Population frequency and age of c.806 C>T mutation in CYB5R3 gene as cause of recessive congenital methemoglobinemia in Yakutia

Type-I recessive congenital methemoglobinemia (RCM) is a rare autosomal disease characterized by a deficiency of the soluble form of nicotineamide adenine dinucleotide (NADH)-cytochrome b5 reductase (b5R) and clinically manifests as cyanosis of skin and mucous membranes. In the Russian Federation, type-I RCM is widely disturbed in Yakutia due to the local founder effect. The molecular genetics cause of type-I RCM in Yakutia is mutation c.806C>T in the CYB5R3 gene. In this work we used 13 polymorphic markers, which flanking the CYB5R3 gene to establish the founder haplotype. The age of the mutation was estimated as about 285 ± 135 years. In this work, we have evaluated the frequency of the c.806C>T mutation in Yakutia, which averaged 55 : 1000 Yakuts. The calculated frequency of disease was 1: 1250 Yakuts.     

Enzymopenic hereditary methemoglobinemia: a clinical/biochemical classification

Recessively inherited NADH-cytochrome B5 reductase deficiency, when present in the homozygous or doubly heterozygous form, is manifested by two different clinical presentations, depending on the nature and cellular distribution of the mutant enzyme. The observations supporting a clinical and biochemical classification of enzymopenic hereditary methemoglobinemia are summarized. Type I, with deficiency demonstrable only in the erythrocytes, presents as uncomplicated, benign methemoglobinemia. Type II, generalized cytochrome B5 deficiency demonstrable in all of the tissues that have been examined, is accompanied by severe, lethal, progressive neurological disability, in addition to methemoglobinemia. Type III deficiency is limited to hematopoietic cells and resembles Type I clinically. Type IV, also clinically like Type I, is associated with deficiency of the cofactor, cytochrome B5. Except for Type IV, the different types appear to be the result of mutations in paired alleles of a gene on chromosome 22 that affect the catalytic activity or stability of the cytochrome B5 reductase.     

Soluble and microsomal forms of NADH-cytochrome beta 5 reductase from human placenta. Similarity with NADH-methemoglobin reductase from human erythrocytes.

In congenital methemoglobinemia associated with mental retardation a generalized deficiency of NADH-cytochrome beta 5 reductase (NADH : ferricytochrome beta 5 oxidoreductase, EC 1.6.2.2) has been found in soluble extracts of red blood cells, as well as in deoxycholate-treated extracts of leukocytes, muscle, liver and fibroblasts (Leroux et al. (1975) Nature 258, 619-620). In the present study the relationship between the microsomal (I) and the soluble (II) NADH-cytochrome beta 5 reductase was investigated, using human placenta as a source of enzyme. Both forms were compared to the human red-cell soluble NADH-methemoglobin reductase (III) and NADH-cytochrome beta 5 reductase (IV). The four entities exhibited great immunological similarities. It is concluded that the three soluble enzymes (II, III and IV) are identical. The detergent-solubilized microsomal NADH-cytochrome beta 5 reductase (I) is immunologically very similar to the soluble enzymes, but presents distinct features possibly due to the presence of a hydrophobic part.     

Clinical and biological forms of cytochrome b5 reductase deficiency]

Twenty-four personal cases of recessive congenital methemoglobinemia (RCM) due to cytochrome b5 reductase deficiency are analysed. They can be divided into two categories: 1) RCM type I, in which cyanosis is the single clinical symptom; 2) RCM type II in which cyanosis is associated with severe mental retardation and bilateral athetosis. The enzyme deficiency is restricted to the red cell soluble cytochrome b5 reductase in RCM type I, whereas in the type II form the enzyme defect is generalized to all tissues, involving both the soluble and the microsomal forms of cytochrome b5 reductase. Different mutations occurring at the same locus might account for this heterogeneity. However the mechanism of brain damage in case of generalized deficiency of cytochrome b5 reductase is still unknown.     

