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Measurement of lysosomal disease (LD) biomarkers can reveal valuable information about disease status. Lyso-globotriaosylceramide (lyso-Gb 3 ), glucosylsphingosine (lyso-Gb 1 ), galactosylsphingosine (psychosine), and glucose tetrasaccharide (Glca1-6Glca1-4Glca1-4Glc, Glc 4 ) are biomarkers associated with Fabry, Gaucher, Krabbe, and Pompe disease, respectively. Clinical biomarker testing is performed to guide patient management, including monitoring disease progression and initiating treatment, and in diagnostic evaluations of either symptomatic patients or asymptomatic individuals with a positive family history or abnormal newborn screen. Biomarker analysis can be performed through independent analysis of a single analyte or as a multiplex assay measuring analytes for more than one disorder utilizing liquid chromatographic separation and tandem mass spectrometric detection. These guidelines were developed to provide technical standards for biomarker analysis, results interpretation, and results reporting, highlighting Fabry, Gaucher, Krabbe, and Pompe diseases as examples., Competing Interests: Conflict of Interest Funding and support listed here did not support development of this document unless included in the acknowledgments section. A.R.S., P.L.H., and T.R.D. direct clinical laboratories that perform analysis of 1 or more biomarkers on a fee-for-service basis. W.R.W. has served as a consultant to Sanofi, Takeda, Chiesi, Spark, Uniqure, and BioMarin, serves on clinical studies for Sanofi, Takeda, Chiesi, Protalix, Sangamo, 4D Molecular Therapeutics, BioMarin, Pfizer, and Amicus, and has received research support from Takeda and Amicus., (Copyright © 2024 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Thiopurine 6-mercaptopurine (6-MP) is metabolized by thiopurine methyl transferase (TPMT). TPMT genetic variation results in some individuals having reduced or absent TPMT enzyme activity. If these individuals take a full thiopurine dose, life-threatening adverse events can occur. Testing identifies patients with reduced or absent TPMT activity and is recommended before initiation of therapy. The TPMT∗8 allele, defined by c.644G>A (p.Arg215His), is common among individuals of African ancestry (approximately 2.3% minor allele frequency) but is not included in genotyping recommendations due to its uncertain function. Here, a clinical TPMT enzyme activity assay was used to assess TPMT activity in red blood cells from 982 patients, including those with ∗1/∗8 (n = 22), ∗3A/∗8 (n = 1), and ∗3C/∗8 (n = 1) TPMT diplotypes. The average production of 6-methylmercaptopurine (primary TPMT product measured clinically) was 3.08 ± 0.16 nmol/mL per hour for ∗1/∗8 individuals, compared with 3.77 ± 0.03 nmol/mL per hour for normal metabolizers (P = 0.0001) and 2.39 ± 0.06 nmol 6-methylmercaptopurine/mL per hour for intermediate metabolizers (P < 0.0001). Individuals with a TPMT∗1/∗8 diplotype displayed reduced 6-MP metabolism between that of normal metabolizers and intermediate metabolizers, suggesting that TPMT∗8 is a reduced function allele., Competing Interests: Disclosure Statement A.M.M. is a member of the College of American Pathologists/American College of Medical Genetics and Genomics Biochemical and Molecular Genetics Committee and Pharmacogenetics Workgroup, the Association for Molecular Pathology Pharmacogenomics Working Group, and the ClinPGx Scientific Advisory Board., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Metachromatic leukodystrophy (MLD) is a rare neurodegenerative lysosomal storage disease resulting from bi-allelic pathogenic variants in the ARSA gene. MLD is distinguished clinically based on the age of onset into late-infantile, juvenile, and adult. The late-infantile type is the most severe phenotype presenting with hypotonia, weakness, gait abnormalities, which progresses to mental and physical decline leading to early death. MLD is considered to be pan-ethnic and no founder variants have previously been described in the Ashkenazi Jewish population. We identified three unrelated individuals of Ashkenazi Jewish descent with homozygosity or compound heterozygosity for the c.178C>T (p.Arg60Trp) variant in the ARSA gene, with a phenotype consistent with late-infantile MLD. The carrier frequency was calculated among 93,293 individuals of Ashkenazi Jewish descent through the Dor Yeshorim screening program and found to have a carrier frequency on 1 in 1554 or 0.06%, which may be representative of a founder variant. Molecular protein modeling showed that the variant affects regional folding. Late-infantile MLD should be considered when the c.178C>T (p.Arg60Trp) variant in the ARSA gene is present in either the homozygous or the compound heterozygous states., (© 2024 Wiley Periodicals LLC.)

