Ann. Hum. Genet. (2000), 64, 479–490

Printed in Great Britain

479

A genome-wide family-based linkage study of coeliac disease

A. L. KING", J. Y. YIANNAKOU", P. M. BRETT#, D. CURTIS$, M.-A. MORRIS", A. M.

DEARLOVE%, M. RHODES%, S. ROSEN-BRONSON&, C. MATHEW', H. J. ELLIS"

P. J. CICLITIRA"

"Department of Gastroenterology (GKT), The Rayne Institute, St. Thomas’ Hospital, London SE1

7EH, UK#Periodontology, Eastman Dental Institute, UCL, 256 Gray’s Inn Road, London. WC1X 8LD, UK

$Academic Department of Psychological Medicine, St Bartholomew’s and the Royal London School of

Medicine and Dentistry, 3rd Floor Alexandra Wing, Turner Street, London E1, UK%UK HGMP Resource Centre, Hinxton, Cambridge CB10 1SB, UK

&Georgetown University Medical Centre, Washington, DC, 20007, USA'Paediatric Research Unit (GKT), Guy’s Hospital, London, SE1 9RT, UK

(Received 4.7.00. Accepted 21.9.00)

The susceptibility to develop coeliac disease (CD) has a strong genetic component, which is not

entirely explained by HLA associations. Two previous genome wide linkage studies have been

performed to identify additional loci outside this region. These studies both used a sib-pair design

and produced conflicting results.

Our aim is to identify non-MHC genetic loci contributing to coeliac disease using a family based

linkage study. We performed a genome wide search in 16 highly informative multiply affected

pedigrees using 400 microsatellite markers with an average spacing of 10 cM. Linkage analysis was

performed using lod score and model free methods.

We identified two new potential susceptibility loci with lod scores of 1±9, at 10q23±1, and 16q23±3.

Significant, but lower lod scores were found for 6q14 (1±2), 11p11 (1±5), and 19q13±4 (0±9), areas

implicated in a previous genome wide study. Lod scores of 0±9 were obtained for both D7S507, which

lies 1 cM from the γT-cell receptor gene, and for D2S364, which lies 12 cM from the CTLA4 gene.

Coeliac disease (CD) is a gluten sensitive

enteropathy in which dietary exposure to wheat,

barley, rye, and possibly oats results in small

bowel mucosal atrophy and consequent mal-

absorption. There is a strong genetic component

to disease development as demonstrated by a

disease concordance among monozygotic twins

of 70–100% (Polanco et al. 1981; Salazar de

Souza et al. 1987), and a 30–50% concordance in

Correspondence: Prof. PJ Ciclitira, Department ofGastroenterology (GKT), The Rayne Institute, St.Thomas’ Hospital, London SE1 7EH, United Kingdom.Tel}Fax: 020-7960-5529.

Email : paul.ciclitira!kcl.ac.uk

HLA identical siblings. In addition, the disease

prevalence among first degree relatives of pro-

bands is 10–15% (Ellis, 1981; Marsh, 1992),

compared with an estimated population preva-

lence in both Europe and in the USA of 1:250

(Greco, 1997; Not et al. 1998).

The contribution of the HLA region to this

genetic component has been well described. The

majority of cases in Northern Europe are

associated with possession of the HLA-B8-DR3-

DQ2 haplotype (Sollid & Thorsby 1993). How-

ever, in Southern Europe some cases are as-

sociated with a heterozygous combination of

HLA-DR5}-DR7 genes. A unifying hypothesis is

that CD is associated with the DQ alleles

DQA1*0501 and DQB1*0201 arranged in either

480 A. L. K

cis or trans configuration (Sollid et al. 1989) since

these alleles are present both in HLA-DR3 and in

HLA-DR5}DR7 subjects. In some ethnic groups

where there is a low prevalence of HLA-DR3 and

-DR7 up to 20% of cases are associated with the

HLA-DR4-DQ8 haplotype (Tighe et al. 1992;

Tighe et al. 1993). Here susceptibility is thought

to be conferred by the alleles DQA1*0301 and

DQB1*0302, although they appear to have less

effect on disease development than DQA1*0501

and DQB1*0201.

