Proficiency testing for HLA-DRB high-resolution typing: a 4-year experience in Italy.

Twenty-one Italian laboratories participated for four consecutive years in the collaborative Italian proficiency testing of HLA class II (DRB1, DRB3, DRB4 and DRB5) high-resolution typing. In this paper the results are analysed. Seven different kinds of errors are described and discussed. The errors were divided into technical errors and errors of allele reporting. Each year, a list of errors made was prepared by our laboratory and discussed collegially with all laboratories. The allele reporting errors diminished over time, as a result of the common discussions. The technical performance of the laboratories did not improve overall for the 21 laboratories participating: for 16 of them results were good or improved in quality, but for the remaining five results deteriorated over time. From this experience, some recommendations for the future emerged. A relevant conclusion was that, to improve the performance of a group of laboratories, the proficiency test is not effective alone, but should be integrated within a framework of continuous collaboration and mutual technical help.

[The Italian Registry of Bone Marrow Donors: genetic structure and recruitment strategy]. / Il Registro Italiano dei Donatori di Midollo Osseo: struttura genetica e strategie di reclutamento.

The genetic structure of the Italian bone marrow donor population was analysed by estimating the HLA-A, -B and -DR gene and haplotype frequencies for the total population and for the Italian administrative regions. The haplotype frequencies were used to predict the probability of finding HLA-compatible donors for Italian patients depending on the registry size, and the probability of recruiting in the different Italian regions a donor with a new phenotype. The analysis of these probabilities allows us to propose strategies for donors recruitment in order to increase the phenotypic variability of the registry, then its efficiency.

HLA polymorphisms in Italian bone marrow donors: a regional analysis.

The aim of this study was to analyse the genetic structure of the Italian bone marrow donor population on the basis of HLA polymorphisms. Maximum likelihood estimates of gene and haplotype frequencies, goodness of fit to Hardy-Weinberg predictions and heterozygosity were calculated for 18 Italian administrative regions. Moreover, the phenotypic peculiarity of the regional populations was assessed by analysing the number of "typical phenotypes" found in each region. Multivariate analyses carried out on HLA-A and HLA-B gene frequencies gave a genetic pattern of the donor pools that reflects the structure of the Italian population determined in previous population genetic studies. Sardinia shows a very large genetic difference with respect to the other regions; of these, the central-southern regions are well-differentiated from the central-northern. Southern regions present higher genetic heterogeneity and a higher probability of providing donors with phenotypes not already present in the Italian bone marrow registry. The large sample size of the bone marrow donor registry allowed us to estimate gene and haplotype frequencies with greater accuracy than in previous studies. Our results may be of use in determining strategies for donor recruitment and selecting unrelated donors for patients requiring bone marrow grafting, as well as for anthropological, epidemiological and population genetics studies.

The natural history of an HLA haplotype and its recombinants.

The presence of haplotype-specific recombination sites can be determined by analyzing the conservation of extended haplotypes in the population. This approach considers all meioses in the history of the population and requires the presence of characteristic markers that easily allow the identification of the haplotype or of its recombined segments. The recombination breakpoint can then be mapped by looking for shared alleles between haplotypes selected through the specific marker/s. We identified a rare perfect tandem duplication of a 145 base pair segment in the LTA promoter, which tags a B60 (B60D) haplotype. The duplication was detected in 16/90 B60+ Europeans, while absent in 101 B60+ Orientals. The conservation of the class I end and the extreme variability of the class II end suggested that the present-day B60D haplotypes originated from an ancestral haplotype by recombination events centromeric to the duplicated sequence. Through a fine mapping using markers of the HLA central region a preferential recombination site was localized in the 60 kilobase interval between TNFd,e, and D6S273/K11 Amicrosatellite loci (i.e., between LST1 and BAT3 genes). This site behaves as a potent recombination enhancer leading to fragmentation in most of the extant B60D haplotypes and can be considered responsible for their "instability". In the relatively recently founded Finnish population, where the LST1/BAT3 interval recombination has probably not yet had the chance to occur, a founder effect can explain the presence of a rare DP (DPB1(*)1601) allele in most B60D haplotypes in this population.

Familial membranous nephropathy.

Numerous HLA studies suggest that genetic factors play an important role in the development of membranous nephropathy (MN). We studied seven patients with idiopathic MN, from three unrelated families of Italian ancestry. Complement phenotype analysis and restriction fragment length polymorphism (RFLP) typing of HLA class II and of the switch region genes were done in family members. In the first family, the father, one son, and one daughter had MN; another daughter had clinical glomerulonephritis. The three members with MN shared one HLA haplotype carrying DR beta 11; in the two siblings with the disease, the second HLA haplotype carried the DR beta 3.2 allele. In families 2 and 3, two brothers had MN: in family 2, they differed in at least one haplotype; in family 3, they differed in both haplotypes. Only family 3 was informative with regard to the RFLP of the switch region genes: the two siblings were identical for both Ig heavy chain haplotypes. No clinical, laboratory or morphologic features consistent with a secondary form of the disease were found. Familial clustering of MN suggests a genetically transmitted mechanism.

