Penetrance of Methylene Tetrahydrofolate Reductase C677T Gene Polymorphism and Karyotypic Variations Associated Increase Genetic Susceptibility in the Cases of Congenital Heart Defects

Congenital heart disease (CHD), a multifaceted disorder occurs during embryogenesis due to exposure of environmental mutagens (teratogens) exposed antenatally leading to high-risk of infant mortality. Present study has been designed with the aims to evaluate the frequency of chromosome variation and corelate to methylene tetrahydrofolate reductase (MTHFR) C677T gene polymorphism as “risk factor” in clinically diagnosed cases of CHD using lymphocytes cultures and ARMS PCR, respectively. FISH analysis was carried for confirmation of chromosome-21. Interestingly, cytogenetics study shows variation in the frequency of structural and numerical chromosome aberrations with frequency in all the cases of CHD. Case-1, showing deletion of short arm of chromosome-18 and Robertsonian translocation between G/G chromosome association (24.00%), Case-2 showing numerical variation (trisomy-21), Case-3, includes dicentric, chromatid break in chromosome-2, deletion of short arm in chromosome-5, reciprocal translocation involving chromosome-6 and 10 and reporting first time appearance of ring of Y-chromosome. Case-4 showing structural variations (16.00%) including dicentric, chromatid breaks and trisomy of chromosome-21. The most common dominant frequency was observed in karyotype trisomy-21(58.30%) in all the four cases of CHD as an end point for genetic bio maker and showing significant differences (p < 0.001) using X2- test between total number of chromosomes and trisomy-21 MTHFR (C677T) gene polymorphism reveals (25.00%) of genetic heterozygosity of CT alleles and 75.00% cases shows homozygosity of wild type (CC) alleles, suggesting the variations in the frequency either in karyotypes or MTHFR C677T alleles are due to unconstitutional penetrance of gene in the genome of CHD cases and increase genetic susceptibility to make the disease more complex. Penetrance of Methylene Tetrahydrofolate Reductase C677T Gene Polymorphism and Karyotypic Variations Associated Increase Genetic Susceptibility in the Cases of Congenital Heart Defects.

In human, MTHFR gene is thermolabile in nature and present on chromosome 1p3 6.3. The polymorphic variation of C677T allele (rs1801133) , the missense (point) mutation is responsible for the reduction enzymatic activity (30% -70%) followed by increase "risk factor" in the variety of disease in heterozygous condition other than CHDs, such as neural tube defects (NTD), mental retardation, miscarriage and cancers [12][13][14][15][16][17]. Only few studies have been demonstrated regarding variations of MTHFR allele in CHD cases in Indian population [18,19]. Therefore, the present study has been designed to evaluate the frequency of karyotypic variations in individual case and try to find out their correlation with penetrance of MTHFR C677T gene polymorphism as "risk factor" in CHDs cases to confirm the hypothesis that congenital anomalies are polygenic in nature.

Cytogenetics Study
Lymphocytes cultures were set up in the media (RPMI 1640) supplemented with foetal calf serum (5%), Phytohaemagglutinin-M and antibiotic (penicillin & streptomycin) for 72hrs in CO 2 incubator at 37°C under sterile conditions. Harvesting was performed after adding colchicine (10 µl) in the cultures to arrest mitosis.
Prewarmed KCL (0.056%) solution was used as hypotonic, and cells were fixed in acetic acid and methanol (1:3) as detailed procedure is illustrated earlier by Saxena et al in 2020. Giemsa stain was used for staining and GTG banding was performed after using (0.001%) trypsin in PBS buffer (pH 7.4) and karyotyping was performed according to the ISCN (2016) [20] using applied spectral imaging software (Genesis, USA).

Fluorescence in-situ Hybridization (FISH).
FISH analysis was carried out for the study of D21S65 specific region for chromosome-21 in both interphase and metaphases plates after using probe LSI 21 spectrum orange having region 21q22.13-q22.2 of 220 kb, obtained from Abbott-Vysis, Inc. (USA).
Chromosome -21 was identified by green signal and nuclear DNA was labelled with DAPI as counter stain (Blue).

Statistical Analysis
Chi square (X2) test was used to find to calculate p value and level of significance (p=value) between normal and abnormal cells during analysis of karyotypes in individual case of CHDs.

Results
Cytogenetics analysis in CHD cases were performed for developing karyotypes individual to find end point according to  Table 1.

Discussion
The etiopathology of CHDs are complex due to the interactions of genetic and environmental factors [4,22]. It has been identified that ~30% of CHD cases are syndromic involving chromosomal aberrations, while non-syndromic are monogenic and shows variety of genes. Now it is evident form preview of the fact that genetic factors become essential to understand the etiopathology in CHD cases and risk assessment before initiation of clinical management and counselling. However, there is lack of the genetic knowledge of CHDs in Indian population, only few studies have been achieved to study an association of genetic factors in CHDs. Therefore, present study was performed to accumulate the data from different approaches like karyotypic pattern and MTHFR (C677T) gene polymorphism to determine "risk factors" in CHD patients.
Epidemiological studies revel that frequency (%) and types of chromosomal abnormalities varies between USA or Europe, but there is a lack of data in Indian population. Hence, the present study on CHDs becomes relevant to assess the risk using MTHFR C677T gene polymorphism analysis and try to correlate with karyotypic variations with agreement by the authors present study is small, but quite interesting. Previous studies identified chromosomal abnormalities with variation in the frequency from 3-18% in CHDs due to different variables such as clinical characteristics, age and ethnicity [22,23]. In the present study, numerical variations 18 are associated with the cases of CHD [23,24]. Previous stud also reported trisomy-13, however, such chromosomal aberrations failed to observe in the present study may be either due to small sample size or different environmental factors [24].
In the present study, frequency of structural chromosomes was varying from 8-30% alone over total cytogenetics alterations and more interesting than numerical end point due to the appearance of "Y-ring chromosome and centromeric breakage reporting first time.
Author failed to interpret the present findings that there might be involvement of conserve DNA sequences exist at centromeric regions and role of sex-chromosome during pathogenesis of CHD cases. Earlier study shows the deletion of the short arm of chromosome -6 and 17, while present study also documented chromatid breaks -unstable regions of chromosomes [22][23][24][25].
Interestingly, present study shows "novel" structural changes has not been reported earlier with high impact on genomic instability of CHDs genome.  [28].
Importantly, studies by Hobbs and co-workers identified Hcy, S-adenosylhomocysteine, and methionine as the most important biomarkers predictive in mothers whose pregnancies were affected by congenital heart defects (224 case subjects) or unaffected by any birth defect (90 control subjects)7. The variations in allele frequency may be either due to different clinical feature or penetrance of mutant allele to the CHDs genome to increase severity of the disease, but simultaneously the epigenetic factors should not be ignored.

Conclusion
From the present study we are reporting first time the

Acknowledgement
AKS thankfully acknowledges to the Director, AIIMS Patna also give special thanks to the patients who participates in the study.