RESUMEN
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.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/diagnóstico , Reacción en Cadena de la Polimerasa Multiplex/métodos , Diagnóstico Preimplantación , Niño , Fibrosis Quística/genética , Fibrosis Quística/patología , Femenino , Francia , Asesoramiento Genético , Genotipo , Haplotipos , Heterocigoto , Humanos , Masculino , EmbarazoRESUMEN
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.
Asunto(s)
Bases de Datos Genéticas , Enfermedades Genéticas Congénitas/genética , Mutación , Enfermedades Raras/genética , Codón de Terminación , Etnicidad/genética , Predicción , Enfermedades Genéticas Congénitas/clasificación , Enfermedades Genéticas Congénitas/terapia , Terapia Genética , Genética Médica/ética , Genotipo , Humanos , Internet , Fenotipo , ARN sin Sentido/uso terapéutico , Enfermedades Raras/clasificación , Enfermedades Raras/terapia , Terminología como Asunto , Transcripción Genética/efectos de los fármacosRESUMEN
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.
Asunto(s)
Fibrosis Quística/diagnóstico , Fibrosis Quística/genética , Asesoramiento Genético , ADN/sangre , Femenino , Feto/metabolismo , Tamización de Portadores Genéticos , Humanos , Intercambio Materno-Fetal , Pruebas Prenatales no Invasivas , Atención Preconceptiva , Embarazo , Diagnóstico Preimplantación , Diagnóstico PrenatalRESUMEN
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.
Asunto(s)
Distrofina/genética , Distrofia Muscular de Duchenne/genética , Reacción en Cadena de la Polimerasa/métodos , Diagnóstico Preimplantación/métodos , Amelogenina/genética , Femenino , Eliminación de Gen , Humanos , Distrofia Muscular de Duchenne/diagnóstico , Linaje , EmbarazoRESUMEN
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.
Asunto(s)
Amniocentesis/métodos , Muestra de la Vellosidad Coriónica/métodos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística , Reacción en Cadena de la Polimerasa/métodos , Adulto , Investigación sobre la Eficacia Comparativa , Fibrosis Quística/sangre , Fibrosis Quística/diagnóstico , Fibrosis Quística/genética , Electroforesis Capilar/métodos , Femenino , Humanos , Mutación , Embarazo , Diagnóstico Prenatal/métodos , Reproducibilidad de los ResultadosRESUMEN
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.
Asunto(s)
Repeticiones de Microsatélite , Recombinación Genética , Proteína de Retinoblastoma/genética , Ligamiento Genético , Humanos , Masculino , EspermatozoidesRESUMEN
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.
Asunto(s)
Cromosomas Humanos Par 21 , Hibridación Fluorescente in Situ/métodos , Secuencia de Bases , Cartilla de ADN , Humanos , Datos de Secuencia MolecularRESUMEN
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.
Asunto(s)
Aneuploidia , Hibridación Fluorescente in Situ/métodos , Reacción en Cadena de la Polimerasa/métodos , Espermatozoides , Adulto , Núcleo Celular/química , ADN Satélite/análisis , Humanos , MasculinoRESUMEN
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.
Asunto(s)
Blastocisto/ultraestructura , Aberraciones Cromosómicas , Desarrollo Embrionario , Aneuploidia , Cromosomas Humanos Par 13 , Cromosomas Humanos Par 16 , Cromosomas Humanos Par 18 , Cromosomas Humanos Par 21 , Cromosomas Humanos Par 9 , Cartilla de ADN , Femenino , Haploidia , Humanos , Hibridación Fluorescente in Situ , Mosaicismo , Embarazo , Cromosoma X , Cromosoma YRESUMEN
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.
Asunto(s)
Transferasas Alquil y Aril , Proteínas Portadoras/genética , Coroideremia/diagnóstico , Proteínas del Ojo/genética , Tamización de Portadores Genéticos/métodos , Pruebas Genéticas/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de Secuencia , Cromosoma X/genética , Proteínas de Unión al GTP rab , Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/análisis , Proteínas Portadoras/química , Coroideremia/genética , Análisis Mutacional de ADN , ADN Complementario/genética , Proteínas del Ojo/análisis , Proteínas del Ojo/química , Femenino , Francia , Genes Recesivos , Humanos , Masculino , Biosíntesis de Proteínas , Análisis de Secuencia de ADNAsunto(s)
Síndrome de Dandy-Walker/genética , Translocación Genética , Pintura Cromosómica , Cromosomas Humanos Par 1/genética , Cromosomas Humanos Par 12/genética , Cromosomas Humanos Par 2/genética , Cromosomas Humanos Par 5/genética , Cromosomas Humanos Par 6/genética , Análisis Citogenético , Síndrome de Dandy-Walker/patología , Humanos , Hibridación Fluorescente in Situ , Recién Nacido , Cariotipificación , MasculinoAsunto(s)
Genes de Retinoblastoma , Asesoramiento Genético , Retinoblastoma/genética , Neoplasias Óseas/epidemiología , Neoplasias Óseas/genética , Preescolar , Cromosomas Humanos Par 13/genética , Análisis Mutacional de ADN , Femenino , Tamización de Portadores Genéticos , Predisposición Genética a la Enfermedad , Haplotipos , Humanos , Lactante , Masculino , Neoplasias Primarias Múltiples/genética , Osteosarcoma/epidemiología , Osteosarcoma/genética , Linaje , Retinoblastoma/clasificación , Retinoblastoma/epidemiología , Retinoblastoma/prevención & controlRESUMEN
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.
