Novel missense mutations (p.T596M and p.P1797H) in NOTCH1 in patients with bicuspid aortic valve

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Abstract

The bicuspid aortic valve (BAV) is the most common congenital cardiac malformation, occurring in 1–2% of the population. In a recent report, mutations in NOTCH1 a signaling and transcriptional regulator have been shown to cause BAV in two families. This study provides data on systematic sequencing in search for novel mutations in NOTCH1 gene in a large sample BAV. For the first time, we report results of a systematic mutation-analysis based on DNA-sequencing of all coding exons and adjacent splice consensus sequences of NOTCH1 gene. Our analyses revealed 57 NOTCH1 sequence variants. Twenty-one variants are located within exons and 36 within intronic or 5′-UTR sequences. Thirty-five variants were described previously as polymorphisms. The remaining 22, however, were neither listed in public SNP databases nor in the literature and were therefore considered novel. Seventeen variants were found only once (MAF = 1%), of these 15 were novel. Two sequence variants led to amino acid substitutions (p.T596M and p.P1797H) and are located in highly conserved regions of the NOTCH1 protein. In addition, these two mutations could not be detected in at least 327 healthy controls by using RFLP-analysis. The functional relevance of the other 13 novel and rare variants could not be proven without further functional examination. In this study, we provide a new evidence that the mutations in the NOTCH1 gene may trigger the underlying mechanism causing the valve calcification, especially in BAV. In conclusion, NOTCH1 gene mutations do not only play a role in familiar BAV, but can also be observed in approximately 4% of sporadic cases.

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Material and methods

DNA samples. Genomic DNA was extracted from peripheral blood cells or calcified aortic tissues obtained during aortic valve replacement surgery from 48 patients with BAV (mean age 53.4 ± 13.9 years; 40 male/8 female) using the QIAamp DNA Mini-Kit according to the manufacturer’s recommendations (Qiagen, Hilden, Germany). All patients were clinically evaluated for evidence of BAV, classified as fusion of right-coronary, left-coronary leaflets (77.1%), and others (22.9%). Clinical characteristics of

Results

We re-sequenced all 34 coding exons including adjacent intronic as well as 5′ and 3′ untranslated sequences from the human NOTCH1 gene in 48 patients with sporadic BAV in order to screen for NOTCH1 variants that might cause BAV.

In total we identified 57 heterozygous variants in the analyzed patients (Table 3). Twenty-one sequence variants are located within exons and 36 within intronic or 5′-UTR sequences. Thirty-five variants were described previously as polymorphisms. The remaining 22,

Discussion

The importance of genetic factors involved in the development of aortic valve disease has become more apparent. Recently, the most common congenital cardiac malformation, the bicuspid aortic valve (BAV), was described as a genetic disorder, which occurs in 1–2% of the general population [2], [7], [9], [15], [16]. Recently, Garg et al., reported mutations in NOTCH1 gene in two families with BAV (R1108X; H1505del) that were associated with the calcification phenotype in BAV [13]. The study by

Acknowledgments

We are grateful to all our patients with BAV for their help and contribution to this study. This work was done in collaborations supported by: Deutsche Forschungsgemeinschaft (Schu672/9-1, Schu672/10-1, Schu672/12-1, and Schu672/14-1) and Bundesministerium für Bildung und Forschung (National Genome Network 2 (01GS0418) H.S., J.E.).

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