Genetics Flashcards
Where in the cell is DNA stored?
In chromosomes found in the nucleus
How many human genes are there?
Approximately 20 000
Where in the cell in mitochondrial DNA stored?
In mitochondria found in the cytoplasm
What are the DNA nucleotides?
Purines - Adenine and Guanine
Pyrimidines - Cytosine and Thymine
Which nucleotides form pairs?
A and T
C and G
How many chromosomes are in a human cell?
46 = 23 pairs
What is a centromere?
Constricted region separating p and q arms of chromosome that helps align chromosomes during cell division
What is a telomere and its code in humans?
Repetitive DNA segment located at the end of a chromosome for protection
TTAGGG
What is an exon?
Protein encoding DNA
What is an intron?
Non-protein encoding DNA, can alter gene expression
How many genes are there in the mitgenome?
37 genes
What is transcription and how is it performed?
Process of turning DNA into RNA.
RNA polymerase synthesises mRNA
What is translation and how is it performed?
Turning mRNA into a protein
At the ribosome mRNA is translated in sets of 3 bases (codon) which correlate to specific amino acids. tRNA facilitates the addition of specific amino acids to the polypeptide chain
What are the different types of genetic diseases?
- Chromosomal - numerical, structural
- Single gene: AD, AR, X-linked
- Mitochondrial
- Imprinting
- Multifactorial (gene + environment)
- Acquired somatic (chromosome or gene)
What is a variant?
A change in DNA
What is the difference between a SNP and a SNV?
Both mean a single substitution in single nucleotide, however SNP must be common (>1%) and only occurs in germline DNA
What is a germline variant?
Inherited variation present in all body cells (including gametes)
What is a somatic variant?
Acquired variation after conception present in some cells and not in gametes
What is Chromosomal variation?
Change in number or structure of chromosomes
What are the different types of chromosomal variation?
- Numerical: gain or loss of entire chromosome or chromosome sets
- structural: deletion, duplication or rearrangement of chromosomes
What is a balanced chromosomal variation?
Rearrangement of genetic material such as through inversion or translocation that results in overall no gain or loss of genetic material
Can produce gametes with incomplete sets of chromosomes
What is an unbalanced chromosome variation?
Gain or loss of genetic material such as through deletion, duplication, trisomy or monosomy
What is tetrasomy?
Having 2 copies of same chromosome
What is triploidy?
Having extra set of chromosomes
What is an Indel?
Small insertion or deletion of <50 bases in a gene
What is a copy number variant?
A deletion or duplication in the number of copies of a DNA segment
What is a missense variant?
A SNV which results in a different amino acid in the protein
What is a nonsense variant?
A SNV which causes the premature termination of a protein
What is a frameshift variant?
An Indel that disrupts the triplet reading frame of DNA
Most commonly results in truncated protein as leads to creation of stop codon
What is a splicing variant?
An alteration in the DNA sequence occurring at the boundary of an exon and intron (splice site)
What are consequences of splicing variant?
Can result in the gain or loss of exons or the inclusion of introns altering the protein coding sequence
What is a trinucleotide repeat?
Sequence of 3 nucleotides repeated in tandem on the same contiguous section of a gene
What is heteroplasmy?
Mixture of mtDNA copies with mutations and normal copies
What is homoplasmy?
All mTDNA copies have mutations
What is imprinting?
Differential expression of a gene according to parent of origin due to methylation status
(e.g paternally imprinted = paternal copy is inactive)
How do imprinting disorders arise?
From an imbalance of imprinted genes such as:
-chromosomal deletion/duplication/rearrangement
-uniparental disomy
-methylation loss/gain
- single gene sequence variants
What is uniparental disomy?
Where an individual receives 2 copies of a chromosome or chromosome section from one parent and none from the other
What do the square, circle, diamond and triangle represent on a pedigree?
Square - male
Circle - female
Diamond - sex unknown
Triangle - miscarriage
What does a coloured in symbol mean on a pedigree?
Affected
What does a struck out symbol mean on a pedigree?
Deceased
What does a double line between 2 individuals mean on a pedigree?
