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Complex I deficiency, the most common respiratory chain defect, is genetically heterogeneous: mutations in 8 nuclear and 7 mitochondrial DNA genes encoding complex I subunits have been described. However, these genes account for disease in only a minority of complex I–deficient patients. We investigated whether there may be an unknown common gene by performing functional complementation analysis of cell lines from 10 unrelated patients. Two of the patients were found to have mitochondrial DNA mutations. The other 8 represented 7 different (nuclear) complementation groups, all but 1 of which showed abnormalities of complex I assembly. It is thus unlikely that any one unknown gene accounts for a large proportion of complex I cases. The 2 patients sharing a nuclear complementation group had a similar abnormal complex I assembly profile and were studied further by homozygosity mapping, chromosome transfers, and microarray expression analysis. NDUFS6, a complex I subunit gene not previously associated with complex I deficiency, was grossly underexpressed in the 2 patient cell lines. Both patients had homozygous mutations in this gene, one causing a splicing abnormality and the other a large deletion. This integrated approach to gene identification offers promise for identifying other unknown causes of respiratory chain disorders.

Kufs disease is the major adult form of neuronal ceroid lipofuscinosis, but is rare and difficult to diagnose. Diagnosis was traditionally dependent on the demonstration of characteristic storage material, but distinction from normal age-related accumulation of lipofuscin can be challenging. Mutation of CLN6 has emerged as the most important cause of recessive Kufs disease but, remarkably, is also responsible for variant late infantile ceroid lipofuscinosis. Here we provide a detailed description of Kufs disease due to CLN6 pathogenic variants. We studied 20 cases of Kufs disease with CLN6 pathogenic variants from 13 unrelated families. Mean age of onset was 28 years (range 12-51) with bimodal peaks in teenage and early adult life. The typical presentation was of progressive myoclonus epilepsy with debilitating myoclonic seizures and relatively infrequent tonic-clonic seizures. Patients became wheelchair-bound with a mean 12 years post-onset. Ataxia was the most prominent motor feature. Dementia appeared to be an invariable accompaniment, although it could take a number of years to manifest and occasionally cognitive impairment preceded myoclonic seizures. Patients were usually highly photosensitive on EEG. MRI showed progressive cerebral and cerebellar atrophy. The median survival time was 26 years from disease onset. Ultrastructural examination of the pathology revealed fingerprint profiles as the characteristic inclusions, but they were not reliably seen in tissues other than brain. Curvilinear profiles, which are seen in the late infantile form, were not a feature. Of the 13 unrelated families we observed homozygous CLN6 pathogenic variants in four and compound heterozygous variants in nine. Compared to the variant late infantile form, there was a lower proportion of variants that predicted protein truncation. Certain heterozygous missense variants in the same amino acid position were found in both variant late infantile and Kufs disease. There was a predominance of cases from Italy and surrounding regions; this was partially explained by the discovery of three founder pathogenic variants. Clinical distinction of type A (progressive myoclonus epilepsy) and type B (dementia with motor disturbance) Kufs disease was supported by molecular diagnoses. Type A is usually caused by recessive pathogenic variants in CLN6 or dominant variants in DNAJC5. Type B Kufs is usually associated with recessive CTSF pathogenic variants. The diagnosis of Kufs remains challenging but, with the availability of genetic diagnosis, this will largely supersede the use of diagnostic biopsies, particularly as biopsies of peripheral tissues has unsatisfactory sensitivity and specificity.

The chromosome 15q13.3 region has been implicated in epilepsy, intellectual disability and neuropsychiatric disorders, especially schizophrenia. Deficiency of the acetylcholine receptor gene CHRNA7 and the partial duplication, CHRFAM7A, may contribute to these phenotypes and we sought to comprehensively analyze these genes in genetic generalized epilepsy. We analyzed using DHPLC, Sanger sequencing and long range PCR, 174 probands with genetic generalized epilepsy with or without intellectual disability or psychosis, including 8 with the recurrent 15q13.3 microdeletion. We searched CHRNA7 and CHRFAM7A for single sequence variants, small copy number variants, and the common 2-bp deletion in CHRFAM7A. We identified two novel and one reported missense variants. The common 2-bp deletion was not enriched in patients compared to controls. Our data suggest that missense mutations in CHRNA7 contribute to complex inheritance in genetic generalized epilepsy in a similar fashion to the 15q13.3 microdeletion. They do not support a pathogenic role for the common 2-bp CHRFAM7A deletion. Refereed/Peer-reviewed

We identified a small family with autosomal recessive, infantile onset epilepsy and intellectual disability. Exome sequencing identified a homozygous missense variant in the gene TNK2, encoding a brain-expressed tyrosine kinase. Sequencing of the coding region of TNK2 in 110 patients with a similar phenotype failed to detect further homozygote or compound heterozygote mutations. Pathogenicity of the variant is supported by the results of our functional studies, which demonstrated that the variant abolishes NEDD4 binding to TNK2, preventing its degradation after epidermal growth factor stimulation. Definitive proof of pathogenicity will require confirmation in unrelated patients.

Glucose transporter 1 (GLUT1) deficiency caused by mutations of SLC2A1 is an increasingly recognized cause of genetic generalized epilepsy. We previously reported that >10% (4 of 34) of a cohort with early onset absence epilepsy (EOAE) had GLUT1 deficiency. This study uses a new cohort of 55 patients with EOAE to confirm that finding. Patients with typical absence seizures beginning before 4 years of age were screened for solute carrier family 2 (facilitated glucose transporter), member 1 (SLC2A1) mutations or deletions. All had generalized spike-waves on electroencephalography (EEG). Those with tonic and/or atonic seizures were excluded. Mutations were found in 7 (13%) of 55 cases, including five missense mutations, an in-frame deletion leading to loss of a single amino acid, and a deletion spanning two exons. Over both studies, 11 (12%) of 89 probands with EOAE have GLUT1 deficiency. Given the major treatment and genetic counseling implications, this study confirms that SLC2A1 mutational analysis should be strongly considered in EOAE.