Transferrin isoform analysis is an established laboratory test for congenital disorders of glycosylation (CDG). Despite its long history of clinical use, little has been published about its empirical sensitivity for specific conditions. We conducted a retrospective analysis of ten years of testing data and report our experience with transferrin testing for type I profiles and its sensitivity for the most common congenital disorder of glycosylation, PMM2-CDG. The data demonstrate 94% overall test sensitivity for PMM2-CDG and importantly demonstrate two known, recurrent variants enriched in false positive cases highlighting an important limitation of the test. The data confirm the clinical validity of transferrin isotype analysis as a screening test for disorders of protein N-linked glycosylation and as functional test for PMM2 genotypes of uncertain significance., Competing Interests: Declaration of competing interest None., (Copyright © 2024. Published by Elsevier Inc.)

Treatment of phenylketonuria (PKU) has evolved since the initial introduction of a phenylalanine (Phe) restricted diet. The most recent option for adults affected with PKU is treatment with an alternate enzyme, phenylalanine ammonia lyase (PAL), that metabolizes excess Phe. Proper management of all patients with PKU relies on accurate measurement of Phe levels in blood, to comply with guidance intended to minimize the neurological symptoms. Recently, our laboratory was notified of discrepant results for a patient with PKU who is treated with pegvaliase. Two specimens were collected at the same time but yielded unexpectedly different Phe concentrations. After exclusion of specimen mix-ups or analytical errors, we suspected that there was residual pegvaliase activity in the specimens continuing to degrade Phe after collection. To investigate this possibility, we performed spiking studies that showed the degradation of Phe over time at ambient temperatures. Sample preparation by protein crash appears to deactivate pegvaliase and prevents further Phe degradation. However, because pegvaliase deactivation would be required immediately following blood collection, appropriate mitigation measures must be implemented, including stringent pre-analytical requirements, alternate sample matrices such as dried blood spots, or point of care testing. Until then, health care professionals need to be cautious in their interpretation of Phe levels in their patients with PKU that are treated with pegvaliase., Competing Interests: The authors declare no conflict of interest related to this work., (© 2024 The Authors. Published by Elsevier Inc.)

Creatine transporter deficiency has been described with normal or uninformative levels of creatine and creatinine in plasma, while urine has been the preferred specimen type for biochemical diagnosis. We report a cohort of untreated patients with creatine transporter deficiency and abnormal plasma creatine panel results, characterized mainly by markedly decreased plasma creatinine. We conclude that plasma should be considered a viable specimen type for the biochemical diagnosis of this disorder, and abnormal results should be followed up with further confirmatory testing., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Since the UK commenced newborn screening for isovaleric acidemia in 2015, changes in prescribing have increased the incidence of false positive (FP) results due to pivaloylcarnitine. A review of screening results between 2015 and 2022 identified 24 true positive (TP) and 84 FP cases, with pivalate interference confirmed in 76/84. Initial C5 carnitine (C5C) did not discriminate between FP and TP with median (range) C5C of 2.9 (2.0-9.6) and 4.0 (1.8->70) µmol/L, respectively, and neither did Precision Newborn Screening via Collaborative Laboratory Integrated Reports (CLIR), which identified only 1/47 FP cases. However, among the TP cases, disease severity showed a correlation with initial C5C in 'asymptomatic' individuals ( n = 17), demonstrating a median (range) C5C of 3.0 (1.8-7.1) whilst 'clinically affected' patients ( n = 7), showed a median (range) C5C of 13.9 (7.7-70) µmol/L. These findings allowed the introduction of dual cut-off values into the screening algorithm to reduce the incidence of FPs, with initial C5C results ≥ 5 µmol/L triggering urgent referral, and those >2.0 and <5.0 µmol/L prompting second-tier C5-isobar testing. This will avoid delayed referral in babies at particular risk whilst reducing the FP rate for the remainder., Competing Interests: The authors declare no conflict of interest.

