ras mutations in endocrine tumors: mutation detection by polymerase chain reaction-single strand conformation polymorphism.

To elucidate the molecular basis for endocrine tumorigenesis, ras mutations in human endocrine tumors were analyzed using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Mutations of the H-, K-, N-ras genes were examined in genomic DNAs from 169 successfully amplified primary endocrine tumors out of 189 samples. Four out of 24 thyroid follicular adenomas analyzed contained mutated N-ras codon 61, and one contained the mutated H-ras codon 61. One of the 19 pheochromocytomas revealed mutation of the H-ras codon 13. No mutations of the ras gene were detected in pituitary adenomas, parathyroid tumors, thyroid cancers, endocrine pancreatic tumors, and adrenocortical tumors. Based on these findings we conclude that activation of the ras gene may play a role in the tumorigenesis of a limited number of thyroid follicular adenomas and pheochromocytomas, and that mutation of the ras gene is not frequent in other human endocrine tumors.     

ras Mutations in Endocrine Tumors: Mutation Detection by Polymerase Chain Reaction-Single Strand Conformation Polymorphism

To elucidate the molecular basis for endocrine tumorigenesis, ras mutations in human endocrine tumors were analyzed using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Mutations of the H-, K-, N- ras genes were examined in genomic DNAs from 169 successfully amplified primary endocrine tumors out of 189 samples. Four out of 24 thyroid follicular adenomas analyzed contained mutated N- ras codon 61, and one contained the mutated H- ras codon 61. One of the 19 pheochromocytomas revealed mutation of the H- ras codon 13. No mutations of the ras gene were detected in pituitary adenomas, parathyroid tumors, thyroid cancers, endocrine pancreatic tumors, and adrenocortical tumors. Based on these findings we conclude that activation of the ras gene may play a role in the tumorigenesis of a limited number of thyroid follicular adenomas and pheochromocytomas, and that mutation of the ras gene is not frequent in other human endocrine tumors.     

Alterations of p53 in tumorigenic human bronchial epithelial cells correlate with metastatic potential.

The cellular and molecular mechanisms of radiation-induced lung cancer are not known. In the present study, alterations of p53 in tumorigenic human papillomavirus-immortalized human bronchial epithelial (BEP2D) cells induced by a single low dose of either alpha-particles or 1 GeV/nucleon (56)Fe were analyzed by PCR-single-stranded conformation polymorphism (SSCP) coupled with sequencing analysis and immunoprecipitation assay. A total of nine primary and four secondary tumor cell lines, three of which were metastatic, together with the parental BEP2D and primary human bronchial epithelial (NHBE) cells were studied. The immunoprecipitation assay showed overexpression of mutant p53 proteins in all the tumor lines but not in NHBE and BEP2D cells. PCR-SSCP and sequencing analysis found band shifts and gene mutations in all four of the secondary tumors. A G-->T transversion in codon 139 in exon 5 that replaced Lys with Asn was detected in two tumor lines. One mutation each, involving a G-->T transversion in codon 215 in exon 6 (Ser-->lle) and a G-->A transition in codon 373 in exon 8 (Arg-->His), was identified in the remaining two secondary tumors. These results suggest that p53 alterations correlate with tumorigenesis in the BEP2D cell model and that mutations in the p53 gene may be indicative of metastatic potential.     

Genetic alterations in N-bis(2-hydroxypropyl)nitrosamine-induced rat transplantable thyroid carcinoma lines: analysis of the TSH-R, G(alpha)s, ras and p53 genes

It has recently been shown that point mutations of the TSH-R or G(alpha)s genes are associated with autonomous hyperfunctioning thyroid adenomas and differentiated carcinomas. We therefore screened for mutations in the TSH-R, G(alpha)s, ras and p53 genes in nine rat transplantable thyroid carcinoma lines derived from tumors induced by DHPN as a chemical carcinogen. Mutations were identified using single- strand conformation polymorphism and DNA sequencing analysis. Point mutations in G(alpha)s codon 201 (CGC-->CAC) were detected in three lines (33%), resulting in a heterozygous alteration (Arg-->His) in the expressed G(alpha)s protein. The mean intracellular cAMP level (2.30 +/- 0.27 nmol/mg) of the three mutated cell lines was significantly increased as compared with that of the lines (1.54 +/- 0.32 nmol/mg) without the G(alpha)s mutation (P < 0.01, by paired t-test). Also, these three cell lines had an activating mutation in Ki-ras codon 12 (GGT-->GAT). One TSH-R gene mutation was found with a base substitution in codon 636 (TGC-->TGT) but no amino acid change. No p53 gene (exons 5- 8) mutations were detected in any of the cell lines analyzed. The results suggest that mutational activation of the G(alpha)s gene may play a tumorigenic role through constitutive activation of the cAMP pathway and that G-->A point mutations in the G(alpha)s and ras genes in thyroid carcinomas directly reflect interaction of the chemical carcinogen with guanine residues in DNA.      

