Constitutional mismatch repair deficiency syndrome: clinical description in a French cohort.

BACKGROUND: Constitutional mismatch repair deficiency (CMMRD) syndrome is a childhood cancer predisposition syndrome involving biallelic germline mutations of MMR genes, poorly recognised by clinicians so far. METHODS: Retrospective review of all 31 patients with CMMRD diagnosed in French genetics laboratories in order to describe the characteristics, treatment and outcome of the malignancies and biological diagnostic data. RESULTS: 67 tumours were diagnosed in 31 patients, 25 (37%) Lynch syndrome-associated malignancies, 22 (33%) brain tumours, 17 (25%) haematological malignancies and 3 (5%) sarcomas. The median age of onset of the first tumour was 6.9 years (1.2-33.5). Overall, 22 patients died, 9 (41%) due to the primary tumour. Median survival after the diagnosis of the primary tumour was 27 months (0.26-213.2). Failure rate seemed to be higher than expected especially for T-cell non-Hodgkin's lymphoma (progression/relapse in 6/12 patients). A familial history of Lynch syndrome was identified in 6/23 families, and consanguinity in 9/23 families. PMS2 mutations (n=18) were more frequent than other mutations (MSH6 (n=6), MLH1 (n=4) and MSH2 (n=3)). CONCLUSIONS: In conclusion, this unselected series of patients confirms the extreme severity of this syndrome with a high mortality rate mostly related to multiple childhood cancers, and highlights the need for its early detection in order to adapt treatment and surveillance.

Evaluation of a new panel of six mononucleotide repeat markers for the detection of DNA mismatch repair-deficient tumours.

BACKGROUND: Microsatellite instability (MSI) is a molecular phenotype due to defective DNA mismatch repair (MMR) system. It is used to predict outcome of colorectal tumours and to screen tumours for Lynch syndrome (LS). A pentaplex panel composed of five mononucleotide markers has been largely recommended for determination of the MSI status. However, its sensitivity may be taken in default in occasional situations. The aim of the study was to optimise this panel for the detection of MSI. METHODS: We developed an assay allowing co-amplification of six mononucleotide repeat markers (BAT25, BAT26, BAT40, NR21, NR22, NR27) and one polymorphic dinucleotide marker (D3S1260) in a single reaction. Performances of the new panel were evaluated on a cohort of patients suspected of LS. RESULTS: We demonstrate that our assay is technically as easy to use as the pentaplex assay. The hexaplex panel shows similar performances for the identification of colorectal and non-MSH6-deficient tumours. On the other hand, the hexaplex panel has higher sensitivity for the identification of MSH6-deficient tumours (94.7% vs 84.2%) and MMR-deficient tumours other than colorectal cancer (92.9% vs 85.7%). CONCLUSION: The hexaplex panel could thus be an attractive alternative to the pentaplex panel for the identification of patients with LS.

Butyrate enemas upregulate Muc genes expression but decrease adherent mucus thickness in mice colon.

Colonic mucosal protection is provided by the mucus gel, mainly composed of mucins. Several factors can modulate the formation and the secretion of mucins, and among them butyrate, an end-product of carbohydrate fermentation. However, the specific effect of butyrate on the various colonic mucins, and the consequences in terms of the mucus layer thickness are not known. Our aim was to determine whether butyrate modulates colonic MUC genes expression in vivo and whether this results in changes in mucus synthesis and mucus layer thickness. Mice received daily for 7 days rectal enemas of butyrate (100 mM) versus saline. We demonstrated that butyrate stimulated the gene expression of both secreted (Muc2) and membrane-linked (Muc1, Muc3, Muc4) mucins. Butyrate especially induced a 6-fold increase in Muc2 gene expression in proximal colon. However, butyrate enemas did not modify the number of epithelial cells containing the protein Muc2, and caused a 2-fold decrease in the thickness of adherent mucus layer. Further studies should help understanding whether this last phenomenon, i.e. the decrease in adherent mucus gel thickness, results in a diminished protective function or not.

