|Ahead of print publication
Review of clinical and hematological profile of hemoglobin D cases in a single centre
Neha Singh1, Tulika Seth2, Seema Tyagi2
1 Centre for Community Medicine, AIIMS, New Delhi, India
2 Department of Hematology, AIIMS, New Delhi, India
|Date of Submission||07-Nov-2022|
|Date of Decision||15-Dec-2022|
|Date of Acceptance||22-Dec-2022|
|Date of Web Publication||18-Feb-2023|
Centre for Community Medicine, AIIMS, New Delhi
Source of Support: None, Conflict of Interest: None
Introduction: Hemoglobin D Punjab is a common hemoglobin D variant, which is essentially known to cause a mild disease even in homozygous state. Heterozygous state of hemoglobin D Punjab with sickle cell is the only presentation when a severe disease may be expected. We aim to study the spectrum of clinical and haematological profile of all case of haemoglobin D, reported at an apex referral centre of the country. Material and Methods: This is a record based retrospective study of cases of hemoglobin D Punjab from hematology OPD of a apex medical care centre. The clinical and hematological findings of 30 cases with genotype/phenotype of hemoglobin D Punjab were recorded from physical records and electronic data maintained at the institute. The cases were divided into homozygous Hb D Punjab, Double heterozygous hemoglobin S and D, Hemoglobin D-β-thalassemia, and Hemoglobin D trait. Results: The reported 30 cases consisted of 3 cases of homozygous Hb D Punjab, 8 cases of co-mutation of Hb D and beta thalassemia, 6 cases of co-mutation of Hb D with sickle cell and 13 cases of Hb D trait. All three cases of Hb D disease were mild to moderately symptomatic with co-existent splenomegaly. Out of the 8 cases reported of Hb D-β-thalassemia, three cases were found to have moderate anemia and one with severe anemia. The MCV of all participants were reduced, while, MCHC of all cases was within normal range. Among the 6 cases of compound heterozygous Hb D and S Disease two had hemolytic faces and three cases had splenomegaly. Blood transfusion was required by four out of six cases at varied frequency. Conclusion: We find the spectrum of symptoms presented by the cases of hemoglobin D Punjab is varied and few cases may require blood transfusion and medical care with follow up even in absence of heterozygous hemoglobin S inheritance.
Keywords: Beta-thalassemia, hemoglobin D-Punjab, hemoglobinopathy
| Introduction|| |
Hemoglobin D (Hb D)-Punjab is a structural variant of hemoglobin resulting from glutamate to glutamine substitution at codon 121 of the beta chain and is commonly distributed worldwide.
Approximately 15 known variants of Hb D are reported in the literature. The same amino acid substitution (Glu → Gln) at β121 position causes the variants Hb D-Punjab, Hb D-Los-Angeles, Hb D-North-Carolina, Hb D-Portugal, and Hb D-Chicago. Among the Hb D variants, Hb D-Punjab (also known as Hb D-Los Angeles) is the most common and affects both sexes equally. The disease occurs most often in people whose ancestors come from Pakistan, Northwestern India, and Iran with the greatest prevalence (2%) in Sikhs in Punjab (India). However, studies from various parts of the country have reported a frequency range of 0.06%–0.2% in the blood samples evaluated for suspicion of hemoglobinopathies at a center.,,
The Hb D-Punjab variant may be genetically inherited as heterozygous trait with normal Hb A, thus affecting only one β globin chain. This condition is known to be asymptomatic both clinically and hematologically. The homozygous genotype Hb D disease (Hb DD) in which both the β globin chains are affected is also not commonly related to symptomatic cases, but occasionally individuals with this profile can develop mild to moderate hemolytic anemia.,
The co-inheritance of Hb D-Punjab and beta-thalassemia is known to manifest as mild microcytic and hypochromic anemia with no severe clinical or hematological changes.
Sometimes, even greater vulnerability for red cell lysis in Hb S/D-Punjab patients is reported in comparison with the Hb SS genotype for vaso-occlusion episodes or required number of blood transfusions.
