Molecular Diagnosis of Thalassemias and Hemoglobinopathies. An ACLPS Critical Review CME/SAM. Daniel E. Sabath, MD, PhD ABSTRACT.

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1 Molecular Diagnosis of Thalassemias and Hemoglobinopathies An ACLPS Critical Review Daniel E. Sabath, MD, PhD From the Department of Laboratory Medicine, University of Washington, Seattle. CME/SAM Key Words: Thalassemia; Hemoglobinopathy; Molecular diagnosis; Genetic counseling Am J Clin Pathol July 2017;148:6-15 DOI: /AJCP/AQX047 ABSTRACT Objectives: To describe the use of molecular diagnostic techniques for patients with hemoglobin disorders. Methods: A clinical scenario is presented in which molecular diagnosis is important for genetic counseling. Globin disorders, techniques for their diagnosis, and the role of molecular genetic testing in managing patients with these disorders are described in detail. Results: Hemoglobin disorders, including thalassemias and hemoglobinopathies, are among the commonest genetic diseases, and the clinical laboratory is essential for the diagnosis of patients with these abnormalities. Most disorders can be diagnosed with protein-based techniques such as electrophoresis and chromatography. Since severe syndromes can result due to inheritance of combinations of globin genetic disorders, genetic counseling is important to prevent adverse outcomes. Protein-based methods cannot always detect potentially serious thalassemia disorders; in particular, α-thalassemia may be masked in the presence of β-thalassemia. Deletional forms of β-thalassemia are also sometimes difficult to diagnose definitively with standard methods. Conclusions: Molecular genetic testing serves an important role in identifying individuals carrying thalassemia traits that can cause adverse outcomes in offspring. Furthermore, prenatal genetic testing can identify fetuses with severe globin phenotypes. Upon completion of this activity you will be able to: describe the genetic basis for α-thalassemia, β-thalassemia, and hemoglobin variants. apply laboratory techniques for the diagnosis of globin genetic disorders. discuss the role for molecular genetic testing in diagnosis and genetic counseling. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Exam is located at Clinical Scenario You are consulted by a genetic counselor who wants to know what laboratory testing you would recommend for her clients. She is advising a couple, each of whom is of Cambodian ancestry. They had a baby who has some form of α-thalassemia based on newborn screening, which detected the presence of hemoglobin Bart s by high-performance liquid chromatography and isoelectric focusing. Because of this, the couple had hemoglobin studies performed on themselves. While neither parent is symptomatic, both are microcytic, and hemoglobin analysis demonstrated that the father is a carrier of hemoglobin E, while the mother has β-thalassemia trait (thalassemia minor). 6 Am J Clin Pathol 2017;148:6-15, All rights reserved. For permissions, please journals.permissions@oup.com

2 Questions 1. What are the likely α-globin (HBA) and β-globin (HBB) genotypes for each parent? 2. What outcomes are possible in offspring from this couple? 3. What additional testing would you recommend for this couple and their child? Hemoglobin disorders are the most frequent genetic diseases in the world, particularly in those parts of the world where malaria has been endemic. These disorders include hemoglobinopathies, which are caused by structural changes in the globin protein chains of hemoglobin, and thalassemias, which are disorders of globin expression. These disorders can result in anemia, shortened RBC life span/hemolysis, and other systemic pathology depending on the specific mutation(s) present. Diagnostic techniques include protein electrophoresis and chromatography, special RBC preparations and stains, and nucleic acid testing. In this review, I describe the diagnostic approach in laboratory testing for globin disorders with a focus on the application of nucleic acid testing. Thalassemia The thalassemias are diseases caused by decreased expression of one of the two globin chains of the hemoglobin molecule, α (HBA) and β (HBB). Decreased expression can result from deletion of the structural gene(s); mutations that result in decreased RNA synthesis, processing, or stability; or mutations resulting in decreased protein synthesis or stability. The decrease in expression of one of the globin chains results in accumulation of excess polypeptides encoded by the unaffected gene. This chain imbalance causes abnormal RBC maturation, resulting in microcytosis as the characteristic laboratory abnormality. Milder forms of thalassemia do not cause anemia; more severe forms can cause microcytic anemia, hemolysis, iron loading, and transfusion dependence in the most severe forms. Thalassemia is a significant public health burden in affected regions, 1 and thus prenatal screening and genetic counseling are important in preventing the most severe forms of thalassemia. α-thalassemia α-thalassemia is caused by deletion in approximately 95% of cases, with the remaining minority due to point mutations Figure 1. The HBA genes consist of two highly similar structural genes that encode identical A B C D E Figure 1 Diagrammatic representation of deletions causing α-thalassemia. In the absence of deletions, there are two HBA genes on each chromosome 16 (A). Deletion of one HBA gene results in α + -thalassemia trait, which generally is not associated with microcytosis (B). Deletion of both HBA genes in cis results in α 0 -thalassemia trait, which is associated with