Most spinal muscular atrophy (SMA) carrier screening assays quantify SMN1 exon 7 copy number, providing limited information about the prevalence or structure of SMN1–SMN2 hybrid alleles generated through gene conversion. As long-read sequencing (LRS) studies begin to resolve these alleles at high resolution, typically in modest cohorts, large population-scale data remain essential for defining the broader allele-frequency context in which such structural signatures occur. We evaluated SMN1 exon 7 and exon 8 copy number in 18,015 individuals undergoing expanded carrier screening using a SNP-based array. Carriers were classified by deletion pattern, and representative cases underwent droplet digital PCR (ddPCR) to distinguish contiguous deletions from gene-conversion–derived hybrid alleles based on coordinated SMN1/SMN2 exon and intron copy number profiles. Among 457 carriers (2.5

COASY-related disorders (CRDs) are a spectrum of autosomal recessive conditions caused by the dysfunction of CoA synthase, an enzyme responsible for the final steps of CoA synthesis. Clinical manifestations of CRDs are highly variable, ranging from perinatal lethal pontocerebellar hypoplasia to childhood-onset neurodegenerative brain iron accumulation, which is often recognized after clinical regression. Recent reports have described a few individuals with CRD who screened positive for carnitine palmitoyltransferase-I deficiency by newborn screening (NBS). However, heterogeneous clinical presentations, conflicting biochemical/molecular sequencing of CPT1A, and a lack of metabolic characterization have led to lengthy, costly diagnostic journeys. To address some of these aspects, this investigation retrospectively evaluated NBS acylcarnitine patterns in five CRD cases using Collaborative Laboratory Integrated Reports (CLIR). A total of 25 metabolites/ratios were identified to deviate significantly from reference ranges and were primarily composed of elevated free carnitine and reduced long-chain acylcarnitine levels. While low acylcarnitine concentrations are often not reported due to a lack of lower reference cutoffs, ratios involving these metabolites relative to short-chain acylcarnitines could aid in identifying CRD cases via NBS. When comparing this pattern to CPT-Ia cases, we confirmed a nearly identical acylcarnitine pattern between these, and thus support the need to consider CRD in cases with NBS results suggestive of CPT-Ia. This study is the first case series to characterize NBS patterns in patients with CRD and highlights the unique opportunity for early detection, particularly in cases that are neonatally asymptomatic and have unremarkable confirmatory biochemical results.

The alpha globin gene cluster on chromosome 16 encodes alpha-like globins whose expression changes during development. HBM located in this cluster was considered a pseudogene until its transcript was discovered in 2005, but without a detectable protein product. Here, we provide the first detailed description of HBM-encoded globin at the protein level (hemoglobin mu chain [HBM]) across different age groups using liquid chromatography coupled with tandem mass spectrometry. Using targeted analysis, we demonstrate that HBM protein is readily detectable in circulation with its levels falling with increasing gestational age. We also report HBM levels in relation to other hemoglobin chains in neonatal, pediatric, and adult blood. Furthermore, analysis of alpha thalassemia patient samples revealed that HBM levels were elevated 3- to 17-fold, indicating a compensatory role, which could be explored for its therapeutic potential. This study enhances our understanding of erythropoiesis and globin chain production through ontogeny.

Purines and pyrimidines are key cellular components for the development, proliferation, and repair of cells and tissues, whose circulating levels are associated with various diseases. Biospecimen processing delays can significantly induce hypoxanthine and inosine levels, among other changes, in blood. We developed a liquid chromatography tandem mass-spectrometry method to quantify metabolites of the purine and pyrimidine pathways and investigated the impact of delayed sample processing on single donor plasma and dried blood spots (DBS). Plasma hypoxanthine increased above the normal reference range to an average 20-fold increase across individual donors when whole blood was kept refrigerated 64 h prior to processing. The same trend was observed in dried blood spots (DBS) prepared from refrigerated whole blood. Plasma inosine also increased above the reference range after a 48-hour delay in processing refrigerated whole blood. In contrast, hypoxanthine remained stable in DBS for up to 48 h, regardless of the card storage temperature. Therefore, DBS is a feasible alternative specimen type to measure hypoxanthine if frozen shipping, delayed centrifugation, or storage interval is a concern. Understanding the impact of specimen handling conditions is essential for successful biomarker discovery, especially when new biomarkers are to be introduced into the clinical laboratory.

