1. Beta Globin and Mutations Alexandra Childs and Ryan Edwards

2. Beta Globin Chromosome position: 11p15.5 DNA nucleotides position: 5246696..5248301 To the left of this gene in HBD or Delta globin and to the right of this gene is OR51v1 or Olfactory Receptor

3. HBB Location http://ghr.nlm.nih.gov/gene=HBB

4. Sequence of Beta Globin Green: introns Red: exons Blue: Everything that is transcribed but non-coding

5. Mature mRNA and Translated protein Sequence Nucleotide Sequence (444 nt): ATGGTGCATCTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGGGCAAGGTGAACGTGGATGAAG TTGGTGGTGAGGCCCTGGGCAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGTTCTTTGAGTCCTTTGG GGATCTGTCCACTCCTGATGCTGTTATGGGCAACCCTAAGGTGAAGGCTCATGGCAAGAAAGTGCTCGGT GCCTTTAGTGATGGCCTGGCTCACCTGGACAACCTCAAGGGCACCTTTGCCACACTGAGTGAGCTGCACT GTGACAAGCTGCACGTGGATCCTGAGAACTTCAGGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCA TCACTTTGGCAAAGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAAT GCCCTGGCCCACAAGTATCACTAA Translation (147 aa): MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLG AFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVAN ALAHKYH

6. Beta Globin Mutations Shepherd’s Bush Hb Mainz M Saskatoon Thalassemia Mutation on the Initiation Codon ATG to ATA Hb Rocky Mountain

7. Shepherd’s Bush

8. Gly and Asp Glycine Aspartic Acid →

9. SB Mutated Sequence Mutated Nucleotide Sequence (444 nt): ATGGTGCATCTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGGGCAAGGTGAACGTGGATGAAG TTGGTGGTGAGGCCCTGGGCAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGTTCTTTGAGTCCTTTGG GGATCTGTCCACTCCTGATGCTGTTATGGGCAACCCTAAGGTGAAGGCTCATGGCAAGAAAGTGCTCGGT GCCTTTAGTGATGACCTGGCTCACCTGGACAACCTCAAGGGCACCTTTGCCACACTGAGTGAGCTGCACT GTGACAAGCTGCACGTGGATCCTGAGAACTTCAGGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCA TCACTTTGGCAAAGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAAT GCCCTGGCCCACAAGTATCACTAA Mutated Translation (147 aa): MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLG AFSDALAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVAN ALAHKYH

10. Shepherds Bush Changes to Protein

11. Shepherds Bush Physical Signs According to the variant database, this mutation manifests itself as an increase in oxygen affinity. It also results in a decreased bpg (2,3-dpg). Moreover, the mutation causes the hemoglobin to be unstable and hemolytic anemia

12. Schematic Diagram of Hb-Mainz

13. Val and Glu Valine Glutamic Acid 

14. Hb-Mainz Mutated Sequence Mutated Nucleotide Sequence (444 nt): ATGGTGCATCTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGGGCAAGGTGAACGTGGATGAAG TTGGTGGTGAGGCCCTGGGCAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGTTCTTTGAGTCCTTTGG GGATCTGTCCACTCCTGATGCTGTTATGGGCAACCCTAAGGTGAAGGCTCATGGCAAGAAAGTGCTCGGT GCCTTTAGTGATGACCTGGCTCACCTGGACAACCTCAAGGGCACCTTTGCCACACTGAGTGAGCTGCACT GTGACAAGCTGCACGAGGATCCTGAGAACTTCAGGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCA TCACTTTGGCAAAGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAAT GCCCTGGCCCACAAGTATCACTAA Mutated Translation (147 aa): MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLG AFSDALAHLDNLKGTFATLSELHCDKLHEDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVAN ALAHKYH

15. Hb-Mainz Changes to Protein

16. Hb-Mainz Physical Effects According to the variant database, Hb-Mainz causes hemoglobin to become unstable and also causes hemolytic severe anemia.