HLA haplotypes and hormonal studies in 25 Italian families of patients with classical and non-classical 21-OH deficiency.

To investigate the genetic polymorphisms of the HLA region and the molecular defect of the P450c21B gene in congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency, we studied 89 individuals from 25 families of CAH patients (14 classical forms, 11 non-classical forms). The following immunogenetic and hormonal investigations were performed: HLA-A and B typing, restriction fragment length polymorphism (RFLP) analysis of 21-hydroxylase A and B genes, and serum 17-OH-progesterone values determined basally and 60 min after ACTH stimulation. In the patients affected by the classical form, RFLP analysis revealed 5 deletions and 1 gene conversion in 6 haplotypes and no molecular defect in the others, who probably carry point mutations. In the patients with non-classical form we found P450c21A duplication in 11/18 haplotypes; 9 of the 11 patients shared the HLA-B14 allele. Utilizing both hormonal and genetic data we identified two cryptic forms; hormonal data alone failed to differentiate heterozygous from normal individuals.

HLA supratypes in an Italian population.

A supratype analysis of a North Italian population was performed, using 16 polymorphisms in the HLA region spanning the HLA-A-DP segment. Fourteen supratypes were identified, mostly corresponding to those found in other Caucasoid populations. The degree of their conservation both within the B-DR/DQ region and in the regions telomeric and centromeric from HLA-A and DP was evaluated and linkage disequilibria among several DR and DP alleles were identified. Notably, the degree of association with DP increased when the DR marker was part of a conserved B-DR/DQ supratype. These data are relevant to the definition of the genetic structure of the population and to the prediction of probabilities of histocompatibility matching between unrelated individuals.

Distribution of tumor necrosis factor alleles (NcoI RFLP) and their relationship to HLA haplotypes in an Italian population.

The NcoI RFLP of the tumor necrosis factor (TNF) beta gene was analyzed in a panel of 105 unrelated healthy Italian blood donors. The gene frequencies of the 10.5 kb and 5.5 kb allele were 0.73 and 0.27, respectively. The 5.5 kb band was significantly positively associated with HLA-A1, B8, DR17.1, and C4AQ0, and negatively associated with DR7.2, DQw9 and C4A6, all being specificities which belong to two well-known Caucasoid ancestral haplotypes. When the population was subdivided on the basis of TNF phenotypes, different linkage disequilibria between HLA alleles were detected in the three phenotypic classes. From this analysis it was possible to relate preferential HLA associations, most of which are characteristic of ancestral haplotypes, to TNF polymorphism.

Hormonal profiles in Italian late-onset adrenal hyperplasia correlate with HLA class III polymorphisms.

To investigate the genetic polymorphisms of the HLA region in late-onset adrenal hyperplasia, 13 Italian patients affected by the disease were analyzed for: (1) HLA-A and -B typing; (2) restriction fragment length polymorphism (RFLP) of DR beta, DQ beta, DQ alpha, 21-hydroxylase A and B genes; (3) fourth complement fraction loci A and B (C4A and C4B), second complement fraction (C2) and properdin B factor (Bf) complement typing; (4) hormonal characteristics associated with some HLA haplotypes. HLA alleles B14 and DR beta 1 were found to be significantly more frequent in patients with respect to controls (relative risk: 8.7 and 7.2, p less than 0.001 and p less than 0.0001, respectively). Also C4B*2, 1 duplication was more frequent in patients than in normal subjects (23% vs. 1.5%, p less than 0.0001). Moreover, patients carrying a duplicated C4B (as well as those having the B14 antigen) showed higher 17-hydroxyprogesterone levels after ACTH stimulation. RFLP analysis of 21-hydroxylase genes with a specific probe revealed a duplication of 21-hydroxylase A gene in 40% of patients. All these individuals carried the C4A*2 B*2,1 phenotype and 75% of them displayed a clearly recognizable duplication at the C4B locus. These data support the hypothesis that in late-onset adrenal hyperplasia the 21-hydroxylase A pseudogene, even if inactive, may play a negative role in the regulation of 21-hydroxylase biosynthesis. Furthermore, we suggest analyzing class III phenotypes to screen the enzymatic defect.

Familial occurrence of primary glomerulonephritis: evidence for a role of genetic factors.

