Biallelic SORD pathogenic variants cause Chinese patients with distal hereditary motor neuropathy

Sorbitol dehydrogenase gene (SORD) has been identified as a novel causative gene of recessive forms of hereditary neuropathy, including Charcot–Marie–Tooth disease type 2 and distal hereditary motor neuropathy (dHMN). Our findings reveal two novel variants (c.404 A > G and c.908 + 1 G > C) and one known variant (c.757delG) within SORD in four Chinese dHMN families. Ex vivo cDNA polymerase chain reaction confirmed that c.908 + 1 G > C variant was associated with impaired splicing of the SORD transcript. In vitro cell functional studies showed that c.404 A > G variant resulted in aggregate formation of SORD and low protein solubility, confirming the pathogenicity of SORD variants. We have provided more evidence to establish SORD as a causative gene for dHMN.

Sorbitol dehydrogenase gene (SORD) encodes a 357 amino acid protein and the protein acts as a key enzyme in the polyol pathway. Very recently, variants in SORD have been identified as a novel cause, and also the most common form in individuals affected by CMT2 or dHMN 6 . Totally, 45 affected individuals from 38 families, including 23 CMT2 (3 from China), 18 dHMN, and 4 intermediate CMT patients were identified to carry homozygous or heterozygous c.757delG (p.Ala253GlnfsTer27) variant in SORD.
Here, we report the identification of two novel variants (c.404 A > G and c.908 + 1 G > C) and one known variant (c.757delG) in SORD in four Chinese dHMN families. This report further establishes SORD as a disease-causing gene for dHMN, and expands both the phenotypic and mutational spectra of the SORD-associated hereditary neuropathy.
To investigate the underlying causative gene in Chinese CMT2 and dHMN, we have collected a cohort of 20 CMT2 and 9 dHMN patients without known genetic cause. Whole-exome sequencing (WES) was conducted on all the patients. After analyzing and filtering, we found three dHMN patients from family 1, 2, and 3 carrying the known homozygous c.757delG (p.Ala253GlnfsTer27) variant in SORD. Furthermore, two novel variants were detected in the proband (dHMN) of family 4, including a missense variant c.404 A > G (p.His135Arg) and a splicing variant c.908 + 1 G > C. The results of Sanger sequencing confirmed the presence of the identified variants in corresponding individuals and showed SORD variants co-segregating with the disease in four families, respectively ( Fig. 1a, b). They were absent in several general population frequency databases, such as the 1000 Genomes Project, Exome Aggregation Consortium (ExAC), and the Genome Aggregation Database (gnomAD). To ascertain the pathogenicity of these two novel variants, we analyzed them using several bioinformatic algorithms. The p.His135Arg was predicted to be damaging by SIFT (score 0.001), disease-causing by MutationTaster (score 1.0), probably damaging by PolyPhen-2 (score 1.0), and damaging by CADD (score 24.7). The c.908 + 1 G > C variant was located within the highly conserved donor splice site of exon 8 and predicted to cause abnormal splicing by abolishing the donor splice site of exon 8 (dbscSNV ada-score = 0.9999, rf-score = 0.934).
To confirm whether the c.908 + 1 G > C variant affects splicing, total RNA was isolated from patient's peripheral blood, and reverse transcribed into cDNA and amplified. Agarose gel electrophoresis showed a normal-sized band and a shorter band in the proband (II-2) of family 4. TA clone and Sanger sequencing of cDNA polymerase chain reaction (PCR) products revealed two abnormal spliced SORD transcripts, an amplicon with four nucleotides inserted prior to exon 9 due to the utilization of a cryptic intronic donor splice site (p.Trp304*), and an amplicon skipping of 35 base pairs at the 3′ end of exon 8 that caused premature stop codon p.Asp293AlafsTer21 (Fig. 1c).
To explore the influence of the variant c.757delG (p.Ala253Glnf-sTer27) on the function of SORD gene, we used quantitative PCR to examine SORD mRNA levels in peripheral blood from the proband of family 2 (II-1), his father (I-2), and the healthy control. Compared to the healthy control, the patient carrying a homozygous c.757delG (p.Ala253GlnfsTer27) variant showed a significant reduction in SORD mRNA expression levels of~70-80% when normalized to the control gene GAPDH (p < 0.001). His father carrying a heterozygous c.757delG (p.Ala253GlnfsTer27) variant exhibited a milder decrease of~45-50% in mRNA levels (p < 0.01; Fig. 1d). It suggested that mRNAs of patients with the c.757delG (p.Ala253GlnfsTer27) variant were unstable on account of the nonsense-mediated mRNA decay (NMD).
