Highlighting the impact of cascade carrier testing in cystic fibrosis families.

BACKGROUND: Cascade carrier testing within cystic fibrosis (CF) affected families offers relatives of CF patients the opportunity to know their status regarding the mutation that segregates within their family, and thus to make informed reproductive choices. As an Australian study has recently shown that this test seemed underused, we searched to assess uptake of this test in a European area where CF is common, and to report its public health implications. METHODS: This study relied on 40 CF-affected families from western Brittany, France. Investigations included drawing of family trees and registration of carrier tests performed in those families. RESULTS: Of the 459 relatives eligible for testing, 185 were tested, leading to an adjusted uptake rate of testing of 40.7% (95% CI: [34.1%; 47.3%]). The main predictors for having testing were being female (p=0.031) and having a high prior risk (p<0.001). Planning a pregnancy or expecting a child (reported in at least 38.4% of tested relatives) also appeared critical in choosing to be tested. Overall, carrier testing allowed to reassure more than 1/4 of the relatives and to detect five new 1-in-4 at-risk couples who then requested prenatal diagnosis. CONCLUSIONS: This observational study assesses, for first time in Europe, uptake of CF cascade carrier testing within CF families, which is a critical tool to reassure non-carriers and to detect early new at-risk couples.

The UMD-APC database, a model of nation-wide knowledge base: update with data from 3,581 variations.

Familial adenomatous polyposis (FAP) is a rare autosomal-inherited disease that highly predisposes to colorectal cancer, characterized by a diffuse duodenal and colorectal polyposis associated with various extradigestive tumors and linked to germline mutations within the APC gene. A French consortium of laboratories involved in APC mutation screening has progressively improved the description of the variation spectrum, inferred functional significance of nontruncating variations, and delineated phenotypic characteristics of the disease. The current version of the UMD-APC database is described here. The total number of variations has risen to 5,453 representing 1,473 distinct variations. The published records initially registered into the database were extended with 3,581 germline variations found through genetic testing performed by the eight licensed laboratories belonging to the French APC network. Sixty six of 149 variations of previously unknown significance have now been classified as (likely) causal or neutral. The database is available on the Internet (http://www.umd.be/APC/) and updated twice per year according to the consensus rules of the network. The UMD-APC database is thus expected to facilitate functional classification of rare synonymous, nonsynonymous, and intronic mutations and consequently improve genetic counseling and medical care in FAP families.

CDH1 germline mutations and the hereditary diffuse gastric and lobular breast cancer syndrome: a multicentre study.

INTRODUCTION: CDH1 predisposes primarily to diffuse gastric cancer (DGC). Multiple DGC cases in a family, DGC at a young age in an individual or the combination of DGC andlobular breast cancer (LBC) in an individual or a family define the hereditary DGC syndrome (HDGC), and testing for germline CDH1 mutations is warranted in HDGC. METHODS AND RESULTS: We report all index cases from Ile-de-France in which a germline CDH1 mutation has been identified. Out of 18 cases, 7 do not fulfil the HDGC-defining criteria. Three of them are women who presented initially with bilateral LBC below age 50, without personal or family history of DGC, and who subsequently developed symptomatic DGC. DISCUSSION: Our series of CDH1 mutation carriers is the largest to date and demonstrates that LBC might be the first manifestation of HDGC. A personal or family history of multiple LBCs at a young age, even without DGC, should prompt CDH1 mutation screening. It is paramount to identify mutation carriers early, so that they can benefit from prophylactic gastrectomy before they develop symptomatic, highly lethal DGC. We recommend a revision of the HDGC-defining criteria and propose for consideration the name 'Hereditary Diffuse Gastric and Lobular Breast Cancer' instead of HDGC.

Genetic and clinical specificity of 26 symptomatic carriers for dystrophinopathies at pediatric age.

