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\begin{document}

\title{Hemochromatosis in a \selectlanguage{greek}β-\selectlanguage{english}thalassemia minor patient with H63D homozygous
mutation: A case report\selectlanguage{english}}



\author[1]{Nishan Pokhrel}%
\author[1]{Shambhu Khanal}%
\author[1]{Parikshit Chapagain}%
\author[1]{Biraj Pokhrel}%
\author[1]{Anjan Shrestha}%
\affil[1]{Tribhuvan University Institute of Medicine}%


\vspace{-1em}



  
  \date{May 07, 2024}


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\selectlanguage{english}
\begin{abstract}
We report the case of a 73-year-old man who was presumed to have iron
deficiency anemia and was treated with iron supplements since
adolescence. His workup revealed \selectlanguage{greek}β-\selectlanguage{english}thalassemia minor and H63D homozygous
hereditary hemochromatosis complicated with liver cirrhosis and
hepatocellular carcinoma.%
\end{abstract}%



\sloppy


\textbf{KEYWORDS}

anemia, \selectlanguage{greek}β-\selectlanguage{english}thalassemia, case report, cirrhosis, h63d mutation, hereditary
hemochromatosis, hepatocellular carcinoma, \emph{HFE} gene, iron
overload

\section*{Introduction}

{\label{introduction}}

Hereditary hemochromatosis (HH) is an autosomal recessive disorder
caused mostly by mutations in the~\emph{HFE~} gene located on chromosome
6. It is characterized by an excessive accumulation of iron in the body,
and affects 0.3\% to 0.5\% of Caucasians of northern European
descent.\textsuperscript{1} Increased intestinal absorption of iron
leads to iron overload in several organs, resulting in various
manifestations such as liver cirrhosis, hepatocellular carcinoma (HCC),
diabetes, arthritis, and cardiomyopathy.\textsuperscript{2}

\selectlanguage{greek}β-\selectlanguage{english}thalassemia is an inherited hemoglobinopathy characterized by reduced
synthesis of one or both of the \selectlanguage{greek}β-\selectlanguage{english}globin chains of adult hemoglobin. Two
\selectlanguage{greek}β-\selectlanguage{english}globin genes are present on chromosome 11. The \selectlanguage{greek}β-\selectlanguage{english}globin genes (\selectlanguage{greek}β/β\selectlanguage{english})
produce the \selectlanguage{greek}β-\selectlanguage{english}globin chains, which compose normal adult hemoglobin.
Their mutations result in an absence (\selectlanguage{greek}β\selectlanguage{english}\textsuperscript{0}) or
diminished production (\selectlanguage{greek}β\selectlanguage{english}\textsuperscript{+}) of the \selectlanguage{greek}β-\selectlanguage{english}globin chain.
\selectlanguage{greek}β-\selectlanguage{english}thalassemia minor (BTM) (\selectlanguage{greek}β\selectlanguage{english}\textsuperscript{+}/\selectlanguage{greek}β,
β\selectlanguage{english}\textsuperscript{0}/\selectlanguage{greek}β\selectlanguage{english}) is the mildest form of the disease, and is
usually an asymptomatic carrier state. It manifests as mild anemia with
microcytosis and hypochromia of the red blood cells (RBCs), and can be
mistaken for iron deficiency anemia (IDA). Individuals with BTM develop
mild~ineffective erythropoiesis, which increases iron absorption;
however, most of them do not develop iron overload.\textsuperscript{3}

The coexistence of HH and \selectlanguage{greek}β-\selectlanguage{english}thalassemia has not been reported from Nepal
to the best of our knowledge. Herein, we describe the case of a
73-year-old man suspected with IDA, who was eventually diagnosed to have
hemochromatosis resulting from coincidental BTM and H63D homozygous HH
and long-term iron supplementation.

