The combined use of enzyme activity and metabolite assays as a strategy for newborn screening of mucopolysaccharidosis type I

Introduction

Mucopolysaccharidoses (MPSs) are a group of rare diseases (lysosomal storage disorders; LSDs) caused by a reduction or lack of the lysosomal enzymes that catalyze the stepwise degradation of glycosaminoglycans (GAGs) [1].

Mucopolysaccharidosis type I (MPS I) involves a deficiency of the lysosomal enzyme α-L-iduronidase (IDUA) resulting in progressive lysosomal accumulation of dermatan and heparan sulfates with clinical manifestations characteristic of the MPS phenotype [2]. Major clinical findings include coarse facial features, skeletal and joint sequelae, a marked disproportionate short stature with short trunk and normal limbs, hepatosplenomegaly and visual impairment secondary to corneal clouding, astigmatism and/or retinopathy. Although MPS I has traditionally been divided into three broad groups based on symptom severity: Hurler (H), Hurler-Scheie (H/S) and Scheie (S) syndromes, it is increasingly considered to have a continuous disease spectrum. Hurler and Scheie syndromes represent phenotypes at the severe and mild extremes of the clinical spectrum, respectively, whereas the Hurler-Scheie syndrome has an intermediate phenotype. Therapeutic approaches for MPS I include enzyme replacement therapy (ERT) with L-iduronidase, allogeneic hematopoietic stem cell transplantation (HSCT) and recently, ex vivo gene therapy in autologous hematopoietic stem cells [3], [4]. The progressive nature of MPS I means that treatment success depends on early initiation. Recently, it has been demonstrated that newborn screening (NBS) for MPS I allows timely initiation of therapy, potentially changing its natural history [5].

Several analytical strategies are available for efficient full population screenings [6], [7], [8], [9], [10].

In Taiwan, a pilot NBS program for MPS I conducted from October 2008 to April 2013 screened 35,285 newborns and identified two patients with confirmed MPS I, estimated incidence 1:17,643 [11]. NBS programs that include MPS I have been described in the USA. In 2013, Missouri was the first state to introduce MPS I and other LSDs in a pilot screening program using a multiplexed fluorometric enzymatic assay (digital microfluidic platform) [12]. In the first 6 months, 43,701 newborns were screened; 32 were investigated for MPS I (recall rate 0.037%). Of these, three were confirmed to have an MPS I genotype (estimated incidence 1:14,567). Of the remaining 29 patients, seven had pseudodeficiency alleles, two were carriers, 16 were false-positives and four had results pending. In Washington State, the NBS Laboratory conducted two pilot studies on anonymous DBS using multiplex-tandem mass spectrometry (MS/MS) enzymatic activity assays that included IDUA [7], [10]. The analytical ranges for lysosomal enzymes were 5- to 15-fold higher with MS/MS compared to the fluorimetric assays using 4-methylumbelliferyl substrates. The rate of screen positive detection was consistently lower for the MS/MS assay compared to the fluorimetric assay using a digital microfluidics platform. In 2017, a screening project conducted in Illinois on Pompe, MPS I, Gaucher, Fabry, and Niemann-Pick diseases identified one confirmed MPS I case estimated incidence 1:219,793 [13]. α-L-iduronidase pseudodeficiencies were detected more often than true deficiencies. A recent North Carolina study employing a 2-tier method for MPS I screening in 62,734 newborns confirmed the need for second-tier testing to reduce the burden of false positives [14]. In early 2016, following systematic evidence-based studies, MPS I neonatal screening was approved by the US Secretary of the Department of Health and Human Services, and added to their Recommended Universal Screening Panel [15].

In Northeastern Italy, an NBS program for detecting LSDs including MPS I using MS/MS began in 2015. It revealed a high frequency of IDUA pseudodeficiency alleles (benign variants) with reduced in vitro IDUA enzyme activity that resulted in a high recall rate for retesting by leucocyte enzyme activity and 24-h urinary GAGs [16], [17]. We found a high incidence of pseudodeficiency alleles (e.g. encoding p.Ala79Thr, p.His82Gln, p.Asp223Asn) in newborns of African origin, confirming previous reports [12], [13], [18].

Here, we report our experience with MPS I screening in over 125,000 newborns, initially conducted with IDUA enzyme activity alone, and subsequently with the introduction of a second-tier test to measure GAGs in DBS to reduce the false positive rate. We also report the clinical outcome of two affected patients diagnosed early through this NBS program.

Materials and methods

Results

Second-tier test by DBS GAGs analysis

To improve the performance of NBS for MPS I and reduce the large number of recalls, we recently developed a second-tier test based on quantification of GAG levels in DBSs by methanolysis followed by LC-MS/MS analysis.

Optimization of the methanolysis reaction

The reaction conditions for GAGs methanolysis were optimized at 45 min at 65 °C to maximize the concentrations of DS and HS. Mass spectra from methanolysates of the GAG standards shown predominantly the protonated form for all the standards; sodiated molecular ions were not detected. Figure 1 shows the chromatographic separation of disaccharides from DS, HS, CS, and the respective internal standards performed on a BEH-Amide column over a run time of 9 min, including the re-equilibration step.

