The diagnosis was based on clinical data as well as reduced GCase activity and molecular analysis. This study conformed to human research guidelines stated in the Declaration of Helsinki and written informed consent was obtained from all subjects. The female patient was a 62-year-old who first visited Shaoxing People's Hospital because of massive splenomegaly and thrombocytopenia with no neurological impairment.

The three-dimensional (3D) protein structures of the wild-type and mutant GCase were predicted using I-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER) and Pymol. ClustalX was used to analyze evolutionary conservation among different paralogs and orthologs.

DNA was extracted from fresh blood using a standard phenol-chloroform extraction method after digestion with proteinase K to ensure that the DNA quality and quantity met the experimental requirements. Specific primers were used to amplify the 8–11 exon of GBA1 gene. The forward and reverse sequences were as follows: forward primer (5′-TGTGTGCAAGGTCCAGGATCAG-3′), reverse primer (5′-ACCACCTAGAGGGGAAAGTG-3′). Sanger sequencing was performed by Sangon Biotech (Shanghai, China). The sequences obtained were aligned to the available reference sequence in The National Centre for Biotechnology Information GeneBank database to detect variants.

The length of the GBA1 cDNA is approximately 1.6 kb. Total RNA from peripheral blood lymphocytes of healthy family members was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, United States) and was reverse transcribed into first-strand cDNA with the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Jiangsu, China) according to the manufacturer's instructions. The wild-type GBA1 cDNA was cloned using primers containing the XhoI and EcoRI restriction sites. The forward and reverse sequences were as follows: forward primer (5′-CCGCTCGAGAGATGGAGTTTTCAAG-3′),reverse primer (5′-GGAATTCCACTGGCGACGCCACAG-3′). To clone the verified PCR products into the pEGFP-N1 vector by double digestion with XhoI and EcoRI. GBA1 mutations were introduced into the wild-type plasmid by PCR-based site-directed mutagenesis to obtain the pEGFP-N1-GBA1-MUT vector (c.1240G > C:forward primer: 5′-AACCTCCTGTACCATCTGGTCGGCTGGACC-3′; reverse primer: 5′-GGTCCAGCCGACCAGATGGTACAGGAGGTT-3′; c.1342G > C: forward primer: 5′-GTAGACATCACC AAGCACACGTTTTACAAA-3′; reverse primer: 5′-TTTGTAAAACGTGTGCTTGGTGATGTCTAC-3′) with the Phanta Max Super-Fidelity DNA polymerase and confirmed by sequencing. The plasmids were extracted with the endotoxin-free plasmid mini-extraction kit (Tiangen, Beijing, China).

HEK 293 T cells were grown in Dulbecco's Modified Eagle Medium (Gibco, NY, United States) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin, and 100 U/ml streptomycin at 37 °C in a humidified atmosphere enriched with 5% CO₂. Cell transfection was performed according to the manufacturer's instructions using Lipofectamine 3,000 transfection reagent (Invitrogen) for the pEGFP-N1 vector, and the GBA1 wild-type (WT) and mutant-type (MT), respectively. After 48 h transfection, cells were collected for GCase enzyme assays and Western blotting. All transfections were performed in duplicate in three individual experiments.

The cells were grown on Petri dishes and fixed with cold 4% formaldehyde for 30 min at −20 °C and blocked in 0.2% Triton X-100 for 5 min at room temperature. The fixed cells were separately stained with DAPI to visualize the nuclear DNA. Fluorescence microscopy was carried out with an LSM880 confocal microscope equipped with appropriate filter sets.

When HEK 293 T cells reached 70% confluence in 6-well plates, 2.5 µg pEGFP-N1-GBA1-WT and MT were transfected into the cells using Lipofectamine 3,000 transfection reagent (Invitrogen). The cells were collected by centrifugation and washed with pre-chilled PBS after 48 h transfection, then 500 µl PBS was added to resuspend the cells, and the total cellular protein was extracted by repeated freezing and thawing. GCase concentrations were detected using the Human Glucosylceramidase (GCase) ELISA Kit (CUSABIO).

When HEK 293 T cells reached 50% confluence in 6-well plates, 2.5 µg pEGFP-N1-GBA1-WT and MT were transfected into the cells using Lipofectamine 3,000 transfection reagent (Invitrogen). 48 h after transfection, cells were collected and washed with cold PBS (Sigma–Aldrich), and the total cell protein was extracted by repeated freezing and thawing before resuspension of the cells in 150 µl PBS containing 1% PMSF. Cell debris was removed by centrifugation, and cell lysates were quantified using the Pierce™ Rapid Gold BCA Protein Assay Kit (Thermo Fisher Scientific). 15 μg protein was mixed with 20 ul 4-methylumbelliferyl-β-glucosidase substrate, incubated at 37 °C for 2 h, and 200 ul glycine buffer was added to terminate the reaction. Sample 4-methylumbelliferone fluorescence intensity was measured using a microplate reader at an excitation wavelength of 355 nm and an emission wavelength of 460 nm.

