Characterization of a Putative Founder Mutation that Accounts for the High Incidence of Cystinosis in Brittany

Abstract

Abstract. Cystinosis is an autosomal recessive disorder, characterized by an accumulation of intralysosomal cystine, with an incidence of 1 in 100,000 to 200,000 live births. A higher incidence of cystinosis, 1 in 26,000 live births, has been reported in the western French province of Brittany. PCR amplification and sequencing has identified a 27-bp deletion starting 3 bp before the end of exon 8 and continuing into intron 8, 898-900+24del27, which has only been detected in families from this region. Reverse transcription—PCR amplification of RNA from an affected individual has shown that this mutation is indeed a splice-site mutation and results in the production of aberrant transcripts. These transcripts are predicted to either severely truncate cystinosin or alter its topology, thus accounting for the severe phenotype of these individuals. The mutation 898-900+24del27 has been identified in 7 of 18 alleles studied. This mutation is likely to be a founder mutation and would account for the higher incidence of cystinosis in Brittany.¹

Cystinosis (MIM 21980) is an autosomal recessive disorder characterized by an accumulation of intralysosomal cystine due to a defective cystine transport across the lysosomal membrane. Schematically, three clinical forms have been defined (¹). The infantile form appears at 6 to 8 mo of age with generalized proximal renal tubular dysfunction (the renal Fanconi syndrome) and progresses, if untreated, to end-stage renal failure before age 10 yr. Other clinical signs, such as retinal blindness, hypothyroidism, diabetes mellitus, swallowing difficulties, and neurologic deterioration, appear because of the accumulation of cystine in different organs. The juvenile form (MIM 219900) is characterized by glomerular damage, which manifests itself around age 10 to 12 yr and slowly progresses to glomerular insufficiency, photophobia, and late development of pigmentary retinopathy. The ocular nonnephropathic form (MIM 219750) is solely characterized by the presence of corneal cystine-crystal deposits that can result in mild photophobia but no renal anomalies.

In 1998, a positional cloning strategy led to the identification of the gene that underlies cystinosis, CTNS. CTNS is composed of 12 exons and extends over 23 kb (²). The detection of mutations in CTNS in affected individuals, which included splice-site, nonsense, and missense mutations, as well as in-and out-of-frame deletions/insertions, demonstrated that the three clinical forms are allelic (^2,3,4,5), as was previously suggested by complementation studies (⁶). The most common CTNS mutation is a 57-kb (⁷) deletion that involves the marker D17S829 and extends upstream from IVS10. This deletion has been detected in the homozygous state in individuals affected with the infantile form and in the heterozygous state in individuals with the juvenile form (²). It is thought to have arisen because of a founder effect that occurred in northern Europe around the middle of the first millennium AD (^8,9).

In North America and France, the incidence of infantile cystinosis has been estimated to between 1 in 100,000 and 200,000 live births (^1,10). However, a higher incidence of cystinosis has been reported in certain subpopulations around the world, in France (^10,11), the United Kingdom (¹²), Germany (¹³), and the French-speaking population of Quebec (¹⁴). The western province of Brittany in France has been reported as having an incidence of cystinosis of 1 in 26,000 live births (¹¹). We report here on the characterization of the mutation segregating in the affected unrelated families from Brittany. This mutation is likely to be a founder mutation and would account for the higher incidence of cystinosis in this region.

Materials and Methods

Participants

We studied five children from families, P8, P38, P43, P63, and P80, who presented with infantile cystinosis and originated from Brittany, for mutations in the CTNS gene. In family P8, it is the paternal grandmother who comes from the northern region of Brittany called Co[Combining Circumflex Accent]tes d'Armor. In families P38 and P43, both sets of parents come from the northwestern region of Brittany called Finistère. In family P63, the father comes from Brittany, and in family P80, the mother is from a town in the southern region of Brittany called Nantes. The clinical features of all affected individuals corresponded to the classical infantile cystinosis phenotype. The first symptoms of the probands were detected between age 6 and 36 mo (mean 15 mo) and were characterized by growth delay, polyuria, and/or rickets. The diagnosis was made between age 11 and 36 mo. At that time, the leukocyte cystine content was 2.01 to 7.5 nmol half-cystine/mg protein (mean 4.45). All probands developed corneal cystine crystal deposits and photophobia. They all received cysteamine throughout the course of the disease, although for two of them this treatment was started at age 22 and 23 yr. Four of these children are now between age 12 and 30 yr (there is no follow up for the fifth one). The three oldest probands reached end-stage renal failure at age 8, 10, and 13 yr, and two of them developed diabetes during renal transplantation and steroid therapy, and, later, cerebral complications.

