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.

Full text

RESEARCH ARTICLE OPEN ACCESS
Biallelic SOX8 Variants Associated With Novel
Syndrome With Myopathy, Skeletal Deformities,
Intellectual Disability, and Ovarian Dysfunction
Jodi Warman-Chardon, MD, MSc, Taila Hartley, MSc, Aren Elizabeth Marshall, PhD, Arran McBride, MSc,
Madeline Couse, MSc, William Macdonald, PhD, Mellissa R.W. Mann, PhD, Pierre R. Bourque, MD,
Ari Breiner, MD, MSc, Hanns Lochm¨
uller, MD, PhD, John Woulfe, MD, PhD, Marcos Loreto Sampaio, MD,
Gerd Melkus, PhD, Bernard Brais, MDCM, PhD, David A. Dyment, DPhil, MD, Kym M. Boycott, MD, PhD, and
Kristin Kernohan, PhD
Neurol Genet 2023;9:e200088. doi:10.1212/NXG.0000000000200088
Correspondence
Dr. Warman-Chardon
jwarman@toh.ca
or Dr. Kernohan
kkernohan@cheo.on.ca
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 pre-
sented 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 lympho-
blasts, 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 mo-
lecular pathogenesis of this disease.
From the Department of Medicine (J.W.-C., P.R.B., A.B., H.L.), Th e Ottawa Hospital; The Ottawa Hospital Research Institut e (J.W.-C., P.R.B., H.L., J.W., M.L.S., G.M.); Faculty of Medicine
(J.W.-C., P.R.B., A.B., H.L., J.W., M.L.S ., D.A.D., K.M.B.); Children’s Hospital of Eastern Ontario Research Institute (J. W.-C., T.H., A.E.M., A.M., H.L., D.A.D., K.M.B., K.K.), University of Ottaw a;
Hospital for Sick Children (M.C.) , Centre for Computational Medicine, Toronto, Canada; Departmen t of Obstetrics (W.M., M.R.W.M.), Gynaecology and Reproductive Sciences,
University of Pittsburgh School of Medi cine; Magee-Womens Research Institute (W.M., M.R.W.M.), Pittsburgh, PA; Department of Pathology and Laboratory Medi cine (A.B., J.W.), The
Ottawa Hospital; Department of Radiology (M.L.S ., G.M.), Radiation Oncology and Medical Physics, University of Ottawa; Department of Neurol ogy and Neurosurgery (B.B.), Montreal
Neurological Institute and Hospital, McGil l University; and Newborn Screening Ontario (K.K .), Children’s Hospital of Eastern Ontario, Ottaw a, Canada.
Go to Neurology.org/NG for full disclosures. Funding information is provided at the end of the article.
The Article Processing charge was funded by the authors.
This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivativesLicense 4.0 (CC BY-NC-ND), which permits downloading
and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.
Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1

Introduction
Congenital myopathies (CMs) are genetically heterogeneous
muscle disorders typically presenting with neonatal or
childhood-onset weakness and hypotonia with a static or
slowly progressive disease course.
1
Patients with CM may
have dysmorphic characteristics secondary to the myopathy,
including high-arched palate, elongated facies, scoliosis, joint
contractures, and foot deformities.
2
Diagnoses of CM can be
challenging because of a range of disease severity, and many
patients may have nonspecific or normal biopsy findings.
3
Advances in molecular genetics have enabled broadening of
the clinical phenotypes in known CM genes, while leading to
the identification of novel genetic etiologies.
Our understanding of the human genome has significantly
advanced in the past decade, but much is left to be discov-
ered.
4
The human genome contains ;20,000 protein-coding
genes, many of which are tightly regulated for precise ex-
pression in each cell type at all developmental stages. In ad-
dition, although most genes have both alleles regulated
concurrently, some are only expressed from one parental al-
lele (imprinted genes). The regulation of genes is highly rel-
evant to human health; the combination of a gene’s function
and expression pattern dictates the phenotypic consequences
of genetic aberrations. These factors also influence the in-
heritance pattern for genetic diseases. Finally, many genes are
associated with multiple different phenotypes depending on
the mutational mechanism and inheritance pattern.
SOX [SRY (sex-determining region on the Y chromosome)-
related HMG (high mobility group)-box] proteins are a family
of transcriptional regulators defined by the presence of a highly
conserved HMG domain that is responsible for sequence-
specific DNA binding and subsequent DNA bending.
5,6
The
human genome contains 20 SOX family members, which have
highly divergent developmental functions in a vast array of
tissues (reviewed in reference 7). This SOX family is further
subdivided into 8 groups on the basis of sequence similarity
(Groups A-H).
8
One of the best characterized groups of SOX
proteins is subgroup E, which includes SOX8, SOX9, and
SOX10. SOX9 is predominantly expressed in chondrocytes and
sertoli cells,
9,10
and mutations cause campomelic dysplasia.