The molecular basis underlying the clinical variability in symptomatic Duchenne muscular dystrophy (DMD) carriers are still to be precised. We report 26 cases of early symptomatic DMD carriers followed in the French neuromuscular network. Clinical presentation, muscular histological analysis and type of gene mutation, as well as X-chromosome inactivation (XCI) patterns using DNA extracted from peripheral blood or muscle are detailed. The initial symptoms were significant weakness (88%) or exercise intolerance (27%). Clinical severity varied from a Duchenne-like progression to a very mild Becker-like phenotype. Cardiac dysfunction was present in 19% of the cases. Cognitive impairment was worthy of notice, as 27% of the carriers are concerned. The muscular analysis was always contributive, revealing muscular dystrophy (83%), mosaic in immunostaining (81%) and dystrophin abnormalities in western blot analysis (84%). In all, 73% had exonic deletions or duplications and 27% had point mutations. XCI pattern was biased in 62% of the cases. In conclusion, we report the largest series of manifesting DMD carriers at pediatric age and show that exercise intolerance and cognitive impairment may reveal symptomatic DMD carriers. The complete histological and immunohistological study of the muscle is the key of the diagnosis leading to the dystrophin gene analysis. Our study shows also that cognitive impairment in symptomatic DMD carriers is associated with mutations in the distal part of the DMD gene. XCI study does not fully explain the mechanisms as well as the wide spectrum of clinical phenotype, though a clear correlation between the severity of the phenotype and inactivation bias was observed.

CFTR mutation combinations producing frequent complex alleles with different clinical and functional outcomes.

Genotype-phenotype correlations in cystic fibrosis (CF) may be difficult to establish because of phenotype variability, which is associated with certain CF transmembrane conductance regulator (CFTR) gene mutations and the existence of complex alleles. To elucidate the clinical significance of complex alleles involving p.Gly149Arg, p.Asp443Tyr, p.Gly576Ala, and p.Arg668Cys, we performed a collaborative genotype-phenotype correlation study, collected epidemiological data, and investigated structure-function relationships for single and natural complex mutants, p.[Gly576Ala;Arg668Cys], p.[Gly149Arg;Gly576Ala;Arg668Cys], and p.[Asp443Tyr;Gly576Ala;Arg668Cys]. Among 153 patients carrying at least one of these mutations, only three had classical CF and all carried p.Gly149Arg in the triple mutant. Sixty-four had isolated infertility and seven were healthy individuals with a severe mutation in trans, but none had p.Gly149Arg. Functional studies performed on all single and natural complex mutants showed that (1) p.Gly149Arg results in a severe misprocessing defect; (2) p.Asp443Tyr moderately alters CFTR maturation; and (3) p.Gly576Ala, a known splicing mutant, and p.Arg668Cys mildly alter CFTR chloride conductance. Overall, the results consistently show the contribution of p.Gly149Arg to the CF phenotype, and suggest that p.[Arg668Cys], p.[Gly576Ala;Arg668Cys], and p.[Asp443Tyr;Gly576Ala;Arg668Cys] are associated with CFTR-related disorders. The present study emphasizes the importance of comprehensive genotype-phenotype and functional studies in elucidating the impact of mutations on clinical phenotype.

Nonvisualization of fetal gallbladder increases the risk of cystic fibrosis.

OBJECTIVE: The aim of our study is to evaluate the prevalence of cystic fibrosis (CF) in fetuses referred for genetic testing because of ultrasonographic sign (nonvisualized fetal gallbladder--NVFGB). METHOD: We reviewed the results of CFTR gene analysis over the period 2002 to 2009 in all consecutive cases referred because of NVFGB in Western France. We correlated these data with the presence of a more classical ultrasonographic finding (fetal echogenic bowel - FEB). RESULTS: Cystic fibrosis was diagnosed in 5 of the 37 fetuses with NVFGB (13.5%, 95% confidence interval (CI): [2.5%; 24.5%]) and in only 9 of the 229 other cases referred because of FEB (3.9%, 95% CI: [3.2%; 14.7%]). In our series, all CF-affected fetuses with NVFGB also had FEB. The risk of CF was 11.6-fold higher in fetuses with both indications (NVFGB + FEB) than in fetuses with isolated FEB (45.5% vs 3.9%, RR = 11.6, 95% CI: [4.7%; 28.8%], p = 0.0001). We also estimated that the residual risk of CF was less than 1 in 68 (1.5%) when a single mutation was identified in the fetus by our molecular protocol. CONCLUSION: Ultrasonographic evidence of NVFGB is an additional risk factor for CF in cases with FEB.

p.Ser1235Arg should no longer be considered as a cystic fibrosis mutation: results from a large collaborative study.

