Real-time polymerase chain reaction (PCR) makes use of a defined threshold
value (CT). To measure the quantitative differences between wild-type
(CTN) and mutant (CTM) alleles, the difference between
the respective CT values (ΔCT) can be used. A, Clear
discrimination of the wild-type allele from the mutant allele on experimental
serial dilutions of the mutant alleles into wild-type DNA. Following the ΔCT(M-N) analysis, wild-type alleles cluster above a ΔCT value of 6,
while mutant alleles cluster at a value of less than 4. The arbitrarily assigned
ΔCT cut-off values between 3.5 to 4.5 (shaded area) were used to distinguish
between the presence of normal and mutant alleles. B, Analysis of size-fractionated
circulatory DNA from maternal plasma samples in combination with the PCR/peptide-nucleic-acids
(PNA) clamping step results in the correct appraisal of the presence (n = 4)
or absence (n = 9) of the mutant allele. Three cases were classified
as uncertain. C, Analysis of total circulatory DNA without size-fractionation
results in the incorrect appraisal of 3 of 4 cases at the presence of the
mutant allele even when using the PCR/PNA clamping step. D, Analysis of size-fractionated
circulatory DNA without the use of the PCR/PNA clamping step results in the
incorrect appraisal of the presence of the mutant allele in 2 cases.
Li Y, Di Naro E, Vitucci A, Zimmermann B, Holzgreve W, Hahn S. Detection of Paternally Inherited Fetal Point Mutations for β-Thalassemia Using Size-Fractionated Cell-Free DNA in Maternal Plasma. JAMA. 2005;293(7):843-849. doi:10.1001/jama.293.7.843
Author Affiliations: Laboratory for Prenatal
Medicine, University Women’s Hospital/Department of Research, University
Hospital, Basel, Switzerland (Drs Li, Zimmermann, Holzgreve, and Hahn); and
Department of Obstetrics and Gynecology (Dr Di Naro) and Division of Hematology
II (Dr Vitucci), University of Bari, Bari, Italy.
Context Currently, fetal point mutations cannot be reliably analyzed from circulatory
fetal DNA in maternal plasma, due to the predominance of maternal DNA sequences.
However, analysis of circulatory fetal DNA sequences in maternal plasma have
been shown to selectively enrich for fetal DNA molecules on the basis of a
smaller molecular size than maternal DNA.
Objective To examine the prenatal analysis of 4 common β-thalassemia point
mutations: IVSI-1, IVSI-6, IVSI-110, and codon 39.
Design, Setting, and Patients A total of 32 maternal blood samples were collected at 10 to 12 weeks
of gestation (mean, 10.7 weeks) between February 15, 2003, and February 25,
2004, in Bari, Italy, from women with risk for β-thalassemia in their
newborns immediately prior to chorionic villous sampling. Samples in which
the father and mother did not carry the same mutation were examined. Circulatory
DNA was size-fractionated by gel electrophoresis and polymerase chain reaction
(PCR) amplified with a peptide-nucleic-acid clamp, which suppresses amplification
of the normal maternal allele. Presence of the paternal mutant allele was
detected by allele-specific real-time PCR.
Main Outcome Measure Detection of paternally inherited β-globin gene point mutations.
Results Presence or absence of the paternal mutant allele was correctly determined
in 6 (86%) of 7 cases with the IVSI-1 mutation, 4
(100%) of 4 with the IVSI-6 mutation, 5 (100%) of
5 with the IVSI-110 mutation, and 13 (81%) of 16
with the codon 39 mutation. One false-positive test result was scored for
the IVSI-1 mutation. Two cases with the codon 39
mutation were classified as uncertain and 1 case was excluded due to lack
of a diagnostic test result at the time of analysis. These results yielded
an overall sensitivity of 100% and specificity of 93.8%, with classified cases
Conclusion Our recently described technique of the size-fractionation of circulatory
DNA in maternal plasma may be potentially useful for the noninvasive prenatal
determination of fetal point mutations.
