Molecular genetic confirmatory testing from newborn screening samples for the common African‐American, Asian Indian, Southeast Asian, and Chinese β‐thalassemia mutations

Abstract

b-Thalassemia is a serious health problem in the United States, especially in California, due to increased Asian immigration. Neonatal screening by using high-performance liquid chromatography (HPLC) or isoelectric focusing (IEF) may lead to confusion due to interactions of various hemoglobinopathies with b-thalassemia. Our purpose was to develop single-tube multiplexed PCR assays using original neonatal screening specimens to identify the mutations responsible for b-thalassemia in order to expedite diagnostic confirmation. Primers were designed for two to six common ethnic-specific mutations using the amplification refractory mutation system (ARMS). This multiplex ARMS approach was standardized using DNA samples with known mutations for b-thalassemia in those of Asian (Southeast Asian, Chinese, and Asian Indian) and African-American descent. Specimens from African-American neonates were tested for two mutations (À88 and À29); Asian Indians for five mutations (IVSI-1, IVSI-5, codons (Cd) 41/42, Cd 8/9, and 619-bp deletion); Chinese, Taiwanese, and Southeast Asians for seven mutations (Cd 41/42, Cd 17, À28, IVSII-654, Cd 71/72, IVSI-5, and IVSI-1). We identified each of these b-thalassemia mutations in multiplexed ARMS from positive control samples. We tested 25 anonymized dried blood specimens from neonates who had been diagnosed with b-thalassemia and who also belonged to these ethnic groups. We detected a mutation specific to the neonate's ethnic group using the ARMS approach in nearly all specimens, and the results were confirmed by sequencing. Multiplexed ARMS for ethnic-specific b-thalassemia mutations from the original newborn screening dried blood specimens is a rapid and efficient approach for diagnostic confirmation. Am.

Systematic prediction and validation of breakpoints associated with copy-number variants in the human genome

“…To evaluate our breakpoint predictions critically, we first focused on breakpoints that were previously precisely mapped in the regions analyzed here. Indeed, we identified all four previously sequenced breakpoints (16,26) in the individuals available to us at nucleotide level ( Table 1): both of the physical boundaries of a 619-bp heterozygous deletion causing ␤-thalassemia (26) and the breakpoints of a 1.4-Mb heterozygous deletion associated previously with 22q11-deletion syndrome (16). Furthermore, the heterozygosity (16, 26) of both deletions was correctly identified by BreakPtr.…”

Birth prevalence of disorders detectable through newborn screening by race/ethnicity

“…Only limited race-specific disorder profiles have been reported elsewhere. Previous research has examined the relationship between ethnicity and a single genetic disorder, including the prevalence of a mutation or disorder within a specific ethnic group, [4][5][6][7][8] within several ethnic groups in a region, [9][10][11][12][13][14][15][16][17] or by geographic region only. 18,19 No studies have published the disorder prevalence rates by specific racial/ethnicity groups in a large US population.…”

Understanding Beta-Thalassemia with Focus on the Indian Subcontinent and the Middle East

“…However, since these techniques require specialized skill and reagents, they are expensive [61]. They are presently not ideal for economically-unstable countries within Indian Subcontinent and the Middle East.…”

“…Unknown HBB mutations must be discovered using screening methods such as direct sequencing of genomic DNA, single strand conformation polymorphism analysis or denaturing gradient gel electrophoresis [61]. However, since these techniques require specialized skill and reagents, they are expensive [61].…”