To date there has been little evidence for the involvement of genetic factors in the development of IBS. Although there have been indications from non-genetic studies that there may be familial associations in adults with IBS, including some twin studies,44,45,54
no candidate genes have been associated with any IBS phenotype. Recently, Pata24
reported no association between SERT-P polymorphisms and the presence of IBS, but did suggest the short/short (s/s
) homozygous polymorphism as a risk factor for the constipation predominant form of the disease and the long/short (l/s
) heterozygous polymorphism similarly for dIBS. Interestingly, the s/s
variants have both been shown to lead to less transporter protein expression and less serotonin reuptake20,22
and thus the apparently opposing associations with cIBS and dIBS reported are difficult to interpret based on predicted functional consequences of the polymorphisms. The authors noted that these conclusions were drawn from very small samples (n
26 for cIBS; n
18 for dIBS) and as such interpretation of the results should be limited. However, in their pilot study involving 173 patients with IBS (mixed subgroup population) or chronic abdominal pain, Kim and colleagues26
also determined a trend towards an association between the s/s
polymorphism and the constipated (IBS) phenotype. It is not immediately obvious how a polymorphism that produces a reduced level of functional reuptake transporter, and elevated 5-HT levels, results in a constipated phenotype but plausible mechanisms do exist (see below).
In stark contrast with these earlier studies, the present data from a North American study population provides evidence of an association between the s/s genotype of the SERT-P polymorphism and dIBS, which is perhaps somewhat more intuitive based on the predicted functional consequence of raised levels of 5-HT in the bowel. This study represents the first evidence for a statistically significant association between the dIBS phenotype and a polymorphism in the SERT gene. It is not known at this time whether the commonly used diagnostic distinction of IBS sufferers into constipation predominant, diarrhoea predominant, or alternators, has any true physiological or genetic basis, but further studies of this type will surely help to define this more precisely.
In our study, the distribution of the genotypes in the dIBS cases clearly reflects an increase of the s/s
genotype when compared with the control population. From the allelic analyses, no such association with the phenotype was found, suggesting that two alleles are required to see the effect of association. In this study and others,55
the distribution of the genotypes in the general population is biased towards the heterozygous l/s
form, with the s/s
genotype being least frequent, accounting for only approximately 18%. The increased risk of carrying this genotype in the dIBS disease population appears to be at the expense of the more common heterozygous form as l/s
frequency is reduced and that of the l/l
form is unchanged. This may explain in part the lack of allelic association described above.
The few studies that have been performed in vitro to date suggest that the s allele is dominant, such that both the heterozygous (l/s
) and homozygous (s/s
) genotypes result in reduced transporter expression and thus function, compared with the l/l
Therefore, if we hypothesise that such reduced SERT expression results in dIBS symptoms, then both genotypes containing the s allele should be associated with the presence of the disease. This was not what we found; indeed, analysis of the differences between the frequency of combined s/s
relative to the l/l
genotype was not performed as the genotype frequencies of these two groups were virtually identical and therefore quite obviously not significantly different. The important point to stress from our analysis, as mentioned above, is the greater proportion of s/s
genotypes in the disease population compared with controls, apparently resulting from a shift from the l/s
form. The overall risk of bearing a copy of the s allele is unchanged in the disease but rather the shift from heterozygous to homozygous forms appears to be the more relevant factor. Perhaps the small trend for a greater functional consequence of the homozygous genotype in vitro (Lesch et al
described a greater significance reported for the s/s
comparison than for the l/s
comparison) could help us understand these disease phenotype findings. However, direct correlation between in vitro findings in isolated cells and disease symptom expression is a huge leap and probably not a sensible link to try to claim.
