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Characterizing single gene disorders

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Title: Characterizing single gene disorders


1
Characterizing single gene disorders
  • Specifics of cloning disease genes
  • positional cloning and CF
  • Duchenne muscular dystrophy
  • Fragile X gene
  • Huntington gene
  • Model organisms
  • XREFdb
  • mouse models
  • information to be gathered from genome analysis

2
Positional cloning
  • Cloning can be done with no information about the
    actual gene product except its approximate gene
    location
  • Very time consuming- by 95 only 50 genes had
    been cloned this way
  • Genes cloned this way all followed similar
    approaches-
  • first mapping disease in affected families
  • Either detect chromosomal rearrangements or use
    markers
  • need to find a marker for which a particular
    allele and the occurrence of the disease are
    co-inherited in a family
  • important to note that between different
    families the marker alleles will not be the same
  • This is because the mutation occurred long ago on
    a particular chromosome and each time the
    mutation occurs spontaneously, it happens on a
    different chromosome (ie. A chromosome with
    different polymorphisms and therefore different
    alleles at marker loci)
  • identifying novel candidate gene and show that
    patients had mutations in that gene

3
Position-independent cloning
  • possible if information about protein product,
    DNA sequence or function is known
  • Partial amino acid sequence can be used to
    predict DNA sequence
  • synthetic oligo can be used to hybridize to
    screen cDNA libraries
  • Or - can raise an antibody against the protein
  • Then screen a cDNA expression library
  • cDNA library in which the inserts are transcribed
    and translated and the protein can be detected
    with an antibody
  • But these cases are rarer in human genetics-
    usually positional cloning is used.

4
Positionally-Cloned Genes Mutated in Human
Disease in the EST division of GenBank
  • Human Disease MIM
    Gene GenBank Exact GenBank

  • Symbol Accession Match(es)
    Accession
  • for cDNA in dbEST for EST
  • Aarskog-Scott syndrome 305400 FGD1 U11690
    Yes F06587
  • Achondroplasia 100800 FGFR3 M58051
    Yes R85021
  • Adenomatous Polyposis Col 175100 APC M74088
    Yes H29191
  • Adrenoleukodystrophy
    300100 ALD Z21876 Yes D31532
  • Alagille Syndrome 118450 JAG1 AF00383
    7 Yes AA046860
  • Alzheimer Disease, type 3 104311 PS1 L76517
    Yes AA533888
  • Alzheimer Disease, type 4 600759 PS2 L44577
    Yes AA602396
  • Ataxia Telangiectasia 208900 ATM U26455
    Yes H43382
  • Barth Syndrome
    302060 BTHS X92762 Yes Z39302

5
Chromosome walks
  • start with a probe detecting a linked marker
  • probe a gene library containing overlapping
    clones
  • carry out a series of southern blots with probes
    derived from each new piece of walk
  • walks often halted due to gaps

6
Chromosome jumps
  • technique for bridging gaps and covering larger
    distances
  • use rare cutters like NotI (sites every 500 kb)
  • linker DNA with a nonsense suppressor ligated to
    NotI ends
  • cut with EcoR1 and ligate into a ? vector
    containing nonsense mutation in essential gene
  • select plaques that form on E. coli host (only
    occurs if linkers present)

7
CF cloning, 1989
  • Lap-Chee Tsui John Riordan (Sick Kids, Toronto)
    and Francis Collins (Michigan)
  • Cystic fibrosis represents the first genetic
    disorder elucidated strictly by the process of
    reverse genetics (later called positional
    cloning), i.e., on the basis of map location but
    without the availability of chromosomal
    rearrangements or deletions
  • By use of a combination of chromosome walking and
    jumping, Rommens et al. (1989) succeeded in
    covering the CF region on 7q.
  • The jumping technique was particularly useful in
    bypassing 'unclonable' regions, which are
    estimated to constitute 5 of the human genome.
  • one clue- the identification of undermethylated
    CpG islands
  • another clue- screening of a cDNA library
    constructed from cultured sweat gland cells of a
    non-CF individual.
  • The CF gene proved to be about 250,000 bp long, a
    surprising finding since the absence of apparent
    genomic rearrangements in CF chromosomes and the
    evidence of a limited number of CF mutations
    predicted a small mutational target.