Krabbe disease (KD) is part of newborn screening (NBS) in 11 states with at least one additional state preparing to screen. In July 2021, KD was re-nominated for addition to the federal Recommended Uniform Screening Panel (RUSP) in the USA with a two-tiered strategy based on psychosine (PSY) as the determinant if an NBS result is positive or negative after a first-tier test revealed decreased galactocerebrosidase activity. Nine states currently screening for KD include PSY analysis in their screening strategy. However, the nomination was rejected in February 2023 because of perceived concerns about a high false positive rate, potential harm to newborns with an uncertain prognosis, and inadequate data on presymptomatic treatment benefit or harm. To address the concern about false positive NBS results, a survey was conducted of the eight NBS programs that use PSY and have been screening for KD for at least 1 year. Seven of eight states responded. We found that: (1) the use of PSY is variable; (2) when modeling the data based on the recommended screening strategy for KD, and applying different cutoffs for PSY, each state could virtually eliminate false positive results without major impact on sensitivity; (3) the reason for the diverse strategies appears to be primarily the difficulty of state programs to adjust screening algorithms due to the concern of possibly missing even an adult-onset case following a change that focuses on infantile and early infantile KD. Contracts with outside vendors and the effort/cost of making changes to a program's information systems can be additional obstacles. We recommend that programs review their historical NBS outcomes for KD with their advisory committees and make transparent decisions on whether to accept false positive results for such a devastating condition or to adjust their procedures to ensure an efficient, effective, and manageable NBS program for KD.

Very-Long-Chain Acyl-CoA Dehydrogenase Deficiency (VLCADD) is a fatty acid oxidation disorder characterized by the decreased ability of the enzyme very-long-chain acyl-CoA dehydrogenase to break down fatty acids with 14 to 20-long carbon chains. The resulting clinical manifestations are variable in severity and include hypoketotic hypoglycemia, rhabdomyolysis, and cardiomyopathy. Treatment can consist of limiting the dietary intake of long-chain fatty acids, the prevention of fasting, and the supplementation of medium-chain fats. This study, conducted in the context of a 5-year long-term follow-up on VLCADD, evaluates how the diagnosis of this fatty acid disorder impacts the family, specifically as it relates to the medical diet and barriers to care. Caregivers ( n = 10) of individuals with VLCADD responded to a survey about how VLCADD potentially impacts their family. The review included the clinical outcomes of the patients ( n = 11), covering instances of rhabdomyolysis, cardiomyopathy, and hospitalizations related to VLCADD. Families affected by VLCADD experience barriers to care, including difficulties with finances, ability to work, and access to nutrition.

Although decreased citrulline is used as a newborn screening (NBS) marker to identify proximal urea cycle disorders (UCDs), it is also a feature of some mitochondrial diseases, including MT-ATP6 mitochondrial disease. Here we describe biochemical and clinical features of 11 children born to eight mothers from seven separate families who were identified with low citrulline by NBS (range 3-5 μM; screening cutoff >5) and ultimately diagnosed with MT-ATP6 mitochondrial disease. Follow-up testing revealed a pattern of hypocitrullinemia together with elevated propionyl-(C3) and 3-hydroxyisovaleryl-(C5-OH) acylcarnitines, and a homoplasmic pathogenic variant in MT-ATP6 in all cases. Single and multivariate analysis of NBS data from the 11 cases using Collaborative Laboratory Integrated Reports (CLIR; https://clir.mayo.edu) demonstrated citrulline <1st percentile, C3 > 50th percentile, and C5-OH >90th percentile when compared with reference data, as well as unequivocal separation from proximal UCD cases and false-positive low citrulline cases using dual scatter plots. Five of the eight mothers were symptomatic at the time of their child(ren)'s diagnosis, and all mothers and maternal grandmothers evaluated molecularly and biochemically had a homoplasmic pathogenic variant in MT-ATP6, low citrulline, elevated C3, and/or elevated C5-OH. All molecularly confirmed individuals (n = 17) with either no symptoms (n = 12), migraines (n = 1), or a neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) phenotype (n = 3) were found to have an A or U mitochondrial haplogroup, while one child with infantile-lethal Leigh syndrome had a B haplogroup., (© 2023 Wiley Periodicals LLC.)

