Positional Cloning

1
See pp. 412-420 (Chapter 14) of book
Positional Cloning of Spinocerebellar Ataxia-1
(SCA-1) Cloning genes (when you know nothing
about the gene product) by determining the exact
location of the locus on the chromosome
2
Ataxia loss of coordination
Nicholas Friedreich (Germany) first described an
inherited ataxia (1860s, 1870s)
Friedreichs Ataxia
3
Pierre Marie (French) observed 4 families with
different symptoms (1893)
Maries Ataxia
but now generally known as SCA
4
Graft-vs-Host Disease involving skin lesions in
a patient following bone marrow transplantation
for myelodysplasia.
Image courtesy of Romeo A. Mandanas
5
White Blood Cells of Immune System
Capable of recognizing HLA proteins that are
located on the outer surfaces of most other
cells Human Leukocyte Antigens HLA
Many different loci With numerous alleles at
each locus A 19 alleles B 20 alleles
6
Japanese researchers in the 1970s found a family
with 5 children, where 3 had Maries
Ataxia. Lets look at their data..
From Hereditary Ataxia and the HLA Genotypes.
New England Journal of Medicine, 1974, Vol.
291154.
7
This result could easily be interpreted if we
assume the ataxia gene locus to be on the sixth
chromosome near the HLA loci. Yakura et
al., 1974 (Japan)
8
From Somatic Cell Hybridization studies it was
shown that all of the HLA loci are on Chromosome
6 !
This result could easily be interpreted if we
assume the ataxia gene locus to be on the sixth
chromosome near the HLA loci. Yakura et
al., 1974 (Japan)
9
1977, Jackson et al., Spinocerebellar Ataxia and
HLA Linkage. University of Mississippi, New
England Journal of Medicine
10
Somatic Cell Hybridization pp. 131-136
http//www.mun.ca/biology/scarr/Somatic_Cell_Hybri
dization.htm
11
6 T 1
6 1T
Translocation Exchange of chromosome parts
Francke et al. 1977 Proceedings of the National
Academy of Sciences (Vol. 741147)
12
6 T 1
6 1T
UC-San Diego,1977 Got cell lines from a family
with a translocation. 6T line no HLA 1T line
had HLA ! Concluded that the HLA genes lie
on that segment of Chromosome 6.
13
HLA gene region on Chromosome 6
MHC Major Histocompatability Complex
14
stopped
Notice that the alleles for Loci A B did not
get swapped, But the alleles for A C did!
15
Lots of loci in the HLA region
16
1977
17
Linkage Analysis
RFLP
RFLP
STRP
Ranum et. al., 1991, American Journal of Human
Genetics, (4931).
Done in Harry Orrs lab.
18
Normal Cerebellar
Ataxia
The Purkinje Cells which normally line up between
the layers of the cerebellum (arrows) are lost in
hereditary ataxia. First observed in
SCA patients (post-mortem) in 1974.
http//www.cvm.missouri.edu/ataxia/causes.htm
19
Annals of Neurology, 1988.
Spinocerebellar
ataxia variable age of onset and linkage to
human leukocyte antigen in a large
kindred. Zoghbi HY, Pollack MS, Lyons LA,
Ferrell RE, Daiger SP, Beaudet A.Department of
Pediatrics, Baylor College of Medicine, Houston,
TX 77030.
We studied a seven-generation kindred with
autosomal dominant spinocerebellar ataxia (SCA)
to assess linkage relationships to multiple human
leukocyte antigen (HLA) loci on the short arm of
chromosome 6. Age at onset, clinical features,
and course of the disease are described. Although
the mean age of onset was 34 years in this
family, in 6 of 41 affected individuals onset was
below 15 years of age and was accompanied by the
unique clinical features of mental retardation
and rapid progression of disease. Linkage studies
were performed on 93 individuals, and the results
show strong evidence for linkage of the SCA locus
to the HLA loci. A maximum logarithm of the odds
score of 5.83 was found at a recombination
fraction of 0.12. This is the first documentation
of childhood onset in the HLA-linked form of SCA.
