Pathogenic TNNI1 variants disrupt sarcomere contractility resulting in hypo- and hypercontractile muscle disease.

Troponin I (TnI) regulates thin filament activation and muscle contraction. Two isoforms, TnI-fast (TNNI2) and TnI-slow (TNNI1), are predominantly expressed in fast- and slow-twitch myofibers, respectively. TNNI2 variants are a rare cause of arthrogryposis, whereas TNNI1 variants have not been conclusively established to cause skeletal myopathy. We identified recessive loss-of-function TNNI1 variants as well as dominant gain-of-function TNNI1 variants as a cause of muscle disease, each with distinct physiological consequences and disease mechanisms. We identified three families with biallelic TNNI1 variants (F1: p.R14H/c.190-9G>A, F2 and F3: homozygous p.R14C), resulting in loss of function, manifesting with early-onset progressive muscle weakness and rod formation on histology. We also identified two families with a dominantly acting heterozygous TNNI1 variant (F4: p.R174Q and F5: p.K176del), resulting in gain of function, manifesting with muscle cramping, myalgias, and rod formation in F5. In zebrafish, TnI proteins with either of the missense variants (p.R14H; p.R174Q) incorporated into thin filaments. Molecular dynamics simulations suggested that the loss-of-function p.R14H variant decouples TnI from TnC, which was supported by functional studies showing a reduced force response of sarcomeres to submaximal [Ca2+] in patient myofibers. This contractile deficit could be reversed by a slow skeletal muscle troponin activator. In contrast, patient myofibers with the gain-of-function p.R174Q variant showed an increased force to submaximal [Ca2+], which was reversed by the small-molecule drug mavacamten. Our findings demonstrated that TNNI1 variants can cause muscle disease with variant-specific pathomechanisms, manifesting as either a hypo- or a hypercontractile phenotype, suggesting rational therapeutic strategies for each mechanism.

Generation of two iPSC lines from adult central core disease patients with dominant missense variants in the RYR1 gene.

RYR1 variants are a common cause of congenital myopathies, including multi-minicore disease (MmD) and central core disease (CCD). Here, we generated iPSC lines from two CCD patients with dominant RYR1 missense variants that affect the transmembrane (pore) and SPRY3 protein domains (p.His4813Tyr and p.Asn1346Lys, respectively). Both lines had typical iPSC morphology, expressed canonical pluripotency markers, exhibited trilineage differentiation potential, and had normal karyotypes. Together with existing RYR1 iPSC lines, these represent important tools to study and develop treatments for RYR1-related myopathies.

Generation of two iPSC lines from patients with inherited central core disease and concurrent malignant hyperthermia caused by dominant missense variants in the RYR1 gene.

RYR1 variants are the most common genetic cause of congenital myopathies, and typically cause central core disease (CCD) and/or malignant hyperthermia (MH). Here, we generated iPSC lines from two patients with CCD and MH caused by dominant RYR1 variants within the central region of the protein (p.Val2168Met and p.Arg2508Cys). Both lines displayed typical iPSC morphology, uniform expression of pluripotency markers, trilineage differentiation potential, and had normal karyotypes. These are the first RYR1 iPSC lines from patients with both CCD and MH. As these are common CCD/MH variants, these lines should be useful to study these conditions and test therapeutics.

Detecting missed diagnoses of spinal muscular atrophy in genome, exome, and panel sequencing datasets.

