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HUMAN MUTATION 26(5), 399^412, 2005


Molecular Diagnosis of Inherited Disorders: Lessons From Hemoglobinopathies

George P . Patrinos,1 Panagoula Kollia,2,3 and Manoussos N. Papadakis4
Erasmus University Medical Center, Faculty of Medicine and Health Sciences, MGC-Department of Cell Biology and Genetics, Rotterdam, The Netherlands; 2Laboratory of Molecular Genetics and Cytogenetics, Larissa, Greece; 3University of Thessaly, School of Medicine, Department of Biology, Larissa, Greece; 4Laikon General Hospital, Center for Thalassemia, Unit of Prenatal Diagnosis, Athens, Greece Communicated by Richard G.H. Cotton Hemoglobinopathies constitute a major health problem worldwide, with a high carrier frequency, particularly in certain regions where malaria has been endemic. These disorders are characterized by a vast clinical and hematological phenotypic heterogeneity. Over 1,200 different genetic alterations that affect the DNA sequence of the human a-like (HBZ, HBA2, HBA1, and HBQ1) and b-like (HBE1, HBG2, HBG1, HBD, and HBB) globin genes are mainly responsible for the observed clinical heterogeneity. These mutations, together with detailed information about the resulting phenotype, are documented in the globin locus-specific HbVar database. Family studies and comprehensive hematological analyses provide useful insights for accurately diagnosing thalassemia at the DNA level. For this purpose, numerous techniques can provide accurate, rapid, and cost-effective identification of the underlying genetic defect in affected individuals. The aim of this article is to review the diverse methodological and technical platforms available for the molecular diagnosis of inherited disorders, using thalassemia and hemoglobinopathies as a model. This article also attempts to shed light on issues closely related to thalassemia diagnostics, such as prenatal and preimplantation genetic diagnoses and r 2005 genetic counseling, for better-quality disease management. Hum Mutat 26(5), 399412, 2005. Wiley-Liss, Inc.

thalassemia; hemoglobinopathies; molecular diagnostics; mutation analysis; prenatal diagnosis; HbVar globin locus-specific database; genetic counseling

INTRODUCTION The physiological status of every mammalian organism depends on the energy supply and continuous oxygen flow. Hemoglobin (Hb), the oxygen transport molecule, consists of two a-like and two b-like globin chains that are covalently linked to heme, the oxygen-binding group. In most animal species, different Hb molecules alternate during the different developmental stages, resulting from the exquisitely coordinated expression of the a -like and a-like globin genes (Fig. 1A). The human a-like globin gene cluster (HBAC) consists of four functional gene copies: HBZ (MIM] 142310), HBA2 (MIM] 141850), HBA1 (MIM] 141800), and HBQ1 (MIM] 142240). These gene copies encode the 141 amino acid z-, a2-, a1-, and y-globin chains, respectively. The HBAC is located on the short arm of human chromosome 16 and is under the regulatory control of the a-globin HS-40 enhancer [Higgs et al., 1990; Liebhaber et al., 1990]. The human b-like globin gene cluster (HBBC) consists of five functional gene copies: HBE1 (MIM] 142100), HBG2 (MIM] 142250), HBG1 (MIM] 142200), HBD (MIM] 142000), and HBB (MIM] 141900). These gene copies encode the 146 amino acid e-, Gg-, Ag-, d-, and b-globin chains, respectively (summarized in Fig. 1B). During development, proper expression of these genes is under the control of the b-globin locus control region (LCR) [Grosveld et al., 1987]. The two globin gene clusters became physically separated following the initial duplication of the ancestral globin gene, approximately 450 million years ago [Goodman et al., 1987]. Two gene copies of each cluster

(HBA2/HBA1 and the HBG2/HBG1) produce identical proteins as a result of gene duplication and conversion events [reviewed in Papadakis and Patrinos, 1999]. Two developmental switches in globin gene expression take place in the HBBC during ontogeny: the embryonic to fetal globin switch, coinciding with the transition from the primitive to definitive hematopoiesis, and the fetal to adult globin switch during the perinatal period. Both of these switches are controlled exclusively at the transcriptional level [Stamatoyannopoulos and Grosveld, 2001]. Recent experimental evidence suggests that b-globin LCR and the globin genes expressed at each developmental stage participate in multiple interactions to form a higherorder chromatin structure, termed the b-globin active chromatin hub (ACH) [Palstra et al., 2003, Patrinos et al., 2004a]. Similar interactions may also occur in the HBAC. Thalassemias and hemoglobinopathies comprise a heterogeneous group of disorders of Hb synthesis that are characterized by either a globin chain imbalance due to the reduced output or
Received 15 December 2004; accepted revised manuscript 16 May 2005. Correspondence to: George P. Patrinos, Erasmus University Medical Center, Faculty of Medicine and Health Sciences, MGC-Department of Cell Biology and Genetics, PO Box 1738, 3000 DR, Rotterdam,The Netherlands. E-mail: DOI 10.1002/humu.20225 Published online 1 September 2005 in Wiley InterScience (www.


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FIGURE 1. A: Structure of the dierent human Hb molecules through development.Together with heme, a-like and b-like globin chains are forming tetramers for oxygen transport in the tissues of the developing embryo. B: Schematic drawing of the human a- and b-globin multigene loci residing on chromosome 16 (16p13.3) and 11 (11p15.4), respectively. Each locus consists of functional genes arranged according to their order of developmental expression (see text for details), and pseudogenes (depicted in gray). a-Globin HS-40 enhancer and b-globin LCR are responsible for high-level expression and proper developmental control of the a-like and b-like globin genes, respectively. Each globin gene is relatively small in size, consisting of 3 exons (solid boxes) and 2 introns (white boxes), anked by 50 and 3 0 untranslated regions (UTRs, gray boxes). Codon numbers are indicated underneath. C: Summary of the various a- and b-thalassemia syndromes, categorized according to a- and b-globin gene expression and globin chain imbalance, respectively.

absence of one or more of the globin chains (in some hemoglobinopathies, globin chain imbalance is not observed), or a variant Hb fraction. These disorders result from mutations in the HBAC or HBBC. They are the most common single gene disorders in humans. About 7% of the world population carries a globin gene mutation, and in the vast majority of cases it is inherited as an autosomal recessive trait [Weatherall and Clegg, 2001]. To date, over 1,200 different mutant alleles have been characterized at the molecular level [Hardison et al., 2002], and each country has its own mutational spectrum of Hb variants and thalassemia mutations [Patrinos et al., 2004b]. Specific mutant alleles have reached high frequencies in certain regions, such as the Mediterranean basin, Africa, and the Middle East, due to malaria endemicity [Flint et al., 1998], and apparently have occurred in more than one mutational event.

