A case of maternal-foetal chimerism identified during routine histocompatibility testing for hematopoietic stem cell transplantation.

This report describes a case of maternal-foetal chimerism identified in a boy diagnosed with SCID, who underwent HLA testing in preparation for HSCT. The first analysis was carried out on DNA from peripheral blood and included HLA-A, HLA-B, HLA-DRB1 typing using PCR-SSO. The patient's HLA-B typing results were noninterpretable. All samples were re-typed for HLA-B using PCR-SSP, again resulting in noninterpretable typing of patient's HLA-B. In both cases, several weak positive probes/reactions interfered with the interpretation when using commercial software. Next round of HLA typing, using PCR-SSP and PCR-SSO methods, included the patient's bone marrow sample and HLA-C locus, but interpretation was again not possible. The PCR-STR analysis performed on both peripheral blood and bone marrow samples revealed seven STRs for which two maternal and one paternal allele were detected. Retrospective manual interpretation of HLA-B and HLA-C typing revealed that weak positive reactions were indeed owed to paternal HLA-B and HLA-C alleles and that the patient had both maternal and one paternal allele. Retyping of HLA-B and HLA-C loci and STR analysis on the patient's buccal cells sample revealed the expected one maternal/one paternal allele pattern. In summary, the combination of several different typing methods and manual interpretation were necessary to obtain the patient's HLA typing results.

The possible role of the tumour necrosis factor polymorphisms and human leucocyte antigens in the development of prostate cancer.

The cause of prostate cancer (PC), one of the most common cancers found among ageing men, remains unclear, but genetic predisposition is believed to play a major role in its aetiology. The aim of the study was to examine HLA genes polymorphism and TNF polymorphisms in PC development. Patients diagnosed with PC (N = 113) and 150 healthy individuals were tested for HLA-A, HLA-B and HLA-DRB1 genes and for TNFa, TNFb and TNFd microsatellites. The comparison of patients and controls revealed a positive association of HLA-DRB1*12, TNFa2 and TNFb5, and a negative association of HLA-DRB1*13 and TNFb4 with PC. A division of patients into groups according to age, pre-operative PSA level, Gleason score (GS) and involvement of prostatic capsule, seminal vesicles or bladder neck and perineural invasion of PC demonstrated the following: a positive correlation of HLA-DRB1*12 and a negative correlation of HLA-DRB1*13 with younger patients (<65 years), GS > 7 and the positive association of prostatic capsule, seminal vesicles, bladder neck and perineural invasion of PC; TNFb4 allele's negative association with older patients displaying higher PSA levels, higher GS and positive surrounding tissue involvement; positive association of TNFb5 allele for both older and younger patients. Investigation of HLA genes and TNF microsatellites demonstrated a possible role of HLA-DRB1 and TNF regions in PC aetiology.

Nonfrequent but well-documented, rare and very rare HLA alleles observed in the Croatian population.

The aim of the study was to evaluate the presence of nonfrequent, rare and very rare alleles among Croats and to estimate whether they are associated with specific alleles at other human leukocyte antigen (HLA) loci. This retrospective study included the typing results from the last 10 years; total number of individuals included was approximately 45,000. Among 17 alleles so far observed only once in our population, 6 (A*24:41, B*07:02:28, B*35:03:03, B*39:40N, DRB1*13:23 and DRB1*14:111) belong to very rare alleles, 2 (B*44:16 and DRB1*01:31) belong to rare alleles according to the 'Rare Alleles Detector' tool ( www.allelefrequencies.net), while for the B*35:101:01 allele published data exist only in the IMGT/HLA database. The remaining eight HLA alleles observed only once among Croats are considered as frequent according to the 'Rare Alleles Detector'. Those 17 HLA alleles are not declared as common well defined (CWD) alleles in the CWD allele catalogue 2.0.0. Haplotype analysis of nonfrequent alleles detected in our sample supports the idea that different populations, although similar in some aspects regarding HLA allele and haplotype distribution, still have some unique characteristics. This is the case for A*01:02, B*39:10 and DRB1*13:32 which form haplotypes unreported to date among our subjects.

