Performance of LIFECODES HLA-DQB1 Typing Kit Using Luminex Platform in Koreans

Intermediate-resolution HLA-DQ typing has gained importance in organ transplantation recently. We evaluated the performance of the LIFECODES HLA-DQB1 typing kit (Immucor, USA) using sequence-specific oligonucleotide (SSO) probe and Luminex platform (Luminex Corp., USA) on 100 samples tested by sequence-based typing (SBT) using the AlleleSEQR HLA-DQB1 kit (Abbott Molecular, USA) in Korean individuals. No sample showed ambiguity in the assignment of 4-digit HLA-DQB1 allele with the LIFECODES HLA-DQB1 SSO typing kit, and the results were fully concordant with those of high-resolution typing of AlleleSEQR HLA-DQB1 SBT up to 4-digit level. Three samples required adjustment of false reactions (3/100, 3.0%): two samples with DQB1*03:03/*06:01 showed false-positive result in probe 253, and 1 sample with DQB1*04:02/*05:02 showed false-negative result in probe 217. We tested an additional sample with DQB1*03:03/*06:01, which showed same false-positivity in probe 253 and 2 samples with DQB1*04:02/*05:02, which showed no false reaction. The false reactions did not result in ambiguity or change in the HLA allele assignment. We could assign HLA-DQB1 alleles to 4 digit-level without ambiguity, with 100% concordance with the SBT results. Thus, LIFECODES HLA-DQB1 SSO typing kit showed good performance for intermediate-resolution HLA-DQB1 typing in clinical laboratory for organ transplantation in Koreans.

Study on the relationships between the HPV infection and race susceptibility in the carcinogenesis of Xinjiang Uighur women with cervical cancer / 中华微生物学和免疫学杂志

Objective To investigate the relationships between the HPV infection and race susceptibility in the carcinogenesis of Uighur women with cervical cancer from the southern Xinjiang, one of the high risk region of cervical cancer in China. Methods To detect 21 subtypes of HPV and the 13 alleles of HLA from 200 cervical cancer cases and 200 normal tissues, by using flow-through hybridization and gene chip (HybriMax) method and polymerase chain reaction sequence-specific oligonucleotide method (PCRSSO). Results ( 1 )The proportion of HPV positive in cervical cancer and control group were 88.5% and 7.0% respecfively;HPV16 was the most common type in HPV positive cervical cancer patients with the rate of 90.96%, following were HPV18 (5.08%), HPV68(3.95% ),HPV45 (3.39%), HPV58 (3.39%),HPV39( 1.69% ), HPV31 ( 1.69% ), HPV56( 1.13% ). In cervical cancer and control group, the positive rate of HPV and HPV16 were significantly higher than that in control group. (2) In cervical cancer group the frequency of HLA-DRB1 * 15 in HPV positive cervical cancer cases was significantly higher than that among the HPV negative cases. While the frequency of HLA-DRB1 * 12 in HPV positive eervical caneer eases was significantly lower than that in the HPV negative e asea. Conclusion HPV16 was the most common type in both cervical cancer and control groups, the frequency of HPV16 in cervical carcinomas was very high, following HPV18 and HPV68, and HPV68 ranked third which was different from the results of other reports,this indicates that Uighur women are infected with this type more common. It appears that HLA-DRB1 * 15may be related to the susceptibility to cervical cancer and the HPV16 infection among the Uighur women,while the HLA-DRB1 * 12 the protective gene to HPV16 infection in Uighur women. The study of HLA alleles in the carcinogenesis of cervical carcinomas may play an important role in the gene intervention research of cervical cancer.

Otimização de metodologia para o estudo de genes KIR / Methodology optimization for KIR genotyping

