Susanne Greber-Platzer

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.

ATP6AP1 deficiency causes an immunodeficiency with hepatopathy, cognitive impairment and abnormal protein glycosylation.

The V-ATPase is the main regulator of intra-organellar acidification. Assembly of this complex has extensively been studied in yeast, while limited knowledge exists for man. We identified 11 male patients with hemizygous missense mutations in ATP6AP1, encoding accessory protein Ac45 of the V-ATPase. Homology detection at the level of sequence profiles indicated Ac45 as the long-sought human homologue of yeast V-ATPase assembly factor Voa1. Processed wild-type Ac45, but not its disease mutants, restored V-ATPase-dependent growth in Voa1 mutant yeast. Patients display an immunodeficiency phenotype associated with hypogammaglobulinemia, hepatopathy and a spectrum of neurocognitive abnormalities. Ac45 in human brain is present as the common, processed ∼40-kDa form, while liver shows a 62-kDa intact protein, and B-cells a 50-kDa isoform. Our work unmasks Ac45 as the functional ortholog of yeast V-ATPase assembly factor Voa1 and reveals a novel link of tissue-specific V-ATPase assembly with immunoglobulin production and cognitive function.

Mutations at the galactose-1-p-uridyltransferase gene in infants with a positive galactosemia newborn screening test.

Newborn screening for galactosemia yields a high number of false-positive results. Confirmatory DNA testing for unknown galactosemia mutations on the initial positive sample using novel techniques of mutation detection tenders itself to reduce the recall rate. The potential benefits of confirmatory DNA testing, however, could be offset by the detection of a high percentage of galactosemia carriers, Duarte/galactosemia compound heterozygotes, and infants with benign sequence changes in the galactose-1-phosphate uridyltransferase (GALT) gene among infants with a positive biochemical screening test. Our aim was to determine the frequency and allelic distribution of all sequence changes in the GALT gene in 110 newborns with a positive total galactose screening test among 43,688 Austrian newborns screened consecutively. We found that only 20 of the 110 probands carried at least one known or novel candidate galactosemia mutation (one galactosemia homozygote, 7 Duarte/galactosemia compounds, 12 carriers) as judged by denaturing gradient gel electrophoresis and cleavage fragment length polymorphism analysis. Four novel galactosemia candidate mutations (Q9H, A46fsdelCAGCT, M129T, L342I) were identified. Sixty-seven probands had no detectable sequence changes and 23 carried only the benign Duarte or Los Angeles variant alleles or silent mutations. We conclude that a rapid and automatable confirmation test for unknown GALT mutations, e.g. on a high-density oligonucleotide array basis, has the potential to lower the recall rate of galactosemia screening in our population by about five-fold from 0.25 to 0.046%. Further research, however, will be required before the development of such a test can be advocated.

Familial cryptic translocation with del 4q34→qter and dup 12pter→p13 in sibs with tracheal stenosis: Clinical, classical and molecular cytogenetic studies and CGH analyses from archival placental tissues evidencing tertiary trisomy 4 in one abortion specimen

We report on two retarded half-sibs of different sex and seemingly normal karyotype who had the same syndrome of minor anomalies, heart defect and a distal tracheal stenosis, and who shared a healthy mother. These findings raised suspicions of a cryptic chromosome translocation. A translocation t(4;12)(q34;p13), balanced in the mother and unbalanced in the sibs with loss of terminal 4q and gain of terminal 12p regions, was verified by FISH using whole chromosome painting, subtelomeric and YAC probes. Clinical features could be explained by partial monosomy 4q and partial trisomy 12p. Tracheal stenosis was interpreted as a consequence of the same developmental disturbance leading to esophageal atresia and tracheo-esophageal fistula. It was attributed to the 4q deletion in which esophageal atresia as also respiratory difficulties and airway obstructions had been described. Paraffin-embedded placental tissues were available from three of the five abortions of the mother allowing DNA extraction and comparative genome hybridization (CGH). Two of the abortion specimens had the same der(4)t(4;12)(q34;p13) unbalanced translocation as identified in the sibs. In the third abortion specimen, suspicious of triploidy because of partial hydatidiform mole, CGH uncovered a tertiary trisomy 4 resulting from a 3:1 segregation of the translocation chromosomes and their homologs during maternal meiosis I. Differences in CGH results using DNA generated directly or after DOP-PCR were explained by DNA fragmentation in paraffin-embedded tissues and unequal amplification. Am. J. Med. Genet. 94:271-280, 2000.

