Devin Oglesbee

Reduction of the false-positive rate in newborn screening by implementation of MS/MS-based second-tier tests: The Mayo Clinic experience (2004–2007)

SummaryThe continued expansion of newborn screening programmes to include additional conditions increases the responsibility of newborn screening laboratories to provide testing with the highest sensitivity and specificity to allow for identification of affected patients while minimizing the false-positive rate. Some assays and analytes are particularly problematic. Over recent years, our laboratory tried to improve this situation by developing second-tier tests to reduce false-positive results in the screening for congenital adrenal hyperplasia (CAH), tyrosinaemia type I, methylmalonic acidaemias, homocystinuria, and maple syrup urine disease (MSUD). Beginning in 2004, this approach was applied to Mayo’s newborn screening programme and resulted in a false-positive rate of 0.09%, a positive predictive value of 41%, and a positive detection rate of 1 affected case in 1672 babies screened.

Combined newborn screening for succinylacetone, amino acids, and acylcarnitines in dried blood spots

BACKGROUND Tyrosinemia type I (TYR 1) is a disorder causing early death if left untreated. Newborn screening (NBS) for this condition is problematic because determination of the diagnostic marker, succinylacetone (SUAC), requires a separate first-tier or only partially effective second-tier analysis based on tyrosine concentration. To overcome these problems, we developed a new assay that simultaneously determines acylcarnitines (AC), amino acids (AA), and SUAC in dried blood spots (DBS) by flow injection tandem mass spectrometry (MS/MS). METHODS We extracted 3/16-inch DBS punches with 300 microL methanol containing AA and AC stable isotope-labeled internal standards. This extract was derivatized with butanol-HCl. In parallel, we extracted SUAC from the residual filter paper with 100 microL of a 15 mmol/L hydrazine solution containing the internal standard 13C5-SUAC. We combined the derivatized aliquots in acetonitrile for MS/MS analysis of AC and AA with additional SRM experiments for SUAC (m/z 155-137) and 13C5-SUAC (m/z 160-142). Analysis time was 1.2 min. RESULTS SUAC was increased in retrospectively analyzed NBS samples of 11 TYR 1 patients (length of storage, 52 months to 1 week; SUAC range, 13-81 micromol/L), with Tyr concentrations ranging from 65 to 293 micromol/L in the original NBS analysis. The mean concentration of SUAC in 13 521 control DBS was 1.25 micromol/L. CONCLUSION The inclusion of SUAC analysis into routine analysis of AC and AA allows for rapid and cost-effective screening for TYR 1 with no tangible risk of false-negative results.

Second-tier test for quantification of alloisoleucine and branched-chain amino acids in dried blood spots to improve newborn screening for maple syrup urine disease (MSUD).

BACKGROUND Newborn screening for maple syrup urine disease (MSUD) relies on finding increased concentrations of the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine by tandem mass spectrometry (MS/MS). d-Alloisoleucine (allo-Ile) is the only pathognomonic marker of MSUD, but it cannot be identified by existing screening methods because it is not differentiated from isobaric amino acids. Furthermore, newborns receiving total parenteral nutrition often have increased concentrations of BCAAs. To improve the specificity of newborn screening for MSUD and to reduce the number of diet-related false-positive results, we developed a LC-MS/MS method for quantifying allo-Ile. METHODS Allo-Ile and other BCAAs were extracted from a 3/16-inch dried blood spot punch with methanol/H2O, dried under nitrogen, and reconstituted into mobile phase. Quantitative LC-MS/MS analysis of allo-Ile, its isomers, and isotopically labeled internal standards was achieved within 15 min. To determine a reference interval for BCAAs including allo-Ile, we analyzed 541 dried blood spots. We also measured allo-Ile in blinded samples from 16 MSUD patients and 21 controls and compared results to an HPLC method. RESULTS Intra- and interassay imprecision (mean CVs) for allo-Ile, leucine, isoleucine, and valine ranged from 1.8% to 7.4%, and recovery ranged from 91% to 129%. All 16 MSUD patients were correctly identified. CONCLUSIONS The LC-MS/MS method can reliably measure allo-Ile in dried blood spots for the diagnosis of MSUD. Applied to newborn screening as a second-tier test, it will reduce false-positive results, which produce family anxiety and increase follow-up costs. The assay also appears suitable for use in monitoring treatment of MSUD patients.