Receptores killer cell immunoglobulin-like (KIRs) são moléculas localizadas na superfície de células natural killer (NK) e em subpopulações de linfócitos T codificadas por genes do cromossomo 19q13.4. A interação entre receptores KIR e moléculas antígeno leucocitário humano (HLA) de classe I determina se células NK exercerão ou não sua função citotóxica e/ou secretora de citocinas ou se esta será inibida. Este trabalho teve por finalidade otimizar a metodologia para a genotipagem KIR, baseando-se nas condições descritas por Martin (2004). A técnica utilizada foi a reação em cadeia da polimerase com primers de sequência específica (PCR-SSP) com iniciadores sintetizados pela Invitrogen® e visualização do produto amplificado em gel de agarose a 2 por cento com brometo de etídio. Adaptações foram realizadas e a concentração de alguns reagentes foi alterada, como a do controle interno de 100 nM para 150 nM, iniciadores específicos senso e antissenso de KIR12.5/12.3, KIR13.5/13.3, KIR14.5/14.3, KIR22.5/22.3 e KIR36.5/36.3 de 500 nM para 750 nM e da solução de MgCl2 de 1,5 mM para 2 mM. As concentrações dos demais reagentes e temperaturas de amplificação foram mantidas. Nessas condições, o uso da Taq DNA polimerase recombinante (Invitrogen®) foi satisfatório. Os resultados das genotipagens de 70 indivíduos foram confirmados por rSSO-Luminex® (One Lambda, Canoga Park, CA, EUA). A tipagem de genes KIR por essa técnica apresentou sensibilidade, especificidade, reprodutibilidade e baixo custo.

The killer cell immunoglobulin-like receptors (KIRs) are molecules expressed on natural killer (NK) cells surface and in T-cell subsets encoded by genes located in chromosome 19q13.4. The interaction between KIR receptors and HLA class I molecules determines if the NK cells will fulfill their cytotoxic function and/or cytokine secretion or if this function will be inhibited. The objective of this work was to optimize KIR genotyping method described by Martin (2004). It was used PCR-SSP (polymerase chain reaction-sequence-specific primers) with primers synthesized by Invitrogen® and visualization of the amplified products on 2 percent agarose gel electrophoresis, containing ethidium bromide. Some adaptations were made and the reagents had their concentrations increased: the internal control from 100 nM to 150 nM, forward and reverse specific primers KIR12.5/12.3, KIR13.5/13.3, KIR14.5/14.3, KIR22.5/22.3 and KIR36.5/36.3 from 500 nM to 750 nM, and MgCl2 solution from 1.5 mM to 2 mM. Other reagent concentrations and amplification temperatures were maintained. Satisfactory results were obtained with Taq DNA Polymerase Recombinant (Invitrogen®). The results of seventy samples were confirmed by rSSO-Luminex® (One Lambda, Canoga Park, CA, USA). This KIR typing method proved to be accurate, specific, reproducible and cost effective.

The correlation between HLA-A allele and anaphylactoid purpura in children of Mongolia / 中国基层医药

ObjectiveTo explore the association between HLA -A gene and anaphylactoid purpura(AP) in children of Mongolia in Inner Mongolia. To find correlated genes and study part of pathogenesis and the method of prevention and cure of AP. MethodsThe method of case control was adopted and selected 56 children with AP as case group and 66 health children as control group in Mongolia,who had resided in Inner Mongolia three generations without consanguinity, history of mixed, marriages, other medical history , and family history of immunity,led into polymerase chain reaction sequence specific oligonucleotide probes technique, analyzed the type of HLA-A gene. The compare of gene frenquency made with logistic regression after χ2 or Fisher test. ResultsThe gene frenquency of HLA- A * 11 ( 16. 1% ) allele in case group compared to that of control group( 9. 1% ) ,Wald of HLA-A * 11 gene was 3. 954 ,P =0. 047, the difference had statistical significance. B = 0. 844 > 0, OR = 2. 325 > 1, it helped development of the disease,which 95％confident interval was 1. 012-5.340,which did not include 1 ,EF =0. 342 >0. ConclusionHLA-A * 11 allele may be the susceptible gene of AP in children of Mongolia in Inner Mongolia.

HLA class-Ⅰ antigens in Gansu Chinese Han nationality with leukemia / 中国免疫学杂志

Objective:Host genetic factors are known to contribute to disease susceptibility.They may also be important in defining the pattern of disease presentation and progression,as well as its overall prognosis.However,no consistent HLA class-Ⅰ associations have been established in leukemia by PCR/SSOP in Gansu Chinese Han.Such studies have been reported in other counties,with conflicting results.This is the first PCR-based HLA class-Ⅰ association study in northwestern Chinese Han nationality leukemia.Methods:Compared HLA class-Ⅰ in 43 Chinese leukemia patients and 66 healthy Chinese controls as determined by polymerase chain reaction and sequence-specific olignucletide probe hybridization(PCR/SSO) DNA analysis.The present findings imply that HLA-associated genetic factors influence the risk for the development of leukemia.

