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Exome Sequencing Study of Rare Syndrome Points to Compound Inheritance Involving Regulatory Variants

By Andrea Anderson

NEW YORK (GenomeWeb News) – In a study appearing online yesterday in Nature Genetics, investigators from the Wellcome Trust Sanger Institute, the University of Cambridge, and elsewhere used exome sequencing to reveal the compound inheritance patterns behind an autosomal recessive condition called thrombocytopenia with absent radii, or TAR, syndrome.

TAR is characterized by low blood platelet counts, blood clotting problems, and skeletal abnormalities that include missing radius bones in the forearms.

Patterns in the five patient exomes sequenced for the study indicated that TAR results from a deletion or another mutation that knocks out one copy of a gene called RBM8A, combined with the presence of one of two regulatory SNPs that affect the expression of the remaining copy of the gene. A similar pattern held in all but two of the 50 cases that the team tested subsequently.

While the RBM8A null mutations are rare, at least one of the regulatory SNPs implicated in TAR appears to be unexpectedly common, with a minor allele frequency of around 3 percent in those of European ancestry.

"The combination of a null allele with a regulatory region SNP — and a regulatory region SNP that is common — that is new," co-senior author Cedric Ghevaert, a hematology researcher at the University of Cambridge, told GenomeWeb Daily News.

Findings from the study are expected to have immediate implications for TAR screening and genetic counseling. By underscoring the potential importance of non-coding, regulatory regions in the genome, Ghevaert noted, the work may also inform studies of other conditions.

"I think that this shows that the cost [of sequencing regulatory regions] actually is necessary if you want to not miss out on genetic information," he said. "And that the functional annotation of the genome in each of the lines that you're interested is important because it tells you where the regulatory regions are."

An array-based comparative genomic hybridization study published by a German-led research team more than five years ago uncovered a chromosome 1 microdeletion in individuals with TAR that affected a number of genes including RBM8A.

But because the condition shows autosomal recessive inheritance, authors of the array CGH study and other researchers studying the disease have been searching for additional genetic contributors to TAR.

"It was quite clear that the deletion wasn't the whole story," Ghevaert explained, "simply because the parents of the affected children had the deletion and were perfectly healthy themselves."

For the current study, Ghevaert and his colleagues used Agilent SureSelect arrays to capture coding sequences from five individuals known to carry the TAR-associated chromosome 1 deletion. They then sequenced the individuals' exomes to between 123- and 127-fold mean coverage with the Illumina GAII.

Analyses of the exomes did not point to any coding mutations beyond the known chromosome 1 deletion. But four of the five individuals had the same SNP in the 5' UTR of RBM8A. The fifth had a SNP in the gene's first intron.

"The advantage of exome sequencing is that you get a little bit of the overhang sequences — sequences that are before and after each of the exons," Ghevaert said. "So you get a little bit of the 5' UTR and the 3' UTR, as well as some of the intron."

Sanger sequencing in another 48 TAR-affected individuals with the chromosome 1 deletion uncovered the same 5' UTR SNP in 35 individuals and the intronic variant in 11 others.

While the intronic SNP is fairly rare in the general population, turning up in just 0.42 percent of the more than 7,500 healthy individuals included in the Cambridge BioResource, the 5' UTR SNP seems to be far more common, with a minor allele frequency of roughly 3 percent.

"One of the SNPs is actually not rare at all," Ghevaert noted. "Three percent is actually not rare, which means that there are homozygous carriers out there."

Two more samples, from a mother and fetus diagnosed with TAR, carried neither SNP, suggesting additional regulatory alleles can contribute to the condition.

Similarly, the well-known TAR-related microdeletion on chromosome 1 does not seem to be the only alteration that can effectively knock out one copy of RBM8A, researchers found.

Through Sanger sequencing of RBM8A exons in two TAR-affected individuals without the characteristic chromosome 1 deletion, they tracked down a frameshift insertion that inactivated the gene in one individual and a missense mutation producing a premature stop codon in RBM8A in the other.

Such findings "confirmed that RBM8A was the gene of interest," Ghevaert said, "because the premature stop codon was in RBM8A specifically and it was not the whole region that was actually affected."

Because RBM8A codes for an exon-junction complex component called Y14, the study authors concluded that TAR stems from defects in the EJC — an RNA processing complex that helps destroy faulty messenger RNAs and directs the cellular localization of some mRNAs.

Consistent with their cell line experiments, the researchers saw that samples from TAR patients who carried the chromosome 1 deletion in combination with either the 5' UTR or intronic SNP had lower-than-usual levels of the Y14 protein, as did a patient with the 5' UTR SNP and a frameshift insertion in RBM8A.

The presence of the 5' UTR SNP alone did not dramatically alter RBM8A gene expression in platelet samples from a dozen healthy individuals who were heterozygous for the variant, the team reported.

The researchers are now looking for additional regulatory variants that contribute to TAR and exploring the tissue-specific effects of the RBM8A alterations already identified.

Regardless of the precise mechanism by which these changes produce TAR, though, an improved understanding of the genetic features behind the condition can now be applied to genetic counseling and TAR screening, which, until now, was limited to testing for the chromosome 1 deletion.

"Obviously we can identify the parent who carries a deletion — that we've been able to do for a long time," Ghevaert said. "But now we are able to look for [regulatory] SNPs in the other parent."

To that end, Ghevaert and his colleagues are collaborating with a clinical lab in Bristol, UK to develop TAR screening strategies for individuals who are known to carry chromosome 1 microdeletions and/or have a family history of TAR.

Those involved in the current study are also considering follow-up research to try to find additional genetic contributors in syndromes that share the same chromosome 1 microdeletion with TAR but involve distinct clinical features.

To look more broadly at rare, inherited bleeding and platelet-related disorders, Ghevaert added, members of the Bleeding and platelet Related Inherited Disorder and Genetic Evaluation, or BRIDGE, study team are establishing a DNA repository to be used in future exome sequencing studies.

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