Identification of novel Asian Indian and Japanese mutations causing β-thalassaemia in the Egyptian population

β-thalassaemia is a major health problem in Egypt. It has been estimated that of the 1.5 million live births, 1000 children with β-thalassaemia major are born annually. Although the available treatment has increased the life expectancy of patients, it is still unsatisfactory and represents a significant drain on the country’s resources. National screening and prenatal diagnosis programmes can be provided in Egypt once the spectrum of β-thalassaemia mutations has been identified within the Egyptian population. We have examined 16 DNA samples with 21 β-thalassaemia mutations that remained unidentified in a study of 54 patients reported by Rady and colleagues in 1996. Using the polymerase chain reaction and single strand conformation analysis we identified the following changes: frameshift (FS) codon (CD) 8/9 (+G), 4 FS CD 29 (–G) and 2 novel mutations in exon I (15 CD 22 A-C and 1 FS CD 28 –C). In addition, a silent, probably polymorphic mutation, CD 17 G-A was present in all chromosomes. Received: 19 August 1996 / Revised: 21 September 1996     

Molecular basis of β-thalassemia in Turkey: detection of rare mutations by direct sequencing

Summary Using restriction endonuclease analysis, oligonucleotide hybridization, and direct sequencing of amplified genomic DNA, we characterized 11 different mutations in the DNA of 26 patients from Turkey homozygous for -thalassemia. We found that mutations IVS-1 nt110, IVS-1 nt6, and the frameshift at codon 8 were the most frequent. By direct sequencing we characterized two very rare mutations not previously reported in the Turkish population: a frameshift +1 at codons 9/10 and a nonsense mutation at codon 15.     

Improved detection of β-thalassaemia carriers by a two-test method

Summary Seven red cell parameters, taken one at a time and in their 21 possible pairs, were investigated for their power to discriminate between adult carriers of the -thalassaemia allele and adult normal subjects. The red blood cell count (RBC), haemoglobin concentration (Hb), haematocrit (Hct), mean cell volume (MCV), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), and haemoglobin A₂(HbA₂) fraction were measured in 24 obligate heterozygotes and in 99 adult controls with comparable age and sex distributions. Quadratic discriminant functions were computed using Bayesian analysis of univariate and bivariate Gaussian density functions. Classification errors were then calculated by integrating the density function for one genotype over the region assigned to the other.In the univariate case, MCH led to the lowest cost of misclassification while MCV was the second best discriminant for all posterior probabilities considered. In the bivariate case, MCV combined with percentage Hb A₂ yielded the best discrimination and generated misclassification costs roughly 1/30 of those generated by the most efficient single parameter. When use of MCV alone cannot classify an individual reliably either as a heterozygote or as homozygous normal, combined use of MCV and percentage Hb A₂ is recommended for maximum accuracy.Application of this screening method to 260 adult subjects at risk for thalassaemia heterozygosity yielded an unbiased frequency of 0.067 for the adult carrier in the Montreal Greek community, a value similar to that reported in the source population in Greece. The improved discriminations thus achieved is particularly useful for sibs of affected subjects whose high prior probability of heterozygosity (0.67) impairs classification.     

Molecular characterization of β-thalassemia mutations in Egypt

Summary The relative frequency of different -thalassemia mutations and their association with -globin haplotypes were studied in patients from the Nile delta region, Egypt, by means of the polymerase chain reaction, oligonucleotide hybridization and restriction analysis. We found that 8 mutations account for 77% of -thalassemia chromosomes in this population, the commonest being IVS-1 nt 110, IVS-1 nt 6 and IVS-1 nt 1. Each mutation was associated with a specific haplotype, with the exception of IVS-1 nt 110, found on 3 different chromosomal backgrounds. Our data show that testing for the 8 detectable mutations makes feasible prenatal diagnosis in 65% of at risk couples and exclusion testing in an additional 25% of cases.     

