thalassemia: prenatal screening & diagnosis - ogshk.org · copyright © 2016. all rights...

Copyright © 2016. All Rights Reserved. Faculty of Medicine, The Chinese University of Hong Kong

Wah Yi Man

Thalassemia:

Prenatal Screening & Diagnosis

• Associate consultant

• Department of Obstetrics & Gynaecology

• Prince of Wales hospital


Thalassaemia

Most common hereditary disease and single-gene defect in Hong Kong

Lau et al NEJAM 1997

Prevalence of alpha thalassaemia 5%


Thalassaemia

Most common hereditary disease and single-gene defect in Hong Kong

Lau et al NEJAM 1997

Prevalence of beta thalassaemia 3.1%


Prenatal Screening and Diagnosis

Pregnant woman

Carrier screen by MCV fetus no risk Mother not carrier

May thal carrier be missed with normal MCV (false –ve)? Possible false -ve: • Single α-globin gene deletion (silent α-carrier; 75%

normal MCV)



Pregnant woman



normal MCV) • Single α –gene mutations

• e.g. Hb Constant Spring, Hb QuongSze

Stop codon mutate



Pregnant woman





• Rare silent β-thalassaemia mutation(β++/βN )

chr 11p15.5

5’ 3’ β LCR δ Aγ ε

Gγ



Pregnant woman





• Rare silent β-thalassaemia mutation(β++/βN ) • Hb E carrier (50% normal MCV) (mutation in β globin gene)

chr 11p15.5


Gγ



Pregnant woman





• Rare silent β-thalassaemia mutation(β++/βN ) • Hb E carrier (50% normal MCV) • Hereditary Persistence of Fetal Hb (HPFH) Does it matter to miss the above?


chr 16p13.3

ζ α2 α1 5’ 3’

----------------------------------30 Kb---------------------------------

chr 11p15.5


Gγ


chr 16p13.3

ζ α2 α1 5’ 3’

----------------------------------30 Kb---------------------------------

chr 11p15.5


Gγ

At birth: 50-80% HbF Adult Hb pattern at ~ 1 year of life HbF <1.1% HbA ~97% HbA2 <3.5%


α- globin gene Deletions


α- globin multigene clusters at chromosome 16p13.3: sites of

common Deletions

chr 16p13.3

ζ α2 α1 5’ 3’

----------------------------------30 Kb---------------------------------

--SEA

--FIL

--THAI

--MED

α0(--/)

α+ (-α/)

-α3.7

-α4.2

(normal MCV)

10 different α+ deletion

> 40 different α0 deletion

Normal Silent Minor HbH Hb Barts (αα/αα) > (-α/αα) > (--/αα), (-α/-α) > (--/-α) > (--/--)


α- globin gene Mutations


α- globin multigene clusters at chromosome 16p13.3:

Mutations

chr 16p13.3

ζ α2 α1 5’ 3’

----------------------------------30 Kb---------------------------------

Mutations affect α2 (αTα/) much more than α1 (ααT/) • At least 90 mutations reported

• e.g. Hb Constant Spring (αCSα/) and Hb Guong Sze (αQSα/) • Produce unstable α- globin chains, worsen phenotype


(--/αTα) Non-deletional HbH hydrops (αTα/αα)


β- globin gene Mutations


β- globin multigene clusters at chromosome 11p15.5:

Mutations

chr 11p15.5

5’ 3’ β LCR δ Aγ ε Gγ

• Mutations may cause ↓β- globin production (>300 variants): complete absence (β0/)

severe reduction (β+/) mild reduction (β++/)

Normal Silent Minor Intermedia Major βN/βN > β ++/βN > β0/βN , β+/βN, > β+/β+ > β+/β0 > β0/β0

Coinherited modifying ameliorating genetic factors could result in milder phenotype



Mutations

chr 11p15.5



Clinical severity depends on the extent of alpha/nonalpha globin chain imbalance Ameliorating genetic factors • Reduce alpha chain output

