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.

Genetic Analysis of 27 Y-STR Haplotypes in 11 Iranian Ethnic Groups.

BACKGROUND: The study of Y-chromosomal variations provides valuable insights into male susceptibility in certain diseases like cardiovascular disease (CVD). In this study, we analyzed paternal lineage in different Iranian ethnic groups, not only to identify developing medical etiology, but also to pave the way for gender-specific targeted strategies and personalized medicine in medical genetic research studies. METHODS: The diversity of eleven Iranian ethnic groups was studied using 27 Y-chromosomal short tandem repeat (Y-STR) haplotypes from Y-filer® Plus kit. Analysis of molecular variance (AMOVA) based on pair-wise RST along with multidimensional scaling (MDS) calculation and Network phylogenic analysis was employed to quantify the differences between 503 unrelated individuals from each ethnicity. RESULTS: Results from AMOVA calculation confirmed that Gilaks and Azeris showed the largest genetic distance (RST=0.35434); however, Sistanis and Lurs had the smallest considerable genetic distance (RST=0.00483) compared to other ethnicities. Although Azeris had a considerable distance from other ethnicities, they were still close to Turkmens. MDS analysis of ethnic groups gave the indication of lack of similarity between different ethnicities. Besides, network phylogenic analysis demonstrated insignificant clustering between samples. CONCLUSION: The AMOVA analysis results explain that the close distance of Azeris and Turkmens may be the effect of male-dominant expansions across Central Asia that contributed to historical and demographics of populations in the region. Insignificant differences in network analysis could be the consequence of high mutation events that happened in the Y-STR regions over the years. Considering the ethnic group affiliations in medical research, our results provided an understanding and characterization of Iranian male population for future medical and population genetics studies.

Identification of a Novel Variant in CC2D1A Gene Linked to Autosomal Recessive Intellectual Disability 3 in an Iranian Family and Investigating the Structure and Pleiotropic Effects of this Gene.

Objectives: Intellectual disability (ID) represents a significant health challenge due to its diverse and intricate nature. A multitude of genes play a role in brain development and function, with defects in these genes potentially leading to ID. Considering that many of these genes have yet to be identified, and those identified have only been found in a small number of patients, no complete description of the phenotype created by these genes is available. CC2D1A is one of the genes whose loss-of-function mutation leads to a rare form of non-syndromic ID-3(OMIM*610055), and four pathogenic variants have been reported in this gene so far. Materials & Methods: n the current study, two affected females were included with an initial diagnosis of ID who were from an Iranian family with consanguineous marriage. Whole-exome sequencing was used to identify the probable genetic defects. The Genotypic and phenotypic characteristics of the patients were compared with a mutation in the CC2D1A gene, and then the structure of the gene and its reported variants were investigated. Results: The patients carried a novel homozygous splicing variant (NM_017721, c.1641+1G>A) in intron 14, which is pathogenic according to the ACMG guideline. Loss-of-function mutations in CC2D1A have severe phenotypic consequences such as ID, autism spectrum disorder (ASD), and seizures. However, missense mutations lead to ASD with or without ID, and in some patients, they cause ciliopathy. Conclusion: This study reports the fifth novel, probably pathogenic variant in the CC2D1A gene. Comparing the clinical and molecular genetic features of the patients with loss-of-function mutation helped to describe the phenotype caused by this gene more precisely. Investigating the CC2D1A gene's mutations and structure revealed that it performs multiple functions. The DM14 domain appears more pivotal in triggering severe clinical symptoms, including ID, than the C2 domain.

Contribution of genetic variants in the development of familial premature coronary artery disease in a cohort of cardiac patients.

