Cystic fibrosis (CF) is the most common severe autosomal recessive disorder of the Caucasian popu... more 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 hypothesi...
Routine determination of mutations in cystic fibrosis requires accurate, rapid, reliable and low-... more 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.
We examined the morphological and functional characteristics of erythroblasts derived from marrow... more We examined the morphological and functional characteristics of erythroblasts derived from marrow erythroid progenitor cells grown in a methylcellulose microculture, which were taken from a female child with rare atypical sideroblastic anaemia (SA) partially responsive to pyridoxine. Colony formation was within the normal range in three successive cultures (median values: 82.25 CFU-E and 16.4 BFU-E derived colonies/6.6 X 10(4) cells) compared to growth by normal cells (65-315 CFU-E and 9-40 BFU-E). We evaluated in vitro differentiation by biochemical microassay of a cytosol enzyme involved in the haem pathway: uroporphyrinogen I synthase (UROS). The UROS values in the erythroid colonies from SA marrow were at the lowere end of the normal range (median values: 6.7 +/- 0.3 and 14.4 +/- 3.8 pmol uroporphyrinogen/h in CFU-E and BFU-E-derived colonies respectively versus 17.4 +/- 7.3 and 25 +/- 7.2 pmol/h in CFU-E and BFU-E colonies from normal subjects. Ultrastructural examination of th...
To screen the BIGH3 gene in three unrelated families with lattice corneal dystrophy (LCD), two of... more To screen the BIGH3 gene in three unrelated families with lattice corneal dystrophy (LCD), two of which disclosed a particular phenotype. Genomic DNA was extracted from peripheral leukocytes of the affected patients and their family members. The entire coding sequence of the BIGH3 gene was screened for mutations by means of transcript analysis on total RNA isolated from peripheral leukocytes by reverse transcription-polymerase chain reaction performed with primers designed for this study. Each mutation was confirmed at the genomic level, by using published primers. One family that had a typical form of LCD, had the described R124C mutation in the BIGH3 gene. Two families with atypical forms of LCD were negative for the previously known mutations of the gene. Direct sequencing of the BIGH3 mRNA in the latter two families allowed us to identify two mutations located in exon 14. They consist of a 9-bp insertion at position 18851886 and one missense mutation at position 1877 of the BIGH...
By using the single strand conformational analysis to search for point mutations in the choroider... more 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.
The aim of this study was to define the RT-PCR-PTT parameters for CHM gene analysis and to evalua... more 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.
... MARIE DESGEORGES, PAULE KJELLBERG, JACQUES DEMAILLE, AND MIREILLE CLAUSTRES Laboratoire de Bi... more ... MARIE DESGEORGES, PAULE KJELLBERG, JACQUES DEMAILLE, AND MIREILLE CLAUSTRES Laboratoire de Biochimie Genitique Institut de Biologie Montpellier ... Am J Hum Genet 47:611-615 Macek M Jr, Ladanyi L, Burger J, Reis A (1992) Missense variations in the cystic ...
Fourteen years ago, the first preimplantation genetic diagnosis was described in the literature. ... more Fourteen years ago, the first preimplantation genetic diagnosis was described in the literature. The aim of the preimplantation genetic diagnosis is to analyse genes or chromosomes of embryos just before their transfer into the uterus. This technique avoids, for an identified pathology, the birth of children suffering from this pathology or repetitive medical abortions after prenatal diagnosis. The preimplantation genetic diagnosis needs to be performed by a multidisciplinary team working in well defined legal conditions. The preimplantation genetic diagnosis procedure includes an in vitro fertilization and intracytoplasmic sperm injection, a biopsy of one embryo cell and the genetic analysis of this cell. However, only a restricted number of genetic pathologies can benefit from such management. Preimplantation genetic diagnosis is a promising procedure for numerous patients. However, the evolution of its indications highlights the importance of informing both the couples and the society.
