Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-08-25
2004-01-06
Kunz, Gary (Department: 1647)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024300, C536S024310
Reexamination Certificate
active
06673909
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to genes involved in the onset of muscular dystrophy.
Muscular dystrophies constitute a heterogeneous group of disorders. Most are characterized by weakness and atrophy of the proximal muscles, although in rare myopathies such as “Miyoshi myopathy” symptoms may first arise in distal muscles. Of the various hereditary types of muscular dystrophy, several are caused by mutations or deletions in genes encoding individual components of the dystrophin-associated protein (DAP) complex. It is this DAP complex that links the cytoskeletal protein dystrophin to the extracellular matrix protein, laminin-2.
Muscular dystrophies may be classified according to the gene mutations that are associated with specific clinical syndromes. For example, mutations in the gene encoding the cytoskeletal protein dystrophin result in either Duchenne's Muscular Dystrophy or Becker's Muscular Dystrophy, whereas mutations in the gene encoding the extracellular matrix protein merosin produce Congenital Muscular Dystrophy. Muscular dystrophies with an autosomal recessive mode of inheritance include “Miyoshi myopathy” and the several limb-girdle muscular dystrophies (LGMD2). Of the limb-girdle muscular dystrophies, the deficiencies resulting in LGMD2C, D, E, and F result from mutations in genes encoding the membrane-associated sarcoglycan components of the DAP complex.
SUMMARY OF THE INVENTION
A novel protein, designated dysferlin, is identified and characterized. The dysferlin gene is normally expressed in skeletal muscle cells and is selectively mutated in several families with the hereditary muscular dystrophies, e.g., Miyoshi myopathy (MM) and limb girdle muscular dystrophy-2B (LGMD2B). These characteristics of dysferlin render it a candidate disease gene for both MM and LGMD2B. An additional novel protein, brain-specific dysferlin, has also been identified. Defects in brain-specific dysferlin may predispose to selected disorders of the central nervous system. Moreover, the expression of brain-specific dysferlin may be important as a marker for normal neural development (e.g., in vivo or in neural cells in culture). Manipulation of levels of expression of brain-specific dysferlin, and of the type of expressed brain-specific dysferlin is of use for analyzing the function of brain-specific dysferlin and related dysferlin-associated molecules.
The invention features an isolated DNA which includes a nucleotide sequence hybridizing under stringent hybridization conditions to a strand of SEQ ID NO:3 or SEQ ID NO:117. SEQ ID NO:117 corresponds to nucleotides 374-6613 of wild type dysferlin.
The invention also features an isolated DNA including a nucleotide sequence selected from SEQ ID NOs:4-12. SEQ ID NOs:4-12 are oligonucleotides that span the mutations of 537insA, Q605X, 5966delG, E1883X, 6391+1G to A, I1298V, R2042C, H1857R, and 6071/2delAG, respectively (Table 2).
Also within the invention is an isolated DNA comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs:22-30.
Also within the invention is an isolated DNA comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs:22-30. SEQ ID NOs:22-30 are oligonucleotides with wild type sequences that span the mutant regions identified in the mutants 537inSA, Q605X, 5966delG, E1883X, 6391+1G to A, I1298V, R2042C, H1857R, and 6071/2delAG, respectively (Table 2).
Also within the invention is a pair of PCR primers consisting of:
(a) a first single stranded oligonucleotide consisting of 14-50 contiguous nucleotides of the sense strand of SEQ ID NO:117; and
(b) a second single stranded oligonucleotide consisting of 14-50 contiguous nucleotides of the antisense strand of SEQ ID NO:117, wherein the sequence of at least one of the oligonucleotides is identical to a portion of a strand of SEQ ID NO:3, and the first oligonucleotide is not complementary to the second oligonucleotide.
Also within the invention is a pair of single stranded oligonucleotides selected from of SEQ ID NOs 130-231, SEQ ID NO:110, and SEQ ID NO:112.
