Three Newly Recognized Likely Pathogenic Gene Variants Associated with Hereditary Transthyretin Amyloidosis.

INTRODUCTION: Hereditary transthyretin amyloidosis (ATTRv [variant]) is a clinically heterogeneous, progressively debilitating, fatal disease resulting from the deposition of insoluble amyloid fibrils in various organs and tissues. Early diagnosis of ATTRv can be facilitated with genetic testing; however, such testing of the TTR gene identifies variants of uncertain significance (VUS) in a minority of cases, a small percentage of which have the potential to be pathogenic. The Akcea/Ambry VUS Initiative is dedicated to gathering molecular, clinical, and inheritance data for each TTR VUS identified by genetic testing programs to reclassify TTR variants to a clinically actionable status (e.g., variant likely pathogenic [VLP]) where appropriate. METHODS: Classification criteria used here, based on recommendations from the American College of Medical Genetics and Genomics, are stringent and comprehensive, requiring distinct lines of evidence supporting pathogenesis. RESULTS: Three TTR variants have been reclassified from VUS to VLP, including c.194C>T (p.A65V), c.172G>C (p.D58H), and c.239C>T (p.T80I). In each case, the totality of genetic, structural, and clinical evidence provided strong support for pathogenicity. CONCLUSIONS: Based on several lines of evidence, three TTR VUS were reclassified as VLP, resulting in a high likelihood of disease diagnosis for those and subsequent patients as well as at-risk family members.

Harmonizing the Collection of Clinical Data on Genetic Testing Requisition Forms to Enhance Variant Interpretation in Hypertrophic Cardiomyopathy (HCM): A Study from the ClinGen Cardiomyopathy Variant Curation Expert Panel.

Diagnostic laboratories gather phenotypic data through requisition forms, but there is no consensus as to which data are essential for variant interpretation. The ClinGen Cardiomyopathy Variant Curation Expert Panel defined a phenotypic data set for hypertrophic cardiomyopathy (HCM) variant interpretation, with the goal of standardizing requisition forms. Phenotypic data elements listed on requisition forms from nine leading cardiomyopathy testing laboratories were compiled to assess divergence in data collection. A pilot of 50 HCM cases was implemented to determine the feasibility of harmonizing data collection. Laboratory directors were surveyed to gauge potential for adoption of a minimal data set. Wide divergence was observed in the phenotypic data fields in requisition forms. The 50-case pilot showed that although demographics and assertion of a clinical diagnosis of HCM had 86% to 98% completion, specific phenotypic features, such as degree of left ventricular hypertrophy, ejection fraction, and suspected syndromic disease, were completed only 24% to 44% of the time. Nine data elements were deemed essential for variant classification by the expert panel. Participating laboratories unanimously expressed a willingness to adopt these data elements in their requisition forms. This study demonstrates the value of comparing and sharing best practices through an expert group, such as the ClinGen Program, to enhance variant interpretation, providing a foundation for leveraging cumulative case-level data in public databases and ultimately improving patient care.

Clinical validity assessment of genes for inclusion in multi-gene panel testing: A systematic approach.

BACKGROUND: Advances in sequencing technology have led to expanded use of multi-gene panel tests (MGPTs) for clinical diagnostics. Well-designed MGPTs must balance increased detection of clinically significant findings while mitigating the increase in variants of uncertain significance (VUS). To maximize clinical utililty, design of such panels should include comprehensive gene vetting using a standardized clinical validity (CV) scoring system. METHODS: To assess the impact of CV-based gene vetting on MGPT results, data from MGPTs for cardiovascular indications were retrospectively analyzed. Using our CV scoring system, genes were categorized as having definitive, strong, moderate, or limited evidence. The rates of reported pathogenic or likely pathogenic variants and VUS were then determined for each CV category. RESULTS: Of 106 total genes, 42% had definitive, 17% had strong, 29% had moderate, and 12% had limited CV. The detection rate of variants classified as pathogenic or likely pathogenic was higher for genes with greater CV, while the VUS rate showed an inverse relationship with CV score. No pathogenic or likely pathogenic findings were observed in genes with a limited CV. CONCLUSION: These results demonstrate the importance of a standardized, evidence-based vetting process to establish CV for genes on MGPTs. Using our proposed system may help to increase the detection rate while mitigating higher VUS rates.

