Transcriptomic analysis of resistance and short-term induction response to pyrethroids, in Anopheles coluzzii legs.

BACKGROUND: Insecticide-treated bed nets and indoor residual spraying comprise the major control measures against Anopheles gambiae sl, the dominant vector in sub-Saharan Africa. The primary site of contact with insecticide is through the mosquitoes' legs, which represents the first barrier insecticides have to bypass to reach their neuronal targets. Proteomic changes and leg cuticle modifications have been associated with insecticide resistance that may reduce the rate of penetration of insecticides. Here, we performed a multiple transcriptomic analyses focusing on An. coluzzii legs. RESULTS: Firstly, leg-specific enrichment analysis identified 359 genes including the pyrethroid-binder SAP2 and 2 other chemosensory proteins, along with 4 ABCG transporters previously shown to be leg enriched. Enrichment of gene families included those involved in detecting chemical stimuli, including gustatory and ionotropic receptors and genes implicated in hydrocarbon-synthesis. Subsequently, we compared transcript expression in the legs of a highly resistant strain (VK7-HR) to both a strain with very similar genetic background which has reverted to susceptibility after several generations without insecticide pressure (VK7-LR) and a lab susceptible population (NG). Two hundred thirty-two differentially expressed genes (73 up-regulated and 159 down-regulated) were identified in the resistant strain when compared to the two susceptible counterparts, indicating an over-expression of phase I detoxification enzymes and cuticular proteins, with decrease in hormone-related metabolic processes in legs from the insecticide resistant population. Finally, we analysed the short-term effect of pyrethroid exposure on An. coluzzii legs, comparing legs of 1 h-deltamethrin-exposed An. coluzzii (VK7-IN) to those of unexposed mosquitoes (VK7-HR) and identified 348 up-regulated genes including those encoding for GPCRs, ABC transporters, odorant-binding proteins and members of the divergent salivary gland protein family. CONCLUSIONS: The data on An. coluzzii leg-specific transcriptome provides valuable insights into the first line of defense in pyrethroid resistant and short-term deltamethrin-exposed mosquitoes. Our results suggest that xenobiotic detoxification is likely occurring in legs, while the enrichment of sensory proteins, ABCG transporters and cuticular genes is also evident. Constitutive resistance is primarily associated with elevated levels of detoxification and cuticular genes, while short-term insecticide-induced tolerance is linked with overexpression of transporters, GPCRs and GPCR-related genes, sensory/binding and salivary gland proteins.

L-2-hydroxyglutaric aciduria: clinical and molecular study in three Tunisian families. Identification of a new mutation and inter-familial phenotype variability.

We report clinical and molecular studies in three unrelated Tunisian families containing seven patients with L2HGA. Although the age of onset is similar in all these patients at nearly 6 years, they progressively developed peculiar clinical phenotypes different from family to family. The three patients of family 1 showed mental retardation, epilepsy, cerebellar ataxia and pyramidal and pseudobulbar syndromes. The two patients of family 2 showed mental retardation and parkinsonism especially extrapyramidal stiffness, dystonia and myoclonus. The two patients of family 3 showed an intermediate phenotype; they share some clinical signs of the patients of family 1 (epilepsy, pyramidal and extrapyramidal syndromes) and some clinical signs of the patients of family 2 (extrapyramidal stiffness and dystonia). Molecular study identified a novel homozygous c.185C>A, p.A62D mutation on the L2HGDH gene in families 1 and 3 and the already known homozygous c.241A>G, p.K81E mutation in family 2. We suppose that the type of mutation in the L2HGDH gene does not play a complete role in the inter-familial phenotype variability. Disturbance of other unknown metabolic pathways related to L2HGA may contribute to this phenomenon.

Variable cardiac involvement in Tunisian siblings harboring FKRP gene mutations.

