For this reason, a number of strategies have been devised for interrogating the exomes of individuals affected with Mendelian disorders in search of disease-causing variations ( 7, 8). The majority of sequence variations that are currently known to cause Mendelian disease in humans is within or immediately adjacent to DNA sequences that are ultimately translated into protein (i.e., the exome) ( 6). When we amplified exon 9, a protein-coding segment of the MAK gene, to confirm the above-mentioned variants, we observed a larger than expected fragment. Mice lacking Mak show elongated connecting cilia, reduced electrical response to light stimulation, protein mislocalization, and ultrastructural defects in outer segment morphogenesis ( 5). In contrast, studies of the retina in Mak −/− mice reveal a dramatic effect of Mak deficiency on photoreceptor cell structure, function, and viability ( 5). Despite its expression during the cell cycle in developing germ cells, targeted deletion of Mak in mice does not result in impaired fertility ( 4). The MAK gene, which encodes the MAK enzyme, has been described as a testis-enriched gene with two transcripts that result in a complex pattern on Western blot. The ABI dataset contained two mutations in the cilia-associated gene male germ cell-associated kinase ( MAK) that were not present in the Illumina dataset. There were five genes with one truncating and one missense variant, and of these 10 variants, only 4 variants were present in the data from both sequencers. Similarly, two genes were each predicted to harbor two different truncating mutations however, only one variant in each gene was confirmed by Sanger sequencing. Three genes were predicted to have homozygous truncating mutations, which would result in a shortened nonfunctional protein, but none of these genes were confirmed to be homozygous by the automated dideoxynucleotide sequencing method. An average of 417 plausible disease-causing sequence variations, in an average of 405 genes, was identified by each sequencing platform. In both experiments, more than 20,000 sequence variations were detected. These sequences were aligned to the reference human genomic sequence ( 3). The Illumina sequencing experiment yielded 71 million uniquely mapped paired-end sequences, with each end being 50 bp in length, whereas the ABI experiment yielded 157 million uniquely mapped single-end sequences, each being 50 bp in length. Genomic DNA from a patient with autosomal recessive RP was sequenced on both Illumina and ABI sequencing instruments. In addition to showing the use of induced pluripotent stem cells to efficiently evaluate the pathogenicity of specific mutations in relatively inaccessible tissues like retina, this study reveals algorithmic and molecular obstacles to the discovery of pathogenic insertions and suggests specific changes in strategy that can be implemented to more fully harness the power of sequencing technologies. However, in the proband with the Alu insertion, the developmental switch to the MAK transcript bearing exons 9 and 12 did not occur.
In the RP control individual, we found that a transcript lacking exon 9 was predominant in undifferentiated cells, whereas a transcript bearing exon 9 and a previously unrecognized exon 12 predominated in cells that were differentiated into retinal precursors.
#FLASHFROZEN 1 3 6 SKIN#
Induced pluripotent stem cells were derived from the skin of the proband and a patient with non- MAK–associated RP (RP control). Several isoforms of MAK that result from alternative splicing were identified. Immunohistochemistry of human donor tissue revealed that MAK is expressed in the inner segments, cell bodies, and axons of rod and cone photoreceptors. MAK encodes a kinase involved in the regulation of photoreceptor-connecting cilium length. All 21 affected probands are of Jewish ancestry. Screening of 1,798 unrelated RP patients identified 20 additional probands homozygous for this insertion (1.2%). We used exome sequencing to identify a homozygous Alu insertion in exon 9 of male germ cell-associated kinase ( MAK) as the cause of disease in an isolated individual with RP. Retinitis pigmentosa (RP) is a genetically heterogeneous heritable disease characterized by apoptotic death of photoreceptor cells.
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