It is believed that the amino acid changes in β−121 Glu → Gln positions of Hb D enhance the interaction of Hb S molecules and subsequently lead to its polymerization. The polymerization of Hb S in turn increases the probability of sickling due to a shorter transit time of these cells in the blood capillaries. This may explain the severity of clinical presentation in Hb S/D-Punjab patients.,,
The known natural history of Hb D cases may have a different presentation due to various factors known and unknown. Hb D variant is usually reported as an asymptomatic condition when inherited as trait or a mild-to-intermediate microcytic condition when inherited in combination with beta-thalassemia. It may manifest as severe symptoms in the compound heterozygous inheritance with Hb S and in the homozygous state, it is reported as a rather asymptomatic condition.,
The aim of this study is to assess and document the spectrum of clinical symptom complex and hematological findings associated with the various forms of Hb D-Punjab.
| Materials and Methods|| |
This is a record-based retrospective study of cases of Hb D-Punjab from the hematology outpatient department (OPD) of an apex medical care center reported between 2005 and 2018. The study subjects were referred for evaluation and management from peripheral health facilities as well as from other OPDs of the institute. Data were extracted from records regarding detailed clinical history. Clinical details included age at onset of symptoms, age at diagnosis, sex, presenting complaints, the onset of complications such as episodes pain crisis or history suggestive of vaso-occlusive crisis, number of blood transfusions, clinical complaints, treatment received, and duration of the last follow-up. Details of the physical examination carried out to evaluate for hemolytic facies, pallor, icterus, and hepatosplenomegaly were also taken.
Complete blood cell count, peripheral blood smear (PBS) for red cell morphology, and reticulocyte count were conducted from blood samples of all the patients. Quantitation of HbA, HbD, HbF, HbS, and HbA2 was carried out with high-performance liquid chromatography (HPLC) which has been found to be a reliable tool for diagnosis of Hb variants. Hemoglobin fractions were identified by their retention time and quantified by computing the area under the corresponding peak in the elution profile. The complete hematological profile of patients was assessed using reports of complete blood count, serum iron studies, and HPLC.
Data regarding clinical presentation, complication, and hematological characteristics were collected from physical records maintained at the hematology department and from electronic database maintained at the institute. The cases were divided into HbDD, Double heterozygous hemoglobin S and D (HbSD), Hb D-beta-thalassemia, and hemoglobin D trait.
| Results|| |
The results of clinical and hematological profile of 30 patients consisting of 3 cases of Hb DD-Punjab, 8 cases of co-mutation of Hb D and beta-thalassemia, 6 cases of co-mutation of Hb D with sickle cell, and 13 cases of Hb D trait are presented in [Table 1], [Table 2], [Table 3], [Table 4].
|Table 3: Clinical characteristics of hemoglobin D sickle compound heterozygous cases|
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|Table 4: Clinical characteristics of hemoglobin D trait/hemoglobin D heterozygous cases|
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Hemoglobin D disease
All three cases of Hb DD were mild to moderately symptomatic with co-existent splenomegaly. Case 2 (33/Male) was incidentally diagnosed during workup for cholestatic jaundice. His serum iron studies and osmotic fragility were within normal range. He later developed acute hepatitis with renal failure, though etiology could not be pointed he improved with dialysis and antibiotics. Case 3 (21/Male) presented with long-standing history (11 year) of joint pain and difficulty in walking along with progressive pallor. On investigation, peripheral smear revealed spherocytes, basophilic stippling, and Cabot's ring along with fragmented cells suggestive of hemolysis. He was diagnosed with a case of hemolytic anemia along with Hb DD.
Out of the 8 cases reported of Hb D-beta-thalassemia one had normal hemoglobin levels (28/Male) and three had mild anemia (14/Female), (24/Male), (34/Male), three cases were found to have moderate anemia while one case presented with severe anemia. These three cases of moderate anemia were also found to have concomitant iron deficiency anemia, which was diagnosed after iron studies. The mean hemoglobin of the reported cases was 9.5 with a standard deviation of ± 2.6 comprising Hb D as the highest fraction with a mean value of 83.55% ± 2.05%. The mean corpuscular volume (MCV) of all participants was lower than normal with a mean value of 64.7 ± 4.4. However, the MCHC of all cases was within normal range.
Double heterozygous hemoglobin D and S
We report 6 cases of compound heterozygous Hb D-Punjab and Hb S inheritance with a range of symptoms from pallor to frequent pain crisis. Two out of six cases had hemolytic faces and three out of 6 cases had splenomegaly. Blood transfusion was required in four out of six cases but the frequency of requirement varied ranging from occasional transfusion to one transfusion every 15–30 days.
The hemoglobin level of all the cases was in the category of moderate-to-severe anemia with the mean level of hemoglobin being 7.05 g/dl ± 1.78. All the cases were started on hydroxy urea with a follow-up period of 12–38 months. The cases responded with decrease in the frequency of pain crisis and reduced requirement of blood transfusion after few months of therapy. The mean hemoglobin level at follow-up rose to 10.8 g/dl ± 1.14, the difference between the hemoglobin levels was significant (P < 0.001). The Hb D fraction of the cases was between 38% and 45% with a mean value of 41.6 ± 2.5, Hb S value ranged between 25% and 44%.