Chronic glomerulonephritis was diagnosed in 23 patients born in a small valley in Northern Italy and in five additional patients with one parent or a more remote ancestor born in the same area, all belonging to three potentially related families. Eighteen patients had biopsy-proven glomerulonephritis: 11 IgA nephropathy, 3 IgM nephropathy, 2 membranoproliferative type I, and 2 mesangial proliferative glomerulonephritis with isolated C3 deposits. Ten had clinical glomerulonephritis. A community screening programme discovered six additional related patients. Two underwent renal biopsy (1 IgA nephropathy; 1 focal glomerulosclerosis); four were diagnosed as having clinical glomerulonephritis. Genealogical investigation identified five deceased family members with diagnoses of chronic nephritis recorded on their death certificates. No environmental nephrotoxic factors were identified. Restriction fragment length polymorphism (RFLP) analysis of HLA-DR beta, HLA-DQ alpha and beta genes, and complement typing for C4A, C4B, and Bf polymorphisms were carried out for 29 patients, 168 healthy relatives, and 24 local controls. The frequency of HLA-Dw8.1 specificity, related to DR beta 8, DQ beta 3b, and DQ alpha 1a RFLPs, was significantly increased more in the affected and unaffected pedigree members than in Italian controls.

HLA class II gene frequencies in Italy.

The frequency of HLA alleles at HLA-DR and DQ loci, and that of the related HLA-D specificities, were estimated in the Italian population. 109 healthy unrelated subjects, born in several Italian regions and living in the district of Torino, were studied. DNA typing was achieved by the restriction fragment length polymorphism (RFLP) analysis of HLA-DR beta, DQ alpha and beta genes, hybridizing specific probes with TaqI digested DNAs. The present study allowed to define in more detail the HLA class II polymorphisms in the Italian population.

HLA in juvenile dermatitis herpetiformis: clinical heterogeneity correlated with DNA and serological polymorphism.

A group of 30 Italian children affected by Dermatitis Herpetiformis (DH) was analysed for HLA region polymorphisms with both serological and DNA methods. Serological typing was performed on HLA-A, B, C, DR, DQ antigens and C4A, C4B, Bf polymorphisms. DNA RFLPs obtained with TaqI enzyme were investigated with cDNA probes specific for DR beta, DQ alpha and DQ beta genes. The results were correlated with intestinal involvement and age at onset of the disease. The following observations were made: (1) the intestinal biopsies revealed a direct correlation between degree of lesions and age at onset of DH; (2) a significantly increased relative risk (RR) was found for the following HLA antigens: A1 (RR = 2.2), B8 (RR = 6.2), Cw7 (RR = 3.9), C4AQ0 (RR = 7.4), DR3 (RR = 5.2), DR7 (RR = 4.4), DRw53 (RR = 4.7), DQw2 (RR = 6.0); (3) B8 and DR3 were significantly more frequent in patients with severe intestinal lesions; and (4) of the two DR3 subtypes revealed by RFLP typing, only 3.1 showed an increased frequency in DH patients (RR = 8.4). It is suggested that the susceptibility to Juvenile DH is determined by the same genes, within the HLA region, that are associated with Coeliac Disease.

Familial IgM mesangial nephropathy: a morphologic and immunogenetic study of three pedigrees.

Eight patients belonging to 3 unrelated families had biopsy-proven IgM mesangial nephropathy. In the first family, the mother and the 2 daughters were affected; in the second, the mother and the son; in the third, 2 sisters and the brother. Two additional sisters of the third family showed a clinical picture consistent with chronic glomerulonephritis. The clinical picture was that of hematuria and/or proteinuria. No patients had nephrotic syndrome. Genealogic investigation enabled us to discover 2 additional affected members in the kindred of the first family (1 with IgA nephropathy, 1 with clinical glomerulonephritis) and 3 other affected members in the pedigree of the third family (1 with IgA nephropathy, 1 with sclerosing glomerulonephritis, 1 with clinical glomerulonephritis). Immunogenetic studies showed the recurrence of an extended haplotype bearing DR beta 11-DQ beta 3B-DQ alpha 2-C4A3-C4B1-BfS in 9 of 10 affected members. Our data suggest that genetic factors may be involved in the mechanism of the disease and support the hypothesis that IgM nephropathy is a distinct disease entity.

A new duplication at the C4B locus associated with the HLA-Aw68, Cw8, Bw65 haplotype.

A family with a cross-over between HLA-B and HLA-DR was analysed for its complement alleles. This allowed location of the cross-over between HLA-B and C4. Furthermore, the same family showed a previously undescribed duplication at the C4B locus (C4B* 2,2) that was associated with the HLA-Aw68, Cw8, Bw65, C2*1, Bf*S, C4A*2, DR7, DQw2 haplotype.

