WO2001085998A1 - Haplotypes du gene crybb1 - Google Patents

Haplotypes du gene crybb1 Download PDF

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Publication number
WO2001085998A1
WO2001085998A1 PCT/US2001/014715 US0114715W WO0185998A1 WO 2001085998 A1 WO2001085998 A1 WO 2001085998A1 US 0114715 W US0114715 W US 0114715W WO 0185998 A1 WO0185998 A1 WO 0185998A1
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Prior art keywords
crybbl
gene
haplotype
seq
individual
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PCT/US2001/014715
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English (en)
Inventor
Julie Y. Choi
Amir Kazemi
Stefanie E. Kliem
Beena Koshy
Eileen Rounds
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Genaissance Pharmaceuticals, Inc.
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Priority to AU2001259595A priority Critical patent/AU2001259595A1/en
Publication of WO2001085998A1 publication Critical patent/WO2001085998A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/20—Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations

Definitions

  • This invention relates to variation in genes that encode pharmaceutically-important proteins.
  • this invention provides genetic variants of the human crystallin, beta B 1 (CRYBB 1 ) gene and methods for identifying which variant(s) of this gene is/are possessed by an individual.
  • haplotype is the ordered combination of polymorphisms in the sequence of each form of a gene that exists in the population. Because haplotypes represent the variation across each form of a gene, they provide a more accurate and reliable measurement of genetic variation than individual polymorphisms. For example, while specific variations in gene sequences have been associated with a particular phenotype such as disease susceptibility (Roses AD supra; Ulbrecht M et al. 2000 Am JRespir Crit Care Med 161: 469-74) and drug response (Wolfe CR et al.
  • CRYBBl is a member of the beta/gamma-crystallin protein superfamily that are structural proteins of the eye lens.
  • CRYBBl, along with CRYBB2 and CRYBB3 form the basic subgroup of the ( ⁇ B-) crystallin family of proteins.
  • the three genes (CRYBBl, -2,-3) are closely linked on chromosome 22q.
  • Members of the beta/gamma-crystallin protein suerfamily, including CRYBBl have a two-domain beta structure which folds into four similar Greek key motifs.
  • CRYBB2 Congenital human cataracts have been described that are associated with an altered form of CRYBB2 (Kramer et al., Genomics 1996; 35:539-542; Litt et al., Hum. Mol. Genet. 1997; 6:665-668). Mutations to this gene result in a truncated polypeptide that would not be expected to fold properly, but instead randomly aggregate and cause light scattering (Graw, B ⁇ ol Chem. 1997; 378:1331-1348).
  • CRYBB2 is the beta- crystallin family member that is most strongly expressed in the adult lens, and was therefore chosen first for mutation screening (OMIM NO: 123620). It is possible that mutations in CRYBBl may also lead to improper protein folding and subsequent cataract development.
  • the crystallin, beta Bl gene is located on chromosome 22ql2.1 and contains 6 exons that encode a 252 amino acid protein.
  • Reference sequences for the CRYBBl gene (Genaissance Reference No. 1223838; SEQ ID NO: 1), coding sequence (GenBank Accession No:NM_001887.1), and protein are shown in Figures 1, 2 and 3, respectively.
  • polymorphic sites correspond to the following nucleotide positions in Figure 1: 15430 (PS1), 15469 (PS2), 15549 (PS3), 15551 (PS4), 21856 (PS5), 21877 (PS6), 25787 (PS7), 25808 (PS8), 26059 (PS9), 26084 (PS10), 32167 (PS11) and 34489 (PS12).
  • PS polymorphic sites
  • guanine or adenine at PS 1 cytosine or thymine at PS2, cytosine or thymine at PS3, cytosine or thymine at PS4, guanine or adenine at PS5, cytosine or adenine at PS6, cytosine or thymine at PS7, thymine or guanine at PS8, guanine or adenine at PS9, cytosine or thymine at PS 10, thymine or adenine at PS 11 and cytosine or thymine at PS 12.
  • the inventors have determined the identity of the alleles at these sites in a human reference population of 79 unrelated individuals self-identified as belonging to one of four major population groups: African descent, Asian, Caucasian and Hispanic/Latino. From this information, the inventors deduced a set of haplotypes and haplotype pairs for PS1-12 in the CRYBBl gene, which are shown below in Tables 4 and 3, respectively. Each of these CRYBBl haplotypes defines a naturally-occurring isoform (also referred to herein as an "isogene") of the CRYBBl gene that exists in the human population. The frequency with which each haplotype and haplotype pair occurs within the total reference population and within each of the four major population groups included in the reference population was also determined.
  • the invention provides a method, composition and kit for genotyping the CRYBBl gene in an individual.
  • the genotyping method comprises identifying the nucleotide pair that is present at one or more polymorphic sites selected from the group consisting of PS1, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS11 and PS12 in both copies of the CRYBBl gene from the individual.
  • a genotyping composition of the invention comprises an oligonucleotide probe or primer which is designed to specifically hybridize to a target region containing, or adjacent to, one of these novel CRYBBl polymorphic sites.
  • a genotyping kit of the invention comprises a set of oligonucleotides designed to genotype each of these novel CRYBBl polymorphic sites. The genotyping method, composition, and kit are useful in determining whether an individual has one of the haplotypes in Table 4 below or has one of the haplotype pairs in Table 3 below.
  • the invention also provides a method for haplotyping the CRYBBl gene in an individual.
  • the haplotyping method comprises determining, for one copy of the CRYBBl gene, the identity of the nucleotide at one or more polymorphic sites selected from the group consisting of PS1, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS11 and PS12.
  • the haplotyping method comprises determining whether one copy of the individual's CRYBBl gene is defined by one of the CRYBBl haplotypes shown in Table 4, below, or a sub-haplotype thereof.
  • the haplotyping method comprises determining whether both copies of the individual's CRYBBl gene are defined by one of the CRYBBl haplotype pairs shown in Table 3 below, or a sub-haplotype pair thereof.
  • the method for establishing the CRYBBl haplotype or haplotype pair of an individual is useful for improving the efficiency and reliability of several steps in the discovery and development of drugs for treating diseases associated with CRYBB 1 activity, e.g., cataract.
  • the haplotyping method can be used by the pharmaceutical research scientist to validate CRYBB 1 as a candidate target for treating a specific condition or disease predicted to be associated with CRYBB 1 activity. Determining for a particular population the frequency of one or more of the individual CRYBBl haplotypes or haplotype pairs described herein will facilitate a decision on whether to pursue CRYBB 1 as a target for treating the specific disease of interest. In particular, if variable CRYBBl activity is associated with the disease, then one or more CRYBBl haplotypes or haplotype pairs will be found at a higher frequency in disease cohorts than in appropriately genetically matched controls.
  • variable CRYBBl activity has little, if any, involvement with that disease.
  • the pharmaceutical research scientist can, without a priori knowledge as to the phenotypic effect of any CRYBBl haplotype or haplotype pair, apply the information derived from detecting CRYBBl haplotypes in an individual to decide whether modulating CRYBB 1 activity would be useful in treating the disease.
  • the claimed invention is also useful in screening for compounds targeting CRYBBl to treat a specific condition or disease predicted to be associated with CRYBB 1 activity. For example, detecting which of the CRYBBl haplotypes or haplotype pairs disclosed herein are present in individual members of a population with the specific disease of interest enables the pharmaceutical scientist to screen for a compound(s) that displays the highest desired agonist or antagonist activity for each of the most frequent CRYBBl isoforms present in the disease population.
  • the claimed haplotyping method provides the scientist with a tool to identify lead compounds that are more likely to show efficacy in clinical trials.
  • the method for haplotyping the CRYBBl gene in an individual is also useful in the design of clinical trials of candidate drugs for treating a specific condition or disease predicted to be associated with CRYBBl activity. For example, instead of randomly assigning patients with the disease of interest to the treatment or control group as is typically done now, determining which of the CRYBBl haplotype(s) disclosed herein are present in' individual patients enables the pharmaceutical scientist to distribute CRYBBl haplotypes and/or haplotype pairs evenly t,o treatment and control groups, thereby reducing the potential for bias in the results that could be introduced by a larger frequency of a CRYBB 1 haplotype or haplotype pair that had a previously unknown association with response to the drug being studied in the trial. Thus, by practicing the claimed invention, the scientist can more confidently rely on the information learned from the trial, without first determining the phenotypic effect of any CRYBBl haplotype or haplotype pair.
  • the invention provides a method for identifying an association between a trait and a CRYBB 1 genotype, haplotype, or haplotype pair for one or more of the novel polymorphic sites described herein.
  • the method comprises comparing the frequency of the CRYBBl genotype, haplotype, or haplotype pair in a population exhibiting the trait with the frequency of the CRYBBl genotype or haplotype in a reference population. A higher frequency of the CRYBBl genotype, haplotype, or haplotype pair in the trait population than in the reference population indicates the trait is associated with the CRYBBl genotype, haplotype, or haplotype pair.
