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What are the three kinds of genetic disorders?

- chromosomal disorders
- single-gene (monogenic) disorders
- polygenic/multifactorial disorders


What are aneuploidies?

- abnormal chromosome number, caused by misassortment (non-disjunction) mainly in meiosis
- extra single chromosome: trisomy (e.g. 47 chrs)
- deletion of single chromosome: monosomy (e.g. 45 chrs)
- polyploidy: multiples of haploid number of chromosomes e.g. triploidy (3n = 69 chrs)

- tend to be very detrimental to life
- exception (to a certain extent) are aneuploidies of the sex chromosomes, but some still result in miscarriage
- aneuploidies of the autosomal chromosomes are highly problematic, the majority will not see to full term, let alone to see the end of the first trimester

exceptions e.g. trisomy 21
trisomy 18 and 13 may also make it to full term, severe disability

can also see mosaicism
- might occur post conception


What are structural abnormalities associated with chromosome disorders?

- rearrangement or deletion of chromosomes, caused by chromosome breakage during crossing over (includes translocations, inversions, duplications)
- can also get mosaicism


What are clinical indications for chromosome analysis?

- problems of early growth and development
- stillbirth and neonatal death
- fertility problems
- family history
- neoplasia
- pregnancy in a woman of advanced age or with an increased risk screening result


What is down syndrome?

most common autosomal chromosomal disorder that we see

major cause of intellectual disability and congenital heart disease
- characteristic set of facial and physical features
- associated with congenital anomalies of the GIT, increased risk of leukaemia, immune system defects, and an Alzheimer-like dementia (premature ageing)


What is the incidence of Down Syndrome?

1 in 660 births


What is classical cytogenetic analysis?

standard trisomy 21: 47,XX+21 or 47,XY+21
taken from dividing cells - have to grow them and have enough of them so can take a few weeks


What is FISH?

- FISHing for chromosomes
- fluorescence in situ hybridisation: chromosome 21 probe on interphase cells
- looking at non-dividing cells
- increasingly used in pregnancy, takes 24-48 hours
- fast test - may not be completely reliable
- offer the woman a karyotype before considering termination or continuing the pregnancy
- partly to do with sampling procedure
- look at the sex chromosomes and the 3 autosomal chromosomes that most commonly give rise to full term aneuploidy


What is chromosomal rearrangement?

- e.g. reciprocal translocation between chromosomes 21 and 3
- part of 21 onto 3, vice versa
- can be seen in a karyotype
- can have an abnormality that might not result in an abnormal phenotype: balanced translocation
- provided there is no net gain or loss of functional genetic material (e.g. enhancer region)
- can be a carrier of a translocation and not know it


What is a robertsonian translocation?

chromosome 21 rearrangement
- this term is used only for translocation between two ACROCENTRIC chromosomes (12, 14, 15, 21, 22)
- lose the short arms
- usually only one centromere
- will this alter phenotype? if no gain or loss, no


What unbalanced translocations in trisomy 21?

- 5% of down syndrome cases due to unbalanced translocations - net gain
- may have occurred during meiosis, or mitosis
- 1-2% of cases because its been inherited from a carrier parent
karyotype: 46,XX,der (14;21)+21

inherit from carrier with balanced translocation between chromosomes 14 and 21
45,XX der (14;21)


How many cases of Down syndrome are inherited?

- 1-2% of cases


What are microarrays?

- DNA chips
- DNA sequences and/or gene expression
- can look at single nucleotide polymorphisms (SNPs)
- can look at larger changes - number of chromosomes and copy number variations (CNVs)


What are chromosome microarrays (CMA)?

- CGH (comparitive genome hybridisation) and SNP arrays
-- also called molecular karyotyping
- probes (e.g. 25 base oligonucleotides) - along entire genome
- variable resolution
- example, for deletions and microdeletions
-- Wolf-Hirschhorn syndrome - 4p terminal deletion

- test of choice for a child with developmental delay
- covered on medicare
- done rather than a standard karyotype
- problem is that the better the resolution the more we see, but are they always pathogenic?


What are single gene disorders?

- pathology can generally be related to mutation/s in a single gene (genotype/phenotype correlation?)

inherited as:
- autosomal dominant, e.g. Huntington disease, osteogenesis imperfecta
- autosomal recessive e.g. cystic fibrosis, thalassaemias, sickle cell disease, hereditary haemochromatosis
- X-linked recessive, e.g. duchenne muscular dystrophy, haemophilia
- mitochondrial (maternal)

these days thinking more about modifier genes, epigenetics, environmental contribution to single gene defects

NB: penetrance, variable expressivity, genetic heterogeneity etc
are they truly monogenic? modifier genes?


What is direct genetic testing?

in pathology settings, main techniques used to look for single gene mutations are:
- polymerase chain reaction (PCR)
-- amplifies large amounts of specific sequences of DNA around the mutation site
- +/– restriction fragment polymorphisms (RFLPs)
-- uses restriction enzymes that cut specific sequences of DNA
-- design test so that cutting with the enzyme distinguishes between normal and mutated allele

now increasingly used:
- disease related panels of genes/gene mutations
- DNA microarrays (pathogenic SNPs)
- DNA sequencing (next generation/massively parallel sequencing)


What is beta-thalassaemia?

- mutations cause decreased synthesis of one or more b-globin chains
- lead to reduced or no production at all
- get imbalance in relative amounts of alpha and beta chains – het homotetramers instead of heterotetramers i.e. alpha-4
- homotetramers precipitate in RBCs and lead to their destruction and subsequent anaemia
- treatments but no cure
- common in people of certain ethnic backgrounds
-- people southern european, middle east, north africa, asia
-- migration patterns
- carrier frequency of up to 1:20, even 1:4
- autosomal recessive


What is a test for one mutation that leads to beta-thalassaemia?

