David Gokhale

Evidence that an HLA-DQA1-DQB1 haplotype influences susceptibility to childhood common acute lymphoblastic leukaemia in boys provides further support for an infection-related aetiology.

Comparison of DQA1 and DQB1 alleles in 60 children with common acute lymphoblastic leukaemia (c-ALL) and 78 newborn infant control subjects revealed that male but not female patients had a higher frequency of DQA1*0101/*0104 and DQB1*0501 than appropriate control subjects. The results suggest a male-associated susceptibility haplotype in c-ALL and supports an infectious aetiology.

Further investigation of the role of HLA-DPB1 in adult Hodgkin's disease (HD) suggests an influence on susceptibility to different HD subtypes.

SummaryIt has been suggested in a number of studies that susceptibility to adult Hodgkin’s disease (HD) is influenced by the HLA class II region, and specifically by alleles at the HLA-DPB1 locus. Since HD is diagnostically complex, it is not clear whether different HLA-DPB1 alleles confer susceptibility to different HD subtypes. To clarify this we have extended a previous study to type DPB1 alleles in 147 adult HD patients from a single centre. We have analysed patients with nodular sclerosing (NS), mixed cellularity (MC) or lymphocyte predominant (LP) HD, and gender in relation to HLA-DPBI type, in comparison with 183 adult controls. The results confirmed previously reported associations of DPB1*0301 with HD susceptibility (relative risk (RR) = 1.42; 95% confidence interval (CI) 0.86–2.36) and DPB1*0201 with resistance to HD (RR = 0.49; CI 0.27–0.90). However, analysis by HD subtype and gender showed that *0301-associated susceptibility was confined to females with HD (RR = 2.46; CI 1.02–5.92), and *0201-associated resistance to females with NS-HD (RR = 0.28; CI 0.10–0.79). Susceptibility to NS-HD was also associated in females with *1001 (RR = 11.73; CI 1.32–104.36), and resistance with *1101 (RR = 0.08; CI 0.01–0.65). In contrast, susceptibility to LP-HD was associated in males with *2001 (RR = 32.14; CI 3.17–326.17), and to MC-HD with *3401 (RR = 16.78; CI 2.84–99.17). Comparison of DPB1-encoded polymorphic amino-acid frequencies in patients and controls showed that susceptibility to MC-HD was associated with Leucine at position 35 of DPB1 (RR = 8.85; CI 3.04–25.77), Alanine-55 (RR = 15.17; CI 2.00–115.20) and Valine-84 (RR = 15.94; CI 3.55–71.49). In contrast, Glutamic acid 69 was significantly associated with resistance to MC-HD (RR = 0.14; CI 0.03–0.60). Certain DPB1 alleles and individual DPβ1 polymorphic amino acid residues may thus affect susceptibility and resistance to specific HD subtypes. This may be through their influence on the binding of peptides derived from an HD-associated infectious agent, and the consequent effect on immune responses to the agent.

Molecular analysis of HLA-DQB1 alleles in childhood common acute lymphoblastic leukaemia

