Genetics Flashcards
Normal human karyotype
46 chromosomes - 2n (22 pairs of autosomes and a pair of sex chromosomes)
Chromosome structure
Each chromosome consists of a short (p) and long (q) arm joined at the centromere
Telomere
Telomere - seal ends of chromosomes and maintain structural integrity (repetitive sequence of thymine)
Telomerase replaces 5’ end of long strand during DNA replication making strand shorter until it can no longer divide
Chromosome classification
• centrally = metacentric
• terminal = acrocentric (13,14,15,21,22 - Robertsonian chromosomes)
• intermediate position = submetacentric
Mitosis
process by which chromatically separate and divide in to two separate cells
Usually lasts 1 to 2 hours
5 distinct stages: prophase, prometaphase, metaphase, anaphase and telophase
Prophase
chromosomes condense, mitotic spindle begins to form, two centrioles begin to form and move to opposite poles (microtubules begin to form from alpha/ beta tubulin)
Prometaphase
nuclear membrane disintergrates and chromosomes attach to microtubules by centromeres
Metaphase
chromosomes line along equatorial plate of cell to form mature spindle- chromosomes maximally contracted so visible X shape
Anaphase
centromere of each chromosome divides longitudinally and 2 daughter chromatids separate to opposite poles of cell
Telophase
2 groups of chromatids become surrounded by nuclear membrane
Cytokinesis
cell cytoplasm divides to form two new diploid daughter cells
Interphase
G1 - chromosomes become thin and extended (variable in length so accounts for change in generation time. Cells that have stopped dividing, e.g. Neurones, arrest in this phase = G0)
S - DNA replication occurs and the chromatin of each chromosome is replicated (homologous pairs replicate in synchrony but 1 of X chromosomes is always late as is inactive X chromosome that forms the sex chromatin which viewed in interphase of female somatic cell)
G2 - chromosomes begin to condense
Importance of mitosis
• producing 2 genetically identical daughter cells to parent cell
• growth
• replace dead cells
Clinical relevance of mitosis
• detecting chromosomal abnormalities
• categorising tumours as benign or malignant
• grading malignant tumours
Meiosis
the process of nuclear division that occurs in final stages of gamete formation
Prophase I
chromosomes are already split longitudinally into 2 chromatids joined at centromere. Homologous chromosomes pair (with exception of X and Y in males where pairing only occurs at tip of shorter arms called pseudoautosomal region) and crossing over may occur, exchange of non-sister chromatid alleles.
Metaphase I
the nuclear membrane disappears and chromosomes become aligned on the equatorial plane of cell- attached to spindle
Anaphase I
chromosomes separate to opposite poles as spindle contracts (independent assortment)
Telophase I
each set of haploid chromosomes separated so cleaves to form 2 new daughter gametes (secondary spermatocytes or oocytes)
Meiosis II
same as mitotic division to form 4 haploid (n) new daughter gametes (spermatids or ova)- genetically different
Preventing mutations during cell replication/division
• genetic material protected in stem cells as rapid cell division occurs in daughter cells not stem cells
• 3 checkpoints: at end of G1 restriction point, needs external growth factor for cell division to continue; in G2 checkpoint looks for damage and unreplicated DNA, can stop cell cycle and kill cell to prevent replication of mutated DNA controlled by P53 but gene for P53 is often mutated in cancer cells leading to uncontrolled cell division; S phase checkpoint by RPA protein itstabilises the replication fork and coordinates repair
3 Main differences between mitosis and meiosis
- Mitosis = diploid cells, meiosis = haploid cells
- Mitosis occurs in somatic cells and early stages of gamete formation, meiosis occurs only at final stage of gametic maturation
- Mitosis = one cell division, meiosis = 2 cell divisions
3 cell populations
• permanent cells- cells that never divide G0 eg neurones
• Labile cells- cells that constantly divide eg epidermis
• stable cells- spend most of time in G0 but can be induced to re-enter cell cycle eg liver cells
Stopping mitosis
Mitotic spindle - taxol or vinca alkaloids (vinblastine, vincristine)
Spindle poles- ispinesib
Anaphase - colchicine-like drugs- form ring structures
Oogenesis
mature ova develop from oogonia which themselves originate from primordial germ cells by a process involving 20-30 mitotic divisions that occur in first few months of embryonic life.
By completion of embryogenesis at 8 months of intrauterine life, the oogonia have begun to mature into primary oocytes that undergo meiosis.
