Specific mutations of the RET proto-oncogene are related to disease phenotype in MEN 2A and FMTC.

We have analysed 118 families with inherited medullary thyroid carcinoma (MTC) for mutations of the RET proto-oncogene. These included cases of multiple endocrine neoplasia types 2A (MEN 2A) and 2B (MEN 2B) and familial MTC (FMTC). Mutations at one of 5 cysteines in the extracellular domain were found in 97% of patients with MEN 2A and 86% with FMTC but not in MEN 2B patients or normal controls. 84% of the MEN2A mutations affected codon 634. MEN 2A patients with a Cys634 to Arg substitution had a greater risk of developing parathyroid disease than those with other codon 634 mutations. Our data show a strong correlation between disease phenotype and the nature and position of the RET mutation, suggesting that a simple, constitutive activation of the RET tyrosine kinase is unlikely to explain the events leading to MEN 2A and FMTC.

A common variant in BRCA2 is associated with both breast cancer risk and prenatal viability.

Inherited mutations in the gene BRCA2 predispose carriers to early onset breast cancer, but such mutations account for fewer than 2% of all cases in East Anglia. It is likely that low penetrance alleles explain the greater part of inherited susceptibility to breast cancer; polymorphic variants in strongly predisposing genes, such as BRCA2, are candidates for this role. BRCA2 is thought to be involved in DNA double strand break-repair. Few mice in which Brca2 is truncated survive to birth; of those that do, most are male, smaller than their normal littermates and have high cancer incidence. Here we show that a common human polymorphism (N372H) in exon 10 of BRCA2 confers an increased risk of breast cancer: the HH homozygotes have a 1.31-fold (95% CI, 1.07-1.61) greater risk than the NN group. Moreover, in normal female controls of all ages there is a significant deficiency of homozygotes compared with that expected from Hardy-Weinberg equilibrium, whereas in males there is an excess of homozygotes: the HH group has an estimated fitness of 0.82 in females and 1.38 in males. Therefore, this variant of BRCA2 appears also to affect fetal survival in a sex-dependent manner.

Heterogeneous mutation of the RET proto-oncogene in subpopulations of medullary thyroid carcinoma.

Mutations in the RET proto-oncogene are associated with the pathogenesis of medullary thyroid carcinoma (MTC). In an attempt to understand this process, we examined microdissected subpopulations from MTC and multiple metastases from these tumors. Approximately 80% of sporadic MTC's had at least one subpopulation with the RET codon 918 mutation, which is a mutation previously detected in sporadic MTC as a somatic mutation and in multiple endocrine neoplasia type 2B as a germline mutation. However, the distribution of this mutation was nonhomogeneous, occurring only in subpopulations in most tumors and among subsets of multiple metastases, thus implying that although the codon 918 mutation could be an early event, it is not necessarily an early or essential event in tumorigenesis. This heterogeneity suggests either that the codon 918 mutation can arise as an event in progression within a metastatic clone or within a single tumor, or that MTC can be of polyclonal origin. Of significance, one of two multiple endocrine neoplasia type 2A MTCs carried a somatic mutation at codon 918, in addition to the RET mutation present in the germline. We found no correlation between the presence of other somatic genetic events, such as loss of heterozygosity on chromosome arms 1p and 22q, and RET mutation status in the various subpopulations of MTC.

Multiple mRNA isoforms of the human RET proto-oncogene generated by alternate splicing.

The RET proto-oncogene encodes a receptor tyrosine kinase. We and others have recently shown that distinct germline mutations of the RET proto-oncogene account for the majority of cases of the dominantly inherited multiple endocrine neoplasia (MEN) type 2 syndromes, and can cause a dominantly inherited form of Hirschsprung disease, a disorder of development of the autonomic innervation of the gut. RET is also oncogenically activated in some sporadic thyroid and adrenal tumours. Here we report the characterisation of multiple mRNA isoforms of RET generated by alternate splicing. Two isoforms are predicted to encode membrane-spanning receptors with a truncated extracellular ligand-binding domain. A third isoform is predicted to encode a soluble, secreted form of the receptor. These mRNA isoforms are expressed in both normal and tumour tissues.

A novel point mutation in the tyrosine kinase domain of the RET proto-oncogene in sporadic medullary thyroid carcinoma and in a family with FMTC.

