Detecting and Reporting Alpha Thalassemia In Newborns

T. Davis, C. Moore, L. Nayak, M.C. Dorley, M. del Pilar Aguinaga, M. Chan, J. Ubaike, C. Yusuf

• Alpha Thalassemia Screening Status in the US

• Clinical Presentation of Alpha Thalassemia

• Hemoglobin Bart’s Percentages

• Hemoglobin Constant Spring

• Hemoglobins on the RUSP

 Alpha Thalassemia Screening in the US

All newborn screening programs are detecting alpha thalassemia.

How many programs are reporting it?

How is it being reported? Hb H Disease, silent carrier, alpha thal trait,

Hb Constant Spring, alpha thal major, Hb Bart’s %

As a workgroup, how can we improve the status of alpha thalassemia

reporting in the US?
 Alpha Thalassemia Survey Data
Response Rate (83%) Number of States that Report Alpha
Thalassemia (Hb Bart's)

95%

Number of Programs that did not Respond = 9 Number of Programs that Report Alpha Thalassemia (Hb Bart's) = 42
Number of Programs that Responded = 44 Number of Programs that Do Not Report Alpha Thalassemia (Hb Bart's) = 2
 Alpha Thalassemia Survey Data
Hemoglobin Screening Method(s)

Method Number of Programs (N=44)

IEF/HPLC 18
IEF Only 9
HPLC/IEF 8
HPLC Only 7
Unknown 2

Two Methods = 26
One Method = 16

IEF/HPLC IEF Only HPLC/IEF HPLC Only Unknown

Alpha Thalassemia Survey Data
Hb Bart’s Percentages Used

Classification Average Min Max

Silent Carrier (N=3) 7 3 11

Alpha Thal Trait (N=10) 9 2 20

Hb H Disease (N=14) 22 11 31

Alpha Thal Major (N=9) 64 25 100

Other (N=9) 15 3 25
 Clinical Presentation of Alpha Thalassemia

Alpha Genes
Classification Clinical Features
Affected

1 Silent Carrier Benign

2 Alpha Thal Trait Mild anemia. Microcytosis.

3 Deletional Hb H Disease Moderate anemia and microcytosis. Transfusion rarely required.

Severe microcytic anemia and microcytosis with delayed growth.

3 Non-Deletional Hb H Disease
20% require transfusion.

4 Alpha Thal Major Fetal demise and high risk to mother.

 Normal Newborn (Birth to approximately 3 weeks of age)

α Fetal Hemoglobin (F) =αγ

γα
α1 α1
β
α αβ β β
Adult Hemoglobin (A) = βα β
The ratio of alphas to beta-like chains should be 1:1!
γA γA
α γ
α2 α2 F = αγ γ
α
γG γG

O O IEF gel O
O
Chromosome 16 Chromosome 11
A = αβ

BioRad vnbs
 Alpha Thalassemia Trait
αγ β
α1 α1 Fetal Hemoglobin (F) = γα β β
α αβ β
Adult Hemoglobin (A) = βα

Hb Bart’s (B) = γγ
γγ γA γA
γ
α2 α2 γ
α
γG γG

O O O
O
13.3% Hb Bart’s
Chromosome 16 Chromosome 11
 Hb Bart’s

• Hb Bart’s is a tetramer of gamma globin chains, along with

acetylated and glycated forms.
• Bart’s is formed when there is a reduction in functional alpha globin
chains.
• On isoelectric focusing gels, Bart’s presents as 2 to 3 bands.

• On cation exchange HPLC, Bart’s presents as 1 to 3 peaks.

• Discovered at St. Bartholomew’s Hospital in London.

• High levels of Bart’s are more common in Asian populations.

 Deletional Hb H Disease
αγ
Fetal Hemoglobin (F) = γα
α1 α1 αβ β
α Adult Hemoglobin (A) = βα β β
β
Hb Bart’s (B) = γγ
γγ Hb H = ββ
ββ
γA γA
γ
α2 α2 γ
γG γG

O O O
O
Chromosome 16 Hb Bart’s = 34.3% Chromosome 11
 Reporting Hb Bart’s from HPLC
Alpha Thalassemia Severity Genes Affected BioRad vnbs HPLC Extended Gradient
FA Normal 0 0-6.4% < 4.9%
Silent Carrier 1 6.5-9.1% 4.9-6.4%
Alpha Thal Trait 2 9.2-24.9% 6.5-17.9%
Hb H Disease 3 >=25% >= 18%
Alpha Thal Major 4 100% 100%

Extended Gradient
BioRad vnbs
Factors that impact %

-Sample Matrix

-Resolution

-Software Integration
 HPLC Bart’s Percentages
Alpha Thalassemia Severity Genes Affected BioRad vnbs HPLC
FA Normal 0 0-6.4%
Silent Carrier 1 6.5-9.1%
Alpha Thal Trait 2 9.2-24.9%
Hb H Disease 3 >=25%
Alpha Thal Major 4 100%

