The multi-faceted nature of 15 CFTR exonic variations: Impact on their functional classification and perspectives for therapy.

BACKGROUND: The majority of variants of unknown clinical significance (VUCS) in the CFTR gene are missense variants. While change on the CFTR protein structure or function is often suspected, impact on splicing may be neglected. Such undetected splicing default of variants may complicate the interpretation of genetic analyses and the use of an appropriate pharmacotherapy. METHODS: We selected 15 variants suspected to impact CFTR splicing after in silico predictions on 319 missense variants (214 VUCS), reported in the CFTR-France database. Six specialized laboratories assessed the impact of nucleotide substitutions on splicing (minigenes), mRNA expression levels (quantitative PCR), synthesis and maturation (western blot), cellular localization (immunofluorescence) and channel function (patch clamp) of the CFTR protein. We also studied maturation and function of the truncated protein, consecutive to in-frame aberrant splicing, on additional plasmid constructs. RESULTS: Six of the 15 variants had a major impact on CFTR splicing by in-frame (n = 3) or out-of-frame (n = 3) exon skipping. We reclassified variants into: splicing variants; variants causing a splicing defect and the impairment of CFTR folding and/or function related to the amino acid substitution; deleterious missense variants that impair CFTR folding and/or function; and variants with no consequence on the different processes tested. CONCLUSION: The 15 variants have been reclassified by our comprehensive approach of in vitro experiments that should be used to properly interpret very rare exonic variants of the CFTR gene. Targeted therapies may thus be adapted to the molecular defects regarding the results of laboratory experiments.

Dermatological and genetic data in tuberous sclerosis: A prospective single-center study of 38 patients.

BACKGROUND: Tuberous sclerosis complex (TSC) is a genetic disorder involving the TSC1 or TSC2 gene. Skin signs are prominent, but dermatological data are scarce. This study aims to describe the cutaneous signs of TSC with the genotype. METHODS: We studied the dermatological characteristics of 38 patients with TSC at the University Hospital of Montpellier. We collected details of genotypic features. RESULTS: All the patients presented at least one cutaneous sign. The dermatological examination alone was sufficient to establish a definite diagnosis of TSC based on the diagnostic criteria for 34/38 patients. No association was found between cutaneous signs and the presence of a TSC1 or TSC2 mutation. We noted skin signs that were poorly described in the disease, namely epidermal nevus in 3 patients, vascular malformation in 2 patients, and keratosis pilaris in 9 patients. DISCUSSION: While several studies demonstrate a more severe neurological phenotype in TSC2 mutated patients, skin expression does not appear to differ according to the mutated gene. Further case reports and molecular genetic studies are needed to determine the link between epidermal nevus, vascular malformations, keratosis pilaris and TSC.

[Prevalence of the tuberous sclerosis complex at patients taken care for a renal angiomyolipoma]. / Prévalence de la sclérose tubéreuse de Bourneville chez des patients pris en charge pour un angiomyolipome rénal.

INTRODUCTION: Bourneville's tuberous sclerosis or Tuberous Sclerosis Complex (TSC) is an autosomal dominant hereditary phakomatosis associated with angiomyolipomas (AML) of the kidney. The aim of this study was to identify the prevalence of TSC in patients diagnosed and cared for AML in our department of urology. MATERIALS AND METHODS: All the patients with AML were included between March 2009 and June 2016 in a French university hospital. Each patient was reviewed in consultation with a clinical examination and imaging. Specific clinical criteria were used to refer patients to genetic analysis. Patients with a high TSC probability had a genetic analysis to search TSC1 and TSC2 genes mutations. RESULTS: In all, 28 patients were included and 3 (11%) were diagnosed TSC. The median age of the patients was 62 years (36-82 years). The most frequent clinical criteria were facial angiofibromas in 7 patients (25%). Among the 8 patients (29%) with evocative clinical criteria, a mutation of the TSC1 and TSC2 genes was identified in 3 patients (11%) with a diagnosis of TSC made before the AML diagnosis. CONCLUSION: In this study, 8 patients (29%) presented clinical criteria suggestive of TSC, preferentially dermatological. The diagnosis was confirmed by screening TSC1 and TSC2 genes mutations in 3 patients (11%), nevertheless prevalence of TSC is most probably underestimated by the genetic mosaïcisme of this pathology.

