A new HLA-C*05 allele, HLA-C*05:156, characterized by full-length hemizygous sequencing.

HLA-C*05:156 allele differs from C*05:01:01:02 by a nucleotide change in exon 2 at codon 9.

Genetic risk for co-occurrence of type 1 diabetes and celiac disease is modified by HLA-C and killer immunoglobulin-like receptors.

The prevalence of celiac disease (CD) in patients with type 1 diabetes (T1D) has been reported to be 5-7 times higher than in the general population. Risk factors for co-occurrence of both diseases have not been entirely established. The aim of our study was to analyze possible impact of human leukocyte antigen (HLA) class I and killer cell immunoglobulin-like receptors (KIRs) on the co-occurrence of T1D and CD. We analyzed 67 patients with T1D, 68 patients with CD, 69 patients with both diseases (T1D+CD) and 130 controls. Statistical analysis was based on two tailed Fisher exact test with corrections for multiple testing. After stratification by DR3-DQ2, an association of HLA class I part of the COX haplotype (A1-B8-Cw7-DR3-DQ2) was not observed with each of the studied diseases separately, but it could be shown in case of the co-occurrence of T1D and CD. Only in the group of patients with coexisting diseases, the presence of HLA-C*07 (P = 8.65×10(-3) ) and HLA-B*08 (P = 0.03) but not HLA-A*01 increased the succeptibility. Our current data indicated that C*07, contributing C1 ligand (Pc = 3.67×10(-5) ) rather than B*08, that possesses no KIR ligand, could have an impact on the innate immunity rout of this susceptibility. The significant combination of C1-KIR2DL3 (Pc = 1.97×10(-4) ) observed in patients with coexisting diseases supports this hypotesis. Interestingly, no association was observed when C1 in combination with its stronger inhibitory receptor KIR2DL2 was investigated. Predominantly, weak inhibition in patients with coexisting T1D and CD could lead to a natural killer cell response, making them vulnerable for developing more than one autoimmune disease.

Resolution of HLA-B*44:02:01G, -DRB1*14:01:01G and -DQB1*03:01:01G reveals a high allelic variability among 12 European populations.

Within the framework of the EU-funded HLA-NET action, an analysis of three G-group alleles, HLA-B*44:02:01G, DRB1*14:01:01G and DQB1*03:01:01G, was undertaken in 12 European populations. Ambiguities were resolved by polymerase chain reaction-sequence-specific amplification (PCR-SSP) or PCR-sequence-based typing (PCR-SBT) in a total of 5095 individuals. The results of the DRB1*14:01/14:54 ambiguity showed high relative ratios (24-53%) of DRB1*14:01 in Bulgarians, Croatians, Greeks, Italians and Slovenians, contrasting with low ratios (6-13%) in Austrians, Finnish, French, Hungarians, Norwegians and Swiss. Resolution of the B*44:02/44:27 ambiguity showed that B*44:27 had a high relative ratio in Slovenians (25.5%) and Bulgarians (37%) and low in French and Swiss (0.02-1%), and was not observed in Greeks and Italians. The highest relative ratio of DQB1*03:19 was found in Portuguese (11%), by contrast with low ratios (0-3%) in the other five populations. Analysis of the A, B, DRB1 phenotypes and family-derived haplotypes in 1719 and 403 individuals positive for either HLA-B*44:02G or DRB1*14:01G ambiguities, respectively, showed some preferential associations, such as A*26∼DRB1*14:01, B*35∼DRB1*14:01, B*38∼DRB1*14:01 and B*44:27∼DRB1*16. Because these ambiguities are located outside the peptide-binding site, they may not be recognized by alloreactive T-cells. However, because of strong linkage disequilibrium (LD), the DRB1*14:01 vs DRB1*14:54 and the B*44:02 vs B*44:27 mismatches are associated to DRB3-, and C-mismatches, respectively. These results are informative for algorithms searching unrelated hematopoietic stem cell donors. For B*44:27-positive patients, searches are expected to be more successful when requesting donors from Southeastern-European ancestry. Furthermore, the introduction of human leukocyte antigen (HLA)-typing strategies that allow resolving exon 4 (for class I) and exon 3 (for class II) polymorphisms can be expected to contribute significantly to population genetics studies.

