Genetics and Reproductive Options for SMA Families · Kandel, JH Schwartz, TM Jessell , eds., 4....

Genetics and Reproductive Options for SMA Families2018 Annual SMA ConferenceDallas, TexasFriday, June 15, 2017

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Part 1: SMA and GeneticsLouise R Simard, PhD

Part 2: SMA Carrier ScreeningMelissa Gibbons, MS CGC

Part 3: Reproductive Options for SMA Families

Harvey J Stern, MD, PhD

Genetics and Reproductive Options for SMA Families

Part 1: SMA and Genetics

Louise R. Simard, PhDUniversity of Manitoba, Faculty of Medicine

Dept. Biochemistry & Medical GeneticsWinnipeg, MB, Canada R3E 0J9

[email protected]

1987 - Ph.D. U Toronto – Medical Genetics. 1988-2006 – Ste. Justine Hospital Research Centre, U Montreal

Independent research program in SMADNA diagnostics Consultant, incl. SMA testing

2006-present – Professor, Department Head, U Manitoba

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Questions are welcome!

mailto:[email protected]


Outline• SMA

Lower Motor Neuron Disease With a wide range of clinical presentation (severity)

• SMA Gene SMN1 and SMN2 SMN1 Mutations SMA severity

• SMA Genetics Chromosomes as vehicles of the DNA genomic blueprint Autosomal Recessive Inheritance

• SMA Molecular Tests Diagnosis Carrier

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SMA is a lower motor neuron disease that recurs in families (inherited).

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Type 2Intermediate SMA

< 18 monthsNever stand unaided

Decreased life expectancy

Type 3Kugelberg-Welander

18 monthsStand alone but loss of mobility

Normal life expectancy

Type 4 Adult SMA≥21 years of age

Progressive muscle weaknessNormal life expectancy

Type 1Werdnig-Hoffman

< 6 monthsNever sit unaided

Death before 2 years

Type 0Prenatal SMA

In uteroRespiratory support

Death before 1 month

Disease Severity

01

23

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Wide ClinicalPresentation!

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



Identifying the SMA gene 1 gene 5 SMA types

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Nature (1990) 345:823-825Nature (1990) 540-541

Nature (1990) 344:767-768 Lancet (1990) 336:271-273

Lefebvre et al., (Melki laboratory, INSERM Institut Necker)



Genetic Code = Blueprint of life~20,000 genes in the human genome

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DNA = four nucleic acids (C, G, T, A)Different sequence of A-C-T-G = geneC (cytosine) pairs with G (guanine)T (thymine) pairs with A (adenine)

CGT

AT A

A

TCG GC

CG

GC

AT TA

www.photoresearchers.com

1953


Geno

me

Deco

ratio

n Pa

ge/N

CBI

https://autisable.com/


SMN1 vs. SMN2 gene Chr 5q13

1 2 3 4 5 6 7 8Promoter

CSTOP

1 2 3 4 5 6 7 8Promoter

TSTOP

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)

proteinunfunctionalprotein

functionalprotein

~10% Full-length transcripts

~90% Nonfunctional SMN protein

100% Full-length transcripts

Functional Wild Type SMN protein

> 99 % identical

SMN2 SMN17T

7

C

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http://www.bbc.com/news/science-environment-27592655

Mutations in SMN1Cause 5q-SMA




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

atie

nts

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

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SMA locus is very variable

But…also find:

SMN2 SMN1

SMN2 SMN1On average:

SMN1

SMN2 SMN1SMN2

SMN1SMN1No apparent Clinical effect

SMN2

SMN2 SMN1 SMN2SMA causing

Chromosomes

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SMN2 is a major modifier geneSMN2 copy number decreases with increasing severity

0

10

20

30

40

50

60

70

80

90

1 2 3 4

type I

type II

type III

% p

atie

nts

# SMN2 copies

n=188n=110n=77

Taken from Feldkotter et al., (2002) Am J Hum Genet 70:358-368

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SMA is inherited as an Autosomal Recessive Disorder

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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 recessivemode 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


http://www.sciencemuseum.org.uk/





SMA is inherited as an Autosomal Recessive Disorder

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• 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.

