Evaluation of two methods for computational HLA haplotypes inference using a real dataset

<p>Abstract</p> <p>Background</p> <p>HLA haplotype analysis has been used in population genetics and in the investigation of disease-susceptibility locus, due to its high polymorphism. Several methods for inferring haplotype genotypic data have been proposed, but it is unclear how accurate each of the methods is or which method is superior. The accuracy of two of the leading methods of computational haplotype inference – Expectation-Maximization algorithm based (implemented in Arlequin V3.0) and Bayesian algorithm based (implemented in PHASE V2.1.1) – was compared using a set of 122 HLA haplotypes (A-B-Cw-DQB1-DRB1) determined through direct counting. The accuracy was measured with the Mean Squared Error (<it>MSE</it>), Similarity Index (<it>I</it>_<it>F</it>) and Haplotype Identification Index (<it>I</it>_<it>H</it>).</p> <p>Results</p> <p>None of the methods inferred all of the known haplotypes and some differences were observed in the accuracy of the two methods in terms of both haplotype determination and haplotype frequencies estimation. Working with haplotypes composed by low polymorphic sites, present in more than one individual, increased the confidence in the assignment of haplotypes and in the estimation of the haplotype frequencies generated by both programs.</p> <p>Conclusion</p> <p>The PHASE v2.1.1 implemented method had the best overall performance both in haplotype construction and frequency calculation, although the differences between the two methods were insubstantial. To our knowledge this was the first work aiming to test statistical methods using real haplotypic data from the HLA region.</p

Uusi DNA:n puhdistusmenetelmä

Nykyiset DNA:n eristämiseen ja puhdistamiseen käytetyt myrkyttömät proteiinien ulossuolausmenetelmät (protein-salting-out) ovat vielä melko aikaavieviä ja eräissä tilanteissa jopa epäluotettavia. Tämän työn tarkoituksena on kehittää luotettava ja entisiä menetelmiä nopeampi proteiinien ulossuolausmenetelmä kromosomaalisen DNA:n eristämiseksi viljellyistä ihmisen soluista (Jurkat, K562 ja U937). Tämän uuden proteiinien ulossuolausmenetelmän avulla voidaan 60 minuutissa eristää puhdasta ja suurimolekyylimassaista DNA:ta erilaisiin vaativiin sovelluksiin, kuten genomisten DNA kirjastojen valmistukseen ja niiden analyysiin. Tässä työssä kehitetty DNA:n eristysmenetelmä validoitiin tekemällä puhdistetusta DNA:sta geenikirjasto, josta vektoretti-PCR:ään perustuvalla kromosomikävelyllä eristettiin ihmisen kasvurajoitegeenin, tp53-geenin, promoottorialue, joka analysoitiin restriktioentsyymikartoituksella ja sekvennoinilla

Análisis de la variabilidad entre especies de camélidos sudamericanos utilizando genes relacionados a la inmunidad

