Human sulfite oxidase R160Q: Identification of the mutation in a sulfite oxidase-deficient patient and expression and characterization of the mutant enzyme

Sulfite oxidase catalyzes the terminal reaction in the degradation of sulfur amino acids. Genetic deficiency of sulfite oxidase results in neurological abnormalities and often leads to death at an early age. The mutation in the sulfite oxidase gene responsible for sulfite oxidase deficiency in a 5-year-old girl was identified by sequence analysis of cDNA obtained from fibroblast mRNA to be a guanine to adenine transition at nucleotide 479 resulting in the amino acid substitution of Arg-160 to Gln. Recombinant protein containing the R160Q mutation was expressed in Escherichia coli, purified, and characterized. The mutant protein contained its full complement of molybdenum and heme, but exhibited 2% of native activity under standard assay conditions. Absorption spectroscopy of the isolated molybdenum domains of native sulfite oxidase and of the R160Q mutant showed significant differences in the 480- and 350-nm absorption bands, suggestive of altered geometry at the molybdenum center. Kinetic analysis of the R160Q protein showed an increase in Km for sulfite combined with a decrease in kcat resulting in a decrease of nearly 1,000-fold in the apparent second-order rate constant kcat/Km. Kinetic parameters for the in vitro generated R160K mutant were found to be intermediate in value between those of the native protein and the R160Q mutant. Native sulfite oxidase was rapidly inactivated by phenylglyoxal, yielding a modified protein with kinetic parameters mimicking those of the R160Q mutant. It is proposed that Arg-160 attracts the anionic substrate sulfite to the binding site near the molybdenum.

Ongoing immunoglobulin somatic mutation in germinal center B cell-like but not in activated B cell-like diffuse large cell lymphomas

B cell diffuse large cell lymphoma (B-DLCL) is a heterogeneous group of tumors, based on significant variations in morphology, clinical presentation, and response to treatment. Gene expression profiling has revealed two distinct tumor subtypes of B-DLCL: germinal center B cell-like DLCL and activated B cell-like DLCL. In a separate study, we determined that B-DLCL can also be subdivided into two groups based on the presence or absence of ongoing Ig gene hypermutation. Here, we evaluated the correlation between these B-DLCL subtypes established by the two different methods. Fourteen primary B-DLCL cases were studied by gene expression profiling using DNA microarrays and for the presence of ongoing mutations in their Ig heavy chain gene. All seven cases classified as germinal center B cell-like DLCL by gene expression showed the presence of ongoing mutations in the Ig genes. Five of the seven cases classified by gene expression as activated B cell-like DLCL had no ongoing somatic mutations, whereas, in the remaining two cases, a single point mutation was observed in only 2 of 15 and 21 examined molecular clones of variable heavy (VH) chain gene, respectively. These two cases were distantly related to the rest of the activated B cell-like DLCL tumors by gene expression. Our findings validate the concept that lymphoid malignancies are derived from cells at discrete stages of normal lymphocyte maturation and that the malignant cells retain the genetic program of those normal cells.

Functional expression of the Wilson disease protein reveals mislocalization and impaired copper-dependent trafficking of the common H1069Q mutation

Wilson disease is an autosomal recessive disorder of hepatic copper metabolism caused by mutations in a gene encoding a copper-transporting P-type ATPase. To elucidate the function of the Wilson protein, wild-type and mutant Wilson cDNAs were expressed in a Menkes copper transporter-deficient mottled fibroblast cell line defective in copper export. Expression of the wild-type cDNA demonstrated trans-Golgi network localization and copper-dependent trafficking of the Wilson protein identical to previous observations for the endogenously expressed protein in hepatocytes. Furthermore, expression of the Wilson cDNA rescued the mottled phenotype as evidenced by a reduction in copper accumulation and restoration of cell viability. In contrast, expression of an H1069Q mutant Wilson cDNA did not rescue the mottled phenotype, and immunofluorescence studies showed that this mutant Wilson protein was localized in the endoplasmic reticulum. Consistent with these findings, pulse–chase analysis demonstrated a 5-fold decrease in the half-life of the H1069Q mutant as compared with the wild-type protein. Maintenance of these transfected cell lines at 28°C resulted in localization of the H1069Q protein in the trans-Golgi network, suggesting that a temperature-sensitive defect in protein folding followed by degradation constitutes the molecular basis of Wilson disease in patients harboring the H1069Q mutation. Taken together, these studies describe a tractable expression system for elucidating the function and localization of the copper-transporting ATPases in mammalian cells and provide compelling evidence that the Wilson protein can functionally substitute for the Menkes protein, supporting the concept that these proteins use common biochemical mechanisms to effect cellular copper homeostasis.