MYLK pathogenic variants aortic disease presentation, pregnancy risk, and characterization of pathogenic missense variants.

PURPOSE: Heritable thoracic aortic disease can result from null variants in MYLK, which encodes myosin light-chain kinase (MLCK). Data on which MYLK missense variants are pathogenic and information to guide aortic disease management are limited. METHODS: Clinical data from 60 cases with MYLK pathogenic variants were analyzed (five null and two missense variants), and the effect of missense variants on kinase activity was assessed. RESULTS: Twenty-three individuals (39%) experienced an aortic event (defined as aneurysm repair or dissection); the majority of these events (87%) were aortic dissections. Aortic diameters were minimally enlarged at the time of dissection in many cases. Time-to-aortic-event curves showed that missense pathogenic variant (PV) carriers have earlier-onset aortic events than null PV carriers. An MYLK missense variant segregated with aortic disease over five generations but decreases MYLK kinase acitivity marginally. Functional Assays fail to identify all pathogenic variants in MYLK. CONCLUSION: These data further define the aortic phenotype associated with MYLK pathogenic variants. Given minimal aortic enlargement before dissection, an alternative approach to guide the timing of aortic repair is proposed based on the probability of a dissection at a given age.

IGFBPL1 Regulates Axon Growth through IGF-1-mediated Signaling Cascades.

Activation of axonal growth program is a critical step in successful optic nerve regeneration following injury. Yet the molecular mechanisms that orchestrate this developmental transition are not fully understood. Here we identified a novel regulator, insulin-like growth factor binding protein-like 1 (IGFBPL1), for the growth of retinal ganglion cell (RGC) axons. Expression of IGFBPL1 correlates with RGC axon growth in development, and acute knockdown of IGFBPL1 with shRNA or IGFBPL1 knockout in vivo impaired RGC axon growth. In contrast, administration of IGFBPL1 promoted axon growth. Moreover, IGFBPL1 bound to insulin-like growth factor 1 (IGF-1) and subsequently induced calcium signaling and mammalian target of rapamycin (mTOR) phosphorylation to stimulate axon elongation. Blockage of IGF-1 signaling abolished IGFBPL1-mediated axon growth, and vice versa, IGF-1 required the presence of IGFBPL1 to promote RGC axon growth. These data reveal a novel element in the control of RGC axon growth and suggest an unknown signaling loop in the regulation of the pleiotropic functions of IGF-1. They suggest new therapeutic target for promoting optic nerve and axon regeneration and repair of the central nervous system.

Retrospective analysis in oculocutaneous albinism patients for the 2.7 kb deletion in the OCA2 gene revealed a co-segregation of the controversial variant, p.R305W.

BACKGROUND: Oculocutaneous albinism (OCA) is an autosomal recessive disorder. A significant portion of OCA patients has been found with a single pathogenic variant either in the TYR or the OCA2 gene. Diagnostic sequencing of the TYR and OCA2 genes is routinely used for molecular diagnosis of OCA subtypes. To study the possibility that genomic abnormalities with single or multiple exon involvement may account for a portion of the potential missing pathogenic variants (the second), we retrospectively analyzed the TYR gene by long range PCR and analyzed the target 2.7 kb deletion in the OCA2 gene spanning exon 7 in OCA patients with a single pathogenic variant in the target genes. RESULTS: In the 108 patients analyzed, we found that one patient was heterozygous for the 2.7 kb OCA2 gene deletion and this patient was positive with one pathogenic variant and one possibly pathogenic variant [c.1103C>T (p.Ala368Val) + c.913C>T (p.R305W)]. Further analysis of maternal DNA, and two additional OCA DNA homozygous for the 2.7 kb deletion, revealed that the phenotypically normal mother is heterozygous of the 2.7 kb deletion and homozygous of the p.R305W. The two previously reported patients with homozygous of the 2.7 kb deletion are also homozygous of p.R305W. CONCLUSIONS: Among the reported pathogenic variants, the pathogenicity of the p.R305W has been discussed intensively in literature. Our results indicate that p.R305W is unlikely a pathogenic variant. The possibility of linkage disequilibrium between p.R305W with the 2.7 kb deletion in OCA2 gene is also suggested.

