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Genetics and Reproductive
Options for SMA Families
2017 Annual SMA Conference Orlando, FL (Disney World) Friday, June 30, 2017
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Genetics and Reproductive Options for SMA Families
Part 1: Genetics
Louise R. Simard, PhD University of Manitoba, Faculty of Medicine
Dept. Biochemistry & Medical Genetics
Winnipeg, MB, Canada R3E 0J9
1987 - Ph.D. U Toronto – Medical Genetics. 1988-2006 – Ste. Justine Hospital Research Centre, U Montreal
Independent research program in SMA DNA diagnostics Consultant, incl. SMA testing
2006-present – Professor and Department Head, U Manitoba
2
Questions are welcome!
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Genetics and Reproductive Options for SMA Families
SMA and Genetics: Outline
• SMA lower Motor Neuron Disease
with a wide range of disease severity
• SMA is an Autosomal Recessive Genetic Disorder
• DNA and the SMA Gene, SMA Mutations & SMA severity
• SMA DNA Molecular Tests ― Diagnosis
― Carrier
― Prenatal
3
Part 1: Genetics
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Genetics and Reproductive Options for SMA Families
SMA is a lower motor neuron disease that
recurs in families (inherited).
4
Type 2
Intermediate SMA < 18 months
Never stand unaided
Decreased life expectancy
Type 3 Kugelberg-Welander
18 months
Stand alone but loss of mobility
Normal life expectancy
Type 4 Adult SMA 21 years of age
Progressive muscle weakness
Normal life expectancy
Type 1 Werdnig-Hoffman
< 6 months
Never sit unaided
Death before 2 years
Type 0 Prenatal SMA
In utero
Respiratory support
Death before 1 month
Disease
Severity
0 1
2 3
4
Wide Clinical
Presentation! Image adapted from from
“Principles of Neural Science”, ER
Kandel, JH Schwartz, TM Jessell,
eds., 4th Ed. McGraw Hill.
Loss of MN
cells leads
to Muscle
Atrophy
Part 1: Genetics
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Genetics and Reproductive Options for SMA Families
SMA is inherited as an
Autosomal Recessive Disorder
5
Part 1: Genetics
The fundamental unit of inheritance is DNA.
Each cell has over 5 feet of DNA!
It must be compacted to fit inside a cell
in the form of chromosomes.
We have 23 pairs of chromosomes; each
set is inherited from our biological parents.
www.sciencemuseum.org.uk
SMA autosomal recessive
mode of inheritance
********************************************
There must be a mutation (error) in
both copies of a gene (maternal and
paternal) for the genetic disorder to be
passed onto a child.
1866
https://www.biography.com/.image/t_share/ MTIwNjA4NjMzNTA5MDg2NzMy/ gregor-mendel-39282-1-402.jpg
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Genetics and Reproductive Options for SMA Families
SMA is inherited as an
Autosomal Recessive Disorder
6
• Affected individuals have mutations in
both copies of a recessive gene.
• Carriers have a mutation in only one
copy of a recessive gene and is
asymptomatic.
• Males and females can be carriers.
• Males and females can be affected.
• If both parents are carriers, the risk of
having a child with the disease is 25%.
Having an affected child is a surprise
because carrier parents are
asymptomatic.
From this point onwards – genetic
counseling becomes possible.
