Abstract:
The present invention relates to the identification of the NSDHL gene product as a 3β-HSD participating in the conversion of 4,4-dimethylcholest-8(9)-en-3β-ol to cholest-8(9)-en-3β-ol in the cholesterol and vitamin D biosynthetic pathway. Based upon this function the present invention contemplates methods for manipulating the biosynthetic pathway at the step of involvement of NSDHL to increase or decrease the levels of cholesterol and/or vitamin D (or downstream products such as steroids) produced by a cell. Also contemplated are methods for manipulating the accumulation of intermediate compounds upstream of the step of NSDHL in the pathway. Diagnostic methods involving identification of mutations in the genes encoding enzymes involved in the conversion of 4,4-dimethylcholest-8(9)-en-3β-ol to cholest-8(9)-en-3β-ol are also provided, as well as diagnostic methods involving detection of abnormal accumulation of sterol intermediates prior to generation of choles-8(9)-en-3β-ol in the pathway.

Description:
[0001]    This is a continuation-in-part of U.S. Patent Application Ser. No. 60/137,020 filed Jun. 1, 1999. 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    The present invention generally relates to 3β-hydroxysteroid dehydrogenase (3β-HSD) enzymes that participate in cholesterol and vitamin D biosynthesis. More particularly, the invention relates to the 3β-HSD enzyme termed NSDHL and to manipulation of the chemical reaction(s) in which it participates.  
         BACKGROUND  
         [0003]    Cholesterol is a key component of cell membranes and is the immediate precursor for the synthesis of all known steroid hormones and bile acids. Vitamin D is important in calcium homeostasis and bone formation. Cholesterol and vitamin D can be synthesized in a multistep pathway from the sterol precursor lanosterol. As illustrated in FIG. 1, lanosterol is typically first converted to 4, 4-dimethylcholest-8(9)-en-3β-ol. Next, 4,4-dimethylcholest-8(9)-en-3 β-ol is converted to 4-methylcholest-8(9)-en-3β-ol and then to cholest-8(9)-en-3β-ol by sequential removal of the two C-4 methyl groups from the sterol backbone. Cholest-8(9)-en-3β-ol is, in turn, converted to lathosterol (cholest-7-en-3β-ol). 7-dehydrocholesterol is then generated from lathosterol and is the immediate major precursor of both cholesterol and vitamin D. Intermediates preceding lanosterol in the cholesterol biosynthetic pathway also serve as precursors for the synthesis of non-sterol isoprenes such as isopentenyl-tRNAs, dolichol, ubiquinone, and haem A. The isopentenyl groups in tRNAs stabilize codon-anticodon interaction contributing to fidelity of protein synthesis from mRNA. Dolichol is required for the synthesis of glycoproteins. Ubiquinone and haem A are important components of the mitochondrial respiratory chain. While some genes encoding enzymes participating in the mammalian biosynthetic pathway have been previously characterized, other genes remain unknown.  
           [0004]    Various disease states have been linked to abnormalities in cholesterol biosynthesis. For example, mevalonic aciduria was the first disorder of cholesterol synthesis to be recognized and is caused by a defect in mevalonate kinase, an enzyme participating in early steps of the pathway. Patients experience developmental delay, failure to thrive, hypotonia, ataxia, hepatosplenomegaly, cataracts, lymphadenopathy, anaemia, myopathy, and enteropathy with fat malabsorption.  
           [0005]    Additionally, defects in later steps of the pathway in the conversion of lanosterol to cholesterol have been described in a single infant with “desmosterolosis” in Clayton,  Archi. Dis. Child.,  78: 185-189 (1998) and in patients with Smith-Lemli-Opitz syndrome (SLOS) in Kelley,  Am. J. Hum. Genet.,  63: 322-326 (1998). The desmosterolosis afflicted infant had a cleft palate, ambiguous genitalia, short limbs, short and unrotated intestines and bilateral renal hypoplasia. SLOS is characterized by mental retardation, microcephaly, failure to thrive, cataracts, cleft palate, postaxial polydactyly and/or syndactyly of the second and third toes, congenital heart disease, genital abnormalities, photosensitivity, and, occasionally, chondrodysplasia punctata. Affected patients accumulate 7-dehydrocholesterol and mutations in the 3β-hydroxysteriod-Δ 7 -reductase gene have recently been identified in numerous SLOS probands. See, e.g., Wassif et al.,  Am. J. Hum. Genet.,  63: 66-62 (1998). Even more recently, Kelley et al.,  Am. J. Med. Gen.,  83: 213-219 (1999) reported that patients with X-linked dominant Conradi-Hünermann-Happle syndrome and nonspecific lethal chondrodysplasia punctata exhibited abnormally increased levels of 8-dehydrocholesterol and cholest-8(9)-en-3β-ol.  
           [0006]    Other disease states have been suggested to involve 3β-HSD abnormalities but the biosynthetic pathway in which those enzymes participated had not previously been demonstrated. For example, Herman et al. [presenting at the 12 th  International Mouse Genome Conference, Garmisch, Germany (September 1998) and at the American Society of Human Genetics Meeting Late Breaking Plenary Session, Denver, Colo. (October 1998)] described an X-linked mouse mutation bare patches (Bpa) occurring in a gene, the Nsdhl gene putatively encoding a 3β-HSD, as being a murine model of the human X-linked dominant disorder chondrodysplasia punctata (CDPX2). The human and murine phenotypes of the two disorders are very similar and include hyperkeratotic skin eruptions and hair loss, a skeletal dysplasia characterized by abnormal epiphyseal calcifications (chondrodysplasia punctata), and frequent cataracts and or microophthalmia. A human genomic DNA containing a gene (NSDHL) corresponding to the murine Nsdhl gene was disclosed in Genbank Accession No. U82671. The human NSDHL gene has been mapped on the X chromosome at Xq28 and comprises eight exons. At the time of the Herman et al. presentations, the substrate of the putative enzyme encoded by the human NSDHL and murine Nsdhl genes was not known. However, Gachotte et al.,  Proc. Natl. Acad. Sci. USA,  95: 13794-13799 (1998) reported that the S. cerevisiae homolog of Nsdhl functions as a C-3 sterol dehydrogenase (C-4 decarboxylase) in the synthesis of ergosterol, the major sterol of yeast and Kelley et al. (1999), supra suggested that Nsdhl gene product may have a role in cholesterol biosynthesis in 4-demethylation of lanosterol.  
           [0007]    There thus continues to exist in the art a need for the identification of genes encoding enzymes that participate in the biosynthetic pathway(s) for cholesterol and vitamin D as well as for the identification of disease states resulting from mutations in those genes or abnormalities in their expression.  
         BRIEF SUMMARY OF THE INVENTION  
         [0008]    The present invention relates to the identification of the NSDHL gene product as a 3β-HSD involved in the conversion of 4,4-dimethylcholest-8(9)-en-3β-ol to cholest-8(9)-en-3β-ol in the cholesterol and vitamin D biosynthetic pathway.  
           [0009]    Based upon this function, the present invention contemplates methods for manipulating the biosynthetic pathway at the step of involvement of NSDHL to increase or decrease the levels of cholesterol and/or vitamin D (or downstream products such as steroids) produced by a cell. Also contemplated are methods for manipulating (increasing or decreasing) the accumulation of intermediate compounds upstream of the step of involvement of NSDHL in the pathway. These methods of manipulation are useful, for example, in in vitro cell culture methods to produce steroids for which cholesterol is a precursor and in in vivo treatments for disease states involving the biosynthetic pathway.  
           [0010]    The present invention contemplates that various disease states including bone, skin and eye disorders are associated with mutations in the NSDHL gene and other genes encoding proteins involved in the conversion of 4,4-dimethylcholest-8(9)-en-3β-ol to cholest-8(9)-en-3β-ol. Thus, detection of mutations in these genes is useful in the initial diagnosis of the disease states and in evaluating treatment options.  
           [0011]    Embodiments of the invention are methods for diagnosing disease states associated with mutations in the NSDHL gene or the other genes encoding proteins involved in the same step of the biosynthetic pathway, that is the step in which 4,4-dimethylcholest-8(9)-en-3β-ol is converted to cholest-8(9)-en-3β-ol. It is contemplated that the step of removal of the two C-4 methyl groups requires the sequential actions of a C-4 sterol methyloxidase, the NSDHL gene product (a 3β-HSD) and a 3-keto reductase (i.e., the sterol-4-demethylase complex). See FIG. 2. In addition, regulatory or other accessory proteins may be required. A cDNA sequence encoding a human C-4 sterol methyloxidase is deposited under GenBank U60205 (SEQ ID NO: 5 herein) and was identified based on homology to yeast ERG25 [Li and Kaplan,  J. Biol. Chem.,  271: 16927-16933 (1996)].  
           [0012]    In the diagnostic methods of the invention, polynucleotides obtained from a patient are analyzed for mutations (i.e., nucleotide differences from wild type) using primers or probes based on the NSDHL polynucleotide sequence set out in SEQ ID NO: 1 or the C-4 sterol methyloxidase sequence set out in SEQ ID NO: 5 using standard polynucleotide sequencing, amplification and/or hybridization techniques. Examples of intronic primer pairs for routine sequencing of and mutation detection in the 5′ noncoding region and eight exons of the human NSDHL gene are:  
                                                                                                                                                           5′ noncoding           5′ AAAGACTGGTGCGCTAAAGC 3′   (SEQ ID NO:7)       5′ CGAGCTTCCTCCACCAAGAG 3′   (SEQ ID NO:8)                    Exon 1 (ending at nucleotide 151 of SEQ ID NO:1)            5′ CCCCGTCTTTATTGGGCAAG 3′   (SEQ ID NO:9)       5′ ACTGCCCAGTCGCTGACACAG 3′   (SEQ ID NO:10)                    Exon 2 (corresponding to nucleotides 152 through       302 of SEQ ID NO:1)            5′ GGCATCTGCCCAAAACACTAAC 3′   (SEQ ID NO:11)       5′ CCACAGGTAAATAGTATCAGCC 3′   (SEQ ID NO:12)                    Exon 3 (corresponding to nucleotides 303 through       461 of SEQ ID NO:1)            5′ TTCCAGTCCTCACTACCCTG 3′   (SEQ ID NO:13)       5′ AGTATCGTGGTTTCCCTTCG 3′   (SEQ ID NO:14)                    Exon 4 (corresponding to nucleotides 462 through       608 of SEQ ID NO:1)            5′ TGCCATTGACCTGTCAAAGC 3′   (SEQ ID NO:15)       5′ CCCTTAGAAAGGGCCATCAC 3′   (SEQ ID NO:16)                    Exon 5 (corresponding to nucleotides 609 through       737 of SEQ ID NO:1)            5′ GGATCATGCACTGTTTGAATTG 3′   (SEQ ID NO:17)       5′ GGATTCTAAACCCTTCAGTC 3′   (SEQ ID NO:18)                    Exon 6 (corresponding to nucleotides 738 through       880 of SEQ ID NO:1)            5′ CTAGGAATTTGCAATGGACG 3′   (SEQ ID NO:19)       5′ TGAATGCGAGCATGGACCAG 3′   (SEQ ID NO:20)                    Exon 7 (corresponding to nucleotides 881 through       983 of SEQ ID NO:1)            5′ AAGACTTGGGAGTGGCCCTG 3′   (SEQ ID NO:21)       5′ AGGCAAGGAGAAGAAACCCG 3′   (SEQ ID NO:22)                    Exon 8 (beginning at nucleotide 984       of SEQ ID NO:1)            5′ TTCAACTTTGGGCAGGTGGG 3′   (SEQ ID NO:23)       5′ CTCCATAGCATCATCCATGG 3′   (SEQ ID NO:24) and       5′ CATTCCACTACTACAGCTGC 3′   (SEQ ID NO:25)       5′ TGTATAAACCAGAAGAGGGG 3′   (SEQ ID NO:26).          
 
