhythmia affecting ~1-3/1,000 individuals. Mutations in PRKAG2 have been described in rare patients in association with cardiomyopathy. However, the genetic basis of WPW in individuals with a structurally normal heart remains poorly understood. Sudden death due to atrial fibrillation (AF) can also occur in these individuals. Several studies have indicated that despite ablation of an accessory pathway, the risk of AF remains high in patients compared to general population. METHODS: We applied exome sequencing in 305 subjects, including 65 trios, 80 singletons, and 6 multiple affected families. We used de novo analysis, candidate gene approach, and burden testing to explore the genetic contributions to WPW. RESULTS: A heterozygous deleterious variant in PRKAG2 was identified in one subject, accounting for 0.6% (1/151) of the genetic basis of WPW in this study. Another individual with WPW and left ventricular hypertrophy carried a known pathogenic variant in MYH7. We found rare de novo variants in genes associated with arrhythmia and cardiomyopathy (ANK2, NEBL, PITX2, and PRDM16) in this cohort. There was an increased burden of rare deleterious variants (MAF ≤ 0.005) with CADD score ≥ 25 in genes linked to AF in cases compared to controls (P = .0023). CONCLUSIONS: Our findings show an increased burden of rare deleterious variants in genes linked to AF in WPW syndrome, suggesting that genetic factors that determine the development of accessory pathways may be linked to an increased susceptibility of atrial muscle to AF in a subset of patients.

Association Study of Exon Variants in the NF-κB and TGFß Pathways Identifies CD40 as a Modifier of Duchenne Muscular Dystrophy.

The expressivity of Mendelian diseases can be influenced by factors independent from the pathogenic mutation: in Duchenne muscular dystrophy (DMD), for instance, age at loss of ambulation (LoA) varies between individuals whose DMD mutations all abolish dystrophin expression. This suggests the existence of trans-acting variants in modifier genes. Common single nucleotide polymorphisms (SNPs) in candidate genes (SPP1, encoding osteopontin, and LTBP4, encoding latent transforming growth factor ß [TGFß]-binding protein 4) have been established as DMD modifiers. We performed a genome-wide association study of age at LoA in a sub-cohort of European or European American ancestry (n = 109) from the Cooperative International Research Group Duchenne Natural History Study (CINRG-DNHS). We focused on protein-altering variants (Exome Chip) and included glucocorticoid treatment as a covariate. As expected, due to the small population size, no SNPs displayed an exome-wide significant p value (< 1.8 × 10-6). Subsequently, we prioritized 438 SNPs in the vicinities of 384 genes implicated in DMD-related pathways, i.e., the nuclear-factor-κB and TGFß pathways. The minor allele at rs1883832, in the 5'-untranslated region of CD40, was associated with earlier LoA (p = 3.5 × 10-5). This allele diminishes the expression of CD40, a co-stimulatory molecule for T cell polarization. We validated this association in multiple independent DMD cohorts (United Dystrophinopathy Project, Bio-NMD, and Padova, total n = 660), establishing this locus as a DMD modifier. This finding points to cell-mediated immunity as a relevant pathogenetic mechanism and potential therapeutic target in DMD.

Congenital Titinopathy: Comprehensive characterization and pathogenic insights.

OBJECTIVE: Comprehensive clinical characterization of congenital titinopathy to facilitate diagnosis and management of this important emerging disorder. METHODS: Using massively parallel sequencing we identified 30 patients from 27 families with 2 pathogenic nonsense, frameshift and/or splice site TTN mutations in trans. We then undertook a detailed analysis of the clinical, histopathological and imaging features of these patients. RESULTS: All patients had prenatal or early onset hypotonia and/or congenital contractures. None had ophthalmoplegia. Scoliosis and respiratory insufficiency typically developed early and progressed rapidly, whereas limb weakness was often slowly progressive, and usually did not prevent independent walking. Cardiac involvement was present in 46% of patients. Relatives of 2 patients had dilated cardiomyopathy. Creatine kinase levels were normal to moderately elevated. Increased fiber size variation, internalized nuclei and cores were common histopathological abnormalities. Cap-like regions, whorled or ring fibers, and mitochondrial accumulations were also observed. Muscle magnetic resonance imaging showed gluteal, hamstring and calf muscle involvement. Western blot analysis showed a near-normal sized titin protein in all samples. The presence of 2 mutations predicted to impact both N2BA and N2B cardiac isoforms appeared to be associated with greatest risk of cardiac involvement. One-third of patients had 1 mutation predicted to impact exons present in fetal skeletal muscle, but not included within the mature skeletal muscle isoform transcript. This strongly suggests developmental isoforms are involved in the pathogenesis of this congenital/early onset disorder. INTERPRETATION: This detailed clinical reference dataset will greatly facilitate diagnostic confirmation and management of patients, and has provided important insights into disease pathogenesis. Ann Neurol 2018;83:1105-1124.

