-thalassemia is a common genetic disorder that is characterized by deficient or absent synthesis of -globin chains of the hemoglobin ( hb ) molecule . the human -globin gene cluster has two  genes ( 2 and 1 ) on each chromosome 16 . a normal person has four functional -globin genes and is designated as /. most -thalassemia determinants are deletions involving one ( / ) or both : loss of either both -genes from a single chromosome ( - - / ) or one gene from both chromosomes ( - / -  ) . an individual with three  genes ( / ) is not anemic but a heterozygote who inherits two functional -globin genes (  / ) has mild hypochromic microcytic anemia . the patients usually have splenomegaly ( which may be severe ) and occasionally this is complicated by hypersplenism . sickle - cell anemia is an autosomal recessive genetic red cell disorder with a worldwide distribution that results from a point mutation at codon 6 of the beta - globin gene , with the replacement of glutamic acid to valine , leading to the production of a defective beta globin chain (  , 6v ) of the hemoglobin . while the coinheritance of another beta - globin gene anomaly increases the proportion of hbs and therefore the risk of sickling , the combination of a deletion of an alpha - gene anomaly reduces intraerythrocytic hbs concentration , attenuating therefore the problems due to the sickle cell trait . since sickle cell trait is mainly seen in black populations , the coinheritance of hbh disease and a sickle cell trait has only been rarely described . usually , in patients with hbh disease , ineffective erythropoiesis in an expanded marrow stimulates iron absorption even if iron stores are adequate , and this stimulation increases the risk of iron excess when transfusions are given . we show here a rare case of a coinheritance of a hbh disease together with a sickle cell trait , presenting with iron deficiency . a 40-year - old turkish woman was referred to our outpatient clinic for evaluation and treatment of an undetermined thalassemia and sickle cell trait . g / l ) with microcytosis ( mcv 55.1 fl ) , hypochromia ( mchc 239 g / l ) , anisocytosis ( rdw 20.5 % ) and hypochromic reticulocytes ( chr 16.2 pg ) ( table 1 ) . leucocyte and platelet counts were within the normal range . on the blood smear we found anisocytosis , poikilocytosis , anisochromia , microcytes and target cells . the patient had severe iron deficiency ( ferritin level 7 ng / ml ; transferrin saturation 7% ) . in brilliant cresyl blue staining more than 50% of the erythrocytes showed typical hbh inclusions . hplc ( high - performance liquid chromatography ) revealed a normal hba2 ( 2.3% ) and hbf ( 0.7% ) , an abnormal hemoglobin s ( 19% ) and persistence of hba ( > 50% ) ( table 2 and figure 1 ) . genomic dna was isolated using manual or automated extraction protocols . using a reverse - hybridization of biotinylated pcr products ( viennalab ) a heterozygous mutation of the  gene at codon 6 ( hbb : c.20a > t ; corresponding to hbs ) , was detected . the -globin genes were amplified by pcr and sequenced using the bigdye terminator cycle sequencing kit , and the abi 3130 automated capillary sequencer ( applied biosystems inc . , foster city , ca , usa ) . we revealed a 5 nucleotide deletion in the ivs - i donor site ( gaggtgagg > this deletion was pseudo - homozygote since we showed later that one allele of the alpha gene complex contained a 6.1 kb deletion eliminating both alpha genes . multiplex ligation - dependent probe amplification ( mlpa ) reactions were performed for detection of copy number variation in the -globin gene clusters . the reaction was performed according to the manufacturers protocol using the salsa mlpa kit p140b2 hba and the servicexs hba kit described by harteveld et al . , 2005 . products were separated by capillary electrophoresis on the abi 3130 ( applied biosystems ) and data analysed using genemarker ( softgenetics ) . threshold ratios for deletion and duplication were set at > 0.75 and > 1.3 , respectively . we found a heterozygous deletion of probes 525 with the p140b2 kit and of probes 815 with the servicexs kit . this corresponds to a more than 6.1 kb long deletion of one allele in the alpha gene complex including the zeta and both alpha genes , leaving the theta gene intact . this type of deletion is called - - med - ii and is rather common in the mediterranean area and was frequently found in the turkish / greek population . 
