Ben Carritt

Serotype Switching in a Partially Deleted RHD Gene

We have studied the RH genes in donors with the RhD‐negative haplotype dCes. In contrast to the usual arrangement of genes in RhD‐negative individuals, where the lack of antigen expression is due to deletion of the entire RHD gene, we find that the dCeshaplotype includes an RHD gene with an internal deletion. Moreover, there appear to be no 5′ sequences characteristic of RHC suggesting that the RhC antigen may be encoded by a truncated RHD or a recombinant RHD/CE gene in these dCes/dce genomes.

DNA‐Based Rhesus Typing: Simultaneous Determination of RHC and RHD Status Using the Polymerase Chain Reaction

We describe a PCR‐based method of performing RHD and C/c typing in a single reaction. The method is based on an earlier observation of a polymorphism in intron 2 of both genes which, in addition to detecting the RHD deletion responsible for most known D‐negative phenotypes, is also associated with C/c serological type. Using this assay, we typed 105 unrelated individuals from at least four different population groups and compared the results to those obtained using conventional serological testing of red cells. An absolute correlation, with no exceptions, was seen. We also showed that the method has potential in the antenatal determination of RH type, as it was possible to type fetal trophoblasts recovered from the endocervical canal at 9 weeks pregnancy.

Direct molecular analysis of a deletion of 3p in tumors from patients with sporadic renal cell carcinoma

Abstract Normal and tumorous nephrectomy specimens from seven renal cell carcinoma patients were subjected to a Southern analysis using chromosome #3-specific polymorphic probes. Three patients were not informative because of homozygosity at all loci studied. One patient showing heterozygosity at 3q in normal tissue had a tumor that remained heterozygous. In three patients the tumor showed loss of heterozygosity for a short arm marker at 3p21. In one of them heterozygosity for a second short arm marker was also lost. Another of these three patients retained heterozygosity for this second short arm marker, as well as for a long arm marker, suggesting a chromosomal breakpoint between the loci for the two short arm markers. Our results demonstrate that the known involvement of a short arm region of chromosome #3 in the development of renal cell carcinoma can readily be further evaluated by direct molecular methods.

LOSS OF HETEROZYGOSITY FOR A CHROMOSOME-3 SEQUENCE PRESUMABLY AT 3P21 IN SMALL-CELL LUNG-CANCER

A recombinant DNA fragment detecting a chromosome #3 restriction fragment length polymorphism presumably at p21 was hybridized to HindIII-digested DNA isolated from the leukocytes of 12 patients of small cell lung cancer. Four of them appeared to be heterozygous. Analysis of tumor material from these four patients revealed homozygosity for either one or the other restriction fragment in every case. Our findings suggest the presence on the short arm of chromosome #3 of a recessive mutant cancer gene contributing to the development of small cell lung cancer.

Denaturing gradient gel electrophoresis: a novel method for determining Rh phenotype from genomic DNA.

Denaturing gradient gel electrophoresis (DGGE) was carried out on PCR products amplified from exons 2 and 5 of RHD and RHCE. Exon 2 of RHD and exon 2 of the C allele of RHCE have an identical sequence, which differs from that of the c allele of RHCE. One band representing D and/or C, and another representing c, could be distinguished by DGGE of exon 2 amplifications of genomic DNA from individuals with the appropriate Rh phenotype. C and c could only be distinguished in D‐negative samples. Exon 5 of RHD and exon 5 of the E and e alleles of RHCE all have different nucleotide sequences. Bands representing D, E and e could be distinguished following DGGE of the products of exon 5 amplification of genomic DNA from individuals with red cells of the appropriate Rh phenotype. In samples from individuals with VS+ red cells (V+ or V−) there was a shift of the band representing e. Sequencing demonstrated that VS is associated with a RHCE e sequence with a single base change predicting a Leu245 → Val substitution in the Rh polypeptide. This substitution may be responsible for the VS and es antigens.

Microsatellite Variation within the Human RHCE Gene

Background and Objectives: This study provides a unique method for identifying individuals carrying the Rh haplotype cDe, and supports a model for the evolution of Rh haplotypes in which cDe is the progenitor. Materials and Methods: DNA from 212 unrelated donors of known Rh serological phenotype was PCR amplified. The resulting products were analysed by denaturing polyacrylamide gel electrophoresis, denaturing gradient gel electrophoresis and DNA sequencing. Results: Two adjacent microsatellite repeat elements of the form (AC)n (GCAC)n were found within the human Rh blood group genes. These display copy number variation which was non–randomly distributed with respect to Rh serological phenotype, and was restricted to alleles of RHCE expressing the c antigen. Conclusion: The predominantly Black African allele cDe displayed a unique set of microsatellite alleles, providing a method of identifying individuals carrying this haplotype.

A PCR-aided transcript titration assay revealing very low expression of a gene at band 3p21 in 33 cells lines derived from all types of lung cancer

We have developed a general PCR-based method to quantify the amount of a specific mRNA present in a given cell line or tissue. We applied this quantitative PCR to analyse the expression of D8, a human gene which we recently identified in the chromosomal region 3p21, the common deletion region of lung cancer. Our PCR-aided assay shows that in most lung-cancer-derived cell lines the amount of D8 transcripts is only 2% or less of that in normal lung tissue. The virtual absence of expression may imply some role of the gene in the development of lung cancer.

Structure of the Human D1F15S1A Locus: A Chromosome 1 Locus with 97% Identity to the Chromosome 3 Gene Coding for Hepatocyte Growth Factor-like Protein

The human chromosome 3 locus coding for hepatocyte growth factor-like protein/macrophage stimulating protein (HGFL/MSP) is homologous to two sets of amplified loci on human chromosome 1 at 1p36. One copy of one of the amplified loci (D1F15S1A) has been further characterized by restriction enzyme and DNA sequence analysis. A total of 8331 bp of continuous sequence was determined for this locus. The first 6878 bp of sequence is 96.1% identical to the HGFL/MSP gene, while there is no homology between the two genes following nucleotide 6878. Based on the presence of a 5 bp deletion in putative exon 2 and several downstream stop codons it is very likely that this gene is a pseudogene. Screening of a human liver cDNA library with a chromosome 1-specific probe indicates that at least several other members of the chromosome 1 loci are transcribed.

A deletion at the short arm of chromosome 3 is common to all major types of lung cancer

Abstract Occurrence of short arm deletions of chromosome 3 as a consistent abnormality of small cell lung cancer (SCLC) has been a matter of some dispute. A cytogenetic analysis of cell lines derived from SCLC tumours from different patients delimited the common deletion to 3p21-22. The DNA clone defining D1S1 contains a repetitive element present on chromosome 1, but has been shown to originate from chromosome 3. We localized the clone by in situ hybridization of some of its single copy sequences at 3p21. One subclone, pH3H2, detects a DNA polymorphism with a frequency of heterozygotes close to the maximum of 0.5. The distribution of alleles among lung cancer patients appeared to be similar to that in the general population. By applying pH3H2 in a Southern analysis of both constitutive and tumor DNA from the same lung cancer patients the possible occurrence of 3p21 deletions in SCLC and other types of lung cancer was investigated. All informative cases of SCLC, squamous carcinoma, and adenocarcinoma studied did show loss of an allele at the 3p21 locus DNF15S2 identified by pH3H2. Therefore, some recessive tumor suppressor gene at 3p21 may be involved in the development of lung cancer in general. This agrees with the concept that the different types of lung cancer originate from one common malignantly transformed stem cell population in which different histological characteristics reflect preferentially expressed differentiation pathways.