Expert Panel Curation of 31 Genes in Relation to Limb Girdle Muscular Dystrophy.

Introduction: Limb girdle muscular dystrophies (LGMDs) are a group of genetically heterogeneous autosomal conditions with some degree of phenotypic homogeneity. LGMD is defined as having onset >2 years of age with progressive proximal weakness, elevated serum creatine kinase levels and dystrophic features on muscle biopsy. Advances in massively parallel sequencing have led to a surge in genes linked to LGMD. Methods: The ClinGen Muscular Dystrophies and Myopathies gene curation expert panel (MDM GCEP, formerly Limb Girdle Muscular Dystrophy GCEP) convened to evaluate the strength of evidence supporting gene-disease relationships (GDR) using the ClinGen gene-disease clinical validity framework to evaluate 31 genes implicated in LGMD. Results: The GDR was exclusively LGMD for 17 genes, whereas an additional 14 genes were related to a broader phenotype encompassing congenital weakness. Four genes (CAPN3, COL6A1, COL6A2, COL6A3) were split into two separate disease entities, based on each displaying both dominant and recessive inheritance patterns, resulting in curation of 35 GDRs. Of these, 30 (86%) were classified as Definitive, 4 (11%) as Moderate and 1 (3%) as Limited. Two genes, POMGNT1 and DAG1, though definitively related to myopathy, currently have insufficient evidence to support a relationship specifically with LGMD. Conclusions: The expert-reviewed assertions on the clinical validity of genes implicated in LGMDs form an invaluable resource for clinicians and molecular geneticists. We encourage the global neuromuscular community to publish case-level data that help clarify disputed or novel LGMD associations.

Genome sequencing enables diagnosis and treatment of SLC5A6 neuropathy.

The sodium-dependent multivitamin transporter encoded by SLC5A6 is responsible for uptake of biotin, pantothenic acid, and α-lipoic acid. Thirteen individuals from eight families are reported with pathogenic biallelic SLC5A6 variants. Phenotype ranges from multisystem metabolic disorder to childhood-onset peripheral motor neuropathy. We report three additional affected individuals with biallelic SLC5A6 variants. In Family A, a male proband (AII:1) presenting in early childhood with gross motor regression, motor axonal neuropathy, recurrent cytopenia and infections, and failure to thrive was diagnosed at 12 years of age via genome sequencing (GS) with a paternal NM_021095.4:c.393+2T>C variant and a maternal c.1285A>G p.(Ser429Gly) variant. An uncle with recurrent cytopenia and peripheral neuropathy was subsequently found to have the same genotype. We also report an unrelated female with peripheral neuropathy homozygous for the c.1285A>G p.(Ser429Gly) recurrent variant identified in seven reported cases, including this study. RT-PCR studies on blood mRNA from AII:1 showed c.393+2T>C caused mis-splicing with all canonically spliced transcripts in AII:1 containing the c.1285A>G variant. SLC5A6 mRNA expression in AII:1 fibroblasts was ~50% of control levels, indicative of nonsense-mediated decay of mis-spliced transcripts. Biotin uptake studies on AII:1 fibroblasts, expressing the p.(Ser429Gly) variant, showed an ~90% reduction in uptake compared to controls. Targeted treatment of AII:1 with biotin, pantothenic acid, and lipoic acid resulted in clinical improvement. Health Economic analyses showed implementation of GS as an early investigation could have saved $ AUD 105,988 and shortened diagnostic odyssey and initiation of treatment by up to 7 years.

Refining clinically relevant parameters for mis-splicing risk in shortened introns with donor-to-branchpoint space constraint.

