Mutation in the gene encoding ferritin light polypeptide causes dominant adult-onset basal ganglia disease.

We describe here a previously unknown, dominantly inherited, late-onset basal ganglia disease, variably presenting with extrapyramidal features similar to those of Huntington's disease (HD) or parkinsonism. We mapped the disorder, by linkage analysis, to 19q13.3, which contains the gene for ferritin light polypeptide (FTL). We found an adenine insertion at position 460-461 that is predicted to alter carboxy-terminal residues of the gene product. Brain histochemistry disclosed abnormal aggregates of ferritin and iron. Low serum ferritin levels also characterized patients. Ferritin, the main iron storage protein, is composed of 24 subunits of two types (heavy, H and light, L) which form a soluble, hollow sphere. Brain iron deposition increases normally with age, especially in the basal ganglia, and is a suspected causative factor in several neurodegenerative diseases in which it correlates with visible pathology, possibly by its involvement in toxic free-radical reactions. We found the same mutation in five apparently unrelated subjects with similar extrapyramidal symptoms. An abnormality in ferritin strongly indicates a primary function for iron in the pathogenesis of this new disease, for which we propose the name 'neuroferritinopathy'.

Both isoforms of ketohexokinase are dispensable for normal growth and development.

Dietary fructose intake has dramatically increased over recent decades and is implicated in the high rates of obesity, hypertension, and type 2 diabetes (metabolic syndrome) in Western societies. The molecular determinants of this epidemiologic correlation are incompletely defined, but high-flux fructose catabolism initiated by ketohexokinase (Khk, fructokinase) is believed to be important. The Khk gene encodes two enzyme isoforms with distinctive substrate preferences, the independent physiological roles of which are unclear. To investigate this question, and for testing the importance of Khk in metabolic syndrome, isoform-selective genetic lesions would be valuable. Two deficiency alleles of the mouse Khk gene were designed. The first, Khk(3a), uses targeted "knock-in" of a premature termination codon to induce a selective deficiency of the minor Khk-A isoform, preserving the major Khk-C isoform. The second, the Khk(Δ) allele, ablates both isoforms. Mice carrying each of these Khk-deficiency alleles were generated and validated at the DNA, RNA, and protein levels. Comparison between normal and knockout animals confirmed the specificity of the genetic lesions and allowed accurate analysis of the cellular distribution of Khk within tissues such as gut and liver. Both Khk(3a/3a) and Khk(Δ/Δ) homozygous mice were healthy and fertile and displayed minimal biochemical abnormalities under basal dietary conditions. These studies are the first demonstration that neither Khk isoform is required for normal growth and development. The new mouse models will allow direct testing of various hypotheses concerning the role of this enzyme in metabolic syndrome in humans and the value of Khk as a pharmacological target.

Inferring relative proportions of DNA variants from sequencing electropherograms.

MOTIVATION: Determination of the relative copy number of single-nucleotide sequence variants (SNVs) within a DNA sample is a frequent experimental goal. Various methods can be applied to this problem, although hybridization-based approaches tend to suffer from high-setup cost and poor adaptability, while others (such as pyrosequencing) may not be accessible to all laboratories. The potential to extract relative copy number information from standard dye-terminator electropherograms has been little explored, yet this technology is cheap and widely accessible. Since several biologically important loci have paralogous copies that interfere with genotyping, and which may also display copy number variation (CNV), there are many situations in which determination of the relative copy number of SNVs is desirable. RESULTS: We have developed a desktop application, QSVanalyzer, which allows high-throughput quantification of the proportions of DNA sequences containing SNVs. In reconstruction experiments, QSVanalyzer accurately estimated the known relative proportions of SNVs. By analyzing a large panel of genomic DNA samples, we demonstrate the ability of the software to analyze not only common biallelic SNVs, but also SNVs within a locus at which gene conversion between four genomic paralogs operates, and within another that is subject to CNV. AVAILABILITY AND IMPLEMENTATION: QSVanalyzer is freely available at http://dna.leeds.ac.uk/qsv/. It requires the Microsoft .NET framework version 2.0, which can be installed on all Microsoft operating systems from Windows 98 onwards. CONTACT: msjimc@leeds.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Isolation of human C-reactive protein complementary DNA and localization of the gene to chromosome 1.

