To sequence the exome or the genome: that is the question

Genome Advance of the Month

To sequence the exome or the genome: that is the question

By Elizabeth Burke, Ph.D.
Intramural Postdoctoral Fellow, NHGRI

Over the past few years, researchers have employed DNA sequencing technology to identify disease-causing mutations like those associated with the most common inherited disease of the retina, autosomal recessive retinitis pigmentosa (ARRP), which ultimately leads to blindness.  Although ARRP patients display almost identical symptoms, this disease has been shown to result from mutations in more than 50 different genes.  What is extraordinary is the fact that roughly half of clinically-diagnosed ARRP patients do not have a mutation in these genes, indicating that there are other mutations yet to be discovered.  This finding has led scientists to reevaluate which of the two main methods of DNA sequencing should be used to identify mutations in diseases such as ARRP.

The two methods differ in the level of DNA coverage they provide.  The first is called whole exome sequencing (WES), which only covers the regions of the genome that contain the actual DNA code for making proteins; these are known as exons. In fact, the word exon was derived from "EXpressed regiON," because these are the regions of the genome that are translated and expressed as proteins.  Exons are important, but they only amount to about 1.5 percent of the genome.  Whole genome sequencing (WGS), in contrast to WES, covers the entire genome, including both the coding and non-coding regions.

Until recently, researchers have opted to use WES partly because it costs less, but also because the functional consequences of mutations occurring within coding regions of genes are much easier to interpret: Mutations in WES sequences would result in abnormal or non-functional proteins.

However, it is likely that many disease-causing mutations are actually located outside of exons and consequently will not be identified by WES.  Structural variations - alterations such as deletions, inversions or duplications of DNA segments larger than 1,000 base pairs - are also generally not detected by this method.

More and more scientists are questioning whether the lower price of WES is worth the loss of coverage.  October's Genome Advance of the Month highlights a study, published in the October 1, 2013, issue of Proceedings of the National Academy of Sciences, which demonstrates the advantages of WGS in the identification of ARRP patient mutations.

A collaborative group of researchers from around the world performed WGS for 16 unrelated ARRP patients from diverse ethnic backgrounds and assessed DNA changes in both the coding and non-coding portions of the genome.  The team began their analysis by first searching the genes previously known to be associated with ARRP.  Of the 16 patients examined, eight were found to contain homozygous (an identical change inherited from both parents) or compound heterozygous (two different changes in the same gene - one from each parent) mutations in seven of the ARRP-associated genes.

While most mutations they found were simple, single base pair substitutions or small insertions or deletions, two of these eight patients were discovered to have pathogenic, structural variations that originated in non-coding regions.  One patient was missing a stretch of 2,300 base pairs that contained an entire exon of the gene called USH2A.  The other patient was found to carry a head-to-head inverted duplication of 446,000 base pairs that contained seven exons of the EYS gene.

After screening all 16 patients for mutations in the ARRP-associated genes, the genomes of the eight patients with unidentified mutations were further examined.  The team found one of these patients carried a homozygous mutation in the NEK2 gene, which created a premature stop signal in the sequence that would result in the expression of a shortened, non-functional version of the protein.  To determine if the loss of NEK2 function could cause ARRP in humans, the research team blocked expression of NEK2 in a type of fish used to model human diseases, called the zebrafish, and assessed whether retinal degeneration had occurred. The zebrafish were found to have an increased amount of cell death in their retina - the cause of retinal disease in humans - confirming that the NEK2 mutation could indeed cause ARRP symptoms.

Without performing both WES and WGS for these patients and comparing the results, it is difficult to estimate how many mutations were found due to the additional coverage provided by WGS.  Nevertheless, the scientists were able to find nine of the 16 patients' mutations (two mutations per patient; a total of 18 mutations) with all but two being novel.  This method also helped them determine for the first time that mutations in the gene NEK2 can cause ARRP.  Moreover, WGS detected two structural variations that began in non-coding regions, which WES would have likely missed.

As sequencing technology becomes cheaper, faster and more accurate over the next few years, it is likely that more scientists and clinical diagnostic laboratories will opt for the higher coverage provided by WGS.   However, until the cost of WGS is reduced, the team of scientists suggests that the choice between WGS and WES depends on the disease that is being investigated and the question that is being answered.  If structural variants or mutations in non-coding regions are suspected to be involved in a disease, WGS is the better choice.

Regardless of whether the exome or genome wins this battle, we will all reap the rewards in the end.  The fast-paced advancement of these technologies guarantees that our ability to identify disease-causing mutations will only improve over time.  This is good news for both scientists and patients alike.

Read the article:

Nishiguchi KM, Tearle RG, Liu YP, Oh EC, Miyake N, Benaglio P, Harper S, Koskiniemi-Kuendig H, Venturini G, Sharon D, Koenekoop RK, Nakamura M, Kondo M, Ueno S, Yasuma TR, Beckmann JS, Ikegawa S, Matsumoto N, Terasaki H, Berson EL, Katsanis N, Rivolta C. Whole genome sequencing in patients with retinitis pigmentosa reveals pathogenic DNA structural changes and NEK2 as a new disease gene. PNAS, 110(40):16139-16144. 2013. [PubMed]

Posted: November 27, 2013