M., Haas, O.A., Burmeister, T., Dingermann, T., Klingebiel, T. &

Marschalek, R. (2006) The MLL recombinome of acute leukemias.

Leukemia, 20, 777–784.

Nachman, J.B., Sather, H.N., Sensel, M.G., Trigg, M.E., Cherlow, J.M.,

Lukens, J.N., Wolff, L., Uckun, F.M. & Gaynon, P.S. (1998) Aug-

mented post-induction therapy for children with high-risk acute

lymphoblastic leukemia and a slow response to initial therapy. New

England Journal of Medicine, 338, 1663–1671.

Pieters, R., Schrappe, M., De Lorenzo, P., Hann, I., De Rossi, G., Felice,

M., Hovi, L., LeBlanc, T., Szczepanski, T., Ferster, A., Janka, G.,

Rubnitz, J., Silverman, L., Stary, J., Campbell, M., Li, C.K., Mann,

G., Suppiah, R., Biondi, A., Vora, A. & Valsecchi, M.G. (2007) A

treatment protocol for infants younger than 1 year with acute

lymphoblastic leukaemia (Interfant-99): an observational study and

a multicentre randomised trial. Lancet, 370, 240–250.

Pocock, C.F., Malone, M., Booth, M., Evans, M., Morgan, G., Greil, J.

& Cotter, F.E. (1995) BCL-2 expression by leukaemic blasts in

a SCID mouse model of biphenotypic leukaemia associated with the

t(4;11)(q21;q23) translocation. British Journal of Haematology, 90,

855–867.

Pui, C.H., Chessells, J.M., Camitta, B., Baruchel, A., Biondi, A., Boyett,

J.M., Carroll, A., Eden, O.B., Evans, W.E., Gadner, H., Harbott, J.,

Harms, D.O., Harrison, C.J., Harrison, P.L., Heerema, N., Janka-

Schaub, G., Kamps, W., Masera, G., Pullen, J., Raimondi, S.C.,

Richards, S., Riehm, H., Sallan, S., Sather, H., Shuster, J., Silverman,

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M. (2003) Clinical heterogeneity in childhood acute lymphoblastic

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Keywords: acute leukaemia, MLL, MLLT1, xenograft model,

translocation.

First published online 23 January 2008

doi:10.1111/j.1365-2141.2007.06966.x

Circulating haematopoietic progenitors are differentiallyreduced amongst subtypes of dyskeratosis congenita

Dyskeratosis congenita (DC) is an inherited multi-system

disorder classically characterized by a triad of abnormal skin

pigmentation, nail dystrophy and leucoplakia. Bone marrow

(BM) failure is the principal cause of mortality and would have

developed in the majority of patients by 30 years of age. DC

has been shown to be linked to mutations in components of

the telomerase complex including the nucleolar protein

dyskerin, the RNA component (TERC) and the reverse

transcriptase component (TERT) (Marrone and Dokal 2006).

While reduced BM haematopoiesis and a reduction in the

numbers of haematopoietic progenitors and their ability to

expand have already been reported in several cases of DC

(Colvin, et al 1984, Friedland, et al 1985, Marley, et al 1999,

Marsh, et al 1992, Yamaguchi, et al 2005), to date no

comparison of progenitors has been made between the

different genetic subtypes.