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.

HLA-A, HLA-B and HLA-DRB1 allele and haplotype diversity among volunteer bone marrow donors from Croatia.

The determination of human leucocyte antigen (HLA)-A, HLA-B and HLA-DRB1 alleles in the routine procedure of a volunteer hematopoietic stem cell (HSC) donor's registration in the Croatian Bone Marrow Donor Registry (CBMDR) is performed to enhance the odds of finding a suitable HLA compatible donor for patients in need of a HSC transplantation worldwide. However, besides its original purpose, it also provides valuable information about the HLA polymorphism among Croats. The aim of the present study was to analyse the HLA allele and haplotype frequencies in a sample of 4000 donors from CBMDR. The distribution of HLA-A, HLA-B and HLA-DRB1 alleles did not demonstrate significant differences from the data reported for other European populations. The higher frequency of B*40:02 allele in comparison with B*40:01 and DRB1*11:04 in comparison with DRB1*11:01 is interesting because it represents a difference in comparison with the Western and Northern European populations which are a main source of donors for Croatian patients. The haplotype frequencies show a greater variation and difference in comparison with data from other registries and populations; however, due to a lack of high-resolution haplotype data, comparison was possible only with a very limited number of other populations.

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.

Heterogeneity of HLA-DRB1*04 alleles and haplotypes in the Croatian population.

Analysis of allele distribution at the HLA-DRB1*04 gene, as one of the frequent ones among Croatians, and their HLA-A-B-DRB1 haplotypes in the Croatian population was performed in this study. Using LABType® SSO and PCR-SSP method, 11 DRB1*04 subtypes were observed, of which DRB1*04:01 was the most frequent (28.0%) followed by DRB1*04:02 (26.3%), DRB1*04:03 (22.3%), and DRB1*04:04 (14.2%). The significant haplotypes (with highest P value) for given DRB1*04 allele were the following combinations: HLA-B*15:01-DRB1*04:01, HLA-B*38:01-DRB1*04:02, HLA-B*35:03-DRB1*04:03, HLA-B*35:03-DRB1*04:08, HLA-B*14:01-DRB1*04:04, and HLA-B*49-DRB1*04:05. Marked differences in the distribution of our most frequent haplotypes of HLA-B-DRB1*04 (HLA-B*38:01-DRB1*04:02 and HLA-B*15:01-DRB1*04:01) were found in comparison to other European populations investigated so far. Additionally, comparison of HLA-A-B-DRB1*04 haplotypes showed that although there are similarities in the haplotype structure between our and other populations, there are also noteworthy differences. In summary, the identification of conserved and unusual DRB1*04 haplotypes in the present study of Croats should have important clinical implications for donor-recipient matching in the hematopoietic stem cell transplantation program, help in the understanding of HLA polymorphisms in different European populations, and also prove to be very useful in the determination of possible susceptibility genes involved in HLA-DRB1*04-associated diseases.

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.

The investigation of HLA microsatellites influence in predisposition to sarcoidosis among Croatians.

