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”
Ataxia = “loss of coordination”
Nicholas Friedreich (Germany) first
described an inherited ataxia (1860s, 1870s):
Friedreich’s Ataxia
Pierre Marie (French) observed 4 families
with different symptoms (1893):
Marie’s Ataxia
…but now generally known as
SCA
“Graft-vs-Host Disease” involving skin lesions in a
patient following bone marrow transplantation for
myelodysplasia.
Image courtesy of Romeo A. Mandanas
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
Many different loci…
With numerous alleles
at each locus…
A = 19 alleles
B = 20 alleles
HLA
Japanese researchers in the 1970s found a family
with 5 children, where 3 had Marie’s Ataxia.
Lets look at their data…..
From: Hereditary Ataxia and the HLA Genotypes.
New England Journal of Medicine, 1974, Vol. 291:154.
“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)
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)
1977, Jackson et al., Spinocerebellar Ataxia and HLA Linkage.
University of Mississippi, New England Journal of Medicine
Somatic
Cell
Hybridization
pp. 131-136
http://www.mun.ca/biology/scarr/Somatic_Cell_Hybridization.htm
6T
1
6
1T
Translocation
Exchange of
chromosome parts
Francke et al. 1977
Proceedings of the
National Academy of
Sciences (Vol. 74:1147)
6T
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.
HLA gene region
on Chromosome 6
MHC =
Major Histocompatability Complex
stopped
Notice that the alleles for
Loci A & B did not get swapped,
But the alleles for A & C did!
Lots of loci in the HLA region
1977
Linkage Analysis
RFLP
STRP
RFLP
Ranum et. al., 1991, American Journal of Human Genetics, (49:31).
Done in Harry Orr’s lab.
Normal
The Purkinje Cells
which normally line up
between the layers of
the cerebellum
(arrows) are lost in
hereditary ataxia.
Cerebellar Ataxia
First observed in SCA
patients (post-mortem)
in 1974.
http://www.cvm.missouri.edu/ataxia/causes.htm
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.
.
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
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
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
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 Huntington’s Disease, Alzheimer’s,
Cystic Fibrosis, and SCA. He had access to his own families of ataxia
patients in Nebraska & Minnesota.
…combined resources
with Zoghbi in 1990
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
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).
Nature Genetics, 1993
Muscular Dystrophy gene =
2.4 million bases
Southern Blot
showing “Anticipation”
Onset =4 years
23 x 3 = 69 bases
Onset =30 years
Normal
6-39 repeats
in healthy people
In: Nature, 1994 (7:513)
mRNA = less than 1% of gene
Where is the Polyglutamine (Poly Q) Tract?
Where is the Polyglutamine (Poly Q) Tract?
30 Q
Lets continue the story of SCA
as told by Dr. Huda Zoghbi
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).
Globin gene
Healthy
Anemic
Use a probe from for this region
For Sickle Cell Anemia
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
And how does that help
Positionally Clone
genes??
A and B are
homologous
chromosomes
EcoRI cuts the “A” allele in
half, and Probe 3 allows
you to visualize that. Lets
pretend the “A” allele is
the diseased allele.
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.
that reveals RFLP
Healthy
The RE site for this disease must be here
Diseased
Diseased
Healthy
Diseased
Healthy
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.
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.
Linked gene here
Different library made
with different RE
Each time you
make a new probe,
use that to look for
RFLPs in healthy vs.
diseased people.
Different library made
with different RE
Chromosome Walking
If an RFLP can’t be found for the disease of interest (for instance,
point mutations wouldn’t 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
If the disease of interest
involves muscle tissue
then this probe might
be important…
especially if it doesn’t
occur in diseased
people.
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.
Southern Blot
showing “Anticipation”
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)