Analysis of Ambiguities of HLA-B Typing using the Dynal RELI(TM) SSO HLA-B Test and Development of an`Interpretation Program for Koreans' / 대한진단검사의학회지

BACKGROUND: In recent years, DNA typing has been increasingly used in HLA-A and B typing, and commercial kits based on the PCR-SSO method are most commonly used in Korea. However, SSO typing kits show ambiguities to some extent in the generic level typing of HLA-B alleles. We analyzed the ambiguities in the Dynal RELI(TM) SSO HLA-B test (Dynal B test) with confirmatory typing results, and developed and evaluated the accuracy and efficacy of an `Interpretation Program for Koreans'. METHODS: A total of 2, 169 Korean marrow donor registry samples were typed for HLA-B alleles using the Dynal B test (56 probes) and all of the 222 cases showing ambiguities were subjected to confirmatory typing. We have developed an `Interpretation Program for Koreans' for the Dynal B test on the basis of the allele frequencies of Korean, Japanese and Asian populations. The samples showing ambiguities in the Dynal B test were interpreted using the `Interpretation Program for Koreans' and the results were compared with confirmatory typing results. RESULTS: The Dynal B test showed 10.2% (222/2, 169) of ambiguities and these ambiguities were classified into 47 different band patterns. These ambiguity patterns were interpreted using the `Interpretation Program for Koreans', which showed ambiguities in 14 band patterns and 3.4% (73/2, 169) of the total samples. Among these ambiguities, 4 band patterns (55 samples) arose from those alleles which are not found in Koreans and rarely found in Japanese or Asians (B*1522, *3521, *7802). Thus, excluding these rarities, only less than 1% (18/2, 169) of samples resulted in ambiguities, and most (16/18) of these were B55 vs. B56 ambiguities. The results from the `Interpretation Program for Koreans' were fully concordant with the confirmatory typing results. CONCLUSIONS: The Dynal B test showed around 10% ambiguities and the `Interpretation Program for Koreans' showed 3.4% of ambiguities. Excluding the ambiguities with extremely low probabilities arising from rare alleles in Japanese or Asians, actually >S99% of the samples could be typed accurately using the program without additional confirmatory tests.

HLA-A, -B, -DR Allele Frequencies and Haplotypic Associations in Koreans Defined by Generic-Level DNA Typing / 대한진단검사의학회지

BACKGROUND: HLA allele and haplotype distribution varies widely among different ethnic groups. For organ transplantation, anthropology and disease association studies, reliable data on the HLA distribution in each ethnic group is needed. In recent years, more accurate DNA typing methods are increasingly used in place of the serologic typing method. METHODS: We examined HLA-A, -B, and -DR alleles at the generic (serologic) level in 1, 600 Koreans registered for the Korea Marrow Donor Program (KMDP) using the PCR-sequence specific oligonucleotide (SSO) method (Dynal RELI(TM) kit). Allele and haplotype frequencies were estimated by the maximum likelihood method using the computer program developed for the 11th International Histocompatibility Workshop. RESULTS: HLA alleles found in Koreans were 13 in A, 31 in B, and 13 in DR locus. Most frequent alleles with frequencies > or =10% were: A2, A24, A33, A11; B62, B44, B51; DR4, DR15, DR13, and DR8 in each locus in decreasing order of frequency. Subtype frequencies of B61 and B75 serologic specificities were identified: B*4002 (51.1%), *4003 (7.6%) and *4006 (41.3%) for B61, and B*1502 (9.5%) and *1511 (90.5%) for B75. Two-locus haplotypes with frequencies> or =0.1% were presented (99 A-B, 115 B-DR), among which those with frequencies> or =1.0% showing significant positive linkage disequilibrium (P or =0.1% were identified in Koreans, among which 38 haplotypes showed frequencies> or =0.5%. We compared the results of this study with those of our previous study of serologically typed HLA-A, -B and DNA typed HLA-DR in 2, 000 Koreans. Results from the two studies were similar, but blank frequencies were decreased to 0% for HLA-A, -B, and -DR locus compared with the frequencies of 0.3-0.8% in the previous study (A, 0.3%; B, 0.8%; DR, 0.3%) and all of the serologic splits could be assigned in this study. CONCLUSIONS: In this study, we provided the allele and haplotype frequencies of HLA-A, -B, and -DR in Koreans defined by a DNA typing method, which can be used as basic data on Koreans for organ transplantation and disease association studies.

