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Positional Cloning of Spinocerebellar Ataxia-1 (SCA-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”. Ataxia = “loss of coordination”.

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Positional Cloning of Spinocerebellar Ataxia-1 (SCA-1)

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  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): Friedreich’s Ataxia

  3. Pierre Marie (French) observed 4 families with different symptoms (1893): Marie’s 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 Marie’s Ataxia. Lets look at their data….. From:Hereditary Ataxia and the HLA Genotypes. New England Journal of Medicine, 1974, Vol. 291:154.

  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_Hybridization.htm

  11. 6 T 1 6 1T Translocation Exchange of chromosome parts Francke et al. 1977 Proceedings of the National Academy of Sciences (Vol. 74:1147)

  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, (49:31). Done in Harry Orr’s 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 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

  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 (7:513) mRNA = less than 1% of gene

  32. Where is the Polyglutamine (Poly Q) Tract?

  33. Where is the Polyglutamine (Poly Q) Tract? 30 Q

  34. Lets continue the story of SCA as told by Dr. Huda Zoghbi

  35. 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).

  36. Globin gene Healthy Anemic Use a probe from for this region For Sickle Cell Anemia

  37. 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

  38. And how does that help Positionally Clone genes??

  39. 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.

  40. that reveals RFLP

  41. The RE site for this disease must be here Healthy Diseased Diseased Healthy Diseased Healthy

  42. 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.

  43. 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.

  44. 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

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