ELearning SCD BTHAL Presentation Nov3 2015

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Sickle Cell Disease & β- Thalassemia Genes, Disease, and Therapeutics Fall 2015

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Sickle Cell Disease Presentation - Genes, Diseases, and Therapeutics

Transcript of ELearning SCD BTHAL Presentation Nov3 2015

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Sickle Cell Disease & β-Thalassemia

Genes, Disease, and TherapeuticsFall 2015

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November 3Genetics, newborn screening,

molecular phenotype, case study, inheritance, prenatal testing, question

and answer

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Hemoglobin

• Hemoglobin (Hb) protein transports oxygen in red blood cells

• Adult Hb is a tetramer with 2 α chains, 2 β chains and 4 heme groups

• α chain encoded on chromosome 16 gene cluster and β chain on chromosome 11 gene cluster

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Adult hemoglobin

• Hemoglobin A (α2β2)– The most common type in adults (about 97%)

• Hemoglobin A2 (α2δ2)– δ chain synthesis begins late in the third trimester

and in adults, it has a normal level of about 2%• Hemoglobin F (α2γ2)

– In adults, restricted to a limited population of red cells called F cells and has a normal level of less than 1%

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Beta-globin LCR

• Upstream of the epsilon-globin gene in the beta-globin gene cluster on chromosome 11

• LCR, in addition to individual promoters for each beta-globin like gene in the beta-globin gene cluster, provides regulatory control of gene expression

• “Switching” of beta-globin like genes during development

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Developmental regulation of expression of beta-globin gene cluster

• Two developmental switches in expression from the cluster– Embryonic to fetal during the first trimester– Fetal to adult around birth

• Processes involved– DNA methylation:

• Hypermethylation of beta-globin promoter during embryonic and fetal stages

• Hypermethylation of gamma-globin promoter during adult stage

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Developmental regulation of expression of beta-globin gene cluster

• Processes involved (cont)– Histone acetylation and deacetylation

• Active globin genes are hyperacytylated• Inactive globin genes are hypoacytylated

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Sickle Cell Disease & β-Thalassemia

• Mutations in either α or β globin gene cluster members can lead to a wide variety of abnormal Hb (structural or numerical chain problems)

• SCD is usually a defect in globin chain structure• β-Thal is usually a defect in globin chain number• Combinations possible - A sickle allele and one

type of β-Thal allele together cause Sβ0-Thal (βSβ0) which has many features of sickle cell disease

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Sickle Cell Disease

• SCD alleles are more common in the following ancestries: African, Mediterranean, Middle Eastern, Indian, Caribbean, and parts of Central and South America

• One in every 300-500 African-Americans born/year has SCD (specifically, Hb SS)

• One in every 36,000 Hispanic-Americans born/year has SCD (specifically, Hb SS)

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Sickle Cell Disease

• SCA allele (βS) provides protection against malaria and thus is more prevalent in populations where malaria is widespread– RBC of infected heterozygotes (βSβwt)

believed to express malarial antigens more effectively than infected homozygote normal (βwtβwt) so immune system will clear infection more rapidly in these RBCs

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β-Thalassemia

• β-Thal alleles are more common in the following ancestries: Mediterranean, Middle Eastern, Central Asian, Indian, Far Eastern, and African.

• Like the SCD allele, βthal alleles (β0 and β+, but note not truly alleles per say) provide protection against malaria and thus are more prevalent in populations where malaria is widespread

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SCD & β-Thalassemia newborn screen

• All 50 U.S. states and D.C. screen for SCD (which includes sickle cell anemia), many also screen for types of β-Thalassemia– SCD screening may include screening for

SCA, sickle cell trait, SC Disease, Sβ0-Thalassemia, Sβ+-Thalassemia, SE Disease, and CC Disease.

