Molecular Genetics

Question 1

Name 2 X-Linked Recessive disorders

Answer 1

Duchenne/Becker Muscular Dystrophy
SBMA
Androgen Insensitivity Syndrome

Question 2

Name 2 X-Linked Dominant disorders

Answer 2

X-linked hypophosphatemia
X-linked Alport Syndrome
Rett Syndrome

Question 3

Name three clinical features associated with CF

Answer 3

Chronic coughing and wheezing
Failure to thrive
Pancreatic insufficiency

Question 4

Name the most common CF mutation

Answer 4

p.Phe508del - 75%

Question 5

Describe the function of CFTR (7q31.2)

Answer 5

Cyclic AMP-activated chloride channel located in the plasma membrane of secretory eithelial cells

Question 6

How many types of SMA are there?

Answer 6

6 - Prenatal, type 1, Type 2, Type 3, Type 4 and atypical SMA

Question 7

Describe 3 characteristic features of SMA

Answer 7

Progressive proximal symmetrical limb and trunk muscle weakness,
Intercostal muscle weakness,
Fine tremor
Facial weakness

Question 8

What is the most common type of SMA? Describe

Answer 8

Type 1 - profound hypotonia, symmetrical flaccid paralysis, progressive, death at early age

Question 9

Give me some molecular facts about SMA

Answer 9

SMN1 and pseudogene SMN2
4% population have 2 copies of SMN1 on one chrm (range 0-5)
95-98% SMA homozygous for deletion of at least exon 7
2-5% are compound heterozygotes for del and pathogenic inactivating mutn

Question 10

How do we interpret SMA carrier testing?

Answer 10

Bayes calculation

Question 11

Briefly describe CRISPR-Cas9

Answer 11

Gene editing - Cas9 protein complex containing specific sequence of RNA - once complimentary sequence is identified the DNA is cut and released into the cell. Cellular DNA repair mechanisms repair the break - but this is prone to error. By introducing templates of ‘corrected’ DNA - these specific sequences can be incorporated to replace mutant alleles

Question 12

What is the clinical significance of 36-39 CAG repeats in Huntington Disease?

Answer 12

Reduced penetrance - may or may not be affected

Question 13

Above what number of CAG repeats would you see fully penetrant development of Huntington Disease?

Answer 13

40 CAG repeats

Question 14

What is the significance of 27-35 CAG repeats in Huntington Disease?

Answer 14

Intermediate allele - will not cause disease but may expand to cause disease if paternally transmitted

Question 15

Name 3 disorders associated with FMR1

Answer 15

Fragile X, FXTAS and POI

Question 16

How is Fragile X caused?

Answer 16

Expansion of unstable 5’ UTR CGG repeat to >200 repeats, causing gene silencing

Question 17

Give 3 clinical features of Fragile X in males

Answer 17

moderate/severe intellectual and social impairment, characteristic facial features, joint laxity, macro-orchidism

Question 18

Give the clinical presentation of Fragile X in females

Answer 18

Variable phenotype - apparently normal approx 50%) through to mild/moderate mental and social impairment

Question 19

What is the significance of repeat size of 55-200 in FMR1?

Answer 19

Premutation - may expand in maternal line to cause FraX in future generations. Patients may develop FXTAS or POI

Question 20

What is the proposed disease mechanism for FXTAS/POI

Answer 20

NOT the same as FraX - ?toxic gain of function?

Question 21

What is the location of FMR1?

Answer 21

Xq27.3

Question 22

What is the role of FMRP protein?

Answer 22

RNA binding protein, present in many tissues including brain, ovaries and testes - thought to act as a shuttle within cells by transporting mRNA from nucleus to areas of cell where proteins assembled. Also helps to control when the instructions in these mRNA molecules are used to build proteins.

Question 23

Name 2 NGS platforms

Answer 23

Agilent Sure Select, ThermoFisher Ion Torrent, Illumina HiSeq/MiSeq SBS

Question 24

What are the mutation ranges for SBMA?

Answer 24

Normal - 34 CAG repeats or less
Questionable - 35 CAG repeats
Reduced penetrance - 36-37 CAG repeats
Affected fully penetrant - 38 CAG repeats or greater
Sequence any 35-37 repeats

Question 25

Name 3 CAG expansion disorders

Answer 25

Huntingtons SBMA SCAs - 7 of them

Question 26

Name 2 CGG expansion disorders

Answer 26

Fragile X | FXTAS

Question 27

Name 3 CTG expansion disorders

Answer 27

Myotonic Dystrophy 1 Huntington Like Disease 2 SCA 8

Question 28

Name a GAA expansion disorder

Answer 28

Friedreich Ataxia

Question 29

Name a CCTG expansion disorder

Answer 29

Myotonic Dystrophy 2

Question 29

What additional features might you see in a juvenile HD patient with a large expansion?

