Violaine Bourdon

Arabidopsis Mutants Impaired in Cosuppression

Post-transcriptional gene silencing (cosuppression) results in the degradation of RNA after transcription. A transgenic Arabidopsis line showing post-transcriptional silencing of a 35S–uidA transgene and uidA-specific methylation was mutagenized using ethyl methanesulfonate. Six independent plants were isolated in which uidA mRNA accumulation and β-glucuronidase activity were increased up to 3500-fold, whereas the transcription rate of the 35S–uidA transgene was increased only up to threefold. These plants each carried a recessive monogenic mutation that is responsible for the release of silencing. These mutations defined two genetic loci, called sgs1 and sgs2 (for suppressor of gene silencing). Transgene methylation was distinctly modified in sgs1 and sgs2 mutants. However, methylation of centromeric repeats was not affected, indicating that sgs mutants differ from ddm (for decrease in DNA methylation) and som (for somniferous) mutants. Indeed, unlike ddm and som mutations, sgs mutations were not able to release transcriptional silencing of a 35S–hpt transgene. Conversely, both sgs1 and sgs2 mutations were able to release cosuppression of host Nia genes and 35S–Nia2 transgenes. These results therefore indicate that sgs mutations act in trans to impede specifically transgene-induced post-transcriptional gene silencing.

SMARCB1/INI1 germline mutations contribute to 10% of sporadic schwannomatosis

BackgroundSchwannomatosis is a disease characterized by multiple non-vestibular schwannomas. Although biallelic NF2 mutations are found in schwannomas, no germ line event is detected in schwannomatosis patients. In contrast, germline mutations of the SMARCB1 (INI1) tumor suppressor gene were described in familial and sporadic schwannomatosis patients.MethodsTo delineate the SMARCB1 gene contribution, the nine coding exons were sequenced in a series of 56 patients affected with a variable number of non-vestibular schwannomas.ResultsNine variants scattered along the sequence of SMARCB1 were identified. Five of them were classified as deleterious. All five patients carrying a SMARCB1 mutation had more multiple schwannomas, corresponding to 10.2% of patients with schwannomatosis. They were also diagnosed before 35 years of age.ConclusionsThese results suggest that patients with schwannomas have a significant probability of carrying a SMARCB1 mutation. Combined with data available from other studies, they confirm the clinical indications for genetic screening of the SMARCB1 gene.

T-cell immune constitution after peripheral blood mononuclear cell transplantation in complete DiGeorge syndrome

Summary.  Complete DiGeorge syndrome (cDGS) is a congenital disorder characterized by typical facies, thymic aplasia, susceptibility to infections, hypoparathyroidism and conotruncal cardiac defect. Fetal thymus or post‐natal thymus tissue transplantations and human leucocyte antigen (HLA)‐genoidentical bone marrow transplantations were followed in a few cases by immune reconstitution. More recently, a peripheral blood mononuclear cell transplantation (PBMCT) was performed with an HLA‐genoidentical donor and followed by a partial T‐cell engraftment and immune reconstitution. We report a boy with cDGS, without cardiac defect, who suffered recurrent severe infections. At the age of 4 years, he underwent PBMCT from his HLA‐genoidentical sister. He received no conditioning regimen, but graft‐versus‐host disease (GVHD) prophylaxis was with oral cyclosporin A and mycophenolate mofetil. Toxicity was mild, with grade I acute GVHD. The patient is currently 2·5 years post‐PBMCT with excellent clinical performances. Mixed chimaerism can only be observed on the T‐cell population (50% donor T cells). T‐lymphocyte count fluctuated (CD3 more than 400 × 106/l at d 84 and CD4 more than 200 × 106/l at d 46). Exclusive memory phenotype T cells and absence of new thymic emigrants suggest expansion of infused T cells. T‐cell mitogen and tetanus antigen responses normalized a few months after transplantation. After immunizations, specific antibodies were produced. PBMCT from an HLA identical sibling could be an efficient treatment of immune deficiency in cDGS.

Spectrum of MECP2 mutations in Rett syndrome.

