CTNS mutations in an American-based population of cystinosis patients.

Nephropathic cystinosis is an autosomal recessive lysosomal storage disease characterized by renal failure at 10 years of age and other systemic complications. The gene for cystinosis, CTNS, has 12 exons. Its 2.6-kb mRNA codes for a 367-amino-acid putative cystine transporter with seven transmembrane domains. Previously reported mutations include a 65-kb "European" deletion involving marker D17S829 and 11 small mutations. Mutation analysis of 108 American-based nephropathic cystinosis patients revealed that 48 patients (44%) were homozygous for the 65-kb deletion, 2 had a smaller major deletion, 11 were homozygous and 3 were heterozygous for 753G-->A (W138X), and 24 had 21 other mutations. In 20 patients (19%), no mutations were found. Of 82 alleles bearing the 65-kb deletion, 38 derived from Germany, 28 from the British Isles, and 4 from Iceland. Eighteen new mutations were identified, including the first reported missense mutations, two in-frame deletions, and mutations in patients of African American, Mexican, and Indian ancestry. CTNS mutations are spread throughout the leader sequence, transmembrane, and nontransmembrane regions. According to a cystinosis clinical severity score, homozygotes for the 65-kb deletion and for W138X have average disease, whereas mutations involving the first amino acids prior to transmembrane domains are associated with mild disease. By northern blot analysis, CTNS was not expressed in patients homozygous for the 65-kb deletion but was expressed in all 15 other patients tested. These data demonstrate the origins of CTNS mutations in America and provide a basis for possible molecular diagnosis in this population.     

Two novel CTNS mutations in cystinosis patients in Thailand

Cystinosis is an autosomal recessive disorder characterized by defective transport of cystine across the lysosomal membrane and resulting in renal, ophthalmic, and other organ abnormalities. Mutations in the CTNS gene cause a deficiency of the transport protein, cystinosin. We performed mutation analysis of CTNS in six cystinosis patients from four families in Thailand. Using PCR sequencing of the entire coding regions, we identified all eight mutant alleles, including two mutations, p.G309D and p.Q284X, that have not been previously reported. This study expands the mutational and population spectrum of nephropathic cystinosis.     

Characterization of seven novel mutations on the HEXB gene in French Sandhoff patients

Sandhoff disease (SD) is an autosomal recessive lysosomal storage disease caused by mutations in the HEXB gene encoding the beta subunit of hexosaminidases A and B, two enzymes involved in GM2 ganglioside degradation. Eleven French Sandhoff patients with infantile or juvenile forms of the disease were completely characterized using sequencing of the HEXB gene. A specific procedure was developed to facilitate the detection of the common 5′-end 16 kb deletion which was frequent (36% of the alleles) in our study. Eleven other disease-causing mutations were found, among which four have previously been reported (c.850C>T, c.793T>G, c.115del and c.800_817del). Seven mutations were completely novel and were analyzed using molecular modelling. Two deletions (c.176del and c.1058_1060del), a duplication (c.1485_1487dup) and a nonsense mutation (c.552T>G) were predicted to strongly alter the enzyme spatial organization. The splice mutation c.558+5G>A affecting the intron 4 consensus splice site led to a skipping of exon 4 and to a truncated protein (p.191X). Two missense mutations were found among the patients studied. The c.448A>C mutation was probably a severe mutation as it was present in association with the known c.793T>G in an infantile form of Sandhoff disease and as it significantly modified the N-terminal domain structure of the protein. The c.171G>C mutation resulting in a p.W57C amino acid substitution in the N-terminal region is probably less drastic than the other abnormalities as it was present in a juvenile patient in association with the c.176del. Finally, this study reports a rapid detection of the Sandhoff disease-causing alleles facilitating genetic counselling and prenatal diagnosis in at-risk families.     

