ALBERT

Description: Hereditary spherocytosis (HS) is a genetically and phenotypically heterogeneous hemolytic anemia caused by deficiency in red blood cell (RBC) cytoskeleton proteins leading to disruptions in the vertical association of the cytoskeleton with the RBC lipid bilayer. Monoallelic mutations in the genes encoding ankyrin (ANK1), beta-spectrin (SPTB) and band 3 (SLC4A1) or biallelic mutations in the genes encoding alpha-spectrin (SPTA1), ankyrin, and protein 4.2 (EPB42) result in HS. Autosomal recessive HS due to compound heterozygous defects in SPTA1 is typically severe and diagnosis based on phenotypic assays like RBC morphology, osmotic fragility or ektacytometry is complicated by transfusion dependence resulting in most of the circulating RBCs to be of donor origin. We have developed a rapid comprehensive next-generation sequencing-based assay that evaluates 27 genes with published disease-causing mutations for RBC cytoskeletal disorders, enzymopathies, and CDAs. We describe here patients with hemolytic anemia due to SPTA1 mutations, identified utilizing this assay, and their phenotype-genotype correlation. Each of these cases, when possible, has been also evaluated with ektacytometry and immunoblotting of RBC ghosts for alpha-spectrin quantitation. Wichterle et al in 1996 had estimated that alphaLEPRA(Low Expression PRAgue) mutation (c.4339-99C〉T) occurs in SPTA1 gene in about 5% of Caucasians. This mutation leads to activation of an alternate acceptor splice site at position -70 of intron 30, causing frame shift and premature termination, thereby leading to decrease in alpha-spectrin production in this allele to about 16% of normal. We have found a cohort of three transfusion-dependent hereditary hemolytic anemia cases where a nonsense mutation in SPTA1 gene has occurred in trans to alphaLEPRA mutation, resulting in premature termination (see Table 1). Transfusion dependence was alleviated in two of these patients after splenectomy; the third one did not have splenectomy yet. RBC phenotype explored after splenectomy revealed an ektacytometry curve indicating spherocytosis (Figure 1A) and severely decreased alpha-spectrin on immunoblotting along with significant decrease of the associated beta-spectrin (Figure 1B). A patient with moderately severe form of HS, maintaining a hemoglobin value greater than 7 g/dL and requiring only occasional transfusions during periods of illness or stress, was found to have alphaLEPRA occurring in trans to an intronic splicing mutation c.1351-1G〉TG where there is substitution at nucleotide-1 of intron positioned between nucleotides 1350 and 1351 of the SPTA1 mRNA. This splicing mutation may allow for some expression of functional alpha-spectrin protein from this allele in contrast to no protein expression in the previous cases of premature termination. Alternatively, other gene mutations, not identified by the next-generation sequencing panel we used, may contribute to this patient's milder phenotype. A couple with history of two fetal losses associated with hydrops fetalis seeked genetic counseling and gave consent to have diagnostic evaluation of genes associated with non-immune hemolytic anemia using targeted next-generation sequencing. Results of the panel revealed a heterozygous frameshift SPTA1 mutation in each of the parents (c.4206delG in the father and c.4180delT in the mother). These mutations in compound heterozygous state in the offspring likely caused total absence of alpha spectrin and fatal hemolytic anemia by the time of birth. Hereditary Spherocytosis is characterized by wide phenotypic variability that will be better understood with studies of genotype-phenotype association. While complete absence of alpha-spectrin expression due to null mutations of both SPTA1 alleles is incompatible with life, a nonsense or splicing SPTA1 mutation in trans to an alphaLEPRA low expression allele causes severe or moderately severe recessive HS, respectively. Targeted next-generation sequencing can be an effective diagnostic tool particularly for patients requiring frequent transfusions that preclude meaningful phenotypical testing of their red blood cells. Figure 1. SPTA1 null mutations occurring in trans to alpha-LEPRA causing severe HS Figure 1. SPTA1 null mutations occurring in trans to alpha-LEPRA causing severe HS Figure 2. Figure 2. Disclosures Begtrup: GeneDx: Employment.

