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\begin{document}

\title{Monolobated Megakaryocytes in Diamond Blackfan Anemia with RPL5 Mutation
at Disease Presentation Mimicking Myelodysplastic Syndrome}



\author[1]{Audi Setiadi}%
\author[1]{Claudia Singh}%
\author[2]{Amanda Li}%
\author[1]{Nicholas Au}%
\author[2]{Ali Amid}%
\affil[1]{The University of British Columbia}%
\affil[2]{BC Children's Hospital}%


\vspace{-1em}



  
  \date{May 18, 2024}


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\textbf{Monolobated Megakaryocytes in Diamond Blackfan Anemia
with\emph{RPL5} Mutation at Disease Presentation Mimicking
Myelodysplastic Syndrome}

\textbf{Audi Setiadi\textsuperscript{1,2} ORCID 0000-0002-2361-7391
(audi.setiadi@cw.bc.ca)}

\textbf{Claudia Singh\textsuperscript{1}(cadsingh@student.ubc.ca)}

\textbf{Amanda Li\textsuperscript{3}(ali3@cw.bc.ca)}

\textbf{Nicholas Au\textsuperscript{1,2}(nau2@cw.bc.ca)}

\textbf{Ali Amid\textsuperscript{3} 0000-0001-6926-7502
(ali.amid@cw.bc.ca)}

\begin{enumerate}
\tightlist
\item
  Department of Pathology and Laboratory Medicine, University of British
  Columbia, Vancouver, BC, Canada
\item
  Department of Pathology and Laboratory Medicine, Division of
  Hematopathology, BC Children's Hospital, Vancouver, BC, Canada
\item
  Division of Hematology \& Oncology, BC Children's Hospital, Vancouver,
  BC, Canada
\end{enumerate}

\textbf{Correspondence to:} Audi Setiadi, BC Children's Hospital, Room
2K29 - 4480 Oak Street, Vancouver BC V6H 3N1. Tel: (604) 875-2345 x2269.
Fax: (604) 875-2815. audi.setiadi@cw.bc.ca

Conflict of interest disclosure: No conflict of interest

Word count: 838

Running title: Atypical Megakaryocytes in Diamond Blackfan Anemia

Figures: 1 (3 panels)

\textbf{Key words:} Diamond Blackfan Anemia; RPL5 mutation; Diamond
Blackfan Anemia type 6, megakaryocytes; dysplastic megakaryocytes;
myelodysplastic syndrome

Diamond-Blackfan anemia (DBA) is congenital erythroid aplasia associated
with defects in ribosomal biogenesis, classically presenting with
macrocytic anemia in infancy and congenital anomalies\selectlanguage{ngerman}¹. Ribosomal
protein S19 (\emph{RPS19)} is the most commonly mutated gene, while
Ribosomal protein L5 (\emph{RPL5)} mutation accounts for the minority
(7\%) of cases and is associated with more severe phenotype
than\emph{RPS19} mutation\textsuperscript{2,3}. Approximately 80\% of
patients with DBA respond to corticosteroid therapy, whereas 20\%
require chronic red blood cell (RBC) transfusion\textsuperscript{4}.
However, they are also at risk of developing myeloid malignancies later
in life, including myelodysplastic syndrome
(MDS)\textsuperscript{4,5,6}. There is an overlap in presentation
between DBA and MDS, therefore careful bone marrow assessment with
ancillary testing are crucial to detect disease transformation.
Megakaryocytic dysplasia at disease presentation in DBA is a highly
atypical finding that has not been well described in the literature. We
report two cases of DBA with \emph{RPL5} mutation (DBA type 6) showing
peculiar megakaryocytic morphology with monolobated nuclei at baseline,
mimicking MDS.

Two unrelated infants presented with reticulocytopenic anemia, mild
dysmorphic features and cardiac anomalies at birth. Patient 1 was a term
male infant with hemoglobin (Hb) of 81 g/L, reticulocyte count of 10 x
10\selectlanguage{english}9/L, and mean corpuscular volume (MCV) of 99.2 fL. Patient 2 was a
female infant born at 36 weeks gestation with Hb of 67 g/L, reticulocyte
count of 56 x 109/\selectlanguage{english}L, and MCV of 110 fL. Both otherwise had normal blood
cell counts and smears. Bone marrow biopsies were first performed at
three months of age for patient 1 and one year of age for patient 2.
Bone marrow in both patients showed normal to mildly increased
cellularity with paucity of erythroid precursors and no dysplasia in
erythroid or granulocytic lineages. Blasts were not increased
(\textless{}1\%). Unexpectedly, however, both marrows showed increased
number of monolobated megakaryocytes, observed in approximately half of
the lineage (Figs. 1A-C). Exome sequencing revealed heterozygous
mutation in \emph{RPL5} gene c.{[}67C\textgreater{}T{]};{[}={]},
p.{[}Arg23*{]};{[}={]} for patient 1 and c.67C\textgreater{}T,
p.{[}Arg23Ter{]} for patient 2, leading to the diagnosis of DBA type 6.
Cytogenetic analysis were normal and next generation sequencing of the
bone marrow samples were negative for mutations in myeloid-malignancy
related genes.

