Modulation of Malaria Phenotypes by Pyruvate Kinase (PKLR) Variants in a Thai Population

Pyruvate kinase (PKLR) is a critical erythrocyte enzyme that is required for glycolysis and production of ATP. We have shown that Pklr deficiency in mice reduces the severity (reduced parasitemia, increased survival) of blood stage malaria induced by infection with Plasmodium chabaudi AS. Likewise, studies in human erythrocytes infected ex vivo with P. falciparum show that presence of host PK-deficiency alleles reduces infection phenotypes. We have characterized the genetic diversity of the PKLR gene, including haplotype structure and presence of rare coding variants in two populations from malaria endemic areas of Thailand and Senegal. We investigated the effect of PKLR genotypes on rich longitudinal datasets including haematological and malaria-associated phenotypes. A coding and possibly damaging variant (R41Q) was identified in the Thai population with a minor allele frequency of ~4.7%. Arginine 41 (R41) is highly conserved in the pyruvate kinase family and its substitution to Glutamine (R41Q) affects protein stability. Heterozygosity for R41Q is shown to be associated with a significant reduction in the number of attacks with Plasmodium falciparum, while correlating with an increased number of Plasmodium vivax infections. These results strongly suggest that PKLR protein variants may affect the frequency, and the intensity of malaria episodes induced by different Plasmodium parasites in humans living in areas of endemic malaria.     

A case of hyperlipidemia with homozygous apolipoprotein E5 (Glu3-->Lys)

In this study, we present clinical feature of a novel case with homozygous apolipoprotein (apo) E5.The patient was a 53-year-old Japanese woman. She was from a small island off the coast of Kagoshima Prefecture, Japan. Her parents were first degree cousins. No corneal opacification, xanthomatosis, lymphadenopathy, or hepatosplenomegaly was observed. There have been no signs of clinically overt atherosclerosis to date. Her serum total cholesterol, triglycerides (TG) and high-density lipoprotein (HDL)-cholesterol levels were 11.6, 6.1 and 1.2 mmol/l, respectively, and apo A-I, A-II, B, C-II, C-III and E levels were 121, 34.8, 269, 10.4, 25.7 and 10.3 mg/dl, respectively. Serum lipoprotein profile analyzed by agarose gel electrophoresis and differential staining revealed markedly increased cholesterol and TG in both beta and prebeta-migrated lipoproteins, whereas alpha-migrated lipoprotein showed decreased cholesterol. Her apo E isoform analyzed by isoelectric focusing (IEF) was found to be homozygous apo E5.Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis of her apo E and lipoprotein lipase (LPL) genes revealed that she had a homozygous apo E (Glu3-->Lys) and heterozygous LPL variant Ser447 to Ter. Her son and daughter, both of whom had hyperlipidemia, were found to have apo E3/5 phenotype. Direct sequencing analysis of her apo E gene confirmed a homozygous one nucleotide change: G to A at nucleotide position of 2836 in the exon 3, resulting in Glu3-->Lys mutation.This is the first report of lipids and lipoprotein profiles in patients with homozygous apo E5 (Glu3-->Lys).     

Association of a Glu92Lys substitution in MC1R with extended brown in Japanese quail (Coturnix japonica)

We investigated melanocortin 1 receptor (MC1R) as a candidate locus for the Extended brown phenotype in quail, in which there is a general darkening throughout the plumage. An initial screen of variation in MC1R in Extended brown and in wild-type quails revealed two polymorphic non-synonymous sites. One of these sites, a G-to-A substitution leading to a Glu92Lys mutation, was perfectly associated with plumage phenotype; all Extended brown birds were homozygous for Lys92. Co-segregation of the Glu92Lys mutation with the Extended brown phenotype was confirmed in 24 progeny of an E/e(+) x E/e(+) cross. Glu92Lys is likely to be the causative mutation for the increased melanism in Extended brown, given that the same mutation is associated with melanic plumage in many breeds of domestic chicken, as well as in a wild passerine bird (the bananaquit, Coereba flaveola) and laboratory mice. Interestingly, the increase in melanization with the Glu92Lys mutation is less marked in quails than in most other birds and mammals. Phylogenetic results indicate that the Glu92Lys mutation has independently occurred in quail and chicken lineages.     

