Experience with the Urinary Tetrasaccharide Metabolite for Pompe Disease in the Diagnostic Laboratory

H OH metabolites OH

Article Experience with the Urinary Tetrasaccharide Metabolite for Pompe Disease in the Diagnostic Laboratory

Jennifer T. Saville 1 and Maria Fuller 1,2,*

1 Genetics and Molecular Pathology, SA Pathology at Women’s and Children’s Hospital, 72 King William Road, North Adelaide, SA 5006, Australia; [email protected] 2 Adelaide Medical School, University of Adelaide, North Terrace, Adelaide, SA 5000, Australia * Correspondence: [email protected]; Tel.: +61-(0)8-8161-6741

Abstract: Following clinical indications, the laboratory diagnosis of the inherited metabolic myopathy, Pompe disease (PD), typically begins with demonstrating a reduction in acid alpha-glucosidase (GAA), the enzyme required for lysosomal glycogen degradation. Although simple in concept, a major challenge is defining reference intervals, as even carriers can have reduced GAA, and pseudodeficiencies complicate interpretation. Here, we developed a mass spectrometric assay for quantification of a urinary glycogen metabolite (tetrasaccharide) and reported on its utility as a confirmatory test for PD in a diagnostic laboratory. Using two age-related reference intervals, eight returned tetrasaccharide concentrations above the calculated reference interval but did not have PD, highlighting non-specificity. However, retrospective analysis revealed elevated tetrasaccharide in seven infantile-onset (IOPD) cases and sixteen late-onset (LOPD) cases, and normal concentrations in one heterozygote. Prospective tetrasaccharide analysis in nine individuals with reduced GAA confirmed IOPD in one, LOPD in six and identified two heterozygotes. Using this metabolite as a biomarker of therapeutic response was not overly informative; although most patients showed an initial drop following therapy initiation, tetrasaccharide concentrations fluctuated considerably and

Citation: Saville, J.T.; Fuller, M. remained above reference intervals in all patients. While useful as a conﬁrmation of PD, its utility as Experience with the Urinary a biomarker for monitoring treatment warrants further investigation. Tetrasaccharide Metabolite for Pompe Disease in the Diagnostic Laboratory. Keywords: Pompe disease; diagnosis; glycogen; lysosomal storage disorder; urinary tetrasaccharide; Metabolites 2021, 11, 446. https:// biomarker; biochemical monitoring; glycogen storage disease; mass spectrometry doi.org/10.3390/metabo11070446

Academic Editor: Martin Krššák 1. Introduction Received: 5 May 2021 Pompe disease (PD) is an inherited, progressive, metabolic myopathy resulting from Accepted: 5 July 2021 Published: 8 July 2021 the lysosomal accumulation of glycogen due to a deﬁciency in the lysosomal hydrolase, acid alpha-glucosidase (GAA; EC 3.2.1.20). The disease is broadly divided into two forms.

Publisher’s Note: MDPI stays neutral Infantile-onset PD (IOPD) typically presents within the first six months of life with car- with regard to jurisdictional claims in diomyopathy, respiratory dysfunction and hypotonia and, if left untreated, premature published maps and institutional affil- death results from cardiorespiratory failure [1,2]. Late-onset PD (LOPD) manifests later in iations. life, with limb and respiratory muscle weakness and a slower progressing disease course [3]. The clinical manifestations of LOPD are highly varied, as is age of symptom onset, and they are often confused with more common muscular dystrophies. Early diagnosis of IOPD is critical for life-saving treatment in the form of enzyme replacement therapy (ERT) that reduces ventilation dependence and mortality risks by up Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. to 99% [4]. Likewise, in LOPD, the mortality rate reportedly decreased five-fold in treated This article is an open access article patients compared to those who were untreated [5]. Confirming a suspected diagnosis of distributed under the terms and PD makes use of the biochemical and genetic characteristics of the disease. Initial testing, conditions of the Creative Commons just like that used in PD newborn screening programs [6], measures GAA activity in dried Attribution (CC BY) license (https:// blood spots (DBS) using artificial substrates and detection, either with fluorescence [7] creativecommons.org/licenses/by/ or mass spectrometry [8,9]. Reduced GAA activity in DBS is reported in both IOPD and 4.0/). LOPD patients [10], but second tier testing is required to confirm a diagnosis due to

Metabolites 2021, 11, 446. https://doi.org/10.3390/metabo11070446 https://www.mdpi.com/journal/metabolites Metabolites 2021, 11, 446 2 of 11

pseudodeficiency alleles which result in a reduction in GAA activity in vitro but no active disease [11]. Molecular screening for GAA genetic variants can be used as confirmation of PD but is complicated by a high number of genetic variants of uncertain significance (VUS) [12,13] that still require phenotype confirmation. Another biochemical feature of PD is the accumulation of lysosomal glycogen; however, this is restricted to biopsied muscle tissue and measurement is not straightforward [14]. Amylolytic degradation of glycogen, followed by α-1,4 glucosidase activity, produces a tetrasaccharide, 6-α-D-glucopyranosyl-maltotriose, a metabolite of glycogen that is excreted in the urine [15]. Present in all urine, this tetrasaccharide has been shown to be elevated in patients with glycogen storage disorders, including Pompe disease [16–19]. Several studies have shown the urine tetrasaccharide is elevated in patients with IOPD and LOPD [20,21] but, importantly, not in individuals carrying the pseudodeficiency allele [18,22]. The urine tetrasaccharide has also shown promise as a biomarker for monitoring patients post initiation of ERT [23–25]. In particular, Young et al. [24] found urine tetrasaccharide concentrations correlated with muscle glycogen content at 52 weeks post- ERT, and that patients with higher pre-treatment tetrasaccharide concentrations had poorer motor function outcomes. Here, we describe the development and validation of a mass spectrometric method for the measurement of the tetrasaccharide in our diagnostic service laboratory, and thereafter report on the outcome of this urinary metabolite for the diagnosis of PD, as well as for biochemical monitoring of patients in receipt of ERT.

2. Results 2.1. Urine Tetrasaccharide Method Validation The internal standard (IS) and tetrasaccharide eluted at the same retention time (2.9 min) on the pentaﬂuorophenyl (PFP) liquid chromatography (LC) column with no interfering peaks (Figure1a), with maltotetraose also eluting at 2.9 min. The calibration curve was linear across the concentration range of 2–300 mmol/mol creatinine (Figure1b). The limit of detection was calculated as three times the signal-to-noise at 0.02 mmol/mol creatinine, whereas the limit of quantiﬁcation was 0.08 mmol/mol creatinine, at which the target concentration was achieved within a 10% margin of error. The interassay CV was 9%, determined using a PD patient sample for 65 separate assays over a twelve-month period. Recovery was 82% (n = 8), determined by measurement of maltotetraose (120 mmol/mol creatinine) from control urine. The tetrasaccharide was stable in urine after three cycles of freeze/thaw, stored at room temperature for up to one week and at −20 ◦C for up to 17 months (data not shown). Prepared derivatized urine samples were stable for up to 36 h in the autosampler at 18 ◦C. Of note, preparation of quality control (QC) material by the addition of maltotetraose to control urine was not stable, with an observed reduction in the calculated concentration following storage at −20 ◦C and −70 ◦C (data not shown). To overcome this, we employed PD patient urine as low (8 mmol/mol creatinine) and high (40 mmol/mol creatinine) QC material. Urine tetrasaccharide concentrations from 190 patients without PD were used to calculate reference intervals using a 95% percentile upper limit. Figure2 shows that tetrasaccharide concentrations were higher in individuals < 2 years of age and, as such, two age-related reference ranges were determined (1) <10 mmol/mol creatinine for patients < 2 years of age (n = 67) and (2) <3 mmol/mol creatinine for patients > 2 years old (n = 123). There were eight patients (4 patients < 2 and 4 > 2 years of age) who returned urine tetrasaccharide concentrations above the calculated reference intervals but did not have PD (Figure2). Metabolites 2021, 11, x 2 of 12