Intronic deletions that critically shorten donor-to-branchpoint (D-BP) distance of a precursor mRNA impose biophysical space constraint on assembly of the U1/U2 spliceosomal complex, leading to canonical splicing failure. Here we use a series of ß-globin (HBB) gene constructs with intron 1 deletions to define D-BP lengths that present low/no risk of mis-splicing and lengths which are critically short and likely elicit clinically relevant mis-splicing. We extend our previous observation in EMD intron 5 of 46 nt as the minimal productive D-BP length, demonstrating spliceosome assembly constraint persists at D-BP lengths of 47-56 nt. We exploit the common HBB exon 1 ß-thalassemia variant that strengthens a cryptic donor (NM_000518.5(HBB):c.79G > A) to provide a simple barometer for the earliest signs of space constraint, via cryptic donor activation. For clinical evaluation of intronic deletions, we assert D-BP lengths > 60 nt present low mis-splicing risk while space constraint increases exponentially with D-BP lengths < 55 nt, with critical risk and profound splicing abnormalities with D-BP lengths < 50 nt.

Genome and RNA sequencing boost neuromuscular diagnoses to 62% from 34% with exome sequencing alone.

OBJECTIVE: Most families with heritable neuromuscular disorders do not receive a molecular diagnosis. Here we evaluate diagnostic utility of exome, genome, RNA sequencing, and protein studies and provide evidence-based recommendations for their integration into practice. METHODS: In total, 247 families with suspected monogenic neuromuscular disorders who remained without a genetic diagnosis after standard diagnostic investigations underwent research-led massively parallel sequencing: neuromuscular disorder gene panel, exome, genome, and/or RNA sequencing to identify causal variants. Protein and RNA studies were also deployed when required. RESULTS: Integration of exome sequencing and auxiliary genome, RNA and/or protein studies identified causal or likely causal variants in 62% (152 out of 247) of families. Exome sequencing alone informed 55% (83 out of 152) of diagnoses, with remaining diagnoses (45%; 69 out of 152) requiring genome sequencing, RNA and/or protein studies to identify variants and/or support pathogenicity. Arrestingly, novel disease genes accounted for <4% (6 out of 152) of diagnoses while 36.2% of solved families (55 out of 152) harbored at least one splice-altering or structural variant in a known neuromuscular disorder gene. We posit that contemporary neuromuscular disorder gene-panel sequencing could likely provide 66% (100 out of 152) of our diagnoses today. INTERPRETATION: Our results emphasize thorough clinical phenotyping to enable deep scrutiny of all rare genetic variation in phenotypically consistent genes. Post-exome auxiliary investigations extended our diagnostic yield by 81% overall (34-62%). We present a diagnostic algorithm that details deployment of genomic and auxiliary investigations to obtain these diagnoses today most effectively. We hope this provides a practical guide for clinicians as they gain greater access to clinical genome and transcriptome sequencing.

Lmod2 is necessary for effective skeletal muscle contraction.

Muscle contraction is a regulated process driven by the sliding of actin-thin filaments over myosin-thick filaments. Lmod2 is an actin filament length regulator and essential for life since human mutations and complete loss of Lmod2 in mice lead to dilated cardiomyopathy and death. To study the little-known role of Lmod2 in skeletal muscle, we created a mouse model with Lmod2 expressed exclusively in the heart but absent in skeletal muscle. Loss of Lmod2 in skeletal muscle results in decreased force production in fast- and slow-twitch muscles. Soleus muscle from rescued Lmod2 knockout mice have shorter thin filaments, increased Lmod3 levels, and present with a myosin fiber type switch from fast myosin heavy chain (MHC) IIA to the slower MHC I isoform. Since Lmod2 regulates thin-filament length in slow-twitch but not fast-twitch skeletal muscle and force deficits were observed in both muscle types, this work demonstrates that Lmod2 regulates skeletal muscle contraction, independent of its role in thin-filament length regulation.

Digenic inheritance involving a muscle-specific protein kinase and the giant titin protein causes a skeletal muscle myopathy.

