Genetic variation screening of TNNT2 gene in a cohort of patients with hypertrophic and dilated cardiomyopathy

Mutations in troponin T (TNNT2) gene represent the important part of currently identified disease-causing mutations in hypertrophic (HCM) and dilated (DCM) cardiomyopathy. The aim of this study was to analyze TNNT2 gene exons in patients with HCM and DCM diagnosis to improve diagnostic and genetic consultancy in affected families. All 15 exons and their flanking regions of the TNNT2 gene were analyzed by DNA sequence analysis in 174 patients with HCM and DCM diagnosis. We identified genetic variations in TNNT2 exon regions in 56 patients and genetic variations in TNNT2 intron regions in 164 patients. Two patients were found to carry unique mutations in the TNNT2 gene. Limited genetic screening analysis is not suitable for routine testing of disease-causing mutations in patients with HCM and DCM as only individual mutation-positive cases may be identified. Therefore, this approach cannot be recommended for daily clinical practice even though, due to financial constraints, it currently represents the only available strategy in a majority of cardio-centers.     

Structural characterization of the human fast skeletal muscle troponin I gene (TNNI2)

Three troponin I genes have been identified in vertebrates that encode the isoforms expressed in adult cardiac muscle (TNNI3), slow skeletal muscle (TNNI1) and fast skeletal muscle (TNNI2), respectively. While the organization and regulation of human cardiac and slow skeletal muscle genes have been investigated in detail, the fast skeletal troponin I gene has to date only been examined in birds. Here, we describe the structure and complete sequence of the human fast skeletal muscle troponin I gene (TNNI2) and identify putative regulatory elements within both the 5' flanking region and the first intron. In particular, a region containing MEF-2, E-box, CCAC and CAGG elements was identified in intron 1 that closely resembles the fast internal regulatory element (FIRE) of the quail intronic enhancer. We have previously shown that the fast skeletal muscle troponin I gene is located at 11p15.5 and noted potential close linkage with the fast skeletal muscle troponin T gene (TNNT3). Here, we have isolated two independent human PAC genomic clones that contain either TNNI2 or TNNT3 and demonstrate by interphase FISH mapping that they are less than 100 kb apart in the genome. The results demonstrate that the human TNNI2 gene is closely related to its avian counterparts with conserved elements within both the putative promoter and first intron. Our data further confirm close physical linkage of TNNI2 and TNNI3 on 11p15.5.     

Assignment of the slow troponin T (TNNT1) gene to chromosome 19 using polymerase chain reaction

Summary Confirmation that the slow troponin (TNNT1) gene lies on chromosome 19 has been obtained by means of the polymerase chain reaction and somatic cell hybrids.     

A novel nemaline myopathy in the Amish caused by a mutation in troponin T1

The nemaline myopathies are characterized by weakness and eosinophilic, rodlike (nemaline) inclusions in muscle fibers. Amish nemaline myopathy is a form of nemaline myopathy common among the Old Order Amish. In the first months of life, affected infants have tremors with hypotonia and mild contractures of the shoulders and hips. Progressive worsening of the proximal contractures, weakness, and a pectus carinatum deformity develop before the children die of respiratory insufficiency, usually in the second year. The disorder has an incidence of approximately 1 in 500 among the Amish, and it is inherited in an autosomal recessive pattern. Using a genealogy database, automated pedigree software, and linkage analysis of DNA samples from four sibships, we identified an approximately 2-cM interval on chromosome 19q13.4 that was homozygous in all affected individuals. The gene for the sarcomeric thin-filament protein, slow skeletal muscle troponin T (TNNT1), maps to this interval and was sequenced. We identified a stop codon in exon 11, predicted to truncate the protein at amino acid 179, which segregates with the disease. We conclude that Amish nemaline myopathy is a distinct, heritable, myopathic disorder caused by a mutation in TNNT1.     