A Search for Mutations in the DIA1 Gene in Case of Hereditary Methemoglobinemia Type I in the Yakut Population

In the patients with enzymopenic hereditary methemoglobinemia type I, a disease widely distributed on the territory of Yakutia, a search for the mutations in exons 3 and 4 of the DIA1 gene encoding NADH-cytochrome b5 reductase was carried out. It was shown that Yakut patients have none of three missence mutations, Arg57Gln, Leu72Pro, and Val105Met, described in case of this disease in the neighboring populations, Chinese and Japanese, inhabiting the territories south of Yakutia.     

Fifty mosses on five trees: comparing phylogenetic information in three types of non-coding mitochondrial DNA and two chloroplast loci

Given the frequent genomic recombinations in plant mitochondrial DNA, intergenic regions of this organelle genome had so far not been considered as loci of potential phylogenetic information. Based on the recent evidence that an evolutionary ancient mitochondrial nad5-nad4 gene continuum is conserved in bryophytes we have compiled a dataset for a phylogenetically wide sampling of 50 mosses covering this intergenic region. The length of the intergenic region was generally in the range of 585 (Diphyscium) to 646 bp (Tomentypnum) with rare exceptions, for example all Polytrichales taxa showing a 200 bp deletion as an apparent synapomorphy of this order. Phylogenetic information in the novel marker sequence was compared with that of a nad5 gene region containing a group I intron and a nad2 gene region containing a group II intron as well as with two widely sampled chloroplast data sets, rbcL and rps4. Indel evolution in the three types of non-coding mitochondrial sequences is obviously more taxon-dependent than locus-dependent, indicating lineage-specific insertion/deletion rates. For example, larger sequence deletions are a general feature in Schistostega and Tetraphis. Although confidence for particular nodes in the phylogeny was found to vary among data sets, gene trees were essentially without conflict with respect to well-supported ones and add up in information towards a reasonably well-resolved moss phylogeny. However, while a consensus on the latter is clearly emerging, sufficient confidence is still lacking for the first dichotomies among the arthrodontous mosses leading into subclasses Bryidae, Dicranidae and Funariidae and the relative placement of nematodontous mosses (Polytrichales and Tetraphidales) on the backbone of early moss phylogeny.     

The structure of the S127P mutant of cytochrome b5 reductase that causes methemoglobinemia shows the AMP moiety of the flavin occupying the substrate binding site

Methemoglobinemia, the first hereditary disease to be identified that involved an enzyme deficiency, has been ascribed to mutations in the enzyme cytochrome b(5) reductase. A variety of defects in either the erythrocytic or microsomal forms of the enzyme have been identified that give rise to the type I or type II variant of the disease, respectively. The positions of the methemoglobinemia-causing mutations are scattered throughout the protein sequence, but the majority of the nontruncated mutants that produce type II symptoms occur close to the flavin adenine dinucleotide (FAD) cofactor binding site. While X-ray structures have been determined for the soluble, flavin-containing diaphorase domains of the rat and pig enzymes, no X-ray or NMR structure has been described for the human enzyme or any of the methemoglobinemia variants. S127P, a mutant that causes type II methemoglobinemia, was the first to be positively identified and have its spectroscopic and kinetic properties characterized that revealed altered nicotinamide adenine dinucleotide hydride (NADH) substrate binding behavior. To understand these changes at a structural level, we have determined the structure of the S127P mutant of rat cytochrome b(5) reductase to 1.8 A resolution, providing the first structural snapshot of a cytochrome b(5) reductase mutant that causes methemoglobinemia. The high-resolution structure revealed that the adenosine diphosphate (ADP) moiety of the FAD prosthetic group is displaced into the corresponding ADP binding site of the physiological substrate, NADH, thus acting as a substrate inhibitor which is consistent with both the spectroscopic and kinetic data.     