\section*{Case report}

{\label{case-report}}

A 73-year-old normotensive Asian man presented with generalized
weakness, dizziness, and palpitations for two weeks. He had experienced
such symptoms at frequent intervals since adolescence. In the past, he
had also been hospitalized on multiple occasions for fever, chills,
rigors, cough, and diarrhea. Seven years ago, he was diagnosed with
diabetes mellitus. He did not report anorexia, nausea, vomiting,
prolonged bleeding, epistaxis, hematemesis, and melena. He consumed a
mixed diet. He had smoked 48 pack-years of cigarettes, and had consumed
seven units of alcohol daily for 33 years, from the age of 20 to 53. His
family did not have history of similar illnesses or consanguinity.

On examination, the patient appeared ill, and had visible muscle
wasting. His vital signs were normal. Pallor was observed; icterus,
cyanosis, clubbing, and lymphadenopathy were absent. Respiratory and
cardiovascular examination findings were normal. Abdominal examination
revealed a distended abdomen with shifting dullness on percussion. The
liver was enlarged, and had an irregular surface. The liver span
measured 19 cm, and the lower edge was palpated 10 cm below the right
subcostal margin. The spleen was enlarged, too, and the splenic notch
was palpated 11 cm below the left subcostal margin. The pubic hair had a
normal pattern of distribution. Digital rectal examination revealed
grade-II prostatomegaly.

The patient was admitted, and investigations were done. Complete blood
count revealed the following findings: hemoglobin (Hb), 6.4 g/dL
(13.5-17.5); mean corpuscular volume (MCV), 69.4
\selectlanguage{greek}µ\selectlanguage{english}m\textsuperscript{3}~(80-100); mean corpuscular Hb (MCH), 20.6 pg/cell
(25.4-34.6); mean corpuscular Hb concentration (MCHC), 29.5\% (31-36);
RBC count, 3.1 million/\selectlanguage{greek}µ\selectlanguage{english}L (4.3-5.9); white blood cell (WBC) count, 3870
cells/\selectlanguage{greek}µ\selectlanguage{english}L (4000-11000); and platelet count 60000 cells/\selectlanguage{greek}µ\selectlanguage{english}L
(150000-450000). Liver function test revealed the following findings:
total bilirubin, 14 \selectlanguage{greek}µ\selectlanguage{english}mol/L (3-21); direct bilirubin, 4 \selectlanguage{greek}µ\selectlanguage{english}mol/L (0-5);
alanine aminotransferase (ALT), 48 IU/L (5-45); aspartate
aminotransferase (AST), 51 IU/L (5-40); alkaline phosphatase (ALP), 48
IU/L (5-45); lactate dehydrogenase (LDH), 422 IU/L (\textless{}460);
albumin 30 g/L (38-49); prothrombin time (PT), 18 s (11-15); and
international normalized ratio (INR), 1.31 (0.8-1.1). Iron profile
revealed the following findings: serum ferritin, 1215.9 ng/mL (22-322);
serum iron, 188 \selectlanguage{greek}µ\selectlanguage{english}g/dL (65-175); transferrin saturation, 94.5\% (25-45);
and total iron binding capacity, 199 \selectlanguage{greek}µ\selectlanguage{english}g/dL (250-450). Other laboratory
findings were: fasting blood glucose, 130 mg/dL (70-110); two hours
postprandial blood glucose, 190 mg/dL (\textless{}140);
HbA\textsubscript{1}C, 5.5 g/dL (4.5-6.4); c-peptide, 3.8 ng/mL
(0.78-5.19); and a normal renal function test. Mentzer index, the ratio
of MCV (in fL) to RBC count (in millions/\selectlanguage{greek}µ\selectlanguage{english}L), was 22.39. Peripheral
blood smear showed microcytic RBCs with anisopoikilocytosis, target
cells, teardrop cells, few microspherocytes, and schistocytes.
High-performance liquid chromatography (HPLC, Bio-Rad D-10 hemoglobin
testing system) revealed 5.9\% HbA\textsubscript{2}~(1.5-3.5) and 1.7\%
HbF (0-2.0). High HbA\textsubscript{2}, low MCV, and low MCHC indicated
BTM. Serological spot tests for human immunodeficiency virus (HIV)
antibody, hepatitis B surface antigen (HBsAg), and hepatitis C virus
(HCV) antibody were negative. The ascitic fluid analysis revealed high
serum ascites albumin gradient (SAAG [?] 1.1 g/dL) and a low total protein
(\textless{} 2.5 g/dL).

