Genome analysis techniques and their applications in elucidation of molecular underpinnings of rare genetic diseases.

Vzácná onemocnění představují heterogenní skupinu více než ~7000 různých onemocnění, která postihují 3,5-5,9 % celosvětové populace. Většina vzácných onemocnění je genetických, ale kauzální geny jsou známy jen u některých z nich. Řada pacientů se vzácným onemocněním zůstává bez diagnózy, která je klíčová pro genetické poradenství, prevenci a léčbu. S rozvojem nových metod analýzy genomu, klesající cenou sekvenování a rostoucími znalostmi o lidském genomu byl nastolen nový koncept identifikace onemocnění podmiňujících genů, založený na porovnávání genetické variability pacienta s genetickou variabilitou běžné populace. Tato disertační práce popisuje nové metody sekvenace genomu (NGS), bioinformatickou analýzu získaných dat a jejich využití ve studiu molekulární podstaty vzácných, geneticky podmíněných onemocnění. Tyto postupy vedly k určení a charakterizaci kauzálních genů a genových mutací u autosomálně dominantního tubulointersticiálního onemocnění ledvin (SEC61A1, MUC1), autosomálně dominantní neuronální ceroidní lipofuscinózy (CLN6, DNAJC5), neurodegenerativního onemocnění neznámé etiologie (VPS15), Akadské varianty Fanconiho syndromu (NDUFAF6) a spinální svalové atrofie (SMN1). Zavedením nových metod analýzy genomu se zvýšila diagnostická výtěžnost vzácných onemocnění z původního 1 % na 50 %....Rare diseases represent a heterogeneous group of more than ~7000 different diseases, affecting 3,5-5,9% of the global population. Most rare diseases are genetic, but causal genes are known only in some of them. Many patients with rare diseases remain without a diagnosis, which is crucial for genetic counseling, prevention, and treatment. With the development of new methods of genome analysis, decreasing cost of sequencing, and increasing knowledge of the human genome, a new concept for identifying disease-causing genes was established. It is based on comparing the patient's genetic variability with the genetic variability of the general population. This dissertation describes next-generation sequencing technologies (NGS), bioinformatic analysis of acquired data and their applications in the elucidation of molecular underpinnings of rare genetic diseases. These procedures have led to the identification and characterization of causal genes and gene mutations in autosomal dominant tubulointerstitial kidney disease (SEC61A1, MUC1), autosomal dominant neuronal ceroid lipofuscinosis (CLN6, DNAJC5), neurodegenerative disease of unknown etiology (VPS15), Acadian variant of Fanconi syndrome (NDUFAF6) and spinal muscular atrophy (SMN1). The application of novel genome analysis techniques increased the...Institute of Inherited Metabolic Disorders First Faculty of Medicine Charles University in PragueÚstav dědičných metabolických poruch 1.LF a VFN v Praze1. lékařská fakultaFirst Faculty of Medicin

Heterozygous Loss-of-Function SEC61A1 Mutations Cause Autosomal-Dominant Tubulo-Interstitial and Glomerulocystic Kidney Disease with Anemia

Autosomal-dominant tubulo-interstitial kidney disease (ADTKD) encompasses a group of disorders characterized by renal tubular and interstitial abnormalities, leading to slow progressive loss of kidney function requiring dialysis and kidney transplantation. Mutations in UMOD, MUC1, and REN are responsible for many, but not all, cases of ADTKD. We report on two families with ADTKD and congenital anemia accompanied by either intrauterine growth retardation or neutropenia. Ultrasound and kidney biopsy revealed small dysplastic kidneys with cysts and tubular atrophy with secondary glomerular sclerosis, respectively. Exclusion of known ADTKD genes coupled with linkage analysis, whole-exome sequencing, and targeted re-sequencing identified heterozygous missense variants in SEC61A1—c.553A>G (p.Thr185Ala) and c.200T>G (p.Val67Gly)—both affecting functionally important and conserved residues in SEC61. Both transiently expressed SEC6A1A variants are delocalized to the Golgi, a finding confirmed in a renal biopsy from an affected individual. Suppression or CRISPR-mediated deletions of sec61al2 in zebrafish embryos induced convolution defects of the pronephric tubules but not the pronephric ducts, consistent with the tubular atrophy observed in the affected individuals. Human mRNA encoding either of the two pathogenic alleles failed to rescue this phenotype as opposed to a complete rescue by human wild-type mRNA. Taken together, these findings provide a mechanism by which mutations in SEC61A1 lead to an autosomal-dominant syndromic form of progressive chronic kidney disease. We highlight protein translocation defects across the endoplasmic reticulum membrane, the principal role of the SEC61 complex, as a contributory pathogenic mechanism for ADTKD

Genome analysis techniques and their applications in elucidation of molecular underpinnings of rare genetic diseases.

Rare diseases represent a heterogeneous group of more than ~7000 different diseases, affecting 3,5-5,9% of the global population. Most rare diseases are genetic, but causal genes are known only in some of them. Many patients with rare diseases remain without a diagnosis, which is crucial for genetic counseling, prevention, and treatment. With the development of new methods of genome analysis, decreasing cost of sequencing, and increasing knowledge of the human genome, a new concept for identifying disease-causing genes was established. It is based on comparing the patient's genetic variability with the genetic variability of the general population. This dissertation describes next-generation sequencing technologies (NGS), bioinformatic analysis of acquired data and their applications in the elucidation of molecular underpinnings of rare genetic diseases. These procedures have led to the identification and characterization of causal genes and gene mutations in autosomal dominant tubulointerstitial kidney disease (SEC61A1, MUC1), autosomal dominant neuronal ceroid lipofuscinosis (CLN6, DNAJC5), neurodegenerative disease of unknown etiology (VPS15), Acadian variant of Fanconi syndrome (NDUFAF6) and spinal muscular atrophy (SMN1). The application of novel genome analysis techniques increased the..