The prevalence of the disease susceptibility

HLA alleles in healthy control populations is

high (25%), suggesting that additional genetic

and}or environmental factors are required for

disease development. Several epidemiologic and

segregation studies have therefore proposed a

genetic model for the inheritance of coeliac

disease based on the involvement of at least one

non-MHC linked gene (Pena et al. 1978; Green-

berg & Lange, 1982; Houlston & Ford, 1996). We

have previously studied microsatellite markers

flanking the class II region of the MHC to

establish the parental origin of the susceptibility

DQ alleles. Results suggested that the HLA

association is probably due to the fact that it is

necessary to have these DQ alleles in order to

develop CD. The study did not support the

presence of either a rare mutation within the DQ

alleles or an HLA-linked gene nearby which

might be in linkage disequillibrium with the DQ

locus (Brett et al. 1999).

Two previous attempts to identify non-HLA

loci using genome wide linkage studies of affected

sib pairs produced conflicting results. Zhong et al.

(1996) performed an autosomal screen using 40

affected sib-pairs and a novel three-stage pro-

tocol. They identified five main areas of interest

apart from HLA on chromosomes 6p23, 7q31±3,

11p11, 15q26, and 22cen. Six further areas

produced weaker evidence for linkage: 3q27,

5q33±3, 6p23, 6p12, 19p13±3, 19q13±1, and

19q13±4. However the study used relatively small

numbers of sib-pairs, many from just one

kindred. The parents were not genotyped, and

this can produce a bias towards false positive

results not only if marker allele frequencies are

misspecified but also if population stratifications

or other causes of increased homozygosity are

present (Curtis & Sham, 1996). A study looking at

all 11 implicated regions failed to confirm

Zhong’s results except at the HLA region and on

chromosome 15q26, where weak evidence of

linkage of linkage was found (Houlston et al.

1997). Our unit also studied the five most positive

regions and failed to support linkage (Brett et al.

1998). Another genome-wide search was carried

out by Greco et al. (1998) using a larger sample of

110 affected sib-pairs. The study also examined

the five main regions proposed by Zhong et al. in

greater detail, and again failed to confirm linkage

in these areas. It did however propose further

areas of interest at 5qter and, in a subgroup of

patients, at 11qter.

The CTLA4}CD28 region on chromosome

2q33 has been independently implicated in an

association study from France (Djilali-Saiah et

al. 1998) and a linkage study from Finland

(Holopainen et al. 1999). These genes have a role

in controlling the T cell proliferative response,

and are associated with other autoimmune

diseases such as Type 1 diabetes and Graves

disease. However, a study of Italian and Tunisian

patients found no evidence of linkage or as-

sociation in this region (Clot et al. 1999).

Families

Families with two or more affected individuals

were recruited with the help of gastroentero-

logists throughout the UK, and as a result of an

advertisement in the UK Coeliac Society news-

letter. The sixteen most informative families

(Figure 1) were selected from a core sample of 21

highly informative pedigrees used in our previous

genetic studies. One hundred and twenty-six

individuals were genotyped of which 47 were

classed as affected on clinical grounds, having

been diagnosed according to the revised

ESPGAN criteria (Walker-Smith et al. 1990).

Unaffected relatives were screened for subclinical

disease using anti-gliadin (AGA) and anti-endo-

mysial antibodies (EmA), a strategy that has

Family-based linkage study of coeliac disease 481

Fig. 1 For legend see p. 482.

been shown to be highly effective (Corrao et al.

1994). IgA and IgG AGA were measured by

ELISA, while EmA were detected by immuno-

fluorescence using human umbilical cord. Both

assays were performed within our laboratory,

and have been previously shown to have sen-

sitivities and specificities in excess of 90%

(Yiannakou et al. 1997). Five individuals tested

positive and were asked to have a duodenal

biopsy. Two individuals accepted and the di-

agnosis was confirmed by the histological finding

of subtotal villous atrophy. Of the three others,

one had a strongly positive IgA and IgG AGA

and a strongly positive EmA, plus the HLA-DQ2

482 A. L. K

Fig. 1. (cont.) 16 Pedigrees used for genome wide linkage study of coeliac disease.

haplotype. This individual is estimated to have

about a 99% chance of having coeliac disease, so

was classed as being affected despite the lack of

histological confirmation. The other two indiv-

iduals also refused biopsy, one of whom had a

weakly positive EmA, and the other a positive

IgA AGA but negative EmA. These individuals

were classed as being of unknown affection

status. Since the original design of the study, all

individuals have been tested using IgA and IgG

tissue transglutaminase (tTG) antibodies (Diet-

rich et al. 1998; Sulkanen et al. 1998). Individuals

with positive IgG, but negative IgA tTG anti-

bodies were screened for IgA deficiency using a

total IgA assay. Total IgA levels and tTG

antibodies were assayed by ELISA. As a result of

this screening, no additional cases of CD, and no

IgA deficient individuals, were identified.

