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Cystathionine β-synthase T833C/844INS68 polymorphism: a family-based study on mentally retarded children

Cystathionine β-synthase T833C/844INS68 polymorphism: a family-based study on mentally retarded... Background: Cystathionine β-synthase (CBS) mediates conversion of homocysteine to cystathionine and deficiency in enzyme activity may lead to hyperhomocysteinemia/homocystinuria, which are often associated with mental retardation (MR). A large number of polymorphisms have been reported in the CBS gene, some of which impair its activity and among these, a T833C polymorphism in cis with a 68 bp insertion at 844 in the exon 8 is found to be associated with mild hyperhomocysteinemia in different ethnic groups. Methods: The present study is aimed at investigating the association between T833C/844ins68 polymorphism and MR. One hundred and ninety MR cases were recruited after psychometric evaluation. Hundred and thirty- eight control subjects, two hundred and sixty-seven parents of MR probands and thirty cardiovascular disorder (CVD) patients were included for comparison. Peripheral blood was collected after obtaining informed written consent. The T833C/844ins68 polymorphism was investigated by PCR amplification of genomic DNA and restriction fragment length polymorphism analysis, followed by statistical analysis. Results: The genotypic distribution of the polymorphism was within the Hardy-Weinberg equilibrium. A slightly increased genotypic frequency was observed in the Indian control population as compared to other Asian populations. Both haplotype-based haplotype relative risk analysis and transmission disequilibrium test reveled lack of association of the T833C/844ins68 polymorphism with MR; nevertheless, the relative risk calculated was higher (>1) and in a limited number of informative MR families, preferential transmission of the double mutant from heterozygous mothers to the MR probands was noticed (χ = 4.00, P < 0.05). Conclusion: This is the first molecular genetic study of CBS gene dealing with T833C/844ins68 double mutation in MR subjects. Our preliminary data indicate lack of association between T833C/844ins68 polymorphism with MR. However, higher relative risk and biased transmission of the double mutation from heterozygous mothers to MR probands are indicative of a risk of association between this polymorphism with mental retardation. Page 1 of 6 (page number not for citation purposes) Behavioral and Brain Functions 2005, 1:25 http://www.behavioralandbrainfunctions.com/content/1/1/25 Background Cystathionine β-synthase (CBS) catalyzes the condensa- tion of serine and homocysteine to form cystathionine and abnormality in CBS activity is manifested in two major clinical conditions, viz. hyperhomocysteinemia and homocystinuria. Insufficiency in CBS activity may lead to hyperhomocysteinemia [1], which is considered to be an independent risk factor for arteriosclerosis [2]. In addition to that, since homocysteine is vasculotoxic as well as neurotoxic, hyperhomocysteinemia predisposes to cardiovascular disorder (CVD) and cognitive dysfunction [3,4]. On the other hand, gross deficiency in CBS activity is associated with homocystinuria, an inborn recessive metabolic disorder [4]. The major pathologic abnormali- ties associated with homocystinuria include thromboem- bolism, ectopia lentis, osteoporosis, mental retardation (MR) and other neurological and psychiatric abnormali- ties [4]. The neurological malfunctioning can be ascribed to the oxidation of excess homocysteine to homocysteic acid, which interacts with the N-methyl-D-aspartate receptor, causing excessive calcium influx and free radical production, thereby leading to neurotoxicity [5]. In addi- Representation Figure 1 of double mutation in exon 8 of CBS gene tion, MR associated with B12 and folate deficiency could Representation of double mutation in exon 8 of CBS be attributed to the neurotoxic effects of homocysteine gene: lane 1 = homozygous genotype without insertion, lane since B12 and folate act as co-factors in the homocysteine 2 = heterozygous genotype with 68 bp insertion, lane 3 = to methionine remethylation pathway. φX174 HaeIII digest showing bands at 310, 281, 234,194 and 118 respectively, lane 4 = BsrI digestion of heterozygous The human CBS gene, located at 21q22.3 [6], is known to PCR product (of lane 2), showing complete digestion of the have a large number of mutations, the majority of which higher allele of 239 bp into 130 bp and 109 bp. are missense in nature [7]. Three different nonsense mutants, as well as some insertion, deletion and splice variants have also been identified, some of which are pol- and a small subset of CVD patients, since hyperhomo- ymorphic in nature. A transition mutation, T833C gener- cysteinemia/homocystinuria is often associated with MR ating a BsrI restriction site [8,9] is known to segregate in as well as CVD [4]. Familial transmission pattern of the cis with 844ins68 mutation in exon 8 [10] and is reported double mutation was analyzed, by haplotype-based hap- to be associated with premature occlusive arterial disease lotype relative risk analysis (HHRR) and transmission dis- [11]. Ethnic heterogeneity of 844ins68 polymorphism is equilibrium test (TDT), using heterozygous parents only. highly prevalent in African, European and North Ameri- can populations [9,12,13]. Methods Study design Since the T833C/844ins68 double mutation was not Our study population consisted of 138 healthy control detected by Franco et al. [13] and Pepe et al. [14] in their individuals, 30 CVD patients (average age 53.5 yrs) and studies on some Asian populations (Japanese, Tharu, Chi- 190 MR children (age range 6–8 yrs) along with their par- nese and Indonesian), this allele has been projected by ents. The cardiologist (J.M) involved in the study helped Pepe et al. [14] as a reliable anthropogenetic