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Pedigrees of genetic disorders - Coggle Diagram
Pedigrees of genetic disorders
PCR
Fundamental basis of methods for detection of
Mutation
Gene structure
Transcriptional activity
Primers
Short, synthetic oligonucleotide
used in molecular techniques from PCR to DNA sequencing
Rules
17-38 bases long
Base composition should be 50-60% G+C
3' end should be G or C
Prevents breathing of ends
Increases efficiency of PCR
T\(_m\) = 55-80\(^o\)C
Primer self-complementarity should be avoided
Ability to form 2\(^o\) structures e.g. hairpins
Runs of 3 or more Cs or Gs at 3'-ends should be avoided
May promote mispriming at G or C rich sequences
Problems
Self dimerisation + hairpin loops
Microarrays
Multiplex assays replacing diagnostic PCR
Same hybridisation rules
Different technology - still reliant on manufacturing good oligonucleotide probes
Patterns of inheritance
Classification of genetic disorders
Single gene disorders
Medical conditions caused by change in one or both copies of a particular pair of genes
3 common types
Autosomal dominant
Heterozygotes
Affected with one copy of altered gene
Examples
Huntingtons
Neurofibromatosis type 1
Marfan syndrome
Arachnodactyly (long fingers)
Dislocated lens
Familial hypercholesterolemia
Familial Adenomatous Polyposis (FAP)
Prader-willi
Autosomal recessive
Homozygotes
Affected with 2 copies of altered gene
Examples
Sickle cell
Cystic fibrosis
Batten disease
Congenital deafness
Phenylketonuria (PKU)
Spinal muscular atrophy
Recessive blindness
Maple syrup urine disease
X-linked
Males with altered gene on X chromosome are always affected
Examples
Fragile X
Haemophilia
Duchenne muscular dystrophy
Fabry disease
Retinitis pigmentosa
Alport syndrome
Hunter syndrome
Ocular albinism
Adrenoleucodystrophy
Multifactorial diseases
Chromosome disorders
Mendelian inheritance
Autosomal vs. sex chromsomes
Autosomal
Genes located on autosomes
2 chromosomes 2 alleles
Heterozygous or homozygous
Dominant or recessive
Sex
Genes located on one of the sex chromosomes
Males are hemizygous
Dominant or recessive
Family studies
Observe
How a trait is passed from one generation to the next
Frequency in relatives
Advice for family on likelihood of family members developing a disease
e.g. child with unexplained fracture - history of fractures after trivial injuries may suggest osteogenesis imperfecta
Pedigree drawing
Helps to determine if a disease has a genetic origin
Predict which individuals may be carriers or go on to develop a condition
Recurrence risk
Probability that the offspring of a couple will express a genetic disease
Autosomal dominant
Affected parent will pass the dominant allele to 50% their offspring
Sex is not relevant
Unaffected individuals do not have the allele so cannot pass it on
Shows vertical inheritance pattern (affected individuals in each generation)
Triple repeat disorders
Triplet repeats show instability
Size of repeat expands in germ line
Repeat expansion may occur in exons, introns, UTRs or non-coding RNA regions of a gene
Many disorders show decreased age of onset when size of repeat expands
Repeat expansion occurs during subsequent generations
Anticipation
Parental origin (maternal or paternal) can often influence anticipation
Most often involves inheritance of mutation from paternal origin
Mechanisms of expansion causing disease
Depends on where the repeat occurs in a gene
Protein coding region
Gain of function
Toxic protein
RNA coding regions (introns and UTRs)
mRNA instability
Translation effects
Non-coding regions
Transcriptional effects
Interference with regulation of nearby genes
Mechanism for triple repeat expansion
Slippage
Daughter stand 'slips'
Unequal crossing over
Huntington's disease
Incidence
3 to 7 per 100,00
typically European descent
Neurodegenerative disease
Characterised by progressive dementia + involuntary movements
Degeneration of neurons in cerebral cortex
Chorea caused by degeneration of basal ganglia
Cognitive and language decline
No cure or treatment
Typical course of disease from 15 onwards
Autosomal dominant
Paternal anticipation
Polyglutamine disease
Expansion of CAG repeat within exon 1 of the Huntington gene
Polyglutamine tract within the Huntington protein
Function of Huntington protein currently unknown but expressed in all cells
Increase in polyglutamine length causes Huntington protein to aggregate
Inclusion bodies formed + protein behaves as toxin
Abnormal Huntington protein has been shown to interact with a number of transcription factor proteins
Deregulation of gene expression
Autosomal recessive
Family tree
Very different to autosomal dominant diseases
Not possible to trace mutant transmission without genetic testing
Carriers have no problems or phenotypes
Few individuals affected
Often only siblings are affected
Must have 2 carrier parents to be affected
1/4 siblings affected