The aim of the present study was to analyze the distribution of HLA alleles (A, B, DRB1, DQB1) and HLA microsatellite alleles (TNFa, TNFb, TNFd, D6S273, D6S1014) in the Croatian patients with acute (N=93), as well as chronic sarcoidosis (N=40), in comparison to healthy controls (N=177), and investigate whether the polymorphism within the HLA region could be associated with different forms of sarcoidosis. Genomic DNA was isolated from peripheral blood. Patients were analyzed for HLA class I loci (A, B) by serology, while PCR-SSP method was used for HLA class II loci (DRB1, DQB1). Five HLA microsatellites were analyzed by PCR and electrophoresis in an automated sequencer. No significant deviation in the distribution of frequencies at HLA class I alleles was observed between the two patients' subgroups and controls. Regarding the HLA class II alleles, a statistically significant increase in frequency of HLA-DRB1*03 and DQB1*0201 allele was found among patients with acute sarcoidosis in comparison to controls as well as in comparison to patients with chronic sarcoidosis. The same finding was observed for HLA-DRB1*03/DQB1*0201 haplotype (Pcorr=0.0168; OR=2.83). In the group of patients with chronic sarcoidosis DRB1*11 (P=0.0219; OR=2.44), DRB1*15 (P=0.0414; OR=2.47) demonstrated statistically significant difference in comparison to controls only, while a lower frequency of DRB1*13 (P=0.0156; OR=0.24) in this group was statistically significant when compared to both patients with acute sarcoidosis and controls. None of the alleles at TNFa microsatellite showed significant difference in distribution among both subgroups of patients and controls. Significant difference between patients with acute form of disease and controls was found for the following alleles: TNFd-2 (Pcorr=0.00007; OR=4.89), D6S273-7 (Pcorr=0.0213; OR=2.96), and D6S1014-7 (Pcorr=0.0028; OR=3.97). On the other hand, patients with chronic sarcoidosis differed from control subjects for D6S1014-8 (Pcorr=0.0296; OR=8.35) allele. This study suggests the existence of an association of non-HLA markers with sarcoidosis and the involvement of the region between HLA-DQB1 and D6S273 loci in its pathophysiology.

High resolution definition of HLA-DRB haplotypes by a simplified microsatellite typing technique.

In humans, the region configurations DR1, DR8, DR51, DR52 and DR53 are known to display copy number as well as allelic variation, rendering high resolution typing of HLA-DRB haplotypes cumbersome. Advantage was taken of microsatellite D6S2878, present in all DRB genes/pseudogenes with an intact exon 2-intron 2 segment. This DRB-STR is highly polymorphic in composition and length. Recently, it was proven that all exon 2 sequences could be linked to a certain DRB-STR that segregates with the respective DRB allele. Because haplotypes show differential copy numbers and compositions of exon 2-positive DRB genes/pseudogenes, unique DRB-STR patterns could be described that appear to be specific for a particular DRB haplotype. The aim of this workshop project was to approve and to qualify this simple typing protocol in a larger panel covering different European populations. All participants succeeded in correctly defining the DRB-STR amplicons varying from 135 to 222 base pair (bp) lengths. The panel of 101 samples covered 50 DRB alleles distributed over 37 different haplotypes as defined by exon 2 sequence-based typing. These haplotypes could be refined into 105 haplotypes by DRB-STR typing. Thus, discrimination of exon 2-identical DRB alleles was feasible, as well as the exact description of three different crossing-over events that resulted in the generation of hybrid DR region configurations. This typing procedure appears to be a quick and highly robust technique that can easily be performed by different laboratories, even without experience in microsatellite typing; thus, it is suitable for a variety of researchers in diverse research areas.

Myoblast fusion and innervation with rat motor nerve alter distribution of acetylcholinesterase and its mRNA in cultures of human muscle.

To elucidate the mechanisms underlying acetylcholinesterase (AChE) localization, we analyzed the distribution of AChE and Ache mRNA during myogenesis in cocultures of human muscle and fetal rat spinal cord. We observed a temporal coincidence in alterations of AChE localization and nuclei expressing the message, suggesting developmental regulation at the mRNA level. Nonuniform mRNA staining among nuclei suggests asynchronous regulation, also supporting an earlier proposal that transcription proceeds intermittently. Asynchrony seems to be overridden by generally acting factors during myoblast fusion, when message is up-regulated, and at the onset of muscle contractions, when it becomes restricted to some nuclei in the junctional region and focal patches of AChE appear near nerve contacts. Coincidence of mRNA down-regulation and synthesis of stable basal lamina-bound AChE suggests coordinated adaptation, so that sufficient enzyme may be derived from low message levels.

HLA-B*27/HLA-B*07 in combination with D6S273-134 allele is associated with increased susceptibility to juvenile spondyloarthropathies.

