A Novel Double Mutation Val648ile and Val804leu of Ret Proto-Oncogene in Multiple Endocrine Neoplasia Type 2

Objective: We report the case of a female patient with multiple endocrine neoplasia type 2A (MEN2A) who was found to have a double mutation in the RET (rearranged during transfection) proto-oncogene.Methods:RET mutational analysis was performed by Sanger DNA sequencing.Results: The proband was a compound heterozygote for the RET germline mutations Val648Ile and Val804Leu on exons 11 and 14, respectively. Genetic analysis of family members showed the presence of the Val648Ile mutation in all except 1 daughter who carried the Val804Leu mutation. However, none of them showed any clinical, biochemical, or histologic signs of neoplastic disease either in the thyroid or adrenal gland. Furthermore, a daughter and the proband's sister who underwent a prophylactic thyroidectomy did not show pathologic evidence of C-cell disease.Conclusions: We hypothesize that the combined effect of the 2 mutations may have induced the development of pheochromocytoma (PHEO) in our patient. Thus, in the presence of single RET-induced mild medullary thyroid cancer (MTC) phenotype, the search for additional genetic anomalies may lead to the discovery of rare but potentially more aggressive double mutation genotypes.Abbreviations: ACTH = adrenocorticotropic hormone ATA = American Thyroid Association CT = calcitonin FMTC = familial medullary thyroid cancer HSCR = Hirschsprung disease MEN2A/B = multiple endocrine neoplasia type 2A/2B MTC = medullary thyroid cancer PHEO = pheochromocytoma RET = rearranged during transfection SDHB/D = succinate dehydrogenase subunits B/D VHL = Von Hippel-Lindau     

A large family with hereditary MTC: role of RET genetic analysis in differential diagnosis between MEN 2A and FMTC.

Germline mutations of the RET proto-oncogene cause three different cancer syndromes: multiple endocrine neoplasia type 2A (MEN 2A), multiple endocrine neoplasia type 2B (MEN 2B) and familial medullary thyroid carcinoma (FMTC). In the absence of biochemical and/or clinical evidence of pheochromocytoma and hyperparathyroidism, patients with MEN 2A disease display the same phenotype of FMTC disease, although prognosis and clinical management in both affected and unaffected familial members are quite different. We studied a family with hereditary MTC, whose proband was referred to us because of enlarged cervical nodes and increased calcitonin serum levels 28 years after the total thyroidectomy for MTC. Cervical node dissection was carried out and subsequently the presence of MTC metastasis was histologically confirmed. A RET genomic mutation at codon 634 (TGC-->TTC) was identified in the proband and in seven out of 19 familial members studied. Accordingly, a hereditary disease was suggested. However, the strong association of RET mutation at codon 634 with the presence of pheochromocytoma in MEN 2 disease suggested a more rigorous management in all gene carriers. Indeed, during the follow-up pheochromocytoma was subsequently identified in the proband. This finding suggests that all families with a pedigree suggestive of FMTC should be regarded at risk from MEN 2A disease, at least when a critical mutation in the RET cysteine domain is detected.     

Analysis of Mutations of the RET Proto-oncogene in Patients with Medullary Thyroid Carcinoma

The spectrum of mutations of the RET proto-oncogene was analyzed in Russian patients with inherited or sporadic medullary thyroid carcinoma (MTC). Four RET exons (11, 13, 15, and 16) were subjected to molecular analysis, and mutations were revealed and identified in 47.4% (9/19) patients with sporadic MTC. In total, six different mutations (including three new ones) were observed. The most common mutation affected codon 918 to cause substitution of methionine with threonine and accounted for 31.6% alleles. Analysis of exons 11 and 16 revealed four types of mutations in patients with inherited multiple endocrine neoplasia type 2 (MEN 2). Mutations were found in each patient. Thyroidectomy was performed in four asymptomatic carriers of RET mutations from three MEN 2A families (in two families, affected relatives had bilateral pheochromocytoma). In two patients, analysis of the surgery material revealed MTC microfoci in both lobes of the thyroid gland. The results provide the ground for constructing a bank of genetic information on Russian MTC patients with the clinically verified diagnosis.     