11,12
SOX10 is expressed in both the peripheral and central nervous
systems,
13
and mutations cause Waardenburg-Hirschsprung
disease.
14-16
SOX8 has been associated with a range of human
reproductive anomalies including 46, XY disorders of (or dif-
ferences in) sex development (DSD), as well as male infertility
and primary ovarian insufficiency (POI) in women.
17,18
While
Sox8 is expressed in the mouse testis, it is also expressed in the
CNS, neural crest derivatives, myotomes, skeletal muscles,
satellite cells, cartilage, and kidney.
19
Given the broad expres-
sion, it remains to be seen if SOX8 is responsible for additional
phenotypes. In mice, loss of Sox8 results is subtle skeletal de-
formities, weight reduction, and osteopenia.
20
Here, we report a female patient with clinical features of a
novel syndrome with severe facial weakness, micrognathia,
high-arched palate, kyphoscoliosis/skeletal dysplasia, con-
tractures, congenital nonprogressive myopathy with proximal
and distal leg weakness, short stature, intellectual delay, and
respiratory failure and amenorrhea. Exome and genome se-
quencing identified compound heterozygous variants in
SOX8.Wefind that the regulation of SOX8 is complex and
that it displays imprinted expression in some cell types, but
not in others, providing molecular evidence that both iden-
tified variants adversely affected gene function. Our findings
expand the disease associations of SOX8 while beginning to
provide molecular insights into disease pathogenesis.
Methods
Standard Protocol Approvals, Registrations,
and Patient Consents
The affected individual presented to the Medical Genetics
service for evaluation. She and her family were enrolled in the
Care4Rare Canada research study because of the lack of a
molecular diagnosis. Approval of the study design was
obtained from the institutional research ethics board
(Children’s Hospital of Eastern Ontario; #1104E and
CTO1577), and free and informed consent was obtained
before enrollment for all participants.
Muscle Biopsy
For muscle biopsy of the lateral quadriceps, immunohisto-
chemical and histologic studies, including the electron mi-
croscopic examination, were performed. Some fragments of
the muscle biopsy were flash frozen in liquid nitrogen at
−80°C while others were immersed in 4% buffered para-
formaldehyde before being embedded in paraffin. Sections of
the flash-frozen muscle were cut at 5 μm on a cryotome and
stained using hematoxylin and eosin, periodic acid Schiff,
modified Gomori trichrome, Masson trichrome, vanGieson,
and oil red O. Histochemical staining for lactate dehydro-
genase, NADH, acid phosphatase, and myophosphorylase
and myofibrillar ATPase (developed at pH 4.3, 4.6, and 9.4)
was also performed on sections, as well as immunostaining
using antibodies α-sarcoglycan, β-sarcoglycan, δ-sarcoglycan,
and γ-sarcoglycan and C-terminal, N-terminal and rod do-
mains of dystrophin, spectrin, emerin, dysferlin, slow and fast
myosin, collagen VI, merosin, αB-crystallin, and β-dystrogly-
can. Electron microscopy was also performed.
Glossary
CM = congenital myopathy; EMG = electromyogram; POI = primary ovarian insufficiency.
2Neurology: Genetics | Volume 9, Number 5 | October 2023 Neurology.org/NG

MRI
Whole-body muscle MRI was performed (1.5T Siemens
Magneton, 5 mm slice thickness). CoronalT1 sequences of the
thorax and abdomen, and axial T1 and STIR-weighted se-
quences of the thorax, abdomen, and upper and lower limbs
were obtained. Multiplanar multisequential MRI of the head
without contrast was also performed.
Identification of Rare Variants by Exome and
Genome Sequencing
For exome sequencing, exonic DNA was selected using the
Agilent SureSelect 50 Mb (V5) All Exon Kit following man-
ufacturer’s instructions and sequenced on an Illumina HiSeq
2500. Read alignment, variant calling, and annotation were
performed as previously described for FORGE and Care4-
Rare Canada projects, with a pipeline based on Burrows-
Wheeler Aligner, Picard, ANNOVAR, and custom annotation
scripts.
21-24
Average coverage for the exomes was 141× for the
affected individual and 114× and 129× for the mother and
father, respectively, and 95% of the CCDS exons in all exomes
were covered at >10×. Details of genome sequencing are
available in the eMethods (links.lww.com/NXG/A629).
Genomic Imprinted Expression, Real-Time PCR,
Minigene, Western Blot, Coimmunoprecipitation,
and Chromatin Immunoprecipitation Analyses
Details of the laboratory assays used in this study are available
in the eMethods (links.lww.com/NXG/A629).
Data Availability
The data are not publicly available because of privacy and
ethical restrictions. The data that support the findings of this
study are available on request from the corresponding author.
Specifically, the data from the family can be accessed by re-
quest through the Genomics4RD platform.
Results
Patient Description
The female proband was born to a 22-year-old, G3P2, French
Canadian mother and a 30-year-old Caucasian father. The
parents and 2 siblings (brother and sister) were healthy.
