Molecular characterization of Joubert syndrome in Saudi Arabia

Joubert syndrome (JS) is a ciliopathy that is defined primarily by typical cerebellar structural and ocular motility defects. The genetic heterogeneity of this condition is significant with 16 genes identified to date. We have used a combination of autozygome‐guided candidate gene mutation analysis and exome sequencing to identify the causative mutation in a series of 12 families. The autozygome approach identified mutations in RPGRIP1L, AHI1, TMEM237, and CEP290, while exome sequencing revealed families with truncating mutations in TCTN1 and C5ORF42. Our study, the largest comprehensive molecular series on JS, provides independent confirmation of the recently reported TCTN1, TMEM237, and C5ORF42 as bona fide JS disease genes, and expands the allelic heterogeneity of this disease. Hum Mutat 33:1423–1428, 2012. © 2012 Wiley Periodicals, Inc.     

Genetics of ataxia telangiectasia in a highly consanguineous population

Ataxia telangiectasia (AT) is a rare autosomal recessive multisystemic disorder. It usually presents in toddler years with progressive ataxia and oculomotor apraxia, or less commonly, in the late-first or early-second decade of life with mixed movement disorders. Biallelic mutations in ataxia telangiectasia mutated gene (ATM) cause AT phenotype, a disease not well documented in Saudi Arabia, a highly consanguineous society. We studied several Saudi AT patients, identified ATM variants, and investigated associated clinical features. We included 17 patients from 12 consanguineous families. All patients had comprehensive clinical and radiological assessment, and most were examined through whole-exome sequencing (WES). Selected individuals were analyzed using various genetic approaches. We identified five different ATM variants in our patients: three previously reported mutations, and two novel variants. Nearly all patients had classical AT presentation except for two patients with a milder phenotype. Among the three known variants, a deletion causing truncation (c.381delA resulting in p.(Val128Ter)) was identified in 13 patients. Two patients harboured the other two truncating variants, (c.9001_9002delAG resulting in p.Ser3001Phefs*6) and (c.9066delA resulting in p.Glu3023Alafs*10) and two patients had novel compound heterozygous variants (NM_000051.3:Paternal Allele:c.8762C > G;p.Thr2921Arg and Maternal Allele:c.1057T > C;p.Cys353Arg). We speculate that c.381delA is a founder mutation in our population. This study provides a genotype–phenotype relationship in a previously unstudied consanguineous population. Our findings contribute to improve local clinical care, therapy, and genetic counseling.     

Anaphylaxis to lidocaine with tolerance to articaine in a 12 year old girl

True allergic reactions to local anesthetics are extremely rare and constitute less than 1% of all reactions. In addition, many of those allergic reactions are caused by the preservative constituents of the local anesthetics. Here we report a 12 year old girl with anaphylaxis to lidocaine (an amide local anesthetic) on two occasions. The allergy was confirmed by positive skin prick test to the drug. Skin testing and challenge to another amide local anesthetic (articaine) were negative. Subsequently, its use was well tolerated in a dental procedure. Up to our knowledge, this is the first report of a patient who is allergic to lidocaine and tolerant to articaine.Abbreviations: LA, local anesthetic; SPT, skin prick test; SQ, subcutaneous     

A comparative analysis approach to determining the pathogenicity of mitochondrial tRNA mutations

Distinguishing pathogenic from polymorphic changes poses significant problems for geneticists and despite 30 years of postgenomic experience this remains the case in mitochondrial genetics. Base substitutions in mitochondrial tRNA (mt-tRNA) genes are particularly difficult, but important, because they are common causes of pathology and associated with high rates of transmission. Providing accurate genetic advice to patients and their families is of paramount importance in disease prevention, and brings into sharp focus the factors used to distinguish pathogenic from polymorphic variants. We have reevaluated our pathogenicity scoring system for mt-tRNA mutations following a considerable increase in the number reported since the system was devised in 2004. This allowed us to address notable issues including the underestimation of "definitely pathogenic" mutations resulting from insufficient data collection. We illustrate the robustness of our revised scoring system using novel pathogenic and previously reported polymorphic changes and conclude that while clear evidence from single-fiber and/or trans-mitochondrial cybrid studies remains the gold standard for assigning pathogenicity, our scoring system is valuable for deciding which mt-tRNA mutations to investigate further using these labor-intensive techniques.     