FISH diagnosis of the common 57-kb deletion in <Emphasis Type="Italic">CTNS</Emphasis> causing cystinosis

Cystinosis is an autosomal recessive lysosomal storage disease caused by mutations in CTNS. The most prevalent CTNS mutation, a 57-kb deletion, occurs in ~60% of patients in the United States and northern Europe and removes exons 1–9, most of exon 10, the CTNS promoter region, and all of an adjacent gene of unknown function called CARKL. CTNS codes for the lysosomal cystine transporter, whose absence leads to intracellular cystine accumulation, widespread cellular destruction, renal Fanconi syndrome in infancy, renal glomerular failure in later childhood, and other systemic complications. Because treatment with oral cysteamine can prevent or delay these complications significantly, early and accurate diagnosis is critical. This study describes the generation of fluorescence in situ hybridization (FISH) probes for the 57-kb deletion in CTNS, enabling cytogenetics laboratories to test for this common mutation. The probes would also be able to detect a less frequent 11.7-kb deletion. A blinded study was performed using multiplex PCR analysis as the gold standard to determine the presence or absence of the 57-kb deletion. The FISH probes, evaluated on 12 lymphoblastoid cell lines from singly deleted, doubly deleted, and nondeleted patients, made the correct diagnosis in every case. This appears to be the first FISH-based diagnostic method described for any lysosomal storage disorder. It can assist in the antenatal and perinatal diagnosis of cystinosis and promote earlier salutary therapy with cysteamine.     

Molecular characterization of CTNS deletions in nephropathic cystinosis: development of a PCR-based detection assay.

Nephropathic cystinosis is an autosomal recessive disorder that is characterized by accumulation of intralysosomal cystine and is caused by a defect in the transport of cystine across the lysosomal membrane. Using a positional cloning strategy, we recently cloned the causative gene, CTNS, and identified pathogenic mutations, including deletions, that span the cystinosis locus. Two types of deletions were detected-one of 9.5-16 kb, which was seen in a single family, and one of approximately 65 kb, which is the most frequent mutation found in the homozygous state in nearly one-third of cystinotic individuals. We present here characterization of the deletion breakpoints and demonstrate that, although both deletions occur in regions of repetitive sequences, they are the result of nonhomologous recombination. This type of mechanism suggests that the approximately 65-kb deletion is not a recurrent mutation, and our results confirm that it is identical in all patients. Haplotype analysis shows that this large deletion is due to a founder effect that occurred in a white individual and that probably arose in the middle of the first millenium. We also describe a rapid PCR-based assay that will accurately detect both homozygous and heterozygous deletions, and we use it to show that the approximately 65-kb deletion is present in either the homozygous or the heterozygous state in 76% of cystinotic patients of European origin.     

Novel mutations in the 3' region of the polycystic kidney disease 1 (PKD1) gene

Mutation screening in 90 unrelated ADPKD1 patients was carried out on some of the exons in the single copy area (37, 38, 39, 44, 45) using genomic PCR and SSCP. Four novel mutations were found: a 15 bp in-frame deletion in exon 39 [nt11449 (del 15)], a 2 bp deletion in exon 44 [nt12252 (del 2)], a G insertion in exon 44 [nt12290 (Ins G)], and a GTT in-frame deletion in exon 45 [nt12601 (del 3)].     

The NF1 gene contains hotspots for L1 endonuclease-dependent de novo insertion

Long interspersed (L1) and Alu elements are actively amplified in the human genome through retrotransposition of their RNA intermediates by the -100 still retrotranspositionally fully competent L1 elements. Retrotransposition can cause inherited disease if such an element is inserted near or within a functional gene. Using direct cDNA sequencing as the primary assay for comprehensive NF1 mutation analysis, we uncovered in 18 unrelated index patients splicing alterations not readily explained at the genomic level by an underlying point-mutation or deletion. Improved PCR protocols avoiding allelic drop-out of the mutant alleles uncovered insertions of fourteen Alu elements, three L1 elements, and one poly(T) stretch to cause these splicing defects. Taken together, the 18 pathogenic L1 endonuclease-mediated de novo insertions represent the largest number of this type of mutations characterized in a single human gene. Our findings show that retrotransposon insertions account for as many as -0.4% of all NF1 mutations. Since altered splicing was the main effect of the inserted elements, the current finding was facilitated by the use of RNA-based mutation analysis protocols, resulting in improved detection compared to gDNA-based approaches. Six different insertions clustered in a relatively small 1.5-kb region (NF1 exons 21(16)-23(18)) within the 280-kb NF1 gene. Furthermore, three different specific integration sites, one of them located in this cluster region, were each used twice, i.e. NM_000267.3(NF1):c.1642-1_1642 in intron 14(10c), NM_000267.3(NF1):c.2835_2836 in exon 21(16), and NM_000267.3(NF1):c.4319_4320 in exon 33(25). Identification of three loci that each served twice as integration site for independent retrotransposition events as well as 1.5-kb cluster region harboring six independent insertions supports the notion of non-random insertion of retrotransposons in the human genome. Currently, little is known about which features make sites particularly vulnerable to L1 EN-mediated insertions. The here identified integration sites may serve to elucidate these features in future studies.     

