Neonatal hemoglobinopathy screening: molecular genetic technologies

Molecular Genetics and Metabolism 80 (2003) 129–137

www.elsevier.com/locate/ymgme

Minireview

Neonatal hemoglobinopathy screening: moleculargenetic technologies

Urvashi Bhardwaj,a,b Yao-Hua Zhang,a,b and Edward R.B. McCabea,b,c,d,e,*

a Department of Pediatrics, 22-412 MDCC, David Geffen School of Medicine at UCLA, 10833 Le Conte Avenue, Los Angeles, CA 90095-1752, USAb Mattel Children�s Hospital at UCLA, 10833 Le Conte Avenue, Los Angeles, CA 90095-1752, USAc Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

d Molecular Biology Institute, Los Angeles, CA, USAe UCLA Center for Society, the Individual and Genetics, Los Angeles, CA, USA

Received 8 August 2003; accepted 12 August 2003

Introduction

Newborn screening is a systematic application of tests

for early detection, diagnosis, and treatment of certain

genetic or metabolic disorders that can lead to mortality,

morbidity, and associated disabilities, if untreated. The

development of a screening test for phenylketonuria

(PKU) in the early 1960s by Dr. Robert Guthrie and a

system for collection and transportation of blood sam-

ples on paper blotters as dried spots, led to the begin-ning of newborn screening programs in the US [1–5].

Newborn screening for a disorder is based on the

frequency of the disorder in the population, and avail-

ability of an effective screening test and treatment.

Additionally, the cost of the test is taken into consid-

eration. In the US, the number of genetic and metabolic

disorders included in the state screening programs varies

from state to state, ranging from 4 to 36, but themajority of the programs screen for eight or fewer

disorders [6]. All states screen for PKU and hypothy-

roidism. Beyond those two, there is no programmatic

uniformity throughout the states [3–7].

Neonatal screening for hemoglobinopathies, partic-

ularly for sickle cell disease, was initiated in several

states in the 1970s [8]. Gaston et al. [9] reported the

reduced mortality of infants with sickle cell disease whentreated with penicillin prophylaxis, and, therefore, ar-

gued for early detection and intervention. An NIH

Consensus Development Conference in 1987 concluded

that universal newborn screening for sickle cell disease

* Corresponding author. Fax: 1-310-206-4584.

E-mail address: [email protected] (E.R.B. McCabe).

1096-7192/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.ymgme.2003.08.014

should be initiated so that penicillin prophylaxis and

comprehensive care could be commenced early to pre-vent overwhelming infections, and their associated

morbidity and mortality [10]. Newborn screening iden-

tifies neonates with a broad range of hemoglobinopa-

thies, which will be valuable not only for care of the

child but also for genetic counseling of the parents,

including potential risks for subsequent pregnancies and

opportunities for antenatal screening [11,12].

In the US, as of December 2002, 44 states had uni-versal screening for hemoglobinopathies, whereas, six

states provided screening for selected populations [6].

Screening methods

State laboratories may choose among a variety of

testing methods to achieve maximum efficiency and ef-

fectiveness in screening. Originally, the methods used to

screen for hemoglobin variants included hemoglobin

electrophoresis in basic and alkaline media [13].

Although, this methodology is still widely practiced, it isa labor-intensive technique with limited sensitivity.

Sickle cell disease may go undetected in the premature

infants because of inability to detect Hb S concentra-

tions less than 10% [14]. In addition, this technique is

less sensitive for the detection and quantification of low

concentrations of hemoglobin variants such as Hb A2,

or the fast moving variants such as Hb H or Hb Barts

[15,16]. In neonatal blood, a major fraction of hemo-globin is fetal hemoglobin (Hb F); therefore, confirma-

tory testing is recommended after 2–3 months when Hb

F is replaced by adult hemoglobin.

mail to: [email protected]

130 U. Bhardwaj et al. / Molecular Genetics and Metabolism 80 (2003) 129–137

With the improvement of techniques, isoelectric fo-cusing (IEF) became more popular due to its lower

cost and better resolution [17]. Although IEF is labor-

intensive and time-consuming, it has more precision

and accuracy than standard electrophoresis, and

therefore has become the method of choice in the

majority of large-scale screening programs [18,19].

Occasionally, the fast moving bands (Hb Barts) may be

missed with IEF particularly when neonatal screeningsamples are analyzed for a-thalassemia [20–22]. To

minimize the chances of human error with IEF, some

programs are converting to a more sophisticated and

fully automated technique, like high performance li-

quid chromatography (HPLC) as the primary or an-

cillary screening technique [8]. Additionally, HPLC has

the capability to quantitate hemoglobin variants;

therefore, this technique has been shown to be equiv-alent to, or more reliable than, IEF [15,23]. However,

IEF is still the method of choice in many of the state

programs.

In general, every system has some limitations due to

the inability to detect all of the hemoglobin variants;

therefore, confirmatory testing is required, particularly

for neonates with sickle cell disease, b-thalassemia, and

Hb Barts. Additionally, confirmatory testing is requiredin neonates who receive blood transfusions prior to

screening [24,25]. The majority of screening laboratories

in the United States use either electrophoresis, IEF or

HPLC as the secondary test. Due to the instability of

hemoglobin, aging of the neonatal sample should be

considered in interpretation [26]. As a result, for the best

interpretation of the results, samples should be tested

within 7 days [27]. Therefore, for confirmatory testing, asecond sample is often required when one of the com-

monly used screening methods is used, and affected in-

fants may not receive penicillin prophylaxis until they

are older than the recommended age for initiation of

prophylaxis.

With the demonstration of DNA microextraction

from the filter paper blotters, the potential applicability

of DNA diagnosis for the newborn screening was real-ized [28]. Application of DNA analysis in the Texas

neonatal screening program demonstrated a marked

reduction in the age at diagnosis for the hemoglobin-

opathies from 4 months of age down to approximately 2

months of age [29]. Additionally, this method avoids the

confusion of diagnosis in transfused infants [24]. To

decrease the labor-intensity and the cost of methodol-

ogy, a direct amplification method was developed thatinvolved PCR of an aliquot of the methanol-fixed dried

blood specimen, thereby eliminating the need of DNA

extraction prior to the amplification [30,31]. This pro-

vides the most accurate diagnosis in a patient of any age

irrespective of transfusion [24]. This innovation reduced

the cost of an analysis from an estimate of $25 to $5–10

[31]. This approach reduced the necessity for, and the

cost of, obtaining a new second sample for confirmatorytesting by 97% [32]. Aging of the dried blood sample

does not affect the stability of DNA, thereby eliminating

the requirement of a second sample for confirmatory

testing.

