Abstract

Chronic lymphocytic leukemia (CLL) shows evidence of familial aggregation, but the genetic basis is poorly understood. The existence of a linkage between HLA and Hodgkin lymphoma, another B-cell disorder, coupled with the fact that CLL is frequently associated with autoimmune disease, led to the question of whether the major histocompatibility complex (MHC) region is involved in familial cases of CLL. To examine this proposition, 5 microsatellite markers on chromosome 6p21.3 were typed in 28 families with CLL, 4 families with CLL in association with other lymphoproliferative disorders, and 1 family with splenic lymphoma with villous lymphocytes. There was no evidence of linkage in these families to chromosome 6p21.3. The best estimates of the proportions of sibling pairs with CLL that share 0, 1, or 2 MHC haplotypes were not significantly different from the null expectation. This implies that genes within the MHC region are unlikely to be the major determinants of familial CLL.

Introduction

In Western countries, leukemia affects approximately 1 in 50 of the population.1 Of the many subtypes, B-cell chronic lymphocytic leukemia (CLL) is the most common, constituting about one third of all cases.2 Its incidence rate increases logarithmically from age 35 years, with the median age at diagnosis being 65 years.3 

Epidemiologic studies strongly suggest that a subset of CLL cases have an inherited basis. More than 40 separate reports have described the clustering of CLL in small families, occasionally in association with other lymphoproliferative disorders (LPDs).4 Systematic analyses of the risk in relatives indicate that the risk of CLL is increased 3-fold in relatives of patients.3,5-9 The genetic basis for predisposition to CLL is unknown. The difficulty of ascertaining large CLL families with multiple available affected members limits the power of a genome-wide screen for linkage and makes evaluation of candidate loci a more feasible approach. The established relation between HLA and Hodgkin lymphoma, another B-cell disorder,10,11 coupled with the fact that CLL is associated with autoimmune disease,12-14led us to examine whether the major histocompatibility complex (MHC) region is involved in familial cases of CLL. In this study, we examined this proposition by conducting an analysis for linkage at chromosome 6p21.3 in families with CLL and associated LPDs.

Study design

Patient selection

Thirty-three families were ascertained by hematologists in the United Kingdom, Norway, Israel, Italy, Ireland, Germany, Portugal, The Netherlands, and Australia. The diagnoses of B-CLL and other LPDs in family members were established in all cases using standard clinicohematologic and immunologic criteria. Samples were obtained with informed consent and ethical review board approval. DNA was salt-extracted from EDTA blood samples using a standard sucrose lysis method.

Genotyping

Polymerase chain reaction (PCR) was performed at the following microsatellite loci: D6S299, D6S464, D6S276, D6S273, and D6S291. PCR primer sequences and reaction conditions were taken from the Marshfield database (http://www.marshmed.org). Dye-labeled PCR products were detected on ABI 377 DNA sequencers and analyzed using Genescan and Genotyper software.

Statistical methods

Multipoint analysis was performed with the program GENEHUNTER15 using the nonparametric LOD (NPL)-all statistic, which calculates approximate P values in a model free analysis. The contribution of the MHC toward the overall susceptibility to CLL was determined from the allele-sharing probabilities between affected sibling pairs using the method of Risch.10 This method assesses the magnitude of linkage of a locus with disease in terms of the ratio λ (the relative risk, ie, the observed frequency of sharing of zero alleles identical by descent [IBD] at a locus with what is expected in the absence of linkage). If Z0 is the proportion of affected sibling pairs sharing zero parental alleles IBD at the locus, the sibling relative risk, λs, is given by 1/4Z0. This formula holds true regardless of the mode of inheritance at the disease locus, the number of alleles and their frequencies, the penetrance, and the population prevalence of disease. The 95% confidence intervals for λs are given by the following16:

 
expln(λs)±1.96×ν1.

In this equation, ν1 is given by (4λs/n) × (1 − 1/4λs); and n indicates the number of affected sibling pairs.

Haplotype-sharing probabilities among affected sibling pairs were assessed for the purpose of deriving λs using the program MAPMAKER-SIBS.17 Marker allele frequencies were obtained from the Marshfield database (http://www.marshmed.org) and were similar to the relative frequencies seen across all the families typed. Distances between markers were also obtained from the Marshfield database.

Results and discussion

Thirty-two families were used in the linkage analysis. Table 1 details their clinical characteristics. The first 28 of these families had at least 2 members affected with CLL. Of these families, numbers 42 and 65 also had family members affected with other LPDs. Families 44, 47, 59, and 80 had one family member affected with CLL and a second with another LPD. Family 102 included 2 sisters with splenic lymphoma with villous lymphocytes.

Table 1.

