1. Direction of DBA Research and Overview Steven R. Ellis, Ph.D. Professor, Department of Biochemistry University of Louisville July 16 th 2015 Camp Sunshine Research Director Diamond Blackfan Anemia Foundation

2. General Trends in DBA Research Focus on translational research: Bench to bedside Continued reliance on private Foundations to support DBA research Improved drugs Genetics; gene discovery, genotype/phenotype relationships Beyond anemia; syndromic DBA 2012 - 3 year, DOD Award to Dr. Lodish -New drugs for anemia treatment based on a new understanding of the mechanisms of stress erythropoiesis April 2014 - bridge grant (DBAF) – between years 1 and 2, $21, 281 August 2014 – program discontinued September 2013 – anticipating funds for year 2 $95,000 awarded to Dr. Lodish (DBAF and DBAC) to continue his work

3. Your Foundations at Work 4/2015 – DBAF and DBAC awarded a grant of $35,000 to Johan Flygare to help start up a new lab and screen a library of over 12,000 chemical compounds to identify potential drugs that alleviate the symptoms of their mouse model of DBA 2008/2009 Johan was a post-doctoral fellow in Harvey Lodish’s laboratory and did the initial work on how glucocorticoids stimulate red cell production Prior to 2008, Johan was a graduate student in the laboratory of Stefan Karlsson helping develop the mouse model for DBA which is being used to test gene therapy approaches for DBA 4/2014 – DBAF awarded a grant of $36,000 to Vijay Sankaran to study the role of GATA1 in red cell production with an eye toward novel therapies for DBA 1/2015 – DBAF and DBA UK awarded a grant of $41,810 to Nickolas Watkins to study the role of RPL5 and RPL11 in signaling p53 activation

4. Physicians like Drs. Vlachos and Lipton will have their hands full with clinical trials Increased number of potential drugs coming down the R&D pipeline Drugs specifically directed at DBA /drugs targeted at the intrinsic nature of genetic diseases Personalized medicine, drugs as diverse as the heterogeneity of DBA patients Direction of DBA Research (2015 Predictions) A drug may target a subset of DBA genes A drug may target a particular type of genetic lesion

5. AUG – UUU – AGG – CUG – GCA – AAG – CUA – AAA – GGA – UCC - UAA Met – Phe – Arg – Leu – Ala – Lys – Leu – Lys – Gly – Ser - Term Central Dogma of Molecular Biology mRNA Protein AUG – UUU – AGG – CUG – GCA – AAG – CUA – AAA – GGA – UCC - UAA aagcuuccaau//gguccaucguac transcription splicing pre-mRNA translation 3.5 billion bases intron exon

6. AUG – UUU – AGG – CUG – GCA – AAG – CUA – AAA – GGA – UCC - UAA Met – Phe – Arg – Leu – Ala – Lys – Leu – Lys – Gly – Ser - Term mRNA Protein translation C Phe silent/synonymous U Term nonsense/nonsynonymous frameshift Mutation Muddle G Glu missense/nonsynonymous Lys NH 3 + Glu COO - Asp COO - *

7. With the Advent of Whole Exome and Whole Genome Sequencing We are Faced with an Increasing Number of Variants of Unknown Significance (VUS) An extended family seen by the bone marrow failure unit at the National Cancer Institute 3 affected family members with a VUS in RPS19 V138L, valine to leucine substitution at position 138 COO - C HH 3 + N CH CH 3 COO - C HH 3 + N CH CH 3 CH 2 V, Valine L, Leucine

8. LVVILLVVIL human Gregory (2007) NAR The in silico prediction programs are not unanimous about whether this would be a potentially disease causing variant. It does not exist in any public databases. V138L The Plot Thickens

9. Pre-rRNA Processing as a Means of Studying Ribosomal Protein Function

10. Patients Display a Pre-rRNA Processing Signature Consistent with V138L Being a Pathogenic Mutation Flygare, Aspesi et al. 2007 Blood RPS19 Knockdown ✔ ✔ PPC ✔

11. FINAL THOUGHTS These Folks Represent Only a Fraction of the People Out There Working to Help Your Kids

12. AUG – UUU – AGG – CUG – GCA – AAG – CUA – AAA – GGA – UCC - UAA Met – Phe – Arg – Leu – Ala – Lys – Leu – Lys – Gly – Ser - Term mRNA Protein translation C Phe silent/synonymous U Term nonsense/nonsynonymous frameshift Mutation Muddle G Glu missense/nonsynonymous Atalurin – a drug that causes ribosomes to read through premature termination codons

13. AUG – UUU – AGG – CUG – GCA – AAG – CUA – AAA – GGA – UCC - UAA Met – Phe – Arg – Leu – Ala – Lys – Leu – Lys – Gly – Ser - Term Mutation Muddle mRNA Protein AUG – UUU – AGG – CUG – GCA – AAG – CUA – AAA – GGA – UCC - UAA aagcuuccaau//gguccaucguac transcription splicing pre-mRNA translation Drugs are in development to fix splicing defects

14. Physicians like Drs. Lipton and Vlachos will have their hands full with clinical trials Increased number of potential drugs coming down the R&D pipeline Drugs specifically directed at DBA /drugs targeted at the intrinsic nature of genetic diseases Personalized medicine, drugs as diverse as the heterogeneity of DBA patients Specific Trends in DBA Research A drug may target a subset of DBA genes A drug may target a particular type of genetic lesion