Universidad Nacional Agraria La Molina. Facultad de Ciencias. Departamento Académico de BiologíaEl presente estudio evaluó la diversidad genética presente en genes relacionados a la inmunidad dentro de 3 especies de camélidos sudamericanos (Vicugna pacos, Lama glama y Vicugna vicugna) con el fin de evidenciar si las especies salvajes presentan una mayor variabilidad en comparación con sus parientes domesticados. Se obtuvo el ADN nuclear mediante la extracción de glóbulos blancos por la metodología Salting out (Aljanabi & Martinez, 1997) modificado por el laboratorio de ecología molecular y biodiversidad de la UNMSM. Posteriormente se amplificaron por PCR de punto final las regiones ricas en Leucinas de los genes Toll Like Receptor y los exones número 2 del MHC clase II mediante cebadores elaborados específicamente para camélidos. Los productos de la amplificación fueron secuenciados obteniéndose 49 secuencias para el gen TLR2, 54 para el gen TLR4 y 20 pertenecientes al gen DRB1 del MHC clase II para posteriormente realizar una estimación de los haplotipos parentales más probables (PHASE) y con ello poder hacer el análisis de la diversidad y diferenciación genética. Se obtuvieron un total de 40 haplotipos diferentes y 25 sitios polimórficos distribuidos entre los tres genes evaluados. El análisis evidencio una variabilidad ligeramente alta del gen TLR2 dentro de la población de vicuñas y una alta variabilidad del gen TLR4 en llamas. La diferenciación genética fue muy elevada entre llamas y vicuñas y moderada entre alpacas y vicuñas. Se concluye que las alpacas, llamas y vicuña presentan una similar variabilidad genética para las regiones LRR en los genes TLR2 y TLR4 y para el exón 2 del gen DRB1. Este el primer trabajo que analiza la diversidad de genes ligados a la inmunidad dentro de la población de camélidos sudamericanos.This study assessed the genetic diversity present in immunity-related genes within 3 South American camelids species (Vicugna pacos, Lama glama and Vicugna vicugna) to show whether wild species have greater variability compared to their domesticated relatives. Nuclear DNA was obtained by the extraction of white blood cells by Salting out (Aljanabi & Martinez, 1997) methodology modified by UNMSM's molecular ecology and biodiversity laboratory. Subsequently, the Leucine-rich regions of the Toll-Like Receptor genes and the number 2 exons of the MHC class II were amplified by end-point PCR by primers made specifically for camelids. The amplification products were sequenced obtaining 49 sequences for the TLR2 gene, 54 for the TLR4 gene, and 20 belonging to the DRB1 gene of the Class II MHC and subsequently perform an estimate of the most likely parental haplotypes (PHASE) and thus be able to analyze diversity and genetic differentiation. A total of 40 different haplotypes and 25 polymorphic sites were obtained distributed among the three genes evaluated. The analysis showed slightly high variability of the TLR2 gene within the vicuña population and high variability of the TLR4 gene on llamas. Genetic differentiation was remarkably high between llamas and vicuñas and moderate between alpacas and vicuñas. It is concluded that alpacas, llamas, and vicuña exhibit a similar genetic variability for the LRR regions in the TLR2 and TLR4 genes and for exon 2 of the DRB1 gene. This is the first work that analyses the diversity of immunity-linked genes within the South American camelid´s population

Noninherited maternal Human Leukocyte Antigens: donor availability and clinical outcome in unrelated cord blood transplantation

Foetal exposure to semi-allogeneic cells from maternal microchimaerism (MMc) has been associated with the development of tolerance towards noninherited maternal antigens (NIMA) through a regulatory T cell response. This concept can be exploited in the selection of permissible Human Leukocyte Antigen (HLA) mismatches in cord blood (CB) transplantation (CBT) due to the availability of maternal samples for HLA typing and the identification of NIMA. CB virtual phenotypes (VPs) were generated by the substitution of 1-3 CB HLA (HLA-A, -B and/or –DRB1) for the corresponding NIMA, to permit the identification of virtual full HLA matches (VFM: 5/6 + 1 NIMA, 4/6 + 2 NIMA or 3/6 + 3 NIMA) for 457 patients. Maternal HLA typing for 4,671 CB donors resulted in the generation of 66,225 VPs and 52,875 of these were unique. When combined with the inherited phenotypes of 21,020 CB donors, the total unique phenotypes to increased to 65,046. VFMs, with adequate cell dose, doubled the cumulative availability of a matched donor for European Caucasoid patients and tripled the availability for patients of other ethnicities. Analyses of NIMA matching and clinical outcomes were performed for 198 transplants but was statistically underpowered to detect an association. High levels of MMc have been associated with transplantation tolerance. HLA qPCR assays with 0.01% sensitivity were optimised. MMc was detected in 27% of 96 samples tested. MMc was more frequent in CB from earlier gestational time points and appeared to be associated with bi-directional maternal-foetal HLA compatibility. The incorporation of NIMA in CB donor selection therefore increases the donor pool available to patients, particularly ethnic minorities, requiring CBT