           [0013]    Techniques contemplated by the invention include, but are not limited to, well-known techniques such as polymerase chain reaction techniques, single-strand conformation polymorphism analysis (SSCP) [Orita et al.,  Proc Natl. Acad. Sci. USA,  86: 2766-2770 (1989)]; heteroduplex analysis [White et al.,  Genomics,  12: 301-306 (1992)]; denaturing gradient gel electrophoresis analysis [Fischer et al.,  Proc. Natl. Acad. Sci. USA,  80: 1579-1583 (1983); and Riesner et al.,  Electrophoresis,  10: 377-389 (1989)]; DNA sequencing; RNase cleavage [Myers et al., Science, 230: 1242-1246 (1985)]; chemical cleavage of mismatch techniques [Rowley et al.,  Genomics,  30: 574-582 (1995); and Roberts et al.,  Nucl. Acids Res.,  25: 3377-3378 (1997)]; restriction fragment length polymorphism analysis; single nucleotide primer extension analysis [Shumaker et al.,  Hum. Mutat.,  7: 346-354 (1996); and Pastinen et al., Genome Res., 7: 606-614 (1997)]; 5′ nuclease assays [Pease et al.,  Proc. Natl. Acad. Sci. USA,  91: 5022-5026 (1994)]; DNA Microchip analysis [Ramsay, G.,  Nature Biotechnology,  16: 40-48 (1999); and Chee et al., U.S. Pat. No. 5,837,832]; and ligase chain reaction [Whiteley et al., U.S. Pat. No. 5,521,065]. See generally, Schafer and Hawkins,  Nature Biotechnology,  16: 33-39 (1998). All of the foregoing documents are hereby incorporated by reference in their entirety.  
           [0014]    In one preferred embodiment the assaying involves sequencing of nucleic acid to determine nucleotide sequence thereof, using any available sequencing technique. [See, e.g., Sanger et al.,  Proc. Natl. Acad. Sci. (USA),  74: 5463-5467 (1977) (dideoxy chain termination method); Mirzabekov,  TIBTECH,  12: 27-32 (1994) (sequencing by hybridization); Drmanac et al.,  Nature Biotechnology,  16: 54-58 (1998) and  Science,  260: 1649-1652 (1993) (sequencing by hybridization); Kieleczawa et al.,  Science,  258: 1787-1791 (1992) (sequencing by primer walking); (Douglas et al.,  Biotechniques,  14: 824-828 (1993) (Direct sequencing of PCR products); and Akane et al.,  Biotechniques  16: 238-241 (1994); Maxam and Gilbert,  Meth. Enzymol.,  65: 499-560 (1977) (chemical termination sequencing)].  
           [0015]    Alternatively, diagnostic methods of the invention involve detection in a patient&#39;s body fluid (e.g., serum, urine, semen, amniotic fluid, saliva, pleural fluid, peritoneal fluid, and cerebrospinal fluid) or cells (e.g., fibroblasts, keratinocytes, chondrocytes, liver tissue or osteoid tissue) of sterol intermediates or their metabolites prior to the step of generation of cholest-8(9)-en-3β-ol in the cholesterol/vitamin D biosynthetic pathway. Preferably detection is by gas chromatography and mass spectrometry (GC/MS) or related mass spectrometric techniques, and more preferably is by selected ion monitoring gas chromatography/mass-spectrometry (SIM-GC/MS) as described in Kelley,  Clinica Chimica Acta,  236: 45-58 (1995) which is incorporated by reference herein. In the methods, a sample obtained from a patient is analzyed by SIN-GC/MS for accumulation of 4,4-dimethylcholest-8(9)-en-3β-ol, 4-methylcholest-8(9)-en-3β-ol (methylsterol-1) and/or 4-methylcholesta-8(9),24-dien-3β-ol (methylsterol-2), sterol intermediates generated prior to or during the NSDHL step of involvement in the biosynthetic pathway. Accumulation of these intermediates is indicative of an abnormality at this step in the pathway because only trace concentrations of the intermediates are detectable in normal individuals.  
           [0016]    Preferred diagnostic methods contemplated by the invention are methods of diagnosis of CHILD syndrome (involving skin and bone), of skin disorders such as psoriasis and ichthyoses, of bone disorders such as osteoporosis and osteosclerosis, of eye disorders such as cataracts and microopthalmia and of arthritis.  
           [0017]    In another embodiment, treatment of skin, bone and eye disorders is contemplated by the invention. Preferred indications are CHILD syndrome (involving skin and bone), skin disorders such as psoriasis and ichthyoses, bone disorders such as osteoporosis and osteosclerosis, eye disorders such as cataracts and microopthalmia, and arthritis. As noted above, manipulation of the biosynthetic pathway at the step of involvement of NSDHL may involve increasing or decreasing downstream products produced by a cell or may involve increasing or decreasing upstream products produced by a cell. This is because mutations in genes encoding proteins involved in the step alter the balance between upstream and downstream products. In disease states such as CHILD syndrome, psoriasis, ichthyoses, osteosclerosis, cataracts, micropthalmia and arthritis, it is contemplated that treatment according to the invention involves decreasing upstream products and increasing downstream products. Tissue abnormalities that arise in these disorders can be caused by adverse effects of intermediate sterols upstream of the step of involvement of NSDHL in the pathway (such as C29 sterols 4,4′-dimethylcholest-en-3β-ol and 4,4′-dimethylcholesta-8,24-dien-3β-ol) and/or deficiency in downstream products. In disease states such as osteoporosis it is contemplated that treatment involves increasing upstream products and decreasing downstream products in order to promote bone formation.  
           [0018]    In one embodiment, agents to be used to therapeutically manipulate the biosynthetic pathway can be identified in assays based on the 3β-HSD activity of the NSDHL (SEQ ID NO: 2) or Nsdhl (SEQ ID NO: 4) enzyme products, or in assays based on the methyloxidase activity of C-4 sterol methyloxidase (SEQ ID NO: 6). The enzyme is made by standard recombinant techniques by expressing human NSDHL (SEQ ID NO: 1) or murine Nsdhl (SEQ ID NO: 3) or C-4 sterol methyloxidase cDNA in an appropriate host cell. Activity of the enzyme is measured in the presence and absence of a test agent and agents which increase or decrease the activity of the enzyme are identified. As one example, the active site of NSDHL enzyme (amino acids 92-96 of SEQ ID NO: 2) exhibits relatively low homology to other mammalian 3β-HSDs and is contemplated as a binding site for a agent that would inhibit the NSDHL enzyme.  
           [0019]    In other embodiments, expression of the NSDHL gene or C-4 sterol methyloxidase gene is directly manipulated in vivo either locally (e.g., topically) or systemically, for example, using antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences or using gene therapy.  
           [0020]    Antisense or sense oligonucleotides, according to the present invention, comprise a fragment of DNA (such as that DNA set forth in SEQ ID NOs: 1 or 5). Such a fragment generally comprises at least about 14 nucleotides, preferably from about 17 to about 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, for example, Stein and Cohen ( Cancer Res.,  48: 2659, 1988) and van der Krol et al. ( BioTechniques,  6: 958, 1988). Binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block or inhibit protein expression by one of several means, including enhanced degradation of the mRNA by RNAseH, inhibition of splicing, premature termination of transcription or translation, or by other means. The antisense oligonucleotides thus may be used to block expression of proteins. Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar-phosphodiesterase backbones (or other sugar linkages) and wherein such sugar linkages are resistant to endogenous nucleases. Such oligonucleotides with resistant sugar linkages are stable in vivo (i.e., capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequence. Other examples of sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties and other moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lysine). Further still, intercalating agents, as ellipticine, and the alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide sequence.  
           [0021]    Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, lipofection, CaPO 4 -mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Barr virus. Sense or antisense oligonucleotides also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell. Alternatively, a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence formation of an oligonucleotide-lipid complex. The sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase.  
           [0022]    Mutations in the genes encoding proteins involved in the conversion of 4,4-dimethylcholest-8(9)-en-3β-ol to cholest-8(9)-en-3β-ol that result in reduction or loss of normal expression or function of the proteins are contemplated to underlie aforementioned disease states. The invention comprehends gene therapy to restore gene function to treat those disease states. For example, delivery of functional NSDHL or C-4 sterol methyloxidase genes to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus or retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments). Delivery may be systemic or local as appropriate. See, for example, Anderson,  Nature,  392(6679 Suppl):25-30 (1998).  
         DETAILED DESCRIPTION OF THE INVENTION  
         [0023]    The invention is illustrated by the following examples wherein Example 1 describes the tissue distribution of Nsdhl mRNA, Example 2 describes abnormal sterol accumulation in Bpa mice, Example 3 reports mutations in the human NSDHL gene in patients with CHILD syndrome and Example 4 describes abnormal sterol accumulation in psoriasis patients.  
       