Establishment of 2 human thyroid-carcinoma cell-lines (8305c, 8505c) bearing p53 gene-mutations

New cell lines, designated 8305C and 8505C, were established from undifferentiated thyroid carcinomas of a 67 year-old-female patient and a 78-year-old-female patient, respectively. Pathologically both these primary undifferentiated carcinoma tissues contained residual well differentiated components, suggesting well differentiated to undifferentiated carcinoma progression. Cell kinetic analysis indicate that the cell population doubling time is 43 h for 8305C and 36 h for 8505C. The saturation density at confluency is 5.7 x 10(4) cells/cm2 for 8305C and 1.1 x 10(5) cells/cm2 for 8505C. To identify genetic changes that may have occurred in these two cell lines, tumor suppressor genes p53, Rb, APC and MCC were analyzed. Sequence analysis confirmed a C:G to T:A transition at the first base of p53 gene codon 273 in 8305C and a C:G to G:C transversion at the first base of p53 codon 248 in 8505C. Polymerase chain reaction-loss of heterozygosity assays confirmed allelic deletion of p53 gene from the 8505C cell line. Loss of heterozygosity of other tumor suppressor genes were not observed. Given that p53 mutations associate with undifferentiated carcinoma but not with well differentiated carcinoma during multistep carcinogenesis of the thyroid, these cell lines should prove useful for research into the role of p53 gene mutations in malignant transformation.     

Analysis of the p53 gene in human uterine carcinoma cell lines.

The inactivation of the tumor suppressor gene p53 has been demonstrated in a variety of human tumors. In this study, we present a p53 gene analysis of 13 uterine carcinoma cell lines. Sequencing analysis of the entire coding region revealed mutations changing the p53 amino acid composition in all six endometrial carcinoma cell lines tested (Ishikawa, Hecl-A, Hecl-B, KLE, RL95-2, and AN-3). Of the seven cervical carcinoma cell lines, two (HT-3 and C-33A) contained p53 codon changes as well. We were unable to detect human papillomavirus in these two cell lines. By contrast, five human papillomavirus-positive cervical carcinoma cell lines (HeLa S-3, Caski, SiHa, C-4I, and ME-180) contained wild-type p53 gene sequences. We suggest that, in the human papillomavirus-positive cervical tumors, p53 inactivation occurred via the known mechanism of viral E6/cellular p53 protein association, whereas in all other tumors p53 function was compromised by changes in the amino acid sequence.     

Mutation and homozygous deletion analyses of genes that control the G1/S transition of the cell cycle in skin melanoma: p53, p21, p16 and p15

Introduction The role of genes involved in the control of progression from the G1 to the S phase of the cell cycle in melanoma tumors is not fully known. Material and methods The aims of our study were to analyse alterations in p53, p21, p16 and p15 genes in melanoma tumors and melanoma cell lines by single strand conformational polymorphism (SSCP), and to detect homozygous deletions. We analysed the DNA from 39 patients with primary and metastatic melanomas, and from 9 melanoma cell lines. Results The SSCP technique showed heterozygous defects in the p53 gene in 8 or 39 (20.5%) melanoma tumors: three point mutations in intron sequences (introns 1 and 2) and exon 10, and three new polymorphisms located in introns 1 and 2 (C to T transition at position 11701 in intron 1; C insertion at position 11818 in intron 2; and C insertion at position 11875 in intron 2). One melanoma tumor exhibited two heterozygous alterations in the p16 exon 1 (stop codon and missense mutation). No defects were found in the remaining genes. Homozygous deletions were more frequent in melanoma cell lines than in melanoma tumors in p21, p16 and p15 (22.2%, 44.4%, and 44.4% versus 7.7%, 2.5%, and 5.1% respectively). TP53 did not show homozygous deletions. Conclusions Our results suggest that these genes are involved in melanoma tumorigenesis; but perhaps not in the major targets. Other suppressor genes that may be informative of the mechanism of tumorigenesis in skin melanomas need to be studied.     