OBJECTIVE: Juvenile spondylarthropathies (jSpA) are polygenic and the clustering of disease in families is caused mainly by genetic factors. Our aim was to look for possible associations of other HLA-A and B specificities, MICA and D6S273 microsatellite polymorphisms that might play a role in determining the susceptibility to jSpA. PATIENTS AND METHODS: jSpA were diagnosed in 74 Croatian children, and 169 healthy unrelated individuals served as the control group. HLA class I (A, B) typing of all individuals was performed, and HLA-B7 and HLA-B27 positive subjects were subtyped by PCR-SSP method. MICA and D6S273 microsatellites alleles were analyzed by electrophoresis in an automated sequencer. RESULTS: We identified 26 HLA-B*07 and 31 HLA-B*27 positive patients with jSpA. DNA subtyping of HLA-B*27 specificity demonstrated only two subtypes, B*2702 (19.35%) and B*2705 (80.65%), among jSpA patients. Subtyping analysis of HLA-B*07 gene showed presence of only one subtype, B*0702. The OR for HLA-B*07 was 2.61, while the highest OR for a single HLA specificity was found for HLA-B*27 (OR=5.60). The HLA-B*07/B*27 combination found in six children showed higher risk (OR=14.82), but the combination of specificities: HLA-B*07/HLA-B*27, and D6S273-134 allele demonstrated the highest risk (OR=26.83). The association with D6S273-134 allele was not a result of the linkage disequilibrium with HLA-B*27 specificity (LD=-0.5). CONCLUSION: Our findings provide evidence that HLA-B*27/HLA-B*07 in combination with D6S273-134 allele is associated with increased susceptibility to jSpA in Croatian children.

Identification of the novel HLA-B*18:37:02 allele in a Croatian individual.

The new allele HLA-B*18:37:02 differs from HLA-B*18:37:01 by one nucleotide substitutions in exon 2.

The distribution of the DRB4*01:03:01:02N null allele in HLA-DRB1~DQB1 haplotypes in the Croatian population.

The aim of the present study was to investigate frequency and haplotype distribution of DRB4 alleles in the Croatian population. The investigated sample consisted of 288 cadaveric donor samples positive for one of the DR53 alleles. HLA-A, -B, -C, -DRB1, and -DQB1 typing was performed using the polymerase chain reaction-sequence specific primers (PCR-SSP) low resolution method, while HLA-DRB4 and selected HLA class II specificities typing was performed using PCR-SSP at the allelic level. Three different DRB4 alleles were observed among DRB1*04 samples; DRB4*01:02 (2.38%), DRB4*01:03 (91.27%), and DRB4*01:03:01:02N (6.35%). The DRB4*01:03:01:02N allele was predominantly observed among DRB1*04:02-positive samples, while DRB4*01:02 and DRB4*01:03 alleles did not associate preferably with any of the DRB1*04 subtypes. Among DRB1*04~DRB4~DQB1 haplotypes, the predominant DQB1 allele was DQB1*03:02 (69.94%). Seven different DRB4 alleles were found among DRB1*07:01-positive samples. The analysis of DRB1*07~DRB4~DQB1 haplotypes showed that DRB4*01:03 was found in the majority of HLA-DRB1*07:01~DQB1*02:02 (49.09%) haplotypes while DRB1*07:01~DQB1*03:03 haplotypes carried the DRB4*01:03:01:02N allele almost exclusively (98.21%). Among six DRB1*09:01-positive samples, HLA-DRB1*09:01~DRB4*01:03~DQB1*03:03 was the only detected haplotype. The extended haplotype analysis showed a high frequency of HLA-B*15(B62)~C*03(Cw9)~DRB1*04:02~DRB4*01:03:01:02N~DQB1*03:02 and HLA-B*57~C*06~DRB1*07:01~DRB4*01:03:01:02N~DQB1*03:03 haplotypes. In conclusion, the data presented in this study should prompt other population studies focused on DRB3/4/5 genes and be used as a basis for future investigations of the clinical relevance of these genes in transplantation setting.

