RESUMO
This study provides an overview of 10 years of experience of preimplantation genetic diagnosis (PGD) for cystic fibrosis (CF) in our center. Owing to the high allelic heterogeneity of CF transmembrane conductance regulator (CFTR) mutations in south of France, we have set up a powerful universal test based on haplotyping eight short tandem repeats (STR) markers together with the major mutation p.Phe508del. Of 142 couples requesting PGD for CF, 76 have been so far enrolled in the genetic work-up, and 53 had 114 PGD cycles performed. Twenty-nine cycles were canceled upon in vitro fertilization (IVF) treatment because of hyper- or hypostimulation. Of the remaining 85 cycles, a total of 493 embryos were biopsied and a genetic diagnosis was obtained in 463 (93.9%), of which 262 (without or with a single CF-causing mutation) were transferable. Twenty-eight clinical pregnancies were established, yielding a pregnancy rate per transfer of 30.8% in the group of seven couples with one member affected with CF, and 38.3% in the group of couples whose both members are carriers of a CF-causing mutation [including six couples with congenital bilateral absence of the vas deferens (CBAVD)]. So far, 25 children were born free of CF and no misdiagnosis was recorded. Our test is applicable to 98% of couples at risk of transmitting CF.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Diagnóstico Pré-Implantação , Criança , Fibrose Cística/genética , Fibrose Cística/patologia , Feminino , França , Aconselhamento Genético , Genótipo , Haplótipos , Heterozigoto , Humanos , Masculino , GravidezRESUMO
New technologies, which constantly become available for mutation detection and gene analysis, have contributed to an exponential rate of discovery of disease genes and variation in the human genome. The task of collecting and documenting this enormous amount of data in genetic databases represents a major challenge for the future of biological and medical science. The Locus Specific Databases (LSDBs) are so far the most efficient mutation databases. This review presents the main types of databases available for the analysis of mutations responsible for genetic disorders, as well as open perspectives for new therapeutic research or challenges for future medicine. Accurate and exhaustive collection of variations in human genomes will be crucial for research and personalized delivery of healthcare.
Assuntos
Bases de Dados Genéticas , Doenças Genéticas Inatas/genética , Mutação , Doenças Raras/genética , Códon de Terminação , Etnicidade/genética , Previsões , Doenças Genéticas Inatas/classificação , Doenças Genéticas Inatas/terapia , Terapia Genética , Genética Médica/ética , Genótipo , Humanos , Internet , Fenótipo , RNA Antissenso/uso terapêutico , Doenças Raras/classificação , Doenças Raras/terapia , Terminologia como Assunto , Transcrição Gênica/efeitos dos fármacosRESUMO
While the goals of genetic counseling for cystic fibrosis - delivering relevant information on the risk of recurrence and nondirectional support of couples at risk in their reproductive choices - have not changed fundamentally, the practice has evolved considerably in the last decade, growing more complex to face new challenges but also proving more effective. Many factors have contributed to this evolution: technical progress in the exploration of the genome (new generation sequencing) and in reproductive medicine, but also societal developments promoting access to genetic information and the professionalization of genetic counselors in France. The prospect of expanded pre-conception screening of at-risk couples makes genetic counselors major actors not only in medical care centers, but also in modern society by contributing to genetic education among citizens. © 2020 French Society of Pediatrics. Published by Elsevier Masson SAS. All rights reserved.
Assuntos
Fibrose Cística/diagnóstico , Fibrose Cística/genética , Aconselhamento Genético , DNA/sangue , Feminino , Feto/metabolismo , Triagem de Portadores Genéticos , Humanos , Troca Materno-Fetal , Teste Pré-Natal não Invasivo , Cuidado Pré-Concepcional , Gravidez , Diagnóstico Pré-Implantação , Diagnóstico Pré-NatalRESUMO
Duchenne muscular dystrophy (DMD) is a common childhood lethal X-linked recessive disorder, resulting from deletions, duplications and point mutations in the dystrophin gene. Single-cell protocols for preimplantation genetic diagnosis (PGD) still remain challenging due to the enormous size of the gene and the high risk of intragenic recombination, limitations that often lead to sex determination and selection of female embryos. This study describes direct and rapid decaplex and dodecaplex polymerase chain reaction protocols enabling the analysis of five or seven exons and four microsatellite markers scattered along the dystrophin gene, chosen to be located in the two deletion hotspots, and the analysis of amelogenin sequences for gender determination. The dodecaplex protocol may be applied to most of the couples requesting PGD for DMD in whom the female partner is a carrier of a deletion. This generic approach will allow prompt response to the PGD referrals by reducing the pre-clinical PGD work-up. It was successfully applied in three DMD families, resulting in the birth of a girl as well as in a healthy ongoing pregnancy.