    
    
     EXAMPLE 1  
       [0024]    Previous Northern analyses with partial human NSDHL cDNA probes (called XAP104 and H105E3) demonstrated ubiquitous expression in adult tissues of a 1.5-2.0 kb transcript. See Levin et al.,  Genome Res.,  6: 465-477 (1996) and Heiss et al,  Genome Res.,  6: 478-491 (1996).  
         [0025]    Expression of wild type murine Nsdhl mRNA was examined and compared to the human tissue distribution results. Northern blots containing 4 μg of mRNA were prepared from cultured undifferentiated embryonic stem cells, from embryos isolated from timed matings, and from other murine tissues. The Northern blots were probed with the 1.4kb Nsdhl EST 605654 or a 780 bp human GAPDH cDNA (control) and exposed under X-ray film. RT-PCR was also performed on bone/cartilage dissected from femurs of newborn mice.  
         [0026]    High levels of expression of Nsdhl transcript were detected in undifferentiated embryonic stem cells and mid and late gestation mouse embryos. In adult mice, expression was detected in all tissues with the highest expression seen in ovary, testis, liver, adrenal, and kidney. Expression was also observed in eye, skin and newborn bone/cartilage, tissues that are affected in surviving Bpa females.  
       EXAMPLE 2  
       [0027]    To determine the substrate of the Nsdhl gene product sterol metabolism was examined in Bpa mice.  
         [0028]    Sterol distributions in cultured skin fibroblasts or tissue samples obtained from Bpa females and control mice were analyzed by gas chromatography and selected-ion mass spectrometry as described in Kelley et al. (1999), supra except that the gas chromatographic matrix was 5% phenylmethylsilicone (HP-2, Hewlett Packard). Primary murine fibroblasts were expanded by weekly subculture (1:2) in RPMI 1640 supplemented with 15% fetal calf serum and incubated at 37° in 5% CO 2  For analysis of sterol metabolism, cells were subcultured 1:3 in T25 flasks and fed at 24 hours and 7 days with RPMI containing 15% delipidated fetal calf serum as described in Gibson et al,  J. Lipid Res.,  31: 515-521 (1990). After 14 days growth in delipidated medium, the cells were harvested and sterols quantitated. Tissues from female Bpa mice were analyzed for sterol content in the same manner as skin fibroblasts following initial homogenization of the tissue in 1 ml of sterol saponification solution in a ground glass homogenizer. Results of the analyses are presented in Table 1 below and in FIG. 3 which shows gas chromatographic flame ionization profiles of the sterol extracts of the normal mouse fibroblasts (upper tracing) and the Bpa fibroblasts (lower tracing). In the figure, the ordinates are detector response and the abscissae are elution time. The identified compounds are: (1) internal standard (epicoprostanol); (2) cholesterol; (3) desmosterol; and (4) lathosterol. Tentatively identified compounds include: (5) 4-methylcholest-8(9)-en-3β-ol; (6) 4-methylcholesta-8(9),24-dien-3β-ol; and (7) 4,4′-dimethylcholest-8(9)-en-3β-ol.  
                                                           TABLE 1                           Sterol Analysis of Cultured Skin Fibroblasts from Bpa Mice                Female   Female   Female               Bpa   Bpa   Bpa   Male Mouse           Culture 1   Culture 2   Mean   Controls (4)       Sterol   % Sterols   % Sterols   % Sterols   % Sterols                    Cholesterol   79.3   63.4   71.4   100.0       Desmosterol    3.3    5.8    4.6   &lt;0.1       Lathosterol    0.6    3.7    2.1   &lt;0.1       4-Methylcholest-    7.9    3.9    5.9   &lt;0.1       8(9)-en-βol       4-Methylcholesta-    6.8   17.8   12.3   &lt;0.1       8(9),24,dien-3β-ol       4,4-Diemthylcholest-    1.1    1.0    1.1   &lt;0.1       8(9)-en-3β-ol                  
 
