Clinical phenotypes and molecular characterization of Hb H-Paksé disease

BACKGROUND AND OBJECTIVES: Hemoglobin Constant Spring (Hb CS), caused by a termination codon mutation (TAA-->CAA) in the a2 gene, is the most common non-deletional type of a thalassemia in Southeast Asia. This mutation can most easily be detected by loss of an MseI-restriction site (T/TAA) spanning the termination codon. Recently, we sequenced the a globin genes from patients with a thalassemia in whom this MseI site was absent. This revealed, a previously described termination codon mutation (TAA-->TAT) associated with Hb Paksé. This prompted us to re-evaluate the molecular basis of a thalassaemia in other Thai patients with non-deletional types of Hb H disease. DESIGN AND METHODS: DNA samples from 30 patients, previously diagnosed as having Hb H-CS disease, were characterized by direct genomic sequencing and by using a mismatched polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Clinical and hematologic data were assessed. RESULTS. Hemoglobin electrophoresis in almost all 30 unrelated patients with non-deletional a thalassemia revealed a slow migrating band resembling Hb CS. Five of these patients were found to have Hb H-Paksé disease and the remainder had Hb H-CS disease. Comparing the hematology in patients with these two genotypes, no significant differences were found except that the proportion of Hb H was higher in patients with Hb H-Paksé disease. INTERPRETATION AND CONCLUSIONS: These results suggest that termination codon mutations may have been previously misidentified in many cases of non-deletional Hb H disease. Findings from six unrelated families described in this study suggest that the proportion of patients with the Hb Paksé mutation might be underestimated and that this mutation could be prevalent in Southeast Asia. Analysis of mismatched-PCR-RFLP, described here, was shown to provide an unequivocal diagnosis and will be applicable in population screening programs.     

First Case of a Compound Heterozygosity for Two Nondeletional α-Thalassemia mutations,Hb Constant Spring and Hb Quong Sze

Nondeletional α-thalassemia (α-thal) is the result of point mutations in critical regions of the α-globin genes, affecting mRNA processing, mRNA translation, or α-globin stability. Hb Constant Spring (Hb CS, HBA2: c.427T?>?C) is the most common nondeletional α-thal that results from a nucleotide substitution at the termination codon of the α2-globin gene. Hb Quong Sze (Hb QS, HBA2: c.377T?>?C) is another nondeletional α-thal in South China with the missense mutation at codon 125 of the α2-globin gene making this hemoglobin (Hb) variant highly unstable. Although homozygosity for Hb CS (α^CSα/α^CSα) or Hb QS (α^QSα/α^QSα) has been reported, clinical pictures vary from severe hemolysis that developed early in life to only mild anemia, no clinical phenotypic data of compound heterozygosity for Hb CS/Hb QS (α^CSα/α^QSα) has been described. In this report we describe an adult case with such a compound heterozygosity who presented with a mild α-thal.     

Molecular screening of the Hbs Constant Spring (codon 142, TAA>CAA, α2) and Paksé (codon 142, TAA>TAT, α2) mutations in Thailand

Hb Constant Spring [Hb CS, α142(H19)Term] and Hb Paksé [α142(H19)Term] occur from the mutation in the termination codon of the α2-globin gene, TAA>CAA (→Gln) and TAA>TAT (→Tyr), respectively. They are the most common nondeletional α-thalassemia (α-thal) variants causing Hb H disease in Southeast Asia. In this study, 587 cord blood samples were screened for the Hb CS and Hb Paksé mutations by a dot-blot hybridization technique using oligonucleotide probes specific for each mutation. The results showed that the prevalence of Hb CS and Hb Paksé in Central Thailand are 5.80 and 0.51%, respectively, which is in concordance with the results from previous studies.     