There was no known consanguinity, and the family histories
were noninformative. The proband had weakness at birth with
poor suck, micrognathia, hypotonia, and talipes. She was
documented to have significant motor delay as a child and
required spinal fixation surgery at age 15 years. She attended
regular school with documented learning difficulties. She re-
presented to the Medical Genetics service at age 27 years with
thin body habitus (weight 26.5 kg, −5.0 SD; BMI 14.1, −3.4
SD), short stature (144 cm, −2.7 SD), and a head circum-
ference of 52 cm (−1.8 SD). She had mild intellectual delay.
She had clinical features of a congenital, nonprogressive my-
opathy with moderate proximal and distal weakness (2- to 4+
diffusely) and scapular winging. She had prominent cranial
weakness, with marked facial paresis with no frontalis con-
traction and incomplete eye closure, upper lid retraction,
narrow mouth, tented upper lip vermilion, micrognathia/
dental crowding, and prominent dysarthria and dysphonia. She
had marked asymmetric contractures in elbows, knees, and
ankles and long, tapered, finger hyperlaxity (Figure 1). She also
had amenorrhea until age 17 years and has mild chronic oli-
gomenorrhea (6–10/y). She has chronic respiratory failure
Figure 1 Diffuse Decreased Muscle Bulk With Contractures and Mild Variation in Fiber Size on Muscle Biopsy
(A–D) Clinical images demonstrated marked de-
crease in muscle bulk with asymmetric contrac-
tures. 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 endo-
mysial 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).
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requiring nocturnal Bilevel Positive Airway Pressure at age 27
years, and pulmonary function testing demonstrated severe
restrictive lung disease. Transthoracic echocardiogram was
normal. Creatine kinase was normal. Electrodiagnostic motor
and sensory nerve conduction studies were normal. Needle
electromyogram (EMG) studies demonstrated short‐duration,
small‐amplitude motor unit action potential, and early re-
cruitment patterns were observed in the involved proximal
muscles and distal muscles, suggesting myopathy, with no
myotonic discharges. Repetitive stimulation studies at 3 Hz of
the abductor digiti minimi and trapezius were normal. Single-
fiber EMG studies of the forehead did not demonstrate any
motor units.
Muscle biopsy of the right medial gastrocnemius at age 1 year
(Figure 1) demonstrated mild variation in fiber size with
scattered, moderately small, rounded polyhedral fibers of both
types. The endomysial connective tissue was mildly increased.
The muscle biopsy did not demonstrate fiber splitting, ring
fibers, nuclear chains, or sarcoplasmic masses. There was no
inflammation degeneration, necrosis, or regeneration identi-
fied. Intramuscular nerve fibers and twigs were normally
myelinated. Repeat muscle biopsy of the lateral vastus medi-
alis at age 33 years revealed mild variation in fiber size at-
tributable to rare, small regenerating fibers in formalin-fixed,
paraffin-embedded specimen with rare angular, atrophic fibers
(Figure 1). There were no significant dystrophic features.
Figure 2 MRI Demonstrates Large Posterior Fossa CSF Spaces and Diffuse Decreased Muscle Volume, With Fatty Re-
placement 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 demon-
strate severe atrophy of the right pectoralis major and minor
(C), with severe fatty infiltration (arrow). (D) Mild-to-moder-
ate 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 gastrocne-
mius (left more than right), with mild atrophy.
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Histochemical staining for myofibrillar ATPase revealed a
predominance of type II muscle fibers. On immunostaining,
normal staining patterns were observed for the following
antigens: α-sarcoglycan, β-sarcoglycan, δ-sarcoglycan, and
γ-sarcoglycan and C-terminal, N-terminal, and rod domains
of dystrophin, spectrin, emerin, dysferlin, slow and fast my-
osin, collagen VI, merosin, αB-crystallin, and β-dystroglycan.
Ultrastructural analysis did not show significant findings.
MRI of the brain demonstrated large posterior fossa CSF
spaces, which appear in direct relationship with enlarged
fourth ventricles, possibly consistent with a mild congenital,
Dandy-Walker malformation (Figure 2, A and B). Muscle
MRI demonstrated fatty infiltration of the paravertebral
muscles, severe atrophy and fatty infiltration of the right
pectoralis major and minor (normal on the left) and serratus
anterior, moderate-to-severe fatty infiltration and moderate
atrophy of the semimembranosus, mild-to-moderate atrophy
and fatty infiltration of the vastus lateralis on the right along its
central and inferior thirds, moderate-to-severe bilateral fatty
infiltration of the soleus and mild fatty infiltration of the
medial head of the gastrocnemius (left more than right), with
mild atrophy. The shoulder girdle and visualized areas of the
upper extremities were reported as presenting minimal fatty
infiltration and mild-to-moderate symmetrical global loss of
volume (Figure 2, C–E). There were no signal changes in the
STIR sequence. X-rays showed skeletal dysplasia, including
cervical thoracic scoliosis and lumbar scoliosis, overtubulated
long bones with distal femoral condyles, which were not well
formed, mild bowing for both tibias and fibulas, and positional
abnormalities of the feet.
