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.     

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.     

Molecular and biochemical screening for the diagnosis and management of medullary thyroid carcinoma in multiple endocrine neoplasia type 2A.

Patients with Multiple Endocrine Neoplasia (MEN) type 2A are at risk for early medullary thyroid carcinoma (MTC). We performed different screening tests for MTC--a recently reported biochemical screening test using omeprazole-induced calcitonin (CT) stimulation and DNA analysis--in fifteen members of two non-consanguineous Brazilian families with MEN 2A. RET proto-oncogene analysis was carried out by direct DNA sequencing of PCR-amplified products for exons 10 and 11. Family 1 showed a germline mutation (C634Y) in three individuals; a sister and a brother with symptomatic MTC; the former also presented with pheochromocytoma and hyperparathyroidism, and her son was a nine-year-old boy of previously unknown status. Family 2 showed the C634R mutation only in the index case, who presented with cutaneous lichen amyloidosis in addition to MTC, pheochromocytoma and hyperparathyroidism. Neither her parents nor her four brothers showed this genetic abnormality, suggesting a de novo RET proto-oncogene mutation in this patient. The controls and patients presented normal basal gastrin levels and a significant increase after omeprazole. Basal CT levels were elevated in patients with MTC and undetectable in control and asymptomatic family members. No subject showed any increase in CT levels after omeprazole treatment. In conclusion, the two most frequent RET proto-oncogene mutations in MEN 2A are present in Brazilian families. In addition, the specificity of basal and omeprazole-stimulated calcitonin is rather limited, and the efficacy of the omeprazole test still needs to be systematically examined. Therefore, RET proto-oncogene analysis must be the first choice for a screening procedure to identify gene carriers in MEN 2A family members and to permit early prophylactic treatment of MTC.     

Medullary thyroid carcinoma: from molecular studies to clinical decision

The paper is focused on guidelines of practice in inherited medullary thyroid cancer, diagnosed on the basis of DNA analysis. Identification of RET mutation implies further steps of diagnostic procedure, some of them - USG, FNAB and calcitonin level tests - are common for all types of mutation, other are related to ascertained type of mutation. In asymptomatic RET mutation carriers, prophylactic thyroidectomy is indicated. In MEN2B inherited cancer reveals its symptoms quickly and shows dynamic progress. In MEN2A/FMTC the clinical picture is diversified - in some patients the course of disease is mild, however in some other cases the progression of disease and even death occur regardless of the proper treatment. Unfortunately, there are no molecular prognostic markers in medullary thyroid carcinoma. Recent papers and also our own unpublished results show that gene expression profile, is similar in MEN2A and sporadic cancer. This group differs from MEN2B by its expression profile. In conclusion it is to be emphasized that although inherited medullary thyroid carcinoma is a rare disease, the diagnostic algorithm is well established and maximizes the chance for early diagnosis. Moreover, it needs to be stressed that DNA analysis results inform us not only about the necessity of further therapy, but also suggest different ways of proceeding in particular type of mutation.     

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.     

Biological effects of the dual phenotypic Janus mutation of ret cosegregating with both multiple endocrine neoplasia type 2 and Hirschsprung's disease

Gain-of-function mutations of ret receptor tyrosine kinase, the signaling receptor for glial cell line-derived neurotrophic factor, cause sporadic thyroid and adrenal malignancies as well as endocrine cancer syndromes, such as multiple endocrine neoplasia types 2A and 2B (MEN 2A and MEN 2B) and familial medullary thyroid carcinoma. Loss-of-function mutations of ret cause Hirschsprung's disease (HSCR) or colonic aganglionosis. In 20-30% of families with a mutation at residues 609, 611, 618, or 620 of RET, MEN 2A and familial medullary thyroid carcinoma cosegregate with HSCR. These mutations constitutively activate RET due to aberrant disulfide homodimerization and diminish the level of RET at the plasma membrane. It is not known how these mutations simultaneously lead to both gain- and loss-of-function RET-associated diseases. We provide an explanation for the dual phenotypic Janus mutation at Cys620 of RET. In Madin-Darby canine kidney (MDCK) cells, the Janus mutation impairs the glial cell line-derived neurotrophic factor-induced effects of RET on cell migration, differentiation, and survival but simultaneously promotes rapid cell proliferation.     

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.     

