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Optimal molecular diagnosis of primary dyslipidemia is challenging to confirm the diagnosis, test and identify at risk relatives. The aim of this study was to test the application of a single targeted next‐generation sequencing (NGS) panel for hypercholesterolemia, hypocholesterolemia, and hypertriglyceridemia molecular diagnosis. NGS workflow based on a custom AmpliSeq panel was designed for sequencing the most prevalent dyslipidemia‐causing genes (ANGPTL3, APOA5, APOC2, APOB, GPIHBP1, LDLR, LMF1, LPL, PCSK9) on the Ion PGM Sequencer. One hundred and forty patients without molecular diagnosis were studied. In silico analyses were performed using the NextGENe software and homemade tools for detection of copy number variations (CNV). All mutations were confirmed using appropriate tools. Eighty seven variations and 4 CNV were identified, allowing a molecular diagnosis for 40/116 hypercholesterolemic patients, 5/13 hypocholesterolemic patients, and 2/11, hypertriglyceridemic patients respectively. This workflow allowed the detection of CNV contrary to our previous strategy. Some variations were found in previously unexplored regions providing an added value for genotype‐phenotype correlation and familial screening. In conclusion, this new NGS process is an effective mutation detection method and allows better understanding of phenotype. Consequently this assay meets the medical need for individualized diagnosis of dyslipidemia. [ABSTRACT FROM AUTHOR]

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%). [ABSTRACT FROM PUBLISHER]

Carrier detection in a mucopolysaccharidosis type II family (Hunter disease) allowed the identification of germline and somatic mosaicism in the patient's 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 proband's 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 proband's mother is difficult to exclude and should be considered in genetic counselling. [ABSTRACT FROM PUBLISHER]

Presents molecular studies performed in patients with mucopolysaccharidosis type II (MPS II). Characterization of the Portuguese iduronate-2-sulfatase gene mutations; Clinical symptoms of MPS II; Application of polymerase chain reaction to identify mutations.

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. [ABSTRACT FROM PUBLISHER]

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. © 2005 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background and Aims: Abetalipoproteinemia (ABL) is a rare recessive monogenic disease due to MTTP (microsomal triglyceride transfer protein) mutations leading to the absence of plasma apoB-containing lipoproteins. Here we characterize a new ABL case with usual clinical phenotype, hypocholesterolemia, hypotriglyceridemia but normal serum apolipoprotein B48 (apoB48) and red blood cell vitamin E concentrations., Methods: Histology and MTP activity measurements were performed on intestinal biopsies. Mutations in MTTP were identified by Sanger sequencing, quantitative digital droplet and long-range PCR. Functional consequences of the variants were studied in vitro using a minigene splicing assay, measurement of MTP activity and apoB48 secretion., Results: Intestinal steatosis and the absence of measurable lipid transfer activity in intestinal protein extract supported the diagnosis of ABL. A novel MTTP c.1868G>T variant inherited from the patient's father was identified. This variant gives rise to three mRNA transcripts: one normally spliced, found at a low frequency in intestinal biopsy, carrying the p.(Arg623Leu) missense variant, producing in vitro 65% of normal MTP activity and apoB48 secretion, and two abnormally spliced transcripts resulting in a non-functional MTP protein. Digital droplet PCR and long-range sequencing revealed a previously described c.1067+1217_1141del allele inherited from the mother, removing exon 10. Thus, the patient is compound heterozygous for two dysfunctional MTTP alleles. The p.(Arg623Leu) variant may maintain residual secretion of apoB48., Conclusions: Complex cases of primary dyslipidemia require the use of a cascade of different methodologies to establish the diagnosis in patients with non-classical biological phenotypes and provide better knowledge on the regulation of lipid metabolism., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Classically, the study of splicing impact of variation located near the splice site is performed by both in silico and mRNA analysis. However, RNA sample was rarely available., Objective: To characterize a panel of putative haemophilia A splicing variations., Materials and Methods: Twenty-six F8 variations identified from a cohort of 2075 haemophilia A families were studied using both bioinformatic tools and in vitro minigene assays in HeLa and Huh7 cells., Results: An aberrant splicing was demonstrated for 21/26 tested sequence variations. A good correlation between in silico and in vitro analysis was obtained for variations affecting donor splice site (12/14) and for the synonymous variations located inside an exon (6/6). Conversely, no concordant results were observed for the six variations affecting acceptor splice sites. The variations resulted more frequently in exon skipping (n = 13) than in activation of nearby cryptic splice sites (n = 5), in use of a de novo splice site (n = 2) or in insertion of large intronic sequences (n = 1). This study allowed to reclassify 5 synonymous substitutions c.1167A>G (p.Gln389Gln), c.1569G>T (p.Leu523Leu), c.1752G>A (p.Gln584Gln), c.5586G>A (p.Leu1862Leu) and c.6066C>T (p.Gly2022Gly) as splicing variations. The pathological significance of five variations remained unclear (c.222G>A [p.Thr74Thr], c.237C>T [p.Asn79Asn], c.240C>T [p.Ile80Ile], c.2113+5_2113+8del and c.2113+5G>A)., Discussion: The minigene assay herein gave additional evidences for the clinical significance of 21/26 F8 putative splice site mutations. Such investigation should be performed for each F8 putative splice site variation for which no mRNA sample is available, notably to greatly improve the genetic counselling given to female carriers., (© 2019 John Wiley & Sons Ltd.)

