Preimplantation genetic diagnosis for single gene disorders: experience with five single gene disorders

We report our experience of 14 preimplantation genetic diagnosis (PGD) cycles in eight couples carrying five different single gene disorders, during the last 18 months. Diagnoses were performed for myotonic dystrophy (DM), cystic fibrosis (CF) [ΔF508 and exon 4 (621+1 G>T)], fragile X and CF simultaneously, and two disorders for which PGD had not been previously attempted, namely neurofibromatosis type 2 (NF2) and Crouzon syndrome. Diagnoses for single gene disorders were carried out on ideally two blastomeres biopsied from Day 3 embryos. A highly polymorphic marker was included in each diagnosis to control against contamination. For the dominant disorders, where possible, linked polymorphisms provided an additional means of determining the genotype of the embryo hence reducing the risk of misdiagnosis due to allele dropout (ADO). Multiplex fluorescent polymerase chain reaction (F-PCR) was used in all cases, followed by fragment analysis and/or single-stranded conformation polymorphism (SSCP) for genotyping. Embryo transfer was performed in 13 cycles resulting in one biochemical pregnancy for CF, three normal deliveries (a twin and a singleton) and one early miscarriage for DM and a singleton for Crouzon syndrome. In each case the untransferred embryos were used to confirm the diagnoses performed on the biopsied cells. The results were concordant in all cases. The inclusion of a polymorphic marker allowed the detection of extraneous DNA contamination in two cells from one case. Knowing the genotype of the contaminating DNA allowed its origin to be traced. All five pregnancies were obtained from embryos in which two blastomeres were biopsied for the diagnosis. Our data demonstrate the successful strategy of using multiplex PCR to simultaneously amplify the mutation site and a polymorphic locus, fluorescent PCR technology to achieve greater sensitivity, and two-cell biopsy to increase the efficiency and success of diagnoses. Copyright © 2002 John Wiley & Sons, Ltd.     

Treatment of food industry waste by bench-scale upflow anaerobic sludge blanket-anoxic-aerobic system.

An upflow anaerobic sludge blanket (UASB)-anoxicaerobic system was used for treatment of tomato and bean processing wastewater. At various hydraulic retention times, ranging from 0.7 to 5 days, excellent removal of chemical oxygen demand (COD), biochemical oxygen demand (BOD), total suspended solids (TSS), ammonia-nitrogen (NH4-N), and total Kjeldahl nitrogen was achieved with final effluent BOD/TSS/NH4N concentrations of less than 15/15/1 mg/L. Biogas yield in the UASB reactor varied from 0.33 to 0.44 m3/kgCODremoved. The kinetics of anaerobic treatment were investigated. The yield coefficient was 0.03 gVSS/gCOD; maximum specific growth rate was 0.24 day(-1); Monod half velocity constant was 135 mgCOD/L; and specific substrate utilization rate was 3.25 gCOD/gVSS x d. Nitrification and denitrification kinetics were studied in batch experiments, and the rates were comparable with those in the continuous flow system.