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.

Standardized practices for RNA diagnostics using clinically accessible specimens reclassifies 75% of putative splicing variants.

PURPOSE: Genetic variants causing aberrant premessenger RNA splicing are increasingly being recognized as causal variants in genetic disorders. In this study, we devise standardized practices for polymerase chain reaction (PCR)-based RNA diagnostics using clinically accessible specimens (blood, fibroblasts, urothelia, biopsy). METHODS: A total of 74 families with diverse monogenic conditions (31% prenatal-congenital onset, 47% early childhood, and 22% teenage-adult onset) were triaged into PCR-based RNA testing, with comparative RNA sequencing for 19 cases. RESULTS: Informative RNA assay data were obtained for 96% of cases, enabling variant reclassification for 75% variants that can be used for genetic counseling (71%), to inform clinical care (32%) and prenatal counseling (41%). Variant-associated mis-splicing was highly reproducible for 28 cases with samples from ≥2 affected individuals or heterozygotes and 10 cases with ≥2 biospecimens. PCR amplicons encompassing another segregated heterozygous variant was vital for clinical interpretation of 22 of 79 variants to phase RNA splicing events and discern complete from partial mis-splicing. CONCLUSION: RNA diagnostics enabled provision of a genetic diagnosis for 64% of recruited cases. PCR-based RNA diagnostics has capacity to analyze 81.3% of clinically significant genes, with long amplicons providing an advantage over RNA sequencing to phase RNA splicing events. The Australasian Consortium for RNA Diagnostics (SpliceACORD) provide clinically-endorsed, standardized protocols and recommendations for interpreting RNA assay data.

WGS and RNA Studies Diagnose Noncoding DMD Variants in Males With High Creatine Kinase.

OBJECTIVE: To describe the diagnostic utility of whole-genome sequencing and RNA studies in boys with suspected dystrophinopathy, for whom multiplex ligation-dependent probe amplification and exomic parallel sequencing failed to yield a genetic diagnosis, and to use remnant normal DMD splicing in 3 families to define critical levels of wild-type dystrophin bridging clinical spectrums of Duchenne to myalgia. METHODS: Exome, genome, and/or muscle RNA sequencing was performed for 7 males with elevated creatine kinase. PCR of muscle-derived complementary DNA (cDNA) studied consequences for DMD premessenger RNA (pre-mRNA) splicing. Quantitative Western blot was used to determine levels of dystrophin, relative to control muscle. RESULTS: Splice-altering intronic single nucleotide variants or structural rearrangements in DMD were identified in all 7 families. Four individuals, with abnormal splicing causing a premature stop codon and nonsense-mediated decay, expressed remnant levels of normally spliced DMD mRNA. Quantitative Western blot enabled correlation of wild-type dystrophin and clinical severity, with 0%-5% dystrophin conferring a Duchenne phenotype, 10% ± 2% a Becker phenotype, and 15% ± 2% dystrophin associated with myalgia without manifesting weakness. CONCLUSIONS: Whole-genome sequencing relied heavily on RNA studies to identify DMD splice-altering variants. Short-read RNA sequencing was regularly confounded by the effectiveness of nonsense-mediated mRNA decay and low read depth of the giant DMD mRNA. PCR of muscle cDNA provided a simple, yet informative approach. Highly relevant to genetic therapies for dystrophinopathies, our data align strongly with previous studies of mutant dystrophin in Becker muscular dystrophy, with the collective conclusion that a fractional increase in levels of normal dystrophin between 5% and 20% is clinically significant.

Neurogenetic fetal akinesia and arthrogryposis: genetics, expanding genotype-phenotypes and functional genomics.

BACKGROUND: Fetal akinesia and arthrogryposis are clinically and genetically heterogeneous and have traditionally been refractive to genetic diagnosis. The widespread availability of affordable genome-wide sequencing has facilitated accurate genetic diagnosis and gene discovery in these conditions. METHODS: We performed next generation sequencing (NGS) in 190 probands with a diagnosis of arthrogryposis multiplex congenita, distal arthrogryposis, fetal akinesia deformation sequence or multiple pterygium syndrome. This sequencing was a combination of bespoke neurogenetic disease gene panels and whole exome sequencing. Only class 4 and 5 variants were reported, except for two cases where the identified variants of unknown significance (VUS) are most likely to be causative for the observed phenotype. Co-segregation studies and confirmation of variants identified by NGS were performed where possible. Functional genomics was performed as required. RESULTS: Of the 190 probands, 81 received an accurate genetic diagnosis. All except two of these cases harboured class 4 and/or 5 variants based on the American College of Medical Genetics and Genomics guidelines. We identified phenotypic expansions associated with CACNA1S, CHRNB1, GMPPB and STAC3. We describe a total of 50 novel variants, including a novel missense variant in the recently identified gene for arthrogryposis with brain malformations-SMPD4. CONCLUSIONS: Comprehensive gene panels give a diagnosis for a substantial proportion (42%) of fetal akinesia and arthrogryposis cases, even in an unselected cohort. Recently identified genes account for a relatively large proportion, 32%, of the diagnoses. Diagnostic-research collaboration was critical to the diagnosis and variant interpretation in many cases, facilitated genotype-phenotype expansions and reclassified VUS through functional genomics.