Introduction: Plasma amino acids profiling can aid in the screening and diagnosis of aminoacidopathies. The goal of the current study was to analyze and report the metabolic profiles of plasma amino acid (PAA) and additionally to compare PAA-reference intervals (RI) from Pakistan with more countries utilizing Clinical Laboratory Integrated Reports (CLIR)., Methods: This was a cross sectional prospective single center study. Twenty-two amino acids were analyzed in each sample received for one year at the clinical laboratory . Data was divided into reference and case data files after interpretation by a team of pathologists and technologists. All PAA samples were analyzed using ion-exchange high-performance chromatography. The CLIR application of Amino Acid in Plasma (AAQP) was used for statistical analysis for both data sets and post-analytical interpretive tools using a single condition tool was applied., Result: The majority of 92% (n = 1913) of PAA profiles out of the total 2081 tests run were non-diagnostic; the PAA values were within the age-specific RI. The PAA median was in close comparison close to the 50th percentile of reference data available in CLIR software. Out of the total 2081 tests run, one hundred and sixty-eight had abnormal PAA levels; 27.38% were labeled as non-fasting samples, and the main aminoacidopathies identified were Phenylketonuria and Maple Syrup Urine Disorder., Conclusion: An agreement of >95% was observed between the reporting done by the pathologists and technologists' team and then after the application of CLIR. Augmented artificial intelligence using CLIR can improve the accuracy of reporting rare aminoacidopathies in a developing country like ours., Competing Interests: Authors state no conflict of interest., (© 2022 Published by Elsevier Ltd on behalf of IJS Publishing Group Ltd.)

The purpose of this pilot project was to evaluate the efficacy of the Collaborative Integrated Laboratory Reports (CLIR) postanalytical tools from Mayo Clinic for detection of newborns with proximal urea cycle disorders (PUCD) in the Georgia newborn screening program that uses the underivatized Neobase2 kit (Perkin Elmer). We evaluated 138,560 newborn screening (NBS) samples (between 125,000 and 130,000 children) and used the CLIR result interpretation guidelines to stratify results. Children at higher risk of having a PUCD received follow-up services including confirmatory lab testing (ammonia, plasma amino acids, urine orotic acid) or a repeat NBS sample. We made multiple adjustments to our CLIR PUCD tool and to our follow-up algorithms in order to reduce false positives. Regardless, a high number of NBS samples resulted with false positives in part due to the glutamine peak also containing lysine. No children were diagnosed with a PUCD during our study period, and the Emory Genetics Metabolic Center is unaware of any children diagnosed outside of the NBS system during that time. Based on our experience, PUCD is not suitable for statewide NBS using Neobase2 and CLIR. Other methodologies that can separate glutamine from other amino acids may have better performance., (© 2022 Wiley Periodicals LLC.)

Healthcare workers have never faced a medical crisis that compares to the coronavirus disease-2019 pandemic. This modern-day pandemic fight draws parallels to a war. Because of these similarities, it would make sense that the experiences frontline providers have when transitioning to a normal healthcare routine would emulate experiences service members voice when reintegrating home from a battlefield. These common experiences include a unified mission, an exhausting, adrenaline-packed responsibility, and a celebrity-like status to the public. The pandemic response consumed healthcare with one united mission, but as we reemerge from the pandemic and restore other aspects of our healthcare system, multiple missions develop and compete. Returning to a common routine and regular status can manifest feelings of disappointment in healthcare workers' everyday lives and career choices and lead to a reexamination of priorities and professions. As an organization with a focus on high reliability, mitigating the harm to staff and delivery system may be our new priority. The risk of not facing this situation head on is a potential exodus of seasoned professionals contemplating their future and selecting other career paths, thus draining the current institutional knowledge and potentially deterring future generations from healthcare. Leaders in the healthcare industry need to take a proactive stance in addressing this reemergence. Integrated, proactive programming is needed utilizing evidence-based resiliency training from professional organizations such as the National Alliance on Mental Illness, the Department of Health and Human Services' Substance Abuse and Mental Health Services Administration, as well as the existing Department of Defense programs. The Veterans Affairs has the backbone to develop and offer these programs and make a positive difference with Employee Whole Health efforts. Organized, evidence-based programming such as second victim education, mindfulness, and other personal skill building could be key to improving the lifelong well-being of our caregivers., (© The Association of Military Surgeons of the United States 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Newborn screening for congenital hypothyroidism remains challenging decades after broad implementation worldwide. Testing protocols are not uniform in terms of targets (TSH and/or T4) and protocols (parallel vs. sequential testing; one or two specimen collection times), and specificity (with or without collection of a second specimen) is overall poor. The purpose of this retrospective study is to investigate the potential impact of multivariate pattern recognition software (CLIR) to improve the post-analytical interpretation of screening results. Seven programs contributed reference data (N = 1,970,536) and two sets of true (TP, N = 1369 combined) and false (FP, N = 15,201) positive cases for validation and verification purposes, respectively. Data were adjusted for age at collection, birth weight, and location using polynomial regression models of the fifth degree to create three-dimensional regression surfaces. Customized Single Condition Tools and Dual Scatter Plots were created using CLIR to optimize the differential diagnosis between TP and FP cases in the validation set. Verification testing correctly identified 446/454 (98%) of the TP cases, and could have prevented 1931/5447 (35%) of the FP cases, with variable impact among locations (range 4% to 50%). CLIR tools either as made here or preferably standardized to the recommended uniform screening panel could improve performance of newborn screening for congenital hypothyroidism.