Consanguineous relationship
What does a diagonal arrow to an individual mean on a pedigree?
That person is providing information
What are features of autosomal dominant inheritance?
- no skipped generations
- Transmitted by males and females
- Affects males and females
- Heterozygotes affected
What is the offspring risk of an affected parent with and AD condition?
50%
What does expression mean in relation to AD inheritance?
The degree to which a disorder is expressed in an individual
What does penetrance mean in relation to AD inheritance?
The proportion of individuals with the gene mutation who are clinically affected
What is anticipation in relation to AD inheritance?
When there is increasing severity and/or earlier onset of disease with each successive generation due to expansion of trinucleotide repeats
What are features of autosomal recessive inheritance?
- needs to have mutation in both copies of the gene to be affected
- usually individuals affected in a single generation
- both parents are carriers and unaffected
- affects Males and Females
What is a compound heterozygote?
A person with 2 different mutations in each copy of a gene
What are the features of X-linked recessive inheritance?
- transmitted through carrier females
- mostly affects males
- usually female carriers unaffected
- affected males cannot transmit disorder to their sons
What are features of mitochondrial inheritance?
- transmitted through females only
- males and females affected
- affected males cannot transmit to their offspring
What are features of imprinted inheritance?
- inherited in similar pattern to AD
- affects males and females
- gender of transmitting parent will determine expression in children
- transmitting parent may show non penetrance
What is the aetiology and incidence of Down syndrome?
Trisomy 21
Results from:
Non-disjunction during cell division (usually egg),
OR
Unbalanced translocation (unaffected parent has blanced translocation)
OR
Mosaic
1/800 births
What is the chance of a sibling being born with Down syndrome?
Low chance if non-disjunction or mosaic
10-15% chance if Mother is carrier of translocation
What are the clinical features of Down Syndrome?
- Developmental delay and ID
- Characteristic facial features
- Muscle hypotonia in infancy
- Congenital heart disease
- GORD
- coeliac disease
- Hypothyroidism
- Leukaemia
- Early onset dementia
What prenatal screening is available for Down Syndrome?
1st trimester (9-13+6): PAPP-A, bHCG, nuchal translucency
OR
NIPT blood test
What is the cause an incidence of Turner Syndrome?
Partial or complete loss of X chromosome
1/2500 lives births
What are consequences of Turner Syndrome?
-Physical: short, shield shaped chest with wide nipples
- lymphoedema
- Cardiac anomalies (coarctation of aorta)
- primary ovarian failure
- neurocognitive difficulties
- endocrinopathies such as hypothyroid
What is the aetiology and incidence of Kleinfelter syndrome?
One or more extra X chromosome
The more X the more severe
1 in 450 men
What are features of Kleinfelter?
Low muscle tone
Developmental delay
Hypoandrogenism
Metabolic syndrome
Hypothyroidism
VTE
Osteoporosis
Increased risk of cancer
What is the aetiology and diagnostic technique of Di George syndrome?
Deletion 22q11.2
Diagnosed on microarray or FISH
What are features of Di George syndrome?
- Congenital heart disease esp conotruncal malformations
- palate abnormalities
- Characteristic face (low set ears, wide eyes)
- Learning difficulties
- immune deficiency
What is Huntington disease and its aetiology?
Triplet repeat expansion (CAG) in HTT gene inherited in autosomal dominant fashion
Progressive neurological disorder onset at 35-44 years of age with 15-18 years survival after onset
What are features of Huntington disease?
- movement disorder: chorea, bradykinesia, rigidity, dystonia
- impaired voluntary motor function
- cognitive decline
- psychiatric disturbance
What is the neuropathology of Huntington disease?
Intraneuronal includions of huntingtin protein in caudate, putamen and cerebral cortex
What number of CAG repeats correlate with disease in Huntington disease?
Normal < 26
27-35 high risk for transmitting disease
36-39 reduced penetrance
> 40 full penetrance
Juvenile onset > 60
Why is anticipation more common with paternal transmission of Huntington Disease?
Instability of CAG repeat during spermatogenesis
What are Spinocerebellar ataxia and their mode of inheritance?
