Sickle Cell Disease Clinicals Presentation
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Transcript of Sickle Cell Disease Clinicals Presentation
Sickle Cell Disease & β-Thalassemia
Genes, Disease, and TherapeuticsFall 2015
November 10Clinical phenotype, differential
diagnosis, diagnosis, management – treatment and therapeutic
approaches, question and answer
SCD – Clinical Phenotype
• Intermittent episodes of small vessel occlusion (caused by the rigid and sickle-shaped RBC being unable to flow properly through the vessel) which leads to:– Tissue ischemia with eventual necrosis– Inflammatory response– Neuropeptide release– Hemolysis
• Chronic hemolysis which contributes to feature of chronic anemia
SCD – Clinical Phenotype
• These events cause pain and dysfunction in a variety of organ systems
• Improved treatment of the disease in childhood has extended survival in the U.S. to over age 60– Main causes of death: infection, acute chest syndrome,
pulmonary artery hypertension, cerebrovascular events– Causes of death more frequent in childhood: infection
and sequestration crises– Causes of death more frequent in adulthood: chronic
end-organ dysfunction, thrombotic disease and complications related to treatment
SCD – Clinical Phenotype
• Early sign of SCD is dactylitis – pain and/or swelling of extremities
• Splenic sequestration– 10-30% of children with SCD– Most common between 6 months and 3 years– Acutely enlarged spleen and a drop in
hemoglobin levels– May require splenectomy and can cause death
SCD – Clinical Phenotype
• Aplastic crisis– More common in children– Period of profound anemia often caused by
infection– The already-taxed RBC production machinery
is temporarily compromised further by, for example, a viral infection and severe anemia results
SCD – Clinical Phenotype
• “Sickle cell crisis”– Vaso-occlusive episode of pain– Can be caused by infection, hypoxia, dehydration,
and others– Acute occlusion of small vessels leads to
ischemia, inflammatory response and neuropeptide release
– Severe pain usually requires opioid administration and is frequent cause of hospital admissions in SCD
SCD – Clinical Phenotype
• Acute chest syndrome– Often caused by vaso-occlusion of pulmonary
vasculature– Characterized via chest x-ray and sign of
acute respiratory symptoms– Etiology varies and may include – infection,
presence of fat emboli in vasculature – Major cause of death in SCD
SCD – Clinical Phenotype
• Cerebrovascular events– Stroke, cerebral hemorrhage, and cerebral
microvascular disease– Up to 50% of SCD patients will have some
sign of cerebrovascular disease by age 14– Stroke is seen in about 11% of children with
SCD
SCD – Clinical Phenotype
• Hemolysis-related issues– Leg ulcers, priapism, pulmonary artery
hypertension (6-35% of adults with SCD), chronic anemia, predisposition to aplastic crisis
• Increased risk for infection– Splenic damage in SCD impairs organ’s ability
to remove bacteria from blood
SCD – Clinical Phenotype
• Clinical phenotype more severe in those with SCA or Sβ0-Thalassemia
• Less severe clinical picture in those with SC Disease or Sβ+-Thalassemia
SCD – Clinical Phenotype
• SC Disease phenotype– Not as severe as in SCA or Sβ0-Thalassemia,
but more severe than in Sickle Cell Trait or in HbC Trait (HbAHbC)
– Painful crises usually appear later in life (often after age 20) and at lower frequency (at about half the frequency as SCA)
– Hemolysis and anemia are more mild, but still present
SCD – Clinical Phenotype
• SC Disease phenotype– RBC have longer lifespan than those in
patients with SCA– Acute chest syndrome is seen in about 30%
of those with SC Disease• Median age of onset is later than in those with SCA
