Selective Screening for Metabolic Disorders in the Slovenian Pediatric Population.

BACKGROUND: Inborn errors of metabolism (IEM) are disorders with a block in the metabolic pathway caused by a genetic defect of a specific enzyme. Although each of these diseases is quite rare, as a group they account for a significant proportion of newborn and childhood morbidity and mortality. Early diagnosis is important to prevent complications or even death of the child. Selective screening is an important diagnostic tool for the diagnosis of IEM.
METHODS: In Slovenia, symptomatic patients with suspected IEM are referred to the University Children's Hospital Ljubljana. Techniques used for selective screening are gas chromatography-mass spectrometry, ion exchange chromatography-post-column derivatization, liquid chromatography-tandem mass spectrometry and isoelectric focusing. Fluorimetric method is used for enzyme activity measurement.
RESULTS: There are 168 patients with amino and organic acidemias, 5 patients with disorders in fatty acids metabolism, 1 patient with a congenital disorder of glycosylation, 42 patients with Fabry disease (of which 37 are adult) and 20 patients with Gaucher disease (of which 18 are adult) in the Slovenian Register for Rare Diseases.
CONCLUSIONS: In Slovenia, management of patients with IEM is centralized at the University Children's Hospital, with the exception of adult patients with Fabry and Gaucher disease. The team work is well organized with close cooperation between the laboratory and pediatricians specialized in metabolic disorders. According to the known frequencies of IEM from the literature, we would expect more positive results than obtained. To evaluate these results, we are planning to perform a pilot study on expanded newborn screening.

Introduction

Selective screening is an important diagnostic tool for the diagnosis of various types of IEM. IEM are individually rare, heterogeneous genetic disorders, but collectively numerous. They mostly occur in early infancy and childhood, with a diverse clinical spectrum. The incidence within different racial and ethnic groups varies, with predominance of certain IEM within particular groups. Late diagnosis and treatment of some metabolic disorders can cause irreversible mental retardation ranging from mild to severe, neurological damage, physical disability and even fatality (1, 2). Early diagnosis is important not only for treatment but also for genetic counseling. In previous years, an increase in recognized IEM has been observed, possibly reflecting the improved diagnostic facilities, better coverage, increased medical awareness and newly discovered diseases (2, 3). Selective screening is performed for patients with clinical signs, findings of routine laboratory tests or family history indicating a metabolic disorder. Basic tests measure metabolites that are elevated due to a block at some point in the metabolic pathway. The techniques used most commonly for metabolites detection are gas chromatography-mass spectrometry (GC-MS) for organic acids determination, ion exchange chromatography-post-column derivatization for amino acids measurement (5) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for acylcarnitine analysis (6). Isoelectric focusing of serum transferrin is a widely used method to screen for congenital disorders of N-glycosylation (7). Additionally, the approach is to screen at the enzyme level, as for galactosemia or Fabry disease (8, 9). An abnormal result from a screening test is followed by a subsequent definitive test to confirm the suspected diagnosis, namely enzyme activity measurement or DNA testing.

In 2000, we initiated selective screening for inborn errors of metabolism in Slovenia by analyzing amino acids and organic acids. Other methods were introduced later-on. In the present work we discuss our experiences from the past 13 years.

Materials and Methods

Patients

Symptomatic patients with suspected IEM from all parts of Slovenia were referred to the University Children’s Hospital in Ljubljana or their samples were sent from other hospitals. Vomiting, convulsion, slow development, mental retardation, abnormal muscular tone, jaundice and hepatomegaly, coma of unexplained etiology, cardiomyopathy, dysmorphic features were the most often signs in patients. Laboratory tests, which indicated the referral, showed metabolic acidosis, hypoglycemia, hyperammonemia, lactic acidemia and positive ketone bodies in urine. Patients’ clinical information was obtained from the request forms.

Organic acids analysis

Random urine samples without addition of preservative were collected; creatinine was measured and urine was stored at −20 °C prior to analysis. Briefly, a urine sample was oximated with hydroxylamine (O-Ethyl hydroxylamine hydrochloride, Aldrich, Germany). An internal standard, 2-phenylbutyric acid (Aldrich, Germany), was added in a concentration of 100 mmol/mol creatinine. Urine was acidified and saturated with NaCl and organic acids were extracted using ethyl-acetate. Organic layer was evaporated under the steam of nitrogen. The dried residue was dissolved in pyridine (Fluka, Germany), derivatized with BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide, Aldrich, Germany) and injected into a GC-MS system (Agilent Technologies, USA). The obtained spectra were compared with the known library spectra.

