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Molecular, cell biological and genetic aspects of diseases 5 ECTs Heli Ruotsalainen [email protected] • Inheritance: patterns, different mutation type, genetic diseases, how to search diseases and possible treatments • Mitochondria and mitochondrial diseases • Golgi and glycosylation linked diseases • Peroxisomes and peroxisomal diseases Content of inheritance part • • • • • • • • • Inheritance patterns of genetic diseases What kind of changes in genome cause diseases? Methods to search a disease gene Chromosome mutations Trinucleotide repeat diseases Prion diseases Development and inheritance of cancer Finnish disease heritage How to diagnose an inherited disease and treat e.g. by using gene therapy? Litterature • Nussbaum, R., McInnes, R.R., Willard, H.F., Boerkoel, C.: Thompson & Thompson Genetics in Medicine, WB Saunders Company, 2007 • Strachan, T., Read, AP: Human Molecular Genetics, Bios Scientific Publishers, 2010 • OMIM.org • www.findis.org • FIN: Norio, R.: Suomi neidon geenit, Otava, 2000 yleiskatsauksia • FIN: Aula, P., Kääriäinen, H., Palotie, A.: Perinnöllisyyslääketiede, Duodecim 2006 Glossary of terms • • • • • • • • • gene locus allele genotype phenotype homozygous (AA, aa, +/+, -/-) heterozygous (Aa, +/-) dominant recessive autosomal Gene locus 2 alleles Glossary of terms • • • • • • • • • penetrance polymorphism X-chromosomal carrier pedigree mitochondrial inheritance monogenic polygenic (multifactorial inheritance) epistasis Pedigree Healthy male Affected male Healthy female Affected female Basis to study the human inherited diseases History of genetics Gregor Mendel (1822-1884) Series of expreiments with pea plants Mendel’s laws of inheritance 1866 foundation of genetics Structure of DNA was solved 1950s Rosalind Franklin: X-ray of DNA 1952 Watson and Crick: discover the double helix structure of DNA ( based on Franklin’s data) 1953 Human genome • Genome has been sequenced 2003 • 3.3 billion basepairs – 20500 gene – Publicly available in gene banks – Individuals are 99.9 % identical – Varying nukleotides in every ~100-300 bp – e.g. genomes of James Watson and Graig Venter have been sequenced Human genome project Genetic diseases Chromosomal disorders 0.19% - translocations - inversions - aneuploidy Changes in genome - mutations - single nucleotide changes - deletions - insertions - inversions - Splicing errors - Nucleotide repeats Monogenic diseases, 0.36% - Mendelian inheritance - recessive - dominant - X-chromosomal Mitochondrial diseases Multifactorial diseases, 4.7% - Several genes predisposing mutations - Environmental factors Somatic mutations and cancer Online Mendelian Inheritance in Man (OMIM) An Online Catalog of Human Genes and Genetic Disorders http://omim.org/ Number of Entries in OMIM (Updated June 27th, 2016) : Prefix * Gene description X Linked 705 Y Linked 48 Mitochondrial Totals 35 15,312 + Gene and phenotype, combined 78 2 0 2 82 # Phenotype description, molecular basis known 4,409 304 4 29 4,746 % Phenotype description or locus, 1,491 molecular basis unknown 125 5 0 1,621 Other, mainly phenotypes with suspected mendelian basis 1,690 112 2 0 1,804 22,192 1,248 59 66 23,565 Totals Autosomal 14,524 Online Mendelian Inheritance in Man (OMIM) #256300 NEPHROTIC SYNDROME, TYPE 1; NPHS1 • nephrotic syndrome • Each OMIM entry is given a unique six-digit number: • 1----- (100000- ) 2----- (200000- ) Autosomal loci or phenotypes • • • • • entries created before May 15, 1994 3----- (300000- ) X-linked loci or phenotypes 4----- (400000- ) Y-linked loci or phenotypes 5----- (500000- ) Mitochondrial loci or phenotypes 6----- (600000- ) Autosomal loci or phenotypes • entries created after May 15, 1994 • Allelic variants are designated by the MIM number of the entry, followed by a decimal point and a unique 4-digit variant number. For example, allelic variants in the factor IX gene (300746) are numbered 