(by about 10 years)• When present, length of hospital stay and death
rate similar to that seen in SCA
SCD – Clinical Phenotype
• SC Disease phenotype– Retinopathy more common in SC Disease than
in other SCD• Often occurs between the ages of 15 and 30, is
progressive, and can culminate in visual loss • One study found that 1/3 of children with SC Disease
had retinopathy, whereas only 3% of children with SCA had this characteristic
• Exact mechanism unknown, but thought to be related to increased cell density and greater blood viscosity seen in SC Disease
SCD – Clinical Phenotype
• SC Disease phenotype– Avascular necrosis (also known as
osteonecrosis) at least as frequently seen in SC Disease as in SCA
• Can be painful and disabling• Often in hips and shoulders (although shoulder
presentation uncommon in patients under 25 years of age)
Sickle Cell Trait – Clinical Phenotype
• Heterozygotes are usually asymptomatic carriers
• Some conditions may induce vaso-occlusive events though: extreme physical exertion, altitude, dehydration
• Particular kidney problems may arise
Impact of Fetal Hb on Clinical Phenotype
• Primary hemoglobin from about 8-11 weeks in utero until birth is fetal Hb (2 a and 2 g chains)
• Fetal Hb persists in appreciable levels until about 6 months of age when adult Hb dominates– Onset of symptoms in SCA often delayed until 2 months of
age• Fetal Hb can disrupt the polymerization of Hb S and
thus reduce the severity of the clinical picture in SCD• One treatment for SCD, hydroxyurea, stimulates
production of fetal Hb• Will discuss more on fetal Hb later
β-Thalassemia – Clinical Phenotype
• β-Thalassemia major, intermedia and minor
• Anemia caused by ineffective erythropoiesis– Reduced β-chain production results in excess
unbound a-chains in erythrocyte precursors in the bone marrow and spleen
• Unbound a-chains precipitate and damage the precursors - the result is ineffective erythropoiesis which leads to anemia
β-Thalassemia – Clinical Phenotype
• β-Thalassemia major (Cooley’s anemia)– Clinical presentation usually between 6-24 months of age
– Anemia, failure to thrive, pale appearance, splenomegaly, feeding issues
– Regular red blood cell transfusion therapy must begin soon to avoid skeletal changes (from bone marrow expansion), osteoporosis, leg ulcers, growth retardation and other problems
• If not regularly transfused, usually die before age 30
• Transfusion may cause iron overload so chelation therapy must also be started to combat this
β-Thalassemia – Clinical Phenotype
• β-Thalassemia intermedia– Clinical presentation usually after 24 months of age
– Anemia, pale appearance, some enlargement of spleen, possible skeletal changes
– Transfusions not normally required or only occasionally required
– Iron overload due to increased intestinal absorption of iron from ineffective erythropoiesis may occur
β-Thalassemia – Clinical Phenotype
• β-Thalassemia minor– Carrier state (one normal allele, one abnormal
β-globin allele)
– Usually just mild anemia, but if have excess alpha chain production, may be intermedia diagnosis
– Transfusion not required
SCD – Differential Dx
• Non-SCD related anemias• Leukemias• Pulmonary embolism unrelated to SCD• Septic arthritis• Some connective tissue diseases
β-Thalassemia – Differential Dx
• Hemolytic anemia – may see signs of anemia such as fatigue and may also see mild splenomegaly; blood tests will indicate hemolysis as cause of anemia (versus inefficient erythropoiesis of β-Thalassemia)
• Congenital Dyserythropoietic Anemia – Anemia caused by inefficient erythropoiesis. Macrocytic anemia (versus microcytic anemia of β-Thalassemia). Different gene involved as well.