We used fibroblast of patient 4 with compound c.908 + 1 G > C/p.His135Arg variants as a model to assess the effects of two variants on SORD protein expression. Western blot showed that SORD protein expression was significantly decreased compared to two healthy controls ( Fig. 1 e, f). A previous study has demonstrated that residues 90-110 and 120-140 in SORD subunit are important for the stability and binding of tetramer 7 . To define whether the variant p.His135Arg and two previously reported missense variants (p.Arg110Pro and p.Ala153Asp) flanking it altered the tetramer-forming interactions, five plasmids expressing H.-L. Dong et al. wild type (WT), R110P, H135R, A153D, or A253Qfs*27 protein were constructed. Immunofluorescence experiment in HeLa cells transiently expressing WT or mutant SORD showed that WT SORD displayed diffuse intracellular staining, whereas mutant SORD displayed granules throughout the cytosol (Fig. 1g). To confirm the presence of SORD aggregates, we investigated the soluble and insoluble SORD fractions by western blot. The levels of mutant SORD protein were markedly reduced in the soluble fractions of transfected cells with an increase in the insoluble fractions ( Fig. 1h-j). Altogether, these studies provided evidence that p.His135Arg caused aggregate forming of SORD due to probable conformational changes in tetrameric state, which indicated the variant was pathogenic.
Altogether, we identified 4 unrelated dHMN patients harboring biallelic SORD pathogenic variants. The detailed clinical features and nerve conduction studies of these patients were shown in Table 1. All four cases were sporadic and only one (Family 1: II-1) was born of a consanguineous marriage. The average age of disease onset was 12.5 ± 3.5 years (range 9-16). All affected individuals complained about muscle weakness in their lower limbs during their teenage years, thus having difficulty in walking, running, and squatting down. The disease presented slowly progressive in all the probands. The distal muscle atrophy began in their feet and legs, followed by intrinsic muscles of the hands. As a consequence, they were not able to make fine and complicated movements using their hands. On electrophysiological examination, the nerve conduction studies showed lengthdependent axonal motor neuropathy with marked decrease in the amplitudes of compound muscle action potentials and mild reduce in the motor nerve conduction velocities in lower limb nerves. The sensory nerve conduction velocities and sensory nerve action potentials were in the normal range.
The SORD catalyzes the interconversion between glucose and fructose via sorbitol together with aldose reductase. The polyol pathway is an important alternate route for sugar metabolism. Therefore, it is believed that SORD is involved in the development of diabetic neuropathy 8,9 . To date, eight pathogenic variants in SORD have been identified in CMT2 or dHMN. The variant c.757delG (p.Ala253GlnfsTer27) is the most common cause with a frequency of up to~10% in undiagnosed CMT2 or dHMN cases 6 . Similarly, the most common pathogenic variant (c.757delG, p. Ala253GlnfsTer27) was identified in three dHMN families in our study, which occupied 33.3% (3/9) of genetically undefined dHMN cases. In addition, the patient carrying a homozygous c.757delG (p.Ala253GlnfsTer27) variant showed a dramatic reduction in SORD mRNA levels. This result was consistent with the finding of the protein expression level in the patient with the c.757delG (p. Ala253GlnfsTer27) variant 6 , indicating a NMD mechanism and loss of function of SORD gene. The absence of SORD protein and an increased level of intracellular sorbitol were found in patientderived fibroblasts 6 . Furthermore, synaptic degeneration and progressive motor impairment appeared in Drosophila with loss of SORD orthologs 6 .
In conclusion, we identified SORD pathogenic variants in four dHMN families, further establishing SORD as a disease-causing gene for dHMN. Our findings provide different mutation types of SORD, expanding the spectrum of pathogenic variants in SORD and highlighting the importance of screening SORD variants among undefined hereditary neuropathy patients.

