Detection of Rare Deletion Mutation in the Alpha-Globin Gene Locus Establishes a Diagnosis of Hb H Disease

reprinted from february 2018

pathology ◆ laboratory medicine ◆ laboratory management Detection of rare deletion mutation in the alpha-globin gene locus establishes a diagnosis of Hb H disease

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Neng Chen, PhD, DABMGG Patients with Hb H disease are confirm a diagnosis of Hb H disease Cathi Rubin Franklin, MS, LCGC, DABMGG often diagnosed after being seen for and provide information for genetic Franklin Quan, PhD, DABMGG microcytic anemia or other indica- counseling. The alpha-globin gene locus (Fig. 1A) tions, such as infection, hepatospleno- Patient case. A 50-year-old Afri- contains two alpha-globin genes: megaly, cholelithiasis, or severe ane- can-American male was evaluated HBA1 and HBA2. An upstream reg- mia during pregnancy. For patients by his primary care physician be- ulatory region, known as HS-40, is with low MCV, Heinz bodies after cause of anemia. Several years prior, essential for alpha-globin gene ex- brilliant cresyl blue staining of blood the patient had gastrointestinal pression.1,2 The loss or inactivation of smears, or Hb H and/or Hb Barts by bleeding and was diagnosed with one or two alpha-globin genes is not high-performance liquid chromatog- colon cancer. He was anemic and clinically significant while the loss or raphy or gel electrophoresis, the dif- transfused at that time as part of his inactivation of all four alpha-globin ferential diagnosis should include Hb treatment. His primary physician genes results in a lethal condition H disease, hemolytic anemia, and ordered blood smears and iron stud- known as hemoglobin (Hb) Barts alpha-thalassemia X-linked intellec- ies to investigate the cause of his hydrops fetalis syndrome, the most tual disability (ATRX) syndrome. anemia. The blood smears revealed severe form of alpha-thalassemia. Molecular genetic testing is used to microcytosis and the presence of The loss or inactivation of three alpha-globin genes results in a form of A. HS-40 Pseudo HBA2 HBA1 alpha-thalassemia known as hemoglobin H (Hb H) disease. Hb H disease has a variable phe- notype. Hb H disease caused by deletions in the alpha-globin gene HS-40 Pseudo -alpha3.7 del B. Alpha-globin locus locus is associated with moderate with -alpha3.7 del anemia, microcytosis, and hypochro- mia. In contrast, nondeletional Hb H Pseudo -alpha3.7 del Alpha-globin locus disease, caused by a point mutation with HS-40 del plus -alpha3.7 del or small insertion/deletion in one of Fig.Figure 1. 1. Structure Structure of the of alpha the- globinalpha-globin gene locus. A gene. Normal locus. arrangement A. Normalof HBA1, HBA2 arrangementand HS-40. Each of alpha HBA1,-globin geneHBA2, locus and contains HS-40. two alpha Each-globin genes, the alpha-globin genes, is associated alpha-globinHBA1 and HBA2. gene HS-40 is locus the upstream contains regulatory two region alpha-globin required for alpha genes,-globin HBA1 gene expression. and HBA2. The alpha HS-40-globin isgene the locus upstream also contains regulatory two non-functional pseudo-genes. B. Genotype for the patient. One chromosome has the –alpha3.7 deletion; the other chromosome has the –alpha3.7 deletion and a HS-40 deletion. with a more severe anemia, and pa- region required for alpha-globin gene expression. The alpha-globin gene locus also contains two non-functional tients are likely to be transfusion pseudogenes. B. Genotype for the patient. One chromosome has the -alpha3.7 deletion; the other chromosome dependent. has the -alpha3.7 deletion and an HS-40 deletion. 2 | CAP TODAY target cells, indicating microcytic rule out alpha-thalassemia as a cause to the nature of the methodology anemia. Iron studies indicated that of this patient’s anemia, molecular used, the precise size and break- all tested parameters were within the testing for mutations in the alpha- points of this deletion could not be normal reference ranges (Table 1). globin locus was performed. determined. However, the data indi- Iron deficiency was ruled out as a The most common mutations cate that the deletion extends at least cause of the patient’s anemia. The causing alpha-thalassemia are large 11-kb upstream of the HS-40 region hemoccult test was positive at the deletions. Moreover, seven common but not into the downstream pseu- time of his visit to the specialist. deletions, -alpha3.7, -alpha4.2, --SEA, dogene region (Fig. 1B). The causes of anemia include se- --MED, --FIL, --THAI, and --20.5,6 The test results indicate that the vere iron deficiency, thalassemia, and account for approximately 85 percent patient has two alpha-globin fusion chronic diseases (e.g. chronic infec- of all alpha-thalassemia mutations.7 genes that should be functional. tion or inflammatory processes).3 Testing for these common deletions However, the HS-40 regulatory re- Anemia may also be idiopathic. The was performed using a multiplex gion is deleted on one chromosome. hematologist ordered a hemoglobin- gap-PCR assay.6 This testing indi- Since the HS-40 regulatory region is opathy evaluation (CBC and hemo- cated that the patient is homozygous required for the expression of the globin HPLC) to investigate the pos- for the -alpha3.7 deletion. The -al- alpha-globin genes on the same chro- sibility of thalassemia as the cause of pha3.7 deletion generates a func- mosome,1,2 the fusion gene on the this patient’s anemia. The RBC indi- tional alpha-globin fusion gene that chromosome with the HS-40 deletion ces (Table 2) clearly showed that this consists of exon 1 of the HBA2 gene is expected to be nonfunctional. Con- patient was severely anemic. and exons 2 and 3 of the HBA1 gene. sequently, this patient has only one The hemoglobin HPLC (Table 3) Therefore, the test results indicated functional alpha-globin gene, which was normal, with the exception of the that the patient has two functional is consistent with a diagnosis of Hb

