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.

Investigating the suitability of high-resolution mass spectrometry for newborn screening: identification of hemoglobinopathies and ß-thalassemias in dried blood spots.

A fast and reliable method for the determination of hemoglobinopathies and thalassemias by high-resolution accurate mass spectrometry (HRAM/MS) is presented. The established method was verified in a prospective clinical study (HRAM/MS vs. high-pressure liquid chromatography [HPLC]) of 5335 de-identified newborn samples from the Hamburg area. The analytical method is based on a dual strategy using intact protein ratios for thalassemias and tryptic digest fragments for the diagnosis of hemoglobinopathies. Due to the minimal sample preparation and the use of flow injection, the assay can be considered as a high-throughput screening approach for newborn screening programs (2 min/sample). Using a simple dried blood spot (DBS) extraction (tryptic digest buffer), the following results were obtained: (1) a carrier incidence of 1:100 newborns (35 FAS, nine FAC, eight FAD and two FAE), and (2) no homozygous affected patient was detected. Using the HRAM/MS protocol, an unknown Hb mutation was identified and confirmed by genetic testing. In addition to greater specificity toward rare mutations and ß-thalassemia, the low price/sample (1-2€) as well as an automated data processing represent the major benefits of the described HRAM/MS method.

Recommendations for clinical monitoring of patients with acid sphingomyelinase deficiency (ASMD).

BACKGROUND: Acid sphingomyelinase deficiency (ASMD), a rare lysosomal storage disease, results from mutations in SMPD1, the gene encoding acid sphingomyelinase (ASM). As a result, sphingomyelin accumulates in multiple organs including spleen, liver, lung, bone marrow, lymph nodes, and in the most severe form, in the CNS and peripheral nerves. Clinical manifestations range from rapidly progressive and fatal infantile neurovisceral disease, to less rapidly progressing chronic neurovisceral and visceral forms that are associated with significant morbidity and shorter life span due to respiratory or liver disease. OBJECTIVES: To provide a contemporary guide of clinical assessments for disease monitoring and symptom management across the spectrum of ASMD phenotypes. METHODS: An international group of ASMD experts in various research and clinical fields used an evidence-informed consensus process to identify optimal assessments, interventions, and lifestyle modifications. RESULTS: Clinical assessment strategies for major organ system involvement, including liver, spleen, cardiovascular, pulmonary, and neurological/developmental are described, as well as symptomatic treatments, interventions, and/or life style modifications that may lessen disease impact. CONCLUSIONS: There is currently no disease-specific treatment for ASMD, although enzyme replacement therapy with a recombinant human ASM (olipudase alfa) is in clinical development. Current monitoring addresses symptoms and multisystem involvement. Recommended interventions and lifestyle modifications are designed to address morbidity and disease complications and improve patient quality of life. While infantile neurovisceral ASMD is uniformly fatal in early childhood, patients with chronic visceral and chronic neurovisceral ASMD require appropriate management throughout childhood and adulthood by an interdisciplinary clinical team.

Obstructive sleep apnea and craniofacial appearance in MPS type I-Hurler children after hematopoietic stem cell transplantation.

OBJECTIVES: Mucopolysaccharidosis type I (MPS I) is an inherited lysosomal storage disorder characterized by severe multi-systemic organ manifestations including obstructive sleep apnea syndrome (OSAS). Hematopoietic stem cell transplantation (HSCT) is the treatment of choice in severe MPS I (MPS IH, Hurler syndrome). However, the effect of HSCT on OSAS in MPS IH still remains unclear. The purpose of this study was to analyze respiratory patterns during sleep following HSCT in MPS IH children and to relate these findings to craniofacial abnormalities. METHODS: Overnight polysomnographies of nine MPS IH children (mean age: 8.2 years) previously treated with HSCT were retrospectively analyzed. Magnetic resonance images of the head were assessed with regard to soft and hard tissue abnormalities of the upper respiratory tract. RESULTS: The mean apnea hypopnea index (AHI) was 5.3 events/h (range, 0.3-12.2), and the majority of apnea/hypopneas were obstructive. Whereas two patients had severe OSAS (AHI > 10) and two moderate OSAS (5 > AHI < 10), five patients had no evidence of OSAS (AHI < 2.0). Donor cell chimerism was significantly lower in MPS IH patients with OSAS as compared to patients without OSAS (p < 0.001). The upper airway space and the maxilla were significantly smaller and the adenoids larger in MPS IH patients with OSAS as compared to those of non-OSAS patients. CONCLUSION: OSAS was only observed in MPS IH patients with graft failure or low donor cell chimerism. Conversely, successful HSCT seems to ameliorate adenoid hyperplasia and maxillary constriction in MPS IH patients and thereby minimizes the risk of OSAS at least at younger ages.

