The Contribution of the DLG5 113A Variant in Early-Onset Inflammatory Bowel Disease

Article Outline

• Abstract
• Methods
  • Disease Phenotyping and Data Collection
  • Deprivation Status
  • Genotyping
  • Statistical Analysis
• Results
  • Transmission Disequilibrium Testing Analysis
  • Association with Affluence
  • Multifactorial Analysis
  • Genotype-Phenotype Analysis: Unifactorial Analysis
    • Growth centiles
  • Sex
    • Extra-intestinal manifestations
    • Age at diagnosis
  • Disease Location and Behavior
    • DLG5-CARD15 interaction
• Discussion
• Acknowledgment
• References
• Copyright

Objective

To assess the contribution of the 113 G→A missense mutation within the discs, large homolog 5 (DLG5) gene in childhood-onset inflammatory bowel disease (IBD) in Scotland.

Study design

Two-hundred and ninety-six children with IBD were studied. Parental DNA was also collected for transmission disequilibrium testing (TDT) analysis. Genotyping was performed by TaqMan®. Genotype-phenotype analysis was also undertaken. Socioeconomic status was assigned using a deprivation category (DepCat) score 1 through 7 (1 = most affluent).

Results

TDT analysis demonstrated a significant association with IBD (P = .045). On unifactorial analysis, 113A carriage was associated with: (1) higher social class (DepCat 1 compared with 2-7, and 1-2 compared with 3-7) (66.7% vs 22.6%, P = .0005, OR 6.84 [1.99-23.55] and 37.2% vs 22.2%, P = .03, OR 2.08 [1.04-4.17], respectively); (2) higher height centile (>75^th centile vs <75^th centile) (42.9% vs 23.1%, P = .01, OR 2.50 [1.18-5.28]); and (3) male sex in Crohn’s disease (CD) (29.3% vs 16.9%, P = .04, OR 2.04 [1.01-4.11]). Multifactorial analysis demonstrated that higher social class (DepCat 1) was independently associated with carriage of variants of 113A (P = .001, OR=6.92 [2.24-21.33]).

Conclusions

DLG5 113A is associated with increased susceptibility to IBD in Scottish children. The effect may be most marked for those children living in relative affluence.

Abbreviations: BMI, Body mass index, CD, Crohn’s disease, DepCat, Deprivation category, DLG5, Discs, large homolog 5, IBD, Inflammatory bowel disease, UC, Ulcerative colitis, TDT, Transmission disequilibrium testing

A strong genetic basis for inflammatory bowel disease (IBD) has been suggested by evidence from twin studies, and family studies,¹ and has been supported by the identification of several genes associated with increased susceptibility to IBD: CARD15,², ³ DLG5,⁴ SLC22A4/5,⁵ MDR1,⁶ and CARD4.⁷ The disease model that has evolved is that ulcerative colitis (UC) and Crohn’s disease (CD) are related polygenic diseases sharing some but not all susceptibility genes.⁸

We have demonstrated a rising incidence of childhood-onset IBD in Scotland⁹, ¹⁰ and within the Scottish population we have demonstrated association between susceptibility to early-onset CD both with northern latitudes and with relative affluence.¹¹ In a study of 580 incident IBD cases over the period 1981 to 1995, the relative risk of developing IBD and CD was significantly increased in areas of relative affluence, compared with areas of socioeconomic deprivation (P = .02 and P = .03, respectively).

