Identification and characterization of three large deletions and a deletion/polymorphism in the CFTR gene.

Cystic fibrosis (CF) is mainly caused by small molecular lesions of the CFTR gene; mutation detection methods based on conventional PCR do not allow the identification of all CF alleles in a population and large deletions may account for a number of these unidentified molecular lesions. It is only recently that the availability of quantitative PCR methodologies made the search for large gene rearrangements easier in autosomal diseases. Using a combination of different methods, nine of the 37 unidentified CF alleles (24%) were found to harbor large deletions in our cohort of 1600 CF alleles. Three are new deletions, and we report the breakpoints of the previously described EX4_EX10del40kb deletion. An intronic deletion polymorphism affecting intron 17b was also found on almost 1% of "normal" chromosomes. Examination of the breakpoint sequences confirmed that intron 17b is indeed a hot spot for deletions, and that most of these rearrangements are caused by non-homologous recombination.

Mucopolysaccharidosis type II--genotype/phenotype aspects.

UNLABELLED: Establishing correlations between a patient's genotype and clinical phenotype is based on the assumption that the same clinical consequences will be observed in individuals with the same residual function of a specific metabolic step. In mucopolysaccharidosis type II (MPS II; Hunter disease), patients present with a wide clinical spectrum. Furthermore, current methods for measuring the activity of the deficient enzyme in MPS II--iduronate-2-sulphatase (IDS)--are insufficiently sensitive to differentiate between complete absence of activity and the presence of residual activity. Attempts have therefore been made to establish genotype-phenotype correlations in order to explain the large degree of heterogeneity and to serve as a better guide to prognosis on which to base genetic counselling and treatment options. Using MPS II as an example, this paper illustrates the difficulties and potential advantages of determining genotype-phenotype correlations in lysosomal storage diseases. The response of patients with MPS II to allogenic bone marrow transplantation provides some insight into the likely influence of certain genotypes on therapeutic efficacy. CONCLUSIONS: Evaluation of residual activity of IDS in MPS II using gene analysis, expression studies and transcript analysis does not always allow prediction of a patient's phenotype. The variable response to bone marrow transplantation, however, illustrates the potential importance of determining the genotype for selecting the most appropriate therapy for individual patients.

Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France.

We have collated the results of cystic fibrosis (CF) mutation analysis conducted in 19 laboratories in France. We have analyzed 7, 420 CF alleles, demonstrating a total of 310 different mutations including 24 not reported previously, accounting for 93.56% of CF genes. The most common were F508del (67.18%; range 61-80), G542X (2.86%; range 1-6.7%), N1303K (2.10%; range 0.75-4.6%), and 1717-1G>A (1.31%; range 0-2.8%). Only 11 mutations had relative frequencies >0. 4%, 140 mutations were found on a small number of CF alleles (from 29 to two), and 154 were unique. These data show a clear geographical and/or ethnic variation in the distribution of the most common CF mutations. This spectrum of CF mutations, the largest ever reported in one country, has generated 481 different genotypes. We also investigated a cohort of 800 French men with congenital bilateral absence of the vas deferens (CBAVD) and identified a total of 137 different CFTR mutations. Screening for the most common CF defects in addition to assessment for IVS8-5T allowed us to detect two mutations in 47.63% and one in 24.63% of CBAVD patients. In a subset of 327 CBAVD men who were more extensively investigated through the scanning of coding/flanking sequences, 516 of 654 (78. 90%) alleles were identified, with 15.90% and 70.95% of patients carrying one or two mutations, respectively, and only 13.15% without any detectable CFTR abnormality. The distribution of genotypes, classified according to the expected effect of their mutations on CFTR protein, clearly differed between both populations. CF patients had two severe mutations (87.77%) or one severe and one mild/variable mutation (11.33%), whereas CBAVD men had either a severe and a mild/variable (87.89%) or two mild/variable (11.57%) mutations.

Combined liver-kidney transplantation in primary hyperoxaluria type 1.

