Molecular diagnosis of known recessive ataxias by homozygosity mapping with SNP arrays.

The diagnosis of rare inherited diseases is becoming more and more complex as an increasing number of clinical conditions appear to be genetically heterogeneous. Multigenic inheritance also applies to the autosomal recessive progressive cerebellar ataxias (ARCAs), for which 14 genes have been identified and more are expected to be discovered. We used homozygosity mapping as a guide for identification of the defective locus in patients with ARCA born from consanguineous parents. Patients from 97 families were analyzed with GeneChip Mapping 10K or 50K SNP Affymetrix microarrays. We identified six families homozygous for regions containing the autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) gene, two families homozygous for the ataxia-telangiectasia gene (ATM), two families homozygous for the ataxia with oculomotor apraxia type 1 (AOA1) gene, and one family homozygous for the AOA type 2 (AOA2) gene. Upon direct gene testing, we were able to identify a disease-related mutation in all families but one of the two kindred homozygous at the ATM locus. Although linkage analyses pointed to a single locus on chromosome 11q22.1-q23.1 for this family, clinical features, normal levels of serum alpha-foetoprotein as well as absence of mutations in the ATM gene rather suggest the existence of an additional ARCA-related gene in that interval. While the use of homozygosity mapping was very effective at pointing to the correct gene, it also suggests that the majority of patients harbor mutations either in the genes of the rare forms of ARCA or in genes yet to be identified.

Ataxia with oculomotor apraxia type 2: clinical, biological and genotype/phenotype correlation study of a cohort of 90 patients.

Ataxia with oculomotor apraxia type 2 (AOA2) is an autosomal recessive disease due to mutations in the senataxin gene, causing progressive cerebellar ataxia with peripheral neuropathy, cerebellar atrophy, occasional oculomotor apraxia and elevated alpha-feto-protein (AFP) serum level. We compiled a series of 67 previously reported and 58 novel ataxic patients who underwent senataxin gene sequencing because of suspected AOA2. An AOA2 diagnosis was established for 90 patients, originating from 15 countries worldwide, and 25 new senataxin gene mutations were found. In patients with AOA2, median AFP serum level was 31.0 microg/l at diagnosis, which was higher than the median AFP level of AOA2 negative patients: 13.8 microg/l, P = 0.0004; itself higher than the normal level (3.4 microg/l, range from 0.5 to 17.2 microg/l) because elevated AFP was one of the possible selection criteria. Polyneuropathy was found in 97.5% of AOA2 patients, cerebellar atrophy in 96%, occasional oculomotor apraxia in 51%, pyramidal signs in 20.5%, head tremor in 14%, dystonia in 13.5%, strabismus in 12.3% and chorea in 9.5%. No patient was lacking both peripheral neuropathy and cerebellar atrophy. The age at onset and presence of occasional oculomotor apraxia were negatively correlated to the progression rate of the disease (P = 0.03 and P = 0.009, respectively), whereas strabismus was positively correlated to the progression rate (P = 0.03). An increased AFP level as well as cerebellar atrophy seem to be stable in the course of the disease and to occur mostly at or before the onset of the disease. One of the two patients with a normal AFP level at diagnosis had high AFP levels 4 years later, while the other had borderline levels. The probability of missing AOA2 diagnosis, in case of sequencing senataxin gene only in non-Friedreich ataxia non-ataxia-telangiectasia ataxic patients with AFP level > or =7 microg/l, is 0.23% and the probability for a non-Friedreich ataxia non-ataxia-telangiectasia ataxic patient to be affected with AOA2 with AFP levels > or =7 microg/l is 46%. Therefore, selection of patients with an AFP level above 7 microg/l for senataxin gene sequencing is a good strategy for AOA2 diagnosis. Pyramidal signs and dystonia were more frequent and disease was less severe with missense mutations in the helicase domain of senataxin gene than with missense mutations out of helicase domain and deletion and nonsense mutations (P = 0.001, P = 0.008 and P = 0.01, respectively). The lack of pyramidal signs in most patients may be explained by masking due to severe motor neuropathy.

Ataxia with oculomotor apraxia type 2: a clinical and genetic study of 19 patients.