Hb A2 level, which was slightly be- alpha-globin genes, a result that was H disease. Since deletions in the al- low the lower limit of the normal inconsistent with his severe anemia. pha-globin gene locus are the cause range. However, unstable Hb vari- To confirm that the patient is ho- of this patient’s Hb H disease, he is ants, such as Hb H, Hb Barts, or Hb mozygous for the -alpha3.7 deletion expected to have the milder form of Constant Spring (Hb CS), are not al- (genotype -alpha3.7/-alpha3.7) and Hb H disease.4 As a result, it would ways detected by hemoglobin HPLC not heterozygous for the -alpha3.7 not be surprising that his anemia was or gel electrophoresis. As a result, to deletion and a rare, two alpha-globin undiagnosed until he presented with gene deletion (genotype GI bleeding and colon cancer. Unfor- Table 1. Iron studies for the patient -alpha3.7/large dele- tunately, we do not have information tion), alpha-globin gene on the time course of his anemia. Parameters Case Normal reference range dosage (i.e. copy num- Nonetheless, the RBC indices in- Ferritin (ng/mL) 203 20–380 ber) was determined us- dicated this patient has anemia that Iron (mcg/dL) 61 50–180 ing a semiquantitative is more severe than expected for an TIBC (mcg/dL) 264 250–425 fluorescent PCR assay.8,9 individual with the deletional form % saturation 23 15–60 This assay measures the of Hb H disease (Table 2). The GI relative dosage of am- bleeding this patient experienced Table 2. Red blood cell indices for the patient plification products de- could cause iron deficiency, which Indices Case Normal Hb H disease rived from the HS-40 would increase the severity of his reference range reference range region, nonfunctional anemia. However, adult patients Hbg (g/dL) 8.9 14.9–16.9 8.7–10.34 pseudogenes, and re- with Hb H disease can have iron MCV (fL) 52.7 83.99–94.01 57–654 gions spanning the overload.4 Because this patient’s iron MCH (pg) 15.7 29.0–32.8 17.2–19.64 HBA2 and HBA1 genes indices were in the normal range 5 RDW (%) 32.9 11.0–15.0 21.2–34.7 (Fig. 1A). This assay (Table 1), iron overload from Hb H confirmed that the pa- disease could be compensating for Table 3. Hemoglobin analysis for the patient tient is homozygous for the iron deficiency caused by GI Hb type Case Normal Hb H disease the -alpha3.7 deletion. bleeding. As a result, it is likely that reference range reference range Unexpectedly, this as- both GI bleeding and Hb H disease Hb A (%) 97.3 96–98 60–90 say also revealed the have contributed to the severity of Hb A (%) 1.7 2–3 <2.0 2 presence of a deletion this patient’s microcytic anemia. Hb F (%) <1 <1 <1 that removes the HS-40 Continued monitoring would be Hb H (%) 0 0 0.8–40 regulatory region on necessary to determine whether this Hb Barts (%) 0 0 2–5 one chromosome. Due individual’s anemia is less severe if CAP TODAY | 3 the GI bleeding resolves. Genetic counseling and testing of at-risk fam- - ily members are recommended. Discussion. It is straightforward to diagnose patients who have microcytic anemia that is caused by a single etiology. However, when there are multiple contributing factors, such as the GI bleeding and Hb H disease in our patient, interpreting the test results is more difficult. An iron study in isolation (Table 1) may be mislead- ing because adult patients with Hb H disease can have iron overload. A comprehensive evaluation of iron studies, RBC indices, hemoglobin HPLC or gel electrophoresis, molecuFigure- 2. MolecularFig. 2. diagnosis Molecular for Hb diagnosisH disease in foralpha Hb-globin H disease gene locus. in alpha-globin gene locus. Testing for common large deletions in the alpha-globin gene locus is the first-tier molecular test for Hb H diseases. If deletions lar testing, and clinical information is often necessary to arrive at the correct removing three copies of alpha-globin genes are detected, the patient is diagnosed with Hb H disease. However, if a deletion or deletions removing only one or two alpha-globin genes are detected, alpha- diagnosis and enable the selection of globin gene sequencing to detect point mutations and small insertion or deletions can be performed the appropriate management/treat- to determine if the remaining alpha-globin genes are functional. In addition, testing for rare large ment plan for the patient. deletions can also be performed to determine if rare deletions contribute to the loss of alpha-globin gene Large deletions are the most com- function. The presence of only one functional alpha-globin gene will confirm a diagnosis of Hb H disease. mon molecular basis for alpha-thalassemia. The -alpha3.7, -alpha4.2, lecular genetic analysis of the alpha- severe case of hemoglobin H disease. Blood. --SEA, --MED, --FIL, --THAI, and and/or beta-globin genes can be 2010;116(12):2193–2194. --20.5 deletions are the most common used to confirm the hemoglobin 3. Van Vranken M. Evaluation of microcytosis. deletions causing alpha-thalas- analysis findings. Therefore, molecu- Am Fam Physician. 