Evaluation of the Diagnostic Accuracy of CareStart G6PD Deficiency Rapid Diagnostic Test (RDT) in a Malaria Endemic Area in Ghana,Africa

Background

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most widespread enzyme defect that can result in red cell breakdown under oxidative stress when exposed to certain medicines including antimalarials. We evaluated the diagnostic accuracy of CareStart G6PD deficiency Rapid Diagnostic Test (RDT) as a point-of-care tool for screening G6PD deficiency.

Methods

A cross-sectional study was conducted among 206 randomly selected and consented participants from a group with known G6PD deficiency status between February 2013 and June 2013. A maximum of 1.6ml of capillary blood samples were used for G6PD deficiency screening using CareStart G6PD RDT and Trinity qualitative with Trinity quantitative methods as the “gold standard”. Samples were also screened for the presence of malaria parasites. Data entry and analysis were done using Microsoft Access 2010 and Stata Software version 12. Kintampo Health Research Centre Institutional Ethics Committee granted ethical approval.

Results

The sensitivity (SE) and specificity (SP) of CareStart G6PD deficiency RDT was 100% and 72.1% compared to Trinity quantitative method respectively and was 98.9% and 96.2% compared to Trinity qualitative method. Malaria infection status had no significant (P=0.199) change on the performance of the G6PD RDT test kit compared to the “gold standard”.

Conclusions

The outcome of this study suggests that the diagnostic performance of the CareStart G6PD deficiency RDT kit was high and it is acceptable at determining the G6PD deficiency status in a high malaria endemic area in Ghana. The RDT kit presents as an attractive tool for point-of-care G6PD deficiency for rapid testing in areas with high temperatures and less expertise. The CareStart G6PD deficiency RDT kit could be used to screen malaria patients before administration of the fixed dose primaquine with artemisinin-based combination therapy.     

A Missense Mutation in a Highly Conserved Region of CASQ2 Is Associated with Autosomal Recessive Catecholamine-Induced Polymorphic Ventricular Tachycardia in Bedouin Families from Israel

Catecholamine-induced polymorphic ventricular tachycardia (PVT) is characterized by episodes of syncope, seizures, or sudden death, in response to physical activity or emotional stress. Two modes of inheritance have been described: autosomal dominant and autosomal recessive. Mutations in the ryanodine receptor 2 gene (RYR2), which encodes a cardiac sarcoplasmic reticulum (SR) Ca(2+)-release channel, were recently shown to cause the autosomal dominant form of the disease. In the present report, we describe a missense mutation in a highly conserved region of the calsequestrin 2 gene (CASQ2) as the potential cause of the autosomal recessive form. The CASQ2 protein serves as the major Ca(2+) reservoir within the SR of cardiac myocytes and is part of a protein complex that contains the ryanodine receptor. The mutation, which is in full segregation in seven Bedouin families affected by the disorder, converts a negatively charged aspartic acid into a positively charged histidine, in a highly negatively charged domain, and is likely to exert its deleterious effect by disrupting Ca(2+) binding.     

Identification and Functional Study of a New Missense Mutation in the Motor Head Domain of Myosin VIIA in a Family with Autosomal Dominant Hearing Impairment (DFNA11)

The MYO7A encodes a protein classified as an unconventional myosin. Here, we present a family with non-syndromic autosomal dominant hearing impairment that clinically resembles other previously published DFNA11 families. Affected members of the family present with an ascending audiogram affecting low and middle frequencies at young ages and then affecting all frequencies with increasing age. Genome-wide linkage analysis using Illumina Cyto-12 Chip mapped the disease locus to the DFNA11 interval in the family. A c.2003G→A (p.R668H) mutation of the MYO7A, is heterozygous in all affected family members and absent in 100 healthy individuals. Arg668His is located in a region of the myosin VIIA motor domain that is highly conserved among different species. Molecular modeling predicts that the conserved R668 residue plays important structural role in linking different lobes of motor domain together. In the actin-activated ATPase activity assay, the rate of NADH oxidation was higher in the wild-type myosin VIIA, indicating that the ATPase activity in the p.R668H mutant myosin VIIA was significantly destroyed.     

