A new allele, HLA-DRB4*010304.

We report here the identification of a novel DRB4*01 allele, DRB4*010304, found in a patient waiting for a liver transplantation. The new allele was detected during a routine DNA-based HLA typing. Sequencing confirmed that the new allele is identical to DRB4*01030101 at exon 2 except for position 216 where the new allele has a T instead of a C.

Genotyping for cytokine polymorphisms: allele frequencies in the Italian population.

It has been demonstrated that many cytokine genes [e.g. tumour necrosis factor alpha (TNF-alpha) and interleukin 10 (IL-10)] show polymorphisms which may affect gene transcription, causing individual variations in cytokine production. The majority of polymorphisms described are single nucleotide polymorphisms (SNPs). In 140 healthy Italian subjects, the allelic and genotype frequencies were determined for the cytokine genes IL-1 alpha (T/C -889), IL-1 alpha (C/T -511, T/C +3962), IL-12 (C/A -1188), interferon (IFN)-gamma (A/T UTR 5644), transforming growth factor (TGF)-alpha (C/T codon 10, G/C codon 25), TNF-alpha (G/A -308, G/A -238), IL-2 (T/G -330, G/T +166), IL-4 (T/G -1098, T/C -590, T/C -33), IL-6 (G/C -174, G/A nt565), IL-10 (G/A -1082, C/T -819, C/A -592), IL-1R (C/T pst11970), IL-1RA (T/C mspa111100) and IL-4RA (G/A +1902). All typings were performed with PCR-SSP assays. Allele and genotype frequencies and linkage disequilibria were calculated and compared with those of other populations.

New HLA-A*11 allele, A*1112, identified by sequence-based typing.

In this report, we describe the identification of HLA-A*1112, a novel HLA-A*11 allele found in two Italian families. The new allele was detected during routine HLA typing by a polymerase chain reaction sequence-specific primer and was confirmed by high-resolution sequencing-based typing. The nucleotide sequences of HLA-A*1112 exons 2 and 3 are identical to HLA-A*11011 except for a single nucleotide substitution in codon 90 (GAC-->GCC).

Comparison of different techniques for detection of anti-HLA antibodies in sera from patients awaiting kidney transplantation.

The aim of the present study was to compare different techniques of HLA antibody detection in patients waiting for a transplant. Two methods of HLA antibody screening were compared: the complement-dependent cytotoxicity (CDC) test and the enzyme-linked immunosorbent solubility assay (ELISA). Three different commercial kits for the latter assay were tested: the kits were produced by One Lambda (O.L.) and SANG STAT for prescreening and specificity determination, and GTI only for prescreening. Of the two techniques, only CDC can detect IgM. The study included 207 serum samples selected from 192 patients waiting for a first kidney transplant. The conclusions are that O.L. is more sensitive than CDC and the other ELISA kits and is much faster for analysing a serum (taking only a few hours), but costs about 16 times more than CDC and does not detect IgM antibodies, so can be used only in support of CDC.

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.

[Pregnancy after kidney transplantation. Review of the international literature and presentation of personal case reports]. / Gravidanza dopo trapianto di rene. Revisione della letteratura internazionale e presentazione della casistica personale.

BACKGROUND: Advances in surgical techniques and immunosuppression have improved the results in organ transplantation. The quality of life in these patients is good in the most of cases and pregnancy, which means for them to resume a normal life, isn't an exceptional event, specially for kidney transplant recipients. METHODS: Retrospective data regarding pregnancies observed at the Dept. of Nephrology and Dialysis of the S. Giovanni Battista Hospital in Turin, have been collected to value the pregnancy frequency and outcome (complications, miscarriage, therapeutic abortion), the mother follow-up as a function of transplant rejection risk, the newborn conditions, their hematological and immunological situation, and the children follow-up. RESULTS: This study includes 9 pregnancy (6 at term and 3 abortions), observed since 1987 in 6 kidney transplant recipients. Congenital malformations or immunological diseases have not been observed; according to the literature, there was a high frequency of preterm delivery and intrauterine grow retardation. Complications during pregnancy were observed in 5 cases (83.3%). CONCLUSIONS: The study confirms that kidney transplant recipients can carry a pregnancy through and give birth to healthy infants, but these pregnancies are to be regarded as being at high risk and require a multi-disciplinary approach.

Cytotoxic T lymphocyte precursor frequency as a predictor of acute graft-versus-host disease in bone marrow transplantation from HLA-identical siblings.

