Higher precision, first tier newborn screening for metachromatic leukodystrophy using 16:1-OH-sulfatide.

Newborn screening (NBS) for metachromatic leukodystrophy (MLD) is based on first-tier measurement of sulfatides in dried blood spots (DBS) followed by second-tier measurement of arylsulfatase A in the same DBS. This approach is very precise with 0-1 false positives per ∼30,000 newborns tested. Recent data reported here shows that the sulfatide molecular species with an α-hydroxyl, 16­carbon, mono-unsaturated fatty acyl group (16:1-OH-sulfatide) is superior to the original biomarker 16:0-sulfatide in reducing the number of first-tier false positives. This result is consistent across 4 MLD NBS centers. By measuring 16:1-OH-sulfatide alone or together with 16:0-sulfatide, the estimated false positive rate is 0.048% and is reduced essentially to zero with second-tier arylsulfatase A activity assay. The false negative rate is predicted to be extremely low based on the demonstration that 40 out of 40 newborn DBS from clinically-confirmed MLD patients are detected with these methods. The work shows that NBS for MLD is extremely precise and ready for deployment. Furthermore, it can be multiplexed with several other inborn errors of metabolism already tested in NBS centers worldwide.

Importance to include differential diagnostics for acid sphingomyelinase deficiency (ASMD) in patients suspected to have to Gaucher disease.

The clinical manifestation of sphingolipidosis leads often to misclassification between acid sphingomyelinase deficiency (ASMD) and Gaucher disease. In this multicenter, prospective study, we investigated a cohort of 31,838 individuals suspected to have Gaucher disease, due to clinical presentation, from 61 countries between 2017 and 2022. For all samples, both Acid-ß-glucocerebrosidase and acid sphingomyelinase enzyme activities were measured in dried blood spot specimens by tandem mass spectrometry followed by genetic confirmatory testing in potential positive cases. In total, 5933 symptomatic cases showed decreased enzyme activities and were submitted for genetic confirmatory testing. 1411/5933 (24%) cases were finally identified with Gaucher disease and 550/5933 (9%) with ASMD. Most of the confirmed ASMD cases were newborns and children below 2 years of age (63%). This study reveals that one in four cases suspected for Gaucher disease is diagnosed with ASMD. An early appropriate diagnostic work-up is essential because of the availability of a recently approved enzyme replacement therapy for ASMD. In conclusion, a diagnostic strategy using differential biochemical testing including genetic confirmatory testing in clinically suspected cases for sphingolipidosis is highly recommended.

Long-Term Evaluation of Biomarkers in the Czech Cohort of Gaucher Patients.

A personalized treatment decision for Gaucher disease (GD) patients should be based on relevant markers that are specific to GD, play a direct role in GD pathophysiology, exhibit low genetic variation, reflect the therapy, and can be used for all patients. Thirty-four GD patients treated with enzyme replacement therapy (ERT) or substrate reduction therapy (SRT) were analyzed for platelet count, chitotriosidase, and tartrate-resistant acid phosphatase activity in plasma samples, and quantitative measurement of Lyso-Gb1 was performed in dried blood spots. In our ERT and SRT study cohorts, plasma lyso-GL1 correlated significantly with chito-triosidase (ERT: r = 0.55, p < 0.001; SRT: r = 0.83, p < 0.001) and TRAP (ERT: r = 0.34, p < 0.001; SRT: r = 0.88, p < 0.001), irrespective of treatment method. A platelet count increase was associated with a Lyso-Gb1 decrease in both treatment groups (ERT: p = 0.021; SRT: p = 0.028). The association of Lyso-Gb1 with evaluated markers was stronger in the SRT cohort. Our results indicate that ERT and SRT in combination or in a switch manner could offer the potential of individual drug effectiveness for particular GD symptoms. Combination of the key biomarker of GD, Lyso-Gb1, with other biomarkers can offer improved response assessment to long-term therapy.

Molecular basis of mucopolysaccharidosis IVA (Morquio A syndrome): A review and classification of GALNS gene variants and reporting of 68 novel variants.

