David C. Kasper

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 Gauchers disease, Pompes disease, Fabrys 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 β-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 Fabrys disease (1 per 3859 births), followed by Pompes disease (1 per 8684), and Gauchers 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.

Quantitative PCR Method for Sensitive Detection of Ruminant Fecal Pollution in Freshwater and Evaluation of This Method in Alpine Karstic Regions

ABSTRACT A quantitative TaqMan minor-groove binder real-time PCR assay was developed for the sensitive detection of a ruminant-specific genetic marker in fecal members of the phylum Bacteroidetes. The qualitative and quantitative detection limits determined were 6 and 20 marker copies per PCR, respectively. Tested ruminant feces contained an average of 4.1 × 109 marker equivalents per g, allowing the detection of 1.7 ng of feces per filter in fecal suspensions. The marker was detected in water samples from a karstic catchment area at levels matching a gradient from negligible to considerable ruminant fecal influence (from not detectable to 105 marker equivalents per liter).

The bacterial load of Ureaplasma parvum in amniotic fluid is correlated with an increased intrauterine inflammatory response

Ureaplasma spp. are the most frequently isolated microorganisms inside the amniotic cavity and have been associated with spontaneous abortion, chorioamnionitis, premature rupture of the membranes (PROM), and preterm labor (PL). We analyzed 118 samples from amniotic fluid of preterm infants before 34 weeks of gestation by quantitative polymerase chain reaction (qPCR). Bacterial load, Ureaplasma biovar discrimination (Ureaplasma urealyticum and Ureaplasma parvum), and the level of inflammation were correlated with short-term clinical outcome. U. parvum was the predominant biovar, and increased bacterial load was significantly linked to histologic chorioamnionitis, PROM + PL, early-onset sepsis, and bronchopulmonary dysplasia. Furthermore, there was a positive correlation between the amount of U. parvum and the magnitude of inflammatory response inside the amniotic cavity observed by elevated interleukin 8 levels. We postulate that the bacterial load of Ureaplasma spp. measured by qPCR should be determined in studies investigating the potential clinical impact of intrauterine Ureaplasma spp. on the outcome of preterm infants.

Quantitative real-time polymerase chain reaction for the accurate detection of Toxoplasma gondii in amniotic fluid

Infection with Toxoplasma gondii during pregnancy is often asymptomatic and may cause severe fetal damage. A quantitative TaqMan minor groove binder real-time polymerase chain reaction (PCR) assay was developed for the specific and sensitive detection of the previously described 529-bp repeat element occurring up to 200 to 300 times in T. gondii genome. The qualitative and quantitative detection limits determined were 6 and 20 marker copies (1/30 to 1/50 of 1 parasite) per PCR, respectively. In addition to standard PCR cycling conditions, 3 different fast PCR protocols were evaluated to minimize run time. A higher variability but no loss of specificity was observed. For the evaluation of clinical applicability, a total of 135 amniotic fluid samples were analyzed targeting both 529-bp and B1 gene. The sensitivity and specificity were 88.0% and 100.0% for B1, and 100.0% and 98.2% for 529-bp PCR assay (positive predictive value and negative predictive value: 100.0% and 97.4%, and 92.6% and 100.0%, respectively). Our results demonstrated an increased sensitivity of the 529-bp PCR assay even in a faster protocol.

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.

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.

In utero exposure to Ureaplasma spp. is associated with increased rate of bronchopulmonary dysplasia and intraventricular hemorrhage in preterm infants.

Abstract Aims: We determined the association between short-term neonatal morbidities, such as bronchopulmonary dysplasia (BPD) and intraventricular hemorrhage (IVH), and Ureaplasma spp. in amniotic fluid, placental and amniotic mem-brane of preterm infants. Methods: This study enrolled 257 patients who were born by cesarean section at <34 weeks’ gestation. Patients were divided into two groups according to detection of Ureaplasma spp. by culture-based and/or polymerase chain reaction (PCR) techniques. Results: Significant differences were observed between both groups for all IVH (P=0.032) and IVH grades III or IV (P=0.013), as wells as for BPD [odds ratio (OR) 5.46, 95% confidence interval (CI) 2.02–14.77], oxygen requirement at 28 days postnatal age (OR 1.93, 95% CI 1.00–3.70), and for death between 28 days and 36 postmenstrual weeks or BPD (OR 4.20, 95% CI 1.77–9.96). Ureaplasma spp. was a significant predictor (P<0.001) of BPD after correcting for birth weight (P=0.003) and positive pressure ventilation (P=0.001). Conclusions: In our study population Ureaplasma spp. was associated with BPD and IVH in preterm infants even after adjustment for multiple risk factors.

