Neurogenetics Flashcards
What are neuromuscular disorders?
Affect the function of voluntary muscles via a defect in either the muscle, the neuromuscular junction or the motor neuron (nerve).
Why is molecular diagnosis important?
- closure
- family planning, genetic counselling
- screening other family members
- prognosis
- increased knowledge of interactions
What are the steps in finding a disease gene?
- affected individual is referred from clinician
- Extract DNA from affected person (and affected/unaffected family members)
- sequence
- filter out common SNPs and poor-quality reads (against databases)
- compare affected to unaffected to identify variant/s that segregates with disease
- If novel variant suspected, can additional unrelated individuals be found?
- if needed, research to identify function of candidate genes
- sequence region surrounding candidate variant using Sanger
- If strong candidate found perform functional studies to confirm
What is a targeted panel?
A neuromuscular disease panel that is constantly growing and contains neurogenetic disease genes. Now split over 2 different panels “nerve” and “muscle”
How do you make a targeted panel?
- 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
What is the sample prep time, run time and initial data processing of making a targeted panel?
- sample prep time = 2 days
- run time = 4 hours
- initial data processing = 1 day
What is the average coverage of the targeted panel?
~300x, with 95% to 20x (therefore, 5% of targets on the panel are not effectively covered
How many variants around are made after the targeted panel process and how many remain after initial filtering?
~1600-1800 variants
- 60-80 remain after initial filtering
How long does variant analysis take for targeted panels?
5 minutes to 6 months
What is the diagnostic success of targeted panels?
~30%
What is the advantage of targeted panels compared to WES?
- panels can diagnose a single affected individual when no other affected individuals are present
- WES and linkage mapping require multiple family members
What are the disadvantages of targeted panels?
- 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
What is whole exome sequencing (WES)?
- Hybridisation probes capture the coding portion of the genome for high-throughput sequencing
What are the advantages and limitations of WES?
- 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
What is whole genome sequencing?
- 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
What is linkage analysis?
- When a large family pedigree is available, can perform linkage mapping in conjunction with sequencing
- generally done with SNP-array
What are the challenges of diagnosing Neuromuscular diseases
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
What are the reasons 40-60% of cases are unsolved?
- synonymous changes
- variants of unknown significant
- novel non-coding genes
- regulatory non coding variants
- large repetitive genes
What are functional studies?
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
What symptoms did the patient in case study present with?
- lower limb weakness
- waddling gait, delayed motor milestones
What was done to determine diagnosis of patient
- 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
What is DYNCH1 associated with?
Known disease causing mutation associated with spinal muscular atrophy (SMA-LED) - a nerve condition
