Hb Mizuho is very rare. To our knowledge, only 6 cases have been reported in the literature [1,2,3,4,5,6], with only one adult patient. Hb Mizuho was first reported in a Japanese girl (1), next in an Italian girl (2), and later in boys from USA (3) and the Netherlands (4). Hb Mizuho has recently been identified in two family members from Sri Lanka (5). Hb Mizuho was mainly reported as a de novo mutation, with the exception of the two children reported here and the Sri Lankan boy.
Here, we describe the courses of three Swiss family members with Hb Mizuho with transfusion dependency since early infancy due to macrocytic, normochromic anemia with severe chronic hemolysis. Interestingly, splenectomy did not lead to resolution of transfusion dependency in any of them. Removal of the spleen ideally prolongs red blood cell survival by reducing red blood cell sequestration and may ultimately result in the reduced need for blood transfusions. Splenectomy has been shown to reduce transfusion requirements in patients with thalassemia [7, 8] as well as sickle cell disease . In unstable hemoglobins, splenectomy should be considered when there is severe anemia and/or massive or symptomatic splenomegaly . In the mother, there was practically no change in transfusion requirement post-splenectomy; the children however have profited mildly.
Variants of unstable hemoglobin represent a diagnostic challenge in rare cases of congenital hemolytic anemia. The lack of previous family history can guide the diagnosis in the wrong direction and delay the diagnosis. Easier access to molecular tests has greatly facilitated diagnosis in recent years. The first of our three cases clearly illustrates this difficulty prior to easy access to molecular diagnosis. Despite the fact that hemolysis was detected right after birth, several rounds of diagnostic work-up remained unsuccessful with a confirmed diagnosis only at the age of 23 years. This was only possible with analysis of fresh blood, sampled directly in the laboratory.
The clinical history of case 1 is unique since this is the first Hb Mizuho presenting with Moyamoya complications and the first case reported with long-term erythrocyte exchange.
Moyamoya disease is a very rare disorder characterized by progressive stenosis and occlusion of the internal carotid arteries and arteries of the circle of Willis and its major branches. Suzuki and Takaku introduced the term “Moyamoya disease” to describe the numerous collateral vessels at the base, which resembled a “puff of smoke” [11,12,13]. A single etiologic agent has not been identified, although toxic, infectious, and inflammatory etiologies have been proposed, but never proven . Moyamoya disease has previously been associated with sickle cell disease . Moyamoya has also been associated with other hemolytic anemias including hemoglobin E-beta thalassemia as well as the rare hemoglobin Fairfax with beta-thalassemia and Southampton hemoglobinopathy [16,17,18,19]. Putting all this into consideration, a correlation between Moyamoya and Hb Mizuho in our case is, though not proven, likely.
Furthermore, patients with chronic hemolytic anemia are known to be at a higher risk of thrombotic complications suggestive of a hypercoagulable state, the mechanism of coagulation activation likely being of multifactorial origin . Regarding the association of Hb Mizuho and thrombosis, we have not found other cases with thrombosis reported in the literature. It must be emphasized that the patient presented here developed Moyamoya disease at the age of 25. Since elevated free plasma hemoglobin caused by hemolysis is toxic for the vascular endothelium, this is a probable cause for the vasoocclusive events in our patient.
This case suggests that patients with Hb Mizuho could be at risk of thromboembolic complications and thrombosis prophylaxis may be warranted in particular cases.
Our index patient has profited from red blood cell exchange apheresis. Recurrent vasoocclusive events have since not occurred, and her iron values are stable. Sufficiently repressing the production of the harmful, aberrant hemoglobin could therefore be considered beneficial as secondary prophylaxis after a thromboembolic event. In red cell exchange, red blood cells (RBCs) containing abnormal Hb are removed and replaced by healthy volunteer donor RBCs. Red cell exchange can be performed by manual exchange or by automated exchange using a blood cell separator (erythrocytapheresis). Compared to simple RBC transfusions, this procedure offers several advantages, such as lower risk for iron accumulation and efficient control of pathological erythrocyte populations. The most frequent indication for RBC exchange is sickle cell disease; this is the standard treatment in patients with a history of or a risk for acute stroke and is a clinical option for other acute complications of sickle cell disease .
In this case, the treatment with hydroxyurea did not bring any clinical benefit; further conclusions are no possible due to the lack of data in the literature reporting experiences using hydroxyurea in this hemoglobinopathy.
Iron overload is of clinical relevance in many hemoglobinopathies. Iron overload develops from increased intestinal iron absorption signaled by hepcidin suppression secondary to ineffective erythropoiesis , while it can also be secondary to regular transfusions. The degree of toxicity may also vary depending on the disease.