 table 1hematological data from the patient.parameter , unitsat 1 presentation3 months after 1500 mg ferric carboxy - maltose infusionreference valueshb , g / l7183120160hct , l / l0.300.330.360.46mcv , fl55.155.17995mch , pg13.213.727.033.2mchc , g / l239250320360hyporbc , % 76.169.8<5.0rdw , % 20.519.911.514.5platelets , 10/l214162150450reticulocytes , 20221027reticulocytes , 10/l10811240140wbc , 10/l4.856.023.5010.00neutrophils , 10/l3.424.381.3006.700ferritin , ng / ml73310200transferrin saturation , % 7141645%str , mg / l23.1111.642.24.5hb , hemoglobin ; hct , hematocrit ; mcv , mean corpuscolar volume ; mchc , mean corpuscolar hemoglobin concentration ; rdw , red cell distribution width ; hyporbc , hypochromic erythrocyte ; str , soluble transferrin receptor ; wbc , white blood cells . hb , hemoglobin ; hct , hematocrit ; mcv , mean corpuscolar volume ; mchc , mean corpuscolar hemoglobin concentration ; rdw , red cell distribution width ; hyporbc , hypochromic erythrocyte ; str , soluble transferrin receptor ; wbc , white blood cells . table 2quantitave values of hemoglobin components from the patient obtained by high - performance liquid chromatography and results of molecular analysis.parametervaluereferenceshb a2 , % 2.32.03.5hb f,%0.7<2hb h , % 110abnormal hbhbsnoneperc . abnormal hb , % 190sickle cell testpositivenegativemutation analysis1.2 ivs - i donor site 5nt / -- med ii deletion2 . figure 1high - performance liquid chromatography pattern of our patient with hemoglobin h ( hbh ) disease . red graph represents our patient with clear hbh / hbbart 's peak at 1.5 minutes and a hbs peak at 11 minutes retention time . blue graph is a control . 
  high - performance liquid chromatography pattern of our patient with hemoglobin h ( hbh ) disease . red graph represents our patient with clear hbh / hbbart 's peak at 1.5 minutes and a hbs peak at 11 minutes retention time . hemizygote deletion of 5 nucleotides at the ivs - i donorsite of the alpha2 gene , hba2:c.95 + 2_95 + 6deltgagg . hemizygote deletion of 5 nucleotides at the ivs - i donorsite of the alpha2 gene , hba2:c.95 + 2_95 + 6deltgagg . thus this patient suffers from hbh disease and a sickle cell trait ; the hbh disease was due to an -thalassemia ( - -med - ii ) / -thalassemia ( 2 ivs - i donor site 5nt ) . the symptomatic anemia with iron deficiency was treated with iron infusion with a total dose of 1500 mg ferric carboxy - maltose . g / l , the ferritin level to 33 g / l and the transferrin saturation to 14% ( table 1 ) . we show here a patient with a rare hereditary constellation of three different genetic anomalies of the haemoglobin genes : the patients presented hbh disease with two deletions on the alpha genes and a sickle cell anomaly on the beta gene . there are only few published case reports about the coincidence of hbh disease with a -- med ii deletion and a hb s heterozygosity . patients with hbh disease have a severe hypochromia with low mcv and mch values , but increased iron stores . in our case , the moderate , microcytic hyochromic anemia and the splenomegaly were the consequences of the hbh disease , and could not be reversed by iron substitution . however , we assume that the structural beta - globin chain variant in our patient leads to lower quantities of unstable hbh , and therefore to a milder form of hbh disease with less ineffective hematopoiesis and only minimal increase of gastrointestinal iron absorption . hypermenorrhoea and two pregnancies were additional reasons for the unusual iron deficiency in this case of thalassemia .
we present a case of a 40-year - old female from turkey , who was referred to our outpatient clinic for an undetermined thalassemia and sickle cell trait . at first consultation hemoglobin 
 was decreased ( 71 
 g / l ) with microcytosis ( mcv 55.1 fl ) , and hypochromia ( mchc 239 
 g / l ) . 
 the patient had severe iron deficiency . 
 brilliant cresyl blue staining showed > 50% of the erythrocytes with typical hemoglobin h ( hbh ) inclusions . 
 high - performance liquid chromatography ( hplc ) revealed normal levels of hba2 and hemoglobin f ( hbf ) , and additionally a hemoglobin s ( 19% ) . 
 molecular diagnostics revealed the mutations 2 ivs - i donor site 5nt and a -- med ii deletion in the alpha gene complex and confirmed the heterozygote mutation of the beta - gene at codon 6 ( hbb : c.20a > t ; hbs ) . in conclusion , we present an extremely rare combination of hbh disease and sickle cell trait . 
 this combination may explain the mild form of the hbh disease , with moderate anemia , splenomegaly but iron deficiency , rather than iron overload , as usually observed in hbh disease .