**5.1 Gene deletions**


*Genetics of Thalassemia DOI: http://dx.doi.org/10.5772/intechopen.106748*

• These events are associated with biochemical and clinical features of hereditary persistence of fetal Hb (HPFH) with the added presence of the structural variant, Hb Kenya. The persistence of high levels of fetal hemoglobin synthesis into adult life may arise because of the removal of stage selector elements and silencers normally positioned between the gamma and delta genes, and the closer apposition of a strong beta globin gene enhancer normally located at the 3′ side of the beta gene to the G (gamma) and Kenya genes. Hb Kenya is rare; the anti-Kenya state has yet not been identified [32, 33].

#### **5.2 Mutations affecting transcription**

Alpha and beta thalassemia have been found to arise from mutations that alter known promoter or enhancer sequences for alpha or beta globin genes [28]. These point mutations alter the efficiency of the promoter or enhancer, while others are small gene deletions or rearrangement that disrupts their spatial integrity [29].

#### **5.3 Mutations affecting Pre-mRNA splicing**

Many mutations have been described that disrupt normal splicing of the mRNA precursor. Among these are some of the most common forms of beta thalassemia and more common varieties of "non-deletion" forms of alpha thalassemia [21, 34].

#### **5.4 Alteration of canonical splice signals**

Some thalassemic splicing mutations directly disrupt the canonical "splicing signals" used to mark the beginning and end of each intron so that normal splicing can occur. These short sequences are required by the splicing machinery. They signify the places at which excision of the intron, and ligation together of the flanking exons, should occur [34, 35].

Certain bases in these splicing signals are "invariant", such as the GT dinucleotide required at the 5′ beginning of the intron and the AG dinucleotide required at the 3′ end of the intron. The several bases to either side of these invariant nucleotides are consensus sequences within which alteration of the base will change the efficiency with which the site is used [36]. Thus, mutations altering these nucleotides can abolish normal splicing or reduce it to a variable degree. Mutations that alter the consensus splice sites reduce production of alpha or beta globin mRNA; the pre-mRNA molecules which are not properly spliced appear to be catabolized rapidly, so that abnormal mRNA products do not accumulate [37].

In some cases, pre-mRNAs that are not spliced at the proper sites are spliced elsewhere by activation of "cryptic" sites, resulting in the production of structurally abnormal, usually non-functioning messenger RNAs [35]. The molecular basis for variability with which mutations reduce the efficiency of normal splicing, or generate production of detectable abnormally spliced products, remains poorly understood and needs to be investigated.

#### **5.5 Activation of cryptic splicing sites**

Another class of splicing mutations, one of which produces a very common form of thalassemia in the Mediterranean region, includes those in which the mutation

activates a "cryptic" splice site. These mutations are not located within the consensus sites at either end of introns. Rather, base substitution, small deletions, or small insertions of DNA, can convert a site within an exon or intron that normally bears only a slight resemblance to a splice site into one containing much stronger consensus signal [37, 38]. Depending upon the consequential "strength" of the signals, the splicing apparatus will utilize that site instead of normal site in a greater or lesser percentage of the pre-mRNA molecules being spliced. At least two spliced mRNA products result, accumulating in varying percentages, depending on efficiency with which the cryptic site is used and the stability of the abnormally spliced product [29, 38].

#### **5.6 Altered mRNA translation and stability**

Mutations that create a premature translation termination codon (nonsense codon) account for the most common forms of thalassemia, in terms of numbers of patients affected. These mutations create translation stop signals prematurely, so that the complete beta globin polypeptide is never made. In the most common type of thalassemia, globin fragments are highly unstable, resulting in the accumulation of protein synthesized from the mutated gene (i.e., beta (0) thalassemia) [38]. As a result, patients homozygous for this defect cannot make any beta chains and suffer from a severe form of beta thalassemia [27].

Premature translation termination can occur by base substitution or deletion of bases in an exon, producing so called "frame shift" mutations. The inserted or deleted stretch of DNA contains a number of bases that is an exact multiple of three, so "open" translation reading frame is maintained. So, the insertion or deletion will cause the ribosome to begin reading the codons out of the normal reading frame. One consequence of this "frame shift" mechanism is that the probability that a UAA, UAG, or UGA translation termination codon will be encountered with the shifted reading frame within 50 or so bases downstream [38, 39].

In the normal reading frame, these three bases (UAA, UAG, UGA) are usually divided among two codons, and thus never "read" as a stop codon by the ribosome. In the shifted reading frame, the three bases will appear as a single codon and translation ceases. Thus frame shifting results into premature translation termination. A reason for occurrences of both alpha and beta thalassemia mutations.

### **6. Nonsense-mediated decay**

The physiologic consequences of translation termination are very clear; functional globin not synthesized and thalassemia results. Less obvious is the phenomenon that these prematurely terminated mRNAs accumulate in greatly reduced amounts. The reason behind impaired accumulation is not clear, since their transcription, processing, and stability elements are intact. Recent research work has shown that there are normal cell defense mechanisms to eliminate abnormally translated mRNAs. They probably exist in order to guard against the accumulation of truncated protein products, which have the potential to interact abnormally with other proteins and damage cells. This protective phenomenon is called nonsense-mediated decay.

Curiously, premature stop codons that occur in the final exon of either the alpha or beta globin gene accumulate at nearly normal levels. Moreover, the truncated polypeptides also accumulate stably and in significant amounts. It appears that the process of nonsense-mediated decay affects only those mRNAs in which the premature stop

codon occurs in the first or second exons. This has been found in other gene systems as well. Indeed, there is increase in substantiation that nonsense-mediated decay and mRNA splicing are interactive processes [32].
