Subtyping for TNFa microsatellite sequence variation.
The microsatellite locus TNFa is frequently used as an additional genetic marker in studies of the major histocompatibility complex (MHC). Novel sequence variations at the TNFa locus have been described, and which may have implications for genetic analyses. In this study, we set up a nested polymerase chain reaction-sequence-specific primer (PCR-SSP) approach to type for these TNFa sequence variations. First, sequencing analysis of workshop B lymphoblastoid cell lines (n=13) showed the presence of three sequence variations upstream of the dinucleotide repeat at TNFa. Using nested PCR-SSP, we were able to detect these variations in a larger B lymphoblastoid cell line panel (n=34). Furthermore, we were able to show that TNFa alleles a7 and a10 are present in two distinct conformations leading to "splitting" of TNFa alleles exhibiting identical fragment lengths. To establish the frequency of the TNFa alleles and their variants, we performed microsatellite typing of a large panel of random individuals from the Dutch population (n=272). Subsequent nested PCR-SSP typing showed the presence of three previously described sequence variations in the Dutch population. Furthermore, the presence of a fourth subtype was established. The described variations of allele TNFa7 and TNFa10 are present in the random population with significant frequencies. Haplotyping analysis between HLA-DR, TNFa, and HLA-B showed that allele TNFa7.2 is present in an extended DR7-TNFa7.2-B13 haplotype. In this way, we were able to show that the additional sequence variations behave like distinct TNFa alleles.
van der Slik
A R
AR
Department of Immunohaematology and Blood Transfusion, Leiden University Medical Centre, The Netherlands.
Shing
D C
DC
Eerligh
P
P
Giphart
M J
MJ
D002460:Cell Line; D014644:Genetic Variation; D006239:Haplotypes; D006801:Humans; D018895:Microsatellite Repeats; D016133:Polymerase Chain Reaction / Q000379:methods; D014409:Tumor Necrosis Factor-alpha / Q000145:classification / Q000235:genetics*
D014409:Tumor Necrosis Factor-alpha