Details of the families studied

Family no. Index case Age at diagnosis, y Relative Age at diagnosis, y 
Female 56 Sister 66 
   Identical twin 59 
Female 54 Brother 46  
   Brother 47 
   Father 71  
Female 42 Brother 40 
   Mother 69  
Male 70 Brother 76 
   Brother 55  
11 Female 69 Sister 61 
13 Female 70 Brother 58 
15 Male 63 Sister 61 
18 Female 74 Sister 73 
20 Male 64 Brother 66 
36 Male 81 Sister 77  
   Brother 64 
37 Male 85 Sister 86  
   Sister 79 
   Brother 60  
38 Male 77 Brother 71 
39 Female 60 Sister 51 
40 Male 71 Sister 48 
41 Male 35 Sister 51  
42 Male 59 Second cousin 43  
   Brother (HD) 52 
45 Male 61 Sister 52 
46 Female 61 Sister 59 
48 Female 45 Sister 51 
49 Male 69 Sister 80 
60 Male 55 Sister 49 
63 Male 57 Sister 43 
64 Male 62 Brother 61 
65 Male 62 Brother 70  
   Sister (myeloma) 75  
78 Male 32 Uncle 54 
   Uncle 50  
85 Male 60 Brother 49 
96 Male 65 Brother 61 
98 Male 67 Brother 64  
44 Female 39 Brother (HCL) 39  
47 Female 36 Male cousin (NHL) 64 
59 Male 44 Brother (NHL) 48 
80 Male 51 Brother 58  
   Male cousin (HCL) 46  
102 Female (SLVL) 70 Sister (SLVL) 65 
Family no. Index case Age at diagnosis, y Relative Age at diagnosis, y 
Female 56 Sister 66 
   Identical twin 59 
Female 54 Brother 46  
   Brother 47 
   Father 71  
Female 42 Brother 40 
   Mother 69  
Male 70 Brother 76 
   Brother 55  
11 Female 69 Sister 61 
13 Female 70 Brother 58 
15 Male 63 Sister 61 
18 Female 74 Sister 73 
20 Male 64 Brother 66 
36 Male 81 Sister 77  
   Brother 64 
37 Male 85 Sister 86  
   Sister 79 
   Brother 60  
38 Male 77 Brother 71 
39 Female 60 Sister 51 
40 Male 71 Sister 48 
41 Male 35 Sister 51  
42 Male 59 Second cousin 43  
   Brother (HD) 52 
45 Male 61 Sister 52 
46 Female 61 Sister 59 
48 Female 45 Sister 51 
49 Male 69 Sister 80 
60 Male 55 Sister 49 
63 Male 57 Sister 43 
64 Male 62 Brother 61 
65 Male 62 Brother 70  
   Sister (myeloma) 75  
78 Male 32 Uncle 54 
   Uncle 50  
85 Male 60 Brother 49 
96 Male 65 Brother 61 
98 Male 67 Brother 64  
44 Female 39 Brother (HCL) 39  
47 Female 36 Male cousin (NHL) 64 
59 Male 44 Brother (NHL) 48 
80 Male 51 Brother 58  
   Male cousin (HCL) 46  
102 Female (SLVL) 70 Sister (SLVL) 65 

Relevant condition is CLL unless indicated. HD indicates Hodgkin disease; HCL, hairy cell leukemia; NHL, non-Hodgkin leukemia; SLVL, splenic lymphoma with villous lymphocytes.

Table 2 shows the nonparametric LOD (NPL) scores for linkage of CLL to chromosome 6p21.3 microsatellite markers for CLL families and for all families. Four of the markers reside within the HLA cluster, and all 5 span a region of 7.23 cM. These data provide no evidence of linkage of CLL to 6p21.3 markers in these families. The maximum NPL score was 0.27 at HLA (P > .2) for the CLL families. The affected-sibling-pair MAPMAKER-SIBS analysis of the CLL families showed that the best estimates of the proportions of sibling pairs that share 0, 1, or 2 haplotypes at the MHC region (at D6S273) were their null expectations (ie, 0.25, 0.50, and 0.25, respectively). On this basis, the sibling relative risk attributable to the MHC region is 1.0. The upper 95% confidence limit for the sibling relative risk, taking into account study size, is 1.8.

Table 2.

Multipoint NPL scores for linkage to chromosome 6p21.3 markers

graphic
 
 
graphic
 
 
*

 Ordered from telomere; relative position given in parentheses.

Vertical bar indicates position of HLA region.