Rapid exome sequencing and adjunct RNA studies confirm the pathogenicity of a novel homozygous ASNS splicing variant in a critically ill neonate.

Rapid genomic diagnosis programs are transforming rare disease diagnosis in acute pediatrics. A ventilated newborn with cerebellar hypoplasia underwent rapid exome sequencing (75 h), identifying a novel homozygous ASNS splice-site variant (NM_133436.3:c.1476+1G>A) of uncertain significance. Rapid ASNS splicing studies using blood-derived messenger RNA from the family trio confirmed a consistent pattern of abnormal splicing induced by the variant (cryptic 5' splice-site or exon 12 skipping) with absence of normal ASNS splicing in the proband. Splicing studies reported within 10 days led to reclassification of c.1476+1G>A as pathogenic at age 27 days. Intensive care was redirected toward palliation. Cost analyses for the neonate and his undiagnosed, similarly affected deceased sibling, demonstrate that early diagnosis reduced hospitalization costs by AU$100,828. We highlight the diagnostic benefits of adjunct RNA testing to confirm the pathogenicity of splicing variants identified via rapid genomic testing pipelines for precision and preventative medicine.

Targeted gene panel use in 2249 neuromuscular patients: the Australasian referral center experience.

OBJECTIVE: To develop, test, and iterate a comprehensive neuromuscular targeted gene panel in a national referral center. METHODS: We designed two iterations of a comprehensive targeted gene panel for neuromuscular disorders. Version 1 included 336 genes, which was increased to 464 genes in Version 2. Both panels used TargetSeqTM probe-based hybridization for target enrichment followed by Ion Torrent sequencing. Targeted high-coverage sequencing and analysis was performed on 2249 neurology patients from Australia and New Zealand (1054 Version 1, 1195 Version 2) from 2012 to 2015. No selection criteria were used other than referral from a suitable medical specialist (e.g., neurologist or clinical geneticist). Patients were classified into 15 clinical categories based on the clinical diagnosis from the referring clinician. RESULTS: Six hundred and sixty-five patients received a genetic diagnosis (30%). Diagnosed patients were significantly younger that undiagnosed patients (26.4 and 32.5 years, respectively; P = 4.6326E-9). The diagnostic success varied markedly between disease categories. Pathogenic variants in 10 genes explained 38% of the disease burden. Unexpected phenotypic expansions were discovered in multiple cases. Triage of unsolved cases for research exome testing led to the discovery of six new disease genes. INTERPRETATION: A comprehensive targeted diagnostic panel was an effective method for neuromuscular disease diagnosis within the context of an Australasian referral center. Use of smaller disease-specific panels would have precluded diagnosis in many patients and increased cost. Analysis through a centralized laboratory facilitated detection of recurrent, but under-recognized pathogenic variants.

Recurrent TTN metatranscript-only c.39974-11T>G splice variant associated with autosomal recessive arthrogryposis multiplex congenita and myopathy.

We present eight families with arthrogryposis multiplex congenita and myopathy bearing a TTN intron 213 extended splice-site variant (NM_001267550.1:c.39974-11T>G), inherited in trans with a second pathogenic TTN variant. Muscle-derived RNA studies of three individuals confirmed mis-splicing induced by the c.39974-11T>G variant; in-frame exon 214 skipping or use of a cryptic 3' splice-site effecting a frameshift. Confounding interpretation of pathogenicity is the absence of exons 213-217 within the described skeletal muscle TTN N2A isoform. However, RNA-sequencing from 365 adult human gastrocnemius samples revealed that 56% specimens predominantly include exons 213-217 in TTN transcripts (inclusion rate ≥66%). Further, RNA-sequencing of five fetal muscle samples confirmed that 4/5 specimens predominantly include exons 213-217 (fifth sample inclusion rate 57%). Contractures improved significantly with age for four individuals, which may be linked to decreased expression of pathogenic fetal transcripts. Our study extends emerging evidence supporting a vital developmental role for TTN isoforms containing metatranscript-only exons.

Detection of variations and identifying genomic breakpoints for large deletions in the LDLR by Ion Torrent semiconductor sequencing.