With a synthetic oligonucleotide mixture as probe, complementary DNA clones of C-reactive protein were isolated from an adult human liver complementary DNA library. The clones ranged in size from 700 to 1100 base pairs and were identified by partial DNA sequence analysis. One complementary DNA clone was used as a probe for hybridization with human-rodent DNA's isolated from somatic cell hybrids and bound to nitrocellulose filters (Southern blot analysis) to assign the human C-reactive protein gene to chromosome 1.

Development of orthotopic tumour models using ultrasound-guided intrahepatic injection.

Mouse models of human diseases are an essential part of the translational pipeline. Orthotopic tumour mouse models are increasingly being used in cancer research due to their increased clinical relevance over subcutaneous xenograft models, particularly in relation to metastatic disease. In this study, we have developed orthotopic colorectal cancer liver metastases (CRCLM) and primary cholangiocarcinoma (CCA) models in BALB/c nude mice using minimally invasive ultrasound-guided intrahepatic injection. Due to its minimally invasive nature, the method reduced risk from surgical complications whilst being fast and easy to perform and resulted in measurable tumour volumes 1 to 3 weeks post-injection. Tumour volumes were monitored in vivo by weekly high-frequency ultrasound (HF-US) and/or twice weekly bioluminescence imaging (BLI) and confirmed with end-point histology. Take rates were high for human CRC cells (>73%) and for CCA cells (90%). We have demonstrated that this method reliably induces CRCLM and CCAs, in which tumour volume can be monitored throughout using HF-US and/or BLI. This provides a promising experimental tool for future testing of cancer therapeutics in an orthotopic model.

Direct detection of the common Mediterranean beta-thalassemia gene with synthetic DNA probes. An alternative approach for prenatal diagnosis.

The most common form of beta-thalassemia among Mediterraneans results from a single nucleotide substitution within the first intervening sequence (IVS-1) of the beta-globin gene. This particular mutation is not detectable in uncloned DNA by restriction enzyme analysis. Using synthetic DNA of 19-nucleotides in length corresponding to the normal and mutant IVS-1 sequences as probes, we have developed a direct assay for this gene defect. Under carefully controlled experimental conditions these synthetic probes detect only their homologous sequences in restriction digests of both cloned and uncloned DNA samples. The method is sufficiently sensitive to establish the genotype of individuals with respect to this defect using approximately 20 micrograms total DNA. This assay provides an alternative to fetal blood and DNA linkage analysis for the prenatal diagnosis of this variety of beta-thalassemia, particularly among Greek families where it is especially common.

Identification of a point mutation in the adenosine deaminase gene responsible for immunodeficiency.

Deficiency of adenosine deaminase (ADA) is the cause of an autosomal recessive form of immunodeficiency. We sought to define, at a molecular level, the mutations responsible for ADA deficiency in the cell line GM-1715, derived from an immunodeficient patient. Full-length complementary DNA (cDNA) for ADA was synthesized and cloned from the cell line. Sequence analysis of the clones revealed a point mutation in codon 101 (CGG to CAG) that predicts an amino acid change from arginine to glutamine. Southern blot analysis, based on silent polymorphisms in the cDNA sequence, indicated that only one of the defective alleles of the GM-1715 line had been sequenced. The mutation that was identified appears to be responsible for the loss of function in this allele, since the predicted primary structure of the enzyme is otherwise entirely normal.

Rheumatoid arthritis synovial T cells regulate transcription of several genes associated with antigen-induced anergy.