SCD - Diagnosis
• Newborn screening followed by diagnostic testing soon after allows for most diagnoses prior to onset of symptoms– Early diagnosis important as prophylactic antibiotics often
started to avoid infections that can be seen in SCD• As discussed in prior lecture, IEF often used in
newborn screen• Positive IEF screen result followed by positive HPLC
result free of ambiguous possibilities can be diagnostic• If second test is DNA test, can also be diagnostic
SCD - Diagnosis
• As illustrated in prior lecture, idea behind diagnosis is to use multiple (different) techniques to determine the diagnosis– Certain techniques can resolve certain
hemoglobin possibilities– Example from prior case study illustrated how
HbCHarlem and HbOArab migrate near, but not with HbC on IEF. Also, cannot resolve these two with IEF, must use something else like up-to-date HPLC
SCD - Diagnosis
• Review of previously discussed means for identifying Hb protein– Cellulose acetate or citrate agar hemoglobin
electrophoresis: Has poorest overall resolution (but can resolve certain things other techniques cannot), but is rapid and inexpensive
– IEF (isoelectric focusing): Less quantitative than HPLC, but can be used in high-throughput screening
– HPLC (high-performance liquid chromatography): More quantitative than IEF and has good protein resolution
SCD - Diagnosis
• Gene testing– Targeted mutational analysis: available for Hb S, C, D, O
and particular β-Thal mutations• RFLP and PCR-based methods available
– Sequence analysis: available if targeted mutational analysis is uninformative
• Important to determine Hb status accurately, keeping in mind the transition from fetal to adult Hb in the newborn period as well as the possibility for both a and b-Thal to be present with a sickle or other structurally variant Hb
SCD - Diagnosis
• Clinical symptoms in infants and young children used in diagnosis:
• Dactylitis• Hemolysis and anemia• Bacterial sepsis or meningitis• Splenic enlargement• Acute chest syndrome
β-Thalassemia – Diagnosis
• Clinical presentation of anemia as described previously– Severe anemia early in life that requires regular
blood transfusion suggests β-Thalassemia major– More mild anemia later in life that may require
occasional transfusion suggests β-Thalassemia intermedia
• Hematologic testing – indicates microcytic anemia
β-Thalassemia – Diagnosis
• Gene testing– Targeted mutational analysis: Although large
number of different mutations may be implicated, some subsets known to be more common in particular ethnic populations
• Usually use primer-specific PCR to detect– Sequence analysis– Deletion/Duplication analysis available – useful
for the complex β-Thalassemia conditions such as db-Thal
SCD - Management
• Once diagnosed (usually in early infancy due to widespread use of newborn screening for SCD), preventative measures should be undertaken– By 2 months of age: Administer pneumococcal
vaccine and begin penicillin prophylaxis • Has been shown to reduce the risk of death for children
with SCA• Is also recommended in cases of Sβ0-Thalassemia
– All normal immunizations as well as seasonal influenza immunization should be administered
SCD - Management
• Lifestyle approaches to prevent symptoms:– In general, maintain hydration, avoid extremes of climate,
avoid excessive physical exertion and high altitudes– School-age children can participate in physical education
• Allow rest periods and encourage fluid intake– Competitive athletics are acceptable if carefully
monitoring for signs of fatigue– Travel above 15,000 feet in nonpressurized vehicles can
induce vaso-occlusive complications• Airplanes usually fine, hiking Mt. Everest is not
SCD - Management
• Treatment of symptoms include:– Use of opioids to treat severe episodes of
pain– Use of oxygen, analgesics, antibiotics, and
possibly transfusions in cases of ACS– Rapid treatment of splenic sequestration,
bacterial infections, and other problems that can worsen quickly
SCD - Management
• More on management of pain episodes– Sickle cell crises are difficult to manage in that
they are somewhat unpredictable and can cause severe pain
– Mild to moderate pain episode• Oral analgesics at home supplemented with massage,
warm showers, and relaxation techniques are often initial recommendation
• If pain becomes severe, or fever, chest pain, breathing difficulty, or pain in unusual location arises, health care provider should be alerted
SCD - Management
• More on management of pain episodes (cont.)– Severe pain episode
• Patient goes to ER or, if available, Sickle Cell Day Treatment Center
• Administration of intravenous or intramuscular pain medications (for example, opioids like morphine)
• Depending on severity/longevity of pain episode and presence of additional complications (possible ACS for example), hospitalization may be required
SCD - Management
