Session 3: Molecular Genetics Flashcards
Name 2 X-Linked Recessive disorders
- Duchenne/Becker Muscular Dystrophy (DMD/BMD)
- Spinal and Bulbar Muscular Atrophy (SBMA)
- Androgen Insensitivity Syndrome (AIS)
Name 2 X-Linked Dominant disorders
X-linked hypophosphatemia
X-linked Alport Syndrome
Rett Syndrome
Name three clinical features associated with CF
Chronic coughing and wheezing
Failure to thrive
Pancreatic insufficiency
Name the most common CF mutation
p.Phe508del - 75%
Describe the function of CFTR (7q31.2)
Cyclic AMP-activated chloride channel located in the plasma membrane of secretory eithelial cells
How many types of SMA are there?
6 - Prenatal, type 1, Type 2, Type 3, Type 4 and atypical SMA
Describe 3 characteristic features of SMA
Progressive proximal symmetrical limb and trunk muscle weakness,
Intercostal muscle weakness,
Fine tremor
Facial weakness
Carrier freq 1:40-60 depending population
What is the most common type of SMA? Describe clin features
Type 1 - profound hypotonia, symmetrical flaccid paralysis, progressive, death at early age
Give me some molecular facts about SMA
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 mutation
How do we interpret SMA carrier testing?
Bayes calculation
Briefly describe CRISPR-Cas9
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
What is the clinical significance of 36-39 CAG repeats in Huntington Disease?
Reduced penetrance - may or may not be affected
Above what number of CAG repeats would you see fully penetrant development of Huntington Disease?
40 CAG repeats
What is the significance of 27-35 CAG repeats in Huntington Disease?
Intermediate allele - will not cause disease but may expand to cause disease if paternally transmitted
Name 3 disorders associated with FMR1
- Fragile X,
- FXTAS,
- POI
How is Fragile X caused?
Expansion of unstable 5’ UTR CGG repeat to >200 repeats, causing gene silencing
Give 3 clinical features of Fragile X in males
moderate/severe intellectual and social impairment, characteristic facial features, joint laxity, macro-orchidism
Give the clinical presentation of Fragile X in females
Variable phenotype - apparently normal approx 50%) through to mild/moderate mental and social impairment
What is the significance of repeat size of 55-200 in FMR1?
Premutation - may expand in maternal line to cause FraX in future generations. Patients may develop FXTAS or POI
What is the proposed disease mechanism for FXTAS/POI
NOT the same as FraX - ?toxic gain of function?
What is the location of FMR1?
Xq27.3
What is the role of FMRP protein?
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.
Name 2 NGS platforms
Agilent Sure Select,
ThermoFisher Ion Torrent,
Illumina HiSeq/MiSeq SBS seq by synthesis
What are the mutation ranges for SBMA?
Spinal and Bulbar Muscular Atrophy
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
Name 3 CAG expansion disorders
- Huntingtons
- Spinal and Bulbar Muscular Atrophy (SBMA)
- SCAs - 7 of them
Name 2 CGG expansion disorders
Fragile X
FXTAS
Name 3 CTG expansion disorders
Myotonic Dystrophy 1
Huntington Like Disease 2
SCA 8
Name a GAA expansion disorder
Friedreich Ataxia
Name a CCTG expansion disorder
Myotonic Dystrophy 2
What additional features might you see in a juvenile HD patient with a large expansion?
Bradykinesia -slowness of movement
Dystonia
What factors can contribute to pathogenic mechanism of repeat expansion diseases
- 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
What causes Myotonic Dystrophy 1?
CCTG repeat in intron 1 of the CNBP gene
What are some clinical features of DM1?
Progressive weakness and myotonia
Cataracts
Cardiac arrhythmias
Endocrinopathy
Cognitive impairment
Name 7 methods for detecting UPD
MS-PCR
MS-Melt Curve Analysis
MS-MLPA
MS-Pyrosequencing
Microsatellite analysis
SNP Arrays
WGS/WES - trios especially powerful
Name 3 methods of detecting UPD that rely on bisulphite conversion
MS-PCR
MS-Melt Curve Analysis
MS-Pyrosequencing
What are the pros and cons of using microsatellite analysis for UPD detection?
