rare β-thalassemia mutations are cause of concern
TRANSCRIPT
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