Correlation between cystathionine beta synthase gene polymorphisms, plasma homocysteine and idiopathic mental retardation in Indian individuals from Kolkata

Neuroscience Letters 453 (2009) 214–218

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Correlation between cystathionine beta synthase gene polymorphisms, plasma homocysteine and idiopathic mental retardation in Indian individuals from Kolkata Samikshan Dutta, Arpita Chatterjee, Swagata Sinha, Anindita Chattopadhyay, Kanchan Mukhopadhyay ∗ Manovikas Biomedical Research and Diagnostic Centre, 482 Madudah, Plot I-24, Sec.-J, E.M. Bypass, Kolkata 700107, India

a r t i c l e

i n f o

Article history: Received 19 November 2008 Received in revised form 12 February 2009 Accepted 13 February 2009 Keywords: CBS Mental retardation T833C/844ins68 31 bp VNTR Homocysteine

a b s t r a c t Deficiency in cystathionine beta synthase (CBS) enzyme sometimes leads to hyperhomocysteinemia/homocystinuria, conditions often associated with mental retardation (MR). In this investigation, association of idiopathic MR (IMR) with six CBS gene polymorphisms and fasting total plasma homocysteine (plHcy) was explored. Nuclear families with IMR probands (N = 180) and control subjects (N = 106) were recruited. Genomic DNA was subjected to PCR amplification and RFLP analysis. plHcy was measured by enzyme immunoassay. Data obtained was subjected to statistical analyses. Linkage disequilibrium between polymorphic sites was computed. T833C/844ins68 polymorphism revealed significant maternal transmission in IMR cases. The 31 bpVNTR 21 repeat allele was significantly higher in male IMR cases as compared to sex-matched controls (P = 0.004). A significant difference was also noticed in genotype frequencies of male IMR cases (P = 0.005). Four other sites, G919A, C1105T, G1316A and G1330A, were not polymorphic in the studied population. While no significant contribution of any particular genotype was observed, plHcy level was significantly higher in male IMR cases as compared to sex-matched controls (P = 0.0001). The data presented here is probably indicative of a higher risk of IMR in male subjects in association with two CBS polymorphisms and mild elevation in plHcy concentration. © 2009 Elsevier Ireland Ltd. All rights reserved.

Cystathionine beta synthase (CBS; EC 4.2.1.22) is an important enzyme for maintaining plasma homocysteine (plHcy). Hcy concentration is increased in brain and cerebrospinal fluid during neuronal diseases with a concurrent increase of plHcy [21] and therefore, increased plHcy concentration could be an indicator of brain hyperhomocysteinemia. Increased Hcy has also been reported to be associated with age related dystrophy; plHcy concentrations above 11.9 ␮M impart nearly 3-fold higher risk for white matter damage as compared to below 8.6 ␮M [30]. Hcy has been shown to be transported into the brain from plasma as well as transported out from the brain via specific bi-directional cellular transporters [11]. Moreover, in vitro study has shown that Hcy can be produced by neuronal cells in the brain [12]. Hcy could act as a potent neurotoxic component both under in vivo and in vitro conditions [6] and CBS deficiency has been found to be associated with risk of several clinical and pathologic abnormalities [20] including mental retardation (MR). Human CBS gene, located at 21q22.3, is known to have a large number of missense mutations [17]. A T833C transition mutation is known to segregate in cis with 844ins68 insertion polymorphism

∗ Corresponding author. Tel.: +91 33 4001 9179; fax: +91 33 2442 8275. E-mail address: [email protected] (K. Mukhopadhyay). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.02.040

