Prenatal diagnosis of cystic fibrosis: 10-years experience.

PURPOSE: We present in this study our 10years experience in prenatal diagnosis of cystic fibrosis performed in the Tunisian population. PATIENTS AND METHODS: Based 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. RESULTS: Thirteen 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%. CONCLUSION: Our diagnostic strategy provides rapid and reliable prenatal diagnosis at risk families of cystic fibrosis.

Absent gallbladder on fetal ultrasound: prenatal findings and postnatal outcome.

OBJECTIVES: Fetal gallbladder non-visualization on prenatal ultrasound in the second trimester is uncommon and in most cases the gallbladder is detected eventually. Associations of gallbladder non-visualization with cystic fibrosis, aneuploidy, agenesis of the gallbladder and biliary atresia have been reported. We present our experience and review the literature. METHODS: During the study period from January 2004 to June 2009 we collected prospectively cases of non-visualization of the fetal gallbladder in the second trimester. In each case the fetus was evaluated by two examiners on at least two occasions, at least a week apart. Cases with no additional sonographic malformations were designated as isolated. Further evaluation included follow-up scans and a meticulous search for fetal anomalies. All patients were offered genetic consultation. Cystic fibrosis testing, amniocentesis for karyotyping and analysis of fetal digestive enzymes in the amniotic fluid were offered. RESULTS: We collected 21 cases of non-visualization of the fetal gallbladder, 16 of which were isolated and five of which had additional malformations. In four of these five, the associated anomalies were severe and the pregnancies were terminated for aneuploidy (two cases of trisomy 18 and one triploidy) or for the severity of the associated anomalies. Associated anomalies included left isomerism with complex cardiac anomaly and intrauterine growth restriction with multisystem anomalies. The fifth fetus had interrupted inferior vena cava with azygos continuation without other anomalies and the child was alive and well at the age of 4 years. In 15 of the 16 isolated cases, antenatal and postnatal development were normal at the last follow-up, ranging from 4 months to 2.5 years. One case of cystic fibrosis was diagnosed prenatally and this pregnancy was terminated. There were no diagnoses of abnormal karyotype or biliary atresia among cases of isolated non-visualization of the gallbladder. CONCLUSIONS: When prenatal non-visualization of the fetal gallbladder is associated with other severe malformation, aneuploidy should be suspected. When it is isolated, if cystic fibrosis is ruled out, the outcome is good.

Prenatal diagnosis in a cystic fibrosis family: a combined molecular strategy for a precise diagnosis.

INTRODUCTION: The high genetic heterogeneity in populations with a wide spectrum of mutations in the CF transmembrane conductance regulator gene (CFTR), makes the detection of mutations a very hard and difficult task, thereby limiting the accurate diagnosis of the disease, mainly in patients with uncharacterized mutations. MATERIAL AND METHODS: Molecular strategies, like targeted identification of the most frequent CFTR mutations in Mexican population combined with linkage analysis using markers, is very useful for carrier detection and for prenatal diagnosis in affected families with CF. In this paper we show that the combination of methodologies was a crucial alternative to reach a precise prenatal CF diagnosis. We documented CF diagnosis in a 14th-week fetus combining the screening of the most common mutations in Mexican population with linkage analysis of two extragenic polymorphisms (XV2C/TaqI and KM19/PstI). RESULTS: We determined that the fetus inherited the PG542X mutation from its mother and an unknown mutation from its father through the chromosomal phases analysis.

Laser-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis.

BACKGROUND: Human embryonic stem cells (hESCs) suitable for future transplantation therapy should preferably be developed in an animal-free system. Our objective was to develop a laser-based system for the isolation of the inner cell mass (ICM) that can develop into hESC lines, thereby circumventing immunosurgery that utilizes animal products. METHODS: Hatching was assisted by micromanipulation techniques through a laser-drilled orifice in the zona pellucida of 13 abnormal preimplantation genetic diagnosed blastocysts. ICMs were dissected from the trophectoderm by a laser beam and plated on feeders to derive hESC lines. RESULTS: eight ICMs were isolated from nine hatched blastocysts and gave rise to three hESC lines affected by myotonic dystrophy type 1, hemophilia A and a carrier of cystic fibrosis 405 + 1G > A mutation. Five blastocysts that collapsed during assisted hatching or ICM dissection were plated whole, giving rise to an additional line affected by fragile X. All cell lines expressed markers of pluripotent stem cells and differentiated in vitro and in vivo into the three germ layers. CONCLUSIONS: These hESC lines can serve as an important model of the genetic disorders that they carry. Laser-assisted isolation of the ICMs may be applied for the derivation of new hESC lines in a xeno-free system for future clinical applications.