16(th) IHIW: analysis of HLA population data, with updated results for 1996 to 2012 workshop data (AHPD project report).

We present here the results of the Analysis of HLA Population Data (AHPD) project of the 16th International HLA and Immunogenetics Workshop (16IHIW) held in Liverpool in May-June 2012. Thanks to the collaboration of 25 laboratories from 18 different countries, HLA genotypic data for 59 new population samples (either well-defined populations or donor registry samples) were gathered and 55 were analysed statistically following HLA-NET recommendations. The new data included, among others, large sets of well-defined populations from north-east Europe and West Asia, as well as many donor registry data from European countries. The Gene[rate] computer tools were combined to create a Gene[rate] computer pipeline to automatically (i) estimate allele frequencies by an expectation-maximization algorithm accommodating ambiguities, (ii) estimate heterozygosity, (iii) test for Hardy-Weinberg equilibrium (HWE), (iv) test for selective neutrality, (v) generate frequency graphs and summary statistics for each sample at each locus and (vi) plot multidimensional scaling (MDS) analyses comparing the new samples with previous IHIW data. Intrapopulation analyses show that HWE is rarely rejected, while neutrality tests often indicate a significant excess of heterozygotes compared with neutral expectations. The comparison of the 16IHIW AHPD data with data collected during previous workshops (12th-15th) shows that geography is an excellent predictor of HLA genetic differentiations for HLA-A, -B and -DRB1 loci but not for HLA-DQ, whose patterns are probably more influenced by natural selection. In Europe, HLA genetic variation clearly follows a north to south-east axis despite a low level of differentiation between European, North African and West Asian populations. Pacific populations are genetically close to Austronesian-speaking South-East Asian and Taiwanese populations, in agreement with current theories on the peopling of Oceania. Thanks to this project, HLA genetic variation is more clearly defined worldwide and better interpreted in relation to human peopling history and HLA molecular evolution.

Strategies to work with HLA data in human populations for histocompatibility, clinical transplantation, epidemiology and population genetics: HLA-NET methodological recommendations.

HLA-NET (a European COST Action) aims at networking researchers working in bone marrow transplantation, epidemiology and population genetics to improve the molecular characterization of the HLA genetic diversity of human populations, with an expected strong impact on both public health and fundamental research. Such improvements involve finding consensual strategies to characterize human populations and samples and report HLA molecular typings and ambiguities; proposing user-friendly access to databases and computer tools and defining minimal requirements related to ethical aspects. The overall outcome is the provision of population genetic characterizations and comparisons in a standard way by all interested laboratories. This article reports the recommendations of four working groups (WG1-4) of the HLA-NET network at the mid-term of its activities. WG1 (Population definitions and sampling strategies for population genetics' analyses) recommends avoiding outdated racial classifications and population names (e.g. 'Caucasian') and using instead geographic and/or cultural (e.g. linguistic) criteria to describe human populations (e.g. 'pan-European'). A standard 'HLA-NET POPULATION DATA QUESTIONNAIRE' has been finalized and is available for the whole HLA community. WG2 (HLA typing standards for population genetics analyses) recommends retaining maximal information when reporting HLA typing results. Rather than using the National Marrow Donor Program coding system, all ambiguities should be provided by listing all allele pairs required to explain each genotype, according to the formats proposed in 'HLA-NET GUIDELINES FOR REPORTING HLA TYPINGS'. The group also suggests taking into account a preliminary list of alleles defined by polymorphisms outside the peptide-binding sites that may affect population genetic statistics because of significant frequencies. WG3 (Bioinformatic strategies for HLA population data storage and analysis) recommends the use of programs capable of dealing with ambiguous data, such as the 'gene[rate]' computer tools to estimate frequencies, test for Hardy-Weinberg equilibrium and selective neutrality on data containing any number and kind of ambiguities. WG4 (Ethical issues) proposes to adopt thorough general principles for any HLA population study to ensure that it conforms to (inter)national legislation or recommendations/guidelines. All HLA-NET guidelines and tools are available through its website http://hla-net.eu.

