Assessment of the functional impact of germline BRCA1/2 variants located in non-coding regions in families with breast and/or ovarian cancer predisposition.

PURPOSE: The molecular mechanism of breast and/or ovarian cancer susceptibility remains unclear in the majority of patients. While germline mutations in the regulatory non-coding regions of BRCA1 and BRCA2 genes have been described, screening has generally been limited to coding regions. The aim of this study was to evaluate the contribution of BRCA1/2 non-coding variants. METHODS: Four BRCA1/2 non-coding regions were screened using high-resolution melting analysis/Sanger sequencing or next-generation sequencing on DNA extracted from index cases with breast and ovarian cancer predisposition (3926 for BRCA1 and 3910 for BRCA2). The impact of a set of variants on BRCA1/2 gene regulation was evaluated by site-directed mutagenesis, transfection, followed by Luciferase gene reporter assay. RESULTS: We identified a total of 117 variants and tested twelve BRCA1 and 8 BRCA2 variants mapping to promoter and intronic regions. We highlighted two neighboring BRCA1 promoter variants (c.-130del; c.-125C > T) and one BRCA2 promoter variants (c.-296C > T) inhibiting significantly the promoter activity. In the functional assays, a regulating region within the intron 12 was found with the same enhancing impact as within the intron 2. Furthermore, the variants c.81-3980A > G and c.4186-2022C > T suppress the positive effect of the introns 2 and 12, respectively, on the BRCA1 promoter activity. We also found some variants inducing the promoter activities. CONCLUSION: In this study, we highlighted some variants among many, modulating negatively the promoter activity of BRCA1 or 2 and thus having a potential impact on the risk of developing cancer. This selection makes it possible to conduct future validation studies on a limited number of variants.

Odontoblastic differentiation of dental pulp stem cells from healthy and carious teeth on an original PCL-based 3D scaffold.

AIMS: To isolate and characterize dental pulp stem cells (DPSCs) obtained from carious and healthy mature teeth extracted when conservative treatment was not possible or for orthodontic reasons; to evaluate the ability of DPSCs to colonize, proliferate and differentiate into functional odontoblast-like cells when cultured onto a polycaprolactone cone made by jet-spraying and prototyped into a design similar to a gutta-percha cone. METHODOLOGY: DPSCs were obtained from nine carious and 12 healthy mature teeth. Then cells were characterized by flow cytometry and submitted to multidifferentiation to confirm their multipotency. These DPSCs were then cultured on a polycaprolactone cone in an odontoblastic differentiation medium. Cell proliferation, colonization of the biomaterial and functional differentiation of cells were histologically assessed. For the characterization, a t-Student test was used to compare the two groups. RESULTS: In all cell cultures, characterization highlighted a mesenchymal stem cell phenotype (CD105+, CD90+, CD73+, CD11b-, CD34-, CD45-, HLA-DR-). No significant differences were found between cultures obtained from carious and healthy mature teeth. DPSCs from both origins were able to differentiate into osteocytes, adipocytes and chondrocytes. Cell colonization was observed both on the surface and in the thickness of polycaprolactone cones as well as a mineralized pericellular matrix deposit composed of type I collagen, alkaline phosphatase, osteocalcin and dentin sialophosphoprotein. CONCLUSIONS: DPSCs were isolated from both carious and healthy mature teeth. They were able to colonize and proliferate within a polycaprolactone cone and could be differentiated into functional odontoblast-like cells.

A treadmill exercise reduced cardiac fibrosis, inflammation and vulnerability to ischemia-reperfusion in rat pristane-induced arthritis.