Early inflammation in the airways of a cystic fibrosis foetus.

In cystic fibrosis patients, inflammation is often considered to be secondary to chronic infections. In the present study, we show increased levels of pro-inflammatory proteins in the lungs of a cystic fibrosis foetus compared to the lungs of two normal foetuses. Our findings suggest therefore the existence of an early intrinsic pro-inflammatory state in cystic fibrosis airways.

[Fetal echogenic bowel by ultrasound: what is the clinical significance?].

Echogenic bowel is diagnosed in 0.2% to 1.4% of second trimester ultrasonographic examinations. This finding occurs as a normal variant in the second trimester but also has been associated with several pathologic conditions that include cystic fibrosis, chromosomal abnormalities and in utero infection with cytomegalovirus and toxoplasmosis. Ultrasound assessment of echogenic bowel is usually subjective by comparing the echogenicity with adjacent bone or liver. The diagnosis of fetal echogenic bowel in the second trimester has significant implications for prenatal management. Fetal echogenic bowel should be considered an important marker of placental damage. This finding in the second trimester is strongly associated with adverse pregnancy outcome due to utero-placental insufficiency, particularly in women with elevated maternal serum alpha-fetoprotein concentration due to severe feto-maternal bleeding. This review focuses on the definition and diagnosis of this entity and problems raised by echogenic bowel due to subjectivity of the diagnosis. It also includes the pathophysiology in the different conditions and the prevalence of each condition. Based on this review, we suggest the evaluation that is needed, as well as the recommendations to follow-up, during the remaining term of pregnancy according to the literature.

Preimplantation genetic diagnosis for cystic fibrosis: a case report.

Cystic fibrosis is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator gene. This disorder produces a variable phenotype including lung disease, pancreatic insufficiency, and meconium ileus plus bilateral agenesis of the vas deferens causing obstructive azoospermia and male infertility. Preimplantation genetic diagnosis is an alternative that allows identification of embryos affected by this or other genetic diseases. We report a case of couple with cystic fibrosis; the woman had the I148 T mutation and the man had the Delta F508 gene mutation. The couple underwent in vitro fertilization, associated with preimplantation genetic diagnosis, and with subsequent selection of healthy embryos for uterine transfer. The result was an uneventful pregnancy and delivery of a healthy male baby.

Generation of Human Induced Pluripotent Stem Cells from Extraembryonic Tissues of Fetuses Affected by Monogenic Diseases.

The generation of human induced pluripotent stem cells (hiPSCs) derived from an autologous extraembryonic fetal source is an innovative personalized regenerative technology that can transform own-self cells into embryonic stem-like ones. These cells are regarded as a promising candidate for cell-based therapy, as well as an ideal target for disease modeling and drug discovery. Thus, hiPSCs enable researchers to undertake studies for treating diseases or for future applications of in utero therapy. We used a polycistronic lentiviral vector (hSTEMCCA-loxP) encoding OCT4, SOX2, KLF4, and cMYC genes and containing loxP sites, excisible by Cre recombinase, to reprogram patient-specific fetal cells derived from prenatal diagnosis for several genetic disorders, such as myotonic dystrophy type 1 (DM1), ß-thalassemia (ß-Thal), lymphedema-distichiasis syndrome (LDS), spinal muscular atrophy (SMA), cystic fibrosis (CF), as well as from wild-type (WT) fetal cells. Because cell types tested to create hiPSCs influence both the reprogramming process efficiency and the kinetics, we used chorionic villus (CV) and amniotic fluid (AF) cells, demonstrating how they represent an ideal cell resource for a more efficient generation of hiPSCs. The successful reprogramming of both CV and AF cells into hiPSCs was confirmed by specific morphological, molecular, and immunocytochemical markers and also by their teratogenic potential when inoculated in vivo. We further demonstrated the stability of reprogrammed cells over 10 and more passages and their capability to differentiate into the three embryonic germ layers, as well as into neural cells. These data suggest that hiPSCs-CV/AF can be considered a valid cellular model to accomplish pathogenesis studies and therapeutic applications.

