Genetic characterization of KCNQ1 variants improves risk stratification in type 1 long QT syndrome patients.

AIMS: KCNQ1 mutations cause QTc prolongation increasing life-threatening arrhythmias risks. Heterozygous mutations [type 1 long QT syndrome (LQT1)] are common. Homozygous KCNQ1 mutations cause type 1 Jervell and Lange-Nielsen syndrome (JLNS) with deafness and higher sudden cardiac death risk. KCNQ1 variants causing JLNS or LQT1 might have distinct phenotypic expressions in heterozygous patients. The aim of this study is to evaluate QTc duration and incidence of long QT syndrome-related cardiac events according to genetic presentation. METHODS AND RESULTS: We enrolled LQT1 or JLNS patients with class IV/V KCNQ1 variants from our inherited arrhythmia clinic (September 1993 to January 2023). Medical history, ECG, and follow-up were collected. Additionally, we conducted a thorough literature review for JLNS variants. Survival curves were compared between groups, and multivariate Cox regression models identified genetic and clinical risk factors. Among the 789 KCNQ1 variant carriers, 3 groups were identified: 30 JLNS, 161 heterozygous carriers of JLNS variants (HTZ-JLNS), and 550 LQT1 heterozygous carriers of non-JLNS variants (HTZ-Non-JLNS). At diagnosis, mean age was 3.4 ± 4.7 years for JLNS, 26.7 ± 21 years for HTZ-JLNS, and 26 ± 21 years for HTZ-non-JLNS; 55.3% were female; and the mean QTc was 551 ± 54 ms for JLNS, 441 ± 32 ms for HTZ-JLNS, and 467 ± 36 ms for HTZ-Non-JLNS. Patients with heterozygous JLNS mutations (HTZ-JLNS) represented 22% of heterozygous KCNQ1 variant carriers and had a lower risk of cardiac events than heterozygous non-JLNS variant carriers (HTZ-Non-JLNS) [hazard ratio (HR) = 0.34 (0.22-0.54); P < 0.01]. After multivariate analysis, four genetic parameters were independently associated with events: haploinsufficiency [HR = 0.60 (0.37-0.97); P = 0.04], pore localization [HR = 1.61 (1.14-1.2.26); P < 0.01], C-terminal localization [HR = 0.67 (0.46-0.98); P = 0.04], and group [HR = 0.43 (0.27-0.69); P < 0.01]. CONCLUSION: Heterozygous carriers of JLNS variants have a lower risk of cardiac arrhythmic events than other LQT1 patients.

Molecular genetic screening after non-ischaemic sudden cardiac arrest and no overt cardiomyopathy in real life: A major tool for the aetiological diagnostic work-up.

BACKGROUND: With the development of advanced sequencing techniques, genetic testing has emerged as a valuable tool for the work-up of non-ischaemic sudden cardiac arrest (SCA). AIMS: To evaluate the effectiveness of genetic testing in patients with unexplained SCA, according to clinical phenotype. METHODS: All patients who underwent molecular genetic testing for non-ischaemic SCA with no left ventricular cardiomyopathy between 2012 and 2021 in two French university hospitals were included. RESULTS: Of 66 patients (mean age 36.7±11.9years, 54.5% men), 21 (31.8%; 95% confidence interval 22.4-45.3%) carried a genetic variant: eight (12.1%) had a pathogenic or likely pathogenic (P/LP) variant and 13 (19.7%) had a variant of uncertain significance (VUS). Among 37 patients (56.1%) with no phenotypic clues, genetic testing identified a P/LP variant in five (13.5%), mainly in RYR2 (n=3) and SCN5A (n=2), and a VUS in nine (24.3%). None of the nine patients with phenotypic evidence of channelopathies had P/LP variants, but two had VUS in RYR2 and NKX2.5. Among the 20 patients with suspected arrhythmogenic cardiomyopathy, three P/LP variants (15.0%) and two VUS (10.0%) were found in DSC2, PKP2, SCN5A and DSG2, TRPM4, respectively. Genetic testing was performed sooner after cardiac arrest (P<0.001) and results were obtained more rapidly (P=0.02) after versus before 2016. CONCLUSION: This study highlights the utility of molecular genetic testing with a genetic variant of interest identified in one-third of patients with unexplained SCA. Genetic testing was beneficial even in patients without phenotypic clues, with one-fourth of patients carrying a P/LP variant that could have direct implications.

