Molecular Basis of Myotonic Disorders & New Diagnostic Techniques

Acta Universitatis Tamperensis No. 1783


By Olayinka Raheem
December 2012
Tampere University Press
Distributed By Coronet Books
ISBN: 9789514489754
143 pages
$92.50 Paper Original

Myotonic disorders are primary diseases of the muscle characterized by myotonia, the delayed relaxation of skeletal muscles after voluntary contraction. Myotonic disorders include the myotonic dystrophies (DMs) and non-dystrophic myotonias. Myotonic dystrophies type 1 (DM1) and type 2 (DM2) are multisystemic disorders caused by tri- (CTG)n or tetranucleotide (CCTG)n repeat expansion mutations in transcribed but not translated regions of the genes DMPK and ZNF9, respectively. Adult onset DM1 and DM2 share some features in the clinical presentation as well as in the molecular genetics and pathomechanisms. However, they also show distinct differences, including disease severity and involvement of muscles and muscle fiber types. So far, over 300 DM2 patients have been identified in Finland, which is in contrast to the prevalence estimate of 1/105 (corresponding to 50 patients) reported previously. DM2 is of particular interest, because it shows a wide range of clinical manifestations and therefore a makes clinical diagnosis extremely difficult. Regarding molecular pathogenesis it has been suggested that ZNF9 is of no significance for the disease pathogenesis, and that the disease is caused solely by RNA toxicity as a result of the underlying repeat expansion mutation. Such an exclusive explanation does not however explain the higher amount of toxic RNA in DM2 than DM1 muscle still resulting in milder clinical manifestations in DM2 compared to DM1. In this thesis work, we were able to show that ZNF9 expression in DM2 patients is in fact altered at multiple levels. While toxic RNA effects likely explain overlapping phenotypic manifestations between DM1 and DM2, abnormal ZNF9 levels in DM2 may account for at least some of the differences.

It is important to improve diagnostic accuracy in order to efficiently identify symptomatic patients for correct final diagnosis and appropriate medical attention and management. The different enzyme histochemical ATPase properties of myosins to separate the muscle fiber types have been utilized in diagnostic muscle biopsy routine for more than four decades. The ATPase staining method is rather laborious and has several disadvantages, such as weakening of staining over time and non-specific staining of capillaries, making the distinction of extremely atrophic muscle fibers difficult. Extremely small atrophic type 2/IIA fibers are characteristic for DM2 and usually remain undetected using the ATPase staining method. A reliable and advanced immunohistochemical myosin double staining method for the identification of fiber types, including these highly atrophic type IIA fibers in routine diagnostics was developed in this thesis work. With this double staining method, it is easily possible to distinguish all different fiber types using a one slide technique.

In addition to the obvious usefulness in DM2 diagnostics we were able to identify a completely new disease with this technique, because of the absence of fast type IIA fibers in patients’ muscle biopsies. The disease is caused by disruptive recessive mutations in the MYH2 gene resulting in total absence of MyHC IIA protein and correspondingly total lack of fast type IIA muscle fibers.

Myotonia congenita is a non-dystrophic myotonia disease caused by mutations in the chloride channel gene (CLCN1). Currently, final diagnosis of patients with symptoms is frequently obtained by molecular genetic DNA testing. However, the increased use of genetic testing also results in many cases where the genetic results do not provide full clarification of the clinical disease.

In this thesis work, the developed immunohistochemical staining method for chloride channel protein (ClC-1) in muscle fibers proved to be a robust method for the assessment of sarcolemmal ClC-1 protein on muscle sections. This method provided means to identify new mutations, to reclassify the W118G CLCN1 change as a moderately pathogenic mutation, and to clarify the final diagnosis in myotonia patients in whom only one recessive mutation had been identified by genetic testing. The methods developed in this thesis work combined with genetic testing are powerful approaches to achieving final diagnosis in patients with myotonic disorders. Comprehensive understanding of the molecular pathomechanisims of genetic diseases is also one of the pre-requisites for the future development of therapeutical options.

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