De Novo SOX6 Variants Cause a Neurodevelopmental Syndrome Associated with ADHD, Craniosynostosis, and Osteochondromas

Introduction: The SOX gene family consists of twenty transcription factors that play a pivotal role in cell fate and differentiation during the development of many organ systems. Within these SRY-related (SOX) genes is a highly conserved high mobility group (HMG) domain that has been shown to be critical for DNA binding and bending, nuclear trafficking, and protein-protein interactions. Mutations within this transcription factor family have been associated with rare congenital disorders, known as SOXopathies. These mutations are commonly de novo, heterozygous and inactivating, and exhibit gene haploinsufficiency. Of these twenty transcription factors, SOX6 is known to be involved in chondrocyte differentiation and development of the central nervous system. Although there have been reports of SOX6 variants causing adult pathological conditions, there has yet to be a well-established association between SOX6 variants and a developmental syndrome. Objectives: The objective of this study was to use clinical and genetic data to examine SOX6 mutations found in 19 individuals demonstrating developmental delay and to test the transcriptional activity of the 4 missense variants in vitro to determine if SOX6 haploinsufficiency leads to a neurodevelopmental SOXopathy. Methods: Nineteen individuals were identified as carriers of SOX6 variants, confirmed by molecular karyotyping, whole-exome sequencing, or whole-genome sequencing. Clinical pathogenicity was predicted and assessed in silico and in vitro. Expression plasmids for SOX6 missense variants were generated by PCR mutagenesis. The four missense variants generated were: p.Trp161Cys, p.Met605Thr, p.Trp639Arg, and p.Ser746Leu, with p.Met605Thr and p.Trp639Arg located within the HMG domain. For reporter assays, HEK293 cells were transfected in triplicate cultures with 3.5 µL ViaFect Transfection Reagent and a total of 1000ng of DNA. SOX6 intracellular localization was tested by transfecting either HEK293 or COS-1 cells and cytoplasmic and nuclear extracts were prepared for Western Blot analysis. Whole cell extracts transfected with respective WT-SOX6 or variant plasmid were also prepared for a dimerization assay. SOX6’s ability to bind DNA was also tested in an electrophoretic mobility shift assay (EMSA). Results: Study cohort consisted of 19 individuals from 17 unrelated families originating in Belgium, Canada, France, Germany, the Netherlands, Slovenia, the UK, and the US. These individuals shared milestone delays and intellectual disability, and exhibited abnormalities including mild dysmorphism, craniosynostosis, and osteochondromas. Immunoblots of nuclear and cytoplasmic extracts showed all variants were efficiently expressed however p.Met605Thr and p.Trp639Arg were not translocated or retained into the nucleus as efficiently as WT-SOX6 and the other two missense variants. The EMSA showed that proteins outside of the HMG domain behaved like WT-SOX6, but p.Met605Thr and p.Trp639Arg failed to bind the DNA probe. Reporter assay activity showed that the two variants outside of the HMG domain p.Trp161Cys and p.Ser746Leu displayed similar or slightly higher activity compared to WT-SOX6 while the two variants p.Met605Thr and p.Trp639Arg showed diminished reporter activity. Conclusions: These findings provide evidence that SOX6 variants cause a SOXopathy, which has been designated in Online Mendelian Inheritance in Man (OMIM) as #618971 Tolchin-Le Caignec syndrome (TOLCAS)

De Novo SOX6 Variants Cause a Neurodevelopmental Syndrome Associated with ADHD, Craniosynostosis, and Osteochondromas.

SOX6 belongs to a family of 20 SRY-related HMG-box-containing (SOX) genes that encode transcription factors controlling cell fate and differentiation in many developmental and adult processes. For SOX6, these processes include, but are not limited to, neurogenesis and skeletogenesis. Variants in half of the SOX genes have been shown to cause severe developmental and adult syndromes, referred to as SOXopathies. We here provide evidence that SOX6 variants also cause a SOXopathy. Using clinical and genetic data, we identify 19 individuals harboring various types of SOX6 alterations and exhibiting developmental delay and/or intellectual disability; the individuals are from 17 unrelated families. Additional, inconstant features include attention-deficit/hyperactivity disorder (ADHD), autism, mild facial dysmorphism, craniosynostosis, and multiple osteochondromas. All variants are heterozygous. Fourteen are de novo, one is inherited from a mosaic father, and four offspring from two families have a paternally inherited variant. Intragenic microdeletions, balanced structural rearrangements, frameshifts, and nonsense variants are predicted to inactivate the SOX6 variant allele. Four missense variants occur in residues and protein regions highly conserved evolutionarily. These variants are not detected in the gnomAD control cohort, and the amino acid substitutions are predicted to be damaging. Two of these variants are located in the HMG domain and abolish SOX6 transcriptional activity in vitro. No clear genotype-phenotype correlations are found. Taken together, these findings concur that SOX6 haploinsufficiency leads to a neurodevelopmental SOXopathy that often includes ADHD and abnormal skeletal and other features