significant microcytosis but little to no anemia (C). Homozygosity for α + -thalassemia results in mild microcytosis generally with minimal anemia (D). Compound heterozygosity for α + -thalassemia and α 0 -thalassemia traits results in hemoglobin H disease, which is associated with significant microcytosis and anemia (E). Not shown is hemoglobin Bart s hydrops fetalis, which results from homozygosity for α 0 -thalassemia. See the main text for full details. protein chains at the far telomeric end of the short arm of chromosome 16. The most common deletions involve loss of 3.7 kb of sequence resulting in a HBA2-HBA1 fusion gene. Several different breakpoints are associated with this mutation, variously referred to as α + -thalassemia or trans deletion α-thalassemia, which is likely due to unequal crossing over during meiosis. The chain imbalance caused by these deletions is relatively mild, so affected individuals may not be microcytic. 2 Since individuals with these deletions by definition will transmit at least one HBA gene to their offspring, there is no risk of having children with a life-threatening form of α-thalassemia (hemoglobin Bart s hydrops fetalis), which occurs due to loss of all four HBA genes. As a result, genetic testing is not necessarily indicated for the purposes of genetic counseling or prenatal diagnosis, although the definite risk of a significant α-thalassemia syndrome in offspring can only be determined by genetic testing of the prospective parents. Diagnosis of α + -thalassemia is often one of exclusion: patients are microcytic with no evidence Am J Clin Pathol 2017;148:6-15 7

3 Sabath / Hemoglobin Disorder Molecular Diagnosis of iron deficiency, but no abnormal findings are present by hemoglobin electrophoresis or chromatography. There may be insufficient excess β-globin to form hemoglobin H (HbH) inclusions, but it is possible to detect the small amount of HbH present in these individuals using special gel electrophoresis and staining techniques. 3 In contrast, deletion of both HBA genes on the same chromosome, referred to as α 0 -thalassemia or cis deletion α-thalassemia, results in a greater degree of chain imbalance, since there is a 50% decrease in HBA expression. This deletion is very common in individuals of Southeast Asian ancestry, and there are various α 0 -thalassemia mutations found in other populations, including Filipino, Thai, and Mediterranean. The main significance of α 0 -thalassemia is that couples each heterozygous for these deletions are at 25% risk of having offspring with hemoglobin Bart s hydrops fetalis. This syndrome manifests as severe anemia and hydrops in affected fetuses, who either die in utero or are stillborn. Mothers carrying these fetuses are at increased risk for preeclampsia and other obstetrical complications. 4,5 For this reason, genetic counseling and prenatal diagnosis are critical for preventing this condition. Compound heterozygosity for α 0 -thalassemia and α + -thalassemia (deletion of three of four HBA genes) is referred to as HbH disease. The significant globin chain imbalance associated with this genotype results in significant microcytosis (mean corpuscular volume [MCV] fl), chronic hemolysis, and splenomegaly, but these individuals generally have a normal life span without significant complications. These individuals are at risk for transmitting hemoglobin Bart s hydops fetalis, so the same genetic counseling and prenatal diagnosis considerations apply as for those with α 0 -thalassemia. A minority of α-thalassemia is due to point mutations affecting the HBA genes. As of this writing, 111 sequence variants are associated with α-thalassemia in the HbVar database ( query category Thalassemias, query chains alpha1 and alpha2, last accessed November 26, 2016). Homozygotes or compound heterozygotes for nondeletional α-thalassemia can resemble HbH disease phenotypically. query chain beta, last accessed November 23, 2016). β-thalassemia can be further subdivided into β 0 -thalassemia, where no HBB protein is produced from the mutated allele, or β + -thalassemia, where reduced quantities of protein are produced. Heterozygotes for either type of allele have microcytosis, clinically referred to as β-thalassemia minor. Compound heterozygotes for two β + -thalassemia alleles or one β + and one β 0 allele have a more severe phenotype termed β-thalassemia intermedia, which includes anemia, hemolysis, iron loading, and occasional requirement for transfusion. Individuals with two β 0 -thalassemia alleles have the most severe form of the disease termed β-thalassemia major, resulting in transfusion-dependent anemia, severe transfusional iron loading requiring chelation therapy, and shortened life expectancy. Laboratory diagnosis of nondeletional β-thalassemia generally relies on the detection of increased levels of hemoglobin A 2, which comprises HBA and the minor adult β-like globin, HBD (δ). Many individuals with the various types of β-thalassemia also exhibit an increase in fetal hemoglobin (hemoglobin F, comprising HBA and HBG [γ]-globin chains), although this is not a universal finding and depends on sequences located on chromosome 11 as well as other locations in the genome. 6-8 A minority of β-thalassemia is due to deletion. Deletions encompassing the HBB gene, both the HBB and HBD genes, and the entire HBB complex have all been described. 9 There are also rare mutations in which the regulatory region of the HBB locus, termed the locus control region (LCR), is deleted, resulting in no expression of the linked β-like globin genes, even though they are present and structurally normal. 