Consensus recommendation for a diagnostic guideline for acid sphingomyelinase deficiency.

Disclaimer:This diagnostic guideline is intended as an educational resource and represents the opinions of the authors, and is not representative of recommendations or policy of the American College of Medical Genetics and Genomics (ACMG). The information should be considered a consensus based on expert opinion, as more comprehensive levels of evidence were not available in the literature in all cases. BACKGROUND: Acid sphingomyelinase deficiency (ASMD) is a rare, progressive, and often fatal lysosomal storage disease. The underlying metabolic defect is deficiency of the enzyme acid sphingomyelinase that results in progressive accumulation of sphingomyelin in target tissues. ASMD manifests as a spectrum of severity ranging from rapidly progressive severe neurovisceral disease that is uniformly fatal to more slowly progressive chronic neurovisceral and chronic visceral forms. Disease management is aimed at symptom control and regular assessments for multisystem involvement. PURPOSE AND METHODS: An international panel of experts in the clinical and laboratory evaluation, diagnosis, treatment/management, and genetic aspects of ASMD convened to review the evidence base and share personal experience in order to develop a guideline for diagnosis of the various ASMD phenotypes. CONCLUSIONS: Although care of ASMD patients is typically provided by metabolic disease specialists, the guideline is directed at a wide range of providers because it is important for primary care providers (e.g., pediatricians and internists) and specialists (e.g., pulmonologists, hepatologists, and hematologists) to be able to identify ASMD.Genet Med advance online publication 13 April 2017.

Diagnosis of neuronal ceroid lipofuscinosis type 2 (CLN2 disease): Expert recommendations for early detection and laboratory diagnosis.

Neuronal ceroid lipofuscinoses (NCLs) are a heterogeneous group of lysosomal storage disorders. NCLs include the rare autosomal recessive neurodegenerative disorder neuronal ceroid lipofuscinosis type 2 (CLN2) disease, caused by mutations in the tripeptidyl peptidase 1 (TPP1)/CLN2 gene and the resulting TPP1 enzyme deficiency. CLN2 disease most commonly presents with seizures and/or ataxia in the late-infantile period (ages 2-4), often in combination with a history of language delay, followed by progressive childhood dementia, motor and visual deterioration, and early death. Atypical phenotypes are characterized by later onset and, in some instances, longer life expectancies. Early diagnosis is important to optimize clinical care and improve outcomes; however, currently, delays in diagnosis are common due to low disease awareness, nonspecific clinical presentation, and limited access to diagnostic testing in some regions. In May 2015, international experts met to recommend best laboratory practices for early diagnosis of CLN2 disease. When clinical signs suggest an NCL, TPP1 enzyme activity should be among the first tests performed (together with the palmitoyl-protein thioesterase enzyme activity assay to rule out CLN1 disease). However, reaching an initial suspicion of an NCL or CLN2 disease can be challenging; thus, use of an epilepsy gene panel for investigation of unexplained seizures in the late-infantile/childhood ages is encouraged. To confirm clinical suspicion of CLN2 disease, the recommended gold standard for laboratory diagnosis is demonstration of deficient TPP1 enzyme activity (in leukocytes, fibroblasts, or dried blood spots) and the identification of causative mutations in each allele of the TPP1/CLN2 gene. When it is not possible to perform both analyses, either demonstration of a) deficient TPP1 enzyme activity in leukocytes or fibroblasts, or b) detection of two pathogenic mutations in trans is diagnostic for CLN2 disease.