The innovation of the polymerase chain reaction

(PCR) technology has tremendously empowered DNA-

based testing for neonatal hemoglobinopathy screening.

Currently many techniques are available for the geno-typing of sickle cell disease and related disorders, e.g.,

Hb C, Hb E, b-thalassemia, a-thalassemia, db-thalas-semia, and hereditary persistence of fetal hemoglobin

(HPFH). The advantages and disadvantages of each

technique are important for their specific application(s).

The main requirements of any of the techniques for

newborn screening are accuracy, speed, cost, and ability

to detect different mutations simultaneously. The arrayof methodologies for detecting hemoglobinopathies

include the traditional techniques such as restriction

enzyme analysis, dot blot analysis with allele-specific

oligonucleotide (ASO) hybridization, reverse dot blot

analysis, and amplification refractory mutation system

(ARMS), as well as more sophisticated techniques like

denaturing gradient gel electrophoresis (DGGE), real-

time PCR with florescent labeled hybridization probes,automated DNA sequencing and oligonucleotide mi-

croarray (‘‘microchip’’) analysis. The large deletions of

the b-globin and a-globin loci can be detected by con-

ventional Southern blot hybridization along with re-

striction mapping. Recently, the gap-PCR approach has

been adopted for the well-characterized deletions of the

globin genes. Mutation detection strategies may be di-

vided into two types: those that identify specific andwell-characterized mutations and those that detect

uncharacterized sequence variations.

Despite the fact that more than 700 hemoglobin

variants and more than 200 b-thalassemia mutations

have been described, only a small number of mutations

are responsible for the majority of the cases in any

ethnic population [33–35]. This has implications in

population-based strategy such as newborn screening.

Molecular analysis of globin gene

Restriction enzyme analysis

Screening by restriction enzyme analysis is the tradi-

tional method to diagnose hemoglobinopathies, in-cluding the thalassemias. This methodology involves

differential allele-specific cleavage depending on the

presence or absence of the mutation that creates or

eliminates a specific restriction site [36,37]. Occasionally,

mismatched primers are designed in order to create a

restriction site that is not originally present in the nat-

urally occurring sequence, and hence can distinguish the

U. Bhardwaj et al. / Molecular Genetics and Metabolism 80 (2003) 129–137 131

wild type allele from the mutant allele using this artifi-cially created site [29,38,39].

This methodology was applied successfully for new-

born screening in the Texas molecular genetic follow-up

laboratory for Hb S, C, and E using the primary

screening sample [29]. For b-thalassemia, many groups

have shown the applicability of this methodology for

detecting common mutations in specific ethnic groups

[40–42]. However, for newborn screening for the b-tha-lassemias, this methodology has less potential because of

the cost and labor due to the variety of the mutations

responsible for this disorder.

Dot blot with allele-specific oligonucleotide hybridization

Allele-specific oligonucleotide hybridization in a dot

blot format is another conventional approach for de-tecting the known point mutations in the globin gene

[43–45]. The method involves PCR amplification of the

gene of interest followed by spotting of amplified

products on the immobilized membrane (e.g., nitrocel-

lulose or nylon). Subsequently the membrane-bound

DNA is allowed to hybridize with the labeled (isotopic

or non-isotopic) oligonucleotide probes. The oligonu-

cleotide probes are complementary to either the normalor mutant sequence at that point and each allele requires

a specific probe. This technique has been used success-

fully for the screening and confirmation of hemoglo-

binopathies with dried blood specimens [32,46].

Although this method is very useful for the confirmation

of Hb S, C, and E, it is not the method of choice for

b-thalassemia mutations for newborn screening using

dried blood spots.

Reverse dot blot

The reverse dot blot technique is based on the same

principle as the dot blot described above, except that the

allele-/sequence-specific oligonucleotide probes are

bound to the membrane and serve as the target for hy-

bridization with amplified DNA. Therefore, the reversedot blot can simultaneously look for all of the common

mutations [47]. This system is very rapid and accurate,

and hence has the potential for large population

screening. This assay is particularly valuable for new-

born screening since it is a non-isotopic methodology

that requires only limited amounts of DNA. In addition,

this technique is very robust and has special applicability

for the detection of high-mutation spectrum disorderssuch as hemoglobinopathies, b-thalassemia and for

point mutations causing a-thalassemia using newborn

screening specimens [48–51]. The critical requirement of

this system is that the oligonucleotide probes should

have similar melting temperatures (Tms) so as to have

uniform hybridization and washing conditions for all

the probes.

Amplification refractory mutation system

ARMS is a direct PCR-based assay to detect point

mutations that does not require further analysis of the

amplified product [52]. This technique detects point

mutations in the presence or absence of allele-specific

primers. The 30 terminal nucleotide in the primer is

either complementary to the normal sequence or the

mutant sequence at a particular position. An additionalmismatch is always introduced three or four nucleotides

from the 30 end to increase the primer specificity.

Therefore, the DNA will be amplified when the 30 ter-minal nucleotide matches perfectly with the target

DNA. The mutant and normal alleles are amplified in

separate reactions, indicating that the DNA is normal,

heterozygous or homozygous for a particular mutation.

In addition, a pair of internal control primers is alwaysincluded in the reaction to indicate successful amplifi-

cation.

ARMS has been widely used for the identification of

b-thalassemia mutations in various ethnocultural groups

[53–55]. There are more than 200 mutations responsible

for b-thalassemia [34]. Although a population typically

has 4–5 common mutations accounting for 85–90% of

the cases, it is labor-intensive to look for each of thesemutations in a sample by ARMS. Therefore, for new-

born screening, this method is not convenient for con-

ditions such as b-thalassemia. To overcome this

problem, multiplex-PCR protocols have been attempted

and tested for b-thalassemia using ARMS, and there are

successful reports of multiplexing the common

mutations [56–59]. However, there is the possibility of

non-specific/false positive bands that can be avoided byselecting the appropriate primer and the optimal primer

concentration. We have successfully developed a multi-

plex-ARMS protocol for the common b-thalassemia

mutations in various ethnic populations (Bhardwaj et

al., submitted). In our experience, multiplexed-ARMS

PCR has proven to be extremely robust, accurate, cost-

effective, and labor-efficient, satisfying the primary

requirements of newborn screening.