OBJECTIVES: The aims of this study were to 1) compare LDLR variant detection between Ion Torrent Personal Genome Machine (PGM) sequencing and conventional methods used for familial hypercholesterolaemia (FH) diagnosis i.e. exon-by-exon sequence analysis and multiplex ligation-dependent probe amplification (MLPA) and 2) identify genomic breakpoints for 12 cases of large deletions in LDLR previously identified by MLPA. METHODS: Thirty FH patient samples were selected, 22 with mutations previously determined. Primers were designed and optimised to generate six amplicons covering the entire LDLR and sequenced on a PGM. An additional twelve samples carrying MLPA variants were sequenced on the PGM followed by Sanger sequencing to establish the breakpoints. RESULTS: A total of 2179 LDLR variants were identified in the 30 samples, with 383 variants in the region sequenced that was common to both PGM and Sanger methods. Three discrepancies were identified; two of these were identified by visual inspection of the BAM files, whilst the remaining discrepancy was likely an artefact of the PCR approach. Approximate genomic breakpoints for the 12 MLPA variants were identified using PGM sequencing, and Sanger sequencing of these regions established causative breakpoints. Eleven different rearrangements/mutational events were found, with eight out of eleven occurring in Alus. Two of the three samples with exons 2-6del had identical breakpoints. Two samples with exons 11-12del had unique breakpoints, indicating separate ancestral origin or mutational events. CONCLUSIONS: This study showed that Ion Torrent PGM sequencing is an accurate and efficient method to detect LDLR variants while providing additional information such as genomic breakpoints.

Molecular pathology of familial hypercholesterolemia, related dyslipidemias and therapies beyond the statins.

The development of the statin class of cholesterol-lowering drugs is one of the most significant success stories of modern pharmacotherapy. World-wide there are an estimated 150 million people on statins, with the emerging economies of India and China predicted to contribute significantly to that number. Notwithstanding their success, a significant number of people cannot tolerate statins because of serious side effects; of equal concern, a substantial proportion of high risk patients fail to reach cholesterol-lowering targets. For these subjects there is an urgent need for new cholesterol-lowering agents to be used alone or in combination with statins. The success of statins has been largely underpinned by knowledge of cholesterol homeostasis at a molecular level, knowledge that was first gleaned in the 1980s from Brown and Goldstein's pioneering studies of familial hypercholesterolemia (FH, OMIM 143890). Follow-up work that has identified a number of intracellular and circulating factors, all capable of disrupting LDL clearance, has revealed that the low-density lipoprotein receptor- (LDLR) mediated clearance pathway is substantially more complex than previously thought. These factors were discovered in studies of individuals with very rare inherited conditions that lead to either hypo- or hypercholesterolemia. These investigations, besides providing clearer insight into the molecular mechanisms regulating plasma LDL concentrations, have also revealed a number of novel therapeutic targets independent from statins. Consequently, a number of novel therapeutic approaches that are based on small interfering bio-molecules, including antisense oligonucleotides, are now in clinical development. These are aimed at impairing the assembly, synthesis and secretion of apolipoprotein B-containing lipoproteins and/or accelerating their hepatic catabolism. The aim of this article is to focus on these recent advances in the understanding of the molecular basis of cholesterol metabolism that should herald novel cholesterol-lowering agents beyond the statins.

Smad2: a candidate gene for the murine autoimmune diabetes locus Idd21.1.

CONTEXT: Congenic NOD.ABH(D18Mit8-D18Mit214) mice, which contain greater than 12.8 Mb of DNA encompassing Idd21.1 from diabetes-resistant Biozzi/ABH mice, have a lower frequency of diabetes compared with the parental nonobese diabetic (NOD) strain, possibly due to reduced pathogenicity of ß-islet-infiltrating immune cells. OBJECTIVE: The objective of the study was to identify an Idd21.1 candidate gene. METHODS: The methods used in the study were adoptive transfer into scid mice lacking an adaptive immune system; dendritic cell phenotyping and gene expression analysis; and fine-mapping Idd21.1 by congenic mapping. RESULTS: Diabetes incidences of NOD.scid.ABH(D18Mit8-D18Mit214) mice receiving splenocytes from NOD and NOD.ABH(D18Mit8-D18Mit214) were similar to that previously observed in NOD.scid recipients, suggesting that the diabetes resistance in NOD.ABH(D18Mit8-D18Mit214) is primarily mediated by the adaptive immune system, findings supported by adoptive transfer of CD4(+) T cells. In activated dendritic cells, there were no conclusive differences in cytokine profiles and activation marker expression. However, microarray analysis comparing gene expression between activated dendritic cells from NOD and NOD.ABH (D18Mit8-D18Mit214) revealed that Smad2, in a maximal 6.5-Mb region to which Idd21.1 was further resolved by congenic mapping, was differentially expressed (increased in NOD). Quantitative real-time PCR confirmed the differential expression of Smad2, and other genes in the TGF-ß signaling pathway, in activated dendritic cells. CONCLUSIONS: These results implicate Smad2 as an Idd21.1 candidate and Smad2 and the TGF-ß signaling pathway in activated dendritic cells in diabetogenesis. With suggestive evidence from human genome-wide association studies supporting a role for SMAD7 in human type 1 diabetes, a comprehensive genetic investigation of the SMAD genes in type 1 diabetes is warranted.