Overhydrated stomatocytosis associated with a complexRHAGgenotype including a novelde novomutation

Abstract

Overhydrated stomatocytosis is a rare autosomal dominant disorder known to cause variably severe haemolytic anaemia due to heterozygous mutations in the gene. We report a 26-year-old man with recurring jaundice, splenohepatomegaly and mild chronic haemolytic anaemia with significant stomatocytosis. Extensive haemolytic work-up including flow cytometry for eosin-5'-maleimide and CD47 expression levels was carried out. Targeted resequencing revealed two probably causative heterozygous mutations in (Leu336Ser and Ile149Met) and one heterozygous mutation in (Glu1046Lys) involvement was confirmed by decreased RhAG macrocomplex component indicated by the reduced CD47 expression on erythrocytes. analysis concordantly flagged:Leu336Ser and :Glu1046Lys as likely deleterious mutation, whereas:Ile149Met was reported as likely neutral by PROVEAN. Family screening by Sanger sequencing revealed :Leu336Ser in a mother and:Glu1046Lys in a father who were both asymptomatic, excluding them as causative dominant events, thus establishing :Ile149Met, novel mutation as probably causative. This case illustrates the importance of family screening in interpreting next-generation sequencing (NGS) data, as in silico analysis alone can be misleading. Erudite generation of diagnostic possibilities based on a thorough baseline clinical and laboratory work-up remains as important as ever, even as NGS brings about a paradigm shift in the diagnostic work-up of rare haemolytic anaemias.

A nonsense variant in the Hexokinase 1 gene (HK1) causing severe non‐spherocytic haemolytic anaemia: genetic analysis exemplifies ambiguity due to multiple Isoforms

“…To further determine the diagnosis, DNA from the patient, his parents and asymptomatic sibling was subjected to targeted resequencing using TruSight One Sequencing Panel (Illumina Inc., San Diego, CA, USA) after obtaining the necessary approval and consents. Preparation of DNA libraries and data analysis used MiSeq Reporter and VariantStudio, as described (Jamwal et al, ), but no potentially pathogenic variant was found. For whole‐exome sequencing (WES), DNA libraries were prepared using the Nextera Rapid Capture Enrichment guide (Illumina) by enzymatic fragmentation as per the manufacturer's instructions (tagmentation) followed by purification to ensure uniform and appropriate fragment size.…”

A Family Based Whole Exome Sequence Study to Indentify Modifier Genes for Phenotype Heterogeneity Between Severe and Non-Severe Thalassemia Patients

“…Mutated RhAG leads to Overhydrated hereditary Stomatocytosis (OHST). Previously, it was reported that reduced or null expression of RhAG is associated with osmotically fragile erythrocyte and exhibit cation leak which leads to mild to moderate macrocytic hemolytic anemia 17 .…”

Next-Generation Sequencing–Based Diagnosis of Unexplained Inherited Hemolytic Anemias Reveals Wide Genetic and Phenotypic Heterogeneity

“…The OHS patient had compensated hemolysis and a complex genotype for the RHAG gene. 13 All three affected siblings in a family with Mediterranean stomatocytosis/macrothrombocytopenia had a phenotype restricted to hematological manifestations along with short stature. 14 All the variants prioritized as potentially causal were predicted to be highly deleterious/harmful by in silico analysis by PolyPhen, SIFT, PROVEAN, MutPred, Mendelian Clinically Applicable Pathogenicity, and Combined Annotation Dependent Depletion.…”

“…25,26 From India, there is only one phenotypic description of OHS, 27 and the patient with a genetic defect in the RHAG gene causing OHS was reported for the first time recently from the country. 13 Figure 2 Lollipop plot highlighting missense (green) and truncation/nonsense (black) variants detected in the primary pyruvate kinase (PK) protein and its domains. The p.Arg486Trp was the most frequently recurring variant (plot generated using cBioPortal MutationMapper, https://www.cbioportal.org/ mutation_mapper, last accessed July 25, 2019).…”