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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.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.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.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.

Denaturing high-performance liquid chromatography (dHPLC) is a new automated sizing method when used in non-denaturing conditions and is a powerful mutation detection tool in denaturing conditions. We introduce three possible forensic applications of this new technology. (1) Determination of age at death. Various mutations accumulate in mtDNA during ageing. According to this, we are developing a new method to determine age at death based on dHPLC ability to detect mtDNA mutations. mtDNA is extracted from autopsy tissues (iliopsoas, liver, kidney, putamen and heart) of numerous individuals representing a wide age spectrum. After amplification and digestion of the entire mtDNA in 90–600 bp fragments, separation is performed by dHPLC at different temperatures. We are studying the qualitative and quantitative differential accumulation of mutations with age among the various tissues. Our methodology is presented. (2) Human identification. In non-denaturing conditions, dHPLC gives size-based separation of DNA fragments. This allows separation of short tandem repeats (STRs) fragments. The different alleles of the HUMTH01, F13A01, vWa31 and FES/FPS loci were separated and sized in 5 min. (3) Gender determination. Used in non-denaturing conditions, dHPLC can separate the two sex-specific alleles of the amelogenin locus in 4 min without any preparation of the PCR product.

We have developed a preimplantation genetic diagnosis (PGD) strategy for Duchenne muscular dystrophy (DMD) allowing the simultaneous amplification of four exons (6, 8, 28 and 32) of the dystrophin gene together with ZFX/ZFY genes for gender determination. Preliminary experiments were carried out on 215 single lymphocytes from male and female individuals. Amplification rates ranged from 90.2% for exon 6 to 96.7% for exons 8 and 32. At least four of the five sequences were successfully amplified in 95.8% of single cells, and sexing was possible in 98.5%. This 5-plex assay was found to be robust enough to be used in a PGD clinical procedure and was therefore applied to a family whose female partner was a heterozygous carrier of a large deletion extending from exon 21 to exon 34 of the dystrophin gene. We have thus analysed two exons located in the deleted region of the gene, two non-deleted exons used as intrasample controls, and ZFX/ZFY genes. Cleavage stage embryo biopsy followed by PCR resulted in transfer of three unaffected embryos. The advantage of the present approach is to identify and subsequently transfer unaffected male embryos in addition to female embryos, and is now applicable to all families displaying a deletion involving at least one of these exons.

La derniere decade a devoile la base genetique de l’absence bilaterale des canaux deferents (ABCD) en demontrant son association avec des mutations dans le gene CFTR, de sorte que, dans la majorite des cas, l’ABCD est maintenant consideree comme une forme incomplete de mucoviscidose. Nous resumons dans cet article de revue les principaux resultats de la compilation de l’analyse du gene CFTR realisee par les laboratoires francais pour 3923 patients atteints de mucoviscidose typique et 800 hommes en bonne sante presentant une ABCD. Le degre d’expression clinique depend de plusieurs variables, en particulier des mecanismes moleculaires par lesquels les diverses mutations alterent ou interrompent l’activite canal ionique de la proteine CFTR. Le spectre clinique varie de la mucoviscidose severe avec deterioration pulmonaire progressive et insuffisance pancreatique (CF-PI), aux formes pulmonaires attenuees avec conservation des fonctions pancreatiques (CF-PS) ou encore des formes mono ou pauci-symptomatiques denommees recemment “maladies du CFTR”. Dans la mucoviscidose typique, 310 mutations CFTR differentes avaient ete identifiees en France en 1999, representant 94% des 7846 alleles CF. Pres de 500 genotypes mutationnels differents avaient ete recenses, composes soit de 2 mutations severes (88%, CF-PI), soit d’une mutation severeen trans d’une moderee (11%, CF-PS) ou de 2 mutations moderees (1% des genotypes identifies). Dans le groupe ABCD, 137 mutations eparpillees sur l’ensemble du gene CFTR avaient ete identifiees dans 60% des 1600 alleles ABCD. Parmi les 150 genotypes caracterises, l’heterozygotie composite etait la regle, les combinaisons les plus frequentes etant ΔF508/5T (35%), ΔF508/autre mutation (30%, incluant ΔF508/R117H-7T, 5,6%), et 5T/autre mutation (17%). Par contraste avec le groupe CF, 88% des genotypes identifies comprenaient une mutation severeen trans d’une moderee, et 12% 2 mutations moderees. Un total de 22 genotypes etaient communs entre les cohortes CF et ABCD. Le role de l’allele 5T comme variant d’epissage a penetrance incomplete est rappele. D’autres variations de sequence, tels l’allele TG12 ou le polymorphisme exonique M470V peuvent influencer l’epissage et/ou la fonction du CFTR. Cette etude confirme la tres grande heterogeneite moleculaire CFTR dans l’ABCD et la necessite d’une etude exhaustive du gene pour detecter les mutations chez ces patients. Des etudes de suivi a long terme des hommes ABCD sont indispensables pour evaluer les consequences phenotypiques de nombreux genotypes mutationnel.