Pros: Will distinguish whole/partial UPD and Hetero/Isodisomy, relatively cheap, no prior conversion step, reliable method
Cons: Need parental samples and informative markers
Describe MS-MLPA
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 needed,
can distinguish UPD from deletion from a small amount of DNA
can’t distinguish UPD and mutation in imprinting centre
How can you use WGS/WES for detection of UPD?
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
What are the pros and cons of using bisulphite conversion for UPD testing?
Pros: Allows for analysis without parental samples,
bisulphite conversion kits readily available,
cheap and effective
Cons: Won’t distinguish segmental/whole UPD, or del from UPD (MS-PCR)
Describe Class 1 CFTR mutations
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
Describe Class 2 CFTR mutations
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
Describe Class 3 CFTR mutations
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
Describe Class 4 CFTR mutations
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.
Describe Class 5 CFTR mutations
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
Describe Class 6 CFTR mutations
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
How many classes of CFTR mutations are there?
6
Curry-Jones syndrome
7q32.1
Mosaisms
Recurrent somatic variant SMO p.L412F
Patchy skin lesions, polysyndactyly, cerebral malform, craniosynostosis, iris colobimas, microphthalmia
Potential treatment for CF
Gene therapy - introducing a normal copy of CFTR
CFTR modulators:
1. Read through -Ataluren
2. Corrector therapy - corrects mts that cause misfolding and degradation eg F508del - Lumacaftor
3. Potentatior therapy - improves CFTR channel function by correction mts thatvreduce gating efficiency eg G551D - Ivacafor
NBS - CF variants
4 variants:
p.Phe508del
p.Gly542X
p.Gly551Asp
c.489+1G>T
CF intron 9 poly T & polyTG tract - significance
Poly T
5T - 90% cases exon 10 skipped
PolyTG
Longer TG associated with disease phenotype
Common TG lenght = 11, 13 and 13
Most common TG11
Increased skipping of ex10 caused by TG12, TG13 and 5T
TG12/T5 assoc. with CBAVD
TG13/T5 assoc. with atypical CF
5 T also modifies expression of p.Arg117His ( variable phenotype)
9T in exclusively seen in cis with p.Phe508del
What is the UK carrier frequency of SMA?
1/50
What are the main clinical features of SMA?
Weakness and paralysis of the voluntary muscles, due to spinal cord and motor neuron degeneration. Muscular atrophy.
Progressive proximal weakness
Intercostal muscle weakness, leading to breathing difficulty.
Fine tremor
Fasciculations in the tongue make feeding difficult
Describe the 5 types of SMA
Prenatal - Arthrogryposis multiplex congenita and congenital axonal neuropathy. Death within 1 month. SMN2 copies: 1
Type I: Acute infant - diagnosed <6 months old. Floppy baby, weak intercostal muscles lead to respiratory failure. 60% of SMA. Death at early age (<2yrs old). Lack of motor development, tongue fasciculation. SMN2 copies: 2
Type II: Chronic infant - diagnosed 6-12months. 27% of SMA, hypotonia, 70% reach adulthood, sit unaided, no walking without support. Progressive muscle weakness. SMN2 copies 3-4
Type III: Juvenile - IIIa diagnosed at <3 years; IIIb diagnosed at >3 years. 12% of SMA. Some ambulation retained. Proximal muscle weakness develops, legs more severely affected than arms. SMN2 copies: 3-4
Type IV: adult - ~1% of patients, onset of muscle weakness in 2nd-3rd decade, legs more affected than arms. Normal life expectancy. SMN2 copies 4-8
How many individuals are thought to carry two copies of SMN1 on a single chromosome
4%
Describe the basic gene structure of SMN1/2
SMN1 and SMN2 (pseudogene). Arranged in tandem.
SMN1 is functional, SMN2 is largely unfunctional and lacks exon 7 in most transcripts. Increased copies of SMN2 can compensate for loss of SMN1 in some cases.
SMN1 - >90% of transcripts are full length; SMN2 90% of transcripts are missing exon 7, due to the c.840C>T silent variant present in an ESE.
How many bases so SMN1 and SMN2 differ by? What are the common variants that allow the two genes to be distinguished?
5.
c.840C>T and c.859G>C in SMN2
What is the SMN protein?
A ubiquitously expressed protein present in abundance in the motor neurons and spinal cord.
It functions to chaperone the assembly of the snRNAs used for splicing. Lack of the protein thought to disrupt mRNA processing in neuromuscular junctions.
