HLA-DRB1, DQA1, and DQB1 alleles associated with giant cell arteritis in northern Italy.

OBJECTIVE: To evaluate by molecular typing the possible associations of HLA-DRB1, DQA1, and DQB1 alleles with biopsy proven giant cell arteritis (GCA) in a Mediterranean country, and to examine possible relationships between these alleles and GCA clinical subsets. METHODS: Thirty-nine patients from the Reggio Emilia area diagnosed over a 12 year period with biopsy proven GCA were studied. The clinical findings at diagnosis and during the followup were evaluated through interviews and by reviewing the medical records. HLA-DRB1, DQA1, and DQB1 alleles were determined in the 39 patients and in 250 healthy controls from the same geographic area by polymerase chain reaction amplification using sequence-specific primers. RESULTS: No associations were found between GCA and the shared epitope, the DRYF epitope, or the DRB1*04 or DQA1 alleles. The only significant association was with DQB1*0302 allele (p = 0.03, RR = 2.2). However, the association was weak and the significance was lost when corrected for the number of antigens tested. The frequencies of DQB1*0301 and 0302 in DR4 patients were not significantly different from those observed in DR4 positive controls. Significant associations were found between DRB1*04 allele and the presence of systemic signs and/or symptoms (p = 0.04, RR = 1.5) and between DRB1*07 allele and the male patients (p = 0.04, RR = 2.6). CONCLUSION: Our data showed no associations of biopsy proven GCA with HLA-DRB1*04 and HLA-DRB1*01 alleles, rheumatoid epitope, or DRYF epitope. Discrepancies with other studies may be related to the different ethnic backgrounds of the populations studied and to differences in the referral patterns of patients with GCA.

HLA-DRB1 alleles associated with polymyalgia rheumatica in northern Italy: correlation with disease severity.

OBJECTIVE: To examine the association of HLA-DRB1 alleles with polymyalgia rheumatica (PMR) in a Mediterranean country and to explore the role of HLA-DRB1 genes in determining disease severity. METHODS: A five year prospective follow up study of 92 consecutive PMR patients diagnosed by the secondary referral centre of rheumatology of Reggio Emilia, Italy was conducted. HLA-DRB1 alleles were determined in the 92 patients, in 29 DR4 positive rheumatoid arthritis (RA) patients, and in 148 controls from the same geographical area by polymerase chain reaction amplification and oligonucleotide hybridisation. RESULTS: No significant differences were observed in the frequencies of HLA-DRB1 types and in the expression of HLA-DRB 70-74 shared motif between PMR and controls. The frequency of the patients with double dose of epitope was low and not significantly different in PMR and in controls. No significant differences in the distribution of HLA-DR4 subtypes were observed between DR4+ PMR, DR+ RA, and DR4+ controls. Results of the univariate analysis indicated that an erythrocyte sedimentation rate (ESR) at diagnosis > 72 mm 1st h, the presence of HLA-DR1, DR10, rheumatoid epitope, and the type of rheumatoid epitope were significant risk factors associated with relapse/recurrence. Cox proportional hazards modelling identified two variables that independently increased the risk of relapse/recurrence: ESR at diagnosis > 72 mm 1st h (RR=1.5) and type 2 (encoded by a non-DR4 allele) rheumatoid epitope (RR=2.7). CONCLUSION: These data from a Mediterranean country showed no association of rheumatoid epitope with PMR in northern Italian patients. A high ESR at diagnosis and the presence of rheumatoid epitope encoded by a non-DR4 allele are independent valuable markers of disease severity.

HLA-DRB1 alleles associated with rheumatoid arthritis in Northern Italy: correlation with disease severity.

The aim of the study was to evaluate the relationship between the presence of the 'rheumatoid epitope', defined by a sequence motif in the HLA-DRB1 alleles, rheumatoid factor and disease severity in Northern Italian patients with rheumatoid arthritis (RA). Twenty-nine DR4-positive and 57 DR4-negative RA patients were studied. Each DR4-positive patient was matched with two DR4-negative controls of similar disease duration and sex. HLA-DRB1 alleles were determined in the 86 patients and 351 controls from the same geographical area. The patients were retrospectively evaluated for extra-articular features (EAF) and radiographic damage. The rheumatoid epitope was expressed in 45% of patients. No significant differences in the presence of rheumatoid factor, EAF and articular damage were observed between patients with no, one or two doses of epitope. However, the patients encoding the epitope by an HLA-DR4 allele had a higher number of eroded joints and a higher Larsen score compared to those without the epitope. No differences were present between patients expressing HLA-DRB1*01 alleles and those lacking the rheumatoid epitope. Even in the absence of expression of the rheumatoid epitope, seropositive patients had more EAF and more erosive disease compared to those who were seronegative. Even if most Northern Italian RA patients do not express the rheumatoid epitope, the radiological severity of disease is associated with HLA-DRB1*04 alleles.

