Identification of an elusive spliceogenic MYBPC3 variant in an otherwise genotype-negative hypertrophic cardiomyopathy pedigree.

The finding of a genotype-negative hypertrophic cardiomyopathy (HCM) pedigree with several affected members indicating a familial origin of the disease has driven this study to discover causative gene variants. Genetic testing of the proband and subsequent family screening revealed the presence of a rare variant in the MYBPC3 gene, c.3331-26T>G in intron 30, with evidence supporting cosegregation with the disease in the family. An analysis of potential splice-altering activity using several splicing algorithms consistently yielded low scores. Minigene expression analysis at the mRNA and protein levels revealed that c.3331-26T>G is a spliceogenic variant with major splice-altering activity leading to undetectable levels of properly spliced transcripts or the corresponding protein. Minigene and patient mRNA analyses indicated that this variant induces complete and partial retention of intron 30, which was expected to lead to haploinsufficiency in carrier patients. As most spliceogenic MYBPC3 variants, c.3331-26T>G appears to be non-recurrent, since it was identified in only two additional unrelated probands in our large HCM cohort. In fact, the frequency analysis of 46 known splice-altering MYBPC3 intronic nucleotide substitutions in our HCM cohort revealed 9 recurrent and 16 non-recurrent variants present in a few probands (≤ 4), while 21 were not detected. The identification of non-recurrent elusive MYBPC3 spliceogenic variants that escape detection by in silico algorithms represents a challenge for genetic diagnosis of HCM and contributes to solving a fraction of genotype-negative HCM cases.

A cryptic splice-altering KCNQ1 variant in trans with R259L leading to Jervell and Lange-Nielsen syndrome.

Here we report an infant with clinical findings suggestive of Jervell and Lange-Nielsen syndrome (JLNS), including a prolonged QT interval (LQTS) and chronic bilateral sensorineural deafness. NGS analysis revealed one known heterozygous pathogenic missense variant, KCNQ1 p.R259L, previously associated with LQTS but insufficient to explain the cardioauditory disorder. In a screening of proximal intronic regions, we found a heterozygous variant, KCNQ1 c.1686-9 T > C, absent from controls and previously undescribed. Several splicing prediction tools returned low scores for this intronic variant. Driven by the proband's phenotype rather than the neutral predictions, we have characterized this rare intronic variant. Family analysis has shown that the proband inherited the missense and the intronic variants from his mother and father, respectively. A minigene splicing assay revealed that the intronic variant induced an additional transcript, arising from skipping of exon 14, which was translated into a truncated protein in transfected cells. The splice-out of exon 14 creates a frameshift in exon 15 and a stop codon in exon 16, which is the last exon of KCNQ1. This mis-spliced transcript is expected to escape nonsense-mediated decay and predicted to encode a truncated loss-of-function protein, KCNQ1 p.L563Kfs*73. The analysis of endogenous KCNQ1 expression in the blood of the proband's parents detected the aberrant transcript only in the patient's father. Taken together, these analyses confirmed the proband's diagnosis of JLNS1 and indicated that c.1686-9 T > C is a cryptic splice-altering variant, expanding the known genetic spectrum of biallelic KCNQ1 variant combinations leading to JLNS1.

Carrier detection and prenatal diagnosis of congenital adrenal hyperplasia must identify 'apparently mild' CYP21A2 alleles which associate neonatal salt-wasting disease.

OBJECTIVE: Couples at risk of severe congenital adrenal hyperplasia (CAH) may be offered prenatal treatment or preimplantation diagnosis. However, proper genetic counselling requires the accurate identification of apparently 'mild alleles' in partners of CAH-carriers/patients. METHODS: CYP21A2 gene analyses were performed in 255 patients with severe 21-hydroxylase deficiency (21-OHD), 94 with mild 21-OHD, 752 parental samples, 233 clinically unaffected partners and 253 historic DNA samples. GENSCAN and ClustalX2.0 software were used for in silico analyses, and Epidat 3.1 software for statistical calculations. RESULTS: Twenty-seven partners were carriers of p.Val282Leu (alias p.Val281Leu, allele frequency 11.7%, 7.4-16.1). 'Val282Leu alleles' were detected in 30 patients with salt-wasting (SW) disease, 21 with other pseudogene-derived and rare coding cis severe mutations, and 9 without. These CYP21A2 genes were compared to those of 94 fully characterized patients with mild deficiency carrying p.Val282Leu in compound heterozygosity with a severe allele. A rare intronic variant, c.292+5G>A, was detected in the nine 'severe Val282Leu alleles' and that was not seen in mild p.Val282Leu alleles, in other deficient alleles or in normal chromosomes. The in silico documented effect on splicing and the clinical association (p < 0.0001) confirmed p.Val282Leu; c.292+5G>A as a severe allele. CONCLUSION: As only severe alleles require clinical intervention, CAH-carrier detection of p.Val282Leu should be followed by the analysis of c.292+5G>A.