Mutations in the melanocortin 1 receptor (MC1R) gene are associated with coat colours in the domestic rabbit (Oryctolagus cuniculus)

We sequenced almost the complete coding region of the MC1R gene in several domestic rabbits (Oryctolagus cuniculus) and identified four alleles: two wild-type alleles differing by two synonymous single nucleotide polymorphisms (c.333A>G;c.555T>C), one allele with a 30-nucleotide in-frame deletion (c.304_333del30) and one allele with a 6-nucleotide in-frame deletion (c.280_285del6). A polymerase chain reaction-based protocol was used to distinguish the wild-type alleles from the other two alleles in 263 rabbits belonging to 37 breeds or strains. All red/fawn/yellow rabbits were homozygous for the c.304_333del30 allele. This allele represents the recessive e allele at the extension locus identified through pioneering genetic studies in this species. All Californian, Checkered, Giant White and New Zealand White rabbits were homozygous for allele c.280_285del6, which was also observed in the heterozygous condition in a few other breeds. Black coat colour is part of the standard colour in Californian and Checkered breeds, in contrast to the two albino breeds, Giant White and New Zealand White. Following the nomenclature established for the rabbit extension locus, the c.280_285del6 allele, which is dominant over c.304_333del30, may be allele E(D) or allele E(S).     

More than one mutant allele causes infantile Tay-Sachs disease in French-Canadians

Two Tay-Sachs disease (TSD) patients of French-Canadian origin were shown by Myerowitz and Hogikyan to be homozygous for a 7.6-kb deletion mutation at the 5' end of the hexosaminidase A α-subunit gene. In order to determine whether all French-Canadian TSD patients were homozygotes for the deletion allele and to assess the geographic origins of TSD in this population, we ascertained 12 TSD families of French-Canadian origin and screened for occurrence of mutations associated with infantile TSD. DNA samples were obtained from 12 French-Canadian TSD families. Samples were analyzed using polymerase-chain-reaction (PCR) amplification followed by hybridization to allele-specific oligonucleotides (ASO) or by restriction analysis of PCR products. In some cases Southern analysis of genomic DNA was performed. Eighteen of the 22 independently segregating mutant chromosomes in this sample carried the 7.6-kb deletion mutation at the 5' end of the gene. One chromosome carried the 4-nucleotide insertion in exon 11 (a “Jewish” mutation). In this population no individuals were detected who had the substitution at the splice junction of exon 12 previously identified in Ashkenazi Jews. One chromosome carried an undescribed B¹ mutation; this allele came from a parent of non-French-Canadian origin. Patients in three families carried TSD alleles different from any of the above mutations. The 5' deletion mutation clusters in persons originating in southeastern Quebec (Gaspé) and adjacent counties of northern New Brunswick.     

Two sisters with Rett syndrome and non-identical paternally-derived microdeletions in the MECP2 gene

The unique case of two sisters with symptoms of RTT and two quite distinct, novel, and apparently de novo microdeletions of the MECP2 gene is described. One sister possessed an 18 base-pair (bp) deletion (c.1155_1172del18) within the deletion hotspot region of exon 4, whereas the other sister exhibited a 43 bp deletion at a different location in the same exon (c.1448_1461del14+29). Although these lesions occurred on the same paternally-derived X chromosome, this is probably due to chance co-occurrence owing to the relatively high mutation rate of the MECP2 gene rather than to a constitutional mutator phenotype.     

