Molecular Genetics

Question 1

Name an X-linked dominant disorder

Answer 1

Fragile X syndrome.

Alports syndrome.

Question 2

Name an X-linked dominant lethal disorder

Answer 2

Rett syndrome.

Incontinentia pigmenti

Question 3

What causes SBMA

Answer 3

CAG trinucleotide expansion (>35 rpts) of exon 1 of the AR gene Xq12

Question 4

What are the features of SBMA

Answer 4

Age of onset: 30-50yrs. Progressive neuromuscular disorder (weakness, atrophy, fasciculations) of the lower motor neurons.
Gynecomastia. Testicular atrophy. Mild androgen insensitivity.

Question 5

Describe features of Alports syndrome

Answer 5

Renal disease (progressive renal failure, haematuria, proteinuria).
Hearing loss.
Ocular lesions.

Question 6

What’s causes Alports.

Answer 6

85% X-linked dominant: COL4A5 mutations.

15% autosomal recessive (10%) and dominant (5%): COL4A3 and COL4A4

Question 7

Describe features of Retts

Answer 7

LoF MECP2 mutations. Usually male lethal.
Progressive neurodegenerative disease. Normal development until 18 months, then regression (language and motor). Characteristic hand flapping. Gait ataxia, panic attacks, autistic features.

Question 8

Name an Autosomal dominant disorder: Gain of Function

Answer 8

Huntingtons.
Myotonic dystrophy.
CMT2A.
BCR-ABL1 fusion.
Spinocerebellar ataxia.
Achondroplasia

Question 9

Name an Autosomal dominant disorder: Loss of Function

Answer 9

Cytogenetics: DiGeorge, Williams, Cri du chat.
Cancer: BRCA, PTEN, TP53.
Single gene disorder: Aniridia, Alagille syndrome.

Question 10

Name an Autosomal dominant disorder: Dominant Negative

Answer 10

TP53.
PML-RARA fusion.
Osteogenesis Imperfecta.
Autosomal dominant myotonia congentia

Question 11

Name disease associated with PMP22 at 17p12

Answer 11

Over expression: Charcot Marie Tooth type 1a/Hereditary Motor & sensory Neuropathy type 1a.
Haploinsufficiency: Hereditary Neuropathy with liability of Pressure Palsies.

Question 12

Name disease associated with AR at Xq12

Answer 12

Mutation: Androgen Insensitivity Syndrome.

CAG expansion: Spinal and Bulbar Muscular Atrophy.

Question 13

Name disease associated with RET 10q22.21

Answer 13

LoF mut: Hirschsprung Disease.
GoF mut: MEN2 (multiple endocrine neoplasia type 2).
Somatic: NSCLC, Thyroid carcinoma.

Question 14

What genes are associated with Lynch syndrome/ hereditary nonpolyposis colorectal cancer

Answer 14

DNA mismatch repair genes: MLH1, MSH2, MSH6, PMS2, EPCAM.

Question 15

What genes are associated with Noonan syndrome

Answer 15

PTPN11 (50%). SOS1 (13%). RAF1 (3-17%). KRAS (1%). NRAS (1%). BRAF (1%).

Question 16

What are the genes involved in SMA and how many copies are there. What do they express

Answer 16

SMN1 and SMN2 (pseudo gene). There’s 1-5 copies of SMN2 in tandem. 4% population have 2 copies of SMN1 in tandem.
SMN1 & 2 differ by 5nt in exon 7 so exon 7 isn’t transcribed from SMN2.
SMN1 expresses a full length transcript. SMN2 expresses a short transcript that’s non functional and rapidly degraded (it does produce a small amount of full length).

Question 17

What’s the new mutation rate for SMN1 gene

Answer 17

2% SMA patients have a de novo mutation

Question 18

What’s cystic fibrosis

Answer 18

Most common autosomal recessive disorder in Caucasians.

A multi system disorder affecting: pancreatic, pulmonary, gastrointestinal, reproductive.

Question 19

Name two treatments for CF

Answer 19

Potentiators (directly activate CFTR protein): (G551D) IVAcaftor.
Correctors (promote correction of protein misfolding): (p.Phe508del) LUMAcaftor.

Question 20

Name a treatment for SMA

Answer 20

Antisense oligonucleotide treatment: corrects SMN2 splicing resulting in restored SMN expression.
Quinazoline related compound: increases SMN2 promoter activity, altered SMN mRNA levels or splicing patterns leading to increased SMN protein levels.
Histone deacetylase inhibitors are being looked at.

Question 21

Discuss SMA testing strategy

Answer 21

SMN1 exon 7 dosage analysis first (often done with exon8 for a control). If have 0 copies of exon 7 that’s the result. If 1 copy: sequence looking for intragenic mutation.

