Adolf Mühl

Arginine:Glycine Amidinotransferase Deficiency: The Third Inborn Error of Creatine Metabolism in Humans

Arginine:glycine amidinotransferase (AGAT) catalyzes the first step of creatine synthesis, resulting in the formation of guanidinoacetate, which is a substrate for creatine formation. In two female siblings with mental retardation who had brain creatine deficiency that was reversible by means of oral creatine supplementation and had low urinary guanidinoacetate concentrations, AGAT deficiency was identified as a new genetic defect in creatine metabolism. A homozygous G-A transition at nucleotide position 9297, converting a tryptophan codon (TGG) to a stop codon (TAG) at residue 149 (T149X), resulted in undetectable cDNA, as investigated by reverse-transcription PCR, as well as in undetectable AGAT activity, as investigated radiochemically in cultivated skin fibroblasts and in virus-transformed lymphoblasts of the patients. The parents were heterozygous for the mutant allele, with intermediate residual AGAT activities. Recognition and treatment with oral creatine supplements may prevent neurological sequelae in affected patients.

Development and Evaluation of Quality Control Dried Blood Spot Materials in Newborn Screening for Lysosomal Storage Disorders

BACKGROUND Lysosomal storage disorders (LSDs) comprise more than 40 genetic diseases that result in the accumulation of products that would normally be degraded by lysosomal enzymes. A tandem mass spectrometry (MS/MS)-based method is available for newborn screening for 5 LSDs, and many laboratories are initiating pilot studies to evaluate the incorporation of this method into their screening panels. We developed and evaluated dried blood spot (DBS) QC materials for LSDs and used the MS/MS method to investigate their suitability for LSD QC monitoring. METHODS We incubated 3.2-mm punches from DBS controls for 20-24 h with assay cocktails containing substrate and internal standard. Using MS/MS, we quantified the resulting product and internal standard. Samples were run in triplicate for 3 consecutive days, and results were reported as product-to-internal standard ratios and enzyme activity units (micromol/L/h). RESULTS Enzyme activity interday imprecision (CV) for the high, medium, and low series were 3.4%-14.3% for galactocerebroside alpha-galactosidase, 6.8%-24.6% for acid alpha-galactosidase A, 7.36%-22.1% for acid sphingomyelinase, 6.2%-26.2% for acid alpha-glucocerebrosidase, and 7.0%-24.8% for lysosomal acid alpha-glucosidase (n = 9). In addition, DBS stored at -20 degrees and 4 degrees C showed minimal enzyme activity loss over a 187-d period. DBS stored at 37 degrees and 45 degrees C had lower activity values over the 187-day evaluation time. CONCLUSIONS Suitable QC materials for newborn screening of LSDs were developed for laboratories performing DBS LSD screening. Good material linearity was observed, with goodness-of-fit values of 0.953 and higher. The QC materials may be used by screening laboratories that perform LSD analysis by MS and/or more conventional fluorescence-based screening methods.

Changes of tissue creatine concentrations upon oral supplementation of creatine-monohydrate in various animal species