AIMS: To explore cardiac structural and functional parameters and myocardial sensitivity to ischemia in a rat model of chronic arthritis, pristane-induced arthritis (PIA), and to investigate the effects of a running exercise protocol on cardiac disorders related to rheumatoid arthritis (RA). MAIN METHODS: 3 groups of male Dark Agouti rats were formed: Controls, PIA and PIA-Exercise. The PIA-Exercise group was subjected to an individualized treadmill running protocol during the remission phase. At acute and chronic phases of PIA, cardiac structure was analyzed by histology. Cardiac function was explored in isolated hearts to measure left ventricular developed pressure (LVDP), cardiac compliance and infarct size before and after ischemia/reperfusion. Cardiac inflammation was evaluated through VCAM-1 mRNA expression by RT-qPCR. Plasma irisin levels were measured by ELISA. KEY FINDINGS: PIA rats exhibited myocardial hypertrophy fibrosis and inflammation at the 2 inflammatory phases of the model. At chronic phase only, LVDP and cardiac compliance were lower in PIA compared to controls. As compared to sedentary PIA, exercise did not change cardiac function but reduced fibrosis, inflammation, infarct size, and arthritis severity and increased irisin levels. Cardiac inflammation positively correlated with fibrosis, while irisin levels negatively correlated with cardiac inflammation and fibrosis. SIGNIFICANCE: In the PIA model that recapitulated most cardiac disorders of RA, a daily program of treadmill running alleviated cardiac fibrosis and inflammation and improved resistance to ischemia. These data provide arguments to promote the practice of exercise in RA patients for cardiac diseases prevention.

Lack of referral for genetic counseling and testing in BRCA1/2 and Lynch syndromes: a nationwide study based on 240,134 consultations and 134,652 genetic tests.

Based on nationwide data from the French national cancer institute (INCa), we analyzed the evolution of cancer genetics consultations and testing over time, and the uptake of targeted tests in relatives of families with BRCA1/2 or MMR genes mutation. Genetic testing and consultations for familial high-risk individuals are exclusively funded and monitored by the INCa in France. All nationwide cancer genetics centers reported annually standardized parameters of activity from 2003 to 2011. The analysis included a total of 240,134 consultations and 134,652 genetic tests enabling to identify 32,494 mutation carriers. Referral for hereditary breast and ovarian cancer (HBOC) or colorectal cancer predisposition syndromes represented 59 % (141,639) and 23.2 % (55,698) consultations, respectively. From 2003 to 2011, we found a dramatic and steady increase of tests performed for BRCA1/2 (from 2,095 to 7,393 tests/year, P < 0.0001) but not for MMR genes (from 1,144 to 1,635/year, P = NS). The overall percentage of deleterious mutations identified in the probands tested was 13.8 and 20.9 % in HBOC and Lynch syndromes, respectively. Pooled analysis for BRCA1/2 and Lynch syndrome tests showed an inverse relationship between the percentage of mutation detected and the absolute number of tests performed over the time (overall Cochran-Armitage test for trend: P < 0.001). In families with BRCA1/2 or MMR identified mutations, there was an average number of 2.94 and 3.28 relatives performing targeted tests, respectively. This nationwide study shows a lack of referral and genetic testing in Lynch as compared to HBOC syndromes. Only a third of relatives of a proband with a predisposing mutation performed a targeted test. Enhanced information about benefit of genetic testing should be given to clinicians and patients for Lynch syndrome and relatives of a proband carrying an identified predisposing mutation.

RB1 and TP53 pathways in radiation-induced sarcomas.

The tumour suppressor genes, TP53 and RB1, and four genes involved in their regulation, INK4a, ARF, MDM2 and MDMX, were analysed in a series of 36 post-radiotherapy radiation-induced sarcomas. One-third of the tumours developed in patients carrying a germline mutation of RB1 that predisposed them to retinoblastoma and radiation-induced sarcomas. The genetic inactivation of RB1 and/or TP53 genes was frequently observed in these sarcomas. These inactivations were owing to an interplay between point mutations and losses of large chromosome segments. Radiation-induced somatic mutations were observed in TP53, but not in RB1 or in the four other genes, indicating an early role of TP53 in the radio-sarcomagenesis. RB1 and TP53 genes were biallelically coinactivated in all sarcomas developing in the context of the predisposition, indicating that both genes played a major role in the formation of these sarcomas. In the absence of predisposition, TP53 was biallelically inactivated in one-third of the sarcomas, whereas at least one allele of RB1 was wild type. In both genetic contexts, the TP53 pathway was inactivated by genetic lesions and not by the activation of the ARF/MDM2/MDMX pathway, as recently shown in retinoblastomas. Together, these findings highlight the intricate tissue- and aetiology-specific relationships between TP53 and RB1 pathways in tumorigenesis.