An intramolecular quadruplex of (GGA)(4) triplet repeat DNA with a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad, and its dimeric interaction.

The structure of d(GGAGGAGGAGGA) containing four tandem repeats of a GGA triplet sequence has been determined under physiological K(+) conditions. d(GGAGGAGGAGGA) folds into an intramolecular quadruplex composed of a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad. Four G-G segments of d(GGAGGAGGAGGA) are aligned parallel with each other due to six successive turns of the main chain at each of the GGA and GAGG segments. Two quadruplexes form a dimer stabilized through a stacking interaction between the heptads of the two quadruplexes. Comparison of the structure of d(GGAGGAGGAGGA) with the reported structure of d(GGAGGAN) (N=G or T) containing two tandem repeats of the GGA triplet revealed that although the two structures resemble each other to some extent, the extension of the repeats of the GGA triplet leads to distinct structural differences: intramolecular quadruplex for 12-mer versus intermolecular quadruplex for 7-mer; heptad versus hexad in the quadruplex; and three sheared G:A base-pairs versus two sheared G:A base-pairs plus one A:A base-pair per quadruplex. It was also suggested that d(GGAGGAGGAGGA) forms a similar quadruplex under low salt concentration conditions. This is in contrast to the case of d(GGAGGAN) (N=G or T), which forms a duplex under low salt concentration conditions. On the basis of these results, the structure of naturally occurring GGA triplet repeat DNA is discussed.     

A New GA-Insensitive Semidwarf Mutant of Rice (Oryza sativa L.) with a Missense Mutation in the SDG Gene

A semidwarf line of Indica rice, Xinguiai, was derived from the progeny of a cross between the double dwarf mutant Xinguiaishuangai and the wild-type variety Nanjing 6. The semidwarf phenotype was controlled by the semidwarf gene, sdg. The second sheath and shoot elongation responses of the dwarf mutant to exogenous gibberellin (GA₃) showed that sdg was insensitive to gibberellin (GA), and its endogenous GAs content was higher than that in wild-type cultivars. The SDG gene was cloned by a map-based cloning method and sequencing analysis revealed that the coding region of sdg had a single nucleotide substitution resulting in a single amino acid change from alanine to threonine. A cleaved amplified polymorphic sequence marker was designed according to sequences from mutant and wild-type materials. This sequence marker could be used to distinguish wild types and mutants, and thus, could be used for molecular marker-assisted selection. The dwarf phenotype of the sdg mutant was restored to a normal phenotype by introducing the wild-type SDG gene. Rice transformation experiments and GUS staining demonstrated that the SDG gene was predominantly expressed in vegetative organs.     

Hemoglobin Kariya [alpha 40 (C5) Lys leads to Glu]: a new hemoglobin variant with an increased oxygen affinity

A new abnormal hemoglobin, Hb Kariya [alpha 40 (C5) Lys leads to Glu], with an amino acid substitution at the alpha 1 beta 2 contact was discovered in a young Japanese man. This variant migrated to the anode faster than Hb A, being nearly the same as Hb I in electrophoretic mobility. It amounted to about 6% of the total hemoglobin of the hemolysate. This hemoglobin showed an increased oxygen affinity, decreased heme-heme interaction and a lowered 2,3-DPG effect.     

