Clinical Chemistry 00:0
1–2 (2023)
Clinical Perspective on Use of Long-Read Sequencing
in Prenatal Diagnosis of Thalassemia
Diana M. Toledoa,* and Katherine A. Laffertya
This is an editorial focusing on the clinical perspective of
a long-read sequencing method in the prenatal diagnosis
of alpha- and beta-thalassemia, including a comparison
between this method and standard PCR-based methods.
Though incremental, the increased sensitivity and speci-
ficity using long-read sequencing is an important advan-
tage of this methodology in the prenatal diagnostic arena
due to false positive or false negative results having great-
er consequence when a family is making decisions about
their pregnancy.
Alpha- and beta-thalassemia are autosomal recessive
disorders that have impaired α-globin or β-globin func-
tion, respectively (1). The carrier frequency for α- and
β-thalassemia are both among the highest for monogenic
genetic diseases, especially for those of Chinese ancestry
(2). Typically, there are four functional α-globin genes
on chromosome 16 (HBA1 and HBA2) and two func-
tional β-globin genes on chromosome 11 (HBB).
Given the high homology between the HBA1 and
HBA2 loci, short-read next-generation sequencing has
not been the best approach for molecular testing of
this region, due to the inability for reads to discriminate
between the two loci during alignment. Standard meth-
ods of molecular testing for these genomic regions have
been PCR-based technologies, though the increasing
availability of long-read sequencing (LRS) has made
this a new potential molecular testing method.
The report by Liang et al. in this issue of Clinical
Chemistry highlights the clinical feasibility and results
of an LRS-based molecular testing method termed
comprehensive analysis of thalassemia alleles (CATSA)
specifically in prenatal diagnosis of α- and/or
β-thalassemia (3). Of note, this method has been in
use for preconception or carrier screening of thalassemia,
and the utility in this prenatal cohort was assessed in this
article. This retrospective clinical study compared the
a
Genomics Platform, The Broad Institute of MIT and Harvard,
Cambridge, MA, USA.
*Address correspondence to this author at: HCLD, 320 Charles St.,
Cambridge, MA 02141, USA. E-mail: dtoledo@broadinstitute.org.
Received December 14, 2022; accepted December 21, 2022
https://doi.org/10.1093/clinchem/hvac223
© American Association for Clinical Chemistry 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
Editorial
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CATSA method (4) results to the PCR-based ap-
proaches used on amniocentesis samples from 278 at-
risk pregnancies from 11 study centers throughout
China. Of the 278 fetuses tested in this cohort, the
standard PCR-based method used during the initial pre-
natal diagnosis accurately identified 191 fetuses with the
correct variant(s). However, CATSA accurately identi-
fied 206 fetuses with the correct variant(s), with further
orthogonal confirmation of these results by independent
PCR and Sanger sequencing. These 15 fetuses with add-
itional and/or corrected variant calls accounted for an in-
crease in yield of 7.9% using CATSA compared to
standard PCR-based methods. This is a substantial in-
crease in detection yield between methods. Of most im-
portance, the CATSA results would have likely changed
the pregnancy outcome in one of these 15 fetuses. This
pregnancy was terminated due to the PCR-based meth-
od resulting in an HBB c.52A > T (B0) homozygous
finding, correlated with a phenotype of β-thalassemia
major. However, the CATSA method identified this
variant in the HBB gene (c.52A > T) in the heterozygous
state which is consistent with β-thalassemia trait having
a milder or asymptomatic phenotype. This false positive
result made the difference between a pregnancy termin-
ation and potential continuation in this case. These
types of scenarios exemplify why incremental increases
in the positive predictive value (PPV) of a method
have a high level of clinical utility.
A major clinical concern with prenatal diagnostics is
the lack of a complete clinical picture to incorporate
with the molecular findings. Particularly in the case of
diagnosing α- and β-thalassemia, unless hydrops has
been diagnosed in utero, no other clinical features of a
thalassemia would be expected on ultrasound or other
available prenatal screening/testing. Therefore, there
are no clinical findings available to increase the confi-
dence of a diagnosis given molecular testing results.
Thalassemia is not unique in this challenge and current-
ly there are many other prenatal screening and testing si-
tuations that occur in the absence of any fetal clinical
findings. A very high PPV is of key importance for
any method used for prenatal screening and testing
where pregnancy decisions are determined solely on
the molecular findings and diagnosis. Emerging meth-
ods that increase PPV, even incremental increases,
should be evaluated, validated, and implemented into
1
Editorial
clinical use and practice. This will ultimately aid and im-
prove patient outcomes and the public’s opinion of gen-
etic testing, particularly in the prenatal period.