  • the trait is susceptibility to a disease, severity of a disease, the staging of a disease or response to a drug.
  • the CRYBBl haplotype is selected from the haplotypes shown in Table 4, or a sub-haplotype thereof. Such methods have applicability in developing diagnostic tests and therapeutic treatments for cataract.
  • the invention provides an isolated polynucleotide comprising a nucleotide sequence which is a polymorphic variant of a reference sequence for the CRYBBl gene or a fragment thereof.
  • the reference sequence comprises SEQ ID NO: 1 and the polymorphic variant comprises at least one polymorphism selected from the group consisting of adenine at PS1, thymine at PS2, thymine at PS3, thymine at PS4, adenine at PS5, adenine at PS6, thymine at PS7, guanine at PS8, adenine at PS9, thymine at PS10, adenine at PS11 and thymine at PS12.
  • a particularly preferred polymorphic variant is an isogene of the CRYBB 1.gene.
  • a CRYBB 1 isogene of the invention comprises guanine or adenine at PS1, cytosine or thymine at PS2, cytosine or thymine at PS3, cytosine or thymine at PS4, guanine or adenine at PS5, cytosine or adenine at PS6, cytosine or thymine at PS7, thymine or guanine at PS8, guanine or adenine at PS9, cytosine or thymine at PS10, thymine or adenine at PS11 and cytosine or thymine at PS12.
  • the invention also provides a collection of CRYBB 1 isogenes, referred to herein as a CRYBB 1 genome anthology.
  • the invention provides a polynucleotide comprising a polymorphic variant of a reference sequence for a CRYBB 1 cDNA or a fragment thereof.
  • the reference sequence comprises SEQ ID NO:2 (Fig.2) and the polymorphic cDNA comprises adenine at a position corresponding to nucleotide 294.
  • a particularly preferred polymorphic cDNA variant comprises the coding sequence of a CRYBBl isogene defined by haplotypes 1- 11 and 13 - 16.
  • Polynucleotides complementary to these CRYBBl genomic and cDNA variants are also provided by the invention. It is believed that polymorphic variants of the CRYBBl gene will be useful in studying the expression and function of CRYBBl, and in expressing CRYBBl protein for use in screening for candidate drugs to treat diseases related to CRYBBl activity.
  • the invention provides a recombinant expression vector comprising one of the polymorphic genomic variants operably linked to expression regulatory elements as well as a recombinant host cell transformed or fransfected with the expression vector.
  • the recombinant vector and host cell may be used to express CRYBBl for protein structure analysis and drug binding studies.
  • the present invention also provides nonhuman transgenic animals comprising one of the CRYBB 1 polymorphic genomic variants described herein and methods for producing such animals.
  • the transgenic animals are useful for studying expression of the CRYBBl isogenes in vivo, for in vivo screening and testing of drugs targeted against CRYBB 1 protein, and for testing the efficacy of therapeutic agents and compounds for cataract in a biological system.
  • the present invention also provides a computer system for storing and displaying polymorphism data determined for the CRYBBl gene.
  • the computer system comprises a computer processing unit; a display; and a database containing the polymorphism data.
  • the polymorphism data includes the polymorphisms, the genotypes and the haplotypes identified for the CRYBB 1 gene in a reference population.
  • the computer system is capable of producing a display showing CRYBBl haplotypes organized according to their evolutionary relationships.
  • Figure 1 illustrates a reference sequence for the CRYBBl gene (Genaissance Reference No. 1223838; contiguous lines; SEQ ID NO:l), with the start and stop positions of each region of coding sequence indicated with a bracket ([ or ]) and the numerical position below the sequence and the polymorphic site(s) and polymorphism(s) identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymorphic site in the sequence.
  • Figure 2 illustrates a reference sequence for the CRYBBl coding sequence (contiguous lines; SEQ ID NO:2), with the polymorphic site(s) . and polymorphism(s) identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymorphic site in the sequence.
  • Figure 3 illustrates a reference sequence for the CRYBBl protein (contiguous lines; SEQ ID NO:3), with the variant amino acid(s) caused by the polymorphism(s) of Figure 2 positioned below the polymorphic site in the sequence.
  • the present invention is based on the discovery of novel variants of the CRYBBl gene.
  • 16 isogenes of the CRYBBl gene by characterizing the CRYBBl gene found in genomic DNAs isolated from an Index Repository that contains immortalized cell lines from one cMmpanzee and 93 human individuals.
  • the human individuals included a reference population of 79 unrelated individuals self-identified as belonging to one of four major population groups: Caucasian (22 individuals), African descent (20 individuals), Asian (20 individuals), or Hispanic/Latino (17 individuals). To the extent possible, the members of this reference population were organized into population subgroups by the self-identified ethnogeographic origin of their four grandparents as shown in Table 1 below.
  • the Index Repository contains three unrelated indigenous American Indians (one from each of North, Central and South America), one three-generation Caucasian family (from the CEPH Utah cohort) and one two-generation African- American family.
  • the CRYBB 1 isogenes present in the human reference population are defined by haplotypes for 12 polymorphic sites in the CRYBB 1 gene, all of which are believed to be novel.
  • the novel CRYBB 1 polymorphic sites identified by the inventors are referred to as PS 1-12 to designate ' the order in which they are located in the gene (see Table 2 below).
  • the inventors herein also determined the pair of haplotypes for the CRYBBl gene present in individual human members of this repository.
  • the human genotypes and haplotypes found in the repository for the CRYBBl gene include those shown in Tables 3 and 4, respectively.
  • the polymorphism and haplotype data disclosed herein are useful for validating whether CRYBBl is a suitable target for drugs to treat cataract, screening for such drugs and reducing bias in clinical trials of such drugs.
  • Allele - A particular form of a genetic locus, distinguished from other forms by its particular nucleotide sequence.
  • Candidate Gene - A gene which is hypothesized to be responsible for a disease, condition, or the response to a treatment, or to be correlated with one of these.
  • Genotype An unphased 5 ' to 3 ' sequence of nucleotide pair(s) found at one or more polymorphic sites in a locus on a pair of homologous chromosomes in an individual.
  • genotype includes a full-genotype and/or a sub-genotype as described below.
  • Sub-genotype The unphased 5' to 3' sequence of nucleotides seen at a subset of the known polymorphic sites examined herein in a locus on a pair of homologous chromosomes in a single individual.
  • Genotyping A process for determining a genotype of an individual.
  • Haplotype A 5' to 3' sequence of nucleotides found at one or more polymorphic sites in a locus on a single chromosome from a single individual.
  • haplotype includes a full-haplotype and/or a sub-haplotype as described below.
  • Full-haplotype The 5 ' to 3 ' sequence of nucleotides found at all known polymorphic sites examined herein in a locus on a single chromosome from a single individual.
  • Sub-haplotype The 5' to 3' sequence of nucleotides seen at a subset of the known polymorphic sites examined herein in a locus on a single chromosome from a single individual.
  • Haplotype pair The two haplotypes found for a locus in a single individual.
  • Haplotyping A process for determining one or more haplotypes in an individual and includes use of family pedigrees, molecular techniques and/or statistical inference.
  • Haplotype data - J_nformation concerning one or more of the following for a specific gene: a listing of the haplotype pairs in each individual in a population; a listing of the different haplotypes in a population; frequency of each haplotype in that or other populations, and any known associations between one or more haplotypes and a trait.
  • Isoform - A particular form of a gene, mRNA, cDNA or the protein encoded thereby, distinguished from other forms by its particular sequence and/or structure.
  • Isogene - One of the isoforms of a gene found in a population.
  • An isogene contains all of the polymorphisms present in the particular isoform of the gene.
  • Isolated - As applied to a biological molecule such as RNA, DNA, oligonucleotide, or protein, isolated means the molecule is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth media. Generally, the term “isolated” is not intended to refer to a complete absence of such material or to absence of water, buffers, or salts, unless they are present in amounts that substantially interfere with the methods of the present invention.
  • Locus - A location on a chromosome or DNA molecule corresponding to a gene or a physical or phenotypic feature.
  • Naturally-occurring A term used to designate that the object it is applied to, e.g., naturally- occurring polynucleotide or polypeptide, can be isolated from a source in nature and which has not been intentionally modified by man.
  • Nucleotide pair The nucleotides found at a polymorphic site on the two copies of a chromosome from an individual.
  • phased As applied to a sequence of nucleotide pairs for two or more polymorphic sites in a locus, phased means the combination of nucleotides present at those polymorphic sites on a single copy of the locus is known.
  • Polymorphic site (PS) - A position within a locus at which at least two alternative sequences are found in a population, the most frequent of which has a frequency of no more than 99%.
  • Polymorphism The sequence variation observed in an individual at a polymo ⁇ hic site.
  • Polymo ⁇ hisms include nucleotide substitutions, insertions, deletions and microsatellites and may, but need not, result in detectable differences in gene expression or protein function.
