LETTERS AND CORRESPONDENCE

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Angiotensinogen Gene Associated Polymorphisms and

Risk of Stroke in Sickle Cell Anemia: Additional Data

Supporting an Association

To the Editor: Cerebrovascular accident (CVA) is a rare feature of childhood

SCA (about 10% of children). Tang et al. [1] reported an odds ratio of 4 for a

pair of alleles of a microsatellite locus within the site of the angiotensinogen

gene (AGT) [1], suggesting both a means of identifying at-risk children as well

as a major role for this gene in the pathogenesis of the complication.

The present study was conducted in order to test this result in children in

Guadeloupe.

PATIENTS AND METHODS

One hundred fifty-six SS children, ages 2–18 years, at the sickle cell center

of Guadeloupe from 1984 to the present were enrolled in the study with

parental consent. Extra blood was taken for DNA extraction at routine

venepuncture.

Definition of CVA was as described elsewhere [2]. Microsatellite alleles

were typed by polymerase chain reaction using a non-radioactive procedure

[3,4]. Sizes and numbers of GT repeats were determined by direct DNA

sequencing of four homozygotes for different alleles using an AB1 Prism 310

sequence detection system (PE Biosystems, Foster City, CA). The nomen-

clature use here for alleles reflects the allele sizes, with a small number

identifying a small fragment.

RESULTS

The DNA of 8 children who suffered one or more CVAs was typed and

compared to those of a random sample of 148 children without strokes

(Table I). Testing the numbers of alleles in the two groups (CVA versus

non-CVA) gives a Pearson statistic of 14.21. Assuming a w2 distribution, n ¼12, provides a P value of 0.287, suggesting no overall evidence of association.

The maximum odds ratio for individual alleles is 3.93 (95% limits of

1.01–15.22, Pearson statistic 3.936, P ¼ 0.047) for allele sz28. These data

imply a risk of childhood CVA (which can be estimated from prospective

data) of 12% for children with this allele and 3% for the others.

The odds ratio for alleles sz22 and/or sz24, those implicated by Tang

et al. [1] in risk development, is 0.159 (0.025–1.033, Pearson 3.7, P ¼ 0.054).

Therefore, the present study provides no evidence of association of these

alleles and risk of CVA in SCA.

DISCUSSION

Tang et al. claimed an overall association between AGT and CVA risk, but

an error with the w2 tables led them to report a P value less than 5%, rather

than the true value of 0.458. Both studies are therefore in agreement. In

Tang’s study, the observed increase in odds ratio for the adjacent pair of

TABLE I. Observed Genotypes of the AGT Microsatellite in SCA Childrena

sz12 {A9} sz14 {A8} sz16 {A7} sz18 {A6} sz20 {A5} sz22 {A4} sz24 {A3} sz26 {A2} sz28 {A1}

sz14 2 4

sz16 3 11* 4

sz18 0 5 4 0

sz20 1 8* 3 2 5

sz22 1 3 7 1 2 4

sz24 1 11 9 4 7 4 5*

sz26 1 4 2 0 2 1 0 1*

sz28 3* 4* 1 1 4 3 6 1* 5*

sz30 0 2* 2 1 1 1 0 0 0

aThe numbers of non-CVA children for a given genotype are shown. If an asterisk is present (*), a child with CVA was also observed with the

genotype. The following genotypes were also observed in the non-CVA group: sz20/sz8, sz28/sz8, sz32/sz16, sz32/sz28, and sz34/sz34. Classical

allele nomenclature is also provided between curly brackets, i.e. {xx}.

American Journal of Hematology 76:310–313 (2004)

ª 2004 Wiley-Liss, Inc.

alleles remains valid with a P < 2%, but caution is required in the presence

of multiple tests. Nonetheless, two studies have now suggested an increased

risk of stroke in SCA associated with particular AGT microsatellite alleles,

albeit weakly and with different alleles. While the most probable explanation

of these disparate results remains the sampling, the genetic heterogeneity of

African populations [5] suggests a gametic association phenomenon, with the

microsatellite allele involved being population-dependent. Given the import-

ance of this complication, there is clearly room for further study of the role

of AGT in risk generation.