Resume L’importante heterogeneite allelique de la mucoviscidose represente la principale difficulte pour l’etablissement de son diagnostic genotypique. De nombreuses etudes ont montre pour certaines mutations du gene CFTR des variations de frequence allelique parfois considerables entre des populations d’origine geographique et ethnique differentes. Materiel et methodes. – Nous avons analyse les genotypes de 207 enfants atteints de mucoviscidose et residant dans le Sud-Ouest de la France. Resultats. – Nous montrons que parmi les 50 mutations identifiees, certaines ont une frequence significativement differente de celle habituellement observee. Ces variations apparaissent plus clairement pour les alleles typiquement originaires du Sud-Ouest de la France. Ainsi, la mutation 1811+1,6kbA>G, rarement observee dans les autres regions ( Conclusion. – Nous montrons qu’il existe dans le Sud-Ouest de la France un spectre mutationnel specifique. Nous considerons que ces donnees regionales sont importantes pour ameliorer la sensibilite du test genetique et preciser le conseil genetique de la mucoviscidose en France.

Purpose To evaluate aortic elasticity with MRI on young asymptomatic individuals with mutation of the smooth muscle myosin heavy chain in whom aortic enlargement is not present. Materials and Methods Aortic compliance, aortic distensibility, and pulse wave velocity (PWV) were semiautomatically measured from MRI in 8 asymptomatic subjects having a mutation of the MYH11 gene (M+) and 21 nonmutated relatives (M−) of similar age, sex, and blood pressure characteristics. Results Despite a similar aortic diameter in both groups, the aortic compliance and distensibility were significantly lower in M+ subjects compared with M− (0.84 ± 0.33 versus 2.03 ± 0.54 mm2/mmHg, 1.18 ± 0.62 10−3 versus 5.11 ± 1.58 10−3 mmHg−1, respectively), and PWV was significantly higher (5.35 ± 1.53 versus 3.60 ± 0.64 m.s−1). A threshold aortic compliance value of 1.3 mm2/mmHg separated the two groups. The receiver operating characteristics curve analysis indicated an optimal threshold of 2.9 10−3 mmHg−1 for aortic distensibility (sensitivity: 87.5%, specificity: 90%), and of 4.4 m.s−1 for PWV (sensitivity: 75%, specificity: 100%). Conclusion Young asymptomatic adults with MYH11 mutation have an aortic compliance impairment which is not detectable by the sole measurement of the aortic size. Aortic compliance measurement might be part of routine examination in patients suspected of inherited aortic disease even with a normal aortic diameter. J. Magn. Reson. Imaging 2008;28:1180–1187. © 2008 Wiley-Liss, Inc.

Resume Deux observations d'hypokaliemie recidivante a I'exercice et a la chaleur, par syndrome de perte de sel d'origine sudorale ont ete rapportes a une forme d'expression phenotypique moderee de la mucoviscidose par mutation du gene CFRT heterozygote composite comportant la mutation L206W, reputee peu severe.

