Kerala Heart Journal - Om Shankar

Kerala Heart Journal 2017; 7(1):1  Original Article

Mutation of Delta-Sarcoglycan Gene in   Dilated Cardiomyopathy Patients in North India Population

Om Shankar1 , Prasenjit Bose2, Barkha Singh3  Rashmi 3 and Royana Singh3                                  

 

1Department of Cardiology and 3 Anatomy, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India

2Department of Anatomy, Raipur Institute of Medical Sciences, Raipur, Chhattisgarh, India

 Corresponding Author:  Dr. Royana Singh, Professor, Department of Anatomy, Institute of Medical Sciences,    Banaras Hindu University, Varanasi, Uttar Pradesh, India

 Phone no: 9450545650

 Email:singhroyana@rediffmail.com. 



Abstract

 

Aim: DCM is a major cause of morbidity and mortality which has a prevalence of one case out of 2500 individuals with an incidence of 7/100,000/year. The chief causes of DCM are heterogeneous including myocarditis, drug toxicity, and ischemia-induced, metabolic, mitochondrial, and genetic abnormalities. The study aims at the illustration of the mutation of the Delta sarcoglycan gene as a causative factor for DCM.

Methods: Primers were designed for the coding region of δ-sarcoglycan gene. The assessment of DCM was based on strict diagnostic criteria requiring the presence of depressed left ventricular systolic function, associated with left ventricular dilation. DNA was extracted from peripheral blood samples. The PCR was carried out followed by DNA sequencing.

Result: Frame shift (deletion of T at position 95) and missense mutations (V140G) and (V156L) in the delta sarcoglycan was observed in patients with dilated cardiomyopathy.

Conclusion: Novel mutations in the δ-sarcoglycan gene maybe associated with DCM in north Indian population 


Key
words: Delta sarcoglycan, Dilated cardiomyopathy, Left ventricle, Right ventricle, Dystrophin-glycoprotein complex
 Abbreviations:
SGCD gene- Sarcoglycan delta gene
DCM Dilated Cardiomyopathy

DGC- Dystrophin Glycoprotein Complex

LGMD -
Limb girdle muscular dystrophy
Chr Chromosome

Ex - Exon

 


INTRODUCTION

Dilated cardiomyopathy is a disease of cardiac muscle, characterized by dilatation and impaired contractility of the left or right ventricles, with progressive development of congestive heart failure. The frequency of DCM was underestimated, because of the lack of diagnostic indices for early diagnosis in asymptomatic relatives and the limited genetic penetration of the disease1,2,3.The DCM incidence is 2.45 cases per 100,000 population4.  The sarcoglycans (α, β, γ &δ) are a group of trans-membrane proteins that interact with dystrophin, they are primarily found in heart and skeletal muscle. The protein is encoded by the SGCD gene (Sarcoglycan delta )  and the gene is one of the four known components of the sarcoglycan complex, which is a subcomplex of the dystrophin glycoprotein complex (DGC).  

Mutations in any of the sarcoglycans may produce an autosomal recessive Limb girdle muscular dystrophy (LGMD) phenotype of progressive early onset muscle weakness, with significant DCM5. Two genes have been identified for the X-linked forms (dystrophin and tafazzin), whereas three other genes (actin, lamin A/C and desmin) cause autosomal dominant DCM. Hypothesizing that DCM is a disease of the cytoskeleton and sarcolemma, focused on candidate genes whose products are found in these structures, the screening of the human δ-sarcoglycan gene, a member of the DGC was  done.