Mutations in the gene encoding fukutin-related protein (FKRP) cause limb-girdle muscular dystrophy 2I (LGMD2I) and congenital muscular dystrophy (MDC1C). Cardiac involvement was frequently reported with numerous mutations including C826A and 1364C > A mutations. The original Tunisian family with LGMD2I included 12 patients sharing the LGMD phenotype and homozygous to the 1486T > A mutation but who did not display any cardiac involvement. In this study, we report the clinical data, cardiac assessment and mutation analysis in four sibs belonging to a second Tunisian LGMD2I family. All patients showed the LGMD phenotype, the oldest brother and sister had mild cardiac involvement, whereas two twin sisters displayed severe cardiomyopathy leading to death. The patients shared the compound heterozygous 1486T > A, 1364C > A mutation in the FKRP gene suggesting that the association of a compound heterozygous state of mutation responsible for LGMD2I and the MDC1C phenotype could lead to cardiac involvement.

PINK1 mutations and parkinsonism.

BACKGROUND: PINK1 loss-of-function causes recessive, early-onset parkinsonism. In Tunisia there is a high rate of consanguineous marriage but PINK1 carrier frequency and disease prevalence have yet to be assessed. OBJECTIVES: The frequency of PINK1 mutations in familial parkinsonism, community-based patients with idiopathic Parkinson disease (PD) (non-familial PD), and control subjects was determined. Demographic and clinical characteristics of individuals with PINK1 homozygous or heterozygous variants, or without PINK1 mutations, were compared. METHODS: A total of 92 kindreds (with 208 affected and 340 unaffected subjects), 240 nonfamilial PD, and 368 control participants were recruited from the Institut National de Neurologie, Tunis. Clinical examinations included Hoehn &Yahr, UPDRS, and Epworth scales. PINK1 sequencing and dosage analysis was performed in familial index patients, the variants identified screened in all subjects. Parkin and LRRK2 genes were also examined. RESULTS: Four PINK1 homozygous mutations, three novel (Q129X, Q129fsX157, G440E, and one previously reported; Q456X), segregate with parkinsonism in 46 individuals in 14 of 92 families (15%). Six of 240 patients with nonfamilial PD were found with either homozygous Q456X or Q129X (2.5%) substitutions. In patients with familial disease, PINK1 homozygotes were younger at disease onset (36 +/- 12 years) than noncarriers (57 +/- 15 years) and more often had an akinetic-rigid presentation at examination and slow progression. CONCLUSIONS: Segregation of PINK1 mutations with parkinsonism within families, and frequency estimates within population controls, suggested only four PINK1 mutations were pathogenic. Several PINK1 sequence variants are potentially benign and there was no evidence that PINK1 heterozygosity increases susceptibility to idiopathic Parkinson disease.

Genetic heterogeneity within a consanguineous family involving the LGMD 2D and the LGMD 2C genes.

The sarcoglycanopathies are a group of autosomal recessive limb girdle muscular dystrophies (AR-LGMD 2) characterised by mutations in gene encoding one of the sarcoglycan subunits. Mutations in SGCA, SGCB, SGCG and SGCD genes are associated with LGMD 2D, 2E, 2C and 2F, respectively. We report three Tunisian patients belonging to the same consanguineous family sharing similar LGMD 2 phenotype but heterogeneous sarcoglycans immunohistochemical patterns. Linkage analysis suggests linkage with the LGMD 2D locus for the two siblings and with LGMD 2C locus for the third patient. Mutation analysis revealed two distinct mutations. A del521T homozygous mutation in exon 6 of the SGCG gene (LGMD 2C), widely distributed in Tunisian patients, was found in one patient, whereas a 157G>A homozygous mutation in exon 2 of the SGCA gene (LGMD 2D) was found in the two siblings. The presence of two distinct genetic forms, LGMD 2C and LGMD 2D in a consanguineous family raises the problem of the complexity of genetic counselling in inbred populations.

Allelic heterogeneity of GNE gene mutation in two Tunisian families with autosomal recessive inclusion body myopathy.

Autosomal recessive hereditary inclusion body myopathy (AR-HIBM), with sparing of the quadriceps, is characterized by adult-onset, with weakness and atrophy of distal lower limb muscles, and typical histopathological findings in muscle biopsy. AR hIBM is associated with mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene on chromosome 9p12-13 . We report two unrelated Tunisian families with clinical and pathological features of AR HIBM. One distinct homozygous GNE missense mutation, M712T, previously reported in Middle Eastern Jewish patients, and a newly identified one, L379H, were found in one patient from each family. We conclude that AR HIBM in Tunisia shows an allelic genetic heterogeneity.

Aprataxin gene mutations in Tunisian families.