The clinical manifestations of b-hemoglobinopathies are rather diverse and are divided into three categories: 1) life-threatening transfusion-dependent b-thalassemia major, resulting from the homozygous or compound heterozygous inheritance of HBB mutation(s); 2) b-thalassemia intermedia, with phenotypes ranging from severe anemia with hepatosplenomegaly to moderate hypochromic microcytic anemia; and 3) b-thalassemia minor or trait, referring to the asymptomatic heterozygous state. Classification can be done according to the clinical symptoms and the degree of severity, the globin chain that is synthesized at a reduced level, or even the mutation responsible for the defective globin chain synthesis. A similar classification system is applicable to a-hemoglobinopathies, ranging from the asymptomatic a-thalassemia trait to the severe Hb H disease and Hb Barts hydrops fetalis syndrome (Fig. 1C). While b -thalassemia symptoms become

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apparent only after birth, a-thalassemia affects all developmental stages. Therefore, a definitive diagnosis of the disease-causing mutation is of the utmost importance for disease management and genetic counseling. This article outlines the basic strategies that are currently available for diagnosing thalassemias and hemoglobinopathies, with a special focus on diagnosis at the DNA level (see http:// Finally, issues related to thalassemia diagnostics, such as prenatal and preimplantation genetic diagnosis and genetic counseling, are discussed. DIFFERENTIAL DIAGNOSIS OF HEMOGLOBINOPATHIES Although they are symptom-free, individuals with thalassemia trait are characterized by a specific hematological profile that can be indicative of their genotype. A number of comprehensive guidelines for laboratory diagnosis of hemoglobinopathies have been published [Thalassemia Working Party of the BCSH General Hematology Task Force, 1994; Working Party of the BCSH General Hematology Task Force, 1998; Old, 2003]; however, they are beyond the scope of this article and will not be discussed in detail. In principle, if an individuals iron status (metabolism) is normal, reduced red blood cell (RBC) indices (i.e., mean corpuscular Hb (MCHo28 pg) and mean corpuscular volume (MCVo81 fl) levels) are suggestive of thalassemia heterozygosity (Fig. 2). If these indices are accompanied by elevated Hb A2 levels, a b-thalassemia diagnosis, coexistent or not with a-thalassemia, is made. Reduced RBC indices accompanied by normal Hb A2 levels are indicative of a-thalassemia trait, coexistence of a- and dthalassemia traits, or coinheritance of d- and b-thalassemia alleles.

In the latter case, caution should be taken, particularly in the case of prenatal diagnosis, as this can easily lead to misdiagnosis of the parents and consequently of the embryos genotype. Also, reduced MCH and MCV values accompanied by elevated Hb F levels are strong indicators of db-thalassemia or compound heterozygosity of b-thalassemia and hereditary persistence of Hb F (HPFH), or coexistence of a-thalassemia with HPFH. Finally, even if the MCH and MCV values are normal, qualitative and quantitative Hb analyses are required in order to distinguish between a normal individual and a HPFH hetero- or homozygote (Hb F45%), or carrier of Hb S or any other structural Hb variant (Hb C, Hb E, etc.). HEMATOLOGICAL AND BIOCHEMICAL SCREENING Aside from neonatal screening, hematological and biochemical investigations of an individual with a suspected hemoglobinopathy can provide useful insights regarding the hematological phenotype. Routine biochemical investigations consist mainly of electrophoretic and/or chromatographic analyses of an individuals Hb fractions and globin chains. Measurement of Hematological Indices An individuals hematological profile consists of measurements of the RBC indices and includes Hb concentration, hematocrit, RBC number, MCH, MCV, and red cell distribution (RDW, an indicator of RBC size variation). Routinely, a blood film accompanies the RBC indices. Depending on the severity of the thalassemia condition, minimal or major decrements in most of the RBC indices are observed. Also, quantitation of the Hb fractions, using a variety of electrophoretic and chromatographic techniques, is an essential part of the hematological profile (see below). Iron deficiency alters RBC indices. If necessary, additional investigations

FIGURE 2. Synopsis of carrier screening for the dierent types of hemoglobinopathies. The primary screen is based mainly on RBC indices and Hb quantitation. Reduced RBC counts (MCHo28 pg, MCVo81 ) are strong indicators of a thalassemia disorder (see text for details), while additional studies are required to characterize a structural Hb variant.


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(e.g., Hb heat stability and sickling tests, oxygen dissociation studies, in vitro globin chain synthesis, etc.) can be performed to detect and/or better characterize a structural Hb variant. Electrophoretic Analytical Methods Electrophoresis has been the method of choice in traditional hematological laboratories for qualitative and quantitative analyses of the various Hb fractions [Marengo-Rowe, 1965, Schmidt, 1993]. Cellulose acetate electrophoresis at alkaline pH (8.28.6), and citrate agar or agarose gel electrophoresis at acid pH (6.06.2) allow the separation of the major Hbs (i.e., Hb A, Hb F, Hb S/D, Hb C/E/O-Arab) and a number of less common Hb variants. Because of its simplicity, cellulose acetate electrophoresis remains one of the most popular methods for Hb screening. However, in addition to being laborious, electrophoretic techniques have the disadvantage of poor precision and accuracy of Hb quantitation using densitometric scanning. Also, urea-triton gel electrophoresis allows the rapid analysis of very small amounts of Hb from hemolysates and permits the examination of globin chain composition and globin synthetic ratios [Alter et al., 1980]. In some cases, mutant globin chains can also be separated with this electrophoretic technique. Finally, isoelectric focusing (IEF) on agarose gels can be used to separate different Hb fractions and variants [Ferrari et al., 1984, Manca et al., 1986] and globin chains [Righetti et al., 1979]. IEF is frequently the first analytical test used for the diagnosis of Hb fractions (Fig. 3A). If a better resolution is required, polyacrylamide gels can be used instead. IEF allows the separation of Hb variants with isoelectric points that differ by as little as 0.02 pH units, and in combination with capillary electrophoresis (capillary IEF) has demonstrated improved resolution, detection sensitivity, and accurate quantitation [Hempe and Craver, 1994].