Both patients were managed with red blood cell transfusions (RBC) as
needed and steroids. Patient 1 had follow-up bone marrow biopsies at age
five (Fig. 1B) and eight years, which revealed similar findings as
previous with persistence of monolobated megakaryocytes and no clonal
evolution. At nine years of age, he had been weaned off steroids and
only required one RBC transfusion in the past 2 years. Patient 2 was
started on steroids at one year of age and was able to discontinue
transfusion therapy.

Both DBA and MDS can present with macrocytosis and reticulocytopenic
anemia. However, bone marrow in DBA is classically characterized by the
absence or \textless{}5\% of erythroid progenitors and absence of
dysplasia at baseline. Megakaryocytic monolobation is considered a
dysplastic feature in MDS, particularly the 5q deletion
subtype\textsuperscript{5}. This observation raised a concern of
transformation to MDS. However, megakaryocytic atypia was present as
early as three months of age for Patient 1, there was no dysplasia in
other cell lineages, genetic testing was negative for MDS-related
mutations, and both patients were eventually weaned off steroids and
became independent of transfusions years later, following the expected
clinical course of DBA. Although megakaryocytes in infants may show less
nuclear segmentation or ploidy compared to older children, the number of
non-lobated megakaryocytes in these cases was out of proportion to
normal age-related changes, and persisted beyond five years of age in
Patient 1. To our knowledge, baseline megakaryocytic dysplasia has never
been published in DBA literature, apart from one poster abstract that
also described an increased number of non-lobulated megakaryocytes in
patients with \emph{RPL5} and \emph{RPL11} mutations\textsuperscript{7}.

These findings shed light into common pathogenesis between the two
disorders. Deletion of 5q in MDS results in haploinsufficiency
of\emph{RPS14} , a ribosomal gene closely related to those implicated in
DBA. \emph{RPS14 is} involved in the MDM2/p53 axis, which is essential
in erythroid and megakaryocytic differentiation\textsuperscript{8,9}.
The inability to assemble ribosomes leads to free ribosomal proteins
which bind Murine Double Minute-2 (MDM2) and in turn suppress
ubiquitination of p53. This allows for accumulation of p53, resulting in
erythroid hypoplasia and dysmegakaryopoiesis\textsuperscript{8,9}.
Interestingly, both RPL5 and RPL11 proteins have also been shown to be
implicated in p53 activation through interaction with the MDM2
protein\textsuperscript{10,11,12}. Through this shared MDM2/p53 pathway,
it is plausible that \emph{RPL5} and \emph{RPL11} haploinsufficiency
leads to dysmegakaryopoiesis similar to that in MDS with 5q deletion.

Childhood MDS is often considered a higher risk disease, with 5-year
overall survival of 63\% and usually requires stem cell
transplant\textsuperscript{5,6}. It is imperative to recognize that the
``dysplastic'' megakaryocytes may represent a baseline finding in DBA
with \emph{RPL5} mutations instead of progression to MDS, since the
long-term prognosis and management for the two conditions are different.
Further studies are needed to investigate whether DBA with
\emph{RPL5}mutations represent a distinct subcategory that shares
similar pathophysiology with MDS with 5q deletion and explore common
therapeutic strategies for both conditions.

\textbf{Consent}

Informed consent was obtained from the patient for publication of this
case report and accompanying images. A copy of the written consent is
available upon request by the Editor-in-Chief of this journal.

\textbf{Competing interests}

The authors declare that they have no competing interests.

\textbf{Authors' contributions}

AS and CS put together the patient data, took pathology images, and
reviewed literature on the topic. AS, NA, AL and AA were all involved in
the patient's care and all authors contributed to writing the
manuscript. All authors read and approved the final manuscript.

\textbf{Acknowledgement}

Dr. Ali Amid's research is supported by Naiman-Vickars Endowment Fund.

\textbf{References}

\begin{enumerate}
\tightlist
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\textbf{Figure Legend:}

FIGURE 1 Bone marrow aspirate slides showing paucity of erythroid
precursors and hypo/monolobated megakaryocytes (pointed by black arrows)
in A) Patient 1 at three months of age (50x objective, Wright stain), B)
Patient 1 at five years of age (50x objective, Wright stain), and C)
Patient 2 at one year of age (50x objective, Wright stain).\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Panel-A-C-300-dpi-PBC/Panel-A-C-300-dpi-PBC}

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\end{figure}

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