joanne mason, registered clinical scientist west midlands regional genetics laboratory,

Acquired isodisomy of chromosome 21 in an acute myeloid leukaemia (AML) patient as an incidental finding during routine chimaerism

analysis, and the introduction of a new RUNX1 screening service.

Joanne Mason, Registered Clinical ScientistWest Midlands Regional Genetics Laboratory, Birmingham Women’s NHS Foundation Trust,

Joanne Mason, WMRGL Birmingham

Introduction• AML is a genetic disease

• Characterised by enhanced proliferation & differentiation block

• ~50% cases have cytogenetically visible aberrations

• The remaining cases have genetic aberrations which are only detectable at the molecular level

• These genetic lesions help to characterise the subtype of leukaemia, and can be used to guide therapeutic decisions and inform prognosis

• Molecularly-targeted therapy (e.g. Glivec in CML)


Patient A

• Diagnosed with AML in May 2006• Karyotype analysis: trisomy 13 (47,XY,+13 [10])• Treated with chemotherapy on the MRC AML15

trial protocol

• Relapsed November 2007 (47,XY,+13)• Salvage chemotherapy, followed by stem cell

transplant (SCT) in March 2008


Chimaerism monitoring post-SCT• Sex-matched SCT patients are monitored for levels of

donor and host DNA post-transplant using polymorphic microsatellite markers.

• A pre-requisite for chimaerism analysis is to find at least one informative marker that distinguishes donor from host.

CAGA CAGA 3-15 CAGA CAGA CAGA


Multiplex microsatellite marker PCR and subsequent fragment analysis


Chimaerism analysis

POST-TRANSPLANT

74% donor26% host

HOST PRE-TRANSPLANT

DONOR


0100002000030000400005000060000700008000090000

100000110000120000130000

100 150 200 250 300 350 400 450 500

Dy

e

Si

gn

al

Size (nt)

133.21134

D21S1437 241.36241

D21S11

245.43246

D21S11

257.62258

D21S11

309.79310

D21S1270

311.85312

D21S1270

327.89328

D21S1270

398.40399

D13S634

415.55416

D13S634

490.06490

D18S535

494.22494

D18S535

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

100 150 200 250 300 350 400 450 500

Dy

e

Si

gn

al

Size (nt)

160.95161

D18S391

246.01246

D13S742

268.35268

D13S742

289.26289

D13S742

379.82380

D18S386 443.85444

D13S305

451.61452

D13S305

0

2500

5000

7500

10000

12500

15000

17500

20000

100 150 200 250 300 350 400 450 500

Dy

e

Si

gn

al

Size (nt)

105.23105

AMEL

111.00111

AMEL

145.93146

D18S1371

150.04150

D18S1371

308.04307

D21S1411

462.61462

D13S628

464.57464

D13S628

Microsatellite results• Pre-transplant

DNA

13 1313

0100002000030000400005000060000700008000090000

100000110000120000130000

100 150 200 250 300 350 400 450 500

Dy

e

Si

gn

al

Size (nt)

241.36241

D21S11

245.43246

D21S11

257.62258

D21S11


Chromosome 21 markers

Average ratio 4:1

D21S11

Penta D D21S1411

D21S1270

Remission DNA

Relapse DNA


• Possible explanations for the discrepancy:

– 1) Sub-microscopic deletion within chromosome 21 (unlikely as multiple deletions would be required)

– 2) A cryptic sub-clone with gain or loss of 21 in some cells, not detected by initial cytogenetic analysis (impossible with a microsatellite ratio of 4:1)

Ch 21 markers : copy number change?

Cytogenetics 2 normal copies Ch 21

– 3) Acquired isodisomy (aka acquired uniparental disomy, or copy number neutral loss of heterozygosity)


Acquired isodisomy (AID)

• Common mechanism of oncogenesis

• Prognostic significance in AML?


