Successes and prospects of molecular diagnosis of the most widespread inherited diseases]

Current state of molecular diagnosis of hereditary diseases most common in the former USSR such as cystic fibrosis, Duchenne muscular dystrophy, haemophilia A and B as well as phenylketonuria is reviewed. Basic results of prenatal diagnosis and carrier detection of the above mentioned diseases in St.-Petersbourg and somewhere else in Russia are presented. The urgent necessity to start an efficient molecular diagnosis of some other widespread hereditary diseases (von Willebrand's disease, Martin-Bell syndrome, polycystic kidney. Huntington chorea, myotonic dystrophy, etc.) is emphasized. Creation of new diagnostic centers dealing with most common diseases as well as complementing each other as to molecular diagnosis of more rare hereditary diseases is substantiated. Prospects of implementation of new molecular methods and novel technical approaches (preimplantation embryos, fetal cells selected from maternal blood) for more efficient diagnosis of hereditary diseases are briefly outlined.     

The dog genome map and its use in mammalian comparative genomics

The dog genome organization was extensively studied in the last ten years. The most important achievements are the well-developed marker genome maps, including over 3200 marker loci, and a survey of the DNA genome sequence. This knowledge, along with the most advanced map of the human genome, turned out to be very useful in comparative genomic studies. On the one hand, it has promoted the development of marker genome maps of other species of the family Canidae (red fox, arctic fox, Chinese raccoon dog) as well as studies on the evolution of their karyotype. But the most important approach is the comparative analysis of human and canine hereditary diseases. At present, causative gene mutations are known for 30 canine hereditary diseases. A majority of them have human counterparts with similar clinical and molecular features. Studies on identification of genes having a major impact on some multifactorial diseases (hip dysplasia, epilepsy) and cancers (multifocal renal cystadenocarcinoma and nodular dermatofibrosis) are advanced. Very promising are the results of gene therapy for certain canine monogenic diseases (haemophilia, hereditary retinal dystrophy, mucopolysaccharidosis), which have human equivalents. The above-mentioned examples prove a very important model role of the dog in studies of human genetic diseases. On the other hand, the identification of gene mutations responsible for hereditary diseases has a substantial impact on breeding strategy in the dog.     

Molecular Genetic Analysis of Hereditary Neurodegenerative Diseases

The review summarizes the results of a decade of molecular genetic studies of several high-incidence hereditary neurodegenerative diseases, including primary parkinsonism, various forms of hereditary dystonia and ataxia, polyglutamine disorders, hepatolenticular degeneration, essential tremor, etc. Various relevant mutations were studied. The character and frequencies of particular mutations and the corresponding genetic disorders were established for the Russian population. Particular genotypes were associated with various clinical variants of the diseases. Genetic loci were identified for several unique hereditary diseases of the nervous system (X-linked cerebellar hypoplasia, an atypical form of autosomal recessive muscular dystrophy, etc.). Nosological positions of the relevant clinical forms were clarified on the basis of the molecular genetic data. Protocols were developed for direct or indirect DNA diagnostics of the diseases under study to improve medical genetic counseling and prevention of new disease cases in affected families.     

Molecular genetic analysis of hereditary neurodegenerative diseases

The review summarizes the results of a decade of molecular genetic studies of several high-incidence hereditary neurodegenerative diseases, including primary parkinsonism, various forms of hereditary dystonia and ataxia, polyglutamine disorders, hepatolenticular degeneration, essential tremor, etc. Various relevant mutations were studied. The character and frequencies of particular mutations and the corresponding genetic disorders were established for the Russian population. Particular genotypes were associated with various clinical variants of the diseases. Genetic loci were identified for several unique hereditary diseases of the nervous system (X-linked cerebellar hypoplasia, an atypical form of autosomal recessive muscular dystrophy, etc.). Nosological positions of the relevant clinical forms were clarified on the basis of the molecular genetic data. Protocols were developed for direct or indirect DNA diagnostics of the diseases under study to improve medical genetic counseling and prevention of new disease cases in affected families.     

Intracellular free calcium concentration in lymphocytes of patients with muscular dystrophies

Intracellular free calcium concentration [( Ca2+]i) of human peripheral blood lymphocytes was determined by fluorescence spectroscopic measurements with quin2 in patients with different types of muscular dystrophy and in controls. The [Ca2+]i level in lymphocytes showed a significant increase in adult type (facioscapulohumeral and limb-girdle) muscular dystrophies, while it showed a decrease in Duchenne dystrophy as compared to the values of age- and sex-matched controls. The data obtained suggest an alteration in the effectiveness of the calcium pump in lymphocytes and may represent a sign of generalized membrane damage in these hereditary muscle diseases.     

