The duplex PCR products of the sample with known Constand spring mutation CS/aa, Quonsze mutation QS/ aa, and Weastmead mutation WS/aa DNA showed significantly different profiles, which
Trang 1mutations and deletions
JINGZHONG LIU, XINGYUAN JIA, NING TANG, XU ZHANG, XIAOYI WU, REN CAI,
LIRONG WANG, QUANZHANG LIU, BAI XIAO, JIM ZHU, and QINGTAO WANG
BEIJING AND GUANGXI, CHINA
Populations in Southeast Asia and South China have high frequencies of a-thalassemia caused by a-globin gene mutations and/or deletions This study was designed to find
an efficient and simple diagnostic test for the mutations and deletions A duplex poly-merase chain reaction (PCR)/denaturing high-pressure liquid chromatography (DHPLC) was used to detect the mutations and deletions A blinded study of 110 samples, which included 92 a-thalassemia samples with various genotypes and 18 normal DNA samples, was carried out by the methods The duplex PCR products of the sample with known Constand spring mutation (CS)/aa, Quonsze mutation (QS)/
aa, and Weastmead mutation (WS)/aa DNA showed significantly different profiles, which suggests that DHPLC analysis at 63.8C can detect potential mutations directly The DHPLC at 50C analysis can distinguish the SEA and nondeletional alleles The new assay is 100% concordant with the original genotype In conclusion, the tech-nique including the duplex PCR assay followed by DHPLC analysis can be used to di-agnose a-thalassemia; this methodology is simple, rapid, accurate, semiautomatic, and high output, and thus, it is suitable for large-scale screening (Translational Re-search 2010;155:148–155)
Abbreviations: CS ¼ Constand spring mutation; DC ¼ dissociation curve analysis; DHPLC ¼ denaturing high-performance liquid chromatography; Duplex PCR ¼ duplex polymerase chain reaction; GC ¼ guanine-cytosine; QS ¼ Quonsze mutation; RDB ¼ reverse dot-blot; TEAA ¼ triethylammonium acetate; WS ¼ Weastmead mutation
a-Thalassemia is the most common recessively inherited
hemoglobin disorder.1 Unlike a-thalassemia, in which
nondeletional mutations predominate, most recognized
a-thalassemia involve deletion of 1 or both a-globin
genes The a-globin gene cluster is located on
chromo-some 16 a2 and a1 are highly homologous, with
se-quence identity greater than 96% between the Z1 and
Z2 boxes and high guanine-cytosine (GC) content.2In
Southeast Asia and southern China, most a-thalassemia
is caused by the deletion of 1 (-a4.2/, -a3.7/; termed a-thalassemia-2) or 2 ( SEA, THAI; termed a-thalasse-mia-1) of the 2 functional a-globin genes3,4 (Fig 1) The SEA is the most common type of
a-thalassemia-1 Even though carriers of the a-thalassemia-1 with SEA type do not manifest any clinical symptoms, cou-ples who are both carriers have a 25% chance of
From the Basic Medical Research Center, Beijing Chaoyang Hospital,
Affiliate of The Capital Medical University, Beijing, China; Institute of
Basic Medical Sciences, Chinese Academy of Medical Sciences &
Peking Union Medical College, Beijing, China; Liuzhou Women and
Children’s Hospital, Liuzhou City, Guangxi, China; Transgenomic
Ltd, Beijing, China; Beijing Deyi Clinical Diagnostic Laboratory,
Beijing, China.
Supported by Grant JS96004 from the Natural Science Foundation of
Beijing, China
Submitted for publication August 4, 2009, revision submitted October
14, 2009; accepted for publication October 16, 2009.
Reprint requests: Jingzhong Liu, PhD, Basic Medical Research Center, Beijing Chaoyang Hospital, Affiliate of Capital Medical University,
8 Gongtinanlu, Chaoyang District, Beijing, China 100020.; e-mail:
liujingzhong090525@hotmail.com ; liujz@public.bta.net.cn (J.L.) 1931-5244/$ – see front matter
Ó 2010 Mosby, Inc All rights reserved.