Clinical genetic testing was noninformative and included
testing for myotonic dystrophy type 1 and type 2, FSHD1,
and a microarray. The microarray did identify a 0.655 Mb
duplication in chromosome 6q22.31 (1118,619,406-
119,274,667 chr37) that involved 7 reference sequence genes
(SLC35F1, CEP85L, BRD7P3, LOC100287632, ASF1A),
but this had no clear clinical significance and was felt to be
noncontributory because the genes involved either had no
known associated phenotypes or were unrelated.
Exome and Genome Sequencing Identified
Biallelic Variants in SOX8
Given the lack of family history of a similar condition, we hy-
pothesized that this rare disease was caused by autosomal re-
cessive or de novo dominant variant(s). Exome sequencing was
performed on genomic DNA from the affected patient and her
parents. Variants present in ≥0.1% minor allele frequency in
gnomAD were excluded. No homozygous or de novo variants
were identified. Three known disease genes were identified with
compound heterozygous variants: NEB (NM_001164507.1:
c.21797C>T; NM_001164507.1:c.402+4_402+27del) while
both variants formally classify as a variant of unknown signifi-
cance (VUS), the patient’s phenotype did not fitbecause
there were no nemaline rods in the muscle biopsy. Therefore,
this gene was eliminated as a candidate. SYNE1 (NM_033071.3:
c.8381C>T, c.16646T>G) and EYS (NM_001142800.1:
c.7796A>G; c.7033C>T) also contained 2 VUS each but were
ruled out as causative because the associated conditions did not
resemble our patient (SYNE1 causes spinocerebellar ataxia, au-
tosomal recessive 8 and arthrogryposis multiplex congenital 3,
myogenic type and EYS causes retinitis pigmentosa 25). Com-
pound heterozygous variants were identified in 2 additional genes
not yet linked to phenotypes: PRUNE2 (NM_001308047.1:
c.8677C>T; c.1132C>G) and SOX8 (NM_014587.3:c.422+5-
G>C; c.583dup). PRUNE2 is a loss-of-function tolerant gene
with a pLi of 0 and has a significant number of individuals that are
homozygous for loss-of-function variants in gnomAD; as such, it
seemed unlikely that this gene caused a loss-of-function autoso-
mal recessive condition. SOX8 is a conserved gene, with only 5
heterozygous loss-of-function alleles in gnomAD (of >200,000
alleles, pLi 0.67), suggesting that this was not tolerant to recessive
loss-of-function variants. SOX8 expression coincides with essen-
tial phases of development in many organs, including the CNS,
neural crest derivatives, myotomes, skeletal muscles, and carti-
lage,
19
many of which overlap with the phenotype presentations
in our patient. The SOX8 splice variant, c.422+5G>C, was pre-
dicted to affect the donor splice site, and the duplication variant,
c.583dup p.(His195ProfsTer11), caused a frameshift and pre-
mature stop codon about half way through the protein. Nei-
ther variant had been seen in gnomAD or our in-house control
database. Interestingly, SOX8 was associated with autosomal
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 repre-
sent the mean of 3 biological replicates, and error bars depict
standard error of the mean. Paired ttest was performed; p=
0.067. (B) Western blot analysis on extracts from fibroblast
cells using both an N-terminal and a C-terminal SOX8 anti-
body in control and affected samples. The N-terminal anti-
body detects a protein of lower molecular weight and lower
abundance in lysate from affected cells, whereas the C-ter-
minal antibody failed to detect any protein in affected
lysates.
Neurology.org/NG Neurology: Genetics | Volume 9, Number 5 | October 2023 5

dominant 46, XY disorders of sex development, as well as in-
fertility in men and POI in women.
17,18
However, this has not
been validated in sufficient independent families to confirm it as a
disease-gene association. Given the compound heterozygous,
highly deleterious variants, we found SOX8 to be the most
compelling candidate. With the advances in genome sequencing,
we then conducted trio whole-genome sequencing to ensure that
there was no other genomic variation that might contribute to the
patient’s condition. SV, CNV, and STR analysis did not identify
any variants of interest, and SNV analysis did not identify any
further variants to consider. We entered this gene in the Match
Maker Exchange via Phenome Central in July 2018. Un-
fortunately, no matches of interest have been identified. We
conclude that SOX8 remains the most compelling candidate for
this patient’s phenotype and set out to investigate the significance
of the SOX8 variants in our functional laboratory.