In digenic inheritance, pathogenic variants in two genes must be inherited together to cause disease. Only very few examples of digenic inheritance have been described in the neuromuscular disease field. Here we show that predicted deleterious variants in SRPK3, encoding the X-linked serine/argenine protein kinase 3, lead to a progressive early onset skeletal muscle myopathy only when in combination with heterozygous variants in the TTN gene. The co-occurrence of predicted deleterious SRPK3/TTN variants was not seen among 76,702 healthy male individuals, and statistical modeling strongly supported digenic inheritance as the best-fitting model. Furthermore, double-mutant zebrafish (srpk3-/-; ttn.1+/-) replicated the myopathic phenotype and showed myofibrillar disorganization. Transcriptome data suggest that the interaction of srpk3 and ttn.1 in zebrafish occurs at a post-transcriptional level. We propose that digenic inheritance of deleterious changes impacting both the protein kinase SRPK3 and the giant muscle protein titin causes a skeletal myopathy and might serve as a model for other genetic diseases.

Biallelic ATP2B1 variants as a likely cause of a novel neurodevelopmental malformation syndrome with primary hypoparathyroidism.

ATP2B1 encodes plasma membrane calcium-transporting-ATPase1 and plays an essential role in maintaining intracellular calcium homeostasis that regulates diverse signaling pathways. Heterozygous de novo missense and truncating ATP2B1 variants are associated with a neurodevelopmental phenotype of variable expressivity. We describe a proband with distinctive craniofacial gestalt, Pierre-Robin sequence, neurodevelopmental and growth deficit, periventricular heterotopia, brachymesophalangy, cutaneous syndactyly, and persistent hypocalcemia from primary hypoparathyroidism. Proband-parent trio exome sequencing identified compound heterozygous ATP2B1 variants: a maternally inherited splice-site (c.3060+2 T > G) and paternally inherited missense c.2938 G > T; p.(Val980Leu). Reverse-transcription-PCR on the proband's fibroblast-derived mRNA showed aberrantly spliced ATP2B1 transcripts targeted for nonsense-mediated decay. All correctly-spliced ATP2B1 mRNA encoding p.(Val980Leu) functionally causes decreased cellular Ca2+ extrusion. Immunoblotting showed reduced fibroblast ATP2B1. We conclude that biallelic ATP2B1 variants are the likely cause of the proband's phenotype, strengthening the association of ATP2B1 as a neurodevelopmental gene and expanding the phenotypic characterization of a biallelic loss-of-function genotype.

Connective tissue presentation in two families expands the phenotypic spectrum of PYROXD1 disorders.

Recessive variants in the oxidoreductase PYROXD1 are reported to cause a myopathy in 22 affected individuals from 15 families. Here, we describe two female probands from unrelated families presenting with features of a congenital connective tissue disorder including osteopenia, blue sclera, soft skin, joint hypermobility and neuromuscular junction dysfunction in addition to known features of PYROXD1 myopathy including respiratory difficulties, weakness, hypotonia and oromotor dysfunction. Proband AII:1 is compound heterozygous for the recurrent PYROXD1 variant Chr12(GRCh38):g.21452130A>G;NM_024854.5:c.464A>G;p.(N155S) and Chr12(GRCh38):g.21462019_21462022del;NM_024854.5:c.892_895del;p.(V298Mfs*4) and proband BII:1 is compound heterozygous for Chr12(GRCh38):g.21468739-21468741del;NM_024854.5:c.1488_1490del;p.(E496del) and Chr12(GRCh38):g.21467619del;NM_024854.5:c.1254+1del. RNA studies demonstrate c.892_895del;p.(V298Mfs*4) is targeted by nonsense mediated decay and c.1254+1delG elicits in-frame skipping of exon-11. Western blot from cultured fibroblasts shows reduced PYROXD1 protein levels in both probands. Testing urine from BII:1 and six individuals with PYROXD1 myopathy showed elevated levels of deoxypyridinoline, a mature collagen crosslink, correlating with PYROXD1-disorder severity. Urine and serum amino acid testing of the same individuals revealed no reportable changes. In contrast to PYROXD1 knock-out, we find no evidence for disrupted tRNA ligase activity, as measured via XBP1 splicing, in fibroblasts expressing PYROXD1 variants. In summary, we expand the clinical spectrum of PYROXD1-related disorders to include an overlapping connective tissue and myopathy presentation, identify three novel, pathogenic PYROXD1 variants, and provide preliminary evidence that elevated urine DPD crosslinks may provide a clinical biomarker for PYROXD1 disorders. Our results advocate consideration of PYROXD1 variants in the differential diagnosis for undiagnosed individuals presenting with a connective tissue disorder and myopathy.

