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The international study ThromboGenomics has evaluated the diagnostic rate using a targeted multigene panel test for the screening of inherited bleeding, thrombotic and platelet disorders.

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.

Encephalitis and encephalopathy in children represent a diagnostic challenge. We describe a patient with relapsing encephalitis in whom the differential diagnosis included acute disseminated encephalomyelitis, human herpesvirus 6 encephalitis, and hemophagocytic lymphohistiocytosis (HLH). Because of its rarity, HLH is often overlooked as a differential diagnosis in encephalitis, especially in the isolated CNS forms. As this case illustrates, inborn errors of immunity can underlie isolated encephalitis and should be included in the differential diagnosis of these presentations.

Brugada syndrome (BrS) is a cardiac arrhythmia disorder associated with sudden death in young adults. With the exception of SCN5A, encoding the cardiac sodium channel NaV1.5, susceptibility genes remain largely unknown. Here we performed a genome-wide association meta-analysis comprising 2,820 unrelated cases with BrS and 10,001 controls, and identified 21 association signals at 12 loci (10 new). Single nucleotide polymorphism (SNP)-heritability estimates indicate a strong polygenic influence. Polygenic risk score analyses based on the 21 susceptibility variants demonstrate varying cumulative contribution of common risk alleles among different patient subgroups, as well as genetic associations with cardiac electrical traits and disorders in the general population. The predominance of cardiac transcription factor loci indicates that transcriptional regulation is a key feature of BrS pathogenesis. Furthermore, functional studies conducted on MAPRE2, encoding the microtubule plus-end binding protein EB2, point to microtubule-related trafficking effects on NaV1.5 expression as a new underlying molecular mechanism. Taken together, these findings broaden our understanding of the genetic architecture of BrS and provide new insights into its molecular underpinnings.

Genetic testing for pathogenic COL4A3–5variants is usually undertaken to investigate the cause of persistent hematuria, especially with a family history of hematuria or kidney function impairment. Alport syndrome experts now advocate genetic testing for persistent hematuria, even when a heterozygous pathogenic COL4A3or COL4A4is suspected, and cascade testing of their first-degree family members because of their risk of impaired kidney function. The experts recommend too that COL4A3or COL4A4heterozygotes do not act as kidney donors. Testing for variants in the COL4A3–COL4A5genes should also be performed for persistent proteinuria and steroid-resistant nephrotic syndrome due to suspected inherited FSGS and for familial IgA glomerulonephritis and kidney failure of unknown cause.

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.

Stringent variant interpretation guidelines can lead to high rates of variants of uncertain significance (VUS) for genetically heterogeneous disease like long QT syndrome (LQTS) and Brugada syndrome (BrS). Quantitative and disease-specific customization of American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines can address this false negative rate.

AbstractBackground:Familial dilated cardiomyopathy (DCM) is genetically heterogeneous and is associated with mutations in at least 40 different genes. Apart from TTNencoding the giant protein Titin, none of these genes have an expected diagnostic yield of more than 5% complicating genetic diagnosis. Whole exome sequencing (WES) is a powerful alternative for the identification of the causal gene, however variant interpretation remains challenging. We report on WES in a large family with autosomal dominant DCM complicated by end stage heart failure and non-sustained ventricular arrhythmias in whom no causative mutation was identified using a targeted gene panel including 28 genes.Methods and results:WES was applied on 2 affected cousins. Stringent filtering of the identified genetic variants was performed including population variant frequencies, in silico analysis, orthologous and paralogous conservation. Subsequently Sanger sequencing was performed for 10 potential disease causing variants in order to confirm the presence of the variant and to evaluate co-segregation. Only one variant in exon 9 of the RBM20 gene (c.2714T > A, p.Met950Lys, NM_001334363) showed full co-segregation in the 7 affected family members resulting in a maximum 2-point LOD score of 2.1 and suggesting this as the pathogenic mutation responsible for the phenotype. Recently mutations in RBM20 have been linked to arrhythmogenic dilated cardiomyopathy caused by defective splicing of the giant sarcomere protein titin and abnormal calcium handling.Conclusions:We report the identification of a novel mutation in RBM20 by WES in a large pedigree with DCM.

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.

We present a new mutation in KCNH2 (c.2038delG) resulting in a frameshift and premature truncation of the IKr channel protein in a large LQTS family with several sudden death cases. This mutation was initially missed by mutation scanning with DHPLC due to allelic dropout and only retrieved after repeat genetic testing with targeted capture and massive parallel sequencing. There was full penetrance of this mutation, only if an individualized QT correction derived from 24-hour Holter data was used. This case again underscores the importance of repeat genetic testing in robust cases of LQTS that remained genotype negative with mutation scanning techniques.

FISH analyses and loss of heterozygosity studies have delineated a commonly deleted region in hematological malignancies flanked byETV6andCDKN1Bon chromosome 12p12.3. The same chromosomal region is also a target for deletions in certain solid tumors. As an initial step toward the cloning of a potential tumor suppressor gene at 12p12.3, we mapped theETV6–CDKN1Bregion physically using bacterial artificial chromosome (BAC) and P1-derived clone (PAC) contigs. The 1.2-Mb high-resolution, contiguous map extends from D12S1095 to D12S929 and consists of 19 PACs and 20 BACs. Pulsed-field gel electrophoresis experiments confirmed the integrity of the clone-based map and identified six CpG islands in the region. A transcript map was generated by performing hybridization selection experiments with the genomic clones, by evaluating known 12p ESTs for their presence in the contig, and by sequence analysis of CpG islands in the region. Altogether evidence was gathered for the presence of the recently publishedLRP6gene and at least seven other new genes in this chromosomal region. TheCLAPS3gene, mapped between D12S391 and D12S358, was reassigned to chromosome 5 since genomic sequencing demonstrated the chromosome 12p sequence to be a pseudogene. Polymorphic CA repeats were identified approximately every 100 kb, which will support future analysis of loss of heterozygosity in tumors. Fluorescencein situhybridization analysis of leukemia patients with del(12p) further refined the commonly deleted segment to 600 kb betweenETV6and D12S358, which apparently excludesCDKN1B.Methylation changes of the CpG islands in theETV6–CDKN1Binterval were assessed by Southern analysis for leukemia patients with hemizygous 12p deletions. A “de novo” methylation was detected only at theLRP6CpG island in 2 of 22 leukemia patients tested and was confirmed by methylation-sensitive PCR and sequencing. The genomic structure ofLRP6was elucidated to allow screening for inactivating mutations, but only intragenic polymorphisms were identified. Hypermethylation of CpG islands associated with gene promoters is reported as a common mechanism for gene silencing and tumor suppressor inactivation. Therefore the consequences of theLRP6CpG island methylation and its role in the observed phenotype need further investigation.