Among the 1700 mutations reported in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, a missense mutation, p.Ser1235Arg, is a relatively frequent finding. To clarify its clinical significance, we collected data from 104 subjects heterozygous for the mutation p.Ser1235Arg from the French CF network, addressed for various indications including classical CF, atypical phenotypes or carrier screening in subjects with or without a family history. Among them, 26 patients (5 having CF, 10 CBAVD (congenital bilateral absence of the vas deferens) and 11 with CF-like symptoms) and 14 healthy subjects were compound heterozygous for a second CFTR mutation. An exhaustive CFTR gene analysis identified a second mutation in cis of p.Ser1235Arg in all CF patients and in 81.8% CBAVD patients. Moreover, epidemiological data from >2100 individuals found a higher frequency of p.Ser1235Arg in the general population than in CF or CBAVD patients. These data, added to the fact that in silico analysis and functional assays suggest a benign nature of this substitution, give several lines of evidence against an association of p.Ser1235Arg with CF or CBAVD.

Focus on cystic fibrosis and other disorders evidenced in fetuses with sonographic finding of echogenic bowel: 16-year report from Brittany, France.

OBJECTIVE: Pregnancies medical follow-up and ultrasonography development have enabled detection of fetal echogenic bowel, a sign associated with various pathologies, including cystic fibrosis. Based on the long experience of a region where cystic fibrosis is frequent (Brittany, France), we describe disorders diagnosed in fetal echogenic bowel fetuses and assess ultrasonography ability in detecting cystic fibrosis in utero. STUDY DESIGN: We reviewed the cases of fetal echogenic bowel diagnosed in pregnant women living in Brittany and referred for CFTR gene analysis over the 1992-2007 period (n = 289). RESULTS: A disorder was diagnosed in 32.2% of the fetuses, cystic fibrosis being the most commonly identified (7.6%). We also found digestive malformations (7.0%), chromosomal abnormalities (3.7%), and maternofetal infections (3.7%). Combining these data with our ongoing newborn screening program since 1989 showed that ultrasonography enabled diagnosis of 10.7% of the cystic fibrosis cases. CONCLUSION: This study highlights the importance of pregnancy ultrasound examinations and their efficiency in detecting cystic fibrosis.

Genotype-phenotype analysis in 2,405 patients with a dystrophinopathy using the UMD-DMD database: a model of nationwide knowledgebase.

UMD-DMD France is a knowledgebase developed through a multicenter academic effort to provide an up-to-date resource of curated information covering all identified mutations in patients with a dystrophinopathy. The current release includes 2,411 entries consisting in 2,084 independent mutational events identified in 2,046 male patients and 38 expressing females, which corresponds to an estimated number of 39 people per million with a genetic diagnosis of dystrophinopathy in France. Mutations consist in 1,404 large deletions, 215 large duplications, and 465 small rearrangements, of which 39.8% are nonsense mutations. The reading frame rule holds true for 96% of the DMD patients and 93% of the BMD patients. Quality control relies on the curation by four experts for the DMD gene and related diseases. Data on dystrophin and RNA analysis, phenotypic groups, and transmission are also available. About 24% of the mutations are de novo events. This national centralized resource will contribute to a greater understanding of prevalence of dystrophinopathies in France, and in particular, of the true frequency of BMD, which was found to be almost half (43%) that of DMD. UMD-DMD is a searchable anonymous database that includes numerous newly developed tools, which can benefit to all the scientific community interested in dystrophinopathies. Dedicated functions for genotype-based therapies allowed the prediction of a new multiexon skipping (del 45-53) potentially applicable to 53% of the deleted DMD patients. Finally, such a national database will prove to be useful to implement the international global DMD patients' registries under development.

Nutritional and genetic determinants of vitamin B and homocysteine metabolisms in neural tube defects: a multicenter case-control study.