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SCD & β-Thalassemia newborn screen

• Currently, most screening done by isoelectric focusing (IEF) from dried blood spot– If IEF test positive, usually a second test is

performed • If second test is HPLC or DNA testing, this usually

allows for a diagnosis to be made (esp. if DNA test, HPLC result may not be reliable as we will discuss later)

• If the second test is positive, often a final test on a fresh blood sample is completed around 6 weeks of age

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SCD & β-Thalassemia newborn screen

• Newborn screening may produce a false negative result if baby has had a transfusion

• Prematurity may also confound newborn screening results– A normal premature infant may only show HbF on

IEF (no HbA made yet)– Premature infant with SCA may also only show

HbF on IEF because of prematurity (the β-globin gene is not yet expressed in large quantities so don’t see that HbS yet)

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Texas newborn screening for SCD

• Texas currently screens for Sickle Cell Anemia (HbSS), SC Disease, Sβ+- Thalassemia, and Sβ0- Thalassemia

• About 1 in 2500 newborns found to have SCD each year via newborn screening in Texas

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Possible IEF results and interpretation

• Result 1: Newborn shows HbF and HbS, but no HbA– What does this suggest?

• Result 2: Newborn shows HbF, HbS, and HbC, but no HbA– What does this suggest?

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Possible IEF results and interpretation

• Result 3: Newborn shows HbF, HbS, and HbA– What does this suggest?

• Result 4: Newborn shows HbS alone (i.e. no HbF, HbA, etc.)– What does this suggest?

• Result 5: Newborn shows HbF alone (i.e. no HbA, HbS, etc.)– What does this suggest?

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SCD - Genetics

• Terminology can be confusing– Most common form of normal adult

hemoglobin is Hb A. When someone has normal adult hemoglobin, they are sometimes referred to as being Hb AA. Genotype is βwtβwt (the α alleles are also wildtype, but they are not usually noted)

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SCD - Genetics

SCD comprises: • Sickle Cell Anemia (involving Hb S with the disease

phenotype sometimes referred to as Hb SS. Genotype βSβS)

– Sickle Cell Trait is Hb AS with genotype βwtβS. This is the carrier heterozygote (the “carrier” state for SCA)

• SC Disease (involving both Hb S & Hb C with the disease phenotype sometimes referred to as Hb SC. Genotype βSβC)

• Sβ0-Thal (involving Hb S with the disease phenotype sometimes referred to as Hb Sβ0. Genotype βSβ0, but note β0 (and β+) not really alleles as discussed previously)

• Other conditions usually involving a sickle allele

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SCD - Genetics

• β globin gene is HBB• Gene is 1.6kb with 3 exons

• Upstream of β globin gene cluster on chromosome 11 is locus control region (LCR)– Controls developmental timing of expression of

the genes in the beta globin gene cluster

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SCD - Genetics

• Numerous types of mutations can lead to numerous types of structural aberrations in the β chain

• SCA (Hb SS) (60-70% of SCD in U.S.)– Both alleles βS. βS results from a point mutation in β globin

gene. GAG (glutamic acid) -> GTG (valine) in 6th codon

– A to T is in nucleotide 20.

• SC Disease (Hb SC)– One allele is βS and other is βC. βC results from a point mutation

in β globin gene. Causes glutamic acid to lysine in 6th codon

– G to A in nucleotide 19.

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Another confusing aspect of Hb literature

• The SCA mutation (and SC disease mutation) is in the “6th codon” of the gene. It’s actually the 7th codon, but because the mature β globin protein does not include the initiating Met, they say the 6th codon.

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SCD - Genetics

• Sβ0-Thalassemia (Hb Sβ0)– One allele is βS and other “allele” is β0. As noted earlier, β0 is

not truly a specific mutation (i.e. particular allele). It refers to the molecular phenotype of the thalassemia. β0 denotes no β chain made from that allele. Numerous different mutations can cause this.

• Sβ+-Thalassemia (Hb Sβ+)– One allele is βS and other “allele” is β+. β+ is not truly a specific

mutation (i.e. particular allele). It refers to the molecular phenotype of the thalassemia. β+ denotes some, but less than wildtype levels, of β chain made from that allele. Numerous different mutations can cause this.