Total RNA was extracted with TRIzol reagent (Invitrogen) from the transfected cells. RT-PCR was performed using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). GBA1 mRNA expression levels were analyzed using the ChamQ SYBR qPCR Master Mix (Vazyme) with the specific primers as follows: forward primer5′-TCCCAAGCCTTTGAGTAGGG-3′, reverse primer 5′-GCCGAAGCTTTTAGGGATGC-3′. Gene expression levels were calculated using the 2^−ΔΔCT method. Expression levels of the target gene was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with the specific primers: forward primer 5′-GTGAAGGTCGGAGTCAACG-3′, reverse primer 5′-TGAGGTCAATGAAGGGGTC-3′.

The transfected cells were lysed using cell lysis buffer for Western blot (Beyotime, Beijing, China) containing 1% PMSF. Protein loading buffer was added to the cell lysates before boiling for 5 min. Protein (25 µg) was loaded for electrophoresis on 10% SDS-PAGE gels and transferred to PVDF membranes. Then, the membranes were blocked with 5% non-fat milk in TBS containing 0.1% Tween 20 for 2 h at room temperature and incubated with antibodies against EGFP (1:2,000, Proteintech, Wuhan, China) at 4 °C overnight. Finally, the membranes were incubated with a secondary goat anti-mouse IgG (1:5,000) for 2 h at room temperature. The signals were detected using the ECL Western blotting detection reagent.

The results are presented as means ± standard deviation of at least three independent biological replicates. At least three technical replicates were used per experiment. The differences between the WT and MT enzyme activities were analyzed using a t-test with GraphPad Prism 5 (GraphPad Software Inc., CA, United States). A p-value of ≤0.05 was considered statistically significant.

The patient was diagnosed with Gaucher disease type I. She was conscious and had no anemic appearance and no ecchymosis or petechiae on the skin of limbs (Figure 1). There was no significant superficial lymphadenopathy. Her breath sounds were clear, without dry or wet sounds. The heart rate was 65 beats/min and was rhythmical. The abdomen was flat and soft, without pressure pain or rebound pain. There was no percussion pain in either kidney and no swelling in the lower limbs. The neurological examination was normal. Routine blood tests showed normal hemoglobin and mild thrombocytopenia. The GCase activity was lower than the reference value. The pathological results showed that the proportion of hematopoietic tissue and fat in the bone and bone marrow was essentially normal. The proportion of granulocytes and erythrocytes in the hematopoietic tissue was approximately normal, and megakaryocytes were occasionally seen. The bone density was generally decreased in the lower and middle femur, knee, pelvis, and both hips. Hyperosteogeny was seen in the knee and both hips. There were no other skeletal abnormalities, fractures, pain and bruising. The physical examination results of the patient are shown in Table 1.

Guangzhou Women's and Children's Hospital (Guangzhou, China) had previously performed sanger sequencing for 11 exons and the exon-intron junction region of the GBA1 gene. DNA sequencing revealed a mutation c.1240G > C (p.Val414Leu) and a known pathogenic mutation c.1342G > C (p. Asp448His) (Figure 2A). The mutations were predicted to alter both the secondary structure and the three-dimensional structures of GCase. In addition, the three-dimensional structures of the WT and MT proteins were visually distinct in both the overall and partial views. The c.1240G > C mutation might affect the catalytic function by disturbing the glycosylation process, and the substitution of the bulky phenylalanine with a small leucine residue might bring in a larger side chain that could form steric effects in the catalytic region (Figure 2).

Evolutionary conservation analysis of multiple sequence alignments of the GCase protein and its homologs showed that the amino acid at residues 414 and 448 are evolutionarily conserved (Figure 3A), suggesting that these amino acids may play an important role in the function of GCase. We verified the constructed plasmids with sequencing, and the results are shown in Figure 3B.

To further study the functional consequences of both GBA1 mutation in vitro, we examined the expression of GBA1 using qPCR and Western blotting. Quantitative detection revealed that the WT GBA1 was more highly expressed than the MT at both the mRNA and protein levels (p < 0.05) (Figures 4A–C).

The subcellular localization analysis showed that both the MT and WT were localized at the cytoplasm, suggesting there is no difference in subcellular localization (Figure 4D). We also detected the GCase concentration, and the results showed that there was a significant difference between the WT and the MT with the V414L mutation, or that with V414L + D448H. However, no significant difference was found between the WT and D448H (Figure 4E). The results of GCase enzyme activity assay showed that there also was a significant difference between the WT and the MT with the V414L mutation, or that with the V414L + D448H mutation (Figure 4F).