Mutation of the gap junction protein alpha 8 (GJA8) gene causes autosomal recessive cataract

Background

GJA8 encodes connexin‐50, a gap junction protein in the eye lens. Mutations in GJA8 have been reported in families with autosomal dominant cataract.

Objective

To identify the disease gene in a family with congenital cataract of autosomal recessive inheritance.

Methods

Eight candidate genes were screened for pathogenic alterations in affected and unaffected family members and in normal unrelated controls.

Results

A single base insertion leading to frameshift at codon 203 of connexin 50 was found to co‐segregate with disease in the family.

Conclusions

These results confirm involvement of GJA8 in autosomal recessive cataract.     

A new locus for autosomal dominant posterior polar cataract in Moroccan Jews maps to chromosome 14q22-23

Background

Posterior polar cataract is a clinically distinctive opacity located at the back of the lens. It is commonly acquired in age related cataract, and may infrequently occur in pedigrees with congenital cataract. To date, five loci for autosomal dominant congenital posterior polar cataract have been identified. These include two genes, CRYAB and PITX3, on chromosomes 11q and 10q respectively, and three loci with as yet unknown genes on chromosomes 1p, 16q and 20p.

Purpose

To find the chromosomal location of a gene causing autosomal dominant congenital posterior polar cataract in three Moroccan Jewish families.

Methods

A whole genome scan was performed using microsatellite markers spaced at approximately 10 cM intervals. For fine mapping, five additional microsatellite markers were genotyped. Two‐point lod scores were calculated using MLINK software, from the LINKAGE program package. After linkage was established, several positional candidate genes were assessed by PCR based DNA sequencing.

Results

The new cataract locus was mapped to an 11.3 cM interval between D14S980 and D14S1069 on chromosome 14q22‐23. A maximum two point lod score of 5.19 at θ = 0 was obtained with the markersD14S274. The positional and functional candidate genes SIX1, SIX4, SIX6, OTX2, and ARHJ were excluded as the cause of cataract in these families.

Conclusion

An as yet unidentified gene associated with posterior polar cataract maps to the long arm of chromosome 14q22‐23.     

Espin gene (ESPN) mutations associated with autosomal dominant hearing loss cause defects in microvillar elongation or organisation

Background

Espins are actin bundling proteins present in hair cell stereocilia. A recessive mutation in the espin gene (Espn) has been detected in the jerker mouse and causes deafness, vestibular dysfunction, and hair cell degeneration. More recently mutations in the human espin gene (ESPN) have been described in two families affected by autosomal recessive hearing loss and vestibular areflexia.

Objective

To report the identification of four additional ESPN mutations (S719R, D744N, R774Q, and delK848) in patients affected by autosomal dominant hearing loss without vestibular involvement.

Results

To determine whether the mutated ESPN alleles affected the biological activity of the corresponding espin proteins in vivo, their ability to target and elongate the parallel actin bundles of brush border microvilli was investigated in transfected LLC‐PK1‐CL4 epithelial cells. For three mutated alleles clear abnormalities in microvillar length or distribution were obtained.

Conclusions

The results further strengthen the causative role of the espin gene in non‐syndromic hearing loss and add new insights into espin structure and function.     

Functional and clinical characterization of a mutation in KCNJ2 associated with Andersen-Tawil syndrome

Background

Andersen‐Tawil syndrome (ATS) is a rare inherited disorder, characterised by periodic paralysis, cardiac dysarrhythmias, and dysmorphic features, and is caused by mutations in the gene KCNJ2, which encodes the inward rectifier potassium channel, Kir2.1. This study sought to analyse KCNJ2 in patients with familial ATS and to determine the functional characteristics of the mutated gene.

