Peter Schielen

Relationship between neonatal screening results by HPLC and the number of α-thalassaemia gene mutations; consequences for the cut-off value

Objectives To evaluate the relationship between FAST peak percentage by adapted Bio-Rad Vnbs analysis using the valley-to-valley integration and genotypes with the aim to improve differentiation between severe α-thalassaemia forms (HbH disease) and the milder disease types. Method DNA analysis for α-thalassaemia was performed on 91 dried blood spot samples presenting normal and elevated FAST peak levels, selected during three years of Dutch national newborn screening. Results Significant differences were found between samples with and without α-thalassaemia mutations, regardless of the genetic profiles. No significant difference was demonstrated between HPLC in -α/αα and -α/-α, between -α/-α and –/αα and between –/αα and –/-α genotypes. Conclusion This study confirms that the percentage HbBarts, as depicted by the FAST peak, is only a relative indication for the number of α genes affected in α-thalassaemia. Based on the data obtained using the modified Bio-Rad Vnbs software, we adopted a cut-off value of 22.5% to discriminate between possible severe α-thalassaemia or HbH disease and other α-thalassaemia phenotypes. Retrospectively, if this cut-off value was utilized during this initial three-year period of neonatal screening, the positive predictive value would have been 0.030 instead of 0.014.

Inborn Errors of Metabolism That Cause Sudden Infant Death: A Systematic Review with Implications for Population Neonatal Screening Programmes

Background: Many inborn errors of metabolism (IEMs) may present as sudden infant death (SID). Nowadays, increasing numbers of patients with IEMs are identified pre-symptomatically by population neonatal bloodspot screening (NBS) programmes. However, some patients escape early detection because their symptoms and signs start before NBS test results become available, they even die even before the sample for NBS has been drawn or because there are IEMs which are not included in the NBS programmes. Objectives and Methods: This was a comprehensive systematic literature review to identify all IEMs associated with SID, including their treatability and detectability by NBS technologies. Reye syndrome (RS) was included in the search strategy because this condition can be considered a possible pre-stage of SID in a continuum of aggravating symptoms. Results: 43 IEMs were identified that were associated with SID and/or RS. Of these, (1) 26 can already present during the neonatal period, (2) treatment is available for at least 32, and (3) 26 can currently be identified by the analysis of acylcarnitines and amino acids in dried bloodspots (DBS). Conclusion: We advocate an extensive analysis of amino acids and acylcarnitines in blood/plasma/DBS and urine for all children who died suddenly and/or unexpectedly, including neonates in whom blood had not yet been drawn for the routine NBS test. The application of combined metabolite screening and DNA-sequencing techniques would facilitate fast identification and maximal diagnostic yield. This is important information for clinicians who need to maintain clinical awareness and decision-makers to improve population NBS programmes.

Nine years of newborn screening for classical galactosemia in the Netherlands: Effectiveness of screening methods, and identification of patients with previously unreported phenotypes

INTRODUCTION Newborn screening (NBS) for classical galactosemia (CG) was introduced in the Netherlands in 2007. Multiple screening methods have been used since, and currently a two-tier system is used, with residual enzyme activity of galactose-1-phosphate-uridyltransferase (GALT) and total galactose concentration in dried blood spots as the primary and secondary markers. As it is essential to monitor effectiveness of NBS programs, we assessed the effectiveness of different screening methods used over time (primary aim), and aimed to identify and investigate patients identified through NBS with previously unreported clinical and biochemical phenotypes (secondary aim). METHODS The effectiveness of different screening methods and their cut-off values (COVs), as used from 2007 through 2015, was determined, and the clinical and biochemical data of all identified patients were retrospectively collected. RESULTS All screening methods and COVs resulted in relatively high false-positive rates and low positive predictive values. Total galactose levels in dried blood spots were far above the COV for NBS in all true positive cases. A total of 31 galactosemia patients were identified, and when corrected for a family with three affected siblings, 14% had a previously unreported phenotype and genotype. These individuals did not demonstrate any symptoms at the time of diagnosis while still being exposed to galactose, had galactose-1-phosphate values below detection limit within months after the start of diet, and had previously unreported genotypes. CONCLUSION Optimization of NBS for CG in the Netherlands is warranted because of the high false-positive rate, which may result in significant harm. Furthermore, a surprising 14% of newborns identified with CG by screening had previously unreported clinical and biochemical phenotypes and genotypes. For them, individualized prognostication and treatment are warranted, in order to avoid unnecessary stringent galactose restriction.