p53 status in spontaneous and dimethylnitrosamine—induced renal cell tumors from rats

Rats carrying the Eker tumor–susceptibility mutation (Eker rats) are predisposed to developing renal cell carcinoma. Rats heterozygous for the Eker mutation develop spontaneous multiple bilateral renal cell tumors by the age of 1 yr. In a previous study, Eker-mutation carrier and noncarrier rats were exposed to the renal carcinogen dimethylnitrosamine (DMN), and male rats carrying the Eker mutation exhibited a 70-fold increase in the induction of renal adenomas and carcinomas when compared with noncarrier rats. In this study, spontaneous and DMN-induced rat renal cell tumors (adenomas and carcinomas) were analyzed for mutations of the p53 gene by direct sequencing of cDNA polymerase chain reaction products. There were no mutations in p53 cDNA derived from renal tumors from six untreated rats. Mutations were found in one of 15 of the DMN-induced tumors: a transition at codon 140, CCT → CTT, in a renal adenoma. Additionally, seven cell lines derived from spontaneous renal cell tumors did not contain mutations in p53. The low frequency of p53 mutations (one of 21 renal cell tumors and none of seven cell lines derived from renal cell tumors) indicates that the development of both spontaneous and carcinogen-induced renal tumors involved a non–p53-dependent pathway. As p53 is infrequently mutated in human renal cell carcinomas and in rat renal mesenchymal tumors, it is likely that a tumor suppressor gene or genes other than p53 are involved in the development of renal cancer. © 1995 Wiley-Liss Inc.     

Point Mutations of ras and Gsα Subunit Genes in Thyroid Tumors

We studied 43 thyroid tumors including 5 adenomatous goiters, 7 follicular adenomas, 22 papillary carcinomas, and 9 medullary carcinomas with regard to the presence of point mutations in the genes of Gs alpha subunit ( Gsα ), Gi2 alpha subunit ( Gi2α ), H- ras , K- ras , and N- ras by a polymerase chain reaction-direct sequencing method. An adenomatous goiter and a follicular adenoma showed double mutations at codon 227 and 231, and 4 papillary carcinomas showed mutation at codon 231 of the Gsα gene. An adenomatous goiter, a follicular adenoma, and a papillary carcinoma showed a missense mutation in codon 13 of the K- ras gene. There were no such missense mutations of these G-protein or ras genes in medullary carcinomas. These data indicate that the genetic events involved in the oncogenesis of parafollicular C-cells are different from those of thyroid follicular cells, in which missense mutations of Gsα and ras genes seem to play important roles in tumorigenesis.     

p53 Gene Mutations Associated with Anaplastic Transformation of Human Thyroid Carcinomas

Anaplastic carcinoma of the thyroid gland, which is one of the most aggressive, malignant tumors in humans, is considered to originate from preexisting differentiated thyroid cancer. To define the genetic alterations associated with such progression, we examined nine cases of anaplastic thyroid carcinoma for mutation in exons 4–9 of the p53 tumor suppressor gene. Preliminary screening for mutation by RNase protection analysis demonstrated that two out of nine anaplastic carcinomas contained sequence alterations in the p53 gene. Subsequent DNA sequencing identified the mutated nucleotides in these two cases; one was a nonsense mutation at codon 165, and the other was a single-base deletion at codon 176 resulting in the creation of a stop codon downstream due to frameshift. The fact that no mutations were detected in coexisting foci of papillary carcinomas from the same patients shows that these mutations of the p53 gene occurred after development of papillary carcinomas. These results suggest that p53 gene mutation triggers the progression from differentiated into anaplastic carcinoma in the human thyroid gland.     

p53 gene mutations associated with anaplastic transformation of human thyroid carcinomas.

