Analysis of single nucleotide polymorphisms of CRYGA and CRYGB genes in control population of western Indian origin

Aim: Polymorphisms in γ-crystallins (CRYG) can serve as markers for lens differentiation and eye disorders leading to cataract. Several investigators have reported the presence of sequence variations within crystallin genes, with or without apparent effects on the function of the proteins both in mice and humans. Delineation of these polymorphic sites may explain the differences observed in the susceptibility to cataract observed among various ethnic groups. An easier Restriction Fragment Length Polymorphism (RFLP)-based method has been used to detect the frequency of four single nucleotide polymorphisms (SNPs) in CRYGA/CRYGB genes in control subjects of western Indian origin. Materials and Methods: A total of 137 healthy volunteers from western India were studied. Examination was performed to exclude volunteers with any ocular defects. Polymerase chain reaction (PCR)-RFLP based method was developed for genotyping of G198A (Intron A), T196C (Exon 3) of CRYGA and T47C (Promoter), G449T (Exon 2) of CRYGB genes. Results: The exonic SNPs in CRYGA and CRYGB were found to have an allele frequency 0.03 and 1.00 for ancestral allele respectively, while frequency of non-coding SNP in CRYGA was 0.72. Allele frequency of T90C of CRYGB varied significantly (P = 0.02) among different age groups. An in-silico analysis reveals that this sequence variation in CRYGB promoter impacts the binding of two transcription factors, ACE2 (Member of CLB2 cluster) and Progesterone Receptor (PR) which may impact the expression of CRYGB gene. Conclusions: This study establishes baseline frequency data for four SNPs in CRYGA and CRYGB genes for future case control studies on the role of these SNPs in the genetic basis of cataract.

causing mutations have been identiÞ ed in the γ-crystallins (CRYG) genes both in mouse and man. [2] Mutations in these genes implicate the CRYG gene cluster as a very critical locus for lens development and diff erentiation. In a review, Graw et al., [2] listed a variety of polymorphic sites that have been identiÞ ed in the mouse Cryga and Crygb genes and showed that some mutations occurring in these genes were associated with diff erent cataract phenotypes. Recently Li et al., [3] reported that a point mutation occurring in the Crygb gene in a mouse causes dominant dense nuclear cataract.
Rogaev et al., [4] studied a tri-nucleotide microsatellite marker for gamma-crystallin B gene (CRYG1) and found it to co-segregate with polymorphic congenital cataract (PCC) yielding a maximum LOD score of 10.62. Santhiya et al., [5] also reported that the variation G198A of Intron A in CRYGA occurred at a fairly high frequency in cases of autosomal dominant cataract cases. In addition to this, the allele -T47C is found to affect the promoter of the CRYGB gene and occurs in Þ ve out of 10 cases in a heterozygous condition in family studies. [5] The CRYGC and CRYGD genes have been extensively studied in humans while the potential role of CRYGA and CRYGB still remains to be ascertained. Thus, for the current study these two genes were chosen for establishing the baseline frequency in western Indians. Information on polymorphic sites in cataract-related genes in the aff ected and unaff ected population, at large, may explain the genetic predisposition to cataract and also the underlying genomic diversity in different ethnic groups. The present study was the Þ rst Polymerase Chain Reaction (PCR)-Restriction Fragment Length Polymorphism (RFLP)-based approach to screen certain single nucleotide polymorphisms (SNPs) at a population level in order to obtain a baseline frequency for use in future case-control studies.