All family members were HLA typed for both

DQ alleles and all but one of the affected

Family-based linkage study of coeliac disease 483

individuals possessed the DQA1*0501

DQB1*0201 heterodimer in either cis or trans

configuration. The other individual possessed the

DQA1*0301 DQB1*0302 haplotype.

The study was approved by the St Thomas’

Hospital Ethics Committee and all subjects

provided informed consent.

HLA typing and Microsatellite Genotyping

DNA was isolated from peripheral blood

leucocytes. Every individual was HLA typed

using PCR-SSP (Olerup et al. 1993). PCR was

performed in 12±5 µl containing: 50 ng DNA,

12±5 pmoles of each primer, 1±0 m MgCl, 10 m

Tris-Cl pH 8±3, 50 m KCl, 200 µ of each

dNTP, 1 unit Taq polymerase (Advanced

Biotechnologies) and 0±01% gelatin. The amplifi-

cation was for 30 cycles of 94 °C for 20 s, 65 °Cfor 50 s and 72 °C for 45 s. The amplified products

were separated by agarose gel electrophoresis

and visualised by ethidium bromide staining and

UV fluorescence.

126 subjects were genotyped with markers

spanning the genome from the ABI LMS2

(MD10) mapping set. This set consists of fluor-

escently labelled PCR primer pairs for 400 highly

polymorphic dinucleotide-repeat microsatellite

markers chosen from the Genethon human

linkage map (Weissenbach et al. 1992; Gyapay et

al. 1994; Dib et al. 1996). The markers have an

average spacing of 10 cM and incorporate

reverse-primertailing chemistry (Brownstein et

al. 1996). PCR reactions were carried out for each

marker individually in a 5 µl reaction volume,

containing 50 ng of DNA, 2±5 m Tris-HCl pH

8±0, 50 m KCl, 250 µ each dNTP, 0±625 pmol

of each primer and 0±25 units of Amplitaq Gold

(Perkin–Elmer).

Reactions were performed on a Perkin Elmer

9600 thermal cycler or using an ABI 877

integrated thermal-cycler robot. A standard

thermocycling profile was used for all markers,

and consisted of an initial denaturation at 95 °Cfor 12 min, which was followed by 10 cycles each

of denaturation at 95 °C for 15 s, annealing at

55 °C for 15 s and synthesis at 72 °C for 30 s. This

was followed by 20 cycles each of denaturation at

89 °C for 15 s, annealing at 55 °C for 15 s and

synthesis at 72 °C for 30 s, and by a final

extension step at 72 °C for 10 min. PCR products

for selected sets of markers were pooled, ethanol

precipitated, and size-fractionated on a 5%

denaturing polyacrylamide gel (Amresco, Ohio)

by electrophoresis on an ABI 377XL sequencer.

PCR products were sized using the Genescan

version 2.1 program, and scored using the

Genotyper version 2.0 program. Genotyping was

checked for Mendelian errors using the

PEDCHECK program (O’Connell & Weeks,

1998).

Linkage analysis

Subjects were classified as affected, unaffected

or of unknown affection status according to their

clinical status. However all unaffected subjects

who were DQ2-negative were also classified as

unknown affection status for purposes of linkage

analysis. This is because we believe that a non-

HLA susceptibility locus cannot exert its effect

on disease development in the absence of a high

risk HLA type. This high risk HLA type is DQ2

in the majority (95%) of coeliac patients in this

population (Sollid & Thorsby, 1993). Of the 126

individuals genotyped, 52 were classed as un-

affected, 50 were classed as affected, and 24 were

classed as ‘unknown’.

Linkage analysis was carried out using stan-

dard lod score methods and using ‘model-free’

likelihood-based analysis. For lod score analyses

the VITESSE program was used (O’Connell &

Weeks, 1995), except for markers on the X

chromosome, for which the FASTLINK program

was used (Cottingham et al. 1993; Schaffer, 1996).