in the exon 8 [26] and was found to be associated with low IQ [2]. This T > C transition probably alters conformation and folding of the enzyme thereby affecting stability of CBS. However, in vitro experiments have shown that effect was corrected by certain mutations in the C-terminal domain or by deletion of this domain [23]. In the Celtic population, a G919A substitution was observed [13] which is located at the active site and is responsible for altered binding of pyridoxal phosphate thereby making patients’ pyridoxine non-responsive [17]. Further, a C > T variation at 369 is associated with pyridoxine-responsive phenotype was first reported in Norwegian population [15]. In pyridoxinenon-responsive homocystinuric patients, a G1316A polymorphism was detected [7] which is associated with reduced CBS activity [7]. Another polymorphism, G1330A at exon 12 was first detected in homocystinuric patients from Netherlands and was reported to modify regulatory domain of functional CBS protein by interfering AdoMet binding site [16]. Further investigations revealed a novel 31 bp variable number of tandem repeat (VNTR) at exon 13–intron 13 junctions, which may generate different splice variants; genomic analysis showed five different alleles with 16–21 repeat units [18]. The higher allele of the VNTR was reported to be associated with increased post-load Hcy in cardiovascular patients from Netherlands [18]. In the present study, we have analyzed these six CBS polymorphisms in the eastern Indian pop-

S. Dutta et al. / Neuroscience Letters 453 (2009) 214–218

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Table 1 Allele frequencies of exon 8 T833C/844ins68 and exon 13–intron 13 VNTR.

IQ (range) Ex 8 ‘a’ allele Ex 8 ‘b’ allele Chi-square; P value VNTR A1 allele VNTR A2 allele VNTR A3 allele VNTR A4 allele VNTR A5 allele LRS; P value

Control (212)

Parents (512)

IMR (360)

DD (76)

Mild (160)

Moderate (118)

Male controls (88)

Male MR cases only (232)

100–120 0.96 0.04 – 0.01 0.12 0.72 0.13 0.02 –

70–120 0.98 0.02 1.8; 0.18 0.00 0.14 0.70 0.12 0.04 7.00; 0.13

40–70 0.97 0.03 0.87; 0.35 0.00 0.13 0.69 0.13 0.05 6.30; 0.18

71–84a 0.95 0.05 0.13; 0.72 0.00 0.17 0.65 0.14 0.04 3.17; 0.53

55–70 0.97 0.03 0.85; 0.36 0.00 0.08 0.74 0.12 0.06 7.29; 0.12

40–54 0.98 0.02 1.70; 0.19 0.00 0.17 0.64 0.14 0.05 5.43; 0.24

100–120 0.94 0.06 – 0.02 0.16 0.65 0.17 0.00 –

40–70 0.97 0.03 1.84; 0.17 0.00 0.14 0.68 0.12 0.06 15.59; 0.004

Number of chromosomes are in parenthesis; LRS = likelihood ratio statistics. Values in bold indicate significant differences. a Developmental Quotients.

ulation from Kolkata and have tried to correlate the data with plHcy. Idiopathic MR (IMR) cases, belonging to Indo-Caucasoid ethnicity, were recruited following the Diagnostic and Statistical Manual of Mental Disorders-IV-TR [1] criteria from the Out-patients department. Intelligence Quotient (IQ) of IMR children above 5 years was determined using WISC [29] and accordingly cases were classified as mild, moderate or severe. Developmental Quotient (DQ) of children below 5 years was assessed separately and grouped under developmental delay category [5]. Cases with history of seizure and/or birth asphyxia, definite genetic and/or chromosomal bases, like fragile X syndrome, Prader-Willi syndrome, Down’s syndrome, Klinefelter’s syndrome, etc. as well as cases with cognitive deficit developed following traumatic head injury, neonatal infection, etc. were excluded. The study population recruited (IMR; N = 180) composed of 80 mild, 59 moderate, 3 severely retarded and 38 with developmental delay; part of these samples were used for previous publications on CBS [9,10]. Age range of probands was between 2.0 and 18 years. Male:female ratio was 1.81. For case–control analysis, 106 healthy volunteers (IQ > 100) also of Indo-Caucasoid ethnicity were recruited following same psychometric analysis method. Age range of controls was between 2.0 and 25 years. Male:female ratio was 0.71. Genomic DNA isolated from peripheral blood [19] was used for PCR amplification. Primer sequences used to amplify six CBS polymorphisms and details of amplification protocol will be available upon request. For analysis of fasting total plHcy, plasma from 51 IMR cases and 33 age-matched (2–18 years) controls were collected in anticoagulant. Human Ethical Committee of the Institute approved the study protocol. Detailed analysis protocol for T833C/844ins68 was published previously [9]. 31 bp VNTR alleles at exon 13–intron 13 junction [18] yielded five fragments with 16 (A1), 17 (A2), 18 (A3), 19 (A4) and 21 (A5) repeats respectively. For restriction fragment length polymorphism (RFLP) analysis of G919A [13], C1105T [15], G1316A [7], G1330A [16], PCR amplicons were digested with AluI, HhaI, MspI, TaqI restriction enzymes (New England Biolab, US; Genei, India) respectively. Digestion products were resolved by polyacrylamide gel electrophoresis and analyzed. Sequences were verified by Applied Biosystems 3130 DNA sequencer using Big Dye v 3.1 chemistry and Sequencing Analysis Software, v 5.2. plHcy was measured by solid phase enzyme immunoassay [28] using kit (Bio-RAD, USA) and optical density was measured at 450 nm using an ELISA reader (Thermo Electron Corp, Germany). Lower absorption indicated higher plHcy in the sample. Genotype frequency was calculated through GENEPOP program. COCAPHASE program v 2.404 [8], which is part of program UNPHASED, was used to compare allelic frequencies as well