ACKNOWLEDGMENTS

We thank the sickle cell patients and their parents who participated in this

study and the medical staff of the Pediatric Department of the ‘‘Centre

Hospitalier Universitaire’’ of Pointe-a-Pitre and of the ‘‘Centre Caribeen de

la Drepanocytose—Guy Merault’’

MARC ROMANA1

JEAN-PIERRE DIARA1,2

LYDIA DOUMBO2

SHANMUGAKONAR MURALITHARAN3

RAJENDRANATH RAMASAWMY3

LYSIANE KECLARD1

VANESSA TARER1

VICKY CHAAR1

JACQUES ELION1,3

RAJAGOPAL KRISHNAMOORTHY1,3

JOHN CLAYTON1

1UMR 458 INSERM, Universite Antilles Guyane, Equipe Guadeloupe,

CHU, Pointe-a-Pitre, Guadeloupe, France2Centre Caribeen de la Drepanocytose «Guy-Merault», CHU,

Pointe-a-Pitre, Guadeloupe, France3UMR 458 INSERM, Universite Paris, 7 Denis Diderot,

Hopital Robert Debre, Paris, France

Contract grant sponsor: European Union;

Contract grant number: TS3*-CT93-0244DG12HSMV;

Contract grant sponsor: Conseil Regional de la Guadeloupe;

Contract grant number: CR 01-01.

Published online in Wiley InterScience (www.interscience.wiley.com).

DOI: 10.1002/ajh.20078

REFERENCES

1. Tang DC, Prauner R, Lui W, et al. Polymorphisms within the angiotensinogen gene

(GT-repeat) and risk of stroke in pediatric patients with sickle cell disease: a case-

control study. Am J Hematol 2001;68:164–169.

2. Gill FM, Sleeper LA, Weiner SJ, et al. Clinical events in the first decade in a cohort

of infants with sickle cell disease. Blood 1995;86:776–783.

3. Caulfield M, Lavender P, Farrall M, et al. Linkage of the angiotensinogen gene to

essential hypertension [see comments]. N Engl J Med 1994;330:1629–1633.

4. Gyapay G, Ginot F, Nguyen S, Vignal A, Weissenbach J. Genotyping procedures in

gene mapping. Methods 1996;9:91–97.

5. Jorde LB, Rogers AR, Bamshad M, et al. Microsatellite diversity and the

demographic history of modern humans. Proc Natl Acad Sci USA 1997;94:

3100–3103.

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Secondary Severe Factor X Deficiency Associated

With Antiphospholipid Syndrome

To the Editor: Lupus is not commonly associated with bleeding. Bleeding in a

case of lupus is usually associated with hypoprothrombinemia [1]. Factor X

deficiency has recently been described in association with antiphospholipid

syndrome (APS) [2]. However, the two cases reported were of a transient

nature. Longstanding factor X deficiency in association with APS has not

been described previously. We recently encountered a case with factor X

deficiency secondary to APS. The case summary is as follows.

A 30-year-old woman presented to the Department of Haematology,

AIIMS, in 1994 with pain and swelling in all limbs, on and off joint pains,

amenorrhea, bleeding from the gums and rectum, and hematuria of 4 years’

duration. There was no prior history of bleeds. A family history of similar

complaints was absent. Laboratory investigations revealed hemoglobin of 10.9

g/dL, total leukocyte count of 9,500/mm3, platelet count of 440,000/mm3,

prothrombin time (PT) of 52 sec (control 37 sec), activated partial thrombo-

plastin time (APTT)of>180 sec (control 37 sec), thrombin time 14 sec (control

16 sec), fibrinogen level 300 mg/dL (normal level 200–450 mg/dL) (Sigma

Diagnostics, St. Louis,MO), and normal platelet aggregation toADP, adrena-

line, and collagen. Factor X activity levels were less than 1%. No inhibitors to

factor X were demonstrated using clotting assays. Mixing studies showed that

the prolonged PT and APTT were corrected by addition of normal plasma.