SpliceVault predicts the precise nature of variant-associated mis-splicing.

Even for essential splice-site variants that are almost guaranteed to alter mRNA splicing, no current method can reliably predict whether exon-skipping, cryptic activation or multiple events will result, greatly complicating clinical interpretation of pathogenicity. Strikingly, ranking the four most common unannotated splicing events across 335,663 reference RNA-sequencing (RNA-seq) samples (300K-RNA Top-4) predicts the nature of variant-associated mis-splicing with 92% sensitivity. The 300K-RNA Top-4 events correctly identify 96% of exon-skipping events and 86% of cryptic splice sites for 140 clinical cases subject to RNA testing, showing higher sensitivity and positive predictive value than SpliceAI. Notably, RNA re-analyses showed we had missed 300K-RNA Top-4 events for several clinical cases tested before the development of this empirical predictive method. Simply, mis-splicing events that happen around a splice site in RNA-seq data are those most likely to be activated by a splice-site variant. The SpliceVault web portal allows users easy access to 300K-RNA for informed splice-site variant interpretation and classification.

Severe NAD(P)HX Dehydratase (NAXD) Neurometabolic Syndrome May Present in Adulthood after Mild Head Trauma.

We have previously reported that pathogenic variants in a key metabolite repair enzyme NAXD cause a lethal neurodegenerative condition triggered by episodes of fever in young children. However, the clinical and genetic spectrum of NAXD deficiency is broadening as our understanding of the disease expands and as more cases are identified. Here, we report the oldest known individual succumbing to NAXD-related neurometabolic crisis, at 32 years of age. The clinical deterioration and demise of this individual were likely triggered by mild head trauma. This patient had a novel homozygous NAXD variant [NM_001242882.1:c.441+3A>G:p.?] that induces the mis-splicing of the majority of NAXD transcripts, leaving only trace levels of canonically spliced NAXD mRNA, and protein levels below the detection threshold by proteomic analysis. Accumulation of damaged NADH, the substrate of NAXD, could be detected in the fibroblasts of the patient. In agreement with prior anecdotal reports in paediatric patients, niacin-based treatment also partly alleviated some clinical symptoms in this adult patient. The present study extends our understanding of NAXD deficiency by uncovering shared mitochondrial proteomic signatures between the adult and our previously reported paediatric NAXD cases, with reduced levels of respiratory complexes I and IV as well as the mitoribosome, and the upregulation of mitochondrial apoptotic pathways. Importantly, we highlight that head trauma in adults, in addition to paediatric fever or illness, may precipitate neurometabolic crises associated with pathogenic NAXD variants.

Case report: Adult-onset limb girdle muscular dystrophy in sibling pair due to novel homozygous LAMA2 missense variant.

Recessive pathogenic variants in the laminin subunit alpha 2 (LAMA2) gene cause a spectrum of disease ranging from severe congenital muscular dystrophy to later-onset limb girdle muscular dystrophy (LGMDR23). The phenotype of LGMDR23 is characterized by slowly progressive proximal limb weakness, contractures, raised creatine kinase, and sometimes distinctive cerebral white matter changes and/or epilepsy. We present two siblings, born to consanguineous parents, who developed adult-onset LGMDR23 associated with typical cerebral white matter changes and who both later developed dementia. The male proband also had epilepsy and upper motor neuron signs when he presented at age 72. Merosin immunohistochemistry and Western blot on muscle biopsies taken from both subjects was normal. Whole exome sequencing revealed a previously unreported homozygous missense variant in LAMA2 [Chr6(GRCh38):g.129297734G>A; NM_000426.3:c.2906G>A; p.(Cys969Tyr)] in the proband. The same homozygous LAMA2 variant was confirmed by Sanger sequencing in the proband's affected sister. These findings expand the genotypic and phenotypic spectrum of LGMDR23.