Methods and results

We screened a family with inherited ATS for the mutation in KCNJ2, using direct DNA sequencing. A missense mutation (T75R) of Kir2.1, located in the highly conserved cytoplasmic N‐terminal domain, was identified in three affected members of this family. Using the Xenopus oocyte expression system and whole cell voltage clamp analyses, we found that the T75R mutant was non‐functional and possessed a strong dominant negative effect when co‐expressed with the same amount of wild type Kir2.1. Transgenic (Tg) mice expressing the mutated form of Kir2.1 in the heart had prolonged QTc intervals compared with mice expressing the wild type protein. Ventricular tachyarrhythmias were observed in 5 of 14 T75R‐Tg mice compared with 1 of 7 Wt‐Tg and none of 6 non‐transgenic littermates. In three of five T75R‐Tg mice with ventricular tachycardia, their ECG disclosed bidirectional tachycardia as in our proband.

Conclusions

The in vitro studies revealed that the T75R mutant of Kir2.1 had a strong dominant negative effect in the Xenopus oocyte expression system. It still preserved the ability to co‐assemble and traffic to the cell membrane in mammalian cells. For in vivo studies, the T75R‐Tg mice had bidirectional ventricular tachycardia after induction and longer QT intervals.     

CRYM mutations cause deafness through thyroid hormone binding properties in the fibrocytes of the cochlea

Background

In a search for mutations of μ‐crystallin (CRYM), a taxion specific crystalline which is also known as an NADP regulated thyroid hormone binding protein, two mutations were found at the C‐terminus in patients with non‐syndromic deafness.

Objective

To investigate the mechanism of hearing loss caused by CRYM mutations

Methods

T3 binding activity of mutant μ‐crystallin was compared with that of wild‐type μ‐crystallin, because μ‐crystallin is known to be identical to T3 binding protein. To explore the sites within the cochlea where μ‐crystallin is functioning, its localisation in the mouse cochlea was investigated immunocytochemically using a specific antibody.

Results

One mutant was shown to have no binding capacity for T3, indicating that CRYM mutations cause auditory dysfunction through thyroid hormone binding properties. Immunocytochemical results indicated that μ‐crystallin was distributed within type II fibrocytes of the lateral wall, which are known to contain Na,K‐ATPase.

Conclusions

CRYM mutations may cause auditory dysfunction through thyroid hormone binding effects on the fibrocytes of the cochlea. μ‐Crystallin may be involved in the potassium ion recycling system together with Na,K‐ATPase. Future animal experiments will be necessary to confirm a causal relation between Na,K‐ATPase, T3, and deafness.     

Is the E133K allele of VG5Q associated with Klippel‐Trenaunay and other overgrowth syndromes?

Background

It has been reported that the activating mutation, E133K, in the angiogenic factor VG5Q (formally named AGGF1) causes Klippel‐Trenaunay Syndrome (KTS), a rare vascular disease associated with asymmetric overgrowth. This proposal followed from the observation that five out of 130 KTS patients were constitutionally heterozygous for VG5Q, E133K.

Objective

To explore the possibility that VG5Q, and specifically E133K, is implicated in other mosaic overgrowth syndromes.

Results

24 patients were analysed for this sequence change.One patient was constitutionally heterozygous for E133K. Analysis of both parents revealed that the patient''s mother, who was healthy, also carried E133K. An analysis of 275 healthy controls showed that 3.3% (9/275) of the population were carriers of E133K.

Conclusions

The findings bring into question the assertion that VG5Q, E133K is a mutation and that it causes KTS.     

Frequency of the mdr-1 C>T gene polymorphism in patients with COPD

BACKGROUND AND AIM:

The multi‐drug resistant‐1 (MDR‐1) gene is located on human chromosome 7 and encodes a glycosylated membrane protein that is a member of the ATP‐binding cassette transporters superfamily. The aim of the study was to reveal the role of the C3435T MDR‐1 gene polymorphism in chronic obstructive pulmonary disease.

METHOD:

DNA samples from 41 patients with chronic obstructive pulmonary disease and 50 healthy control participants were used to compare MDR‐1 gene profiles. Genotyping assays were performed using the StripAssay technique that is based on reverse‐hybridization.

RESULTS:

The T allele polymorphism in the MDR‐1 gene located at position 3435 in exon 26 was shown to correlate with chronic obstructive pulmonary disease.

CONCLUSION:

These preliminary results suggest that the T allele polymorphism of the MDR‐1 gene is associated with chronic obstructive pulmonary disease.     