Iron overload in Hb Mizuho patients seems to be mainly attributed to transfusions and has less to do with ineffective erythropoiesis. The degree of iron overload seems to present itself with different patterns. The type and dosing of chelation therapy should be adapted according to the individual’s course. Both oral and intravenous iron chelators are effective.
Due to the scarce information available on this unusual hemoglobinopathy, we aimed to review all the cases in order to delineate the most relevant characteristics of the published cases to date. Thus, the patients described in the literature had severe hemolytic anemia and with the exception of one; all were regularly transfused (Table 1). Remarkable basophilic stippling, Heinz bodies, and reticulocytosis seem to be a common feature in all these cases. All patients underwent splenectomy at a median age of 4 years (range 3–7) and had variable clinical improvement after splenectomy. One patient did reasonably well until the age of 4 years (3) and was not transfusion dependent even before splenectomy. Splenectomy in this case, which was performed due gradually worsening anemia (hemoglobin 72 g/l at the age of 4 years) and marked splenomegaly (spleen size 349 gr), improved his condition considerably. Three patients achieved complete resolution of transfusion dependency after splenectomy (cases 1, 2, and 4), supporting the benefit of this procedure.
Hemoglobin F level seems to be helpful in the follow-up of Hb Mizuho. A reported case with mild clinical presentation had a large quantity of HbF up to the age of 5 years (HbF levels of 35% and 22% at the ages of 2 and 4 respectively) (3). The Dutch boy described by Harthoorn et.al. (4) similarly had relatively high HbF levels, which could explain why transfusion dependency began much later at the age of 2 years. In the two of our patients, where HbF was measured, levels were normal, nicely explaining transfusion dependency in these patients.
Information regarding the clinical courses of Hb Mizuho patients through to adulthood is scarce. This forth reported patient from the Netherlands was diagnosed with papillary thyroid carcinoma, for which he was curatively treated with a total thyroidectomy plus lymph node dissection and radioactive iodine therapy at the age of 13 years. He later developed fulminant hepatic failure requiring liver transplantation at the age of 25 years. Interestingly, Gilbert syndrome was also diagnosed. The authors describe a possible connection between the hemoglobinopathy and the liver disease, as it is known in intrahepatic cholestasis of sickle cell disease .
A normal hemoglobin electrophoresis should not rule out the diagnosis of unstable hemoglobin in patients with otherwise unexplained hemolytic anemia, since unstable hemoglobin variants that undergo rapid denaturation or are represented in the HbA fraction might not be detected by this method . This could lead to under- or misdiagnosis. Though repeat testing should be encouraged in unclear cases, this series shows the enormous utility of using molecular techniques for diagnosis. The numerous tests initially performed in our reported cases, including isopropanol stability tests, erythrocyte fragility tests, heat denaturation test, high performance liquid chromatography, and hemoglobin electrophoresis, did not yield conclusive results. Examination of hemoglobin stability by heat or isopropanol is a useful screening test in detection of unstable hemoglobins. Stability testing is performed by incubating with isopropanol, whereby unstable hemoglobins yield visible precipitates. Similarly, in heat stability testing, hemolysate is incubated for 1–2 h at 50 °C. Unstable hemoglobins yield a visible precipitate, while normal hemoglobins do not [24, 25]. Correcting pre-analytic errors in our index patient however led to the diagnosis. With this exception, identification of the variant required sequencing of a segment of amplified DNA that included the beta-globin gene. Bone marrow examinations did not contribute to the diagnosis either. The only relevant finding in the five cases where bone marrow examination was performed was erythroid hyperplasia.
It must be highlighted that the first Hb Mizuho case was reported in 1977 with the following 3 cases reported in the early 1990s. The algorithm for hemoglobinopathy testing has since evolved enormously . Red cell indices and morphology followed by separation and measurement of Hb fractions using protein-based methods (Hb electrophoresis and high performance liquid chromatography (HPLC)) are nowadays the methods of choice for initial qualitative and quantitative Hb analyses. Hb variants can usually be detected using these methods. A survey done by van Zwieten et al.  involving 20,000 blood samples showed that high performance liquid chromatography analysis followed by globin gene sequencing of rare variants is an effective method to reveal Hb variants. DNA-based methods are used as the following step for variants with ambiguous or unusual results from protein analysis. Cappellini et al.  illustrate the recommended diagnostic flowchart for identification of thalassemia, which can be applied for suspected Hb variants.
Molecular diagnostic possibilities have grown enormously in the past decades. This paper aims to raise awareness that rarer and not necessarily inherited diseases can nowadays easily be diagnosed.