METHODS Subjects
A cohort of 20 CMT2, 9 dHMN patients, and available unaffected relatives were enrolled consecutively in this study between June 4, 2008 and October 6, 2019 from southeastern China. They have been excluded other inherited peripheral neuropathies by genetic screening of the known causative genes in our previous reports [10][11][12][13] . Each patient received clinical evaluation and diagnosis from at least two senior neurologists, according to clinical features and electrophysiological studies described in our previous reports 11,14 . In addition, the criteria for the enrollment were as follows: (1) autosomal recessive or sporadic; (2) slowly progressive weakness and atrophy of the distal limb muscles; and (3) electrophysiological results showed preserved or only mildly slowed nerve conduction velocities (>38 m/s) and showed primarily axonal damage (CMT2) or a pure motor axonal neuropathy without sensory involvement (dHMN). Written informed consents were obtained from all participants. The study was approved by the ethics board of Second Affiliated Hospital, Zhejiang University School of Medicine, and Huashan Hospital of Fudan University.

Sanger sequencing of SORD and SORD2P
Exonic and intronic areas of SORD and exon 7 of SORD2P were amplified by PCR followed using the primers described previously 6 . Sanger sequencing was performed to validate the candidate variants analyzed by WES and cosegregation of the pedigrees among all available family members.
RNA isolated, splicing analysis, and quantitative PCR Total RNA was isolated from peripheral blood treated with RNAiso Plus (Takara, Japan). RNA was reverse transcribed into cDNA using the PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, Japan). For the analysis of the splicing variant, PCR products of cDNA were visualized in Fig. 1 Genetic and functional findings in dHMN patients with SORD variants. a Pedigrees of four Chinese dHMN families carrying SORD variants. Circle: females; square: males; open symbol: unaffected; filled symbol: affected; arrow: proband of each family. b Sequencing chromatograms of three variants within SORD. The lower chromatogram represents the variant and the upper one represents the normal sequence. c SORD cDNA products were separated by agarose gel electrophoresis and confirmed by Sanger sequencing. Lane 1: marker; lane 2: control; lane 3: subject II-2 (family 4). d Relative SORD mRNA expression levels from peripheral blood in subjects with a heterozygous or homozygous c.757delG (p.Ala253GlnfsTer27) compared with the healthy controls (n = 3 experimental repeats, data are shown as mean ± SD. **p < 0.01; ***p < 0.001). e Fibroblast SORD protein expression levels in two normal controls and patient 4 with compound c.908 + 1 G > C/p. His135Arg variants. f The graph shows β-tubulin normalized SORD expression levels (n = 3 biological repeats, data are shown as mean ± SD. ****p < 0.0001). g Immunofluorescence analysis of HeLa cells expressing WT or mutant SORD. Cells were stained by mouse anti-Myc antibody followed by anti-mouse Alexa Fluor 488 (green)-conjugated secondary antibody and DAPI (blue). Scale bars = 10 μm. h Soluble (S) and insoluble (P) protein fractions per variant with western blot probing for Myc-SORD and β-Tubulin (loading control). The graphs show β-tubulin normalized Myc-SORD expression levels per variant in soluble protein fractions (i) and insoluble protein fractions (j) (n = 3 biological repeats, data are shown as mean ± SD. *p < 0.05; ***p < 0.001; ****p < 0.0001). All blots were derived from the same experiment and were processed in parallel.

Cell culture and plasmids transfection
Primary fibroblasts were obtained from skin biopsy of patient 4 and two normal controls and grown in Dulbecco's Modified Eagle Medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) at 37°C in 5% CO 2 . HEK-293T and Hela cell are from CBTCCCAS (The Cell Bank of Type Culture Collection of Chinese Academy of Sciences). For immunofluorescence analysis, Hela cells were cultured in glass-bottomed 24-well plates and transfected with 500 ng of plasmid DNA, using Lipofectamine 3000 reagent (Invitrogen, USA) for 24 h. For protein expression analysis, HEK-293T cells were cultured in 6 cm dishes and transfected with 4000 ng of plasmid DNA, using Lipofectamine 3000 reagent for 48 h.
Protein solubility assay, SDS-PAGE, and immunoblotting Forty-eight hours after transfection, transfected cells were lysed in the mild lysis buffer (CelLytic TM M, Sigma, USA). Lysates were centrifuged for 15 min

Statistical analyses
All the variables were presented as mean ± s.d. The data were compared with a two-tailed Student's t test or a one-way ANOVA using GraphPad Prism (GraphPad Software Inc.). P values <0.05 were considered significant.

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

DATA AVAILABILITY
The data that support the findings of this study are available from the corresponding author upon reasonable request. Whole-exome sequencing data for the patient are deposited in the Sequence Read Archive (SRA) under accession code number PRJNA672732.