Gene transfer to human fetal pulmonary tissue developed in immunodeficient SCID mice.

Human fetal lung rudiments (8-12 weeks of development) undergo considerable growth upon microsurgical ectopic implantation in the xenograft-tolerant SCID mouse, and differentiate into a lung-like tissue that includes: (i) bronchial structures lined with pseudostratified, secretory, ciliated epithelium surrounded by smooth muscle and cartilage rings, (ii) submucosal glands, and (iii) alveolar sacs. Normal expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein was detected by immunostaining in those grafts, and similar differentiation was observed from either normal or cystic fibrosis (CF) fetal lung rudiments. Upon microinjection into human CF or normal lung grafts in SCID mice, beta-galactosidase-adenovirus gene constructs were efficiently transduced into epithelial and glandular cells. Such an in vivo replica of the human respiratory tissue may be a useful experimental model to study normal and pathologic lung development, and to assay candidate therapeutic gene constructs preclinically.

Progress toward generating a ferret model of cystic fibrosis by somatic cell nuclear transfer.

Mammalian cloning by nuclear transfer from somatic cells has created new opportunities to generate animal models of genetic diseases in species other than mice. Although genetic mouse models play a critical role in basic and applied research for numerous diseases, often mouse models do not adequately reproduce the human disease phenotype. Cystic fibrosis (CF) is one such disease. Targeted ablation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in mice does not adequately replicate spontaneous bacterial infections observed in the human CF lung. Hence, several laboratories are pursuing alternative animal models of CF in larger species such as the pig, sheep, rabbits, and ferrets. Our laboratory has focused on developing the ferret as a CF animal model. Over the past few years, we have investigated several experimental parameters required for gene targeting and nuclear transfer (NT) cloning in the ferret using somatic cells. In this review, we will discuss our progress and the hurdles to NT cloning and gene-targeting that accompany efforts to generate animal models of genetic diseases in species such as the ferret.

Abnormal fucosylation of ileal mucus in cystic fibrosis: I. A histochemical study using peroxidase labelled lectins.

Peroxidase conjugated lectins were used to analyse the glycoproteins of small intestinal mucins in normal infants and those with cystic fibrosis to ascertain whether there are any detectable histochemical differences in saccharide composition. A significant decrease in Lotus tetragonolobus (LTG) binding fucose was shown in normal small intestinal mucin starting around 36 weeks' gestation with total absence of staining at term and beyond. In contrast, the age matched patients with cystic fibrosis showed persistent and intense LTG binding of fucose. These results provide the first clear histochemical evidence that cystic fibrosis mucin is abnormal and confirm the findings of previous biochemical studies.

Congenital mesonephric defects in male infants with mucoviscidosis.

Reports that men with mucoviscidosis were sterile and that portions of their genital tracts were atretic prompted us to investigate the genital tracts of 15 male infants with mucoviscidosis who died within the first year of life and came to necropsy. The genital tracts of all of these infants were abnormal, the abnormalities being confined to mesonephric derivatives. Hypoplastic or absent ducti efferentia, ducti epididymides, or ducti deferentia were found in all 28 specimens of epididymides, and the ducti deferentia were missing from 25 of 27 examples of spermatic cord. The seminal vesicles and the ejaculatory ducts were less frequently hypoplastic or absent. Because these abnormalities of mesonephric derivatives were present so early in life and inflammatory and obstructive changes were absent we believe that they resulted from a failure of development.

Localization of expression of the cystic fibrosis gene in human pancreatic development.