Prevalence and phenotypes associated with ALPK3 null variants in a large French multicentric cohort: Confirming its involvement in hypertrophic cardiomyopathy.

Biallelic disease-causing variants in the ALPK3 gene were first identified in children presenting with a severe cardiomyopathy. More recently, it was shown that carriers of heterozygous ALPK3 null variants are at risk of developing hypertrophic cardiomyopathy (HCM) with an adult onset. Since the number of reported ALPK3 patients is small, the mutational spectrum and clinical data are not fully described. In this multi-centric study, we described the molecular and clinical spectrum of a large cohort of ALPK3 patients. Genetic testing using targeted next generation sequencing was performed in 16 183 cardiomyopathy index cases. Thirty-six patients carried at least one null ALPK3 variant. The five paediatric patients carried two ALPK3 variants, all presented an HCM phenotype with severe outcomes (one transplantation, one heart failure and one cardiac arrest). The 31 adult patients carried heterozygous variants and the main phenotype was HCM (n = 26/31); including 15% (n = 4) presented with an apical or a concentric form of hypertrophy. Reporting a large cohort of ALPK3 patients, this collaborative work confirmed a strong association with HCM and suggesting his screening in the context of idiopathic HCM.

Type 3 long QT syndrome: Is the effectiveness of treatment with beta-blockers population-specific?

BACKGROUND: The efficacy of beta-blocker treatment in type 3 long QT syndrome (LQT3) remains debated. OBJECTIVES: The purpose of this study was to test the hypothesis that beta-blocker use is associated with cardiac events (CEs) in a French cohort of LQT3 patients. METHODS: All patients with a likely pathogenic/pathogenic variant in the SCN5A gene (linked to LQT3) were included and followed-up. Documented ventricular tachycardia/ventricular fibrillation, torsades de pointes, aborted cardiac arrest, sudden death, and appropriate shocks were considered as severe cardiac events (SCEs). CEs also included syncope. RESULTS: We included 147 patients from 54 families carrying 23 variants. Six of the patients developed symptoms before the age of 1 year and were analyzed separately. The 141 remaining patients (52.5% male; median age at diagnosis 24.0 years) were followed-up for a median of 11 years. The probabilities of a CE and an SCE from birth to the age of 40 were 20.5% and 9.9%, respectively. QTc prolongation (hazard ratio [HR] 1.12 [1.0-1.2]; P = .005]) and proband status (HR 4.07 [1.9-8.9]; P <.001) were independently associated with the occurrence of CEs. Proband status (HR 8.13 [1.7-38.8]; P = .009) was found to be independently associated with SCEs, whereas QTc prolongation (HR 1.11 [1.0-1.3]; P = .108) did not reach statistical significance. The cumulative probability of the age at first CE/SCE was not lower in patients treated with a beta-blocker. CONCLUSION: In agreement with the literature, proband status and lengthened QTc were associated with a higher risk of CEs. Our data do not show a protective effect of beta-blocker treatment.

Titin copy number variations associated with dominant inherited phenotypes.