doi:10.1016/j.trsl.2009.10.003
148
Trang 2conceiving a homozygous fetus, which manifests as
Bart’s hydrops fetalis, the most severe thalassemic
syn-drome All these fetuses diein utero or soon after birth
In addition, approximately 75% of mothers who carry
fe-tuses with homozygous for the thalassemia-1 SEA type
will develop toxemia of pregnancy An investigation of
the thalassemia-1 SEA type is therefore essential for
carrier couples and for prenatal diagnosis of conception
by couples who are both carriers of this type of gene
de-letion Diagnostic assays of the a-thalassemia-2 are also
important for genetic counseling, because when
occurring in compound geterozygosity with SEA, the
2a4.2/ or 2a3.7/ will cause HbH disease with a
thalas-semia intermedia phenotype
Nondeletional a-thalassemia, a more severe
expres-sion, may also be induced by point mutation of the a2
gene (Constand spring mutation [CS], Quonsze mutation
[QS], and Weastmead mutation [WS])1,5,6(see the lower
part ofFig 1) In an investigation of 59 cases of HbH
diseases from Guangxi, China, 27 cases (45.8%) were
confirmed to be nondeletional.7To date, techniques for
the detection of a-globin gene deletion have included
Southern blot analysis,8multiplex gap-polymerase chain
reaction (mPCR),3,4,9 and real-time PCR with SYBR
Green1 combined with dissociation curve (DC)
analysis.10
The techniques for the detection of a-globin gene
mutations include PCR product sequencing, reverse
bin gene mutations This lack of research may be largely because of the high homology of the a2 and a1 genes and difficulties in designing specific primers and PCR amplifications based on high GC content Rapid and accurate testing methods are needed to address the diag-nostic challenges of identifying both deletions and muta-tions using the same assay This study reports on 1 such method We have developed a duplex PCR followed by the DHPLC for detecting the SEA and the nondele-tional alleles under 50C condition, and for detecting the 3 known point mutations under 63.8C condition
METHODS Samples. In all, 34 DNA samples with known geno-types including aCSa/aa (4 cases), aQSa/aa (2 cases), aWSa/aa (2 cases), /aa (5 cases), / (2 cases), 3.7/
aa (10 cases), 3.7/ (2 cases), 4.2/aa (5 cases), and 4.2/ (2 cases) were used to establish the methodology
In all, 110 blood samples were collected from Liuzhou city in southern China from January 2007 to May 2008, and they were submitted to the laboratory for the blinded study Informed consent was obtained from the subjects The study conformed to the Chinese ethical guidelines for human and animal research, and it was approved by the Beijing Chaoyang Hospital Ethics Committee
Primer design and duplex PCR assay. Given the minor sequence differences of IVS-II and the 3’untranslated re-gion between the 2 functional a-globin genes a2 and a1,
a 206-bp fragment, including the third exon and both flanking sequences, was designed for amplification (Fig 1) The upstream primer P1 used the difference in
7 bp between the a2 and 1 sequences; thus, 3 bases in the 3’ end of P1 differed from those in the a1 sequence The downstream primer P2 sequence had more bases that differed between the a2 and the a1 Both the P1 and the P2 ensure a specific amplification of a 206-bp segment from the a2 The amplified product was designed as short as 206 bp that will facilitate mutation detection using the DHPLC The most frequently found mutations (CS, QS, and WS) were all in this 206-bp
turing high-performance liquid chromatography
(DHPLC) was used to detect the mutations and
de-letions A blinded study of 110 samples including
92 a-thalassemia with various genotypes was
con-ducted The new assay is 100% concordant with the
original genotypes The new technique can be used
to diagnose a-thalassemia because it is simple,
rapid, accurate, semiautomatic, and high-output;
thus, it is suitable for large-scale screening
Trang 3fragment, which allowed rapid detection of the 3
poten-tial mutation types using DHPLC under parpoten-tially
dena-tured conditions The sequences the P1 and P2 are
shown in blue in the lower part ofFig 1 Given the
dif-ficulties in the above primer design, it was easier to
de-sign the 2 primers that represented the SEA deletion
according to the principles of gap-PCR Primer
se-quences that satisfied the following conditions were
qualified: good specificity for amplification, length
shorter than 206 bp, ability to resolve the peak
associ-ated with this fragment in the DHPLC profile at 50C,
and noninterference with recognition of the specific
mu-tation profile at 63.8C The P3 and P4 primers that were