Hereditary ataxias characterised by degeneration of cerebellum +/- spinal cord
AD inheritance: over 40 types which can be repeat expansion with anticipation or non-repeat expansion
What are the clinical features of spinocerebellar ataxia?
-onset age 30-40 years (some in childhood)
- start with unsteady gait
- difficulty with co-ordination
- Dysarthria and dysphagia
- oculomotor abnormalities
- Non-Ataxic: movement disorder, epilepsy, motor/somatic, urinary symptoms, sleep disorders
What are the diagnostic and predictive testing for spinocerebellar ataxia?
Diagnostic = repeat expansion panels and exome sequencing
Predictive = targeted testing for familial pathogenic variant in unaffected
What are features of myotonic dystrophy?
Mild - cataract, mild myotonia, diabetes
Classic - muscle weakness + wasting, myotonia, cataract, cardiac conduction abnormality, endocrinopathy, shortened life span
Congenital - hypotonia, generalised weakness at birth, respiratory insufficiency, ID, premature death
How is myotonic dystrophy diagnosed?
Expansion of CTG trinucleotide repeat in DMPK gene
How many repeats in DMPK gene correlate with symptoms?
38-49 = premutation
50-150 = mild
50-1000 = classical
> 800 = childhood onset
>1000 = congenital
What are the inheritance features of myotonic dystrophy?
AD
High penetrance
Demonstrates anticipation (maternal > paternal)
Symptom onset correlates with repeat size
What are dystrophinopathies and the different types?
X-linked diseases caused by pathogenic variants in DMD gene
DMD = duchenne muscular dystrophy
BMD = Becker muscular dystrophy
DMD-associated dilated cardiomyopathy
Mild disease with elevated CK, muscle cramps and myoglobinuria
Why is DMD more severe than BMD?
DMD results from frameshift or nonsense mutation causing early truncation of protein, where as BMB results from in-frame mutation
What are features of Duchenne Muscular Dystrophy?
Presents in childhood with delayed motor milestones
Proximal weakness
Elevated CK (10xULN)
Rapidly progressive
Associated with dilated cardiomyopathy
What are features of Becker Muscular dystrophy?
Later onset muscle weakness, proximal > distal
Calf muscle hypertrophy
Elevated CK (5x ULN)
Preserved neck flexor strength
Associated with dilated cardiomyopathy
What are feature of DMD-associated dilated cardiomyopathy?
LV dilatation and CHF
Presents in males 20-40s, rapid progressing
Later onset and slower progression in women
CK increased
What is Marfan syndrome?
AD inherited FBN1 gene mutation resulting in systemic connective tissue disorder
Cardinal features: eyes, skeletal and cardiovascular (aorta)
What are the clinical features of Marfan syndrome?
Eyes: myopia and ectopia lentis, increased risk of retinal detachment, glaucoma and cataracts
Skeletal: bone overgrowth, joint laxity and reduced joint mobility, long extremities, chest deformity, scoliosis, flat feet, protrusio acetabuli
Cardiovascular: Aortic dilatation, MV prolapse +/-MR, TV prolapse, proximal PA enlargement
Other: striae, bulla
What is the biggest intrapartum risk with Marfan syndrome?
Rapid progression of aortic root dilatation can result in dissection or rupture during pregnancy, delivery and post partum
What are the diagnostic criteria of Marfan syndrome in someone WITHOUT a family history?
- Aortic Z-score > 2 and ectopia lentis
- Aortic Z-score > 2 and FBN-1 mutation associated with aortic aneurysm
- Aortic Z-score > 2 and systemci score > 7
- Ectopia lentis AND FBN-1 mutation associated with aortic aneurysm
What is the diagnostic criteria of Marfan syndrome for someone WITH a family history?
- Ectopia lentis and 1st degree relative with Marfan
- Systemtic score >7 and 1st degree relative with Marfan
- Aortic Z-score > 2 and 1st degree relative with Marfan
What are differences between Loeys-Dietz and Marfan syndromes?