Description of the first two seemingly unrelated Greek Cypriot families with a common C618R RET proto-oncogene mutation

Germ-line mutations of the RET proto-oncogene cause three different cancer syndromes: multiple endocrine neoplasia type 2A (MEN2A), multiple endocrine neoplasia type 2B, and familial medullary thyroid carcinoma (FMTC). The objective of the present study was the clinical and molecular characterization of the first two Greek Cypriot families diagnosed with MEN2A and FMTC. The clinical diagnosis of the probands was based on clinical presentation and supported with laboratory findings (calcitonin and carcinoembryonic antigen tumor marker levels). We screened the RET gene by direct DNA sequencing of exons 10, 11, and 16 using genomic DNA as templates. After identification of the mutation, we also developed the amplification refractory mutation system (ARMS) as an alternative method to direct sequencing for genetic diagnosis of 22 additional individuals from both families. We identified the germ-line missense mutation T --> C of codon 618 of exon 10 (C618R) in the probands of both families. By using ARMS, two members of the MEN2A family and five members of the FMTC family were also found positive for the C618R mutation. These are the first seemingly unrelated families in Cyprus investigated clinically and molecularly in detail and shown to transmit this common RET proto-oncogene mutation.     

Mutation analysis of the RET proto-oncogene in Dutch families with MEN 2A, MEN 2B and FMTC: two novel mutations and one de novo mutation for MEN 2A

Hereditary C-cell carcinoma is encountered in multiple endocrine neoplasia type 2A (MEN 2A), MEN 2B, and familial medullary thyroid carcinoma (FMTC). Mutations of the RET proto-oncogene are associated with all three diseases. To obtain an insight into the molecular heterogeneity of MEN 2 syndromes and FMTC in the Netherlands, probands of 20 MEN 2A families, two FMTC families, and seven MEN 2B families were analyzed by the polymerase chain reaction (PCR), DNA sequencing, and restriction enzyme digestion for abnormalities in the RET proto-oncogene. RET mutations were found in all cases. All MEN 2A families had a mutation involving one of five cysteine codons in exons 10 and 11 of RET. Two novel dinucleotide mutations and a de novo mutation were found. Both FMTC families had a mutation of the Cys at codon 618. All MEN 2B probands carried a Met to Thr mutation in exon 16. All mutations could be confirmed by restriction enzyme digestion of PCR amplicons. Identification of the RET mutation in the Dutch population with hereditary C-cell carcinoma facilitates genetic testing for families or individuals at risk for MEN 2A, FMTC, and MEN 2B.     

RET germline mutations identified by exome sequencing in a Chinese multiple endocrine neoplasia type 2A/familial medullary thyroid carcinoma family

Background

Whole exome sequencing provides a labor-saving and direct means of genetic diagnosis of hereditary disorders in which the pathogenic gene harbors a large cohort of exons. We set out to demonstrate a suitable example of genetic diagnosis of MEN 2A/FMTC (multiple endocrine neoplasia type 2/familial medullary thyroid carcinoma) using this approach.

Methodology/Principal Findings

We sequenced the whole exome of six individuals from a large Chinese MEN2A/FMTC pedigree to identify the variants of the RET (REarranged during Transfection) protooncogene and followed this by validation. Then prophylactic or surgical thyroidectomy with modified or level VI lymph node dissection and adrenalectomy were performed for the carriers. The cases were closely followed up. Massively parallel sequencing revealed four missense mutations of RET. We unexpectedly discovered that the proband''s daughter with MEN 2A-related MTC presented a novel p.C634Y/V292M/R67H/R982C compound mutation, due to the involvement of p.C634Y in the proband with MEN 2A and p.V292M/R67H/R982C in the proband''s husband with FMTC. In the maternal origin, p.C634Y caused bilateral MTC in all 5 cases and bilateral pheochromocytoma in 2 of the 5; the earliest onset age was 28 years. In the paternal origin, one of the six p.V292M/R67H/R982C carriers presented bilateral MTC (70 years old), one only had bilateral C-cell hyperplasia (44 years), two had bilateral multi-nodules (46 and 48 years) and two showed no abnormality (22 and 19 years).

Conclusions/Significance

The results confirmed the successful clinical utility of whole exome sequencing, and our data suggested that the p.C634Y/V292M/R67H/R982C mutation of RET exhibited a more aggressive clinical phenotype than p.C634Y or p.V292M/R67H/R982C, while p.V292M/R67H/R982C presented a relatively milder pathogenicity of MTC and likely predisposed to FMTC.     