Germline mutations within one of six codons of the RET proto-oncogene account for the majority of cases of multiple endocrine neoplasia (MEN) type 2A and type 2B and familial medullary thyroid carcinoma (FMTC). MEN 2A and FMTC mutations characterised thus far occur exclusively in the cysteine-rich domain of the extracellular region of RET. We now report a missense mutation in the intracellular tyrosine kinase domain of RET in the germline of a family with FMTC that does not have a cysteine codon mutation. In this family, the mutation, which alters GAG (Glu) to GAC (Asp) at codon 768, segregates with the FMTC phenotype. The same mutation was also detected in sporadic MTC but not in corresponding constitutional DNA, confirming that it is likely to be of pathological significance rather than a rare polymorphism.

Mutation of the RET proto-oncogene is correlated with RET immunostaining in subpopulations of cells in sporadic medullary thyroid carcinoma.

Mutations in the RET proto-oncogene, which encodes a receptor tyrosine kinase, are associated with the pathogenesis of medullary thyroid carcinoma (MTC). Somatic mutations in RET, predominantly at codon 918, and very rarely at codon 883, have been found in a proportion of sporadic MTC. We have previously shown that approximately 80% of sporadic MTCs had at least one subpopulation with a somatic RET mutation. Uneven distribution of somatic mutation within a single tumor or among metastases from a single individual was notable. In the present study, we sought to correlate RET expression, as demonstrated by RET immunohistochemistry, with mutation status in sporadic MTC for each tumor. Seventy evaluable subpopulations, belonging to 28 unrelated sporadic cases, comprising primary MTC and metastases, were immunostained with two different polyclonal antibodies raised against the C-terminus of RET. The regional presence of codon 918 or 883 seemed to coincide with increased RET immunopositivity in at least 62 of 70 (89%, P < 0.000001) tumor subpopulations. The reasons for this concordance are not entirely clear but could be related to either RNA or protein stability. Preliminary studies have suggested that the presence of somatic codon 918 mutation in MTC has a prognostic significance. If these preliminary results prove true, then given our data, we can further explore the feasibility of RET immunocytochemistry as a rapid assessment for the presence of somatic codon 918 for molecular diagnostic and prognostic purposes.

A systematic review of genetic polymorphisms and breast cancer risk.

Studies investigating the relationship between common genetic variants and cancer risk are being reported with rapidly increasing frequency. We have identified 46 published case-control studies that have examined the effect of common alleles of 18 different genes on breast cancer risk. Of these, 12 report statistically significant associations, none of which were reported by more than one study. However, many of the studies were small: 10 of the 46 had 80% power or greater to detect a rare allele homozygote relative risk <2.5. We therefore combined the results of individual studies to obtain more precise estimates of risk. Statistically significant differences in genotype frequencies were found in three case-control comparisons of unselected cases. These were for CYP19 (TTTA)n polymorphism [(TTTA)10 carrier odds ratio (OR) = 2.33; P = 0.002], the GSTP1 Ile105Val polymorphism (Val carrier OR = 1.60; P = 0.02), and the TP53 Arg72Pro polymorphism (Pro carrier OR = 1.27; P = 0.03). In addition, the GSTM1 gene deletion was found to be significantly associated with postmenopausal breast cancer (null homozygote OR = 1.33; P = 0.04). There was also some evidence that homozygotes for the PR PROGINS allele are protected against breast cancer, although this result was of borderline statistical significance. For polymorphisms in BRCA1, COMT, CYP17, CYP1A1, NAT1, and NAT2, the best estimate of risk either from the individual studies or the meta-analyses was sufficiently precise to exclude a relative risk of 1.5 or greater. For the polymorphisms in EDH17B2, ER, CYP2D6, CYP2E1, GSTT1, HSP70, and TNFalpha, the risk estimates, although nonsignificant, were insufficiently precise to exclude a moderate risk (>1.5). Precise estimation of the risks associated with these and other as yet untested genes, as well as investigation of more complex risks arising from gene-gene and gene-environment interactions, will require much larger studies.

Localisation of the gene for multiple endocrine neoplasia type 2A to a 480 kb region in chromosome band 10q11.2.

A YAC contig has been constructed spanning 1.1 Mb of human chromosome band 10q11.2 which encompasses three markers (D10S141, RET, D10S94) closely linked to the gene for MEN 2A. This physical linkage group is ordered cen-D10S141-RET-D10S94-qter, and must include MEN2A, which lies in a 480 kb region between flanking markers D10S141 and D10S94.

