Details of 2092 random population samples from India used to study the 5 bp Deletion Polymorphism.

<p>I/D Insertion/Deletion, D/D Deletion/Deletion, I/I Insertion/Insertion.</p

A Novel Arginine to Tryptophan (R144W) Mutation in Troponin T (<i>cTnT</i>) Gene in an Indian Multigenerational Family with Dilated Cardiomyopathy (FDCM)

<div><p>Cardiomyopathy is a major cause of heart failure and sudden cardiac death; several mutations in sarcomeric protein genes have been associated with this disease. Our aim in the present study is to investigate the genetic variations in Troponin T (<i>cTnT</i>) gene and its association with dilated cardiomyopathy (DCM) in south-Indian patients. Analyses of all the exons and exon-intron boundaries of <i>cTnT</i> in 147 DCM and in 207 healthy controls had revealed a total of 15 SNPs and a 5 bp INDEL; of which, polymorphic SNPs were compared with the HapMap population data. Interestingly, a novel R144W mutation, that substitutes polar-neutral tryptophan for a highly conserved basic arginine in <i>cTnT</i>, altering the charge drastically, was identified in a DCM, with a family history of sudden-cardiac death (SCD). This mutation was found within the tropomyosin (<i>TPM1</i>) binding domain, and was evolutionarily conserved across species, therefore it is expected to have a significant impact on the structure and function of the protein. Family studies had revealed that the R144W is co-segregating with disease in the family as an autosomal dominant trait, but it was completely absent in 207 healthy controls and in 162 previously studied HCM patients. Further screening of the proband and three of his family members (positive for R144W mutant) with eight other genes <i>β-MYH7, MYBPC3, TPM1, TNNI3, TTN, ACTC, MYL2</i> and <i>MYL3</i>, did not reveal any disease causing mutation, proposing the absence of compound heterozygosity. Therefore, we strongly suggest that the novel R144W unique/private mutant identified in this study is associated with FDCM. This is furthermore signifying the unique genetic architecture of Indian population.</p></div

Three SNPs, [rs3729547 (C/T), rs3729843 (G/A), rs3729842 (C/T)], (Table.1; Fig.1D, 1G, 1J) in TNNT2 gene observed in the present study, which were about 2.0 kb apart had shown high Linkage disequilibrium (LD).

<p>The bright red color indicates very strong LD (LOD = 2D′ = 1), white color no LD (LOD<2D′<1), and pink (LOD = 2D′<1) and blue (LOD<2D′ = 1) indicate intermediate LD (the standard color scheme is used to display LD). The values in the LD blocks show the r2 values in percentages or multiplied by 100.</p

A. The genotype frequency of 5(<i>cTnT</i>) gene of DCM, HCM and controls in the present study were compared with the randomly selected individuals from 19 states of India.

<p><b>B</b>. The Allele frequency of 5 bp polymorphism observed in Troponin T (<i>cTnT</i>) gene of DCM, HCM and controls in the present study were compared with the randomly selected individuals from 19 states of India. Individuals from Rajasthan showed high frequency of Deletion allele, whereas the individuals of northeastern states and HER-YORUB-PANEL of Hap Map population showed high frequency of Insertion allele. AP, Andhra Pradesh; KA, Karnataka; TN, Tamil Nadu; MP, Madhya Pradesh; UP, Uttar Pradesh; MH, Maharashtra; GJ, Gujarat; RJ, Rajasthan; CG, Chhattisgarh; WB, West Bengal; HR, Haryana; NL, Nagaland; MZ, Mizoram; JH, Jharkhand; UK, Uttaranchal; JK, Jammu & Kashmir; OD, Orissa; AI, Andaman Islands; NI, Nicobar Islands; AS, Arunachal Pradesh; KE, Kerala; CN, normal controls; HC, Hypertrophic Cardiomyopathy; DC, Dilated Cardiomyopathy.</p

Clinical details of the family members carrying R144W mutation.

<p>SCD- Sudden cardiac death; NYHA-New York Heart Association; LVIDd- left ventricular internal diastolic dimension; LVEF- left ventricular ejection fraction.</p

The hn-RNP's and SR-proteins binding site sequences in controls and DCM as predicted by “Splicing Rainbow” tool.

<p>The hn-RNP's and SR-proteins binding site sequences in controls and DCM as predicted by “Splicing Rainbow” tool.</p

Chi-square and Fisher Exact Probability Test for SNP's found in this study.

<p>*SNP- single nucleotide polymorphism, DCM- Dilated cardiomyopathy.</p

1A-1M: Electropherograms showing SNPs of cTnT gene, observed in the present study on South Indian dilated cardiomyopathy patients.