         [0029]    Cultured fibroblasts from an affected adult Bpa female accumulated large amounts of two C28 sterols and a smaller amount of a C29 sterol (Table 1 and FIG. 2). Similar abnormal accumulations in kidney and liver tissue from Bpa females, ages 6 days, 18 days, and 3 months were also found (data not shown). Although the exact isomeric structures of the accumulated C28 sterols remain to be determined, their fragmentation patterns and retention times are consistent with C-4 methylsterols having double bonds in, respectively, the 8(9) and 8(9), 24 positions. The C29 sterol as was tentatively identified as 4,4′-dimethylcholest-8(9)-en-3β-ol. The abnormal sterol profile is consistent with the function of Nsdhl as a C-3 sterol dehydrogenase involved in the complex series of reactions that result in the sequential removal of the two C-4 methyl groups from the sterol backbone of the cholesterol precursor lanosterol.  
         [0030]    [0030]FIG. 1 is a schematic representation of the enzymatic pathway for conversion of lanosterol to cholesterol in which sterol intermediates that are increased in Bpa mice are shown in bold type and underlined. The saturation of the C-24 methyl groups may occasionally precede that of the C-24 double bond or may occur at different points in the pathway, and removal of the C-4 methyl groups may occasionally precede that of the C-14 methyl group (not shown). FIG. 4 depicts additional steps in the cholesterol biosynthetic pathway that could be affected by the accumulation of sterol intermediates.  
       EXAMPLE 3  
       [0031]    Mutations in the human NSDHL gene have been identified in patients with CHILD syndrome (congenital hemidysplasia, ichthyosiform erythroderma and limb defects), a rare X-linked dominant genodermatosis characterized by unilateral ichthyosiform skin lesions, ipsilateral anomalies of limbs and internal organs, and punctate calcifications of the epiphyses and other cartilaginous structures on the affected side.  
         [0032]    CHILD Syndrome  
         [0033]    In CHILD syndrome patients, there is unilateral distribution of ichthyotic skin at birth, with a sharp line of demarcation between normal and abnormal skin at the midline of the trunk. In most patients, large areas of skin are diffusely abnormal, often on the trunk, but also on the limbs. However, some skin lesions on the affected side may follow lines of Blaschko with interspersed streaks of normal skin. The skin of the face is usually spared, although scalp alopecia may be present. The skin lesions are usually most severe at or shortly after birth and often improve spontaneously, but some patients have persistent patches of involved skin that do not resolve and respond poorly to therapy. Clinically, the skin appears scaly and erythematous. The lesions may be pruritic. Skin lesions in NSDHL deficiency CHILD syndrome are characterized by waxy yellow plaques with a propensity for skin folds. Histologically, the epidermis shows orthohyperkeratosis, parakeratosis, and marked acanthosis with inflammatory infiltrates of lymphocytes, histiocytes and neutrophils. Accumulations of neutrophils may form in the stratum comeum. Verruciform xanthoma, characterized by foamy, lipid-filled histiocytes in the dermal papillae, has also been observed in several reported cases of CHILD syndrome. New patches of involved skin, on the same or on the contralateral side, can develop. Lesions may appear and disappear spontaneously, and usually do not respond well to most therapeutic trials of emollients, topical steroids, etc.  
         [0034]    Patients with skin disorders such as ichthyosiform nevus, ichthyosis, inflammatory linear vernucous epidermal nevus (ILVEN), linear epidermal nevus, epidermal nevus, inflammatory epidermal nevus, epidermal hyperplasia, psoriasiform erythroderma and psoriasiform epidermal nevus, which are clinically very similar to the skin abnormality seen in NSDHL deficiency CHILD syndrome, may have NSDHL deficiency or other sterol biosynthetic defects at the same enzymatic step. ILVEN may represent the mild end of the spectrum of the same defect as that in CHILD syndrome.  
         [0035]    Limb defects may range from mild hypoplasia of digits to agenesis of an entire limb. Punctate calcifications of cartilaginous structures are often observed in newborns or young infants, but usually resolve over time. Finger and toenails may be dysplastic. In addition to the skeletal and cutaneous manifestations, patients may have ipsilateral visceral anomalies, including brain, renal, and cardiac defects. The right side is involved more frequently than the left. If the left side is affected, there is a higher risk for a serious or lethal cardiac defect.  
         [0036]    Identification of Mutations  
         [0037]    A distinct splicing mutation was identified in two unrelated CHILD syndrome patients. The mutation IVS7-2 A to C, alters the splice acceptor site at the beginning of exon 8 of the NSDHL gene. The presence of the heterozygous mutation was confirmed by sequencing PCR products. The mutation was also found to be present in one of the patients&#39; asymptomatic mother by direct sequencing. Confirmation of the mutation was performed by RT-PCR performed as described in Levin et al.,  Genome Res.,  6: 465-477 (1996) on patient lymphoblast DNA using the primers shown below that amplify between exon 7 and exon 8 of the DNA sequence.  
                               Forward   5′ ACGTGGTCCATGGACACA 3′   (SEQ ID NO:27)               Reverse   5′ CATCCATGGTCACTAGTGGC 3′   (SEQ ID NO:28)          
 
         [0038]    PCR conditions were 94° C., 7 minutes; 94° C., 30 seconds; 55° C., 30 seconds; 72° C., 30 seconds; then forty cycles of 72° C., 7 minutes and 4° C. The RT-PCR product was excised from a gel and directly sequenced. It indicated that the mutation results in abnormal splicing and the in frame deletion of the first fifty-six amino acids in exon 8 of the protein.  
         [0039]    Other mutations have been identified in the NSDHL gene of CHILD patients and are described in König et al.,  Am. J. Med. Genet.,  90: 339-346 (2000), which is incorporated by reference herein.  
         [0040]    Sterol Analysis  
         [0041]    Sterol analysis of the serum and/or skin fibroblasts of the patients and mother demonstrated the accumulation of 4-methyl sterols consistent with the mutation in the NSDHL gene.  
       EXAMPLE 4  
       [0042]    Abnormal sterol accumulation consistent with defects in the genes encoding enzymes in the NSDHL step was also observed in psoriasis patients.  
         [0043]    Psoriasis  
         [0044]    Psoriasis is a chronic papulosquamous disorder that undergoies spontaneous remissions and exacerbations. In psoriasis, skin lesions are sharply demarcated with distinct borders. The lesions of psoriasis demonstrate a variety of morphologic types and areas of distribution on the body. The surface of the lesions is covered with silvery scales, and under the scale, there is glossy, homogeneous erythema. Nails are frequently involved in psoriasis; the abnormalities can include nail pits, brownish or yellowish discoloration beneath the nail plate, or severe onychodystrophy. Histopathology of psoriasis is characterized by thickened (3-5×normal) epidermis (acanthosis), parakeratosis, and a lymphohistiocytic infiltrate in the papillary dermis. The dermal papillae become thin and elongated, and contain tortuous blood vessels in an edematous stroma. Psoriasis, its diagnosis, and its treatment are discussed in Abel, Psoriasis 8/97, 2. Dermatology III, pp. Psoriasis-1 to Psoriasis-14, in  Scientific American Medicine , Scientific American, Inc., New York, N.Y. (1973-2000), which is incorporated by reference herein.  
         [0045]    Differences between psoriasis and the NSDHL deficiency skin lesions include an apparent difference in the clinical appearance of the skin scales, in that the scales in psoriasis are silvery and those of NSDHL deficiency are waxy and yellow, and the lack of verruciform xanthomatous changes in the dermis in psoriasis on biopsy. However, in psoriasis and other ichthyoses, such as harlequin ichthyosis, lipid vacoules are seen in lesional keratinocytes ultrastructurally. Keratinocytes may be vacoulated in CHILD syndrome as well.  
         [0046]    Sterol Analysis  
         [0047]    To determine if common psoriasis is associated with the same abnormalities of cholesterol biosynthesis that are characteristic of both human NSDHL- and murine Nsdhl-deficiency, sterols were quantified in the scales of human psoriatic skin and from forearm skin of human adult controls. Presented in Table 2 below are representative data summarizing sterol levels in the skin of three adult psoriasis samples, three normal adult controls, and one of the CHILD syndrome patients discussed in Example 3, wherein all values are percent of total sterols  
                                                           TABLE 2                           Sterol Analysis in Psoriasis                        4-Methyl-   4-Methyl-           Cholesterol   Squalene   sterol-1   sterol-2                        Psoriasis-1   90    0.9   0.3   1.2       Psoriasis-2   88.6    0.6   0.2   2.0       Psoriasis-3   89.4    0.5   1.0   3.7       Control 1   42.0   42.0   &lt;0.1   &lt;0.1       Control 2   57.0   37.1   &lt;0.1   &lt;0.1       Control 3   56.0   36.9   &lt;0.1   &lt;0.1       CHILD   96    0.4   1.3   0.3                  
 
         [0048]    All psoriasis samples tested had increased levels of the same two 4-methylsterols that are most prominent in Nsdhl-deficient mouse plasma, tissues, and cultured cells: 4-methylcholest-8(9)-en-3beta-ol (methylsterol-1) and 4-methylcholesta-8(9),24-dien-3beta-ol (methylsterol-2). These are the sterols that are predicted herein to be increased because of the deficient activity of the NSDHL-encoded 3β-HSD of the sterol-4-demethylase complex. Only trace amounts of these compounds are present in normal skin. Another important characteristic of the skin of the human psoriasis is the near absence of squalene, a isoprenoid precursor of lanosterol and one of the most abundant lipids in normal skin.  
         [0049]    Essentially the same abnormal pattern of sterols was found in the skin of the CHILD syndrome patient with the established NSDHL mutation described in Example 3 above. One difference between the sterol profile of CHILD syndrome skin and that of psoriasis skin is the reversed ratio of the two 4-methylsterols. Nevertheless, the similarity of the two sterol profiles indicates that dysfunction of the sterol-4-demethylase enzyme complex is a characteristic of human psoriasis in the patients examined.  
         [0050]    Diagnostic methods based on the analysis of genes encoding enzymes participating in the sterol-4-demethylase complex or on analysis of sterol accumulation are therefore contemplated as useful to clinicians in the initial diagnosis of psoriasis and also in determining treatment course as different psoriasis patients are not necessarily responsive to the same therapies.  
         [0051]    While the present invention has been described in terms of exemplary methods, it is understood that variations and modifications will occur to those skilled in the art. Therefore, only such limitations as appear in the claims should be placed on the invention.  
     