Over the last decade, genome-wide scanning in IBD has been remarkably successful, leading to the identification of several susceptibility loci, satisfying rigorous criteria for definite linkage.¹² We have shown that IBD1 and IBD5 both contain determinants of susceptibility and disease severity in early-onset IBD in Scotland, but these loci alone do not account for the majority of the genetic risk in our population.¹³, ¹⁴

A European genome-wide scan in 353 IBD sibling pairs (including UK patients) identified an area on chromosome 10 that achieved criteria for “suggestive linkage” in patients with CD.¹², ¹⁵ Fine mapping of this region identified variants of the discs, large homolog 5 (Drosophila) (DLG5) gene situated within this locus that may be associated with increased susceptibility to IBD.⁴ The 113 G→A variant (a missense mutation in exon 30) encoding for the amino acid substitution R30Q was associated with susceptibility to IBD and CD.⁴ Further studies have yielded inconsistent results.¹⁶, ¹⁷, ¹⁸, ¹⁹, ²⁰, ²¹, ²², ²³ Thus the effect appears inconsistent, relatively weak, and population-specific. We have rigorously assessed the contribution of the DLG5 113G/A mutation with respect to IBD susceptibility and phenotype in the high incidence Scottish population.

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Methods 

Patients diagnosed with IBD younger than 16 years of age were studied (197 CD, 73 UC, and 26 indeterminate colitis [IC]). There were 161 males and 135 females (Table I). These persons were recruited from pediatric gastroenterology centers across Scotland and from the Western General Hospital, Edinburgh. Parental DNA was also collected where available to allow transmission disequilibrium testing (TDT) to be performed (DNA from both parents was available in 69% of cases).

Table I. Demographics of inflammatory bowel disease patients at diagnosis

Sex (M/F)	161/135
Median age at diagnosis (y)	11.00(IQR8.42-12.92)
Current smoker	7/295(2.4%)
Family history of IBD	98/292(33.5%)
Caucasian (%)	288(97.3%)
Extraintestinal manifestations
Joint	23/282(8.2%)
Erythema nodosum	26/282(9.2%)
Location according to the Vienna classification25
Terminal ileum (L1)	9(4.6%)
Colon (L2)	48(24.3%)
Ileocolonic (L3)	38(19.3%)
Upper gastrointestinal (L4)	93(47.2%)
None⁎	9(4.6%)
Behavior according to the Vienna classification25
Inflammatory (B1)	163(82.7%)
Stricturing (B2)	7(3.6%)
Penetrating (B3)	27(13.7%)
Ulcerative colitis disease extent
Extensive colitis⁎⁎	52(71.2%)
Anthropometric centiles†
Weight (75-91st)	22/288(7.6%)
Height (75-91st)	28/272(11.1%)
BMI (75-91st)	27/270(10.0%)

The phenotypic data at diagnosis represent 296 patients with IBD (including 197 CD and 73 UC) unless otherwise stated.

Disease Phenotyping and Data Collection 

The diagnosis of IBD was based on standard criteria.²⁴ Comprehensive clinical phenotypic data were obtained using retrospective case note review and personal interview. The complete set of phenotypic variables collected in this cohort has been described previously and includes growth measurements and socioeconomic data assessing relative affluence/deprivation.¹³ The anthropometric information collected was weight and height of patients with a body mass index (BMI) calculated thereafter. The data were plotted on a standard UK centile chart of the UK 1990 population data (© Child Growth Foundation 1996) and an appropriate centile band was allocated. The Vienna classification together with a more extensive phenotypic description for each patient were used to describe the phenotype of patients with CD.¹³, ²⁵

Deprivation Status 

Socioeconomic status of the patient’s postcode was allocated using the Carstairs score. This score is a measure of socioeconomic deprivation derived from levels of male unemployment, head of the household’s social class, the level of overcrowding in households, and car ownership generated from census data.²⁶ Data used to calculate the scores for this study were based on the UK 2001 census data (www.groscotland.gov.uk/grosweb/grosweb.nsf/pages/censushm). The Carstairs score was then assigned a numerical deprivation category (DepCat score) based on the postcode (scored 1-7; 1 = no deprivation and 7 = maximum deprivation). The data for the DepCat score were not calculated by us as part of this study; we simply matched the DepCat scores with the patient postcode (the DepCat scores and matching postcodes were obtained from the Edinburgh University data library). In cases where a patient’s postcode fell between two different DepCat categories, no score was allocated. Thus, this score is derived from a person’s postcode and reflects the socioeconomic status of the area in which a patient lives rather than an individual case-specific score. The Carstairs score has been widely used to study the association between socioeconomic conditions and disease.