Specific secretion of gel-forming mucins and TFF peptides in HT-29 cells of mucin-secreting phenotype.

Trefoil factor family (TFF) peptides are typical secretory products of mucin-producing cells, e.g. of the gastrointestinal tract. Here, the expression and secretion of mucins and TFF peptides was studied in the HT-29 cell line throughout cellular growth and differentiation in relation to a mucin-secreting (HT-29 MTX) or an enterocyte-like (HT-29 G(-)) phenotype. mRNAs of several MUC and TFF genes were expressed in both cell subpopulations. However, for most MUC and TFF genes, the expression appeared strongly induced with the differentiation into the mucin-secreting phenotype. On the other hand, TFF2 was specifically expressed in the mucin-secreting HT-29 MTX cells. The differentiation of HT-29 MTX cells into the mucin-secreting phenotype was characterised by secretion of the gel-forming mucins MUC2, MUC5AC, and MUC5B, however, according to a different pattern in the course of differentiation. A significant amount of TFF1 and TFF3 was secreted after differentiation, also according to a different pattern, whereas TFF2 was only faintly detected. Secretagogues, known to induce the secretion of mucus, increased the secretion of all three TFF peptides. In contrast, neither a secretory mucin nor a TFF peptide was found in the culture medium of HT-29 G(-) cells. Overlay assays indicated that HT-29 MTX mucins bound to secretory peptides of HT-29 MTX cells with relative molecular mass similar to TFF peptides. TFF1 and TFF3 were specifically localised in the mucus layer of HT-29 MTX cells by confocal microscopy. Finally, the secretion of TFF peptides and mucins appears as a co-ordinated process which only occurs after differentiation into goblet cell-like phenotype.

A novel 9-base pair duplication in RET exon 8 in familial medullary thyroid carcinoma.

Familial medullary thyroid carcinoma (FMTC) and multiple endocrine neoplasia type 2A syndromes are dominantly inherited diseases caused by activating germline mutations of the RET protooncogene. The majority of these patients carry a germline point mutation affecting one of five cysteine residues encoded by exon 10 (codon 609, 611, 618, or 620) or 11 (codon 634). In a few FMTC families, point mutations involving noncysteine codons in exon 13 (codons 768, 790, and 791), 14 (codon 804), or 15 (codon 891) have been reported. Hirschsprung's disease is a nonneoplastic disorder associated with RET mutations leading to a loss of function effect. Mutations are identified in 50% of the familial cases and are scattered along the gene. We now report the study of a FMTC family with four affected members and a history of fatal neonatal intestinal obstruction in the sister of the proband. Genetic analysis demonstrated the absence of an usual FMTC mutation and the presence of a germline 9-bp duplication in RET exon 8 in the heterozygous state in all patients with MTC. This new mutation creates an additional cysteine residue in the extracellular cysteine-rich domain of RET. Further studies are warranted to confirm whether this new mutation is causing MTC only or could be associated with Hirschsprung's disease.

Normal respiratory mucosa, precursor lesions and lung carcinomas: differential expression of human mucin genes.

Mucins are glycoproteins synthesized by epithelial cells and thought to promote tumor-cell invasion. Eight human mucin genes have been well characterized: MUC2, MUC5AC, MUC5B, MUC6 map to 11p15.5 and encode secretory gel forming mucins while MUC1, MUC3, MUC4, MUC7 are scattered on different chromosomes and encode membrane-bound or secreted mucins. The expression pattern of the mucin genes is complex in normal airways involving six genes, mainly MUC5AC and MUC5B in mucus-producing cells and MUC4 in a wide array of epithelial cells. MUC5AC overexpression in metaplasia, dysplasia and normal epithelium adjacent to squamous cell carcinoma provides additional arguments for a mucous cell origin of preneoplastic squamous lesions. MUC5AC and MUC5B expression is related to mucus formation in adenocarcinomas. Mucinous bronchioloalveolar carcinoma (BAC) has a particular pattern of mucin gene expression indicating that it has sustained a well-differentiated phenotype similar to the goblet cell, correlated with distinctive features i.e. a noninvasive pattern and a better prognosis than nonBACs. MUC4 is the earlier mucin gene expressed in the foregut, before epithelial differentiation and is expressed independently of mucus secretion both in normal adult airways and carcinomas. These findings are in favor the histogenetic theory of non-small-cell carcinoma originating from a pluripotent mucous cell.

