Band 3, also known as anion exchange 1 (AE1), is a membrane glycoprotein that mediates chloride/ bicarbonate exchange. It is encoded by the human solute carrier family 4 anion exchange member 1 (SLC4A1) gene, which is expressed both at the red blood cell membrane (eAE1) and at the basolateral membrane of alpha intercalated cells in the distal tubules of the kidney (kAE1) [1, 2]. Mutations in SLC4A1 may cause both a renal acidification defect manifesting as distal renal tubular acidosis (dRTA), as well as red cell dysmorphology which include hereditary spherocytosis, hereditary stomatocytosis and South East Asian ovalocytosis [3]. The co-existence of dRTA and ovalocytosis is common in certain geographical areas; however, co-existence of dRTA with hereditary spherocytosis has rarely been reported [4-8]. Here we report 3 siblings in a Southern Indian family with dRTA and hereditary spherocytosis. The patients described here were listed in a review of tropical dRTA [9], but a full description of these unusual cases has not been reported.

The first of the twin, offspring of non-consanguineous parentage, presented at 2 years and 10 months of age with failure to thrive and features of rickets. Evaluation, including frusemide challenge test, showed normal vitamin D levels and renal functions, normal anion gap hypokalemic metabolic acidosis, with high urine pH (pH 6.0 following frusemide administration), hypercalciuria and nephrocalcinosis and was conclusive of dRTA. Complete blood count revealed compensated hemolysis (reticulocyte count 5.4%, hemoglobin level 10.5 g/dL) with presence of spherocytes and acanthocytes. The diagnosis of hereditary spherocytosis was confirmed by positive osmotic fragility test. Her twin sister was consulted shortly afterwards, with similar complaints and clinical picture and was also found to have dRTA (normal anion gap metabolic acidosis with urine pH of 6.1 following frusemide challenge) and hereditary spherocytosis (reticulocyte count 4.7%, hemoglobin 9.1 g/dL and spherocytes and acanthocytes).

Both children responded to alkali supplementation (2.5 to 3 mmoml/Kg of bicarbonate) with normalisation of blood chemistry and significant improvement of their growth. Their younger sibling, 14-months-old boy, evaluated for poor weight gain, was found to have similar clinical features and was also diagnosed to have dRTA (hypokalemic metabolic acidosis, with high urine pH); and hereditary spherocytosis. Physical growth and development improved significantly, following alkali therapy, within weeks.

The gene encoding AE1 (SLC4A1) was analysed (Fig. 1) and a c2573a mutation found, resulting in the substitution of alanine by aspartic acid, Ala858Asp. All patients were homozygous for this substitution while their parents were heterozygous for the same mutation. The parents had clinically normal blood picture and renal functions.

(A) Parents C2537 A858D	(B) Children A2537 D858
Fig. 1 DNA sequencing of SLC4A1 shows that the parents have the heterozygous mis-sense mutation C2537A which leads to amino acid substitution A858D (A) whereas their children were homozygous for the same point mutation A2537/D858 (B).

The rarity of the coexistence of both HS and dRTA in the same patient despite sharing a common protein has been a source of various postulations. Presence of glycophorin A in the red cells in contrast to its absence in the alpha – intercalated cells has been postulated as one of the explanations. Glycophorin acts as a chaperone and improves eAE1 trafficking to the red cell membrane. Mutations in SLC4A1 that specifically cause dRTA affect trafficking of kAE1 in the internal membranes, kAE1 is either held up in the endoplasmic reticulum or Golgi body or delivered to the wrong plasma membrane, i.e. the apical membrane instead of the basolateral membrane in alpha-intercalated cells [10].

The Ala858Asp mutation is effectively a mild mutation. A previous report showed that heterozygous Ala858Asp individuals express 80% AE1 in their red cell membranes, compared to normal controls, not low enough to cause hereditary spherocytosis [4]. However homozygous Ala858Asp patients will therefore have d" 60% AE1, a point at which the membrane becomes unstable and hereditary spherocytosis ensues.

Acknowledgements: Professor Oliver Wrong initiated the collaboration that made　this study possible　but passed away in February 2012 before the work was completed. We acknowledge the contribution of Dr LK Joseph and Dr Pragatheesh P from CMC, Vellore, India in collecting the data for the patient and their family. The work was supported by the UK National Health Service R & D Directorate (LJB, WMB).

Contributors: All the authors have contributed, designed and approved the study.

Funding: None; Competing interests: None stated.

1. Schofield AE, Martin PG, Spillett D, Tanner MJ. The structure of the human red blood cell anion exchanger (EPB3, AE1, band 3) gene. Blood. 1994; 84:2000-12.

2. Wagner S, Vogel R, Lietzke R, 　Koob R,　Drenckhahn D. Immunochemical characterization of a band 3-like anion exchanger in collecting duct of human kidney. Am J Physiol. 1987;253:F213–F21.

3. Stewart AK, Kurschat CE, Alper SL. The SLC4 anion exchanger gene family, In: Alpern RJ, Hebert S. (Eds), Selden and Giebisch’s "The Kidney, Physiology and Patholophysiology" 4th. Ed Amsterdam: Elsevier Academic Press; 2008, p.1499-1537.

4. Bruce LJ, Wrong O, Toye AM, Young MT, Ogle G, Ismail Z, et al. Band 3 mutations, renal tubular acidosis and South-East Asian ovalocytosis in Malaysia and Papua New Guinea: loss of up to 95% band 3 transport in red cells. Biochem J. 2000;350:41-51.

5. Ribeiro ML, Alloisio N, Almeida H, Gomes C, Texier P, Lemos C, et al. Severe hereditary spherocytosis and distal renal tubular acidosis associated with the total absence of band 3. Blood. 2000;96:1602-4.

6. Chu C, Woods N, Sawasdee N, Guizouarn H, Pellissier B, Borgese F, et al. Band 3 Edmonton I, a novel mutant of the anion exchanger 1 causing spherocytosis and distal renal tubular acidosis. Biochem J. 2010;426:379-88.

7. Shmukler BE, Kedar PS, Warang P, Desai M, Madkaikar M, Ghosh K, et al. Hemolytic anemia and distal renal tubular acidosis in two Indian patients homozygous for SLC4A1/AE1 mutation A858D. Am J Hematol. 2010;85:824-8.

8. Fawaz NA, Beshlawi IO, Al Zadjali S, Al Ghaithi HK, Elnaggari MA, Elnour I, et al. dRTA and hemolytic anemia: first detailed description of SLC4A1 A858D mutation in homozygous state. Eur J Haematol. 2012;88:350-5.

9. Khositseth S, Bruce LJ, Walsh SB, Bawazir WM, Ogle GD, Unwin RJ, et al. Tropical distal renal tubular acidosis: clinical and epidemiological studies in 78 patients. QJM. 2012;105:861-77.