Transcript Families of SMA - Children with Spinal Muscular Atrophy

Genetics: The Whole Picture
SMA Takes the Hill 2003
Debra G.B. Leonard, M.D., Ph.D.
Director, Molecular Pathology Laboratory
University of Pennsylvania Health System
Philadelphia, PA
Objectives


Explain the genetic
testing options for SMA
Leave no one behind
What We Will Talk About

Basic Clinical Features of SMA

Basics of Genetics

SMN Gene Structure

SMA Diagnostic Test

SMA Carrier Test

Questions and Discussion
Spinal Muscular Atrophy





The brain makes the body move by sending nerve signals
from the brain to nerve cells in the spinal cord called the
anterior horn motor neurons
These motor neurons relay signals to the muscles which
cause the muscles to contract
Movement occurs when muscles contract
The anterior horn motor neurons no longer function in
individuals with SMA
Since muscles are not signaled to contract and are not used,
the muscles atrophy or get smaller
Clinical Types of SMA

SMA Type I: Werdnig-Hoffmann

SMA Type II: Intermediate

SMA Type III: Kugelberg-Welander
 Most severe form of SMA
 Onset at birth to 3 months, death by ~2 yrs
 Symptoms begin at infancy to toddler age
 Survive beyond 4 yrs of age
 Onset after age 2 yrs to adult
Basics of Genetics

Genetic Information

DNA and Chromosomes

Genes

Messenger RNA (mRNA)

Proteins

Inheritance

Pedigrees
Genetic Information



Genetic information directs growth and
development, and determines physical
characteristics
Every cell in the human body has the same
genetic information
Each cell uses a different part of the genetic
information to perform that cell’s function,
e.g. skin, blood, muscle, nerve, etc.
DNA and Chromosomes

Genetic information is encoded by DNA

Pieces of DNA in cells are called chromosomes

There are 24 kinds of human chromosomes:

Each normal cell has 46 chromosomes:
 1 through 22 (1 is the longest; 22 is the shortest)
 X and Y are the “sex” chromosomes
 2 copies of 1 through 22, plus 2 sex chromosomes
 XX is female, XY is male
Chromosomes

Chromosomes consist of DNA plus proteins

The proteins help to organize the DNA pieces

Each chromosome has a centromere

The centromere divides the DNA into two parts

Each part has a centromeric end and a free end

The free end is called the telomeric end
Chromosome Structure
One Chromosome
p
q
Arm
Arm
Centromeric
Telomeric
Centromeric
Telomeric
Centromere
If arms are of unequal length:
short arm is called p (petite)
long arm is called q
What Makes Each Person Unique?

Each egg or sperm contains 23 chromosomes

One egg and one sperm combine to make a fetus


 One of each pair of chromosomes 1 to 22, at random
 One of the two sex chromosomes, at random
Each person gets half their chromosomes from their
mother and half from their father
Siblings are similar because they share some of the
same chromosomes, but different because they have
some different chromosomes
DNA Encodes Genetic Information



DNA is a chain of four different building blocks (or
bases) called A, C, G and T
A, C, G and T are the letters of the genetic
alphabet
Some parts of each DNA chain encode instructions
which the cell uses to make proteins, that do work
in cells

Protein-coding parts of DNA are called genes

Other parts of each DNA chain are nonsense
Genes - 1




One gene encodes one protein, more or
less
Each gene has regulatory regions, protein
coding regions and nonsense regions
Coding parts of genes are called exons
Noncoding, nonsense parts of genes are
called introns
Gene Structure
Gene
Promoter
Intron
Exon
Intron
Exon
Exon
Promoter region regulates gene expression,
i.e., controls when a gene will be used to
make the protein it encodes
Genes - 2




Genes are located on the arms of the
chromosomes
Each kind of chromosome contains a different set
of genes
Because each cell contains two of each kind of
chromosome, each cell contains two copies of all
the human genes, except the genes on the X and
Y chromosomes in males
There are ~25,000 human genes
Gene Expression:
How Are Proteins Made from Genes?