Mutations of the ATM gene detected in Japanese ataxia-telangiectasia patients: possible preponderance of the two founder mutations 4612del165 and 7883del5

The ATM (A-T, mutated) gene on human chromosome 11q22.3 has recently been identified as the gene responsible for the human recessive disease ataxia-telangiectasia (A-T). In order to define the types of disease-causing ATM mutations in Japanese A-T patients as well as to look for possible mutational hotspots, reverse-transcribed RNA derived from ten patients belonging to eight unrelated Japanese A-T families was analyzed for mutations by the restriction endonuclease fingerprinting method. As has been reported by others, mutations that lead to exon skipping or premature protein truncation were also predominant in our mutants. Six different mutations were identified on 12 of the 16 alleles examined. Four were deletions involving a loss of a single exon: exon 7, exon 16, exon 33 or exon 35. The others were minute deletions, 4649delA in exon 33 and 7883del5 in exon 55. The mutations 4612del165 and 7883del5 were found in more than two unrelated families; 44% (7 of 16) of the mutant alleles had one of the two mutations. The 4612del165 mutations in three different families were all ascribed to the same T→A substitution at the splice donor site in intron 33. Microsatellite genotyping around the ATM locus also indicated that a common haplotype was shared by the mutant alleles in both mutations. This suggests that these two founder mutations may be predominant among Japanese ATM mutant alleles. Received: 15 September 1997 / Accepted: 12 January 1998     

汉族马凡综合征(MFS)患者FBN1基因两种新发突变分析

为调查马凡综合征(Marfan syndrome, MFS)患者的原纤维蛋白-1(Fibrillin-1, FBN1)基因突变情况, 应用聚合酶链反应(PCR)和变性高效液相色谱法(Denaturing high-performance liquid chromatography, DHPLC)对MFS患者的FBN1基因进行突变筛查, 对DHPLC初筛异常的DNA片段进行测序分析。结果在两个MFS家系中发现FBN1基因两种新的突变: 一种为复合突变包含第55号外显子的缺失突变c.6862_6871delGGCTGTGTAG (p.Gly2288MetfsX109)、同义突变c.6861A>G和内含子的突变c.[6871+1_6871+11delGTAAGAGGATC; 6871+34dupCATCAGAAGTGACAGTGGACA]; 另一种为第20号外显子的错义突变c.2462G>A(p.Cys821Tyr)。研究表明, FBN1基因的缺失突变c.[6862_6871delGGCTGTGTAG; 6871+1_6871+11delGTAAGAGGATC] (p.Gly2288MetfsX109)和错义突变c.2462G>A(p.Cys821Tyr)可能分别是这两个家系患者的致病原因。     

The c.469+46_56del mutation in the homeobox MSX1 gene—A novel risk factor in breast cancer?

Purpose: The aim of this study was to investigate the association of a 11 nucleotide deletion, the c.469+46_56del mutation, in the intron of the homeobox MSX1 gene and breast cancer occurrence and characteristics. Methods: The mutation was genotyped in peripheral blood lymphocytes of 200 breast cancer patients and 203 controls by single-strand conformational PCR and DNA sequencing. Results: The del/del variant of the c.469+46_56del mutation increased the risk of breast cancer occurrence (OR 2.20; 95% CI 1.41–3.44, p < 0.05). We did not observe any association between genotypes of this mutation and lymph node status, Bloom–Richardson grading, estrogen and progesterone receptors and HER2 expression. Conclusions: The del/del genotype of the c.469+46_56del mutation in the MSX1 gene may be associated with the increased risk of breast cancer in Polish population and may be considered as an early marker in this disease.     

Screening of Nijmegen breakage syndrome 1 mutations in four unrelated families by polymerase chain reaction using sequence-specific primers

Nijmegen breakage syndrome (NBS) is an autosomal recessive disorder characterized by a marked predisposition to lymphoreticular malignancies. The rarity of the disease and the presence, in several cases, of a mild clinical phenotype make diagnosis difficult. The underlying gene, NBS1, consists of 16 exons and encodes nibrin, a member of the hMRE11/hRAD50/hNBS1 protein complex. In addition to the "Slavic mutation," 657del5, identified in more than 100 patients with NBS, 9 other mutations have been found in families of different ethnic origin. We have developed a polymerase chain reaction (PCR) method to rapidly detect the private mutations, 742insGG and 835del4, in exon 7 and the 900del25 mutation in exon 8 of the NBS1 gene. In particular, we designed NBS1-specific primers for wild-type and mutated alleles, and optimized a specific PCR protocol for each mutation. We used this method to analyze 4 unrelated NBS families, 3 from Italy and 1 from Morocco. We believe it could be a useful tool for: (1) confirming the NBS diagnosis in the presence of clinical signs of the disease; (2) identifying NBS heterozygotes and performing prenatal diagnosis in families with affected members; and (3) screening selected populations in which the frequency of NBS might be higher because of a founder effect.     

