Last data update: May 06, 2024. (Total: 46732 publications since 2009)
Records 1-10 (of 10 Records) |
Query Trace: Mueller PW[original query] |
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Fragile X Syndrome: Scientific Background and Screening Technologies.
Lyons JI , Kerr G , Mueller PW . J Mol Diagn 2015 17 (5) 463-71 Fragile X is the most common inherited cause of mental retardation with a prevalence of 1 in 4000 for males and 1 in 5000 to 8000 for females. The American College of Medical Genetics and Genomics has recommended diagnostic testing for fragile X in symptomatic persons, women with ovarian dysfunction, and persons with tremor/ataxia syndrome. Although medical and scientific professionals do not currently recommend screening nonsymptomatic populations, improvements in current treatment approaches and ongoing clinical trials have generated growing interest in screening for fragile X. Here, we briefly review the relevant molecular basis of fragile X and fragile X testing and compare three different molecular technologies available for fragile X screening in both males and females. These technologic approaches include destabilizing the CGG-repeat region with betaine and using chimeric CGG-targeted PCR primers, using heat pulses to destabilize C-G bonds in the PCR extension step, and using melting curve analysis to differentiate expanded CGG repeats from normals. The first two-step method performed with high sensitivity and specificity. The second method provided agarose gel images that allow identification of males with expanded CGG repeats and females with expanded CGG-repeat bands which are sometimes faint. The third melting curve analysis method would require controls in each run to correct for shifting optimal cutoff values. |
Detection of antibodies directed to the N-terminal region of GAD is dependent on assay format and contributes to differences in the specificity of GAD autoantibody assays for type 1 diabetes
Williams AJ , Lampasona V , Schlosser M , Mueller PW , Pittman DL , Winter WE , Akolkar B , Wyatt R , Brigatti C , Krause S , Achenbach P . Diabetes 2015 64 (9) 3239-46 Autoantibodies to glutamate decarboxylase (GADA) are sensitive markers of islet autoimmunity and type 1 diabetes. They form the basis of robust prediction models and are widely used for recruitment of subjects at high risk of type 1 diabetes to prevention trials. However GADA are also found in many individuals at low risk of diabetes progression. To identify the sources of diabetes irrelevant GADA reactivity therefore, we analyzed data from the 2009 and 2010 Diabetes Autoantibody Standardization Program GADA workshop and found that binding of healthy control sera varied according to assay type. Characterization of control sera found positive by radiobinding assay, but negative by ELISA showed that many of these sera reacted to epitopes in the N-terminal region of the molecule. This finding prompted development of an N-terminally truncated GAD65 radiolabel, 35S-GAD65(96-585), which improved the performance of most GADA radiobinding assays (RBAs) participating in an Islet Autoantibody Standardization Program GADA substudy. These detailed workshop comparisons have identified a source of disease-irrelevant signals in GADA RBAs and suggest that N-terminally truncated GAD labels will enable more specific measurement of GADA in type 1 diabetes. |
Novel method to characterize CYP21A2 in Florida patients with congenital adrenal hyperplasia and commercially available cell lines.
Greene CN , Cordovado SK , Turner DP , Keong LM , Shulman D , Mueller PW . Mol Genet Metab Rep 2014 1 312-323 Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder and affects approximately 1 in 15,000 births in the United States. CAH is one of the disorders included on the Newborn Screening (NBS) Recommended Uniform Screening Panel. The commonly used immunological NBS test is associated with a high false positive rate and there is interest in developing second-tier assays to increase screening specificity. Approximately 90% of the classic forms of CAH, salt-wasting and simple virilizing, are due to mutations in the CYP21A2 gene. These include single nucleotide changes, insertions, deletions, as well as chimeric genes involving CYP21A2 and its highly homologous pseudogene CYP21A1P. A novel loci-specific PCR approach was developed to individually amplify the CYP21A2 gene, the nearby CYP21A1P pseudogene, as well as any 30 kb deletion and gene conversion mutations, if present, as single separate amplicons. Using commercially available CAH positive specimens and 14 families with an affected CAH proband, the single long-range amplicon approach demonstrated higher specificity as compared to previously published methods. |
Standard enrichment methods for targeted next-generation sequencing in high-repeat genomic regions.
