Biallelic mutations in SORD cause a common and potentially treatable hereditary neuropathy with implications for diabetes

Abstract

Here we report biallelic mutations in the sorbitol dehydrogenase gene (SORD) as the most frequent recessive form of hereditary neuropathy. We identified 45 individuals from 38 families across multiple ancestries carrying the nonsense c.757delG (p.Ala253GlnfsTer27) variant in SORD, in either a homozygous or compound heterozygous state. SORD is an enzyme that converts sorbitol into fructose in the two-step polyol pathway previously implicated in diabetic neuropathy. In patient-derived fibroblasts, we found a complete loss of SORD protein and increased intracellular sorbitol. Furthermore, the serum fasting sorbitol levels in patients were dramatically increased. In Drosophila, loss of SORD orthologs caused synaptic degeneration and progressive motor impairment. Reducing the polyol influx by treatment with aldose reductase inhibitors normalized intracellular sorbitol levels in patient-derived fibroblasts and in Drosophila, and also dramatically ameliorated motor and eye phenotypes. Together, these findings establish a novel and potentially treatable cause of neuropathy and may contribute to a better understanding of the pathophysiology of diabetes.

Fig. 1.. Biallelic mutations in SORD cause autosomal recessive dHMN/CMT2

A) Representative pedigrees of dHMN/CMT2 families carrying biallelic mutations in SORD. A full set of pedigrees is provided in Figure S1. The squares indicate males and the circles females. The diagonal lines are used for deceased individuals. Patients are indicated with filled shapes. B) Schematic diagram showing all exons, introns and untranslated regions (UTRs) of SORD based on NCBI reference sequence NM_003104.6. The gray and white boxes represent the coding sequence and UTRs of SORD, respectively. The variants identified in the CMT families considered in the present study map throughout the coding region of the gene. The nonsense variant c.753delG; p.Ala253GlnfsTer27 on exon 7, highlighted, was identified at particular high frequency. C) Distribution of mutations across SORD protein domains. D) SORD protein orthologs alignments showing that the four missense substitutions identified in dHMN/CMT2 families in this study are located at highly conserved residues across species from human to drosophila (sodh2). E) Magnification of the nucleotide sequence of a highly homologous region in exon 7 in SORD (reverse strand) and SORD2P (forward strand). Nucleotides differing in SORD2P from SORD are highlighted in yellow, including a C deletion in SORD2P. Representative electropherograms show that the c.753delG; p.Ala253GlnfsTer27 SORD variant is found in a homozygous state in dHMN/CMT2 patients (red box, lower plot) and in a heterozygous state in available parents (red box, upper plot), and is absent in biallelic state from healthy controls (red box), but it is fixed in SORD2P (blue box).

Fig. 2.. Decreased SORD expression and sorbitol accumulation in patients fibroblasts.

A) Schematic representation of the two-step polyol pathway converting glucose to fructose B) Representative immunoblot showing the levels of SORD and tubulin protein in four healthy controls (lane 1–4), four patients carrying homozygous p.Ala253GlnfsTer27 alleles (lane 5–8), one compound heterozygous p.Ala253GlnfsTer27/ p.Arg299Ter patient (lane 9), and two heterozygous carriers of p.Ala253GlnfsTer27 (lane 10–11). C) Levels of intracellular sorbitol was measured by ultra-performance liquid chromatography and normalised to protein content in cultured fibroblasts from five unrelated healthy controls and five patients carrying biallelic p.Ala253GlnfsTer27 mutations in SORD. The graphs show the mean ± s.d. and data distribution (dots), and the p-value of two-tailed t-tests comparing SORD protein and sorbitol levels across groups - * p<0.05, ** p<0.01, and *** p<0.001. All experiments were twice repeated independently.

Fig. 3.. Loss of Drosophila Sodh2 causes age-dependent synaptic degeneration.