pseudodeficiency alleles which result in a reduction in GAA activity in vitro but no active disease [11]. Molecular screening for GAA genetic variants can be used as confirmation of PD but is complicated by a high number of genetic variants of uncertain significance (VUS) [12,13] that still require phenotype confirmation. Another biochemical feature of PD is the accumulation of lysosomal glycogen; however, this is restricted to biopsied muscle tissue and measurement is not straightforward [14]. Amylolytic degradation of glycogen, followed by α-1,4 glucosidase activity, produces a tetrasaccharide, 6-α-D-glucopyranosyl-maltotriose, a metabolite of glycogen that is excreted in the urine [15]. Present in all urine, this tetrasaccharide has been shown to be elevated in patients with glycogen storage disorders, including Pompe disease [16–19]. Several studies have shown the urine tetrasaccharide is elevated in patients with IOPD and LOPD [20,21] but, importantly, not in individuals carrying the pseudodeficiency allele [18,22]. The urine tetrasaccharide has also shown promise as a biomarker for monitoring patients post initiation of ERT [23–25]. In particular, Young et al. [24] found urine tetrasaccharide concentrations correlated with muscle glycogen content at 52 weeks post- ERT, and that patients with higher pre-treatment tetrasaccharide concentrations had poorer motor function outcomes. Here, we describe the development and validation of a mass spectrometric method for the measurement of the tetrasaccharide in our diagnostic service laboratory, and thereafter report on the outcome of this urinary metabolite for the diagnosis of PD, as well as for biochemical monitoring of patients in receipt of ERT.

2. Results 2.1. Urine Tetrasaccharide Method Validation The internal standard (IS) and tetrasaccharide eluted at the same retention time (2.9 min) on the pentafluorophenyl (PFP) liquid chromatography (LC) column with no interfering peaks (Figure 1a), with maltotetraose also eluting at 2.9 min. The calibration curve was linear across the concentration range of 2–300 mmol/mol creatinine (Figure 1b). The limit of detection was calculated as three times the signal-to-noise at 0.02 mmol/mol creatinine, whereas the limit of quantification was 0.08 mmol/mol creatinine, at which the target concentration was achieved within a 10% margin of error. The interassay CV was 9%, determined using a PD patient sample for 65 separate assays over a twelve-month period. Recovery was 82% (n = 8), determined by measurement of maltotetraose (120 mmol/mol creatinine) from control urine. The tetrasaccharide was stable in urine after three cycles of freeze/thaw, stored at room temperature for up to one week and at −20 °C for up to 17 months (data not shown). Prepared derivatized urine samples were stable for up to 36 h in the autosampler at 18 °C. Of note, preparation of quality control (QC) material by the addition of maltotetraose to control urine was not stable, with an observed Metabolites 2021, 11, 446 reduction in the calculated concentration following storage at −20 °C and −70 °C (data3 of not 11 shown). To overcome this, we employed PD patient urine as low (8 mmol/mol creatinine) and high (40 mmol/mol creatinine) QC material.

20 R2 = 0.9994

Metabolites 2021, 11, x 15 3 of 12

10 Figure 1. Liquid chromatography tandem mass spectrometry of urine tetrasaccharide: (a) extracted ion chromatogram of the tetrasaccharide and internal standard (IS); (b) calibration curve5 of maltotetraose concentration plotted against the ratio of the peak area of the analyte to that of the IS. 0 Urine tetrasaccharide concentrations0 from 100 190 patients 200 without 300 PD 400 were used to cal- Maltotetraose (mmol/mol creat.)

culate reference intervals using a 95% percentile upper limit. Figure 2 shows that tetrasaccharide(a) concentrations were higher in individuals < (2b )years of age and, as such, two age- related reference ranges were determined (1) <10 mmol/mol creatinine for patients < 2 years Figure 1. Liquid chromatographyof age tandem(n = 67) massand (2) spectrometry <3 mmol/mol of urine creatinine tetrasaccharide: for patients (a) extracted> 2 years ionold chromatogram(n = 123). There of were the tetrasaccharide and internaleight standard patients (IS); (4 patients (b) calibration < 2 and curve 4 > of2 years maltotetraose of age) who concentration returned plotted urine tetrasaccharide against the ratio con-

of the peak area of the analytecentrations to that of theabove IS. the calculated reference intervals but did not have PD (Figure 2).

Figure 2. Age-related reference intervals. Urine tetrasaccharide concentrations for 190 individualsindividuals without Pompe disease. Inset shows expanded results for individuals < 5 years old. White squares indicate individuals with concentrations above the reference interval. RR, reference range. indicate individuals with concentrations above the reference interval. RR, reference range.

2.2. Diagnosis Diagnosis of PD Archived DBSDBS and and spot spot urine urine samples samples were were available available from from 23 genetically 23 genetically confirmed confirmed cases casesof PD. of Seven PD. patientsSeven withpatients IOPD with returned IOPD reduced returned GAA reduced activities GAA (0.0–0.9 activities pmol/spot/h), (0.0–0.9 pmol/spot/h),which were well which below were the well normal below reference the norm rangeal reference of 2.9–23.4 range pmol/spot/h of 2.9–23.4(Table pmol/spot/h1). To- (Tabletal α-glucosidase 1). Total α-glucosidase activity is included activity is in included the GAA in assay the GAA protocol assay as protocol a measure as a of measure sample ofintegrity, sample withintegrity, an elevated with an totalelevatedα-glucosidase/GAA total α-glucosidase/GAA ratio also ratio providing also providing an additional an ad- ditionalindicator indicator of PD, rangingof PD, ranging from 32 from to 482 32 forto 482 the for seven the IOPDseven patientsIOPD patients (reference (reference range range2.5–14.6 2.5–14.6).). Urine Urine tetrasaccharide tetrasaccharide concentrations concentrations were were also clearlyalso clearly elevated elevated above above the ref-the referenceerence range range for for all all IOPD IOPD patients patients (Table (Table1). 1). Sixteen Sixteen conﬁrmed confirmed LOPD LOPD patients patients were were demarcated fromfrom controlscontrols by by reduced reduced GAA GAA activity activity and and elevated elevated urine urine tetrasaccharide tetrasaccharide con- concentrationscentrations (Table (Table1 ). Another 1). Another patient, patient, heterozygous heterozygous for the for c.-32-13T>G the c.-32-13T>G variant variant had a had GAA a GAAactivity activity of 1.7 pmol/spot/hof 1.7 pmol/spot/h and a and neutral/GAA a neutral/GAA ratio ofratio 18, of but 18, the but urinary the urinary tetrasaccharide tetrasac- charideconcentration concentration was within was the within normal the range.normal range. Since the introduction of the urine tetrasaccharide mass spectrometry assay within diagnostic service, seven prospective diagnoses of PD patients have been reported in the two-year period (Table 1). This included one 3-month-old presenting with cardiomyopathy and diagnosed with IOPD, and a remaining six patients diagnosed with LOPD. Two patients with low GAA but normal urine tetrasaccharide were confirmed heterozygotes by molecular testing, and a further five were shown to have reduced GAA activity (0.5– 2.5 pmol/spot/h). The latter returned normal urine tetrasaccharide concentrations with no further follow-up because the diagnosis of PD was no longer considered.

