Core monogenic disorders

Question 1

CF incidence & carrier frequency (UK)

Answer 1

Incidence = 1 in 2600
Carrier freq = 1 in 25

Question 2

CF clinical features

Answer 2

Chronic cough
Failure to thrive
Pancreatic insufficiency
Alkalosis and hypotonic dehydration
Neonatal intestinal obstruction
Clubbing of fingers
Recurrent infections
Electrolytes in sweat
Absence of vas deferens
Sputum with Staph or Pseudomonas

Question 3

CFTR protein structure & function

Answer 3

Expressed in plasma membranes of secretory epithelial cells in pancreatic, respiratory, GI and reproductive systems

Conducts flow to chloride irons across cell membrane

Regulatory domain is phosphorylated to open channel. 2x transmembrane domains bind chloride ions, making the channel negatively charged and creating an ionic gradient allowing water to exit cell by osmosis

Question 4

5 classes of CF variants

Answer 4

1. No synthesis, usually protein truncating nonsense/frameshift/splicing e.g. Gly542X
2. Block in processing leading to protein degradation e.g. Phe508del
3. Block in regulation, usually in R domain e.g. G551D
4. Altered conductance, usually in transmembrane domains e.g. Arg1152His
5. Regulation of other ion channels

Question 5

Severe / mild classes

Answer 5

Class 1/2 homozygotes or compound hetz are more severely affected with worse survival. No or severely reduced CFTR protein

Class 4 usually have mild symptoms (more panc/GI, less respiratory). Some functional CFTR protein

Question 6

CF testing strategy

Answer 6

Level 1 = multiplex commercial kit e.g. ARMS. Allele specific PCR assay. 2 reactions, common primer + WT or mutant specific primer. Sizing and use of fluorophores allows detection of many muts in one test. Cross reactivity can occur at codons with multiple muts

Level 2 = sequencing and CNV analysis (if non-Caucasian population or 1 variant identified by level 1)

Question 7

Explanation for a homozygous proband with CF but one parent is not a carrier?

Answer 7

UPD(7), hetz deletions of locus or non-paternity

Question 8

2 examples of CFTR modulator therapy

Answer 8

Ataluren (stop codon readthrough)

Ivacaftor

Question 9

What is the prior chance of an unaffected person being a CF carrier if they have an affected sibling?

Answer 9

2/3

Question 10

SMA incidence & carrier frequency (UK)

Answer 10

Incidence = 1 in 6000-10000
Carrier freq = 1 in 50

Question 11

SMA clinical features

Answer 11

Widespread synaptic defects at NMJs in anterior horn motor neurons of spinal cord

Progressive proximal and symmetrical limb/trunk weakness, breathing difficulties due to intercostal muscle paralysis, finger tremors, poor suck/swallow, aspiration pneumonia, neonatal hypotonia

Question 12

5 types of SMA

Answer 12

Prenatal
Arthrogryposis and no movements in utero due to contractures. Lethal in neonatal period if birth survived

Type 1, acute infantile - 60%
Dx <6m, death early childhood. Hypotonia & symmetrical floppy paralysis, little motor development

Type 2, chronic infantile - 27%
Dx 6-12m, 70% reach adulthood
Unable to walk unaided

Type 3, chronic juvenile - 12%
3a = Dx <3yo; 3b = Dx >3yo
Usually sit/walk unaided but with leg weakness

Type 4, adult - 1%
Similar to type 3 with later onset. Normal life expectancy

Question 13

Describe the 5q12.2q13.3 region

Answer 13

Complex unstable region

SMN1 and SMN2 pseudogene, originate from inverted duplication in tandem

Differ by 5bp (same aa sequence). Key change in SMN2 = c.840C>T disrupts an ESE and decreases splicing of exon 7 = truncated protein that is rapidly degraded. Can produce some functional protein

Can have up to 5 copies of SMN2 in tandem

4% of general population have 2 copies of SMN1 on same chromosome - silent carriers

Question 14

SMN protein function

Answer 14

Molecular chaperone for assembly of small nuclear riboproteins and vital for spliceosome function

Question 15

SMA genetic cause

Answer 15

95-98% = homozygous SMN1 deletion (of at least exon 7)

True deletion or gene conversion event (non-reciprocal exchange of DNA with SMN2 = hybrid SMN with splicing defective exon 7 from SMN2)

2-5% = compound hetz for SMN1 deletion and SNV

1% = homozygous SNV

Question 16

What is the de novo mutation rate for SMA?