At birth, all enter a maturation arrest phase (dictyotene) in which remain suspended until meiosis I is completed at time of ovulation, when a single secondary oocyte is formed that receives most of cytoplasm. Other daughter cell is a polar body (largely consists of nucleus). Meiosis II then occurs (fertilisation can occur) resulting in 2 more polar bodies- meiosis II only completed if fertilisation occurs
Older mothers and non-disjunction
Lengthy interval between onset of meiosis and completion accounts for increased incidences of chromosomal abnormalities in offspring of older mothers: damage to cell’s spindle formation and repair mechanisms, leading to non-disjunction
When does meiosis I complete for oogenesis
Time of ovulation
When does meiosis II complete for oogenesis
Fertilisation
Spermatogenesis
at puberty spermatogonia begin to mature into primary spernatocytes which enter meiosis I and emerge as haploid secondary spermatocytes. Then undergo second mitotic division to form spermatids, which develop into mature spermatozoa
Continuous process so many mitotic divisions leading to new dominant mutations due to consequences of DNA copy errors in interphase (s)
Numerical chromosomal abnormalities
loss or gain of one or more chromosomes (aneuploidy) or the addition of one or more complete haploid components (polyploidy). Loss of a single chromosome - monosomy
Trisomy
presence of an extra chromosome eg downs syndrome is presence of additional 21 chromosome. Usually caused by failure of separation of one of pairs of homologous chromosomes during anaphase I or less often when sister chromatids fail to separate in anaphase II- non-disjunction
Trisomy conditions
Patau syndrome (trisomy 13)
Edward’s syndrome (trisomy 18)
1st trimester miscarriage (trisomy 16)
Down’s syndrome (trisomy 21)
Monosomy
absence of a single chromosome. For an autosome, usually not carried to full term. Turner syndrome - lack of X or Y chromosome resulting in 45,X karyotype. Can be caused by non-disjunction or anaphase lag (loss of chromosome during anaphase)
Polyploidy
cells contain multiple of the haploid number of chromosomes
Triploidy (69) can be caused by failure of maturation meitotic division in an ovum or sperm(eg retention of polar body or diploid can be caused by fertilisation of an ovum by 2 sperm (dispermy)
2 paternal = swollen placenta/ 2 maternal = small placenta - not carried to term
Down syndrome
• learning problems
• short stature
• characteristic facial appearance
• congenital heart disease
Additional 21 chromosome (trisomy)
47, XX/XY +21
Mosaicism
the presence in an individual or tissue of 2 or more cell lines that differ in the genetic constitution but are derived from a single zygote. Usually results from non-disjunction in early embryo mitotic division or can exist if a new mutation arises in a somatic or early germline cell division. Germline/ gonadal mosaicism = duchenne muscular dystrophy
Chimerism
the presence in an individual of 2 or more genetically distinct cell lines derived from more than one zygote. Dispermic chimeras - result of double fertilisation whereby 2 sperm fertilise 2 ova and the resulting 2 zygotes fuse to form one embryo (if different sex, XX/XY karyotype so hermaphroditism). Blood chimeras - exchange of cells, via the placenta, between non-identical twins in utero
robertsonian translocation
results from the breakage of 2 acrocentric chromosomes (13, 14,15, 21 and 22) at or close to their centromeres with subsequent fusion of their long arms - centric fusion. The short arms are lost so the total chromosome number is reduced to 45. (No gain or loss of genetic material as short arms only code for rRNA). Can predispose to birth of babies with Down syndrome as a result of embryo inheriting 2 normal 21 chromosomes and a translocation chromosome involving a 21 chromosome
dosage sensitive
Genes are dosage sensitive (normal dose is 2)- deletion or duplication causes an imbalance, causing a disease
deletion
loss of part of a chromosome and results in monosomy for that segment. Large deletion - Wolf-Hirschhorn and cri du chat. Submicroscopic microdeletions- Prader-Willi and Angelman syndrome
Insertion
when a segment of one chromosome becomes inserted into another
Inversion
a two-break rearrangement when a segment is reversed in position. If involves centromere, pericentric inversion. If only one arm, paracentric inversion- results in recombinant chromosomes (acentric cannot undergo mitotic division. Dicentric are unstable during cell division)
Ring chromosomes
when a break occurs on each arm of a chromosome leaving two ‘sticky’ ends on the central portion that reunite as a ring. The two distal fragments are lost so if an autosome, serious effects
Isochromosome
loss of one arm with duplication of the other as centromere divided transversely not longitudinally (eg 2 long X chromosome arms = Turner syndrome)
Duplication
section is copied
Translocation
the transfer of genetic material from one chromosome to another. A reciprocal translocation is formed when a break occurs in each of 2 chromosomes with the segments being exchanged to form 2 new derivative chromosomes- a Robertsonian translocation is when the breakpoints are located at, or close to, the centromeres of 2 acrocetric chromosomes. Segregation at meiosis: they can segregate to generate significant chromosome imbalance leading to early pregnancy loss or birth of an infant with multiple abnormalities. Problems arise at meiosis as they cannot pair normally to form bivalents- instead form a cluster called a pachytene quadrivalent. When they separate they can:
1. If alternate chromosomes segregate, the gamete will carry a normal/ balanced haploid complement
2. If adjacent chromosomes segregate together, the gamete will acquire an unbalanced chromosome complement, leading to non-disjunction at fertilisation
3. 3 chromosomes can segregate to one gamete with only one chromosome in the other gamete. - tertiary trisomy
Balanced translocation
no gain or loss of DNA (same number of genes just swapped), so healthy human
Unbalanced translocation
loss or gain of DNA, causing a disease
Mosaicism
the presence in an individual or tissue of 2 or more cell lines that differ in the genetic constitution but are derived from a single zygote. Usually results from non-disjunction in early embryo mitotic division or can exist if a new mutation arises in a somatic or early germline cell division
Example of condition caused by Gondal mosaicism
duchenne muscular dystrophy
Chimerism
presence in an individual of 2 or more genetically distinct cell lines derived from more than one zygote.
