An Intriguing High Performance Liquid Chromatogram of a Double Heterozygosity for Hb Q-India/Hb D-Punjab

Abstract

Cation exchange high performance liquid chromatography (HPLC) is commonly utilized as the first method of screening for thalassemias and hemoglobinopathies worldwide. This method of diagnosis requires knowledge of the clinical background and complete blood counts as well as skill and experience in interpreting the sometimes complex results produced. An asymptomatic 27-year-old pregnant North Indian woman was found to have a highly unusual chromatographic pattern with multiple unexpected peaks during routine antenatal screening. Most concerning was a C-window peak as Hb C (HBB: c.19G>A) is rare in ethnic Asian Indian populations. Cellulose acetate electrophoresis at alkaline pH (8.6) and parental screening were performed. These revealed the correct diagnosis to be a double heterozygosity for Hb Q-India (HBA1: c.193G>C) (an uncommon asymptomatic α-globin chain variant) plus Hb D-Punjab (HBB: c.364G>C) (a β-globin chain variant that is common in this region and is asymptomatic in the heterozygous state). The unexpected C-window peak was the hybrid of the abnormal α-Q-India and β-D-Punjab globin chains. Another small peak was explained as a variant Hb A2 formed by the combination of α-Q-India and δ-globin chains. Hematopathologists should be cognizant of the complex pattern resulting from coinheritance of both α- and β-globin structural variants. Second-line testing and parental testing are invaluable in resolving unknown peaks, especially if rare or unexpected variants are being considered. Although both Hb Q-India and Hb D-Punjab are relatively common in northwestern India, to the best of our knowledge, only two recent reports describe a total of three cases of such diagnostically puzzling coinheritance.

The phenotypic and molecular diversity of hemoglobinopathies in India: A review of 15 years at a referral center

“…While Hb SD Punjab disease most often has a severe clinical presentation and these patients are transfusion dependent, Hb SE disease and Hb ED Punjab disease are usually milder. Hb Q India‐Hb D Punjab is a very rare combination of an α and a β chain variant giving an unusual hybrid peak on HPLC, and only a few cases have been described earlier apart from the present case which was also reported by us …”

“…Hb Q India-Hb D Punjab is a very rare combination of an α and a β chain variant giving an unusual hybrid peak on HPLC, and only a few cases have been described earlier apart from the present case which was also reported by us. [23][24][25] Hb Lepore is not very common, and there are sporadic reports from India. [26][27][28] The interaction of Hb Lepore with β thalassemia can result in a thalassemia intermedia-like presentation or a more severe disorder.…”

C-Window Peaks on CE-HPLC are Extremely Rare in Northern India, and Only Infrequently Represent HbC

“…Double heterozygosity for HbD-Punjab and HbQ-India produces an interesting pattern on HbHPLC with four predominant peaks corresponding to HbA, HbD-Punjab, an unknown peak corresponding to HbQ-India and a peak in the C-window corresponding to the D-Q hybrid. In all the cases reported till date on HbHPLC, the D-Q hybrid ranged between 6 and 8% while the HbQ-India varies between 9 and 10.5% and HbD-Punjab varies from 32 to 34% including our 4 cases [10][11][12][13][14][15]. It is important to note that, normally HbQ-India constitutes *15-20% in heterozygous state if not associated with iron deficiency or a concomitant b-thalassemia trait [16,17] and in cases with double heterozygous HbD-Punjab/Q-India, the total amount of HbQ-India and D-Q hybrid also reaches an almost similar value.…”

“…The subsequent series of two cases was published from New Delhi [10]. Since then, there have been a few case report describing this interesting co-inheritance of the a-chain variant HbQ-India and the b-chain variant, HbD-Punjab [11][12][13][14][15]. All these cases have been reported from India which is largely explained due to the geographical pattern of inheritance of these hemoglobins.…”

“…In addition, there are a few case reports of double heterozygous Q-India/D-Punjab cases in which the D-Q hybrid eluted in the C-window with presence of additional peaks of HbQ-India, HbD-Punjab and HbA [10][11][12][13][14][15].…”