Metabolites 2021, 11, 446 4 of 11

Table 1. GAA activity, urine tetrasaccharide concentration and genotype of patients previously conﬁrmed with Pompe disease, as well as those diagnosed since the initiation of the urine tetrasaccharide assay within the diagnostic laboratory.

Age Urine Tetrasaccharide Group GAA Activity (pmol/spot/h) a Ratio Total/GAA Activity b Genotype (years) (mmol/mol creatinine) c Conﬁrmed IOPD 0.6 0.0 482 50 c.953T>C/c.953T>C IOPD 0.7 0.9 32 57 c.266G>T/c.2815_2816del IOPD 0.7 0.7 89 77 Genotype not known † IOPD 0.25 0.9 59 46 Genotype not known † IOPD 0.8 0.3 142 46 c.323C>A homozygous IOPD 0.7 0.8 62 185 c.[2155G>C; 1726C>A; 2065G>A] homozygous IOPD 0.5 0.7 65 106 Genotype not known LOPD 47 0.9 57 10 c.-32-13T>G/c.1827del LOPD 69 0.7 39 9 c.-32-13T>G/c.953T>C LOPD 7 0.0 4417 8 c.-32-13T>G/c.1735G>A LOPD 41 0.8 31 8 c.-32-13T>G/c.307T>G LOPD 4 0.2 260 8 c.1827del/c.[-32-13T>G; 2275G>A] d LOPD 14 0.3 101 61 Genotype not known e LOPD 14 0.6 95 38 Genotype not known e LOPD 2 0.5 88 12 c.1827del/c.[-32-13T>G; 2275G>A] d LOPD 19 0.5 116 7 c.-32-13T>G/c.307T>G LOPD 58 0.9 51 9 c.-32-13T>G/c.1827del LOPD 12 0.4 69 15 Genotype not known LOPD 19 0.4 132 6 c.-32-13T>G/c.482_483del LOPD 35 0.5 61 13 c.-32-13T>G/c.482_483del LOPD 39 0.6 34 12 Genotype not known LOPD 19 0.8 37 6 Genotype not known LOPD 60 1.0 43 10 c.-32-13T>G/c.1441T>C Het 69 1.7 18 1 c.-32-13T>G Diagnosed IOPD 0.25 0.2 200 34 Genotype not known † LOPD 33 1.1 56 9 Genotype not known LOPD 48 0.2 149 5 Genotype not known LOPD 57 0.9 84 5 c.-32-13T>G/c.1075+1G>T LOPD 49 1.9 52 9 c.-32-13T>G/c.2481+109_2646+38del LOPD 63 0.2 246 6 Genotype not known LOPD 55 0.7 113 4 Genotype not known Het 1 2.7 14 1 c.-32-13T>G Het 27 2.4 13 1 c.2481+109_2646+38del a Reference range (RR) 2.9–23.4 pmol/spot/h; b RR 2.5–14.6; c RR < 2 years < 10, >2 years < 3 mmol/mol creatinine; d,e siblings; Het, heterozygote; † cardiomyopathy at presentation. All results measured at diagnosis. Metabolites 2021, 11, 446 5 of 11

Since the introduction of the urine tetrasaccharide mass spectrometry assay within diagnostic service, seven prospective diagnoses of PD patients have been reported in the two-year period (Table1). This included one 3-month-old presenting with cardiomyopathy and diagnosed with IOPD, and a remaining six patients diagnosed with LOPD. Metabolites 2021, 11, x Two patients with low GAA but normal urine tetrasaccharide were conﬁrmed heterozy-6 of 12 gotes by molecular testing, and a further ﬁve were shown to have reduced GAA activity (0.5–2.5 pmol/spot/h). The latter returned normal urine tetrasaccharide concentrations with no further follow-up because the diagnosis of PD was no longer considered. Figure 3 shows that there was an inverse relationship between age at diagnosis and Figure3 shows that there was an inverse relationship between age at diagnosis and urine tetrasaccharide concentration (Pearson’s r = −0.543; p < 0.01), but no correlation was urine tetrasaccharide concentration (Pearson’s r = −0.543; p < 0.01), but no correlation was observedobserved between between age age of of onset onset and and GA GAAA activity activity (Pearson’s (Pearson’s rr == 0.358; 0.358; pp == 0.05) 0.05) or or between between GAAGAA activity activity and and urine urine tetrasaccharide tetrasaccharide concentration concentration (Pearson’s (Pearson’s r r== −0.011;−0.011; p =p 0.59).= 0.59).

FigureFigure 3. 3. RelationshipRelationship between between patient patient age age at at diagnosi diagnosiss and and urine urine tetrasaccharide tetrasaccharide concentrations. concentrations. Pearson’sPearson’s r r= =−0.543;−0.543; p =p 0.002.= 0.002. 2.3. Longitudinal Monitoring of PD Patients 2.3. Longitudinal Monitoring of PD Patients For nine PD patients, urine samples were available pre- and post-ERT (Figure4). For nine PD patients, urine samples were available pre- and post-ERT (Figure 4). Three patients, diagnosed with IOPD between 7 and 11 months of age, continued to have Three patients, diagnosed with IOPD between 7 and 11 months of age, continued to have elevated urinary tetrasaccharide concentrations following ERT, despite an initial drop elevated urinary tetrasaccharide concentrations following ERT, despite an initial drop in in concentration in the ﬁrst urine collected post-treatment. For the remaining six LOPD concentration in the first urine collected post-treatment. For the remaining six LOPD pa- patients, urine tetrasaccharide concentrations were variable in response to treatment. Only tients, urine tetrasaccharide concentrations were variable in response to treatment. Only one patient was an adult at the time of diagnosis (58 years of age), and the urine tetrasac- one patient was an adult at the time of diagnosis (58 years of age), and the urine tetrasaccharide concentration was the second highest for LOPD patients at time of diagnosis charide concentration was the second highest for LOPD patients at time of diagnosis (9 (9 mmol/mol creatinine) but dropped to normal 2.5 years after treatment. A second pa- mmol/mol creatinine) but dropped to normal 2.5 years after treatment. A second patient, tient, diagnosed at 11 years, continued to have elevated tetrasaccharide concentrations up diagnosed at 11 years, continued to have elevated tetrasaccharide concentrations up to 3 to 3 years post-treatment; these reduced to within normal limits following 6 years of ERT. years post-treatment; these reduced to within normal limits following 6 years of ERT. The The youngest LOPD patient at diagnosis was 1.9 years of age, who was diagnosed due youngestto an older LOPD sibling. patient Both at werediagnosis heterozygous was 1.9 years for three of age, pathogenic who was variantsdiagnosed c.[-32-13T>G; due to an older2275G>A(p.Gly759Arg)]/c.1827delC(p.Tyr609*) sibling. Both were heterozygous for three and hadpathogenic the highest variants urine tetrasaccharidec.[-32-13T>G; 2275G>A(p.Gly759Arg)]/c.1827delC(p.Tyr609*)concentration at diagnosis (12 mmol/mol creatinine) and had that the remained highest urine elevated tetrasaccharide after 4 years concentrationof ERT. The olderat diagnosis sibling (3.8(12 mmol/mol years at diagnosis) creatinine) had that variable remained urine elevated tetrasaccharide after 4 years con- ofcentrations ERT. The older post-treatment, sibling (3.8 which years reducedat diagnosis) to normal had variable at 5.8 years urine of agetetrasaccharide before increasing con- centrationsto 6 mmol/mol post-treatment, by 7 years. which Similarly, reduced a patient to normal diagnosed at 5.8 years at 17.5 of age years before of age increasing returned tonormal 6 mmol/mol tetrasaccharide by 7 years. concentrations Similarly, a 2patient years post-ERTdiagnosed before at 17.5 increasing years of to age 4 mmol/mol returned normalcreatinine tetrasaccharide the following concentrations year. The ﬁnal 2 patient,years post-ERT diagnosed before at 7.6 increasing years of age, to 4 continued mmol/mol to creatininehave elevated the following urine tetrasaccharide year. The final up patien to 3 yearst, diagnosed post-ERT. at 7.6 years of age, continued to have elevated urine tetrasaccharide up to 3 years post-ERT.