OTOF mutations revealed by genetic analysis of hearing loss families including a potential temperature sensitive auditory neuropathy allele

Introduction

The majority of hearing loss in children can be accounted for by genetic causes. Non‐syndromic hearing loss accounts for 80% of genetic hearing loss in children, with mutations in DFNB1/GJB2 being by far the most common cause. Among the second tier genetic causes of hearing loss in children are mutations in the DFNB9/OTOF gene.

Methods

In total, 65 recessive non‐syndromic hearing loss families were screened by genotyping for association with the DFNB9/OTOF gene. Families with genotypes consistent with linkage or uninformative for linkage to this gene region were further screened for mutations in the 48 known coding exons of otoferlin.

Results

Eight OTOF pathological variants were discovered in six families. Of these, Q829X was found in two families. We also noted 23 other coding variant, believed to have no pathology. A previously published missense allele I515T was found in the heterozygous state in an individual who was observed to be temperature sensitive for the auditory neuropathy phenotype.

Conclusions

Mutations in OTOF cause both profound hearing loss and a type of hearing loss where otoacoustic emissions are spared called auditory neuropathy.     

Fanconi anaemia complementation group B presenting as X linked VACTERL with hydrocephalus syndrome

Background

The VACTERL with hydrocephalus (VACTERL‐H) phenotype is recognised to be a severe manifestation of autosomal recessive Fanconi anaemia. Several families have been described in which the VACTERL‐H phenotype segregates as an X linked syndrome. The mutations which cause X linked VACTERL‐H syndrome are not known.

Objective

To determine if mutations in FANCB, which are known to cause Fanconi anaemia complementation group B, are a cause of X linked VACTERL‐H syndrome.

Methods

A three generation pedigree with X linked VACTERL‐H syndrome was investigated. X inactivation was tested in carrier females, and fibroblasts from an affected male fetus were analysed for increased sensitivity to diepoxybutane. FANCB coding exons and flanking splice sites were screened for mutations by direct sequencing of polymerase chain reaction (PCR) fragments amplified from genomic DNA. cDNA from affected fetal fibroblasts was analysed by PCR and direct sequencing using specific exonic primers.

Results

A FANCB mutation which results in a premature stop codon by causing skipping of exon 7 was identified. Chromosomes from the affected fetus showed increased sensitivity to diepoxybutane, and carrier women were found to have 100% skewed X inactivation in blood.

Conclusions

Mutations in FANCB are a cause of X linked VACTERL‐H syndrome. The data presented are of relevance to the genetic counselling of families with isolated male cases of VACTERL‐H and Fanconi anaemia.     

Hot water extract of Chlorella vulgaris induced DNA damage and apoptosis

OBJECTIVES:

The aim of this study was to determine the antiproliferative and apoptotic effects of hot water extracts of Chlorella vulgaris on hepatoma cell line HepG2.

INTRODUCTION:

The search for food and spices that can induce apoptosis in cancer cells has been a major study interest in the last decade. Chlorella vulgaris, a unicellular green algae, has been reported to have antioxidant and anti‐cancer properties. However, its chemopreventive effects in inhibiting the growth of cancer cells have not been studied in great detail.

METHODS:

HepG2 liver cancer cells and WRL68 normal liver cells were treated with various concentrations (0‐4 mg/ml) of hot water extract of C. vulgaris after 24 hours incubation. Apoptosis rate was evaluated by TUNEL assay while DNA damage was assessed by Comet assay. Apoptosis proteins were evaluated by Western blot analysis.

RESULTS:

Chlorella vulgaris decreased the number of viable HepG2 cells in a dose dependent manner (p < 0.05), with an IC₅₀ of 1.6 mg/ml. DNA damage as measured by Comet assay was increased in HepG2 cells at all concentrations of Chlorella vulgaris tested. Evaluation of apoptosis by TUNEL assay showed that Chlorella vulgaris induced a higher apoptotic rate (70%) in HepG2 cells compared to normal liver cells, WRL68 (15%). Western blot analysis showed increased expression of pro‐ apoptotic proteins P53, Bax and caspase‐3 in the HepG2 cells compared to normal liver cells WRL68, and decreased expression of the anti‐apoptotic protein Bcl‐2.

