Prenatal Diagnosis Of Cystic Fibrosis Research Articles

Prenatal Diagnosis of Cystic Fibrosis by Celocentesis.

Celocentesis is a new sampling tool for prenatal diagnosis available from 7 weeks in case of couples at risk for genetic diseases. In this study, we reported the feasibility of earlier prenatal diagnosis by celocentesis in four cases of cystic fibrosis and one case of cystic fibrosis and β-thalassemia co-inherited in the same fetus. Celomic fluids were aspired from the celomic cavity between 8+2 and 9+3 weeks of gestation and fetal cells were picked up by micromanipulator. Maternal DNA contamination was tested and target regions of fetal DNA containing parental pathogenetic variants of CFTR and HBB genes were amplified and sequenced. Four of the five fetuses resulted as being affected by cystic fibrosis and, in all cases, the women decided to interrupt the pregnancy. In the other case, the fetus presented a healthy carrier of cystic fibrosis. The results were confirmed in three cases on placental tissue. In one case, no abortive tissue was obtained. In the last case, the woman refused the prenatal diagnosis to confirm the celocentesis data; the pregnancy is ongoing without complications. This procedure provides prenatal diagnosis of monogenic diseases at least four weeks earlier than traditional procedures, reducing the anxiety of patients and providing the option for medical termination of the affected fetus at 8-10 weeks of gestation, which is less traumatic and safer than surgical termination in the second trimester.

Genetic attributes of Iranian cystic fibrosis patients: the diagnostic efficiency of CFTR mutations in over a decade.

Objectives: Cystic fibrosis (CF) is the most prevalent autosomal recessive disorder among Caucasians. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause this pathology. We, therefore, aimed to describe the CFTR mutations and their geographical distribution in Iran. Method: The mutation spectrum for 87 families from all Iranian ethnicities was collected using ARMS PCR, Sanger sequencing, and MLPA. Results: Mutations were identified in 95.8% of cases. This dataset revealed that the most frequent mutations in the Iranian population were F508del, c.1000C>T, c.1397C>G, c.1911delG, and c.1393-1G>A. In addition, we found weak evidence for Turkey being the possible geographical pathway for introducing CFTR mutations into Iran by mapping the frequency of CFTR mutations. Conclusion: Our descriptive results will facilitate the genetic detection and prenatal diagnosis of cystic fibrosis within the Iranian population.

P025 Possibility and reality of prenatal diagnosis of cystic fibrosis in Bulgaria

P025 Possibility and reality of prenatal diagnosis of cystic fibrosis in Bulgaria

Noninvasive Prenatal Diagnosis for Cystic Fibrosis: Implementation, Uptake, Outcome, and Implications.

Noninvasive prenatal diagnosis (NIPD) for monogenic disorders has a high uptake by families. Since 2013, our accredited public health service laboratory has offered NIPD for monogenic disorders, predominantly for de novo or paternally dominantly inherited mutations. Here we describe the extension of this service to include definitive NIPD for a recessive condition, cystic fibrosis (CF). Definitive NIPD for CF was developed using next-generation sequencing. Validation was performed on 13 cases from 10 families before implementation. All cases referred for CF NIPD were reviewed to determine turnaround times, genotyping results, and pregnancy outcomes. Of 38 referrals, 36 received a result with a mean turnaround of 5.75 days (range, 3-11 days). Nine cases were initially inconclusive, with 3 reported unaffected because the low-risk paternal allele was inherited and 4 cases in which the high-risk paternal allele was inherited, receiving conclusive results following repeat testing. One case was inconclusive owing to a paternal recombination around the mutation site, and one case was uninformative because of no heterozygosity. Before 2016, 3 invasive referrals for CF were received annually compared with 38 for NIPD in the 24 months since offering a definitive NIPD service. Timely and accurate NIPD for definitive prenatal diagnosis of CF is possible in a public health service laboratory. The method detects recombinations, and the service is well-received as evidenced by the significant increase in referrals. The bioinformatic approach is gene agnostic and will be used to expand the range of conditions tested for.