The Prevalence of Sickle Cell Disease and Its Implication for Newborn Screening in Germany (Hamburg Metropolitan Area).

Sickle cell disease is among hereditary diseases with evidence that early diagnoses and treatment improves the clinical outcome. So far sickle cell disease has not been included in the German newborn screening program despite immigration from countries with populations at risk. To determine the birth prevalence we tested 17,018 newborns. High pressure liquid chromatography and subsequent molecular-genetic testing were used for the detection and confirmation of hemoglobin variants. The frequency of sickle cell disease-consistent genotypes was one in 2,385 newborns. Duffy-blood group typing showed evidence that affected children were likely of Sub-Saharan ancestry. An inclusion of sickle cell disease into the German newborn screening seems reasonable.

Congenital Adrenal Hyperplasia with Non-functional Mutations in Both Alleles in a Clinically Unaffected Infant.

BACKGROUND: Results in neonatal screening programs aiming at detection of congenital adrenal hyperplasia (CAH) can only report elevated levels of 17-hydroxy-progesterone (17-OHP), without being able to differentiate presence or absence of salt loss. AIM: To predict presence or absence of salt loss in newborn infants with CAH. METHODS: The first specimen of suspected CAH in samples sent from People's Democratic Republic of Laos (Lao PDR) was investigated for known mutations in CAH associated with salt loss. RESULTS: Molecular genetic diagnosis revealed mutations associated with loss of function in both alleles; however, the infant was clinically unaffected even without any corticosteroid substitution therapy. CONCLUSIONS: Although molecular genetic methods can theoretically predict loss of function in CAH, our infant was clinically unaffected even without therapy at 6 years of age. We speculate that in CAH, remaining enzyme activity can be sufficiently high, despite the presence of loss of function mutations, which do not affect infants clinically.

Combined Serological Detection of Circulating Angiostrongylus vasorum Antigen and Parasite-specific Antibodies in Dogs from Hungary.

The occurrence of the nematode Angiostrongylus vasorum, also known as the French heartworm, is increasingly being reported from various European countries. The adults of this parasite species live in the pulmonary arteries and right cardiac ventricle of wild canids and domestic dogs. Larval stages and eggs in the lungs induce inflammatory verminous pneumonia, causing severe respiratory disease in dogs. Furthermore, haematological and neurological signs and even death may occur. In Hungary, A. vasorum has been identified in red foxes, golden jackals and in two dogs and some slugs. In this first large-scale survey, 1247 sera from pet dogs were collected and tested by an ELISA for the detection of circulating antigen of A. vasorum and by a separate ELISA to detect specific antibodies against the parasite. A total of 1.36% (n = 17, 95 % confidence intervals, CI: 0.80 - 2.17 %) of the animals were positive in both ELISAs, while 1.76 % (n = 22, CI: 1.11 - 2.66 %) of the tested dogs were antigen-positive only and 2.73 % (n = 34, CI: 1.90 - 3.79 %) were positive for specific antibodies only. Regions with antigen- and antibody-positive animals overlapped and were distributed over nearly the whole sampled areas of the country. A considerable number of cases was observed in Budapest and also in the southern part of the country bordering Croatia, while in the most eastern part bordering Ukraine no positive samples were detected. These results confirm the endemic occurrence of A. vasorum in dogs originating from different parts of Hungary and the significant advantages of A. vasorum serology in epidemiological studies.

Combined D2-/L2-hydroxyglutaric aciduria (SLC25A1 deficiency): clinical course and effects of citrate treatment.