marker for in collection of samples from CVD patients. MR cases discriminating between two major human groups – Afri- were selected from the Outpatient Department of Mano- cans and Asians. Later on, the 844ins68 variant was vikas Kendra Rehabilitation and Research Institute, Kolk- observed in Han Chinese population with a very low fre- ata and diagnosed by mental health professionals (child quency [15]; however, the T833C polymorphism was not psychiatrist, child psychologist and neurologist) accord- studied. In India, study on T833C mutation revealed ing to the DSM – IV [17] followed by an IQ assessment 4.76% and 9.99% heterozygotes in control and coronary with Wechsler Intelligence Scale for Children [18]. Both heart disease patients respectively [16]. In the present patients and controls were from different parts of India study, we have looked into the frequency of the double and belonged to no particular ethnic group. Peripheral mutant in control population. We have also investigated blood was collected in EDTA, after obtaining informed the occurrence of this double mutant in MR individuals written consent from control individuals, CVD patients, Page 2 of 6 (page number not for citation purposes) Behavioral and Brain Functions 2005, 1:25 http://www.behavioralandbrainfunctions.com/content/1/1/25 Table 1: Global variation of CBS 844ins68 polymorphism in control individuals. Population 2n Heterozygosity (%) Reference US (mixed) 144 11.7 Tsai et al. [9] Dutch 214 14 Kluijtmans et al. [22] Japanese 80 0.00 Franco et al. [13] Chinese 26 0.00 Pepe et al. [14] Indonesian 98 0.00 Pepe et al. [14] Ethiopian 108 11.11 Pepe et al. [14] Spanish 54 25.9 Pepe et al. [14] Sub-Saharan African 180 66.66 Pepe et al. [14] German 400 1.5 Linnebank et al. [23] Chinese (Southern China) 200 5 Zhang & Dai [15] Italian 820 13.7 Grossmann et al. [24] Chinese (Northern China) 248 2.97 Li et al. [25] Indian 276 7.97 Present study 2n = No. of chromosomes Restriction fragment length polymorphism (RFLP) analysis and parents of MR probands. Genomic DNA was isolated by standard high salt precipitation method [19]. The BsrI digestion was carried out for all samples to detect Human Ethical Committee of the Institute approved the T833C mutation [9]. PCR products (3 µL) were digested at study protocol. 65°C for 3 hrs. in a final reaction volume of 25 µL, con- taining 1 U of BsrI (New England Biolabs; NEB) and NEB PCR amplification Buffer 4. Since this point mutation occurs in cis with The primer sequence to amplify the 844ins68 polymor- 844ins68, only the higher allele of 239 bp (A2) was phism in the exon 8 and flanking intron 7 of the CBS gene cleaved by BsrI yielding two fragments of 130 bp and 109 were: sense, 5'-GTTGTTAACGGCGGTATTGG-3', and anti- bp respectively (Fig 1); wild type allele of 171 bp (A1), sense, 5'- GTTGTCTGCTCCGTCTGGTT-3'. PCR amplifica- however, remained undigested. tion was carried out using Perkin Elmer thermal cycler (Gene Amp #2400) in a reaction volume of 20 µL contain- Statistical analysis ing 75–100 ng of genomic DNA, 20 pmoles of each Frequency of the T833C/844ins68 polymorphism in the primer, 1.0 U Taq polymerase (Bangalore Genei, India), control population and patients was calculated for the 200 µM dNTP mix, 0.001% gelatin and 10 mM Tris buffer Hardy-Weinberg equilibrium. Haplotype-based haplo- (Bangalore Genei, India) with 50 mM KCl and 1.0 mM type relative risk (HHRR) analysis [20] as well as transmis- MgCl . After an initial denaturation at 95°C for 3 min, sion disequilibrium test (TDT) [21] was used to ascertain amplification was performed at: 35 cycles of denaturation association between MR and transmission of the double at 94°C for 45 sec, annealing at 58°C for 40 sec and exten- mutation. For TDT, trios with at least one parent having sion at 72°C for 40 sec, followed by a final extension for heterozygous genotype were selected. 5 min at 72°C. PCR products were separated by 12% poly- acrylamide gel electrophoresis at 200V for 3 hrs, stained Results with ethidium bromide and documented in a Gel Doc™ Distribution of 844ins68 heterozygote in various control EQ (BIO-RAD). The wild type allele gave 171 bp fragment populations studied worldwide is presented in Table I. It (A1), and insertion variant gave 239 bp fragment (A2) was reported earlier that this variant is absent in the Asian (Fig. 1). population [13,14]. Later studies involving larger sample size revealed the occurrence of 844ins68 variant in the Chinese population [15,25] with a very low frequency of Table 2: Distribution of CBS T833C/844ins68 polymorphism in different groups. Groups No of chromosome No of mutants Mutant allele frequency ± SE Control 276 11 0.0398 ± 0.0117 CVD Patients 60 1 0.0166 ± 0.0166 MR probands 380 12 0.0316 ± 0.0089 Parents of probands 534 15 0.028 ± 0.0071 Page 3 of 6 (page number not for citation purposes) Behavioral and Brain Functions 2005, 1:25 http://www.behavioralandbrainfunctions.com/content/1/1/25 Table 3: HHRR and TDT Analysis for the CBS T833C/844ins68 polymorphism in nuclear families with MR probands. Allele Transmitted Non-transmitted χ P value Relative risk (RR) HHRR analysis 1 259 264 1.714 0.1905 1.346 TDT analysis 1 3 6 1.00 0.3173 heterozygote as compared to the European and African Saharan population [14]. Two earlier independent studies subjects. We have analyzed the 844ins68 variant in asso- on Japanese and other Asian populations failed to detect ciation with the T833C mutation and our data showed a the mutated allele [13,14] and this led to the proposal higher rate of occurrence of the double mutant in the that this unique double mutation could be used as a reli- Indian control population (7.97%) as compared to the able anthropogenetic marker for differentiating Asians Chinese population. and Africans. However, the validity of the proposal is questioned by subsequent findings of the 844ins68 poly- Frequency of the double mutant