[Aquagenic palmar keratoderma in a patient heterozygous for the mutation c.3197G>C in the CFTR gene]. / Kératodermie aquagénique palmaire chez une patiente hétérozygote pour la mutation c.3197G>C du gène CFTR.

BACKGROUND: Aquagenic palmar keratoderma is an entity recently described in the literature by English and McCollough in 1996. It is a rare condition affecting young women and is of unknown incidence. It causes a wrinkled and oedematous appearance in the skin of the hands that may be seen a few minutes after immersion in water. This condition may be associated with a heterozygous mutation in CFTR, the gene involved in cystic fibrosis. We report the first case of aquagenic keratoderma associated with a new mutation in the CFTR gene. PATIENTS AND METHODS: An 18-year-old patient with no particular history was referred for a painful rash on both palms occurring whenever she showered, and which had been ongoing for several months. The clinical examination was normal except for an appearance of moderate palmar hyperhidrosis. Following a test in which both hands were immersed in cold water for 5minutes, the patient presented itching, burning and pain localized to the hands. The palms were wrinkled and oedematous with white, translucent and confluent papules. A clinical diagnosis of aquagenic palmar keratoderma was made. Since this condition may be associated with mutations in the CFTR gene, a genetic study was performed for this patient and revealed the presence of a new mutation in the CFTR gene for cystic fibrosis in the heterozygous state inherited from her mother: c.3197G>C or p.Arg1066.Pro and a heterozygous polypyrimidic 5T variant inherited from her father. DISCUSSION: We report a new case of aquagenic palmar keratoderma in a patient heterozygous for a new mutation of the gene involved in cystic fibrosis. Several studies have shown association of aquagenic keratoderma with the CFTR gene for heterozygotes (carriers without cystic fibrosis), for patients with cystic fibrosis and for a patient presenting CFTRopathy with pancreatic insufficiency.

Hypochloremia and hyponatremia as the initial presentation of cystic fibrosis in three adults.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Most diagnoses of CF are made during infancy or childhood, and are based on respiratory or digestive involvement. Initial extracellular dehydration leading to the diagnosis of CF is usual in infants but has only exceptionally been reported in adults. We describe three new adult cases of CF initially presenting with depletive hyponatremia and hypochloremia following exposure to heat. At first consultation, these patients had no symptoms suggestive of CF. One patient presented with a seizure induced by hyponatremia. The two other patients were siblings carrying a novel c.4434insA mutation in exon 24 of CFTR. Acute dehydration is a very rare initial manifestation of CF but may be life-threatening. The possibility of CF should not be ignored in cases of depletive hyponatremia, hypochloremia or hypokalemic metabolic alkalosis, even in otherwise healthy patients.

Mitochondrial DNA A3243G mutation involved in familial diabetes, chronic intestinal pseudo-obstruction and recurrent pancreatitis.

AIMS: To report on a family with five members who carry the A3243G mutation in mitochondrial tRNA for leucine 1 (MTTL1) and present with diabetes, chronic intestinal pseudo-obstruction (CIPO) and recurrent pancreatitis, and to screen for this mutation in a cohort of 36 unrelated patients with recurrent pancreatitis. METHODS: The mutation was quantified in several tissue samples from patients. Respiratory chain activity was studied in muscle biopsies and fibroblast cultures. In addition, the thymidine phosphorylase gene (TP) involved in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) and three genes involved in chronic pancreatitis - PRSS1, SPINK1 and CFTR - were sequenced in affected patients. Finally, the MTTL1 gene was examined in 36 unrelated patients who had recurrent pancreatitis, but no mutations in the PRSS1 and SPINK1 genes. RESULTS: Heteroplasmy for the mtDNA A3243G mutation was found in all tissue samples from these patients, but no mutations were found in the genes coding for thymidine phosphorylase, PRSS1, SPINK1 and CFTR. Also, none of the 36 unrelated patients with recurrent pancreatitis were carrying any MTTL1 mutations. CONCLUSION: The mtDNA A3243G mutation associated with the gastrointestinal manifestations observed in the affected family should be regarded as a possible cause of CIPO and unexplained recurrent pancreatitis. However, the mutation is probably only weakly involved in cases of isolated recurrent pancreatitis.

