OF PHOSPHOLAMBAN AND RENINANGIOTENSIN SYSTEM GENES MUTATIONS AND CLINICAL EPIDEMIOLOGY IN HUMAN CARDIOMYOPATHY 1

11 Background 12 Cardiomyopathy is commonly observed disease that may occurs due to mutations in 13 either susceptible genes or modifier gene. People with broad age group are affected 14 either attributable to spontaneous or inherited mutations of these genes. Various gene 15 mutations are reported so far but only few of them were studied in detail. 16 Methods 17 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 5 July 2018 doi:10.20944/preprints201807.0093.v1 © 2018 by the author(s). Distributed under a Creative Commons CC BY license. 2 In the current study, we evaluated epidemiological variables like age, sex, familial status, 18 parental consanguinity. We also described specific clinical symptoms associated with the 19 cardiomyopathy condition in Indian population. 20 Results 21 Our studies on mutation screening of phospholamban gene revealed two transitions 22 (4880 C/T, 4887 T/G) in 5’ flanking region which might cause inherited dilated 23 cardiomyopathy with refractory congestive heart failure are We further deliberated the 24 gene polymorphism of renin angiotensin system gene angiotensin-1-converting enzyme 25 as an associated marker/ modifier in cardiomyopathy patients and their family members. 26 Conclusions 27 Information on epidemiological, clinical statistics, phospholamban gene mutation analysis 28 and angiotensin-1-converting enzyme gene polymorphism is essential to guide the 29 successful execution for future therapies and benefits us to identify those patients at risk 30 for faster disease progression, congestive heart failure, and arrhythmia. 31 32


INTRODUCTION
In the present scenario, the debilitating nature of cardiovascular disorder is alarming and needs a constant watch on the premature morbidity and mortality status.Cardiomyopathy is the heart muscle disease and most common genetic disease of the heart, characterized by heterogeneous morphologic expression and clinical condition (1).It can manifest negligible to extreme hypertrophy, minimal to extensive fibrosis, myocyte disarray, absent to severe left ventricular outflow tract obstruction, distinct septal morphologies, or hypocontractile (2).Cardiomyopathy can cause heart failure (HF), which in most cases leads to sudden cardiac death (SCD).Hypertrophic Cardiomyopathy (HCM), Dilated Cardiomyopathy (DCM), Restrictive Cardiomyopathy (RCM) and Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) are four types of cardiomyopathies reported (3).These types can be primary myocardial disorders or at times develop as a secondary consequence of a variety of conditions viz., myocardial ischemia, inflammation, viral infection, increased myocardial pressure or volume load, and toxic agents (4).The etiology of both HCM and DCM involve cardiac energy imbalances and the clinical expressions of them are based on the addictive factors that are involved in it.
The prevalence of the dilated cardiomyopathy has formerly been estimated at 36.5/100,000, with an incidence of 4-8/100,000 persons-year (5).An echocardiographic analysis of 4111 subjects in CARDIA study by (6) revealed hypertrophic cardiomyopathy affects 1 in 500 people, a prevalence similar to familial hypercholesterolemia. Recent study estimates prevalence of dilated and hypertrophic Cardiomyopathy as 36 cases per 100,000 people and 10-20 cases per 100,000 people respectively (7).Most of the dilated Cardiomyopathy cases are sporadic; although 20-35% of them are familial (8).The incidence of dilated cardiomyopathy varies in men and women.However, in general, heart failure is more common in men (9).The treatment arm of the Studies on Left Ventricular Dysfunction (SOLVD), in which only 15% of the 4 patients were women, reported no sex-related difference in survival in either the placebo group or the Enalapril group (10,11).Age distribution depends on prognosis, diagnosis or onset of any underlying disease.However, advancing age is reported as an independent risk factor for mortality in several studies (12).Phospholamban (PLN) is a small transmembrane phosphoprotein of 52 amino acids that plays an important role in cardiac contraction and relaxation.Phospholamban, expressed in the sarcoplasmic reticulum membrane controls cellular calcium levels by a mechanism that depends on its phosphorylation (13).The human ventricle and quadriceps displayed high levels of phospholamban transcripts and proteins (14), whereas lower expression in smooth muscles and right atrium.DCM patients with a phospholamban gene mutation have a chronically inhibited Ca2+-ATPases pump, which leads to DCM in their teenage (15,16).Susceptibility genes have a role in the development of cardiomyopathies, whereas modifier genes have a role in the evolution or prognosis of the disease.In most studies due to limited sample sizes, the role of susceptibility and modifier genes have been only suggestive (17).Various genes underlying cardiomyopathy have been identified from linkage as well as candidate gene studies, and include those coding for proteins involved in the cytoskeleton, the Z-disk, the nuclear envelop, ion conduction and calcium handling proteins (18).This variability in expression of these causal genes is also seen among family members sharing the same mutation.This variable expressivity, which confuses genotype/phenotype correlations, could be partially explained by both environmental influences and genetic modifiers (19).The angiotensin-1-converting enzyme (ACE) polymorphism has been commonly studied with variable results.It's noteworthy to study and understand the importance of modifier genes.The genetic polymorphisms of the renin-angiotensin-aldosterone system (RAAS) are found to influence the progression to cardiac disorders (20).Angiotensin-1-converting enzyme (ACE), a modifier gene was perceived to have insertion/Insertion (I/I) genotype associated with low serum ACE activity levels, Insertion/Deletion (I/D) genotype with intermediate levels and Deletion/Deletion (D/D) high serum ACE activity levels.The effect of ACE polymorphism on survival in patients with DCM with a DD genotype had poorer prognosis than other genotypes (5-year survival rate 49 vs.72%, p=0.001) (21).The DD genotype was also associated with an increase in left ventricular mass (22).
The present study investigates the association of spectrum of clinical symptoms, the epidemiological variables like age, sex, familial status, parental consanguinity and the mutations in the gene encoding phospholamban cardiac protein and to establish the genotype -phenotype correlations, to identify the modes of inheritance and the risk stratification in a group of clinically well characterized patients and their relatives associated with the cardiomyopathy condition in Indian population.We further studied the role of angiotensin-1-converting enzyme gene polymorphism as an associated marker/ modifier in cardiomyopathy patients and their family members.

