Australian Journal of Basic and Applied Sciences, 5(6): 1-5, 2011 ISSN 1991-8178

Genetic Analysis of Phosphatase 1 Regulatory Subunit 1B (PPP1R1B) among Autistic Patients

Nabil S. Awad and Adel E. El-Tarras

College of Medicine, Biotechnology and Genetic Engineering Unit, Taif University, Taif, Kingdom of Saudi Arabia.

Abstract: Several lines of evidence have demonstrated that genetic factors contribute to the development of autism. During this study, the association between three polymorphisms of , regulatory subunit 1B (PPP1R1B) gene with autism was investigated via PCR-RFLP technique. Three polymorphisms (rs1495099, rs907094 and rs3764352) of PPP1R1B gene were genotyped among 50 autistic patients in comparison with 50 healthy individuals in case control study. The rs1495099 polymorphism was genotyped with PvuII and three genotypes were produced GG, GC and CC. MseI was used to genotype rs907094 and CC, CT and TT genotypes were detected. Three genotypes GG, GA and AA of rs3764352 polymorphism were generated by BccI enzyme. Only one significant association was found between CT genotype of rs907094 polymorphism and autism. The obtained results suggested that PPP1R1B polymorphism (rs1495099, rs907094 and rs3764352) are not related to the development of autism in Saudi autistic patient except CT genotype which belongs to rs907094 polymorphism is statistically correlated with autism.

Key words: polymorphism, PPP1R1B gene, autism, PCR-RFLP.

INTRODUCTION

Autism is a debilitating disorder with heritability of more than 90% (Bailey et al., 1996) and characterized by presence of social deficits, language abnormalities, stereotypes, and repetitive behavior (American Psychiatric Association, 1994). According to the World Health Organization, autism affects about 67 million people worldwide. Data from molecular epidemiological studies provides substantial evidences for genetic component in the vulnerability to autism (Bolton et al., 1994; Folstein and Rosen-Sheidley, 2001 and Sykes and Lamb, 2007). Molecular genetic association studies are a valuable research tool to identify the involved in the etiology of autism. The protein Phosphatase 1, regulatory subunit 1B (PPP1R1B) gene is located at 17q12 and consists of 7 exons (http://www.ncbi.nlm.nih.gov; GeneID 84152) and encodes for DARPP-32. DARPP-32 is a bifunctional signal transducing molecule which controls a serine threonine kinase, as well as a serine threonine phosphatase, modulating the activity of several classes of receptors and ion channels, including and glutamate (Albert et al., 2002). Dopamine (DA) modulates learning, executive functions, emotional processing and social cognition, all of which are impaired in individuals with autism spectrum disorders (Hettinger, 2009). DARPP-32 is expressed in dopaminoceptive (DAceptive) neurons. Rs1495099 is located in intron 1; rs907094 and rs3764352 are located in intron 5 Dopamine-and-cAMP- regulated neuronal phosphoprotein (32 kDa) (DARPP-32), encoded by PPP1R1B, is expressed in brain regions receiving dopamineergic projections, including the prefrontal cortex (PFC), and is implicated in the pathophysiology of (Feldcamp et al., 2008). Several association studies have tested the relevance of polymorphisms on the PPP1R1B gene for the dopamine related phenotypes schizophrenia, bipolar disorders, nicotine dependence, and cognitive functioning (Beuten et al., 2007; Frank et al., 2007; Hu et al., 2007; Li et al., 2006; Manuck et al., 2009 and Volavka et al., 2004). The aim of the present study is to investigate of association between three single nucleotide polymorphisms rs1495099 G/C (intron 1), rs907094 T/C and rs3764352 A/G (intron 5) of PPP1R1B gene and autism among Saudi autistic children based on PCR-RFLP case control study.

MATERIALS AND METHODS

Subjects: The study group consists of one hundred children. Fifty autistic patients and 50 control subjects are recruited. All cases and control subjects are in the age matched and the same geographical and socioeconomics

Corresponding Author: Adel A. El-Tarras, College of Medicine, Biotechnology and Genetic Engineering Unit, Taif University, Taif, Kingdom Saudi Arabia. E-mail: [email protected] 1 Aust. J. Basic & Appl. Sci., 5(6): 1-5, 2011 conditions. Children with any neurological condition suspected to be associated with autism were excluded from control group. The diagnosis of autism was confirmed for all cases using the Autism Diagnostic Interview-Revised (Lord,1997) and the Autism Diagnostic Observation Schedule, modules 1, 2 or 3 (DiLavore et al., 1995; Joseph et al., 2002 and Lord et al., 2002). Verbal consent was obtained from all participants or their relatives and from control subjects prior to blood samples collection and all institutional requirements were met. Blood samples were collected from all participants in anticoagulants EDTA tubes.