Using rare mutations to estimate population divergence times: A maximum likelihood approach

In this paper we propose a method to estimate by maximum likelihood the divergence time between two populations, specifically designed for the analysis of nonrecurrent rare mutations. Given the rapidly growing amount of data, rare disease mutations affecting humans seem the most suitable candidates for this method. The estimator RD, and its conditional version RDc, were derived, assuming that the population dynamics of rare alleles can be described by using a birth–death process approximation and that each mutation arose before the split of a common ancestral population into the two diverging populations. The RD estimator seems more suitable for large sample sizes and few alleles, whose age can be approximated, whereas the RDc estimator appears preferable when this is not the case. When applied to three cystic fibrosis mutations, the estimator RD could not exclude a very recent time of divergence among three Mediterranean populations. On the other hand, the divergence time between these populations and the Danish population was estimated to be, on the average, 4,500 or 15,000 years, assuming or not a selective advantage for cystic fibrosis carriers, respectively. Confidence intervals are large, however, and can probably be reduced only by analyzing more alleles or loci.

A point mutation in the MET oncogene abrogates metastasis without affecting transformation

The MET oncogene encodes the tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF), known to stimulate invasive growth of epithelial cells. MET is overexpressed in a significant percentage of human cancers and is amplified during the transition between primary tumors and metastasis. To investigate whether this oncogene is directly responsible for the acquisition of the metastatic phenotype, we exploited a single-hit oncogenic version of MET, able to transform and to confer invasive and metastatic properties to nontumorigenic cells, both in vitro and in nude mice. We mutagenized the signal transducer docking site of Met (Y1349VHVX3Y1356VNV), which has the uncommon property of binding and activating multiple src homology region 2 (SH2)-containing intracellular effectors. Notably, a point mutation (H1351 → N) increased the transforming ability of the oncogene but abolished its metastatic potential. This mutation duplicates the Grb2 binding site, super-activating the Ras pathway and preventing the binding of the other intracellular transducers. Complementation in trans with another nonmetastatic mutant (N1358 → H), recruiting all the transducers downstream to Met except Grb2, rescued the invasive–metastatic phenotype. It is concluded that the metastatic potential of the MET oncogene relies on the properties of its multifunctional docking site, and that a single point mutation affecting signal transduction can dissociate neoplastic transformation from metastasis.

Both hypertrophic and dilated cardiomyopathies are caused by mutation of the same gene, δ-sarcoglycan, in hamster: An animal model of disrupted dystrophin-associated glycoprotein complex

Cardiomyopathy (CM) is a primary degenerative disease of myocardium and is traditionally categorized into hypertrophic and dilated CMs (HCM and DCM) according to its gross appearance. Cardiomyopathic hamster (CM hamster), a representative model of human hereditary CM, has HCM and DCM inbred sublines, both of which descend from the same ancestor. Herein we show that both HCM and DCM hamsters share a common defect in a gene for δ-sarcoglycan (δ-SG), the functional role of which is yet to be characterized. A breakpoint causing genomic deletion was found to be located at 6.1 kb 5′ upstream of the second exon of δ-SG gene, and its 5′ upstream region of more than 27.4 kb, including the authentic first exon of δ-SG gene, was deleted. This deletion included the major transcription initiation site, resulting in a deficiency of δ-SG transcripts with the consequent loss of δ-SG protein in all the CM hamsters, despite the fact that the protein coding region of δ-SG starting from the second exon was conserved in all the CM hamsters. We elucidated the molecular interaction of dystrophin-associated glycoproteins including δ-SG, by using an in vitro pull-down study and ligand overlay assay, which indicates the functional role of δ-SG in stabilizing sarcolemma. The present study not only identifies CM hamster as a valuable animal model for studying the function of δ-SG in vivo but also provides a genetic target for diagnosis and treatment of human CM.