Assuntos
Distrofina/genética , Distrofia Muscular de Duchenne/genética , Reação em Cadeia da Polimerase/métodos , Diagnóstico Pré-Implantação/métodos , Amelogenina/genética , Feminino , Deleção de Genes , Humanos , Distrofia Muscular de Duchenne/diagnóstico , Linhagem , GravidezRESUMO
BACKGROUND: Analysis of cell-free foetal DNA (cff-DNA) in maternal plasma is very promising for early diagnosis of monogenic diseases; in particular, cystic fibrosis (CF). However, NIPD of single-gene disorders has been limited by the availability of suitable technical platforms and the need to set up patient or disease-specific custom-made approaches. METHODS: To make research applications more readily accessible to the clinic, we offer a simple assay combining two independent methods to determine the presence or absence of paternally inherited foetal allele p.Phe508del (the most frequent mutation in CF patients worldwide). The first method detects the presence or absence of a p.Phe508del allele by Mutant Enrichment with 3'-Modified Oligonucleotide PCR coupled to Fragment Length Analysis (MEMO-PCR-FLA). The second method detects the p.Phe508del allele with classical Multiplex Fluorescent PCR including five intragenic and extragenic STR markers of the CFTR locus and a specific SRY sequence. RESULTS: We collected 24 plasma samples from 23 women carrying foetuses at risk for CF and tested each sample using both methods. Our new procedures were successfully applied to 10 couples where fathers carried the p.Phe508del mutation and mothers were carrying a different mutation in the CFTR gene. These simple tests provided clear positive or negative results from the maternal plasma of the pregnant women. We confirmed the presence of cff-DNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit. All results were correlated with chorionic villus sampling or amniocentesis analyses. CONCLUSIONS: This NIPD approach, easily set up in any clinical laboratory where prenatal diagnosis is routinely performed, offers many advantages over current methods: it is simple, rapid, and cost-effective. It opens up the possibility for testing a large number of couples with offspring at risk for CF.
Assuntos
Amniocentese/métodos , Amostra da Vilosidade Coriônica/métodos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística , Reação em Cadeia da Polimerase/métodos , Adulto , Pesquisa Comparativa da Efetividade , Fibrose Cística/sangue , Fibrose Cística/diagnóstico , Fibrose Cística/genética , Eletroforese Capilar/métodos , Feminino , Humanos , Mutação , Gravidez , Diagnóstico Pré-Natal/métodos , Reprodutibilidade dos TestesRESUMO
In this study, single sperm typing has been used for high-resolution recombination analysis between the retinoblastoma gene and two closely linked extragenic microsatellites (D13S284 and D13S1307). The analysis of 1198 single sperm from three donors allowed the determination of recombination fractions between RB1.20 and D13S284 and RB1.20 and D13S1307 of 0.022 and 0.033, respectively. These results show that RB1 gene and the two microsatellites are closely linked, which validates their potential use in indirect genetic diagnosis of retinoblastoma.
Assuntos
Repetições de Microssatélites , Recombinação Genética , Proteína do Retinoblastoma/genética , Ligação Genética , Humanos , Masculino , EspermatozoidesRESUMO
The "PRimed IN Situ labeling" (PRINS) method is an interesting alternative to in situ hybridization for chromosomal detection. In this procedure, chromosome labeling is performed by in situ annealing of specific oligonucleotide primers, followed by primer elongation by a Taq polymerase in the presence of labeled nucleotides. Using this process, we have developed a simple and semi-automatic method for rapid in situ detection of human chromosome 21. The reaction was performed on a programmable temperature cycler, with a chromosome 21 specific oligonucleotide primer. Different samples of normal and trisomic lymphocytes and amniotic fluid cells were used for testing the method. Specific labeling of chromosome 21 was obtained in both metaphases and interphase nuclei in a 1 hour reaction. The use of oligonucleotide primer for in situ labeling overcomes the need for complex preparations of specific DNA probes. The present results demonstrate that PRINS may be a simple and reliable technique for rapidly detecting aneuploidies.