  • Polymorphism data Information concerning one or more of the following for a specific gene: location of polymo ⁇ hic sites; sequence variation at those sites; frequency of polymo ⁇ hisms in one or more populations; the different genotypes and/or haplotypes determined for the gene; frequency of one or more of these genotypes and/or haplotypes in one or more populations; any known association(s) between a trait and a genotype or a haplotype for the gene.
  • Polymorphism Database A collection of polymo ⁇ hism data arranged in a systematic or methodical way and capable of being individually accessed by electronic or other means.
  • Polynucleotide A nucleic acid molecule comprised of single-stranded RNA or DNA or comprised of complementary, double-stranded DNA.
  • Reference Population A group of subjects or individuals who are predicted to be representative of the genetic variation found in the general population.
  • the reference population represents the genetic variation in the population at a certainty level of at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 99%.
  • SNP Single Nucleotide Polymorphism
  • Subject A human individual whose genotypes or haplotypes or response to treatment or disease state are to be determined.
  • Treatment A stimulus administered internally or externally to a subject.
  • Unphased - As applied to a sequence of nucleotide pairs for two or more polymo ⁇ hic sites in a locus, unphased means the combination of nucleotides present at those polymo ⁇ hic sites on a single copy of the locus is not known.
  • the invention also provides compositions and methods for detecting the novel CRYBBl polymo ⁇ hisms and haplotypes identified herein.
  • compositions comprise at least one CRYBBl genotyping oligonucleotide.
  • a CRYBBl genotyping oligonucleotide is a probe or primer capable of hybridizing to a target region that is located close to, or that contains, one of the novel polymo ⁇ hic sites described herein.
  • the term "oligonucleotide” refers to a polynucleotide molecule having less than about 100 nucleotides.
  • a preferred oligonucleotide of the invention is 10 to 35 nucleotides long. More preferably, the oligonucleotide is between 15 and 30, and most preferably, between 20 and 25 nucleotides in length.
  • oligonucleotide may be comprised of any phosphorylation state of ribonucleotides, deoxyribonucleotides, and acyclic nucleotide derivatives, and other functionally equivalent derivatives.
  • oligonucleotides may have a phosphate-free backbone, which may be comprised of linkages such as carboxymethyl, acetamidate, carbamate, polyamide (peptide nucleic acid (PNA)) and the like (Varma, R. in Molecular Biology and Biotechnology, A Comprehensive Desk Reference, Ed. R. Meyers, VCH Publishers, Inc.
  • Oligonucleotides of the invention may be prepared by chemical synthesis using any suitable methodology known in the art, or may be derived from a biological sample, for example, by restriction digestion.
  • the oligonucleotides may be labeled, according to any technique known in the art, including use of radiolabels, fluorescent labels, enzymatic labels, proteins, haptens, antibodies, sequence tags and the like.
  • Genotyping oligonucleotides of the invention must be capable of specifically hybridizing to a target region of a CRYBBl polynucleotide, i.e., a CRYBBl isogene.
  • specific hybridization means the oligonucleotide forms an anti-parallel double-stranded structure with the target region under certain hybridizing conditions, while failing to form such a structure when incubated with a non-target region or a non-CRYBBl polynucleotide under the same hybridizing conditions.
  • the oligonucleotide specifically hybridizes to the target region under conventional high stringency conditions.
  • the skilled artisan can readily design and test oligonucleotide probes and primers suitable for detecting polymo ⁇ hisms in the CRYBB 1 gene using the polymo ⁇ hism information provided herein in conjunction with the known sequence information for the CRYBBl gene and routine techniques.
  • a nucleic acid molecule such as an oligonucleotide or polynucleotide is said to be a "perfect” or “complete” complement of another nucleic acid molecule if every nucleotide of one of the molecules is complementary to the nucleotide at the corresponding position of the other molecule.
  • a nucleic acid molecule is "substantially complementary” to another molecule if it hybridizes to that molecule with sufficient stability to remain in a duplex form under conventional low-stringency conditions. Conventional hybridization conditions are described, for example, by Sambrook J. et al., in Molecular Cloning, A Laboratory Manual, 2 nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) and by Haymes, B.D.
  • an oligonucleotide primer may have a non-complementary fragment at its 5 ' end, with the remainder of the primer being complementary to the target region.
  • non-complementary nucleotides may be interspersed into the oligonucleotide probe or primer as long as the resulting probe or primer is still capable of specifically hybridizing to the target region.
  • Preferred genotyping oligonucleotides of the invention are allele-specific oligonucleotides.
  • ASO allele-specific oligonucleotide
  • allele-specificity will depend upon a variety of readily optimized stringency conditions, including salt and formamide concentrations, as well as temperatures for both the hybridization and washing steps.
  • Allele-specific oligonucleotides of the invention include ASO probes and ASO primers.
  • ASO probes which usually provide good discrimination between different alleles are those in which a central position of the oligonucleotide probe aligns with the polymo ⁇ hic site in the target region (e.g., approximately the 7 th or 8 th position in a 15mer, the 8 th or 9 th position in a 16mer, and the 10 th or 11 th position in a 20mer).
  • An ASO primer of the invention has a 3 ' terminal nucleotide, or preferably a 3 ' penultimate nucleotide, that is complementary to only one nucleotide of a particular SNP, thereby acting as a primer for polymerase-mediated extension only if the allele containing that nucleotide is present.
  • ASO probes and primers hybridizing to either the coding or noncoding strand are contemplated by the invention.
  • a preferred ASO probe for detecting CRYBBl gene polymo ⁇ hisms comprises a nucleotide sequence, listed 5' to 3', selected from the group consisting of:
  • ACAAAACRCTTCTGT (SEQ ID NO: 4) and its complement, ATGGCATYCAACAGG (SEQ ID NO: 5) and its complement, CACACAGYGCGCAGT (SEQ ID NO: 6) and its complement, CACAGCGYGCAGTGG (SEQ ID NO: 7) and its complement, TCTCCGCRGGACCGT (SEQ ID NO: 8) and its complement, TGGGCGGMGGAGGGG (SEQ ID NO: 9) and its complement, GTTCAGCYCCCTTTG (SEQ ID NO: 10) and its complement, CCTACTGKCCTCAGG (SEQ ID NO: 11) and its complement, GCTCTCCRGCTGGGG (SEQ ID NO: 12) and its complement, AGCCAGGYGGGGCAG (SEQ ID NO: 13) ' and its complement, ⁇ GGCCCCTWCCACAGT (SEQ ID NO: 14) and its complement, and TCATGTTYAATTATT (SEQ ID NO: 15) and its complement.
  • a preferred ASO primer for detecting CRYBBl gene polymo ⁇ hisms comprises a nucleotide sequence, listed 5 ' to 3 ', selected from the group consisting of:
  • GCCTTTACAAAACRC (SEQ ID NO 16), ATGAGCACAGAAGYG (SEQ ID NO 17); TTCTATATGGCATYC (SEQ ID NO 18), CGTGTCCCTGTTGRA (SEQ ID NO 19) ; AAAAGCCACACAGYG (SEQ ID NO 20), TCAGCCACTGCGCRC (SEQ ID NO 21); AAGCCACACAGCGYG (SEQ ID NO 22) , ACTCAGCCACTGCRC (SEQ ID NO 23) ; T €ATTGTCTCCGCRG (SEQ ID NO 24), GTACTCACGGTCCYG (SEQ ID NO 25); GAGTACTGGGCGGMG (SEQ ID NO 26), AGTGTGCCCCTCCKC (SEQ ID NO 27); AGACATGTTCAGCYC (SEQ ID NO 28), AAAGTCCAAAGGGRG (SEQ ID NO 2 ' 9) ; GACTTTCCTACTGKC (SEQ ID NO 30) , GGCAGCCCTGAGGMC (SEQ ID NO 31)
  • genotyping oligonucleotides of the invention hybridize to a target region located one to several nucleotides downstream of one of the novel polymo ⁇ hic sites identified herein. Such oligonucleotides are useful in polymerase-mediated primer extension methods for detecting one of the novel polymo ⁇ hisms described herein and therefore such genotyping oligonucleotides are referred to herein as "primer-extension oligonucleotides”.
  • the 3 '-terminus of a primer- extension oligonucleotide is a deoxynucleotide complementary to the nucleotide located immediately adjacent to the polymo ⁇ hic site.
  • a particularly preferred oligonucleotide primer for detecting CRYBBl gene polymo ⁇ hisms by primer extension terminates in a nucleotide sequence, listed 5 ' to 3 ', selected from the group consisting of:
  • TTTACAAAAC AGCACAGAAG (SEQ ID NO: 41); ATATGGCAT (SEQ ID NO 42) ; GTCCCTGTTG (SEQ ID NO 43);
  • CCACACAGCG SEQ ID NO 46
  • CAGCCACTGC SEQ ID NO 47
  • TTGTCTCCGC (SEQ ID NO 48) • CTCACGGTCC (SEQ ID NO 49);
  • TACTGGGCGG (SEQ ID NO 50) ⁇ GTGCCCCTCC (SEQ ID NO 51);
  • CATGTTCAGC (SEQ ID NO 52) • GTCCAAAGGG (SEQ ID NO 53);
  • TTTCCTACTG SEQ ID NO 54
  • AGCCCTGAGG- SEQ ID NO: 55
  • ATCAGCCAGG SEQ ID NO 58
  • GCCCTGCCCC SEQ ID NO: 59
  • CATGGCCCCT (SEQ ID NO 60) ; GAGACTGTGG (SEQ ID NO 61);
  • a composition contains two or more differently labeled genotyping oligonucleotides for simultaneously probing the identity of nucleotides at two or more polymo ⁇ hic sites. It is also contemplated that primer compositions may contain two or more sets of allele-specific primer pairs to allow simultaneous targeting and amplification of two or more regions containing a polymo ⁇ hic site.