Monogenic disorders frequently involve point mutations. This single
nucleotide exchange makes the analysis of point mutations more complex as
stringent assays need to be established that permit a clear distinction between
normal and mutant alleles. The prenatal diagnosis of this multitude of hereditary
genetic disorders currently relies on invasive procedures,1 such
as amniocentesis or chorionic villous sampling, which are associated with
a small but significant risk of fetal loss.2,3 To
avoid this procedure-related risk, several strategies have been considered
for noninvasive assessment of fetal genetic traits, including the isolation
of rare fetal cells from the maternal circulation and the analysis of circulatory
fetal DNA in maternal plasma.1,4- 6
Although proof-of-principle studies have indicated that the analysis
of isolated fetal cells by single-cell polymerase chain reaction (PCR) can
be used for the noninvasive prenatal diagnosis of hemoglobinopathies,7,8 this strategy is too complex, labor
intensive, and not sufficiently efficient for routine clinical settings. The
analysis of fetal genetic traits by the analysis of cell-free fetal DNA in
maternal plasma has proven to be remarkably reliable for the assessment of
fetal loci absent from the maternal genome, such as Y-chromosome–specific
sequences or the RhD gene in pregnant women who are Rh-negative, especially
in European medical centers.1,4 This
approach, however, is unsuitable for the analysis of fetal loci that do not
differ largely from the maternal alleles, due to the vast predominance of
cell-free maternal DNA in the maternal samples.9 As
such, the analysis of fetal point mutations has been restricted to single-case
It has recently been shown that circulatory fetal DNA sequences are
generally smaller (<300 base pairs [bp]) than comparable circulatory maternal
DNA species (>500 bp).12,13 By
exploiting this observation, we have previously shown that this phenomenon
can be used to selectively enrich for fetal DNA molecules, which permitted
the detection of otherwise masked highly polymorphic fetal microsatellite
markers.12 We examined whether this approach
will permit the detection of fetal point mutations. The advantage of such
a development is that it would permit the detection of paternal mutations,
which could be used to determine which pregnancies are at risk for a compound
heterozygous genetic disorder. We have focused on one of the most common monogenic
disorders, β-thalassemia, and have examined 4 point mutations, which
occur with high frequency in the Mediterranean population.14,15
Following ethical approval from both participating institutions’
review boards and written informed consent from all participants, blood samples
were obtained from 32 pregnant women with risk for β-thalassemia in their
newborns between February 15, 2003, and February 25, 2004, in Bari, Italy.
No one refused to participate and all women were self-declared white (southern
Italian origin). Approximately 18-mL maternal blood samples were collected
into two 9-mL EDTA blood collection tubes (Sarstedt, Sevelen, Switzerland)
at 10 to 12 weeks of gestation (mean, 10.7 weeks; median, 11.2 weeks) before
chorionic villous sampling. Initially, 21 samples were sent as whole blood
by overnight commercial express courier service. Because of concern that this
24-hour delay before processing of the maternal plasma sample might be detrimental,
the remaining 11 samples were processed directly on-site in Bari, Italy, and
the plasma was shipped frozen to Basel, Switzerland.
All samples were sent coded and examined to Basel in a blinded manner.
None of the samples examined have been used in any prior investigations. Plasma
was prepared from the maternal blood samples by high-speed centrifugation
as described previously and stored at –70°C before analysis.12 In addition, the frozen plasma samples shipped from
Bari were again subjected to high-speed centrifugation (16 000g for 10 minutes) before analysis.16 We
focused exclusively on samples in which the father was a carrier for 1 of
the 4 following β-globin gene mutations (IVSI-1, IVSI-6, IVSI-110, and codon 39)
and the mother had been genotyped to carry another β-globin gene mutation.