as haplotype frequencies. Moreover, the “rxc” contingency table (http://www.physics.csbsju.edu/stats) was used for comparing allelic and genotypic frequencies in controls, parents and IMR cases. Independent t-test was performed to compare the plHcy level in cases and control. Haplotype-based haplotype relative risk (HHRR) analysis [25] as well as multi-allelic extended transmission disequilibrium test (ETDT) [22] was performed using UNPHASED v 2.404 [8]. HAPLOVIEW program [3] was used to calculate pair-wise linkage disequilibrium (LD). Genotypic analysis revealed that the population is well within Hardy–Weinberg equilibrium. RFLP analysis for G919A, C1105T, G1316A and G1330A revealed that these are not polymorphic in the studied population. Analysis of T833C/844ins68 showed that derived allele frequency was 0.04 in controls and 0.03 in cases (Table 1, 2 = 0.87, P = 0.35). None of the individuals were found to be homozygous for 833C/844ins68. For simplicity in defining the alleles, wild type allele of T833C/844ins68 will be recognized as “a” and derived allele as “b” throughout rest of the article. In case of the 31 bp VNTR, 18 repeat allele was most common in the studied population (0.72 and 0.69 in controls and cases respectively). Case–control analysis failed to show any difference in overall allele frequency (Table 1; LRS = 6.30, P = 0.18). However, sex-matched comparison between male IMR cases and controls revealed a statistically significant difference in allele (Table 1; LRS = 15.59, P = 0.004) as well as genotype frequency (Table 2; 2 = 21.8, P = 0.005). No such difference was noticed in allele (LRS = 1.82, P = 0.61) and genotype frequency (2 = 8.04, P = 0.33) between female cases and sex-matched controls. Further, no significant difference in genotype frequencies were noticed when groups were analyzed as a whole (Table 2, 2 = 5.00, P = 0.76). Haplotype comparison between cases and controls revealed that a-A3 haplotype (i.e. wild type T833C/844ins68 and 18 repeat of VNTR) was most common in both controls (0.75) and IMR cases (0.72). Pair-wise LD measured for two CBS polymorphisms revealed high D value in case (1.00) and control (0.82) populations; however, r2 was insignificant (0.01 for both groups). Family-based study for T833C/844ins68 revealed significant preferential maternal transmission of the derived ‘b’ allele to IMR cases while no bias in transmission was observed for the VNTR alleles (Table 3). Comparison of fasting plHcy concentration in IMR cases (8.32 ± 0.39 ␮mole/L) and age-matched controls (5.78 ± 0.5 ␮mole/L) revealed statistically significant difference (P = 0.0001). Sex-matched analysis revealed that difference was mainly between male IMR cases and controls (P = 0.0001) rather than between female cases and controls (P = 0.19). However, no

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Table 2 Genotype frequencies of exon 8 T833C/844ins68 and exon 13–intron 13 VNTR.