• More on management of pain episodes (cont.)– Tolerance, physical dependence, and addiction to
opioids used in management of SCD• Tolerance: First sign is usually decrease in time
medication is effective. Larger doses or shorted intervals between doses usually necessary to achieve same level of analgesia
• Physical dependence: Risk for physical dependence varies among individuals, but can often arise after 5-7 days of opioid use. Discontinuation of opioids leads to withdrawal symptoms (ex – nausea, vomiting, sweating, dysphoria)
SCD - Management
• More on management of pain episodes (cont.)– Tolerance, physical dependence, and addiction
to opioids used in management of SCD (cont.)• Addiction: Psychological dependence of complex
etiology.– From NIH guidelines: “Patients with SCD do not appear to be
more likely than others to develop addiction. The denial of opioids to patients with SCD due to fear of addiction is unwarranted and can lead to inadequate treatment. “
SCD - Management
• More on management of ACS– Second most common cause of hospitalization
in sickle cell patients (first is pain crisis)– One main goal is to prevent progression of
ACS to acute respiratory failure– Supplemental oxygen often administered– Transfusion may be given– Pain medications administered– Antibiotics administered
SCD - Management
• More on management of ACS (cont.)– Frequent ACS episodes (or pain episodes)
are associated with shorter lifespans, so important to attempt to reduce frequency of ACS when possible
• One study showed that the frequency of ACS can be reduced by about 50% when hydroxyurea treatment is used
• Some studies suggest that transfusion regimens can also reduced the frequency of ACS episodes
SCD - Management
• More on management of splenic sequestration– Acute splenic sequestration complication (ASSC)
is a leading cause of death in children with SCD– ASSC caused by trapping of RBC in spleen which
leads to a rapid fall in body’s hemoglobin level– Typical clinical manifestations:
• sudden weakness, pallor, tachycardia, rapid breathing, and abdominal fullness.
SCD - Management
• More on management of splenic sequestration (cont.)– ASSC can be fatal if not treated within a few
hours• Immediate RBC transfusion is indicated
– Body hemoglobin levels will then increase, the trapped RBC in the spleen will be remobilized and the spleen will regress down from its enlarged state
SCD - Management
• More on management of splenic sequestration (cont.)– Of those that survive first episode, splenic
sequestration recurs in about 50%• In these cases, management options include
observation, chronic transfusion therapy, or splenectomy– Observation alone not currently recommended by NIH– Conflicting study results regarding the ability of chronic
transfusion therapy to prevent recurrence– Although splenectomy removes possibility of sequestration
problem, possibility for increased infection due to absence of organ (although some studies do not show an increase)
SCD - Management
• Fetal Hb (Hb F) is desirable:– it disrupts the polymerization of Hb S– in the absence of β-chains, γ-chains can bind
to free a-chains• Condition called “hereditary persistence of
fetal hemoglobin” (HPFH) actually rescues one from more severe clinical consequences of βS allele or β0 “allele”
HPFH
• HbF production persists at appreciable levels through childhood and adulthood
• Many ways this can occur, for example:– Point mutations in the promoter regions of the two
γ-globin genes in the β-globin gene cluster can cause high and sustained expression of these genes
– Large deletions that include both the δ and β-globin genes lead to sustained use of γ-globin gene
HPFH
• The mutation leading to HPFH can rescue one from the severe phenotype that usually results from “β0β0” genotype– For example, someone with β0β0 who also has
a point mutation in the promoter of a γ-globin gene causing sustained expression of γ-globin
HPFH
• Example - In some Sardinian populations, the following haplotype is found– -196 C->T A-gamma with a nonsense mutation in the β-globin
gene• This haplotype results in HPFH (from the point mutation
that activates expression of the A-gamma gene) and β0 (nonsense mutation leads to no production of β-chain from this allele)
• Because of the HPFH characteristic, one with this haplotype for both chromosomes is usually clinically normal– Clinically normal in the presence of β0β0
HPFH
• The mutation leading to HPFH can rescue one from the severe phenotype that usually results from βSβS genotype– For example, someone with βSβS who also
has a point mutation in the promoter of a γ-globin gene causing sustained expression of γ-globin
HPFH
• Example - In some Saudi populations, the following haplotype is found– -158 C->T G-gamma plus the SCA mutation in the β-globin gene
• This haplotype results in HPFH (possibly* from the promoter point mutation activating expression of the G-gamma gene) and βS (standard SCA point mutation in β-globin gene )