Contrast enhanced computed tomography scan of the abdomen and pelvis
revealed HCC in segment IVb of liver along with cirrhosis and portal
hypertension (Figure~1). The Child-Pugh score for the severity of liver
disease was 10 (Child class C). As the mass was in contact with anterior
abdominal wall with loss of fat plane, it was unresectable, and liver
transplantation was not possible. The serum alpha-fetoprotein (AFP) was
600 ng/mL (\textless{}10).

To rule out hereditary hemochromatosis, the~\emph{HFE~} gene was
genotyped by multiplex polymerase chain reaction method, which showed
H63D homozygous mutation. Thus, the final diagnosis of BTM with H63D
homozygous HH complicated with liver cirrhosis and HCC was made. Body
iron stores could not be estimated with magnetic resonance imaging (MRI)
due to financial constraints.

Following this, the patient was started on iron chelation therapy with
subcutaneous deferoxamine infusion (1 g/day). After one month of
treatment, his iron profile was: iron, 170 \selectlanguage{greek}µ\selectlanguage{english}g/dL; TIBC, 220 \selectlanguage{greek}µ\selectlanguage{english}g/dL; and
ferritin, 800 ng/mL. As tumor resection and liver transplantation were
not possible, he was discharged on palliative care. His symptoms had
improved marginally at the time of discharge. The patient had a son
whose genetic analysis could not be performed due to financial
constraints, but his iron profile was normal. He was counseled about its
necessity to diagnose the disease early.

The patient followed up twice in the next month. His symptoms had not
improved further. Unfortunately, he passed away after one month of the
last follow-up, at his own home. The immediate cause of death could not
be established.

\section*{Discussion}

{\label{discussion}}

There are four types of HH based on the genetic defects leading to the
disorder. Type 1 hemochromatosis results from mutations in
the~\emph{HFE} ~gene; type 2 from mutations in either the~\emph{HJV~}
or~\emph{HAMP~} gene; type 3 from mutations in the~\emph{TFR2~} gene;
and type 4 from mutations in the~\emph{SLC40A1~} gene. Type 1 is the
commonest form of HH, and results mostly from two mutations in
the~\emph{HFE~} gene: C282Y mutation and H63D
mutation.\textsuperscript{4} C282Y mutation occurs when there is guanine
to adenine change at nucleotide 845 in the~\emph{HFE~} gene, whereas
H63D mutation occurs due to cytosine to guanine change at nucleotide 187
in the~\emph{HFE~} gene.\textsuperscript{5} The normal~\emph{HFE~} gene
product functions, primarily in hepatocytes, to increase hepcidin
transcription and reduce serum iron levels. When the~\emph{HFE~} gene is
mutated, the abnormal protein does not function properly, resulting in
increased absorption of iron.

The most common HH genotype is homozygosity for the C282Y variant
(C282Y/C282Y), and is mainly responsible for clinical hemochromatosis in
Caucasians.\textsuperscript{6,7} In non-Caucasians, the C282Y homozygous
mutation is less prevalent.\textsuperscript{7} Clinical manifestations
and a spectrum of risk for iron overload have been observed most
commonly among C282Y homozygotes followed by C282Y/H63D, C282Y/wt,
H63D/H63D, and H63D/wt genotypes in the descending
order.\textsuperscript{6} H63D/wt or H63D/H63D genotypes are not found
to be associated with iron loading or manifest only mild to moderate
iron overload unless they are associated with C282Y as a compound
heterozygote: C282Y/H63D.\textsuperscript{2}