Mucopolysaccharidosis IVA (MPS IVA, Morquio A syndrome) is a rare autosomal recessive lysosomal storage disorder caused by mutations in the N-acetylgalactosamine-6-sulfatase (GALNS) gene. We collected, analyzed, and uniformly summarized all published GALNS gene variants, thus updating the previous mutation review (published in 2014). In addition, new variants were communicated by seven reference laboratories in Europe, the Middle East, Latin America, Asia, and the United States. All data were analyzed to determine common alleles, geographic distribution, level of homozygosity, and genotype-phenotype correlation. Moreover, variants were classified according to their pathogenicity as suggested by ACMG. Including those previously published, we assembled 446 unique variants, among which 68 were novel, from 1190 subjects (including newborn screening positive subjects). Variants' distribution was missense (65.0%), followed by nonsense (8.1%), splicing (7.2%), small frameshift deletions(del)/insertions(ins) (7.0%), intronic (4.0%), and large del/ins and complex rearrangements (3.8%). Half (50.4%) of the subjects were homozygous, 37.1% were compound heterozygous, and 10.7% had only one variant detected. The novel variants underwent in silico analysis to evaluate their pathogenicity. All variants were submitted to ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) to make them publicly available. Mutation updates are essential for the correct molecular diagnoses, genetic counseling, prenatal and preimplantation diagnosis, and disease management.

α-Mannosidosis - An underdiagnosed lysosomal storage disease in individuals with an 'MPS-like' phenotype.

Individuals affected by alpha-Mannosidosis suffer from similar clinical symptoms such as respiratory infections, skeletal changes as patients with mucopolysaccharidoses (MPS). α-Mannosidosis is considered as an ultra-rare disorders and also diagnostic testing is often limited. With the availability of novel therapies and easy-to-access diagnostic tests (e.g. Tandem mass spectrometry) using dried blood spots for both enzymatic and genetic testing, the chance for the development of a better understanding of disease and awareness may be triggered. In a pilot study, we have investigated 1010 residual dried blood spot samples from individuals suspicious to MPS. In these study cohort, 158/1010 individuals were genetically confirmed for MPS. Additional biochemical and genetic confirmatory testing for α-mannosidases revealed four individuals with a final diagnosis of α-mannosidosis. This unexpected high number of individuals with α-mannosidosis demonstrated the urgent need of taking this rare disorder in clinical and diagnostic consideration particularly in patients suspicious to MPS.

Diagnostic strategy for females suspected of Fabry disease.

A total of 11 948 females suspicious of Fabry disease were tested by a combined biochemical and genetic approach. The enzyme activity, together with the concentration of lyso-GL-3 (lyso-Gb3) biomarker in dried blood spots (DBS), substantially improved the diagnostic detection of Fabry disease in females compared to the enzyme activity alone. Abnormal values for both were highly suspicious of Fabry disease (97% positive predictive value [PPV], similar to PPV in males). In cases with one abnormal biochemical value, elevated lyso-GL-3 is a far more important indicator than low enzyme activity (39% PPV vs 6% PPV). Cases with clearly negative results for both biochemical parameters are unlikely to have Fabry disease, even in clinically highly suspicious cases.

Vaginal Ureaplasma parvum serovars and spontaneous preterm birth.

