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WO1994028128A1 - Essai et modele destines a la polykystose ovarienne et a l'alopecie regionale masculine - Google Patents

Essai et modele destines a la polykystose ovarienne et a l'alopecie regionale masculine Download PDF

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Publication number
WO1994028128A1
WO1994028128A1 PCT/GB1994/001142 GB9401142W WO9428128A1 WO 1994028128 A1 WO1994028128 A1 WO 1994028128A1 GB 9401142 W GB9401142 W GB 9401142W WO 9428128 A1 WO9428128 A1 WO 9428128A1
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WIPO (PCT)
Prior art keywords
pco
cyp17
mpb
gene
mutation
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Ceased
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PCT/GB1994/001142
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English (en)
Inventor
Stephen Franks
Robert Williamson
Adam Henry Carey
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Imperial College of London
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Imperial College of London
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Publication date
Application filed by Imperial College of London filed Critical Imperial College of London
Priority to AU68008/94A priority Critical patent/AU6800894A/en
Priority to EP94916302A priority patent/EP0705336A1/fr
Publication of WO1994028128A1 publication Critical patent/WO1994028128A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)

Definitions

  • This invention is based on the identification of a genetic cause of polycystic ovary syndrome and male pattern baldness and relates to the application of that discovery for diagnostic, therapeutic and modelling purposes.
  • PCOS Polycystic ovary syndrome
  • chat PCO has an underlying genetic cause and that the male phenotype of the disorder is male pattern baldness (MPB) . It has also been discovered that a polymorphism in the gene CYP17 demonstrates an association with the disease. The association of the mutation with PCO and/cr MPB is short of complete segregation, which suggests that CYP17 is one of the major modulating genes for polycystic ovaries and male pattern baldness, but is not causative alone.
  • the CYP17 gene also designated P450XVIIA1
  • the gene was cloned and sequenced by Picado- Leonard & Miller (DNA 6(5) 439-448 (1987)), whose numbering system, as laid out in Figure 3 of their paper, is adopted in this specification.
  • ?450cl7 acts as a 17 ⁇ .-hydroxyiase on pregnenolone and progesterone to form 170H-pregnenolone and 170H- progesterone, respectively. It further acts, as a 17,20 lyase, en 170H-pregnenolone and 170H-progesterone to form DHEA and androstenedione. P450cl7 is believed to be the rate-limiting enzyme in androgen biosynthesis.
  • the invention generally provides model systems of polycystic ovary syndrome (PCO) and/or male pattern baldness (MPB) and DNA molecules which are useful in such model systems and/or in diagnosis or genetic therapy; the model systems and DNA molecules comprise a DNA sequence including at least part of the CYP17 gene which includes a mutation leading to increased androgen biosynthesis.
  • PCO polycystic ovary syndrome
  • MPB male pattern baldness
  • an isolated cr recombinant DNA encoding a mutant CYP17 allele which is associated with polycystic ovary syndrome (PCO) and/or male pattern baldness (MPB) .
  • PCO polycystic ovary syndrome
  • MPB male pattern baldness
  • the mutation may be in the protein-coding region of the CYP17 gene and may be responsible for increased P450cl7 enzymic activity.
  • the mutation may be in the promoter region, which is untranslated and lies 5' to the protein-coding region. Such mutations may lead to increased expression of the gene product.
  • the DNA sequence includes at least part of the 5' untranslated region of the CYP17 gene in which T is replaced by C at position -34 from the translation initiation site. The T ⁇ C change results in the generation of an SP-1 type site and is believed to up-regulate the gene by this means. Consequently, the mutation results in increased androgen biosynthesis.
  • the DNA sequence may include the entire functional promoter, in some embodiments preferably with the protein-coding region. cDNAs and genomic DNAs are encompassed within the scope of the invention.
  • a particular embodiment of the invention relates to an isolated or recombinant DNA including a mutant CYP17 allele having a T ⁇ C mutation at position -34.
  • Recombinant DNA in accordance with the invention may include a mutant CYP17 promoter operatively coupled to a gene (a "reporter gene") to which the promoter is not normally operatively coupled and which encodes a detectable protein (a "reporter”) , which may be an enzyme catalysing a detectable change, for the purpose of studying the promoter.
