A multi-site study using high-resolution HLA genotyping by next generation sequencing.

The high degree of polymorphism at human leukocyte antigen (HLA) class I and class II loci makes high-resolution HLA typing challenging. Current typing methods, including Sanger sequencing, yield ambiguous typing results because of incomplete genomic coverage and inability to set phase for HLA allele determination. The 454 Life Sciences Genome Sequencer (GS FLX) next generation sequencing system coupled with conexio atf software can provide very high-resolution HLA genotyping. High-throughput genotyping can be achieved by use of primers with multiplex identifier (MID) tags to allow pooling of the amplicons generated from different individuals prior to sequencing. We have conducted a double-blind study in which eight laboratory sites performed amplicon sequencing using GS FLX standard chemistry and genotyped the same 20 samples for HLA-A, -B, -C, DPB1, DQA1, DQB1, DRB1, DRB3, DRB4, and DRB5 (DRB3/4/5) in a single sequencing run. The average sequence read length was 250 base pairs and the average number of sequence reads per amplicon was 672, providing confidence in the allele assignments. Of the 1280 genotypes considered, assignment was possible in 95% of the cases. Failure to assign genotypes was the result of researcher procedural error or the presence of a novel allele rather than a failure of sequencing technology. Concordance with known genotypes, in cases where assignment was possible, ranged from 95.3% to 99.4% for the eight sites, with overall concordance of 97.2%. We conclude that clonal pyrosequencing using the GS FLX platform and CONEXIO ATF software allows reliable identification of HLA genotypes at high resolution.

Pituitary control of growth in the neonatal rat: effects of neonatal hypophysectomy on somatic and organ growth, serum insulin-like growth factors (IGF)-I and -II levels, and expression of IGF binding proteins.

The neonatal period is a time of transition between pituitary-independent fetal growth and the pituitary-dependent growth seen in older mammals. To evaluate pituitary-dependent neonatal growth, Wistar rats were hypophysectomized (Hx) on postnatal day 6. Nineteen days post-Hx, body weight and tail length were inhibited 48% and 34%, respectively, compared with sham-Hx controls. Organ weights determined on days 10, 15, 20, 25, and 30 revealed three patterns of pituitary-dependence: 1) pituitary-independent growth in the brain and lung; 2) moderate pituitary-dependent growth in the heart, liver, kidney, and intestine; and 3) marked pituitary-dependent growth in the adrenals, spleen, and testes. Both serum insulin-like growth factor (IGF)-I and -II levels fell significantly in Hx pups by 54 h after Hx (P = 0.0005), and Northern analysis on day 15 showed a significant decrease in liver messenger RNA (mRNA) for IGF-II. Analysis of the major IGF binding proteins (BPs) was performed by Western ligand blots. Hx performed on day 6 resulted in a linear decrease in the amount of the 22k BP from day 10 to day 30. In contrast, the major neonatal BP (IGFBP-2, a 29.5k molecule) showed a biphasic response to neonatal Hx. On postnatal day 10, 4 days after Hx, a significant decrease in IGFBP-2 occurred, which persisted through day 15; by postnatal day 20 and continuing through postnatal day 30, the amount of IGFBP-2 in the serum dramatically increased. The 40 to 50k fraction of IGFBP-3 first appeared in significant quantities by postnatal day 20, and after Hx dropped to 10% of sham-control values. Similarly, Northern analysis on day 15 demonstrated a significant decrease in liver, but not brain, mRNA for IGFBP-2 after Hx, whereas on postnatal day 25, liver mRNA for IGFBP-2 was increased in Hx pups compared with sham controls. We conclude that the pituitary gland exerts significant but selective effects on neonatal growth, with the notable exception of brain growth. Serum levels of both IGF-I and IGF-II, as well as their BPs, are pituitary dependent in the neonatal period. Pituitary-dependent neonatal growth thus appears to be mediated by IGF and modulated by IGF-binding proteins. On the other hand, that portion of the persistent growth in the neonatal Hx rat that is independent of the pituitary-IGF axis may be a good model for investigation of fetal growth.