1 in 4 Chance25%

1 in 4 Chance25%

1 in 2 Chance (50%)

Taken from ghr.nlm.nih.gov


>95% of patients have deleterious mutations in the SMN1 gene(deletions, gene conversions, point mutations)

SMN1 7SMN27T C

X

point mutation

deletion

gene conversion SMN1 SMN2SMN27T T

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Molecular Diagnostic Tests

• Detection of homozygous mutations presence/absence of SMN1 Ex7 = diagnosis

• Detection of heterozygotes Number of copies of exon 7 = carrier testing

• Analysis of point mutations = diagnosis Not yet routine, specialized labs only

SMN2 SMN17T

7

C

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(164 pb)SMN2(187 pb)SMN1

Van der Steege et al. (1996) Am J Hum Genet 58:834-838.

All or None test!

Diagnostic Tests: SMN1 mutationsDiagnosis 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.

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Taken from Passon et al. (2010) Mol Cell Probes 24:310


Diagnostic Tests: Carrier Detection2 vs. 1 copy of SMN1 exon 7 test (dosage)

• 95% of SMA patients are lacking SMN1 exon 7

• Because SMA is an autosomal recessive disorder, most SMA carriers should have only 1 copy of SMN1 exon 7

SMN1

SMN2SMN1:SMN2 copies

2:1 2:21:31:20:4 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 heterozygotesSMN quantitative test

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Taken from Passon et al. (2010) Mol Cell Probes 24:310


Diagnostic Challenges Rarely able to give a 100% Yes or No result.

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These tests do not detect small mutations in the SMN1 gene.

Taken from Alías et al. (2009) Human Genetics 125:29

2 to 5% of individuals have both SMN1 genes on the same chromosome

(2+0 genotype) - so are carriers

On average:SMN2 SMN1

SMN2 SMN1

SMN2

SMN2 SMN1 SMN1

But….


A Genetic Counselor is a valuable resource to help you understand the genetics of SMA, opportunities for testing (diagnosis, carrier status, prenatal) and how to interpret test

results.

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Part 2: SMA Carrier ScreeningMelissa Gibbons, MS CGCCertified Genetic Counselor

Neuromuscular Clinic Children’s Hospital Colorado

Assistant ProfessorUniversity Colorado School of Medicine

MemberCure SMA Medical Advisory Committee


Genetic Counselors are:

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• Master’s-trained health care professionals who combine their knowledge of: ―Basic science―Medical genetics―Epidemiological principles―Counseling theory

• With their skills in: ―Genetic risk assessment―Education―Interpersonal communication and counseling

• To provide services to clients and their families for a diverse set of genetic or genomic indications


Genetic Counselors work in:

• Clinical Setting―Preconception―Prenatal―Pediatric―Adult―Cancer

• Genetic Testing Laboratory• Pharmaceutical Companies

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Genetic Counselor can:

• Explain your/your child’s results• Discuss different genetic testing options• Assist with coordinating carrier testing for

―You―Extended Family

• Provide family education • Discuss treatment options• Discuss reproductive options

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Understanding Your Results

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http://www.curesma.org/documents/support--care-documents/genetics-of-sma.pdf


Understanding Your Results

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Positive Deletion Results:

Inconclusive Deletion Results:

Additional Types of Sequencing Results:

Sequencing Results:

Carrier Testing Affected Individual has

2 SMN1 Deletions

Parents and Extended Family can be tested by

Deletion Studies

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Affected Individual has1 SMN1 Deletion &

1 SMN1 Point Mutation

Parents should be tested first to determine

targeted testing for the extended family


Different Types of Carriers“1 + 0”

“2 + 0”

5q

5q

SMN1

5q

5q

SMN1SMN1

5q

5q

SMN1 SMN1

5q

5q

SMN1SMN1

SMN1

*“2 + 0*”

*Point mutation or microdeletion.Adapted from PMCID: PMC3234503

“1 + 1*”*


What does negative mean?