Dispermic chimeras
result of double fertilisation whereby 2 sperm fertilise 2 ova and the resulting 2 zygotes fuse to form one embryo (if different sex, XX/XY karyotype so hermaphroditism)
Blood chimeras
exchange of cells, via the placenta, between non-identical twins in utero
DNA composition
nucleic acid is a long polymer of nucleotides (each composed of a nitrogenous base, deoxyribose, and a phosphate molecule) with phosphodiester bonds between 3’ and 5’ carbons on adjacent sugars
• purine bases: guanine, adenine. Pyrimidine bases: cytosine, thymine, uracil
• 2 anti-parallel chains in a double helix joined by hydrogen bonds between complementary bases- a purine always pairs to a pyrimidine: G&C (3 hydrogen bonds) and A&T (2 hydrogen bonds)
DNA semiconservative replication
allows replication and self-repair
1. Helix unwound by topoisomerase and strands separated by DNA helicase breaking hydrogen bonds
2. Single stranded binding (SSB) protein coat the strand to prevent team taking or snapping back together and exposed bases act as a template for free DNA nucleotides to bond to by complementary base pairing
3. Primate enzyme synthesises a short RNA primer
4. DNA polymerase joins nucleotides together by forming phosphodiester bonds in 5’ to 3’ direction. Replication fork= leading strand synthesised in continuous process. Lagging strand synthesised in Okazaki fragments which are then joined by DNA ligase
5. DNA replication progresses in both directions from points of origin to form replication bubbles which fuse to form 2 identical daughter molecules
6. (RNAse H recognises RNA primers bound to the DNA template and hydrolyses them and DNA polymerase can fill in the gaps)
Which enzyme unwinds DNA helix
Topoisomerase
Which enzyme breaks hydrogen bonds
DNA helipads
Which enzyme synthesises short RNA primer
Primate enzyme
Which enzyme joins together nucleotide bases
DNA polymerase
Which enzyme joins Okazaki fragments
DNA ligase
Which enzyme hydrolyses RNA primers
RNAse H
Coiling of DNA
DNA is coiled around a histone to form nucleosomes (8 histone proteins)
Tertiary coiling of nucleosomes forms chromatin fibres that form long loops which coil further- the solenoid model. Chromatin condenses into visible aggregates (chromosomes)
Who suggested dna structure
Watson and Crick in 1953 based on X-ray diffraction studies
Telomere
seal ends of chromosomes and maintain structural integrity (repetitive sequence of thymine)
Telomerase replaces 5’ end of long strand during DNA replication making strand shorter until it can no longer divide
Degenerate but unambiguous
amino acids coded for by more than one codon but each codon is specific to one amino acid
Almost universal
same in all organisms apart from fewer than 10 exceptions
Non-overlapping
Each nucleotide is only read once
Telomerase
replaces 5’ end of long strand during DNA replication making strand shorter until it can no longer divide
Nuclear DNA sequence
genes, unique single copy (code for polypeptides), multigene families (arisen through gene duplication eg alpha and beta globin gene), classic gene families (high sequence homology eg numerous copies of genes coding for rRNA), gene superfamilies (limited sequence homology but functionally related)
Extragenic DNA
tandem repeat, satellite, minisatellite, telomeric
Mitochondrial DNA
2 rRNA genes and 22 tRNA genes
RNA
Single-stranded molecule that forms an alpha helix and is relatively short
Contains uracil instead of thymine and ribose instead of deoxyribose
mRNA
formed by transcription of DNA and allows flow of genetic material from nucleus to ribosome. Consists of a leader sequence (with a guanosine cap) at the 5’ end, a coding region and a trailer sequence at the 3’ end with a poly(A) tail
tRNA
single-stranded 3D RNA (clover -shaped formed by hydrogen bonds) that carries amino acids to ribosomes during translation through base pairing the anticodon with the codon of mRNA
- at least 20 types
rRNA
folded RNA which forms aggregates with ribonuclease proteins in ribosomes. Contains many loops and exhibit extensive base pairing in the regions between loops. It has enzymatic properties that catalyse the formation of peptide bonds between amino acids
Allele
alternative form of a gene at a specific locus
Law of uniformity
when 2 homozygotes with different alleles are crossed, all of the offspring in the F1 generation are identical and heterozygous
Law of segregation
each person possesses 2 genes for a particular characteristic, only one of which can be transmitted at any one time
Law of independent assortment
(Mendel’s 2nd law) = members of different gene pairs separate to offspring independently of one another
Genome
all the genes and non-coding DNA in the body
3 types of genome
- Germline - genome in the sperm/eggs. Passed from parent to child- heritable
- Somatic - genome found in every other tissue. Not heritable
- Mitochondrial - found only within the mitochondria. Heritable
Non-coding DNA
promoter sequence (transcription factors binds), introns, enhancers, terminators
Phenotype
the physical or behavioural characteristics of an organism that results from the interaction between its genotype and environment
Mutagenesis
an alteration to the genomic code by exposure to a substance- mutation. Can be in the womb or post natal eg in carcinogenesis, exposure to radiation
Teratogenesis
a damaging effect on embryonic/ fetal development by exposure to a substance eg virus causing cell death, toxin interrupting blood supply. Some teratogens are also mutagens. Teratogens eg smoking, alcohol
Monogenic
caused by a single gene mutation
Somatic
disease causing mutations are found in the affected tissue (cancer)
Malformation
intrinsic issue with development of an organ or tissue eg congenital heart disease. Commonly genetic
Deformation
extrinsic factors impinge upon development of an organ eg compression from womb due to no amniotic fluid (due to no kidneys in baby so no urine), blood clot leading to loss of limb. Less commonly genetic
Minor malformation
more than 2, consider underlying genetic condition
Major malformation
consider underlying genetic condition
Teratogens mechanism
Affects development of tissues not genes
Splice site