A newborn screening method for cerebrotendinous xanthomatosis using bile alcohol glucuronides and metabolite ratios

Cerebrotendinous xanthomatosis (CTX) is a treatable neurodegenerative metabolic disorder of bile acid synthesis in which symptoms can be prevented if treatment with chenodeoxycholic acid supplementation is initiated early in life, making CTX an excellent candidate for newborn screening. We developed a new dried blood spot (DBS) screening assay for this disorder on the basis of different ratios between the accumulating cholestanetetrol glucuronide (tetrol) and specific bile acids/bile acid intermediates, without the need for derivatization. A quarter-inch DBS punch was extracted with methanol, internal standards were added, and after concentration the extract was injected into the tandem mass spectrometer using a 2 min flow injection analysis for which specific transitions were measured for cholestanetetrol glucuronide, taurochenodeoxycholic acid (t-CDCA), and taurotrihydroxycholestanoic acid (t-THCA). A proof-of-principle experiment was performed using 217 Guthrie cards from healthy term/preterm newborns, CTX patients, and Zellweger patients. Using two calculated biomarkers, tetrol:t-CDCA and t-THCA:tetrol, this straightforward method achieved an excellent separation between DBSs of CTX patients and those of controls, Zellweger patients, and newborns with cholestasis. The results of this small pilot study indicate that the tetrol:t-CDCA ratio is an excellent derived biomarker for CTX that has the potential to be used in neonatal screening programs.

An evaluation of the TREC assay with regard to the integration of SCID screening into the Dutch newborn screening program

Newborn screening of severe combined immunodeficiency through the detection of T-cell receptor excision circles will provide the opportunity of treating before the occurrence of life-threatening infections. With the EnLite Neonatal TREC assay (PerkinElmer) and end-point PCR, 39 samples (3.0%) of 1295 heel prick cards of the Dutch newborn screening program required a retest after initial analysis. After retest, 21 samples (1.62%) gave TREC levels below cut-off. A significant reduction in TREC levels was observed in heel prick cards stored for three months (n=33) and one year (n=33). Preterm newborns (n=155) showed significantly lower TREC levels and a higher retest-rate than full-term newborns. Peripheral blood spots of 22 confirmed SCID patients and 17 primary immunodeficiency patients showed undetectable or low TREC-levels. These findings suggest that the EnLite Neonatal TREC assay is a suitable method for SCID-screening in the Netherlands, thereby providing guidance in the decisions concerning implementation into the Dutch program.

Comparison of C26:0-carnitine and C26:0-lysophosphatidylcholine as diagnostic markers in dried blood spots from newborns and patients with adrenoleukodystrophy

X-linked adrenoleukodystrophy (ALD) is the most common leukodystrophy with a birth incidence of 1:14,700 live births. The disease is caused by mutations in ABCD1 and characterized by very long-chain fatty acids (VLCFA) accumulation. In childhood, male patients are at high-risk to develop adrenal insufficiency and/or cerebral demyelination. Timely diagnosis is essential. Untreated adrenal insufficiency can be life-threatening and hematopoietic stem cell transplantation is curative for cerebral ALD provided the procedure is performed in an early stage of the disease. For this reason, ALD is being added to an increasing number of newborn screening programs. ALD newborn screening involves the quantification of C26:0-lysoPC in dried blood spots which requires a dedicated method. C26:0-carnitine, that was recently identified as a potential new biomarker for ALD, has the advantage that it can be added as one more analyte to the routine analysis of amino acids and acylcarnitines already in use. The first objective of this study was a comparison of the sensitivity of C26:0-carnitine and C26:0-lysoPC in dried blood spots from control and ALD newborns both in a case-control study and in newborns included in the New York State screening program. While C26:0-lysoPC was elevated in all ALD newborns, C26:0-carnitine was elevated only in 83%. Therefore, C26:0-carnitine is not a suitable biomarker to use in ALD newborn screen. In women with ALD, plasma VLCFA analysis results in a false negative result in approximately 15-20% of cases. The second objective of this study was to compare plasma VLCFA analysis with C26:0-carnitine and C26:0-lysoPC in dried blood spots of women with ALD. Our results show that C26:0-lysoPC was elevated in dried blood spots from all women with ALD, including from those with normal plasma C26:0 levels. This shows that C26:0-lysoPC is a better and more accurate biomarker for ALD than plasma VLCFA levels. We recommend that C26:0-lysoPC be added to the routine biochemical array of diagnostic tests for peroxisomal disorders.