Primary hyperoxaluria type 1 (PH1) is a rare autosomal recessive disorder characterised by an increased urinary excretion of calcium oxalate, leading to recurrent urolithiasis, nephrocalcinosis and accumulation of insoluble oxalate throughout the body (oxalosis) when the glomerular filtration rate falls to below 40-20 mL/min per 1.73 m(2). The disease is due to a functional defect of the liver-specific peroxisomal enzyme alanine: glyoxylate aminotransferase (AGT), the gene of which is located on chromosome 2q37.3. The diagnosis is based on increased urinary oxalate and glycollate, increased plasma oxalate and AGT measurement in a liver biopsy. AGT mistargeting may be investigated by immuno-electron microscopy and DNA analysis. End-stage renal failure is reached by the age of 15 years in 50% of PH1 patients and the overall death rate approximates 30%. The conservative treatment includes high fluid intake, pyridoxine and crystallisation inhibitors. Since the kidney is the main target of the disease, isolated kidney transplantation (Tx) has been proposed in association with vigorous peri-operative haemodialysis in an attempt to clear plasma oxalate at the time of Tx. However, because of a 100% recurrence rate, the average 3-year graft survival is 15%-25% in Europe, with a 5-10-year patient survival rate ranging from 10% to 50%. Since the liver is the only organ responsible for the detoxification of glyoxylate by AGT, deficient host liver removal is the first rationale for enzyme replacement therapy. Subsequent orthotopic liver Tx aims to supply the missing enzyme in its normal cellular and subcellular location and thus can be regarded as a form of gene therapy. Because of the usual spectrum of the disease, isolated liver Tx is limited to selected patients prior to having reached an advanced stage of chronic renal failure. Combined liver-kidney Tx has therefore become a conventional treatment for most PH1 patients: according to the European experience, patient survival approximates 80% at 5 years and 70% at 10 years. In addition, the renal function of survivors remains stable over time, between 40 and 60 mL/min per 1.73 m(2) after 5 to 10 years. In addition, liver Tx may allow the reversal of systemic storage disease (i.e. bone, heart, vessels, nerves) and provide valuable quality of life. Whatever the transplant strategy, the outcome is improved when patients are transplanted early in order to limit systemic oxalosis. According to the European experience, it appears that combined liver-kidney Tx is performed in PH1 patients with encouraging results, renal Tx alone has little role in the treatment of this disease, and liver Tx reverses the underlying metabolic defect and its clinical consequences.

The 2.1-, 5.4- and 5.7-kb transcripts of the IDS gene are generated by different polyadenylation signals.

Deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS) is responsible for mucopolysaccharidosis type II (OMIM 309900). The IDS gene (Xq28) has been completely sequenced (accession number L35485). Northern blot analysis of poly(A(+)) RNA from different tissues, hybridized with the total IDS cDNA, has revealed three major species of 2.1, 5.4 and 5.7 kb and one minor of 1.4 kb. The 1.4-kb mRNA has been previously described and we show that the three major IDS mRNA are the result of alternative polyadenylation site selection: a non-canonical ATTAAA signal at genomic position 23631 for the 2.1-kb mRNA, a AATAAA signal at position 27156 for the 5.4-kb mRNA and a AATAAA signal at position 27399 for the 5.7-kb mRNA. The different IDS mRNA encode for the same polypeptide and the most abundant transcripts have a long 3'-untranslated region (3'-UTR). The absence of obvious correlation between transcripts content and size, IDS protein amount and IDS activity in the four human fetal tissues tested suggests that it is IDS protein processing that may be regulated rather than IDS gene transcription.

Complex allele [-102T>A+S549R(T>G)] is associated with milder forms of cystic fibrosis than allele S549R(T>G) alone.

We recently reported a novel complex allele in the cystic fibrosis transmembrane regulator (CFTR) gene, combining a sequence change in the minimal CFTR promoter (-102T>A) and a missense mutation in exon 11 [S549R(T>G)]. Here we compare the main clinical features of six patients with cystic fibrosis (CF) carrying the complex allele [-102T>A+S549R(T>G)] with those of 16 CF patients homozygous for mutation S549R(T>G) alone. Age at diagnosis was higher, and current age was significantly higher (P=0.0032) in the group with the complex allele, compared with the S549R/S549R group. Although the proportion of patients with lung colonization was similar in both groups, the age at onset was significantly higher in the group with the complex allele (P=0.0022). Patients with the complex allele also had significantly lower sweat test chloride values (P=0.0028) and better overall clinical scores (P=0.004). None of the 22 patients reported in this study had meconium ileus. All 16 patients homozygous for S549R(T>G), however, were pancreatic insufficient, as compared with 50% of patients carrying the complex allele (P=0.013). Moreover, the unique patient homozygous for [-102T>A+S549R(T>G)] presented with a mild disease at 34 years of age. These observations strongly suggest that the sequence change (-102T>A) in the CFTR minimal promoter could attenuate the severe clinical phenotype associated with mutation S549R(T>G).

COS cell expression studies of P86L, P86R, P480L and P480Q Hunter's disease-causing mutations.