Amino acids analysis

Blood samples (2 mL) were collected into a heparinized vacutainer and centrifuged. Plasma was separated, proteins from plasma were precipitated (Seraprep, Pickering Laboratories, USA), supernatant was filtered through a 0.2 μm syringe filter (Sartorious Minisart, Germany) and stored at −20 °C till the analysis. Urine and cerebrospinal fluid (CSF) samples were treated with Uriprep (Pickering Laboratories, USA), supernatants filtered and stored at −20 °C. The plasma, urine and CSF amino acids concentrations were analyzed by ion exchange chromatography with ninhydrin detection (HPLC pump Series 1200 from Agilent Technologies, and Pinnacle PCX from Pickering Laboratories, USA). All buffers and ninhydrin reagents were obtained from Pickering Laboratories (USA). Amino acids standard solution used for calibration was purchased from Sigma (Germany) and prepared according to the manufacturer’s protocol.

Acylcarnitine analysis

Capillary blood was spotted directly on filter paper (Whatman 903) for the MS/MS analysis. Blood samples were allowed to dry at room temperature for at least 4 hours. They were stored at 8 °C prior to analysis. For the determination of acylcarnitines, Chromsystems MassChrom® Aminoacid and Acylcarnitines from dried blood spot reagent kit was used. The sample preparation was based on extraction followed by derivatization to butyric esters. The dried blood spot controls level I and level II were included in every analytical batch to monitor accuracy and precision within the system. The MS/MS measurements were performed using 3200 QTrap AB SCIEX (USA) and the Perkin Elmer Series 200 HPLC system (USA). ESI-positive ionization and MRM mode were used. The application did not require an HPLC column.

Isoelectric focusing (IEF) of transferrin

Serum was collected and stored at −20 °C prior to analysis. Total iron saturation is necessary prior to IEF. Hydrated Immobiline Dry gel pH 4.0–7.0 (GE Healthcare, Sweden) and Pharmalyte pH 2.5–5 and pH 5–8 (GE Healthcare, Sweden) on a Multiphor II (Pharmacia Biotech, Sweden) system were used; immunofixation was done by exposure to anti-transferrin antibody (Dako, Denmark). Detection was performed by Bomasie Blue solution. Positive controls from CDG type I and CDG type II patients were included in each batch.

Enzyme activity of α-galactosidase A (α-GalA) and glucocerebrosidase in leukocytes

Peripheral venous blood samples (5 mL) were obtained by venepuncture into EDTA tubes. Leukocytes were isolated from whole blood by the ammonium chloride method. The leukocyte pellet was washed with phosphate buffered saline (PBS) and resuspended in 0.25% triton X-1000T. The suspension was sonicated on ice. After centrifugation, the supernatants were used as the cell lysate. Supernatants were stored at −20 °C prior to analysis.

The activity of leukocyte α-GalA and glucocerebrosidase was determined by fluorimetric measurement of the amount of 4-methylumbelliferone released by hydrolysis of a synthetic substrate 4-methylumbelliferyl-α-D-galactopyranoside and 4-methylumbelliferyl-β-D-glucopyranoside, respectively.

Quality control

We are participating in the ERNDIM (European Research Network for Evaluation and Improvement of Screening, Diagnosis and Treatment of Inherited Disorders of Metabolism) schemes for qualitative organic acids, quantitative amino acids, qualitative blood spot acylcarnitine, congenital disorders of glycosylation (http://cms.erndimqa.nl/) and the IN-STAND (Institute for Standardization and Documentation in the Medical Laboratory) scheme for Neonatal Screening – Inborn Errors of Metabolism (http://www.instandev.de/).

Results

The number of measured samples has increased since the beginning of implementation of individual methods and has stabilized in the past years. In the year 2013, around 800 samples were measured for organic acids, 1100 samples for amino acids, 350 samples for acylcarnitines, 50 samples for glycosylation disorders, 13 for Fabry disease and 2 for Gaucher disease. Confirmation of positive results was primarily performed in a different specialized laboratory elsewhere in the EU, a few being confirmed in USLD (mostly genetic tests). Until April 2014, 168 patients with amino and organic acidemias (of which 140 were PKU patients, diagnosed on newborn screening), 5 patients with disorders in fatty acid metabolism, 1 patient with a congenital disorder of glycosylation, 42 patients with Fabry disease (of which 37 are adults) and 20 patients with Gaucher disease (of which 18 are adults) were identified, as presented in Table I. Some of them were diagnosed prior to introducing the abovementioned methods. A list of the DNA tests currently available for confirmation of metabolic disorders in USLD can be found in Table II.

Table I

Part of the Slovenian Register of Rare Disorders.