Mutations in the mitochondrial tRNA Ser(AGY) gene are associated with deafness, retinal degeneration, myopathy and epilepsy

Although over 200 pathogenic mitochondrial DNA (mtDNA) mutations have been reported to date, determining the genetic aetiology of many cases of mitochondrial disease is still not straightforward. Here, we describe the investigations undertaken to uncover the underlying molecular defect(s) in two unrelated Caucasian patients with suspected mtDNA disease, who presented with similar symptoms of myopathy, deafness, neurodevelopmental delay, epilepsy, marked fatigue and, in one case, retinal degeneration. Histochemical and biochemical evidence of mitochondrial respiratory chain deficiency was observed in the patient muscle biopsies and both patients were discovered to harbour a novel heteroplasmic mitochondrial tRNA (mt-tRNA)(Ser(AGY)) (MTTS2) mutation (m.12264C>T and m.12261T>C, respectively). Clear segregation of the m.12261T>C mutation with the biochemical defect, as demonstrated by single-fibre radioactive RFLP, confirmed the pathogenicity of this novel variant in patient 2. However, unusually high levels of m.12264C>T mutation within both COX-positive (98.4 ± 1.5%) and COX-deficient (98.2 ± 2.1%) fibres in patient 1 necessitated further functional investigations to prove its pathogenicity. Northern blot analysis demonstrated the detrimental effect of the m.12264C>T mutation on mt-tRNA(Ser(AGY)) stability, ultimately resulting in decreased steady-state levels of fully assembled complexes I and IV, as shown by blue-native polyacrylamide gel electrophoresis. Our findings expand the spectrum of pathogenic mutations associated with the MTTS2 gene and highlight MTTS2 mutations as an important cause of retinal and syndromic auditory impairment.     

A Novel Homozygous Mutation in <Emphasis Type="Italic">SPTBN2</Emphasis> Leads to Spinocerebellar Ataxia in a Consanguineous Family: Report of a New Infantile-Onset Case and Brief Review of the Literature

The objective of this study was the identification of likely genes and mutations associated with an autosomal recessive (AR) rare spinocerebellar ataxia (SCA) phenotype in two patients with infantile onset, from a consanguineous family. Using genome-wide SNP screening, autozygosity mapping, targeted Sanger sequencing and nextgen sequencing, family segregation analysis, and comprehensive neuropanel, we discovered a novel mutation in SPTBN2. Next, we utilized multiple sequence alignment of amino acids from various species as well as crystal structures provided by protein data bank (PDB# 1WYQ and 1WJM) to model the mutation site and its effect on β-III-spectrin. Finally, we used various bioinformatic classifiers to determine pathogenicity of the missense variant. A comprehensive clinical and diagnostic workup including radiological exams were performed on the patients as part of routine patient care. The homozygous missense variant (c.1572C>T; p.R414C) detected in exon 2 was fully segregated in the family and absent in a large ethnic cohort as well as publicly available data sets. Our comprehensive targeted sequencing approaches did not reveal any other likely candidate variants or mutations in both patients. The two male siblings presented with delayed motor milestones and cognitive and learning disability. Brain MRI revealed isolated cerebellar atrophy more marked in midline inferior vermis at ages of 3 and 6.5 years. Sequence alignments of the amino acids for β-III-spectrin indicated that the arginine at 414 is highly conserved among various species and located towards the end of first spectrin repeat domain. Inclusive bioinformatic analysis predicted that the variant is to be damaging and disease causing. In addition to the novel mutation, a brief literature review of the previously reported mutations as well as clinical comparison of the cases were also presented. Our study reviews the previously reported SPTBN2 mutations and cases. Moreover, the novel mutation, p.R414C, adds up to the literature for the infantile-onset form of autosomal recessive ataxia associated with SPTBN2. Previously, few SPTBN2 recessive mutations have been reported in humans. Animal models especially the β-III^?/? mouse model provided insights into early coordination and gait deficit suggestive of loss-of-function. It is expected to see more recessive SPTBN2 mutations appearing in the literature during the upcoming years.     

Neuromuscular disease presentation with three genetic defects involving two genomes

An extensive range of molecular defects have been identified in the human mitochondrial genome (mtDNA), many associated with well-characterised, progressive neurological syndromes. We describe a patient who presented to a mitochondrial clinic with progressive bilateral ptosis, external opthalmoplegia and increasing difficulty with walking. He had previously been diagnosed with a dominant, demyelinating polyneuropathy due to PMP22 gene duplication and had also developed gout, presenting in acute renal failure, due to an X-linked recessive HPRT gene mutation. Muscle biopsy revealed many COX-deficient fibres which we show contain high levels of a third genetic defect – a novel, mitochondrial tRNA^Leu(CUN) (MTTL2) gene mutation.     