Three missense mutations identified in the IDS gene of our Hunter's disease patients (P86L, P480L and P480Q) and the previously described P86R mutation were expressed in COS cells to evaluate their functional consequence on iduronate-2-sulfatase (IDS) activity and processing. The 86-proline residue belongs to the highly conserved pentapeptide C-X-P-S-R in which cysteine modification to a formylglycine is required for sulfatase activity. The substitution of the 86-proline residue led to a severe mutation as no mature form was targeted to the lysosome in agreement with the severe phenotype observed in patients carrying P86L and P86R mutations. Expression studies with P480L and P480Q mutant cDNAs showed the presence of a small amount of 55 kDa mature form in the lysosomes of transfected COS cells. IDS activity of the P480L and P480Q mutants in cell extracts represents 16.6% and 5.4% of the wild-type, respectively.

Characterization of iduronate sulphatase mutants affecting N-glycosylation sites and the cysteine-84 residue.

Iduronate sulphatase (IDS) is responsible for mucopolysaccharidosis type II, a rare recessive X-linked lysosomal storage disease. The aim of this work was to evaluate the functional importance of each N-glycosylation site, and of the cysteine-84 residue. IDS mutant cDNAs, lacking one of the eight potential N-glycosylation sites, were expressed in COS cells. Although each of the potential sites was used, none of the eight glycosylation sites appeared to be essential for lysosomal targeting. Another important sulphatase co- or post-translational modification for generating catalytic activity involves the conversion of a cysteine residue surrounded by a conserved sequence C-X-P-S-R into a 2-amino-3-oxopropionic acid residue [Schmidt, Selmer, Ingendoh and von Figura (1995) Cell 82, 271-278]. This conserved cysteine, located at amino acid position 84 in IDS, was replaced either by an alanine (C84A) or by a threonine (C84T) using site-directed mutagenesis. C84A and C84T mutant cDNAs were expressed either in COS cells or in human lymphoblastoid cells deleted for the IDS gene. C84A had a drastic effect both for IDS processing and for catalytic activity. The C84T mutation produced a small amount of mature forms but also abolished enzyme activity, confirming that the cysteine residue at position 84 is required for IDS activity.

IDS transfer from overexpressing cells to IDS-deficient cells.

Iduronate sulfatase (IDS) is responsible for mucopolysaccharidosis type II, a rare recessive X-linked lysosomal storage disease. The aim of this work was to test the ability of overexpressing cells to transfer IDS to deficient cells. In the first part of our work, IDS processing steps were compared in fibroblasts, COS cells, and lymphoblastoid cell lines and shown to be identical: the two precursor forms (76 and 90 kDa) were processed by a series of intermediate forms to the 55- and 45-kDa mature polypeptides. Then IDS transfer to IDS-deficient cells was tested either by incubation with cell-free medium of overexpressing cells or by coculture. Endocytosis and coculture experiments between transfected L beta and deleted fibroblasts showed that IDS transfer occurred preferentially by cell-to-cell contact as IDS precursors are poorly secreted by transfected L beta. The 76- and 62-kDa IDS polypeptides transferred to deleted fibroblasts were correctly processed to the mature 55- and 45-kDa forms. L beta were not able to internalize the 90-kDa phosphorylated precursor forms excreted in large amounts in the medium of overexpressing fibroblasts. Enzyme transfer occurred only by cell-to-cell contact, but the precursor forms transferred in L beta after cell-to-cell contact were not processed. This absence of maturation was probably due to a mistargeting of IDS precursors in these cells.

Processing of iduronate 2-sulphatase in human fibroblasts.

Iduronate 2-sulphatase (IDS) is a lysosomal enzyme involved in degradation of dermatan sulphate and heparan sulphate. Antigenic material was obtained either by purification of placental IDS (A and B forms) or by expression of three different fusion peptides in Escherichia coli allowing the production of five specific antibodies. Pulse-chase-labelling experiments in over-expressing fibroblasts showed poor IDS processing but large amounts of precursors were secreted into the medium. The endocytosis of the 35S- or 33P-labelled precursors by deleted fibroblasts together with glycosylation studies and proteolysis inhibition by leupeptin allowed better elucidation of IDS maturation. The initial 73-78 kDa form is converted into a phosphorylated 90 kDa precursor after modification of its oligosaccharide chains in the Golgi apparatus. This precursor is processed by proteolytic cleavage through various intermediates to a major 55 kDa intermediate, with the release of an 18 kDa polypeptide. Further proteolytic cleavage by a thiol protease gives the 45 kDa mature form containing hybrid and complex-type oligosaccharide chains.