Metabolites 2021, 11, 446 6 of 11

Metabolites 2021, 11, x 7 of 12 Metabolites 2021, 11, x Metabolites 2021, 11, x 7 of 12 7 of 12

6 0 2 0 2 0 2 0 6 0 6 0 (a ) (b ) (a ) (a ) (b ) (b )

) 1 5

)

e )

1 5 t

e )

) 1 5

)

e t

4 0 i

r d

4 0 i i

4 0 i

c c

c 1 0

c c 1 0 /

c 1 0

e t

t 2 0

(

e ( e 2 0

2 0 t

( (

( 5 ( 5 5

0 0 0 0 0 0 g 2 4 6 g 1 2 3 . . . n 2 4 6 g in 1 0 2 0 3 0 i g .2 g .4 . .6 g . 1 . 2 t 3 t n 0 in 0 0 0 n 0 0 in n n ti t ti t e e n n n n s s T im e (y e a rs ) e e e e e T im e (y e a rs ) e s s s s r T im e (y e a rs ) r e e T im e (y e a rs ) T im e (yee a rsp) p r T im e (yr e a rs ) r r p p p p

1 0 0 0 1 0 0 0 1 0 0 0 (c )

(c ) (c )

)

t r

t 1 0 0

a a

1 0 0 r

1 0 0 c

e h

e IO P D

IO P Dl c

IO P D a

1 0 R R < 2 y r s

0 m 0 R R < 2 y r s

1 0 R R < 2 y r s ( g

1 0 1

(

( g

l L O P D

o o

l L O P D

l L O P D R R > 2 y r s R R > 2 y r s R R > 2 y r s

1 1 1 g 2 4 6 n g 2 g 4 2 6 4 6 i n t in i n t t e n n s T im e (y e a rs ) e e T im e (y e a rs ) e s s T im e (y e a rs ) r e e p r r p p Figure 4. Urine tetrasaccharideFigure 4. Urineconcentrations tetrasaccharide following concentrations enzyme replacement followingFigure 4. therapyenzymeUrine tetrasaccharide: replacement(a) IOPD and therapy concentrations: (a) IOPD following and enzyme replacement therapy: (a) IOPD and Figure 4. Urine tetrasaccharide concentrations following(b) LOPD enzymepatients replacementat diagnosis therapy:and first (urinea) IOPD post and-treatment. (b) LOPD (c) patients Long-term at monitoring of patients (b) LOPD patients at diagnosis(b) LOPD4 andpatients first aturine diagnosis post-treatment. and first urine(c) Long post-term-treatment. monitoring (c) Long of patients-term monitoring of patients  4  following treatment. followingdiagnosis RR, reference and treatment. first range. urine RR, post-treatment. c.953T>C reference homozygous, range. (c) Long-term followingc.953T>C 0.6 yearsmonitoring treatment.homozygous, at diagnosis; of RR, patients 0.6 reference  years following at range. diagnosis; treatment. c.953T>C  RR, reference homozygous, range. 0.6 years at diagnosis;   c.323G>A homozygous,cc.953T>C.323G>A 0.9 years homozygous, at diagnosis; 0.60.9  yearsc.266G>T/c.2815_2816del,years atatdiagnosis; diagnosis; cc.323G>A.323G>A c.266G>T/c.2815_2816del, 0.7 years homozygous, homozygous, at diagnosis; 0.9 0.9 years0.7 years years at diagnosis; at diagnosis; c.266G>T/c.2815_2816del,c.266G>T/c.2815_2816del,  0.7 years at diagnosis; ▼ c.[-32-13T>G; 2275G>A]/c.1827delC,c.[0.7-32 years-13T>G; at diagnosis; 2275G>A]/c.1827delC, 4 yearsN atc.[-32-13T>G; diagnosis; 4▼ 2275G>A]/c.1827delC,years c.[-32 at-#13T>G; c.[diagnosis;-32- 13T>G;2275G>A]/c.1827delC, ▼ 2275G>A]/c.1827delC,c.[ 4 years-32-13T>G; at diagnosis; 2275G>A]/c.1827delC, 2 H 4c.[-32-13T>G; years at diagnosis; 2275G>A]/c.1827delC,2 c.[-32-13T>G; 2 2275G>A]/c.1827delC, 2 years at diagnosis;  c.2297A>C/c.-32-13T>G, 11 years at diagnosis;  c.-32-13T>G/c.1735G>A, 8 years at diagnosis; years yearsc.2297A>C/c. at diagnosis;-32-13T>G,  c.2297A>C/c.c.2297A>C/c.-32-13T>G, 11 years at-32 diagnosis;-13T>G, 11 11 years yearsc.-32 - at13T>G/c.1735G>A, at diagnosis; diagnosis; • c.-32-13T>G/c.1735G>A,c.-32 8 -13T>G/c.1735G>A, 8 8 years at diagnosis; years at diagnosis;  c.-32-13T>G/c.1827delC, 58 years at diagnosis;  c.-32-13T>G/c.1548G>A, 17 years at diagnosis; years c.-32-13T>G/c.1827delC,c.-32 -at13T>G/c.1827delC, diagnosis;  c.-32 58 58-13T>G/c.1827delC, years at diagnosis; 58  years c.c.-32-13T>G/c.1548G>A,-32 -at13T>G/c.1548G>A, diagnosis;  c.-32 17 17-13T>G/c.1548G>A, years at diagnosis. 17 years at diagnosis. years at diagnosis. years at diagnosis. 3. Discussion 3. Discussion 3. Discussion 3. Discussion To address the need for secondary confirmation of reduced GAA for diagnosing PD, To address the need for secondary confirmation of reduced GAA for diagnosing PD, To address the needTo for address secondary the needconfirmation wefor developedsecondary of reduced confirmation a simple GAA assay for of diagnosing toreduced quantify GAA PD, the for amount diagnosing of a tetrasaccharide PD, in urine, which we developed a simple assay to quantify the amount of a tetrasaccharide in urine, which we developed a simplewe developed assay to quantify a simple the assayis amount presumed to quantify of a totetrasaccharide bethe aamount metabolite of in a urine, tetrasaccharide of glycogen. which This in urine, tetrasaccharide which is excreted at higher is presumed to be a metabolite of glycogen. This tetrasaccharide is excreted at higher con- is presumed to be ais metabolite presumed of to glycogen. be a metabolite Thisconcentrations tetrasaccharide of glycogen. in patientsThis is excreted tetrasaccharide with at PD higher compared is con- excreted with at individuals higher con- harboring the pseudodefi- centrations in patients with PD compared with individuals harboring the pseudodefi- centrations in patientscentrations with PD in compared patients with withciency, PD in whodividuals compared have noharboring with glycogen individuals the accumulation pseudodefi- harboring [22 ].the Here, pseudodefi- we show that measuring the urine ciency, who have no glycogen accumulation [22]. Here, we show that measuring the urine ciency, who have nociency, glycogen who accumulation have no glycogen [22].tetrasaccharide Here,accumulation we show following [22]. that Here,measuring low bloodwe show the GAA urinethat activity measuring is a fast the and urine informative method