Incorporation of distant intronic sequences in mature mRNA is an underappreciated cause of genetic disease. Several disease-causing pseudoexons have been found to contain repetitive elements such as Alu elements. This study describes an original pathological mechanism by which a small intronic deletion leads to Alu exonization. We identified an intronic deletion, c.2113+461_2113+473del, in the F8 intron 13, in two individuals with mild hemophilia A. In vivo and in vitro transcript analysis found an aberrant transcript, with an insertion of a 122-bp intronic fragment (c.2113_2114ins2113+477_2113+598) at the exon 13-14 junction. This out-of-frame insertion is predicted to lead to truncated protein (p.Gly705Aspfs ∗ 37). DNA sequencing analysis found that the pseudoexon corresponds to antisense AluY element and the deletion removed a part of the poly(T)-tail from the right arm of these AluY. The heterogenous nuclear riboprotein C1/C2 (hnRNP C) is an important antisense Alu-derived cryptic exon silencer and binds to poly(T)-tracts. Disruption of the hnRNP C binding site in AluY T-tract by mutagenesis or hnRNP C knockdown using siRNA in HeLa cells reproduced the effect of c.2113+461_2113+473del. The screening of 114 unrelated families with mild hemophilia A in whom no genetic event was previously identified found a deletion in the poly(T)-tail of AluY in intron 13 in 54% of case subjects (n = 61/114). In conclusion, this study describes a deletion leading to Alu exonization found in 6.1% of families with mild hemophila A in France., (Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Autosomal dominant hypercholesterolemia (ADH) is a human disorder characterized phenotypically by isolated high-cholesterol levels. Mutations in the low density lipoprotein receptor (LDLR), APOB, and proprotein convertase subtilisin/kexin type 9 (PCSK9) genes are well known to be associated with the disease. To characterize the genetic background associated with ADH in France, the three ADH-associated genes were sequenced in a cohort of 120 children and 109 adult patients. Fifty-one percent of the cohort had a possible deleterious variant in LDLR, 3.1% in APOB, and 1.7% in PCSK9. We identified 18 new variants in LDLR and 2 in PCSK9. Three LDLR variants, including two newly identified, were studied by minigene reporter assay confirming the predicted effects on splicing. Additionally, as recently an in-frame deletion in the APOE gene was found to be linked to ADH, the sequencing of this latter gene was performed in patients without a deleterious variant in the three former genes. An APOE variant was identified in three patients with isolated severe hypercholesterolemia giving a frequency of 1.3% in the cohort. Therefore, even though LDLR mutations are the major cause of ADH with a large mutation spectrum, APOE variants were found to be significantly associated with the disease. Furthermore, using structural analysis and modeling, the identified APOE sequence changes were predicted to impact protein function., (Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.)

We describe a new family with cathecholaminergic polymorphic ventricular tachycardia (CPVT) linked to the Triadin gene. This is the second report of such a CPVT of autosomal recessive inheritance. Using an NGS panel including 42 genes involved in cardiac sudden death, 2 heterozygous pathogenic mutations (c.613C> T/p.Gln205* and c.22 + 29 A>G) were identified in the Triadin gene in 2 sibs who experienced early severe arrhythmias without evidence of CPVT diagnosis at first cardiac evaluation. However, significant arrhythmias occurred after catecholaminergic stimulation. Each of the TRDN mutations was inherited from a healthy parent. In this family, genetic studies permit confirmation of the CPVT diagnosis in the 2 affected sibs and permit the early diagnosis of the third asymptomatic child. It also helped guide the therapeutic strategy in this family., (© 2015 Wiley Periodicals, Inc.)

