Rinaldo, Piero

Homocystinuria (HCU) due to cystathionine-β-synthase deficiency is generally regarded as a rare disease, but within the Qatari population has an incidence of 1 in 1800 live births. Most newborn screening methods for HCU using dried blood spots (DBS) rely on the detection of an elevated methionine level or a rapid screen for total homocysteine (tHCY). However, screening based on methionine levels alone lacks specificity and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for tHCY exhibit variable results with high false positive rates. This report describes a LC-MS/MS method for detection of tHCY on DBS, with improved specificity. tHCY was extracted from DBS with a solution containing dithiothreitol and subsequently butylated with hydrochloric acid in n-butanol. The butyl esters were separated by liquid chromatography on a reverse-phase column and the homocysteine (HCY), detected by tandem mass spectrometry. The butyl ester of HCY eluted at 1.8 min. Total analysis time was 6.1 min per sample, including column flush and equilibration. This method allows for the quantification of tHCY over a linear range from 0.3 to 200 µM. Intraassay and interassay imprecision and recoveries were acceptable. Good concordance was observed with another LC-MS/MS method. Application of this method improves specificity and reduces false positive rates in screening for HCU.

AbstractBackgroundElevated plasma and urine formiminoglutamic acid (FIGLU) levels are commonly indicative of formiminoglutamic aciduria (OMIM #229100), a poorly understood autosomal recessive disorder of histidine and folate metabolism, resulting from formiminotransferase‐cyclodeaminase (FTCD) deficiency, a bifunctional enzyme encoded by FTCD.MethodsIn order to further understanding about the molecular alterations that contribute to FIGLU‐uria, we sequenced FTCD in 20 individuals with putative FTCD deficiency and varying laboratory findings, including increased FIGLU excretion.ResultsIndividuals tested had biallelic loss‐of‐function variants in protein‐coding regions of FTCD. The FTCD allelic spectrum comprised of 12 distinct variants including 5 missense alterations that replace conserved amino acid residues (c.223A>C, c.266A>G, c.319T>C, c.430G>A, c.514G>T), an in‐frame deletion (c.1373_1375delTGG), with the remaining alterations predicted to affect mRNA processing/stability. These included two frameshift variants (c.990dup, c.1366dup) and four nonsense variants (c.337C>T, c.451A>T, c.763C>T, c.1607T>A).ConclusionWe observed additional FTCD alleles leading to urinary FIGLU elevations, and thus, providing molecular evidence of FTCD deficiency in cases identified by newborn screening or clinical biochemical genetic laboratory testing.

Transplantation of gene-corrected autologous hepatocytes can cure metabolic disease in a preclinical pig model of hereditary tyrosinemia type 1.

Abstract BACKGROUND Newborn screening for lysosomal storage disorders (LSD) has revealed that late-onset variants of these conditions are unexpectedly frequent and therefore may evade diagnosis. We developed an efficient and cost-effective multiplex assay to diagnose six LSDs and several peroxisomal disorders in patients presenting with diverse phenotypes at any age. METHODS Three 3-mm dried blood spot (DBS) punches were placed into individual microtiter plates. One disc was treated with a cocktail containing acid sphingomyelinase-specific substrate and internal standard (IS). To the second DBS we added a cocktail containing substrate and IS for β-glucosidase, acid α-glucosidase, α-galactosidase A, galactocerebrosidase, and α-L-iduronidase. The third DBS was extracted with methanol containing d4-C26 lysophosphatidylcholine as IS and stored until the enzyme plates were combined and purified by liquid–liquid and solid-phase extraction. The extracts were evaporated, reconstituted with the extract from the lysophosphatidylcholine plate, and analyzed by flow injection tandem mass spectrometry. RESULTS Reference intervals were determined by analysis of 550 samples from healthy controls. DBS from confirmed patients with 1 of the 6 LSDs (n = 33), X-adrenoleukodystrophy (n = 9), or a peroxisomal biogenesis disorder (n = 5), as well as carriers for Fabry disease (n = 17) and X-adrenoleukodystrophy (n = 5), were analyzed for assay validation. Prospective clinical testing of 578 samples revealed 25 patients affected with 1 of the detectable conditions. CONCLUSIONS Our flow injection tandem mass spectrometry approach is amenable to high-throughput population screening for Hurler disease, Gaucher disease, Niemann–Pick A/B disease, Pompe disease, Krabbe disease, Fabry disease, X-adrenoleukodystrophy, and peroxisomal biogenesis disorder in DBS.

AbstractMedium‐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.

