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Microarray method for combined CGH, methylation, and SNP analysis

IP.com Disclosure Number: IPCOM000200950D
Publication Date: 2010-Nov-01
Document File: 6 page(s) / 192K

Publishing Venue

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Abstract

Genomic imprinting refers to the process of methylating cytosine bases in CpG dinucleotides within genomic DNA, and it can affect the expression of genes. Even if the canonical nucleotide sequence is correct, defects in imprinting can cause a variety of disorders in humans (for review, see ref 1.) Although advances in DNA sequencing technology have accelerated understanding of human genome sequences, there remains a need for new methods to study imprinting in humans. Currently, imprinting disorders such as Silver Russell Syndrome, Prader-Willi syndrome, and Angelman syndrome are measured with array CGH, SNP microarrays, microsatellite analysis, methylation-sensitive PCR, or a combination of methods.

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Microarray method for combined CGH, methylation, and SNP analysis

Brian J. Peter and Nick Sampas

Background

Genomic imprinting refers to the process of methylating cytosine bases in CpG dinucleotides within genomic DNA, and it can affect the expression of genes.  Even if the canonical nucleotide sequence is correct, defects in imprinting can cause a variety of disorders in humans (for review, see ref 1.)  Although advances in DNA sequencing technology have accelerated understanding of human genome sequences, there remains a need for new methods to study imprinting in humans. Currently, imprinting disorders such as Silver Russell Syndrome, Prader-Willi syndrome, and Angelman syndrome are measured with array CGH, SNP microarrays, microsatellite analysis, methylation-sensitive PCR, or a combination of methods. 

However, no single methodcan detect all these defects and variations, and measurements that measure methylation compromise copy number measurements.

Specifically, methods which do not directly detect methylation (array CGH, SNP microarrays, microsatellite analysis) generally do not detect “heterodisomy,” the case where two different chromosomes are inherited from a single parent (see Fig 1, illustrating the case of maternal disomy).  As illustrated in Fig. 1, in heterodisomic cases, the two chromosomes have identical imprinting, and thus can still lead to a clinical phenotype (such as Prader-Willi syndrome) but the two chromosomes will exhibit different SNP and microsatellite genotypes.  In contrast, methylation-sensitive PCR could detect cases of heterodisomy, but PCR will be limited to the targeted loci, and may not distinguish them from copy number changes such as deletions.

Furthermore, microarray-based methods for measuring DNA methylation generally require amplification of a subset of the DNA or immunoprecipitation with antibodies to methylcytosine.  However, these methods may compromise accurate copy-number detection.  High throughput sequencing methods (i.e., so-called “next-generation sequencing technologies) can be used to measure DNA methylation (through bisulfate sequencing), but accurate measurements of methylation, SNP genotype, and DNA copy number in the same DNA sample would require a high depth of coverage and may be cost-prohibitive.  Furthermore, there are few regions of the genome that are known to be imprinted.  A microarray can offer a focused measurement of methylation in the regions of interest, or a combination of methylation measurements in imprinted regions and genome-wide coverage for SNP and copy number measurements. Here we describe a method that uses microarrays to simultaneously measure copy number, SNP genotype, and CpG methylation (imprinting) at selected genomic loci in a highly parallel, genome wide manner. 

Description of Method

In describing the method, it is understood that the exact way in which the method may be performed may deviate from that set forth below. Specifically, many of...