The distribution of HLA-DRB3 alleles among HLA-DRB1*03:01-positive haplotypes.

HLA-DRB3 allelic polymorphism on HLA-DRB1*03:01-positive haplotypes was investigated among 104 cadaveric donors typed for HLA-A, -B, -DRB1, and -DRB3. Only HLA-DRB3*01:01:02 and -DRB3*02:02:01:01 alleles were detected among HLA-DRB1*03:01-positive individuals and their distribution depended on HLA-B*08 presence: nearly all HLA-B*08-positive samples carried DRB3*01:01:02, while HLA-DRB3*02:02:01:01 was more frequent among HLA-B*08-negative subjects.

Decreased peak bone mass is associated with a 3-bp deletion/insertion of the CYP19 intron 4 polymorphism: preliminary data from the GOOS study.

Finding that estrogen plays an important role in bone homeostasis in men prompted research on relationship of polymorphism at the CYP19 gene and the bone mass. Therefore, influence of 3-bp deletion/insertion polymorphism of CYP19 (TTTA)7 allele on the peak bone mass attainment in males was studied. Fifty-eight unrelated male participants, aged 21-35, were selected depending on the presence of (TTTA)7 (no.=19) or (TTTA)7-3 (no.=39) alleles from the initial cohort of 92 young males. Heterozygotes (TTTA)7/(TTTA)7-3 (no.=13) were not included in the analysis. Serum levels of estradiol, free testosterone, 25-hydroxyvitamin D, bone alkaline phosphatase, osteocalcin, and beta-crosslaps were measured. Bone mass was measured by DXA at the hip and at the spine. (TTTA)7-3 allele was associated with significantly lower femoral neck bone mineral density (BMD) (p=0.02). Logistic regression model indicated strong association of (TTTA)7-3 allele with low BMD in the range of osteopenia/osteoporosis (p=0.014, odds ratio 12.36, confidence intervals 1.65-92.46). In the present study association of 3-bp deletion polymorphism of the (TTTA)7 allele with decreased peak bone mass in males is reported for the first time. However, further studies are necessary to elucidate the functional relevance of this polymorphism.

Common and well-documented HLA alleles over all of Europe and within European sub-regions: A catalogue from the European Federation for Immunogenetics.

BACKGROUND: A catalogue of common and well-documented (CWD) human leukocyte antigen (HLA), previously established by the American Society for Histocompatibility and Immunogenetics (ASHI), is widely used as indicator for typing ambiguities to be resolved in tissue transplantation or for checking the universality of any HLA allele in the world. However, European population samples, which are characterized by a substantial level of genetic variation, are underrepresented in the ASHI catalogue. Therefore, the Population Genetics Working Group of the European Federation for Immunogenetics (EFI) has facilitated data collection for an European CWD catalogue. MATERIALS AND METHODS: To this end, 2nd-field HLA-A, -B, -C,- DRB1,- DQA1,- DQB1 and -DPB1 data of 77 to 121 European population samples (21 571-3 966 984 individuals) from 3 large databases, HLA-net/Gene[VA], allelefrequencies.net and DKMS, were analysed. RESULTS: The total number of CWD alleles is similar in the EFI (N = 1048) and ASHI (N = 1031) catalogues, but the former counts less common (N = 236 vs 377) and more well-documented (N = 812 vs 654) alleles than the latter, possibly reflecting differences in sample numbers and sizes. Interestingly, approximately half of the CWD alleles reported by EFI were not reported by ASHI and vice-versa, underlining the distinct features of the two catalogues. Also, although 78 common alleles are widely distributed across Europe, some alleles are only common within specific sub-regions, showing regional variability. CONCLUSION: Although the definition of CWD alleles itself is affected by different parameters, calling for current updates of the list, the EFI CWD catalogue provides new insights into European population genetics and will be a very useful tool for tissue-typing laboratories in and beyond Europe.