Compound heterozygous splicing variants expand the genotypic spectrum of EMC1-related disorders.

EMC1 encodes subunit 1 of the endoplasmic reticulum (ER) membrane protein complex (EMC), a transmembrane domain insertase involved in membrane protein biosynthesis. Variants in EMC1 are described as a cause of global developmental delay, hypotonia, cortical visual impairment, and commonly, cerebral atrophy on MRI scan. We report an individual with severe global developmental delay and progressive cerebellar atrophy in whom exome sequencing identified a heterozygous essential splice-site variant in intron-3 of EMC1 (NM_015047.3:c.287-1G>A). Whole genome sequencing (WGS) identified a deep intronic variant in intron-20 of EMC1 (NM_015047.3:c.2588-771C>G) that was poorly predicted by in silico programs to disrupt pre-mRNA splicing. Reverse Transcription-PCR (RT-PCR) revealed stochastic activation of a pseudo-exon associated with the c.2588-771C>G variant and mis-splicing arising from the c.287-1G>A variant. This case highlights the utility of WGS and RNA studies to identify and assess likely pathogenicity of deep intronic variants and expands the genotypic and phenotypic spectrum of EMC1-related disorders.

Expanding the Allelic Heterogeneity of ANO10-Associated Autosomal Recessive Cerebellar Ataxia.

Background and Objectives: The term autosomal recessive cerebellar ataxia (ARCA) encompasses a diverse group of heterogeneous degenerative disorders of the cerebellum. Spinocerebellar ataxia autosomal recessive 10 (SCAR10) is a distinct classification of cerebellar ataxia caused by variants in the ANO10 gene. Little is known about the molecular role of ANO10 or its role in disease. There is a wide phenotypic spectrum among patients, even among those with the same or similar genetic variants. This study aimed to characterize the molecular consequences of variants in ANO10 and determine their pathologic significance in patients diagnosed with SCAR10. Methods: We presented 4 patients from 4 families diagnosed with spinocerebellar ataxia with potential pathogenic variants in the ANO10 gene. Patients underwent either clinical whole-exome sequencing or screening of a panel of known neuromuscular disease genes. Effects on splicing were studied using reverse transcriptase PCR to analyze complementary DNA. Western blots were used to examine protein expression. Results: One individual who presented clinically at a much earlier age than typical was homozygous for an ANO10 variant (c.1864A > G [p.Met622Val]) that produces 2 transcription products by altering an exonic enhancer site. Two patients, both of Lebanese descent, had a homozygous intronic splicing variant in ANO10 (c.1163-9A > G) that introduced a cryptic splice site acceptor, producing 2 alternative transcription products and no detectable wild-type protein. Both these variants have not yet been associated with SCAR10. The remaining patient was found to have compound heterozygous variants in ANO10 previously associated with SCAR10 (c.132dupA [p.Asp45Argfs*9] and c.1537T > C [p.Cys513Arg]). Discussion: We presented rare pathogenic variants adding to the growing list of ANO10 variants associated with SCAR10. In addition, we described an individual with a much earlier age at onset than usually associated with ANO10 variants. This expands the phenotypic and allelic heterogeneity of ANO10-associated ARCA.

Minimal expression of dysferlin prevents development of dysferlinopathy in dysferlin exon 40a knockout mice.