We report the identification of three new mouse models, baringo, nice, and stitch, with recessively inherited sensorineural deafness due to novel mutations in the transmembrane channel-like gene 1 (Tmc1). These strains were generated by N-ethyl-N-nitrosourea mutagenesis. DNA sequence analysis revealed changes in c.545A>G, c.1345T>C, and c.1661G>T, causing p.Y182C, p.Y449H, and p.W554L amino acid substitutions in baringo, nice, and stitch mutants, respectively. The mutations affect amino acid residues that are evolutionarily conserved across species. Similar to the previously reported Beethoven Tmc1 mutant, both p.Y182C and p.W554L are located outside a predicted transmembrane domain, whereas the p.Y449H mutation resides in the predicted transmembrane domain 4. Homozygous stitch-mutant mice have severe hearing loss at the age of 4 weeks and are deaf by the age of 8 weeks, whereas both baringo and nice mutants are profoundly deaf at the age of 4 weeks. None of the strains displays signs of vestibular dysfunction. Scanning electron microscopy revealed degeneration of outer hair cells in the basal region of baringo, nice, and stitch mutants. Immunolocalization studies revealed expression of TMC1 protein in the hair cells, spiral ganglion neurons, supporting cells, and stria ligament in the inner ear. Reduced levels of TMC1 protein were observed in the spiral ligament of mutants when compared with wild-type animals. These three allelic mutants provide valuable models for studying nonsyndromic recessive sensorineural hearing loss (DFNB7/11) in humans.

Mammalian mitochondrial DNA (mtDNA) is inherited principally down the maternal line, but the mechanisms involved are not fully understood. Females harboring a mixture of mutant and wild-type mtDNA (heteroplasmy) transmit a varying proportion of mutant mtDNA to their offspring. In humans with mtDNA disorders, the proportion of mutated mtDNA inherited from the mother correlates with disease severity. Rapid changes in allele frequency can occur in a single generation. This could be due to a marked reduction in the number of mtDNA molecules being transmitted from mother to offspring (the mitochondrial genetic bottleneck), to the partitioning of mtDNA into homoplasmic segregating units, or to the selection of a group of mtDNA molecules to re-populate the next generation. Here we show that the partitioning of mtDNA molecules into different cells before and after implantation, followed by the segregation of replicating mtDNA between proliferating primordial germ cells, is responsible for the different levels of heteroplasmy seen in the offspring of heteroplasmic female mice.

Recent developments in molecular genetics, including progress in the human genome project, have allowed identification of genes at an unprecedented rate. To date gene expression profiling studies have focused on identifying transcripts that are specifically or preferentially enriched within the inner ear on the assumption that they are more likely to be important for auditory and vestibular function. It is now apparent that some genes preferentially expressed in the cochleo-vestibular system are not crucial for hearing or balance or their functions are compensated for by other genes. In addition, transcripts expressed at low abundance in the inner ear are generally under-represented in gene profiling studies. In this review, we highlight the limitations of current gene expression profiling strategies as a discovery tool for genes involved in cochleo-vestibular development and function. We argue that expression profiling based on hierarchical clustering of transcripts by gene ontology, combined with tissue enrichment data, is more effective for inner ear gene discovery. This approach also provides a framework to assist and direct the functional characterization of gene products.

Keipert syndrome is a rare condition comprising sensorineural deafness associated with facial and digital abnormalities. To date, Keipert syndrome has been reported in six male patients including two sib pairs; however the genetic basis of Keipert syndrome is yet to be elucidated. We report on the diagnosis of Keipert syndrome in the nephew of the brothers in the first report of Keipert syndrome, with a pedigree consistent with X-linked recessive inheritance. Linkage analysis using microsatellite markers along the X-chromosome suggests that the gene for Keipert syndrome is located in the region Xq22.2-Xq28. We postulate the Keipert syndrome is caused by a novel gene at Xq22.2-Xq28.

OBJECTIVE: Nocturnal frontal lobe epilepsy (NFLE) can be sporadic or autosomal dominant; some families have nicotinic acetylcholine receptor subunit mutations. We report a novel autosomal recessive phenotype in a single family and identify the causative gene. METHODS: Whole exome sequencing data was used to map the family, thereby narrowing exome search space, and then to identify the mutation. RESULTS: Linkage analysis using exome sequence data from two affected and two unaffected subjects showed homozygous linkage peaks on chromosomes 7, 8, 13, and 14 with maximum LOD scores between 1.5 and 1.93. Exome variant filtering under these peaks revealed that the affected siblings were homozygous for a novel splice site mutation (c.93+2T>C) in the PRIMA1 gene on chromosome 14. No additional PRIMA1 mutations were found in 300 other NFLE cases. The c.93+2T>C mutation was shown to lead to skipping of the first coding exon of the PRIMA1 mRNA using a minigene system. INTERPRETATION: PRIMA1 is a transmembrane protein that anchors acetylcholinesterase (AChE), an enzyme hydrolyzing acetycholine, to membrane rafts of neurons. PRiMA knockout mice have reduction of AChE and accumulation of acetylcholine at the synapse; our minigene analysis suggests that the c.93+2T>C mutation leads to knockout of PRIMA1. Mutations with gain of function effects in acetylcholine receptor subunits cause autosomal dominant NFLE. Thus, enhanced cholinergic responses are the likely cause of the severe NFLE and intellectual disability segregating in this family, representing the first recessive case to be reported and the first PRIMA1 mutation implicated in disease.

Background Nonsyndromic autosomal-dominant, adult-onset sensorineural hearing loss resulting from DFNA17 was described in a single American kindred in 1997, and the causative gene was subsequently identified as MYH9. Objective The objective of this study was to report clinical and genetic analyses of an Australian family with nonsyndromic adult-onset sensorineural hearing loss. Methods The clinical presentation of the family was detailed and identification of the causative gene was conducted by SNP genotyping and direct sequencing. Results Sequence analysis of the MYH9 gene revealed the same missense mutation as in the original DFNA17 family. We are not aware of a link between the two kindreds, making the present one only the second DFNA17 family to be reported. Conclusions One important point of clinical relevance is the excellent outcome with cochlear implants in the Australian family compared with a "poor" response in the American family. Thus, cochlear implants should be strongly considered for clinical management of patients with DFNA17 deafness.