Disabilities caused by unstable mutations in Costa Rica

Motonic dystrophy and fragile X syndrome are two genetically determined relatively common disabilities. Both are examples of a new type of mutation mechanism called unstable or dynamic mutations, triple repeats expansions or DNA amplification. Fragile X syndrome is recognized as the main cause of hereditary mental retardation and myotonic dystrophy is considered the most common muscular dystrophy of adults. This is a prospective non randomized study of clinically affected people, in order to confirm the diagnosis with molecular techniques (Southern blot and PCR) and to perform cascade screening of the rest of the family to offer them adequate genetic counseling. We were able to corroborate the initial diagnosis in most clinical cases of myotonic dystrophy, but in the cases of mental retardation more than half studies were negative for fragile X syndrome, stressing the difficulties encountered by medical practitioners to diagnose this syndrome. The reasons for this are several; probable the main culprit is the subtle and unspecific clinical picture affected individuals exhibit, particularly children before puberty. Cascade screening, genetic counseling and selective abortion are the only tools available to prevent these disabling diseases for the moment.     

Dog as a model in studies on human hereditary diseases and their gene therapy

During the last 15 years spectacular progress has been achieved in knowledge on the dog genome organization and the molecular background of hereditary diseases in this species. A majority of canine genetic diseases have their counterparts in humans and thus dogs are considered as a very important large animal model in human biomedicine. Among canine monogenic diseases with known causative gene mutations there are two large groups classified as retinal dystrophies and lysosomal storage diseases. Specific types of these diseases are usually diagnosed in a single or several breeds. A well known disorder, restricted to a single breed, is congenital stationary night blindness described in Briards. This disease is a counterpart of Leber amaurosis in children. On the other hand, one of the most common monogenic human diseases (Duchenne muscular dystrophy), has its canine counterparts in several breeds (e.g., the Golden retriever, Beagle and German short-haired pointer). For some of the canine diseases gene therapy strategy was successfully applied, e.g., for congenital stationary night blindness, rod-cone dystrophy and muccopolysaccharydoses type I, IIIB and VII. Since phenotypic variability between the breeds is exceptionally high, the dog is an interesting model to study the molecular background of congenital malformations (e.g., dwarfism and osteoporosis imperfecta). Also disorders of sexual development (DSD), especially testicular or ovotesticular DSD (78,XX; SRY-negative), which is widely distributed across dozens of breeds, are of particular interest. Studies on the genetic background of canine cancers, a major health problem in this species, are also quite advanced. On the other hand, genetic studies on canine counterparts of major human complex diseases (e.g., obesity, the metabolic syndrome and diabetes mellitus) are still in their infancy.     

Glycobiology of neuromuscular disorders

There has been a recent explosion in the identification of neuromuscular diseases caused by mutations in genes that affect carbohydrate metabolism or protein glycosylation. A number of these findings relate to defects in the glycosylation of alpha dystroglycan. Alpha dystroglycan is an essential component of the dystrophin-glycoprotein complex, and aberrant glycosylation of alpha dystroglycan is associated with multiple forms of muscular dystrophy in mice and humans. We review the evidence that defects in dystroglycan glycosylation cause muscular dystrophy. In addition, we review evidence that glycobiology is important in other disorders that affect muscle, including hereditary inclusion body myopathy type II and congenital disorders of glycosylation. Finally, we discuss the long-term potential of glycotherapies for muscle disorders.     

Skeletal Muscle Diseases: Recent Advances and Some Related Basic Problems

Recent advances in the knowledge of the structure and function of voluntary muscle and some of its diseases are reviewed on the basis of a recently published international symposium. Among the subjects discussed are: the specific metabolic role of various muscle cell constituents (e.g. sarcosomes, microsomes, sarcoplasmic reticulum); the relaxing factor system and the dual (tonic and phasic) innervation of muscle fibres; observations on the physiology of muscle training; the morphologic differential diagnosis of degenerative skeletal muscle diseases; the value of serum enzyme determinations in the early detection of muscular dystrophy and in the identification of dystrophic carriers; the classification and diagnosis of muscular hypotonias of infancy; the role of inactivity and the trophic control of the nervous system in the development of neural (or denervation) atrophies; factors influencing regeneration of muscle fibres; the significance, as a research tool, of the identification of hereditary primary muscle disease in laboratory animals.     

Genomic structure and DNA diagnosis of hereditary monogenic diseases in the Volga-Ural region

The review considers the main results of molecular analysis of the genes responsible for cystic fibrosis, phenylketonuria, Wilson-Konovalov disease, Duchenne-Becker progressive muscular dystrophy, myotonic dystrophy, Huntington's disease, and nonsyndromic hereditary hypoacusis in populations of the Volga-Ural region. The results were obtained in the past ten years within the framework of the Russian program Human Genome. The mutation spectra and frequencies of these genes were characterized in the major ethnic groups (Bashkirs, Tatars, Russians) of Bashkortostan. Several diseases were associated with particular alleles or haplotypes of polymorphic loci of relevant genes. The results were used to develop DNA diagnostic procedures optimal for the region and to establish the origin of the mutations involved.     

