De novo mutations in the BMPR2 gene in patients with heritable pulmonary arterial hypertension.

A substantial proportion of patients with pulmonary arterial hypertension (PAH) have mutations in the Bone Morphogenetic Protein Receptor type-2 (BMPR2) gene. PAH due to BMPR2 mutations is inherited as an autosomal dominant trait with several unique features, including a wide variety of mutations, reduced penetrance, a skewed gender ratio, variable expressivity and genetic anticipation. To address the genetic background of these unique features of BMPR2 mutation, we conducted a systematic analysis of 15 PAH families with BMPR2 mutation. The exonic protein coding sequence of BMPR2 was amplified by polymerase chain reaction and the products were sequenced directly to detect point mutations in BMPR2. Parental identification was carried out to confirm the parental relationship using multiplex 15 loci analysis. Combining mutation detection in family members with parental identification, we described three cases of de novo mutation in the BMPR2 gene by different modes in a PAH family. These de novo mutations may account for the wide variety of mutations in BMPR2. Taken together with the juvenile onset of the disease, there is possibly some balance of de novo mutations and untransmittable mutations which keeps the frequency of PAH low in the general population.

Overexpression of an antisense RNA, ArrS, increases the acid resistance of Escherichia coli.

The antisense RNA ArrS is complementary to a sequence in the 5' untranslated region of the gadE T3 mRNA, the largest transcript of gadE, which encodes a transcriptional activator of the glutamate-dependent acid resistance system in Escherichia coli. Expression of arrS is strongly induced during the stationary growth phase, particularly under acidic conditions, and transcription is dependent on σ(S) and GadE. The aim of the present study was to clarify the role of ArrS in controlling gadE expression by overexpressing arrS in E. coli. The results showed a marked increase in the survival of arrS-overexpressing cells at 2 h after a shift to pH 2.5. This was accompanied by increased expression of gadA, gadBC and gadE. The level of gadE T3 mRNA decreased markedly in response to arrS overexpression, and was accompanied by a marked increase in gadE mRNA T2. T2 mRNA had a monophosphorylated 5' terminus, which is usually found in cleaved mRNAs, and no T2 mRNA was observed in an RNase III-deficient cell strain. In addition, T2 mRNA was not generated by a P3-deleted gadE-luc translational fusion. These results suggest strongly that T2 mRNA is generated via the processing of T3 mRNA. Moreover, the T2 mRNA, which was abundant in arrS-overexpressing cells, was more stable than T3 mRNA in non-overexpressing cells. These results suggest that overexpression of ArrS positively regulates gadE expression in a post-transcriptional manner.

Alu-mediated nonallelic homologous and nonhomologous recombination in the BMPR2 gene in heritable pulmonary arterial hypertension.

PURPOSE: The purpose of this study was to undertake thorough genetic analysis of the bone morphogenetic protein type 2 receptor (BMPR2) gene in patients with pulmonary arterial hypertension. METHODS: We conducted a systematic analysis for larger gene rearrangements together with conventional mutation analysis in 152 pulmonary arterial hypertension patients including 43 patients diagnosed as having idiopathic pulmonary arterial hypertension and 10 diagnosed as having familial pulmonary arterial hypertension. RESULTS: Analysis of the BMPR2 gene revealed each of the four kinds of nonsense and frameshift mutations, one missense mutation, one splice-site mutation, and two types of exonic deletion. For cases in which exons 1-3 were deleted, the 5' and 3' break points were located in the AluY repeat sequences in the 5' side of the adjacent NOP58 gene and in the AluY repeat sequences in intron 3, suggesting an AluY-mediated nonallelic homologous recombination as the mechanism responsible for the deletion. For the case in which exon 10 was deleted, nonhomologous recombination took place between the AluSx site in intron 9 and a unique sequence in intron 10. CONCLUSION: Exonic deletions of BMPR2 account for at least part of BMPR2 mutations associated with heritable pulmonary arterial hypertension in Japan, as previously reported in other populations. One of our cases was mediated via Alu-mediated nonallelic homologous recombination and another was mediated via nonhomologous recombination.