When the protein encoded by a gene is needed by the
cell, RNA copies of the gene are made
DNA and RNA are both called nucleic acids
RNA uses bases A, C, G and U, that correspond to the
A, C, G and T bases of DNA
The RNA copy is processed to remove the introns and
is then called messenger RNA or mRNA
mRNA is the blueprint used to make the protein
The Genetic Code

A protein is a chain of amino acids

3 mRNA bases code for one amino acid


Therefore, the mRNA is used as the blueprint to
make a protein by the protein-making or translation
machinery of a cell
While DNA is very stable, the mRNA is short-lived
(minutes to hours), so the cell can change its gene
expression, and therefore what it is doing, as needed
Gene Expression
DNA
TRANSCRIPTION
RNA
RNA PROCESSING
mRNA
TRANSLATION
Protein
Cell Work
TRANSCRIPTION
Nucleic acid  Nucleic acid
(DNA)
(RNA)
Same Language
TRANSLATION
Nucleic acid  Protein
(RNA)
(Amino Acid)
Different Language
Genetic Diseases



A genetic disease is due to a change in the DNA
sequence of a gene
Because DNA in chromosomes is passed from
parent to child, genetic diseases are also passed
from parent to child
A change in the DNA sequence of a gene is called
a mutation
Examples of Gene Mutations



A change of one base of a gene can change an
amino acid in the protein or can shorten (or
truncate) the protein, affecting the function of
the protein
Deletion of part or all of the gene sequence, so
the protein is not made
Change sequences that direct intron removal,
so the mRNA is not correctly made, so the
protein is not made
Types of Inheritance


Single gene diseases are caused by
mutation of one gene, e.g. cystic
fibrosis, SMA, Huntington disease
Multi-gene diseases are caused by a
combination of mutations in several
genes, e.g. heart disease, asthma,
arthritis
Types of Inheritance

Single gene diseases are caused by
mutation of one gene, e.g. cystic
fibrosis, SMA, Huntington disease
 Dominant inheritance:
Mutation of one gene copy causes disease
 Recessive inheritance:
Mutation of both gene copies causes
disease
Genetic Terminology

Affected:
 Someone who has a genetic disease
 Can be either a dominant or recessive
disease

Carrier:
 Someone who has a gene mutation for a
recessive disease in only one gene copy
 Person does not have disease symptoms,
but may pass on mutation to their children
Pedigrees
= Male
= Female
= Carrier
= Affected
= Fetus
= Deceased
= Marriage
= Children
Used to Describe
Family Relationships
and Diseases
Dominant Disease Risk
Family 1
Family 2
Family 3
A/A
A/a
A/A
A/A
A/A
A/a
A/A A/A A/A
A/A
A/a
A/A
A/A
Mother
A/A
A/A
A = Normal copy
a = Mutant copy
Father
A a
A A/A A/a
A A/A A/a
2 of 4 or 50% risk
of having an
affected child
Recessive Disease Risk
B/b
B/B
B/b
Family 2
B/B
B/b
b/b B/B B/B
Family 3
B/b
B/B
B/B
B/b
B/B
B/B
B/b
Father
Family 1
B = Normal gene
b = Mutant gene
Mother
B b
B B/B B/b
b B/b b/b
1 of 4 or 25% risk of having
an affected child
2 of 4 or 50% risk of having
a child who is a carrier
Spinal Muscular Atrophy


Single gene recessive disease
Second most common lethal recessive
disease after cystic fibrosis

Carrier frequency of ~1 in 50

Incidence of ~1 in 10,000 births
1995: Identification of Gene for SMA
Lefebvre, S, et al., Cell 80: 155, 1995.



SMN gene (Survival of Motor Neurons) located on long
arm of chromosome 5 (5q)
SMN gene has 9 exons & encodes a 294 aa protein
In addition to the SMN gene, a copy of the SMN gene is
present on 5q, located centromeric to the SMN gene
 SMNt for telomeric or SMN1 is mutated to cause SMA
 SMNc for centromeric or SMN2 may alter severity of SMA

SMN1 and SMN2 have only two base differences located
in exons (one in exon 7 & one in exon 8)
Structure of SMN Gene Region
SMNc or 2
SMNt or 1
RNA
1 2a
mRNA
Protein
2b 3 4 5 6
7
8
2 base differences in exons
between SMN1 and SMN2
SMN Gene Mutation Causes SMA
Lefebvre, S, et al., Cell 80: 155, 1995.