Background: Glutaric acidemia type I (GA1) is an organic acidemia that is often unrecognized in the newborn period until patients suffer an acute encephalopathic crisis, which can be mistaken for nonaccidental trauma. Presymptomatic identification of GA1 patients is possible by newborn screening (NBS). However, the biochemical "low-excretor" (LE) phenotype with nearly normal levels of disease metabolites can be overlooked, which may result in untreated disease and irreversible neurological sequelae. The LE phenotype is also a potential source of false negative (FN) NBS results that merits further investigation., Methods: Samples from six LE GA1 patients were analyzed by biochemical and molecular methods and newborn screen outcomes were retrospectively investigated., Results: Five LE GA1 patients were identified that had normal NBS results and three of these presented clinically with GA1 symptoms. One additional symptomatic patient was identified who did not undergo screening. Semiquantitative urine organic acid analysis was consistent with a GA1 diagnosis in two (33%) of the six patients, while plasma glutarylcarnitine was elevated in four (67%) of the six and urine glutarylcarnitine was elevated in four (80%) of five patients. Five GCDH variants were identified in these patients; three of which have not been previously linked to the biochemical LE phenotype., Conclusions: The data presented here raise awareness of potential FN NBS results for LE GA1 patients. The LE phenotype is not protective against adverse clinical outcomes, and the possibility of FN NBS results calls for high vigilance amongst clinicians, even in the setting of a normal NBS result., Competing Interests: The authors declare no potential conflict of interest., (© 2021 The Authors. JIMD Reports published by John Wiley & Sons Ltd on behalf of SSIEM.)

We screened 51,081 newborns for X-linked adrenoleukodystrophy (ALD) using a two-tiered strategy quantifying very long chain lysophosphatadylcholines (LPC). Our testing strategy used flow injection tandem mass spectrometry for the first-tier analysis of LPCs, and second-tier quantification of C26:0 LPC using liquid chromatography tandem mass spectrometry. There were 364 specimens considered abnormal using our first-tier algorithm that relied on the four LPC measurements and post-analytical tools. Second-tier test results were reported as normal or abnormal based on a cutoff for the single analyte, C26:0 LPC. Eleven cases were reported as abnormal based on second-tier test results. One male with ALD was identified, and two females with peroxisomal biogenesis disorders were also identified. A single female case remains unresolved, due to a loss to follow up after a negative molecular test result for ABCD1 gene sequencing. The positive predictive value for confirmed, clinically relevant disorders during this pilot study was 27.3%. Challenges identified during the study period were based around coverage for confirmatory testing, particularly if family members needed molecular testing, which is an ongoing issue with newborn screening in Georgia. We also encountered issues with the follow up for a patient who remained asymptomatic. Due to the different timelines involved with clinical findings in ALD, follow-up coordination may be more difficult, particularly if the child identified by newborn screening (NBS) is the only member of the family affected, or able to be tested.

Georgia uses post-analytical tools through Collaborative Laboratory Integrated Reports (CLIR) to triage abnormal newborn screening (NBS) results for follow-up. Condition specific tools are used to assign each case a risk level, which is used to guide follow-up recommendations. Follow-up recommendations include assessment by the child's primary care provider as well as testing, either a repeat NBS or confirmatory testing. Triaging abnormal cases using these tools has been advantageous in managing the workflow for the follow-up team, as well as prioritizing cases that appropriately require more attention and resources. The initial goal in utilizing these tools was to reduce the amount of confirmatory testing, particularly for disorders where there are many false positives. We assessed the performance of these tools retrospectively for three of the most commonly detected conditions by tandem mass spectrometry in Georgia: phenylketonuria, medium chain acyl-CoA dehydrogenase deficiency and very long chain dehydrogenase deficiency. The post-analytical tools appropriately assigned all true positive cases to the higher levels of follow-up testing and reduced the level of intervention for a significant number of cases as well. Based on the experience gained from our utilization of the tools in the follow-up program, we are well situated to move forward with using the tools in a more prospective manner, and reduce the number of cases that will be reported, rather than just assigning resources appropriately at follow-up. Post-analytical tools are an improvement over trying to capture the variation in the newborn population using multiple cutoffs. It also easily identifies significant abnormalities that are unrelated to inherited disease, such as large amino acid elevations due to total parenteral nutrition., Competing Interests: Conflicts of InterestThe authors declare no conflicts of interest., (© 2020 by the authors.)

