Ugrás a tartalomhoz

Molecular diagnostics

Dr. István Balogh, Dr. János Kappelmayer, Dr. József Tőzsér (2011)

University of Debrecen

Chapter 9. 9. Examples for monogenic diseases II

Chapter 9. 9. Examples for monogenic diseases II

Table of Contents

One of the most prevalent severe monogenic inherited diseases is cystic fibrosis (CF). CF is caused by the abnormal function of the CFTR chloride ion channel. The CFTR protein consists of 1480 amino-acids with the domain organization shown in Figure 9.1. The largest part of the protein is located intracellularly. The two transmembrane domains (TM1, TM2, each composed of 6 transmembrane units) form the chloride channel. The nucleotide binding domains (NBD1, NBD2) have an important role in the opening of the channel. The carboxy terminal part of the protein (TRL, with threonine, arginine, leucine amino acids) makes contact with numerous other proteins and the cytoskeleton. These proteins significantly affect the function of the CFTR, the conductance, the localization and the mediation of other ion channels. The amino acid residue, which is affected by the most frequent CF-causing mutation p.F508del, is located in the NBD1.

Figure 9.1. Figure 9.1. CFTR protein

Figure 9.1. CFTR protein

CF is caused by loss-of-function mutations in the CFTR gene that result in abnormal function of the encoded chloride ion channel. CF is inherited in an autosomal recessive way. To date, more than 1600 different mutations have been described in the CFTR gene. One particular mutation, the deletion of phenylalanine residue at position 506 is the most frequent mutation, being the underlying cause of approximately 70% of the cases. The other alterations show a large interethnic variability, which makes the search for population-specific mutations necessary. CF is the most common severe monogenic disease with a prevalence of 1:3000 and a carrier frequency (heterozygosity for one null allele) of 1:25. The classification of the molecular consequences of the different mutations can be seen in Figure 9.2. Class I mutations will result in the complete or almost complete abolishment of protein synthesis. These mutations usually cause frameshift, affect promoter or cause premature termination, and they are sometimes large deletions and insertions affecting several thousands of nucleotides. Class II mutations affect the folding of the expressed protein. The molecular consequence of these mutations will be the proteasomal degradation of the mutant CFTR proteins, without reaching the cell membrane. Class III mutations affect the regulation of CFTR necessary to its function. These mutations interfere with the nucleotide binding domains. Class IV mutations will cause impaired channel function. In this case, the channel either does not conduct or it is open for too short a time period. Some mutations might affect the turnover of the CFTR protein. The above-mentioned mutations will lead to the development of the disease when they are inherited either in homozygous or compound heterozygous form. Understanding the molecular background of the disease is very important as a novel class of drugs are being tested according to the mutational status (for example in the case of p.Gly551Asp), which means that personalized therapy might be possible in the near future.

Figure 9.2. Figure 9.2. Monogenic disorders: cystic fibrosis (CF)

Figure 9.2. Monogenic disorders: cystic fibrosis (CF)

The most frequent CF causing mutation, p.F508del is a deletion of three nucleotides in the CFTR gene with a consequence of the deletion of a phenylalanine amino-acid at the position 508. Figure 9.3. shows the maturation of the CFTR protein both in the case of wild genotype and of p.F508del. The immature B CFTR protein is located in the endoplasmic reticulum. In the case of the wild genotype a fraction of the newly synthesized CFTR to the status of stable B (although a small fraction is needed therefore approximately 70-80% will be directed to the proteasomal degradation way). In the case of p.F508del mutation all CFTR molecules are degraded. Wild type CFTR protein will acquire complex glycosilation in the Golgi and reaches the cell membrane. The middle part of the picture shows the immunoblotting detection of the CFTR proteins according to their maturation status. The detected molecular weights represent the difference in glycosylation, so it aptly represents the maturation status of the proteins.

Figure 9.3. Figure 9.3. CF. Effect of the p.F508del mutation

Figure 9.3. CF. Effect of the p.F508del mutation