BACKGROUND: Ureaplasma species (spp) are the bacteria most often isolated from the amniotic cavity of women with preterm labor or preterm premature rupture of membranes; thus, the link between intrauterine Ureaplasma spp infection and adverse pregnancy outcome clearly is established. However, because vaginal Ureaplasma spp colonization is very common in pregnant women, the reason that these microorganisms cause ascending infections in some cases but remain asymptomatic in most pregnancies is not clear. Previous studies suggested an association between vaginal colonization with Ureaplasma parvum as opposed to U urealyticum and preterm delivery. However, because of the high frequency of vaginal Ureaplasma spp colonization during pregnancy, additional risk factors are needed to select a group of women who might benefit from treatment. OBJECTIVE: To further identify pregnant women who are at increased risk for preterm delivery, the aim of the present study was to investigate U parvum serovar-specific pathogenicity in a large clinical cohort. STUDY DESIGN: We serotyped 1316 samples that were positive for U parvum using a high-resolution melt polymerase chain reaction assay, and results were correlated with pregnancy outcome. RESULTS: Within U parvum positive samples, serovar 3 was the most common isolate (43.3%), followed by serovar 6 (31.4%) and serovar 1 (25.2%). There was a significantly increased risk for spontaneous preterm birth at very low (<32 weeks gestation; P<.005) and extremely low (<28 weeks gestation; P<.005) gestational age in the group with vaginal U parvum serovar 3 colonization compared with the control group of pregnant women who tested negative for vaginal Ureaplasma spp colonization. This association was found for neither serovar 1 nor serovar 6. The combination of vaginal U parvum serovar 3 colonization and diagnosis of bacterial vaginosis in early pregnancy or a history of preterm birth further increased the risk for adverse pregnancy outcome. CONCLUSION: Colonization with U parvum serovar 3, but not serovar 1 or serovar 6, in early pregnancy is associated with preterm delivery at very and extremely low gestational age. The combination of U parvum serovar 3 colonization and a history of preterm birth or bacterial vaginosis further increases the risk for spontaneous preterm birth at low gestational age and may define a target group for therapeutic intervention studies.

Genotype, phenotype and disease severity reflected by serum LysoGb3 levels in patients with Fabry disease.

BACKGROUND: Fabry disease (FD) is a rare X-linked lysosomal storage disease caused by mutations in the α-galactosidase A (GLA) gene causing deficiency of α-galactosidase A which results in progressive glycosphingolipid accumulation, especially globotriaosylceramide (Gb3), in body liquids and lysosomes. In a large cohort of FD patients, we aimed to establish genotype/phenotype relations as indicated by serum LysoGb3 (deacylated Gb3). METHODS: In 69 consecutive adult FD patients (males: n=28 (41%)) with a GLA-mutation confirmed diagnosis, we conducted a multidisciplinary clinical characterization during their routine annual examinations, and measured serum LysoGb3 levels by high-sensitive electrospray ionization liquid chromatography tandem mass spectrometry. RESULTS: Serum levels of LysoGb3 were significantly higher in Classic compared with Later-Onset phenotype and higher in the latter compared with controls, both in males (52 [40-83] vs 9.5 [4.5-20] vs 0.47 [0.41-0.61] ng/ml, P<0.001) and in females (9.9 [7.9-14] vs 4.9 [1.6-4.9] vs 0.41 [0.33-0.48] ng/ml, P<0.001), respectively. Multivariate linear regression analysis showed that LysoGb3 levels were independently associated with, serum creatinine (ß=0.09, 95%CI 0.04-0.13, P<0.001) and the presence of cardiomyopathy (ß=25, 95%CI 9.8-41, P=0.002). LysoGb3 levels were higher in males with frame-shift and nonsense mutations than in males with missense mutations (84 [72-109] vs 41 [37-52] ng/ml, P=0.002). CONCLUSION: LysoGb3 relates to disease severity, enzyme replacement response, and to the genotype severity in males. LysoGb3 supports identifying patients at risk who require intensive monitoring and treatment. LysoGb3 appears to be one marker of metabolic phenotyping of FD.

Correlation of Lyso-Gb3 levels in dried blood spots and sera from patients with classic and Later-Onset Fabry disease.

BACKGROUND: Fabry disease (FD), an X-linked lysosomal storage disorder, results from the deficient activity of α-galactosidase A (α-Gal A) and the accumulation of its substrates, globotriaosylceramide (Gb3) and its deacylated derivative, globotriaosyl-sphingosine (Lyso-Gb3). Here, we compared the levels of Lyso-Gb3 in dried blood spots (DBS) and sera in affected males and heterozygotes with the "Classic" and "Later-Onset" phenotypes. METHODS: The Lyso-Gb3 concentrations in DBS and sera from 56 FD patients were determined by highly sensitive electrospray ionization liquid chromatography tandem mass spectrometry. RESULTS: The serum Lyso-Gb3 levels in 18 and 5 affected males with the Classic and Later-Onset phenotypes, were 61±38 and 14±12ng/mL, respectively. Lyso-Gb3 levels in 30 females from Classic families and three females from Later-Onset families were 10±5.4 and 2.4±1.0ng/mL, respectively. The linear regression model with serum Lyso-Gb3 as the dependent variable and DBS Lyso-Gb3 an independent variable was described by the function y=-1.83+1.68∗x and showed a high coefficient of determination, R2=0.976. The overall correlation between the Lyso-Gb3 levels in DBS and sera was high (R=0.99; p<0.001). CONCLUSION: DBS provides a convenient, sensitive, and reproducible source to measure Lyso-Gb3 levels for diagnosis, initial phenotypic assignment, and therapeutic monitoring in patients with Fabry disease.

