SMA

Question 1

Question

Answer 1

Answer

Question 2

4 subtypes of SMA

Answer 2

TYPE I - severe; <6 mnths, never sit, death <2yearsTYPE II - intermediate; 7-18 months, sit but never walkTYPE III - mild; >18 mnths, stand and walk independentlyTYPE IV - very mild; 20-30s

Question 3

Clinical features of SMA (generally)

Answer 3

NAME?

Question 4

Cause of SMA

Answer 4

SMN1 exon 7 deletion/gene conversion (98%) Point mutations (2%)

Question 5

Underlying pathogenesis of SMA

Answer 5

SMN protein important in survival of anterior horn motor neurons of the spinal cord, which send signals to muscles to contract. Lack of SMN causes anterior horn cells to degenerate –> fewer, weaker signals

Question 6

% identity between SMN1 and SMN2

Answer 6

99.9%, 5bp different

Question 7

Copy number of SMN1 and SMN2 in normal individuals

Answer 7

0-4 per chromosome for each SMN1 and SMN2

Question 8

Functional difference between SMN1 and SMN2

Answer 8

Synonymous C>T change in exon 7 of SMN2 (pseudogene of SMN1) > inactivates exonic splicing enhancer > leaky splicing.90% transcripts lack exon 7; truncated and degraded. SMN2 only produces 10% of the protein SMN1 does

Question 9

How does SMN2 copy number affect phenotype

Answer 9

DOSAGE DEPENDENT DISEASE MODIFIERMultiple copies of SMN2 can increase SMN protein levels and reduce severity of phenotype HOWEVER not reliable so not used diagnostically

Question 10

Genetics of type 1 SMA

Answer 10

Caused by homozygous deletion of at least exon 7 of SMN1.| 20% of SMN protein produced

Question 11

Genetics of type 2 SMA

Answer 11

Comp het for del of at least exon 7 of SMA1 and gene conversion event. 30% of SMN protein produced

Question 12

Genetics of type 3 SMA

Answer 12

Homozygous for gene conversion event| 40% SMN protein produced

Question 13

Can genotype be used to predict phenotype

Answer 13

No - as its really difficult to determine SMN1 and SMN2 copy number due to variable copy number per allele in normal individuals. PLUS there are modifier genes

Question 14

Principles of gene conversion

Answer 14

OCCURS IN CIS1. double strand breaks 2. 5’ and 3’ ends are moved away from each other3. broken strands align with donor sequence of high homology4. mismatch repair occurs; broken strand is remade using donor strand as template

Question 15

Genetic testing in SMA

Answer 15

MLPA - uses probes specific to small number of differences in seq between SMN1 and SMN2

Question 16

What proportion of NORMAL people lack SMN2

Answer 16

10%

Question 17

What proportion of normal chromosomes have two copies of SMN1? and are the limitations of this?

Answer 17

4%| could mask a carrier of a deletion (2:0)

Question 18

What proportion of mutations in SMA are de novo

Answer 18

2%

Question 19

What is the UK carrier frequency of SMA?

Answer 19

Jan-50

Question 20

What are the main clinical features of SMA?

Answer 20

Weakness and paralysis of the voluntary muscles, due to spinal cord and motor neuron degeneration. Muscular atrophy.Progressive proximal weaknessIntercostal muscle weakness, leading to breathing difficulty.Fine tremorFasciculations in the tongue make feeding difficult

Question 21

Describe the 5 types of SMA

Answer 21

Prenatal - Arthrogryposis multiplex congenita and congenital axonal neuropathy. Death within 1 month. SMN2 copies: 1Type 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: 2Type II: Chronic infant - diagnosed 6-12months. 27% of SMA, hypotonia, 70% reach adulthood, sit unaided, no walking without support. Progressive muscle weakness. SMN2 copies 3-4Type 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: 3-4Type 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

Question 22

How many individuals are thought to carry two copies of SMN1 on a single chromosome

Answer 22

4%

Question 23

Describe the basic gene structure of SMN1/2.

Answer 23

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.

Question 24

How many bases so SMN1 and SMN2 differ by? What are the common variants that allow the two genes to be distinguished?

Answer 24

5.c.840C>T and c.859G>C in SMN2

Question 25

What is the SMN protein?

Answer 25

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.

Question 26

What is the common mutation found in 95-98% of all SMA individuals?

Answer 26

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)

Question 27

What is the mutational spectrum of SMA?

Answer 27

95-98% deletion of exon 72-5% deletion + SNP<1% homozygous SNP - majority located in exons 3 and 6. Some can be deep intronic

Question 28

What is the rate of new mutation?

Answer 28

2% de novo.

Question 29

Describe the genotype/phenotype correlations found in SMA

Answer 29

Type I: homozygous deletion of exon 7Type II: SMN1 > SMN2 gene conversion plus a hemizygous gene deletion of the other allele.Type III: 2 gene conversion event.

Question 30

How can SMN2 copy number compensate, in part, for lack of SMN1?

Answer 30

Disease is caused by low levels of protein, rather than complete lack of protein.SMA I: 9% normal active proteinSMA 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.

Question 31

List some modifier genes involved in the phenotypic severity of SMA

Answer 31

NAIPSERF1Plastin 3ZPR1PTEN

Question 32

Describe the diagnostic workflow for SMA mutation detection

Answer 32

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.