The chorionic villus sampling sample was obtained by transabdominal
puncture with a 23-gauge needle under ultrasonic guidance. The samples were
processed and analyzed at the diagnostic laboratory at the University of Bari,
using an allele refractory mutation system and PCR procedure, followed by
combined reverse dot blot analysis.7,8
Circulatory DNA was extracted from 5- to 10-mL maternal plasma using
commercial column technology (Roche High Pure Template DNA Purification Kit;
Roche, Basel, Switzerland) in combination with a vacuum pump.12 After
extraction, the DNA was separated by agarose gel (1%) electrophoresis (Invitrogen,
Basel, Switzerland), and the gel fraction containing circulatory DNA with
a size of approximately 300 bp was carefully excised. The DNA was extracted
from this gel slice by using an extraction kit (QIAEX II Gel Extraction Kit;
Qiagen, Basel, Switzerland) and eluted into a final volume of 40-μL sterile
10-mM tris -hydrochloric acid, pH 8.0 (Roche).12 Strict
anticontamination procedures were used throughout the procedure, including
the analysis of on average 2 blank gel slices per samples examined, which
were all negative.
Peptide-nucleic-acids (PNAs) bind with very high affinity to specific
DNA sequences (eg, to a wild-type or mutant allele), which may differ by as
little as a single-base change.17 These molecules
can be used when examining DNA samples that contain a mixture of wild-type
and mutant alleles to suppress the specific amplification of either allele.17 In this manner, the mutant or wild-type allele can
be selected specifically from a mixture of both alleles. We used a PNA sequence
specific for the maternal normal allele to suppress amplification of the wild-type
maternal allele, thereby enriching for the presence of paternally inherited
mutant sequences. The PCR/PNA clamping reactions were performed in a total
volume of 30 μL, consisting of 8-μL size-separated circulatory DNA,
1 × buffer with 3.5-mM magnesium, 0.2-mM dNTPs (nucleotides),
0.13-μM of each primer (all the primers used in this study were synthesized
by Microsynth, Basel, Switzerland, and high performance liquid chromatography [HPLC]
purified), and 0.6-U TaqGold DNA polymerase (Applied Biosystems, Rotkreuz,
Switzerland), using the following PNA probe concentrations (Applied Biosystems):
0.67-μM for the IVSI-1 mutation, 0.5-μM for IVSI-6 mutation, 1-μM for IVSI-110
mutation, and 1-μM for codon 39 mutation. The detailed primer sequences
and PCR/PNA clamping reactions are shown in Table
1. The clamping reaction was performed in a thermal cycler (Mastercycler,
Eppendorf, Hamburg, Germany).
Following the PCR/PNA clamping step, the presence of the mutant paternal
allele was detected by a real-time allele-specific PCR reaction, which was
performed on a sequence detector (Perkin Elmer Applied Biosystems 7000 Sequence
Detector, Applied Biosystems). A total of 1 μL of the PCR clamping product
was amplified in duplicate in a final reaction volume of 25 μL containing
160 nM of each primer, and a mixed solution (1X SYBR Green Master Mix, Applied
Biosystems). The specificity of each of the allele-specific assays for the
4 β-globin gene mutations was optimized by evaluating a series of conditions
concerning buffers composition (magnesium ions), temperature and length of
PCR amplification cycles, as well as use of different oligonucleotide primers.18
These experiments were performed on artificial mixtures of mutant DNA
diluted into wild-type DNA. The final conditions are listed in Table 1. For the real-time PCR analysis, the mixed solution (1X
SYBR Green Master Mix) was used to monitor the PCR reaction. The quantitative
process used by real-time PCR makes use of a defined threshold value, which
is determined by the crossing of a defined threshold by the accumulated PCR
product,19 which is termed the threshold value or CT. This value can be used for the accurate
determination of the exact amount on specific input template DNA, by comparison
with a standard curve. To measure the quantitative differences between 2 genetic
loci (eg, wild-type and mutant), the difference between the respective CT values (ΔCT) can be used.20 We
used this ΔCT system to determine the ratio of wild-type to
mutant, whereby the extent of the amplification of the normal wild-type allele
(CTN) was subtracted from that of the mutant allele (CTM).
By the use of this ΔCT(M-N) approach, we observed a clear
discrimination of normal wild-type DNA samples from those samples heterozygous
for the mutant allele, even with experimental conditions in which the mutant
allele constituted less than 10% of the total DNA examined. This analysis
also permitted us to assign arbitrary ΔCT(M-N) cut-off areas
for the 4 allele-specific PCR assays; the normal allele yielding higher and
the mutant allele yielding lower values (Figure).
