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  • 1
    ISSN: 1432-1203
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Deficiency of β-glucuronidase is the cause of the human lysosomal storage disorder mucopolysaccharidosis type VII (MPS VII). The wide interfamilial variation in the presentation of this disorder complicates clinical diagnosis. Since greatly reduced β-glucuronidase enzyme activity may also be found in healthy individuals (pseudodeficiency), diagnosis based on the biochemical phenotype is also difficult. This is illustrated by the patients studied here, who had extremely mild symptoms confined to the spine, or tachycardia, or upper respiratory infection, and who had low β-glucuronidase activity, and excessive granulation of granulocytes and monocytes on routine blood smears. Low enzyme activity was caused by mutations in the β-glucuronidase gene in all cases. One patient was homozygous for the previously described D152N allele. Family information and 35SO4-uptake studies clearly demonstrated that he was pseudodeficient, with symptoms unrelated to his low β-glucuronidase activity. Two patients of another family were compound heterozygotes for a C38G and a Y626H allele, and were probably extremely mild MPS VII patients. The low β-glucuronidase activity in another mild MPS VII patient was due to reduced biosynthesis of stable mRNA from one allele, and a W446X mutation on the second. Extremely low β-glucuronidase enzyme activity was also found in the serum of a carrier of a 1801ΔT allele, possibly as a consequence of a dominant-negative effect. A combination of investigations is necessary in order to differentiate between mild disease and pseudodeficiency in individuals with enzyme activities close to the threshold.
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  • 2
    ISSN: 1432-1203
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Osteogenesis imperfecta (OI) is an autosomal dominant genetic disorder characterized by the presence of brittle bones and decreased bone mass (osteopenia), as a result of mutations in the genes that encode the chains of type I collagen, the major protein of bone. The clinical features of the disease range from death in the perinatal period to normal life span with minimal increase in fractures. The present report describes two polymerase chain reaction (PCR)-based assays allowing preimplantation genetic diagnosis (PGD) on the one hand for OI type I, the mildest form, and on the other hand for OI type IV, which is intermediate in severity between OI type I and OI type III. In the couple referred for PGD for OI type I, the female partner carried a 1-bp deletion in exon 43 of the COL1A1 gene, resulting in a premature stop codon in exon 46. The synthesis of too little type I procollagen results from such a non-functional or COL1A1 null allele. In the other couple, referred for PGD for OI type IV, the male partner carried a G to A substitution in exon 19 of the COL1A2 gene, which results in an abnormal gene product due to an αGly247 (GGT) to Ser (AGT) substitution (G247S). Both mutations result in the loss of a specific restriction enzyme recognition site and can therefore be detected by PCR amplification followed by restriction fragment analysis. PCR amplification of genomic DNA of the parents-to-be with one of the two primers fluorescently labelled, followed by automated laser fluorescence (ALF) gel electrophoresis of the amplified and restricted fragments, allowed a distinction between the healthy and affected genotypes. PCR on single Epstein-Barr-virus (EBV)-transformed lymphoblasts resulted in acceptable amplification efficiencies (87% and 85% for OI type I and OI type IV respectively) and the allele drop-out (ADO) rate was assessed at 11.5% and 11.1% for OI type I and OI type IV respectively. With research blastomeres, 100% amplification rates were obtained and no contamination was observed in the blank controls, which validated the tests for clinical application. Embryos obtained after intracytoplasmic sperm injection (ICSI) were evaluated for the presence of the normal genotype of the non-affected parent. For OI type I, two frozen-thawed ICSI-PGD cycles and two fresh ICSI-PGD cycles were carried out for the same couple. The transfer of two unaffected embryos in the last cycle resulted in a twin pregnancy. A twin pregnancy was also achieved in one clinical ICSI-PGD cycle for OI type IV.
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  • 3
    ISSN: 1432-1203
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The cystic fibrosis (CF) gene deletion F508 was studied in a Belgian population of 74 families and their 83 CF children. The haplotypes for CF and normal chromosomes had previously been determined with several linked DNA probes. In our CF population, the gene deletion F508 was found in 76% of the mutant alleles. Of the deletion F508, 97% segregated with the highest risk haplotype for the CF carrier status. Some 61% of our families were found to be homozygous for this major CF mutation. Each of our three pancreatic sufficiency patients (two of whom were siblings) was heterozygote for the F508 deletion.