- No ectopia lentis in LD
- More severe aneurysms in LD
What are the clinical features of Noonan syndrome?
-Short stature
-Characteristics: broad neck, wide spaced nipples, ptosis, low set ears, short wide nose
-CV: PV stenosis, HCM
- developmental delay
- coagulation defects
- lymphatic dysplasia
- Cryptorchidism
-Neoplasms: neuroblastoma, ALL, AML, JMML, low grade glioma, rhabdomyosarcoma
What is the inheritance pattern and genes involved in Noonan syndrome?
- AD: PTPN11 (others such as SOS1, RAF1, RIT1, BRAF, MAP2K1, NRAS, CBL, MRAS, SHOC2
-AD or AR: LZTR1
What are clinical features of RASopathies?
-craniofacial malformation
-congenital heart disease
- short stature, failure to thrive
- neurocognitive impairment
- cancer predisposition
What is the inheritance pattern of NF-1?
AD, mutation in NF1 gene encoding Neurofibromin protein with variable expression
Prevalence of 1 in 4000-5000
How is NF1 diagnosed?
Clinical diagnosis:
2 or more of:
- 6 or more cafe au lait spot (>15 mm)
- 2 or more neurofibroma OR 1 plexiform neurofibroma
- Axillary or groin freckling
- Lisch nodules (brown nodules in eyes)
- optic pathway glioma
- 1st degree relative with NF1
- Sphenoid wing dysplasia OR thinking of lone bone cortex
Genetic diagnosis if uncertain or for reproductive considerations
What cancers are associated with NF1?
- optic nerve glioma
- Breast cancer (women only)
- phaeochromocytoma
- malignant peripheral nerve sheath tumour
What is the inheritance pattern of TSC?
AD inheritance of TSC1 (hamartin) or TSC2 (tuberin) gene mutations
What are clinical features of TSC?
Tumours of heart, skin, brain, lungs and kidneys
Seizures
TSC-associated neuropsychiatric disorder
How is TSC diagnosed?
- genetic test (10-25% have no mutation identified)
- clinical diagnosis
What are the reproductive options available to couples in genetic counselling where there is no specific familial variant and couple not interested in pregnancy testing?
- No intervention: indicated when low risk to children
- Sperm/egg donation:
- Detailed anomaly scans via maternal-fetal-medicine: for structural anomalies detectable antenatally
What types of prenatal invasive testing are available and their risks (with a specific familial variation known)?
- Chorionic villus sampling (CVS): samples placenta from 11 weeks gestation, miscarriage risk of 1-2%
- Amniocentesis: sample of amniotic fluid from 16 weeks, 0.5-1% miscarriage risk
- Pre-implantation genetic diagnosis (PGD): privately funded in specialist centres, testing of IVF embryo before implantation. Costs of finances, emotional and psychological.
What is carrier testing and in what inheritance patterns is it indicated?
A genetic test in an asymptomatic individual to clarify their genetic status for a condition, with a known pathological variant in family
Indicated in autosomal recessive and x-linked recessive conditions
In asymptomatic patients with a positive family history, when is carrier test NOT offered?
- for variants of uncertain significance
- for variants with limited clinical utility
- for variants identified in unaccredited labs
- for children/minors who cannot consent and does not impact their immediate clinical management
What is a predictive genetic test and when is it indicated?
A genetic test in a pre-symptomatic individual to clarify their risk of developing a condition
Must have a known pathological variant in family
When is predictive testing not indicated in an individual with a positive family history?
- for variants of uncertain significance
- for variants with limited clinical utility
- for variants identified in unaccredited labs
- for children/minors who cannot consent and does not impact their immediate clinical management
What are advantages of predictive testing to individuals?
- implement medical management such as surveillance
- plan/modify career and lifestyle
- make reproductive decisions
- relieve anxiety related to uncertainty of risk
What are disadvantages of predictive testing to individuals?
- No change to medical management if no treatment or surveillance exists
- create anxiety to uncertainty of symptom development
- change in family dynamics and relationships (e.g. if discrepent results)
- create sense of genetic guilt for children/parents
- discrimination from employment and insurance providers
- loss in autonomy for children
What is the process for predictive testing for neurodegenerative disorders?