Creatine is a nutritional supplement with major application as ergogenic and neuroprotective substrate. Varying supplementation protocols differing in dosage and duration have been applied but systematic studies of total creatine (creatine and phosphocreatine) content in the various organs of interest are lacking. We investigated changes of total creatine concentrations in brain, muscle, heart, kidney, liver, lung and venous/portal plasma of guinea pigs, mice and rats in response to 2-8 weeks oral creatine-monohydrate supplementation (1.3-2 g/kg/d; 1.4-2.8% of dietary intake). Analysis of creatine and phosphocreatine content was performed by high performance liquid chromatography. Total creatine was determined as the sum of creatine and phosphocreatine. Presupplementation total creatine concentrations were high in brain, skeletal and heart muscle (10-22 micromol/g wet weight), and low in liver, kidney and lung (5-8 micromol/g wet weight). During creatine supplementation, the relative increase of total creatine was low (15-55% of presupplementation values) in organs with high presupplementation concentrations, and high (260-500% of presupplementation values) in organs with low presupplementation concentrations. The increase of total creatine concentrations was most pronounced after 4 weeks of supplementation. In muscle, brain, kidney and lungs, an additional increase (p<0.01) was observed between 2-4 and 2-8 weeks of supplementation. Absolute concentrations of phosphocreatine increased, but there was no increase of the relative (percentual) proportion of phosphocreatine (14-45%) during supplementation. Statistical comparison of total creatine concentrations across the species revealed no systematically differences in organ distribution and in time points of supplementation. Results suggest that in organs with low presupplementation creatine levels (liver, kidney), a major determinant of creatine uptake is an extra-intracellular concentration gradient. In organs with high presupplementation total creatine levels like brain, skeletal and heart muscle, the maximum capacity of creatine accumulation is low compared to other organs. A supplementation period of 2 to 4 weeks is necessary for significant augmentation of the creatine pool in these organs.

Newborn Screening for Pompe Disease by Measuring Acid α-Glucosidase Activity Using Tandem Mass Spectrometry

BACKGROUND Pompe disease, caused by the deficiency of acid alpha-glucosidase (GAA), is a lysosomal storage disorder that manifests itself in its most severe form within the first months of life. Early detection by newborn screening is warranted, since prompt initiation of enzyme replacement therapy may improve morbidity and mortality. We evaluated a tandem mass spectrometry (MS/MS) method to measure GAA activity for newborn screening for Pompe disease. METHODS We incubated 3.2-mm punches from dried blood spots (DBS) for 22 h with the substrate [7-benzoylamino-heptyl)-{2-[4-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy)-phenylcarbamoyl]- ethyl}-carbamic acid tert-butyl ester] and internal standard [7-d(5)-benzoylamino-heptyl)-[2-(4-hydroxy-phenylcarbamoyl)-ethyl]-carbamic acid tertbutyl ester]. We quantified the resulting product and internal standard using MS/MS. We assessed inter- and intrarun imprecision, carryover, stability, and correlation between enzyme activities and hematocrit and punch location and generated a Pompe disease-specific cutoff value using routine newborn screening samples. RESULTS GAA activities in DBS from 29 known Pompe patients were <2 micromol/h/L. GAA activities in routine newborn screening samples were [mean (SD)] 14.7 (7.2) micromol/h/L (n = 10,279, median 13.3, 95% CI 14.46-14.74 micromol/h/L) and in normal adult samples 9.3 (3.3) micromol/h/L (n = 229, median 9, 95% CI 8.88-9.72 micromol/h/L). GAA activity was stable for 28 days between 37 degrees C and -80 degrees C. Carryover could not be observed, whereas intrarun and interrun imprecision were <10%. The limit of detection was 0.26 micromol/h/L and limit of quantification 0.35 micromol/h/L. CONCLUSIONS The measurement of GAA activities in dry blood spots using MS/MS is suitable for high-throughput analysis and newborn screening for Pompe disease.

Newborn screening for Fabry disease by measuring GLA activity using tandem mass spectrometry

BACKGROUND Fabry disease (FD) is an X-linked lysosomal storage disorder caused by the deficiency of alpha-galactosidase A (GLA). We evaluated a tandem mass spectrometry method to measure GLA activity. METHODS One 3.2mm punch from a dried blood spot sample (DBS) was incubated with substrate and internal standard in the reaction buffer for 22 h. The resulting product was quantified against internal standard using MS/MS. RESULTS The median GLA activity of male newborn DBS (N=5025) was 9.85 + or - 6.4 micromol/h/l (CI 95% is 9.67-10.02 micromol/h/l); The median GLA activity of female newborns (N=4677) was 10.2 + or - 6.3 micromol/h/l (CI 95% is 10.02-10.38 micromol/h/l). The difference between the two subgroups is within assay analytical variation. The GLA activities in the DBS samples from 9 juvenile and adult males with previously identified FD were below 1.64 micromol/h/l. The GLA activities from 32 juvenile and adult females with confirmed FD were below 4.73 micromol/h/l. In 5 (16%) females GLA activities were above the 0.5th percentile of lower limit of CI 95% at 3.18 micromol/h/l. CONCLUSIONS The MS/MS method for Fabry disease newborn screening is robust and can be readily multiplexed with other lysosomal disorders such as Pompe, Gaucher, Niemann-Pick, and Krabbe diseases.