The χ2 test was used to evaluate whether a significant
difference existed between the results obtained by the analysis of size-fractionated
circulating DNA and by the analysis of total-circulatory DNA. The analysis
was performed by using Stata version 8.0 (StataCorp LP, Lausanne, Switzerland). P<.05 was considered statistically significant.
The laboratory components of the study were performed from October 1,
2003, through May 30, 2004, in Basel, Switzerland. Four distinct point mutations
of the β-globin gene—IVSI-1 (n = 7), IVSI-6 (n = 4), IVSI-110
(n = 5), and codon 39 (n = 16)—were examined. For
each of these mutations, an allele-specific real-time PCR assay was developed.
In the case of the codon 39 mutation, the development of an allele-specific
assay was more complex due to the number of repetitive sequences in the vicinity
of the mutation, which initially hindered the specificity of the PCR amplification.
Because we were concerned that circulatory fetal DNA sequences may still
be outnumbered by maternal DNA sequences, even after selective enrichment
on the basis of size, an additional PCR step was used before the allele-specific
PCR assay to ensure that the presence of mutant fetal alleles could be detected
in a mixture of mutant and wild-type alleles. In this additional PCR step,
a PNA sequence was used that binds with high affinity to the wild-type maternal
allele, thereby blocking its amplification during the PCR procedure.17 In this manner, an additional selective enrichment
was performed for the paternally inherited mutant fetal allele. This step
effectively suppressed the amplification of the wild-type allele by at least
a factor of 1000-fold, whereas the mutant allele was amplified with normal
Because a mixture of wild-type and mutant alleles was still present,
a further safety measure was added to our analysis to ensure accurate determination
of the presence of the paternal mutant allele. This was achieved by subtracting
the extent of amplification of CTN from that of CTM in
the subsequent real-time PCR assay. This approach permitted a clear discrimination
between the 2 alleles, even with experimental conditions whereby the mutant
allele was diluted in normal wild-type DNA of the order expected in maternal
plasma samples (Figure, A). These assessments
also permitted establishment of tentative cut-off ranges that could serve
to distinguish between samples in which the mutant allele was present and
those samples in which it was absent.
Confident that our assays were functional, we next tested a series of
32 clinical samples in a blinded manner, after which these results were compared
with those obtained from the analysis of chorionic villous material that had
been performed by an independent routine diagnostic laboratory at the University
These results (Table 2 and Table 3) and the codon 39 mutation (Figure, B) indicated the presence or absence
of the mutant paternal allele in 6 (86%) of 7 cases for IVSI-1, 4 (100%) of 4 cases of IVSI-6, 5 (100%)
of 5 cases for IVSI-110, and 13 (81%) of 16 cases
for the codon 39 mutation. One false-positive test result was scored for our
analysis of a IVSI-1 mutation. Because multiple negative
controls and stringent anticontamination procedures were used throughout our
analysis, it is unlikely that this is due to a contamination, but is most
likely attributable to the concentration of input template DNA being too low
to permit accurate analysis (40 genome-equivalents), as these conditions may
lead to abnormalities during the PCR/PNA clamping step.17
For this reason, in the remaining analysis we examined total cell-free
DNA levels in the majority of cases. If these levels were too low (arbitrary
value of 40 genome-equivalents input DNA), the results were classified as
being uncertain. For this reason, the input DNA concentrations were used within
a certain range (50-700 genome-equivalents; mean, 392 genome-equivalents)
in order for the assay to function reliably. This amount is usually obtainable
from 20-mL maternal blood samples.
Sixteen cases in which the father was a carrier for the β-globin
codon 39 mutation were next examined. Three cases were classified as uncertain
(Figure, B), because the concentration
of input DNA (obtained from 3-mL maternal plasma samples) was too low to permit
accurate analysis (44, 37, and 31 genome-equivalents, respectively). One of
these cases, labeled as uncertain, had to be excluded because the diagnostic
result from the chorionic villus sampling analysis was not available at the
time of our analysis and preparation of our data set.
Our analysis of all 4 paternal mutant loci provide an overall sensitivity
of 92.8% and specificity of 88.2% (Table 3).