Coronary artery disease (CAD), the most prevalent cardiovascular disease, is the leading cause of death worldwide. Heritable factors play a significant role in the pathogenesis of CAD. It has been proposed that approximately one-third of patients with CAD have a positive family history, and individuals with such history are at ~1.5-fold increased risk of CAD in their lifespans. Accordingly, the long-recognized familial clustering of CAD is a strong risk factor for this disease. Our study aimed to identify candidate genetic variants contributing to CAD by studying a cohort of 60 large Iranian families with at least two members in different generations afflicted with premature CAD (PCAD), defined as established disease at ≤45 years in men and ≤55 years in women. Exome sequencing was performed for a subset of the affected individuals, followed by prioritization and Sanger sequencing of candidate variants in all available family members. Subsequently, apparently healthy carriers of potential risk variants underwent coronary computed tomography angiography (CCTA), followed by co-segregation analysis of the combined data. Putative causal variants were identified in seven genes, ABCG8, CD36, CYP27A1, PIK3C2G, RASSF9, RYR2, and ZFYVE21, co-segregating with familial PCAD in seven unrelated families. Among these, PIK3C2G, RASSF9, and ZFYVE21 are novel candidate CAD susceptibility genes. Our findings indicate that rare variants in genes identified in this study are involved in CAD development.

Systemic delivery of specific and efficient CRISPR/Cas9 system targeting HPV16 oncogenes using LL-37 antimicrobial peptide in C57BL/6 mice.

Human papillomavirus (HPV) type 16 is the most common sexually transmitted virus related to cervical cancer. Among different types of advanced novel therapies, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas-mediated gene editing holds great promise for cancer treatment. In this research, optimal gRNA sequences targeting HPV16 E5, E6, E7, and p97 promoter for CRISPR/Cas9-mediated genome editing were designed by in silico prediction. After cloning, delivery of the recombinant vectors into C3, TC1 and HeLa tumor cells was evaluated by Lipofectamine 2000, and LL-37 antimicrobial peptide. Then, the levels of cell cycle proteins (p21, p53, and Rb) were investigated after treatment by western blot analysis. Finally, C57BL/6 mice were inoculated with C3 tumor cells, and treated with recombinant vectors and cisplatin. Based on the tumor size reduction and IHC results, the E6 + E7-treated group with a high percentage of cleaved caspase-3 positive cells (45.75%) and low mitotic index of 2-3 was determined as the best treatment among other groups. Moreover, the potential of LL-37 peptide to overcome the CRISPR/Cas9 delivery challenge was shown for the first time. Overall, our study suggests that the CRISPR/Cas9-mediated gene editing of pre-existing tumors is effective, specific and nontoxic, and the outlook for precise gene therapy in cancer patients is very bright.

Effect of B12 and folate deficiency in hypomethylation of Angiotensin I converting enzyme 2 gene and severity of disease among the acute respiratory distress syndrome patients.

BACKGROUND: Angiotensin I converting enzyme 2 (ACE-2) is the most important receptor and has important role in the entry of corona virus to the host cells. The present study aimed to investigate the different mechanisms involved in the expression regulation of this gene among the COVID-19 patients. METHODS: A total of 140 patients with COVID-19 (n = 70 mild COVID-19, n = 70 ARDS) and 120 controls were recruited. The expression of ACE-2 and miRNAs was evaluated by quantitative real-time PCR (QRT-PCR), and methylation of CpG dinucleotides in the ACE2 promoter was quantified using bisulfite pyro-sequencing. Finally, different polymorphisms of the ACE-2 gene were studied by Sanger sequencing. RESULTS: Our results showed a significant high expression of the ACE-2 gene in the blood samples of acute respiratory distress syndrome (ARDS) patients (3.8 ± 0.77) in comparison with controls (0.88 ± 0.12; p < 0.03). The methylation rate of the ACE-2 gene in ARDS patients was 14.07 ± 6.1 compared with controls (72.3 ± 5.1; p < 0.0001). Among the four studied miRNAs, only miR200c-3p showed significant downregulation in ARDS patients (0.14 ± 0.1) in comparison with controls (0.32 ± 0.17; p < 0.001). We did not see a substantial difference in the frequency of rs182366225 C>T and rs2097723 T>C polymorphisms between patients and controls (p > 0.05). There was a significant correlation between B12 (R = 0.32, p < 0.001), folate (R = 0.37, p < 0.001) deficiency, and hypo-methylation of the ACE-2 gene. CONCLUSION: These results for the first time indicated that among the different mechanisms of ACE-2 expression regulation, its promoter methylation is very crucial and can be affected by factors involved in one-carbon metabolisms such as B9 and B12 vitamins deficiency.

