Chromosome 18p11.2 harbors susceptibility marker: D18S452, for bipolar affective disorder.

AIM: The aim of our study was to investigate whether the tandem repeat polymorphism in D18S452 microsatellite marker at locus 18p11.2 is a risk factor of bipolar affective disorder (BPAD) in Kashmiri population.
MATERIALS AND METHODS: The repeat polymorphism in D18S452 was evaluated by polymerase chain reaction (PCR) analysis of in 74 diagnosed BPAD patients and 74 controls subjects.
RESULTS: Tandem repeat (300 bp*) allele frequency was found to be 1.35% in controls and 8.108% in cases. The tandem repeat (250 bp*) allele frequency was found to be in 91.89% in cases and 98.65% in controls. The 252 bp/252 bp genotype was found to be present in 89.18% of cases and 98.64% of controls, the 300 bp/300 bp genotype in 5.40% of cases and 1.35% of controls and the 252 bp/300 bp variant in 5.40% of cases and none among the controls. Although the proportion of patients homozygous for tandem repeat (300 bp/300 bp) was higher in cases than in controls, the difference was not statistically significant when 252 bp/252 bp genotype was taken as reference (odds ratio [OR]=4.4242; 95% confidence interval [CI] 0.4822-40.5924); P=0.1529). However, when the frequency of heterozygous genotype (252 bp/300 bp) was compared with 252 bp/252 bp statistical significance was observed (OR=8.0603; 95% CI 1.1112-58.4646; P=0.0383).
CONCLUSION: This is the first study reporting a significant association between D18S452 maker with tandem repeat polymorphism in heterozygous condition (252 bp/300 bp) and the development of BPAD in Kashmiri population.

INTRODUCTION

Bipolar affective disorder (BPAD) is a major psychiatric problem with multifactorial etiology affecting approximately 1% of population[1] and with all other bipolar spectrum disorders the prevalence will be as high as 5%.[2] Bipolar disorder is the sixth leading cause of medical disability world-wide among people 15-44 years of age and the estimated disability-adjusted life years of bipolar disorder outrank all cancers and primary neurologic disorders.[3] It is equally prevalent in males and females.[1] Family[4], twin[5] and adoption studies[6] have established that there is a multiple genetic contribution to bipolar disorder and regarding the mechanism of transmission it is thought to be consistent with the epistatic action of several genes with modest individual effect sizes.[789] Although the limits of phenotypic expression are not known, the family studies suggest that bipolar I, bipolar II recurrent major (unipolar) depression and schizoaffective disorder are part of a bipolar spectrum.[4]

Since most psychiatric disorders show a complex mode of inheritance and transmission, the BPAD is no more an exception. Without identifying the genes involved, the mechanism that underlies the pathophysiology of bipolar disorder cannot be elucidated and therefore it is not possible to think of any relief and treatment to victims of the disorder. In the direction of identifying causative genetic markers, various linkage studies on large pedigree cohorts have led to reproducible identification of several susceptibility loci. These include regions on chromosomes 4q, 12q, 13q, 18, 21q and 22q.[101112131415161718] Various types of studies have identified chromosome 18p as a highly potential and susceptible locus, which carry a few candidate genes for affective mood disorder.[91920212223242526] To investigate this region, further studies like case-control for a marker D18S452 (locus 18p11.2) association with BPAD is warranted. Microsatellite marker D18S452 contains 26 dinucleotide repeats (cytosine-adenine (CA)) (http://genome.ucsc.edu/cgi-bin/hgTracks?org=human). Though, the incidence of BPAD in Kashmir is significant yet there has been no genetic study from Kashmir until date and the existence of any genetic predisposition in this population remains totally untouched and unexplored. The molecular and epidemiological studies remain open for research and seem to be promising in addressing the various queries of BPAD etiology in this area. Therefore, the current work was undertaken to determine the structure of the marker and evaluation of any association between the chromosomal susceptibility locus and BPAD.

MATERIALS AND METHODS

Study subjects

The BPAD seems very frequent and a serious health problem in Kashmir Valley. All people who suffer from such ailments report to the Psychiatric Disease Hospital at Srinagar, Kashmir for diagnosis and treatments. Patients were diagnosed with the disorder by psychiatrists who used diagnostics and statistical manual IV based instrument called Mini-International Neuropsychiatric interview at the hospital Srinagar. Those confirmed to have BPAD without having any comorbid condition such as thyroid disorder, migraine, panic disorder and/or eating disorder were enrolled as cases. All the diagnosed patients were included irrespective of any grade (whether or not on medication), age or gender. Controls were recruited from the general medicine wards of Shri Maharaja Hari Singh Hospital, Srinagar following the referral pattern of sex and age matched patients. None of the controls had a personal or family history of any psychiatric disorder which was confirmed by a consultant psychiatrist. The study was approved by the Departmental Research Committee of the Department of Biochemistry University of Kashmir Srinagar and Board of Research Studies, University of Kashmir. A total of 148 subjects were taken in this study out of which 74 were cases and rests 74 were controls. After the detailed explanation of the study, all the subjects provided written informed consent.