METHODS

This study was approved by the Ethical Institutional Committee. Written informed consent was obtained from all participants in accordance with requirements of the ethical committee. Patients and their family members were evaluated by history taking and physical examination. Familial DCM was diagnosed according to previously described and strict diagnostic criteria6,7,8,9, requiring the presence of depressed left ventricular systolic function (fractional shortening <25% and/or ejection fraction <45%), associated with left ventricular dilation (left ventricular end-diastolic diameter >117% of the predicted value corrected for the age and body surface area)10, in the absence of any known cause of heart disease. Exclusion criteria moderate to severe arterial hypertension, coronary artery disease, arrhythmogenic right ventricular dysplasia, high rate supraventricular arrhythmia and cor-pulmonale. For each DCM index patient, a detailed two to three generation pedigree was constructed. A noninvasive familial screening was offered, regardless of family history and then performed in all available relatives by means of clinical examination, electrocardiography and echocardiography. Relatives showing signs of myocardial disease underwent full invasive evaluation. Blood samples were taken from all index patients and relatives and they were examined of routine clinical workup. Seven familial and sporadic patients ranging in age from 2 years to 14 years (mean 5.85 years) with clinically apparent DCM presenting with heart failure and evaluated by history taking and physical examination, 12-lead electrocardiography and trans-thoracic echocardiography for DCM were identified. For the genetic evaluation,  DNA was extracted from peripheral blood samples according to the Sodium perchlorate method (Salting out method)  The SGCD gene  located on the long (q) arm of chromosome 5 between positions 33 and 34. The SGCD gene contains 8 Exons (coding) and 2 Introns (noncoding) region. Primers were designed to amplify DNA across each of the Exons including Exon 9. Gel results of amplified genomic DNA of Exon 3 to Exon 9 was collected (24 DCM and 27 Controls) and subsequently 7 infantile DCM patients and 12 Controls were selected. Sequencing analysis was done to determine polymorphism/mutations in all the patients and control for Exon 3-9.

RESULTS

            A male child of 5 years age was detected with a familial history of elder brother who died of similar symptoms. DNA sequence analysis identified a single base change, 156021981T>G in Exon 6, which changes the amino acid at codon 140 from Valine to Glycine (V140G). Provean and polyphen studies showed that the polymorphic site is deleterious. Both the amino acids were aliphatic and nonpolar in nature however, this change was from a Valine to Glycine.

2. A female child 5 years of age and having no any familial history. DNA sequence analysis identified a deletion at 156021981_156021981delT in Exon 6, which changes the amino acid at codon 140 from Valine (non-polar, neutral) to Glutamate (polar, acidic amino acid) (V140E). Provean result showed that the polymorphic site is deleterious.

3. A female child, 4 years of age with familial history. The patient having symptoms of dilated cardiomyopathy whose polymorphic study was done. Familial history showed that relative of patient had different genetic disorder and other disorder or disease was running the family also, the family has consanguinity.

The electrophoregram and multiple sequence alignment showed change of sequence at site 156022028G>C. This change in amino acid was at codon 156 from Valine to Leucine (V156L). Both the amino acids were non polar and aliphatic in nature.


A)





B)

Fig(A) & (B). Showing gel picture of amplified genomic DNA (Exon 6 of δ-Sarcoglycan Gene) of DCM samples.


MUTATION ANALYSIS

 BHU/13/02



Fig 1(A) Showing picture of DCM Patient BHU/13/02, (B) Pedigree of BHU/13/02 (C) Sequence analysis demonstrates a T to G substitution at position 95 which changes the wild type Valine to Glycine (V140G), (D) Sequence analysis



Sequence analysis of DNA was aligned through Clustal X in which we observed the change of sequence site (chr5:156021981T>G). After that with the help of mutation taster, missense mutation and position(s) of altered Amino acidV140G (Valine to Glycine) were detected.

 BHU/14/350


 


Fig 2- (A) Showing picture of DCM Patient BHU/14/350, (B) Pedigree of BHU/14/350, and (C) Sequence analysis demonstrates deletion of T at position 95, (D) Sequence analysis

Sequence analysis of DNA was aligned through Clustal X in which we observed the change of sequence site (chr5:156021981_156021981delT). After that with the help of mutation taster, frame shift mutation and position(s) of altered Amino acid V140Efs*18 were detected.

 BHU/13/03




Fig 3- (A) Showing picture of DCM Patient BHU/13/03 (B) Pedigree of BHU/14/350, (C) Sequence analysis demonstrates a G to C substitution at position 139 which changes the wild type Valine to Leucine (V156L), (D) Sequence analysis of BHU/13/03

 

Sequence analysis of DNA was aligned through Clustal X in which we observed the change of sequence site (chr5:156022028G>C). After that with the help of mutation taster, missense mutation and position(s) of altered Amino acidV156L (Valine to Leucine) were detected.


 


The missense mutation was detected in one patient named (BHU/13/02) where amino acid changes at V140G (Valine 140 Glycine). Second mutation, the frameshift mutation was detected in patient (BHU/14/350) where amino acid changes at V140Efs* (Valine 140 delete A.A) and the third mutation, missense mutation was detected in patient (BHU/13/3) where amino acid changes at V156L (Valine 156 Leucine).
All these were depicted in a tabular form (Table 1).

