A genetic epidemiology study of congenital adrenal hyperplasia in Italy.

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21OHD-CAH) is an autosomal recessive disorder affecting steroidogenesis, due to mutations in CYP21A2 (6p21.3). 21OHD-CAH neonatal screening is based on 17-hydroxyprogesterone (17OHP) serum levels, showing high type I error rate and low sensitivity to mild CAH forms. Here, we used an epidemiological approach, which estimates the allelic frequency (q) of an autosomal recessive disorder using the proportion of homozygous patients, the mutational spectrum and the inbreeding coefficient in a sample of affected individuals. We applied this approach to 2 independent Italian cohorts of patients with both clinical and molecular diagnosis of 21OHD-CAH from mainland Italy (N = 240) and Sardinia (N = 53). We inferred q estimates of 2.87% and 1.83%, corresponding to a prevalence of 1/1214 and 1/2986, respectively. CYP21A2 mutational spectra were quite discrepant between the 2 cohorts, with V281L representing 74% of all the mutations detected in Sardinia vs 37% in mainland Italy. These findings provide an updated fine-grained picture of 21OHD-CAH genetic epidemiology in Italy and suggest the need for a screening approach suitable to the detection of the largest number of clinically significant forms of CAH.

HLA-G expression and role in advanced-stage classical Hodgkin lymphoma.

Non-classical human leucocyte antigen (HLA)-G class I molecules have an important role in tumor immune escape mechanisms. We investigated HLA-G expression in lymphonode biopsies taken from 8 controls and 20 patients with advanced-stage classical Hodgkin lymphoma (cHL), in relationship to clinical outcomes and the HLA-G 14-basepair (14-bp) deletion-insertion (del-ins) polymorphism. Lymphnode tissue sections were stained using a specific murine monoclonal HLA-G antibody. HLA-G protein expression was higher in cHL patients than controls. In the group of PET-2 positive (positron emission tomography carried out after 2 cycles of standard chemotherapy) patients with a 2-year progression-free survival rate (PFS) of 40%, we observed high HLA-G protein expression within the tumor microenvironment with low expression on Hodgkin and Reed-Sternberg (HRS) cells. Conversely, PET-2 negative patients with a PFS of 86% had higher HLA-G protein expression levels on HRS cells compared to the microenvironment. Lower expression on HRS cells was significantly associated with the HLA-G 14-bp ins/ins genotype. These preliminary data suggest that the immunohistochemical pattern of HLA-G protein expression may represent a useful tool for a tailored therapy in patients with cHL, based on the modulation of HLA-G expression in relation to achievement of negative PET-2.These preliminary data suggest that the immunohistochemical pattern of HLA-G protein expression may represent a useful tool for a tailored therapy in patients with cHL, based on the modulation of HLA-G expression in relation to achievement of negative PET-2.

The distribution of KIR-HLA functional blocks is different from north to south of Italy.

The killer cell immunoglobulin-like receptor (KIR)-human leukocyte antigen (HLA) interaction represents an example of genetic epistasis, where the concomitant presence of specific genes or alleles encoding receptor-ligand units is necessary for the activity of natural killer (NK) cells. Although KIR and HLA genes segregate independently, they co-evolved under environmental pressures to maintain particular KIR-HLA functional blocks for species survival. We investigated, in 270 Italian healthy individuals, the distribution of KIR and HLA polymorphisms in three climatic areas (from cold north to warm south), to verify their possible geographical stratification. We analyzed the presence of 13 KIR genes and genotyped KIR ligands belonging to HLA class I: HLA-C, HLA-B and HLA-A. We did not observe any genetic stratification for KIR genes and HLA-C ligands in Italy. By contrast, in a north-to-south direction, we found a decreasing trend for the HLA-A3 and HLA-A11 ligands (P = 0.012) and an increasing trend for the HLA-B ligands carrying the Bw4 epitope (P = 0.0003) and the Bw4 Ile80 epitope (P = 0.0005). The HLA-A and HLA-B KIR ligands were in negative linkage disequilibrium (correlation coefficient -0.1211), possibly as a consequence of their similar function in inhibiting NK cells. The distribution of the KIR-HLA functional blocks was different along Italy, as we observed a north-to-south ascending trend for KIR3DL1, when coupled with HLA-B Bw4 ligands (P = 0.0067) and with HLA-B Bw4 Ile80 (P = 0.0027), and a descending trend for KIR3DL2 when coupled with HLA-A3 and HLA-A11 ligands (P = 0.0044). Overall, people from South Italy preferentially use the KIR3DL1-HLA-B Bw4 functional unit, while those from the North Italy equally use both the KIR3DL2-HLA-A3/A11 and the KIR3DL1-HLA-B Bw4 functional units to fight infections. Thus, only KIR3DL receptors, which exert the unique role of microbial sensors through the specific D0 domain, and their cognate HLA-A and HLA-B ligands are selectively pressured in Italy according to geographical north-to-south distribution.