CONCLUSIONS:

Chlorella vulgaris may have anti‐cancer effects by inducing apoptosis signaling cascades via an increased expression of P53, Bax and caspase‐3 proteins and through a reduction of Bcl‐2 protein, which subsequently lead to increased DNA damage and apoptosis.     

A novel locus for autosomal dominant non-syndromic deafness, DFNA53, maps to chromosome 14q11.2-q12

Background

Non‐syndromic hearing loss is among the most genetically heterogeneous traits known in humans. To date, at least 50 loci for autosomal dominant non‐syndromic sensorineural hearing loss (ADNSSHL) have been identified by linkage analysis.

Objective

To report the mapping of a novel autosomal dominant deafness locus on the long arm of chromosome 14 at 14q11.2‐q12, DFNA53, in a large multigenerational Chinese family with post‐lingual, high frequency hearing loss that progresses to involve all frequencies.

Results

A maximum multipoint LOD score of 5.4 was obtained for marker D14S1280. The analysis of recombinant haplotypes mapped DFNA53 to a 9.6 cM region interval between markers D14S581 and D14S1021. Four deafness loci (DFNA9, DFNA23, DFNB5, and DFNB35) have previously been mapped to the long arm of chromosome 14. The critical region for DFNA53 contains the gene for DFNA9 but does not overlap with the regions for DFNB5, DFNA23, or DFNB35. Screening of the COCH gene (DFNA9), BOCT, EFS, and HSPC156 within the DFNA53 interval did not identify the cause for deafness in this family.

Conclusions

Identifying the DFNA53 locus is the first step in isolating the gene responsible for hearing loss in this large multigeneration Chinese family.     

Metallo-beta-lactamases among imipenem-resistant Pseudomonas aeruginosa in a Brazilian university hospital

INTRODUCTION:

Imipenem‐resistant Pseudomonas aeruginosa resulting from metallo‐β‐lactamases has been reported to be an important cause of nosocomial infection and is a critical therapeutic problem worldwide, especially in the case of bacteremia.

OBJECTIVES:

To determine the frequency of metallo‐β‐lactamases among imipenem‐resistant Pseudomonas aeruginosa isolates and to compare methods of phenotypic and molecular detection.

METHODS:

During 2006, 69 imipenem‐resistant Pseudomonas aeruginosa samples were isolated from blood and tested for metallo‐β‐lactamase production using phenotypic methods. Minimal Inhibitory Concentratrions (MIC) (µg/mL) was determined with commercial microdilution panels. Pulsed Field Gel Electrophoresis (PFGE) was performed among metallo‐β‐lactamase producers.

RESULTS:

Of all the blood isolates, 34.5% were found to be imipenem‐resistant Pseudomonas aeruginosa. Positive phenotypic tests for metallo‐β‐lactamases ranged from 28%‐77%, and Polymerase Chain Reaction (PCR) were positive in 30% (of note, 81% of those samples were bla_SPM‐1 and 19% were bla_VIM‐2). Ethylenediamine tetracetic acid (EDTA) combinations for the detected enzymes had low kappa values; thus, care should be taken when use it as a phenotypic indicator of MBL. Despite a very resistant antibiogram, four isolates demonstrated the worrisome finding of a colistin MIC in the resistant range. PFGE showed a clonal pattern.

CONCLUSION:

Metallo‐β‐lactamases among imipenem‐resistant Pseudomonas aeruginosa were detected in 30.4% of imipenem‐resistant Pseudomonas aeruginosa isolates. This number might have been higher if other genes were included. SPM‐1 was the predominant enzyme found. Phenotypic tests with low kappa values could be misleading when testing for metallo‐β‐lactamases. Polymerase Chain Reaction detection remains the gold standard.     

Congenital hyperinsulinism and mosaic abnormalities of the ploidy

Background

Congenital hyperinsulinism and Beckwith‐Wiedemann syndrome both lead to β islet hyperplasia and neonatal hypoglycaemia. They may be related to complex genetic/epigenetic abnormalities of the imprinted 11p15 region. The possibility of common pathophysiological determinants has not been thoroughly investigated.