DNA Extraction and Mutation Screening

Blood samples were obtained after informed consent from the probands, and parents, of all the families and DNA extracted by standard procedures. PCR detection of the 57-kb European deletion was carried out with the primers 65A and 65A2, which amplify a 360-bp junction fragment, and primers for the microsatellite D17S829, as described elsewhere (⁹). Mutation screening in the CTNS gene was initially performed by PCR amplification of all exons followed by single-strand conformation polymorphism analysis, as described previously (²). Exon 8 was subsequently amplified by use of the reported exon 8 forward primer (5′-CCCTGCCCTGTCTTGTCC-3′) coupled with the exon 9 reverse primer (5′-GCGTGTCTTCTGTCAAAGGT-3′) and the resulting PCR product directly sequenced.

RNA Isolation and Reverse Transcription—PCR Amplification

RNA was isolated from peripheral blood leukocytes from probands P38 and P43 by use of the RNAeasy mini kit (Qiagen). cDNA synthesis from approximately 1 μg of mRNA was carried out with the Superscript kit (Stratagene, La Jolla, CA) and a reverse primer, cDNAL/5 (5′-GTGGCCTTCAGAGAAAGAGC-3′), situated in the 3′ untranslated region of CTNS, in a total volume of 40 μl. Amplification of the critical region was carried out by nested PCR: the first amplification was performed with 1 μl of cDNA template and the primers A0187/1 (5′-ATGATAAGGAATTGGCTGAC-3′), situated in exon 3, and cDNAL/5; the second round of amplification was performed with 1/25th of the first PCR and the nested primers AO7.U (5′-TAAATGCAACCCTGGTGATC-3′) and AO7.6L (5′-CCAGGAGCACGTTGCCAATG-3′) situated in exons 5 and 11, respectively. PCR conditions were composed of 1 cycle of 94°C for 5 min, 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, followed by a final elongation step of 72°C for 5 min. Reverse transcription (RT)—PCR amplified fragments were gel extracted by use of the QiaexII Gel Extraction Kit (Qiagen, Venlo, The Netherlands), subcloned into the PCR product cloning plasmid pGEM-T Easy (Promega, Madison, WI), and sequenced.

Results

As a first step toward mutation screening, all affected individuals and their parents were tested by PCR for the presence of the marker D17S829 and the 360-bp junction fragment, to determine whether they were carriers of the 57-kb deletion. Probands from families P8, P63, and P80 tested positive for both D17S829 and the 360-bp junction fragment, which indicates that they were heterozygous carriers (data not shown). In contrast, probands from families P38 and P43 were found to be positive for D17S829 and negative for the junction fragment, which demonstrates that they did not carry the 57-kb deletion. Subsequently, the 10 coding CTNS exons were submitted to PCR amplification and single-strand conformation polymorphism analysis, to identify the mutation segregating in these families. All exons amplified and displayed a normal band pattern, with the exception of exon 8, for which no amplification product was detected when the flanking primers situated in intron (IVS) 7 and 8 were used (data not shown). This exon was subsequently amplified by the use of the same forward primer situated in IVS7 coupled with a reverse primer situated in IVS9 (see Materials and Methods section). Direct sequencing of the resultant PCR product demonstrated the existence of a 27-bp deletion beginning 3 bp before the end of exon 8 and continuing into IVS8 (898-900+24del27) (Figure 1). These results indicate that the patients either carry the 898-900+24del27 mutation in the homozygous state (P38 and P43) or are compound heterozygotes for the 898-900+24del27 mutation and the 57-kb deletion (P8, P63, and P80). In the latter cases, the transmission of the 898-900+24del27 mutation was found to be paternal in the first two cases and maternal in the third. The 898-900+24del27 mutation, detected in only one individual (P8) at that time, was included in the list of mutations that underlie classical infantile cystinosis compiled by Attard et al. (⁵).

