We describe a two-year-oldwith mild anemia and marked microcytosis of unclear etiology. Hematologic evaluation suggests an apparent HbCC disease or HbCβ0 thalassemia. Parental testing and routine genetic analysis could not provide a definitive diagnosis. Advanced genetic analyses with SOMA and qPCR were required to uncover a large de novo deletion of the beta globin locus revealing HbCεγδβ0 thalassemia. This case exemplifies the importance of advanced genetic technology in hemoglobinopathies that are inconsistent with routine molecular and laboratory testing. Accurate molecular analyses are necessary to facilitate precise determination of genotype-phenotype correlations and, in some cases, are critical to documenting de novo variants.

Arnold SD, Northrop LE, Green NS, Francis R, Levy B (2014) The Role of Advanced Genetic Analyses to Determine a Complex Hemoglobinopathy. Ann Clin Pathol 2(6): 1040.

•    Hemoglobinopathy
•    SOMA
•    qPCR
•    Thalassemia

HPLC: high performance liquid chromatography; Hb: Hemoglobin; MCV: mean corpuscular volume; SOMA: single nucleotide polymorphism oligonucleotide microarray analysis; SNP: single nucleotide polymorphism; qPCR: quantitative PCR; aCGH: array comparative genomic hybridization

Hemoglobin gene variants are commonly diagnosed via hemoglobin electrophoresis or high performance liquid chromatography (HPLC). These methods are limited in determining novel or complexglobin gene mutations, specifically those associated with thalassemia phenotypes and/or de novo features. Advanced genetic analyses and sequencing bridge the gaps in diagnostic ability allowing for more accurate genotypephenotype correlation. This technology has identified rare cases of spontaneous or inherited deletions in the beta globin locus including εγδβ globin mutations [1-4]. However, the resultant phenotype of these mutations in conjunction with common hemoglobin variants remains to be well described.

A 2 year oldLatino male was seen in consultation for severe microcytosis without anemia. Hewas born full term with an uncomplicated pregnancy and delivery. His hemoglobin (Hb) was 13.7 and mean corpuscular volume (MCV) 66 at 25 days of life (normal Hb 12 MCV 96). Newborn screen showed HbF, HbC, and no HbA. Analysis by HPLC and hemoglobin electrophoresis at 30 days of life revealed: HbF 71.4%, HbC 29.1%, HbA and HbA2 both 0%, suggesting homozygous C disease, HbCC.

He remained asymptomatic with normal growth and development. Over the next two years, his Hb decreased to a baseline of 10 with a MCV of ~50. Two months prior to presentation, he began to develop symptoms of exertional fatigue, ashis mother noticedthat he would often have to take breaks when running and playing with friends. There was no pallor, jaundice, palpitations, dark urine, fevers, headache, or other clinically significant illnesses. Blood counts at this time were significant for Hb 9.7 and MCV 52, with normal iron studies, prompting referral for hematologic evaluation.

Upon presentation to our Pediatric Hematology clinic, his physical examination was normal without tachycardia, jaundice, pallor, or hepatosplenomegaly. His blood counts showed a normal Hb 11, MCV 51.3, RBC 6.24, RDW 21, reticulocyte count 1.4, platelet 247, and WBC 10. He had not been prescribed or administered any iron supplementation or other medications. Peripheral smear revealed a mixed erythrocyte population of target cells, spherocytes, and anisocytosis with microcytic cells; normal platelets of varying sizes, normal granulation patterns; normal size and distribution of leukocytes (Figure 1). A repeat HPLCshowed HbC 93.4%, HbF 3.2%, and A2 3.1%, again consistent with HbCC disease or compound heterozygosity for HbC and beta zero (β0 ) thalassemia (Figure 2). Marked microcytosis also supported a concomitant thalassemia.Initial thalassemia gene testing by an outside commercial laboratory indicated a one alpha globin 3.7gene deletion and homozygous HbCC, with a caveat that a large deletion may invalidate their findings.

Family history was significant only for maternal anemia of pregnancy (Hb 9.3, MCV 91), and she was known to have HbAA by prenatal HPLC. This raised concern for HbC homozygosity secondary to uniparental disomy, and testing of both parents was performed. Resultsconfirmed maternal studies and demonstrated paternal microcytosis (Hb 15.8, MCV 77.4) associated with HbC trait andthe same α-thalassemiatrait mutation (Table 1).

These results seemed to confirm diagnosis of HbCC disease viapaternal uniparental disomy. Due to diagnostic uncertainty, single nucleotide polymorphism (SNP) oligonucleotide microarray (SOMA) was performed. Molecular cytogenetic studies identified a de novo heterozygous 60kB interstitial deletion in the short arm of chromosome 11. Specifically, SOMA indicated a loss on chromosome 11 from positions 5,248,426- 5,308,253 (Hg19). This region contains 5 of the genes (HBD, HBBP1, HBG1, HBG2, HBE1) that comprise the beta globin locus on chromosome 11 (Figure 3). Given the absent probe coverage of the SNP microarray for the HBB gene, the deletion was believed to be slightly larger than indicated by SOMA, and quantitative PCR (qPCR) probes were designed to cover regions within the beta globin gene cluster including the HBB gene. The qPCR analysis revealed that the de novo deletion was slightly larger than that indicated by SOMA, and in fact extended to include the HBB gene (Figure 3). These results confer a diagnosis of HbCεγδβ thalassemia, or clinically described HbCβ0 thalassemia due to a paternally inherited HbCtrait and an extensive, de novo beta globin locus deletion.

