Role of cystathionine β synthase gene variant in Sudanese population
- *Corresponding Author:
- Taghreed Mohamed Osman Derar
College of Applied Studies and Community Services
King Saud University, Saudi Arabia.
Accepted date: June 13, 2017
Several studies showed the relation between renal and Cardiovascular Diseases (CVD) with distinct genotypes. The current study aims to investigate the association with 844ins68 polymorphism on Cystathionine β-synthase (CβS) gene in renal and CVD with Sudanese population. In this pilot study, we have opted 150 cases from Khartoum state hospitals, Khartoum, Sudan. We have selected equally controls, renal and CVD (n=50). Peripheral blood was collected to perform the biochemical and molecular analysis. Genomic DNA was isolated to perform the polymerase chain reaction analysis. The current results showed the non-significant analysis with the combination of renal versus controls (OR-0.81 (0.31, 2.10); p=0.66) and CVD versus controls (OR-0.65 (0.26, 1.67); p=0.37). When we correlated renal samples and CVD cases with biochemical data and 844ins68, we found Vitamin B12, D, TP, URCA and Folic acid, cholesterol to be associated (p<0.05). In conclusion, our results confirm the role of the negative association of 844ins68 polymorphism in the Sudanese population. It may be due to low sample number, and future studies should be performed with larger sample size with multiple CβS gene SNPs in the world's people.
Renal, Cardiovascular, CβS gene, 844ins68.
Cardiovascular Disease (CVD) is leading cause of death, irrespective of race and ethnicity, and is mostly precipitated by cardio metabolic risk and Chronic Kidney Disease (CKD) or End Stage Renal Disease (ESRD) . ESRD patients have high mortality risk as compared with general population. CVD is also a major cause of death in these patients, accounting for 40-50% of total mortality . An earlier study has confirmed patients on chronic dialysis had an 8.8-times increased cardiovascular mortality risk as compared with the general population . Genome-Wide Association Studies (GWASs) have been conducted in recent years and have identified common genetic variants associated with all the main risk factor traits for CVD . Cystathionine β-synthase (CβS) is one of the key enzymes present in transsulfuration pathway, catalysing initial rate-limiting step by converting homocysteine to cystathionine . Metabolism of homocysteine via the transsulfuration pathway is crucial as this helps in maintaining intracellular redox balance. Enzyme defects involved in transsulfuration pathway would lead to homocysteine accumulation of resulting in intracellular redox imbalance and could potentially lead to disease conditions . There are several mutations and polymorphisms in the CβS gene, associated with its function and Homocysteine plasma concentrations . The CβS gene reveals common genetic polymorphism consist 68bp insertion at position 844 (844ins68) . This polymorphism was initially described in homocystinuria patients . Inherited CβS deficiency is acknowledged to be the most frequent cause of homocystinuria in humans . There are no studies carried out in Sudanese population, and this current study aims to investigate the association with 844ins68 polymorphism on CβS gene in renal and CVD with the Sudanese population.
Materials and Methods
This is a case-control study designed for the descriptive and qualitative approach in Sudanese population. All the analyses were limited to our study population. Accurately, 150 subjects were included in this study and all of them were divided into three categories as (i) 50 subjects with cardiovascular diseases (ii) 50 Subjects with Renal diseases and (iii) remaining 50 subjects are matching healthy control subjects. Complete samples were recruited from the Khartoum state hospitals, Khartoum, Sudan. Concern clinicians have confirmed the CVD and renal samples based on the design of the study. Healthy controls were collected from central blood bank of the similar hospital. Inclusion criteria for both the subjects are age restriction (18-60 y), local ethnicity and effected with CVD and renal diseases. The exclusion criteria were the lack of vitamin supplementation, the presence of any form of cancer, liver disease, primary renal disease or any collagenase diseases. A data collection form was designed for the recording of the patient's physical and routine laboratory data. Ethical approvals were granted by the Directorate of Research in Khartoum state in Sudan, and all subjects provided written informed consent. From each patient, 6 ml of blood sample was collected, and 4 ml of coagulant blood sample was used for biochemical and 2 ml for molecular analysis for DNA isolation .