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SCD - Genetics

• Of most common 4 SCD, SCA and Sβ0-Thalassemia are more severe phenotypes versus SC Disease or Sβ+-Thalassemia

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SCD - Genetics

• Numerous other SCD mutations possible. Examples:– SD Disease (Hb SD)

• One allele is βS and other is βD. βD is Glu->Gln at codon 121. Sometimes referred to as D-Punjab reflecting higher incidence in Indian population

– SO Disease (Hb SO)• One allele is βS and other is βo (not to be confused with form

of thalassemia which is “zero”). βo is Glu->Lys at codon 121. Sometimes referred to as O-Arab reflecting higher incidence in Middle Eastern population

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SCD - Genetics

• Numerous other SCD mutations possible. Examples (cont.):– SE Disease (Hb SE)

• One allele is βS and other is βE. βE is Glu->Lys at codon 26. βE found in Sri Lanka, Eastern India, Southeast Asia, and Southwest China

– CC Disease (Hb CC)• Both alleles βC. βC results from a point mutation in β

globin gene. Causes glutamic acid to lysine in 6th codon. CC disease is homozygosity for this mutation

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Case study

• 17 year-old male with severe clinical course– frequent pain crises of increasing intensity– 2 episodes of acute chest syndrome requiring

hospitalization and multiple blood transfusions• Had been diagnosed with SC Disease at

age 6 with a confirmation diagnosis of SC Disease at age 11

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Case study

• SC Disease is normally associated with a less severe clinical picture compared to, for example, SCA

• SC Disease – Painful crises usually appear later in life (often

after age 20)– Less frequent episodes of crises

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Case study

• Methodology used to make the initial diagnosis in patient (at age 6) was unknown

• Methodology used to make diagnosis at age 11 was IEF and an older version of HPLC instrument

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Case study

• DNA sequencing also performed and indicated presence of O-Arab β-globin mutation (versus C Harlem which was in the differential here)

• Discussion with patient also revealed a relevant piece of family history– Patient’s brother was recently diagnosed with S/O-

Arab Disease after an earlier misdiagnosis with SC Disease (methodology in misdiagnosis unknown)

• Patient actually has S/O-Arab Disease (also known as SO Disease), not SC Disease

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Case study

• S/O-Arab Disease– More clinically severe than SC Disease

• S/O-Arab Disease shows earlier onset, more frequent, and more severe clinical characteristics

• hemolytic anemia, jaundice, vaso-occlusive complications such as pain crises and stroke, pneumonia, acute chest syndrome, and sepsis

– Patient had frequent pain crises of increasing intensity and 2 episodes of acute chest syndrome requiring hospitalization and multiple blood transfusions all by age 17

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Case study

• Revised diagnosis led to revised treatment protocol for patient– Patient was started on hydroxyurea (1000

mg/day) in accordance with NIH consensus documents

• As we will discuss in next lecture, hydroxyurea has been shown to reduce episodes of pain crises and acute chest syndrome

• At follow-up, patient reported improved health and anemia had improved somewhat

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Case study

• Easy to misinterpret the IEF result and older version of HPLC did not allow one to distinguish between HbC and HbO-Arab

• So understandable that he was misdiagnosed

• However, the emergence of such a severe clinical picture should have prompted physicians to question diagnosis perhaps earlier

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Case study

• This case also emphasizes importance of using more than one methodology to determine diagnosis in these conditions, especially if there is known possibility of ambiguity

• Investigators noted that if a well-known (albeit not often used now) hemoglobin electrophoretic technique with citrate agar had been used initially, could have distinguished between HbC, HbO-Arab, and HbC-Harlem

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SE Disease case study

• IEF newborn screen of male infant suggested SE Disease

• At age 14 months, presented with fever and strong pain in thighs

• Investigators sought to confirm diagnosis (unclear how diagnosis was initially made after the positive newborn screen)

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• HPLC also suggested SE Disease

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HPLC results of boy’s parents

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SE Disease case study

• Investigators noted that often times it is difficult to distinguish various hemoglobins depending on the technique used– They noted that HbE comigrates with HbO-Arab

(among others) in IEF– HbE comigrates with HbC and HbO-Arab (among

others) in cellulose acetate hemoglobin electrophoresis at alkaline pH

– HbE and HbA2 have similar retention times in HPLC

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SE Disease case study

• How to resolve these issues?• DNA analysis is useful• They used the primer specific PCR-based

system called “Amplification Refractory Mutation System” (ARMS)

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ARMS

• Two different tubes used for PCR analysis– Tube 1: Patient sample, a common primer and a

mutation-specific primer• Often looking for a point mutation. The mutation-specific

primer’s 3’ end is complementary to the mutant nucleotide– HbS and HbE both due to point mutations, so technique is

applicable here

– Tube 2: Patient sample, same common primer as was used in tube 1 and a normal primer