Genomic and Yeast Artificial Chromosome Long-Range Regular Article Linking Six Loci in 10q11.2 and Spanning the Multiple Endocrine Neoplasia Type 2A (MEN2A) Region

Multiple endocrine neoplasia types 2A and 2B (MEN 2A and MEN 2B) and familial medullary thyroid carcinoma (FMTC) are dominantly inherited cancers that have in common the clinical feature of medullary thyroid carcinoma (MTC). We have performed both genomic long-range restriction mapping and yeast artificial chromosome (YAC) contig assembly and restriction mapping to establish physical linkage, order, and distances between six loci in 10q11.2 near the genes responsible for these hereditary cancers. RET, D10S94, D10S182, and D10S102 have been mapped in genomic DNA. RET, D10S94, D10S182, D10F3853, and the 10q11.2 sequences detected by DNA marker DM124 are encompassed by a 1-Mb YAC contig. Six physically linked loci are within 1.4 Mb and have an order and orientation of 10cen, D10F38S3, DM124, RET, D10S94, D10S182, D10S102, 10qter. Mutations in the RET proto-oncogene have recently been demonstrated to be associated with MEN 2A and FMTC. RET is located within a genetically defined MEN2A candidate interval between D10S141 and D10S94; MEN2B has been mapped to a larger, overlapping region between D10S141 and a more distal locus, RBP3. Both our genomic physical map and our YAC contig span the entire MEN2A candidate region and overlap with that of MEN2B . These maps will facilitate the identification of genes that can be considered candidates for MEN2B and the identification of tumor-specific alterations important in sporadic MTC.     

Detection of a germline mutation at codon 918 of the RET proto-oncogene in French MEN 2B families

Multiple endocrine neoplasia type 2A (MEN2A), type 2B (MEN 2B), and familial medullary thyroid carcinoma (FMTC) are three dominantly inherited disorders linked to the same disease locus on chromosome 10. Two types of germline mutation of the RET proto-oncogene, which codes for a transmembrane tyrosine kinase, are associated with MEN 2. Missense mutations at cysteine residues in the extra-cytoplasmic domain are exclusively associated with MEN 2A and FMTC. In MEN 2B patients, a single point mutation at codon 918 has recently been characterized, leading to the replacement of a methionine by a threonine within the RET tyrosine kinase domain. We now report the identification of a mutation at codon 918 in the germline of 16 patients out of 18 unrelated MEN 2B families analyzed. In these families we have been able to demonstrate that, in five cases, the mutation arose de novo, and that, in one kindred, it was coinherited with the disease. These results indicate that a unique mutation at codon 918 of the RET gene is the most prevalent genetic defect causing MEN 2B, but also that rare MEN 2B cases are associated with different mutations yet to be defined.     

Clinical and genetic profile of patients with medullary thyroid cancer treated in the Cancer Centre--Institute of Oncology in Warsaw

INTRODUCTION: The aim of this study was to analyse the distribution and frequency of mutations and their correlations with clinical phenotypes of patients with MTC, to reveal the differences between sporadic and familial type of MTC, and to describe the phenotypes of patients. MATERIALS AND METHODS: 212 patients with medullary thyroid cancer (MTC) were treated in Cancer Centre in Warsaw between 1997 and 2005. In most patients, DNA isolated from peripheral blood leukocytes was tested for RET gene mutations by sequencing and accordingly MTC form was assessed. Genetic testing was performed in the relatives of patients with familial MTC in order to distinguish asymptomatic mutation carriers from noncarriers. RESULTS: RET gene mutations were identified in 46 patients (22%). The others were found noncarriers and sporadic MTC was diagnosed. MEN 2A/FMTC syndrome (multiple endocrine neoplasia type 2A/ familial type of MTC) was diagnosed in 44 patients, MEN 2B syndrome (multiple endocrine neoplasia type 2B) in 2 patients. In patients with sporadic and familial MTC, age at diagnosis and multifocal occurrence was analysed, and the results were found to be in accordance with those of other research centres. However, the distribution and frequency of mutations, as well as some clinical data, such as the frequency of pheochromocytoma occurrence as the first manifestation of MEN syndrome, differed from the published data, and further studies are necessary to reveal the reasons of these differences. CONCLUSIONS: DNA testing for RET gene mutations is reliable as a diagnostic tool and therefore it should be performed for screening of all patients with MTC or other diseases of MEN syndrome.     