Severe episodes of extra cellular dehydration: an atypical adult presentation of cystic fibrosis.

Cystic fibrosis (CF) is usually diagnosed during childhood by respiratory or gastro-intestinal symptoms. Hyponatremic hypochloremic dehydration with metabolic alkalosis is a rare but typical presentation of CF in infants. In contrast, only 3 cases have been described in adults. We report a case of CF in a 33-year-old Caucasian female presenting with a severe sodium and chloride depletion caused by inappropriate sweating. She experienced three episodes of severe dehydration before the diagnosis was suspected. Sweat chloride test was pathological and mild pulmonary involvement was found on CT scan. Delta F508 mutation and a rare mutation (3849+40 A/G) on the intron 19 of CFTR gene were found. Interestingly, our patient has a heterozygote twin sister, carrier of the same mutations of CFTR gene who also developed CF but with a different phenotype. We suspect modifier genes to be implicated in the differences observed between the two phenotypes. We discuss the physiopathology of electrolyte disturbance and review the other similar adults cases.

Cognitive changes in asymptomatic carriers of the Huntington disease mutation gene.

Huntington disease (HD) is a neurodegenerative disorder due to an excessive number of CAG repeats in the IT15 gene on chromosome 4. Studies of cognitive function in asymptomatic gene carriers have yielded contradictory results. This study compared cognitive performance in 44 subjects with the HD mutation (group of carriers) who had no clinical signs of HD and 39 at-risk individuals without HD mutation (group of non-carriers). Neuropsychological evaluation focused on global cognitive efficiency, psychomotor speed, attentional, executive and memory functions. Significant differences, with lower performances in the group of gene carriers, were detected for some measures of psychomotor speed, attention and executive functioning (all P < 0.01). More differences between groups were observed for memory measures, in particular on the California Verbal Memory Test. Complementing these observations, cognitive scores were correlated with age in the group of gene carriers, but not in the group of non-carriers. This suggests that the cognitive changes precede the appearance of the motor and psychiatric symptoms in HD and that tests proved to be sensitive to early HD deficiencies are better suited than global cognitive efficiency scales to observe them.

Bilateral hand amyotrophy with PMP-22 gene deletion.

Hereditary neuropathy with liability to pressure palsies (HNPP) phenotypes are heterogeneous. We report the case of a 52-year-old woman without medical history, who complained of bilateral hand weakness suggestive first of a motor neuron disorder. The presence of a diffuse predominant distal demyelinating neuropathy suggested a deletion of PMP-22 gene, which was confirmed by genetic analysis. This case report underlines a novel phenotype related to the deletion of PMP-22 gene.

Hyperechogenic fetal bowel: a large French collaborative study of 682 cases.

Hyperechogenic fetal bowel is detected in 0.1-1.8% of pregnancies during the second or third trimester. This ultrasound sign is associated with cystic fibrosis or other conditions (e.g., chromosomal anomalies, viral infection) but no large-scale prospective studies have been conducted. This 1997-1998 multicenter study in 22 molecular biology laboratories identified 682 cases of hyperechogenic fetal bowel detected by routine ultrasound examination during the second (86%) or third trimester. The fetal bowel was considered hyperechogenic when its echogenicity was broadly similar to, or greater than, that of the surrounding bone. Karyotyping, screening for viral infection, and screening for cystic fibrosis mutations were performed in all cases. Pregnancy outcome and postnatal follow-up were obtained in 656 of the 682 cases (91%). In 447 cases (65.5%), a normal birth was observed. Multiple malformations were observed in 47 cases (6.9%), a significant chromosomal anomaly was noted in 24 (3.5%), cystic fibrosis in 20 (3%), and viral infection in 19 (2.8%). In utero unexplained fetal death occurred in 1.9% of cases, toxemia in 1.2%, IUGR in 4.1%, and premature birth in 6.2%. This study demonstrates that this ultrasound sign is potentially associated with medically significant outcomes. Having established that the bowel is hyperechogenic, recommended investigations should include a detailed scan with Doppler measurements, fetal karyotyping, cystic fibrosis screening, and infectious disease screening. After birth, newborns require pediatric examination because a surgical treatment may be necessary. This should be combined with clear counseling of the parents.