Patient

Chromosome

Exon

Nucleotide position

Protein position

Mutation type

Provean

BHU/13/DCM02

chr5

Ex 6

156021981T>G

V140G

Missense mutation

Damaging  score-5.55

BHU/14/DCM 350

chr5

Ex 6

156021981_156021981delT

V140Efs*18

Frame- shift mutation

Damaging  score-9.95

BHU/13/DCM 03

chr5

Ex 6

156022028G>C

V156L

Missense mutation

Damaging  score-2.11

Table 1.Shows mutation types in DCM patients along with the damaging scores.

CONCLUSION

Defined by ventricular dilation and diminished contractile function, dilated cardiomyopathy is a prevalent world-wide disorder that is estimated to affect 36.5 per 100,000 individuals12. This complex is formed by the dystroglycan subcomplex (α-dystroglycan and β-dystroglycan), sarcoglycan subcomplex (α-, β-, γ-, and δ- sarcoglycan), caveolin-3, syntrophin, dystrobrevin, and sarcospan and serves as a link between cytoplasmic actin, the membrane, and the extracellular matrix of muscle via laminin-α213,14. Mutations in dystrophin, the DAG subcomplexes, or laminin result in a wide spectrum of skeletal myopathy and/or cardiomyopathy in humans and animal models such as the mouse or hamster15,16,17,18,19,20,21. Furthermore, mutations in this gene have previously been shown to cause either dilated or hypertrophic cardiomyopathies in the hamster and mouse, providing support that this gene, which maps to chromosome 5q33-34 in humans23, is disease-causing in the patients described in this report. Multiple animal models with sarcoglycan deficiency have been produced or are naturally occurring, including the cardiomyopathic hamster due to deletion of Exons 1 and 2 of δ-sarcoglycan20. Hack et al.26 generated a γ-sarcoglycandeficient mouse that developed progressive muscular dystrophy, pronounced cardiac muscle degeneration, and reduced survival. These mice also had reduced levels of β- and δ-sarcoglycan staining of muscle (with normal dystrophin, dystroglycan, and laminin-α2). Coral-Vazquez et al.2

DCM is the most common cardiomyopathy, occurring primarily due to genetic defects or secondarily as a consequence of multiple factors, including long-standing hypertension, ischemic heart disease, infection and sarcoidosis. In this study we found that mutations in δ-sarcoglycan genes are found to be responsible for dilated cardiomyopathy in North-Indian population. In conclusion, the description of mutations in δ-sarcoglycanin patients with DCM provides further support for the concept that the final common pathway for DCM is the cyto-architecture, comprising the cytoskeleton, sarcolemma, and interacting components. In addition,the fact that mutation in δ-sarcoglycanand dystrophin. Current progress in DCM genetics has been significant, although much remains to be learned. Clinical genetic testing is quickly rising, and this new technology now allows patients to undergo clinical genetic testing for many genes at reduced cost. With the discovery of new DCM genes and other DCM genetic causes, will lead to knowledge of the remainder of the genetic makeup of familial DCM and idiopathic DCM. The advantages of genetic testing are (1) Confirmation of diagnosis in patients, (2) Early detection and prevention in relatives and (3) The exclusion of causative mutation in relatives. The result can either terminate clinical follow-up of relatives or institute follow-up.

Our genetic screening of the δ-sarcoglycan gene in North-Indian DCM cases identified missense and frameshift mutations. In conclusion, we describe here novel DCM-associatedmutations in the δ-sarcoglycan gene, missense mutation (V140G) which was found in one patient named BHU/13/DCM02,frameshift mutation (deletion of T at position 95),(V140E),  in second patient named BHU/13/DCM350 and again a missense mutation (V156L) in the third patient named BHU/13/DCM03.


Funding: This work was supported by the Indian Council of Medical research, New Delhi India.


Acknowledgment

This study is a part of my thesis and I am grateful to ICMR (Indian Council of Medical Research) for providing the grant for my thesis and for their full support and cooperation.

 

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8.      Manolio TA, Baughman KL, Rodeheffer R,et al., Prevalence and etiology of idiopathic dilated cardiomyopathy (summary of a National Heart, Lung, and Blood Institute Workshop). Am J Cardiol. 1992; 69:1458-66.

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