A) 3D structure of the Drosophila visual system stained with Horseradish peroxidase (HRP, green; marks neuronal membranes) and bruchpilot (BRP, magenta; marks synaptic active zones), showing the lamina, medulla, and lobula. The xy- and xz-planes showing the photoreceptor terminals and lamina neurons are indicated. B) Optical sections of the laminae of yw control flies at 2 DAE, stained with HRP (green) and BRP (magenta). The organized lamina cartridges and columnar photoreceptor neurons are shown in the xy-plane and xz-plane, respectively. C) Optical sections of the laminae at the level of the terminals of Sodh2^{MB01265/MB01265} homozygous flies at 2 DAE and 10 DAE, stained with HRP (green) and BRP (magenta). Yellow arrowheads indicate the lamina vacuole-like structures that correspond to missing terminals. The areas outlined by yellow boxes are shown at higher magnification. The intensity of BRP is indicated using a red spectrum. Dotted lines indicate the area of lamina vacuole-like structures. Scale bar: 30 μm. D) Quantification of the vacuole-like structures number, size, and BRP intensity. A total of 3 laminae of each group were quantified. Data are presented as mean ± s.d. Statistical analysis was performed using two-way ANOVA followed by post-hoc Tukey’s multiple comparison test. *p<0.05, **p<0.01, ****p<0.0001. E-F) Locomotor activity of control flies (yw) and Sodh2^{MB01265/MB01265} (E) or Sodh1 and Sodh2 pan-neuronal double knockdown (RNAi) (F) flies. n=10 in each group. Data are presented as mean ± s.d. Statistical analysis was performed using two-way ANOVA followed by post-hoc Tukey’s multiple comparison test. ****p<0.0001

Figure 4.. Treatment with aldose reductase inhibitors Epalrestat and Ranirestat decrease sorbitol levels and prevent functional losses.

A) Intracellular sorbitol levels were measured by ultra-performance liquid chromatography and normalized to protein content in fibroblasts from healthy controls (N=5, circle dots) and patients carrying biallelic nonsense mutations in SORD (N=5, square dots) after three days of treatment with 100 μM Epalrestat (blue dots), 10 μM Ranirestat (green dots) or DMSO (red dots). B) Sorbitol level were measured in brain/head homogenates and normalized to protein concentration from wild-type (yw, empty circle dots), Sodh2^{MB01265/MB01265} (full circle dots) and neuron-specific knock-down of Sodh1 and Sodh1 by RNAi (square dots) Drosophilae at 10 days after eggs enclosure (DAE). Sodh2^{MB01265/MB01265} and Sodh1/Sodh2 RNAi were fed Epalrestat (blue dots) or Ranirestat (green dots) at a final concentration of 80 μg/ml, or DMSO (red dots). The graphs show the mean ± s.d. A two-tailed t-test was performed to compare sorbitol level. Statistical significance is indicated as *, ** or *** if p-value <0.05, <0.01 or <0.001, respectively, unless otherwise specified. All experiments were repeated independently twice with similar results. C) Locomotor activity of control flies (yw) feeding with DMSO (black dots), Sodh2^{MB01265/MB01265} flies feeding with DMSO (red dots), 80 μM Epalrestat (blue dots), or 80 μM Ranirestat (green dots). N=10 in each group. Data are presented as mean ± s.d. Statistical analysis was performed using Two-Way ANOVA followed by post-hoc Tukey’s multiple comparison test. * p<0.05, *** p<0.001 D-F) Laminae of Sodh2^{MB01265/MB01265} homozygous flies at 10 DAE and 40 DAE fed with DMSO (D), 80 μM Epalrestat (E), or 80 μM Ranirestat (F) were stained with HRP (green) and BRP (magenta). Yellow arrowheads indicate the lamina vacuole-like structures. The areas outlined by yellow boxes are shown at higher magnification. The intensity of BRP is indicated using a red spectrum. Dotted lines indicate the area of lamina vacuole-like structures. Scale bar: 30 μm. G) Quantification of the vacuole-like structures number, size, and BRP intensity of (D-F). N=3. Data are presented as mean ± s.d. Statistical analysis was performed using two-way ANOVA followed by post-hoc Tukey’s multiple comparison test. *p<0.05, **p<0.01, ****p<0.0001.