Coagulant and anticoagulant blood analysis
Serum sample was separated from 4 ml of coagulant blood for biochemical analysis and measured the Homocysteine (HCV), vitamin-D, B12, folic acid, albumin, blood urea nitrogen, creatinine, cholesterol, triglyceride, Urine Random Calcium/ Creatine Ratio (URCA) and Total Protein (TP) .
Polymerase Chain Reaction (PCR) was performed with the designed primers for CBS (844 in s68) variants based on earlier publications . The primers were redesigned based on the selected variants to confirm the nucleotide sequences. Three steps (denaturation, annealing and extension) with direct PCR method was applied for selected variants for genotyping. PCR amplification was performed with 35 cycles (95°C for 30 s, 60°C for 30 s, 72°C for 45 s) followed by the final extension step at 72°C for 5 min. The reaction volume contained 100 pmol of each primer, 6 μL of sterile water, 10 μL of the 2X master mix (included MgCl2, 10X Taq buffer, 10 unit of Taq DNA polymerase) and 2 μL template DNA. The PCR product was directly separated by electrophoresis in 3.0% Agarose Gel (AGE) and visualised in UVI gel doc.
Statistical significance was examined by two-sided tests; statistical analyses were performed with SPSS version 21.0 software. Mean ± SD were used to measure the clinical characteristics between cases and controls with unpaired Student t-test. Allele frequencies were estimated by the gene counting method, and the chi-square (χ2) test was used to identify departures from Hardy-Weinberg equilibrium. Genotype distribution of several polymorphisms was compared between patients and control subjects by the chi-square test. Yates correction was also carried out for genotype and allele frequencies.
Biochemical and clinical analysis
The baseline characteristic features of control subjects with Renal and cardiovascular subjects have been described in Table 1. The mean age of the renal subjects was found to be 46.6 ± 16.94 compared with CVD, i.e., 54.18 ± 18.24. However, when compared with control subjects (31.92 ± 1.04) both the renal and CVD was identified as statistically significant and elder (p<0.0001). The gender differences between renal, CVD and control subjects are almost similar with height and weight (p>0.05), but BMI compared between the cases and controls were turned to be statistically significant (p=0.04 in renal and p=0.01 in CVD subjects). SBP was found to be positively associated in Renal subjects (p=0.003), whereas DBP was not associated in both the renal and CVD subjects.
|Renal (n=50)||Controls (n=50)||p value||Cardiovascular (n=50)||p value|
|Age (Years)||46.6 ± 16.94||31.92 ± 1.04||<0.0001||54.18 ± 18.24||<0.0001|
|Weight (kg)||56.2 ± 15.86||67.98 ± 12.77||0.13||64.34 ± 10.66||0.2|
|Height (cm)||166.4 ± 9.32||166.8 ± 9.93||0.65||168.86 ± 6.31||0.01|
|BMI (kg/m2)||20.25 ± 5.12||24.37 ± 3.81||0.04||22.44 ± 2.67||0.01|
|HTN (SBP)||145.66 ± 31.97||120 ± 17.37||0.003||113.86 ± 21.64||0.12|
|HTN (DBP)||81.32 ± 15.98||80 ± 13.24||0.19||73.1 ± 15.24||0.32|
|HCY||23.78 ± 13.34||10.97 ± 3.29||<0.0001||27.30 ± 15.14||<0.0001|
|B12||389.43 ± 249.39||200.96 ± 92.81||<0.0001||523.57 ± 393.66||<0.0001|
|FA||17.09 ± 10.61||13.73 ± 4.63||<0.0001||14.76 ± 8.2||0.0001|
|VIT D||56.22 ± 33.64||51.10 ± 21.91||0.003||46.37 ± 30.74||0.01|
|ALB||33.73 ± 6.95||41.71 ± 4.41||0.001||30.31 ± 5.93||0.04|
|BUN||21.67 ± 6.45||3.90 ± 0.91||<0.0001||13.20 ± 11.46||<0.0001|
|CHOL||3.39 ± 0.98||3.82 ± 0.88||0.45||3.25 ± 1.42||0.001|
|CREA||886.39 ± 343.53||70.47 ± 15.50||<0.0001||127.86 ± 68.22||<0.0001|
|TP||71.45 ± 12.32||78.16 ± 4.83||<0.0001||77.06 ± 9.96||0.0001|
|TGL||1.28 ± 0.91||1.41 ± 0.69||0.05||1.19 ± 0.71||0.84|
|URCA||369.12 ± 167.58||253.28 ± 72.15||<0.0001||464.94 ± 156.80||<0.0001|
Table 1: Clinical data between the renal and CVD cases with controls.
