Exome sequencing and cis-regulatory mapping identify mutations in MAK, a gene encoding a regulator of ciliary length, as a cause of retinitis pigmentosa

Abstract

A fundamental challenge in analyzing exome-sequence data is distinguishing pathogenic mutations from background polymorphisms. To address this problem in the context of a genetically heterogeneous disease, retinitis pigmentosa (RP), we devised a candidate-gene prioritization strategy called cis-regulatory mapping that utilizes ChIP-seq data for the photoreceptor transcription factor CRX to rank candidate genes. Exome sequencing combined with this approach identified a homozygous nonsense mutation in male germ cell-associated kinase (MAK) in the single affected member of a consanguineous Turkish family with RP. MAK encodes a cilium-associated mitogen-activated protein kinase whose function is conserved from the ciliated alga, Chlamydomonas reinhardtii, to humans. Mutations in MAK orthologs in mice and other model organisms result in abnormally long cilia and, in mice, rapid photoreceptor degeneration. Subsequent sequence analyses of additional individuals with RP identified five probands with missense mutations in MAK. Two of these mutations alter amino acids that are conserved in all known kinases, and an in vitro kinase assay indicates that these mutations result in a loss of kinase activity. Thus, kinase activity appears to be critical for MAK function in humans. This study highlights a previously underappreciated role for CRX as a direct transcriptional regulator of ciliary genes in photoreceptors. In addition, it demonstrates the effectiveness of CRX-based cis-regulatory mapping in prioritizing candidate genes from exome data and suggests that this strategy should be generally applicable to a range of retinal diseases.

Figure 1

cis-Regulatory Mapping at the RHO Locus (A) The genomic interval including 25 genes on either side of the human RHO locus (pink oval) spans 4.8 Mb. (B) There are eight CBRs in the immediate vicinity of the RHO locus that were assigned to nearby genes as indicated by the arrows. The number of raw sequence reads corresponding to each of the CBRs is shown. (C) The 51 genes shown in (A) were ranked according to the number of raw sequence reads assigned to them. The RHO locus (green arrow), which was assigned a total of 579 raw sequence reads, ranked number one out of all 51 genes in this interval. Only the ten highest ranking genes are shown. Portions of the images in (A) and (B) are adapted from the UCSC Genome Browser.

Figure 2

cis-Regulatory Mapping Identifies MAK as a Cause of Retinitis Pigmentosa (A) The family tree of the Turkish subject with RP. (B) The 38 genes with putative homozygous mutations in the exome-sequence dataset were ranked according to their number of raw sequence reads. MAK (green arrow) ranked fourth. Only the ten highest ranking genes are shown. (C) CBRs in the vicinity of the MAK locus. (D) A PCR product encompassing MAK-CBR3 was cloned into a DsRed reporter vector and coelectroporated along with a ubiquitously expressing CAG-eGFP loading control into explanted P0 mouse retinas. The retinas were grown for 8 days and then imaged in both red and green channels in flat-mount and as cross-sections. MAK-CBR3 drives strong, photoreceptor-specific expression restricted to the outer nuclear layer. The following abbreviations are used: ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

Figure 3

Analysis of Additional MAK Mutations (A) Schematic of the MAK protein shows the location of six identified disease-associated mutations. Cross-species comparison of two regions of MAK indicate that all the identified missense mutations affect highly-conserved residues. (B) In vitro kinase assay showing that two mutations in MAK, p.Gly13Ser and p.Asn130His, result in a loss of kinase activity. Note that there is a modest background level of phosphorylation of myelin basic protein (MBP) even in the empty vector control condition. The graph at the bottom shows results for two biological replicates of the kinase assay. The amount of phospho-MBP is normalized to the amount of kinase expressed. Error bars represent standard deviation.

Figure 4

The Predominant Retinal Transcript Isoform of MAK Includes an Alternative Exon (A) Pattern of phylogenetic conservation within the alternative exon of MAK. The images are adapted from the UCSC Genome Browser.³⁸ (B) PCR results assessing the presence of the alternative exon in cDNA derived from the human retina and testis.