8
Positional cloning to map CF gene
- found linkage between CF and 2 RFLPs- MET and
D7S8 - then found closer markers DS340/DS122
380 kb SalI fragment
9
CFTR gene
  • extremely large gene- covers 230 kb, encodes 6.5
    kb transcript
  • gene expressed in tissues affected by CF- lungs,
    pancreas, sweat glands, liver and others
  • based on clinical phenotypes- prediction was that
    the gene was involved in ion transport
  • CFTR protein- 1480 AA
  • similar to ABC superfamily of membrane
    transporters
  • responsible for pumping ions in and out of cells

10
CF mutations
  • over 550 mutations have been documented
  • Kerem et al. (1989) found that approximately 70
    of the mutations in CF patients correspond to a
    specific deletion of 3 basepairs, which results
    in the loss of a phenylalanine residue at amino
    acid position 508 of the putative product of the
    CF gene (?F508).
  • speculated to have arisen in one of the first
    modern human living in Europe 50,000 years ago
  • Haplotype data based on DNA markers closely
    linked to the putative disease gene locus
    suggested that the remainder of the CF mutant
    gene pool consists of multiple, different
    mutations.

11
CF heterozygosity
  • proposed that in heterozygous state mutations of
    the CFTR gene provide increased resistance to
    infectious diseases, thereby maintaining mutant
    CFTR alleles at high levels in selected
    populations (northern Europeans).
  • Pier et al. (1998) investigated whether typhoid
    fever could be one such disease. This disease is
    initiated when Salmonella typhi enters
    gastrointestinal epithelial cells for submucosal
    translocation. They found that S. typhi uses CFTR
    for entry into epithelial cells. Cells expressing
    wildtype CFTR internalized more S. typhi than
    isogenic cells expressing the most common CFTR
    mutation, ?F508.
  • Heterozygous ?F508 Cftr mice translocated 86
    fewer S. typhi into the gastrointestinal
    submucosa than did wildtype Cftr mice no
    translocation occurred in ?F508 Cftr homozygous
    mice. Pier et al. (1998) concluded that
    diminished levels of CFTR in heterozygotes
    decreases susceptibility to typhoid fever.

12
DMD cloning 1987
  • Cloning approached from various ways
  • all visible cytogenetic rearrangements causing
    DMD have a breakpoint in Xp21
  • many DMD males carried small deletions that
    always spanned Xp21.2
  • linkage data mapped a marker within 8 map units
    of DMD locus

13
DMD cloning
- affected females with balanced translocations-
break at Xp21.2 - X inactivation favours survival
of X/A chromosome - predominant inactivation of
the normal X chromosome in a twin with DMD
14
DMD-cloning of translocation breakpoint
  • Wortons group in Toronto
  • one X21 translocation in an affected female
  • 21p contains many repeated rRNA genes
  • prepared a genomic library from affected patient
    (must contain DNA spanning translocation
    breakpoint)
  • isolated clones containing rRNA DNA and tested
    for hybridization with X chromosome specific DNA
    (could have only come from translocation region)
  • XJ1.1 (X junction) was characterized
  • turns out to be located in intron 17 of
    dystrophin gene

15
DMD cloning
  • Kunkels group (Harvard)
  • subtractive hybridization
  • patient B.B.had 3 disorders that were closely
    linked- likely to carry deletion
  • regions of normal DNA with no match in BB sample
    will hybridize together and can be cloned
  • found one clone, pERT87, that was missing in
    other DMD DNA samples

16
from pERT87 to dystrophin
  • insert was only 200 bp
  • pERT87 detected deletions in 7 of
    cytogenetically normal patients
  • detected polymorphisms that were found to be
    tightly linked to DMD
  • used pERT87 to isolate larger genomic clones in
    walk and probed for exons
  • used clones to probe muscle-specific cDNA
    libraries
  • found a 14 kb transcript that is absent in DMD
    patients
  • encoded large protein, dystrophin, 3685 amino
    acids

17
Dystrophin
  • part of large protein complex
  • links extracellular matrix with internal
    cytoskeleton of cell
  • prevents stress-induced fractures of sarcolemma
    during muscle contraction
  • DMD patients have elevated creatine
    phosphokinase, a sign of muscle degeneration