Cystic fibrosis (CF) is a disease that affects the function of specialized epithelial cells within the lungs, gut, pancreas, sweat glands, and male genital ducts. Following the cloning of the CF gene, it is possible to examine tissue-specific expression of this gene. To investigate the onset of the CF disease process, expression of the CF gene in midtrimester human fetal tissues has been analyzed by in situ hybridization using antisense RNA probes. The major site of CF gene expression at this stage of development is in the pancreas. Within the pancreas, CF mRNA is seen to be largely restricted to the epithelium of intralobular and small interlobular ducts.

Case report: cystic fibrosis and embryonal carcinoma of the testis.

The authors describe a patient with cystic fibrosis and a stage III testicular embryonal cell cancer. Because cystic fibrosis occurs in approximately 1 of 2,500 births and embryonal carcinoma in 3 of 100,000, the likelihood of concurrence for both disorders in the same patient is approximately 1 in 80 million. Involvement of the vas deferens in cystic fibrosis raises the possibility of a fundamental embryologic basis that explains the pathogenesis of this association.

Jejunoileal atresia and associated malformations: correlation with the timing of in utero insult.

PURPOSE: Duodenal atresia is associated with a higher incidence of associated congenital malformations than jejunoileal atresia, supporting the hypothesis that the duodenal obstruction occurs early in fetal life. In this study, the authors analyzed the incidence of major associated malformations in jejunal atresia (JA) and ileal atresia (IA) to determine if there is a positive correlation between the proximity of the intestinal atresia and the association of other major anomalies. METHODS: Records of all patients with jejunoileal atresias treated at the authors' institution between 1980 and 1997 were examined. RESULTS: There were 83 patients with jejunoileal atresias, 38 with JA, and 45 with IA. Sixteen (42%) of the JA patients had an associated major congenital malformation, whereas only 1 (2%) of the IA patients had an associated malformation. A single atresia was found in 18 (47%) of JA patients and 41 (91%) of IA patients. Twenty (53%) of the JA patients had either multiple or apple-peel atresia. Thirteen patients (16%) died, 11 with JA, and 2 with IA. Of the 11 patients with JA who died, 6 had multiple atresias, 4 had cystic fibrosis, and 1 had small bowel volvulus. CONCLUSION: The higher incidence of associated major congenital extraintestinal malformations in JA compared with IA patients suggests that some cases of JA may arise from a malformative process.

[Prenatal molecular diagnosis of cystic fibrosis. Report of 3 cases]. / Diagnóstico molecular prenatal de fibrosis quística. Reporte de tres casos.

Cystic Fibrosis (CF) is a severe and relatively common autosomic recessive disease caused by a variety of mutations in the CFTR gene. The most frequent mutation worldwide, consists of the deletion of the phenylalanine codon at position 508 (delta F508). Here we report the first cases of prenatal diagnosis of CF by DNA analysis in couples at risk in Venezuela. The study focused on the detection of delta F508 alleles analyzing DNA recovered directly from amniocytes or from their cultures, using the polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis. Two of three fetuses resulted homozygotic for the delta F508 allele and the third one turned out to be a delta F508 carrier. This information sustained the genetic counseling of the couples and allowed them to take objective reproductive decisions, a direct consequence of the availability of gene analysis at the DNA level.

[Pathohistological changes in fetuses with cystic fibrosis].

INTRODUCTION: Cystic fibrosis or mucoviscidosis is a genetically caused disease. The intensity of disease and histopathological changes grow throughout the life. According to the literature, pathological changes characteristic of cystic fibrosis become noticeable around the sixth month of life. CASE REPORT: After amniocentesis of a 5-lunar month-old fetus had been done, which confirmed cystic fibrosis, the Ethics Commission approved the preterm labor. The autopsy and histopathological analysis demonstrated the existence of typical histopathological changes in the pancreas and intestines. DISCUSSION: In the late fetal period or during the period around the delivery, cystic fibrosis is usually manifested as meconial cap with or without obstruction of the intestinal lumen. Morphological changes in the exocrine glands usually develop only after birth. In this case, the existence of meconial obstruction, as well as the typical acidofil content in the secretory ducts and acini of the pancreas was confirmed, which is unusual for the fetal age of five months.

Cystic fibrosis transmembrane conductance regulator protein expression in the male excretory duct system during development.