The methodology used to define North and South Scotland was based on patients’ postcodes.¹¹ Written informed consent from all patients and controls was obtained. All relevant Research Ethics Committees approved the study protocol.

Genotyping 

Genomic DNA was extracted from peripheral venous blood in all patients by a modified salting-out technique,²⁷ and it was re-suspended in 1xTE (10 mM Tris [pH 8.0], 1 mM EDTA [pH 8.0]) at a final concentration of 100 ng/μL. The SNP 113G/A (rs1248696) was typed using the TaqMan® system (Applied Biosystems, Foster City, CA) in the Wellcome Trust Clinical Research Facility, Western General Hospital in January 2005 by personnel blinded to the project aims. The genotyping methods used for the three CARD15 mutations (Gly908Arg, Arg702Trp, and Leu1007fsinsC) have been described previously.²⁸

Statistical Analysis 

TDT was performed in IBD trios using TRANSMIT.²⁸ This method makes full use of the data available using inferred genotypes if data from both parents are not available. The PedCheck software program (Department of Human Genetics, University of Pittsburgh, Pittsburgh, PA) was used to exclude any potential cases of nonpaternity or genotyping error.²⁹ Genotype-phenotype associations were analyzed by χ² test, or Fisher’s exact test where appropriate, using the Minitab statistical software package (Minitab Ltd., Coventry, UK). To identify significant independent variables associated with genotype, unifactorial and multifactorial logistic regression analyses were carried out.

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Results 

Transmission Disequilibrium Testing Analysis 

In total, 97% of samples were successfully genotyped. TDT analysis based on 270 families demonstrated an association of allelic variants of the 113A allele with IBD only (χ² = 4.01, P = .045) (Table II).

Table II. Transmission disequilibrium testing results for the DLG5-113 mutant allele

The result of transmission disequilibrium testing for the 113 G→A in 270 families demonstrates association with IBD but not Crohn’s disease or ulcerative colitis.

Association with Affluence 

A higher rate of carriage of the 113A variant was present in the patients with DepCat score 1 (n = 12) compared with those with DepCat score 2 through 7 (n = 252) (66.7% vs 22.6%, P = .0005, OR 6.84 [1.99-23.55]) and in comparing allele frequency between the two groups (33.3% vs 13.3%, P = .006, OR = 3.26 [1.34-7.92]) (Table III). Higher carriage of 113A variants was also demonstrated if patients with DepCat scores 1 and 2 were analysed together (n = 43) and compared with patients with DepCat scores 3 through 7 (n = 221) (37.2% vs 22.2%, P = .03, OR 2.08 [1.04-4.17], respectively). There was no significant difference between northern (n = 35) and southern latitudes (n = 259) in carriage of variant 113A alleles (34.4% vs 23.9%, P = .20, respectively).

Table III. DepCat scores in patients with IBD together with DLG5 113A carriage rates

There is a significant difference in DLG 113A variant carriage in patients in DepCat score 1 v. 2-7 as well as 1 and 2 combined compared with scores 3-7 (P = .001, OR = 6.92 [2.24-21.33], and P = .02, OR = 2.49 [1.13-5.47], respectively). All patients in DepCat 1 were Caucasian (7 males and 5 females).

Multifactorial Analysis 

Binary logistic regression analysis combining family history of IBD, sex, weight, height, BMI centile (as either 75^th-91^st or other), and social class (as either DepCat score 1 or 2-7) together with joint disease in all patients with IBD was performed.

Carriage of the 113A variant was associated with DepCat score 1 taking into account all of the above factors (P = .001, OR = 6.92 [2.24-21.33]). If the same model was run combining DepCat 1 and 2 together compared with 3 through 7 and with all other factors, the association between higher socioeconomic class and 113A carriage was confirmed (P = .02, OR = 2.49 [1.13-5.47]).

