The First Korean Case of SLC12A3 Aberrant Skipping of Two Exons Detected by RNA Splicing Analysis

Gitelman syndrome is a salt-losing tubular disorder that is transmitted as an autosomal recessive trait. Variants in the <i>SLC12A3</i> gene are found in the majority of Gitelman syndrome patients. A 26-year-old woman visited the genetic counseling clinic. Her fiancé was a known Gitelman syndrome patient who was previously diagnosed with 2 pathogenic variants in <i>SLC12A3</i>. In advance of marriage and future family planning, she wanted to perform genetic testing of <i>SLC12A3</i>. A silent exonic variant c.1050G&#x3e;A was found, and multiple splice site in silico algorithms predicted this variant to have potential alteration of splicing. This variant was classified as “variant of uncertain significance,” and RNA splicing analysis was additionally performed. RNA splicing analysis showed aberrant splicing of exon 7–8 skipping. The result points out the potential pathogenicity of this variant, which should be considered a candidate of variant reclassification in the future. We highly recommend the performance of additional RNA splicing analysis, especially for silent variants predicted to have potential alteration of splicing.

False negatives in GBA1 sequencing due to polymerase dependent allelic imbalance

A variant in the GBA1 gene is one of the most common genetic risk factors to develop Parkinson’s disease (PD). Here the serendipitous finding is reported of a polymerase dependent allelic imbalance when using next generation sequencing, potentially resulting in false-negative results when the allele frequency falls below the variant calling threshold (by default commonly at 30%). The full GBA1 gene was sequenced using next generation sequencing on saliva derived DNA from PD patients. Four polymerase chain reaction conditions were varied in twelve samples, to investigate the effect on allelic imbalance: (1) the primers (n = 4); (2) the polymerase enzymes (n = 2); (3) the primer annealing temperature (Ta) specified for the used polymerase; and (4) the amount of DNA input. Initially, 1295 samples were sequenced using Q5 High-Fidelity DNA Polymerase. 112 samples (8.6%) had an exonic variant and an additional 104 samples (8.0%) had an exonic variant that did not pass the variant frequency calling threshold of 30%. After changing the polymerase to TaKaRa LA Taq DNA Polymerase Hot-Start Version: RR042B, all samples had an allele frequency passing the calling threshold. Allele frequency was unaffected by a change in primer, annealing temperature or amount of DNA input. Sequencing of the GBA1 gene using next generation sequencing might be susceptible to a polymerase specific allelic imbalance, which can result in a large amount of flase-negative results. This was resolved in our case by changing the polymerase. Regions displaying low variant calling frequencies in GBA1 sequencing output in previous and future studies might warrant additional scrutiny.

Inter-laboratory proficiency testing scheme for tumour next-generation sequencing in Ontario: a pilot study

Background A pilot inter-laboratory proficiency scheme for 5 Ontario clinical laboratories testing tumour samples for the Ontario-wide Cancer Targeted Nucleic Acid Evaluation (octane) study was undertaken to assess proficiency in the identification and reporting of next-generation sequencing (ngs) test results in solid tumour testing from archival formalin-fixed, paraffin-embedded (ffpe) tissue.Methods One laboratory served as the reference centre and provided samples to 4 participating laboratories. An analyte-based approach was applied: each participating laboratory received 10 ffpe tissue specimens profiled at the reference centre, with tumour site and histology provided. Laboratories performed testing per their standard ngs tumour test protocols. Items returned for assessment included genes and variants that would be typically reported in routine clinical testing and variant call format (vcf) files to allow for assessment of ngs technical quality.Results Two main aspects were assessed:■ Technical quality and accuracy of identification of exonic variants■ Site-specific reporting practicesTechnical assessment included evaluation of exonic variant identification, quality assessment of the vcf files to evaluate base calling, variant allele frequency, and depth of coverage for all exonic variants. Concordance at 100% was observed from all sites in the technical identification of 98 exonic variants across the 10 cases. Variability between laboratories in the choice of variants considered clinically reportable was significant. Of the 38 variants reported as clinically relevant by at least 1 site, only 3 variants were concordantly reported by all participating centres as clinically relevant.Conclusions Although excellent technical concordance for ngs tumour profiling was observed across participating institutions, differences in the reporting of clinically relevant variants were observed, highlighting reporting as a gap where consensus on the part of Ontario laboratories is needed.