Developmental mucin gene expression in the gastroduodenal tract and accessory digestive glands. I. Stomach. A relationship to gastric carcinoma.

Studies were undertaken to provide information regarding cell-specific expression of mucin genes in stomach and their relation to developmental and neoplastic patterns of epithelial cytodifferentiation. In situ hybridization was used to study mRNA expression of eight mucin genes (MUC1-4, MUC5AC, MUC5B, MUC6, MUC7) in stomach of 13 human embryos and fetuses (8-27 weeks' gestation), comparing these with normal, metaplastic, and neoplastic adult tissues. These investigations have demonstrated that MUC1, MUC4, MUC5AC, MUC5B, and MUC6 are already expressed in the embryonic stomach at 8 weeks of gestation. MUC3 mRNA expression can be observed from 10.5 weeks of gestation. MUC2 is expressed at later stages, concomitant with mucous gland cytodifferentiation. Normal adult stomach is characterized by strong expression of MUC1, MUC5AC, and MUC6, less prominent MUC2, and sporadic MUC3 and MUC4, without MUC5B and MUC7. Intestinal metaplasia is characterized by an intestinal-type pattern with MUC2 and MUC3 mRNA expression. Gastric carcinomas exhibit altered mucin gene expression patterns with disappearance of MUC5AC and MUC6 mRNAs in some tumor glands, abnormal expression of MUC2, and reappearance of MUC5B mRNAs. In conclusion, we have observed that patterns of mucin gene expression in embryonic and fetal stomach could show similarities with some gastric carcinomas in adults. Differences in mucin gene expression in developmental, metaplastic, and neoplastic stomach compared to normal adult stomach suggest a possible regulatory role for their products in gastric epithelial cell proliferation and differentiation.

Developmental mucin gene expression in the gastroduodenal tract and accessory digestive glands. II. Duodenum and liver, gallbladder, and pancreas.

Studies were undertaken to provide information regarding cell-specific expression of mucin genes and their relation to developmental and neoplastic patterns of epithelial cytodifferentiation. In situ hybridization was used to study mRNA expression of mucin genes in duodenum and accessory digestive glands (liver, gallbladder, pancreas) of 13 human embryos and fetuses (6. 5-27 weeks' gestation), comparing these with normal and neoplastic adult tissues. These investigations demonstrated that the pattern of mucin gene expression in fetal duodenum reiterated the patterns we observed during gastric and intestinal ontogenesis, with MUC2 and MUC3 expression in the surface epithelium and MUC6 expression associated with the development of Brünner's glands. In embryonic liver, MUC3 was already expressed at 6.5 weeks of gestation in hepatoblasts. As in adults, MUC1, MUC2, MUC3, MUC5AC, MUC5B, and MUC6 were expressed in fetal gallbladder, whereas MUC4 was not. In contrast, MUC4 was strongly expressed in gallbladder adenocarcinomas. MUC5B and MUC6 were expressed in fetal pancreas, from 12 weeks and 26 weeks of gestation, respectively. Surprisingly, MUC3 which is strongly expressed in adult pancreas, was not detected in developmental pancreas. Taken together, these data show complex spatio-temporal regulation of the mucin genes and suggest a possible regulatory role for mucin gene products in gastroduodenal epithelial cell differentiation.

Transdermal nicotine decreases mucosal IL-8 expression but has no effect on mucin gene expression in ulcerative colitis.

