Isolation of new nonsense and frameshift mutants in the immunoglobulin μ heavy-chain gene of hybridoma cells

In order to expand the experimental material available for genetic and biochemical analyses of the natural immunoglobulin genes, we have isolated a variety of mutant mouse hybridoma cell lines. Some of these mutants have partial or complete deletions of the gene. Other mutants have nonsense or frameshift mutations in the exons encoding the variable and the second and third constant region domains of the heavy chain. When combined with earlier mutant data, this collection of genotypically and phenotypically tight mutants of known sequence spans most of the 10 kb of the gene, providing material for a variety of studies of genetic recombination and mRNA metabolism.     

High-frequency loss of specific immunoglobulin production in hybridoma cell lines bearing a chromosomal immunoglobulin κ gene modified by homologous recombination

We have examined the stability of trinitrophenyl (TNP)-specific IgM production in hybridoma cell lines in which homologous recombination was used to change the variable region of an endogenous chromosomal immunoglobulin gene to one specific for TNP. Mutant hybridomas that have lost TNP-specific IgM production are detected with a frequency of approximately 1%. Characterization of the mutant cells reveals a variety of gross rearrangements in the recombinant TNP gene as well as in the endogenous and TNP genes.     

Characterization of the 5′-flanking region and chromosomal assignment of the human brain natriuretic peptide gene

Brain natriuretic peptide (BNP) is a cardiac hormone that occurs predominantly in the ventricle, and synthesis and secretion of BNP are greatly augmented in patients with congestive heart failure and in animal models of ventricular hypertrophy. In order to elucidate the molecular mechanisms underlying the human BNP gene expression in the heart, the human BNP gene was isolated from a size-selected genomic minilibrary. The 1.9-kb human BNP 5-flanking region (–1813 to +110) contained an array of putative cis-acting regulatory elements. Various lengths of the cloned 5-flanking sequences were linked upstream to the bacterial chloramphenicol acetyltransferase (CAT) gene, and their promoter activities were assayed. The 1.9-kb promoter region showed a high-level CAT activity in cultured neonatal rat ventricular cardiocytes. When the CT-rich sequences (–1288 to –1095) were deleted, the high-level activity was reduced to approximately 30%. The 399-bp BNP 5 flanking region (–289 to +110) showed approximately 10% activity of the 1.9-kb region. Furthermore, using human-rodent somatic hybrid cell lines, the BNP gene was assigned to human chromosome 1, on which the atrial natriuretic peptide gene is localized. The present study leads to a better understanding of the molecular mechanisms for the human BNP gene expression in the heart.Abbreviations ANP Atrial natriuretic peptide - AP-1 Activator protein-1 - BNP Brain natriuretic peptide - CAT Chloramphenicol acetyltransferase     

Association of a single-nucleotide polymorphism in the immunoglobulin μ-binding protein 2 gene with immunoglobulin A nephropathy

Immunoglobulin A (IgA) nephropathy is the most common form of primary glomerulonephritis worldwide. The pathogenesis of IgA nephropathy is unknown, but it is certain that some genetic factors are involved in susceptibility to the disease. Employing a large-scale, case–control association study using gene-based single-nucleotide polymorphism (SNP) markers, we previously reported four candidate genes. We report here an additional significant association between IgA nephropathy and an SNP located in the gene encoding immunoglobulin -binding protein 2 (IGHMBP2) at chromosome 11q13.2–q13.4. The association (² =17.1, p=0.00003; odds ratio of 1.85 with 95% confidence interval of 1.39–2.50 in a dominant association model) was found using DNA from 465 affected individuals and 634 controls. The SNP (G34448A) caused an amino acid substitution from glutamine to lysine (E928K). As the gene product is involved in immunoglobulin-class switching and patients with the A allele revealed higher serum levels of IgA (p=0.048), the amino acid change might influence a class switch to increase serum IgA levels, resulting in a higher risk of IgA nephropathy.     

Rearrangement and expression of the immunoglobulin μ-chain gene in human myeloid cells

Immunoglobulin （Ig）, a characteristic marker of B cells, has been reported to be expressed in epithelial cells, with a suggested role in their growth and survival. We have previously reported that IgG heavy chain is expressed in acute myeloid leukemia （AML）, but not in the monocytes or neutrophils from patients with non-hematopoietic neoplasms or healthy controls. In the present study, we assessed IgM heavy chain expression and repertoire in human myeloid cells. We detected VHIzDJHp- rearrangement and expression in 7/7 AML cell lines, 7/14 primary myeloblasts from AML patients, and interestingly, 8/20 monocytes and 3/20 neutrophils from patients with non-hematopoietic neoplasms and healthy individuals. We also found evidence of somatic hypermutation of the variable （V） gene segments in AML-derived IgM gene rearrangements but not in IgM from monocytes or neutrophils from patients with non-hematopoietic neoplasms and healthy individuals. Furthermore, IgM VH~DJH~ gene rearrangements in AML cell lines, primary myeloblasts, and monocytes and neutrophils from patients with non-hematopoietic neoplasms showed a restricted V usage and repertoire, whereas the VH~DJH~ gene rearrangements in monocytes and neutrophils from healthy individuals displayed more diversity. Anti-human IgM inhibited cell proliferation, but did not induce apoptosis in AML cell lines. Our findings suggest that AML-derived IgM might be a novel AML-related molecule that is involved in leukemogenesis and AML progression and might serve as a useful molecular marker for designing targeted therapy and monitoring minimal residual disease.     

