Download I Lecture and part of II lecture

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
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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
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gene locus
allele
genotype
phenotype
homozygous (AA, aa, +/+, -/-)
heterozygous (Aa, +/-)
dominant
recessive
autosomal
Gene locus
2 alleles
Glossary of terms
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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
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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
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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
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> 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:
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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%
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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
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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
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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
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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