x
x x
x
Part 1: Genetics
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Genetics and Reproductive Options for SMA Families
Identifying the SMA gene: 1 gene 5 SMA types
7
Nature (1990) 345:823-825 Nature (1990) 540-541
Nature (1990) 344:767-768 Lancet (1990) 336:271-273
Part 1: Genetics
Lefebvre et al., (Melki laboratory, INSERM Institut Necker)
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Genetics and Reproductive Options for SMA Families
Genetic Code = Blueprint of life ~25,000 genes in the human genome
8
DNA
RNA/mRNA
protein
http://www.bbc.com/news/science-environment-27592655
STOP
1 2 3 4 5 6 7 8
Start
1 2 3 4 5 6 7 8 Promoter
STOP
On/ Off switch
Part 1: Genetics
1953
DNA = four nucleic acids (C, G, T, A)
Different sequence of A-C-T-G = gene
C (cytosine) pairs with G (guanine)
T (thymine) pairs with A (adenine)
C G T
A T A
A
T C G G C
C G
G C
A T T A
www.photoresearchers.com
1953
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Genetics and Reproductive Options for SMA Families
SMN1 vs. SMN2 gene: Chromosome 5q13
Mutations in SMN1 = SMA
1 2 3 4 5 6 7 8 Promoter
C
STOP
1 2 3 4 5 6 7 8 Promoter
T
STOP
1 2 3 4 5 6 8
STOP
Start
STOP
1 2 3 4 5 6 7 8
Start
DNA (a)
RNA (b)
mRNA (c)
protein unfunctional protein
functional protein
~10% Full-length transcripts
~90% Nonfunctional SMN protein
100% Full-length transcripts
Functional Wild Type SMN protein
> 99 % identical
SMN2 SMN1 7
T 7
C
Part 1: Genetics
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Genetics and Reproductive Options for SMA Families
Mutations disrupt a gene’s function
Point Mutations
*AAA to AAT Lysine to Asparagine
**AAA to TAA Lysine to STOP
* Missense mutation
** Nonsense mutation
Mistakes in copying the nucleic acids that make up a gene are called mutations
Promoter STOP
1 2 3 4 5 6 7 8 X * ** 4
Splicing Deletion Duplication
STOP
On/Off switch
Part 1: Genetics
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Genetics and Reproductive Options for SMA Families
Deletions (missing DNA)
is a common mutation in SMA
SMN1 7 8
Very large deletions are often
associated with type I and type II
SMA. Type I SMAs most often
cannot make ANY SMN.
In Type II & III SMA, the mutation
is often confined to SMN1 exon 7.
% p
atients
Simard et al., (1997) Am J Med Genet 72:51-58
0
10
20
30
40
50
60
70
80
90
100
type I type II type III
SMN null SMN E7
Part 1: Genetics
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Genetics and Reproductive Options for SMA Families
SMA locus is very variable
But… also find:
SMN2 SMN1
SMN2 SMN1 On average:
SMN1
SMN2 SMN1 SMN2
SMN1 SMN1 No apparent Clinical effect
SMN2
SMN2 SMN1 SMN2
SMA causing Chromosomes
Part 1: Genetics
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Genetics and Reproductive Options for SMA Families
SMN2 is a major modifier gene why Type 1 or Type 2 or Type 3?
0
10
20
30
40
50
60
70
80
90
1 2 3 4
type I
type II
type III
% p
atient
s
# SMN2 copies
n=188 n=110 n=77
Taken from Feldkotter et al., (2002) Am J Hum Genet 70:358-368
Part 1: Genetics
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Genetics and Reproductive Options for SMA Families
~94% patients = SMN1 exon 7 missing on both chromosomes
~3-4% patients = SMN1 exon 7 missing and a point mutation
~4-5% patients do not have 5q-SMA
SMN1 7 SMN2 7 T C
X
point mutation
deletion
gene conversion SMN1 SMN2 SMN2 7 T T
Part 1: Genetics
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Genetics and Reproductive Options for SMA Families
Molecular Diagnostic Tests
• Detection of homozygous mutations
o presence/absence of SMN1 Ex7 = diagnosis
• Detection of heterozygotes
o quantification of exon 7 = carrier testing
• Analysis of point mutations = diagnosis
o (research labs only)
SMN2 SMN1 7
T 7
C
Part 1: Genetics
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Genetics and Reproductive Options for SMA Families
Diagnostic Tests: SMN mutations Diagnosis of SMA patients
Compared to EMG & muscle biopsy, the DNA test is:
• non-invasive
• sensitive, detects >95% of all SMAs
• accurate, all lacking SMN1 exon 7 have SMA
• ideal for newborn screening
Limitation: Some SMA individuals have a point mutation in the SMN1 gene.
Such mutations are not detected by the deletion or quantitative PCR assay.
(164 pb) SMN2
(187 pb) SMN1
Van der Steege et al. (1996) Am J Hum Genet 58:834-838.
All or None test!
Part 1: Genetics
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Genetics and Reproductive Options for SMA Families
Diagnostic Tests: Carrier Detection 2 vs. 1 SMN1 gene test
• 95% of SMA patients
are lacking SMN exon 7
• Because SMA is an
autosomal recessive
disorder, most SMA
carriers should have
only 1 copy of SMN1
exon 7
SMN1
SMN2 SMN1:SMN2 copies
2:1 2:2 1:3 1:2 0:4 0:4 0:3
McAndrew et al. (1997) Am J Hum Genet 60:1411-1422
Rochette et al., (1997) Neurogenetics 1:141-147.
Detection of heterozygotes SMN quantitative test
Part 1: Genetics
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Genetics and Reproductive Options for SMA Families
Limitations with the SMA Carrier
Test
SMN1 SMN1
Some individuals (2 to 5%) with 2 SMN1
copies are still carriers (2+0 genotype).