Figure 1: Identification of 898-900+24del27 by sequencing. The upper panel shows the sequence of the end of exon 8 and the start of intron (IVS) 8 as obtained by direct sequencing of PCR amplified DNA from a control individual. The arrow marks the exon-intron boundary. The lower panel shows the sequence of the corresponding PCR product after amplification of DNA from an affected individual. The overlining indicates the extent of 898-900+24del27.

Because the 898-900+24del27 mutation spans the donor splice site of exon 8, it was predicted that it would likely affect splicing of CTNS. To test this hypothesis, RNA was isolated from peripheral blood leukocytes of the probands from families P38 and P43 and amplified by RT-PCR with nested primers situated in exons 5 and 11. This resulted in the amplification of mainly three products, a major band of 826 bp (transcript a) and two minor bands of 800 and 664 bp (transcripts b and c, respectively) (Figure 2); additional faint bands were amplified from proband P43 but could not be readily sequenced. RT-PCR amplification of RNA from a control individual with the same primers resulted in a 764-bp product. Sequencing of transcript a demonstrated that it arose from the retention of the remainder of IVS8 (which in its entirety has a size of 89 bp) starting from the end of the 27-bp deletion (Figure 3A). Transcript b arises from a missplicing of exon 7. A cryptic splice site (CAG/gtgatc) 26 bp before the end of exon 7 is used that deletes the end of exon 7. However, the region of exon 8 before the deletion and of IVS8 after the deletion are retained. Finally, transcript c arises from the splicing out of exon 8.

Figure 2: Detection of aberrant transcripts by reverse transcription (RT)—PCR amplification. Lane 1: RT-PCR amplification of RNA from an affected individual results in the detection of three products of 826, 800, and 664 bp (arrowheads). Lane 3: RT-PCR amplification of RNA from a control individual results in the detection of a 764-bp product. Lanes 2 and 4: as negative controls, reverse transcriptase was not added to the reactions.

Figure 3: Schematic representation of the aberrant transcripts and their encoded proteins. (A) The blue box corresponds to exon 7, the green box to exon 8, and the yellow box to exon 9. IVS7 and IVS8 are indicated by dashed lines. Sizes are to scale, 1 cm = 50 bp, with the exception of IVS7, as indicated by the parallel lines. Transcript a contains exon 7, the truncated exon 8 as indicated by a dashed line, part of IVS8 as indicated by a striped gray box, and exon 9. Transcript b contains the partially deleted exon 7, because of the use of a cryptic splice site (CAG/gtgatc) situated 27 bp before the end, as indicated by a dashed blue line, the out-of-frame exon 8 as indicated by a hatched green box, part of IVS8 as indicated by a striped gray box, and exon 9. Transcript c is missing exon 8 but contains an intact exon 7 and exon 9. (B) Cystinosin is composed of seven transmembrane domains, a novel lysosomal sorting motif (in red), a GYDQL lysosomal targeting signal (in blue) oriented toward the cytoplasm, and seven N-glycosylation sites located intralysosomally. The predicted protein A encoded by transcript a would terminate after the second transmembrane domain. The predicted protein B would be missing the end of the first transmembrane domain, the first cytoplasmic loop, and the second transmembrane domain. This region would be replaced by an insertion of 54 aa (in black) that does not form a transmembrane domain, thus altering the topology of cystinosin and reorienting the two sorting motifs intralysosomally. The predicted protein C would terminate at the end of the first cytoplasmic loop.

Discussion

We have characterized a germline mutation, 898-900+24del27, at the end of exon 8 of CTNS in five families with infantile cystinosis from Brittany. This mutation deletes the last 27 bp of exon 8 and was hypothesized to result in a splicing defect as the entire donor consensus splice site was removed. By RT-PCR amplification of RNA isolated from affected individuals who carry the mutation in the homozygous state, we confirmed this mutation as a splice site mutation and found that it resulted in the production of aberrant mRNA transcripts.

