1. 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

2. Objectives Explain the genetic testing options for SMA
Leave no one behind

3. 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

4. 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

5. Clinical Types of SMA SMA Type I: Werdnig-Hoffmann
Most severe form of SMA
Onset at birth to 3 months, death by ~2 yrs
SMA Type II: Intermediate
Symptoms begin at infancy to toddler age
Survive beyond 4 yrs of age
SMA Type III: Kugelberg-Welander
Onset after age 2 yrs to adult

6. Basics of Genetics Genetic Information DNA and Chromosomes Genes
Messenger RNA (mRNA)
Proteins
Inheritance
Pedigrees

7. 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.

8. DNA and Chromosomes Genetic information is encoded by DNA
Pieces of DNA in cells are called chromosomes
There are 24 kinds of human chromosomes:
1 through 22 (1 is the longest; 22 is the shortest)
X and Y are the “sex” chromosomes
Each normal cell has 46 chromosomes:
2 copies of 1 through 22, plus 2 sex chromosomes
XX is female, XY is male

9. 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

10. Chromosome Structure One Chromosome p Arm q Centromere
If arms are of unequal length:
short arm is called p (petite)
long arm is called q
Centromeric
Telomeric
Centromere

11. What Makes Each Person Unique?
Each egg or sperm contains 23 chromosomes
One of each pair of chromosomes 1 to 22, at random
One of the two sex chromosomes, at random
One egg and one sperm combine to make a fetus
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

12. 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

13. 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

14. Gene Structure Gene Promoter
Promoter region regulates gene expression, i.e., controls when a gene will be used to make the protein it encodes
Intron
Exon

15. 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

16. 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

17. 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

18. Gene Expression DNA RNA mRNA Protein Cell Work TRANSCRIPTION
RNA PROCESSING
Protein
TRANSLATION
Cell Work

19. Nucleic acid  Nucleic acid
TRANSCRIPTION
Nucleic acid  Nucleic acid
(DNA) (RNA)
Same Language
TRANSLATION
Nucleic acid  Protein
(RNA) (Amino Acid)
Different Language

20. 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

21. 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

22. 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

23. 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

24. Genetic Terminology Affected: Carrier:
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

25. Pedigrees = Male = Female = Carrier = Affected = Fetus = Deceased
= Marriage
= Children
Used to Describe
Family Relationships
and Diseases

26. Dominant Disease Risk Family 1 Family 2 Family 3 A = Normal copy
a = Mutant copy
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
Mother
Father
2 of 4 or 50% risk
of having an
affected child
A/A A/A A/a A/A A/A
A/A A/A A/A A/a

27. Recessive Disease Risk
B = Normal gene
b = Mutant gene
Family 1
Family 2
Family 3
B/b B/B B/B B/b B/B B/B
B b
B B/B B/b
b B/b b/b
Mother
Father
2 of 4 or 50% risk of having
a child who is a carrier
1 of 4 or 25% risk of having an affected child
B/b B/b B/b B/B B/B
b/b B/B B/B B/b

28. 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

29. 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)

30. Structure of SMN Gene Region
SMNc or 2
SMNt or 1
RNA
1 2a 2b
2 base differences in exons
between SMN1 and SMN2
mRNA
Protein

31. 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 on both SMN1 genes

32. 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)

33. 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

34. Gel electrophoresis to examine intact SMN1 and cut SMN2 PCR copies
SMA Diagnostic Test
Gel electrophoresis to examine
intact SMN1 and cut SMN2 PCR copies
Normal Normal
(95%) (5%) SMA
SMN1 (200 bp)
SMN2 (176 bp)
SMN2 (24 bp)

35. 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

36. 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

37. SMA Diagnostic Test: The Limitations
SMA SMA Non-
(94%) (6%) Carrier 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.

38. Family 1: Requesting Prenatal Counseling
SMA Type II
Diagnosed 1995
10 weeks
What choices does this family have?

39. 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

40. 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

41. SMA Diagnostic Test Results
Son Fetus
SMN1 (200 bp)
SMN2 (176 bp)
SMN2 (24 bp)
SMA
Diagnosis
Confirmed
(2 Deletions)
Will Not Be
Affected

42. 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?

43. 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

44. 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

45. 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)

46. SMA Carrier Test: Method
Two PCRs done in one test:
Exon 7 of SMN1 and SMN2 genes
Part of the CFTR gene
Cut SMN2 PCR copies
Quantify SMN1 and CFTR PCR copies
Calculate SMN1 gene copies:
Number of SMN1 copies
Number of CFTR copies
SMN1 Gene Copy # = X 2

47. SMA Carrier Test: Gel Analysis
Normal Carrier Affected Normal Normal
(2 SMN1) (1 SMN1) (0 SMN1) (3 SMN1) (0 SMN2)
CFTR
SMN1
SMN2

48. SMN Gene Region Possibilities
NORMAL CHROMOSOMES
SMN2
SMN1
SMN1
SMN1
SMN1

49. 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)

50. 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

51. 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?

52. 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

53. 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

54. 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.

55. 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

56. Family 1: SMA Carrier Test Results
CFTR
SMN1
SMN2

57. Family 1: SMA Carrier Test Results
2 copies copy copy copies
Brother and sister are both carriers.
Brother’s risk before testing was 1 in 2, and now is 1
0 copies Not tested

58. Family 1: SMA Carrier Test Results
2 copies copy copy copies
What do carrier results
mean for brother’s wife?
0 copies Not tested

59. 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)

60. Carrier with 2 SMN1 Gene Copies
Non-deletion Mutation Carrier
SMN2
SMN1
SMN2
SMN1

61. 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

62. Family 1: SMA Carrier Test Results
2 copies copy copy copies
What is this couple’s risk
of having a child with SMA?
0 copies Not tested

63. 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)

64. Family 1: SMA Carrier Test Results
2 copies copy copy copies
Why was fetus not tested?
0 copies Not tested

65. 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

66. Family 1: SMA Carrier Test Results?
2 copies copy copy copies
Why does “obligate carrier”
have 2 copies?
0 copies Not tested

67. 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
Two SMN1 copies on one chromosome and none on other
Frequency ~8% of people not affected with SMA
Gonadal Mosaicism: Some but not all sperm have deletion
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

68. 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

69. 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

70. 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)

71. SMA Genetic Testing Summary
SMA Diagnostic Test
Use for diagnosis of SMA
Only positive for ~94% of people with SMA
Cannot distinguish SMA carrier from non-carrier
SMA Carrier Test
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

72. Questions ?