Abstract
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.
Dehydrated hereditary stomatocytosis with new missense mutations in PIEZO1 through the use of next-generation sequencing panel