Asunto(s)
Síndrome de Angelman/diagnóstico , Mutación , Diagnóstico Preimplantación/métodos , Ubiquitina-Proteína Ligasas/genética , Alelos , Femenino , Eliminación de Gen , Humanos , Intrones , Repeticiones de Microsatélite , Linaje , Reacción en Cadena de la Polimerasa , EmbarazoRESUMEN
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.
Asunto(s)
Abstinencia Sexual , Espermatozoides/fisiología , Adulto , Fertilidad/fisiología , Humanos , Masculino , Análisis Multivariante , Análisis de Regresión , Semen/citología , Semen/fisiología , Recuento de Espermatozoides , Motilidad Espermática/fisiología , Espermatozoides/citología , Factores de TiempoRESUMEN
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.
Asunto(s)
Cromosomas Humanos Par 13 , ADN Satélite/genética , Técnicas Genéticas , Polimorfismo Genético , Secuencia de Bases , Cartilla de ADN , Humanos , Datos de Secuencia MolecularRESUMEN
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.
Asunto(s)
Blastocisto/citología , Blastocisto/fisiología , Cromosomas Humanos , Ploidias , Aneuploidia , Aberraciones Cromosómicas , Trastornos de los Cromosomas , Diploidia , Femenino , Fertilización In Vitro , Haploidia , Humanos , Mosaicismo , Poliploidía , TrisomíaRESUMEN
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.
Asunto(s)
Cromosomas Humanos Par 21 , Cartilla de ADN , Hibridación Fluorescente in Situ , Amniocentesis , Líquido Amniótico/citología , Secuencia de Bases , ADN Satélite/análisis , ADN Polimerasa Dirigida por ADN , Síndrome de Down/diagnóstico , Síndrome de Down/genética , Enfermedades Fetales/diagnóstico , Humanos , Linfocitos/ultraestructura , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos Nucleicos , Sensibilidad y Especificidad , Polimerasa Taq , Factores de TiempoRESUMEN
We report the use of the PRimedIN Situ (PRINS) labelling technique for the direct estimation of disomy rates for various chromosomes in human sperm. The PRINS technique provides a rapid and reliable method for chromosome screening since specific labelling may be obtained in less than 2 h. In the present study, the disomy rates of chromosomes 2, 5, 9, 12 and 18 were investigated. The incidences of disomy are similar for all these chromosomes, ranging from 0.27% to 0.33%. These findings suggest an equal distribution of aneuploidies among autosomes in human sperm.
Asunto(s)
Aneuploidia , Espermatozoides/citología , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 12 , Cromosomas Humanos Par 18 , Cromosomas Humanos Par 2 , Cromosomas Humanos Par 5 , Cromosomas Humanos Par 9 , Cartilla de ADN , Humanos , Hibridación Fluorescente in Situ , Masculino , Datos de Secuencia MolecularRESUMEN
The incidence of aneuploidy was estimated for chromosomes 8, 9, 13, 16, and 21 in mature human spermatozoa by primed in situ (PRINS) labeling technique. This method allows us to perform a chromosome-specific detection by in situ annealing of a centromeric specific primer. A dual color PRINS protocol was adapted to human sperm. The decondensation and the denaturation of sperm nuclei were simultaneously performed by 3-M NaOH treatment. Double labeling of spermatozoa was obtained in <2 h. A total of 96,292 sperm nuclei were analyzed by two independent observers. The estimates of disomy were 0.31% for chromosome 8, 0.28% for chromosome 9, 0.28% for chromosome 13, 0.26% for chromosome 16, and 0.32% for chromosome 21. These homogeneous findings suggest an equal distribution of aneuploidies among autosomal chromosomes in males.
Asunto(s)
Aneuploidia , Cromosomas Humanos , Hibridación Fluorescente in Situ/métodos , Espermatozoides , Adulto , Secuencia de Bases , Cromosomas Humanos Par 13 , Cromosomas Humanos Par 16 , Cromosomas Humanos Par 21 , Cromosomas Humanos Par 8 , Cromosomas Humanos Par 9 , Cartilla de ADN , Diploidia , Humanos , Masculino , Datos de Secuencia MolecularRESUMEN
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.