ACE-2 Expression and Methylation Pattern in Bronchoalveolar Lavage Fluid and Bloods of Iranian ARDS Covid-19 Patients.

The aim of the present study was to investigate the expression and methylation pattern of the angiotensin I converting enzyme 2 (ACE-2) in acute respiratory distress syndrome (ARDS) covid-9 patients. A total of 25 patients with covid-19 ARDS and 20 controls were recruited. Expression of the ACE-2 gene was evaluated by quantitative real time PCR, and methylation of CpG dinucleotides, in the ACE-2 promoter, was quantified using bisulfite pyro-sequencing. Our results showed high expression of the ACE-2 gene in the blood samples of ADRS patients (1.93± 0.67) in comparison to controls (0.62±0.35) (P = 0.03). Correspondingly, in ARDS bronchoalveolar lavage fluid (BALF) samples, there was a high expression of this gene (1.8±0.78) in comparison to controls (0.58±0.2) (p <0.05). Moreover, the methylation rate of the ACE-2 gene in blood samples of ARDS patients was 64.07 ±6.1 in comparison to controls (80.3 ±7.3) (p<0.0001). In BALF samples, there was this pattern too (55.07 ±3.1 vs. 72.35±5.1) (p<0.0001). Finally, a significant correlation was found between expression and methylation in BALF (R= -0.54, P= 0.002) and blood (R= -0.321, P= 0.013) samples. These results indicated that aberrant methylation of the ACE-2 promoter might be associated with high expression of this gene and the occurrence of ARDS in covid-19 patients.

Screening for differentially expressed microRNAs in BALF and blood samples of infected COVID-19 ARDS patients by small RNA deep sequencing.

BACKGROUND: The pandemic COVID-19 has caused a high mortality rate and poses a significant threat to the population of the entire world. Due to the novelty of this disease, the pathogenic mechanism of the disease and the host cell's response are not yet fully known, so lack of evidence prevents a definitive conclusion about treatment strategies. The current study employed a small RNA deep-sequencing approach for screening differentially expressed microRNA (miRNA) in blood and bronchoalveolar fluid (BALF) samples of acute respiratory distress syndrome (ARDS) patients. METHODS: In this study, BALF and blood samples were taken from patients with ARDS (n = 5). Control samples were those with suspected lung cancer candidates for lung biopsy (n = 3). Illumina high-throughput (HiSeq 2000) sequencing was performed to identify known and novel miRNAs differentially expressed in the blood and BALFs of ARDS patients compared with controls. RESULTS: Results showed 2234 and 8324 miRNAs were differentially expressed in blood and BALF samples, respectively. In BALF samples, miR-282, miR-15-5p, miR-4485-3p, miR-483-3p, miR-6891-5p, miR-200c, miR-4463, miR-483-5p, and miR-98-5p were upregulated and miR-15a-5p, miR-548c-5p, miR-548d-3p, miR-365a-3p, miR-3939, miR-514-b-5p, miR-513a-3p, miR-513a-5p, miR-664a-3p, and miR-766-3p were downregulated. On the contrary, in blood samples miR-15b-5p, miR-18a-3p, miR-486-3p, miR-486-5p, miR-146a-5p, miR-16-2-3p, miR-6501-5p, miR-365-3p, miR-618, and miR-623 were top upregulated miRNAs and miR-21-5p, miR-142a-3p, miR-181-a, miR-31-5p, miR-99-5p, miR-342-5p, miR-183-5p, miR-627-5p, and miR-144-3p were downregulated miRNAs. Network functional analysis for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), in ARDS patients' blood and BALF samples, showed that the target genes were more involved in activating inflammatory and apoptosis process. CONCLUSION: Based on our results, the transcriptome profile of ARDS patients would be a valuable source for understanding molecular mechanisms of host response and developing clinical guidance on anti-inflammatory medication.