Pretransplant soluble CD30 serum concentration does not affect kidney graft outcomes 3 years after transplantation.

INTRODUCTION: An elevated serum concentration of soluble the form of CD30 (sCD30), an activation marker of mainly T(H)2-type cytokines producing T lymphocytes, has been reported as a predictive factor for acute cellular rejection episodes and poor graft outcomes in kidney transplantation. This historic cohort study investigated the association of a pretransplant sCD30 serum concentrations with kidney graft function and graft survival 3 years posttransplantation in adult recipients of deceased donor kidney grafts, treated with monoclonal anti-CD25 antibodies as an induction treatment combined with a cyclosporine (CsA)-based maintenance triple therapy. MATERIALS AND METHODS: The pretransplant sera of 296 recipients were tested for sCD30 content using a microsphere flow-cytometry assay. The estimated glomerular filtration rate (eGFR) was determined by the 4-variable Modification of Diet in Renal Disease equation. The incidences of graft loss were calculated with the use of Kaplan-Meier survival analysis and compared using the log-rank test. RESULTS: According to the distribution of the pretransplant sCD30 levels concentration ≥2700 pg/mL was defined as high (n = 146) and concentration <2700 pg/mL as low (n = 150). Three years posttransplantation, the eGFR was not significantly different in the recipients in high and low sCD30 groups (65 ± 24 vs 67 ± 21 mL/min/1.73 m(2); P = .43); there was no association between the eGFR 3 years after transplantation and the pretransplant sCD30 levels (r(2) = 0.002; P = .49). Graft survival 3 years after transplantation was also not different in the recipients in high and low sCD30 groups (P = .52). CONCLUSION: In our adult deceased-donor kidney graft recipients, the pretransplant sCD30 serum concentration was not a predictive factor of immunologic risk associated with the kidney graft function 3 years posttransplantation; neither did it affect graft survival 3 years after transplantation. The immunosuppression with anti-CD25 antibodies as an induction treatment combined with the CsA-based maintenance triple therapy could possibly be decisive for our findings.

An influence of HLA-A, B, DR, DQ, and MICA on the occurrence of Celiac disease in patients with type 1 diabetes.

Celiac disease (CD) is more common in individuals with insulin dependent diabetes mellitus (T1D) than in the general population. HLA class II molecules DQ8 (DQB1*0302-DQA1*0301) and DQ2 (DQB1*0201-DQA1*0501) have been identified as key genetic risk factors in both diseases. While DQ8 conveys a higher risk for T1D, DQ2 is more frequent in CD. Less is known about the contribution of HLA class I. The gut immune system has been implicated in the pathogenesis of both diseases. The MICA, which is mainly expressed in the gastrointestinal epithelium and recognized by gammadeltaT lymphocytes and natural killer (NK) cells via the NKG2D, might play a role. The aim of our study was to identify possible HLA class I and MICA alleles and conserved extended haplotypes as risk factors for the development of CD in T1D. Three groups consisting of 37 individuals with T1D and CD, 67 individuals with only T1D and 70 controls were analyzed. HLA class I and MICA alleles were determined using Luminex technology. An occurrence of CD in individuals with T1D was most significantly associated with B*08 (P = 7.3 x 10(-13)), contributing more than any of the HLA class II alleles (DRB1*0301, P = 5.00 x 10(-10); DQB1*0201, P = 7.65 x 10(-8)). Moreover, the association with CD became stronger when B*08(B*08-DQA*0501-DQB1*0201-DRB1*0301, P = 5.07 x 10(-12)) was present in the DRB1*0301-DQB1*0201-DQA1*0501 (P = 5.00 x 10(-10)) extended haplotype. We suggest a combined influence of alleles present in the MICA*008-B*08-A1-DR3-DQ2 extended haplotype on the development of CD in Slovenian individuals with T1D, where B*08 or/and a gene located close to it may play an important role, independently of HLA class II.