Materials and Methods
A total of 137 unrelated healthy volunteers comprising 90 males, 47 females (age range 2.5-67 years) who visited the local eye hospital for an annual eye checkup during the period May 2005 to December 2006 were recruited for the study. The study was approved by the Institutional Ethical Review Committ ee (IERC). A subject qualiÞ ed as a control if (a) both the pupils could be dilated to at least 6 mm, (b) both lenses were graded as having no nuclear, posterior sub-capsular, cortical opacities including Grade I or II opacities. Venous peripheral blood samples were collected from the subjects aft er obtaining an informed consent. Genomic DNA was extracted from the collected samples using a standard protocol. [6] Primer sequences as reported by Santhiya et al., [5,7] were used for ampliÞ cation of the target regions by PCR. The obtained amplicon was divided into two parts and while one part was digested with appropriate restriction endonuclease the other undigested part was used as reference to compare with the fragments generated aft er digestion with the restriction endonuclease. SpeciÞ c restriction endonucleases (procured from Fermentas) were used to study the restriction site aff ected by the reported nucleotide variations based on the restriction maps generated using New England Biolabs (NEB) cutt er soft ware. [8] The digested and undigested PCR products were analyzed using 12% Polyacrylamide Gel Electrophoresis (PAGE) in 1XTBE. Table 1 lists the scheme of restriction endonuclease used and the DNA fragments obtained aft er digestion of PCR amplicon with respective restriction endonuclease at conditions as per the manufacturer 's guidelines (incubation at 37°C for NmuCI, HaeIII and PstI, and 65°C for TaqI overnight). All PCR-RFLP-based analysis was conÞ rmed with DNA sequencing in representative cases.
Allele frequencies were estimated by allele counting method and diff erences in frequencies between the two age groups were determined using two-way contingency table and Chi square test. Hardy-Weinberg estimates were performed using the Michael Court online calculator. The putative changes in the transcription factor binding sites were studied, using AliBaba soft ware [9] that scans for potential transcription factor binding sites, for sequence variations in the promoter region of CRYGB gene.

Results
Four SNPs, namely G198A and T196C in Intron A and Exon 3 of CRYGA, T47C in promoter and G449T in Exon 2 of CRYGB were studied and the sequence variations could be easily identiÞ ed on the basis of the restriction fragments obtained in each case as evident from the gel images shown in Fig. 1. The observed genotype frequencies satisfy Hardy Weinberg Equilibrium for all polymorphisms studied Tables 2 and 3. Out of 137 volunteers, 40% were found to be heterozygous for G198A CRYGA polymorphism (frequency of "A" allele = 0.28) [ Table 2]. The   Table 3]. As the frequency of TT allele was found to be the same in all subjects above the age of 10 years all further analysis was done using the age stratiÞ cation of <12 years and >12 years of age which is the norm for segregating pediatric and adult cases in the medical profession. [10,11] The allele frequency for "T" was 0.23 in <12 year olds and 0.11 in those >12 years Tables  3 and 4. No sequence variation was observed at Nucleotide 449 in Exon 2 of CRYGB as all 121 subjects analyzed were found to have "GG" genotype [ Table 2]. The allele frequencies obtained were compared with frequencies reported for other populations worldwide [ Table 5] and are signiÞ cantly diff erent from those reported by Santhiya et al., [5,7] in families with history of autosomal dominant congenital cataract.
The sequence variation in CRYGB promoter region was also analyzed for change in transcription factor binding sites using the AliBaba soft ware. While the sequence containing the "C" allele at nucleotide position 47 has binding sites for transcription factor ACE2 and PR, the substitution by "T" at this position results in the loss of both these binding sites [ Fig. 2].