Genetics of Behçet's disease in Sardinia: two distinct extended HLA haplotypes harbour the B*51 allele in the normal population and in patients.

OBJECTIVES: To define the contribution of HLA genes and extended HLA haplotypes to the susceptibility to Behçet's disease (BD) in Sardinia. METHODS: Forty-five unrelated Sardinian patients with BD, diagnosed according to the ISG criteria, 45 HLA-B*51 positive and 185 unselected healthy controls were enrolled in the study. DNA samples were typed for HLA class I and class II alleles and genotyped for microsatellites (MICA-TM) and single-nucleotide polymorphisms (rs1264457 HLA-E; rs2281820 motilin; rs1799724 at -857, rs361525 at -238 TNF-alpha) spanning the HLA region. RESULTS: HLA-B*5101 was confirmed as conferring susceptibility to BD (pc=0.0042; OR=4.4; 95% CI=2.0 to 9.6). It is noteworthy that in Sardinia this allele was found more frequently within a haplotype (HLA-A2; -Cw2; -B*5101; -DRB1*11; -DQA1*05; - DQB1*03) that reached its highest frequency in patients with BD. Linkage disequilibrium analysis showed the existence of an additional B*51 haplotype (HLA-A2; -Cw2; -B*5101; -DRB1*04; -DQA1*03; -DQB1*03) not associated with susceptibility to the disease. CONCLUSIONS: In Sardinia, the BD-associated HLA-B*5101 allele is inherited as part of two distinctive haplotypes differently distributed in patients and controls. These findings can be interpreted as suggestive of the presence of additional genes within the MHC region conferring susceptibility to BD. The hypothesis that an environmental pressure could have contributed to the preservation of the BD-associated HLA haplotype in Sardinia is also discussed.

Susceptibility to ankylosing spondylitis but not disease outcome is influenced by the level of HLA-B27 expression, which shows moderate variability over time.

OBJECTIVE: Previous reports have highlighted the relevance of HLA-B27 expression in the pathogenesis of ankylosing spondylitis (AS). The aim of the current study was to estimate the level of HLA-B27 expression on the cell surface of ex vivo monocytes and lymphocytes by a quantitative method and to correlate this with AS disease susceptibility, disease clinical indexes, and the occurrence of acute anterior uveitis (AAU). METHOD: We recruited 32 B27-positive patients with AS and 32 B27-positive healthy normal controls (NCs) for evaluation at different time points. The expression of HLA-B27 molecules was quantified by flow cytometry on ex vivo peripheral blood mononuclear cells (PBMCs). Patients were also evaluated by scores on the Bath AS disease activity (BASDAI), functional (BASFI), and metrology (BASMI) indexes. RESULTS: The expression of HLA-B27 molecules was significantly higher in patients with AS than in B27-matched controls in the case of both monocytes [219K (IQR 174K-308K) vs. 137K (IQR 96K-170K), p < 0.0001] and lymphocytes [82K (IQR 58K-118K) vs. 54K (IQR 44K-61K), p < 0.0001]; AS only vs. AS with AAU: p = 0.744 in monocytes and p = 0.701 in lymphocytes. Comparisons with metrology and functional indexes were also not significant (BASMI: r = 0.05, p = 0.77; BASFI: r = -0.09, p = 0.67). The overexpression of HLA-B27 molecules was stable after 1 week of follow-up. At 3 years follow-up, the variability was moderate and did not correlate with variations in disease activity (BASDAI: r = -0.01, p = 0.92 ns). CONCLUSIONS: The level of HLA-B27 expression in PBMCs correlates with the susceptibility to AS but not with the disease outcome, nor with the occurrence of extra-articular manifestations such as AAU.