A premature-termination mutation in the Mus musculus cyclin-dependent kinase 3 gene

Our understanding of the mammalian cell cycle is due in large part to the analysis of cyclin-dependent kinase (CDK) 2 and CDK4/6. These kinases are regulated by E and D type cyclins, respectively, and coordinate the G1/S-phase transition. In contrast, little is known about CDK3, a homolog of CDK2 and cell division cycle kinase 2 (CDC2). Previous studies using ectopic expression of human CDK3 suggest a role for this kinase in the G1/S-phase transition, but analysis of the endogenous kinase has been stymied by the low levels of protein present in cells and by the absence of an identifiable cyclin partner. Herein we report the presence of a single point mutation in the CDK3 gene from several Mus musculus strains commonly used in the laboratory. This mutation results in the replacement of a conserved tryptophan (Trp-187) within kinase consensus domain IX with a stop codon. The protein predicted to be encoded by this allele is truncated near the T loop, which is involved in activation by CDK-activating kinase. This mutation also deletes motif XI known to be required for kinase function and is, therefore, expected to generate a null allele. In stark contrast, CDK3 from two wild-mice species (Mus spretus and Mus mus castaneus) lack this mutation. These data indicate that CDK3 is not required for M. musculus development and suggest that any functional role played by CDK3 in the G1/S-phase transition is likely to be redundant with another CDK.

Untargeted mutation of the maternally derived mouse hypervariable minisatellite allele in F1 mice born to irradiated spermatozoa

Length change mutation at the Ms6hm hypervariable mouse minisatellite locus was analyzed in C57BL/6N × C3H/HeN F1 mice and the F1 of the reciprocal cross born to irradiated male parents. Spontaneous mutant frequencies were 8.4% and 9.8% for the paternally derived and maternally derived C3H/HeN alleles, respectively. The mutant frequencies for the paternally derived allele increased to 22% and 19% when the male parents were irradiated with 6 Gy at the postmeiotic spermatozoa stage and the spermatogonia stage, respectively. These increases in the mutant frequency were at least 10 to 100 times higher than those expected from the frequency of hits to the 3- to 4-kb allele, suggesting that the length change mutation at this minisatellite locus was not a targeted event due directly to DNA damage in the region. Further analysis demonstrated that the mutant frequency increased also at the maternally derived C3H/HeN allele to 20% when the male parents were irradiated at the spermatozoa stage. This increase in the maternal allele mutation was not observed in F1 born to irradiated spermatogonia. The present study suggests that introduction of DNA damage by irradiated sperm triggers genomic instability in zygotes and in embryos of subsequent developmental stages, and this genomic instability induces untargeted mutation in cis at the paternally derived minisatellite allele and in trans at the maternally derived unirradiated allele. Untargeted mutation revealed in the present study defines a previously unnoticed genetic hazard to the maternally derived genome by the paternally introduced DNA damage.