18
Dystrophin mutations
  • DMD- severe form, wheelchair bound by age 12
  • Becker (BMD)- milder form
  • Davies et al. (1988) concluded that severity of
    phenotype could not be correlated with the size
    of the deletion (most common defect).
  • One mildly affected BMD patient possessed a
    deletion of at least 110 kb including exons
    deleted in many DMD patients.
  • Monaco et al. (1988) propose that although no
    fundamental difference in the size of deletions
    appeared to be present in the 2 forms of disease,
    the deletions in DMD caused frameshifts while
    those in BMD did not.
  • Most patients with DMD are found to have no
    dystrophin protein in muscle, whereas BMD
    patients have an abnormally short variety of
    dystrophin. Presumably the dystrophin that is
    formed is partially functional.
  • they also showed that patients with similar
    in-frame deletions and even similar protein
    levels may have significantly different clinical
    presentations, suggesting that epigenetic and
    environmental factors play a significant role in
    determining the severity of a patient's disease.
  • epigenetic- heritable but not caused by a change
    in DNA sequence- ex. methylation

19
Unstable expanding repeats
  • Novel cause of disease identified in 1991
  • hallmark is anticipation
  • two different classes of expansion
  • very large expansion regions outside of coding
    regions, often also have intermediate
    non-pathogenic but unstable alleles
  • fragile X
  • myotonic dystrophy (unique- no other mutation
    ever found in MD patients)
  • modest expansions of CAG repeats within coding
    sequences
  • found in 8 late onset neurodegenerative diseases,
    including HD
  • no other mutations known to cause disease
  • repeat encodes poly glutamine
  • the larger the repeat, the earlier the onset

20
Fragile sites
  • fragile sites non-staining gaps visible in
    metaphase spreads of cultures grown under
    conditions such as inhibition of DNA synthesis or
    folate deficiency
  • found in trinucleotide repeat expansions
    involving (CCG)n
  • 5 known- 3 on X, 11, 16
  • (3 assoc. with mental retardation)

21
Fragile X Syndrome
  • FRAXA (fragile site associated with syndrome)
    mapped to Xq27.3
  • FMR1 gene (fragile-X mental retardation 1)
  • FMR1 gene product
  • function unknown
  • shares homology with RNA-binding proteins
  • binds to 4 of mRNAs found in brains
  • expansion of the CCG repeat in the 5 UTR region
  • normal range is 6-52 copies of repeat
  • full mutation has 230-1000 copies
  • causes methylation and leads to inactivation of
    FMR1 gene
  • Also see incomplete penetrance
  • some boys carry deletion of gene- proves cause is
    inactivation of gene due to expansion, not some
    other effect of expansion
  • Also see partial dominance as 30 of female
    heterozygotes are affected

22
Cloning FMR1
  • Using a 275-kb fragment of human DNA isolated in
    a YAC and thought to span the fragile site, Yu et
    al. (1991) derived 2 probes that spanned the
    fragile site as demonstrated by in situ
    hybridization.
  • Mapping delineated the sequences that span the
    fragile site to 15 kb.
  • A 5-kb EcoRI fragment was found to contain
    fragile site breakpoints. When used as a probe on
    the chromosomal DNA of normal and fragile X
    individuals, alterations in the mobility were
    found only in fragile X DNA- they were of an
    increased size and varied within families (see
    next slide)
  • Morton and Macpherson (1992) proposed a model in
    which the fragile X mutation is postulated to
    occur as a multistep process.
  • Oudet et al. (1993) observed that a limited
    number of primary events may have been at the
    origin of most present-day fragile X chromosomes
    in Caucasians
  • proposed a putative scheme -6 founder chromosomes
    from which most of the observed fragile X-linked
    haplotypes can be derived directly or by a single
    event at one of the marker loci. May have carried
    a number of CGG repeats in an upper-normal range,
    from which recurrent multistep expansion
    mutations have arisen.

23
Fragile X gene diagnosis
  • premutations
  • number of repeats varies from 60-230
  • can expand to full mutations during meiosis in a
    carrier female, not in a transmitting male
  • affected females always receive allele from their
    mother
  • Southern blotting (DNA of inactiv. X and any full
    mutation X is methylated)
  • use EclXI- methylation-sensitive enzyme
  • N normal
  • P premutation, unmethylated
  • NM methylated normal
  • F methylated full mutation

