Study population and sample collection
The study population consisted of 297,387 newborns from Kumamoto and Fukuoka prefectures between April 2013 and October 2020. Dried-blood spot (DBS) samples were prepared in each maternity clinic or obstetric department using a heel prick procedure 4–6 days after birth for newborn mass screening across all municipalities. After blotting with blood spots (Toyo Roshi Kaisha, Ltd., Tokyo, Japan), the filter paper was dried for at least 4 h at room temperature (i.e., 20 ± 5°C), and the samples were sent to the Newborn Screening Center at KM Biologics Co., Ltd. (Kumamoto, Japan), where publicly-funded newborn mass screening was conducted, within 1 week of collection. After analysis, the DBSs after testing were transferred to Kumamoto University to assay AαGlu activity.
NBS program for Pompe disease
Our proposed NBS for Pompe disease utilizing AαGlu assays on DBSs involves three steps (Fig. 1). First, DBS samples whose AαGlu activity was under the cutoff value (< 6.5 pmol hr− 1 disk− 1 in Method I; < 3.5 pmol hr− 1 disk− 1 in Methods II, III) were recalled, and DBSs were prepared again for a second AαGlu assay. Second, newborns whose AαGlu activity was under the cutoff value were referred to the hospital within two months for clinical examination, and physical and biochemical assays were performed to confirm the symptomatic signs of IOPD. The new DBS samples were subjected to hemoglobin precipitation using the Ba/Zn method to considerably improve the 4MU fluorescence intensity and a third AαGlu assay was also performed. Finally, GAA gene sequencing was performed in newborns whose AαGlu activity was under the cutoff value following the third AαGlu assay to confirm the presence of GAA gene variants. Additionally, the AαGlu activity of the fibroblasts of the newborns was measured between April 2013 and November 2016 (11). The results of the first AαGlu assay was acquired 1–2 weeks after birth; the second AαGlu assay, within 4 weeks after birth; clinical examination, within 2 months after birth; GAA gene analysis and final diagnosis, up to 6 months after birth.
AαGlu assay
Method I
AαGlu assays on DBSs and fibroblasts were performed as previously described (11). Briefly, one disk (3.2 mm in diameter) was punched from DBS cards and placed into a well of a 96-well clear microwell plate (Corning, NY, USA) with 100 µL of 0.8 mM citrate in 24 mM potassium phosphate buffer (pH 6.0) containing 0.1% Triton X-100 for 1 h at room temperature with gentle mixing. In a 96-well black microwell-plate (PerkinElmer, Waltham, MA, USA), a 20 µL aliquot of the extract was then added to 40 µL of 2.0 mM 4MU-αGlc in 0.12 M citrate/0.15 M potassium phosphate buffer (pH 4.0) containing 4.5 µM acarbose, incubated at 37°C for 24 h, and quenched by adding 190 µL of 0.2M glycine/NaOH buffer (pH 10.7) containing 0.1% Triton X-100 and 0.2% SDS to measure fluorescence intensity.
For the Ba/Zn method, a 3.2 mm diameter disk was placed in a 1.5 mL reaction tube with 60 µL of 0.12 M citrate/0.15 M potassium phosphate buffer (pH 4.0) containing 2.0 mM 4MU-αGlc and 3.0 µM acarbose and gently mixed for 10 min at room temperature. The reaction mixture was incubated at 37°C for 24 h, added with 30 µL of 0.15 M barium hydroxide, vortexed, and further incubated at room temperature for 5 min. Then, 30 µL of 0.15 M zinc sulfate was added, and the tubes were mixed by vortexing, incubated for 10 min at room temperature, and centrifuged for 5 min at 12,000 rpm and 5°C. Finally, 90 µL of the supernatant was transferred to a 96-well black microwell plate, and 160 µL of 0.4 M glycine/NaOH buffer (pH 10.7) containing 0.1% Triton X-100 was added to measure fluorescence intensity. Stock solutions of 0, 6.25, 12.5, 25, 50, and 100 µM 4-methylumbelliferone (4MU) in 20 mM sodium phosphate buffer (pH 7.0) were used for standardization of the liberated 4MU concentration. Enzyme activity was expressed as pmol of 4MU released per hour per disk (pmol h− 1 disk− 1). Each assay was performed in duplicate.
Method II
Method II for multiple assays was developed in collaboration with KM Biologics Co., Ltd. (see details at JP6360848B) and implemented in December 2016. Briefly, a single 3.2-mm diameter disk punched from DBSs was incubated in the well of a 96-well clear microwell plate (Corning, NY, USA) with 100 µL of 5 mM MgCl2, 0.5 mM dithiothreitol, 0.05% NaN3, and 0.1% Triton X-100 in 0.8 mM citrate/24 mM potassium phosphate buffer (pH 6.0) for 1 h at room temperature with gentle mixing. A 20 µL aliquot of the extract was added to 40 µL of 2.0 mM 4MU-αGlc in 0.12 M citrate/0.15 M potassium phosphate buffer (pH 4.0) containing 4.5 µM acarbose in a 96-well black microwell-plate (PerkinElmer, Waltham, MA, USA). The reaction mixture was incubated at 37°C for 4 h and quenched by adding 200 µL of 0.3 M glycine/NaOH buffer (pH 10.6) containing 10 mM EDTA to measure fluorescence intensity.