Early somatic mosaicism is a rare cause of long-QT syndrome.

Somatic mosaicism, the occurrence and propagation of genetic variation in cell lineages after fertilization, is increasingly recognized to play a causal role in a variety of human diseases. We investigated the case of life-threatening arrhythmia in a 10-day-old infant with long QT syndrome (LQTS). Rapid genome sequencing suggested a variant in the sodium channel NaV1.5 encoded by SCN5A, NM_000335:c.5284G > T predicting p.(V1762L), but read depth was insufficient to be diagnostic. Exome sequencing of the trio confirmed read ratios inconsistent with Mendelian inheritance only in the proband. Genotyping of single circulating leukocytes demonstrated the mutation in the genomes of 8% of patient cells, and RNA sequencing of cardiac tissue from the infant confirmed the expression of the mutant allele at mosaic ratios. Heterologous expression of the mutant channel revealed significantly delayed sodium current with a dominant negative effect. To investigate the mechanism by which mosaicism might cause arrhythmia, we built a finite element simulation model incorporating Purkinje fiber activation. This model confirmed the pathogenic consequences of cardiac cellular mosaicism and, under the presenting conditions of this case, recapitulated 2:1 AV block and arrhythmia. To investigate the extent to which mosaicism might explain undiagnosed arrhythmia, we studied 7,500 affected probands undergoing commercial gene-panel testing. Four individuals with pathogenic variants arising from early somatic mutation events were found. Here we establish cardiac mosaicism as a causal mechanism for LQTS and present methods by which the general phenomenon, likely to be relevant for all genetic diseases, can be detected through single-cell analysis and next-generation sequencing.

Characterization of novel RS1 exonic deletions in juvenile X-linked retinoschisis.

PURPOSE: X-linked juvenile retinoschisis (XLRS) is a vitreoretinal dystrophy characterized by schisis (splitting) of the inner layers of the neuroretina. Mutations within the retinoschisis (RS1) gene are responsible for this disease. The mutation spectrum consists of amino acid substitutions, splice site variations, small indels, and larger genomic deletions. Clinically, genomic deletions are rarely reported. Here, we characterize two novel full exonic deletions: one encompassing exon 1 and the other spanning exons 4-5 of the RS1 gene. We also report the clinical findings in these patients with XLRS with two different exonic deletions. METHODS: Unrelated XLRS men and boys and their mothers (if available) were enrolled for molecular genetics evaluation. The patients also underwent ophthalmologic examination and in some cases electroretinogram (ERG) recording. All the exons and the flanking intronic regions of the RS1 gene were analyzed with direct sequencing. Two patients with exonic deletions were further evaluated with array comparative genomic hybridization to define the scope of the genomic aberrations. After the deleted genomic region was identified, primer walking followed by direct sequencing was used to determine the exact breakpoints. RESULTS: Two novel exonic deletions of the RS1 gene were identified: one including exon 1 and the other spanning exons 4 and 5. The exon 1 deletion extends from the 5' region of the RS1 gene (including the promoter) through intron 1 (c.(-35)-1723_c.51+2664del4472). The exon 4-5 deletion spans introns 3 to intron 5 (c.185-1020_c.522+1844del5764). CONCLUSIONS: Here we report two novel exonic deletions within the RS1 gene locus. We have also described the clinical presentations and hypothesized the genomic mechanisms underlying these schisis phenotypes.

Biolistic transfection of cultured myotubes.

Transfection of cells in culture with cDNA constructs is a powerful tool in cell biology, but postmitotic cells, including myotubes, can be hard to transfect with classic methods. Biolistics provides an alternative. We have used this biolistic technique to introduce cDNAs into cultured rat, chick, and C2C12 myotubes. This protocol results in efficient (20 to 70%, depending on cell type) transfection of myotubes, high levels of cDNA expression in individual myotubes, and little cellular damage. Using this procedure, we have expressed different muscle-specific cDNAs as green fluorescent protein (GFP) fusions. This technique is rapid, reliable, uses minimal amounts of reagent per transfection, and yields high transfection rates in a previously hard-to-transfect cell type. Its efficiency and reliability are high, regardless of plasmid size or epitope tag. Muscle cell biologists may now perform experiments in mature myotubes rather than relying on transfection of myoblast cultures or heterologous expression systems.