1. Pediatr Blood Cancer 2012;59:941–944
BRIEF REPORT
Severe Fetal and Neonatal Hemolytic Anemia Due to a 198 kb Deletion
Removing the Complete b-Globin Gene Cluster
Madeleine Verhovsek, MD,1,2
* Nirmish R. Shah, MD,3
** Ibifiri Wilcox,1
Sara C. Koenig, MD,1
Tiago Barros,1
Courtney D. Thornburg, MD,3
Martin H. Steinberg, MD,1
Hong-yuan Luo, MB, PhD,1
and David H.K. Chui, MD
1
INTRODUCTION
Hemolytic anemia of the newborn is often due to fetal/maternal
blood group incompatibility, unstable hemoglobinopathies, hered-
itary erythroid enzymopathies, and cell membrane abnormalities.
We report a newborn with life-threatening in utero and neonatal
hemolytic anemia due to a large deletion of the b-globin gene
cluster. By 1 year of age, the severe hemolysis subsided.
CASE REPORT
A 28-year-old woman of Irish/Scottish descent was pregnant
for the 3rd time. Her first two pregnancies ended with therapeutic
abortion in the first trimester. She, her mother, and other family
members were reported to be heterozygous for a large deletion
of the entire b-globin gene cluster [1]. She herself was delivered
by Caesarian section at the 37th week of gestation. At birth, her
hemoglobin (Hb) was 8.6 g/dl; mean corpuscular volume (MCV)
90 fl; reticulocyte count 12.2%; and there were 1,580 nucleated
erythrocytes/100 leukocytes [1]. She was transfused once. The
hemolysis later subsided, and she had persistent microcytic hypo-
chromic anemia but did not require transfusions.
At 29-weeks gestation in her third pregnancy, her hematocrit
was 22%. She was transfused with two units of packed red blood
cells. At delivery (32-weeks gestation), her Hb was 8.3 g/dl,
hematocrit 26%, and MCV 72 fl.
Two days prior to delivery, fetal ultrasound showed thickened
fetal myocardium and elevated middle cerebral artery velocities.
A fetal echocardiogram showed mildly hypertrophic fetal myo-
cardium and dilated ventricles. There was no evidence of conges-
tive heart failure. On the day of delivery, middle cerebral artery
velocities were again elevated. Percutaneous umbilical cord blood
sampling revealed severe anemia. Three in utero transfusions
were given, following which decreased fetal heart tones were
noted and emergent Caesarian section was done. An infant female
was delivered. Her Apgar scores were 2 at 1 minute, and 8 at
5 minutes. The newborn weighed 1.21 kg (25th percentile
corrected for gestational age), and had a head circumference of
26.5 cm (12th percentile).
The newborn’s Hb was 10.3 g/dl, MCV 86 fl, reticulocyte
count 21%, platelet count 223 Â 109
/L, leukocyte count
6.5 Â 109
/L and total bilirubin 4.1 mg/dl. The mother’s blood
groups were Rh positive, type O and the infant’s were Rh nega-
tive, type O. Direct and indirect antiglobulin tests were
negative. Peripheral blood smear showed prominent microcytosis,
hypochromia, poikilocytosis, polychromasia, and nucleated red
blood cells. Hemoglobin electrophoresis revealed Hb F and
A. At birth, the newborn did not have jaundice, or hepato-
splenomegaly, but had respiratory distress and was admitted to
the intensive care unit (ICU).
The infant remained in the ICU for 62 days. Peak bilirubin was
7.8 g/dl at 2½ days after birth. She received phototherapy for
unconjugated hyperbilirubinemia until day 12. On four different
occasions during the first 2 months after birth, her Hb dropped
below 9 and she was transfused (Table I).