Fluorescence-based directed termination PCR: direct mutation characterization without sequencing

We describe a fluorescence-based directed termination PCR (fluorescent DT–PCR) that allows accurate determination of actual sequence changes without dideoxy DNA sequencing. This is achieved using near infrared dye-labeled primers and performing two PCR reactions under low and unbalanced dNTP concentrations. Visualization of resulting termination fragments is accomplished with a dual dye Li-cor DNA sequencer. As each DT–PCR reaction generates two sets of terminating fragments, a pair of complementary reactions with limiting dATP and dCTP collectively provide information on the entire sequence of a target DNA, allowing an accurate determination of any base change. Blind analysis of 78 mutants of the supF reporter gene using fluorescent DT–PCR not only correctly determined the nature and position of all types of substitution mutations in the supF gene, but also allowed rapid scanning of the signature sequences among identical mutations. The method provides simplicity in the generation of terminating fragments and 100% accuracy in mutation characterization. Fluorescent DT–PCR was successfully used to generate a UV-induced spectrum of mutations in the supF gene following replication on a single plate of human DNA repair-deficient cells. We anticipate that the automated DT–PCR method will serve as a cost-effective alternative to dideoxy sequencing in studies involving large-scale analysis for nucleotide sequence changes.

Branched co-polymers of histidine and lysine are efficient carriers of plasmids

We previously determined that a linear co-polymer of histidine and lysine (HK) in combination with liposomes enhanced the transfection efficiency of cationic liposomes. In the current study, we designed a series of HK polymers with increased branching and/or histidine/lysine ratio to determine if either variable affects transfection efficiency. In the presence of liposomes, the branched polymer with the highest number of histidines, HHK4b, was the most effective at enhancing gene expression. Furthermore, when serum was added to the medium during transfection, the combination of HHK4b and liposomes as a gene-delivery vehicle increased luciferase expression 400-fold compared to liposomes alone. In contrast to linear HK polymers, the higher branched HHK polymers were effective carriers of plasmids in the absence of liposomes. Without liposomes, the HHK4b carrier enhanced luciferase expression 15-fold in comparison with the lesser branched HHK2b carrier and increased expression by 5-logs in comparison with the HHK or HK carrier. The interplay of several parameters including increased condensation of DNA, buffering of acidic endosomes and differential binding affinities of polymer with DNA have a role in the enhancement of transfection by the HK polymers. In addition to suggesting that branched HK polymers are promising gene-delivery vehicles, this study provides a framework for the development of more efficient peptide-bond-based polymers of histidine and lysine.

The KMDB/MutationView: a mutation database for human disease genes

The KMDB/MutationView is a graphical database of mutations in human disease-causing genes and its current version consists of nine category-based sub-databases including diseases of eye, heart, ear, brain, cancer, syndrome, autoimmunity, muscle and blood. The KMDB/MutationView stores mutation data of 97 genes involved in 87 different disease and is accessible through http://mutview.dmb.med.keio.ac.jp.

Defective trafficking and function of KATP channels caused by a sulfonylurea receptor 1 mutation associated with persistent hyperinsulinemic hypoglycemia of infancy

The ATP-sensitive potassium channel (KATP) regulates insulin secretion in pancreatic β cells. Loss of functional KATP channels because of mutations in either the SUR1 or Kir6.2 channel subunit causes persistent hyperinsulinemic hypoglycemia of infancy (PHHI). We investigated the molecular mechanism by which a single phenylalanine deletion in SUR1 (ΔF1388) causes PHHI. Previous studies have shown that coexpression of ΔF1388 SUR1 with Kir6.2 results in no channel activity. We demonstrate here that the lack of functional expression is due to failure of the mutant channel to traffic to the cell surface. Trafficking of KATP channels requires that the endoplasmic reticulum-retention signal, RKR, present in both SUR1 and Kir6.2, be shielded during channel assembly. To ask whether ΔF1388 SUR1 forms functional channels with Kir6.2, we inactivated the RKR signal in ΔF1388 SUR1 by mutation to AAA (ΔF1388 SUR1AAA). Inactivation of similar endoplasmic reticulum-retention signals in the cystic fibrosis transmembrane conductance regulator has been shown to partially overcome the trafficking defect of a cystic fibrosis transmembrane conductance regulator mutation, ΔF508. We found that coexpression of ΔF1388 SUR1AAA with Kir6.2 led to partial surface expression of the mutant channel. Moreover, mutant channels were active. Compared with wild-type channels, the mutant channels have reduced ATP sensitivity and do not respond to stimulation by MgADP or diazoxide. The RKR → AAA mutation alone has no effect on channel properties. Our results establish defective trafficking of KATP channels as a molecular basis of PHHI and show that F1388 in SUR1 is critical for normal trafficking and function of KATP channels.