  • CRYBBl genotyping oligonucleotides of the invention may also be immobilized on or synthesized on a solid surface such as a microchip, bead, or glass slide (see, e.g., WO 98/20020 and WO 98/20019). Such immobilized genotyping oligonucleotides may be used in a variety of polymo ⁇ hism detection assays, including but not limited to probe hybridization and polymerase extension assays.
  • Immobilized CRYBBl genotyping oligonucleotides of the invention may comprise an ordered array of oligonucleotides designed to rapidly screen a DNA sample for polymo ⁇ hisms in multiple genes at the same time.
  • the invention provides a kit comprising at least two genotyping oligonucleotides packaged in separate containers.
  • the kit may also contain other components such as hybridization buffer (where the oligonucleotides are to be used as a probe) packaged in a separate container.
  • the kit may contain, packaged in separate containers, a polymerase and a reaction buffer optimized for primer extension mediated by the polymerase, such as PCR.
  • CRYBBl genotype and “CRYBBl haplotype” mean the genotype or haplotype contains the nucleotide pair or nucleotide, respectively, that is present at one or more of the novel polymo ⁇ hic sites described herein and may optionally also include the nucleotide pair or nucleotide present at one or more additional polymo ⁇ hic sites in the CRYBBl gene.
  • the additional polymo ⁇ hic sites may be currently known polymo ⁇ hic sites or sites that are subsequently discovered.
  • One embodiment of the genotyping method involves isolating from the individual a nucleic acid sample comprising the two copies of the CRYBBl gene, or a fragment thereof, that are present in the individual, and determining the identity of the nucleotide pair at one or more polymo ⁇ hic sites selected from the group consisting of PS1, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS11 and PS12 in the two copies to assign a CRYBBl genotype to the individual.
  • the two "copies" of a gene in an individual may be the same allele or may be different alleles.
  • the genotyping method comprises determining the identity of the nucleotide pair at each of PSl-12.
  • the nucleic acid sample is isolated from a biological sample taken from the individual, such as a blood sample or tissue sample.
  • tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair.
  • the nucleic acid sample may be comprised of genomic DNA, mRNA, or cDNA and, in the latter two cases, the biological sample must be obtained from a tissue in which the CRYBBl gene is expressed.
  • mRNA or cDNA preparations would not be used to detect polymo ⁇ hisms located in introns or in 5 ' and 3 ' untranslated regions. If a CRYBB 1 gene fragment is isolated, it must contain the polymo ⁇ hic site(s) to be genotyped.
  • One embodiment of the haplotyping method comprises isolating from the individual a nucleic acid sample containing only one of the two copies of the CRYBBl gene, or a fragment thereof, that is present in the individual and determining-in that copy the identity of the nucleotide at one or more polymo ⁇ hic sites selected from the group consisting of PS1, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS 10, PS11 and PS 12 in that copy to assign a CRYBBl haplotype to the individual.
  • the nucleic acid may be isolated using any method capable of separating the two copies of the CRYBBl gene or fragment such as one of the methods described above for preparing CRYBBl isogenes, with targeted in vivo cloning being the preferred approach.
  • any individual clone will only provide haplotype information on one of the two CRYBBl gene copies present in an individual. If haplotype information is desired for the individual's other copy, additional CRYBBl clones will need to be examined. Typically, at least five clones should be examined to have more than a 90% probability of haplotyping both copies of the CRYBBl gene in an individual.
  • the nucleotide at each of PSl-12 is identified.
  • the haplotyping method comprises determining whether an individual has one or more of the CRYBBl haplotypes shown in Table 4. This can be accomplished by identifying, for one or both copies of the individual's CRYBBl gene, the phased sequence of nucleotides present at each of PSl-12.
  • the present invention also contemplates that typically only a subset of PSl-12 will need to be directly examined to assign to an individual one or more of the haplotypes shown in Table 4. This is because at least one polymo ⁇ hic site in a gene is frequently in strong linkage disequilibrium with one or more other polymo ⁇ hic sites in that gene (Drysdale, CM et al.
  • a CRYBBl haplotype pair is determined for an individual by identifying the phased sequence of nucleotides at one or more polymo ⁇ hic sites selected from the group consisting of PS1, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS11 and PS12 in each copy of the CRYBBl gene that is present in the individual.
  • the haplotyping method comprises identifying the phased sequence of nucleotides at each of PSl-12 in each copy of the CRYBBl gene. When haplotyping both copies of the gene, the identifying step is preferably performed ' with each copy of the gene being placed in separate containers.
  • first and second copies of the gene are labeled with different first and second fluorescent dyes, respectively, and an allele-specific oligonucleotide labeled with yet a third different fluorescent dye is used to assay the polymo ⁇ hic site(s), then detecting a combination of the first and third dyes would identify the polymo ⁇ hism in the first gene copy while detecting a combination of the second and third dyes would identify the polymo ⁇ hism in the second gene copy.
  • the identity of a nucleotide (or nucleotide pair) at a polymo ⁇ hic site(s) may be determined by amplifying a target region(s) containing the polymo ⁇ hic site(s) directly from one or both copies of the CRYBBl gene, or a fragment thereof, and the sequence of the amplified region(s) determined by conventional methods. It will be readily appreciated by the skilled artisan that only one nucleotide will be detected at a polymo ⁇ hic site in individuals who are homozygous at that site, while two different nucleotides will be detected if the individual is heterozygous for that site.
  • the polymo ⁇ hism may be identified directly, known as positive-type identification, or by inference, referred to as negative-type identification.
  • a site may be positively determined to be either guanine or cytosine for an individual homozygous at that site, or both guanine and cytosine, if the individual is heterozygous at that site.
  • the site may be negatively determined to be not guanine (and thus cytosine/cytosine) or not cytosine (and thus guanme/guanine).
  • the target region(s) may be amplified using any oligonucleotide-directed amplification method, including but not limited to polymerase chain reaction (PCR) (U.S. Patent No. 4,965,188), ligase chain reaction (LCR) (Barany et al., Proc. Natl. Acad. Sci. USA 88:189-193, 1991; WO90/01069), and oligonucleotide ligation assay (OLA) (Landegren et al., Science 241:1077-1080, 1988).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • OLA oligonucleotide ligation assay
  • nucleic acid amplification procedures may be used to amplify the target region including transcription-based amplification systems (U.S. Patent No. 5,130,238; EP 329,822; U.S. Patent No. 5,169,766, WO89/06700) and isothermal methods (Walker et al., Proc. Natl. Acad. Sci. USA 89:392- 396, 1992).
  • a polymo ⁇ hism in the target region may also be assayed before or after amplification using one of several hybridization-based methods known in the art.
  • allele-specific oligonucleotides are utilized in performing such methods.
  • the allele-specific oligonucleotides may be used as differently labeled probe pairs, with one member of the pair showing a perfect match to one variant of a target sequence and the other member showing a perfect match to a different variant.
  • more than one polymo ⁇ hic site may be detected at once using a set of allele-specific oligonucleotides or oligonucleotide pairs.
  • the members of the set have melting temperatures within 5°C, and more preferably within 2°C, of each other when hybridizing to each of the polymo ⁇ hic sites being detected.
  • Hybridization of an allele-specific oligonucleotide to a target polynucleotide may be performed with both entities in solution, or such hybridization may be performed when either the oligonucleotide or the target polynucleotide is covalently or noncovalently affixed to a solid support. Attachment may be mediated, for example, by antibody-antigen interactions, poly-L-Lys, streptavidin or avidin-biotin, salt bridges, hydrophobic interactions, chemical linkages, UV cross-linking baking, etc. Allele-specific oligonucleotides may be synthesized directly on the solid support or attached to the solid support subsequent to synthesis.
  • Solid-supports suitable for use in detection methods of the invention include substrates made of silicon, glass, plastic, paper and the like, which may be formed, for example, into wells (as in 96-well plates), slides, sheets, membranes, fibers, chips, dishes, and beads.
  • the solid support may be treated, coated or derivatized to facilitate the immobilization of the allele-specific oligonucleotide or target nucleic acid.
  • the genotype or haplotype for the CRYBBl gene of an individual may also be determined by hybridization of a nucleic acid sample containing one or both copies of the gene, or fragment(s) thereof, to nucleic acid arrays and subarrays such as described in WO 95/11995.
