Evaluation of candidate genes in the absence of positional information: a poor bet on a blind dog!

More than 350 inherited diseases have been reported in dogs and at least 50% of them have human counterparts. To remove the diseases from dog breeds and to identify canine models for human diseases, it is necessary to find the mutations underlying them. To this end, two methods have been used: the functional candidate gene approach and linkage analysis. Here we present an evaluation of these in canine retinal diseases, which have been the subject of a large number of molecular genetic studies, and we show the contrasting outcomes of these approaches when dealing with genetically heterogeneous diseases. The candidate gene approach has led to 377 published results with 23 genes. Most of the results (66.6%) excluded the presence of a mutation in a gene or its coding region, while only 3.4% of the results identified the mutation causing the disease. On the other hand, five linkage analysis studies have been done on retinal diseases, resulting in three identified mutations and two mapped disease loci. Mapping studies have relied on dog research colonies. If this favorable application of linkage analysis can be extended to dogs in the pet population, success in identifying canine mutations could increase, with advantages to veterinary and human medicine.     

Genetic and phenotypic variations of inherited retinal diseases in dogs: the power of within- and across-breed studies

Considerable clinical and molecular variations have been known in retinal blinding diseases in man and also in dogs. Different forms of retinal diseases occur in specific breed(s) caused by mutations segregating within each isolated breeding population. While molecular studies to find genes and mutations underlying retinal diseases in dogs have benefited largely from the phenotypic and genetic uniformity within a breed, within- and across-breed variations have often played a key role in elucidating the molecular basis. The increasing knowledge of phenotypic, allelic, and genetic heterogeneities in canine retinal degeneration has shown that the overall picture is rather more complicated than initially thought. Over the past 20?years, various approaches have been developed and tested to search for genes and mutations underlying genetic traits in dogs, depending on the availability of genetic tools and sample resources. Candidate gene, linkage analysis, and genome-wide association studies have so far identified 24 mutations in 18 genes underlying retinal diseases in at least 58 dog breeds. Many of these genes have been associated with retinal diseases in humans, thus providing opportunities to study the role in pathogenesis and in normal vision. Application in therapeutic interventions such as gene therapy has proven successful initially in a naturally occurring dog model followed by trials in human patients. Other genes whose human homologs have not been associated with retinal diseases are potential candidates to explain equivalent human diseases and contribute to the understanding of their function in vision.     

Role of mitochondrial DNA in diabetes Mellitus Type I and Type II

Morbidity and mortality from diabetes mellitus and associated illnesses is a major problem across the globe. Anti-diabetic medicines must be improved despite existing breakthroughs in treatment approaches. Diabetes has been linked to mitochondrial dysfunction. As a result, particular mitochondrial diabetes kinds like MIDD (maternally inherited diabetes & deafness) and DAD (diabetic autonomic dysfunction) have been identified and studied (diabetes and Deafness). Some mutations as in mitochondrial DNA (mtDNA), that encodes for a significant portion of mitochondrial proteins as well as mitochondrial tRNA essential for mitochondrial protein biosynthesis, are responsible for hereditary mitochondrial diseases. Tissue-specificity and heteroplasmy have a role in the harmful phenotype of mtDNA mutations, making it difficult to generalise findings from one study to another. There are a huge increase in the number for mtDNA mutations related with human illnesses that have been identified using current sequencing technologies. In this study, we make a list on mtDNA mutations linked with diseases and diabetic illnesses and explore the methods by which they contribute to the pathology's emergence.     

Molecular genetic analysis of neurologic diseases]

With the recent progress in molecular genetics, our understanding of neurologic diseases on molecular basis has improved tremendously. Molecular analyses of gene mutations in hereditary neurologic diseases bring us not only the identification of mutations but also better understanding of molecular mechanisms involved in the neurologic diseases. We have identified a missense mutation (444Leu----Pro) in glucocerebrosidase gene of a type 2 Gaucher patient. The mutant glucocerebrosidase carrying 444Pro is unstable and degraded rapidly before the mutant protein is targetted to lysosomes. Detailed analysis on the 444Leu----Pro mutations among three phenotypes (types 1, 2 and 3) of Gaucher disease, it has been demonstrated that patients homozygous for the mutation frequently have the neurologic involvement (types 1 and 2). Very recently we have identified a mutation in tRNA(Lys) gene of mitochondrial genome of patients with myoclonus epilepsy associated with ragged-red fibers. The pathophysiologic mechanism with the tRNA(Lys) mutation is considered to be based on the impaired function of tRNA(Lys).     

