Search

Fragile X syndrome is the most common monogenic cause of mental retardation in boys. It is always characterized clinically by moderate mental retardation and often by a long face with large everted ears and macro-orchidism. The causal mutation is an expansion of a CGG triplet repeat in a 5' exon of the FMR-1 gene in Xq27.3. We report here for the first time a method for preimplantation genetic diagnosis (PGD) for fragile X syndrome based on the amplification of the CGG triplet in the normal allele. Our candidate-patient population, as well as two clinical preimplantation genetic diagnosis (PGD) cycles which led to a pregnancy with an unaffected fetus, are presented in this paper.

Defects in the pyruvate dehydrogenase (PDH) complex are an important cause of primary lactic acidosis, a frequent manifestation of metabolic disease in children. Clinical symptoms can vary considerably in patients with PDH complex deficiencies, and almost equal numbers of affected males and females have been identified, suggesting an autosomal recessive mode of inheritance of the disease. However, the great majority of PDH complex deficiencies result from mutations in the X-linked pyruvate dehydrogenase (E1) alpha subunit gene (PDHA1). The major factors that contribute to the clinical variation in E1alpha deficiency and its resemblance to a recessive disease are developmental lethality in some males with severe mutations and the pattern of X-inactivation in females. To date, 37 different missense/nonsense and 39 different insertion/deletion mutations have been identified in the E1alpha subunit gene of 130 patients (61 females and 69 males) from 123 unrelated families. Insertion/deletion mutations occur preferentially in exons 10 and 11, while missense/nonsense mutations are found in all exons. In males, the majority of missense/nonsense mutations are found in exons 3, 7, 8 and 11, and three recurrent mutations at codons R72, R263 and R378 account for half of these patients with missense/nonsense mutations (25 of 50). A significantly lower number of females is found with missense/nonsense mutations (25). However, 36 females out of 55 affected patients have insertion/deletion mutations. The total number of female and male patients is thus almost the same, although a difference in the distribution of the type of mutations is evident between both sexes. In many families, the parents of the affected patients were studied for the presence of the PDHA1 mutation. The mutation was never present in the somatic cells of the father; in 63 mothers studied, 16 were carriers (25%). In four families, the origin of the new mutation was determined to be twice paternal and twice maternal.

It has previously been shown that defects in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are largely responsible for the condition of congenital bilateral absence of the vas deferens (CBAVD), without associated renal abnormalities, in Caucasian populations. To assess the involvement of the CFTR in CBAVD in a population with presumed low cystic fibrosis (CF) frequency, we have analysed 20 CBAVD males from Egypt for the presence of 12 common Caucasian CFTR mutations and the intron 8 5T splice variant, IVS-5T, known to be a major cause of CBAVD in Caucasian patients. In 16 of the males without associated renal abnormalities only one deltaF508 carrier was identified, but an exceptionally high frequency of the IVS-5T variant was found (14 of 32 alleles or 43.7%), confirming that this variant is involved in many cases of CBAVD, even in populations where CF is rare. CFTR mutations or the IVS-5T variant were found neither in the remaining four patients with associated renal abnormalities nor in the spouses of the 20 CBAVD patients. However, one patient was homozygous for a leucine to proline substitution at amino acid position 541 (L541P) of the CFTR. It is as yet not clear whether this change is involved in CBAVD in this male.

Objectives Mutations in the APC, NF2 and BRCA1 genes cause adult-onset cancer predisposition syndromes. Prenatal diagnosis (PND) and selective pregnancy termination for adult-onset disorders is emotionally difficult and, in some cases, socially not well accepted. Preimplantation genetic diagnosis (PGD) appears as an attractive alternative to PND, as it ensures the establishment of a pregnancy free of the mutation from the onset, circumventing the potentially difficult decision of termination of pregnancy. Methods Development of single-cell PCRs using Epstein-Barr virus transformed lymphoblasts as single-cell model, followed by clinical application in PGD. Results A total of five duplex-PCRs were developed, three for adenomatous polyposis of the colon (APC), one for neurofibromatosis type 2 (NF2) and one for inherited breast and ovarian cancer caused by BRCA1 mutations. Eleven clinical cycles were performed, resulting in the birth of an unaffected girl. For one of the couples undergoing PGD for NF2, a spontaneous pregnancy ensued after five unsuccessful PGD cycles. The couple underwent chorionic villus sampling (CVS) and the application of the same protocol as used during PGD showed an unaffected fetus. Conclusion In this work, we present the development and clinical application of PGD for three cancer predisposition syndromes. Copyright © 2007 John Wiley & Sons, Ltd.