	Amino acid and protein metabolism	No. of patients
1	Phenylketonuria	140
2	BH4 metabolism – PTPS deficiency	2
3	Propionic acidemia	2
4	Methylmalonic acidemia	1
5	Glutaric aciduria type I	2
6	Glycerol kinase deficiency	1
7	Nonketotic hyperglycinemia	3
8	Urea cycle disorder – OTC deficiency	7
9	Homocystinuria	7
10	Canavan disease	1
11	Alkaptonuria	1
12	Barth syndrome	1
	Lysosomal diseases
13	Fabry disease	5 (+37 adults)
14	Gaucher disease type I	2 (+18 adults)
	Fatty acids metabolism
15	LCHAD deficiency	3
16	SCAD deficiency	1
17	GA II	1
	Congenital disorders of glycosylation
18	CDG type II	1

Table II

Genetic tests available in USLD for confirmation of metabolic disorders.

Gene	Disease
PHA	Phenylketonuria
GLA	Fabry disease
CBS	Homocystinuria
ASPA	Canavan disease
ACADS	SCAD
SH2D1A	X-linked adrenoleukodystrophy
TAZ	Barth syndrome
MVK	Mevalonic aciduria

Discussion

The number of live births in Slovenia between 2000 and 2012 was on average 19.634 per year, as reported by the Statistical Office of the Republic of Slovenia (www.stat.si). Regarding the number of births and 2 millions of inhabitants in Slovenia, management of the patients with IEM is reasonably centralized in one metabolic centre at the University Children’s Hospital to ensure a sufficient level of experience; the exception are adult patients with Fabry and Gaucher disease.

The introduction of selective screening in Slovenia enabled the measurement of more patient samples, leading to an increased number of identified patients with metabolic disorders. Some of the samples came from acutely ill patients, but the majority came from patients with nonspecific clinical signs. Most of the patients were diagnosed within the first years of life. Marked exceptions were present, for example, a case of propionic acidemia, where the patient was diagnosed at the age of 15 with mild symptoms and only slightly increased metabolites and a case of late-onset OTC deficiency diagnosed at the age of 13. The most common lysosomal storage diseases are Fabry and Gaucher disease. Fabry patients were mostly diagnosed as adults in a specialized centre for Fabry disease in Slovenj Gradec Hospital. Diagnosis of Fabry disease is frequently delayed due to uncharacteristic symptoms (10–12). Adult Gaucher patients are managed at the Hematology Department, UMC.

The most patients were diagnosed with PKU through the newborn screening program at the Department of Nuclear Medicine, UMC (0–6 confirmed cases per year). The incidence of PKU in Slovenia is around 1:6,000 (13), while in Europe it is between 1:3,000 and 1:30,000 (14, 15). Homocystinuria was diagnosed in 7 patients from 5 unrelated non-consanguineous families with the calculated prevalence estimated to 1 in 300,000. This is similar to the report from former Czechoslovakia, with the calculated frequency of detected homocystinuria of 1 in 349,000 (16). Based on data from countries that consistently screen newborns, the estimated worldwide frequency of homocystinuria ranges from 1 case per 58,000 to 1 case per 1,000,000. Significant variability in the frequency of homocystinuria has been observed (17). OTC deficiency as the most common urea cycle defect was detected in 7 patients. Therefore, the prevalence was similar as for homocystinuria and less than estimated by Testai and Gorelick (15). They estimated the prevalence of OTC to range from 1 in 40,000 to 1 in 80,000. Based on the conditions mentioned in Table I (not taking PKU into account), 1–2 cases were confirmed per year. According to the reported frequencies of IEM, we would expect more identified cases of IEM in the Slovenian population. In particular, more cases of congenital disorders of glycosylation (18) and MCAD deficiency disorders (19). MCAD deficiency is the most common disorder of mitochondrial β-oxidation and has been part of many screening programs for the past ten years (20–22). Reports from newborn screening programs stated that the prevalence of MCAD deficiency was higher than expected (23). Harms and Olgemöller (24) showed that expansion of a screening program resulted in a 57% increase in the overall number of detected cases. Early detection of IEM improves the outcomes of the patients (25). Introduction of MS/MS enables the detection of several diseases in a single run and is nowadays widely used for newborn screening. Many countries have incorporated newborn screening in their public health programs (26–28). In Slovenia, only PKU and CH have been screened for since 1979 and 1982, respectively (29, 30). Based on the positive experiences in selective screening and newborn screening for PKU and CH, expanded screening should be implemented into the Slovenian public health program. To facilitate this, we are planning to perform a pilot study to estimate the incidence of newborn errors of metabolism in Slovenia.