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Case Studies

Detecting somatic mosaicism using massively parallel sequencing

Introduction Discussion Conclusions Methods References

Introduction  

A rite of passage for many new parents is to discover with dismay that their new baby has suddenly developed some mysterious rash. Newborn skin is a delicate thing indeed. First-time moms and dads are may be surprised to find that infant complexion can spontaneously erupt in bumps punctuated with varying shades of red, white, brown, yellow, or pink hues. Many times the rash disappears as quickly as it came, brought on by something as simple as laundry detergent, the normal fluctuations of hormones in newborn babies, heat, or skin irritation from diapers.

In one out of a million babies, though, the rash isn't innocuous. It's a harbinger of Neonatal-Onset Multisystem Inflammatory Disease (NOMID), also referred to as Chronic Infantile Neurologic Cutaneous and Articular syndrome (CINCA).

Discussion  

What is NOMID?

NOMID is at the most severe end of the spectrum of a family of diseases referred to as Cryopyrin-Associated Periodic Syndromes (CAPS). All of the CAPS conditions involve mutations in NLRP3, which encodes cryopryin. Cyropyrin is a NOD-like receptor that is a critical element of the body's innate immune response against infectious agents present in the environment. This receptor oligimerizes and interacts with ASC and pro-caspase-1 to form inflammasomes that affect production of Interleukin 1-beta (IL-1beta). IL-1beta in turn mediates the inflammatoryNLRP3 inflammasome responses that the body uses to defend itself.

In most individuals, the inflammatory response is only mounted on an as-needed basis because cyropyrin assembly into inflammasomes is triggered by signals such microbial molecules referred to as PAMPs. However, in NOMID and other CAPS diseases, this regulation is lost. The inflammatory system flares up repeatedly, causing chronic bouts of rash, fever, headaches, and joint pain. NOMID, an autosomal dominant disease, is the most profound of the CAPS conditions. In NOMID, the inflammatory response occurs over and over again in continuous, unrelenting surges. The toll is devastating: chronic aseptic meningitis due to inflammation in tissues surrounding the brain, vision problems due to high intercranial pressure on the optical nerves, joint problems, sensiorineural hearing loss, the buildup of insoluble forms of protein in tissues and organs (amyloidosis), and mental or cognitive impairment. The mortality rate is 20%. Recently, however, some success has emerged through treatment with IL-1beta blocking drugs, which—if administered early enough—may mitigate progression of NOMID (although not without side effects). For this reason, early detection is critical.

NLRP3 somatic mosaicism: finding the needle in the haystack

But therein lies an extraordinarily complex problem: 60% of NOMID patients are relatively easy to assess using molecular genetic assays because they have germline mutations in NLRP3. For the other 40%, however, NLRP3 mutations are non-germline. This latter group of patients exhibits somatic mosaicism, and detection of mutant alleles in samples is the literal equivalent of trying to find a needle in a haystack. Mutant allele frequency may be as low as 1%, with mosaicism typically ranging between 4.2 to 35.8%. Such patients may be referred to as "mutation-negative NOMID" individuals because—despite presence of clinical symptoms of NOMID—the level of mutant allele copies is below the limit of detection by conventional means. In an attempt to definitively analyze such cases, researchers have resorted to sequencing hundreds of individual subclones from a single patient—a labor-intensive, expensive, and time consuming process that leaves much room for improvement.

Conclusions  

Bringing clarity to ambiguous cases

So how well did the method work? When the technique was used for samples from 10 different mutation-negative NOMID patients, somatic mosaicism was identified in four out of 10. One of the mutations that was identified (p.Phe302Leu) was novel, and was confirmed to be pathogenic through two different cell-based assays.

The authors concluded that "...read depth of approximately 350 for each strand of each amplicon would be sufficient to detect somatic mosaicism as low as 1% with statistical confidence," in theory allowing up to 100 patient samples to be analyzed on a single 454 GS-FLX over approximately 10 hours.

More than NOMID

Importantly, the study impact isn't limited to NOMID/CINCA, but may have implications for other situations in which low-frequency somatic mosaicism plays a role, such as analysis of tumors. Additionally, the Izawa et al. research was cited by scientists investigating somatic alpha-synuclein mutations in Parkinson's disease (Proukakis et al. 2013). Should methods such as these shed light on previously intractable molecular analyses, it would have the potential to spread hope to some of the most difficult biomedical problems currently faced by translational medicine researchers, clinicians, and patients.

Methods  

Massively parallel sequencing to analyze somatic mosaicism

Improvement came in the form of a publication (Izawa et al. 2012) by researchers at the Kyoto University Graduate School of Medicine, the RIKEN Research Center for Allergy and Immunology in Yokohama, the Center for iPS Cell Research and Application at Kyoto University, the Translational Autoinflammatory Disease Section at the NIH, and Kazusa DNA Research Institute in Chiba, Japan. A multi-disciplinary team of scientists including co-first authors Kazushi Izawa and Atsushi Hijikata and co-corresponding authors Ryuta Nishikomori and Osamu Ohara decided to apply next-generation sequencing techniques to the problem of analyzing "mutation-negative NOMID" patient. The major concern, however, was whether the presence of a low-level allele could be discerned with statistical significance among the noise inherent in NGS data.

