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\begin{document}

\title{Congenital dyserythropoietic anemia type IV in the genetic era: A rare
neonatal case report of rapid identification with a review of the
literature}



\author[1]{Sarah Blain}%
\author[1]{Marc-Olivier Deguise}%
\author[1]{Ewurabena Simpson}%
\author[1]{Mira Liebman}%
\author[1]{Emanuela Ferretti}%
\affil[1]{University of Ottawa Faculty of Medicine}%


\vspace{-1em}



  
  \date{May 25, 2024}


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\selectlanguage{english}
\begin{abstract}
Congenital dyserythropoietic anemia type IV (CDAIV) is a rare inherited
hematological disorder presenting severe anemia due to altered
erythropoiesis and hemolysis, with variable needs for recurrent
transfusions. We present a case of a transfusion-dependant male newborn
who required an intrauterine transfusion and presented at birth with
severe hemolytic anemia. Genetic testing rapidly identified a KLF1 gene
mutation, a CDAIV variant. This case highlights the advantages of
next-generation sequencing testing for congenital hemolytic anemia:
diagnostic speed, guidance on natural history, and optimized clinical
management and anticipatory guidance for parents and clinicians. We
reviewed the literature for all CDAIV cases.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Congenital dyserythropoietic anemia (CDA) is an aggregation of rare
inherited hematological disorders characterized by a hallmark feature of
altered erythropoiesis. CDA can be subdivided into 5 categories, namely
type I, II, III, transcription factor related CDA and CDA variants(1).
Each type is characterized by varying hematopathological findings,
clinical presentation, and underlying genetic basis (1). CDAIV (Online
Mendelian Inheritance in Man (OMIM): 613673) is part of the
transcription factor-related CDA family and accounts for only 0.4\% of
all patients with CDA (1). To date, 12 patients have been identified
with CDAIV (see table 1). Patients are found to have a mutation in the
transcription factor KLF1, which has been implicated in various mild
hematological disorders (see supplemental table 1) (2). This is likely
due to the KLF1 role at multiple levels of the erythropoietic process,
and varying mutations may lead to different functional consequences on
the transcription factor (3-5). CDAIV is an autosomal dominant condition
in which most patients harbour a \emph{de novo} heterozygous mutation
c.973G.A (p.E325K) (see table 1) (3-5). Most CDAIV patients present
severe anemia, hemolysis, hepatosplenomegaly, hyperbilirubinemia, and
persistence of fetal hemoglobin. They are often transiently
transfusion-dependant. Many patients present in the neonatal period but
are only diagnosed later in life, because of the non-specific
symptomatology of hemolytic anemias.

We present a case of CDAIV, which presented with severe neonatal anemia
and hyperbilirubinemia, with a subsequent rapid diagnosis through a
commercially available next-generation sequencing (NGS) panel for
congenital hemolytic anemias. We highlight the importance of considering
early genetic testing for unexplained severe and refractory congenital
anemias. Additionally, we advocate for pre-transfusion blood storage in
cases of unexplained anemia in newborns to prevent delays in diagnosis
due to the presence of circulating donor's RBC. Of note, we remind
clinicians that it is routine practice for laboratories to store
pre-transfusion blood samples for 1 week.

\textbf{Case presentation}

This term baby boy was born from a healthy mother with an obstetrical
history notable for three early spontaneous abortions, two healthy-term
babies, and one child with Axenfield Riegers Syndrome (OMIM: 180500).
The prenatal course was complicated by severe fetal anemia at 26 weeks'
gestation (fetal hemoglobin of 39 g/L) requiring one intrauterine
transfusion. The mother's blood group was A\textsuperscript{+}.
Investigations for parvovirus, toxoplasmosis and cytomegalovirus were
negative. Routine serologies were protective. Non-invasive prenatal
testing (NIPT), rapid aneuploidy detection and chromosomal microarray
were normal. Several maternal IAT, done before and after the
intrauterine transfusion, were negative. The etiology of fetal anemia
remained unclear to the maternal-fetal medicine team. The patient was
not referred to hematology.

The baby was born at 37 weeks GA through induced vaginal delivery for
decreased fetal movement in the context of fetal anemia. Initial
complete blood count (CBC) showed hemoglobin of 65 g/L with an increased
MCV of 118 fL and reticulocytosis (574.2 X10\textsuperscript{9}/L). His
blood group was A\textsuperscript{+}. The blood smear suggested
hemolysis (see figure 1A). LDH was elevated at 10 446 U/L (normal
\textless{} 1128 U/L). DAT and IAT were negative. He was transfused pRBC
(see Figure 1B). Pre-transfusion hemoglobin investigations were not
ordered. He developed early onset severe refractory unconjugated
hyperbilirubinemia (see Figure 1D), requiring 3 double volume exchange
transfusions in the first two days of life while under high-intensity
phototherapy. He required another pRBC transfusion after the first
exchange transfusion for mild anemia. He developed thrombocytopenia
within the first 24h, for which he received two platelet transfusions
(see Figure 1B). The thrombocytopenia resolved by DOL 4. An abdominal
ultrasound showed mild hepatosplenomegaly. Given the transfused status,
hemoglobin investigations and enzymopathy testing were not reliable. The
Kleihauer-Betke test from the placenta was negative for feto-maternal
hemorrhage. Further blood bank testing was obtained to rule out
alloimmunization. Maternal serum from the time of delivery was negative
for antibodies against low-frequency antigens. An eluate performed on
cord blood from delivery was negative. The maternal plasma was first
cross-matched against cord RBC, and then cross-matched against paternal
RBC, both of which showed no agglutination. Considering the inability to
test the baby's endogenous blood for hemoglobinopathies, parents were
investigated with hemoglobin electrophoresis, CBC, reticulocyte and
ferritin levels; all tests were unremarkable.