  • the arrays would contain a battery of allele-specific oligonucleotides representing each of the polymo ⁇ hic sites to be included in the genotype or haplotype.
  • polymo ⁇ hisms may also be determined using a mismatch detection technique, including but not limited to the RNase protection method using riboprobes (Winter et al., Proc. Natl. Acad. Sci. USA 82:7575, 1985; Meyers et al., Science 230:1242, 1985) and proteins which recognize nucleotide mismatches, such as the E. coli mutS protein (Modrich, P. Ann. Rev. Genet. 25:229-253, 1991).
  • riboprobes Winter et al., Proc. Natl. Acad. Sci. USA 82:7575, 1985; Meyers et al., Science 230:1242, 1985
  • proteins which recognize nucleotide mismatches such as the E. coli mutS protein (Modrich, P. Ann. Rev. Genet. 25:229-253, 1991).
  • variant alleles can be identified by single strand conformation polymo ⁇ hism (SSCP) analysis (Orita et al., Genomics 5:874-879, 1989; Humphries et al., in Molecular Diagnosis of Genetic Diseases, R. Elles, ed., pp. 321-340, 1996) or denaturing gradient gel electrophoresis (DGGE) (Wartell et al., Nucl. Acids Res. 18:2699-2706, 1990; Sheffield et al, Proc. Natl. Acad. Sci. USA 86:232- 236, 1989).
  • SSCP single strand conformation polymo ⁇ hism
  • DGGE denaturing gradient gel electrophoresis
  • a polymerase-mediated primer extension method may also be used to identify the polymo ⁇ hism(s).
  • Several such methods have been described in the patent and scientific literature and include the "Genetic Bit Analysis” method (W092/15712) and the ligase/polymerase mediated genetic bit analysis (U.S. Patent 5,679,524. Related methods are disclosed in W091/02087, WO90/09455, W095/17676, U.S. Patent Nos. 5,302,509, and 5,945,283. Extended primers containing a polymo ⁇ hism may be detected by mass spectrometry as described in U.S. Patent No. 5,605,798.
  • Another primer extension method is allele-specific PCR (Ruano et al., Nucl.
  • the identity of the.allele(s) present at any of the novel polymo ⁇ hic sites described herein may be indirectly determined by genotyping another polymo ⁇ hic site that is in linkage disequilibrium with the polymo ⁇ hic site that is of interest.
  • an individual's CRYBBl haplotype pair is predicted from its CRYBBl genotype using information on haplotype pairs known to exist in a reference population.
  • the haplotyping prediction method comprises identifying a CRYBBl genotype for the individual at two or more CRYBBl polymo ⁇ hic sites described herein, enumerating all possible haplotype pairs which are consistent with the genotype, accessing data containing CRYBBl haplotype pairs identified in a reference population, and assigning a haplotype pair to the individual that is consistent with the data.
  • the reference haplotype pairs include the CRYBBl haplotype pairs shown in Table 3.
  • the reference population should be composed of randomly-selected individuals representing the major ethnogeographic groups of the world.
  • a preferred reference population allows the detection of any haplotype whose frequency is at least 10% with about 99% certainty and comprises about 20 unrelated individuals from each of the four population groups named above.
  • a particularly preferred reference population includes a 3-generation family representing one or more of the four population groups to serve as controls for checking quality of haplotyping procedures.
  • the haplotype frequency data for each ethnogeographic group is examined to determine whether it is consistent with Hardy- Weinberg equilibrium. Hardy- Weinberg equilibrium (D.L.
  • haplotyping the individual using a direct haplotyping method such as, for example, CLASPER System technology (U.S. Patent No. 5,866,404), single molecule dilution, or allele-specific long-range PCR (Michalotos-Beloin et al., NMc/etc ⁇ c; ⁇ ' i-e5'. 24:4841-4843, 1996).
  • CLASPER System technology U.S. Patent No. 5,866,404
  • single molecule dilution single molecule dilution
  • allele-specific long-range PCR Michalotos-Beloin et al., NMc/etc ⁇ c; ⁇ ' i-e5'. 24:4841-4843, 1996.
  • the assigning step involves performing the following analysis. First, each of the possible haplotype pairs is compared to the haplotype pairs in the reference population. Generally, only one of the haplotype pairs in the reference population matches a possible haplotype pair and that pair is assigned to the individual. Occasionally, only one haplotype represented in the reference haplotype pairs is consistent with a possible haplotype pair for an individual, and in such cases the individual is assigned a haplotype pair containing this known haplotype and a new haplotype derived by subtracting the known haplotype from the possible haplotype pair.
  • the individual is preferably haplotyped using a direct molecular haplotyping method such as, for example, CLASPER System TM technology (U.S. Patent No. 5,866,404), SMD, or allele-specific long-range PCR (Michalotos-Beloin et al., supra).
  • CLASPER System TM technology U.S. Patent No. 5,866,404
  • SMD SMD
  • allele-specific long-range PCR Moichalotos-Beloin et al., supra.
  • a preferred process for predicting CRYBB 1 haplotype pairs from CRYBB 1 genotypes is described in U.S. Provisional Application Serial No. 60/198,340 and the corresponding International Application filed April 18, 2001.
  • the invention also provides a method for determining the frequency of a CRYBBl genotype, haplotype, or haplotype pair in a population.
  • the method comprises, for each member of the population, determining the genotype or the haplotype pair for the novel CRYBBl polymo ⁇ hic sites described herein, and calculating the frequency any particular genotype, haplotype, or haplotype pair is found in the population.
  • the population may be a reference population, a family population, a same sex population, a population group, or a trait population (e.g., a group of individuals exhibiting a trait of interest such as a medical condition or response to a therapeutic treatment).
  • frequency data for CRYBBl genotypes, haplotypes, and/or haplotype pairs are determined in a reference population and used in a method for identifying an association between a trait and a CRYBBl genotype, haplotype, or haplotype pair.
  • the trait may be any detectable phenotype, including but not limited to susceptibility to a disease or response to a treatment.
  • the method involves obtaining data on the frequency of the genotype(s), haplotype(s), or haplotype pair(s) of interest in a reference population as well as in a population exhibiting the trait.
  • Frequency data for one or both of the reference and trait populations may be obtained by genotyping or haplotyping each individual in the populations using one of the methods described above.
  • the haplotypes for the trait population may be determined directly or, alternatively, by the predictive genotype to haplotype approach described above.
  • the frequency data for the reference and/or trait populations is obtained by accessing previously determined frequency data, which may be in written or electronic form.
  • the frequency data may be present in a database that is accessible by a computer. Once the frequency data is obtained, the frequencies of the genotype(s), haplotype(s), or haplotype pair(s) of interest in the reference and trait populations are compared. In a preferred embodiment, the frequencies of all genotypes, haplotypes, and/or haplotype pairs observed in the populations are compared.
  • the trait is predicted to be associated with that CRYBBl genotype, haplotype or haplotype pair.
  • the CRYBBl genotype, haplotype, or haplotype pair being compared in the trait and reference populations is selected from the full-genotypes and full-haplotypes shown in Tables 3 and 4, or from sub-genotypes and sub-haplotypes derived from these genotypes and haplotypes.
  • the trait of interest is a clinical response exhibited by a patient to some therapeutic treatment, for example, response to a drug targeting CRYBBl or response to a therapeutic treatment for a medical condition.
  • medical condition includes but is not limited to any condition or disease manifested as one or more physical and/or psychological symptoms for which treatment is desirable, and includes previously and newly identified diseases and other disorders.
  • clinical response means any or all of the following: a quantitative measure of the response, no response, and adverse response (i.e., side effects).
  • clinical population In order to deduce a correlation between clinical response to a treatment and a CRYBB 1 • genotype, haplotype, or haplotype pair, it is necessary to obtain data on the clinical responses exhibited by a population of individuals who received the treatment, hereinafter the "clinical population".
  • This clinical data may be obtained by analyzing the results of a clinical trial that has already been run and/or the clinical data may be obtained by designing and carrying out one or more new clinical trials.
  • the term "clinical trial” means any research study designed to collect clinical data on responses to a particular treatment, and includes but is not limited to phase I, phase II and phase III clinical trials. Standard methods are used to define the patient population and to enroll subjects.
  • the individuals included in the clinical population have been graded for the existence of the medical condition of interest. . This is important in cases where the symptom(s) being presented by the patients can be caused by more than one underlying condition, and where treatment of the underlying conditions are not the same. An example of this would be where patients experience breathing difficulties that are due to either asthma or respiratory infections. If both sets were treated with an asthma medication, there would be a spurious group of apparent non-responders that did not actually have asthma. These people would affect the ability to detect any correlation between haplotype and treatment outcome.
  • This grading of potential patients could employ a standard physical exam or one or more lab tests. Alternatively, grading of patients could use haplotyping for situations where there is a strong correlation between haplotype pair and disease susceptibility or severity.