Our goal was to determine the effect of transdermal nicotine on cytokine and mucin gene transcription in ulcerative colitis (UC). Sixty-four nonsmoking patients with active UC were randomly assigned to transdermal nicotine (maximum dose 22 mg/day) or placebo for 4 weeks. Clinical assessment and colonic mucosal biopsies were obtained at entry and after 4 weeks. Inflammatory and immunoregulatory cytokines were assessed by qualitative reverse transcriptase-polymerase chain reaction (RT-PCR). Based on this initial screen. IL-8 mRNA levels were measured by RT-competitive PCR. MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, and MUC6 mRNA concentrations were measured by quantitative dot blot analysis. Cytokine mRNA expression, except for IL-8, was similar in all patients. IL-8 mRNA levels were significantly decreased in the colonic mucosa of nicotine-treated patients who improved (p = 0.04). IL-8 mRNA values were similar before and after treatment in nonresponding nicotine-treated patients and in all placebo-treated patients. Mucin gene expression was similar in all patient groups. Beneficial effects of transdermal nicotine in active UC may result from decrease of IL-8 expression at the transcriptional level. Transdermal nicotine has no effect on mucin gene transcription.

Abnormalities in mucin gene expression in Crohn's disease.

Alterations in the structure and/or quantity of mucins could alter the barrier function of mucus and play a role in initiating and maintaining mucosal inflammation in Crohn's disease. To investigate the hypothesis of a mucin gene defect in Crohn's disease, we analyzed the expression of the different mucin genes in the ileal mucosa of patients with Crohn's disease and controls. mRNA expression levels were assessed by a quantitative dot blot analysis and compared (i) between healthy and involved ileal mucosa of patients with Crohn's disease and (ii) between healthy mucosa of patients with Crohn's disease and controls. Expression of the different mucin genes was heterogeneous among controls and patients with Crohn's disease, except for MUC6 in controls. Nevertheless, MUC1 mRNA expression was significantly decreased in the involved ileal mucosa of patients with Crohn's disease when compared to the healthy mucosa (p = 0.02). Moreover, the expression levels of MUC3, MUC4, and MUC5B were significantly lower in both healthy and involved ileal mucosa of patients with Crohn's disease compared to controls (p < or = 0.05). The decrease of expression levels of some mucin genes (more particularly MUC3, MUC4, and MUC5B) in both healthy and involved ileal mucosa suggests a primary or very early mucosal defect of these genes in CD.

Mucinous bronchioloalveolar carcinomas display a specific pattern of mucin gene expression among primary lung adenocarcinomas.

Lung adenocarcinomas are heterogeneous clinically and histologically. Expression of the mucin genes was analyzed as a molecular marker of glandular cytodifferentiation in primary lung adenocarcinomas. Expression was correlated with histopathologic subtypes of World Health Organization classification with the aim of investigating the histogenesis of primary lung adenocarcinomas. Thirty-four primary lung adenocarcinomas were examined by in situ hybridization for mucin gene expression (MUC1-4, MUC5AC, MUC5B, MUC6-7) and by immunohistochemistry for MUC5AC and MUC5B apomucin expression. Mucinous bronchioloalveolar carcinoma (BAC) had a homogeneous pattern of mucin gene expression different from those of other types of lung adenocarcinoma, involving secreted mucins (MUC5AC, MUC5B, and MUC6) and membrane-bound mucins (MUC1, MUC3, and MUC4). Non-BAC adenocarcinoma and mucinous BAC aberrantly expressed mucin genes MUC3, and MUC3 and MUC6, respectively, which are undetectable in normal fetal and adult lung. Our results show the particular phenotype of mucin gene expression in mucinous type of BACs and the heterogeneous expression of respiratory and nonrespiratory mucins in the other types. This finding supports the theory of a common progenitor cell with the potential of multicellular differentiation. From a practical point of view, the aberrant expression of MUC3 and MUC6 could serve as a diagnostic marker in the management of the mucinous type of bronchioloalveolar carcinomas. HUM PATHOL 32:274-281.