BACKGROUND: Titinopathies are caused by mutations in the titin gene (TTN). Titin is the largest known human protein; its gene has the longest coding phase with 364 exons. Titinopathies are very complex neuromuscular pathologies due to the variable age of onset of symptoms, the great diversity of pathological and muscular impairment patterns (cardiac, skeletal muscle or mixed) and both autosomal dominant and recessive modes of transmission. Until now, only few CNVs in TTN have been reported without clear genotype-phenotype associations. METHODS: Our study includes eight families with dominant titinopathies. We performed next-generation sequencing or comparative genomic hybridisation array analyses and found CNVs in the TTN gene. We characterised these CNVs by RNA sequencing (RNAseq) analyses in six patients' muscles and performed genotype-phenotype inheritance association study by combining the clinical and biological data of these eight families. RESULTS: Seven deletion-type CNVs in the TTN gene were identified among these families. Genotype and RNAseq results showed that five deletions do not alter the reading frame and one is out-of-reading frame. The main phenotype identified was distal myopathy associated with contractures. The analysis of morphological, clinical and genetic data and imaging let us draw new genotype-phenotype associations of titinopathies. CONCLUSION: Identifying TTN CNVs will further increase diagnostic sensitivity in these complex neuromuscular pathologies. Our cohort of patients enabled us to identify new deletion-type CNVs in the TTN gene, with unexpected autosomal dominant transmission. This is valuable in establishing new genotype-phenotype associations of titinopathies, mainly distal myopathy in most of the patients.

Novel pathogenic variants in SLCO2A1 causing autosomal dominant primary hypertrophic osteoarthropathy.

Primary hypertrophic osteoarthropathy (PHO), or pachydermoperiostosis, is characterized by a clinical association including digital clubbing, periostosis and pachydermia. SLCO2A1 and HPGD genes are both responsible for PHO. The pathology is classically defined as an autosomal recessive disorder with clinical variability ranging from a mild to more severe phenotype. However, the hypothesis for an autosomal dominant form suggested for a long time was only demonstrated for the first time in 2021 for SLCO2A1. We aimed to detect a second pathogenic variant by a deep sequencing of the entire SLCO2A1 and HPGD genes, associated with functional transcription analysis in PHO patients harboring only one heterozygous variant. Among 10 PHO patients, 4 presented a single pathogenic or probably pathogenic novel variant in SLCO2A1 in heterozygous status (NM_005630.3: c.234+1G > A, c.1523_1524delCT, c.1625G > A and c.31delC), and the others carried homozygous pathogenic variants. For heterozygous forms, we found no additional pathogenic variant in HPGD or SLCO2A1. PHO can be a dominant form with age at disease onset later than that for the recessive form. This dominant form is not exceptional in young adults. In conclusion, both modes of inheritance of PHO explain the clinical variability and the difference in age at disease onset. Molecular analysis is especially required in the incomplete form to distinguish it from secondary hypertrophic osteoarthropathy.

Detailed cell-level analysis of sperm nuclear quality among the different hypo-osmotic swelling test (HOST) classes.

PURPOSE: We studied the quality differences between the different hypo-osmotic swelling test (HOST) classes, as measured by criteria of DNA fragmentation, DNA decondensation, and nuclear architecture. The aim was to find particular HOST classes associated with good-quality metrics, which may be potentially used in ICSI (intra-cytoplasmic sperm injection). METHODS: Ten patients from the Department of Reproductive Medicine at Tenon Hospital (Paris, France) were included. Their semen samples were collected and divided into two fractions: one was incubated in a hypo-osmotic solution as per HOST protocol and sorted by sperm morphology, and a second was incubated without undergoing the HOST protocol to serve as an unsorted baseline. Three parameters were assessed: DNA fragmentation (TUNEL assay), DNA decondensation (chromomycin A3 assay), and nuclear architecture (FISH, with telomeric and whole chromosome painting probes). The different HOST classes were evaluated for these three parameters, and statistical analysis was performed for each class versus the unsorted non-HOST-treated sperm. Results with p<0.05 were considered statistically significant. RESULTS: For each of the parameters evaluated, we found significant differences between HOST-selected spermatozoa and non-selected spermatozoa. Overall, spermatozoa of HOST classes B and B+ exhibited the highest quality based on four metrics (low DNA fragmentation, low DNA decondensation, short inter-telomeric distance, and small chromosome 1 territory area), while spermatozoa of HOST classes A and G exhibited the poorest quality by these metrics. CONCLUSION: In addition to their pathophysiological interest, our results open possibilities of sperm selection prior to ICSI, which may allow for optimization of reproductive outcomes in heretofore unstudied patient populations.