Mueller PW , Lyons J , Kerr G , Haase CP , Isett RB . Genet Med 2013 15 (11) 910-1 In addition to the increasing use of whole-exome and whole-genome sequencing for diagnosis of symptomatic recessive genetic diseases, researchers are developing targeted disease and gene panels using next-generation sequencing (NGS) for potential screening applications in adults and for newborn screening.1 Because significant actions could be taken based on the results of such screens, validation of the methods is necessary. To test the accuracy in a repeat genomic region of a published targeted NGS screening test for 448 childhood recessive diseases, we sequenced three samples reported to be positive by the authors for mutations in a newborn screening disease gene. This complex published method generated raw data from Agilent SureSelect hybrid capture enrichment of sheared DNA that was filtered using a variety of approaches. This method specified a minimum number of uniquely aligned reads of quality score >20; it specified that >14% of reads were needed to call a variant; and it restricted mutation calls to those associated with severe disease phenotypes in databases such as the Human Gene Mutation Database (HGMD). Using this highly curated approach, the authors reported mutations found in 104 Coriell samples, including two congenital adrenal hyperplasia (CAH) mutations in CYP21A2 in three samples clinically affected with diseases other than CAH. HGMD mutation CM071683, Ala392Thr (1174G>A), was reported in samples NA01881 and NA03365, and mutation CM920233, Pro454Ser (1360C>T), was reported in sample NA00059. The second mutation, CM920233, is a recognized cause of mild, nonclassical CAH,2 but the former, CM071683, was reported in 2006 as a novel mutation by Robins and coworkers, who were studying a Caucasian child presenting with premature adrenarche at 6 years of age and advanced bone age of 8.5 years.3 |
CFTR mutation analysis and haplotype associations in CF patients.
Cordovado SK , Hendrix M , Greene CN , Mochal S , Earley MC , Farrell PM , Kharrazi M , Hannon WH , Mueller PW . Mol Genet Metab 2012 105 (2) 249-54 Most newborn screening (NBS) laboratories use second-tier molecular tests for cystic fibrosis (CF) using dried blood spots (DBS). The Centers for Disease Control and Prevention's NBS Quality Assurance Program offers proficiency testing (PT) in DBS for CF transmembrane conductance regulator (CFTR) gene mutation detection. Extensive molecular characterization on 76 CF patients, family members or screen positive newborns was performed for quality assurance. The coding, regulatory regions and portions of all introns were sequenced and large insertions/deletions were characterized as well as two intronic di-nucleotide microsatellites. For CF patient samples, at least two mutations were identified/verified and four specimens contained three likely CF-associated mutations. Thirty-four sequence variations in 152 chromosomes were identified, five of which were not previously reported. Twenty-seven of these variants were used to predict haplotypes from the major haplotype block defined by HapMap data that spans the promoter through intron 19. Chromosomes containing the F508del (p.Phe508del), G542X (p.Gly542X) and N1303K (p.Asn1303Lys) mutations shared a common haplotype subgroup, consistent with a common ancient European founder. Understanding the haplotype background of CF-associated mutations in the U.S. population provides a framework for future phenotype/genotype studies and will assist in determining a likely cis/trans phase of the mutations without need for parent studies. |
Genetic variants associated with fasting blood lipids in the U.S. population: Third National Health and Nutrition Examination Survey.
