Detection of CRISPR/Cas9-Mediated Fetal Hemoglobin Reactivation in Erythroblasts Derived from Cord Blood-Hematopoietic Stem Cells.

Reactivation of the fetal hemoglobin (HbF) in adult erythroid cells via genome editing is a strategy for the treatment of ß-thalassemia and sickle cell disease. In related reports, the reactivation of HbF is regularly examined in erythroblasts which are generated from the adult CD34+ hematopoietic stem and progenitor cells (HSPCs). However, the procurement of adult HSPCs, either from the bone-marrow (BM) or from mobilized peripheral-blood (mPB), is difficult. Cord-blood (CB) is a readily available source of HSPCs. CB-HSPCs, however, produce high quantities of HbF following differentiation into the erythroid lineage-a potential drawback in such studies. Here, we have edited the BCL11A enhancer (a well-characterized HbF-quantitative trait loci or QTL) via CRISPR/Cas9 in order to determine whether HbF reactivation could be detected in CB-HSPC-derived erythroblasts. In the edited erythroblasts, insertion/deletion (indel) frequencies of 74.0-80.4% and BCL11A RNA reduction levels of 92.6 ± 5.1% (P < 0.0001) were obtained. In turn, the γ/ß-globin transcript ratios were increased from 11.3 ± 1.1-fold to 77.1 ± 2.0-fold, i.e., by 6.8-fold (P < 0.0001)-and the HbF% levels increased from 34.3% in the control population to 43.5% in the BCL11A edited erythroblasts. Our results suggest that γ-globin/HbF reactivation via genome editing can be detected in CB-HSPCs generated erythroblasts-rendering CB-HSPCs a useful model for similar studies.

Characterizing Homozygous Variants in Bardet-Biedl Syndrome-Associated Genes Within Iranian Families: Unveiling a Founder Variant in BBS2, c.471G>A.

Bardet-Biedl syndrome (BBS) is a rare inherited ciliopathy disorder characterized by a broad spectrum of clinical symptoms such as retinal dystrophy, obesity, polydactyly, genitourinary and kidney anomalies, learning disability, and hypogonadism. The understanding of the variants involved in BBS-causing genes remains incomplete, highlighting the need for further research to develop a molecular diagnostic strategy for this syndrome. Singleton whole-exome sequencing (WES) was performed on sixteen patients. Our study revealed (1) nine patients carried eight homozygous pathogenic variants with four of them being novel (2) Specifically, a synonymous splicing variant (c.471G > A) in BBS2 gene in six patients with Baloch ethnicity. The identification of runs of homozygosity (ROH) calling was performed using the BCFtools/RoH software on WES data of patients harboring c.471G > A variant. The presence of shared homozygous regions containing the identified variant was confirmed in these patients. In-silico analysis predicted the effect of the c.471G > A variants on BBS2 mRNA splicing. This variant results in disrupted wild-type donor site and intron retention in the mature mRNA. (3) And a deletion of exons 14 to 17 in the BBS1 gene was identified in one patient by Copy-Number Variation (CNV) analysis using the ExomeDepth pipeline. Our results identified the founder variant c.471G > A in the BBS2 gene in the Baloch ethnicity of the Iranian population. This finding can guide the diagnostic approach of this syndrome in future studies.

Comparing four laboratory three-parent techniques to construct human aged non-surrounded nucleolus germinal vesicle oocytes: A case-control study.

