The role of cytochemistry in human genetic research

Summary The role of cytochemistry in human genetics is reviewed. In basic research, autoradiography and cytochemical staining procedures for DNA, RNA, proteins and other cell constituants have contributed to the understanding of the way DNA is localized, duplicated and translated. The development of new banding techniques for the identification of human chromosomes and parts of these together with somatic cell hybridization procedures have significantly contributed to the mapping of the human genome.Cytochemical methods have also been of great help in the elucidation of the responsible molecular defects in Mendelian disorders based on a single gene mutation. The combination of immunological methods and electron-microscopical cytochemistry now enables different posttranslational processing defects to be related to various subcellular compartments. Cytochemistry is also likely to be of importance for the direct demonstration of gene mutations using recombinant DNA technology.Examples are given of contributions of cytochemical methods to the early diagnosis and prevention of congenital disorders. The main examples are the early diagnosis of patients with a chromosomal aberration and of carriers with a balanced translocation. Early genetic counseling of couples at risk forms the basis for prevention of subsequent affected children. Cytochemistry also contributes to the early detection of heterozygotes of X-linked mutations. Finally, autoradiography and ultramicrochemical procedures have been of great help in improving the prenatal diagnosis of genetic metabolic diseases.In honour of Prof. P. van Duijn     

Mapping Approaches to Gene Identification in Humans

Although a number of human genes that cause disease have been traced through the defective product, most genetic defects are recognized only by phenotype. When the biochemical defect is unknown, a gene can be located only through molecular approaches based on coinheritance (genetic linkage) of the disease phenotype with a particular allele of a polymorphic DNA marker that has already been mapped to a specific chromosomal region. Linkage studies in affected families have already localized genes for several important diseases, including cystic fibrosis. Finding a genetic linkage in families in which a disease segregates requires that the human genetic map have a large number of polymorphic markers; when the map is dense enough, any disease gene can be located by linkage to a known marker. Many DNA segments with a high degree of polymorphism are being found and mapped as markers in normal reference pedigrees. Genetic linkage mapping has implications even broader than its application to prenatal diagnosis or therapeutic strategy; analyzing mutations in important genes will illuminate basic mechanisms in molecular biology and the early events that lead to cancer and other disorders.     

Mitochondrial Diseases: Therapeutic Approaches

Therapy of mitochondrial encephalomyopathies (defined restrictively as defects of the mitochondrial respiratory chain) is woefully inadequate, despite great progress in our understanding of the molecular bases of these disorders. In this review, we consider sequentially several different therapeutic approaches. Palliative therapy is dictated by good medical practice and includes anticonvulsant medication, control of endocrine dysfunction, and surgical procedures. Removal of noxious metabolites is centered on combating lactic acidosis, but extends to other metabolites. Attempts to bypass blocks in the respiratory chain by administration of electron acceptors have not been successful, but this may be amenable to genetic engineering. Administration of metabolites and cofactors is the mainstay of real-life therapy and is especially important in disorders due to primary deficiencies of specific compounds, such as carnitine or coenzyme Q10. There is increasing interest in the administration of reactive oxygen species scavengers both in primary mitochondrial diseases and in neurodegenerative diseases directly or indirectly related to mitochondrial dysfunction. Aerobic exercise and physical therapy prevent or correct deconditioning and improve exercise tolerance in patients with mitochondrial myopathies due to mitochondrial DNA (mtDNA) mutations. Gene therapy is a challenge because of polyplasmy and heteroplasmy, but interesting experimental approaches are being pursued and include, for example, decreasing the ratio of mutant to wild-type mitochondrial genomes (gene shifting), converting mutated mtDNA genes into normal nuclear DNA genes (allotopic expression), importing cognate genes from other species, or correcting mtDNA mutations with specific restriction endonucleases. Germline therapy raises ethical problems but is being considered for prevention of maternal transmission of mtDNA mutations. Preventive therapy through genetic counseling and prenatal diagnosis is becoming increasingly important for nuclear DNA-related disorders. Progress in each of these approaches provides some glimmer of hope for the future, although much work remains to be done.     

