Dominique Bozon

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. Hum Mutat 16:143–156, 2000.

Combined liver-kidney transplantation in primary hyperoxaluria type 1

Abstract 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 m2. 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 m2 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.

Identification of iduronate sulfatase gene alterations in 70 unrelated Hunter patients

We studied 70 unrelated Hunter patients and found a gene alteration in every patient. The molecular heterogeneity was very important. Large gene rearrangements were identified in 14 patients. Forty‐three different mutations were identified in the 56 other patients and 31 were not previously described. Deletions and insertions, splice site mutations were associated with a severe phenotype as nonsense mutations except Q531X. Only a few mutations were present in several patients making difficult genotype‐phenotype correlations. Mutation identification allows accurate carrier detection improving prenatal diagnosis. The mother was not found to be a carrier in five cases among the 44 sporadic cases. Haplotype analysis demonstrated a higher frequency of mutations in male meiosis.

Mutation analysis in 24 French patients with glycogen storage disease type 1a.

Both alleles of 24 French glycogen storage disease type 1a patients were sequenced: 14 different mutations allowed the identification of complete genotypes for all the patients. Nine new gene alterations are reported. Five mutations, Q347X, R83C, D38V, G188R, and 158 del C, account for 75% of the mutated alleles. These data show that the molecular pathology of the glucose-6-phosphatase gene is heterogeneous in this population. Complete genotyping of the index case by systematic sequencing is necessary to allow prenatal diagnosis in chorionic villi for at risk couples.

Complete loss of expression of the ANT1 gene causing cardiomyopathy and myopathy

Background The ANT1 gene, encoding ADP/ATP translocase 1, was investigated in an adult patient with an autosomal recessive mitochondrial disorder characterised by congenital cataracts, hypertrophic cardiomyopathy, myopathy and lactic acidosis. Methods and results ANT1 sequencing showed that the patient was homozygous for a new nucleotide variation, c.111+1G→A, abolishing the invariant GT splice donor site of intron 1. The ANT1 transcript was undetectable in both muscle and skin fibroblasts. A markedly abnormal metabolic profile was found, and skeletal muscle showed a dramatic proliferation of abnormal mitochondria, increased mitochondrial mass, and multiple mitochondrial DNA deletions. No compensating increase in the transcript level of the ANT3 gene, which encodes the human ubiquitous isoform of the ADP/ATP translocase, was observed. The patients heterozygous mother had normal clinical, biochemical and pathological features. Conclusions Complete loss of expression of the ANT1 gene causes a clinical syndrome mainly characterised by cardiomyopathy and myopathy. This report expands the clinical spectrum of ANT1-related human diseases, and emphasises the crucial role of the mitochondrial ADP/ATP carriers in muscle function and pathophysiology of human myopathies.

MPS II in females: molecular basis of two different cases

Editor—Hunter disease or mucopolysaccharidosis type II (MPS II, MIM 309900) is an X linked recessive disease resulting from deficiency of the lysosomal enzyme iduronate-2-sulphatase (IDS, E.C.3.1.6.13).1 The IDS cDNA has been isolated2 and the genomic region containing the IDS gene and pseudogene has been completely sequenced.3 Phenotypic expression of X linked disorders in females may be the result of an X chromosome anomaly or homozygosity for the mutated gene, but is most frequently the result of skewed X chromosome inactivation. We describe two affected girls, case 1 and case 2, with a mild and a severe form of MPS II, respectively. Both have a normal karyotype but increased dermatan sulphate and heparan sulphate excretion in urine, a marked deficiency of IDS activity, and normal α-L-iduronidase, β-D-glucuronidase, and arylsulphatase A activities in leucocytes and cultured skin fibroblasts ruling out MPS I, MPS VII, and multiple sulphatase deficiency. Molecular studies showed that case 1 is the first case of female MPS II with two mutated IDS genes and that case 2 has a de novo gene rearrangement on the paternal allele (a 3254 bp deletion from intron 7 to intron 8 with an insertion of 20 bp) associated with skewed X inactivation of the normal maternal X chromosome. Case 1 was born into a French gypsy family. At 11 years of age, she presented with hepatomegaly and growth retardation, but had no dysmorphic features, no multiplex dysostosis, no corneal clouding, no cardiovascular disease, no splenomegaly, and no mental retardation, in agreement with a mild form of MPS II. A unique IDS transcript with a T to C substitution at cDNA position 246 in exon 2 (accessionM38371 2) was found in fibroblasts, changing codon CTC (leucine) to CCC (proline), L41P. Sequencing of the PCR products from intron …

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%).

Germline and somatic mosaicism in a female carrier of Hunter disease.

Carrier detection in a mucopolysaccharidosis type II family (Hunter disease) allowed the identification of germline and somatic mosaicism in the patients mother: the R443X mutation was found in a varying proportion in tested tissue (7% in leucocytes, lymphocytes, and lymphoblastoid cells, and 22% in fibroblasts). The probands sister carries the at risk allele (determined by haplotype analysis), but not the mutation. In sporadic cases of X linked diseases, germline mosaicism of the probands mother is difficult to exclude and should be considered in genetic counselling.

IDS gene-pseudogene exchange responsible for an intragenic deletion in a hunter patient

Hunter disease or mucopolysaccharidosis type II is an X‐linked disease caused by the deficiency of the lysosomal enzyme iduronate‐2‐sulfatase (IDS). The IDS gene (24 kb) contains nine exons and has been completely sequenced. A pseudogene (IDS‐2 locus) distal to the functional IDS gene has recently been identified.

Partial Deletion of the AGXT Gene (EX1_EX7del) : A New Genotype in Hyperoxaluria Type 1

Primary hyperoxaluria type 1 (PH1) is a rare autosomal (2q37.3) recessive metabolic disease caused by a deficiency of the hepatic peroxisomal enzyme alanine:glyoxylate amino transferase. Molecular heterogeneity is important in PH1 as most of the patients (if the parents are unrelated) are compound heterozygotes for rare mutations. We describe the first large deletion in the AGXT gene, removing exons 1 to 7 (EX1_EX7del) that was responsible for one case of severe PH1. This 10 kb deletion was identified by Southern blotting of genomic DNA digested by Xba I and hybridized with different exonic probes. Both parents (from Turkey) are first cousin and carry the deletion. It is of note that the presently reported patient did not exhibit any AGT catalytic activity and even so, he progressed towards end‐stage renal disease only at 19 years old. Hum Mutat 15:384–385, 2000.