Assuntos
Cromossomos Humanos Par 21 , Hibridização in Situ Fluorescente/métodos , Sequência de Bases , Primers do DNA , Humanos , Dados de Sequência MolecularRESUMO
Rapid and specific identification of chromosomes can be attained in situ using the PRimed IN Situ (PRINS) labelling technique. We have adapted this technique to mature human sperm in combination with a protocol for simultaneous decondensation and denaturation of sperm nuclei. This strategy allowed us to obtain double labelling of human spermatozoa in a < 2-hr reaction. In the present study, we report the estimates of disomy for chromosomes 3, 7, 10, 11, and 17 on 64,642 spermatozoa from 2 normal males. The incidences of disomy ranged from 0.28-0.34%. There were no significant interindividual or interchromosomal differences in disomy rates.
Assuntos
Aneuploidia , Hibridização in Situ Fluorescente/métodos , Reação em Cadeia da Polimerase/métodos , Espermatozoides , Adulto , Núcleo Celular/química , DNA Satélite/análise , Humanos , MasculinoRESUMO
OBJECTIVE: To present the use of primed in situ labeling method in preimplantation diagnosis. DESIGN: Double- and triple-primed in situ labeling were performed on 10 morphologically abnormal preimplantation embryos, using combinations of specific primers for chromosomes 9, 13, 16, 18, 21, X, and Y. SETTING: Embryos were obtained from patients at the Montpellier University Hospital. PATIENT(S): Seven women undergoing IVF at the Montpellier University Hospital. INTERVENTION(S): Isolated interphase nuclei from poor quality preimplantation embryos were prepared for primed in situ labeling technique. MAIN OUTCOME MEASURE(S): Numerical abnormalities assessed by primed in situ labeling analysis. RESULT(S): Using directly fluorescent-labeled nucleotides, the labeling reaction for three chromosomes did not exceed 2.30 hours. Only three analyzed embryos appeared to be chromosomally normal. Mosaicism, aneupoidy, and haploidy were observed in the seven other embryos. CONCLUSION(S): The primed in situ labeling method offers a simple and reliable screening tool for gender determination and aneuploidy detection. The use of this technique may contribute to significantly improve the procedure of preimplantation diagnosis.
Assuntos
Blastocisto/ultraestrutura , Aberrações Cromossômicas , Desenvolvimento Embrionário , Aneuploidia , Cromossomos Humanos Par 13 , Cromossomos Humanos Par 16 , Cromossomos Humanos Par 18 , Cromossomos Humanos Par 21 , Cromossomos Humanos Par 9 , Primers do DNA , Feminino , Haploidia , Humanos , Hibridização in Situ Fluorescente , Mosaicismo , Gravidez , Cromossomo X , Cromossomo YRESUMO
PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
Assuntos
Alquil e Aril Transferases , Proteínas de Transporte/genética , Coroideremia/diagnóstico , Proteínas do Olho/genética , Triagem de Portadores Genéticos/métodos , Testes Genéticos/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Deleção de Sequência , Cromossomo X/genética , Proteínas rab de Ligação ao GTP , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/análise , Proteínas de Transporte/química , Coroideremia/genética , Análise Mutacional de DNA , DNA Complementar/genética , Proteínas do Olho/análise , Proteínas do Olho/química , Feminino , França , Genes Recessivos , Humanos , Masculino , Biossíntese de Proteínas , Análise de Sequência de DNAAssuntos
Síndrome de Dandy-Walker/genética , Translocação Genética , Coloração Cromossômica , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 2/genética , Cromossomos Humanos Par 5/genética , Cromossomos Humanos Par 6/genética , Análise Citogenética , Síndrome de Dandy-Walker/patologia , Humanos , Hibridização in Situ Fluorescente , Recém-Nascido , Cariotipagem , MasculinoAssuntos
Genes do Retinoblastoma , Aconselhamento Genético , Retinoblastoma/genética , Neoplasias Ósseas/epidemiologia , Neoplasias Ósseas/genética , Pré-Escolar , Cromossomos Humanos Par 13/genética , Análise Mutacional de DNA , Feminino , Triagem de Portadores Genéticos , Predisposição Genética para Doença , Haplótipos , Humanos , Lactente , Masculino , Neoplasias Primárias Múltiplas/genética , Osteossarcoma/epidemiologia , Osteossarcoma/genética , Linhagem , Retinoblastoma/classificação , Retinoblastoma/epidemiologia , Retinoblastoma/prevenção & controleRESUMO
Angelman syndrome (AS) is a neurodevelopmental disorder associated with the loss of maternal gene expression in chromosome region 15q11-q13. AS is caused by a wide variety of genetic mechanisms, including mutations in the UBE3A gene that have been identified in 10-15% of patients; when the mother is heterozygous for the causative mutation, the risk of recurrence in subsequent pregnancies is 50%. The present authors have developed a preimplantation genetic diagnosis (PGD) assay for a family displaying a 10 bp deletion in exon 9 of the UBE3A gene, which was shared by two affected children and their phenotypically normal mother. A duplex polymerase chain reaction protocol was established, allowing the efficient amplification of the mutation together with an informative microsatellite marker (D15S122) located in intron 1 of the UBE3A gene. As most of UBE3A mutations identified so far are unique to one family, the present authors have also developed an indirect single cell protocol based upon the co-amplification of two microsatellite markers located within (D15S122) and close to the UBE3A gene (D15S1506). This strategy may be applied to all informative families requesting PGD for Angelman syndrome associated with mutations in the UBE3A gene.
