Background-Mutations in the plakophilin-2 gene (PKP2) have been found in patients with arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVC). Hence, genetic screening can potentially be a valuable tool in the diagnostic workup of patients with ARVC. Methods and Results-To establish the prevalence and character of PKP2 mutations and to study potential differences in the associated phenotype, we evaluated 96 index patients, including 56 who fulfilled the published task force criteria. In addition, 114 family members from 34 of these 56 ARVC index patients were phenotyped. In 24 of these 56 ARVC patients (43%), 14 different (11 novel) PKP2 mutations were identified. Four different mutations were found more than once; haplotype analyses revealed identical haplotypes in the different mutation carriers, suggesting founder mutations. No specific genotype-phenotype correlations could be identified, except that negative T waves in V 2 and V 3 occurred more often in PKP2 mutation carriers (PϽ0.05). Of the 34 index patients whose family members were phenotyped, 23 familial cases were identified. PKP2 mutations were identified in 16 of these 23 ARVC index patients (70%) with familial ARVC. On the other hand, no PKP2 mutations at all were found in 11 probands without additional affected family members (PϽ0.001). Conclusions-PKP2 mutations can be identified in nearly half of the Dutch patients fulfilling the ARVC criteria. In familial ARVC, even the vast majority (70%) is caused by PKP2 mutations. However, nonfamilial ARVC is not related to PKP2. The high yield of mutational analysis in familial ARVC is unique in inherited cardiomyopathies. (Circulation.
Mutation detection through exome sequencing allows simultaneous analysis of all coding sequences of genes. However, it cannot yet replace Sanger sequencing (SS) in diagnostics because of incomplete representation and coverage of exons leading to missing clinically relevant mutations. Targeted next-generation sequencing (NGS), in which a selected fraction of genes is sequenced, may circumvent these shortcomings. We aimed to determine whether the sensitivity and specificity of targeted NGS is equal to those of SS. We constructed a targeted enrichment kit that includes 48 genes associated with hereditary cardiomyopathies. In total, 84 individuals with cardiomyopathies were sequenced using 151 bp paired-end reads on an Illumina MiSeq sequencer. The reproducibility was tested by repeating the entire procedure for five patients. The coverage of ≥30 reads per nucleotide, our major quality criterion, was 99% and in total ∼21,000 variants were identified. Confirmation with SS was performed for 168 variants (155 substitutions, 13 indels). All were confirmed, including a deletion of 18 bp and an insertion of 6 bp. The reproducibility was nearly 100%. We demonstrate that targeted NGS of a disease-specific subset of genes is equal to the quality of SS and it can therefore be reliably implemented as a stand-alone diagnostic test.
We identified, by homozygosity mapping, a novel locus on 10q21.3-q22.1 for Goldberg-Shprintzen syndrome (GOSHS) in a consanguineous Moroccan family. Phenotypic features of GOSHS in this inbred family included microcephaly and mental retardation, which are both central nervous system defects, as well as Hirschsprung disease, an enteric nervous system defect. Furthermore, since bilateral generalized polymicogyria was diagnosed in all patients in this family, this feature might also be considered a key feature of the syndrome. We demonstrate that homozygous nonsense mutations in KIAA1279 at 10q22.1, encoding a protein with two tetratrico peptide repeats, underlie this syndromic form of Hirschsprung disease and generalized polymicrogyria, establishing the importance of KIAA1279 in both enteric and central nervous system development.
Biallelic truncating mutations in the newly identified gene ALPK3 give rise to severe, early-onset cardiomyopathy in humans. Our findings highlight the importance of transcription factor pathways in the molecular mechanisms underlying human cardiomyopathies.
This novel LMNA deletion causes a distinct, highly malignant cardiomyopathy with early-onset primary cardiac fibrosis likely due to an effect of the shortened mutant protein, which secondarily leads to arrhythmias and end-stage cardiac failure.
Natural isolates of Bacillus subtilis are often difficult to transform due to their low genetic competence levels. Here we describe two methods that stimulate natural transformation. The first method uses plasmid pGSP12, which expresses the competence transcription factor ComK and stimulates competence development about 100-fold. The second method stimulates Campbell-type recombination of DNA ligation mixtures in B. subtilis by the addition of polyethylene glycol. We employed these novel methods to study the regulation of the synthetases for the lipopeptide antibiotics mycosubtilin (myc) and surfactin (srfA) in B. subtilis strain ATCC 6633. By means of lacZ reporter fusions, it was shown that the expression of srfA is >100 times lower in strain ATCC 6633 than in the laboratory strain B. subtilis 168. Expression of the myc operon was highest in rich medium, whereas srfA expression reached maximal levels in minimal medium. Further genetic analyses showed that the srfA operon is mainly regulated by the response regulator ComA, while the myc operon is primarily regulated by the transition-state regulator AbrB. Although there is in vitro evidence for a synergistic activity of mycosubtilin and surfactin, the expression of both lipopeptide antibiotics is clearly not coordinated.
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