The study was approved by the local ethics committee of the Federal State Budgetary Institution “Research Centre for Medical Genetics” (the approval number 2015-5/3). The written informed consent was obtained from the patients’ parents and the protocol was approved by the local Institutional Review Board.

The activity of lysosomal enzymes was measured in dried blood spot samples by LC-MS/MS method. The internal standards and substrates for GLB1, I2S, NAGLU, GALNS, ARSB, GUSB, and TPP1 were commercially purchased from PerkinElmer, Inc. (Waltham, MA, USA).

The multiplex assay was performed as follows. A 3-mm punch of dried blood spot was incubated in the buffer containing four substrates and internal standards overnight. A liquid–liquid extraction by using aqueous NaCl and ethyl acetate was performed. Subsequently, the ethyl acetate layer was then collected and dried. The sediment was consequentially resuspended in solvent for auto-sampling for tandem mass spectrometry analysis.

Samples were measured using a LC-30 Nexera System (Shimadzu Corporation, Kyoto, Japan) and a tandem mass spectrometer QTrap 4500 (ABSciex, USA) equipped with an positive electrospray ionization. The LC column was a Phenomenex Fusion-RP 50 × 2.1 mm, 4 µm (Phenomenex, Torrance, CA, USA), and the column oven temperature was 50 °C.

Genomic DNA was extracted from whole blood with EDTA using GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA). Sanger sequencing was performed on ABI PRISM 3500xL Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA). Whole-genome sequencing of the patient’s DNA was performed with TruSeq DNA PCR-Free sample preparation kit on NovaSeq 6000 (Illumina, San Diego, CA, USA) with mean coverage of 42X.

Bioinformatics pipeline: sequence reads were aligned to the human reference genome GRCh37 (hg19) using Burrows-Wheeler Aligner v.0.7.17-r1188 (http://bio-bwa.sourceforge.net, accessed on 13th Jun 2022). Single-nucleotide variants and small insertions and deletions (indels) were called with Strelka2 Small Variant Caller v.2.9.10 (https://github.com/Illumina/strelka, accessed on 13th Jun 2022) and the Genome Analysis Toolkit v.4 (https://gatk.broadinstitute.org, accessed on 13th Jun 2022). Structural variants were called with Manta v. 1.6.0 (https://github.com/Illumina/manta, accessed on 13th Jun 2022). The reported variants were annotated with their genomic coordinates, allele frequency (gnomAD database, http://gnomad.broadinstitute.org, accessed on 13th Jun 2022), functional consequence, and impact level on the gene product using SnpEff v5 (http://pcingola.github.io/SnpEff, accessed on 13th Jun 2022). Variants were prioritized by the consensus score of the set of bioinformatic tools, which predict the pathogenicity of the variant and the deleterious effect on protein (SIFT, SIFT4G, Polyphen2, MutationAssessor, FATHMM, PROVEAN, DEOGEN2, LRT, PrimateAI, MetaSVM, MetaLR, SpliceAI, MMsplice, SPiP, Spidex). Data analysis was performed with custom web-based NGS-data-Genome interface.

Variants were named according to the GLB1 reference sequence NM_000404.4 and GRCh37.p13 (hg19) genome assembly.

The patient’s total RNA was isolated from cultured fibroblasts using Total RNA Purification Plus Kit (Norgene, Thorold, ON, Canada). The first strand of cDNA was synthesized using ImProm-II™ Reverse Transcriptase (Promega, Madison, WI, USA) and oligo(dT) primers. Overlapping fragments of the GLB1 cDNA were amplified by PCR and Sanger sequenced.

The 505 bp fragment of the (WT) GLB1 intron 5 (NC_000003.11:g.33099863_33100367) and the ~1850 bp fragment with PP insertion were placed between two constitutively spliced exons (V1 and V2) of the pSpl3-Flu2-mTK vector. pSpl3-Flu2-mTK is a modification of the pSpl3-Flu vector33, in which the CMV promoter was changed to the Minick promoter (the −33 to +32 region of the Herpes simplex thymidine kinase promoter) and the strong cryptic splice site downstream of the multiple cloning site was deleted. These modifications were made as they improved the recognition of a number of previously studied exons (unpublished data).

The 425 bp fragment of the mouse U7-snRNA gene containing promoter and terminator sequences was amplified with tailed primers 5′-ttaaAGATCTtaacaacataggagctgtg-3′ and 5′-ttaaCTCGAGcacatacgcgtttcctagg-3′ and cloned into pcDNA3.1 vector between BglII and XhoI restriction sites. Overlap-extension PCR was used to introduce several modifications. At first, U7-specific Sm binding site (AATTTGTCTAG) was replaced by the consensus Sm binding site (AATTTTTGGAG), thus incorporating the modified snRNA into the snRNP complex targeting the spliceosome34. For a number of constructs, the sequence, containing heterogeneous ribonucleoprotein A1 (hnRNPA1) binding sites (ATGATAGGGACTTAGGGTG) was added at the 5′ end of the coding sequence to improve the efficiency of splicing inhibition35. The 18 bp sequence, which is complementary to the histone pre-mRNA (AAGTGTTACAGCTCTTTT) is replaced by various antisense sequences, targeting the studied pre-mRNA region. Sequences of primers used for creating modified U7-snRNA.

Identification of splicing regulatory motifs was performed with HExoSplice36 (http://bioinfo.univ-rouen.fr/HExoSplice_submit/inputs.php, accessed on 13th Jun 2022). Scores of splice sites were calculated by MaxEntScan (http://hollywood.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html, accessed on 13th Jun 2022).

Plasmids with modified U7-snRNA genes were co-transfected with minigenes (250 ng of both plasmids in 24-well plate cell) into HEK293T cells (ATCC CRL-3216™) using Lipofectamine 3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA). After 48 h, cells were harvested for RNA isolation and reverse transcription. Minigene-specific primers with 6-FAM modification located in the exons V1 and V2 were used to amplify the splicing products, which were further visualized by polyacrylamide gel electrophoresis and quantitatively analyzed by fragment analysis. Fragment analysis was performed using ABI PRISM 3500xL Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) and Coffalyser.Net software v.220513.1739 (https://support.mrcholland.com/downloads/coffalyser-net, accessed on 13th Jun 2022).

Sequences of primers used in this study are listed in Supplementary Note 3.

Further information on research design is available in the Nature Research Reporting Summary linked to this article.