In the Affected Individual, the Maternal SOX8
mRNA Produced a Truncated Protein While the
Paternal Variant Potentially Affected
mRNA Splicing
We began by assessing SOX8 expression levels in cells from
the affected individual compared with age-matched and sex-
matched control fibroblast cells. Muscle tissue was not used
for functional studies because, unfortunately, there was no
material available from previous muscle biopsies available for
this purpose. Real-time PCR revealed a modest increase in
SOX8 mRNA transcript abundance in fibroblast cells from the
affected individual (Figure 3A). Western blot analysis using a
SOX8 N-terminal antibody detected a protein of reduced
molecular weight in affected cells, which also showed de-
creased protein abundance compared with the wild-type form
of this protein in control fibroblast cells (Figure 3B). Immu-
nodetection of SOX8 at the C-terminal domain revealed an
absence of SOX8 protein in patient cells (Figure 3B). Given
the presence of only one band on the western blot, we se-
quenced the cDNA and identified transcripts containing only
the maternal frameshift variant, c.583dup p.(His195Prof-
sTer11), which corresponded appropriately to the protein
size observed by western blot analysis. Therefore, we only
detected the maternal allele in both the cDNA and protein
assays. At this time, SOX8 was computationally predicted to
be imprinted, with paternal-specific expression, but this
finding was not confirmed.
25
Given our findings, we pursued
the hypothesis that the SOX8 gene was in fact imprinted, with
only the maternal allele expressed. We assessed allelic SOX8
expression in 3 control fibroblast and 2 lymphoblast samples
for whom we had available paternal and maternal SNPs within
cell lines and confirmed that SOX8 is maternally expressed in
fibroblasts and lymphoblasts in all samples (data not shown).
We conclude that SOX8 is maternally expressed in fibroblasts
and lymphoblasts and that the maternally inherited variant,
c.583dup p.(His195ProfsTer11), produced a truncated pro-
tein in our patient. Using a lymphoblast cell line derived from
the patient’s father, we revealed that the c.422+5C>G variant
allele was not expressed and therefore was likely grand-
paternally inherited, supporting maternal-specificSOX8 ex-
pression in these cells. We note that SOX8 has a neighboring
gene, LMF1, which also displayed imprinted expression in
some tissues (data mined from
26
), and that there are maternal
and paternal gametic differentially methylated regions nearby,
suggesting that SOX8 and LMF1 may be part of an imprinted
domain.
The finding that SOX8 is imprinted was intriguing in light of
the biallelic variants and the reports of autosomal dominant
conditions in the literature. We hypothesized that if SOX8
was expressed from both alleles in other cell types or during
particular developmental stages, it would produce a more
extensive phenotype. We mined data in the literature and
found evidence, where SNPs were informative, of tissue-
specificSOX8 imprinting. Namely, a study by Baran et al.
26
found some tissues with allelic SOX8 expression while others
exhibited biallelic expression. Given that the paternal allele
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 coim-
munoprecipitated both SOX9 and SOX10, using lysates of
control and affected fibroblast cells.
6Neurology: Genetics | Volume 9, Number 5 | October 2023 Neurology.org/NG

was expressed in some cell types and/or developmental
windows,
26
we sought to assess if the paternally inherited
c.422+5C>G variant had an effect on mRNA splicing, which
was predicted to affect the donor splice site. Given the in-
ability to assess the paternal c.422+5C>G in the available
patient-derived cells because it was silenced, we cloned this
variant into a minigene splicing construct and assessed for
splicing. We observed that this variant produced 3 different
products confirmed by sequencing, a normal length transcript
and 2 other transcripts which were missing regions of the
adjacent exon, indicating an adverse effect on mRNA splicing.
Given the absence of significant coding portions, it is antici-
pated that these transcripts would lead to a protein with
functional deficits.
Both of the proband’s parents carry a variant in SOX8 gene.
If both parents inherited the SOX8 variant from their fa-
thers, the variant would have no effect on the parent’s
phenotypebecausethepaternalalleleissilenced.Intissue
where there may be biallelic expression, the parents would
have one wild-type and one variant allele. Because both
parents had no phenotype, it suggests that these variants do
not have an effect in a heterozygous state and that other
alleles that have been associated with a dominant condition
intheliteratureeitherhaveadifferent mechanism or that
SOX8 variation was not likely the explanation for their
findings. We note these variants are missense variants
and so a gain-of-function mechanism is possible. Taken
together, we propose that SOX8 has both biallelic and
maternal expression, depending on the cell type, and that
both variants observed in our patient could have an effect
on the SOX8 product.
SOX10 is Upregulated in SOX8-Deficient Cells
Reports from SOX8-deficient mice suggest that there may be
functional compensation for loss of SOX8 by SOX9 and/or
SOX10 in some tissues.
19,27
As such, we assessed the ex-
pression patterns of SOX9 and SOX10 in patient fibroblast
cells to determine whether this effect is occurring in our pa-
tient. While SOX9 mRNA levels were similar between affected
and control cells, SOX10 mRNA levels were increased in af-
fected cells (Figure 4A). Congruently, western blot analysis
showed no significant differences in SOX9 protein levels,
whereas there was an increase in SOX10 in the affected cells
(Figure 4B). In addition to potential functional redundancy
between SOX8, SOX9, and SOX10 proteins, it has been
shown that the SOXE transcription factors not only homo-
dimerize but can also heterodimerize with each other.