Proteomic Identification of DNA-PK Involvement within the RET Signaling Pathway

Constitutive activation of the Rearranged during Transfection (RET) proto-oncogene leads to the development of MEN2A medullary thyroid cancer (MTC). The relatively clear genotype/phenotype relationship seen with RET mutations and the development of MEN2A is unusual in the fact that a single gene activity can drive the progression towards metastatic disease. Despite knowing the oncogene responsible for MEN2A, MTC, like most tumors of neural crest origin, remains largely resistant to chemotherapy. Constitutive activation of RET in a SK-N-MC cell line model reduces cell sensitivity to chemotherapy. In an attempt to identify components of the machinery responsible for the observed RET induced chemoresistance, we performed a proteomic screen of histones and associated proteins in cells with a constitutively active RET signaling pathway. The proteomic approach identified DNA-PKcs, a DNA damage response protein, as a target of the RET signaling pathway. Active DNA-PKcs, which is phosphorylated at site serine 2056 and localized to chromatin, was elevated within our model. Treatment with the RET inhibitor RPI-1 significantly reduced s2056 phosphorylation in RET cells as well as in a human medullary thyroid cancer cell line. Additionally, inhibition of DNA-PKcs activity diminished the chemoresistance observed in both cell lines. Importantly, we show that activated DNA-PKcs is elevated in medullary thyroid tumor samples and that expression correlates with expression of RET in thyroid tumors. These results highlight one mechanism by which RET signaling likely primes cells for rapid response to DNA damage and suggests DNA-PKcs as an additional target in MTC.     

Recurrent mutations in the factor IX gene: founder effect or repeat de novo events

The investigation of 114 unrelated patients, representing about half the sample of the German haemophilia B population, enabled us to delineate the causative mutation in 103 (90.4%) haemophilic factor IX genes. Of these 103 cases 84 (81.6%) turned out to be unique molecular events, the remainder being repeats. Haplotype analysis revealed that the great majority, if not all, of these recurrent observations occurred independently. This conclusion is supported by our finding that three de novo mutations could be demonstrated at two sites of frequent mutation. A further 20 de novo events could be established in an unselected sample of 37 families with sporadic haemophilia B and 37 families with a history of the disease. Altogether, the germ line of origin could be determined in 21 of these 23 cases, thereby indicating a ratio of male to female mutation rates close to 2. On the basis of the data available, it is becoming clear that rearrangements in the factor IX gene (35.4% of de novo cases) are responsible for haemophilia B at a higher frequency than has been observed today (12.3%). More than two-thirds of the de novo cases cause the severe form of the disease, thereby reflecting the deficit of these haemophilic genes in the actual gene pool because of excess mortality in the past. In addition 40% (12/30) of the de novo single-base mutations were transitions at CpG dinucleotides. Compared with the expected at-random frequency, this observation indicates an 83-fold enhancement of mutation at CpG.     

C-cell hyperplasia, pheochromocytoma and sympathoadrenal malformation in a mouse model of multiple endocrine neoplasia type 2B

Dominantly inherited multiple endocrine neoplasia type 2B (MEN2B) is characterized by tumors of the thyroid C-cells and adrenal chromaffin cells, together with ganglioneuromas of the gastrointestinal tract and other developmental abnormalities. Most cases are caused by substitution of threonine for Met918 in the RET receptor tyrosine kinase, which is believed to convert the RET gene to an oncogene by altering the enzyme's substrate specificity. We report the production of a mouse model of MEN2B by introduction of the corresponding mutation into the ret gene. Mutant mice displayed C-cell hyperplasia and chromaffin cell hyperplasia progressing to pheochromocytoma. Homozygotes did not develop gastrointestinal ganglioneuromas, but displayed ganglioneuromas of the adrenal medulla, enlargement of the associated sympathetic ganglia and a male reproductive defect. Surprisingly, homozygotes did not display any developmental defects attributable to a loss-of-function mutation. Thus, while our results support the conclusion that the Met918Thr substitution is responsible for MEN2B, they suggest that the substrate specificity of the RET kinase does not interfere with its normal role in the development of the kidneys and enteric nervous system.     

A two-hit model for development of multiple endocrine neoplasia type 2B by RET mutations

Multiple endocrine neoplasia (MEN) type 2B mutations have been reported at methionine 918 or alanine 883 in the tyrosine kinase domain of the RET proto-oncogene. Recently, a new combination of two germline missense mutations at valine 804 and tyrosine 806 was identified in a patient with MEN 2B-like clinical phenotypes including medullary thyroid carcinoma, mucosal neuroma, and marfanoid habitus. In this case, valine 804 and tyrosine 806 were replaced with methionine and cysteine, respectively. In the present study, biological activities of RET with these new mutations were compared with those with known MEN 2A or MEN 2B mutations. The transforming activity of RET with the V804M/Y806C mutation was about 8- to 13-fold higher than that of RET with a single V804M or Y806C mutation. Like RET with the M918T or A883F MEN 2B mutation, the transforming activity of RET with the V804M/Y806C mutation was not affected by substitution of phenylalanine for tyrosine 905 that abolished the activity of RET with the MEN 2A mutation. On the other hand, substitution of phenylalanine for tyrosines 864 and 952 drastically diminished the activity of RET with the V804M/Y806C, M918T or A883F mutation, suggesting that these three mutant proteins have similar biological properties.     