A novel break point of the BMPR2 gene exonic deletion in a patient with pulmonary arterial hypertension.

The presence of genetic rearrangements of bone morphogenetic protein type 2 receptor (BMPR2) was identified in pulmonary arterial hypertension (PAH) patients as the deletion or duplication of one or more exons of the gene. We recently investigated the deletion break points in exonic deletions of BMPR2 in two Japanese familial cases with PAH, and found that these were Alu-mediated via either non-allelic homologous recombination or non-homologous recombination. We herein report the third case of exonic deletion, which was in a 25-year-old female PAH patient with a deletion of BMPR2 exon 3. The break point in this case was not located in an Alu sequence. The 5'- and 3'-break point maps between the inverted Alu sequences in intron 2 and in exon 3, respectively, resulted in a 759-bp deletion. This novel exonic deletion in this PAH case may be a unique and non-recurrent rearrangement, and appears to be of a different size from that in other patients.

Transcription of an antisense RNA of a gadE mRNA is regulated by GadE, the central activator of the acid resistance system in Escherichia coli.

6H57, a 69-nucleotide-long small RNA, was isolated in shotgun cloning using an RNA sample derived from early stationary-phase cells. The 6H57 gene is located in a 798-bp intergenic region between two acid resistance-related genes, hdeD and gadE, and is encoded on the strand opposite these flanking genes. In this study, we carried out stringent Northern blotting to determine target mRNAs of 6H57. A band approximately 1300 nucleotides in length was detected using a probe containing a partial sequence of 6H57 and was confirmed to be the gadE mRNA T3, which has a 566-nucleotide-long 5' untranslated region. These results show that 6H57 is an antisense RNA of gadE mRNA T3 and can base pair with a -380 to -312 region of the translation initiation site of gadE. We analyzed the transcription of 6H57 and showed that 6H57 transcription is dependent on GadE in the early stationary phase. Furthermore, 6H57 is induced in the exponential growth phase by an acid stimulus of pH 5.5. A 189-bp DNA fragment containing the upstream region of the 6H57 gene showed clear promoter activities in these culture conditions. These results suggest that 6H57 plays several roles in acid resistance, and we renamed it acid resistance-related small RNA.

Poor outcomes in carriers of the RNF213 variant (p.Arg4810Lys) with pulmonary arterial hypertension.

BACKGROUND: A variant of c.14429G>A (p.Arg4810Lys, rs112735431) in the ring finger protein 213 gene (RNF213; NM_001256071.2) has been recently identified as a risk allele for pulmonary arterial hypertension (PAH). PAH can be added as a new member of RNF213-associated vascular diseases, which include Moyamoya disease and peripheral pulmonary stenosis. Our aim was to identify the clinical features and outcomes of PAH patients with this variant. METHODS: Whole-exome sequencing was performed in 139 idiopathic (or possibly heritable) PAH patients. RESULTS: The RNF213 p.Arg4810Lys variant was identified in a heterozygous state in 11 patients (7.9%). Time-course changes in hemodynamics after combination therapy in the patients with the RNF213 p.Arg4810Lys variant were significantly poorer compared with those carrying the bone morphogenic protein receptor type 2 (BMPR2) mutation (n = 36) (comparison of changes in mean pulmonary arterial pressure, p = 0.007). The event-free rate of death or lung transplantation was significantly poorer in RNF213 p.Arg4810Lys variant carriers than in BMPR2 mutation carriers (5-year event-free rate since the introduction of prostaglandin I2 infusion, 0% vs 93%, respectively; p < 0.001). CONCLUSIONS: Idiopathic PAH patients with the RNF213 p.Arg4810Lys variant are associated with poor clinical outcomes even in recent times. Earlier consideration of lung transplantation might be required for RNF213 p.Arg4810Lys variant carriers who are developing PAH. Documentation of the RNF213 p.Arg4810Lys variant, as well as already known pathogenic genes, such as BMPR2, can provide clinically relevant information for therapeutic strategies, leading to a personalized approach for the treatment of PAH.