The 312N variant of the luteinizing hormone/choriogonadotropin receptor gene (LHCGR) confers up to 2·7-fold increased risk of polycystic ovary syndrome in a Sardinian population.

OBJECTIVE: Polycystic ovary syndrome (PCOS) is a frequent condition, affecting about 15% of women of reproductive age. Because of its familial occurrence, a multifactorial model of susceptibility, including both genetic and environmental factors, has been proposed. However, the identification of genetic factors has been elusive. DESIGN: Case-control study aimed at evaluating possible associations between functionally relevant variants of the luteinizing hormone/choriogonadotrophin receptor gene (LHCGR) and PCOS phenotype. PATIENTS: A total of 198 PCOS and 187 non-PCOS women, aged 14-35 years, of Sardinian origin, were referred to the outpatient clinic of the Department of Obstetrics and Gynaecology of the University of Cagliari (Sardinia). PCOS diagnosis was based on the Rotterdam criteria. MEASUREMENTS: We determined the genotype of ins18LQ, S291N and S312N variants at the LHCGR locus. Genotype was related to the presence or absence of PCOS and to several clinical and biochemical characteristics. RESULTS: The presence of at least one 312N allele was strongly associated with PCOS risk (OR, 2·04; 95% CI, 1·32-3·14; χ(2) , 10·47; P = 0·001). 312N homozygosity was associated with a further risk increase (OR, 2·73; 95% CI, 1·25-5·95; χ(2) , 6·65; P = 0·01). The number of ins18LQ alleles was associated with LH serum levels in controls (χ(2) , 8·04, P = 0·017). CONCLUSIONS: For the first time, we have identified a genetic variant that is strongly associated with PCOS in an isolated population. These results, if confirmed in other cohorts, may provide the opportunity to test the S312N genotype at the LHCGR locus in fertile women to assess the risk of PCOS. The avoidance of triggering factors like weight increase may improve the reproductive outcome of potentially at-risk subjects.

High frequency of the TARDBP p.Ala382Thr mutation in Sardinian patients with amyotrophic lateral sclerosis.

Recently, rare mutations in the TARDBP gene have been identified in familial and sporadic amyotrophic lateral sclerosis (ALS) patients. The purpose of this study was to characterize the genetic variability of the TARDBP gene in a cohort of Sardinian ALS patients. The coding region of the gene was analyzed in 97 unrelated patients previously tested negative for superoxide dismutase (SOD1) mutations. The p.Ala382Thr (c.1144G>A) mutation was found in 30 patients (30.9%). The mutation was predominant in familial ALS patients (FALS) as it was represented in 24 of 30 FALS cases (80%) (p < 0.0003). Six cases were apparently sporadic (9% of sporadic ALS patients). No further mutation of TARDBP was found in our cohort of ALS patients. Patients carrying the mutation showed spinal site of onset in 24 cases (80%), an average age at onset of 54.7 ± 11.1 years, not significantly different from patients not harboring TARDBP mutations (56.7 ± 9.6) and a female:male gender ratio of 1:1.1. The haplotype analysis carried out using eight microsatellite markers flanking the gene showed a founder effect for this mutation. Finally, we estimated the age-specific penetrance of the TARDBP p.Ala382Thr mutation in an additional sample of 47 carriers (20 affected and 27 unaffected). The average penetrance to 70 years was 60% (95% confidence interval 41-79%). A trend toward a higher penetrance in males was observed. Even in the presence of a causal mutation, most of the ALS clinical heterogeneity, however, draws upon from a multifactorial context.

Distribution of HLA alleles and haplotypes in the Maldivian population.

The study of human leukocyte antigen (HLA), allele and haplotype frequencies within populations provides an important source of information for anthropological investigation, organ and hematopoietic stem-cell transplantation purposes as well as disease association studies. As of today, there are no data available in the literature on the HLA structure of the Maldivian population. Altogether 106 families were studied. We used the parents of each family (212 unrelated individuals) to analyze the frequencies of HLA class I and class II allele groups and haplotypes.

The conundrum of HLA-DRB1*14:01/*14:54 and HLA-DRB3*02:01/*02:02 mismatches in unrelated hematopoietic SCT.

