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\begin{document}

\title{Double Trouble: A Case of Fraternal Twins with Iron-Refractory
Iron-Deficiency Anaemia}



\author[1]{Jacques Malherbe}%
\author[2]{Catherine Cole}%
\affil[1]{Fiona Stanley Hospital}%
\affil[2]{Saturn Pathology}%


\vspace{-1em}



  
  \date{May 25, 2024}


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\selectlanguage{english}
\begin{abstract}
Iron-refractory iron-deficiency anaemia (IRIDA) is a rare autosomal
recessive disease that presents in childhood. We report the case of
fraternal twins presenting with severe hypochromic microcytic anaemia
and hypoferritinemia. Two missense mutations affecting the
\emph{TRMPSS6} gene were identified, consistent with IRIDA. Subsequent
parenteral iron therapy improved clinical and blood parameters.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION:}

Iron deficiency anaemia in children is usually attributed to poor
dietary intake of oral iron, malabsorption secondary to inhibitory
substances (\emph{e.g.} cow's milk) and chronic inflammatory conditions
affecting the gastrointestinal tract (\emph{e.g.} coeliac
disease,\emph{Strongyloides stercoralis} ) {[}1-3{]}. Upon treatment of
the underlying cause, most individuals respond to oral iron therapy with
marked improvements in their clinical symptomatology and red cell
indices {[}3{]}. However, patients who are refractory to oral
supplementation should provoke further investigation into uncommon and
rare disorders affecting iron absorption, metabolism and erythropoiesis
(\emph{i.e.} heme and globin manufacture). These disorders include
thalassaemia, sideroblastic anaemia and iron-refractory iron-deficiency
anaemia (IRIDA) {[}1,2{]}.

IRIDA is a rare, autosomal recessive disorder and usually presents in
early childhood {[}1,2{]}. Such patients present with severe microcytic
anaemia, profound hypoferremia, low transferrin saturation, elevated
hepcidin levels and negligible responses to oral iron replacement
therapy {[}2{]}. The disorder arises from mutations affecting
the\emph{TMPRSS6} gene located on the long arm of chromosome 22q12.3
{[}2,4{]}. The gene encodes matriptase-2 (MT-2), a transmembrane serine
protease expressed by hepatocytes that once activated, downregulates
hepcidin expression by interfering with hemojuvelin-hepcidin coupling.
This abrogates the inhibitory functions of hepcidin, thereby augmenting
iron absorption from the gut and facilitating the mobilisation of iron
stores from macrophages {[}2,4{]}.

Mutations affecting the \emph{TMPRSS6} gene were first discovered in
five multiplex kindreds by Finberg and colleagues in 2008 {[}4{]}. Since
then, several missense, nonsense, frameshift and splicing mutations
presenting in compound heterozygote or single homozygote forms have been
identified among an array of ethnicities and across all continents
except Australasia {[}5-12{]}. Missense mutations
affecting\emph{TMPRSS6} are generally associated with less severe
microcytic anaemias and lower hepcidin levels in comparison to those
IRIDA patients with other mutation types {[}4,6,7,12{]}. These patients
also tend to show some clinical response to oral iron replacement
therapy. IRIDA patients refractory to oral iron therapy require parental
supplementation {[}2,6{]}. Newer agents targeting hepcidin signalling
cascades have shown promising therapeutic benefits in murine models
{[}13{]}.

We report IRIDA in fraternal twins who failed to respond to oral
replacement therapy. Massively parallel gene sequencing of\emph{TMPRSS6}
confirmed the presence of two missense mutations expressed in a compound
heterozygous genotype. The case highlights the genetic and clinical
heterogeneity of patients with IRIDA despite common mutations affecting
\emph{TMPRSS6} and the occasional need for parenteral iron
supplementation.