The use of dried blood spot samples in the diagnosis of lysosomal storage disorders — Current status and perspectives

Dried blood spot (DBS) methods are currently available for identification of a range of lysosomal storage disorders (LSDs). These disorders are generally characterized by a deficiency of activity of a lysosomal enzyme and by a broad spectrum of phenotypes. Diagnosis of LSD patients is often delayed, which is of particular concern as therapeutic outcomes (e.g. enzyme replacement therapy) are generally more favorable in early disease stages. Experts in the field of LSDs diagnostics and screening programs convened and reviewed experiences with the use of DBS methods, and discuss the diagnostic challenges, possible applications and quality programs in this paper. Given the easy sampling and shipping and stability of samples, DBS has evident advantages over other laboratory methods and can be particularly helpful in the early identification of affected LSD patients through neonatal screening, high-risk population screening or family screening.

Newborn Screening for Lysosomal Storage Disorders in Hungary

Even though lysosomal storage disorders (LSDs) are considered to be orphan diseases, they pose a highly relevant cause for morbidity and mortality as their cumulative prevalence is estimated to be 1:4,000. This is especially important as treatment in form of enzyme replacement therapy, substrate reduction therapy or stem cell transplantation is amenable for some LSDs. It is plausible that an early start of treatment might improve the overall prognosis and, even more important, prevent irreversible damage of key organs. To get a more precise insight into the real frequency of some LSDs in the general population, we screened 40,024 samples from the Hungarian newborn screening (NBS) program in Szeged for Fabry disease (FD), Gaucher disease (GD), Pompe disease (PD), and Niemann-Pick A/B (NPB) disease using tandem mass spectrometry. Altogether, 663 samples (1.66%) were submitted for retesting. Genetic confirmation was carried out for 120 samples with abnormal screening results after retesting, which identified three cases of GD, three cases of FD, nine cases of PD, and two cases with NPB. In some cases, we detected up to now unknown mutations - one in NPB and seven in PD - which raise questions about the clinical consequences of a NBS in the sense of late-onset manifestations. Overall, we conclude that screening for LSDs by tandem MS/MS followed by a genetic workup in identified patients is a robust, easy, valid, and feasible technology in newborn screening programs. Furthermore, early diagnosis of LSDs gives a chance to early treatment, but needs more clinical long-term data especially regarding the consequence of private mutations.

Clinical and neuropsychological outcome in 33 patients with biotinidase deficiency ascertained by nationwide newborn screening and family studies in Austria

Abstract Newborn screening for biotinidase deficiency (BD) provides prevention of neurological sequelae in patients with low residual enzyme activity by early treatment with oral biotin substitution. Screening 1.1 million newborns in Austria and consecutive family studies led to the identifcation of 21 patients with profound BD (residual activity <10%) (incidence: 1:59,800) and to 12 patients with partial BD (residual activity 10%–30%) (incidence 1:89,700). Application of an HPLC assay using the natural substrate biocytin allowed exact quantification of extremely low residual biotinidase activities and thus subdivision of patients with profound BD into a group with a residual activity 0%–1% of normal activity (n=5) and >1%–<10% (n=16) respectively. Evaluation of clinical and neuropsychological outcome showed that only patients with a biotinidase activity <1% (n=3/5) exhibited characteristic clinical symptoms within the first weeks of life, while five patients with a residual activity of 1.2%–4.6% did not develop clinical symptoms even when not treated until 3.5–21 years. In all patients with residual activity <10% and biotin substitution within the first weeks of life, neuropsychological outcome was normal, while abnormal in three out of five patients tested for IQ and treated after the age of 3.5 years. In five out of nine patients with poor compliance or delayed or no treatment, visual and brainstem auditory evoked potentials were measured and were within age-related normal values. All patients with partial BD available for follow-up remained clinically and neuropsychologically asymptomatic without treatment at ages 2.5–10 years. Conclusion The incidence of biotinidase deficiency in Austria is comparable to other European countries. Subdivision of the group of patients with profound biotinidase deficiency suggests that only patients with residual activities <1% are prone to develop clinical symptoms early in life, while patients with residual activities >1% may remain asymptomatic even without treatment, as do patients with partial deficiency. Moderate mental retardation might represent a possible manifestation of cerebral dysfunction in patients with profound biotinidase deficiency.