The frequency of the 2+0 genotype varies
between ethnic groups
Therefore, we can never
give a 100% Yes or No
result.
Population Frequency Risk
Caucasian 0.0009 1:632
Ashkenazi Jewish 0.0019 1:350
Asian 0.0011 1:628
African American 0.0041 1:121
Hispanic 0.0007 1:1061
But, …….
Part 1: Genetics
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Genetics and Reproductive Options for SMA Families
Limitations with the SMA Carrier
Test
This test does not detect the rare small mutations in the gene.
Part 1: Genetics
19
Taken from Alías et al. (2009) Human Genetics 125:29
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Genetics and Reproductive Options for SMA Families
Prenatal SMA testing
• Prenatal diagnosis is offered when the SMA
index case has a homozygous absence of SMN1
exon 7 or if the parents of an affected, deceased
child have been shown to be carriers by
quantitative PCR.
• Given the highly unstable nature of the 5q-SMA
locus, surprises do occur.
Part 1: Genetics
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Genetics and Reproductive Options for SMA Families
Should I have prenatal testing? Depends on your genotype! Carrier status is VIP.
Each baby = unique event = own combination of paternal/maternal genes.
N / N N / m
N / m m / m
Norm SMA carrier
mutated
25% risk of having
SMA baby
Norm
mutated
SMA
Carrier
50% risk of having
SMA baby
SMA individual mutated mutated
mutated
Norm
SMA
Carrier
N / m N / m
m / m m / m
Part 1: Genetics
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Genetics and Reproductive Options for SMA Families
Should I have prenatal testing? Depends on your genotype! Carrier status is VIP.
Prenatal testing not necessary
but … carrier risk = 50%.
Prenatal testing not necessary but
… carrier risk = 100%.
N / N N / N
N / m N / m
Normal non-carrier
Normal Normal
mutated
Normal N / m N / m
N / m N / m
SMA
Carrier
Normal non-carrier
Normal Normal
mutated
mutated
SMA
individual
Part 1: Genetics
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Genetics and Reproductive Options for SMA Families
Should I have prenatal testing? Depends on your genotype!
Knowing carrier status can avoid unnecessary prenatal
testing.
Knowing carrier status before pregnancy can facilitate
choice of type of prenatal testing.
If carrier status is unknown – and you are expecting a
child – the faster you get testing – the faster you will
know if prenatal testing is necessary.
Part 1: Genetics
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Part 2
24
Harvey J. Stern MD, PhD
Director, Reproductive Genetics
Genetics & IVF Institute
Reproductive Options for
Sma Families
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Genetics and Reproductive Options for SMA Families
Workshop Goals
What are the genetic aspects of
SMA especially as they relate to
reproduction?
What are the reproductive choices
for families at-risk for SMA?
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RECESSIVE DISEASE
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Genetics and Reproductive Options for SMA Families
Reproductive Options for SMA Families
Very difficult decisions about
family building
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Genetics and Reproductive Options for SMA Families
Reproductive Options for SMA Families
• Decisions regarding childbearing are personal
and reflect our own ethical, moral and religious
views. This is not a “one size fits all” type of
issue.
• Couples who are at-risk for a genetic disorder
should be allowed to make up their own minds
after considering all their reproductive options.
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Genetics and Reproductive Options for SMA Families
Reproductive Options for SMA Families
• Decisions are influenced by the couple’s
experience with SMA. Those who have had a
child or sib affected will react differently from a
couple identified to be at-risk by genetic
screening.
• Decisions are also influenced by couple’s
attitudes towards prenatal diagnosis and
assisted reproduction (“playing God”)
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Genetics and Reproductive Options for SMA Families
Reproductive Options for SMA Families
Most families do not wish to bring another
child with SMA into the world.
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Genetics and Reproductive Options for SMA Families
• Have no or no more children. (Most Common
Choice)
• Adoption.
• Use a gamete donor. (pre-tested for SMA)
Reproductive Options for SMA Families
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Genetics and Reproductive Options for SMA Families
Reproductive Options for SMA Families
Natural conception with 1st or 2nd
trimester prenatal testing.
Preimplantation Genetic Diagnosis
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Genetics and Reproductive Options for SMA Families
What is PGD?
• Involves the use of assisted reproduction
technologies (IVF) to provide a method
of prenatal diagnosis.