.
20
Zoghbi HY, O'Brien WE, Ledley FD. Linkage
relationships of the human methylmalonyl CoA
mutase to the HLA and D6S4 loci on chromosome 6.
Genomics. 1988 Zoghbi HY, Daiger SP, McCall A,
O'Brien WE, Beaudet AL. Extensive DNA
polymorphism at the factor XIIIa (F13A) locus and
linkage to HLA. Am J Hum Genet. 1988 Ledley FD,
Lumetta MR, Zoghbi HY, VanTuinen P, Ledbetter SA,
Ledbetter DH. Mapping of human methylmalonyl CoA
mutase (MUT) locus on chromosome 6. Am J Hum
Genet. 1988 Ballantyne CM, Zoghbi HY, Grzeschik
KH, O'Brien WE, Beaudet AL. A human single copy
DNA probe (ZB6-1) detects multiple polymorphisms
on 6q. Nucleic Acids Res. 1988 Bibbins KB,
Tsai JY, Schimenti J, Sarvetnick N, Zoghbi HY,
Goodfellow P, Silver LM. Human homologs of two
testes-expressed loci on mouse chromosome 17 map
to opposite arms of chromosome 6. Genomics. 1989
Zoghbi HY, Sandkuyl LA, Ott J, Daiger SP,
Pollack M, O'Brien WE, Beaudet AL. Assignment of
autosomal dominant spinocerebellar ataxia (SCA1)
centromeric to the HLA region on the short arm of
chromosome 6, using multilocus linkage analysis.
Am J Hum Genet. 1989 Zoghbi HY, McCall AE.
TaqI polymorphism at the D6S91 locus. Nucleic
Acids Res. 1990 Zoghbi HY, McCall AE. BclI and
MspI polymorphisms at the D6S90 locus. Nucleic
Acids Res. 1990 Zoghbi HY, Ballantyne CM,
O'Brien WE, McCall AE, Kwiatkowski TJ Jr,
Ledbetter SA, Beaudet AL. Deletion and linkage
mapping of eight markers from the proximal short
arm of chromosome 6. Genomics. 1990 Zoghbi HY,
McCall AE, LeBorgne-Demarquoy F. Sixty-five
radiation hybrids for the short arm of human
chromosome 6 their value as a mapping panel and
as a source for rapid isolation of new probes
using repeat element-mediated PCR. Genomics. 1991
Ranum LP, Chung MY, Duvick LA, Zoghbi HY, Orr
HT. Dinucleotide repeat polymorphism at the
D6S109 locus. Nucleic Acids Res. 1991 Blanche H,
Zoghbi HY, Jabs EW, de Gouyon B, Zunec R, Dausset
J, Cann HM. A centromere-based genetic map of the
short arm of human chromosome 6. Genomics.
1991 Weber JL, Kwitek AE, May PE, Zoghbi HY.
Dinucleotide repeat polymorphism at the D6S105
locus. Nucleic Acids Res. 1991
21
Zoghbi HY, McCall AE. TaqI polymorphism at the
D6S91 locus. Nucleic Acids Res. 1990 Zoghbi HY,
McCall AE. BclI and MspI polymorphisms at the
D6S90 locus. Nucleic Acids Res. 1990 Zoghbi HY,
Ballantyne CM, O'Brien WE, McCall AE, Kwiatkowski
TJ Jr, Ledbetter SA, Beaudet AL. Deletion and
linkage mapping of eight markers from the
proximal short arm of chromosome 6. Genomics.
1990 Zoghbi HY, McCall AE, LeBorgne-Demarquoy
F. Sixty-five radiation hybrids for the short arm
of human chromosome 6 their value as a mapping
panel and as a source for rapid isolation of new
probes using repeat element-mediated PCR.