GENETIC ANALYSIS
In the present study, the index cases are categorized into two groups viz., familial case showing the incidence of the disease in first and second-degree relatives and in sporadic cases lack of any familial incidence, presumably non-genetic in origin.The present study examined a comprehensive screening of Phospholamban and a key determinant/modifier angiotensin-1-converting enzyme gene polymorphism.

DNA ISOLATION and QUANTIFICATION
Genomic DNA was extracted from the peripheral blood following the protocol of (23).Briefly, the collected blood was mixed with an equal volume of TKM1 buffer (10mM Tris-HCl, 10mM KCl,10mM MgCl 2 and 2mM EDTA) and 100 μl Triton X.
The contents were centrifuged and the pellet was washed with TKM1 repeatedly until the cell debris is washed out.The pellet is suspended in 800 μl of TKM2 solution (10mM Tris-HCl,10mM KCl, 10mM MgCl 2 , 2mM EDTA and 0.4M NaCl) followed by centrifugation and precipitation of the supernatant in ethanol.The DNA samples were then stored at -20°C for subsequent analyses.The DNA was quantified using spectrophotometer.The DNA was diluted in TE to yield 50ng/μl concentration.

ANGIOTENSIN-1-CONVERTING ENZYME GENOTYPING
The primers of the hotspots exons of the Angiotensin-1-Converting Enzyme (ACE) forward primer 5'-tatttttctcataattaaaattcctgc-3' and reverse primer 5'-aaagtaagaattaccaaagtcagcg-3' were used (25).Angitensin-1-converting enzyme genotyping was based on amplification of genomic DNA by PCR and the products were detected on 2% agarose gel.Amplification products 490 bp and 190 bp corresponding to the I and D alleles respectively (26).