allele in different types of morphism in different Chinese populations [15,25] and subjects is presented in Table II. Eleven out of 138 control T833C transition mutation in Indian population [16,26]. individuals (n = 276) appeared heterozygous for the The present study clearly demonstrates that the T833C/ mutation. On the contrary, only one person out of 30 844ins68 polymorphism may not be suitable as an CVD patients showed heterozygous genotype. In the MR anthropogenetic marker for differentiating Asians and group, 12 out of 190 MR probands showed presence of Africans. the polymorphism and no significant deviation in allele frequencies was noted in the MR probands and their par- Among Indians, presence of T833C heterozygote has been ents (Table II). reported in 1 out of 21 (4.76%) [16] and 3 out of 100 (3%) [26] control individuals. However, in both studies HHRR analysis for the T833C/844ins68 polymorphism the association of 844ins68 mutation with the T833C showed lack of significant association between MR and transition mutation has not been mentioned, while in the transmission of the double mutant (χ = 1.714, P = present investigation we have observed invariable segrega- 0.1905) (Table III). TDT analysis also revealed lack of tion of these two mutations in cis, which is consistent with association between the T833C/844ins68 polymorphism earlier observations [10,13,14,22]. and MR (χ = 1.00, P = 0.3173) (Table III). However, transmission from the mother (Table IV), heterozygous Hyperhomocysteinemia is hypothesized as an independ- for the alleles, revealed that the A2 allele was preferen- ent risk factor for CVD [22,27] and the 844ins68 variant tially transmitted to the MR probands (χ = 4.00, P = was previously reported to be associated with premature 0.0455), while no significant contribution of the paternal occlusive arterial disease [11]. A recent article on Chinese allele was noticed (data not presented). congenital heart disease (CHD) patients have shown that the 844ins68 could be a risk factor for CHD, and the inser- Discussion tion especially in mothers could increase the risk in off- The present finding of 11 out of 138 Indian control indi- spring [25]. On the other hand, the findings of Zhang and viduals with the T833C/844ins68 polymorphism reveal a Dai [15] on adult Chinese patients have suggested that the higher percentage of heterozygotes (7.97%) as compared 844ins68 could provide protection to vascular throm- to the Chinese control population (Table I); but, this boembolic disease. The mutant allele frequencies among value is significantly lower than those quoted for Sub- the Indian controls and the CVD patients are 0.0398 and Table 4: Transmission pattern of CBS T833C/844ins68 from mother to MR proband. Allele Transmitted Non-transmitted χ P value HHRR analysis (all 1 176 180 2.0449 0.1527 mothers) HHRR analysis 1 2 6 4.00 0.0455 (heterozygous mothers only) Page 4 of 6 (page number not for citation purposes) Behavioral and Brain Functions 2005, 1:25 http://www.behavioralandbrainfunctions.com/content/1/1/25 0.0166 respectively (Table II) and no association was associated with transmission of the mutant allele. The observed between T833C/844ins68 polymorphism and preferential transmission of T833C/844ins68 polymor- CVD. Heterozygous T833C/844ins68 alleles were phism from the heterozygous mother to the MR proband observed at higher percentage in Indian CVD patients (χ = 4.00, P = 0.0455) also supports the above possibility. (3.33%) as compared to Chinese ischemic patients (~1%). The lower value of 3.33% for CVD patients as Conclusion compared to that of the control (7.97%) seems to support This is the first report on CBS T833C/844ins68 polymor- the idea of a possible protective role of the double muta- phism in association with MR and to the best of our tion to vascular thromboembolic disease, as proposed by knowledge, this is also the first information on the occur- Zhang and Dai [15]; however, such a conclusion is not rence of the CBS double mutation in the Indian popula- called for due to the relatively small CVD sample size in tion. Our preliminary data indicate lack of significant the present study. association between T833C/844ins68 polymorphism with MR; however, the relative risk calculated and the While the T833C mutation results in Ile278Thr change in preferential transmission of the double mutation from the mutant protein, in the case of T833C/844ins68, the 68 informative mothers to MR probands do suggest a risk of bp insertion results in an alternative splice site generating association of this polymorphism with MR and warrants mostly wild type mRNA with traces of another transcript, further investigation. detectable only in the nucleus [9,12]. This accounts for the rescue of the transition mutation at 278 in the case of the List of Abbreviations used double mutant [9,10]. However, these observations can- MR (Mental retardation), CBS (Cystathionine β-syn- not account for the conflicting reports on the association thase), CVD (cardiovascular disorder), HHRR (haplotype- between cardiovascular disease in different ethnic popula- based haplotype relative risk), TDT (transmission disequi- tions and the mutant allele [28,29]. Case control studies librium test), CHD (congenital heart disease). showed that this double mutation might be a neutral pol- ymorphism in the CBS gene and splicing of the intron 7 Competing interests eliminates the mutant allele carrying the T833C [22]. On The author(s) declare that they have no competing inter- ests. the other hand, in Northern Europe, the T833C mutation is common in patients presenting clinically with homo- cystinuria and is associated with a B6-responsive pheno- Contributors type [22,28]. SD (JRF, DBT Grant) was responsible for acquisition of data and analysis and drafting the article. SS performed The CBS T833C/844ins68 polymorphism has been psychometric analysis and clinical