Genomics. 1991 Ranum LP, Chung MY, Duvick LA,
Zoghbi HY, Orr HT. Dinucleotide repeat
polymorphism at the D6S109 locus. Nucleic Acids
Res. 1991 Blanche H, Zoghbi HY, Jabs EW, de
Gouyon B, Zunec R, Dausset J, Cann HM. A
centromere-based genetic map of the short arm of
human chromosome 6. Genomics. 1991 Weber JL,
Kwitek AE, May PE, Zoghbi HY. Dinucleotide repeat
polymorphism at the D6S105 locus. Nucleic Acids
Res. 1991 10 Orr HT, Chung MY, Banfi S,
Kwiatkowski TJ Jr, Servadio A, Beaudet AL, McCall
AE, Duvick LA, Ranum LP, Zoghbi HY. Expansion of
an unstable trinucleotide CAG repeat in
spinocerebellar ataxia type 1. Nat Genet.
1993 11 Zoghbi HY, Frontali M, Orr HT,
Sandkuijl L, Cann H, Sasaki H, Chamberlain S,
Terrenato L, Rich SS. Linkage studies in
dominantly inherited ataxias. Adv Neurol.
1993 12 Eng CM, Durtschi BA, Zoghbi HY, Beaudet
AL. Isolation, mapping, and characterization of
two cDNA clones expressed in the cerebellum.
Genomics. 1992 13 Meese EU, Witkowski CM,
Zoghbi HY, Stanbridge EJ, Meltzer PS, Trent JM.
Development and utilization of a somatic cell
hybrid mapping panel to assign NotI linking
probes to the long arm of human chromosome 6.
Genomics. 1992 14 Summers KM, Tam KS, Bartley
PB, Drysdale J, Zoghbi HY, Halliday JW, Powell
LW. Fine mapping of a human chromosome 6 ferritin
heavy chain pseudogene relevance to
haemochromatosis. Hum Genet. 1991 15 Le
Borgne-Demarquoy F, Kwiatowski TJ Jr, Zoghbi HY.
Two dinucleotide repeat polymorphisms at the
D6S202 locus. Nucleic Acids Res. 1991 16 Keats
BJ, Pollack MS, McCall A, Wilensky MA, Ward LJ,
Lu M, Zoghbi HY. Tight linkage of the gene for
spinocerebellar ataxia to D6S89 on the short arm
of chromosome 6 in a kindred for which close
linkage to both HLA and F13A1 is excluded. Am J
Hum Genet. 1991 17 Ellison KA, Fill CP, Zoghbi
HY. MspI and MboI polymorphisms at the DXS704
locus. Nucleic Acids Res. 1991 18 Kwiatkowski
TJ Jr, Beaudet AL, Trask BJ, Zoghbi HY. Linkage
mapping and fluorescence in situ hybridization of
TCTE1 on human chromosome 6p analysis of
dinucleotide polymorphisms on native gels.
Genomics. 1991 19 Zoghbi HY, Jodice C,
Sandkuijl LA, Kwiatkowski TJ Jr, McCall AE,
Huntoon SA, Lulli P, Spadaro M, Litt M, Cann HM.