Exon 8 genotypes

Control (106)

Parents (256)

IMR (180)

DD (38)

Mild (80)

Moderate (59)

Male controls (44)

Male MR cases only (116)

aa ab

0.92 0.08 –

0.95 0.05 1.98; 0.16

0.94 0.06 0.93; 0.34

0.89 0.11 0.14; 0.71

0.95 0.05 0.85; 0.36

0.97 0.03 1.58; 0.21

0.89 0.11 –

0.95 0.05 1.91; 0.17

A1A2 A2A2 A2A3 A2A4 A2A5 A3A3 A3A4 A3A5 A4A4 A4A5

0.02 0.04 0.09 0.06 0.00 0.57 0.14 0.05 0.03 0.00

0.00 0.05 0.11 0.06 0.01 0.53 0.14 0.07 0.02 0.01

0.00 0.04 0.12 0.04 0.01 0.50 0.16 0.09 0.03 0.01

0.00 0.05 0.21 0.03 0.00 0.42 0.16 0.08 0.05 0.00

0.00 0.01 0.09 0.04 0.01 0.55 0.18 0.11 0.01 0.00

0.00 0.08 0.12 0.05 0.00 0.46 0.15 0.08 0.04 0.02

0.04 0.02 0.11 0.11 0.00 0.52 0.14 0.00 0.04 0.00

0.00 0.07 0.13 0.01 0.01 0.46 0.17 0.11 0.03 0.00

–

3.56; 0.89

5.00; 0.76

5.92; 0.43

6.23; 0.51

5.47; 0.60

–

Chi-square; P value

VNTR genotypes

Chi-square; P value

21.8 0.005

Number of individuals are in parenthesis. Values in bold indicate significant differences.

Table 3 Family-based analysis for exon 8 T833C/844ins68 and exon 13–intron 13 VNTR.

Exon 8 T833C/844ins68

Number of families

Alleles

Transmitted

Not transmitted

LRS

P value

HHRR

180

250 3 6 3 6 1

0.31

180

247 6 3 6 1 6

1.04

TDT

a b a b a b

1.02

0.31

7.14

0.007

A2 A3 A4 A5 A2 A3 A4 A5

23 168 19 7 9 31 15 7

29 157 20 11 15 20 16 11

1.99

0.57

3.09

0.38

HHRR (heterozygous mothers only)

Exon 13–intron 13 31 bp VNTR

7

HHRR

180

TDT

180

Values in bold indicate significant differences.

significant difference in plHcy level was observed between male and female IMR probands (P = 0.57). Genotype-plHcy correlation analysis also revealed significant difference in Hcy level between IMR cases and controls (Table 4). Distribution of T833C/844ins68 polymorphism is extremely heterogeneous in the world population. In the present investigation, we have observed a low frequency of the derived allele while no significant differences in allelic and genotypic frequencies were observed by case–control analysis (Tables 1 and 2). Familial analysis also failed to show any bias in transmission of alleles to IMR probands (P = 0.31 in both HHRR and TDT analysis). However, an odd ratio of 2.02 (CI95% = 0.5–8.14) and relative risk of 2.0 (CI95% = 0.71–5.62) for the derived allele may be indicative of a risk of IMR associated with the derived allele. We have already reported

earlier, using a smaller sample size, that the T833C/844ins68 (‘b’ allele) confers a risk of MR [9]. In the present study, using a wellcategorized group of IMR probands, preferential transmission of the ‘b’ allele was noticed when only maternal transmission was considered (P = 0.007) which is supporting our previous observation. For the 31 bp VNTR, case–control analysis revealed lack of significant association with IMR (Tables 1 and 2). VNTR repeat frequencies are almost comparable to American and European populations (∼0.77) [18,31]. Homozygous genotype with 18 repeat allele was predominant in cases (0.55–0.42) and controls (0.57), though differences were statistically not significant. Our earlier investigation also revealed lack of significant differences in allelic as well as genotypic frequencies between IMR cases and controls in the same ethnic population [10]. However, in the present investiga-

Table 4 Correlation between genotypes and plasma Hcy concentration. Polymorphisms

IMR cases

Controls

P value

Genotype

plHcy (␮mole/L) Mean ± SE

Genotype

plHcy (␮mole/L) Mean ± SE

Exon 8 T833C/844ins68

aa (43) ab (8)