Prenatal Diagnosis of Cystic Fibrosis and Hemophilia: Incidental Findings and Weak Points.

Because of the progression of genetics and genomics, the demand for prenatal diagnosis (PD) for inherited genetic diseases has increased. However, several incidental findings may emerge during PD, like misattributed paternity, the evidence of disease in a parent, and the possible misinterpretation of the results because of complex alleles or de novo mutations that have several implications. In a retrospective observational study on all the couples referred to our Medical School (1993–2018) for PD of genetic inherited diseases (n = 1502), we selected the cases of PD for cystic fibrosis (CF, n = 239) and hemophilia A and B (HA, HB, n = 47), revising all incidental findings previously mentioned. We found one case in which a technical error led to PD of carrier in two siblings that were born affected by CF, four cases of misattributed paternity, eight cases of asymptomatic parents revealed as affected by CF transmembrane regulator (CFTR)-related disorders, a case of a novel complex allele that could have caused the diagnosis of CF in a carrier fetus, and a case of a de novo mutation in a mother (already a carrier) that caused hemophilia in a child that PD had revealed as healthy. We present these conditions as clinical cases and discuss the technical, clinical, ethical, and legal aspects to be considered.

Prenatal Diagnosis of Cystic Fibrosis.

Cystic fibrosis (CF) is an inherited disease characterized by the accumulation of thick, sticky mucus which damages epithelia in organs such as the lungs, pancreas, liver, intestines, and other parts of the body. The most common symptoms are sinopulmonary disease and chronic gastrointestinal tract problems resulting from decreased mucociliary clearance and inflammation. CF is the most common life-limiting autosomal recessive disorder in people of northern European ancestry and it affects other populations with different prevalence. CF can be diagnosed by many methods including testing for blood immunoreactive trypsin, sweat chloride, transepithelial nasal potential difference, and molecular genetic testing.

WS10.1 Isolated non-visualisation of foetal gallbladder should be considered for the prenatal diagnosis of cystic fibrosis

WS10.1 Isolated non-visualisation of foetal gallbladder should be considered for the prenatal diagnosis of cystic fibrosis

Prenatal diagnosis of cystic fibrosis in combination with herpetic infection in parents with uncompensated family history

Cystic fibrosis (CF) is a chronic genetic disorder that affects the exosecretory glands responsible for the production of mucus and sweat; as well as respiratory, digestive and reproductive systems. The article describes a brief description of this disease, the features of intrauterine manifestations and differential diagnosis with other conditions.In most cases, prenatal diagnosis of CF is offered to a family that already has children with CF. Regarding the prospective prenatal diagnosis of CF in families without CF history, as well as in couples with established status of heterozygous carrier of mutations in the CFTR gene, it is presented mainly in sporadic cases. In all other cases, prenatal diagnosis of CF is based on ultrasound detection of increased intestinal echogenicity. The hyperechogenic intestine is observed in 50–75% fetuses with CF. The incidence of intrauterine infection in fetuses with the hyperechogenic intestine is varies from 0 to 10%. The spectrum of perinatal infections is more often represented by cytomegalovirus, herpes, syphilis, toxoplasmosis, rubella and parvovirus B19.The second trimester amniotic fluid contains two main enzymes (alkaline phosphatase and gamma-glutamyltranspeptidase), whose decline of level is a reliable marker for CF. The article presents a clinical case of prenatal diagnosis of CF in combination with intrauterine infection with herpes simplex virus in 20 weeks pregnancy in a fetus with a hyperechoic intestine. Whenthe amniotic fluid was investigated in a fetus with a normal karyotype are revealed decline of alkaline phosphatase and gamma-glutamyltranspeptidase levels, the mutation delF508 CFTR in the homozygous state and the DNA regions homologous HSV I, II types. Taking into account the unfavorable morbid prognosis, the fetus with CF was eliminated for 22 weeks pregnancy at the family’s request. At autopsy were found the typical manifestations of CF in the female abortus. The molecular gene study of both parents confirmed the heterozygous carrier state. The family received recommendations on the mandatory prenatal diagnosis in subsequent pregnancies.

