Willy Lissens

Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens

BACKGROUND Congenital bilateral absence of the vas deferens (CBAVD) is a form of male infertility in which mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been identified. The molecular basis of CBAVD is not completely understood. Although patients with cystic fibrosis have mutations in both copies of the CFTR gene, most patients with CBAVD have mutations in only one copy of the gene. METHODS To investigate CBAVD at the molecular level, we have characterized the mutations in the CFTR gene in 102 patients with this condition. None had clinical manifestations of cystic fibrosis. We also analyzed a DNA variant (the 5T allele) in a noncoding region of CFTR that causes reduced levels of the normal CFTR protein. Parents of patients with cystic fibrosis, patients with types of infertility other than CBAVD, and normal subjects were studied as controls. RESULTS Nineteen of the 102 patients with CBAVD had mutations in both copies of the CFTR gene, and none of them had the 5T allele. Fifty-four patients had a mutation in one copy of CFTR, and 34 of them (63 percent) had the 5T allele in the other CFTR gene. In 29 patients no CFTR mutations were found, but 7 of them (24 percent) had the 5T allele. In contrast, the frequency of this allele in the general population was about 5 percent. CONCLUSIONS Most patients with CBAVD have mutations in the CFTR gene. The combination of the 5T allele in one copy of the CFTR gene with a cystic fibrosis mutation in the other copy is the most common cause of CBAVD: The 5T allele mutation has a wide range of clinical presentations, occurring in patients with CBAVD or moderate forms of cystic fibrosis and in fertile men.

Microsurgical epididymal sperm aspiration and intracytoplasmic sperm injection : a new effective approach to infertility as a result of congenital bilateral absence of the vas deferens

OBJECTIVE To present and assess the efficacy of a new approach for the treatment of infertility due to congenital bilateral absence of the vas deferens. DESIGN A retrospective study of consecutive trials. SETTING Centre for Reproductive Medicine, which is a tertiary referral institution. PATIENTS Twelve couples suffering from infertility because of congenital bilateral absence of the vas deferens. INTERVENTIONS A microsurgical epididymal sperm aspiration procedure was performed in the husbands, followed by intracytoplasmic sperm injection of oocytes recovered from the wives. Cleaving embryos were transferred to the uterine cavity 48 hours after the intracytoplasmic sperm injection procedure. MAIN OUTCOME MEASURES Sperm parameters after microsurgical epididymal sperm aspiration, fertilization, cleavage, and pregnancy rates. RESULTS In all 14 microsurgical epididymal sperm aspiration procedures, sperm was retrieved. Notwithstanding the poor quality of this epididymal sperm, a fertilization rate of 58% was achieved after intracytoplasmic sperm injection. On 10 occasions, embryos were transferred and five patients became pregnant, i.e., an overall pregnancy rate of 35.7% per started trial and 50.0% per transfer. Another two patients became pregnant after replacement of frozen-thawed embryos, which increases the pregnancy rate to 50.0% per microsurgical epididymal sperm aspiration procedure. Early pregnancy wastage was 57%, limiting the ongoing pregnancy rate to 21.4% per microsurgical epididymal sperm aspiration procedure. CONCLUSION This study shows the combined microsurgical epididymal sperm aspiration-intracytoplasmic sperm injection procedure to be highly efficient in achieving fertilization in vitro, even after recovery of grossly impaired epididymal sperm.

Mutations in the gene encoding the latency-associated peptide of TGF-β1 cause Camurati-Engelmann disease

Camurati-Engelmann disease (CED; MIM 131300), or progressive diaphyseal dysplasia, is a rare, sclerosing bone dysplasia inherited in an autosomal dominant manner. Recently, the gene causing CED has been assigned to the chromosomal region 19q13 (refs 1–3). Because this region contains the gene encoding transforming growth factor-β1 (TGFB1), an important mediator of bone remodelling, we evaluated TGFB1 as a candidate gene for causing CED.

Mutations in the X-linked pyruvate dehydrogenase (E1) α subunit gene (PDHA1) in patients with a pyruvate dehydrogenase complex deficiency

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) α subunit gene (PDHA1). The major factors that contribute to the clinical variation in E1α 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.

Respiratory chain complex V deficiency due to a mutation in the assembly gene ATP12

In patients with mitochondrial encephalomyopathies an increasing number of causative gene defects have been detected. The number of identified pathogenic mitochondrial DNA mutations has largely increased over the past 15 years. Recently, much attention has turned to the investigation of nuclear oxidative phosphorylation (OXPHOS) gene defects. Within the OXPHOS defects, complex V deficiency is rarely found and, so far, these defects have only been attributed to mutations in the mitochondrial MTATP6 gene. Mutation analysis of the complete coding regions at the cDNA level of the nuclear ATP11, ATP12, ATPα, ATPβ and ATPγ genes and the mitochondrial MTATP6 and MTAT8 genes was undertaken in two unrelated patients. Blue Native polyacrylamide gel electrophoresis followed by catalytic staining had already documented their complex V decreased activity. Extensive molecular analysis of five nuclear and two mitochondrial genes revealed a mutation in the ATP12 assembly gene in one patient. This mutation is believed to be the cause of the impaired complex V activity. To our knowledge, this is the first report of a pathogenic mutation in a human nuclear encoded ATPase assembly gene.