diagnosis, intellectual reported in homozygous condition, with a very low fre- contribution. AC helped in psychological evaluation and quency, from American black artheroslerosis patients [9]. IQ determination. PKG was responsible for neurological We have failed to observe any subject homozygous for the investigations and intellectual contribution. JG diagnosed mutation in our study group, which possibly points the CVD cases. MS revised the manuscript critically. KM towards the deleterious effect of this mutation in was responsible for concept and study design, interpreta- homozygous condition. tion of data and revising the article critically. Final manu- script was approved by all authors. The present finding of preferential transmission of T833C/844ins68 polymorphism to the MR proband from Acknowledgements Authors are thankful to the families who have participated in the study. This the heterozygous mother is the first evidence of any asso- work was partly sponsored by a grant from the Department of Biotechnol- ciation of CBS gene with MR, although the association of ogy, Government of India; Grant No. BT/PR4269/SPD/11/478/2003. hyperhomocysteinemia/homocystinuria with psychiatric manifestations including MR is well established [3,4,30]. References Among different markers for cobalamine/folate status, 1. Clarke R, Daly L, Robinson K, Naughten E, Cahalane S, Fowler B, Gra- plasma homocysteine showed the best association with ham I: Homocysteinemia: A risk factor for vascular disease. N Eng J Med 1991, 325:967. aging related neuropsychiatric dysfunction [31] and an 2. Boushey CJ, Beresford SAA, Omenn GS, Motulsky AG: A quantita- earlier investigation revealed that the CBS 844ins68 allele tive assessment of plasma homocysteine as a risk factor for frequency was lower in children with high IQ as com- vascular disease. Probable benefits of increasing folic acid intakes. J Am Med Assoc 1995, 274:1049-57. pared to those with average IQ [32]. In the present inves- 3. Carson NA, Neill DW: Metabolic abnormalities detected in a tigation, though the HHRR and TDT analysis failed to survey of mentally backward individuals in North Ireland. Arch Dis Child 1962, 37:505-13. show any significant transmission of the mutant allele to 4. Mudd SH, Levy HL, Kraus JP: From Disorders of transulfuration. the MR probands (χ = 1.714 and 1.00 respectively), the In The metabolic and molecular bases of inherited disease 8th edition. relative risk (RR = 1.346) calculated showed a risk of MR Page 5 of 6 (page number not for citation purposes) Behavioral and Brain Functions 2005, 1:25 http://www.behavioralandbrainfunctions.com/content/1/1/25 Edited by: Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Kinzler 25. Li Y, Cheng J, Zhu WL, Dao JJ, Yan LY, Li MY, Li SQ: Study of serum KW, Vogelstein B. New York: McGraw-Hill; 2001:2007-2056. Hcy and polymorphisms of Hcy metabolic enzymes in 192 5. Lipton SA, Kim WK, Choi YB, Kumar S, D'Emilia DM, Rayudu PV, families affected by congenital heart disease. Beijing Da Xue Arnelle DR, Stamler JS: Neurotoxicity associated with dual Xue Bao 2005, 37:75-80. actions of homocysteine at the N-methyl-D-aspartate recep- 26. Khare A, Ghosh K, Shetty S, Kulkarni B, Mohanty D: Combination tor. Proc Natl Acad Sci 1997, 94:5923-28. of thrombophilia markers in acute myocardial infarction of 6. Munke M, Kraus JP, Ohura T, Francke U: The gene for cystathio- the young. Ind J Med Sci 2004, 58:381-86. nine β-synthase (CBS) maps to the subtelomeric region on 27. Boers GHJ, Smals AGH, Trijbels FJM, Fowler B, Bakkeren JAJM, human chromosome 21q and to proximal mouse chromo- Schoonderwaldt HC, Kleijer WJ, Kloppenborg PWC: Heterozygos- some 17. Am J Hum Genet 1988, 42:550-59. ity for homocystinuria in premature peripheral and cerebral 7. Kraus JP, Oliveriusova J, Sokolova J, Kraus E, Vlcek C, de Franchis R, occlusive arterial disease. N Eng J Med 1985, 313:709-15. Maclean KN, Bao L, Bukovska G, Patterson D, Paces V, Ansorge W, 28. Gaustadnes M, Ingerslev J, Rutiger N: Prevalence of congenital Kozich V: The human cystathionine β-synthase (CBS) gene: homocystinuria in Denmark. N Engl J Med 1999, 340:1513. complete sequence, alternative splicing, and polymor- 29. Kluijtmans LAJ, Boers GHJ, Kraus JP, van den Heuvel LPWJ, Cruys- phisms. Genomics 1998, 52:312-24. berg JRM, Trijbels FJM, Blom HJ: The molecular basis of Cystath- 8. Kozich V, Kraus JP: Screening for mutations by expressing ionine β-Synthase deficiency in Dutch patients with patient cDNA segments in E. coli: homocystinuria due to homocystinuria: Effect of CBS genotype on biochemical and cystathionine β-synthase deficiency. Hum Mutat 1992, 1:113-23. clinical phenotype and on response to treatment. Am J Hum 9. Tsai MY, Bignell M, Schwichtenberg K, Hanson Q: High prevalence Genet 1999, 65:59-67. of a mutation in the cystathionine β-synthase gene. Am J Hum 30. Abbott MH, Folstein SE, Abbey H, Pyeritz RE: Psychiatric manifes- Genet 1996, 59:1262-67. tations of homocystinuria due to cystathionine beta-syn- 10. Sebastio G, Sperandeo MP, Panico M, de Franchis R, Kraus JP, Andria thase deficiency: prevalence, natural history, and G: The molecular basis of homocystinuria due to cystathio- relationship to neurologic impairment and vitamin B6- nine β-synthase deficiency in Italian families, and report of responsiveness. Am J Med Genet 1987, 26:959-69. four novel mutations. Am J Hum Genet 1995, 56:1324-33. 31. Robins Wahlin RB, Wahlin A, Winblad B, Backamn L: The influence 11. Orendac M, Muskova B, Richterova F, Zvarova J, Stefek M, Zaykova of serum vitamin B12 and folate status on cognitive function- E, Stribrny J, Hyanek J, Kraus JP, Kozich V: Mutation C677T in ing in very old age. Biol Psychol 2001, 56:247-65. MTHFR gene and polymorphism 844ins68 in the CBS gene: 32. Barboux S, Plomin R, Whitehead AS: Polymorphisms of genes Risk factor for peripheral arterial occlusive disease? Neth J controlling homocysteine/folate metabolism and cognitive Med 1998:S47. function. Neuroreport 2000, 11:1133-36. 