Suzanne K. Cordovado

Genetics of Kidneys in Diabetes (GoKinD) Study: A Genetics Collection Available for Identifying Genetic Susceptibility Factors for Diabetic Nephropathy in Type 1 Diabetes

The Genetics of Kidneys in Diabetes (GoKinD) study is an initiative that aims to identify genes that are involved in diabetic nephropathy. A large number of individuals with type 1 diabetes were screened to identify two subsets, one with clear-cut kidney disease and another with normal renal status despite long-term diabetes. Those who met additional entry criteria and consented to participate were enrolled. When possible, both parents also were enrolled to form family trios. As of November 2005, GoKinD included 3075 participants who comprise 671 case singletons, 623 control singletons, 272 case trios, and 323 control trios. Interested investigators may request the DNA collection and corresponding clinical data for GoKinD participants using the instructions and application form that are available at http://www.gokind.org/access. Participating scientists will have access to three data sets, each with distinct advantages. The set of 1294 singletons has adequate power to detect a wide range of genetic effects, even those of modest size. The set of case trios, which has adequate power to detect effects of moderate size, is not susceptible to false-positive results because of population substructure. The set of control trios is critical for excluding certain false-positive results that can occur in case trios and may be particularly useful for testing gene-environment interactions. Integration of the evidence from these three components into a single, unified analysis presents a challenge. This overview of the GoKinD study examines in detail the power of each study component and discusses analytic challenges that investigators will face in using this resource.

Improving newborn screening for cystic fibrosis using next-generation sequencing technology: a technical feasibility study

Purpose:Many regions have implemented newborn screening (NBS) for cystic fibrosis (CF) using a limited panel of cystic fibrosis transmembrane regulator (CFTR) mutations after immunoreactive trypsinogen (IRT) analysis. We sought to assess the feasibility of further improving the screening using next-generation sequencing (NGS) technology.Methods:An NGS assay was used to detect 162 CFTR mutations/variants characterized by the CFTR2 project. We used 67 dried blood spots (DBSs) containing 48 distinct CFTR mutations to validate the assay. NGS assay was retrospectively performed on 165 CF screen–positive samples with one CFTR mutation.Results:The NGS assay was successfully performed using DNA isolated from DBSs, and it correctly detected all CFTR mutations in the validation. Among 165 screen-positive infants with one CFTR mutation, no additional disease-causing mutation was identified in 151 samples consistent with normal sweat tests. Five infants had a CF-causing mutation that was not included in this panel, and nine with two CF-causing mutations were identified.Conclusion:The NGS assay was 100% concordant with traditional methods. Retrospective analysis results indicate an IRT/NGS screening algorithm would enable high sensitivity, better specificity and positive predictive value (PPV). This study lays the foundation for prospective studies and for introducing NGS in NBS laboratories.Genet Med 18 3, 231–238.

CFTR mutation analysis and haplotype associations in CF patients.

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 Preventions 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.

Nephropathy in Type 1 Diabetes Is Diminished in Carriers of HLA- DRB1*04: The Genetics of Kidneys in Diabetes (GoKinD) Study

OBJECTIVE—The purpose of this study was to examine whether known genetic risk factors for type 1 diabetes (HLA-DRB1, -DQA1, and -DQB1 and insulin locus) play a role in the etiology of diabetic nephropathy. RESEARCH DESIGN AND METHODS—Genetic analysis of HLA-DRB1, -DQA1, -DQB1 and the insulin gene (INS) was performed in the Genetics of Kidneys in Diabetes (GoKinD) collection of DNA (European ancestry subset), which includes case patients with type 1 diabetes and nephropathy (n = 829) and control patients with type 1 diabetes but not nephropathy (n = 904). The availability of phenotypic and genotypic data on GoKinD participants allowed a detailed analysis of the association of these genes with diabetic nephropathy. RESULTS—Diabetic probands who were homozygous for HLA-DRB1*04 were 50% less likely to have nephropathy than probands without any DRB1*04 alleles. In heterozygous carriers, a protective effect of this allele was not as clearly evident; the mode of inheritance therefore remains unclear. This association was seen in probands with both short (<28 years, P = 0.02) and long (≥28 years, P = 0.0001) duration of diabetes. A1C, a marker of sustained hyperglycemia, was increased in control probands with normoalbuminuira, despite long-duration diabetes, from 7.2 to 7.3 to 7.7% with 0, 1, and 2 copies of the DRB1*04 allele, respectively. This result is consistent with a protective effect of DRB1*04 that may allow individuals to tolerate higher levels of hyperglycemia, as measured by A1C, without developing nephropathy. CONCLUSIONS—These data suggest that carriers of DRB1*04 are protected from some of the injurious hyperglycemic effects related to nephropathy. Interestingly, DRB1*04 appears to be both a risk allele for type 1 diabetes and a protective allele for nephropathy.