Chromatographic Analysis of Hbs and Globin Chains Chromatographic methods are also widely used for Hb quantitation and initial screening of Hb variants. Microcolumn chromatography is a satisfactory method for Hb A2 quantitation to pinpoint individuals with b-thalassemia trait [Efremov and Huisman, 1974]. However, the presence of Hb variants can alter the results, and therefore it cannot be considered as a general method for Hb quantitation. Consequently, cation-exchange highperformance liquid chromatography (CE-HPLC) has become the method of choice to quantify the various normal and abnormal Hb fractions [Rogers et al., 1985]. This is currently the general method used to measure Hb A2 and Hb F levels and detect several abnormal Hbs. This method tends to replace electrophoretic techniques for primary screening of Hbs of clinical significance, and to be at least an additional tool for the presumptive identification of Hb variants [Joutovsky et al., 2004]. Several automated apparatuses have been developed for high-throughput measurement [Ou and Rognerud, 1993]. In recent years, reversed-phase HPLC (RP-HPLC) of globin chains has become an important tool for the study of Hb abnormalities [Leone et al., 1985]. It has been used mostly to measure the g -globin chain ratios in various Hb disorders, but because of its high sensitivity this method may be also useful in the diagnosis of hemoglobinopathies and the detection and study of Hb variants (Fig. 3B)even those that are indistinguishable by the battery of electrophoretic tests. MOLECULAR DIAGNOSIS OF HEMOGLOBINOPATHIES Although a number of low- (i.e., o2030 samples/day), medium- (o100 samples/day), and high- (4200 samples/day)

FIGURE 3. A: Separation of Hb molecules [Hb A, Hb S, Hb F, and Hb Fac (i.e., the acetylated fraction of Hb F) by isoelectric focusing for prenatal diagnosis of b-thalassemia at week 20 of gestation. Lane 1: Normal embryo. Lane 2: Hb S homozygous sample. Lane 3: Hb S heterozygous sample. B: RP-HPLC chromatogram showing the separation of the various globin chains of a carrier for the structural variant Hb F-Lesvos.The variant globin chain is eluted before the normal a-globin chain (adapted from Papadakis et al. [1996], with permission of the publisher).

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throughput molecular diagnostic techniques are available for genetic testing of Hb disorders, DNA sequencing is still the ultimate method for definitively identifying unknown sequence alterations. However, it should always be coupled with additional mutation scanning methods (see below). As far as the globin genes are concerned, caution should be taken when HBA2/HBA1 and HBG2/HBG1 gene sequencing comes into question, owing to their high sequence homology [Michelson and Orkin, 1983, Patrinos et al., 2001]. However, the detection of known thalassemia mutations and globin chain variants using well-established protocols, and in populations with a preestablished globin gene mutation spectrum, does not necessarily require DNA sequencing, particularly for small molecular-diagnostic laboratories with a limited budget and a small-to-medium sample volume. DNA Sources In the vast majority of analyses for molecular diagnosis of thalassemias, DNA is extracted from peripheral blood leucocytes. For prenatal diagnosis, fetal DNA is mostly isolated from chorionic villus samples obtained at 912 weeks of gestation by transcervical or transabdominal aspiration during an ultrasound examination [Old et al., 1986]. Also, fetal DNA can be extracted from amniotic fluid cells. In recent years, noninvasive methods for collecting samples have emerged. The most attractive approach so far is to recover fetal cells from the maternal circulation [Cheung et al., 1996]. Alternatively, fetal DNA can be isolated from maternal plasma and serum [Lo et al., 1997, Chiu et al., 2002]. Although they are highly attractive, such noninvasive sampling methods are still under development and hence are not widely offered for globin gene mutation screening. Finally, if preimplantation genetic diagnosis (PGD) is required, polar bodies or blastomeres, at either the cleavage or blastocyst stage, are isolated from preimplantation embryos, and these cells are used for DNA extraction to perform the genetic diagnosis. Short History of Molecular Diagnostics for Thalassemias Although the concept of employing molecular diagnostics for hemoglobinopathies was established in 1949, when Linus Pauling and his colleagues [1949] introduced the term molecular disease in the medical vocabulary for sickle cell anemia (SCA), the first practical seeds were sown in the early days of recombinant DNA technology. cDNA cloning and sequencing revealed the primary globin gene sequence [Liebhaber et al., 1980, Fritsch et al., 1980, Efstratiadis et al., 1980]. This in turn provided a number of DNA sequences to use as probes, which allowed the analysis of genomic regions via Southern blotting and led to the concept and application of restriction fragment length polymorphism (RFLP) analysis to track a mutant allele for pre- and postnatal diagnosis [Little et al., 1980]. Kan and colleagues [1976] were the first to carry out prenatal diagnosis of a-thalassemia, using hybridization on DNA isolated from fetal fibroblasts. Later, Kan and Dozy [1978] implemented RFLP analysis to pinpoint SCA alleles of African descent. At that time, however, a significant methodological bottleneck had to be overcome. The disease-causing mutation could only be identified through the construction of a genomic DNA library from the affected individual, in order to first clone the mutated allele and then determine its nucleotide sequence. Many human globin gene mutations have been identified through such approaches [Busslinger et al., 1981; Treisman et al., 1983]. Orkin and his colleagues [1982] demonstrated that a number of sequence variations were linked

