CANDIDATE GENE STUDIES AND IBS

Serotonin and IBS

Many investigators have focused on specific candidate genes and whether variation in these genes is associated with IBS. Serotonin (5-hydroxytryptamine, 5-HT) is the most widely studied molecule due to its role in brain-gut axis and abundance in the gastrointestinal tract. Majority of body serotonin is present in gastrointestinal tract where it is synthesized mainly by enterochromaffin cells (EC cells) and some of it by myenteric plexus neurons. Serotonin is a known neurotransmitter and a local hormone in the enteric nervous system. It is released from EC cells in response to variety of stimuli and exerts its local paracrine effects through serotonin receptors. There are seven serotonin receptors identified so far named as 5-HT1 to 5-HT7. Out of these seven receptors, 5HT1 to 5-HT4 and 5HT7 play a key role in mediating intestinal responses. The effect of serotonin molecule initiated after its binding to serotonin receptor is eventually terminated by its uptake through a serotonin reuptake transporter (SERT)[16].

There is evidence that patients with IBS have defects in serotonergic signaling. Almost four decades ago, it was discovered that patients with IBS have more number of EC cells as compared to controls[17,18]. Since then numerous studies have explored the relationship between EC cells, serotonin and IBS. Study by Bearcroft et al[19] demonstrated that patients with D-IBS have higher blood levels of serotonin. Similarly studies by Dunlop et al[20] and Atkinson et al[21] have shown higher blood levels of serotonin in D-IBS patients and lower blood levels of serotonin in C- IBS patients. These authors also measured the levels of 5-HIAA (5-hydroxyindoleacetic acid), a 5-HT metabolite, in the rectal mucosal biopsy specimens and blood to obtain 5-HT/5-HIAA ratios as a surrogate marker for serotonin turnover. Based on analysis of 5-HT/5-HIAA ratios in blood and mucosal biopsies, serotonin release defects in C-IBS and serotonin uptake defects in D-IBS were suggested. In the search for an explanation to these differences SERT functions, polymorphism of SERT gene has been extensively investigated.

The SERT gene, also known as solute carrier family 6 member 4 (SLC6A4), was mapped to chromosome 17q11.2 by Ramamoorthy et al[22] in 1993. Polymorphic loci that affect the expression and function of SERT gene have been identified. There is a GC base pair rich repetitive sequence located at 5’ regulatory end of the SERT gene and is labeled as 5-HT transporter linked promoter region (5-HTT LPR). Polymorphism due to deletion or insertion of 44 base pairs in this region resulting in a long (L) and short (S) allele was first discovered by Heils et al[23]. Another common polymorphism of SERT gene results from a variable number of 17 base pair repeats (VNTR: variable number tandem repeats) in the intron 2 of gene[24]. VNTR has 4 alleles described with 9, 10, 11 and 12 repeats.

The evidence on the relationship between different genotypes and their phenotypic expression comes from a study conducted by Lesch et al[25]. Lesch et al[25] found that S/S genotype as compared to other genotypes L/L and S/L had less 5-HT uptake resulting in higher blood levels of 5-HT. Based on studies from Dunlop et al[20] and Atkinson et al[21] where defects in serotonin uptake were suggested in D-IBS, S/S genotype was expected to be associated with D-IBS. Several researchers have explored the association between IBS and SERT gene since then but have come up with conflicting results. In 2002 for the first time, Pata et al[26] demonstrated higher percentage of C-IBS patients to have S/S genotype and D-IBS to have L/S genotype. In the same year Camilleri et al[27] investigated the association between SERT polymorphism and response to Alosetron (5-HT3 receptor antagonist) and found higher response rates to the drug in IBS individuals with L/L genotype. According to the study by Lee et al[28] in the Korean population there was no association between IBS and SERT gene polymorphism. However, another Korean study by Park et al[29] found significantly higher frequency of S/S genotype among patients with D-IBS, contrary to results of study by Pata et al[26] mentioned above. Similarly, in the North American study by Yeo et al[30] S/S genotype was found to be associated with D-IBS female patients but Saito et al[31] found a significantly higher number of S/S genotype with M-IBS patients. In the study involving Indian IBS patients a significant association was found between S/S genotype and C-IBS[32]. In a Chinese study by Li et al[33] allele frequency of the L/L genotype was significantly higher in the C-IBS group. In the same study, researchers also demonstrated poor response to Tegaserod (5HT4 partial selective agonist) associated with L/L genotype.

Another polymorphism pertaining to a single nucleotide polymorphism locus, rs25531 that is located immediately upstream of 5-HTTLPR was described by Kohen et al[34]. It has two - A and G alleles. Hu et al[35] showed that A variant of L allele (designated as L A ) yields higher SERT expression as compared to G variant of L allele (designated as L G ). This implies that L G (G variant of rs25531 with L allele) actually behaves as the low expressing S allele. With respect to VNTR polymorphism, one study[36] found a significant association between the VNTR polymorphism and IBS but other studies by Yeo et al[30], Pata et al[26] and Li et al[33] did not find any significant association.

To investigate the possible association between IBS subtypes and SERT polymorphism, Van Kerkhoven et al[37] conducted a meta-analysis of eight studies in 2007 that found no significant association between SERT polymorphism and IBS subtypes. Another meta-analysis conducted in 2013 that included more recent studies conducted since the first meta-analysis by Van Kerkhoven et al in 2007, concluded a positive association between SERT polymorphism and C-IBS[38]. In another meta-analysis from 2013 performed by Areeshi et al[39] that included twelve studies comprising 2068 IBS patients, no association was found between SERT polymorphism and IBS overall. However, when the studies were stratified according the country of origin, significant association was found in American and Asian studies. The most recent meta-analysis with the largest sample size, involving 25 studies comprising of 3443 IBS patients found a positive association between SERT polymorphism and IBS but this association was found only in the East Asian population and not in the Caucasian population[40]. One probable explanation for this ethnic difference, as evidenced by this meta-analysis, could be the significantly lower frequency of L allele among the East Asian population as compared to Caucasian controls.

Besides the most widely studied polymorphism involving SERT LPR discussed above, several other polymorphism loci have been explored. These include polymorphisms of gene involving other serotonin receptors - 5-HT2A gene[41-43] and 5-HT3E gene[44] and yet again the studies have yielded either conflicting results or need further validation in other ethnic groups.

Tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT biosynthetic pathway has two isoforms - TPH1 and TPH2 encoded by genes on chromosome 11 and 12 respectively. Jun et al[45] found no association between TPH1 gene single nucleotide polymorphism and risk of developing IBS, however found significant association with severity as well as number of days with diarrhea in IBS patients. TPH2 gene polymorphism was also tested and shown to be associated with reduced risk of having IBS but statistically this difference was barely significant. Similar to this study by Jun et al, no association was found between genotype frequencies and IBS in the study conducted by Grasberger et al[46]. But the CC genotype was found to be more prevalent in IBS-D patients in the study. Researchers that investigated the colonic mucosa levels of TPH1 mRNA in IBS patients found significantly reduced TPH1 mRNA levels in both IBS subtypes[47].