<p>Mutation sites were shown with arrows. <b>Fig. 1A</b><b>. R144W</b> [rs483352832]: Electropherogram (arrow) showing a novel missense mutation (R144W) at the nucleotide position g.14351 of human cTnT gene. The upper lane showing sequences of homozygous wild type allele ‘C’ in a control individual. The middle and the lower lanes were showing the sequences of heterozygous (C/T  = Y) alleles in two individuals (a DCM patient and his relative, respectively). <b>Fig. 1B. G>A [IVS11-1G]</b> [rs483352835]: Electropherogram (arrow) showing a variant at splice acceptor site of human <i>cTnT</i> gene at nucleotide position g.16283, the electropherogram of a upper lane showing sequence of heterozygous (A/G = R) variant in a DCM patient, the lower lane showing sequence of control individual having wild type allele ‘G’ (homozygous). <b>Fig. 1C. N164N</b> [rs483352833]: Electropherogram (arrow) showing a novel synonumous mutation (N164) at the nucleotide position g.15304 of human <i>cTnT</i> gene in 2 DCM patients. The upper lane shows the sequences of heterozygous (C/T  = Y) transition in a DCM patient. The middle lane was the sequences of a control individual showing the wild type allele ‘C’ (homozygous). The lower lane sequences showing heterozygous (C/T  = Y) transition was from a 2^nd DCM patient. <b>Fig. 1D</b>. [rs3729842]: Electropherogram showing (arrow) a single nucleotide polymorphism at the nucleotide position g.10636 (C/T = Y) in intron 5 of human <i>cTnT</i> gene. The upper and the middle lanes were sequences showing heterozygous (C/T = Y) transition in DCM patients, the lower lane showing homozygous wild type (C/C) allele in a control individual. <b>Fig. 1E</b>. [rs3729845]: Electropherogram showing (arrow) at the nucleotide position g.13011 of human <i>cTnT</i> gene. The upper lane showing sequences of the heterozygous (A/G  = R) transition, and the lower lane showing homozygous wild type (G/G) allele of a control. <b>Fig. 1F</b>. [rs1104859]: Electropherogram showing (arrow) at the nucleotide position <b>g.11643</b> (A/C = M) in Intron 11 of human <i>cTnT</i> gene. The upper lane sequences showing the heterozygous (A/C = M) transversion, the middle lane showing homozygous wild type (G/G), and the lower lane sequences showing mutant homozygous (C/C) allele. <b>Fig.1G</b><b>. SNP-rs3729843</b>: Electropherogram showing (arrow) a SNP at the nucleotide position g.10822 (G/A = R) in intron 5 of human <i>cTnT</i> gene. The upper lane sequences showing mutant homozygous (A/A) allele. The middle lane sequences showing heterozygous (G/A = R) transition allele, and the lower lane showing sequences of homozygous wild type (G/G) allele in a control individual. <b>Fig. 1H</b>. [rs45576939]: Electropherogram showing (arrow) a novel mutation G>A at nucleotide position g.10370 in intron 4 of human <i>cTnT</i> gene, the upper lane displaying homozygous mutant (A/A) allele, and the lower lane showing sequences of a wild type allele (G/G). <b>Fig. 1I</b>. [rs45576635]: Electropherogram showing (arrow) a SNP at the nucleotide position g.14492 (C/T = Y) in intron 15 of human <i>cTnT</i> gene, the upper and the middle lanes sequences displaying heterozygous (C/T = Y) transition, and the lower lane sequences showing homozygous wild type (C/C) allele. <b>Fig. 1J</b>. [rs3729547]: Electropherogram showing (arrow) a polymorphic variant at the nucleotide position g.13424 of human <i>cTnT</i> gene, the upper lane displaying sequences of the heterozygous (C/T  = Y) transition, the middle lane sequences showing homozygous wild type (C/C) allele, and the lower lane displaying sequences of the homozygous mutant (T/T) allele. <b>Fig. 1K</b>. [rs483352834]. Electropherogram (arrow) showing a novel mutation at the nucleotide position g.15179 C>T in intron 11 of human <i>cTnT</i> gene, the upper lane displaying sequences of a DCM patient having heterozygous (C/T) transition, and the lower lane exhibiting sequences of a control individual having homozygous wild type allele (C/C). <b>Fig. 1L. K276K</b>. [rs483352836]: Electropherogram (arrow) exhibiting novel synonumous (K276) variant at the nucleotide position g.19429 of human <i>cTnT</i> gene in a DCM patient, the DCM patient displaying heterozygous (G/A  = R) transition. <b>Fig. 1M</b>. Sequence electropherogram showing (CTTCT) 5 bp Polymorphism. <b>Ma</b>. Presence of two copies of CTTCT (Insertion/Insertion – homozygous insertion) in both the chromosomes, <b>Mb</b>. Absence of one copy of CTTCT (Deletion/Deletion – homozygous deletion in both the chromosomes, <b>Mc</b>. Presence of 2 copies of CTTCT in one chromosome and presence of one copy of CTTCT in another chromosome (Insertion/deletion – heterozygous allele). g.6626-30 (5 bp).</p

The amino acid arginine at residue 144 in human Troponin T (<i>cTnT</i>) is highly conserved across many species, including mouse, rat, chicken, rabbit, sheep and bovine.

<p>The amino acid arginine at residue 144 in human Troponin T (<i>cTnT</i>) is highly conserved across many species, including mouse, rat, chicken, rabbit, sheep and bovine.</p

The codon usage in human cTnT (Genbank NO. NM_000364) gene.

<p>Chr- Chromosome, SNP- Single Nucleotide Polymorphism, Ref- References, Nt. Nucleotide, A.A-Amino Acid.</p