10,11 When the HBD gene (or the LCR) is deleted, there is no diagnostic increase in hemoglobin A 2, and a diagnosis of β-thalassemia is difficult to make. Most HBB-HBD deletions are associated with significant increases in hemoglobin F, providing a clue as to the diagnosis, 9 but if hemoglobin F is not elevated, it is difficult to distinguish deletional β-thalassemia from α-thalassemia. Molecular techniques are the only way to make this distinction. β-thalassemia In contrast to α-thalassemia, 95% of β-thalassemias are due to point mutations that cause abnormal RNA transcription, processing or stability, or nonsense mutations resulting in production of abnormal proteins or nonsense-mediated RNA decay. Currently, 279 sequence variants are associated with β-thalassemia in the HbVar database ( hbvar/query_vars3, query category Thalassemias, Hemoglobinopathies Structural variants of the globin genes are termed hemoglobinopathies. Some variants are associated with clinical diseases such as sickle cell anemia and related sickling disorders, hemolysis due to unstable hemoglobins, hemoglobins with altered oxygen affinity, and hemoglobins in which iron cannot be maintained in the ferrous (Fe 2+ ) state. However, most structural variants are clinically silent 8 Am J Clin Pathol 2017;148:6-15

4 and are only discovered incidentally, often during the measurement of hemoglobin A 1c in diabetics by high-performance liquid chromatography (HPLC) or capillary zone electrophoresis. In addition, when the amino acid change of a variant results in no difference in charge, the variant may not be detected by any chromatographic technique. Protein-Based Diagnostic Modalities Hemoglobin Electrophoresis Separation of hemoglobins by various electrophoretic techniques is a mainstay of diagnosis of hemoglobin disorders; these techniques and their advantages and limitations are listed in Table 1. The most common electrophoretic method currently in use is isoelectric focusing, which has largely replaced (other than on proficiency testing surveys and board examinations) cellulose acetate alkaline electrophoresis. This method easily separates the most common normal hemoglobins, including hemoglobin A, hemoglobin F, and hemoglobin A, as well as the common variants hemoglobins S and C. The elevated hemoglobin A 2 associated with β-thalassemia and excess HBB chains associated with HbH disease are also visualized by isoelectric focusing. A number of other relatively common variants are difficult to resolve from hemoglobin S (eg, hemoglobin D or hemoglobin G) or hemoglobin C (eg, hemoglobin E or hemoglobin O Arab ) by isoelectric focusing, so another electrophoretic technique Table 1 Various Techniques Used for the Diagnosis of Hemoglobin Disorders Method Advantages Limitations Cellulose acetate electrophoresis alkaline ph Citrate agar electrophoresis acidic ph Supravital staining with brilliant cresyl blue Isoelectric focusing High-performance liquid chromatography Low cost, extensive laboratory experience Can distinguish uncommon variants from hemoglobin S or hemoglobin C, low cost When positive, definitive diagnosis of α-thalassemia, semiquantitative results allow presumptive genotyping Good resolution, widely used, relatively low cost Quantitative, good resolution, rapid, widely adopted Capillary zone electrophoresis High resolution, can resolve hemoglobin A 2 from hemoglobin E, quantitative Mass spectrometry DNA blot analysis Gap polymerase chain reaction Multiplex ligase-dependent probe amplification Comparative genomic hybridization Sanger sequencing Allele-specific methodologies (allele-specific polymerase chain reaction, reverse dot-blot, arrays, etc) Next-generation sequencing Useful for rapidly characterizing hemoglobin variants Can detect large deletions, specific breakpoints do not need characterization Rapid, can be multiplexed, good for common deletions, especially in α-thalassemia Can cover large chromosomal regions for deletion analysis, quantitative, exact breakpoints do not need characterization Can cover large chromosomal regions for deletion analysis, high resolution, exact breakpoints do not need characterization Best current method for characterizing globin variants and point mutations causing thalassemia Useful in genetically homogeneous populations, high throughput, economical Has potential to characterize mutations and deletions throughout all globin genes in parallel Low resolution, not generally used in modern laboratories, may miss β-thalassemia in newborn period Low resolution, high-performance liquid chromatography can usually substitute May be negative in milder forms of α-thalassemia, labor intensive Cannot definitively identify some variants, not sensitive for α-thalassemia, not quantitative, may miss β-thalassemia in newborn period Cannot definitively identify some variants, not sensitive for α-thalassemia, may miss β-thalassemia in newborn period Cannot definitively identify some variants, not sensitive for α-thalassemia, may miss β-thalassemia in newborn period No role in diagnosis of thalassemia Low resolution, requires specific probes, labor intensive, largely replaced by newer techniques, cannot detect point mutations (except for those affecting restriction sites) Cannot detect point mutations, will miss deletions lacking specific primers Low resolution, cannot detect point mutations or small deletions Cannot reliably detect single α-globin deletions due to cross-hybridization Not useful for detecting deletions Less useful in ethnically diverse populations Not yet developed for this application, may be subject to problems in repetitive sequences, particularly α-globin genes Am J Clin Pathol 2017;148:6-15 9