Predicting the risk of cystic fibrosis with abnormal ultrasound signs of fetal bowel: results of a French molecular collaborative study based on 641 prospective cases.

Hyperechogenic fetal bowel is prenatally detected by ultrasound during the second trimester of pregnancy in 0.1-1.8% of fetuses. It has been described as a normal variant but has often been associated with severe diseases, notably cystic fibrosis (CF). The aim of our study was to determine the risk of CF in a prospective study of 641 fetuses with ultrasonographically abnormal fetal bowel and the residual risk when only one mutation is detected in the fetus. Fetal cells and/or parental blood cells were screened for CFTR mutations. Two screening steps were used, the first covering the mutations most frequently observed in French CF patients (mutation detection rate of 70-90%) and, when a CF mutation was detected, a DGGE-sequencing strategy. We observed a 3.1% risk of CF when a digestive tract anomaly was prenatally observed at routine ultrasound examination. The risk was higher when hyperechogenicity was associated with bowel dilatation (5/29; 17%) or with the absence of gall bladder (2/8; 25%). The residual risk of CF was 11% when only one CF mutation was detected by the first screening step, thereby justifying in-depth screening. Mutations associated with severe CF (DeltaF508 mutation) were more frequently observed in these ultrasonographically and prenatally detected CF cases. However, the frequency of heterozygous cases was that observed in the normal population, which demonstrates that heterozygous carriers of CF mutations are not at increased risk for hyperechogenic bowel. In conclusion, fetal bowel anomalies indicate a risk of severe cystic fibrosis and justify careful CFTR molecular analysis.

[Genetic male infertility and medically assisted reproduction]. / Infertilité masculine d'origine génétique et assistance médicale à la procréation.

About half the cases of infertility have their origin in the male partner. Infertility due to males has several possible aetiologies. In about 30% of cases, genetic disorders are suspected of being the main cause. They could interfere with the development of the male gonads, the urogenital tract or the hypothalamo-hypophyseal axes. Such disorders could also stop germ cell generation and maturation or lead to the production of non-functional spermatozoa. Genetic disorders of chromosomal origin could give rise to abnormal karyotypes or germinal mosic figure. They could involve gene abnormalities affecting numerous genes localized on several chromosomes, in particular the Y chromosome. The physiopathologic identification of male infertility is interesting because of the risk of the genetic factors involved being transmitted to the offspring. The subject is of importance, specially because of the increasing use of intracytoplasmic sperm injections. Couples should therefore be precisely counselled to enable them to make a well-informed choice among various solutions, e.g. ART, with or without sperm donation, or adoption.

Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France.

We have collated the results of cystic fibrosis (CF) mutation analysis conducted in 19 laboratories in France. We have analyzed 7, 420 CF alleles, demonstrating a total of 310 different mutations including 24 not reported previously, accounting for 93.56% of CF genes. The most common were F508del (67.18%; range 61-80), G542X (2.86%; range 1-6.7%), N1303K (2.10%; range 0.75-4.6%), and 1717-1G>A (1.31%; range 0-2.8%). Only 11 mutations had relative frequencies >0. 4%, 140 mutations were found on a small number of CF alleles (from 29 to two), and 154 were unique. These data show a clear geographical and/or ethnic variation in the distribution of the most common CF mutations. This spectrum of CF mutations, the largest ever reported in one country, has generated 481 different genotypes. We also investigated a cohort of 800 French men with congenital bilateral absence of the vas deferens (CBAVD) and identified a total of 137 different CFTR mutations. Screening for the most common CF defects in addition to assessment for IVS8-5T allowed us to detect two mutations in 47.63% and one in 24.63% of CBAVD patients. In a subset of 327 CBAVD men who were more extensively investigated through the scanning of coding/flanking sequences, 516 of 654 (78. 90%) alleles were identified, with 15.90% and 70.95% of patients carrying one or two mutations, respectively, and only 13.15% without any detectable CFTR abnormality. The distribution of genotypes, classified according to the expected effect of their mutations on CFTR protein, clearly differed between both populations. CF patients had two severe mutations (87.77%) or one severe and one mild/variable mutation (11.33%), whereas CBAVD men had either a severe and a mild/variable (87.89%) or two mild/variable (11.57%) mutations.