Association of IL6 and CCL2 gene polymorphisms with the outcome of allogeneic haematopoietic stem cell transplantation.

Various polymorphisms of non-HLA genes have recently been investigated as candidate risk factors in allogeneic haematopoietic SCT (aHSCT). Our study aimed at exploring possible associations of IL6 and CCL2 single nucleotide polymorphisms (SNP) with aHSCT outcome. A total of 166 HLA-identical aHSCT pairs recruited in were genotyped for IL6 -174 G/C, IL6 -597 G/A, CCL2 -2518 A/G and CCL2 -2076 A/T SNPs by PCR with sequence-specific primers (PCR-SSP). The association between IL6 -174 GG genotype and increased risk of acute GVHD was found in whole study group (P=0.03) and in the subgroup of related aHSCT (P=0.01), association between IL6 -597 GG genotype and the occurrence of acute GVHD was detected only in the related aHSCT pairs (P=0.02). Furthermore, reduction in OS was revealed among recipients possessing IL6 -174(*)G allele in the group of related aHSCT pairs (P=0.04). Presence of CCL2 -2076 TT genotype was associated with decrease of OS (P=0.04) and increase of TRM (P=0.02) in patients transplanted by related donor. These results, in the context of previous findings, suggest that IL6 gene polymorphisms may be associated with aHSCT outcome, particularly in patients transplanted from a related donor.

Does pretransplant soluble CD30 serum concentration affect deceased-donor kidney graft function 3 years after transplantation?

Elevated serum concentrations of soluble CD30 molecule (sCD30) have been related to acute cellular rejection and poor graft outcomes in kidney transplantation. This historical cohort study investigated the association of pretransplant sCD30 serum concentrations with kidney graft function expressed as estimated glomerular filtration rate (GFR) at 3 years after transplantation. Pretransplant sera from 176 adult deceased-donor kidney graft recipients were tested for sCD30 content using a commercially available automated enzyme-linked immunosorbent assay. The immunosuppression consisted of induction therapy with monoclonal anti-CD25 antibodies and a maintenance regimen of cyclosporine (CsA)-based therapy. GFR was estimated (eGFR) by the four-variable Modification of Diet in Renal Disease (MDRD) Study equation. According to the distribution of pretransplant sCD30 levels (median 66.7 U/mL; interquartile range, 46.6 to 98.6 U/mL), a concentration of 66 U/mL or higher was defined as high (n = 89) and below 66 U/mL as low (n = 87). Three years after transplantation, eGFR was not significantly different among recipients in high versus low sCD30 groups (69 +/- 23 mL/min/1.73m2 vs 66 +/- 21 mL/min/1.73m2; P = .327) and there was no correlation between eGFR and pretransplant sCD30 levels (r2 = 0.001; P = .73). Upon multivariate regression analysis, donor age, recipient body mass index at transplantation, and acute rejection episodes were independent variables affecting eGFR at 3 years after transplantation. This study showed that pretransplant sCD30 serum concentrations were not associated with deceased-donor kidney graft function at 3 years after transplantation. The immunosuppression with anti-CD25 antibodies and a triple CsA-based maintenance regimen could possibly be decisive for our findings.

A novel homozygous frameshift deletion c.471del of HFE associated with hemochromatosis.