8-hydroxydeoxyguanosine generated in the earthworm Eisenia fetida grown in metal-containing soil.

Heavy metal pollution of soil causes biological problems, such as mutagenicity to living organisms, including human beings. However, few methods have been developed to assess metal mutagenicity in soil. To avoid metal mutagenicity, an adequate bio-monitoring method is required. In the present study, to determine if the analysis of oxidative DNA damage generated in the earthworm is a useful bio-monitoring method for soil mutagenicity, the accumulation of 8-hydroxydeoxyguanosine (8-OH-dG), a major form of oxidative DNA damage, in Eisenia fetida (Savigny, 1826) treated with cadmium chloride (CdCl2) or nickel chloride (NiCl2) was analyzed. E. fetida was treated with Cd (10 or 200 microg/g soil) or Ni (10 or 200 microg/g soil) for 1, 2, and 3 weeks or 3 months. After metal exposure, the metal concentration in E. fetida was analyzed by atomic absorption spectrometry and the 8-OH-dG accumulated in E. fetida was analyzed by HPLC analyses and immunohistochemistry. Atomic absorption spectrometry revealed that Cd, but not Ni, accumulated within E. fetida. The 8-OH-dG levels in the DNA of E. fetida treated with Cd for 3 months were significantly higher than those in control E. fetida. Moreover, immunohistochemical analyses revealed that positive signals for 8-OH-dG accumulation in seminal vesicles were detected only in E. fetida treated with 10 microg of Cd for 3 months. Although some points remain unresolved, a bio-monitoring system analyzing the DNA damage generated in the earthworm might be useful for the assessment of the mutagenicity of soil contaminated with various heavy metals, such as Cd.

Valosine-containing proteins (VCP) in an annelid: identification of a novel spermatogenesis related factor.

Two cDNAs similar to mammalian valosine-containing proteins (VCPs) were isolated from the common lumbricid earthworm Eisenia fetida (Savigny, 1826). The primary sequences, referred to as eVCP-1 and eVCP-2, display a similarity of 74%. Despite of the variable C-termini, both eVCPs have a conserved intron/exon organization spanning 14 kb, which is also conserved to their mammalian counterparts. Although this finding strongly suggests VCPs have a common ancestral origin, phylogenetic analysis predicts that eVCP-2 may be distinct. An investigation by reverse transcription-polymerase chain reaction (RT-PCR) revealed that, whilst evcp-1 was ubiquitously expressed during all developmental stages, evcp-2 was specifically expressed in the anterior segments of sexually mature earthworms. In situ hybridization clearly demonstrated that evcp-2 is expressed in the seminal vesicles, the location of spermatogenesis, and more precisely within the cytophores surrounded by secondary spermatocytes or spermatids. Taken together, this evidence leads to the notion that eVCP-2 is a likely component involved in the final modulation of spermatogenesis.

Keratinocyte growth inhibition by high-dose epidermal growth factor is mediated by transforming growth factor beta autoinduction: a negative feedback mechanism for keratinocyte growth.