Molecular heterogeneity of RET loss of function in Hirschsprung's disease.

The RET proto-oncogene encodes a receptor with tyrosine kinase activity (RET) that is involved in several neoplastic and non-neoplastic diseases. Oncogenic activation of RET, achieved by different mechanisms, is detected in a sizeable fraction of human thyroid tumors, as well as in multiple endocrine neoplasia types 2A and 2B (MEN2A and MEN2B) and familial medullary thyroid carcinoma tumoral syndromes. Germline mutations of RET have also been associated with a non-neoplastic disease, the congenital colonic aganglionosis, i.e. Hirschsprung's disease (HSCR). To analyse the impact of HSCR mutations on RET function, we have introduced into wild-type RET and activated RET(MEN2A) and RET(MEN2B) alleles three missense mutations associated with HSCR. Here we show that the three mutations caused a loss of function of RET when assayed in two model cell systems, NIH 3T3 and PC12 cells. The effect of different HSCR mutations was due to different molecular mechanisms. The HSCR972 (Arg972-->Gly) mutation, mapping in the intracytoplasmic region of RET, impaired its tyrosine kinase activity, while two extracellular mutations, HSCR32 (Ser32-->Leu) and HSCR393 (Phe393-->Leu), inhibited the biological activity of RET by impairing the correct maturation of the RET protein and its transport to the cell surface.     

Differential effects of leukocyte common antigen-related protein on biochemical and biological activities of RET-MEN2A and RET-MEN2B mutant proteins

Protein-tyrosine-phosphatases (PTPs), in conjunction with protein-tyrosine kinases, play essential regulatory roles in diverse cellular activities by modulating the phosphorylation state of target proteins. Leukocyte common antigen-related (LAR) protein is a widely expressed receptor-type protein-tyrosine-phosphatase that is implicated in the regulation of intracellular signaling triggered by both cell adhesion and peptide growth factors. The gene for LAR is localized to human chromosome 1p32, a region frequently deleted in tumors of neuroectodermal origin, including neuroblastoma, pheochromocytoma, and medullary thyroid carcinoma. On the other hand, the RET gene codes for a transmembrane tyrosine kinase and is responsible for the development of multiple endocrine neoplasia (MEN) 2A and 2B. To explore the potential role of LAR in RET tyrosine kinase activity and RET-induced signal transduction, we cotransfected LAR and RET with a MEN2A or MEN2B mutation (designated RET-MEN2A or RET-MEN2B) into the NIH 3T3 cell line. Here we show that LAR reduces the constitutive tyrosine autophosphorylation and kinase activity of RET-MEN2A but not RET-MEN2B, accompanying a significant decrease of phosphorylation of phospholipase Cgamma, AKT, and ERK1/2. Interestingly, LAR expression significantly decreased the levels of disulfide-linked RET-MEN2A dimerization. Moreover, reduced oncogenic activity of RET-MEN2A by overexpression of LAR was observed both by an in vitro colony formation assay and by in vivo tumorigenicity in scid mice. These results thus suggest that LAR may contribute to deactivation of the RET-MEN2A mutant protein and reduction of its oncogenic activity in vivo.     

Positive selection for new disease mutations in the human germline: evidence from the heritable cancer syndrome multiple endocrine neoplasia type 2B

Multiple endocrine neoplasia type 2B (MEN2B) is a highly aggressive thyroid cancer syndrome. Since almost all sporadic cases are caused by the same nucleotide substitution in the RET proto-oncogene, the calculated disease incidence is 100-200 times greater than would be expected based on the genome average mutation frequency. In order to determine whether this increased incidence is due to an elevated mutation rate at this position (true mutation hot spot) or a selective advantage conferred on mutated spermatogonial stem cells, we studied the spatial distribution of the mutation in 14 human testes. In donors aged 36-68, mutations were clustered with small regions of each testis having mutation frequencies several orders of magnitude greater than the rest of the testis. In donors aged 19-23 mutations were almost non-existent, demonstrating that clusters in middle-aged donors grew during adulthood. Computational analysis showed that germline selection is the only plausible explanation. Testes of men aged 75-80 were heterogeneous with some like middle-aged and others like younger testes. Incorporating data on age-dependent death of spermatogonial stem cells explains the results from all age groups. Germline selection also explains MEN2B's male mutation bias and paternal age effect. Our discovery focuses attention on MEN2B as a model for understanding the genetic and biochemical basis of germline selection. Since RET function in mouse spermatogonial stem cells has been extensively studied, we are able to suggest that the MEN2B mutation provides a selective advantage by altering the PI3K/AKT and SFK signaling pathways. Mutations that are preferred in the germline but reduce the fitness of offspring increase the population's mutational load. Our approach is useful for studying other disease mutations with similar characteristics and could uncover additional germline selection pathways or identify true mutation hot spots.     