Molecular diagnosis of cardiomyopathies remains difficult not only because of the large number of causative genes and the high rate of private mutations but also due to the large number of unclassified variants (UVs) found in patients' DNA. This study reports the functional splicing impact of nine novel genomic variations previously identified in unrelated patients with cardiomyopathies. To identify splice variants among these UVs, a combination of in silico and in vitro hybrid minigene tools was used as transcript is not available. Using this two-step approach, these UVs were reclassified as splicing mutations (MYBPC3-c.655-25A>G, MYBPC3-c.1790G>A (p.Arg597Gln), MYBPC3-c.2414-36G>T) or as mutations with a majority of abnormally spliced transcripts (MYBPC3-c.1182C>A, TNNT2-c.460G>A (p.Glu154Lys), and TNNT2-c.822-3C>A) or as variations with a weak splicing effect (TNNT2-c.1000-38C>A). For the two remaining variations in intron 11 of the TNNT2 gene in the vicinity of the acceptor splice site (c.571-7G>A, c.571-29G>A), a minigene assay was inconclusive as exon 12 is neither recognized as an exon by HeLa nor by H9c2 cells. Our study highlights the importance of the combined use of in silico and in vitro splicing assays to improve the prediction of the functional splicing impact of identified genetic variants if the RNA sample from the patient is not easily available.

Objective: To investigate whether mutations in the SURF1 gene are a cause of Charcot-Marie-Tooth (CMT) disease., Methods: We describe 2 patients from a consanguineous family with demyelinating autosomal recessive CMT disease (CMT4) associated with the homozygous splice site mutation c.107-2A>G in the SURF1 gene, encoding an assembly factor of the mitochondrial respiratory chain complex IV. This observation led us to hypothesize that mutations in SURF1 might be an unrecognized cause of CMT4, and we investigated SURF1 in a total of 40 unrelated patients with CMT4 after exclusion of mutations in known CMT4 genes. The functional impact of c.107-2A>G on splicing, amount of SURF1 protein, and on complex IV activity and assembly was analyzed., Results: Another patient with CMT4 was found to harbor 2 additional SURF1 mutations. All 3 patients with SURF1-associated CMT4 presented with severe childhood-onset neuropathy, motor nerve conduction velocities <25 m/s, and lactic acidosis. Two patients had brain MRI abnormalities, including putaminal and periaqueductal lesions, and developed cerebellar ataxia years after polyneuropathy. The c.107-2A>G mutation produced no normally spliced transcript, leading to SURF1 absence. However, complex IV remained partially functional in muscle and fibroblasts., Conclusions: We found SURF1 mutations in 5% of families (2/41) presenting with CMT4. SURF1 should be systematically screened in patients with childhood-onset severe demyelinating neuropathy and additional features such as lactic acidosis, brain MRI abnormalities, and cerebellar ataxia developing years after polyneuropathy.

Abetalipoproteinemia (ABL) is an inherited disease characterized by the defective assembly and secretion of apolipoprotein B-containing lipoproteins caused by mutations in the microsomal triglyceride transfer protein large subunit (MTP) gene (MTTP). We report here a female patient with an unusual clinical and biochemical ABL phenotype. She presented with severe liver injury, low levels of LDL-cholesterol, and subnormal levels of vitamin E, but only mild fat malabsorption and no retinitis pigmentosa or acanthocytosis. Our objective was to search for MTTP mutations and to determine the relationship between the genotype and this particular phenotype. The subject exhibited compound heterozygosity for two novel MTTP mutations: one missense mutation (p.Leu435His) and an intronic deletion (c.619-5&#95;619-2del). COS-1 cells expressing the missense mutant protein exhibited negligible levels of MTP activity. In contrast, the minigene splicing reporter assay showed an incomplete splicing defect of the intronic deletion, with 26% of the normal splicing being maintained in the transfected HeLa cells. The small amount of MTP activity resulting from the residual normal splicing in the patient explains the atypical phenotype observed. Our investigation provides an example of a functional analysis of unclassified variations, which is an absolute necessity for the molecular diagnosis of atypical ABL cases.

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 patient's 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.

The POLG genes were sequenced in two unrelated patients presenting with Alpers syndrome. The novel c.3626&#95;3629dupGATA and the c.3643+2T>C alleles were associated in trans with p.A467T and p.[W748S;E1143G], respectively. POLG transcripts from skin fibroblasts showed complete exon 22 skipping for patient 2, but surprisingly partial exon 22 skipping from the c.3626&#95;3629dupGATA for patient 1. The creation of a putative exonic splicing silencer could be responsible for the splicing anomaly observed in patient 1. Both c.3643+2T>C and c.3626&#95;3629dupGATA create a premature termination codon and a low polymerase γ activity in skin fibroblasts is responsible for the severe phenotype in these patients., (Copyright © 2010 Mitochondria Research Society. Published by Elsevier B.V. All rights reserved.)