The biochemical analysis such as Homocysteine (HCY), Vitamin B12, folic acid, albumin, blood urea nitrogen, cholesterol, creatinine, total protein, triglycerides and URCA were performed between renal, CVD and control subjects (Table 2). When comparison was carried out between renal and control subjects HCY, B12, FA, VIT-D, Alb, Bun, Crea, TP, URCA was significantly associated (p<0.05). The other tests such as Chol and Trig was not associated (p>0.05). The same tests were performed in CVD disease and compared with the similar controls performed in renal subjects and results confirmed HCY, B12, FA, VIT-D, Alb, Bun, Crea, TP, URCA and Chol was significantly associated (p<0.05) and Trig was negatively associated (p>0.05).
|Genotype||Renal (n=50)||Controls (n=50)||OR (95% CI)||P value|
|(844 in s68) II||00 (00)||01 (02%)||Reference|
|(844 in s68) ID||12 (24%)||10 (20%)||1.00 (0.00, 255.5)||0.99|
|(844 in s68) DD||38 (76%)||39 (72%)||0.81 (0.31, 2.10)||0.66|
|(844 in s68) I||12 (0.12)||12 (0.12)||Reference|
|(844 in s68) D||88 (0.88)||88 (0.88)||1.00 (0.00-255.5)||1|
|Genotype||CVD (n=50)||Controls (n=50)||OR (95% CI)||P value|
|(844 in s68) II||00 (00)||01 (02%)||Reference|
|(844 in s68) ID||14 (28%)||10 (20%)||4.14 (0.15, 112)||0.36|
|(844 in s68) DD||36 (72%)||39 (72%)||0.65 (0.26, 1.67)||0.37|
|(844 in s68) I||14 (0.14)||12 (0.12)||Reference|
|(844 in s68) D||86 (0.86)||88 (0.88)||0.83 (0.36-1.91)||0.67|
Table 2: Genotype analysis with renal and controls and CVD versus controls.
The genotype and allele frequencies were carried out between renal, CVD compared with control subjects. HWE was found to be significant in both renal and CVD diseases. Table 2 describe the genotype association between renal cases and control subjects. The gene CBS (844 in s68) genotype significant association was not appeared between cases and controls (ID vs. II; OR-1.00 (0.00, 255.5), p=0.99, DD vs. ID OR-0.81 (0.31, 2.1), p=0.66). The Table 2 consist of allele frequencies calculated between renal cases and control subjects. C allele of C677T variants was found to be high in renal cases (95%); T allele in controls i.e., 6% (T vs. C: OR-0.82 (0.24-2.79); p=0.75). ID polymorphism alleles of both the cases and controls were found to be similar, i.e., D vs. I: OR-1.00 (0.00-255.5), p=0.00). The 844 in s68polymorphisms of CBS gene showed the negative association after performing the Yate's correction within the genotypes (ID vs. II; OR-4.14 (0.15, 112); p=0.36; DD vs. ID; OR-0.65 (0.26, 1.67); p=0.37). The 844 in s68 polymorphism were not associated after incorporation of Yate's correction (T vs. C; OR-1.54 (0.53-4.52); p=0.42; G vs. A; OR-1.54 (0.53-4.52); p=1.00 and D vs. I; OR-0.83 (0.36-1.91); p=0.67). Statistical significant differences in allele frequencies were not observed with CVD cases and control subjects.
This is a pilot study carried out in Khartoum state hospitals, Khartoum, Sudan and totally, 150 subjects were recruited. The results of this current study concluded there is no significant association in our population. CVD is known as the combination of heart cum blood vessels consist of arteries and veins which can effect with the heart attack, heart failure and stroke. Renal patients are prone to develop stroke or heart attack than they are to receive dialysis. Diabetes is a common risk factor for both renal and CVD’s.