Also within the invention is an isolated DNA including a nucleotide sequence that encodes a protein that shares at least 70% sequence identity with SEQ ID NO:2, or a complement of the nucleotide sequence.
Also within the invention is an isolated DNA including a nucleotide sequence which hybridizes under stringent hybridization conditions to a strand of a nucleic acid, the nucleic acid having a sequence selected from SEQ ID NOs:31-79 and 90-100. SEQ ID NOs:90-100 are intron sequences from a dysferlin gene. Specifically, SEQ ID NOs:90-100 are intron sequence 5′ of exon 50, intron sequence 3′ of exon 50, intron sequence 5′ of exon 51, intron sequence 3′ of exon 51, intron sequence 5′ of exon 52, intron sequence 3′ of exon 52, intron sequence 5′ of exon 53, intron sequence 3′ of exon 53, intron sequence 5′ of exon 54, intron sequence 3′ of exon 54, and intron sequence 5′ of exon 55.
Also within the invention is a single stranded oligonucleotide of 14-50 nucleotides in length having a nucleotide sequence which is identical to a portion of a strand of a nucleic acid selected from SEQ ID NOs:31-79 and 90-100.
Also within the invention is a pair of PCR primers consisting of:
(a) a first single stranded oligonucleotide consisting of 14-50 contiguous nucleotides of the sense strand of a nucleic acid selected from SEQ ID NOs:31-85; and
(b) a second single stranded oligonucleotide consisting of 14-50 contiguous nucleotides of the antisense strand of a nucleic acid selected from SEQ ID NOs:31-85, wherein the sequence of at least one of the oligonucleotides includes a sequence identical to a portion of a strand of a nucleic acid selected from SEQ ID NOs: 31-79 and 90-100, and the first oligonucleotide is not complementary to the second oligonucleotide.
Also within the invention is a pair of single stranded oligonucleotides selected from SEQ ID NOs 101-116, SEQ ID NOs 184-185, SEQ ID NOs 188-191, SEQ ID NOs 210-213, and SEQ ID NOs 216-217.
Also within the invention is a substantially pure protein that has an amino acid sequence sharing at least 70% sequence identity with SEQ ID NO:2.
Also within the invention is a substantially pure protein the sequence of which includes amino acid residues 1-500, 501-1000, 1001-1500, or 1501-2080 of SEQ ID NO:2.
Also within the invention is a substantially pure protein including the amino acid sequence of SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, or SEQ ID NO:89.
In another aspect, the invention features a transgenic non-human mammal having a transgene disrupting or interfering with the expression of a dysferlin gene, the transgene being chromosomally integrated into the germ cells of the animal.
Another embodiment of the invention features a method of decreasing the symptoms of muscular dystrophy in a mammal by introducing into a cell of the mammal (e.g., a muscle cell or a muscle precursor cell) an isolated DNA which hybridizes under stringent hybridization conditions to a strand of SEQ ID NO:3.
Another aspect of the invention provides a method for identifying a patient, a fetus, or a pre-embryo at risk for having a dysferlin-related disorder by (a) providing a sample of genomic DNA from the patient, fetus, or pre-embryo; and (b) determining whether the sample contains a mutation in a dysferlin gene.
In another aspect, the invention provides a method for identifying a patient, a fetus, or a pre-embryo at risk for having a dysferlin-related disorder by (a) providing a sample including dysferlin mRNA from the patient, fetus, or pre-embryo; and (b) determining whether the dysferlin mRNA contains a mutation.
Methods of identifying mutations in a dysferlin sequence are useful for predicting (e.g., predicting whether an individual is at risk for developing a dysferlin-related disorder) or diagnosing disorders associated with dysferlin, e.g., MM and LGMD2B. Such methods can also be used to determine if an individual, fetus, or a pre-embryo is a carrier of a
Aoki Masashi
Brown, Jr. Robert H.
Ho Meng F.
Liu Jing
Matsuda-Asada Chie
Fish & Richardson P.C.
Hayes Robert C.
Kunz Gary
The General Hospital Corporation
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