5 Sabath / Hemoglobin Disorder Molecular Diagnosis or HPLC is required to definitively distinguish among these possibilities. Electrophoresis using citrate agar at ph 6.2 allows hemoglobin S or hemoglobin C to be distinguished from other variants that migrate similarly by isoelectric focusing. Even greater resolution is now possible using capillary zone electrophoresis, which can, for example, separate hemoglobin E from hemoglobin A 2, something not possible with the techniques mentioned above or with HPLC. 12 Capillary zone electrophoresis has the potential to replace older electrophoresis methods in many laboratories in the near future. HPLC A number of HPLC methods are available from various manufacturers that separate hemoglobins based on cation exchange chromatography. These methods are approved by the US Food and Drug Administration for quantification of hemoglobin F and hemoglobin A 2 as well as hemoglobin A 1c (glycated hemoglobin) for monitoring therapy in patients with diabetes. 13 HPLC allows separation of hemoglobin S and hemoglobin C from hemoglobins that have similar migration on electrophoresis. It also allows quantification of the variant hemoglobins. HPLC cannot separate hemoglobin E from hemoglobin A 2. Although this is not generally of diagnostic significance, the ability to separate these two hemoglobins can be useful in distinguishing homozygosity for hemoglobin E from compound heterozygosity for hemoglobin E and β 0 -thalassemia. 14 Separation of hemoglobins E and A 2 can be accomplished by capillary zone electrophoresis. Other chromatographic techniques are available for separating hemoglobin species. Notably, boronate affinity chromatography is useful for separating glycated from nonglycated hemoglobin species for monitoring diabetic patients, even those with hemoglobin variants, 15 but this technique has no utility for diagnosing thalassemias or hemoglobinopathies. Use of Electrophoresis and Chromatography for Diagnosing Hemoglobin Disorders Diagnosis of β-thalassemia is usually accomplished by demonstrating increased hemoglobin A 2 by electrophoresis and/or chromatography, although in unusual circumstances such as coexistent δ-thalassemia or severe iron deficiency, the level of hemoglobin A 2 may not be increased. The diagnostic increase in hemoglobin A 2 will also not be observed in β-thalassemia due to deletion of the HBB and HBD genes, although hemoglobin F is often elevated, 9 as seen in δβ-thalassemia and hereditary persistence of fetal hemoglobin (HPFH). Protein-based techniques may also not detect the presence of β-thalassemia in the newborn period, since hemoglobin A 2 does not reach adult levels until about 6 months of age. However, thalassemia major due to homozygosity/compound heterozygosity for two β 0 -thalassemia alleles would be suspected due to a lack of hemoglobin A at birth. Hemoglobin electrophoresis and HPLC generally do not contribute to the diagnosis of the milder forms of α-thalassemia, in which one or two (cis or trans) HBA genes are deleted. The excess HBB chains in these disorders are not abundant enough to visualize by standard techniques, partially due to the instability of HbH. HbH does form inclusions in rare RBCs, which can be visualized by supravital staining with brilliant cresyl blue, 16 but this technique is not available in many laboratories. Thus, α-thalassemia is often a diagnosis of exclusion, where a patient with microcytosis, normal iron studies, and normal hemoglobin electrophoresis/ HPLC is presumed to have some form of α-thalassemia, although methods have been described to detect the small amounts of HbH that are present in milder forms of α-thalassemia. 3 For these patients, molecular diagnosis may be the only means of definitive diagnosis, and this can be critical for genetic counseling. In the setting of HbH disease, where three HBA genes are deleted or where there is a combination of HBA deletions and point mutations, there is enough chain imbalance to visualize HbH by electrophoresis or HPLC. Presumptive identification of a variant hemoglobin can be accomplished by gel electrophoresis, isoelectric focusing, HPLC, and/or capillary zone electrophoresis. Comparison with samples containing a previously documented variant can be analyzed in parallel for identification. Prior to the availability of DNA sequencing, hemoglobin variants were characterized by mass spectrometry of proteolytic global peptides. 17,18 Although DNA sequencing is the most common means of identifying variant hemoglobins, newer techniques of mass spectrometry offer the prospect of rapidly identifying hemoglobin variants based on the mass of the intact molecule or defined globin fragments. Molecular Diagnostic Techniques for Globin Disorders α-thalassemia Since most α-thalassemias are due to deletion of one or more HBA genes, and since these deletions are 10 Am J Clin Pathol 2017;148:6-15

