Genetics: The Whole Picture

SMA Takes the Hill 2003Debra G.B. Leonard, M.D., Ph.D.Director, Molecular Pathology LaboratoryUniversity of Pennsylvania Health SystemPhiladelphia, PA

ObjectivesExplain the genetic testing options for SMALeave no one behind

What We Will Talk AboutBasic Clinical Features of SMABasics of GeneticsSMN Gene StructureSMA Diagnostic TestSMA Carrier TestQuestions and Discussion

Spinal Muscular AtrophyThe brain makes the body move by sending nerve signals from the brain to nerve cells in the spinal cord called the anterior horn motor neuronsThese motor neurons relay signals to the muscles which cause the muscles to contractMovement occurs when muscles contract The anterior horn motor neurons no longer function in individuals with SMASince muscles are not signaled to contract and are not used, the muscles atrophy or get smaller

Clinical Types of SMASMA Type I: Werdnig-HoffmannMost severe form of SMAOnset at birth to 3 months, death by ~2 yrsSMA Type II: IntermediateSymptoms begin at infancy to toddler ageSurvive beyond 4 yrs of ageSMA Type III: Kugelberg-Welander Onset after age 2 yrs to adult

Basics of GeneticsGenetic InformationDNA and ChromosomesGenesMessenger RNA (mRNA)ProteinsInheritancePedigrees

Genetic InformationGenetic information directs growth and development, and determines physical characteristicsEvery cell in the human body has the same genetic informationEach cell uses a different part of the genetic information to perform that cells function, e.g. skin, blood, muscle, nerve, etc.

DNA and ChromosomesGenetic information is encoded by DNAPieces of DNA in cells are called chromosomesThere are 24 kinds of human chromosomes:1 through 22 (1 is the longest; 22 is the shortest)X and Y are the sex chromosomesEach normal cell has 46 chromosomes:2 copies of 1 through 22, plus 2 sex chromosomesXX is female, XY is male

ChromosomesChromosomes consist of DNA plus proteinsThe proteins help to organize the DNA piecesEach chromosome has a centromereThe centromere divides the DNA into two partsEach part has a centromeric end and a free endThe free end is called the telomeric end

Chromosome Structure

What Makes Each Person Unique?Each egg or sperm contains 23 chromosomesOne of each pair of chromosomes 1 to 22, at randomOne of the two sex chromosomes, at randomOne egg and one sperm combine to make a fetusEach person gets half their chromosomes from their mother and half from their fatherSiblings are similar because they share some of the same chromosomes, but different because they have some different chromosomes

DNA Encodes Genetic InformationDNA is a chain of four different building blocks (or bases) called A, C, G and TA, C, G and T are the letters of the genetic alphabetSome parts of each DNA chain encode instructions which the cell uses to make proteins, that do work in cellsProtein-coding parts of DNA are called genesOther parts of each DNA chain are nonsense

Genes - 1One gene encodes one protein, more or lessEach gene has regulatory regions, protein coding regions and nonsense regionsCoding parts of genes are called exonsNoncoding, nonsense parts of genes are called introns

Gene Structure

Genes - 2Genes are located on the arms of the chromosomesEach kind of chromosome contains a different set of genesBecause 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 malesThere 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 acidsRNA uses bases A, C, G and U, that correspond to the A, C, G and T bases of DNAThe RNA copy is processed to remove the introns and is then called messenger RNA or mRNAmRNA is the blueprint used to make the protein

The Genetic CodeA protein is a chain of amino acids3 mRNA bases code for one amino acidTherefore, the mRNA is used as the blueprint to make a protein by the protein-making or translation machinery of a cellWhile 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 ExpressionDNA

TRANSCRIPTIONNucleic acid Nucleic acid (DNA) (RNA)Same Language

Genetic DiseasesA genetic disease is due to a change in the DNA sequence of a geneBecause DNA in chromosomes is passed from parent to child, genetic diseases are also passed from parent to childA change in the DNA sequence of a gene is called a mutation

