Application of diagnostic methods and molecular diagnosis of hemoglobin disorders in Khuzestan province of Iran

BACKGROUND:

The hemoglobinopathies refer to a diverse group of inherited disorders characterized by a reduced synthesis of one or more globin chains (thalassemias) or the synthesis of structurally abnormal hemoglobin (Hb). The thalassemias often coexist with a variety of structural Hb variants giving rise to complex genotypes and an extremely wide spectrum of clinical and hematological phenotypes. Hematological and biochemical investigations and family studies provide essential clues to the different interactions and are fundamental to DNA diagnostics of the Hb disorders. Although DNA diagnostics have made a major impact on our understanding and detection of the hemoglobinopathies, DNA mutation testing should never be considered a shortcut or the test of first choice in the workup of a hemoglobinopathy.

MATERIALS AND METHODS:

A careful three-tier approach involving: (1) Full blood count (2) Special hematological tests, followed by (3) DNA mutation analysis, provides the most effective way in which to detect primary gene mutations as well as gene-gene interactions that can influence the overall phenotype. With the exception of a few rare deletions and rearrangements, the molecular lesions causing hemoglobinopathies are all identifiable by PCR-based techniques. Furthermore, each at-risk ethnic group has its own combination of common Hb variants and thalassemia mutations. In Iran, there are many different forms of α and β thalassemia. Increasingly, different Hb variants are being detected and their effects per se or in combination with the thalassemias, provide additional diagnostic challenges.

RESULTS:

We did step-by-step diagnosis workup in 800 patients with hemoglobinopathies who referred to Research center of Thalassemia and Hemoglobinopathies in Shafa Hospital of Ahwaz Joundishapour University of medical sciences, respectively. We detected 173 patients as iron deficiency anemia (IDA) and 627 individuals as thalassemic patients by use of different indices. We have successfully detected 75% (472/627) of the β-thalassemia mutations by using amplification refractory mutation system (ARMS) technique and 19% (130/627) of the β-thalassemia mutations by using Gap-PCR technique and 6% (25/627) as Hb variants by Hb electrophoresis technique. We did prenatal diagnosis (PND) for 176 couples which had background of thalassemia in first pregnancy. Result of PND diagnosis in the first trimester was 35% (62/176) affected fetus with β-thalassemia major and sickle cell disease that led to termination of the pregnancy.

CONCLUSION:

Almost all hemoglobinopathies can be detected with the current PCR-based assays with the exception of a few rare deletions. However, the molecular diagnostic service is still under development to try and meet the demands of the population it serves. In the short term, the current generation of instruments such as the capillary electrophoresis systems, has greatly simplified DNA sequence analysis.     

Identification and Characterization of Thioredoxin H-Type Gene Family in Triticum turgidum ssp. durum in Response to Natural and Environmental Factor-Induced Oxidative Stress

Plant Molecular Biology Reporter - Key message: Thioredoxin h-type isoforms are tissue-specific, differentially expressed in germinating seeds and under salinity stress and highly regulated by...     

Variation in Capsidiol Sensitivity between Phytophthora infestans and Phytophthora capsici Is Consistent with Their Host Range

Plants protect themselves against a variety of invading pathogenic organisms via sophisticated defence mechanisms. These responses include deployment of specialized antimicrobial compounds, such as phytoalexins, that rapidly accumulate at pathogen infection sites. However, the extent to which these compounds contribute to species-level resistance and their spectrum of action remain poorly understood. Capsidiol, a defense related phytoalexin, is produced by several solanaceous plants including pepper and tobacco during microbial attack. Interestingly, capsidiol differentially affects growth and germination of the oomycete pathogens Phytophthora infestans and Phytophthora capsici, although the underlying molecular mechanisms remain unknown. In this study we revisited the differential effect of capsidiol on P. infestans and P. capsici, using highly pure capsidiol preparations obtained from yeast engineered to express the capsidiol biosynthetic pathway. Taking advantage of transgenic Phytophthora strains expressing fluorescent markers, we developed a fluorescence-based method to determine the differential effect of capsidiol on Phytophtora growth. Using these assays, we confirm major differences in capsidiol sensitivity between P. infestans and P. capsici and demonstrate that capsidiol alters the growth behaviour of both Phytophthora species. Finally, we report intraspecific variation within P. infestans isolates towards capsidiol tolerance pointing to an arms race between the plant and the pathogens in deployment of defence related phytoalexins.     

Large-Scale Gene Disruption in Magnaporthe oryzae Identifies MC69, a Secreted Protein Required for Infection by Monocot and Dicot Fungal Pathogens

To search for virulence effector genes of the rice blast fungus, Magnaporthe oryzae, we carried out a large-scale targeted disruption of genes for 78 putative secreted proteins that are expressed during the early stages of infection of M. oryzae. Disruption of the majority of genes did not affect growth, conidiation, or pathogenicity of M. oryzae. One exception was the gene MC69. The mc69 mutant showed a severe reduction in blast symptoms on rice and barley, indicating the importance of MC69 for pathogenicity of M. oryzae. The mc69 mutant did not exhibit changes in saprophytic growth and conidiation. Microscopic analysis of infection behavior in the mc69 mutant revealed that MC69 is dispensable for appressorium formation. However, mc69 mutant failed to develop invasive hyphae after appressorium formation in rice leaf sheath, indicating a critical role of MC69 in interaction with host plants. MC69 encodes a hypothetical 54 amino acids protein with a signal peptide. Live-cell imaging suggested that fluorescently labeled MC69 was not translocated into rice cytoplasm. Site-directed mutagenesis of two conserved cysteine residues (Cys36 and Cys46) in the mature MC69 impaired function of MC69 without affecting its secretion, suggesting the importance of the disulfide bond in MC69 pathogenicity function. Furthermore, deletion of the MC69 orthologous gene reduced pathogenicity of the cucumber anthracnose fungus Colletotrichum orbiculare on both cucumber and Nicotiana benthamiana leaves. We conclude that MC69 is a secreted pathogenicity protein commonly required for infection of two different plant pathogenic fungi, M. oryzae and C. orbiculare pathogenic on monocot and dicot plants, respectively.     