• Because of the HPFH characteristic, one with this haplotype for both chromosomes is mostly clinically normal– Clinically normal in the presence of βsβs
SCD - Management
• Hydroxyurea– Drug that stimulates production of fetal Hb– It is a ribonucleotide reductase inhibitor and
thus is cytotoxic• Leads to rapid erythropoiesis, a state which
encourages HbF production
SCD - Management
• Hydroxyurea (cont.)– Also reduces vascular inflammation– Has been shown to reduce frequency of painful crises
and acute chest syndrome• Trials underway to determine if cerebrovascular complications
also decreased– Long term adverse effects are not yet well established– Some indication that the combination of erythropoietin
and hydroxyurea increase Hb F to levels higher than that achieved when using hydroxyurea alone
SCD - Management
• Other drugs that promote fetal Hb– Hydroxyurea is primary drug in clinical use, but the
following other drugs are still under investigation– 5-azacytidine
• Nucleoside analog that inhibits methylation of cytosine in DNA
• First “hemoglobin switching” agent used• Idea is that gamma globin methylation is inhibited by 5-
azacytidine and thus gene is active – Fetal Hb production is increased
SCD - Management
• Other drugs that promote fetal Hb– Butyrate derivatives
• Modulates globin gene expression in various ways, one being inhibition of histone deacetylase (HDAC)
• Inhibition of HDAC on gamma globin gene allows for “open” chromatin structure there and active gene expression
• One study– Arginine butyrate given once or twice monthly– 11 of 15 sickle cell patients responded with a mean rise in Hb
F from 7% to 21%– Some maintained this level for 1-2 years
SCD - Management
• In patients with increased risk for stroke or for those that have had stroke, ACS, pulmonary hypertension, ASSC, or severe end-organ damage, chronic red blood cell transfusion therapy is often used– Goal is to maintain HbS below 30%– Possible iron overload must be addressed
with concurrent chelation therapy
SCD - Management
• Stem Cell Transplantation– Usually performed in patients that exhibit
severe phenotype (for example, have had a cerebrovascular event or recurrent episodes of ACS) and have an HLA matched sibling available
• About 85% of these individuals have had disease-free survival
SCD - Management
• Stem Cell Transplantation– Among patients who had stable engraftment
of donor cells• No subsequent sickle cell-related clinical events• Stabilization of preexisting sickle cell-related organ
damage• Recovery of splenic function• Some patients developed mixed donor-host
hematopoietic chimerism– these patients also had no symptoms from SCD
SCD - Management
• SCD and pregnancy– Pregnancy is not contraindicated in women with SCD,
but medical team must be aware of problems that may arise
• Increased risk for preeclampsia, preterm delivery, babies with intrauterine growth retardation and low birth weight
• Sometimes increased risk for thrombosis, infection, and painful crises over non-pregnant SCD state
– Hydroxyurea use must be stopped prior to conception– Possible opiate withdrawal in newborns of mothers
treated with high doses of medication
β-Thalassemia - Management
• Transfusion therapy initiated at diagnosis– Regular transfusions for major form and as-
needed transfusions for intermedia form• corrects anemia among other benefits
• Risk for transfusion-based iron overload– Iron overload can cause growth retardation,
delay of sexual maturation, cardiac, liver and endocrine problems
β-Thalassemia - Management
• Risk for transfusion-based iron overload lowered by concurrent use of chelation therapy– Iron chelators bind the excess iron and allow
it to be removed from the body– Chelation therapy often initiated after 10-12
transfusions, but use serum ferritin concentration to determine when to begin
β-Thalassemia - Management
• Desferal (deferoxamine mesylate)– Commonly used iron chelator– Typical administration is over 8-24 hours via a
small portable pump capable of providing continuous mini-infusion
– Since it is administered over a long period of time (and sometimes over multiple days), patient compliance is sometimes an issue
β-Thalassemia - Management
• Bone marrow transplantation– Disease-free survival in children who undergo
BMT and do not have hepatomegaly, liver fibrosis, or iron accumulation pre-transplant is about 90%
– If have all three risk factors, disease-free survival drops to about 60%
SCD and β-Thalassemia - Management
• Particular application of umbilical cord blood transplantation – Prenatal HLA and globin gene testing in a
pregnancy where the parents already have an affected child (SCD or β-Thalassemia)
• If fetus is globin normal and HLA-compatible, subsequent collection of umbilical cord blood and transplantation into the affected child is possible
• Ethics – conceive solely to treat the affected child?