Association of the ACE-II genotype with the risk of nephrotic syndrome in Pakistani children

Nephrotic syndrome is a common pediatric glomerular disease associated with heavy proteinuria. Since, the angiotensin converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism is a putative genetic risk factor for NS, in this study, ACE (I/D) polymorphism was analyzed in 268 NS and 223 control samples by a PCR-based method. The genotypic and allelic frequencies were determined and the association between ACE I/D polymorphism and NS was evaluated. The frequency distribution of the II, ID and DD genotypes was 82 (30.6%), 128 (47.8%) and 58 (21.6%) in the NS patients and 9 (4.0%), 171 (76.7%) and 43 (19.3%) in the control samples respectively. In the Pakistani pediatric NS population, the II genotypic and allelic frequencies were found to be significantly associated with the disease (OR = 6.755; C.I = 3-14.9). No significant association was found between this polymorphism and the response to standard steroid therapy. Thus, in contrast to reports from other parts of the world, the II genotype was found to be significantly associated with NS in the Indian and Malay populations and in the Pakistani population described here. To our knowledge, this is the first report from Pakistan describing the association of the ACE I/D polymorphism with pediatric NS. On the basis of these results, it is suggested that analysis of the ACE (I/D) polymorphism should be performed for the early diagnosis in the high risk NS patients in South Asia.     

Exonic point mutations in NADH-cytochrome B5 reductase genes of homozygotes for hereditary methemoglobinemia, types I and III: Putative mechanisms of tissue-dependent enzyme deficiency

We analyzed the NADH-cytochrome b5 reductase gene of hereditary methemoglobinemia type I and type III, by using PCR-related techniques. The mutation in type I is a guanine-to-adenine substitution in codon 57 of exon 3 of the NADH-cytochrome b5 reductase gene, and the sense of this codon is changed from arginine to glutamine. In type III the mutation is a thymine-to-cytosine transition in codon 148 of exon 5, causing leucine-to-proline replacement in type III. The former mutation abolishes the MspI recognition site. Homozygosity for the former mutation in a patient with type I was confirmed by restriction analysis of PCR-amplified fragments and by dot blot hybridization of amplified products with allele-specific oligonucleotide probes. The latter mutation generates a recognition site for MspI. Amplification of exon 5 by PCR followed by digestion with MspI revealed homozygosity for this mutation in patients with type-III. Putative mechanisms of tissue-dependent enzyme defects in hereditary methemoglobinemia are discussed.     

Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5

The nucleotide sequence of 1200 bp from the unique region of transposon Tn5 containing the neomycin phosphotransferase gene (neo) was determined, and the location of the neo gene was identified by deletion mutants in a translational reading frame of 792 bp. The derived gene product, an aminoglycoside 3′-phosphotransferase (APH) II, consists of 264 amino acid residues and has a calculated M_r of 29053. Its amino acid sequence shows sequence homologies to the APH type I enzyme coded for by transposon Tn903 (Oka et al., 1981).     

The MerE protein encoded by transposon Tn21 is a broad mercury transporter in Escherichia coli

In order to clarify the physiological role of the merE gene of transposon Tn21, a pE4 plasmid that contained the merR gene of plasmid pMR26 from Pseudomonas strain K-62, and the merE gene of Tn21 from the Shigella flexneri plasmid NR1 (R100) was constructed. Bacteria with plasmid pE4 (merR-o/p-merE) were more hypersensitive to CH₃Hg(I) and Hg(II), and took up significantly more CH₃Hg(I) and Hg(II), than the isogenic strain. The MerE protein encoded by pE4 was localized in the membrane cell fraction, but not in the soluble fraction. Based on these experimental results, we suggest for the first time that the merE gene is a broad mercury transporter mediating the transport of both CH₃Hg(I) and Hg(II) across the bacterial membrane.     