for the tetrasaccharide following low blood GAA activity is a fast and informative method for the tetrasaccharide followingtetrasaccharide low blood following GAA activity diagnosislow blood is a fast of GAA IOPDand activity informative and LOPD. is a fast method The and use informative for of derivatizingthe method reagents for the shortens chromatographic diagnosis of IOPD anddiagnosis LOPD. of The IOPD use and of deriva LOPD.runtizing times The reagentsuse and, of althoughderivadiagnosis shortenstizing our ofchromatographic reagents runIOPD time and shortens at LOPD. 13 min chromatographic The is longer use of thanderiva the tizing recent reagents 5 min shortens reported chromatographic run times and, althoughrun times our runand ,time although at 13 minourusing runis butyllonger time 4-aminobenzoate atthan 13run themin times recent is longer and 5 [min,19 althoughthan], reported we the do recentour not run require 5 mintime reportedsolid-phaseat 13 min is extractionlonger than to the remove recent 5 min reported using butyl 4-aminobenzoate [19], we do notusing require butyl solid 4-aminobenzoate-phase extraction [19], to we remove do not 1 require- solid-phase extraction to remove 1- using butyl 4-aminobenzoate [19], we do not1-phenyl-3-methyl-5-pyrazolone require solid-phase extraction to (PMP), remove significantly 1- simplifying sample preparation. phenyl-3-methyl-5-phenylpyrazolone-3-methyl (PMP-5),- pyrazolonesignificantlyOur ( PMPsimplifying results), sign concurphenylificantly sample with-3 -simplifying methylpreparation. previous-5-pyrazolone methods sample reportingpreparation. (PMP), sign urinary ificantly tetrasaccharide simplifying elevations sample preparation. Our results concur Ourwith results previous concur methods within IOPD reportingprevious and LOPD methodsurinary patientsOur tetrasaccharide reporting results compared urinaryconcur eleva- with withtetrasaccharide control previous subjects. methods eleva- However, reporting we urinary made use tetrasaccharide of elevations in IOPD and LOPDtions inpatients IOPD andcompared LOPD withpatientsjust twocontrol compared reference subjects.tions intervals with However, in control IOPD for controland subjects.we madeLOPD subjects, However, use patients <2 years comparedwe made of age use with of 10 control mmol/mol subjects. creatinine However, we made use of just two referenceof intervals just two forreference control intervals subjeandcts, <3for <2 mmol/mol controlyears of subje ofage creatininejust cts,of two10 <2 mmol/mol referenceyears for theof age cohort intervalscreati- of 10 aged mmol/mol for over control two. creati- subje Previouscts, <2 methods, years of age using of the10 mmol/mol creatinine and <3 mmol/molnine creatinine and <3 mmol/mol for the cohort creatininederivatizing aged forover the reagent two cohortnine. Previous butyl and aged 4-aminobenzoate<3 over methods,mmol/mol two. Previoususing creatinine [ 20] andmethods, for without the usingcohort derivatization aged over [two17],. required Previous methods, using the derivatizing reagentthe derivatizing butyl 4-aminobenzoate reagent butylfive sets[20] 4-aminobenzoate ofand reference withoutthe intervalsderivatizing derivatization [20] and from without reagent <1 [17], to >20 derivatization re-butyl years 4- ofaminobenzoate age. [17], Using re- just [20] two and reference without intervals, derivatization [17], re- 1 quired five sets of referencequired five intervals sets of freferencerom <1the to intervals >20 diagnostic years f romof sensitivity age. quired<1 toUsing >20 five of yearsjust our sets two method ofof age.reference Using was 100%, intervals just two while freferencerom specificity <1 to >20 was years 96%; of theseage. Using results just two reference intervals, the diagnosticintervals, sensitivity the diagnostic of our method sensitivityare similar was of to,100% our although, intervals, methodwhile slightlyspecificity was the 100%diagnostic improve was, while 96% upon,sensitivity specificity; the 98.5% of was our sensitivity96% method; was and 100% 92%,specificity while specificity was 96%; these results are similarthese to, results although are similar slightly to, improvepreviously although upon, slightly reported thethese 98.5%improve [17 results]. sensitivity Elevations upon, are similar the and in 98.5% urinary 92% to, although sensitivity tetrasaccharide slightly and 92% improve in individuals upon, the without 98.5% PD sensitivity and 92% specificity previouslyspecificity reported previously [17]. Elevations reported in urinary[17]. Elevations tetrasaccharidespecificity in urinary previously in individualstetrasaccharide reported [17]. in individuals Elevations in urinary tetrasaccharide in individuals without PD are not withoutunexpected PD aregiven not that unexpected this metabolite given that is not withoutthis specific metabolite PD to are PD isnot and not unexpected eleva-specific to given PD and that eleva- this metabolite is not specific to PD and elevations have also beentions observed have alsoin other been glycogen observed storage in other diseases glycogentions [17], havestorage Duchenne also diseases been muscular observed [17], Duchenne in other muscular glycogen storage diseases [17], Duchenne muscular dystrophy [26] anddystrophy in patients [26] with and acute in patients pancreatitis, with acutemusculardystrophy pancreatitis, trauma, [26] urinary muscular and in tract patients trauma, with urinary acute tract pancreatitis, muscular trauma, urinary tract

