Heterozygous truncating variants in SUFU cause congenital ocular motor apraxia

Purpose This study aimed to delineate the genetic basis of congenital ocular motor apraxia (COMA) in patients not otherwise classifiable. Methods We compiled clinical and neuroimaging data of individuals from six unrelated families with distinct clinical features of COMA who do not share common diagnostic characteristics of Joubert syndrome or other known genetic conditions associated with COMA. We used exome sequencing to identify pathogenic variants and functional studies in patient-derived fibroblasts. Results In 15 individuals, we detected familial as well as de novo heterozygous truncating causative variants in the Suppressor of Fused (SUFU) gene, a negative regulator of the Hedgehog (HH) signaling pathway. Functional studies showed no differences in cilia occurrence, morphology, or localization of ciliary proteins, such as smoothened. However, analysis of expression of HH signaling target genes detected a significant increase in the general signaling activity in COMA patient–derived fibroblasts compared with control cells. We observed higher basal HH signaling activity resulting in increased basal expression levels of GLI1, GLI2, GLI3, and Patched1. Neuroimaging revealed subtle cerebellar changes, but no full-blown molar tooth sign. Conclusion Taken together, our data imply that the clinical phenotype associated with heterozygous truncating germline variants in SUFU is a forme fruste of Joubert syndrome.

regions were enriched using the SureSelect XT All Exon V7 in-solution technology (Agilent, Santa Clara, USA) and were sequenced using the Illumina NovaSeq system (Illumina, San Diego, USA).Illumina bcl2fastq2 was used to demultiplex sequencing reads.Adapter removal was performed with Skewer.The trimmed reads were mapped to the human reference genome (hg19) using the Burrows Wheeler Aligner and variants were called using inhouse software.Only SNVs and small indels in the coding regions and the flanking intronic regions (±8 bp) with a MAF <1.5% were evaluated.Minor allele frequencies were taken from public databases (gnomAD, dbSNP) and an in-house database.Known disease-causing variants (according to HGMD®) were evaluated in up to ±30 bp of flanking regions and up to 5% MAF.Evaluation was based on the ACMG guidelines for the interpretation of sequence variants.In the affected subject II.1 of family 5, all exons and adjacent exon-intron boundaries of SUFU were analyzed by PCR and subsequent, bidirectional Sanger sequencing.In family 6, ES was performed for individual II.1 using the SureSelect V6 enrichment kit (Agilent), paired-end sequenced on an Illumina HiSeq4000 and analyzed using the pipeline of the Department of Human Genetics, Technical University of Munich, Germany, as described previously. 13l detected SUFU variants were confirmed by PCR amplification and subsequent Sanger sequencing on an independent DNA sample and tested for co-segregation within the respective families using the BigDye terminator v3.1 chemistry (Thermo Fisher Scientific) on a 3500 Genetic Analyzer (Thermo Fisher Scientific).

Cell culture and treatments
Primary dermal fibroblasts established from affected subjects III.3 (family 1), II.1 (family 2), II.1 (family 3), II.1 (family 4) and five healthy control subjects were cultured in Dulbecco's modified Eagle medium (DMEM, Gibco) supplemented with 10% fetal calf serum (FCS, Gibco) and antibiotics at 5% CO2 and 37°C.Before treatment, cells were starved in DMEM supplemented with 0.1% FCS and antibiotics for 24 hrs.Treatment of cells was carried out with 100 nM Smoothened Agonist (SAG, Cayman Chemical), 1 µM vismodegib (Selleckchem) or a combination of both reagents for 16 hrs.SAG and vismodegib were solved in DMSO.DMSO-treated cells served as controls.Experiments were performed in biological triplicates.

Cilia formation and immunofluorescence staining
For analysis of cilia formation, fibroblasts were grown on coverslips to 90%-95% confluency.

Real-time quantitative PCR
Total RNA was extracted from dermal fibroblasts using TRIzol Reagent (Thermo Fisher Scientific).Synthesis of cDNA was performed using the Superscript II reverse transcriptase (Invitrogen) and random hexamer primers.Gene expression for GLI1, GLI2, GLI3, HIP1, and PTCH1 was quantified by SYBR Green-based qRT-PCR assays on an ABI Prism HT 7900 Detection System instrument (Applied Biosystems) by using the primer pairs listed in Table S1.Data were analyzed by the standard curve method for relative quantification.
Experiments were performed with two different passages per fibroblasts each analyzed in biological triplicates, which were measured in technical triplicates.Amplification of 18S rRNA and HPRT were used as endogenous controls for the normalization of target gene expression.

Statistical analysis
qRT-PCR data analysis and determination of statistical differences were performed using the software GraphPad Prism 6 by nonparametric Mann-Whitney testing.Values were considered significant when p < 0.05.
Figure S1Expression of Hedgehog signaling signature genes in COMA-patient derived dermal fibroblasts A This questionnaire used in this study was published previously as additional file with reference 12: Wente S., et al.Nosological delineation of congenital ocular motor apraxia type Cogan: an observational study.Orphanet J Rare Dis.2016;11(1),104.doi:10.1186/s13023-016-0486-z