Within-assessor reliability and minimal detectable change of gait kinematics in a young obese demographic

INTRODUCTION Three-dimensional gait analysis (3DGA) in obese populations is a difficult task due to a great amount of subcutaneous fat. This makes it more challenging to identify anatomical landmarks, thus leading to inconsistent marker placement. Therefore, the purpose of this study was to investigate the test-retest reliability for kinematic measurements of obese children and adolescents. METHODS Nine males and two females with an age-based BMI above the 97th percentile (age: 14.6±2.6years, BMI: 33.4±4.4kg/m2) were administered to two 3DGA sessions. To quantify reliability of discrete parameters the intraclass correlation coefficient (ICC2,k), standard error of measurement (SEM) and minimal detectable change (MDC) were calculated. To quantify waveform similarity, the coefficient of multiple correlation (CMC) and the linear fit method (LFM) were used. RESULTS From 28 kinematic parameters, 23 showed acceptable ICCs (≥0.70) and the remaining parameters demonstrated moderate values. These were peak hip extension during stance (0.58), mean pelvis rotation (0.60), mean anterior pelvic tilt (0.64), peak knee flexion during swing (0.67) and peak hip abduction during swing (0.69). The SEM was below 5° for all parameters. The MDC for the sagittal, frontal, and transversal plane were on average 7.5°±2.2, 4.6°±1.3 and 6.0°±0.9 respectively. Both the LFM and CMC showed, in general, moderate to good reliability except for pelvis tilt and hip rotation. CONCLUSION Data demonstrated acceptable error margins especially for the sagittal and frontal plane. Low reliability for the pelvis tilt indicates that great effort is necessary to position the pelvic markers consistently during repeated sessions.

Chondroitin Sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1) Deficiency Results in a Mild Skeletal Dysplasia and Joint Laxity.

Mutations in genes encoding enzymes responsible for the biosynthesis and structural diversity of glycosaminoglycans (GAGs) cause a variety of disorders affecting bone and connective tissues, including Desbuquois dysplasia (DD). In an infant with prenatal‐onset disproportionate short stature, joint laxity, and radiographic findings typical for DD compound‐heterozygosity for a large intragenic deletion, and a p.Pro384Arg missense mutation in CSGALNACT1 was found. CSGALNACT1 encodes chondroitin sulfate N‐acetylgalactosaminyltransferase‐1 (CSGalNAcT‐1, ChGn‐1), which initiates chondroitin sulfate (CS) chain biosynthesis on the so‐called GAG‐protein linker region tetrasaccharide. Biochemical studies revealed a reduced GalNAc‐transferase activity of the Arg‐384 mutant protein, whereas no differences in proteoglycan synthesis in fibroblasts and the GAG content in the urine were found between patient and controls. This is the first description of bi‐allelic loss‐of‐function mutations in CSGALNACT1 that produce a skeletal dysplasia reminiscent of the skeletal dysplasia of Csgalnact1–/– mice, and adds to the genetic heterogeneity of DD.

The effects of a strength and neuromuscular exercise programme for the lower extremity on knee load, pain and function in obese children and adolescents: study protocol for a randomised controlled trial

BackgroundChildhood obesity is one of the most critical and accelerating health challenges throughout the world. It is a major risk factor for developing varus/valgus misalignments of the knee joint. The combination of misalignment at the knee and excess body mass may result in increased joint stresses and damage to articular cartilage. A training programme, which aims at developing a more neutral alignment of the trunk and lower limbs during movement tasks may be able to reduce knee loading during locomotion. Despite the large number of guidelines for muscle strength training and neuromuscular exercises that exist, most are not specifically designed to target the obese children and adolescent demographic. Therefore, the aim of this study is to evaluate a training programme which combines strength and neuromuscular exercises specifically designed to the needs and limitations of obese children and adolescents and analyse the effects of the training programme from a biomechanical and clinical point of view.Methods/DesignA single assessor-blinded, pre-test and post-test randomised controlled trial, with one control and one intervention group will be conducted with 48 boys and girls aged between 10 and 18 years. Intervention group participants will receive a 12-week neuromuscular and quadriceps/hip strength training programme. Three-dimensional (3D) gait analyses during level walking and stair climbing will be performed at baseline and follow-up sessions. The primary outcome parameters for this study will be the overall peak external frontal knee moment and impulse during walking. Secondary outcomes include the subscales of the Knee injury and Osteoarthritis Outcome Score (KOOS), frontal and sagittal kinematics and kinetics for the lower extremities during walking and stair climbing, ratings of change in knee-related well-being, pain and function and adherence to the training programme. In addition, the training programme will be evaulated from a clinical and health status perspective by including the following analyses: cardiopulmonary testing to quantify aerobic fitness effects, anthropometric measures, nutritional status and psychological status to characterise the study sample.DiscussionThe findings will help to determine whether a neuromuscular and strength training exercise programme for the obese children population can reduce joint loading during locomotion, and thereby decrease the possible risk of developing degenerative joint diseases later in adulthood.Trial registrationClinicalTrials NCT02545764, Date of registration: 24 September 2015.

