The gene for autosomal dominant retinitis pigmentosa in a large pedigree of Irish origin has recently been found to be linked to an anonymous polymorphic sequence, D3S47 (C17), from the long arm of chromosome 3. As the gene coding for rhodopsin is also assigned to the long arm of chromosome 3 and is expressed in rod photoreceptors that are affected early in this blinding disease, we searched for a mutation of the rhodopsin gene in patients with autosomal dominant retinitis pigmentosa. We found a C----A transversion in codon 23 (corresponding to a proline----histidine substitution) in 17 of 148 unrelated patients and not in any of 102 unaffected individuals. This result, coupled with the fact that the proline normally present at position 23 is highly conserved among the opsins and related G-protein receptors, indicates that this mutation could be the cause of one form of autosomal dominant retinitis pigmentosa.

Single-strand conformation polymorphism analysis (SSCP) is a rapid method for detection of minor sequence changes in polymerase chain reaction-amplified DNA. Since the first reported use of SSCP in 1989 (Orita et al., 1989), this technique has been used widely to detect mutations in oncogenes, tumor suppressor genes, and genes responsible for genetic diseases. Published mutations that have been detected using this technique include base substitutions, small insertions and deletions, and rearrangements. This technique has also been applied for the detection of DNA polymorphisms at various loci of the human genome (reviewed by Hayashi, 1991; Hayashi, 1993). However, many factors can influence the sensitivity of SSCP, and its optimization is highly empirical. In this review, we estimate the percentage of mutations that can be detected by this technique under various controlled conditions, and describe some critical elements affecting sensitivity.

We have used polyclonal anti-synthetic peptide serum to study the role of retinoblastoma gene (RB) inactivation in a variety of human tumor cell lines. Our analysis indicates that inactivation of the RB protein, p105-Rb, is universal in retinoblastoma cells, vindicating the predictions of the Knudson "two-hit" hypothesis. In addition, our analysis has shown that inactivations of the RB gene are nearly as frequent in a more common human tumor, small cell lung carcinoma. One-third of bladder carcinomas surveyed also carry altered or absent p105-Rb. Other human tumors by contrast demonstrate only infrequent inactivation of the RB gene. These results suggest that inactivation of the RB gene is a critical step in the pathogenesis of a subset of human tumors.The recent isolation of molecular clones of the retinoblastoma gene (RB) has made it possible to evaluate the role of RB gene inactivation in the genesis of a variety of human tumors (1-3). These molecular analyses have confirmed and extended earlier karyotypic and genetic studies that had implied that both copies ofthis gene suffer inactivation during the formation of retinoblastomas and several types of sarcoma (4-10). Since the inactivation ofRB function appears to trigger tumorigenesis, the RB gene has been termed a "tumor suppressor" gene, whose expression is required to constrain normal cellular proliferation.Analysis of the protein product encoded by the RB gene, p105-Rb, has shown it to be a nuclear phosphoprotein having an affinity for DNA (11,12 . This complex formation is apparently central to the ability of these oncoproteins to transform primary cells (14,17).In the initial studies designed to detect inactivation of chromosomal copies of the RB gene, cloned segments of the RB gene were used as probes in Southern blot analysis of tumor DNAs (1)(2)(3)(18)(19)(20)(21) MATERIALS AND METHODSPreparation of Cell Lysates and Immunoprecipitation of the RB Protein from Human Tumor Cells. Tumor cell cultures were incubated for 3-5 hr with [35S~methionine, and cell lysates were prepared as described (13). Immunoprecipitations were performed with rabbit serum no. 147, 144, or 140 (13), and precipitates were resolved by electrophoresis for 15 hr on SDS/polyacrylamide gels, processed for fluorography, and exposed for 1-3 days at -70'C. Bladder, colon, and breast carcinoma cultures were acquired from the American Type Culture Collection as were two retinoblastoma cultures (Y79 and WERI-1). The derivation ofall other retinoblastoma cultures and small cell lung carcinoma (SCLC) cultures has been described (1,23,24). Melanoma tumor cells were a kind gift of Nick Dracopoli of the Massachusetts Institute of Technology.Amplification ofTumor Cell mRNA by Using the Polymerase Chain Reaction (PCR) Technique and DNA Sequence Analysis. RB mRNA was specifically amplified by using oligonucleotide primers from the RB cDNA sequence and the PCR as described (12). Amplified fragments were cloned into M13 and sequenced on both strands by the dideoxy chain termination techniq...