Associations between 60 SNPs identified by APEX microarray and growth rate,meatiness and selection index in boars

A total of 312 boars (201 Landrace and 111 Large White) were genotyped with a custom-made low throughput genotyping microarray (called SNiPORK) based on array primer extension (APEX) technology. The results were used to association studies between genotyped SNP markers and daily gains, meat content and selection index. Among the 60 SNP markers analyzed, 14 of them showed statistically significant associations between the genotype and the level of at least one trait. In order to find extremely beneficial or unfavorable intergenic diplotype combinations, 5 SNP markers were selected: CAST A499C, MYF6 T255C, PKLR C384T, SFRS1 C1146T and TNNT3 T153C, which showed statistically significant associations at P ≤ 0.01 within one of the traits and the frequency of homozygotes with a minor allele of at least 0.1. Among 10 possible permutations, statistically significant associations were found only for a combination of SNP markers in TNNT3 × SFRS1 genes and, interestingly, for all combinations with SNP located within the calpastatin (CAST) gene commonly known as a gene influencing pork quality traits. This study also found that CAST allele A (which is beneficial for pork tenderness) is also favorable for growth rate. This effect is clearly increased with additive alleles C of myogenic factor MYF6 and troponin T3 (TNNT3) and is decreased with each allele T from serine rich splicing factor 1 (SFRS1) gene. For meat content, the most favorable genotype of calpastatin gene was AC, the effect of which was generally increased with each C allele of MYF6 and TNNT3 and decreased with each allele T from SFRS1 and PKLR (puruvate kinase) genes, respectively. The calpastatin AC genotype seemed to be beneficial for selection index, although in combination with the CC genotype of troponin T3, calpastatin genotypes AA and CC reached higher values. In the case of the combination of genotypes TNNT3 × SFRS1, the most preferable for all analyzed traits is the CC genotype of TNNT3, especially in combination with CT or CC genotypes in the SFRS1 gene. We conclude that searching for interaction effects between candidate SNPs (even of moderate influence) may lead to interesting and valuable findings enabled better understanding and applications of SNPs in pork yield and quality improvement programmes.     

Assignment of the Human Fast Skeletal Troponin T Gene (TNNT3) to Chromosome 11p15.5: Evidence for the Presence of 11pter in a Monochromosome 9 Somatic Cell Hybrid in NIGMS Mapping Panel 2

Human fast skeletal troponin T (TnT_f), the tropomyosin binding component of the multisubunit troponin complex, plays an important role in the Ca²⁺regulation of striated muscle contraction. Specific primers designed from the 3′ end of human TnT_fcDNA were used to amplify an intronic region by polymerase chain reaction (PCR). This TnT_f-specific PCR product was detected from two somatic cell hybrids containing human chromosomes 9 and 11, respectively, in NIGMS mapping panel 2. However, further studies with other somatic hybrid cell lines (Bios Laboratory) localized the TnT_fgene (HGMW-approved symbol TNNT3) only to chromosome 11. This observation was further confirmed by fluorescencein situhybridization with a 12-kb TnT_fgenomic probe generated by extended PCR, showing the sublocalization of the gene to band p15.5 on chromosome 11. This locus is of specific interest, as Beckwith–Wiedemann syndrome and various childhood and adult tumor-related abnormalities have been mapped to this region. The study also indicates the presence of an 11pter region in the NIGMS cell hybrid GM10611, which has previously been reported to contain only human chromosome 9.     

Close physical linkage of human troponin genes: organization, sequence, and expression of the locus encoding cardiac troponin I and slow skeletal troponin T

Based on chromosomal mapping data, we recently revealed an unexpected linkage of troponin genes in the human genome: the six genes encoding striated muscle troponin I and troponin T isoforms are located at three chromosomal sites, each of which carries a troponin I-troponin T gene pair. Here we have investigated the organization of these genes at the DNA level in isolated P1 and PAC genomic clones and demonstrate close physical linkage in two cases through the isolation of individual clones containing a complete troponin I-troponin T gene pair. As an initial step toward fully characterizing this pattern of linkage, we have determined the organization and complete sequence of the locus encoding cardiac troponin I and slow skeletal troponin T and thereby also provide the first determination of the structure and sequence of a slow skeletal troponin T gene. Our data show that the genes are organized head to tail and are separated by only 2.6 kb of intervening sequence. In contrast to other troponin genes, and despite their close proximity, the cardiac troponin I and slow skeletal troponin T genes show independent tissue-specific expression. Such close physical linkage has implications for the evolution of the troponin gene families, for their regulation, and for the analysis of mutations implicated in cardiomyopathy.     