To this end, peripheral blood (PB) was obtained from nine

patients with DC (seven males, two females; mean age

24 years) of different subtypes and eight healthy volunteers

(seven males, one female; mean age 39 years) attending the

Royal London Hospital with informed, written consent and

approval from Research Ethics Committee (Table I). Within

6 h of venesection, mononuclear cells (MNCs) were separated

using Lymphoprep, washed twice with phosphate-buffered

saline, resuspended at 1Æ5 · 106 cells/ml in Iscove’s modified

Dulbecco’s medium + 2% fetal calf serum and incubated in

tissue culture plates for 2 h at 37�C in 5% CO2 humidified

atmosphere to remove plastic-adherent cells. Non-adherent

cells were plated at 105 MNCs/1Æ1 ml in methylcellulose-

containing medium (StemCell Technologies 0435, London,

UK) containing stem cell factor, granulocyte-macrophage

colony-stimulating factor, interleukin-3, interleukin-6, granu-

locyte colony-stimulating-factor, erythropoietin and serum in

35-mm Petri dishes and incubated at 37�C in a 5% CO2

humidified atmosphere for 12 days. Progenitor colonies were

identified and enumerated by light microscopy using visual

identification guidelines set out by StemCell Technologies. For

all statistical analyses, P-values <0Æ05 were considered to be

significant.

Overall, numbers of myeloid and erythroid progenitors were

reduced in all DC patients (Fig 1A). However, the genotype

of the patient correlated with the extent of reduction

with DKC1 mutations resulting in approximately fivefold

(P < 0Æ01), fourfold (P = 0Æ07) and twofold (P = 0Æ07) reduc-

tions in myeloid (granulocyte/macrophage colony-forming

units; CFU-GM), late erythroid CFUs and early erythroid

(erythroid burst-forming units; BFU-E) colonies respectively,

while TERC mutations resulted in the production of almost

Correspondence

ª 2008 The AuthorsJournal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 140, 712–727 719

Tab

leI.

Cli

nic

ald

etai

lso

fp

atie

nts

ino

ur

coh

ort

.

Mu

tate

d

gen

e

Nu

cleo

tid

e

mu

tati

on

Age

(yea

rs)

Cli

nic

al

pre

sen

tati

on

Mu

cocu

tan

eou

s

feat

ure

s

Hb

(g/l

)

Hb

F

(%)

MC

V

(fl)

WB

C

(·10

9/l

)

Neu

t

(·10

9/l

)

Lym

ph

(·10

9/l

)

Plt

(·10

9/l

)Dt

elR

efer

ence

DK

C1

G12

05A

43C

lass

ical

DC

Cla

ssic

altr

iad

142

––

5Æ7

3Æ4

1Æ7

192

)2Æ

9(H

eiss

,et

al19

98)

DK

C1

C10

58T

27C

lass

ical

DC

Cla

ssic

altr

iad

145

–89

Æ46Æ

83Æ

32Æ

419

9)

0Æ6

(Kn

igh

t,et

al19

99)

DK

C1

C10

58T

18C

lass

ical

DC

Cla

ssic

altr

iad

151

–98

Æ45Æ

12Æ

91Æ

492

)1Æ

2(K

nig

ht,

etal

1999

)

DK

C1

G12

05A

14Id

enti

fied

asp

art

of

fam

ily

stu

dy

Sub

tle

nai

lan

dsk

in

abn

orm

alit

ies

136

0Æ8

88Æ6

6Æ3

3Æ9

1Æ5

247

)5Æ

3(H

eiss

,et

al19

98)

TE

RC

110–

113d

el18

AA

Sub

tle

skin

pig

men

tati

on

abn

orm

alit

ies

117

3Æ4

103Æ

93Æ

41Æ

61Æ

490

)5Æ

3(M

arro

ne,

etal

2007

)

TE

RC

53–8

7del

26A

ASu

btl

esk

inp

igm

enta

tio

n

abn

orm

alit

ies

106

2Æ9

125Æ

23Æ

31Æ

41Æ

535

)2Æ

8(M

arro

ne,

etal

2007

)

TE

RC

G17

8A6

AA

No

ne

116

5Æ8

98Æ6

7Æ1

2Æ2

3Æ8

24)

2Æ2

(Mar

ron

e,et

al20

07)

TE

RC

C18

0T35

AA

Gre

yh

air

at16

year

s11

23Æ

111

9Æ3

3Æ9

2Æ1

1Æ3

24)