codes in DNA to move a portion of RNA. Second most common section for mutations
Major histocompatibility complex
located on chromosome 6
• group of genes that code for proteins found on the surfaces of cells that help the immune system recognize foreign substances
DNA repair
• mismatch repair
• DNA base excision
Dual excision
a short section of single stranded DNA (25ish nucleotides) containing the lesion is removed
Micro satellite instability
condition that arises when a mutation develops in the mismatch repair genes so the cell can no longer repair errors (insertions or deletions) leading to an increased mutation rate
Proband
individual of interest on pedigree drawing
Indicated by an arrow
Pedigree chart: square
Male
Pedigree chart: circle
Female
Pedigree chart: diamond
Gender unknown
Pedigree chart: diagonal line through symbol
Person deceased
Pedigree chart: brackets around a symbol and a dashed line
Adopted
Pedigree chart: P in symbol
Pregnant
Pedigree chart: 2 lines from same symbol
Non-identical twins
Pedigree chart: 2 lines from same symbol with horizontal line joining
Identical twins
Pedigree chart: triangle
Pregnancy loss eg miscarriage, still birth, elective abortion
Pedigree chart: line through connecting line
Estranged
Pedigree chart: number in symbol
Multiple of same gender and generation
Pedigree chart: shaded in symbol
Affected by disease
Pedigree chart: half shaded symbol
Carrier of disease
Percentage of DNA that is coding
1.5%
20000-22000 genes coding for proteins in humans
Autosomal dominant inheritance
manifests in the heterozygous state (tends to be toxic gain-of-function variants) eg toxic mRNA molecule in cell which is too long and doesn’t break down so poisons the cell
Usually multiple generations affected
Transmission by individuals of both sexes to both sexes
Males + females affected in equal proportions (but can be more common in one sex eg breast cancer)
What causes variety in frequency of genetic disorders
environment (eg sickle cell disease and malaria), isolation and cultural differences
Carrier frequency for recessive conditions varies by population
Autosomal inheritance
Inheritance of a trait or disorder by a gene on an autosome- usually more than one gene
Pleiotropy
- A single gene that may give rise to 2 or more apparently unrelated effects eg tuberous sclerosis affects learning difficulties, a facial rash (adenoma sebaceum), epilepsy or subungual fibromas
Variable expressivity
dominant disorders can show variation from person to person
Penetrance
percentage of individuals who have a certain genetic variant who develop a condition because of it
Age-related penetrance
percentage of individuals with a genetic variant who develop a condition at a given age- important for diagnosis and preventative measures
Reduced penetrance
individuals with a heterozygous gene mutation that show very few abnormal clinical features, may be due to modifying effects of other genes and interaction of gene with environmental factors.
Non-penetrance
no features of a disorder
Recurrence risk
an affected person has 50% chance of having an affected child (for each pregnancy)
Homozygosity- autosomal dominant
may be more/less severely affected or have an earlier age of onset
Anticipation
onset of the disease occurs at an earlier stage in offspring than parents or disease occurs with increasing severity in subsequent generations. As a result of the expansion of unstable triplet repeat sequences- larger sequence, more severe
De novo mutations
newly arised mutation- the disease causing genetic variant occurs in either the sperm or the egg, or during early embryo cell division (in zygote). An unaffected parent has a child with an autosomal dominant condition- not inherited- mutation in germline (more common in sperm due to more cell divisions so mutations more likely)
Associated with increased age of the father as a result of large number of mitotic divisions male gametes undergo
Co-dominance
two allelic traits that are both expressed in the heterozygous state eg blood group
Autosomal recessive inheritance
manifest when mutant allele is homozygous. Heterozygous individuals are carriers but unaffected. (Tends to be loss of function gene eg loss of function of enzyme)
Usually one generation affected
Parents can be related (consanguineous)
Males + females affected in equal proportions (but can be more common in one sex eg breast cancer)
Carrier frequency
If 2 carriers, 25% chance of being affected. If not affected, 2/3 chance of being a carrier (if have affected sibling)
Consanguinity
the rarer the recessive trait, the greater the frequency of related parents (consanguinity)- increase chance of recessive conditions
Pedigree chart: double line between symbols
Married but related
Pseudodominace
- if an homozygous and heterozygous have offspring, 50% chance of being affected
Locus heterogeneity
conditions due to mutations in more than one gene/ same condition by different gene mutations
Genocopies
disorders with same phenotype from different loci
Compound heterogeneity
two different mutations at the same locus causing the disease as both genes non-functional so same as Homozygosity
Allelic polymorphism
When more than one allele can be found for a given gene within the normal population
Locus heterogeneity
An allele is one of a number of alternative forms of the same gene found at the same genetic locus
Allelic/mutational heterogeneity
Lots of different mutations in one gene cause a condition eg cystic fibrosis
Cystic fibrosis
• most common recessive condition affecting Northern European population
• incidence of ~ 1 in 2500
• carrier frequency 1/25
• F508 mutation in CFTR gene on 7q31.2
• over 1000 mutations (mutational heterogeneity)
• standard carrier testing detects top 29 mutations (~90%)
• sweat testing (salt levels in babies sweat) = diagnostic test (not genetic analysis)
Neurofibromatosis type 1 (NF1)
• Dominant disorder
• Most affected people only have skin signs (café-au-lait macules and neurofibromas)
• Small % have epilepsy and learning problems
• Different seventy of symptoms with same genetic variant (variable expressivity)
X-linked inheritance
The pattern of inheritance shown by genes that are located on either of the sex chromosomes