Neural tube defects (NTDs) are severe congenital malformations due to failure of neural tube formation in early pregnancy. The proof that folic acid prevents NTDs raises the question of whether other parts of homocysteine (Hcy) metabolism may affect rates of NTDs. This French case-control study covered: 77 women aged 17-42 years sampled prior to elective abortion for a severe NTDs (cases) and 61 women aged 20-43 years with a normal pregnancy. Plasma and erythrocyte folate, plasma B6, B12 and Hcy were tested as five polymorphisms MTHFR 677 C --> T, MTHFR 1298 A --> C, MTR 2756 A --> G, MTTR 66 A --> G and TCN2 776 C --> G. Cases had significantly lower erythrocyte folate, plasma folate, B12 and B6 concentrations than the controls, and higher Hcy concentration. The odds ratio was 2.15 (95% CI: 1.00-4.59) for women with the MTRR 66 A --> G allele and it was decreased for mothers carrying the MTHFR 1298 A --> C allele. In multivariate analysis, only the erythrocyte folate concentration (P = 0.005) and plasma B6 concentration (P = 0.020) were predictors. Red cell folate is the main determinant of NTDs in France. Folic acid supplement or flour fortification would prevent most cases. Increased consumption of vitamins B12 and B6 could contribute to the prevention of NTDs. Genetic polymorphisms played only a small role. Until folic acid fortification becomes mandatory, all women of reproductive age should consume folic acid in a multivitamin that also contains B12 and B6.

Prenatal diagnosis of cystic fibrosis: the 18-year experience of Brittany (western France).

OBJECTIVE: This study reports 18 years of experience in prenatal diagnosis (PD) of cystic fibrosis (CF) in a region where CF is frequent and the uptake of PD is common (Brittany, western France). METHOD: All PDs made over the period 1989-2006 in women living in Brittany were collected. RESULTS: We recorded 268 PDs made in 1 in 4 risk couples, plus 22 PDs directly made following the sonographic finding of echogenic bowel. Most of the 268 PDs were done in couples already having CF child(ren) (n = 195, 72.8%). Close to one-fifth followed cascade screening (n = 49, 18.3%), which identified 26 new 1 in 4 risk couples among the relatives of CF patients or of carriers identified through newborn screening (NBS). The remaining PDs were mainly made in couples whose 1 in 4 risk was evidenced following the diagnosis of echogenic bowel in a previous pregnancy (n = 22, 8.2%). Although patients' life expectancy has considerably improved, in our population the great majority of couples chose pregnancy termination when PD indicated that the foetus had CF (95.9%). CONCLUSION: This study describes the distribution of PDs according to the context in which the 1 in 4 risk was discovered and highlights the real decisions of couples as regards pregnancy termination after a positive PD.

Colorectal adenomatous polyposis Associated with MYH mutations: genotype and phenotype characteristics.

PURPOSE: Recent literature reports that several digestive diseases are associated with mutations in the base excision repair gene MYH. This study was designed to establish the prevalence of germ-line MYH mutations in a series of 56 consecutive patients with no detectable APC mutation and describe the phenotype of those with MYH mutations. METHODS: MYH mutations were screened by DNA sequencing after polymerase chain reaction amplification of each exon. Clinical, endoscopic, and surgical data were collected for the tested patients. RESULTS: MYH mutations were identified only in the group of patients with attenuated adenomatous polyposis with ten or more adenomatous polyps. The prevalence of MYH mutations was 34.4 percent (11 cases) in this subgroup of 30 patients. There were two homozygotes and eight compound heterozygotes. Only one patient had a monoallelic mutation. At least one of two mutational hot spots was identified in ten patients. Three patients presented with a family history of adenomatous polyposis in siblings, without vertical transmission. The median number of colorectal adenomatous polyps was 53 without preferential localization. Colorectal cancer was associated with polyposis in seven patients. Gastric and duodenal adenomas were diagnosed in one case. Ten of 11 patients underwent colectomy. CONCLUSIONS: MYH mutations have been observed in one-third of patients with attenuated polyposis. The phenotype of the disease is similar to attenuated familial adenomatous polyposis. Upper gastrointestinal endoscopy also should be recommended. However, its transmission shows evidence of a recessive pattern.