300746.0001 through 300746.0101. Monogenic traits • Only one gene affected • Mendelian inheritance simple pattern of inheritance • A disease can be caused several, differents mutations in a single gene • Environmental effects, for example phenylketouria • Inheritance patterns: – Autosomal recessive – Autosomal dominant – X-chromosomal – Mitochondrial (Alex Kastaniotios will talk more about this) Autosomal recessive inheritance 25% 50% 50% 25% Affected individuals are indicated by solid black symbols and unaffected carriers are indicated by the half black symbols. • manifests only when an individual has two copies of the mutant allele. • parents are healthy carriers • 25 % chance of being homozygous mutant (affected) or wild type; 50 % chance to be a carrier • rare ( 30 % of inherited diseases) • consanguinity increases the risk of manifestation • isolated populations, finnish disease heritage • cystic fibrosis 1:2500 in Europe, carrier frequency 1:20; in Finland incidence is 1:30000 OMIM #219700 Cystic fibrosis • Autosomal recessive • Complex, multiorgan disease • Mutation in Cystic fibrosis transmembrane conductance regulator (CFTR) gene • > 1000 different mutations Dependent cAMP phosporylation Binding of ATP opens the channel Video of CFTR http://www.mun.ca/biology/scarr/Cystic_Fibrosis_&_CF_Protein.html Cystic fibrosis – effet of mutations Affect the function of CFTR ( e.g. phosphorylation site mutated) Prevent the transport CFTR to membrane of epithelial cell Reduce partially or block totally the synthesis of CFTR:n Mutations prevent the transport of Cl- ions out from epithelial cells Cystic fibrosis • Movement of Cl- ions through epithelial cell membrane is disturbed movement of Na+ and H2O is also affected hyperabsorbed • CFTR regulates also the activity of epithelial Na+ channel and transport of other electrolytes sticky mucus accumulates on cell surface movement of cilia affected removal of bacteria and other particles restricted infections Cystic fibrosis • Function of alveolar and pancreatic ducts disturbed – Secretion of sticky mucus – In the lungs, mucus clogs the airways and traps bacteria leading to infections, extensive lung damage and eventually, respiratory failure – In the pancreas, the mucus prevents the release of digestive enzymes that allow the body to break down food and absorb vital nutrients – Lung symptoms severe , lead to premature death (> 40 years) – Phe 508 (ATP binding domain) most common mutation, in 70 % of cases • incidence 1:2000-2500 – high incidence in Europe, not in Finland http://learn.genetics.utah.edu/content/disorders/singlegene/cf/ Phenylketonuria OMIM #261600 • Autosomal recessive • Learning disability, begins at 6 months of age, progressive • Entsymopathy: defective enzyme phenylalaninehydroxylase – Phe accumulates into tissues, 30 normal level → toxic to central nervous system – Synthesis of tyrosine is reduced – Transport of other large, neutral amino acids to brain is decreased synthesis of proteins and neurotransmitters in disturbed • Part of Phe is transaminated to phenylpyruvate phenylketone urine Phenylketonuria • Can be treated with diet, if started early enough – Screening of newborns (not in Finland) • Incidence 1: 10 000 (in Turkey 1:600) – Rare in Finns (1:100 000-1:200 000), Jews and Afro-Americans • Typical features: light skin colour, blond hair, blue eyes (less melanin pigment) • Four main mutations in Northern Europe: – – – – Arg261-Gln, mild, 18 % of cases Arg408-Trp, clear disease, 38% of cases Exon 12 skipped, clear disease, 38% of cases Arg158-Gln, mild, 14% of cases Duodecim 2009;125(10):1069-75 Autosomal dominant inheritance 50% 0% • • • • • • 0% 50% one copy of the mutant allele causes the disease 50% chance to inherit the mutant allele Phenotypically healthy does not transfer the trait Manifests in every generation Over 2000 diseases are known E.g. Huntington’s disease OMIM #143890 Familial