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CNV Analysis Unearths Compound Gene Mutations Behind Congenital Scoliosis

NEW YORK (GenomeWeb) – In a study appearing online last night in the New England Journal of Medicine, an international team led by investigators at Fudan University and Peking Union Medical College describes TBX6 null mutations that contribute to congenital scoliosis in the Han Chinese population when found in combination with a specific haplotype of the gene.

The researchers searched for culprits in the vertebral malformation condition by doing copy number profiling on hundreds of Han Chinese individuals with or without sporadic congenital scoliosis, including members of 20 parent-child trios.

Using a combination of array comparative genome hybridization, quantitative PCR, and DNA sequencing, they narrowed in on TBX6 null mutations in around 11 percent of those with sporadic congenital scoliosis.

But the team's follow-up analyses indicated that the condition does not arise when these mutations are present in just one copy of the TBX6 gene. Rather, the patterns identified in the affected families pointed to compound inheritance involving null mutations affecting one copy of the TBX6 and the presence of so-called hypomorphic haplotype — a version of the gene containing variants that dial down TBX6 activity.

"[T]he effect of diminished TBX6 dosage is complex," Fudan University's Feng Zhang and Peking Union Medical College orthopedic surgery researcher Guixing Qiu, the study's senior authors, and their colleagues explained. "We found that an additional TBX6 hypomorphic allele is required to cause a further decrement in gene-expression dosage beyond haploinsufficiency for penetrance of the congenital scoliosis phenotype."

While past research points to a genetic component for congenital scoliosis, most of the mutations implicated in the condition so far have exhibited low penetrance, the researchers noted, hinting at the complexity of the condition's genetic underpinnings.

For instance, vertebral problems have been found in a subset of individuals with duplications or deletions involving a chromosome 16 region known as 16p11.2, they explained. But while copy number alterations in that region are associated with conditions ranging from autism to obesity, their potential role in congenital scoliosis is unclear.

The researchers used Agilent 1x1M CGH microarrays to assess samples from 161 Han Chinese individuals with sporadic congenital scoliosis diagnosed at Peking Union Medical College Hospital between the fall of 2010 and spring 2014. Among them were 20 affected individuals from trios that included two unaffected parents, who also were tested for the study.

When the team compared copy number patterns in the sporadic congenital scoliosis patients with those found in 166 healthy control individuals from the same population, it uncovered null mutations affecting one copy of TBX6 in 17 of the affected individuals tested — just shy of 11 percent.

A dozen of the TBX6 alterations involved chromosome 16 deletions that overlapped with the gene, while the remaining five were frameshift or nonsense mutations involving individual nucleotides in the gene. Such glitches did not turn up in individuals without sporadic congenital scoliosis, the team noted.

By taking a closer look at families affected by chromosome 16 deletions, though, the researchers determined that sporadic congenital scoliosis likely stems from more than just heterozygous null mutations in TBX6.

Indeed, by focusing in on two families that contained affected and unaffected individuals with the same chromosome 16p11.2 deletion, they found three SNPs in TBX6 that were shared in all of the individuals with sporadic congenital scoliosis, comprising a haplotype that appeared to modify the risk associated with deletions involving the other copy of the gene.

The team found further support for this inheritance model when they scrutinized samples from 76 more Han Chinese individuals with sporadic congenital scoliosis and another 42 individuals from China or the US with documented deletions involving the suspicious chromosome 16 region.

In the first of these follow-up groups, some 8 percent of individuals with congenital scoliosis showed signs of compound inheritance involving TBX6 null mutations combined with the risky TBX6 haplotype.

Compound inheritance of these TBX6 glitches was even more common amongst individuals with both 16p11.2 deletions and congenital scoliosis, researchers reported, turning up in 83 percent of those congenital scoliosis cases.

Meanwhile, functional experiments done in vitro on human or mouse cell lines indicated that the risky haplotype — which includes two non-coding TBX6 variants — likely diminishes the gene's expression, leading to congenital scoliosis when inherited in combination with null mutations on the alternative copy of TBX6.

The study's authors argued that this newly described inheritance model "will enable the molecular diagnosis of the disorder and genetic counseling for some persons who have or are at risk for congenital scoliosis."