Identification of a bovine β-mannosidosis mutation and detection of two β-mannosidase pseudogenes

β-Mannosidase deficiency results in β-mannosidosis, a severe neurodegenerative lysosomal storage disease identified in cattle, goats, and humans. To more fully understand the molecular pathology of this disease, the mutation associated with bovine β-mannosidosis was identified by sequence analysis of cDNA from an affected calf. A transition mutation of G to A at position 2574 of the cDNA coding sequence creates a premature stop codon near the 3′ end of the protein coding region. To aid commercial breeders of Salers cattle, a PCR-based test was developed to detect the mutation for β-mannosidosis carrier screening. Application of this test also revealed the presence of two β-mannosidase pseudogenes. Portions of the pseudogenes were amplified with allele-specific primers and then sequenced. One pseudogene was highly homologous (>99% sequence identity) to the expressed cDNA sequence over the 1292 bp that were sequenced, while the other showed more divergence (83% sequence identity) in the 477 bp that were sequenced. Both are processed pseudogenes that are not expressed. The severity of the bovine β-mannosidosis phenotype suggests that the 22 C-terminal amino acids of β-mannosidase play an important role in the function of this enzyme. Received: 18 June 1999 / Accepted: 13 August 1999     

Observation of a parental inversion variant in a rare Williams–Beuren syndrome family with two affected children

The Williams–Beuren syndrome (WBS) region at 7q11.23 is subject to several genomic rearrangements, one of which, the WBSinv-1 variant, is an inversion polymorphism. The WBSinv-1 chromosome has been shown to occur frequently in parents of individuals with WBS, implying that it predisposes the region to the WBS deletion. Here we investigate two WBS families with multiple affected children, and show that in one family, both siblings have a deletion on a WBSinv-1 chromosome background that arose due to interchromosomal recombination. These results suggest that the two WBS deletions in this family were independent events, and that there is likely a significant increase in the risk of deletion of the WBS region associated with the WBSinv-1 chromosome. The rarity of multiplex WBS families would suggest that the overall risk of having a child with WBS is still relatively low; however, families with an existing member with WBS may choose to opt for WBSinv-1 testing and genetic counseling.     

Unique pattern of mutations in β-thalassemia patients in Western Uttar Pradesh

CONTEXT:

β-thalassemia is one of the most common heterogeneous inherited single gene disorders. The disease results from one or more of 380 different mutations in the β-globin gene. Uttar Pradesh (U.P.) is the most populous state of India, comprising various ethnic groups and Bareilly is one of the largest cities situated in Western U.P.

AIMS:

To examine the prevalence of five common β-thalassemian mutations: Intervening Sequence IVS 1-5 (c. 92 + 5 G > C), codon 8/9 (c. 27_28insG), codon 41/42 (c. 124_127delTTCT), IVS 1-1 (c. 92 + 1 G > T) and codon 26 G-A (c. 79G > A) in Western U.P.

SETTINGS AND DESIGN:

Patients attending camps organized by the Thalassemia Society, Bareilly were selected for the study.

MATERIALS AND METHODS:

A total of 48 blood samples were collected from the patients of transfusion dependent β-thalassemia from July 2011 to May 2012. All the samples were analyzed for five common mutations by using the Amplification Refractory Mutation System (ARMS)-hot start-polymerase chain reaction (PCR) technique.

RESULTS:

Among the five common mutations prevalent in India, we were able to detect all except codon 26 G-A (c. 79G > A), which is prevalent in northeast India. These four mutations accounted for 58% of the total number of our patients. The IVS 1-5 (G-C) was found to be the most common mutation with a frequency of 46% and the 2 ^ndmost common mutation was Fr8/9 (+G) with a frequency of 21%. The frequency of other mutations was IVS1-1 (12%) and Cd 41/42 (4%).