Human embryonic stem cells (hESC) are considered to be an indefinite source of self-renewing cells that can differentiate into all types of cells of the human body and could be used in regenerative medicine, drug discovery and as a model for studying early developmental biology. hESC carrying disease-causing mutations hold promise as a tool to investigate mechanisms involved in the pathogenesis of the disease. In this report, we describe the behaviour of an expanded CTG repeat in the 3 0 untranslated region of the DMPK gene in VUB03_DM1, a hESC line carrying the myotonic dystrophy type 1 (DM1) mutation compared with the normal CTG repeat in two hESC lines VUB01 and VUB04_CF. Expanded CTG repeats were detected by small amount PCR, small pool PCR and Southern blot analysis in consecutive passages of VUB03_DM1. An important instability of the CTG repeat was detected during prolonged in vitro culture, showing stepwise increases of the repeat number in consecutive passages as well as a higher range of variability. This variability was present in cells of different colonies of the same passage and even within single colonies. The high repeat instability is in contrast to the previously observed stability of the repeat in preimplantation embryos and in fetuses during the first trimester of pregnancy. This in vitro culture of affected hESC represents a valuable model for studying the biology of repeat instability.

Huntington disease (HD) is an autosomal dominant, lethal neurodegenerative disorder of the central nervous system, caused by an uncontrolled expansion of a CAG dynamic mutation in the coding region of the IT15gene. Although a majority of patients have a midlife onset of the disease, in a small number of patients the disease manifests before 20 years of age. In adults, HD is mainly characterised by involuntary movements, personality changes and dementia. By contrast, in children a dominant picture of bradykinesia, rigidity, dystonia and epileptic seizures is noticed. The earlier onset is often associated with a paternal transmission of the disease allele to the offspring. We report here a rather unusual infantile onset of the disease in a little girl who presented with a history of seizures and psychomotor regression starting at the age of 3 years. A progressive cortical-subcortical atrophy, progressive cerebellar atrophy and lesions in the basal ganglia were found on MRI. An important expansion, of 214 triplet numbers, of the CAG repeat size associated with HD, was observed. Conclusion:Juvenile Huntingdon disease should be considered in children suffering from a progressive neurodegenerative disease.

Intracytoplasmic sperm injection (ICSI) involves the insertion of a single spermatozoon into the oocyte, bypassing all the egg coat penetration and gamete fusion steps characteristic of natural fertilization. This was first achieved in the sea urchin, in the mouse, and later in hamster eggs. This micromanipulation approach was also plagued by oocyte injury and lysis, with only about 30% of injected mouse eggs surviving the procedure. Because the sperm-egg fusion step is bypassed in ICSI, male pronucleus development generally required oocyte activation in most species tested. This was achieved by the vigorous suction of ooplasm prior to sperm nucleus insertion or by exposure to A23187. The first ICSI offspring were obtained in the rabbit following the transfer of sperm-injected eggs into the oviduct of a pseudopregnant female, with a bovine live birth reported soon thereafter. Although applied to human gametes some years earlier, the first human pregnancies from ICSI was established only in 1992, and since then, hundreds of thousands of ICSI babies have been born. ICSI has made conception and parenthood possible for couples with many forms of male factor infertility and at rates similar to those in patients treated by standard IVF with apparently normal gametes.

The clinical phenotype of cystic fibrosis (CF) patients is very variable and it has been suggested that patients lacking the cystic fibrosis transmembrane conductance regularor(CFTR) have milder lung disease than those having an altered CFTR. However, on the basis of the large variation in lung function in patients homozygous for the most common CF mutation, deltaF508, and W1282X homozygotes, it was concluded that most CF patients have a common phenotype, but that other genetic and environmental factors may be important for the clinical phenotype. We describe a patient, homozygous for a frameshift mutation in the regulatory (R)domain of the CFTR, who presented with mild lung disease but severe hepatic and pancreatic involvement. The mutation, 2184delA (deletion of A at position 2184 together with an A to G substitution atposition 2183 in exon 13) was originally characterised by D Bozon and L-C Tsui (personal communication) and was found in both parents of our patient in a screening programme of non-deltaF508 CF chromosomes with denaturing gradient gel electrophoresis, followed by sequencing. The boy was born at term in December 1977, birth weight 3500g, to healthy, nonconsanguineous parents. Cystic fibrosis presented neonatally with meconium ileus which was treated surgically. CF was confirmed by positive pilocarpine iontophoresis sweat test at 10 days. Conventional treatment for CF was given. The clinical course of the lung disease was mild. At the age of 5 years a nasal polypectomy was performed. Liver function tests altered from this age onwards. Hepatomegaly was observed two years later. He was admitted to hospital at the age of 11,5 years for intravenous antibiotic treatment because of pulmonary infection. Pseudomonas aeruginosa was isolated from sputum cultures soon after this, but not repeatedly. At 13,5 years he was asymptomatic with discrete clubbing, hepatomegaly of 4 cm, and pubertal state A1P1G2. Weight and height were between the 10th and 25th centiles. Respiratory function tests for FVC, FEV, and PEFR were 89%, 83%, and 76% of predicted, respectively. Schachman score was good (80/100) as was the Chrispin-Norman score at 6/38. ALT (71 IU/l, normal