The measurement of precursor frequencies of donor anti-recipient cytotoxic T lymphocytes (CTL-p) has been shown to predict the incidence and the severity of acute graft-versus-host disease (aGVHD) in unrelated donor bone marrow transplantation (BMT). In HLA-identical sibling BMT, where aGVHD is most likely caused by minor histocompatibility antigen mismatches, this assay did not appear to be sensitive enough to provide similar predictive information. In this study, the CTL-p frequencies and the incidence and severity of aGVHD in 51 onco-hematological patients transplanted from HLA-identical siblings were compared. Sibling donors were selected on the basis of HLA identity using serological typing for HLA-A, B, C antigens, whereas HLA-DRB was tested by molecular analysis. Sibling identity was also confirmed by DNA heteroduplex analyses. Fifteen out of 21 (71%) patients with high precursor frequency (>1:100 x 10(3)) and 12 out of 30 (40%) with low precursor frequency (<1:100 x 10(3)) experienced clinically significant (II-IV) aGVHD. A significant correlation (P = 0.04) between CTL-p frequency and severe aGVHD was demonstrated. Moreover there was a positive trend for a high frequency response according to an increasing grade of aGVHD, which was statistically significant (P = 0.04). In our experience the CTL-p assay is a helpful predictive test for aGVHD in HLA-identical sibling BMT, indicating high risk patients possibly requiring additional prophylaxis.

Analysis of the turin umbilical cord blood bank registry.

BACKGROUND: The polymorphic nature of the HLA system reduces a patient's probability of finding an HLA-compatible unrelated bone marrow (BM) donor, even though more than 6 million individuals are enrolled in international registries. Recently, umbilical cord blood (UCB) has been successfully employed as a source of HPCs. The use of such cells reduces the risk of GVHD and allows transplants with one or two HLA mismatches. UCB represents an expensive resource: therefore, it is necessary to carefully manage the UCB unit inventory. STUDY DESIGN AND METHODS: The current study analyzed the genetic heterogeneity of HLA-A, -B, and -DR gene frequencies between pools of UCB and unrelated-donor BM in the Piedmont (an administrative region of Italy). An Italian hematology patient's probability of finding complete or partial matches as a function of donor pool size was determined by considering subsamples randomly selected from the local unrelated BM donors. RESULTS: The HLA gene frequencies in UCB and unrelated-donor BM pools were not significantly different. The search simulation, based on actual HLA phenotypes, showed that the percentage of Italian patients matched with an HPC unit increases remarkably if 1 or 2 mismatches are accepted, reaching a proportion of 90 percent with an inventory of only about 500 units, while the increment is not so remarkable if the number of UCB units is greater. CONCLUSION: To optimize economic resources and to be internationally competitive, UCB banks should aim to increase the genetic heterogeneity of their units rather than increasing the UCB inventory, acquire efficient quality control systems, and acquire and preserve UCB units with a greater number of nucleated cells.

[Transplantation collaborative networks]. / Le reti collaborative per i trapianti.

The collaborative networks in Italy are described, with their objectives and the geographical areas covered. Their main features are: function of the reference centres, waiting lists, criteria for organ assignment, common programmes for some patients, services offered to the network members, organ procurement activity. The analysis of the activities performed included organ exchanges within and outside the network, common activities for tasks that could not be satisfactorily attained by a single centre or region, the collaborative studies. Finally, the role of the networks and of a national coordination, in order to better develop transplants nationwide, is discussed.

[Waiting lists for transplantation in Italy]. / Le liste di attesa trapianto in Italia.

To plan health services it is essential to gauge the needs. In transplant field in Italy, the first suitable data for waiting lists were collected in 1998. The data collected by Istituto Superiore di Sanità gave us a shot of patients field in waiting list at that time. We here analyse more significant data about heart, liver and kidney waiting lists. The situation is very different among north, centre and south of Italy: in South, where transplant activity is low, we found rare transplant centres, and most of patients prefer north centres. In kidney waiting list we found 1100 patients living in southern regions but registered in the waiting list of a different region. These data can help in planning development lines in Italy.

Waiting list for kidney transplant. Some patients wait too long.

The selection of a kidney graft recipient should be made not only taking into account biological and clinical parameters, for assuring the maximum possible clinical success; the ethical objective to allow every patient equal opportunity of receiving a transplant should also be pursued. In every waiting list of transplant candidates a proportion of patients remains in the list for a particularly long time. The present analysis aimed to find out the factors associated with a prolonged waiting time, in order to allow the implementation of patient selection criteria able to balance unfavourable factors. The analysis of the waiting list of our kidney transplant centre allowed to observe that blood group 0, anti-HLA immunisation, presence of rare HLA antigens and, at a lesser extent, HLA homozygosity are associated with a longer waiting time for a kidney transplant.