  • a reporter gene operatively coupled to a gene to which the promoter is not normally operatively coupled and which encodes a detectable protein (a "reporter”) , which may be an enzyme catalysing a detectable change, for the purpose of studying the promoter.
  • Recombinant DNA of the invention may be in the form of a vector, such as a plasmid, cosmid, phagemid or virus, which may be used to transform or transfect a host cell.
  • a vector such as a plasmid, cosmid, phagemid or virus, which may be used to transform or transfect a host cell.
  • Vectors including a promoter operatively coupled to protein-coding DNA may be useful as expression vectors; vectors unable to express protein-encoding DNA may still be useful as cloning vectors.
  • a host cell transformed or transfected with a vector as described above.
  • the host cell may be prokaryotic or eukaryotic.
  • a suitable prokaryotic host may be Escherichia coli .
  • Suitable eukaryotic hosts include yeast (such as Saccharomyces cerevi ⁇ iae) , insect cells (such as ⁇ podoptera frugiperda) and mammalian cells, which may be primary cells or an immortalised cell line.
  • Recombinant or isolated DNA in accordance with the invention may be in the form of a construct suitable for generating a transgenic animal.
  • transgenic animals of the invention therefore include those having a transgene encoding a mutant CYP17 allele which is associated with PCO and/or MPB.
  • Such transgenic animals may be of any desired species and may be prepared by methods known in the art. For mice, the standard Hogan microinjection protocol (Hogan et al., "Manipulating the Mouse Embryo: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA (1986)) may be used.
  • Transformed or transfected cells and transgenic animals in accordance with the invention are useful not only for studying the genetic basis of PCO/MPB and its associated biochemistry but also for screening for drugs and evaluating drug effectiveness.
  • a cell or animal in a screen for a compound potentially useful in the treatment or prophylaxis of PCO and/or MPB.
  • a compound under test may be provided or administered to the cell or animal; androgen biosynthesis, particularly as mediated by ?450cl7, may be monitored.
  • the invention also has application in determining a genetic predisposition to PCO and/or MPB and other genetic analysis.
  • a method of genetic analysis particularly for the purpose of . determining a genetic predisposition of a subject to PCO and/or MPB, the method comprising detecting in the subject's DNA the presence of a CYP17 allele. Detection, which may involve DNA sequencing, may be carried out on material removed from, and not returned to, the subject's body. Detection may alternatively or additionally involve the use of probes, in which case oligo- or poly-nucleotides as described below may be useful.
  • a oligo- or poly-nucleotide specifically hybridising to a mutant CYP17 allele having a T ⁇ C mutation at position -34.
  • the hybridisation conditions will be sufficiently stringent to result in specific hybridisation.
  • the oligo- or poly-nucleotide which may be at least 10, 15, 20 or 25 nucleotides in length, in increasing order of preference, may be labelled with a detectable label, such as a radioisotope. However, that is not essential.
  • the oligo- or polynucleotide may be used as a primer in a polymerase chain reaction-based detection method.
  • the fact that the T ⁇ C mutation at position -34 results in a Msp l restriction site means that, for this specific mutation at least, the PCR reaction product may be analysed by restriction enzyme digestion.
  • Such a detection method could also be used in a PCR-based method which used primers based on sequences nearby the site of the mutation, rather than taken from the mutation site itself.
  • gene therapy in the form of gene implants, to prevent the development of PCO/MPB.
  • a variety of ways may be used to restore normal, or at least acceptable, levels of CYP17 expression.
  • a normal CYP17 gene or promoter may form the basis of a gene implant; alternatively, the production of P450cl7 by a mutant CYP17 gene, or the activity of the P450cl7 so produced, may be modulated.
  • Embodiments directed to modulation of the production of P450cl7 include methods that employ specific antisense polynucleotides complementary to all or part of a variant CYP17 gene or, more specifically, promoter.