Anaplastic carcinoma of the thyroid gland, which is one of the most aggressive, malignant tumors in humans, is considered to originate from preexisting differentiated thyroid cancer. To define the genetic alterations associated with such progression, we examined nine cases of anaplastic thyroid carcinoma for mutation in exons 4-9 of the p53 tumor suppressor gene. Preliminary screening for mutation by RNase protection analysis demonstrated that two out of nine anaplastic carcinomas contained sequence alterations in the p53 gene. Subsequent DNA sequencing identified the mutated nucleotides in these two cases; one was a nonsense mutation at codon 165, and the other was a single-base deletion at codon 176 resulting in the creation of a stop codon downstream due to frameshift. The fact that no mutations were detected in coexisting foci of papillary carcinomas from the same patients shows that these mutations of the p53 gene occurred after development of papillary carcinomas. These results suggest that p53 gene mutation triggers the progression from differentiated into anaplastic carcinoma in the human thyroid gland.     

Wild-type p53 suppresses growth of human prostate cancer cells containing mutant p53 alleles

Evidence supporting a broad role for the inactivation of the p53 gene in human tumorigenesis has been provided by studies showing that the p53 gene is mutated in many human cancers. In this study, we report on the mutational status of the p53 gene in prostate cancer cells and provide functional evidence that the wild-type p53 gene may have a role in suppressing prostatic tumorigenesis. Sequence analysis of exons 5-8 of the p53 gene reveals that three of five prostate cancer cell lines (TSUPr-1, PC3, DU145) contain mutations which alter the amino acid sequence of this most highly conserved portion of the gene. One of two primary prostatic cancer specimens examined also contained a mutation in this region. Transfection of the wild-type p53 gene versus a mutated p53 gene into two cell lines with p53 mutations results in reduced colony formation. Wild-type p53 gene expression is apparently incompatible with continued growth of these tumor cells inasmuch as none of the colonies which formed after wild-type transfections retain the transfected p53 sequences. Immunocytochemical data indicate that prostate carcinoma cells expressing the transfected wild-type p53 gene are growth arrested because they exhibit a reduced level of thymidine incorporation into DNA. This study is the first report of p53 gene mutations in prostate cancer cells and suggests a functional role for the p53 gene in suppressing prostatic tumorigenesis.     

Similar incidence of K-ras mutations in lung carcinomas of FVB/N mice and FVB/N mice carrying a mutant p53 transgene

Mutated p53 genes are capable of complementing activated ras genes in the transformation of primary rat embryo fibroblasts in vitro. Mutations in both genes have also been found in several human cancers, including lung carcinomas. We generated transgenic mice containing a p53 construct with a missense mutation in exon 5 (ala135val) to study the role of p53 mutations in lung tumorigenesis, and to facilitate identification of other genetic events that might complement p53 mutations in mouse lung carcinogenesis. The p53 transgenic lines exhibited a higher frequency of lethal lung tumors than the parental FVB/N strain. We examined the spontaneously-arising lung carcinomas from mice expressing the mutated p53 transgene for K-ras mutations using single-stranded conformation polymorphism (SSCP) and/or direct sequencing approaches. Fifteen of 29 (52%) carcinomas contained mutations in the K-ras oncogene. Six of 15 of the K-ras mutations were in codon 61 and 9/15 were in codon 12. Subsequent analysis of spontaneous lung carcinomas from mice of the FVB/N parental strain showed that 9/12 (75%) carcinomas examined contained K-ras mutations. Two of these were in codon 12, one in codon 13, and 6 were in codon 61. These results demonstrate that the frequency of ras mutations does not differ between the p53 FVB/N transgenic mice and their parental FVB/N strain but suggest that a high frequency of mutations K-ras can be correlated with lung tumorigenesis in both groups of mice.      

p53 mutations are absent from carcinogen-induced mouse liver tumors but occur in cell lines established from these tumors.

Mutations in the p53 gene are frequent genetic alterations in human hepatocellular carcinomas. We have examined, by single-strand conformation polymorphism analysis of polymerase chain reaction products, a total of 93 carcinogen-induced liver tumors from mice of three different strains (C3H/He, C57BL/6J, and B6C3F1) for the presence of p53 aberrations. Hepatoma lines, established from 12 liver tumors, were also included in the analysis. While structural aberrations of the p53 gene were not detected in any of the primary mouse liver tumors analyzed, single-base substitutions occurred at different locations within the p53 gene in three of the cell lines during in vitro propagation. One hepatoma line carried two point mutations on separate alleles. All four mutations were either G:C----C:G or C:G----G:C transversions. Mutations at codon 61 of the c-Ha-ras gene, which were frequent in primary liver tumors from C3H/He and B6C3F1 mice, were not detected in the hepatoma lines. Our data indicate (i) that c-Ha-ras but not p53 mutations play an important role during the early stages of mouse hepatocarcinogenesis and (ii) that p53 mutations confer a selective growth advantage to the mutated hepatoma cells in vitro.     