Mitochondrial DNA mutations and human disease

Mitochondrial disorders are a group of clinically heterogeneous diseases, commonly defined by a lack of cellular energy due to oxidative phosphorylation (OXPHOS) defects. Since the identification of the first human pathological mitochondrial DNA (mtDNA) mutations in 1988, significant efforts have been spent in cataloguing the vast array of causative genetic defects of these disorders. Currently, more than 250 pathogenic mtDNA mutations have been identified. An ever-increasing number of nuclear DNA mutations are also being reported as the majority of proteins involved in mitochondrial metabolism and maintenance are nuclear-encoded. Understanding the phenotypic diversity and elucidating the molecular mechanisms at the basis of these diseases has however proved challenging. Progress has been hampered by the peculiar features of mitochondrial genetics, an inability to manipulate the mitochondrial genome, and difficulties in obtaining suitable models of disease. In this review, we will first outline the unique features of mitochondrial genetics before detailing the diseases and their genetic causes, focusing specifically on primary mtDNA genetic defects. The functional consequences of mtDNA mutations that have been characterised to date will also be discussed, along with current and potential future diagnostic and therapeutic advances.     

The human mitochondrial transport/carrier protein family. Nonsynonymous single nucleotide polymorphisms (nsSNPs) and mutations that lead to human diseases

There are 67 proteins in the human mitochondrial transport protein family. They have been identified from among the proteins of the RefSeq database on the basis of sequence similarity to proteins that have been functionally identified as mitochondrial transport proteins. They have also been identified by matching their predicted structure to the high resolution structure of the bovine ADP/ATP T1 transporter subunit/carboxyatractyloside complex. 74 nonsynonymous single nucleotide polymorphisms (nsSNP) have been identified in their gene sequences. These nsSNPs are present in genes of 30 of the proteins. No nsSNP has been found in 24 of the protein genes and no search has as yet been carried out on the rest (13) of them. The largest number of nsSNPs are in the ADP/ATP T3 transporter, the uncoupling protein 3 L, and the phosphate transporter genes with 7, 6, and 6, respectively. nsSNPs are located in groups along the protein sequence suggesting that certain protein domains are too critical for transport function to tolerate mutations. This interpretation has been validated with mutation and function studies of the phosphate transporter. Human diseases have been identified with replacement mutations in seven of these proteins. Their genes are not abnormally susceptible to mutations since they have the smallest number of nsSNPs. Disease causing mutations have also been observed as: substitution, silent (may affect stability of messages), frameshift (protein truncation or elongation), splicing (exon skipping), residue deletion. Disease causing mutations have only been identified in few transporter genes because others do not yield dramatic symptoms or are essential and thus lethal. Mutations in other transporter genes may also only have a major impact through their combination with other genes and their nsSNPs.     

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.     

Contribution of rare and common variants determine complex diseases—Hirschsprung disease as a model

Finding genes for complex diseases has been the goal of many genetic studies. Most of these studies have been successful by searching for genes and mutations in rare familial cases, by screening candidate genes and by performing genome wide association studies. However, only a small fraction of the total genetic risk for these complex genetic diseases can be explained by the identified mutations and associated genetic loci. In this review we focus on Hirschsprung disease (HSCR) as an example of a complex genetic disorder. We describe the genes identified in this congenital malformation and postulate that both common ‘low penetrant’ variants in combination with rare or private ‘high penetrant’ variants determine the risk on HSCR, and likely, on other complex diseases. We also discuss how new technological advances can be used to gain further insights in the genetic background of complex diseases. Finally, we outline a few steps to develop functional assays in order to determine the involvement of these variants in disease development.     