1 Implementing prenatal diagnosis of cystic fibrosis based on cell-free fetal DNA

1 Implementing prenatal diagnosis of cystic fibrosis based on cell-free fetal DNA

Prenatal diagnosis of cystic fibrosis: 10-years experience

PurposeWe present in this study our 10years experience in prenatal diagnosis of cystic fibrosis performed in the Tunisian population. Patients and methodsBased on family history, 40 Tunisian couples were selected for prenatal diagnosis. Fetal DNA was isolated from amniotic fluid collected by transabdominal amniocentesis or from chronic villi by transcervical chorionic villus sampling. The genetic analysis for cystic fibrosis mutations was performed by denaturant gradient gel electrophoresis and denaturing high-pressure liquid phase chromatography. We performed microsatellites analysis by capillary electrophoresis in order to verify the absence of maternal cell contamination. ResultsThirteen fetuses were affected, 21 were heterozygous carriers and 15 were healthy with two normal alleles of CFTR gene. Ten couples opted for therapeutic abortion. The microsatellites genotyping showed the absence of contamination of the fetal DNA by maternal DNA in 93.75%. ConclusionOur diagnostic strategy provides rapid and reliable prenatal diagnosis at risk families of cystic fibrosis.

322 Prenatal diagnosis of cystic fibrosis: Parental opinion, clinical course in the first year of life and influence of having a sibling with the disease

322 Prenatal diagnosis of cystic fibrosis: Parental opinion, clinical course in the first year of life and influence of having a sibling with the disease

Prenatal diagnosis of cystic fibrosis: an experience of 181 cases

The demand for prenatal diagnosis (PD) of cystic fibrosis (CF) is increasing. We performed pre-test multidisciplinary counselling for 192 couples at CF reproductive risk. In 11/192 (5.7%) cases PD was not performed mainly because counselling revealed a reproductive risk for atypical (mild) CF, while 181 PDs were performed in couples revealed at high risk for CF mainly because they already had a CF child (148/181, 81.8%) or had been identified through cascade screening (28/181, 15.5%). In 167/181 (92.3%) cases (including two dichorionic twin pregnancies), PD was performed on chorionic villi, and in 14 on amniocyte DNA. Only 1/181 PD was unsuccessful. In all other cases, single tandem repeat analysis excluded maternal contamination, and PD was made within 7 days of sampling. In total 116/180 (64.4%) PDs were made with dot-blot analysis; 40 (22.2%) required gene sequencing; in 4/180 cases we tested the gene for large rearrangements; in 23/180 (12.8%) cases linkage analysis was necessary because parental mutation(s) were unknown. Forty-two out of 180 (23.3%) PDs revealed an affected foetus. All couples but one interrupted pregnancy. The first twin PD revealed the absence (1 foetus) and the presence of one mutation (the other foetus); the second twin PD revealed one parental mutation (1 foetus) and both parental mutations (the other foetus); the couple planned selective interruption. PD for CF should be performed in reference laboratories equipped for gene scanning and linkage analysis, with a multidisciplinary staff able to offer counselling to couples during all phases of PD.

C37 Clinical validation of a non-invasive test for prenatal diagnosis of cystic fibrosis and spinal muscular atrophy based on genetic analysis of circulating trophoblastic cells

C37 Clinical validation of a non-invasive test for prenatal diagnosis of cystic fibrosis and spinal muscular atrophy based on genetic analysis of circulating trophoblastic cells

Amniotic Fluid Digestive Enzyme Analysis Is Useful for Identifying CFTR Gene Mutations of Unclear Significance