Spinal muscular atrophy (SMA) is a genetic disorder that causes progressive degeneration of lower motor neurons and the subsequent loss of muscle function throughout the body. It is the second most common recessive disorder in individuals of European descent and is present in all populations. Accurate tools exist for diagnosing SMA from short read and long read genome sequencing data. However, there are no publicly available tools for GRCh38-aligned data from panel or exome sequencing assays which continue to be used as first line tests for neuromuscular disorders. We therefore developed and extensively validated a new tool - SMA Finder - that can diagnose SMA not only in genome, but also exome and targeted sequencing samples aligned to GRCh37, GRCh38, or T2T-CHM13. It works by evaluating aligned reads that overlap the c.840 position of SMN1 and SMN2 in order to detect the most common molecular causes of SMA. We applied SMA Finder to 16,626 exomes and 3,911 genomes from heterogeneous rare disease cohorts sequenced at the Broad Institute Center for Mendelian Genomics as well as 1,157 exomes and 8,762 targeted sequencing samples from Tartu University Hospital. SMA Finder correctly identified all 16 known SMA cases and reported nine novel diagnoses which have since been confirmed by clinical testing, with another four novel diagnoses undergoing validation. Notably, out of the 29 total SMA positive cases, 21 had an initial clinical diagnosis of muscular dystrophy, congenital myasthenic syndrome, or congenital myopathy. This underscored the frequency with which SMA can be misdiagnosed as other neuromuscular disorders and confirmed the utility of using SMA Finder to reanalyze phenotypically diverse neuromuscular disease cohorts. Finally, we evaluated SMA Finder on 198,868 individuals that had both exome and genome sequencing data within the UK Biobank (UKBB) and found that SMA Finder's overall false positive rate was less than 1 / 200,000 exome samples, and its positive predictive value (PPV) was 96%. We also observed 100% concordance between UKBB exome and genome calls. This analysis showed that, even though it is located within a segmental duplication, the most common causal variant for SMA can be detected with comparable accuracy to monogenic disease variants in non-repetitive regions. Additionally, the high PPV demonstrated by SMA Finder, the existence of treatment options for SMA in which early diagnosis is imperative for therapeutic benefit, as well as widespread availability of clinical confirmatory testing for SMA, may warrant the addition of SMN1 to the ACMG list of genes with reportable secondary findings after genome and exome sequencing.

Loss-of-function variants in JPH1 cause congenital myopathy with prominent facial involvement.

Background: Weakness of facial, ocular, and axial muscles is a common clinical presentation in congenital myopathies caused by pathogenic variants in genes encoding triad proteins. Abnormalities in triad structure and function resulting in disturbed excitation-contraction coupling and Ca 2+ homeostasis can contribute to disease pathology. Methods: We analysed exome and genome sequencing data from three unrelated individuals with congenital myopathy characterised by striking facial, ocular, and bulbar involvement. We collected deep phenotypic data from the affected individuals. We analysed the RNA-seq data of one proband and performed gene expression outlier analysis in 129 samples. Results: The three probands had remarkably similar clinical presentation with prominent facial, ocular, and bulbar features. Disease onset was in the neonatal period with hypotonia, poor feeding, cleft palate and talipes. Muscle weakness was generalised but most prominent in the lower limbs with facial weakness also present. All patients had myopathic facies, bilateral ptosis, ophthalmoplegia and fatiguability. While muscle biopsy on light microscopy did not show any obvious morphological abnormalities, ultrastructural analysis showed slightly reduced triads, and structurally abnormal sarcoplasmic reticulum. DNA sequencing identified three unique homozygous loss of function variants in JPH1 , encoding junctophilin-1 in the three families; a stop-gain (c.354C>A; p.Tyr118*) and two frameshift (c.373del p.Asp125Thrfs*30 and c.1738del; p.Leu580Trpfs*16) variants. Muscle RNA-seq showed strong downregulation of JPH1 in the F3 proband. Conclusions: Junctophilin-1 is critical to the formation of skeletal muscle triad junctions by connecting the sarcoplasmic reticulum and T-tubules. Our findings suggest that loss of JPH1 results in a congenital myopathy with prominent facial, bulbar and ocular involvement. Key message: This study identified novel homozygous loss-of-function variants in the JPH1 gene, linking them to a unique form of congenital myopathy characterised by severe facial and ocular symptoms. Our research sheds light on the critical impact on junctophilin-1 function in skeletal muscle triad junction formation and the consequences of its disruption resulting in a myopathic phenotype. What is already known on this topic: Previous studies have shown that pathogenic variants in genes encoding triad proteins lead to various myopathic phenotypes, with clinical presentations often involving muscle weakness and myopathic facies. The triad structure is essential for excitation-contraction (EC) coupling and calcium homeostasis and is a key element in muscle physiology. What this study adds and how this study might affect research practice or policy: This study establishes that homozygous loss-of-function mutations in JPH1 cause a congenital myopathy predominantly affecting facial and ocular muscles. This study also provides clinical insights that may aid the clinicians in diagnosing similar genetically unresolved cases.

Role of the repeat expansion size in predicting age of onset and severity in RFC1 disease.