Les conséquences fonctionnelles des variations nucléotidiques situées dans les régions promotrice... more Les conséquences fonctionnelles des variations nucléotidiques situées dans les régions promotrices des gènes sont à ce jour très peu étudiées. Notre laboratoire a initié l'étude fonctionnelle des variations de séquence situées dans le promoteur minimal du gène CFTR. En effet, plusieurs d'entre elles ont particulièrement retenu notre attention, puisqu'elles ont été essentiellement identifiées chez des patients atteints soit de bronchiectasie isolée, soit de mucoviscidose. Des analyses in silico ont mis en évidence la présence de sites de fixation pour de nombreux facteurs de transcription. Nous avons choisi d'étudier plus particulièrement le rôle de deux facteurs, Sp1 (Specificity protein 1) et FoxA2 (Forkhead box A2). En effet, il a été montré que ces deux facteurs agissaient en synergie pour activer la transcription du promoteur de l'uteroglobin/CC10 dans les poumons [1]. Méthodes : L'expression des protéines Sp1 et FoxA2 dans des lignées cellulaires pulmonaires a été évaluée par Western blot et immunofluorescence indirecte. Afin de montrer les liaisons spécifiques de ces deux facteurs sur les séquences d'ADN d'intérêt, des expériences d'interactions ADN-protéines in vitro ont été réalisées. Des expériences de co-transfections transitoires avec les vecteurs d'expression des protéines d'intérêt et de leurs dominants négatifs respectifs, et l'utilisation d'activateurs ou inhibiteurs spécifiques, ont été initiées afin de déterminer l'effet de ces facteurs sur la régulation transcriptionnelle du gène CFTR. Résultat : Après avoir vérifié l'expression des protéines Sp1 et FoxA2 dans des cellules épithéliales d'origine pulmonaire, nous avons montré la fixation in vitro des facteurs Sp1 et FoxA2 sur la séquence promotrice du gène CFTR pour les différentes variations de séquence étudiées. L'analyse des premières expériences de cotransfections transitoires montre que le facteur Sp1 active la transcription du gène CFTR. L'étude de l'effet du facteur FoxA2 est en cours de réalisation. Conclusions : Les études fonctionnelles des variations de séquence associées exclusivement à une atteinte pulmonaire chronique devraient permettre, au-delà de la détermination du caractère délétère de ces variations, une meilleure caractérisation des transfacteurs susceptibles de diriger l'expression tissu-spécifique du gène CFTR. Ces travaux devraient également permettre d'entrevoir de nouvelles approches thérapeutiques pour les maladies des voies respiratoires. Référence
Cystic fibrosis (CF) is the most common severe autosomal recessive disorder of the Caucasian popu... more 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 hypothesi...
Routine determination of mutations in cystic fibrosis requires accurate, rapid, reliable and low-... more 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.
We examined the morphological and functional characteristics of erythroblasts derived from marrow... more We examined the morphological and functional characteristics of erythroblasts derived from marrow erythroid progenitor cells grown in a methylcellulose microculture, which were taken from a female child with rare atypical sideroblastic anaemia (SA) partially responsive to pyridoxine. Colony formation was within the normal range in three successive cultures (median values: 82.25 CFU-E and 16.4 BFU-E derived colonies/6.6 X 10(4) cells) compared to growth by normal cells (65-315 CFU-E and 9-40 BFU-E). We evaluated in vitro differentiation by biochemical microassay of a cytosol enzyme involved in the haem pathway: uroporphyrinogen I synthase (UROS). The UROS values in the erythroid colonies from SA marrow were at the lowere end of the normal range (median values: 6.7 +/- 0.3 and 14.4 +/- 3.8 pmol uroporphyrinogen/h in CFU-E and BFU-E-derived colonies respectively versus 17.4 +/- 7.3 and 25 +/- 7.2 pmol/h in CFU-E and BFU-E colonies from normal subjects. Ultrastructural examination of th...
To screen the BIGH3 gene in three unrelated families with lattice corneal dystrophy (LCD), two of... more To screen the BIGH3 gene in three unrelated families with lattice corneal dystrophy (LCD), two of which disclosed a particular phenotype. Genomic DNA was extracted from peripheral leukocytes of the affected patients and their family members. The entire coding sequence of the BIGH3 gene was screened for mutations by means of transcript analysis on total RNA isolated from peripheral leukocytes by reverse transcription-polymerase chain reaction performed with primers designed for this study. Each mutation was confirmed at the genomic level, by using published primers. One family that had a typical form of LCD, had the described R124C mutation in the BIGH3 gene. Two families with atypical forms of LCD were negative for the previously known mutations of the gene. Direct sequencing of the BIGH3 mRNA in the latter two families allowed us to identify two mutations located in exon 14. They consist of a 9-bp insertion at position 18851886 and one missense mutation at position 1877 of the BIGH...