Inter-species comparative antioxidant assay and HPTLC analysis of sakuranetin in the chloroform and ethanol extracts of aerial parts of Rhus retinorrhoea and Rhus tripartita

Context: Extensive research on Rhus (Anacardiaceae) shows their antioxidant potential, which warrants further evaluation of its other species.

Objective: To perform a comparative antioxidant assay on extracts of R. retinorrhoea and R. tripartita, including sakuranetin quantification by a validated HPTLC method.

Materials and methods: In vitro antioxidant assay was performed on chloroform and ethanol extracts of R. retinorrhoea Steud. ex Oliv. (RRCE and RREE) and R. tripartita (Ucria) Grande (RTCE and RTEE) by DPPH radical scavenging (at 31.25, 62.5, 125, 250 and 500?μg/mL concentrations) and β-carotene-linoleic acid bleaching methods at 500?μg/mL concentration. Densitometric HPTLC method was developed and validated using toluene: ethyl acetate: methanol (8:2:0.2; v/v/v) as mobile phase, executed on glass-backed silica gel F₂₅₄ plate and scanned at 292?nm.

Results: Antioxidant activity of Rhus extracts tested by the two methods (DPPH/BCB) was found in order of RTEE?>?RREE?>?RTCE?>?RRCE with IC₅₀ 118.67/256.26, 315.75/82.35, 827.92/380.0 and 443.69/292.75, respectively. Scanning of the HPTLC plate provided an intense peak of sakuranetin at R_f =?0.59. The estimated sakuranetin content in the dry weight of the extracts was highest in RREE (27.95?μg/mg) followed by RRCE (25.22?μg/mg), RTEE (0.487?μg/mg) and RTCE (0.0?μg/mg). Presence of sakuranetin in RREE, RRCE and RTEE supported the highest antioxidant property of the two Rhus species. Nonetheless, low sakuratenin in R. tripartita indicated the presence of other bioactive constituents responsible for synergistic antioxidant activity.

Conclusion: The developed HPTLC method therefore guarantees its application in quality control of commercialized herbal drugs and formulations containing sakuranetin.     

Physiological and oxidative stress responses of Solanum lycopersicum (L.) (tomato) when exposed to different chemical pesticides

Pesticide overuse can have negative effects on developmental processes of non-target host plants. By increasing reactive oxygen species (ROS) levels, pesticides negatively affect cellular metabolism, biochemistry and physiological machinery of plants. Considering these problems, the current study was planned to assess the effect of three different groups of pesticides, namely diazinon (DIZN), imidacloprid (IMID) and mancozeb (MNZB) on Solanum lycopersicum L. (tomato). In general, pesticides resulted in a progressive decrease in physiological and biometric parameters of S. lycopersicum (L.), which varies significantly among concentrations and species of pesticides. Among them, 200 μg_MNZB mL⁻¹ had the most severe negative impact and reduced germination rate, root biomass, chl a, chl b, total chlorophyll and carotenoids by 62, 87, 90, 88, 92 and 90%, respectively. In addition, higher doses of pesticides greatly reduced the flowering, fruit attributes and lycopene content. Furthermore, plants exposed to 200 μg_DIZN mL⁻¹ showed a progressive drop in root cell viability (54% decrease), total soluble sugar (TSS) (64% decrease) and total soluble protein (TSP) (67% decrease) content. Data analysis indicated that greater doses of pesticides dramatically raised ROS levels and induced membrane damage through production of thiobarbituric acid reactive substances (TBARS), as well as increased cell injury. To deal with pesticide-induced oxidative stress, plants subjected to greater pesticide dosages, showed a substantial increase in antioxidant levels. For instance, ascorbate peroxidase (APX), catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) and guaiacol peroxidase (GPX) were maximally increased by 48, 93, 71, 52 and 94%, respectively following 200 μg_MNZB mL⁻¹ soil exposures. Additionally, under a confocal laser scanning microscope (CLSM), pesticide exposed S. lycopersicum (L.) roots stained with 2′,7′-dichlorodihydrofluorescein diacetate (2′7′-DCF) and 3,3′-diaminobenzidine, exhibited an increased ROS production in a concentration-dependent manner. Further, elevated pesticide concentrations resulted in alterations in mitochondrial membrane potential (ΔΨ_m) and cellular death in roots, as evidenced by increased Rhodamine 123 (Rhd 123) and Evan''s blue fluorescence, respectively. These findings clearly showed that applying pesticides in excess of permissible amounts might induce oxidative stress and cause oxidative damage in non-target host plants. Overall, the current study indicates that a thorough and secure method be used before selecting pesticides for increasing production of agronomically important vegetable crops in various agro-climatic zones.

Pesticide overuse can have a negative effect on the development processes of non-target plants.