8.43 ± 0.43 7.76 ± 1.21

aa (32) ab (1)

4.73 ± 0.35 11

0.0001 –

Exon 13–intron 13 31 bp VNTR

A1A1 (34) A1A2 (16) A2A2 (1)

8.22 ± 0.51 8.61 ± 0.71 7.5

A1A1 (16) A1A2 (17) A2A2 (0)

5.72 ± 0.20 5.84 ± 0.17 0.00

0.0034 0.0007 –

Number of individuals are in parenthesis; ‘a’ allele of CBS T833C/844ins68 represents wild type whereas ‘b’ represents derived allele. The 31 bp VNTR was analyzed as bi-allelic marker, where A1 was considered as ≤18 repeats allele and A2 was considered as 19 and higher repeats allele. Values in bold indicate significant differences.

S. Dutta et al. / Neuroscience Letters 453 (2009) 214–218

tion, sex-matched analysis revealed significant difference in allelic as well as genotypic frequencies between male IMR cases and male controls which is basically due to the absence of the A5 allele in male control subjects (Table 2). It has already been reported that with increase in repeat number there is a chance of formation of CBS splices variants [18]. Whether higher frequency of A5 repeat in male IMR cases is indicative of a risk of IMR in male subjects due to the presence of different CBS splice variants is yet to be proved. Higher repeats of VNTR alleles along with T833C/844ins68 were shown to confer risk of Alzheimer’s disease after the age of 75 years, and 21st repeat VNTR allele was speculated as a risk factor for Alzheimer’s disease after 64 years [4]. In this study, using a well-defined MR population, significant difference in A5 (21 repeat) allele frequency was observed between male IMR cases and controls (Table 1; P = 0.004). Since we have noticed significant bias in maternal transmission of “b” allele of T833C/844ins68 and a higher occurrence of A5 allele in male IMR cases, it may be speculated that there is a risk of IMR in association with these alleles in the population under investigation. Haplotype analysis did not reveal any significant difference between cases and controls. High D value between T833C/844ins68 and 31 bp VNTR in case and control groups along with low r2 may indicate limited chances for these polymorphic regions to be transmitted together. Current study showed significant difference in total fasting plHcy level between cases and age-matched controls (P = 0.0001). Moreover, when comparison was carried out for subjects matched for sex, only male IMR cases showed significant difference in Hcy (P = 0.0001). These results may indicate an association between mild elevation in Hcy (8.32 ± 0.39) and risk of IMR in male individuals recruited for this study since none of the IMR cases had plHcy level >14 ␮mole/L. Genotype–phenotype correlation analysis also revealed differences among various ethnic populations. Under heterozygous condition, T833C/844ins68 polymorphism showed a decrease in fasting plHcy level in IMR cases as compared to the homozygous genotype (Table 4). Tsai et al. [27] also have reported similar association in US Caucasian population. However, in a case–control analysis on Chinese stroke patients, the insertion variant was found to have significant association with higher fasting plHcy level [24]. For the 31 bp VNTR, no positive association with plHcy level was ascertained, which is similar to our previous finding [10]. Further, no correlation could be drawn between plHcy level and CBS polymorphisms. Comparative analysis between ‘ab’ genotype of T833C/844ins68 as well as ‘A2A2’ genotype of 31 bp VNTR and plHcy level could not be performed due to insufficient sample size. The significant difference in plHcy level between male IMR cases and controls, not high enough to be considered as hyperhomocysteinemia, could be due to some factors that influence Hcy level since even reduced urinary excretion may increase plHcy concentration [14]. From this first molecular genetic study on six CBS polymorphisms and plHcy in association with IMR it can be speculated that 833C/844ins68 and A5 allele of exon 13–intron 13 VNTR together with mild increase in plHcy may confer some risk of IMR in male subjects in this population. However, for confirming the above-mentioned notion, it would be important to recruit a larger population with various ethnic groups.

Acknowledgements Authors are thankful to all families for their participation in study. This investigation was partly sponsored by a grant from Department of Biotechnology, Government of India; Grant No.

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