We conducted a pilot newborn screening (NBS) study for Pompe disease (PD) and mucopolysaccharidosis type I (MPS I) in the multiethnic population of Georgia. We screened 59,332 infants using a two-tier strategy of flow injection tandem mass spectrometry (FIA-MSMS) enzyme assays. The first tier of testing was a 2-plex assay measuring PD and MPS I enzyme activity, followed by a second-tier test with additional enzymes to improve specificity. Interpretation of results was performed using post-analytical tools created using Collaborative Laboratory Integrated Reports (CLIR). We identified a single case of infantile onset PD, two cases of late onset PD, and one pseudodeficiency. The positive predictive value (PPV) for PD screening during the study was 66.7%. No cases of MPS I were identified during the study period, but there were 2 confirmed cases of pseudodeficiency and 6 cases lost to follow up. The two-tier screening strategy was successful in reducing false positive results and allowed for the identification and early treatment of a case of infantile PD but the frequency of pseudodeficiency in MPS I is problematic. Molecular testing is required and should be covered by the screening program to avoid delays in case resolution., Competing Interests: Conflicts of Interest: The authors declare no conflict of interest.

The long-term consequences and need for therapy in children with short-chain acyl-CoA dehydrogenase deficiency (SCADD) or isobutyryl-CoA dehydrogenase deficiency (IBDD) identified via newborn screening (NBS) remains controversial. Initial clinical descriptions were severe; however, while most cases identified through NBS have remained asymptomatic, clinical concerns have been raised in these populations. It is not clear whether these children are asymptomatic because of the success of NBS, or because the normal clinical course of these disorders is relatively benign. To evaluate these possibilities in our program, we evaluated the clinical outcomes of children with SCADD or IBDD identified by the Georgia NBS compared to the health status of a healthy age-matched control group. We also assessed parental anxiety during a phone interview both subjectively and objectively using the Pediatric Inventory for Parents (PIP), a validated measure of illness-related parental stress. The general health of 52 SCADD and nine IBDD cases from 2007 to 2016 were compared to the general health of unaffected control children obtained through the Centers for Disease Control and Prevention (CDC) parent listserv. We also collected statements from parents who participated in a phone survey regarding events they experienced during and after their diagnostic process. Overall, the children with SCADD and IBDD had no major health problems. There was no significant difference in cognitive development (p = .207). We identified a slightly higher incidence of reported neonatal hypoglycemia in the SCADD group; two of these occurred in the context of maternal diabetes. All interviewed parents reported extreme anxiety during the diagnostic period and current feelings of uncertainty about their child's future. PIP scores for all six caregivers who responded to that portion of the survey were consistent with some degree of parental stress. The greatest reported stressor was the unknown long-term impact of the illness. All children with SCADD and IBDD had no significant long-term sequelae. The phone interviews revealed substantial parental anxiety about the identification and follow-up of SCADD and IBDD. Based on our findings, the anxiety parents experience may be unwarranted given that we see no disease-associated morbidity or mortality in these children. Consideration should be given to the removal of these conditions from NBS panels, or if that is not possible, clinicians could educate parents on the benign nature of these diagnoses and release them from follow-up without treatment., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.