This was improved to a sensitivity of 100% and specificity of 93.8%, if the
2 cases classified as uncertain and the case without a diagnostic test result
from the chorionic villus sampling analysis were excluded.
To verify the validity of our approach concerning the enrichment of
circulatory fetal DNA sequences on the basis of their smaller size vs maternal
DNA sequences, a parallel assessment of maternal plasma DNA samples that had
not been subjected to size-fractionation was performed. This led to the incorrect
evaluation of 6 (46%) of 13 cases that had inherited the paternal mutation
for all 4 mutations examined, which is significantly different from our results
obtained using size-fractionated DNA (χ2 test, P = .02). For the codon 39 mutation, these 3 cases are represented
in the Figure, C. We also evaluated
the value of the PCR/PNA clamping step to suppress amplification of the normal
maternal allele on some of our samples (n = 16). This analysis indicated
that in the absence of this suppression step incorrect results were recorded
in 2 of 4 cases, even when using size-separated circulatory DNA (codon 39
mutation: Figure, D).
We initially only examined 21 maternal whole blood samples shipped directly
from Bari to Basel by express courier. Nevertheless, it was quite apparent
that the samples had been altered considerably during the 24-hour freight
period, which has been suggested to hinder the analysis of fetal alleles in
maternal plasma.11 Therefore, we also examined
11 plasma samples that had been directly prepared on site in Bari and shipped
frozen to Basel for analysis. An examination of the total circulatory DNA
in these 2 sets of samples indicated that considerable apoptosis of maternal
cells had occurred in the samples shipped as whole blood (data not shown).
Because this apoptotic process increases the amount of maternal DNA present
in the gel fraction selected for the enrichment of fetal DNA species, it could
thereby hinder the analysis of the mutant fetal allele. Our analysis of these
samples indicated that the presence of this increased amount of maternal DNA
did not hinder the detection of the mutant fetal allele with the conditions
we had chosen.
Based on our results, which made use of a combination of size-separation
and PCR/PNA clamping step, our strategy probably can be used for samples that
have been shipped considerable distances (>1600 kilometers) by express courier
service (<24 hours). The advantage of detecting these paternal mutations
in compound heterozygous Mendelian disorders is that their absence can be
used to exclude pregnancies at risk for these disorders, such as β-thalassemia
major, thereby obviating the need for an invasive prenatal diagnostic procedure.
Furthermore, a decrease of these risk-associated procedures can be achieved.9
Our study indicates that fetal genetic traits involving point mutations
can be detected from the analysis of circulatory fetal DNA in maternal plasma
by selecting circulatory DNA sequences with a size of less than 300 bp. This
step permits a selective enrichment of the fetal DNA species.12 Nevertheless,
we have determined that despite this step, the reliable detection of fetal
mutant alleles involving point mutations requires an additional safe-guard
to ensure their optimal analysis by allele-specific PCR. In our case, this
was achieved by using a PNA probe that binds with high affinity to the normal
allele, thereby suppressing the amplification of maternal wild-type sequences.17 We showed that the combination of these 2 procedures
permits the ready detection of paternally inherited fetal mutant alleles for
4 common β-thalassemia mutations.
Our study indicates that an analysis of 32 maternal blood samples, taken
at 10 to 12 weeks of gestation, resulted in only 1 misdiagnosis, provided
the 2 cases classified as uncertain, as well as the case with no clinical
diagnostic result, are excluded. This yielded a sensitivity of 100% and a
specificity of 93.8%. The goal of 100% sensitivity is far-reaching, as even
the analysis of simple fetal genetic loci completely absent from the maternal
genome, such as the RhD gene in pregnancies at risk for hemolytic disease
of the newborn, cannot be achieved with 100% sensitivity on first trimester
samples, with 2 of 9 RhD fetuses not being detected correctly.21,22
The 1 false-positive test result observed for the detection of the IVSI-1 mutant allele was probably due to an insufficient
amount of sample DNA (<50 genome-equivalents), and these conditions may
lead to abnormal amplification during the PNA-clamping process.17 Alerted
that low-input DNA levels may be a problem, our subsequent analysis of 16
samples at risk for paternal inheritance for a codon 39 mutation contained
3 cases that were identified as uncertain because the quantity of the target
DNA was low and on this basis reliable diagnostic results could not be determined.