to specific b-globin gene mutations. These groups of RFLPs, termed haplotypes (both intergenic and intragenic), provided a first screening approach to determine which HBB gene is mutated. At the same time, a number of exploratory methods for identifying mutations in patients DNA were developed to provide a shortcut to DNA sequencing. The first methods involved mismatch detection in DNA/DNA or RNA/DNA heteroduplexes [Myers et al., 1985]. With these laborious and time-consuming approaches, a number of sequence variations were identified, which made possible the design of short synthetic oligonucleotides as allele-specific probes for genomic Southern blots. This experimental design was quickly implemented for the detection of HBB gene mutations [Orkin et al., 1983; Pirastu et al., 1983]. Methodology Overview: Symphony of aThousand With the advent of PCR, the battery of diagnostic tools for globin gene mutation screening was significantly enriched. As with many other genetic disorders, DNA amplification is coupled to a rich repertoire of methodologies (summarized in Table 1) for detecting known mutations or screening for unknown sequence alterations inside the human globin loci. The descriptions and underlying principles of these methods have been published elsewhere [Xiao and Oefner, 2001, Patrinos and Ansorge, 2005] and will not be detailed here, as they lie outside the scope of this article. Detection of known globin gene mutations. Restriction endonuclease analysis and allele-specific mutation detection or amplification are the most widely used techniques to detect known globin gene mutants. Enzymatic amplification combined with restriction enzyme analysis was among the first techniques reported for prenatal diagnosis of SCA [Saiki et al., 1985]. With this method, numerous globin gene mutant alleles can be easily screened using specific restriction endonucleases (Fig. 4A). In cases in which the mutation fails to create or abolish a restriction site, the latter can be artificially created by incorporating the necessary nucleotide change(s) in the amplification primer. Allele-specific amplification using the amplification refractory mutation system (ARMS) [Newton et al., 1989] is probably one of the most popular detection methods for known HBA2/HBA1 [Eng et al., 2001] and HBB gene point mutations [Old et al., 1990, Tan et al., 1994], since it has the advantage of being able to detect virtually all known sequence variations. False-negative results due to amplification failure can be easily monitored using an internal control of an irrelevant genomic region, while the single-tube assay allows for the simultaneous detection of both wild-type and mutant alleles [Ye et al., 2001]. Competitive oligonucleotide priming (COP) is also based on allele-specific amplification, but it differs from ARMS in the sense that in the former, mismatch prevents annealing rather than extension of the mismatched primer. Genotyping is achieved through differential labeling of the allele-specific competitive primers, and requires somewhat elaborate detection systems because the PCR product size is the same. The latter presumably has prevented wide applicability of this method, which has only been described for the detection of common b-thalassemia mutations [Athanassiadou et al., 1995]. Allele-specific mutation detection of amplified DNA offers an attractive alternative to ARMS and is based on hybridization of PCR products to allele-specific oligonucleotide probes (ASO). This method can be applied in two formats. The first is the forward ASO approach, whereby PCR products are immobilized on a membrane and hybridized to labeled ASO probes. The use of ASO probes actually predates PCR [Pirastu et al., 1983, Orkin et al.,


HUMAN MUTATION 26(5), 399^412, 2005 Summary of the Most Widely Used Molecular DiagnosticTechniques to Screen forThalassemia and Hemoglobinopathies Methodology

Globin Genes HBA1 , HBA2


Mutation type Deletions Point mutations Point mutations Point mutations Point mutations Deletions Point mutations Point mutations Deletions Deletions Point mutations Point mutations Point mutations/small frameshifts Point mutations Point mutations Point mutations/small frameshifts Point mutations/small frameshifts Point mutations/small frameshifts Point mutations Point mutations/small frameshifts Point mutations Deletions Deletions Point mutations/small frameshifts Deletions No mutations detected

References Chu et al. [2000] Eng et al. [2001] Traeger-Synodinos et al. [1993] Chan et al. [1999]; Foglietta et al. [2003] Harteveld et al. [1996]; Jorge et al. [2003] Ou-Yang et al. [2004] Lacerra et al. [2004] Harteveld et al. [1996]; Lacerra et al. [2004] Bowie et al. [1994]; Liu et al. [2000]; Chong et al. [2000];Tan et al. [2001] Sun et al. [2001] Chan et al. [2004] Old et al. [1990];Tan et al. [1994] Cai et al. [1994]; Ugozzoli et al. [1998]; Winichagoon et al. [1999]; Lappin et al. [2001] Athanassiadou et al. [1995] Takahashi-Fujii et al. [1994]; Gupta and Agarwal [2003] Losekoot et al. [1990]; Ghanem et al. [1992]; Vrettou et al. [1999] Shaji et al. [[2003] Colosimo et al. [2002];Wu et al. [2003b] Moreno et al. [2002];Vrettou et al. [2003] Gemignani et al. [2002]; Foglieni et al. 2004]; Chan et al. [2004] Papadakis et al. [1997] Craig et al. [1994] De Andrade et al. [2003] Gottardi et al. [1992]; Patrinos et al. [1995] Craig et al. [1994] Papachatzopoulou et al. (in press)

Southern blot ARMS ASO Reverse dot-blot SSCP DHPLC DGGE Gap^PCR Real-time PCR Microarrays


ARMS Reverse dot-blot Competitive oligopriming SSCP DGGE TTGE DHPLC Real-time PCR Microarrays



HBG1e, HBG2f HBE1g

The experimental protocols summarized herewith report general scanning methods for the entire genomic and regulatory regions of the human globin genes. However, it should be stated that there are many more dierent experimental conditions, which have been reported for globin gene mutation screening but only for individual mutants, which are not summarized in this table. a NM ^ 000558.3. b NM ^000517.3. c NM ^ 000518.4. d NM^ 000519.2. e NM^ 000184.2. f NM ^ 000184.2. g NM^ 005330.3.

