DNA Sequencing and Disease Causation

Question 1

What are the advantages and disadvantages of array CGH sequencing compared to karyotyping?

Answer 1

Advantages:
Higher resolution (~100x)
Automated
Can be performed on stored DNA

Disadvantages:
No direct visualization of chromosomes means no info on structure (only copy number).
Copy numbers are relative so you would miss triploidy (rare)

Question 2

How can changes in the genetic sequence lead to hypertrophic cardiomyopathy?

Answer 2

Nonsense mutations in myosin binding protein C and missense mutations in the Beta myosin heavy chain create changes in the structure and thus function of these proteins. This increases the risk of HCM

Question 3

Outline the process of PCR

Answer 3

Mix the double stranded DNA sample, primers, free nucleotides and DNA polymerase. Heat the mixture to 95 degrees to denature the DNA, separating the 2 strands.

Cool to 55 degrees to allow primers to anneal to the strands. Increase to 70 degrees (optimum for Taq polymerase).

The Taq polymerase forms comp base pairs w each DNA sample strand using the free nucleotides. This produces TWO double stranded DNA samples. This cycle is repeated to give rise to an amount of DNA sufficient to create a DNA profile.

Question 4

Describe the process of Sanger sequencing

Answer 4

Hay a specific primer and DNA polymerase which adds nucleotides into a corresponding sequence.

To find the exact composition of the DNA sequence, hay que stop the reaction to identify the end base of this particular DNA fragment. This is done w a dideoxynucleotide (removing an oxygen atom from the ribonucleotide).

The polymerase enzyme can no longer add normal nucleotides onto this DNA chain. We identify the chain terminating nucleotide by a specific fluorescent dye, 4 specific colors for each base to produce the sequence below:

Question 5

Describe the steps in next generation sequencing

Answer 5

Extracted DNA is randomly broken into <1000 bp fragments and bound to known adaptor sequences:

Question 6

Give pros and cons of single based sequencing

Answer 6

•Single gene: used for single gene disorders eg: Neurofibromatosis type 1 is due to a variant in NF1.

• Cheap, fast, reliable. Low risk of incidental findings and/or VUS (variant of uncertain significance)
• Cons: imited to 1 gene. Single read so difficult to detect mosaicism.
• Does not detect structural rearrangements
• Misses non-coding regions

Question 7

Describe gene panel sequencing

Answer 7

• Gene panel: used when a disease has many genes linked to it, like Lynch syndrome
• Fairly cheap, reads multiple genes so good read depth (e.g. can detect mosaicism)
• Cons: limited to genes selected for panel
• Difficult to update
• Does not detect structural rearrangements
• Misses non-coding regions

Question 8

Describe whole exome sequencing

Answer 8

Whole exome sequencing is just of the exons, whole genome is everything)

• Can identify novel gene-disease associations
• No need to update (can choose genes)
• Can be performed rapidly if indicated (e.g. prenatal)
• Cons: limited read depth. Risk of VUS
• Can’t detect structural rearrangements. Misses non-coding regions

Question 9

Describe whole genome sequencing

Answer 9

Entire genome, inc non-coding regions. V expensive, takes time.

• Can identify novel gene-disease associations and structural rearrangements
• Uniform coverage (incl. non-coding regions)
• Cons: poor read depth, high risk of VUS*
• Large data volume is difficult to interpret

Question 10

What is Huntington’s disease?

Answer 10

Neurological features: Chorea (>90% individuals). Impaired voluntary movements. Gait disturbance. Dysphagia/dysarthria

Psychiatric Features: Change in personality, increased incidence of suicide, cognitive decline

No cure, but:

• Medications to help chorea
• Antidepressants and antipsychotics
• Therapies – OT, SLT, physiotherapy

Question 11

What is a triplet repeat and how does it rleate to Huntingtons disease?

Answer 11

A triplet repeat= 3 bases of DNA coding for an amino acid repeated over and over again. In Huntington’s disease, the amino acid which is repeated is glutamine coded by C A G within the Huntington gene (HTT.)

It is normal to carry up to 27 repeats of glutamine in your Huntington’s gene, but if you have more than 39 repeats then you will develop HD

Question 12

What is anticipation and how is it linked to HD?

Answer 12

Anticipation: successive generations of a family may present symptoms at increasingly early ages due to an increased size a triplet repeat.

DNA repair/replication apparatus “slips” on CAG repeats, so the size of the CAG repeat increases and thus so does liklehood of developing HD

Occurs more commonly in paternal transmission. This is bc sperm precursors constantly replicating = more opportunities for errors. Only one round of DNA replication in oogenesis.

Question 13

Describe fragile X syndrome

Answer 13

Caused by CGG trinucleotide repeat (>200 repeats) in the 5’-UTR of FMR1 on the X- chromosome.

