Abstract • 97

In many hereditary disorders, the analysis of genotype-phenotype correlations has lead to the identification of genetic variants of a gene locus and of modifying factors thus revolutionizing the pathophysiological understanding. β-thalassemia represents a prominent example in this respect. The clinical variability of this disorder can be explained on a molecular level in most cases. The careful analysis of a rare variant of β-thalassemia resulted in the appreciation of the medical importance and in the characterisation of NMD that represents a basic mechanism for the quality control of gene expression. This mechanism enables the identification and elimination of mRNA molecules that contain premature translation stop codons (PTCs) and code for C-terminally truncated polypeptides. The dominant negative effects of such pathological protein fragments can thus be avoided. If NMD fails in the context of β-thalassemia, clinically relevant symptoms occur in heterozygotes and affected families display an unusual dominant mode of inheritance.

The molecular mechanism of NMD was characterized by expression analysis of recombinant β-globin genes in transfected cell lines. The analysis of genes with an array of PTCs established a positional boundary ∼50 nucleotides 5′ of the terminal exon/intron juncture. mRNAs with a PTC 5′ of this boundary are eliminated by NMD, whereas those with a PTC 3′ of this boundary are stable. These results suggested an involvement of splicing in NMD. This hypothesis was directly tested by the introduction of an intron into the 5′ UTR, which results in the decay of a structurally normal mRNA. Splicing thus represents a nuclear component of the NMD pathway.

The role of translation in NMD was analyzed by the introduction of an iron-responsive element (IRE) into the 5′ UTR of normal and PTC mutated β-globin genes, which enables the iron dependent regulation of translatability of the specific mRNA via binding of the iron regulatory protein (IRP) to the IRE. The results of these analyses demonstrated that translation represents a necessary cytoplasmic component of the NMD pathway.

The suggested model for NMD involves the spliceosome dependent tagging of the exon/intron junctures and the ribosome dependent recognition of the relative position of the translation termination codon and the tag. If the elongating ribosome encounters all the tags before translation is terminated, the mRNA is stable. On the other hand, if translation is terminated before the ribosome has encountered all the tags, the mRNA is destabilized by a ribosomal post-termination activity. This model is referred to as the post-termination surveillance model or as the binary specification model of NMD. In β-thalassemia, NMD appears to protect the vast majority of heterozygotes from clinical manifestations and seems to represent an important mechanism to explain the common recessive mode of inheritance of this disorder.