       
       
         1 
         
           
             28  
           
           
             1  
             1563  
             DNA  
             Homo sapiens  
             
               CDS  
               (195)..(1313)  
             
           
            1 

cgccgagctg ggccaatcct cttggtggag gaagctcggc tgattctcgg ctcacgcggg     60 

aggggagtaa agggtggcgg tccgggcctg gagttcagtg ggtgcagcct gcttgcgagc    120 

tgaggccaga caggggggcg cctacggacg gaaaagaaaa gttgattaca aacgggacca    180 

tattttgctt cgaa atg gaa cca gca gtt agc gag cca atg aga gac caa      230 
                Met Glu Pro Ala Val Ser Glu Pro Met Arg Asp Gln 
                  1               5                  10 

gtc gca cgg act cat ttg aca gag gac act ccc aaa gtg aat gct gac      278 
Val Ala Arg Thr His Leu Thr Glu Asp Thr Pro Lys Val Asn Ala Asp 
         15                  20                  25 

ata gaa aag gtt aac cag aat cag gcc aag aga tgc aca gtg atc ggt      326 
Ile Glu Lys Val Asn Gln Asn Gln Ala Lys Arg Cys Thr Val Ile Gly 
     30                  35                  40 

ggc tct gga ttc ctg ggg cag cac atg gtg gag cag ttg ctg gca aga      374 
Gly Ser Gly Phe Leu Gly Gln His Met Val Glu Gln Leu Leu Ala Arg 
 45                  50                  55                  60 

gga tat gct gtc aat gta ttt gat atc cag caa ggg ttt gat aat ccc      422 
Gly Tyr Ala Val Asn Val Phe Asp Ile Gln Gln Gly Phe Asp Asn Pro 
                 65                  70                  75 

cag gtg cgg ttc ttt ctg ggt gac ctc tgc agc cga cag gat ctg tac      470 
Gln Val Arg Phe Phe Leu Gly Asp Leu Cys Ser Arg Gln Asp Leu Tyr 
             80                  85                  90 

cca gct ctg aaa ggt gta aac aca gtt ttc cac tgt gcg tca ccc cca      518 
Pro Ala Leu Lys Gly Val Asn Thr Val Phe His Cys Ala Ser Pro Pro 
         95                 100                 105 

cca tcc agt aac aac aag gag ctc ttt tat aga gtg aat tac att ggc      566 
Pro Ser Ser Asn Asn Lys Glu Leu Phe Tyr Arg Val Asn Tyr Ile Gly 
    110                 115                 120 

acc aag aat gtc att gaa act tgc aaa gag gct ggg gtt cag aaa ctc      614 
Thr Lys Asn Val Ile Glu Thr Cys Lys Glu Ala Gly Val Gln Lys Leu 
125                 130                 135                 140 

att tta acc agc agt gcc agt gtc atc ttt gag ggc gtc gat atc aag      662 
Ile Leu Thr Ser Ser Ala Ser Val Ile Phe Glu Gly Val Asp Ile Lys 
                145                 150                 155 

aat gga act gaa gac ctt ccc tat gcc atg aaa ccc att gac tac tac      710 
Asn Gly Thr Glu Asp Leu Pro Tyr Ala Met Lys Pro Ile Asp Tyr Tyr 
            160                 165                 170 

aca gag act aag atc tta cag gag agg gca gtt ctg ggc gcc aac gat      758 
Thr Glu Thr Lys Ile Leu Gln Glu Arg Ala Val Leu Gly Ala Asn Asp 
        175                 180                 185 

cct gag aag aat ttc tta acc aca gcc atc cgc cct cat ggc att ttc      806 
Pro Glu Lys Asn Phe Leu Thr Thr Ala Ile Arg Pro His Gly Ile Phe 
    190                 195                 200 

ggc cca agg gac ccg cag ttg gta ccc atc ctc atc gag gca gcc agg      854 
Gly Pro Arg Asp Pro Gln Leu Val Pro Ile Leu Ile Glu Ala Ala Arg 
205                 210                 215                 220 

aac ggc aag atg aag ttc gtg att gga aat ggg aag aac ttg gtg gac      902 
Asn Gly Lys Met Lys Phe Val Ile Gly Asn Gly Lys Asn Leu Val Asp 
                225                 230                 235 

ttc acc ttt gtg gag aac gtg gtc cat gga cac atc ctg gcg gca gag      950 
Phe Thr Phe Val Glu Asn Val Val His Gly His Ile Leu Ala Ala Glu 
            240                 245                 250 

cag ctc tcc cga gac tcg aca ctg ggt ggg aag gca ttt cac atc acc      998 
Gln Leu Ser Arg Asp Ser Thr Leu Gly Gly Lys Ala Phe His Ile Thr 
        255                 260                 265 

aat gat gag ccc atc cct ttc tgg aca ttc ctg tct cgc atc ctg aca     1046 
Asn Asp Glu Pro Ile Pro Phe Trp Thr Phe Leu Ser Arg Ile Leu Thr 
    270                 275                 280 

ggc ctc aat tat gag gcc ccc aag tac cac atc ccc tac tgg gtg gcc     1094 
Gly Leu Asn Tyr Glu Ala Pro Lys Tyr His Ile Pro Tyr Trp Val Ala 
285                 290                 295                 300 

tac tac ctg gcc ctc ctg cta tcc ctg ctg gtg atg gtg atc agt cct     1142 
Tyr Tyr Leu Ala Leu Leu Leu Ser Leu Leu Val Met Val Ile Ser Pro 
                305                 310                 315 

gtc atc cag ctg cag ccc acc ttc aca ccc atg cgg gtc gca ctg gct     1190 
Val Ile Gln Leu Gln Pro Thr Phe Thr Pro Met Arg Val Ala Leu Ala 
            320                 325                 330 

ggc aca ttc cac tac tac agc tgc gag aga gcc aaa aag gcc atg ggc     1238 
Gly Thr Phe His Tyr Tyr Ser Cys Glu Arg Ala Lys Lys Ala Met Gly 
        335                 340                 345 

tac cag cca cta gtg acc atg gat gat gct atg gag agg acc gtg cag     1286 
Tyr Gln Pro Leu Val Thr Met Asp Asp Ala Met Glu Arg Thr Val Gln 
    350                 355                 360 

agc ttt cgc cac ctg cgg agg gtc aag tgagggacac tggaggctgg           1333 
Ser Phe Arg His Leu Arg Arg Val Lys 
365                 370 

gctctctcga cacgttgctc agccagtcac tccttcccct gtggattgat gaaataacat   1393 

cctttgaatg agtttgctct gagcctgtga ctccttctgc taggcagaga gcgcacccta   1453 

ctctttccgt gacgatgagg gcggcaaaaa cagacatttc ttccttcatg gaactggatt   1513 

tggatttctt gaagcaggca gcttcatatt ataccgattt gttctctgtc              1563 

 
           
             2  
             373  
             PRT  
             Homo sapiens  
           
            2 

Met Glu Pro Ala Val Ser Glu Pro Met Arg Asp Gln Val Ala Arg Thr 
  1               5                  10                  15 

His Leu Thr Glu Asp Thr Pro Lys Val Asn Ala Asp Ile Glu Lys Val 
             20                  25                  30 

Asn Gln Asn Gln Ala Lys Arg Cys Thr Val Ile Gly Gly Ser Gly Phe 
         35                  40                  45 

Leu Gly Gln His Met Val Glu Gln Leu Leu Ala Arg Gly Tyr Ala Val 
     50                  55                  60 

Asn Val Phe Asp Ile Gln Gln Gly Phe Asp Asn Pro Gln Val Arg Phe 
 65                  70                  75                  80 