Rheumatoid arthritis (RA) is a chronic, inflammatory synovitis whose pathogenesis may involve autoimmune mechanisms. Anergy is a state of T-cell nonresponsiveness characterized by downregulated IL-2 production. Paradoxically, RA T cells are hyporesponsive and proliferate poorly to antigens and mitogens, thus sharing some characteristics with anergic T cells. We analyzed the molecular basis of anergy in cloned human CD4+ T cells using differential display RT-PCR and subsequently examined the levels of differentially expressed transcripts in RA and, as control, reactive arthritis (ReA) synovium. Several transcriptional events were common to anergic T cells and RA synovium. These included downregulation of CALMODULIN:, which is critical to T-cell activation, and of cellular apoptosis susceptibility protein, which may mediate resistance to apoptosis in RA. Transcription of CALMODULIN: in RA synovium was less than 1% of that in ReA and was lower in RA synovial fluid mononuclear cells than in paired PBMCs. Following anti-TNF-alpha therapy in vivo, RA PBMC CALMODULIN: transcripts increased five- to tenfold. Pharmacological calmodulin blockade in vitro impaired antigen-specific proliferation. These data provide a link between reduced CALMODULIN: transcription and impaired T-cell responsiveness in RA. The identification of transcriptional changes common to anergic and RA synovial T cells should help interpret some of the characteristic RA cellular defects.

Human menopausal and pregnant mare serum gonadotrophins in murine superovulation regimens for transgenic applications.

Superovulation is a fundamental procedure for generating transgenic rodents. While various methods exist, zygote yield/quality remain suboptimal, making these techniques open to refinement. All require a follicle stimulating and a luteinising effect. The former can be induced by pregnant mare serum gonadotrophin (PMSG) or other compounds like human menopausal gonadotrophin (HMG). While HMG can double zygote yield compared to PMSG, no study has compared their effects on embryo quality. Embryo yield could also be increased with PMSG: timing administration at estrus may further improve follicular recruitment. This study compared: (i) the efficacy of HMG/PMSG for producing viable embryos for microinjection; and (ii) the effect of HMG/PMSG administration at estrus on embryo yield. Whitten effect-induced estrous C57/Bl6xCBA F(1) hybrid mice were superovulated as follows: PMSG (day 1; 5 IU intraperitoneally) or HMG (days 1 and 2; 1 IU intramuscularly); all received human chorionic gonadotrophin (hCG) on day 3 (5 IU, intraperitoneally). Zygotes were retrieved following mating, morphologically assessed and microinjected with innocuous ZhAT1R construct (expressing LacZ reporter and human angiotensin II type 1 receptor) before transfer to pseudopregnant recipients. Pups were tested for the transgene by Southern blot. Neither HMG nor PMSG proved superior in improving embryo yield, morphology and short-term post-microinjection survival. However, HMG group micromanipulated embryos all failed to establish a pregnancy/generate transgenic pups, unlike their PMSG counterparts. While HMG can be used for superovulation, it appears to increase embryo vulnerability to the long-term effects of microinjection. Furthermore, the embryo yields associated with HMG can be replicated by timing PMSG injection to coincide with Whitten effect-induced estrus.

Regulation and function of the interaction between the APC tumour suppressor protein and EB1.

The interaction between the adenomatous polyposis coli (APC) tumour suppressor and the microtubule-associated protein EB1 was examined. Immunoprecipitation suggested that APC and EB1 were not associated in cultures of HCT116 cells arrested in mitosis. The C-terminal 170 amino acids of APC, purified as a bacterial fusion protein, precipitated EB1 from cell extracts, significantly refining the location of the EB1 interaction domain in APC. In vitro phosphorylation of this fusion protein by either protein kinase A or p34cdc2 reduced its ability to bind to EB1. Expression of GFP fusions to C-terminal APC sequences lacking or including the APC basic domain but encompassing the EB1 binding region in SW480 cells revealed a microtubule tip association which co-localized with that of EB1. Expression of the basic domain alone revealed a non-specific microtubule localization. In vitro interaction studies confirmed that the APC basic domain did not contribute to EB1 binding. These findings strongly suggest that the interaction between APC and EB1 targets APC to microtubule tips, and that the interaction between the two proteins is down-regulated during mitosis by the previously described mitotic phosphorylation of APC.

EB1, a protein which interacts with the APC tumour suppressor, is associated with the microtubule cytoskeleton throughout the cell cycle.