Mutations in TRIM63 cause an autosomal-recessive form of hypertrophic cardiomyopathy.

OBJECTIVE: Up to 50% of patients with hypertrophic cardiomyopathy (HCM) show no disease-causing variants in genetic studies. TRIM63 has been suggested as a candidate gene for the development of cardiomyopathies, although evidence for a causative role in HCM is limited. We sought to investigate the relationship between rare variants in TRIM63 and the development of HCM. METHODS: TRIM63 was sequenced by next generation sequencing in 4867 index cases with a clinical diagnosis of HCM and in 3628 probands with other cardiomyopathies. Additionally, 3136 index cases with familial cardiovascular diseases other than cardiomyopathy (mainly channelopathies and aortic diseases) were used as controls. RESULTS: Sixteen index cases with rare homozygous or compound heterozygous variants in TRIM63 (15 HCM and one restrictive cardiomyopathy) were included. No homozygous or compound heterozygous were identified in the control population. Familial evaluation showed that only homozygous and compound heterozygous had signs of disease, whereas all heterozygous family members were healthy. The mean age at diagnosis was 35 years (range 15-69). Fifty per cent of patients had concentric left ventricular hypertrophy (LVH) and 45% were asymptomatic at the moment of the first examination. Significant degrees of late gadolinium enhancement were detected in 80% of affected individuals, and 20% of patients had left ventricular (LV) systolic dysfunction. Fifty per cent had non-sustained ventricular tachycardia. Twenty per cent of patients suffered an adverse cerebrovascular event (20%). CONCLUSION: TRIM63 appears to be an uncommon cause of HCM inherited in an autosomal-recessive manner and associated with concentric LVH and a high rate of LV dysfunction.

A novel ABCA12 pathologic variant identified in an Ecuadorian harlequin ichthyosis patient: A step forward in genotype-phenotype correlations.

BACKGROUND: Autosomal recessive congenital ichthyoses (ARCI) have been associated with different phenotypes including: harlequin ichthyosis (HI), congenital ichthyosiform erythroderma (CIE), and lamellar ichthyosis (LI). While pathogenic variants in all ARCI genes are associated with LI and CIE phenotypes, the unique gene associated with HI is ABCA12. In HI, the most severe ARCI form, pathogenic variants in both ABCA12 gene alleles usually have a severe impact on protein function. The presence of at least one non-truncating variant frequently causes a less severe congenital ichthyosis phenotype (LI and CIE). METHODS: We report the case of a 4-year-old Ecuadorian boy with a severe skin disease. Genetic diagnosis was performed by NGS. In silico predictions were performed using Alamut software v2.11. A review of the literature was carried out to identify all patients carrying ABCA12 splice-site and missense variants, and to explore their genotype-phenotype correlations. RESULTS: Genetic testing revealed a nonsense substitution, p.(Arg2204*), and a new missense variant, p.(Val1927Leu), in the ABCA12 gene. After performing in silico analysis and a comprehensive review of the literature, we conclude that p.(Val1927Leu) affects a well conserved residue which could either disturb the protein function or alter the splicing process, both alternatives could explain the severe phenotype of our patient. CONCLUSION: This case expands the spectrum of ABCA12 reported disease-causing variants which is important to unravel genotype-phenotype correlations and highlights the importance of missense variants in the development of HI.

Targeted resequencing of regulatory regions at schizophrenia risk loci: Role of rare functional variants at chromatin repressive states.

There is mounting evidence that regulatory variation plays an important role in genetic risk for schizophrenia. Here, we specifically search for regulatory variants at risk by sequencing promoter regions of twenty-three genes implied in schizophrenia by copy number variant or genome-wide association studies. After strict quality control, a total of 55,206bp per sample were analyzed in 526 schizophrenia cases and 516 controls from Galicia, NW Spain, using the Applied Biosystems SOLiD System. Variants were filtered based on frequency from public databases, chromatin states from the RoadMap Epigenomics Consortium at tissues relevant for schizophrenia, such as fetal brain, mid-frontal lobe, and angular gyrus, and prediction of functionality from RegulomeDB. The proportion of rare variants at polycomb repressive chromatin state at relevant tissues was higher in cases than in controls. The proportion of rare variants with predicted regulatory role was significantly higher in cases than in controls (P=0.0028, OR=1.93, 95% C.I.=1.23-3.04). Combination of information from both sources led to the identification of an excess of carriers of rare variants with predicted regulatory role located at polycomb repressive chromatin state at relevant tissues in cases versus controls (P=0.0016, OR=19.34, 95% C.I.=2.45-2495.26). The variants are located at two genes affected by the 17q12 copy number variant, LHX1 and HNF1B. These data strongly suggest that a specific epigenetic mechanism, chromatin remodeling by histone modification during early development, may be impaired in a subset of schizophrenia patients, in agreement with previous data.