p53 protein accumulation and gene mutations in human glioma cell lines

Mutations in, and aberrant expression of, the p53 tumor suppressor gene were assessed in 17 cell lines derived from human malignant brain tumors (glioblastoma multiforme). Ex-ons 5 through 8 were screened by single strand conformational polymorphism analysis (SSCP), followed by direct DNA sequencing. Mutations were found in 6 of 17 glioma cell lines, i.e., at a frequency similar to that found in primary malignant gliomas. Loss of the wild type allele was observed in 4 of the mutated cell lines. Two cell lines had the same mutation (CGG → TGG; Arg → Trp) in codon 248. Five of 6 mutations were transitions, 4 of which occurred at CpG dinucleotides. In one cell line a 10-bp deletion at the intron 4/exon 5 junction was found. Five of 6 glioma cell lines contained a mutation identical to that in the respective primary tumor despite prolonged in vitro culture (140-221 passages). Thus, the acquisition of p53 mutations during culture appears to be infrequent. Two cell lines derived from heterozygous tumors maintained the wild type p53 allele during long term culture. p53 protein levels were assessed by immunofluorescence cytochemistry and immunoprecipitation followed by Western blot analysis and revealed elevated levels of the p53 protein, although to a variable extent, in all cell lines with p53 mutations. A marked p53 protein accumulation was also observed in two cell lines lacking p53 mutations in exons 5 through 8, indicating that a prolonged half life of the gene product is not solely dependent on an aberrant coding sequence. The remaining cell lines had either low levels or no detectable p53 protein; one of the latter contained a gross rearrangement of the p53 gene. Our results suggest that with respect to p53 gene status, glioma cell lines usually resemble the original tumors and may, therefore, be suitable for studying the biological changes associated with p53 mutations in glial tumors.     

Tobacco smoke-induced lung tumorigenesis in mutant A/J mice with alterations in K-ras, p53, or Ink4a/Arf

A/J mice with genetic alterations in K-ras, p53, or Ink4a/Arf were employed to investigate whether mice carrying these germline mutations would be susceptible to tobacco smoke-induced lung tumorigenesis. Transgenic mice of both genders and their wild-type littermates were exposed to environmental cigarette smoke for 6 months, followed by recovery in air for 5 months. A significant increase of lung tumor multiplicity was observed in K-ras, p53, or Ink4a/Arf mutant mice when compared with wild-type mice. Furthermore, an additive effect was observed between the mice with a mutant p53 transgene and an Ink4A/Arf deletion during tobacco smoke-induced lung tumorigenesis. Sequence analysis of the K-ras gene indicated that the mutations had occurred at either codon 12/13 or 61 in both spontaneously occurring (air control) and tobacco smoke-induced lung tumors. K-ras mutations were found in 62% of the tumors from air-control animals and 83% in those exposed to tobacco smoke. The mutation spectrum found in tumors from mice exposed to tobacco smoke is somewhat similar to that in tumors from air-control mice. In addition, we identified three novel mutations at codon 12: GGT (Gly) --> TTT (Phe), ATT (Ile), and CTT (Leu). These findings provide evidence that K-ras, p53, and Ink4a/Arf mutations play a role in tobacco smoke-related lung carcinogenesis. The similarity of the mutation spectra in the K-ras oncogene observed in tobacco smoke-induced tumors, as compared to spontaneous tumors, suggests that tobacco smoke enhances lung tumorigenesis primarily through promoting spontaneously occurring K-ras mutations.     