The Genome Structure and DNA Diagnosis of Monogenic Hereditary Disorders in the Volga–Ural Region

The review considers the main results of molecular analysis of the genes responsible for cystic fibrosis, phenylketonuria, Wilson–Konovalov disease, Duchenne–Becker progressive muscular dystrophy, myotonic dystrophy, Huntington's disease, and non-syndromic hereditary deafness in populations of the Volga–Ural region. The results were obtained in the past ten years within the framework of the Russian program Human Genome. The mutation spectra and frequencies of these genes were characterized in the major ethnic groups (Bashkirs, Tatars, Russians) of Bashkortostan. Several diseases were associated with particular alleles or haplotypes of polymorphic loci of relevant genes. The results were used to develop DNA diagnostic procedures optimal for the region and to establish the origin of the mutations involved.     

Allele-specific therapy: suppression of nonsense mutations by readthrough inducers

Ten percent of human hereditary diseases are linked to nonsense mutations (premature termination codon). These mutations lead to premature translation termination, trigger the synthesis of a truncated protein and possibly lead to mRNA degradation by the NMD pathway (nonsense mediated mRNA decay). For the past ten years, therapeutic strategies have emerged which attempt to use molecules that facilitate tRNA incorporation at premature stop codon (readthrough), thus allowing for the synthesis of a full length protein. Molecules currently used for this approach are mostly aminoglycoside antibiotics (gentamicin, amikacin…) that bind the decoding center of the ribosome. This therapeutic approach has been studied for various genetic diseases including Duchenne muscular dystrophy (DMD) and cystic fibrosis. The feasibility of this approach depends on induced readthrough level, mRNA quantity, re-expressed protein functionality and characteristics of each disease.     

Living with myotonic dystrophy; what can be learned from couples? a qualitative study

Background  

Myotonic dystrophy type 1 (MD1) is one of the most prevalent neuromuscular diseases, yet very little is known about how MD1 affects the lives of couples and how they themselves manage individually and together. To better match health care to their problems, concerns and needs, it is important to understand their perspective of living with this hereditary, systemic disease.     

Genetic diseases of the Kennedy pathways for membrane synthesis

The two branches of the Kennedy pathways (CDP-choline and CDP-ethanolamine) are the predominant pathways responsible for the synthesis of the most abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, respectively, in mammalian membranes. Recently, hereditary diseases associated with single gene mutations in the Kennedy pathways have been identified. Interestingly, genetic diseases within the same pathway vary greatly, ranging from muscular dystrophy to spastic paraplegia to a childhood blinding disorder to bone deformations. Indeed, different point mutations in the same gene (PCYT1; CCTα) result in at least three distinct diseases. In this review, we will summarize and review the genetic diseases associated with mutations in genes of the Kennedy pathway for phospholipid synthesis. These single-gene disorders provide insight, indeed direct genotype-phenotype relationships, into the biological functions of specific enzymes of the Kennedy pathway. We discuss potential mechanisms of how mutations within the same pathway can cause disparate disease.     

Exclusion of the gelsolin gene on 9q32-34 as the cause of familial lattice corneal dystrophy type I.

Familial lattice corneal dystrophy type I (LCD1) is a localized form of inherited amyloidosis limited to the corneal stroma. Recently the Finnish form of hereditary amyloidosis with lattice corneal dystrophy has been shown to be due to a mutation in the gelsolin gene (G654----A; Asp187----Asn). In this paper we exclude the gelsolin gene as the cause of the autosomal dominant form of isolated LCD1.     

Suppression of nonsense mutations in the Dystrophin gene by a suppressor tRNA gene

Nonsense mutations in the dystrophin gene are the cause of Duchenne muscular dystrophy (DMD) in 10-15% of patients. In such an event, one approach to gene therapy for DMD is the use of suppressor tRNAs to overcome the premature termination of translation of the mutant mRNA. We have carried out cotransfection of the HeLa cell culture with constructs containing a suptRNA gene (pcDNA3suptRNA) and a marker LacZ gene (pNTLacZhis) using their polymer VSST-525 complexes. It was found that the number of cells producing beta-galactosidase depends inversely on the dose of the suptRNA gene. A single in vivo injection of the construct providing for expression of the suptRNAochre gene into mdx mouse muscle resulted in the production of dystrophin in 2.5% of fibers. This suggests that suppressor tRNAs are applicable in gene therapy for hereditary diseases caused by nonsense mutations.     

Genetics and epigenetics of facio-scapulohumeral progressive (Landouzy-Dejerine) muscular dystrophy

Landouzy-Dejerine muscular dystrophy is a rare hereditary disease with prevalence of 0.9 to 1.4 in 100,000. Clinically the disease is characterized by weakness and atrophy of the facial and shoulder girdle muscles. It is caused by partial deletion of the 3.3-kb subtelomeric D4Z4 repeat on chromosome 4 (locus 4q35). This paper presents a critical review of the literature data and hypotheses explaining molecular mechanisms of progressive fascioscapulohumeral muscular dystrophy.