Genotype–phenotype correlation in patients with deletional and nondeletional mutations of Hb H disease in Southwest of Iran

We studied the alpha-globin gene genotypes, hematologic values, and transfusion-dependence of patients with Hb H disease. Molecular characterization of alpha-thalassemia was performed. We identified 120 patients with Hb H disease. Of these patients, 35 (29.16%) had deletional form of Hb H disease, and 85 (70.83%) had different form of non-deletional Hb H disease. The most frequently observed Hb H genotypes were --Med/–α3.7 in 33 patients (27.5%), αCD19(-G) α/αCD19(-G) α in 25 cases (20.83%), αpolyA2α/αpolyA2α in 15 (12.5%), and αpolyA1α/αpolyA1α in 13 (10.83%) respectively. The probability of receiving at least one transfusion blood in deletional form was observed in 3 of 35 (8.57%) patients which just seen in 3 of 33 (9%) patients with --Med/–α3.7 genotype. This form was also observed in 8 of 85 (9.4%) patients in non-deletional Hb H diseases which five of them had Med deletion in compound with alpha globin point mutations. Nondeletional Hb H disease was more severe than deletional Hb H disease requiring more blood transfusions. We can recommend that Med deletion in compound with alpha-globin point mutations, polyA1 and constant spring in homozygous form needs to be taken into consideration when offering counseling to high-risk couples.

www.nature.com/scientificreports/ relatively rare, usually caused by homozygosity for non-deletional alleles, and sometimes by compound heterozygosity for a double α-globin gene deletion on one chromosome and a point mutation of either the α1 or α2 globin gene on the other chromosome 8 . In general, the clinical phenotypes of Hb H disease are variable, ranging from asymptomatic to harsh forms as in some patients on regular/irregular blood transfusion 4 . Non-deletional Hb H disease has more severe clinical symptoms and they are more anemic, prone to hepatosplenomegaly and transfusion dependency 7,9 . Because of high number of α-thalassemia carriers and consanguineous marriages in Iranian families, the prevalence of individuals with Hb H or even hydrops fetalis is increased 10 .
We evaluated Hb H disease in Iranian patients in Khuzestan Province in order to arrange a sensible prevention and management approach for the disease. Consanguineous and ethnic marriages in this region makes the controlling of disease more complicated and bring the necessity of clinical follow-up and routine screening for anemia at birth, during infancy and childhood.

Methods
Patients. We followed medical records of 120 patients with Hb H disease. These patients referred to the Narges Prenatal Diagnostics and Medical Genetics Laboratory as part of a national program for the prevention of thalassemia. Informed consent was obtained from the parents or the patients participating in this study. The red blood cell indices were automatically measured on a Coulter Counter ABX Micros 60 (Helena Laboratories, Beaumont, TX, USA). Hemoglobin H value was measured by high performance liquid chromatography (HPLC) using the VARIANT ™ HPLC system (Bio-Rad Laboratories, Hercules, CA, USA). The HbA2 band was measured by column chromatography (Beta-Thal HbA2 Quik Column Kit, Helena Laboratories) although HbF was performed by hemoglobin electrophoresis on cellulose acetate. Patients came from different cities of Khuzestan province with different ethnics. Phenotypic analysis was performed based on some routine analysis of polar, splenomegaly, Hepatomegaly, transfusion histories, and whether the patient had undergone splenectomy. This study was also reviewed and approved by the Ethics Committee of Pasteur Institute of Iran. All methods were carried out in accordance with relevant guidelines and regulations.
Genotypic analysis. Molecular studies were conducted on genomic DNA isolated from peripheral blood cells by salting-out procedure 11 . For identifying α-thalassemia genotype, investigation of common mediterranean -globin gene deletions (-3.7 , -4.2 -20.5 and --MED ) was performed by multiplex gap polymerase chain reaction as described previously; 12 the entire α and β-globin genes was amplified and DNA sequenced, ABI -3130 (Applied Biosystems, Foster City, CA, USA). In order to detect non common alpha deletions, multiplex ligation-dependent probe amplification (MLPA assay) was performed using the SALSA MLPA kit (MRC-Holland, Amsterdam, Netherlands). Then amplified fragments were separated by capillary electrophoresis, on an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems, Foster city, CA, USA) and analysis was performed by gene marker software v.1.6 (Soft Genetics, State College, PA, USA).

Results
The cohort. Of the 120 patients with Hb H disease, 50 were male and 70 were female. The average age was 23.0 ± 7 years.
In deletional form of Hb H disease, three patients were received blood who inherited --Med /-α 3.7 genotype. The first patient was a 60 year old woman who was receiving blood every month and who had a hemoglobin level (Hb) of 7.1 g per deciliter (g/dl). She underwent splenectomy because of the need for frequent blood transfusion. Second patient was a 28 year old woman who had Hb of 7.8 g/dl receiving blood for two times during pregnancy and also underwent splenectomy. Third patient was a 24 year old woman who had Hb of 7.9 g/dl receiving blood just once during pregnancy.
In non-deletional form of Hb H disease, eight patients were received blood which five of them had Med deletion in compound with alpha globin point mutations, especially during pregnancy. Interestingly, two patients with the --Med /α polyA1 α genotype received once transfusion had undergone splenectomy (Table 1).

Discussion
Hb H disease displayed by a varied clinical and hematologic phenotypic heterogeneity which is mostly seen in some regions of Southeast Asia, the Middle East and the Mediterranean countries 13 . The phenotypic variability of Hb H disease is ranging from asymptomatic, to need for periodic transfusions, to severe anemia with hemolysis 4 .
Of the 120 patients we studied, 29.16% had the deletional genotype and 70.83% had the non-deletional genotype. As in many other genetic diseases, the incidence of Hb H disease varies in different ethnic groups. By contrast with our patients from Khuzestan province, which more ethnic background was Arab (73.33%), the most common α-globin genotype in our Hb H patients was --Med /-α 3.7 (27.5%) that was similar to other reports of Iran and other populations with different ethnic backgrounds [14][15][16][17][18][19] . This genotype frequency followed by α CD19(-G) α / α CD19(-G) α (20.83%), α polyA2 α/α polyA2 α (12.5%), and α polyA1 α/α polyA1 α (10.83%) genotypes which is almost similar to previous reported study from Khuzestan province 16 . Although, this frequencies of Hb H genotypes are in contrast to Arabian Peninsula countries where the majority of Hb H disease cases are actually due to homozygosity for polyA1 mutation 20 but of the 13 α polyA1 α/α polyA1 α mutations detected 12 were from Arab ethnic group.
On the other hand, red cells hemolysis in Hb Constant Spring is maybe because of precipitation and aggregation of mRNAs that affecting the red cell membrane and producing visible basophilic stippling 30 .
According to this study, patients with non deletional/deletional and nondeletional Hb H disease usually are more anemic, and more likely to require transfusions suggested that Hb H disease is not as benign a disorder.
The diagnosis of Hb H disease at the molecular level is important for genetic counseling and the identification of families at risk for having pregnancies affected with Hb H disease. Regarding the need for blood transfusion in deletional and non-deletional Hb H disease, most of deletional Hb H cases were managed without blood transfusion. Nondeletional Hb H disease was more severe than deletional Hb H disease, with patients undergoing lower Hb levels and higher HB H percentage, requiring more blood transfusions and should be monitored closely. Therefore, we can recommend that Med deletion in compound with alpha-globin point mutations, polyA1 and constant spring in homozygous form needs to be taken into consideration when offering counseling to high-risk couples.

Data availability
All data generated during and/or analyzed during the current study are available upon request by contact the corresponding author.