The penetrance was set to 0±8 with a phenocopy

risk of 0±0001. Each analysis was carried out

using a dominant and a recessive model, with the

disease allele frequency set to 0±01 and 0±14

respectively. These values were chosen fairly

arbitrarily to provide a plausible mode of

transmission, and a population prevalence that

conforms to the observed one. For the initial

484 A. L. K

screening analyses a set of two-point analyses

was carried out with each marker and a set of

three-point analyses was carried out with each

pair of adjacent markers. Overall lod scores were

calculated under the assumption of admixture to

produce a heterogeneity lod score (HLOD).

The ‘model-free’ analyses were carried out

using the MFLINK program (Curtis & Sham,

1995) and the accompanying MFMAP utility.

MFLINK calculates the likelihood of the data

with the disease locus at a given map position

using a range of different dominant and recessive

transmission models, all yielding the same dis-

ease prevalence (Kp) and parameterised using a

single variable, the heterozygote penetrance (f",

which is varied from 0 to 1). The MLOD is the

maximum lod score obtained for any of these

transmission models (maximised over f"). The

MALOD is the maximum admixture lod ob-

tained for any model (maximised over f"and the

proportion of families linked, alpha). The

MFLOD is the difference between the log like-

lihood maximised over both f"and alpha and the

log likelihood maximised over f"

but with alpha

constrained to 0. Two-point analyses were car-

ried out with each marker using a test position at

a recombination fraction of 0±05 with the marker,

and three-point analyses were carried out with

each pair of adjacent markers testing a position

midway between them.

An initial screen of all the markers and pairs of

markers was carried out using the above meth-

ods. Each marker and each pair of markers

yielded two admixture lod scores and three lod

scores from the MFLINK analyses (MLOD,

MALOD and MFLOD for the core and spectrum

models). Each type of lod score was converted to

a likelihood ratio statistic by multiplying by

2 ln(10)¯ 4±6. The statistic derived from the

conventional admixture lod score (HLOD) was

taken to be distributed as a 50:50 mixture of

χ##

and χ#!. As originally described (Curtis &

Sham, 1995), the likelihood ratio statistic from

the MFLOD was taken to be distributed as a

50:50 mixture of χ#"and χ#

!. Subsequently (Curtis

& Sham, 1999), it has been shown that

2 ln(10)*MLOD can be taken to be distributed as

χ#"and that 2 ln(10)*MALOD can conservatively

be taken to be distributed as a 50:50 mixture of

χ##

and χ#!. Using these distributions allows p

values to be derived so that the different types of

lod score can be compared more easily. All

regions which yielded a result significant at p!0±05 using any of the methods of analysis were

selected for further study.

Additional analyses from regions highlighted

by the screening analyses consisted of carrying

out overlapping five-point linkage analyses using

sets of four adjacent markers at a time. Het-

erogeneity lod scores were calculated assuming a

dominant and recessive transmission model as

described above.

When all of the two-point and three-point

heterogeneity lods and MFLINK lods were

scrutinised, regions containing the following

markers produced at least one statistic signif-

icant at p! 0±05: D1S214, D1S213, D2S364,

D4S412, D4S1572, D6S460, D7S507, D8S284,

D9S167, D9S158, D10S597, D11S904, D11S925,

D12S85, D14S288, D15S153, D16S3091,

D17S798, D18S452, D18S70, D19S210,

D22S423. These results are detailed in Table 1.

In several of these regions more than one marker

and}or method of analysis yielded positive lod

scores. The full set of results can be inspected

at web-site: www.mds.qmw.ac.uk.statgen}dcurtis}cdscan.html (embargoed till publication).

The regions highlighted by the two-point and

three-point screening analyses were investigated

more intensively using overlapping five-point

analyses and the results of these are presented in

Table 2. For some of the regions evidence for

linkage is maintained, while for others the five-

point lod score analyses do not support linkage.

The highest lod scores were produced by regions

around D10S597 and D16S3091, both of which

produced admixture lod scores approaching 2.