Genotyping for the SERT-P polymorphism was performed in duplicate for all samples and the genotypes were scored in duplicate by different individuals, and thus it is unlikely that these results are due to genotyping error. This study has not determined how far the block of LD containing the SERT-P polymorphism extends upstream of the SERT gene and no LD map is available for the area surrounding the gene. Therefore, we cannot rule out the possibility that another polymorphism upstream of the SERT gene, which is in high LD with the SERT-P polymorphism, may also be associated with the dIBS phenotype. The genotypic association is also reflected in the HWE deviation observed for dIBS cases, and this method of detecting disease association in a heterogeneous disease such as IBS has been reported in the literature.56
There can be no doubt that genetic studies on populations collected during clinical trials are unfortunately far from perfectly structured due to the strict limitation on the individuals that can be collected and their willingness to be involved in a genetic analysis. In the case of this IBS study, it was not possible to collect data from cIBS patients and so the analysis and conclusions are restricted solely to the dIBS population. Collection of matched controls was also problematic but this problem was addressed by increasing the number of controls used to over twice the number of cases. We have demonstrated (unpublished data) that when matched controls are not available, an increased number of controls from a general ethnic match increases study power and suppresses confounding effects such as population stratification and admixture. However, as the SERT-P polymorphism is known to show frequency variability between ethnic groups,57,58
additional studies based around the genetic hypothesis formulated in this work should be undertaken to replicate the association demonstrated.
It is interesting to speculate how the evidence of association between the s/s
genotype of the SERT-P polymorphism with diarrhoea predominant IBS might provide some insight into the underlying pathophysiology of the disease, at least in this subgroup of female patients. As described above, reduced transcription of the 5-HT transporter would be expected to lead to enhanced levels of 5-HT in the bowel of these patients, which in turn might be predicted to result in increased bowel motility and secretion, and subsequent diarrhoea; a situation not dissimilar to that described for SERT knockout mice.19
Furthermore, as observed in the SERT−/− mice, it is intriguing to consider the possibility that such increased 5-HT may also lead to a constipated phenotype, as suggested by earlier studies.24,26
This may occur following 5-HT receptor desensitisation in the presence of high levels of agonist for long periods, or indeed could be produced by excessive stimulation of the 5-HT receptor bearing enteric neurones such that the normal motility pattern is stimulated to such an extent that coordination of enteric reflexes breaks down and contractile and secretory function fail. At a cellular level, such excessive neuronal depolarisation may result in “depolarisation block”. In this condition, the neuronal membrane potential remains depolarised above the threshold for sodium channel activation, and further action potential generation is not possible. In this way, varying degrees of excitation of enteric neurones might contribute to both hyper- and hypomotility and secretion.
The potential effects of reduced uptake of 5-HT are not however restricted to the neurones of the enteric nervous system. Excessive or prolonged stimulation of extrinsic afferents may also result in the development of neuronal sensitisation, at peripheral, spinal, or higher CNS levels, such that perception of sensations from the bowel is heightened, resulting in symptoms of urgency, bloating, and pain. Such neuronal hypersensitivity is thought to be a key factor in the generation of sensory IBS symptoms,59,60
and can be mimicked in animals by repetitive physical stimulation of the bowel, or by periods of infection,61,62
In a similar way, repeated stimulation of either extrinsic afferent nerves by excessive 5-HT in patients with impaired reuptake may also provide another method by which sensory perception of events in the gastrointestinal tract may be inappropriately enhanced.
This study shows that the SERT polymorphism, or a polymorphism in linkage disequilibrium with the SERT polymorphism, may play a role in the development of IBS. We do not believe that the reduction in SERT is the sole cause of dIBS in our population but we do believe it might be a contributing factor. Perhaps a combination of this polymorphism with many other factors, such as raised EC cell number, could explain the variety of symptom patterns observed across the disease population. The recent finding of reduced SERT expression in IBS patient biopsies,18
coupled with our own findings, does encourage speculation that there is a role for altered transporter function in the disease. While research continues into the variety of possible factors which initiate, maintain, and exacerbate the symptoms suffered in IBS, the data reported in the present study clearly provide further support for the key role of the serotonin system in IBS pathophysiology and will hopefully stimulate further effort towards identifying and understanding further potential genetic and other factors that might contribute to the disease.