- Genetic counselling to provide information and clarify motivation
- Psychologist review to perform mental state assessment and impact of result
- Geneticist review to perform clinical exam and obtain consent
- Geneticist review to provide results
What are the 3 different types of gene variants, their heritability and risk to relatives?
- Germline: heritable, a risk to relatives
- Acquired: somatic, no risk to relatives
- Mosaic: potentially heritable, risk to offspring only
Mutations in what types of genes cause cancer?
Proto-oncogenes
Tumour suppressor genes
Mismatch repair genes
What are gain of function variants and their heritability?
Variants in a gene (proto-onogene) or its promotor which increase the number and/or function of protein
Rarely inherited and typically acquired
What are 2 examples of inherited gain of function variants?
-RET gene in MEN2
-MET gene in Herediatry papillary renal cell carcinoma type 1
What are loss of function variants and how many copies need to be affected?
Decreased expression of a gene encoding for protein involved in DNA damage repair, regulation of cell division OR promotion of apoptosis
Need to lose function fo both copies (either inherited and/or acquired)
What are methods of tumour testing for loss of function gene variants?
-IHC
- testing for consequence of loss of function (Microsatellite instability)
-Tumour gene testing: methylation, sequencing and copy number analysis
What indicates an abnormal result on IHC for loss of function testing?
Loss of protein
What are limitations of IHC when used for assessing loss of function variants?
- does not inform mechanism of protein loss
- may be normal but protein is dysfunctional
What familial cancer syndromes have IHC screening tests available?
- Lynch syndrome: MLH1, PMS2, MSH2, MSH6
-HPT-JTS: parafibromin
-Phaeo-paraganglioma predisposition syndrome: SDHA, SDHB - HLRCC: fumarate hydrase
What is the two hit phenomenon of hereditary cancer?
Inheriting a pathogenic variant in a single copy and then losing the wild type allele (somatic loss) resulting in genomic instability
What is the risk to family members with AD inheritance in familial cancer syndromes?
- 50% risk
- Except in de novo mutation, where 1% risk to siblings due to gonadal mosaicism
What is the risk of autosomal inheritance if both parents carry a copy of the pathogenic variant?
50% risk of being a carrier
25% risk of being affected
What are 2 examples of common AR inherited familial cancer syndromes?
- MUTYH associated polyposis (MAP)
- Fanconi anaemia
What defines a clinically actionable variant?
- high risk
- inform the management of patient
- genetic testing will change management of unaffected relatives
- implications for related bone marrow donors
- family planning implications
What proprtion of bowel cancers are due to an inheritable predisposition?
5-10%
What is Lynch syndrome, its prevalance and surveillance/treatment?
Pathogenic variant in one of the MMR genes MLH1, PMS2, MSH6, MSH2/EPCAM
Associated with increased risk of colorectal, endometrial, ovarian and other GI cancers
1/440
Surveillance colonoscopies yearly from age 25
TAH + BSO age 40
Aspirin
Enrolment in familial cancer registry
What are the characteristic patterns of mismatch protein repair loss on IHC of colorectal cancer?
-MSH2 and MSH6
-MSH6 alone
-MLH1 and PMS2
-PMS2 alone
What patterns of mismatch repair protein loss on IHC of colorectal tumours should be referred to genetic cancer service for consideration of Lynch syndrome?
-PMS2 alone
-MSH6 alone
-MSH2 and MSH6
- MLH1 and PSM2 if BRAF wildtype
What are the classification of variants and their meaning?
Class 1 = not pathogenic
Class 2 = unlikely pathogenic
Class 3 = uncertain
Class 4 = likely pathogenic (clinically significant)
Class 5 = pathogenic (clinically significant)
When should a genetic basis of hypertrophic cardiomyopathy be suspected?
Asymmetric thickening of myocardium >15 mm not attributable to hypertension or valve disease
What is the genetic basis of HCM?
Pathologic variants in MYBPC3 and MYH7 most common, affect sarcomere function
What is the treatment of hypertrophic cardiomyopathy?