An abbreviated tract of five thymidines (5T) in intron 8 of the cystic fibrosis transmembrane conductance regulator ( CFTR) gene is found in similar to 10% of individuals in the general population. When found in trans with a severe CFTR mutation, 5T can result in male infertility, nonclassic cystic fibrosis, or a normal phenotype. To test whether the number of TG repeats adjacent to 5T influences disease penetrance, we determined TG repeat number in 98 patients with male infertility due to congenital absence of the vas deferens, 9 patients with nonclassic CF, and 27 unaffected individuals ( fertile men). Each of the individuals in this study had a severe CFTR mutation on one CFTR gene and 5T on the other. Of the unaffected individuals, 78% ( 21 of 27) had 5T adjacent to 11 TG repeats, compared with 9% ( 10 of 107) of affected individuals. Conversely, 91% ( 97 of 107) of affected individuals had 12 or 13 TG repeats, versus only 22% ( 6 of 27) of unaffected individuals (P < .00001). Those individuals with 5T adjacent to either 12 or 13 TG repeats were substantially more likely to exhibit an abnormal phenotype than those with 5T adjacent to 11 TG repeats ( odds ratio 34.0, 95% CI 11.1 - 103.7, P < .00001). Thus, determination of TG repeat number will allow for more accurate prediction of benign versus pathogenic 5T alleles.

Abnormal movements are not unusual in childhood. Recent genetic progresses provide a new approach of childhood movement disorders. Several loci have been identified in paroxysmal dyskinesia, or in Gilles de la Tourette syndrome. A gene has been cloned in Hallervorden-Spatz syndrome, and a gene has recently been implicated in benign hereditary chorea. Considerable advances concern the genetic of dystonic syndromes: several chromosomal localizations have been identified, and several genes have been cloned. Genetic advances allow nosographic reclassification of some entities and offer new molecular tools for a more appropriate diagnosis. The increasing wealth of genetic knowledge will provide further insight in the understanding of abnormal movement disorders in childhood.

Retinoblastoma is a malignant intra-ocular tumour of developing retina initiated by inactivation of both alleles of the retinoblastoma susceptibility (RB1) gene. This paper reports the first clinical experience of preimplantation genetic diagnosis (PGD) for hereditary retinoblastoma using two highly polymorphic microsatellite markers RB1.20 and D13S284, located within and close to the RB1 gene respectively. Duplex PCRs were tested on more than 300 single lymphocytes from heterozygous individuals at both loci, in order to test the accuracy and reliability of the single-cell protocol. This procedure requires a nested PCR and the analysis of fluorescently labelled PCR products on an automatic DNA sequencer. Amplification efficiency and allele drop-out rates ranged from 96.7 to 98.4%, and 3.7 to 5.4% respectively. This test was found to be accurate and reliable enough to be applied to the study of human blastomeres. Subsequently, this approach was used in a PGD treatment cycle for a couple who already had a child affected with hereditary retinoblastoma and found to be informative for both microsatellite markers.

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, 1717-1GA, G551D, W1282X, R553X, deltaI507, 1078delT, 2183AAG, 3849+10kbCT, R1162X, 621+1GT, 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.

The large molecular heterogeneity in cystic fibrosis (CF) represents the main difficulty for the genotype characterization. Moreover, numerous studies have reported considerable variations in frequencies of cystic fibrosis transmembrane conductance regulator (CFTR) mutations in different populations.We analyzed the genotype of 207 CF children living in southwest France.Among 50 identified mutations, we report for some of them a widely modified incidence compared with those observed in other regions of France. These differences were more significant in the subset of the CF chromosomes originating in southwest France. Thus, the 1811 + 1.6 kbAG mutation, rarely observed in the other French regions (0.5%), proved to be, with a frequency of 8.8%, the most frequent mutation after the F508 deletion (57%). The frequencies of N1303K, 1811 + 1.6 kbAG and R334W mutations were also clearly increased: 7.9 and 2.6%, respectively.We show that the southwest of France is characterized by a specific mutational spectrum. We consider that these regional data on the spectrum of CF mutations are crucial to develop more accurate and less expensive molecular screening strategies for cystic fibrosis in France.