Metabolites 2021, 11, 446 7 of 11

are not unexpected given that this metabolite is not specific to PD and elevations have also been observed in other glycogen storage diseases [17], Duchenne muscular dystrophy [26] and in patients with acute pancreatitis, muscular trauma, urinary tract infections and some cancers [27]. Kumlien et al. [27] also determined that a high carbohydrate diet could elevate the urine tetrasaccharide up to three-fold. In this study, one neonate who returned a tetrasaccharide concentration of 21 mmol/mol creatinine was hypoglycemic, with standard treatment involving glucose supplementation. Among the other seven individuals that returned urine tetrasaccharide concentrations above the reference interval, four were only marginally elevated. Two individuals aged 6 and 8 months returned a concentration of 10 and 12 mmol/mol creatinine, respectively, whereas a 4 year old and a 46 year old both had tetrasaccharide concentrations of 4 mmol/mol creatinine. Given these outliers, we assessed whether more defined age-related reference intervals would have normalized these samples, noting that other studies had five groups [17,19,20]. Using a 90% upper percentile (required due to smaller sample numbers in each group), we calculated 11 false positives over the age ranges reported by both An et al. [20] and Sluiter et al. [17], and 16 false positives over the five age ranges reported by Piraud et al. [19]. Using only two age ranges, therefore, allowed a greater specificity while still enabling 100% sensitivity. We note that the method described here cannot distinguish between isomers; while, in glycogen storage diseases, including PD, this tetrasaccharide is commonly referred to as a glucose tetrasaccharide [27], this is an assumption only. While the mass spectrometry method measures a mass to charge ratio which could include any four hexose moieties, An et al. [20] used high-performance liquid chromatographic separation to show that PD patient urine predominantly contained glucose tetrasaccharide with no appreciable amounts of the isomer maltotetraose. Previous studies support the hypothesis that tetrasaccharide is derived from muscle glycogen by showing a statistically significant correlation between tetrasaccharide concentration and glycogen storage in the muscles of PD patients [28,29]. In contrast to An et al., Sluiter et al. [17] reported that maltotetraose was present in ap- proximately 5% of urine samples, predominantly in newborns. In our study, 27 control urine samples were tested from infants younger than 3 months of age, and only one had elevated urine tetrasaccharide concentrations, which could be explained by glucose supplementation. The primary role of the urinary tetrasaccharide resides in the diagnostic trajectory of PD to validate low GAA determinations (Figure5). Although no individuals with the pseudodeficiency were identified in our cohort, three individuals aged 1, 27 and 69 years of age, with GAA below the reference interval, were carriers of PD variants; the normal urinary tetrasaccharide virtually excluded a PD diagnosis, highlighting its utility (Table1). Therefore, if the urinary tetrasaccharide is within the normal range, a diagnosis of PD may be dismissed, thus avoiding unnecessary molecular testing and allowing other avenues of investigation to be pursued to explain symptomology. Although classic IOPD, which presents within the first three months of life, has prototypical clinical presentations that rarely go unrecognized, there is a broad spectrum of phenotypes which is influenced by a number of factors, including residual enzyme activity. Residual enzyme activity is typically < 3% of the mean of normal in classic IOPD, but in non-classical/juvenile OPD and LOPD, residual activity is 3–30% [30,31], meaning GAA is a poor predictor of disease severity. In our cohort of PD patients, we also showed that there was no correlation between age at diagnosis and GAA activity, but we did show a correlation with urine tetrasaccharide concentrations. Figure3 shows that higher urine tetrasaccharide concentrations were associated with younger patients, and that older patients (LOPD) had lower concentrations. Our data therefore support previous findings reporting correlations with urinary tetrasaccharide, age of symptom onset and PD phenotype [19,29].

Metabolites 2021, 11, 446 4 8 of 11

Figure 5. Laboratory diagnostic pathway for Pompe disease. a pmol/spot/h; b ratio of total α- glucosidase/GAA; c mmol/mol creatinine.

Inadequate monitoring and clinical data were available from our small set of PD patients on ERT to fully assess the utility of the urine tetrasaccharide as a response biomarker. 1Longitudinal monitoring of PD patients post-ERT revealed that urinary tetrasaccharide concentrations did not normalize in all patients. In particular, IOPD patients, despite showing an initial drop, still had signiﬁcant urinary tetrasaccharide concentrations up to four years following ERT. Chien et al. [32] recently reported correlations between urinary tetrasaccharide and ERT outcomes in patients with IOPD, with ambulate patients having signiﬁcantly lower urinary tetrasaccharide concentrations compared with patients unable to walk. Of note, they found that all patients sustained concentrations above the normal reference range, in agreement with our data. Little data are currently available on ERT outcomes in patients with LOPD or on urinary tetrasaccharide concentrations, and our data suggest that there is a general trend of reducing concentrations following the initiation of therapy (Figure4).

4. Materials and Methods 4.1. Materials

All solvents were purchased from Merck (Darmstadt, Germany). NH4OH (30% v/v), HCOOH (98% v/v) and CHCl3 (containing 1% CH3CH2OH) were reagent grade, and CH3OH, H2O, (CH3)2CHOH and CH3CN LiChroSolv were gradient grade. NH4COOH (HPLC grade) and chondroitin disaccharide ∆di-0S were purchased from Sigma Aldrich (St. Louis, MO, USA). 1-phenyl-3-methyl-5-pyrazolone (PMP) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Maltotetraose was purchased from Carbosynth Ltd. (Crompton, Witham, UK).

4.2. Patient Samples All DBS and urine samples were submitted to our department for the diagnosis and biochemical monitoring of metabolic diseases. The use of patient samples in this study was approved by the Institutional Human Research Ethics Committee (HREC/15/WCHN/69). All DBS were stored at room temperature and urine samples were stored at −20 ◦C and thawed at room temperature prior to analysis.

4.3. Urinary Tetrasaccharide Determination A seven-point standard curve was prepared with each assay by addition of maltotetraose in 0.01 mL of H2O to give ﬁnal concentrations of 2, 5, 15, 30, 50, 100, 300 mmol/mol creatinine. Patient urine (0.05 µmol creatinine equivalent) and standards were lyophilized before reconstitution with 1 nmol of chondroitin disaccharide ∆di-0S (IS) in 0.1 mL of PMP ◦ (0.25 M in 0.4 M of NH4OH; pH 9.5). Samples were incubated at 70 C for 90 min prior to Metabolites 2021, 11, 446 9 of 11

acidification with 0.5 mL of 0.2 M HCOOH. Excess PMP was removed by shaking with CHCl3 (0.5 mL) for 1 min prior to centrifugation for 5 min (13,000× g; RT). The organic phase was removed and the wash step was repeated a further three times. The aqueous phase (0.1 mL) was removed and placed in the well of a 96-well microtiter plate, which was heat-sealed for analysis by liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Desalting and partial separation of samples was achieved on a Pursuit 3 pentafluorophenyl (PFP) column (2.0 × 100 mm; 3 µm; Agilent Technologies, Santa Clara, CA, USA) maintained at 30 ◦C with an Agilent Affinity 1290 inline filter containing a 0.3 µm of frit placed in front of the column. Samples were maintained at 18 ◦C in the autosampler with 2 µL injected into the mobile phase, which was at a flow rate of 0.3 mL/min. Mobile phase A consisted of H2O with 0.1% HCOOH and mobile phase B contained CH3CN with 0.1% HCOOH. The column was equilibrated at 20% mobile phase B with a linear ramp to 22.5% mobile phase B over 5 min, followed by an increase to 90% mobile phase B at 5.5 min. This was maintained for 2 min before being decreased to 20% mobile phase B at 8 min, where the column was re-equilibrated for 4 min prior to injection of the next sample. The first 2.1 min was sent to waste before being directed into the ESI source (ES 5000 V) of an AB SCIEX QTRAP 6500 tandem mass spectrometer in positive ion mode and with an ion source temperature of 300 ◦C. Nitrogen was used for curtain gas, 25 units; collision gas, high; nebulizer gas 1, 20 units and auxiliary gas 2, 40 units. Analytes were measured by multiple reaction monitoring with the transitions 997/175 and 710/253 for the tetrasaccharide and IS, respectively. Urinary tetrasaccharide concentrations were determined using a calibration curve created in MultiQuant (SCIEX; v. 3.0.1) of the maltotetraose concentration and the ratio of chromatographic peak area of maltotetraose and the IS, using a 1/x2 weighting. Acceptance for the calibration curve was a coefficient of determination r2 ≥ 0.99, and each standard had to be within 15% of the target concentration. Tetrasaccharide concentration was normalized to creatinine concentration and expressed as mmol/mol creatinine.