OBJECTIVE. The goal was to determine the prevalence and effects of slight/mild bilateral sensorineural hearing loss among children in elementary school. METHODS. A cross-sectional, cluster-sample survey of 6581 children (response: 85%; grade 1: n = 3367; grade 5: n = 3214) in 89 schools in Melbourne, Australia, was performed. Slight/mild bilateral sensorineural hearing loss was defined as a low-frequency pure-tone average across 0.5, 1, and 2 kHz and/or a high-frequency pure-tone average across 3, 4, and 6 kHz of 16 to 40 dB hearing level in the better ear, with air/bone-conduction gaps of RESULTS. Fifty-five children (0.88%) had slight/mild bilateral sensorineural hearing loss. Children with and without sensorineural hearing loss scored similarly in language (mean: 97.2 vs 99.7), reading (101.1 vs 102.8), behavior (8.4 vs 7.0), and parent- and child-reported child health-related quality of life (77.6 vs 80.0 and 76.1 vs 77.0, respectively), but phonologic short-term memory was poorer (91.0 vs 102.8) in the sensorineural hearing loss group. CONCLUSIONS. The prevalence of slight/mild bilateral sensorineural hearing loss was lower than reported in previous studies. There was no strong evidence that slight/mild bilateral sensorineural hearing loss affects adversely language, reading, behavior, or health-related quality of life in children who are otherwise healthy and of normal intelligence.

The pathways responsible for sound perception in the cochlea involve the coordinated and regulated expression of hundreds of genes. By using microarray analysis, we identified several transcripts enriched in the inner ear, including the maternally expressed gene 3 (Meg3/Gtl2), an imprinted noncoding RNA. Real-time PCR analysis demonstrated that Meg3/Gtl2 was highly expressed in the cochlea, brain, and eye. Molecular studies revealed the presence of several Meg3/Gtl2 RNA splice variants in the mouse cochlea, brain, and eye. In situ hybridizations showed intense Meg3/Gtl2 RNA staining in the nuclei of type I spiral ganglion cells and in cerebellum near the dorsal vestibular region of the cochlea. In embryonic mouse head sections, Meg3/Gtl2 RNA expression was observed in the otocyst, brain, eye, cartilage, connective tissue, and muscle. Meg3/Gtl2 RNA expression increased in the developing otocyst and localized to the spiral ganglion, stria vascularis, Reissner's membrane, and greater epithelial ridge (GER) in the cochlear duct. RT-PCR analysis performed on cell lines derived from the organ of Corti, representing neural, supporting, and hair cells, showed significantly elevated levels of Meg3/Gtl2 expression in differentiated neural cells. We propose that Meg3/Gtl2 RNA functions as a noncoding regulatory RNA in the inner ear and that it plays a role in pattern specification and differentiation of cells during otocyst development, as well as in the maintenance of a number of terminally differentiated cochlear cell types. Copyright 2005 Wiley-Liss, Inc.

Our study outlines an alternative approach for the selection and investigation of genes involved in inner ear function.To gain understanding of the gene pathways involved in the development of the normal cochlea.Microarray technology currently offers the most efficient approach to investigate gene expression and identify pathways involved in cell differentiation. Epidermal growth factor (EGF) induces cultures derived from the organ of Corti to proliferate and produce new hair cells. Since pluripotent embryonic stem (ES) cells have the capacity to generate all tissues, we induced murine ES cells to differentiate towards ectodermal and neuroectodermal cell types and from there investigated their commitment towards the hair cell lineage in the presence of EGF. Cells were collected at three points along the differentiation pathway and their expression profiles were determined using the Soares NMIE mouse inner ear cDNA library printed in microarray format.Three genes up-regulated after addition of EGF (serine (or cysteine) proteinase inhibitor, clade H, member 1 (Serpinh1), solute carrier family 2 (facilitated glucose transporter), member 10 (Slc2a10) and secreted acidic cysteine-rich glycoprotein (Sparc)) were selected for further analysis and characterization. Of the three genes, Serpinh1 and Slc2a10 have never been implicated in the hearing process.

The cochlear portion of the inner ear converts movements produced by sound waves into electrical impulses. Transcripts enriched in the cochlea are likely to have an important role in hearing. In this paper, we report that microarray analyses of the Soares NMIE inner ear library revealed cochlear enriched expression of apolipoprotein D (apoD), a glycoprotein and member of the lipocalin family that transport small hydrophobic ligands. The cochlear enriched expression of Apod was validated by quantitative real time PCR analysis. To investigate the function of apoD in the inner ear the transcript and protein were localised in the cochlea. Apod messenger RNA (mRNA) expression was localised to the spiral ligament and spiral limbus, particularly in the suprastrial and supralimbral regions. The apoD protein was detected in the spiral ligament, spiral limbus and also in the outer hair cells of the organ of Corti. Investigation of cell lines exhibiting characteristics of hair and supporting cells revealed no Apod mRNA expression in these cells. This suggests transport of the protein within the cochlea, followed by internalisation into outer hair cells. The spiral limbus and ligament contain subpopulations of fibrocytes that are intimately involved in regulation of ion balance in the cochlear fluids and type I, II and III fibrocytes of the spiral ligament were all shown to be positive for apoD protein. On the basis of these results it was hypothesised that apoD could be involved in maintaining cochlear fluid homeostasis. To determine whether the apoD gene product was important for normal auditory function the hearing ability of an apoD knockout mouse was tested. The mouse was found to have a hearing threshold that was not significantly different to the control strain.

Mutations in GJB2, the gene encoding connexin-26 at the DFNB1 locus on 13q12, are found in as many as 50% of subjects with autosomal recessive, nonsyndromic prelingual hearing impairment. However, genetic diagnosis is complicated by the fact that 10%-50% of affected subjects with GJB2 mutations carry only one mutant allele. Recently, a deletion truncating the GJB6 gene (encoding connexin-30), near GJB2 on 13q12, was shown to be the accompanying mutation in approximately 50% of these deaf GJB2 heterozygotes in a cohort of Spanish patients, thus becoming second only to 35delG at GJB2 as the most frequent mutation causing prelingual hearing impairment in Spain. Here, we present data from a multicenter study in nine countries that shows that the deletion is present in most of the screened populations, with higher frequencies in France, Spain, and Israel, where the percentages of unexplained GJB2 heterozygotes fell to 16.0%-20.9% after screening for the del(GJB6-D13S1830) mutation. Our results also suggest that additional mutations remain to be identified, either in DFNB1 or in other unlinked genes involved in epistatic interactions with GJB2. Analysis of haplotypes associated with the deletion revealed a founder effect in Ashkenazi Jews and also suggested a common founder for countries in Western Europe. These results have important implications for the diagnosis and counseling of families with DFNB1 deafness.