A Missense Mutation in the Rabbit Melanocortin 4 Receptor (MC4R) Gene is Associated with Finisching Weight in a Meat Rabbit Line

In this study we resequenced 1729 bp of the rabbit melanocortin 4 receptor (MC4 R) gene in 31 rabbits from different breeds/lines and identified ten polymorphisms: one was an indel and 9 were single nucleotide polymorphisms (SNPs). The indel and 5 SNPs were in the 5′-flanking region, 3 were synonymous SNPs and one was a missense mutation (c.101G>A; p.G34D), located in a conserved position of the extracellular tail of the MC4 R protein. The missense mutation was analyzed in a panel of 74 rabbits of different breeds and in 516 performance tested rabbits of a commercial paternal line under selection for growth efficiency. Association analysis indicated that rabbits with the less frequent genotype in this population (DD) had a lighter weight at 70 postnatal days than animals with genotype GD (P < 0.10) and animals with genotype GG (P < 0.05). This is the third study on candidate genes, after those on GH1 and IGF2 that reported a marker associated with finishing weight. Therefore, it seems that a candidate gene approach in rabbit based on previous information accumulated in other livestock species could be useful to identify genes explaining a fraction of variability of performance traits with potential application on rabbit breeding and selection.     

Mutation in Integrin-Linked Kinase (ILKR211A) and Heat-Shock Protein 70 Comprise a Broadly Cardioprotective Complex

Rationale

Integrin-linked kinase (ILK) has been proposed as a novel molecular target that has translational potential in diverse cardiac diseases, since its upregulation promotes a broadly cardioprotective phenotype. However, ILK has been implicated as both a cardioprotective and oncogenic target, which imposes therapeutic constraints that are generally relevant to the translational potential of many kinases.

Objective

To study the cardioprotective properties of the activation-resistant, non-oncogenic, mutation of ILK (ILK^R211A) against experimental MI in vivo and Doxorubicin induced apoptosis in vitro and it’s relationships to stress induced heat shock proteins.

Methods/Results

The transgenic mouse heart over-expressing a point mutation in the ILK pleckstrin homology (PH) domain (Tg^R211A) exhibits a highly cardioprotective phenotype based on LAD-ligation-induced MI reduction in vivo, and on protection against doxorubicin (DOX)-induced cardiomyocyte apoptosis when overexpressed in human induced pluripotent stem cell (iPS)-derived cardiomyocytes in vitro. Intriguingly, the degree of cardioprotection seen with the ILK^R211A mutation exceeded that with the ILK^S343D mutation. Microarray and immunoprecipitation analyses revealed upregulation of expression levels and specific binding of ILK^WT, ILK^S343D and ILK^R211A to both constitutively active heat-shock protein 70 (Hsc70) and inducible Hsp70 in response to MI, and to acute ILK overexpression in iPSC-cardiomyocytes. ILK-mediated cardioprotection was shown to depend upon Hsp70 ATPase activity.

Conclusions

These findings indicate that wild type ILK and the non-oncogenic ILK^R211A mutation comprise a cardioprotective module with Hsp/c70. These results advance a novel target discovery theme in which kinase mutations can be safely engineered to enhance cardioprotective effects.     

Prevalence of Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency among Malaria Patients in Southern Thailand: 8 Years Retrospective Study

Erythrocytes deficient in glucose-6-phosphate dehydrogenase (G6PD) is more susceptible to oxidative damage from free radical derived compounds. The hemolysis triggered by oxidative agents such as primaquine (PQ) is used for the radical treatment of hypnozoites of P. vivax. Testing of G6PD screening before malaria treatment is not a common practice in Thailand, which poses patients at risk of hemolysis. This retrospective study aimed to investigate the prevalence of G6PD in malaria patients who live in Southern Thailand. Eight hundred eighty-one malaria patients were collected for 8-year from 2012 to 2019, including 785 (89.1%) of P. vivax, 61 (6.9%) of P. falciparum, 27 (3.1%) of P. knowlesi, and 8 (0.9%) of mixed infections. The DiaPlexC genotyping kit (Asian type) and PCR-RFLP were employed to determine the G6PD variants. The result showed that 5 different types of G6PD variants were identified in 26 cases (2.9%); 12/26 (46.2%) had Mahidol (487G>A) and 11/26 (42.3%) had Viangchan (871G>A) variants, while the rest had Kaiping (1388G>A), Union (1360C>T), and Mediterranean (563C>T) variants. G6PD Songklanagarind (196T>A) variant was not found in the study. Our result did not show a significant difference in the malaria parasite densities in patients between G6PD-deficient and G6PD-normal groups. According to our findings, testing G6PD deficiency and monitoring the potential PQ toxicity in patients who receive PQ are highly recommended.     