Molecular Characterization of β-Thalassemia Intermedia in Southeast Iran

Inheritance of mild mutations within the β-globin gene and coinheritance of α-thalassemia (α-thal) are known as two important genetic modifiers in β-thalassemia (β-thal) intermedia (β-TI). We aimed to evaluate the spectrum of β- and α-thal mutations in β-TI patients in Southeast Iran. Common β- and α-globin gene mutations were detected by amplification refractory mutation system–polymerase chain reaction (ARMS–PCR) and multiplex gap-PCR, respectively. There were 26 male (57.8%) and 19 female (42.2%) patients. HBB: c.92?+?5T?>?C [IVS-I-5 (G?>?C)] and HBB: c.?138C?+?1G?>?A [IVS-II-I (G?>?A)] represented the prevalent alleles with respective frequencies of 60.0 and 10.0%. Other β-globin mutations included HBB: c.-138C?>?T [–88 (C?>?T)], HBB: c.27_28insG [frameshift codons (FSC) 8/9 (+G)], HBB: c.46delT [codon 15 (–T)], HBB: c.93-22_95del (IVS-I, 25 del), and the 619?bp deletion (NG_000007.3: g.71609_72227del619). The predominant genotypic combinations were β⁰/β⁰ (68.9%), β⁰/β^+?(8.9%) and β⁺/β^+?(2.2%). Coinheritance of α-thal was observed in 33.0% of the patients, with the –α^3.7 (rightward) (NG_000006.1: g.34164_37967del3804) as the most common deletion (86.0%). One patient was diagnosed with the –α^4.2 (leftward) (AF221717) and one with the – –^MED (g.24664_41064del16401) deletions, while no patients carried the –(α)^20.5 (g.15164_37864del22701), α^–5?nt (HBA2: c.95?+?2_95_6delTGAGG) or codon 19 (–G) (HBA2: c.56delG) mutations. The alleviating molecular mechanism was not explainable by β^+?or concurrent α-thal in more than half of our β-TI patients. This encourages conducting more studies to identify other contributing factors, especially Hb F-inducing genetic modifiers.     

Infectious origins of, and molecular mimicry in, Guillain-Barré and Fisher syndromes

Guillain-Barré syndrome (GBS), characterised by limb weakness and areflexia, is the prototype of postinfectious autoimmune diseases, and Campylobacter jejuni is the most frequent antecedent pathogen. GBS subsequent to C jejuni enteritis is associated with a severe, pure motor axonal variant and IgG antibodies against GM1, GM1b, GD1a, or GalNAc-GDla, gangliosides expressed in human peripheral nerves. Lipopolysaccharides of C jejuni isolated from GBS patients have ganglioside-like epitopes. Cytomegalovirus is the most common viral antecedent infection. Patients with demyelinating GBS who have had a recent CMV infection have severe sensory deficits and anti-GM2 IgM antibody. CMV-infected fibroblasts express the GM2 epitope. Fisher syndrome (FS), characterised by ophthalmoplegia, ataxia, and areflexia, is a GBS variant associated with anti-GQ1b IgG antibody. GQ1b is enriched in the cranial nerves that innervate the extraocular muscles. Some patients develop FS after C jejuni infection, and the lipopolysaccharide present bears the GQ1b epitope. Molecular mimicry is a possible cause of GBS and FS.     

Molecular characterization of deletional forms of β-thalassemia in Taiwan

beta-Thalassemia is one of the most common genetic diseases in Taiwan. The most common mutations of beta-globin are point mutations, and six mutations account for over 90% of cases. Less than 5% of the cases with beta-globin gene deletion result in beta-thalassemia minor. The mutational type of the deletion is not clear in Taiwanese. We used polymerase chain reaction (PCR)-based methods to detect the breakpoint junctions of different deletional types of beta-thalassemia. In total, six cases of clinically suspected deletional type of beta-thalassemia were studied. The results showed that there were three types of deletions in these cases: two cases each for hereditary persistent fetal hemoglobinemia (HPFH) of the Southeast Asian (SEA) type, HPFH of the Yunnanese type, and gamma(G)+(gamma(A)deltabeta)(0)deletions, respectively. The clinical features of these deletional mutations are milder than the beta(o) types of the point mutation. The patients with compound heterozygous mutations of the point mutation and the deletional mutation are always transfusion independent.     

Lack of evidence for an association between α-adducin and blood pressure regulation in Asian populations

Recent studies have found the tryptophan allele of a glycine to tryptophan polymorphism at position 460 (G460W) of the α-adducin protein to be associated with essential hypertension in European populations. We examined whether the tryptophan allele is associated with hypertension in a different population, comprised of subjects of Chinese origin from Taiwan, and Chinese and Japanese origin from the San Francisco Bay area and Hawaii. We adapted the 5′ allelic discrimination assay or TaqMan to type individuals for the G460W polymorphism, and using this method we typed more than 1000 individuals. The frequency of the W allele was slightly increased in the treated subjects in the Chinese population (0.458 v 0.423) but not the Japanese population (0.549 v 0.558). We considered dominant, recessive, and additive models in our analysis. There was a significant result for a recessive model for systolic blood pressure in the Chinese population (χ² 6.84, df = 2, P < .05), but only suggestive evidence for diastolic blood pressure (χ² 3.30). In contrast, in the Japanese population, there was no evidence for a positive association under any model. For the combined Chinese and Japanese samples, the evidence for association with α-adducin was not significant.     