27,28
Based on these findings, we investigated the interaction be-
tween SOX8-SOX9 and SOX8-SOX10 in control and affected
fibroblast cells. Coimmunoprecipitation experiments using a
SOX8 N-terminal antibody detected SOX8-SOX9 and SOX8-
SOX10 protein-protein interactions in both control and pa-
tient cells were approximately equivalent (Figure 4C).
Overall, SOX8 deficiency in patient cells results in potential
compensatory SOX10 regulation, although the truncated
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 in-
dicated CTNNB1 locations from the
schematic for affected and control cells.
Enrichment at site 2 was found to
be significantly decreased (*p<0.05;
paired ttest). (B) Western blot analysis
on extracts from fibroblast cells show-
ing a decrease in total and activated
β-catenin protein levels in affected cells
compared with controls while axin pro-
tein levels remained unchanged. (C)
Real-time PCR analysis on cDNA from
fibroblast cells showed altered expres-
sion 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;pairedttest).
Neurology.org/NG Neurology: Genetics | Volume 9, Number 5 | October 2023 7

SOX8 protein can heterodimerize with the other SOXE
members.
Downstream SOX8 Targets Are Misregulated
Given that the maternally expressed truncated protein can
bind other SOXE members, we wondered whether down-
stream function was affected, especially considering that
SOX8 is a transcription factor with a number of previously
defined targets.
5,6
We began by using quantitative ChIP
analysis to assess SOX8 binding near the β-catenin gene
(CTNNB1). We found that the highest level of occupancy was
close to the transcriptional start site and that this occupancy
was decreased in patient cells (Figure 5A). We conclude that
the truncated SOX8 protein has impaired binding capacity at
this site, thereby potentially affecting transcription at other
known SOX8 target sites. To test this, we assessed expression
levels at SOX8 target genes. We found that the overall mRNA
transcript abundance was altered for components of the Wnt/
β-catenin pathway and various other SOX targets, with no-
table increases for LHX2,SOX10,WNT5A,WNT6, and APC
and decreases for FZD7,LRP5,WNT2, and COL2A1 ex-
pression levels. Furthermore, we found a decrease in both
total and active β-catenin protein levels (Figure 5, B and C).
We conclude that the maternal SOX8 truncated protein has
impaired function.
Discussion
We have identified an autosomal recessive condition charac-
terized by congenital myopathy with skeletal dysplasia, re-
spiratory failure, intellectual delay, contractures, short stature,
and amenorrhea. The affected female patient carries com-
pound heterozygous variants in SOX8. Molecular profiling of
affected cells showed both variants have a functional effect.
In the literature, there was a previously reported female pa-
tient with delayed growth and development and skeletal de-
fects, with early failure of gonad development with a large
duplication immediately upstream of SOX8.
29
The phenotype
of this patient overlaps with the one reported here, includ-
ing short stature, distinctive craniofacial abnormalities, de-
velopmental delay, and gonadal dysfunction. Heterozygous
SOX8 variants have been noted in a handful of individuals
with reproductive anomalies, although the mechanism for
these phenotypes remains unknown.
17,18
SOX8 is a tran-
scription factor expressed in many tissues, including muscle,
testis, nervous system, and cartilage, and involves evolutionary
conserved regulatory elements.
30
SOX8 appears to display
tissue-specific imprinting. However, the presence of dual loss-
of-function variants and lack of a phenotype in her parents
suggests that our patient truly has an autosomal-recessive
condition.
SOX8 is expressed in many organs, including skeletal muscles,
cartilage, the CNS, kidneys, and gonads/testis,
19
and is a known
regulator of differentiation of skeletal muscle, possibly by
impairing myogenic basic helix-loop-helix protein function.
30
SOXproteinsperformuniquefunctionsindifferent cell types
through interactions with different binding partners (reviewed
in reference 31). As a well-characterized SOX8 target, we
showed that the WNT/β-catenin pathway was misregulated in
fibroblasts. Notably, the WNT pathway plays important roles in
bone and muscle development.
32
Although at the moment we
cannot directly link SOX8 and other genes implicated in con-
genital myopathies, these genes play critical roles in muscle
structure and function,
33
which SOX8 is predicted to influence
through the WNT/β-catenin pathway. Similarly, with respect to
the brain MRI findings,SOX8isexpressedandplaysarolein
neuroepithelial and glial precursor cells
34
;ofthemorethan100
genes associated with malformations of cortical development,
the biological pathways they are involved with include cell cycle
regulation, cell fate specification, and neuronal migration and
basement membrane function, among others.
35
In addition, it is
anticipated that there are many more genes affected by SOX8
regulation and that this may vary by cell type, depending on the
interacting cofactors.