The contribution of germline rearrangements to the spectrum of BRCA2 mutations.

BACKGROUND: Few germline BRCA2 rearrangements have been described compared with the large number of germline rearrangements reported in the BRCA1 gene. However, some BRCA2 rearrangements have been reported in families that included at least one case of male breast cancer. OBJECTIVE: To estimate the contribution of large genomic rearrangements to the spectrum of BRCA2 defects. METHODS: Quantitative multiplex PCR of short fluorescent fragments (QMPSF) was used to screen the BRCA2 gene for germline rearrangements in highly selected families. QMPSF was previously used to detect heterozygous deletions/duplications in many genes including BRCA1 and BRCA2. RESULTS: We selected a subgroup of 194 high risk families with four or more breast cancers with an average age at diagnosis of < or = 50 years, who were recruited through 14 genetic counselling centres in France and one centre in Switzerland. BRCA2 mutations were detected in 18.6% (36 index cases) and BRCA1 mutations in 12.4% (24 index cases) of these families. Of the 134 BRCA1/2 negative index cases in this subgroup, 120 were screened for large rearrangements of BRCA2 using QMPSF. Novel and distinct BRCA2 deletions were detected in three families and their boundaries were determined. We found that genomic rearrangements represent 7.7% (95% confidence interval 0% to 16%) of the BRCA2 mutation spectrum. CONCLUSION: The molecular diagnosis of breast cancer predisposition should include screening for BRCA2 rearrangements, at least in families with a high probability of BRCA2 defects.

The rostromedial zona incerta is involved in attentional processes while adjacent LHA responds to arousal: c-Fos and anatomical evidence.

Neurons producing melanin-concentrating hormone (MCH) are located in the tuberal lateral hypothalamus (LHA) and in the rostromedial part of the zona incerta (ZI). This distribution suggests that rostromedial ZI shares some common features with the LHA. However, its functions with regard to arousal or feeding, which are often associated with the LHA, have not been thoroughly investigated. This study analyses the responses in the tuberal LHA and adjacent rostromedial ZI after experiments related to arousal, exploration, food teasing and ingestive behavior. Specific aspects of the connections of the rostromedial ZI were also studied using retrograde and anterograde tract-tracing approaches. The rostromedial ZI is activated during exploratory and teasing experiments. It receives specific projections from the frontal eye field and the anterior pole of the superior colliculus that are involved in gaze fixation and saccadic eye movements. It also receives projections from the laterodorsal tegmental nucleus involved in attention/arousal. By contrast, the tuberal LHA is activated during wakefulness and exploratory behavior and reportedly receives projections from the medial prefrontal and insular cortex, and from several brainstem structures such as the periaqueductal gray. We conclude that the rostromedial ZI is involved in attentional processes while the adjacent tuberal LHA is involved in arousal.

Multiplex PCR/liquid chromatography assay for detection of gene rearrangements: application to RB1 gene.

Screening for large gene rearrangements is established as an important part of molecular medicine but is also challenging. A variety of robust methods can detect whole-gene deletions, but will fail to detect more subtle rearrangements that may involve a single exon. In this paper, we describe a new, versatile and robust method to assess exon copy number, called multiplex PCR/liquid chromatography assay (MP/LC). Multiple exons are amplified using unlabeled primers, then separated by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC), and quantitated by fluorescent detection using a post-column intercalation dye. The relative peak intensities for each target directly reflect exon copy number. This novel technique was used to screen a panel of 121 unrelated retinoblastoma patients who were tested previously using a reference strategy. MP/LC correctly scored all deletions and demonstrated a previously undetected RB1 duplication, the first to be described. MP/LC appears to be an easy, versatile, and cost-effective method, which is particularly relevant to denaturing HPLC (DHPLC) users since it broadens the spectrum of available applications on a DHPLC system.