Molecular analysis for mitochondrial DNA disorders

In this article, we review the current methodologies used for the molecular diagnosis of mitochondrial DNA defects. Definition of mitochondrial disorders at the molecular level has been difficult because of both clinical and genetic heterogeneity. Direct DNA analysis for common point mutations and large mtDNA deletions is readily performed and can be done routinely. However, a large number of patients who have the clinical manifestations and muscle pathology findings consistent with mitochondrial DNA disorders do not have detectable common mutations. Additional mutation screening methods are required for the detection of rare and previously undescribed mutations in the mitochondrial genome.     

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.     

DNA single-strand breaks and neurodegeneration

The association of human genetic disorders with defects in the DNA damage response is well established. Most of the major DNA repair pathways are represented by diseases in which that pathway is absent or impaired, including those responsible for repairing DNA double-strand breaks. Conspicuous by their absence, however, have been human disorders associated with defects in the repair or response to DNA single-strand breaks (SSBs). However, three papers have recently associated hereditary spinocerebellar ataxia with mutations in genes connected with SSBR. The emerging links between SSBR and neurodegeneration are discussed.     

Diagnostic challenges of mitochondrial DNA disorders

Although mitochondrial disorders are increasingly being recognized, confirming a specific diagnosis remains a great challenge due to the genetic and clinical heterogeneity of the disease. The heteroplasmic nature of most pathogenic mitochondrial DNA mutations and the uncertainties of the clinical significance of novel mutations pose additional complications in making a diagnosis. Suspicion of mitochondrial disease among patients with multiple, seemingly unrelated neuromuscular and multi-system disorders should ideally be confirmed by the finding of deleterious mutations in genes involving mitochondrial biogenesis and functions. The genetics are complex, as the primary mutation can be either in the nuclear or the mitochondrial DNA (mtDNA). MtDNA mutations are often maternally inherited, but can also be sporadic or secondary to mutations in nuclear-encoded mitochondrial-targeted genes. Several well-defined clinical syndromes associated with specific mutations have been described, yet the genotype-phenotype correlation is often unclear and most patients do not fit within any defined syndrome and even within a family the expressivity of the disease can be extremely variable. This article describes examples representing diagnostic challenges of mitochondrial diseases that include the limitations of the mutation detection method, the occurrence of mitochondrial disease in families with another known neuromuscular disorder, atypical clinical presentation, the lack of correlation between the degree of mutant heteroplasmy and the severity of the disease, variable penetrance, and nuclear gene defects causing mtDNA depletion.     

Human genetic disorders, a phylogenetic perspective

When viewed from the perspective of time, human genetic disorders give new insights into their etiology and evolution. Here, we have correlated a specific set of Alu repetitive DNA elements, known to be the basis of certain genetic defects, with their phylogenetic roots in primate evolution. From a differential distribution of Alu repeats among primate species, we identify the phylogenetic roots of three human genetic diseases involving the LPL, ApoB, and HPRT genes. The different phylogenetic age of these genetic disorders could explain the different susceptibility of various primate species to genetic diseases. Our results show that LPL deficiency is the oldest and should affect humans, apes, and monkeys. ApoB deficiency should affect humans and great apes, while a disorder in the HPRT gene (leading to the Lesch-Nyhan syndrome) is unique to human, chimpanzee, and gorilla. Similar results can be obtained for cancer. We submit that de novo transpositions of Alu elements, and saltatory appearances of Alu-mediated genetic disorders, represent singularities, places where behavior changes suddenly. Alus' propensity to spread, not only increased the regulatory and developmental complexity of the primate genome, it also increased its instability and susceptibility to genetic defects and cancer. The dynamic spread not only provided markers of primate phylogeny, it must have actively shaped the course of that phylogeny.     

