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