Co-inherited alpha thalassaemia • β0/β0 plus -α/-α or ααT/αα more likely thalassaemia

intermedia phenotype • β0/β0 plus -α/αα more likely thalassaemia major phenotype

• High HbF production • Deltabeta thalassaemia, beta promoter deletion • Hereditary persistent fetal haemoglobin

Involve hematologist for counselling

Milder phenotype resulting from coinherited modifying ameliorating genetic factors



Mutations

chr 11p15.5


• Mutations may cause ↓β- globin production (>300 variants): complete absence (β0/)

severe reduction (β+/) mild reduction (β++/)

• Mutations may produce abnormal or unstable Hb variants, e.g. HbE (βE), Hb Lepore (δβ+ thal)


50% HbE/βN 50% HbE/HbE> HbE/β+ HbE/β0

may need repeated transfusion in childhood，phenotype become more stable later in adolescent


β- globin gene Deletions



Deletion

β-deletion

(δβ)-deletion

(εγδβ)-deletion

chr 11p15.5


Gγ

(δβ)0: increased HbF, reduced HbA2

MCV HbF HbA2 ↓ →/slight ↑ ↑ similar to mutation carrier

↓ ↑ → be careful

↓ → → missed by Hb pattern A

s ca

rrie

r o

f th

e d

elet

ion

s



Deletion

β-deletion

(δβ)-deletion

(εγδβ)-deletion

chr 11p15.5


Gγ





s ca

rrie

r o

f th

e d

elet

ion

s

Heterozygote εγδβ deletion Adult phenotype: similar to β trait



Deletion

β-deletion

(δβ)-deletion

(εγδβ)-deletion

chr 11p15.5


Gγ





s ca

rrie

r o

f th

e d

elet

ion

s

more than 40 types of deletion

→ ↑ → Hereditary Persistence of Fetal Hemoglobin (HPFH)

↑γ-expression (Not deletion)



Deletion

β-deletion

(δβ)-deletion

(εγδβ)-deletion

chr 11p15.5


Gγ





s ca

rrie

r o

f th

e d

elet

ion

s

more than 40 types of deletion

→ ↑ → Hereditary Persistence of Fetal Hemoglobin (HPFH)

↑γ-expression (Not deletion)

Homozygote δβ-thalassemia 100% HbF Phenotype: thalassemia intermedia rather than thalassemia major Homozygote HPFH Phenotype: benign



Pregnant woman

Carrier screen by MCV fetus minimal risk Mother not carrier

Hb pattern, Fe status, Father of preg Hb status

Not carriers of the same type

fetus no risk

May thalassaemia carrier be missed by Hb pattern?

May miss rare types of thalassaemia carriers that may cause fetal anaemia, and thalassaemia intermedia postnatally


‘Hb Pattern’

High-performance liquid chromatography (HPLC)

Hb Electrophoresis


H Inclusion bodies

• Inclusion bodies = HbH：β4 – β chains excess relatively to α chains

• Presence of HbH indicates α-thal carrier (3 genes or 2 genes deletion) – in HK: mostly (--/αα), less likely (-α/-α)

• Absence of HbH inclusions does not preclude α-carrier state

– 1 gene deletion, mutation, or even in 2 genes deletion, esp in Fe deficiency

– αβ carrier (in 7% of β carrier): no relative β chains excess

• Specific but not very sensitive


Hb Electrophoresis

Barrett et al Best Pr OG 2017

Cellulose acetate Citrate agar

• Different hemoglobins run at different

speed under different electrophoresis

HbA2 false –ve • Fe def may mask β carriers • (δβ)-deletion carriers (HbF ↑) • (εγδβ)-deletion carriers (HbF→) • β++ (silent carrier) benign • HPFH (HbF ↑) benign


Maternal Paternal Fetus Remark

α MCV=N α-carrier / N If paternal αTα: fetal (--/αTα)