Molecular characterisation of 34 patients with biotinidase deficiency ascertained by newborn screening and family investigation.

This study characterises the spectrum of biotinidase mutations in 21 patients (17 families) with profound biotinidase deficiency (BD) and 13 unrelated patients with partial BD using a denaturing gradient gel electrophoretic mutation screening and selective sequencing approach. In 29 from 30 unrelated families we found biallelic mutations including four common mutations, D444H (frequency 23.3%), G98:d7i3(20.0%), Q456H(20.0%), T532M (15.0%) and nine rare mutations (V62M, R157H, A171T+D444H, C423W, D543H, L279W, N172S, V109G, 12236G-A) with frequencies less than 5.0%. Only three profound BD patients with G98:d7i3/G98:d7i3 and Q456H/Q456H genotypes and residual biotinidase activities of 0.0%, and 0.9% of normal activity developed clinical symptoms before biotin supplementation at 8 weeks of age. All other patients remained asymptomatic within the first months of life or even longer without treatment. Two patients homozygous for the frameshift mutation G98:d7i3 had no measurable residual enzyme activity. Twelve patients with partial BD had the D444H mutation in at least one allele. We conclude that, based on mutation analysis and biochemical examinations of the enzyme, it is currently not clearly predictable whether an untreated patient will develop symptoms or not, although it seems that patients with activities lower than 1% are at a high risk for developing symptoms of the disease early in life.

Molecular characterisation and neuropsychological outcome of 21 patients with profound biotinidase deficiency detected by newborn screening and family studies.

Early recognition by newborn screening and oral biotin supplementation may prevent clinical and neurological deficits in profound biotinidase deficiency (residual plasma biotinidase activity <10%). In order to evaluate possible correlations of molecular characteristics, onset and continuation of treatment and clinical outcome, we investigated 21 patients detected by newborn screening and consecutive family investigations. In 18 patients found by newborn screening, the range of biotinidase activities was 0%–9% residual activity. Application of a sensitive HPLC assay enabled us to discriminate five patients with residual biotinidase activities <1%. Two patients with zero activities were homozygous for the G98:d7i3 mutation and three patients with activities <1% carried mutations G98:d7i3, R157H, and Q456H. The mutation spectrum of the remaining patients included T532M, A171T+D444H, V62M,C432W, and D444H. Evaluation of clinical and neuropsychological outcome showed that only patients with biotinidase activities <1% exhibited characteristic clinical symptoms within the first weeks of life whereas five patients with residual activities of 1.2%–4.6% did not develop clinical symptoms even when not treated until 3.5–21 years. In all patients treated with biotin within the first weeks of life, neuropsychological outcome was normal whereas abnormal in three out of five patients tested for IQ and treated after the age of 3.5 years. Conclusion:the clinical and molecular spectrum of profound biotinidase deficiency is heterogeneous. Early onset of symptoms is predicted by the presence of zero residual activity as measured by sensitive assays and by homozygosity for the G98:d7i3 mutation. In patients with higher residual activities and variable mutational spectrum, correlation with the onset and severity of symptoms cannot be made.