Sunday, August 16, 2009

INDELS, gene expression and hereditary cancer

INDELs (the insertion or deletion of nucleotides) comprise an emergent and increasingly important class of sequence variants in the human genome. While there is no fixed limit as to the number of nucleotides that can be inserted or deleted, the vast majority are around 1-4 nucleotides. Although initially, it was thought that both insertions and deletions arose through the same mechanisms, recent research has noted that motifs indicative of replication events were frequently found near insertions, while recombination motifs were more commonly found near deletions, which has now led to the conclusion that they arise independently.

INDELS are important since they play a pathological role in many human genetic diseases. In most cases that have been studied, INDELS occur in the open reading frames of mRNAs. This alteration can lead to termination of protein translation and loss of gene function. For example, out of frame insertions in the ribosomal gene RPS19 lead to Diamond-Blackfan Anemia Disease. In other diseases, the insertion of a triplet repeat preserves the frame, but leads to the reiteration of an amino acid. For example, in Kennedy’s disease the insertion of CAG repeats leads to a polyglutamine segment in the Androgen Receptor.

However, the recent comprehensive sequencing of the human genome has shown that INDELs are much more densely populated in the non-protein encoding regions of the human genome. Since these regions contain gene regulatory elements, this suggests that INDELs may aberrantly regulate gene expression. In this report, using a bioinformatics approach we have examined whether INDELs might regulate expression by creating novel targets for microRNAs. MicroRNAs are small RNAs consisting of twenty-one to twenty-two nucleotides. To date, around 800 microRNAs have been cloned and sequenced from the human genome. MicroRNAs down regulate gene expression by annealing to complementary sequence in the untranslated regions of mRNA. Thus, an INDEL could either provide critical nucleotides to support the binding of the microRNA or delete nucleotides that preclude the contiguous binding of the seed sequence.

We reasoned that if INDELs indeed could create microRNA target sites and down regulate expression, this might be readily apparent in genes whose lack of expression leads to cell proliferation. Thus, we examined the INDELs found in the untranslated regions of BCRA1 mRNA. BRCA1 mRNA encodes a human tumor suppressor protein whose loss of expression confers a significant risk of cancer. Using RNA: RNA interaction prediction software, we found an INDEL (out of a total of 4 reported INDELs) that was capable of significantly strengthening a microRNA target site. As shown in figure 1 the deletion of two residues (CU) creates a contiguous sequence fully complementary to the seed sequence of microRNA.

Although this concept needs to be functionally tested, it is interesting to reflect on its possible disease significance. Such a phenomena might explain the well known clinical heterogeneity observed in patients who test positive in the BCRA1 diagnostic test. Although the population who harbor a structural mutation in one allele of BCRA1 has a very high overall risk of breast and ovarian cancer, some patients get cancer whilst others don’t. The molecular mechanism that underlies this heterogeneity is not well understood.

Our observation that INDELs could create microRNA target sites suggests a plausible mechanism. BRCA1 positive patients that get cancer might do so because their “normal “ allele is in fact abnormal and contains an INDEL that can down regulate its expression. Thus, activation of the microRNA will lead to down regulation of BCRA1 and tumorigenesis. Importantly, the activation of the microRNA may itself be regulated by other cellular and environmental factors. Thus, this may provide an important link to environmental factors (hormones, carcinogens) that may promote tumorigenesis in the BRCA1 positive population. In sum, sequencing the noncoding regions of the ‘normal’ allele might provide answers to why some carriers of BRCA1 mutations get cancer, and others don’t.

Guest Contributor : Elizabeth Dietz

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