Embryo implantation after biopsy of one or two cells from cleavage-stage embryos with a view to preimplantation genetic diagnosis

Preimplantation genetic diagnosis (PGD) can be offered as an alternative to prenatal diagnosis (PND) to couples at risk of having a child with a genetic disease. The affected embryos are detected before implantation by fluorescent in situ hybridisation (FISH) for sexing (X‐linked diseases) and chromosomal disorders (numerical and structural) or by polymerase chain reaction (PCR) for monogenic disorders (including some X‐linked diseases). The accuracy and reliability of the diagnosis is increased by analysing two blastomeres of the embryo. However, the removal of two blastomeres might have an effect on the implantation capacity of the embryo. We have evaluated the implantation of embryos after the removal of one, two or three cells in 188 PGD cycles where a transfer was done. The patients were divided into five groups: a first group which received only embryos from which one cell had been removed, a second group which received only embryos from which two cells had been removed, a third group which received a mixture of embryos from which one and two cells had been taken, a fourth group where two and three cells had been removed, and a fifth group where three cells had been removed. The overall ongoing pregnancy rate per transfer was 26.1%, the overall implantation rate per transfer was 15.2% and the overall birth rate was 14.2%. Although pregnancy rates between the groups cannot be compared because the second group (two cells removed) contains more rapidly developing and therefore ‘better quality’ embryos, an ongoing pregnancy rate of 29.1% and an implantation rate of 18.6% per transferred embryo in this group is acceptable, and we therefore advise analysing two cells from a ≥7‐cell stage embryo in order to render the diagnosis more accurate and reliable. Copyright

Genetic causes of spermatogenic failure

Approximately 10%-15% of couples are infertile, and a male factor is involved in almost half of these cases. This observation is due in part to defects in spermatogenesis, and the underlying causes, including genetic abnormalities, remain largely unknown. Until recently, the only genetic tests used in the diagnosis of male infertility were aimed at detecting the presence of microdeletions of the long arm of the Y chromosome and/or chromosomal abnormalities. Various other single-gene or polygenic defects have been proposed to be involved in male fertility. However, their causative effects often remain unproven. The recent evolution in the development of whole-genome-based techniques and the large-scale analysis of mouse models might help in this process. Through knockout mouse models, at least 388 genes have been shown to be associated with spermatogenesis in mice. However, problems often arise when translating this information from mice to humans.

Bilateral striatal necrosis with a novel point mutation in the mitochondrial ATPase 6 gene

A 2.5-year-old boy with bilateral striatal lesions is reported. Using polymerase chain reaction-single-strand conformation polymorphism analysis and direct DNA sequencing, a novel point mutation (T to C) at nucleotide 8851 of the mitochondrial DNA (mtDNA) was identified. This mutation changes a highly conserved tryptophan to arginine in subunit 6 of the mtATPase gene. The mutation was nearly homoplasmic and maternally inherited. This is the first published report of a mutation in the mtDNA in bilateral striatal degeneration. It is possible that other cases of bilateral striatal degeneration have been caused by mutations in the mtATPase 6 gene or genes encoding other subunits of the mtATPase; and therefore the mtATPase genes should be examined in children with this condition.

Possible role of USP26 in patients with severely impaired spermatogenesis

The ubiquitin-specific protease 26 (USP26) gene is an X-linked gene specifically expressed in testis tissue. This gene is therefore a potential infertility gene. In order to analyse its possible involvement in spermatogenesis and infertility, 42 patients with Sertoli cell-only syndrome were analysed for mutations in this gene. We found four patients with exactly the same three changes of the nucleotide and therefore also amino acid sequence. These patients showed 370-371insACA, 494T>C and 1423C>T causing T123-124ins, L165S and H475Y, respectively. These changes were not found in 10 control samples. Furthermore, two polymorphisms were observed which do not alter the amino-acid sequence. A restriction analysis that can make a distinction between a T and a C, at position 494, was set up in order to examine more patient and control samples. Another 69 patients with Sertoli cell-only syndrome, 32 patients with maturation arrest and 142 control samples were analysed using this method. None of the control samples or patients with maturation arrest featured the change at position 494. However, four more patients with Sertoli cell-only syndrome were identified with the three alterations. The frequency of alterations in this group is therefore 7.2% (8/111). One of the patients had a deletion of the long arm of the Y chromosome, while another patient had a varicocoele. These results indicate that these alterations might be involved in male infertility or might increase the risk of male infertility.

Evaluation of parental mitochondrial inheritance in neonates born after intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is now used when severe male-factor infertility has been documented. Since defective mitochondrial functions may result in male hypofertility, it is of prime importance to evaluate the risk of paternal transmission of an mtDNA defect to neonates. DNA samples from the blood of 21 infertile couples and their 27 neonates born after ICSI were studied. The highly polymorphic mtDNA D-loop region was analyzed by four PCR-based approaches. With denaturing gradient gel electrophoresis (DGGE), which allows 2% of a minor mtDNA species to be detected, the 27 newborns had a DGGE pattern identical to that of their mother but different from that of their father. Heteroplasmy documented in several parents and children supported an exclusive maternal inheritance of mtDNA. The parental origin of the childrens mtDNA molecules also was studied by more-sensitive assays: restriction-endonuclease analysis (REA) of alpha[32P]-radiolabeled PCR products; paternal-specific PCR assay; and depletion of maternal mtDNA, followed by REA. We did not detect paternal mtDNA in nine neonates, with a sensitivity level of 0.01% in five children, 0.1% in two children, and 1% in two children. The estimated ratio of sperm-to-oocyte mtDNA molecules in humans is 0.1%-1.5%. Thus, we conclude that, in these families, the ICSI procedure performed with mature spermatozoa did not alter the uniparental pattern of inheritance of mtDNA.