Disease-causing mitochondrial heteroplasmy segregated within induced pluripotent stem cell clones derived from a patient with MELAS.

Mitochondrial diseases display pathological phenotypes according to the mixture of mutant versus wild‐type mitochondrial DNA (mtDNA), known as heteroplasmy. We herein examined the impact of nuclear reprogramming and clonal isolation of induced pluripotent stem cells (iPSC) on mitochondrial heteroplasmy. Patient‐derived dermal fibroblasts with a prototypical mitochondrial deficiency diagnosed as mitochondrial encephalomyopathy with lactic acidosis and stroke‐like episodes (MELAS) demonstrated mitochondrial dysfunction with reduced oxidative reserve due to heteroplasmy at position G13513A in the ND5 subunit of complex I. Bioengineered iPSC clones acquired pluripotency with multilineage differentiation capacity and demonstrated reduction in mitochondrial density and oxygen consumption distinguishing them from the somatic source. Consistent with the cellular mosaicism of the original patient‐derived fibroblasts, the MELAS‐iPSC clones contained a similar range of mtDNA heteroplasmy of the disease‐causing mutation with identical profiles in the remaining mtDNA. High‐heteroplasmy iPSC clones were used to demonstrate that extended stem cell passaging was sufficient to purge mutant mtDNA, resulting in isogenic iPSC subclones with various degrees of disease‐causing genotypes. On comparative differentiation of iPSC clones, improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared with isogenic clones with high heteroplasmy. Thus, mtDNA heteroplasmic segregation within patient‐derived stem cell lines enables direct comparison of genotype/phenotype relationships in progenitor cells and lineage‐restricted progeny, and indicates that cell fate decisions are regulated as a function of mtDNA mutation load. The novel nuclear reprogramming‐based model system introduces a disease‐in‐a‐dish tool to examine the impact of mutant genotypes for MELAS patients in bioengineered tissues and a cellular probe for molecular features of individual mitochondrial diseases. STEM Cells2013;31:1298–1308

Determination of Total Homocysteine, Methylmalonic Acid, and 2-Methylcitric Acid in Dried Blood Spots by Tandem Mass Spectrometry

BACKGROUND Newborn screening (NBS) for inborn errors of propionate, methionine, and cobalamin metabolism relies on finding abnormal concentrations of methionine and propionylcarnitine. These analytes are not specific for these conditions and lead to frequent false-positive results. More specific markers are total homocysteine (tHCY), methylmalonic acid (MMA), and methylcitric acid (MCA), but these markers are not detected by current NBS methods. To improve this situation, we developed a method for the detection of tHCY, MMA, and MCA in dried blood spots (DBSs) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHODS The analytes were extracted from a single 4.8-mm DBS punch with acetonitrile:water:formic acid (59:41:0.42) containing dithiothreitol and isotopically labeled standards (d(3)-MMA, d(3)-MCA, d(8)-homocystine). The extract was dried and treated with 3 N HCl in n-butanol to form butylesters. After evaporation of the butanol, the residue was reconstituted and centrifuged and the supernatant was subjected to LC-MS/MS analysis. Algorithms were developed to apply this method as an efficient and effective second-tier assay on samples with abnormal results by primary screening. RESULTS The 99th percentiles determined from the analysis of 200 control DBSs for MMA, MCA, and HCY were 1.5, 0.5, and 9.8 μmol/L, respectively. Since 2005, prospective application of this second-tier analysis to 2.3% of all NBS samples led to the identification of 13 affected infants. CONCLUSIONS Application of this assay reduced the false-positive rate and improved the positive predictive value of NBS for conditions associated with abnormal propionylcarnitine and methionine concentrations.

Newborn screening for lysosomal storage disorders

Every newborn in the U.S. is screened for at least 29 disorders, where evidence suggests that early detection is possible and beneficial. With new or improved treatment options and development of high-throughput screening tests, additional conditions have been proposed for inclusion in newborn screening programs. Among those are several lysosomal storage disorders that have been evaluated in limited pilot studies or that are already included in a few national or international newborn screening programs. These conditions include Pompe disease, Niemann-Pick type A/B disease, Fabry disease, Krabbe disease, Mucopolysaccharidoses types I and II, and Gaucher disease. Here, we review the current state of newborn screening for these lysosomal storage disorders.