X chromosomes = X-linked
Y chromosome = Y-linked or holandric inheritance
Male-male transmission
in a family shows condition is not X- linked
Duchenne muscular dystrophy
affected men (mostly)
• mutation in the dystrophin gene on the X chromosome
• absence of dystrophin protein in skeletal muscle
• limb weakness in males
• eventual use of a wheelchair
• Raised serum creatine kinase CK
Gondal mosaicism
de novo mutation only found in the ovary, not in blood DNA of mother. No Clinical test for this. Could offer a prenatal test (amniocentesis)
X-linked recessive inheritance
a trait determined by a gene carried on the X chromosome and usually manifests only in males (a male with a mutant allele on his single X chromosome is hemizygous)
• transmitted by usually healthy heterozygous female carriers to affected males, as well as by affected males to their obligate carrier daughters (consequent risk to male grandchildren - diagonal pattern of transmission)
• male cannot transmit an X-linked trait to his son as receives Y sex chromosome
• for a carrier female, each son has a 50% chance of being affected and each daughter has a 50% chance of being a carrier
• many diseases eg Duchenne muscular dystrophy transmitted via female carriers or new mutations as males rarely survive to reproductive age
Variable expression
in several X-linked disorders heterozygous females have a mosaic phenotype with a mixture of features of the normal and mutant alleles. Due to random process of X-inactivation
Females affected with X-linked recessive disorders can be due to:
Homozygosity
Skewed x-inactivation
Numerical x-chromosome abnormalities
X-autosome translocations
Skewed x-inactivation/ lyonisation
Lyonisation (random X-inactivation) = female cells randomly inactivate one of their X chromosomes during embryogenesis. If through chance, the healthy X chromosome is inactivated more than the mutated X chromosome in a given tissue it will cause disease. Skewed X inactivation - 80% of cells show preferential inactivation of one X chromosome (should be 50:50), can do on blood or affected tissue eg muscle
Numerical x-chromosome abnormalities
if only has one X chromosome and it is mutated eg Turner syndrome
X-autosome translocations
if breakpoint of translocation disrupts gene on X chromosome female can be affected
Barr body
transcriptionally inactive X chromosome
X-linked dominant inheritance
both males and females (generally less severely) are affected
• Affected males can transmit the disorder to their daughters but not sons
• eg hypophosphatemia (vitamin D-resistant rickets) - affected with short stature
Examples of X-linked dominant inheritance
Rett syndrome or Alport’s syndrome
Non-Mendelian inheritance
a disease not explained by a dominant, recessive or x-linked mode of inheritance.
SNP
single nucleotide polymorphism: a genomic variant at a single base position in the DNA
Multi factorial inheritance
the risk of the condition in relatives of an affected individual is much higher than in the general population
• the incidence of the condition is greatest amongst relatives of the most severely affected patients
• risk is greatest for first degree relatives and decreases for more distant relatives
• if more than one affected close relative then the risks for other relatives are increased
Liability
genetic risk interacting with environmental exposure- can be considered as a single entity. Continuous normal distribution- a threshold exists above which the disease occurs
GWAS (genetic wise association)
based on SNPs genetic sequencing to see if certain SNPs are more common in healthy or diseased population to identify location of genetic variation causing disease- can then tell risk- association between genotypes and phenotypes
Somatic mosaicism
certain issues have genetic variant and some do not
• Not inherited
• can form cancer after exposure to a mutagen (mutation occurs in stem cell within a tissue). More serve genetic changes occur as tumour grows and progresses
• can cause a developmental disorder (localised overgrowth) for autosomal dominant genes
Mitochondrial disease
16.5kB mitochondrial chromosome- mtDNA and some genes on chromosome 1 code for respiratory enzymes
• Mitochondrial DNA has a higher rate of spontaneous mutation that nuclear DNA
• mitochondria produce energy in form of ATP- symptoms occur due to a lack of energy to drive cellular functions
• greater proportion of mutant mitochondria in a cell/tissue the more likely there is to be disease
• only ova large enough to contain significant numbers of mitochondria so all mitochondria derived from mother
• some ova can contain more mutated mitoxhrobdria than others in same female so offspring affected differently
• male with a mitochondrial disease cannot have an affected child
Homoplasmy
All mitochondria in cell have same genetic code
Heteroplasmy
certain proportion of mitochondria in a cell has genetic variant
Imprinting disorders
2 copies of every gene- for certain genes either the maternal or paternal copy is ‘switched off’ - imprinted genes due to methylation of DNA
• disease can be caused when imprinting is altered or genes are switched on/off inappropriately
• deletion of active gene leads to disease
• eg Prader-Willi syndrome- deletion of male paternal gene at chromosome 15p, floppy baby, learning problems, obesity. Angelman syndrome - deletion of female paternal gene at chromosome 15p, below average size, epilepsy, learning problems
Leber’s optic neuropathy
mutations in mitochondrial DNA which encode complex 1 (mitochondrial enzyme for generating ATP)
• gradual onset of painless visual loss- thinning of optic nerve
• males much more likely to be affected than females
Mutation
heritable alteration or change in the genetic material
Can arise through exposure to mutagenic agents eg radiation/ benzopyrene (product of incomplete combustion of hydrocarbons, it is a DNA-adduct so reacts with bases to form a bulky group disrupting replication, or spontaneously through errors in DNA replication and repair
Ionising radiation: can damage bases, causes breaks in phosphate backbone
UV : damaged bases- forms thymine dimers
Single nucleotide variants
change of one nucleotide (wild-type) to another nucleotide (mutant).