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.     

Role of Dok1 in cell signaling mediated by RET tyrosine kinase

Using a yeast two-hybrid screen, we identified Dok1 as a docking protein for RET tyrosine kinase. Dok1 bound more strongly to RET with a multiple endocrine neoplasia (MEN) 2B mutation than RET with a MEN2A mutation and was highly phosphorylated in the cells expressing the former mutant protein. Analysis by site-directed mutagenesis revealed that tyrosine 361 in mouse Dok1 represents a binding site for the Nck adaptor protein and tyrosines 295, 314, 361, 376, 397, and 408 for the Ras-GTPase-activating protein. We replaced tyrosine 361 or these six tyrosines with phenylalanine (designated Y361F or 6F) in Dok1 and introduced the mutant Dok1 genes into the cells expressing the wild-type RET or RET-MEN2B protein. Overexpression of Dok1 or Dok1-Y361F, but not Dok1-6F, suppressed the Ras/Erk activation induced by glial cell line-derived neurotrophic factor or RET-MEN2B, implying that this inhibitory effect requires the Ras-GTPase-activating protein binding to Dok1. In contrast, overexpression of Dok1, but not Dok1-Y361F or Dok1-6F, enhanced the c-Jun amino-terminal kinase (JNK) and c-Jun activation. This suggested that the association of Nck to tyrosine 361 in Dok1 is necessary for the JNK and c-Jun activation by glial cell line-derived neurotrophic factor or RET-MEN2B. Because a high level of the JNK phosphorylation was observed in the cells expressing RET-MEN2B, its strong activation via Nck binding to Dok1 may be responsible for aggressive properties of medullary thyroid carcinoma developed in MEN 2B.     

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.     

Parental origin of factor IX gene mutations, and their distribution in the gene.

Genomic amplification followed by direct sequencing enabled us to establish the causative mutation in 67 unrelated hemophilia B patients of predominantly German origin. With the detection of the mutation, extensive pedigree analysis has become feasible. We therefore anticipated that determination of the origin of mutation could be achieved in a comparatively great number of families. Although these investigations often were restricted by the availability of blood samples from the maternal grandparents or great-grandparents, we were able to prove a de novo mutation in 9 of 20 families with sporadic hemophilia B and in 3 of 20 families with a history of the disease. This could be achieved with the aid of RFLP analysis and, in one case, where the mutation is still unknown, with the aid of biochemical and immunological factor IX assays. Since the maternal grandfather was decreased in two of these families, the germ line of origin could not be determined precisely. In the remaining families, the female and male germ lines turned out to be the origin of mutation in six and four cases, respectively, and an effect of paternal age on the mutations observed could not be excluded. Furthermore, our data indicate that the hemophilia B gene pool is mainly renewed by variable mutations.     

A novel polymorphism in the coding sequence of the human RET proto-oncogene

A novel polymorphism in the coding sequence of the human RET proto-oncogene is described. The RET proto-oncogene maps to chromosome 10q11.2, and is involved in multiple endocrine neoplasia (MEN 2A, MEN 2B), familial medullary thyroid carcinoma and Hirschsprung's disease.     

MECP2 mutations in sporadic cases of Rett syndrome are almost exclusively of paternal origin

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder that apparently is lethal in male embryos. RTT almost exclusively affects female offspring and, in 99.5% of all cases, is sporadic and due to de novo mutations in the MECP2 gene. Familial cases of RTT are rare and are due to X-chromosomal inheritance from a carrier mother. We analyzed the parental origin of MECP2 mutations in sporadic cases of RTT, by analysis of linkage between the mutation in the MECP2 gene and intronic polymorphisms in 27 families with 15 different mutations, and we found a high predominance of mutations of paternal origin in 26 of 27 cases (P<.001). The paternal origin was independent of type of mutation and was found for single-base exchanges as well as for deletions. Parents were not of especially advanced age. We conclude that de novo mutations in RTT occur almost exclusively on the paternally derived X chromosome and that this is most probably the cause for the high female:male ratio observed in patients with RTT. Affected males recently have been described in a few cases of familial inheritance. Identification of the parental origin may be useful to distinguish between the sporadic form of RTT and a potentially familial form. This distinction will allow geneticists to offer more-specific counseling and discriminate between higher (maternal origin) and lower (paternal origin) recurrence risk.