4Æ5

(Mar

ron

e,et

al20

07)

TE

RT

G18

92A

33T

hro

mb

ocy

top

enia

,

pu

lmo

nar

yfi

bro

sis

Gre

yh

air

at17

year

s13

90Æ

992

Æ83Æ

32Æ

40Æ

756

–U

np

ub

lish

edm

uta

tio

n

No

rmal

ran

ges:

135–

175

0–1

80–9

64–

112–

7Æ5

1Æ5–

415

0–40

0)

3Æ8

–+

5Æ7

–

Hb

,hae

mo

glo

bin

;Hb

F,f

oet

alh

aem

ogl

ob

in;M

CV

,mea

nco

rpu

scu

lar

volu

me;

WB

C,w

hit

eb

loo

dce

llco

un

t;N

eut,

neu

tro

ph

ilco

un

t;L

ymp

h,l

ymp

ho

cyte

cou

nt;

Plt

,pla

tele

tco

un

t;Dt

el,c

han

gein

telo

mer

e

len

gth

rela

tive

toco

ntr

ol

ases

tab

lish

edfr

om

hea

lth

yw

ild

-typ

eco

ntr

ol

dat

ain

ou

rla

bo

rato

ry;D

C,D

yske

rato

sis

con

gen

ita;

AA

,ap

last

ican

aem

ia.‘

–’

ind

icat

esd

ata

un

avai

lab

le.‘

Age

’is

the

age

of

the

pat

ien

t

wh

enth

eb

loo

dsa

mp

lew

asta

ken

.

Blo

od

cou

nt

figu

res

inb

old

hig

hli

ght

valu

eso

uts

ide

the

no

rmal

ran

ge.

‘Cla

ssic

altr

iad

’co

mp

rise

sab

no

rmal

skin

pig

men

tati

on

,n

ail

dys

tro

ph

yan

dle

uco

pla

kia.

Th

ere

fere

nce

sq

uo

ted

no

teth

efi

rst

do

cum

ente

dre

po

rto

fth

ed

isea

se-c

ausi

ng

mu

tati

on

.

Correspondence

ª 2008 The Authors720 Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 140, 712–727

no countable colonies. Only the CFU-GM colony counts for

DKC1 mutant samples reached a statistically significant

difference (P < 0Æ05) using a Mann–Whitney U-test, perhaps

because of the small numbers. All P-values for TERC mutant

samples were <0Æ05. The only TERT mutant sample available

for analysis gave a count intermediate between that of the

DKC1 and TERC mutants.

Dyskeratosis congenita is such a rare disorder that large

numbers of patient samples will always be difficult to assemble

(particularly where it is important to set up cultures within

12 h of venesection) and as such, the small numbers here

prevent the drawing of rigorous conclusions. However, given

that the patients were not selected on any other basis than they

presented consecutively at the clinic, the trend is unlikely to be

an experimental or statistical artefact.

There is an apparent link between the critically low

progenitor colonies from the PB of patients with TERC

mutations and the disease phenotype – DC patients with TERC

mutations tend to present initially with aplastic anaemia (AA).

BM progenitors from AA patients have been shown to be

particularly deficient in proliferation and potential for expan-

sion (Martinez-Jaramillo, et al 2002) and in this context the

link between progenitor count and clinical presentation is

logical enough. However, the findings were surprising as the

X-linked form of DC is generally considered to be more severe.

Taking our cohort into consideration, though the patients with

DKC1 mutations had relatively mild haematological deficien-

cies compared with those with TERC mutations, their muco-

cutaneous abnormalities were more severe. This dissociation

might suggest that a telomerase defect is primarily responsible

for the haematological aspects of the disorder while other

functions associated with dyskerin (e.g. ribosome biogenesis)

might play a greater role in development of mucocutaneous

abnormalities.

