Expanded newborn screening program in Slovenia using tandem mass spectrometry and confirmatory next generation sequencing genetic testing

The NBS program was first introduced in Slovenia in 1979 with screening for phenylketonuria (PKU) (12). Two years later, screening for congenital hypothyroidism (CH) was added (13). From 1993 to 2012, 358,831 newborns were screened for PKU, and 57 were diagnosed with the disease. A total of 427,396 newborns were screened for CH between the years 1991 and 2012,and the disease was confirmed in 184 cases (14). The incidence of PKU in the stated period was, therefore, 1:6769 for PKU and 1:2323 for CH, which meets the estimated incidence of those diseases in Europe (1:3000 – 1:30,000 for PKU and 1:1300 – 1:13,000 for CH) (14, 15).

In 2000, selective screening for IEMs was initiated, starting with analyses of amino acids and organic acids in symptomatic patients with suspected IEMs or patients with a positive family history for IEMs. Different chromatographic methods were used for the detection of metabolites, such as gas chromatography-mass spectrometry (GC-MS) for organic acid determination and ion exchange chromatography-post-column derivatization or liquid chromatography-tandem mass spectrometry (LC-MS/MS) for amino acid measurement. Liquid chromatography-tandem mass spectrometry was also used for acylcarnitine analysis. As of April 2014, a total of 168 patients with organic acid disorders and amino acid disorders (of them 140 with PKU diagnosed on NBS) and five with fatty acid oxidation disorders were in the Slovenian Register of Inborn Errors of Metabolism (16). Comparing these results with the data from those countries with expanded NBS screening programs showed that we should expect more identified cases (16). Therefore, a pilot study to estimate the incidences of IEMs and support the expansion of the NBS program in Slovenia was performed in 2014–2016 (10, 11).

To prepare an optimal strategy for the organization of the expanded NBS for IEMs in Slovenia, a pilot study was performed analysing 10,048 Slovenian newborns (10). In the initial stage, each IEM that was included in the study was identified by acylcarnitine and amino acid analysis with the MS/MS method for screening and confirmed with confirmatory testing. NGS was included for confirmatory testing in combination with other established tests for IEM detection, such as organic acid determination, amino acid analysis and enzyme activity testing. NGS allows the simultaneous analysis of all genes associated with IEMs included in the screening, and thus represents the method of choice for the confirmation of the diseases identified in the NBS. Out of 10,048 results, 113 follow-ups (newborns with the ten highest concentrations of metabolites included in the pilot study) were evaluated at an outpatient clinic, where additional confirmatory analyses were performed. With confirmatory tests, four new patients with IEMs were detected, namely a patient with glutaric aciduria type 1, a patient with very long-chain-acyl-CoA-dehydrogenase deficiency, and two patients with 3-methylcrotonyl-CoA-carboxylase deficiency. In all cases, the NGS approach identified causative genetic variants, such as those that have not yet been described in the literature (10, 11). Based on our results and comparisons with other European countries, the cut-off values and ratios of individual metabolites were determined.

The process begins with informing parents about the goals and the course of the NBS program. Parents are informed about the NBS procedure and its advantages by a neonatologist at the first medical examination of the newborn. At the same time, an online information leaflet is available (18). The collection of the samples and input of patient data into an electronic medical record (EMR) are performed next, with the generation of a barcode and QR code, followed by the transport of samples to the laboratory, where they are automatically processed, tested, analysed, and confirmed. In cases of a positive screening result, the family is informed by one of the paediatricians subspecialized in paediatric endocrinology and inborn errors of metabolism at the University Medical Centre (UMC) – University Children’s Hospital Ljubljana (UCHL), who also provide family counselling, treatment and further management (9, 17).

Written informed permission is not required to perform the NBS in Slovenia, because the program is a mandatory nationwide population program, which is required by national legislation. If the parents refuse to take part in the program, the neonatologist performs additional counselling before the discharge of the newborn from the hospital. A written refusal statement must be signed on a blank sample card, and the child’s paediatrician is informed. A blank card with maternal data is sent to the laboratory and archived (9).