Gap-PCR

Conventionally, large DNA deletions are studied by

the traditional method of Southern blotting. This

method can identify both new as well as known, well-

characterized deletions. However, recently, gap-PCR

has begun to be preferred over Southern blotting todetect the well-characterized gene deletions [60–62]. In

this technique, three oligonucleotide primers are de-

signed to amplify deletion-specific products in the

presence of the deletion or the normal allele. Two

primers flank each of the deletion breakpoints, and

hence can amplify the target only in the presence of the

deletion. Since the primers are spaced widely apart in the


normal DNA, therefore, they cannot amplify the normalallele. This approach has been used for the detection of

large deletions of the a- and b-globin genes causing

a-thalassemia, HPFH and db-thalassemia [61,62]. We

have successfully demonstrated the utility of this meth-

odology using original newborn screening specimens to

detect common a-thalassemia and HPFH deletions

[63,64].

Single stranded confirmation polymorphism and hetero-

duplex analysis

Single stranded confirmation polymorphism (SSCP)

is an electrophoretic technique to detect known as well

as unknown mutations. In this method, the PCR-am-

plified product is denatured with heat and formamide,

and is run on a denaturing polyacrylamide gel [65]. Se-quence variation alters three-dimensional folding of the

single stranded DNA and affects electrophoretic mo-

bility. Even the variation of a single nucleotide may

result in a difference in folding and mobility on the gel.

SSCP is widely used due to its simplicity; however, it has

limitations in terms of the length of the fragment to be

analyzed, difficulty in interpretation and a frequent re-

quirement for multiple conditions to detect all of themutations [66]. Therefore, it has less applicability for

newborn screening.

Heteroduplex analysis is similar in principle to SSCP.

This technique consists of the analysis of duplex DNA

in a native gel. The presence of even a single nucleotide

mismatch and the heteroduplex formation between the

normal and mutant DNA strands typically results in

retardation of mobility through a polyacrylamidegel [67].

These systems seem promising, yet are not 100%

sensitive or specific in detection of mutations. However,

the combination of both SSCP and heteroduplex

analysis has been reported to detect all the known

mutations [68].

Denaturing gradient gel electrophoresis

Denaturing gradient gel electrophoresis (DGGE) is a

very sensitive methodology to detect single nucleotide

variations. This method allows the detection of single

nucleotide variations on the basis of differences in hy-

bridization stability of the target DNA. It involves the

electrophoretic mobility of double-stranded DNA

through a gradient of increasing denaturants (urea/formamide). When the concentration of the denaturing

agents in the gradient is sufficient to overcome the sta-

bility of double stranded conformation in the lowest

melting domain, it leads to partial denaturation of the

fragment. Consequently, there is branching of the DNA

strands, leading to the retardation of its electrophoretic

mobility. Therefore, any sequence variation in the DNA

fragment may be identified, theoretically at least, on thebasis of its position in the gel [69].

Whenever, the mutation is present within a high

melting temperature domain, however, its identification

can be problematic due to complete strand dissociation.

This situation can be avoided by introduction of a GC-

clamp to the target fragment through one of the PCR

primers [70]. The addition of a GC-clamp can prevent

the complete denaturation of the DNA fragment,thereby increasing the sensitivity of the system. Addi-

tionally, the target fragment should be designed in such

a way that the entire fragment behaves as one melting

domain, otherwise only the mutations in the lowest

melting domain are readily detectable [71].

Mutations in the b-globin gene (b-thalassemia,

HPFH point mutations) have been demonstrated using

this methodology [72,73]. To detect b-thalassemia, theb-globin gene is amplified in five fragments. The analysis

of normal, heterozygous, and homozygous DNA is done

on the basis of their patterns. Usually, the homozygous

wild type or mutant fragment will produce a single band

due to the formation of a homoduplex, while heterozy-

gous fragments will produce four bands due to the for-

mation of two homoduplexes and two heteroduplexes.

Since the melting behavior of the fragment depends onthe base-composition of the DNA fragment, the reso-

lution is usually very good with a single nucleotide

variation. Sequencing may be done for the further

characterization of the sequence variant. Therefore, this

method has the potential of detecting new mutations of

the b-globin gene.

Application of DGGE to dried blood spots has been

successfully reported in cystic fibrosis [74], indicatingthat this approach would also be applicable for follow-

up to hemoglobinopathy screening. This approach

would be labor-intensive and therefore costly if used for

routine confirmatory testing.

Manual and automated sequencing

The developments of DNA sequencing [75,76], PCR[37] and automated fluorescent DNA sequencing [77–80]

have revolutionized DNA analysis. Traditionally, DNA

sequencing was performed by either chemical cleavage

or dideoxy chain termination. Both methods had similar

popularity initially, but through the course of time, the

chain termination approach became more widely used.

Chain termination sequencing is based on the syn-

thesis of single stranded DNA by means of a single la-beled primer. Four different PCR reactions are

performed, all with the single primer and the four de-

oxynucleoside triphosphates (dNTPs), and each indi-

vidual reaction with the 20; 30 dideoxy analog of one of

the four dNTPs. Since the dideoxy nucleoside lacks the

3-hydroxyl necessary for further extension of the primer,

the incorporation of a molecule of the dideoxy analog


terminates further extension. Therefore, the reaction ineach tube is blocked by a specific analog generating a

series at various lengths due to incorporation of that

dideoxy nucleoside by the polymerase enzyme. These

four sets of fragments are then separated side-by-side on

a polyacrylamide gel, which is capable of separating the

fragments with the difference of a single nucleotide.

Depending on whether the primer is radioactively, flu-

orescently, or chemiluminiscently labeled, the sequenc-ing gel can be analyzed with the appropriate detector

and the DNA sequence is determined by the band

pattern.

