Application of an expanded multiplex genotyping assay for the simultaneous detection of Hemoglobin Constant Spring and common deletional α‐thalassemia mutations

Abstract

Hemoglobin Constant Spring (HbCS) is the most common nondeletional alpha-thalassemia variant causing HbH disease, making its detection crucial in populations at risk. Universal newborn screening for HbH is carried out in California. Identification of alpha-thalassemia genotypes responsible for HbH and HbH-CS requires rapid, accurate and cost-effective genotyping methods suitable for population screening. We incorporated the HbCS mutation into our existing seven-plex genotyping assay for common alpha-thalassemia deletions. To assess the feasibility and diagnostic utility of this expanded multiplex gap-PCR assay, we determined genotypic frequencies of HbCS in samples referred for alpha-thalassemia testing between 1 January 2006 and 31 December 2008. During the 3-year study period, 1436 samples were genotyped for alpha-thalassemia. HbH-CS accounted for 23 (13%) of the 176 cases of HbH disease identified. In a subset of 145 newborns referred by the California NBS program with an elevated Hb Bart's level at birth, HbH disease was confirmed in 134 (93%) and HbH-CS identified in 13 (10%) of these. This expanded genotyping assay has proven to be a rapid, reliable and clinically useful diagnostic tool for the detection of HbH-CS disease.

Associations of α-thalassemia and BCL11A with stroke in Nigerian, United States, and United Kingdom sickle cell anemia cohorts

“…7 Multiplex PCR reactions on 100–200ng of gDNA were used to detect α-thalassemia status using methods previously described in the University of Ibadan and UIC cohorts. 8 To avoid primer dimers, only primers for the HBA2 gene, α-3.7K deletion and α-4.2K deletion were used in the PCR reaction. Genotyping for BCL11A and α-thalassemia status for the University of Ibadan and UIC subjects was performed centrally at UIC.…”

Clinical Manifestations of -Thalassemia

“…There are also many different nondeletional forms of a-thalassemia, many of which are also named after their place of origin. The clinical phenotypes of hemoglobin H disease that result from the interaction of nondeletional mutations ( -/a ND a) are more severe and less common than the deletional forms (-a/ -) Lorey et al 2001a;Ne et al 2008;Michlitsch et al 2009;Singer et al 2009;Kidd et al 2010;Lal et al 2011;Vichinsky 2012). The most common interaction of this type is with the a-globin chain-termination mutant, Hb Constant Spring ( -/a CS a), which accounts for approximately 27% of hemoglobin H diseases in California (Table 2) and which occurs frequently in many countries in Southeast Asia.…”

“…Hemoglobin A 2 levels are lower in a-thalassemia disorders. Automated high-performance liquid chromatography (HPLC) is an excellent screening method for high-risk populations and in newborn screening programs (Lorey et al 2001a;Hoppe 2009;Michlitsch et al 2009;Kidd et al 2010). In the neonatal period, hemoglobin Bart's is commonly seen on HPLC or hemoglobin electrophoresis.…”

“…Definitive diagnosis requires molecular genetic testing. Multiplex genotyping assays using gap polymerase chain reaction can rapidly diagnose most deletional and nondeletional mutations (Kidd et al 2010). Confirmatory parental studies should always be performed in newborns and children identified with hemoglobin H. Patients with a-thalassemia disorders often coinherit other mutations including hemoglobin E, b-thalassemia, and hemoglobin S (Li et al 2006;Liao et al 2007;Hoppe 2009).…”

Prenatal and newborn screening for hemoglobinopathies

“…The majority of cases are South East Asian, with a Laotian, Vietnamese or Cambodian ancestry (40%), and the remaining are Filipino (15%), Chinese (13%), or other Asian (9%). The distribution of Hb H genotypes among California newborns has been previously reported [6,8]. The majority of cases are caused by deletions in the a-gene locus, with the --SEA/-a3.7 genotype constituting 61% of cases, followed by the --FIL/-a3.7 and the --SEA/-a4.2 constituting another 23% cases.…”