The spectrum of CFTR mutations in three South African populations is presented. To date. a total of 192 white patients (384 chromosomes) with confirmed CF have been tested. deltaF508 accounts for 76% of the CF chromosomes in this group, with 3272-26A--G, 394delTT and G542X occurring at the following frequencies: 4, 3.6 and 1.3%, respectively. A further 11 mutations account for 6% of CF chromosomes. A total of 91% of the CF-causing mutations can now be detected in the South African white population. Haplotype analysis suggests a founder effect in South Africans of European origin for the two common CFTR mutations, 3272-26A--G and 394delTT. The diagnosis of CF has been confirmed in 14 coloured and 12 black CF patients. In the coloured population, both the deltaF508 and 3120 + 1G--A mutations occur at appreciable frequencies of 43 and 29%, respectively. In the black population, the most common CF-causing mutation, the 3120 + 1G--A mutation, occurs at an estimated frequency of 46%. Four other mutations have been detected, resulting in the identification of a total of 62.5% of mutations in this population.

To evaluate the occurrence and inheritance of various types of pigmentary retinopathy in patients followed at the outpatient clinic in the university hospital, Montpellier, France. To characterize genes and mutations causing these conditions.Ophthalmic examination and various visual tests were performed. Mutations were sought from genomic DNA by PCR amplification of exons associated with single-strand conformation analysis and/or direct sequencing.Among 315 patients over an 8-year period, cases of retinitis pigmentosa (63.2%), Usher's syndrome (10.2%), Stargardt's disease (5.4%), choroideremia (3.2%), Leber's congenital amaurosis (3.2%), congenital stationary night blindness (2.9%), cone dystrophy (2.5%), dominant optic atrophy (1.9%), X-linked juvenile retinoschisis (1.6%), Best's disease (1.6%), and others (4.3%) were diagnosed. In retinitis pigmentosa, inheritance could be determined in 54.2% of the cases including dominant autosomic (26.6%), recessive autosomic (22.6%), and X-linked cases (5%) while it could not be confirmed in 45.7% of the cases (simplex cases in the majority). For the 6 examined genes, mutations were found in 22 out of 182 propositus (12.1%). Analysis of phenotype-genotype correlations indicates that in retinitis pigmentosa, RDS is more frequently associated with macular involvement and retinal flecks, RHO with regional disease, and RPE65 with the great severity of the disease with some cases of Leber's congenital amaurosis.Identification of genes may help in diagnosis and in genetic counseling, especially in simplex cases with retinitis pigmentosa. In this latter condition, molecular diagnosis will be necessary to rationalize future treatments.

We report a large genomic deletion of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, viz., a deletion that is frequently observed in Central and Eastern Europe. The mutation, termed CFTRdele2, 3(21 kb), deletes 21, 080 bp spanning introns 1-3 of the CFTR gene. Transcript analyses have revealed that this deletion results in the loss of exons 2 and 3 in epithelial CFTR mRNA, thereby producing a premature termination signal within exon 4. In order to develop a simple polymerase chain reaction assay for this allele, we defined the end-points of the deletion at the DNA sequence level. We next screened for this mutation in a representative set of European and European-derived populations. Some 197 CF patients, including seven homozygotes, bearing this mutation have been identified during the course of our study. Clinical evaluation of CFTRdele2, 3(21 kb) homozygotes and a comparison of compound heterozygotes for deltaF508/CFTRdele2, 3(21 kb) with pairwise-matched deltaF508 homozygotes indicate that this deletion represents a severe mutation associated with pancreatic insufficiency and early age at diagnosis. Current data show that the mutation is particularly common in Czech (6.4% of all CF chromosomes), Russian (5.2%), Belorussian (3.3%), Austrian (2.6%), German (1.5%), Polish (1.5%), Slovenian (1.5%), Ukrainian (1.2%), and Slovak patients (1.1%). It has also been found in Lithuania, Latvia, Macedonia and Greece and has sporadically been observed in Canada, USA, France, Spain, Turkey, and UK, but not in CF patients from Bulgaria, Croatia, Romania or Serbia. Haplotype analysis has identified the same extragenic CF-haplotype XV-2c/KM. 19 "A" and the same infrequent intragenic microsatellite haplotype 16-33-13 (IVS8CA-IVS 17bTA-IVS 17bCA) in all examined CFTRdele2, 3(21 kb) chromosomes, suggesting a common origin for this deletion. We conclude that the 21-kb deletion is a frequent and severe CF mutation in populations of Eastern- and Western-Slavic descent.

