Genetic counseling for cystic fibrosis: A basic model with new challenges.

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.

Non-invasive prenatal diagnosis (NIPD) of cystic fibrosis: an optimized protocol using MEMO fluorescent PCR to detect the p.Phe508del mutation.

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.

Preimplantation genetic diagnosis for cystic fibrosis: the Montpellier center's 10-year experience.

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.

[Genetic mutation databases: stakes and perspectives for orphan genetic diseases]. / Banques de données de mutations: enjeux et perspectives pour les maladies génétiques orphelines.

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.

Rapid and powerful decaplex and dodecaplex PGD protocols for Duchenne muscular dystrophy.

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.

Strategies for preimplantation genetic diagnosis of Angelman syndrome caused by mutations in the UBE3A gene.

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.

Specific detection of deleted and non-deleted dystrophin exons together with gender assignment in preimplantation genetic diagnosis of Duchenne muscular dystrophy.

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.

First preimplantation genetic diagnosis of hereditary retinoblastoma using informative microsatellite markers.

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.

Meiotic segregation analysis of RB1 alleles in retinoblastoma pedigrees by use of single-sperm typing.

In hereditary retinoblastoma, different epidemiological studies have indicated a preferential paternal transmission of mutant retinoblastoma alleles to offspring, suggesting the occurrence of a meiotic drive. To investigate this mechanism, we analyzed sperm samples from six individuals from five unrelated families affected with hereditary retinoblastoma. Single-sperm typing techniques were performed for each sample by study of two informative short tandem repeats located either in or close to the retinoblastoma gene (RB1). The segregation probability of mutant RB1 alleles in sperm samples was assessed by use of the SPERMSEG program, which includes experimental parameters, recombination fractions between the markers, and segregation parameters. A total of 2,952 single sperm from the six donors were analyzed. We detected a significant segregation distortion in the data as a whole (P=.0099) and a significant heterogeneity in the segregation rate across donors (.0092). Further analysis shows that this result can be explained by segregation distortion in favor of the normal allele in one donor only and that it does not provide evidence of a significant segregation distortion in the other donors. The segregation distortion favoring the mutant RB1 allele does not seem to occur during spermatogenesis, and, thus, meiotic drive may result either from various mechanisms, including a fertilization advantage or a better mobility in sperm bearing a mutant RB1 gene, or from the existence of a defectively imprinted gene located on the human X chromosome.

Direct estimation of the recombination frequency between the RB1 gene and two closely linked microsatellites using sperm typing.

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.

[Update on a diagnostic test for choroideremia: the protein truncation test (PTT)]. / Mise au point d'un test diagnostique de la choroïdérémie: le test de troncation des protéines (PTT).

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.

Refinement of genetic localization of the Alström syndrome on chromosome 2p12-13 by linkage analysis in a North African family.

Alström syndrome is a rare autosomal recessive disorder characterized by retinal pigment degeneration, neurogenic deafness, infantile obesity, hyperlipidemia, and non-insulin-dependent diabetes mellitus. While the disease-related gene remains unknown, studies of the genetic isolate of French Acadians provisionally locate the Alström syndrome on chromosome 2p12-13 within a 14.9-cM interval. To confirm this finding in another ethnic population and refine the candidate region we investigated by linkage analysis a consanguineous family of North African origin, in which three of seven siblings displayed all major neurological and metabolic features of Alström syndrome. Genotyping was performed on an ABI377 DNA automatic sequencer and LOD scores were obtained with the Fastlink program. Five markers previously investigated in French Acadians confirmed the involvement of the candidate region, although pairwise LOD scores were of poor significance (Zmax = 2.9). To further confirm homogeneity and refine the candidate region, 20 additional markers were investigated. Haplotype analysis and allele segregation revealed that affected children shared a single haplotype and were homozygous for the eight most centromeric markers (D2S291-D2S2114), over a 6.1-cM interval. Significative multipoint LOD scores (Zmax = 3.96) were obtained between markers D2S2110/145 and D2S286. Two clusters of known genes are present in this refined region of chromosome 2p, the most attractive candidate being the hexokinase II gene. However, except for several known polymorphisms, no mutations were detected in the coding region of this gene. In conclusion, the location of Alström syndrome on chromosome 2p12-13 is confirmed, reducing the genetic interval to 6.1 cM.

Incidence of chromosome 1 disomy in human sperm estimated by the primed in situ (PRINS) labeling technique.

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.

Cytogenetic analysis of meiotic segregation in sperm from two males heterozygous for reciprocal translocations using PRINS and humster techniques.

The meiotic segregation patterns of 2 reciprocal translocations t(7;9)(q33;p21) and t(7;18)(q35;q11) were analyzed in sperm of 2 heterozygote carriers. Both sperm karyotyping and in situ PRINS labeling of sperm nuclei were performed on a sperm sample from each subject. Using the humster technique, 54 and 72 sperm chromosome complements were successfully analyzed for the t(7;9) and the t(7;18) respectively. The frequencies of alternate, adjacent 1, adjacent 2 and 3:1 segregations were 44.44%, 37.04%, 12.96% and 5.56% for the t(7;9) and 33.33%, 43.05%, 19.45% and 4.17% for the t(7;18). The PRINS procedure allowed the rapid screening of large samples of spermatozoa. However, alternate and adjacent 1 segregants were not discriminated because of the generation of centromeric signals. The segregation pattern was determined on 10,658 spermatozoa for the t(7;9) and 10,462 for the t(7;18). The distributions of segregants were similar to those obtained by sperm karyotyping. These data were pooled with results from 37 reciprocal translocations previously studied by sperm karyotyping and 6 recently investigated by FISH. The analysis of these compiled data demonstrates the particularity of the production of imbalances in male gametes; independent of the predisposition for a type of imbalance at term, there is a preferential production of adjacent 1 imbalance in sperm.

Preimplantation embryo chromosome analysis by primed in situ labeling method.

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.