designed in this study satisfied the above conditions and
formed a successful duplex PCR assay with the P1 and
P2 primers SEA/ and aa/ or aTa/ were detected by
the duplex PCR The 25-mL PCR mixture contained
2.5 mL 103 buffer, 2.0 mL deoxyribonucleotide
triphos-phate, 2.5 mL MgCl2, 5.0 mL betaine, 0.5 mL SYBR
Green1, 0.36 mg primers 1–3, 0.26 mg primer 4, 1 mg
Gold-Taq DNA polymerase, and 3 mL/0.6 mg of DNA
The negative and positive controls were included PCR assays were performed using an ABI Prism 5700 ther-macycler (Applied Biosystems, Foster City, Calif) PCR conditions were 94C for 10 min followed by 38 cycles of 94C for 20 s, 65C for 30 s, and 72C for
40 s The annealing temperature was decreased by 1C each cycle until it reached 61C (34 cycles), and the final extension was conducted at 72C for 6 min Samples were then placed on ice for DHPLC analysis
Two gap-PCRs. 2a3.7 and 2a4.2 were detected by
2 gap-PCR using primer pairs (p5 and p6) and (p7 and p8), respectively The sequences of the 4 primers were shown in previously published paper.4The conditions were the same as the duplex PCR, except that extension time was 2 min
DHPLC analysis.DHPLC analysis was conducted on the WAVE nucleic acid fragment analysis system (Trans-genomic, Omaha, Nebr) as previously described.12The duplex PCR products were analyzed at 50C in a linear acetonitrile gradient with triethylammonium acetate (TEAA) as the mobile phase, using buffer A (0.1 mol/L
Fig 1 Designing the primers P1 and P2 as well as P3 and P4 The upper part shows locations of the 4 primers in the
a-gene cluster and the 3 deletions The lower part shows sequences around the amplicon from the P1 and P2 The
bases of the HBA1, which are different from the HBA2, are in red The sequences in blue are primer P1 and the
complementary sequence of primer P2 The 3 HBA2 bases in bold blue are the potentially mutable bases Compared
with nt1839-1845 of HBA1, HBA2 deletes 7 bp (Color version of the figure is available online.)
Trang 4TEAA) and buffer B (0.1 mol/L TEAA with 25%
aceto-nitrile) (Transgenomic) The initial buffer concentrations
were 49.8% B, with a gradient over the 12.5-min run time
to 65%, and the flow rate was 0.9 mL/min The duplex
PCR products were also analyzed for mutations at
63.8C using the same method The initial buffer
concen-trations were 51.1% B, with a gradient over the 4.5-min
run time to 60.1%, and the flow rate was 0.9 mL/min
For deletion detection of the 2 gap-PCR products, the
ini-tial buffer concentrations were 65.7% B with a linear
gra-dient over the 4.5-min run time to 74.7%, and the flow
rate was 0.9 mL/min The eluted DNA fragments were
detected at a wavelength of 260 nm
The blinded study. A cohort of 110 blinded samples
in-cluding patients and normal individuals were detected
and analyzed with the methods described in this article
(Fig 2) The results were compared with their original genotype with the multiplex PCR/agarose gel electro-phoresis, RDB, and sequencing by different investiga-tors from Liuzhou Women and Children’s Hospital
A concordance was calculated
RESULTS Single-tube duplex PCR detection for SEA/ and nondeletional a-globin alleles (aa/ or aTa/). Duplex PCR product results are shown inFig 3 An absorption peak appeared at 5.1 6 0.1 min in all samples with aa, aCSa, or aQSa alleles (Fig 3, 2–7), which indicates that they were positive for the 206-bp product An ab-sorption peak appeared at 1.0 6 0.1 min in all samples with SEA deletion alleles (Fig 3, 1–3), which indicates that they were positive in the 78-bp product A second peak appeared simultaneously in samples 2 and 3, which were in accordance with the known genotype ( /aa and /aCSa) The peak shapes of other samples were also in accordance with their known genotypes
Rapid detection of CS, QS, and WS point mutations. With DHPLC, potential mutations in the du-plex PCR product can be detected rapidly according to profile differences.Figure 4shows the analysis profiles for the duplex PCR products of 6 known genotypes at 63.8C aa/aa had no mutated samples; thus, only
1 peak appeared at 4.8 min (samples 5 and 6) Homodu-plex double peaks were found at 4.7–4.8 min in the CS/aa sample, and heteroduplex double peaks appeared simultaneously at 4.1–4.2 min (Fig 4, 1 and 2) A single peak appeared at 4.8 6 0.1 min in the QS/aa samples (Fig 4, 3 and 4), and a heteroduplex double peak appeared simultaneously at 4.5–4.6 min The 3 continu-ous peaks that appeared at 4.25–4.45 min in the WS/aa samples (Fig 5, 1 and 2) had completely different profiles than those of the carriers with the 2 above mutations
China and Southeast Asia.