DNA SEQUENCING
Genomic DNA from individuals with different SSCP patterns was amplified and sequenced with using Applied Biosystems 3730xl DNA Analyzer.The sequencing PCR was carried on 96 well micro-titer plates in a 5 μl reaction volume containing nuclease free water, the amplified template, "BigDye" (fluorescently labeled ddNTPs, dNTPs) and primers.The amplified DNA was precipitated by incubating at room temperature with 25μl of 3M-sodium acetate in ethanol (120 μl of 3M-sodium acetate in 3 ml of 100% ethanol) for 15min.The DNA was made single stranded by adding Hi-Di Form amide and sequenced on ABI3730xl automated DNA analyzer.The chromatograms obtained were analyzed on "Auto assembler" chromatogram analyzer on a Macintosh operating system.
Complete sequencing work was carried out at the "Center for Cellular and Molecular Biology" Hyderabad.

BIOINFORMATICS
Multiple Sequence Analysis (MSA) was used for sequence comparison between generated sequences of the present study and to the corresponding reference sequences obtained from GenBank.The generated gene sequences were translated as protein sequences for future analysis (ExPASY Tool).Using Conserved Domains Database and Motif Finder Database alteration in the conserved domains and motifs as a consequence of mutation were identified for the generated sequences.

STATISTICAL ANALYSES
Statistical analyses were carried out using Statistical Analysis Solutions (SAS 9.2).The mean and standard deviation were computed for various quantitative parameters and calculated.P-value <0.05 was considered significant.Association and relative risk estimates were carried out using Chi square test for the qualitative parameters at 1% and 5% levels of significance.The Allele frequencies were computed by gene counting method.Departure from Hardy-Weinberg equilibrium was tested by chi-square test.The odds ratio was computed using SAS.

Patients and Baseline Characteristics
A total of 109 unrelated index patients who have echocardiographically and electrocardiographically assessed for cardiomyopathy and 100 aged matched control subjects were studied.About 73 percent of the patients had dilated cardiomyopathy and 27 percent hypertrophic cardiomyopathy.Details on age, body mass index, blood pressure, blood biochemical profile, associated clinical and non-clinical features of the patients, control subjects were given in (Table 1).Males were significantly higher among the patients (73%).The mean age of all patients was 37.22 ± 12.43 years.Occurrence was familial in 24% of cases, but sporadic in the other 76% of patients.In patients group about 30% of them had family history of sudden cardiac death and 14% of them showed parental consanguinity.Mean value of body mass index and body surface area was same in patients and control (p=0.085,p=0.515).

Hemodynamic and Biochemical Parameters
Diastolic blood pressure, heart rate and systolic blood pressure of cardiomyopathy patients was extremely significant when compared with the control subjects (p<0.05).
Cardiomyopathy patients were characterized with high atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) level (p<0.001).Contrastingly creatinine phosphokinase (CpK), serum aspartate transaminase (SGOT), serum alanine transaminase (SGPT), glucose, urea and creatinine levels did not show much variation between patients and control group (Table 1).Similarly, co-morbid factors such as diabetes, obesity, smoking and alcoholism did not show any influence on patient population.

CLINICAL CHARACTERISTICS
The electrocardiographic and echocardiographic characteristics of the cardiomyopathic patients and control group were given in

Phospholamban Gene Mutation
Phospholamban gene is located on chromosome 6q22.1.Genomic sequence of the gene is 12146 bps nucleotides long which encodes 1742 bps mRNA coding region comprises 159 bps which encodes 2exons.The protein is a pentamer and is a major substrate for cAMP-dependent protein kinase in the cardiac muscle.The protein inhibits Ca2+-ATPase in unphosphorylated state.The protein is a key regulator of cardiac diastolic function.
Table 3 shows the detailed description of the mutations in phospholamban gene.Seven mutations -two in the 5' flanking region, two in exon 1 and three in the intron1 region (Figure 1 (a-f)).Transition T/G in 4887 nucleotide regions of 5' flanking region was observed in two HCM probands with four affected family members and also in one dilated cardiomyopathic proband with no affected relatives.15511-15512 insertion T was observed in one dilated cardiomyopathic proband with 2 affected family members and one dilated cardiomyopathic proband with no affected family members.