Current and future direction in treatment of HPV-related cervical disease.

Human papillomavirus (HPV) is the most common sexually transmitted virus in the world. About 70% of cervical cancers are caused by the most oncogenic HPV genotypes of 16 and 18. Since available prophylactic vaccines do not induce immunity in those with established HPV infections, the development of therapeutic HPV vaccines using E6 and E7 oncogenes, or both as the target antigens remains essential. Also, knocking out the E6 and E7 oncogenes in host genome by genome-editing CRISPR/Cas system can result in tumor growth suppression. These methods have shown promising results in both preclinical and clinical trials and can be used for controlling the progression of HPV-related cervical diseases. This comprehensive review will detail the current treatment of HPV-related cervical precancerous and cancerous diseases. We also reviewed the future direction of treatment including different kinds of therapeutic methods and vaccines, genome-editing CRISPR/Cas system being studied in clinical trials. Although the progress in the development of therapeutic HPV vaccine has been slow, encouraging results from recent trials showed vaccine-induced regression in high-grade CIN lesions. CRISPR/Cas genome-editing system is also a promising strategy for HPV cancer therapy. However, its safety and specificity need to be optimized before it is used in clinical setting.

Vitexin Induces Apoptosis in MCF-7 Breast Cancer Cells through the Regulation of Specific miRNAs Expression.

In this research, we investigated microRNAs (miRNAs) expression profile in MCF-7 breast cancer cell line which treated with 150 µM vitexin. Profiling of miRNAs expression was performed using TaqMan MiRNA Array. Apoptosis was analyzed by flow cytometry and the expression of some genes involved in "anti-proliferative" signaling pathways were evaluated by western blotting and real time PCR methods. Twenty microRNAs were differentially expressed in vitexin treated cells compared to the control. Among them, let-7- b, c were up regulated while miRNA-17-5p was down regulated with highest score. Also, we detected the expression changes of mentioned miRNAs target genes as well as genes involved in caspase apoptosis pathways. Our results provide the first evidence that vitexin can effect miRNA expression in MCF-7 cells. Also based on our finding, vitexin can be an attractive miRNA mediated chemo preventive and therapeutic agent in breast cancer.

SARS-CoV-2 outbreak in Iran: The dynamics of the epidemic and evidence on two independent introductions.

The SARS-CoV-2 virus has been rapidly spreading globally since December 2019, triggering a pandemic, soon after its emergence. While Iran was among the first countries confronted with rapid spread of virus in February 2020, no real-time SARS-CoV-2 whole-genome tracking in early phase of outbreak was performed in the country. To address this issue, we provided 50 whole-genome sequences of viral isolates ascertained from different geographical locations in Iran during March-July 2020. The corresponding analysis on origins, transmission dynamics and genetic diversity of SARS-CoV-2 virus, represented at least two introductions of the virus into the country, constructing two major clusters defined as B.4 and B.1*. The first entry of the virus might have occurred around very late 2019/early 2020, as suggested by the time to the most recent common ancestor, followed by a rapid community transmission that led to dominancy of B.4 lineage in early epidemic till the end of June. Gradually, reduction in dominancy of B.4 occurred possibly as a result of other entries of the virus, followed by surge of B.1* lineages, as of mid-May. Remarkably, variation tracking of the virus indicated the increase in frequency of D614G mutation, along with B.1* lineages, which showed continuity till October 2020. The increase in frequency of D614G mutation and B.1* lineages from mid-May onwards predicts a rapid viral transmission that may push the country into a critical health situation followed by a considerable change in composition of viral lineages circulating in the country.

A novel variant of C12orf4 linked to autosomal recessive intellectual disability type 66 with phenotype expansion.