PCR Amplification and Genotyping Assays: In the present work, three polymorphisms, rs1495099 G/C, rs907094 T/C and rs3764352 A/G, at the PPP1R1B gene were genotyped via PCR-RFLP technique. PCR reactions of three studied polymorphisms were conducted using Phusion Blood Direct PCR kit (Finnzymes). PCR reactions was performed in a reaction volume of 20 ìl containing 1 ìl blood, 10 Pmol of each of primers; rs1495099 F 5'- TTGTTGCTGAGCTGAGATGC-3' and R 5'-CTCCAGGGAAATGCACAAAG-3'; rs907094 F 5'- ACCTGATTGGGAGAGGGACT-3' and R 5'-GTAAGCTGAGGGGCCTGTG-3'; and rs3764352 F 5'- CTGTTTTGGAGGGGTCTCAG-3' and R 5'-TGGGAATACTGAAGAGTCAACC-3'. F 5' - GGATCCTTCGTGGCCGTCGTTCAAGC-3' (Jena Bioscience, Jena, Germany), 10ìl of 2x phusion Blood PCR Buffer (Finnymes) and 0.4ìl phusion Blood DNA Polymerase. PCR amplification was carried out via Phusion Blood Direct PCR kit according to the manufacturer’s instructions. Briefly, Lysis of cells at 95 ºC for 5 min, followed by 35 cycles each at 98 ºC for 1 sec, annealing temperature at 60ºC for 5 Sec, extension temperature at 72 ºC for 30 Sec, and final extension at 72 ºC for 1 min. The amplified DNA fragments were separated on 1.5% agarose gel (Bioshop Canada, Burlington, Ontario, Canada), with DNA ladder standard 100 bp (Bioshop Canada). All gels were stained with ethidum bromide 0.5 ìg/ml (Bioshop Canada), visualized on a UV Transilluminator and photographed by GeneSnap 4.00- Gene Genius Bio Imaging System (Syngene, Frederick, Maryland, USA). The digital image files were analyzed using Gene Tools software from Syngene. PCR product of rs1495099, rs907094 and rs3764352 polymorphisms were digested by 0.3 Unit of each restriction enzymes PvuII, MseI and BccI respectively using the buffers recommended by the manufacturer (Bioshop Canada). After digestion with restriction enzyme each amplicon may be cleaved into cut (C allele) or uncut (G allele) for rs1495099 polymorphism, cut (T allele) or uncut (C allele) for rs907094 and cut (A allele) or uncut (G allele) for rs3764352. The cleaved bands were visualized after electrophoresis on 2.5% agarose gel with DNA ladder standard 50 bp (Bioshop Canada).

Statistical analysis: Allele frequencies of the three studied polymorphisms, rs1495099, rs907094 and rs3764352 were calculated by single gene counting method. The crude measure of association between studied SNPS and Autism was expressed as odds ratio (OR) and its 95% confidence interval (95% CI) was calculated. Allelic and genotypic associations were evaluated by Chi-square test to compare the genotype frequencies between cases and controls. Statistical packages used for the analysis were Epi-Info-version 6 and SPSS version 16.

RESULTS AND DISCUSSION

Genomic DNA from 50 autistic patients and 50 control subjects were used to carry out PCR amplifications for rs1495099, rs907094 and rs3764352.Electrophoretic banding patterns of 230bp amplified bands were identified (Fig. 1). Amplified bands for rs1495099, rs907094 and rs3764352 polymorphisms were cleavage with PvuII, MseI and BccI restriction enzymes respectively. Figs. (2 & 3) showed electrophoretic banding pattern and Table (1) illustrate distribution of different three genotypes for each polymorphism among autistic patients and control subject groups as well as alleles frequency for each polymorphism. Neither the genotypes nor the allele frequency of PPP1R1B rs1495099 and rs3764352 SNPs examined in the autistic patients differ significantly from those observed for the control subjects Tables (1 & 2). Among detected three genotypes of rs907094 polymorphism only TC genotype showed significant association with autism.