Substitution mutation
replacement of a single nucleotide by another.
• most common type
• transition = replaced by same type of nucleotide (pyrimidine or purine) eg G and A or C and T
• transversion = replaced by other type of nucleotide
• transitions are more common than transversions
Transition mutation
replaced by same type of nucleotide (pyrimidine or purine) eg G and A or C and T
Transversion mutation
replaced by other type of nucleotide
Silent/ synonymous mutations
the mutation does not alter the polypeptide product of the gene due to genetic code for some amino acids being degenerate. Usually substitution mutation
Types of non synonymous mutations
Missense
Nonsense
Frameshift
Deletion mutation
the loss of one or more nucleotides. Causes a frameshift and larger deletions may result in partial or whole gene deletions and may arise through unequal crossover between repeat sequences
Insertion mutation
the addition of one or more nucleotides into a gene. Causes a frameshift.
Missense mutation
single base-pair substitution can result in coding for a different amino acid and synthesis of an altered protein (a nonconservative substitution occurs if amino acid is chemically dissimilar so it leads to a reduction or loss of biological activity. A conservative substitution is the replacement with a chemically similar amino acid, and may have no functional effect.)
Nonsense mutation
-a substitution that leads to a STOP codon, resulting in termination of translation, producing non-functional polypeptides. mRNA containing premature termination codons are frequently degraded by nonsense-mediated decay. Loss-of-function variant
Frameshift mutation
a mutation involving insertion or deletion of nucleotides that are not a multiple of 3, disrupts reading frame so amino acid sequence is subsequently altered. Loss-of-function variant
Splicing mutations
mutations of the highly conserved splice donor (GT) and splice acceptor (AG) sites usually result in different splicing. Can result in loss of coding sequence (exon skipping) or retention of intronic sequence (leading to frameshift mutations)- intron translated into protein. Loss-of-function variant
Canonical splice sites
GT
AG
Splice acceptor site
CGAT
Copy number variants
deletion or duplication of a segment of chromosome. Can affect single exon or hundreds of genes. Deletions more likely harmful than duplications
Trinucleotide repeat expansion
if triplet repeats expand in non-coding parts above a certain threshold RNA can’t be broken down, causing disease eg >30 CAG causes Huntingdon’s diseases. Forms an elongated, toxic mRNA which resists degradation
Out-of-frame mutation
leads to formation of premature STOP codon. Nonsense-mediated decay. More likely to cause disease
STOP codons
UAA
UAG
UGA
In-frame mutations
multiple of 3 nucleotides. Lose/gain single amino acid can have few effects
Variant is pathogenic if
• a de novo variant is more likely to be causing a medical condition
• The variant is found in several people in the family who have the disease (positive segregation)
• Absence if SNV from ‘healthy’ populations
• Computational tools predict damaging effect
Variant is benign if
• variant is found in an unaffected parent
• Variant is found commonly in healthy populations
• Computational tools predict variant has no effect on gene function
Trio testing
(testing parents) is important as variant inherited from a normal parent unlikely to be causal- de novo mutation instead. Compared to database of similar ancestry
Different genetic variants may need different tests to detect
Comparative genetic hybridisation
can detect CNVs involving single exons (like ELISA test)
Exome = exons plus some splice sites (miss non-coding variants and trinucleotide repeats)
Genome = exons, promoters/enhancers, splice sites, trinucleotide repeats (can also detect CNVs)
American college of medical genetics criteria (ACMG)
Pathogenic variant
Likely pathogenic variant
Variant of uncertain significance
Likely benign variant
Benign variant
Why is genome sequencing the most comprehensive test
Genome gives more data than exomes but might miss mosaic changes (in blood) that exomes can detect as DNA is sequenced fewer times. Exomes don’t detect changes in non-coding regions
Chorionic villous sampling
taking a sample of trophoblastic cells adjacent to placenta to aid genetic screening
Exome
exons plus some splice sites (miss non-coding variants and trinucleotide repeats)
Genome
exons, promoters/enhancers, splice sites, trinucleotide repeats (can also detect CNVs)
Nonsense-mediated decay
mRNA containing premature termination codons are frequently degraded
P53 gene
• DNA damage detected- initiate repair mechanisms
• Pause cell cycle until repair is carried out
• Halt cell cycle if DNA not repaired
• Apoptosis- command cell to commit suicide if DNA damage not repaired
Tumour suppressor genes
code for proteins that carry out DNA repair, slowing the cell cycle, signalling apoptosis
A normal gene maintains constant rate of cell division so prevents tumour formation
Hypermethylation of DNA can occur preventing a transcription factor binding, meaning the gene is not transcribed and so leading to uncontrolled cell division
A mutation can produce non-functional polypeptides
Must inherit 2 mutated alleles as it is recessive
Oncogenes
proto-oncogenes stimulate a cell to divide when growth factors attach to a cell membrane receptor which activates genes that cause DNA replication and mitosis