1983], and because of its simplicity it has become one of the most widely adopted methods in hemoglobinopathy diagnostics, using either radioactive (Fig. 4B) [Saiki et al., 1986] or nonradioactive [Saiki et al., 1988] probes. PCR-ASO is most useful when large numbers of samples are being screened for a small number of mutant alleles (e.g., for point mutations in the HBA2/HBA1 genes [Traeger-Synodinos et al., 1993]). The second format is the reverse ASO approach, or reverse dot-blot, whereby ASO probes are immobilized on a membrane and hybridized to labeled PCR products. The latter can be considered the founding principle behind genotyping microarrays (see below). The reverse dot-blot is a widely used tool for routine screening of numerous mutant alleles in the HBA2/HBA1 [Chan et al., 1999, Foglietta et al., 2003] and HBB genes [Cai et al., 1994, Winichagoon et al., 1999]. Automated platforms for preparing the reverse dot-blot membranes (strips) have been reported that allow printing of large numbers of strips with higher-density arraying [Lappin et al., 2001] and hence commercialization of the entire process. Today there are a number of commercially available mutation detection assays for a- and b-thalassemia, such as the b-globin strip assay (22 mutations; ViennaLab, Vienna, Austria; www., the BeTha GeneTM 1 Kit for Mediterranean

b-thalassemia mutations [Ugozzoli et al., 1998], the BeThaTM Gene 2 Kit for Asian b-thalassemia mutations, and the Alpha Gene 1 Kit for the most common HBA2/HBA1 gene mutations (all in microtiter plate formats; Biorad Laboratories, Hercules, CA; and the b-hemoglobinopathy mutation assay (six b-globin variants and 42 b-thalassemia mutations; Roche Molecular Diagnostics, Pleasanton, CA; [Jarvis et al., 2004], which has yet to be offered commercially. Finally, real-time PCR has recently emerged for rapid genotyping in the HBAC [Sun et al., 2001] and HBBC [Moreno et al., 2002, Vrettou et al., 2003] without the need for post-PCR sample manipulation. The method is based on the use of fluorescently labeled hybridization probes that are specific for each mutation. Each probe yields a different melting curve, and genotyping is performed on the basis of a melting-curve analysis. This allows one to quickly assign hetero- or homozygosity for the wild-type and/or mutant allele, and at the same time monitor for falsepositive or negative results. Although it is expensive and difficult to standardize, the assay is very fast, simple, and highthroughput, and allows the reliable detection of several mutations simultaneously.

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Scanning methods for unknown globin gene sequence alterations. Denaturing gradient gel electrophoresis (DGGE) and single-stranded conformation polymorphism (SSCP) analysis are two of the most commonly used methods for screening for both known and unknown mutations in human globin loci. The hallmark of both techniques is their high discriminatory potential between the wild-type and different mutant alleles, since even a single base difference anywhere in the amplified DNA fragment

will theoretically yield a different electrophoretic pattern. A high mutation detection efficiency (i.e., 70100%) has been reported for both techniques, which makes them extremely advantageous for fast mutation screening. The small size of all globin genes, and the various optimizations steps required for SSCP , such as gel conditions and electrophoresis parameters, have established DGGE as the universal scanning method for unknown mutations for all human globin genes on a routine basis (Table 1; Fig. 5). For laboratories with a small sample volume, DGGE is a valuable tool for pre- and postnatal diagnosis of hemoglobinopathies. Alternatively, temporal temperature gel electrophoresis (TTGE), which relies on a temporal temperature gradient instead of the chemical gradient used in DGGE, has also been reported to be easier to perform and more reproducible (compared to DGGE) in the routine molecular diagnosis of HBB gene mutations [Shaji et al., 2003]. This technique circumvents the cumbersome gel casting with chemical denaturing gradient gels required for DGGE.

4. A: Identication of the Cd27 G 4T mutation (NM^ 000519.2:p.A28S), the most frequent mutation in the Hellenic population leading to d-thalassemia, using a restriction enzyme-based strategy of amplied DNA. The 522 bp band corresponds to the diagnostic fragment for the mutant allele (indicated with an asterisk). Lanes 1 and 5: Normal individuals. Lanes 2 and 4: Heterozygotes. Lane 3: Homozygote. C: Nondigested PCR product, M: uv174/HaeIII size marker. B: Identication of the IVS I-110 G4A mutation (NM^ 000518.4:c.202G4A) in couples at risk for thalassemia, using PCR-ASO. Samples B2, C1, and D1 are heterozygotes for the aforementioned mutation. FIGURE

5. Identication of the A c^117 G 4A mutation (NM^ 000184.2:c.^170G4A), which is responsible for the Greek type of nondeletional HPFH, using PCR/DGGE analysis. Lanes 2 and 5: Normal individuals. Lanes 1 and 4: Heterozygotes. Lane 3: Homozygote.The 40^70% denaturing gradient is depicted at the left. FIGURE


A: Schematic representation of the gap-PCR concept for rapid detection of known large deletions and/or genomic rearrangements.Two oligonucleotide primer pairs (Pr1/Pr2 and Pr1/Pr3) are designed to detect the wild-type allele and that with the deletion (depicted in gray), respectively. The deletion-specic primer set (Pr1/Pr3) is designed such that a PCR product (Del, usually smaller than the wt) is generated only in the presence of the deletion, and at the same time no amplication occurs at the wild-type allele, since the complementary sequence of Pr2 will be missing due to the deletion. Similarly, amplication in the wild-type allele from the wild-type-specic primer pair (Pr1/Pr2) yields a dierently-sized PCR product (wt); however, no amplication occurs with the deletion-specic primer set, because Pr3 is located far away from Pr1. A similar strategy is implemented for the detection of gross rearrangements. B: Identication of theTurkish type of inversion/deletion db-thalassemia by gap-PCR. For breakpoint A, lanes 1 and 4 correspond to wild-type individuals, and lanes 2 and 3 correspond to heterozygotes for the aforementioned deletion. Identication of breakpoint B in these samples with dierent primer sets (lanes 1 and 2) conrmed the presence of this deletion. M: uv174/HaeIII size marker. C: Identication of the 13.4 kb deletion, which is responsible for Sicilian db-thalassemia. Lane 1: Normal individual. Lanes 2 and 3: Heterozygous and homozygous sample for the deletion, respectively. M: jw174/HaeIII size marker.