Fig 3 DHPLC profiles for the duplex PCR products of 7 DNA samples
of known genotypes at 50C 1 5 SEA/SEA; 2 5 SEA/aa; 3 5
SEA/aCSa; 4 5 aa/aa; 5 5 23.7/aa; 6 5 24.2/aa; 7 5 aQSaa/
aa The peak appeared at 5.1 6 0.1 min, which indicates aa, aCSa,
or aQSa alleles, whereas a peak appeared at 1.0 6 0.1 min, which
indicates the positive SEA allele (Color version of figure is available
online.)
Fig 4 DHPLC profiles for the duplex PCR products of 6 samples at 63.8 C Four had mutations and 2 did not 1 and 2 5 aCSa/aa;
3 and 4 5 aQSa/aa; 5 and 6 5 aa/aa (Color version of figure is available online.)
Trang 5Table Isummarized the criteria for the diagnosis of the 3
mutations according to DHPLC profiles at 63.8C from
the 36 known samples with well-known different
geno-types
Detection of 2a3.7 and 2a4.2 with DHPLC. Figure 6
shows the DHPLC profile for the gap-PCR product
(1.6 kb) that was used to detect 2a3.7 at 50C A
peak can be found at 2.5 6 0.1 min for all samples
with 2a3.7 alleles (Fig 6, 1–5); no corresponding
peak was found in the samples without -a3.7 alleles
(Fig 6, 6 and 7) Figure 7 shows the DHPLC profile
for a second gap-PCR product (1.5 kb) that was used
to detect 2a4.2 at 50C A peak can be found at
2.3 6 0.1 min (Fig 7, 1–5) The detected 2a3.7 and
24.2 genotypes by DHPLC were fully concordant
with by the DC analysis (Figs 8 and 9), and the previ-ously published results.10
DC analysis of duplex PCR and gap-PCR products. The products from the 3 PCR assays were also subjected to
DC analysis to obtain the genotype diagnosis rapidly for various types of deletion mutations within a-thalassemia
Figure 8shows the DC profiles for the duplex PCR prod-ucts of 4 different genotype samples The peak at
85 6 0.2C represents the 78-bp product (ie, the SEA positive identifying with the absorption peak appeared at 1.0 6 0.1 min in DHPLC) (Fig 3) The peak at
89 6 0.2C represents the 206-bp product (ie, aa or aTa positive identifying with the absorption peak appeared at 5.1 6 0.1 min in DHPLC) (Fig 3).Figure 9
shows the DC profiles for the gap-PCR products to detect 2a3.7 or 2a4.2 deletions A broad peak appeared at 84–
86C in all samples with the 2a3.7 allele (Fig 9, A), whereas a peak appeared at 82 6 0.2C in all samples with the 2a4.2 allele (Fig 9,B) These results were in accordance with those by the DHPLC method
The blinded study results of 110 DNA samples. A total of
110 DNA samples with various a-thalassemia–causing deletions and mutations identified by 4 gap-PCRs as well as the DC analysis were compared to test the speci-ficity and sensitivity of the new assay by blind analysis
Table II shows the results for 110 DNA samples analyzed by using the duplex PCR and the 2 gap-PCRs combined with DHPLC The data indicate that the
3 PCR and DHPLC methods in this investigation can
be used to carry out accurate and rapid diagnosis of all deletion-type a-thalassemia Normal allele and mutant allele (aTa) were distinguished by DHPLC at 63.8C According to the profiles inFigs 4 and 5, this investiga-tion detected 9 cases of nondeleinvestiga-tional HbH and 12 cases
Fig 5 DHPLC profiles for the duplex PCR products of DNA from 4
known mutation carriers and 1 negative control at 63.8 C 1 5 aWSa/
aa; 2 5 aCSa/aa; 3 5 aQSa/aa; 4 5 aa/aa; 5 5 negative control.
(Color version of figure is available online.)
Fig 6 DHPLC profiles for the gap-PCR products to detect 2a3.7 for 7
DNA samples of known genotypes at 50 C 1 and 2 5 2a3.7/aa;
3 5 2a3.7/ 4 5 2a3.7/-a3.7; 5 5 2a3.7/-a4.2; 6 5 aa/aa;
7 5 2a4.2/aa All samples carrying 2a3.7 allele showed a peak at
2.5 6 0.1 min (Color version of figure is available online.)
Fig 7 DHPLC profiles for the gap-PCR products to detect -a4.2 in 7 DNA samples of known genotypes at 50C 1 and 2 5 a4.2/aa; 3 5 -a4.2/ 4 5 -a4.2/-a4.2; 5 5 -a3.7/-a4.2; 6 5 aa/aa; 7 5 -a3.7/aa All samples carrying -a4.2 allele showed a peak at 2.3 6 0.1 min (Color version of figure is available online.)