Ataxia with oculo-motor apraxia type 2 (AOA2) is a recently described autosomal recessive cerebellar ataxia (ARCA) caused by mutations in the senataxin gene (SETX). We analysed the phenotypic spectrum of 19 AOA2 patients with mutations in SETX, which seems to be the third most frequent form of ARCA in Algeria after Freidreich ataxia and Ataxia with vitamin E deficiency. In AOA2 patients, the mean age at onset for all families was in the second decade. Cerebellar ataxia was progressive, slowly leading to disability which was aggravated by axonal polyneuropathy present in almost all the patients. Mean disease duration until wheelchair was around 20 years. Oculo-motor apraxia (OMA) was present in 32% of the patients while convergent strabismus was present in 37%. Strabismus is therefore also very suggestive of AOA2 when associated with ataxia and polyneuropathy even in the absence of OMA. Cerebellar atrophy was more severe in the eldest patients; however it may also be an early sign since it was present in the youngest and paucisymptomatic patients. The initial sign was gait ataxia in all but two patients who presented with head tremor and writer cramp, respectively. Serum alpha-fetoprotein, which was elevated in all tested patients, was a good marker to suggest molecular studies of the SETX gene.

Prevalence and characterization of extended-spectrum beta-lactamases in Klebsiella pneumoniae in Algiers hospitals (Algeria).

AIM OF THE STUDY: To determine the prevalence and the diversity of extended-spectrum beta-lactamases (ESBLs) in 196 Klebsiella pneumoniae clinical isolates collected from three hospitals in Algiers. MATERIALS AND METHODS: Antibiograms were done on Mueller-Hinton agar plates with the disc-diffusion method and MICs were determined by agar-dilution method. Mating experiments were performed in agar medium. Plasmid DNA was extracted by the alcalin-lysis method. Total DNA was extracted with a Qiagen mini kit and screened for bla(TEM) and bla(CTX-M) genes by PCR. Linkage of bla(CTX-M) genes with insertion sequence ISEcp1B and class 1 integrons was investigated by PCR. PCR products were sequenced by the Sanger method. The epidemiological relationships between ESBL-producing K. pneumoniae isolates were analyzed by ERIC-PCR. RESULTS: Thirty-nine (19.9%) isolates were found to produce ESBLs belonging to CTX-M-1 group and TEM penicillinases (CTX-M-3, CTX-M-15 and TEM-1). ERIC-PCR analysis showed that the isolates are genetically unrelated. The bla(TEM) and bla(CTX-M) genes as well as aminoglycosides and sulfonamides resistance determinants were found located in self-transferable plasmids of approximately 85 kb. The class 1 integrons and the insertion sequence ISEcp1B were present in the isolates and in their transconjugants. ISEcp1B was found genetically linked to the bla(CTX-M) genes and located 127bp upstream, with the presence of the V and W sequences. CONCLUSION: The study revealed a high rate of ESBL-producing K. pneumoniae in Algerian hospitals, resulting from horizontal dissemination of mobile bla(CTX-M) genes.

Founder effect and estimation of the age of the c.892C>T (p.Arg298Cys) mutation in LMNA associated to Charcot-Marie-Tooth subtype CMT2B1 in families from North Western Africa.

CMT2B1, an axonal subtype (MIM 605588) of the Charcot-Marie-Tooth disease, is an autosomal recessive motor and sensory neuropathy characterized by progressive muscular and sensory loss in the distal extremities with chronic distal weakness. The genetic defect associated with the disease is, to date, a unique homozygous missense mutation, p.Arg298Cys (c.892C>T), in the LMNA gene. So far, this mutation has only been found in affected individuals originating from a restricted region of North Western Africa (northwest of Algeria and east of Morocco), strongly suggesting a founder effect. In order to address this hypothesis, genotyping of both STRs and intragenic SNPs was performed at the LMNA locus, at chromosome 1q21.2-q21.3, in 42 individuals affected with CMT2B1 from 25 Algerian families. Our results indicate that the affected individuals share a common ancestral haplotype in a region of about 1.0 Mb (1 cM) and that the most recent common ancestor would have lived about 800-900 years ago (95% confidence interval: 550 to 1300 years).

Prevalence of beta-lactams resistance among Escherichia coli clinical isolates from a hospital in Algiers.