Genotype-Phenotype Analysis: Unifactorial Analysis 

On unifactorial analysis, carriage of the 113A allele was associated with the higher height centiles (>75^th centile vs <75^th centile) (42.9% vs 23.1%, P = .01 OR 2.50 [1.18-5.28]) at disease diagnosis. Carriage of variants was highest in the 75^th through 91^st centile for weight and height at diagnosis compared with those in the nine other centile bands (56.2% vs 23.4%, P = .004, OR 4.20 [1.49-11.81] and 48.0% vs 22.4%, P = .006 OR 3.19 [1.36-7.47], respectively).

Sex 

In CD, the carriage rate of 113A variant alleles was higher in males than in females (29.3% vs 16.9%, P = .04, OR 2.04 [1.01-4.11]). Allele frequency showed a similar trend (male 15.1% vs female 9.0%, P = .07, OR 1.79 [0.94-3.40]), but this difference was not statistically significant. There was no sex-related difference between allele frequency and carriage rates of 113A variants in patients with UC or IBD.

The carriage of 113A variant alleles was uncommon in patients with joint disease or symptoms but did not reach statistical significance (13.0% vs 25.9%, P = .21, OR 0.42 [0.12-1.49], respectively).

There was no difference in the median ages at diagnosis of carriers of 113A variant alleles versus noncarriers for patients with CD at 11.4 years versus 11.2 years (P = .91) and with IBD at 11.3 years versus 10.9 years (P = .73).

Disease Location and Behavior 

There was no difference in allele frequency or carriage rates of this 113A DLG5 variant in disease location or behavior in patients with either CD or UC at diagnosis.

There was no difference in carriage of the DLG5 113A variant when patients were stratified based on carriage of at least one of the three common CARD15 variants (Gly908Arg, Arg702Trp, and Leu1007fsinsC). The carriage of CARD15 variant alleles compared with noncarriage in patients with IBD or CD was 14.0% versus 20.5% (P = .30, OR 0.63 [0.26-1.53]) and 14.7% versus 20.5% (P = .43, OR 0.66 [0.23-1.88]), respectively.

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Discussion 

Our data suggest that the penetrance of the DLG5 genetic variants, and thus genetic associations with disease, may be influenced by environmental factors present in affluent families in Scotland. Thus although the overall association of the DLG5 113 variant alleles with early-onset IBD in the Scottish population appears modest, the association with children living in affluence is strong. These findings suggest a potentially novel insight into gene-environmental interactions in IBD.

We have previously demonstrated an association between higher social class (DepCat 1) and early-onset IBD in Scotland by Poisson regression analysis in a study of 580 patients with IBD; these data clearly demonstrated that the relative risk of IBD and CD is highest in the most affluent families.¹¹ The association of CD with affluence has also been demonstrated in other childhood IBD populations.³⁰ Our findings complement previous UK data, suggesting that the presence of a hot water tap and a separate bathroom increase the risk of developing CD.³¹, ³² The reason for these associations with affluence and household hygiene remain unclear, but the role of water quality,³² diet (cold-chain hypothesis), hygiene, and infection rates³³ are potentially plausible.

We now present evidence suggesting that the most susceptible persons within these affluent regions may also possess a risk genotype, supporting a gene-environment interaction as an explanation for the association with affluence. The association with the most affluent appears robust in the present study—the relationship is present when DLG5 113A carriage rate is compared between patients with DepCat score 1 and DepCat 2 through 7 moreover, the association holds on multifactorial analysis notwithstanding the small number of patients in DepCat 1.

The potential mechanisms whereby DepCat 1 children should differ from children with other DepCat scores and how this influences the development of IBD remain uncertain and a matter for investigation, but it is interesting to note some of the differences highlighted from published studies. Children from lower DepCat scores have higher rates of dental caries, are more likely to be colonized with yeasts, and have higher rates of Helicobacter pylori infection.³⁴, ³⁵ Breast-feeding incidence and duration are highest in DepCat 1 and 2 together in one study,³⁶ and they are higher in DepCat 1 compared with 2 and other categories in a second study.³⁷ These data highlight the fact that the early exposure to microbes in each DepCat category is likely to be different and present a possible mechanism of how DepCat score, environment, and genotype interact.