Background: Attention deficit hyperactivity disorder (ADHD) is a complex condition with high heritability. However, both biochemical investigations and association and linkage studies have failed to define fully the underlying genetic factors associated with ADHD. We have identified a family co-segregating an early onset behavioural/developmental condition, with features of ADHD and intellectual disability, with a pericentric inversion of chromosome 3, 46N inv(3)(p14:q21). Methods: We hypothesised that the inversion breakpoints affect a gene or genes that cause the observed phenotype. Large genomic clones (P1 derived/yeast/bacterial artificial chromosomes) were assembled into contigs across the two inversion breakpoints using molecular and bioinformatic technologies. Restriction fragments crossing the junctions were identified by Southern analysis and these fragments were amplified using inverse PCR. Results: The amplification products were subsequently sequenced to reveal that the breakpoints lay within an intron of the dedicator of cytokinesis 3 ( DOCK3 ) gene at the p arm breakpoint, and an intron of a novel member of the solute carrier family 9 (sodium/hydrogen exchanger) isoform 9 ( SLC9A9 ) at the q arm. Both genes are expressed in the brain, but neither of the genes has previously been implicated in developmental or behavioural disorders. Conclusion: These two disrupted genes are candidates for involvement in the pathway leading to the neuropsychological condition in this family.

The development and function of the inner ear is complex requiring the correct and coordinated expression of many genes. The recent progress in the analyses of the human and other genomes has provided tools for identification of genes involved in hearing. As more and more nucleotide sequence information accumulates, experimental methods of molecular biology are rapidly being supplemented, and partially supplanted, by computational methods. In this study we present comprehensive in silico analyses of a cDNA library representing almost 1600 transcripts isolated from mouse inner ear. By mining the public databases we were able to rapidly and efficiently identify numerous transcripts likely to have a specific role in the auditory or vestibular function of the inner ear. Analyses revealed about 600 known genes and almost 100 inner-ear specific transcripts. Almost 50 of these are candidate genes for hearing impairment based on their chromosomal localization and inner-ear expression pattern. We describe a powerful approach to identify novel genes associated with hearing and vestibular function, further increasing our understanding of the molecular biology of the inner ear.

This study addressed the question of whether a hearing loss caused by mutations in the connexin 26 gene had a significant effect on language and speech perception outcomes in children using cochlear implants or hearing aids. The families of children who had participated in two previous studies of language development were invited to participate in this genetic study. From the 52 children whose families agreed to participate, 15 children with connexin 26 mutations in both chromosomes were identified. After taking into account other factors which are known to affect language development and speech perception in children with impaired hearing, no significant differences were found between the 15 children where connexin 26 was known to be the cause of deafness and the other 37 children in the study.

We have analyzed Twinkle, the causative gene for autosomal dominant progressive external ophthalmoplegia (adPEO) on chromosome 10, in 11 Australian autosomal dominant progressive external ophthalmoplegia families of Caucasian origin, and investigated whether there are distinct molecular and clinical features associated with mutations in this gene. We found two new mutations in Twinkle, in 3 of the 11 pedigrees examined. One resides in the linker region of this gene while the other is in the primase domain. Both regions are highly conserved between species. Multiple deletions in the mtDNA from muscle are not always prominent and there are significant variations in the clinical presentation within and between families with mutations in the Twinkle gene. Therefore, genotype/phenotype predictions are difficult. No mutations were found in adenine nucleotide translocator 1 (ANT1), another known adPEO causative gene, in four of the seven remaining families investigated. Thus, Twinkle appears to be the most common gene associated with adPEO in Australian families.

Two loci for nonsyndromic recessive deafness located on chromosome 21q22.3 have previously been reported, DFNB8 and DFNB10. Recently a gene which encodes a transmembrane serine protease, TMPRSS3 or ECHOS1, was found to be responsible for both the DFNB8 and DFNB10 phenotypes. To determine the contribution of TMPRSS3 mutations in the general congenital/childhood nonsyndromic deaf population we performed mutation analysis of the TMPRSS3 gene in 448 unrelated deaf patients from Spain, Italy, Greece, and Australia who did not have the common 35delG GJB2 mutation. From the 896 chromosomes studied we identified two novel pathogenic mutations accounting for four mutant alleles and at least 16 nonpathogenic sequence variants. The pathogenic mutations were a 1-bp deletion resulting in a frameshift and an amino acid substitution in the LDLRA domain of TMPRSS3. From this and another study we estimate the frequency of TMPRSS3 mutations in our sample as 0.45%, and approximately 0.38% in the general Caucasian childhood deaf population. However, TMPRSS3 is still an important contributor to genetic deafness in populations with large consanguineous families.

Rolandic epilepsy (RE) is the most common idiopathic focal childhood epilepsy. Its molecular basis is largely unknown and a complex genetic etiology is assumed in the majority of affected individuals. The present study tested whether six large recurrent copy number variants at 1q21, 15q11.2, 15q13.3, 16p11.2, 16p13.11 and 22q11.2 previously associated with neurodevelopmental disorders also increase risk of RE. Our association analyses revealed a significant excess of the 600 kb genomic duplication at the 16p11.2 locus (chr16: 29.5-30.1 Mb) in 393 unrelated patients with typical (n = 339) and atypical (ARE; n = 54) RE compared with the prevalence in 65,046 European population controls (5/393 cases versus 32/65,046 controls; Fisher's exact test P = 2.83 × 10(-6), odds ratio = 26.2, 95% confidence interval: 7.9-68.2). In contrast, the 16p11.2 duplication was not detected in 1738 European epilepsy patients with either temporal lobe epilepsy (n = 330) and genetic generalized epilepsies (n = 1408), suggesting a selective enrichment of the 16p11.2 duplication in idiopathic focal childhood epilepsies (Fisher's exact test P = 2.1 × 10(-4)). In a subsequent screen among children carrying the 16p11.2 600 kb rearrangement we identified three patients with RE-spectrum epilepsies in 117 duplication carriers (2.6%) but none in 202 carriers of the reciprocal deletion. Our results suggest that the 16p11.2 duplication represents a significant genetic risk factor for typical and atypical RE.

