Constitutional 9p22q translocation in a patient with melanoma, deafness and DNA repair deficiency disrupts p14arf and down-regulates TBX1

Xiaohui Tan1, Sarah Anzick2, Sikandar G. Khan1, Takahiro Ueda1,3, Gary Stone2, John J. DiGiovanna1,4, Deborah Tamura1, Daniel Wattendorf5, Carmen Brewer6, Chris Zalewski6, Robert Walker2, John A Butman7, Andrew Griffith6, Paul Meltzer2, Paul Bergstresser8 and Kenneth H. Kraemer1

1DNA Repair Section, Dermatology Branch, CCR, NCI 2Genetics Branch, NCI 3Pharm/ Medical Device Agency, Tokyo, Japan 4 Derm, Brown Med School, Providence, RI 5Office of the Air Force Surgeon General 6Otolaryngology Br, NIDCD 7Radiology, CC 8Derm, Univ of Texas SW, Dallas, TX DNA REPAIR DEFICIENCY, MELANOMA, DEAFNESS, AND 9p22q TRANSLOCATION – A NEW SYNDROME?

Low set ears Contractures

Posterior fifth toes

12 y/o Patient DD129BE Sensorineural deafness, DiGeorge Syndrome / Velo-cardio-facial Syndrome like features Melanoma 0.25 mm

14 y/o CLINICAL EXAMINATION

• Audiologic assessment No measureable hearing bilaterally and absent vestibular reactivity

• CT and MRI Bilaterally symmetric cochlear hypoplasia and vestibular dysplasia with absence of the cochlear nerve. ANALYSIS

t(9;22)(p21;q11.2)

Reciprocal translocation between the short arm of and the long arm of THE AIM

1. To map the chromosome breakpoints in cells from patient DD129BE 2. To develop a detailed molecular characterization of the candidate for his clinical syndrome of melanoma, defective DNA repair and deafness. FISH analysis of t(9;22) with whole chromosome painting Chromosome 9 Chromosome 22

CHROMOSOME 9 and 22 PROBES ARE SPLIT BY THE TRANSLOCATION FISH analysis of the translocation on chr9 q21.33 q21.13 p21.3 p21.1 p12 p22.3 p24.1 q34.3 q33.1 p24.2 q31.1 q12

9p11-q11CEP 9p21 LSI p16 Normal 9

der 22

der 9

CHROMOSOME 9 p16 PROBE IS SPLIT BY THE TRANSLOCATION Array Comparative Hybridization (aCGH) of Total Genomic DNA

No detectable changes BIVARIATE FLOW SORTING TO ISOLATE DERIVATIVE

Normal chromosomes Patient chromosomes

derivative chromosome 9

derivative chromosome 22 Derivative Chromosomes can be Separated By Bivariate Flow Sorting Breakpoint Located Within CDKN2A Using aCGH On Flow Sorted Derivative Chromosome 9

Area 1 derivative BREAKPOINT chromosome 9

Break on Chromosome 9 splits CDKN2A gene

BREAKPOINT

TEL CEN

Intron 1 Breakpoint on Chromosome 9 Interrupts p14/ARF Leading to Melanoma Laser Capture Microdissected Melanoma Tissue from patient DD129BE

Two UV-type Missense in Exon 1 β of p14arf Gene

Wild type gCAG>CGG pQ57R gAGA>GGA pR62G Knudson Model for Cancer

The melanoma in patient DD129BE may have resulted when one copy of p14arf was disrupted by the translocation followed by somatic mutations in the other copy. DNA REPAIR DEFICIENCY IN PATIENT DD129BE FIBROBLASTS

Reduced HCR is not corrected by any of the known XP complementation groups Increased HCR by Co-transfection with p14

DNA repair defect improved by transfection with p14 cDNA vector FISH Analysis of DiGeorgechr22:17,300,000-20,000,000 Syndrome Critical Region on Chr22

3 megabase DiGeorge Syndrome Critical Region

17500000 18,000,000 18,500,000 GAP 19,000,000 19,500,000 3090o16 278E23 VCFS probe 138C22 3148D21 586I18 562F10 - - - + + +

586I18 Normal 22 3090o16 562F10

Der 9 Der 22 Der 22 Der 22 Normal 9

Der 9 Normal 22

Normal 9 Normal 22 Der 9

562F10, 3090o16 and 586I18 PROBES ARE SPLIT BY TRANSLOCATION Array CGH On Flow Sorted Derivative 22

Areas 3-8 BREAKPOINT pooled derivative chromosome 22 980kb

CHROMOSOME 22 BREAKPOINT LIES WITHIN A BREAKPOINT HOTSPOT REGION IN A GAP IN THE MAP

Breakpoint hotspot 22d 22c 22b 22b 22c 22a PATRR22 GAP GAP PATRR22(1kb)

22q11.2 17500000 18,000,000 18,500,000 19,000,000 19,500,000

VCFS probe 138C22 3148D21 586I18 562F10 278E23 BACs - - + + ISOLATION OF TRANSLOCATION JUNCTION FROM Derivative 9 BY PCR