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Although SSCP does not enjoy wide applicability in globin gene genetic screening, as in a variety of other genomic loci [Garinis et al., 2005], a number of SSCP-based mutation-screening strategies have been described for the HBA2/HBA1 [Harteveld et al., 1996, Jorge et al., 2003], HBB [Takahashi-Fujii et al., 1994, Gupta and Agarwal, 2003], and HBE1 genes (Papachatzopoulou et al., in press). However, careful adjustment of the experimental conditions is required in order to obtain reproducible results between different runs, particularly for the purposes of prenatal diagnosis. In recent years, denaturing HPLC (DHPLC) has been gradually adopted for use in several diagnostic laboratories because it provides a semiautomated, fast, and reliable alternative to DGGE. DHPLC uses an ion-pair chromatography separation principle, combined with precise control of the column temperature and optimized mobile phase gradient for separation of mutant DNA molecules [reviewed in Xiao and Oefner, 2001]. Different experimental protocols have been described for a- and bthalassemia (Table 1), showing 100% sensitivity and specificity in detecting point mutations or even large deletions, even in the case of the GC-rich and highly homologous HBA2 and HBA1 genes [Lacerra et al., 2004]. Moreover, no false-negative or falsepositive results have been reported. Considering the abovementioned advantages, together with the high initial investment costs for purchasing the DHPLC setup, this method seems appropriate for diagnostic laboratories that have large test volumes and are involved in routine carrier identification and mutation screening of thalassemia patients. It should be noted that ideally the above-mentioned methodologies should be coupled with DNA sequencing, for either the definitive identification of unknown globin gene sequence variations or the confirmation of inconclusive results, such as neutral gene variants, or ambiguous chromatograms and/or electrophoretic patterns. Rapid detection of large genomic rearrangements by gap-PCR. Most of the common a-thalassemia mutant alleles are due to deletions or gross rearrangements of large genomic fragments containing the genes and their regulatory sequences [reviewed in Weatherall and Clegg, 2001], and db-, gdb-, egdbthalassemia and deletional HPFH conditions have a similar molecular etiology. Gap-PCR provides an alternative to Southern blot analysis [Chu et al., 2000] for the routine detection of such mutants because it is a rapid, simple to use, and cost-effective approach. The rationale behind gap-PCR is the generation of wildtype and deletion-specific amplification products from two oligonucleotide primer pairs respectively, from which one primer is common (Fig. 6A). In addition to the above-mentioned advantages, the overall principle of gap-PCR allows easy monitoring of falsenegative results. Today, gap-PCR is the ultimate method for reliable detection of the common deletional a-thalassemia alleles [Liu et al., 2000, Chong et al., 2000, Tan et al., 2001]. In addition, Hb Lepore or several deletion mutants in the HBBC, which lead to dbthalassemia or deletional HPFH, can be easily detected using this technique (Fig. 6B and C) [Craig et al., 1994]. An alternative approach to gap-PCR that employs fluorescent PCR to identify quantitative differences in the amplification product, which are directly proportional to the initial amount of genomic copies in the DNA template, was recently proposed [De Andrade et al., 2003]. Although this technique is potentially useful for identifying unknown deletions, the requirement of a specialized setup makes it less applicable than gap-PCR. Precise mapping of the various HBBC deletions and recording their exact breakpoints in the HbVar database for Hb variants and thalassemia mutations are currently being performed (Ross C.

Hardison, unpublished results). This will eventually allow additional gap-PCR screening strategies to be designed and implemented for each deletional mutant separately. High-Throughput Globin Gene Mutation Screening Recent developments in automation and miniaturization technologies have created new standards and changed the philosophy of molecular diagnostics in the postgenomic era. DNA microarrays have become synonymous with high-throughput mutation detection and large-scale DNA sequencing [reviewed in Southern, 1996]. A practical system that allows high-throughput genotyping and mutation detection by employing allele-specific extension on oligonucleotide arrays has been reported [Shumaker et al., 1996]. This method, known as allele-specific arrayed primer extension (AS-APEX; see also Pastinen et al. [2000]), relies on the sequence-specific extension by reverse transcriptase of two immobilized allele-specific oligonucleotide primers (approximately 3040 bp in length) that differ at their 30 -nucleotide, defining the alleles. Based on this principle, a microarray consisting of allelespecific primers to detect the most common 15 nondeletional HBA2/HBA1 and 23 HBB gene mutations in southeast Asia was constructed [Chan et al., 2004], which eliminates the need for multiple reverse dot-blot analyses. A similar microarray, with the 10 HBB and seven glucose-6-phosphate dehydrogenase (G6PD) common gene mutations in the Mediterranean population, has also been constructed for b-thalassemia and G6PD deficiency mutation screening, respectively (Thalassochip microarray [Gemignani et al., 2002]). A different methodology, which resembles PCR-ASO, has also been employed to screen for mutations leading to b-thalassemia, in an array format [Foglieni et al., 2004). A microelectronic DNA chip was constructed that contained PCR-amplified fragments from a large number of b-thalassemia homozygous and compound heterozygous samples, using primer pairs, one of which was biotinylated at its 50 end [Santacroce et al., 2002]. Following denaturation, the biotinylated strand was electronically spotted to discrete sites on streptavidin-coated gel pad surfaces, and allelespecific dye-labeled oligonucleotide probes were used to detect wild-type and nine different mutants. For all of the array-based genotyping platforms, sensitivity and specificity of 90100% have been reported, which together with the low-cost reagents and short processing times strongly indicate a potential use for this technology in screening programs for large populations with a high incidence of different types of thalassemia. However, this technology is still used on an experimental basis and has not yet been implemented for routine globin gene mutation detection. Finally, pyrosequencing is also gaining momentum for resequencing of genomic regions [Ronaghi, 2001, and references therein]. Pyrosequencing has the potential advantages of flexibility, accuracy, and parallel processing, and can be easily automated. Pyrosequencing technology is relatively new, and has only recently been implemented to quantify the relative levels of normal and sickle HBB mRNA in patients [Wu et al., 2003a]. The technology is already time- and cost-competitive, compared to existing sequencing methods, and given the potential for further developments and improvements in chemistry, instrumentation, and integration with sample preparation, it could be a promising alternative for resequencing of the compact human globin gene loci. Selecting the Optimum Mutation Detection Strategy Before one selects a method to detect globin gene mutations in a diagnostic laboratory, several issues must be considered,