BACKGROUND: Cationic liposomes represent an important gene delivery system due to their low immunogenicity, but are relatively inefficient, with optimisation of DNA-liposome complexes (lipoplexes) for transfection necessary for each cell type of interest. There have been few studies examining optimisation in neuronal cell types or determining how the structure of lipoplexes affects transfection efficiency. METHODS: Four commercially available cationic liposome formulations were used to optimise transfection efficiency in neuronal cells. The DNA to liposome ratio and the amount of DNA used in transfections were varied. Transfection efficiency was determined by the percentage of cells positive for the micro-galactosidase reporter gene product. The structure of lipoplexes was studied using atomic force microscopy. Lipoplexes were characterised further using dynamic light scattering to determine size and fluorescence techniques to show DNA compaction. RESULTS: Optimal transfection conditions were found to differ between immortalised cell lines and primary cells. High transfection efficiencies in immortalised cell lines were achieved predominantly with multivalent cationic liposomes while primary neuronal cells showed optimal transfection efficiency with monovalent cationic liposomes. The structure of lipoplexes was observed with atomic force microscopy and showed globular complexes for multivalent cationic liposomes, while monovalent liposomes gave less compact structures. In support of this finding, high levels of DNA compaction with multivalent liposomes were observed using fluorescence quenching measurements for all DNA to liposome ratios tested. One monovalent liposome showed increasing levels of compaction with increasing liposome amount. Dynamic light scattering showed little change in complex size when the different lipoplexes were studied. CONCLUSIONS: Optimisation of transfection efficiency was different for cell lines and primary neurons. Immortalised cells showed optimal transfection with multivalent liposomes while primary neurons showed optimal transfection with monovalent liposomes. The charge ratio of the monovalent liposome was below one, suggesting a different mechanism of lipoplex binding and uptake in primary neurons. The structure of lipoplexes, as

Mutations in the human gap junction β-2 gene (GJB2) that encodes connexin-26 have been shown to cause non-syndromic sensorineural hearing loss (NSSNHL) at the DFNB1 locus on 13q11. Functional and genetic data regarding the disease causing potential of one particular GJB2 sequence variant, 101 T→C (M34T), have proven contradictory. In this study, we found the prevalence of the M34T allele in a cohort of white sib pairs and sporadic cases with NSSNHL from the United Kingdom and Ireland to be 3.179% of chromosomes screened. Significantly, we identified the first M34T/M34T genotype cosegregating in a single family with mid to high frequency NSSNHL. Screening a control population of 630 subjects we identified 25 M34T heterozygotes; however, no M34T homozygotes were detected. Surprisingly, the majority of M34T alleles (88%) were in cis with a 10 bp deletion in the 5' non-coding sequence. This non-coding deletion was also homozygous in the homozygous M34T subjects. Microsatellite analysis of flanking loci in M34T heterozygotes and controls does not define an extensive ancestral haplotype but preliminary data suggest two common alleles in subjects with the M34T allele. In summary, we provide data that support M34T acting as a recessive GJB2 allele associated with mild-moderate prelingual hearing impairment. Keywords: GJB2; connexin-26; M34T; hearing loss

Copper homeostasis in mammals is maintained by the balance of dietary intake and copper excretion via the bile. Sheep have a variant copper phenotype and do not efficiently excrete copper by this mechanism, often resulting in excessive copper accumulation in the liver. The Wilson disease protein (ATP7B) is a copper transporting P-type ATPase that is responsible for the efflux of hepatic copper into the bile. To investigate the role of ATP7B in the sheep copper accumulation phenotype, the cDNA encoding the ovine homologue of ATP7B was isolated and sequenced and the gene was localised by fluorescence in situ hybridisation to chromosome 10. The 6.3 kb cDNA encoded a predicted protein of 1444 amino acids which included all of the functional domains characteristic of copper transporting P-type ATPases. ATP7B mRNA was expressed primarily in the liver with lower levels present in the intestine, hypothalamus and ovary. A splice variant of ATP7B mRNA, which was expressed in the liver and comprised approximately 10% of the total ATP7B mRNA pool, also was isolated. The results suggest that ATP7B is produced in the sheep and that the tendency to accumulate copper in the liver is not due to a gross alteration in the structure or expression of ATP7B.

Background: The most common form of nonsyndromic neurosensory autosomal recessive deafness, DFNB1, is caused by mutations in the connexin 26 gene ( GJB2 ) on chromosome 13. One mutation, in which one guanosine (G) residue is deleted from a run of 6 Gs (35delG), is found in 40% to 70% of DFNB1 cases and has an expected population frequency of one in 40 to one in 100. Methods and Results: Polymerase chain reaction (PCR)-based tests for the 35delG mutation were developed. They are based on mismatched PCR primers that produce novel Eco RII or Dde I restriction enzyme sites depending on the number of Gs at the 35delG locus. An Eco RII site is generated in the wild-type sequence (6 Gs), but not when the 35delG mutation is present. Alternatively, a Dde I site can be generated so that this enzyme cuts the PCR product when the 35delG mutation is present, but not the wild-type sequence. Conclusions: These tests enable a quick and reliable screen for the common 35delG mutation.

SummaryMitochondrial genetics is complicated by heteroplasmy, or mutant load, which may be from 1%–99%, and thus may produce a gene dosage–type effect. Limited data are available for genotype/phenotype correlations in disorders caused by mtDNA mutations; therefore, prenatal diagnosis for mtDNA mutations has been hindered by an inability to predict accurately the clinical severity expected from a mutant load measured in fetal tissue. After reviewing 44 published and 12 unpublished pedigrees, we considered the possibility of prenatal diagnosis for two common mtDNA mutations at nucleotide 8993. We related the severity of symptoms to the mutant load and predicted the clinical outcome of a given mutant load. We also used the available data to generate empirical recurrence risks for genetic counseling, which may be used in conjunction with prenatal diagnosis.

In 1951, Denis Leigh first described a striking neuropathology that occured in a child who died of a neurodegenerative disease (Leigh 1951). The paper focused on the patient's distinct neuropathological abnormalities, which we now associate with Leigh disease, or Leigh syndrome (MIM 256000). Definite diagnosis of this neurodegenerative disorder still depends on the identification, either in postmortem samples or by use of neuroimaging techniques, of the characteristic neuropathology of focal, bilaterally symmetrical spongiform lesions, especially in the thalamus and brain stem regions.

