Hypertrophic cardiomyopathy (HCM) is a heterogeneous autosomal dominant cardiac disorder with a prevalence of 1 in 500. Over 450 different pathogenic mutations in at least 16 genes have been identified so far. The large allelic and genetic heterogeneity of HCM requires high-throughput, rapid, and affordable mutation detection technologies to efficiently integrate molecular screening into clinical practice. We developed a custom DNA resequencing array that contains both strands of all coding exons (160), splice-site junctions, and 5'UTR regions of 12 genes that have been clearly implicated in HCM (MYH7, MYBPC3, TNNT2, TPM1, TNNI3, MYL3, MYL2, CSRP3, PLN, ACTC, TNNC1, and PRKAG2). We analyzed a first series of 38 unrelated patients with HCM (17 familial, 21 sporadic). A total of 953,306 bp across the 38 patients were sequenced with a mean nucleotide call rate of 96.92% (range: 93-99.9%). Pathogenic mutations (single nucleotide substitutions) in MYH7, MYBPC3, TNNI3, and MYL3 (six known and six novel) were identified in 60% (10/17) of familial HCM and 10% of sporadic cases (2/21). The high-throughput HCM resequencing array is the most rapid and cost-effective tool for molecular testing of HCM to date; it thus has considerable potential in diagnostic and predictive testing, and prognostic stratification.

Overall, mutations on both alleles of DNAH5 were identified in 15% of our clinically heterogeneous cohort of patients. Although genetic alterations remain to be identified in most patients, DNAH5 is to date the main PCD gene.

Background: Primary ciliary dyskinesia (PCD) is a rare recessive hereditary disorder characterized by dysmotility to immotility of ciliated and flagellated structures. Its main symptoms are respiratory, caused by defective ciliary beating in the epithelium of the upper airways (nose, bronchi and paranasal sinuses). Impairing the drainage of inhaled microorganisms and particles leads to recurrent infections and pulmonary complications. To date, 5 genes encoding 3 dynein protein arm subunits (DNAI1, DNAH5 and DNAH11), the kinase TXNDC3 and the X-linked RPGR have been found to be mutated in PCD. Objectives: We proposed to determine the impact of the DNAI1 gene on a cohort of unrelated PCD patients (n = 104) recruited without any phenotypic preselection. Methods: We used denaturing high-performance liquid chromatography and sequencing to screen for mutations in the coding and splicing site sequences of the gene DNAI1. Results: Three mutations were identified: a novel missense variant (p.Glu174Lys) was found in 1 patient and 2 previously reported variants were identified (p.Trp568Ser in 1 patient and IVS1+2_3insT in 3 patients). Overall, mutations on both alleles of gene DNAI1 were identified in only 2% of our clinically heterogeneous cohort of patients. Conclusion: We conclude that DNAI1 gene mutation is not a common cause of PCD, and that major or several additional disease gene(s) still remain to be identified before a sensitive molecular diagnostic test can be developed for PCD.

Although next-generation sequencing will replace all large-scale sequencing platforms for inherited cardiac disorders in the near future, this HCM resequencing array is currently the most rapid, cost-effective and reasonably efficient technology for first-tier mutation screening of HCM in clinical practice. Because of its design, the array is also an appropriate tool for initial screening of other inherited forms of cardiomyopathy.

Mendelian cardiomyopathies and arrhythmias are characterized by an important genetic heterogeneity, rendering Sanger sequencing very laborious and expensive. As a proof of concept, we explored multiplex targeted high-throughput sequencing (HTS) as a fast and cost-efficient diagnostic method for individuals suffering from Mendelian cardiac disorders. We designed a DNA capture assay including all exons from 130 genes involved in cardiovascular Mendelian disorders and analysed simultaneously four samples by multiplexing. Two patients had familial hypertrophic cardiomyopathy (HCM) and two patients suffered from long QT syndrome (LQTS). In patient 1 with HCM, we identified two known pathogenic missense variants in the two most frequently mutated sarcomeric genes MYH7 and MYBPC. In patient 2 with HCM, a known acceptor splice site variant in MYBPC3 was found. In patient 3 with LQTS, two missense variants in the genes SCN5A and KCNQ were identified. Finally, in patient 4 with LQTS a known missense variant was found in MYBPC3, which is usually mutated in patients with cardiomyopathy. Our results showed that multiplex targeted HTS works as an efficient and cost-effective tool for molecular diagnosis of heterogeneous disorders in clinical practice and offers new insights in the pathogenesis of these complex diseases.