X linked recessive inheritance. Females can be affected due to skewed X inactivation

Aberrant hypermethylation of the expanded repeat leads to decrease in/silencing of FMR1 transcription

Question 14

What is imprinting? Draw a diagram to explain it

Answer 14

Imprinting is a process which leads to genes being expressed in a parent of origin specific manner

Takes place in gametes, maintained throughout mitotic divisions

~80 human genes are involved in imprinting, many of which are for embryonic growth and placental development

Imprinting disorders result from changes in the expression of imprinted genes—either through genetic changes in the genes themselves, or epigenetic changes in their control. Hay a number of different imprinting disorders with distinct phenotypes

Question 15

Describe the symptoms of Prader Willi Syndrome and what its caused by

Answer 15

Infancy: Hypotonia with history of poor suck. Poor feeding. Global developmental delay

Childhood: Excessive eating with central obesity if uncontrolled

Adulthood: Cognitive impairment, usually mild intellectual disability. Excessive eating with central obesity if uncontrolled. Hypothalamic hypogonadism and/or typical behaviour problems

Caused by an absence of imprinted gene expression in the paternally derived region of chromosome 15. In most cases, this is caused by a maternally derived deletion on 15 Q. Can also be caused by maternal disomy

Question 16

Describe Angelman’s syndrome and what it is caused by

Answer 16

• Severe developmental delay or intellectual disability, severe speech impairment.
• Gait ataxia and/or tremulousness of the limbs
• Typical behavioural profile - happy demeanor- frqnt laughing, smiling, and excitability
• Microcephaly
• Seizures are also common

Cause: abnormal methylation at 15q11.2-q13 due to:

Deletion of 15q11.2-q13 on maternal chromosome 15. Paternal disomy of paternal chromosome 15. Imprinting defect on maternal chromosome 15

Question 17

Describe genetic counselling with respect to imprinting disorders

Answer 17

Risk to other family members dependent on mechanism by which disease is caused

Low risk of having another affected child if confirmed imprinting defect

If caused by a deletion that is proved to be de novo still a risk of germline mosaicism

Other mechanisms such as balanced translocations may have higher risk

Question 18

Describe Beckwith-Wiedemann Syndrome

Answer 18

Beckwith-Wiedemann Syndrome: Neonatal hypoglycaemia, Macrosomia, Macroglossia, Hemihypertrophy, Embryonal tumours, Renal abnormalities

Silver Russell Syndrome: Asymmetric IUGR with relative macrocephaly, Triangular face, Frontal bossing, Body asymmetry, Growth Failure.

Both of these conditions are caused by changes affecting a set of imprinted genes on chromosome 11

Question 19

Features of mitochondrial disease?

Answer 19

● Mitochondrial diseases cause defects in energy production. They are clinically heterogeneous but usually severe diseases

● Include deafness, blindness, diabetes, loss of skills, and heart and liver failure

● Mitochondrial diseases often do not have a clear genotype/phenotype correlation

Question 20

Give an example of a mitochondrial disorder

Answer 20

Leber Hereditary Optic Neuropathy (LHON)

● Bilateral, painless, subacute visual failure

● Males are four to five times more likely than females to be affected

● In about 25% of cases, visual loss is bilateral at onset.

● Neurologic abnormalities such as postural tremor, peripheral neuropathy, myopathy, and movement disorders have been reported

Question 21

How are mitochondrial disorders passed on?

Answer 21

Maternally inherited

Note mitochondrial diseases can be inherited in a Mendelian fashion as the majority of mitochondrial proteins are encoded in the nuclear genome

Question 22

How can a mother with a mitochondrial disorder have one child with and one child without the disorder?

Answer 22

It could be that this disorder has reduced penetrance i.e not everybody who carries the underlying genetic change will develop the disorder.

Or that the condition has variable expressivity whereby two people with the same genetic change may have very different phenotypes- some are affected so mildly that it appears sub clinical

Question 23

What is meant by Homoplasmy and Heteroplasmy in mitchondrial DNA?

Answer 23

Question 24

How does Homoplasmy and Heteroplasmy in mitchondrial DNA affect inheritance of mitochondrial disorders?

Answer 24

Unaffected carrier mothers can transmit mitochondrial diseases due to uneven distribution of mutant mitochondria during oogenesis:

Question 25

Describe

Answer 25

Multisystem disorder characterized by myoclonus (often the first symptom) followed by generalized epilepsy, ataxia, weakness, and dementia.

Onset is usually in childhood, occurring after normal early development.

Common findings= hearing loss, lipomas, short stature, optic atrophy, and cardiomyopathy with Wolff-Parkinson-White (WPW) syndrome.