The gene for autosomal dominant spinocerebellar
ataxia (SCA1) maps telomeric to the HLA complex
and is closely linked to the D6S89 locus in three
large kindreds. Am J Hum Genet. 1991
22
Summers KM, Tam KS, Bartley PB, Drysdale J,
Zoghbi HY, Halliday JW, Powell LW. Fine mapping
of a human chromosome 6 ferritin heavy chain
pseudogene relevance to haemochromatosis. Hum
Genet. 1991 Le Borgne-Demarquoy F, Kwiatowski
TJ Jr, Zoghbi HY. Two dinucleotide repeat
polymorphisms at the D6S202 locus. Nucleic Acids
Res. 1991 Keats BJ, Pollack MS, McCall A,
Wilensky MA, Ward LJ, Lu M, Zoghbi HY. Tight
linkage of the gene for spinocerebellar ataxia to
D6S89 on the short arm of chromosome 6 in a
kindred for which close linkage to both HLA and
F13A1 is excluded. Am J Hum Genet. 1991 Ellison
KA, Fill CP, Zoghbi HY. MspI and MboI
polymorphisms at the DXS704 locus. Nucleic Acids
Res. 1991 Kwiatkowski TJ Jr, Beaudet AL, Trask
BJ, Zoghbi HY. Linkage mapping and fluorescence
in situ hybridization of TCTE1 on human
chromosome 6p analysis of dinucleotide
polymorphisms on native gels. Genomics. 1991
Zoghbi HY, Jodice C, Sandkuijl LA, Kwiatkowski
TJ Jr, McCall AE, Huntoon SA, Lulli P, Spadaro M,
Litt M, Cann HM. The gene for autosomal dominant
spinocerebellar ataxia (SCA1) maps telomeric to
the HLA complex and is closely linked to the
D6S89 locus in three large kindreds. Am J Hum
Genet. 1991 Eng CM, Durtschi BA, Zoghbi HY,
Beaudet AL. Isolation, mapping, and
characterization of two cDNA clones expressed in
the cerebellum. Genomics. 1992 Meese EU,
Witkowski CM, Zoghbi HY, Stanbridge EJ, Meltzer
PS, Trent JM. Development and utilization of a
somatic cell hybrid mapping panel to assign NotI
linking probes to the long arm of human
chromosome 6. Genomics. 1992 Orr HT, Chung MY,
Banfi S, Kwiatkowski TJ Jr, Servadio A, Beaudet
AL, McCall AE, Duvick LA, Ranum LP, Zoghbi HY.
Expansion of an unstable trinucleotide CAG repeat
in spinocerebellar ataxia type 1. Nat Genet.
1993 Zoghbi HY, Frontali M, Orr HT, Sandkuijl L,
Cann H, Sasaki H, Chamberlain S, Terrenato L,
Rich SS. Linkage studies in dominantly inherited
ataxias. Adv Neurol. 1993
23
Eng CM, Durtschi BA, Zoghbi HY, Beaudet AL.
Isolation, mapping, and characterization of two
cDNA clones expressed in the cerebellum.
Genomics. 1992 Meese EU, Witkowski CM, Zoghbi
HY, Stanbridge EJ, Meltzer PS, Trent JM.
Development and utilization of a somatic cell
hybrid mapping panel to assign NotI linking
probes to the long arm of human chromosome 6.
Genomics. 1992 Zoghbi HY, Frontali M, Orr HT,
Sandkuijl L, Cann H, Sasaki H, Chamberlain S,
Terrenato L, Rich SS. Linkage studies in
dominantly inherited ataxias. Adv Neurol. 1993

.and finally (after 22 publications on
linkage) Orr HT, Chung MY, Banfi S, Kwiatkowski
TJ Jr, Servadio A, Beaudet AL, McCall AE, Duvick
LA, Ranum LP, Zoghbi HY. Expansion of an unstable
trinucleotide CAG repeat in spinocerebellar
ataxia type 1. Nature Genetics 1993
24
Dr. Harry Orr
Director,
Institute of Human Genetics
University of Minnesota His
original specialty were the HLA genes themselves,
which expanded into an interest in genetic
diseases like Huntingtons Disease, Alzheimers,
Cystic Fibrosis, and SCA. He had access to his
own families of ataxia patients in Nebraska
Minnesota.
combined resources with Zoghbi in 1990
25
Blazar BR, Lasky LC, Perentesis JP, Watson KV,
Steinberg SE, Filipovich AH, Orr HT, Ramsay NK.
Successful donor cell engraftment in a recipient
of bone marrow from a cadaveric donor. Blood.