A gene locus for progressive familial heart block type II (PFHBII) maps to chromosome 1q32.2-q32.3

Cardiac conduction defects that are associated with dilated cardiomyopathy (DCM) are generally considered to be sporadic clinical entities, although familial forms of disorders with these clinical features have been identified in a number of families in different countries. An autosomal dominant cardiac disorder characterised by conduction abnormalities and DCM, termed progressive familial heart block type II (PFHBII) (OMIM 140400), has been described in a South African Caucasian family of Northern European descent. Known candidate loci for isolated conduction disorders, isolated DCM and conduction disorders complicated by DCM were excluded from disease causation in this family by linkage analysis, with the exception of the DCM-associated (CMD1D) locus on chromosome 1q32, where a maximum multipoint lod score of 3.7 in the interval between D1S3753 and D1S414, was generated. This region encompassed the troponin T gene (TNNT2), however, genetic fine mapping and haplotype analysis excluded TNNT2 as cause of PFHBII and placed the disease-causative gene within a 3.9 cM (2.85 Mb) interval, flanked by D1S70 and D1S505. Analysis of KCNH1, KIAA0205, LAMB3 and PPP2R5A, which map within the critical interval, indicated that the PFHBII-causative mutation does not lie within the coding regions or splice junctions of these plausible candidate genes. The data indicate the existence of a novel locus involved in the pathogenesis of cardiac conduction abnormalities and DCM.     

Hypertrophic cardiomyopathy: two homozygous cases with "typical" hypertrophic cardiomyopathy and three new mutations in cases with progression to dilated cardiomyopathy

About 10% of cases of hypertrophic cardiomyopathy (HCM) evolve into dilated cardiomyopathy (DCM) with unknown causes. We studied 11 unrelated patients (pts) with HCM who progressed to DCM (group A) and 11 who showed "typical" HCM (group B). Mutational analysis of the beta-myosin heavy chain (MYH7), myosin-binding protein C (MYBPC3), and cardiac troponin T (TNNT2) genes demonstrated eight mutations affecting MYH7 or MYBPC3 gene, five of which were new mutations. In group A-pts, the first new mutation occurred in the myosin head-rod junction and the second occurred in the light chain-binding site. The third new mutation leads to a MYBPC3 lacking titin and myosin binding sites. In group B, two pts with severe HCM carried two homozygous MYBPC3 mutations and one with moderate hypertrophy was a compound heterozygous for MYBPC3 gene. We identified five unreported mutations, potentially "malignant" defects as for the associated phenotypes, but no specific mutations of HCM/DCM.     

心脏肌球蛋白重链基因c.1273G>A突变与肥厚型心肌病的关联分析

为研究中国人家族性肥厚型心肌病(HCM)的致病基因突变位点, 分析基因型与临床表型的相互关系, 文章在1个中国汉族HCM家系中进行心脏肌钙蛋白T (TNNT2) 基因、心脏肌球蛋白结合蛋白C (MYBPC3) 基因和心脏β-肌球蛋白重链 (MYH7) 基因的突变筛查, 聚合酶链式反应(PCR)扩增基因功能区外显子片段并对PCR产物进行测序分析。结果表明: 在该家系接受调查的7名成员中有4名成员携带MYH7基因c.1273G>A杂合突变, 该突变位点位于MYH7基因的14号外显子并使425位的甘氨酸(Gly)转换为精氨酸(Arg)。该突变首次在国内HCM家系中发现, 突变携带者的临床表型在家系内部呈现明显的异质性。该家系成员TNNT2及MYBPC3基因未发现突变且正常对照组相同位置未发现异常。MYH7基因是我国家族性 HCM的致病基因之一, 携带c.1273G>A突变的肥厚型心肌病患者临床表型差异明显, 提示可能有其它因素参与了肥厚型心肌病的发展过程。     

Targeted disruption of the cardiac troponin T gene causes sarcomere disassembly and defects in heartbeat within the early mouse embryo