Combined D,L-2-hydroxyglutaric aciduria (DL-2HGA; OMIM #615182) is a rare neurometabolic disorder clinically characterized by muscular hypotonia, severe neurodevelopmental dysfunction, and intractable seizures associated with respiratory distress. Biochemically, DL-2HGA patients excrete increased amounts of D- and L-2-hydroxyglutarate (D2HG and L2HG, respectively), with predominance of D2HG, and α-ketoglutarate, and show a decrease in urinary citrate. Impaired function of the mitochondrial citrate carrier (CIC) due to pathogenic mutations within the SLC25A1 gene has been identified as the underlying molecular cause of the disease. CIC mediates efflux of the mitochondrial tricarboxylic acid (TCA) cycle intermediates citrate and isocitrate in exchange for cytosolic malate. Thus, depletion of cytosolic citrate as well as accumulation of citrate inside mitochondria have been considered to play a role in the pathophysiology of DL-2HGA. Here, we report for the first time on a patient with a genetically confirmed diagnosis of DL-2HGA and treatment with either malate or citrate. During malate treatment, urinary malate concentration increased, but beyond that, neither biochemical nor clinical alterations were observed. In contrast, treatment with citrate led to an increased urinary excretion of TCA cycle intermediates malate and succinate, and by trend to an increased concentration of urinary citrate. Furthermore, excretion of D2HG and L2HG was reduced during citrate treatment. Clinically, the patient showed stabilization with regard to frequency and severity of seizures. Treating DL-2HGA with citrate should be considered in other DL-2HGA patients, and its effects should be studied systematically.

Late-onset Pompe disease is prevalent in unclassified limb-girdle muscular dystrophies.

OBJECTIVE: Late-onset Pompe disease is a rare, but potentially treatable metabolic myopathy, and therefore should not be overlooked. However, it is not unusual that patients go undiagnosed for many years. We hypothesized that patients with late-onset Pompe disease may have been overlooked in a population of patients with unclassified neuromuscular disease. METHODS: We used DBS (dried blood spots) to screen for Pompe disease in the two largest neuromuscular clinics and one of the main respiratory centers in Denmark. We selected patients with unclassified LGDM (limb-girdle muscular dystrophy), idiopathic elevation of creatine kinase, unexplained myopathy on muscle biopsy, unexplained restrictive respiratory insufficiency or unspecified myopathy for screening. RESULTS: 177 patients were found eligible for inclusion, and 103 (58.2%) patients accepted screening. Three patients with Pompe disease were identified with DBS, and subsequent genetic testing revealed known pathogenic mutations in the GAA gene. All three patients were found among 38 patients with unclassified LGMD (8%). CONCLUSION: Our findings indicate that a DBS should be considered early in the diagnostic work-up of patients with an LGMD phenotype, to rule out Pompe disease. Retrospectively, all 3 patients presented with "red flags" more compatible with Pompe disease than LGMD, including; 1) mild non-dystrophic, myopathic features on muscle biopsy, 2) creatine kinase levels below 1000, and 3) disproportionate axial and respiratory muscle involvement in comparison with limb muscle involvement.

Diagnosing mucopolysaccharidosis IVA.

Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is an autosomal recessive lysosomal storage disorder resulting from a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) activity. Diagnosis can be challenging and requires agreement of clinical, radiographic, and laboratory findings. A group of biochemical genetics laboratory directors and clinicians involved in the diagnosis of MPS IVA, convened by BioMarin Pharmaceutical Inc., met to develop recommendations for diagnosis. The following conclusions were reached. Due to the wide variation and subtleties of radiographic findings, imaging of multiple body regions is recommended. Urinary glycosaminoglycan analysis is particularly problematic for MPS IVA and it is strongly recommended to proceed to enzyme activity testing even if urine appears normal when there is clinical suspicion of MPS IVA. Enzyme activity testing of GALNS is essential in diagnosing MPS IVA. Additional analyses to confirm sample integrity and rule out MPS IVB, multiple sulfatase deficiency, and mucolipidoses types II/III are critical as part of enzyme activity testing. Leukocytes or cultured dermal fibroblasts are strongly recommended for enzyme activity testing to confirm screening results. Molecular testing may also be used to confirm the diagnosis in many patients. However, two known or probable causative mutations may not be identified in all cases of MPS IVA. A diagnostic testing algorithm is presented which attempts to streamline this complex testing process.

Establishment of the first newborn screening program in the People's Democratic Republic of Laos.

