Biallelic SOX8 Variants Associated With Novel Syndrome With Myopathy, Skeletal Deformities, Intellectual Disability, and Ovarian Dysfunction

Abstract

Background and objectives: The human genome contains ∼20,000 genes, each of which has its own set of complex regulatory systems to govern precise expression in each developmental stage and cell type. Here, we report a female patient with congenital weakness, respiratory failure, skeletal dysplasia, contractures, short stature, intellectual delay, respiratory failure, and amenorrhea who presented to Medical Genetics service with no known cause for her condition.

Methods: Whole-exome and whole-genome sequencing were conducted, as well as investigational functional studies to assess the effect of SOX8 variant.

Results: The patient was found to have biallelic SOX8 variants (NM_014587.3:c.422+5G>C; c.583dup p.(His195ProfsTer11)). SOX8 is a transcriptional regulator, which is predicted to be imprinted (expressed from only one parental allele), but this has not yet been confirmed. We provide evidence that while SOX8 was maternally expressed in adult-derived fibroblasts and lymphoblasts, it was biallelically expressed in other cell types and therefore suggest that biallelic variants are associated with this recessive condition. Functionally, we showed that the paternal variant had the capacity to affect mRNA splicing while the maternal variant resulted in low levels of a truncated protein, which showed decreased binding at and altered expression of SOX8 targets.

Discussion: Our findings associate SOX8 variants with this novel condition, highlight how complex genome regulation can complicate novel disease-gene identification, and provide insight into the molecular pathogenesis of this disease.

Figure 1. Diffuse Decreased Muscle Bulk With Contractures and Mild Variation in Fiber Size on Muscle Biopsy

(A–D) Clinical images demonstrated marked decrease in muscle bulk with asymmetric contractures. Muscle biopsy at age 1 year (E, F) demonstrated mild variation in fiber size with scattered moderately small rounded polyhedral fibers of both types. The fiber size variation was most prominent just under the fascia. The endomysial connective tissue was mildly increased (arrow). (G) Repeat muscle biopsy at age 33 years revealed mild variation in fiber size attributable to rare, small regenerating fibers with rare angular, atrophic fibers (arrow).

Figure 2. MRI Demonstrates Large Posterior Fossa CSF Spaces and Diffuse Decreased Muscle Volume, With Fatty Replacement of Most Prominent in the Right Pectoralis, Vastus Lateralis, Semimembranosus, and Soleus

(A) Head MRI with sagittal T1 FLAIR and (B) axial T2 FLAIR demonstrate large posterior fossa CSF spaces, which appear in direct relationship with enlarged fourth ventricle (arrow). There is also slight deformity of the cerebellum. (C, D and E) Whole-body muscle MRI T1-weighted axial images demonstrate severe atrophy of the right pectoralis major and minor (C), with severe fatty infiltration (arrow). (D) Mild-to-moderate atrophy and fatty infiltration of the vastus lateralis on the right along its central and inferior thirds (arrow) and bilateral moderate-to-severe fatty infiltration and moderate atrophy of the semimembranosus (chevron). There were no signal changes in the STIR sequence (not shown). (E) Moderate-to-severe bilateral fatty infiltration of the soleus (arrow) and mild fatty infiltration of the medial head of the gastrocnemius (left more than right), with mild atrophy.

Figure 3. Expression of SOX8 mRNA Was Elevated and Produced a Truncated Protein in Affected Cells

(A) Real-time PCR analysis on cDNA from fibroblast cells showing increased SOX8 transcript abundance in affected cells (Aff) compared with control (Ctrl). Graphed data represent the mean of 3 biological replicates, and error bars depict standard error of the mean. Paired t test was performed; p = 0.067. (B) Western blot analysis on extracts from fibroblast cells using both an N-terminal and a C-terminal SOX8 antibody in control and affected samples. The N-terminal antibody detects a protein of lower molecular weight and lower abundance in lysate from affected cells, whereas the C-terminal antibody failed to detect any protein in affected lysates.

Figure 4. SOX8/SOX9/SOX10 Complex Was Maintained in Affected Cells, Although SOX10 Was Upregulated, Possibly Providing Some Compensation for SOX8 Deficiency

(A) Real-time PCR analysis on cDNA from fibroblast cells showed no change in SOX9 levels and an increase in SOX10 mRNA abundance in affected cells compared with control. Graphed data represent the mean of 3 biological replicates, and error bars depict standard error of the mean. Paired t test was performed; *p < 0.05. (B) Western blot analysis on extracts from fibroblast cells showed a decrease in SOX9 protein and an increase in SOX10 protein in affected compared with control cells. (C) Immunoprecipitation was performed using a SOX8 N-terminal antibody, which coimmunoprecipitated both SOX9 and SOX10, using lysates of control and affected fibroblast cells.

Figure 5. SOX8 Mutations in Affected Cells Lead to Misregulation of SOX8 Targets and Defective DNA Binding of SOX8 at Some Target Sites

(A) Quantitative SOX8 ChIP analysis was performed at the β-catenin gene, CTNNB1. A schematic of this gene is shown with numerically labeled black bars above the gene representing qPCR amplicons that were interrogated for ChIP-qPCR analysis. Graphed data on the right are the results of SOX8 quantitative ChIP analysis at the indicated CTNNB1 locations from the schematic for affected and control cells. Enrichment at site 2 was found to be significantly decreased (*p < 0.05; paired t test). (B) Western blot analysis on extracts from fibroblast cells showing a decrease in total and activated β-catenin protein levels in affected cells compared with controls while axin protein levels remained unchanged. (C) Real-time PCR analysis on cDNA from fibroblast cells showed altered expression of many components of the Wnt/β-catenin pathway and various other SOX targets in affected compared with control cells. Graphed data represent the mean of 3 biological replicates, and error bars depict standard error of the mean (*p < 0.05; paired t test).