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.

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.

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.