Figure 1:

Chromatographic separation of GAG disaccharides and internal standards in a high QC DBS on the BEH Amide column over a 9-min run.

MRM acquisition window 2.5–8 min. DS, dermatan sulfate; CS, chondroitin sulfate; HS, heparan sulfate; DS ²H₆, DS internal standard; CS ²H₆, CS internal standard IS; HS ²H₆, HS internal standard; MRM, multiple reaction monitoring.

Method validation

Results of method validation studies conducted on DBS are summarized in Table 2. Bias and precision were evaluated by analyzing QC samples at low and high concentrations. Evaluation of within-day and between-day precision demonstrated CVs <15% at two concentration levels. LODs for DS, HS, and CS disaccharides were 0.1, 0.2 and 0.2 mg/L, respectively, whereas LLOQs were 0.4, 0.5 and 0.7 mg/L. Good linearity was obtained in the range of investigated concentrations for the three standards (HS, r²=0.998; DS, r²=0.998; CS, r²=0.992) (Supplementary Figure S4). DBS specimens were useful for HS and DS determination for at least 6 months of storage under ambient conditions, refrigerated, frozen (−20 °C) or frozen (−80 °C). Specimens were also subjected to up to six freeze/thaw cycles.

Table 2:

Analytical performance of the LC-MS/MS method: intra- and inter-assay precision (percent coefficient of variation, CV %) at three concentration levels and recovery %.

	QC endogenous		Medium QC (endogenous+10 mg/L)			High QC3 (endogenous+40 mg/L)
	Mean±SD, mg/L	Precision CV, %	Mean±SD, mg/L	Precision CV, %	Recovery, %	Mean±SD, mg/L	Precision CV, %	Recovery, %
Intra-assay, (n=11)
DS	1.60±0.13	8.1	10.9±1.2	11	93	42.9±2.9	9.7	103.2
HS	0.9±0.2	22.2	10.1±0.95	9.41	92	39.6±3.2	9.8	96.7
Inter-assay, (n=7)
DS	1.80±0.29	16.3	11.2±1.32	11.9	93.9	42.9±4.9	11.4	102.7
HS	0.72±0.17	23.8	10.4±1.30	12.4	96.6	35.6±3.5	9.8	87.3

1. CV%, percentage coefficient of variation; LC-MS/MS, liquid chromatography tandem mass spectrometry; SD, standard deviation.

Reference ranges were determined in 40 healthy newborns (HS mean 1 mg/L, range 0–3.2; DS mean 1.5 mg/L, range 0.5–2.7).

We measured GAGs in DBS from 22 previously identified newborns with low IDUA enzyme activity and known genomic status. Two of the patients had two pathological alleles (true positives), 17 had pseudodeficiency, one was a carrier and two carried variants of unknown significance). Only the two neonates with two pathogenic alleles had high levels of HS (4.9–10.4 mg/L) and DS (7.4–8.8 mg/L); all other samples had GAGs within the normal range (mean HS 1.7 mg/L, range 1.1–3.1; mean DS 1.9 mg/L, range 1.5–2.9) (Figure 2).

Figure 2:

HS and DS concentrations in DBS of controls (black dots), false positive MPS I newborns (empty square) and newborns with confirmed MPS I (black square).

Potential interference

Potential interference due to the presence of heparin anticoagulant in venous blood draws was evaluated by preparing DBS specimens from venous blood collected with lithium heparin (Hep DBS) versus blood collected via a finger prick from the same donor (n=4). HS was 100-fold higher in the DBS spotted from heparin blood, compare to the finger prick DBS (Supplementary Table S3). Heparin interferes with the measurement of HS because methanolysis produces a common IdoA-GlcN disaccharide that is monitored by LC-MS/MS.

Discussion

The recent introduction of MS/MS methods to analyze metabolites in DBS has facilitated and expanded the number of inherited diseases detectable in newborn screening programs. Pilot screening programs for lysosomal disorders have been implemented in several countries worldwide [9].

In 2014, expanded neonatal screening for amino acid, organic acid and β-oxidation disorders was established in our laboratory in Northeastern Italy, and recently a lysosomal diseases panel has been proposed [16], [17]. Our experience shows that NBS for LSDs can be beneficial where there is an established treatment that can delay or halt progression of the disease and/or improve long-term outcomes, particularly when treatment is initiated before damage becomes irreversibly. We also underscore the need to reduce the number of false positives, which lead to unnecessary anxiety and expensive follow-up. In order to achieve this, new strategies that include second-tier tests have been employed [22], [23]. Second-tier biochemical tests that measure lysosphingolipids such as LysoGb1 in Gaucher disease, LysoGb3 in Fabry disease and psychosine in Krabbe disease clearly reduce the number of false positives in LSD newborn screening [17], [24], [25].

Our MPS I screening has identified a high number of false positives, mainly due to the presence of pseudodeficiency alleles [13], [16], [18]. The frequency of pseudodeficiency varies by population: a high incidence of was found in African and African-American populations [5], [17], [18], conversely, no pseudodeficiency was detected in a recent study in Taiwan [26].