By using the single strand conformational analysis to search for point mutations in the choroider... more 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.
The aim of this study was to define the RT-PCR-PTT parameters for CHM gene analysis and to evalua... more 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.
... MARIE DESGEORGES, PAULE KJELLBERG, JACQUES DEMAILLE, AND MIREILLE CLAUSTRES Laboratoire de Bi... more ... MARIE DESGEORGES, PAULE KJELLBERG, JACQUES DEMAILLE, AND MIREILLE CLAUSTRES Laboratoire de Biochimie Genitique Institut de Biologie Montpellier ... Am J Hum Genet 47:611-615 Macek M Jr, Ladanyi L, Burger J, Reis A (1992) Missense variations in the cystic ...
Fourteen years ago, the first preimplantation genetic diagnosis was described in the literature. ... more Fourteen years ago, the first preimplantation genetic diagnosis was described in the literature. The aim of the preimplantation genetic diagnosis is to analyse genes or chromosomes of embryos just before their transfer into the uterus. This technique avoids, for an identified pathology, the birth of children suffering from this pathology or repetitive medical abortions after prenatal diagnosis. The preimplantation genetic diagnosis needs to be performed by a multidisciplinary team working in well defined legal conditions. The preimplantation genetic diagnosis procedure includes an in vitro fertilization and intracytoplasmic sperm injection, a biopsy of one embryo cell and the genetic analysis of this cell. However, only a restricted number of genetic pathologies can benefit from such management. Preimplantation genetic diagnosis is a promising procedure for numerous patients. However, the evolution of its indications highlights the importance of informing both the couples and the society.
Les conséquences fonctionnelles des variations nucléotidiques situées dans les régions promotrice... more Les conséquences fonctionnelles des variations nucléotidiques situées dans les régions promotrices des gènes sont à ce jour très peu étudiées. Notre laboratoire a initié l'étude fonctionnelle des variations de séquence situées dans le promoteur minimal du gène CFTR. En effet, plusieurs d'entre elles ont particulièrement retenu notre attention, puisqu'elles ont été essentiellement identifiées chez des patients atteints soit de bronchiectasie isolée, soit de mucoviscidose. Des analyses in silico ont mis en évidence la présence de sites de fixation pour de nombreux facteurs de transcription. Nous avons choisi d'étudier plus particulièrement le rôle de deux facteurs, Sp1 (Specificity protein 1) et FoxA2 (Forkhead box A2). En effet, il a été montré que ces deux facteurs agissaient en synergie pour activer la transcription du promoteur de l'uteroglobin/CC10 dans les poumons [1]. Méthodes : L'expression des protéines Sp1 et FoxA2 dans des lignées cellulaires pulmonaires a été évaluée par Western blot et immunofluorescence indirecte. Afin de montrer les liaisons spécifiques de ces deux facteurs sur les séquences d'ADN d'intérêt, des expériences d'interactions ADN-protéines in vitro ont été réalisées. Des expériences de co-transfections transitoires avec les vecteurs d'expression des protéines d'intérêt et de leurs dominants négatifs respectifs, et l'utilisation d'activateurs ou inhibiteurs spécifiques, ont été initiées afin de déterminer l'effet de ces facteurs sur la régulation transcriptionnelle du gène CFTR. Résultat : Après avoir vérifié l'expression des protéines Sp1 et FoxA2 dans des cellules épithéliales d'origine pulmonaire, nous avons montré la fixation in vitro des facteurs Sp1 et FoxA2 sur la séquence promotrice du gène CFTR pour les différentes variations de séquence étudiées. L'analyse des premières expériences de cotransfections transitoires montre que le facteur Sp1 active la transcription du gène CFTR. L'étude de l'effet du facteur FoxA2 est en cours de réalisation. Conclusions : Les études fonctionnelles des variations de séquence associées exclusivement à une atteinte pulmonaire chronique devraient permettre, au-delà de la détermination du caractère délétère de ces variations, une meilleure caractérisation des transfacteurs susceptibles de diriger l'expression tissu-spécifique du gène CFTR. Ces travaux devraient également permettre d'entrevoir de nouvelles approches thérapeutiques pour les maladies des voies respiratoires. Référence
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