Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-12-21
2002-12-03
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S029000
Reexamination Certificate
active
06489115
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the fields of molecular biology and molecular genetics. More specifically, the invention relates to methods for identifying genetic alterations associated with cancer, Fragile X syndrome, Huntington's disease, myotonic dystrophy and other disorders.
BACKGROUND OF THE INVENTION
Several publications are referenced by numerals in parentheses in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found at the end of the specification. The disclosure of each of these publications is incorporated by reference herein.
Trinucleotide repeat (TNR) instability has recently been recognized as the mutational cause of at least 13 different inherited diseases (1-3). Increases in TNR lengths provide the molecular alterations associated with Huntington's disease (HD), Fragile X syndrome, myotonic dystrophy, SBMA (spinal and bulbar muscular atrophy), SCA I (spinocerebellar ataxia type I) and other syndromes.
Huntington's disease (HD) provides a paradigm of a genetic disease caused by TNR instability. HD is a progressive neurodegenerative malady that results in a number of symptoms, including choreic movement disorder and dementia (4). The disease typically first manifests itself in individuals in their 30's and 40's, and culminates in premature death 10-20 years later. The disease is inherited in an autosomal dominant manner, with particularly severe effects when inherited from the father.
The genetic phenomenon known as anticipation has also been associated with TNR diseases, including HD. Anticipation is found when the disease presents more severely or occurs with earlier onset with each generation. This non-Mendelian inheritance pattern was puzzling for many years. It has now been elucidated by molecular analysis of the mutated loci.
It is now accepted that the vast majority of TNR disease cases result from the expansion of naturally occurring TNR's. The HD gene, for example, contains a series of CAG's within the coding region of the corresponding protein, huntingtin (4). This repeat tract of CAG codons encoding glutamine residues, normally occurs 10-29 times in unaffected populations. However, 36-121 copies have been observed in patients afflicted with HD (4,5). The correlation between TNR length and the disease state is extremely high (>95%), lending strong support to the hypothesis that this mutation is intimately linked to the disease (4,5). The correlation between TNR length and HD has been verified using a transgenic mouse model for HD (6) in which a transgene containing the expanded CAG tract was sufficient to induce symptoms in mice similar to those observed in HD.
Examination of human families with a history of HD indicates that increases in TNR's occur both in the germline and in somatic tissue (4,5,7). Gains in TNR length can be quite large, even between successive generations, especially in cases where the parent harbors 30-35 repeats, a number that is intermediate between normal and diseased states (8).
As mentioned previously, TNR instability is now known to be a causative factor in at least 12 other genetic diseases (1-3). In each case, a distinct gene containing a TNR has increased in length in diseased individuals. The triplet sequences known to undergo TNR expansions are CNG (where N is any nucleotide) or GAA(9).
Instability appears to be restricted to these repeats, as there is no evidence to date to suggest instability of other triplet sequences, such as TAG or GAC. A number of molecular characteristics varies with each disease. For example, the TNR tract can reside within or outside of a structural gene. The number of repeats in the diseased versus normal populations varies widely between diseases. For example, this number can be in excess of 2,000 repeats for myotonic dystrophy (10). The mutant gene is expressed in some diseases but not in others. The encoded proteins have widely different biochemical properties. Additionally, the pattern of germline and somatic variation differs.
In addition to genetic disorders, emerging evidence supports an important connection between TNR instability and prostate and testicular cancer. In prostate cancer, TNR length affects cancer risk (11) due to the presence of an unstable CAG repeat in the androgen receptor (AR) gene (12). Deletions of the CAG tract are sometimes associated with prostate tumor formation (13). A molecular explanation for these findings is provided by mutational studies (14) that have shown AR transactivation of important AR-responsive genes is directly related to the number of CAG repeats. Clearly AR function depends on TNR length and hence is directly affected by the genetic stability (or instability) of the tract. For testicular cancer, expansion of CAG tracts was observed in five different families predisposed to this malignancy (15). That study concluded that CAG expansion may play an important role in testicular tumorigenesis. However, the gene or genes responsible for CAG instability in prostate and testicular tumor cells have not yet been identified.
Given the medical importance of TNR mutations and the novel genetic behavior of these elements, intense efforts are underway to elucidate the mechanism (or mechanisms) underlying TNR instability in human cells. However, to date there are at least three major experimental limitations which have hampered progress of these efforts. First, nearly all investigations have been limited to tissue samples from affected human kindreds. Second, analysis has typically been confined to endogenous (naturally occurring) DNA sequences, as opposed to test sequences that are more easily manipulated. Third, it has been difficult, if not impossible to identify individual cells which have undergone expansions. Instead, physical methods such as PCR or Southern blotting have been performed on unselected cell populations.
To further confound analysis, transgenic mice strains have been established that harbor human TNR-containing genes but do not appear to have large, frequent TNR expansions. Surprisingly, the TNR sequences in transgenic mice are very stable (16-19). In these studies, parts or all of human genes (HD, SCA I, etc.) that include CAG/CTG tracts of 55-162 repeats were integrated into the mouse genome at the corresponding loci. The genetic stability of these sequences was monitored both in somatic tissue and through intergenerational transmission. The TNRs in these transgenes show no alterations (l1) or small changes of 1-8 repeats in tract size (17-19). Approximately equal numbers of expansions and contractions have been observed. Perhaps TNR expansions appear at higher rates in humans due to some aberrant DNA metabolic event that is absent in mice. These results serve to underscore the importance of using human cells for studies on the stability of TNRs.
SUMMARY OF THE INVENTION
In accordance with the present invention, methods are provided for the rapid and efficient analysis of trinucleotide repeat (TNR) tract alterations in mammalian cells. An exemplary method of the invention entails contacting mammalian cells with a shuttle vector under conditions whereby the shuttle vector enters the cells and replicates therein. Following replication, the shuttle vector is recovered and transfected into yeast cells under selection pressure. Alterations of the TNR tract in the mammalian cell results in a restoration of histidine or uracil expression for example, from the shuttle vector, thereby allowing the transfected yeast cells to survive in the absence of these agents. Only those yeast cells containing altered TNR tract lengths survive in the presence of the selection agent. The shuttle vector DNA may optionally be isolated. Alteration in TNR tract length DNA is then characterized using conventional molecular biology techniques. Such methods include, without limitation, polymerase chain reaction, nucleotide sequencing and gel electrophoresis. The shuttle vector DNA comprises TNR tracts having trinucleotides sel
Lahue Robert S.
Miret Juan J.
Pelletier Richard
Dann Dorfman Herrell and Skillman
McKelvey Terry
The Board of Regents of the University of Nebraska
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