Repeated investigations were negative for the presence of lupus anticoagulant

and antinuclear and anticardiolipin antibodies. However, the antibodies to

IgM b2-glycoprotein I was present in the patient (12.6 IU/mL; normal 0–5

IU/mL). Doppler could not demonstrate a thrombus. However, technetium-

99m scanning of the lower limbs revealed thinning and obliteration of peroneal

veins and posterior tibial veins at several points, suggestive of vasculitis with

possible obstruction. In view of the patient having bleeding as well as vasculitis

with possible thrombotic manifestations, a diagnosis of antiphospholipid syn-

drome (APS) was made. She had earlier received steroids, danazol, and fresh

frozenplasma forher bleeds, but nosubstantial responsewasobserved. Shewas

given cyclophosphamide 1 g on first day followed by cyclosporine 150mg/day.

Since then, there has been no bleeding or limb swelling. Moreover, the PT,

APTT, and factor X activity levels are normal.

Antiphospholipid syndrome (APS) with presence of antibodies to

b2-glycoprotein I (IgG or IgM) alone, without any other antiphospholipid

antibodies, have been described earlier [3]. In a large study, 5% of the

patients with APS had antibodies solely to b2-glycoprotein I [4]. Although

steroids have been used successfully in bleeding associated with lupus anti-

coagulant [1], in our case, they were not very useful. The patient responded

well to cyclosporine. Although this has not yet been reported, cyclosporine

may prove to be a potent drug in such cases.

P. MISHRA

T. CHATTERJEE

A. DIXIT

V.P. CHOUDHRY

RAJAT KUMAR

R. SAXENA

Department of Hematology, All India Institute of Medical Sciences,

Ansarinagar, New Delhi

Published online in Wiley InterScience (www.interscience.wiley.com).

DOI: 10.1002/ajh.20085

REFERENCES

1. Vivaldi P, Rossetti G, Monica G, Finazzi G. Severe bleeding due to acquired

hypoprothrombinemia—lupus anticoagulant syndrome. Case report and review of

literature. Haematologica 1997;82:345–347.

2. Ashrani AA, Aysola A, Al-Khatib H, Nichols WL, Key NS. Lupus anticoagulant

associated with transient severe factor X deficiency: a report of two patients

presenting with major bleeding complications. Br J Haematol 2003;121:639–642.

3. Alarcon-Segovia D, Mestanza M, Cabiedes J, Cabral AR. The antiphospholipid

syndromes. A variant in patients with systemic lupus erythematosus with antibodies

to b2-glycoprotein but no antibodies detectable in standard antiphospholipid assays.

J Rheumatol 1997;25(7):1545–1551.

4. Day HM, Thiagarajan P, Ahn C, Reveille JD, Tinker KF, Arnett FC. Autoantibodies

to b2-glycoprotein I in systemic lupus erythematosus and primary antiphospholipid

antibody syndrome: clinical correlations in comparison with other antiphospholipid

antibody tests. J Rheumatol 1998;25(4):667–674.

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Letters and Correspondence 311

Rare b-Thalassemia Mutations Are Cause of Concern

To the Editor: In India, 10,000 b-thalassemics are born every year, which

spells the reason of its importance in the country (http://nagpurcity.net/

netzine/010898a.html). Due to genetic predisposition, the disease does not

have a permanent cure but only a prevention and management regime. The

available remedies for managing thalassemics are costly and have limitations.

Prevention, on the other hand, includes antenatal genetic testing, which may

be applied in a variety of clinical situations, including preconception coun-

seling and prenatal diagnosis (PND).

MATERIALS, METHODS, AND RESULTS

The DNA samples of patients coming for PND were processed as

described previously [1].

SSCP analysis of sample 1, localizedmutation in the fourth fragment of the

b-globin gene, was carried out. Sequencing of the same showed a 17-bp

deletion in exon 3(126–131). The mutation was found associated with haplo-

type III (�+�+0+0). Because the CVS sample was heterozygous for CD

30 (G-C) mutation, the pregnancy was continued and completed to term.