H63D mutation, in isolation, does not cause significant iron overload
despite being associated with increased serum transferrin saturation,
and requires additional modifying factors to express overt iron
overload.\textsuperscript{8} Several authors have studied how H63D
mutations affect iron load in \selectlanguage{greek}β-\selectlanguage{english}thalassemia carriers. In their study,
Wilson et al\textsuperscript{9} found that Egyptian \selectlanguage{greek}β-\selectlanguage{english}thalassemia
patients and carriers, who had homozygous H63D mutation, had
significantly higher serum ferritin levels than those who did not. In
another study, Melis et al\textsuperscript{10}~found that \selectlanguage{greek}β-\selectlanguage{english}thalassemia
carriers, who were homozygous for H63D mutations, had higher ferritin
levels than those who were heterozygous for this~\emph{HFE~} allele, or
those who did not have it. They found the mean ferritin levels to be
389\selectlanguage{ngerman}±75 mcg/L (mean±SD) in homozygotes for the H63D mutation. However,
Yang et al\textsuperscript{11} found differing conclusions in a review
of similar studies that investigated the effect of H63D mutations on
iron load in \selectlanguage{greek}β-\selectlanguage{english}thalassemia major or carrier conditions. Some studies
from Italy, Portugal, India, and Egypt suggested that the interacting
effect of \selectlanguage{greek}β-\selectlanguage{english}thalassemia with homozygous or even heterozygous H63D
mutations might lead to iron overload; other reports from Italy, India,
Thailand, Brazil, and Spain indicated that the iron status was not
related to H63D mutation.

BTM, which results from heterozygosity for \selectlanguage{greek}β-\selectlanguage{english}thalassemia, is clinically
asymptomatic, and is defined by characteristic hematological features:
microcytosis, hypochromia, and increased HbA\textsubscript{2~}level. The
hemoglobin pattern of \selectlanguage{greek}β-\selectlanguage{english}thalassemia heterozygotes is characterized by
92\% to 95\% HbA, \textgreater{}3.8\% HbA\textsubscript{2}, and a
variable amount of HbF (0.5\% to 4\%). BTM is identified by determining
MCV, MCHC, and HBA\textsubscript{2}.\textsuperscript{12} Mentzer index
is one of several discrimination indices that can be calculated from RBC
indices during routine complete blood count. It is regarded as an
inexpensive measure to differentiate BTM from IDA, especially in
settings where iron profile and HPLC are unavailable. It is less time
consuming, and does not necessitate additional laboratory work. Mentzer
index greater than 13 is compatible with IDA, and a value less than 13
is suggestive of BTM.\textsuperscript{13} In our case, the Mentzer index
was 22.39, which would indicate IDA, but HPLC findings suggested the
diagnosis of BTM.

Individuals with BTM tend to increase iron absorption because of mild
anemia and slightly increased erythropoiesis, and the level of ferritin
increases moderately with age. Only a minority of them develop iron
overload, depending on the presence of other acquired or genetic
factors. Some cases develop iron overload due to additional genetic
determinants, whereas others do so due to long-term iron
supplementation.\textsuperscript{10,14}~Owing to mild anemia and
microcytosis,~BTM is often mistaken as IDA, and supplementary iron is
prescribed. Our patient, too, was repeatedly prescribed iron supplement
with the suspicion of IDA because of which he might have developed iron
overload; H63D homozygous mutation might have played an augmenting role.
Knox Macaulay et al\textsuperscript{15} have reported a similar
occurrence as ours in a 68-yr-old woman who received iron intermittently
for 15 years due to the misdiagnosis of BTM as IDA. On presentation, she
had a fully saturated iron binding capacity and clinical evidence of
iron overload. Therefore, serum iron studies must be done in an
individual with microcytic anemia to distinguish thalassemia from IDA
(low ferritin suggests iron deficiency), and to look for iron overload
in individuals with thalassemia.\textsuperscript{3}

