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NCGR Advances Sequencing-Based Carrier Screening Test for Recessive Genetic Disorders


By Julia Karow

One year after teaming up with the Beyond Batten Disease Foundation to develop a genetic carrier screening test for severe recessive genetic disorders, researchers at the National Center for Genome Resources have chosen a panel of more than 400 diseases and have tested several DNA enrichment and sequencing platforms.

Santa Fe, NM-based NCGR is also in the process of forming collaborations with research and clinical groups to validate the test in a large number of samples with the goal of offering it to patients by the end of this year.

The aim of the test is to tell prospective parents what severe genetic diseases they are carriers for, diseases that together they might pass on to their children. "The goal here is to solve a horrible medical nightmare," said Stephen Kingsmore, NCGR's president.

A year ago, the researchers had not decided whether to develop the test on a microarray or a sequencing platform (see In Sequence 3/10/2010). In the meantime, they have come to the conclusion that sequencing is the way to go. "An array platform would be very static," said Kingsmore, and would not allow them to add newly found mutations, or to discover new mutations.

Though an array-based test would cost less and have a faster development cycle than a sequencing-based test, he said, multiplexing will bring the cost within the same range. For example, if about 100 samples were pooled in a single sequencing run, "then the costs become fairly comparable," he said. Currently, the aim is to offer the carrier screening test for under $500.

So far, NCGR has chosen 448 genetic diseases to be included in the test, a mix of autosomal recessive and X-linked recessive disorders that are "fairly catastrophic" in children, for example juvenile Batten disease.

Genes involved in some of these diseases are protected by patents, and before the test is offered commercially, NCGR will have to think about its strategy regarding these genes. "I'm very hopeful that we will be able to offer all the catastrophic diseases," Kingsmore said. "I think it would be a tragedy if we were blocked" because of patents.

In the process of developing the test, Kingsmore and colleagues have also found that databases of mutations involved in genetic diseases — such as the Human Gene Mutation Database — are both incomplete and contain misannotations. Some so-called mutations, for example, appear to be merely polymorphisms. "There is clearly a need to clean up the databases and have the ability to identify new mutations," he said, adding that no single comprehensive database exists, and that many testing labs maintain their own internal databases for individual conditions.

NCGR's test will combine targeted enrichment of DNA with next-generation sequencing, and the center recently compared four enrichment technologies to see how well they were able to detect a spectrum of known mutations: Agilent's SureSelect; RainDance Technologies' microdroplet PCR; Febit's HybSelect; and Olink Genomics' Selector Technology.

According to Callum Bell, program lead at NCGR, the scientists enriched DNA from 24 samples from the Coriell repository, representing 18 diseases and a total of 42 mutations, and sequenced the DNA on the Illumina platform. In order to maximize the variety of mutations, they chose mostly compound heterozygotes.

They "quickly eliminated" both Febit and Olink, he said, Febit because the enrichment was "very poor" and Olink because the coverage was very uneven, with a small number of targets being highly over-represented. RainDance and Agilent's SureSelect both performed well in the assay, Bell said, and from a technical perspective, would both meet the researchers' criteria.

According to Kingsmore, RainDance has better coverage than hybridization-based approaches and minimizes allelic bias better than the other methods. NCGR, which has its own RainDance instrument in house, has been using the platform to enrich about 2 megabases of DNA, or 8,000 amplicons, he said.

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The researchers are currently in the process of optimizing the test further, in order to make a final decision on both the enrichment method and the sequencing platform. So far, they have used their in-house Illumina platform and have tested SOLiD 3 by sending samples to Life Technologies. They also hope to be able to try the Ion Torrent sequencing platform soon.

The next phase of the project will be a larger validation study in about 1,000 samples by the end of this year, according to Kingsmore. "What we are proposing to do is characterize a very large number of samples to validate just about all of the disease states and most of the common mutations," he said.

For that study, NCGR is partnering with Hilger Ropers, head of the department of human molecular genetics at the Max Planck Institute for Molecular Genetics in Berlin.

Ropers' group has been working on a similar genetic carrier test, but focusing on X-linked as well as autosomal recessive forms of mental retardation. "We will profit from our mutual experience with these techniques, and we will jointly negotiate with these companies," Ropers told In Sequence last week. "I'm pretty optimistic that by the end of this year, there will be a test." The plan is to validate the test both in a US and in a German population, he said, probably next year rather than this year.

NCGR is also looking for collaborators to offer the test. So far, the center has talked to the Mayo Clinic, as well as Children's Mercy Hospital and Quest Diagnostics in Kansas City.

NCGR plans to bring the test and software into CLIA compliance, and to set up a CLIA-certified lab, possibly in collaboration with one of these institutions. "It makes sense for us to try and partner with someone who already has a high-volume reference laboratory," Kingsmore said, and is familiar with sample handling, billing, and other issues. "We don't really want to build it from scratch."

Kingsmore said the test would likely be first released at a small number of initial adopter sites in the US and Europe. These sites could include in vitro fertilization clinics as well as centers with a good test interpretation infrastructure.

NCGR's carrier test would differ in several ways from a multiplexed genetic carrier test already sold by Counsyl, a California-based startup that recently began offering a "Universal Genetic Test" that covers variants involved in more than 100 life-threatening genetic diseases.

While Counsyl's test is based on a microarray platform and is offered directly to consumers, NCGR's will be based on sequencing and offered through the medical establishment. Also, unlike Counsyl, NCGR is a not-for-profit institute.

Several attempts to contact Counsyl president Balaji Srinivasan to find out whether the company is also working on a more comprehensive sequencing-based carrier test were unsuccessful. According to filings with the US Securities and Exchange Commission, the company raised almost $2.2 million from an equity offering earlier this month, and almost $2.7 million last year.

Another company, Boston-based Good Start Genetics, is also working on a pre-pregnancy test for multiple genetic disorders (see In Sequence 3/31/2009). Company officials did not respond to several interview requests.

Researchers at Cold Spring Harbor Laboratory have been working with Dor Yeshorim, a New York-based organization that provides genetic carrier screening to orthodox Jewish communities, for genetic diseases prevalent in these populations, in order to develop multiplexed testing on a next-gen sequencing platform using a new multiplexing method called DNA Sudoku (see In Sequence 6/9/2009).

Yaniv Erlich, a CSHL researcher working on the project, told In Sequence last week that he has received more than 1,000 samples from Dor Yeshorim and plans to test them for about 15 autosomal recessive diseases, including Tay Sachs disease and cystic fibrosis, within the next few months, aiming to run up to 1,000 samples per Illumina sequencing run.

The goal is to reduce the cost of the test from a current level of several hundred dollars to tens of dollars, he said, as well as to be able to capture new mutations.