Concomitantly, the baby's refractory unconjugated hyperbilirubinemia
progressed to significant combined hyperbilirubinemia within the first
24 hours of life (see figure 1D, E). An extensive
gastroenterologic/metabolic work-up showed no primary liver disease. The
conjugated hyperbilirubinemia was thought to be inspissated bile duct
syndrome caused by overwhelming hemolysis. It improved with ursodiol. He
was discharged on DOL 10 with no transfusion requirement for one week.

The patient required pRBCs at two weeks of age and every two weeks
thereafter. At two weeks old, we performed alpha thalassemia gene
mapping, which came back normal, and sent a Invitae Hereditary Hemolytic
Anemia Panel (test code: 55679) genetic panel on mucosal cells. This
panel uses next-generation sequencing to analyze 40 genes involved in a
vast array of hereditary hemolytic anemias. At one month of age, his
genetic results yielded a KLF1 gene mutation C.973G\textgreater{}A, a
pathologic variant known to cause CDAIV. A bone marrow transplant
consultation was obtained. A fully matched sibling hematologic stem cell
transplant is planned once the patient is 1 year old.

\textbf{Discussion}

This patient presented with congenital anemia of unknown cause with a
history of intrauterine transfusion requirement. Hemolytic markers were
strikingly elevated. Reticulocytosis explained the high MCV. The
differential diagnosis of congenital hemolytic anemia is wide
(supplemental table 2). Maternal-fetal alloimmunization is the most
common etiology. There are case reports of fetuses with hydrops fetalis
secondary to alloimmunization that were born with a negative IAT (17).
We therefore performed extensive blood bank testing that completely rule
out alloimmunization. We sent a mucosal DNA NGS inherited hemolytic
anemia panel. This test is not affected by the post-transfusion status.
CDA was historically diagnosed by RBC property testing and bone marrow
morphology. However, with the advent of genetic testing, NGS has become
the gold standard to diagnose rare congenital hemolytic anemia, like
CDA. The genetic basis of CDAIV was first sequenced nearly ten years ago
by Arnaud \& \emph{al.} (2010), who identified two patients with a KLF1
mutation. Twelve patients have been described since. Most show
consistent features with onset in the neonatal period (8/12 patients),
but delayed diagnoses occur despite accessible technologies (see table
1). We reached a diagnosis within one month of life, which is the
fastest CDA diagnosis ever described. In summary, our case highlights a
patient with CDA4 \emph{de novo} mutation with \emph{in
utero}erythropoietic disturbances requiring transfusion, post-natal
hemolytic anemia and combined hyperbilirubinemia transiently refractory
to exchange transfusions. Thrombocytopenia has been rarely described in
CDAI and CDAIV(18). The transient thrombocytopenia of this patient was
considered secondary to exchange transfusions. This case demonstrates
the importance of a prenatal referral to hematology and neonatology when
hydrops fetalis of unknown etiology is detected for diagnostic and
postnatal planning purposes. It is a reminder that it is optimal
practice to obtain pre-transfusion blood sampling in an EDTA tube in
patients with severe anemia. Finally, it illustrates that the use of NGS
technologies can expedite early diagnosis of rare congenital anemia,
like CDAIV, allows for anticipatory guidance and consequently reduces
anxiety of clinicians and families, while optimizing health care
utilization.



\textbf{Acknowledgements}

We would like to extend our gratitude to Dr. Ruth Padmore for assistance
with photomicroscopy and her review of the manuscript. We want to thank
the patient and their family for their inclusion in the study.

\textbf{Declarations}

\emph{Ethics approval, consent to participate and consent for
publication}

No ethics approval was required by our center for the description of
case reports requiring one patient. The patient legal guardian's
consented for participation and publication of this case report.

\emph{Availability of data and materials}

The datasets used and/or analyzed during the current study are available
from the corresponding.

\textbf{References:}

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2. Belgemen-Ozer T, Gorukmez O. A Very Rare Congenital Dyserythropoietic
Anemia Variant-Type IV in a Patient With a Novel Mutation in the KLF1
Gene: A Case Report and Review of the Literature. J Pediatr Hematol
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3. Ilsley MD, Huang S, Magor GW, Landsberg MJ, Gillinder KR, Perkins AC.
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\textbf{Figure 1} : \textbf{Patterns of hemoglobin, reticulocytes,
platelets and bilirubin levels in the first 96 hours and onwards.} (A)
Peripheral blood smear (60X) showing non-specific variable morphology:
anisocytosis with microcytes, spherocytes, schistocytes, elliptocytes,
macrocytes given elevated reticulocyte count, tear-drop cells, all
co-occurring with transfused red blood cells of normal

appearance. (B) Early anemia \& thrombocytopenia associated with brief
reticulocytosis requiring

multiple blood transfusions, pRBCs transfusions (green arrowhead) and
platelet transfusions (black arrowhead). (C) pRBCs transfusion (green
arrowhead) requirement and hemoglobin maintenance with reticulocytopenia
and rapidly resolving thrombocytopenia over time. (D) Refractory
hyperbilirubinemia despite phototherapy and exchange transfusions (red
arrowhead) and a progressive conjugated fraction over time. (E)
Resolution of conjugated and unconjugated hyperbilirubinemia over time.

\textbf{Table 1:} Previous CDA4 cases described in the literature.\selectlanguage{english}
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\textbf{Hosted file}

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