Analysis of Ambiguities of HLA-DR Typing using the Dynal RELI(TM) SSO HLA-DRB Kit and Development of an 'Interpretation Program for Koreans' / 대한진단검사의학회지

BACKGROUND: HLA-DR typing kits using reverse-SSO (sequence specific oligonucleotide) method show considerable ambiguities in HLA-DRB1 generic typing. We analyzed the ambiguities of the Dynal RELI(TM) SSO HLA-DRB test (Dynal DRB test) and developed an 'Interpretation Program for Koreans'. METHODS: A total of 3,000 Koreans were typed for HLA-DRB1/B3/B4/B5 using the 36 probe Dynal DRB test and all of the cases showing ambiguities in HLA-DRB1 generic typing were subjected to confirmatory typing using the PCR-single strand conformation polymorphism (SSCP) method. On the basis of these results, an 'Interpretation Program for Koreans'was developed for the 45 probe Dynal DRB test. RESULTS: Among 3,000 Koreans tested by the 36 probe Dynal DRB test, 456 cases (15.2%) showed ambiguities. In 95% of the ambiguity cases (433/456) and 99.2% of the total cases tested (433/3,000), the'most probable type'could be expected from the DRB1 gene frequencies and DRB1-B3/B4/B5 associations in Koreans and these results were in accordance with the confirmatory typing results as well as the results given by the 'Interpretation Program for Koreans'. Similarly, the 'Most Probable'could be assigned by the program in 99.4% (348/350) of the cases tested with the 45 probe Dynal DRB test. CONCLUSIONS: Ambiguity in the Dynal DRB test was observed in >15% of the Korean samples tested. The majority (95%) of the ambiguities could be resolved on the basis of HLA-DRB1 gene frequencies and DRB1-B3/B4/B5 associations in Koreans. Furthermore, using the program developed in this study, the correct assignment of DRB1 generic types was possible without additional typing in the majority (>99%) of the cases tested.

Geostatistics as a tool to improve sampling and statistical analysis in wetlands: a case sudy on dynamics of organic matter distribution in the Pantanal of Mato Grosso, Brazil

The Pantanal of Mato Grosso presents distinct landscape units: permanently, occasionally and periodically flooded areas. In the last ones, sampling is especially difficult due to the high heterogeneity occurring inter and intrastratas. This paper presents a comparison of different methodological approaches showing that they can influence decisively the knowledge of distribution organic matter dynamics. In such an area in order to understand the role of the flood pulse in the distribution dynamics of organic matter in a wetland at the Pantanal, we considered that there is spatial dependence between points. This consideration contradicts the classical statistic principle that focuses on the aleatority, and allowed the obtainment of a larger volume of information from a minor sampling effort, which means better performance, with time and money economy.

O Pantanal de Mato Grosso apresenta unidades distintas em sua paisagem: áreas permanentemente alagadas, áreas eventualmente alagáveis e áreas periodicamente alagáveis. Nestas últimas, as amostragens são particularmente difíceis, dada a heterogeneidade não apenas entre os estratos a serem amostrados como também dentro dos mesmos. Este trabalho usa como exemplo a avaliação do papel do pulso de inundação na dinâmica de distribuição de matéria orgânica em um campo inundável pantaneiro para mostrar que diferentes enfoques metodológicos e delineamentos amostrais podem influenciar decisivamente. Partimos do princípio de que há dependência espacial entre os pontos, contrariando a estatística clássica que enfoca a aleatoriedade. Este procedimento permite a obtenção de maior volume de informações a partir de menor esforço amostral, o que significa melhor desempenho, com economia de tempo e recursos.

Identification and sequence analysis of a novel allele HLA-B~* 9537 / 中国输血杂志

Tin Exon3,resulting in 4 amino acid changed from Glu to Asp(E103D),Thr to Lys(T113K),Gln to Glu(Q114E) and Ser to Phe(S116F),respectively.Conclusion The novel allelewas identified,and was assigned the name B*9537 officially by the WHO Nomenclature Committee.