The DRPLA CAG repeats in an Italian population sample: evaluation of the polymorphism for forensic applications.

The DRPLA CAG repeats polymorphism has been studied in an Italian population sample. PCR amplification, manual PAGE and silver staining were employed. A total of 16 different alleles, spanning the range from 5 to 21 CAG triplettes, was observed. The heterozygosity was 0.81 and no significant deviation from Hardy-Weinberg equilibrium was found 81 meioses from parentage testing were also analyzed and a Mendelian pattern of inheritance was observed in all cases. In addition, we could successfully type DRPLA locus in some forensic specimens, 1 ng of DNA allowing clear definition of alleles. The authors conclude that the DRPLA CAG repeats analysis may be useful for forensic applications.

Molecular analysis of HLA genes for the selection of unrelated bone marrow donor.

The selection of fully matched unrelated volunteer donors (UVD) in BMT requires a molecular characterization of MHC polymorphism, since most phenotypically HLA-identical donors can be non-identical when analyzed at a genomic level. The present report describes a molecular typing protocol for HLA genes developed for the selection of UVD, and its application to some donor-recipient pairs. The protocol involves three successive steps. Firstly, PCR with sequence-specific primers for HLA-DRB1 and -DQB1 genes is performed to identify the major alleles of the recipient. PCR-fingerprint matching is then introduced for HLA-A, B, C and DRB, DQB and DPB genes to screen prospective donors. Those showing matched fingerprinting patterns are finally submitted to direct sequencing of the DRB1 gene. DPB compatibility is assessed by oligotyping when there are several potential class I and DRB matched donors. This strategy was applied retrospectively to three BMT recipients and their previously selected donors. Three other patients and their 12 prospective donors were submitted to our protocol before BMT. Clinical evaluation of transplant outcomes indicates the primary importance of complete DRB and class I matching, while DQB and DPB compatibility seems to be less critical.

Retinitis pigmentosa, ataxia, and mental retardation associated with mitochondrial DNA mutation in an Italian family.

An Italian pedigree including two sisters and their mother affected by a neuro-ophthalmic disease characterised by retinitis pigmentosa, ataxia, and psychomotor retardation is reported. Molecular analysis of mitochondrial DNA showed the presence of heteroplasmic 8993 point mutation in the subunit 6 of the ATPase gene. The clinical features and genetic findings in this family were comparable with those recently described in an English family. The mitochondrial DNA analysis of the family showed a correlation between the amount of mutated DNA and the disease severity in the probands, and indicated the presence of a threshold amount of mutated genome inducing ophthalmic defects. Moreover, the comparative analysis of blood, hairs, muscle, and urinary tract epithelia of two probands revealed an essentially similar distribution of mutated and wild type mitochondrial genomes. Our results suggest that the 8993 mitochondrial DNA mutation characterises a disease with similar clinical features in different populations.

New strategies for selection of unrelated bone marrow donors.

An important problem in the selection of unrelated donors for bone marrow transplantation (UD-BMT), is HLA matching, between selected donor and recipient. Serological screening, mixed lymphocyte culture (MLC), and sequence specific oligonucleotide genotyping (PCR-SSO) are the methods commonly used for typing of HLA-genes. These ways to select donor candidates are time-expensive. We set up new applications of the "fingerprinting-PCR" technique, to analyse the polymorphic second exon of DRB, DQB, DQA, DPB of HLA Class II and second exon A, B, C HLA-Class I genes, and to search for identity between patient and serologically selected unrelated donors. In an assessment of the technique, 50 normal samples, and 4 unrelated HLA-A and HLA-B serological matched patient-donor pairs were analysed for HLA polymorphic regions. In 3 of the 4 cases (UD-BMT) at least HLA-DRB mismatched different donor-transplanted patterns were identified. In all cases PCR-SSO analysis was performed as control. Based on our data, we suggest that identification of UD for allogeneic BMT should follow these steps: 1) serological HLA-Class I and II genes screening; 2) HLA-Class II DRB gene PCR fingerprinting; 3) confirmation by SSO analysis in case of fingerprinting identity. 4) HLA-Class II DQA, DQB, DPB PCR fingerprinting. Moreover, confirmation by PCR fingerprinting or protein isoelectrofocusing of HLA-Class I identity is recommended. This "strategy" may contribute to rapid and specific selection of unrelated marrow donors.

Asp57-negative HLA DQ beta chain and DQA1*0501 allele are essential for the onset of DQw2-positive and DQw2-negative coeliac disease.