Metapopulation extinction caused by mutation accumulation

Theory suggests that the risk of extinction by mutation accumulation can be comparable to that by environmental stochasticity for an isolated population smaller than a few thousand individuals. Here we show that metapopulation structure, habitat loss or fragmentation, and environmental stochasticity can be expected to greatly accelerate the accumulation of mildly deleterious mutations, lowering the genetic effective size to such a degree that even large metapopulations may be at risk of extinction. Because of mutation accumulation, viable metapopulations may need to be far larger and better connected than would be required under just stochastic demography.

A single nucleotide polymorphism in the RAD51 gene modifies cancer risk in BRCA2 but not BRCA1 carriers

BRCA1 and BRCA2 carriers are at increased risk for both breast and ovarian cancer, but estimates of lifetime risk vary widely, suggesting their penetrance is modified by other genetic and/or environmental factors. The BRCA1 and BRCA2 proteins function in DNA repair in conjunction with RAD51. A preliminary report suggested that a single nucleotide polymorphism in the 5′ untranslated region of RAD51 (135C/G) increases breast cancer risk in BRCA1 and BRCA2 carriers. To investigate this effect we studied 257 female Ashkenazi Jewish carriers of one of the common BRCA1 (185delAG, 5382insC) or BRCA2 (6174delT) mutations. Of this group, 164 were affected with breast and/or ovarian cancer and 93 were unaffected. RAD51 genotyping was performed on all subjects. Among BRCA1 carriers, RAD51-135C frequency was similar in healthy and affected women [6.1% (3 of 49) and 9.9% (12 of 121), respectively], and RAD-135C did not influence age of cancer diagnosis [Hazard ratio (HR) = 1.18 for disease in RAD51-135C heterozygotes, not significant]. However, in BRCA2 carriers, RAD51-135C heterozygote frequency in affected women was 17.4% (8 of 46) compared with 4.9% (2 of 41) in unaffected women (P = 0.07). Survival analysis in BRCA2 carriers showed RAD51-135C increased risk of breast and/or ovarian cancer with an HR of 4.0 [95% confidence interval 1.6–9.8, P = 0.003]. This effect was largely due to increased breast cancer risk with an HR of 3.46 (95% confidence interval 1.3–9.2, P = 0.01) for breast cancer in BRCA2 carriers who were RAD51-135C heterozygotes. RAD51 status did not affect ovarian cancer risk. These results show RAD51-135C is a clinically significant modifier of BRCA2 penetrance, specifically in raising breast cancer risk at younger ages.

A splicing switch and gain-of-function mutation in FgfR2-IIIc hemizygotes causes Apert/Pfeiffer-syndrome-like phenotypes

Intercellular signaling by fibroblast growth factors plays vital roles during embryogenesis. Mice deficient for fibroblast growth factor receptors (FgfRs) show abnormalities in early gastrulation and implantation, disruptions in epithelial–mesenchymal interactions, as well as profound defects in membranous and endochondrial bone formation. Activating FGFR mutations are the underlying cause of several craniosynostoses and dwarfism syndromes in humans. Here we show that a heterozygotic abrogation of FgfR2-exon 9 (IIIc) in mice causes a splicing switch, resulting in a gain-of-function mutation. The consequences are neonatal growth retardation and death, coronal synostosis, ocular proptosis, precocious sternal fusion, and abnormalities in secondary branching in several organs that undergo branching morphogenesis. This phenotype has strong parallels to some Apert's and Pfeiffer's syndrome patients.