Sterility due to bilateral destruction in utero or in early infancy resulting in congenital absence of the vas deferens is the rule in male patients with cystic fibrosis. To understand the developmental pattern of this anomaly, the microscopic morphology of the male excretory system was analyzed during development and the expression of the cystic fibrosis transmembrane conductance regulator protein was explored by immunohistochemistry. We observed that cystic fibrosis fetuses had no excretory ducts agenesis or obstruction until 22 weeks of gestation. However, a focal inflammatory pattern and mucinous plugs in the oldest cystic fibrosis case suggested a disruptive mechanism. Immunolabeling of cytoplasmic epithelial cystic fibrosis transmembrane conductance regulator protein was demonstrated in all cystic fibrosis and control cases with a similar pattern of expression of the protein between age-matched controls and cystic fibrosis cases. At midgestation, an apical intensification appeared in both cystic fibrosis and control cases and was stable during the remainder of fetal life. No gradient of intensity could be detected between the different segments of the excretory tract. These findings are different from those reported in adults. The absence of any morphologic anomaly until 22 weeks of gestation, the focal destruction of the epithelial structures during the second trimester, and the chronological pattern of expression of cystic fibrosis transmembrane conductance regulator are of interest for a better understanding of the pathophysiology of this disease.

Preimplantation genetic diagnosis for cystic fibrosis: a case report / Diagnóstico genético pré-implantacional na fibrose cística: relato de caso

Cystic fibrosis is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator gene. This disorder produces a variable phenotype including lung disease, pancreatic insufficiency, and meconium ileus plus bilateral agenesis of the vas deferens causing obstructive azoospermia and male infertility. Preimplantation genetic diagnosis is an alternative that allows identification of embryos affected by this or other genetic diseases. We report a case of couple with cystic fibrosis; the woman had the I148 T mutation and the man had the Delta F508 gene mutation. The couple underwent in vitro fertilization, associated with preimplantation genetic diagnosis, and with subsequent selection of healthy embryos for uterine transfer. The result was an uneventful pregnancy and delivery of a healthy male baby.

A fibrose cística é uma doença autossômica recessiva causada por mutações no gene regulador de condutância transmembrana na fibrose cística. Produz fenótipo variável, incluindo doença pulmonar, insuficiência pancreática, íleo meconial, além de agenesia bilateral dos ductos deferentes, causando azoospermia obstrutiva e infertilidade masculina. O diagnóstico genético pré-implantacional é uma alternativa diagnóstica, que permite identificar embriões portadores de fibrose cística e outras doenças genéticas. Relatamos o caso de um casal portador de fibrose cística, sendo a mulher portadora da mutação I148 T e o homem da mutação gênica Delta F508. O casal foi submetido a técnicas de fertilização in vitro associadas ao diagnóstico genético pré-implantacional, com consequente seleção de embriões saudáveis, que foram transferidos para o útero, resultando em gravidez sem intercorrências e com feto saudável, do sexo masculino.

Notable contribution of large CFTR gene rearrangements to the diagnosis of cystic fibrosis in fetuses with bowel anomalies.

Grade III fetal bowel hyperechogenicity and/or loop dilatation observed at the second trimester of pregnancy can be due to several disease conditions, including cystic fibrosis (CF). Screening for frequent CF mutations is performed as a first step and, in certain situations, such as when a frequent CF mutation is found in the fetus, the increased risk of CF justifies an in-depth study of the second allele. To determine the contribution of large CFTR gene rearrangements in such cases, detected using a semiquantitative fluorescent multiplex PCR (QFM-PCR) assay, we collated data on 669 referrals related to suspicion of CF in fetuses from 1998 to 2009. Deletions were found in 5/70 cases in which QFM-PCR was applied, dele19, dele22_23, dele2_6b, dele14b_15 and dele6a_6b, of which the last three remain undescribed. In 3/5 cases, hyperechogenicity was associated with dilatation and/or gallbladder anomalies. Of the total cases of CF recognized in the subgroup of first-hand referrals, deletions represent 16.7% of CF alleles. Our study thus strengthens the need to consider large CFTR gene rearrangements in the diagnosis strategy of fetal bowel anomalies, in particular in the presence of multiple anomalies.