CONCLUSION:

This study provides evidence that the pattern of mutations in Western U.P. is different from the rest of India and even from the neighboring states (Delhi and Punjab). To the best of our knowledge, mutation Fr8/9, the 2^ndmost common mutation in our study has never been reported to be so common from anywhere in India. Some mutations, which are prevalent in other regions are absent in our region (mutation for ε-globin). Hence, these findings can be called unique to Western U.P.     

A comparison of ten digestive enzymes reveals a lack of chitinase in a phasmid and the loss of two β-glucanases in a mantid

In all developmental stages, the phasmid Peruphasma schultei (Conle & Hennemann, 2005) is an obligate herbivore, whereas the mantid Hierodula membranacea (Burmeister, 1838) is an obligatory carnivore. In P. schultei, the luminal activity of all enzymes is approxximately 50% in the crop and 50% in the midgut, which corresponds to the approximate 50 : 50 ratio of volumes of these two regions. These ratios would be expected in insects with a constant feeding rate on an unvaried diet. The enzyme activity and volume ratios in Hierodula membranacea vary considerably because of the irregular feeding habits. These differences in activity ratios between phasmids and mantids are not associated with the obligate phytophagous or carnivorous diet. The ratio of membrane bound to luminal aminopeptidases and disaccharidases in the midgut of both species are not significantly different and are within the normal range of other paurometabolous insects. Cellobiase and other plant cell wall digesting enzymes, laminarinase and cellobiase, are present in the phasmid but totally lacking in the mantid. The obligate carnivorous feeding habits of mantids could represent a selective factor leading to the loss of the ability to produce β-glucanases. Chitinase is a moulting enzyme in all insects, whereas, in H. membranacea, chitinase also occurs as a luminal digestive enzyme. This modified enzyme function requires production and secretion in another tissue, namely the midgut.     

The spectrum of β-thalassemia mutations in northern and northeastern Thailand

Summary A total of 123 -thalassemia genes from northern (n = 113) and northeastern (n = 10) Thailand were examined. Using five oligonucleotide probes, the mutation in 108 genes (88%) was identified: 50 nonsense 17, 49 frameshift 41-42, 4-28(AG), 2IVS1 nt5(GC), 2IVS2 nt654, and 1 deletion removing the entire -globin gene. The nonsense 17 mutation (n = 39) was linked to a single haplotype, whereas the frameshift 41-42 mutation occurred with several haplotypes. The results of the present study indicate that prenatal diagnosis of clinically important -thalassemia syndromes using a limited set of oligonucleotides is feasible in approximately 80% of affected families in northern Thailand and most of the families with -thalassemia-Hb E disease in northeastern Thailand.     

Analysis of β-globin gene haplotypes in Asian Indians: origin and spread of β-thalassaemia on the Indian subcontinent

-globin gene haplotypes were determined for 196 normal (-A) and 419 thalassaemia (-Th) chromosomes of individuals from four different regions of the Indian subcontinent; North-west Pakistan, Gujarat, Punjab and Sindh. Analysis of -A and -Th haplotypes and haplotype-mutation associations in each regional group along with a consideration of Indian history provided information about the origin and spread of -thalassaemia mutations on the Indian subcontinent. The data are consistent with relatively recent and local origins for most -thalassaemia mutations. The frequencies of particular alleles differ markedly in various regions and these may be useful population markers. Of the high frequency alleles, intervening sequence 1 (IVS-1) nucleotide 5 (G-C) and codons 41/42 (-CTTT) appear to be older as suggested by multiple haplotype associations and a widespread geographical distribution. The microepidemiology of -thalassaemia in this region reflects considerable ethnic diversity, gene flow from population migration and natural selection by malaria infection.     

A novel mutation in exon 17 of the β-subunit of rod phosphodiesterase in two RP sisters of a consanguineous family

We report the molecular analysis of the subunit of the rod phosphodiesterase (PDEB) gene in a consanguineous autosomal recessive retinitis pigmentosa family that shows homozygosity for polymorphisms in the genomic region comprising this gene, and positive linkage between a PDEB marker and the diesease. The two affected sisters are homozygous for a T to G transversion in codon 699 of the PDEB gene, leading to the substitution of a leucine by an arginine residue. This change, enclosed in the catalytic domain of the PDEB, could result in a modification of the protein structure preventing the physiological hydrolysis of cGMP.     