An automated sequencer runs on the same principle

of chain termination. The dideoxy nucleosides are

labeled with four different fluorescent dyes and the four

reactions are performed in a single tube. In the original

systems, the reaction is analyzed on an acrylamide geland fluorescently labeled fragments are separated on the

basis of their sizes [78]. A laser beam at the bottom of

the gel excites the fluorophors and each labeled frag-

ment emits light at a distinct wavelength due to excita-

tion by laser beam. The light is detected and recorded,

and the sequence is reconstructed from the series of

colored fragments. Innovations to this technology con-

tinue to increase the throughput of this assay, includingsubstitution of capillary electrophoresis for the gel

separation [81,82].

We have demonstrated automated sequencing di-

rectly from newborn screening cards for the b-globingene to analyze b-thalassemia as well as non-deletional

HPFH (Bhardwaj et al., unpublished data). These re-

sults are very promising with regard to identification of

unknown or unusual mutations in follow-up to newbornscreening for hemoglobinopathies.

Real-time PCR

Real-time PCR with fluorometry is an amplification-

based technology, which detects point mutations in the

b-globin gene as well as in other genes. This technology

includes fluorescence monitoring of the amplificationreaction to quantitate and/or characterize the PCR

product(s) without the need for post-PCR analysis (e.g.,

by restriction enzyme digestion, electrophoresis, etc.)

[83,84]. This technique detects mutations by differences

in the melting temperature of fluorescently labeled oli-

gonucleotide probes when hybridized to different am-

plified alleles. Two fluorescently labeled probes are used

in the system, one of which, ‘‘the anchor probe,’’ is la-beled at the 50 end and lies in close proximity to the

mutation, while the other ‘‘sensor’’ probe is placed over

the mutation at a distance of 2–5 nucleotides from the

anchor probe [85]. The probes are designed to have

different Tms, such that the sensor probe will have the

lower Tm. As the temperature increases, the fluores-

cence emission is monitored. Since the sensor probe has

a known Tm, in the event of a single base mismatch,there is an alteration of Tm by 5–10 �C, thereby allowingeasy discrimination of the mutant from the wild type

allele. The use of two different probes allows the detec-

tion of two alleles in a single reaction. This technique

has been successfully applied to common variants of

hemoglobin and b-thalassemia [85–87]. Therefore, it

may be applicable for newborn screening programs,

although it might not be cost-effective for screening.

Oligonucleotide microarray analysis

Microarray based hybridization is an emerging

technology for detection of single nucleotide polymor-

phisms of target DNA and mutations [88,89]. This

technology is based on the principle of hybridization

with immobilized oligonucleotide probes as in a reversedot blot. Unlike the traditional reverse dot blot, an ar-

ray consists of hundreds to thousands of oligonucleo-

tides immobilized on a solid surface such as glass slides,

polypropylene sheets or gel pads [90–92]. Therefore, the

DNA chip is capable of identifying many mutations/

polymorphisms simultaneously. Oligonucleotide arrays

can be manufactured using two different approaches,

one with the oligonucleotide solution deposited onto thesurface by either spotting techniques or by microfabri-

cated ink jet pumps. The other method involves the

synthesis of oligonucleotides directly on the chip sur-

face. To enhance the specificity of hybridization for an

array testing a single nucleotide substitution, each target

is allowed to hybridize to a set of overlapping oligonu-

cleotide probes that contain the mismatch [93]. The

target DNA is prepared by nested PCR to produce asingle stranded fragment labeled with fluorescent dyes or

haptens on either end [94]. In order to get efficient dif-

fusion of the target, single stranded DNA is randomly

fragmented to minimize inter- and intramolecular

structures. After the hybridization of the labeled target

DNA on the microchip, the fluorescent pattern is

monitored on an epifluorescent microscope equipped

with a charged couple device (CCD) [95]. The imagefrom the microchip is analyzed and processed digitally.

In order to achieve a specificity approaching 100%

and to minimize non-specific signal, several modifica-

tions are being done to improve this technique. With

these modifications, there are successful reports of mu-

tation analysis in many diseases, including the hemo-

globinopathies [96]. Gemignani et al. [97] have reported

reliable detection of the 17 b-thalassemia and glucose6-phosphate dehydrogenase mutations commonly found

in a Mediterranean population. Their ‘‘Thalassochip’’

was based on primer extension along with the allele-

specific primed extension. The arrayed primer extension

approach consists of a sequencing reaction that is initi-

ated by an oligonucleotide attached to the solid support,

in this case a glass surface [98,99]. The reaction is


terminated before the mutation site, which is extended bythe addition of fluorescently labeled dideoxynucleoside

triphosphate complementary to the mutant nucleotide.

The results are interpreted by reading the incorporated

fluorescent base in the target sequence. In another tech-

nique, the oligonucleotide is allele-specific and can

extend only when it perfectly matches the target.

Although, these technologies can prove to be pow-

erful tools to detect particular sequence variants forhemoglobinopathies, including the thalassemias, they

are not yet 100% sensitive or specific (see Fig. 1).

Summary and conclusions

The development of PCR and sequencing has revo-

lutionarized the molecular diagnosis of genetic disor-ders. Although some of the recently developed

techniques have proven to be robust with high

throughput, they are not yet sufficiently sensitive or

Fig. 1. Diagrammatic representation of hemoglobin indicating a- and b-globithalassemia and hereditary persistence of fetal hemoglobin (HPFH) deletion

dicated as: initiation codon mutation, frameshift, splice site, RNA c

mutations of a- and b-globin genes.

specific to replace existing conventional PCR basedtechniques, such as dot blot, ARMS, etc. No single

method has the capability to detect each and every

mutation, yet the combination of two or more may be

sufficient to give a reliable diagnosis.

For newborn screening, there is a need for a tech-

nique, which is simple, accurate, labor-efficient, and

cost-effective, and can utilize the original screening

sample (dried blood spot). In our experience, reverse dotblot and multiplexed-ARMS PCR can be candidate

techniques for detecting the known point mutations

causing hemoglobinopathies (such as Hb S, C, and E),

b-thalassemia, a-thalassemia, and non-deletional

HPFH. Occasionally, sequencing is also required for the

rare or uncharacterized mutations. Large deletions of

the a- and b-globin gene can be identified by gap-PCR

directly from dried blood spots. However, unknowndeletions may require extensive analysis by traditional

Southern blotting. Microarray approaches promise

improved high-throughput analysis for the future.

n genes, with approximate location of various types of a-, b-, db-, ecdb-s indicated by solid lines while non-deletional point mutations are in-

leavage, cap-site, promoter region, unstable globin, non-sense


References

[1] R. Guthrie, A. Susi, A simple method for detecting phenyketon-

uria in large populations of newborn infants, Pediatrics 32 (1963)

338–343.

