Screening of all the exons including the exon-intron boundaries of the TNNI3
gene in 101 individuals with HCM (Table) along with 160 healthy controls from India revealed a total of 16 mutations, including 15 SNPs, and a 4
bp deletion/insertion polymorphism (Table). Of the 15 SNPs, 7 were exonic (one novel, 3 reported non-synonymous and 3 synonymous mutations), and 9 were intronic mutations (Table). Interestingly, we found three heterozygous arginine to glutamine (R to Q) substitution at 3 positions 98, 141 and 162 in TNNI
3 FigureA, B, C, of these R98Q in exon 6 of TNNI3
gene is novel (FigureA) observed in a 28 years old severe hypertrophic obstructive cardiomyopathy patient (HOCM) with interventricular septum (IVS) thickness of 25
mm and his 5 years old asymptomatic son, with the family history of sudden cardiac deaths (Figure). The dominant R141Q mutation in exon 7 of TNNI3
gene (FigureB), lies within the “minimum inhibitory sequence” (residues 137 to 148) region, was observed in two individuals with severe familial asymmetric septal hypertrophy (ASH+), with interventricular septum (IVS) thickness of 25 and 28
mm, respectively. The dominant R162Q mutation (FigureC) in exon 7 of TNNI3
gene was observed in an individual with severe asymmetric septal hypertrophy (ASH) with mean thickness of 29
mm had abnormal echocardiogram/ECG. Screening for this mutation (R162Q) in all the available family members (Figure) revealed its presence in 9 individuals (FigureC). Seven out of 9 individuals with R162Q mutation showed allelic heterogeneity with having a synonymous mutation at g.4797: G
A: E179E (FigureF) in exon 7, which replaces a very frequent codon (GAG: 85%) with rare codon (GAA: 14%) (Table). Four out of 7 Individuals with allelic heterogeneity (R162Q and E179E) (FigureC, F) had presented with severe septal hypertrophy (ASH++) with the mean thickness of 27, 28, 29, 32
mm and the ECGs were abnormal in all the four individuals. History of sudden cardiac death was also been recorded in this family (Figure).
Clinical features exhibited by hypertrophic cardiomyopathy (HCM) patients
Mutations observed in troponin I (TNNI3) gene of the cases/controls
Figure 1 (A - F). Sequence electropherograms of TNNI3 gene. Upper panel representing the control sequences, whereas the lower panel showing the mutations observed in the HCM patients. The mutations sites are shown with arrows. A. Novel missense heterozygous mutation (more ...)
A pedigree of a hypertrophic cardiomyopathy (HCM) family with R98Q mutation in the exon 6 of cardiac troponin ITNNI3gene is depicted.
A pedigree of a four-generation hypertrophic cardiomyopathy (HCM) family with R162Q and E179E mutations in the exon 7 of cardiac (TNNI3)Troponin I gene.
The codon usage in human cTnI (GenBank No. NM_000363) gene
T transition resulting in the replacement of proline with arginine (P82R) in exon 5, lies within the troponin T binding domain (61-112), was observed in 2 HCM patients and a control individual. A total of three synonymous mutations (g.2560; G
T; g.2563; C
A; g.4797; G
A) were observed exclusively in HCM patients (Tables
, ). The G
T mutation at g.2560 (R68R), observed in 4 HCM patients, replaces a frequent codon (CGG: 24%) with a less frequent one (CGT: 4%) (Table). The C
A mutation at g.2563 (R69R), observed in 2 HCM patients, replaces the frequent codon (CGC: 36%) with rare codon (CGA: 12%) (Table). The 4
bp deletion/insertion polymorphism in TNNI3
gene was observed almost in equal frequency in both the patients and the controls, suggesting that it was not associated with HCM.
analysis of 2 novel SNPs using Splicing Rainbow tool predicted abnormal splicing pattern (Table) by removing or creating binding sites for hnRNPs or the SR proteins (http://www.ebi.ac.uk/asd-srv/wb.cgi?method
). A novel splice acceptor site mutation at g.2653; G
A (Table; Figure), was found exclusively in a HCM patient, had been predicted to abort a binding site for hnRNP.U1
and one more novel mutation at g.4003 C
T (Table; Figure), was observed exclusively in the two severe HCM patients, revealed a drastic change in the binding sites for hnRNPs and SR proteins (2 sites in hnRNPs and 3 sites in SR proteins). The disturbed binding sites due to g.4003 C
T mutation were hnRNP-E1
, SRp20, SC35, U2AF65 (Table; Figure), further emphasize its regulatory role, however it needs further investigation. In addition to the novel mutations, we have also observed SNPs reported elsewhere; rs11667847, rs3729836, rs3729837, rs11671293, rs3729838, rs3729711, rs3729841. The allele frequencies of the SNPs were comparable to the HapMap populations (http://www.HapMap.org
The hn RNPs and SR proteins binding site sequences in normal and mutant as predicted by “Splicing Rainbow” tool
Our concern is that the difference in frequencies of the alleles observed between the cases and controls are associated with disease or a difference unrelated to disease arising from underlying genetic differences between the populations from which the case and control samples were drawn: ‘population stratification’. In order to maximize our chances detecting such stratification, we genotyped cases and control samples using a panel of 50 ancestry-informative markers (AIMs) for inferring ancestry [7
] and performed a principal components analysis on the data together with HapMap samples of Chinese, European, Yoruban [9
], and found no significant difference in ancestry between cases and controls (Figure). On the other hand, the Chinese, European, Yoruban, the cases/controls were making clusters among themselves, confirming that the 50 AIMs are sufficient for detecting whether or not ancestry differences along this axis are present. Thus, the population stratification along the axis was ruled out as the cause of the disease association.