BACKGROUND: The presence of chromosomal abnormalities could have a negative impact for human embryonic stem cell (hESC) applications both in regenerative medicine and in research. A biomarker that allows the identification of chromosomal abnormalities induced in hESC in culture before they take over the culture would represent an important tool for defining optimal culture conditions for hESC. Here we investigate the expression of CD30, reported to be a biomarker of hESCs with abnormal karyotype, in undifferentiated and spontaneously differentiated hESC. METHODS AND RESULTS: hESC were derived and cultured on mouse fibroblasts in KO-SR containing medium (serum free media) and passaged mechanically. Our results based on analysis at mRNA (RT-PCR) and protein (fluorescence-activated cell sorting and immunocytochemistry) level show that CD30 is expressed in undifferentiated hESC, even at very early passages, without any correlation with the presence of chromosomal anomalies. We also show that the expression of CD30 is rapidly lost during early spontaneous differentiation of hESC. CONCLUSION: We conclude that CD30 expression in hESC cultures is probably a consequence of culture conditions, and that KO-SR may play a role. In addition, the expression of so-called 'stemness' markers does not change in undifferentiated hESC during long-term culture or when cells acquire chromosomal abnormalities.

Since the birth of Louise Brown in July 1978 and the birth of the first intracytoplasmic sperm injection (ICSI) child in January 1992 many couples with longstanding female-factor or male-factor infertility can be helped to overcome their infertility resulting in a delivery and birth of a child. The final and ultimate goal of all infertility treatments has been to give the large population of infertile couples a chance to fulfil their childwish and experience the happiness of having a healthy child. Major advances have been made in the different treatment protocols for infertility during the last 25 years. It is, however, surprising that only a limited number of studies have been carried out assessing the health of the children born after ART. In this review we shall comment on the limitations of follow-up studies on ART children and we shall review existing data on the outcome of in vitro fertilization (IVF) and ICSI pregnancies. The most important outcome data consist of information on minor and major congenital malformations obtained prenatally or after birth, as well as on the further development of the children.

Background Human embryonic stem cells (hESC) have the capacity to differentiate in vivo and in vitro into cells from all three germ lineages. The aim of the present study was to investigate the effect of specific culture conditions on the differentiation of hESC into lung epithelial cells. Methods Undifferentiated hESC, grown on a porous membrane in hESC medium for four days, were switched to a differentiation medium for four days; this was followed by culture in air-liquid interface conditions during another 20 days. Expression of several lung markers was measured by immunohistochemistry and by quantitative real-time RT-PCR at four different time points throughout the differentiation and compared to appropriate controls. Results Expression of CC16 and NKX2.1 showed a 1,000- and 10,000- fold increase at day 10 of differentiation. Other lung markers such as SP-C and Aquaporin 5 had the highest expression after twenty days of culture, as well as two markers for ciliated cells, FOXJ1 and β-tubulin IV. The results from qRT-PCR were confirmed by immunohistochemistry on paraffin-embedded samples. Antibodies against CC16, SP-A and SP-C were chosen as specific markers for Clara Cells and alveolar type II cells. The functionality was tested by measuring the secretion of CC16 in the medium using an enzyme immunoassay. Conclusion These results suggest that by using our novel culture protocol hESC can be differentiated into the major cell types of lung epithelial tissue.