AbstractNewborn screening (NBS) is justified if early intervention is effective in a disorder generally not detected early in life on a clinical basis, and if sensitive and specific biochemical markers exist. Experience with NBS for homocystinurias and methylation disorders is limited. However, there is robust evidence for the success of early treatment with diet, betaine and/or pyridoxine for CBS deficiency and good evidence for the success of early betaine treatment in severe MTHFR deficiency. These conditions can be screened in dried blood spots by determining methionine (Met), methionine‐to‐phenylanine (Met/Phe) ratio, and total homocysteine (tHcy) as a second tier marker. Therefore, we recommend NBS for cystathionine beta‐synthase and severe MTHFR deficiency. Weaker evidence is available for the disorders of intracellular cobalamin metabolism. Early treatment is clearly of advantage for patients with the late‐onset cblC defect. In the early‐onset type, survival and non‐neurological symptoms improve but the effect on neurocognitive development is uncertain. The cblC defect can be screened by measuring propionylcarnitine, propionylcarnitine‐to‐acetylcarnitine ratio combined with the second tier markers methylmalonic acid and tHcy. For the cblE and cblG defects, evidence for the benefit of early treatment is weaker; and data on performance of Met, Met/Phe and tHcy even more limited. Individuals homozygous or compound heterozygous for MAT1A mutations may benefit from detection by NBS using Met, which on the other hand also detects asymptomatic heterozygotes. Clinical and laboratory data is insufficient to develop any recommendation on NBS for the cblD, cblF, cblJ defects, glycineN‐methyltransferase‐, S‐adenosylhomocysteinehydrolase‐ and adenosine kinase deficiency.

AbstractBackgroundNewborn screening (NBS) for Krabbe disease (KD) in New York and Missouri is conducted by measuring galactocerebrosidase (GALC) activity using tandem mass spectrometry (MS/MS). These NBS efforts have shown that the incidence of KD is unexpectedly low (1:400,000) while many individuals (ca. 1:6000) with reduced GALC activity and genotypes of uncertain significance are detected and subjected to follow up testing. Psychosine (PSY) is a putative marker of KD progression and can be measured in dried blood spots (DBS). We sought to determine the role that PSY levels play in NBS for KD, follow up, and treatment monitoring.MethodsPSY was eluted from DBS with methanol containing N,N‐dimethyl‐D‐erythro‐sphingosine as internal standard (IS). Liquid chromatography‐MS/MS was conducted over 17 minutes in the multiple reaction monitoring positive mode to follow the precursor to product species transitions for PSY and IS. Separation of the structural isomers PSY and glucosylsphingosine was accomplished by hydrophilic interaction liquid chromatography.ResultsPre‐analytical and analytical factors were studied and revealed satisfactory results. PSY was also measured in DBS collected from controls (range: <8 nmol/L, N = 220), KD patients at various disease stages (range: 8–112, N = 26), and GALC mutation carriers (range: <15 nmol/L, N = 18).ConclusionsPSY measurement in DBS could serve as a 2nd tier assay in NBS for KD, simplify and reduce the cost of follow up protocols, help determine disease progression, and be used to monitor KD patients following hematopoietic stem cell transplantation. However, additional chronological measurements of PSY in KD patients are required to confirm these possibilities.

Abstract 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.

BACKGROUND Friedreich ataxia (FRDA) is caused by reduced frataxin (FXN) concentrations. A clinical diagnosis is typically confirmed by DNA-based assays for GAA-repeat expansions or mutations in the FXN (frataxin) gene; however, these assays are not applicable to therapeutic monitoring and population screening. To facilitate the diagnosis and monitoring of FRDA patients, we developed an immunoassay for measuring FXN. METHODS Antibody pairs were used to capture FXN and an internal control protein, ceruloplasmin (CP), in 15 μL of whole blood (WB) or one 3-mm punch of a dried blood spot (DBS). Samples were assayed on a Luminex LX200 analyzer and validated according to standard criteria. RESULTS The mean recovery of FXN from WB and DBS samples was 99%. Intraassay and interassay imprecision (CV) values were 4.9%–13% and 9.8%–16%, respectively. The FXN limit of detection was 0.07 ng/mL, and the reportable range of concentrations was 2–200 ng/mL. Reference adult and pediatric FXN concentrations ranged from 15 to 82 ng/mL (median, 33 ng/mL) for DBS and WB. The FXN concentration range was 12–22 ng/mL (median, 15 ng/mL) for FRDA carriers and 1–26 ng/mL (median 5 ng/mL) for FRDA patients. Measurement of the FXN/CP ratio increased the ability to distinguish between patients, carriers, and the reference population. CONCLUSIONS This assay is applicable to the diagnosis and therapeutic monitoring of FRDA. This assay can measure FXN and the control protein CP in both WB and DBS specimens with minimal sample requirements, creating the potential for high-throughput population screening of FRDA.