Characterizing the morbid genome of ciliopathies

Abstract

Background: Ciliopathies are clinically diverse disorders of the primary cilium. Remarkable progress has been made in understanding the molecular basis of these genetically heterogeneous conditions; however, our knowledge of their morbid genome, pleiotropy, and variable expressivity remains incomplete.

Results: We applied genomic approaches on a large patient cohort of 371 affected individuals from 265 families, with phenotypes that span the entire ciliopathy spectrum. Likely causal mutations in previously described ciliopathy genes were identified in 85% (225/265) of the families, adding 32 novel alleles. Consistent with a fully penetrant model for these genes, we found no significant difference in their "mutation load" beyond the causal variants between our ciliopathy cohort and a control non-ciliopathy cohort. Genomic analysis of our cohort further identified mutations in a novel morbid gene TXNDC15, encoding a thiol isomerase, based on independent loss of function mutations in individuals with a consistent ciliopathy phenotype (Meckel-Gruber syndrome) and a functional effect of its deficiency on ciliary signaling. Our study also highlighted seven novel candidate genes (TRAPPC3, EXOC3L2, FAM98C, C17orf61, LRRCC1, NEK4, and CELSR2) some of which have established links to ciliogenesis. Finally, we show that the morbid genome of ciliopathies encompasses many founder mutations, the combined carrier frequency of which accounts for a high disease burden in the study population.

Conclusions: Our study increases our understanding of the morbid genome of ciliopathies. We also provide the strongest evidence, to date, in support of the classical Mendelian inheritance of Bardet-Biedl syndrome and other ciliopathies.

Keywords: Acrocallosal; Bardet-Biedl; Cilia; Founder; Joubert; Meckel-Gruber; Modifier; Nephronophthisis; Oligogenic; Oral-facial-digital; Polycystic kidney; Senior-Loken; Variability.

Fig. 1

A pie chart representation of the distribution of the different ciliopathy syndromes included in the study cohort

Fig. 2

TXNDC15 is a novel gene that causes Meckel-Gruber syndrome. a Pedigree of the three families showing the consanguineous nature of the parents. The index is indicated in each pedigree by a black arrow. b Upper panel: sequence chromatogram for the three homozygous mutant alleles in TXNDC15 and their locations indicated in a schematic of TXNDC15. Lower panel: schematic of the TXNDC15 protein and the location of the mutations in each specific domain. c Multisequence alignment of the deleted five amino acid residues (p. (Ser225_His229del)) showing high conservation of this part of amino acids down to Taeniopygia guttata (boxed in red)

Fig. 3

Mutation in TXNDC15 causes ciliogenesis defect in patient cells and aberrant localization of TMEM67 using TXNDC15 RNAi. a and b Immunofluorescence images of serum-starved fibroblasts from the affected individual in family 3 (II:1) and control fibroblasts (a) and TXNDC15 siRNA in human hTERT-RPE1 cells (b) stained for the ciliary marker ARL13B (green), gamma tubulin (red), and DNA (blue). Compared to controls, fibroblasts from II:1 in family 3 and TXNDC15 siRNA knockdown showed a marked ciliogenesis defect. c Bar graph showing the significant reduction in the number of ciliated fibroblast cells derived from individual II:1 from family 3. d Bar graph showing a significant reduction in the number of ciliated TXNDC15 siRNA cells compared to negative control scrambled siRNA. e Bar graph showing the efficiency of TXNDC15 siRNA compared to negative control scrambled siRNA (siScr) as quantified by qRT-PCR for the TXNDC15 transcript. f Bar graph showing the average increase in the cilium length following TXNDC15 siRNA knockdown compared to negative control scrambled siRNA (siScr). g Immunofluorescence microscopy images of serum-starved hTERT-RPE1 cells following TXNDC15 siRNA knockdown stained for the ciliary marker ARL13B (red), the transition zone marker TMEM67 (green), and DNA (blue) showing the incorrect localization of the TMEM67 ciliary receptor to the transition zone compared to negative control scrambled siRNA (siScr). h Bar graph showing an elongation in the total cilia length and elongation of the transition zone due to mislocalization of TMEM67 (arrowheads in g). Statistical significance of pair-wise comparisons are indicated by * p < 0.05, ** p < 0.01, and **** p < 0.0001 (panels c–f, Student’s paired t test for n = 3 biological replicates; and panel h, Pearson’s chi-squared test for n = 3 biological replicates)

Fig. 4

Circos image showing the different ciliopathy disorders in relation to known ciliopathy genes

Fig. 5

Circos image showing the different ciliopathy disorders in relation to ciliary compartment