Angiotensin I-converting enzyme genotype significantly affects progression of IgA glomerulonephritis in an italian population.

To evaluate the role of angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism in the progression of immunoglobulin A glomerulonephritis (IgA-GN), genotype distribution in 81 biopsy-proven cases of IgA-GN was studied. A logistic regression model showed that the risk for homozygous DD was not significantly elevated in patients with IgA-GN compared with healthy subjects (odds ratio = 1.16; confidence interval [CI], 0.4 to 3.3). However, the 5-year (78% v 90%) and 10-year (52% v 82%) renal survival rates for 47 patients with serum creatine (Cr) levels of 1.5 mg/dL or less at biopsy was significantly less in DD patients (n = 18; chi2 = 5.41; P = 0.02). The hazard ratio (HR) for DD (multivariate analysis from Cox proportional model after adjustment for known factors of progression, such as hypertension [HPT] and proteinuria [PTO]) was 3.07 (CI, 1.1 to 9.4). The HR for heavy PTO was 6.1 (CI, 1.9 to 19). The association of DD genotype with progression was even more striking when patients with other risk factors (heavy proteinuria) were excluded, as shown by DD-related risk in the absence (HR = 3.6; CI, 1.1 to 12) and presence (HR = 2; CI, 0.4 to 10) of PTO. The risk ratio was further increased by the coexistence of DD + PTO (HR = 9.16; CI, 1.8 to 15.7). Furthermore, in a cross-sectional study among patients with IgA-GN, a logistic regression model showed that the risk for homozygous DD was greater, although not at a statistically significant level in the end-stage renal failure subgroup compared with the normal renal function subgroup (odds ratio = 3.16; CI, 0.7 to 13.7) after adjustment by sex, age at biopsy, HPT, PTO, and therapy. Last, DD was significantly more frequent in those patients who started hemodialysis at an earlier age (chi2 for trend = 6.81; P = 0.009). Our study further supports that ACE genotype is a risk factor not for the development, but for the worsening of IgA-GN clinical course. However, on the basis of current knowledge, we cannot exclude that I/D polymorphism may simply serve as a prognostic marker, eventually linked with other discrete loci involved in the progression of renal damage.

[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.

[Genetic analysis of Italian hematologic patients candidates for bone marrow transplantation from unrelated donors]. / Analisi genetica di pazienti ematologici italiani candidati al trapianto di midollo osseo da donatore non consanguineo.

Frequencies of HLA-A, -B and -DR antigens and haplotypes were determined in 1945 Italian patients suffering from hematologic diseases and requiring bone marrow transplantation from unrelated donors. These frequencies were compared with those obtained from the Italian bone marrow donor population. No significant differences were found when considering the number of comparisons made, suggesting that the genetic structure of the Italian patients is not different from that of the Italian donor population.

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.

Human leukocyte antigen class II polymorphisms and genetic susceptibility of IDDM in Egyptian children.

OBJECTIVE: To analyze the association between human leukocyte antigen (HLA) and insulin-dependent diabetes mellitus (IDDM) in the Egyptian population for the first time and, thus, to determine the frequency of risk-associated alleles identified by a genomic HLA class II typing. Egyptians are genetically classified as North Africans and considered to be between Caucasoids and Africans (closer to Caucasoids). RESEARCH DESIGN AND METHODS: HLA class II typing was performed for 50 IDDM patients and 50 healthy control subjects by a restriction fragment-length polymorphism (RFLP) technique. The analysis of position 57 of the DQB1 molecules was conducted by polymerase chain reaction and specific sequence oligonucleotide hybridization. RESULTS: The frequency of DRB1*0301-DRB3*0201-DQA1*0501-DQB1*0201 haplotype was 43.9% in the IDDM patients and 7.1% in the control subjects (P < 0.00001), reflecting the increased prevalence of DQA1*0501 susceptibility allele coding for arginine (Arg) in position 52 and DQB1*0201 susceptibility allele non-coding aspartic acid (Asp) at position 57. Alleles DQB1*0601 and 0603, both carrying Asp at position 57 of the beta-chain, and DQA1*0103, encoding a non-Arg 52 alpha-chain, were significantly decreased among the IDDM patients. The presence of four susceptibility residues (two DQA1 Arg 52+ and two DQB1 Asp 57-) conferred the highest relative risk at 20.2. On the other hand, homozygous genotypes for DQA1 non-Arg 52 and DQB1 Asp 57 were found only in the control group. CONCLUSIONS: IDDM susceptibility and resistance in the Egyptian population is strongly associated with the expressed DQ alpha- and beta-heterodimers in a dose-effective manner, as already defined in many different ethnic groups.

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.