Multiexon skipping leading to an artificial DMD protein lacking amino acids from exons 45 through 55 could rescue up to 63% of patients with Duchenne muscular dystrophy.

Approximately two-thirds of Duchenne muscular dystrophy (DMD) patients show intragenic deletions ranging from one to several exons of the DMD gene and leading to a premature stop codon. Other deletions that maintain the translational reading frame of the gene result in the milder Becker muscular dystrophy (BMD) form of the disease. Thus the opportunity to transform a DMD phenotype into a BMD phenotype appeared as a new treatment strategy with the development of antisense oligonucleotides technology, which is able to induce an exon skipping at the pre-mRNA level in order to restore an open reading frame. Because the DMD gene contains 79 exons, thousands of potential transcripts could be produced by exon skipping and should be investigated. The conventional approach considers skipping of a single exon. Here we report the comparison of single- and multiple-exon skipping strategies based on bioinformatic analysis. By using the Universal Mutation Database (UMD)-DMD, we predict that an optimal multiexon skipping leading to the del45-55 artificial dystrophin (c.6439_8217del) could transform the DMD phenotype into the asymptomatic or mild BMD phenotype. This multiple-exon skipping could theoretically rescue up to 63% of DMD patients with a deletion, while the optimal monoskipping of exon 51 would rescue only 16% of patients.

Late ovarian relapse of TEL/AML1 positive ALL confirming that TEL deletion is a secondary event in leukemogenesis.

We describe here a late extramedullary ovarian relapse in an 18-year-old female who was diagnosed with hypotetraploid cell acute lymphoblastic leukaemia (cALL) at the age of 6. At both occurrences of the disease cells were analyzed by morphology, immunophenotyping, cytogenetics and molecular methods. TEL/AML1 was detected by RT-PCR and FISH analysis in both events. We demonstrated, using detection of IGH/TCR rearrangements and TEL/AML1 breakpoints sequencing that the cells were clonally related. Moreover, interphasic FISH using TEL and AML1 probes showed the loss of a second TEL at the time of relapse. This observation confirms that TEL/AML1 alone is not sufficient to trigger ALL and that TEL deletion is a secondary event in leukemogenesis. To our knowledge, it is the first complete description of extramedullary ALL relapse combining all methodologies.

Phenotypic and molecular variability of the holoprosencephalic spectrum.

Since 1996, a European network has been organized from Rennes, France and holoprosencephalic files were collected for clinical and molecular study. Familial instances of typical and atypical holoprosencephaly (HPE) were found in 30% of cases. All affected children had psychomotor delay with microcephaly, often associated with endocrine, digestive, and respiratory abnormalities, and thermal dysregulation. Among 173 subjects in the molecular study, 28 heterozygous mutations were identified (16%): 15 SHH mutations, 6 ZIC2 mutations, 5 SIX3 mutations, and 2 TGIF mutations.

Molecular screening of SHH, ZIC2, SIX3, and TGIF genes in patients with features of holoprosencephaly spectrum: Mutation review and genotype-phenotype correlations.

Holoprosencephaly (HPE; 1 out of 16,000 live births; 1 out of 250 conceptuses) is a complex brain malformation resulting from incomplete cleavage of the prosencephalon, affecting both the forebrain and the face. Clinical expressivity is variable, ranging from a single cerebral ventricle and cyclopia to clinically unaffected carriers in familial dominant autosomic HPE. The disease is genetically heterogeneous, but additional environmental agents also contribute to the etiology of HPE. In our cohort of 200 patients, 34 heterozygous mutations were identified, 24 of them being novel ones: 13 out of 17 in the Sonic hedgehog gene (SHH); 4 out of 7 in ZIC2; and 7 out of 8 in SIX3. The two mutations identified in TGIF have already been reported. Novel phenotypes associated with a mutation have been described, such as abnormalities of the pituitary gland and corpus callosum, colobomatous microphthalmia, choanal aperture stenosis, and isolated cleft lip. This study confirms the great genetic heterogeneity of the disease, the important phenotypic variability in HPE families, and the difficulty to establish genotype-phenotype correlations.