Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-10-02
2004-11-16
Falk, Anne-Marie (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S325000, C435S455000, C435S461000
Reexamination Certificate
active
06818401
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for detection and interpretation of disease related mutations through the combination of haploid gene transfer with functional, immunological or other analysis of the gene product.
BACKGROUND OF INVENTION
Detection of disease-causing mutations is a complex and challenging task in medical and veterinary genetics and research. Unfortunately, loss-of-function mutations, including partial loss-of-function mutation, or gain-of-function mutations, including alteration of function and dominant negative mutations, causing inherited genetic diseases are a common problem for humans and other animals. Complete and effective detection of these mutations presents enormous possibilities as a diagnostic, preventative, or research tool.
Currently genomic sequencing of peripheral blood DNA is widely used for identification of genetic mutations associated with various diseases. In particular, it may be used to detect mutations in individuals for inherited genetic diseases. For example, Myriad Genetics, Inc. (Salt Lake City, Utah) has developed a genetic test for detection of loss-of-function mutations in BRCA1 and BRCA2, genes which have been linked to breast cancer. This test sequences all coding exons of BRCA1 and BRCA2, making it labor-intensive and costly. In addition, it cannot detect deleted exons, inversions, mutations causing loss of transcriptional activity, etc. As a result, many mutations in these two genes cannot be meaningfully detected by genomic sequencing. Table 1 displays the types and frequencies of mutations found in the BRCA1 and BRCA2 genes. Furthermore, when diploid cells that are heterozygous for a loss-of-function or a gain-of-function mutation are tested, the wild type allele can often mask the mutant allele. As a result, this test may not be accurate in detecting single mutant alleles. The usefulness of this and other such tests to the medical and veterinary professions and research scientists is therefore limited by their diagnostic shortcomings and prohibitive costs.
TABLE 1
Frequency and Type of Mutations
in the BRCA1 and BRCA2 Genes
Mutation Type
BRCA1 Gene
BRCA2 Gene
Frameshift
195 (42.5%)
126 (53.5%)
Nonsense
55 (12%)
20 (7.8%)
Splice
16 (3.5%)
4 (1.6%)
Missense
21 (4.6%)
12 (4.7%)
Large Deletion
3 (0.7%)
—
Polymorphism
37 (8%)
6 (2.4%)
Yet Unclassified
132 (28.7%)
76 (30%)
Total Number
459 (100%)
254 (100%)
The Protein Truncation Test (PTT) is another diagnostic test available for the detection of loss-of-function alleles, which involves in vitro transcription and translation of the gene of interest, followed by gel electrophoretic analysis. This test is designed to detect mutations that produce a truncated protein. While this test provides an efficient means of detecting nonsense mutations, it is of no real use for detection of many other common mutations, such as frameshift, missense, inversions, and other mutations that have no detectable effect on the size of the transcribed protein.
Microarrays present another means of detecting mutations. In these assays thousands of specific oligonucleotides complementary to all known base substitutions, insertions and deletions for a gene of interest are bound to glass slides. Fluorescently labeled PCR-amplified fragments from the gene of interest are then hybridized to the microarray and binding to a particular oligonucleotide is detected. Microarrays have high up-front costs and are also not accurate at detecting heterozygous mutations. They are further limited to detection of mutations represented in the oligonucleotides.
A number of indirect methods for molecular detection of mutations exist. These include single-strand conformation polymorphism, denaturing gradient gel electrophoresis, denaturing high-performance liquid chromatography and other electrophoretic or enzymatic-based methods. Each of these methods is limited in the types of mutations it can detect and in its ability to detect heterozygous mutations in general.
To overcome the difficulty in the detection of heterozygote genotypes for inherited genetic disorders, Yan., “Conversion of diploidy to haploidy”,
Nature
403: 723-724 (February, 2000) (Yan (1)), Yan et al., “Genetic testing-Present and Future”,
Science
298: 1890-1891 (September, 2000) (Yan (2)), and Zoghbi et al., “Assignment of Autosomal Dominant Spinocerebellar Ataxia (SCA1) Centromeric to the HLA Region on the Short Arm of Chromosome 6, Using Multilocus Linkage Analysis”,
Am. J. Hum. Genet
. 44: 255-263 (1989) have all proposed a method of genetic testing using somatic cell hybrids haploid for a chromosome of interest. This method manipulates the two copies (alleles) of a gene of interest from a donor cell by separating the two chromosomes so that each can be analyzed individually. Detection of heterozygous mutations by these methods is improved in such cells because the wild type allele has been eliminated and cannot mask the mutated allele. However, the method described requires extremely labor intensive and impractical techniques for the isolation and segregation of haploid hybrids bearing the desired chromosome in a haploid state. Further, while the nucleic acid analysis of the haploid cells would facilitate detection of exon deletions, inversions, and transcriptional defects, the approach does not offer a significant advantage over traditional methods. Yan (2) admit that “[i]t is important to note that Conversion [the Yan et al. approach] is not a substitute for the [traditional] detection methods described above, but rather is an adjunct that provides improved nucleic acid templates that can maximize the sensitivity of conventional methods”,
Science
289, p.1892. Yan (2) further admit that “[d]isadvantages of the Conversion [Yan et al.] approach include the increased time and expense associated with the hybrid generation and screening process”,
Science
289, p.1892. Thus, while the proposed method offers an improvement over the conventional screening methods, reliance on the conventional methods is not abolished and the improvement in detection is slight, especially in light of the dramatic increases in time and expense associated with the method.
Several other methods of transferring one or multiple chromosomes to a host cell have been previously described (U.S. Pat. No. 4,806,476; WO 00/34436; U.S. Pat. No. 6,077,697). This method, microcell-mediated chromosome transfer (MMCT) was first described by Fournier and Ruddle for the transfer of murine chromosomes from one cell to another (PNAS 74: 319-323 (1977)) and by McNeill and Brown for the transfer of single human chromosomes from one cell to another (PNAS 77:5394-5398 1980). MMCT describes a way of generating microcells, by prolonged colcemid and cytochalsin B treatment of donor cells, which contain one or more chromosomes or chromosomal fragments from donor cells, and fusing them using polyethylene glycol (PEG) with target cells to generate microcell hybrids, haploid for the desired chromosome/chromosomal fragment from the donor cell (FIG.
2
). While these papers presents an efficient means of generating haploid cells, they fail to describe a method employing easily obtainable donor cells. In the paper of Fournier and Ruddle, mouse embryo fibroblasts were used as donors for microcell-mediated chromosome transfer. McNeill and Brown utilized human foreskin fibroblasts as donors for human chromosome transfer.
Therefore, there is a need for a medically, veterinarily, or scientifically useful method of detecting loss-of-function mutations, including partial loss-of-function mutations, or gain-of-function mutations, including alteration of function and dominant negative mutations, in any of a variety of genes. The present invention addresses the deficiencies of the prior art by providing a method for genetic testing using easily obtainable sources of genetic material that can 1) detect many types of mutations, including nonsense, missense, frameshif
Beaudet Arthur
Bodamer Olaf
Killary Ann
Lovell Maria Mercedes
Baker & Botts LLP
Board of Regents University of Texas System
Falk Anne-Marie
Sullivan Daniel M.
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