Dataset Information

Genotyping: the HLA system and embryo development.

ABSTRACT: The human major histocompatibility complex (MHC), in addition to its role in the regulation of cell-cell interactions in the immune response, also influences reproductive success. Human leukocyte antigen-G (HLA-G) is an MHC class I gene of particular interest in reproductive biology because of its specific expression on fetal cytotrophoblast cells, and its reported involvement both in protection of the developing fetus from destruction by the maternal immune response and in the prevention of maternal pre-eclampsia. HLA-G has 15 known alleles at the DNA level, and allelic frequency varies among ethnic groups. This study describes the results of an inaugural attempt to correlate an HLA-G genetic polymorphism with pregnancy outcome in a patient population undergoing IVF. The study group was composed of 102 Caucasian women. A maternal HLA-G genetic polymorphism was investigated by polymerase chain reaction (PCR) analysis of DNA collected from granulosa cells surrounding oocytes harvested for the IVF procedure. While no statistically significant correlation was identified in this initial study, larger studies examining DNA from trios of mother, father and offspring are planned.

SUBMITTER: Warner CM

PROVIDER: S-EPMC2557436 | biostudies-literature | 2002 Mar-Apr

REPOSITORIES: biostudies-literature

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Genotyping: the HLA system and embryo development.

Warner Carol M CM Tyas David A DA Goldstein Carmit C Comiskey Martina M Cohen Jacques J Brenner Carol A CA

Reproductive biomedicine online 20020301 2

The human major histocompatibility complex (MHC), in addition to its role in the regulation of cell-cell interactions in the immune response, also influences reproductive success. Human leukocyte antigen-G (HLA-G) is an MHC class I gene of particular interest in reproductive biology because of its specific expression on fetal cytotrophoblast cells, and its reported involvement both in protection of the developing fetus from destruction by the maternal immune response and in the prevention of mat ...[more]

PMID: 12470575

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Project description:Although human leukocyte antigen (HLA) DQ and DR loci appear to confer the strongest genetic risk for type 1 diabetes, more detailed information is required for other loci within the HLA region to understand causality and stratify additional risk factors. The Type 1 Diabetes Genetics Consortium (T1DGC) study design included high-resolution genotyping of HLA-A, B, C, DRB1, DQ, and DP loci in all affected sibling pair and trio families, and cases and controls, recruited from four networks worldwide, for analysis with clinical phenotypes and immunological markers.In this article, we present the operational strategy of training, classification, reporting, and quality control of HLA genotyping in four laboratories on three continents over nearly 5 years.Methods to standardize HLA genotyping at eight loci included: central training and initial certification testing; the use of uniform reagents, protocols, instrumentation, and software versions; an automated data transfer; and the use of standardized nomenclature and allele databases. We implemented a rigorous and consistent quality control process, reinforced by repeated workshops, yearly meetings, and telephone conferences.A total of 15,246 samples have been HLA genotyped at eight loci to four-digit resolution; an additional 6797 samples have been HLA genotyped at two loci. The genotyping repeat rate decreased significantly over time, with an estimated unresolved Mendelian inconsistency rate of 0.21%. Annual quality control exercises tested 2192 genotypes (4384 alleles) and achieved 99.82% intra-laboratory and 99.68% inter-laboratory concordances.The chosen genotyping platform was unable to distinguish many allele combinations, which would require further multiple stepwise testing to resolve. For these combinations, a standard allele assignment was agreed upon, allowing further analysis if required.High-resolution HLA genotyping can be performed in multiple laboratories using standard equipment, reagents, protocols, software, and communication to produce consistent and reproducible data with minimal systematic error. Many of the strategies used in this study are generally applicable to other large multi-center studies.

Project description:Genotyping of the human leucocyte antigen (HLA) is indispensable for various medical treatments. However, unambiguous genotyping is technically challenging due to high polymorphism of the corresponding genomic region. Next-generation sequencing is changing the landscape of genotyping. In addition to high throughput of data, its additional advantage is that DNA templates are derived from single molecules, which is a strong merit for the phasing problem. Although most currently developed technologies use genomic DNA, use of cDNA could enable genotyping with reduced costs in data production and analysis. We thus developed an HLA genotyping system based on next-generation sequencing of cDNA.Each HLA gene was divided into 3 or 4 target regions subjected to PCR amplification and subsequent sequencing with Ion Torrent PGM. The sequence data were then subjected to an automated analysis. The principle of the analysis was to construct candidate sequences generated from all possible combinations of variable bases and arrange them in decreasing order of the number of reads. Upon collecting candidate sequences from all target regions, 2 haplotypes were usually assigned. Cases not assigned 2 haplotypes were forwarded to 4 additional processes: selection of candidate sequences applying more stringent criteria, removal of artificial haplotypes, selection of candidate sequences with a relaxed threshold for sequence matching, and countermeasure for incomplete sequences in the HLA database.The genotyping system was evaluated using 30 samples; the overall accuracy was 97.0% at the field 3 level and 98.3% at the G group level. With one sample, genotyping of DPB1 was not completed due to short read size. We then developed a method for complete sequencing of individual molecules of the DPB1 gene, using the molecular barcode technology.The performance of the automatic genotyping system was comparable to that of systems developed in previous studies. Thus, next-generation sequencing of cDNA is a viable option for HLA genotyping.

Dataset Information

Genotyping: the HLA system and embryo development.

Publications

Genotyping: the HLA system and embryo development.

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