- Treatment of heart failure via medications
- Treatment of obstruction via surgery, alcohol ablation
- Treatment of arrhythmia via medications
- ICD for primary or secondary prevention (guided by sudden cardia death risk assessment)
What is the commonest known genetic causes of DCM?
Titin (TTN) gene mutation, seen in 20% cases
LMNA seen in 5% cases
Both involved in sarcomere function
What is AVRC and its characteristic ECG and MRI findings?
Arrhythmogenic right ventricular cardiomyopathy. Fatty fibrous tissue replaces normal heart muscle predisposing to arrhythmia and heart failure
ECG = epsilon wave (positive inflection buried in QRS)
MRI = bright signal in RV free wall due to myocardial atrophy + fatty replacement
What is the most common genetic cause of Arrhythmogenic cardiomyopathy?
PKP2 pathologic variants, affect desmosome function
What are common features of familial cardiomyopathies?
- most autosomal dominant
-show age related and reduced penetrance
What specific treatment should an LMNA subtype DCM receive?
ICD
What are clinical features of long QT syndrome?
Prolongation of QTc represents delayed ventricular prolongation
Predisposes to malignant ventricular arrhythmias (Torsades, VFib)
What is the genetic basis of long QT syndrome?
Either:
- loss of function in cardiac K+ channel genes (KCNQ1 = LQT1, KCNH2 = LQT2)
- gain of function in cardiac Na+ channel genes (SCN53A = LQT3)
What is catecholaminergic polymorphic ventricular tachycardia?
Exercise or emotion induced bidirectional or polymorphic VT
Most commonly gain of function RyR2 gene mutation (affects calcium flow) inherited in AD fashion
Rarely CASQ2 gene inherited in AR fashion
Structurally normal heart and normal resting ECG
What is Brugada syndrome?
AD loss of function mutations in SCN5A seen in 20-30%
Characteristic coved ST segment elevations in V1-V3 similar to RBBB - may require provocation test
What is the genetic basis and features of Loeys Dietz syndrome?
AD TGFBR-2 or TGFBR-1 gene mutations
Arterial aneurysms or totuosity
Hypertelorism (increased distance between body parts)
Cleft palate, bifid uvula
What is the definition of gene therapy?
The treatment of genetic disease by transferring genetic material (DNA or RNA) into cels of the patient
What is gene augmentation therapy?
Introduction of a functional copy of a gene to a cell with a non-functioning gene copy
What is gene silencing therapy?
RNA interference - silencing of gene expression through the introduction of dsRNA which is processed to microRNA which results in the degradation of mRNA, preventing translation into a functional protein
What is gene editing as a gene therapy strategy?
Introduction of the desired gene via guide RNA and DNA-as such as CRISPR-CAS9
Compare ex vivo and in vivo gene therapies
In vivo = direct administration of delivery vector, less complex but need to consider toxicity or on-target effects in other tissues
Ex vivo = generation of genetically modified cells that are administered, more complex but can control which cells are affected
What is the role of Nucleic acid vectors?
To protect plasmids from degradation
To deliver nucleic acids to cytoplasm or nucleus
To control insertion point and limit immunogenecity
To give numerous copies in a single treatment
What are 4 types of viral vectors and their pros and cons?
- Adenovirus: large packaging size, fast expression, limited insertional mutagenesis, transietn expression and immunogenic
- HSV: transynaptic transmission, no risk of insertional mutagenesis, cytotoxic
-Adeno-associated virus: borad tropism, long expression, low risk insertional mutagenesis, can cross blood brain barrier, pre-existing antibodies, small size
-Lentivirus: long term expression, affects slowly dividing cells, risk of insertional mutagenesis and germ line transmission
What are pros and cons of non-integrating vectors for gene therapy?
No risk of insertional mutagenesis as not inserted into chromosome
Useful for post-mitotic cells
Exist as episome so not replicated in daughter cells
What are risks with gene therapy?