Discussion
Crystallins in lens do not turn over and must serve the lens for the lifetime of a person. Thus, the lens is even more dependent than most other tissues on protection from any kind of damage. Besides maintaining lens transparency, βγ-crystallins (Beta and gamma crystallins), may also function as stress protection proteins that are induced during periods of critical stress on the retina. [1] Sequence changes occurring in the form of nucleotide polymorphisms in these protective systems could critically lead to accumulation of abnormally folded proteins eventually leading to disease. [12,13] γ-crystallins may also have developmental roles and numerous SNPs in their genes have been linked to hereditary cataracts. Santhiya et al., [5,7] have reported a co-segregation of SNPs in CRYG, CRYBB2 and GJA8 genes with familial congenital cataract. At the same time there are other contradictory reports both in mice and in humans on polymorphic sites within these genes with no apparent eff ects on the function of the respective proteins. [14,15]  SD -Standard deviation, HWE -Hardy weinberg equilibrium, "1" = P value between age group 0-10 and 11-20 years, "2" = P value between age group 11-20 and 21-30 years, "3" = P value between age group 21-30 and 31-40 years, "4" = P value between age group 31-40 and 41-50 years, "5" = P value between age group 41-50 and 51-60 years, "6" = P value between age group 51-60 and >60 years, "7" = P value between age group 0-10 and >11 years In the present study, the Þ rst of its kind in India, the baseline frequency for four SNPs in CRYGA and CRYGB genes has been studied in healthy Indian volunteers with no history of any eye disease including cataract. A review of literature reveals that 12 years is the given norm for categorization of patients into pediatric cases. [10,11] The observed diff erence in the allele distribution of CRYGB promoter region, T47C, with age may be due to the inherent inability to exclude the subjects susceptible to age-related cataract from the younger group (<12 years of age) while all such individuals in the older group would have been excluded from the study due to the rigorous exclusion criteria followed during this study. Selection of "control or unaff ected" population is an important aspect of case-control design for studying genetic markers for age-related disorders. Therefore special att ention must be paid to patient/subject recruitment as certain nucelotide changes may play a critical role during perinatal and/or paediatric growth phase only.
It is also interesting to note that the same SNP when analyzed for putative transcription factor binding site (through AliBaba soft ware) shows altered binding for two transcription factors. While the T47 allele looses the binding site for transcription factors ACE 2 and PR, the 47C allele retains both these binding sites. This is an important Þ nding in the light of reports showing that progesterone leads to glucocorticoidlike eff ects in various tissues [16,17] and the long-term use of glucocorticoids induces cataract. As the CRYGB gene has not been characterized well in humans, [3] our observation on the putative alteration of transcription factor binding sites warrants future studies to delineate the speciÞ c role of this allele in the etiology of eye disorders and disease progression.
When the frequencies obtained in the present study were compared with those reported in diff erent populations of the world by NCBI SNP database (dbSNP), [18] the allele frequency for 198G→A in CRYGA gene was found to be similar to that observed in Africans. Frequency of T47C in the promoter region of CRYGB is similar to those reported for Africans, Chinese and Japanese. It is noteworthy that allele frequencies for both these polymorphisms differ within Japanese sub-populations, emphasizing the fact that diff erences do exist within a geographical region. [19] No database records are available for frequencies of CRYGA T196C (Exon 3) and CRYGB G449T (Exon 2). The observed frequency   for T196C is similar to that reported earlier by Santhiya et al., [5] in congenital cataract cases, indicating that this polymorphism may have no role in cataractogenesis. When the present Þ ndings (in healthy volunteers) are compared with the incidence of these polymorphisms in cataract probands studied by Santhiya et al., [5,7] a signiÞ cant diff erence in the frequency of CRYGA 198A (Odds ratio = 7.1, 95% CI = 1.57-31.9) and CRYGB 47C mutation (Odds ratio = 22.5, 95% CI = 3.7-135.4) is observed, implicating a role of these mutations in cataractogenesis. The CRYGC and CRYGD genes are already well studied in case of humans, and the potential role of CRYGA and CRYGB is yet to be explored. The current study establishes the baseline frequency for speciÞ c sequence variations in CRYGA-B genes which will be useful for future case-control studies in this ethnic group. It has yet to be experimentally proved that functional promoter variation in CRYGB and non-coding variant of CRYGA may aff ect expression or generate splice variants of CRYG genes. These Þ ndings will give insights into genetics of cataract/s. These kinds of studies will be of paramount importance in order to guide development of a medical therapy that will prevent or delay the onset of adult cataract, lessening the burden on the aging population and the consequent requirement for large numbers of surgical procedures. The present study needs to be extended in cataract patients to ascertain the association with the etiology of cataractogenesis.