Participants and ethical statement
The proband patient was admitted to Xin Hua Hospital in 2018 with proteinuria two years. Written informed consent was obtained from the involved subjects and all procedures were performed in accordance with the Declaration of Helsinki. The study was performed under the approval of the Ethics Committee from Xin Hua Hospital, Shanghai Jiao Tong University School of Medicine (approval No.XHEC-D-2020-002).
DNA and RNA isolation
Blood samples were collected from the patient, his family members and normal controls using test tubes containing EDTA anticoagulant. Genomic DNA was extracted from PBMCs using DNA Midi Kit (Qiagen, Milano, Italy) according to the manufacturer’s protocol. Total RNA in PBMCs was collected in Tempus™ blood RNA tubes (Biotek, Canada) and isolated with Tempus™ Spin RNA Isolation kit (Biotek, Canada) according to the manufacturer’s protocol.
Genomic DNA sequencing analysis
The full length genomic DNA of GLA were amplified by nested polymerase chain reaction (PCR). Primers are designed according to the genomic GLA reference sequence (NCBI reference sequence NM_000169) listed in Supplement Table 1. Sanger sequencing was performed to verify the sequences.
Whole exome sequencing (WES)
Whole-exome sequencing was performed on the patient as described [16] , and the results were analyzed to verify the absence of mutations in genes associated with steroid-resistant nephrotic syndrome or focal segmental glomerulosclerosis [17]. The non-neutral variant p.Arg229Gln in the NPHS2 gene and the high-risk alleles G1 and G2 in the APOL1 gene were manually inspected. The entire exome was analyzed for variations in GLA copy number as follows. First, the coverage of each GLA exon was normalized by the average sequencing depth of the sample. Next, the normalized coverage data were compared with our in-house database of sequencing data of a healthy population, and then the copy numbers of each exon were estimated.
Reverse transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR (qRT-PCR)
To evaluate the range of transcripts produced from the GLA gene, total RNA was extracted from PBMCs from the patient, his mother, and healthy controls as described above. First-strand cDNA synthesis was performed using the HifairTM 1st Strand cDNA Synthesis SuperMix Kit for qPCR (Yeasen, Shanghai, China). Then PCR was performed using the PrimerSTAR MAX DNA Polymerase (TaKaRa, Beijing, China), and amplicons were visualized on 1.5% agarose gels and sequenced on an ABI PRISM 3100 Genetic Analyzer. Primer sequences were listed in Supplement Table 1.
To quantify the effect of IVS4+1326C>T on the expression of GLA mRNA level in PBMCs, qRT-PCR were performed using 2 pairs of cross-exon primers (listed in Supplement Table 1) and Green Master Mix (Yeasen, Shanghai, China) to specifically amplify the full-length transcript of GLA, and qRT-PCR was normalized to that of GAPDH. Data was analyzed using software Geneious version 10.2.3 (Biomatters, Auckland, New Zealand).
Plasmid construction
Full-length human GLA cDNA was subcloned into the PHAGE vector at the BamHI and SalI sites, upstream from a hemagglutinin (HA) tag. The resulting PHAGE-GLA plasmid was then subjected to multiple PCR using mutated primers (listed in Supplement Table 1) and PrimerSTAR MAX DNA Polymerase (TaKaRa Bio, Beijing, China) to give rise to mutant PHAGE-GLA plasmids encoding aberrantly spliced transcripts of GLA. All constructs were verified by sequencing.
Cell culture and transfection
HEK293T cells were maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin at 37℃ with 5% CO2. HEK293T cells were transfected with the wild-type PHAGE-GLA plasmid or mutant PHAGE-GLA plasmids using Lipofectamine 2000 (Invitrogen, California, USA) according to the manufacturer’s instructions. Transfected cells were incubated for 24-48 hours post transfection and proteins were analyzed by Western blot analysis.
Western blot analysis
Cell lysates were prepared using Cell Lysis Buffer (Beyotime, Shanghai, China). Protein (10-40 µg) was fractionated on 8-12.5% SDS-polyacrylamide gels and transferred to 0.22-µm polyvinylidene fluoride membranes. After 1 hour of incubation at room temperature in 5% milk, the membrane was incubated with the primary antibody for 2 hours followed by incubation with the appropriate secondary antibodies for 2 hours. Antibody binding was detected using the ECL Plus Western Blotting System (GE, Buckinghamshire, UK). GAPDH was detected as an internal control. The primary antibodies were mouse monoclonal anti-HA antibody (1:2000, Cell Signal Technology, Massachusetts, USA) and rabbit polyclonal anti-GAPDH antibody (1:1000, Cell Signal Technology, Massachusetts, USA).
α-galactosidase (α-GalA) enzyme assay
Activities of α-GalA in cell lysates of HEK293T cells transfected with the wild-type or mutant PHAGE-GLA plasmids were measured using the Micro α-Galactosidase Assay Kit (Solarbio, Beijing, China) according to the manufacturer’s instructions.
Statistical analysis
Continuous data that were normally distributed were expressed as mean ± standard deviation. Intergroup differences were assessed for significance using ANOVA with Bonferroni-Holm post-test correction or Student’s t test, as appropriate. Differences associated with P < 0.05 were considered statistically significant.