The Relationship of Cytomorphology of Medullary Thyroid Carcinomas between Family Members with the Same RET Proto-Oncogene Mutation

Objective: To evaluate the relationship of the cytomorphology of medullary thyroid carcinomas (MTC) between family members with the same RET proto-oncogene mutation. Study Design: Review of the fine-needle aspiration slides of 13 cases with MTC proven by surgery and pathology from 5 unrelated families with either multiple endocrine neoplasia (MEN) type 2A or familial MTC (FMTC). Results: Small, round, and abundant large oval-to-polygonal cells were major cytomorphologic findings in 66.7% of family members with exon 11, codon 634 TGC → CGC germline mutation. Small, round cells and only a few or no polygonal cells were found in 66.7% of family members with exon 11, codon 634 TGC → TTC germline mutation and in 100% of family members with codon 634 TGC → TGG germline mutations, as well as in 100% of family members with exon 10, codon 620 TGC → GGC germline mutation. Conclusions: The high rate of similarity of cytomorphology (66.7-100%) in the family members with MEN type 2A or FMTC might be related to the same etiology in the production of MTC in the same family. The relationship of the respective cytomorphology with the long-term prognosis is worth elucidating further.     

Point mutagenesis in mouse reveals contrasting pathogenetic effects between MEN2B- and Hirschsprung disease-associated missense mutations of the RET gene

Missense mutations of the RET gene have been identified in both multiple endocrine neoplasia (MEN) type 2A/B and Hirschsprung disease (HSCR: congenital absence of the enteric nervous system, ENS). Current consensus holds that MEN2A/B and HSCR are caused by activating and inactivating RET mutations, respectively. However, the biological significance of RET missense mutations in vivo has not been fully elucidated. In the present study, we introduced one MEN2B-associated (M918T) and two HSCR-associated (N394K and Y791F) RET missense mutations into the corresponding regions of the mouse Ret gene by genome editing (Ret^M919T, Ret^N396K and Ret^Y792F) and performed histological examinations of Ret-expressing tissues to understand the pathogenetic impact of each mutant in vivo. Ret^M919T/+ mice displayed MEN2B-related phenotypes, including C-cell hyperplasia and abnormal enlargement of the primary sympathetic ganglia. Similar sympathetic phenotype was observed in Ret^M919T/- mice, demonstrating a strong pathogenetic effect of the Ret M918T by a single-allele expression. In contrast, no abnormality was found in the ENS of mice harboring the Ret N394K or Y791F mutation. Most surprisingly, single-allele expression of RET N394K or Y791F was sufficient for normal ENS development, indicating that these RET mutants exert largely physiological function in vivo. This study reveals contrasting pathogenetic effects between MEN2B- and HSCR-associated RET missense mutations, and suggests that some of HSCR-associated RET missense mutations are by themselves neither inactivating nor pathogenetic and require involvement of other gene mutations for disease expressivity.     

The minisequencing method: a simple strategy for genetic screening of MEN 2 families

Background  

Multiple endocrine neoplasia type 2 is an autosomal dominant disorder. MEN 2A is characterized by medullary thyroid carcinoma, pheochromocytoma and hyperparathyroidism; MEN 2B by medullary thyroid carcinoma, pheochromocytoma and characteristic stigmata. Activating germline mutations of the RET proto oncogene are responsible for this hereditary syndrome. Codon 634 mutations are the most common mutations occurring in MEN 2A families whereas a specific mutation at codon 918 is observed in the great majority of MEN 2B families. Analysis of these codons will provide a final diagnosis in the great majority of affected families making unnecessary further studies. To specifically study the codons 634 and 918 we used a minisequencing method as an alternative method to complete sequencing.     

RET tyrosine kinase signaling in development and cancer

The variety of diseases caused by mutations in RET receptor tyrosine kinase provides a classic example of phenotypic heterogeneity. Gain-of-function mutations of RET are associated with human cancer. Gene rearrangements juxtaposing the tyrosine kinase domain to heterologous gene partners have been found in sporadic papillary carcinomas of the thyroid (PTC). These rearrangements generate chimeric RET/PTC oncogenes. In the germline, point mutations of RET are responsible for multiple endocrine neoplasia type 2 (MEN 2A and 2B) and familial medullary thyroid carcinoma (FMTC). Both MEN 2 mutations and PTC gene rearrangements potentiate the intrinsic tyrosine kinase activity of RET and, ultimately, activate the RET downstream targets. Loss-of-function mutations of RET cause Hirschsprung's disease (HSCR) or colonic aganglionosis. A deeper understanding of the molecular signaling of normal versus abnormal RET activity in cancer will enable the development of potential new treatments for patients with sporadic and inherited thyroid cancer or MEN 2 syndrome. We now review the role and mechanisms of RET signaling in development and carcinogenesis.     