17. Hb M-Saskatoon Mutation

18. M-Saskatoon Detailed Sequence (Mutated) Nucleotide Sequence (444 nt): ATGGTGCATCTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGGGCAAGGTGAAC GTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGCTGCTGGTGGTCTACCCTTGGACCCAG AGGTTCTTTGAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGGCAACCCTAAGGT GAAGGCTTATGGCAAGAAAGTGCTCGGTGCCTTTAGTGATGGCCTGGCTCACCTGGACAA CCTCAAGGGCACCTTTGCCACACTGAGTGAGCTGCACTGTGACAAGCTGCACGTGGATCCT GAGAACTTCAGGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAA GAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAATGCCC TGGCCCACAAGTATCACTAA (Mutated) Translation (147 aa): MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKV KAYGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTP PVQAAYQKVVAGVANALAHKYH CAT becomes TAT H becomes Y

19. Amino Acid Change HistidineTyrosine →

20. 3D Protein Sequence of Hemoglobin Histidine is a positively charged hydrophilic amino acid Tyrosine is an uncharged hydrophilic amino acid What do you notice about where this mutation is near to? We will come back to this…

21. M-Saskatoon, Methemoglobinemia Special class of low O 2 affinity hemoglobin variant Iron in the heme is in an abnormal oxidation state Known as methemoglobin. – useless in O 2 binding. Has a brown pigment Causes cyanosis Mutation may also cause Dissociation of Dimers

22. Thalassemias Mutation of Initiation Codon ATG -> ATA beta0 (Original) Nucleotide Sequence (444 nt): ATGGTGCATCTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGGGCAAGGTGAACGTGGATGAAGTT GGTGGTGAGGCCCTGGGCAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGTTCTTTGAGTCCTTTGGGGAT CTGTCCACTCCTGATGCTGTTATGGGCAACCCTAAGGTGAAGGCTCATGGCAAGAAAGTGCTCGGTGCCTTTA GTGATGGCCTGGCTCACCTGGACAACCTCAAGGGCACCTTTGCCACACTGAGTGAGCTGCACTGTGACAAGCT GCACGTGGATCCTGAGAACTTCAGGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAA GAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAATGCCCTGGCCCACAAGT ATCACTAA (Original) Translation (147 aa): MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFS DGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH RED – Initiation Codon that changes to ATA which no longer begins the reading frame Blue- Region of nucleotide base pairings, amino acids, and protein that is not read because of the mutation to the START codon Orange- Codon which becomes the START codon but originally is split into two codons before the mutation. All subsequent amino acids change in the translation Green- New Stop Codon

23. Amino Acid Change MethionineIsoleucine →

24. New Sequence Details (Mutated) Nucleotide Sequence (444 nt): ATGAAGTTGGTGGTGAGGCCCTGGGCAGGCTGCTGGTGGTCTA CCCTTGGACCCAGAGGTTCTTTGAGTCCTTTGGGGATCTGTCCA CTCCTGATGCTGTTATGGGCAACCCTAAGGTGAAGGCTCATGGC AAGAAAGTGCTCGGTGCCTTTAGTGATGGCCTGGCTCACCTGG ACAACCTCAAGGGCACCTTTGCCACACTGAGTGAGCTGCACTG TGACAAGCTGCACGTGGATCCTGAGAACTTCAGGCTCCTGGGC AACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATT CACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGT GTGGCTAATGCCCTGGCCCACAAGTATCACTAA (Mutated) Translation (aa): MKLVVRPWAGCWWSTLGPRGSLSPLGICPLLMLLWATLR(STOP)