[Ovarian mucinous tumor of gastric and intestinal type associated with Peutz-Jeghers syndrome: in situ hybridization study of apomucin gene transcripts]. / Tumeur mucineuse ovarienne de type gastrique et intestinal associée au syndrome de Peutz-Jeghers; étude en hybridation in situ des transcrits des gènes d'apomucines.

Occurrence of mucinous tumors is favored by Peutz-Jeghers syndrome (PJS). A case of bilateral ovarian mucinous tumor associated with ovarian mature teratoma occurring in a 22-year-old woman with PJS was reported. Tumor cells included 5 cell types: tall columnar mucinous pale cells with neutral mucins; goblet cells with acidic nonsulfated mucins; non mucinous columnar cells; mucinous cuboidal cells lining small glands; endocrine cells. Expression of the MUC2, MUC3, MUC5AC and MUC6 genes was demonstrated by in situ hybridization according to cell type. Some atypia and numerous mitotic figures were observed in basal glands. Diagnosis was ovarian borderline mucinous tumor with gastric and intestinal phenotype associated with PJS.

[Urothelial tumor and colonic cancer in the context of a syndrome of hereditary predisposition to HNPCC colonic cancer]. / Tumeur urothéliale et cancer colique dans le cadre d'un syndrome de prédisposition héréditaire au cancer colique HNPCC.

The authors report the association of ureteric tumour and colon carcinomas in the context of hereditary predisposition to HNPCC colon cancer (hereditary non polyposis colon cancer). The recall the diagnostic criteria of HNPCC syndrome and emphasize the importance of guiding the clinical interview of patients with upper urinary tract tumours in order to detect a family history and the presence of gastrointestinal tumours.

Frequencies of KIT and PDGFRA mutations in the MolecGIST prospective population-based study differ from those of advanced GISTs.

Gastrointestinal stromal tumors (GISTs) are the most common human sarcoma. Most of the data available on GISTs derive from retrospective studies of patients referred to oncology centers. The MolecGIST study sought to determine and correlate clinicopathological and molecular characteristics of GISTs. Tumor samples and clinical records were prospectively obtained and reviewed for patients diagnosed in France during a 24-month period. Five hundred and ninety-six patients were included, of whom 10% had synchronous metastases. GISTs originated from the stomach, small bowel or other site in 56.4, 30.2 and 13.4% of cases, respectively. The main prognostic markers, tumor localization, size and mitotic index were not independent variables (P < 0.0001). Mutational status was determined in 492 (83%) patients, and 138 different mutations were identified. KIT and PDGFRA mutations were detected in 348 (71%) and 74 (15%) patients, respectively, contrasting with 82.8 and 2.1% in patients with advanced GIST (MetaGIST) (P < 0.0001). Further comparison of localized GISTs in the MolecGIST cohort with advanced GISTs from previous clinical trials showed that the mutations of PDGFRA exon18 (D842V and others) as well as KIT exon11 substitutions (W557R and V559D) were more likely to be seen in patients with localized GISTs (odds ratio 7.9, 3.1, 2.7 and 2.5, respectively), while KIT exon 9 502_503dup and KIT exon 11 557_559del were more frequent in metastatic GISTs (odds ratio of 0.3 and 0.5, respectively). These data suggest that KIT and PDGFRA mutations and standardized mitotic count deserve to be investigated to evaluate the relapse risk of GISTs.

Genomic organization of the human mucin gene MUC5B. cDNA and genomic sequences upstream of the large central exon.