Looking more closely at the DKC1 mutant samples of the

three progenitor colony types assayed here, the BFU-E colonies

were the least reduced. As this colony type represents a more

primitive erythroid lineage, this is consistent with the

hypothesis that much of the DC phenotype is because of

premature death or senescence of haematopoietic cells. More

primitive cells might therefore have greater potential to expand

before reaching a critical check-point such as critically short

telomeres.

To determine the ability of progenitor cells to self-renew,

replica dishes were set up on the day of the initial colony count

for colony replating assays. After 7 days incubation, primary

myeloid colonies were picked and transferred to methylcellu-

lose-containing medium in 96-well plates. Plates were incu-

bated as above for a further 7 days before enumeration of

secondary colonies.

Interestingly, the proportion of primary colonies giving

rise to secondary colonies was broadly similar for DKC1

mutations and wild-type controls (Fig 1B) as were the average

numbers of secondary colonies produced by each primary

colony (Fig 1C). For the single sample with a TERT mutation,

both parameters were slightly below those for wild-type

and DKC1 mutant samples. As TERC mutants produced

few or no colonies, it was not possible to perform a reliable

replating assay.

The similar results between wild-type and DKC1 mutations

are intriguing, suggesting that the potential for renewal of

individual progenitors is not significantly reduced, only that

there are either fewer progenitors present in the PB of DC

patients or that fewer of those present are capable of initial

expansion.

Fig 1. Progenitor colony counts from the peripheral blood of

patients with dyskeratosis congenita (DC) and healthy controls: A)

Average granulocyte/macrophage colony-forming units (CFU-GM),

erythroid-colony forming units (CFU-E) and erythroid burst-forming

units (BFU-E) progenitor colony counts from 5 · 105 mononuclear

cells from peripheral blood of normal, wild-type controls and DC

patients with DKC1, TERC or TERT mutations. B) Percentage of

primary CFU-GM colonies which when transferred to fresh medium

gave rise to secondary colonies. C) The average number of secondary

colonies produced from each primary CFU-GM colony when trans-

ferred to fresh medium.

Correspondence

ª 2008 The AuthorsJournal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 140, 712–727 721

This report highlights another differentiating factor among

the various subtypes of DC, demonstrating a new link between

genotype and phenotype in the disease and has implications

for the diagnosis and treatment of DC patients dependent

upon the causative mutation in each case.

Michael Kirwan1

Tom Vulliamy1

Richard Beswick1

Amanda J. Walne1

Colin Casimir2

Inderjeet Dokal1

1Academic Unit of Paediatrics, Institute for Cell and Molecular Science,

Barts and The London, Queen Mary’s School of Medicine and Dentistry,

University of London, and 2School of Life Sciences, Kingston University,

London, England

Email: m.j.kirwan@qmul.ac.uk

Acknowledgements

This work was supported by the Medical Research Council and

The Wellcome Trust.

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doi:10.1111/j.1365-2141.2008.06991.x

Platelet count is an IPSS-independent risk factor predictingsurvival in refractory anaemia with ringed sideroblasts

Recently, Bowles et al (2006) reported that a low platelet mass

[i.e., mean platelet volume (MPV) · platelet count] is a marker

for poor survival in myelodysplastic syndromes (MDS), and

that the MPV provides independent prognostic information.

In their 58-patient cohort, median survival was 5 months for

platelet mass <0Æ6 ml/l, >82 months for platelet mass >1Æ2 ml/

l, and 30 months for intermediate values. MPV is easily

obtainable, so if the prognostic value of this measurement

could be independently confirmed, platelet mass and MPV

might prove clinically useful.

Platelet parameters are of particular interest in refractory

anaemia with ringed sideroblasts (RARS), especially with

respect to the provisional entity RARS with thrombocytosis

(RARS-T) (Szpurka et al, 2006). Therefore, we evaluated the

validity of MPV and platelet mass as prognostic indicators in

RARS.

Correspondence

ª 2008 The Authors722 Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 140, 712–727