  • Such complementary antisense polynucleotides may include nucleotide substitutions, additions, deletions or transpositions, so long as specific hybridisation to the relevant target sequence, which in preferred embodiments will be in the region of position -34, is retained as a property of the polynucleotide.
  • An antisense polynucleotide should preferentially bind to a variant CYP17 gene sequence as opposed to wild type CYP17.
  • EXAMPLE 1 Evidence for a Single Gene Effect causing Polycystic Ovary Syndrome and Male Pattern Baldness Patients were recruited prospectively and consecutively from the endocrine and infertility clinics at the Samaritan and St. Mary's Hospitals, London (probands) . Each proband had been diagnosed as having polycystic ovaries (PCO) by pelvic ultrasound scan and was known to have at least one sister, whether or not she was thought to be affected.
  • PCO polycystic ovaries
  • the ultrasound scan was performed using an ACUSON 128 ultrasound machine and the ovarian morphology defined as previously described (Adams et al , The Lancet (ii):1375-78 (1985), Adams et al, Bri tish Medical Journal 293:355-59 (1986)) .
  • the probands were of several racial and national origins which were noted, and presented with a variety of symptoms.
  • Nine of the 14 women had irregular menses (four of these were also hirsute) four presented with hirsutis but regular cycles and one had a history of primary infertility.
  • PCOS menstrual irregularity (prior to commencing a combined oral contraceptive pill) and/or unwanted hair, with or without acne.
  • polycystic ovary is an autosomal dominantly inherited disorder with a male phenotype of male pattern baldness.
  • the underlying biochemical abnormality may affect the regulation of the enzyme P450c 17 ⁇ , which catalyses the rate limiting step in androgen biosynthesis in the ovary and adrenal.
  • This enzyme is coded for by the gene CYP17.
  • a YAC clone containing CYP17 has been isolated; its presence was confirmed by restriction mapping and sequencing of the PCR products from exons two and five. Purified YAC DNA was used as a template for Alu PCR, the products of which were labelled to map the human CYP17 gene to chromosome 10q24 - 25 by fluorescent in si tu hybridisation.
  • markers covering the region 10q23 - 26 were used to identify linkage with the disease locus. These markers are PCR based polymorphic loci containing short tracts of
  • the PCR for each marker were conducted in a total reaction volume of 25 ⁇ l containing, 20ng of human DNA, 50pmol of each primer, CA BIO reaction buffer with lOO ⁇ M of each ⁇ P 32 -dATP/dCTP/dGTP/dTTP (Pharmacia) and 1 unit of Tag polymerase (Cambio) .
  • the reaction was performed in an automated thermal cycler (Perkin-Elmer/Cetus) with 35 cycles of denaturing at 94°C (1 min) , annealing at 56°C (1 min) and extension of 72°C (1 min) .
  • An initial denaturing step of 10 mins at 94°C was employed with a final extension at 72°C for 10 mins.
  • a polymorphism in which a T is replaced by a C, was found to exist within the 5' transcribed region of the cypl7 gene at -34bp from the printed sequence (Picado-Lenard S. Miller, DNA 6(5) 439- 448 (1987) ) .
  • This base change creates a recognition site for the restriction enzyme MspKL (NEB) .
  • a PCR fragment of 459bps containing the polymorphism is generated using the following primers:
  • PCR Polymerase chain reaction
  • the gel was subsequently stained with ethidium bromide (lOO ⁇ ls of lOmg/ml Sigma) in lxTBE (0.9M tris, 0.9M boric acid, 0.5M EDTA) for 30 minutes and then destained in lxTBE for 10 minutes.
  • ethidium bromide lOO ⁇ ls of lOmg/ml Sigma
  • alleies homozygote affected individuals
  • fragments will be generated of 124 and 335bp.
  • Heterozygous individuals for this mutation will have all three fragments present; 459, 335 and 124bp.
  • a homozygous unaffected individual would only demonstrate the uncut product of 459bp.
  • EXAMPLE 5 Genetic Segregation Analysis A two point analysis shows that five families generate a positive lod score of 1.8, twelve families generate a negative lod score of -2.65 (ie, exclusion) and five are uninformative. Within the five families generating the positive lod score, the mutation segregation is for the most part consistent. This suggests:
  • the mutation in CYP17 is associated with PCO/MPB; (2) for a number of families, this mutation may be causative; and
  • PCO/MPB may be caused by CYP17 mutations alone in some families or individuals, by the effect of the mutation causing an up-regulation in the rate limiting step in androgen biosynthesis. It is likely that one or two other genes may be causally associated with PCO/MPB. These other genes may be involved in androgen synthesis or may affect insulin levels or action.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