Identification of seven novel mutations in LH β-subunit gene by SSCP

Seven new point mutations have been identified from LH -subunit gene by PCR-mediated SSCP, and sequencing. One mutation was found changing amino acid from Gln₁₀₂ to Ser₁₀₂. The remaining six mutations, which did not change the codings, were in complete linkage disequilibrium. SSCP can be used in the diagnosis of LH-related disorders.     

Molecular basis of β-thalassemia in Thailand: analysis of β-thalassemia mutations using the polymerase chain reaction

Summary -Thalassemia mutations in 71 chromosomes of Thai patients from the northeast, the middle and the south of the country were investigated using dot blot hybridization of PCR (polymerase chain reaction)-amplified DNA with allelespecific oligonucleotide probes. Eight different known molecular defects were detected, at different frequencies. There was an amber mutation in codon 17, a C-T transversion at position 654 of IVS-2, a frameshift mutation between codons 71 and 72, an A-G transition at nucleotide -28 within the TATA box (known as Chinese mutations), a G-T transversion at position 1 of IVS-1 (an Indian mutation), a 4bp deletion in codons 41/42 and a G-C transversion at position 5 of IVS-1 (described as both Chinese and Indian mutations) and a Thai original mutation, an ochre mutation in codon 35. Analysis of the three unknown alleles by DNA sequencing of the cloned DNA fragment amplified by PCR revealed an A-G substitution at the second position of the codon for amino acid 19 (AAC-AGC). The analytic approach used in the present study and the characteristic distribution of mutations in each region of Thailand will prove useful for setting up a prenatal diagnosis program.     

Surface histidine mutations for the metal affinity purification of a β-carbonic anhydrase

Metal affinity chromatography using polyhistidine tags is a standard laboratory technique for the general purification of proteins from cellular systems, but there have been no attempts to explore whether the surface character of a protein may be engineered to similar affinity. We present the Arg160His mutation of Haemophilus influenzae carbonic anhydrase (HICA), which mimics the endogenous metal affinity of Escherichia coli carbonic anhydrase (ECCA). The purity and activity of the mutant are reported, and the purification is discussed. This is the first step toward developing a general method to engineer surface metal affinity for use in purification and metal labeling techniques.     

Profiling β-thalassaemia mutations in India at state and regional levels: implications for genetic education, screening and counselling programmes

Thalassaemia and sickle cell disease have been recognized by the World Health Organization as important inherited disorders principally impacting on the populations of low income countries. To create a national and regional profile of β-thalassaemia mutations in the population of India, a meta-analysis was conducted on 17 selected studies comprising 8,505 alleles and offering near-national coverage for the disease. At the national level 52 mutations accounted for 97.5% of all β-thalassaemia alleles, with IVSI-5(G>C) the most common disease allele (54.7%). Population stratification was apparent in the mutation profiles at regional level with, for example, the prevalence of IVSI-5(G>C) varying from 44.8% in the North to 71.4% in the East. A number of major mutations, such as Poly A(T>C), were apparently restricted to a particular region of the country, although these findings may in part reflect the variant test protocols adopted by different centres. Given the size and genetic complexity of the Indian population, and with specific mutations for β-thalassaemia known to be strongly associated with individual communities, comprehensive disease registries need to be compiled at state, district and community levels to ensure the efficacy of genetic education, screening and counselling programmes. At the same, time appropriately designed community-based studies are required as a health priority to correct earlier sampling inequities which resulted in the under-representation of many communities, in particular rural and socioeconomically under-privileged groups. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11568-010-9132-3) contains supplementary material, which is available to authorized users.     