Examples of Gene MutationsA 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 proteinDeletion of part or all of the gene sequence, so the protein is not madeChange sequences that direct intron removal, so the mRNA is not correctly made, so the protein is not made

Types of InheritanceSingle gene diseases are caused by mutation of one gene, e.g. cystic fibrosis, SMA, Huntington diseaseMulti-gene diseases are caused by a combination of mutations in several genes, e.g. heart disease, asthma, arthritis

Types of InheritanceSingle gene diseases are caused by mutation of one gene, e.g. cystic fibrosis, SMA, Huntington diseaseDominant inheritance: Mutation of one gene copy causes diseaseRecessive inheritance: Mutation of both gene copies causes disease

Genetic TerminologyAffected: Someone who has a genetic disease Can be either a dominant or recessive diseaseCarrier: Someone who has a gene mutation for a recessive disease in only one gene copyPerson does not have disease symptoms, but may pass on mutation to their children

Pedigrees= Male= Female = Carrier= Affected= Fetus= Deceased= Marriage= ChildrenUsed to DescribeFamily Relationshipsand Diseases

Dominant Disease RiskFamily 1Family 2Family 3A/A A/A A/a A/A A/AA/AA/AA/a A/A A/A A/AA = Normal copya = Mutant copyA/A A/A A/A A/a

Recessive Disease RiskB = Normal geneb = Mutant geneB/bB/B B/B B/b B/B B/BB/b B/b B/b B/B B/BFamily 1Family 2Family 3b/b B/B B/B B/b

Spinal Muscular AtrophySingle gene recessive disease Second most common lethal recessive disease after cystic fibrosisCarrier frequency of ~1 in 50Incidence of ~1 in 10,000 births

1995: Identification of Gene for SMASMN gene (Survival of Motor Neurons) located on long arm of chromosome 5 (5q)SMN gene has 9 exons & encodes a 294 aa proteinIn addition to the SMN gene, a copy of the SMN gene is present on 5q, located centromeric to the SMN geneSMNt for telomeric or SMN1 is mutated to cause SMASMNc for centromeric or SMN2 may alter severity of SMASMN1 and SMN2 have only two base differences located in exons (one in exon 7 & one in exon 8)Lefebvre, S, et al., Cell 80: 155, 1995.

Structure of SMN Gene RegionSMNc or 2SMNt or 11 2a 2b 3 4 5 6 7 82 base differences in exons between SMN1 and SMN2ProteinRNAmRNA

SMN Gene Mutation Causes SMADeletion of exon 7 or 7 & 8 associated with SMA229 Patients: 103 Type I, 91 Type II, 35 Type III213/229 (93%): exon 7 & 8 deleted on both SMN1 copies13/229 (5.6%): only exon 7 deleted on both SMN1 copies2/229 (0.9%): exon 7 deletion on one SMN1 gene copy and a smaller mutation on the other SMN1 gene copy1/229 (0.4%) had point mutation on one gene only246 Controls: None with deletion of exon 7 + 8 on both SMN1 genesLefebvre, S, et al., Cell 80: 155, 1995.

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 copyRarely, SMA patients may have non-deletion mutations on both SMN1 gene copies (estimated to be ~1 in 1,000 people with SMA)

SMA Diagnostic TestDiagnosis of SMA is by absence of SMN1 exon 7Testing complicated by presence of SMN2 gene which has an exon 7 with only 1 base difference from SMN1Diagnostic test uses PCR method to make millions of copies of exon 7 from both the SMN1 and SMN2 genesThe 1 base difference allows the SMN2 PCR copies to be cut into 2 pieces, but not the SMN1 PCR copiesThe 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 TestNormalNormal (95%) (5%) SMASMN1 (200 bp)SMN2 (176 bp)