N-glycanase deficiency (NGLY1 deficiency, NGLY1-CDDG), the first autosomal recessive congenital disorder of N-linked deglycosylation (CDDG), is caused by pathogenic variants in NGLY1. The majority of affected individuals have been identified using exome or genome sequencing. To date, no reliable, clinically available biomarkers have been identified. Urine oligosaccharide analysis was included as part of a routine evaluation for possible biomarkers in patients with confirmed NGLY1-CDDG. During the qualitative review of oligosaccharide profiles by an experienced laboratory director an abnormal analyte with a proposed structure of Neu5Ac1Hex1GlcNAc1-Asn was identified in NGLY1-CDDG patient urine samples. The same species has been observed in profiles from individuals affected with aspartylglucosaminuria, although the complete spectra are not identical. Additional studies using tandem mass spectrometry confirmed the analyte's structure. In addition to the known NGLY1-CDDG patients identified by this analysis, a single case was identified in a population referred for clinical testing who subsequently had a diagnosis of NGLY1-CDDG confirmed by molecular testing. Urine oligosaccharide screening by MALDI-TOF MS can identify individuals with NGLY1-CDDG. In addition, this potential biomarker might also be used to monitor the effectiveness of therapeutic options as they become available., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Hereditary fructose intolerance (HFI) is an autosomal recessive disorder caused by aldolase B (ALDOB) deficiency resulting in an inability to metabolize fructose. The toxic accumulation of intermediate fructose-1-phosphate causes multiple metabolic disturbances, including postprandial hypoglycemia, lactic acidosis, electrolyte disturbance, and liver/kidney dysfunction. The clinical presentation varies depending on the age of exposure and the load of fructose. Some common infant formulas contain fructose in various forms, such as sucrose, a disaccharide of fructose and glucose. Exposure to formula containing fructogenic compounds is an important, but often overlooked trigger for severe metabolic disturbances in HFI. Here we report four neonates with undiagnosed HFI, all caused by the common, homozygous mutation c.448G>C (p.A150P) in ALDOB, who developed life-threatening acute liver failure due to fructose-containing formulas. These cases underscore the importance of dietary history and consideration of HFI in cases of neonatal or infantile acute liver failure for prompt diagnosis and treatment of HFI., (Copyright © 2018 Elsevier Inc. All rights reserved.)

GM2 activator (GM2A) deficiency (OMIM 613109) is a rare lysosomal storage disorder, with onset typically in infancy or early childhood. Clinically, it is almost indistinguishable from Tay-Sachs disease (OMIM 272800) or Sandhoff disease (OMIM 268800); however, traditionally available biochemical screening tests will most likely reveal normal results. We report a 2-year-old male with initially normal development until the age of 9 months, when he presented with developmental delay and regression. Workup at that time was unrevealing; at 15 months, he had abnormal brain MRI findings and a cherry red spot on ophthalmological examination. Family history and all laboratory studies were uninformative. The combination of a cherry red spot and developmental regression was strongly suggestive of a lysosomal storage disorder. Sequence analysis of GM2A did not reveal any pathogenic variants; however, exon 2 of GM2A could not be amplified by PCR, raising suspicion for a large, homozygous deletion. Subsequent copy number analysis confirmed a homozygous deletion of exon 2 in GM2A. This is the first reported case of GM2A deficiency being caused by a whole exon deletion. We describe previously unreported electron microscopy findings in this disease, thus expanding the clinical and variant spectrum for GM2 activator deficiency. These findings demonstrate the increased degree of suspicion required for diagnosis of this rare disorder. Brief Summary: This case of GM2 activator deficiency was caused by a homozygous deletion in GM2A, demonstrating the need to include exon level copy number analysis in any workup to fully exclude this disorder.

Background: Outbreaks of unexplained illness frequently remain under-investigated. In India, outbreaks of an acute neurological illness with high mortality among children occur annually in Muzaffarpur, the country's largest litchi cultivation region. In 2014, we aimed to investigate the cause and risk factors for this illness., Methods: In this hospital-based surveillance and nested age-matched case-control study, we did laboratory investigations to assess potential infectious and non-infectious causes of this acute neurological illness. Cases were children aged 15 years or younger who were admitted to two hospitals in Muzaffarpur with new-onset seizures or altered sensorium. Age-matched controls were residents of Muzaffarpur who were admitted to the same two hospitals for a non-neurologic illness within seven days of the date of admission of the case. Clinical specimens (blood, cerebrospinal fluid, and urine) and environmental specimens (litchis) were tested for evidence of infectious pathogens, pesticides, toxic metals, and other non-infectious causes, including presence of hypoglycin A or methylenecyclopropylglycine (MCPG), naturally-occurring fruit-based toxins that cause hypoglycaemia and metabolic derangement. Matched and unmatched (controlling for age) bivariate analyses were done and risk factors for illness were expressed as matched odds ratios and odds ratios (unmatched analyses)., Findings: Between May 26, and July 17, 2014, 390 patients meeting the case definition were admitted to the two referral hospitals in Muzaffarpur, of whom 122 (31%) died. On admission, 204 (62%) of 327 had blood glucose concentration of 70 mg/dL or less. 104 cases were compared with 104 age-matched hospital controls. Litchi consumption (matched odds ratio [mOR] 9·6 [95% CI 3·6 - 24]) and absence of an evening meal (2·2 [1·2-4·3]) in the 24 h preceding illness onset were associated with illness. The absence of an evening meal significantly modified the effect of eating litchis on illness (odds ratio [OR] 7·8 [95% CI 3·3-18·8], without evening meal; OR 3·6 [1·1-11·1] with an evening meal). Tests for infectious agents and pesticides were negative. Metabolites of hypoglycin A, MCPG, or both were detected in 48 [66%] of 73 urine specimens from case-patients and none from 15 controls; 72 (90%) of 80 case-patient specimens had abnormal plasma acylcarnitine profiles, consistent with severe disruption of fatty acid metabolism. In 36 litchi arils tested from Muzaffarpur, hypoglycin A concentrations ranged from 12·4 μg/g to 152·0 μg/g and MCPG ranged from 44·9 μg/g to 220·0 μg/g., Interpretation: Our investigation suggests an outbreak of acute encephalopathy in Muzaffarpur associated with both hypoglycin A and MCPG toxicity. To prevent illness and reduce mortality in the region, we recommended minimising litchi consumption, ensuring receipt of an evening meal and implementing rapid glucose correction for suspected illness. A comprehensive investigative approach in Muzaffarpur led to timely public health recommendations, underscoring the importance of using systematic methods in other unexplained illness outbreaks., Funding: US Centers for Disease Control and Prevention., (Copyright © 2017 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND license. Published by Elsevier Ltd.. All rights reserved.)

