Blood samples of five families (39 individuals) with a positive family history of myocardial infarction were collected from the areas of Lahore, Bahawalpur and Khanewal Punjab, Pakistan. Among thirty-nine individuals of the diseased families, there were five probands (affected from MI), one deceased, two parents, three siblings, eight members were outside of families, thirteen first degree relatives and seven-second degree relatives. There were eighteen females and twenty-one males who participated in this study. The ratio of males was high as compared to females. The mean age of probands was ±50.83 years whereas; mean BMI of the probands was ±30.20 Kg/m2. The clinical characteristics of the families are presented in Table 1 (A) and other clinical characteristics of diseased patients which include environmental factors and other complications associated with MI presented in Table 1 (B).
Phenotypic analysis of families
Family no. 1 consisted of eight members. Blood samples and demographic data were collected from each member for genetic analysis. The proband (father) was diagnosed with MI at the age of 54yrs while other members of the family were healthy. Family no. 2 consists of eleven members. Proband and his brother were affected with MI. At the age of 56yrs and 52yrs, the proband and his brother were affected with MI, respectively. The remaining nine members of the family were healthy. The family no. 3 consists of eight members. The proband (mother) had MI at the age of 48yrs while other remaining members of the family were healthy. Family no. 4 consists of seven members. The proband (daughter) was diagnosed with MI at the age of 40yrs heaving BMI value of 36.6 kg/m² which shows that proband was obese while other members of the family were healthy. Family no. 5 consists of five members (Fig. 1). The proband (father) was diagnosed with MI at the age of 55yrs while other members of the family were healthy. Blood samples of all the members with demographic data were collected.
Primer Optimization and Polymerase Chain Reaction (PCR)
PCR amplification was performed on DNA samples of all the diseased families for both SNPs (rs17576 and rs3918242). The PCR products of 243bp and 369bp of both rs17576 and rs3918242 were obtained by using gradient PCR. The amplicons were confirmed by running 2% agarose gel electrophoresis, observed under the gel documentation system. The PCR amplicons of a single-family are shown in Figure 2A, B.
Genetic analysis of RFLP Sma I and Pedigree analysis
As a result of Sma I enzyme digestion, DNA fragments of 85bp, 145bp and 243bp were produced. For homozygous GG alleles 130bp and 243bp, for homozygous AA alleles 243bp and heterozygous AG alleles 85bp, 145bp and 243bp fragments were yielded (Fig. 3A, B and Fig. 4A, B).
Genetic analysis of RFLP Sph I and Pedigree analysis
As a result of Shp I enzyme digestion, DNA fragments of 131bp, 209bp and 369bp were produced. For homozygous TT alleles 131bp and 209 bp, for homozygous CC alleles 369bp and heterozygous TC alleles 131bp, 209bp and 369 bp fragments were yielded (Fig. 5A, B and Fig. 6A, B).
Genotyping of All Selected Families
Genotyping of families was done and most of the members showed no mutation having genotypes AA and CC for rs17576 and rs3918424 respectively. In the family MI1 (diseased) show heterozygous mutation (AG) with rs17576 and homozygous mutation (TT) with rs3918424. While in the same family, one member M5(healthy) was also having a CT mutation for rs3918242. In another family MI 4, the proband was having mutation AG and CT for both SNPs respectively. In family MI 5 the member 1 (proband) shows the heterozygous mutation (CT) with rs3918242 and A/G for rs17576 (Table 2).
Allelic and genotypic frequencies through statistical analysis
The allelic and genotypic frequencies analysis was done of all patients and control members whose data was collected from five families. The genotype and allelic frequencies of all 38 subjects were analyzed and represented in Tables 3 and 4 respectively. MMP9 SNP identified T/T and C/T genotypes rs3918242 in patients and C/C in controls while A/G genotype rs17576 in patients and A/A in controls. The SNPs followed the Hardy Weinberg equilibrium and rs17576 & rs3918242 were significantly associated with the onset of MI in the Pakistani population at genotypic as well as the allelic level (p<0.05). We found the T allele as a risk allele having a significant association (p< 0.05) of variant rs3918242 with MI because its allelic frequency is higher among patients than in control. Moreover, allele G of rs17576 was found as a risk allele having significant association (p<0.05) with MI because of its higher allelic frequency among patients than control. The p values were calculated from Fisher's test.