Mutations in the MECP2 (Methyl-CpG-binding protein) gene recently have been reported to cause Rett syndrome (RTT), an X-linked progressive encephalopathy. We have collected the results of MECP2 analysis conducted in four laboratories in France. A total of 301 RTT alleles have been analyzed, demonstrating a total of 69 different mutations so far observed and accounting for 64% of MECP2 genes in RTT patients living in France. R168X (11.5%) is the most common of MECP2 mutations, followed by R255X (10.9%), R270X (10.5%), T158M (7.8%), and R306C (6.8%). Only 10 mutations had a relative frequency > 2%. A total of 59 mutations were found in a small number of RTT alleles (from 1 to 2). These data demonstrate the high allelic heterogeneity of RTT in France and provide information relevant to the development of strategies for molecular diagnosis and genetic counseling in RTT families.

Germline APC mutation spectrum derived from 863 genomic variations identified through a 15-years medical genetics service to French FAP patients

Heterozygous APC germline alteration is responsible for familial adenomatous polyposis, a colon cancer predisposition with almost complete penetrance. Point mutations generally lead to truncated proteins or no protein at all. They mainly involve exon 3 to codon 1700 (exon 15). The work presented here delineates precisely the APC mutation spectrum from 15 years of systematic molecular screening which identified 863 independent alterations in the French population.

Evidence of somatic mosaicism for a MECP2 mutation in females with Rett syndrome: diagnostic implications.

Editor—Rett syndrome (RTT) (MIM 312750) is an X linked dominant neurodevelopmental disorder that occurs almost exclusively in females. Affected girls are considered to have a normal perinatal period followed by a period of regression, loss of acquired purposeful manual and speech skills, hand wringing, gait disturbance, and growth retardation.1 2 A gene for RTT has been identified in the Xq28 region which encodes the methyl-CpG binding protein 2 (MeCP2) involved in transcriptional silencing.3 4 This disorder most frequently occurs sporadically and results from a de novo mutation, although a few familial cases have been reported. Many studies5-16 have shown that the MECP2 gene is mutated in approximately 80% of patients with classical RTT and the MECP2 mutation spectrum includes missense, nonsense, and frameshift mutations, as well as larger rearrangements like deletions encompassing a few hundred bp.16 The failure to detect MECP2 mutations in the remaining 20% may indicate the presence of mutations in unexplored regions of the MECP2 gene, such as regulatory elements or non-coding regions, notably in the new first exon17 or in an additional RTT locus. Here, we report for the first time mosaicism for a somatic MECP2 mutation found in two unrelated females affected with RTT. These two girls were diagnosed according to the international criteria of the Rett Syndrome Diagnostic Criteria Work Group.18 The first patient (case 1) is 13 years old. She suffers from classical Rett syndrome with 7/9 of the necessary criteria, 4/8 of the supportive criteria, and none of the exclusion criteria.18More specifically, she had a normal neonatal period and head circumference at birth and a phase of social withdrawal at the age of 12 months when she lost purposeful hand skills and developed stereotypic hand movements, ataxia, and apraxia. She suffered from …

Deletion screening by fluorescence in situ hybridization in Rett syndrome patients

Mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene have been found to be a cause of Rett syndrome (RTT). Mutation screening was based on various techniques including denaturing gradient gel electrophoresis, single-strand conformation polymorphism analysis, heteroduplex analysis, DNA sequencing and recently Southern Blot analysis. Mutation detection was achieved in 80% of typical RTT with a high prevalence of recurrent mutations. In order to provide further insights into the spectrum of MECP2 rearrangements in patients without any point mutation or small deletion/insertion in the coding region MECP2 gene, we screened 25 classical RTT females using fluorescence in situ hybridization analysis. No deletion were found in our group, suggesting that MECP2 gross rearrangements are a rare cause of Rett syndrome.