Identification of a pathogenic PMP2 variant in a multi-generational family with CMT type 1: Clinical gene panels versus genome-wide approaches to molecular diagnosis.

Charcot-Marie-Tooth (CMT) disease type 1 is an inherited peripheral neuropathy characterized by demyelination and reduced nerve conduction velocities. We present a multi-generational family with peripheral neuropathy in whom clinical CMT panel testing failed to conclude a molecular diagnosis. We found a PMP2 pathogenic variant c.155T > C, p.(Ile52Thr) that segregates with disease suggesting that PMP2 variants should be considered in patients with neuropathy and that it may be prudent to include in clinical CMT gene panels.

Diagnosis and etiology of congenital muscular dystrophy: We are halfway there.

OBJECTIVE: To evaluate the diagnostic outcomes in a large cohort of congenital muscular dystrophy (CMD) patients using traditional and next generation sequencing (NGS) technologies. METHODS: A total of 123 CMD patients were investigated using the traditional approaches of histology, immunohistochemical analysis of muscle biopsy, and candidate gene sequencing. Undiagnosed patients available for further testing were investigated using NGS. RESULTS: Muscle biopsy and immunohistochemical analysis found deficiencies of laminin α2, α-dystroglycan, or collagen VI in 50% of patients. Candidate gene sequencing and chromosomal microarray established a genetic diagnosis in 32% (39 of 123). Of 85 patients presenting in the past 20 years, 28 of 51 who lacked a confirmed genetic diagnosis (55%) consented to NGS studies, leading to confirmed diagnoses in a further 11 patients. Using the combination of approaches, a confirmed genetic diagnosis was achieved in 51% (43 of 85). The diagnoses within the cohort were heterogeneous. Forty-five of 59 probands with confirmed or probable diagnoses had variants in genes known to cause CMD (76%), and 11 of 59 (19%) had variants in genes associated with congenital myopathies, reflecting overlapping features of these conditions. One patient had a congenital myasthenic syndrome, and 2 had microdeletions. Within the cohort, 5 patients had variants in novel (PIGY and GMPPB) or recently published genes (GFPT1 and MICU1), and 7 had variants in TTN or RYR1, large genes that are technically difficult to Sanger sequence. INTERPRETATION: These data support NGS as a first-line tool for genetic evaluation of patients with a clinical phenotype suggestive of CMD, with muscle biopsy reserved as a second-tier investigation. Ann Neurol 2016;80:101-111.

Novel Col12A1 variant expands the clinical picture of congenital myopathies with extracellular matrix defects.

INTRODUCTION: Mutations in the COL12A1 (collagen, type XII, alpha 1) gene have been described in a milder Bethlem-like myopathy in 6 patients from 3 families (dominant missense), and in a severe congenital form with failure to attain ambulation in 2 patients in a single pedigree (recessive loss-of-function). METHODS: We describe an 8-year-old girl of Polish origin who presented with profound hypotonia and joint hyperlaxity at birth after a pregnancy complicated by oligohydramnios and intrauterine growth retardation. RESULTS: We identified a novel, potentially pathogenic heterozygous missense COL12A1 c.8329G>C (p.Gly2777Arg) variant using a targeted sequencing panel. Patient fibroblast studies confirmed intracellular retention of the COL12A1 protein, consistent with a dominant-negative mutation. CONCLUSIONS: As our patient showed a more intermediate phenotype, this case expands the phenotypic spectrum for COL12A1 disorders. So far, COL12A1 disorders seem to cover much of the severity range of an Ehlers-Danlos/Bethlem-like myopathy overlap syndrome associated with both connective tissue abnormalities and muscle weakness. Muscle Nerve 55: 277-281, 2017.

Mutations in GDP-mannose pyrophosphorylase B cause congenital and limb-girdle muscular dystrophies associated with hypoglycosylation of α-dystroglycan.