4.4. GAA Activity in Dried Blood Spots GAA activity was measured from 3 mm DBS, as previously described [7]. In brief, blood was eluted from the DBS and α-glucosidase measured using the ﬂuorometric substrate 4-methylumbelliferyl-α—D-glucopyranoside. Acarbose was added under acidic conditions to inhibit maltase-glucoamylase and allow for the measurement of GAA activity by release of the 4-methylumbelliferyl (4-MU) moiety. Total neutral α-glucosidase (pH 7.0) was used as a measure of sample integrity. A calibration curve consisting of 4-MU (0–1 nM) was used to calculate enzyme activity following background subtraction to account for autoﬂuorescence.

5. Conclusions Following initial enzymatic investigations suggestive of PD, the urinary tetrasaccharide is a useful metabolite in a second-tier laboratory test to conﬁrm or exclude a diagnosis of PD. This report provides additional and much needed data, supporting the utility of the urinary tetrasaccharide in the diagnostic setting of PD, especially given that there are very few literature reports on the measurement of this metabolite. As a biomarker to monitor treatment response in PD patients, further work is clearly needed, although our data does show that the tetrasaccharide responds, at least to some extent, to ERT.

Author Contributions: Conceptualization, M.F.; methodology, J.T.S.; validation, M.F. and J.T.S.; formal analysis, J.T.S.; investigation, J.T.S. and M.F.; data curation, J.T.S.; writing—original draft preparation, M.F. and J.T.S.; writing—review and editing, M.F. and J.T.S.; supervision, M.F.; project administration, M.F. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Metabolites 2021, 11, 446 10 of 11

Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Human Research Ethics Committee of the Women’s and Children’s Health Network (HREC/15/WCHN/69). Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within the article. Acknowledgments: The authors would like to thank Belinda McDermott and Elaine Ravenscroft for technical assistance and Emily Button for helpful discussions. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References 1. van den Hout, H.M.; Hop, W.; van Diggelen, O.P.; Smeitink, J.A.M.; Smit, G.P.A.; Poll-The, B.T.T.; Bakker, H.D.; Loonen, M.C.B.; de Klerk, J.B.C.; Reuser, A.J.J.; et al. The natural course of infantile Pompe’s disease: 20 original cases compared with 133 cases from literature. Pediatrics 2003, 112, 332–340. [CrossRef] 2. Kishnani, P.S.; Hwu, W.L.; Mandel, H.; Nicolino, M.; Yong, F.; Corzo, D. Infantile-Onset Pompe Disease Natural History Study Group. A retrospective, multinational, multicenter study on the natural history of infantile-onset Pompe disease. J. Pediatr. 2006, 148, 671–676. [CrossRef] 3. Wokke, J.H.J.; Escolar, D.M.; Pestronk, A.; Jaffe, K.M.; Carter, G.T.; van den Berg, L.H.; Florence, J.M.; Mayhew, J.; Skrinar, A.; Corzo, D.; et al. Clinical features of late-onset Pompe disease: A prospective cohort study. Muscle Nerve 2008, 38, 1236–1245. [CrossRef] 4. Kishnani, P.S.; Corzo, D.; Nicolino, M.; Byrne, B.; Mandel, H.; Hwu, W.L.; Leslie, N.; Levine, J.; Spencer, C.; McDonald, M.; et al. Recombinant human acid α-glucosidase: Major clinical benefits in infantile-onset Pompe disease. Neurology 2007, 68, 99–109. [CrossRef] 5. Schoser, B.; Stewart, A.; Kanters, S.; Hamed, A.; Jansen, J.; Chan, K.; Karamouzian, M.; Toscano, A. Survival and long-term outcomes in late-onset Pompe disease following alglucosidase alfa treatment: A systematic review and meta-analysis. J. Neurol. 2017, 264, 621–630. [CrossRef] 6. Burton, B.K.; Kronn, D.F.; Hwu, W.L.; Kishnani, P.S.; Pompe Disease Newborn Screening Working Group. The initial valuation of patients after positive newborn screening: Recommended algorithms leading to a confirmed diagnosis of Pompe disease. Pediatrics 2017, 140, S14–S23. [CrossRef][PubMed] 7. Zhang, H.; Kallwass, H.; Young, S.P.; Carr, C.; Dai, J.; Kishnani, P.S.; Millington, D.S.; Keutzer, J.; Chen, Y.-T.; Bali, D. Comparison of maltose and acarbose as inhibitors of maltase-glucoamylase activity in assaying acid alpha-glucosidase activity in dried blood spots for the diagnosis of infantile Pompe disease. Genet. Med. 2006, 8, 302–306. [CrossRef] 8. Li, Y.; Scott, C.R.; Chamoles, N.A.; Ghavami, A.; Pinto, B.M.; Turecek, F.; Gelb, M.H. Direct multiplex assay of lysosomal enzymes in dried blood spots for newborn screening. Clin. Chem. 2004, 50, 1785–1796. [CrossRef][PubMed] 9. Dajnoki, A.; Mühl, A.; Fekete, G.; Keutzer, J.; Orsini, J.; Dejesus, V.; Zhang, X.K.; Bodamer, O.A. Newborn screening for Pompe disease by measuring acid alpha-glucosidase activity using tandem mass spectrometry. Clin. Chem. 2008, 54, 1624–1629. [CrossRef][PubMed] 10. Kallwass, H.; Carr, C.; Gerrein, J.; Titlow, M.; Pomponio, R.; Bali, D.; Dai, J.; Kishnani, P.; Skrinar, A.; Corzo, D.; et al. Rapid diagnosis of late-onset Pompe disease by fluorometric assay of α-glucosidase activities in dried blood spots. Mol. Genet. Metab. 2007, 90, 449–452. [CrossRef][PubMed] 11. Tajima, Y.; Matsuzawa, F.; Aikawa, S.I.; Okumiya, T.; Yoshimizu, M.; Tsukimura, T.; Ikekita, M.; Tsujino, S.; Tsuji, A.; Edmunds, T.; et al. Structural and biochemical studies on Pompe disease and a “pseudodeficiency of acid α-glucosidase”. J. Hum. Genet. 2007, 52, 898–906. [CrossRef][PubMed] 12. Niño, M.Y.; In’t Groen, S.L.; Bergsma, A.J.; van der Beek, N.A.M.E.; Kroos, M.; Hoogeveen-Westerveld, M.; van der Ploeg, A.T.; Pijnappel, W.W.M.P. Extension of the Pompe mutation database by linking disease-associated variants to clinical severity. Hum. Mutat. 2019, 40, 1954–1967. [CrossRef] 13. Ficicioglu, C.; Ahrens-Nicklas, R.C.; Barch, J.; Cuddapah, S.R.; DiBoscio, B.S.; DiPerna, J.C.; Gordon, P.L.; Henderson, N.; Menello, C.; Luongo, N.; et al. Newborn screening for Pompe disease: Pennsylvania experience. Int. J. Neonatal Screen. 2020, 13, 89. [CrossRef][PubMed] 14. Fuller, M.; Duplock, S.; Turner, C.; Davey, P.; Brooks, D.A.; Hopwood, J.J.; Meikle, P.J. Mass spectrometric quantification of glycogen to assess primary substrate accumulation in the Pompe mouse. Anal. Biochem. 2012, 421, 759–763. [CrossRef][PubMed] 15. Hallgren, P.; Hansson, G.; Henriksson, K.G.; Häger, A.; Lundblad, A.; Svensson, S. Increased excretion of a glucose-containing tetrasaccharide in the urine of a patient with glycogen storage disease type II (Pompe’s disease). Eur. J. Clin. Investig. 1974, 4, 429–433. [CrossRef] 16. Lennartsson, G.; Lundblad, A.; Sjöblad, S.; Svensson, S.; Öckerman, P.-A. Quantitation of a urinary tetrassacharide by gas chromatography and mass spectrometry. Biomed. Mass Spectrom. 1976, 3, 51–54. [CrossRef] Metabolites 2021, 11, 446 11 of 11