Strategies to reduce recall rates in MPS I screening have included the use of second-tier biochemical metabolite assays, genetic testing and post-analysis of NBS data with statistical tools. The multivariate pattern recognition software Collaborative Laboratory Integrated Reports (CLIR) is freely available and widely used for postanalytical interpretation. CLIR can improve the quality of newborn screening conducted with tandem mass spectrometry [27], and can be used as a decision tool to identify candidate patients for second-tier testing. IDUA gene sequencing has been proposed as a second-tier option. The North Carolina NBS program includes CLIR analysis and DNA sequencing in their screening algorithm [14], [28]. They successfully demonstrate the utility of a two-tier approach to reduce the number of newborns referred for follow-up by excluding specimen that have low IDUA activity but carry only pseudodeficiency variants.

Biochemical quantification of the primary GAG storage products HS and DS in DBS has been proposed. Until recently, there was limited knowledge of the quantity of disaccharides in DBS. In 2010, Tomatsu et al. reported a method to quantify HS and DS in DBS by LC-MS/MS after enzymatic digestion [29]. This methodology allows sensitive quantification of GAGs, but involves long incubation periods and expensive reagents. In 2012, De Ruijter et al. used this method to show that GAGs were elevated in newborn DBS of patients with MPS I, MPS II and MPS III [30], suggesting that GAG determination could be a potential first-tier test for MPS newborn screening. In 2017, a similar study by Kubaski et al. confirmed the validity of measuring GAGs for identifying neonates with MPSs [31]. However, application as a first-tier test NBS is limited by the long run time and high reagent costs. Rinaldo et al. used GAG quantification as a second-tier test on specimens that had been identified as at risk for MPS I by post-analysis biostatistical analysis (CLIR) among 55,161 neonates screened in Kentucky [22]. This second-tier assay uses enzymatic digestion followed by MS. Implementing it in the workflow reduced the number of recalls to only two newborns; MPS I was confirmed in one.

Two-tier screening with sequential determination of enzyme activity and multiple GAGs has been proposed as the gold standard for the diagnosis of patients affected by mucopolysaccharidosis [32], [33]. More recently, Peck et al. used GAG quantification in DBS as a second-tier biomarker test for 1213 newborns identified as at risk of MPS I based on first-tier NBS [34]. GAGs analysis accurately discriminated between patients with confirmed MPS I and false-positive cases due to pseudodeficiency or heterozygosity, and increased the specificity of newborn screening for MPS I.

Our NBS program for MPS I revealed a high incidence of IDUA pseudodeficiency in the screened population. In February 2019, we introduced a second-tier test to measure HS and DS in DBSs by LC-MS/MS after chemical hydrolysis (methanolysis). This technique was originally developed elsewhere for use on CSF and urine [20], [35], [36], [37]; we have optimized it for DBS. The simple and rapid method entails direct incubation of DBS samples in methanolic-HCl, without an extraction step. We validated the assay for clinical application by establishing the overall precision, accuracy, linearity and potential limits. Intra- and between-day reproducibility of HS and DS quantification is high. We propose this method as an economical and rapid alternative to the enzymatic digestion assay currently used.

After introducing this second-tier test into our MPS I screening algorithm, the recall rate decreased from 0.046% to 0.006%. Retrospective testing of GAGs in DBS from patients known to carry single pathogenic alleles or to have pseudodeficiency alleles was able to exclude any severe form of MPS I with a negative predictive value of 100%. This is consistent with reports that sequential IDUA enzyme activity and GAGs analysis reduce false positives.

This method may be applicable to second-tier testing for MPS II, MPS III and MPS VI in NBS; however, its use for MPS IV might be limited by poor quantification of KS using methanolysis [36].

This method has several potential limitations. First, the use of heparin as an anticoagulant in sample collection interferes with HS quantification because common species are generated during methanolysis [19], a phenomenon not observed with enzymatic digestion. Second, the reference ranges identified in this study may be limited to our laboratory as the purity of standard used for the calibration curves is not reported for all analytes. Moreover, biochemical analysis may differ in terms of the targeted metabolites (GAGs vs. non-reducing end oligosaccharides) and analytical methods used (GAG analysis after methanolysis vs. enzymatic digestion; LC-MS/MS vs. fluorimetric analysis). Zhang et al. compared urinary GAGs concentrations obtained with methanolysis with those with enzymatically derived dimers. They concluded that reported reference values should only be used as a guide, because inter-laboratory and inter-method differences in accuracy may affect results [19]. It is recommended that laboratory-specific age-matched control ranges be established from a normal population. Third, newborns with attenuated MPS I may be misdiagnosed using this screening algorithm; more data must be collected on GAGs in attenuated MPS I.

In conclusion, although experience with MPS I screening is still limited, early identification and intervention has improved the outcome of our patients with MPS I. GAGs analysis in DBSs discriminates patients with confirmed MPS I and false positives, thereby increasing the specificity of MPS I newborn screening.