Gene Therapy for SCD and β-Thalassemia
• Lentiviral-mediated delivery of normal β-globin gene has been investigated (among other gene therapy approaches)– In prior discussion on viral vectors, we
discussed insertional mutagenesis and also how different retroviruses sometimes show preference for particular integration sites, but if integrate randomly:
• Your transgene may be inserted into a region of heterochromatin and become silenced
Gene Therapy for SCD and β-Thalassemia
• The silencing may be “permanent” in that all progeny cells from a particular integration event will be similarly silenced or silencing may fluctuate between progeny that have the same integration event (position effect variegation or PEV)
• Attempts to combat transgene silencing include use of insulator elements
Gene Therapy for SCD and β-Thalassemia
• Insulator element– Protects the transgene from the spread of
heterochromatin– Example is 5’ HS ankyrin insulator– Possible to also prevent insertional
mutagenesis since the transgene’s promoter elements may also be insulated away from nearby genes?
Gene Therapy for SCD and β-Thalassemia
• Another aspect to consider, especially in the hemoglobinopathies, is size of cargo– LCR along with globin transgene usually
required for optimal expression– Lentivirus is able to handle larger inserts and
is more stable (versus, for example, gammaretrovirus)
Gene Therapy for SCD and β-Thalassemia
• Lentiviral-mediated delivery of normal β-globin gene for SCA– Since sickle allele still present and expressed, this
is of somewhat limited use in SCA• For SCA – deliver g-globin gene or attempt to correct
the mutant transcript instead– Delivering g-globin gene would require same large LCR
elements etc, but would provide better defense against sickle β-globin chain compared to delivery of normal β-globin gene
– Correcting sickle β-globin transcript would be useful and would not require delivery of large cargo, however would require efficient manipulation of splicing in one approach
EXON 1 EXON 2 EXON 35’ss 3’ss 5’ss 3’ss
B.D.
Spacer5’ss.THERAPEUTIC
5’ EXON 45’ss 3’ss
THERAPEUTIC 3 4
3’
5’ Exon Replacement via Trans-Splicing
Attempting to correct the mutant transcript
Spliceosome Mediated RNA Trans-Splicing (SMaRT)
5’
Mutation
1 2 35’ 4
RNA is Repaired Mutation Still Present
Endogenous Cis-Splicing
Point mutation in SCA
IVS1 +5 G->T
IVS1 +5 G->C
IVS1 +1 G->T
IVS1 +1 G->A
Some, but also cryptic.b+-thal
Some, but mostly cryptic.Severe b+-thal
No - cryptic only.b0-thal
No - cryptic only.b0-thal
Correctly Spliced b-globin mRNA Produced?b-thalassemia Mutation
Mediterranean, Northern European
Asian Indians
Jordanians, Egyptians,SyriansPalestinians
Ethnic Group
Asian Indians
Four Different Splicing Mutations Implicated in b-Thalassemia
5’PTM1
5’ss 3’ss
Binding
Spacer5’ss A
Tag
5’ C
ap
AN5’
Cap
Domain
EXON 1
EXON 1
*//