Abstract
Polycystic kidney disease (PKD) is a genetic disorder that affects between 1 in 400 to in 1000 people worldwide. PKD is a ciliopathy and is characterized by fluid-filled cysts that form in both kidneys, causing significant kidney enlargement and eventually leading to kidney failure. There are two main types of PKD: autosomaldominant polycystic kidney disease (ADPKD) and autosomal recessive kidney disease (ARPKD). The disease can be associated with many complex syndromes, for example, Bardet-Biedl Syndrome (BBS), and Meckel-Gruber Syndrome (MKS). MKS is a lethal malformation disorder characterized by bilateral cystic dysplasia, hepatic developmental defects, polydactyly, and brain abnormalities. MKS3/TMEM67 has been identified as one of the MKS-causing genes. We have found 2 sequence variants causing amino acid changes in exon 20 of the ovine MKS3/TMEM67 gene, p.[(Ile680Asn; Ile686Ser)]. This is associated with polycystic kidneys in newborn lambs, and has been observed in two different flocks with distinct genetic backgrounds. This finding leads to our hypothesis that the sheep might suffer from MKS, caused by these amino acid changes in MKS3.
To investigate whether the amino acid changes in the ovine MKS3 gene were the cause of the PKD in our sheep, the current project utilized an anti-sense mks3-targetted morpholino knockdown methodology in zebrafish in order to produce mks3-knockdown zebrafish embryos, followed by a rescue experiment with either the mutant (OaMKS3(I680N;I686S) or the wild-type ovine MKS3 mRNA (OaMKS3). The knockdown phenotypes included shortened body axis, thin and wide somites, undulated notochords in 11-12 somite embryos, cardiac oedema, body axis anomalies, possible brain defects, and notochord deformities in the 60-72 hours postfertilization (hpf) embryos. Zebrafish embryos were either injected with morpholino alone, or co-injected with both the morpholino and OaMKS3 or OaMKS3(I680N;I686S) mRNA in order to carry out the rescue experiments.
As indicated above, we investigated the pathogenicity of the two ovine missense mutations by using the OaMKS3 and the OaMKS3(I680N;I686S) mRNA, which contains the two mutations of interest, to rescue the mks3-knockdown embryos. The MKS3 functional studies were performed in zebrafish at the 11-12-somite (14 hpf) stage, resulting in a statistically significant rescue by OaMKS3 mRNA (**p-value < 0.01). In contrast, the OaMKS3(I680N;I686S) construct showed a significant reduction in ability to rescue the morphological phenotype compared to the wildtype rescue (*p-value < 0.05) suggesting that the two mutations have pathogenic potential. This data was supported by the results obtained from 11-12-somite embryos stained by in situ hybridization and 60-72 hpf embryos that presented similar rescue trends. Furthermore, as the majority of the mutant mRNA-rescued population presented with either grade 1 or normal phenotype, rather than a grade 2 phenotype, which was the most common phenotype presenting in the mks3 knockdown embryos, this suggested that there was possibly only a partial rescue induced by the mutant construct. Therefore, in summary, the investigations we have presented in this thesis suggest that the two missense mutations, p.[(Ile680Asn; Ile686Ser)], are indeed pathogenic and were the likely cause of Meckel-Gruber syndrome in our sheep.