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. 2000 Feb;10(2):165-73.
doi: 10.1101/gr.10.2.165.

The genomic region encompassing the nephropathic cystinosis gene (CTNS): complete sequencing of a 200-kb segment and discovery of a novel gene within the common cystinosis-causing deletion

J W Touchman  1 , Y Anikster, N L Dietrich, V V Maduro, G McDowell, V Shotelersuk, G G Bouffard, S M Beckstrom-Sternberg, W A Gahl, E D Green
Affiliations

The genomic region encompassing the nephropathic cystinosis gene (CTNS): complete sequencing of a 200-kb segment and discovery of a novel gene within the common cystinosis-causing deletion

J W Touchman et al. Genome Res. 2000 Feb.

Abstract

Nephropathic cystinosis is an autosomal recessive disorder caused by the defective transport of cystine out of lysosomes. Recently, the causative gene (CTNS) was identified and presumed to encode an integral membrane protein called cystinosin. Many of the disease-associated mutations in CTNS are deletions, including one >55 kb in size that represents the most common cystinosis allele encountered to date. In an effort to determine the precise genomic organization of CTNS and to gain sequence-based insight about the DNA within and flanking cystinosis-associated deletions, we mapped and sequenced the region of human chromosome 17p13 encompassing CTNS. Specifically, a bacterial artificial chromosome (BAC)-based physical map spanning CTNS was constructed by sequence-tagged site (STS)-content mapping. The resulting BAC contig provided the relative order of 43 STSs. Two overlapping BACs, which together contain all of the CTNS exons as well as extensive amounts of flanking DNA, were selected and subjected to shotgun sequencing. A total of 200,237 bp of contiguous, high-accuracy sequence was generated. Analysis of the resulting data revealed a number of interesting features about this genomic region, including the long-range organization of CTNS, insight about the breakpoints and intervening DNA associated with the common cystinosis-causing deletion, and structural information about five genes neighboring CTNS (human ortholog of rat vanilloid receptor subtype 1 gene, CARKL, TIP-1, P2X5, and HUMINAE). In particular, sequence analysis detected the presence of a novel gene (CARKL) residing within the most common cystinosis-causing deletion. This gene encodes a previously unknown protein that is predicted to function as a carbohydrate kinase. Interestingly, both CTNS and CARKL are absent in nearly half of all cystinosis patients (i.e., those homozygous for the common deletion). [The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AF168787 and AF163573.]

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Figures

Figure 1
Figure 1
BAC-based STS-content map of the region of chromosome 17p13 containing CTNS. A fully contiguous BAC contig map spanning the genomic segment encompassing CTNS is depicted, oriented with 17pter leftward and 17cen rightward. Shown along the top are the deduced positions of 43 STSs (spaced in an equidistant fashion from one another). Information about the STSs and their corresponding PCR assays is available in GenBank and/or GDB. Genetic markers are indicated by their D17S numbers, the one gene-specific STS by its assigned abbreviation (ASPA), and all the other STSs by their GenBank accession numbers. BACs are depicted as horizontal lines, with the length of each line reflecting the clone's STS content (as opposed to its insert size). The BAC names include the following prefixes reflecting the clone's library of origin: (RG) Research Genetics human BAC library; (GS) Genome Systems human BAC library; and (NH) Roswell Park Cancer Institute human BAC library RPCI-11. (●) The STS was verified to be present in that clone by PCR testing; (█) the STS was derived from the insert end of that BAC. The two BACs subjected to complete sequencing (see Fig. 2), which together contain the entire CTNS gene, are contained within a dashed box.
Figure 2
Figure 2
Long-range organization of genes within the 200-kb interval encompassing CTNS. The positions and intron/exon organization of five genes detected in the genomic sequence are schematically depicted, with the 17p telomere (Tel) leftward and the centromere (Cen) rightward. In each case, the introns and exons are drawn to scale, with vertical bars reflecting individual exons and arrows indicating the direction of transcription. The general position of a sixth gene, the vanilloid receptor/SIC gene, is also depicted; intron/exon organization is not shown due to the lack of available human cDNA sequence. The positions of the two sequenced BACs (RG147P12 and RG87B10) and the common 57-kb cystinosis-causing deletion are also shown. Additional structural details about this sequence, including the location of human repetitive elements, are provided in GenBank accession no. AF168787.
Figure 3
Figure 3
Genomic structure of CARKL. A detailed view of the intron/exon organization of CARKL is provided. Each of the seven exons is depicted to scale, with the hatched regions corresponding to the predicted ORF. Available 3′ and 5′ ESTs from the terminal exon (GenBank accession nos. indicated) are depicted. GRAIL2- and GENSCAN-predicted exons are indicated (top). The asterisks indicate cases where GRAIL2 incorrectly predicts the location of utilized splice sites. The CARKL mRNA sequence is provided in GenBank accession no. AF163573.
Figure 4
Figure 4
Expression profile of CARKL. A Northern blot containing 2 μg of poly(A)+ RNA from the indicated tissues was hybridized with a 1072-bp CARKL cDNA-specific probe spanning exons 2–7 (a) and then with a human β-actin-specific probe (b). Autoradiography was performed for 24 and 2 hr, respectively.
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