We report the molecular characterization of two splice mutations in two different French families affected with a late onset form of Charcot-Marie-Tooth disease type 1B (CMT1B), an autosomal dominant inherited disorder caused by mutations in the myelin protein zero gene. The first substitution, c.306G>A, located in exon 3, does not change the codon p.Val102Val but is co-transmitted with the disease in the first family. The second substitution, c.675+3dup, is an insertion of a T at position +3 of intron 5. To identify the functional impact of these nucleotide changes on splicing and because no RNA sample was available, we used in silico prediction and in vitro splicing assay. Mutation c.306G>A increases the strength of a preexisting cryptic donor site at position c.304 which becomes stronger than the normal donor site of intron 3. This variation creates a sequence that better matches the U1 small nuclear RNA (snRNA) binding consensus, and HeLa cells, transfected with the mutant minigene, produce a truncated exon 3 messenger RNA (mRNA). Mutation c.675+3dup was predicted to abolish the donor site of intron 5, and, indeed, HeLa cells transfected with the mutant minigene completely skip exon 5 from the transcript. The mutated sequence abolishes U1 snRNA binding and co-transfection of a mutated complementary U1 snRNA restored exon 5 inclusion in the mRNA. This work provides valuable information regarding the molecular basis of two forms of late onset of CMT1B, U1 snRNA mis-binding, and provides more evidence that a "silent" polymorphism may be a disease causing mutation.

We studied a series of 42 cases of transposition of the great arteries (TGA), a complex heart defect (CHD) that is two times more prevalent in males than in females. A mutation in the X chromosome at the ZIC3 gene was found in two affected siblings (one male, one female) and their unaffected mother. A second factor, skewed X-inactivation pattern explained the discrepancy between the daughter/mother phenotype. In this family, the missense mutation (p.W255G) was found in the first zinc finger of ZIC3, a domain that is relatively specific to each of the five human ZIC genes. It was tested further along with two other mutations of this domain (p.C253S and p.H286R). In transfected 3T3 cells, mutants p.W255G and p.H286R expressed lower protein levels, and an increased protein degradation (p.W255G only). Moreover, mutants p.C253S and p.W255G had a decreased transcription activation of the TK-luciferase reporter gene. Nuclear translocation of the three ZIC3 mutants varied considerably depending on the experimental models. Finally, p.W255G and p.H286R showed diminished activities for both left-right axis disturbance and neural crest induction in Xenopus embryos. These results suggest that mutations in the first zinc finger of ZIC3 mildly affect several functions of the protein., ((c) 2007 Wiley-Liss, Inc.)

DGUOK [dG (deoxyguanosine) kinase] is one of the two mitochondrial deoxynucleoside salvage pathway enzymes involved in precursor synthesis for mtDNA (mitochondrial DNA) replication. DGUOK is responsible for the initial rate-limiting phosphorylation of the purine deoxynucleosides, using a nucleoside triphosphate as phosphate donor. Mutations in the DGUOK gene are associated with the hepato-specific and hepatocerebral forms of MDS (mtDNA depletion syndrome). We identified two missense mutations (N46S and L266R) in the DGUOK gene of a previously reported child, now 10 years old, who presented with an unusual revertant phenotype of liver MDS. The kinetic properties of normal and mutant DGUOK were studied in mitochondrial preparations from cultured skin fibroblasts, using an optimized methodology. The N46S/L266R DGUOK showed 14 and 10% residual activity as compared with controls with dG and deoxyadenosine as phosphate acceptors respectively. Similar apparent negative co-operativity in the binding of the phosphate acceptors to the wild-type enzyme was found for the mutant. In contrast, abnormal bimodal kinetics were shown with ATP as the phosphate donor, suggesting an impairment of the ATP binding mode at the phosphate donor site. No kinetic behaviours were found for two other patients with splicing defects or premature stop codon. The present study represents the first characterization of the enzymatic kinetic properties of normal and mutant DGUOK in organello and our optimized protocol allowed us to demonstrate a residual activity in skin fibroblast mitochondria from a patient with a revertant phenotype of MDS. The residual DGUOK activity may play a crucial role in the phenotype reversal.