A custom DBS card with space for barcode and QR code with the information of the newborn and the mother is used for blood sampling. Blood samples are taken 48 to 72 hours after birth from the newborn’s heel or by venepuncture, according to the technical requirements (9).

Sample information and newborn identification data are entered into the hospital EMR, and an order for the NBS testing is created. The sample must be sent from the nursery within 24 hours by mail or courier service to the Department of Nuclear Medicine (DNM), UMC Ljubljana, where all the samples are accepted. Half of the sample card is used at DNM, where the fluorometric method is used for PKU screening and dissociation-enhanced lanthanide fluorescence immunoassay is used for CH screening, as described earlier (14). The other half is sent to the Clinical Institute for Special Laboratory Diagnostics, UMC – University Children’s Hospital Ljubljana (CISLD), where the expanded NBS is performed. In the DNM and CISLD laboratory, sample barcodes are scanned, and the orders made by the hospitals are accepted and automatically transferred into the laboratory information system. Inadequate samples are declined, the nursery is informed, and a new sample is requested (9)

The algorithm for reporting and confirming results of the expanded NBS has been prepared, depending on the level of disease suspicion, mainly based on the degree of metabolite elevation and profiles of their combinations (Fig 1). The nursery is informed about the results of the screening automatically through the information system as soon as the laboratory specialist or/and clinician confirms the results. In the case of borderline or positive results, a new additional sample must be tested. For newborns with borderline results the nursery collects a second sample on the screening card and sample goes through the same initial screening procedure again. When the second testing is indicative of a disease, a newborn is referred to the UCHL for further diagnostics. Newborns with a positive result on initial screening are referred directly to the UCHL. Depending on the disease in question, a sample of urine, plasma or blood may also be requested. All positive results are tested using NGS and/or other confirmatory methods. Additionally, enzymatic activity is determined when indicated (performed at the Amsterdam Medical Center laboratory). The newly detected patients are treated and followed up at the UCHL (9).

Figure 1

Confirmatory testing for borderline and positive CH results is done at DNM, while PKU borderline results are retested at DNM, final confirmation of positive result is done at CISLD (14).

Samples are stored in the laboratory for an indefinite period and can be reused for the selective diagnostics if permitted by official state regulations. Quality indicators for the NBS program are evaluated once per year (9). CISLD is participating in different external quality control schemes, the Newborn Screening Quality Assurance Program (NSQAP) at the US Centers for Disease Control and Prevention (CDC) (https://www.cdc.gov/labstandards/nsqap.html) and European Research Network for evaluation and improvement of screening, Diagnosis and treatment of Inherited disorders of Metabolism (ERNDIM) (https://www.erndimqa.nl/)

When the diagnosis is confirmed, the patient is included in the Slovenian Register of Rare Diseases, and the registration needs to be initiated by the physician who confirmed the diagnosis (9).

A MS/MS (Waters Xevo TQD LC-MS/MS) is used for expanded screening (9). It quantifies the tested metabolites with stable-isotope-labelled internal standards, and allows the detection of many metabolites simultaneously in a single run (19). It can accommodate a high throughput of samples (2–3 min per sample) and has low reagent cost at the same time as being sensitive and specific (20).

For NGS confirmatory testing, an in-house panel of 72 genes has been developed by the newborn screening team, which includes a geneticist (Table 1). The NGS panel includes causative genes for the 18 diseases included in the NBS program (Table 2), with the addition of genes for the conditions that present with the increase of the same metabolites as the targeted disease and are therefore invaluable for differential diagnostics. Variants detected with NGS are confirmed by Sanger sequencing and reported to the NBS clinical team, which includes paediatricians subspecialized in the paediatric endocrinology and inborn errors of metabolism.

If one of the diseases not included in expanded screening is detected, the full NBS team (which includes both laboratory and clinical NBS teams) could make a case-by-case decision to proceed with diagnostics and reporting if that would be in the presumed clinical benefit of the newborn (21).

During the first year of the expanded NBS program, all 14 nurseries were gradually connected in the same laboratory information system.