Capillary electrophoretic analysis of DNA restriction fragments and PCR products for polymorphism and mutation studies in cystic fibrosis and Gaucher's disease.

Two methods are described for the analysis of DNA restriction fragments and PCR products in studies on polymorphism and mutation in cystic fibrosis and Guacher's disease, based on capillary electrophoresis. In one CE system, a Beckman kit for producing a chemical gel (polymerized within the capillary) is used for single-stranded DNA fragments from 10 to 300 bases in size. Its performance was demonstrated on the separation of a mixture of polydeoxyadenylic acids p(dA)40-60 at 30 degrees C. Electrokinetic injection was used (5-7 kV for 5-20 s), the applied field being 300 V cm-1 for an effective length of 7, 20 or 30 cm and 100 microns i.d., with Tris-borate buffer containing urea. Typical electropherograms are presented for the analysis of CF mutation delta F508 in PCR products from homozygous and heterozygous individuals, illustrating the resolution of two complementary single strands (95b and 95b) of a DNA fragment. DNA fragments differing in size by only one base could also be resolved, as shown for the 105b and 106b fragments obtained from a heterozygote for 3905 insT CF mutation, with a run time of ca-45 min. If discrimination were only required between fragments differing by two or more bases, run times could be reduced by 6 when using a capillary length of only 7 cm x 100 microns i.d.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutation analysis in 600 French cystic fibrosis patients.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene of 600 unrelated cystic fibrosis (CF) patients living in France (excluding Brittany) was screened for 105 different mutations. This analysis resulted in the identification of 86% of the CF alleles and complete genotyping of 76% of the patients. The most frequent mutations in this population after delta F508 (69% of the CF chromosomes) are G542X (3.3%), N1303K (1.8%), W1282X (1.5%), 1717-1G-->A (1.3%), 2184delA + 2183 A-->G (0.9%), and R553X (0.8%).

Analysis of CFTR transcripts in nasal epithelial cells and lymphoblasts of a cystic fibrosis patient with 621 + 1G-->T and 711 + 1G-->T mutations.

We have analyzed the CFTR mRNA populations in a cystic fibrosis patient heterozygous for the 621 + 1G-->T and 711 + 1G-->T mutations. Total RNA isolated from the nasal epithelial cells and Epstein-Barr virus-transformed lymphoblasts derived from this patient was reversely transcribed and a region extending from exon 3 to exon 7 of the gene was amplified by the polymerase chain reaction and analyzed. Three abnormal products were identified, suggesting the presence of three aberrant transcripts, and their profiles were identical in both cell types. Two of the products were found to be missing either exon 4 or exon 5 as anticipated from the transcripts from the 621 + 1G-->T or 711 + 1G-->T alleles, respectively. The third product was apparently derived from an alternatively spliced mRNA species in the absence of the nominal splice site (in 621 + 1G-->T) through the use of a cryptic splice donor sequence (TT528/GTGAGG) within exon 4. Although reading frames appeared to be preserved in all three putative transcripts, significant portions of the presumed first and second transmembrane spans as well as the immediately following cytoplasmic domain would be deleted from the mutant CFTR polypeptides, if made. These observations are consistent with a loss of CFTR function in this cystic fibrosis patient.

Genetic determination of exocrine pancreatic function in cystic fibrosis.

We showed elsewhere that the pancreatic function status of cystic fibrosis (CF) patients could be correlated to mutations in the CF transmembrane conductance regulator (CFTR) gene. Although the majority of CF mutations--including the most common, delta F508--strongly correlated with pancreatic insufficiency (PI), approximately 10% of the mutant alleles may confer pancreatic sufficiency (PS). To extend this observation, genomic DNA of 538 CF patients with well-documented pancreatic function status were analyzed for a series of known mutations in their CFTR genes. Only 20 of the 25 mutations tested were found in this population. They accounted for 84% of the CF chromosomes, with delta F508 being the most frequent (71%), and the other mutations accounted for less than 5% each. A total of 30 different, complete genotypes could be determined in 394 (73%) of the patients. The data showed that each genotype was associated only with PI or only with PS, but not with both. This result is thus consistent with the hypothesis that PI and PS in CF are predisposed by the genotype at the CFTR locus; the PS phenotype occurs in patients who have one or two mild CFTR mutations, such as R117H, R334W, R347P, A455E, and P574H, whereas the PI phenotype occurs in patients with two severe alleles, such as delta F508, delta I507, Q493X, G542X, R553X, W1282X, 621 + 1G----T, 1717-1G----A, 556delA, 3659delC, I148T, G480C, V520F, G551D, and R560T.