Other regions produced moderate or only weakly

positive lod scores. In some cases it can be seen

that most of the overall positive lod score results

Fam

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Table 1. Results significant at p! 0±05 from initial screen of two- and three-point conventional and model-free linkage analysis

Market at or nearto peak lod

Mapposition

(cM)Cytogenetic

locationa Most significant result obtainedNominalp value Comments

D1S214 9±5 1p36±3 Recessive three-point HLOD of 1±7 0±01 Nearby markers also moderately positiveD1S213 245±7 1q41 Three-point MALOD of 1±1 0±04D2S364 192±1 2q31±1 Two-point MALOD of 1±4 0±02D4S412 0±0 4p16±3 Three-point MALOD of 1±3 0±03D4S1572 67±4 4q22-4q24 Two-point MALOD of 1±6 0±01 More distal markers also positiveD6S460 81±1 6q14-6q15 Three-point MLOD of 1±7 0±005D7S507 31±1 7p15 Three-point MFLOD of 0±7 0±04D8S284 140±3 8q23-8q24±1 Three-point MLOD of 1±4 0±01D9S167 74±0 9q21±32-9q21±33 Three-point MFLOD of 0±8 0±03D9S158 158±8 9q34±3 Three-point dominant HLOD of 1±2 0±03D10S597 129±6 10q23±1 Three-point MALOD of 1±6 0±01 Positive results over wide regionD11S904 32±1 11p13 Three-point MALOD of 1±6 0±01D11S925 118±8 11q23±3 Three point MLOD of 1±2 0±02D12S85 66±5 12q13 Two-point recessive HLOD of 1±2 0±03D14S288 40±9 14q13-14q21 Two-point MFLOD of 1±1 0±01D15S153 62±9 15q21-15q22 Two-point MLOD of 0±9 0±04D16S3091 102±5 16q23±3 Two-point MALOD of 2±6 0±001D17S798 57±6 17q21±31 Three-point dominant HLOD of 1±2 0±03D18S452 17±2 18p11±31 Two-point MALOD of 1±0 0±05D18S70 136±1 18q23 Three-point dominant HLOD of 1±1 0±04D19S210 88±2 19q13±4 Two-point MFLOD of 1±1 0±01D22S423 42±3 22q13±1 Two-point MLOD of 1±5 0±009 Nearby markers also positive

a Approximate cytogenetic location derived from The Genome Database, http:}}www.gdb.org}

486

A.L

.K

Table 2. Results of five-point lod score analysis of the regions highlighted by initial screening analyses*

Marker ator near

to peak lod

Mapposition

(cM)Cytogeneticlocation†

Most significantresult obtained

Nominalp value Comments

D1S214 9±5 1p36±3 Recessive HLOD of 1±3 0±03 Lod 0±5 in F1, 0±8 in F2, 0±7 in F5, 0±7 in F8, 0±7 in F12, 0±9 in F14D1S413 213±8 1q31-1q32±1 Recessive HLOD of 0±8 0±08 Lod of 1±3 in F3, 0±7 in F5, 0±7 in F8, 0±7 in F9, 0±5 in F11D2S364 192±1 2q31±1 Recessive HLOD of 0±9 0±06 Lod of 0±5 in F1, 0±5 in F11, 0±9 in F14, 1±5 in F16D4S2935 9±1 4p16±1 Dominant HLOD of 0±7 0±1 Lod 1±8 in F3D4S406 78±0 4q26 Recessive HLOD of 1±2 0±03 Dominant HLOD is 1±0 with more proximal markersD6S460 81±0 6q14-6q15 Recessive HLOD 1±2 0±03 Moderately positive lods in about half the familiesD7S507 31±1 7p15 Dominant HLOD of 0±9 0±06 Lod of 1±2 in F1, 0±5 in F6, 0±8 in F9D8S284 140±3 8q23-8q24±1 Recessive HLOD of 0±6 0±1 Little support for linkageD9S283 85±3 9q22±1 Dominant HLOD of 1±0 0±05 Lod 1±4 in F2D9S158 158±8 9q34±3 Dominant HLOD of 0±6 0±1 Little support for linkageD10S597 129±6 10q23±1 Recessive HLOD of 1±9 0±006 Lod 0±5 in F1, 2±1 in F2, 0±9 in F3, 0±5 in F9, 0±7 in F12, 0±5 in F15D11S935 43±8 11p11 Recessive HLOD of 1±5 0±02 Lod 0±5 in F5, 0±5 in F15, 1±8 in F16D11S925 118±8 11q23±3 Dominant HLOD 0±5 NS‡ Little support for linkageD12S1617 48±0 12p12±1 Recessive HLOD of 0±4 NS Little support for linkageD14S275 20±2 14q11±2 Recessive HLOD of 0±5 NS Little support for linkageD15S153 62±9 15q21-15q22 Recessive HLOD of 0±2 NS No support for linkageD16S3091 102±5 16q23±3 Recessive HLOD of 1±9 0±006 Lod 0±5 in F1, 0±8 in F3, 0±5 in F4, 0±5 in F11, 0±7 in F12, 0±7 in F14, 1±3 in F16D17S798 57±6 17q21±31 Dominant HLOD of 1±0 0±05 Lod 0±9 in F3, 0±5 in F6, 0±9 in F16D18S452 17±2 18p11±31 Recessive HLOD of 0±7 0±1 Lod 0±7 in F5, 0±5 in F10, 0±5 in F11, 1±8 in F16D18S70 136±1 18q23 Dominant HLOD of 1±3 0±03 Lod 1±1 in F16D19S210 88±2 19q13±4 Dominant HLOD of 0±9 0±06 Lod 0±6 in F1, 0±5 in F6, 1±2 in F7D22S274 50±2 22q13±33 Recessive HLOD of 1±2 0±03 Dominant HLOD of 1±1 with more proximal markers