RFC1 disease, caused by biallelic repeat expansion in RFC1, is clinically heterogeneous in terms of age of onset, disease progression and phenotype. We investigated the role of the repeat size in influencing clinical variables in RFC1 disease. We also assessed the presence and role of meiotic and somatic instability of the repeat. In this study, we identified 553 patients carrying biallelic RFC1 expansions and measured the repeat expansion size in 392 cases. Pearson's coefficient was calculated to assess the correlation between the repeat size and age at disease onset. A Cox model with robust cluster standard errors was adopted to describe the effect of repeat size on age at disease onset, on age at onset of each individual symptoms, and on disease progression. A quasi-poisson regression model was used to analyse the relationship between phenotype and repeat size. We performed multi-variate linear regression to assess the association of the repeat size with the degree of cerebellar atrophy. Meiotic stability was assessed by Southern blotting on first-degree relatives of 27 probands. Finally, somatic instability was investigated by optical genome mapping on cerebellar and frontal cortex and unaffected peripheral tissue from four post-mortem cases. A larger repeat size of both smaller and larger allele was associated with an earlier age at neurological onset (smaller allele HR = 2.06, p < 0.001; larger allele HR = 1.53, p < 0.001) and with a higher hazard of developing disabling symptoms, such as dysarthria or dysphagia (smaller allele HR = 3.40, p < 0.001; larger allele HR = 1.71, p = 0.002) or loss of independent walking (smaller allele HR = 2.78, p < 0.001; larger allele HR = 1.60; p < 0.001) earlier in disease course. Patients with more complex phenotypes carried larger expansions (smaller allele: complex neuropathy RR = 1.30, p = 0.003; CANVAS RR = 1.34, p < 0.001; larger allele: complex neuropathy RR = 1.33, p = 0.008; CANVAS RR = 1.31, p = 0.009). Furthermore, larger repeat expansions in the smaller allele were associated with more pronounced cerebellar vermis atrophy (lobules I-V ß=-1.06, p < 0.001; lobules VI-VII ß=-0.34, p = 0.005). The repeat did not show significant instability during vertical transmission and across different tissues and brain regions. RFC1 repeat size, particularly of the smaller allele, is one of the determinants of variability in RFC1 disease and represents a key prognostic factor to predict disease onset, phenotype, and severity. Assessing the repeat size is warranted as part of the diagnostic test for RFC1 expansion.

Generation of two induced pluripotent stem cell lines from a 33-year-old central core disease patient with a heterozygous dominant c.14145_14156delCTACTGGGACA (p.Asn4715_Asp4718del) deletion in the RYR1 gene.

Central core disease (CCD) is a congenital disorder that results in hypotonia, delayed motor development, and areas of reduced oxidative activity in the muscle fibre. Two induced pluripotent stem cell (iPSC) lines were generated from the lymphoblastoid cells of a 33-year-old male with CCD, caused by a previously unreported dominant c.14145_14156delCTACTGGGACA (p.Asn4715_Asp4718del) deletion in the RYR1 gene. Both lines demonstrated typical morphology, pluripotency, trilineage differentiation, and had a normal karyotype. As the first published iPSC model of CCD caused by an RYR1 variant these lines are a potential resource for further investigation of RYR1-related myopathies in a human context.

RFC1 in an Australasian neurological disease cohort: extending the genetic heterogeneity and implications for diagnostics.

Cerebellar ataxia, neuropathy and vestibular areflexia syndrome is a progressive, generally late-onset, neurological disorder associated with biallelic pentanucleotide expansions in Intron 2 of the RFC1 gene. The locus exhibits substantial genetic variability, with multiple pathogenic and benign pentanucleotide repeat alleles previously identified. To determine the contribution of pathogenic RFC1 expansions to neurological disease within an Australasian cohort and further investigate the heterogeneity exhibited at the locus, a combination of flanking and repeat-primed PCR was used to screen a cohort of 242 Australasian patients with neurological disease. Patients whose data indicated large gaps within expanded alleles following repeat-primed PCR, underwent targeted long-read sequencing to identify novel repeat motifs at the locus. To increase diagnostic yield, additional probes at the RFC1 repeat region were incorporated into the PathWest diagnostic laboratory targeted neurological disease gene panel to enable first-pass screening of the locus for all samples tested on the panel. Within the Australasian cohort, we detected known pathogenic biallelic expansions in 15.3% (n = 37) of patients. Thirty indicated biallelic AAGGG expansions, two had biallelic 'Maori alleles' [(AAAGG)exp(AAGGG)exp], two samples were compound heterozygous for the Maori allele and an AAGGG expansion, two samples had biallelic ACAGG expansions and one sample was compound heterozygous for the ACAGG and AAGGG expansions. Forty-five samples tested indicated the presence of biallelic expansions not known to be pathogenic. A large proportion (84%) showed complex interrupted patterns following repeat-primed PCR, suggesting that these expansions are likely to be comprised of more than one repeat motif, including previously unknown repeats. Using targeted long-read sequencing, we identified three novel repeat motifs in expanded alleles. Here, we also show that short-read sequencing can be used to reliably screen for the presence or absence of biallelic RFC1 expansions in all samples tested using the PathWest targeted neurological disease gene panel. Our results show that RFC1 pathogenic expansions make a substantial contribution to neurological disease in the Australasian population and further extend the heterogeneity of the locus. To accommodate the increased complexity, we outline a multi-step workflow utilizing both targeted short- and long-read sequencing to achieve a definitive genotype and provide accurate diagnoses for patients.