What is the common mutation found in 95-98% of all SMA individuals?
A deletion encompassing at least exon 7 of SMN1 - deletion can occur by homozygous deletion, or by gene conversion to SMN2 exon 7 (contains the c.840G>C resulting in exon skipping)
What is the mutational spectrum of SMA?
95-98% deletion of exon 7
2-5% deletion + SNP
<1% homozygous SNP - majority located in exons 3 and 6. Some can be deep intronic
What is the rate of new mutation?
2% de novo.
Describe the genotype/phenotype correlations found in SMA
Type I: homozygous deletion of exon 7
Type II: SMN1 > SMN2 gene conversion plus a hemizygous gene deletion of the other allele.
Type III: 2 gene conversion event.
How can SMN2 copy number compensate, in part, for lack of SMN1?
Disease is caused by low levels of protein, rather than complete lack of protein.
SMA I: 9% normal active protein
SMA II: 14%
SMA III: 18%
Full length SMN levels of 23% are needed for normal function.
The presence of 3+ SMN2 copies can make the phenotype milder.
The c.859G>C variant creates an new ESE in exon 7 of SMN2, to enhance exon 7 inclusion.
List some modifier genes involved in the phenotypic severity of SMA
NAIP
SERF1
Plastin 3
ZPR1
PTEN
Describe the diagnostic workflow for SMA mutation detection
Copy number analysis using specific probes for SMN2 c.840G>C and SMN1 WT sequences. (QPCR, RT-PCR, MLPA)
Sequencing using LR-PCR or cDNA analysis to ID point mutations on the second allele.
What project is ongoing and looks to introduce an NIPD test for SMA?
NIPSIGEN
Why is interpreting SMA carrier tests tricky?
6% of parents of a simplex case will have normal SMN1 dosage:
- 4% have two copies of SMN1 on a single chromosome.
- 2% of patients have a de novo deletion of exon 7 - only 1 parent is a carrier
What therapies are currently being trialled to treat SMA?
- increase expression of SMN proteins by correcting SMN2 splicing using antisense oligos - ISIS-SMNrx is currently in phase 3 clinical trials - enhances inclusion of exon 7.
- Gene conversion from SMN2 to SMN1
- Use stem cell therapy to compensate for lack of SMN protein - AveXis is currently in phase 1 clinical trials.
What is the locus of Cri-du-chat?
5p15.3-p15.2
What is the locus of Sotos?
5q35
What is NSD1 associated with?
Sotos
What is the locus of Cornelia de Lange?
5p13.2
What is NIPBL associated with?
Cornelia de Lange
What is the locus of WAGR?
11p13
What is the PWS/AS locus?
15q11-q13
What is the locus of Rubinstein-Taybi?
16p13.3
What is CREBBP associated with?
Rubinstein Taybi
What is the locus of Smith Magenis?
17p11.2
What is RAI1 associated with?
Smith Magenis/Potocki Lupski
What is the locus of Potocki-Lupski?
17p11.2
What is the locus of HNPP?
17p12
What is the locus of CMT1A?
17p12
What is PMP22 associated with?
HNPP/CMT1A
What is the location of NF1?
17q11.2
What is the location of SRY?
Yp11.31
What is the location of SHOX?
Xp22.33
What is the locus of Bloom Syndrome?
15q26.1
What is the locus of Ataxia Telangietasia?
11q22.3
What is BLM associated with?
Bloom Syndrome
What is ATM associated with?
Ataxia Telangiectasia
What is the locus of Nijmegan BS?
8q21.3
What is the location of FMR1?
Xq27.3
What is the location of XIST?
Xq13
3 X-linked recessive disorders
DMD, BMD, SBMA, Androgen Insensitivity Syndrome
3 X-linked dominant disorders
X-linked Alport Syndrome, Intercontinentia Pigmentia, Rett Syndrome
Expansion size for Fragile X - full
> 200 rpts
Premutation expansion for Fragile X
55-200 rpts
Normal allele size for Huntingtons
Less than 27 rpts
Intermediate expansion size for Huntingtons
27-35 rpts
Incomplete penetrance expansion for Huntingtons
36-39 rpts
Fully penetrant expansion for Huntingtons
40+rpts
Expansions for juvenile onset Huntingtons
60+rpts
