Enzymology and genetic analysis results of the proband and the family members
The patient’s GCase activity of peripheral blood leukocytes was 3.8 nmol/(mg·h) (reference range: 10-25 nmol/[mg·h]). GBA1 gene test showed heterozygous mutations of c.1448T>C (p.L483P)/c.928A>G (p.S310G) (Fig. 2&3). The diagnosis was GD (type 1). The father was a p.L483P heterozygote carrier, and the mother a p.S310G heterozygote carrier. The younger sister did not carry either mutation. Predictions made by PolyPhen-2, SIFT and Mutation Taster suggested that the p.L483P and p.S310G mutations might be pathogenic. According to ACMG guideline, the p.L483P was evaluated as pathogenic variant, and the p.S310G was probably pathogenic variant.
Fig.2 Family pedigree of the proband. The first-generation members have no information. The second-generation is the father of proband is heterozygous for exon 10 c.1448T>C (p.L483P) mutation, and the mother is heterozygous for exon 7 c.928A>G (p.S310G) mutation. The third generation is the proband who was compound heterozygous for p.L483P/p.S310G . Her sister was normal and did not carry either mutation.
Fig.3 DNA sequencing analysis of GBA1 gene. a exon 7 c.928A>G (p.S310G) novel heterozygous missense mutation compared to b the corresponding wild-type sequence. c exon 10 c.1448T>C (p.L483P) heterozygous mutation, compared to d the corresponding wild type sequence.
Protein structure analysis
The GCase protein belongs to the GH30 family of glycoside hydrolases. Different from the traditional three-domain classification method, domain I and domain II were reclassified as domain 2, and the previous domain III was classified as domain 1[12,13].The GCase protein contains two domains, in which the active sites E274 and E379 are located in the β -folded plates β4 and β7 of Domain 1. The mutation point p.S310G was located at α-helix α4. Although it appeared to be closer to E274 in the secondary structure. In the three-dimensional structure, p.S310G was actually located at the bottom of α4 and E274 at the top of β4. The distance between the two was relatively far. The mutation point p.L483P was located at the β pleated-sheet βs6 in Domain 2, which was far away from the two active centers. Therefore, being far away from the GCase active centers, the mutation points p.S310G and p.L483P were predicted not to have a significant impact on the catalytic properties of the protein.
The p.S310G mutation was located at α-helix α4. In wild type, the C-β atom of the side chain of Ser residue formed the van der Waals force with the C-γ2 atom of the Val253 side chain and the C atom of the Leu307 main chain. In addition, the N atom of the Ser residue main chain formed a hydrogen bond with a length of 3.5 Å and 3.2 Å with the O atom of the Thr306 and Leu307 main chains, respectively; the O atom of the main chain formed a hydrogen bond with a length of 3.3 Å with the N atom of the His313 main chain. After Ser310 mutated into Gly310, the hydrogen bond of the main chain remained, while the van der Waals force formed by the side chain and surrounding residues disappeared. The p.S310G mutation weakened the force between this point and Val253, which was located above the α helix α3, therefore might affect the stability between α2 and α3(Fig. 4).
Fig.4 Molecular contacts of residue 310. a wild type GCase protein. b Mutant Type.
p.L483P was located in the loop region connecting βs5 and βs6 (near βs6). In wild type, the side chain of Leu483 formed abundant van der Waals forces with the surrounding residues. The C-δ1 atom of its side chain could form van der Waals forces with C-δ2 atom of Leu104, C-γ1 atom of Val499, C-γ1 atom of Val507, and C-δ2 atom of Leu509, and the C-δ2 atom could form van der Waals forces with C-γ atom of Asn501 and C-β atom of Ser523. Those van der Waals forces were formed due to the interactions between the side chains. When Leu483 mutated into Pro483, the N-Cα rotation of Pro was bound by the pyrrolidine ring in its structure, thereby having less conformational freedom. This structure limited the diversity of its spatial conformation, especially in the loop region. The existence of Proline could help to stabilize the loop region which was originally more flexible. Therefore, theoretically, the p.L483P mutation located in the loop region, should have enhanced the stability of this region. But due to side chain changes, the original 6 van der Waals forces were reduced to 3, and the retained van der Waals forces were formed between C-γ of Pro483 and C-δ2 of Leu104 and C-γ1 of Val507, and one between C-β and C-γ1 of Val499. Thus，it can be seen that the introduction of Pro stabilized the conformation of the loop region, but the reduced van der Waals forces of the side chain might also affect the function of the protein structure (Fig. 5).
Fig.5 Molecular contacts of residue 483.a wild type GCase protein. b Mutant Type.