Phe Leu Gly Asp Leu Cys Ser Arg Gln Asp Leu Tyr Pro Ala Leu Lys 
                 85                  90                  95 

Gly Val Asn Thr Val Phe His Cys Ala Ser Pro Pro Pro Ser Ser Asn 
            100                 105                 110 

Asn Lys Glu Leu Phe Tyr Arg Val Asn Tyr Ile Gly Thr Lys Asn Val 
        115                 120                 125 

Ile Glu Thr Cys Lys Glu Ala Gly Val Gln Lys Leu Ile Leu Thr Ser 
    130                 135                 140 

Ser Ala Ser Val Ile Phe Glu Gly Val Asp Ile Lys Asn Gly Thr Glu 
145                 150                 155                 160 

Asp Leu Pro Tyr Ala Met Lys Pro Ile Asp Tyr Tyr Thr Glu Thr Lys 
                165                 170                 175 

Ile Leu Gln Glu Arg Ala Val Leu Gly Ala Asn Asp Pro Glu Lys Asn 
            180                 185                 190 

Phe Leu Thr Thr Ala Ile Arg Pro His Gly Ile Phe Gly Pro Arg Asp 
        195                 200                 205 

Pro Gln Leu Val Pro Ile Leu Ile Glu Ala Ala Arg Asn Gly Lys Met 
    210                 215                 220 

Lys Phe Val Ile Gly Asn Gly Lys Asn Leu Val Asp Phe Thr Phe Val 
225                 230                 235                 240 

Glu Asn Val Val His Gly His Ile Leu Ala Ala Glu Gln Leu Ser Arg 
                245                 250                 255 

Asp Ser Thr Leu Gly Gly Lys Ala Phe His Ile Thr Asn Asp Glu Pro 
            260                 265                 270 

Ile Pro Phe Trp Thr Phe Leu Ser Arg Ile Leu Thr Gly Leu Asn Tyr 
        275                 280                 285 

Glu Ala Pro Lys Tyr His Ile Pro Tyr Trp Val Ala Tyr Tyr Leu Ala 
    290                 295                 300 

Leu Leu Leu Ser Leu Leu Val Met Val Ile Ser Pro Val Ile Gln Leu 
305                 310                 315                 320 

Gln Pro Thr Phe Thr Pro Met Arg Val Ala Leu Ala Gly Thr Phe His 
                325                 330                 335 

Tyr Tyr Ser Cys Glu Arg Ala Lys Lys Ala Met Gly Tyr Gln Pro Leu 
            340                 345                 350 

Val Thr Met Asp Asp Ala Met Glu Arg Thr Val Gln Ser Phe Arg His 
        355                 360                 365 

Leu Arg Arg Val Lys 
    370 

 
           
             3  
             2218  
             DNA  
             Mus musculus  
             
               CDS  
               (216)..(1301)  
             
           
            3 

gagcgtcaat tggttgcgcc agagccaagc tagaccaatc aacattatga aggaagctct     60 

gctgattgtg ggctcatatc tatcagtaga aaagggtggc gggtgttcag ccagacttct    120 

ctggttgccg gttgtctgca agctgaggtc gatcatttga gtgtctaaac cgggaagaag    180 

agttgattgc aaacgaaacc atactttgag ccata atg gaa caa gct gtt cat       233 
                                       Met Glu Gln Ala Val His 
                                         1               5 

ggt gaa tca aag cga ggc cag gtc aca gga aca cat ttg aca aat gac      281 
Gly Glu Ser Lys Arg Gly Gln Val Thr Gly Thr His Leu Thr Asn Asp 
             10                  15                  20 

att tcc aaa gct aag aag tgc aca gtg att gga ggc tct ggg ttc ctg      329 
Ile Ser Lys Ala Lys Lys Cys Thr Val Ile Gly Gly Ser Gly Phe Leu 
         25                  30                  35 

ggg cag cac atg gtg gag cag ttg ctg gag cga ggc tat act gtc aat      377 
Gly Gln His Met Val Glu Gln Leu Leu Glu Arg Gly Tyr Thr Val Asn 
     40                  45                  50 

gta ttt gat atc cac caa ggc ttt gat aac ccc cgg gtg cag ttc ttt      425 
Val Phe Asp Ile His Gln Gly Phe Asp Asn Pro Arg Val Gln Phe Phe 
 55                  60                  65                  70 

ata ggc gac ctg tgc aac caa cag gac ctg tac cca gct ctc aaa ggt      473 
Ile Gly Asp Leu Cys Asn Gln Gln Asp Leu Tyr Pro Ala Leu Lys Gly 
                 75                  80                  85 

gta agc aca gtt ttc cac tgc gcg tcc cct ccg ccg tac agt aac aac      521 
Val Ser Thr Val Phe His Cys Ala Ser Pro Pro Pro Tyr Ser Asn Asn 
             90                  95                 100 

aag gag ctc ttt tat aga gtg aat ttc att ggc acc aag act gtc att      569 
Lys Glu Leu Phe Tyr Arg Val Asn Phe Ile Gly Thr Lys Thr Val Ile 
        105                 110                 115 

gaa acc tgc aga gag gcc gga gtt cag aaa ctc att tta acc agc agt      617 
Glu Thr Cys Arg Glu Ala Gly Val Gln Lys Leu Ile Leu Thr Ser Ser 
    120                 125                 130 

gcc agt gtt gtc ttt gag ggt gtt gac ata aaa aat gga act gaa gac      665 
Ala Ser Val Val Phe Glu Gly Val Asp Ile Lys Asn Gly Thr Glu Asp 
135                 140                 145                 150 

ctc cct tac gcc atg aag cct att gac tat tac aca gag acc aag atc      713 
Leu Pro Tyr Ala Met Lys Pro Ile Asp Tyr Tyr Thr Glu Thr Lys Ile 
                155                 160                 165 

ttg cag gag aga gca gta ctg gat gcc aac gac cct aag aag aat ttt      761 
Leu Gln Glu Arg Ala Val Leu Asp Ala Asn Asp Pro Lys Lys Asn Phe 
            170                 175                 180 

tta acc gca gcc att cgt cct cat ggc att ttc ggc cca agg gac ccc      809 
Leu Thr Ala Ala Ile Arg Pro His Gly Ile Phe Gly Pro Arg Asp Pro 
        185                 190                 195 

cag ttg gtc cca atc cta att gat gca gct aga aag ggc aaa atg aag      857 
Gln Leu Val Pro Ile Leu Ile Asp Ala Ala Arg Lys Gly Lys Met Lys 
    200                 205                 210 

ttc atg att gga aat ggg gaa aac ctg gtg gac ttc acc ttc gtg gag      905 
Phe Met Ile Gly Asn Gly Glu Asn Leu Val Asp Phe Thr Phe Val Glu 
215                 220                 225                 230 

aat gtg gtt cat gga cac atc tta gcc gct gag cac ctc tcc caa gat      953 
Asn Val Val His Gly His Ile Leu Ala Ala Glu His Leu Ser Gln Asp 
                235                 240                 245 

gca gct cta ggt gga aag gca ttt cac atc acc aac gat gaa cca atc     1001 
Ala Ala Leu Gly Gly Lys Ala Phe His Ile Thr Asn Asp Glu Pro Ile 
            250                 255                 260 

cct ttc tgg acg ttc ctg tcc cgc att ctg aca ggc ctc aat tat gag     1049 
Pro Phe Trp Thr Phe Leu Ser Arg Ile Leu Thr Gly Leu Asn Tyr Glu 
        265                 270                 275 

gcc cct aag tac cac atc ccc tac tgg atg gcc tat tac ctt gct ttc     1097 
Ala Pro Lys Tyr His Ile Pro Tyr Trp Met Ala Tyr Tyr Leu Ala Phe 
    280                 285                 290 

ctg cta tct cta ctg gtg atg gtg gtc agc cct ctc atc caa atc cag     1145 
Leu Leu Ser Leu Leu Val Met Val Val Ser Pro Leu Ile Gln Ile Gln 
295                 300                 305                 310 

cca acc ttt aca cca att cga gtg gca ttg gct gga aca ttc cac tat     1193 
Pro Thr Phe Thr Pro Ile Arg Val Ala Leu Ala Gly Thr Phe His Tyr 
                315                 320                 325 

tac agt tgt gaa aaa gcc aaa aag ctc ttt ggg tac cgg cca ctg gtc     1241 
Tyr Ser Cys Glu Lys Ala Lys Lys Leu Phe Gly Tyr Arg Pro Leu Val 
            330                 335                 340 

