Hematological characteristics and effective screening for compound heterozygosity for Hb constant spring and deletional α<sup>+</sup>‐thalassemia
Authors: Noppacharn Uaprasert, Ponlapat Rojnuckarin, Rung Settapiboon, Supaporn Amornsiriwat, Pranee Sutcharitchan
Journal: American Journal of Hematology (2011)

Hemoglobin Constant Spring (HbCS) is an unstable α-globin variant causing α-thalassemia phenotypes. The prevalence of compound heterozygosity for HbCS and deletional α+-thalassemia (HbCS/α+-thal) and its characteristics compared with HbCS heterozygosity have not been reported. We performed molecular analysis to detect α+-thal alleles in 75 HbCS heterozygotes and correlated with hematological characteristics. There were 34 HbCS heterozygotes (45.3%) carrying α+-thal. Hematological parameters of HbCS/α+-thal demonstrated lower Hb, MCV, MCH, and MCHC than HbCS heterozygotes, but higher HbCS levels. MCH was the most helpful variable in differentiating these two groups with ROC area under curve (AUC) of 0.927. MCH > 27 pg was able to exclude the presence of α+-thal, while MCH < 25.5 pg yielded the greatest diagnostic performance with sensitivity and specificity of 97.1% and 80.5%, respectively. The validation in another sample set of 69 HbCS heterozygotes confirmed that MCH > 27 pg was able to exclude the coinheritance with α+-thal, while MCH < 25.5 pg was an effective cut-off for predicting α+-thal with sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 95.2%, 70.8%, 58.8%, and 97.1%, respectively. Therefore, this could be a cost-effective screening criterion to identify deletional α+-thal in HbCS heterozygotes avoiding expensive molecular testing. Thalassemia syndromes are the commonest monogeneic diseases in the world. In Thailand where thalassemia syndromes are highly prevalent, the gene frequencies are 20–30% for α-thalassemia, 3–9% for β-thalassemia, up to 52% for hemoglobin E (HbE) and 3–6% for HbCS [1-3]. Combined different mutations are very common resulting in various clinical syndromes with considerable variability in severity [4-6]. The rapid growth of Asian population in the US has increased the prevalence of nonsickling hemoglobin disorders in the US health care system [7]. Therefore, thalassemia syndromes have expanded towards a global public health. HbCS is the most common nondeletional α-thalassemia caused by the mutation at the stop codon on α2-globin gene leading to insertion of glutamine and a total extension of 31 amino acid residues [1, 2, 8, 9]. HbCS is an unstable hemoglobin detectable only 1–2% in HbCS heterozygotes and frequently undetectable by gel electrophoresis. In HbCS homozygotes, it comprises approximately 5–6% of total hemoglobins [8, 10]. Hemoglobin H (HbH) disease is the most severe nonfatal form of α-thalassemia. The nondeletional HbH disease, of which HbCS accounts for the majority, is implicated in as much as 60% [8, 11, 12]. The unawareness of the coexisting deletional α+-thal could mislead genetic counseling. Clinical and hematological characteristics of heterozygosity and homozygosity for HbCS have been previously reported. Notably, the characteristic feature of all HbCS variants were overhydrated red blood cells relative to deletional α-thalassemia variants resulting in normalized red cell volumes [9]. This was found in both heterozygous and homozygous states of HbCS, as well as in coinheritance with α0-thalassemia that results in HbHCS [1, 8, 9]. Although HbCS heterozygotes supposedly have normal MCV, we have encountered approximately 40–50% of Thai cases showing low MCV and MCH compared to reference values from previous reports [1, 13]. The coinherited deletional α+-thal has been hypothesized. Out of 5,196 hemoglobin analyses performed in 2005, there were 114 cases (2.2%) showing an abnormal band suggesting HbCS: 77 HbCS heterozygotes, 21 HbHCS diseases, 7 HbEABart's diseases, 7 HbCS homozygotes, and 2 double heterozygotes for HbCS and HbE. Out of 77 cases (1.5%) identified as HbCS heterozygotes, two cases were Hb Pakse on the single-tube multiplex amplification refractory mutation system (ARMS) and excluded from the study. From 75 proven HbCS heterozygotes, the DNA analysis based on multiplex gap polymerase chain reaction revealed 34 (45.3%) compound heterozygotes for HbCS/α+-thal. Hematological parameters and hemoglobin analysis of each group were summarized as means and standard deviations (Table I). Statistical analysis of hematological parameters and hemoglobin analysis demonstrated that Hb, MCV, MCH, and MCHC of compound heterozygotes for HbCS/α+-thal were significantly lower than those of HbCS heterozygotes, while HbCS levels of compound heterozygotes for HbCS/α+-thal were significantly higher than those of HbCS heterozygotes. Among these parameters, MCH was the most helpful indicator of the coexistence of deletional α+-thal with the AUC of 0.927. In addition, MCV and MCHC demonstrated high diagnostic performance with the AUC of 0.881 and 0.879, respectively, while Hb demonstrated the lower AUC of 0.657. Therefore, MCH and MCV were selected to test their effectiveness for identifying the coexisting deletional α+-thal (Fig. 1). Various cut-offs of these hematological parameters were selected and tested to yield at least 95% sensitivity for the coexistence with deletional α+-thal. Either MCH > 27 pg or MCV > 84 fL was able to exclude the presence of deletional α+-thal. However, MCH demonstrated a greater diagnostic yield due to higher specificity and PPV. MCH < 25.5 pg yielded the most accurate cut-off to predict the presence of deletional α+-thal with promising sensitivity, specificity, PPV, NNV and accuracy of 97.1%, 80.5%, 80.5%, 97.1%, and 88%, respectively. Combined MCH and MCV did not increase diagnostic value compared to MCH alone. Therefore, MCH lower than 27 and 25.5 pg were selected to validate their effectiveness for predicting the coinheritance with deletional