Duchenne (DMD) and Becker (BMD) are allelic forms of a X-linked neuromuscular disorder. Both are caused by mutations arising in the gene encoding dystrophin, a cytoskeletal protein. Two-thirds of DMD/BMD patients have large deletions localised in two hot spots, and the remaining cases are presumed to be caused by point mutations. Since Duchenne muscular dystrophy is a serious disorder for which at present there is no effective treatment, much emphasis has been given to prevention. This involves the ascertainment of women likely to have an affected son, and the provision of genetic counselling and prenatal diagnosis for such women. Accurate carrier detection and genetic counselling depend upon identifying the mutation itself in the proband. Large deletions are easily identified using multiplex polymerase chain reaction (PCR) whereas detection of point mutations is restricted to a few number of specialized laboratories. Hence carrier and prenatal diagnosis in 40% of families rely heavily on indirect approaches which presents major drawbacks and are not applicable in sporadic cases of DMD or BMD. We developped a strategy for searching small alterations in the dystrophin gene. As most of the non-deletion mutations cause a premature termination of translation, we have used the Protein Truncation Test to scan specifically the dystrophin transcripts isolated from muscle biopsies. This approach allowed to detect the disease-causing mutations in more than 90% of the patients who have been investigated; his efficiency is thus significantly higher than DNA-based strategies. The identification of the mutation in non deleted sporadic cases allows the at-risks females to benefit from an accurate diagnosis. Also, the characterization of the molecular defects provides a better understanding of the molecular pathology of the dystrophin gene.

Congential bilateral aplasia of vas deferens (CBAVD), a form of male sterility, has been suggested to represent a "genital" form of cystic fibrosis (CF), as mutations in the CFTR gene have been identified in most patients with this condition. Interestingly, the 5T allele in intron 8 appeared to be the most frequent mutation associated with CBAVD. However, the molecular basis of CBAVD is not completely understood. We have analysed the complete coding and flanking CFTR sequences by PCR-DGGE in 64 men with CBAVD from southern France with the aim to list any sequence alteration. Fourty-two of the 64 patients (65.6%) had mutations on both copies of the CFTR gene, including one patient with two mutations in the same copy (DF508 + A1067T). The 5T allele was present in 21/64 cases (33%). Six of the 28 different mutations identified in this study had never been described previously, and appeared to be specific to CBAVD (P111L, M244K, A1364V, G544V, 2896insAG,-33G-A).

By using the single strand conformational analysis to search for point mutations in the choroideremia gene, we have identified an intronic polymorphism within the intron 2 of the CHM gene. We have studied the frequency of this polymorphism in the population from South of France.

We describe particular clinical features in a three-generation family with X-linked CSNBi and present the genetic analysis.The diagnosis of CSNBi was established on clinical and electrophysiological criteria. Polymorphic DNA markers of the Xp region were analyzed by fluorescent polymerase chain reaction.Clinical findings evidenced an atypical association of both myopia and hyperopia in the same brotherhood. The most interesting feature in this family was the observation of major worsening of the clinical shape between the first and the third generation of affected individuals. DNA analysis did not show significant linkage between the disease and markers of the Xp11-p21 region. Southern analysis did not show expansion of trinucleotide repeat CAG/CTG and CCG/CGG over the three generation.Haplotypic analysis together with clinical observations allow to exclude the existence of a myopia gene closely linked to the CSNB2 locus. The clinical anticipation observed in this family does not seem to be linked with trinucleotide repeat expansion CAG/CTG or CCG/CGG.

The aim of this study was to define the RT-PCR-PTT parameters for CHM gene analysis and to evaluate its interest as a method for CHM mutation screening.The entire CHM coding region was reversed-transcribed in three overlapping cDNA segments (RT-PCR) which were amplified and further analyzed by PTT after in vitro transcription/translation.This strategy enabled us to detect a truncated peptide in each of the 6 unrelated patients from southern France who were investigated. The mutation was further characterized by direct sequencing of the RT-PCR product.In CHM gene, all conditions are present to make the RT-PCR-PTT strategy the method of choice for mutation screening. As a result of the simplified protocol described in this study, the families of the patients could benefit from accurate carrier-status assessment.

By using the single strand conformational analysis (SSCA) to search for point mutations in the choroideremia gene, we have identified a previously undescribed polymorphism within exon 5a (381A/G). We have studied the frequency of this polymorphism in a population from Southern France. The sequence variation creates a new restriction site for HhaI, allowing a convenient DNA-based genetic counseling in families in which the causal disease mutation is unknown.

