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4.1.1.1. Failure to extend the 3p linkage region

To better stratify the numerous IBD linkage regions by their effect on the disease

population, it is important that extension studies are conducted on genome scan linkage

regions when the size of the ASP cohort increases. With the exception of the IBD

linkage regions on 14q and 19p, all other significant (LOD > 3.59) IBD linkages have

resulted from studies across suggestive loci in expanded cohorts (Duerr et al., 2000; Hampe et a i , 1999b; Hugot et al., 1996; Rioux et al., 2000; Satsangi et al., 1996b). In the present study, a suggestive IBD loci on 3p from the Satsangi et al. (1996) genome

scan was re-analysed by conducting a study in the region with an expanded population

of 284 IBD ASPs (Table 2.1.). It was hoped that, like the loci mentioned above, the

extension study would result in significant linkage. However, linkage was not detected

in the region for any phenotype model (Table 3.9.).

4.1.1.2. Genetic heterogeneity and differences in methodology

Due to the difference in results from this extension study and genome scan by Satsangi

for linkage (Table 3.10.). Since the additional families used in the present study were

ascertained using the same criteria as used previously (re: clinical diagnosis, family size,

ethnicity), the difference in results may be caused by both genetic heterogeneity and

differences in the methodology of analysis. The analysis showed that for SIBS1&2 the

observed increase in sharing within the 3p region (IBD MAS of 0.55), was more

consistent with the sharing observed in the present extension study rather than the

previous genome scan (IBD MAS of 0.54 and 0.63, respectively). If the difference in

results between the present extension study and the previous genome scan were caused

by genetic heterogeneity, then sharing at the level observed in the previous genome scan

should have been detected when SIBS1&2 were analysed separately. Since SIBS1&2

data was not consistent with the previous genome scan data, the differences between the

two studies are more likely to be caused by differences in methodology of linkage

analysis. In the previous genome scan, two-point linkage analysis rather than mulitpoint

analysis was used. In two-point analysis, because sharing status is not inferred by using

data of surrounding markers, increased bias caused by the proportion of informative

families is observed (Section 1.4.1.3.). If a marker by chance is more informative in

families sharing the region, increased sharing and thus linkage is observed for the

marker despite the fact that if all families were informative no increase in sharing would

have been detected. It is this bias caused by using two-point analysis that may account

for most of the difference in the results seen between the two studies.

4.1.1.3. Relative significance of the 3p putative IBD susceptibility region

Sinee the publication of the Satsangi et al. (1996) IBD genome scan, other independent

region-specific studies in the 3p IBD linkage region have been conducted (Annese et al., 1999; Brant et a i, 1998; Rioux et a i , 1998; Vermeire et al., 2000). These studies also failed to see linkage to IBD. To date, 5 genome scans and 4 replication studies

have failed to detect linkage to chromosome 3p. It has been noted that suggestive

linkage will occur by chance once per genome scan and thus the linkage to 3p

previously reported by Satsangi et al. (1996) may be a false positive. However, a recent

genome scan by Rioux et al. (2000) has reported linkage to the chromosome 3 region,

with a LOD o f 2.4, which suggests that within the 3p region the possibility a gene of

small effect on IBD aetiology remains.

4.1.2. Chromosome 6p replication study

4.1.2.1. Replication of the IBD3 susceptibility locus

IBD genome scans have resulted in the identification of several regions of linkage.

However, only two regions have been subsequently replicated by independent region-

specific studies. Recently, Hampe et al. (1999b) reported linkage to 6p in an extension

study using the largest IBD population to date (428 ASPs). In the present study, the 6p

linkage region was replicated in the Oxford IBD cohort containing 284 ASPs (Table

2.1.). The nonparametric peak multipoint LOD score of 3.04 neai* D6S291 (Table 3.29.)

exceeds the proposed criteria for independent replication of significant linkage (LOD =

1.5) (Lander and Kruglyak, 1995). However, while this represents the first region-

specific replication study of the IBD3 locus, both a recent genome scan by Rioux et al. (2000) and an earlier candidate gene study by Yang et al. (1999) also reported

suggestive linkage to 6p between D6S1281 and D6S1019 in a cohort of 183 IBD ASPs

(LOD = 2.3) and around TNF in a cohort of 70 CD ASPs (P = 0.002), respectively. All

three IBD studies showed linkage contributed by both the UC and the CD populations.

However, both the linkage peak from the Rioux et al. (2000) study and the peak from

this study were approximately 15 cM proximal to the initial reports of linkage (Hampe

between actual gene location and peak linkage are not unexpected in complex traits

(Kruglyak and Lander, 1996; Roberts et a i , 1999). Taken together the results support the existence of an IBD susceptibility locus at 6p {IBD3).