α+-thal in the other set of HbCS heterozygotes. Diagnostic performance of selected cut-offs of MCH (A) and MCV (B) in identifying the coexistence of deletional α+-thal with HbCS heterozygotes. There were 69 confirmed HbCS heterozygotes for validation analysis. The molecular test revealed 21 (30.43%) compound heterozygotes for HbCS/α+-thal. MCH > 27 pg was able to exclude the coinherited α+-thal, while MCH < 25.5 pg has consistently displayed very good diagnostic performance with sensitivity, specificity, PPV, NPV, and accuracy of 95.2%, 70.8%, 58.8%, 97.1%, and 78.3%, respectively. In our analysis, we identified 34 cases (43.3%) of compound heterozygotes for HbCS/α+-thal in 75 cases of molecularly confirmed HbCS heterozygotes. Therefore, this compound heterozygote is common in Thailand, but usually missed by a routine hemoglobin analysis possibly causing incorrect genetic counseling. Compound heterozygotes for HbCS/α+-thal have lower Hb, MCV, MCH, and MCHC than those of HbCS heterozygotes. The decreased Hb levels, red cell volumes and hemoglobin contents are due to more severe α-globin gene defects leading to reduced α-globin synthesis and an augmented imbalance between α-globin and β-globin chains. The defects could explain an increase in HbCS levels in the compound heterozygous state from an increase in αCS-globin to α-globin chain ratio. However, this effect is very modest since αCS-globin mRNA is very unstable and accounts for only 1–2% of protein output of a normal α2-globin gene [8]. Conventionally, MCH and MCV are the most common red cell indices using in the thalassemia screening program. MCH > 27 pg and MCV > 84 fL were able to exclude the coinherited deletional α+-thal with 100% sensitivity. However, MCH < 25.5 pg yielded the highest specificity and PPV to predict compound heterozygosity for HbCS/α+-thal. Hence, this level may be used to screen cases that do not need DNA testing. To prove the effectiveness of the criteria, an independent test set was used to validate the cut-offs derived from the training set. MCH > 27 pg was able to exclude the coexisted deletional α+-thal, while MCH < 25.5 pg consistently showed great diagnostic values. Using MCH < 25.5 pg as the screening criteria for HbCS/α+-thal was able to reduce 51% of cases requiring genetic tests. Only 3% of compound heterozygotes will be missed. However, a concomitant of iron deficiency anemia or anemia of chronic disorders could interfere with red cell parameters and affect diagnostic value of these screening criteria. Therefore, appropriate tests are required based on clinical settings. Sanchaisuriya et al. demonstrated that MCV < 74 fL, MCH < 24 pg and HbE < 26% levels were very helpful for screening of double heterozygosity for HbE and α0-thalassemia (HbE/α0-thal) [14]. As double heterozygotes for HbE/α0-thal are at the risk of fatal Hb Bart's hydrops fetalis, a cut-off point yielding 100% sensitivity is an optimal goal. On the other hand, HbH and HbHCS disease are nonfatal conditions and a screening parameter yielding greater than 95% sensitivity is sufficient. This study demonstrated that MCH < 25.5 pg yielded high diagnostic performance in both the training and the test set. In conclusion, this study is the first report of hematological characteristics of compound heterozygosity for HbCS/α+-thal compared with pure HbCS heterozygotes. This common coexistence of deletional α+-thal resulted in depressed Hb, MCV, MCH, and MCHC, but elevated HbCS levels. MCH > 27 pg was able to exclude coinherited deletional α+-thal, while MCH < 25.5 pg could be an effective screening cut-off to identify compound heterozygosity for HbCS/α+-thal. Blood specimens of patients suspected for hemoglobinopathies or premarriage/prenatal testing were collected and analyzed at King Chulalongkorn Memorial Hospital, Bangkok, Thailand. The samples of proven HbCS heterozygotes collected in 2005 were assigned as the training set, while the other set of molecular confirmed HbCS heterozygotes identified between 2008 and 2010 was assigned as the test set. Complete blood count was measured by an automated cell counter (Haematologie, Montpellier, France). Hemoglobin analysis was performed using both Isoelectric focusing (IEF) method with IsoScan Imaging System for Hemoglobin Testing (Perkin–Elmer Life and Analytical Sciences, Turku, Finland) and high performance liquid chromatography (HPLC) with the β-thalassemia short program on Variant Hemoglobin Testing System (Bio-Rad Laboratories, CA) to determine the quantity of each hemoglobin type. Cases showing an abnormal band suggesting HbCS on IEF and/or HPLC were confirmed using a single-tube multiplex ARMS method to differentiate HbCS [Termination codon (TAA→CAA)] from Hb Pakse [HbPS; Termination codon (TAA→TAT)] with a few modifications [4, 15]. The α-globin genes were analyzed based on multiplex gap polymerase chain reaction method for detection of the majority of deletional α-thalassemia determinants as previously described [16]. Red cell parameters and the quantity of HbCS were demonstrated as means and standard deviations. The comparison of means was analyzed by independent sample t-test. The P < 0.05 was considered statistically significant. Sensitivity, specificity, PPV, NPV and accuracy of each selected cut-off was calculated each time on the total number of individuals. The receiver operating curve (ROC) analysis was performed to compare the diagnostic performance of each hematological parameters in detection of the coexisting deletional α+-thal. The selected cut-offs demonstrating the greatest diagnostic performance were further validated in the test set. All statistic parameters were computed using the SPSS version 16 for Window. Noppacharn Uaprasert*, Ponlapat Rojnuckarin*, Rung Settapiboon*, Supaporn Amornsiriwat*, Pranee Sutcharitchan*, * Department of Medicine, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, Thailand.