Epigenetic mechanisms in neurological disease

The exploration of brain epigenomes, which consist of various types of DNA methylation and covalent histone modifications, is providing new and unprecedented insights into the mechanisms of neural development, neurological disease and aging. Traditionally, chromatin defects in the brain were considered static lesions of early development that occurred in the context of rare genetic syndromes, but it is now clear that mutations and maladaptations of the epigenetic machinery cover a much wider continuum that includes adult-onset neurodegenerative disease. Here, we describe how recent advances in neuroepigenetics have contributed to an improved mechanistic understanding of developmental and degenerative brain disorders, and we discuss how they could influence the development of future therapies for these conditions.     

Mouse models of abnormal skeletal development and homeostasis

Studies of a number of mouse mutations with skeletal defects have contributed significantly to the understanding of bone development and homeostasis. In many cases, such mutants are also genetic models of disorders in humans, characterized by reduced bone mass (osteoporosis), increased bone mass (osteopetrosis), or abnormalities in endochondral ossification (chondrodysplasias).     

Prenatal diagnosis--principles of diagnostic procedures and genetic counseling

The frequency of inherited malformations as well as genetic disorders in newborns account for around 3-5%. These frequency is much higher in early stages of pregnancy, because serious malformations and genetic disorders usually lead to spontaneous abortion. Prenatal diagnosis allowed identification of malformations and/or some genetic syndromes in fetuses during the first trimester of pregnancy. Thereafter, taking into account the severity of the disorders the decision should be taken in regard of subsequent course of the pregnancy taking into account a possibilities of treatment, parent's acceptation of a handicapped child but also, in some cases the possibility of termination of the pregnancy. In prenatal testing, both screening and diagnostic procedures are included. Screening procedures such as first and second trimester biochemical and/or ultrasound screening, first trimester combined ultrasound/biochemical screening and integrated screening should be widely offered to pregnant women. However, interpretation of screening results requires awareness of both sensitivity and predictive value of these procedures. In prenatal diagnosis ultrasound/MRI searching as well as genetic procedures are offered to pregnant women. A variety of approaches for genetic prenatal analyses are now available, including preimplantation diagnosis, chorion villi sampling, amniocentesis, fetal blood sampling as well as promising experimental procedures (e.g. fetal cell and DNA isolation from maternal blood). An incredible progress in genetic methods opened new possibilities for valuable genetic diagnosis. Although karyotyping is widely accepted as golden standard, the discussion is ongoing throughout Europe concerning shifting to new genetic techniques which allow obtaining rapid results in prenatal diagnosis of aneuploidy (e.g. RAPID-FISH, MLPA, quantitative PCR).     

Individualized medicine 2010

Molecular and cell biology have revolutionized not only diagnosis, therapy and prevention of human diseases but also greatly contributed to the understanding of their pathogenesis. Based on modern molecular and biochemical methods it is possible to identify on the one hand point mutations and single nucleotide polymorphisms. On the other hand, using high throughput array technologies, it is possible to analyse thousands of genes or gene products simultaneously, resulting in an individual gene or gene expression profile (signature). These data increasingly allow to define the individual risk for a given disease and to predict the individual prognosis of a disease as well as the efficacy of therapeutic strategies (individualized medicine). In the following sections some of the recent advances of predictive medicine and their clinical relevance will be addressed.     

The inheritance of pathogenic mitochondrial DNA mutations

Mitochondrial DNA mutations cause disease in > 1 in 5000 of the population, and ～ 1 in 200 of the population are asymptomatic carriers of a pathogenic mtDNA mutation. Many patients with these pathogenic mtDNA mutations present with a progressive, disabling neurological syndrome that leads to major disability and premature death. There is currently no effective treatment for mitochondrial disorders, placing great emphasis on preventing the transmission of these diseases. An empiric approach can be used to guide genetic counseling for common mtDNA mutations, but many families transmit rare or unique molecular defects. There is therefore a pressing need to develop techniques to prevent transmission based on a solid understanding of the biological mechanisms. Several recent studies have cast new light on the genetics and cell biology of mtDNA inheritance, but these studies have also raised new controversies. Here we compare and contrast these findings and discuss their relevance for the transmission of human mtDNA diseases.     

