DSpace community: 附設醫院基因醫學部

Determination of Smni/Smn2 Gene Dosage by a Quantitative Genotyping Platform Combining Capillary Electrophoresis and Maldi-Tof Mass Spectrometry

title: Determination of Smni/Smn2 Gene Dosage by a Quantitative Genotyping Platform Combining Capillary Electrophoresis and Maldi-Tof Mass Spectrometry abstract: Background: Spinal muscular atrophy (SMA) is a common inherited and fatal neuromuscular disease caused by deletions and/or mutations that lead to altered concentrations of proteins encoded by the survival motor neuron genes SMN1 and SMN2. Because of the high incidence ( at least 1 in 10 000 live births and a carrier frequency of 1 in 35 to 1 in 50) and severity of the disease, precise quantification of SMN1 and SMN2 gene copy numbers is essential for diagnosis and genetic counseling. Methods: We developed a genotyping platform combining capillary electrophoresis and matrix- assisted laser desorption/ ionization time-of-flight mass spectrometry ( MALDI-TOF MS) to quantify absolute gene dosage. The absolute gene dosage can be determined by a multiplexed competitive PCR protocol followed by capillary electrophoresis analysis. The relative SMN1/SMN2 ratio can be analyzed by PinPoint assay followed by MALDI-TOF MS analysis. Results: The complementary assays were evaluated in confirmed cases including 9 affected patients, 33 carriers, and 478 healthy individuals from the general population. We were able to determine all genotypes with different SMN1/SMN2 gene copy number ratios, which unambiguously diagnosed carrier status and the severity of SMA with 100% specificity. Conclusions: This quantitative genotyping platform is suitable for detection of SMA. The described approach may serve as a general quantitative genotyping method for molecular diagnosis of other inheritable diseases. (C) 2006 American Association for Clinical Chemistry.

Decreased Maternal Serum Placenta Growth Factor in Early Second Trimester and Preeclampsia

title: Decreased Maternal Serum Placenta Growth Factor in Early Second Trimester and Preeclampsia abstract: Objective: To compare early second-trimester maternal serum placenta growth factor concentrations in patients with subsequent development of preeclampsia and those with normal pregnancies.Methods: We conducted a case-control analysis of stored maternal serum of 27 women who subsequently developed preeclampsia and 227 randomly selected normal controls during the gestational period of 14-19 weeks. Using such a sample size, there was a greater than 95% power to test a difference in the primary study interest. A quantitative sandwich enzyme immunoassay was used to measure the maternal serum placenta growth factor concentration. For statistical analysis, Mann-Whitney U tests, multiple linear regression analysis, multivariable logistic regression model, and receiver-operating characteristic (ROC ) curve were used. P < .05 was considered statistically significant.Results: Maternal serum placenta growth factor concentration was associated with the occurrence of subsequent preeclampsia (P < .001) and gestational age (P <. 001). The median ( interquartile range) of multiples (MoM) of the gestational age stratified median for placenta growth factor in preeclampsia was 0.55 (0.33, 0.85). The ROC curve revealed that the specificity was 70% when the diagnostic sensitivity was 70%, and the optimal cutoff value of placenta growth factor MoM was 0.76. The risk of developing preeclampsia subsequently was increased 2.5-fold for maternal serum placenta growth factor concentration decrements of 0.1 MoM.Conclusion: A decreased maternal serum placenta growth factor concentration in the early second trimester is highly associated with the subsequent development of preeclampsia, but a large prospective study is needed to explore its use as an early predictor for the condition. (Obstet Gynecol 2001;97:898-904. (C) 2001 by The American College of Obstetricians and Gynecologists.).

Raised Maternal Serum Placenta Growth Factor Concentration during the Second Trimester Is Associated with down Syndrome

title: Raised Maternal Serum Placenta Growth Factor Concentration during the Second Trimester Is Associated with down Syndrome abstract: Objectives To compare early second-trimester maternal serum placenta growth factor concentrations in Down syndrome pregnancies and those in normal pregnancies.Methods A case- control study was performed to evaluate the maternal serum placenta growth factor concentrations in 36 Down syndrome and 320 normal pregnancies with matched gestational age during the second trimester. For the detection of serum concentrations of placenta growth factor, a quantitative sandwich enzyme immunoassay technique (R & D Systems Inc., Minneapolis, Minnesota, USA) was performed. Results Using a multiple linear regression model, maternal serum placenta growth factor level was associated with gestational age (p < 0.001) and the existence of Down syndrome pregnancy (p < 0. 001). After converting maternal serum placenta growth factor concentrations of each analyte to multiples of the appropriate gestational median (MoM), placenta growth factor MoM (p < 0.001) was revealed to be an independent variable for Down syndrome pregnancies after adjusting for the effects of maternal age (p < 0.001), free beta-hCG (p < 0. 001) and AFP (p = 0.014) by multivariate logistic regression analysis.Conclusions Maternal serum placenta growth factor concentration was elevated in Down syndrome pregnancies during the early second trimester. Placenta growth factor might be a novel marker for maternal serum Down syndrome screening. Copyright (C) 2002 John Wiley Sons, Ltd.

Rapid Detection of Beta-Globin Gene (Hbb) Mutations Coupling Heteroduplex and Primer-Extension Analysis by Dhplc

title: Rapid Detection of Beta-Globin Gene (Hbb) Mutations Coupling Heteroduplex and Primer-Extension Analysis by Dhplc abstract: Beta-thalassemia is a common inherited disease, resulting from one or more of a total of more than 200 different mutations in the beta-globin gene (111313). Efficient and reliable mutation. screening methods are essential in order to establish appropriate prevention programs for at, risk populations based upon a molecular diagnosis. We have developed a rapid and highly-specific mutation screening test for the diagnosis of beta-thalassemia by coupling heteroduplex and primer. extension analysis based on the denaturing high performance liquid chromatography (DHPLC) system. A total of 161 healthy heterozygous Taiwanese carriers featuring 10 different HBB mutations and 30 patients exhibiting 12 different compound heterozygous or homozygous HBB mutations were subjected to DHPLC. The elution profile for the heteroduplex analysis of DHPLC could be successfully used to identify the common disease-causing mutations of HBB. To further confirm the sequence variants, we developed a technique combining multiplex primer- extension analysis coupled with DHPLC for the genotyping of eight common disease,causing mutations in the HBB gene. Overall, by coupling heteroduplex and primer-extension analysis based upon DHPLC, we were able to unambiguously identify the most common beta- thalassemia mutations corresponding to more than 99% of HBB alleles among the Taiwanese population. In conclusion, compared to classic approaches to mutation screening for this malady, we suggest that DHPLC is an excellent technique to be applied to the genetic screening of prenatal and postnatal individuals as a part of a diagnosis program for beta- thalassemia and provides a more-efficient, economic, and sensitive means to undertake such a screening program. (C) 2003 Wiley-Liss, Inc .