case_images: PMC10644772_01
case_images: Clinical data of the family with HSP. (A) Brain MRI of the proband. The spinal cord MRI scans showed the (B) cervical spinal cord, (C) thoracic spinal cord, and (D) lumbar spinal cord of the proband. (E) The EEG of the proband. Red lines represent the 3-7 Hz medium-high amplitude theta rhythm. (F) Major events in the medical record of the proband. (G) Genealogy of this family having HSP. Squares and circles indicate male and female, respectively. Filled symbols indicate affected individuals. Black indicates an affected individual observed by clinical and molecular examination. Gray indicates an individual suspected of having spastic paraplegia, but that was not examined clinically and molecularly. Open symbols indicate unaffected individuals. Slashes indicate the individuals who are deceased. The arrow indicates the proband. "A/G" indicates the individuals who carried the heterozygous splice variant (c.712-2A>G) in the WASHC5 gene. "A/A" indicates the individuals without the c.712-2A>G variant. (H) Schematic representation of the filtering strategies employed in the WES analysis. (I) Sanger sequencing showed the c.712-2A>G variant in the WASHC5 gene (left) and the characterization of the anomalous splicing induced by the c.712-2A>G variant (right). (J) Agarose gel electrophoresis showed the PCR amplification of the WASHC5 cDNA of the region from exon 6 to exon 7. The upper band corresponds to the mutant transcript. Lane 1: DNA size markers; lane 2: healthy family member (II-1) without the variant; and lane 3: the proband with the heterozygous c.712-2A>G variant. (K) Schematic representation of the anomalous mRNA splicing caused by the c.712-2A>G variant.
case_images: fgene-14-1205052-g001.jpg
case_images: PMC10644772_01_fgene-14-1205052-g001.jpg
case_images: F1
case_images: The brain magnetic resonance imaging (MRI) of the proband showed mild brain atrophy (Figure 1A). The spinal cord MRI scans were normal (Figures 1B-D). The electroencephalogram (EEG) showed mild abnormalities (Figure 1E). The major events as per medical records during 2018-2022 have been shown in Figure 1F. The proband was initially clinically diagnosed with spastic paraplegia. Other family members showed no obvious abnormalities (Figure 1G). All variants detected in the proband were initially screened and further filtered based on a gene panel of movement disorders, as outlined in Figure 1H; Supplementary Table S5. After analysis, a heterozygous splice variant (chr8:126088744T>C/NM_014846.4: c.712-2A>G) in WASHC5 was identified in the proband (Figure 1I). Co-segregation analysis showed that the c.712-2A>G variant was not present in the healthy members (II-1, III-1, III-2, and IV-1) in this family (Figure 1G). Consequently, the diagnosis of the proband was determined to be SPG8 type. In contrast, relatives without the c.712-2A>G variant only had a single PCR product of the expected size (307 nucleotides) (Figure 1J). The retention of 30 nucleotides leads to the insertion of 10 aberrant amino acids (LSRGVVLTLR) at position 237 of the WASHC5 protein, thus resulting in an abnormally prolonged protein (1,169 aa) (Figures 1I, K).
case_images: Bioinformatics analysis of the identified WASHC5 splice-altering variant. (A) Structure prediction of the wildtype WASHC5 (WASHC5-WT) protein structure and the c.712-2A>G mutant WASHC5 (WASHC5-MUT) protein structure. (B) Conservation analysis of the related amino acid region was predicted by ConSurf Server software. (C) The affected amino acid region was predicted as "slight intolerant" by MetaDome server software. Real-time PCR analysis of the proband (II-3) and controls (II-1 and III-2). The relative mRNA expression levels of the genes encoding members of the WASH complex (D) VCP, ARPC2, and ARPC3 (E) in peripheral blood lymphocytes (PBLs) are depicted. mRNA abundance of endosomal-related genes (F) and lysosome-related genes (G) in PBL. The expression levels of each gene in the proband were set to "1". The experiment was repeated more than three times. Data are presented as mean +- standard error of mean (SEM). Two groups were compared using the unpaired Student's t-test. A p-value <0.05 was considered statistically significant. *p < 0.05, the healthy family member (III-2) vs. the proband; #p < 0.05, the healthy family member (II-1) vs. the proband.
case_images: fgene-14-1205052-g002.jpg
case_images: PMC10644772_01_fgene-14-1205052-g002.jpg
case_images: F2
case_images: SWISS-MODEL software (https://swissmodel.expasy.org/) showed that the splice variant may lead to an insertion of 10 amino acids behind Q237 in the mutated WASHC5 protein (Figure 2A). In addition, ConSurf Server software (http://consurf.tau.ac.il/) predicted that the 10 amino acids were inserted in the conserved region of the WASHC5 protein (Figure 2B). Furthermore, MetaDome software (https://stuart.radboudumc.nl/metadome/dashboard) indicated that the affected residues are located in the slightly intolerant region of the WASHC5 protein (Figure 2C). We set the mRNA levels of each gene in the proband (II-3) as "1." qPCR analysis indicated that the expression of WASH complex member-related genes in the proband, including WASHC1 and WASHC3, was significantly decreased when compared with the healthy family members (Figure 2D). The results showed that there was a significant change in the proband's ARPC3 gene expression (Figure 2E). In addition, the transcriptional analysis revealed that the expression of endosomal-related genes (RAB5A, RAB5C, RAB11A, and RAB11B) and lysosome-related genes (LAPTM4B and LAPTM5) was significantly upregulated in the proband when compared to the healthy family members (Figures 2F, G).