Objective

To report abnormalities of the ploidy in two unrelated patients with congenital hyperinsulinism.

Methods

Two patients with severe congenital hyperinsulinism, one overlapping with Beckwith‐Wiedemann syndrome, had pancreatic histology, ex vivo potassium channel electrophysiological studies, and mutation detection of the encoding genes. The parental genetic contribution was explored using genome‐wide polymorphism, fluorescent in situ hybridisation (FISH), and blood group typing studies.

Results

Histological findings diverged from those described in focal congenital hyperinsulinism or Beckwith‐Wiedemann syndrome. No potassium channel dysfunction and no mutation of its encoding genes (SUR1, KIR6.2) were detected. In patient 1 with congenital hyperinsulinism and Beckwith‐Wiedemann syndrome, paternal isodisomy for the whole haploid set was homogeneous in the pancreatic lesion, and mosaic in the leucocytes and skin fibroblasts (hemihypertrophic segment). Blood group typing confirmed the presence of two erythroid populations (bi‐parental v paternal only contribution). Patient 2 had two pancreatic lesions, both revealing triploidy with paternal heterodisomy. Karyotype and FISH analyses done on the fibroblasts and leucocytes of both patients were unremarkable (diploidy).

Conclusions

Diploid (biparental/paternal‐only) mosaicism and diploid/triploid mosaicism were present in two distinct patients with congenital hyperinsulinism. These chromosomal abnormalities led to paternal disomy for the whole haploid set in pancreatic lesions (with isodisomy or heterodisomy), thereby extending the range and complexity of the mechanisms underlying congenital hyperinsulinism, associated or not with Beckwith‐Wiedemann syndrome.     

Evaluation of antianxiety and sedative effects of essential oil of Ducrosia anethifolia in mice

INTRODUCTION:

We investigated the antianxiety and sedative effects of the essential oil of Ducrosia anethifolia. Boiss. (Apiaceae).

METHODS:

We used elevated plus maze, spontaneous motor activity and ketamine‐induced sleep tests in mice. In addition, the essential oil was analyzed by GC/MS. Twenty compounds were identified, and n‐decanal (70.1%) and alpha‐pinene (12.4%) constituted the major components.

RESULTS:

In elevated plus maze, Ducrosia anethifolia essential oil at doses of 25–200 mg/kg increased the percentage of open arm time and entries. Unlike diazepam, ducrosia anethifolia essential oil could not suppress spontaneous motor activity and did not alter ketamine‐induced sleep parameters. These results are indicative of antianxiety effect of Ducrosia anethifolia essential oil without sedative effect.     

Biallelic mutation of MSH2 in primary human cells is associated with sensitivity to irradiation and altered RAD51 foci kinetics

Background

Reports of differential mutagen sensitivity conferred by a defect in the mismatch repair (MMR) pathway are inconsistent in their conclusions. Previous studies have investigated cells established from immortalised human colorectal tumour lines or cells from animal models.

Methods

We examined primary human MSH2‐deficient neonatal cells, bearing a biallelic truncating mutation in MSH2, for viability and chromosomal damage after exposure to DNA‐damaging agents.

Results

MSH2‐deficient cells exhibit no response to interstrand DNA cross‐linking agents but do show reduced viability in response to irradiation. They also show increased chromosome damage and exhibit altered RAD51 foci kinetics after irradiation exposure, indicating defective homologous recombinational repair.

Discussion

The cellular features and sensitivity of MSH2‐deficient primary human cells are broadly in agreement with observations of primary murine cells lacking the same gene. The data therefore support the view that the murine model recapitulates early features of MMR deficiency in humans, and implies that the variable data reported for MMR‐deficient immortalised human cells may be due to further genetic or epigenetic lesions. We suggest caution in the use of radiotherapy for treatment of malignancies in individuals with functional loss of MSH2.     

Penetrance of adrenocortical tumours associated with the germline TP53 R337H mutation

Background

An inherited germline P53 mutation has been identified in cases of childhood adrenocortical carcinoma (ACT), a neoplasm with a high incidence in southern Brazil. The penetrance of ACT in carriers of the point mutation, which encodes an arginine‐to‐histidine substitution at codon 337 of TP53 (R337H), has not been determined.