von Recklinghausen neurofibromatosis (NF1) is a common hereditary disorder characterized by neural crest-derived tumors, particularly benign neurofibromas whose malignant transformation to neurofibrosarcomas can be fatal. The NF1 gene has been mapped to a small region of chromosome 17q, but neither the nature of the primary defect nor the mechanisms involved in tumor progression are understood. We have tested whether NF1 might be caused by the inactivation of a tumor suppressor gene on 17q, analogous to that on chromosome 22 in NF2, by searching for deletions of chromosome 17 in NF1-derived tumor specimens. Both neurofibrosarcomas from patients with "atypical" NF and 5 of 6 neurofibrosarcomas from NF1 patients displayed loss of alleles for polymorphic DNA markers on chromosome 17. However, the common region of deletion was on 17p and did not include the NF1 region of 17q. Since no loss of markers on chromosome 17 was observed in any of 30 benign tumors from NF1 patients, the 17p deletions seen in neurofibrosarcomas are probably associated with tumor progression and/or malignancy. This region contains a candidate gene for tumor progression, p53, which has recently been implicated in the progression of a broad array of human cancers. In a preliminary search for p53 aberrations by direct sequencing of polymerase chain reaction-amplified DNA from 7 neurofibrosarcomas, 2 tumors that contained point mutations in exon 4 of the p53 gene were found, suggesting a role for this gene in at least some neurofibrosarcomas. Thus the formation of malignant neurofibrosarcomas may result from several independent genetic events including mutation of the NF1 gene, whose mechanism of tumorigenesis remains uncertain, and subsequent loss of a "tumor suppressor" gene on 17p, most likely p53.

The retinoblastoma (Rb) antioncogene encodes a nuclear phosphoprotein, p105-Rb, that forms protein complexes with the adenovirus E1A and SV40 large T oncoproteins. A novel, aberrant Rb protein detected in J82 bladder carcinoma cells was not able to form a complex with E1A and was less stable than p105-Rb. By means of a rapid method for the detection of mutations in Rb mRNA, this defective Rb protein was observed to result from a single point mutation within a splice acceptor sequence in J82 genomic DNA. This mutation eliminates a single exon and 35 amino acids from its encoded protein product.

Mutations of the p53 gene on chromosome 17p are a common genetic change in the malignant progression of many cancers. We have analyzed 38 malignant tumors of ovarian or peritoneal mullerian type for evidence of p53 variations at either the DNA or protein levels. Genetic studies were based on single-strand conformation polymorphism analysis and DNA sequencing ofexons 2 through 11 of the p53 gene; mutations were detected in 79% of the tumors. These data show a statistically significant association between mutations at C-G pairs and a history of estrogen therapy. Two of 20 patients whose normal tissue could be studied carried germ-line mutations of p53. Immunohistochemical analysis of the p53 protein was carried out using monoclonal antibody PAb1801. Ninetysix percent of the missense mutations were associated with abnormal accumulation of p53 protein, but nonsense mutations, a splicing mutation, and most deletions did not result in p53 protein accumulation. A statistically significant association between p53 protein accumulation in poorly differentiated stage Im serous carcinomas and small primary tumor size at diagnosis was found, perhaps suggesting that p53 protein accumulation accelerates the metastatic spread from a primary tumor. Overall, our findings indicate that alterations of p53 play a major role in ovarian cancer, including predisposition to the disease in some patients, and suggest a possible mechanism for somatic mutations leading to this cancer.

New germline mutations of the p53 gene are rare among patients with "sporadic" sarcoma but may be common in patients with sarcoma whose background includes either multiple primary cancers or a family history of cancer. Diverse mutations of this gene were associated with an increased likelihood of cancer; hence, the entire gene should be considered a target for heritable mutation. It appears that the group of patients with cancer who carry germline mutations of the p53 gene is more diverse than is suggested by the clinical definition of the Li-Fraumeni syndrome. The identification of carriers could be of substantial clinical importance.

Loss of chromosome 13q occurs in up to 50% of human astrocytomas, suggesting the presence of an astrocytoma tumor suppressor gene on that chromosome. To determine whether the retinoblastoma susceptibility gene (Rb) on 13q14 contributes to the formation of astrocytomas, we examined 85 tumors for loss of heterozygosity (LOH) at the intragenic Rb 1.20 locus. LOH was detected in 16 of 54 informative high-grade astrocytomas (30%), but was not detected in 12 low-grade gliomas. Deletion mapping with flanking markers on 13q revealed that the Rb 1.20 region was preferentially targeted by the deletions. Tumors with LOH at Rb 1.20 were examined for mutations in the remaining Rb allele using single-strand conformational polymorphism (SSCP) analysis and direct DNA sequencing. Mutations were detected in exon 8 (1 tumor), exon 24 (2 tumors), and intron 24 (1 tumor). Rb protein expression, as assessed by immunohistochemistry, was altered in 3 of 9 cases with LOH and in 1 tumor without LOH. Our results demonstrate that Rb inactivation contributes to the formation of high-grade astrocytomas, and therefore implicate a second, known tumor suppressor gene in astrocytoma tumorigenesis.