In CβS gene, a 68 bp insertion between base 844 and 845 (844ins68) at the junction of intron 7 and exon eight has been associated with lower post-methionine load increase in total Hcy concentrations . The CβS 844ins68 polymorphism was firstly reported as a novel mutation in an Italian patient with classic homocystinuria due to CBS deficiency. The patient is heterozygous (I/N) for the mutation. Because the insertion (I allele) introduces a premature termination codon in the CβS mRNA, it is assumed that the truncated CBS protein is nonfunctional. However, a subsequent report  showed that individuals carrying the I allele of the mutation have normal size mRNA. Later, large quantities of studies showed that the 844ins68 was commonly distributed among humans as a polymorphism. The current study was found to be significantly associated with specific diseases [15-18] and with some of the studies concluded to be non-significant associations [6,10,19-25]. An earlier study  performed meta-analysis studies in Down syndrome offsprings in MTHFR and MTR gene and found to be the negative association. However, studies were found to be associated with a reduced risk of cleft palate in French and Belgian populations . However, this study concludes the prominent involvement of the vitamin B6- dependent transsulfuration pathway of homocysteine in OFC risk and the interest for evaluating vitamin B6 status in further population studies. A 68 bp insertion in CβS gene (844ins68) and A2756G transition of MS gene has been found to be associated with low levels of plasma homocysteine in a population in the Midwestern region of the USA .
When we correlated renal samples with biochemical data and 844ins68, we found Vitamin B12, D, TP and URCA to be associated (p<0.05; Table 3) and in Table 4, we have compared with CVD cases and found Folic acid and cholesterol to be associated (p<0.05). The strength of our study was, we selected purely Sudanese subjects to get the outcome more accurately, and we followed our inclusion and exclusion criteria. The limitations of this study were low sample size, selection of single SNP and excluded the correlation with combined renal and CVD diseases. In conclusion, our results confirm the role of the negative association of 844ins68 polymorphism in the Sudanese population. Future studies should be performed with larger sample size with multiple CβS gene SNPs in the global population.
|S. no||Biochemical variables||844 ins68 (DD=38)||844 ins68 (ID=12)||P value|
|1||Homocysteine||23.7 ± 13.1||23.9 ± 15.0||0.51|
|2||Vitamin B12||407.3 ± 277.6||332.7 ± 136.7||0.01|
|3||Folic acid||16.9 ± 10.7||17.5 ± 11.2||0.78|
|4||Vitamin D||57.4 ± 37.6||52.5 ± 19.3||0.02|
|5||Albumin||34.0 ± 7.4||32.8 ± 5.8||0.44|
|6||Blood urea nitrogen||21.7 ± 6.8||21.3 ± 5.6||0.5|
|7||Cholesterol||3.2 ± 1.0||3.7 ± 0.9||0.73|
|8||Creatinine||882.7 ± 332.2||897.9 ± 406.2||0.34|
|9||TP||70.8 ± 13.8||73.3 ± 6.7||0.01|
|10||Triglyceride||1.2 ± 0.8||1.4 ± 1.0||0.3|
|11||URCA||365.2 ± 76.6||381.4 ± 122.0||0.03|
Table 3: Biochemical analysis with 844 ins68 polymorphism in ESRD patients.
|S. no||Biochemical variables||844 in s68 (DD=36)||844 in s68 (ID=14)||P value|
|1||Homocysteine||27.0 ± 11.1||28.0 ± 14.5||0.22|
|2||Vitamin B12||525.9 ± 400.2||517.5 ± 390.8||0.99|
|3||Folic acid||14.0 ± 6.1||16.6 ± 12.1||0.0006|
|4||Vitamin D||47.7 ± 32.3||42.8 ± 26.9||0.52|
|5||Albumin||29.4 ± 5.4||32.4 ± 6.7||0.31|
|6||Blood urea nitrogen||12.4 ± 10.8||15.1 ± 13.1||0.36|
|7||Cholesterol||2.9 ± 0.7||4.0 ± 2.2||0.0001|
|8||Creatinine||122.2 ± 59.9||142.3 ± 86.8||0.09|
|9||TP||75.9 ± 10.2||79.9 ± 8.8||0.61|
|10||Triglyceride||1.0 ± 0.6||1.6 ± 0.8||0.18|
|11||URCA||470.1 ± 159.5||451.6 ± 154.3||0.96|
Table 4: Biochemical analysis with 844 ins68 polymorphism in CVD patients.
