Laboratory analysis of acylcarnitines, 2020 update: a technical standard of the American College of Medical Genetics and Genomics (ACMG)

Acylcarnitine analysis is a useful test for identifying patients with inborn errors of mitochondrial fatty acid β-oxidation and certain organic acidemias. Plasma is routinely used in the diagnostic workup of symptomatic patients. Urine analysis of targeted acylcarnitine species may be helpful in the diagnosis of glutaric acidemia type I and other disorders in which polar acylcarnitine species accumulate. For newborn screening applications, dried blood spot acylcarnitine analysis can be performed as a multiplex assay with other analytes, including amino acids, succinylacetone, guanidinoacetate, creatine, and lysophosphatidylcholines. Tandem mass spectrometric methodology, established more than 30 years ago, remains a valid approach for acylcarnitine analysis. The method involves flow-injection analysis of esterified or underivatized acylcarnitines species and detection using a precursor-ion scan. Alternative methods utilize liquid chromatographic separation of isomeric and isobaric species and/or detection by selected reaction monitoring. These technical standards were developed as a resource for diagnostic laboratory practices in acylcarnitine analysis, interpretation, and reporting

CAP/ACMG proficiency testing for biochemical genetics laboratories: a summary of performance

Testing for inborn errors of metabolism is performed by clinical laboratories worldwide, each utilizing laboratory-developed procedures. We sought to summarize performance in the College of American Pathologists’ (CAP) proficiency testing (PT) program and identify opportunities for improving laboratory quality. When evaluating PT data, we focused on a subset of laboratories that have participated in at least one survey since 2010

A missense variant in <i>FTCD</i> is associated with arsenic metabolism and toxicity phenotypes in Bangladesh

Inorganic arsenic (iAs) is a carcinogen, and exposure to iAs via food and water is a global public health problem. iAs-contaminated drinking water alone affects >100 million people worldwide, including ~50 million in Bangladesh. Once absorbed into the blood stream, most iAs is converted to mono-methylated (MMA) and then di-methylated (DMA) forms, facilitating excretion in urine. Arsenic metabolism efficiency varies among individuals, in part due to genetic variation near AS3MT (arsenite methyltransferase; 10q24.32). To identify additional arsenic metabolism loci, we measured protein-coding variants across the human exome for 1,660 Bangladeshi individuals participating in the Health Effects of Arsenic Longitudinal Study (HEALS). Among the 19,992 coding variants analyzed exome-wide, the minor allele (A) of rs61735836 (p.Val101Met) in exon 3 of FTCD (formiminotransferase cyclodeaminase) was associated with increased urinary iAs% (P = 8x10-13), increased MMA% (P = 2x10-16) and decreased DMA% (P = 6x10-23). Among 2,401 individuals with arsenic-induced skin lesions (an indicator of arsenic toxicity and cancer risk) and 2,472 controls, carrying the low-efficiency A allele (frequency = 7%) was associated with increased skin lesion risk (odds ratio = 1.35; P = 1x10-5). rs61735836 is in weak linkage disequilibrium with all nearby variants. The high-efficiency/major allele (G/Valine) is human-specific and eliminates a start codon at the first 5´-proximal Kozak sequence in FTCD, suggesting selection against an alternative translation start site. FTCD is critical for catabolism of histidine, a process that generates one-carbon units that can enter the one-carbon/folate cycle, which provides methyl groups for arsenic metabolism. In our study population, FTCD and AS3MT SNPs together explain ~10% of the variation in DMA% and support a causal effect of arsenic metabolism efficiency on arsenic toxicity (i.e., skin lesions). In summary, this work identifies a coding variant in FTCD associated with arsenic metabolism efficiency, providing new evidence supporting the established link between one-carbon/folate metabolism and arsenic toxicity

Moonlighting Newborn Screening Markers: The Incidental Discovery of a Second-Tier Test for Pompe Disease

Purpose: To describe a novel biochemical marker in dried blood spots suitable to improve the specificity of newborn screening for Pompe disease. Methods: The new marker is a ratio calculated between the creatine/creatinine (Cre/Crn) ratio as the numerator and the activity of acid α-glucosidase (GAA) as the denominator. Using Collaborative Laboratory Integrated Reports (CLIR), the new marker was incorporated in a dual scatter plot that can achieve almost complete segregation between Pompe disease and false-positive cases. Results: The (Cre/Crn)/GAA ratio was measured in residual dried blood spots of five Pompe cases and was found to be elevated (range 4.41–13.26; 99%ile of neonatal controls: 1.10). Verification was by analysis of 39 blinded specimens that included 10 controls, 24 samples with a definitive classification (16 Pompe, 8 false positives), and 5 with genotypes of uncertain significance. The CLIR tool showed 100% concordance of classification for the 24 known cases. Of the remaining five cases, three p.V222M homozygotes, a benign variant, were classified by CLIR as false positives; two with genotypes of unknown significance, one likely informative, were categorized as Pompe disease. Conclusion: The CLIR tool inclusive of the new ratio could have prevented at least 12 of 13 (92%) false-positive outcomes