Answer 29

Bradykinesia | Dystonia

Question 30

What factors can contribute to pathogenic mechanism of repeat expansion diseases

Answer 30

``` Sequence of repeat Size of repeat Location of repeat within gene Whether repeat encodes RNA or protein Function of repeat-containing gene Extent of meiosis and somatic instability ```

Question 31

What causes Myotonic Dystrophy 1?

Answer 31

CTG expansion in the 3’UTR of the DMPK gene

Question 32

What causes Myotonic Dystrophy 2?

Answer 32

CCTG repeat in intron 1 of the CNBP gene

Question 33

What are some clinical features of DM1?

Answer 33

``` Progressive weakness and myotonia Cataracts Cardiac arrhythmias Endocrinopathy Cognitive impairment ```

Question 34

Name 7 methods for detecting UPD

Answer 34

``` MS-PCR MS-Melt Curve Analysis MS-Pyrosequencing Microsatellite analysis MS-MLPA SNP Arrays WGS/WES - trios especially powerful ```

Question 35

Name 3 methods of detecting UPD that rely on bisulphite conversion

Answer 35

MS-PCR MS-Melt Curve Analysis MS-Pyrosequencing

Question 36

What are the pros and cons of using microsatellite analysis for UPD detection?

Answer 36

Pros: Will distinguish whole/partial UPD and Hetero/Isodisomy, relatively cheap, no prior conversion step, reliable method Cons: Need parental samples and informative markers

Question 37

Describe MS-MLPA

Answer 37

2 tubes, 1 for CNV, 1 for methylation specific enzyme digest. Methylated DNA won’t digest, and then will amplify during MLPA. Semi quantitative, no parental bloods need, can distinguish UPD from deletion from a small amount of DNA. No need for parental samples, can’t distinguish UPD and mutation in imprinting centre

Question 38

How can you use WGS/WES for detection of UPD?

Answer 38

Trio analysis - bioinformatic pipelines will check for identity by looking for biparental inheritance Can detect mosaics and can distinguish full range of del/UPD/IC defect Expensive and time consuming, requires parental samples

Question 39

What are the pros and cons of using bisulphite conversion for UPD testing?

Answer 39

Allows for analysis without parental samples, bisulphite conversion kits readily available, cheap and effective Won’t distinguish segmental/whole UPD, or del from UPD (MS-PCR)

Question 40

Describe Class 1 CFTR mutations

Answer 40

Nonsense, most frameshift mutns and large del’s create premature stop codons causing defective protein synthesis and no CFTR protein expressed. Severe phenotype, W128X, R553X, G542X. Treatments - readthrough drugs - Ataluren phase 3 trials

Question 41

Describe Class 2 CFTR mutations

Answer 41

Some missense mutns and in frame del’s disrupt CFTR protein folding and trafficking to the surface. F506del, N1303K. Treatments: correctors to promote folding - Lumacafter

Question 42

Describe Class 3 CFTR mutations

Answer 42

Some missense mutns result in substitution of aa’s, disrupting regulation of CFTR channel, which no longer opens in response to channel agonists. G551D, G551S, G1349D. Treatment Ivacafter being trialled.

Question 43

Describe Class 4 CFTR mutations

Answer 43

Some missense mutations result in changes to CFTR protein structure that forms the pore of the channel which can restrict the movement of Cl- ions through the channel - conductance defect. Eg R117H, R334W, R347P. Ivacafter being trialled.

Question 44

Describe Class 5 CFTR mutations

Answer 44

Some missense mutations result in alternative splicing that disrupts mRNA processing - extremely reduced amounts of normal CFTR protein are synthesised, less protein at cell surface. 2789+5G>A, A455E. Treatments are focussed on compounds that enhance CFTR retention/anchoring

Question 45

Describe Class 6 CFTR mutations

Answer 45

Different types of mutations increase the turnover of CFTR protein at the cell surface, quickly removed and degraded by cell machinery. Egs include Rescued F508del, 120del23, N287Y, 4326delTC, 4279insA

Question 46

How many classes of CFTR mutations are there?

Answer 46

6