Plasma LysoGb3: A useful biomarker for the diagnosis and treatment of Fabry disease heterozygotes.

BACKGROUND: Fabry disease (FD) is a rare X-linked lysosomal storage disorder due to mutations in the α-galactosidase A gene (GLA) that result in absent or markedly reduce α-galactosidase A (α-GalA) enzymatic activity. As a result, the major glycosphingolipid substrates, globotriaosylceramide (Gb3) and globotriaosylsphingosine (LysoGb3) accumulate in plasma, urine and tissue lysosomes. In females, the diagnosis can be complicated by the fact that 40-50% of GLA-mutation confirmed heterozygotes have normal or only slightly decreased leukocyte α-GalA activities. Recently, LysoGb3 has been appreciated as a novel FD biomarker, especially for therapeutic monitoring. METHODS: Among our GLA-mutation proven FD patients, we screened 18 heterozygotes whose leukocyte α-GalA activity was determined at initial diagnosis. For these females, we measured their serum LysoGb3 levels using highly-sensitive electrospray ionization liquid chromatography tandem mass spectrometry. RESULTS: We identified three unrelated females in whom the accumulating LysoGb3 was increased, whereas their leukocyte α-GalA activities were in the normal range. CONCLUSION: LysoGb3 serves as an useful biomarker to improve the diagnosis of FD heterozygotes and for therapeutic evaluation and monitoring.

Quantification of excess risk for diabetes for those born in times of hunger, in an entire population of a nation, across a century.

Based on a unique dataset comprising all 325,000 Austrian patients that were under pharmaceutical treatment for diabetes during 2006 and 2007, we measured the excess risk of developing diabetes triggered by undernourishment in early life. We studied the percentage of all diabetes patients in the total population specifically for each year of birth, from 1917 to 2007. We found a massive excess risk of diabetes in people born during the times of the three major famines and immediately after, which occurred in Austria in the 20th century: 1918-1919, 1938, and 1946-1947. Depending on the region, there was an up to 40% higher chance of having diabetes when born in 1919-1921, compared with 1918 or 1922, where age-specific typical diabetes ratios are observed. The excess risk for diabetes was practically absent in those provinces of Austria that were less affected by the famines. We show that diabetes rates exhibit nontrivial, age-specific sex differences, and correlate with the economic wealth of the region. Our results might be of relevance for establishing higher awareness in the health system for those born in high-risk years, and underline the importance of ensuring sufficient nutrition in prenatal and early stages of life.

The Austrian Toxoplasmosis Register, 1992-2008.

BACKGROUND: We aimed to determine the incidence of primary gestational infections with Toxoplasma gondii and congenital toxoplasmosis in Austria, a country with a nationwide prenatal serological screening program since 1974. METHODS: We analyzed retrospective data from the Austrian Toxoplasmosis Register of pregnant women with Toxoplasma infection and their offspring with births between 1992 and 2008, identified by the prenatal mandatory screening program. Treatment was administered to women from diagnosis of a Toxoplasma infection until delivery. Infected infants were treated up to 1 year of life routinely. Clinical manifestations in infected infants were monitored at least for 1 year and documented in the register. RESULTS: The Austrian Toxoplasmosis Register included 2147 pregnant women with suspected Toxoplasma infection. Annually, 8.5 per 10 000 women acquired Toxoplasma infection during pregnancy, and 1.0 per 10 000 infants had congenital toxoplasmosis (13% mean transmission rate). Our data showed that women treated according to the Austrian scheme had a 6-fold decrease in the maternofetal transmission rate compared to women without treatment. CONCLUSIONS: Results from the Austrian Toxoplasmosis Register show the efficiency of the prenatal screening program. Our results are of clinical relevance for infants, healthcare systems, and policy makers to consider preventive Toxoplasma screening as a potential tool to reduce the incidence of congenital toxoplasmosis.