6 common in the affected populations, molecular diagnostic techniques are focused on demonstrating these common deletions. DNA blot analysis has been used in the past, but polymerase chain reaction (PCR) methods using gap-pcr are most commonly used currently. 19 PCR primers that flank the common breakpoints can demonstrate deletions by generating PCR products of specific sizes. The specific primers used in a given laboratory depend on the common deletions in the population being served. Several comprehensive sets of PCR primers to detect most HBA deletions have been described. 19 An alternative technique for characterizing deletions is multiplex ligation-dependent probe amplification (MLPA). 20 This is a technique that can quantify gene copy numbers and thus can detect gene deletions based on reduction in gene dosage. In MPLA, a series of oligonucleotide probe pairs that anneal to DNA in adjacent sites is designed to cover the chromosomal area of interest at convenient intervals. When the oligonucleotides anneal to their target sequences, DNA ligase will covalently link the probes that anneal at adjacent sites, and the resulting ligated sequences are amplified by PCR. The quantity of PCR product is proportional to the target copy number, so deleted regions are identified by decreased signal for a given probe pair. Unlike with PCR, where the exact chromosomal breakpoint is required to design PCR primers, MLPA is capable of detecting unusual deletions that have not been previously described or are not covered by whatever primer sets a given laboratory uses based on its patient population. This technique has been used successfully to characterize known and novel deletions of the HBA locus that cause α-thalassemia, although MLPA had difficulty in consistently detecting trans HBA deletions. 21 In our laboratory, we have experimented with using array-comparative genomic hybridization (array-cgh) to characterize globin gene deletions. By creating a custom oligonucleotide array that densely tiles the HBA locus, we were able to detect heterozygotes for the common Filipino and Southeast Asian cis HBA deletions based on a 50% reduction in signal relative intensity in the deleted regions. The array-cgh method can also detect heterozygotes with the single-gene (trans) α 3.7 and α 4.2 deletions based on a reproducible pattern of array-cgh results, although the results are difficult to interpret a priori due to cross-hybridization between HBA1 and HBA2 sequences. 22 Others have used a similar approach For nondeletional α-thalassemia, (Sanger) DNA sequencing is the most comprehensive method to detect all mutations to avoid having to design mutation-specific assays. Allele-specific PCR has a role in areas where certain mutations are common, such as hemoglobin Constant Spring in Southeast Asia. 26 A reverse dot-blot strategy has also been described that can detect common deletions and point mutations. 27 Sanger sequencing of HBA genes is somewhat complex methodologically since the two HBA genes on chromosome 16 are nearly identical for a stretch of over 1 kbp that includes the 5' and 3' flanking regions, coding sequences, and introns. To selectively amplify the HBA1 and HBA2 genes, one needs to use sequences at least 1,000 base pairs 5' to the structural genes to design specific PCR primers for each HBA gene. These 5' primers can be combined with a common 3' primer. In addition, the HBA sequences are relatively guanine-cytosine rich, so standard PCR conditions need to be optimized to efficiently amplify these sequences. 19 Once successfully amplified, the PCR products can be sequenced directly using standard Sanger methods. Sequences obtained can be compared with those in the HbVar database to determine clinical significance ( β-thalassemia and HPFH Since most β-thalassemias are due to point mutations, Sanger sequencing is the most practical current method to detect all possible mutations in an individual. 28 DNA sequencing of the HBB gene is less technically complex than that for the HBA genes, since PCR amplification is easier due to there being only a single HBB gene on each chromosome 11 and since the sequence is less guanine-cytosine rich than the HBA genes. A number of different PCR and sequencing strategies have been described. 29 Since some populations have a relatively small number of mutations causing β-thalassemia, a number of methods targeting a limited set of mutations have been developed that are less expensive and allow higher throughput than Sanger sequencing. Many of these more ethnically homogeneous populations are in resource-limited regions of the world, so more cost-effective methods are badly needed. Reverse dot-blot methods, in which biotinylated patient DNA is hybridized to membranes on which mutation-specific oligonucleotides are immobilized and is then detected by enzyme-linked streptavidin, can be more economical than direct sequencing and can screen for multiple mutations Rather than using membranes, oligonucleotides can also be applied to an array substrate and used in a similar fashion. 33 Another strategy is to use allele-specific PCR to detect specific mutations, as described for common Southeast Asian and Indian mutations. 34 An approach casting a wider net to detect mutations is heteroduplex analysis using denaturing HPLC, which can distinguish various mutations based on the chromatographic characteristics of PCR products containing various HBB mutations, either as homoduplexes in the homozygous state or as heteroduplexes in Am J Clin Pathol 2017;148:

