NEW YORK (GenomeWeb News) – A research team studying a rare condition called "congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies," or CLOVES, has tracked down somatic mutations in the cancer-related gene PIK3CA that turn up with varying frequency in some, but not all, patient tissues.
As they reported online yesterday in the American Journal of Human Genetics, researchers from the US and Brazil used targeted and whole-exome sequencing to look for genetic glitches present specifically in affected tissues from half a dozen children with CLOVES.
The search led to mosaic, activating mutations in PIK3CA that patients had not inherited from their parents, but which appear to have arisen during development. The precise nature of the PIK3CA alterations varied from one individual to the next — as did affected tissues and mutant allele frequencies within them — but included missense mutations previously described in breast, lung, and other cancers.
In addition to offering clues for understanding other excess growth conditions, the findings are expected to inform efforts to come up with more effective treatments for CLOVES, which is currently managed surgically.
"Having now found these driving mutations in CLOVES, we have a good starting point from which to both develop models to understand how mutations in PIK3CA cause malformation and overgrowth and to determine which drugs and other therapies can be used safely and successfully to improve the lives of individuals with CLOVES and other conditions with similar clinical characteristics," corresponding author Matthew Warman, director of Children's Hospital Boston's orthopedic research laboratories, said in a statement.
Since CLOVES was first defined in the mid-2000s, some 90 children around the world have been diagnosed with the condition, characterized by variable symptoms that can include limb or spine deformity, fatty tissue growths, and abnormalities in the skin and vasculature.
Given the patchy presentation of these malformations, researchers suspected that the condition stemmed from postzygotic rather than inherited genetic changes, though the identity of the gene or genes involved remained a mystery.
In an effort to track down the genetic culprit in CLOVES, Warman and his colleagues did sequencing on fresh, frozen, and fixed tissue samples from six children with CLOVES who ranged in age from one to 18 years old, looking for differences in gene-coding sequences between affected and unaffected tissues.
The team used whole-exome and messenger RNA sequencing to test affected tissue samples from four individuals for whom fresh and frozen samples were available. Patterns in the protein-coding sequencing, captured using Agilent's SureSelect kit, were then compared to those in unaffected samples from two of the same individuals.
For two more patients, researchers did exon sequencing experiments focused on 77 genes believed to be involved in growth factor signaling pathways. For those experiments, they used custom arrays to capture exon sequence for the genes of interest from formalin-fixed, paraffin-embedded tissue samples. A matched normal saliva sample from one of the two individuals was also tested.
After sorting through their sequence data and sifting out SNPs found in dbSNP, 1000 Genomes Project data, and the National Heart, Lung, and Blood Institute's whole-exome database, investigators ranked variants with respect to their frequency in affected tissues.
In all six of the CLOVES patients, the team saw missense mutations in PIK3CA in affected tissues that were absent from unaffected samples tested for a subset of the same individuals. In the affected tissues, mutant PIK3CA alleles turned up at frequencies as high as 30 percent and as low as 3 percent.
Consistent with the prediction that these alterations lead to enhanced activity for PIK3CA's protein product, follow-up experiments using overgrowth samples from some of the individuals with CLOVES contained higher-than-usual levels of phosphorylation on proteins downstream of activated PIK3CA.
In cancer, study authors explained, comparable mutations to PIK3CA appear to spur on tumor tenacity and aggressiveness, while enhanced expression of missense mutation-containing versions of the genes have been implicated in transformation.
Moreover, they noted that one of the CLOVES patients tested for the current study was also diagnosed with Wilms tumor, a form of childhood kidney cancer, though most individuals with the overgrowth condition are not.
Based on this pattern, the researchers speculated that "endogenous expression of missense PIK3CA mutants could be transformative in some human cell types."
"We hypothesize that the low rate of malignant transformation in individuals with CLOVES syndrome is due to the low level of endogenous PIK3CA expression in most cells."