OBJECTIVES: The People's Democratic Republic of Laos belongs to the minority of countries worldwide without an established newborn bloodspot screening (NBS) system. METHODS: In 2008, we initiated a pilot project of a neonatal screening system in the delivery suites of the Laotian capital, Vientiane. Samples were analysed for thyrotropin-stimulating hormone and 17-hydroxyprogesterone. RESULTS: Altogether 11 362 samples were taken; an initially high recall rate dropped eventually to just above 4%. Two cases of hypothyroidism and one case of congenital adrenal hyperplasia were identified and received timely treatment. CONCLUSIONS: In summary, we have demonstrated the feasibility of establishing an NBS system in a low-resource setting as prevalent in Laos. Obstacles for the establishment of a general NBS covering the whole country include the question of financial cover, treatment costs, and adequate teaching and supervision of technicians and doctors.

Fat and carbohydrate metabolism during exercise in late-onset Pompe disease.

Pompe disease is caused by absence of the lysosomal enzyme acid alpha-glucosidase. It is generally assumed that intra-lysosomal hydrolysis of glycogen does not contribute to skeletal muscle energy production during exercise. However, this hypothesis has never been tested in vivo during exercise. We examined the metabolic response to exercise in patients with late-onset Pompe disease, in order to determine if a defect in energy metabolism may play a role in the pathogenesis of Pompe disease. We studied six adult patients with Pompe disease and 10 healthy subjects. The participants underwent ischemic forearm exercise testing, and peak work capacity was determined. Fat and carbohydrate metabolism during cycle exercise was examined with a combination of indirect calorimetry and stable isotope methodology. Finally, the effects of an IV glucose infusion on heart rate, ratings of perceived exertion, and work capacity during exercise were determined. We found that peak oxidative capacity was reduced in the patients to 17.6 vs. 38.8 ml kg(-1) min(-1) in healthy subjects (p = 0.002). There were no differences in the rate of appearance and rate of oxidation of palmitate, or total fat and carbohydrate oxidation, between the patients and the healthy subjects. None of the subjects improved exercise tolerance by IV glucose infusion. In conclusion, peak oxidative capacity is reduced in Pompe disease. However, skeletal muscle fat and carbohydrate use during exercise was normal. The results indicate that a reduced exercise capacity is caused by muscle weakness and wasting, rather than by an impaired skeletal muscle glycogenolytic capacity. Thus, it appears that acid alpha-glucosidase does not play a significant role in the production of energy in skeletal muscle during exercise.

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.

Tandem mass spectrometry screening for very long-chain acyl-CoA dehydrogenase deficiency: the value of second-tier enzyme testing.

OBJECTIVE: To evaluate newborn screening (NBS) for very long-chain acyl-CoA dehydrogenase deficiency (VLCADD), we further characterized newborns with elevation of one or all C14-carnitine derivatives on NBS from a total of 90 338 newborns. STUDY DESIGN: Palmitoyl-CoA oxidation was performed in lymphocytes to define very long-chain acyl-CoA dehydrogenase function. Molecular analysis followed in children with residual activities<50%. The acylcarnitine pattern on days 2 to 3 of life was evaluated thoroughly to define possible discrimination markers. RESULTS: Forty newborns with increased C14:1-carnitine were identified (1:2500). In 2 newborns, VLCADD was confirmed with enzyme and molecular analyses (prevalence, 1:50,000). One of these newborns had normal results on a second screening. Also, the combination of absolute acylcarnitine values and acylcarnitine ratios did not allow correct identification of the newborn as a patient with VLCADD. CONCLUSIONS: Reliable diagnosis is not feasible with acylcarnitine analysis alone. Enzyme analysis in lymphocytes is a reliable and rapid method for correctly assessing all newborns with VLCADD and should be carried out in all newborns identified during the first screening, regardless of the results of a later acylcarnitine profile.

Diagnostic efficacy of the fluorometric determination of enzyme activity for Pompe disease from dried blood specimens compared with lymphocytes-possibility for newborn screening.