Nephropathy in Type 1 Diabetes is Diminished in Carriers of HLA DRB1 04: The GoKinD Study

OBJECTIVE—The purpose of this study was to examine whether known genetic risk factors for type 1 diabetes (HLA-DRB1, -DQA1, and -DQB1 and insulin locus) play a role in the etiology of diabetic nephropathy. RESEARCH DESIGN AND METHODS—Genetic analysis of HLA-DRB1, -DQA1, -DQB1 and the insulin gene (INS) was performed in the Genetics of Kidneys in Diabetes (GoKinD) collection of DNA (European ancestry subset), which includes case patients with type 1 diabetes and nephropathy (n = 829) and control patients with type 1 diabetes but not nephropathy (n = 904). The availability of phenotypic and genotypic data on GoKinD participants allowed a detailed analysis of the association of these genes with diabetic nephropathy. RESULTS—Diabetic probands who were homozygous for HLA-DRB1*04 were 50% less likely to have nephropathy than probands without any DRB1*04 alleles. In heterozygous carriers, a protective effect of this allele was not as clearly evident; the mode of inheritance therefore remains unclear. This association was seen in probands with both short (<28 years, P = 0.02) and long (≥28 years, P = 0.0001) duration of diabetes. A1C, a marker of sustained hyperglycemia, was increased in control probands with normoalbuminuira, despite long-duration diabetes, from 7.2 to 7.3 to 7.7% with 0, 1, and 2 copies of the DRB1*04 allele, respectively. This result is consistent with a protective effect of DRB1*04 that may allow individuals to tolerate higher levels of hyperglycemia, as measured by A1C, without developing nephropathy. CONCLUSIONS—These data suggest that carriers of DRB1*04 are protected from some of the injurious hyperglycemic effects related to nephropathy. Interestingly, DRB1*04 appears to be both a risk allele for type 1 diabetes and a protective allele for nephropathy.

Assessment of DNA contamination from dried blood spots and determination of DNA yield and function using archival newborn dried blood spots

BACKGROUND Residual dried blood spots (DBS) from newborn screening programs are often stored for years and are sometimes used for epidemiological studies. Because there is potential for DNA cross-contamination from specimen-to-specimen contact, we determined contamination levels following intentional contact and assessed archival DBS DNA degradation after storage in an uncontrolled environment. METHODS DBS from healthy adult females were rubbed with DBS from healthy or cystic fibrosis (CF)-affected adult males. Total human and male DNA was measured from the female DBS. Contamination levels were assessed using short tandem repeats (STRs). Female DBS contaminated with CF male DNA containing the F508del were analyzed for presence of this mutation. Archival DBS DNA amplification efficiency was determined using STR analysis. RESULTS Most female DBS were contaminated, however only one specimen showed an incomplete STR profile consistent with contaminating CF-affected male DNA. Further testing by CF mutation screening was negative. DNA extracted from archival DBS showed robust amplification (range 100 bp-320 bp). CONCLUSIONS Lightly abrasive contact between DBS resulted in DNA cross-contamination. The contaminating DNA did not interfere in CF-mutation tests; however this should be determined for individual assays. Since DNA from archival DBS robustly amplifies, newborn DBS could provide an invaluable resource for public health studies.