Unaffected siblings have 2/3 chance of being carriers
Consanguinity
Mating within the family
Often first cousins mating
Greatly increases chance of 2 carriers mating
Sickle cell
Single point gene mutation
Glutamic acid substituted with valine at position 6 of \(\beta\)-globin chain
GAG \(\rightarrow\) GTG
Homozygous inheritance (HbSS) causes sickle cell disease
Individuals suffer sickle cell crisis episodes
Mutant \(\beta\)6 has poor solubility leading to problems
Cystic fibrosis
Genetic basis
Locus 7q31.2
Region q31.2 on long (q) arm of chromosome 7
Protein size
1480 amino acids
MW = 170kDa
N-glycosylation results in apparent 180kDa size
Cystic fibrosis transmembrane conductance regulator (CFTR) gene
Protein function
CFTR closely resembles solute transporter proteins + places CFTR as ATP binding cassette (ABC) transporter
Normal protein product is chloride channel protein in membranes of cells lining passageways of lungs, liver, pancreas, intestines, reproductive tract and skin
Also involved in regulation of other transport pathways
Controls chloride ion movement in and out of cells
Requires ATP to pump Cl\(^-\) across cell membrane
\(\Delta\)F508 is the predominant mutation in CF patients
66% of all CF patients
Prevalence varies between populations
Results from 3bp deletion eliminating a phenylalanine at position 508
1000 known mutations in the CFTR gene
Tests designed to detect most common mutations + identify carriers
OLA kit
Phenotype + biochemical testing
Problem
Mutations have different frequencies in diff. populations
Screening for 25 most common mutations will only detect ~57% carriers in Hispanic American populations
X-linked recessive disorders
Sex chromosomes
1095 genes mapped to X chromosome
Females have 2 x copies of ~950 genes that males only have 1 copy of
Pseudoautosomal region
Region of X and Y chromosomes that match
Necessary to line up correctly during mitosis
Inheritance pattern
Male to female to male
Cannot be male to male or female to female
Males are vulnerable because they only have one X
Females are protected by heterozygous advantage
Carrier mother + normal father
50% males affected
50% males unaffected
50% females carriers
50% females unaffected
Normal mother + affected father
All daughters are carriers
All sons are normal
Duchennes Muscular Dystrophy
DMD gene encodes dystrophin
Located close to the muscle cell membrane
Links actin to muscle cell cytoskeleton + membranes
Provides integrity to cell
2/3 cases transmit down lineages from carrier mothers
1/3 cases caused by new mutations (de novo)
No previous family history of disease
Usually lethal before 30
Due to pathology very few males are able to reproduce (low genetic fitness)
Mutations
Deletion correlates with severity of muscular dystrophy
If a deletion leads to Becker it tends to be less severe (typically in frame effect)
Allelic variants of DMB + BMD
Duchenne
Very large deletions/ out of frame changes to exons
Frame shift results in premature termination codons + truncated proteins
Frame shift from splicing mutations, deletions or duplications
Becker
Deletion or duplication
In-frame changes to exons
Protein produced with reduced expression or partially functional
Becker phenotype is less severe so ~90% mutations are inherited rather than 2/3
Gross pathology
Yellowish-white fat replaces normally reddish-brown skeletal muscle
Western and immunofluorescence
DMD
Almost no dystrophin present or severely reduced abundance
BMD
Dystrophin has reduced abundance
May appear normal or reduced size
Diagnosis/detection
Multiplex PCR detects mutations in dystrophin gene
Allows coverage of all exons
Fragile X
Triple repeat expansion (CGG) on X chromosome
Higher no. repeats = more severe disease
Characteristic features
Mental retardation
Learning difficulties
Prominent ears
Elongated face
Macro orchidism (enlarged testis)
Females are less severely affected than males (mothers with pre-mutation may be normal)
Only 50% of females with full mutation may manifest with learning difficulties
Shows anticipation in successive generations
Repeat expansion occurs during oogenesis
More severe when transmitted from a female
Analysis of mothers X demonstrates pre-mutation
Expands to give her son the disease
Diagnostic tests
Southern blot analysis of the triplet repeat gives indication of no. repeats
X chromosome shows breakage in a folate deficiency medium
Fragile site only in individuals with full mutation
Cytogenic test
Not explanation of disease mechanism
Genetic mechanism
CGG expansions cause >99% of fragile X syndrome cases
FMR1 gene has 10-50 copies of CGG in 5'UTR
In fragile X can be >200
High CGG allows methylation (expanded CpG island)
FMR1 expression silenced
FMRP protein deficiency
FMRP protein involved in normal miRNA function
Mis-processing of miRNAs may be responsible for mental retardation
Normal pathway is important in miRNA regulation of transcripts in the brain