  • the therapeutic treatment of interest is administered to each individual in the trial population and each individual's response to the treatment is measured using one or more predetermined criteria. It is contemplated that in many cases, the trial population will exhibit a range of responses and that the investigator will choose the number of responder groups (e.g., low, medium, high) made up by the various responses. In addition, the CRYBBl gene for each individual in the trial population is genotyped and/or haplotyped, which may be done before or after administering the treatment.
  • correlations between individual response and CRYBBl genotype or haplotype content are created. Correlations may be produced in several ways. In one method, individuals are grouped by their CRYBBl genotype or haplotype (or haplotype pair) (also referred to as a polymo ⁇ hism group), and then the averages and standard deviations of clinical responses exhibited by the members of each polymo ⁇ hism group are calculated.
  • a second method for finding correlations between CRYBBl haplotype content and clinical responses uses predictive models based on error-minimizing optimization algorithms.
  • One of many possible optimization algorithms is a genetic algorithm (R. Judson, "Genetic Algorithms and Their Uses in Chemistry” in Reviews in Computational Chemistry, Vol. 10, pp. 1-73, K. B. Lipkowitz and D. B. Boyd, eds. (VCH Publishers, New York, 1997).
  • Simulated annealing Press et al., "Numerical Recipes in C: The Art of Scientific Computing", Cambridge University Press (Cambridge) 1992, Ch. 10), neural networks (E. Rich and K.
  • the correlation is found using a genetic algorithm approach as described in PCT Application Serial No. Correlations may also be analyzed using analysis of variation (ANONA) techniques to determine how much of the variation in the clinical data is explained by different subsets of the polymo ⁇ hic sites in the CRYBBl gene.
  • ANONA analysis of variation
  • ANONA is used to test hypotheses about whether a response variable is caused by or correlated with one or more traits or variables that can be measured (Fisher and vanBelle, supra, Ch. 10).
  • a mathematical model may be readily constructed by the skilled artisan that predicts clinical -response as a function of CRYBBl genotype or haplotype content.
  • the model is validated in one or more follow-up clinical trials designed to test the model.
  • the identification of an association between a clinical response and a genotype or haplotype (or haplotype pair) for the CRYBBl gene may be the basis for designing a diagnostic method to determine those individuals who will or will not respond to the treatment, or alternatively, will respond at a lower level and thus may require more treatment, i.e., a greater dose of a drug.
  • the diagnostic method may take one of several forms: for example, a direct D ⁇ A test (i.e., genotyping or haplotyping one or more of the polymo ⁇ hic sites in the CRYBBl gene), a serological test, or a physical exam measurement.
  • a direct D ⁇ A test i.e., genotyping or haplotyping one or more of the polymo ⁇ hic sites in the CRYBBl gene
  • serological test i.e., a serological test
  • a physical exam measurement i.e., a physical exam measurement.
  • this diagnostic method uses the predictive haplotyping method described above.
  • the invention provides an isolated polynucleotide comprising a polymo ⁇ hic variant of the CRYBBl gene or a fragment of the gene which contains at least one of the novel polymo ⁇ hic sites described herein.
  • the nucleotide sequence of a variant CRYBBl gene is identical to the reference genomic sequence for those portions of the gene examined, as described in the Examples below, except that it comprises a different nucleotide at one or more of the novel polymo ⁇ hic sites PS1, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS 10, PS 11 and PS 12.
  • nucleotide sequence of a variant fragment of the CRYBBl gene is identical to the corresponding portion of the reference sequence except for having a different nucleotide at one or more of the novel polymo ⁇ hic sites described herein.
  • the invention specifically does not include polynucleotides comprising a nucleotide sequence identical to the reference sequence of the CRYBBl gene, which is defined by haplotype 12, (or other reported CRYBBl sequences) or to portions of the reference sequence (or other reported CRYBBl sequences), except for genotyping oligonucleotides as described above.
  • the location of a polymo ⁇ hism in a variant gene or fragment is identified by aligning its sequence against SEQ ID ⁇ O:l.
  • the polymo ⁇ hism is selected from the group consisting of adenine at PS1, thymine at PS2, thymine at PS3, thymine at PS4, adenine at PS5, adenine at PS6, thymine at PS7, guanine at PS8, adenine at PS9, thymine at PS10, adenine at PS11 and thymine at PS12.
  • the polymo ⁇ hic variant comprises a naturally-occurring isogene of the CRYBBl gene which is defined by any one of haplotypes 1- 11 and 13 - 16 shown in Table 4 below.
  • Polymo ⁇ hic variants of the invention may be prepared by isolating a clone containing the CRYBBl gene from a human genomic library.
  • the clone may be sequenced to determine the identity of the nucleotides at the novel polymo ⁇ hic sites described herein. Any particular variant claimed herein could be prepared from this clone by performing in vitro mutagenesis using procedures well-known in the art.
  • CRYBBl isogenes may be isolated using any method that allows separation of the two "copies" of the CRYBBl gene present in an individual, which, as readily understood by the skilled artisan, may be the same allele or different alleles. Separation methods include targeted in vivo cloning (TINC) in yeast as described in WO 98/01573, U.S. Patent No. 5,866,404, and U.S. Patent No. 5,972,614. Another method, which is described in U.S. Patent No. 5,972,614, uses an allele specific oligonucleotide in combination with primer extension and exonuclease degradation to generate hemizygous DNA targets.
  • TTC targeted in vivo cloning
  • Another method which is described in U.S. Patent No. 5,972,614, uses an allele specific oligonucleotide in combination with primer extension and exonuclease degradation to generate hemizygous DNA targets.
  • the invention also provides CRYBBl genome anthologies, which are collections of CRYBBl isogenes found in a given population.
  • the population may be any group of at least two individuals, including but not limited to a reference population, a population group, a family population, a clinical population, and a same sex population.
  • a CRYBBl genome anthology may comprise individual CRYBBl isogenes stored in separate containers such as microtest tubes, separate wells of a microtitre plate and the like. Alternatively, two or more groups of the CRYBBl isogenes in the anthology may be stored in separate containers. Individual isogenes or groups of isogenes in a genome anthology may be .
  • a preferred CRYBBl genome anthology of the invention comprises a set of isogenes defined by the haplotypes shown in Table 4 below.
  • An isolated polynucleotide containing a polymo ⁇ hic variant nucleotide sequence of the invention may be operably linked to one or more expression regulatory elements in a recombinant expression vector capable of being propagated and expressing the encoded CRYBBl protein in a prokaryotic or a eukaryotic host cell.
  • expression regulatory elements which may be used include, but are not limited to, the lac system, operator and promoter regions of phage lambda, yeast promoters, and promoters derived from vaccinia virus, adenovirus, retroviruses,Or SN40.
  • regulatory elements include, but are not limited to, appropriate leader sequences, termination codons, polyadenylation signals, and other sequences required for the appropriate transcription and subsequent translation of the nucleic acid sequence in a given host cell.
  • the expression vector contains any additional elements necessary for its transfer to and subsequent replication in the host cell. Examples of such elements include, but are not limited to, origins of replication and selectable markers.
  • Such expression vectors are commercially available or are readily constructed using methods known to those in the art (e.g., F. Ausubel et al., 1987, in "Current Protocols in Molecular Biology", John Wiley and Sons, New York, New York).
  • Host cells which may be used to express the variant CRYBBl sequences of the invention include, but are not limited to, eukaryotic and mammalian cells, such as animal, plant, insect and yeast cells, and prokaryotic cells, such as E. coli, or algal cells as known in the art.
  • the recombinant expression vector may be introduced into the host cell using any method known to those in the art including, but not limited to, microinjection, electroporation, particle bombardment, transduction, and transfection using DEAE-dextran, lipofection, or calcium phosphate (see e.g., Sambrook et al. (1989) in "Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Press, Plainview, New York).
  • eukaryotic expression vectors that function in eukaryotic cells, and preferably mammalian cells, are used.
  • Non-limiting examples of such vectors include vaccinia virus vectors, adenovirus vectors, he ⁇ es virus vectors, and baculovirus transfer vectors.
  • Preferred eukaryotic cell lines include COS cells, CHO cells, HeLa cells, NIH/3T3 cells, and embryonic stem cells (Thomson, J. A. et al., 1998 Science 282:1145-1147).
  • Particularly preferred host cells are mammalian cells.
  • polymo ⁇ hic variants of the CRYBBl gene will produce CRYBBl mRNAs varying from each other at any polymo ⁇ hic site retained in the spliced and processed mRNA molecules.
  • mRNAs can be used for the preparation of a CRYBBl cDNA comprising a nucleotide sequence which is a polymo ⁇ hic variant of the CRYBBl reference coding sequence shown in Figure 2.
  • the invention also provides CRYBBl mRNAs and corresponding cDNAs which comprise a nucleotide sequence that is identical to SEQ ID NO:2 (Fig.
  • a particularly preferred polymo ⁇ hic cDNA variant comprises the coding sequence of a CRYBBl isogene defined by haplotypes 1- 11 and 13 - 16. Fragments of these variant mRNAs and cDNAs are included in the scope of the invention, provided they contain the novel polymo ⁇ hism described herein.