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive disorder of glyoxylate metabolism, in which excessive oxalates are formed by the liver and excreted by the kidneys, causing a wide spectrum of disease, ranging from renal failure in infancy to mere renal stones in late adulthood. This disease is caused by a deficiency of alanine:glyoxylate aminotransferase (AGT), which is encoded by a single copy gene, AGXT, located in 2q37.3. We identified an apparently homozygous, loss-of-function, mutation in a patient; the gene defect was present in the heterozygous mother but not in the patient's father. We performed a microsatellite repeat analysis using 13 specific chromosome 2 markers and non-chromosome 2 minisatellites. Six specific chromosome 2 markers showed an apparently homozygous maternal inheritance while four showed a biparental transmission consistent with paternity (confirmed by minisatellite analysis). Quantitative PCR of AGXT exons 1 and 3 on the patient's and parents genomic DNA revealed the presence of two copies of the gene. This is the first case of PH1 caused by segmental maternal isodisomy of 2q37.3., ((c) 2004 Wiley-Liss, Inc.)

In recent years, some patients bearing "atypical" forms of cystic fibrosis (CF) with normal sweat chloride concentrations have been described. To identify the spectrum of mutant combinations causing such atypical CF, we collected the results of CFTR (ABCC7) mutation analysis from 15 laboratories. Thirty patients with one or more typical symptoms of the disease associated with normal or borderline sweat chloride levels and bearing two CFTR mutations were selected. Phenotypes and genotypes of these 30 patients are described. A total of 18 different CFTR mutations were observed in the 60 chromosomes analysed. F508del was present in 31.6 % of the mutated chromosomes and 3849+10kbC>T in 13.3 %. R117H, D1152H, L206W, 3272-26A>G, S1235R, G149R, R1070W, S945L, and the poly-T tract variation commonly called IVS8-5T were also observed. The relative frequency of CFTR mutations clearly differed from that observed in typical CF patients or in CBAVD patients with the same ethnic origin. A mild genotype with one or two mild or variable mutations was observed in all the patients. These findings improve our understanding of the distribution of CFTR alleles in CF with normal or borderline sweat chloride concentrations and will facilitate the development of more sensitive CFTR mutation screening., (Copyright 2003 Wiley-Liss, Inc.)

Mitochondrial DNA depletion is a well established cause of severe liver failure in infancy. The autosomal inheritance of this quantitative mitochondrial DNA defect supports the involvement of a nuclear gene in the control of mitochondrial DNA level. We previously described a case of a 28-month-old child presenting with a progressive liver fibrosis due to a mitochondrial DNA depletion (85% at 12 months of age). As this syndrome was clinically liver-restricted, a liver transplant was initially discussed. We report the clinical, biochemical and molecular follow-up of this child, now 6 years old. The patient displayed a spontaneous gradual improvement of his liver function with continuous increment of clotting factor values since 32 months of age. A marked reduction of the previous extensive fibrosis was evidenced on a liver biopsy performed at 46 months of age associated with a dramatic decrease of the mitochondrial DNA depletion (35%). Consequently, an almost complete restoration of respiratory chain activities containing mitochondrial DNA-encoded subunits was observed. This is the first report of a revertant phenotype in liver mitochondrial DNA depletion syndrome.

Creatine kinases (CK) play a prominent role in cell energy distribution through an energy shuttle between mitochondria and other organelles. Human brain CK was cloned and overexpressed in COS-7 cells. We then deleted His-65 and/or Pro-66 situated near the center of a flexible loop as shown by X-ray crystallography on mitochondrial and cytosolic CK. The DeltaH65 mutant had nearly the same affinity for its substrates as wild isoenzyme, but its stability was very low. Unlike DeltaH65, DeltaH65P66 had a eightfold decreased affinity for creatine phosphate and was unable to dephosphorylate cyclocreatine phosphate. Our results demonstrate that, despite an overall similar shape of the proteins, this loop accounts for some subtle differences in isoenzyme functions.

In order to identify additional genotypes in primary hyperoxaluria type 1, we sequenced the AGXT genes of 9 patients. We report 5 new mutations. Three are splice-site mutations situated at the end of intron 4 and 8 (647-1G>A, 969-1G>C, 969-3C>G), one is a missense mutation in exon 5 (D183N), and one is a short duplication in exon 2 (349ins7). Their consequence is always a lack of enzymatic activity of the Alanine-Glyoxylate Aminotransferase (AGT); for 4 of them, we were able to deduce that they were associated to the absence of AGT protein. These mutations are rare, as they have been found on one allele in our study (except 969-3C>G present in 2 unrelated families), and have not been previously reported.

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&#95;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., (Copyright 2000 Wiley-Liss, Inc.)

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., (Copyright 2000 Wiley-Liss, Inc.)

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.