Following discharge from the hospital, she was not given any
more transfusions. At 8½ months of age, her MCV decreased to a
nadir of 52 fl (Table I), attributable to normal physiologic postna-
tal changes, elimination of transfused erythrocytes and decreased
reticulocytosis. Over the next few years, her MCV should reach
the level similar to her mother’s. At 2 years of age, her Hb was
10.2 g/dl and MCV 58 fl (Table I); and she met all developmental
Fetal and neonatal hemolytic anemia can be caused by (gdb)0
-
thalassemia deletions of the b-globin gene cluster. Many of these
deletions have not been well characterized, and diagnostic tests
are not readily available, thus hampering carrier detection, family
counseling, and antenatal diagnosis. We report and define a 198 kb
deletion removing the entire b-globin gene cluster, which was
found in members of a multigeneration family of Irish/Scottish
descent. The proband had life-threatening fetal and neonatal
hemolytic anemia which subsided by 1 year of age. Pediatr Blood
Cancer 2012;59:941–944. ß 2012 Wiley Periodicals, Inc.
Key words: fetal anemia; neonatal hemolytic anemia; hereditary anemia; b-globin gene deletion; (gdb)0
-thalassemia
1
Hematology/Oncology, Department of Medicine, Boston University
School of Medicine, Boston, MA; 2
Department of Medicine, McMaster
University, Hamilton, ON, Canada; 3
Hematology/Oncology, Depart-
ment of Pediatrics, Duke University School of Medicine, Durham,
NC
Conflict of interest: Nothing to report.
Dr. Madeleine Verhovsek and Dr. Nirmish R. Shah contributed equally
to this work.
Sara C. Koenig present address is Department of Pathology, Univer-
sity of New Mexico School of Medicine, Albuquerque, NM 87131.
*Correspondence to: Dr. Madeleine Verhovsek, MD, St. Joseph’s
Healthcare, 50 Charlton Avenue East, Room L208, Hamilton, ON,
Canada L8N 4A6. E-mail: verhovm@mcmaster.ca
**Correspondence to: Dr. Nirmish R. Shah, MD, Department of
Pediatric Hematology/Oncology, Duke University Medical Center,
Durham, NC 27710. E-mail: nirmish.shah@duke.edu
Received 23 November 2011; Accepted 6 January 2012
ß 2012 Wiley Periodicals, Inc.
DOI 10.1002/pbc.24094
Published online 31 January 2012 in Wiley Online Library
(wileyonlinelibrary.com).

2. milestones and was between 25th and 50th percentiles for both
her height and weight.
MOLECULAR TESTING
Multiplex Ligation-Dependent Probe Amplification
(MLPA)
MLPA on her b-globin gene cluster [2] showed a large dele-
tion spanning from 50
to the HS 5 of the b-locus control region
(LCR) to 30
of the b-globin gene (Fig. 1A), consistent with the
previously published report [1].
Identification of the Deletion Breakpoints
Several PCR primers were designed to amplify and sequence
the region 5–10 kb downstream of the b-globin gene. The results
revealed that the putative deletion breakpoint resides within
6–9 kb downstream of the b-globin gene (data not shown).
Subsequently, a common PCR primer (50
-GGCTTGGCTCCTG-
TTTAGTATTGC-30
) at 10 kb downstream of the b-globin gene
was used to pair with a series of PCR primers spaced approxi-
mately 10 kb apart at 10–140 kb upstream of the b-globin gene
cluster to amplify the intervening DNA fragments by long-range
PCR.
In an amplification using the above PCR primer and another
primer (50
-CCTGAGCCTCT GGAAACACTAAGA-30
), a unique
amplicon of $7 kb in length was found in the patient’s genomic
DNA, but not in a normal individual. This amplicon was
sequenced, and the results showed that the (gdb)0
-thalassemia
deletion was from nt 5178571, 8.4 kb downstream of the b-globin
gene, to nt 5376341, $123.8 kb upstream of the b-globin LCR
(Fig. 1B). The deletion spanned 197,770 bp that removed the
entire b-globin gene cluster including the LCR, and olfactory
receptor genes, OR51VI, OR51B4, OR51B2, OR51B5, and
OR51B6. We name this the Duke (gdb)0
-thalassemia deletion.