Dynamic regulation of inter-organelle communication by ubiquitylation controls skeletal muscle development and disease onset.

Ubiquitin-proteasome system (UPS) dysfunction is associated with the pathology of a wide range of human diseases, including myopathies and muscular atrophy. However, the mechanistic understanding of specific components of the regulation of protein turnover during development and disease progression in skeletal muscle is unclear. Mutations in KLHL40, an E3 ubiquitin ligase cullin3 (CUL3) substrate-specific adapter protein, result in severe congenital nemaline myopathy, but the events that initiate the pathology and the mechanism through which it becomes pervasive remain poorly understood. To characterize the KLHL40-regulated ubiquitin-modified proteome during skeletal muscle development and disease onset, we used global, quantitative mass spectrometry-based ubiquitylome and global proteome analyses of klhl40a mutant zebrafish during disease progression. Global proteomics during skeletal muscle development revealed extensive remodeling of functional modules linked with sarcomere formation, energy, biosynthetic metabolic processes, and vesicle trafficking. Combined analysis of klh40 mutant muscle proteome and ubiquitylome identified thin filament proteins, metabolic enzymes, and ER-Golgi vesicle trafficking pathway proteins regulated by ubiquitylation during muscle development. Our studies identified a role for KLHL40 as a regulator of ER-Golgi anterograde trafficking through ubiquitin-mediated protein degradation of secretion-associated Ras-related GTPase1a (Sar1a). In KLHL40-deficient muscle, defects in ER exit site vesicle formation and downstream transport of extracellular cargo proteins result in structural and functional abnormalities. Our work reveals that the muscle proteome is dynamically fine-tuned by ubiquitylation to regulate skeletal muscle development and uncovers new disease mechanisms for therapeutic development in patients.

A common flanking variant is associated with enhanced meiotic stability of the FGF14 -SCA27B locus.

The factors driving initiation of pathological expansion of tandem repeats remain largely unknown. Here, we assessed the FGF14 -SCA27B (GAA)•(TTC) repeat locus in 2,530 individuals by long-read and Sanger sequencing and identified a 5'-flanking 17-bp deletion-insertion in 70.34% of alleles (3,463/4,923). This common sequence variation was present nearly exclusively on alleles with fewer than 30 GAA-pure repeats and was associated with enhanced meiotic stability of the repeat locus.

Ablation of the carboxy-terminal end of MAMDC2 causes a distinct muscular dystrophy.

The extracellular matrix (ECM) has an important role in the development and maintenance of skeletal muscle, and several muscle diseases are associated with the dysfunction of ECM elements. MAMDC2 is a putative ECM protein and its role in cell proliferation has been investigated in certain cancer types. However, its participation in skeletal muscle physiology has not been previously studied. We describe 17 individuals with an autosomal dominant muscular dystrophy belonging to two unrelated families in which different heterozygous truncating variants in the last exon of MAMDC2 co-segregate correctly with the disease. The radiological aspect of muscle involvement resembles that of COL6 myopathies with fat replacement at the peripheral rim of vastii muscles. In this cohort, a subfascial and peri-tendinous pattern is observed in upper and lower limb muscles. Here we show that MAMDC2 is expressed in adult skeletal muscle and differentiating muscle cells, where it appears to localize to the sarcoplasm and myonuclei. In addition, we show it is secreted by myoblasts and differentiating myotubes into to the extracellular compartment. The last exon encodes a disordered region with a polar residue compositional bias loss of which likely induces a toxic effect of the mutant protein. The precise mechanisms by which the altered MAMDC2 proteins cause disease remains to be determined. MAMDC2 is a skeletal muscle disease-associated protein. Its role in muscle development and ECM-muscle communication remains to be fully elucidated. Screening of the last exon of MAMDC2 should be considered in patients presenting with autosomal dominant muscular dystrophy, particularly in those with a subfascial radiological pattern of muscle involvement.