Biochemical and molecular diagnosis of mitochondrial respiratory chain disorders

Biochemical diagnosis of mitochondrial respiratory chain disorders requires caution to avoid misdiagnosis of secondary enzyme defects, and can be improved by the use of conservative diagnostic criteria. Pathogenic mutations causing mitochondrial disorders have now been identified in more than 30 mitochondrial DNA (mtDNA) genes encoding respiratory chain subunits, ribosomal- and t-RNAs. mtDNA mutations appear to be responsible for most adult patients with mitochondrial disease and approximately a quarter of paediatric patients. A family history suggesting maternal inheritance is the exception rather than the norm for children with mtDNA mutations, many of whom have de novo mutations. Prenatal diagnosis and pre-implantation genetic diagnosis can be offered to some women at risk of transmitting a mtDNA mutation, particularly those at lower recurrence risk. Mutations in more than 30 nuclear genes, including those encoding for respiratory chain subunits and assembly factors, have now been shown to cause mitochondrial disorders, creating difficulties in prioritising which genes should be studied by mutation analysis in individual patients. A number of approaches offer promise to guide the choice of candidate genes, including Blue Native-PAGE immunoblotting and microarray expression analysis.     

Human diseases with impaired mitochondrial protein synthesis

Mitochondrial respiratory chain deficiencies represent one of the major causes of metabolic disorders that are related to genetic defects in mitochondrial or nuclear DNA. The mitochondrial protein synthesis allows the synthesis of the 13 respiratory chain subunits encoded by mtDNA. Altogether, about 100 different proteins are involved in the translation of the 13 proteins encoded by the mitochondrial genome emphasizing the considerable investment required to maintain mitochondrial genetic system. Mitochondrial protein synthesis deficiency can be caused by mutations in any component of the translation apparatus including tRNA, rRNA and proteins. Mutations in mitochondrial rRNA and tRNAs have been first identified in various forms of mitochondrial disorders. Moreover abnormal translation due to mutation in nuclear genes encoding tRNA-modifying enzymes, ribosomal proteins, aminoacyl-tRNA synthetases, elongation and termination factors and translational activators have been successively described. These deficiencies are characterized by a huge clinical and genetic heterogeneity hampering to establish genotype-phenotype correlations and an easy diagnosis. One can hypothesize that a new technique for gene identification, such as exome sequencing will rapidly allow to expand the list of genes involved in abnormal mitochondrial protein synthesis.     

DNA-Reparaturdefekte und Krebs

A peculiarity of the lymphatic system is its high rate of somatic recombination with associated hypermutability. In this process, DNA double-strand breaks are generated and processed, whereby the genes responsible are often also involved in general double-strand break repair. Germline mutations in these genes are responsible for a particularly high risk for lymphoma. The study of such genetic disorders and the characteristic somatic mutations in lymphoma have led to major contributions to our understanding of DNA repair defects and carcinogenesis in general.     

Analysis of the molecular pathophysiology of sleep disorders relevant to a disturbed biological clock

Genetic studies have revealed several clock gene variations/mutations involved in the manifestation of sleep disorders or interindividual differences in sleep–wake patterns, but only part of the genetic risk can be explained by the gene variations/mutations identified to date. Recent progress in research into circadian rhythm generation has provided efficient tools for eliciting the molecular basis of clock-relevant sleep disorders, complementing traditional genetic analysis. While the human master clock resides in the suprachiasmatic nucleus of the hypothalamus (central clock), peripheral tissue cells also generate self-sustained circadian oscillations of clock gene expression (peripheral clock), enabling estimation of individual human clock properties through a single collection of skin fibroblasts or venous blood cells. Some of the established cell lines exhibit autonomous circadian oscillations of clock gene expression, and introduction of clock gene variations into these cell lines by gene targeting makes it possible to investigate changes in the circadian phenotype induced by these variations/mutations without the need for generating transgenic animals. Estimation of human clock properties using peripheral tissue cells, in addition to genetic analysis, will facilitate comprehensive explication of the genetic risk of a variety of disorders relevant to biological clock disturbances, including sleep disorders, mood disorders, and metabolic diseases.     

