FIGURE LEGENDS
Figure 1. Phenotypes and genotypes in patients carrying biallelicCC2D2A variants
A. Distribution of phenotypes associated with CC2D2A biallelic variants in reported patients. n indicates total number of patients. ASD, autism spectrum disorder; Cogan, Cogan-type congenital oculomotor apraxia; JBTS, Joubert syndrome; JSRD, Joubert syndrome related disorders; MKS, Meckel syndrome; ML, Meckel-like syndrome; RCD, rod cone dystrophy; ?, not unequivocally described. B. Distribution ofCC2D2A variant consequences detected in index patients. n indicates total number of alleles. AAdel, single amino acid deletion; Large ins/del, large insertions/deletion including retrotransposon insertion. C. Distribution of CC2D2A allelic status detected in patients with Meckel syndrome or Meckel-like syndrome. D. Distribution of CC2D2A allelic status detected in patients with Joubert syndrome or Joubert syndrome related disorders. E. Distribution ofCC2D2A allelic status detected in patients with kidney disease. F. Distribution of CC2D2A allelic status detected in patients without kidney disease. Truncating indicates either a nonsense or a frameshift variant.
Figure 2. Exon usage and tissue specific transcript expression ofCEP120 and CC2D2A
Ai. CEP120 predicted protein coding transcript isoforms with highest expression levels in kidney medulla (red) und cerebellar hemisphere (blue) based on RNA sequencing data from the Genotype-Tissue Expression (GTEx) Project. Aii. CEP120 genomic localization, the different exons detected in GTEx data with the different imputed splice junctions and the three transcripts detected at highest levels in kidney medulla and cerebellar hemisphere. Exons are labelled with respect to transcript ENST00000328236. The open reading frame is shown in dark grey with the start codon marked with an arrowhead. Aiii. Predicted protein products from the three analyzed transcripts. Bi. CC2D2Apredicted protein coding transcript isoforms with highest expression levels in kidney medulla (red) and cerebellar hemisphere (blue) based on RNA sequencing data from the Genotype-Tissue Expression (GTEx) Project. Bii. CC2D2A genomic localization, the different exons detected in GTEx data with the different imputed splice junctions and the three transcripts detected at highest levels in kidney medulla and cerebellar hemisphere. Exons are labelled with respect to transcript ENST00000503292. The open reading frame is shown in dark grey with the start codon marked with an arrowhead. Junction reads enriched in the kidney medulla (compared to cerebellum) or marked in red and junction reads enriched in the cerebellum (compared to kidney) are marked in blue. Biii. Predicted protein products from the three analyzed transcripts, sequences deviating from reference sequence are depicted in orange. The Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. The data used for the analyses described in this manuscript were obtained from the GTEx Portal on 15/05/2020.
Figure 3. Basal exon skipping of CC2D2A exon 30 in kidney and human urine-derived renal epithelial cells (hURECs) and correlation with tissue specific disease expression
A. RT-PCR using RNA isolated from human kidney, whole blood and hURECs.CC2D2A primer pair (arrows) designed to detect exon 30 skipping illustrated on the right. Note shortened transcript at ~150bp (*asterisk) detected in kidney and hURECs suggesting basal exon 30 skipping. B. Prevalence of kidney disease associated with truncating CC2D2A variants in different exons. Exons are labelled according to transcript ENST00000328236. The different exons and possible splice junctions detected in GTEx are shown below transcript ENST00000328236. The specific splice junction leading to basal exon 30 skipping in the kidney is marked in diagonal shading. N indicates the total number of patients (with and without kidney disease) harboring at least one truncating variants in the corresponding exons. 35/48 (73%) present with kidney disease. Note that both patients with a truncating variant in exon 30, undergoing basal exon skipping in the kidney, have no reported kidney disease (0%).
Figure 4: Distribution of mutations in CEP120 and CC2D2Aand identification of potential targets for exon skipping
A. CEP120 mRNA (NM_153223.3) and exon structure with UTR in grey. Exon numbers are shown below exons with nucleotide numbers in multiples of three below the exon numbers. Exact multiples of three are shown in green. Protein domains are shown in color-code for coiled-coil domain (CC) and the 3 C2 domains. Detected CEP120 variants are painted above the mRNA structure with respect to their location and allelic frequency in index patients. Variant consequences are color-coded as indicated. Exon numbers that appear as candidates for exons skipping based on nucleotide numbers and domain functions are shaded in green, while candidates arising from tissue-specific transcript analysis are shaded in blue. B. CC2D2A mRNA (NM_001080522.2) and exon structure with UTR in grey. Exon numbers are shown below exons with nucleotide numbers in multiples of three below the exon numbers. Exact multiples of three are shown in green. Protein domains are shown in color-code for coiled-coil domain 1 and 2 (CC) and the C2 domain. Exon numbers that appear as candidates for exons skipping based on nucleotide numbers and domain functions are shaded in green, candidates arising from tissue-specific transcript analysis are shaded in blue and possible candidate based on conflicting domain annotation shaded in turquoise. Only truncating CC2D2A variants that are reported in candidate exons for exon skipping are painted above the mRNA structure with respect to their location and allelic frequency in index patients. Variant consequences are color-coded as indicated. The retrotransposon insertion described in one family is not represented.