Question 22

Discuss testing strategy form CF

Answer 22

Test using mutation panel (commercial usually) of approx 30 mutations - detects about 90% mutations.
Could sequence CFTR if haven’t detected mutation from panel.
PolyTtract for 5T if testing for CVAD.
could test for TG (12/13) repeat if 5T is detected in CVAD.

Question 23

When test for CF

Answer 23

Neonate with meconium ileus.
Neonate with elevated IRT.
Positive sweat test.
Features of CF.

Question 24

Name an X-linked recessive disorder

Answer 24

Duchenne muscular dystrophy/Becker muscular dystrophy.
Spinal and bulbar muscular atrophy.
Androgen insensitivity syndrome.

Question 25

What are the features of duchenne muscular dystrophy

Answer 25

Progressive and symmetrical muscle weakness often with CALF HYPERTROPHY and SEVERE JOINT CONTRACTURES. Classic feature: gowers sign.Age of onset: 2-5yrs. 1st signs: impaired motor development and delayed milestones. 5th affected makes have MR. Wheelchair bound by 12yrs and dies of respiratory failure/cardiomyopathy late teens/early twenties (have cardiomyopathy by 18yrs). Referral reason: delayed milestones, waddling gait, difficulty climbing.

Question 26

What are the features of Becker muscular dystrophy

Answer 26

Similar progression to DMD (progressive symmetrical weakness, often calf hypertrophy. Older onset to DMD: 5-15yrs Slower rate of disease progression (wheelchair bound by 16yrs). Milder phenotype: activity induced cramps, flexion contractures of elbows, preservation of neck flexor muscle. Heart failure is the most common cause of death at about 40yrs.

Question 27

What are the features of DMD-associated dilated cardiomyopathy (DCM)

Answer 27

Dilated cardiomyopathy with congestive heart failure. NO skeletal involvement. Typically males 20-40yrs (females in later life). Rapid progression to death in males/ slower (over a decade) in females.

Question 28

What causes DCM

Answer 28

caused by DMDmut that affects muscle promotor and associated with exon 1 only which results in no dystrophin transcripts produced in cardiac muscle.

Question 29

What causes DMD/BMD, discuss the gene

Answer 29

DMD Xq21 dystrophin. 2.4Mb. 79 exons. Expression controlled by 3 independently regulated promoters (B- brain, M-muscle, P-purkinje). ``` 2 deletion hotspots: (screening 19 exons account for 98% all deletions) Central Region (exon 44-53) (80% del/ 20% dup). 5' region (exon2-20) (20% del/80%dup) ```

Question 30

What is the general DMD mutation rule for DMD and BMD

Answer 30

90%cases: Out-of-frame mutation : DMD. In-frame mutation : BMD. single point mutation/ small in/del: 25% DMD and 10-20% BMD.

Question 31

Treatment for DMD

Answer 31

Not curative. Aims to alleviate symptoms with steroids, physio, anticongestives. ``` Dystrophin restoration approaches: Stop codon read through (GENTAMICIN). Exon skipping (MORPHOLINO OLIGONUCLEOTIDE). ```

Question 32

What's the difference in terms of proteins between DMD and BMD

Answer 32

DMD: lack dystrophin protein. BMD: reduction in amount of protein, alteration in size of protein. Lack of dystrophin protein causes progressive fibre damage and membrane leakage.

Question 33

What's the role of dystrophin

Answer 33

One of the major roles of dystrophin is a glycoprotein complex where it stabilises sarcolemma and protects muscle fibres from long term contraction induced damage and necrosis.

Question 34

What can cause of female DMD Carrie to exhibit a phenotype

Answer 34

Non random X inactivation. X-auto some translocation. UPD of X chromosome. Deletion involving other X / compound heterozygote of two DMD pathogenic variants

Question 35

What type of mutations are seen in the DMD gene in DMD

Answer 35

60-65% are large insertions or deletions that cause a frameshift. 40% are point mutations or small frameshift rearrangements including pure intron deletions.

Question 36

How are DMD mutations tested for

Answer 36

Most widely used method now is MLPA. Analyse all exons which increases mutation detection rate. It'll detect whole exon deletions and duplications and will characterise breakpoints to an exon level and can be used for female carriers.

Question 37

What influences the severity of the DMD/BMD phenotype

Answer 37

The principle factor is the reading frame. Severity of clinical symptoms does vary according to which exons and how many are involved.

Question 38

Discuss CBAVD and infertility

Answer 38

Congenital bilateral absence of the vas deferens. OBSTRUCTIVE AZOOSPERMIA. 1-2% of all infertility. Caused by mucus clogging the vas deferens as they're for wing leading to deterioration before birth. CFTR: mutated in 70-90% cases. 88%: 1 severe mutation (p.Phe508del, R117H) and 1 mild mutation (5T polyT tract allele). 12%: 2 mild mutations. Using standard CF mutation detection methods only 47% CBAVD must will be detected.