A high prevalence of beta-lactams resistance among Enterobacteriaceae have been reported worldwide; however, there are not sufficient data on this issue in Algeria. beta-Lactams susceptibility of 203 Escherichia coli clinical isolates was determined by agar diffusion method, and production of extended-spectrum beta-lactamases (ESBL) was screened by double-disk synergy test. This analysis showed five well-defined phenotypes: 1) 62 isolates (30.5%) were susceptible to all beta-lactams; 2) 135 isolates (66.5%) presented a broad-spectrum beta-lactamases phenotype (BSBL); 3) three isolates (1.5%) were defined as producing ESBLs; 4) two isolates (1%) were AmpC cephalosporinase producers; and 5) one isolate (0.5%) presented a phenotype of cell-decreased permeability to beta-lactams. Isoelectric focusing revealed beta-lactamases with isolectric points of 5.4 or 7.6 for isolates with BSBL phenotype; approximately 9.0 for two ESBL isolates; 5.4, 7.6 and approximately 9.0 for the remaining ESBL isolate; and 5.4 and approximately 9.0 for the AmpC isolates. The cefotaxime hydrolysis corresponds to the basic bands with an isoelectric point of approximately 9.0. Conjugation assay showed transfer of penicillinase and AmpC resistance phenotypes and their corresponding beta-lactamases to recipient E. coli BM21 in association with plasmids of 71.4 kb for the AmpC isolates and from 40-56 kb for penicillinase isolates. This result showed that the AmpC phenotype is plasmid mediated. ESBL isolates were found not to transfer their resistance through conjugation experiment. Polymerase chain reaction (PCR) experiments using primers specific to blaTEM, blaAmpC and blaCTX-M genes showed specific amplification with blaCTX-M primer for two ESBL isolates; blaTEM and blaCTX-M for the remaining ESBL isolate; and blaTEM and blaAmpC for the AmpC isolates and their corresponding transconjugants. The study showed a high rate of isolates producing penicillinase, and low frequencies of AmpC and ESBL phenotypes. AmpC beta-lactamases were plasmid mediated, and ESBLs belong to the CTM-M type.

Prevalence of β - lactams resistance among Escherichia coli clinical isolates from a hospital in Algiers

La prevalencia de la resistencia a los betalactámicos entre las enterobacterias es alta en todo el mundo, pero en Argelia no se dispone desuficientes datos. Se determinó la sensibilidad a los betalactámicos de 203 cepas clínicas de Escherichia coli mediante difusión en agar y seanalizó la producción de betalactamasas de espectro extendido (BLEE) mediante la técnica de la sinergia del doble disco. Este análisis mostrócinco fenotipos bien definidos: 1) 62 cepas (30,5%) fueron sensibles a todos los betalactámicos; 2) 135 cepas (66,5%) presentaron unfenotipo caracterizado por betalactamasas de amplio espectro (BLEA); 3) 3 cepas (1,5%) se definieron como productoras de BLEE; 4) 2 cepas(1%) fueron productoras de cefalosporinasa tipo AmpC; y 5) una (0,5%) presentó un fenotipo de disminución de la permeabilidad celulara los betalactámicos. La determinación del punto isoeléctrico mostró betalactamasas con puntos isoeléctricos de 5,4 o 7,6 para las cepascon fenotipo BLEA; ∼9,0 para 2 cepas productoras de BLEE; 5,4, 7,6 y ∼9,0 para la tercera cepa productora de BLEE; y 5,4 y ∼9,0 para lascepas productoras de cefalosporinasa AmpC. La hidrólisis de cefotaxima se corresponde con las bandas básicas con un punto isoeléctricode ∼9,0. El ensayo de conjugación mostró una transferencia de los fenotipos de resistencia de penicilinasas y cefalosporinasa AmpC y suscorrespondientes betalactamasas a E. coli BM21 en asociación con plásmidos de 71,4 kb para las cepas productoras de cefalosporinasaAmpC y de 40-56 kb para las productoras de penicilinasas. Este resultado mostró que el fenotipo productor de cefalosporinasa AmpC estámediado por plásmidos. Las cepas productoras de BLEE no transfirieron su resistencia mediante el ensayo de conjugación. La reacción encadena de la polimerasa utilizando primers específicos para los genes blaTEM, blaAmpC y blaCTX-M mostró una amplificación específica con elprimer para blaCTX-M en dos cepas productoras de BLEE; los primers para blaTEM y blaCTX-M para la tercera cepa productora de BLEE; y losprimers para blaTEM y blaAmpC para las cepas productoras de cefalosporinasa AmpC y sus cepas transconjugantes correspondientes. El estudiomostró una alta tasa de cepas productoras de penicilinasas y una baja frecuencia de fenotipos productores de AmpC y BLEE. Las betalactamasasproductoras de cefalosporinasa AmpC estaban mediadas por plásmidos y las BLEE pertenecieron al tipo CTM-M