Background: Two of the most common mutations in the mitochondrial DNA (mtDNA) of children occur at nucleotide 8993 (nt8993). The base substitutions of T to G (T8993G) and T to C (T8993C) are known to cause neurologic disorders and are routinely screened for in patients suspected of having a mitochondrial disorder. Methods and Results: Both mutations at nt8993 create a novel HpaII restriction endonuclease site and are usually detected by polymerase chain reaction (PCR) amplification of a section of the mtDNA containing nt8993, followed by HpaII digestion. The resulting fragment sizes are then analyzed by agarose gel electropho resis. Initial testing on a child referred for analysis suggested that the proband and his maternal relatives all had the common mtDNA mutation T8993C; however, subsequent restriction endonuclease and DNA sequencing analysis showed that the proband and his maternal relatives were homoplasmic for a novel variant at nt8856. This variant also creates an Hpa II restriction endonuclease site, and the fragments generated by the site are almost identical in size to those generated as a result of the nt8993 mutation when commonly used primers amplify the PCR product. Conclusions: A novel mutation in the mtDNA at nt8856 creates an HpaII restriction endonuclease site that has the potential to generate false positives when PCR products are tested for mutations at nt8993. This emphasizes the need for restriction endonuclease-based diagnostic tests for mtDNA mutations to account for the highly polymorphic nature of the mtDNA sequence and the importance of confirming a mutation by a second method.

We have identified a new pathogenic mechanism for an inherited muscular dystrophy in which functional haploinsufficiency of the extracellular matrix protein collagen VI causes Bethlem myopathy. The heterozygous COL6A1 mutation results in a single base deletion from the mRNA and a premature stop codon. The mutant mRNA is unstable, subject to nonsense-mediated mRNA decay, and is almost completely absent both from patient fibroblasts and skeletal muscle, resulting in haploinsufficiency of the alpha1(VI) subunit and reduced production of structurally normal collagen VI. This is the first example of a muscular dystrophy caused by haploinsufficiency of a structural protein or member of the dystrophin-glycoprotein complex, and identifies collagen VI as a critical contributor to cell-matrix adhesion in skeletal muscle.

Prelingual non-syndromic (isolated) deafness is the most frequent hereditary sensory defect. In >80% of the cases, the mode of transmission is autosomal recessive. To date, 14 loci have been identified for the recessive forms (DFNB loci). For two of them, DFNB1 and DFNB2, the genes responsible have been characterized; they encode connexin 26 and myosin VIIA, respectively. In order to evaluate the extent to which the connexin 26 gene (Cx26) contributes to prelingual deafness, we searched for mutations in this gene in 65 affected Caucasian families originating from various countries, mainly tunisia, France, New Zealand and the UK. Six of these families are consanguineous, and deafness was shown to be linked to the DFNB1 locus, 10 are small non consanguineous families in which the segregation of the trait has been found to be compatible with the involvement of DFNB1, and in the remaining 49 families no linkage analysis has been performed. A total of 62 mutant alleles in 39 families were identified. Therefore, mutations in Cx26 represent a major cause of recessively inherited prelingual deafness since according to the present results they would underlie approximately half of the cases. In addition, one specific mutation, 30delG, accounts for the majority (approximately 70%) of the Cx26 mutant alleles. It is therefore one of the most frequent disease mutations so far identified. Several lines of evidence indicate that the high prevalence of the 30delG mutation arises from a mutation hot spot rather than from a founder effect. Genetic counseling for prelingual deafness has been so far considerably impaired by the difficulty in distinguishing genetic and non genetic deafness in families presenting with a single deaf child. Based on the results presented here, the development of a simple molecular test could be designed which should be of considerable help.

Deficiency of complex I (reduced nicotinamide adenine dinucleotide dehydrogenase-ubiquinone oxidoreductase) of the mitochondrial respiratory chain may be seen as a pure myopathy or as a neuromuscular disorder at presentation. Efficacy of long- term therapy for these disorders is yet to be established. We report the case of a female patient with complex I deficiency and skeletal myopathy, who has had a sustained clinical response to riboflavin during 3 years of therapy. Molecular studies found no mutations in the putative flavin mononucleotide binding site in the 51 kd subunit of complex I, but a T-to-C transition at nucleotide 3250 in the mitochondrial DNA tRNA(Leu(UUR)) gene was identified. This mutation has been reported in one other family in that five members had fatigue with or without muscle weakness. There were also five cases of unexplained infant deaths in that family and two cases in the family reported here. Riboflavin therapy should be attempted in all patients with complex I deficiency when the clinical presentation is one of isolated skeletal myopathy.

The availability of glucose, and its glycolytic product lactate, for cerebral energy metabolism is regulated by specific brain transporters. Inadequate energy delivery leads to neurologic impairment. Haploinsufficiency of the glucose transporter GLUT1 causes a characteristic early onset encephalopathy, and has recently emerged as an important cause of a variety of childhood or later-onset generalized epilepsies and paroxysmal exercise-induced dyskinesia. We explored whether mutations in the genes encoding the other major glucose (GLUT3) or lactate (MCT1/2/3/4) transporters involved in cerebral energy metabolism also cause generalized epilepsies. A cohort of 119 cases with myoclonic astatic epilepsy or early onset absence epilepsy was screened for nucleotide variants in these five candidate genes. No epilepsy-causing mutations were identified, indicating that of the major energetic fuel transporters in the brain, only GLUT1 is clearly associated with generalized epilepsy.

