Disease resulting from genetic changes

Question 1

Which genes are affected in duchenne muscular dystrophy (DMD)

Answer 1

• X-linked recessive
• Mutation in the DMD gene @ Xp21 => incomplete, non-functional dystrophin protein
• frameshift mutations (insertion/deletion of 1+ exons/dNTP) => premature stop codon = shortened protein

Question 2

Which genes are affected in heriditary hemochromatosis

Answer 2

• autosomal recessive occuring mainly in caucasian male

- mutations in HFE gene located @ 6p22.2

Question 3

Due to mutations in the DMD gene what happens next?

Answer 3

• absent/non-functional dystrophin = sarcolemma not stabilised = compromised
  = influx of Ca2+ in muscle fibres
  = protease activation (bc Ca2+ co factor)
  = Muscle fibre destruction

Question 4

What lab findings to expect in diagnosing DMD

Answer 4

• Markly elevated serum CK
• Abnormal muscle histology:
- Fibre hypertrophy, degeneration, atrophy
- Inc endomysial CT

Question 5

Which tests used to diagnose DMD (briefly describe)

Answer 5

• Southern Blotting: genomic DNA digested w/ HindIII, Bgl II or EcoRI cut exons => cDNA probes used to detect deletions
• multiplex PCR: 6 diff primer sets amplify regions from exons of genomic DNA. may be detected by gel or capillary electrophoresis
• Sequencing to detect point mutations

Question 6

Due to mutations in the HFE gene what happens next?

Answer 6

• HFE protein bound to transferrin-receptors 1& 2 @ cell surface (iron sensing)
• non-functional HFE protein ≠ not response to transferrin-bound iron ≠ hepcidin not produced = transferring becomes saturated = inc free Fe3+ = destruction of cells

Question 7

mutatons for HFE gene have _ _ meaning it _ risk of having the disease. & list the other risks

Answer 7

incomplete penetrance meaning inc risk of having a particular disease
Risks:
- Male
- Alcohol intake
- Dietary iron & Vit c intake
- Blod donation

Question 8

What lab findings to expect in diagnosing hereditary hemochromatosis

Answer 8

• Iron studies 
= inc serum ferritin
= Increased transferring sauturation
•HFE mutation analysis
• liver biopsy used if other mutations present & if ferittin markedly elevated

Question 9

Which test used to diagnose hereditary hemochromatosis (briefly describe?)

Answer 9

PCR-RFLP

• H63D mutation destroys Bcl I R.site = 207bp after digestion
• C282Y mutation creates extra Rsa I site = 110bp product

Question 10

Which genes are affected in Cystic fibrosis (CF)

Answer 10

• mutation in CFTR gene @ 7q31.2 (27 exons)

- can be expressed homozygous or heterozygous (diff mutation that causes CF)

Question 11

Most common mutation of CF is c.1521_1523delCTT => p.Phe508del (what does this mean)

Answer 11

• CTT deletion in the coding region of NA from 1521-1523

=> deletion of Phe @ 508 on protein

Question 12

Due to mutations in the CFTR gene what happens next?

Answer 12

• CFTR = CF transmembrane conductance regulator > transports Cl- out of cells = maintain vol of mucus on ciliated epithelia in lungs so cilia can move
• CF = dec mus vol ≠ cilia move & inc mucus [ ] = bacterial growth

Question 13

What lab findings to expect in diagnosing CF

Answer 13

1. Test for immunoreactive Trypsinogen (elevated in newborns)
2. Sweat test (Gold std) determines [Cl-].
   If [Cl-] > 60mM = CF

Question 14

Which tests used to diagnose CF

Answer 14

1. Oligonucleotide Ligation Assay
2. Temporal Temperature Gradient Electrophorhesis
3. Bead-based arrays
4. Next-gen sequencing

Question 15

Describe the Oligonucleotide Ligation Assay

Answer 15

• 3 diff probes for each allele
• 1x (fluoresenctly-labelled) common probe binds to 3’ to mutation site
• 1x probe for normal allele (shorter)
• 1x probe for mutant allele (longer)
  => mutant allele detected coz migrate slower

Question 16

Describe Temporal Temperature Gradient Electrophorhesis

Answer 16

• based on DNA denaturation at diff temps during electrophorhesis
• Mutations melt @ diff temp. = affect migration

Question 17

Which genes are affected in alpha thalassemia

Answer 17

• mutation in the alpha globin gene @ 16p13.3
• 2 pairs of alpha gene inhireited from each parent
• mutation in one or more alpha globin gene => alpha thalassemia
• inc deletion = inc severity

Question 18

Principle of Gap PCR to detect alpha thalassemia (popular molecular method for detecting a thal.)

Answer 18

• deletions of lrg regions of alpha-globin gene cluster make it possible to be amplified

Question 19

Why do you need a LIS region?

Answer 19

> amplification control = ensure that PCR works

Question 20

Principle of the Multiplex Ligation-Dependent Probe Amplification (MLPA)

Answer 20

• probes designed to anneal next to each other on DNA seq
• 1x LH probe contains F primer binding region
• 1x RH probe contains stuffer sequ. & R primer binding region
  1. DNA denatured & primers bind
  2. DNA ligase ligate adjacent probes tog.
  3. Probes are amplified by PCR
  4. products analysed in capillary electro.
  = length of stuffer is varied so multiple targets can be analysed in same rxn

Question 21

Interpret the results of MLPA

Answer 21

• Ratio of ~1 : copy # of target region = in test & normal samples
• Ratio >1 : duplication
• ~0.5 : heterozygous deletion of that area of gene
• ~0 : Homozygous deletion of that area of gene

Question 22

Explain the terminology of gene position
• p arm
• q arm
• template
• explain this CFTR gene is located @ 7q31.2

Answer 22

• p arm = short arm
• q arm = long arm
• chromo, arm, regions, bands & sub-bands
• 7q31.2 = chrom. 7, long arm, 3-1, 2

Question 23

when is molecular testing used to dx CF

Answer 23

• Pre-natal screening
• confirm dx
• family member screen

Question 24

Difference b/w OLA & MLPA*

Answer 24

• OLA: 3 probes, ligation & PCR?*

- MLPA: 3 probes (each w/ a primer), Hybridisation, ligation & PCR

Question 25

in alpha thalassemia, how many mutations comprise the majority of all causative mutations?*

Answer 25

7*