Genetic, immunologic, and environmental heterogeneity of IDDM. Incidence and 12-mo follow-up of an Italian population.

The 1-yr incidence of insulin-dependent diabetes mellitus (IDDM) in a population of the Piedmont and Aosta Valley area of Italy was recorded. Anti-virus antibodies (e.g., Coxsackie B1-6, mumps, cytomegalovirus), islet cell antibodies (ICAs), and HLA-A, -B, -C, and -DR were determined in 74 IDDM patients (38 males, 36 females) and in controls. Total IDDM incidence was 5.0/100,000, and the incidence for those less than 20 yr of age was 11.6/100,000. Anti-virus antibody frequency was not different in IDDM patients and controls. ICAs were present in 58% of IDDM patients at onset and in 30% after 12 mo, and complement-fixing ICAs were found in 39 and 17%, respectively. IDDM was significantly and positively associated with DR3/DR4 and negatively associated with DR2 and DR5. ICA frequency was significantly higher in DR3/DR4 heterozygote patients than in patients without DR3 and DR4. These results suggest that in this IDDM population viral etiology is not evident, ICAs offer only a partial pathogenetic explanation, and genetic and immunologic heterogeneity is evident.

Properdin factor B polymorphism in four Sardinian villages.

A sample of healthy unrelated individuals was typed for properdin factor B (Bf) polymorphism in four Sardinian villages. Two villages, Desulo and Tonara, are located in the highlands; the other two, Orosei and Galtellì, are located in the lowlands. No heterogeneity was found between the highland and the lowland villages, whereas a significant difference was found between the Sardinian villages and continental Italy. The allele Bf-F1 shows the highest gene frequency so far observed. Typically Sardinian is the gametic association (haplotype) HLA-A30, Cw5, B18, BfF1, DR3.

Genetic and population structure of four Sardinian villages.

Data on microgeographic population structure on four neighbouring villages of Sardinia island (Italy) are presented and discussed. Two villages are located in the lowlands where malaria from Plasmodium falciparum was endemic until the eradication of paludism. The other two villages are located in the highlands and they were malaria-free because of the altitude. Census data, inbreeding, migration matrices and surname distributions have been collected. The genetic differentiation of the four villages, tested for 31 genetic polymorphisms (106 alleles), is only in part compatible with migration rates inferred from demographic data. The possible adaptive nature of some genetic markers with respect to malarial resistance is discussed. Ambiguous results from population genetics quantitative methods do not support definite answers.

HLA-DR antigens in HBsAg-positive chronic active liver disease with and without associated delta infection.

The A, B, C and DR locus specificities of the human leukocyte antigens system (HLA) were determined in 45 delta-positive and 44 delta-negative Italian patients, all with HBsAg-positive chronic active liver disease; controls were 526 healthy Italian blood donors matched for age, sex and geographical origin. HLA-A, B, C gene frequencies were not significantly changed. In delta-positive patients, the frequencies of the DR locus specificities were: DR2, 37.8%; DR3, 20%; DR4, 11.1%. In the delta-negative patients, the frequencies were: DR2, 13.6%; DR3, 36.4%; DR4, 0%. Control frequencies were: DR2, 19.4%; DR3, 17.1%; DR4, 18.5%. The corrected p values of the differences between controls and delta-positive patients were: DR2, pc = 0.046; DR3, pc = NS (not significant); DR4, pc = NS. The corrected p values of the differences between controls and delta-negative patients were: DR2, pc = NS; DR3, pc = 0.03; DR4, pc = 0.002. These findings show that: (a) DR3, a genetic marker of autoimmunity, might assist the establishment of chronic HBsAg liver disease in the absence of delta superinfection; (b) DR2 is linked with failure to clear the delta agent, and (c) DR4 may protect from virus B persistence. Identification of adventitious factors such as delta may help uncover a subgroup of HBsAg carriers who are genetically predisposed to develop chronic liver disease.

HLA-DR3 and DR7 in coeliac disease: immunogenetic and clinical aspects.

The association of HLA-A,B,C, DR polymorphisms and of Bf and GLO with coeliac disease was analysed in 100 Italian children. Primary involvement of HLA-DR3 and DR7 is apparent, while specificities of nearby loci are probably associated secondarily, because of linkage disequilibrium. Direct assessment of D/DR genotype through family studies and mixed lymphocyte cultures led to the recognition of two high risk genotypes DR3/3 and DR3/7, and of two lower risk genotypes DR3/X and DR7/X. The different weight of the HLA-dependent genetic factors is to some extent correlated with the clinical and immunological parameters, suggesting that the low-risk genotypes induce a milder expression of coeliac disease. Furthermore, other genetic factors, such as sex, appear to contribute to the penetrance of the disease, especially in the case of DR3/X and DR7/X.