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as discussed briefly below. Although not every methodological platform is applicable in every laboratory involved in molecular testing for thalassemias, the golden rule is to use at least two different methods that are technically sound and yield unambiguous results, so that consistency and accuracy are ensured. Scientic and nancial issues. The expected test volume and the allocated budget are two equally important parameters that a laboratory providing molecular diagnostic services for thalassemias must consider. The choice of method may also depend on the nature of the molecular analysis the laboratory performs (i.e., prenatal and/or postnatal diagnosis, etc.). Most of the time, the number of tests performed is rather small, which dictates the adoption of a low- or medium-throughput methodology. In these cases, carrier screening or prenatal diagnosis of known mutations can be performed using reverse dot-blot or restriction enzyme analysis, which are reliable and cost-effective (e.g., less than 100h per sample for SCA mutation restriction enzyme-based detection). Scanning for unknown mutations in cases in which the underlying globin gene mutation is not found can be performed using, e.g., DGGE or DNA sequence analysis (400h and 600h for a full HBB gene scanning, respectively) or even DHPLC. Although the latter is significantly faster than DGGE analysis, it has the disadvantage of requiring a high initial investment. Automation, which helps to minimize analytical errors, is appropriate only for larger laboratories or reference centers that can handle large sample volumes. In such laboratories, the purchase of a sophisticated molecular diagnostic infrastructure, such as robotics for DNA preparation/amplification, automated DNA sequencers, or even microarray-related equipment, can be justified. Furthermore, the nature of the expected mutation itself (i.e., point mutation or deletion) can determine the optimum screening strategy. For example, a gap-PCR screening assay is recommended for use on a routine basis in a molecular diagnostic laboratory to identify the most frequent HBA2/HBA1 gene deletion mutants. Finally, prior knowledge of the spectrum and frequencies of the various globin gene mutations in the population to be tested greatly facilitates the choice of the mutation screening strategy. In several at-risk populations, the reported globin gene mutation spectrum is extremely heterogeneous. Relevant information is recorded in the HbVar database [Patrinos et al., 2004b] (see next paragraph) and the National Mutation Databases [Patrinos and Brookes, in press; Patrinos et al., 2005]. Legal, ethical, and administrative issues. Ideally, each laboratory should be quality accredited (by Clinical Laboratory Improvement Amendment (CLIA) approval or ISO17025 certification), e.g., to comply with competent genetic testing procedures and personnel qualifications. The adoption of home-brew diagnostic assays can be cost-effective, but on the other hand has the inherent danger that the laboratory may fail the accreditation process, as these tests cannot be easily standardized. The purchase of well-established globin gene screening assays is therefore highly recommended. Also, the full integration of all facets of the diagnostic laboratory (i.e., general administration, comprehensive electronic patient records, DNA banking, etc.) using custom-based enterprise resource planning (ERP) systems is becoming increasingly necessary for fruitful everyday operations. There are also several ethical issues pertaining to hemoglobinopathy genetic testing. Each thalassemia patient needs to understand why the test is being offered, as well as its implications for disease prevention and management, and the patients

psychosocial well-being (see below). Written informed consent should be obtained from the patient, parent, or guardian prior to the test, and strict confidentiality regarding the patients genetic and/or medical records and test results must always be ensured. HbVar Database for Hb Variants and Thalassemia Mutations In the late 1990s, Huisman et al. [1997, 1998] published two books that recorded information on Hb variants and thalassemias. These books are a rich source of information about not only the mutations, but also the methods used in detection and analysis, their biochemical properties, associated clinical effects, ethnic distribution, and other data. The sheer amount of information tabulated, and the continuous accumulation of new mutation data and their complexity dictated the construction of a globin-specific database as an up-to-date and accessible repository of this information, which has been done in three discrete stages. The first stage involved the conversion of these resources to an HTML format that could be freely accessed on the World Wide Web [Chui et al., 1998; Hardison et al., 1998]. During the second stage, much of the information contained in these syllabi was converted into an SRS database at the European Bioinformatics Institute (accessible at [Hardison et al., 2001]. Finally, in 2002, HbVar, the first true locus-specific database for the globin genes, was realized as a result of a multicenter academic initiative. This database (accessible at includes detailed information on pathology, hematology, clinical presentation, and laboratory findings (range of Hb levels, hematocrit, etc.) on most of the published and unpublished Hb variants and thalassemia mutations. A considerable amount of biochemical data on the variants are also recorded, including the techniques used to identify, isolate, and determine their structure, stability, function, and qualitative distribution in ethnic groups and geographic locations [Hardison et al., 2002]. These data can be easily accessed through summary listings or user-generated queries, which can be highly specific. HbVar is useful not only for the research community, geneticists, and physicians as an aid in diagnosis, but also to other interested individuals, such as patients and their parents, people involved in providing genetic services and counseling, pharmaceutical industries, etc. Since the official announcement of HbVar in 2002, users have requested information on the frequencies of thalassemia mutations in different populations, which significantly simplifies genetic testing. To provide the requisite information, the frequency spectrum in 48 countries or ethnic backgrounds for 121 HBB gene mutations has been extracted from the literature and incorporated into HbVar records. Therefore, a user can query not only for all of the b-thalassemia mutations found in a given population, but also to specify the frequency range in which they occur and hence to focus a search on common or rare alleles [Patrinos et al., 2004b]. Also, HbVar has been linked with the GALA database [Giardine et al., 2003], since for several fields of study the information in HbVar must be combined with the wealth of information about features of genomic DNA, such as gene structures, conservation among species, etc. HbVar is currently in its second stage of being upgraded to increase its capabilities. To keep pace with user requirements, an online repository for experimental protocols to detect the globin gene variation is being constructed (George P . Patrinos, unpublished results), while automated routines have been implemented to scan for single nucleotide polymorphisms (SNPs) across the


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globin gene loci. Also, HbVar is currently being updated with a- and b-thalassemia mutation frequency data for more at-risk populations.