12. Sperandeo MP, de Franchis R, Andria G, Sebastio G: A 68-bp inser- tion found in a homocystinuric patient is a common variant and is skipped by alternative splicing of the cystathionine β- synthase mRNA. Am J Hum Genet 1996, 59:1391-93. 13. Franco RF, Elion J, Lavinha J, Krishnamoorthy R, Tavella MH, Zago MA: Heterogeneous ethnic distribution of the 844ins68 in the cystathionine β-synthase gene. Hum Hered 1998, 48:338-42. 14. Pepe G, Camacho Vanegas O, Rickards O, Giusti B, Comeglio P, Brunelli T, Marcucci R, Prisco D, Gensini GF, Abbate R: World dis- tribution of the T833C/844INS68 CBS in cis double muta- tion: a reliable anthropological marker. Hum Genet 1999, 104:126-29. 15. Zhang G, Dai G: Gene polymorphisms of homocysteine metabolism-related enzymes in Chinese patients with occlu- sive coronary artery or cerebral vascular diseases. Thromb Res 2001, 104:187-95. 16. Nair KG, Nair SR, Ashavaid TF, Dalal JJ, Eghlim FF: Methylenetet- rahydrofolate reductase gene mutation and hyperhomo- cysteinemia as a risk factor for coronary heart disease in the Indian population. J Assoc Physicians India 2002:9-15. 17. American Psychiatric Association: Diagnostic and statistical manual of mental disorders, Text Revised: DSM-IV, Washington, D.C 1994. 18. Wechsler D: Wechsler Intelligence Scale for Children-Third Edition: Man- ual San Antonio, TX: Psychological Corporation; 1991. 19. Miller SA, Dykes DD, Polesky HF: A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acid Res 1988, 16:1215. 20. Terwilliger JD, Ott J: A haplotype-based "haplotype relative risk" approach to detecting allelic association. Hum Heredity 1992, 42:337-46. 21. Spielman RS, McGinnis RE, Ewens WJ: Transmission test for link- Publish with Bio Med Central and every age disequilibrium: The insulin gene region and insulin- scientist can read your work free of charge dependent diabetes mellitus (IDDM). Am J Hum Genet 1993, 52:506-16. "BioMed Central will be the most significant development for 22. Kluijtmans LAJ, Boers GHJ, Trijbels FJM, Van Lith-Zanders HMA, van disseminating the results of biomedical researc h in our lifetime." den Heuvel LPWJ, Blom HJ: A common 844ins68 insertion vari- Sir Paul Nurse, Cancer Research UK ant in the cystathionine β-synthase gene. Biochem and Mol Med 1997, 62:23-25. Your research papers will be: 23. Linnebank M, Homberger A, Junker R, Nowak-Goettl U, Harms E, available free of charge to the entire biomedical community Koch HG: High prevalence of the I278T mutation of the human cystathionine β-synthase detected by a novel screen- peer reviewed and published immediately upon acceptance ing application. Thromb Haemost 2001, 85:986-88. cited in PubMed and archived on PubMed Central 24. Grossmann R, Schwender S, Geisen U, Schambeck C, Merati G, Wal- ter U: CBS 844ins68, MTHFR TT677 and EPCR 4031ins23 yours — you keep the copyright genotypes in patients with deep-vein thrombosis. Thromb Res BioMedcentral 2002, 107:13-15. Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 6 of 6 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Behavioral and Brain Functions Springer Journals

Cystathionine β-synthase T833C/844INS68 polymorphism: a family-based study on mentally retarded children

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Biomedicine; Neurosciences; Neurology; Behavioral Therapy; Psychiatry
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Abstract

Background: Cystathionine β-synthase (CBS) mediates conversion of homocysteine to cystathionine and deficiency in enzyme activity may lead to hyperhomocysteinemia/homocystinuria, which are often associated with mental retardation (MR). A large number of polymorphisms have been reported in the CBS gene, some of which impair its activity and among these, a T833C polymorphism in cis with a 68 bp insertion at 844 in the exon 8 is found to be associated with mild hyperhomocysteinemia in different ethnic groups. Methods: The present study is aimed at investigating the association between T833C/844ins68 polymorphism and MR. One hundred and ninety MR cases were recruited after psychometric evaluation. Hundred and thirty- eight control subjects, two hundred and sixty-seven parents of MR probands and thirty cardiovascular disorder (CVD) patients were included for comparison. Peripheral blood was collected after obtaining informed written consent. The T833C/844ins68 polymorphism was investigated by PCR amplification of genomic DNA and restriction fragment length polymorphism analysis, followed by statistical analysis. Results: The genotypic distribution of the polymorphism was within the Hardy-Weinberg equilibrium. A slightly increased genotypic frequency was observed in the Indian control population as compared to other Asian populations. Both haplotype-based haplotype relative risk analysis and transmission disequilibrium test reveled lack of association of the T833C/844ins68 polymorphism with MR; nevertheless, the relative risk calculated was higher (>1) and in a limited number of informative MR families, preferential transmission of the double mutant from heterozygous mothers to the MR probands was noticed (χ = 4.00, P < 0.05). Conclusion: This is the first molecular genetic study of CBS gene dealing with T833C/844ins68 double mutation in MR subjects. Our preliminary data indicate lack of association between T833C/844ins68 polymorphism with MR. However, higher relative risk and biased transmission of the double mutation from heterozygous mothers to MR probands are indicative of a risk of association between this polymorphism with mental retardation. Page 1 of 6 (page number not for citation purposes) Behavioral and Brain Functions 2005, 1:25 http://www.behavioralandbrainfunctions.com/content/1/1/25 Background Cystathionine β-synthase (CBS) catalyzes the condensa- tion of serine and homocysteine