Expanding the phenotype of DNMT3A as a cause a congenital myopathy with rhabdomyolysis.

Pathogenic variants in DNMT3A are most commonly associated with Tatton-Brown-Rahman Syndrome (TBRS), but includes other phenotypes such as Heyn-Sproul-Jackson syndrome and acute myeloid leukemia (AML). We describe a patient presenting to the neuromuscular clinic with a de novo missense variant in DNMT3A where the striking clinical feature is that of a congenital myopathy with associated episodes of rhabdomyolysis, severe myalgias and chest pain along with phenotypic features associated with TBRS. Muscle biopsy showed minor myopathic features and cardiac investigations revealed mildly impaired bi-ventricular systolic function. We confirmed the DNA methylation profile matched haplo-insufficient TBRS cases, consistent with a loss of methyltransferase activity. Our report emphasizes the phenotypic overlap of patients with syndromic disorders presenting to neuromuscular clinics and limitations of gene panels in establishing a molecular diagnosis.

Novel SERAC1 Variant Presenting With Adult-Onset Extrapyramidal Dystonia-Parkinsonism Phenotype: A Case Report.

Objectives: To report a novel likely pathogenic variant in the SERAC1 gene associated with early adult-onset parkinsonism and progressive dystonia. Methods: Clinical, biochemical, and imaging assessments were performed on 2 affected adult brothers with a genetically unsolved progressive neurologic disorder followed by whole-genome sequencing. Results: A homozygous likely pathogenic variant in the SERAC1 gene (c.[129-2A > C], p.[(?)];[(?)]) was discovered. Discussion: We describe a novel homozygous variant in the serine active site-containing protein 1 gene (SERAC1) in 2 brothers with a progressive extrapyramidal movement disorder of early onset parkinsonism and dystonia. Previous variants have been associated with a severe 3-methylglutaconic aciduria with dystonia, deafness, hepatopathy, encephalopathy and Leigh-like syndrome, or juvenile onset complicated spastic paraparesis. Our cases expand the phenotype of SERAC1 variants, with an adult-onset presentation of dystonia-parkinsonism.

Transcriptome and Genome Analysis Uncovers a DMD Structural Variant: A Case Report.

Objective: Duchenne muscular dystrophy (DMD) is caused by pathogenic variants in the dystrophin gene (DMD). Hypermethylated CGG expansions within DIP2B 5' UTR are associated with an intellectual development disorder. Here, we demonstrate the diagnostic utility of genomic short-read sequencing (SRS) and transcriptome sequencing to identify a novel DMD structural variant (SV) and a DIP2B CGG expansion in a patient with DMD for whom conventional diagnostic testing failed to yield a genetic diagnosis. Methods: We performed genomic SRS, skeletal muscle transcriptome sequencing, and targeted programmable long-read sequencing (LRS). Results: The proband had a typical DMD clinical presentation, autism spectrum disorder (ASD), and dystrophinopathy on muscle biopsy. Transcriptome analysis identified 6 aberrantly expressed genes; DMD and DIP2B were the strongest underexpression and overexpression outliers, respectively. Genomic SRS identified a 216 kb paracentric inversion (NC_000023.11: g.33162217-33378800) overlapping 2 DMD promoters. ExpansionHunter indicated an expansion of 109 CGG repeats within the 5' UTR of DIP2B. Targeted genomic LRS confirmed the SV and genotyped the DIP2B repeat expansion as 270 CGG repeats. Discussion: Here, transcriptome data heavily guided genomic analysis to resolve a complex DMD inversion and a DIP2B repeat expansion. Longitudinal follow-up will be important for clarifying the clinical significance of the DIP2B genotype.

A novel phenotype of AChR-deficiency syndrome with predominant facial and distal weakness resulting from the inclusion of an evolutionary alternatively-spliced exon in CHRNA1.