Conflict of Interest
We are thankful to Dr. Salah Al Basha (AL Sha’ab Teaching Hospital- Khartoum, Mr. Maher AL Johani (KKUH, Riyadh) and Mr. Alyassa Jubartallah (Dr. Salma Centre for Renal Diseases, Khartoum).
- Liu M, Li X, Lu L, Cao Y, Sun R, Chen S, Zhang P. Cardiovascular disease and its relationship with chronic kidney disease. Eur Rev Med Pharmacol Sci 2014; 18: 2918-2926.
- Rothuizen TC, Ocak G, Verschuren JJ, Dekker FW. Candidate gene analysis of mortality in dialysis patients. PLoS One 2015; 10: 0143079.
- de Jager DJ, Grootendorst DC, Jager KJ, Van Dijk PC, Tomas LM, Ansell D, Collart F, Finne P, Heaf JG, de Meester J. Cardiovascular and noncardiovascular mortality among patients starting dialysis. JAMA 2009; 302: 1782-1789.
- Benton MC, Lea RA, Macartney-Coxson D, Hanna M, Eccles DA, Carless MA, Chambers GK, Bellis C, Goring HH, Curran JE. A phenomic scan of the norfolk island genetic isolate identifies a major pleiotropic effect locus associated with metabolic and renal disorder markers. PLoS Genet 2015; 11: 1005593.
- Holwerda KM, Weedon-Fekjer MS, Staff AC, Nolte IM, Van Goor H, Lely AT, Faas MM. The association of single nucleotide polymorphisms of the maternal cystathionine-β-synthase gene with early-onset preeclampsia. Pregn Hypertens Int J Womens Cardiovasc Health 2016; 6: 60-65.
- Kumar J, Garg G, Karthikeyan G, Sengupta S. Cystathionine β-synthase 844Ins68 polymorphism is not associated with the levels of homocysteine and cysteine in an Indian population. Biomarkers 2010; 15: 283-287.
- Sponholz C, Kramer M, Schoneweck F, Menzel, U, Rahatloo KI, Giamarellos-Bourboulis EJ, Papavassileiou V, Lymberopoulou K, Pavlaki M, Koutelidakis I. Polymorphisms of cystathionine beta-synthase gene are associated with susceptibility to sepsis. Eur J Human Gene 2015.
- Popp RA, Crisan TO, Rotar I, Farcas MF, Trifa AP, Militaru MS, Pop IV. Cystathionine beta-synthase 844ins68 genetic polymorphism in spontaneous abortion susceptibility. Appl Med Inform 2011; 29: 34.
- Sebastio G, Sperandeo MP, Panico M, de Franchis R, Kraus JP, Andria G. The molecular basis of homocystinuria due to cystathionine beta-synthase deficiency in Italian families, and report of four novel mutations. Am J Human Gene 1995; 56: 1324.
- Bi XH, Zhao HL, Zhang ZX, Liu Q, Zhang JW. Association analysis of CβS 844ins68 and MTHFD1 G1958A polymorphisms with Alzheimers disease in Chinese. J Neural Trans 2010; 117; 499-503.
- Khan IA, Shaik NA, Kamineni V, Jahan P, Hasan Q, Rao P. Evaluation of gestational diabetes mellitus risk in south Indian women based on MTHFR (C677T) and FVL (G1691A) mutations. Front Pediatr 2015; 3: 34.
- Khan IA, Vattam KK, Jahan P, Hasan Q, Rao P. Importance of glucokinase-258G/A polymorphism in Asian Indians with post-transplant and type 2 diabetes mellitus. Intractable Rare Dis Res 2016; 5: 25-30.
- Yakub M, Moti N, Parveen S, Chaudhry B, Azam I. Polymorphisms in MTHFR, MS and CBS genes and homocysteine levels in a Pakistani population. PLoS One 2012; 7: 33222.
- Chen C, Gan YY. The allele frequencies of three polymorphisms in genes involved in homocysteine metabolism in a group of unrelated healthy Singaporeans. Dis Markers 2010; 29: 111-119.