to form cystathionine and abnormality in CBS activity is manifested in two major clinical conditions, viz. hyperhomocysteinemia and homocystinuria. Insufficiency in CBS activity may lead to hyperhomocysteinemia [1], which is considered to be an independent risk factor for arteriosclerosis [2]. In addition to that, since homocysteine is vasculotoxic as well as neurotoxic, hyperhomocysteinemia predisposes to cardiovascular disorder (CVD) and cognitive dysfunction [3,4]. On the other hand, gross deficiency in CBS activity is associated with homocystinuria, an inborn recessive metabolic disorder [4]. The major pathologic abnormali- ties associated with homocystinuria include thromboem- bolism, ectopia lentis, osteoporosis, mental retardation (MR) and other neurological and psychiatric abnormali- ties [4]. The neurological malfunctioning can be ascribed to the oxidation of excess homocysteine to homocysteic acid, which interacts with the N-methyl-D-aspartate receptor, causing excessive calcium influx and free radical production, thereby leading to neurotoxicity [5]. In addi- Representation Figure 1 of double mutation in exon 8 of CBS gene tion, MR associated with B12 and folate deficiency could Representation of double mutation in exon 8 of CBS be attributed to the neurotoxic effects of homocysteine gene: lane 1 = homozygous genotype without insertion, lane since B12 and folate act as co-factors in the homocysteine 2 = heterozygous genotype with 68 bp insertion, lane 3 = to methionine remethylation pathway. φX174 HaeIII digest showing bands at 310, 281, 234,194 and 118 respectively, lane 4 = BsrI digestion of heterozygous The human CBS gene, located at 21q22.3 [6], is known to PCR product (of lane 2), showing complete digestion of the have a large number of mutations, the majority of which higher allele of 239 bp into 130 bp and 109 bp. are missense in nature [7]. Three different nonsense mutants, as well as some insertion, deletion and splice variants have also been identified, some of which are pol- and a small subset of CVD patients, since hyperhomo- ymorphic in nature. A transition mutation, T833C gener- cysteinemia/homocystinuria is often associated with MR ating a BsrI restriction site [8,9] is known to segregate in as well as CVD [4]. Familial transmission pattern of the cis with 844ins68 mutation in exon 8 [10] and is reported double mutation was analyzed, by haplotype-based hap- to be associated with premature occlusive arterial disease lotype relative risk analysis (HHRR) and transmission dis- [11]. Ethnic heterogeneity of 844ins68 polymorphism is equilibrium test (TDT), using heterozygous parents only. highly prevalent in African, European and North Ameri- can populations [9,12,13]. Methods Study design Since the T833C/844ins68 double mutation was not Our study population consisted of 138 healthy control detected by Franco et al. [13] and Pepe et al. [14] in their individuals, 30 CVD patients (average age 53.5 yrs) and studies on some Asian populations (Japanese, Tharu, Chi- 190 MR children (age range 6–8 yrs) along with their par- nese and Indonesian), this allele has been projected by ents. The cardiologist (J.M) involved in the study helped Pepe et al. [14] as a reliable anthropogenetic marker for in collection of samples from CVD patients. MR cases discriminating between two major human groups – Afri- were selected from the Outpatient Department of Mano- cans and Asians. Later on, the 844ins68 variant was vikas Kendra Rehabilitation and Research Institute, Kolk- observed in Han Chinese population with a very low fre- ata and diagnosed by mental health professionals (child quency [15]; however, the T833C polymorphism was not psychiatrist, child psychologist and neurologist) accord- studied. In India, study on T833C mutation revealed ing to the DSM – IV [17] followed by an IQ assessment 4.76% and 9.99% heterozygotes in control and coronary with Wechsler Intelligence Scale for Children [18]. Both heart disease patients respectively [16]. In the present patients and controls were from different parts of India study, we have looked into the frequency of the double and belonged to no particular ethnic group. Peripheral mutant in control population. We have also investigated blood was collected in EDTA, after obtaining informed the occurrence of this double mutant in MR individuals written consent from control individuals, CVD patients, Page 2 of 6 (page number not for citation purposes) Behavioral and Brain Functions 2005, 1:25 http://www.behavioralandbrainfunctions.com/content/1/1/25 Table 1: Global variation of CBS 844ins68 polymorphism in control individuals. Population 2n Heterozygosity (%) Reference US (mixed) 144 11.7 Tsai et al. [9] Dutch 214 14 Kluijtmans et al. [22] Japanese 80 0.00 Franco et al. [13] Chinese 26 0.00 Pepe et al. [14] Indonesian 98 0.00 Pepe et al. [14] Ethiopian 108 11.11 Pepe et al. [14] Spanish 54 25.9 Pepe et al. [14] Sub-Saharan African 180 66.66 Pepe et al. [14] German 400 1.5 Linnebank et al. [23] Chinese (Southern China) 200 5 Zhang & Dai [15] Italian 820 13.7 Grossmann et al. [24] Chinese (Northern China) 248 2.97 Li et al. [25] Indian 276 7.97 Present study 2n = No. of chromosomes Restriction fragment length polymorphism (RFLP) analysis and parents of MR probands. Genomic DNA was isolated by standard high salt precipitation method [19]. The BsrI digestion was carried out for all samples to detect Human Ethical Committee of the Institute approved the T833C mutation [9]. PCR products (3 µL) were digested at study protocol. 