The 2.6-kb CTNS transcript encodes a 367 aa protein named cystinosin (²). Cystinosin is composed of seven transmembrane domains preceded by seven potential glycosylation sites at the amino-terminal end, and followed by a tyrosine-based lysosomal targeting signal (GYDQL) at the carboxy-terminal end (see Figure 3B). The lysosomal localization of cystinosin has recently been shown by in vitro immunofluorescence studies, and this targeting requires the presence of the GYDQL signal and a novel sorting motif situated in the cytoplasmic loop (aa 280 to 288) formed between the third and fourth transmembrane domains (¹⁵).

The largest of the three amplified aberrant transcripts created by 898-900+24del27, transcript a, would result in a frameshift that leads to the introduction of a stop codon 33 aa after the last intact codon before the deletion (aa position 185). The predicted truncated protein (A) would be 218 aa, terminating after the end of the second transmembrane domain (aa position 183) (Figure 3B). Transcript b would delete aa 146 to 154, induce a frame shift of 32 aa (the previous exon 8), and introduce 22 aa (retention of IVS8), before going back into frame at the start of exon 9 (aa position 188). Thus, the region 146 to 187 aa of cystinosin, which encodes the end of the first transmembrane domain, the first cytoplasmic loop and the second transmembrane domain, is essentially removed from the predicted elongated 379-aa protein (B). Finally, transcript c introduces a stop codon 10 aa after the deletion site, which results in a predicted 164-aa protein (C) that terminates after the end of the first cytoplasmic loop (aa 154).

Predicted proteins A and C are missing both the GYDQL tyrosine-based lysosomal targeting signal and the second lysosomal sorting motif (Figure 3B). Thus, these proteins would presumably never make it to the lysosome, probably being degraded within the cell, thus explaining the severe cystinosis phenotype observed in these families. The replacement of 42 aa with 54 others, creating protein B, would completely alter the topology of cystinosin, because the new aa residues do not form a transmembrane domain.

The mutation 898-900+24del27 has only been detected in five families from Brittany and not in other French or British families. All of these children are either homozygous or heterozygous for this mutation, the other mutation, in the latter case, being the 57-kb European deletion. Three of the five children have both parents originating from Brittany, whereas the other two have only one parent, the one carrying 898-900+24del27, coming from this region. At least five other children with infantile cystinosis and with both parents originating from Brittany exist, as was determined by the French epidemiologic survey of cystinosis (¹⁰). Four of these children carry the 57-kb deletion in the homozygous state, and the fifth child is from a consanguineous family and carries a homozygous splice site mutation, 1310-12G→A (⁵). On the whole, 7 of 18 mutant alleles from Brittany display the 898-900+24del27 mutant allele.

The identification of the same mutation in seemingly unrelated families from the same closed community is indicative of a common founder effect. Several founding mutations have now been found in the French Canadian population of Quebec, thus accounting for the higher incidence of cystinosis in this subpopulation, and these mutations have been found to be of both French and Irish origin (¹⁶). Interestingly, though, it is the “Celtic” mutation that has made the most significant contribution to cystinosis in the present-day population of French Canada. This mutation, W138X, has been detected in approximately half of the alleles studied, as is roughly the case for the 898-900+24del27 mutation. However, confirmation and dating of this novel putative founder mutation requires haplotype analysis, but this is hindered because of the small size and number of the families carrying the mutation. Nevertheless, the identification of the 898-900+24del27 mutation facilitates molecular diagnosis in these and future families from the Brittany area. Indeed, a simple diagnostic test would consist of an initial screening for the 57-kb deletion, as described elsewhere (⁹). The DNA of heterozygous carriers or individuals who do not carry this deletion would then be subsequently amplified by use of the forward primer situated in IVS7 coupled with the reverse primer situated in IVS9. In this way, the carriers of the 898-900+24del 27 mutation can be simply, rapidly, and efficiently identified.

We thank Yves Deris for assistance with artwork and all the physicians who referred the patients for this study. This work was supported by Vaincre les Maladies Lysosomales, the Association Franc[COMBINING CEDILLA]aise contres les Myopathies, and the Association pour l'Utilization du Rein Artificiel.