Identification of a Novel G2073A Mutation in 23S rRNA in Amphenicol-Selected Mutants of Campylobacter jejuni

Objectives

This study was conducted to examine the development and molecular mechanisms of amphenicol resistance in Campylobacter jejuni by using in vitro selection with chloramphenicol and florfenicol. The impact of the resistance development on growth rates was also determined using in vitro culture.

Methods

Chloramphenicol and florfenicol were used as selection agents to perform in vitro stepwise selection. Mutants resistant to the selective agents were obtained from the selection process. The mutant strains were compared with the parent strain for changes in MICs and growth rates. The 23S rRNA gene and the L4 and L22 ribosomal protein genes in the mutant strains and the parent strain were amplified and sequenced to identify potential resistance-associated mutations.

Results

C. jejuni strains that were highly resistant to chloramphenicol and florfenicol were obtained from in vitro selection. A novel G2073A mutation in all three copies of the 23S rRNA gene was identified in all the resistant mutants examined, which showed resistance to both chloramphenicol and florfenicol. In addition, all the mutants selected by chloramphenicol also exhibited the G74D modification in ribosomal protein L4, which was previously shown to confer a low-level erythromycin resistance in Campylobacter species. The mutants selected by florfenicol did not have the G74D mutation in L4. Notably, the amphenicol-resistant mutants also exhibited reduced susceptibility to erythromycin, suggesting that the selection resulted in cross resistance to macrolides.

Conclusions

This study identifies a novel point mutation (G2073A) in 23S rRNA in amphenicol-selected mutants of C. jejuni. Development of amphenicol resistance in Campylobacter likely incurs a fitness cost as the mutant strains showed slower growth rates in antibiotic-free media.     

P450C17 (CYP17) Deficiency in Native Mexican Patient with a Novel CYP17A1 Mutation

ObjectiveTo report a case of congenital adrenal hyperplasia due to CYP17 deficiency caused by a novel CYP17A1 mutation.MethodsWe describe the clinical, biochemical, genetic, and radiologic findings of a sporadic case of congenital adrenal hyperplasia due to CYP17 deficiency in a young patient.ResultsAn 18-year-old woman presented with hypogonadism and progressive muscle weakness and had not yet undergone thelarche, adrenarche, and menarche. Blood pressure was 155/90 mm Hg, she had no axillary or pubic hair, breasts were Tanner stage 1, and female genitalia were Tanner stage 1. Further laboratory studies showed hypokalemia with metabolic alkalosis, hypergonadotropic hypogonadism, a 46,XY karyotype, a low 17-hydroxyprogesterone level, and a high deoxycorticosterone level. Sequencing of the CYP17A1 gene demonstrated homozygous transversion of cytosine to adenine (TCAàTAA) in exon 5, which causes a premature stop codon at position 288 (Ser288X). Imaging studies showed large adrenal glands, cystic picture in the inguinal canal (suggestive of intra-abdominal testes), and absent Müllerian structures. Exploratory laparotomy was performed to remove the remaining gonads, and the final histologic examination showed atrophic testes.ConclusionsCongenital adrenal hyperplasia due to CYP17 deficiency should be suspected in patients with hypertension, hypokalemic alkalosis, and hypogonadism. In such cases, it is mandatory to assess the karyotype and perform hormonal and molecular genetic studies. (Endocr Pract. 2011;17:99-103)     

Maternally inherited cardiomyopathy and hearing loss associated with a novel mutation in the mitochondrial tRNA(Lys) gene (G8363A).