The conformation and thermal stability of soluble cytochrome P-450 and cytochrome P-450 incorporated into liposomal membranes

The α-helix content of the cytochrome P-450 incorporated into the liposomal membranes of either phosphatidylcholine or microsomal phospholipid insignificantly differed from that of the soluble one. The binding of both type I and type II substrates with cytochrome P-450 incorporated into phosphatidylcholine and microsomal phospholipid membranes did not change the conformation of the polypeptide chain. In contrast to this the type II substrates increased the α-helix content of soluble hemoprotein about 3–5%. Dithionite reduction of the cytochrome P-450 haem increased the degree of α spiralization up to 10% for soluble hemoprotein and up to 5% for the membrane-bound enzyme only. The investigation of the thermal stability of the soluble and liposomal forms of cytochrome P-450 showed that the enzyme incorporated into phospholipid vesicles was highly stable. The heating of the enzyme was followed by a slightly pronounced cooperative transition in contrast to the well-pronounced transition for the soluble cytochrome P-450. Hence, the incorporation of the soluble cytochrome P-450 into phospholipid bilayer does not result in significant change of α-helix content, but the increasing of rigidity and thermal stability of the membrane-bound hemoprotein molecule is observed.     

Molecular analysis of survival motor neuron (SMN) and neuronal apoptosis inhibitory protein (NAIP) genes of spinal muscular atrophy patients and their parents

We have assayed deletions of two candidate genes for spinal muscular atrophy (SMA), the survival motor neuron (SMN) and neuronal apoptosis inhibitory protein (NAIP) genes, in 101 patients from 86 Chinese SMA families. Deletions of exons 7 and 8 of the telomeric SMN gene were detected in 100%, 78.6%, 96.6%, and 16.7%, in type I, II, III, and adult-onset SMA patients, respectively. Deletion of exon 7 only was found in eight type II and one type III patient. One type II patient did not have a deletion of either exon 7 or 8. The prevalence of deletions of exons 5 and 6 of the NAIP gene were 22.5% and 2.4% in type I and II SMA patients, respectively. We also examined four polymorphisms of SMN genes and found that there were only two, SMN-2 and ^CBCD541-2, in Chinese subjects. In our study, analysis of the ratio of the telomeric to centromeric portion (T/C ratio) of the SMN gene after enzyme digestion was performed to differentiate carriers, normals, and SMA patients. We found the T/C ratio of exon 7 of the SMN gene differed significantly among the three groups, and may be used for carrier analysis. An asymptomatic individual with homozygous deletion of exons 7 and 8 of the SMN gene showed no difference in microsatellite markers in the SMA-related 5q11.2–5q13.3. In conclusion, SMN deletion in clinically presumed child-onset SMA should be considered as confirmation of the diagnosis. However, adult-onset SMA, a heterogeneous disease with phenotypical similarities to child-onset SMA, may be caused by SMN or other gene(s). Received: 13 November 1996 / Accepted: 13 May 1997     

Genetisches Modell der autosomal-rezessiv erblichen proximalen spinalen Muskelatrophie

Proximal spinal muscular atrophy (SMA) is one of the most common autosomal recessive diseases. According to the achieved milestones, SMA is divided into 3 groups: SMA types I–III. SMA is caused by mutations in the survival motor neuron 1 (SMN1) gene, which is located on chromosome 5. Wild type alleles usually have one or two SMN1 gene copies, disease alleles may show deletions, large scale deletions, or point mutations. The proposed genetic model is based on published data on SMA types I–III. The complex genetic model of SMA allows all parameters—even those which have not been assessed so far—to be calculated. The SMN1 allele frequencies included the following: normal allele b (1 copy of SMN1): ≈?0.9527; normal allele c (2 copies of SMN1): ≈?0.0362; deletion a (0 copies of SMN1): ≈?0.0104; point mutation d (1 copy of SMN1): ≈?0.0003; large scale deletion g (0 copies of SMN1): ≈?0.0004. The result is a gene frequency of approximately 1:90 and a carrier frequency of about 1:46.     