Answer 16

2%

Due to instability and unequal Xover of region

Explains high incidence of disease with very high lethality (most affected do not reproduce)

Question 17

How does SMN2 copy number affect SMA presentation?

Answer 17

Directly impacts disease severity

SMN2 produces some functional protein that can compensate for SMN1 deletions

e.g. Type 1 SMA has ~9% full length protein

SMN protein levels of 23% required for normal motor neuron function

Reports of 5 copies of SMN2 and 0 copies of SMN1 in people with no SMA symptoms

Question 18

How does c.859G>C affect SMN2?

Answer 18

Introduces ESE in exon 7, increasing exon 7 inclusion and increasing full length protein translation

Attenuates disease

Question 19

What method is required to sequence the 5q12.2q13.3 region?

Answer 19

Allele specific long range PCR to distinguish SMN1/2

Question 20

2 examples of SMA treatments

Answer 20

Nusinersen/Spinraza and Risdiplam- ASOs that promotes inclusion of exon 7 in SMN2 to increase full length protein expression

Zolgensma - SMN1 transgene with synthetic promoter

Question 21

Describe XLR inheritance

Answer 21

Vertical transmission of mutant X from carrier mother to son

Usually family history of affected male relatives on maternal side

All daughter of affected males are obligate carriers

Females can be affected if X-inactivation is skewed, UPD(X), Turner syndrome, deletion or compound hetz.

Question 22

DMD clinical features

Answer 22

Progressive and symmetrical muscle weakness. Proximal > distal and lower > upper

Calf hypertrophy, Gowers sign and raised CK (enzyme released from damaged muscles)

Death by 20-30yo due to respiratory complications and cardiomyopathy

Question 23

BMD clinical features

Answer 23

Milder and later onset compared to DMD

Skeletal muscle weakness, neck flexor strength is intact and remain ambulatory into 30’s. Death by mid-40s due to cardiomyopathy

Question 24

What clinical features do female carriers exhibit?

Answer 24

Manifesting carriers have DMD related DCM later in life but no skeletal muscle weakness

Question 25

What is the function of dystrophin?

Answer 25

Rod shaped cytoskeletal protein essential for sarcolemma stability in muscle. Localises to cytoplasm, forms part of the dystrophin-associated protein complex (DAPC), links actin cytoskeleton to the extracellular matrix

Decreased dystrophin = instability of DAPC, membrane disruption and muscle fibre damage

Question 26

Explain the molecular difference between DMD and BMD

Answer 26

Affect on open reading frame (caution for dups as unknown if in tandem or not)

DMD is the complete loss of protein due to OUT OF FRAME CNVs

BMD is reduced amount of functional protein due to IN FRAME CNVs

Question 27

What are the most common variants in DMD?

Answer 27

Deletions = 61-66%
Duplications = 11-13%
Complex/SNV(nonsense) = remainder

Deletions are more common in BMD

Question 28

Where are the deletion hotspots in DMD?

Answer 28

Proximal exons 2-19 (30%)

Distal exons 45-55 (70%) with a common breakpoint in intron 44

Question 29

2x dystrophin-restoring treatments for DMD

Answer 29

Exondys51 - exon skipping ASO to restore ORF

Translara - stop codon readthrough to promote expression of some functional protein

Question 30

What does 4u represent in Bayes calculations for XL diseases?

Answer 30

Quantifies the small chance a female with no family history of XL disease is a carrier

Takes into account the de novo mutation rate in each generation

This is used as the ‘Prior’ chance of a female being a carrier in a Bayes calculation

1u is the ‘Conditional’ chance of a non-carrier female having an affected son