36
In Sox8-deficient mice, major de-
velopmental defects do not occur because many SOX8 targets
overlap with those for SOX9, SOX10, or both proteins, sug-
gesting that a compensatory mechanism exists between these
proteins.
19,30,37
Additional evidence of functional compensation
between SOXE proteins comes from mouse models deficient
for Sox10 and Sox9, where additional loss of Sox8 led to
worsening phenotypes.
38,39
In the patient’sfibroblasts, we ob-
served upregulation of SOX10, possibly representing a com-
pensatory mechanism. Increase SOX10 expression, but not
SOX9,mayreflect a cell-specificeffect in fibroblasts; we cannot
exclude the possibility that other tissues or cell types may show
different patterns of SOX9 and SOX10 expression on SOX8
disruption because both proteins have the capacity to hetero-
dimerize with SOX8.
In summary, we have used exome sequencing to identify a
novel autosomal-recessive condition associated with SOX8
loss-of-function. The existence of tissue-specific imprinted
genes raises an interesting concern in the gene discovery field
in which gene candidates are assessed based on their pre-
sumed inheritance pattern, and it is significantly more difficult
to identify conditions with more complex underlying genetics.
Better resources defining imprinted genes are required for
optimal gene discovery research. The phenotype of this
condition includes congenital, nonprogressive disorder of
CNS, skeletal muscles/cartilage, skeletal and gonadal system
with congenital myopathy with contractures, scoliosis, in-
tellectual delay, and amenorrhea. This phenotype may rep-
resent a novel condition or phenotypic extension of the
previously described Marden-Walker syndrome.
40
Marden-
Walker syndrome is characterized by psychomotor re-
tardation, kyphoscoliosis, contractures, and a facial paresis
with blepharophimosis, micrognathia, and a high-arched
palate. Further phenotypes may include Dandy-Walker mal-
formation with hydrocephalus and vertebral malformation.
The proband reported here has additional atypical features of
amenorrhea and nocturnal respiratory failure not commonly
reported in the Marden-Walker syndrome. The molecular
8Neurology: Genetics | Volume 9, Number 5 | October 2023 Neurology.org/NG

cause of Marden-Walker syndrome is currently unknown and
may represent a genetically heterogeneous condition, and
molecular assessment of additional patients will be in-
formative. Additional patients with biallelic variants in SOX8
are required to confirm this novel disease association and
delineate the phenotypic spectrum. More broadly further
studies looking at tissue-specific imprinted genes are also re-
quired to assess the relevance for rare diseases.
Acknowledgment
K.M. Boycott is supported by a CIHR Foundation Grant
(FDN-154279) and a Tier 1 Canada Research Chair in Rare
Disease Precision Health. T. Hartley is supported by a CIHR
Doctoral Award–Frederick Banting and Charles Best Canada
Graduate Scholarship. A.E. Marshall is supported by a
Canadian Institutes of Health Research (CIHR) fellowship
award (MFE-176616). J.W. Chardon is supported by a
Department of Medicine University of Ottawa Clinical
Research Chair, and this work was also supported by
Physician Services Incorporated Grant (19-28). H. Lochm¨ul-
ler receives support from the Canadian Institutes of Health
Research (Foundation Grant FDN-167281), the Canadian
Institutes of Health Research and Muscular Dystrophy
Canada (Network Catalyst Grant for NMD4C), the Canada
Foundation for Innovation (CFI-JELF 38412), and the
Canada Research Chairs program (Canada Research Chair
in Neuromuscular Genomics and Health, 950-232279).
Study Funding
This work was performed under the Care4Rare Canada
Consortium funded by Genome Canada and the Ontario
Genomics Institute (OGI-147), the Canadian Institutes of
Health Research (GP1-155867), Ontario Research Fund,
Genome Alberta, Genome British Columbia, Genome Que-
bec, and Children’s Hospital of Eastern Ontario Foundation.
Disclosure
The authors report no relevant disclosures. Go to Neurology.
org/NG for full disclosures
Publication History
Received by Neurology: Genetics December 19, 2022. Accepted in final
form May 30, 2023. Submitted and externally peer reviewed. The
handling editor was Associate Editor Margherita Milone, MD, PhD.