Breast and ovarian cancer predisposition due to de novo BRCA1 and BRCA2 mutations.

BRCA1 and BRCA2 are the two major genes predisposing to breast and ovarian cancer. Whereas high de novo mutation rates have been demonstrated for several genes, only 11 cases of de novo BRCA1/2 mutations have been reported to date and the BRCA1/2 de novo mutation rate remains unknown. The present study was designed to fill this gap based on a series of 12 805 consecutive unrelated patients diagnosed with breast and/or ovarian cancer who met the inclusion criteria for BRCA1/2 gene analysis according to French guidelines. BRCA1/2 mutations were detected in 1527 (12%) patients, and three BRCA1 mutations and one BRCA2 mutation were de novo. The BRCA1/2 de novo mutation rate was estimated to be 0.3% (0.1%; 0.7%). Although rare, it may be useful to take the possibility of de novo BRCA1/2 mutation into account in genetic counseling of relatives and to improve the understanding of complex family histories of breast and ovarian cancers.

Evaluation of a French ELISA for the detection of Salmonella Enteritidis and Salmonella Typhimurium in flocks of laying and breeding hens.

In France, the regular and compulsory detection of Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) in flocks of breeding and laying hens is based on bacteriological examination of environmental swabs and faeces samples. The aim of this study was to compare this bacteriological examination with a serological method (ELISA) developed in our laboratory. This ELISA was first evaluated by use of artificially infected hens. During these experimental infection studies, several groups of hens were inoculated with SE, ST, different vaccines and different Salmonella serovars to calculate the experimental parameters of our ELISA. Then, in a field study, 43 flocks were followed monthly using two bacteriological samples (environmental swab and pool of faeces) and 20 serological samples (sera or yolks). Twenty-seven flocks without SE or ST gave a negative serological response throughout their surveillance. Among the 10 various serovars different from SE and ST isolated in this study, S. Heidelberg, S. Agona and S. Hadar gave seropositive results in seven flocks. Consequently, this ELISA was not specific of SE and ST as it detected serovars sharing or not common antigens with SE and ST. Seropositive results were also obtained each month for two flocks where no Salmonella could be isolated. Finally, in seven flocks found infected with SE or ST, the positive ELISA results appeared later than the bacteriological detection. Therefore, for the detection of chicken flocks recently infected with SE or ST, bacteriological examination currently used in France seems to be more appropriate than this ELISA.

Comprehensive screening for constitutional RB1 mutations by DHPLC and QMPSF.

Constitutional mutations of the RB1 gene are associated with a predisposition to retinoblastoma. It is essential to identify these mutations to provide appropriate genetic counseling in retinoblastoma patients, but this represents an extremely challenging task, as the vast majority of mutations are unique and spread over the entire coding sequence. Since 2001, we have implemented RB1 testing on a routine basis as part of the clinical management of retinoblastoma. As most screening techniques do not meet the requirements for efficient RB1 testing, we have devised a semi-automated denaturing high-performance liquid chromatography (DHPLC) method for point mutation detection combined with a quantitative multiplex PCR of short fluorescent fragments (QMPSF) approach to screen for gene rearrangements. We report the results of this comprehensive screening of all exons and promoter of RB1 in 192 unrelated patients, mostly of French origin. Among 102 bilateral and/or familial cases and 90 unilateral sporadic probands, mutations were identified in 83 (81.5%) and 5 (5.5%) cases, respectively. A total of 43 mutations have not been previously reported. The mutational spectrum was found to be significantly different from previous published series, displaying a surprising amount of splice mutations and large deletions. This study demonstrates the reliability of DHPLC for RB1 analysis, but also illustrates the need for a deletion scanning approach. Finally, considering the benefits to retinoblastoma patients, RB1 testing should be widely implemented in routine healthcare because our study clearly illustrates its feasibility.

