Overhydrated Hereditary Stomatocytosis

A number sign (#) is used with this entry because of evidence that overhydrated hereditary stomatocytosis (OHST) is caused by heterozygous mutation in the RHAG gene (180297) on chromosome 6p12.

Description

Overhydrated hereditary stomatocytosis is a variably compensated macrocytic hemolytic anemia of fluctuating severity, characterized by circulating erythrocytes with slit-like lucencies (stomata) evident on peripheral blood smears. OHST red cells exhibit cation leak, resulting in elevated cell Na+ content with reduced K+ content, with increased ouabain-resistant cation leak fluxes in the presence of presumably compensatory increases in ouabain-sensitive Na(+)-K(+) ATPase activity, and red cell age-dependent loss of stomatin/EBP7.2 (EBP72; 133090) from the erythroid membrane. Clinically, patients with OHST exhibit overhydrated erythrocytes and a temperature-dependent red cell cation leak. The temperature dependence of the leak is 'monotonic' and has a steep slope, reflecting the very large leak at 37 degrees centigrade (summary by Bruce, 2009 and Stewart et al., 2011).

For a discussion of clinical and genetic heterogeneity of the hereditary stomatocytoses, see 194380.

Clinical Features

Lock et al. (1961) described a 'new' hereditary red cell anomaly associated with hemolytic anemia. They referred to it as stomatocytosis because of a pale-staining band in the erythrocytes. Erythrocytes showed shortened survival and increased osmotic fragility. Stomatocytes are uniconcave with a slit-like rather than a circular area of central pallor in stained preparations. It is clear that there is more than one disorder manifested by stomatocytosis and hemolytic anemia. In the family reported by Lock et al. (1961), Mabin and Chowdhury (1990) and Barton et al. (2003) described a hemolytic aplastic crisis with death in an adult from parvovirus infection. Barton et al. (2003) discussed problems of management associated with members of the Jehovah's Witnesses sect opposing transfusion for religious reasons.

Meadow (1967) studied a 5-year-old girl who presented at age 3.75 years with recurrent episodes of jaundice, each time preceded by a cough, fever, and malaise. Examination revealed a pale, slightly jaundiced girl with marked splenomegaly as well as moderate hepatomegaly; she was anemic with 30% reticulocytes, and spherocytes were present in the blood film as well. She underwent splenectomy, and histology was compatible with congenital hemolytic anemia. Postoperatively she continued to be anemic, with reticulocytosis and abnormal red cell appearance, and she also had occasional bouts of jaundice. Blood films at age 5 revealed stomatocytosis, with as many as 30 to 40% stomatocytes in some films. Meadow (1967) stated that this was the fourth reported case of stomatocytic congenital hemolytic anemia; however, noting that the presence of stomatocytes was initially missed in this patient as well as in the 2 cases described by Lock et al. (1961), Meadow (1967) proposed that the condition might not be as rare as its documentation would suggest.

In a child with hemolytic anemia, Zarkowsky et al. (1968) found high sodium (100 mEq per liter) and low potassium (40 mEq per liter) in the red cells. Splenectomy was beneficial. Both parents were of Hungarian descent. They and a female sib had normal blood studies.

In 3 males in 3 successive generations, Oski et al. (1969) found hemolytic anemia, stomatocytic red cells, and increased red cell fragility. Old cells were less dense than young cells and had a high sodium/low potassium content.

Mentzer et al. (1975) studied red blood cells from a 6-year-old boy of Portuguese ancestry with hereditary stomatocytosis and severe hemolytic anemia, who presented with jaundice, hyperbilirubinemia, chronic anemia, and hepatosplenomegaly, and who underwent splenectomy at 4.5 years of age. Blood smear showed 10 to 15% stomatocytes presplenectomy, with an increase to 75% postsplenectomy. Osmotic fragility of both fresh and incubated erythrocytes was greatly increased. Deformability was reduced, and ferrokinetic studies showed that these rigid cells were sequestered by the spleen. The patient's high sodium/low potassium erythrocytes were markedly permeable to both sodium and potassium, and active cation transport was more than 10 times normal, sustained by an increase of similar magnitude in glycolysis. Deprived of glucose, cells rapidly became swollen and lysed; Mentzer et al. (1975) noted that prolonged entrapment in acidic, hypoglycemic regions of the spleen would recapitulate these unfavorable events in vivo. Splenectomy was followed by an improvement in red cell survival, although there was evidence of continuing hemolysis. The boy's parents and 4 sibs were clinically and hematologically normal, with normal red cell Na+ and K+ concentrations.

Mentzer et al. (1976) found that the extreme defect in cation permeability of hereditary stomatocytosis could be corrected in vitro by a bifunctional imidoester, dimethyl adipimidate. After restoration of normal permeability, membrane rigidity, morphology, and cell cation and water content were corrected also. Sheep show a polymorphism of red cell potassium and sodium concentration. So-called LK sheep have low potassium and high sodium, whereas HK sheep have the converse. Low potassium is dominant to high potassium. A precisely comparable situation has not been found in man. (See review by Lush, 1966).