BACKGROUND: The three-parent assisted reproductive technique may increase oocyte competence. OBJECTIVE: In this case-control study, the suitability of germinal vesicle transfer (GVT), synchronous ooplasmic transfer (sOT), asynchronous ooplasmic transfer using cryopreserved MII oocyte (caOT), and asynchronous ooplasmic transfer using waste MII oocyte (waOT) for maturation of the human-aged non-surrounded nucleolus germinal vesicle-stage (NSN-GV) oocyte were investigated. MATERIALS AND METHODS: NSN-GV oocytes were subjected to four methods: group A (GVT), B (sOT), C (caOT) D (waOT), and E (Control). The fusion rates, MI, MII, ICSI observations and cleavage at 2-cell, 4-cell, and 8-cell stages were compared in the groups. RESULTS: In GVT, none of the oocytes fused. In sOT, all oocytes fused, 20 achieved the MI, 14 progressed to MII, 8 fertilized, 6 cleaved and 5, 4, and 3 achieved the 2-cells, 4-cells and 8-cells, respectively. In caOT, all oocytes fused and achieved the MI, 8 progressed to MII and fertilized, 6 cleaved and 6, 5, and 5 achieved the 2-cells, 4-cells, and 8-cells respectively. In waOT, all oocytes fused, 5 and 3 progressed to MI and MII, respectively, but only one fertilized, cleaved and reached a 4-cells stage. In group E, 6 and 2 oocytes progressed to MI and MII, respectively, and only one fertilized but arrested at the zygote stage. caOT had the highest survival rate when compared to sOT (p = 0.04), waOT (p = 0.002), and control (p = 0.001). CONCLUSION: The caOT method was beneficial over sOT, waOT, and GVT in supplementing the developmental capacity of human-aged NSN-GV oocytes.

The effect of paternal age on semen quality and fertilization outcome in men with normal sperm DNA compaction, reactive oxygen species, and total antioxidant capacity levels.

OBJECTIVE: Studies showed a decrease of the semen analysis parameters and an increase in the average age of first-time fathers over the past several decades. The aim of the present study was to assess the influence of paternal age on semen quality and fertilization outcomes in men with normal sperm DNA fragmentation and chromatin maturation index (DFI and CMI), reactive oxygen species (ROS), and total antioxidant capacity (TAC) levels. MATERIAL AND METHODS: The study was performed on 70 men with their wife's age ≤38 years and normal sperm DFI, CMI, ROS, and TAC levels. None of the couples had a history of genital inflammation, chronic diseases, endocrine abnormality, chromosomal aberrations, Y chromosome microdeletion, azoospermia, and leukocytospermia. These men were separated into 2 groups according to their age (group A: age <45 years and group B: age ≥45 years). Semen analysis and fertilization outcome after using the intracytoplasmic sperm injection were assessed in both groups. RESULT: Sperm concentration showed a significant reduction in group B (p=0.04). Although semen volume, sperm normal morphology, and progressive motility were decreased in group B, the reduction was not significant when compared with group A (p=0.09, p=0.47, and p=0.77, respectively). In addition, the differences of embryo quality with grades A, B, and C and 8-cell embryo formation were not statistically significant between the 2 groups. CONCLUSION: These results demonstrated that in men with normal sperm DFI, CMI, ROS, and TAC levels, there were no significant changes in semen parameters and fertilization outcomes with an increasing age.

A panel of noncoding RNAs in non-small-cell lung cancer.

Non-small-lung cancer (NSCLC) is the leading cause of cancer death. Early detection of NSCLC could pave the way for effective therapies. Analysis of molecular genetic biomarkers in biological fluids has been proposed as a useful tool for cancer diagnosis. Here, we aimed to develop a panel of noncoding RNAs (ncRNAs) in sputum for NSCLC early detection. Expression of 11 ncRNAs were analyzed by real-time polymerase chain reaction in sputum samples of 30 NSCLC patients and 30 sex- and age-matched cancer-free controls. Stability of endogenous microRNAs (miRNAs) in sputum was evaluated after 3 and 6 days at 4°C, 6 months, and 1 year at -80°C. Nine ncRNAs showed significant differences of their expression in sputum between NSCLC patients and controls. A logistic regression model with the best prediction was built based on miR-145, miR-126, and miR-7. The composite of the three miRNAs produced 90% sensitivity and specificity in distinguishing NSCLC patients from the controls. Results indicate that miRNAs could be useful biomarkers based on their stability under various storage conditions and maintain differential changes between cancer and control groups. Moreover, measurement of miRNAs in sputum could be a noninvasive approach for detection of lung cancer.

Clinically Significant Dysregulation of hsa-miR-30d-5p and hsa-let-7b Expression in Patients with Surgically Resected Non-Small Cell Lung Cancer.

BACKGROUND: The cyclin E2 (CYCE2) is an important regulator in the progression and development of NSCLC, and its ectopic expression promoted the proliferation, invasion, and migration in several tumors, including Non-Small Cell Lung Cancer (NSCLC). However, the upregulation of CYCE2 in NSCLC cells suggested that it has a key role in tumorigenicity. In addition, the RAS family proteins as oncoproteins were activated in many major tumor types and its suitability as the therapeutic target in NSCLC was proposed. Considering the crucial role of microRNAs, it was hypothesized that altered expression of hsa-miR-30d-5p and hsa-let-7b might provide a reliable diagnostic tumor marker for diagnosis of NSCLC. METHOD: Real-time RT-PCR approach could evaluate the expression alteration of hsa-miR-30d-5p and hsa-let-7b and it was related to the surgically resected tissue of 24 lung cancer patients and 10 non-cancerous patients. The miRNAs expression was associated with clinicopathological features of the patients. RESULTS: Hsa-miR-30d showed a significant downregulation (p=0.0382) in resected tissue of NSCLC patients compared with control group. Its expression level could differentiate different stages of malignancies from each other. The ROC curve analysis gave it an AUC=0.73 (p=0.037) which was a good score as a reliable biomarker. In contrast, hsa-let-7b was significantly overexpressed in tumor samples (p=0.03). Interestingly, our findings revealed a significant association of hsa-let-7b in adenocarcinoma tumors, compared to Squamous Cell Carcinomas (SCC) (p<0.05). Also, analysis of ROC curve of hsa-let-7b (AUC=0.74, p-value=0.042) suggests that it could be as a suitable biomarker for NSCLC. CONCLUSION: Together, these results suggest a possible tumor suppressor role for hsa-miR-30d in lung tumor progression and initiation. Moreover, upregulation of hsa-let-7b was associated with the tumor type.

A new hereditary colorectal cancer network in the Middle East and eastern mediterranean countries to improve care for high-risk families.

Colorectal cancer (CRC) has a very high incidence in the western world. Data from registries in the Middle East showed that the incidence of CRC is relatively low in these countries. However, these data also showed that CRC incidence has increased substantially over the past three decades and that a high proportion of cases are diagnosed at an early age (<50 years). In view of these findings, more attention should be paid to prevention. Because of the often limited financial resources, focused screening of individuals with hereditary CRC, in particular those with Lynch syndrome, appears to be the most cost-effective strategy. During recent meetings of the Palestinian Society of Gastroenterology and the Mediterranean Task force for Cancer Control (MTCC) in Jericho, and the Patient's Friends Society of Jerusalem in Hebron the issue of hereditary CRC in the Middle East was discussed and the idea was conceived to establish a network on hereditary colorectal cancer (HCCN-ME) with the goal of improving care for high-risk groups in the Middle East and (Eastern) Mediterranean Countries.

Experimental strategies towards increasing intracellular mitochondrial activity in oocytes: A systematic review.

PURPOSE: The mitochondrial complement is critical in sustaining the earliest stages of life. To improve the Assisted Reproductive Technology (ART), current methods of interest were evaluated for increasing the activity and copy number of mitochondria in the oocyte cell. METHODS: This covered the researches from 1966 to September 2015. RESULTS: The results provided ten methods that can be studied individually or simultaneously. CONCLUSION: Though the use of these techniques generated great concern about heteroplasmy observation in humans, it seems that with study on these suggested methods there is real hope for effective treatments of old oocyte or oocytes containing mitochondrial problems in the near future.

Association between polymorphisms in Interleukin-16 gene and risk of late-onset Alzheimer's disease.

Cytokines play important roles in the inflammation pathways. Alzheimer's disease (AD) is an inflammatory disease, and it is suggested that cytokines are able to influence AD. We investigated the association between IL16 polymorphisms and risk of AD in an Iranian population. The case group consisted of 144 individuals with AD and the control group included 173 healthy individuals. Genotyping of the IL16 rs4072111 C>T and rs1131445 T>C polymorphisms was determined using PCR-RFLP method. The frequency of rs4072111 CT genotype was significantly lower (P=0.007; OR=0.5, 95% CI: 0.3-0.8) in the patients (17.3%) than the control group (30%). The rs4072111 T allele was significantly lower (P=0.008; OR=0.5, 95% CI: 0.3-0.9) in the cases (8.6%) compared with the control group (15.6%). The IL16 rs4072111 polymorphism may be associated with susceptibility to AD and the T allele may have a protective role in the progression of AD in an Iranian population.

Mutation Analysis of SLC20A2 and SPP2 as Candidate Genes for Familial Idiopathic Basal Ganglia Calcification.

BACKGROUND: Familial Idiopathic Basal Ganglia Calcification (IBGC) is a rare neurodegenerative disorder which is usually transmitted as an autosomal dominant trait. IBGC is genetically heterogeneous and SLC20A2, on chromosome 8p21.1-8q11.23, is the first gene found in IBGC-affected patients with varied ancestry. On the other hand, several candidate genes for IBGC on chromosome 2q37, including the SPP2 gene, may play a role in inhibiting calcification. METHODS: Totally, 22 members of a three generational Iranian family affected by IBGC, with an autosomal dominant pattern of inheritance were included in this study. DNA was extracted from the whole blood using standard salting out method. To find a mutation responsible for IBGC, we sequenced the coding region of SLC20A2 as well as promoter and coding region of SPP2 in the index subject of IBGC-affected family. RESULTS: Pathogenic mutation was found neither in SLC20A2 nor in SPP2. CONCLUSION: Our results strengthen genetic heterogeneity of this condition. Additional mutation studies are necessary to find a gene or genes responsible for IBGC in this affected family.

Molecular analysis of a consanguineous Iranian polycystic kidney disease family identifies a PKD2 mutation that aids diagnostics.

BACKGROUND: Polycystic kidney diseases (PKD) are a group of monogenic disorders that are inherited dominantly (autosomal dominant PKD; ADPKD) or recessively, including, autosomal recessive PKD (ARPKD). A number of recessive, syndromic disorders also involve PKD but have a range of pleiotropic phenotypes beyond the kidney, and are enriched in consanguineous families. CASE PRESENTATION: We describe here a consanguineous Iranian pedigree in which PKD was diagnosed in four generations, but also included cases with additional abnormalities, including mental retardation. We employed molecular screening to reveal the etiology of the PKD. Since the PKD seemed to be dominantly inherited, molecular diagnostics was performed by direct sequencing of the ADPKD genes, PKD1 and PKD2. Clinical and imaging data was collected on family members. The sequence analysis revealed a PKD2 single base-pair deletion, c.1142delG, and segregation was demonstrated in 16 PKD patients from different branches of the family. In keeping with other reports, the PKD2 phenotype in this family was overall mild, and characterized by conserved kidney function, although 12 cases had some evidence of renal insufficiency. Several younger mutation carriers had borderline or no clinical characteristics of ADPKD, while a patient that required a renal transplant at 14 y did not have the PKD2 mutation. CONCLUSIONS: The molecular analysis of an Iranian family showed that the PKD was due to a PKD2 mutation. The identification of the causative mutation allowed an accurate diagnosis in a number of individuals with equivocal imaging data. Consequently, these patients could be followed appropriately as at-risk individuals. In addition, the PKD2 diagnosis ruled out a syndromic form of PKD as the cause of the additional phenotypes in the family.

Post marketing surveillance on safety and efficacy of IMOD in Iranian patients with HIV/AIDS.

Acquired immune deficiency syndrome is one of the world's serious health problems. Immune-based therapy is a new approach in the treatment of HIV infected patients. IMOD™ with the ability to correct immune deficiencies has been introduced for the management of HIV infection. In the phase IV trial study the main objectives were to assess the possible side effects, evaluate its effect on CD4⁺ T lymphocyte count and patients' and physicians' satisfactions for 600 HIV infected patients in 13 centers during 2007. The observed adverse events in patients included: headache and vertigo (1.2%), nausea (1.2%), gastritis (1.2%), phlebitis (1%) and mild rash (1%); serious adverse events were not observed in any of IMOD™ recipients. Therefore it was not needed to terminate the treatment in any of patient. The results of this study demonstrated that daily prescription of IMOD™ significantly increases T lymphocyte CD4⁺ and total lymphocyte count in HIV-positive patients. In addition, nearly 90% of the patients and 70% physicians are satisfied by IMOD™ treatment.

Screening for large rearrangements of the RB1 gene in Iranian patients with retinoblastoma using multiplex ligation-dependent probe amplification.

PURPOSE: To screen deletions/duplications of the RB1 gene in a large cohort of Iranian patients using the multiplex ligation-dependent probe amplification (MLPA) technique. METHODS: A total of 121 patients with retinoblastoma, involving 55 unilateral and 66 bilateral or familial retinoblastomas, were included in this study. Among these patients, 121 blood and 43 tissue samples were available. DNA was extracted from the blood and tissue samples and analyzed with an RB1-specific MLPA probe set. The mutation findings were validated with SYBR Green Real-Time PCR. RESULTS: Twenty-two mutations were found in 21 patients; of these, ten mutations were detected in patients with isolated unilateral retinoblastoma. CONCLUSIONS: Our results suggested that MLPA is a fast, reliable, and powerful method for detecting deletions/duplications in patients with retinoblastoma.

Genotyping of Polymorphic Microsatellite Markers Linked to RB1 Locus in Iranian Population.

BACKGROUND: Retinoblastoma is the most common intraocular tumor in childhood and mutation in the RB1 gene will trigger the tumorigenesis. So far, a wide range of the mutations along the length of RB1 gene have been reported. However, some could not be detected by common detection methods. In such condition, linkage analysis using microsatellite markers is suggested to trace unknown RB1 mutations in the affected family. The aim of the present study was to evaluate the heterozygosity rates and genotyping of three microsatellite markers near or inside of the RB1 gene. METHODS: Totally, 120 unrelated healthy people from Fardis, Karaj, Iran were recruited and from each participant genomic DNA was extracted from 5 ml of peripheral blood. Three microsatellite markers D13S153, D13S156 and D13S128 located within or adjacent to the RB1 gene were selected for linkage analysis. The reliability of microsatellite markers and linkage analysis were investigated in 10 members of 2 families with familial retinoblastoma. RESULTS: Our results showed that heterozygosity rates for the three markers D13S153, D13S156 and D13S128 were 74, 70 and 78%, respectively. On the other hand, 2 and 36 out of 120 people were homozygote and heterozygous for all loci, respectively. CONCLUSION: Given the heterozygosity rates, it may be concluded that all microsatellite markers D13S153, D13S156 and D13S128 are informative and have a high rate of heterozygosity and sensitivity. Therefore, tracing the unknown RB1 mutated alleles using linkage analysis in Iranian family with familial retinoblastoma could be recommended by means of these three microsatellite markers.

RB1 gene mutations in Iranian patients with retinoblastoma: report of four novel mutations.

Mutations in the RB1 gene lead to retinoblastoma, which is the most common intraocular tumor in children under the age of 6. In the present survey, the mutations of 18 unrelated Iranian retinoblastoma patients were characterized. Mutation analysis of the RB1 gene was performed in patients by sequencing all coding regions and by multiplex ligation probe-dependent amplification analysis. Clinical signs and symptoms of the retinoblastoma patients were similar to those of previously described patients with retinoblastoma. Eight known mutations and four novel mutations (c.832_833insT, c.1943delC, c.1206C>T, and c.2029delG) were determined. In silico analysis of the c.1206C>T variant showed that exon 12 contained an SC-35 consensus sequence, and this variation disrupted the splicing enhancer element and caused skipping of exon 12. Molecular genetic testing of retinoblastoma patients greatly affects the genetic counseling of the families involved, as well as the management of the disease in patients and at-risk relatives.