1. RESEARCH FUNDED BY DBAF AND DBA CANADA: ONE RESEARCHER’S EXPERIENCE

2. LIFE CHANGING MOMENT: 2009

3. GENETICS OF DBA

4. DIAMOND BLACKFAN ANEMIA MUTATIONS - 2009
genotype unknown
RESEQUENCING PROJECT

5. DBAR RESEQUENCING PROJECT
Diamond Blackfan Anemia Registry
n > 700 patients
Jeff Lipton
Adrianna Vlachos
Mutation analysis
n = 115 patients
North American DBA Registry
Patients with known DBA-associated RP mutations n = 40
Patients without defined mutations
n = 75 patients
There are 609 patients enrolled in the Diamond Blackfan Anemia Registry. A subset of 155 patients underwent standard mutation analysis by gene sequencing. 40 of these patients were found to have previously identified RP gene mutations patients did not have any of the known gene mutations or had not been fully genotyped. 75 of these were submitted to the NIH’s DNA Resequencing and Genotyping Service. Resequencing of the 80 RP genes was performed on these samples. 24 patients were found to have defined mutations in previously published RP genes. 23 patients were found to have RP sequence changes whose significance has not been confirmed and 28 patients were found to have no identifiable sequence changes. Sufficient DNA was available on 23 of these 28 patients for further study.
Resequencing = We amplify the specified gene(s) or region(s) requested by the investigator using PCR. The PCR primers are designed to specifically target the gene(s) or region(s) requested. They are Tm matched and designed from a masked reference sequence to exclude design in problematic regions such as repeat motifs and high GC content. Each primer pair is "tailed" with a universal M13 forward and reverse sequencing primer to enable subsequent sequencing. Optimal PCR conditions are determined empirically or computationally and confirmed by multiple quality assurance steps. Once the regions are amplified, each PCR product is sequenced from the forward and reverse direction to provide double-stranded coverage. Sequencing is carried out with Sanger Big-Dye Terminator sequencing, and detection with capillary-based sequencing machines.
Resequencing of 80 ribosomal genes
n = 75
Defined mutations found
n = 24 (32%)
Changes of unknown significance
n = 23 (31%)
No identifiable sequence changes
n = 28 (37%)

6. COST OF SEQUENCING A GENOME
Gordon Moore: Over the history of computing hardware, the computational capacity of a computer doubles approximately every two years

7. HUMAN CHROMOSOMES
But as we learn about the structure of genomes, we have learned that all of us have places where we are missing a part of a chromosome. If we sequence in that region, we only get the sequence of one chromosome. If the deletion causes the disease, we miss it because you can’t sequence what is not there, and we only see the sequence from the complete chromosome.

8. HUMAN CHROMOSOMES
But as we learn about the structure of genomes, we have learned that all of us have places where we are missing a part of a chromosome. If we sequence in that region, we only get the sequence of one chromosome. If the deletion causes the disease, we miss it because you can’t sequence what is not there, and we only see the sequence from the complete chromosome.

9. DETECTION TECHNOLOGY
Because we know the sequence of the human genome, we can make little DNA molecules from any place we want in the genome, and attach them to beads. The bead is the blue ball here, and the DNA in blue is attached to that bead. You can think of these little DNA pieces as the dots on a map indicating where different cities, towns or points of interest are. It is relatively easy to find a million of these little markers that are spread out all over the genome short distances apart. But it requires robots to make all of these DNAs, attach them to the beads and place them in a grid so we know where each one is. These robots were first developed to help with the sequencing, but now have been refined for new jobs.
Once you get your grid ready, you can take DNA from a DBA patient break it up into small pieces and because DNA is double stranded, it will find it’s perfect match and stick there. In the example shown here, the first bead shows a sequence that is represented on both chromosomes, so all the targets are filled. The middle one is from a patient with a deletion in this region and you can see that DNA from one chromosome finds a home, but because the other is missing this region, there is less DNA stuck.

10. DECIPHERING THE DATA

11. DELETION OF RPS19 IN DBA Frequency B allele Log R ratio
Single-copy deletions of RPS19 and RPS26 in DBA probands. Log2 intensity ratio of SNP probes surrounding deletions of RPS19 on chromosome 19 (A) and RPS26 on chromosome 12 (B). The regions displayed include chromosome 19 from and chromosome 12 from , corresponding to the boxed region in the chromosome ideogram above each panel. The region of single-copy loss is shaded. The locations of RPS19 and RPS26 are indicated by the vertical line through the deletion regions.

12. 1.6 Mb RPS17 deletion
Log R
ratio
15 kb RPS26 deletion
Log R
ratio

13. DELETION OF RPL15 AND RPL31 IN DBA
Frequency
B allele
Log R
ratio
Farrar JE et al. Am J Hematol. 89:985-91, 2014.

15. SEQUENCING PIPELINE
COST OF SEQUENCING A GENOME

16. Whole Exome sequencing Whole Genome sequencing
Mutation Analysis of DBA Patients
Common DBA mutations
negative
Whole Exome sequencing
Whole Genome sequencing
Hypothesis: Mutations in genes involved in ribosome assembly and function
Large deletion analysis
negative
Hypothesis: Small deletions, mutations in regulatory regions

17. SUMMARY OF EXOME SEQUENCING RESULTS
12 Families completed
20 candidate variants analyzed
Functional Validation negative
Variants tested were not found in ~200 patients in DBAR
Conclusion: Mutations in the coding regions of non RP genes are not a frequent cause of DBA

18. Whole Genome Sequencing Analysis
Summary of Genome Sequencing Results
Small deletions detected in 3/5

19. SMALL MOLECULE SCREEN FOR DBA

20. REPORTER ASSAY FOR RP GENE EXPRESSION
mRNA (90%): ANNNNN….ATG
TOP mRNA (10%): CYYYYYY…ATG
Renilla
Firefly
TRE
L32 5’UTR
CUCUCUUCCUC
pTET2-L32TOP 2A
AAGAGAAGGAG
pTET2-L32noTOP
Crysti Tsujiura

21. INHIBITION OF TRANSLATION
L32TOP
L32noTOP
Fold increase in
luc expressin
+ serum
- serum
dox concentration (mM)

22. AUTOMATED SCREENING: 1536 AT A TIME
Robotics
Nanoliter Dispensing 1 2
Microscopy
So knowing the targets, we can design experiments that can see if we can use a drug to make the target do what WE want instead of what IT wants to do. Robots that can make the screening of 300,000 new compounds for effects on the target genes associated with DBA in a matter of hours.
But in reality is is not the robots, and it is not the people operating them that is going to provide a cure for DBA or any other disease.
It is people like you who care enough to come out and support these kind of endeavors that will enable the robots and their operators to help. I am honored to be with you tonight.
Laser Cytometry
Current avg ~ 2.2 million wells/wk 22 22 22

23. DBA Patients and Families:
FINAL THOUGHTS
DBA Patients and Families:
YOU ARE NOT ALONE!
Each of the investigators in this photo represent teams of dedicated people who are trying to understand, treat and cure DBA.

24. FINAL THOUGHTS

25. THANK YOU
Thanks to all of you for being here. You are providing the most valuable contribution of all:
Hope, Support and a Sense of Community.
No DBA patient or family could feel alone in the presence of people like you.
No researcher could not be inspired by people like you.