Oncogenes can become permanently activated-
• receptor protein activated, even when no growth factor
• oncogene may code for a growth factor that is produced in excessive amounts
Results in excessive cell division
Only inherit 1 mutated allele as dominant
Cancer cells have decreased methylation causing activation of genes that promote cellgrowth, loss of imprinting and chromosome instability (highly active = more likely to mutate)
Therapeutic drugs targeting DNA replication
Inhibitors of nucleotide synthesis
DNA polymerase inhibitors
DNA template damaging agents
Inhibitors of DNA topoisomerase
From genome sequence can infer:
• age, ethnicity, sexuality, reconstruct facial appearance
• Can use Y chromosome genetic markers to connect surnames to people
De-identification → criminal activity/ genomic inference → discrimination
Newborns genome project
proposed to undertake genome sequencing at birth to screen for a greater range of treatable genetic disorders- population screening
• as don’t understand genetic variants, babies would require further testing
• May not develop disease, penetrance; variability; unfounded anxiety
Predicative genetic testing
• individual without symptoms requests test for highly penetrant genetic variant causing a disease
• Autosomal dominant neurological conditions
• In mentally competent adults this can be seen to promote their autonomy provided no evidence of coercion by a third party
• For children (under 16) UK guidelines, under Gillick competence, are to only perform diagnostic tests in children or for conditions in which preventive treatment is required eg child bowel cancers. Parental anxiety is often main reason for requests. A non-competent child cannot make an autonomous decision so should not have presymptomatic tests
Direct to consumer testing
• saliva sample
• Uses SNP ChIP, less commonly exome sequencing
• Rarely any clinical input and no consideration of family history
• Negative doesn’t mean zero risk- means you have no elevated risk compared to general population
• Associated with dustress due to lack of clinical advice; could result in inappropriate management or screening; can increase healthcare costs through additional referrals needed to manage direct to consumer results
SNP microarray
uses known nucleotide sequences as probes to hybridise with the tested DNA sequences, allowing qualitative and quantitative single nucleotide polymorphisms analysis through signal detection
Current issues for newborn genome sequencing
• what conditions to screen for?
• how to deal with adult onset genetic conditions?
• how reliable are genetic variants in predicting disease onset in these contexts?
• What if untreatable conditions are diagnosed?
Advantages of screening
• informed choice
• Improved understanding
• Early treatment when available
• Reduction in births of affected homozygotes
Ethical considerations of screening
• attendant risk and awareness of prenatal diagnosis may create a sense of guilt, especially if decision involves possible pregnancy termination (prognosis of disease cannot be stated with certainty due to variability or reduced penetrance or if hope of a treatment developed)
• pressure to participate causing mistrust and suspicion
• stigmatisation of carriers (social, insurance, employment)- discrimination
• irrational anxiety in carriers
• inappropriate reassurance if test is not 100% sensitive
• Confidentiality?
• Short circuits natural selection
Invasive prenatal diagnosis
(amniocentesis)
Ending a pregnancy affected by familial genetic condition- but what is defined as a serious condition, perspectives of clinicians and patients vary
People with objectively ‘severe’ disease still report a good quality of life not a cause of suffering- many disagreed with prenatal testing for their condition
Non-invasive prenatal diagnosis
(testing free fetal DNA extracted from maternal serum)- no increased risk of miscarriage, trisomy screening, bespoke sequencing for single gene disorders (usually paid for privately)
Advantages of prenatal diagnosis
potential to facilitate autonomy by increasing information available to pregnant women;
more cost efffective than invasive tests
Disadvantages of prenatal screening
potential to increase number of terminations;
increased chance of terminating healthy pregnancy;
equity of access;
screen out disabilities eg Downs Syndrome
Preimplantation genetic testing
routine NHS procedure for genetic conditions before IVF- downplays risk of IVF
Potential to select based on physical traits using PGT;
Genetic variants identified in GWAS as being associated with height, IQ, athleticism are used to select embryos- PGT-P (polygenic risk scores)
Tests for polymorphisms (likelihood of traits) not genetic variants that cause disease
Methods of chromosome analysis:
Any tissue with living nucleated cells that undergo division can be used eg most commonly lymphocytes, skin, bone marrow
1. Sample added to a small volume of nutrient medium containing phytohemagglutinin which stimulates T lymphocytes to divide
2. Cells cultured in sterile conditions at 37°C for 3 days then colchicine added to each mixture (prevents spindle formation so cells arrested during metaphase when chromosomes maximally condensed)
3. Hypotonic saline added causing blood cells to lyse and results in spreading of chromosomes which are then fixed, mounted on a slide and stained.