Hb Paksé [(alpha2) codon 142 (TAA-->TAT or Term-->Tyr)J in Thai patients with EAbart's disease and Hb H Disease

Hb Paksé is caused by an alpha2-globin gene termination codon mutation, TAA-->TAT or Term-->Tyr, initially described in a Laotian family. We now report for the first time that the same mutation has been found in 14 Thai patients, seven with EABart's disease, four with Hb H disease, and three with alpha-thalassemia trait who were initially diagnosed as having Hb Constant Spring (Hb CS; alpha2-globin gene termination codon mutation TAA-->CAA or Term-->Gln). Co-inheritance of this mutation with alpha-thalassemia-1 (SEA type) leads to Hb H disease (hereafter designated as Hb H-Paksé disease) and to a complex thalassemia syndrome, namely EABart's-Paksé disease. Hematological data of these patients were compared with those of classical Hb H-CS and the EABart's patients. To facilitate epidemiological and diagnostic screening of Hb Paksé, a simple assay procedure based on allele specific polymerase chain reaction (PCR) amplifications was developed and validated. Using this allele specific PCR as a screening method, five additional individuals with Hb Paksé were found among 71 Thai subjects previously thought to have Hb CS.     

Molecular Characterization of β-Thalassemia in the Czech and Slovak Populations: Mediterranean,Asian and Unique Mutations

β-Thalassemia (β-thal) is considered rare in Central Europe. As in other malaria-free regions, the presence of β-thal in Central Europe reflects historical and recent immigration, and demographic changes that have influenced the genetic variability of the current populations living in this area. This study assesses the frequency and spectrum of mutations on the β-globin gene in Czech and Slovak subjects with clinical symptoms of thalassemia. The results of the initial part of this research were published more than two decades ago; the aim of this study was to update these original reports. During the period from 2002 to 2015, 400 cases from Czech and Slovak hematological centers were analyzed. Twenty-nine β-thal mutations, identified in 356 heterozygotes from 218 unrelated families, involve five unique mutations including a recently described insertion of a transposable L1 element into the β-globin gene. One mutation described here is reported for the first time. Most of the mutations were of Mediterranean origin and accounted for 82.0% of cases. All but one case studied were heterozygous carriers, manifesting β-thal minor, with rare exceptions represented by the rare (β⁰) codons 46/47 (+G) (HBB: c.142_142dupG) mutation associated with an α-globin gene quadruplication and by dominantly inherited β-thal with a more severe phenotype. One double heterozygous β-thal patient was a recent immigrant from Moldavia. The list of δβ-thal alleles (26 carriers, 16 families) contains Hb Lepore and two types of δβ⁰-thal deletions. In the past, genetic drift and migration as well as recent immigrations were responsible for the introduction of Mediterranean alleles, while several mutations described in single families were of local origin.     

Meta-analysis of TNF-�� promoter �C308A/G polymorphism and SLE susceptibility in Asian populations

The aim of this study was to summarize results on the association of tumor necrosis factor-α (TNF-α) promoter –308A/G polymorphism with systemic lupus erythematosus (SLE) susceptibility in Asian populations by using the meta-analysis. We searched all the publications about the association between TNF-α promoter –308A/G polymorphism and SLE in Asian populations from PubMed, Elsevier Science Direct, Chinese Biomedical Literature Database (CBM), Chinese National Knowledge Infrastructure (CNKI), and Wanfang (Chinese). Meta-analysis was performed for genotypes AA versus GG, GA versus GG, AA versus GG + GA, GA + AA versus GG, and A allele versus G allele in a fixed/random effect model. A total of 12 studies (1017 cases and 1086 controls) were included in the current meta-analysis (Chinese, Japanese, and Thai). When all groups were pooled, a significant association of A allele and increased SLE risk was found (OR = 1.44, 95%CI = 1.04–2.01, P = 0.03). When analyses were restricted to more ethnically homogeneous populations, similar result was found in Chinese population (OR = 1.59, 95%CI = 1.12–2.26, P = 0.009). But the association between TNF-α promoter –308 polymorphism and SLE was not observed when examining the contrast of G/A + A/A versus G/G, A/A versus A/G + G/G, A/A versus G/G, and G/A versus G/G. This meta-analysis demonstrates the association between TNF-α promoter –308A/G polymorphism and SLE in Asian populations, especially in Chinese population.     