Case 2 (20/Male) and Case 6 (21/Male) were initially diagnosed with Sickle cell anemia at the peripheral center and were later found to be heterozygote with Hb D and S. The blood picture of case 2 (20/Male) and case 6 (18/Male) showed mild polychromasia and presence of Howel-Jolly bodies on peripheral smear indicative of auto splenectomy. Five out of four cases were found to have splenomegaly and Case 1 (16/Female) had a hepatosplenomegaly with 3 cm of liver and 15 cm of spleen palpable below the costal margin.
Hemoglobin D trait/hemoglobin D heterozygote
We report 13 cases of Hb D trait, most of the cases were asymptomatic or mildly symptomatic with pallor and generalized weakness as the commonest presentation. The hemoglobin levels of most of the cases were in the range of normal-to-mild anemia with a mean value of 8.86 ± 1.8 g/dl. The hemoglobin comprised of Hb D fraction in the range of 28%–42% with a mean value of 34.2 ± 4.1.
Case 1 (1.5/Female) reported with pallor and low hemoglobin level comprising of HbD 28.3% and Hb A2 level of 3.3. On investigation, her serum ferritin was found to be low and spleen was not palpable. The Hb A2 level was borderline for the diagnosis of compound heterozygous Hb D and beta-thalassemia, these may be due to compromised production of A2 levels in view of low iron levels in the body. The alpha-thalassemia report of this patient was pending and a concomitant alpha-thalassemia may also explain the presence of such low hemoglobin in an apparent Hb D trait patient.
Case 2 (10/female) and Case 6 (40/male) reported with low hemoglobin levels, their PBS revealed the presence of spherocytes and on investigation, both were diagnosed as cases of hereditary spherocytosis along with Hb D trait.
Case 3 (38/male) and Case 8 (2/female) had anemia due to blood loss. Case 3 was diagnosed with hemorrhoids and was taken up for surgery subsequently, whereas, case 8 was diagnosed with the presence of helminthic infection (Ancylostoma duodenale) and had suffered multiple episodes of bloody diarrhea. She was managed medically and both patients reported normal hemoglobin levels on follow-up.
Case 7 (1.5/male), Case 8 (2/female), Case 9 (2/male), Case 10 (5/male), and Case 11 (7/male) reported with low hemoglobin level and on the investigation were found to have low serum ferritin levels and were diagnosed as cases of iron deficiency anemia. They responded well with iron supplements and on follow-up reported rise of hemoglobin levels.
Case 12 (2.5/Male) presented with low hemoglobin levels and showed the presence of frontal bossing and malar prominence of the face along with hepatosplenomegaly (Liver − 4 cm, Spleen – 8 cm). His iron studies were normal, alpha mutation study and G6PD deficiency study were found negative. On PBS there was the presence of large histocytes along with cells appearing like crumpled tissue paper. He was diagnosed as a case of Gaucher's Disease along with Hb D trait.
| Discussion|| |
The cases reported in this study, Hb D-Punjab homozygous (n = 3), compound heterozygous Hb D-beta-thalassemia (n = 8), compound heterozygous Hb D-Sickle cell (n = 6), Hb D trait (n = 13) presented with a spectrum of signs and symptoms. All cases were registered and followed up hematology OPD of the institute.
Hemoglobin D disease
Clinical manifestations of sickle cell Hb S D-Pubjab exhibit slightly milder outcome compared to common sickle cell anemia (HbSS). However, Splenomegaly is more common in HbSD-Punjab than in HbSS disease .
The cases of Hb DD reported in literature are largely asymptomatic. All 3 reported cases of homozygous Hb D in the present study had hemoglobin level falling in mild anemia and were found to be having splenomegaly. Two out of three cases had comorbidities contributing to the symptoms at presentation; however, we report one case with no diagnosed co-morbidity and presenting with symptoms of anemia. There are reported with presentation of moderate anemia, moderate splenomegaly, and mild hepatomegaly with cholelithiasis. The case was diagnosed to have co-existing hemolytic anemia.
HbF levels of all 3 reported HbD cases were normal; however, the HbA2 values were on the lower side with a range of 0.9%–2.5%. Similar findings of Hb variant fraction in cases diagnosed as HbD are reported in literature.