Outcome of Whole Exome Sequencing for Diagnostic Odyssey Cases of an Individualized Medicine Clinic: The Mayo Clinic Experience

OBJECTIVE To describe the experience and outcome of performing whole-exome sequencing (WES) for resolution of patients on a diagnostic odyssey in the first 18 months of an individualized medicine clinic (IMC). PATIENTS AND METHODS The IMC offered WES to physicians of Mayo Clinic practice for patients with suspected genetic disease. DNA specimens of the proband and relatives were submitted to WES laboratories. We developed the Genomic Odyssey Board with multidisciplinary expertise to determine the appropriateness for IMC services, review WES reports, and make the final decision about whether the exome findings explain the disease. This study took place from September 30, 2012, to March 30, 2014. RESULTS In the first 18 consecutive months, the IMC received 82 consultation requests for patients on a diagnostic odyssey. The Genomic Odyssey Board deferred 7 cases and approved 75 cases to proceed with WES. Seventy-one patients met with an IMC genomic counselor. Fifty-one patients submitted specimens for WES testing, and the results have been received for all. There were 15 cases in which a diagnosis was made on the basis of WES findings; thus, the positive diagnostic yield of this practice was 29%. The mean cost per patient for this service was approximately

Confirming Variants in Next-Generation Sequencing Panel Testing by Sanger Sequencing.

8000. Medicaid supported 27% of the patients, and 38% of patients received complete or partial insurance coverage. CONCLUSION The significant diagnostic yield, moderate cost, and notable health marketplace acceptance for WES compared with conventional genetic testing make the former method a rational diagnostic approach for patients on a diagnostic odyssey.

Design and Analytical Validation of Clinical DNA Sequencing Assays

Current clinical laboratory practice guidelines for next-generation sequencing (NGS) do not provide definitive guidance on confirming NGS variants. Sanger confirmation of NGS results can be inefficient, redundant, and expensive. We evaluated the accuracy of NGS-detected single-nucleotide variants (SNVs) and insertion/deletion variants (indels) and the necessity of NGS variant confirmation using four NGS target-capture gene panels covering 117 genes, 568 Kbp, and 77 patient DNA samples. Unique NGS-detected variants (1080 SNVs and 124 indels) underwent Sanger confirmation and/or were compared to data from the 1000 Genomes Project (1000G). Recurrent variants in unrelated samples resulted in 919 comparisons between NGS and Sanger, with 100% concordance. In a second comparison, 762 unique NGS results (736 SNVs, 26 indels) from seven 1000G samples were found to have 97.1% concordance with 1000G phase 1 data. Sanger sequencing and 1000G phase 3 data confirmed the accuracy of the NGS results for all 1000G phase 1 discrepancies. In all samples, the depth of coverage exceeded 100× in >99.7% of bases in the target regions. In conclusion, confirmatory analysis by Sanger sequencing of SNVs detected via capture-based NGS testing that meets appropriate quality thresholds is unnecessarily redundant. In contrast, Sanger sequencing for indels may be required for defining the correct genomic location, and Sanger may be used for quality-assurance purposes.

Development of a newborn screening follow-up algorithm for the diagnosis of isobutyryl-CoA dehydrogenase deficiency

CONTEXT DNA sequencing is the method of choice for mutation detection in many genes. OBJECTIVES To demonstrate the analytical accuracy and reliability of DNA sequencing assays developed in clinical laboratories. Only general guidelines exist for the validation of these tests. We provide examples of assay validation strategies for DNA sequencing tests. DESIGN We discuss important design and validation considerations. RESULTS The validation examples include an accuracy study to evaluate concordance between results obtained by the newly designed assay and analyzed by another method or laboratory. Precision (reproducibility) studies are performed to determine the robustness of the assay. To assess the quality of sequencing assays, several sequence quality measures are available. In addition, assessing the ability of primers to specifically and robustly amplify target regions before sequencing is important. CONCLUSION Protocols for validation of laboratory-developed sequencing assays may vary between laboratories. An example summary of a validation is provided.