Assuntos
Síndrome de Angelman/diagnóstico , Mutação , Diagnóstico Pré-Implantação/métodos , Ubiquitina-Proteína Ligases/genética , Alelos , Feminino , Deleção de Genes , Humanos , Íntrons , Repetições de Microssatélites , Linhagem , Reação em Cadeia da Polimerase , GravidezRESUMO
OBJECTIVE: To evaluate the effects of long abstinence periods on semen characteristics. DESIGN: Semen analysis was performed on six men after various sexual abstinence periods (from 2 to 18 days). The variations in semen parameters were analyzed statistically as a function of the duration of abstinence. SETTING: The Reproductive Biology Laboratory at the University of Montpellier Medical School. RESULTS: Lengthy sexual abstinence was found to affect all semen characteristics. Semen volume and concentration and total sperm count showed significant increases, whereas motility and normal morphology decreased significantly with duration of abstinence. Significant changes in the percentage of normal sperm forms were observed after more than seven days' abstinence. CONCLUSION: The study indicates that the influence of long sexual abstinence on semen quality varies with the variable considered. With regard to fertility, a long abstinence period might induce senescence of spermatozoa.
Assuntos
Abstinência Sexual , Espermatozoides/fisiologia , Adulto , Fertilidade/fisiologia , Humanos , Masculino , Análise Multivariada , Análise de Regressão , Sêmen/citologia , Sêmen/fisiologia , Contagem de Espermatozoides , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/citologia , Fatores de TempoRESUMO
The centromeric alpha satellite DNA subfamilies from chromosomes 13 and 21 are almost identical in sequence and cannot be easily distinguished by mean of probes for Southern blot or in situ hybridisation. We have used the oligonucleotide-primed in situ (PRINS) labelling technique with primers defined from the alpha satellite sequence of chromosome 13. One primer was found to label specifically the centromeric region of chromosomes 13 and allowed the detection of a polymorphism between two chromosome 13 homologues in one individual.
Assuntos
Cromossomos Humanos Par 13 , DNA Satélite/genética , Técnicas Genéticas , Polimorfismo Genético , Sequência de Bases , Primers do DNA , Humanos , Dados de Sequência MolecularRESUMO
The PRINS technique has been used to rapidly detect chromosomes 21 in both metaphases and interphase nuclei. A specific labeling was obtained in preparations from lymphocytes and amniotic fluid cells. The method is based on annealing of specific oligonucleotide primers and subsequent primer extension by a Taq DNA polymerase. PRINS is an interesting alternative to in situ hybridization for cytogenetic diagnosis and physical mapping.