Trang 6of mutation carrier samples, as follows: 5 cases of aCSa/
aa, 4 cases of aQSa/aa, and 3 cases of aWSa/aa
(Table I) These results were in accordance with those
from the mPCR, RDB, and sequencing methods In
a word, the blinded study results of the 110 DNA samples
using the new assay is 100% concordant with the
genotypes detected by the current standard methods
DISCUSSION
This investigation described a novel technology that
could be adopted into the clinical laboratory because it
has several features The technology includes 1 duplex
PCR and 2 gap-PCRs followed by DHPLC that can
detect 3 deletions and 3 mutations at same time The
206-bp product replaced the 1.9-kb amplicon in the
pre-vious methods,4,10and it can be used to identify whether
nondeletion alleles (aa or aTa) exist The homozygous
and heterozygous deletions can thus be distinguished,
and a complete diagnosis can be made for the genotypes
of all deletion-type a-thalassemia (Table II)
Further-more, the 3 kinds of potential point mutations that cause
nondeletional a-thalassemia can be detected by DHPLC
at 63.8C (Table I), which takes only about 10 min
More than 100 samples can be processed overnight
auto-matically Thus, this methodology was more convenient,
for 4 min and then renatured by decreasing the tempera-ture to room temperatempera-ture The 2 samples were analyzed
by DHPLC at 63.8C; the same profiles as the CS muta-tion carrier were obtained as expected
These results indicate that this methodology has predic-tive values The optimization of reaction conditions is im-portant to achieve excellent detection results Particularly important factors include primer design, the concentration ratio between the 2 pairs of primers, the proper amount of DMSO, the amount of betaine (which will damage the DHPLC analytical column if too much is used), and the application of a touchdown heat cycle The DNA extrac-tion method and the quality of the extract significantly influenced the effectiveness of PCR amplification and the results of DHPLC detection In all, 11 samples had low peaks After purification of the DNA samples, the low peak problem was resolved The cutoff values of the peaks for a positive aa and positive SEA were 0.4 and 0.5, respectively Therefore, it is likely that the low peak was caused by low DNA quality or concentration
An integrative diagnosis can be carried out on deletional a-thalassemia by observing comprehensively the loca-tions of the 4 potential peaks in the DHPLC or DC anal-yses for the 3 PCR products Both DHPLC and DC can
be used to obtain accurate diagnostic results rapidly and may be used according to a given laboratory’s available equipment If the laboratory is equipped for both instru-ments, then we recommend using the real-time PCR and DC to diagnose deletion types, followed by a DHPLC analysis of the duplex PCR products at 63.8C for sam-ples that require mutation detection If a confident diagno-sis cannot be carried out with DHPLC or DC profiles, this indicates poor quality of the DNA sample, and purifica-tion procedures or reextracpurifica-tion should be carried out be-fore the method is repeated
CONCLUSION Use of the duplex PCR and the 2 gap-PCR assays designed in this investigation in combination with DHPLC and/or DC analysis allows the complete
Fig 8 DC profiles for the duplex PCR products of 4 DNA samples of
known genotypes: aa/aa, / , /aa, /aCSa The peak at
85 6 0.2 C represents the SEA allele; the peak at 89 6 0.2 C
repre-sents the aa or aTa alleles (Color version of figure is available
online.)
Table I Criteria for the diagnosis of mutated
a-thalassemia according to DHPLC profiles at 63.8C
4.05–
4.20min
4.2–
4.4min
4.55–
4.62min
4.75–
4.85min
Gene mutant
Trang 7diagnosis of the 3 kinds of mutations and 3 kinds of
de-letions for a-thalassemia that are common in China and
Southeast Asia The new assay is 100% concordant with
the original genotype The method has predictive values and is simple, rapid, accurate, semiautomatic, and cost effective
Fig 9 A, DC profiles for the gap-PCR products to detect 2a3.7 in 4 DNA samples of known genotypes: 2a3.7/
aa,2a3.7/ ,aa/aa, /aa B, DC profiles for the gap-PCR products to detect 2a4.2 in 4 DNA samples of 4
known genotypes: 2a4.2/aa,2a4.2/ , aa/aa, /aa (Color version of figure is available online.)
Table II Diagnosis results of deletional a-globin genotypes for 110 DNA samples by DHPLC at 50C followed by DHPLC at 63.8C for the duplex PCR products
Diagnosis
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