CLINICAL CASES
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that are attributed to larger deletions ranging from 290 base pairs (bp) to greater
than 60 kbp. One is a family of upstream deletions, which down regulates the
locus control region, and the other affects the promoter region of P-globin
gene. The promoter region deletions typically include the mRNA CAP site,
TATA box, and CAAT elements. Apart from deletions, there are insertions of
6 to 7 kbp retrotransposons in the P-globin gene that decrease the transcript
level to 15 percent in comparison to the control. In addition, there are also a
few thalassemia mutations that segregate independently of the P-globin gene,
such as those involving the trans-acting regulatory factors that affect the
expression of the P-globin gene. One can thus broadly classify the different
classes of mutations which result in P-thalassemia as follows:
1.
Transcription (deletion, insertion, etc.)
2.
Messenger RNA (mRNA) processing (cryptic splice sites, consensus
sequence, poly A addition site, etc.)
3.
Translation (initiation, nonsense, frameshift mutations)
4.
Posttranslation stability (highly unstable P-chain)
5.
Determinants unlinked to the P-globin gene cluster
The most common point mutations occur in the CAAT or TATA box in
the promoter sequence. The single base substitution A to G at position -29
in individuals of African descent leads to a mild form of the disease, whereas
the substitution A to C in those of Chinese descent leads to thalassemia major.
Also, a single nucleotide substitution in the GT/AG splice junction can lead
to a misspliced mRNA that does not allow the translation of a functional
P-globin chain. The C to T mutation at position -101 causes an extremely
mild deficit in P-globin gene. The effect is so mild that it is a “silent” or benign
mutation in heterozygotes. All the point mutations that prevent the translation
of the P-globin mRNA (for example, changing the start codon ATG to GTG,
or TGT to the stop codon TGA) or a single nucleotide frame shift lead to
P0-phenotype.
There are several biochemical techniques that can be used to accurately
determine the genetic defect that results in a particular thalassemia. Many of
these techniques make use of the polymerase chain reaction (PCR), which
amplifies the DNA and allows detection of multiple mutations at once.
The
amplification refractory mutation system
(ARMS) is a technique in
which the target DNA is amplified using a common primer and either muta-
tion specific primer for P-thalassemia or the correct sequence primer to the tar-
get. This method provides quick screening of the DNA to detect if the patient
carries the mutant gene or not. When both the primers are used in the same PCR
reaction, they compete to amplify the target, and the technique is termed COP
(competitive oligonucleotide priming), and the primers are labeled with different
fluorescent dyes to allow detection.
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