Array comparative genomic hybridisation analysis of boys with X linked hypopituitarism identifies a 3.9 Mb duplicated critical region at Xq27 containing SOX3.

INTRODUCTION: Array comparative genomic hybridisation (array CGH) is a powerful method that detects alteration of gene copy number with greater resolution and efficiency than traditional methods. However, its ability to detect disease causing duplications in constitutional genomic DNA has not been shown. We developed an array CGH assay for X linked hypopituitarism, which is associated with duplication of Xq26-q27. METHODS: We generated custom BAC/PAC arrays that spanned the 7.3 Mb critical region at Xq26.1-q27.3, and used them to search for duplications in three previously uncharacterised families with X linked hypopituitarism. RESULTS: Validation experiments clearly identified Xq26-q27 duplications that we had previously mapped by fluorescence in situ hybridisation. Array CGH analysis of novel XH families identified three different Xq26-q27 duplications, which together refine the critical region to a 3.9 Mb interval at Xq27.2-q27.3. Expression analysis of six orthologous mouse genes from this region revealed that the transcription factor Sox3 is expressed at 11.5 and 12.5 days after conception in the infundibulum of the developing pituitary and the presumptive hypothalamus. DISCUSSION: Array CGH is a robust and sensitive method for identifying X chromosome duplications. The existence of different, overlapping Xq duplications in five kindreds indicates that X linked hypopituitarism is caused by increased gene dosage. Interestingly, all X linked hypopituitarism duplications contain SOX3. As mutation of this gene in human beings and mice results in hypopituitarism, we hypothesise that increased dosage of Sox3 causes perturbation of pituitary and hypothalamic development and may be the causative mechanism for X linked hypopituitarism.

A new autosomal dominant pure cerebellar ataxia.

A kindred is described with a dominantly inherited "pure" cerebellar ataxia in which the currently known spinocerebellar ataxias have been excluded. In the eight subjects studied, a notable clinical feature is slow progression, with the three least affected having only a mild degree of gait ataxia after three or more decades of disease duration. Pending an actual chromosomal locus discovery, the name spinocerebellar ataxia (SCA)15 is expectantly applied.

Atomic force microscopy imaging of DNA-cationic liposome complexes optimised for gene transfection into neuronal cells.

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

A novel deletion in the flavin-containing monooxygenase gene (FMO3) in a Greek patient with trimethylaminuria.

Mutations of the flavin-containing monooxygenase type 3 gene (FMO3) that encode the major functional form present in adult human liver, have been shown to cause trimethylaminuria. We now report a novel homozygous deletion of exons 1 and 2 in an Australian of Greek ancestry with TMAuria, the first report of a deletion causative of trimethylaminuria. The deletion occurs 328 bp upstream from exon 1. The 3'-end of the deletion occurs in intron 2, 10013 base pairs downstream from the end of exon 2. The deletion is 12226 bp long. For the proband homozygous for the human FMO3 gene deletion, it is predicted that in addition to loss of monooxygenase function for human FMO3 substrates, such as TMA and other amines, the proband will exhibit decreased tolerance of biogenic amines, both medicinal and those found in foods.

Genes for left-handedness: how to search for the needle in the haystack?

Although several genes that determine left-right asymmetry for structural syndromes such as situs inversus have been characterised in recent years (Supp, Witte, Potter, & Brueckner, 1997), there has been little progress in determining which genes or loci predispose to left-right handedness in humans. Linkage analysis has been used widely for the localisation of genes followed by their positional cloning. The complex genetics of handedness is one of the greatest problems for standard linkage analysis. Several genetic models have been proposed for the inheritance of handedness in humans. On the basis of these models, left-handedness can be considered a common single gene trait with a high gene frequency and a non-mendelian inheritance pattern. We report here a possible strategy, using these genetic models, that can be applied for the identification for genes determining handedness in humans.

Profuse familial adenomatous polyposis with an adenomatous polyposis coli exon 3 mutation.

The attenuated form of familial adenomatous polyposis coli (AAPC) is associated with mutations in the adenomatous polyposis coli (APC) gene which cluster in the 5' region of the gene. It has been proposed that a 'genotype-phenotype boundary' exists at codons 159-163, and mutations that are 5' of this boundary will produce AAPC. Herein we document a three-generation family with an exon 3 mutation well to the 5' side of the proposed boundary, in which two affected individuals have had, in their 40s, a profuse form of familial adenomatous polyposis coli. We conclude that the codon 159-163 'boundary' is indicative rather than definitive. These two patients also had postoperative intra-abdominal adhesions, severely so in one.

Friedreich ataxia: an overview.

Friedreich ataxia, an autosomal recessive neurodegenerative disease, is the most common of the inherited ataxias. The recent discovery of the gene that is mutated in this condition, FRDA, has led to rapid advances in the understanding of the pathogenesis of Friedreich ataxia. About 98% of mutant alleles have an expansion of a GAA trinucleotide repeat in intron 1 of the gene. This leads to reduced levels of the protein, frataxin. There is mounting evidence to suggest that Friedreich ataxia is the result of accumulation of iron in mitochondria leading to excess production of free radicals, which then results in cellular damage and death. Currently there is no known treatment that alters the natural course of the disease. The discovery of the FRDA gene and its possible function has raised hope that rational therapeutic strategies will be developed.