A gap-PCR test was designed capable of definitive identification
of this deletion (Fig. 1C).
DISCUSSION
The syndrome of fetal and neonatal hemolytic anemia due to
(gdb)0
-thalassemia deletion was first described in 1972 [3]. The
affected neonates often were from ethnic backgrounds where
thalassemia was not prevalent [34]. Most infants had brisk hemo-
lysis and erythroblastosis during the perinatal period, and required
blood transfusions. Several reported cases, including the present
one, were given in utero transfusions. Of nine infants with this
syndrome in a large Dutch family [5], one was stillborn, two died
shortly after birth, five received one or more exchange transfu-
sions, and one did not require transfusion. However, all reports
confirmed a relatively stable microcytic hypochromic anemia later
in childhood and adulthood with RBC indices similar to common
b-thalassemia trait.
Some deletions remove either part or all of the b-LCR leaving
the downstream b-like globin genes intact; other deletions remove
the LCR and some or all of the downstream b-like globin genes
[reviewed in ref. 4]. In the present family, the deletion is exten-
sive. The long interspersed elements (LINE) of the L1 family are
found in large numbers in this region of chromosome 11p15. The
nucleotide sequences of both the 50
and 30
junctions of this dele-
tion are homologous to the L1 family, suggesting that these L1
repetitive DNA elements might be responsible for the recombina-
tion resulting in the deletion.
It is conceivable that the switch from g- to b-globin chain
production in utero might be relatively uncoordinated resulting in
a transitory excess of a-globin chains, which is further exacerbat-
ed by the (gdb)0
-thalassemia deletion. Globin chain synthesis was
undertaken in the first reported case [3] showing a (g þ b)/a ratio
of 0.39 in the newborn period, much less than that found (0.70) in
newborns with b-thalassemia trait alone. These excess a-globin
chains form insoluble aggregates of multimers leading to
TABLE I. Hematology Results
Age after birth
Gestational age
(weeks) Hb (g/dl) MCV (fL)
Reticulocyte
counts (%) Transfusion
Total bilirubin
(mg/dl)
1 day before birth 32 Hct. 14% — — In utero transfusion —
1 day after birth 32 10.3 86 21.5 — 4.1
3 days 32.5 9.5 86 23.3 — 7.8
6 days 33 10 82 — — 5.1
11 days 33.5 8.5 76 — Transfusion —
13 days 34 13.9 80 — — 3.7
21 days 35 8.7 80 1.5 Transfusion —
28 days 36 10.2 84 1.6 — —
35 days 37 8.5 83 1.6 Transfusion —
36 days 37 13.1 83 — — —
42 days — 10.7 83 0.9 — —
55 days — 7.1 81 — Transfusion —
2.5 months — 8.2 80 4.4 — —
5.5 months — 8.9 55 1.6 — —
8.5 months — 9.4 52 1.6 — —
11.5 months — 9.7 53 1.1 — —
13 months — 9.2 59 1.1 — —
18 months — 10.0 58 — — —
24 months — 10.2 58 — — —
942 Verhovsek et al.
Pediatr Blood Cancer DOI 10.1002/pbc

3. Fig. 1. Molecular characterization of the Duke (gdb)0
-thalassemia deletion: MLPA showing the signals of all except two probes (colored red)
in the b-globin gene cluster equal to half of the signals of probes (colored blue) for genes on other chromosomes which serve as control (A).
The first probe on the left is located $1 Mb upstream of the b-LCR. The last probe of the b-globin gene cluster on the right is located $9 kb
downstream from the b-globin gene. Nucleotide sequences across the Duke (A
gdb)0
-thalassemia deletion breakpoints, are shown in the
underlined middle row (B). The upper row shows the nucleotide sequences from nt 5178539 to nt 5178604. The lower row shows the
nucleotide sequences from nt 5376308 to nt 5376373. The deletion breakpoints are nt 5178571, $8.4 kb downstream of the b-globin gene, and
nt 5376341, $123.8 kb upstream of the b-globin LCR. The extent of the Duke (gdb)0
-thalassemia deletion is 197,770 bp or $198 kb.
