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. 2017 Aug;38(6):1232-1240.
doi: 10.1007/s00246-017-1650-5. Epub 2017 Jun 12.

Hypoplastic Left Heart Syndrome Sequencing Reveals a Novel NOTCH1 Mutation in a Family with Single Ventricle Defects

Matthew D Durbin  1 , Adrian G Cadar  2 , Charles H Williams  2 , Yan Guo  3 , David P Bichell  4 , Yan Ru Su  2 , Charles C Hong  5   6   7
Affiliations

Hypoplastic Left Heart Syndrome Sequencing Reveals a Novel NOTCH1 Mutation in a Family with Single Ventricle Defects

Matthew D Durbin et al. Pediatr Cardiol. 2017 Aug.

Abstract

Hypoplastic left heart syndrome (HLHS) has been associated with germline mutations in 12 candidate genes and a recurrent somatic mutation in HAND1 gene. Using targeted and whole exome sequencing (WES) of heart tissue samples from HLHS patients, we sought to estimate the prevalence of somatic and germline mutations associated with HLHS. We performed Sanger sequencing of the HAND1 gene on 14 ventricular (9 LV and 5 RV) samples obtained from HLHS patients, and WES of 4 LV, 2 aortic, and 4 matched PBMC samples, analyzing for sequence discrepancy. We also screened for mutations in the 12 candidate genes implicated in HLHS. We found no somatic mutations in our HLHS cohort. However, we detected a novel germline frameshift/stop-gain mutation in NOTCH1 in a HLHS patient with a family history of both HLHS and hypoplastic right heart syndrome (HRHS). Our study, involving one of the first familial cases of single ventricle defects linked to a specific mutation, strengthens the association of NOTCH1 mutations with HLHS and suggests that the two morphologically distinct single ventricle conditions, HLHS and HRHS, may share a common molecular and cellular etiology. Finally, somatic mutations in the LV are an unlikely contributor to HLHS.

Keywords: Congenital heart disease; HAND1 and NOTCH1; Hypoplastic left heart syndrome; Somatic mutation; Whole exome sequencing.

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Figures

Figure 1
Figure 1. No mutation was found in HAND1 in left ventricle of HLHS patients
(A) Using the Vanderbilt Congenital Heart Disease Tissue Bank, we isolated fresh frozen left ventricular (LV) and right ventricular (RV) tissue from 14 HLHS patients. (B) We amplified and sequenced a 1996 bp segment including HAND1’s entire coding region and a majority of the noncoding region. This included the Exon 1, which encodes the bHLH domain, previously reported to be the site of a recurrent somatic mutation (A126fs) in 24 of 31 HLHS LVs examined (17). (C) Shown are sequences corresponding to A126 in 9 HLHS LV and 5 HLHS RV samples. There were no mutations detected in this region or elsewhere in the HAND1 coding region.
Figure 2
Figure 2. Exome wide search for somatic mutation in four HLHS patients
Right, Schema for validation of putative somatic mutations identified by WES. We examined 10 tissue samples including 4 patients’ LVs and PBMCs, and additional outflow tract (OFT) tissue in 2 of those patients. WES at the 100× coverage identified 271 sequence discrepancies between LV or OFT and the corresponding PBMCs. All were in areas of low sequence coverage. 71 discrepant sequences had a read depth > 4,0. Of these, only 24 had MAF <1%. Sanger sequencing of these 24 samples failed to confirm a somatic mutation. We also conducted Sanger sequencing on 40 additional discrepant variants based on biological plausibility (see text), and found no somatic mutation. Left, breakdown of WES results by patients.
Figure 3
Figure 3. Search for mutations in genes previously implicated in HLHS reveals a novel truncation mutation in NOTCH1
(A) In WES of 10 samples of HLHS patients’ left ventricular tissue, outflow tract, and blood, we focused on 20 genes previously associated with to HLHS or ventricular development. We detected total of 41 SNVs (Table 2). Of these, 10 were nonsynonymous changes, and only 1 had a MAF <1%. This mutation was a premature stop codon in the NOTCH1 gene. (B) Sanger sequencing of patient 111’s PBMC confirmed the mutation, a heterozygous A to C transversion, creating a stop codon at C1554. (C) The predicted mutant Notch1 protein was truncated at the Lin/NOTCH repeat (LNR) region of the extracellular domain, hence lacking the transmembrane (TM) and intracellular signaling domains. Also shown are RBP J-associated molecule (RAM) domain, ankyrin (ANK) domain and PEST domain.
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