The characteristics of the adenomatous polyposis coli (APC) associated protein EB1 were examined in mammalian cells. By immunocytochemistry EB1 was shown to be closely associated with the microtubule cytoskeleton throughout the cell cycle. In interphase cells EB1 was associated with microtubules along their full length but was often particularly concentrated at their tips. During early mitosis, EB1 was localized to separating centrosomes and associated microtubules, while at metaphase it was associated with the spindle poles and associated microtubules. During cytokinesis EB1 was strongly associated with the midbody microtubules. Treatment with nocodazole caused a diffuse redistribution of EB1 immunoreactivity, whereas treatment with cytochalasin D had no effect. Interestingly, treatment with taxol abolished the EB1 association with microtubules. In nocodazole washout experiments EB1 rapidly became associated with the centrosome and repolymerizing microtubules. In taxol wash-out experiments EB1 rapidly re-associated with the microtubule cytoskeleton, resembling untreated control cells within 10 min. Immunostaining of SW480 cells, which contain truncated APC incapable of interaction with EB1, showed that the association of EB1 with microtubules throughout the cell cycle was not dependent upon an interaction with APC. These results suggest a role for EB1 in the control of microtubule dynamics in mammalian cells.

Promoter analysis of the human ubiquitin-conjugating enzyme gene family UBE2L1-4, including UBE2L3 which encodes UbcH7.

UbcH7 is a ubiquitin-conjugating enzyme mediating c-fos degradation, transcription factor NF-kappaB maturation, human papilloma virus-mediated p53 and Myc protein degradation, in vitro. Previously, we characterised a highly dispersed gene family, UBE2L1-UBE2L4, whose members could potentially encode different isoforms of the UbcH7 protein. UBE2L3, located at chromosome 22q11.2, is the only identified family member with introns and encodes a polypeptide sequence identical to that of UbcH7. Promoter characterisation of UBE2L1, UBE2L3 and UBE2L4 5'-upstream regions was performed to establish which are transcribed under normal physiological conditions and after heat shock. Promoter activity was observed only with the UBE2L3 construct, the minimal promoter lying within a region 100 bp upstream of the transcriptional start site. No evidence for the presence of UBE2L1 or UBE2L4 transcripts was observed in human or murine tissues and cell lines. These data strongly suggest that UBE2L1 and UBE2L4 are likely to encode pseudogenes. Sequencing revealed that the UBE2L3 promoter contained no TATA or CCAAT boxes. Protein:DNA interaction studies confirmed the presence of binding sites for the transcription factors AP2 and Sp1 in the UBE2L3 minimal promoter. Deletion of these binding sites indicated that these factors are crucial for transcription of this gene.

Isolation and characterisation of a cDNA encoding the precursor for a novel member of the acyl-CoA dehydrogenase gene family.

A gene encoding the precursor for a novel member of the human acyl-CoA dehydrogenase (ACD) gene family has been isolated which maps to human chromosome 11q25. The cDNA contains an open reading frame of 1248 nucleotides encoding a predicted 415-amino-acid peptide, and shares considerable sequence similarity with other members of the ACD family.

The isolation and characterisation of cDNA and genomic clones for human lecithin: cholesterol acyltransferase.

The protein sequencing of tryptic peptides from purified human lecithin: cholesterol acyltransferase (LCAT) identified sufficient amino-acid sequence to construct a corresponding mixed oligonucleotide probe. This was used to screen an adult human cDNA liver library, from which incomplete cDNA clones were isolated. The DNA sequence of these clones allows the prediction of the entire amino-acid sequence of the mature LCAT enzyme. The mature protein consists of 416 amino acids and contains several marked stretches of hydrophobic residues and four potential glycosylation sites. The cDNA probe detects LCAT mRNA sequences approx. 1500 bases long in human liver, but not intestine, RNA. The cDNA probe was used to isolate LCAT genomic recombinants from a human genomic library. Southern blotting data, and restriction site mapping, suggest that there is a single human LCAT structural gene between 4.3 and 5.5 kb in size.

Application of PCR to human gene detection.

The advent of the polymerase chain reaction has stimulated the development of a number of rapid methods for characterizing human genes. In addition, the unprecedented level of sensitivity offered by some of these methods may prove to be of great value in the detection of minority cell populations. Over the past year, technical developments have been made in this area.