Description: Background: The hemoglobinopathies are a genetically complex group of blood disorders that includes sickle cell disease (SCD), thalassemia, and other structural and/or functional hemoglobin (Hb) variants. For decades, the mainstay of diagnostic testing for hemoglobinopathies has been Hb separation techniques (e.g., electrophoresis, chromatography), sometimes paired with functional assays of Hb (e.g., solubility, instability, oxygen affinity). Such protein-based testing cannot detect or adequately differentiate a number of clinically significant hemoglobinopathies. Nevertheless, most clinicians continue to rely primarily on protein-based diagnostic methods despite the availability of genetic testing. The reasons for this are not fully understood, but include concerns about the cost of genetic testing and the perception that protein-based methods are usually sufficient for clinical care. Objective: To determine the clinical utility of genetic testing for the diagnosis of hemoglobinopathies by quantifying how often a suspected clinical diagnosis is changed, clarified or excluded by genetic testing. Methods: We reviewed the results of 500 sequential orders for clinical genetic testing for known or suspected hemoglobinopathies that were performed and interpreted at Cincinnati Children's Hospital Medical Center between 1/2013 and 5/2015. This comprehensive genetic testing service is a collaboration between the divisions of Hematology and Human Genetics. For this analysis we reviewed orders for copy number variation analysis (CNV) of the α-globin gene cluster, sequencing of the α-globin genes (HBA1, HBA2), CNV of the β-globin gene cluster, and/or sequencing of the β-globin genes (HBB). An order for testing could include any combination of these 4 tests. We compared the stated indications and suspected diagnoses for testing provided by the ordering physician with the final results of the genetic testing. For each order, the results of this comparison (suspected diagnosis vs. genetic diagnosis) were classified into 1 of 4 mutually exclusive groups: (1) suspected diagnosis confirmed, (2) suspected diagnosis excluded, (3) suspected diagnosis clarified, or (4) new or unexpected diagnosis. The category "suspected diagnosis clarified" includes cases where the overall suspected diagnosis was confirmed, but additional clinically meaningful genetic data was also uncovered (e.g., homozygous sickle cell anemia was confirmed, but co-inherited α-globin gene deletions were also identified). Results: 500 serial genetic testing panels were performed on 475 unique individuals (7 specimens were from an embryo or fetus). These 500 panels included 1-4 individual tests: HBB sequencing in 423, α-globin CNV in 475, β-globin CNV in 345, and HBA1/2 sequencing in 164. The final diagnosis was a specific genotype of SCD (± α-thalassemia and/or gene-deletion HPFH) in 234; thalassemia (α, β, or δβ) in 139; Hb S trait (± α-thalassemia) in 18; Hb C trait or disease (± α-thalassemia) in 13; Hb E trait or disease (± α-thalassemia) in 9; another named or novel Hb variant in 22; and normal Hb in 65. Overall, the suspected diagnosis was confirmed in 246 (49.2%), clarified in 156 (31.2%), excluded in 77 (15.4%), and in 21 (4.2%) a new or unsuspected diagnosis was made. For patients with a suspected diagnosis of SCD, the diagnosis was mostly confirmed (66.7%) and never excluded by genetic testing, but it was clarified in 31.6%, and an unsuspected genotype of SCD was identified in 1.8%. For patients with a suspected diagnosis other than SCD, the diagnosis was confirmed in 34%, clarified in 30.6%, excluded in 29.1%, and an unsuspected genotype was identified in 6.4%. Not considering the clinical utility of the "suspected diagnosis clarified" category, a suspected diagnosis was excluded or determined to be a new or unsuspected diagnosis in 98/500 (19.6%) of all cases. Additionally, follow-up genetic testing was recommended for 31/500 (6.2%). Conclusion: In half (50.8%) of all cases, genetic testing provided new or additional diagnostic information that was not apparent on protein-based diagnostic methods. Genetic testing excluded or substantially changed a suspected diagnosis in one-quarter (19.6%) of all cases. Therefore, genetic testing for hemoglobinopathies has very high clinical utility, and we propose that it should be considered a standard of care for all patients with known or suspected hemoglobinopathies. Disclosures Quinn: Amgen: Research Funding; Eli Lilly: Research Funding; MAST Therapeutics: Research Funding; Glycomimetics: Research Funding; Silverlake Research: Consultancy. Begtrup:GeneDx: Employment.