Gene expression of parathyroid tumors: molecular subclassification and identification of the potential malignant phenotype

Parathyroid tumors are heterogeneous, and diagnosis is often difficult using histologic and clinical features. We have undertaken expression profiling of 53 hereditary and sporadic parathyroid tumors to better define the molecular genetics of parathyroid tumors. A class discovery approach identified three distinct groups: (1) predominantly hyperplasia cluster, (2) HRPT2/carcinoma cluster consisting of sporadic carcinomas and benign and malignant tumors from Hyperparathyroidism-Jaw Tumor Syndrome patients, and (3) adenoma cluster consisting mainly of primary adenoma and MEN 1 tumors. Gene sets able to distinguish between the groups were identified and may serve as diagnostic biomarkers. We demonstrated, by both gene and protein expression, that Histone 1 Family 2, amyloid beta precursor protein, and E-cadherin are useful markers for parathyroid carcinoma and suggest that the presence of a HRPT2 mutation, whether germ-line or somatic, strongly influences the expression pattern of these 3 genes. Cluster 2, characterized by HRPT2 mutations, was the most striking, suggesting that parathyroid tumors with somatic HRPT2 mutation or tumors developing on a background of germ-line HRPT2 mutation follow pathways distinct from those involved in mutant MEN 1-related parathyroid tumors. Furthermore, our findings likely preclude an adenoma to carcinoma progression model for parathyroid tumorigenesis outside of the presence of either a germ-line or somatic HRPT2 mutation. These findings provide insights into the molecular pathways involved in parathyroid tumorigenesis and will contribute to a better understanding, diagnosis, and treatment of parathyroid tumors.     

Mutations and expression of the p53 gene in rat bladder carcinomas and cell lines

Abnormalities of the p53 gene are frequently observed in human tumors, including urinary bladder carcinoma, suggesting that p53 plays an important role in human carcinogenesis. However, its role in rat bladder carcinogenesis is unclear. We investigated p53 gene mutations and expression in rat urinary bladder carcinogenesis in vivo and in Vitro. Fifteen urothelial cell lines, including six untransformed (nontumorigenic) ones, six transformed (tumorigenic) in vitro, and three derived from tumors induced in vivo, were examined for p53 expression by immunochemical analysis and for p53 mutations; in addition, 81 rat bladders were analyzed immunohistochemically for p53 expression, and 23 rat bladder tumors were analyzed for p53 mutations. Four cell lines had mutations in the p53 gene. Two of these were missense point mutations, and the other two were splicing mutations. On the other hand, no mutations were found in the bladder tumors induced in rats. By immunoprecipitation with PAb240, which is supposed to be specific for mutant p53, we detected mutations in three of the cell lines; PAb240 did not react with wild-type p53. However, in all cell lines and in growing populations of primary cultured bladder urothelial cells, p53 expression was detected immunohistochemically or by western blotting using PAb240 or PAb 421 monoclonal antibodies. In a high percentage of transitional cell carcinomas, wild-type p53 expression was detected by immunohistochemical analysis with PAb240. These results suggest that p53 gene mutations may not occur frequently in rat bladder carcinogenesis in vivo but may occur in vitro and that p53 overexpression detected immunohistochemically is common and may be related to cell proliferation rather than to the presence of mutations in rat bladder carcinogenesis. © 1994 Wiley-Liss, Inc.     

Common tumour p53 mutations in immortalized cells from Hupki mice heterozygous at codon 72

Codon 72 of human p53 gene is polymorphic, encoding arginine or proline. Here we report construction of a human p53 knock-in (Hupki) mouse encoding the codon 72(pro) variant. The new strain was crossed with the original Hupki mice (codon 72(arg/arg)) to obtain primary embryonic fibroblasts polymorphic at codon 72 or homozygous for codon 72(pro). The fibroblasts, cultured under standard conditions, immortalized within 12 weeks and acquired p53 mutations similarly to Hupki codon 72(arg/arg) cells investigated previously. Sequencing of human p53 exons 4-9 in immortalized cultures revealed missense mutations found repeatedly in human tumours. In cell lines ensuing from benzo(a)pyrene-treated cultures the combined p53 mutation pattern from experiments with the 3 codon 72 genotypes showed a predominance of strand-biased G to T transversions (18 of 36 mutations), and mutations recurring at smokers' lung tumour hotspot codons 157 and 273, supporting involvement of tobacco carcinogens in shaping the mutation signature in lung cancers of smokers. Mutations in cell lines from unexposed cultures did not cluster at these codons and G to T transversions were uncommon (2 of 52 mutations) (Fisher's exact test P<0.0001). Most mutations (13/16) in cell lines derived from cells polymorphic at codon 72 were found on the proline allele, with loss of the arginine allele.