Appendix Authors
Name Location Contribution
Jodi
Warman-
Chardon,
MD, MSc
Department of Medicine, The
Ottawa Hospital; The Ottawa
Hospital Research Institute;
Faculty of Medicine;
Children’s Hospital of
Eastern Ontario Research
Institute, University of
Ottawa, Canada
Drafting/revision of the
manuscript for content,
including medical writing for
content; major role in the
acquisition of data; study
concept or design; analysis
or interpretation of data
Appendix (continued)
Name Location Contribution
Taila
Hartley, MSc
Children’s Hospital of
Eastern Ontario Research
Institute, University of
Ottawa, Canada
Drafting/revision of the
manuscript for content,
including medical writing for
content; major role in the
acquisition of data; study
concept or design; analysis
or interpretation of data
Aren
Elizabeth
Marshall,
PhD
Children’s Hospital of
Eastern Ontario Research
Institute, University of
Ottawa, Canada
Drafting/revision of the
manuscript for content,
including medical writing for
content; major role in the
acquisition of data; analysis
or interpretation of data
Arran
McBride,
MSc
Children’s Hospital of
Eastern Ontario Research
Institute, University of
Ottawa, Canada
Major role in the acquisition
of data; analysis or
interpretation of data
Madeline
Couse, MSc
Hospital for Sick Children,
Centre for Computational
Medicine, Toronto, Canada
Major role in the acquisition
of data; analysis or
interpretation of data
William
Macdonald,
PhD
Department of Obstetrics,
Gynaecology and Reproductive
Sciences, University of
Pittsburgh School of Medicine;
Magee-Womens Research
Institute, Pittsburgh, PA
Major role in the acquisition
of data; analysis or
interpretation of data
Mellissa
R.W. Mann,
PhD
Department of Obstetrics,
Gynaecology and
Reproductive Sciences,
University of Pittsburgh
School of Medicine; Magee-
Womens Research Institute,
Pittsburgh, PA
Drafting/revision of the
manuscript for content,
including medical writing for
content; major role in the
acquisition of data; analysis
or interpretation of data
Pierre R.
Bourque,
MD
Department of Medicine, The
Ottawa Hospital; The Ottawa
Hospital Research Institute;
Faculty of Medicine,
University of Ottawa, Canada
Drafting/revision of the
manuscript for content,
including medical writing for
content; analysis or
interpretation of data
Ari Breiner,
MD, MSc
Department of Medicine, The
Ottawa Hospital; Faculty of
Medicine, University of Ottawa;
Department of Pathology and
Laboratory Medicine, The
Ottawa Hospital, Canada
Major role in the acquisition
of data; analysis or
interpretation of data
Hanns
Lochm¨
uller,
MD, PhD
Department of Medicine, The
Ottawa Hospital; The Ottawa
Hospital Research Institute;
Faculty of Medicine;
Children’sHospital of
Eastern Ontario Research
Institute, University of
Ottawa, Canada
Drafting/revision of the
manuscript for content,
including medical writing for
content; major role in the
acquisition of data; analysis
or interpretation of data
John
Woulfe, MD,
PhD
The Ottawa Hospital
Research Institute; Faculty of
Medicine, University of
Ottawa; Department of
Pathology and Laboratory
Medicine, The Ottawa
Hospital, Canada
Major role in the acquisition
of data; analysis or
interpretation of data
Marcos
Loreto
Sampaio,
MD
The Ottawa Hospital Research
Institute; Faculty of Medicine;
Department of Radiology,
Radiation Oncology and
Medical Physics, University of
Ottawa, Canada
Major role in the acquisition
of data; analysis or
interpretation of data
Continued
Neurology.org/NG Neurology: Genetics | Volume 9, Number 5 | October 2023 9

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Appendix (continued)
Name Location Contribution
Gerd
Melkus, PhD
The Ottawa Hospital
Research Institute;
Department of Radiology,
Radiation Oncology and
Medical Physics, University
of Ottawa, Canada
Major role in the acquisition
of data; analysis or
interpretation of data
Bernard
Brais,
MDCM, PhD
Department of Neurology
and Neurosurgery, Montreal
Neurological Institute and
Hospital, McGill University,
Canada
Major role in the acquisition
of data; analysis or
interpretation of data
David A.
Dyment,
DPhil, MD
Faculty of Medicine;
Children’s Hospital of
Eastern Ontario Research
Institute, University of
Ottawa, Canada
Major role in the acquisition
of data; analysis or
interpretation of data
Kym M.
Boycott,
MD, PhD
Faculty of Medicine;
Children’s Hospital of
Eastern Ontario Research
Institute, University of
Ottawa, Canada
Drafting/revision of the
manuscript for content,
including medical writing for
content; major role in the
acquisition of data; study
concept or design; analysis
or interpretation of data
Kristin
Kernohan,
PhD
Children’s Hospital of
Eastern Ontario Research
Institute, University of
Ottawa; Newborn Screening
Ontario, Children’s Hospital
of Eastern Ontario, Ottawa,
Canada
Drafting/revision of the
manuscript for content,
including medical writing for
content; major role in the
acquisition of data; study
concept or design; analysis
or interpretation of data
10 Neurology: Genetics | Volume 9, Number 5 | October 2023 Neurology.org/NG

DOI 10.1212/NXG.0000000000200088
2023;9; Neurol Genet
Jodi Warman-Chardon, Taila Hartley, Aren Elizabeth Marshall, et al.
Deformities, Intellectual Disability, and Ovarian Dysfunction
Variants Associated With Novel Syndrome With Myopathy, SkeletalSOX8Biallelic
This information is current as of September 19, 2023
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