7 Sabath / Hemoglobin Disorder Molecular Diagnosis heterozygotes Alternatively, similar results can be obtained using single-stranded conformational polymorphism analysis. 38 An analogous method for detecting β-thalassemia mutations that screens PCR products for base substitutions is high-resolution melting curve analysis. 39 As another way to detect multiple mutations simultaneously, a method using melting curve analysis was developed to detect 24 β-thalassemia mutations common in the southern Chinese population using a multiplexing strategy that allows numerous mutations to be detected in a single tube. 40 A completely alternative method involves resolving DNA fragments by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. 41,42 Deletions of the HBB locus that cause δβ-thalassemia or HPFH, as well as rarer deletions, can be detected by gap- PCR if the deletion breakpoints are known. 43 Strategies using MLPA have also been described 20,44 using similar strategies as those for the HBA locus. More recently, our laboratory and others have demonstrated the utility of array- CGH for characterizing deletions of the HBB locus Because these arrays use probes spaced at short intervals throughout the locus, it is possible to finely map deletion breakpoints, design PCR primers to amplify the breakpoint region, and determine the sequences flanking the breakpoints. We used this technique to confirm the breakpoints for several known mutations causing δβ-thalassemia and HPFH as well as previously unmapped deletions. 22 When to Use Molecular Testing for Patients With Hemoglobin Disorders? Patients suspected of having hemoglobin disorders should initially be screened with a CBC to detect the presence of anemia with or without microcytosis. Regardless of MCV, patients should have hemoglobin analysis by isoelectric focusing, capillary zone electrophoresis, and/ or HPLC to detect hemoglobin variants and to quantify hemoglobin F and hemoglobin A 2. Microcytic patients should in addition have measurement of iron supply parameters (serum iron, iron binding capacity, ferritin, etc) and detection of HbH inclusions by brilliant cresyl blue staining if available. Depending on the results and whether there are genetic counseling issues to consider, molecular testing for globin abnormalities may be indicated. An algorithm for deciding whom to test and what molecular tests to perform is illustrated in Figure 2, and common presentations and suggested diagnostic approaches are listed in Table 2. Patients with ambiguous hemoglobin testing results or unusual phenotypes are candidates for DNA testing. Couples seeking genetic counseling should be offered DNA testing when both prospective parents are suspected of having cis α-globin deletions or both have β-thalassemia traits. In addition, it should be kept in mind that individuals with β-thalassemia trait or hemoglobin E trait may have coincident α-thalassemia trait that is masked by the microcytosis associated with these β-globin disorders, and thus α-globin DNA testing should be performed to evaluate risk to offspring of hemoglobin Bart s hydrops fetalis. Prenatal genetic testing should be offered to couples at risk for producing offspring with clinically severe hemoglobin disorders. Conclusions Hemoglobinopathies and thalassemias remain a worldwide cause of morbidity and mortality. 1 Although historically limited to regions with endemic malaria, individuals with hemoglobin disorders now live throughout the world, and laboratories in regions previously unaffected by malaria, such as North America, northern Europe, Japan, and Australia, are now called upon to diagnose hemoglobinopathies and thalassemias. In this review, I have summarized the current standard diagnostic methods as well as more novel molecular techniques that have recently become available. One challenge in this area is that much of the disease burden of hemoglobinopathies and thalassemias is in parts of the world with limited resources for health care. Some of the methods that focus on mutations that are frequent in a given region may allow relatively inexpensive diagnoses, but additional work remains to provide molecular diagnostic techniques for these populations. There are also different considerations for pediatric diagnostic laboratories, where the focus is generally on making diagnoses for children with abnormal newborn screens or unexplained anemias vs adult diagnostic laboratories, where genetic counseling is often the focus in addition to making new diagnoses. Molecular diagnostic techniques are less commonly needed in the pediatric setting, whereas these techniques are frequently critical in the setting of genetic counseling and prenatal diagnosis. The next advance in molecular diagnostics for hemoglobin disorders will be next-generation sequencing. There will be some technical challenges in implementing next-generation sequencing, especially with respect to the HBA genes, which, because of the nearly identical sequence between the HBA1 and HBA2 genes, will make it challenging to determine whether a given mutation belongs to one or the other of these genes. Aside from this challenge however, various strategies now in place for next-generation sequencing, including hybrid capture and whole-genome 12 Am J Clin Pathol 2017;148:6-15

8 Figure 2 An algorithm for the use of molecular diagnostic testing for individuals with hemoglobin abnormalities. If the diagnosis of thalassemia or hemoglobinopathy is apparent based on hemoglobin studies and there are no genetic counseling issues, additional genetic testing is generally not needed. When there are genetic counseling issues or when the diagnosis is unclear, DNA testing may be offered as illustrated in this flow diagram. approaches, should be applicable. It is my hope that the cost of these technologies will eventually decrease enough to make them available to resource-limited settings where diagnosis of hemoglobin disorders will be most valuable. Case Summary Since this couple had a child with α-thalassemia, one or both must be carriers for this condition. Since both parents have a β-globin phenotype that would mask an underlying α-thalassemia, molecular testing is required to make a diagnosis and to determine risk to future offspring. Being of Southeast Asian ancestry, the most likely genotype is the common Southeast Asian cis α-globin deletion (α 0 -thalassemia). α-globin genotyping by gap-pcr would be the most cost-effective way to determine