Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-10-17
2003-07-01
Horlick, Kenneth R. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S024300, C536S024320, C536S024330, C536S025400, C210S600000
Reexamination Certificate
active
06586181
ABSTRACT:
This invention relates to a diagnostic method for detecting genetic changes associated, inter alia, with the development of cancer. The method detects allelic imbalance (AI), such as loss of heterozygosity (LOH), in nucleic acid from an individual and can be applied to samples in which only a small proportion of the cells display AI, thereby allowing the early identification of mutations which lead to the progression of cancer. The invention also relates to amplification primers for use in the method and to diagnostic kits containing them.
The term “allelic imbalance” refers to the chromosomal loss or gain of a region of a chromosome when the partner chromosome (in a diploid cell) is unaltered. Allelic imbalance is typically found in tumour cells. Allelic imbalance may be due to selective loss of a region of DNA derived from a single chromosome and is referred to as loss of heterozygosity (LOH) when the partner chromosome is varied in some form, most commonly by microsatellite size. Other causes of AI include: gene amplification (i.e. myc oncogene may be amplified 5-10 fold), heteroplasmy or, at the transcript level, differential allelic expression (i.e. maternal allele vs paternal allele).
Heteroplasmy most commonly refers to imbalanced ratios of maternal mitochondrial DNA and may be associated with specific pathogenic effects. The phenotype is dependent not just on homo/heterozygosity, but the relative frequencies of both “alleles”. The inheritance of allele sequences does not occur in a Mendelian manner, and the ratio of maternal “mutant” and maternal “WT” alleles can vary between tissue/cell types. Heteroplasmy may be involved in a wide range of pathological conditions (eg mitochondrial, cardiac and encephalic myopathies) including a possible role in the pathology of Parkinson's and Alzheimer's disease (Suomalainen A, Annals of Medicine. 29:235-246, 1997).
Loss of a particular region of a chromosome is a frequent event in the development and progression of cancer. Different types of tumour have been found to exhibit loss of different chromosomal regions, strongly suggesting that the regions contain genes which are essential for the prevention of neoplasia. These genes, called tumour suppressor genes, function primarily in the regulation of the cell cycle. Inactivation of tumour suppressor genes is believed to be one of the earliest cellular events which lead to the development of cancer.
Identification of genetic changes involved in the development of cancer is a promising area for the early diagnosis of the disease, and improved techniques for detecting these genetic changes are required.
The loss of one variant form of the two copies of a given DNA, or potentially, transcribed RNA sequence, normally present in diploid cells is defined herein as loss of heterozygosity (LOH). LOH reflects part of the process which ultimately results in the inactivation of tumour suppressor genes. Somatic mutation, defined herein as acquired small DNA sequence changes such as point mutation, small insertions or deletions, frequently results in the inactivation of the remaining functional allele.
Detection of such alterations in nucleic acid sequence may be difficult for two reasons. Firstly, the affected cells may be very rare within the tissue sample of interest. Secondly, the exact position of the mutation within this sequence of interest may not easily be predicted, thus requiring detailed and time consuming analysis, a process incompatible with the development of a reliable and cost effective diagnostic test. Although several techniques have been used to detect LOH as a diagnostic indicator of cancer, available methods suffer from a number of disadvantages and generally lack sensitivity. As a consequence, there is a significant need for more sensitive methods for the identification of somatic nucleic acid sequence changes.
A fundamental requirement for any method to detect AI is a procedure to distinguish between the two alleles (chromosomal regions of interest) within a cell. Techniques to achieve this fall into two main categories, cytogenetic and molecular.
Cytogenetic techniques directly visualise, by microscopy based techniques, the loss of a chromosome or a chromosomal region within a cell. A normal cell should contain two copies of each autosome one of maternal origin and one of paternal origin. The principal requirement to detect AI (such as LOH) cytogenetically is a probe which will specifically hybridise to the particular chromosome or region of interest. The identification of AI is achieved by counting the number of signals associated with each nucleus using microscopy.
In contrast, molecular techniques analyse a population of cells and require a probe or marker which differs between the maternal and paternal alleles. Traditionally, microsatellite markers have been used for this purpose. As long as a chromosome pair differs in the number of repeat units at the relevant microsatellite then loss of one of the chromosomes is indicated by loss of a microsatellite of the appropriate size.
With AI detection, in a situation where all of the cells in the sample to be analysed contain the alteration then the analysis is fairly straightforward, with the proviso that the region of interest is either large enough to allow cytogenetic analysis or appropriate sequence information is available for molecular analysis. Indeed, with substantially homogeneous populations of tumour cells, simple one round heteroduplex analysis has been proposed as a means of detecting the LOH at tumour suppressor gene loci (Mansukhani et al (1997, Diag. Mol. Pathol., 6, 229-237). However, AI analysis is much more difficult in mixed samples, i.e. those in which only a proportion of the cells comprising the sample display AI. For example, if only 5% of the cells in a population display LOH of, say the maternal allele, then the ratio of paternal to maternal alleles in the sample would be 51:49. It is often the case that the clinical samples available for analysis in cancer diagnosis are not homogeneous and the proportion of cells containing any given mutation can vary from 100% to less than 1%.
Existing techniques are not well suited to LOH analysis in mixed populations. Although cytogenetic techniques have the potential to detect LOH in a small proportion of a sample, in practice they are technically difficult and not well developed to routine clinical use, and are limited to the detection of relatively large sequence changes. Currently available molecular techniques are more amenable to clinical application but they are not able to detect small changes in the relative level of maternal to paternal alleles within an under represented population. Mansukhani et al (1997, Diag. Mol. Pathol., 6, 229-237) developed a method based on the formation of heteroduplexes between PCR amplified alleles which could be used to identify recurrent mutations in the BRCA1 and BRCA2 genes. However, the analytical sensitivity of the method was very low, severely limiting its use in clinical diagnosis. Indeed, the authors report that use of heteroduplex analysis for detecting the loss of the remaining allele is only possible if the sample contains no more than 3-10% normal issue.
Similarly, the sensitivity of other currently available molecular techniques (i.e. microsatellite LOH analysis) is such that when the rarer population, i.e. the population with LOH, is less than about 25% of the total sample, LOH is not easily detectable.
Denaturing high performance liquid chromatography (DHPLC) has been shown recently to be a useful method for detecting single nucleotide polymorphisms and inherited mutations by detecting heteroduplex DNA (Liu et al. Nucleic Acids Research. 26(6):1396-1400, 1998; O'Donovan et al. Genomics 52:44-49, 1998). U.S. Pat. No. 5,795,976 also discloses a method for separating heteroduplex and homoduplex molecules in a mixture using high performance liquid chromatography. Separation of heteroduplexes and homoduplexes by DHPLC is particularly useful in the method of the present invention.
The present inven
Fox Jayne C
Haque Kemal
Little Stephen
Horlick Kenneth R.
Ropes & Gray
Syngenta Limited
Wilder Cynthia
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