A number of rare diseases have been associated with cilial dysfunction, including spina bifida, some forms of retinitis pigmentosa, some obesity, some diabetes and liver disease, some breathing disorders, and so forth. A paper in last week's Science by Lee et al. describes one ciliopathy, Joubert's syndrome, a rare genetic disorder that affects the cerebellum, and thus balance and coordination. This is of general interest because, as a commentary in the same issue points out, it elucidates just one aspect of why complex diseases can be so difficult to understand.
Lee et al. identified a gene, a TMEM (transmembrane) gene, that seemed to be responsible for Joubert's syndrome in 5 of the 10 families in their study. The disorder in the other families, who did not carry the same gene variant, seemed to be phenotypically identical, so they resequenced the area around the gene in question to look for possible causative variants nearby. Sequencing of the 'exome' has become de riguer in recent years (that is, all the exons, or coding regions, in a genome; as this is only ~1% of the genome, it's a lot cheaper and faster than sequencing the entire genome). But, as this paper and commentary point out, restricting the search only to exons can miss important variants.
Indeed, Lee et al. found mutations in the neighboring related TMEM gene. Both genes, TMEM138 and TMEM216, encode transmembrane proteins, that is, proteins that rest across cell membranes with part sticking out into the space surrounding the cell where it can monitor aspects of the environment, and the other part remaining inside the cell. But the authors found no homologous regions in the genes or the resulting proteins, and thus nothing that explained why the disease could be the same in all families. This prompted them to look for shared sequence in the regulation of the expression of the two genes.
To test for coordinated expression, we examined tissue-expression patterns of human TMEM138 and TMEM216 using the microarray database and in situ hybridization of human embryos. We found tight coexpression values of human TMEM138 and TMEM216 across the major tissues, including the brain and kidneys, and similar expression patterns in various tissues, including the kidneys, cerebellar buds, and telencephalon, at 4 to 8 gestational weeks (gw) of human embryos. To test whether this coordinated expression was due to the adjacent localization, we compared mRNA levels in zebrafish versus mice, representing species before and after the gene rearrangement event. Using quantitative polymerase chain reaction (qPCR), we detected tightly coordinated expression levels in mice compared with those in zebrafish (correlation coefficient r = 0.984 versus 0.386), which suggests that TMEM138 and TMEM216 might share regulatory elements (REs) within the ~23-kb intergenic region. We further examined several experimental features and found that regulatory factor X 4, a transcription factor regulating ciliary genes, binds a RE conserved in the noncoding intergenic region to mediate coordinated expressions of TMEM138 and TMEM216Further analysis leads them to suggest that both genes are necessary for normal development of the cilium, and that this is because they are regulated by a shared intergenic region, a 'cis-regulatory module', or CRM, a binding site for transcription factors that regulate nearby genes but that is not itself part of those genes. How these modules arise or how the coordinated expression of genes evolves is not well-understood, but this CRM seems to explain the pattern Lee et al. found in the Joubert syndrome families they studied.
Aravinda Chakravarti and Ashish Kapoor say in their commentary on this paper, and Mendelian disease in general, that this work represents a maturing of the understanding of complex genetic disease. The genetics community should no longer be focused on single gene mutations, or even exomes (the protein-coding sections of 'genes'), but instead should recognize that complex diseases will require complex explanations.
Mutation analyses of single-gene defects have identified two puzzles: One is that not all individuals with a specific disorder have identifiable coding mutations; the other is that not all individuals with identical mutations, even in the same family, are equally affected, and some may be symptom-free. The first mystery has many suspected causes: The disorder may be due to another gene—even the adjacent one, as Lee et al. demonstrate—or arise from mutations in a gene's regulatory sequences, or be a phenocopy (a trait that is not of genetic origin but is environmentally induced and mimics the phenotype produced by a gene). This is a persistent challenge in studying an outbred organism like humans; just because a disorder is monogenic does not imply that it is monocausal. The second problem is more mysterious and far less understood. Phenotypic discordance, or variation in disease penetrance, between identical mutation bearers could result from differential environmental exposures (such as normal intelligence versus mental retardation in diet-treated versus untreated phenylketonuria).The goal remains to determine causation as well as to predict disease. The pendulum keeps swinging between the search for common and rare variants with which to do this -- as this commentary says, "Studies of Mendelian disease should also move from its preoccupation with rare variants to a focus on common polymorphisms, particularly at regulatory sequences affecting either rare disorders like Hirschsprung disease or common disorders like myocardial infarction."
One reason for the current focus -- should we call it a 'fad'? -- on rare variants is that the heavily touted promise that everything in the universe would be explained as being due to common genetic variants (and hence attractive to pharmaceutical companies and useful for widespread risk prediction) was that the theory has proven largely to be a bust. So since nobody will give up on predictive genotyping, the move was to rare variants which, not incidentally, will require extensive DNA sequencing, data bases, analysis and the grants that go with them, to find and document. It's difficult not to wax cynical in this way.
As we have written many times, here and elsewhere, when there are many pathways to the same phenotype, including gene by environmental interactions, and when everyone is genetically unique, the idea that most cases of rare or common diseases can be explained or predicted is likely to be an unattainable goal. As a rule, causation involves a spectrum of strong and weak, common and rare, interacting effects.
Still, it is a sign of progress when major players, not just those of us working on a smaller scale or even those on the sidelines, are cautioning about the ineffectiveness of looking for answers only at single genes or coding regions, or in enormous studies.