1986 Koller BH, Geraghty D, Orr HT, Shimizu Y,
DeMars R. Organization of the human class I major
histocompatibility complex genes. Immunol Res.
1987 Rich SS, Wilkie P, Schut L, Vance G, Orr
HT. Spinocerebellar ataxia localization of an
autosomal dominant locus between two markers on
human chromosome 6. Am J Hum Genet. 1987 Duvick
L, Rich SS, Orr HT. A polymorphic DNA probe,
p1-10-2, from chromosome 6. Nucleic Acids Res.
1990 Ranum LP, Chung MY, Duvick LA, Zoghbi HY,
Orr HT. Dinucleotide repeat polymorphism at the
D6S109 locus. Nucleic Acids Res. 1991 Ranum LP,
Duvick LA, Rich SS, Schut LJ, Litt M, Orr HT.
Localization of the autosomal dominant HLA-linked
spinocerebellar ataxia (SCA1) locus, in two
kindreds, within an 8-cM subregion of chromosome
6p. Am J Hum Genet. 1991 Feddersen RM,
Ehlenfeldt R, Yunis WS, Clark HB, Orr HT.
Disrupted cerebellar cortical development and
progressive degeneration of Purkinje cells in
SV40 T antigen transgenic mice. Neuron.
1992 Ranum LP, Rich SS, Nance MA, Duvick LA,
Aita JF, Orr HT, Anton-Johnson S, Schut LJ.
Autosomal dominant spinocerebellar ataxia locus
heterogeneity in a Nebraska kindred. Neurology.
1992 Zoghbi HY, Frontali M, Orr HT, Sandkuijl L,
Cann H, Sasaki H, Chamberlain S, Terrenato L,
Rich SS. Linkage studies in dominantly inherited
ataxias. Adv Neurol. 1993 Banfi S, Chung MY,
Kwiatkowski TJ Jr, Ranum LP, McCall AE, Chinault
AC, Orr HT, Zoghbi HY. Mapping and cloning of the
critical region for the spinocerebellar ataxia
type 1 gene (SCA1) in a yeast artificial
chromosome contig spanning 1.2 Mb. Genomics.
1993 Orr HT, Chung MY, Banfi S, Kwiatkowski TJ
Jr, Servadio A, Beaudet AL, McCall AE, Duvick LA,
Ranum LP, Zoghbi HY. Expansion of an unstable
trinucleotide CAG repeat in spinocerebellar
ataxia type 1. Nat Genet. 1993
26
Feddersen RM, Ehlenfeldt R, Yunis WS, Clark HB,
Orr HT. Disrupted cerebellar cortical development
and progressive degeneration of Purkinje cells in
SV40 T antigen transgenic mice. Neuron.
1992 Ranum LP, Rich SS, Nance MA, Duvick LA,
Aita JF, Orr HT, Anton-Johnson S, Schut LJ.
Autosomal dominant spinocerebellar ataxia locus
heterogeneity in a Nebraska kindred. Neurology.
1992 Zoghbi HY, Frontali M, Orr HT, Sandkuijl L,
Cann H, Sasaki H, Chamberlain S, Terrenato L,
Rich SS. Linkage studies in dominantly inherited
ataxias. Adv Neurol. 1993 Banfi S, Chung MY,
Kwiatkowski TJ Jr, Ranum LP, McCall AE, Chinault
AC, Orr HT, Zoghbi HY. Mapping and cloning of the
critical region for the spinocerebellar ataxia
type 1 gene (SCA1) in a yeast artificial
chromosome contig spanning 1.2 Mb. Genomics.
1993 Orr HT, Chung MY, Banfi S, Kwiatkowski TJ
Jr, Servadio A, Beaudet AL, McCall AE, Duvick LA,
Ranum LP, Zoghbi HY. Expansion of an unstable
trinucleotide CAG repeat in spinocerebellar
ataxia type 1. Nat Genet. 1993
27
Once they found an RFLP probe that appeared to
be close to the SCA gene they used that same
probe to screen Genomic Libraries from healthy
and ataxic people, as well as to screen a cDNA
Library (made from fetal brain tissue).