[2] President�s commission for the study of ethical problems in

medicine and biomedical research. Screening and counseling for

genetic conditions: a report on the ethical, social and legal

implications of genetic screening, counseling and education

programs. Washington, DC: US Government Printing Office,

1983.

[3] R. Guthrie, Organization of a regional newborn screening

laboratory, in: H. Bickel, R. Guthrie, G. Hemmersen (Eds.),

National Screening for Inborn Errors of Metabolism, Springer,

Berlin, 1980, pp. 259–270.

[4] L.L. McCabe, B.L. Therrell Jr., E.R. McCabe, Newborn screen-

ing: rationale for a comprehensive, fully integrated public health

system, Mol. Genet. Metab. 77 (2002) 267–273.

[5] M.J. Khoury, L.L. McCabe, E.R. McCabe, Population screening

in the age of genomic medicine, N. Engl. J. Med. 348 (2003) 50–58.

[6] GAO, United States General Accounting Office. Newborn

screening, characteristics of state programs, March 2003.

[7] Newborn Screening Task Force. Serving the family from birth to

the medical home. Newborn screening: a blueprint for the future—

a call for a national agenda on state newborn screening programs.

Pediatrics 106S (2000) 386–427.

[8] Newborn Screening Committee, The Council of Regional Net-

works for Genetic Services (CORN), National Newborn Screen-

ing Report-1995, CORN, Atlanta, Sept 1999.

[9] M.H. Gaston, J.I. Verger, G. Woods, et al., Prophylaxis with oral

penicillin in children with sickle cell anemia: a randomized trial,

N. Eng. J. Med. 314 (1986) 1593–1599.

[10] D.L. Wethers, Panel, Newborn screening for sickle cell disease and

other hemoglobinopathies, National Institutes of Health Consen-

sus Development Conference Statement 6 (1987) 1–22.

[11] D. Zeuner, A.D. Ades, J. Karnon, J. Brown, C. Dezaterux, E.N.

Anionwu, Antenatal and neonatal hemblobinopathies screening in

the UK: review and economic analysis, Health Technol. Assess 3

(1999).

[12] F. Lorey, G. Cunningham, E.P. Vichinsky, B.H. Lubin, H.E.

Witkowska, A. Matsunaga, M. Azimi, J. Sherwin, J. Eastman, F.

Farina, J.S. Waye, D.H.K. Chui, Universal Newborn Screening

for Hb H disease in California, Genet. Test. 5 (2001) 93–100.

[13] International committee for standardization in hematology. Rec-

ommendations for neonatal screening for hemoglobinopathies,

Clin. Lab. Hematol. 10 (1988) 335–345.

[14] C.S. Chapman, Neonatal screening for hemoglobinopathies, Clin.

Lab. Hematol. 21 (1999) 229–234.

[15] J. Lafferty, College of American Pathologists Hemoglobinopathy

Survey HG-B, College of American Pathologists, Chicago, IL,

1999.

[16] G.M. Clarke, T.N. Higgins, Laboratory investigation of hemo-

globinopathies and thalassemia: review and update, Clin. Chem.

46 (2000) 1284–1290.

[17] F. Galacteros, K. Kleman, J. Caburi-Martin, J. Rosa, B. Lubin,

Cord blood screening for hemoglobin abnormalities by thin layer

isoelectric focusing, Blood 56 (1980) 1068–1078.

[18] M. Campbell, J.S. Henthorn, S.C. Davies, Evaluation of cation-

exchange HPLC compared with isoelectric focusing for neonatal

hemoglobinopathy screening, Clin. Chem. 45 (1999) 969–975.

[19] Sickle Cell Disease Guideline Panel. Sickle cell disease: screening,

diagnosis, management and counseling in newborns and infants

(Clinical Practice Guideline 6) Rockville MD: US Department of

Health and Human Sciences, 1993.

[20] T. Kinney, M. Sawtschenko, M. Whorton, J. Shearin, C. Stine, L.

Hofman, R. Safko, T. Vitaglione, R.E. Kaufman, Technique,

comparison and report of North Carolina experience, Pediatrics

83 (Suppl.) (1989) 843–848.

[21] S.J. Henderson, K. Fishlock, M.E. Horn, L. Oni, A.J. Bellingham,

Neonatal screening for the hemoglobin variants using filter-paper

dried blood specimens, Clin. Lab. Hematol. 13 (1991)

327–334.

[22] K.M. Kleman, E. Vichinski, B.H. Lubin, Experience with

newborn screening using isoelectric focusing, Pediatrics 83

(Suppl.) (1989) 852–854.

[23] F.P.L. Van den Jijs, G.A. Van den Verg, J.G. Schermer, F.D.

Muskiet, H. Landman, F.A.J. Muskiet, Screening of cord blood

for hemoglobinopathies and thalassemia by HPLC, Clin. Chem.

38 (1992) 1864–1869.

[24] M. Wilborn, B.L. Therrell, E.R.B. McCabe, Newborn screening

for sickle cell disease in transfused infants (Abstract) National

Newborn Screening Meeting. June 7–11, Seattle, Washington,

1994.

[25] W. Reed, P.A. Lane, F. Lorey, J. Bojanowski, M. Glass, R.R.

Louie, B.H. Lubin, E.P. Vichinsky, Sickle cell disease not

identified by newborn screening because of prior transfusion, J.

Pediatr. 136 (2000) 145–146.

[26] B.A. Frost, A.J. Bellingham, Neonatal hemoglobinopathy screen-

ing, Acta Hematol. 78 (1987) 142–143.

[27] British Committee for Standards in Haematology, The laboratory

diagnosis of hemoglobinopathies, Br. J. Hematol. 101 (1998) 783–

792.

[28] E.R.B. McCabe, S.-Z. Huang, W.K. Seltzer, M.L. Law, DNA

microextraction from dried blood spots on the filter paper

blotters: potential applications to newborn screening, Hum.

Genet. 75 (1987) 213–216.

[29] Y.-H. Zhang, L. McCabe, M. Wilborn, B.L. Therrell, E.R.B.