Analysis of MAT1A gene mutations in a child affected with simple hypermethioninemia北大核心CSCD

Objective To detect potential mutations of MATl A gene in a child suspected with simple hypermethioninemia by MS/MS neonatal screening. Methods Clinical data of the child was collected. Genomic DNA was extracted by astandard method and subjected to targeted sequencing using an Ion Ampliseq™ Inherited Disease Panel. Detectedmutations were verified by Sanger sequencing. Results The child showed no clinical features except evaluatedmethionine. A novel compound mutation of the MATIA gene, i. e. , c. 345delA and c. 529C>T, was identified in the child. His father and mother were found to be heterozygous for the c. 345delA mutation and c. 529C > Tmutation, respectively. Conclusion The compound mutation c. 345delA and c. 529C>T of the MATIA gene probably underlie the disease in the child. The semi-conductor sequencing has provided an important means for thediagnosis of hereditary diseases.     

Multiple-copy integration of the α-galactosidase gene from Cyamopsis tetragonoloba into the ribosomal DNA of Kluyveromyces lactis

Summary We have developed a vector system for high-copy-number integration into the ribosomal DNA of the yeast Kluyveromyces lactis. This system is analogous to the pMIRY-system developed for Saccharomyces cerevisiae. Plasmids containing a portion of K. lactis rDNA for targeted homologous recombination, as well as the S. cerevisiae TRP1 gene with various promoter deletions, were constructed and, after transformation to K. lactis, analyzed for both copy number and stability. These plasmids were found to be present in about 60 copies per cell and were stably maintained during growth under non-selective conditions. Using this vector system, we expressed a fusion construct containing the S. cerevisiae GAL7 promoter, the SUC2 (invertase) signal sequence and the gene coding for -galactosidase from the plant Cyamopsis tetragonoloba. Although the maximum copy number of these integrated plasmids was only about 15, we nevertheless obtained a high level of -galactosidase production (250 mg/l) with a secretion efficiency of about 95%. When compared to extrachromosomal K. lactis vectors containing the same fusion construct, the multicopy integrants showed a much higher -galactosidase production level and a considerably higher stability under non-selective conditions.     

Are single nucleotide polymorphisms of the immunoglobulin A Fc receptor gene associated with allergic asthma?

BACKGROUND: Eosinophils are important components of allergic inflammation. The immunoglobulin A (IgA) Fc receptor (FcalphaRI), encoded by the FCAR gene, is a possible candidate for eosinophil activation at mucosal surfaces, where IgA is abundant. Both elevated cell surface expression of FcalphaRI and increased avidity for IgA were described on eosinophils from allergic subjects. The aim of our study was to examine the possible association of FCAR gene polymorphisms with allergic asthma. METHODS: We screened three regions of the FCAR gene: (1) the promoter region, (2) exon 3, encoding the first extracellular domain (EC1), and (3) exon 5, coding for the transmembrane and cytoplasmic domain, for new and published polymorphisms using a sensitive temperature gradient gel electrophoresis technique and compared their frequencies in 112 patients diagnosed with allergic asthma and 100 healthy controls. RESULTS: Six polymorphisms, including two novel ones, were detected. No differences between patients and controls were found in the distribution of any of these polymorphisms. CONCLUSION: FcalphaRI polymorphism does not seem to be a risk factor in allergic asthma. Nevertheless, this is the first report on the distribution of 6 single nucleotide polymorphisms of the FCAR gene in a human population and the first study on FCAR polymorphism in allergic asthma.     