Epidemiological studies suggest that childhood common acute lymphoblastic leukaemia (c-ALL) may be the rare outcome of early post-natal infection with a common infectious agent. One of the factors that may determine whether a child succumbs to c-ALL is how it responds to the candidate infection. Since immune responses to infection are under the partial control of (human leucocyte antigen) HLA genes, an association between an HLA allele and c-ALL could provide support for an infectious aetiology. To define the limit of c-ALL susceptibility within the HLA region, we have compared HLA-DQB1 allele frequencies in a cohort of 62 children with c-ALL with 76 newborn controls, using group-specific polymerase chain reaction (PCR) amplification, and single-strand conformation polymorphism (SSCP) analysis. We find that a significant excess of children with c-ALL type for DQB1*05 [relative risk (RR): 2.54, uncorrected P=0.038], and a marginal excess with DQB1*0501 (RR: 2.18; P=0.095). Only 3 of the 62 children with c-ALL have the other susceptibility allele, DPB1*0201 as well as DQB1*0501, whereas 15 had one or the other allele. This suggests that HLA-associated susceptibility may be determined independently by at least two loci, and is not due to linkage disequilibrium. The combined relative risk of the two groups of children with DPB1*0201 and/or DQB1*0501 is 2.76 (P=0.0076). Analysis of amino acids encoded by exon 2 of DQB1 reveal additional complexity, with significant (P<0.05) or borderline-significant increases in Gly26, His30, Val57, Glu66-Val67 encoding motifs in c-ALL compared with controls. Since these amino acids are not restricted to DQB1*0501, our results suggest that, as with DPB1, the increased risk of c-ALL associated with DQB1 is determined by specific amino acid encoding motifs rather than by an individual allele. These results also suggest that HLA-associated susceptibility to c-ALL may not be restricted to the region bounded by DPB1 and DQB1.

Pseudoautosomal linkage of familial hodgkin's lymphoma: molecular analysis of a unique family with leri–weill dyschondrosteosis and hodgkins lymphoma

Hodgkin’s lymphoma (HL) is generally a sporadic disease that may be caused by an infectious agent, possibly Epstein‐ Barr virus (EBV). However, there is consistent evidence of familial clustering in HL (Ferraris et al, 1997), suggesting a role for heritable predisposition in a proportion of patients. Although progress with the identification of an HL-predisposing gene has been hindered by the rarity of multiplepatient HL families, our description of a family in which two sisters with the skeletal dysplasia Leri‐Weill dyschondrosteosis (LWD) both developed HL in adolescence led us to suggest that an HL-predisposing gene might be linked to the LWD gene (Gokhale et al, 1995). Subsequent identification of the LWD gene as the pseudoautosomal short stature homeobox gene SHOX (Shears et al, 1998) led to the hypothesis, based on observations in our HL-LWD family, that an HL-predisposing gene might be located in the major pseudoautosomal region (PAR1) of the X and Y chromosomes (Horowitz & Wiernik, 1999). We have studied further the PAR1 in the HL-LWD family using microsatellite marker analysis and fluorescence in-situ hybridization (FISH). Fluorescent polymerase chain reaction (PCR) primers were used to amplify the pseudoautosomal microsatellite markers DXYS233, DXS6814, DXYS228, DXYS230 and the SHOX CA repeat [located approximately 15 kilobases (kb) upstream of SHOX]. PCR products were analysed using an ABI Prism TM 377 Genetic Analyser and allele sizes calculated using GENOTYPER TM software (Applied Biosystems, Warrington, UK). The microsatellite marker DXYS232 and the 4 basepair (bp) insertion ⁄ deletion polymorphism at the MIC2 locus were amplified using nonfluorescent primers, and PCR products resolved using polyacrylamide gel electrophoresis and silver staining. FISH was performed on metaphase spreads of lymphocytes using a panel of cosmid probes from the Xp ⁄ Yp telomere spanning PAR1. One of these, LLNOYCO3¢M¢34F5, encompasses the SHOX gene. Cosmids were labelled using the Bio-Nick labelling system (Life Technologies). The results of the microsatellite and FISH analysis are summarized in Fig 1A and B. The individuals tested included the probands P1 and P2, both affected with LWD and Hodgkin’s lymphoma, their mother (M), who only had LWD, their unaffected father (F) and their maternal grandmother (GM). Our results confirmed that the siblings with HL-LWD each harboured a maternally inherited microdeletion within PAR1 encompassing SHOX, estimated to be 900 kb, and located between 200 kb and 1100 kb from the Xp telomere. Three genes of potential importance in HL are located within the pseudoautosomal region, including the interleukin 3 receptor a chain (IL3RA), the granulocyte‐ macrophage colony stimulating factor receptor a chain (CSF2RA) and MIC2, which encodes the CD99 cell adhesion protein. These genes were not, however, within the deleted region in the two siblings (Fig 1C). While it is not yet possible to exclude haploinsufficiency due to the deletion of some unknown tumour suppressor gene, an intriguing possibility is that the PAR1 deletion caused a long-range position effect by downregulating expression of one of the above genes. MIC2 is a particularly strong candidate as decreased expression is associated with the development of Hodgkin’s Reed‐Sternberg cells (H-RS cells; Kim et al, 1998) and is downregulated by EBV latent membrane protein 1, which is highly expressed in H-RS cells (Lee et al, 2001). Against this hypothesis is the fact that PAR1 microdeletions of a similar size are the most common defect in LWD, but there is, as yet, no report of an association between LWD and HL. The existence of an HL-predisposing gene in PAR1 (Horwitz & Wiernik, 1999) thus remains speculative, but further studies of genes in this region in familial Hodgkin’s lymphoma should help to resolve this issue.