Mitotic Recombination

21 21

AID 21


Acquired isodisomy (AID)

• AID is a mechanism by which homozygosity for a mutation can be achieved without detrimental loss or gain of contiguous chromosome material

• It is cytogenetically invisible (both chromosomes look the same) and therefore very difficult to detect unless you specifically look for it.

• DNA microarrays – sub-microscopic & cryptic changes


AID21: What genes might be affected?

• RUNX1 21q22.3• Transcription factor• Most frequent target for chromosomal

translocation in leukaemia

• Point mutations – in sporadic AML – In familial platelet disorder/AML (FPD/AML)


RUNX1 point mutations in sporadic AML• 1.2% of all AML• Highly associated with

– AML FAB M0– trisomy 21 – trisomy 13 (80-100%) [Patient AS 47,XY,+13]

• RUNX1 mutation associated with a poor prognosis in MDS (prognosis in AML not yet known)

• Discovery of mutations has implications for – Risk adapted therapy– Molecularly targeted therapy


Familial Platelet Disorder with Predisposition to Acute Myeloid Leukaemia (FPD/AML)

• Rare autosomal dominant disorder• Characterised by inherited thrombocytopenia,

platelet function defect and a lifelong risk of myelodysplastic syndrome (MDS) and AML

• Caused by heterozygous germline mutations in RUNX1

• Worldwide, only fifteen pedigrees have been reported to date.

• In November 2008, request for ?FPD/AML in a West Midlands AML patient.


RUNX1 Point Mutations

• RUNX1 mutation screening service– AID21 patient – AML cases with a strong association with

RUNX1 mutations (FAB M0, +13)– FPD/AML patient

• Sequencing of the entire coding region


RUNX1 mutation screening service

• cDNA template• PCR under same conditions (‘touchdown PCR’)• M13 tag to facilitate high-throughput sequencing

a

b

c

d

Primer sequences courtesy of Dicker et al, Leukemia 2007


RUNX1 sequencing results.....so far

• Patient A: p.Asp171Gly (D171G, homozygous)• DNA binding domain

• Previously reported in two AML patients

• 26% of mutations in RUNX1 are homozygous (wild-type RUNX1 is lost)

Wild-type

Patient AS


• SNP-based DNA microarrays to investigate cytogenetically cryptic areas of somatically acquired homozygosity (AID)

• Postulated that such regions contain homozygous mutations in genes known to be mutational targets in leukaemia.

• In 7 of 13 cases with acquired isodisomy, homozygous mutations were identified at four distinct loci (WT1, FLT3, CEBPA, and RUNX1)

• The mutation precedes mitotic recombination, which acts as a "second hit" responsible for removal of the remaining wild-type allele.


RUNX1 sequencing results.....so far• ?FPD/AML patient and three AML patients with trisomy

13 (i.e. highly likely to have RUNX1 mutations)

• Patient B AML 47,XX +13– p.Val137_Gly138insThr

wt

B

wt

C

• Patient C AML 50,XY +8,+9,+13,+21– p.Met25Lys– p.Arg135Lys

• All de novo, but two other mutations involving arginine 135 have been reported before


Further work• Complete the sequence analysis of all four

fragments comprising the coding region of RUNX1

• Effect of mutations?

– Inheritance pattern in familial cases

– Confirm RUNX1 mutations are acquired and not constitutional by sequencing stored remission DNA


SummaryUnexpected microsatellite pattern in pre-transplantDNA taken at relapse

Molecular data + cytogenetic data = acquired isodisomy 21

Candidate gene = RUNX1

RUNX1 mutation D171G

Sequencing service for other sporadic AML patients,and for suspected FPD/AML referrals.


Acknowledgements

• Birmingham, WMRGL:– Val Davison– Mike Griffiths– Fiona Macdonald– Susanna Akiki– Paula White– Natalie Morrell– Charlene Crosby

• Birmingham Clinicians:– Dr Prem Mahendra– Prof Charlie Craddock

Thank you for your attention