A high prevalence of β-lactams resistance among Enterobacteriaceae have been reported worldwide; however, there are not sufficient dataon this issue in Algeria. β-Lactams susceptibility of 203 Escherichia coli clinical isolates was determined by agar diffusion method, and productionof extended-spectrum β-lactamases (ESBL) was screened by double-disk synergy test. This analysis showed five well-defined phenotypes:1) 62 isolates (30.5%) were susceptible to all β-lactams; 2) 135 isolates (66.5%) presented a broad-spectrum β-lactamases phenotype(BSBL); 3) three isolates (1.5%) were defined as producing ESBLs; 4) two isolates (1%) were AmpC cephalosporinase producers; and 5) one isolate(0.5%) presented a phenotype of cell-decreased permeability to β-lactams. Isoelectric focusing revealed β-lactamases with isolectric pointsof 5.4 or 7.6 for isolates with BSBL phenotype; ∼9.0 for two ESBL isolates; 5.4, 7.6 and ∼9.0 for the remaining ESBL isolate; and 5.4 and ∼9.0for the AmpC isolates. The cefotaxime hydrolysis corresponds to the basic bands with an isoelectric point of ∼9.0. Conjugation assay showedtransfer of penicillinase and AmpC resistance phenotypes and their corresponding β-lactamases to recipient E. coli BM21 in association withplasmids of 71.4 kb for the AmpC isolates and from 40–56 kb for penicillinase isolates. This result showed that the AmpC phenotype is plasmidmediated. ESBL isolates were found not to transfer their resistance through conjugation experiment. Polymerase chain reaction (PCR) experimentsusing primers specific to blaTEM , blaAmpC and blaCTX-M genes showed specific amplification with blaCTX-M primer for two ESBL isolates;blaTEM and blaCTX-M for the remaining ESBL isolate; and blaTEM and blaAmpC for the AmpC isolates and their corresponding transconjugants. Thestudy showed a high rate of isolates producing penicillinase, and low frequencies of AmpC and ESBL phenotypes. AmpC β-lactamases wereplasmid mediated, and ESBLs belong to the CTM-M type

The Rhizobium etli rpoN locus: DNA sequence analysis and phenotypical characterization of rpoN, ptsN, and ptsA mutants.

The rpoN region of Rhizobium etli was isolated by using the Bradyrhizobium japonicum rpoN1 gene as a probe. Nucleotide sequence analysis of a 5,600-bp DNA fragment of this region revealed the presence of four complete open reading frames (ORFs), ORF258, rpoN, ORF191, and ptsN, coding for proteins of 258, 520, 191, and 154 amino acids, respectively. The gene product of ORF258 is homologous to members of the ATP-binding cassette-type permeases. ORF191 and ptsN are homologous to conserved ORFs found downstream from rpoN genes in other bacterial species. Unlike in most other microorganisms, rpoN and ORF191 are separated by approximately 1.6 kb. The R. etli rpoN gene was shown to control in free-living conditions the production of melanin, the activation of nifH, and the metabolism of C4-dicarboxylic acids and several nitrogen sources (ammonium, nitrate, alanine, and serine). Expression of the rpoN gene was negatively autoregulated and occurred independently of the nitrogen source. Inactivation of the ptsN gene resulted in a decrease of melanin synthesis and nifH expression. In a search for additional genes controlling the synthesis of melanin, an R. etli mutant carrying a Tn5 insertion in ptsA, a gene homologous to the Escherichia coli gene coding for enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system, was obtained. The R. etli ptsA mutant also displayed reduced expression of nifH. The ptsN and ptsA mutants also displayed increased sensitivity to the toxic effects of malate and succinate. Growth of both mutants was inhibited by these C4-dicarboxylates at 20 mM at pH 7.0, while wild-type cells grow normally under these conditions. The effect of malate occurred independently of the nitrogen source used. Growth inhibition was decreased by lowering the pH of the growth medium. These results suggest that ptsN and ptsA are part of the same regulatory cascade, the inactivation of which renders the cells sensitive to toxic effects of elevated concentrations of malate or succinate.

[Genetic study of the autosomal recessive form of Charcot-Marie-Tooth in an Algerian family]. / Etude génétique de la maladie de Charcot-Marie-Tooth forme autosomique récessive chez une famille algérienne.

Charcot-Marie-Tooth disease (CMT) is a hereditary neuropathy characterized by muscular atrophy and progressive sensitive alterations that affect limbs. The CMT is one of the most heterogenous diseases, clinically as well as genetically. At least twelve loci are responsible for the CMT phenotype, four of them for the autosomal recessive form. The aim of our work was to determinate the implication/exclusion of these four loci in an Algerian family by linkage analysis using microsatellites markers. We have tested the four loci on 8q13-21.1 (CMT4A), 11q23 (CMT4B), 5q23-33 (CMT4C) 8q24 (CMTAR). The haplotype reconstruction allowed us to exclude all the loci in this family, suggesting that the locus (gene) responsible for this form of CMT is localized elsewhere in the genome, thus providing an other observation of the great heterogeneity of the CMT, particularly autosomal recessive.