BTM is usually associated with mild anemia.\textsuperscript{12} Our
patient's hemoglobin on presentation was 6.4 gm/dL. One reason for such
low hemoglobin could be the physiological effect of iron overload on
heme synthesis. It has been suggested that increased iron concentration
at the site of hemoglobin synthesis in patients with iron overload forms
an iron-pyridoxal complex, and prevents pyridoxal-5-phosphate from
serving as a coenzyme in heme synthesis.\textsuperscript{16}

Chronic alcohol consumption, in moderate to excessive amounts, is
associated with elevated levels of serum ferritin and transferrin
saturation, and can result in increased hepatic iron
stores.\textsuperscript{17} Ethanol induces the downregulation of the
transcription factor that regulates hepcidin expression. This effect
results in the upregulation of the intestinal iron transporters, thereby
increasing intestinal iron absorption.\textsuperscript{17} Iron overload
occurs not only in patients with HH but also in individuals with
alcoholic liver disease, nonalcoholic fatty liver disease, and hepatitis
C infection. Chronic liver disease decreases the synthetic function of
the liver, including the production of hepcidin. Lower levels of
hepcidin increase the iron load.\textsuperscript{17}

Hemochromatosis requires a high degree of suspicion for clinical
identification as most of the patients present with symptoms like
extreme fatigue, arthralgia, and loss of libido, which are usually
attributed to other diseases. Skin pigmentation, diabetes mellitus, and
liver cirrhosis, which are thought to be the usual manifestations of
hemochromatosis, occur infrequently.\textsuperscript{18} Our patient
reported easy fatigability, and had diabetes mellitus and liver
cirrhosis. He did not report arthralgia or loss of libido.
Hemochromatosis might have eluded suspicion for a long time in our
patient because fatigue is more likely to be attributed to anemia than
to hemochromatosis, and liver cirrhosis to chronic alcohol consumption.
Additionally, patients with iron overload are more prone to infections
due to organisms whose virulence is increased in the presence of excess
iron, such as~\emph{Listeria monocytogenes} ,~\emph{Yersinia
enterocolitica} ,~\emph{Escherichia coli,~} and~\emph{Vibrio vulnificus}
.\textsuperscript{4} In our patient, we could only suspect that these
infections might have caused the past episodes of recurrent fever and
diarrhea.

Patients with iron overload are treated with phlebotomy. Those who
cannot tolerate phlebotomy due to concomitant anemia can be treated with
iron chelation if the iron load is significant.\textsuperscript{4} Iron
chelation therapy is not recommended in individuals with isolated
ferritin elevations below 1000 ng/mL because the adverse effects are
likely to outweigh the benefits.\textsuperscript{19} As our patient had
severe anemia with iron overload (ferritin 1215.9 ng/mL), we avoided
phlebotomy, and offered iron chelation with deferoxamine~instead.

Kowdley et al\textsuperscript{4} recommend performing~\emph{HFE~}
mutation analysis and iron studies of all first-degree relatives of
patients with~\emph{HFE} -related HH to detect disease early and prevent
complications. Detection of C282Y homozygosity or compound
heterozygosity in the relative, in the presence of increased serum
ferritin levels, requires the initiation of therapeutic phlebotomy and
yearly follow-up with iron-studies. C282Y heterozygotes, H63D
heterozygotes, and H63D homozygotes can be reassured that they are not
at risk for developing progressive or symptomatic iron overload. Our
patient had a son, who was counseled for~\emph{HFE~} mutation analysis
and iron studies.~\emph{HFE~} mutation analysis could not be performed
due to financial limitation, but the iron profile was normal.