4.1.2.2. Relative significance of fBD3

IBD3 is the only locus where positive linkage has been reported in each of the three largest independent IBD studies (Table 1.1.). Both IBDl and IBD2 have been explored in independent linkage studies with greater power than the original studies reporting

linkage to the regions. However, these later studies of greater power have all failed to

show evidence of linkage to the regions (Cho et a i , 1998; Duerr et al., 2000; Hampe et a l , 1999a; Lesage et al., 2000; Rioux et a i , 2000). One study even excluded the possibility of a gene in the IBDl and 1BD2 intervals with a A,s of 2.0 or greater (Rioux et a i , 1998). The differences between these studies may be because of genetic

heterogeneity or the difficulty in detecting genes of modest effect in complex traits

(Risch and Merikangas, 1996). In the first instance, since linkage cohorts are frequently

ascertained differently and consist of different ethnicity, a linkage region detected in one

study may contain a gene that only affects people with the same ethnicity and severity

of disease and would thus be undetected in a population of different composition. In the

second instance, if a cohort analysed for linkage were biased in favour of families

containing a particular disease gene, increased linkage would be detected compared to a

non-biased population. If the disease gene in question also had a low displacement (t)

in the overall population, than the actual increase in sharing (Y or MAS) caused by the

gene could be insufficient to detect linkage above a LOD of 2.19 (Risch, 2000).

Therefore, as a result of bias in the initial study, subsequent studies containing larger

D 6 S 2 7 S HLA-DPB1 m a jor h isto co m p a tib ility c o m p le x , c la s s II. D P b e ta 1

Z N F 204 zinc fin g er p ro tein 204 NFKBIL1 n u c le a r fa c to r of k a p p a light p o ly p e p tid e g e n e e n h a n c e r in B c ells inhibitor like 1

D 6 S 2 5 5 P P T 2 p a lm itoyl-prote in t h io e s te r a s e 2

H 1F 5 H I h is to n e family, m e m b e r 5 IT PR 3 inosrtol 1 .4 ,5 - tn p h o s p h a te re c e p to r , tyjne 3 P R S S # th y m u s sp e c if ic s e n n e p e p tid a s e A G ER a d v a n c e d g ty c o sy ta tio n e n d p ro d u c t-sp e c ific

to b family to b family H L A -D 0 8 m a jor h isto co m p a tib ility c o m p lex c la s s 11. D O b e ta

z147 zinc fin g e r p ro tie n C 0 L 1 1 A 2 c o lla g e n , ty p e XI, a lp h a 2

C 0 X 1 1 c y to c h r o m e C o x id a s e su b u n it 11 SA C M 2L s u p p r e s s o r of a ctin m u ta tio n s 2, y e a s t, hom olog-like

H 2B F R H 2B h is to n e fam ily, m e m b e r R PH F 1 PH D fin g er p rotein

SR E -Z B P zinc finger p ro te in (S R E -Z B P ) CR EBL1 cA M P r e s p o n s i v e e le m e n t b in d in g p ro tein like 1 H 2A F P H2A h is to n e fam ily m e m b e r P P S M B 8 p r o te a s o m e su b u n it, b e ta ty p e 8

H 3FK H 3 h is to n e fam ily, m e m b e r K HLA-DMA m a jo r h isto co m p a tib ility c o m p le x , c la s s II, DM a lp h a

HLA-A m a jor h isto co m p a tib ility c o m p le x , c la s s 1 A BF B -factor p ro p erd in

Z N F 1 7 3 z inc fin g e r p ro te in 173 R D R a d in b lo o d g ro u p

HLA-F g e n e for h u m a n le u k o c y te a n tig e n F R N F 3 o r R I N G S f e m a le s te rile h o m e o tic -r e la te d g e n e t or ring finger p ro tein 3

KIAA 0036 KIA A 0036 g e n e p ro d u c t R P S I O r ib o so m a l p ro tein 8 1 0

H C G V h e m o c h r o m a to s is c a n d id a te g e n e V TAF2I TATA box b in d in g p ro tein ( T B P ) - a s s o c ia te d f a c to r. RNA p o ly m e r a s e II, 1 28kD

CAK cell a d h e s io n k in a se SR PK 1 S F R S p ro te in k in a s e 1

hcgVIll hcgV lll FK B P5 F K 5 0 6 -b in d in g p ro tein 5

P P P 1 R 1 0 p ro tein p h o s p h a ta s e 1 re g u la to ry s u b u n it 10 P P A R D p e ro x is o m e pro lite ra to r a c tiv a te d re c e p to r , d e lta