We performed this linkage analysis of chromosome 6p21.3 to determine whether genes within the MHC region are implicated in familial CLL for 2 reasons. First, an association between CLL and autoimmune disease has been reported.12 Patients with CLL frequently share common HLA haplotypes with relatives who have autoimmune disease. The majority of B-cell CLL is CD5+, and B cells are implicated in autoimmunity. Hence, genetic determinants of CD5+ B-cell proliferation or differentiation are likely to be involved in both B-CLL and autoimmune disease.12 The notion of a relation between CLL and autoimmune disease is supported by animal studies using congenic New Zealand mouse strains.13,14 Second, Hodgkin lymphoma shows a strong linkage to HLA.10,11 The underlying basis of linkage is not through a common haplotype, but it appears that certain HLA-DPB1 alleles may affect susceptibility and resistance to specific subtypes of Hodgkin lymphoma.11,18 In Hodgkin disease, the allele-sharing probabilities between affected siblings suggest that the HLA locus is likely to explain a 2-fold sibling relative risk, with more than half of all cases arising in susceptible individuals. Although our linkage analysis does not support a similar conclusion for CLL, the 95% confidence limit for the estimate of the sibling relative risk attributable to HLA does not preclude that variation within HLA or MHC is a determinant of CLL susceptibility in some instances. Our findings do, however, imply that genes within the MHC region are unlikely to be the sole or major determinants of familial CLL.

Acknowledgments

We thank the families who took part in this study, and we also are grateful to Benjamin Hilditch for data management.

Supported by the Leukaemia Research Fund and BREAKTHROUGH Breast Cancer.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

References

References
1
Miller BA, Ries LAG, Hankey BF, Kosary CL, Harras A, Devesa SS, eds. Cancer Statistics Review 1973-90. NIH Publication No. 93. National Cancer Institute; 1993:2789-2794.
2
Gale
RP
Foon
KA
Biology of chronic lymphocytic leukemia.
Semin Hematol.
24
1987
209
229
3
Linet
MS
Blattner
WA
The epidemiology of chronic lymphocytic leukemia.
Chronic Lymphocytic Leukemia.
Polliack
A
Catovsky
D
1988
11
32
Harwood Academic Publishers
Chur, Switzerland
4
Horwitz
M
The genetics of familial leukemia.
Leukemia.
11
1997
1347
1359
5
Radovanovic
Z
Markovic-Denic
L
Jankovic
S
Cancer mortality of family members of patients with chronic lymphocytic leukaemia.
Eur J Epidemiol.
10
1994
211
213
6
Pottern
LM
Linet
M
Blair
A
et al
Familial cancers associated with subtypes of leukaemia and non-Hodgkin's lymphoma.
Leuk Res.
15
1991
305
314
7
Cartwright
RA
Bernard
SM
Bird
CC
et al
Chronic lymphocytic leukaemia: case-control epidemiological study in Yorkshire.
Br J Cancer.
56
1987
79
82
8
Gunz
FW
Gunz
JP
Veale
AM
Chapman
CJ
Houston
IB
Familial leukaemia: a study of 909 families.
Scand J Haematol.
15
1975
117
131
9
Goldgar
DE
Easton
DF
Cannon-Albright
LA
Skolnick
MH
Systematic population-based assessment of cancer risk in first-degree relatives of cancer probands.
J Natl Cancer Inst.
86
1994
1600
1608
10
Risch
N
Assessing the role of HLA-linked and unlinked determinants of disease.
Am J Hum Genet.
40
1987
1
14
11
Klitz
W
Aldrich
CL
Fildes
N
Horning
SJ
Begovich
AB
Localization of predisposition to Hodgkin disease in the HLA class II region.
Am J Hum Genet.
54
1994
497
505
12
Shirai
T
Genetic predisposition of autoimmune disease and B-cell chronic lymphocytic leukemia (B-CLL).
Tohoku J Exp Med.
173
1994
133
140
13
Hirose
S
Hamano
Y
Shirai
T
Genetic factors predisposing to B-CLL and to autoimmune disease in spontaneous murine model.
Leukemia.
11(suppl 3)
1997
267
270
14
Okada
T
Takiura
F
Tokushige
K
et al
Major histocompatibility complex controls clonal proliferation of CD5+ B cells in H-2-congenic New Zealand mice: a model for B cell chronic lymphocytic leukemia and autoimmune disease.
Eur J Immunol.
21
1991
2743
2748
15
Kruglyak
L
Daly
MJ
Reeve-Daly
MP
Lander
ES
Parametric and nonparametric linkage analysis: a unified multipoint approach.
Am J Hum Genet.
58
1996
1347
1363
16
Cordell
H
Olsen
J
Confidence interval calculation from relative risk estimates.
Genet Epidemiol.
14
1997
593
598
17
Kruglyak
L
Lander
ES
Complete multipoint sib-pair analysis of qualitative and quantitative traits.
Am J Hum Genet.
57
1995
439
454
18
Taylor
GM
Gokhale
DA
Crowther
D
et al
Further investigation of the role of HLA-DPB1 in adult Hodgkin's disease (HD) suggests an influence on susceptibility to different HD subtypes.
Br J Cancer.
80
1999
1405
1411

Author notes

R. S. Houlston, Institute of Cancer Research, 15 Cotswold Rd, Sutton, United Kingdom; e-mail:r.houlston@icr.ac.uk.