acc atg gat gaa gct gtg gaa agg act gtg cag agt ttc cac cac ctg     1289 
Thr Met Asp Glu Ala Val Glu Arg Thr Val Gln Ser Phe His His Leu 
        345                 350                 355 

cgg aag gac aag tgaagttccc cagtgccctc cccagtaact ttctcctctc         1341 
Arg Lys Asp Lys 
    360 

tgttctaaag aacgcatatc cttaactgag tttctcttga cctgtgtgtc ccttgctggt   1401 

tagatggtag catacctgac accctccatg acattggagg tgacaacagg agacatttct   1461 

cccaggatag cattgaactt ctcaggcaga gtcataatct actgcttggg tctttttctc   1521 

tcccccacac ccctacttct gtctcctggg ttcattatca gaaagacagc actaaagtga   1581 

agtctttatc tggggtctta aaaattgaag caaaaccaga aattgtaaac acacagtaag   1641 

ccttcagaca tacattttat atgatcacag tacaatagct caaagtattg atgaatgtaa   1701 

tccccaatcc ttaaggataa atccactgct ggttccttgc ccctcacata ctgtctaggt   1761 

ctctttcaaa gatggttgca gtgtctgcct ctattgtttt tccataaatc atttcaattt   1821 

atccacagac tctggtgtgg tctttattgc tcagtgatgg tgcaatgcaa tggtacagtg   1881 

tgtgctgtgt tagctggtca gctcctaata aacaggagat gatggctgct tggcaataag   1941 

ccaaaaagag tggtctcctt catcgacatg atgtttgagt agaaactcct ccgtgagatg   2001 

gggcagaatg ggttacagac atctttgcag ctcatttcac agaacattat taggccactt   2061 

tccaaagagt acaggccact aacaatccag ccatttctgt tctctgaact cattgaaatg   2121 

tattcttgaa gaattctaag ttcttcttca tcactgttca acagtaagat gattggggga   2181 

aaataacaaa gaaataaaag ccatacagtt tgtgttg                            2218 

 
           
             4  
             362  
             PRT  
             Mus musculus  
           
            4 

Met Glu Gln Ala Val His Gly Glu Ser Lys Arg Gly Gln Val Thr Gly 
  1               5                  10                  15 

Thr His Leu Thr Asn Asp Ile Ser Lys Ala Lys Lys Cys Thr Val Ile 
             20                  25                  30 

Gly Gly Ser Gly Phe Leu Gly Gln His Met Val Glu Gln Leu Leu Glu 
         35                  40                  45 

Arg Gly Tyr Thr Val Asn Val Phe Asp Ile His Gln Gly Phe Asp Asn 
     50                  55                  60 

Pro Arg Val Gln Phe Phe Ile Gly Asp Leu Cys Asn Gln Gln Asp Leu 
 65                  70                  75                  80 

Tyr Pro Ala Leu Lys Gly Val Ser Thr Val Phe His Cys Ala Ser Pro 
                 85                  90                  95 

Pro Pro Tyr Ser Asn Asn Lys Glu Leu Phe Tyr Arg Val Asn Phe Ile 
            100                 105                 110 

Gly Thr Lys Thr Val Ile Glu Thr Cys Arg Glu Ala Gly Val Gln Lys 
        115                 120                 125 

Leu Ile Leu Thr Ser Ser Ala Ser Val Val Phe Glu Gly Val Asp Ile 
    130                 135                 140 

Lys Asn Gly Thr Glu Asp Leu Pro Tyr Ala Met Lys Pro Ile Asp Tyr 
145                 150                 155                 160 

Tyr Thr Glu Thr Lys Ile Leu Gln Glu Arg Ala Val Leu Asp Ala Asn 
                165                 170                 175 

Asp Pro Lys Lys Asn Phe Leu Thr Ala Ala Ile Arg Pro His Gly Ile 
            180                 185                 190 

Phe Gly Pro Arg Asp Pro Gln Leu Val Pro Ile Leu Ile Asp Ala Ala 
        195                 200                 205 

Arg Lys Gly Lys Met Lys Phe Met Ile Gly Asn Gly Glu Asn Leu Val 
    210                 215                 220 

Asp Phe Thr Phe Val Glu Asn Val Val His Gly His Ile Leu Ala Ala 
225                 230                 235                 240 

Glu His Leu Ser Gln Asp Ala Ala Leu Gly Gly Lys Ala Phe His Ile 
                245                 250                 255 

Thr Asn Asp Glu Pro Ile Pro Phe Trp Thr Phe Leu Ser Arg Ile Leu 
            260                 265                 270 

Thr Gly Leu Asn Tyr Glu Ala Pro Lys Tyr His Ile Pro Tyr Trp Met 
        275                 280                 285 

Ala Tyr Tyr Leu Ala Phe Leu Leu Ser Leu Leu Val Met Val Val Ser 
    290                 295                 300 

Pro Leu Ile Gln Ile Gln Pro Thr Phe Thr Pro Ile Arg Val Ala Leu 
305                 310                 315                 320 

Ala Gly Thr Phe His Tyr Tyr Ser Cys Glu Lys Ala Lys Lys Leu Phe 
                325                 330                 335 

Gly Tyr Arg Pro Leu Val Thr Met Asp Glu Ala Val Glu Arg Thr Val 
            340                 345                 350 

Gln Ser Phe His His Leu Arg Lys Asp Lys 
        355                 360 

 
           
             5  
             1751  
             DNA  
             Homo sapiens  
             
               CDS  
               (27)..(908)  
             
           
            5 

gcgagatgac tgcagagatt tgaaaa atg gca aca aat gaa agt gtc agc atc      53 
                             Met Ala Thr Asn Glu Ser Val Ser Ile 
                               1               5 

ttt agt tca gca tcc ttg gct gtg gaa tat gta gat tca ctt tta cct      101 
Phe Ser Ser Ala Ser Leu Ala Val Glu Tyr Val Asp Ser Leu Leu Pro 
 10                  15                  20                  25 

gag aat cct ctg caa gaa cca ttt aaa aat gct tgg aac tat atg ttg      149 
Glu Asn Pro Leu Gln Glu Pro Phe Lys Asn Ala Trp Asn Tyr Met Leu 
                 30                  35                  40 

aat aat tat aca aag ttc cag att gca aca tgg gga tcc ctt ata gtt      197 
Asn Asn Tyr Thr Lys Phe Gln Ile Ala Thr Trp Gly Ser Leu Ile Val 
             45                  50                  55 

cat gaa gcc ctt tat ttc tta ttc tgt tta cct gga ttt tta ttt caa      245 
His Glu Ala Leu Tyr Phe Leu Phe Cys Leu Pro Gly Phe Leu Phe Gln 
         60                  65                  70 

ttt ata cct tat atg aaa aaa tac aaa att caa aag gat aag cca gag      293 
Phe Ile Pro Tyr Met Lys Lys Tyr Lys Ile Gln Lys Asp Lys Pro Glu 
     75                  80                  85 

aca tgg gaa aac caa tgg aag tgt ttc aaa gtt ctt ctc ttt aat cac      341 
Thr Trp Glu Asn Gln Trp Lys Cys Phe Lys Val Leu Leu Phe Asn His 
 90                  95                 100                 105 

ttc tgt atc cag ctg cct ttg att tgt gga acc tat tat ttt aca gag      389 
Phe Cys Ile Gln Leu Pro Leu Ile Cys Gly Thr Tyr Tyr Phe Thr Glu 
                110                 115                 120 

tat ttc aat att cct tat gat tgg gaa aga atg cca aga tgg tat ttt      437 
Tyr Phe Asn Ile Pro Tyr Asp Trp Glu Arg Met Pro Arg Trp Tyr Phe 
            125                 130                 135 

ctt ttg gca aga tgc ttt ggt tgt gca gtc att gaa gat act tgg cac      485 
Leu Leu Ala Arg Cys Phe Gly Cys Ala Val Ile Glu Asp Thr Trp His 
        140                 145                 150 

tat ttt ctg cat aga ctc tta cac cac aaa aga ata tac aag tat att      533 
Tyr Phe Leu His Arg Leu Leu His His Lys Arg Ile Tyr Lys Tyr Ile 
    155                 160                 165 

cat aaa gtt cat cat gag ttt cag gct cca ttt gga atg gaa gct gaa      581 
His Lys Val His His Glu Phe Gln Ala Pro Phe Gly Met Glu Ala Glu 
170                 175                 180                 185 

tat gca cat cct ttg gag act cta att ctt gga act gga ttt ttc att      629 
Tyr Ala His Pro Leu Glu Thr Leu Ile Leu Gly Thr Gly Phe Phe Ile 
                190                 195                 200 

