18 Abstracts

B-2.2

#23

HLA-C LOCUS POLYMORPHISM ANALYZED BY MOLECULAR APPROACH. M Falco,

L Delfino, A Longo, A Morabito, M Colonna and GB Ferrara, Istituto

Nazionale per la Ricerca sul Cancro, Genova, Centro Ricerche Im~un£

ematologiche AVIS, Bergamo, Italy and Harvard University, Cambridge,

MA, USA.

We analyzed the HLA-C locus polymorphism using polymerase chain

reaction and hybridization with sequence specific oligonucleotides

(PCR-SSOs). A total of 51 homozygous B-cell lines from the 10th

International Histocompatibility Workshop and 47 healthy unrelated

italian individuals were considered for the present study. The ser E

logical HLA Class I typing of these samples was available. PCR ampl!

fication of HLA-C was carried out using three primer pairs. Two sets

of primers were used to selectively amplify the first and the second

exon of this locus. A third primer combination amplify part of the

third exon of HLA-C alleles (with exception of HLA-C*0401 and BeWoCI)

and few HLA-B variants. This molecular approach provides a simple

and informative method for HLA-C analysis allowing the typing of all

the known serological specificities with almost all their subtypes.

In addition serological blanks alleles, previously defined by DNA

sequencing, are now easily detected. Our data reveal a new hybridiza

tion pattern that suggests the presence of a novel Cw7 allele (prov!

sionally called Cw7.3). This allele shares sequences, in the probed

regions, with HLA-C*0701 in the first and third exon, and with HLA-C

*0702 in the second one. The molecular typing is generally in agree

ment with the serological results.

B-2.2 #24

THE COMPLEXITY OF DEFINING CLASS I ALLELES IN AFRICAN AMERICANS. S G Rodriquez, ML landoli, AG Wagner, S Rosen-Bronson, AH Johnson and CK Hurley, Departments of Microbiology and Pediatrics, Georgetown University Medical School, Washington DC.

The lack of alloantisera to define specific antigens and di f f icul t ies in interpretation of typing results with current reagents remain as major limitations for transplantation matching in African Americans (AA). The B70 antigenic specificity is among the most common antigens present in AA; however, monospecific B70 typing sera are not available and sera which detect B70 and its serologic subtypes, B71 and B72, are crossreactive with antigens of the B3S, B15 and B5 CREGs. B71 and B72 di f fer in their serologic similarity to other antigens. For example, sera defining B71 mainly crossreact with the B35 antigen, while the sera defining B72 have a greater degree of crossreactivity with B62. To better define the alleles in the B70 family, we have sequenced a B71 allele from an AA individual. The sequence most closely resembles the sequence of B'1503, an allele encoding B72. This similarity extends to other B15 alleles encoding molecules typing as B62. The B71 sequence differs by at most 7-8 amino acids from these antigens differing only in the regions that encode for the peptide binding groove. In spite of the close serologic relationship, the B71 sequence differs more extensively from the B35 al le l ic products, and these 10-12 amino acid substitutions are located throughout the heavy chain. In addition, we are currently sequencing a BS3 allele, a member of the B5, B3S CREG with an unusual serologic pattern. These are examples of the complexity of HLA polymorphism among AA which l ikely result in inaccurate antigen assignments. This d i f f icu l ty in identifying HLA alleles may explain, in part, the high rate of graft loss and highly sensitized patients in this population.