PRENATAL AND PREIMPLANTATION GENETIC DIAGNOSIS OF THALASSEMIA Prenatal and preimplantation genetic diagnoses of hemoglobinopathies are two sides of the same coin, which are both aimed at reducing the incidence of thalassemia and the number of affected individuals in at-risk populations. Prenatal diagnosis became possible in the early 1970 s, and in most European countries prenatal diagnosis for couples at risk for Hb disorders is available, with the option of selective abortion [reviewed in Petrou and Modell, 1995]. However, it is widely accepted that the latter cannot be considered an optimal or easy solution. Chorionic villus sampling under ultrasound guidance in the first trimester of pregnancy (1012 weeks of gestation) usually provides sufficient amounts of DNA for further analysis, with a relatively low risk of procedure-related pregnancy loss (0.6% [Seeds, 2004]). Virtually all of the DNA diagnostic methods reported for thalassemia diagnosis are suitable for prenatal diagnosis. It is highly recommended, however, that fetal DNA analysis be performed by two independent DNA diagnostic techniques that yield unambiguous results before a diagnosis is made. Maternal contamination of the fetal tissue is the most common problem, which can lead to false-positive or (even worse) false-negative results. Analysis of fetal polymorphic markers, such as microsatellites, short tandem repeats (STRs), etc., are always used as a control for contamination and are evaluated together with the respective maternal and paternal patterns. PGD is a very early form of prenatal diagnosis in which oocytes or preimplantation embryos are genetically analyzed, so that only those that are judged to be free of the genetic defect under consideration are transferred. Detection of mutations in the HBB and HBA2/HBA1 genes, leading to SCA and b- and athalassemia, has received considerable attention from the PGD community because of their high frequency in certain populations. The strategies developed for thalassemia PGD ideally aim to detect as many different mutations as possible with the same assay, because of the variety of HBB gene mutations. However, only a handful of protocols reported so far meet this goal, using either DGGE analysis [Vrettou et al., 1999] or reverse dot-blot to screen for b-thalassemia [Jiao et al., 2003]. Several other protocols employ restriction enzyme digestion to screen for SCA [Xu et al., 1999] and b-thalassemia [Kuliev et al., 1999; De Rycke et al., 2001], SSCP analysis for PGD of b-thalassemia [El-Hashemite et al., 1997], and a single fluorescent gap-PCR protocol for PGD of a-thalassemia [Piyamongkol et al., 2001].

health professionals as far as prenatal diagnosis and, eventually, termination of pregnancy are concerned. Nevertheless, under certain conditions the couple can be reassured that the phenotype of their affected child will be mild, such as 1) when the parents are silent b-globin gene mutation (i.e., the b1101 C4T mutation (NM000518.4:c.151 C4T)) carriers, resulting in very mild clinical phenotypes in the homozygous state; 2) when mild and severe b -thalassemia mutations are likely to be interacting in the compound heterozygous state; or 3) when b-thalassemia is coinherited with a-globin gene triplication (aaa) or a HPFH condition. Prenatal diagnosis is not offered for such cases [Petrou, 2005, and references therein]. Counseling for couples that are at risk for SCA is often perceived as a relatively simple procedure, but in fact it can be significantly more complex because of variations in the severity of SCA, which can range from very mild to very severe [reviewed in Serjeant, 1993]. However, counseling for couples at risk for a-thalassemia is more straightforward because of the usually hopeless prognosis for an affected fetus and the likelihood of lifethreatening obstetric risks for the mother [Petrou et al., 1992].

CONCLUSIONS With existing mutation detection technologies, most of which rely on DNA amplification, almost every known sequence variation that leads to an Hb disorder can be identified. However, apart from the disease-specific investigations used as primary screening tools, very few approaches can distinguish hemoglobinopathies from the vast majority of other inherited disorders for which the same spectrum of techniques has been applied (with generally the same advantages and disadvantages). In the foreseeable future, integration of sample preparation and mutation analysis, as well as higher-throughput approaches, will be necessary to meet the anticipated high demand for thalassemia screening programs. Recent developments in automated high-throughput mutation detection technologies, and the fast pace of discoveries of new globin gene sequence variations dictates the design and implementation of a general screening method with the capacity to identify every globin gene mutation and/or genomic rearrangement possible in both human globin gene clusters and their flanking regions. The construction of globin gene microarrays for resequencing or comparative genomic hybridization (i.e., in the format of short (500600 bp) PCR-amplified fragments or oligonucleotides) could be the solution. Currently, very few genes can be screened with such an approach, which requires an expensive and highly sophisticated hybridization and signal detection infrastructure. Affymetrix (Santa Clara, CA; www. has developed the p53 GeneChips for resequencing purposes, and together with NimbleGen Systems (Madison, WI; offers solutions for both constructing and analyzing custom-made resequencing arrays. The small size of the human globin genes compared to others (i.e., p53 or CFTR genes) can be extremely advantageous and may potentially allow for the incorporation of the entire HBAC and/or HBBC genomic sequences onto one single array.

GENETIC COUNSELING FOR HEMOGLOBINOPATHIES Genetic counseling is very important in view of the phenotypic diversity of thalassemia. Generally, the inheritance of two b-globin gene mutations results in a blood transfusion-dependent thalassemia. However, there are mild b-thalassemia mutations that result in thalassemia intermedia. For instance, when both parents carry the mild b188 C4T mutation (NM_000518.4:c.138C4T, using the cDNA numbering designation), the homozygous state generally results in a very mild clinical phenotype. This type of mild thalassemia poses an ethical dilemma to both parents and

ACKNOWLEDGMENT We are indebted to Dr. Claire Wyman for critically reading this manuscript and offering valuable comments.

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