Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-07-30
2002-11-05
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S024300, C536S024310
Reexamination Certificate
active
06475739
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to disease diagnosis and to the identification of disease carriers. More specifically, the present invention provides methods for identifying individuals who are afflicted with or carriers of diseases associated with one or more genomic deletion.
2. Description of the Related Art
Van Buchem's disease (VBD) is a rare autosomal recessive disorder that results in a bone dysplasia referred to as craniotubular hyperostosis. VBD was first described in 1962 as including osteosclerosis of the skull, mandible, clavicles, ribs, and diaphysis of the long bones beginning during puberty and, in some cases, leading to optic atrophy and perceptive deafness from nerve pressure. Van Buchem et al.,
Am. J. Med
. 33:387-397 (1962).
More recently, additional occurrences of VBD have been reported. In 1988, Fryns et al. described a 7.5-year-old boy with VBD. This patient had presented at 2 months of age with left-side peripheral facial nerve palsy but had, at that time, no radiologically visible signs of sclerosis of the skull.
Europ. J. Pediat
. 147:99-100 (1988).
In 1997, Balemans et al. studied 11 VBD patients from a highly inbred and geophraphically isolated Dutch family. Each of these patients shared a common ancestor from 9 preceding generations. By applying a genome wide search for linkage using more than 300 microsatellite markers having an average spacing of 10 cM, these authors found a maximum lod score of 9.33 at theta=0.01 with marker D17S1299 and narrowed the assignment to a region of less than 1 cM between markers D17S1787 and D17S934
. Am. J. Hum. Genet
. 61(Suppl.):A12 (1997); See, also, Van Hul et al.,
Am. J. Hum. Genet
. 62:391-399 (1998).
A related disease sclerosteosis is an autosomal semi-dominant disease that shares some of the clinical symptoms of VBD. The term “sclerosteosis” has been applied to a disorder similar to Van Buchem hyperostosis corticalis generalisata but differing in the radiologic appearance of the bone changes and in the presence of asymmetric cutaneous syndactyly of the index and middle fingers in many cases. In
Handbuch der Kinderheilkunde
351-355 (Opitz, H. et al., Berlin: Springer (pub.), 1967). More specifically, this disease resembles VBD in that it comprises a progressive sclerosing bone dysplasia characterized by generalized osteosclerosis and hyperostosis of the skeleton, affecting mainly the skull and mandible thereby causing facial paralysis and hearing loss. In contrast to VBD, however, sclerosteosis is further characterized by gigantism and hand abnormalities.
The rare genetic mutation responsible for the sclerosteosis syndrome has been localized to the region of human chromosome 17 that encodes a novel member of the TGF-beta binding-protein family (one representative example of which is designated “hSOST”). In 1999, Balemans et al. assigned the locus for sclerosteosis to 17q12-q21 which is the same general region as the locus for VBD.
Am. J. Hum. Genet
. 64:1661-1669 (1999). Due to the clinical similarities between VBD and sclerosteosis, Beighton et al. suggested that these conditions might be caused by mutations within the same gene.
Clin. Genet
25:175-181 (1984). This hypothesis was further supported by the genetic experimentation later performed by Balemans et al. Supra.
Traditional methodologies for identifying genomic deletions such as, for example, restriction fragment length polymorphism (RFLP), fluorescence in situ hybridization (FISH) and Southern blotting permit the identification of individuals who are homozygous for a genomic deletion and, as a consequence, are afflicted with the associated genetic disease. Because these methods are time consuming and/or require high-quality DNA samples or live cells, they are of limited use in the identification of individuals who are heterozygous for and, therefore, carriers of a genetic disease. What is needed in the art are methods that permit the rapid identification of genetic disease carriers, which methods distinguish between individuals who are homozygous for a genomic deletion, individuals who are heterozygous for a genomic deletion and individuals who do not possess a given genomic deletion. As described in detail herein, the present invention fulfills this and other related needs.
BRIEF SUMMARY OF THE INVENTION
The present invention is directed generally to disease diagnosis and to the identification of disease carriers. More specifically, the invention disclosed herein provides methods for identifying chromosomal deletions that are associated with a disease phenotype. Particular methods within the scope of the present invention are directed to the identification of individuals who are afflicted with or carriers for the genomic deletion associated with Van Buchem's disease. By alternate embodiments, the present invention also provides methods having general utility in the detection of a wide variety of diseases characterized by genomic deletions.
The present invention provides, in one embodiment, methods for distinguishing between an individual who is homozygous for a genomic deletion, an individual who is heterozygous for a genomic deletion and an individual who is negative for a genomic deletion.
A first method comprises: (a) obtaining a sample of genomic DNA from an individual; (b) performing a first amplification reaction with a first oligonucleotide primer pair comprising a first oligonucleotide primer and a second oligonucleotide primer wherein the first oligonucleotide primer is complementary to the nucleotide sequence upstream of the genomic deletion and the second oligonucleotide primer is complementary to the nucleotide sequence downstream of said genomic deletion; (c) performing a second amplification reaction with a second oligonucleotide primer pair comprising a third oligonucleotide primer and a fourth oligonucleotide primer wherein the third oligonucleotide primer is complementary to the nucleotide sequence either upstream or downstream of the genomic deletion and the fourth oligonucleotide primer is complementary to the nucleotide sequence comprising the genomic deletion; and (d) detecting the product of the amplification reactions of (b) and (c).
By this first type method, a positive amplification reaction of (b) and a negative amplification reaction of (c) indicates an individual who is homozygous for the large genomic deletion; a positive amplification reaction of (b) and a positive amplification reaction of (c) indicates an individual who is heterozygous for the large genomic deletion; and a negative amplification reaction of (b) and a positive amplification reaction of (c) indicates an individual who is negative for the large genomic deletion.
A second method comprises: (a) obtaining a sample of genomic DNA from said individual; (b) performing an amplification reaction employing at least two oligonucleotide primer pairs in which an oligonucleotide primer is common to both said primer pairs, wherein a first primer pair has a first oligonucleotide primer complementary to a nucleotide sequence that flanks said genomic deletion upstream of said genomic deletion and a second oligonucleotide primer complementary to a nucleotide sequence that flanks said genomic deletion downstream of said genomic deletion, and a second primer pair has third oligonucleotide primer complementary to a nucleotide sequence within said genomic deletion and either said first or second oligonucleotide primer; and (c) detecting an amplified product of said amplification reaction.
By this second method, a positive amplification reaction of said first primer pair and a negative amplification reaction of said second primer pair indicates an individual that is homozygous for said large genomic deletion; a positive amplification reaction of said first primer and a positive amplification reaction of said second primer pair indicates an individual that is heterozygous for said large genomic deletion; and a negative amplification reaction of said first primer pair and a positive amplification reac
Brunkow Mary E.
Paeper Bryan
Proll Sean
Staehling-Hampton Karen
Celltech R&D, Inc.
Horlick Kenneth R.
Seed Intellectual Property Law Group PLLC
Stzelecka Teresa
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