Cardiac troponin T (cTnT) is a component of the troponin (Tn) complex in cardiac myocytes, and plays a regulatory role in cardiac muscle contraction by anchoring two other Tn components, troponin I (TnI) and troponin C, to tropomyosin (Tm) on the thin filaments. In order to determine the in vivo function of cTnT, we created a null cTnT allele in the mouse TNNT2 locus. In cTnT-deficient (cTnT^−/−) cardiac myocytes, the thick and thin filaments and α-actinin-positive Z-disk-like structures were not assembled into sarcomere, causing early embryonic lethality due to a lack of heartbeats. TnI was dissociated from Tm in the thin filaments without cTnT. In spite of loss of Tn on the thin filaments, the cTnT^−/− cardiac myocytes showed regular Ca²⁺-transients. These findings indicate that cTnT plays a critical role in sarcomere assembly during myofibrillogenesis in the embryonic heart, and also indicate that the membrane excitation and intracellular Ca²⁺ handling systems develop independently of the contractile system. In contrast, heterozygous cTnT^+/− mice had a normal life span with no structural and functional abnormalities in their hearts, suggesting that haploinsufficiency could not be a potential cause of cardiomyopathies, known to be associated with a variety of mutations in the TNNT2 locus.     

The highly conserved COOH terminus of troponin I forms a Ca2+-modulated allosteric domain in the troponin complex

The primary structure of the COOH-terminal region of troponin I (TnI) is highly conserved among the cardiac, slow, and fast skeletal muscle TnI isoforms and across species. Although no binding site for the other thin filament proteins is found at the COOH terminus of TnI, truncations of the last 19-23 amino acid residues reduce the activity of TnI in the inhibition of actomyosin ATPase and result in cardiac muscle malfunction. We have developed a specific monoclonal antibody (mAb), TnI-1, against the conserved COOH terminus of TnI. Using this mAb, isolation of the troponin complex by immunoaffinity chromatography from muscle homogenate and immunofluorescence microscopic staining of myofibrils indicate that the COOH terminus of TnI forms an exposed structure in the muscle thin filament. Binding of this mAb to the COOH terminus of cardiac TnI induced extensive conformational changes in the protein, suggesting an allosteric role of this region in the functional integrity of troponin. In the absence of Ca2+, the binding of troponin C and troponin T to TnI had very little effect on the conformation of the COOH terminus of TnI as indicated by the unaffected mAb affinity for the TnI-1 epitope. However, Ca2+ significantly increased the accessibility of the TnI-1 epitope on TnI in the presence of troponin C and troponin T. The results provide evidence that the COOH terminus is an essential structure in TnI and participates in the allosteric switch during Ca2+ activation of contraction.     

A novel TNNI2 mutation causes Freeman–Sheldon syndrome in a Chinese family with an affected adult with only facial contractures

Distal arthrogryposes (DAs), a clinically and genetically heterogeneous group of disorders characterized by congenital contractures with predominant involvement of the hands and feet, can be classified into at least 12 different forms. These autosomal dominant disorders are of variable expressivity and reduced penetrance. Mutations in sarcomeric protein genes, including troponin I2 (TNNI2), troponin T3 (TNNT3), tropomyosin 2 (TPM2), embryonic myosin heavy chain 3 (MYH3), and myosin binding protein C1 (MYBPC1), have been identified in distal arthrogryposis type 1 (DA1, MIM 108120), type 2B (DA2B, MIM 601680) and type 2A (DA2A)/Freeman–Sheldon syndrome (FSS, MIM 193700). However, mutations causing FSS have only been reported in MYH3. Herein we describe a Chinese DA family whose members meet classical strict criteria for FSS, as well as one member of the family who has isolated facial features consistent with FSS. No disease-causing mutation was found in MYH3. Segregation of microsatellite markers flanking the TNNI2 and TNNT3 genes at 11p15.5 was compatible with linkage. Subsequent sequencing of TNNI2 revealed a novel mutation, c.A493T (p.I165F), located in the C-terminal region, which is critical for proper protein function. This mutation was found to cosegregate with the FSS phenotype in this family, and assessment using SIFT and PolyPhen-2 predicted a damaging effect. To the best of our knowledge, we report the first TNNI2 mutation in classical FSS and describe an atypical adult FSS case with only facial contractures resulting from somatic mosaicism. We infer that DA1, DA2B and FSS represent a phenotypic continuum of the same disorder and provide further genetic evidence for this hypothesis.