Genotype–phenotype correlations analysis of mutations in the phenylalanine hydroxylase (PAH) gene

The aims of our research were to define the genotype–phenotype correlations of mutations in the phenylalanine hydroxylase (PAH) gene that cause phenylketonuria (PKU) among the Israeli population. The mutation spectrum of the PAH gene in PKU patients in Israel is described, along with a discussion on genotype–phenotype correlations. By using polymerase chain reaction/denaturing high-performance liquid chromatography (PCR/dHPLC) and DNA sequencing, we screened all exons of the PAH gene in 180 unrelated patients with four different PKU phenotypes [classic PKU, moderate PKU, mild PKU, and mild hyperphenylalaninemia (MHP)]. In 63.2% of patient genotypes, the metabolic phenotype could be predicted, though evidence is also found for both phenotypic inconsistencies among subjects with more than one type of mutation in the PAH gene. Data analysis revealed that about 25% of patients could participate in the future in (6R)-l-erythro-5, 6, 7, 8-tetrahydrobiopterin (BH₄) treatment trials according to their mutation genotypes. This study enables us to construct a national database in Israel that will serve as a valuable tool for genetic counseling and a prognostic evaluation of future cases of PKU.     

Lysosomal storage disease in the brain: mutations of the β-mannosidase gene identified in autosomal dominant nystagmus

PurposeGenetic etiology of congenital/infantile nystagmus remains largely unknown. This study aimed to identify genomic mutations in patients with infantile nystagmus and an associated disease network.MethodsPatients with inherited and sporadic infantile nystagmus were recruited for whole-exome and Sanger sequencing. β-Mannosidase activities were measured. Gene expression, protein–protein interaction, and nystagmus-associated lysosomal storage disease (LSD) genes were analyzed.ResultsA novel heterozygous mutation (c.2013G>A; p.R638H) of MANBA, which encodes lysosomal β-mannosidase, was identified in patients with autosomal-dominant nystagmus. An additional mutation (c.2346T>A; p.L749H) in MANBA was found by screening patients with sporadic nystagmus. MANBA was expressed in the pretectal nucleus of the developing midbrain, known to be involved in oculomotor and optokinetic nystagmus. Functional validation of these mutations demonstrated a significant decrease of β-mannosidase activities in the patients as well as in mutant-transfected HEK293T cells. Further analysis revealed that nystagmus is present in at least 24 different LSDs involving the brain.ConclusionThis is the first identification of MANBA mutations in patients with autosomal-dominant nystagmus, suggesting a new clinical entity. Because β-mannosidase activities are required for development of the oculomotor nervous system, our findings shed new light on the role of LSD-associated genes in the pathogenesis of infantile nystagmus.     

The suppression of the μ rhythm during the creation of imagery representation of movement

The aim of this study was to answer the following question: are there differences between the attenuation of μ rhythms, recorded with EEG in the parietal area during observation of movement and the creation of its imaginative representation? In addition, we checked the extent to which the μ rhythm suppression depends on whether the observed and the imagined movement is performed by a human or is artificial. As a result of the experiment a significant difference in μ rhythm suppression between the conditions “Observation,” “Imagery,” and “White noise” was recorded. It did not matter whether the motion was carried out by a human being or performed by a machine. The results are discussed in the light of findings which relate to the mirror neuron system.     

When and how should new technology be introduced into the IVF laboratory?

There are many examples in assisted reproduction technology, where new technology and methods have been introduced into the clinical setting without appropriate development and evidence-based medicine to show that the procedure is safe and beneficial to the patient. Examples include preimplantation genetic screening, assisted hatching, in vitro maturation, blastocyst transfer and vitrification. Changes to culture media composition, stimulation regimes and laboratory protocols are also often established internationally without adequate validation. More recently, novel equipment that needs to be validated before it enters routine clinical use is being developed for IVF. With technologies such as producing gametes from stem cells around the corner, it is vital to ensure that the necessary research and development is conducted before bringing new techniques into clinical practice. Ideally, this should include preliminary work on animal models, such as mice/rats/rabbits/larger mammals, followed by studies on human embryos donated for research and finally well-designed RCTs with a follow up of all children born from the procedure. If such preliminary studies are not performed and published, it is possible that technology bringing no clinical benefit or leading to adverse health outcomes in the children born by these practices may be introduced. All IVF clinics need to consider the safety and efficacy of new technologies before introducing them.     

Identification of three missense mutations in the peroxisome proliferator-activated receptor α gene in Japanese subjects with maturity-onset diabetes of the young

We screened the protein-coding region of the peroxisome proliferator-activated receptor α gene (PPARA) and the flanking intron sequences for mutations in 57 unrelated Japanese subjects with maturity-onset diabetes of the young (MODY). We found three missense mutations, designated P22R, D140Y, and V227A. The D140Y and V227A mutations were found at similar frequencies in MODY and in nondiabetic Japanese subjects, suggesting that they were unlikely to be pathogenic. The P22R mutation was found in a single female subject with MODY. Two of her four siblings, all of whom were diagnosed with diabetes before age 35 years, also inherited the P22R mutation. However, two other diabetic siblings had not inherited the mutant allele, implying that the P22R mutation was not the cause of MODY in this family. Variation in the coding region of PPARA is unlikely to be a major cause of MODY in Japanese people. Received: January 11, 2001 / Accepted: February 9, 2001     