Question 39

What percentage of female permutation carriers have POI

Answer 39

21%

Question 40

What are the 2 disorders associated with having an pre-mutation of FMR1

Answer 40

FXTAS: fragile X associated tremor-ataxia syndrome. FXPOI: fragile X associated premature ovarian insufficiency.

Question 41

How is Kallman linked to infertility and what genes are involved

Answer 41

Endocrine disorder. So reduced t Levels of GnRH- hypogonadotrophic hypogonadism: undeveloped gonads and incomplete secondary sexual maturation. Males: undeveloped testes/ lack second sex charc/ infertile. Females: little or no breast development/ primary amenorrhea. Gens: KAL1: Xp22.33. FGFR1 (8p21 (AD)). GnRHreceptor (4q13.2 (AR)).

Question 42

How is Noonan associated to infertility.

Answer 42

Males: uni and bi-lateral cryptorchidism (77%) resulting in azoospermia/oligospermia. Female: normally fertile.

Question 43

In terms of fertility discuss SBMA.

Answer 43

Progressive androgen insensitivity. Severe oligospermia. Infertility. Testicular atrophy.

Question 44

What are the symptoms of Huntingtons

Answer 44

Progressive neurodegenerative disorder. Motor symptoms. Cognitive impairment. Psychiatric disturbances. First symptoms manifest: 35-60yrs. Disease duration: 15-20yrs.

Question 45

Discuss HTT huntingtin

Answer 45

It's ubiquitously expressed and required for normal development. 2 alternatively polyadenylated forms: larger 13.7Kb expressed in adult and fetal brain; smaller 10.3Kb s widely expressed. Expansion causes of gain of function: acquires a deleterious effect casting neuronal dysfunction and degeneration.

Question 46

Discuss HTT expansions

Answer 46

CAG expansion Less than 27 rpts: normal. 27-35 rpts: intermediate (family at increased risk) 36-39 rpts: incomplete penetrance (diagnosis confirmed) (family at increased risk) 40 and over: pathogenic (diagnosis confirmed) (family at increased risk) Repeat expansion are usually paternal (risk of offspring developing HD is highest in dads carrying intermediate rpts and over 35yrs old).

Question 47

Discuss HD and anticipation

Answer 47

Increase in disease severity/reduction image of onset is observed in successive generations. Repeat numbers correlate inversely with mean age of onset. Longer repeats= earlier onset. ``` Very large expansions (>60) present with juvenile HD - before 20yrs. Shorter expansions (36-39) can be asymptomatic.. ``` Number of repeats accounts for about 70% of variance in age of onset

Question 48

What considerations for HD if getting homozygous result

Answer 48

Genotype to establish of real. Consider age of onset (large expansion: Southern blot). STR allele analysis (check for two allele). Polymorphism under biding site

Question 49

What causes phenotype on FXTAS and FXPOI

Answer 49

Excess of FMRI mRNA causes a toxic build up- gain of function phenotype.

Question 50

Where's the expansion in FMR1

Answer 50

CCG repeat in the first untranslated exon of FMR1

Question 51

What are the expansion sizes in fragile x

Answer 51

Normal: 6-45 rpt. Stable allele (98% popn: model number: 30rpts). Intermediate: 46-58 rpt: less than 50: likely stable; 50-58: may show instability. Premutation: 59-200rpt UNMETHYLATED: distinction between pre and full is methylation status.. Full mutation: 200-1000 rpts METHYLATED: 20% full muts are mosaic

Question 52

What's the significance of AGG repeats in CGG repeats of FMR1

Answer 52

Stability of intermediate expansion correlates to the presence of two or more AGG interspersions. Unstable repeats tend to late AGG or have only one. Therefor it's assumed it's the presence of these that maintain the stability.

Question 53

What percentages of permutation carriers manifest FXPOI and FXTAS

Answer 53

FXTAS: 16.5% women; 45,0.5% men. FXPOI: 20% women.

Question 54

What's the phenotype of a female full FMR1 expansion carrier

Answer 54

Variable: 50% apparently normal- 50% moderate mental and social impairment Due to differences in proportions of active and inactive normal and mutation X chromosome in relevant tissues.

Question 55

What the phenotype of FXPOI

Answer 55

Amenorrhea before 40yrs for over 4 months in association with FSH level in menopausal range. Varying degree of ovarian function seen in 50% women.

Question 56

What's the phenotype of FXTAS

Answer 56

Late onset neurodegenerative disorder. Late onset cerebellar ataxia and intention tremor. Severity of symptoms and reduction in age of onset proportional to increase number of repeats. Clinical symptoms less severe in females

Question 57

What tests are carried out to detect fragile x

Answer 57

Most common: fluorescent PCR across CGG repeat. Southern blotting. Long range PCR.