The complete structure of the DNA encoding the polypeptide chain of human mucin MUC5B has been determined. In this paper, we report the full-length cDNA (3886 bp) and genomic (15,143 bp) sequences upstream of the unusually large central exon of the human mucin gene MUC5B. This region, composed of 29 exons, encodes 1283 amino acid residues. Exon sizes vary from 44 to 262 bp, and intron sizes range from 87 to 1703 bp. We determined the 5'-end of MUC5B by performing rapid amplification of cDNA ends-polymerase chain reaction experiments leading to the same length of the amplified product and by using primer extension experiments. A putative translation start site was found at nucleotide +37. We compared the amino-terminal region of MUC5B with those of pro-von Willebrand Factor, MUC2 and MUC5AC, and animal mucins, RMuc2, PSM, and FIM-B.1. The primary amino acid sequence with a high content of cysteine residues demonstrates a high degree of similarity with other members of the 11p15 mucin gene family, particularly MUC5AC. The complete genomic organization and both full-length genomic and cDNA sequences of MUC5B have been elucidated. This gene contains 48 exons and encodes 5662 amino acid residues to give a polypeptide with a Mr approximately 600,000.

Human mucin gene MUC5AC: organization of its 5'-region and central repetitive region.

Human mucin gene MUC5AC is clustered with MUC2, MUC5B and MUC6 on chromosome 11p15.5. We report here the full length cDNA sequence upstream of the repetitive region of human MUC5AC. We have also determined the sequence of its large central tandem repeat array. The 5'-region reveals high degree of sequence similarity with MUC2 and MUC5B and codes for 1336 amino acids organized into a signal peptide, four pro-von Willebrand factor-like D domains (D1, D2, D' and D3) and a short domain which connects to the central repetitive region. In the central region, 17 major domains have been identified. Nine code for cysteine-rich domains (Cys-domains 1-9) and exhibit high sequence similarity to the cysteine-rich domains described in the central region of MUC2 and MUC5B. Cys-domains 1-5 are interspersed by domains enriched with serine, threonine, and proline residues. Cys-domains 1-9 are interspersed by four domains (TR1-TR4) composed of various numbers of MUC5AC-type repeats. Southern-blot analyses reveal allelic variations both in length and nucleotide sequence. The length polymorphism which is due to variable numbers of tandem repeats is located in TR1 and TR4, whereas a mutation polymorphism detected with TaqI is located in Cys-domain 6. In this study, the organization of MUC5AC has been entirely elucidated showing extensive similarity to the other chromosome 11p15 MUC genes, particularly MUC5B, and providing additional arguments for common evolution from a single ancestral gene.

Mucin gene transcripts in benign and borderline mucinous tumours of the ovary: an in situ hybridization study.

Mucinous tumours of the ovary are characterized by mucin-secreting cells exhibiting a variable endocervical, intestinal, gastric or pancreatobiliary phenotype as ascertained by microscopy, electron microscopy, histochemistry or immunohistochemistry. The molecular mechanisms underlying the tumourigenesis process are not well understood. The mucin glycoproteins expressed by ovarian mucinous tumours have not been fully characterized, but mucins are known to be implicated in tumour progression in various epithelial neoplasms. The purpose of this study was to evaluate the expression of mucin genes (MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6) in ovarian mucinous tumour cells, to relate MUC gene expression to the histological diagnosis, and to compare the expression patterns with those observed in normal tissues. The expression of mucin genes was evaluated by in situ hybridization in 21 mucinous tumours (11 adenomas and ten borderline tumours). Heterogeneity of expression correlated with morphological heterogeneity. Intense expression of the MUC5AC gene, suggesting a gastric surface cell phenotype, was demonstrated in 18/21 tumours (86%). Goblet cells expressing the MUC2 gene and columnar cells expressing the MUC3 gene were consistent with an intestinal phenotype, which was observed in 15 tumours (71%) including nine adenomas and six borderline tumours. Major expression of MUC4 and MUC5B consistent with an endocervical phenotype was observed in seven benign (64%) and three borderline (30%) tumours. In all, the MUC profiles suggested gastrointestinal-type cells in 13 cases (62%), gastric-type cells in five cases (24%), and intestinal-type cells in two cases (one benign, one borderline) (9%); the results were inconclusive in one borderline tumour (5%). It is concluded that gastric and, to a lesser degree, intestinal differentiation are early and almost constant events in ovarian mucinous tumourigenesis.