Chromosome G-banding
treated with typsin to denature protein content and then stained with a DNA binding dye (Giemsa) to give a pattern of light and dark bands (400-500 bands per set)
Each band = 6-8 Mbp
Metaphase spreads
counting number of chromosomes
Idiogram
chromosome banding pattern
Fluorescence in-situ hybridisation (FISH)
can be used to detect and characterise subtle chromosome abnormalities: DNA probe is labeled with a fluorochrome which after hybridisation allows it to be visualised using a fluorescence microscope
Types of FISH probes:
• centromic probes- consist of repetitive DNA sequences found in and around centromere (useful for aneuploidy syndromes)
• chromosome specific unique-sequence probes - specific for a particular locus (useful for deletions and duplications)
• whole-chromosome paint probes - a mix of probes to fluoresce an entire chromosome (useful for translocations)
Euchromatin
stains lightly and consists of actively expressed genes
Increased acetylation of histones
Heterochromatin
stains darkly and is made up largely of inactive, unexpressed, repetitive DNA
Increased methylation if DNA
Inaccessible gene
Decreased acetylation
Increased methylation
More condensed
Heterochromatin
No access to TF
Inactive
Accessible gene
Increased acetylation
Decreased methylation
Less condensed
Euchromatin
Access to TF
Active
Epigenetics
How environmental influences, such as diet, stress and toxins, can subtly alter the genetic inheritance of an organism’s offspring, without changes to the DNA base sequence
Epigenome
the second layer of chemical tags that cover DNA and histones, and so determines the shape of the DNA-histone complex
It is flexible so can be reversed
It is independent so occurs in different forms at different areas of the DNA
Epigenetic imprinting
only inherit one working copy of a gene as one copy is epigenetically silenced through increased methylation of DNA during formation of oocytes and sperm
If a loss of imprinting- overproduction of proteins due to 2 active copies of gene
Acetylation of histones
acetyl groups can be added to lysine amino acids on histone proteins- lysine has a positively charged R group, which forms ionic bonds with the negatively charged phosphate backbone of DNA
Acetylation to lysine residues removes the positive ion and removes a bond between the histone protein and DNA so the complex is less condensed -TF and RNA polymerase can bind more easily so gene transcribed and expressed
Deacetylation returns lysine to its positively charged state which has a stronger attraction to the DNA molecule and inhibits transcription
Methylation of DNA
methyl groups can be added to a C atom on a cytosine base within
sequences with multiple C and G bases
The addition of methyl groups prevents the TF binding and attracts proteins that condense the complex by inducing deacetylation of histones so gene is not transcribed
Penetrance
An index of the proportion of individuals with a gene mutation who show it
Haploinsufficiency
Where a diploid organism only has a single functional copy of a gene (the other is inactive due to a mutation) and the single functional gene does not produce enough gene product to bring about a wild-type phenotype , resulting in disease
When is anticipation seen
Trinucleotide repeat disorders
ACMG Criteria
The American College of Medical Genetics and Genomics are used for the interpretation of sequence variants in Mendelian disorders. Variants are classified into five categories: pathogenic, likely pathogenic, uncertain significance (VUS), likely benign and benign.
Down Syndrome
A congenital condition caused by trisomy 21 (an extra copy of all or part of chromosome 21).
Edward’s syndrome
A congenital condition caused by trisomy 18 (an extra copy of all or part of chromosome 18).
Eugenics
pseudoscience that promotes the improvement of a species or race by means of selecting for particular inherited characteristics.
Hemizgous
term used to describe the genotype of a male with an X-linked trait (because males only have one X chromosome).
Lyonisation
process of inactivation of one of the X chromosomes in females
Patau syndrome
A congenital condition caused by trisomy 13 (an extra copy of all or part of chromosome 13).
Polymorphism
The presence of two or more variant forms of a specific genetic sequence in the genome. The sequences may vary by only a single nucleotide (called a single-nucleotide polymorphism) or may involve longer stretches of DNA.
Recurrence risk
statistic that estimates the probability that a condition present in one or more family members will recur in another relative in the same or future generations.
Imprinting
1 parental allele expressed, other suppressed by epigenetics
Prophase
Nuclear membrane: starts disintegrating
Spindle fibres: centrosome microtubules move to poles
Prometaphase
Nuclear membrane: dissolves
Spindle fibres: form and attach
Telophase
Nuclear membrane: reforms
Spindle fibres: disintegrate
Assortative mating
Choose mates with similar or dissimilar phenotypes
Knudson multiple hit hypothesis
> ## 2 defective alleles for disease eg cancer
What is southern blotting used for
DNA
What is northern blotting used for
RNA
What is western blotting used for
Proteins
What type of genetic condition is Huntington’s disease
Autosomal dominant
What are the 3 genetic mechanisms for Down syndrome
Gamete non-disjunction
Robertsonian translocation
Mosaic
What is the major genetic mechanism responsible for Prader-Willi syndrome
Micro-deletion of the parental chromosome
What is a reciprocal translocation
Transfer of genetic material between 2 non-homologous chromosomes caused by break points in each
Pleiotropy
A condition where a single mutation causes more than one observable phenotypic effect
Example of a pleiotropy condition
Phenylketonuria
What is the transcriptome
Composed of all RNA present in a cell
- Where does alternative splicing producing different gene products occur?
IN mRNA