Population-specific polymorphisms of the human FMO3 gene: significance for detoxication.

Flavin-containing monooxygenase form 3 (FMO3) is one of the major enzyme systems that protect humans from the potentially toxic properties of drugs and chemicals. FMO3 converts nucleophilic heteroatom-containing chemicals and endogenous materials to polar metabolites, which facilitates their elimination. For example, the tertiary amine trimethylamine is N-oxygenated by human FMO3 to trimethylamine N-oxide, and trimethylamine N-oxide is excreted in a detoxication and deoderation process. In normal humans, virtually all trimethylamine is metabolized to trimethylamine N-oxide. In a few humans, trimethylamine is not efficiently metabolized to trimethylamine N-oxide, and those individuals suffer from trimethylaminuria, or fishlike odor syndrome. Previously, we identified mutations of the FMO3 gene that cause trimethylaminuria. We now report two prevalent polymorphisms of this gene (K158E and V257M) that modulate the activity of human FMO3. These polymorphisms are widely distributed in Canadian and Australian white populations. In vitro analysis of wild-type and variant human FMO3 proteins expressed from the cDNA for the two naturally occurring polymorphisms showed differences in substrate affinities for nitrogen-containing substrates. Thus, for polymorphic forms of human FMO3, lower k(cat)/K(m) values for N-oxygenation of 10-(N, N-dimethylaminopentyl)-2-(trifluoromethyl) phenothiazine, trimethylamine, and tyramine were observed. On the basis of in vitro kinetic parameters, human FMO1 does not significantly contribute to human metabolism of trimethylamine or tyramine. The results imply that prevalent polymorphisms of the human FMO3 gene may contribute to low penetrance predispositions to diseases associated with adverse environmental exposures to heteroatom-containing chemicals, drugs, and endogenous amines.

Frequency of spinocerebellar ataxia types 1, 2, 3, 6, and 7 in Australian patients with spinocerebellar ataxia.

The frequencies of various genetically defined spinocerebellar ataxias (SCAs) vary in different populations presumably due to founder effects. No data have been published on the Australian population. Although predominantly of Anglo-Celtic extraction, Australia has also received considerable influx from southeastern Europe and more recently eastern and southeastern Asia. We examined the frequency of mutations for SCA types 1, 2, 3, 6, and 7 in southeastern Australia. Of 88 pedigrees with multiple-affected members, SCA type 1 (SCA1) accounted for 16%, SCA2 for 6%, SCA3 for 12%, SCA6 for 17%, SCA7 for 2%, and 47% (41 pedigrees) were negative for each of SCA1, 2, 3, and 6. Twenty of the 41 negative pedigrees were also negative for dentatorubralpallidoluysian atrophy, and indeed dentatorubralpallidoluysian atrophy has not been reported in Australia. In addition, no pedigree information was available on a further four patients with SCA1, three patients with SCA2, three patients with SCA3, and three patients with SCA6. One SCA1 and two SCA2 patients had no other known affected family members. In total, of 63 pedigrees or individuals with positive tests, 30% were those with SCA1, 15% with SCA2, 22% with SCA3, 30% with SCA6, and 3% with SCA7. Judging by pedigree names, four of the nine SCA2 positive individuals/pedigrees were of Italian extraction, and four of the 14 SCA3 positive individuals/pedigrees were of Chinese descent, whereas only 1 of the 20 SCA1 positive individuals/pedigrees were non-Anglo-Celtic. These results are in accordance with the known ethnic composition of the Australian population and with gene frequencies in these constituent ethnic groups reported by others. The frequency of large-normal alleles for SCA1 and SCA3 in the population reflects the prevalence of these two diseases, supporting the hypothesis that disease alleles arise by expansion of large-normal alleles.

G130V, a common FRDA point mutation, appears to have arisen from a common founder.

Friedreich ataxia (FRDA) is the most common inherited ataxia. About 98% of mutant alleles have an expansion of a GAA trinucleotide repeat in intron 1 of the affected gene, FRDA. The other 2% are point mutations. Of the 17 point mutations so far described, three appear to be more common. One of these is the G130V mutation in exon 4 of FRDA. G130V, when present with an expanded GAA repeat on the other allele, is associated with an atypical FRDA phenotype. Haplotype analysis was undertaken on the four families who have been described with this mutation. The results suggest a common founder for this mutation. Although marked differences in extragenic marker haplotypes were seen in one family, similar intragenic haplotyping suggests the same mutation founder for this family with the differences explicable by two recombination events.

Clinical and genetic study of Friedreich ataxia in an Australian population.