Chang MH , Yesupriya A , Ned RM , Mueller PW , Dowling NF . BMC Med Genet 2010 11 62 BACKGROUND: The identification of genetic variants related to blood lipid levels within a large, population-based and nationally representative study might lead to a better understanding of the genetic contribution to serum lipid levels in the major race/ethnic groups in the U.S. population. METHODS: Using data from the second phase (1991-1994) of the Third National Health and Nutrition Examination Survey (NHANES III), we examined associations between 22 polymorphisms in 13 candidate genes and four serum lipids: high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), and triglycerides (TG). Univariate and multivariable linear regression and within-gene haplotype trend regression were used to test for genetic associations assuming an additive mode of inheritance for each of the three major race/ethnic groups in the United States (non-Hispanic white, non-Hispanic black, and Mexican American). RESULTS: Variants within APOE (rs7412, rs429358), PON1 (rs854560), ITGB3 (rs5918), and NOS3 (rs2070744) were found to be associated with one or more blood lipids in at least one race/ethnic group in crude and adjusted analyses. In non-Hispanic whites, no individual polymorphisms were associated with any lipid trait. However, the PON1 A-G haplotype was significantly associated with LDL-C and TC. In non-Hispanic blacks, APOE variant rs7412 and haplotype T-T were strongly associated with LDL-C and TC; whereas, rs5918 of ITGB3 was significantly associated with TG. Several variants and haplotypes of three genes were significantly related to lipids in Mexican Americans: PON1 in relation to HDL-C; APOE and NOS3 in relation to LDL-C; and APOE in relation to TC. CONCLUSIONS: We report the significant associations of blood lipids with variants and haplotypes in APOE, ITGB3, NOS3, and PON1 in the three main race/ethnic groups in the U.S. population using a large, nationally representative and population-based sample survey. Results from our study contribute to a growing body of literature identifying key determinants of plasma lipoprotein concentrations and could provide insight into the biological mechanisms underlying serum lipid and cholesterol concentrations. |
Diabetes Antibody Standardization Program: evaluation of assays for insulin autoantibodies
Schlosser M , Mueller PW , Torn C , Bonifacio E , Bingley PJ . Diabetologia 2010 53 (12) 2611-20 AIMS/HYPOTHESIS: Insulin autoantibodies (IAA) are important in type 1 diabetes risk assessment. However, their determination varies more between laboratories than other diabetes autoantibodies. The Diabetes Antibody Standardization Program (DASP) aims to improve and standardise measurement of autoantibodies associated with type 1 diabetes. We report the results of measurement of IAA from DASP workshops in 2002, 2003 and 2005. METHODS: Up to 32 laboratories in 14 countries participated in each workshop. Aliquots of coded sera from 50 patients with newly diagnosed type 1 diabetes and 100 blood donor controls were circulated to participating laboratories. Reported results were analysed using receiver operator characteristic (ROC) curves. We compared concordance of antibody levels by ranking, IAA and insulin antibody (IA) indices and units derived from an IA standard curve. RESULTS: In all three workshops IAA assay performance had improved compared with DASP 2000. The median area under the ROC curve was 0.73 in DASP 2002, 0.78 in 2003 and 0.80 in 2005 (p = 0.0012), and median laboratory-assigned sensitivity was 26% in 2002, 36% in 2003 and 45% in 2005 (p < 0.0001). There was, however, marked variation between assays. The range of AUC was 0.36-0.91 and that of laboratory-assigned sensitivity was 22-57%. Concordance of ranking of patient serum samples was related to AUC (p < 0.001). Using an index related to common IAA and IA-positive or -negative control sera improved the concordance between assays (p < 0.0001). CONCLUSIONS/INTERPRETATION: The overall performance of IAA assays has improved but there is still wide variation between laboratories. Concordance between assays would be improved by the use of a common reference reagent. |
Perturbations in the lipid profile of individuals with newly diagnosed type 1 diabetes mellitus: lipidomics analysis of a Diabetes Antibody Standardization Program sample subset