A 47-year-old white male patient who manifested biochemical evidence of iron overload was found not to be a carrier of the three most common mutations, C282Y, H63D and S65C, of the HFE gene. Sequencing of the patient's entire HFE-coding region revealed a presence of a previously undescribed frameshift deletion c.471del in exon 3 resulting in a premature termination of a nonsense HFE protein. Interestingly, the patient was a homozygous carrier of this novel mutation and his hemochromatosis phenotype can be explained by the fact that he has no intact HFE protein. To the best of our knowledge, this is the first description of a complete loss of function of the HFE gene because of a homozygous mutation. The patient's son was found to be a heterozygous carrier of the mutation and has so far exhibited no indications of iron overload. Similarly, A*02-B*40/A*02-B*40 homozygous human leukocyte antigen (HLA) genotype was determined in the patient and heterozygous A*02-B*40/A*03-B*35 HLA genotype in his son. Thus, the novel HFE frameshift deletion c.471del was linked to the HLA-A*02-B*40 haplotype.

A novel HLA-B*2730 allele found in a Slovene patient affected with IgA nephropathy.

A novel allele HLA-B*2730 and the haplotype HLA-A*03-B*2730-Cw*02-DRB1*16-DRB5 have been found in a Slovene patient and his mother. The exon 2 sequence is identical to that of HLA-B*2702, and the exon 3 sequence is identical to that of HLA-B*2719. A possible mechanism for the generation of B*2730 is a gene conversion event.

Genetic risk factors associated with Creutzfeld-Jakob disease in Slovenians and a rapid typing for PRNP codon 129 single nucleotide polymorphism.

PRNP has been the most informative marker for the predisposition to variant Creutzfeld-Jakob disease (vCJD). All victims of the vCJD carried methionine (M) at the position 129 of the PrP. Prions could travel through the immune system to get from the gut to the brain, and human leucocyte antigens (HLAs) could be involved in this carriage, with HLA-DQ7 being less efficient. Contradictory reports have raised the question of the influence of sampling in population studies. We developed a fast and reliable real-time polymerase chain reaction for codon 129 single nucleotide polymorphism (SNP) using TaqMan technology, which overcomes the main drawbacks of other methods and analysed Slovenian population (n = 97). The comparison with other populations served for the estimation of the genetic risk for the development of vCJD in Slovenians. The frequencies at the codon 129 SNP in the Slovenian population were 43.3% M, 45.4% M/V 11.3% V. Considerable differences between the DQ7 frequencies in diverse samples from the same population can be seen, especially when compared to Slovenian population. This could be because of the diverse criteria for including subjects into the study and the sampling of geographically distinct subpopulations. Analysing the adequacy of HLA-DQ7 as a possible predictive factor for developing Creutzfeld-Jakob disease (CJD) by case - control studies could be improved with exact and equal sampling of groups of patients and controls. CJD genetic risk factors in the Slovenians were not found significantly different than those in British.

The HLA-DRB, -DQB polymorphism and anti-insulin antibody response in Slovenian patients with type 1 diabetes.