- insertional mutagenesis: results from random integration into genome disrupting nearby genes
-host immune responses: can result in SIRS, destruction of transduced cells
-Editing accuracy: “off target” edits may create functional impairments or cells with oncogenic potential
What is Voretigene neparvovec (Luturna)?
Gene augmentation therapy to treat RPE65-related retinal dystrophies
Adeno-associated viral vector containing functional copy of gene that is not integrated but is expressed
Currently not funded
What is onasemnogene abeparvovec (Zolgensma)?
Gene therapy that introduces functioning copy of SMN1 gene in AAV-9 vector administered as a single infusion for patients under 9 months
(Funded in Australia)
What is Nusinersen (Spinraza)?
Gene therapy of complimentary RNA sequence that binds to SMN2 gene facilitating increased SMN protein production
(Funded in Australia)
Requires q4monthly infusion
What is Inclisiran (leqvio)?
RNA silencing gene therapy that inhibits LDL receptor degradation used for treating familial hypercholesterolaemia
Q6monthly dosing
What types of genetic testing are available for chromosomal abnormalities?
- conventional (G-banded) karyotype
- FISH
-microarray
What types of genetic testing are available for Gene abnormalities?
- sanger sequencing
- multiplex ligation-dependent probe amplification (MLPA)
- southern blotting/triplet primed PCR
- genomic testing (targeted panel, whole exome, whole genome)
What types of genetic testing are available for mitochondrial abnormalities?
- sanger sequencing
- mitochondrial DNA genomic sequencing
What types of genetic testing are available for epigenetic abnormalities?
- microarray
- methylation specific mulitplex- dependent probe amplification (MS-MLPA)
-sanger sequencing
What is karyotype testing and what can it detect?
Disruption of cells during cell division (metaphase) and visualisation of chromosomes with special stains through light microscope
Polyploidy, anueploidy
Translocations, inversions, rings
CNVs 4-6Mb
What is FISH and what are its benefits?
Use of fluorescently labelled DNA probes to identify specific chromosomal abnormalities (2 probes used control and target)
Simple and quick, able to target region, detects smaller chromosome deletions/duplications not visible on karyotype
Unable to detect very small CNVs or gene sequence variants
What is a microarray and what are its benefits?
Chip of thousands of DNA probes hybridises with digested and labelled patient DNA
Able to detect CNVs including small ones, copy neutral changes such as seen in parental disomy
Unable to detect structural rearrangements or sequence variants
What is PCR?
Producing copy of target region identified by primers of DNA via DNA polymerase and amplifying it for detection
What is the gold standard test for single genes?
Sanger sequencing
How is Sanger sequencing performed?
Amplification of target DNA with fluorescently-labelled dideoxynucleotides to allow for detection
What is next generation sequencing?
PCR amplification of DNA applied to set of primers which is read and analysed
Often applied in targeted panel of exons
Identifies SNVs
What are the key limitations of gene testing?
Unable to assess for CNVs which account for approx 10% diseases (Can be detected on MPLA, whole genome sequencing but not used clinically)
Unable to detect nucleotide repeat expansions (use southern blot or triplet primed PCR)
What is MLPA?
Multiplex-ligation-dependent probe amplification
Quantitative method of analysis small CNVs (deletions/duplications) through the use of custom probes
What is Southern Blot?
Gel electrophoresis of digested DNA that is then hybridised to labelled probes and exposed to x-ray
Now superseded by triplet-primed PCR
How are imprinting inherited disorders tested?
MS-MLPA, amplification of digested DNA which is then analysed
Also detects CNVs
What are the roles of diagnostic genetic testing?
- In diagnostic uncertainty
- Management and surveillance planning
- Genetic counselling
What classifications of variants are reported on genetic testing?
-Variant of uncertain significance (class 3)
-Likely pathogenic (class 4)
-Pathogenic (class 5)
Does a pathogenic variant result on genetic test confirm a diagnosis?
Not necessarily
Need to clarify:
1. patient phenotype matches condition
2. Number and location of variants match mode of inheritance
Does a negative diagnostic genetic test exclude a genetic cause for a phenotype?
No - test selection and quality, patient characteristics and existing literature may limit ability to detect genetic basis