\textbf{CASE PRESENTATION:}

A nine-year-old, non-Indigenous, Caucasian Australian boy (twin \#1) was
referred by his general practitioner to the paediatric gastroenterology
service at our State's children hospital for investigation and workup of
his severe microcytic anaemia that was not responding to a one-month
trial of oral 5mg/kg of ferrous sulphate liquid (30mg/mL) therapy. His
weight, height and growth were stable and normal for his age. His diet
was varied and included regular red meat intake without excess cow's
milk consumption. Despite the anaemia (Hb 69g/L), he was asymptomatic
and his mother reported no history of unexplained fevers or drenching
night sweats He had no personal or family history of chronic
inflammatory disorders.

On examination, his vital signs were within the normal range and his
oxygen saturation was \textgreater{}95\% on room air. He exhibited
marked pallor of the palmar creases, conjunctiva and face. A
comprehensive systems examination was otherwise unremarkable with no
organomegaly or lymphadenopathy. Initial full blood count and serum iron
studies (\emph{table 1} ) showed a profound hypochromic microcytic
anaemia (Hb 69g/L, MCV 56fL), normal erythrocyte sedimentation rates
(7.0 mm/hour), an increased red cell distribution width (22.0\%), severe
hypoferremia (\textless{}2.0\selectlanguage{greek}μ\selectlanguage{english}mol/L) with very low transferrin
saturations (0.03\%) and normal serum ferritin levels (32.0\selectlanguage{greek}μ\selectlanguage{english}g/L).
Gastroenterological investigations were undertaken, including a
gastroscopy, which elicited no evidence for \emph{Helicobacter pylori}or
\emph{Strongyloides stercoralis} infections, coeliac disease, autoimmune
gastritis or evidence of other chronic inflammatory diseases. The
patient was subsequently referred to the paediatric haematology service
for further investigation and continued with oral iron replacement
therapy.

He was reviewed five months later in the outpatient haematology clinic
alongside his fraternal twin brother (twin \#2), who was noted to
exhibit similar clinical features of central and peripheral pallor.
Peripheral blood sampling of the second twin identified similar
abnormalities to his brother (\emph{table 1} ) and he were also
commenced on oral iron replacement therapy. The blood films from both
twins showed persisting moderate erythrocyte anisopoikilocytosis, pencil
cells and elliptocytes despite ongoing oral iron therapy (\emph{figure
1a,b} ) with negligible improvements in red cell indices, serum iron and
ferritin. Haemoglobin electrophoresis studies excluded a
haemoglobinopathy in both siblings. Bone marrow biopsies were performed
and showed normocellular trilineage haematopoiesis with no ringed
sideroblasts or neoplasia. Occasional mild dyserythropoiesis
characterised by nuclear budding, few binucleate forms and poor
haemoglobinisation were seen (\emph{figure 1c,d} ). Cytogenetic analyses
were normal although iron stores within the marrow were absent
(\emph{figure 1e,f} ). Hepcidin levels were not measured as an approved
assay for this hormone was not available in our laboratory.

Given the ongoing poor response in both twins to oral iron replacement
therapy, a possible diagnosis of IRIDA was considered. Massively
parallel gene sequencing was performed on a peripheral blood sample of
twin \#1 one month later to assess for a \emph{TMPRSS6} mutation. Two
compound heterozygous c.(1324G\textgreater{}A; 1564G\textgreater{}A),
p.(G442R; E522K) mutations were identified, confirming a diagnosis of
IRIDA. No other novel mutations were identified in the
\emph{TMPRSS6}gene. Both twins were commenced on parenteral ferrous
carboxymaltose 500mg administered over 15 minutes one month later with
concurrent cessation of oral iron therapy. This resulted in marked
improvements in red cell indices and serum ferritin parameters. At five
years following their initial diagnosis, both twins remain clinically
well and have not required further parenteral iron supplementation.