  • the invention specifically excludes polynucleotides identical to previously identified and characterized CRYBBl cDNAs and fragments thereof.
  • Polynucleotides comprising a variant RNA or DNA sequence may be isolated from a biological sample using well-known molecular biological procedures or may be chemically synthesized.
  • a polymo ⁇ hic variant of a CRYBBl gene fragment comprises at least one novel polymo ⁇ hism identified herein and has a length of at least 10 nucleotides and may range up to the full length of the gene.
  • such fragments are between 100 and 3000 nucleotides in length, and more preferably between 200 and 2000 nucleotides in length, and most preferably between 500 and 1000 nucleotides in length.
  • nucleic acid molecules containing the CRYBB 1 gene may be complementary double stranded molecules and thus reference to a particular site on the sense strand refers as well to the corresponding site on the complementary antisense strand.
  • reference may be made to the same polymo ⁇ hic site on either strand and an oligonucleotide may be designed to hybridize specifically to either strand at a target region containing the polymo ⁇ hic site.
  • the invention also includes single-stranded polynucleotides which are complementary to the sense strand of the CRYBBl genomic variants described herein.
  • Polynucleotides comprising a polymo ⁇ hic gene variant or fragment may be useful for therapeutic pu ⁇ oses.
  • an expression vector encoding the isoform may be administered to the patient.
  • the patient may be one who lacks the CRYBB 1 isogene encoding that isoform or may already have at least one copy of that isogene.
  • CRYBBl isogene In other situations, it may be desirable to decrease or block expression of a particular CRYBBl isogene.
  • Expression of a CRYBBl isogene may be turned off by fransforming a targeted organ, tissue or cell population with an expression vector that expresses high levels of untranslatable mRNA for the isogene.
  • oligonucleotides directed against the regulatory regions (e.g., promoter, introns, enhancers, 3' untranslated region) of the isogene may block transcription. Oligonucleotides targeting the transcription initiation site, e.g., between positions -10 and +10 from the start site are preferred.
  • inhibition of transcription can be achieved using oligonucleotides that base-pair with region(s) of the isogene DNA to form triplex DNA (see e.g., Gee et al. in Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y., 1994).
  • Antisense oligonucleotides may also be designed to block translation of CRYBB 1 mRNA transcribed from a particular isogene. It is also contemplated that ribozymes may be designed that can catalyze the specific cleavage of CRYBB 1 mRNA transcribed from a particular isogene.
  • the oligonucleotides may be delivered to a target cell or tissue by expression from a vector introduced into the cell or tissue in vivo or ex vivo.
  • the oligonucleotides may be formulated as a pharmaceutical composition for administration to the patient.
  • Oligoribonucleotides and/or oligodeoxynucleotides intended for use as antisense oligonucleotides may be modified to increase stability and half-life.
  • Possible modifications include, but are not limited to phosphorothioate or 2' 0- methyl linkages, and the inclusion of nontraditional bases such as inosine and queosine, as well as acetyl- , methyl-, thio-, and similarly modified forms of adenine, cytosine, guanine, thymine, and uracil which are not as easily recognized by endogenous nucleases.
  • Effect(s) of the polymo ⁇ hisms identified herein on expression of CRYBBl may be investigated by preparing recombinant cells and/or nonhuman recombinant organisms, preferably recombinant animals, containing a polymo ⁇ hic variant of the CRYBBl gene.
  • expression includes but is not limited to one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into CRYBBl protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.
  • the desired CRYBBl isogene may be introduced into the cell in a vector such that the isogene remains extrachromosomal. In such a situation, the gene will be expressed by the cell from the extrachromosomal location.
  • the CRYBBl isogene is introduced into a cell in such a way that it recombines with the endogenous CRYBBl gene present in the cell. Such recombination requires the occurrence of a double recombination event, thereby resulting in the desired CRYBBl gene polymo ⁇ hism.
  • Vectors for the introduction of genes both for recombination and for extrachromosomal maintenance are known in the art, and any suitable vector or vector construct may be used in the invention. Methods such as electroporation, particle bombardment, calcium phosphate co-precipitation and viral transduction for introducing DNA into' cells are known in the art; therefore, the choice of method may lie with the competence and preference of the skilled practitioner.
  • Examples of cells into which the CRYBBl isogene may be introduced include, but are not limited to, continuous culture cells, such as COS, NIH/3T3, and primary or culture cells of the relevant tissue type, i.e., they express the CRYBBl isogene. Such recombinant cells cari be used to compare the biological activities of the different protein variants.
  • Recombinant nonhuman organisms i.e., transgenic animals, expressing a variant CRYBBl gene are prepared using standard procedures known in the art.
  • a construct comprising the variant gene is introduced into a nonhuman animal or an ancestor of the animal at an embryonic stage, i.e., the one-cell stage, or generally not later than about the eight-cell stage.
  • Transgenic animals carrying the constructs of the invention can be made by several methods known to those having skill in the art.
  • One method involves transfecting into the embryo a retrovirus constructed to contain one or more insulator elements, a gene or genes of interest, and other components known to those skilled in the art to provide a complete shuttle vector harboring the insulated gene(s) as a transgene, see e.g., U.S. Patent No. 5,610,053.
  • Another method involves directly injecting a transgene into the embryo.
  • a third method involves the use of embryonic stem cells. Examples of animals into which the CRYBBl isogenes may be introduced include, but are not limited to, mice, rats, other rodents, and nonhuman primates (see "The Introduction of Foreign Genes into Mice" and the cited references therein, In: Recombinant DNA, Eds. J.D. Watson, M.
  • Transgenic animals stably expressing a human CRYBBl isogene and producing human CRYBB 1 protein can be used as biological models for studying diseases related to abnormal CRYBB 1 expression and/or activity, and for screening and assaying various candidate drugs, compounds, and treatment regimens to reduce the symptoms or effects of these diseases.
  • compositions for treating disorders affected by expression or function of a novel CRYBB 1 isogene described herein.
  • the pharmaceutical composition may comprise any of the following active ingredients: a polynucleotide comprising one of these novel CRYBBl isogenes; an antisense oligonucleotide directed against one of the novel CRYBBl isogenes, a polynucleotide encoding such an antisense oligonucleotide, or another compound which inhibits expression of a novel CRYBB 1 isogene described herein.
  • the composition contains the active ingredient in a therapeutically effective amount.
  • composition also comprises a pharmaceutically acceptable carrier, examples of which include, but are not limited to, saline, buffered saline, dextrose, and water.
  • a pharmaceutically acceptable carrier examples of which include, but are not limited to, saline, buffered saline, dextrose, and water.
  • Those skilled in the art may employ a formulation most suitable for the active ingredient, whether it is a polynucleotide, oligonucleotide, protein, peptide or small molecule antagonist.
  • the pharmaceutical composition may be administered alone or in combination with, at least one other agent, such as a stabilizing compound.
  • Administration of the pharmaceutical composition may be by any number of routes including, but not limited to oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, intradermal, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).
  • the dose can be estimated initially either in cell culture assays or in animal models.
  • the animal model may also be used to determine the appropriate concentration range and route of administration.
  • Such information can then be used to determine useful doses and routes for administration in humans.
  • the exact dosage will be determined by the practitioner, in light of factors relating to the patient requiring treatment, including b ⁇ t not limited to severity of the disease state, general health, age, weight and gender of the patient, diet, time and frequency of administration, other drugs being taken by the patient, and tolerance/response to the treatment.
  • any or all analytical and mathematical operations involved in practicing the methods of the present invention may be implemented by a computer.
  • the computer may execute a program that generates views (or screens) displayed on a display device and with which the user can interact to view and analyze large amounts of information relating to the CRYBB 1 gene and its genomic variation, including chromosome location, gene structure, and gene family, gene expression data, polymo ⁇ hism data, genetic sequence data, and clinical data population data (e.g., data on ethnogeographic origin, clinical responses, genotypes, and haplotypes for one or more populations).
  • the CRYBBl polymo ⁇ hism data described herein may be stored as part of a relational database (e.g., an instance of an Oracle database or a set of ASCII flat files).
  • polymo ⁇ hism data may be stored on the computer's hard drive or may, for example, be stored on a CD-ROM or on one or more other storage devices accessible by- the computer.
  • the data may be stored on one or more databases in communication with the computer via a network.
  • EXAMPLE 1 This example illustrates examination of various regions of the CRYBBl gene for polymo ⁇ hic sites.
  • the following target regions were amplified using either the PCR primers represeneted below or 'tailed' PCR primers, each of which includes a universal sequence forming a noncomplementary 'tail' attached to the 5 ' end of each unique sequence in the PCR primer pairs.
  • the universal 'tail' sequence for the forward PCR primers comprises the sequence 5 '-TGTAAAACGACGGCCAGT-3 ' (SEQ ID NO:64) and the universal 'tail' sequence for the reverse PCR primers comprises the sequence 5'- AGGAAACAGCTATGACCAT-3' (SEQ ID NO:65).