this couple s α-globin genotypes and risk to future offspring. Any offspring who coinherit one of the parent s β-globin disorders and a cis α-globin deletion would not be expected to Table 2 Common Presentations With Genetic Counseling Implications, Risks for Offspring, and Suggested Workups Prospective Parent With Consider in Partner Risk to Offspring Workup for Partner Hemoglobin S trait Hemoglobins S, C, E, D; O Arab traits; β-thalassemia trait Hemoglobin C, E, D or O Arab trait α 0 -Thalassemia trait β-thalassemia trait (cannot exclude coincident α 0 - thalassemia trait) Hemoglobin E trait (cannot exclude coincident α 0 - thalassemia trait) Clinically significant sickling disorder Hemoglobin analysis (HPLC and/or IEF), HbA 2 quantification Hemoglobin S trait Clinically significant sickling disorder Hemoglobin analysis α 0 - or α + -thalassemia trait α 0 - and/or β-thalassemia traits, hemoglobin S trait α 0 - and/or β-thalassemia traits, hemoglobin S trait Hemoglobin H disease, hemoglobin Bart s hydrops fetalis β-thalassemia intermedia or β-thalassemia major, hemoglobin Bart s hydrops fetalis, clinically significant sickling disorder (with S trait) Hemoglobin Bart s hydrops fetalis, E/β 0 - thalassemia, clinically significant sickling disorder (with S trait) BCB, brilliant cresyl blue; HbA 2, hemoglobin A 2 ; HPLC, high-performance liquid chromatography; IEF, isoelectric focusing. Hemoglobin analysis, BCB inclusion body study; α-globin deletion analysis if inconclusive Hemoglobin analysis, BCB inclusion body study; α-globin deletion analysis for both parents if inconclusive Hemoglobin analysis, BCB inclusion body study; α-globin deletion analysis for both parents if inconclusive Am J Clin Pathol 2017;148:

9 Sabath / Hemoglobin Disorder Molecular Diagnosis have a syndrome any more severe than either thalassemia trait alone. Since the father has hemoglobin E trait and the mother has some type of β-thalassemia trait, it is important to determine the specific β-thalassemia mutation that the mother carries, since if it is a β 0 -thalassemia mutation, there is a 25% chance that future offspring would be compound heterozygotes for hemoglobin E/β 0 -thalassemia. This would result in a thalassemia intermedia phenotype, with iron loading with possible transfusion dependence. The mother s genotype would generally be determined by β-globin gene sequencing. Finally, depending on the α- and β-globin genotypes of the parents, it is even possible that future offspring could have the combination of hemoglobin E/β 0 -thalassemia/hbh disease, which can result in a severe transfusion-dependent thalassemia. Thus, the recommendation would be for both parents to have α-globin DNA analysis with additional β-globin DNA sequencing for the mother. Since newborn screening by hemoglobin analysis would not determine the child s α-globin genotype, α-globin DNA analysis could be performed on the child to determine likely outcomes. Alternatively, the child could be evaluated at age 8 to 9 months to determine the thalassemia phenotype, from which one could possibly infer the α-globin genotype. Prenatal genetic testing would allow the parents to determine possible adverse outcomes in future pregnancies. Corresponding author: Daniel E. Sabath, MD, PhD, Dept of Laboratory Medicine, University of Washington, Box Harborview Medical Center 3NJ-345.1, 325 Ninth Ave, Seattle, WA 98104; dsabath@uw.edu. References 1. Weatherall DJ. The inherited diseases of hemoglobin are an emerging global health burden. Blood. 2010;115: Higgs DR. The pathophysiology and clinical features of α thalassemia. In: Steinberg MH, Forget BG, Higgs DR, et al, eds. Disorders of Hemoglobin: Genetics, Pathophysiology, and Clinical Management. New York, NY: Cambridge University Press; 2009: Agarwal AM, Nussenzveig RH, Hoke C, et al. Identification of one or two α-globin gene deletions by isoelectric focusing electrophoresis. Am J Clin Pathol. 2013;140: Liang ST, Wong VC, So WW, et al. Homozygous alpha-thalassaemia: clinical presentation, diagnosis and management: a review of 46 cases. Br J Obstet Gynaecol. 1985;92: Tan SL, Tseng AM, Thong PW. Bart s hydrops fetalis clinical presentation and management an analysis of 25 cases. Aust N Z J Obstet Gynaecol. 1989;29: Garner CP, Tatu T, Best S, et al. Evidence of genetic interaction between the β-globin complex and chromosome 8q in the expression of fetal hemoglobin. Am J Hum Genet. 2002;70: Menzel S, Garner C, Gut I, et al. A QTL influencing F cell production maps to a gene encoding a zinc-finger protein on chromosome 2p15. Nat Genet. 2007;39: Wahlberg K, Jiang J, Rooks H, et al. The HBS1L-MYB intergenic interval associated with elevated hbf levels shows characteristics of a distal regulatory region in erythroid cells. Blood. 2009;114: Thein SL, Wood WG. The molecular basis of β thalassemia, δβ thalassemia, and hereditary persistence of fetal hemoglobin. In: Steinberg MH, Forget BG, Higgs DR, et al, eds. Disorders of Hemoglobin: Genetics, Pathophysiology, and Clinical Management. New York, NY: Cambridge University Press; 2009: Curtin P, Pirastu M, Kan YW, et al. A distant gene deletion affects β-globin gene function in an atypical γδβ-thalassemia. J Clin Invest. 1985;76: Bender MA, Byron R, Ragoczy T, et al. Flanking HS-62.5 and 3' HS1, and regions upstream of the LCR, are not required for β-globin transcription. Blood. 2006;108: Borbely N, Phelan L, Szydlo R, et al. Capillary zone electrophoresis for haemoglobinopathy diagnosis. J Clin Pathol. 2013;66: Rhea JM, Molinaro R. Pathology consultation on HbA 1c methods and interferences. Am J Clin Pathol. 