Adaptive mutation related to cellulose producibility in <Emphasis Type="Italic">Komagataeibacter medellinensis</Emphasis> (<Emphasis Type="Italic">Gluconacetobacter xylinus</Emphasis>) NBRC 3288

Gluconacetobacter xylinus (formerly Acetobacter xylinum and presently Komagataeibacter medellinensis) is known to produce cellulose as a stable pellicle. However, it is also well known to lose this ability very easily. We investigated the on and off mechanisms of cellulose producibility in two independent cellulose-producing strains, R1 and R2. Both these strains were isolated through a repetitive static culture of a non-cellulose-producing K. medellinensis NBRC 3288 parental strain. Two cellulose synthase operons, types I and II, of this strain are truncated by the frameshift mutation in the bcsBI gene and transposon insertion in the bcsCII gene, respectively. The draft genome sequencing of R1 and R2 strains revealed that in both strains the bcsBI gene was restored by deletion of a nucleotide in its C-rich region. This result suggests that the mutations in the bcsBI gene are responsible for the on and off mechanism of cellulose producibility. When we looked at the genomic DNA sequences of other Komagataeibacter species, several non-cellulose-producing strains were found to contain similar defects in the type I and/or type II cellulose synthase operons. Furthermore, the phylogenetic relationship among cellulose synthase genes conserved in other bacterial species was analyzed. We observed that the cellulose genes in the Komagataeibacter shared sequence similarities with the γ-proteobacterial species but not with the α-proteobacteria and that the type I and type II operons could be diverged from a same ancestor in Komagataeibacter.     

A search for mutations in the DIA1 gene in case of hereditary methemoglobinemia type I in the iakut population

In the patients with enzymopenic hereditary methemoglobinemia type I, a disease widely distributed on the territory of Yakutia, a search for the mutations in exons 3 and 4 of the DIA1 gene encoding NADH-cytochrome b5 reductase was carried out. It was shown that Yakut patients have none of three missence mutations, Arg57Gln, Leu72Pro, and Val105Met, described in case of this disease in the neighboring populations, Chinese and Japanese, inhabiting the territories south of Yakutia.     

Invertase activity and the kinetin-stimulated enlargement of detached radish cotyledons

Cytokinin treatment is known to promote expansion of light-grown excised radish (Raphanus sativus L. cv Crimson Giant) cotyledons. This expansion, at least in part, seems to be related to an increased accumulation of osmotically active reducing sugars. Kinetin treatment did not cause increased levels of isocitrate lyase activity over the controls, but stimulated increased levels of two invertase forms, designated types I and II. Type I was soluble and type II was insoluble after homogenization in 10 millimolar tris(hydroxymethyl)aminomethane-HCl (pH 7.0). Both types were soluble after homogenization in 300 millimolar NaCl. At low salt concentration, type II was retained on a diethylamioethyl-cellulose column and type I was not. Type II was then eluted from the column at high salt concentration. Types I and II exhibited pH optima of 5.3 and 4.3, Michaelis constants of 4.96 and 1.23 millimolar sucrose, and molecular weights of 65,000 and 57,000 daltons, respectively. The kinetin promotion of reducing sugar accumulation may be related to increased levels of the two invertase forms, but is probably not a result of direct cytokinin-stimulated glyoxysomal activity.     

Evidence for Contribution of Neutral Trehalase in Barotolerance of Saccharomyces cerevisiae

In yeast, trehalose accumulation and its hydrolysis, which is catalyzed by neutral trehalase, are believed to be important for thermotolerance. We have shown that trehalose is one of the important factors for barotolerance (resistance to hydrostatic pressure); however, nothing is known about the role of neutral trehalase in barotolerance. To estimate the contribution of neutral trehalase in resisting high hydrostatic pressure, we measured the barotolerance of neutral trehalase I and/or neutral trehalase II deletion strains. Under 180 MPa of pressure for 2 h, the neutral trehalase I deletion strain showed higher barotolerance in logarithmic-phase cells and lower barotolerance in stationary-phase cells than the wild-type strain. Introduction of the neutral trehalase I gene (NTH1) into the deletion mutant restored barotolerance defects in stationary-phase cells. Furthermore, we assessed the contribution of neutral trehalase during pressure and recovery conditions by varying the expression of NTH1 or neutral trehalase activity with a galactose-inducible GAL1 promoter with either glucose or galactose. The low barotolerance observed with glucose repression of neutral trehalase from the GAL1 promoter was restored during recovery with galactose induction. Our results suggest that neutral trehalase contributes to barotolerance, especially during recovery.