Primary acetylcholine receptor deficiency is the most common subtype of congenital myasthenic syndrome, resulting in reduced amount of acetylcholine receptors expressed at the muscle endplate and impaired neuromuscular transmission. AChR deficiency is caused mainly by pathogenic variants in the ε-subunit of the acetylcholine receptor encoded by CHRNE, although pathogenic variants in other subunits are also seen. We report the clinical and molecular features of 13 patients from nine unrelated kinships with acetylcholine receptor deficiency harbouring the CHRNA1 variant NM_001039523.3:c.257G>A (p.Arg86His) in homozygosity or compound heterozygosity. This variant results in the inclusion of an alternatively-spliced evolutionary exon (P3A) that causes expression of a non-functional acetylcholine receptor α-subunit. We compare the clinical findings of this group to the other cases of acetylcholine receptor deficiency within our cohort. We report differences in phenotype, highlighting a predominant pattern of facial and distal weakness in adulthood, predominantly in the upper limbs, which is unusual for acetylcholine receptor deficiency syndromes, and more in keeping with slow-channel syndrome or distal myopathy. Finally, we stress the importance of including alternative exons in variant analysis to increase the probability of achieving a molecular diagnosis.

Loss of function variants in DNAJB4 cause a myopathy with early respiratory failure.

DNAJ/HSP40 co-chaperones are integral to the chaperone network, bind client proteins and recruit them to HSP70 for folding. We performed exome sequencing on patients with a presumed hereditary muscle disease and no genetic diagnosis. This identified four individuals from three unrelated families carrying an unreported homozygous stop gain (c.856A > T; p.Lys286Ter), or homozygous missense variants (c.74G > A; p.Arg25Gln and c.785 T > C; p.Leu262Ser) in DNAJB4. Affected patients presented with axial rigidity and early respiratory failure requiring ventilator support between the 1st and 4th decade of life. Selective involvement of the semitendinosus and biceps femoris muscles was seen on MRI scans of the thigh. On biopsy, muscle was myopathic with angular fibers, protein inclusions and occasional rimmed vacuoles. DNAJB4 normally localizes to the Z-disc and was absent from muscle and fibroblasts of affected patients supporting a loss of function. Functional studies confirmed that the p.Lys286Ter and p.Leu262Ser mutant proteins are rapidly degraded in cells. In contrast, the p.Arg25Gln mutant protein is stable but failed to complement for DNAJB function in yeast, disaggregate client proteins or protect from heat shock-induced cell death consistent with its loss of function. DNAJB4 knockout mice had muscle weakness and fiber atrophy with prominent diaphragm involvement and kyphosis. DNAJB4 knockout muscle and myotubes had myofibrillar disorganization and accumulated Z-disc proteins and protein chaperones. These data demonstrate a novel chaperonopathy associated with DNAJB4 causing a myopathy with early respiratory failure. DNAJB4 loss of function variants may lead to the accumulation of DNAJB4 client proteins resulting in muscle dysfunction and degeneration.

Deep Intronic FGF14 GAA Repeat Expansion in Late-Onset Cerebellar Ataxia.

BACKGROUND: The late-onset cerebellar ataxias (LOCAs) have largely resisted molecular diagnosis. METHODS: We sequenced the genomes of six persons with autosomal dominant LOCA who were members of three French Canadian families and identified a candidate pathogenic repeat expansion. We then tested for association between the repeat expansion and disease in two independent case-control series - one French Canadian (66 patients and 209 controls) and the other German (228 patients and 199 controls). We also genotyped the repeat in 20 Australian and 31 Indian index patients. We assayed gene and protein expression in two postmortem cerebellum specimens and two induced pluripotent stem-cell (iPSC)-derived motor-neuron cell lines. RESULTS: In the six French Canadian patients, we identified a GAA repeat expansion deep in the first intron of FGF14, which encodes fibroblast growth factor 14. Cosegregation of the repeat expansion with disease in the families supported a pathogenic threshold of at least 250 GAA repeats ([GAA]≥250). There was significant association between FGF14 (GAA)≥250 expansions and LOCA in the French Canadian series (odds ratio, 105.60; 95% confidence interval [CI], 31.09 to 334.20; P<0.001) and in the German series (odds ratio, 8.76; 95% CI, 3.45 to 20.84; P<0.001). The repeat expansion was present in 61%, 18%, 15%, and 10% of French Canadian, German, Australian, and Indian index patients, respectively. In total, we identified 128 patients with LOCA who carried an FGF14 (GAA)≥250 expansion. Postmortem cerebellum specimens and iPSC-derived motor neurons from patients showed reduced expression of FGF14 RNA and protein. CONCLUSIONS: A dominantly inherited deep intronic GAA repeat expansion in FGF14 was found to be associated with LOCA. (Funded by Fondation Groupe Monaco and others.).