One of these cases had to be excluded from our analysis due to lack of a diagnostic
result at the time of our data set preparation.
Our method also functioned in samples that had been shipped as whole
blood, indicating that this approach probably would be suited for the analysis
of clinical samples, which frequently have to be sent to specialized laboratories
for analysis. In our opinion, a 20-mL maternal blood sample should be sufficient
to permit an efficacious analysis.
In the context of detecting paternally inherited fetal point mutations,
the use of mass spectrometry for the analysis of fetal point mutations from
circulatory plasma DNA has recently been reported.23 Although
the determination of the presence of the paternal β-globin gene point
mutation could be determined with high degrees of accuracy, these results
were based on only 5 pertinent cases. An advantage of the mass spectrometry
approach over our analysis is that it does not require any additional processing
of the sample, such as size-fractionation. It is also much more amenable to
high-throughput automated analysis. A disadvantage of this alternative development
is that it requires sophisticated and expensive equipment not readily available
to the majority of diagnostic or research laboratories.
Our approach, in contrast, is relatively simple and can be performed
without the need for complex machinery, as it relies on technologies consistent
with those currently used in many routine diagnostic and research laboratories.
Moreover, we have estimated that the cost of the single analysis may be as
low as US $8. This low-cost and use of simple equipment is especially suitable
for the screening of at-risk pregnancies in developing countries. This method
is also useful for detection of other fetal single-gene mutations, such as
Size-fractionation may potentially provide an alternative approach for
the noninvasive prenatal assessment of fetal single-gene disorders involving
compound heterozygous mutations. The fetal genotype in those cases in which
both partners are carriers for the same disease allele, frequently the case
for cystic fibrosis,24 could be determined
by an analysis of paternally and maternally inherited single-nucleotide polymorphisms
associated with the mutant allele, as had recently been shown for a similar
case at risk for β-thalassemia.23 In the
near future, the approach we have outlined may bring this desired goal in
prenatal medicine closer to a clinical application.
Corresponding Author: Sinuhe Hahn, PhD,
Laboratory for Prenatal Medicine, University Women’s Hospital/Department
of Research, Spitalstrasse 21, CH 4031 Basel, Switzerland (firstname.lastname@example.org).
Author Contributions: Dr Li had full access
to all of the data in the study and takes responsibility for the integrity
of the data and the accuracy of the data analysis.
Study concept and design: Li, Di Naro, Vitucci,
Acquisition of data: Li, Di Naro, Vitucci,
Analysis and interpretation of data: Li, Zimmermann,
Drafting of the manuscript: Li, Hahn.
Critical revision of the manuscript for important
intellectual content: Li, Di Naro, Vitucci, Zimmermann, Holzgreve,
Statistical analysis: Li.
Obtained funding: Holzgreve, Hahn.
Administrative, technical, or material support:
Di Naro, Vitucci, Zimmermann, Hahn.
Study supervision: Hahn.
Financial Disclosures: Drs Li, Zimmerman, Holzgreve,
and Hahn have filed for a patent covering the use of size-fractionated DNA
for noninvasive prenatal diagnosis. This patent is owned by the University
Hospital, Basel, Switzerland. Drs Di Naro and Vitucci reported no financial
Funding/Support: This study was supported by
the European Union FP6 Network of Excellence “SAFE,” as well as
funds from the University Women’s Hospital/Department of Research, University
Hospital, Basel, Switzerland.
Role of the Sponsor: The European Union FP6
Network of Excellence “SAFE” and the University Women’s
Hospital/Department of Research did not participate in the design and conduct
of the study, in the collection, analysis, and interpretation of the data,
or in the preparation, review, or approval of the manuscript.
Acknowledgment: We thank Susanne Mergenthaler,
PhD, and Corinne Rusterholz, PhD, for valuable discussions and critical evaluation
of the final manuscript.