Deletion of exon 7 or 7 & 8 associated with SMA
229 Patients: 103 Type I, 91 Type II, 35 Type III
 213/229 (93%): exon 7 & 8 deleted on both SMN1 copies
 13/229 (5.6%): only exon 7 deleted on both SMN1 copies
 2/229 (0.9%): exon 7 deletion on one SMN1 gene copy and
a smaller mutation on the other SMN1 gene copy
 1/229 (0.4%) had point mutation on one gene only

246 Controls:
 None with deletion of exon 7 + 8 on both SMN1 genes
Mutation Types in SMA



~94% of SMA patients have deletion of
exon 7 from both of their SMN1 genes
~6% of SMA patients have an exon 7 deletion
on one SMN1 gene copy and a small mutation
on the second SMN1 copy
Rarely, SMA patients may have non-deletion
mutations on both SMN1 gene copies
(estimated to be ~1 in 1,000 people with SMA)
SMA Diagnostic Test





Diagnosis of SMA is by absence of SMN1 exon 7
Testing complicated by presence of SMN2 gene which has an
exon 7 with only 1 base difference from SMN1
Diagnostic test uses PCR method to make millions of copies of
exon 7 from both the SMN1 and SMN2 genes
The 1 base difference allows the SMN2 PCR copies to be cut
into 2 pieces, but not the SMN1 PCR copies
The PCR copies are examined and an absence of the intact
SMN1 PCR copies is diagnostic of SMA for 94% of individuals
with SMA
SMA Diagnostic Test
Gel electrophoresis to examine
intact SMN1 and cut SMN2 PCR copies
Normal
(95%)
SMN1 (200 bp)
SMN2 (176 bp)
SMN2 (24 bp)
Normal
(5%)
SMA
Specimens for SMA Diagnostic Test


All cells of the body have the same DNA
Therefore, SMA testing can be performed on
any cells from a person who needs to be
tested

Generally, a tube of blood is used

Prenatal specimens can also be used
Method for SMA Diagnostic Test


DNA is purified from the cells of the
specimen
DNA is used for PCR of SMN1 and SMN2
exon 7

The SMN2 PCR copies are cut

The PCR products are examined on a gel

Absence of SMN1 exon 7 copies confirms
SMA diagnosis
SMA Diagnostic Test:
The Limitations
SMA
(94%)
SMA
(6%)
NonCarrier Carrier
SMN1 (200 bp)
SMN2 (176 bp)
SMN2 (24 bp)
Only positive for ~94% of individuals with SMA.
Cannot distinguish SMA carrier from non-carrier.
Family 1:
Requesting Prenatal Counseling
SMA Type II
Diagnosed 1995
10 weeks
What choices does this family have?
Family 1: The Options




Can use direct amniotic fluid, cultured
amniocytes or CVS to test the fetus
Does the affected son have an exon 7 SMN1
deletion on both his SMN1 gene copies?
If not known, testing the son will increase
the predictive value of fetal testing
Can do tests for son and fetus at the same
time or sequentially
Family 1: The Decision
The family chooses to:
 Use an amniotic fluid specimen so
do not have to wait for culturing
the amniocytes
 Have the son and the fetus tested
at the same time
SMA Diagnostic Test Results
Son
Fetus
SMN1 (200 bp)
SMN2 (176 bp)
SMN2 (24 bp)
SMA
Will Not Be
Diagnosis
Affected
Confirmed
(2 Deletions)
Family 1: Extended Family
Wife’s brother and his wife
want to know their risk of having
a child affected with SMA
What can be done?
Family 1: Extended Family



The SMA Diagnostic Test can only be used
to diagnose people with SMA symptoms
The brother and his wife are not affected,
but may be carriers
Need a test that can detect SMA carriers
SMA Carrier Test