The authors report clinical and genetic study of 13 patients from three unrelated Tunisian families with an early onset cerebellar ataxia associated with oculomotor apraxia. Cerebellar ataxia with oculomotor apraxia 1 (AOA1) represents a clinically heterogeneous disease caused by mutations in the aprataxin gene. Two novel mutations were identified, the complete deletion of the gene, which seems to not correlate with an increased severity of the disease, and a splice mutation on the acceptor splice site of exon 7.

[Lateral gaze palsy and progressive scoliosis in 4 Tunisian families]. / Paralysie du regard latéral et scoliose progressive: à propos de 4 familles tunisiennes.

In 1975, Sharpe and Silversides described a neurological entity in a Chinese family. Clinical picture was characterized by paralysis of horizontal gaze, pendular nystagmus and progressive scoliosis. To date, 43 cases have been reported. The pathogenesis remains unclear. The Authors report four Tunisian families with 12 affected individuals. The age of patients ranges from 6 to 34 Years. All examined patients have complete lateral gaze palsy, pendular nystagmus and progressive scoliosis. Blood routine tests, cerebrospinal fluid (CSF), evoked potentials, electromyography (EMG), muscle biopsy, CT scan and cerebral MRI were normal. Autosomal recessive (AR) mode of inheritance is the most probable pattern.

Phenotype and sarcoglycan expression in Tunisian LGMD 2C patients sharing the same del521-T mutation.

Limb-girdle muscular dystrophy type 2C is an autosomal recessive muscular disorder caused by mutations in the gene encoding the gamma-sarcoglycan subunit. This gamma-sarcoglycanopathy is prevalent in Tunisia where only one homozygous mutation a 521-T deletion has been identified. The aim of this study was to carry out a comparative clinical and immunocytochemical analysis of Tunisian patients sharing the same gamma-sarcoglycan gene mutation. One hundred and thirty-two patients were classified as severe, moderate or mild according to a calculated severity score. Heterogeneous phenotypes between siblings were encountered in 75% of the families. The severity of the disease was not found to be related to the age of onset. Immunohistochemical studies of muscle biopsy showed a total absence of gamma-sarcoglycan, a normal or slightly reduced alpha and delta-sarcoglycans whereas the expression of beta-sarcoglycan was variable. The residual sarcoglycan expression was not related to the clinical phenotype. In conclusion, the phenotypic variability in sarcoglycanopathies in Tunisia seems to involve a modifying gene controlling the course of the disease.

Fukutin-related protein gene mutated in the original kindred limb-girdle MD 2I.

The authors mapped an autosomal recessive form of limb-girdle MD on chromosome 19q13.3 (LGMD2I), further narrowed down the candidate region to 1.1 Mb, and identified one new homozygous mutation in the fukutin-related protein (FKRP) gene on patients of the original Tunisian family. Immunohistochemical and immunoblot analysis showed abnormal expression of alpha-dystroglycan and laminin-alpha2 supporting the hypothesis that FKRP has a role in the interaction between the extracellular matrix components.

Effect of vitamin E supplementation in patients with ataxia with vitamin E deficiency.

Ataxia with vitamin E (Vit E) defciency (AVED) is an autosomal recessive disorder caused by mutations of the alpha tocopherol transfer protein gene. The Friedreich ataxia phenotype is the most frequent clinical presentation. In AVED patients, serum Vit E levels are very low in the absence of intestinal malabsorption. As Vit E is a major antioxidant agent, Vit E deficiency is supposed to be responsible for the pathological process. Twenty-four AVED patients were fully investigated (electromyography, nerve conduction velocity (NVC) studies, somatosensory evoked potentials, cerebral computed tomography scan, sural nerve biopsy, genetic studies) and supplemented with Vit E (800 mg daily) during a 1-year period. Clinical evaluation was mainly based on the Ataxia Rating Scale (ARS) for cerebellar ataxia assessment and serum Vit E levels were monitored. Serum Vit E levels normalized and ARS scores decreased moderately but significantly suggesting clinical improvement. Better results were noted with mean disease duration < or = 15 years. Reflexes remained abolished and posterior column disturbances unchanged. Vitamin E supplementation in AVED patients stabilizes the neurological signs and can lead to mild improvement of cerebellar ataxia, especially in early stages of the disease.