hypercholesterolemia • Dominant inheritance • Mutation in a gene codes for LDL receptor – Normally participates in the endocytosis of LDL from the blood stream to liver – 2-10% of mutations are large insertions, deletions and re-arrangements due to Alu recombination events • 7 mutations cover 93% of Finnish cases: – FH-Helsinki (large deletion), FH-Pohjois-Karjala (small deletion), FH-Turku (G823D), FH-Pori (L380H), FH-Pogosta (R574Q), FH-11 (D558N) ja FH-12 (C331W) • Homozygote very ill (incidence 1: 1000 000): coronary disease, heart infarct early, rarely survive over 30 years • Lot of heterozygotes, incidence 1:500 Reseptors in clathrin coated pits on the cell surface Familial hypercholesterolemia • A a consequence of LDL receptor mutation the level of LDL-cholesterol is high in blood circulation (1) LDL particles penetrate the arterial wall and accumulate within the blood vessel endothelium and are oxidized (2) trigger an inflammatory response Monocytes migrate below endothelium, differentiate into macrophages, and ingest LDL transform into “foam cells” (3) Smooth muscle cells proliferate and form a “fibrous cap”(4) Plaque reduces blood flow and may lead to blood clots http://www.utm.utoronto.ca/~w3bio315/RME/plaque.html Familial hypercholesterolemia 1. Receptor is not produced 3. 4. 2. 5. Below is a pedigree of three generations for some human disease. a) What is a pattern of inheritance? b) Based on the pedigree who are carriers, heterozygous, for the disease ? c) What is the chance that III:2 is heterozygous for the disease ? d) If III:3 and III:4 would have a child together, what would be the chance that their first child would be affected I 1 2 II 1 2 3 4 4 5 III 1 2 3 X-chromosomal inheritance • About 500 disease known • Inheritance and phenotype differ between different sexes • Mutation in females X-chromosome is inherited to 50% of the daughters and 50% of the sons • Mutation in males X-chromosome is inherited to all daughters, but not to any of the sons a. b. Dominant inheritance Dominant – lethal in males Recessive http://www.glowm.com/?p=glowm.cml/section_view&articleid=342 X-chromosomal recessive inheritance Mother: carrier Father: affected • E.g. fragile X, hemophilia A, Duchenne muscular dystrophy – Females healthy carriers carrier’s son has 50% chance to be affected, daughter’s 50% chance to be a carrier http://www.genetics.com.au/factsheet/fs42.asp X-chromosomal recessive inheritance Mother: affected Father: affected • E.g. Alport’s kidney disease (type IV collagen, 5-chain), • Condition worse in males http://www.genetics.com.au/factsheet/fs42.asp X-chromosomal inactivation = Lyonization • Some X-chromosomal gene products are needed in equal amounts both in males and females = inactivation is necessary • random, in females • X chromosome is silenced by packaging it into a transcriptionally inactive structure called heterochromatin • irreversible → cell herited – Reversed during oogenesis • Females are mosaics: X-chromosome from mother and father • Not all genes of X-chromosome are inactivated homologs in Ychromosome • X-chromosome in inherited diseases : heterozygous carriers symptoms vary • Inactive X forms a discrete body within the nucleus called a Barr body– inactivated Xchromosome Journal of Cell Biology, Vol 135, 1427-1440, 1996 OMIM #300377 Muscular dystrophy (lihasrappeuma) • X-chromosomal recessive • DMD1-gene, 2,4 Mb, 8 promoters • DMD1-gene codes for dystrophin protein – Part of a protein complex that links cytoskeleton to extracellular matrix – In the absence of dystrophin myofiber is torn and scared contraction force declines • 60% of mutations are deletions (from 1 exon to whole gene), 2 deletion hot spot regions http://physrev.physiology.org/content/82/2/291 http://www.ncbi.nlm.nih.gov/gene/1756 Muscular dystrophy (lihasrappeuma) • Duchenne and Becker muscular dystrophies • Duchenne more severe and more common (15% of cases) • Muscle weakness, death