ABSTRACT
To enable the efficient analysis of a highly polymorphic swine major histocompatibility complex (MHC) class II gene, swine leukocyte antigen (SLA)-DQB1, we developed a simple and comprehensive high-resolution genotyping protocol. To obtain sufficient sequence information to design a set of common genotyping primers for SLA-DQB1, we cloned SLA-DQB1 introns 1 and 2 from 11 alleles with official four-digit allelic designations and sequenced the regions directly surrounding the SLA-DQB1 exon 2. Significant intronic nucleotide variations, including several deletions, were identified. Based on 733-bp assembled genomic sequences including introns 1 and 2 and exon 2 from 11 different alleles, a primer set was identified that allowed the ubiquitous amplification and analysis of the complete SLA-DQB1 exon 2 sequence. We then developed a method to directly sequence the amplified polymerase chain reaction (PCR) products without further experimental steps. We especially focused on avoiding superimposed peaks, which arose from the presence of allelic deletions, in the sequencing electropherogram of SLA-DQB1 heterozygous animals. The genotyping accuracy was evaluated by comparing the results of genomic sequence-based typing (GSBT) with those of other available methods, including cDNA sequence-based typing (SBT), low-resolution PCR typing with sequence-specific primers, allelic segregation analysis, and heterozygote simulation typing. In all cases, the results were consistent between SLA-DQB1 GSBT and previously reported methods or expected results. We applied it to genotype 350 animals from seven pig breeds. The observed level of heterozygosity from our genotyping was â¼51%, reflecting that a large portion of the animals were inbred miniature pigs. Among the seven pig breeds tested, the allelic diversity of SLA-DQB1 was highest in Berkshire pigs. In conclusion, we have developed a simple and effective SLA-DQB1 GSBT method by combining simple genomic DNA PCR and direct sequencing. Our new method may aid in the study of SLA diversity and disease resistance and susceptibility.
Subject(s)
HLA-DQ Antigens/genetics , Histocompatibility Antigens Class II/genetics , Polymerase Chain Reaction/veterinary , Sequence Analysis, DNA/veterinary , Swine , Animals , Base Sequence , DNA Primers , Genetic Variation , Genotype , HLA-DQ beta-Chains , Histocompatibility Antigens Class I , Molecular Sequence Data , Reproducibility of Results , Sequence Alignment , Swine/geneticsABSTRACT
BACKGROUND: The treatment of corneal perforations due to corneal ulceration is a serious challenge in countries where there are no eye banks. The author presents an original surgical procedure for treating these cases: lamellar autografting. METHODS: The corneal transplant was taken at 12 h; size was 2 to 4 mm and thickness 2/3 of the stroma. Twelve patients were treated. RESULTS: The results are satisfactory and the grafting successful; no complication of significant importance has been noted. CONCLUSION: Lamellar autografting is an effective method for treating small corneal perforations. It can be temporary in case of central localization, or permanent in case of paracentral perforation.