Epilepsy comprises several syndromes, amongst the most common being mesial temporal lobe epilepsy with hippocampal sclerosis. Seizures in mesial temporal lobe epilepsy with hippocampal sclerosis are typically drug-resistant, and mesial temporal lobe epilepsy with hippocampal sclerosis is frequently associated with important co-morbidities, mandating the search for better understanding and treatment. The cause of mesial temporal lobe epilepsy with hippocampal sclerosis is unknown, but there is an association with childhood febrile seizures. Several rarer epilepsies featuring febrile seizures are caused by mutations in SCN1A, which encodes a brain-expressed sodium channel subunit targeted by many anti-epileptic drugs. We undertook a genome-wide association study in 1018 people with mesial temporal lobe epilepsy with hippocampal sclerosis and 7552 control subjects, with validation in an independent sample set comprising 959 people with mesial temporal lobe epilepsy with hippocampal sclerosis and 3591 control subjects. To dissect out variants related to a history of febrile seizures, we tested cases with mesial temporal lobe epilepsy with hippocampal sclerosis with (overall n = 757) and without (overall n = 803) a history of febrile seizures. Meta-analysis revealed a genome-wide significant association for mesial temporal lobe epilepsy with hippocampal sclerosis with febrile seizures at the sodium channel gene cluster on chromosome 2q24.3 [rs7587026, within an intron of the SCN1A gene, P = 3.36 × 10 -9, odds ratio (A) = 1.42, 95% confidence interval: 1.26-1.59]. In a cohort of 172 individuals with febrile seizures, who did not develop epilepsy during prospective follow-up to age 13 years, and 6456 controls, no association was found for rs7587026 and febrile seizures. These findings suggest SCN1A involvement in a common epilepsy syndrome, give new direction to biological understanding of mesial temporal lobe epilepsy with hippocampal sclerosis with febrile seizures, and open avenues for investigation of prognostic factors and possible prevention of epilepsy in some children with febrile seizures. © 2013 The Author (2013). Published by Oxford University Press on behalf of the Guarantors of Brain., 0, SCOPUS: ar.j, info:eu-repo/semantics/published

Recent advances in molecular genetics have translated into the increasing utilisation of genetic testing in the routine clinical practice of neurologists. There has been a steady, incremental increase in understanding the genetic variation associated with epilepsies. Genetic testing in the epilepsies is not yet widely practiced, but the advent of new screening technologies promises to exponentially expand both knowledge and clinical utility. To maximise the value of this new genetic insight we need to rapidly extrapolate genetic findings to inform patients of their diagnosis, prognosis, recurrence risk and the clinical management options available for their specific genetic condition. Comprehensive, highly specific and sensitive genetic test results improve the management of patients by neurologists and clinical geneticists. Here we discuss the latest developments in clinical genetic testing for epilepsy and describe new molecular genetics platforms that will transform both genetic screening and novel gene discovery.

Kufs disease, an adult-onset neuronal ceroid lipofuscinosis, is challenging to diagnose and genetically heterogeneous. Mutations in CLN6 were recently identified in recessive Kufs disease presenting as progressive myoclonus epilepsy (Type A), whereas the molecular basis of cases presenting with dementia and motor features (Type B) is unknown. We performed genome-wide linkage mapping of two families with recessive Type B Kufs disease and identified a single region on chromosome 11 to which both families showed linkage. Exome sequencing of five samples from the two families identified homozygous and compound heterozygous missense mutations in CTSF within this linkage region. We subsequently sequenced CTSF in 22 unrelated individuals with suspected recessive Kufs disease, and identified an additional patient with compound heterozygous mutations. CTSF encodes cathepsin F, a lysosomal cysteine protease, dysfunction of which is a highly plausible candidate mechanism for a storage disorder like ceroid lipofuscinosis. In silico modeling suggested the missense mutations would alter protein structure and function. Moreover, re-examination of a previously published mouse knockout of Ctsf shows that it recapitulates the light and electron-microscopic pathological features of Kufs disease. Although CTSF mutations account for a minority of cases of type B Kufs, CTSF screening should be considered in cases with early-onset dementia and may avoid the need for invasive biopsies.

Animal models that recapitulate human disease are proving to be an invaluable tool in the identification of novel disease-associated genes. These models can improve our understanding of the complex genetic mechanisms involved in disease and provide a basis to guide therapeutic strategies to combat these conditions. We have identified a novel mouse model of non-syndromic sensorineural hearing loss with linkage to a region on chromosome 18. Eeyore mutant mice have early onset progressive hearing impairment and show abnormal structure of the sensory epithelium from as early as 4 weeks of age. Ultrastructural and histological analyses show irregular hair cell structure and degeneration of the sensory hair bundles in the cochlea. The identification of new genes involved in hearing is central to understanding the complex genetic pathways involved in the hearing process and the loci at which these pathways are interrupted in people with a genetic hearing loss. We therefore discuss possible candidate genes within the linkage region identified in eeyore that may underlie the deafness phenotype in these mice. Eeyore provides a new model of hereditary sensorineural deafness and will be an important tool in the search for novel deafness genes.

Myosin VI (Myo6) is known to play an important role in the mammalian auditory and vestibular systems. We have identified a novel N-ethyl-N-nitrosourea mutagenised mouse strain, charlie, carrying an intronic Myo6 splice site mutation. This mutation (IVS5+5G > A) results in skipping of exon 5, and is predicted to cause a frameshift and premature termination of the protein. We detected essentially no Myo6 transcript in tissue from charlie homozygous mutant mice (Myo6chl/chl). Myo6chl/chl mice exhibit vestibular dysfunction and profound hearing impairment when first tested at four weeks of age. Analysis of vestibular and cochlear hair cells by scanning electron microscopy and immunohistochemistry revealed highly disorganised hair bundles with irregular orientation and kinocilium position at postnatal stage P2–P3. Within a few weeks, the majority of hair cell stereocilia are missing, or fused and elongated, and degeneration of the sensory epithelium occurs. This novel mouse strain will be an important resource in elucidating the role myosin VI plays in the mammalian auditory system, as well as its non-auditory functions.

Glucose transporter 1 (GLUT1) deficiency caused by mutations of SLC2A1 is an increasingly recognized cause of genetic generalized epilepsy. We previously reported that10% (4 of 34) of a cohort with early onset absence epilepsy (EOAE) had GLUT1 deficiency. This study uses a new cohort of 55 patients with EOAE to confirm that finding. Patients with typical absence seizures beginning before 4 years of age were screened for solute carrier family 2 (facilitated glucose transporter), member 1 (SLC2A1) mutations or deletions. All had generalized spike-waves on electroencephalography (EEG). Those with tonic and/or atonic seizures were excluded. Mutations were found in 7 (13%) of 55 cases, including five missense mutations, an in-frame deletion leading to loss of a single amino acid, and a deletion spanning two exons. Over both studies, 11 (12%) of 89 probands with EOAE have GLUT1 deficiency. Given the major treatment and genetic counseling implications, this study confirms that SLC2A1 mutational analysis should be strongly considered in EOAE.