  • the nucleotide positions of the first and last nucleotide of the forward and reverse primers for each region amplified are presented below and correspond to positions in Figure 1.
  • Fragment 1 15478-15500 complement of 16110-16088 634 nt
  • Fragment 2 15154-15178 complement of 15761-15739 608 nt
  • Fragment 4 17523-17544 complement of 17978-17955 456 nt
  • Fragment 5 21579-21600 complement of 22138-22116 560 nt
  • Fragment 7 31730-31751 complement of 32242-32217 513 nt
  • Fragment 8 34344-34363 complement of 34811-34790 468 nt
  • Fragment 9 34131-34154 complement of 34549-34526 419 nt
  • Amplification profile 97°C - 2 min. 1 cycle
  • the PCR products were purified using a Whatman/Polyfiltronics 100 ⁇ l 384 well unifilter plate essentially according to the manufacturers protocol.
  • the purified DNA was eluted in 50 ⁇ l of distilled water.
  • Sequencing reactions were set up using Applied Biosystems Big Dye Terminator chemistry essentially according to the manufacturers protocol.
  • the purified PCR products were sequenced in both directions using either the primer sets represented below with the positions of their first and last nucleotide corresponding to positions id Figure 1, or the appropriate universal 'tail' sequence as a primer.
  • Reaction products were purified by isopropanol precipitation, and run on an Applied Biosystems 3700 DNA Analyzer.
  • Polyld is a unique identifier assigned to each PS by Genaissance Pharmaceuticals, Inc.
  • This example illustrates analysis of the CRYBBl polymo ⁇ hisms identified in the Index Repository for human genotypes and haplotypes.
  • the different genotypes containing these polymo ⁇ hisms that were observed in the reference population are shown in Table 3 below, with the haplotype pair indicating the combination of haplotypes determined for the individual using the haplotype derivation protocol described below.
  • Table 3 homozygous positions are indicated by one nucleotide and heterozygous positions are indicated by two nucleotides. Missing nucleotides in any given genotype in Table 3 were inferred based on linkage disequilibrium and/or Mendelian inheritance.
  • haplotype pairs shown in Table 3 were estimated from the unphased genotypes using a computer-implemented extension of Clark's algorithm (Clark, A.G. 1990 Mol Bio Evol 7, 111-122) for assigning haplotypes to unrelated individuals in a population sample, as described in U.S. Provisional Application Serial No. 60/198,340 entitled "A Method and System for Determining Haplotypes from a Collection of Polymo ⁇ hisms" and the corresponding International Application filed April 18, 2001.
  • haplotypes are assigned directly from individuals who are homozygous at all sites or heterozygous at no more than one of the variable sites.
  • haplotypes This list of haplotypes is augmented with haplotypes obtained from two families (one three-generation Caucasian family and one two-generation African- American family) and then used to deconvolute the unphased genotypes in the remaining (multiply heterozygous) individuals.
  • Isogenes of the CRYBBl gene are identical to the reference sequence at the regions examined except where they vary at the polymo ⁇ hic site.
  • a specific CRYBBl isogene identified by a haplotype in Table 4 comprises each oligo in the set of oligos shown in Table 5 for that haplotype.
  • PS1 ACAAAACGCT-TCTGT (SEQ ID NO: 67)
  • PS2 ATGGCATCCAACAGG (SEQ ID NO: 69)
  • PS3 CACACAGCGCGCAGT (SEQ ID NO: 71)
  • CACACAGTGCGCAGT (SEQ ID NO: 72)
  • PS4 CACAGCGCGCAGTGG (SEQ ID NO: 73)
  • PS5 TCTCCGCGGGACCGT (SEQ ID NO: 75)
  • PS6 TGGGCGGCGGAGGGG (SEQ ID NO: 77)
  • PS7 GTTCAGCCCCCTTTG (SEQ ID NO: 79)
  • PS8 CCTACTGTCCTCAGG (SEQ ID NO: 81)
  • PS9 GCTCTCCGGCTGGGG (SEQ ID NO: 83)
  • GCTCTCCAGCTGGGG (SEQ ID NO: 84)
  • PS10 AGCCAGGCGGGGCAG (SEQ ID NO: 85)
  • PS11 GGCCCCTTCCACAGT (SEQ ID NO: 87)
  • PS12 TCATGTTCAATTATT (SEQ ID NO: 89)
  • CRYBBl coding sequence isogenes are identical at the regions examined to the reference CRYBBl coding sequence, SEQ ID NO:2 (Fig 2.), except where they vary a pa polymo ⁇ hic site.
  • CRYBBl coding sequence isogenes are defined by subhaplotypes of the full CRYBBl haplotypes shown in Table 5.
  • a specific CRYBBl coding sequence isogene identified by a sub-haplotype of a haplotype in Table 5 comprises each oligo in the set of oligos shown in Table 6 for that CRYBBl sub-haplotype.
  • Table 7 shows the percent of the chromosomes characterized by a given haplotype for all unrelated individuals in the Index Repository for which haplotype data was obtained.
  • the percent of unrelated individuals who have a given CRYBB 1 haplotype pair is shown in Table 8.
  • the "Total" column shows this frequency data for all of these unrelated individuals, while the other columns show the frequency data for these unrelated individuals categorized according to their self- identified ethnogeographic origin.
  • Abbreviations used in Tables 7 and 8 are: AF, African or African- American; AS, Asian; CA, Caucasian; HL, Hispanic-latino; and AM, Native Americans.
  • HAP1 HAP2 Total CA AF AS HL AM
  • the size and composition of the Index Repository were chosen to represent the genetic diversity across and within four major population groups comprising the general United States population. For example, as described in Table 1 above, this repository contains approximately equal sample sizes of
  • African-descent, Asian-American, European-American, and Hispanic-Latino population groups Almost all individuals representing each group had all four grandparents with the same ethnogeographic background.
  • the number of unrelated individuals in the Index Repository provides a sample size that is sufficient to detect SNPs and haplotypes that occur in the general population with high statistical certainty. For instance, a haplotype that occurs with a frequency of 5% in the general population has a probability higher than 99.9% of being observed in a sample of 80 individuals from the general population. Similarly, a haplotype that occurs with a frequency of 10% in a specific population group has a 99% probability of being observed in a sample of 20 individuals from that population group.
  • the size and composition of the Index Repository means that the relative frequencies determined therein for the haplotypes and haplotype pairs of the THPO gene are likely to be similar to the relative frequencies of these THPO haplotypes and haplotype pairs in the general U.S. population and in the four population groups represented in the Index Repository.
  • the genetic diversity observed for the three THPO haplotypes and haplotype pairs of the THPO gene are likely to be similar to the relative frequencies of these THPO haplotypes and haplotype pairs in the general U.S. population and in the four population groups represented in the Index Repository. The genetic diversity observed for the three

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Abstract

L'invention concerne de nouvelles variantes génétiques du gène Cristallin, Beta B1 (CRYBB1). Elle traite également de différents génotypes, haplotypes et paires d'haplotypes pour le gène CRYBB1 qui existent dans la population des Etats-Unis en général. Elle concerne en outre des compositions et des méthodes d'haplotypage et/ou de génotypage du gène CRYBB1 chez un individu, ainsi que de polynucléotides définis par la séquence de ces haplotypes.
PCT/US2001/014715 2000-05-05 2001-05-07 Haplotypes du gene crybb1 WO2001085998A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102382889A (zh) * 2011-11-10 2012-03-21 武汉大学 人crybb1基因突变及其用途
CN106521020A (zh) * 2017-01-04 2017-03-22 青岛大学 一种crybb1基因的snp位点
NO20170739A1 (en) * 2017-05-04 2018-11-05 Patogen As Novel virus in Fish and Method for detection
CN113186192A (zh) * 2021-05-06 2021-07-30 潍坊医学院 Crybb2基因突变体、多肽、试剂盒、构建体及重组细胞

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HULSEBOS ET AL.: "Assignment of the BB1 crystallin gene (CRYBB1) to human chromosome 22 and mouse choromosome 5", GENOMICS, vol. 29, 1995, pages 712 - 718, XP002945646 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102382889A (zh) * 2011-11-10 2012-03-21 武汉大学 人crybb1基因突变及其用途
CN102382889B (zh) * 2011-11-10 2014-08-13 武汉大学 人crybb1基因突变及其用途
CN106521020A (zh) * 2017-01-04 2017-03-22 青岛大学 一种crybb1基因的snp位点
CN106521020B (zh) * 2017-01-04 2019-07-19 青岛大学 一种crybb1基因的snp位点
NO20170739A1 (en) * 2017-05-04 2018-11-05 Patogen As Novel virus in Fish and Method for detection
NO344051B1 (en) * 2017-05-04 2019-08-26 Patogen As Novel virus in Fish and Method for detection
CN113186192A (zh) * 2021-05-06 2021-07-30 潍坊医学院 Crybb2基因突变体、多肽、试剂盒、构建体及重组细胞

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