4887(T/G) mutation:
In the pedigree of the H9 proband (male, 21years) (Figure 2), the proband's father and his two uncles died young of sudden cardiac arrest.The younger brother of the proband's father has an affected son.The mutation was in heterozygotic state and it is not observed in unaffected.In the pedigree of H10 proband (Figure 3), the T to G transition was observed in two family members The H10 proband (Male, age: 41years) had an affected offspring who was deceased subsequently to this study.The proband's parents are consanguineous and his mother died of cardiac arrest.The proband's brother had also carried this mutation and his daughter died unexpectedly at the age of 18 years.

ins T mutation:
The intronic mutation in PLN gene was observed in one dilated cardiomyopathy patient and in his family members (Figure 4).The D15 proband (Male, age: 37 years) who carried this mutation has an affected father (66 years).The proband's father's brother died of sudden cardiac arrest at the age of 22 years.The mutation in their family members were in heterozygotic state and not found in unaffected.

Angiotensin-1-Converting Enzyme Gene Polymorphism
The present study reports on the molecular screening of a population of 109 unrelated index patients and 100 controls.Mutational screening was performed in one modifier gene Angiotensin-1-converting enzyme (ACE) i.e., zinc metallopeptidase widely distributed on the surface of endothelial and epithelial cells (27).When in demand, renin activity leads to the conversion of angiotensinogen to angiotensin I. ACE then converts angiotensin I to angiotensin II, which have been implicated in the modulation of cardiac growth (28) illustrates variation in the genotype and allele frequency of the patient and control.Most notably the deletion homozygotes found to be higher in the patients.Also, the percent of heterozygote varied significantly among the two groups.Similarly, the deletion allele frequency was considerably higher in patients about 60% than controls (44%).The chi square value in patients and controls was 0.109 and 1.148 respectively.
The genotypes DD vs ID, DD vs II, ID vs II, DD + ID vs II, DD vs ID + II and D vs I was taken for the odds ratio comparison.All the odds ratio values were higher than the threshold value of 1.The odds ratio was highest for the genotype DD vs II (3.82) with a χ2 value of 10.59.This was followed by DD + ID vs II and ID vs II genotypes having an odds ratio of 2.79 (χ2 =8.60) and 2.27 (χ2=4.48)respectively.The DD vs ID genotype comparison had an odds ratio of 0.60 (χ2 =1.94).It should be noted that the χ2 value is taken to evaluate the significance of the genotypes.Patients with D allele tend to be a higher risk and modify the disease pattern when compared with I allele.The odds ratio was found out to be 1.14 with a χ2 value 12.09.In Table 5 the chi square and the odds ratio values were calculated to prove the significance of the genotype of patients and reference to the control.

DISCUSSION
Cardiomyopathies are diseases of the heart muscle and a cause of concern.They exhibit a wide spectrum in disease onset, manifestation as well as in progression (30).Significant differences were observed for age and sex ratio between control and patients with cardiomyopathy.Mean age of diagnosis was higher in the patient data of the present study than in studies of HCM and DCM previously reported (31,32).This may be due to mutation carriers screened in a predictive setting represent an asymptomatic subgroup within the total population of affected.Besides age, gender was an important cofactor in the clinical manifestation of HCM and DCM (33).The cardiomyopathy patients of the present study were characterized with significant increase in atrial natriuretic peptide and brain natriuretic peptide, which were correlated with left ventricular ejection fraction, mean pulmonary arterial pressure and pulmonary artery wedge pressure.Present data provides evidence that ANP and BNP are the best indicators for heart failure in cardiomyopathy patients.An elevated mean diastolic blood pressure (81.41 ± 4.76) and systolic blood pressure (125.0 ± 9.17) were observed in cardiomyopathy patient's data of the present study.A higher diastolic and systolic blood pressure has been observed in Caucasians (34), Chinese (35) and Japanese (36) origin.The mutation that is located near the promoter region has been defined as the fragment with maximal transcriptional activity (44).The increase or the decrease in phospholamban activity due to the disruption around the promoter region can lead to cardiomyopathy (41).