Lande et al. (1982) found absent band 7 membrane protein (stomatin; 133090) in 2 patients with high sodium/low potassium erythrocytes, 1 of whom was the Portuguese boy originally studied by Mentzer et al. (1975). They focused on a 28-kD component of normal erythrocyte membranes.

Eber et al. (1989) studied red cell membrane composition in 4 patients with hereditary stomatocytosis, including a 22-year-old mother previously reported by Schroter et al. (1981) and her affected 2-year-old daughter, a 24-year-old Hungarian man originally described by Zarkowsky et al. (1968), and a 23-year-old German man previously reported by Bienzle et al. (1975). All 4 patients exhibited congenital hemolytic anemia and splenomegaly, with partial improvement after splenectomy in the 3 affected adults. Hematology showed stomatocytosis, with low hemoglobin, elevated reticulocyte count, elevated mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and high sodium/low potassium RBCs. Gel electrophoresis in all 4 patients demonstrated complete absence of the same 28-kD integral protein 7.2 that was found to be lacking in 2 patients by Lande et al. (1982); Eber et al. (1989) suggested that absence of stomatin might be a unifying feature of hereditary stomatocytosis.

Argent et al. (2004) described a boy, born to consanguineous Tunisian parents, who showed a severe multisystem disease with dyserythropoietic sideroblastic anemia of neonatal onset, delayed neurologic development with hypotonia and convulsions, salt-losing nephropathy, chronic watery diarrhea, lacticacidosis with mitochondrial dysfunction, brittle hair, hypergammaglobulinemia, fatty liver with intermittent transaminasemia, and terminal pulmonary fibrosis. The boy had a total of 5 sibs of both sexes; 2 were stillborn and 2, both born with hydrops fetalis, lived only a short time. One sister was alive and well. The severe red cell defect in the patient described by Argent et al. (2004) prompted them to perform SDS gel analysis of the red cell membranes, which showed a deficiency within 'band 7' at 32 kD. Analysis of the gene encoding stomatin revealed a complex series of aberrant splice forms centered around exon 3, most missing some or all of exon 2, all of exon 3, and some or all of exon 4. Argent et al. (2004) could find no genomic lesion to explain the aberrant splice forms, and analysis of parental DNA showed no abnormalities. The red cell abnormalities in this patient included marked poikilocytosis, some stomatocytes, polychromasia, anisocytosis, some schizocytes, target cells, and red cells with basophilic granulations. He was transfusion-dependent from birth. He had persistently high aldosterone and renin (179820) and high ferritin (see 134790) at age 2.5 years. Argent et al. (2004) noted that the multisystem pathology in this patient did not conform to any previously described syndrome. Although the disorder differed clinically from OHST, the stomatin defect was comparable to that observed in OHST (Eber et al., 1989).

Morle et al. (1989) reported a French boy with hemolytic anemia who exhibited pallor, intermittent jaundice, respiratory infections, and abdominal pain. Hematology showed 15 to 20% stomatocytes, a marked increase in red cell volume, and an increased reticulocyte count. At age 6 years, he underwent splenectomy, which partially corrected the anemia, but macrocytosis and increased reticulocyte count persisted. Osmotic resistance and density were reduced, but deformity was unchanged in isotonicity. Erythrocyte Na+ was high and K+ low, and membrane band 7 was reduced by 72% compared to controls. Morle et al. (1989) excluded the diagnosis of cryohydrocytosis (185020) because of the absence of significant change in the osmotic ektacytometric gradient curve after storage in the cold. The proband's deceased father was apparently also affected: he underwent splenectomy at age 14 years, and had persistent anemia and macrocytosis. He died of embolism to the basilar artery. The proband's mother and a brother were asymptomatic and hematologic analysis showed no abnormalities.

Kanzaki and Yawata (1992) studied clinical heterogeneity and the role of red cell membrane protein band 7 in membrane transport in 44 patients with hereditary stomatocytosis with normal red cell membrane lipids. These patients were arbitrarily categorized into 3 phenotypes, based on the extent of sodium influx: hereditary stomatocytosis type I, with markedly increased Na influx; type II, with moderately increased Na influx; and type III, with normal Na influx. The extent of anemia and jaundice was almost identical in the 3 groups. Approximately one-third of the cases (14/44) showed no overt hemolysis, even with marked stomatocytosis. Cell hydration was abnormal in type I (MCV = 119.6) but normal in types II and III (MCV = 98.2 and 94.1). The studies suggested that band 7 may not be involved in an essential way in the formation of the stomatocytic changes.

Huppi et al. (1991) observed marked stomatocytosis in an infant female born at 31 weeks' gestation who showed severe hemolytic anemia and hyperbilirubinemia for which exchange transfusion was performed. The mother also had congenital stomatocytosis.