Objective

To investigate the penetrance of childhood ACT in carriers of the R337H TP53 mutation.

Methods

The family histories of 30 kindreds of 41 southern Brazilian children with ACT were obtained. A PCR based assay was used to detect this P53 mutation in a large number of relatives of children with ACT. In all, 927 individuals were tested for the mutation, 232 from the non‐carrier and 695 (including the 40 probands) from the carrier parental lines.

Results

40 children with ACT carried the TP53 R337H mutation; the remaining child with ACT was not tested. There was no evidence of Li‐Fraumeni syndrome in any of the kindreds; however, seven met the criteria for Li‐Fraumeni‐like syndrome. The carrier parental line was identified in each kindred. Of the 695 individuals tested in the carrier parental line, 240 (34.5%) were positive for the mutation, while none of the 232 individuals in the other parental line carried the mutation. The penetrance of ACT was 9.9% (95% confidence interval, 8.7% to 11.1%).

Conclusions

The TP53 R337H mutation dramatically increases predisposition to childhood ACT but not to other cancers, and explains the increased frequency of ACT observed in this geographic region.     

Cowden syndrome and Bannayan Riley Ruvalcaba syndrome represent one condition with variable expression and age-related penetrance: results of a clinical study of PTEN mutation carriers

Background

The most commonly reported phenotypes described in patients with PTEN mutations are Bannayan–Riley–Ruvalcaba syndrome (BRRS), with childhood onset, macrocephaly, lipomas and developmental delay, and Cowden Syndrome (CS), an adult‐onset condition recognised by mucocutaneous signs, with a risk of cancers, in particular those of the thyroid and breast. It has been suggested that BRRS and CS are the same condition, but the literature continues to separate them and seek a genotype–phenotype correlation.

Objective

To study the clinical features of patients with known PTEN mutations and observe any genotype–phenotype correlation.

Methods

In total, 42 people (25 probands and 17 non‐probands) from 26 families of all ages with PTEN mutations were recruited through the UK clinical genetics services. A full clinical history and examination were undertaken.

Results

We were unable to demonstrate a genotype–phenotype correlation. Furthermore, our findings in a 31‐year‐old woman with CS and an exon 1 deletion refutes previous reports that whole exon deletions are only found in patients with a BRRS phenotype.

Conclusion

Careful phenotyping gives further support for the suggestion that BRRS and CS are actually one condition, presenting variably at different ages, as in other tumour‐suppressor disorders such as neurofibromatosis type 1. This has important counselling implications, such as advice about cancer surveillance, for children diagnosed with BRRS.     

Epsilon sarcoglycan mutations and phenotype in French patients with myoclonic syndromes

Background

Myoclonus dystonia syndrome (MDS) is an autosomal dominant movement disorder caused by mutations in the epsilon‐sarcoglycan gene (SGCE) on chromosome 7q21.

Methods

We have screened for SGCE mutations in index cases from 76 French patients with myoclonic syndromes, including myoclonus dystonia (M‐D), essential myoclonus (E‐M), primary myoclonic dystonia, generalised dystonia, dystonia with tremor, and benign hereditary chorea. All coding exons of the SGCE gene were analysed. The DYT1 mutation was also tested.

Results

Sixteen index cases had SGCE mutations while one case with primary myoclonic dystonia carried the DYT1 mutation. Thirteen different mutations were found: three nonsense mutations, three missense mutations, three splice site mutations, three deletions, and one insertion. Eleven of the SGCE index cases had M‐D and five E‐M. No SGCE mutations were detected in patients with other phenotypes. The total number of mutation carriers in the families was 38, six of whom were asymptomatic. Penetrance was complete in paternal transmissions and null in maternal transmissions. MDS patients with SGCE mutation had a significantly earlier onset than the non‐carriers. None of the patients had severe psychiatric disorders.

Conclusion

This large cohort of index patients shows that SGCE mutations are primarily found in patients with M‐D and to a lesser extent E‐M, but are present in only 30% of these patients combined (M‐D and E‐M).