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Molecular diagnostics

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

University of Debrecen

Chapter 8. 8. Examples for monogenic diseases

Chapter 8. 8. Examples for monogenic diseases

Table of Contents

Duchenne/Becker muscular dystrophy (DMD-BMD) shows X chromosome-linked recessive inheritance. The disease is characterized by progressive muscle weakness and it is caused by mutations in one of the largest human gene, dystrophin. Dystrophin gene is 100-times bigger than an average human gene, its size is half of the entire E. coli genome. It is larger than any chromosome in the yeast. Its transcription takes 16 hours and provides the first mammalian example of the co-transcriptional splicing. It spans 2400 kb in the chromosome X and contains 79 exons. The encoded protein with the same name contains 3685 amino acids, its molecular mass is 427 kDa. The major function of the dystrophin is the mechanical reinforcement of the sarcolemma, by creating a physical link between the intracellular contractile elements and the components of the extracellular matrix. It binds with its N-terminal to actin and has contact with the dystroglycans. Two allelic diseases, Duchenne and Becker, are caused by different mutations in the dystrophin gene. These mutations in 2/3 of the cases are large deletions affecting one or more exons. Mutations that do not lead to the complete loss of dystrophin protein cause the much more benign Becker muscular dystrophy, which was described as an independent clinical entity before the identification of their common molecular background. The status of the open reading frame is an important prognostic factor. If the open reading frame is not changed, the muscular dystrophy will be Becker-type. In such a case, although the protein function is impaired, it is not completely abolished.

One third of the Duchenne/Becker muscular dystrophy cases are the consequence of novel mutations. The proportion is similar for point mutations and for gene segment duplications.

The laboratory diagnosis of Duchenne/Becker muscular dystrophy can be performed at different levels. Serum creatin kinase activity (released from the necrotizing muscle cells) is elevated in the patients, this overlaps, however, with the normal range in carriers. In the case of clinical suspicion, it is possible to analyze muscle biopsy for the presence of dystrophin protein. It can be done either by immunohistochemistry or SDS-PAGE followed by immunoblotting with a mono- or polyclonal antibody. The latter might help in determining the size (i.e. the level of truncation) of the mutant dystrophin and is able to give a “yes/no” answer as well (dystrophin present or absent).

Molecular genetic testing involves special PCR assays that are able to detect the presence or absence of the exons located at the deletion hot spots (17 exons and the promoter can be tested this way). These tests, however, cannot be used for carrier testing. To do so, another special amplification assay can be utilized, the multiplex ligation dependent probe amplification (MLPA, see later).

When it was determined that these two disorders are caused by mutations in a single gene, the clinical category of dystrophinopathy was created. It was soon realized that a third disorder, the X-linked cardiomyopathy also belongs to this group. This disease is caused by specific mutations that occur in the heart specific promoter of the dystrophin gene. The consequence of these mutations will be the absence of dystrophin in the heart tissue while the skeletal muscle will have a normal or close to normal dystrophin level (Figure 8.1.). Therefore, the discovery, cloning and characterization of the dystrophin gene helped to set up the molecular associations in three different clinical diseases and in the development of the molecular genetic diagnosis.

Figure 8.1. Figure 8.1. Allelic disorders: one gene, three diseases

Figure 8.1. Allelic disorders: one gene, three diseases

Duchenne/Becker muscular dystrophy provides a good example of the presentation of the indirect molecular testing as well. Linkage analysis can be performed using intragenic or adjacent polymorphic markers when samples from members of more generations of the family are available and no information is available about the exact site of the disease-causing mutation. The testing shown on Figure 8.2. uses the a1, a2, b1, b2 polymorphic markers. In the case of this family, the a1-b1 marker combination defines the disease-causing X chromosome. In the third generation, the sisters of the affected male child can be tested for their possible mutation carrier status. III/2 member of the family is a carrier, therefore she can inherit the disease, while III/1 sister has not inherited the disease-causing chromosome.

Figure 8.2. Figure 8.2. Linkage analysis for the identification of carrier status

Figure 8.2. Linkage analysis for the identification of carrier status