Positional Cloningof 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”

See pp. 412-420 (Chapter 14) of book

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

HLAMany different loci…

With numerous allelesat each locus…

A = 19 allelesB = 20 alleles

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 onthe sixth chromosome near the HLA loci.” Yakura et al., 1974 (Japan)

“This result could easily be interpreted if we assume the ataxia gene locus to be onthe 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 !

1977, Jackson et al., Spinocerebellar Ataxia and HLA Linkage.University of Mississippi, New England Journal of Medicine

http://www.mun.ca/biology/scarr/Somatic_Cell_Hybridization.htm

SomaticCell

Hybridization

pp. 131-136

6 T 1 6 1T

Translocation

Exchange ofchromosome parts

Francke et al. 1977Proceedings of the

National Academy of Sciences (Vol. 74:1147)

6 T 1 6 1T

UC-San Diego,1977

Got cell lines froma 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 regionon Chromosome 6

MHC = Major Histocompatability Complex

Notice that the alleles forLoci A & B did not get swapped,

But the alleles for A & C did!

stopped

Lots of loci in the HLA region

1977

Linkage Analysis

RFLP RFLPSTRP

Ranum et. al., 1991, American Journal of Human Genetics, (49:31).Done in Harry Orr’s lab.

The Purkinje Cells which normally line up between the layers of

the cerebellum (arrows) are lost in hereditary ataxia.

First observed in SCAFirst observed in SCApatients (post-mortem) patients (post-mortem)

in 1974.in 1974.

Normal

Cerebellar Ataxia

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

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

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

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. 1993Banfi 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

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 andataxic 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 =30 years

Onset =4 years

Normal

23 x 3 = 69 bases

6-39 repeatsin 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).

For Sickle Cell Anemia

Use a probe from for this region

Globin gene

HealthyAnemic

You can thinkof “B” as “little a”

Not cut here

Southern Blots(of genomic DNA) following digestionwith EcoRI enzyme

Probes are valuable for identifying the

mutations in a well-characterized gene

A and B are homologous chromosomes

And how does that help Positionally Clone

genes??

You can thinkof “B” as “little a”

Not cut here

If you made agenomic libraryof a person witha RFLP-mapped

disease, you could use Probe 3

to screen the library.

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

The other two probes would work too, but be further away from mutation.

that reveals RFLP

Diseased

Diseased

Diseased

Healthy

Healthy

Healthy

The RE site for this disease must be here

If this band is alwayspresent in people with thedisease then the probe couldbe useful in screening alibrary.

So, the hard partis finding the rightcombination of RE and probe….which is one reason why

Postional Cloning isso slow and expensive.

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.

Different library madewith different RE

Different library madewith different RE

Linked gene here

Each time youmake a new probe,use that to look for

RFLPs in healthy vs.diseased people.

Chromosome Walking

If an RFLP can’t be found for the disease of interest (for instance,point mutations wouldn’t reveal themselves as RFLPs unlessthe 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 interestinvolves muscle tissuethen this probe might

be important…especially if it doesn’t

occur in diseasedpeople.

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)