Drs. Tom Prior and Arthur Burghes from
Ohio State University first reported SMA
Carrier Test method in 1997
Non-radioactive adaptation of the their
method developed at UPenn
McAndrew et al., Am J Hum Genet 60: 1411, 1997
SMA Carrier Test: Theory


The goal is to determine the number of SMN1 exon 7
copies a person has
The number of PCR copies made depends on the number
of gene copies in the DNA used for PCR
 More SMN1 gene copies produce more SMN1 PCR copies
 Fewer SMN1 gene copies produce fewer SMN1 PCR copies

The number of SMN1 PCR copies made is compared to
the number of PCR copies made from a gene “always”
present in 2 copies (CFTR gene)
SMA Carrier Test: Method

Two PCRs done in one test:

Cut SMN2 PCR copies

Quantify SMN1 and CFTR PCR copies

Calculate SMN1 gene copies:
 Exon 7 of SMN1 and SMN2 genes
 Part of the CFTR gene
Number
of
SMN1
copies
SMN1 Gene Copy # =
X2
Number of CFTR copies
SMA Carrier Test: Gel Analysis
Normal
Carrier Affected Normal Normal
(2 SMN1) (1 SMN1) (0 SMN1) (3 SMN1) (0 SMN2)
CFTR
SMN1
SMN2
SMN2
SMN Gene Region Possibilities
NORMAL CHROMOSOMES
SMN2
SMN1
SMN1
SMN1
SMN1
SMA Carrier Test: Limitations

Carrier test will not detect 3% of SMN1 gene
mutations that are not SMN1 exon 7 deletions
 6% of SMA patients have one non-deletion mutation
 This equals 3% of the SMN1 gene copies

Carrier test cannot differentiate:
 One SMN1 gene copy on each of 2 chromosomes (not a
carrier), from
 2 SMN1 gene copies on one chromosome and no SMN1
gene copies on the second chromosome (carrier)
Two SMN1 Copies by Carrier Test
1 Copy on Each Chromosome 5 (Not a Carrier)
SMN2
SMN1
SMN2
SMN1
2 Copies on One Chromosome 5 with a Deletion (Carrier)
SMN2
SMN1
SMN1
Family 1: Extended Family
Wife’s brother and his wife
want to know their risk of having
a child affected with SMA
What can be done?
Family 1: The Choices



The wife can be tested by the SMA Carrier Test
to determine her SMN1 gene copy #
The brother can be tested by the SMA Carrier
Test, but his carrier risk would be reduced if his
sister is shown to have an exon 7 SMN1 deletion
Most likely sister is a carrier since her son has
two deletion mutations, although new mutation
frequency is high
New Mutations in SMA



Approximately 2% of SMA patients have a
new mutation on one of their SMN1 genes
This means that one parent was not a
carrier
The majority of new mutations occur in
the SMN1 gene copy inherited from the
father
Family 1: The Choices



The wife can be tested by the SMA Carrier Test
to determine her SMN1 gene copy #
The brother can be tested by the SMA Carrier
Test, but his carrier risk would be reduced if his
sister is shown to have an exon 7 SMN1
deletion.
The sister and her husband could be tested to
rule out a new mutation in their son.
Family 1: The Decision
The family chooses to:
Test both the brother and his wife
Test both the sister and her husband to:
 Improve the interpretation of testing for the
brother
 Check for a possible new mutation in their son
Family 1: SMA Carrier Test Results
CFTR
SMN1
SMN2
SMN2
Family 1: SMA Carrier Test Results
2 copies
0 copies
1 copy
Not tested
1 copy
2 copies
Brother and sister are
both carriers.
Brother’s risk before
testing was 1 in 2,
and now is 1
Family 1: SMA Carrier Test Results
2 copies
1 copy
1 copy
2 copies
What do carrier results
mean for brother’s wife?
0 copies
Not tested
Family 1: Married into SMA Family