Possible relationship between the van der Woude syndrome (vWS) locus and nonsyndromic cleft lip with or without cleft palate (NSCL/P).

Cleft lip with or without cleft palate (CL/P) is one of the most common congenital malformations in humans occurring with a birth prevalence of approximately 1:1,000. CL/P may be part of a defined syndrome, sequence or association, although most individual or familial cases present as an isolated (nonsyndromic) malformation (NSCL/P). Inheritance is generally regarded as multigenic although, in some families, NSCL/P seemingly segregates as a monogenic trait. On the other hand, van der Woude syndrome (vWS) is a rare autosomal dominant with cardinal features of lower-lip pits (LLP) and CL/P or cleft palate (alone). Since none of these traits is present in all mutation carriers, some individual or familial vWS cases, especially those lacking LLP, are indiscernible from NSCL/P, raising the question whether allelic variation at the vWS locus could underlie NSCL/P. This question was addressed using parametric linkage (LOD score) analysis in 21 multiplex NSCL/P families based on a tightly linked microsatellite marker (D1S3753), and nonparametric analysis using the transmission/disequilibrium test (GTDT) in 106 NSCL/P triads and selecting markers D1S205, D1S491, and D1S3753. No evidence for linkage of NSCL/P to vWS was found on the 21 families using the LOD score approach. In contrast, TDT yielded a significant P value of 0.04 for D1S205, supporting involvement of vWS in NSCL/P in a complex, modifying/polygenic manner rather than as a monogenic/major disease locus.

Novel recurrent nonsense mutation causing neurofibromatosis type 1 (NF1) in a family segregating both NF1 and Noonan syndrome.

Neurofibromatosis type 1 (NF1), a genetic disorder with neuroectodermal involvement, demonstrates phenotypic overlap in some patients with Noonan syndrome (NS), ultimately resulting in the so-called neurofibromatosis-Noonan syndrome (NF-NS). A strong association of the two phenotypic traits was recently illustrated by a four-generation family, although NF1 and NS were eventually demonstrated to segregate independently on the basis of polymorphic DNA markers [Bahuau et al., 1996: Am J Med Genet 66:347-355]. Identification of the causal NF1 mutation seemed a prerequisite to further dissecting this singular familial association. Using the protein truncation assay, a nonsense mutation (C2446T-->R816X) of the neurofibromin gene was evidenced. This mutation occurred on a CpG dinucleotide within exon 16 and 5' to the GAP domain-specifying region of the gene. R816X creates a recognition site for endonuclease HphI, absent in 2 individuals with NS only. Screening 184 unrelated NF1 patients, three novel occurrences of the mutation were found in individuals diagnosed with classical NF1. Based on the assumption of genotype-phenotype correlation in these individuals, clinical and molecular analyses of this four-generation family demonstrated that the NF-NS phenotype was additive, being the result of both classical NF1 and NS. This particular observation also suggests the presence of an NS locus on 17q, which might be of interest for further linkage studies.

Pierre Robin sequence and interstitial deletion 2q32.3-q33.2.

Pierre Robin sequence (PRS) consists of the nonrandom association of micrognathia, cleft palate (CP), and glossoptosis. It also includes respiratory and feeding difficulties that appear to be neurogenic rather than mechanical in causation. Genetic determinants are thought to underlie this functional and morphological entity, based on the existence of Mendelian syndromes with PRS, and the rare observations of familial nonsyndromic PRS, in which some of the affected individuals have isolated CP. We report the association of PRS with deletion 2q32.3-q33.2 due to an unbalanced reciprocal translocation 46,XX, t(2;21), del 2(q32.3q33.2), and we refine the deletion interval with regard to YAC probes and polymorphic DNA markers. The deletion was shown to be flanked by D2S369 (telomeric) and D2S315 (centromeric), thus it maps to a recently determined chromosomal region known to be nonrandomly associated with CP. This observation supports the hypothesis for the genetic bases of nonsyndromic PRS, strengthens its possible genetic association with isolated CP, and provides a candidate PRS locus, in chromosomal region 2q32.3-q33.2.