A KLHL40 3' UTR splice-altering variant causes milder NEM8, an under-appreciated disease mechanism.

Nemaline myopathy 8 (NEM8) is typically a severe autosomal recessive disorder associated with variants in the kelch-like family member 40 gene (KLHL40). Common features include fetal akinesia, fractures, contractures, dysphagia, respiratory failure and neonatal death. Here, we describe a 26-year-old man with relatively mild NEM8. He presented with hypotonia and bilateral femur fractures at birth, later developing bilateral Achilles' contractures, scoliosis, and elbow and knee contractures. He had walking difficulties throughout childhood and became wheelchair bound from age 13 after prolonged immobilization. Muscle magnetic resonance imaging at age 13 indicated prominent fat replacement in his pelvic girdle, posterior compartments of thighs and vastus intermedius. Muscle biopsy revealed nemaline bodies and intranuclear rods. RNA sequencing and western blotting of patient skeletal muscle indicated significant reduction in KLHL40 mRNA and protein, respectively. Using gene panel screening, exome sequencing and RNA sequencing, we identified compound heterozygous variants in KLHL40; a truncating 10.9 kb deletion in trans with a likely pathogenic variant (c.*152G > T) in the 3' untranslated region (UTR). Computational tools SpliceAI and Introme predicted the c.*152G > T variant created a cryptic donor splice site. RNA-seq and in vitro analyses indicated that the c.*152G > T variant induces multiple de novo splicing events that likely provoke nonsense mediated decay of KLHL40 mRNA explaining the loss of mRNA expression and protein abundance in the patient. Analysis of 3' UTR variants in ClinVar suggests variants that introduce aberrant 3' UTR splicing may be underrecognized in Mendelian disease. We encourage consideration of this mechanism during variant curation.

Unique Capabilities of Genome Sequencing for Rare Disease Diagnosis.

Background: Causal variants underlying rare disorders may remain elusive even after expansive gene panels or exome sequencing (ES). Clinicians and researchers may then turn to genome sequencing (GS), though the added value of this technique and its optimal use remain poorly defined. We therefore investigated the advantages of GS within a phenotypically diverse cohort. Methods: GS was performed for 744 individuals with rare disease who were genetically undiagnosed. Analysis included review of single nucleotide, indel, structural, and mitochondrial variants. Results: We successfully solved 218/744 (29.3%) cases using GS, with most solves involving established disease genes (157/218, 72.0%). Of all solved cases, 148 (67.9%) had previously had non-diagnostic ES. We systematically evaluated the 218 causal variants for features requiring GS to identify and 61/218 (28.0%) met these criteria, representing 8.2% of the entire cohort. These included small structural variants (13), copy neutral inversions and complex rearrangements (8), tandem repeat expansions (6), deep intronic variants (15), and coding variants that may be more easily found using GS related to uniformity of coverage (19). Conclusion: We describe the diagnostic yield of GS in a large and diverse cohort, illustrating several types of pathogenic variation eluding ES or other techniques. Our results reveal a higher diagnostic yield of GS, supporting the utility of a genome-first approach, with consideration of GS as a secondary or tertiary test when higher-resolution structural variant analysis is needed or there is a strong clinical suspicion for a condition and prior targeted genetic testing has been negative.

STRling: a k-mer counting approach that detects short tandem repeat expansions at known and novel loci.

Expansions of short tandem repeats (STRs) cause many rare diseases. Expansion detection is challenging with short-read DNA sequencing data since supporting reads are often mapped incorrectly. Detection is particularly difficult for "novel" STRs, which include new motifs at known loci or STRs absent from the reference genome. We developed STRling to efficiently count k-mers to recover informative reads and call expansions at known and novel STR loci. STRling is sensitive to known STR disease loci, has a low false discovery rate, and resolves novel STR expansions to base-pair position accuracy. It is fast, scalable, open-source, and available at: github.com/quinlan-lab/STRling .