Germline mutations of the RET proto-oncogene in eight Japanese patients with multiple endocrine neoplasia type 2A (MEN2A)

Multiple endocrine neoplasia type 2A (MEN2A) is a dominantly inherited cancer syndrome characterized by medullary thyroid carcinoma, pheochromocytoma, and parathyroid hyperplasia. The gene responsible for MEN2A was localized by linkage analysis to chromosome 10q11.2 in 1987, and recently mutations in RET, a proto-oncogene in the candidate region, were discovered in patients with MEN. The majority of mutations found so far in MEN2A patients have been located in nucleotide sequences encoding cysteine residues in the extracellular domain of RET. To characterize MEN2A germline alterations in the Japanese population, we screened DNA from eight unrelated patients for mutations in exons 10 and 11 of the RET proto-oncogene and found mutations in all eight patients, at codons 618, 620, or 634; each of these sites encodes a cysteine residue in the extracellular domain of RET. The mutations were confirmed in other affected individuals in the respective families by digestion of polymerase chain reaction (PCR) products containing the mutated codons with restriction enzymes (RsaI, CfoI, or AluI) for which cleavage sites had been generated by the specific genetic alteration. These PCR-restriction enzyme systems will be useful for genetic diagnosis in members of families carrying these mutations.     

The rare intracellular RET mutation p.S891A in a Chinese Han family with familial medullary thyroid carcinoma

We report intracellular RET mutation in a Han Chinese pedigree with familial medullary thyroid carcinoma (FMTC). Direct sequencing of RET proto-oncogene identified a missense c.2671T>G (p.S891A) mutation in 6 of 14 family members. The single nucleotide polymorphisms c. 135A>G (p.A45A), IVS4+48A>G, c. 1296A>G (p.A432A), c. 2071G>A (p.G691S), c. 2307T>G (p.L769L) and a variant c. 833C>A (p.T278N) were also found in 6 carriers. Among 5 of the 6 carriers presented medullary thyroid carcinoma (MTC) as an isolated clinical phenotype, with elevated basal serum calcitonin (Ct). Two underwent non-normative thyroidectomy either two or four times without physician awareness or diagnosis of this disease at initial treatment, but with elevated Ct. One with elevated pre-Ct accepted total thyroidectomy (TT) with modified bilateral neck dissection (MBiND), and whose seventh posterior rib MTC metastases was confirmed 5 months after surgery. Moreover, results of two affected individuals with elevated Ct were reduced to normal after TT with MBiND or prophylactic VI compartmental dissection. However, only another carrier with the variant p.T278N had slightly elevated Ct rejected surgery and was strictly monitored. Given these case results, we suggest that screening of RET and pre-surgical Ct levels in the management of MTC patients is essential for earlier diagnosis and more normative initial treatment, that FMTC patients with cervical lymph nodes metastases may be cured by TT with MBiND, and that prophylactic VI compartmental dissection should be avoided when Ct levels are low.     

Transforming ability of MEN2A-RET requires activation of the phosphatidylinositol 3-kinase/AKT signaling pathway

The RET gene codes for a receptor tyrosine kinase that plays a crucial role during the development of both the enteric nervous system and the kidney. Germ line missense mutations at one of six codons specifying extracytoplasmic cysteines are responsible for two related cancer disorders as follows: multiple endocrine neoplasia type2A (MEN2A) and familial medullary thyroid carcinoma (FMTC). MEN2A and FMTC mutations result in a constitutive catalytic activity and as a consequence convert RET into a dominantly acting transforming gene. Although it has been shown that RET-MEN2 mutants activate several transduction pathways, their respective contribution to the neoplastic phenotype remains poorly understood. Over the past few years, it has become increasingly clear that the transforming ability of several viral and cellular oncoproteins depends on their capacity to activate phosphatidylinositol 3-kinase (PI3K). We now report that RET carrying a representative MEN2A mutation at Cys-634 (termed RET-MEN2A) activates PI3K and its downstream effector, the serine/threonine kinase AKT/protein kinase B. Previous studies have demonstrated that mutation of Tyr-1062, which is the intracellular docking site for Shc and Enigma on RET, abolishes the RET-MEN2A transforming activity. We provide evidence that mutation of Tyr-1062 abrogates the binding of the p85 regulatory subunit of PI3K to RET-MEN2A and the subsequent stimulation of the PI3K/AKT pathway. Furthermore, infection of rat fibroblasts with a retrovirus expressing a dominant-interfering form of PI3K suppresses RET-MEN2A-dependent transformation, whereas overexpression of AKT enhances the RET-MEN2A oncogenic potential. In summary, these data are consistent with the notion that RET-mediated cell-transforming effect is critically dependent on the activation of the PI3K/AKT pathway.     