Austrian Newborn Screening Program: a perspective of five decades.

In 1966, the National Austrian Newborn Screening Program for inherited metabolic and endocrine disorders was initiated. In the last five decades, around four million babies were screened and in more than 2600 babies, various inborn errors of metabolism and endocrine disorders were detected. This health-preventive program was continuously expanded from phenylketonuria and galactosemia to congenital hypothyroidism, biotinidase deficiency, cystic fibrosis, and congenital adrenal hyperplasia. In 2002, the introduction of tandem mass spectrometry substantially increased the number of detectable rare diseases, and now includes disorders of fatty acid oxidation, organic acidurias, and various disorders of amino acid metabolism. In this review, we highlight the development of the Austrian screening program, and pinpoint future disorders and challenges.

Neonatal screening for lysosomal storage disorders: feasibility and incidence from a nationwide study in Austria.

BACKGROUND: The interest in neonatal screening for lysosomal storage disorders has increased substantially because of newly developed enzyme replacement therapies, the need for early diagnosis, and technical advances. We tested for Gaucher's disease, Pompe's disease, Fabry's disease, and Niemann-Pick disease types A and B in an anonymous prospective nationwide screening study that included genetic mutation analysis to assess the practicality and appropriateness of including these disorders in neonatal screening panels. METHODS: Specimens from dried blood spots of 34,736 newborn babies were collected consecutively from January, 2010 to July, 2010, as part of the national routine Austrian newborn screening programme. Anonymised samples were analysed for enzyme activities of acid ß-glucocerebrosidase, α-galactosidase, α-glucosidase, and acid sphingomyelinase by electrospray ionisation tandem mass spectrometry. Genetic mutation analyses were done in samples with suspected enzyme deficiency. FINDINGS: All 34,736 samples were analysed successfully by the multiplex screening assay. Low enzyme activities were detected in 38 babies. Mutation analysis confirmed lysosomal storage disorders in 15 of them. The most frequent mutations were found for Fabry's disease (1 per 3859 births), followed by Pompe's disease (1 per 8684), and Gaucher's disease (1 per 17,368). The positive predictive values were 32% (95% CI 16-52), 80% (28-99), and 50% (7-93), respectively. Mutational analysis detected predominantly missense mutations associated with a late-onset phenotype. INTERPRETATION: The combined overall proportion of infants carrying a mutation for lysosomal storage disorders was higher than expected. Neonatal screening for lysosomal storage disorders is likely to raise challenges for primary health-care providers. Furthermore, the high frequency of late-onset mutations makes lysosomal storage disorders a broad health problem beyond childhood. FUNDING: Austrian Ministry of Health, Family, and Women.

Urinary metabolomic markers of aminoglycoside nephrotoxicity in newborn rats.

BACKGROUND: Aminoglycoside exposure is a common cause of acute kidney injury (AKI). Delay in the diagnosis of AKI using conventional biomarkers has been one of the important obstacles in applying early effective interventions. We tested the hypothesis that urinary metabolomics could identify novel early biomarkers for toxic renal injury. METHODS: Three-day-old rats were divided into three groups; they received a single daily injection of vehicle (0.9% NaCl solution) or gentamicin at a dose of 10 or 20 mg/kg/d for 7 d. Urine and blood were collected after 3 and 7 d of injections. Urinary metabolites were evaluated using high-performance liquid chromatography and gas chromatography/mass spectrometry. RESULTS: A distinct urinary metabolic profile characterized by glucosuria, phosphaturia, and aminoaciduria was identified preceding changes in serum creatinine. At both the gentamicin doses, urinary tryptophan was significantly (P < 0.05) increased (fold change: 1.91 and 2.31 after 3 d; 1.81 and 1.93 after 7 d). Similarly, kynurenic acid, a tryptophan metabolite, showed a significant (P < 0.05) decrease (fold change: 0.26 and 0.24 after 3 d; 0.21 and 0.52 after 7 d), suggesting an interruption of the normal tryptophan metabolism pathway. CONCLUSION: We conclude that urinary metabolomic profiling provides a robust approach for identifying early and novel markers of gentamicin-induced AKI.