红鳍东方鲀(Takifugu rubripes)MC4R基因的多态性分析

采用PCR-SSCP(single strand conformation polymorphism)技术和DNA测序方法分析红鳍东方鲀MC4R(Melanocortin-4receptor)基因编码区多态性。在MC4R基因编码区48 nt和264 nt均发生了碱基的转换突变(G→A),两个突变位点分别位于M1和M2引物扩增产物中。引物M1扩增产物SSCP分析得到两种基因型:AA基因型和AB基因型,并且AA基因型和A等位基因频率明显高于AB基因型和B等位基因。引物M2扩增产物也得到两种基因型:CC基因型和CD基因型,CC基因型和C等位基因频率明显高于CD基因型和D等位基因。遗传变异结果分析表明,两个突变位点均属于低度多态性,而且群体遗传杂合度较低,反映了该群体的遗传一致性较高。     

Abnormal mitochondrial autophagy in nephropathic cystinosis

Cystinosis, which is characterized by lysosomal accumulation of cystine in many tissues, was the first known storage disorder caused by defective metabolite export from the lysosome. The molecular and cellular mechanisms underlying nephropathic cystinosis, the most severe form, which exhibits generalized proximal tubular dysfunction and progressive renal failure, remain largely unknown. We used renal proximal tubular epithelial (RPTE) cells and fibroblasts from patients with three clinical variants of cystinosis: nephropathic, intermediate and ocular to explore the specific injury mechanism in nephropathic cystinosis. We demonstrate enhanced autophagy of mitochondria, increase in apoptosis and mitochondrial dysfunction in the nephropathic cystinosis phenotype. Furthermore, specific inhibition of autophagy results in significant attenuation of cell death in nephropathic cystinosis. This study provides ultrastructural and functional evidence of abnormal mitochondrial autophagy in nephropathic cystinosis, which may contribute to renal Fanconi syndrome and progressive renal injury.Key words: cystinosis, autophagy, mitochondria, kidney, lysosome, apoptosis, cell death, mitophagyCystinosis is an autosomal recessive metabolic disorder caused by mutations in the CTNS gene, which encodes a 7-transmembrane domain protein, cystinosin, a lysosomal cystine transporter. Cystinosis belongs to the family of lysosomal storage disorders (LSDs) characterized by the tissue accumulation of cystine crystals leading to multiple organ dysfunction. The three types of cystinosis, i.e., nephropathic (classic renal and systemic disease), intermediate (a late-onset variant of nephropathic cystinosis) and non-nephropathic (clinically affecting only the cornea) are allelic disorders caused by CTNS mutations. Children affected with nephropathic cystinosis present with the Fanconi syndrome and usually develop progressive renal failure within the first decade of life. The mechanism linking lysosomal cystine storage to pathological manifestations, in particular to the prominent proximal tubular defect and renal injury, remains unclear. Renal injury in nephropathic cystinosis may not simply be caused just by cystine accumulation, as disruption of the ctns gene in mice induces cystine storage in many tissues but does not result in signs of tubulopathy or renal failure; renal injury is not seen in other human forms of cystinosis and progressive renal injury occurs despite cystine depletion therapy.The purpose of our study was to investigate the specific mechanism leading to tubulopathy and end stage renal injury in nephropathic cystinosis. We used primary fibroblast and renal proximal tubular epithelial (RPTE) cells derived from patients with three clinical phenotypes of cystinosis. Our data show an abnormal increase in macroautophagy (hereafter referred to as autophagy), specific to the nephropathic variant of cystinosis. We also demonstrate that specific inhibition of autophagy rescues cell death in nephropathic cystinotic RPTE cells. Our results indicate that mitochondrial autophagy may be a critical mechanism contributing to renal Fanconi syndrome and progressive renal injury in nephropathic cystinosis.Abnormal autophagy was also recently observed in other types of lysosomal storage diseases (LSD). However, our study provides the first evidence supporting the extensive involvement of autophagy in nephropathic cystinosis pathogenesis. Abundant vacuolization and abnormal mitochondria are detected by electron microscopy (EM) in nephropathic cystinotic cells. Additionally, elevated levels of LC3-II and Beclin 1 are also observed in nephropathic cystinotic RPTE cells, indicating a role of Beclin 1-mediated autophagy in cystinosis. These results altogether establish an abnormal increase in autophagy in nephropathic cystinotic cells.Renal biopsies from patients with nephropathic cystinosis can reveal abnormally large mitochondria, but the relevance of this finding and other ultrastructural abnormalities is unclear. Our study further demonstrates a significant decrease in mitochondrial ATP generation with an increase in reactive oxygen species (ROS) in cystinotic cells. To further dissect the association of abnormal mitochondria with increased autophagy in cystinosis, we carefully examined the electron micrographs