• “normal primer” = primer with the 3’ end complementary to the normal nucleotide

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ARMS

• PCR reaction run and result run on a gel• If patient is homozygous normal, will only

see a PCR product on the gel from tube 2• If patient is homozygous mutant, will only

see a PCR product on the gel from tube 1• If patient is heterozygous with one normal

and one mutant allele, will see PCR products from both tubes 1 and 2

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This is not from the case study, but is conventional ARMS

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ARMS in the case study

• They used a single tube ARMS variation• Can detect mutant and normal in single tube• Still “ARMS” in that 3’ end of primer is the key

– use different primers for mutant versus normal and the 3’ end nucleotide reflects the difference between two

• Add another primer set so can differentiate mutant versus normal also by size on electrophoresis

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SE Disease case study

• Boy’s SE Disease diagnosis could be confirmed– Investigators noted that DNA analysis is useful in diagnosis

of hemoglobinopathies where ambiguity may arise when using other techniques (IEF, HPLC, etc)

• Investigators reviewed a number of SE Disease case studies and noted:– Although HbS and HbE have higher incidence in somewhat

distinct populations, population migrations and racial intermarriages over the last century have led to increasing numbers of individuals who are compound heterozygotes for Hb S and Hb E

– SE Disease not a benign disease

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β-Thalassemia - Genetics

• Terminology is clinically based – β-Thalassemia major, intermedia and minor

• Numerous types of mutations can lead to numerous types of numerical aberrations in the β chain

• β-Thalassemia major possible genotypes (again, note that the thalassemia “allele” represents one of many different mutations that may cause either the absence or reduction of β chain production from that “allele”)– β0β0, β0β+, β+β+ (note β+β+ may also cause intermedia, it

depends on the severity of the particular mutation)

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β-Thalassemia - Genetics

• β-Thalassemia intermedia possible genotypes – β+β+, β0βwt (for example, if have also have

increased amount of α-globin – to be discussed later)

• β-Thalassemia minor possible genotypes – β0βwt (especially if also have compensatory α-

Thal), β+βwt

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β-Thalassemia - Genetics

• Prior are all considered simple β-Thalassemia

• Complex β-Thalassemia – β globin gene and one or more other members of chromosome 11 β globin gene cluster involved (may also include LCR)

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Hb Lepore and Hb Miyada

• Meiotic recombination from mispairing between the similar d and β globin genes creates two fusion strands, Lepore and Miyada (aka a type of anti-Lepore)

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Hb Lepore and Hb Miyada

• Sometimes classified as Hb structural variant with β-Thalassemia phenotype– Not only does it produce abnormal beta globin chain, it also has

reduced beta globin chain synthesis• Fusion chain in each (Lepore and Miyada) has reduced

expression (not completely absent) so usually β+

• Homozygous mutant or compound heterozygote with a severe β-Thalassemia allele leads to β-Thalassemia major

• Compound heterozygote with βs : HbS/HbLepore, for example, is considered more of a “structural variant” of hemoglobin disorder

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β-Thalassemia - Genetics

• The location and type of mutation in the β-globin gene sometimes allows you to predict phenotype

• Examples that lead to β-Thalassemia major – Homozygosity (or compound het with another β0

“allele”) for IVS1 +1 G->T• Mutation found in Asian Indian and Chinese population

• Mutation is in splice donor site and results in no normal β-globin mRNA formed – is a β0 “allele”

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Reminder on normal splicing

• Consensus splice site sequences for major class of introns (U2 introns)– MAG/gtragt for EXON/intron (5’ss) junction– cag/G for intron/EXON (3’ss) junction

• Where M = A or C and R = A or G

• “gt” in red is the core splice donor and “ag” in blue is the core splice acceptor

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β-Thalassemia - Genetics

• Examples that lead to β-Thalassemia major (cont)– Homozygosity (or compound het with another

β0 “allele”) for IVS1 +1 G->A• Mutation found in Jordanian, Egyptian, Syrian, and

Palestinian populations

• Mutation is in splice donor site and results in no normal β-globin mRNA formed – is a β0 “allele”

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β-Thalassemia - Genetics

• Examples that lead to β-Thalassemia major (cont)– Homozygosity (or compound het with another

β0 “allele”) for MET1ARG• Mutation found in Chinese population

• Initiator codon mutation, ATG to AGG – is a β0 “allele”