Question 33

What project is ongoing and looks to introduce an NIPD test for SMA?

Answer 33

NIPSIGEN.

Question 34

Why is interpreting SMA carrier tests tricky?

Answer 34

6% of parents of a simplex case will have normal SMN1 dosage:1. 4% have two copies of SMN1 on a single chromosome.2. 2% of patients have a de novo deletion of exon 7 - only 1 parent is a carrier.

Question 35

What therapies are currently being trialled to treat SMA?

Answer 35

1. 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 7.2. Gene conversion from SMN2 to SMN13. Use stem cell therapy to compensate for lack of SMN protein - AveXis is currently in phase 1 clinical trials.

Question 36

what is the SMA carrier frequency in uk?

Answer 36

Jan-50

Question 37

what is the main clinical symptom of SMA?

Answer 37

#NAME?

Question 38

describe the different SMA types? what is the most likely cause?

Answer 38

- type 1 = most common form <6 months- floppy babydeath at early age- poor head control and unable to sit- swallowing and feeding difficulties- homozygous deletiontype 2 = <1 year- low muscle tone- may be able to sit, never able to walk- 70% reach adulthood- gene conversion in one allele and hemi deletion in other- type 3 = >1 until adulthood- can stand and walkprobability of being ambulatory decreases with age- gene conversion in both allelestype 4 = adult onsetnormal life expectancy<1% of patientsaytypical - non chromosome 5 relatedfloppiness, multiple fractures, high CK

Question 39

if a baby presents as floppy, what is the testing triad?

Answer 39

SMA, myotonic dystrophy and PWSnew TD = R70 (separate referral from hypotonic infants which go for R69 DM1, PWS and microarray so now the consultant has to specify SMA)

Question 40

what causes SMA?

Answer 40

homozygous or compound het deletions and mutations of SMN1- 98% homozygous deletion of SMN1 exon 7 (deletions or gene conversions)- 2% compound het for a deletion and mutation- <1% have homozygous pathogenic mutation in SMN1mutations may be deep intronic. 2% of deletions are de novo (unequal crossing over)Incidence of SMA remains high due to high new mutation rate- germline and somatic mosaicism have been described

Question 41

why is the SMA gene difficult to test

Answer 41

- has SMN2 (centromeric) pseudogene- complex region of high instability with repetitive sequences, retrotransposable elements, deletions and inverted duplications- 4% of population have two copies of SMN1 on one chromosome (can have up to 5 copies)- SMN1 and 2 differ by 5 base pairs- critical difference is a synonymous variant in SMN2 exon 7 which alters ESE and 90% of transcripts lack exon 7 in SMN2 and produce truncated product and so is degraded- SMN2 still produces some full length SMN protein (10%) but not sufficient to compensate for lack of SMN1- homozygous SMN1 del patients have at least one copy of SMN2 as SMN null mice is embryonic lethal- higher copies of SMN2 leads to milder phenotypes

Question 42

what is the SMN protein a component of?

Answer 42

spliceosome

Question 43

how do you test for SMA?

Answer 43

-98% are deletions so MLPA or real-time PCR for SMN1 exon 7 & 8 deletion - MLPA uses probes specific to the SNV differences between SMN1 and SMN2- multiplex real time PCR uses TaqMan probes specific to exon 7 SMN1 and a control probe- 2% sequence variant - NGS, allele specific long range PCR and RT-PCR/cDNA sequencing are used for specific analysis of SMN1

Question 44

why might a parent of a child with SMA have normal SMN1 dosage? how can this be confirmed?

Answer 44

- 4% of population have two copies of SMN1 on one chromosome - linkage analysis- de novo deletion - 2% of patients, can do bayes calculation for normal dosage parent.

Question 45

what therapies are available for SMA?what are issues with these treatments?

Answer 45

drugs work by stabilising the SMN protein or modulating SMN2 expression- Increasing protein: Zolgensma - virus vector delivers SMN1 transgene to affected motor neuronsmodulate SMN2:Nusinersen/Spinraza is an antisense oligo to promote inclusion of exon 7 in SMN2 transcripts by blocking intron 7 splice site to increase SMN protein levels. highly stable and not toxic. Risdiplam - oral (not injected)promotes inclusion of exon 7 in SMN2Issues - usually diagnosedin advanced stages where motor degeneration has occured. screened for?current pilot study to include SMA in UK newborn screening - March 2022

Question 46

How many types of SMA are there?

Answer 46

6 - Prenatal, type 1, Type 2, Type 3, Type 4 and atypical SMA

Question 47

Describe 3 characteristic features of SMA

Answer 47

Progressive proximal symmetrical limb and trunk muscle weakness,Intercostal muscle weakness,Fine tremorFacial weaknessCarrier freq 1:40-60 depending population

Question 48

What is the most common type of SMA? Describe clin features

Answer 48

Type 1 - profound hypotonia, symmetrical flaccid paralysis, progressive, death at early age

Question 49

Give me some molecular facts about SMA

Answer 49

SMN1 and pseudogene SMN24% population have 2 copies of SMN1 on one chrm (range 0-5)95-98% SMA homozygous for deletion of at least exon 72-5% are compound heterozygotes for del and pathogenic inactivating mutation

Question 50

How do we interpret SMA carrier testing?

Answer 50

Bayes calculation