65°C for 3 hrs. in a final reaction volume of 25 µL, con- taining 1 U of BsrI (New England Biolabs; NEB) and NEB PCR amplification Buffer 4. Since this point mutation occurs in cis with The primer sequence to amplify the 844ins68 polymor- 844ins68, only the higher allele of 239 bp (A2) was phism in the exon 8 and flanking intron 7 of the CBS gene cleaved by BsrI yielding two fragments of 130 bp and 109 were: sense, 5'-GTTGTTAACGGCGGTATTGG-3', and anti- bp respectively (Fig 1); wild type allele of 171 bp (A1), sense, 5'- GTTGTCTGCTCCGTCTGGTT-3'. PCR amplifica- however, remained undigested. tion was carried out using Perkin Elmer thermal cycler (Gene Amp #2400) in a reaction volume of 20 µL contain- Statistical analysis ing 75–100 ng of genomic DNA, 20 pmoles of each Frequency of the T833C/844ins68 polymorphism in the primer, 1.0 U Taq polymerase (Bangalore Genei, India), control population and patients was calculated for the 200 µM dNTP mix, 0.001% gelatin and 10 mM Tris buffer Hardy-Weinberg equilibrium. Haplotype-based haplo- (Bangalore Genei, India) with 50 mM KCl and 1.0 mM type relative risk (HHRR) analysis [20] as well as transmis- MgCl . After an initial denaturation at 95°C for 3 min, sion disequilibrium test (TDT) [21] was used to ascertain amplification was performed at: 35 cycles of denaturation association between MR and transmission of the double at 94°C for 45 sec, annealing at 58°C for 40 sec and exten- mutation. For TDT, trios with at least one parent having sion at 72°C for 40 sec, followed by a final extension for heterozygous genotype were selected. 5 min at 72°C. PCR products were separated by 12% poly- acrylamide gel electrophoresis at 200V for 3 hrs, stained Results with ethidium bromide and documented in a Gel Doc™ Distribution of 844ins68 heterozygote in various control EQ (BIO-RAD). The wild type allele gave 171 bp fragment populations studied worldwide is presented in Table I. It (A1), and insertion variant gave 239 bp fragment (A2) was reported earlier that this variant is absent in the Asian (Fig. 1). population [13,14]. Later studies involving larger sample size revealed the occurrence of 844ins68 variant in the Chinese population [15,25] with a very low frequency of Table 2: Distribution of CBS T833C/844ins68 polymorphism in different groups. Groups No of chromosome No of mutants Mutant allele frequency ± SE Control 276 11 0.0398 ± 0.0117 CVD Patients 60 1 0.0166 ± 0.0166 MR probands 380 12 0.0316 ± 0.0089 Parents of probands 534 15 0.028 ± 0.0071 Page 3 of 6 (page number not for citation purposes) Behavioral and Brain Functions 2005, 1:25 http://www.behavioralandbrainfunctions.com/content/1/1/25 Table 3: HHRR and TDT Analysis for the CBS T833C/844ins68 polymorphism in nuclear families with MR probands. Allele Transmitted Non-transmitted χ P value Relative risk (RR) HHRR analysis 1 259 264 1.714 0.1905 1.346 TDT analysis 1 3 6 1.00 0.3173 heterozygote as compared to the European and African Saharan population [14]. Two earlier independent studies subjects. We have analyzed the 844ins68 variant in asso- on Japanese and other Asian populations failed to detect ciation with the T833C mutation and our data showed a the mutated allele [13,14] and this led to the proposal higher rate of occurrence of the double mutant in the that this unique double mutation could be used as a reli- Indian control population (7.97%) as compared to the able anthropogenetic marker for differentiating Asians Chinese population. and Africans. However, the validity of the proposal is questioned by subsequent findings of the 844ins68 poly- Frequency of the double mutant allele in different types of morphism in different Chinese populations [15,25] and subjects is presented in Table II. Eleven out of 138 control T833C transition mutation in Indian population [16,26]. individuals (n = 276) appeared heterozygous for the The present study clearly demonstrates that the T833C/ mutation. On the contrary, only one person out of 30 844ins68 polymorphism may not be suitable as an CVD patients showed heterozygous genotype. In the MR anthropogenetic marker for differentiating Asians and group, 12 out of 190 MR probands showed presence of Africans. the polymorphism and no significant deviation in allele frequencies was noted in the MR probands and their par- Among Indians, presence of T833C heterozygote has been ents (Table II). reported in 1 out of 21 (4.76%) [16] and 3 out of 100 (3%) [26] control individuals. However, in both studies HHRR analysis for the T833C/844ins68 polymorphism the association of 844ins68 mutation with the T833C showed lack of significant association between MR and transition mutation has not been mentioned, while in the transmission of the double mutant (χ = 1.714, P = present investigation we have observed invariable segrega- 0.1905) (Table III). TDT analysis also revealed lack of tion of these two mutations in cis, which is consistent with association between the T833C/844ins68 polymorphism earlier observations [10,13,14,22]. and MR (χ = 1.00, P = 0.3173) (Table III). However, transmission from the mother (Table IV), heterozygous Hyperhomocysteinemia is hypothesized as an independ- for the alleles, revealed that the A2 allele was preferen- ent risk factor for CVD [22,27] and the 844ins68 variant tially transmitted to the MR probands (χ = 4.00, P = was previously reported to be associated with premature 0.0455), while no significant contribution of the paternal occlusive arterial disease [11]. A recent article on Chinese allele was noticed (data not presented). congenital heart disease (CHD) patients have shown that the 844ins68 could be a risk factor for CHD, and the inser- Discussion tion especially in mothers could increase the risk in off- The present finding of 11 out of 138 Indian control indi- spring [25]. On the other hand, the findings of Zhang and viduals with the T833C/844ins68 polymorphism reveal a Dai [15] on adult Chinese patients have suggested that the higher percentage of heterozygotes (7.97%) as compared 844ins68 could provide protection to vascular throm- to the Chinese control population (Table I); but, this boembolic disease. The mutant allele frequencies among value is significantly lower than those quoted for Sub- the Indian controls and the CVD patients are 0.0398 