Lack of association between childhood common acute lymphoblastic leukaemia and an HLA-C locus dimorphism influencing the specificity of natural killer cells

Previous serological studies documenting an association between acute lymphoblastic leukaemia (ALL) and HLA‐Cw antigens suggested that the HLA‐C locus might influence susceptibility to ALL. However, associations with more than one Cw antigen suggest that polymorphic variants shared by more than Cw allele could be involved. Recent studies have shown that the HLA‐C locus encodes two ligands (NK1 and NK2) recognized by receptors on natural killer (NK) cells. HLA‐Cw alleles encoding these ligands are dimorphic, dependent on whether they encode one or other NK ligand. To determine whether susceptibility to the common (CD10+) form of childhood ALL (c‐ALL) is associated with NK1 or NK2, we carried out a molecular analysis of 94 childhood c‐ALL patients and 136 infant controls. We found no difference in the frequency of NK1 and NK2 alleles, phenotypes or genotypes between the patients and controls, suggesting that this does not explain the role of the HLA‐C locus in susceptibility to childhood c‐ALL.

Evidence of an Increased Frequency of HLA-DPB1*0301 in Hodgkin’s Disease Supports an Infectious Aetiology

Hodgkin’s disease (HD) may be the rare outcome of a common infection which is influenced by host genetic susceptibility. We have analysed this hypothesis by determining the frequency of HLA-DPB1 alleles in two series of HD patients using molecular typing methods. One series consisted of a retrospective/prospective group of 118 patients over the age of 15 years, and the other a prospective group of 45 patients between the ages of 16 and 24 years. In both series, the proportion of patients typing for HLA-DPB1 *0301 was greater than that for the controls, suggesting that this may be an HD-susceptibility allele. Analysis of DPB1 alleles in relation to HD subtype also showed that the increase in *0301 was present in nodular sclerosing HD (HDNS) patients, as well as in mixed cellularity HD (HDMC) and lymphocyte predominant HD (HDLP) patients. Preliminary evidence was obtained suggesting an increase in *0401, and possibly *0501, in HDMC and HDLP. Analysis of *0301-like hypervariable region (HVR) associations with HD subtypes indicated an increase in an *0301-like HVR-C motif in HDNS, but not in non-HDNS. The frequency of *0301- and *0401 -like HVR-C and HVR-F amino acid residues also differed in HDNS and non-HDNS patients. The frequency of the HVR-C amino acid residues Asp55, Glu56 (*0301) was increased in HDNS, but not in non-HDNS patients, whereas the frequency of Ala55, Ala56 (*0401) was increased in non-HDNS, but not in HDNS patients. In addition, the frequency of the HVR-F amino acid residues Asp84, Glu85 and Ala86 (*0301) was greater in the HDNS than non-HDNS patients, whereas there was no increase in Gly84, Gly85, Pro86 (*0401). Although these are preliminary findings, they suggest that genetic susceptibility to an infectious aetiology in HD may reside at the level of DPB peptide-binding residues rather than with a specific allele.