Uncertainty still exists on the role of polymorphisms outside the HLA-DRB1 binding site or inside the HLA-DRB3 binding groove in unrelated hematopoietic SCT (HSCT). The ideal model to solve the conundrum consists of the transplants mismatched for HLA-DRB1*14:01/*14:54 and/or for HLA-DRB3*02:01/*02:02. A task force was set up in Italy to recruit transplanted pairs defined as HLA-DRB1*14:01 before 2006, the year crucial for the proper definition of the HLA-DRB1*14:54 allele in molecular biology. Out of 2723 unrelated pairs, 189 transplanted in Italy from 1995 to 2006 were HLA-DRB1*14:01 positive; 103/189 pairs with good historical DNA were retyped for HLA-DRB1*14 and HLA-DRB3 at-high resolution level; 31/103 pairs had HLA-DRB1*14 and/or HLA-DRB3 mismatched; 99/103, having complete clinical data, underwent statistical analysis for OS, TRM, disease-free survival and acute and chronic GvHD. No significant involvement of HLA-DRB1*14:01/*14:54 or HLA-DRB3*02:01/*02:02 mismatches was found, either alone or combined. Our findings suggest that disparities at exon 3 of the HLA-DRB1 gene seem unlikely to influence the outcome after HSCT. The same may be envisaged for HLA-DRB3(*)02:01 and (*)02:02 alleles which, although differing in the Ag binding site, seem unable to modulate an appreciable immune response in an HSCT setting.

Association between different morphological types and abnormal karyotypes in early pregnancy loss.

OBJECTIVES: To identify the abnormal transvaginal ultrasound (TVS) findings typical of aneuploidic pregnancies that end with early pregnancy loss (EPL). METHODS: This was a prospective clinical trial over a 2½-year period from January 2004 to June 2006 at the University Hospital of Cagliari, Italy. One hundred and fifty-six singleton pregnancies with EPL were evaluated by TVS, both before and at the moment of EPL diagnosis. Fetal karyotyping was performed on products of conception using microsatellite analysis to exclude maternal contamination in 46,XX cases. The proportions of abnormal karyotypes were compared among different groups of EPLs characterized by different morphological features. RESULTS: Six morphological types were identified in EPLs, one normal and five abnormal (small gestational sac, small embryo/fetus, early symmetrical arrested growth, enlarged yolk sac and empty sac). The highest rate of chromosomal abnormalities was observed in the early symmetrical arrested growth group (100%), followed by small embryo/fetus (94.1%), enlarged yolk sac (93.3%) and empty sac (72.2%) groups. The majority of cases of trisomy 22 (92.3%) were in the enlarged yolk sac group and the majority of cases with a 45,X karyotype were in the small embryo/fetus group (77.8%). CONCLUSIONS: There is an association in EPLs between some abnormal karyotypes and some morphological types. The demonstration by TVS of abnormalities in the development of early pregnancy structures could be helpful for predicting aneuploidy in EPLs.

The role of killer immunoglobulin-like receptor haplotypes on the outcome of unrelated donor haematopoietic SCT for thalassaemia.

Recent insight into the pathophysiology of acute GVHD after allogeneic haematopoietic SCT has led to a growing interest in the role of natural killer (NK) cells. NK cell cytotoxicity is mainly regulated by the interaction of activating and inhibitory killer immunoglobulin-like receptors (KIRs) with their respective ligands. To investigate the impact of KIRs and their ligands on haematopoietic SCT outcome, we performed a retrospective study of 78 transfusion-dependent thalassaemia patients (median age 10 years, range 1-29 years) transplanted from an unrelated donor selected using high-resolution molecular typing for both class I and II loci after a myeloablative conditioning regimen. GVHD prophylaxis consisted of CsA, short-term MTX and anti-thymocyte globulin in all patients. We found that patients transplanted from donors homozygous for KIR haplotype A had a greater risk of developing grade II-IV acute GVHD compared with those transplanted from a donor carrying at least one B haplotype (hazard ratio=4.5, 99% confidence interval=1.2-17.1, P=0.003). Our study suggests that KIR genotyping of donor and recipient pairs could contribute to the identification of patients at high risk for developing severe complications of haematopoietic SCT and thus may help with the choice of intensity of GVHD prophylaxis.

Identification of a new HLA DRB1 allele (HLA-DRB1*1167) in a potential hematopoietic stem cell donor from Iraqi Kurdistan.