at higher magnifications. We discovered various stages of degradation of mitochondria by autophagy (hereafter referred to as mitophagy). To further validate mitophagy in cystinosis, we used an immunofluorescence (IF) approach to capture colocalization images of LC3, LAMP-2 (lysosomal marker) and ATP5H (mitochondrial marker). Intriguingly, an increase in LAMP-2 perinuclear staining is detected by IF assay in cystinotic cells. This observation may also denote enhanced active autophagy as LAMP-2 is involved in lysosomal biogenesis and/or the fusion between autophagosomes and lysosomes. Alternatively, LAMP-2 accumulation could be a manifestation of retarded autophagic flux in cystinotic cells. A decreased ability of lysosomes to fuse with autophagosomes has been reported in various LSDs. However, the colocalization of LC3 and LAMP-2 in nephropathic cystinotic RPTE cells argues against this possibility. Nevertheless, the possibility of autophagic flux blockade after autophagosome-lysosome fusion leading to detrimental effects is yet to be investigated. Interestingly, previously published EM reports of the renal biopsies of patients with nephropathic cystinosis show only the nucleus and a thin rim of cytoplasm as remnants in a proximal tubular cell, while mitochondria and lysosomes completely disappear.Conventionally, autophagy has been suggested as a cytoprotective mechanism to ensure cell survival during starvation. In contrast, several forms of cell death have been associated with the appearance of autophagic vesicles. To gain insight into the role of autophagy as regards to cell death or cell survival in nephropathic cystinosis, we used 3-methyladenine (3MA), a specific inhibitor of autophagy and assayed cell viability and apoptosis in cystinotic cells. Increased apoptosis has been previously reported in cultured cystinotic fibroblasts and RPTE cells. Treatment with 3MA in cystinotic cells significantly rescues cell death, thus suggesting a synergistic role of apoptosis and autophagy in cystinosis.In conclusion, as illustrated in Figure 1, we speculate that there is a multifaceted impact of autophagy in nephropathic cystinosis as follows: (1) the mechanism linking autophagy to lysosomal cystine or apoptosis in cystinotic cells could potentially be related to lysosomal membrane permeabilization (LMP), proposed as an early step in apoptosis in cystinosis. We hypothesize that abnormal induction of autophagy besides providing more cargo to be digested in the lysosomes, leads to increased fusion of autophagosomes with cystine-laden lysosomes, rendering them more sensitive to membrane destabilization, and thus making them readily enter the apoptotic pathway; (2) the second most important question is the link between abnormal mitochondria and mitophagy in cystinosis. A decreased level of cytosolic glutathione in cystinotic cells is one of the known factors responsible for generating damaged mitochondria. Our data also indicate an impairment of complex I activity, an increase in ROS and a decrease in mitochondrial ATP generation in cystinotic cells. We hypothesize that the abnormal induction of autophagy leads to depletion of mitochondria, forcing cells to enter the ‘starvation mode,’ thereby leading to an uncontrolled autophagy and cell death; (3) the third key question yet to be answered is the link between autophagy and renal injury in nephropathic cystinosis. Skeletal muscles and neuronal tissues are the primary organs where autophagy is physiologically enhanced. Recently, it has been shown that mouse kidneys exert a high level of autophagy under basal conditions, influencing the susceptibility to glomerular disease and renal failure. Thus, we postulate an organ- and tissue-specific effect of abnormally induced autophagy in nephropathic cystinosis, causing severe injury to kidneys leading to loss of renal function, ultimately culminating in end-stage renal disease.Open in a separate windowFigure 1A schematic view of the interplay between autophagy, abnormal mitochondria and cell death in cystinosis. Abnormal induction of autophagy, typically mitophagy, forces cells into a starvation mode leading to cell death; and renders cystine-laden lysosomes sensitive to lysosomal membrane permeabilization (LMP) making it readily enter the apoptosis pathway. A potential block in autophagic flux, after autophagosome-lysosome fusion, remains to be elucidated. Preferential severe kidney damage in nephropathic cystinosis may be due to the tissue- and organ-specific injury effect of autophagy.The recent progress in autophagy research has increased the need for additional studies so that we can fully understand the underlying pathological mechanisms and the significance of the lysosomal cell death axis in lysosomal storage disorders.     