FA Normal Silent Carrier

6.4% FA Normal

6.7% Silent Carrier 6.4% 6.7%

17.7% Alpha Thal Trait

41.3% Hb H Disease
 HPLC Bart’s Percentages
Alpha Thalassemia Severity Genes Affected BioRad vnbs HPLC
FA Normal 0 0-6.4%
Silent Carrier 1 6.5-9.1%
Alpha Thal Trait 2 9.2-24.9%
Hb H Disease 3 >=25%
Alpha Thal Major 4 100%

Silent Carrier Alpha Thal Trait

6.4% FA Normal

6.7% Silent Carrier 6.7% 17.7%

17.7% Alpha Thal Trait

41.3% Hb H Disease
 HPLC Bart’s Percentages
Alpha Thalassemia Severity Genes Affected BioRad vnbs HPLC
FA Normal 0 0-6.4%
Silent Carrier 1 6.5-9.1%
Alpha Thal Trait 2 9.2-24.9%
Hb H Disease 3 >=25%
Alpha Thal Major 4 100%

Alpha Thal Trait Hb H Disease

6.4% FA Normal

6.7% Silent Carrier 17.7% 41.3%

17.7% Alpha Thal Trait

41.3% Hb H Disease
 Hb Bart’s Percentages (N=4000) May 2016 through April 2017, Washington State
45

40

35

30

25
25.0%
20

15

10
9.2%
5

0
0 500 1000 1500 2000 2500 3000 3500 4000
 FREQUENCY

0
20
40
60
80
100
120
2.6 140
3
3.4
3.8
4.2
4.6
5
5.4
5.8
6.2
6.6
7
7.4
7.8
8.2
8.7
9.1
9.5 9.2%
9.9
10.3
10.7
11.1
11.5
11.9
12.3
12.7
13.1
13.5
13.9
14.3
14.7
15.1
BART'S %

15.5
15.9
16.3
16.7
17.1
17.5
17.9
18.3
Alpha Thal Trait

18.7
19.1
19.5
19.9
20.3
20.7
21.1
Hb Bart’s (N=4000) May 2016 through April 2017, Washington State

21.5
21.9
22.3
22.7
23.1
23.5
23.9
24.3
24.7
25.1
25.5
25.0%

25.9
26.3
26.7
Hb H
 Hb Constant Spring

• Most common of the non-deletional alpha thalassemias.

• Hb Constant Spring is a point mutation on the alpha 2 gene at stop codon 142
TAA →CAA (Term → Glutamine). Causes the alpha chain to be extended to 172
amino acids.

• Long peptide chain is unstable and there is a very low percentage of protein
present.

• Detectable in very low levels on IEF and not on HPLC, except with whole blood.

• Seen more at early age on IEF. 24 hours vs 7 days.

 HS-40 (Super Enhancer)
Normal Deletional Hb H Hb H/Constant Spring

α1 α1 α1 α1
α1 α1

α2 α2 α2 α2 α2 α2CS
 Hb E trait and Hb H/Constant Spring

αγ
Fetal Hemoglobin (F) = γα Hb E = αβ E

α1 βα E
β
α1 Adult Hemoglobin (A) = αβ Hb Bart’s (B) = γγ
γγ
β β
α1 βα β
Hb Constant Spring = αCS γ + αCS β
γα βα γA γA
γ
α2 α2CS γ
γG γG

O O O
O
Chromosome 16 Hb Bart’s = 40.5% Chromosome 11
 Alpha Thalassemia Silent Carrier/Hb Constant Spring

Hb Bart’s

Hb Bart’s Peak Area = 7.6%

Hb Constant Spring bands

 Real Time PCR Allelic Discrimination

Hb H + Hb CS

HPLC or IEF Hb H

Hb H
 10 Years, 2005 - 2014

Hb H Detected Hb H/Constant Spring % Asian Population

State per 100,000 births per 100,000 births 2010 Census
Washington 6.3 0.6 7.2
Tennessee 2.7 N/A 1.4
Texas 4.8 N/A 3.8
 Sickle cell Disease Sickle C Disease
Sickle Beta Zero Thalassemia

Core Conditions

Hemoglobinopathies on the RUSP

Secondary Conditions (Various Other Hemoglobinopathies)
Hemoglobin H Disease Hemoglobin E Beta Zero Thalassemia
Beta Zero Thalassemia
Database of Human Hemoglobin
Sickle Beta Plus Thalassemia Variants and Thalassemias Variants with High Oxygen Affinity
Beta Gene Entries = 895
Hemoglobin E Disease Variants with Low Oxygen Affinity
Alpha 1 Gene Entries = 350

Hemoglobin SV Disease Alpha 2 Gene Entries = 431 Beta Thalassemia Variants

Thalassemia Entries = 487
Hemoglobin SE Disease Unstable Hemoglobin Variants

Hemoglobin C Disease Hemoglobin D Disease Hemoglobin SD Disease Alpha Thalassemia Variants

Sickling Disorders

Thalassemia Syndromes (α and β)

Unstable Hemoglobin Variants

Altered Oxygen Affinity Variants