Sample collection and deoxyribonucleic acid extraction

Around (3 ml) peripheral blood was collected from each subject in sterilized plastic vials containing ethylenediaminetetraacetic acid (0.5M; pH-8.0) and stored at −20°C for further analysis. DNA was isolated by a standard phenol-chloroform extraction method.[27] The quality of the genomic DNA was examined by gel electrophoresis using 0.8% agarose gel. The quantity of the DNA was estimated by making appropriate dilutions to determine the optical density at 260 nm and 280 nm by double beam spectrophotometer (spectron 2206) and the concentration was determined using equation:

DNA (μg/ml) = A260 × 50 × dilution factor

The ratio of A260/A280 was calculated and the DNA samples for which the ratio was 1.7-1.9 were considered for the future use.

Genotyping

Polymerase chain reaction (PCR) was performed in total volume of 25 μl. The PCR reactions mixture composed of 50-150 ng genomic DNA; 0.2 mM dNTPs; 0.4 pmoles/μl of each primer; 1.5 U of Taq polymerase in 1 × PCR buffer and 200 nM each of the forward primer: 5′-TGG GGC ATA CAT AGT GCA AA-3′ and reverse primer: 5′-AGG CCT TTT GCT AGT TGG GT-3′. PCR cycling parameters were a 4 min denaturation cycle at 94°C and 34 cycles of the following: 94°C for 45 s 59.2°C for 30 s and 72°C for 45 s and a final extension at 72°C for 5 min. The amplified products were visualized on a 3% agarose gel using ethidium bromide.

Statistical analysis

The χ2 test was used for the comparison of the allele and genotype frequency between the cases and controls. The distribution of the genotype frequencies in both groups did not deviate from the Hardy-Weinberg equilibrium. The odds ratio (OR) were calculated as estimates of relative risk for disease and 95% confidence intervals (CIs) were calculated for all observed allele frequencies. P < 0.05 was considered as statistically significant. The SPSS statistical software package (version 11.5 SPSS Chicago IL) was used for the statistical analysis.

RESULTS

Genotypic and allelic frequencies

In the present study, 74 BPAD cases and 74 matched controls belonging to the Kashmir division were analyzed for D18S452 polymorphism. We found six types of alleles corresponding to base pair size: 200 bp; between 200 and 252 bp; 252 bp; between 252 and 300 bp; 300 bp; between 300 and 350 bp. On analyzing the allelic frequency two main groups turned out – rare and frequent genotype with respect to the locus. Only two subjects (one from each case [1.35%] and control group [1.35%]) had allelotype corresponding to 200 bp. There was just one case representation from each 200 bp to 250 bp; between 252 bp and 300 bp and 300-350 bp alleotypes. A single subject had from controls had 300 bp allelotype. 63 patients (87.16%) and 72 controls (97.297%) had allelotype corresponding to 252 bp. Seven patients (7.43%) had allelotype corresponding to 300 bp [Figure 1 and Table 1].

Figure 1

Representative gel picture showing 3% agarose gel electrophoresis after polymerase chain reaction lane 1 represents negative control; lane 2 and 4 represents homozygous condition (300 bp); lane 3 represents homozygous condition (252-300 bp) lane 5 represents homozygous condition (200 bp); lane 6 represents heterozygous condition (252 bp/300-350 bp); lane 7-10, 12 and 14 represents homozygous condition (252 bp); lane 13 represents homozygous condition (200-252 bp). Lane 11 represents 50 bp ladders

Table 1

Frequency of D18S452 alleles (major and minor alleles) in the study population

Since the alleles corresponding to bp size 200, between 200 and 252 bp, between 252 and 300 bp, between 300 and 350 bp were very rare we considered them as minor alleles and grouped them under two main classes of major alleles; 252 bp* (containing alleles of size up to 252 bp) and 300 bp* (containing alleles corresponding to more than 252 bp).

The tandem repeat (300 bp*) allele frequency was found to be 1.35% in the controls and 8.108% in cases. The tandem repeat (252 bp*) allele frequency was found to be 91.89% in cases and 98.65% in controls. The 252 bp/252 bp genotype was found to be present in 89.18% of the cases and 98.64% of the controls the 252 bp/300 bp variant in 5.40% of the cases and none among the controls and the 300 bp/300 bp genotype in 5.40% of cases and 1.35% of controls [Tables 2 and 3].