1179A novel exonic variant in TRIM55 gene predisposes to heart failure

Background/Introduction Heart failure is a potentially lethal cardiac disorder. The genetic variants as well as the molecular signaling pathways accounting for the majority of the heritability of sporadic heart failure are unknown. Here, we screened for novel genetic variants associated with heart failure in the Finnish population. A previously uncharacterized variant in TRIM55 was selected for further validation. Purpose To identify and characterize heart failure-associated genetic variants. Methods Heart failure-associated genetic variants were screened from the FINRISK survey study data by the National Institute of Health and Welfare of Finland (genotype data available for approx. 28,000 individuals, 1,485 individuals with heart failure). A probably damaging heart failure-associated exonic variant in TRIM55 gene (rs138811034), encoding an E140K variant, was functionally characterized in HL-1 mouse cardiomyocytes using CRISPR/Cas9 technology to introduce the alteration in the endogenous locus. Variant cell lines were analyzed with RNA sequencing. The effect of the variant on cellular viability was tested with CTG assays. Hypertrophic response after isoproterenol treatment was studied by quantitating cell surface area with immunofluorescence microscopy. Western analyses of p21 expression and flow cytometry-based cell cycle analyses were carried out to assess effects on cell cycle progression. In zebrafish embryos in vivo, cardiac contractility was videomicroscopically measured after transient CRISPR-mediated knockdown of the trim55a gene. Results HL-1 cells harboring Trim55 E140K/− genotype demonstrated significantly reduced viability as compared to the WT/WT cell (P=0.006), similarly to the cell line with double knockout (−/−) of the gene. E140K/−, −/− and WT/− cell lines demonstrated a significant hypertrophic response to isoproterenol stimulation (P=0.007, P=0.007 and P=0.018, respectively), while the WT/WT cells did not. In the E140K/− and −/− cell lines, p21 protein expression was increased and there were significantly fewer cells in the mitotic G2/M phase of the cell cycle, as compared to the WT/WT cells (P=0.031 and P=0.024, respectively). In the E140K/− and −/− cell lines, RNA sequencing revealed systemic alterations in the genes related to cardiac contractility and stress. In zebrafish embryos with deleted trim55a gene, ejection fraction was reduced as compared to control sgRNA-injected embryos (30.8% ± 6.0% vs 43.9%± 2.2% (95% CIs), respectively, P=0.019). Conclusions TRIM55 E140K variant was statistically associated with heart failure in the Finnish population. In HL-1 cardiomyocytes in vitro, the variant reduced cellular viability and cell cycle progression and promoted hypertrophy. In zebrafish embryos, loss of trim55a caused a significant reduction in cardiac contractility. These findings are consistent with a novel role for TRIM55 polymorphism in predisposing to heart failure. Acknowledgement/Funding Finnish Cultural Foundation (Varsinais-Suomi Regional Fund), Inkeri ja Mauri Vänskän säätiö, Academy of Finland [310507], Sigrid Jusélius Foundation

Genetic architecture and adaptations of Nunavik Inuit

The Canadian Inuit have a distinct population background that may entail particular implications for the health of its individuals. However, the number of genetic studies examining this Inuit population is limited, and much remains to be discovered in regard to its genetic characteristics. In this study, we generated whole-exome sequences and genomewide genotypes for 170 Nunavik Inuit, a small and isolated founder population of Canadian Arctic indigenous people. Our study revealed the genetic background of Nunavik Inuit to be distinct from any known present-day population. The majority of Nunavik Inuit show little evidence of gene flow from European or present-day Native American peoples, and Inuit living around Hudson Bay are genetically distinct from those around Ungava Bay. We also inferred that Nunavik Inuit have a small effective population size of 3,000 and likely split from Greenlandic Inuit ∼10.5 kya. Nunavik Inuit went through a bottleneck at approximately the same time and might have admixed with a population related to the Paleo-Eskimos. Our study highlights population-specific genomic signatures in coding regions that show adaptations unique to Nunavik Inuit, particularly in pathways involving fatty acid metabolism and cellular adhesion (CPNE7, ICAM5, STAT2, and RAF1). Subsequent analyses in selection footprints and the risk of intracranial aneurysms (IAs) in Nunavik Inuit revealed an exonic variant under weak negative selection to be significantly associated with IA (rs77470587; P = 4.6 × 10−8).

Mutation Analysis ofHTRA2Gene in Chinese Familial Essential Tremor and Familial Parkinson’s Disease

Background.HTRA2has already been nominated as PARK13 which may cause Parkinson’s disease, though there are still discrepancies among these results. Recently, Gulsuner et al.’s study found thatHTRA2p.G399S is responsible for hereditary essential tremor and homozygotes of this allele develop Parkinson’s disease by examining a six-generation family segregating essential tremor and essential tremor coexisting with Parkinson’s disease. We performed this study to validate the condition ofHTRA2gene in Chinese familial essential tremor and familial Parkinson’s disease patients, especially essential tremor.Methods. We directly sequenced all eight exons, exon-intron boundaries, and part of the introns in 101 familial essential tremor patients, 105 familial Parkinson’s disease patients, and 100 healthy controls.Results. No exonic variant was identified, while one exon-intron boundary variant (rs2241028) and one intron variant (rs2241027) were detected, both with no clinical significance and uncertain function. There was no difference in allele, genotype, and haplotype between groups.Conclusions.HTRA2exonic variant might be rare among Chinese Parkinson’s disease and essential tremor patients with family history, andHTRA2may not be the cause of familial Parkinson’s disease and essential tremor in China.