A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells

Autism spectrum disorders (ASD) are complex neurodevelopmental diseases in which different combinations of genetic mutations may contribute to the phenotype. Using Rett syndrome (RTT) as an ASD genetic model, we developed a culture system using induced pluripotent stem cells (iPSCs) from RTT patients' fibroblasts. RTT patients' iPSCs are able to undergo X-inactivation and generate functional neurons. Neurons derived from RTT-iPSCs had fewer synapses, reduced spine density, smaller soma size, altered calcium signaling and electrophysiological defects when compared to controls. Our data uncovered early alterations in developing human RTT neurons. Finally, we used RTT neurons to test the effects of drugs in rescuing synaptic defects. Our data provide evidence of an unexplored developmental window, before disease onset, in RTT syndrome where potential therapies could be successfully employed. Our model recapitulates early stages of a human neurodevelopmental disease and represents a promising cellular tool for drug screening, diagnosis and personalized treatment.     

Molecular diagnosis of Duchenne/Becker muscular dystrophy by polymerase chain reaction and microsatellite analysis

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive genetic disorders resulting from mutations in the dystrophin gene. About two-thirds of the affected patients have large deletions or duplications, which occur in the 5' and central region of the gene. The remaining DMD/BMD cases show no deletions, so they cannot be easily identified by current strategies. In these DMD/BMD families, a linkage analysis that involves DNA markers of the flanking and intragenic dystrophin gene are necessary for carrier and prenatal diagnosis. We analyzed eighteen deletion-prone exons of the gene by a polymerase chain reaction (PCR) in order to characterize the molecular defects of the dystrophin gene in Korean DMD/BMD families. We also performed a linkage analysis to assess the usefulness and application of six short tandem repeat markers for molecular diagnosis in the families. We observed a deletion that eliminated the exon 50. Also, a linkage analysis in the families with six short tandem repeat (STR) markers showed heterozygosity at most of the STR markers. The haplotype analysis was useful for detecting the carrier status. This study will be helpful for a molecular diagnosis of DMD/BMD families in the Korean population.     

Molecular biology of disorders of sex differentiation.

Sexual ambiguity can be a difficult and sometimes confusing diagnostic problem in children. Recent developments in molecular biology have provided the opportunity to analyze the gene responsible for testicular determination, SRY, the androgen receptor gene and the gene encoding the cP450 enzyme specific for 21-hydroxylation, CYP21B, whose defects are responsible for congenital adrenal hyperplasia. Southern-blotting studies and PCR analyses of SRY, androgen receptor and CYP21B genes can be routinely used for the direct diagnosis of gonadal dysgenesis, androgen insensitivity syndromes and congenital adrenal hyperplasia, respectively. In sex-reversed XY females, several de novo mutations or deletions in the SRY gene have been reported. Defects in the human androgen receptor cause a spectrum of defects in male phenotypic sexual development associated with abnormalities in the receptor protein. Analyses of the androgen receptor gene structure have identified the causative mutation in some families: mutations that result in large-scale alterations of the structure of the androgen receptor, mRNA or gene mutations that alter the primary structure of the androgen receptor protein and mutations that alter the level of mRNA. The diversity of clinical phenotypes, apparent in 21-hydroxylase deficiency, is paralleled by a considerable degree of mutational heterogeneity in the CYP21 gene locus. Various changes causing severe 21-hydroxylase deficiency have been reported: point mutations, gene conversions and gene deletions. In conclusion, substantial progress has been made elucidating genetic defects causing sex reversal in XY females, the androgen insensitivity syndrome and congenital adrenal hyperplasia. Molecular genetics can also be applied for carrier identification and prenatal diagnosis.