What is the sudden death syndrome in Southeast Asian males?

Sudden unexplained death syndrome describes the death of apparently healthy individuals--usually young men--in whom postmortem examination does not reveal the cause of death. The victims are in apparently good health and usually die at night while sleeping. They die within minutes after the onset of agonal respiration. Patients who have been resuscitated were found to have ventricular fibrillation and inducible polymorphic ventricular tachycardia in the electrophysiologic laboratory. This syndrome has been most frequently described in young Southeast Asian men. In this review, the epidemiology, clinical and electrophysiologic manifestations, pathology and risk factors, prognosis, and treatments for sudden unexplained death syndrome are described.     

Fetal Anemia and Hydrops Fetalis Associated with Homozygous Hb Constant Spring (HBA2: c.427T > C)

Hb Constant Spring (Hb CS, HBA2: c.427T?>?C) is a common nondeletional α-thalassemia (α-thal) that results from a nucleotide substitution at the termination codon of the α2-globin gene. Homozygosity for Hb CS (α^CSα/α^CSα) is relatively rare, and generally characterized with mild hemolytic anemia, jaundice, and splenomegaly. In this report we present a fetus with cardiomegaly, pericardial effusion, enlarged placenta and increased middle cerebral artery-peak systolic velocity (MCA-PSV) at 24 weeks’ gestation. Fetal blood sampling revealed the severe anemia [hemoglobin (Hb) level being 4.8 g/dL] and Hb H (β4) disease-like hematological findings with Hb Bart’s (γ4) level of 17.9%. DNA sequencing of the α-globin genes found that both partners were Hb CS carriers and the fetus was an Hb CS homozygote. Therefore, this was a rare case of homozygous Hb CS which demonstrated an unusual and serious anemia and hydrops fetalis in utero.     

Does genetic heterogeneity account for the divergent risk of type 2 diabetes in South Asian and white European populations?

Aims/hypothesis

South Asians are up to four times more likely to develop type 2 diabetes than white Europeans. It is postulated that the higher prevalence results from greater genetic risk. To evaluate this hypothesis, we: (1) systematically reviewed the literature for single nucleotide polymorphisms (SNPs) predisposing to type 2 diabetes in South Asians; (2) compared risk estimates, risk alleles and risk allele frequencies of predisposing SNPs between South Asians and white Europeans; and (3) tested the association of novel SNPs discovered from South Asians in white Europeans.

Methods

MEDLINE, Embase, the Cumulative Index to Nursing and Allied Health Literature (CINAHL) and the Cochrane registry were searched for studies of genetic variants associated with type 2 diabetes in South Asians. Meta-analysis estimates for common and novel bi-allelic SNPs in South Asians were compared with white Europeans from the DIAbetes Genetics Replication And Meta-analysis (DIAGRAM) consortium. The population burden from predisposing SNPs was assessed using a genotype score.

Results

Twenty-four SNPs from 21 loci were associated with type 2 diabetes in South Asians after meta-analysis. The majority of SNPs increase odds of the disorder by 15–35% per risk allele. No substantial differences appear to exist in risk estimates between South Asians and white Europeans from SNPs common to both groups, and the population burden also does not differ. Eight of the 24 are novel SNPs discovered from South Asian genome-wide association studies, some of which show nominal associations with type 2 diabetes in white Europeans.

Conclusions/interpretation

Based on current literature there is no strong evidence to indicate that South Asians possess a greater genetic risk of type 2 diabetes than white Europeans.     

