Neurogenetics

Question 1

What are neuromuscular disorders?

Answer 1

Affect the function of voluntary muscles via a defect in either the muscle, the neuromuscular junction or the motor neuron (nerve).

Question 2

Why is molecular diagnosis important?

Answer 2

• closure
• family planning, genetic counselling
• screening other family members
• prognosis
• increased knowledge of interactions

Question 3

What are the steps in finding a disease gene?

Answer 3

1. affected individual is referred from clinician
2. Extract DNA from affected person (and affected/unaffected family members)
3. sequence
4. filter out common SNPs and poor-quality reads (against databases)
5. compare affected to unaffected to identify variant/s that segregates with disease
6. If novel variant suspected, can additional unrelated individuals be found?
7. if needed, research to identify function of candidate genes
8. sequence region surrounding candidate variant using Sanger
9. If strong candidate found perform functional studies to confirm

Question 4

What is a targeted panel?

Answer 4

A neuromuscular disease panel that is constantly growing and contains neurogenetic disease genes. Now split over 2 different panels “nerve” and “muscle”

Question 5

How do you make a targeted panel?

Answer 5

• gDNA fragmented
• hybridised with RNA library baits designed to anneal to your genes of interest
• baits contain biotin label
• target DNA purified from total DNA by addition of streptavidin coated magnetic beads
• purified target DNA then amplified and sequenced

Question 6

What is the sample prep time, run time and initial data processing of making a targeted panel?

Answer 6

• sample prep time = 2 days
• run time = 4 hours
• initial data processing = 1 day

Question 7

What is the average coverage of the targeted panel?

Answer 7

~300x, with 95% to 20x (therefore, 5% of targets on the panel are not effectively covered

Question 8

How many variants around are made after the targeted panel process and how many remain after initial filtering?

Answer 8

~1600-1800 variants

- 60-80 remain after initial filtering

Question 9

How long does variant analysis take for targeted panels?

Answer 9

5 minutes to 6 months

Question 10

What is the diagnostic success of targeted panels?

Answer 10

~30%

Question 11

What is the advantage of targeted panels compared to WES?

Answer 11

• panels can diagnose a single affected individual when no other affected individuals are present
• WES and linkage mapping require multiple family members

Question 12

What are the disadvantages of targeted panels?

Answer 12

• Can be gaps in coverage (meaning disease variant is missed)
• sequencing errors can occur (especially in homopolymer regions)
• if variant not previously reported, then it’s not on the panel

Question 13

What is whole exome sequencing (WES)?

Answer 13

• Hybridisation probes capture the coding portion of the genome for high-throughput sequencing

Question 14

What are the advantages and limitations of WES?

Answer 14

• useful when you have a whole family, uninformative with single individual
• can pick up mutations in genes not already known to cause disease
• reduced cost compared to whole genome

Question 15

What is whole genome sequencing?

Answer 15

• Sequencing the whole genome
• not commonly used as expensive
• ALL data is there, picks up splice variants
• can be used in conjunction with RNA-seq
• limitation is determining effect of non-coding sequence variants

Question 16

What is linkage analysis?

Answer 16

• When a large family pedigree is available, can perform linkage mapping in conjunction with sequencing
• generally done with SNP-array

Question 17

What are the challenges of diagnosing Neuromuscular diseases

Answer 17

Even though Mendelian inheritance, answer not always there:

• incomplete penetrance
• genetic heterogeneity (large no of causative genes)
• phenotypic heterogeneity (multiple genes with overlapping phenotype)
• allelic heterogeneity (variety of mutations in each gene)
• accurate clinical info needed
• lack of knowledge about certain proteins
• dominant disease genes difficult

Question 18

What are the reasons 40-60% of cases are unsolved?

Answer 18

• synonymous changes
• variants of unknown significant
• novel non-coding genes
• regulatory non coding variants
• large repetitive genes

Question 19

What are functional studies?

Answer 19

Once you identify variant that segregates with the disease in a family, functional studies “prove” the variant causes the disease. Using null mutations in an animal model

Question 20

What symptoms did the patient in case study present with?

Answer 20

• lower limb weakness

- waddling gait, delayed motor milestones

Question 21

What was done to determine diagnosis of patient

Answer 21

• Ran the patient on the neuromuscular panel - didn’t get a result
• then linkage analysis from WES: found 2 regions (Chr14 and 9) that were linked with the disease (LOD = 2)
• WES identified a variant in Cytoplasmic dynein 1 - DYNC1H1c, located within chr14 linkage peak (confirmed by Sanger)
• Cytoplasmic dynein 1 acts as a motor neuron for the motility of vesicles and organelles along microtubules

Question 22

What is DYNCH1 associated with?

Answer 22

Known disease causing mutation associated with spinal muscular atrophy (SMA-LED) - a nerve condition