Medullary thyroid carcinoma: the comparison of the hereditary and sporadic types of cancer

INTRODUCTION: The assessment of frequency and type of mutation and differences in prognosis between sporadic and hereditary type of medullary thyroid carcinoma (MTC), based on own DNA analysis, was performed. MATERIAL AND METHODS: The group of 190 persons with hereditary MTC or asymptomatic mutation carriers was analyzed. Patients with sporadic MTC without RET gene mutation were included into control group (708 persons). The recognition of MTC type was based on assessment of family history, physical examination and genetic analysis. The family history consisted of information about MTC, pheochromocytoma and other neoplasms and hyperparathyroidism in relatives. RESULTS: The mutations located in codon 634 of exon 11 were the most often (43% of all mutations and 49% of mutations in syndrome MEN 2A/FMTC). The age of diagnosis was ranged between 7 and 71 years (mean age: 39 +/- 15.2 years, median age: 41 years). In hereditary MTC the mean age of diagnosis was 27 +/- 13.9 years and was significantly lower than in sporadic one, where it was 45.7 +/- 14.3 years. The relationship between diagnosis, age and subtypes of hereditary MTC was assessed--no significant differences in examined subgroups were observed. The mean age of diagnosis in MEN 2A/FMTC and MEN 2A syndrome was 28-29 years, in MEN 2B - 21 years. The overall survival in sporadic MTC after 5 years was 97%, in hereditary MTC - 79%. Analysis performed after excluding suprarenal causes of death revealed no statistically significant differences in overall survival between both subtypes of MTC. CONCLUSIONS: 1. Hereditary MTC is still diagnosed too late, besides of DNA analysis. 2. In hereditary and sporadic MTC the prognosis is comparable.     

Sequence analysis of the MEN I gene in two patients with multiple cutaneous lipomas and endocrine tumors.

The discovery of mutations of the menin gene in a few multiple endocrine neoplasma type 1 (MEN I)-associated lipomas and loss of heterozygosity (LOH) on chromosome 11q13 in some sporadic lipomas has stimulated the hypothesis that lipomas may belong to the group of sporadic tumors caused by defects of the gene responsible for MEN I. Since it is unclear if the above hypothesis applies to all patients with lipoma or just to specific subsets, we searched to enlarge the database on this topic. For this purpose, we identified two patients with multiple cutaneous lipomas. One had an additional pituitary adenoma and familial presentation of multiple lipomas, the other had recurrent goiter in the setting of a family history of adenomatous goiter. Deoxyribonucleic acid (DNA) was analyzed by complete direct DNA sequencing of all coding exons and splice junctions of the MEN I gene. No mutation was identified in the coding exons of the menin gene. In contrast to former data on sporadic lipomas, these data are the first to render evidence that mutations of the MEN I gene may not be responsible for the formation of multiple lipomas, even if they appear in the context of other endocrine tumors.     

A comprehensive,sensitive and economical approach for the detection of mutations in the <Emphasis Type="Italic">RB1</Emphasis> gene in retinoblastoma

Retinoblastoma (Rb) is the most common primary intraocular malignancy in children. It is brought about by the mutational inactivation of both alleles of RB1 gene in the developing retina. To identify the RB1 mutations, we analysed 74 retinoblastoma patients by screening the exons and the promoter region of RB1. The strategy used was to detect large deletions/duplications by fluorescent quantitative multiplex PCR; small deletions/insertions by fluorescent genotyping of RB1 alleles, and point mutations by PCR-RFLP and sequencing. Genomic DNA from the peripheral blood leucocytes of 74 Rb patients (53 with bilateral Rb, 21 with unilateral Rb; 4 familial cases) was screened for mutations. Recurrent mutations were identified in five patients with bilateral Rb, large deletions in 11 patients (nine with bilateral Rb and two with unilateral Rb), small deletions/insertions were found in 12 patients all with bilateral Rb, and point mutations in 26 patients (14 nonsense, six splice site, five substitution and one silent change). Three mutations were associated with variable expressivity of the disease in different family members. Using this method, the detection rates achieved in patients with bilateral Rb were 44/53 (83%) and with unilateral Rb, 5/21 (23.8%). This approach may be feasible for clinical genetic testing and counselling of patients.