Matrix-assisted laser desorption/ionization for simultaneous quantitation of (acyl-)carnitines and organic acids in dried blood spots.

RATIONALE: Screening for inborn errors of metabolism using mass spectrometry is part of nationwide newborn screening programs and involves the detection of disease relevant (acyl-)carnitines and organic acids from dried blood spots. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) is a well-established tool for proteomics approaches. In recent years, this technique has become more and more integrated in analysis and identification of small metabolites and disease biomarkers in daily clinical laboratories. METHODS: We used a combination of both MALDI and high-resolution accurate mass (HR/AM) mass spectrometry using a linear ion trap-Orbitrap for the identification of small molecules from dried blood spots that serve as biomarkers for inborn errors of metabolism. The levels of detected metabolite species were compared between healthy newborns and affected patients with various inborn errors of metabolism using isotopically labeled internal standards and new bioinformatics software, respectively. RESULTS: (Acyl-)carnitine levels from normal and affected patients could be quantified and differentiated. Additionally, using the high resolving power of full scan Orbitrap mass spectrometry and novel software tools we demonstrated the identification and quantification of disease-specific organic acids. CONCLUSIONS: MALDI-HR/AM and full scan spectra to obtain information for the metabolic status of patients is a promising complementary approach to electrospray ionization mass spectrometry by simplified sample preparation, facilitating the screening of hundreds of metabolites from small sample volumes.

The predictive value of immature granulocyte count and immature myeloid information in the diagnosis of neonatal sepsis.

BACKGROUND: To determine the predictive value of the immature granulocyte count and the immature myeloid information in neonatal early onset sepsis we examined 133 blood samples of patients admitted to our neonatal intensive care unit. METHODS: Measurements were performed using the Sysmex XE-2100, an automated hematological analyzer. Patients were divided into two groups: 1) symptomatic neonates with diagnosis of early onset sepsis; and 2) controls including asymptomatic neonates who were admitted because of prematurity, low birth weight, or delayed postnatal transition. RESULTS: The number of immature granulocytes and the immature myeloid information were significantly elevated in neonates with early onset sepsis compared to controls (median 280/µL vs. 50/µL, p=0.049 and 639/µL vs. 89/µL, p<0.0001, respectively). CONCLUSIONS: Automated determinations of immature granulocytes and immature myeloid information seem to be useful adjunctive methods in the diagnosis of neonatal early onset sepsis.

Simplified newborn screening protocol for lysosomal storage disorders.

BACKGROUND: Interest in lysosomal storage disorders, a collection of more than 40 inherited metabolic disorders, has increased because of new therapy options such as enzyme replacement, stem cell transplantation, and substrate reduction therapy. We developed a high-throughput protocol that simplifies analytical challenges such as complex sample preparation and potential interference from excess residual substrate associated with previously reported assays. METHODS: After overnight incubation (16-20 h) of dried blood spots with a cassette of substrates and deuterated internal standards, we used a TLX-2 system to quantify 6 lysosomal enzyme activities for Fabry, Gaucher, Niemann-Pick A/B, Pompe, Krabbe, and mucopolysaccharidosis I disease. This multiplexed, multidimensional ultra-HPLC-tandem mass spectrometry assay included Cyclone P Turbo Flow and Hypersil Gold C8 columns. The method did not require offline sample preparation such as liquid-liquid and solid-phase extraction, or hazardous reagents such as ethyl acetate. RESULTS: Obviating the offline sample preparation steps led to substantial savings in analytical time (approximately 70%) and reagent costs (approximately 50%). In a pilot study, lysosomal enzyme activities of 8586 newborns were measured, including 51 positive controls, and the results demonstrated 100% diagnostic sensitivity and high specificity. The results for Krabbe disease were validated with parallel measurements by the New York State Screening Laboratory. CONCLUSIONS: Turboflow online sample cleanup and the use of an additional analytical column enabled the implementation of lysosomal storage disorder testing in a nationwide screening program while keeping the total analysis time to <2 min per sample.