SSCP analysis of sample 2 revealed a mutation in the first fragment

(322 bp) of the b-globin gene encompassing the promoter and exon 1.

Sequencing analysis of the same showed a single-base substitution (A-G) at

position �29 of the b-globin gene. The mutation was associated with the

haplotype (+�+� � �+) in combination with CD16 (+� �� �+�).

CVS diagnosis identified a carrier.

DISCUSSION

In this letter, we describe two different rare b-thal mutations that were

encountered in two families of UP, India. The first, a 17-bp deletion at

exon 3(126–131), causes a shift in the open reading frame, introducing a

stop codon ‘‘TAA’’ at CD 133 and leading to premature truncation of the

b-globin chain. This mutation has been independently described in three

Indian families, suggesting its presence in this population [2], but its origin

remains unknown.

In the second case, another rare mutation, �29 (A-G), was seen. The

first child was compound heterozygous for CD16 (�C) and �29 (A-G)

mutation and CVS heterozygous for �29; �29 (A-G), a mild mutation, is

characterized by a residual high b-chain production. This mutation is not

reported in Indians but has been documented in American Blacks [3] and in

Chinese [4]. In all three studies, the mutation was found to be associated

with different haplotypes, showing that the mutation arose independently.

Because promoter mutations are mild/silent type, when present with other

types of mutations (b0/b+/b++), they result in the phenotypes being more

mild. This becomes clear in our cases, where the first child of the proband

had both b-globin gene defects (�29/Cd16) yet had only four transfusions

in a span of 2 years and a hemoglobin level of 6–7 g %. This highlights the

importance of the characterization and elucidation of unknown mutations

in patients and the resulting phenotypes. Knowledge of the relationship

between genotype and phenotype has important implications for the

screening of b-thalassemia carriers, genetic counseling, and PND and for

the proper treatment of the patients.

ACKNOWLEDGMENTS

The authors are thankful to DST, ICMR, New Delhi, for their financial

assistance in carrying out the work and to JICA for providing the infra-

structure for the Thalassemia Screening Program at SGPGIMS, Lucknow.

The authors are also thankful to Dr. Y. Hattori, Department of Clinical

Laboratory Sciences, Yamaguchi University School of Medicine, Ube,

Japan, for providing sequencing analysis of these samples.

ANJU GUPTA

SARITA AGARWAL

Department of Genetics, Sanjay Gandhi Post Graduate Institute of

Medical Sciences, Lucknow, India

Published online in Wiley InterScience (www.interscience.wiley.com).

DOI: 10.1002/ajh.20084

REFERENCES

1. Gupta A, Hattori Y, Agarwal S. Initiation codon mutation in an Asian Indian

family. Am J Hematol 2002;7:134.

2. Nadkarni A, Sakaguchi T, Takaku H, et al. A novel b0-thalassemia mutation at

codon 55 (�A) and a rare 17-bp deletion at codons 126–131 in the Indian

population. Hemoglobin 2002;26:41.

3. Antonarakis SE, Irkin SH, Cheng TC, et al. b-Thalassemia in American Blacks: novel

mutations in the ‘‘TATA’’ box and an acceptor splice site. Proc Natl Acad Sci USA

1984;81:1154.

4. Huang SZ, Xu YH, Zeng FY, Wu DF, Ren ZR, Zeng YT. The same TATA box

b-thalassemia mutation in Chinese and US Blacks: another example of independent

origin. Hum Genet 1986;74:152.

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Sometimes Appendicitis Can Wait

To the Editor: A pregnant 30-year-old woman was diagnosed with acute

myelocytic leukemia (AML-M). Initial treatment was therapeutic interrup-

tion of pregnancy followed by standard induction chemotherapy with

daunorubicin and cytosine arabinoside (ara-C). She achieved complete remis-

sion and received two courses of consolidation chemotherapy. On day 20

after the second course consisting of ara-C (3 g/m2 twice daily for 4 days) she

became febrile and complained of pain limited in right side of the abdomen.

Physical examination showed local defense without any signs of peritonitis.