Precision Newborn Screening for Lysosomal Disorders

Purpose: The implementation of newborn screening for lysosomal disorders has uncovered overall poor specificity, psychosocial harm experienced by caregivers, and costly follow-up testing of false-positive cases. We report an informatics solution proven to minimize these issues. Methods: The Kentucky Department for Public Health outsourced testing for mucopolysaccharidosis type I (MPS I) and Pompe disease, conditions recently added to the recommended uniform screening panel, plus Krabbe disease, which was added by legislative mandate. A total of 55,161 specimens were collected from infants born over 1 year starting from February 2016. Testing by tandem mass spectrometry was integrated with multivariate pattern recognition software (Collaborative Laboratory Integrated Reports), which is freely available to newborn screening programs for selection of cases for which a biochemical second-tier test is needed. Results: Of five presumptive positive cases, one was affected with infantile Krabbe disease, two with Pompe disease, and one with MPS I. The remaining case was a heterozygote for the latter condition. The false-positive rate was 0.0018% and the positive predictive value was 80%. Conclusion: Postanalytical interpretive tools can drastically reduce false-positive outcomes, with preliminary evidence of no greater risk of false-negative events, still to be verified by long-term surveillance

A tailored approach to fusion transcript identification increases diagnosis of rare inherited disease.

BackgroundRNA sequencing has been proposed as a means of increasing diagnostic rates in studies of undiagnosed rare inherited disease. Recent studies have reported diagnostic improvements in the range of 7.5-35% by profiling splicing, gene expression quantification and allele specific expression. To-date however, no study has systematically assessed the presence of gene-fusion transcripts in cases of germline disease. Fusion transcripts are routinely identified in cancer studies and are increasingly recognized as having diagnostic, prognostic or therapeutic relevance. Isolated reports exist of fusion transcripts being detected in cases of developmental and neurological phenotypes, and thus, systematic application of fusion detection to germline conditions may further increase diagnostic rates. However, current fusion detection methods are unsuited to the investigation of germline disease due to performance biases arising from their development using tumor, cell-line or in-silico data.MethodsWe describe a tailored approach to fusion candidate identification and prioritization in a cohort of 47 undiagnosed, suspected inherited disease patients. We modify an existing fusion transcript detection algorithm by eliminating its cell line-derived filtering steps, and instead, prioritize candidates using a custom workflow that integrates genomic and transcriptomic sequence alignment, biological and technical annotations, customized categorization logic, and phenotypic prioritization.ResultsWe demonstrate that our approach to fusion transcript identification and prioritization detects genuine fusion events excluded by standard analyses and efficiently removes phenotypically unimportant candidates and false positive events, resulting in a reduced candidate list enriched for events with potential phenotypic relevance. We describe the successful genetic resolution of two previously undiagnosed disease cases through the detection of pathogenic fusion transcripts. Furthermore, we report the experimental validation of five additional cases of fusion transcripts with potential phenotypic relevance.ConclusionsThe approach we describe can be implemented to enable the detection of phenotypically relevant fusion transcripts in studies of rare inherited disease. Fusion transcript detection has the potential to increase diagnostic rates in rare inherited disease and should be included in RNA-based analytical pipelines aimed at genetic diagnosis

An activating germline IDH1 variant associated with a tumor entity characterized by unilateral and bilateral chondrosarcoma of the mastoid

Chondrogenic tumors involving the temporal bone are rare and typically arise spontaneously with unilateral presentation. Somatic IDH mutations are common in these tumors, but germline inheritance has not been documented to our knowledge. We describe familial chondrosarcoma, grade 1, of the mastoid with unilateral presentation in the mother and bilateral presentation in each of her two children. Each individual presented with headaches, facial paresis, and conductive hearing loss between the ages of 9–12. Exome sequencing of all three affected family members identified a shared germline heterozygous c.299G>A (p.Arg100Gln) missense variant in IDH1. The p.Arg100Gln variant has only rarely been observed as a somatic mutation in glial tumors, and previous in vitro experiments have shown that p.Arg100Gln produces small amounts of the oncometabolite D-2-hydroxyglutarate (D2HG). Biochemical testing in all three affected family members on urine and plasma was unable to detect increases in D2HG in these sample types. Due to insufficient tumor for methylation studies, we performed genome-wide methylation analysis of an IDH1 p.Arg100Gln mutant brain tumor from an unrelated individual to functionally evaluate this variant. These studies demonstrated a global hypermethylation phenotype consistent with other known isocitrate dehydrogenase (IDH) mutant brain tumors, suggesting that this variant has neomorphic activity despite low-level production of D2HG. The bones of the facial skeleton are formed by membranous ossification and we hypothesize that abnormal embryonic cartilage that rests within the suture lines may be involved in this tumor entity. Testing of additional individuals with similar presentations is needed to confirm this finding and clarify the associated phenotypes