Table 1: Genotypes and allele frequencies of SNPs and association analysis of each SNP in population –based samples. Polymorphism n Genotype P-value Allele frequency ------rs1495099 100 GG (230,180 bp) CG (230,180,50 bp) CC (230 bp) 0.76 G C ------n%n % n % Autistic 50 20 40 15 30 15 30 0.55 0.45 Control 50 23 46 12 24 15 30 0.58 0.42

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Table 1: Continue rs907094 100 TT (230,180 bp) TC (230,180,50 bp) CC (230 bp) 0.027 F(T) F(C) ------n%n % n % Autistic 50 18 36 15 30 17 34 0.51 0.49 Control 50 15 30 6 12 29 58 0.36 0.64 rs3764352 100 AA (230,180 bp) AG (230,180,50 bp) GG (230 bp) 0.565 F(A) F(G) ------n%n % n % Autistic 50 6 12 18 36 26 52 0.30 0.7 Control 50 6 12 23 46 21 42 0.35 0.65

Table 2: Odds ratio and confidence interval for association of genotypes within PPP1R1B gene polymorphisms and risk of autism. Polymorphism Genotype Case (n =50) Control (n =50) OR (95% CI) Significant rs907094 CC 18 15 1.17 (0.47, 2.90) NS TC 15 6 3.14 (1.00, 10.24*) Significant risk TT 17 29 0.37 (0.15, 0.91) NS rs907099 GG 20 23 0.78 (0.33, 1.87) NS GC 15 12 1.36 (0.51, 3.62) NS CC 15 15 1.00 (0.39, 2.56) NS Rs3764352 GG 6 6 1.00 (0.26, 3.86) NS GA 18 23 0.66 (0.27, 1.59) NS AA 26 21 1.50 (0.63, 3.56) NS

Fig. 1: PCR amplification of rs907094 polymorphism of PPP1R1B gene among autistic patients (P) and healthy control subjects (C).

Fig. 2: PCR-RFLP analysis of rs3764352 polymorphism of PPP1R1B gene with Bccl enzyme among autistic patients (P) and healthy control subjects (C).

Fig. 3: PCR-RFLP analysis of rs1495099 polymorphism of PPP1R1B gene with Pvull enzyme among autistic patients (P) and healthy control subjects (C).

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Three polymorphisms (rs1495099G/C, rs907094T/C and rs3764352A/G) in the PPP1R1B gene were tested in 112 male-only affected sib-pair families and a comparison group of 443 males and females (Hettinger, 2009), based on single marker and haplotype case-control comparisons as well as family-based tests including quantitative transmission disequilibrium tests (QTDT). The obtained results indicated that there was an increased frequency of rs1495099 C alleles (P=0.001) and CC genotypes (P=0.001) in affected males compared to the comparison group. Family-based tests performed under a recessive model showed over-transmission of the C allele (P=0.0009). QTDT analyses showed associations between rs1495099 C and more severe problems with social interaction (P=0.0016) and nonverbal communication (P=0.0046), as well as increased stereotypic behaviours (P=0.0072) as determined using Autism Diagnostic Interview-Revised subdomain scores. The obtained data suggested that the rs1495099 C allele, or functional variants in linkage disequilibrium with this polymorphism, cause changes either in PPP1R1B expression or DARPP-32 function in DAceptive neurons in brain regions which have a role in social interaction, communication and the pathophysiology of stereotypes. Two of these three SNPs (rs1495099 and rs3764352), however, did not show any significant association in present case control study. Among rs3764352 genotypes only TC genotype showed significant risk for autism while other two genotypes (TT and CC) did not show any significant association with autism among Saudi autistic patients. This finding suggesting that studied PPP1R1B SNPs unlikely to be related to be development of Autism in Saudi patients except TC genotype among rs907094 SNP. This might be due to differences in ethnic background, study design, sampling size. Although the present results did not provide evidence that the PPP1R1B is a susceptibility gene for autism in the studied Saudi population, PPP1R1B may contribute to Autism by interacting with other genes, or it may be a target of other Autism-related genes. Further functional analysis and genetic association studies are needed to determine the interaction of the loci between PPP1R1B and other genes, and their potential roles in the pathophysiology of Autism. Various studies have been reported that the DARPP-32 function is dependent on its phosphorylation status which regulated by three different pathways. Many candidate genes have been involved in such pathway (Svenningsson et al., 2003, 2002a and 2002b). The interaction between the loci within those genes and PPP1R1B may reduce the power to detect their effects in association studies. Future studies may detect and characterize interactions among the loci in these genes using multifactor statistical methods (Jian et al., 2007). However, since DARPP-32 occupies a unique position as a central molecular switch, integrating multiple information streams at dopaminoceptive neurons and converging through a variety of neurotransmitters, neuromodulators, neuropeptides, and steroid hormones (Jian et al., 2007 and Svenningsson et al., 2004) further case-control and family-based association studies with a larger sample size are needed to determine whether polymorphisms of PPP1R1B genes are associated with Autism.

Conclusion: In conclusion, the findings of the present investigation indicated that except CT genotype among (rs907094 SNP) genotypes other studied SNPs within PPP1R1B gene do not elevate the risk for autism in the studied Saudi population. Further functional analysis and genetic association studies using larger samples are needed to clarify the exact role of this gene in the pathophysiology of the autism.

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