Can incorporating molecular testing improve the accuracy of newborn screening for congenital adrenal hyperplasia?

BACKGROUND: Single-tier newborn screening (NBS) for CAH using 17-hydroxyprogesterone (17OHP) measured by fluoroimmunoassay (FIA) in samples collected at 24-48 hours produces a high false-positive rate (FPR). 2nd tier steroid testing can reduce the FPR and has been widely implemented. We investigated the accuracy of an alternative multi-tier CAH NBS protocol that incorporates molecular testing of the CYP21A2 gene and reduces the 1st tier 17OHP cutoff to minimize missed cases. METHODS: Created a Minnesota-specific CYP21A2 pathogenic variants panel; develop a rapid, high-throughput multiplex, allele-specific-primer-extension assay; perform 1-year retrospective analysis of Minnesota NBS results comparing metrics between a conventional steroid-based two-tier protocol and a molecular-based multi-tier NBS protocol, applied post-hoc. RESULTS: CYP21A2 gene sequencing of 103 Minnesota families resulted in a Minnesota-specific panel of 21 pathogenic variants. Centers for Disease Control and Prevention (CDC) created a molecular assay with 100% accuracy and reproducibility. Two-tier steroid-based screening of 68,659 live births during 2015 resulted in 2 false negatives (FNs), 91 FPs, and 1 true positive (TP). A three-tier protocol with a lower 1st-tier steroid cutoff, 2nd-tier 21-variant CYP21A2 panel and 3rd-tier CYP21A2 sequencing would have resulted in 0 FNs, 52 FPs and 3 TPs. CONCLUSIONS: Incorporation of molecular testing could improve the accuracy of CAH NBS, although some distinct challenges of molecular testing may need to be considered before implementation by NBS programs.

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.

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.

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.

Genetic studies of a cluster of acute lymphoblastic leukemia cases in Churchill County, Nevada.

OBJECTIVE: In a study to identify exposures associated with 15 cases of childhood leukemia, we found levels of tungsten, arsenic, and dichlorodiphenyldichloroethylene in participants to be higher than mean values reported in the National Report on Human Exposure to Environmental Chemicals. Because case and comparison families had similar levels of these contaminants, we conducted genetic studies to identify gene polymorphisms that might have made case children more susceptible than comparison children to effects of the exposures. DESIGN: We compared case with comparison children to determine whether differences existed in the frequency of polymorphic genes, including genes that code for enzymes in the folate and purine pathways. We also included discovery of polymorphic forms of genes that code for enzymes that are inhibited by tungsten: xanthine dehydrogenase, sulfite oxidase (SUOXgene), and aldehyde oxidase. PARTICIPANTS: Eleven case children were age- and sex-matched with 42 community comparison children for genetic analyses. Twenty parents of case children also contributed to the analyses. RESULTS: One bilalleleic gene locus in SUOX was significantly associated with either case or comparison status, depending on which alleles the child carried (without adjusting for multiple comparisons). CONCLUSIONS: Although genetic studies did not provide evidence that a common agent or genetic susceptibility factor caused the leukemias, the association between a SUOXgene locus and disease status in the presence of high tungsten and arsenic levels warrants further investigation. RELEVANCE: Although analyses of community clusters of cancer have rarely identified causes, these findings have generated hypotheses to be tested in subsequent studies.

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.