due to breathing problems • Incidence 1:3500 in males – Becker: no frameshift change milder – Carriers (females) mild symptoms (depends on X inactivation) Monogenic vs Multifactorial In multifactorial diseases inheritance pattern is not so clear, because many genes and environmental factors affect the manifestation Multifactorial diseases Effect of many genes Gene + Gene+ Gene - Gene Gene + Disease phenotype Gene + other Activity Stress Smoking “Environmental factors” S. Solovieva - Example of epistasis • E gene: pigment or no pigment work first • B gene: the amount of pigment effect depends on E gene Features of multifactorial inheritance • Appears in families inheritance doesn’t follow any pattern • The number of affected in a family influences the risk of recurrence • Severity affects recurrence • Consanguinity of parents Genomic imprinting (Perimän leimautuminen ) • Expression of a gene is regulated by a parent-of-origin-specific manner (linked to sex of parent) • Methylation (inactivation) of other allele during female/male meiosis (epigenetic regulation) – Many are tissue specific • About 100 genes known (> ?), as large groups in genome – For example genes encoding RNA modifying proteins, proteins regulating the tissue growth and brain functions http://www.geneimprint.com Nature Reviews Neuroscience 8, 832-843 (2007) Genomic imprinting 15q11-q12 ATP10A + • In chromosomal region 15q11-12, 6 imprinted genes: inactivation of genes depends whether it is inherited from mother or father • Paternal deletion in region 15q11-12 causes Prader-Willi syndome and maternal deletion Angelman’s syndrome – Prader-Willi syndrome (PWS): • PWS gene normally paternally active, maternal inactive • Deletion in paternal allele or both alleles from mother disease • mild to moderate intellectual impairment and learning disabilities, obesity, hypogenitals – Angelman syndrome (AS): • Normally maternal gene active, paternal inactive • Deletion in maternal allele, or both alleles from father disease • delayed development, intellectual disability, severe speech impairment, and problems with movement and balance PWS & AS are caused by deletions, mutations or by uniparental disomy (defect in chromosomal segregation) Uniparental disomy (UPD) • Person has two copies of a chromosome from one parent and no copy from the other parent • Arises from a meiotic chromosome segration defect (I/II) • Trisomy rescue (loss of one homologue) can lead to UPD • UPD can arise also in fertilization, if one gamete is disomic and other nullisomic for same chromosome • homozygous for recessive trait, eventhough only one parent has the gene defect Cystic fibrosis Factors that complicate the determination of inheritance pattern • Onset age varies, individual differences • Incomplete penetrance: for some reason disease doesn’t manifest despite the gene defect (fig) The autosomal dominant condition is usually represented in each generation, but with reduced penetrance, a generation may appear to be "skipped" because of the lack of phenotypic expression. Factors that complicate the determination of inheritance pattern • Single gene influences more than one character different symptoms in different tissues in different individuals = pleiotrophy Factors that complicate the determination of inheritance pattern • anticipation = the symptoms of the genetic disorder become apparent at an earlier age and severity of symptoms increases with each generation • Different gene defect, single disorder – Locus heterogeneity (BRCA1, BRCA2; retinitis pigmentosa) – Allelic heterogeneity • One gene, one mutation, one disease – rare – Finnish disease heritage • One gene, many mutations, one disease – common – Cystic fibrosis • One gene, many mutations, many diseases – β-globin mutation: Glu6Val sickle cell anemia, Phe42Ser hemolytic anemia, Asp99Asn polycytemia • Many genes, many mutation, one diseases – Polycystic kidney disease , cyst in liver