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Hereditary xerocytosis (HX), also known as dehydrated stomatocytosis (DHSt) is a dominantly inherited genetic disorder exhibiting red cell membrane dehydration caused by the loss of the monovalent cation K+ and water. Variants in mechanosensitive Piezo ionic channels of the PIEZO1 gene are the primary cause of HX. We have utilized high throughput and highly precise next-generation sequencing (NGS) to make a diagnosis and examine the genotype-phenotype relationship in inflexible HX cases. Seven unrelated patients with unexplained hemolytic anemia were scrutinized with a panel probing 8000 genes related to congenital anemia. Targeted next-generation sequencing identified 8 missense variants in the PIEZO1 gene in 7 unrelated Indian patients. Three of the 8 variants are novel (c.1795G > C, c.2915G > A, c.7372 T > C) and the remaining five (c.4082A > G, c.6829C > A, c.7374C > G, c.7381G > A, c.7483_7488dup) are previously reported. The variants have been validated by Sanger sequencing. One patient with autosomal dominant mutation (c.7372 T > C) is associated with iron refractory iron deficiency anemia. Of the 7 patients, one has HX in combination with a novel homozygous variant (c.994G > A) in the PKLR gene causing PK deficiency resulting in severe clinical manifestations with phenotypic variability. In silico prediction using bioinformatics tools were used to study the possible damaging effects of the novel variants. Structural-functional analysis of the novel variants was investigated by molecular modeling software (PyMOL and Swiss PDB). These results encompass the heterogeneous behavior of mechano-sensitive Piezo1 protein observed in HX patients in India. Moreover, NGS imparted a subtle, economical, and quick tool for understanding the genetic cause of undiagnosed cases of congenital hemolytic anemia. NGS grants a potential technology integrating clinical history together with molecular report profiting in such patients and their families.

Most patients with anemia are diagnosed through clinical phenotype and basic laboratory testing. Nonetheless, in cases of rare congenital anemias, some patients remain undiagnosed despite undergoing an exhaustive workup. Genetic testing is complicated by the large number of genes that are involved in rare anemias, due to similarities in the clinical presentation. We sought to enhance the diagnosis of patients with congenital anemias by using targeted next-generation sequencing. The genetic diagnosis was performed by gene capture followed by next-generation sequencing of 76 genes known to cause anemia syndromes. Genetic diagnosis was achieved in 17 of 21 transfusion-dependent patients and undiagnosed by conventional workup. Four cases were diagnosed with red cell membrane protein defects, four patients were diagnosed with pyruvate kinase deficiency, one case of adenylate kinase deficiency, one case of glucose phosphate isomerase deficiency, one case of hereditary xerocytosis, three cases having combined membrane and enzyme defect, two cases with Diamond-Blackfan anemia (DBA) and 1 with CDA type II with 26 different mutations, of which 21 are novel. Earlier incorporation of this NGS method into the workup of patients with congenital anemia may improve patient care and enable genetic counselling.

Mitochondrial DNA (mtDNA) deletion is associated with a variety of clinical entities. In addition to progressive external ophthalmoplegia and Kearns-Sayre syndrome, mtDNA deletions have been demonstrated in Pearson's syndrome. We report an mtDNA deletion in an infant with a variant of Pearson's syndrome. Not only does she have congenital anemia, severe tubulopathy, and exocrine pancreas insufficiency, but she also has diabetes and cerebral atrophy. However, there are no signs of gut or liver involvement. Bone marrow improved while new tissues were involved, thus showing variability in progression of the disease. Decreased respiratory chain enzyme activities were demonstrated in muscle, and an mtDNA deletion was demonstrated in muscle, kidney, leukocytes, and fibroblasts.

A child is described who suffered from a severe congenital anemia. The anemia persisted and a regular transfusional regimen became necessary. Bone marrow aspirates showed an erythroblastic hyperplasia associated with some dyserythropoietic features. A most striking and constant finding was the presence of many inter-erythroblastic chromatin bridges. The reticulocyte count was always low, in spite of the increased erythropoietic activity. A beta-thalassemia trait inherited from the mother was demonstrated. The hypothesis of dyserythropoiesis/thalassemia syndrome was put forward, based on the assumption that the two genes may have interacted with each other.

The two main goals of this study were: to determine how O2 uptake, ventilation, and CO2 production during exercise were acutely affected by transfusion in children with congenital anemia (thalassemia major and Diamond-Blackfan syndrome) requiring hypertransfusion and chelation therapy and to compare gas exchange responses to exercise of the anemic patients to normal values. Thirteen patients (age range 7-27) performed cycle ergometry with a progressively increasing work rate. Gas exchange was measured breath-by-breath. Tests were done before and after routine transfusion (mean increase in hematocrit 22%). The results were compared to 10 age-matched normal children who performed the same protocol on two occasions separated by a 2-day interval, and to the results of 109 normal children studied in this laboratory. Transfusion resulted in: a small, but significant increase in the anaerobic threshold (9%) and an increase in the slope of the relationship between O2-uptake and heart rates. Despite these improvements, the majority of the patients had abnormally low values of maximal O2 uptake, anaerobic threshold, and slope of the O2 uptake-heart rate relationship. The abnormalities were more marked in the older patients. Measurement of gas exchange during exercise may be helpful in determining an optimal hematocrit for patients on hypertransfusion regimens.

A 12 year old Caucasian girl with congenital anemia and episodic jaundice was studied. Hemolysis was not present as evidenced by a normal Cr51 red cell survival time. Congenital dyserythropoietic anemia type II (Heimpel) was diagnosed after finding a positive acidified serum test of the circulating red cells and marked erythroid hyperplasia with erythroblastic multinuclearity in a bone marrow aspirate. A bone marrow specimen was labeled with H3T in vitro and autoradiographs prepared. Electron microscopy was also done on the same specimen. The percent of uninucleated normoblasts labeling with H3T indicated normal DNA synthesis. However, only 2% of the binucleated and none of the multinucleated polychromatophilic normoblasts labeled with H3T, indicating decreased DNA synthesis in these forms. By electron microscopy, excessive membrane structures forming invaginations or cisternae and encompassing the circumference of the cell in varying degrees were seen in the majority of the normoblasts. The nuclear membrane appeared normal. Despite the abnormality, nuclear extrusion was noted in these cells. Small cisternae were also found at the periphery of the cell in about 1–2% of the mature erythrocytes. These findings are suggestive that the cells with more severe structural abnormalities and/or decreased DNA synthesis are destroyed intramedullary and the circulating red cells are derived from a less abnormal portion of precursors.