What does negative mean?g.27134T>G


Options for Carrier Testing • Single gene testing

―Testing for one condition―Also referred to as known familial mutation testing

• Targeted carrier screening―Testing based on your ethnicity or family history―Also referred to as ethnic-based carrier screening

• Expanded carrier screening―Many disorders are screened for using a single sample―Test selection is done without regard to race or ethnicity―Each company determines the genes on their panel ―Screening panels usually focus on severe disorders that

affect a person’s quality of life from an early age

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Carrier Testing

• Allows for more informative preconception counseling

− Reproductive risks for the couple− Discussion of preimplantation/prenatal testing options− Development of PGD for your family

• Provides information to family members− Who should be tested− What testing do they need

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Resources

• Genetic Counselors on the Cure SMA Medical Advisory Board

− Jin Yun (Helen) Chen, Massachusetts General Hospital− Khalida Liaquat, Quest Diagnostics− Melissa Gibbons, Children’s Hospital Colorado

• National Society of Genetic Counselors―NSGC.org

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Part 3: Reproductive Options for SMA Families

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Harvey J. Stern MD, PhDDirector, Reproductive GeneticsGenetics & IVF Institute


Workshop GoalsWhat 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


Very difficult decisions about family building

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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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• 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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Most families do not wish to bring another child with SMA into the world.

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• Have no or no more children (Most Common Choice)

• Adoption• Use a gamete donor (pre-tested for SMA)


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Natural conception with 1st or 2nd

trimester prenatal testing.Preimplantation Genetic Diagnosis

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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.

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Development of PGD

• This year represents the 28th 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 affectedaffected

PGD for Genetic Disease

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In-Vitro Fertilization (IVF)

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Questions About PGD

Why do I need to do IVF, I have no trouble getting pregnant?

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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


IVF/PGD Procedure

Components of IVF/PGD Cycle:1. Down-regulation (preparation)2. Ovarian Stimulation - FSH3. Egg Retrieval4. Fertilization (ICSI)5. Embryo biopsy and testing6. Embryo transfer to uterus

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Parts Of An IVF Cycle

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Normal Ovulation

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Embryo DevelopmentDay 2Day 3Day 4Day 5

Fertilization

2 cell4 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


Stimulated Ovary

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http://www.google.com/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwi6mJv96YnTAhWGSSYKHYCgBEUQjRwIBw&url=http://www.advancedfertility.com/ivfprocedures.htm&bvm=bv.151426398,d.ZGg&psig=AFQjCNEDDuxbmGCba0QzKbCEFHlMu2NGqQ&ust=1491361290085263

Egg Retrieval

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Egg Aspiration

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Egg Ready For Fertilization

Fertilization by IntracytoplasmicSperm Injection

ICSI

ICSI60

Embryo Development

2 cell

3 cell

4 cell

8 cell

61From: Veeck LL, An Atlas of Human Gametes and Conceptuses, Parthenon Publishing, 1999

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Embryo Development

Morula stageembryos

Blastocyst stage embryos

From: Veeck LL, An Atlas of Human Gametes and Conceptuses, Parthenon Publishing, 1999

Embryo Transfer

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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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PGD for SMA

EmbryoBiopsy

SMN gene

SMN 1 & 2 = unaffected

SMN 2 only = affected

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NO SMN1Affected

SMN1 PresentSMN 1 & 2

Genetics and Reproductive Options for SMA Families 70

24 Chromosome Analysis by MicroarrayPerformed concurrently with SMA testing to identify the embryos with the best potential to make a baby as well as being free of SMA.


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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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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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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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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