SMN2 (24 bp)Gel electrophoresis to examine intact SMN1 and cut SMN2 PCR copies

Specimens for SMA Diagnostic TestAll cells of the body have the same DNATherefore, SMA testing can be performed on any cells from a person who needs to be testedGenerally, a tube of blood is usedPrenatal specimens can also be used

Method for SMA Diagnostic TestDNA is purified from the cells of the specimenDNA is used for PCR of SMN1 and SMN2 exon 7The SMN2 PCR copies are cut The PCR products are examined on a gelAbsence of SMN1 exon 7 copies confirms SMA diagnosis

SMA Diagnostic Test: The Limitations SMA SMA Non- (94%) (6%) Carrier CarrierCannot distinguish SMA carrier from non-carrier.SMN1 (200 bp)SMN2 (176 bp)

SMN2 (24 bp)Only positive for ~94% of individuals with SMA.

Family 1: Requesting Prenatal CounselingSMA Type IIDiagnosed 1995What choices does this family have?10 weeks

Family 1: The OptionsCan use direct amniotic fluid, cultured amniocytes or CVS to test the fetusDoes 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 testingCan do tests for son and fetus at the same time or sequentially

Family 1: The DecisionThe family chooses to:Use an amniotic fluid specimen so do not have to wait for culturing the amniocytesHave the son and the fetus tested at the same time

SMA Diagnostic Test ResultsSon FetusSMA DiagnosisConfirmed(2 Deletions)Will Not BeAffectedSMN1 (200 bp)SMN2 (176 bp)

SMN2 (24 bp)

Family 1: Extended FamilyWifes brother and his wife want to know their risk of having a child affected with SMAWhat can be done?

Family 1: Extended FamilyThe SMA Diagnostic Test can only be used to diagnose people with SMA symptomsThe brother and his wife are not affected, but may be carriersNeed a test that can detect SMA carriers

SMA Carrier TestDrs. Tom Prior and Arthur Burghes from Ohio State University first reported SMA Carrier Test method in 1997Non-radioactive adaptation of the their method developed at UPennMcAndrew et al., Am J Hum Genet 60: 1411, 1997

SMA Carrier Test: TheoryThe goal is to determine the number of SMN1 exon 7 copies a person hasThe number of PCR copies made depends on the number of gene copies in the DNA used for PCRMore SMN1 gene copies produce more SMN1 PCR copiesFewer SMN1 gene copies produce fewer SMN1 PCR copiesThe 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: MethodTwo PCRs done in one test:Exon 7 of SMN1 and SMN2 genes Part of the CFTR geneCut SMN2 PCR copiesQuantify SMN1 and CFTR PCR copies Calculate SMN1 gene copies:Number of SMN1 copiesNumber of CFTR copiesSMN1 Gene Copy # = X 2

SMA Carrier Test: Gel Analysis CFTR

SMN1 SMN2

SMN2 Normal Carrier Affected Normal Normal(2 SMN1) (1 SMN1) (0 SMN1) (3 SMN1) (0 SMN2)

SMN Gene Region PossibilitiesSMN2SMN1SMN1SMN1SMN1NORMAL CHROMOSOMES

SMA Carrier Test: LimitationsCarrier test will not detect 3% of SMN1 gene mutations that are not SMN1 exon 7 deletions 6% of SMA patients have one non-deletion mutationThis equals 3% of the SMN1 gene copiesCarrier test cannot differentiate: One SMN1 gene copy on each of 2 chromosomes (not a carrier), from2 SMN1 gene copies on one chromosome and no SMN1 gene copies on the second chromosome (carrier)

Two SMN1 Copies by Carrier TestSMN2SMN1SMN1 2 Copies on One Chromosome 5 with a Deletion (Carrier)SMN2 1 Copy on Each Chromosome 5 (Not a Carrier)SMN1SMN1SMN2

Family 1: Extended FamilyWifes brother and his wife want to know their risk of having a child affected with SMA

What can be done?