High-resolution polymerase chain reaction sequence-specific primer typing and sequence-based typing of the human leukocyte antigen (HLA) gene DRB1 in a potential hematopoietic stem cell donor of Kurdish ethnicity revealed a new allelic variant of HLA-DRB1*11. The sequence was named DRB1*1167, and comparison with previously described DRB1 alleles demonstrated a mixed pattern shared with some DRB1*08 alleles.

Association of MICA alleles with psoriatic arthritis and its clinical forms. A multicenter Italian study.

OBJECTIVE: Analysis of the association between psoriatic arthritis (PsA) clinical forms and MICA gene transmembrane polymorphisms. METHODS: Patients were classified as having peripheral asymmetric oligoarthritis (AO), peripheral symmetric poly-arthritis (PA) and spondylitis (SP), or disease combinations (PA/SP, OA/SP). Two hundred and twenty-six patients with PsA were typed for MICA exon 5 microsatellite (TM) by heteroduplex analysis and compared with 225 normal controls. RESULTS: MICA-TM microsatellite typing revealed that, among the different clinical forms of PsA, only the combined PA/SP subset shows a significant positive association with MICA-A9 and a lower frequency of MICA-A4, A5 genotype in PsA patients with a decrease, only in the PA/SP cohort, of all MICA-A5 combinations except MICA-A5, -A9. CONCLUSION: These results suggest a role for genes within the HLA region in the pathogenesis of PsA, and reinforce the idea that the different forms of PsA may have heterogeneous genetic basis.

Distribution of killer cell immunoglobulin-like receptors genes in the Italian Caucasian population.

BACKGROUND: Killer cell immunoglobulin-like receptors (KIRs) are a family of inhibitory and activatory receptors that are expressed by most natural killer (NK) cells. The KIR gene family is polymorphic: genomic diversity is achieved through differences in gene content and allelic polymorphism. The number of KIR loci has been reported to vary among individuals, resulting in different KIR haplotypes. In this study we report the genotypic structure of KIRs in 217 unrelated healthy Italian individuals from 22 immunogenetics laboratories, located in the northern, central and southern regions of Italy. METHODS: Two hundred and seventeen DNA samples were studied by a low resolution PCR-SSP kit designed to identify all KIR genes. RESULTS: All 17 KIR genes were observed in the population with different frequencies than other Caucasian and non-Caucasian populations; framework genes KIR3DL3, KIR3DP1, KIR2DL4 and KIR3DL2 were present in all individuals. Sixty-five different profiles were found in this Italian population study. Haplotype A remains the most prevalent and genotype 1, with a frequency of 28.5%, is the most commonly observed in the Italian population. CONCLUSION: The Italian Caucasian population shows polymorphism of the KIR gene family like other Caucasian and non-Caucasian populations. Although 64 genotypes have been observed, genotype 1 remains the most frequent as already observed in other populations. Such knowledge of the KIR gene distribution in populations is very useful in the study of associations with diseases and in selection of donors for haploidentical bone marrow transplantation.

Systemic lupus erythematosus in Europe at the change of the millennium: lessons from the "Euro-Lupus Project".

The "Euro-Lupus Cohort" is composed by 1000 patients with systemic lupus erythematosus (SLE) that have been followed prospectively since 1991. These patients have been gathered by a European consortium--the "Euro-Lupus Project Group". This consortium was originated as part of the network promoted by the "European Working Party on SLE", a working group created in 1990 in order to promote research in Europe on the different problems related to this disease. The "Euro-Lupus Cohort" provides an updated information on the SLE morbidity and mortality characteristics in the present decade as well as defines several clinical and immunological prognostic factors.

Identification of a new allele, HLA-DRB1*1366*.

High-resolution polymerase chain reaction sequence-specific primer typing of the human leucocyte antigen (HLA)-DRB1 gene of an Italian patient candidate for bone marrow transplantation revealed a new allelic variant of HLA-DRB1*13. The sequence was named DRB1*1366, and comparison with previously described DRB1 alleles demonstrated the two closely related sequences were HLA-DRB1*1330 and HLA-DRB1*130302.

Association of HLA-DRB3*0202 and serum IgG antibodies to Chlamydia pneumoniae with essential hypertension in a highly homogeneous population from Majorca (Balearic Islands, Spain).