Two mutations of the Gsα gene in two Japanese patients with sporadic pseudohypoparathyroidism type Ia

Pseudohypoparathyroidism Ia (PHP-Ia), is an inherited disease with clinical hypoparathyroidism caused by parathyroid hormone resistance (PTH), and shows the phenotype of Albright hereditary osteodystrophy (AHO), including short stature, obesity, round face, brachydactyly, and subcutaneous ossification. This disease is caused by mutation that inactivates the α-subunit of Gs, the stimulatory regulator of adenylyl cyclase. Here, a novel frameshift mutation (delG at codon 88) in exon 4, and a missense mutation (R231H) in exon 9 of the Gsα gene were identified in two Japanese patients with sporadic PHP-Ia. Deletion of a G in exon 4 at codon 88 in the first patient produced a premature stop codon, resulting in the truncated protein. The second patient had a previously reported R231H mutation. Because this amino acid is located in a region, switch 2, that is thought to interact with the βγ subunit of Gsα protein, this mutation may impair Gs protein function. We report here one novel Gsα mutation, and note that mutations in Japanese patients with PHP-Ia are probably heterogeneous. Received: November 27, 2000 / Accepted: March 21, 2001     

Does the genotype of HHT patients with mutations of the ENG and ACVRL1 gene correlate to different expression levels of the angiogenic factor VEGF?

The aim of this study was to determine in what way HHT (hereditary hemorrhagic telangiectasia) patients with mutations for the endoglin (ENG) or activin receptor-like kinase 1 (ACVRL1) gene show different expression levels of the angiogenic factor VEGF (vascular endothelial growth factor) by correlating VEGF to the HHT genotype. In 18 HHT patients, who were screened for ENG and ACVRL1 gene mutations and 25 healthy controls the VEGF plasma level as well as the VEGF tissue expression were determined by ELISA technique and cryostat sections of the nasal mucosa. In general, the VEGF plasma levels as well as the VEGF tissue expression were significantly higher in HHT patients compared to healthy controls. However, the correlation of VEGF to the HHT genotype did not show any significant differences, i.e. the VEGF plasma levels as well as the VEGF tissue expression in HHT patients with ENG gene mutations did not differ significantly to those of HHT patients with ACVRL1 gene mutations or mutations for both the genes. In spite of the fact that the angiogenic factor VEGF seems to play an important role in the pathogenesis of HHT, it cannot serve as a specific diagnostic screening marker. These results underline the importance and necessity of molecular analyses in HHT patients.     

Allelic exclusion of immunoglobulin gene rearrangement and expression: why and how?

Since the discovery of the allelic exclusion of immunoglobulin (Ig) gene expression by Pervis in the 1960s [J. Exp. Med. 122 (1965) 853], much attention has been focused on its mechanism. Much less attention has been paid, however, to the question of why B cells demonstrate such unusual genetic regulation of antigen receptor gene expression. A large body of literature implicates the Ig gene products as feedback regulators of their own genetic rearrangement [Adv. Immunol.78 (2001)169; Science 236 (1987)816]. While a role for Ig gene products in the regulation of V(D)J recombination is beyond debate, it is extremely unlikely that such a feedback mechanism would be fast enough to avoid occasional near-simultaneous rearrangement of allelic loci leading to dual receptor gene expression. This review will suggest an hypothesis to answer the 'why bother' aspect of allelic exclusion and then go on to propose a mechanism, distinct from feedback regulation, which may contribute to the allelic exclusion of Ig gene expression.     

Formation and repair of DNA lesions in the p53 gene: Relation to cancer mutations?

The number and diversity of mutations in the p53 mutation data base provides indirect evidence that implicates environmental mutagens in human carcinogenesis. The p53 gene has a large mutational target size; more than 280 out of 393 amino acids are found mutated in tumors. We argue that there is possibly a limited involvement of selection for specific mutations in the central domain of the protein, and that the distribution of DNA damage along the p53 gene caused by environmental carcinogens can be correlated with the mutational spectra, i.e., hotspots and types of mutations, of certain cancers. This concept has been validated by experiments with sunlight and the cigarette smoke component benzo[a]pyrene representing the polycyclic aromatic hydrocarbon class of carcinogens. The damage/repair data obtained for these mutagens can predict certain parameters of the mutational spectra including the distribution of hotspots in human nonmelanoma skin cancers and lung cancers from smokers. Future studies with suspected mutagens may help to implicate causative agents involved in other cancers, such as colon and breast cancer, where the exact carcinogen has not yet been identified but an environmental factor is suspected. Environ. Mol. Mutagen. 31:197–205, 1998 © 1998 Wiley-Liss, Inc.