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We prospectively compared a single-tube multiplex polymerase chain reaction (PCR) for detecting alpha-thalassemia with our current approach using 452 blood samples. Initial evaluation of 89 specimens revealed sensitivity and specificity, respectively, for the hemoglobin H inclusion body test (HbH prep) vs PCR for detecting alpha-thalassemia carriers of 0.79 and 0.96 and for a mean corpuscular volume (MCV) of 82 µm (82 fL) or less, 1.0 and 0.45. Detection of all alpha-thalassemia genotypes was significantly lower for HbH prep and MCV (sensitivity and specificity, respectively: HbH prep, 0.48 and 0.96; MCV, 0.87 and 0.47). In a follow-up evaluation of patients with positive HbH prep results or suspected alpha-thalassemia prescreened by low MCV, the sensitivity and specificity, respectively, of HbH prep vs PCR increased to 0.97 and 0.93 for alpha-thalassemia and 0.83 and 0.92 for any alpha-thalassemia. PCR detected alpha-thalassemia in 37.2% of 298 suspected alpha-thalassemia cases with suggestive indices but negative HbH prep results and no detectable hemoglobinopathy. This multiplex approach was more sensitive than the HbH prep for detecting all alpha-thalassemia genotypes, particularly alpha-thalassemia; was particularly valuable for identifying carriers of alpha-thalassemia at risk for offspring with hemoglobin Bart hydrops fetalis, regardless of other diagnosed hemoglobinopathies; and is an ideal adjunct to standard clinical screening protocols for detecting alpha-globin deletions.
Thalassemia is a group of genetic disorders characterized by quantitative defects in globin chain synthesis. The subsequent absence or decrease of hemoglobin production results in microcytosis with varying degrees of anemia. These conditions commonly are found in people of Mediterranean, African, Middle Eastern, Indian, Chinese, or Southeast Asian origin. Some evidence suggests that natural selection may have elevated and maintained the high frequency of globin deletions as protection against a severe form of malaria. While the heterozygous state for thalassemia (thalassemia trait) is a benign condition, the homozygous state is associated with more severe disorders that cause substantial morbidity and mortality worldwide.
In the healthy adult, hemoglobin A is the most prevalent form, composed of 2 alpha-globin and 2 beta-globin chains. Two minor hemoglobins also are present: hemoglobin A2 (2 alpha- and 2 delta-globin chains) and hemoglobin F (2 alpha- and 2 gamma-globin chains). There are 4 alpha-globin genes, 2 of which are encoded in tandem (in cis) on each chromosome 16p13.33 (
/
), and 2 beta-globin genes, 1 on each chromosome 11. beta-Thalassemia often is due to a point mutation or a small deletion or insertion mutation in one of the beta-globin genes, leading to decreased or absent beta-globin chain synthesis. On the other hand, alpha-thalassemia usually is caused by deletion of one or more alpha-globin genes. The rate of synthesis of alpha-globin chains is related to the number of genes deleted. Loss of 1 alpha-globin gene (heterozygous alpha -thalassemia; -
/
) results in a silent carrier, not manifesting abnormalities other than mild microcytosis. Loss of 2 alpha-globin genes on 1 chromosome (heterozygous alpha -thalassemia; - -/
), or on opposite chromosomes (homozygous alpha -thalassemia; -
/-
) may result in a mild microcytic hypochromic anemia. Loss of 3 genes (- -/-
) results in an intermediate thalassemia disease state known as hemoglobin H (HbH) disease, and may be associated with reduced growth rate, iron overload unrelated to transfusion history, and other complications. Complete deletion of all 4 alpha-globin genes results in severe hypoxia and anemia in utero (hemoglobin Bart hydrops fetalis; - -/- -), a lethal condition. The laboratory identification of people with alpha-thalassemia is important for 2 reasons: (1) for the identification of patients of reproductive age who are carriers of serious or life-threatening hemoglobinopathies and (2) for the investigation of microcytic anemia to prevent erroneous and unnecessary medical intervention for iron deficiency.
People with heterozygous alpha-thalassemia and HbH disease are carriers of hemoglobin Bart hydrops fetalis, as they both carry the alpha-thalassemia mutation. Parents who both carry this mutation have a 25% risk of conceiving a fetus with hemoglobin Bart hydrops fetalis. People who are heterozygous and homozygous for alpha mutations are not carriers of hemoglobin Bart hydrops fetalis but are carriers of HbH disease.
The most common inherited disorder of hemoglobin synthesis is alpha-thalassemia. More than 95% of alpha-thalassemia cases are deletional. The most common alpha-thalassemia deletions are the Southeast Asian (- -), Mediterranean (- -), 20.5 kilobase (kb) (- -20.5), and Filipino (- -). The most common alpha-thalassemia deletions are the rightward 3.7 kb (-
) and the leftward 4.2 kb (-
). The - - and - - deletions are found in Southeast Asian and Chinese populations. A majority of these, more than 90% in most populations, are the - - deletion, which occurs with a frequency of 4.5% to 14%. The - - and - -20.5 deletions are found in Mediterranean and Arab populations with a frequency ranging from less than 0.1% to 0.7%. A majority of these, more than 70% in most populations, are the - - deletion. The -
and -
deletions are found in Mediterranean, Arab, Indian, Southeast Asian, Chinese, and Oceanian populations, with a frequency ranging from 2.2% to more than 26% in some regions. A majority of these, more than 80% in most populations, are the -
deletion. The population served by our facility has a large proportion of people of Asian ethnicity. In Washington State, which has similar demographics to those of southern British Columbia, anomalies of the alpha-globin gene complex were detected in 66% of Asian American patients referred for reproductive reasons. The importance of genotyping to family planning is significant, particularly in Asian ethnic communities, owing to the high frequency of alpha -thalassemia mutations in this population.
In general, laboratory diagnosis of alpha-thalassemia carriers is performed by the HbH inclusion body test (HbH prep). This test is laborious, observer-dependent, and reported to have poor sensitivity. Initial screening for other causes of microcytosis, such as iron deficiency, beta-thalassemia trait, or thalassemic hemoglobin variants, can improve sensitivity. The HbH prep test is seldom positive in alpha-thalassemia, and laboratory diagnosis in these cases relies on a high index of suspicion, based on a decreased mean corpuscular volume (MCV) and the absence of other causes of microcytosis, followed by DNA studies. Molecular methods for detecting and typing the alpha-thalassemia deletions typically have required the use of Southern blot analysis. The advent of nonradioactive labeling methods improved the safety of the procedure; however, it remains a time-consuming, labor-intensive, and expensive technique. Polymerase chain reaction (PCR) methods developed to specifically amplify individual deletions have required multiple reactions owing to different reagent and cycling conditions appropriate to each deletion. Recently, a single-tube multiplex PCR screen was developed to identify the 6 most common deletional mutations (described in the preceding paragraph). The present study examined the effectiveness of the multiplex PCR for the detection of alpha-thalassemia compared with our current screening approach of low MCV, exclusion of other causes of microcytosis, and HbH prep.
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