17. Sluiter, W.; van den Bosch, J.C.; Goudriaan, D.A.; van Gelder, C.M.; de Vries, J.M.; Huijmans, J.G.M.; Reuser, A.J.J.; van der Ploeg, A.T.; Ruijter, G.J.G. Rapid ultraperformance liquid chromatography-tandem mass spectrometry assay for a characteristic glycogen-derived tetrasaccharide in Pompe disease and other glycogen storage diseases. Clin. Chem. 2012, 58, 1139–1147. [CrossRef] 18. Manwaring, V.; Prunty, H.; Bainbridge, K.; Burke, D.; Finnegan, N.; Franses, R.; Lam, A.; Vellodi, A.; Heales, S. Urine analysis of glucose tetrasaccharide by HPLC; a useful marker for the investigation of patients with Pompe and other glycogen storage diseases. J. Inherit. Metab. Dis. 2012, 35, 311–316. [CrossRef] 19. Piraud, M.; Pettazzoni, M.; de Antonio, M.; Vianey-Saban, C.; Froissart, R.; Chabrol, B.; Young, S.; Laforêt, P.; French Pompe Study Group. Urine glucose tetrasaccharide: A good biomarker for glycogenoses type III and III? A study of the French cohort. Mol. Genet. Metab. Rep. 2020, 23, 100583. [CrossRef][PubMed] 20. An, Y.; Young, S.P.; Hillman, S.L.; Van Hove, J.L.; Chen, Y.T.; Millington, D.S. Liquid chromatography assay for a glucose tetrasaccharide, a putative biomarker for the diagnosis of Pompe disease. Anal. Biochem. 2000, 287, 136–143. [CrossRef] 21. Canbay, E.; Vural, M.; Uçar, S.K.; Sezer, E.D.; Karasoy, H.; Vüceyar, A.N.; Çoker, M.; Sözmen, E.Y. The decision-making levels of urine tetrasaccharide for the diagnosis of Pompe disease in the Turkish population. J. Pediatr. Endocrinol. Metab. 2020, 33, 391–395. [CrossRef] 22. Chien, Y.H.; Goldstein, J.L.; Hwu, W.L.; Smith, P.B.; Lee, N.C.; Chiang, S.C.; Tolun, A.A.; Zhang, H.; Vaisnins, A.E.; Millington, D.S.; et al. Baseline urinary tetrasaccharide concentrations in patients with infantile- and late-onset Pompe disease identiﬁed by newborn screening. JIMD Rep. 2015, 19, 67–73. 23. An, Y.; Young, S.P.; Kishnani, P.S.; Millington, D.S.; Amalﬁtano, A.; Corz, D.; Chen, Y.T. Glucose tetrasaccharide as a biomarker for monitoring the therapeutic response to enzyme replacement therapy for Pompe disease. Mol. Genet. Metab. 2005, 85, 247–254. [CrossRef][PubMed] 24. Young, S.P.; Zhang, H.; Corzo, D.; Thurberg, B.L.; Bali, D.; Kishnani, P.S.; Millington, D.S. Long-term monitoring of patients with infantile-onset Pompe disease on enzyme replacement therapy using a urinary glucose tetrasaccharide biomarker. Genet. Med. 2009, 11, 536–541. [CrossRef][PubMed] 25. Khan, A.A.; Case, L.E.; Herburt, M.; DeArmey, S.; Jones, H.; Crisp, K.; Zimmermand, K.; ElMallah, M.; Young, S.P.; Kishnani, P.S. Higher dosing of alglucosidase alfa improves outcomes in children with Pompe disease: A clinical study and review of the literature. Genet. Med. 2020, 22, 898–907. [CrossRef][PubMed] 26. Lundblad, A.; Svensson, S.; Yamashina, I.; Ohta, M. Increased urinary excretion of a glucose-containing tetrasaccharide in patients with Duchenne muscular dystrophy. FEBS Lett. 1979, 97, 249–252. [PubMed] 27. Heiner-Fokkema, M.R.; van der Krogt, J.; de Boer, F.; Fokkert-Wilts, M.J.; Maatman, R.G.; Hoogeveen, I.J.; Derks, T.G. The multiple faces of urinary glucose tetrasaccharide as biomarker for patients with hepatic glycogen storage diseases. Genet. Med. 2020, 22, 1915–1916. [CrossRef] 28. Kumlien, J.; Chester, M.A.; Lindberg, B.S.; Pizzo, P.; Zopf, D.; Lundblad, A. Urinary excretion of a glucose-containing tetrasaccharide. A parameter for increased degradation of glycogen. Clin. Chim. Acta 1988, 176, 39–48. [CrossRef] 29. Young, S.P.; Piraud, M.; Goldstein, J.L.; Zhang, H.; Rehder, C.; Laforet, P.; Kishnani, P.S.; Millington, D.S.; Bashir, M.R.; Bali, D.S. Assessing disease severity in Pompe disease: The roles of a urinary glucose tetrasaccharide biomarker and imaging techniques. Am. J. Med. Genet. 2012, 160, 50–58. [CrossRef] 30. Chan, J.; Desai, A.K.; Kazi, Z.B.; Corey, K.; Austin, S.; Hobson-Webb, L.D.; Case, L.E.; Jones, H.N.; Kishnani, P.S. The emerging phenotype of late-onset Pompe disease: A systematic literature review. Mol. Genet. Metab. 2017, 120, 163–172. [CrossRef] 31. van Capelle, C.I.; van der Meijden, J.C.; van den Hout, J.M.; Jaeken, J.; Baethmann, M.; Voit, T.; Kroos, M.A.; Derks, T.G.; Rubio-Gozalbo, M.E.; Willemsen, M.A.; et al. Childhood Pompe disease: Clinical spectrum and genotype in 31 patients. Orphanet J. Rare Dis. 2016, 11, 65. [CrossRef][PubMed] 32. Chien, Y.H.; Tsai, W.H.; Chang, C.L.; Chiu, P.C.; Chou, Y.Y.; Tsai, F.J.; Wong, S.L.; Lee, N.C.; Hwu, W.L. Earlier and higher dosing of alglucosidase alfa improve outcomes in patients with infantile-onset Pompe disease: Evidence from real-world experiences. Mol. Genet. Metab. Rep. 2020, 23, 100591. [CrossRef][PubMed]