Before testing, the wife had ~1 in 50 chance of
being a carrier (carrier frequency in general
population)
She has 2 copies, but still has a small risk of
carrying a non-deletion mutation or having 2
SMN1 copies on one chromosome and a deletion
on the other chromosome (2+0 Carrier)
Carrier with 2 SMN1 Gene Copies
2 + 0 Carrier
SMN2
SMN1
SMN1
Non-deletion Mutation Carrier
SMN2
SMN1
SMN2
SMN1
Family 1: Married into SMA Family



Before testing, the wife had ~1 in 50 chance of
being a carrier (carrier frequency in general
population)
She has 2 copies, but still has a small risk of
carrying a non-deletion mutation or having 2
SMN1 copies on one chromosome and a deletion
on the other chromosome (2 + 0 Carrier)
By Bayesian analysis, wife’s carrier risk is
reduced from ~1 in 50 to ~1 in 800
Family 1: SMA Carrier Test Results
2 copies
1 copy
1 copy
2 copies
What is this couple’s risk
of having a child with SMA?
0 copies
Not tested
Family 1: Couple’s Combined Risk


Before testing, the couple’s risk of having a child
with SMA was ~1 in 400 (1/2 X 1/50 X 1/4)
After testing know:
 Brother is a carrier (risk of 1)
 Wife’s risk of being a carrier is ~1 in 800 without
including risk of a new mutation since she is female

Therefore, the risk of having an affected child is
reduced to ~1 in 3200 (1 X 1/800 X 1/4)
Family 1: SMA Carrier Test Results
2 copies
1 copy
1 copy
2 copies
Why was fetus not tested?
0 copies
Not tested
Family 1: Prenatal SMA Testing




In general, the SMA Carrier Test is not used for
prenatal diagnosis
Use SMA Diagnostic Test to test if fetus has
deletion of SMN1
Individual can choose to have Carrier testing in the
future as an adult
May use SMA Carrier Test for testing of a fetus in a
family with a non-deletion mutation
Family 1: SMA Carrier Test Results
?
2 copies
1 copy
1 copy
2 copies
Why does “obligate carrier”
have 2 copies?
0 copies
Not tested
Family 1: “Carrier” with 2 Copies

New Mutation
 SMN1 new mutation rate estimated at ~2% (7 in 340 SMA families)
 11 of 15 cases had new mutation on father’s chromosome revealing a high
incidence of rearrangement during spermatogenesis
 Son may have a new mutation, reducing couple’s future risk

2+0 Carrier

Gonadal Mosaicism: Some but not all sperm have deletion
 Two SMN1 copies on one chromosome and none on other
 Frequency ~8% of people not affected with SMA
 We have seen 1 case with 2 copies in blood and <2 copies in sperm
Resolve new mutation from 2+0 by linkage analysis
If new mutation, test father’s sperm for mosaicism
Linkage Analysis





Method for tracking chromosomes in a family
For SMA, track chromosome 5q
Must include affected family member to define
which 5q’s have mutated SMN1 genes
In combination with Carrier Test, can distinguish
2+0 from new mutation, but requires extended
family members
Can be used to identify carriers in families with
non-deletion mutations
Uses of SMA Carrier Test

Family member of person with SMA (parents,
sibling, aunt, uncle, cousin, grandparent, etc.)

Married into family with SMA

Married to someone affected with SMA

Symptomatic with negative SMA Direct Test

Parents of one child with SMA to potentially
identify a new mutation and decrease future risk

Sperm donors and/or recipients

Prenatal diagnosis for non-deletion mutation
Non-Deletion Mutation Testing




Most non-deletion mutations occur in exon 6 of the
SMN1 gene
Sequence analysis of the SMN1 gene can sometimes
identify the mutation
Can use the known mutation to track the mutated
gene through a family and for prenatal diagnosis
Not currently available except for research
(Dr. Gonzalez, Dupont Children’s Hospital, DE)
SMA Genetic Testing Summary

SMA Diagnostic Test

SMA Carrier Test

 Use for diagnosis of SMA
 Only positive for ~94% of people with SMA
 Cannot distinguish SMA carrier from non-carrier
 Determines SMN1 gene copy number
 Cannot detect non-deletion or 2:0 carriers
Further clarification by linkage analysis by
tracking chromosome 5 in a family
Questions
?