The large number of CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] mutations and the existence of variants of unclear significance complicate the prenatal diagnosis of cystic fibrosis (CF). The aim of this study was to determine whether the pattern of amniotic fluid digestive enzymes (AF-DEs) could be correlated with the severity of CFTR mutations. The AF-DE pattern (gamma-glutamyltranspeptidase, aminopeptidase M, and the intestinal isoform of alkaline phosphatase) was retrospectively analyzed in 43 AF samples. All fetuses presented 2 CFTR mutations, which were classified according to the severity of the disease: CF/CF (n = 38); CF/CFTR-related disorders (n = 1); and CF/unknown variant (n = 4). The relationships between clinical CF status, CFTR mutations, and AF-DE pattern were studied. Of 38 severely affected CF fetuses, an "obstructive" AF-DE pattern was observed in 15 of 15 samples collected before 22 weeks, irrespective of the CFTR mutation (diagnostic sensitivity, 100%; diagnostic specificity, 99.8%). In the 23 fetuses evaluated after 22 weeks, the AF-DE pattern was abnormal in 7 cases and noncontributive in 16 (diagnostic sensitivity, 30.4%; diagnostic specificity, 99.8%). Of the 5 questionable cases (F508del/N1224K, F508del/L73F, 3849+10kbC>T/G1127E, F508del/S1235R, F508del/G622D), all were CF symptom free at 2-4 years of follow-up. The AF-DE pattern (<22 weeks) was typical in 3 cases but abnormal in the last 2 cases. AF-DE analysis is of value for prenatal CF diagnosis in classic forms of CF and could be helpful in nonclassic CF.

Three Novel CFTR Polymorphic Repeats Improve Segregation Analysis for Cystic Fibrosis

Molecular diagnosis for cystic fibrosis (CF) is based on the direct identification of mutations in the CFTR gene [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] (detection rate about 90% with scanning procedures) and on segregation analysis of intragenic polymorphisms for carrier and prenatal diagnosis in about 20% of CF families in which 1 or both causal mutations are unknown. We identified 3 novel intragenic polymorphic repeats (IVS3polyA, IVS4polyA, and IVS10CA repeats) in the CFTR gene and developed and validated a procedure based on the PCR followed by capillary electrophoresis for large-scale analysis of these polymorphisms and the 4 previously identified microsatellites (IVS1CA, IVS8CA, IVS17bTA, and IVS17bCA repeats) in a single run. We validated the procedure for both single- and 2-cell samples (for a possible use in preimplantation diagnosis), and on a large number of CF patients bearing different genotypes and non-CF controls. The allelic distribution and heterozygosity results suggest that the 3 novel polymorphisms strongly contribute to carrier and prenatal diagnosis of CF in families in which 1 or both causal mutations have not been identified. At least 1 of the 4 previously identified microsatellites was informative in 78 of 100 unrelated CF families; at least 1 of all 7 polymorphisms was informative in 98 of the families. Finally, the analysis of haplotypes for the 7 polymorphisms revealed that most CF mutations are associated with different haplotypes, suggesting multiple slippage events but a single origin for most CFTR mutations. The analysis of the 7 polymorphisms is a rapid and efficient tool for routine carrier, prenatal, and preimplantation diagnosis of CF.

Strategy for prenatal diagnosis of cystic fibrosis in Russia

Strategy for prenatal diagnosis of cystic fibrosis in Russia

New strategy for the prenatal detection/exclusion of paternal cystic fibrosis mutations in maternal plasma

Background Since the presence of fetal DNA was discovered in maternal blood, different investigations have focused on non-invasive prenatal diagnosis. The analysis of fetal DNA in maternal plasma may allow the diagnosis of fetuses at risk of cystic fibrosis (CF) without any risk of fetal loss. Here, we present a new strategy for the detection of fetal mutations causing CF in maternal plasma. Methods We have used a mini-sequencing based method, the SNaPshot®, for fetal genotyping of the paternal mutation in maternal blood from three pregnancies at risk of CF. Results The paternal mutation was detected in the analysis of plasma samples from cases 1 and 3 but not in case 2. Results of a posterior conventional molecular analysis of chorionic biopsies were in full agreement with those obtained from analysis of the plasma samples. Conclusions The knowledge about the inheritance of the paternal mutation in a fetus may avoid the conventional prenatal diagnosis in some cases. The SNaPshot® technique has been shown to be a sensitive and accurate method for the detection of fetal mutations in maternal plasma. Its ease handling, rapid and low cost makes it appropriate for a future routine clinical use in non-invasive prenatal diagnosis of cystic fibrosis.