MECP2 Mutations or Polymorphisms in Mentally Retarded Boys

AbstractBackground: Among the well characterized X-linked conditions causing mental retardation, mutations in the methyl-CpG-binding protein 2 gene (MECP2) in Xq28 have been found in up to 85% of patients with Rett syndrome, a neurologic disorder which, in addition to other symptoms, severely affects higher cognitive functions in females. Mutations in the MECP2 gene are involved in a broad spectrum of phenotypes from classical Rett syndrome to mild intellectual difficulties in females and neonatal encephalopathy in males. Recently, mutations in the MECP2 gene were reported in males with non-specific mental retardation suggesting that defects in MECP2 could be responsible for up to 2% of X-linked mental retardation. Methods: We screened by denaturing high-pressure liquid chromatography the entire coding region and flanking intronic sequences of the MECP2 gene in a cohort of 354 mentally retarded males found negative for an expansion across the FRAXA CGG repeat and in a family in which a boy and his sister were mentally retarded. Results: We identified mainly silent polymorphisms within the MECP2 gene, together with four sequence alterations of unknown significance, i.e. three missense mutations (T197M, T228S, and P376S) and one substitution at position −19 in intron 3 (378-19delT). Further familial investigations allowed us to ruled out a pathogenic effect for the intronic variant, the T228S and the P376S missense mutations. Conclusions: These results confirm that MECP2 mutations in males are far more rare than initially thought and call for a careful evaluation of the pathogenicity of the MECP2 missense mutations identified in mentally retarded males before genetic counseling is proposed to the relatives.

Prediction of BRCA1 Germ-Line Mutation Status in Patients with Breast Cancer Using Histoprognosis Grade, MS110, Lys27H3, Vimentin, and KI67

Family structure, lack of reliable information, cost, and delay are usual concerns when deciding to perform BRCA analyses. Testing breast cancer tissues with four antibodies (MS110, lys27H3, vimentin, and KI67) in addition to grade evaluation enabled us to rapidly select patients for genetic testing identification. We constituted an initial breast cancer tissue microarray, considered as a learning set, comprising 27 BRCA1 and 81 sporadic tumors. A second independent validation set of 28 BRCA1 tumors was matched to 28 sporadic tumors using the same original conditions. We investigated morphological parameters and 21 markers by immunohistochemistry. A logistic regression model was used to select the minimal number of markers providing the best model to predict BRCA1 status. The model was applied to the validation set to estimate specificity and sensibility. In the initial set, univariate analyses identified 11 markers significantly associated with BRCA1 status. Then, the best multivariate model comprised only grade 3, MS110, Lys27H3, vimentin, and KI67. When applied to the validation set, BRCA1 tumors were correctly classified with a sensitivity of 83% and a specificity of 81%. The performance of this model was superior when compared to other profiles. This study offers a new rapid and cost-effective method for the prescreening of patients at high risk of being BRCA1 mutation carriers, to guide genetic testing, and finally to provide appropriate preventive measures, advice, and treatments including targeted therapy to patients and their families.

Novel diagnostic tool for prediction of variant spliceogenicity derived from a set of 395 combined in silico/in vitro studies: an international collaborative effort

Abstract Variant interpretation is the key issue in molecular diagnosis. Spliceogenic variants exemplify this issue as each nucleotide variant can be deleterious via disruption or creation of splice site consensus sequences. Consequently, reliable in silico prediction of variant spliceogenicity would be a major improvement. Thanks to an international effort, a set of 395 variants studied at the mRNA level and occurring in 5′ and 3′ consensus regions (defined as the 11 and 14 bases surrounding the exon/intron junction, respectively) was collected for 11 different genes, including BRCA1, BRCA2, CFTR and RHD, and used to train and validate a new prediction protocol named Splicing Prediction in Consensus Elements (SPiCE). SPiCE combines in silico predictions from SpliceSiteFinder-like and MaxEntScan and uses logistic regression to define optimal decision thresholds. It revealed an unprecedented sensitivity and specificity of 99.5 and 95.2%, respectively, and the impact on splicing was correctly predicted for 98.8% of variants. We therefore propose SPiCE as the new tool for predicting variant spliceogenicity. It could be easily implemented in any diagnostic laboratory as a routine decision making tool to help geneticists to face the deluge of variants in the next-generation sequencing era. SPiCE is accessible at (https://sourceforge.net/projects/spicev2-1/).