All 8 reported cases had high Hb D fraction in the range of 80%–90%, low Hb A fraction (3%–5%), and elevated Hb A2 fraction (3.9% and 5.5%). All the cases had low levels of MCV. Similar levels of hemoglobin fraction and MCV levels have been reported for cases of double heterozygous Hb D and beta-thalassemia.,
Hemoglobin level of severe anemia along with reduced erythrocytic indices and a hypochromic, microcytic red cell picture is also reported in literature for Hb D-beta-thalassemia cases. In contrast to this, a case series of three compound heterozygous individuals for Hb D-Punjab and beta-thalassemia reported that out of the three only one case had been diagnosed based on clinical symptoms while the other two cases were found in routine prenatal screening. This demonstrates the high range of variability in the clinical presentation of cases with coinheritance of Hb D-Punjab with beta-thalassemia.
Double heterozygous hemoglobin D and S (HbSD)
Clinical manifestations of sickle cell Hb D/S exhibit slightly milder outcome compared to homozygous state of Hb S but server to any other form of Hb D inheritance. However, Splenomegaly is encountered more frequently among Hb S/D-Punjab than in homozygous Hb S. The severity of the symptoms and complications of Hb SS genotype are modified and reduced to a certain extend in presence of elevated blood HbF. Hydroxyurea is known to induce the production of Hb F among patients with Hb S variant of hemoglobin but the results of hydroxyurea treatment are yet to be established for Hb SD-Punjab disease. In our study, all the 8 cases received low doses of hydroxyurea and experienced decrease in vaso-occlusive symptoms along with requirement for blood transfusion, similar results with hydroxy urea therapy are reported by other researchers.,
The cases reported in our study had Hb D levels in the range of 38%–45% and their Hb S levels in the range of 24%–45%. The clinical presentation at the time of reporting was diverse ranging from mild symptoms to frequent vaso-occlusive pain crisis. A similar spectrum of symptoms was reported in literature., Their prognosis is better than that of patients with Hb SS, and the unpredictability of presentation is affected by the Hb F levels, presence of alpha-thalassemia, HBB haplotype, age, and environmental factors.
Splenomegaly in compound heterozygous Hb D/Hb S cases has been variably reported, five out of six cases in our study were having splenomegaly. Patients with Hb S and D from Kuwait and Arabia have not reported splenomegaly. Reports of Hb D/Hb S from India have found splenomegaly in more than half of the patients and also a high level of Hb F is found to be associated with the presence of splenomegaly.,
Hemoglobin D trait/heterozygous
Hb D trait on its own is not known to cause any symptom or any hematologically deranged findings. In our study, the patients were mostly symptomatic due to co-existing anemia, which is a major public health problem in India. The manifestation of symptoms among cases with Hb A2 in the range of 3%–4% may be attributed to underlying iron deficiency anemia, which may have negatively affected the production of hemoglobin A2 and subsequently led to misdiagnosis of heterozygous Hb D-beta-thalassemia to Hb D trait. Furthermore, the comorbidities of the patients may have contributed to the symptoms at the time of presentation.
A mini-review from UAE reported findings of five cases with Hb D trait, their hemoglobin level was normal or slightly decreased and MCV was also reduced. The fraction of Hb D in the cases was between 29% and 39% similar to our study. Low MCV level among Hb D heterozygotes is often related to coinheritance of alpha-thalassemia among the cases; while in population without alpha-thalassemia, Hb D-Punjab heterozygotes have normal hemoglobin levels and MCV.,
Strengths and limitation of the study
The currents study adds to the understanding of the clinical spectrum of Hb D cases found in the country by reporting clinical and hematological profiles of 30 cases. We report cases other than compound heterozygote Hb DS state presenting with anemia and requiring blood transfusion. Thus, have flagged the requirement of detailed hematological investigation and follow-up even in cases of known Hb D variant hemoglobin.
We present results from retrospective data and have tried our best to present retract and report available information. However, molecular analysis to identify the mutations was not done for the given set of patients and hence conclusive decision regarding the variability of presentation cannot be made.
| Conclusion|| |
Hb D-Punjab hemoglobin variants are largely known as asymptomatic and clinically benign in the absence of a compound heterozygous state with another variant. However, demographic changes such as population migration cause new spectrum of inherited hemoglobin disorders to emerge along with variability in clinical presentation. Hb D-Punjab in its various genotype/phenotype manifestations is believed to be asymptomatic or mildly symptomatic. We highlight in our study that the presentation of Hb D-Punjab cases has a spectrum of severity and moderate–severe symptoms along with vaso-occlusive episodes and requirement of multiple blood transfusion may be encountered.
Therefore, it is important to undertake a detailed hematological investigation in suspected cases of Hb D-Punjab to diagnose any underlying erythrocytic manifestation and facilitate error-free counseling and proper management Hb DD.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]