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Whereas truncating variants of the giant protein Titin (TTNtv) are the main cause of familial dilated cardiomyopathy (DCM), recently Filamin C truncating variants (FLNCtv) were identified as a cause of arrhythmogenic cardiomyopathy (ACM). Our aim was to characterize and compare clinical and MRI features of TTNtvand FLNCtvin the Belgian population. In index patients referred for genetic testing of ACM/DCM, FLNCtvand TTNtvwere found in 17 (3.6%) and 33 (12.3%) subjects, respectively. Further family cascade screening yielded 24 and 19 additional truncating variant carriers in FLNCand TTN, respectively. The main phenotype was ACM in FLNCtvcarriers whereas TTNtvcarriers showed either an ACM or DCM phenotype. Non-sustained Ventricular Tachycardia was frequent in both populations. MRI data, available in 28/40 FLNCtvand 32/52 TTNtvpatients, showed lower Left Ventricular (LV) ejection fraction and lower LV strain in TTNtvpatients (p< 0.01). Conversely, both the frequency (68% vs 22%) and extent of non-ischemic myocardial late gadolinium enhancement (LGE) was significantly higher in FLNCtvpatients (p< 0.01). Hereby, ring-like LGE was found in 16/19 (84%) FLNCtvversus 1/7 (14%) of TTNtvpatients (p< 0.01). In conclusion, a large number of FLNCtvand TTNtvpatients present with an ACM phenotype but can be separated by cardiac MRI. Whereas FLNCtvpatients often have extensive myocardial fibrosis, typically following a ring-like pattern, LV dysfunction without or limited replacement fibrosis is the common TTNtvphenotype.

The recent Chandos House meeting of the Alport Variant Collaborative extended the indications for screening for pathogenic variants in the COL4A5, COL4A3and COL4A4genes beyond the classical Alport phenotype (haematuria, renal failure; family history of haematuria or renal failure) to include persistent proteinuria, steroid-resistant nephrotic syndrome, focal and segmental glomerulosclerosis (FSGS), familial IgA glomerulonephritis and end-stage kidney failure without an obvious cause. The meeting refined the ACMG criteria for variant assessment for the Alport genes (COL4A3–5). It identified ‘mutational hotspots’ (PM1) in the collagen IV α5, α3 and α4 chains including position 1 Glycine residues in the Gly-X-Y repeats in the intermediate collagenous domains; and Cysteine residues in the carboxy non-collagenous domain (PP3). It considered that ‘well-established’ functional assays (PS3, BS3) were still mainly research tools but sequencing and minigene assays were commonly used to confirm splicing variants. It was not possible to define the Minor Allele Frequency (MAF) threshold above which variants were considered Benign (BA1, BS1), because of the different modes of inheritances of Alport syndrome, and the occurrence of hypomorphic variants (often Glycine adjacent to a non-collagenous interruption) and local founder effects. Heterozygous COL4A3and COL4A4variants were common ‘incidental’ findings also present in normal reference databases. The recognition and interpretation of hypomorphic variants in the COL4A3–COL4A5genes remains a challenge.

Deletions on chromosome 15q14 are a known chromosomal cause of cleft palate, typically co-occurring with intellectual disability, facial dysmorphism, and congenital heart defects. The identification of patients with loss-of-function variants in MEIS2, a gene within this deletion, suggests that these features are attributed to haploinsufficiency of MEIS2. To further delineate the phenotypic spectrum of the MEIS2-related syndrome, we collected 23 previously unreported patients with either a de novo sequence variant in MEIS2(9 patients), or a 15q14 microdeletion affecting MEIS2(14 patients). All but one de novo MEIS2variant were identified by whole-exome sequencing. One variant was found by targeted sequencing of MEIS2in a girl with a clinical suspicion of this syndrome. In addition to the triad of palatal defects, heart defects, and developmental delay, heterozygous loss of MEIS2results in recurrent facial features, including thin and arched eyebrows, short alae nasi, and thin vermillion. Genotype–phenotype comparison between patients with 15q14 deletions and patients with sequence variants or intragenic deletions within MEIS2, showed a higher prevalence of moderate-to-severe intellectual disability in the former group, advocating for an independent locus for psychomotor development neighboring MEIS2.

In inherited primary arrhythmia syndromes (PAS) and cardiomyopathies (CMP), the yield of genetic testing varies between 20 and 75% in different diseases according to studies performed in the pre next-generation sequencing (NGS) era. It is unknown whether retesting historical negative samples with NGS techniques is worthwhile. Therefore, we assessed the value of NGS-based panel testing in previously genotype negative–phenotype positive probands. We selected 107 patients (47 PAS and 60 CMP) with a clear phenotype who remained genotype negative after genetic analysis of the main genes implicated in their specific phenotype. Targeted sequencing of the coding regions of 71 PAS- and CMP-related genes was performed. Variant interpretation and classification was done according to a cardiology-specific scoring algorithm (‘Amsterdam criteria’) and the ACMG-AMP criteria. Co-segregation analysis was performed when DNA and clinical data of family members were available. Finally, a genetic diagnosis could be established in 21 patients (20%), 5 PAS (11%) and 16 CMP (27%) patients, respectively. The increased detection rate was due to sequencing of novel genes in 52% of the cases and due to technical failures with the historical analysis in 48%. A total of 118 individuals were informed about their carrier state and either reassured or scheduled for proper follow-up. To conclude, genetic retesting in clinically overt PAS and CMP cases, who were genotype negative with older techniques, resulted in an additional genetic diagnosis in up to 20% of the cases. This clearly supports a policy for genetic retesting with NGS-based panels.

We present, on behalf of EuroGentest and the European Society of Human Genetics, guidelines for the evaluation and validation of next-generation sequencing (NGS) applications for the diagnosis of genetic disorders. The work was performed by a group of laboratory geneticists and bioinformaticians, and discussed with clinical geneticists, industry and patients’ representatives, and other stakeholders in the field of human genetics. The statements that were written during the elaboration of the guidelines are presented here. The background document and full guidelines are available as supplementary material. They include many examples to assist the laboratories in the implementation of NGS and accreditation of this service. The work and ideas presented by others in guidelines that have emerged elsewhere in the course of the past few years were also considered and are acknowledged in the full text. Interestingly, a few new insights that have not been cited before have emerged during the preparation of the guidelines. The most important new feature is the presentation of a ‘rating system’ for NGS-based diagnostic tests. The guidelines and statements have been applauded by the genetic diagnostic community, and thus seem to be valuable for the harmonization and quality assurance of NGS diagnostics in Europe.