No evidence for antibodies to mycobacterial A60 antigen in Crohn's disease sera by enzyme-linked immunoabsorbent assay (ELISA).

Sera from patients with Crohn's disease were tested with an ELISA system for antibodies to antigen A60 prepared from Mycobacterium bovis (BCG), M. avium and M. paratuberculosis. No evidence was found for significantly high titres of antibody to these antigens.

Study of the delivery of the gD gene of pseudorabies virus to one-day-old piglets by adenovirus or plasmid DNA as ways to by-pass the inhibition of immune response by colostral antibodies.

In the present study, it was shown that piglets with maternal antibodies, which had been primed with a replication-defective adenovirus that expresses the pseudorabies virus (PRV) glycoprotein gD and boosted with the Bartha vaccine strain at 10 weeks of age are equally protected clinically upon a challenge as piglets without maternal antibodies vaccinated with the same approach or with the Bartha vaccine strain alone. Priming with a plasmid that expresses gD was less efficient.

Longitudinal serological responses to Salmonella enterica of growing pigs in a subclinically infected herd.

A longitudinal survey was conducted in France in a subclinically Salmonella-infected farrow-to-finish pig farm to describe the time-course of the serological response to Salmonella enterica in growing pigs. We used three batches of sows and their corresponding litters (n = 31 litters). Among these, 256 pigs randomly selected and individually identified were followed from the first week of age until slaughter. Serial individual blood samples were submitted to indirect Salmonella-ELISA testing. Salmonella shedding was investigated by bacteriological testing of faecal material regularly collected from the sows and pigs and by environment swabs taken from the pens. Caecal contamination was checked at the slaughterhouse. Information about litter composition (filiation), location of the pigs in post-weaning and fattening pens, sanitary events, sex and body weights was recorded. 11.6% of the pigs shed S. enterica; 52% of pigs seroconverted before slaughter. The age-related variation of the natural logarithm of calibrated optical densities (COD) of pigs was described with two linear mixed models. From 8 to 61 days of age, the decrease in COD with age was fitted with a model including random effects of the animal and the dam on the intercept and slope, a batch random effect on the intercept and an individual birth-weight fixed effect on the intercept. The dam random effect was explained by the parity of the sow, Salmonella shedding by the sow during the farrowing phase and the value of the optical density of colostrum collected at parturition. A second model fitting the increase in COD from 61 days of age until slaughter included the random effect on intercept of the batch and the random effects on slope and intercept of the animal, the dam and the pen in which the followed animals were located during the fattening phase and the environmental contamination as fixed effect. In this second model, no relation was found between individual slaughter-bacteriological results and increasing COD values. Considering seroconversion time between 61 days of age and slaughter, survival function were constructed using the Cox proportional-hazards model. Both methods suggested that seroconversions generally occurred during the last third of the fattening phase (from 140 days of age to slaughter), while shedding was observed during the first half of the fattening period. The fitted models suggest the existence of clusters (such as pen and litter of origin).

[Palliative treatment of cancers of the esophagus and cardia with neodymium YAG laser: short-term results and multidimensional analysis of factors related to functional improvement and tumor destruction]. / Traitement palliatif des cancers de l'oesophage et du cardia par le laser YAG néodyme: Résultats à court terme et analyse multidimensionnelle des facteurs liés à l'amélioration fonctionnelle et à la destruction tumorale.