\textbf{DISCUSSION:}

We report IRIDA in fraternal twins who presented with severe microcytic
anaemia, hypoferremia, hypoferritinemia and low transferrin saturation
states. Two missense mutations (p. G442R; p.E522K) in the
\emph{TMPRSS6}gene presenting in a compound heterozygous form were
detected using massively parallel gene sequencing. The mutations
sequenced in our Australian twins have been previously identified in
several other IRIDA patients {[}4,7,12{]}. To date, only one other case
report of a French-Canadian kindred has identified the precise compound
heterozygous missense mutations of \emph{TMPRSS6} seen in our patients
{[}7{]}. In parallel with the findings reported by Khuong-Quang \emph{et
al.} (2013) {[}7{]}, our patients presented with severe hypochromic
microcytic anaemia, hypoferremia and negligible transferrin saturation.
Similar blood count and serum iron abnormalities have been seen in other
IRIDA patients with either the p.G442R or the p.E522K missense mutation
{[}4,12{]}. However, unlike the French-Canadian siblings {[}7{]}, our
twins showed borderline normal to low serum ferritin levels and failed
to respond to simple oral iron replacement therapies. It was only after
the administration of parenteral iron infusions that profound and
sustained improvements in the haemoglobin and serum ferritin of our
patients were observed.

Reasons for these clinical differences despite a similar genotype has
been a contentious issue within the IRIDA arena. Genotype-phenotype
studies of a large European cohort of IRIDA patients across multiple
unrelated families have proposed that missense mutations affecting
the\emph{TMPRSS6} mutation through a compound heterozygous phenomenon
typically present with less severe anaemia and milder hypoferremia
{[}6{]}. In addition, they usually respond to oral iron therapy in
comparison to patients with single homozygous mutations or other lesion
types (\emph{e.g.} frameshift, nonsense). However, this appears to be at
odds with our patients. The clinical heterogeneity of IRIDA been
attributed to diverse \emph{TMPRSS6} mutations affecting variable
domains within the encoded MT-2 product, leading to different degrees of
dysfunction {[}6,12{]}. The p.G442R mutation of the \emph{TMPRSS6} gene
is known to target and alter the activity of the second C1r/C1s, urchin
embryonic growth factor and bone morphogenetic protein 1 (CUB) domain of
MT-2, which is involved in its autoactivation through autocatalytic
cleavage {[}4,6,12{]}. In contrast, the p.E522K mutation disrupts the
second class A low-density lipoprotein receptor (LDLRA) domain further
downstream {[}6,12{]}. This domain has been proposed to assist with the
conformational folding of MT-2 to achieve autocatalytic cleavage, whilst
also facilitating interactions between hemojuvelin and MT-2. Silvestri
and co-workers (2009) {[}12{]} showed through a series of separately
transfected HeLa cell lines with multiple missense mutations targeting
the \emph{TMPRSS6} gene, including p.G442R and p.E522K, that the latter
was more mutagenic. LDLRA domain alterations propagated through the
p.E522K lesion showed reduced expression of MT-2 on the cell membrane of
transfected cells and was unable to interfere with hemojuvelin {[}12{]}.
In contrast, the p.G442R mutation and subsequent aberrations in the CUB
domain did not alter the expression of the MT-2 protein on the cell
membrane {[}12{]}. Moreover, CUB mutations still retained partial,
albeit lowered hemojuvelin cleavage capabilities {[}12{]}. A compound
heterozygous inheritance mechanism for both missense mutations may
therefore cause a severe IRIDA phenotype that does not respond to oral
iron therapy through complete dysfunction of the mutated MT-2 encoded
product.