Assuntos
Cromossomos Humanos Par 21 , Primers do DNA , Hibridização in Situ Fluorescente , Amniocentese , Líquido Amniótico/citologia , Sequência de Bases , DNA Satélite/análise , DNA Polimerase Dirigida por DNA , Síndrome de Down/diagnóstico , Síndrome de Down/genética , Doenças Fetais/diagnóstico , Humanos , Linfócitos/ultraestrutura , Dados de Sequência Molecular , Sequências Repetitivas de Ácido Nucleico , Sensibilidade e Especificidade , Taq Polimerase , Fatores de TempoRESUMO
In in vitro fertilization (IVF) procedures, morphologic embryo grading is the sole criteria for selection of embryos transferable in utero. Cytogenetic analysis of preimplantation embryos was performed to investigate the relationship between chromosomal status and morphologic quality of preimplantation eggs. Aneuploidy was the most frequently observed abnormality. In addition, various types of aberrations such as polyploidy, haploidy, mosaicism, and fragmentation were also found. Our results, pooled with data drawn from previous reports, demonstrated the prognostic value of the embryo grading system as a means for eliminating chromosomally abnormal embryos. In contrast, data suggested that some aspects of the IVF process might be responsible for the occurrence of these abnormalities.
Assuntos
Blastocisto/citologia , Blastocisto/fisiologia , Cromossomos Humanos , Ploidias , Aneuploidia , Aberrações Cromossômicas , Transtornos Cromossômicos , Diploide , Feminino , Fertilização in vitro , Haploidia , Humanos , Mosaicismo , Poliploidia , TrissomiaRESUMO
The primed in-situ labelling (PRINS) method is an alternative to in-situ hybridization for chromosomal detection based on the use of chromosome-specific oligonucleotide primers. Using this process, we have developed a simple and semi-automatic method for rapid in-situ detection of human chromosomes. The reaction was performed on a programmable temperature cycler. Specific labelling was obtained in < 2 h reaction. Double PRINS techniques were performed on six morphologically abnormal preimplantation embryos using primers specific for chromosomes 9, 16, 18, 21, X and Y. The majority of these embryos displayed chromosomal abnormalities. The present results demonstrate that PRINS may be a simple and reliable technique applicable in human preimplantation diagnosis.
Assuntos
Blastocisto/ultraestrutura , Aberrações Cromossômicas/diagnóstico , Citodiagnóstico/métodos , Testes Genéticos , Hibridização In Situ/métodos , Blastocisto/citologia , Blastômeros/ultraestrutura , Transtornos Cromossômicos , Cromossomos Humanos/ultraestrutura , Primers do DNA , DNA Satélite , Fertilização in vitro , Corantes Fluorescentes , Marcadores Genéticos , Testes Genéticos/métodos , Humanos , Reação em Cadeia da PolimeraseRESUMO
PURPOSE: The analysis of the chromosomal constitution of human gametes and embryos is of particular importance for investigation of aneuploidy occurrence and diagnostic purposes. The PRINS method constitutes an alternative to FISH for in situ chromosomal identification. We have adapted this method to human gametes, zygotes, and preimplantation embryos. RESULTS: Chromosome-specific labeling was obtained in gametes, zygotes, and isolated blastomeres. Simultaneous detection of two or three chromosomes can be completed in less than 3 hr using fluorochrome-labeled nucleotides. CONCLUSIONS: The PRINS technique appears to be more efficient than FISH for detection and discrimination of alpha-satellite DNA sequences. The present study demonstrates the usefulness of PRINS for chromosomal screening and preimplantation diagnosis.
Assuntos
Aneuploidia , Blastocisto/ultraestrutura , Cromossomos Humanos , DNA Satélite/análise , Hibridização de Ácido Nucleico , Óvulo/ultraestrutura , Espermatozoides/ultraestrutura , Zigoto/ultraestrutura , Cromossomos Humanos/genética , Primers do DNA , DNA Polimerase Dirigida por DNA , Estudos de Avaliação como Assunto , Feminino , Humanos , Masculino , Taq PolimeraseRESUMO
The primed in situ (PRINS) labeling technique was used to determine the rate of disomy of chromosomes 1 and 16 in sperm of two normal subjects. Two different but specific primers (alpha-satellite and satellite II) for chromosome 1 were used in parallel experiments to test the efficiency of PRINS labeling in sperm nuclei. A minimum of 10,000 sperm nuclei per chromosome primer was analyzed, leading to a total number of 41,651 scored spermatozoa. Similar rates of chromosome 1 disomy (mean values, 0.18% and 0.20%) were found in both donors when the alpha-satellite and satellite II primers were used, demonstrating the reliability of PRINS labeling on sperm nuclei. For chromosome 16, the disomy rate among the two donors ranged from 0.20% to 0.24%. This study confirms that PRINS provides a rapid and efficient method for in situ chromosomal screening of sperm nuclei.