- Dutta S, Chatterjee A, Sinha S, Chattopadhyay A, Mukhopadhyay K. Correlation between cystathionine beta synthase gene polymorphisms, plasma homocysteine and idiopathic mental retardation in Indian individuals from Kolkata. Neurosci Lett 2009; 453: 214-218.
- Golimbet V, Korovaitseva G, Abramova L, Kaleda V. The 844ins68 polymorphism of the cystathionine beta-synthase gene is associated with schizophrenia. Psychiatry Res 2009; 170: 168-171.
- Golimbet VE, Lebedeva IS, Alfimova MV, Barkhatova AN, Lezheiko TV, Kolesina N, Borozdina SA, Abramova LI. Homocysteine-related genes and attention in patients with schizophrenia and schizoaffective psychosis. Zh Nevrol Psikhiatr Im SS Korsakova 2010; 110: 86-89.
- Murthy J, Lakkakula S, Gurramkonda VB, Pathapati RM, Maram R. CBS c.844ins68 polymorphism frequencies in control populations: implications on nonsyndromic cleft lip with or without cleft palate. Cleft Palate Craniofac J 2015; 52: 49-53.
- Ayala C, Garcia R, Cruz E, Prieto K, Bermudez M. Homocysteine levels and polymorphisms of MTHFR and CBS genes in Colombian patients with superficial and deep venous thrombosis. Biomedica 2010; 30: 259-267.
- Fintelman-Rodrigues N, Correa JC, Santos JM, Pimentel MM, Santos-Reboucas CB. Investigation of CBS, MTR, RFC-1 and TC polymorphisms as maternal risk factors for Down syndrome. Dis Markers 2009; 26: 155-161.
- Grobelny BT, Ducruet AF, Derosa PA, Kotchetkov IS, Zacharia BE, Hickman ZL, Fernandez L, Narula R, Claassen J, Lee K, Badjatia N, Mayer SA, Connolly ES. Gain-of-function polymorphisms of cystathionine beta-synthase and delayed cerebral ischemia following aneurysmal subarachnoid hemorrhage. J Neurosurg 2011; 115: 101-107.
- Houcher B, Bourouba R, Djabi F, Yilmaz E, Egin Y, Akar N. Polymorphisms of 5, 10-methylenetetrahydrofolate reductase and cystathionine beta-synthase genes as a risk factor for neural tube defects in Setif, Algeria. Pediatr Neurosurg 2009; 45: 472-477.
- Hozyasz KK, Mostowska A, Szaflarska-Poplawska A, Lianeri M, Jagodzinski PP. Polymorphic variants of genes involved in homocysteine metabolism in celiac disease. Mol Biol Rep 2012; 39: 3123-3130.
- Izci AYO, Ay ME, Erdal ME, Cayan F, Tekin S, Soylemez F, Sungur MA, Derici Yildirim D. Folate metabolism gene polymorphisms and risk for down syndrome offspring in Turkish women. Genet Test Mol Biomarkers 2015; 19: 191-197.
- Ouyang S, Liu Z, Li Y, Ma F, Wu J. Cystathionine beta-synthase 844ins68 polymorphism is unrelated to susceptibility to neural tube defects. Gene 2014; 535: 119-123.
- Yang M, Gong T, Lin X, Qi L, Guo Y, Cao Z, Shen M, Du Y. Maternal gene polymorphisms involved in folate metabolism and the risk of having a Down syndrome offspring: a meta-analysis. Mutagene 2013; 28: 661-671.
- Goffinet L, Oussalah A, Guéant-Rodriguez RM, Chery C, Basha M, Avogbe PH, Josse T, Jeannesson E, Rouyer P, Flayac J. Cystathionine β-synthase genetic variant rs2124459 is associated with a reduced risk of cleft palate in French and Belgian populations. J Med Gene 2016; 104111.
- Zhang Y, Wang H, Sun HW, Chen YL, Ouyang JY, Wang Y, Wang L, Zhang XY. Correlation between cystathionine beta-synthase T883C genetic polymorphism and primary hypertension. Exp Ther Med 2014; 8: 713-718.