Genomic structure of the human congenital chloride diarrhea (CLD) gene

Abstract

Congenital chloride diarrhea (CLD) is caused by mutations in a gene which encodes an intestinal anion transporter. We report here the complete genomic organization of the human CLD gene which spans approximately 39 kb, and comprises 21 exons. All exon/intron boundaries conform to the GT/AG rule. An analysis of the putative promoter region sequence shows a putative TATA box and predicts multiple transcription factor binding sites. The genomic structure was determined using DNA from several sources including multiple large-insert libaries and genomic DNA from Finnish CLD patients and controls. Exon-specific primers developed in this study will facilitate mutation screening studies of patients with the disease. Genomic sequencing of a BAC clone H_RG364P16 revealed the presence of another, highly homologous gene 3′ of the CLD gene, with a similar genomic structure, recently identified as the Pendred syndrome gene (PDS).

Introduction

Congenital chloride diarrhea (CLD) is an autosomal recessive disorder of intestinal anion transport (Norio et al., 1971). It manifests as a lifelong acid diarrhea with high chloride concentration and if left untreated, the loss of fluids and electrolytes leads to death in early infancy. A defect has been suggested in ileal and colonic absorption of Cl⁻ in exchange for HCO₃⁻ (Holmberg, 1986; Holmberg et al., 1977). By a positional candidate gene strategy, the previously cloned gene Down-regulated in adenoma (DRA) (Schweinfest et al., 1993) was implicated as a candidate for CLD (Höglund et al., 1995, Höglund et al., 1996a). The finding of missense and frameshift mutations in 32 Finnish and four Polish CLD patients confirmed the identity of DRA as the CLD gene (Höglund et al., 1996b). The protein is most related to the sulfate transporter protein encoded by the human diastrophic dysplasia gene (DTDST) (Hästbacka et al., 1994, GenBank number: U14528) and to members of the sulfate permease gene family in other species (Table 1). In vitro, the CLD protein is a Na⁺-independent transporter of chloride (Moseley et al., submitted), sulfate and oxalate (Silberg et al., 1995).

The full coding region of the human DRA cDNA has been determined previously (Schweinfest et al., 1993, GenBank number L02785). Its open reading frame of 764 amino acids predicts a protein of 84.5 kDa. CLD has 10, 12, or 14 putative hydrophobic membrane spanning domains (Bairoch and Apweiler, 1996; Bissig et al., 1994; Byeon et al., 1996; Hästbacka et al., 1994). Amino- and carboxy-terminal ends are intracellular (Byeon et al., 1996).

To allow for comprehensive mutation screening of congenital chloride diarrhea patients and to understand the evolution of the sulfate transporter gene, the complete genomic structure of the CLD gene was determined and exon-specific primers were designed.