A combination of specific HLA class II antigens and the presence of type 1 diabetes (T1D)-related antibodies has a high positive predictive value for T1D but low sensitivity. The aim of the present study was to determine the frequencies of HLA-DRB-DQB deduced haplotypes associated with susceptibility and protection in Slovenian patients with established T1D, to evaluate the relationship between the HLA-DRB1-QBP-DQB1 haplotypes and the presence of insulin autoantibodies (IAA) and glutamic acid decarboxylase antibodies (GADA), and to access the possible impact of polymorphic QBP promoters on this relationship. A cohort of 135 patients with T1D (age 17.5 +/- 7.0 years, duration of T1D 9.14 +/- 6.3 years) was investigated. HLA-DRB1 and DQB1 alleles were typed using the polymerase chain reaction (PCR)-reverse line blot method. QBP promoter region alleles were determined using PCR-sequence-specific oligonucleotide hybridization (SSO) and PCR-sequence-specific primers (SSP). IAA and GADA antibodies were determined by enzyme-linked immunosorbent assay (ELISA). The chi-square test with Yates' correction was used for statistical analysis. Deduced haplotypes DRB1*0301-DQB1*0201 (P = 0.0001, OR = 3.4), DRB1*0401-DQB1*0302 (P = 0.0001, OR = 29.8), and DRB1*0402-DQB1*0302 (P = 0.008, OR = 4.7) were significantly more common, and DRB1*1501-DQB1*0602 (P = 0.0001, OR = 0.03) significantly less common in the investigated cohort than in a Slovenian control group. The highest risk and the strongest protective HLA-DR-DQ haplotypes found in Slovenian patients with T1D did not differ from those found in other Caucasian populations. While the DRB1*0301-QBP2.1-DQB1*0201 haplotype, where QBP2.1 did not help to further distinguish DQB1*0201-possessing haplotypes in IAA-positive and IAA-negative patients, was strongly associated with the presence of IAA, the DRB1*0101-QBP5.12-DQB1*0501 haplotype, although not protective compared to the control population, was associated with an absence of IAA in the investigated cohort. It is suggested that there may be a combined influence of the QBP5.12 promoter and the DQB1*0501 functional molecule on reduced IAA production.

The contribution of HLA-DQB1 coding and QBP promoter alleles to anti-Ro alone autoantibody response in systemic lupus erythematosus.

OBJECTIVE: To analyse the influence of HLA-DR, DQ and corresponding DQA1 and DQB1 promoter alleles (QAP and QBP) on the anti-Ro alone autoantibody response in systemic lupus erythematosus (SLE). METHODS: Sixty-five unrelated anti-La antibody-negative SLE patients, 37 of them with and 28 without anti-Ro antibodies, were included. Anti-Ro antibodies were determined by both counter-immunoelectrophoresis and enzyme-linked immunosorbent assay. Seventy-four healthy individuals were selected as controls. The patients and controls were analysed for HLA-DRB1, QAP, DQA1, QBP and DQB1 alleles by DNA typing. The allelic frequencies of anti-Ro alone-positive and anti-Ro-negative SLE patients and healthy controls were compared using the chi(2) test or Fisher's exact test as appropriate. RESULTS: The DQB1*0202 allele showed a significant positive correlation with anti-Ro alone antibodies [odds ratio (OR)=16.949, P=0.0015, corrected P=0.018], while the QBP5.11 allele and the combination of DQB1*0301 and its promoter QBP3.1 were under-represented in anti-Ro-alone-positive SLE patients (P=0.01, corrected P=0.048 and corrected P=0.048 respectively). CONCLUSIONS: The above-mentioned alleles may contribute to the presence or absence of anti-Ro alone autoantibodies in SLE patients.

The contribution of HLA-Class II antigens in humoral non-response and delayed response to HBsAG vaccination.

The variability in the immune response modulated by HLA alleles may be an important factor for the induction of the protective effect of HBsAg vaccines. We present here the analysis of HLA-DRB1, DQB1 and DQA1 alleles and their combinations in the group of 36 individuals with poor humoral immmune response to HBsAg vaccination. Comparison with the control group, consisted of 60 randomly choosen healthy subjects, revealed that the DRB1*1601, DQB1*0502, DQA1*0102 haplotype is overrepresented in the group of hyporesponders and may therefore be regarded as a factor influencing poor antibody responsiveness. We observed that after revaccination two of three individuals who failed to develop anti-HBs antibodies carry the same phenotype DRB1*0101,DRB1*0301;DQB1*0501,DQB1*0201;DQA1+ ++*0101,DQA1*0501, which supports the conjecture that immunogenicity of the HBsAg vaccine depends on specific combination of HLA DR and DQ molecules on antigen presenting cells.

Possible influence of genes located on chromosome 6 within or near to the major histocompatibility complex on development of essential hypertension.