[Usefulness of combined sequencing of the mitochondrial genome and a targeted panel of nuclear genes involved in mitochondrial diseases]. / Intérêt du séquençage combiné du génome mitochondrial et d'un panel ciblé de gènes nucléaires impliqués dans les maladies mitochondriales.

The molecular study of mitochondrial diseases, essential for diagnosis, is special due to the dual genetic origin of these pathologies: mitochondrial DNA and nuclear DNA. Complete mtDNA sequencing still remains the first line diagnostic test followed if negative, by resequencing panels of several hundred mitochondrially-encoded nuclear genes. This strategy, with an initial entire mtDNA sequencing, is currently justified by the presence of nuclear mitochondrial DNA sequences (NUMTs) in the nuclear genome. We designed a resequencing panel combining the mtDNA and 135 nuclear genes which was evaluated compared to the performances of the standard mtDNA sequencing. Method validation was performed on the reading depth and reproducibility of the results. Thirty patients were analyzed by both methods. We were able to demonstrate that NUMTs did not impact the mtDNA sequencing quality, as the identified variants and mutant loads were identical with the reference mtDNA sequencing method. Reading depths were higher than the recommendations of the MitoDiag French diagnostic network, for the entire mtDNA for muscle and for 70% of the mtDNA for blood. These results highlight the usefulness of combining both mtDNA and mitochondrially nuclear-encoded genes and thus obtain more complete results and faster turnaround time for mitochondrial disease patients.

A novel deep intronic variant in ATP7B in five unrelated families affected by Wilson disease.

BACKGROUND: Wilson disease is an autosomal recessive metabolic disorder resulting from accumulation of excess copper especially in the liver and brain. This disease is mainly characterized by hepatic disorders and less frequently by neuro-psychiatric disturbances. This recessive disease is due to mutation in ATP7B, which codes for an ATPase involved in copper-transport across the plasma membrane. Molecular diagnosis of WD is positive in approximately 98% of cases. Also, in few cases, WD patients present a single deleterious mutation (heterozygous) or no mutation after sanger and NGS standard sequencing analysis of ATP7B. Therefore, in these problematic WD cases, we hypothesized that deleterious mutations reside in intronic regions of ATP7B. METHODS: Complete ATP7B gene was sequenced by Next Generation Sequencing including its promoter. RESULTS: Five unrelated families with Wilson disease shared the same novel, deep intronic NG_008806.1 (ATP7B_v001):c.2866-1521G>A variant in ATP7B. Analysis of RNA transcripts from primary fibroblasts of one patient confirmed the deleterious impact of the intronic variant on splicing and its likely pathologic effect in this compound heterozygote. CONCLUSION: This discovery of a novel intronic mutation in ATP7B has improved the molecular diagnosis of WD in the French patient cohort to greater than 98%. Thus, we recommend complete sequencing of ATP7B gene, including introns, as a molecular diagnostic approach in cases of clinically confirmed WD which lack pathogenic exon or promoter variants in one or both alleles.

Double chromosomal translocation in an infertile man: one-step FISH meiotic segregation analysis and reproductive prognosis.

BACKGROUND: The prevalence of chromosomal translocations is 1/500 in the general population. While in the vast majority of cases, carriers have a normal phenotype; they can present with difficulty conceiving due to the presence of a proportion of unbalanced gametes as a consequence of abnormal chromosomal segregation during meiosis. Since complex translocations involve three or more chromosomes, meiotic segregation leads to a greater number of possible combinations which effectively complicate both their study and therapeutic care. CASE PRESENTATION: We report on the case of a male carrier of a complex homogeneous double Robertsonian translocation: 44, XY, der(13;14)(q10;q10),der(21;22)(q10;q10). We studied his meiotic segregation by FISH on spermatozoa from the initial sample, as well as following discontinuous gradient centrifugation and after incubation in an hypo-osmotic solution. CONCLUSION: We report a method to study in a simple single-step manner the meiotic segregation of double Robertsonian translocations in spermatozoa. Further, our results suggest that reproductive prognosis of affected individuals may be markedly improved by HOST-based sperm selection (HBSS).