and kidney, kidney dysfunction • Gene loci chr. 16 ja 4 • One gene, one mutation, many diseases – Apert , Crouzon, Jackson –Weiss and Pfeiffer syndrome • Mutation in gene encoding for fibroblast growth factor receptor 2 DNA damage • DNA polymerase has proofreading activity – – – – Overall error rate about 1 /100 milj nt 99.9 % repaired Errors less than 1 / cell, about 1017 cell division Most of the errors in somatic cells not inherited Worst mutation prevent fertilization and are never detected • Mutation hot spots in DNA: – GC-dinucleotide islands (hot spot) – Methylation of C deamination leads to C-T change What kind of gene defects? Polymorphic change - harmless, individual variation • genetic polymorphisms are interindividual, functionally silent differences in DNA sequence that make each human genome unique – SNP must be present in at least one percent of the general population • Differences between individuals, ~10 milj SNP/genome; allelic heterogeneity • Non-coding regions • SNP= single nucleotide polymorphism • e.g. C/T, A/G http://learn.genetics.utah.edu/content/pharma/snips/ • RFLP (restriction fragment length polymorphism) • SNP creates or destroys restriction site – e.g. GAATTC → GATTC http://www.allanwilsoncentre.ac.nz/massey/learning/departments/centres-research/allan-wilson-centre/ourresearch/resources/recreate-the-research/where-did-i-come-from/polymorphism.cfm • Tandem repeats e.g. TTTTTTT ; CACACACACA; GACGACGACGAC http://www.slideshare.net/hhalhaddad/the-human-genome-project-part-iii Mutation = inherited change in DNA structure that can cause a disease Point mutation/substitution: • Transition mutation (A-G, C-T) • Transversion mutation (A-C, G-T) • Lenght of DNA is not altered Normal • Effect: Silent mutation Missense mutation Nonsense mutation Deletion and frameshift in reading frame DNA:n pituus Length of DNA muuttuu altered Insertion and frameshift in reading frame • Deletion Indels • Insertion • Duplication Length of DNA altered • Inversion → Leads to frameshift mutation in coding region Mutations outside the coding region • Mutations in promoter area Gene is not transcribed OR it is continuously transcribed OR no effect • Mutation in intronic region splicing defect /no effect Normal splicing *)Mutation in acceptor site of intron I exon 2 is skipped frameshift **)Mutation in donor site of intron II translation continues to intron II Splice site mutations • Point mutation in conserved region: at the end of intron, in the branch point of intron or in exon – Exon skipping: deletion – New splice site: insertion – nonsense mutation often leads to exon skipping • Frameshift premature STOP codon • Reduction in mRNA level (increase in degradation, processing prevented) Deletions and duplications caused by long repeats • Homologous pairing – Alu-repeats • Unequal crossing over gene duplication or deletion event that deletes a sequence in one strand and replaces it with a duplication from its sister chromatid in mitosis or from its homologous chromosome during meiosis Unequal crossing over happens between homologous sequences that are not paired precisely • Recombinations can increase or decrease gene number Gene dosage – Charcot- Marie-Tooth disease 1A caused by duplication – Hereditary neuropathy with liability to pressure palsies (HNPP) deletion 1,5 kb Charcot- Marie-Tooth disease 1A HNPP http://www.ncbi.nlm.nih.gov/dbvar/content/overview/ • Inversion makes a break in gene • hemofilia A J Genet Med. 2010 Jun;7(1):1-8 What kind of outcome a gene defect can have in the body? • Most severe defects are lethal • Affects the gene product: RNA or protein – Mutation in mitochondrial DNA change in tRNA • tRNA(leu): MELAS (Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes ) • tRNA(lys): MERRF (Myoclonic Epilepsy and Ragged Red Fibers) http://www.sec.gov/Archives/edgar/data/1043961/0001144 20411056965/v236826_ex99-1.htm TRENDS in Genetics Vol.20 No.12 December 2004