28
Nature Genetics, 1993
Muscular Dystrophy gene 2.4 million bases
29
Southern Blot showing Anticipation
Onset 4 years
23 x 3 69 bases
Onset 30 years
Normal
30
6-39 repeats in healthy people
31
In Nature, 1994 (7513)
mRNA less than 1 of gene
32
Where is the Polyglutamine (Poly Q) Tract?
33
Where is the Polyglutamine (Poly Q) Tract?
30 Q
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Lets continue the story of SCA as told by Dr.
Huda Zoghbi
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Nature Genetics, 1993
So, Positional Cloning techniques were used to
isolate a 1,200,000 bp piece of Chromosome 6.
Less than 1 of this region actually codes for
the SCA-1 transcript (mRNA).
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Globin gene
Healthy Anemic
Use a probe from for this region
For Sickle Cell Anemia
43
Probes are valuable for identifying the
mutations in a well-characterized gene
A and B are homologous chromosomes
Not cut here
You can think of B as little a
Southern Blots (of genomic DNA) following
digestion with EcoRI enzyme
44
And how does that help Positionally Clone
genes??
45
EcoRI cuts the A allele in half, and Probe 3
allows you to visualize that. Lets pretend the
A allele is the diseased allele.
A and B are homologous chromosomes
Not cut here
You can think of B as little a
If you made a genomic library of a person with a
RFLP-mapped disease, you could use Probe 3 to
screen the library.
The other two probes would work too, but be
further away from mutation.
46
that reveals RFLP
47
Healthy
Diseased
Diseased
Healthy
Diseased
Healthy
48
So, the hard part is finding the
right combination of RE and probe.which is
one reason why Postional Cloning is so slow and
expensive.
If this band is always present in people with
the disease then the probe could be useful in
screening a library.
49
One way of finding the best probe is
Chromosome Walking
If linkage (by studying pedigree analysis) can be
shown for a disease (that is already cloned),
then begin there, and walk to the gene of
interest.
50
Linked gene here
Different library made with different RE
Different library made with different RE
Each time you make a new probe, use that to look
for RFLPs in healthy vs. diseased people.
51
Chromosome Walking
52
If an RFLP cant be found for the disease of
interest (for instance, point mutations wouldnt
reveal themselves as RFLPs unless the single
mutation was exactly on a RE site) you can look
at transcription. mRNA can be isolated from
healthy vs. sick people (using Poly-A
chromatography) and then ran on a gel,
transferred to a membrane, and probed just like a
Southern Blot. NORTHERN BLOT
53
If the disease of interest involves muscle
tissue then this probe might be
important especially if it doesnt occur in
diseased people.
54
Northern blot showing the presence of mRNA
hybridizing to sadA cDNA in different types of
tissue. 1, Dry seeds 2, seeds after 16 h of
soaking in tap water 3, shoots 9 d after sowing
4, cotyledons 14 d after sowing 5, leaf buds 14
d after sowing 6, cotyledons 21 d after sowing
7, second leaf pairs 21 d after sowing 8, third
leaf pairs 21 d after sowing 9, fourth leaf
pairs 21 d after sowing 10, fifth leaf pairs 21
d after sowing 11, roots from plants grown in
vermiculite 14 d after sowing 12, roots from
plants grown in vermiculite 21 d after sowing
13, roots from plants grown in vermiculite 42 d
after sowing 14, stems 21 d after sowing 15,
tendrils 16, flowers (white) 17, flowers
(purplish) and 18, pods.
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Southern Blot showing Anticipation
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The length of a centiMorgan (in terms of DNA
bases) is different for each species In
Humans 1 cM 1 million DNA bases (on average)
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