In reviewing hereditary stomatocytosis and stomatin, Stewart et al. (1993) defined 2 common forms of hereditary spherocytosis: 'overhydrated' and 'dehydrated.' Although stomatin, an integral membrane protein which acts as a channel regulator, is deficient in the 'overhydrated' form, Northern blots showed the presence of normal-sized mRNA, and RT-PCR sequencing of the patients' RNA revealed only a conservative third base position change (Stewart and Argent, 1992), presumably reflecting a polymorphism. Carella et al. (1998) referred to hereditary xerocytosis (194380) as dehydrated hereditary stomatocytosis (DHS); they referred to the disorder discussed here as overhydrated hereditary stomatocytosis (OHST). The state of hydration of the cells reflects the relative permeability of Na to K. Cases in which the increase in K-permeability outweighs that of Na, as well as those in which the reverse is true, show dehydration.

Fricke et al. (2003) studied red cell membrane stomatin levels in affected individuals from 2 previously reported OHST families (Lock et al., 1961; Meadow, 1967). Using an antistomatin antibody, Fricke et al. (2003) found that stomatin was present in some red cells from these patients, and flow cytometry analysis indicated that younger cells had more stomatin. Immunocytochemistry and Western blotting revealed that in OHST patients, the protein was present in spleen, liver, neutrophils, platelets, monocytes, and approximately 50% of peripheral lymphocytes, with the same distribution as in controls. In addition, there was no significant abnormality in the stomatin gene sequence in these patients. Fricke et al. (2003) concluded that the deficiency of stomatin from OHST red cells is due to loss of stomatin as an 'innocent victim' from abnormal erythrocytes, upon their maturation in the bone marrow and in the circulation.

Fricke et al. (2004) reported a female patient of Somalian descent and a French male patient with OHST, as well as an affected Hispanic mother and 2 children. Red cell analysis showed that all 5 affected individuals had elevated intracellular Na+ with a reciprocal decrease in K+. The probands from all 3 families showed a more than 20-fold increase in the K+ leak at 37 degrees centigrade compared to control, with simple monotonic temperature dependence. However, the NaK pump acceleration in the Somalian patient was only about 3 times normal, whereas that of the French patient was 15 times normal (NaK pump rate was not measured in the Hispanic proband). Stomatin was not completely absent from patient erythrocytes, but was present at variable levels, e.g., 23% of normal in the Somalian patient and 5.6% in the French patient, compared to 3.4% and 15.7% in the previously reported Stockport (Lock et al., 1961) and Brighton (Meadow, 1967) patients. Immunocytochemistry showed that nearly all stomatocytes were stomatin-negative in the French and Hispanic probands, whereas there was only a minority of stomatin-deficient red cells in the Somalian patient. Fricke et al. (2004) concluded that stomatin is deficient only in very Na(+)-K(+) leaky examples of the hereditary stomatocytoses, with all of the stomatin-deficient cases showing higher leak K+ flux than nonstomatin-deficient cases.

Clinical Management

Stewart et al. (1996) documented postsplenectomy thrombotic complications in affected individuals from 4 families with OHST, including the families previously reported by Lock et al. (1961), Meadow (1967), Mentzer et al. (1975), and Eber et al. (1989), as well as in patients from 3 families with DHS. Stewart et al. (1996) stated that because splenectomy is only of limited therapeutic benefit in stomatocytosis, it should not be performed without careful consideration. The authors also noted that a tendency to iron overload is evident in many of these patients, even without hypertransfusion and irrespective of splenectomy.

Molecular Genetics

In 7 OHST kindreds, including 6 that had previously been reported and designated as the Stockport (Lock et al., 1961), Brighton (Meadow, 1967), Grenoble (Morle et al., 1989), and Harrow, Toulouse, and Albuquerque (Fricke et al., 2004) kindreds, Bruce et al. (2009) analyzed the candidate gene RHAG and identified heterozygosity for the same missense mutation in affected individuals from 6 of the families (F65S; 180297.0011). The remaining patient (Harrow; of Somalian descent) was heterozygous for a different missense mutation (I61R; 180297.0012). Neither mutation was found in 56 controls. Bruce et al. (2009) stated that all affected individuals exhibited 'classic' OHS, with very leaky red cells showing an approximately 40-fold increase in Na+ and K+ transport rates, resulting in moderate hemolytic anemia. Mean red cell volumes were abnormally large, and blood smears showed stomatocytes, echinocytes, target cells, and macrocytes, as well as punctate red cells in a splenectomized patient. Osmotic ektacytometry displayed a shifted curve typical of OHS. Expression in Xenopus laevis oocytes demonstrated that the mutant proteins induced a large monovalent cation leak.

Stewart et al. (2011) identified heterozygosity for the RHAG F65S mutation in 4 unrelated individuals with overhydrated stomatocytosis, including the patient originally reported by Mentzer et al. (1975). The mutation was shown to have arisen de novo in the 1 patient for whom parental DNA was available. Severity of anemia varied among the 4 patients; only some patient red cells showed stomatocytic morphology, but all patients had macrocytosis beyond that expected for the degree of reticulocytosis. Noting that 17 of 18 OHST patients with published RHAG mutations harbored a heterozygous F65S mutation, Stewart et al. (2011) concluded that F65S represents a mutation hotspot for RHAG-associated OHS.

In a mother and daughter with OHST previously reported by Eber et al. (1989), Shmukler et al. (2013) identified heterozygosity for the F65S mutation in the RHAG gene. The mutation was also present in the daughter's affected son, but was not found in 3 unaffected family members.