Dysferlin is a Ca2+-activated lipid binding protein implicated in muscle membrane repair. Recessive variants in DYSF result in dysferlinopathy, a progressive muscular dystrophy. We showed previously that calpain cleavage within a motif encoded by alternatively spliced exon 40a releases a 72 kDa C-terminal minidysferlin recruited to injured sarcolemma. Herein we use CRISPR/Cas9 gene editing to knock out murine Dysf exon 40a, to specifically assess its role in membrane repair and development of dysferlinopathy. We created three Dysf exon 40a knockout (40aKO) mouse lines that each express different levels of dysferlin protein ranging from ~ 90%, ~ 50% and ~ 10-20% levels of wild-type. Histopathological analysis of skeletal muscles from all 12-month-old 40aKO lines showed virtual absence of dystrophic features and normal membrane repair capacity for all three 40aKO lines, as compared with dysferlin-null BLAJ mice. Further, lipidomic and proteomic analyses on 18wk old quadriceps show all three 40aKO lines are spared the profound lipidomic/proteomic imbalance that characterises dysferlin-deficient BLAJ muscles. Collective results indicate that membrane repair does not depend upon calpain cleavage within exon 40a and that ~ 10-20% of WT dysferlin protein expression is sufficient to maintain the muscle lipidome, proteome and membrane repair capacity to crucially prevent development of dysferlinopathy.

Factors associated with smokefree rules in the homes of Black/African American women smokers residing in low-resource rural communities.

Little is known about the implementation of voluntary policies in the homes of Black/African American women smokers who live in rural areas where health care access is limited. This paper examines 1) the sample's prevalence of comprehensive smoke-free rules; 2) sociodemographic, social, and smoking characteristics of women by home rule type; and 3) the association of social indicators with the outcome complete ban on smoked tobacco use in the home (n = 191). Families Rising to Enforce Smokefree Homes collected baseline data from 2019 to 2021 prior to randomization to an intervention that aimed to increase comprehensive smokefree policies in the homes African American women living in the rural Delta region of Arkansas. The primary outcome was implementation of a complete ban on all smoked tobacco products anywhere inside the home. Results showed that 26% of women had a rule that completely banned all smoked tobacco products in the home. Women who reported having no ban were more likely to be employed part-time (50.0%), while women with a partial (66.9%) or complete ban (60.0%) were more likely to not currently work for pay. Women who indicated that they just meet basic expenses and meet needs with little left had significantly lower odds of having a complete ban on smoked tobacco in the home than women who indicated that they live comfortably. Perceived financial security may be a motivating factor that helps women keep their homes free from all smoked tobacco products (# NCT03476837).

A genetic basis is identified in 74% cases of paediatric hyperCKaemia without weakness presenting to a tertiary paediatric neuromuscular centre.

Paediatric hyperCKaemia without weakness presents a clinical conundrum. Invasive investigations with low diagnostic yields, including muscle biopsy, may be considered unjustifiable. Improved access to genome-wide genetic testing has shifted first-line investigations towards genetic studies in neuromuscular disease. This research aims to provide an evidence-based diagnostic approach to paediatric hyperCKaemia without weakness, a current gap in the literature. We identified 47 individuals (10-months to 16-years-old; 34 males, 13 females) from 43 families presenting with hyperCKaemia on two or more occasions, without weakness, from The Children's Hospital at Westmead Neuromuscular Clinic Database. Clinical features, investigations and outcomes were analysed via retrospective chart review. Genetic testing has been performed in 34/43. Genetic variants explaining hyperCKaemia were identified in 25/34 (74%) using multiplex ligation-dependent probe amplification, massive parallel sequencing, single gene testing and exome sequencing. Pathogenic/likely pathogenic variants were identified in 19 neuromuscular disease genes and six metabolic myopathy genes. Individuals with metabolic diagnoses had higher peak creatine kinase levels that sometimes normalized. Conversely, creatine kinase levels remained persistently elevated those with neuromuscular diagnoses. In summary, a genetic cause is found in most paediatric patients with hyperCKaemia without weakness informing clinical management and counselling. Thus, we propose a diagnostic algorithm for this cohort.

Prevalence, parameters, and pathogenic mechanisms for splice-altering acceptor variants that disrupt the AG exclusion zone.