The genetic predisposition to coeliac disease is associated with the HLA DQw2 allele. Coeliac patients lacking the DQw2 allele are very rare and always exhibit the DR4-DQw3 haplotype. We performed oligotyping of polymerase chain reaction (PCR)-amplified DQA1 and DQB1 genes in six DQw2-negative and 30 DQw2-positive coeliac patients. The DQB analysis showed that all six DQw2-negative patients possessed the DQB1*0302 allele. The other DQB alleles found in five of these patients were DQB1*0501, DQB1*0604 and DQB1*0302. The DQ beta chains encoded from all these alleles have the replacement of aspartic acid residue at position 57 (Asp57), as well as the DQB1*0201 allele which was found in all 30 DQw2-positive coeliac patients. The DQw2-negative proband who lacked the homozygous Asp57 replacement exhibited the DQA1*0501 allele in the DQA1 gene. The DQA1*0501 allele was also found in 27 of the 30 DQw2-positive coeliac patients. Among this group of coeliacs, the four cases lacking the DQA1*0501 allele exhibited the homozygous Asp57 replacement in the DQ beta chain. Our results indicate that Asp57-negative DQ beta alleles are involved in both DQw2-positive and -negative coeliac patients. Moreover, when the Asp57-negative DQ beta chain is encoded from only one of the two DQB1 genes the DQA1*0501 allele is always present.

Structural organization of BCR-ABL gene in chronic phase and blast transformation in chronic myeloid leukemia patients.

We studied 36 DNA samples of 18 patients affected with chronic myeloid leukemia (CML) for the presence of mutations in the first exon of the BCR gene was divided into four regions amplified by polymerase chain reaction (PCR). By single strand conformation polymorphism analysis (SSCP) and direct sequencing of amplified fragments, we found different banding profiles in 9 out of 18 patients in the PCR fragment spanning nucleotide 506-826. In one patient, sequence analysis revealed the presence of a point mutation at nucleotide 669 (A-T; Gln-Leu). No difference was found between DNA samples collected during the chronic phase and the blastic transformation. No different mobility shifts of single stranded PCR products were found in the other amplified fragments. The activation of BCR-ABL involves direct interaction between BCR first exon sequences and the tyrosine kinase regulatory domains of ABL. In the first BCR exon, and around the mutated sequences two SH-2-binding sites, are retained. These domains are essential for BCR-ABL-mediated transformation. Our results demonstrate the presence of point mutation in this regulatory region, which may suggest a role for the altered BCR sequence in activation of the BCR-ABL oncogene.

Mitochondrial DNA analysis in Leber's hereditary optic neuropathy.

A mitochondrial DNA (mtDNA) mutation at nucleotide 11778 has been reported as the genetic defect associated with Leber's hereditary optic neuropathy (LHON), but some pedigrees failed to reveal this mutation. The authors present the genetic analysis of a large Italian LHON family with three probands and 16 asymptomatic maternal relatives. The 11778 mtDNA mutation was present in this family and absent in 52 Italian healthy controls, confirming the association between this genetic defect and LHON. In addition, they found a variable proportion of mutated and wild-type mtDNA (heteroplasmy) in every maternally related family member, including the probands. Different patterns of heteroplasmy were present in the pedigree and lower levels of wild-type mtDNA seemed to correlate with the disease status. Moreover, the authors evaluated the mitotic segregation of mt genomes in blood, hair and urinary tract epithelia of the three patients and they found a similar level of heteroplasmy in these tissues.

HLA-DP polymorphism in northern Italian celiac patients.

The contribution of HLA-DP genes to celiac disease susceptibility has been investigated in 95 Italian patients, 41 with childhood and 54 with adult disease onset. Polymerase chain reaction amplification, sequence-specific oligonucleotide probe hybridization and restriction fragment length polymorphism analyses have been carried out. All celiac patients and 56 out of 128 random healthy controls were DQw2-positive. The frequency of the DPB1*0101 allele was significantly increased (pc = 0.002, relative risk 5.21) in patients with celiac disease (23.2%) compared to the whole panel of controls (5.5%), but not to the 56 controls bearing DQw2 (10.7%). No significant difference in the frequency of DPB1*0101 was found between celiac patients with pediatric (24.4%) or adult (22.2%) onset. The DPB1*0101 allele was associated with both the DR3-DQw2 and DR7-DQw2 haplotypes. Moreover, our study has not confirmed the association with DPB1*0402 and DPB1*0301 previously reported in celiac children from southern Italy. The linkage of the DPB1*0101 allele with the DQ locus and the observation that the DP but not the DQ association appears to be ethnically dependent strongly support a secondary role of DP molecules in celiac disease susceptibility.