Molecular analysis of 53 fragile X families with the probe StB12.3.

Fifty-three pedigrees with the fragile X syndrome have been studied for amplification of the CGG repeat sequence adjacent to the CpG island in the FMR1 gene. Probe StB12.3 allowed direct detection of affected males, carrier females, normal transmitting males, as well as prenatal diagnosis. Comparison of our molecular data with our previous linkage data from 38 families indicates the effectiveness of direct DNA analysis. A total of 325 individuals were studied and no new mutation was found. All daughters of males with a premutation had a premutation. When the mother had a full mutation no children had a premutation. In premutated mothers, the size of the premutation seems to be a determining factor for the transition to the full mutation. All affected males had a full mutation or mosaicism and only 42% of the females with a full mutation were mentally impaired. Analysis of large families over 3 generations illustrated clearly the Sherman paradox. Furthermore, the analysis of these families is in reasonable agreement with the multiallelic model of Morton and Macpherson [Proc Natl Acad Sci USA 89:4215-4217, 1992]. Mosaic cases in the offspring of the mothers with a full mutation suggest a maternal germinal mosaicism. Then an abnormal methylation and a somatic heterogeneity established in very early steps of embryogenesis could explain these cases.

Quantitative determination of the hybrid Bcr-Abl RNA in patients with chronic myelogenous leukaemia under interferon therapy.

In vitro amplification of the Bcr-Abl cDNA has been widely used to assess for the presence of minimal residual disease in patients with chronic myelogenous leukaemia (CML) presenting with complete clinical and cytogenetic remission. However, the level of sensitivity achieved in different laboratories remains largely unknown. Moreover, the results are usually expressed as positive or negative, making a precise follow-up of the patients difficult. In an attempt to overcome these limitations, we devised a quantitative method to measure the amount of Bcr-Abl mRNA in clinical samples. This methodology involves a single reverse transcription step, followed by separate amplifications of Bcr-Abl and total Abl mRNA. These two amplifications are performed in the presence of different dilutions of a same internal standard. This standard consists of Bcr-Abl sequences with an eight bases deletion in exon 2 of Abl. One of the primers used in each separate reaction is labelled with fluorescein. Following amplification, PCR products derived from cellular RNA and those from the internal standard are separated and their relative fluorescence is determined using a laser fluorescent DNA sequencer (ALF, Pharmacia). The number of Bcr-Abl and total Abl mRNA molecules initially present in each sample is then calculated. The accuracy and reproducibility of this method was assessed by studying serial dilutions of K562 RNA into normal RNA. Blood samples from 10 patients in cytogenetic remission under interferon therapy were studied. Only one sample was found negative while the others contained between 0.05 and 17 hybrid Bcr-Abl mRNA molecules per 1000 molecules of Abl mRNA. These results suggest that a variable number of malignant cells are present in the majority of CML patients in cytogenetic remission following interferon therapy.

A simplified method for determination of specific DNA or RNA copy number using quantitative PCR and an automatic DNA sequencer.

Quantification of specific RNA or DNA molecules that are present in minute amounts in biological samples has previously been performed using PCR in the presence of an internal standard. We have adapted this concept by introducing several modifications that facilitate the quantification of the products and obviate the need for radioisotopes. After amplification, individual products are separated on sequencing gels and directly quantified using a fluorescent automated DNA sequencer. We describe two applications of this approach: the quantitation of minute amounts of bcr-abl hybrid mRNA from malignant cells and the determination of gene copy number in cells stably transfected with a plasmid bearing a chloramphenicol acetyltransferase gene.