Identification of the novel HLA-A*01:200 allele by sequence-based typing in a Croatian individual.

The new allele HLA-A*01:200 differs from A*01:12 by four nucleotide substitutions in exon 3.

Association of HLA alleles and haplotypes with CYP21A2 gene p. V282L mutation in the Croatian population.

The CYP21A2 mutations that are in linkage disequilibrium with particular HLA-A, -B, -DRB1 alleles/haplotypes, cause deficiency of the 21-hydroxylase enzyme (21-OHD) and account for the majority of congenital adrenal hyperplasia (CAH) cases. The aim of this study was to investigate those associations with the p.V282L mutation linked to the non-classical (NC) form of CAH among Croatians. The study included parents of patients with the NC form of CAH, positive for the p.V282L mutation (N = 55) and cadaveric donor samples (N = 231). All subjects were HLA-A, -B, and -DRB1 typed and tested for the presence of the p.V282L mutation. Among parents of patients, 92.73% of subjects were positive for the B*14:02 allele and almost half of them carried the HLA-A*33:01-B*14:02-DRB1*01:02 haplotype. Among cadaveric samples 77 out of 96 subjects positive for the B*14:02 allele had the p.V282L mutation. Among them, 37 were positive for the HLA-A*33:01-B*14:02-DRB1*01:02 haplotype, 23 had the HLA-A*33:01-B*14:02-DRB1*03:01 haplotype, 8 had the B*14:02-DRB1*01:02 combination and 5 were carrying the HLA-A*68:02-B*14:02-DRB1*13:03 haplotype. Only 4 of these subjects were positive for the B*14:02 allele. HLA-B*14:02 was the only single allele with association that reached statistically significant P value (RR = 12.00; P = 0.0024). Haplotypes B*14:02-DRB1*01:02 (P < 0.001) and HLA-A*68:02-B*14:02-DRB1*13:03 (P < 0.001) as well as HLA-A*33:01-B*14:02-DRB1*01:02 and HLA-A*33:01-B*14:02-DRB1*03:01 showed high relative risks (RR = 45.00, RR = 41.63 and RR = 36.96, respectively). Our data support the previously documented association of the HLA-A*33:01-B*14:02-DRB1*01:02 haplotype with the p.V282L mutation, but also point out a high frequency of the p.V282L mutation among Croatians with HLA-A*33:01-B*14:02-DRB1*03:01 and HLA-A*68:02-B*14:02-DRB1*13:03 haplotypes.

Electron paramagnetic resonance reveals altered topography of the active center gorge of acetylcholinesterase after binding of fasciculin to the peripheral site.

Fasciculin, a peptidic toxin from snake venom, inhibits mammalian and fish acetylcholinesterases (AChE) by binding to the peripheral site of the enzyme. This site is located at the rim of a narrow, deep gorge which leads to the active center triad, located at its base. The proposed mechanisms for AChE inhibition by fasciculin include allosteric events resulting in altered conformation of the AChE active center gorge. However, a fasciculin-induced altered topography of the active center gorge has not been directly demonstrated. Using electron paramagnetic resonance with the spin-labeled organophosphate 1-oxyl-2,2,6, 6-tetramethyl-4-piperidinylethylphosphorofluoridate (EtOSL) specifically bound to the catalytic serine of mouse AChE (mAChE), we show that bound fasciculin on mAChE slows down, but does not prevent phosphorylation of the active site serine by EtOSL and protects the gorge conformation against thermal denaturation. Most importantly, a restricted freedom of motion of the spin label bound to the fasciculin-associated mAChE, compared to mAChE, is evidenced. Molecular models of mAChE and fasciculin-associated mAChE with tethered EtOSL enantiomers indicate that this restricted motion is due to greater proximity of the S-EtOSL nitroxide radical to the W86 residue in the fasciculin-associated enzyme. Our results demonstrate a topographical alteration indicative of a restricted conformation of the active center gorge of mAChE with bound fasciculin at its rim.