Connexin gene mutations in human genetic diseases

Rapid advances in understanding the molecular biology of the gap junctional proteins - connexins (Cx) - have revealed that these proteins are indispensable for various cellular functions. Recent findings that mutational alterations of Cx genes leads to several quite different human diseases provide additional evidence that these proteins possess several not yet fully understood functions. Many different mutations of Cx32 have been found in the hereditary peripheral neuropathy - X-linked Charcot-Marie-Tooth syndrome and several mutations of Cx26 and Cx31 have been detected in deafness. Individual mutations of Cx46, Cx50 and Cx43 have been found in cataract or heart malformations. In this review, we analyzed the functional importance of mutations of different Cx described in different human diseases. Topological comparison of mutations in different Cx species has revealed several hot spots, where mutations are common for two different Cx or diseases. The value of Cx mutations associated with diseases for understanding Cx functions is discussed.     

Genetics and aetiology of Pagetic disorders of bone

Paget’s disease of bone (PDB) is a late-onset disorder characterised by focal areas of increased bone turnover containing enlarged hyperactive osteoclasts. The disease has a strong genetic predisposition and mutations in SQSTM1 have been associated with familial and sporadic disease in up to 40% of cases. Additional genetic loci have been associated in other cases, but genes are yet to be identified. Earlier-onset conditions with similar bone pathology (familial expansile osteolysis, expansile skeletal hyperphosphatasia and early-onset PDB) are caused by mutations in TNFRSF11A (RANK). The syndrome of inclusion body myositis, Paget’s disease and frontotemporal dementia is caused by mutations in VCP. Despite the increased knowledge about genes involved in PDB and related disorders, the etiology of the diseases remains puzzling. Presence of inclusion bodies appears to link Pagetic diseases mechanistically to diseases associated with presence of misfolded proteins or abnormalities in the ubiquitin-proteasomal, or autophagy pathways. Juvenile PDB, caused by osteoprotegerin deficiency, appears mechanistically distinct from the other Pagetic diseases. This review will discuss evidence from recent studies, including new animal models for Pagetic diseases.     

Readthrough compounds for nonsense mutations: bridging the translational gap

Approximately 10% of all pathological mutations are nonsense mutations that are responsible for several severe genetic diseases for which no treatment regimens are currently available. The most widespread strategy for treating nonsense mutations is by enhancing ribosomal readthrough of premature termination codons (PTCs) to restore the production of the full-length protein. In the past decade several compounds with readthrough potential have been identified. However, although preclinical results on these compounds are promising, clinical studies have not yielded positive outcomes. We review preclinical and clinical research related to readthrough compounds and characterize factors that contribute to the observed translational gap.     

Suppression of premature termination codons as a therapeutic approach

In this review, we describe our current understanding of translation termination and pharmacological agents that influence the accuracy of this process. A number of drugs have been identified that induce suppression of translation termination at in-frame premature termination codons (PTCs; also known as nonsense mutations) in mammalian cells. We discuss efforts to utilize these drugs to suppress disease-causing PTCs that result in the loss of protein expression and function. In-frame PTCs represent a genotypic subset of mutations that make up ~11% of all known mutations that cause genetic diseases, and millions of patients have diseases attributable to PTCs. Current approaches aimed at reducing the efficiency of translation termination at PTCs (referred to as PTC suppression therapy) have the goal of alleviating the phenotypic consequences of a wide range of genetic diseases. Suppression therapy is currently in clinical trials for treatment of several genetic diseases caused by PTCs, and preliminary results suggest that some patients have shown clinical improvements. While current progress is promising, we discuss various approaches that may further enhance the efficiency of this novel therapeutic approach.     

Suppression of premature termination codons as a therapeutic approach

In this review, we describe our current understanding of translation termination and pharmacological agents that influence the accuracy of this process. A number of drugs have been identified that induce suppression of translation termination at in-frame premature termination codons (PTCs; also known as nonsense mutations) in mammalian cells. We discuss efforts to utilize these drugs to suppress disease-causing PTCs that result in the loss of protein expression and function. In-frame PTCs represent a genotypic subset of mutations that make up ~11% of all known mutations that cause genetic diseases, and millions of patients have diseases attributable to PTCs. Current approaches aimed at reducing the efficiency of translation termination at PTCs (referred to as PTC suppression therapy) have the goal of alleviating the phenotypic consequences of a wide range of genetic diseases. Suppression therapy is currently in clinical trials for treatment of several genetic diseases caused by PTCs, and preliminary results suggest that some patients have shown clinical improvements. While current progress is promising, we discuss various approaches that may further enhance the efficiency of this novel therapeutic approach.     