MED20 mutation associated with infantile basal ganglia degeneration and brain atrophy

AbstractInfantile movement disorders are rare and genetically heterogeneous. We set out to identify the disease-causing mutation in siblings with a novel recessive neurodegenerative movement disorder. Genetic linkage analysis and whole-exome sequencing were performed in the original family. A cohort of six unrelated patients were sequenced for further mutations in the identified candidate gene. Pathogenicity of the mutation was evaluated by in silico analyses and by structural modeling. We identified the first and homozygous mutation (p.Gly114Ala) in the Mediator subunit 20 gene (MED20) in siblings presenting with infantile-onset spasticity and childhood-onset dystonia, progressive basal ganglia degeneration, and brain atrophy. Mediator refers to an evolutionarily conserved multi-subunit RNA polymerase II co-regulatory complex. Pathogenicity of the identified missense mutation is suggested by in silico analyses, by structural modeling, and by previous reporting of mutations in four distinct Mediator subunits causing neurodegenerative phenotypes. No further MED20 mutations were detected in this study. Conclusion: We delineate a novel infantile-onset neurodegenerative movement disorder and emphasize the Mediator complex as critical for normal neuronal function. Definitive proof of pathogenicity of the identified MED20 mutation will require confirmation in unrelated patients.

Is the reliability of 3D kinematics of young obese participants dependent on the hip joint center localization method used

The aim of this study was to investigate if the test-retest reliability for three-dimensional (3D) gait kinematics in a young obese population is affected by using either a predictive (Davis) or a functional (SCoRE) hip joint center (HJC) localization approach. A secondary goal was to analyze how consistent both methods perform in estimating the HJC position. A convenience sample of ten participants, two females and eight males with an age-based body mass index (BMI) above the 97th percentile (mean±SD: 34.2±3.9kg/m2) was recruited. Participants underwent two 3D gait analysis sessions separated by a minimum of one day and a maximum of seven days. The standard error of measurement (SEM) and the root mean square error (RMSE) of key kinematic parameters along with the root mean square deviation (RMSD) of the entire waveforms were used to analyze the test-retest reliability. To get an estimate of the consistency of both HJC localization methods, the HJC positions determined by both methods were compared to each other. SEM, RMSE, and RMSD results indicate that the HJC position estimations between both methods are not different and demonstrate moderate to good reliability to estimate joint kinematics. With respect to the localization of the HJC, notable inconsistencies ranging from 0 to 5.4cm were observed. In conclusion, both approaches appear equally reliable. However, the inconsistent HJC estimation points out, that accuracy seems to be a big issue in these methods. Future research should attend to this matter.

Demethylation of the promoter region of GPX3 in a newborn with classical phenylketonuria

OBJECTIVES Phenylketonuria (PKU) is characterized by a high phenylalanine (phe) in plasma and oxidative stress. However, the monitoring of oxidative stress in newborns with PKU using the activity levels of antioxidant enzymes is not optimal. We investigated the possibility of monitoring an increased reactive oxygen species (ROS) production using DNA methylation changes of an oxidative stress response element in the promoter region of an enzymatic antioxidant gene. DESIGN AND METHODS Using DNA extracted from blood leukocytes, the cytosine phosphodiester bond guanine positions of an overlapping CCAAT box/metal response element (CGATTGGCTG) of the glutathione peroxidase 3 promoter activated by oxidative stimuli and expressed in plasma were analysed for methylation changes in 20 newborns with hyperphenylalaninemia and 20 healthy controls. RESULTS A demethylated allele was detected in a PKU patient at a phe level of 465μmol/L on day 2 after birth, but not in other patients (phe<465μmol/L, ≥day 2 after birth; phe>465μmol/L, ≥day 3 after birth) and healthy controls (phe<465μmol/L, ≥day 2 after birth). CONCLUSIONS The detection of the demethylated allele could be time and phe concentration dependent. The demethylated allele is suggested as an early epigenetic marker for an extracellular monitoring of an increased ROS production in newborns with PKU.