Transferability of thermostabilizing mutations between β-adrenergic receptors

Abstract

In previous work we described six point mutations that thermostabilised the turkey β₁-adrenergic receptor (tβ₁AR). The thermostable mutant, tβ₁AR-m23, had an apparent T_m 21°C higher than the native protein when solubilized in dodecylmaltoside (DDM) and, in addition, was significantly more stable in short chain detergents, which allowed its crystallization and structure determination. Identification of thermostabilizing mutations in tβ₁AR was performed by systematic mutagenesis followed by expressing and assaying each of the 318 mutants for their thermostability. This is time-consuming, so to facilitate studies on related receptors, we have studied the transferability of these mutations to the human adrenergic receptors, hβ₁AR and hβ₂AR, which have, respectively, 76% and 59% sequence identity to tβ₂AR, excluding the N- and C-termini. Thermostability assays revealed that hβ₁AR was much more unstable than tβ₂AR, whereas hβ₂AR was more stable than tβ₁AR. Addition of the 6 thermostabilizing mutations in tβ₂AR-m23 into both hβ₂AR and hβ₂AR increased their apparent T_ms by 17°C and 11°C, respectively. In addition, the mutations affected the global conformation of the human receptors so that they were predominantly in the antagonist bound form, as was originally observed for tβ₂AR-m23. Thus, once thermostabilizing mutations have been identified in one G protein-coupled receptor, stabilization of close members within the subfamily is rapidly obtainable.     

Screening for mutations in human cardiomyopathy- is RBM24 a new but rare disease gene?

With great interest we have read the study of Liu et al.(2018) revealing the role of RNA binding protein 24 (RBM24) on global alternative splicing and dilated cardiomyopathy (DCM) in mice. As suggested previously, deficiency of Rbm24 causes embryonic lethality limiting the functional analyses (Yang et al., 2014). To circumvent this limitation the authors generated cardiac specific Rbm24 deficient mice and showed that homozygous deletion of Rbm24 at postnatal stage leads to rapidly progressive DCM and heart failure (Liu et al., 2018).     

First detection of OKP-A β-lactamase in two Serratia marcescens isolates in China

Two strains of Enterobacteriaceae producing prodigiosin were isolated from meat in the Sichuan province of China in 2010. The strains were identified by Vitek system, 16S rDNA, rpoB, pfs and luxS genes. Minimum inhibitory concentrations were determined using the broth microdilution method. The two strains were screened for the presence of β-lactamase genes (blaTEM, blaSHV, blaOKP, and blaCTX-M genes). Based on PCR amplification and 16S rDNA sequencing the analysed strains were identified as Serratia marcescens. In addition, morphological and biochemical identification showed that the two stains were definitely S. marcesens. Antimicrobial susceptibility test showed that both strains were resistant to ampicillin and first-generation cephalosporins while being susceptible to cefotaxime, ceftiofur, ceftriaxone, imipenem and aztreonam. It was found that blaOKP had been identified first from the two S. marcescens strains, ch1 and ch2. The isolates were closely related as shown by pulsed-field gel electrophoresis (PFGE). The narrow-spectrum OKP-A β-lactamase gene blaOKP-A-13 was found to be chromosomally located in S. marcescens. The isolates produced a β-lactamase with a pI of approximately 8.2, which corresponds to the OKPA family. Findings indicate that OKP enzymes are not Klebsiella pneumoniae-specific chromosomal ?-lactamases, and the first isolation of S. marcescens producing OKP-A ?-lactamase suggests that the blaOKP gene may be disseminated between different species.     

Heterologous co-expression of two β-glucanases and a cellobiose phosphorylase resulted in a significant increase in the cellulolytic activity of the Caldicellulosiruptor bescii exoproteome

Journal of Industrial Microbiology & Biotechnology - The ability to deconstruct plant biomass without conventional pretreatment has made members of the genus Caldicellulosiruptor the target of...