Oomycetes, effectors, and all that jazz

Plant pathogenic oomycetes secrete a diverse repertoire of effector proteins that modulate host innate immunity and enable parasitic infection. Understanding how effectors evolve, translocate and traffic inside host cells, and perturb host processes are major themes in the study of oomycete-plant interactions. The last year has seen important progress in the study of oomycete effectors with, notably, the elucidation of the 3D structures of five RXLR effectors, and novel insights into how cytoplasmic effectors subvert host cells. In this review, we discuss these and other recent advances and highlight the most important open questions in oomycete effector biology.     

Genome sequencing reveals agronomically important loci in rice using MutMap

The majority of agronomic traits are controlled by multiple genes that cause minor phenotypic effects, making the identification of these genes difficult. Here we introduce MutMap, a method based on whole-genome resequencing of pooled DNA from a segregating population of plants that show a useful phenotype. In MutMap, a mutant is crossed directly to the original wild-type line and then selfed, allowing unequivocal segregation in second filial generation (F(2)) progeny of subtle phenotypic differences. This approach is particularly amenable to crop species because it minimizes the number of genetic crosses (n = 1 or 0) and mutant F(2) progeny that are required. We applied MutMap to seven mutants of a Japanese elite rice cultivar and identified the unique genomic positions most probable to harbor mutations causing pale green leaves and semidwarfism, an agronomically relevant trait. These results show that MutMap can accelerate the genetic improvement of rice and other crop plants.     

Impact of single-nucleotide polymorphisms at the TP53-binding and responsive promoter region of BCL2 gene in modulating the phenotypic variability of LGMD2C patients

Apoptosis of skeletal muscle fibers is a well-known event occurring in patients suffering from muscular dystrophies. In this study, we hypothesized that functional polymorphisms in genes involved in the mitochondrial apoptotic pathway might modulate the apoptotic capacity underlying the muscle loss and contributing to intrafamilial and interfamilial variable phenotypes in LGMD2C (Limb Girdle Muscular Dystrophy type 2C) patients sharing the same c.521delT mutation in SGCG gene. Detection of apoptosis was confirmed on muscle biopsies taken from LGMD2C patients using the TUNEL method. We genotyped then ten potentially functional SNPs in TP53, BCL-2 and BAX genes involved in the mitochondrial apoptotic pathway. Potential genotype-dependent Bcl-2 and p53 protein expressed in skeletal muscle was investigated using western blot and ELISA assays. The result showed that muscle cells carrying the TP53-R72R and TP53-16?bp del/del genotypes displayed an increased p53 level which could be more effective in inducing apoptosis by activation of the pro-apoptotic gene expression. In addition, the BCL2-938 AA genotype was associated with increased Bcl-2 protein expression in muscle from LGMD2C patients compared to -938CC genotype, while there was no evidence of significant difference in the BAX haplotype. Our findings suggest that increased Bcl-2 protein expression may counteract pro-apoptotic pathways and thus reduce the muscle loss. To the best of our knowledge, this is a pioneer study evaluating the role of apoptotic BCL-2 and TP53 genes in contributing to the phenotypic manifestation of c.521delT mutation in LGMD2C patients. Larger studies are needed to validate these findings.     

Effector genomics accelerates discovery and functional profiling of potato disease resistance and phytophthora infestans avirulence genes

Potato is the world's fourth largest food crop yet it continues to endure late blight, a devastating disease caused by the Irish famine pathogen Phytophthora infestans. Breeding broad-spectrum disease resistance (R) genes into potato (Solanum tuberosum) is the best strategy for genetically managing late blight but current approaches are slow and inefficient. We used a repertoire of effector genes predicted computationally from the P. infestans genome to accelerate the identification, functional characterization, and cloning of potentially broad-spectrum R genes. An initial set of 54 effectors containing a signal peptide and a RXLR motif was profiled for activation of innate immunity (avirulence or Avr activity) on wild Solanum species and tentative Avr candidates were identified. The RXLR effector family IpiO induced hypersensitive responses (HR) in S. stoloniferum, S. papita and the more distantly related S. bulbocastanum, the source of the R gene Rpi-blb1. Genetic studies with S. stoloniferum showed cosegregation of resistance to P. infestans and response to IpiO. Transient co-expression of IpiO with Rpi-blb1 in a heterologous Nicotiana benthamiana system identified IpiO as Avr-blb1. A candidate gene approach led to the rapid cloning of S. stoloniferum Rpi-sto1 and S. papita Rpi-pta1, which are functionally equivalent to Rpi-blb1. Our findings indicate that effector genomics enables discovery and functional profiling of late blight R genes and Avr genes at an unprecedented rate and promises to accelerate the engineering of late blight resistant potato varieties.