Method III
Method III for more high-throughput assays was also developed in collaboration with KM Biologics Co., Ltd. (see details at P2017-245523) and implemented in February 2019. A single 3.2 mm diameter disk punched from DBSs was incubated in the wells of a 96-well clear microwell plate (Corning, NY, USA) with 200 µL of 38 mM KCl, 5 mM MgCl2, 0.05% NaN3, and 0.1% Triton X-100 in 5 mM sodium acetate buffer (pH 5.2) for 1 h at room temperature with gentle mixing. A 20-µL aliquot of the extract was added to 40 µL of 2.0 mM 4MU-αGlc in 0.12 M citrate/0.15 M potassium phosphate buffer (pH 4.0) containing 4.5 µM acarbose in a 96-well black microwell-plate (PerkinElmer, Waltham, MA, USA). The reaction mixture was incubated at 38°C for 3 h, and the reaction was stopped by adding 200 µL of 0.3 M glycine/NaOH buffer (pH 10.6) containing 10 mM EDTA to measure fluorescence intensity.
AαGlu assay of fibroblasts
AαGlu assays of fibroblasts were conducted between April 2013 and November 2016. Briefly, fibroblasts were collected from a skin biopsy and cultured under standard conditions in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum and antibiotics (50 kU/L penicillin and 50 mg/L streptomycin). After growing to 100% confluence, fibroblasts were harvested and washed with phosphate-buffered saline. The cell pellet was stored at -80°C until use. Fibroblasts (2 to 4 × 106 cells) were homogenized in 150 µL of water using sonication on ice, and 10 µL of the cell homogenate was added to 40 µL of the substrate solution containing 2.0 mM 4MU-αGlc in 0.12 M citrate in 0.15 M potassium phosphate buffer (pH 4.0) containing 3.75 µM acarbose (final concentration: 3.0 µM) in a well of a 96-well black microwell-plate. The reaction mixture was incubated at 37°C for 1 h, and the reaction was stopped by the addition of 200 µL of 0.2 M glycine/NaOH buffer (pH 10.7) containing 0.1% Triton X-100 to measure fluorescence intensity, with blank correction. A stock solution of 250 µM 4MU in 20 mM sodium phosphate buffer (pH 7.0) was used for calibration of the liberated 4MU concentration. Enzyme activity was expressed as nanomoles of 4MU released per hour per milligram of cellular protein (nmol h− 1 mg− 1 protein). Each assay was performed in duplicate.
GAA sequencing using NGS
GAA sequencing using NGS was conducted as described previously (31). Briefly, genomic DNA was extracted from total blood using a Gentra Puregene Blood Kit (Qiagen, Hilden, Germany) and stored at -80°C until use. The 22-kbp region, including the GAA, was amplified by dividing the genomic DNA into three fragments using long-range polymerase chain reaction (Supplementary Fig. S2) with DNA polymerase (KOD FX; Toyobo, Osaka, Japan) on a Veriti Thermal Cycler (Applied Biosystems, Foster City, CA, USA). PCR products were purified using an Agencourt AMP XP PCR Purification Kit (Beckman Coulter, Brea, CA, USA) and quantified with a Qubit dsDNA HS Assay Kit (Life Technologies, Carlsbad, CA, USA) using a Qubit 2.0 Fluorometer (Life Technologies). Simultaneous fragmentation of PCR products and adaptor ligation were performed using a Nextera XT Kit (Illumina, San Diego, CA, USA). Indexed DNA was purified using an Agencourt AMP XP PCR Purification Kit (Beckman Coulter). Each library was validated using High Sensitivity D1000 ScreenTape (Agilent Technologies, Santa Clara, CA, USA) with an Agilent 2200 TapeStation and quantified using a Qubit dsDNA HS Assay Kit with a Qubit 2.0 Fluorometer to allow for library normalization. Sequencing was performed with a MiSeq Reagent Kit v3 on MiSeq sequencer (Illumina) using the “paired-end” sequencing run method. Data were aligned to target sequences on the human reference genome sequence using the MiSeq Reporter software (Illumina). Sequence data analysis, mapping, and variant calling were streamlined using MiSeq Reporter v2 (Illumina). Briefly, reads were aligned to the reference sequence from 80,101,882 to 80,123,207 of the genome sequence of chromosome 17 (NC_000017.11) using bwa-0.6.1. Single-nucleotide polymorphism and insertion/deletion identifications were performed using the Genome Analysis Toolkit (GATK v1.6; Broad Institute, Cambridge, MA, USA), and visualization was performed using IGV_2.3.40 (Broad Institute).
GAA resequencing using the Sanger method
Variants detected in the GAA gene using NGS were re-sequenced using the Sanger method (12). Briefly, a region including the variant was amplified using PCR with an appropriate set of primers. PCR products were sequenced on an ABI3500xl auto sequencer (Applied Biosystems) and analyzed using Sequencher 5.0 (Gene Codes Corporation, Foster City, CA, USA).
Mutation analysis of the variants
The mRNA reference sequence (RefSeq) NM_000152.4 was used, whereby the “A” nucleotide of the ATG codon at nucleotide position 398 of the RefSeq constituted + 1 numbering of the cDNA sequence. The ATG codon was also represented as + 1 for amino acid numbering as set forth by the AαGlu preprotein sequence NP_000143.2. Mutation nomenclature followed the guidelines established by the Human Genome Variation Society (http://varnomen.hgvs.org/). The Pompe disease GAA variant database (12) (http://www.pompevariantdatabase.nl; Accessed March 3, 2021) and the ClinVar Miner (13) (https://clinvarminer.genetics.utah.edu/; Accessed March 3, 2021) were to classify each variant. PolyPhen-2 (32) (http://genetics.bwh.harvard.edu/pph2; Accessed March 3, 2021) was used to predict the potential effect of an amino acid alteration on the function of AαGlu. The online bioinformatics tool, Human Splicing Finder (14) (http://www.umd.be/HSF3/; Accessed March 3, 2021) was used to estimate the effects of mutations on splicing signals.