2014;141: Keren DF, Hedstrom D, Gulbranson R, et al. Comparison of Sebia Capillarys capillary electrophoresis with the Primus high-pressure liquid chromatography in the evaluation of hemoglobinopathies. Am J Clin Pathol. 2008;130: Flückiger R, Mortensen HB. Glycated haemoglobins. J Chromatogr. 1988;429: Sabath DE, Cross ST, Mamiya LY. An improved method for detecting red cells with hemoglobin H inclusions that does not require glass capillary tubes. Clin Lab Haematol. 2003;25: Wada Y, Hayashi A, Fujita T, et al. Structural analysis of human hemoglobin variants with field desorption mass spectrometry. Biochim Biophys Acta. 1981;667: Shackleton CH, Falick AM, Green BN, et al. Electrospray mass spectrometry in the clinical diagnosis of variant hemoglobins. J Chromatogr. 1991;562: Liu YT, Old JM, Miles K, et al. Rapid detection of α-thalassaemia deletions and α-globin gene triplication by multiplex polymerase chain reactions. Br J Haematol. 2000;108: Harteveld CL, Voskamp A, Phylipsen M, et al. Nine unknown rearrangements in 16p13.3 and 11p15.4 causing α- and β-thalassaemia characterised by high resolution multiplex ligation-dependent probe amplification. J Med Genet. 2005;42: Kipp BR, Roellinger SE, Lundquist PA, et al. Development and clinical implementation of a combination deletion PCR and multiplex ligation-dependent probe amplification assay for detecting deletions involving the human α-globin gene cluster. J Mol Diagn. 2011;13: Sabath DE, Bender MA, Sankaran VG, et al. Characterization of deletions of the HBA and HBB loci by array comparative genomic hybridization. J Mol Diagn. 2016;18: Blattner A, Brunner-Agten S, Ludin K, et al. Detection of germline rearrangements in patients with α- and β-thalassemia using high resolution array CGH. Blood Cells Mol Dis. 2013;51: Am J Clin Pathol 2017;148:6-15

10 24. Phylipsen M, Chaibunruang A, Vogelaar IP, et al. Fine-tiling array CGH to improve diagnostics for α- and β-thalassemia rearrangements. Hum Mutat. 2012;33: Sankaran VG, Xu J, Byron R, et al. A functional element necessary for fetal hemoglobin silencing. N Engl J Med. 2011;365: Pichanun D, Munkongdee T, Klamchuen S, et al. Molecular screening of the Hbs Constant Spring (codon 142, TAA>CAA, α2) and Paksé (codon 142, TAA>TAT, α2) mutations in Thailand. Hemoglobin. 2010;34: Lin M, Zhu JJ, Wang Q, et al. Development and evaluation of a reverse dot blot assay for the simultaneous detection of common alpha and beta thalassemia in Chinese. Blood Cells Mol Dis. 2012;48: Wong C, Dowling CE, Saiki RK, et al. Characterization of β-thalassaemia mutations using direct genomic sequencing of amplified single copy DNA. Nature. 1987;330: Chan OT, Westover KD, Dietz L, et al. Comprehensive and efficient HBB mutation analysis for detection of β-hemoglobinopathies in a pan-ethnic population. Am J Clin Pathol. 2010;133: Maggio A, Giambona A, Cai SP, et al. Rapid and simultaneous typing of hemoglobin S, hemoglobin C, and seven Mediterranean β-thalassemia mutations by covalent reverse dot-blot analysis: application to prenatal diagnosis in Sicily. Blood. 1993;81: Cai SP, Wall J, Kan YW, et al. Reverse dot blot probes for the screening of β-thalassemia mutations in Asians and American blacks. Hum Mutat. 1994;3: D Souza E, Sawant PM, Nadkarni AH, et al. Evaluation of the use of monoclonal antibodies and nested PCR for noninvasive prenatal diagnosis of hemoglobinopathies in India. Am J Clin Pathol. 2008;130: Chan K, Wong MS, Chan TK, et al. A thalassaemia array for Southeast Asia. Br J Haematol. 2004;124: Wang W, Kham SK, Yeo GH, et al. Multiplex minisequencing screen for common Southeast Asian and Indian β-thalassemia mutations. Clin Chem. 2003;49: Wu G, Hua L, Zhu J, et al. Rapid, accurate genotyping of β-thalassaemia mutations using a novel multiplex primer extension/denaturing high-performance liquid chromatography assay. Br J Haematol. 2003;122: Su YN, Lee CN, Hung CC, et al. Rapid detection of β-globin gene (HBB) mutations coupling heteroduplex and primer-extension analysis by DHPLC. Hum Mutat. 2003;22: Li Q, Li LY, Huang SW, et al. Rapid genotyping of known mutations and polymorphisms in β-globin gene based on the DHPLC profile patterns of homoduplexes and heteroduplexes. Clin Biochem. 2008;41: Chinchang W, Viprakasit V, Pung-Amritt P, et al. Molecular analysis of unknown β-globin gene mutations using polymerase chain reaction single strand conformation polymorphism (PCR- SSCP) technique and its application in Thai families with β-thalassemias and β-globin variants. Clin Biochem. 2005;38: Shih HC, Er TK, Chang TJ, et al. Rapid identification of HBB gene mutations by high-resolution melting analysis. Clin Biochem. 2009;42: Xiong F, Huang Q, Chen X, et al. A melting curve analysis-based PCR assay for one-step genotyping of β-thalassemia mutations a multicenter validation. J Mol Diagn. 2011;13: Liao HK, Su YN, Kao HY, et al. Parallel minisequencing followed by multiplex matrix-assisted laser desorption/ionization mass spectrometry assay for β-thalassemia mutations. J Hum Genet. 2005;50: Thongnoppakhun W, Jiemsup S, Yongkiettrakul S, et al. Simple, efficient, and cost-effective multiplex genotyping with matrix assisted laser desorption/ionization time-of-flight mass spectrometry of hemoglobin beta gene mutations. J Mol Diagn. 2009;11: Craig JE, Barnetson RA, Prior J, et al. Rapid detection of deletions causing δβ thalassemia and hereditary persistence of fetal hemoglobin by enzymatic amplification. Blood. 1994;83: Phylipsen M, Prior JF, Lim E, et al. Thalassemia in Western Australia: 11 novel deletions characterized by multiplex ligation-dependent probe amplification. Blood Cells Mol Dis. 2010;44: Am J Clin Pathol 2017;148: