Specific Targeting of Zinc Transporter LIV-1 with Immunocytokine Containing Anti-LIV-1 VHH and Human IL-2 and Evaluation of its In vitro Antitumor Activity.

BACKGROUND: Interleukin 2 (IL-2) is a vital cytokine in the induction of T and NK cell responses, the proliferation of CD8+ T cells, and the effective treatment of human cancers, such as melanoma and renal cell carcinoma. However, widespread use of this cytokine is limited due to its short half-life, severe toxicity, lack of specific tumor targeting, and activation of Treg cells mediated by high-affinity interleukin-2 receptors. OBJECTIVE: In this study, a tumor-targeting LIV-1 VHH-mutIL2 immunocytokine with reduced CD25 (α chain of the high-affinity IL-2 receptor) binding activity was developed to improve IL-2 half-life by decreasing its renal infiltration in comparison with wild and mutant IL-2 molecules. METHODS: The recombinant immunocytokine was designed and expressed. the biological activity of the purified fusion protein was investigated in in vitro and in vivo experiments. RESULTS: The fusion protein represented specific binding to MCF7 (the breast cancer cell line) and more efficient cytotoxicity than wild-type IL-2 and mutant IL-2. the PK parameters of the recombinant immunocytokine were also improved in comparison to the IL-2 molecules. CONCLUSION: The observed results showed that LIV1-mIL2 immunocytokine could be considered an effective agent in the LIV-1-targeted treatment of cancers due to its longer half-life and stronger cytotoxicity.

MicroRNA-561-3p indirectly regulates the PD-L1 expression by targeting ZEB1, HIF1A, and MYC genes in breast cancer.

Globally, breast cancer is the second most common cause of cancer-related deaths among women. In breast cancer, microRNAs (miRNAs) are essential for both the initiation and development of tumors. It has been suggested that the tumor suppressor microRNA-561-3p (miR-561-3p) is crucial in arresting the growth of cancer cells. Further research is necessary to fully understand the role and molecular mechanism of miR-561 in human BC. The aim of this study was to investigate the inhibitory effect of miR-561-3p on ZEB1, HIF1A, and MYC expression as oncogenes that have the most impact on PD-L1 overexpression and cellular processes such as proliferation, apoptosis, and cell cycle in breast cancer (BC) cell lines. The expression of ZEB1, HIF1A, and MYC genes and miR-561-3p were measured in BC clinical samples and cell lines via qRT-PCR. The luciferase assay, MTT, Annexin-PI staining, and cell cycle experiments were used to assess the effect of miR-561-3p on candidate gene expression, proliferation, apoptosis, and cell cycle progression. Flow cytometry was used to investigate the effects of miR-561 on PD-L1 suppression in the BC cell line. The luciferase assay showed that miRNA-561-3p targets the 3'-UTRs of ZEB1, HIF1A and MYC genes significantly. In BC tissues, the qRT-PCR results demonstrated that miR-561-3p expression was downregulated and the expression of ZEB1, HIF1A and MYC genes was up-regulated. It was shown that overexpression of miR-561-3p decreased PD-L1 expression and BC cell proliferation, and induced apoptosis and cell cycle arrest through downregulation of candidate oncogenes. Furthermore, inhibition of candidate genes by miR-561-3p reduced PD-L1 at both mRNA and protein levels. Our research investigated the impact of miR-561-3p on the expression of ZEB1, HIF1A and MYC in breast cancer cells for the first time. Our findings may help clarify the role of miR-561-3p in PD-L1 regulation and point to this miR as a potential biomarker and novel therapeutic target for cancer immunotherapy.

DNA repair-related heritable photosensitivity syndromes: Mutation landscape in a multiethnic cohort of 17 multigenerational families with high degree of consanguinity.

Inherited photosensitivity syndromes are a heterogeneous group of genetic skin disorders with tremendous phenotypic variability, characterized by photosensitivity and defective DNA repair, especially nucleotide excision repair. A cohort of 17 Iranian families with heritable photosensitivity syndromes was evaluated to identify their genetic defect. The patients' DNA was analyzed with either whole-exome sequencing or RNA sequencing (RNA-Seq). The interpretations of the genomic results were guided by genome-wide homozygosity mapping. Haplotype analysis was performed for cases with recurrent mutations. RNA-Seq, in addition to mutation detection, was also utilized to confirm the pathogenicity. Thirteen sequence variants, including six previously unreported pathogenic variants, were disclosed in 17 Iranian families, with XPC as the most common mutated gene in 10 families (59%). In one patient, RNA-Seq, as a first-tier diagnostic approach, revealed a non-canonical homozygous germline variant: XPC:c.413-9 T > A. The Sashimi plot showed skipping of exon 4 with dramatic XPC down-expression. Haplotype analysis of XPC:c.2251-1 G>C and XPC:1243 C>T in four families showed common haplotypes of 1.7 Mb and 2.6 Mb, respectively, denoting a founder effect. Lastly, two extremely rare cases were presented in this report: a homozygous UVSSA:c .1990 C>T was disclosed, and ERCC2-related cerebro-oculo-facio-skeletal (COFS) syndrome with an early childhood death. A direct comparison of our data with the results of previously reported cohorts demonstrates the international mutation landscape of DNA repair-related photosensitivity disorders, although population-specific differences were observed.

First report of a patient with homozygous hemoglobin Ernz: Evidence to support a non-pathogenic variant.

Hemoglobin Ernz (Hb Ernz) is a missense variant in ß-globin caused by a Threonine to Asparagine substitution at the 123rd amino acid position and HBB c.371C > A in gene level. Hb Ernz has been classified as Uncertain Significance (VUS) by ACMG due to limited reports and the absence of any homozygote genotypes. In our study, we found eight cases of Hb Ernz by DNA sequencing of the ß-globin gene during >20 years of Thalassemia Screening in individuals with borderline hematological parameters who were possible carriers of thalassemia or their spouses. We also report the first homozygote variant of Hb Ernz. Our findings suggest that the changes in hematological parameters observed in individuals with Hb Ernz are likely due to α-globin gene mutations rather than Hb Ernz itself. These findings support the reclassification of Hb Ernz as a benign variant in variant classification.

Low incidence of microsatellite instability in gastric cancers and its association with the clinicopathological characteristics: a comparative study.

Gastric cancer is a complex heterogeneous disease with different molecular subtypes that have clinical implications. It is characterized by high mortality rates and limited effective therapies. Microsatellite instability (MSI) has been recognized as a subgroup with a good prognosis based on TCGA and ACRG categorizations. Besides its prognostic and predictive value, gastric cancers with high MSI exhibit different clinical behaviors. The prevalence of high MSI has been assessed in gastric cancer worldwide, especially in East Asia, but there is a lack of such information in the Middle East. Therefore, this study aimed to investigate the incidence and status of MSI in Iranian gastric cancer patients using 53 samples collected from 2015 to 2020 at Taleghani Hospital Medical Center. DNA from tumoral and normal tissues were extracted and assessed through multiplex-PCR based on five mononucleotide repeats panel. Clinicopathological variables, including age, sex, Lauren classification, lymph node involvement, TNM stage, differentiation, localization, and tumor size, were also analyzed. With 2 males and 2 females, high microsatellite instability represented a small subgroup of almost 7.5% of the samples with a median age of 60.5 years. High microsatellite instability phenotypes were significantly associated with patients aged 68 years and older (p­value of 0.0015) and lower lymph node involvement (p­value of 0.0004). Microsatellite instability was also more frequent in females, with distal gastric location, bigger tumor size, and in the intestinal type of gastric cancer rather than the diffuse type.

Identification of Novel Mutations in the MMAA and MUT Genes among Methylmalonic Aciduria Families.

Background: Methylmalonic aciduria is a rare inherited metabolic disorder with autosomal recessive inheritance pattern. There are still MMA patients without known mutations in the responsible genes. This study aimed to identify mutations in Iranian MMA families using autozygosity mapping and NGS. Methods: Multiplex PCR was performed on DNAs isolated from 12 unrelated MMA patients and their family members using 19 STR markers flanking MUT, MMAA, and MMAB genes, followed by Sanger sequencing. WES was carried out in the patients with no mutation. Results: Haplotype analysis and Sanger sequencing revealed two novel, mutations, A252Vf*5 and G87R, within the MMAA and MUT genes, respectively. Three patients showed no mutations in either autozygosity mapping or NGS analysis. Conclusion: High-frequency mutations within exons 2 and 3 of MUT gene and exon 7 of MMAB gene are consistent with the global expected frequency of genetic variations among MMA patients.

Mutational Analysis and Genotype Investigation of Less Known Gaucher Mutations through Haplotype Analysis in Iranian Gaucher Patients.

Gaucher's disease (GD) is the most frequent lysosomal storage disorder resulting from a deficiency of the enzyme glucocerebrosidase (GBA) which causes the accumulation of glucocerebroside. More than 500 mutations have been reported on the GBA gene so far. In this study, we aimed to investigate more on the genotype of less known mutations through haplotype analysis to explain their disease-causing inheritance. Eight patients and three carriers from nine different families were enrolled in the study. DNA sequencing of all GBA gene's exons was performed and pathogenicity of the mutations was investigated. Using GBA gene-linked STR markers, allele segregations were determined in some families. A total of six different mutations were determined. Five and three patients were identified to carry mutations in homozygous and compound heterozygote patterns respectively, three participants also were identified as carriers. The most prevalent mutations were c.1448 T>C and RecNcil, however, three less common mutations were identified (i.e., c.1223 C>T, c.1315 A>G, and c.1214 G>C). In conclusion, we evaluated six different mutations in Iranian patients and elucidated the inheritance of the three less-known mutations by linkage analysis.

In silico Analysis of Two Novel Variants in the Pyruvate Carboxylase (PC) Gene Associated with the Severe Form of PC Deficiency.

Background: Inborne errors of metabolism are a common cause of neonatal death. This study evaluated the acute early-onset metabolic derangement and death in two unrelated neonates. Methods: Whole-exome sequencing (WES), Sanger sequencing, homology modeling, and in silico bioinformatics analysis were employed to assess the effects of variants on protein structure and function. Results: WES revealed a novel homozygous variant, p.G303Afs*40 and p.R156P, in the pyruvate carboxylase (PC) gene of each neonate, which both were confirmed by Sanger sequencing. Based on the American College of Medical Genetics and Genomics guidelines, the p.G303Afs*40 was likely pathogenic, and the p.R156P was a variant of uncertain significance (VUS). Nevertheless, a known variant at position 156, the p.R156Q, was also a VUS. Protein secondary structure prediction showed changes in p.R156P and p.R156Q variants compared to the wild-type protein. However, p.G303Afs*40 depicted significant changes at C-terminal. Furthermore, comparing the interaction of wild-type and variant proteins with the ATP ligand during simulations, revealed a decreased affinity to the ATP in all the variants. Moreover, analysis of Single nucleotide polymorphism impacts on PC protein using Polyphen-2, SNAP2, FATHMM, and SNPs&GO servers predicted both R156P and R156Q as damaging variants. Likewise, free energy calculations demonstrated the destabilizing effect of both variants on PC. Conclusion: This study confirmed the pathogenicity of both variants and suggested them as a cause of type B Pyruvate carboxylase deficiency. The results of this study would provide the family with prenatal diagnosis and expand the variant spectrum in the PC gene,which is beneficial for geneticists and endocrinologists.

Genetic attributes of Iranian cystic fibrosis patients: the diagnostic efficiency of CFTR mutations in over a decade.

Objectives: Cystic fibrosis (CF) is the most prevalent autosomal recessive disorder among Caucasians. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause this pathology. We, therefore, aimed to describe the CFTR mutations and their geographical distribution in Iran. Method: The mutation spectrum for 87 families from all Iranian ethnicities was collected using ARMS PCR, Sanger sequencing, and MLPA. Results: Mutations were identified in 95.8% of cases. This dataset revealed that the most frequent mutations in the Iranian population were F508del, c.1000C>T, c.1397C>G, c.1911delG, and c.1393-1G>A. In addition, we found weak evidence for Turkey being the possible geographical pathway for introducing CFTR mutations into Iran by mapping the frequency of CFTR mutations. Conclusion: Our descriptive results will facilitate the genetic detection and prenatal diagnosis of cystic fibrosis within the Iranian population.

Cohort profile update: Tehran cardiometabolic genetic study.

The Tehran cardiometabolic genetic study (TCGS) is a large population-based cohort study that conducts periodic follow-ups. TCGS has created a comprehensive database comprising 20,367 participants born between 1911 and 2015 selected from four main ongoing studies in a family-based longitudinal framework. The study's primary goal is to identify the potential targets for prevention and intervention for non-communicable diseases that may develop in mid-life and late life. TCGS cohort focuses on cardiovascular, endocrine, metabolic abnormalities, cancers, and some inherited diseases. Since 2017, the TCGS cohort has augmented by encoding all health-related complications, including hospitalization outcomes and self-reports according to ICD11 coding, and verifying consanguineous marriage using genetic markers. This research provides an update on the rationale and design of the study, summarizes its findings, and outlines the objectives for precision medicine.

Identifying and predicting the pathogenic effects of a novel variant inducing severe early onset MMA: a bioinformatics approach.

BACKGROUND: Methylmalonic acidemia (MMA) is a rare metabolic disorder resulting from functional defects in methylmalonyl-CoA mutase. Mutations in the MMAB gene are responsible for the cblB type of vitamin B12-responsive MMA. RESULTS: This study used Whole-exome sequencing (WES), Sanger sequencing, linkage analysis, and in-silico evaluation of the variants' effect on protein structure and function to confirm their pathogenicity in a 2-day-old neonate presenting an early-onset metabolic crisis and death. WES revealed a homozygous missense variant on chromosome 12, the NM_052845.4 (MMAB):c.557G > A, p.Arg186Gln, in exon 7, a highly conserved and hot spot region for pathogenic variants. After being confirmed by Sanger sequencing, the wild-type and mutant proteins' structure and function were modeled and examined using in-silico bioinformatics tools and compared to the variant NM_052845.4 (MMAB):c.556C > T, p.Arg186Trp, a known pathogenic variant at the same position. Comprehensive bioinformatics analysis showed a significant reduction in the stability of variants and changes in protein-protein and ligand-protein interactions. Interestingly, the variant c.557G > A, p.Arg186Gln depicted more variations in the secondary structure and less binding to the ATP and B12 ligands compared to the c.556C > T, p.Arg186Trp, the known pathogenic variant. CONCLUSION: This study succeeded in expanding the variant spectra of the MMAB, forasmuch as the variant c.557G > A, p.Arg186Gln is suggested as a pathogenic variant and the cause of severe MMA and neonatal death. These results benefit the prenatal diagnosis of MMA in the subsequent pregnancies and carrier screening of the family members. Furthermore, as an auxiliary technique, homology modeling and protein structure and function evaluations could provide geneticists with a more accurate interpretation of variants' pathogenicity.

Identification and characterization of the largest deletion in the PCCA gene causing severe acute early-onset form of propionic acidemia.

Whole-exome sequencing (WES) is an excellent method for the diagnosis of diseases of uncertain or heterogeneous genetic origin. However, it has limitations for detecting structural variations such as InDels, which the bioinformatics analyzers must be aware of. This study aimed at using WES to evaluate the genetic cause of the metabolic crisis in a 3-day-old neonate admitted to the neonatal intensive care unit (NICU) and deceased after a few days. Tandem mass spectrometry (MS/MS) showed a significant increase in propionyl carnitine (C3), proposing methylmalonic acidemia (MMA) or propionic acidemia (PA). WES demonstrated a homozygous missense variant in exon 4 of the BTD gene (NM_000060.4(BTD):c.1330G > C), responsible for partial biotinidase deficiency. Segregation analysis of the BTD variant revealed the homozygous status of the asymptomatic mother. Furthermore, observation of the bam file, around genes responsible for PA or MMA, by Integrative Genomics Viewer (IGV) software displayed a homozygous large deletion in the PCCA gene. Comprehensive confirmatory studies identified and segregated a novel outframe deletion of 217,877 bp length, "NG_008768.1:g.185211_403087delinsTA", extended from intron 11 to 21 of the PCCA, inducing a premature termination codon and activation of nonsense-mediated mRNA decay (NMD). Homology modeling of the mutant PCCA demonstrated eliminating the protein's active site and critical functional domains. Thereupon, this novel variant is suggested as the largest deletion in the PCCA gene, causing an acute early-onset PA. These results could expand the PCCA variants spectrum, and improve the existing knowledge on the molecular basis of PA, as well as provide new evidence of pathogenicity of the variant (NM_000060.4(BTD):c.1330G > C.

Aberrant promoter hypermethylation of miR-335 and miR-145 is involved in breast cancer PD-L1 overexpression.

PD-L1 is one of the most important immune checkpoint molecules in breast cancer that plays an important role in suppressing the immune system when confronted with tumor cells and is regulated by various microRNAs. Among them, microRNA-335-3p and microRNA-145-5p, regulated by DNA methylation, have tumor suppressor activities. We studied the role of miR-335 and -145 on PD-L1 suppression in breast cancer. The expression of miR-355 and miR-145 was significantly downregulated in BC tissues and cell lines compared to their controls, and their downregulation was negatively correlated with PD-L1 overexpression. In-silico and luciferase reporter systems confirmed that miR-335 and -145 target PD-L1. In BC tissues and cell lines, cancer-specific methylation was found in CpG-rich areas upstream of miR-335 and-145, and up-regulation of PD-L1 expression was connected with hypermethylation (r = 0.4089, P = 0.0147, and r = 0.3373, P = 0.0475, respectively). The higher levels of miR-355 and -145 in BC cells induced apoptosis, arrested the cell cycle, and reduced proliferation significantly. In summary, we found that miR-335 and -145 are novel tumor suppressors inactivated in BC, and these miRs may serve as potential therapeutic targets for breast cancer treatment.

Development of a genetic risk score for obesity predisposition evaluation.

Obesity is a major public health issue resulting from an interaction between genetic and environmental factors. Genetic risk scores (GRSs) are useful to summarize the effects of many genetic variants on obesity risk. In this study, we aimed to assess the association of previously well-studied genetic variants with obesity and develop a genetic risk score to anticipate the risk of obesity development in the Iranian population. Among 968 participants, 599 (61.88%) were obese, and 369 (38.12%) were considered control samples. After genotyping, an initial screening of 16 variants associated with body mass index (BMI) was performed utilizing a general linear model (p < 0.25), and seven genetic variants were selected. The association of these variants with obesity was examined using a multivariate logistic regression model (p < 0.05), and finally, five variants were found to be significantly associated with obesity. Two gene score models (weighted and unweighted), including these five loci, were constructed. To compare the discriminative power of the models, the area under the curve was calculated using tenfold internal cross-validation. Among the studied variants, ADRB3 rs4994, FTO rs9939609, ADRB2 rs1042714, IL6 rs1800795, and MTHFR rs1801133 polymorphisms were significantly associated with obesity in the Iranian population. Both of the constructed models were significantly associated with BMI (p < 0.05) and the area under the mean curve of the weighted GRS and unweighted GRS were 70.22% ± 0.05 and 70.19% ± 0.05, respectively. Both GRSs proved to predict obesity and could potentially be utilized as genetic tools to assess the obesity predisposition in the Iranian population. Also, among the studied variants, ADRB3 rs4994 and FTO rs9939609 polymorphisms have the highest impacts on the risk of obesity.

Mutation update: The spectra of PLEC sequence variants and related plectinopathies.

Plectin, encoded by PLEC, is a cytoskeletal linker of intermediate filaments expressed in many cell types. Plectin consists of three main domains that determine its functionality: the N-terminal domain, the Rod domain, and the C-terminal domain. Molecular defects of PLEC correlating with the functional aspects lead to a group of rare heritable disorders, plectinopathies. These multisystem disorders include an autosomal dominant form of epidermolysis bullosa simplex (EBS-Ogna), limb-girdle muscular dystrophy (LGMD), aplasia cutis congenita (ACC), and an autosomal recessive form of EBS, which may associate with muscular dystrophy (EBS-MD), pyloric atresia (EBS-PA), and/or congenital myasthenic syndrome (EBS-MyS). In this study, genotyping of over 600 Iranian patients with epidermolysis bullosa by next-generation sequencing identified 15 patients with disease-causing PLEC variants. This mutation update analyzes the clinical spectrum of PLEC in our cohort and in the literature and demonstrates the relationship between PLEC genotype and phenotypic manifestations. This study has integrated our seven novel PLEC variants and phenotypic findings with previously published data totaling 116 variants to provide the most complete overview of pathogenic PLEC variants and related disorders.

Induction of apoptosis and cell cycle arrest in colorectal cancer cells by novel anticancer metabolites of Streptomyces sp. 801.

BACKGROUND: Colorectal cancer is the third and most significant cause of death and fourth most common cancer in the world. Chemotherapy can be introduced in the cases of locally or distantly invasive colorectal cancer. In recent years Actinomycetes, especially the genus Streptomyces, contain numerous bioactive compounds, some of which are known as important anti-tumor chemotherapy drugs. In this research, we aimed to explore the anti-cancer mode of action of Streptomyces sp. 801 on colorectal cancer cells in vitro conditions. METHODS: Fermented supernatant of strain Streptomyces sp. 801 isolated from soil showed maximum growth inhibition on human colorectal cancer cells. The cytotoxic effects of various concentrations of EtOAc extract from bacterial culture supernatant on HT-29, HCT 116 and SW480 cancer cells were surveyed using the MTT assay. Moreover, flow cytometry assays and Bax, Bcl-2, Cyclin D1 and P21 gene expressions were carried out to assess the apoptotic and cell cycle effects. Also, the scratch assay was performed to measure migration. Finally, Ethyl acetate (EtOAc) extract was analyzed by LC-MS to identify anti-cancer compounds. RESULTS: The cell viability of all three cell lines were decreased in a dose-dependent manner. The successful induction of apoptosis and cell cycle arrest at IC50 values, were confirmed by flow cytometry as well as by the mRNA expression levels of the genes involved in these processes. Scratch assays indicated the inhibition of cell migration in the cancer cell lines treated by Streptomyces sp. 801. Nine anti-cancer compounds of Streptomyces sp. 801 were detected by liquid chromatography-mass spectrometry (LC-MS) analysis. CONCLUSIONS: These findings suggest that Streptomyces sp. 801 can be a source of promising anticancer metabolites.

Reporting Two Novel Mutations in Two Iranian Families with Cystic Fibrosis, Molecular and Bioinformatic Analysis

Background: Cystic fibrosis (CF) is the most common heredity disease among the Caucasian population. More than 350 known pathogenic variations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (NM_000492.4) cause CF. Herein, we report the outcome of our investigation in two unrelated Iranian families with CF patients. Methods: We conducted phenotypic examination, segregation, linkage analysis, and CFTR gene sequencing to define causative mutations. Results: We found two novel mutations in the present study. The first one was a deletion causing frameshift, c.299delT p.(Leu100Profs*7), and the second one was a missense mutation, c.1857G>T, at nucleotide binding domain 1 of the CFTR protein. Haplotype segregation data supported our new mutation findings. Conclusion: Findings of this study expand the spectrum of CFTR pathogenic variations and can improve prenatal diagnosis and genetic counseling for CF.

Ichthyosis follicularis syndromes in patients with mutations in GJB2.

Ichthyosis follicularis (IF) manifests as generalized spiny follicular projections found in syndromic diseases secondary to SREBF1 and MBTPS2 mutations. We sought the genetic cause of IF in two distinct families from a cohort of 180 patients with ichthyosis. In Family 1, the proband (Patient 1) presented with IF, bilateral sensorineural hearing loss and punctate palmoplantar keratoderma. Using DNA from peripheral blood lymphocytes, two compound heterozygous mutations, c.526A>G and c.35delG, were discovered in GJB2. In Family 2, the proband (Patient 2) presented with a previously unreported IF phenotype in the context of keratitis-ichthyosis-deafness syndrome, and whole-exome sequencing found a de novo heterozygous mutation, c.148G>A in GJB2. Histopathology was consistent with porokeratotic eccrine ostial and dermal duct naevus (PEODDN) and IF in Patients 1 and 2, respectively. Our findings add to the clinical and histopathological spectrum of IF and emphasize the association of PEODDN-like entities with GJB2 variants.

A decade of molecular preimplantation genetic diagnosis of 350 blastomeres for beta-thalassemia combined with HLA typing, aneuploidy screening and sex selection in Iran.

BACKGROUND: Preimplantation genetic diagnosis (PGD) has been developed to detect genetic disorders before pregnancy which is usually done on blastomeres biopsied from 8-cell stage embryos obtained from in vitro fertilization method (IVF). Here we report molecular PGD results for diagnosing of beta thalassemia (beta-thal) which are usually accompanied with evaluating chromosomal aneuploidies, HLA typing and sex selection. METHODS: In this study, haplotype analysis was performed using short tandem repeats (STRs) in a multiplex nested PCR and the causative mutation was detected by Sanger sequencing. RESULTS: We have performed PGDs on 350 blastomeres from 55 carrier couples; 142 blastomeres for beta-thal only, 75 for beta-thal and HLA typing, 76 for beta-thal in combination with sex selection, and 57 for beta-thal and aneuploidy screening. 150 blastomeres were transferable, 15 pregnancies were happened, and 11 babies born. We used 6 markers for beta-thal, 36 for aneuploidy screening, 32 for sex selection, and 35 for HLA typing. To our knowledge combining all these markers together and the number of STR markers are much more than any other studies which have ever done. CONCLUSIONS: PGD is a powerful diagnostic tool for carrier couples who desire to have a healthy child and wish to avoid medical abortion.

Efficacy and antitumor activity of a mutant type of interleukin 2.

Interleukin-2 (IL-2) is an important cytokine in survival, expansion, function of CD8+ T cells and natural killer cells in immunotherapy of melanoma and renal cell carcinomas. Its severe toxicity following binding to its high affinity IL-2 receptor alpha (IL-2Rα) has restricted its application in cancer patients. In the present study, we investigated the antitumor efficacy and cytotoxicity of a mutated human IL-2 previously designed by selective amino acid substitutions, and its reduced affinity towards high-affinity IL-2Rα (CD25) was approved compared to the wild type IL-2 (wtIL-2). Furthermore, their ability to induce PBMC cell proliferation, and interferon-gamma secretion was compared. The mutant IL-2 also represented higher antitumor activity and more efficient cytotoxicity than wild type hIL-2. The developed mutant IL-2 can be an alternative tool in IL-2 associated immunotherapy of various cancers.