Separate studies investigating the relationship of essential hypertension (EH) with the HLA system and with Chlamydia pneumoniae (C. pneumoniae) infection have given conflicting results. Our aim was to clarify these relationships and determine whether the HLA system and C. pneumoniae infection interact with respect to the risk for EH. An association study (110 essential hypertensives and 107 controls) was conducted in a highly homogeneous population in the Balearic Island of Majorca (Spain). Molecular typing of HLA-B and HLA-DRB and quantification of serum levels of IgG antibodies to C. pneumoniae (sIgGa-Cp) were determined. Student's t-test, chi(2)-statistics, logistic regression analysis, and general linear model ANOVA were used for statistical analysis. The results showed that EH was related with HLA-DRB3*0202 in the whole study population, and with levels of sIgGa-Cp>63.5 BU/ml in the group of individuals with sIgGa-Cp>30 BU/ml (OR (95% CI) adjusted for obesity, familial history of EH and diabetes=2.06 (1.07-3.97), P=0.03, and =4.60 (1.06-19.90), P=0.04, respectively). The association between EH and sIgGa-Cp was observed in the DRB3*0202(+) individuals, but not in the DRB3*0202(-) subgroup (OR (95% CI)=11.14 (1.92-64.54), P=0.004, and =0.98 (0.22-4.43), P=0.64, respectively (P of the Mantel-Haenszel test for homogeneity of OR=0.06)). In our population, EH was positively associated with HLA-DRB3*0202 and with high levels of sIgGa-Cp. Moreover, a significant interaction of DRB3*0202 on the effect of sIgGa-Cp was observed, as the association of EH with these antibodies depended on the presence of DRB*0202.

A new germline RET mutation apparently devoid of transforming activity serendipitously discovered in a patient with atrophic autoimmune thyroiditis and primary ovarian failure.

Gain-of-function RET mutations are responsible for multiple endocrine neoplasia syndromes (MEN) 2A and 2B and familial medullary thyroid carcinoma (FMTC), whereas loss-of-function mutations are found in Hirschsprung disease. We report a new RET point mutation [R694Q (CGG-->CAG)], serendipitously found in a 23-yr-old woman with hypothyroidism due to atrophic Hashimoto's thyroiditis and primary ovarian failure, without altered calcitonin secretion. Familial history and clinical and biochemical evaluation of first-degree relatives were negative for FMTC, MEN 2A and 2B, and Hirschsprung disease. Genetic analysis showed that the mutation was inherited from the mother, who was submitted 2 yr before to thyroidectomy for goitrous Hashimoto's thyroiditis. Histological revision and immunohistochemical studies documented normal C cell number and morphology. We cloned the mutation in an expression vector encoding a full-length RET protein. The construct was transiently expressed in 293T cells in parallel with a wild-type RET and a C634R MEN 2A-associated RET mutant. Proteins were harvested from transfected cells, and tyrosine phosphorylation levels were assayed. The mutation did not exert significant potentiating effects on RET kinase. A focus assay was also performed on NIH3T3 fibroblasts; the mutant did not exert significant transforming activity. In conclusion, a new RET mutation was found in two subjects without any evidence of MEN and FMTC. In keeping with clinical data, transfection studies confirmed lack of activating activity. This serendipitous discovery, apparently devoid of oncogenic potential, underscores the problems that may be encountered in genomic studies on RET.

A psoriasis vulgaris protective gene maps close to the HLA-C locus on the EH18.2-extended haplotype.

We determined the molecular haplotypes of the HLA-A, HLA-C and HLA-B loci and the MHC class I-B-related (MIB) microsatellite in 179 unrelated psoriatic patients (72 familial cases) and in 120 controls. The HLA-A*3002-Cw*0501-B*1801-MIB1 haplotype showed a strong negative association with psoriasis vulgaris (PV) and in particular with familial PV, revealing the presence of a PV-protective gene. Analysis of association and linkage disequilibrium of the single alleles and the various two-three-four-locus segments of this haplotype indicated the presence of a protective gene telomeric to the HLA-C locus. This finding was confirmed in 13 informative multiplex PV families, in which at least one parent carried the EH18.2 haplotype. In two families, an affected sibling presented HLA-A/C recombination on the EH18.2 haplotype. A study of 12 polymorphic microsatellites in all members of the informative families, 145 PV patients, 120 controls and 32 EH18.2 homozygous healthy individuals demonstrated that the protection conferred by the EH18.2 haplotype lies within a 170 kb interval between the C143 and C244 loci, most probably in a 60 kb segment between the C132 and C244 loci.