2010;82(9):1117–1122. semia.6,7 As a result, testing for these lar analysis is crucial to confirm or 4. Chui DH, Fucharoen S, Chan V. Hemo- globin H disease: not necessarily a benign deletions is usually the first-tier mo- rule out thalassemia as a cause of disorder. Blood. 2003;101(3):791–800. lecular test when alpha-thalassemia anemia. Without molecular analysis, 5. Venugopal S, Dhuri S, Al Jabal KB, Shaju A. is suspected (Fig. 2). In our case, the the diagnosis of Hb H disease would Hemoglobin H disease in Muscat, Oman—a first-tier testing indicated the patient not have been made for this patient. 5 year study. Oman Med J. 2008;23(2):82–85. is homozygous for the -alpha3.7 dele- In summary, molecular analysis is 6. Chong SS, Boehm CD, Higgs DR, Cutting tion. Additional testing for rare dele- critical in confirming the diagnosis of GR. Single-tube multiplex-PCR screen for tions resulted in the detection of a Hb H disease, although the diagnosis common deletional determinants of alpha- deletion involving the HS-40 regula- must be supported by the clinical thalassemia. Blood. 2000;95(1):360–362. tory region, which enabled the diag- presentation and other laboratory 7. Origa R, Moi P. Alpha-Thalassemia. Nov. 1, 2005. Updated Dec. 29, 2016. In: Adam MP, nosis of Hb H disease for this patient. tests. Determining the genotype of an Ardinger HH, Pagon RA, et al., eds. GeneRe- Thus, it is necessary to perform ad- individual affected by Hb H disease views [database online]. Seattle: University ditional testing for rare deletions and using molecular methods provides of Washington; 1993–2017. www.ncbi.nlm.nih.gov/ other types of mutations before ex- information for genetic counseling books/NBK1435/. cluding a diagnosis of Hb H disease. and reproductive planning and as- 8. Liu ET, He M, Rajgopal U. Differential poly- In addition, when investigating sists in predicting the severity of the merase chain reaction in the analysis of gene dosage. Semin Cancer Biol. 1993;4(1):47–58. the possibility that thalassemia is the disease in affected individuals. 9. Hantash FM, Redman JB, Starn K, et al. cause of microcytic anemia, hemo- 1. Coelho A, Picanço I, Seuanes F, Seixas MT, Novel and recurrent rearrangements in the globin HPLC or gel electrophoresis Faustino P. Novel large deletions in the hu- CFTR gene: clinical and laboratory implica- provides clues to the etiology of the man alpha-globin gene cluster: clarifying the tions for cystic fibrosis screening.Hum Genet. anemia by detecting abnormal hemo- HS-40 long-range regulatory role in the native 2006;119(1-2):126–136. chromosome environment. Blood Cells Mol globins. However, some unstable Dis. 2010;45(2):147–153. hemoglobin variants, such as Hb H, Dr. Chen and Dr. Quan are senior directors, 2. Sollaino MC, Paglietti ME, Loi D, Con- Department of Molecular Genetics, and Cathi Hb Barts, and Hb CS, may be diffi- giu R, Podda R, Galanello R. Homozygous Franklin is manager of the Genetic Counselor cult to detect or not be detected by deletion of the major alpha-globin regula- Organization—all at Quest Diagnostics, San Juan HPLC or gel electrophoresis. Mo- tory element (MCS-R2) responsible for a Capistrano, Calif. 4 | CAP TODAY

case report Test yourself Here are three questions taken from the case report. Answers are online now at www.amp.org/casereports and will be published next month in CAP TODAY.

1. Hb H disease is usually caused by the loss of expression of what number of alpha-globin genes? a) one b) two c) three d) four

2. Which of the following is not one of the seven common alpha- thalassemia deletion mutations? a) -alpha3.7 deletion b) --SEA deletion c) --FIL deletion d) HS-40 deletion

3. Hemoglobin HPLC analysis may not be able to detect Hb H, due to its: a) low molecular weight b) poor stability c) high molecular weight d) low quantity