By using the single strand conformation analysis to search for point mutations in the choroideremia gene, we had previously identified the first truncative mutation responsible for CHM in France. The aim of the present study was to perform a simple and nonisotopic routine test to identify carriers and non carriers in the relevant family.We used a PCR-based restriction analysis to detect the presence or absence of the mutation in the family members, as the mutation creates a restriction site in the coding sequence of the CHM gene.We could follow the segregation of the mutation in the pedigree, and unambiguously determine the genetic status of the females.When a mutation responsible for choroideremia modifies a restriction site, the PCR-restriction provides an efficient and unexpensive one-day test to detect heterozygosity in the family.

Cystic fibrosis (CF) is the most common severe autosomal recessive disorder of the Caucasian population. Since the identification of the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene in 1989, over 550 mutations have been characterized. CFTR can function as a chloride channel with unique properties. It has become clear, however, that CFTR can also regulate a variety of other cationic and anionic channels. The CFTR gene screening in men with bilateral congenital absence of the vas deferens (CBAVD) led to speculation that these patients have a specific mild form of CF. Recent studies allow patients with CBAVD to be classified in four categories: patients with two CFTR mutations (19%); patients with one CFTR mutation and the 5T allele in trans (33%); patients with only one CFTR mutation or only the 5T allele (27%); patients without CFTR mutations and 5T allele (21% The high proportion of patients with CBAVD who do not have CFTR mutations allows to suggest two hypothesis : other undetected changes in CFTR may be involved in CBAVD ; another gene or genes could be responsible for CBAVD.

Accurate estimation of age at death is a critical problem in forensic sciences. The macroscopic methods (using teeth and bones) which are commonly used, provide results with too wide a confidence interval in individuals older than 45. Several publications have shown that various mutations accumulate in mitochondrial DNA during ageing. According to these results, we are developing a new and original method to determine age at death by mutation detection. Actually, when used in denaturing conditions, dHPLC provides a rapid, automated scanning method for mutations, even when the nature and location of the mutation is unknown.

Samples from 30 members of a french cystic fibrosis (CF) family had to be typed with probes for restriction fragment length polymorphisms (RFLPs) known to be linked to the CF gene, to fulfill the expectations of twenty-two low-risk relatives who were asking for carrier testing. Classical linkage-disequilibrium data between KM-19 and XV-2c polymorphisms and the CF locus were not informative enough for some individuals, and other RFLPs had to be analyzed to determine which chromosomes carried the deficient gene in the family. We report the retrospective screening for delta F508 mutation in this extended family to illustrate the drastic improvements that the direct detection of the major mutation responsible for CF has on genetic counselling of relatives of patients with cystic fibrosis.

The effects of natural and synthetic androgens on erythroid colony formation in children's bone marrow cultures were studied using a methylcellulose microculture assay. In an attempt to predict the clinical response to androgens in two children with Fanconi anaemia (FA) and two children with Diamond-Blackfan syndrome (DB), we tested the hormonal stimulation of testosterone, nortestosterone and etiocholanolone on CFU-E, BFU-E and uroporphyrinogen I synthase activity (UROS). We observed that colony formation and UROS activity were reduced when compared to values obtained with normal children's bone marrow cultures. The addition of steroids to the cultures significantly enhanced the numbers of CFU-E and BFU-E derived colonies and their UROS activity in marrow from patients with FA and one patient with DB. The strong depletion of marrow progenitor cells in the unresponsive marrow from child 4 with DB could explain the absence of hormonal response. Whereas the responsiveness to steroids varied according to the individual, the in vitro testing of erythroid differentiation in the presence of androgens theoretically may lead to an effective prediction of response to therapy in children with hypoplastic anaemia.

