by ecancer reporter Clare Sansom

Chronic lymphocytic leukaemia (CLL) is the most common form of leukaemia in adults, affecting between one and five per 100,000 worldwide.

However, in contrast to chronic myeloid leukaemia, the genetic changes that are involved in the development of this leukaemia are still very poorly understood.

A group of researchers led by Carlos Lopez-Otin from the University of Oviedo, Oviedo, Spain have now used whole exome sequencing to investigate the genetic origins of CLL.

Lopez-Otin and his colleagues sequenced the exomes of matched leukaemia and normal cells from 127 individuals who had been diagnosed with CLL but not yet received any treatment.

Somatic mutations in the tumour tissue were identified using the Sidrón algorithm and a number of genes found to be frequently mutated; most had previously been implicated in leukaemia development.

One gene was noted as of particular interest, however: this was POT1, which was the second most frequently affected by point mutations and had not previously been linked to CLL.

This gene encodes the protein POT1 (Protection of telomeres protein 1) which is a nuclear protein that, as its name implies, is involved in maintaining the structure of telomeres.

Telomeres are regions of repetitive DNA at the ends of chromosomes that protect those chromosome ends from degradation and from fusion with other chromosomes.

The POT1 gene was found to be mutated in about 5% of leukaemia cases, but, crucially, it was only mutated in individuals in which the immunoglobulin gene IGHV@ had not been mutated.

The researchers then validated their findings by Sanger sequencing of the POT1 gene in tumour and matched normal tissue from a further 214 individuals diagnosed with CLL.

A total of 3.5% of all individuals with CLL, and about 9% of those carrying the wild type IGHV@ gene, were found to have mutations in POT1.

Twelve different mutations were observed in this gene, three of which were predicted to result in a truncated protein; the remaining mutations were non-synonymous and were predicted to be deleterious using bioinformatics tools.

The protein encoded by POT1 is one component of the shelterin complex of proteins that are involved in maintaining the integrity of telomeres, and it is the only shelterin protein that contains the OB fold that binds single-stranded DNA.

Nine of the 12 POT1 mutations observed in patients with CLL were found to be in the OB region, which is involved in protein-telomere interactions.

These mutations are therefore likely to destabilize this interaction and diminish the ability of the protein to bind telomeres; the other three are truncations that will produce truncated protein that is unlikely to be functional.

A more detailed structural analysis of the mutated residues confirmed that many of the point mutations observed would disrupt electrostatic interactions between the POT1 protein and telomere DNA.

Mutations in this region of POT1 have previously been observed in and functionally linked to the development of other human cancers.

Constructs containing wild type and mutant POT1 cDNA were translated in vitro, and the resulting proteins incubated with single-stranded telomeric DNA.

The wild type protein formed complexes with the telomeric DNA efficiently, whereas none of the mutated proteins were able to do so; they were, however, able to localize to the telomeres with the other shelterin proteins.

Expression of mutant POT1 in human cells, and of its mouse homolog in mouse cells, resulted in elongated telomeres with unprotected ends that led eventually to defects in the chromosomes.

Unprotected telomere ends can lead to an accumulation of genomic aberrations, which is a key event in tumour formation.

The researchers used FISH analysis to examine genomic aberrations in leukaemia cells in metaphase, and discovered that more aberrations and particularly more end-to-end chromatid fusions occurred in cells bearing POT1 mutations.

Taken together, these results suggest that POT1 – the first shelterin gene to be discovered to be mutated in human cancer – is frequently mutated in CLL, particularly in individuals with wild type IGHV@, and that mutations in this gene drive genetic instability and cancer development.

This finding may have implications for the development of novel treatments for this relatively common disease.

Reference

Ramsay, A.J., Quesada, V., Foronda, M. and 13 others (2010). POT1 mutations cause telomere dysfunction in chronic lymphocytic leukemia. Nature Genetics, published online ahead of print 17 March 2013. doi:10.1038/ng.2584