Family 1: The ChoicesThe 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 SMAApproximately 2% of SMA patients have a new mutation on one of their SMN1 genesThis means that one parent was not a carrierThe majority of new mutations occur in the SMN1 gene copy inherited from the father

Family 1: The ChoicesThe 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 DecisionThe family chooses to:Test both the brother and his wifeTest both the sister and her husband to: Improve the interpretation of testing for the brotherCheck for a possible new mutation in their son

Family 1: SMA Carrier Test Results CFTR

SMN1 SMN2

SMN2

Family 1: SMA Carrier Test Results2 copies 1 copy 1 copy 2 copies0 copies Not testedBrother and sister are both carriers.Brothers risk before testing was 1 in 2, and now is 1

Family 1: SMA Carrier Test ResultsWhat do carrier results mean for brothers wife?2 copies 1 copy 1 copy 2 copies0 copies Not tested

Family 1: Married into SMA FamilyBefore 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 CopiesSMN2SMN2SMN1SMN1 2 + 0 Carrier Non-deletion Mutation CarrierSMN1SMN1SMN2

Family 1: Married into SMA FamilyBefore 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, wifes carrier risk is reduced from ~1 in 50 to ~1 in 800

Family 1: SMA Carrier Test ResultsWhat is this couples risk of having a child with SMA?2 copies 1 copy 1 copy 2 copies0 copies Not tested

Family 1: Couples Combined RiskBefore testing, the couples 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)Wifes risk of being a carrier is ~1 in 800 without including risk of a new mutation since she is femaleTherefore, 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 ResultsWhy was fetus not tested?2 copies 1 copy 1 copy 2 copies0 copies Not tested

Family 1: Prenatal SMA TestingIn general, the SMA Carrier Test is not used for prenatal diagnosisUse SMA Diagnostic Test to test if fetus has deletion of SMN1Individual can choose to have Carrier testing in the future as an adultMay use SMA Carrier Test for testing of a fetus in a family with a non-deletion mutation

Family 1: SMA Carrier Test ResultsWhy does obligate carrier have 2 copies?2 copies 1 copy 1 copy 2 copies0 copies Not tested?

Family 1: Carrier with 2 CopiesNew MutationSMN1 new mutation rate estimated at ~2% (7 in 340 SMA families) 11 of 15 cases had new mutation on fathers chromosome revealing a high incidence of rearrangement during spermatogenesisSon may have a new mutation, reducing couples future risk2+0 CarrierTwo SMN1 copies on one chromosome and none on other Frequency ~8% of people not affected with SMAGonadal Mosaicism: Some but not all sperm have deletionWe have seen 1 case with 2 copies in blood and

Linkage AnalysisMethod for tracking chromosomes in a familyFor SMA, track chromosome 5q Must include affected family member to define which 5qs have mutated SMN1 genesIn combination with Carrier Test, can distinguish 2+0 from new mutation, but requires extended family membersCan be used to identify carriers in families with non-deletion mutations

Uses of SMA Carrier TestFamily member of person with SMA (parents, sibling, aunt, uncle, cousin, grandparent, etc.)Married into family with SMAMarried to someone affected with SMASymptomatic with negative SMA Direct TestParents of one child with SMA to potentially identify a new mutation and decrease future riskSperm donors and/or recipientsPrenatal diagnosis for non-deletion mutation

Non-Deletion Mutation TestingMost non-deletion mutations occur in exon 6 of the SMN1 geneSequence 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 diagnosisNot currently available except for research (Dr. Gonzalez, Dupont Childrens Hospital, DE)

SMA Genetic Testing SummarySMA Diagnostic TestUse for diagnosis of SMAOnly positive for ~94% of people with SMACannot distinguish SMA carrier from non-carrierSMA Carrier TestDetermines SMN1 gene copy numberCannot detect non-deletion or 2:0 carriersFurther clarification by linkage analysis by tracking chromosome 5 in a family

Questions?