24
Huntington Disease (HD) gene- 1993
  • 1983- first disease to be localized to a
    chromosome- 4p16.3- using RFLP linkage analysis
  • using physical maps and obtaining large numbers
    of probes- detailed haplotyping narrowed region
    down to 500 kb
  • used exon trapping to identify a number of
    interesting genes
  • IT15 (interesting transcript 15) showed
    consistent difference between HD and normal DNA
  • in DHD patients, one IT15 gene had no more than
    35 CAG repeats, the other had 40 CAG repeats
  • found that normal allele has 9-35 repeats
  • alleles with 36-39 repeats- could be affected or
    normal
  • gene called HD, encodes huntingtin
  • expressed in a wide range of cell types,
    including neurons
  • found in cytoplasm associated with membranes,
    vesicles and cytoskeleton (may be acting in
    vesicle trafficking)
  • poly Glu expansion in coding region leads to
    protein aggregates that eventually kill the cell

25
Huntington Disease
  • PCR of gene fragment containing CAG repeat
  • bands are silver stained to detect DNA
  • lanes 1, 2, 6,10 are from unaffected people
  • 3, 4, 5, 7, 8 from affected people
  • lane 5 juvenile onset case (note father)
  • lane 9 affected fetus

26
XREFdb
  • XREFdb is a publicly accessible database that is
    a component of a research project (the XREF
    project), which is devoted to cross-referencing
    the genetics of model organisms with mammalian
    phenotypes and accelerating the identification of
    genes mutated in human diseases.
  • To cross-reference homologous genes in yeast and
    human, all yeast ORF protein sequences from the
    Saccharomyces Genome Database (SGD) were used as
    queries against a six-frame translation of the
    human EST subset of the Database of Expressed
    Sequence Tags (dbEST) maintained at the National
    Center for Biotechnology Information (NCBI) .
    Search hits are ranked by their BLAST score and
    the best human EST hit is recorded for each yeast
    ORF

27
Human genes with yeast homologs
  • Human Disease Gene Yeast homolog Function
  • Hereditary non-polyposis colon cancer
    120436 MSH2 3.2e-254 MSH2 DNA repair
    protein
  • Hereditary non-polyposis colon cancer
    120436 MLH1 7.7e-190 MLH1 DNA repair
    protein
  • Cystic fibrosis
    219700 CFTR 4.3e-162 YCF1 Metal
    resistance protein
  • Wilson disease
    277900 WND 2.1e-152 CCC2 Copper
    transporter
  • Glycerol kinase deficiency 307030 GK
    5.4e-124 GUT1 Glycerol kinase
  • Bloom syndrome
    210900 BLM 3.1e-112 SGS1 Helicase
  • Adrenoleukodystrophy, X-linked 300100 ALD
    1.0e-101 PXA1 Peroxisomal ABC
    transporter
  • Ataxia telangiectasia
    208900 ATM 7.9e-85 TEL1 PI3 kinase
  • Myotonic dystrophy 160900 DM 1.7e-79
    N1727 Hypothetical protein
  • Neurofibromatosis, type 1 162200 NF1
    3.1e-40 IRA2 Inhibitory regulator
    protein

28
Mouse model of CF
  • Van Doorninck et al. (1995) generated a mouse
    model of CF with the phe508del mutation
  • In this model of CF the mutant CFTR was not
    processed efficiently to the fully glycosylated
    form in vivo
  • However, the mutant protein was expressed as
    functional chloride channels in the plasma
    membrane of cells cultured at reduced
    temperature.
  • Furthermore, they could show that the
    electrophysiologic characteristics of the mouse
    phe508del-CFTR channels were indistinguishable
    from normal. In homozygous mutant mice they did
    not observe a significant effect of genetic
    background on the level of residual chloride
    channel activity. The data showed that like its
    human homolog, the mouse mutant CFTR is a
    temperature-sensitive processing mutant, and
    therefore an authentic model for study of
    pathophysiology and therapy.

29
Rescue of CF mice with human gene
  • Using an intact human CFTR gene, Manson et al.
    (1997) generated transgenic mice carrying a
    320-kb YAC.
  • Mice that only expressed the human transgene were
    obtained by breeding with Cambridge-null CF mice.
  • One line had approximately 2 copies of the intact
    YAC.
  • Mice carrying this transgene and expressing no
    CFTR appeared normal and bred well, in marked
    contrast to the null mice, where 50 died by
    approximately 5 days of age.
  • Expression of the transgene was highly cell-type
    specific and matched that of the endogenous mouse
    gene in the crypt epithelia throughout the gut
    and in salivary gland tissue. However, there was
    no transgene expression in some tissues, such as
    the Brunner glands, where it would be expected.
    Where there were differences between the mouse
    and human pattern of expression, the transgene
    followed the mouse pattern.

30
From mice to humans- an example
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