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.

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 () gene. Centers for Disease Control and Prevention's Newborn Screening Quality Assurance Program houses a dried blood spot repository of samples containing mutations to assist newborn screening laboratories and ensure high-quality mutation detection in a high-throughput environment.

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 21 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.

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.

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.

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.

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.

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.

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.

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.

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.

Identification of two novel DQA1 alleles, DQA1*0107 and DQA1*0602, by sequence-based typing in the GoKinD population.

Two novel DQA1 alleles, DQA1*0107 and DQA1*0602, were discovered using DQA1 sequence-based typing (SBT) in participants in the Genetics of Kidneys in Diabetes (GoKinD) Study. The DQA1*0107 allele, found in three unrelated Caucasian participants, contains a novel polymorphism at codon 79 of exon 2 (CGC-->TGC), which results in an amino acid change from an arginine to a cysteine. The participants containing this novel polymorphism also had a 1-bp insertion in intron 2 that is common to the *01 alleles.

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.