Z N F 165 zinc fin g e r p ro tein 165 KN SL2 K inesin-iike 2

P5-1 P5-1 C L P S c o lip a s e , p a n c r e a tic

Z N F 184 k ru p p e i- r e la te d zinc fin g er p rotein HLA-DNA m a jo r h isto co m p a tib ility c o m p le x c la s s II. DM a lp h a

C D S N c o rn e o d e s m o s in D G S 2 9 ■

KIAA 0170 KIA A017 0 g e n e p ro d u ct C S B P 1 c y to k in e s u p p r e s s iv e an ti-in flam m ato ry bin d in g p ro tein 1 {p38 MAP k in a se ) ABC 50 A T P -b in d in g c a s s e t t e 5 0 (TN F a lp h a stim u la tin g KIA A 0229 KIA A0229 g e n e p ro d u ct

D IF2 D if fe re n tia tio n -d e p e n d e n t g e n e 2 G L P 1 R g lu c a g o n -lik e p e p tid e 1 r e c e p to r D D X16 DEAD /H (A sp-G lu-A la-A sp/H is) box p o lp e p tid e 16 K IAA 0082 K IAA 0082 g e n e p ro d u ct

HLA-Bw72 a n iig e n H LA -Bw 72 a n tig e n N dr p ro te in k in a se Ndr p rotein k in a s e

HLA-C M ajor h isto co m p a tib ility c o m p le x , c la s s 1. C C OX 6A 1 c y to c h r o m e c o x id a s e s u b u n it Via p o lp e p tid e 1 MICA MHC c l a s s 1 p o ly p e p tid e -re la te d s e q u e n c e A K IAA 0646 C 3 H C 4 -ty p e z inc fin g er p ro tein HLA-E M ajor h isto co m p a tib ility c o m p le x c la s s 1. E CFLAR C A S P 8 a n d FAD D-like a p c p to s i s reg u la to r G N U g u a n in e n u c le o tid e b in d in g protein-like l O R P 1 5 0 150 kD a o x y g e n -r e g u la te d p ro tein MOG m yelin o lig o d e n d ro c y te g lycoprotein CD KN 1A cyclin d e p e n d e n t k in a s e inhibitor 1A (p21 C ip l )

F L 0 T 1 floliftin 1 S F R S 3 splicing fa c to r a rg in in e /s e r in e - r ic h 3

S C I p u ta tiv e tr a n s -a c tin g fac to r in volve d in cell cy cle c o n tro l P B S 1 3 p B 8 l 3 p r e c u rs o r p o ly p e p tid e , te stis - s p e c if ic R X R -b e ta retin o id X r e c e p to r b e ta Z N F 7 6 zinc finger p ro te in 76 ( e x p r e s s e d in te s tis )

HLA-G HLA-G h isto co m p a tib ility a n tig e n c la s s 1, G PIM1 p im -1 o n c o g e n e

R P S 1 9 rib o so m a l p ro te in S I 9 S N R P C sm a ll n u c le a r n b o n u c le o p r o te in p o ly p e p tid e 0 z inc lin g e r p ro tein z inc fin g er p ro tein a p o lip o p ro te in M a p o lip o p ro te in M

MICB MHC c l a s s 1 p o ly p e p tid e -re la te d s e q u e n c e B T A P2 tr a n s p o rte r 2, ABC (A TP b in d in g c a s s e tte ) HLA-J M ajor h isto co m p a tib ility c o m p le x , c la s s 1, J C Y P 21 c y to c h r o m e P 4 5 0 su b fam ily XXI

C 2 c o m p le m e n t c o m p o n e n t 2 T S B P te n s sp e cific b a s ic p rotein

b e ta-tu b u lin c lo n e 2 4 7 0 3 b e ta-tu b u lin G S N g e tso lin (am y lo id o sis. F in n ish ty p e ) A RA 24 a n d r o g e n r e c e p to r a s s o c i a te d p rotein 24 M O C S 1 m o ly b d e n u m c o fa c to r s y n th e s is 1

H2BFN H 2B h is to n e fam ily m e m b e r N K IA A0349 KIA A 0349 g e n e p ro d u ct

h s p 70 h e a t s h o c k p ro tein K IA A0240 KIA A 0240 g e n e p ro d u ct

HLA-DRBI M ajor h isto co m p a tib ility c o m p le x c l a s s II. DR b e ta i P P P 2 R 5 0 p ro tein p h o s p h a ta s e 2. reg u la to ry s u b u n it B (B56), d e lta iso lo rm

N 0 T C H 4 N otch (d ro so p h ila ) h o m o lo g 4 K IA A 0708 K IA A0708 g e n e p ro d u ct