HLA-DR genotyping and clinical application using Microarray DNA Chip / 中国免疫学杂志

Objective:To establish a new method performed on an DNA chip for genotyping HLA DR and supply a new view.Methods:According to the particular sequence of HLA DR exon2,HLA DR genotyping Chip was made,then the labeled PCR products hybridized with them,the signals were sanned by sanner and analyzed by Imagene software.70 standard DNA and 200 donor recipients have been genotyped and some of samples have been sequenced.Results:The experimental results showed that the HLA DR genotyping chip made are accurate and sensitive.Thirty alleles of HLA DR were accurately distinguished and its overall time of DNA typing was 3 hours.Conclusion:This proved that the DNA Microarray technique is good for DR genotyping and high resolution,high specificity,well reproducibility.Compared with PCR SSP and PCR SSO methods,the genotyping chip method is more intuitionistic and has the advantage of integration.It can also genotype HLA A,B alleles and many persons in a chip at the same time.It is more suitable for clinical application and establishment of marrow bank and umbilical cord bank.

Expression of Skin Sulfhydryl Oxidase in Normal Skin , Cultured Skin and Several Abnormally Keratinizing Dermatoses / 대한피부과학회지

BACKGROUND: Skin sulfhydryl oxidase(SSO) is the enzyme catalyzing the covalent crosslinking of the protein molecules via disulfide bond formation, which is probably responsible for the formation of tight and stable structures in the skin tissues, However, the lack of its gene cloning and antibody preparation limited the study for the role of the enzyme in the tissues. OBJECTIVE: It is required to study the role of the enzyme in the normal skin tissues and several dermatosis with abnormal keratinization and its regulation by several materials and drugs in the culture models. METHODS: We performed immunohistochemical analysis to determine the localization and expression of the enzyme in the normal skin and mucosa and hair tissues and in the skin explant cultures and artificial skins with irritant- and differentiation modifieradded conditions. RESULTS: In the skin, SSO was most strongly expressed in the granular layers and mostweakly in the basal layers. The similar but weaker expression pattern was observed in the mucosa with stronger expression in the upper flattened layers compared to the lower layers. In the hair, it was expressed in the inner and outer root sheaths at the isthmus portion in weaker stainable patterns. The strong expression of SSO was not observed beneath the Monro microabscess or parakeratosis in the involved aren of psoriasis. In actinic keratosis and Bowen disease, it was expressed more strongly in the dyskeratotic cells or parakeratotic areas. And in the horn pearls of the squamous cell carcinoma, the strong expression of SSO was observed. In the skin ezplant culture, the expression of SSO is induced by treatment with sodium lauryl sulfate or retinoic acid with more extended stained areas compared to the control. In contrast, its expression is not basically modified on the cellular level but inhibited dynamically by a decrease of the granular layers with retinoic acid in the artificial skin(raft culture). CONCLUSION: And the inducibility of the enzyme by the irritating agents in the skin organ explant culture suggests the role of the enzyme as one of skin defense system. The decrease of granular layers by retinoic acid in the artificial skin(raft culture) reflects the fact that its expression can be inhibited indirectly during the keratinization process. Therefore, it can be summarized that SSO is the enzyme involved in the keratinizing process of skin tissues as well as the protective function for the skin tissues.

Identification of a novel HLA class Ⅰ allele,B~*1316 / 中国输血杂志

Objective To analyze the nucleotide sequences of novel HLA class I le,B*1316.Methods Routine sequence-specific oligonucleotide(SSO) typing and sequencing based typing(SBT) was used.Results The B*1316 allele differs from B*1302 by one nucleotide substitution in exon 3: T to A at nt position 184,which results in an amino acid substitution at codon 62 from Val to Glu.Conclusion A novel HLA class I allele,B*1316 has been identified,and was officially recognized by WHO Nomenclature Committee in April 2006.

Correlation between gene HLA-class I polymorphism and susceptibility to leukemia / 西安交通大学学报(医学版)

Objective To explore the correlation between gene HLA-class I polymorphism and susceptibility to leukemia in Chinese Gansu Han people and search for the genes susceptible to leukemia.Methods HLA-A and B alleles polymorphism in 65 patients with leukemia and 48 normal subjects were determined by PCR with sequence specific oligucleotide probe(PCR-SSO).Results The allele frequencies of HLA-A01 and B38 were increased (P