Human MYO7A mutations can cause a variety of conditions involving the inner ear. These include dominant and recessive non-syndromic hearing loss and syndromic conditions such as Usher syndrome. Mouse models of deafness allow us to investigate functional pathways involved in normal and abnormal hearing processes. We present two novel mouse models with mutations in the Myo7a gene with distinct phenotypes. The mutation in Myo7a(I487N/I487N) ewaso is located within the head motor domain of Myo7a. Mice exhibit a profound hearing loss and manifest behaviour associated with a vestibular defect. A mutation located in the linker region between the coiled-coil and the first MyTH4 domains of the protein is responsible in Myo7a(F947I/F947I) dumbo. These mice show a less severe hearing loss than in Myo7a(I487N/I487N) ewaso; their hearing loss threshold is elevated at 4 weeks old, and progressively worsens with age. These mice show no obvious signs of vestibular dysfunction, although scanning electron microscopy reveals a mild phenotype in vestibular stereocilia bundles. The Myo7a(F947I/F947I) dumbo strain is therefore the first reported Myo7a mouse model without an overt vestibular phenotype; a possible model for human DFNB2 deafness. Understanding the molecular basis of these newly identified mutations will provide knowledge into the complex genetic pathways involved in the maintenance of hearing, and will provide insight into recessively inherited sensorineural hearing loss in humans.

Brain malformations and neurological dysfunctions are often seen in pyruvate dehydrogenase (PDH) deficient patients. To understand these clinical presentations, we have analyzed the localization and developmental expression of PDH in the embryonic mouse nervous system. Immunostaining was performed to localize PDH E1 alpha protein. PDH activities were measured before and after activation. PDH E1 alpha mRNA levels were quantitated by reverse transcriptase-polymerase chain reaction. Abundant PDH E1 alpha protein was localized in the central nervous system and other neural tissues in embryos at embryonic day (E) 11 onwards. The PDH activity was very low in E9 brain and it increased continuously until the end of gestation. The proportion of active form of PDH increased significantly in E15 brain. Analysis of the PDH E1 alpha mRNA showed that only the X-linked form of the gene was transcribed. The overall mRNA level of E9 brain was approximately 93% of the adult value. It decreased gradually during embryogenesis. A large increase took place at the end of gestation. The mRNA level of PDH was approximately 100 times higher than that of the acetoacetyl-CoA thiolase gene. These results suggest that PDH E1 alpha transcripts of E9 brain are not translated at a high level. The appearance of PDH activity and its increase during E11 and E15 are mainly due to increased levels of translation and activation of PDH. Increased PDH activity at the end of gestation is attributed to an increase in transcription. Our data to a large extent explain pathological presentations in PDH E1 alpha deficient patients with congenital brain disorders.

Mutations in the SLC26A4 gene can cause both Pendred syndrome and nonsyndromic enlargement of the vestibular aqueduct, two conditions associated with sensorineural hearing loss. We analyzed the SLC26A4 gene in 44 hearing-impaired patients by nested polymerase chain reaction followed by high-resolution melt analysis. We also used this approach to scan for mutations in KCNJ10 and FOXI1, two genes reported to play a role in the pathogenesis of Pendred syndrome and enlarged vestibular aqueduct. Seven patients with known SLC26A4 mutations were included as controls. All previously identified mutations were detected by high-resolution melt analysis. Of the patients with no known mutations, we detected two SLC26A4 mutations in 5 probands (12%), one mutation in 9 probands (21%), and no mutations in 29 probands (67%). We identified two novel SLC26A4 mutations, p.T485M and p.F718S, and found no evidence of a digenic contribution of KCNJ10 and FOXI1 mutations.

Osmium tetroxide and hydroxylamine are used to detect mutations in DNA and RNA after hybridization of mutant and wild-type DNA. Mismatched T and C bases, respectively, are modified by these reagents and the DNA strand cleaved at the mismatched bases by subsequent treatment with piperidine. This allows detection and location of the mutation. Although most T.G mismatches have been reported to be reactive to osmium tetroxide, some have been reported to be unreactive. The aim of this study was to collect and analyze the reactive and unreactive T.G mismatches. We have collected sequence contexts of all reactive and unreactive T.G mismatches for analysis. This involves 10 unreactive T.G mismatches (plus one T.C) and 19 reactive T.G mismatches. Sequence effects of bases surrounding these mismatches must influence this reactivity. There must be many types of such sequence effects. We postulate that because of the dominance of 5' G bases near the T of unreactive T.G mismatches and the absence of 5' G bases in reactive T.G mismatches that the stacking of the 5' G on the mismatched T is the reason for this lack of reactivity in the majority of the cases studied here.

We have determined by deletion analysis that the most proximal region of the Pdha-2 promoter between nucleotide position -187 to +22 harbors a transcriptionally active core. This "core" promoter directs high levels of CAT (chloramphenicol acetyltransferase) reporter gene transcription in HeLa cells. DNase I footprinting of the proximal promoter revealed four regions of protection. One of these contains the consensus sequence for the Sp1 binding site and another the ATF/CREB binding site. The cis-sequences of the remaining two protected regions (designated MEP-2 and MEP-3; Mouse E1 alpha Promoter site) show no apparent consensus homology with cis-elements of other known transcription factors. Results of electrophoretic mobility shift assays confirm that the ATF/CREB and MEP binding sites interact in a characteristic and specific manner with factors present in nuclei of both testis and somatic tissue. The factor which recognizes the MEP-3 motif appears to be ubiquitous, whereas the MEP-2-protein complexes were tissue-specific. Interestingly, formation of a complex involving MEP-2 and a putative testis-specific binding factor (tau-MEP-2BF) is first observed in the testis of 2-week-old mice, this correlates with the expression of Pdha-2. In contrast, the formation of complexes between the MEP-2 binding site and a somatic variant of MEP-2BF (sigma-MEP-2BF) decreases in the testis as spermatogenesis proceeds. Our results suggest that 1) the MEP-2 binding factors are temporally regulated during spermatogenesis, and 2) interactions involving these factors with the MEP-2 cis-element may be important for modulating Pdha-2 expression.