* The highest admixture lod score obtained from any of the six models tested (core, spectrum or combined; dominant or recessive) is shown, together with informationregarding the model which produces this score and the pedigree(s) which appear to make the main contribution to it.

† Approximate cytogenetic location derived from The Genome Database, http :}}www.gdb.org}‡ NS, not significant.

Family-based linkage study of coeliac disease 487

mainly from one or two families while in others a

number of families each makes a contribution.

The results are compatible with some of the

regions identified harbouring susceptibility loci,

some of which appear to be active in different

subsets of families. Nonetheless, it is impossible

to draw any firm conclusions as to which findings

represent genuine linkage.

Several regions were identified as being poss-

ibly of interest from the initial two- and three-

point analyses, but the five-point analyses serve

to focus attention on those which are most likely

to harbour susceptibility loci. When two-point

linkage analyses are carried out there may be

large differences between the results obtained

from adjacent markers which can simply reflect

random variation in the subset of meioses for

which each marker is informative. If one marker

happens to be uninformative for more of the

meioses which are recombinant than another

then it will provide more evidence for linkage. By

contrast, multipoint analysis can provide almost

complete information regarding the inheritance

of each chromosomal segment and should be far

less sensitive to this random noise. A disad-

vantage of multipoint analysis may be that

model mis-specifications are more likely to

produce incorrect results, especially spurious

exclusions (Risch & Giuffra, 1992). However

recently it has been suggested that such concerns

may be exaggerated (Greenberg et al. 1998), and

our own results include a number of positive lod

scores. We regard the positive lod scores we have

obtained from five-point analyses as being more

likely to reflect true linkages than would similar

lod scores derived from two-point analyses.

Coeliac disease is genetically a complex disease

in that there are likely to be several genes in

addition to the HLA locus, which may contribute

to disease development, either separately or

interactively. We have chosen to implement a

family-based design, because multiply affected

pedigrees have more power to detect linkage in

the presence of genetic heterogeneity than sib-

pairs (Risch, 1989) and seem generally powerful

unless susceptibility alleles are very common

(Durner et al. 1999). None of our lod scores

provides definitive evidence for linkage, although

this is hardly unexpected in a complex disease

where each individual gene may make only a

relatively small contribution to susceptibility.

Our most positive admixture lod scores, each

of 1±9, were found using markers on chromosome

10q23 and 16q23, and it may be that these areas

contain novel susceptibility genes not detected in

the two previous genome wide studies. Less

strongly positive lod scores were obtained from

several other regions, some of which are note-

worthy because of proximity to potential can-

didate genes or because they have supported

linkage in other studies.

The region on chromosome 2q gives only

modest evidence of linkage with a recessive

HLOD of 0±9 at D2S364. However this finding

may be of interest because D2S634 lies only

12 cM from the CTLA4 gene, which has been

implicated in previous association and linkage

studies of coeliac disease.