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  • 4
    ISSN: 1432-1203
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary We report the molecular characterization of a case of a functional PDH-El (E1 subunit of pyruvate dehydrogenase) deficiency, a cause of severe congenital lactic acidosis. Residual PDH-El activity was reduced to 10% of normal values, although the subunit appeared to be quantitatively and qualitatively normal at the protein level as determined by Western blotting. The sequence of PDH-Elα mRNA and the corresponding genomic DNA revealed an in-frame 21-bp insertion between codons 305 and 306 of the normal Ela cDNA. The mutational insert commences with a novel GAT codon and is a nearly perfect tandem duplication of the wild type DNA sequence. A serine phosphorylation site regulating the activity of the PDH complex is altered by this insertion, which in all likelihood is responsible for the functional enzymatic deficiency leading to lactic acidosis.
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  • 5
    ISSN: 1432-1203
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract We used polymerase chain reaction (PCR)/single-strand conformation polymorphism analysis and direct sequencing of the coding region of the β-glucuronidase cDNA and gene to detect mutations causing β-glucuronidase enzyme deficiency in five MPS VII patients. Four patients presented with hydrops fetalis, one with an early infantile form of the disease. Genetic heterogeneity of MPS VII alleles was further confirmed in this study. Recurrent mutations were observed in patients of related origin. Previously unknown alleles detected were R110X, F361Δ9, 1270 + 1G→A, S52F and 1480Δ4. Reverse transcription/PCR analysis of the 1270 + 1G→A messenger showed aberrant splicing: inclusion of intron 7 or skipping of exons 6–7 and 9. Messenger RNA transcribed from the R110X and 1480Δ4 alleles was unstable. We detected a 2154A/G change in the 3′ non-coding region of the gene, in the neighbourhood of the two consensus polyadenylation sites. 3′-Rapid amplification of cDNA ends/ PCR of fibroblast cDNA revealed equal usage of two alternative polyadenylation sites. The 2154A/G substitution did not influence adenylation-site choice, nor the amount of stable messenger produced. The finding that 2 out of 30 normal controls carried the 2154G allele indicated that the 2154A/G substitution is a harmless polymorphism. The S52F and F361Δ9 cDNAs were constructed in vitro and used to transfect COS cells transiently. Both mutations completely abolished enzyme activity.
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  • 6
    ISSN: 1432-1203
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract We have previously sequenced the complete coding region and the promoter region of the β-glucuronidase gene of a patient with mild mucopolysaccharidosis type VII (MPS VII) and identified a nonsense mutation in the gene inherited from her mother. The mutation inherited from her father was not found. Here, we have extended the sequence analysis of the introns to cover all putative lariat branch points and putative intronic enhancers, although no nucleotide changes have been found in these regions. Careful analysis of mRNA structure by reverse transcription/polymerase chain reaction (RT-PCR) and direct sequencing has revealed the inclusion of a new exon derived from an antisense Alu-repeat in intron 8 and the skipping of exon 9 in a large proportion of the mRNA of our patient. A 2-bp deletion creating a strong 5’-splice site has subsequently been identified in the paternal gene of the patient (IVS8+0.6kbdelTC). With a sensitive RT-PCR assay, we demonstrate that both the inclusion of the Alu-cassette and the skipping of exon 9 are minor events in control samples and that mRNA with both alterations is only found in the IVS8+0.6kbdelTC carrier. The increased proportion of exon 9 skipping seems to be related to the premature termination of translation. This is the third report of a human disease mutation that creates a splice site and activates an antisense Alu-cassette; the question rises as to how these apparently strong cryptic exons are generally excluded from coding sequences.
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  • 7
    ISSN: 1573-7330
    Keywords: congenital anomalies ; preimplantation genetic diagnosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Background: Preimplantation genetic diagnosis is an exciting advance in prenatal diagnosis. However, the safety of embryo biopsy must be determined with respect to both pregnancy rate and cogenital anomalies. Analysis: Too few pregnancies have been reported to allow meaningful inferences to be drawn, for which reason data on pregnancy losses and anomalies after conventional IVF were first reviewed. Loss rates are approximately 25%, and anomaly rates are not increased over that observed in the general population. Unfortunately, considerable methodological problems exist in published surveys: lack of proper controls, failure to take into account potential confounding variables, anomaly surveillance that is inconsistent with respect to the vigor with which anomalies are sought, inclusion or exclusion of minor anomalies, inclusion or exclusion of anomalies evident only on ultrasound, and even inclusion or exclusion of anomalies present in terminated pregnancies. We recommend prospective surveillance for major anomalies, defined as those causing death, major handicap or requiring surgery. Prospective surveillance ideally dictates collection of intake information at the time pregnancy is diagnosed, surveillance during pregnancy to exclude teratogenic influences, and systematic neonatal anomaly surveillance.
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