BACKGROUND: Pompe disease is a rare, autosomal-recessive disorder which results from a defect in the lysosomal enzyme acid alpha-glucosidase (GAA). The onset of this disease is highly variable, with infantile types being the most severe. Traditionally, lymphocytes, fibroblasts or muscle biopsies were necessary for enzyme activity measurement, because these materials do not express maltase-glucoamylase (MGA) that interferes with the assay. Recently, acarbose was found to inhibit MGA activity selectively, so that dried blood became accessible for GAA assessment. AIM: To evaluate the diagnostic efficacy of GAA measurement in dried blood specimens (DBSs) in comparison with lymphocytes. If DBSs provided reliable results, the diagnosis of Pompe disease could be facilitated, and high-throughput screening would become possible. METHODS AND RESULTS: GAA activity was measured in DBSs of known patients at pH 3.8 (with and without acarbose) and at pH 7.0. Additionally, lymphocytes were obtained from the same patients, and the enzyme activity was determined at pH 4 to pH 7. In total, seven infantile patients and 29 patients with late-onset variants were investigated. All patients were reliably identified by both methods. Furthermore, a simplified protocol was established for neonatal screening. CONCLUSION: The fluorometric technique for the assessment of GAA activity in DBS provides a reliable diagnosis for all variants of Pompe disease. The assay protocol could be simplified for neonatal screening, without increasing the false positive rate significantly or burdening the laboratory with time-consuming procedures.

Elimination of Dirofilaria (syn. Nochtiella) repens microfilariae in dogs with monthly treatments of moxidectin 2.5%/imidacloprid 10% (Advocate, Bayer) spot-on.

Elimination of microfilaria in dogs infected with zoonotic Dirofilaria repens would be desirable to reduce further spread. Moxidectin has demonstrated efficacy against microfilariae and safety in dogs infected with Dirofilaria immitis and could be an option for controlling D. repens microfilariae. A field study with 64 dogs previously confirmed positive for D. repens microfilaria was conducted in Hungary, in which a spot-on product (Advocate, Bayer) was tested. Treatments were applied to 44 dogs once a month for 3 months (five dogs) or 6 months (22 dogs), alternatively every 2 weeks for 6 months (17 dogs). Twenty dogs remained untreated. Microfilaria counts were performed once a month and for a further 6 months following the last treatment. Two weeks after the first treatment, 38 of 44 dogs were microfilaria negative. Four weeks after the initial treatment, one dog still showed a low microfilaria count. Following the second treatment, all treated dogs were negative. This status was maintained during the 6-month observation period after the last treatment. These data demonstrate the successful long-lasting elimination of microfilariae. Moreover, it may be supposed that adult D. repens were killed based on the observation that no further microfilariae were seen up to 6 months after the end of the treatment period.

At-Risk Testing for Pompe Disease Using Dried Blood Spots: Lessons Learned for Newborn Screening.

Pompe disease (GSD II) is an autosomal recessive disorder caused by deficiency of the lysosomal enzyme acid-α-glucosidase (GAA, EC 3.2.1.20), leading to generalized accumulation of lysosomal glycogen especially in the heart, skeletal, and smooth muscle, and the nervous system. It is generally classified based on the age of onset as infantile (IOPD) presenting during the first year of life, and late onset (LOPD) when it presents afterwards. In our study, a cohort of 13,627 samples were tested between January 2017 and December 2018 for acid-α-glucosidase (GAA, EC 3.2.1.20) deficiency either by fluorometry or tandem mass spectrometry (MS). Testing was performed for patients who displayed conditions of unknown etiology, e.g., CK elevations or cardiomyopathy, in the case of infantile patients. On average 8% of samples showed activity below the reference range and were further assessed by another enzyme activity measurement or molecular genetic analysis. Pre-analytical conditions, like proper drying, greatly affect enzyme activity, and should be assessed with measurement of reference enzyme(s). In conclusion, at-risk testing can provide a good first step for the future introduction of newborn screening for Pompe disease. It yields immediate benefits for the patients regarding the availability and timeliness of the diagnosis. In addition, the laboratory can introduce the required methodology and gain insights in the evaluation of results in a lower throughput environment. Finally, awareness of such a rare condition is increased tremendously among local physicians which can aid in the introduction newborn screening.