Long-term survival in patients with hemochromatosis is dependent on the
degree and the duration of iron overload.\textsuperscript{20} The most
important prognostic factor at the time of diagnosis is the presence of
liver fibrosis or cirrhosis. Patients treated before the occurrence of
liver cirrhosis have a normal life expectancy, whereas untreated
patients die, most frequently from heart failure, cirrhosis,
or~HCC.\textsuperscript{19} We encountered our patient at a late stage
of illness when hemochromatosis was already complicated with liver
cirrhosis and HCC. Though liver transplantation is indicated in patients
with HH who develop HCC,\textsuperscript{4} it was not possible in our
patient due to the adherence of the tumor with the anterior abdominal
wall. He might have died as a consequence of HCC.

\section*{Conclusion}

{\label{conclusion}}

Serum iron profile should be routinely measured in all patients with
microcytic anemia. BTM may often be erroneously diagnosed as IDA, and
should be suspected in any patient unresponsive to iron therapy.
Hemochromatosis should be considered in anemic individuals with high
serum transferrin saturation. Sometimes BTM may be accompanied by
coexisting~\emph{HFE~} gene mutations, which aggravate iron overload on
the background of \selectlanguage{greek}β-\selectlanguage{english}thalassemia.

\textbf{List of Abbreviations}

AFP: Alpha Ferroprotein

ALP: Alkaline Phosphatase

ALT: Alanine Aminotransferase

AST: Aspartate Aminotransferase

BTM: \selectlanguage{greek}β-\selectlanguage{english}Thalassemia Minor

Hb: Hemoglobin

HBsAg: Hepatitis B Surface Antigen

HCC: Hepatocellular Carcinoma

HCV: Hepatitis C Virus

HH: Hereditary Hemochromatosis

HIV: Human Immunodeficiency Virus

HPLC: High-Performance Liquid Chromatography

IDA: Iron Deficiency Anemia

INR: International Normalized Ratio

LDH: Lactate Dehydrogenase

MCH: Mean Corpuscular Hemoglobin

MCHC: Mean Corpuscular Hemoglobin Concentration

MCV: Mean Corpuscular Volume

PT: Prothrombin Time

RBC: Red Blood Cell

SAAG: Serum Ascites Albumin Gradient

TIBC: Total Iron Binding Capacity

TSAT: Transferrin Saturation

\textbf{ACKNOWLEDGEMENTS}

The authors are grateful to Dr. Pradeep Raj Regmi, Pediatric Imaging
European School of Radiology (ESOR), University Hospital of Ioannina,
Greece for his insights on the radiological images; the patient; and his
family for their valuable support in the preparation of this manuscript.

\textbf{Declaration}

\textbf{Ethics approval and consent to participate}

Need for ethical approval waived. Consent from the patient's daughter
deemed to be enough.

\textbf{Consent for publication}

Written informed consent was obtained from the patient for publication
of this case report and any accompanying images. A copy of the written
consent is available for review by the editor-in-chief of this journal.

\textbf{Availability of data and material}

Not applicable

\textbf{Conflict of interest}

The authors declare that there is no conflict of interest regarding the
publication of this paper.

\textbf{Author contributions}

NBP, SK, and BP: wrote the initial draft of the manuscript. AS reviewed
the manuscript. NBP, PC, and BP: edited the draft and reshaped it into
this manuscript. All authors approved the final version of the
manuscript and agree to be accountable for all aspects of the work in
ensuring that questions related to the accuracy or integrity of any part
of the work are appropriately investigated and resolved.

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\textbf{Caption of figure}

\textbf{Figure 1} CECT scan of abdomen and pelvis (A-axial, B-coronal,
C-sagittal) showing 4.6 \selectlanguage{ngerman}× 3 cm well-circumscribed, hypodense mass in
segment IVb of liver in subcapsular region (red arrows). The mass is in
contact with anterior abdominal wall with loss of fat plane. CECT,
contrast-enhanced computed tomography\selectlanguage{english}
\begin{figure}[H]
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\includegraphics[width=0.70\columnwidth]{figures/Figure-CECT-SuppInfo/Figure-CECT-SuppInfo}

\end{center}
\end{figure}

\selectlanguage{english}
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