Of interest, we observed that despite a reasonable trial of oral iron
replacement therapy, neither child showed any clinical improvement in
red cell indices or serum iron levels. Previously published reports of
IRIDA patients with missense mutations affecting the CUB and LDLRA
domains, including the French-Canadian kindred, have successfully
treated their patients with oral iron supplements {[}6,7{]}.
Interestingly, the French-Canadian siblings only showed clinical
improvements after one year of oral iron therapy in combination with
ascorbic acid {[}7{]}. The shorter time course and exclusive use of oral
iron supplementation without ascorbic acid may account for the lack of
response seen in our patients. Nevertheless, parenteral iron infusions
are reported to yield clinical benefits. It has been previously shown
that recovery of red cell indices and ferritin is often slow and
prolonged with an ongoing need for parenteral iron administration
{[}2,6{]}. Surprisingly, we observed marked and sustained improvements
in red cells indices and ferritin levels in our patients after a single
iron infusion\textbf{.}

In summary, we have reported IRIDA in paediatric, fraternal twins with
compound heterozygous missense mutations in the \emph{TMPRSS6} gene. We
show that despite knowledge of these \emph{TMPRSS6} mutations
{[}4,6,7,12{]}, they confer extensive clinical heterogeneity. Therapy
should therefore be directed on a case-by-case basis. Currently, the
frequency of parenteral iron administration in IRIDA cases refractory to
oral iron replacement therapy is not established. Future prospective
cohort studies will hopefully elicit appropriate therapeutic guidelines
for IRIDA.

\textbf{ACKNOWLEDGEMENTS:}

The authors wish to express their gratitude to Dr. Sarah Steed for
sampling the bone marrow material, Dr. John Beilby for facilitating the
genetic testing and PathWest Laboratory Medicine for their technical
assistance.

\textbf{DECLARATIONS:}

Consent:

Written informed consent for the publication of this case and the
accompanying images was obtained from the patients' legal guardian.

Conflicts of Interest:

The authors declare that they have no conflicts of interest.

Funding \& Financial Support:

No funding or financial support was received for the study.

Author Contributions:

CHC cared for the patient. All authors collected, analysed and
synthesised all longitudinal data. JAJM undertook a comprehensive
literature review. JAJM wrote the manuscript. All authors critically
revised and approved the final manuscript.

Data Availability Statement:

All data generated and pertaining to this case report are included in
the manuscript.

\textbf{REFERENCES}

\begin{enumerate}
\tightlist
\item
  Pippard MJ. Microcytic anaemias in childhood and iron-refractory iron
  deficiency anaemia. \emph{Br J Haematol} 2017;177:167-168.
\item
  Henney MM, Finberg KE. Iron-refractory iron deficiency anemia
  (IRIDA).\emph{Hemat Oncol Clin North Am} 2014;28:637-652.
\item
  Hershko C, Camaschella C. How I treat unexplained refractory iron
  deficiency anemia. \emph{Blood} 2014;123:326-335.
\item
  Finberg KE, Henney MM, Campagna DR, \emph{et al}. Mutations
  in\emph{TMPRSS6} cause iron-refractory iron deficiency anemia
  (IRIDA).\emph{Nat Genet} 2008;40:569-571.
\item
  De Falco L, Totaro F, Nai A, \emph{et al}. Novel
  \emph{TMPRSS6}mutations associated with iron-refractory iron
  deficiency anemia (IRIDA). \emph{Hum Mutat} 2010;31:e1390-1405.
\item
  De Falco L, Silverstri L, Kannengiesser C, \emph{et al}. Functional
  and clinical impact of novel \emph{Tmprss6} variants in
  iron-refractory iron-deficiency anemia patients and genotype-phenotype
  studies. \emph{Hum Mutat} 2014;35:1321-1329.
\item
  Khuong-Quang D, Schwartzentruber J, Westerman M, \emph{et al}. Iron
  refractory iron deficiency anemia: Presentation with hyperferritinemia
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\item
  Choi HS, Yang HR, Song SH, \emph{et al}. A novel mutation Gly603Arg
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\item
  Sato T, Iyama S, Murase K, \emph{et al}. Novel missense mutation in
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\item
  Guillem F, Lawson S, Kannengiesser C, \emph{et al}. Two nonsense
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\item
  Pellegrino RM, Coutinho M, D'Ascola D, \emph{et al}. Two novel
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\item
  Silvestri L, Guillem F, Nai A, \emph{et al.} Molecular mechanisms of
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\item
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\end{enumerate}