Application of novel DNA sequencing techniques in biomedical research

Next generation sequencing technologies are changing the way scientific experiments and diseases diagnostics are performed and thus will allow what is called personalized medicine. The sense of presented thesis is to make survey of new approaches to DNA sequencing and demonstrate usage and constraints of bioinformatic analytical tools available to day. Discussed techniques are then applied to the case study of finding molecular basis for rare hereditary disease. Introductory part deals with overview of commercially available sequencing techniques (454 Life Science, Applied Biosystems, Illumina, Helicos). Fundamentals of each method are described and possible further development is outlined. Post sequencing data analysis is than discussed in details. In practical section we demonstrate genome analysis techniques successfully used to reveal causal mutation in the gene responsible for adult form of autozomal neuronal ceroid lipofuscinosis (ANCL). Combination of linkage analysis (Merlin), copy number variant analysis (Genome-Wide Human SNP Array 6.0), analysis of expression profiles (HumanRef-8 v2 Expression BeadChips) and exome sequencing (SOLiD™ 4 System) has been applied to members of one ANCL family. We also paid attention to comparison, evaluation and selection of available mapping algorithms used in..

Next Generation Sequencing Technologies: Applications in Human Genome Research and Potential Impact on the Field of Molecular Medicine

Ústav biofyziky a informatiky 1. LF UK v PrazeInstitute of Biophysics and Informatics First Faculty of Medicine Charles University in PragueFirst Faculty of Medicine1. lékařská fakult

Návrh Real-Time PCR testu pro detekci Aphibiocystidium ranae jako modelového druhu pro studium rhinosporidiózy

The aim of this thesis is to develop species-specific Real-Time PCR assay for detection of frog parasite Amphibiocystidium ranae as a model approach for studying rhinosporidiosis in human, caused by Rhinosporidium seeberi. Similarities of these parasites allow to study human rhinosporidiosis by analogy. Sequences of the gene for 18S rRNA of Amphibiocystidium ranae were analysed by multiple sequence alignment with sequences of closely related organisms found in GenBank nucleotide database. Amphibiocystidium ranae-specific regions were found and three primer sets were designed, two of them together with probe to increase specificity. Specificity was checked against GenBank nucleotide database and ribosomal RNA database SILVA. Primer sets were tested on samples taken from frogs. Specificity was confirmed by melting curve analysis. Amphibiocystidium ranae-specific Real-Time PCR assay was developed and can be used for detection of this parasite

Cerebellar dysfunction in a family harboring the PSEN1 mutation co-segregating with a Cathepsin D variant p.A58V

Presenile dementia may be caused by a variety of different genetic conditions such as familial Alzheimer's disease, prion disease as well as several hereditary metabolic disorders including adult onset neuronal ceroid lipofuscinosis. We report a multigenerational family with autosomal dominant presenile dementia harboring a cerebellar phenotype. Longitudinal clinical work-up in affected family members revealed ataxia accompanied by progressive cognitive decline, rapid loss of global cognition, memory, visuospatial and frontal-executive functions accompanied by progressive motor deterioration and early death. Linkage analysis and exome sequencing identified the p.S170F mutation of Presenilin 1 in all affected individuals, which is known to be associated with very early onset Alzheimer's disease. Additional search for potentially modifying variants revealed in all affected individuals of the third generation a paternally inherited variant p.A58V (rs17571) of Cathepsin D which is considered an independent risk factor for Alzheimer's disease. Involvement of cerebellar and brainstem structures leading to functional decortication in addition to rapid progressive presenile dementia in this PSEN1 family may therefore indicate an epistatic effect of the p.A58V Cathepsin D variant on the deleterious course of this disease

Mutations in DNAJC5, Encoding Cysteine-String Protein Alpha, Cause Autosomal-Dominant Adult-Onset Neuronal Ceroid Lipofuscinosis

Autosomal-dominant adult-onset neuronal ceroid lipofuscinosis (ANCL) is characterized by accumulation of autofluorescent storage material in neural tissues and neurodegeneration and has an age of onset in the third decade of life or later. The genetic and molecular basis of the disease has remained unknown for many years. We carried out linkage mapping, gene-expression analysis, exome sequencing, and candidate-gene sequencing in affected individuals from 20 families and/or individuals with simplex cases; we identified in five individuals one of two disease-causing mutations, c.346_348delCTC and c.344T>G, in DNAJC5 encoding cysteine-string protein alpha (CSP alpha). These mutations-causing a deletion, p.Leu116del, and an amino acid exchange, p.Leu115Arg, respectively-are located within the cysteine-string domain of the protein and affect both palmitoylation-dependent sorting and the amount of CSP alpha in neuronal cells. The resulting depletion of functional CSP alpha might cause in parallel the presynaptic dysfunction and the progressive neurodegeneration observed in affected individuals and lysosomal accumulation of misfolded and proteolysis-resistant proteins in the form of characteristic ceroid deposits in neurons. Our work represents an important step in the genetic dissection of a genetically heterogeneous group of ANCLs. It also confirms a neuroprotective role for CSP alpha in humans and demonstrates the need for detailed investigation of CSP alpha in the neuronal ceroid lipofuscinoses and other neurodegenerative diseases presenting with neuronal protein aggregation