The epidermal growth factor receptor and its ligands initiate a major signaling pathway that regulates keratinocyte growth in an autocrine manner. It is well known that high doses of epidermal growth factor receptor ligands inhibit keratinocyte growth. Recently, signal transducers and activators of transcription 1-dependent p21Waf1/Cip1 induction were reported to be involved in high-dose epidermal growth factor-dependent cell growth arrest in the A431 squamous cell carcinoma cell line; however, transfection of dominant-negative signal transducers and activators of transcription 1 adenovirus vector did not block epidermal growth factor-induced growth inhibition in normal human keratinocytes. As transforming growth factor beta is a potent inhibitor of keratinocyte proliferation, we hypothesized that transforming growth factor beta contributes to epidermal growth factor-mediated keratinocyte growth inhibition. Epidermal growth factor concentrations of 10 ng per ml enhanced transforming growth factor beta1 mRNA expression from 3 to 6 h poststimulation. Enzyme-linked immunosorbent assay analysis detected 150 pg per ml of transforming growth factor beta1 in the culture medium of keratinocytes incubated with 10 and 100 ng per ml epidermal growth factor, whereas 0.1 and 1.0 ng per ml epidermal growth factor slightly enhance transforming growth factor beta1 production. Epidermal growth factor (100 ng per ml) upregulated luciferase activity of p3TP-lux, which contains three tandem transforming growth factor beta-Smad signaling responsive elements, 6-fold compared with unstimulated cells. The epidermal growth factor-dependent induction of p3TP-lux luciferase activity was disrupted by transfection of the dominant negative form of transforming growth factor beta type I receptor adenovirus vector (AxdnALK5), which suggests that epidermal growth factor-induced transforming growth factor beta acts in an autocrine manner in keratinocytes. Moreover, transfection of AxdnALK5 completely blocked the growth inhibition induced by 100 ng per ml of epidermal growth factor in normal keratinocytes. These data demonstrate that an autocrine transforming growth factor beta1-ALK5 pathway is a negative feedback mechanism for epidermal growth factor-induced normal human keratinocyte growth.

Evolution of the cytoplasmic and mitochondrial phosphagen kinases unique to annelid groups.

Creatine kinase (CK) is a member of a group of phosphoryl transfer enzymes called phosphagen kinases that play a key role in cellular energy transactions in animals. Three CK isoform gene families are known-cytoplasmic CK (CK), flagellar CK (fCK), and mitochondrial CK (MiCK). Each of the isoforms has a unique gene structure (intron/exon organization). A broad array of other phosphagen kinases is present in animals. Some of these enzymes are found only in annelids and closely related groups including glyocyamine kinase (GK), lombricine kinase (LK), taurocyamine kinase (TK), and a unique arginine kinase (AK) restricted to annelids. Phylogenetic analyses of these annelid phosphagen kinases indicate that they appear to have evolved from a CK-like ancestor. To gain a greater understanding of the relationship of the CK isoforms to the annelid enzymes, we have determined the intron/exon organization of the genes for the following phosphagen kinases: Eisenia LK, Sabellastarte AK, and Arenicola mitochondrial TK (MiTK). Analysis of genomic database for the polychaete Capitella sp. yielded two putative LK genes [cytoplasmic LK and mitochondrial LK (MiLK)]. The intron/exon organization of these genes was compared with available data for cytoplasmic and mitochondrial CKs, and an annelid GK. Surprisingly, these annelid genes, irrespective of whether they are cytoplasmic (LK, AK, and GK) or mitochondrial (MiTK and MiLK), had the same 8-intron/9-exon organization and were strikingly similar to MiCK genes sharing seven of eight splice junctions. These results support the view that the MiCK gene is basal and ancestral to the phosphagen kinases unique to annelids.

DMRT-1 expression during NEC8 human embryonic carcinoma cell differentiation.

To elucidate the relationship between dsx and mab-3 related transcription factor 1 (Dmrt-1) and differentiation, alteration in mRNA levels during differentiation of NEC8 human embryonic carcinoma cells was investigated. After stimulation with 50 nM phorbol 12-myristate 13-acetate (PMA), the cells differentiated into cells with mesenchymal characteristics and upregulated Dmrt-1 mRNA, possibly through the protein kinase C/mitogen-activated protein kinase/activated protein-1 signaling pathway. Conversely, knockdown of Dmrt-1 by small interfering RNA resulted in cell morphology that was different from that after PMA treatment. These results indicated that Dmrt-1 expression was apparently associated with the differentiation of NEC8, and this cell line may be a helpful in vitro tool to clarify the role of Dmrt-1 in the differentiation process.