25. Hb Rocky Mountain

26. Sequence Detail of Hb Rocky Mountain (Mutated)Nucleotide Sequence (444 nt): ATGGTGCATCTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGGGCAAGGTGAAC GTGGTTGAAGTTGGTGGTGAGGCCCTGGGCAGGCTGCTGGTGGTCTACCCTTGGACCCAG AGGTTCTTTGAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGGCAACCCTAAGGT GAAGGCTCATGGCAAGAAAGTGCTCGGTGCCTTTAGTGATGGCCTGGCTCACCTGGACAA CCTCAAGGGCACCTTTGCCACACTGAGTGAGCTGCACTGTGACAAGCTGCACGTGGATCCT GAGAACTTCAGGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAA GAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAATGCCC TGGCCCACAAGTATCACTAA (Original)Translation (147 aa): MVHLTPEEKSAVTALWGKVNVVEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKV KAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFT PPVQAAYQKVVAGVANALAHKYH GAT becomes GTT Aspartic Acid (D) becomes Valine (V)

27. Amino Acid Change Aspartic AcidValine →

28. Protein Structure and Description of Effects of Mutation Aspartic Acid is hydrophilic and negatively charged polar Valine is hydrophobic and non polar making it mildly unstable The change in the protein sequence may affect Hb function in cells as it causes an alteration in the protein shape

29. Effects of Hb Rocky Mountain Those with an Hb Rocky Mountain mutation (a.k.a. Hb Paddington) develop Microcytosis Smaller red blood cells Those with mutation are at higher risk of being anemic Why do you think that is if the red blood cell count is the same as a healthy person?

30. Useful Sites for Sheperds Bush/ Biblography http://globin.bx.psu.edu/cgi- bin/hbvar/query_vars3?mode=output&display_f ormat=page&i=387 http://globin.bx.psu.edu/cgi- bin/hbvar/query_vars3?mode=output&display_f ormat=page&i=387 http://globin.cse.psu.edu/html/huisman/variants /beta/Shepherds.Bush.html http://globin.cse.psu.edu/html/huisman/variants /beta/Shepherds.Bush.html http://books.google.com/books?id=H85dwxYTKL wC&lpg=RA1- PA1623&ots=A9ufCRxiMW&dq=Shepherds%20Bu sh%20mutat&pg=RA1- PA1623#v=onepage&q=shepherds%20bush&f=fal se http://books.google.com/books?id=H85dwxYTKL wC&lpg=RA1- PA1623&ots=A9ufCRxiMW&dq=Shepherds%20Bu sh%20mutat&pg=RA1- PA1623#v=onepage&q=shepherds%20bush&f=fal se

31. Sources for Hb-Mainz http://www.umm.edu/blood/anehemol.htm http://globin.bx.psu.edu/cgi- bin/hbvar/query_vars3?mode=output&displa y_format=page&i=451 http://globin.bx.psu.edu/cgi- bin/hbvar/query_vars3?mode=output&displa y_format=page&i=451 http://www.ncbi.nlm.nih.gov/pubmed/17356 397 http://www.ncbi.nlm.nih.gov/pubmed/17356 397

32. Useful Sites of Hb Rocky Mountain http://globin.bx.psu.edu/cgi- bin/hbvar/query_vars3?mode=output&displa y_format=page&i=925 http://globin.bx.psu.edu/cgi- bin/hbvar/query_vars3?mode=output&displa y_format=page&i=925 http://www.pdb.org/pdb/explore/jmol.do?str uctureId=2HHB&bionumber=1 http://www.pdb.org/pdb/explore/jmol.do?str uctureId=2HHB&bionumber=1 http://en.wikipedia.org/wiki/Mean_corpuscul ar_volume http://en.wikipedia.org/wiki/Mean_corpuscul ar_volume http://en.wikipedia.org/wiki/Microcytosis

33. Sites On Hb M-Saskatoon http://web2.airmail.net/uthman/hemoglobin opathy/hemoglobinopathy.html http://web2.airmail.net/uthman/hemoglobin opathy/hemoglobinopathy.html http://www.ncbi.nlm.nih.gov/pubmed/76630 00 http://www.ncbi.nlm.nih.gov/pubmed/76630 00 http://globin.bx.psu.edu/cgi- bin/hbvar/query_vars3?mode=output&displa y_format=page&i=359 http://globin.bx.psu.edu/cgi- bin/hbvar/query_vars3?mode=output&displa y_format=page&i=359