Sorensen CM , Ding J , Zhang Q , Alquier T , Zhao R , Mueller PW , Smith RD , Metz TO . Clin Biochem 2010 43 (12) 948-56 OBJECTIVES: To characterize the lipid profile of individuals with newly diagnosed type 1 diabetes mellitus using LC-MS-based lipidomics and the accurate mass and time (AMT) tag approach. DESIGN AND METHODS: Lipids were extracted from plasma and sera of 10 subjects from the Diabetes Antibody Standardization Program (years 2000-2005) and 10 non-diabetic subjects and analyzed by capillary liquid chromatography coupled with a hybrid ion-trap-Fourier transform ion cyclotron resonance mass spectrometer. Lipids were identified and quantified using the AMT tag approach. RESULTS: Five hundred fifty-nine lipid features differentiated (q<0.05) diabetic from healthy individuals in a partial least-squares analysis, characterizing individuals with recently diagnosed type 1 diabetes mellitus. CONCLUSIONS: A lipid profile associated with newly diagnosed type 1 diabetes may aid in further characterization of biochemical pathways involved in lipid regulation or mobilization. |
Harmonization of glutamic acid decarboxylase and islet antigen-2 autoantibody assays for National Institute of Diabetes and Digestive and Kidney Diseases Consortia
Bonifacio E , Yu L , Williams AK , Eisenbarth GS , Bingley PJ , Marcovina SM , Adler K , Ziegler AG , Mueller PW , Schatz DA , Krischer JP , Steffes MW , Akolkar B . J Clin Endocrinol Metab 2010 95 (7) 3360-7 BACKGROUND/RATIONALE: Autoantibodies to islet antigen-2 (IA-2A) and glutamic acid decarboxylase (GADA) are markers for diagnosis, screening, and measuring outcomes in National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) consortia studies. A harmonization program was established to increase comparability of results within and among these studies. METHODS: Large volumes of six working calibrators were prepared from pooled sera with GADA 4.8-493 World Health Organization (WHO) units/ml and IA-2A 2-235 WHO units/ml. Harmonized assay protocols for IA-2A and GADA using (35)S-methionine-labelled in vitro transcribed and translated antigens were developed based on methods in use in three NIDDK laboratories. Antibody thresholds were defined using sera from patients with recent onset type 1 diabetes and healthy controls. To evaluate the impact of the harmonized assay protocol on concordance of IA-2A and GADA results, two laboratories retested stored TEDDY study sera using the harmonized assays. RESULTS: The harmonized assays gave comparable but not identical results in the three laboratories. For IA-2A, using a common threshold of 5 DK units/ml, 549 of 550 control and patient samples were concordantly scored as positive or negative, specificity was greater than 99% with sensitivity 64% in all laboratories. For GADA, using thresholds equivalent to the 97th percentile of 974 control samples in each laboratory, 1051 (97.9%) of 1074 samples were concordant. On the retested TEDDY samples, discordance decreased from 4 to 1.8% for IA-2A (n = 604 samples; P = 0.02) and from 15.4 to 2.7% for GADA (n = 515 samples; P < 0.0001). CONCLUSION: Harmonization of GADA and IA-2A is feasible using large volume working calibrators and common protocols and is an effective approach to ensure consistency in autoantibody measurements. |
Novel human leukocyte antigen class I and class II alleles identified by sequence-based typing in the Genetics of Kidneys in Diabetes (GoKinD) study population
Cordovado SK , Hancock LN , Hendrix M , Greene CN , Mueller PW . Hum Immunol 2009 70 (9) 747-9 Nine novel HLA class I and class II alleles were identified by sequence-based typing (SBT) in Caucasian participants from the Genetics of Kidneys in Diabetes (GoKinD) study. All novel alleles were single nucleotide substitutions. Seven alleles resulted in an amino acid change and two alleles were silent substitutions. The new alleles are as follows: five HLA-A alleles (*0132, *020121, *0344, *030107, *2507), one HLA-C allele (*0619), two HLA-DQB1 alleles (*0204, *0318), and one HLA-DPB1 allele (*1802). Eight of these new alleles were identified in participants with type 1 diabetes, three of whom also had diabetic nephropathy, and one new allele was identified in an unaffected parent of a participant with type 1 diabetes. All new alleles were isolated and characterized by use of single allele amplification (SAA) SBT; the new alleles were confirmed by sequence-specific primer (SSP) amplification. |
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