β‐Tubulin mutations that cause severe neuropathies disrupt axonal transport

Microtubules are fundamental to neuronal morphogenesis and function. Mutations in tubulin, the major constituent of microtubules, result in neuronal diseases. Here, we have analysed β‐tubulin mutations that cause neuronal diseases and we have identified mutations that strongly inhibit axonal transport of vesicles and mitochondria. These mutations are in the H12 helix of β‐tubulin and change the negative charge on the surface of the microtubule. This surface is the interface between microtubules and kinesin superfamily motor proteins (KIF). The binding of axonal transport KIFs to microtubules is dominant negatively disrupted by these mutations, which alters the localization of KIFs in neurons and inhibits axon elongation in vivo. In humans, these mutations induce broad neurological symptoms, such as loss of axons in the central nervous system and peripheral neuropathy. Thus, our data identified the critical region of β‐tubulin required for axonal transport and suggest a molecular mechanism for human neuronal diseases caused by tubulin mutations.     

How to assess the pathogenicity of mutations in Charcot-Marie-Tooth disease and other diseases?

Knowledge whether a certain DNA variant is a pathogenic mutation or a harmless polymorphism is a critical issue in medical genetics, in which results of a molecular analysis may serve as a basis for diagnosis and genetic counseling. Due to its genetic heterogeneity expressed at the levels of loci, genes and mutations, Charcot-Marie-Tooth (CMT) disease can serve as a model group of clinically homogenous diseases for studying the pathogenicity of mutations. Close to a 17p11.2-p12 duplication occurring in 70% of patients with the demyelinating form of CMT disease, numerous mutations have been identified in poorly characterized genes coding for proteins of an unknown function. Functional analyses, segregation analyses of large pedigrees, and inclusion of large control groups are required to assess the potential pathogenicity of CMT mutations. Hence, the pathogenicity of numerous CMT mutations remains unclear. Some variants detected in the CMT genes and originally described as pathogenic mutations have been shown to have a polymorphic character. In contrast, polymorphisms initially considered harmless were later reclassified as pathogenic mutations. However, the process of assessing the pathogenicity of mutations, as presented in this study for CMT disorders, is a more general issue concerning all disorders with a genetic background. Since the number of DNA variants is still growing, in the near future geneticists will increasingly have to cope with the problem of pathogenicity of identified genetic variants.     

Mutations causing neurodegenerative tauopathies

Tau is the major component of the intracellular filamentous deposits that define a number of neurodegenerative diseases. They include the largely sporadic Alzheimer's disease (AD), progressive supranuclear palsy, corticobasal degeneration, Pick's disease and argyrophilic grain disease, as well as the inherited frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). For a long time, it was unclear whether the dysfunction of tau protein follows disease or whether disease follows tau dysfunction. This was resolved when mutations in Tau were found to cause FTDP-17. Currently, 32 different mutations have been identified in over 100 families. About half of the known mutations have their primary effect at the protein level. They reduce the ability of tau protein to interact with microtubules and increase its propensity to assemble into abnormal filaments. The other mutations have their primary effect at the RNA level and perturb the normal ratio of three-repeat to four-repeat tau isoforms. Where studied, this resulted in a relative overproduction of tau protein with four microtubule-binding domains in the brain. Individual Tau mutations give rise to diseases that resemble progressive supranuclear palsy, corticobasal degeneration or Pick's disease. Moreover, the H1 haplotype of Tau has been identified as a significant risk factor for progressive supranuclear palsy and corticobasal degeneration. At a practical level, the new work is leading to the production of experimental animal models that reproduce the essential molecular and cellular features of the human tauopathies, including the formation of abundant filaments made of hyperphosphorylated tau protein and nerve cell degeneration.