Nucleotide numbering is done according to NCBI Reference Sequence NT_009237.18, chromosome 11 genomic contig, GRCh37.p5 Primary
Assembly. (Reference sequence for Chromosome 11:1-50723853, essentially the beginning part of chromosome 11 up to the centromere. This
is the v37 or hg19 version of the human genome reference.) Gap-PCR test designed to identify the Duke (gdb)0
-thalassemia deletion (C). Lane
M, molecular size markers; Lane 1, patient heterozygous for the deletion shown by the mutant 1.6 kb and normal 0.8 kb bands; Lane 2, a
normal individual with the 0.8 kb band. PCR primers to detect the deletion: 50
-GGCTTGGCTCCTGTTTAGTATTGC-30
and 50
-GCAGTTTT-
GAGTGAGTTTCTTAATCCTCAG-30
. PCR primers to detect the normal: 50
-TCATTCGTCTGTTTCCCATT-30
and 50
-TCCTAAGCCAGTGC-
CAGAAG-30
. This amplicon encompasses the b-globin gene from promoter nt-161 to IVS II-115.
Pediatr Blood Cancer DOI 10.1002/pbc

4. hemolysis. It can be made worse if the proteolytic machinery in
fetal and neonatal erythroid cells is not optimal [6], or if the
production of a-hemoglobin stabilizing protein (AHSP) in fetal
and newborn erythroid cells is less than robust [7]. With time, the
unaffected b-globin gene achieves appropriate expression and
possibly both erythroid proteolytic capacity and production of
AHSP attain normal levels. The affected children then have he-
matologic phenotypes similar to common b-thalassemia trait.
This report reaffirms that (gdb)0
-thalassemia should be consid-
ered as one of the differential diagnoses in fetal and newborn
hemolytic anemia often with normoblastosis, even among families
with ethnic backgrounds not commonly associated with thalassemia.
REFERENCES
1. Pirastu M, Kan YW, Lin CC, et al. Hemolytic disease of the newborn caused by a new deletion of the
entire b-globin cluster. J Clin Invest 1983;72:602–609.
2. Koenig SC, Becirevic E, Hellberg MSC, et al. Sickle cell disease caused by heterozygosity for Hb S and
novel LCR deletion: Report of two patients. Am J Hematol 2009;84:603–606.
3. Kan YW, Forget BG, Nathan DG. gb-Thalassemia: A cause of hemolytic disease of the newborn.
N Engl J Med 1972;286:129–134.
4. Thein SL, Wood WG. The molecular basis of b thalassemia, db thalassemia, and hereditary persistence
of fetal hemoglobin. In: Steinberg MH, Forget BG, Higgs DR, Weatherall DJ, editors. Disorders of
hemoglobin genetics, pathophysiology, and clinical management. Cambridge, UK: Cambridge Univer-
sity Press; 2009. pp 323–356.
5. Oort M, Herrspink W, Roos D, et al. Haemolytic disease of the newborn and chronic anaemia induced
by gb-thalassaemia in a Dutch family. Br J Haematol 1981;48:251–262.
6. Ho JP, Hall GW, Watt S, et al. Unusually severe heterozygous b-thalassemia: Evidence for an interacting
gene affecting globin translation. Blood 1998;92:3428–3435.
7. Kong Y, Zhou S, Kihm AJ, et al. Loss of a-hemoglobin-stabilizing protein impairs erythropoiesis and
exacerbates b-thalassemia. J Clin Invest 2004;114:1457–1466.
944 Verhovsek et al.
Pediatr Blood Cancer DOI 10.1002/pbc