Normal Chr9-1R Chr9-5R Chr9-6R Chr9-5F Chr9-6F Chr9-1F Chromosome 9

Derivative 9 Chr9-1R Chr9-5R Chr9-6R 22b 22c 22d

Centromere telomere

TA repeat variable palindrome in PATRR22 TA-rich region flanking the TA repeat variable region on chr 22 Breakpoint JunctionPATRR22 is present region on chr 9 Lymphoblast Genomic DNA Genomic DNA only in cells from patient DD129BE

(bp) (bp) 2000 2000 1000 1000 300 300 100

(bp)

800 300 100 Junction Fragment Sequences of Der9

Chr9-1R Chr9-5R Chr9-6R 5F3R 6F 4R 4F 3F Chr9-1F Chr 9 Centromere Chr9-1R Chr9-5R Chr9-6R 22b22c 22d telomere Der 9

SINE (88% similarity to regions TA repeat variable region on chr 22 palindrome in PATRR22 on PATRR22 and chr9) TA repeat variable region on chr 9 TA-rich region flanking TA-rich region flanking the TRANSLOCATION BREAKPOINTthe PATRR22 LIES PATRR22WITHIN AT-RICH REGIONS ON CHR 9 AND CHR 22

Sequence of normal chromosome 9 Sequence of der 9

Bold lettering indicates the sequence on der(9) Localization of Translocation Breakpoints Within PATRR22

t(11;22)

t(9;22)

-31

9,22 TRANSLOCATION BREAKPOINT IS IN A BREAKPOINTKurahashi, HOTSPOT et al. REGIONGenome Research, WITHIN (2007) PATRR22 17:461-469 AK12956719,985,286

AK129567 19, 852,118 278E23

19,809,922 DKF2p434k 19119, 802,941 BCR 19, 787,305

LZTR1 19, 663,751 DKF2p761 18,404,283

SERPIND1 19,458,383 PI4KA 19, 391,979

RTN4R 19,257,965 22a PCQAP 19, 191,886 KLHL22 19, 125,806 SCAF2 19, 108,875

22c ZNF74 19, 078,480 19,057,981 562F10

19,022,374 USP41 19,047,911 19,000,000 19,500,000 19,000,000 22b

18,940,000 AK129567 18,962,132 (1kb) 18,890,000 GAP 18,859,173 50kb 586I18 KIAA1666 18,835,994 22b PATRR22 PI4KAP2 18, 763,731

18,696,552 AK129567 18, 706,399 22c DGCR6L 18,682,046 RTN4R 18,608,938 t(1;22) t(4;22) t(11;22) t(17;22) t(17;22) t(11;22) t(4;22) t(1;22) 22d 3148D21 18,444,588 DGCR8 18,453,210 HTF9C 18,479,398

800 KB C22orf25 18,388,637 2655G7 18,325,388 NT-011519 138C22 18,259,187

22q11.2 C22orf29 18,213,661 18,259,187 186o8 TBX1 18,124,226 18,077,328 TBX1 probe TBX1 18068734-18,298734 LOC150185-17,918,882 3 megabase DiGeorge Syndrome Critical Region

HIRA 17,698,224-17799219 1057H19 17,690,710

CLTCL17,546,987 is 800kb away from the breakpoint in gap 17500000 18,000,000 18,000,000 17500000 18,500,000 t(X;22) TBX1 PATRR22 22q11.2 BACs Breakpoint hotspot Genes FISH analysis of the translocation on chr22 with TBX1 probe

Chromosome 9

Chromosome 22 Der 9

Der 22

TBX1 PROBE IS NOT SPLIT BY THE TRANSLOCATION No mutations in promoter or exons in three isoforms of TBX1 Dramatically reduced TBX1 Expression in Patient’s cells

Semiquantitative RT-PCT Lymphoblast Fibroblast

t r l t r l r n e r n e r a e r e e a e e k e i th i th h m r t t th rm t t r a a a o a o a o P a o P M W M F N M F N TBX1 650bp β actin 300bp No detectable TBX1 expression in DD129BE cells by PCR or qRT-PCR Real Time T-PCT Fold Change Relative to Normal Fibroblasts (AG13354) Fibroblasts Normal Patient Mother Father Microarray Analysis of Expression of the Genes Around the Translocation

Low expression of CDKN2A and TBX1 by microarray SUMMARY

• We have demonstrated that the constitutional t(9;22) translocation occurs in AT-rich repeat intervals on both chromosomes 9 and 22

• The rearrangement was mediated by a 6 bp CACGTG palindromic sequence between the breakpoints. Chromosome 9 had a of 71 bp in an AT-rich repeat region in intron 1 of CDKN2A gene, and chromosome 22 had a deletion of 62 bp in PATRR22 in an uncloned gap.

• The melanoma in this patient may have arisen according to the Knudson model in which one copy of p14arf is disrupted by the translocation followed by a somatic mutations in the other copy CONCLUSIONS

These rearrangements on chromosomes 9 and 22 in patient DD129BE resulted in: 1. down regulation of CDKN2A on chr 9 that contributes to the melanoma susceptibility and reduced DNA repair and 2. down regulation of TBX1 on chr 22 that contributes to his deafness.