Molecular Diagnosis Of Alphathalassemia

The presence of alpha-thalassemia, suspected on the basis of hematological and/or clinical findings [i.e., micro-cytosis with normal HbA2 and F and normal serum iron status in carriers; microcytic and hypocromic hemolytic anemia with RBC inclusion bodies and a fast-moving band (HbH) at the hemoglobin electrophoresis; presence of Hb Bart's in the newborn], should be confirmed, if requested, by globin gene synthesis or even better by globin gene DNA analysis.

The exact definition of the genotype with molecular methods is relevant for genetic counseling in carriers and for prognosis in HbH disease patients. In the last few years several approaches based on polymerase chain reaction (PCR) have been developed for the detection of the most common deletional and nondeletional types of alpha-thalassemia (for references, see Ref. [16]).

The common deletion alpha0- or alpha+-thalassemias are detected using two primers (specific for each deletion type), which flank the deletion breakpoints. Amplification occurs only in the presence of the deletion, whereas it does not occur in normal subjects because the two primers are separated by too great a distance. This approach is known as GAP-PCR. As a control, DNA from a normal subject is simultaneously amplified using one of the primers flanking the breakpoint and a primer homologous to a DNA region deleted by the mutation. The less common deletion alleles have to be identified by Southern blot analysis.

Nondeletional forms of alpha thalassemia are detected by selective amplification of alpha1 and alpha2 genes, followed by restriction enzyme analysis, when the mutation creates or abolishes a cleavage site [i.e., NcoI for T! G alpha2 initiation codon mutation, HphI for the - 5 bp (donor) alpha2 IVSI-1 deletion, Msel for Hb Constant Spring] or by Dot Blot analysis or ARMS with specific oligo-probes. A strategy for the diagnosis of all the known point mutations, which involves the combined application of denaturing gradient gel electrophoresis (DGGE) and single-strand conformational analysis (SSCA), followed by direct DNA sequencing, has been reported.[17]

Multiplex PCR assays including up to seven alpha-thalassemia deletional alleles or common Southeast Asian nondeletional alleles have been recently described and seem to give robust and reproducible results.[18-20]

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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