The application of multiplexed, multi-dimensional ultra-high-performance liquid chromatography/tandem mass spectrometry to the high-throughput screening of lysosomal storage disorders in newborn dried bloodspots

Lysozomal storage disorders are just beginning to be routinely screened using enzyme activity assays involving dried blood spots and tandem mass spectrometry (MS/MS). This paper discusses some of the analytical challenges associated with published assays including complex sample preparation and potential interference from excess residual substrate. Solutions to these challenges are presented in the form of on-line two-dimensional chromatography to eliminate off-line liquid-liquid extraction (LLE) and solid-phase extraction (SPE), the use of ultra-high-performance liquid chromatography (UHPLC) to separate excess substrate from all other analytes and multiplexed sample introduction for higher throughput required of a population screening assay. High sensitivity, specificity and throughput were demonstrated using this novel method.

MicroRNA-146: Tiny Player in Neonatal Innate Immunity?

Background: Concise regulation of the Toll signaling pathway is mandatory in neonatal innate immunity. The microRNA-146 family (miR-146a/b) was recently reported to be a regulator of Toll-like receptor 4 (TLR4) through a negative feedback loop mechanism. Acting as a potent regulator, miRNA helps to protect the organism from developing overwhelming proinflammatory immune responses leading to septic shock or chronic inflammatory diseases. Objective: We investigated for the first time whether miRNA-146a/b plays a regulatory role in human monocytes derived from infant cord or adult blood, and whether differences in miRNA-146 expression exist. Methods: Expression profiles of miR-146a/b and TLR4 were studied by real-time PCR upon stimulation with lipopolysaccharide. Results: Both members of the miRNA-146 family showed a time-dependent upregulation. For miR-146a, a statistically higher significant increase was found after 24 h of stimulation in monocytes from cord blood compared to those derived from adults. In contrast, no differences were found for miR-146b and TLR4, respectively. Conclusion: We conclude that differences between the negative regulatory role for miR-146a obviously exist in neonatal and adult TLR4 signaling, and suggest that more intense research in the involvement of miRNA in immune regulation will facilitate the understanding of the development and function of the innate immune system of neonates.

Small sizes and indolent evolutionary dynamics challenge the potential role of P2RY8-CRLF2–harboring clones as main relapse-driving force in childhood ALL

The P2RY8-CRLF2 fusion defines a particular relapse-prone subset of childhood acute lymphoblastic leukemia (ALL) in Italian Association of Pediatric Hematology and Oncology Berlin-Frankfurt-Münster (AIEOP-BFM) 2000 protocols. To investigate whether and to what extent different clone sizes influence disease and relapse development, we quantified the genomic P2RY8-CRLF2 fusion product and correlated it with the corresponding CRLF2 expression levels in patients enrolled in the BFM-ALL 2000 protocol in Austria. Of 268 cases without recurrent chromosomal translocations and high hyperdiploidy, representing approximately 50% of all cases, 67 (25%) were P2RY8-CRLF2 positive. The respective clone sizes were ≥ 20% in 27% and < 20% in 73% of them. The cumulative incidence of relapse of the entire fusion-positive group was clone size independent and significantly higher than that of the fusion-negative group (35% ± 8% vs 13% ± 3%, P = .008) and primarily confined to the non-high-risk group. Of 22 P2RY8-CRLF2-positive diagnosis/relapse pairs, only 4/8 had the fusion-positive dominant clone conserved at relapse, whereas none of the original 14 fusion-positive small clones reappeared as the dominant relapse clone. We conclude that the majority of P2RY8-CRLF2-positive clones are small at diagnosis and virtually never generate a dominant relapse clone. Our findings therefore suggest that P2RY8-CRLF2-positive clones do not have the necessary proliferative or selective advantage to evolve into a disease-relevant relapse clone.