The aim of this study was to report initial results of endoscopic Nd YAG laser therapy in palliative treatment of esophageal and cardial carcinoma and to define parameters that could predict initial outcome. Of 116 patients, 95 men and 21 women (mean age 68 years, range 33-94 years), 6 patients presented with inaccessible, completely obstructing squamous cell carcinoma. Of these 6 tumors, 5 were located in the cervical esophagus. Seventy-two patients with squamous cell carcinoma and 38 patients with adenocarcinoma were treated. Improvement of dysphagia was achieved in 90 of 110 patients (82 p. 100) after 2.8 sessions (range 2-7 sessions). Total destruction of the intraluminal tumor was achieved in 66 patients (60 p. 100) after 3.3 sessions, partial destruction in 36 patients (33 p. 100) after 4.3 sessions and treatment had no effect in 8 patients (7 p. 100) after 3.8 sessions. Forty-four of the 52 impassable stenoses with the Olympus GIFP3 endoscope were passed through with the Olympus GIFQ endoscope after 3.7 sessions. Six complications occurred: 1 esophageal perforation and 5 esobronchotracheal fistulas. Improvement of dysphagia and tumoral destruction were analyzed in two manners. Of 13 selected variables, 3 were significantly related with improvement of dysphagia after stepwise logistic regression: destruction of intraluminal tumor (p = 0.003), endoscopic fungating appearance (p = 0.03) and the age related type of surgical contraindication (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Use of muscle exudates for the detection of anti-gE antibodies to Aujeszky's disease virus.

A commercial ELISA test to detect serum anti-gE antibodies to Aujeszky's disease virus was adapted for use with muscle exudates. The muscle samples were taken from the diaphragm of pig carcasses at the slaughterhouse. Three hundred and eighty-nine pairs of samples of serum and muscle exudate were compared to determine the possibility of using muscle exudate samples in a programme to control Aujeszky's disease. Taking the serum samples as the reference, the individual sensitivity of the test was 93.2 per cent and the individual specificity was 98.3 per cent. The concentration of antibodies in the muscle exudates was on average 20 times lower than that in the serum samples.

[Genetic testing for cystic fibrosis: evaluation of the Elucigene CF20 kit in blood and buccal cells]. / Diagnostic moléculaire de la mucoviscidose: evaluation de la trousse Elucigene CF20 dans le sang et les cellules buccales.

Routine determination of mutations in cystic fibrosis requires accurate, rapid, reliable and low-cost methods, permitting the simultaneous detection of multiple mutations. The Elucigene CF20 kit developped by Cellmark Diagnostics, uses multiplex ARMS, which allows the screening for 20 CFTR gene mutations (deltaF508, G542X, N1303K, 1717-1G>A, G551D, W1282X, R553X, deltaI507, 1078delT, 2183AA>G, 3849+10kbC>T, R1162X, 621+1G>T, R334W, R347P, 3659delC, R117H, S1251N, E60X, A455E ) in a work day without specific instrumentation. The kit distinguishes between homozygotes and heterozygotes for deltaF508, but not for rare mutations. The kit detects from 68 to 92% of defective alleles in Caucasians. We evaluate the kit in a blind study in two independent laboratories. Thirty blood samples and thirty mouthwash samples from CF patients, carriers and unaffected individuals were analysed by the Elucigene CF20 kit. All the samples were previously analysed by denaturing gradient gel electrophoresis and sequencing. The Elucigene CF20 kit consists of three multiplexes. Each mutiplex contains ARMS specific primers for six to eight mutations and two control reactions. The absence of the upper control fragment indicates that a repeat test is required. We demonstrated a first time amplification rate of 98.3%: of the 60 samples tested, one required a reamplification. Results compared with the reference method demonstrated that in all cases where one or more of the 20 mutations detected by the kit were present in the test set, the kit accurately identified them. Reproducibility was assessed by repeating the analysis of a blood and mouthwash sample five times. Cross reactivity between R117C and R117H, R117P and R117H, R347P and R347H, deltaI507 and deltaF508, G551D and R553X were evaluated. Only a cross reactivity between R347P and R347H was observed. The kit is specially useful for first line study of patients and carrier identification.