The new techniques of culture of bone marrow have shown that androgens and 5 beta steroids exert a direct effect on erythroid precursor cells from human or animal bone marrow. By contrast, the mechanisms of the intracellular actions of those compounds are poorly understood. Tritiated methyltrienolone (R 1881), a synthetic androgen that highly binds to androgen receptor, has been used for the study of a binding activity in nuclear extracts of cultured erythroblasts from human bone marrow. The nuclear extracts contain binding sites saturable at low concentrations of 3H-R 1881 (20-30 nM). Scatchard analysis revealed that the R 1881-nuclear complex has a dissociation constant (Kd) of 25-50 nM, and the number of binding sites was 235-370 fmoles/mg protein. The complex sedimented on 5-20% sucrose gradient in the 3.9 S region and 5 beta dihydrotestosterone compete strongly with R 1881 for binding sites. This binding component has characteristics of high affinity, low-capacity, sedimentation behaviour, and specificity commonly attributed to "androgen receptors".

Studies of human breast cancers show that tumoral tissues have a much higher ferritin content (mean : 4,5 times) than normal tissues. However, only 10% of patients without metastasis present raised levels of serum ferritin, whereas hyperferritinemia is observed in almost 95% of patients with liver, bone or lung metastasis.

The effect of human somatomedin C/insulin-like growth factor I(SM-C/IGF-1) and human growth hormone (hGH) on colony formation by erythroid precursor cells (CFU-E and BFU-E) from children's bone marrow or blood was studied by methylcellulose cloning assay. We found that physiological concentrations of IGF-1, but not of hGH, stimulated erythropoiesis in vitro in the presence of erythropoietin, as demonstrated by the increased activity of a cytosolic enzyme of the heme pathway (uroporphyrinogen I synthase). The results suggest that IGF-I could be involved in the regulation of erythroid differentiation.

The highly specific polymerase chain reaction recently described can be used to amplify selectively several polymorphic regions of DNA genetically close to the cystic fibrosis gene. This method, providing automated, revolutionizes the classical methods of prenatal diagnosis and carrier detection.

We describe a simple spectrophotometric microassay to quantify the proliferation and the differentiation of human bone marrow or blood erythroid progenitor cells CFU-E and BFU-E. These precursors give rise, in culture, to colonies and bursts with markedly variations in size and hemoglobinization, which cannot be accurately evaluated by the usual method of scoring. We then developed a sensitive biochemical microassay to measure the uroporphyrinogen I synthase activity of progenitors grown in small wells. This assay is a valuable index of erythroid differentiation in vitro. This method can offer the opportunity to test the efficiency in vitro of various therapeutic agents in patients with hemopoietic disorders.

Ferritin, iron storage protein, presents two types of subunits, H and L, the respective proportions of which varying with the tissue of origin and defining molecules called isoferritins. This work attempts to compare four human ferritins (from liver, spleen, heart and placenta) by means of the microcomplement fixation technique. Results show that placenta and liver ferritins are closely related, while slight but significant differences appear between liver, spleen and heart ferritins. These differences are obviously less important compared to those observed between ferritins from different species of origin, as shown by the results expressed in terms of index of dissimilarity or immunological distance. Those results are fully consistent with the elementary aminoacid composition as well as with the relative proportions of H and L subunits among the various types of human isoferritins we have studied.

We studied the role of androgens on children erythropoietic precursors cells in culture. Cultures of normal marrow from surgical intervention (informed consent) were carried out using a miniaturized methyl-cellulose method in the presence of erythropoietin (11U/ml). Effects of testosterone (T), 5 -dihydrotestosterone (DHT) and nor-testosterone (nor-T) were evaluated on colony forming units erythroid (CFU-E) after 7days of incubation at 37°C CFU-E were quantified by scoring directly colonies and by a biochemical determination of the uroporphyrinogen I synthase activity (UROS). Results are given as number of CFU-E per 32.000 nucleated cells plated. UROS activity is expressed as pmoles of uroporphyrinogen formed per hour and per well. T,DHT and nor-T significantly stimulate children marrow progenitor cells in culture.Androgens act on both growth and maturation of CFU-E.A direct effect of androgens on erythroid differentiation (via an androgen receptor?) in children bone marrow culture may explain the efficiency of androgen treatment in aplastic anemia.

COMPARATIVE EFFECTS OF HUMAN GROWTH HORMONE (GH) AND INSULIN-LIKE GROWTH FACTOR I/SOMATOMEDIN C (IGF I/ SMC) ON ERYTHROPOIETIC CULTURES FROM NORMAL CHILDREN