Friedreich ataxia is an autosomal recessive disorder caused by mutations in the FRDA gene that encodes a 210-amino acid protein called frataxin. An expansion of a GAA trinucleotide repeat in intron 1 of the gene is present in more than 95% of mutant alleles. Of the 83 people we studied who have mutations in FRDA, 78 are homozygous for an expanded GAA repeat; the other five patients have an expansion in one allele and a point mutation in the other. Here we present a detailed clinical and genetic study of a subset of 51 patients homozygous for an expansion of the GAA repeat. We found a correlation between the size of the smaller of the two expanded alleles and age at onset, age into wheelchair, scoliosis, impaired vibration sense, and the presence of foot deformity. There was no significant correlation between the size of the smaller allele and cardiomyopathy, diabetes mellitus, loss of proprioception, or bladder symptoms. The larger allele size correlated with bladder symptoms and the presence of foot deformity. The duration of disease is correlated with wheelchair use and the presence of diabetes, scoliosis, bladder symptoms and impaired proprioception, and vibration sense but no other complications studied.

Direct evidence that mitochondrial iron accumulation occurs in Friedreich ataxia.

Friedreich ataxia (FRDA) is due to mutations in the FRDA gene (FRDA). When the gene homologous to FRDA is knocked out in yeast, there is accumulation of iron in mitochondria and reduced respiratory function. So far, there is only indirect evidence to support the hypothesis that FRDA is due to accumulation of mitochondrial iron leading to increased production of free radicals. We show here that mitochondrial iron is significantly higher in fibroblasts from patients with FRDA than in control fibroblasts. This is the first direct evidence that the findings in yeast are reproducible in cells from patients with FRDA.

Genotype and intellectual phenotype in untreated phenylketonuria patients.

Previous studies have shown that genotype correlates with biochemical phenotype in treated phenylketonuria. If there is a strong correlation between genotype and intellectual phenotype of untreated patients, it would be possible to determine which individuals would have normal intelligence without treatment. In this study, 42 families with untreated phenylketonuria were analyzed to examine whether there was an association between genotype and untreated intellectual phenotype. Previously 12 of the 42 families were genotyped; now the genotyping of these patients is almost complete (40/42), a more thorough investigation was possible. Although the predicted phenylalanine hydroxylase (PAH) enzyme activity, based on genotype, showed an association with the patients' intellectual phenotype, the extensive overlap between the groups means the association is of little clinical value. Unrelated individuals with the same genotype and also siblings were found to have very different intellectual phenotypes. These phenotypic differences could not be explained by a difference in diet; therefore, we propose that another gene or genes may be modifying the intellectual phenotype of untreated patients. A preliminary search for possible modifying genes was performed. The possibility that a modifying gene was linked to the PAH gene on chromosome 12 was investigated using markers closely linked to the gene; however, no evidence for a modifying gene close to the PAH gene was found. Tyrosine hydroxylase was chosen as a candidate gene, because it can perform the same reaction as PAH. Using a common polymorphism within the gene, we found that this gene did not cause the discordant results and thus, did not modify the PAH phenotype.

Early-onset Alzheimer's disease caused by a novel mutation at codon 219 of the presenilin-1 gene.

Mutations in the presenilin 1 (PS1) gene are responsible for approximately 50% of early onset autosomal-dominant Alzheimer's disease cases. A PCR based mutation detection method, chemical cleavage of mismatch, was used to detect a novel PS1 mutation in the coding sequence of the PS1 gene. Sequencing confirmed a T to C transition altering a leucine to proline at codon 219 of the PS1 gene. This is a novel mutation in exon 7 of the PS1 gene occurring outside the transmembrane regions of IV and V.

Spinocerebellar ataxia type 2: clinical features of a pedigree displaying prominent frontal-executive dysfunction.

BACKGROUND: Spinocerebellar ataxia type 2 (SCA2) is a recently delineated cause of autosomal dominant cerebellar ataxia type I. The basic clinical neurologic features of SCA2 have been described in the literature, but neuropsychological features have not, despite statements that some patients became demented. OBJECTIVE: To describe the clinical and neuropsychological features of patients from a pedigree with SCA2. PATIENTS AND METHODS: We studied 8 affected members of an Australian pedigree of northern Italian origin with autosomal dominant cerebellar ataxia type I caused by SCA2. Patients underwent clinical neurologic examination and abbreviated neuropsychological testing, while some also underwent magnetic resonance imaging. The results were compared with pooled results from previously published studies of patients with SCA2. RESULTS: The pedigree displayed anticipation, with earlier onset in later generations, and there was an inverse correlation between repeat number and age at onset. The principal difference from other clinical reports of SCA2 was our finding of unequivocal frontal-executive dysfunction in 5 of 6 individuals who could be tested quantitatively, despite Mini-Mental State Examination scores in the nondemented range. This feature did not appear to correlate with either repeat size or duration of illness. CONCLUSIONS: In light of a recent report of frontal-executive dysfunction in spinocerebellar ataxia type III, we postulate that this pattern may be common to the autosomal dominant cerebellar ataxias and frequently may be overlooked because of the insensitivity of routine screening tests such as the Mini-Mental State Examination.