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β-Thalassemia - Genetics

• Examples that may lead to β-Thalassemia intermedia (depending on the other allele)– IVS1 +5 G->C

• Mutation found in Asian Indian population

• Mutation is in splice donor region of β-globin intron 1 and results in reduced normal β-globin mRNA production– is a β+ “allele”

• Note that the consensus sequence on the earlier slide is variable depending on the gene and intron we are talking about

– IVS1 of normal β-globin begins with GTTGGT - the nucleotides that differ from the consensus sequence are in green

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β-Thalassemia - Genetics

• Examples that may lead to β-Thalassemia intermedia (depending on the other allele) (cont)– IVS1 +5 G->T

• Mutation found in Mediterranean and Northern European populations

• Mutation is in splice donor region of β-globin intron 1 and results in reduced normal β-globin mRNA production– is a β+ “allele”

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β-Thalassemia - Genetics

• Examples that may lead to β-Thalassemia intermedia (depending on the other allele) (cont)– IVS1 +110 G->A

• Mutation found in Mediterranean populations

• Mutation is in β-globin intron 1 and creates a new splice acceptor site. This results in reduced normal β-globin mRNA production– is a β+ “allele”

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β-Thalassemia - Genetics

– IVS1 +110 G->A (cont.)• Mutation is in β-globin intron 1 and creates a new

splice acceptor site. This results in reduced normal β-globin mRNA production– is a β+ “allele”

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SCD – Molecular Pathology SCA

• Under deoxygenated state, the codon 6 valine binds in a hydrophobic pocket of another β chain and rigid strands of hemoglobin polymers form

• Attempts of these RBC to flow through narrow vessels causes them to adopt a sickle shape and become stuck in the vessel (vascular occlusion)

• Ischemia (and eventual necrosis) followed by inflammation and neuropeptide release cause damage (hemolysis) and pain

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SCD – Molecular Pathology SC Disease

• Oxygenated Hb C tends to crystallize, causing RBC to be somewhat rigid

• Hb C also induces RBC to dehydrate– K-Cl co-transport thought to be involved

• In SC Disease, Hb C enhances pathogenic properties of the Hb S that is also present– Dehydrated RBC (from presence of Hb C) has an

increased hemoglobin concentration which is a favorable environment for Hb S polymerization to occur

– Leads to a significant disorder, albeit not as severe as SCA

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SCD – Molecular Pathology SC Disease

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β-Thalassemia – Molecular Pathology

• Normal hemoglobin has an equal balance of α and β chains

• In β-Thalassemia, reduced or no β chains creates an excess of free α chains

• These free α chains are insoluble and precipitate in RBC precursors which are destroyed in bone marrow so have ineffective erythropoiesis

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SCD and β-Thalassemia - Inheritance

• Inheritance is autosomal recessive • Parents of an affected individual are usually

heterozygotes (carriers) and not severely affected– Carriers of SCA allele – Have Sickle Cell Trait.

Usually asymptomatic, but may show signs of disease under extreme exertion, at high altitude or if severely dehydrated

– Carriers of β-Thal allele – Usually have β-Thal minor with mild anemia, however…(next slide)

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Carrier of β-Thal allele

• What do we mean by this? – β0βwt or β+βwt: The person has at least one wild

type β-globin allele• Usually β-Thal minor, but in some cases

may be β-Thal intermedia– If person also has triplicated or quadrupled

alpha globin gene rearrangement

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SCD and β-Thalassemia - Inheritance

• Carrier parents have a 25% chance of having an affected child, 50% chance of having a carrier and 25% chance of having normal non-carrier

• All children of an affected individual will be at least carriers

• Sibling of an affected person - if the sibling is known via clinical diagnosis to be unaffected, what is the chance is a carrier?

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SCD and β-Thalassemia – Prenatal testing

• Chorionic villus sampling (CVS) at 10-12 weeks gestation

• Amniocentesis at 15-18 weeks gestation

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SCD and β-Thalassemia – Prenatal testing

• CVS or amnio– Risk of both is loss of pregnancy– Gene testing from these samples may include

targeted mutation analysis (RFLP often used for SCA) and sequencing analysis (often used for β-Thalassemia since numerous mutations possible)

• Desirable to have parents’ mutations identified prior to prenatal testing