Haplotype analysis of MEN 2 mutations.

Multiple endocrine neoplasia type 2 (MEN 2) is a dominantly inherited cancer syndrome which affects thyroid C cells, and with variable frequency, the adrenal medulla, parathyroid and enteric autonomic ganglia. The syndrome is due to germline mutation in the receptor tyrosine kinase gene, RET. We have recently shown an unexpected correlation between one particular RET mutation, cys634-->arg, and the probability of parathyroid involvement in families with MEN 2A. Here we use haplotype analysis in the families to show that this correlation is not explained by a single founder chromosome which carries both the cys634-->arg mutation and a separate allele conferring susceptibility to parathyroid abnormality, but is probably due to the cys634-->arg mutation itself. The results also indicate that new mutations to MEN 2 are not infrequent.

Mutation of the RET protooncogene in sporadic medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC) occurs sporadically or as part of the inherited cancer syndrome multiple endocrine neoplasia (MEN) type 2. In MEN 2A, germline missense mutations are found in one of five cysteine codons within exons 10 and 11 in the extracellular domain of the RET protooncogene. In MEN 2B, germline mutations occur in codon 918 (exon 16) within the catalytic core of the tyrosine kinase domain. To determine if RET mutations similar to those in MEN 2A and 2B play a role in the pathogenesis of sporadic MTC, we analysed 71 sporadic tumours comprising 68 primary tumours and three cell lines, for mutations in RET exons 10, 11, and 16. We found that 23% of sporadic MTC had RET codon 918 mutations, while only 3% had exon 10 mutations, and none had mutations in exon 11. We found no exon 16 mutations in MTC from 14 MEN 2A cases. Thus, exon 10 and 11 mutations, commonly found in familial MTC and MEN 2A, rarely occur in sporadic MTC; somatic mutation of RET codon 918 appears to play a role in the tumourigenesis of a significant minority of sporadic MTC but not MEN 2A tumours. In addition to their biological interest, these findings may have some clinical application in determining whether a patient presenting with isolated MTC is truly sporadic or is part of an inherited cancer syndrome.

Low frequency of germline mutations in the RET proto-oncogene in patients with apparently sporadic medullary thyroid carcinoma.

BACKGROUND AND OBJECTIVES: Medullary thyroid carcinoma (MTC) occurs both sporadically and in the autosomal dominantly inherited multiple endocrine neoplasia (MEN) type 2 syndromes. The distinction between true sporadic MTC and a new mutation familial case is important for future clinical management of both the patient and family. The susceptibility gene for MEN 2 is the RET proto-oncogene. Systematic analysis for germline mutations of the RET proto-oncogene was performed in a series of 67 patients with apparently sporadic MTC to determine whether they were true sporadic cases or unsuspected de novo MEN 2 cases. DESIGN AND PATIENTS: Sixty-seven unselected patients with sporadic MTC were randomly ascertained from clinic patients from four centres. The diagnosis of MTC was confirmed by histopathology. Germline DNA was extracted from peripheral blood leucocytes or from paraffin-embedded tissue and subsequently used for polymerase chain reaction amplification. MEASUREMENTS: Polymerase chain reaction based RET mutation analysis was performed by direct double-stranded cycle sequencing of exons 10, 11, 13 and 16, within which the majority of MEN2 mutations have been shown to occur. RESULTS: In this series, there was one proven case of germline mutation in RET codon 620, which previously has been shown to be responsible for MEN 2, thus indicating heritable disease. No germline mutation in codon 918, typical of MEN 2B, was found. CONCLUSIONS: A figure of 1.5% germline mutations in 67 apparently sporadic MTC is lower than the incidence of familial disease reported in previous series involving clinical and biochemical screening. The presence of a germline mutation in the RET proto-oncogene in a patient with MTC indicates heritable disease. The absence of germline RET exon 10, 11, 13 or 16 mutation appears to rule out MEN 2A to a high probability, although the presence of a familial form of MTC other than classical MEN 2A cannot be excluded conclusively.

Polymorphisms in the human aromatase cytochrome P450 gene (CYP19) and breast cancer risk.