• Offers an alternative to traditional
methods of prenatal diagnosis including
chorionic villous sampling and
amniocentesis, with the option of
termination of affected pregnancies. 33
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Genetics and Reproductive Options for SMA Families
Development of PGD
• This year represents the 27th anniversary of
the first application of PGD in humans.
―(Handyside et al. Nature 244 1990)
• Since 1990, approximately 400,000 PGD
cycles have been performed worldwide.
• This has resulted in over 100,000 PGD babies
born after the procedure.
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Transfer unaffected embryos
to the patient
affected affected affected
PGD for Genetic Disease
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In-Vitro Fertilization (IVF)
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Genetics and Reproductive Options for SMA Families
Questions About PGD
Why do I need to do IVF, I
have no trouble getting
pregnant?
37
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Genetics and Reproductive Options for SMA Families
Questions About PGD
• IVF is used to increase the odds of producing
a healthy child.
• There is no change in the genetic material
(egg or sperm).
• By creating multiple embryos, one increases
the chance that good quality embryos which
are not affected with SMA will be present and
available to be returned to the uterus.
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IVF
Procedure
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Genetics and Reproductive Options for SMA Families
IVF/PGD Procedure
Components of IVF/PGD Cycle:
1. Down-regulation (preparation)
2. Ovarian Stimulation - FSH
3. Egg Retrieval
4. Fertilization (ICSI)
5. Embryo biopsy and testing
6. Embryo transfer to uterus 40
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Genetics and Reproductive Options for SMA Families
Parts Of An IVF Cycle
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Genetics and Reproductive Options for SMA Families
Normal Ovulation
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Embryo Development Day 2 Day 3 Day 4 Day 5
Fertilization
2 cell 4 cell
8 cell
Morula
Blastocyst
Adapted from Moore and Persaud, Saunders, 1993
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IVF: Monitoring Visit
0
500
1000
1500
2000
2500
3000
0 2 4 6 8 10 12 14
Estradiol levels
Follicle measurement
Days of FSH Stimulation
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Genetics and Reproductive Options for SMA Families
Stimulated Ovary
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Egg Retrieval
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Egg Aspiration
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Egg Ready For Fertilization
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Fertilization by Intracytoplasmic
Sperm Injection
ICSI
ICSI 50
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Embryo Development
2 cell
3 cell
4 cell
8 cell
51 From: Veeck LL, An Atlas of Human Gametes and Conceptuses, Parthenon Publishing, 1999
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Embryo Development
Morula stage
embryos
Blastocyst stage
embryos
From: Veeck LL, An Atlas of Human Gametes and Conceptuses, Parthenon Publishing, 1999
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Embryo Transfer
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Genetics and Reproductive Options for SMA Families
PGD for Single Gene Disorders
• Analysis involves whole genome amplification
with multiplex PCR amplification of the mutation
along with 3-4 linked polymorphic markers
which generate a chromosomal haplotype.
• At the same time, the embryos are also tested
by a chromosomal microarray for abnormalities
(ie. Down syndrome) which can lead to failed
implantation or pregnancy loss.
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Genetics and Reproductive Options for SMA Families
PGD for SMA
Embryo Biopsy
SMN gene
SMN 1 & 2 = unaffected
SMN 2 only = affected
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NO
SMN1
Affected
SMN1
Present SMN 1 & 2
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Genetics and Reproductive Options for SMA Families 60
24 Chromosome Analysis by
Microarray
Performed concurrently with SMA testing to
identify the embryos with the best potential to
make a baby as well as being free of SMA.
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Genetics and Reproductive Options for SMA Families
Accuracy of PGD
• Using the combination of mutational
analysis and linked markers the
accuracy of the test 98-99%.
• Contamination with external DNA is a
major concern.
• Analyses done in surgical clothing in
biohazard hood.
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Genetics and Reproductive Options for SMA Families
Specific Issues with IVF/PGD
• Patient Discomfort: Medications are given by
injection (subQ).
• Abdominal distention and discomfort and some
nausea are common.
• Egg retrieval done under anesthesia.
• Ovarian hyperstimulation syndrome 1-2%.
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Genetics and Reproductive Options for SMA Families
Questions about IVF/PGD
• Is IVF Safe?
• What will I feel?
• Does it increase cancer risk?
• Are the babies born normal?
• What are the costs?
• What about insurance?
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Genetics and Reproductive Options for SMA Families
Questions About IVF/PGD
What is the success rate of IVF/PGD?
• Varies, particularly with maternal age, but in
large series 50-60% of patients became
pregnant per IVF/PGD cycle.
• Improved with concurrent testing for
chromosome abnormalities.
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Thank you!!!
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