Tortorelli, Silvia

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.

AbstractThe porphyrias are a group of disorders characterized by defects in the heme biosynthesis pathway. Many present with skin findings including photosensitivity, bullae, hypertrichosis, and scarring. Systemic symptoms may include abdominal pain, neuropsychiatric changes, anemia, and liver disease. With advances in DNA analysis, researchers are discovering the underlying genetic causes of the porphyrias, enabling family members to be tested for genetic mutations. Here we present a comprehensive review of porphyria focusing on those with cutaneous manifestations. In Part I, we have included the epidemiology, pathogenesis, presentation, diagnosis, and histopathology. Treatment and management options will be discussed in Part II.

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.

Newborn screening (NBS) is a public health program aimed at identifying treatable conditions in presymptomatic newborns to avoid premature mortality, morbidity, and disabilities. Currently, every newborn in the Unites States is screened for at least 29 conditions 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 into NBS programs. Among those are several conditions with a strong neuronopathic component. Some of these conditions have already been added to a few national and international screening programs, whereas others are undergoing pilot studies to determine the test performance metrics. Here, we review the current state of NBS for 13 lysosomal storage disorders, X‐adrenoleukodystrophy, Wilson disease, and Friedreich ataxia. © 2013 Wiley Periodicals, Inc. Dev Disabil Res Rev 2013;17:247–253.

Abstractα‐Methyl‐acyl‐CoA‐racemase (AMACR) deficiency (OMIM 604489) is a rare peroxisomal disorder with a variable age of onset from infancy to late adulthood. We describe a 45‐year‐old male with a history of seizures who presented with relapsing encephalopathy. Laboratory studies revealed an elevated serum pristanic acid concentration, an elevated pristanic/phytanic acid ratio, as well as the previously described homozygous mutation in the AMACR gene, c.154T>C, consistent with AMACR deficiency. This homozygous mutation is associated with a variable phenotype ranging from neonatal cholestasis to late‐onset sensorimotor neuropathy. Dietary pristanic acid restriction was attempted to improve clinical status and the patient has remained in remission for more than 16 months.

BACKGROUNDNewborn 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).METHODSThe 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 (d3-MMA, d3-MCA, d8-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.RESULTSThe 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.CONCLUSIONSApplication 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.

SummaryWe report a 16‐month‐old asymptomatic male with enzyme confirmed isovaleric acidaemia (IVA; isovaleryl‐CoA dehydrogenase deficiency; OMIM 243500) who, upon routine nutritional follow‐up, presented evidence of peroxisomal dysfunction. The newborn screen (2 days of life) revealed elevated C5‐carnitine (2.95 μmol/L; cutoff <0.09 μmol/L) and IVA was subsequently confirmed by metabolic profiling and in vitro enzymology. Plasma essential fatty acid (EFA) analysis, assessed to evaluate nutritional status during protein restriction and l‐carnitine supplementation, revealed elevated C26:0 (5.0 μmol/L; normal <1.3). Subsequently, metabolic profiling and molecular genetic analysis confirmed X‐linked adrenoleukodystrophy (XALD). Identification of co‐inherited XALD with IVA in this currently asymptomatic patient holds significant treatment ramifications for the proband prior to the onset of neurological sequelae, and critically important counselling implications for this family.

Abstract 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 μL 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 μL 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 μmol/L), with Tyr concentrations ranging from 65 to 293 μmol/L in the original NBS analysis. The mean concentration of SUAC in 13 521 control DBS was 1.25 μmol/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.