On a découvert que la polykystose ovarienne (PCO) est due à une cause génetique sous-jacente, en outre, chez les hommes, il a été démontré que le phénotype de ce trouble est l'alopécie régionale masculine (MPB). Une mutation du gène CYP17, qui code l'enzyme P450c17, présente une association avec le phénotype: la mutation comprend le remplacement d'un T par un C dans la région transcrite en position 5' du gène. Des systèmes d'ADN recombinant, permettant la modélisation, le diagnostic et le traitement de la maladie ont été développés en fonction de ces découvertes.
PCT/GB1994/001142 1993-05-26 1994-05-26 Essai et modele destines a la polykystose ovarienne et a l'alopecie regionale masculine Ceased WO1994028128A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU68008/94A AU6800894A (en) 1993-05-26 1994-05-26 Test and model for polycystic ovary syndrome and male pattern baldness
EP94916302A EP0705336A1 (fr) 1993-05-26 1994-05-26 Essai et modele destines a la polykystose ovarienne et a l'alopecie regionale masculine

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9310842.1 1993-05-26
GB939310842A GB9310842D0 (en) 1993-05-26 1993-05-26 Gene identification

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WO1994028128A1 true WO1994028128A1 (fr) 1994-12-08

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PCT/GB1994/001142 Ceased WO1994028128A1 (fr) 1993-05-26 1994-05-26 Essai et modele destines a la polykystose ovarienne et a l'alopecie regionale masculine

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EP (1) EP0705336A1 (fr)
AU (1) AU6800894A (fr)
GB (1) GB9310842D0 (fr)
WO (1) WO1994028128A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU703139B2 (en) * 1995-02-09 1999-03-18 Icn Pharmaceuticals, Inc. Methods and compositions for regulation of CD28 expression
WO2001081628A1 (fr) * 2000-04-25 2001-11-01 Lifespan Biosciences Inc Sequences d'acides nucleiques associees a la calvitie

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108004313B (zh) * 2017-12-20 2021-03-16 新疆医科大学第一附属医院 早发冠心病致病基因及其体外检测的试剂、制剂或试剂盒和应用

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
A.H. CAREY ET AL.;: "Evidence for a single gene effect causing polycystic ovaries and male pattern baldness", CLIN. ENDOCRINOL., vol. 38, 1993, pages 653 - 658 *
D. FERRIMAN AND A.W. PURDIE: "The inheritance of polycystic ovarian disease and a possible relationship to premature balding", CLIN. ENDOCRINOL., vol. 11, 1979, pages 291 - 300 *
J. PICADO-LEONARD AND W.L. MILLER: "Cloning and sequencing of the human gene for P450c17 (steroid 17-alpha-hydroxylase/ 17,20 lyase): similarity with the gene for P450c21", DNA, vol. 6, 1987, pages 439 - 448 *
R.L. ROSENFIELD ET AL.;: "Dysregulation of cytochrome P450c17alpha as the cause of polycystic ovarian syndrome", FERTIL. STERIL, vol. 53, 1990, pages 785 - 791 *
T. YANASE ET AL.;: "Combined 17alpha-hydroxylase/17,20-lyase deficiency due to a 7-basepair duplication in the N-terminal region of the cytochrome P450-17alpha (CYP17) gene", J. CLIN. ENDOCRINOL. METAB., vol. 70, 1990, pages 1325 - 1329 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU703139B2 (en) * 1995-02-09 1999-03-18 Icn Pharmaceuticals, Inc. Methods and compositions for regulation of CD28 expression
WO2001081628A1 (fr) * 2000-04-25 2001-11-01 Lifespan Biosciences Inc Sequences d'acides nucleiques associees a la calvitie

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GB9310842D0 (en) 1993-07-14
EP0705336A1 (fr) 1996-04-10
AU6800894A (en) 1994-12-20

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