α α α-major / carrier / N

α β Carrier of either α, β, or αβ / N

If paternal αβ carrier: fetal ¼ α-major; So need paternal α-gene study

α Inconclusive α-carrier / N If paternal (αTα/αα) : fetal (--/αTα)

β MCV=N β-carrier / N If paternal HbE: fetal HbE/β0 If maternal αβ carrier, Paternal αTα: fetal (--/αTα)

β β β-major / carrier/ N Rarely both αβ carriers (7%*7%),* ¼ α-major

β α Carrier of either α, β, or αβ / N

If maternal αβ carrier: fetal ¼ α-major; So need paternal α-gene study

β Inconclusive β-carrier / N If paternal (εγδβ)0-carrier: fetal ¼ β-major If paternal (αTα/αα) + maternal αβ carrier: fetal (--/αTα)

Inconclusive: low MCV but no inclusion bodies and HbA2 / HbF not increased, no Fe def


Unusual Case 1

Wife: low MCV; confirmed alpha thal carrier

Husband: normal MCV

Fetal cardiomegaly; anaemia (increased MCA-PSV)

• DDx: – Husband not the father

– Other causes of fetal anaemia / cardiomegaly (Rh, Parvovirus etc)

– Husband is non-deletional a thal carrier (constant spring / QuongSze) with normal MCV

– Uniparental disomy (chr 16)


Non-Deletional HbH disease

α1

α2

α1

QS

α1

α2

Father normal MCV mother

fetus

(--/αQSα) May cause in utero anaemia &

hydrops but not lethal



Unusual Case 2

30 year old P0, single and unemployed Mother: alpha-thal carrier but ‘father of pregnancy’ ’s MCV normal Fetal hydrops at 20 weeks


Unusual Case 2

• Cordocentesis: Hb and Hb pattern, karyotype • Paracentesis: Viral isolation and CMV • Maternal urine: CMV • Maternal blood:

• Parvovirus IgG & IgM • CMV IgG, Rubella IgM

• Result: • Fetal anaemia, Hb Barts • 46XY, Other results negative

• Insisted her boyfriend is the father of the pregnancy • TOP after KCL injection at 22 weeks • Fetal blood

• Finger printing confirmed paternity • No alpha-genes detected, only one abn PCR band correspond to SEA deletion • Uniparental Disomy


• One parent is (--/αα) while other is normal • Transmission from the carrier-parent the abnormal chromosome (--),

which then duplicate, but not from the normal parent • Chr 16 is a common site for trisomic rescue and UPD

Uniparental Disomy chr 16

α1

α2

α1

α2

father mother

fetus

α1

α2

UPD


• 29 year old P0, normal MCV, Rh+ve • 20wk USG: Fetal cardiomegaly, high MCA PSV • Further maternal Ix:

– Hb pattern normal – Kleihauer-Betke test, screening for parvovirus, CMV and toxoplasmosis all –ve

• Father of pregnancy: – Unremarkable prenatal course, age 13 incidental finding of low MCV – Hb A2 2.8%, Hb F 0.8%, but no Hb H inclusion bodies were identified ; could not rule out a-thal – No alpha gene deletion or mutation

• Amniocentesis: CMA deletion:

Unusual case 3

chr 11p15.5


εγδβ0/βN carrier: fail to produce enough fetal Hb, need in-utero transfusion After birth: beta minor only (inherited from father)

Hui et al hematol 2017


Conclusion

Primary carrier screening base on MCV level

May miss rare types of thalassaemia carriers which may

cause fetal anaemia, and thalassaemia intermedia

postnatally

Secondary screening by Hb pattern

Need parental genetic studies to rule out occult carrier

status in some special conditions

Inconclusive Hb pattern result may miss rare types of

thalassaemia


Thank you

thalassemia: prenatal screening & diagnosis - ogshk.org · copyright © 2016. all rights...

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