Congenital muscular dystrophies with hypoglycosylation of α-dystroglycan (α-DG) are a heterogeneous group of disorders often associated with brain and eye defects in addition to muscular dystrophy. Causative variants in 14 genes thought to be involved in the glycosylation of α-DG have been identified thus far. Allelic mutations in these genes might also cause milder limb-girdle muscular dystrophy phenotypes. Using a combination of exome and Sanger sequencing in eight unrelated individuals, we present evidence that mutations in guanosine diphosphate mannose (GDP-mannose) pyrophosphorylase B (GMPPB) can result in muscular dystrophy variants with hypoglycosylated α-DG. GMPPB catalyzes the formation of GDP-mannose from GTP and mannose-1-phosphate. GDP-mannose is required for O-mannosylation of proteins, including α-DG, and it is the substrate of cytosolic mannosyltransferases. We found reduced α-DG glycosylation in the muscle biopsies of affected individuals and in available fibroblasts. Overexpression of wild-type GMPPB in fibroblasts from an affected individual partially restored glycosylation of α-DG. Whereas wild-type GMPPB localized to the cytoplasm, five of the identified missense mutations caused formation of aggregates in the cytoplasm or near membrane protrusions. Additionally, knockdown of the GMPPB ortholog in zebrafish caused structural muscle defects with decreased motility, eye abnormalities, and reduced glycosylation of α-DG. Together, these data indicate that GMPPB mutations are responsible for congenital and limb-girdle muscular dystrophies with hypoglycosylation of α-DG.

Genetic modifiers of ambulation in the Cooperative International Neuromuscular Research Group Duchenne Natural History Study.

OBJECTIVE: We studied the effects of LTBP4 and SPP1 polymorphisms on age at loss of ambulation (LoA) in a multiethnic Duchenne muscular dystrophy (DMD) cohort. METHODS: We genotyped SPP1 rs28357094 and LTBP4 haplotype in 283 of 340 participants in the Cooperative International Neuromuscular Research Group Duchenne Natural History Study (CINRG-DNHS). Median ages at LoA were compared by Kaplan-Meier analysis and log-rank test. We controlled polymorphism analyses for concurrent effects of glucocorticoid corticosteroid (GC) treatment (time-varying Cox regression) and for population stratification (multidimensional scaling of genome-wide markers). RESULTS: Hispanic and South Asian participants (n = 18, 41) lost ambulation 2.7 and 2 years earlier than Caucasian subjects (p = 0.003, <0.001). The TG/GG genotype at SPP1 rs28357094 was associated to 1.2-year-earlier median LoA (p = 0.048). This difference was greater (1.9 years, p = 0.038) in GC-treated participants, whereas no difference was observed in untreated subjects. Cox regression confirmed a significant effect of SPP1 genotype in GC-treated participants (hazard ratio = 1.61, p = 0.016). LTBP4 genotype showed a direction of association with age at LoA as previously reported, but it was not statistically significant. After controlling for population stratification, we confirmed a strong effect of LTBP4 genotype in Caucasians (2.4 years, p = 0.024). Median age at LoA with the protective LTBP4 genotype in this cohort was 15.0 years, 16.0 for those who were treated with GC. INTERPRETATION: SPP1 rs28357094 acts as a pharmacodynamic biomarker of GC response, and LTBP4 haplotype modifies age at LoA in the CINRG-DNHS cohort. Adjustment for GC treatment and population stratification appears crucial in assessing genetic modifiers in DMD.

Facile synthesis of nucleoside 5'-(α-P-seleno)-triphosphates and phosphoroselenoate RNA transcription.

Phosphoroselenoate RNA (PSe-RNA) is nuclease resistant and has great potentials in X-ray crystal structure and function studies of noncoding RNAs and protein-RNA interactions. In order to conveniently synthesize PSe-RNA via transcription, we have developed a one-pot synthetic method for the nucleoside 5'-(α-P-seleno)-triphosphates (NTPαSe) analogs without protecting any functionality of the ribonucleosides. The NTPαSe diastereomers have been purified, fully characterized, and incorporated into RNAs by T7 RNA polymerase. The transcribed RNAs are diastereomerically pure, and the Se-derivatized ribozymes are generally active. Furthermore, we have established an affinity purification strategy by using immobilized boronate to conveniently purify NTPαSe analogs. Though the affinity-purified NTPαSe analogs are diastereomeric mixtures, they can be directly used in transcription without a significant impact on the transcription efficiency. Moreover, we found that the PSe-nucleotide is stable during polyacrylamide gel purification, indicating that the PSe-RNAs can be purified straightforwardly for crystal structural and functional studies.