Molecular characterization of estrogen receptor genes in loach Paramisgurnus dabryanus and their expression upon 17α-ethinylestradiol exposure in juveniles

The full-length cDNAs for estrogen receptor 1 (esr1), esr2a and esr2b were isolated and characterized from the loach (Paramisgurnus dabryanus, Cobitidae, cypriniformes). P. dabryanus Esr1, Esr2a and Esr2b share high amino acids identities with their counterparts of cyprinid species. Quantitative real-time PCR (qRT-PCR) was used to analyze the tissue distribution of esr mRNAs in one-year-old P. dabryanus. The mRNA expression of esr1 in female liver was extremely higher than that in other tissues. esr2a mRNA expression in female intestine and in male muscle was higher than that in other tissues. esr2b mRNA expression was the highest in both male and female intestine. Two-month-old P. dabryanus were exposed to 17α-ethinylestradiol (EE2) for 3weeks and the changes of esr mRNA expression in brain, gonad and liver were analyzed by qRT-PCR. Results showed that EE2 at 1, 5 and 25ng/L significantly suppressed testicular esr1 mRNA expression in male. The ovarian esr2a mRNA expression was significantly up-regulated at 1ng/L EE2. In female brain, esr1 mRNA expression was significantly down-regulated at 5ng/L EE2. Both in males and females, EE2 exposure increased the hepatic esr1 mRNA expression in a concentration-dependent manner. The present study suggests that different esrs in different tissues have differential responsiveness to EE2 and the hepatic esr1 is a sensitive biomarker to EE2 at environmental concentrations in P. dabryanus juveniles. So, the loach P. dabryanus, a typical demersal fish, is a promising ecological model organism to detect estrogenic chemicals in the sediment of aquatic environment by using molecular biomarkers.     

Molecular analysis of DHFR and DHPS genes in P. falciparum clinical isolates from the Haut--Ogooué region in Gabon

The main objective of this work was to determine the prevalence of mutations in genes coding for the dihydropteroate synthase (DHPS) and the dihydrofolate reductase (DHFR) enzymes which are implicated in resistance of P. falciparum to antifolate (pyrimethamine-sulfadoxine (P/S)). In this study, 117 human blood samples were collected at Franceville located in the region of Haut-Ogooué (South-eastern Gabon). In this area, a relatively low level of sensitivity of Plasmodium falciparum to P/S has been reported with 18.2% of RII and 12.1% of RI resistance. A nested polymerase chain reaction was used to amplify a fragment of the DHFR gene containing codon 108, where a point mutation causing a Serine (wild type) to Asparagine or to a Threonine (resistant types) change occurs in pyrimethamine resistant parasites. Eleven DHFR fragments were sequenced and mutations occurring at codons 51, 59 and 108 were analysed. The DHPS gene was amplified by polymerase chain reaction (PCR) and sequenced directly or after cloning. Variant amino acid residues 436, 437, 540, 581, 613 associated with sulfadoxine resistance were analysed. The analysis of codon 108 of the DHFR gene was undertaken for 81 isolates. More than one DHFR P. falciparum genotype was present in 64% of the samples. We showed that 47% of 141 DHFR gene PCR products had Serine (wild phenotype), and 52% had Asparagine. We found one isolate with the Thr-108 confirmed by sequencing of the PCR product. Triple, double and single DHFR mutant at positions 51, 59 and 108 were found. Only codons 436 and 437 of the 38 analysed sequences of the DHPS gene revealed point mutations. These results have been compared with those reported from different sites in Africa, Asia or South-America.     

Analysis of Real-Time SYBR-Polymerase Chain Reaction Cycle Threshold for Diagnosis of the α-Thalassemia-1 Southeast Asian Type Deletion: Application to Carrier Screening and Prenatal Diagnosis of Hb Bart's Hydrops Fetalis

Without gel electrophoresis and specific probes, the two tubes real-time SYBR-polymerase chain reaction (SYBR-PCR) was setup by using different primer sets: P1/P2 for the detection of wild type α-globin gene alleles and P1/P3 for detection of the allele bearing the Southeast Asian (SEA) type (– –^SEA) deletion. Analyses of the cycle threshold (C_T) values obtained by each primer set together with a delta-cycle threshold (ΔC_T) and C_T ratio, showed that lower C_T values generated by primer sets P1/P2 and P1/P3 were observed in normal and Hb Bart's hydrops fetalis subjects, respectively. In heterozygous subjects the C_T values generated by both sets of primers were similar to each other. There was no overlapping of ΔC_T and C_T ratio between normal, heterozygous and Hb Bart's hydrops fetalis subjects. Therefore, the two tubes real-time SYBR-PCR could represent a rapid, cost effective, high-throughput assay for screening of carriers and prenatal diagnosis of α-thalassemia-1 (α-thal-1) with the SEA type (– –^SEA) deletion.