1. Understanding RRP a genetic undertaking
Farrel J. Buchinsky, MD
Pediatric Otolaryngologist
Allegheny General Hospital
Pittsburgh, PA
Center for Genomic Sciences
Allegheny Singer Research Institute

2. BLUF (bottom line up front)
Enrollment tripled since Los Angeles focus meeting
Discovered transmission disequilibrium in 2 candidate genes

3. What is the cause of RRP? HPV 6 & 11 Necessary Is it sufficient?
Put the fetus and the vagina here
Necessary
Is it sufficient?

4. Many exposed
Only few get the disease
**correct the spacing

5. Genetic Susceptibility
higher prevalence in relations
But still very rare
HLA DRB1*0301 and DQB1*0201 disproportionately present in RRP
associated with decreased interferon γ expression
Partial reproduc of Gelder
Also assoc in cervical cancer
Epidermodysplasia verruciformis
Bonagura; Hum Immunol. 2004
Gelder; J. of Vir. 2003
Madeleine; JID. 2002
Ramoz; Nature G. 2002

6. Epidermodysplasia Verruciformis
Rare, autosomal recessive, skin disease
Abnormal susceptibility to specific HPVs (5 and 8)
persistent lesions that do not clear virus
4 specific mutations found in EVER1
Dr. Mark Naylor, Electronic Textbook of Dermatology

7. More Genetic Evidence Rabbit papilloma data HIV/AIDS
14 AIDS restriction genes explains some of variability rate at which a population progresses from HIV infection to AIDS
Malaria, Otitis Media
Mortality by infectious disease in adoptees more associated with biologic parents than seen for cardiovascular and cancer
O'Brien; Nature G. 2004
Sørensen; NEJM. 1988

8. From who?

9. So what’s needed
Specimen of Blood
2 specimens
1 mm * 1 mm OR
HPV type

10. Acquisition
IRB
IRB
IRB

11. Enrolment
92 from Patient Support Groups

12. Recruitment Source Count Institution City PI 92
Allegheny Singer Research Institute
Pittsburgh
Buchinsky 23
Children's Medical Center of Dallas
Dallas
McClay 21
Cincinnati Children's Hospital Medical Center
Cincinnati
Myer 17
The Hospital for Sick Children
Toronto
Campisi 14
Children's Hospital of Alabama
Birmingham
Wiatrak 12
Children's Hospital of Wisconsin
Milwaukee
Conley 11
Children's Hospital of the King's Daughters
Norfolk
Derkay
Children's Healthcare of Atlanta at Egleston
Atlanta
Sobol
Children's National Medical Center
Washington
Choi 10
University of Mississippi Medical Center
Jackson
Schweinfurth 7
Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo
Sao Paulo
Tsuji 5
SUNY Upstate Medical Center
Syracuse
Mortelliti 4
Johns Hopkins Children's Center
Baltimore
Tunkel
Le Bonheur Children's Medical Center
Memphis
Thompson
Children's Hospital of Philadelphia
Philadelphia
Elden 3
Children's Hospital of Pittsburgh
Dohar 1
University of Pittsburgh Medical Center
Rosen
Allegheny General Hospital
British Columbia's Children's Hospital
Vancouver
Kozak

13. Which gene or genes 3x109 bases Candidate Gene Approach
Genomic Scan Approach

14. Candidates MHC Innate Immunity Known Cell Biology Interactions
Toll-like receptors
Cytokines regulating the inflammation
Transcription factors regulating immune response
Known Cell Biology Interactions
E5 inhibits H+ATPase from acidifying vesicles
HPV cell receptor
Other Diseases
Epidermodysplasia Verruciformis
Cervical Cancer

15. First Candidate Gene Batching
5 genes
214 SNPs on Sequenom MassARRAY
212 cases + relatives

16. Transmission Disequilibrium Test
Allele 1 = 76%, Allele 2 = 24%
Many trios uninformative
Allele 2 transmitted 16 times, untransmitted 1

17. TDTae in 2 candidate genes
But first...
Quality control scrutiny
Additional SNP genotyping
Sequencing
Gordon; Euro J. Hum. Gen. 2004

18. Whole Genome Illumina SNP Assay 96 humans at a time
SNPs at a time
/\/\/\/
illumiCode
#561
#217
#1024
A/A
T/T
A/T
After amplification the products are hybridized to the Sentrix array for detection
The internal illumiCode that is specific for each locus binds only to its complementary bead
(the position of which was previously identified by decoding)
The genotype is then easily and automatically called as
Loci that are homozygous of an allele will show signal in either the correlated green (Cy3) or red (Cy5) channel and those that are heterozygous show signal in both channels
Illumina SNP Assay

19. Acknowledgments Thank You! CGS Rockefeller University
Garth Ehrlich, Chris Post
Joseph Donfack, Marilyn Smith, Fen Hu
Rockefeller University
Jurg Ott, Sara Hamon
Earliest Collaborators
Craig Derkay, RRP Task Force
Patient Support Groups
Michael Green, Bill Stern
Funding
NIH – Mentored Clinical Scientist Development Award (Mentored Clinical Scientist Development Award (K08))
RRP ISA – IRB assistant
(412)
Thank You!