The evidence for linkage on chromosome 6 is

maximal around D6S460 with a recessive HLOD

of 1±2. This marker lies 8 cM from D6S430 and

19 cM from D6S465, markers spanning the region

on 6p12 implicated by Zhong et al. (1996). This

area produced a multipoint maximum lod score

(MLS) of 1±4 in their study. D6S460 lies 47 cM

from HLA, suggesting that the positive lod score

results from a separate susceptibility locus rather

than being due to an effect of HLA. This

hypothesis is supported by the fact that D6S276,

the marker in closest proximity to HLA, pro-

duces lower lod scores in our families. Zhong et al.

also reported an area on chromosome 19q13±4which produce an MLS of 1±8 around D19S418.

We have found modest evidence for linkage

around D19S210, which lies 6 cM telomeric of

D19S418, with a five-point dominant HLOD of

0±9. The region around D11S871 on chromosome

11p11 was one of the most positive in the Zhong

study, with an MLS of 3±9. In an earlier study

using a slightly different set of pedigrees we

reported negative results with two markers from

488 A. L. K

this region (Brett et al. 1998), but with the

current pedigrees and using 5-point analysis with

a fuller marker set we now obtain a recessive 5-

HLOD of 1±5 around D11S935, which lies 3 cM

proximal of D11S871. On both 19q13±4 and

11p11 our positive lod scores arise mostly from

non-overlapping sets of 3 the 16 families, which

might reflect locus heterogeneity although of

course such results could easily arise by chance.

Another strongly positive region in the Zhong et

al. (1996) study was an area on chromosome 22

near the centromere (D22S420). Our most posi-

tive result on chromosome 22 is a recessive

HLOD of 1±2, occurring at D22S274 which is

51 cM telomeric of D22S420. However more

proximal markers are also modestly positive and

it seems possible that the regions implicated by

both studies may overlap to some extent.

Overall our findings tend to agree with those of

Zhong et al. (1996) with respect to regions on 11p,

19q, 6q and 22q. Although these same areas

produced only negative results in Houlston et al.

(1997) sample of families such inconsistencies are

only to be expected when linkage studies of

diseases with complex inheritance are attempted,

and it may well be that at least some of these

regions do harbour susceptibility genes.

We have previously investigated the T-cell

receptor (TCR) genes, on account of the key role

forT-cell activation in coeliac disease (Yiannakou

et al. 1999). We used a slightly different pedigree

set and obtained largely negative results with

single intragenic markers at the α, β, and δ TCR

genes and with two markers flanking the γ TCR

gene. However in the current study we obtain a

five-point dominant HLOD of 0±9 around

D7S507, which is situated 1 cM from the TCR γ

gene. There is no suggestion of linkage at the

TCR β locus on chromosome 7q, and the region

around marker D14S275, which is situated 14 cM

from the TCR α and δ genes, is only very weakly

positive with five-point analysis. Although the

evidence for linkage to TCR γ is only modest it

nonetheless remains of some interest, given that

the γ}δ T cell populations are increased in the

epithelium of the small intestine in individuals

with untreated coeliac disease.

A search for other candidate genes in all the

areas achieving positive linkage results was

conducted using the National Centre for Bio-

technology Information website http :}}www.

ncbi.nlm.nih.gov}, and the associated utility

LocusLink http :}}www.ncbi.nlm.nih.gov}LocusLink. We did not find any known genes in

any of these areas with obvious immunological or

gut-related functions.

When dealing with complex diseases there are

limited conclusions that can be drawn from

linkage studies. We expect that most results

significant at p! 0±05 will have occurred simply

by chance, especially those obtained only from

two- or three-point analysis, those occurring

with only one marker and those obtained from

only one method of analysis. More highly

significant results obtained from five-point ana-

lysis may be more likely to reflect true linkage, as

may those results which replicate the findings of

previous studies. A number of our results seem

promising and we aim to follow them up by

investigating these regions in a new sample of 34

multiply-affected pedigrees, using additional and

more finely-spaced markers as appropriate.

The genome screen was performed at the MedicalResearch Council’s UK Human Genome Mapping ProjectResource Centre Linkage Hotel, and was funded by anMRC Linkage Hotel grant. A. L. King is funded by aBritish Society of Gastroenterology}Digestive DisordersFoundation 2 year Research Training Fellowship. P. M.Brett was funded by the National Institute of Healthgrant [ROI DK47716], and J. Y. Yiannakou and M.-A.-Morris were funded by the St Thomas’ Research andEndowment committee.

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