Studies both in spontaneously hypertensive rats and in humans have suggested that genes within or near to the HLA complex on chromosome 6p may be associated and linked to the regulation of blood pressure. The aim of this study was to determine whether HLA alleles and their combinations contribute to increased blood pressure, as well as to identify chromosome region that may contain genes involved in the pathogenesis of essential hypertension. Our results suggest that presence of HLA-DRB1*0101/2 DQB1*0501/2 DQA1*0102 allelic combination represents risk factor for development of essential hypertension in Slovenians, while the risk is decreased in individuals possessing HLA-DRB1*1601/2 DQB1*0502 DQA1*0102 or DRB3*. The linkage study indicates a possibility that at least one of the genes responsible for increased blood pressure is located near or within the HLA complex. A possible candidate is human endotelin-1 gene encoding a highly potent vasoactive peptide.

Possible influence of genes located on chromosome 6 within or near to the major histocompatibility complex on development of essential hypertension.

Studies both in spontaneously hypertensive rats and in humans have suggested that genes within or near to the HLA complex on chromosome 6p may be associated and linked to the regulation of blood pressure. The aim of this study was to determine whether HLA alleles and their combinations contribute to increased blood pressure, as well as to identify chromosome region that may contain genes involved in the pathogenesis of essential hypertension. Our results suggest that presence of HLA-DRB1*0101/2 DQB1*0501/2 DQA1*0102 allelic combination represents risk factor for development of essential hypertension in Slovenians, while the risk is decreased in individuals possessing HLA-DRB1*1601/2 DQB1*0502 DQA1*0102 or DRB3*. The linkage study indicates a possibility that at least one of the genes responsible for increased blood pressure is located near or within the HLA complex. A possible candidate is human endotelin-1 gene encoding a highly potent vasoactive peptide.

Adrenal 21-hydroxylase gene mutations in Slovenian hyperandrogenic women: evaluation of corticotrophin stimulation and HLA polymorphisms in screening for carrier status.

OBJECTIVE: To study the incidence of 21-hydroxylase deficiency in Slovenian hyperandrogenic women, at the gene level. Previous endocrine studies indicated large differences in the incidence of 21-hydroxylase deficiency in hyperandrogenic women. The predictive values of the 17-hydroxyprogesterone (17-OHP) response to ACTH stimulation and of HLA typing in screening for carrier status were re-evaluated. DESIGN: Molecular analysis of CYP21 gene, ACTH stimulation and human leucocyte antigen (HLA) typing were performed in 83 consecutive Slovenian hyperandrogenic women. MEASUREMENTS: Cortisol and 17-OHP concentrations were measured at baseline and 60 min after ACTH stimulation. Basal adrenal androgen concentrations were also measured. RESULTS: None of 83 hyperandrogenic patients was affected with non-classical 21-hydroxylase deficiency, but 12 of 81 patients (14.8%) had high concentrations of 17-OHP after stimulation, indicative of carrier status. The increase in 17-OHP concentrations could be explained by a carrier status for CYP21 gene mutations in only three of 12 patients (25%), whereas seven of 69 patients (10. 1%) with normal concentrations of 17-OHP after stimulation were found to be carriers of CYP21 gene mutations, indicating low positive predictive values of ACTH stimulation as a screening test for carriers of 21-hydroxylase deficiency. In total, 11 carriers were identified among 83 patients: seven CYP21 gene deletions/conversions, two Gln(318)Stop and one Val(281)Leu mutation and one gene conversion extending from exon 4 to exon 7 were found. The association between Val(281)Leu mutation and HLA-B14 antigen was confirmed in this Slovenian population. CONCLUSIONS: Basal or ACTH-stimulated 17-OHP concentrations are not a good indicator of the carrier status for 21-hydroxylase deficiency among Slovenian hyperandrogenic patients. Reliable screening for carriers of 21-hydroxylase deficiency is possible only by molecular analysis of the CYP21 gene.