Implementation of the first worldwide quality assurance program for cystic fibrosis multiple mutation detection in population-based screening☆

BACKGROUND CDCs Newborn Screening Quality Assurance Program collaborated with several U.S. Cystic Fibrosis Care Centers to collect specimens for development of a molecular CFTR proficiency testing program using dried-blood spots for newborn screening laboratories. METHODS Adult and adolescent patients or carriers donated whole blood that was aliquoted onto filter paper cards. Five blind-coded specimens were sent to participating newborn screening laboratories quarterly. Proficiency testing results were evaluated based on presumptive clinical assessment. Individual evaluations and summary reports were sent to each participating laboratory and technical consultations were offered if incorrect assessments were reported. RESULTS The current CDC repository contains specimens with 39 different CFTR mutations. Up to 45 laboratories have participated in the program. Three years of data showed that correct assessments were reported 97.7% of the time overall when both mutations could be determined. Incorrect assessments that could have lead to a missed case occurred 0.9% of the time, and no information was reported 1.1% of the time due to sample failure. CONCLUSIONS Results show that laboratories using molecular assays to detect CFTR mutations are performing satisfactorily. The programmatic results presented demonstrate the importance and complexity of providing proficiency testing for DNA-based assays.

Proficiency Testing of Human Leukocyte Antigen-DR and Human Leukocyte Antigen-DQ Genetic Risk Assessment for Type 1 Diabetes Using Dried Blood Spots

Background: The plurality of genetic risk for developing type 1 diabetes mellitus (T1DM) lies within the genes that code for the human leukocyte antigens (HLAs). Many T1DM studies use HLA genetic risk assessment to identify higher risk individuals, and they often conduct these tests on dried blood spots (DBSs) like those used for newborn bloodspot screening. One such study is The Environmental Determinants of Diabetes in the Young (TEDDY), a long-term prospective study of environmental risk factors. To provide quality assurance for T1DM studies that employ HLA genetic risk assessment, the Centers for Disease Control and Prevention (CDC) conducts both a voluntary quarterly Proficiency testing (VQPT) program available to any laboratory and a mandatory annual Proficiency testing (PT) challenge for TEDDY laboratories. Methods: Whole blood and DBS samples with a wide range of validated HLA-DR and HLA-DQ genotypes were sent to the participating laboratories. Results were evaluated on the basis of both the reported haplotypes and the HLA genetic risk assessment. Results: Of the reported results from 24 panels sent out over six years in the VQPT, 94.7% (857/905) were correctly identified with respect to the relevant HLA-DR or HLA-DQ alleles, and 96.4% (241/250) were correctly categorized for risk assessment significant improvement was seen over the duration of this program, usually reaching 100% correct categorization during the last three years. Of 1154 reported results in four TEDDY PT challenges, 1153 (99.9%) were correctly identified for TEDDY eligibility. Conclusions: The different analytical methods used by T1DM research centers all provided accurate (>99%) results for genetic risk assessment. The two CDC PT programs documented the validity of the various approaches to screening and contributed to overall quality assurance.

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.

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.

Newborn Screening Quality Assurance Program for CFTR Mutation Detection and Gene Sequencing to Identify Cystic Fibrosis

All newborn screening laboratories in the United States and many worldwide screen for cystic fibrosis. Most laboratories use a second-tier genotyping assay to identify a panel of mutations in the CF transmembrane regulator (CFTR) gene. Centers for Disease Control and Prevention’s Newborn Screening Quality Assurance Program houses a dried blood spot repository of samples containing CFTR mutations to assist newborn screening laboratories and ensure high-quality mutation detection in a high-throughput environment. Recently, CFTR mutation detection has increased in complexity with expanded genotyping panels and gene sequencing. To accommodate the growing quality assurance needs, the repository samples were characterized with several multiplex genotyping methods, Sanger sequencing, and 3 next-generation sequencing assays using a high-throughput, low-concentration DNA extraction method. The samples performed well in all of the assays, providing newborn screening laboratories with a resource for complex CFTR mutation detection and next-generation sequencing as they transition to new methods.