Absolute neutrophil count was 0.56� 109/L. Streptococcus a-hemolyticuswas

found in blood cultures. Thin-section contrast-enhanced helical dual-phase

scanning (CT scan) showed aspect of typhlitis (dilatation of ascending colon,

spread liquid around the caecum) with thickened appendix. Intravenous

broad-spectrum antibiotics (ticarcillin, clavulanic acid, tobramycin, and

vancomycin) were started on day 11, with close monitoring by the surgical

team. Fever disappeared within 6 days, but local symptoms remained poor.

Antibiotherapy was maintained during 2 weeks. CT scan after the end of

aplasia (white blood cell count of 9.2 � 109/L) was performed and showed

aspect of appendicitis with local abscess. Two weeks after the end of aplasia,

laparoscopy with appendectomy and abscess drainage were performed.

Perforated appendicitis was confirmed by histologic examination. Postopera-

tive courses were uneventful with hospital discharge at day 5.

The diagnosis of a ‘‘surgical abdomen’’ in a patient with acute leukemia is

difficult because of the complications of hematologic malignancy and the

patient’s treatment which may actually mask the common signs and symp-

toms of an acute abdomen. The risk is that of underdiagnosing and under-

treating a potentially life-threatening complication. Surgical exploration had

to be the exception because of the high risk of general sepsis andmortality due

to immunosuppression. The principal diagnosis is a necrotizing process of the

caecum and ascending colon, called typhlitis [1]. The pathogenesis is multi-

factorial, involving poor arterial perfusion and a swarm of bacteria during

neutropenia. Treatment usually calls for broad-spectrum antibiotherapy. In

0.5% of patients treated for acute leukemia, essentially children, right-side

abdominal pain is induced by appendicitis [2]. Up to the 1990s, hesitation in

diagnosis was partly due to lack of efficiency of radiologic exploration. CT

scanning is now the best method of diagnosis for acute appendicitis in usual

situation [3], but clear differentiation between typhlitis and appendicitis is

difficult in leukemic patients because neutropenic enterocolitis could involve

the appendix miming aspect of appendicitis [4]. In both cases, treatment is

independent of diagnosis. Clinical status and kinetics of white cell blood

count guided the course of treatment. Thus some patients with local abscesses

underwent surgical exploration for typhlitis and some with unrecognized

appendicitis were treated by intravenous antibiotherapy and close clinical

312 Letters and Correspondence

monitoring. CT scan showing deep collection 2 weeks after end of aplasia

signed complicated appendicitis because typhlitis typically recedes when a

normal white cell blood count is attained. We support that absolute radio-

logic differentiation between typhlitis and appendicitis is not indispensable.

Good clinical status and ascending white cell blood count direct a non-

immediate surgical attitude.

OLIVIER TURRINI1

VINCENT MOUTARDIER1

NORBERT VEY2

JEROME GUIRAMAND1

BERNARD LELONG1

JEAN-ROBERT DELPERO1

1Department of Surgical Oncology, Institut Paoli Calmettes and

Universite de la Mediterranee, Marseille, France2Department of Hematology, Institut Paoli Calmettes and Universite de

la Mediterranee, Marseille, France

Published online in Wiley InterScience (www.interscience.wiley.com).

DOI: 10.1002/ajh.20086

REFERENCES

1. Wagner ML, Rosenberg HS, Fernbach DJ, Singleton EB. Typhlitis: a complication

of leukemia in childhood. Am J Roentgenol Radium Ther Nucl Med 1970;109:

341–350.

2. Angel CA, Rao BN, Wrenn E Jr, Lobe TE, Kumar AP. Acute appendicitis in

children with leukemia and other malignancies: still a diagnostic dilemma. J Pediatr

Surg 1992;27:476–479.

3. Rao PM, Rhea JT, Novelline RA, et al. Helical CT technique for the diagnosis of

appendicitis. Radiology 1997;202:139–144.

4. McCarville MB, Thompson J, Li C, et al. Significance of appendiceal thickening

in association with typhlitis in pediatric oncology patients. Pediatr Radiol

2004;34(3):245–249.

Letters and Correspondence 313