Comparative genetic mapping for the identification of novel diagnostic and therapeutic targets.

The past year has seen significant advances in the genetic mapping of human and mouse genomes, with the publication of the most detailed and informative genetic maps compiled to date. These maps, together with those for several other species, provide a wealth of information for comparative mapping, with a view to identifying new human disease genes.

A novel mutation in the mitochondrial tRNA(Ser(UCN)) gene in a family with non-syndromic sensorineural hearing impairment.

We describe a family with non-syndromic sensorineural hearing impairment inherited in a manner consistent with maternal transmission. Affected members were found to have a novel heteroplasmic mtDNA mutation, T7510C, in the tRNA(Ser(UCN)) gene. This mutation was not found in 661 controls, is well conserved between species, and disrupts base pairing in the acceptor stem of the tRNA, making it the probable cause of hearing impairment in this family. Sequencing of the other mitochondrial tRNA genes did not show any other pathogenic mutations. Four other mutations causing hearing impairment have been reported in the tRNA(Ser(UCN)) gene, two having been shown to affect tRNA(Ser(UCN)) levels. With increasing numbers of reports of mtDNA mutations causing hearing impairment, screening for such mutations should be considered in all cases unless mitochondrial inheritance can be excluded for certain.

Maternally inherited hearing impairment in a family with the mitochondrial DNA A7445G mutation.

Despite the increasing number of reports of families with hearing impairment and mitochondrial DNA (mtDNA) mutations, the frequency of these mutations as causes of non-syndromic sensorineural hearing impairment (NSSHI) remains unknown. Mutations such as A1555G, A7445G and 7472insC have been found in several unrelated families implying they are more frequent than initially thought. We describe a family with NSSHI due to the presence of the homoplasmic mtDNA A7445G mutation in the tRNASer(UCN) gene. This is the fourth such family described with this mutation, all of different genetic backgrounds. Our study also demonstrates the difficulties sometimes encountered in establishing mitochondrial inheritance of hearing impairment in some families.

A gene for ataxic cerebral palsy maps to chromosome 9p12-q12.

Cerebral palsy (CP) has an incidence of approximately 1 in 750 births, although this varies between ethnic groups. Genetic forms of the disease account for about 2% of cases in most countries, but contribute a larger proportion in certain sub-types of the condition and in populations with a large proportion of consanguineous marriages. Ataxic cerebral palsy accounts for 5-10% of all forms of CP and it is estimated that approximately 50% of ataxic cerebral palsy is inherited as an autosomal recessive trait. We have identified a complex consanguineous Asian pedigree with four children in two sibships affected with ataxic cerebral palsy and have used homozygosity mapping to map the disorder in this family. A genome-wide search was performed using 343 fluorescently labelled polymorphic markers and linkage to chromosome 9p12-q12 was demonstrated. A maximum Lod score of 3.4 was observed between the markers D9S50 and D9S167 using multipoint analysis, a region of approximately 23cM. We have identified a family that segregates both ataxic CP and ataxic diplegia and have mapped the genetic locus responsible in this family to chromosome 9p12-q12. The identification of gene(s) involved in the aetiology of CP will offer the possibility of prenatal/premarital testing to some families with children affected with the disorder and will greatly increase our understanding of the development of the control of motor function.

A new locus for autosomal recessive non-syndromal sensorineural hearing impairment (DFNB27) on chromosome 2q23-q31.

Non-syndromic sensorineural deafness is an extremely genetically heterogeneous condition. We have used autozygosity mapping in a large consanguineous United Arab Emirate family to identify a novel locus for autosomal recessive non-syndromic sensorineural deafness, DFNB27, on chromosome 2q23-q31, with a maximum two-point lod score of 5.18 at theta = 0 for marker D2S2257. The DFNB27 locus extends over a 17 cM region between D2S2157 and D2S2273, and may overlap the DFNA16 locus for dominantly inherited, fluctuating, progressive non-syndromal hearing loss. However, genotype data suggests that the locus is likely to be refined to between D2S326 and D2S2273 and thus distinct from the DFNA16 locus.