PURPOSE:Infantile Krabbe disease (IKD) is a severe, progressive neurological disorder that was recently added to the Recommended Uniform Screening Panel in the United States. IKD is a critical condition that requires a hematopoietic stem cell transplant, preferably by 30 days of age. This study examines whether the 2-tier newborn screening (NBS) strategy (psychosine [PSY] analysis when enzyme activity is low) causes clinically relevant delays compared with PSY measured at a first clinical follow-up visit. METHODS:We reviewed the PSY analyses performed over a 6-year period on dried blood spots on infants < 28 days old. Results were sorted by the ordering site and final diagnosis. RESULTS:On average, infants with a positive NBS for IKD that included abnormal PSY received their diagnosis 7 days sooner than whose PSY testing was initiated during follow-up. Hematopoietic stem cell transplant occurred by the 36th day of life, on average, for cases with IKD detected by 2-tiered NBS. CONCLUSION:Effective NBS for Krabbe disease requires the use of PSY as a second-tier test, allowing for timely initiation of treatment while virtually eliminating false-positive results. PSY should be included in NBS algorithms for Krabbe disease, and timeliness metrics need to be adjusted.

ObjectivesIn this study, we describe a 54-year-old Indian woman who presented with clinical features of Kufs syndrome A (KSA) and Kufs syndrome B (KSB), as well as neuropathologic and genetic findings consistent with neuronal ceroid lipofuscinosis type 13 (CLN13). Subsequently, we review the clinicopathologic features of 20 patients with CLN13 reported in the literature.MethodsData and imaging were obtained from the patient's medical records. The patient was examined neuropathologically, and next-generation sequencing was performed.ResultsClinical radiologic scans revealed bilateral cortical atrophy, ventriculomegaly, a thin corpus callosum, and cerebellar vermian atrophy. Pathologic examination was remarkable for NCL. Postmortem genetic testing revealed a homozygous cathepsin F (CTSF) indel variant. A review of 20 reported CLN13 patients revealed novel clinical subtypes, including KSB type I (KSB-I), KSB type II (KSB-II), and Kufs syndrome C (KSC).DiscussionCLN13 was clinically heterogeneous. Most patients with CLN13 (14/20) did not present with classic KSB (KSB-I). Instead, 6 patients presented with KSB-II, 4 patients presented with KSC (including the present patient), and 3 patients presented with dementia. Our results expand the CLN13 clinical spectrum and emphasize the importance of screening CTSF variants in clinical dementia and movement disorder cohorts.

High-resolution urine oligosaccharide screening with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been clinically available for 10 years and has been used to help diagnose a wide spectrum of disorders. The untargeted nature of MALDI-TOF MS analysis gives it broad clinical utility. Here we report characteristic profiles for several disorders that had not previously been described using this method, including lysosomal disorders, congenital disorders of glycosylation, and glycogen storage disorders. In addition, we review the clinical performance of the assay in our laboratory and provide basic test performance information for each condition. A total of 4445 analyses performed with 4145 unique patient samples generated 139 confirmed and presumptive positive results and 16 false-positive results. The disorder with the highest rate of false positivity was Pompe disease, which has an oligosaccharide profile similar to that seen in formula-fed infants. The positive predictive value of urine oligosaccharide screening with MALDI-TOF MS was 89.7 % across all conditions. For 10 of the 16 conditions detected during the study period, no false-positive results occurred. No known false-negative results were reported for any of the targeted conditions. Urine oligosaccharide analysis with MALDI-TOF MS, which uses an accessible specimen type and offers quick turnaround time, is an effective initial screening method for patients with a clinical presentation suggestive of a lysosomal disorder.

These data demonstrate the clinical utility of urine sorbitol and xylitol analysis in the screening and diagnosis of SORD deficiency-related neuropathy. In addition, this is the first description of elevated xylitol in SORD deficiency as a clinically informative biomarker, which may increase the specificity of biochemical testing.

More than 60 lysosomal disorders have been described to date, and continued advancements in molecular (i.e., next generation sequencing) and biochemical (i.e., mass spectrometry) genetic testing will increase this number. In parallel, the same advancements have improved laboratory efficiency by allowing the simultaneous measurement of multiple enzyme activities and/or biomarker concentrations, as well as the rapid generation of genomic information with fewer tests. Here, we provide an overview of currently available biochemical and molecular genetic tests, and how they and their correlation to each other can support screening, diagnosis, and monitoring of patients with lysosomal disorders.