When the sum is better than the parts: combining the power of comparative genomics and experiments on model organisms

I have been doing some reading for a while now on the ethics of research involving model organisms, particularly the potential for studies on lower species to offer insights into human disease (and thereby contribute to the 3Rs). Some of my musings on the topic can be found here.

Aware of this interest, a colleague recommended that I read a 2004 paper published in the journal Cell. I am very grateful that he did, since the study really has the “wow” factor – demonstrating beautifully the potential of comparative genomics, experiments on model organisms and knowledge of human disease to work together to produce new insights that would have been much harder if any one component was missing. The paper is Comparative genomics identifies a flagellar and basal body proteome that includes the BBS5 human disease gene by Li JB et al. The following notes are my attempt to summarise the best bits.

The importance of cilia and basal bodies in disease
The role of cilia in respiration (and the detrimental effects of smoking on their function) were features of the school biology curriculum when I was a child. However, research over the last ten years or so has demonstrated that cilia have surprisingly diverse roles in development, from determination of left-right symmetry in the body, through to formation and function of specific organs such as the kidneys (for more detail see the Wikipedia entry on Ciliopathy or, if you have access permissions,  Badano et al (2006), The ciliopathies: an emerging class of human genetic disorders Annual Review of Genomics and Human Genetics 7:125-148). Bardet-Biedl syndrome (BBS) is one disorder associated with non-functional or malfunctional cilia. The clinical features can be varied, but include obesity, mental retardation, progressive-onset blindness and polydactylism (i.e. possession of extra digits).

Cilia, and flagella in organisms that possess these related structures, develop from, and remain attached to, modified centrioles called basal bodies. These complex structures involve upwards of 150 proteins.

Comparative genomics
The complete genome sequence for a growing collection of organisms is now known. The authors of the Cell paper – headed up by Susan Dutcher of Washington University, St Louis –  reasoned that they could identify novel genes involved in basal body development by judicious use of such genomic information.

The logic goes like this: basal bodies are needed for both cilia and flagella, therefore genes necessary for proteins in the basal body should be present in the genomes of an organism with cilia and an organism with flagella – even if otherwise morphologically dissimilar – but not in the genome of a species with neither cilia nor flagella. A Venn diagram can be helpful in visualising this subset of genes.

genomevenn

researchers were interested in finding genes expressed in humans (an organism with cilia) and Chlamydomonas (an organism with a flagellum) but not Arabidopsis (which has neither cilia nor flagella) - see text for details

When they looked at the genomes of humans (ciliated, approx 32,ooo genes) and Chlamydomonas (a unicellular flagellate with approximately 20,000 genes) they homed in on a set of about 4,350 genes that were common to both species despite their obvious diversity.

Next, they subtracted from that list all of the genes that were also present in the genome of the thale cress Arabidopsis (approximately 29,000 genes in total).  This plant species has neither cilia nor flagella and therefore ought not to have the genes necessary for their development.

It turned out that a total of 686 genes fit into the subset “in human and in Chlamydomonas but not in Arabidopsis“, termed the flagellar apparatus-basal body (FABB) group by the researchers, and corresponding to the yellow sector in the Venn Diagram above. This may still seem a large number, but when compared with the total of at least 20,000 genes it represents elimination of over 96% of the genome as not being involved in basal bodies.  In this way they hoped that they had shortened the odds on identifying the role(s) played by genes of previously unknown function.

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Is this a valid approach?
If this methodology was to prove successful, a number of predictions about the membership of the FABB sub-group ought to hold true:
  • It ought to contain many of the genes that are already known to be involved in the formation and functioning of flagella and/or cilia
  • It ought to contain unusually high numbers of proteins whose predicted sub-structure fits with features already known amongst basal body proteins
  • Genes in this sub-group ought to be upregulated in Chlamydomonas after they have treated in a way that removed their pre-existing flagellum
  • Interference with the expression of these genes (either at the DNA or mRNA level) ought to cause problems with the functioning of flagella or cilia
  • It ought to include previously unidentified genes which, when mutated, lead to cilia-related disorder in man
By combining a variety of approaches, the authors of this study were able to confirm each of these presuppositions.

Representation of known genes: 52 out of 58 known genes affecting flagellar formation or function in Chlamydomonas were included in the FABB set. In addition 5 out of 6 genes causing Bardet-Biedl Syndrome in humans were within this group.

Characteristic protein sub-structure: The researchers used powerful computer-based structure prediction to compare the human versions of the 688 proteins encoded by genes in the FABB group against 785 random human proteins not in this collection. They found recognised structural features (‘motifs’) associated with cilia were over-represented in the FABB group and that motifs associated with non-cilia proteins were under-represented.

Impact of flagella removal on gene expression: Removal of the flagella of Chlamydomonas can be achieved by sudden change to the pH of the solution in which the cells are suspended. After such a procedure, it is logical that the expression of genes involved in the (re)construction of flagella will be increased. This proved to be the case, with 39 out of the 103 FABB genes tested (38%) demonstrating at least 3-fold upregulation after deflagellation, compared with only 1 in 10 non-FABB genes found in all three organisms.

Gene knockdown leads to flagellar phenotypes: RNA interference (RNAi) in Chlamydomonas was used to reduce the levels of mRNA for six of the FABB genes identified as upregulated after deflagellation. If they genuine encode flagellar proteins then lowering their expression via RNAi ought to lead to phenotypes akin to known flagella mutations. As it transpired, five of the six genes tested demonstrated phenotypes ranging from slow swimming through to a complete lack of flagella.

Confirmation of identity of human disease genes within the FABB set: In addition to the five known Bardet-Biedl genes within the shared set “in humans and in Chlamydomonas but not in Arabidopsis“, one further gene was identified in this study. On the basis of studies involving a Canadian family with an inheritable version of BBS, a gene BBS5 had been mapped to a 14 Mb region of human chromosome 2, but the specific gene responsible had not been identified (there are 230 predicted genes in this stretch). Two genes encoded within this region were represented in the FABB group, one of which was already known to be encode a protein involved in cilia-related disorders. The other gene, however, had not previously been implicated in cilia or flagella function.

In analysing the newly identified gene, the authors were able to identify an A to G point mutation which segregated with the BBS phenotype within the extended family – two suffers in the study group were homozygous G/G, their parents were heterozygous A/G and unaffected relatives were all A/A or A/G. Armed with this information they then examined inheritance of the same gene in different families (from Saudia Arabia and Turkey). All confirmed that the errors in the new gene were responsible for the BBS phenotype, meaning that the gene can be identified as BBS5.

Exemplifying the beauty of combining the different approaches, the team were then able to carry their new information about BBS5 back into studies involving Chlamydomonas. RNAi experiments targeting the BBS5 homolog confirmed the essential role this gene plays in flagella formation and function.

In conclusion, the authors note that this same approach might be applied to genes conserved across different phyla in order to shine a spotlight on particular subsets of functionally-related genes. They also predict that their FABB collection will ultimately be shown to include genes involved in other cilia-related disorders such as retinal degeneration and nephropathies.


 


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