Phospholamban is an inhibitor of endogenous sarcoplasmic reticulum calcium
Some carriers do not exhibit clinical conditions and mutation may be due to other genetic and environmental factors.This study concludes that PLN gene mutations are not frequent cause of hypertrophic or dilated cardiomyopathy in Indian population.
Although similar data were lacking, it was observed that Indians show a greater variation and this may be due to the polymorphism in ACE gene (modifier gene).Further, role of a dual peptide system viz., brain, and atrial natriuretic peptides in sodium balance and blood pressure regulation in those patients cannot be ruled out (45).Angiotensin-1-converting enzyme gene insertion/deletion (I/D) polymorphism is considered as an important modifier gene, which may influence the clinical phenotype of the cardiovascular disorders (46).The present data revealed that the frequencies of the DD genotype and D allele were significantly higher in patients compared with controls, and were associated with increased risk of HCM and DCM.Furthermore, regression analysis revealed that the genotypes DD and D allele were independent risk factors for these cardiomyopathies in Indian population.Patients with the DD genotype had the highest odds ratio of disease susceptibility and the subjects with II genotype have a lower risk of developing cardiomyopathies that may possibly through a cardio protective effect.The D allele compared with I allele has more than 25 percent increased risk to cardiomyopathy.On comparison, the prevalence of D allele in the present study (Indian population) was slightly higher (about 60%) than Tunisian and Turkish populations (47,48).Association of DD genotype / D allele with HCM and DCM have been reporting in many studies (49,50) and the polymorphism considered as a modifier gene marker to cardiomyopathy.The DD genotype was also found to be associated with hypertension, restenosis, diabetes and myocardial infarction (51,52).However, absences of association (53,54) or influencing the cardiac phenotype (55) were reported in few other studies.These variations were partly accounted for ethnicity of the patients and to the sampling of the patients (56, 57).

CONCLUSION:
Among the two types of cardiomyopathies; dilated cardiomyopathy (73%) was found to be most predominant, whereas hypertrophic cardiomyopathy accounts for 27%.In general, increased male predominance (73%) was observed in both the types of cardiomyopathies.Familial occurrence was in 24% of the patients, parental consanguinity was 14% and 30% had family history of sudden cardiac death.Diastolic blood pressure of the cardiomyopathy patients was significant (p<0.05)than the systolic blood pressure and heart rate.Also, they were characterized with higher Atrial Natriuretic Peptide and Brain Natriuretic Peptide (p<0.001).Contrastingly co-morbid factors and other enzymes did not show much influence between patients and control subjects.Mutation screening of phospholamban gene revealed two transitions (4880 C/T, 4887 T/G) in 5' flanking region.Among them 4887 T/G transition was inherited in a hypertrophic cardiomyopathy ATPase in dephosphorylated condition.It plays a regulatory role in the calcium handling during the process of cardiac contraction/relaxation.Mutations in this gene shown to associate with elevated cytosol calcium concentration.Phospholamban is phosphorylated by protein kinase A to increase the reuptake of calcium into sarcoplasmic reticulum (37,38,39).Besides dilated cardiomyopathy though this is the first report to show Phospholamban gene mutation associated with hypertrophic cardiomyopathy, none of the all identified mutations falls neither within the coding region nor at conserved domain.Similar to this study there are few other studies had shown flanking regions and promoter variants of PLN associated with DCM/heart failure and HCM (40, 41).Alternatively, there are reports Preprints (www.preprints.org)| NOT PEER-REVIEWED | Posted: 5 July 2018 doi:10.20944/preprints201807.0093.v1that show no associations (42,43).It is possible that PLN gene mutations can express at a lower level leading to a smaller pathogenic effect during sixth decade of life in these individuals.Contrastingly two familial cases showed mutation of PLN (4887 T/G) possibly decrease the transcriptional activity of promoter and associated with hypertrophic cardiomyopathy.

BLOOD SAMPLE COLLECTION Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 5 July 2018 doi:10.20944/preprints201807.0093.v1 Five
milliliters of peripheral blood were drawn in EDTA coated vacutainer from patients, family members and control subjects and stored at 4°C until DNA extraction.DATA COLLECTION Epidemiology parameters such as height, weight, sex, age at onset, dietary habits, addictions to smoking and alcohol were collected during personal and clinical history.Each of the subjects met the Clinical diagnostic criteria viz., 12 lead electrocardiograms, echocardiogram, clinical symptoms, risk factors, medication and outcome of the disease etc.

Table 1 . Baseline, Hemodynamic and Biochemical Characteristics Characteristics Cases n=109 Control n=100 Cases vs Control P value
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