McCabe, Application of molecular genetics in public health:

improved follow-up in a neonatal hemoglobinopathy screening

program, Biochem. Med. Metab. Biol. 52 (1994) 27–35.

[30] D.C. Jinks, M. Minter, D.A. Tarver, M. Vanderford, J.F.

Hejtmancik, E.R. McCabe, Molecular genetic diagnosis of sickle

cell disease using dried blood specimens on blotters used for

newborn screening, Hum. Genet. 81 (1989) 363–366.

[31] E.R.B. McCabe, Utility of PCR for DNA analysis from dried

blood spots on filter paper blotters, PCR Methods Appl. 1 (1991)

99–106.

[32] M. Descartes, Y. Huang, Y.-H. Zhang, L. McCabe, R. Gibbs,

B.L. Therrell, E.R.B. McCabe, Genotypic confirmation from

original dried blood specimens in a neonatal hemoglobinopathy

screening program, Ped. Res. 31 (1991) 217–221.

[33] M. Angastiniotis, B. Modell, Global epidemiology of hemoglobin

disorders, Ann. NY. Acad. Sci. 850 (1998) 251–259.

[34] D.J. Weatherall, J.B. Clegg (Eds.), The Thalassemia Syndromes,

fourth ed., Blackwell Science, Oxford, 2001, pp. 237–286.

[35] T.H.J. Huisman, M.F.H. Carver, E. Baysal, A Syllabus of

Thalassemia Mutations 1–309, The Sickle Cell Anemia Founda-

tion, Augusta, Georgia, 1997.

[36] M. Pirastu, M.S. Ristaldi, A. Cao, Prenatal diagnosis of

b-thalassemia based on restriction endonuclease analysis of

amplified fetal DNA, J. Med. Genet. 26 (1989) 363–367.

[37] R.K. Saiki, S. Scharf, F. Faloona, K.B. Mullis, G.T. Horn, H.A.

Erlich, N. Arnheim, Enzymatic amplification of beta-globin

genomic sequences and restriction site analysis for diagnosis of

sickle cell anemia, Science 230 (1985) 1350–1354.

[38] A. Haliassos, J.C. Chomel, L. Tesson, M. Baudis, J. Kruh, J.C.

Kaplan, A. Kitzis, Modification of the enzymatically amplified

DNA for the detection of point mutations, Nucleic Acids Res. 17

(1989) 3606.

[39] E.J. Sorscher, Z. Huang, Diagnosis of genetic disease by primer-

specified restriction map modification, with application to cystic

fibrosis and retinitis pigmentosa, Lancet 337 (1991) 1115–1118.


[40] J.-G. Chang, P.-H. Chen, S.-S. Chiou, L.-S. Lee, L.-I. Perng, I.T.-

C. Liu, Rapid diagnosis of b-thalassemia mutations in Chinese by

naturally and amplified created restriction sites, Blood 8 (1992)

2092–2096.

[41] R. Lindeman, S.P. Hu, F. Valpato, R.J. Trent, Polymerase chain

reaction mutagenesis enabling rapid non-radioactive detection of

common b-thalassemia in Mediterraneans, Br. J. Hematol. 78

(1991) 100–104.

[42] X.-M. Xu, W.-F. Ma, L.-L. Song, Q. Xu, J.-Z. Zhang, Direct

genotyping and prenatal diagnosis b-thalassemia in Chinese by

polymerase chain reaction mediated restriction fragment length

polymorphism method, Clin. Biochem 26 (1993) 497–503.

[43] M.S. Ristaldi, M. Pirastu, C. Rosatelli, G. Monni, H. Erlich, R.

Saiki, A. Cao, Prenatal diagnosis of beta-thalassemia in Mediter-

ranean populations by dot blot analysis with DNA amplification

and allele specific oligonucleotide probes, Prenat. Diagn. 9 (1989)

629–638.

[44] D. Sylvester-Jackson, S.L. Page, J.M. White, H.M. McCabe, Y.-

H. Zhang, B.L. Therrell, E.R.B. McCabe, Unbiased analysis of

the frequency of b-thalassemia point mutations in a population of

African–American newborns, Arch. Path. Lab. Med. 117 (1993)

1110–1114.

[45] S.L. Thein, P. Winichagoon, C. Hesketh, S. Best, S. Fucharoen, P.

Wasi, D.J. Weatherall, The molecular basis of b-thalassemia in

Thailand: application to prenatal diagnosis, Am. J. Hum. Genet.

47 (1990) 369–375.

[46] S.-Z. Huang, X.-D. Zhou, H. Zhu, Z.-R. Ren, Y.-T. Zeng,

Detection of b-thalassemia mutations in the Chinese using

amplified DNA from dried blood specimens, Hum. Genet. 84

(1990) 129–131.

[47] R.K. Saiki, P.S. Walsh, C.H. Levenson, H.A. Erlich, Genetic

analysis of amplified DNA with immobilized sequence-specific

oligonucleotide probes, Proc. Natl. Acad. Sci. USA 86 (1989)

6230–6234.

[48] X. Xu, C. Liao, Z. Liu, Y. Huang, J. Zhang, J. Li, Z. Peng, L. Qiu,

Q. Xu, Antenatal screening and fetal diagnosis of b-thalassemia in

a Chinese population: prevalence of the b-thalassemia trait in the

Guangzhou area of China, Hum. Genet. 98 (1996) 199–202.

[49] A. Maggio, A. Giambona, S.P. Cai, J. Wall, Y.W. Kan, F.F.

Chehab, Rapid and simultaneous typing of hemoglobin S,

hemoglobin C and seven Mediterranean beta-thalassemia muta-

tions by covalent reverse dot-blot analysis: application to prenatal

diagnosis in Sicily, Blood 81 (1993) 239–242.

[50] P. Sutcharitchan, R. Saiki, T.H. Huisman, A. Kutlar, V. McKie,

H. Erlich, S.H. Embury, Reverse dot-blot detection of the

African–American beta-thalassemia mutations, Blood 86 (1995)

1580–1585.

[51] V. Chan, I. Yam, F.E. Chan, T.K. Chan, A reverse dot blot

method for rapid detection of non-deletional a-thalassemia, Br. J.

Hematol. 104 (1999) 513–515.

[52] C.R. Newton, A. Graham, L.E. Heptinstall, S.J. Powell, C.