A novel G8363A mutation in the mtDNA tRNA(Lys) gene was associated, in two unrelated families, with a syndrome consisting of encephalomyopathy, sensorineural hearing loss, and hypertrophic cardiomyopathy. Muscle biopsies from the probands showed mitochondrial proliferation and partial defects of complexes I, III, and IV of the electron-transport chain. The G8363A mutation was very abundant (>95%) in muscle samples from the probands and was less copious in blood from 18 maternal relatives (mean 81.3% +/- 8.5%). Single-muscle-fiber analysis showed significantly higher levels of mutant genomes in cytochrome (c) oxidase-negative fibers than in cytochrome (c) oxidase-positive fibers. The mutation was not found in >200 individuals, including normal controls and patients with other mitochondrial encephalomyopathies, thus fulfilling accepted criteria for pathogenicity.     

An exploration of diversity among the Ostertagiinae (Nematoda: Trichostrongyloidea) in ungulates from Sub-Saharan Africa with a proposal for a new genus

Abomasal nematodes (Ostertagiine: Trichostrongyloidea) representing a previously unrecognized genus and species are reported in African buffalo (Syncerus caffer caffer) from Kenya, Uganda, and South Africa. Africanastrongylus buceros gen. nov. et sp. nov. is characterized by a symmetrical tapering synlophe in the cervical region and a maximum of 60 ridges in males and females. Bursal structure is 2-2-1, with subequal Rays 4/5, massive Rays 8, and Rays 9/10, and a massive dorsal lobe that is reduced in length, laterally and dorsally inflated, and positioned ventral to externodorsal rays. Spicules are tripartite, and the gubernaculum is broadly alate in the anterior. A proconus is present. Among ostertagiines with a 2-2-1 bursa (Cervicaprastrongylus, Hyostrongylus, Mazamastrongylus, Sarwaria, Spiculopteragia, and Teladorsagia) specimens of Africanastrongylus are differentiated from respective genera based on the structure of the cervical synlophe, patterns of dorsal, externodorsal, lateral, and ventral rays, and configuration of the genital cone, gubernaculum, and spicules. Among 13 genera of the Ostertagiinae in the global fauna, 3 are entirely limited in distribution to Africa, including Africanastrongylus, Longistrongylus, and Pseudomarshallagia. Another 5 genera including Cervicaprastrongylus, Hyostrongylus, Marshallagia, Ostertagia, and Teladorsagia are represented as mosaics, with diversity centered in Eurasia or the Holarctic. Genera not represented in the African fauna include Camelostrongylus among Caprinae and some Antelopinae from Eurasia, Mazamastrongylus and Spiculopteragia in Cervidae from the Holarctic and Eurasia, respectively, Orloffia in Cervidae and Bovidae from the Holarctic, and Sarwaria among Tragulidae and Bovinae in southern Asia. The diverse nature of the ostertagiine fauna, with a disproportionate number of endemic genera relative to other regions of the northern hemisphere, may reflect the timing of episodic expansion events for artiodactyls into Africa from Eurasia during the Tertiary and Quaternary.     

Genetic variation atapolipoprotein E locus in Ethiopia: an E5 variant corresponds to two different mutant alleles: E*5 (Glu212Lys) and E*5 (Gln204Lys; Cys112Arg)

A previous investigation on apolipoprotein E polymorphism in the Ethiopian population highlighted the presence of a further variant allele named E*5 in addition to the three common alleles. The variant is considered rare elsewhere but has a frequency of more than 1% in this population. Now characterized by gene sequencing and restriction isotyping in many members of the families of the original carriers, the variant isoform has actually been found to be determined by two different gene mutations. Effectively rare in Ethiopians, one of the two, E5 (Gln204Lys, Cys 112Arg), has never been described before. The other, E5 (Glu212Lys), previously described in a subject of Turkish origin, is present at the polymorphic level only in the Ethiopian population. No subjects bearing these variants had anomalous lipid or apolipoprotein patterns. In the course of the present investigation both have been found to occur as rare variants in the southern Italian population as well. The occurrence of the two variants in the populations of Ethiopia and of the Mediterranean basin could be explained by taking into account the relevant Caucasoid contribution to the Ethiopian gene pool.