Purpose: As exome and genome sequencing using high-throughput sequencing technologies move rapidly into the diagnostic process, laboratories and clinicians need to develop a strategy for dealing with uncertain findings. A commitment must be made to minimize these findings, and all parties may need to make adjustments to their processes. The information required to reclassify these variants is often available but not communicated to all relevant parties., Methods: To illustrate these issues, we focused on three well-characterized monogenic, metabolic disorders included in newborn screens: classic galactosemia, caused by GALT variants; phenylketonuria, caused by PAH variants; and medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, caused by ACADM variants. In 10 years of clinical molecular testing, we have observed 134 unique GALT variants, 46 of which were variants of uncertain significance (VUS). In PAH, we observed 132 variants, including 17 VUS, and for ACADM, we observed 64 unique variants, of which 33 were uncertain., Conclusion: After this review, 17 VUS (37%; 7 in ACADM, 9 in GALT, and 1 in PAH) were reclassified from uncertain (6 to benign or likely benign and 11 to pathogenic or likely pathogenic). We identified common types of missing information that would have helped make a definitive classification and categorized this information by ease and cost to obtain.Genet Med 19 1, 77-82.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a rare but important component of the differential diagnosis for adults with a history of premortem mental status changes and the postmortem finding of hepatic steatosis. This case report describes a 30-year-old white man who, following a period of nausea and vomiting, was admitted to the hospital with sudden mental status deterioration followed rapidly by clinical deterioration and death. Treating physicians in this case suspected acute illicit drug toxicity with synthetic cathinones based on social history. Clinicians and medical examiners should be aware that the presentation, signs, and symptoms described may indicate an underlying inborn error of metabolism such as MCAD deficiency and take action accordingly., (© 2015 American Academy of Forensic Sciences.)

Background: Urinary concentrations of creatine and guanidinoacetic acid divided by creatinine are informative markers for cerebral creatine deficiency syndromes (CDSs). The renal excretion of these substances varies substantially with age and sex, challenging the sensitivity and specificity of postanalytical interpretation., Methods: Results from 155 patients with CDS and 12 507 reference individuals were contributed by 5 diagnostic laboratories. They were binned into 104 adjacent age intervals and renormalized with Box-Cox transforms (Ξ). Estimates for central tendency (μ) and dispersion (σ) of Ξ were obtained for each bin. Polynomial regression analysis was used to establish the age dependence of both μ[log(age)] and σ[log(age)]. The regression residuals were then calculated as z-scores = {Ξ - μ[log(age)]}/σ[log(age)]. The process was iterated until all z-scores outside Tukey fences ±3.372 were identified and removed. Continuous percentile charts were then calculated and plotted by retransformation., Results: Statistically significant and biologically relevant subgroups of z-scores were identified. Significantly higher marker values were seen in females than males, necessitating separate reference intervals in both adolescents and adults. Comparison between our reconstructed reference percentiles and current standard age-matched reference intervals highlights an underlying risk of false-positive and false-negative events at certain ages., Conclusions: Disease markers depending strongly on covariates such as age and sex require large numbers of reference individuals to establish peripheral percentiles with sufficient precision. This is feasible only through collaborative data sharing and the use of appropriate statistical methods. Broad application of this approach can be implemented through freely available Web-based software., (© 2015 American Association for Clinical Chemistry.)