Recommendations for newborn screening for galactokinase deficiency: A systematic review and evaluation of Dutch newborn screening data.

INTRODUCTION Galactokinase (GALK) deficiency causes cataract leading to severe developmental consequences unless treated early. Because of the easy prevention and rapid reversibility of cataract with treatment, the Dutch Health Council advised to include GALK deficiency in the Dutch newborn screening program. The aim of this study is to establish the optimal screening method and cut-off value (COV) for GALK deficiency screening by performing a systematic review of the literature of screening strategies and total galactose (TGAL) values and by evaluating TGAL values in the first week of life in a cohort of screened newborns in the Netherlands. METHODS Systematic literature search strategies in OVID MEDLINE and OVID EMBASE were developed and study selection, data collection and analyses were performed by two independent investigators. A range of TGAL values measured by the Quantase Neonatal Total Galactose screening assay in a cohort of Dutch newborns in 2007 was evaluated. RESULTS Eight publications were included in the systematic review. All four studies describing screening strategies used TGAL as the primary screening marker combined with galactose-1-phosphate uridyltransferase (GALT) measurement that is used for classical galactosemia screening. TGAL COVs of 2200 μmol/L, 1665 μmol/L and 1110 μmol/L blood resulted in positive predictive values (PPV) of 100%, 82% and 10% respectively. TGAL values measured in the newborn period were reported for 39 GALK deficiency patients with individual values ranging from 3963 to 8159 μmol/L blood and 2 group values with mean 8892 μmol/L blood (SD ± 5243) and 4856 μmol/L blood (SD ± 461). Dutch newborn screening data of 72,786 newborns from 2007 provided a median TGAL value of 110 μmol/L blood with a range of 30-2431 μmol/L blood. CONCLUSION Based on TGAL values measured in GALK deficiency patients reported in the literature and TGAL measurements in the Dutch cohort by newborn screening we suggest to perform the GALK screening with TGAL as a primary marker with a COV of 2500 μmol/L blood, combined with GALT enzyme activity measurement as used in the classical galactosemia screening, to ensure detection of GALK deficiency patients and minimize false positive referrals.

Newborn blood spot screening for cystic fibrosis with a four-step screening strategy in the Netherlands.

BACKGROUND Newborn screening for cystic fibrosis (NBSCF) was introduced in the Dutch NBS program in 2011 with a novel strategy. METHODS Dutch NBSCF consisted of four steps: immuno-reactive trypsin (IRT), Pancreatitis-associated Protein (PAP), DNA analysis by Inno-LiPa (35 mutations), extended gene analysis (EGA) as fourth step and as safety net. Only samples with two CFTR-variants were considered screen-positive, but samples with one disease-causing variant were considered also screen-positive from April 2013. The first 5 years of NBSCF were evaluated during a follow-up ranging from 2 to 6.8 years for sensitivity, specificity, positive predictive value (PPV), ratio of CF/Cystic Fibrosis Screen Positive infants with an Inconclusive Diagnosis (CFSPID) and median age at diagnosis, and were compared to other novel strategies for NBSCF and European Cystic Fibrosis Society (ECFS) Best Practice Standards of Care. RESULTS NBSCF achieved a sensitivity of 90% (95% CI 82%-94%), specificity of 99.991% (95% CI 99.989%-99.993%), PPV of 63% (95% CI 55%-69%), CF/CFSPID ratio of 4/1, and median age at diagnosis of 22 days, if samples with two variants as well as samples with one disease-causing variant were considered screen-positive. CONCLUSION The program achieved the goal to minimize the number of false positives and showed a favourable performance but sensitivity and CF/CFSPID ratio did not meet criteria of EFCS Best Standards of Care. Changed cut-off values for PAP and IRT and classification of R117H-7T/9T to non-pathogenic may improve sensitivity to ≥95% and CF/CFSPID ratio to 10/1. PPV is estimated to be around 60%.