So the authors started by constructing error rate maps of 14 PCR products covering the entire NLRP3 coding region. Their data set included approximately 1 million reads from 50 control samples thought to be free of somatic mosaicism. This allowed them to analyze patterns of sequencing errors during massively parallel sequencing (MPS) with a Roche 454-FLX sequencer, and adjust for nucleotide position- and DNA strand-specific error rates when assessing mutation-negative NOMID patient MPS data. Articulating the error rate map was critical, because it made a discrimination pipeline for somatic mosaicism determination possible. To generate the data, the authors used a two-step PCR assay and pooled sample libraries for MPS. NLRP3 coding exonic regions and flanking intronic regions were covered by 14 amplicons that were designed to be as long as an average read length for a Roche 454 GS-FLX sequencer (up to 450 bases) and were amplified from each genomic DNA sample using PrimeSTAR GXL DNA Polymerase. The second round of PCR amplification, again performed with PrimeSTAR GXL enzyme, added 3' adaptor sequences and 5' Multiplex Identifier (MID) tags, then samples were applied to a Roche 454 Genome Sequencer (GS)-FLX system, amplified by emPCR, and sequenced in multiplex.

References  

Izawa, et al., Detection of base substitution- type somatic mosaicism of the NLRP3 gene with >99.9% statistical confidence by massively parallel sequencing. DNA Res. 19(2):143–152 (2012).

Proukakis, et al., Somatic alpha-synuclein mutations in Parkinson's disease: Hypothesis and preliminary data. Mov. Disord., 28:705–712 (2013).

NOMID Alliance

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A hot-start, high-fidelity PCR enzyme, PrimeSTAR GXL DNA Polymerase excels in reactions with GC-rich templates, excess template, and long amplicons up to 30 kb (GXL). The polymerase is supplied with separate tubes of optimized buffer (Mg2+ plus), and dNTPs.

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PrimeSTAR GXL DNA Polymerase amplifies products up to 30 kb (human genomic DNA template), 40 kb (lambda DNA), or 13.5 kb (human cDNA)

PrimeSTAR GXL DNA Polymerase amplifies products up to 30 kb (human genomic DNA template), 40 kb (lambda DNA), or 13.5 kb (human cDNA)
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PrimeSTAR GXL DNA Polymerase shows efficient amplification in reaction mixes with a wide range of template quantity, including high levels of template that inhibit the activity of other commercially available high fidelity DNA polymerases

PrimeSTAR GXL DNA Polymerase shows efficient amplification in reaction mixes with a wide range of template quantity, including high levels of template that inhibit the activity of other commercially available high fidelity DNA polymerases
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PrimeSTAR GXL DNA Polymerase: 250 Units (R050A)

PrimeSTAR GXL DNA Polymerase: 250 Units (R050A)
R050B PrimeSTAR® GXL DNA Polymerase 1,000 Units $671.00

License Statement

ID Number  
M54 This product is covered by the claims of U.S. Patent Nos. 7,704,713 and its foreign counterparts. 

A hot-start, high-fidelity PCR enzyme, PrimeSTAR GXL DNA Polymerase excels in reactions with GC-rich templates, excess template, and long amplicons up to 30 kb (GXL). The polymerase is supplied with separate tubes of optimized buffer (Mg2+ plus), and dNTPs. Cat. # R050B contains 4 of Cat. # R050A. Please refer to Cat. # R050A for complete product documentation and resources.


Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Examples of product yield on GC-rich templates: comparison of PrimeSTAR GXL DNA Polymerase with four other commercially available high-fidelity DNA polymerases

Examples of product yield on GC-rich templates: comparison of PrimeSTAR GXL DNA Polymerase with four other commercially available high-fidelity DNA polymerases
Examples of product yield on GC-rich templates: comparison of PrimeSTAR GXL DNA Polymerase with four other commercially available high-fidelity DNA polymerases. Company T’s enzyme includes buffers optimized for GC-rich templates.

Back

PrimeSTAR GXL DNA Polymerase amplifies products up to 30 kb (human genomic DNA template), 40 kb (lambda DNA), or 13.5 kb (human cDNA)

PrimeSTAR GXL DNA Polymerase amplifies products up to 30 kb (human genomic DNA template), 40 kb (lambda DNA), or 13.5 kb (human cDNA)
PrimeSTAR GXL DNA Polymerase amplifies products up to 30 kb (human genomic DNA template), 40 kb (lambda DNA), or 13.5 kb (human cDNA).

Back

PrimeSTAR GXL DNA Polymerase shows efficient amplification in reaction mixes with a wide range of template quantity, including high levels of template that inhibit the activity of other commercially available high fidelity DNA polymerases

PrimeSTAR GXL DNA Polymerase shows efficient amplification in reaction mixes with a wide range of template quantity, including high levels of template that inhibit the activity of other commercially available high fidelity DNA polymerases
PrimeSTAR GXL DNA Polymerase shows efficient amplification in reaction mixes with a wide range of template quantity, including high levels of template that inhibit the activity of other commercially available high fidelity DNA polymerases.


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