Assessing the Performance of Dried-Blood-Spot DNA Extraction Methods in Next Generation Sequencing.

An increasing number of newborn screening laboratories in the United States and abroad are moving towards incorporating next-generation sequencing technology, or NGS, into routine screening, particularly for cystic fibrosis. As more programs utilize this technology for both cystic fibrosis and beyond, it is critical to identify appropriate DNA extraction methods that can be used with dried blood spots that will result in consistent, high-quality sequencing results. To provide comprehensive quality assurance and technical assistance to newborn screening laboratories wishing to incorporate NGS assays, CDC's Newborn Screening and Molecular Biology Branch designed a study to evaluate the performance of nine commercial or laboratory-developed DNA extraction methods that range from a highly purified column extraction to a crude detergent-based no-wash boil prep. The DNA from these nine methods was used in two NGS library preparations that interrogate the CFTR gene. All DNA extraction methods including the cruder preps performed reasonably well with both library preps. One lower-concentration, older sample was excluded from one of the assay evaluations due to poor performance across all DNA extraction methods. When 84 samples, versus eight, were run on a flow cell, the DNA quality and quantity were more significant variables.

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.

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.

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

Abstract 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.

The Newborn Screening Quality Assurance Program at the Centers for Disease Control and Prevention: Thirty-five Year Experience Assuring Newborn Screening Laboratory Quality.

Newborn screening is the largest genetic testing effort in the United States and is considered one of the ten great public health achievements during the first 10 years of the 21st century. For over 35 years, the Newborn Screening Quality Assurance Program (NSQAP) at the US Centers for Disease Control and Prevention has helped NBS laboratories ensure that their testing does not delay diagnosis, minimizes false-positive reports, and sustains high-quality testing performance. It is a multi-component program that provides comprehensive quality assurance services for dried blood spot testing. The NSQAP, the Biochemical Mass Spectrometry Laboratory (BMSL), the Molecular Quality Improvement Program (MQIP) and the Newborn Screening Translation Research Initiative (NSTRI), aid screening laboratories achieve technical proficiency and maintain confidence in their performance while processing large volumes of specimens daily. The accuracy of screening tests could be the difference between life and death for many babies; in other instances, identifying newborns with a disorder means that they can be treated and thus avoid life-long disability or severe cognitive impairment. Thousands of newborns and their families have benefited from reliable and accurate testing that has been accomplished by a network of screening laboratories and the NSQAP, BMSL, MQIP and NSTRI.

Novel method to characterize CYP21A2 in Florida patients with congenital adrenal hyperplasia and commercially available cell lines.

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.

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.

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.

Sequence variants in the PLEKHH2 region are associated with diabetic nephropathy in the GoKinD study population.

Nephropathy is a common microvascular complication of diabetes with a genetic component for disease development. Genetic analyses have implicated multiple chromosomal regions for disease susceptibility but no single locus can account for the majority of the genetic component. Here, we report a genetic analysis of the PLEKHH2 gene that was identified through a single nucleotide polymorphism (SNP) genome-wide association study (GWAS) for association with the development of diabetic nephropathy (DN) in the Genetics of Kidneys in Diabetes (GoKinD) study population. We initially examined the GWAS results from a subset of the GoKinD singleton population based on the two most common HLA diplotypes consisting of 112 cases and 148 controls. We observed two-adjacent markers mapping to the PLEKHH2 locus, rs1368086 and rs725238, each associated at P < 0.001. Additional SNPs were selected for linkage disequilibrium mapping and transmission disequilibrium testing (TdT) in 246 case trio families. A single marker, rs11886047, located upstream of the PLEKHH2 promoter was associated with DN by TdT in the case trios (P = 0.0307), and there was a increase of heterozygous genotypes in cases, relative to controls, from the 601 case and 577 control GoKinD singleton case/control population (P = 0.00256). These findings suggest that PLEKHH2, which has mRNA and protein expression exclusively in the glomerulus, may be a genetic risk factor for susceptibility to DN in the GoKinD population.

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.

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.

Polymorphism scan for differences between transmitted and nontransmitted DRB1*030101 alleles outside of exon 2 for type 1 diabetes: the frequency of polymorphisms is similar.

DRB1*030101 is a major genetic risk factor for type 1 diabetes mellitus (T1DM) and is the only DRB1*03 allele usually seen in T1DM probands. Approximately 16% of parental DRB1*030101 alleles were not transmitted to T1DM probands in our Genetics of Kidneys and Diabetes study trio families. We performed a polymorphism screen to determine whether variations exist in DRB1*030101 alleles outside of exon 2 that may modify risk for developing T1DM. A combination of long-range and sequence-specific priming polymerase chain reaction was used to amplify a hemizygous template from both transmitted and nontransmitted parental DRB1*030101 chromosomes. Exon 2 DRB1*030101-specific and flanking DRB1-specific primers amplified the entire genomic locus as a 10.6-kb 5' fragment and a 5.3-kb 3' fragment, respectively. All exons and intron/exon borders of introns 1 and 2, all of introns 3-5, and flanking regulatory regions of 32 transmitted and 31 nontransmitted alleles (99% power to detect a 5% minimal allele frequency) were analyzed through fluorescent DNA sequencing. The only polymorphic sites detected, a previously described intron 2 complex dinucleotide repeat and an additional complex repeat approximately 1.8 kb downstream of exon 6, do not significantly differ between T1DM patients and controls in this small data set.