Summers, Kalsheker, J.C. Smith, A.F. Markham, Analysis of any

point mutation in DNA. The amplification refractory mutation

system (ARMS), Nucleic Acids Res. 17 (1989) 2503–2516.

[53] J.M. Old, N.Y. Varawalla, D.J. Weatherall, Rapid detection and

prenatal diagnosis of b-thalassemia: studies in Indian and Cypriot

population in the UK, Lancet 336 (1990) 834–837.

[54] N.Y. Varawalla, J.M. Old, R. Sarkar, R. Venkatesan, D.J.

Weatherall, The spectrum of b-thalassemia mutations on the

Indian subcontinent: the basis of prenatal diagnosis, Br. J.

Hematol. 78 (1991) 242–247.

[55] J.A. Tan, J.S. Tay, L.I. Lin, S.K.Kham, J.N.Chia, T.M.Chin,N.B.

Aziz, H.B. Wong, The amplification refractory mutation system

(ARMS); a rapid and direct prenatal diagnostic technique for beta-

thalassemia in Singapore, Prenat. Diagn. 14 (1994) 1077–1082.

[56] P. Fortina, G. Dotti, R. Conant, G. Monokian, T. Parella, W.

Hitchcock, E. Rappaport, E. Schwartz, S. Surrey, Detection of

most common mutations causing b-thalassemia in Mediterraneans

using a multiplex amplification refractory mutation system

(MARMS), PCR Methods Appl. 2 (1992) 163–166.

[57] S. Ahmed, M. Saleem, N. Sultana, Y. Raashid, A. Waqar, M.

Anwar, B. Modell, K.A. Karamat, M. Petrou, Prenatal diagnosis

of beta-thalassemia in Pakistan: experience in a Muslim country,

Prenat. Diagn. 20 (2000) 378–383.

[58] J.G. Chang, H.J. Liu, H.M. Huang, T.Y. Yang, C.P. Chang,

Multiplex mutagenically separated PCR: diagnosis of b-Thalas-semia and hemoglobin variants, Biotechniques 22 (1997) 520–527.

[59] K.L. Tan, J.A. Tan, Y.C. Wong, Y.C. Wee, M.K. Thong, S.F.

Yap, Combine- ARMS: A rapid and cost effective protocol for

molecular characterization of b-Thalassemia in Malaysia, Genet.

Test. 5 (2001) 17–22.

[60] J.S. Wayne, B. Eng, J.A. Hunt, D.H. Chui, Filipino beta-

thalassemia due to a large deletion: identification of the deletion

endpoints and polymerase chain reaction (PCR)-based diagnosis,

Hum. Genet. 94 (1994) 530–532.

[61] D.K. Bowden, M.A. Vickers, D.R. Higgs, A PCR-based strategy

to detect the common severe determinants of alpha thalassaemia,

Br. J. Haematol. 81 (1992) 104–108.

[62] J.E. Craig, R.A. Barnetson, J. Prior, J.L. Raven, S.L. Thein,

Rapid detection of deletions causing db thalassemia and hered-

itary persistence of fetal hemoglobin by enzymatic amplification,

Blood 83 (1994) 1673–1682.

[63] U. Bhardwaj, Y.-H. Zhang, W. Blackburn, L.L. McCabe, E.R.B.

McCabe, Rapid confirmation of Southeast Asian and Filipino

a-thalassemia genotypes from newborn screening specimens, Am.

J. Hematol. 71 (2002) 56–58.

[64] U. Bhardwaj, Y.-H. Zhang, D. Sylvester-Jackson, G.R. Bu-

chanan, B.L. Therrell, L.L. McCabe, E.R.B. McCabe, DNA

diagnosis confirms hemoglobin deletion in newborn screen follow-

up, J. Pediatr. 142 (2003) 346–348.

[65] M. Orita, H. Iwahana, H. Kanazawa, K. Hayashi, T. Sekiya,

Detection of polymorphisms of human DNA by gel electropho-

resis as single-strand conformation polymorphisms, Proc. Natl.

Acad. Sci. USA 86 (1989) 2766–2770.

[66] R.G.H. Cotton, Mutation Detection, Oxford University Press,

New York, 1997.

[67] M.C. Romey, P. Aguilar-Martinez, J. Demaille, M. Claustress,

Rapid detection of single nucleotide deletions: application to the

beta 6 (-A) mutation of the beta-globin gene and to cystic fibrosis,

Hum. Genet. 92 (1993) 627–628.

[68] A.J. Nataraj, I. Olivos-Glander, N. Kusukawa, W.E. Highsmith

Jr., Single-strand conformation polymorphism and heteroduplex

analysis for gel-based mutation detection, Electrophoresis 20

(1999) 1177–1185.

[69] R. Fodde,M.Losekoot,Mutation detection bydenaturing gradient

gel electrophoresis (DGGE), Hum. Mutat. 3 (1994) 83–94.

[70] U.C. Sheffield, D.R. Cox, L.S. Lerman, R.M. Myers, Attachment

of a 40-base-pair G+C-rich sequence (GC-clamp) to genomic

DNA fragment by the polymerase chain reaction results in

improved detection of single base change, Proc. Natl. Acad. Sci.

USA 86 (1989) 232–236.

[71] J. Vijg, N.J. van Orsouw, Two-dimensional gene scanning: explor-

ing human genetic variability, Electrophoresis 20 (1999) 1239–1249.

[72] M. Losekoot, R. Fodde, C.L. Harteveld, H. Van Heeren, P.C.

Giorano, L.F. Bernini, Denaturing gradient gel electrophoresis

and direct sequencing of the PCR amplified genomic DNA: a

rapid and reliable diagnostic approach to beta-thalassemia, Br. J.

Hematol. 76 (1990) 269–274.

[73] E. Gottardi, M. Losekoot, R. Fodde, G. Saglio, C. Camaschella,

L.F. Bernini, Rapid identification by denaturing gradient gel

electrophoresis of mutation in c-globin gene promoters in non-

deletion type of HPFH, Br. J. Hematol. 80 (1992) 533–538.

[74] M.P. Audrezet, B. Costes, N. Ghanem, P. Fanen, C. Verlingue,

J.F. Morin, B. Mercier, M. Goossens, C. Ferec, Screening for


cystic fibrosis in dried blood spots of newborns, Mol. Cell. Probes

7 (1993) 497–502.