- pcr + restriction enzyme for a beta-globin mutation
- mutation is in transcription start site: T changed to C
- leads to loss of recognition sequence for NcoI
- perform PCR first, then cut with NcoO
- analyse products after gel electrophoresis
- homozygous for the mutation = product does not cut at all so single band, thicker
- heterozygous = three bands
- homozygous not the mutation = two bands

- use a test like this when you know the mutation in the family
- e.g. during prenatal testing
- nowadays if you didn't know the mutation you would just sequence the whole genotype
- heterozygous result (or even homozygous negative) could still have other mutations (compound heterozygote)


What are polygenic/multifactorial disorders?

polygenic multifactorial trait/condition - additive contribution of several genes
- each gene confers a degree of susceptibility
- input of these genes not necessarily equal, often quite small
- continuously variable phenotypes
- e.g. height, body weight, cholesterol levels, diabetes, asthma, heart disease

- still don't really have a good diagnostic test for these
- that's because we don't know what all the different genes are


How do we test for complex diseases?

- testing still same as for single-gene conditions
- which genes?
- data based on genome wide association studies (GWAS)
-- SNPs that are shared with much greater frequency among individuals with the same phenotype than among others (controls)
- clinical utility?


Why do we do genetic testing?

- clinical diagnosis
- i.e. symptoms present (e.g. down syndrome, thalassaemia; all ages including prenatal)
- carrier testing (e.g. autosomal recessive, X-linked; cascade/families)
- pre-symptomatic/predictive testing (typically autosomal dominant, late-onset disorders, e.g. Huntington disease, BRAC1/2) -- lots of issues associated e.g. if one family member wants to know and another doesn't etc
- genetic screening?
-- across populations or targeted


What is prenatal diagnosis?

- helps families to make informed choices during pregnancy
- provides reassurance when result is normal
- provides risk information to couples who, otherwise, would not choose to begin pregnancy
- gives couple choice of termination of severely affected fetus
- allows couple to prepare psychologically for birth of affected baby
- helps doctor plan delivery, management and care of affected baby

- there are reasons that a couple might choose to have prenatal testing and that is absolutely their right
- about giving information to make informed decisions


What does prenatal diagnosis require?

- requires sample of foetal cells/DNA/RNA


What is chorionic villus sampling?

- from 11 weeks gestation usually
- placental tissue
- ultrasound
- invasive; risk of miscarriage ~1% > background risk
- if termination of pregnancy (TOP) requested, by aspiration under general anaesthetic


What is amniocentesis?

- 15 - 16 weeks gestation usually
- amniotic fluid (~20 mL), containing sloughed off fetal cells removed
- ultrasound
- invasive; risk of miscarriage ~0.5% > background
- if TOP requested, by prostaglandin induction of labour


What are alternative foetal samplings for testing?

- pre-implantation genetic diagnosis (PGD)
-- one or two cells from dividing zygote at 8 cell (or 100 cell) stage
-- requires IVF
-- unaffected embryos implanted
- expensive: 10 - 20,000

cell free fetal DNA/RNA in maternal blood - called non-invasive prenatal testing (NIPT)
- introduced in clinical settings in Australia in 2013; from ~ 10 weeks gestation; based on next generation sequencing; currently marketed as screening test


What is genetic screening?

- identifies a subset of individuals at high risk of having, or of transmitting to children, a specific genetic disorder
-- general population
-- at risk groups, targeted (ethnicity?)
- often not definitive (but is changing with advances in gene technology)
-- not all genes
-- not all mutations
- false positives and false negatives


Why do we do genetic screening?

- to prevent disease
- for early treatment
- for future reproductive options
- to decrease social and financial burdens (doesn't take into account the whole disability critic debate)


What are the criteria (principles) of screening for a genetic condition?

- must be an important health problem, severe and/or common
- must be preventable or treatable by acceptable methods (includes prenatal)
- screening test must be simple, safe, reliable and acceptable, relatively inexpensive
- education and counselling facilities must be generally available - informed decision-making


What genetic screening is done in victoria?

prenatal screening
- foetus: chromosomal ± neural tube defects
- parents: carrier status for haemoglobinopathies and cystic fibrosis (CF) in high risk ethnic groups

newborn screening
- CF, phenylketonuria (PKU), congenital hypothyroidism and ~20 metabolic (rare) conditions

preconception carrier screening
- ashkenazi jewish high school students (tay-sachs disease plus others with higher carrier frequencies in this ethnic group)
- ad hoc (haemoglobinopathies, CF, FXS?) – often testing occurs during pregnancy


What is prenatal screening?

- prevalence of birth defects: 4 out of 100 births
- offered to all pregnant women

i) ultrasound/nuchal translucency (oedema) - soft signs

ii) maternal serum screening
- chemicals in blood - biomarkers
1st trimester (2 analytes plus nuchal translucency - not neural tube defects)
2nd trimester (4 analytes)

iii) NIPT screening

about 80% of women in victoria have prenatal screening

these are non-invasive procedures and give RISK value only
diagnostic testing requires sample of foetal cells/DNA/RNA


What is personalised medicine?

- individualised care (treatment) based on genotype
-- whole genome/exome sequencing
-- lab-on-a-chip:
> DNA extraction, PCR, array hybridisation and detection on the one microfluidic platform - way of the future?