and Table 4: Transmission pattern of CBS T833C/844ins68 from mother to MR proband. Allele Transmitted Non-transmitted χ P value HHRR analysis (all 1 176 180 2.0449 0.1527 mothers) HHRR analysis 1 2 6 4.00 0.0455 (heterozygous mothers only) Page 4 of 6 (page number not for citation purposes) Behavioral and Brain Functions 2005, 1:25 http://www.behavioralandbrainfunctions.com/content/1/1/25 0.0166 respectively (Table II) and no association was associated with transmission of the mutant allele. The observed between T833C/844ins68 polymorphism and preferential transmission of T833C/844ins68 polymor- CVD. Heterozygous T833C/844ins68 alleles were phism from the heterozygous mother to the MR proband observed at higher percentage in Indian CVD patients (χ = 4.00, P = 0.0455) also supports the above possibility. (3.33%) as compared to Chinese ischemic patients (~1%). The lower value of 3.33% for CVD patients as Conclusion compared to that of the control (7.97%) seems to support This is the first report on CBS T833C/844ins68 polymor- the idea of a possible protective role of the double muta- phism in association with MR and to the best of our tion to vascular thromboembolic disease, as proposed by knowledge, this is also the first information on the occur- Zhang and Dai [15]; however, such a conclusion is not rence of the CBS double mutation in the Indian popula- called for due to the relatively small CVD sample size in tion. Our preliminary data indicate lack of significant the present study. association between T833C/844ins68 polymorphism with MR; however, the relative risk calculated and the While the T833C mutation results in Ile278Thr change in preferential transmission of the double mutation from the mutant protein, in the case of T833C/844ins68, the 68 informative mothers to MR probands do suggest a risk of bp insertion results in an alternative splice site generating association of this polymorphism with MR and warrants mostly wild type mRNA with traces of another transcript, further investigation. detectable only in the nucleus [9,12]. This accounts for the rescue of the transition mutation at 278 in the case of the List of Abbreviations used double mutant [9,10]. However, these observations can- MR (Mental retardation), CBS (Cystathionine β-syn- not account for the conflicting reports on the association thase), CVD (cardiovascular disorder), HHRR (haplotype- between cardiovascular disease in different ethnic popula- based haplotype relative risk), TDT (transmission disequi- tions and the mutant allele [28,29]. Case control studies librium test), CHD (congenital heart disease). showed that this double mutation might be a neutral pol- ymorphism in the CBS gene and splicing of the intron 7 Competing interests eliminates the mutant allele carrying the T833C [22]. On The author(s) declare that they have no competing inter- ests. the other hand, in Northern Europe, the T833C mutation is common in patients presenting clinically with homo- cystinuria and is associated with a B6-responsive pheno- Contributors type [22,28]. SD (JRF, DBT Grant) was responsible for acquisition of data and analysis and drafting the article. SS performed The CBS T833C/844ins68 polymorphism has been psychometric analysis and clinical diagnosis, intellectual reported in homozygous condition, with a very low fre- contribution. AC helped in psychological evaluation and quency, from American black artheroslerosis patients [9]. IQ determination. PKG was responsible for neurological We have failed to observe any subject homozygous for the investigations and intellectual contribution. JG diagnosed mutation in our study group, which possibly points the CVD cases. MS revised the manuscript critically. KM towards the deleterious effect of this mutation in was responsible for concept and study design, interpreta- homozygous condition. tion of data and revising the article critically. Final manu- script was approved by all authors. The present finding of preferential transmission of T833C/844ins68 polymorphism to the MR proband from Acknowledgements Authors are thankful to the families who have participated in the study. This the heterozygous mother is the first evidence of any asso- work was partly sponsored by a grant from the Department of Biotechnol- ciation of CBS gene with MR, although the association of ogy, Government of India; Grant No. BT/PR4269/SPD/11/478/2003. hyperhomocysteinemia/homocystinuria with psychiatric manifestations including MR is well established [3,4,30]. References Among different markers for cobalamine/folate status, 1. Clarke R, Daly L, Robinson K, Naughten E, Cahalane S, Fowler B, Gra- plasma homocysteine showed the best association with ham I: Homocysteinemia: A risk factor for vascular disease. N Eng J Med 1991, 325:967. aging related neuropsychiatric dysfunction [31] and an 2. Boushey CJ, Beresford SAA, Omenn GS, Motulsky AG: A quantita- earlier investigation revealed that the CBS 844ins68 allele tive assessment of plasma homocysteine as a risk factor for frequency was lower in children with high IQ as com- vascular disease. 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Linnebank M, Homberger A, Junker R, Nowak-Goettl U, Harms E, available free of charge to the entire biomedical community Koch HG: High prevalence of the I278T mutation of the human cystathionine β-synthase detected by a novel screen- peer reviewed and published immediately upon acceptance ing application. Thromb Haemost 2001, 85:986-88. cited in PubMed and archived on PubMed Central 24. Grossmann R, Schwender S, Geisen U, Schambeck C, Merati G, Wal- ter U: CBS 844ins68, MTHFR TT677 and EPCR 4031ins23 yours — you keep the copyright genotypes in patients with deep-vein thrombosis. Thromb Res BioMedcentral 2002, 107:13-15. Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 6 of 6 (page number not for citation purposes)

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