[75] A.M. Maxam, W. Gilbert, A new method for sequencing DNA,

Proc. Natl. Acad. Sci. USA 74 (1977) 560–564.

[76] F. Sanger, S. Nicklen, A.R. Coulson, DNA sequencing with chain

terminating inhibitors, Proc. Natl. Acad. Sci. USA 74 (1977)

5463–5467.

[77] L.M. Smith, S. Fung, M.W. Hunkapiller, T.J. Hunkapiller, L.

Hood, The synthesis of oligonucleotides containing an aliphatic

amino group at the 50 terminus: synthesis of fluorescent DNA

primers for use in DNA sequence analysis, Nucleic Acids Res. 13

(1985) 2399–2412.

[78] L.M. Smith, J.Z. Sanders, R.J. Kaiser, P. Hughes, C. Dodd, C.R.

Connell, C. Einer, S.B. Kent, L.E. Hood, Fluorescence detection

in automated DNA sequence analysis, Nature 321 (1986) 674–679.

[79] L.E. Hood, M.W. Hankapiller, L.M. Smith, Automated DNA

sequencing and analysis of human genome, Genomics 1 (1987)

201–212.

[80] T. Hunkapiller, R.J. Kaiser, B.K. Koop, L. Hood, Large scale and

automated sequence determination, Science 254 (1991) 59–67.

[81] M.N. Albarghouthi, A.E. Barron, Polymeric matrices for DNA

sequenced by capillary electrophoresis, Electrophoresis 21 (2000)

4096–4111.

[82] H. Zhou, A.W. Miller, Z. Sosic, B. Buchholz, A.E. Barron, L.

Kotler, B.L. Karger, DNA sequencing up to 1300 bases in 2 h by

capillary electrophoresis with mixed replaceable linear polyacryl-

amide solutions, Anal. Chem. 72 (2000) 1045–1052.

[83] C.P. Ward, A.H. Fensom, P.H. Green, Biallelic discrimination

assays for the three common Ashkenazi Jewish mutation and a

common non-Jewish mutation, in Tay-sachs disease using Taq-

Man probes, Genet. Test. 4 (2000) 351–358.

[84] S. Ballerini, L. Bellincampi, S. Bernardini, S. Cascianis, C. Motti,

C. Cortese, G. Federici, Apolipoprotein E genotyping: a compar-

itive study between restriction endonuclease mapping and allelic

discrimination with the Lightcycler, Clin. Chem. Acta 317 (2002)

71–76.

[85] I. Moreno, P. Bolufer, M.L. Perez, E. Barragan, M.A. Sanz,

Rapid detection of major Mediterranean b-thalassemia mutation

by real-time polymerase chain reaction using fluorophore-labelled

hybridization probes, Br. J. Hematol. 119 (2002) 554–557.

[86] M.G. Herrmann, S.F. Dobrowolski, C.T. Wittwer, Rapid

b-globin genotyping by multiplexing probe melting temperature

and color, Clin. Chem. 46 (2000) 424–428.

[87] C. Vrettou, J. Traeger-Synodinos, M. Tzetis, G. Malamis, E.

Kanavakis, Rapid screening of multiple b-globin gene mutations

by real-time PCR on the Light cycler: application to carrier

screening and prenatal diagnosis of thalassemia syndromes, Clin.

Chem. 49 (2003) 769–776.

[88] G. Ramsay, DNA chips: state-of-the-art, Nat. Biotechnol. 16

(1998) 40–44.

[89] E.M. Southern, DNA chips: analysis sequence by hybridization

on a large scale, Trends Genet. 12 (1996) 110–115.

[90] E.M. Southern, U. Maskos, J.K. Elder, Analysis and comparing

nucleic acid sequencing by hybridization to arrays of oligonucle-

otides: evaluation using experimental models, Genomics 13 (1992)

1008–1017.

[91] S.P.A. Fodor, J.L. Read, M.C. Pirrung, L. Stryer, A.T. Lu, D.

Solas, Light directed spatially addressable parallel chemical

synthesis, Science 251 (1991) 767–773.

[92] K.L. Beattie, W.G. Beattie, L. Meng, S.L. Turner, R. Coral-

Vazquez, D.D. Smith, P.M. McIntyre, D.D. Dao, Advances in

genosensor research, Clin. Chem. 41 (1995) 700–706.

[93] J.G. Hacia, F.S. Collins, Mutational analysis using oligonucleo-

tide microarrays, J. Med. Genet. 36 (1999) 730–736.

[94] M.T. Cronin, R.V. Fucini, S.M. Kim, R.S. Masino, R.M. Waspi,

C.G. Miyada, Cystic fibrosis mutation detection by hybridization

to light generated DNA probe array, Hum. Mutat. 7 (1996)

244–255.

[95] D.D. Bowtell, Options available-from start to finish-for obtaining

expression data by micro array, Nat. Genet. Suppl. 21 (1999)

25–32.

[96] G. Yershov, V. Barsky, Y.A. Belgoveki, E. Kirillov, E. Kreindlin,

I. Ivanov, S. Parinov, D. Guschin, A. Drobishev, S. Dubiley, A.

Mirzabekov, DNA analysis and diagnostics on oligonucleotide

microchips, Proc. Natl. Acad. Sci. USA 93 (1996) 4913–4918.

[97] F. Gemignani, P. Chiara, S. Landi, F. Canzian, A. Kurg, N.

Tonisson, R. Galanello, A. Cao, A. Metspaulu, G. Romeo,

Reliable detection of b-thalassemia and G6PD mutations by a

DNA microarray, Clin. Chem. 48 (2002) 2051–2054.

[98] J.M. Shumaker, A. Metspalu, C.T. Caskey, Mutation detection

by solid phase primer extension, Hum. Mutat. 7 (1996)

346–354.

[99] T. Pastinen, M. Raitio, K. Lindroos, P. Tainola, L. Peltonen, A.C.

Syvanen, A system for specific, high-throughput genotyping by

allele-specific primer extension on microarrays, Genome Res. 10

(2000) 1031–1042.

Neonatal hemoglobinopathy screening: molecular genetic technologies

Documents

Transcript of Neonatal hemoglobinopathy screening: molecular genetic technologies