Table 2

Frequency of D18S452 alleles (after grouping) in the study population

Table 3

Genotypic frequencies of D18S452 polymorphism in the study population

It was observed that although the proportion of individuals who were homozygous for the tandem repeat (300 bp/300 bp) was higher in cases than in controls the difference was not statistically significant when using 252 bp/252 bp genotype as a reference (OR=4.4242; 95% CI 0.4822-40.5924); P=0.1529). However, it was observed that the frequency of the heterozygous genotype (252 bp/300 bp) when compared with 252 bp/252 bp showed statistical significance (OR=8.0603; 95% CI 1.1112-58.4646; P=0.0383).

General characteristics of the study population

Various general characteristics of the study population like mean age gender dwelling and socio-economic status were analyzed between cases and controls, but no statistical significance was observed [Table 4].

Table 4

General characteristics of the study population

D18S452 genotypes and clinic pathological variables

Table 5 shows the genotype frequencies of D18S452 polymorphism relating to age gender dwelling socio-economic status BPAD subtype family history and age at onset. No statistical significance was attained.

Table 5

Correlation between D18S452 polymorphism and clinico-pathological characteristics

DISCUSSION

Overall results suggests that there is a significant association between D18S452 maker with tandem repeat polymorphism in heterozygous condition (252 bp/300 bp) and the development of BPAD in Kashmiri population. The susceptible marker was not explored earlier and its analysis reveals important information regarding the types and frequency of alleles. On the basis of the frequency in the subject, the identified six alleles can be sorted into two categories – frequent and rare one. The allele size determined by the number of dinucleotide (CA) ranged from 200 to 350 bp. The allele 252 bp was predominant in both cases and controls but the 252/300 bp allele was found only in cases (5.40%) and importantly in none of the controls. Most of the alleles are more or less similar in the cases and a significant association between tandem repeat polymorphism in heterozygous condition (252 bp/300 bp) and the development of BPAD in Kashmiri population turns out. There can be many possible ways by which the genotype can contribute to the development of BPAD. It can influence at organizational level of chromosome, its accessibility to various factors and regulatory proteins. Chromosomal locus 18p11.2 harbors many candidate genes like NAPG[28] GNAL[29] NDUFV2[24] and VAPA.[30] It may be possible that this marker is directly or indirectly involved in the regulation of neighboring genes. It is also possible that this locus may be in linkage disequilibrium with other genes.

The result of our study further adds to the data from several other studies which have also shown an association between chromosomal locus 18p and the risk of BPAD. In one study, chromosome 18p has been shown to be a highly susceptible locus for the affective mood disorder[19] Further, support for this region comes from evidence suggesting potential overlap with schizophrenia; Schwab et al., reported a possible schizophrenia locus on 18p11.2 and demonstrated that evidence for linkage in these schizophrenia families increased when individuals with affective diagnoses were also included in the analysis.[31] A susceptibility gene on chromosome 18 and a parent-of-origin effect have been suggested for BPAD.[13] In studies of persons with chromosome 18p - deletions, there have been reports of paranoid psychosis, depression and subclinical obsessive compulsive disorder.[32] In studies of 18qsyndromes, there have also been individual reports of violent/aggressive behaviors.[33] There is one report of a behavioral/psychiatric problem in a case of chromosome 18p tetrasomy; an individual who had a history of aggressive, self-injurious and destructive behavior.[34] There is also a report of a family with a pericentric inversion of chromosome 18, in which three individuals (two with dup (18p)/del (18q) and one with dup (18q)/del (18p), all displayed depression and anxiety.[35]

We also tried to correlate many clinico-phenotypic conditions like BPAD subtype, age at on set and other general characteristics with D18S452 polymorphism, but we could not find any significant association [Table 5].

It may pave a way to understand the development of the BPAD. It can serve us a lead from where we can further explore the biological significance of the identified genotypes and their association with BPAD. The management and therapeutic targets may be identified by designing additional studies on such findings. Bigger sample size, biological characterization of the genotypes in the susceptible and sequencing of such repetitive DNA sequences by modern sequencing technology seems inevitable for further substantiation of our findings.

CONCLUSION

Overall our study suggests that there is a significant association between tandem repeat polymorphism in heterozygous condition (252 bp/300 bp) and the development of BPAD in Kashmiri population. We identified the alleles of the marker D18S452 most of which are a frequent. There are various possible ways by which the 252/300 bp genotype can influence the development of the BPAD. Further studies are required for better understanding of the roles of the genotypes in BAPD pathophysiology.