Raising Awareness of False Positive Newborn Screening Results Arising from Pivalate-Containing Creams and Antibiotics in Europe When Screening for Isovaleric Acidaemia

While the early and asymptomatic recognition of treatable conditions offered by newborn screening confers clear health benefits for the affected child, the clinical referral of patients with screen positive results can cause significant harm for some families. The use of pivalate-containing antibiotics and more recently the inclusion of neopentanoate as a component within moisturising creams used as nipple balms by nursing mothers can result in a significant number of false positive results when screening for isovaleric acidaemia (IVA) by measuring C5 acylcarnitine. A recent survey conducted within centres from nine countries indicated that this form of contamination had been or was a significant confounding factor in the detection of IVA in seven of the nine who responded. In three of these seven the prominent cause was believed to derive from the use of moisturising creams and in another three from antibiotics containing pivalate; one country reported that the cause was mixed. As a result, four of these seven centres routinely perform second tier testing to resolve C5 isobars when an initial C5 result is elevated, and a fifth is considering making this change within their national programme. The use of creams containing neopentanoate by nursing mothers and evolving patterns in the prescription of pivalate-containing antibiotics during pregnancy require those involved in the design and operation of newborn screening programmes used to detect IVA and the doctors who receive clinical referrals from these programmes to maintain an awareness of the potential impact of this form of interference on patient results.

Response to Goodness et al.

Goodness et al. from the Michigan Department of Community Health (MDCH) report their experience of a change of the resin lot of the BioRad Variantnbs reagents causing a dramatic drop in FAST-peak values of the high-performance liquid chromatography (HPLC) analysis. Further to their findings, we present the analytical consequences of a gradient change in the Dutch screening programme. In 2011 BioRad introduced a new gradient (method 1.013 ‘new’, replacing method 1.08 ‘old’) on the Vnbs System to produce a sharper and higher F1 peak (containing acetylated HbF). In our hands, using the Vnbs system, the percentage HbF (‘total F’) is calculated by adding the F1 peak percentage with the F peak percentage. To determine the effect of the new gradient, we re-analysed the samples from our previous paper with both method 1.08 (old) and 1.013 (new). We also analysed 186 random fresh samples of the routine screening programme, using both methods. Method 1.013 (new) produced clearly higher F1 peak percentages and slightly lower F peak percentages. There was a drastic decline in the FAST peak percentage, which contains the HbBart’s peak used in screening for HbH disease. The HbA peak percentages were unaffected. Re-analysis of the samples from our previous study with method 1.013 (new) showed a good correlation (r 1⁄4 0.98) between methods, with FAST peak percentages that were, on average, 37% lower than those obtained with method 1.08. With fresh samples from the routine screening programme, average FAST peak percentages achieved with method 1.013 (new) were 44% lower compared with the analysis with method 1.08 (old). A change of aspects of the HPLC procedure by a manufacturer, whether a change in resin, gradient or some other aspect, will lead to changes in the Hb peak percentages. Inevitably, a re-evaluation of the cut-off values for the various haemoglobinopathies is needed, involving a validation consisting of a comparison of results obtained with the old and new methods. Because of the scarcity of cases, establishing cut-off values will ultimately be a combination of this analytical validation and the original cut-off values, rendering the establishment of new cut-off values, in part, arbitrary. To illustrate this, the Dutch newborn population has a low prevalence of HbH disease, so for alpha-thalassaemia screening, method 1.013 (new) was introduced concomitantly with a lower cut-off value for the FAST peak (from 22.5% to 16%). This cut-off value would be expected to result in a similar number of referrals (N 1⁄4 11, with no known missed patients) to the old cut-off value of 22.5% (N 1⁄4 13). The new cut-off value was introduced on 1 October 2011. Initial results indicate that the number of referrals decreased slightly, from 41 to 24 annually, with no known falsenegative results. In the Netherlands we will continue to monitor the referrals and false-negatives, and plan to publish our results.