In addition to the false-positive result identified in
this cohort, there were two false-negative results for
common β-globin variants. These findings are consistent
with false-positive and -negative results for thalassemia
variants identified through PCR-based methods seen
in a previous study (4). The CATSA method showed
100% accuracy with confirmation using a tertiary meth-
od. Compared to PCR-based methods, CATSA pro-
vides a higher level of sensitivity, specificity, PPV, and
negative predictive value. Other advantages of CATSA
over PCR-based testing methodologies include the abil-
ity to uncover new variations within these genomic re-
gions. Since CATSA is utilizing the PacBio platform
and providing long-read sequencing, any variation with-
in this region can be detected using this method.
PCR-based technologies used in current thalassemia
testing only target specific known common copy num-
ber variation or small variants. Discovering variants of
uncertain significance will likely increase with the
CATSA method but so will the ability to learn more
about these genomic regions and better classify the vari-
ation in these regions that is currently unknown.
CATSA improves prognostic capabilities because it can
identify triplications of the alpha-globin genes, which
modifies β-thalassemia phenotypes due to increased le-
vels of imbalance of the α/β-globin chain. Diagnostic
yield and efficiency are also increased with CATSA since
analysis of both the α- and β-globin chains can be per-
formed within the same assay, rather than having to per-
form at least two PCR-based assays to achieve the same
comprehensiveness. Additionally, CATSA identifies
whether variants are in cis or trans configuration, which
is another advantage over PCR-based methods.
While CATSA is innovative and provides many ad-
vantages, there are some disadvantages. There is a higher
level of complexity associated with LRS protocols, data
processing, and data review compared to other methods.
In addition to complexity, PacBio instruments can be
much more costly than PCR instrumentation, potential-
ly creating barriers for some to adopt these techniques.
Larger amounts of data are generated with LRS methods
compared to PCR, and this data storage also has an in-
creased cost associated with it. The CATSA method has
a longer turnaround time (TAT) of approximately eight
days from sample collection to reporting, whereas
PCR-based methods have a shorter TAT of three days.
These additional five days are less of a concern for pre-
conception or carrier screening but can be critical during
the time-sensitive prenatal period. A potential solution
to overcome this difference in TAT is for both method-
ologies to be performed in parallel, and preliminary
2 Clinical Chemistry 00:0 (2023)
results would be returned within 3 days, though preg-
nancy decision-making should be encouraged to wait
for complete results after the full eight days. This would
provide most patients with the accurate result within
three days, but for the cases that are falsely positive or
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negative, they would receive those results with higher ac-
curacy several days later. This combination approach for
prenatal diagnosis is costly but provides the highest level
of confidence in the results within a rapid TAT that may
ultimately affect the outcome of a pregnancy.
LRS is a powerful tool that offers certain advantages
over short-read sequencing platforms and PCR-based
technologies. CATSA is a comprehensive technique to
more accurately identify and detect common and rare
variants within the α- and β-globin genes that have
high homology and can be difficult to sequence and as-
say. Efforts to validate and implement new tools that in-
crease testing sensitivity and PPV must be prioritized
when they are utilized in the prenatal space. Potential so-
lutions to overcome barriers such as cost and optimiza-
tion of models with rapid turnaround time could
ultimately impact the accuracy and accessibility of thal-
assemia diagnoses to those at high risk, leading to fam-
ilies being able to make more informed decisions
about their pregnancies.
Nonstandard Abbreviations: LRS, long-read sequencing; CATSA,
Comprehensive Analysis of Thalassemia Alleles; PPV, positive predict-
ive value; TAT, turnaround time.
Author Contributions: The corresponding author takes full responsibil-
ity that all authors on this publication have met the following required cri-
teria of eligibility for authorship: (a) significant contributions to the
conception and design, acquisition of data, or analysis and interpretation
of data; (b) drafting or revising the article for intellectual content; (c) final
approval of the published article; and (d) agreement to be accountable for
all aspects of the article thus ensuring that questions related to the accuracy
or integrity of any part of the article are appropriately investigated and re-
solved. Nobody who qualifies for authorship has been omitted from the list.
Authors’ Disclosures or Potential Conflicts of Interest: No authors
declared any potential conflicts of interest.
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