The aromatase enzyme catalyses the conversion of androgens to oestrogens in the oestrogen biosynthesis pathway. Because increased exposure to oestrogens is considered to be a risk factor for breast cancer, the human aromatase gene (CYP19) is a plausible candidate for low penetrance breast cancer susceptibility. Preliminary reports have suggested that specific alleles of a TTTA repeat may be associated with differences in breast cancer risk. We have identified two new polymorphisms in the CYP19 gene: a TCT insertion/deletion in intron 4 and a G-->T substitution in intron 6, which have rare allele frequencies of 0.35 and 0.45, respectively, in the British population. Comparison was made between the frequencies of these alleles and those of the TTTA repeat in up to 599 breast cancer cases and 433 normal controls from the East Anglian, British population. We found strong linkage disequilibrium between the alleles of these three loci, but no significant association of any alleles with breast cancer risk. The maximum odds ratios observed were: 1.03 (95% CI 0.68-1.55) for the intron 4 TCT insertion/deletion polymorphism [del/del versus ins/ins]; 1.56 (95% CI 0.63-3.83) for the intron 4 [TTTA](10) allele; 1.29 (95% CI 0. 75-2.21) for the intron 6 G-->T polymorphism [TT versus GG]. We conclude that the CYP19 gene has no major role in common breast cancer incidence in the British population.

Direct, non-radioactive detection of mutations in multiple endocrine neoplasia type 2A families.

We have designed PCR primers that permit the rapid non-isotopic detection of mutations in codon 634 of the RET proto-oncogene, the causative mutations in over 80% of MEN 2A and 50% of FMTC families. In this paper we report the investigation of eleven MEN 2A families referred to the East Anglian Regional Genetics Service. Nine of these families carry codon 634 mutations. We were able to confirm the diagnosis of MEN 2A in twenty six affected individuals and to determine the carrier status of forty one individuals thought to be at risk of developing the disease. Of those at risk, thirty one patients lacked the familial mutation and ten were presymptomatic carriers of MEN 2A. In five cases the direct test proved that patients who had been treated by thyroidectomy but who lacked confirmed cancer, did not carry the familial mutation, removing the perceived risk of MEN 2A from their children. This group included one patient who had been diagnosed as having mild C-cell hyperplasia, confirming that in MEN 2A families C-cell hyperplasia can result from causes other than the presence of the MEN 2A mutation.

Mutation analysis of the c-mos proto-oncogene and the endothelin-B receptor gene in medullary thyroid carcinoma and phaeochromocytoma.

The characteristic tumours of MEN 2 are medullary thyroid carcinoma (MTC) and phaeochromocytoma. Somatic RET mutations have been found in only 23-40% of sporadic MTC and 10% of sporadic phaeochromocytomas. Thus, we sought other genes which may play a role in the pathogenesis of these tumours. We carried out direct sequence analysis of human c-mos and human ENRB in a series of sporadic MTC and phaeochromocytomas to determine if somatic mutations in these two genes could account for some of the sporadic MEN 2-related tumours in which no RET mutations are detected. No somatic mutations were found.

Polymorphisms in CYP1A1 and smoking: no association with breast cancer risk.

Several studies have investigated polymorphisms in CYP1A1 and breast cancer risk with inconsistent results. We have carried out a population based case-control study of the Thr461Asn and Ile462Val polymorphisms in CYP1A1 to clarify their importance in determining breast cancer susceptibility. A total of 1873 cases and 712 controls were genotyped for Thr461Asn and 1948 cases and 1355 controls were genotyped for Ile462Val. We have also investigated a putative interaction between smoking and CYP1A1 genotype and breast cancer risk using a case only study design. The genotype distribution of Thr461Asp in controls was close to that expected under Hardy-Weinberg equilibrium (HWE). We detected no significant differences in genotype frequencies between breast cancer cases and controls (P = 0.68). Compared with the Thr/Thr homozygotes there was no significant risk for either the Thr/Asp heterozygote [OR = 1.1 (95% CI 0.8-1.4)] or the Asp/Asp homozygote [OR = 0.4 (0.02-6.1)]. The genotype distribution of Ile462Val in controls was also close to that expected under HWE with no significant differences between breast cancer cases and the controls (P = 0.44). No significant risk was found for either the Ile/Val heterozygote [OR = 0.8 (0.6-1.1)] or the Val/Val homozygote [OR = 2.7 (0.3-24)] compared with the Ile/Ile homozygotes. Furthermore, subgroup analyses revealed no effect of age or menopausal status on genotypic risks, and we found no evidence for an interaction between genotype and smoking habit or alcohol consumption and susceptibility to breast cancer. We combined our data for the Ile462Val polymorphism with those from four other published studies, but even with >5000 subjects, none of the genotype-associated risks achieved statistical significance, and there was no consistent pattern to the risks associated with increasing Val allele dosage [Ile/Val OR = 0.9 (0.7-1.1), Val/Val OR = 2.3 (0.4-12), and Val carrier OR = 1.0 (0.9-1.1)].

Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A.

Multiple endocrine neoplasia type 2A (MEN 2A) is a dominantly inherited cancer syndrome that affects tissues derived from neural ectoderm. It is characterized by medullary thyroid carcinoma (MTC) and phaeochromocytoma. The MEN2A gene has recently been localized by a combination of genetic and physical mapping techniques to a 480-kilobase region in chromosome 10q11.2 (refs 2,3). The DNA segment encompasses the RET proto-oncogene, a receptor tyrosine kinase gene expressed in MTC and phaeochromocytoma and at lower levels in normal human thyroid. This suggested RET as a candidate for the MEN2A gene. We have identified missense mutations of the RET proto-oncogene in 20 of 23 apparently distinct MEN 2A families, but not in 23 normal controls. Further, 19 of these 20 mutations affect the same conserved cysteine residue at the boundary of the RET extracellular and transmembrane domains.

Mutations in the RET proto-oncogene and the von Hippel-Lindau disease tumour suppressor gene in sporadic and syndromic phaeochromocytomas.

Phaeochromocytomas may occur sporadically, or as part of the inherited cancer syndromes multiple endocrine neoplasia (MEN) type 2, von Hippel-Lindau disease (VHL), and, rarely, in type 1 neurofibromatosis. In MEN 2, germline missense mutations have been found in one of eight codons within exons 10, 11, 13, 14, and 16 of the RET proto-oncogene. In VHL, germline mutations within one of the three exons are responsible for the majority of cases. To determine if somatic mutations similar to those seen in the germline in MEN 2 or VHL disease play a role in the pathogenesis of sporadic or familial phaeochromocytomas, we analysed 48 sporadic tumours and tumours from 17 MEN 2 and five VHL patients for mutations in RET exons 9, 10, 11, 13, 14, 15, and 16, and the entire coding sequence of VHL. Five of 48 sporadic phaeochromocytomas had RET mutations within exons 10, 11, and 16. Of these, one was proven to be germline and two were proven to be somatic mutations. Four of 48 had VHL mutations; these included both the bilateral cases in the series (one was proven to be a germline mutation) and two others, of which one was proven somatic.

Point mutation within the tyrosine kinase domain of the RET proto-oncogene in multiple endocrine neoplasia type 2B and related sporadic tumours.

The susceptibility loci for the three multiple endocrine neoplasia (MEN) type 2 syndromes have been mapped to the region of chromosome 10q11.2 containing the RET proto-oncogene, which codes for a receptor tyrosine kinase. The majority of MEN 2A and familial medullary thyroid carcinoma results from missense mutations within one of five cysteine codons in the extracellular domain of the RET proto-oncogene. We now report a missense mutation, resulting in the substitution of a threonine for a methionine at codon 918 in the tyrosine kinase catalytic domain, in the germline of 26 of 28 apparently distinct families with MEN 2B. DNA from five of 13 apparently sporadic MTC and one of 12 apparently sporadic phaeochromocytomas harboured a similar mutation, but the corresponding germline DNA was wildtype in each case.

No association between a polymorphism in the steroid metabolism gene CYP17 and risk of breast cancer.

A recent study showed an association between a single base substitution, T-->C, in the promotor region of the CYP17 gene, the risk of breast cancer and age at menarche in Asian, African-American and Latino women from California and Hawaii. The C allele was associated with increased risk of breast cancer, significantly so for patients presenting with advanced disease, whereas the TT genotype was associated with later age at menarche in control subjects. We attempted to confirm these findings in a large case-control study in East Anglia, England (835 cases and 591 control subjects). We found no evidence of an increased risk of breast cancer [odds ratio (OR) 1.10, confidence interval (CI) 0.89-1.37] or advanced breast cancer (OR 0.88, CI 0.38-2.01) in C allele carriers, nor any association between age at menarche and genotype. We conclude that these alleles do not significantly alter breast cancer risk in the English population.