Predicting the pathogenicity of acceptor splice-site variants outside the essential AG is challenging, due to high sequence diversity of the extended splice-site region. Critical analysis of 24,445 intronic extended acceptor splice-site variants reported in ClinVar and the Leiden Open Variation Database (LOVD) demonstrates 41.9% of pathogenic variants create an AG dinucleotide between the predicted branchpoint and acceptor (AG-creating variants in the AG exclusion zone), 28.4% result in loss of a pyrimidine at the -3 position, and 15.1% result in loss of one or more pyrimidines in the polypyrimidine tract. Pathogenicity of AG-creating variants was highly influenced by their position. We define a high-risk zone for pathogenicity: > 6 nucleotides downstream of the predicted branchpoint and >5 nucleotides upstream from the acceptor, where 93.1% of pathogenic AG-creating variants arise and where naturally occurring AG dinucleotides are concordantly depleted (5.8% of natural AGs). SpliceAI effectively predicts pathogenicity of AG-creating variants, achieving 95% sensitivity and 69% specificity. We highlight clinical examples showing contrasting mechanisms for mis-splicing arising from AG variants: (1) cryptic acceptor created; (2) splicing silencer created: an introduced AG silences the acceptor, resulting in exon skipping, intron retention, and/or use of an alternative existing cryptic acceptor; and (3) splicing silencer disrupted: loss of a deep intronic AG activates inclusion of a pseudo-exon. In conclusion, we establish AG-creating variants as a common class of pathogenic extended acceptor variant and outline factors conferring critical risk for mis-splicing for AG-creating variants in the AG exclusion zone, between the branchpoint and acceptor.

Clinical impact of whole-genome sequencing in patients with early-onset dementia.

BACKGROUND: In the clinical setting, identification of the genetic cause in patients with early-onset dementia (EOD) is challenging due to multiple types of genetic tests required to arrive at a diagnosis. Whole-genome sequencing (WGS) has the potential to serve as a single diagnostic platform, due to its superior ability to detect common, rare and structural genetic variation. METHODS: WGS analysis was performed in 50 patients with EOD. Point mutations, small insertions/deletions, as well as structural variants (SVs) and short tandem repeats (STRs), were analysed. An Alzheimer's disease (AD)-related polygenic risk score (PRS) was calculated in patients with AD. RESULTS: Clinical genetic diagnosis was achieved in 7 of 50 (14%) of the patients, with a further 8 patients (16%) found to have established risk factors which may have contributed to their EOD. Two pathogenic variants were identified through SV analysis. No expanded STRs were found in this study cohort, but a blinded analysis with a positive control identified a C9orf72 expansion accurately. Approximately 37% (7 of 19) of patients with AD had a PRS equivalent to >90th percentile risk. DISCUSSION: WGS acts as a single genetic test to identify different types of clinically relevant genetic variations in patients with EOD. WGS, if used as a first-line clinical diagnostic test, has the potential to increase the diagnostic yield and reduce time to diagnosis for EOD.

Empirical prediction of variant-activated cryptic splice donors using population-based RNA-Seq data.

Predicting which cryptic-donors may be activated by a splicing variant in patient DNA is notoriously difficult. Through analysis of 5145 cryptic-donors (versus 86,963 decoy-donors not used; any GT or GC), we define an empirical method predicting cryptic-donor activation with 87% sensitivity and 95% specificity. Strength (according to four algorithms) and proximity to the annotated-donor appear important determinants of cryptic-donor activation. However, other factors such as splicing regulatory elements, which are difficult to identify, play an important role and are likely responsible for current prediction inaccuracies. We find that the most frequently recurring natural mis-splicing events at each exon-intron junction, summarised over 40,233 RNA-sequencing samples (40K-RNA), predict with accuracy which cryptic-donor will be activated in rare disease. 40K-RNA provides an accurate, evidence-based method to predict variant-activated cryptic-donors in genetic disorders, assisting pathology consideration of possible consequences of a variant for the encoded protein and RNA diagnostic testing strategies.