A large Alu-mediated deletion, identified by PCR, as the molecular basis for glycogen storage disease Type II (GSDII)

Glycogen storage disease type II (GSDII) is an autosomal recessive disorder resulting from inherited deficiency of the enzyme lysosomal acid α-glucosidase. Over 40 different mutations have been described but no large deletions have been previously identified. We now describe a homozygous large (9-kb) deletion extending from IVS15 to 4 kb downstream of the terminal exon (exon 20), detected by polymerase chain reaction (PCR)-based methods. The deletion was initially suspected because of failure to amplify a contiguous group of exons by PCR. We hypothesized an Alu/Alu recombination, based on our prior demonstration by Southern blotting of Alu elements in the regions potentially flanking the deletion. Additional sequence analysis of genomic fragments confirmed the presence of Alu elements and allowed the design of flanking primers for PCR amplification. Amplification resulted in a smaller than normal fragment (0.7 vs 10 kb) in homozygosity in the proband and in heterozygosity in her parents. Cloning and sequencing of the smaller than normal 0.7-kb deletion fragment revealed an Alu/Alu deletion junction. In heterozygosity this deletion would not be detected by currently standard PCR mutation detection methods. Based on other Alu-mediated deletions, this deletion is likely to be recurrent and should be screened for in all non-consanguineous GSDII patients, particularly when only one mutation has been identified and none of the 12 single-nucleotide polymorphisms in the deleted region are heterozygous. These observations also suggest that initial characterization of genes at disease-causing loci should include a search for Alu and other repetitive elements to facilitate subsequent PCR-based mutation analysis. Received: 24 August 1998 / Accepted: 13 November 1998     

ERS1 encodes a functional homologue of the human lysosomal cystine transporter

Cystinosis is a lysosomal storage disease caused by an accumulation of insoluble cystine in the lumen of the lysosome. CTNS encodes the lysosomal cystine transporter, mutations in which manifest as a range of disorders and are the most common cause of inherited renal Fanconi syndrome. Cystinosin, the CTNS product, is highly conserved among mammals. Here we show that the yeast Ers1 protein and cystinosin are functional orthologues, despite sharing only limited sequence homology. Ers1 is a vacuolar protein whose loss of function results in growth sensitivity to hygromycin B. This phenotype can be complemented by the human CTNS gene but not by mutant ctns alleles that were previously identified in cystinosis patients. A genetic screen for multicopy suppressors of an ers1Delta yeast strain identified a novel gene, MEH1, which is implicated in regulating Ers1 function. Meh1 localizes to the vacuolar membrane and loss of MEH1 results in a defect in vacuolar acidification, suggesting that the vacuolar environment is critical for normal ERS1 function. This genetic system has also led us to identify Gtr1 as an Meh1 interacting protein. Like Meh1 and Ers1, Gtr1 associates with vacuolar membranes in an Meh1-dependent manner. These results demonstrate the utility of yeast as a model system for the study of CTNS and vacuolar function.