Huntington's disease is an autosomal dominant, late-onset disorder, for which the gene and the causative mutation have been known since 1993. Some at-risk patients choose for presymptomatic testing and can make reproductive choices accordingly. Others however, prefer not to know their carrier status, but may still wish to prevent the birth of a carrier child. For these patients, exclusion testing after prenatal sampling has been an option for many years. A disadvantage of this test is that unaffected pregnancies may be terminated if the parent at risk (50%) has not inherited the grandparental Huntington gene, leading to serious moral and ethical objections. As an alternative, preimplantation genetic diagnosis (PGD) on embryos obtained in vitro may be proposed, after which only embryos free of risk are replaced. Embryos can then be selected, either by the amplification of the CAG repeat in the embryos without communicating results to the patients (ie non-disclosure testing), which brings its own practical and moral problems, or exclusion testing. We describe here the first PGD cycles for exclusion testing for Huntington's disease in five couples. Three couples have had at least one PGD cycle so far. One pregnancy ensued and a healthy female baby was delivered.

BACKGROUND: Mucopolysaccharidosis type IIID (MPS-IIID), or Sanfilippo syndrome type D, is a rare autosomal recessive lysosomal storage disorder caused by mutations in the N-acetylglucosamine-6-sulfatase (GNS) gene, leading to impaired degradation of heparan sulfate. OBJECTIVES: To report the natural history of MPS-IIID in 2 siblings described by Kaplan and Wolfe in 1987 and to study the phenotype in 2 other unrelated families with MPS-IIID. Design, Setting, and Patients Case series of 4 patients with MPS-IIID: 2 siblings followed up at the Montreal Neurological Hospital and Institute, 1 patient followed up at the UZ Brussel, and 1 patient recruited through the prenatal counseling program at the UZ Brussel. MAIN OUTCOME MEASURES: Clinical and molecular data collected from 3 families with enzyme-based diagnosis of MPS-IIID. RESULTS: The course of the disease was characteristic of MPS-IIID in all patients, although survival may be longer than was previously reported. In family 1, both siblings were homozygous for a novel nonsense mutation in the GNS gene (c.1168C>T). In family 2, the proband carried a heterozygous mutation occurring in a splice recognition site in the intron 7 boundary (c.876-2A>G). The second mutation in this patient remains to be identified. In family 3, the proband was homozygous for a novel frameshift mutation in GNS due to the insertion of 5 nucleotides (c.1138_1139insGTCCT). CONCLUSIONS: Major issues in the care of patients with MPS-IIID include behavioral problems, sleep problems, recurrent infections, dysphagia, and pain from orthopedic complications. To date, all mutations in GNS predict protein truncation, and there is no obvious genotype-phenotype correlation.

Primer extension preamplification (PEP) was first described as a method for whole genome amplification, starting from a single cell, originally a spermatozoon, in order to perform genetic recombination studies. Its usefulness for preimplantation diagnosis was shown soon after; the only drawback was the length of the procedure (>14 h). We have developed a shorter PEP protocol for single human blastomeres, enabling us to examine several genetic loci of interest in human genetic diseases with a good amplification efficiency. © European Society for Human Reproduction and Embryology.

Myotonic dystrophy (DM), or Steinert's disease, is an autosomal dominant disease characterized by myotonia, muscular weakness and atrophy, as well as lens opacities, cardiomyopathy and mild endocrine changes. The gene for DM located on 19q contains a triplet repeat at the 3' end of the gene. In DM patients, this repeat is found to be expanded. We have previously described a preimplantation genetic diagnosis (PGD) for DM using polymerase chain reaction (PCR) followed by conventional analysis on ethidium bromide-stained gels. The major drawback of this system was that allelic dropout occurred in >20% of the cells, leading to the loss of healthy embryos for transfer. To resolve this problem, we developed a PGD for DM using fluorescent PCR followed by fragment analysis on an automated DNA sequencer and made a comparison between the conventional PCR described earlier and fluorescent PCR, which turned out to be superior in accuracy and efficiency. Three PGD cycles were performed using fluorescent PCR and are described here.

This male infant was first brought to attention in the neonatal period because he presented clinical and radiological evidence of multiple bone deformities. He was readmitted at 2 1/2 months for hydrocephaly, hepatosplenomegaly and poor somatic and psychomotor development. In addition, coarse facies, corneal opacities and stiff joints were noticed. Bone X-ray anomalies and vacuolized lymphocytes supported the clinical presumption of lysosomal storage disorder. The diagnosis of multiple sulphatase deficiency rests on the presence of MPS and sulphatides in the urine, the finding of a mixed storage process in conjunctival biopsy and the demonstration of deficiencies in arylsulphatases A, B, C, iduronate sulphatase and heparan sulphatase in serum, leukocytes and cultured fibroblasts.