In previous studies, intensive analyses were performed on an interval on Chromosome 4 that modulates the severity of renal cystic and biliary phenotypes. Intensive analyses of the interval using (C57BL/6J-Cys1cpk x CAST/Ei)F2 mice revealed three individual QTL effects that were designated as Mpkd1, Mpkd2 and Mpkd3, [J:160284].
In the current study, congenic strain analyses were used to fine-map the predicted renal cystic disease-modulating effects of the Mpkd loci and provide further supportive evidence implicating Kif12 as the candidate Mpkd2 locus based upon genetic, informatic, and immunolocalization analyses.
A congenic line homozygous for the CAST/EiJ derived proximal-medial segment of Chr 4 spanning the Mpdk1, Mpdk2 and Mpkd3 loci on the C56BL/6J background was developed, B6.CAST-(D4Mit314-D4Mit11). To more precisely evaluate the CAST-derived effects, the Cys1cpk mutation was introgressed into the congenic line using a (B6.CAST-(D4Mit314-D4Mit11 x Bg(CG)-Cys1cpk/Cys1+/J) backcross, creating a new line of B6.Cg-(D4Mit314-D4Mit11)CAST/EiJCys1cpk mice. A control line was also generated in which the Chr 4 segment was derived from the B6 strain, B6.Cg-(D4Mit314-D4Mit11)<C57BL/6J<Cys1cpk. Previous studies suggested strong interactions among the individual Mpkd1-3 loci.
As the first step towards determining whether these loci may also act independently, a new Cys1cpk/+ congenic line was generated to establish the congenic line B6.CAST-(D4Mit314-D4Mit80) in which the CAST-derived Chr 4 interval included only the proximal segment of Chr 4 that contained Mapkd1 and Mapkd2, but not Mapkd3. To evaluate a subset of dominant renal cystic disease-promoting effects associated with
CAST-derived Mpkd1-2 loci, Cys1cpk/cpk mice heterozygous for the CAST-derived Mpkd1-2 loci were generated and their renal cystic disease severity was compared with Cys1cpk/cpk mice homozygous for B6-derived Mpkd1-2 loci. The two groups were generated in the same (B6.CAST-(D4Mit314-D4Mit80) x B6(Cg)-Cys1cpk/Cys1+/J) backcross.
Phenotypic analysis at post-natal day 10 revealed strong disease-accelerating effects of CAST-derived Mpkd1-2 loci. However, the CAST derived Mpkd1-2 locus had no effect on total cyst number or number of cysts in either the medulla or the cortex [Table 1]. The data suggested that the Mpkd1-2 loci do not promote renal cystogenesis per se, but rather modulate renal cystic disease severity.
A deletion mapping approach was used to fine map the renal cystic disease-modulating
effects of the Mpkd2 locus using 10-d old Cys1cpk/cpk Mpkd1-2 recombinants that were
generated from the (B6.CAST-(D4Mit314-D4Mit80) x B6(Cg)-Cys1cpk/Cys1+/J backcross. The Mpkd2 interval was narrowed to 14 Mbp defined by D4Mit288 and D4Mit83 (Chr 4 position 56,769,379 and 70.962.376).
A complementary set of strategies were developed to assess whether the Mpkd2 effect could result from a coding sequence variant within a subset of positional gene candidates that are expressed in kidney and liver, the two organs that predominantly express the recessive PKD phenotypes. The approach identified 11 promising positional Mpkd2 candidates. [Table 2].
While multiple coding variant changes were present within the 11 genes that mapped to the the interval, only the expression of the previously described candidate, Kif12 [J:160284], was strongly correlated with the expression of Cys1 across the multiple anatomical nephron structures and developmental time points catalogued in the GUDMAP Database. Kif12, is a primary cilia-associated protein.