\textbf{TABLES:}

\emph{Table 1.} Haematological and biochemical parameters in both
fraternal twins at diagnosis and four months post-parenteral iron
administration.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\textbf{Parameter} & \textbf{Paediatric Reference Range} & \textbf{Twin
\#1} & \textbf{Twin \#1} & \textbf{Twin \#2} & \textbf{Twin
\#2}\tabularnewline
\midrule
\endhead
& & \textbf{Diagnosis} & \textbf{Four Months Post-Parenteral Iron
Therapy} & \textbf{Diagnosis} & \textbf{Four Months Post-Parenteral Iron
Therapy}\tabularnewline
\textbf{WCC} & \textbf{4.50-14.50 x 10\textsuperscript{9}/L} & 2.80 &
4.90 & 5.70 & 5.70\tabularnewline
\textbf{Platelets} & \textbf{150-400 x 10\textsuperscript{9}/L} & 241 &
292 & 536 & 368\tabularnewline
\textbf{RCC} & \textbf{4.00-6.00 x 10\textsuperscript{12}/L} & 4.1 &
5.26 & 5.17 & 5.59\tabularnewline
\textbf{Hb} & \textbf{115-155g/L} & 61 & 94 & 76 & 100\tabularnewline
\textbf{Hct} & \textbf{0.35-0.45} & 0.26 & 0.32 & 0.27 &
0.33\tabularnewline
\textbf{MCV} & \textbf{75-92fL} & 54 & 60 & 52 & 58\tabularnewline
\textbf{MCH} & \textbf{25.0-33.0pg} & 15.0 & 18.0 & 14.6 &
17.8\tabularnewline
\textbf{MCHC} & \textbf{320-360g/L} & 283 & 299 & 283 &
306\tabularnewline
\textbf{RDW} & \textbf{9.0-15.0\%} & 17.9 & 20.5 & 19.4 &
22.9\tabularnewline
\textbf{Iron} & \selectlanguage{greek}\textbf{7-25μmol/L}\selectlanguage{english} & \textless{} 2 & 4 & 3 &
4\tabularnewline
\textbf{Transferrin} & \selectlanguage{greek}\textbf{20-41μmol/L}\selectlanguage{english} & 35 & 27 & 35 &
30\tabularnewline
\textbf{Transferrin Saturation} & \textbf{12-45\%} & 0.03 & 7 & 4 &
7\tabularnewline
\textbf{Ferritin} & \selectlanguage{greek}\textbf{15-100μg/L}\selectlanguage{english} & 32 & 157 & 9 &
142\tabularnewline
\bottomrule
\end{longtable}

\emph{Abbreviations:} fL -- femtolitre; g -- grams; Hb -- haemoglobin;
Hct -- haematocrit; L -- litre; MCH -- mean cell haemoglobin; MCHC --
mean cell haemoglobin concentration; MCV -- mean cell volume; pg --
picogram; RBC -- red cell count; RDW -- red cell distribution width; WCC
-- white cell count; \selectlanguage{greek}μ -- \selectlanguage{english}micro; \% -- percent.

\textbf{FIGURE LEGENDS:}

\textbf{Figure 1:} Representative blood films and bone marrow aspirate
samples (600x) obtained from both twins prior to parenteral iron
supplementation therapy. The upper and lower rows of images relate to
twin \#1 and twin \#2 respectively.

\textbf{(A, B)} Blood films from both twins show profound hypochromic
erythrocytes with anisopoikilocytosis, occasional pencil cells and
elliptocytes. \textbf{(C, D)} Bone marrow aspirate smears show normal
trilineage haematopoiesis with corresponding \textbf{(E, F)} absent iron
stores.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}

\selectlanguage{english}
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