Prenatal diagnosis of cystic fibrosis: the 18‐year experience of Brittany (western France)

This study reports 18 years of experience in prenatal diagnosis (PD) of cystic fibrosis (CF) in a region where CF is frequent and the uptake of PD is common (Brittany, western France). All PDs made over the period 1989-2006 in women living in Brittany were collected. We recorded 268 PDs made in 1 in 4 risk couples, plus 22 PDs directly made following the sonographic finding of echogenic bowel. Most of the 268 PDs were done in couples already having CF child(ren) (n = 195, 72.8%). Close to one-fifth followed cascade screening (n = 49, 18.3%), which identified 26 new 1 in 4 risk couples among the relatives of CF patients or of carriers identified through newborn screening (NBS). The remaining PDs were mainly made in couples whose 1 in 4 risk was evidenced following the diagnosis of echogenic bowel in a previous pregnancy (n = 22, 8.2%). Although patients' life expectancy has considerably improved, in our population the great majority of couples chose pregnancy termination when PD indicated that the foetus had CF (95.9%). This study describes the distribution of PDs according to the context in which the 1 in 4 risk was discovered and highlights the real decisions of couples as regards pregnancy termination after a positive PD.

Analysis of extra- and intragenic marker haplotypes as part of molecular diagnosis of cystic fibrosis in patients from Serbia

Cystic fibrosis (CF) is the most common fatal autosomal recessive disease in Caucasians. Since characterization of F508del, the predominant mutation in different countries, more than 1500 mutations have been discovered in the CFTR gene, including a large number of polymorphisms. After molecular screening of 222 CF patients from Serbia, we detected 21 different CFTR mutations, F508del being the most frequent (69.59% of CF alleles). A total of 21 mutations cover almost 80% of CF alleles in this group. Since the molecular basis of CF is highly heterogeneous in our population, studying the haplotype association with normal and CF chromosomes could be very helpful in all cases where one or both mutations remain unidentified. Haplotype analysis was done using six diallelic sites and one tetranucleotide repeat (XV2C-KM19-MP6D9-J44-IVS6a(GATT)-M470V-T854T) on 99 F508del, 90 non-F508del, and 105 normal chromosomes. Strong linkage disequilibrium was observed for CFTR mutations and one haplotype (1-2-2-1-6(2)-1-1), while normal chromosomes were mostly associated with another (1-1-2-1-6(2)-1-2). The obtained results show that in most cases it would be possible with this group of polymorphisms to separate normal chromosomes from chromosomes which carry the CFTR mutation. As far as prenatal diagnosis of cystic fibrosis is concerned, haplotype analysis can be used as a helpful method of indirect diagnosis in families at risk with one or both CF alleles uncharacterized.

Genetic characterisation of circulating fetal cells allows non‐invasive prenatal diagnosis of cystic fibrosis

Cystic fibrosis (CF) is an autosomal recessive disease due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The purpose of this study was to develop a molecular method to characterise both paternal and maternal CFTR alleles in DNA from circulating fetal cells (CFCs) isolated by ISET (isolation by size of epithelial tumour/trophoblastic cells). The molecular protocol was defined by developing the F508del mutation analysis and addressing it both to single trophoblastic cells, isolated by ISET and identified by short tandem repeats (STR) genotyping, and to pooled trophoblastic genomes, thus avoiding the risk of allele drop out (ADO). This protocol was validated in 100 leucocytes from F508del carriers and subsequently blindly applied to the blood (5 mL) of 12 pregnant women, at 11 to 13 weeks of gestation, whose offspring had a 1/4 risk of CF. Ten couples were carriers of F508del mutation, while two were carriers of unknown CFTR mutations. Results showed that one fetus was affected, seven were heterozygous carriers of a CFTR mutation, and four were healthy homozygotes. These findings were consistent with those obtained by chorionic villus sampling (CVS). Our data show that the ISET-CF approach affords reliable prenatal diagnosis (PND) of cystic fibrosis and is potentially applicable to pregnant women at risk of having an affected child, thus avoiding the risk of iatrogenic miscarriage.