Growth and development are critical aspects of childhood-onset IBD. Growth failure is a severe manifestation of CD that we have recently shown to be strongly linked to carriage of disease associated variants from within the IBD5 locus.¹⁴ However, in the present study the association of DLG5 variants is with higher height and weight centiles leading to the hypothesis that this variant may act in the opposite manner to IBD5 variants and confers a protective effect against growth failure. A similar protective role has been previously suggested for the –238 tumor necrosis factor-α variant in a pediatric CD population.³⁸ These data therefore also provide indirect support for the concept that this growth variant leads to disease expression in children lacking socioeconomic deprivation.

The 113A variant was commoner in male compared with female children with CD in our study. It has been previously reported that the DLG5 variants are more common in males.³⁹ It is entirely plausible that DLG5 studies in early-onset populations are more likely to give positive results because of the male predominance in childhood-onset CD (contrasting with the female predominance in adult populations).⁴⁰, ⁴¹

What then of the overall contribution of this DLG5 variant to disease susceptibility? Our TDT analysis (P = .045) reveals data similar to those of Stoll et al and subsequent publications, which have suggested a weak overall association of variant alleles of DLG5 113A with IBD.⁴, ¹⁹ In Stoll’s original study, initial TDT analysis in 457 IBD trios (302 CD) demonstrated association with IBD (P = .004) and CD (P = .04),⁴ but a replication cohort of 485 IBD trios only suggested a trend toward significance for IBD and CD (P = .09 and P = .07, respectively). Daly et al reported TDT data in two further IBD populations, one demonstrating significant association (P = .02) but the second showing no association.¹⁹ In the Flemish population, contrasting results to those of Stoll and colleagues are reported with significant undertransmission of 113A variants.¹⁷

Case-control studies have yielded inconsistent results,⁴, ¹⁷, ¹⁸, ¹⁹, ²⁰, ²¹, ²², ²³ with heterogeneity among controls more than cases providing the differences between study populations.¹⁶, ⁴² This heterogeneity between different study populations matches data in the Scottish population for CARD15 and TLR4.¹³, ⁴³, ⁴⁴

Our positive TDT results and the conflicting data from other series are nonetheless consistent with the hypothesis that DLG5 may represent a gene of relatively modest effect in IBD.¹⁶ The population differences can be explained by a combination of factors—heterogeneity between geographically/ethnically distinct populations and, heterogeneity within subgroups of IBD patients with specific differences in the sex and age of patients. It is interesting to note that in our own Scottish population using TDT analysis, a method that protects against the problems of population stratification, we have shown a positive association when a case-control study in a Scottish adult population has been negative.²² An adequately powered case-control study to prove beyond doubt a definite role for DLG5 variants in IBD etiology may necessitate studies of greater size than any yet carried out in CD or UC, requiring several thousand patients and controls.¹⁶, ¹⁹ This strategy is similar to that applied to many other polygenic diseases, in which genetic determinants of low genotype relative risk have been identified.⁴⁵

In summary, we provide preliminary evidence that suggests that a novel environmental modifier, affluence, may affect gene penetrance and disease expression in early-onset IBD. We suggest deprivation status represents a potentially important confounding variable that needs to be investigated in studies of the genetics of IBD and perhaps other common immune-mediated illnesses.

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The authors would like to acknowledge the help of all patients and parents who participated in the study together with the specialist nurses, dieticians, and secretaries in each of the teaching hospitals as well as the pediatricians, practice nurses, and GPs throughout Scotland whose support for the study was invaluable. We thank Robin Rice in the University of Edinburgh data library for supplying the DepCat data and matching postcodes. We would also like to thank the staff at the Wellcome Trust Clinical Research Facility Edinburgh.

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