At presentation, the patientwas started on folic acid supplementation, and serial clinical evaluations to present age 5reveal his baseline at Hb 9. He also developed splenomegaly on exam confirmed on abdominal ultrasound measuring ~13cm. He continues to be followed regularly for surveillance for other phenotypic manifestations.

This case report identifies inconsistencies in diagnosis of hemoglobinopathies using routine parental HPLC testing and standard genetic analyses. This case demonstrates the benefits of advanced genetic testing to identify complex hemoglobinopathies and provide an accurate diagnosis that mayhave otherwise been obscured. Chromosomal microarray analysis, either by array comparative genomic hybridization (aCGH) or SOMA technology, offers significant advantages over traditional molecular technologies such as fluorescence in situ hybridization, Southern blot and Gap-PCR. The primary benefit is the ability to perform a high resolution scan of the entire genome for submicroscopic imbalances and to more specifically map the breakpoints of regions identified with microdeletions and microduplications. Phylipsen et al. recently reported the use of aCGHfor high-resolution breakpoint characterization of small and large rearrangements in patients with α- and β-globin gene cluster rearrangements [5].The absence of this technology could have clinical ramifications.

Homozygous HbC disease has a fairly benign phenotype characterized by mild hemolytic anemia and splenomegaly. This is in large part due to the increased erythrocyte stability and the presence of large, less osmotically fragile spherocytes characteristic of HbCC compared to other hemoglobinopathies. However, occasionally older children and adults with HbCC can experience hyperbilirubinemia and cholelithiasis [6].

HbCβ0 thalassemia generally lacks clinically significant sequelaebeyond mild anemia, microcytosis, andonlyrarely splenomegaly [7]. Mild manifestations are generally a result of a compensatory increased HbA2 or HbF.In our patient, lack of a compensatory response may result from his heterozygous deletion of the corresponding globin exons with εγδβ thalassemiaand may lead to a more severe phenotype.

At the same time, comparable genotype-phenotype correlations were described in individuals where the mutation is limited to the beta globin gene and do not have a concomitant α-mutation. Our patient’sα-thalassemia mutationmay mitigate the concomitantβ globin mutations by reducing the number of freeα−globin chains [8,9]. This constellation of factors with his newly described genotype may predict a long-term clinically milder phenotype of anemia and splenomegaly.

Table 1: Parental laboratory testing.

Abbreviations: β - beta, α - alpha, Hb – hemoglobin, MCV – mean corpuscular volume

The authors would like to thank all the patients and families cared for in the Division of Pediatric Hematology, Oncology, and Stem Cell Transplant as well as all of the faculty and staff in the Department of Pathology and Cell Biology at Columbia University Medical Center. Special thanks to Dr. Patricia M. Raciti who demonstrated the immense learning to be gained from this case.

1. Harteveld CL, Osborne CS, Peters M, van der Werf S, Plug R, Fraser P. Novel 112 kb (epsilonGgammaAgamma) deltabeta-thalassaemia deletion in a Dutch family. Br J Haematol. 2003; 122: 855-858.

2. Verhovsek M, Shah NR, Wilcox I, Koenig SC, Barros T, Thornburg CD. Severe fetal and neonatal hemolytic anemia due to a 198 kb deletion removing the complete β-globin gene cluster. Pediatr Blood Cancer. 2012; 59: 941-944.

3. Brantberg, A. Severe intrauterine anemia: a new form of epsilongammagammadeltabeta thalassemia presenting in utero in a Norwegian family. Haematologica. 2009; 94: 1157-1159.

4. Audino AN, Yeager ND. εGγAγδβ0-thalassemia: a rare but clinically significant cause of hemolytic anemia in infants. J Pediatr Hematol Oncol. 2014; 36: e49-50.

5. Phylipsen M, Chaibunruang A, Vogelaar IP, Balak JR, Schaap RA, Ariyurek Y. Fine-tiling array CGH to improve diagnostics for α- and β-thalassemia rearrangements. Hum Mutat. 2012; 33: 272-280.

6. Fairhurst RM, Casella JF. Images in clinical medicine. Homozygous hemoglobin C disease. N Engl J Med. 2004; 350: e24.

7. Cook CM, Smeltzer MP, Mortier NA, Kirk SE, Despotovic JM, Ware RE. The clinical and laboratory spectrum of Hb C [β6(A3)Glu→Lys, GAG>AAG] disease. Hemoglobin. 2013; 37: 16-25.

8. Cao A, Galanello R, Rosatelli MC. Genotype-phenotype correlations in beta-thalassemias. Blood Rev. 1994; 8: 1-12.

9. Gringras P, Wonke B, Old J, Fitches A, Valler D, Kuan AM. Effect of alpha thalassaemia trait and enhanced gamma chain production on disease severity in beta thalassaemia major and intermedia. Arch Dis Child. 1994; 70: 30-34.