gga atc gtg ctt ttg tgt gat cat gta att ctt ctt tgg gca tgg gtg      677 
Gly Ile Val Leu Leu Cys Asp His Val Ile Leu Leu Trp Ala Trp Val 
            205                 210                 215 

acc att cgt tta tta gaa act att gat gtc cat agt ggt tat gat att      725 
Thr Ile Arg Leu Leu Glu Thr Ile Asp Val His Ser Gly Tyr Asp Ile 
        220                 225                 230 

cct ctc aac cct tta aat ctg atc cct ttc tat gct ggt tct cgg cat      773 
Pro Leu Asn Pro Leu Asn Leu Ile Pro Phe Tyr Ala Gly Ser Arg His 
    235                 240                 245 

cat gat ttc cac cac atg aac ttc att gga aac tat gct tca aca ttt      821 
His Asp Phe His His Met Asn Phe Ile Gly Asn Tyr Ala Ser Thr Phe 
250                 255                 260                 265 

aca tgg tgg gat cga att ttt gga aca gac tct cag tat aat gcc tat      869 
Thr Trp Trp Asp Arg Ile Phe Gly Thr Asp Ser Gln Tyr Asn Ala Tyr 
                270                 275                 280 

aat gaa aag agg aag aag ttt gag aaa aag act gaa taa atatctcacg       918 
Asn Glu Lys Arg Lys Lys Phe Glu Lys Lys Thr Glu 
            285                 290 

taaaccttcc tgaaagataa acgttttcct gaattcagaa actagtagct aacattgctt    978 

ctggagagca gaaataagca tgtcttctgg ctactaagtg ataaaaagaa cattaacaac   1038 

ctttaattac cttcctagtg ggaacttttt ctactttacc tacaagttct atatatgtag   1098 

aaatgaataa atatatattt aagtacagtt ttcatgagga agttttaaaa gaccatgttc   1158 

ctaagcttcc aagaaggttt tggatactag aagtattaat ctatggcttt tctcccagta   1218 

aaaccatagg cctgaagttc acattgggtc tttaaatctt ttagatatat actggtcatt   1278 

tcagaaaatt cttcatagtg gtattggcct tatatttaac ttttttttta tttttttttt   1338 

gagacaaagc cacactctgt ctccttgtct ggagtgtggt ggcacagtct cagctcactg   1398 

caacctctgc ctcccagttc aagcaattct tctgcctcag cctcccaagt agctgggatt   1458 

acaggcaccc gccaccacgc ccagctaatt tttgtatttt tgtagagatg gggtttctcg   1518 

atgttggcca ggctggtctc aaacttctga cctcaagtga tctgcccacc ttggcctccc   1578 

aaagtgctgg gattacaggt gtaagccact gcgcccggcc tttttaactt taaacatgtt   1638 

ttagaattca cctaaagatc aaaatatcat ggattgaacc tcatcaattg atagcagtga   1698 

gtgactgaag cttccaaatc aagaaaagcc ggcaccaaga acttccattc taa          1751 

 
           
             6  
             293  
             PRT  
             Homo sapiens  
           
            6 

Met Ala Thr Asn Glu Ser Val Ser Ile Phe Ser Ser Ala Ser Leu Ala 
  1               5                  10                  15 

Val Glu Tyr Val Asp Ser Leu Leu Pro Glu Asn Pro Leu Gln Glu Pro 
             20                  25                  30 

Phe Lys Asn Ala Trp Asn Tyr Met Leu Asn Asn Tyr Thr Lys Phe Gln 
         35                  40                  45 

Ile Ala Thr Trp Gly Ser Leu Ile Val His Glu Ala Leu Tyr Phe Leu 
     50                  55                  60 

Phe Cys Leu Pro Gly Phe Leu Phe Gln Phe Ile Pro Tyr Met Lys Lys 
 65                  70                  75                  80 

Tyr Lys Ile Gln Lys Asp Lys Pro Glu Thr Trp Glu Asn Gln Trp Lys 
                 85                  90                  95 

Cys Phe Lys Val Leu Leu Phe Asn His Phe Cys Ile Gln Leu Pro Leu 
            100                 105                 110 

Ile Cys Gly Thr Tyr Tyr Phe Thr Glu Tyr Phe Asn Ile Pro Tyr Asp 
        115                 120                 125 

Trp Glu Arg Met Pro Arg Trp Tyr Phe Leu Leu Ala Arg Cys Phe Gly 
    130                 135                 140 

Cys Ala Val Ile Glu Asp Thr Trp His Tyr Phe Leu His Arg Leu Leu 
145                 150                 155                 160 

His His Lys Arg Ile Tyr Lys Tyr Ile His Lys Val His His Glu Phe 
                165                 170                 175 

Gln Ala Pro Phe Gly Met Glu Ala Glu Tyr Ala His Pro Leu Glu Thr 
            180                 185                 190 

Leu Ile Leu Gly Thr Gly Phe Phe Ile Gly Ile Val Leu Leu Cys Asp 
        195                 200                 205 

His Val Ile Leu Leu Trp Ala Trp Val Thr Ile Arg Leu Leu Glu Thr 
    210                 215                 220 

Ile Asp Val His Ser Gly Tyr Asp Ile Pro Leu Asn Pro Leu Asn Leu 
225                 230                 235                 240 

Ile Pro Phe Tyr Ala Gly Ser Arg His His Asp Phe His His Met Asn 
                245                 250                 255 

Phe Ile Gly Asn Tyr Ala Ser Thr Phe Thr Trp Trp Asp Arg Ile Phe 
            260                 265                 270 

Gly Thr Asp Ser Gln Tyr Asn Ala Tyr Asn Glu Lys Arg Lys Lys Phe 
        275                 280                 285 

Glu Lys Lys Thr Glu 
    290 

 
           
             7  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence CH-F primer  
             
           
            7 

aaagactggt gcgctaaagc                                                 20 

 
           
             8  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence CH-R primer  
             
           
            8 

cgagcttcct ccaccaagag                                                 20 

 
           
             9  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence hH105-1F 
      primer  
             
           
            9 

ccccgtcttt attgggcaag                                                 20 

 
           
             10  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence hH105-1R 
      primer  
             
           
            10 

actgcccagt cgctgacaca g                                               21 

 
           
             11  
             22  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence hH105-2F 
      primer  
             
           
            11 

ggcatctgcc caaaacacta ac                                              22 

 
           
             12  
             22  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence hH105-2R 
      primer  
             
           
            12 

ccacaggtaa atagtatcag cc                                              22 

 
           
             13  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence hH105-3F 
      primer  
             
           
            13 

ttccagtcct cactaccctg                                                 20 

 
           
             14  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence hH105-3R 
      primer  
             
           
            14 

agtatcgtgg tttcccttcg                                                 20 

 
           
             15  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence hH105-4F 
      primer  
             
           
            15 

tgccattgac ctgtcaaagc                                                 20 

 
           
             16  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence hH105-4R 
      primer  
             
           
            16 

cccttagaaa gggccatcac                                                 20 

 
           
             17  
             22  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence hH105-5F 
      primer  
             
           
            17 

ggatcatgca ctgtttgaat tg                                              22 

 
           
             18  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence hH105-5R 
      primer  
             
           
            18 

ggattctaaa cccttcagtc                                                 20 

 
           
             19  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence hH105-6F 
      primer  
             
           
            19 

ctaggaattt gcaatggacg                                                 20 

 
           
             20  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence hH105-6R 
      primer  
             
           
            20 

tgaatgcgag catggaccag                                                 20 

 
           
             21  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence hH105-7F 
      primer  
             
           
            21 

aagacttggg agtggccctg                                                 20 

 
           
             22  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence hH105-7R 
      primer  
             
           
            22 

aggcaaggag aagaaacccg                                                 20 

 
           
             23  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence hH105-8F1 
      primer  
             
           
            23 

ttcaactttg ggcaggtggg                                                 20 

 
           
             24  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence hH105-8R1 
      primer  
             
           
            24 

ctccatagca tcatccatgg                                                 20 

 
           
             25  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence hH105-8F2 
      primer  
             
           
            25 

cattccacta ctacagctgc                                                 20 

 
           
             26  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence hH105-8R2 
      primer  
             
           
            26 

tgtataaacc agaagagggg                                                 20 

 
           
             27  
             18  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence forward 
      primer  
             
           
            27 

acgtggtcca tggacaca                                                   18 

 
           
             28  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence reverse 
      primer  
             
           
            28 

catccatggt cactagtggc                                                 20