BACKGROUND: Intellectual disability (ID) is a hallmark of many rare disorders that are highly heterogeneous and complex. A large number of specific genes are involved in development of this heterogeneity, and each of these genes is only found in a small number of patients. This weakens the definition of the predominant genotype and the phenotypic characteristics associated with that gene. Autosomal recessive ID type 66 (OMIM #618221) is one of these very rare diseases created by defects in the C12orf4 gene. METHODS: The present study included two patients from an Iranian family with initial diagnosis of non-syndromic ID, aiming to identify the possible genetic cause(s), and whole-exome sequencing (WES) was performed for the proband. The obtained variant was confirmed by Sanger sequencing and co-segregated in the family. RESULTS: The patients carried a novel pathogenic splicing variant called c.1441-1G>A in exon 12 of the C12orf4 gene (NM_001304811). They predominantly manifested ID, behavioral problems, speech impairment and dysmorphic facial features, some of which had not been reported in previous studies. CONCLUSIONS: A novel pathogenic splicing variant was identified named c.1441-1G>A in the C12orf4 gene. To date, only seven families have been reported with defects in this gene. Previous studies have not highlighted the exact clinical manifestations of these patients; thus, the present study could contribute to a better delineation of the genotype-phenotype correlation and interpretation of very rare variants of the gene.

The PTRHD1 Mutation in Intellectual Disability.

BACKGROUND: Intellectual disability (ID) is a heterogonous disorder with complex etiology. The frequency of autosomal recessive inheritance defects was elevated in a consanguineous family. METHODS: In this study, high-throughput DNA sequencing was performed in an Iranian consanguineous family with two affected individuals to find potential causative variants. Whole-exome sequencing was carried out on the proband and Sanger sequencing was implemented for validation of the likely causative variant in the family members. RESULTS: A novel homozygous missense mutation (p.Arg122Trp) was detected in the PTRHD1 gene. CONCLUSION: PTRHD1 has been recently introduced as a candidate ID and Parkinsonism causing gene. Our findings are in agreement with the clinical spectrum of PTRHD1 mutations; however, our affected individuals suffer from ID manifestations.

Underexpression of hsa-miR-449 family and their promoter hypermethylation in infertile men: A case-control study.

BACKGROUND: Post-transcriptional microRNAs (miRNAs) have a impotrant pattern in the spermatogenesis process. OBJECTIVE: Study of the expression and methylation of hsa-miR-449 family in sperm samples of infertile men. MATERIALS AND METHODS: In this case-control study, we recruited 74 infertile men (with asthenozoospermia, teratozoospermia, asthenoteratozoospermia, and oligoasthenoteratozoospermia) and 30 control samles. Methylation-specific PCR (MSP) method was used for methylation evaluation of hsa-miR-449 a, b, c promoter. By Real time PCR (qRT-PCR) method,we showed downregulation of hsa-miR-449 a, b, c in the sperm samples of infertile men and compared it to their fertile counterparts. RESULTS: There was significant underexperssion, in hsa-miR-449-b in oligoasthenoteratospermic samples (p = 0.0001, F = 2.9). About the methylation pattern, infertile men showed high frequency of methylation in the promoter of hsa-miR-449 a, b, c in comparison to controls (60.8% vs 23.3%), the highest amount of methylation was observed in oligoasthenoteratospermia samples (81.2%). CONCLUSION: In this study, low expression and high methylation of hsa-miR-449-b were observed in infertile men in compared to control samples, which can be one of the causes of defective spermatogenesis.

Clinical and Genetic Characteristics of Splicing Variant in CYP27A1 in an Iranian Family with Cerebrotendinous Xanthomatosis.

Background: Cerebrotendinous xanthomatosis (CTX) is a rare congenital lipid-storage disorder, leading to a progressive multisystem disease. CTX with autosomal recessive inheritance is caused by a defect in the CYP27A1 gene. Chronic diarrhea, tendon xanthomas, neurologic impairment, and bilateral cataracts are common symptoms of the disease. Methods: Three affected siblings with an initial diagnosis of non-syndromic intellectual disability were recruited for further molecular investigations. To identify the possible genetic cause(s), whole exome sequencing was performed on the proband. Sanger sequencing was applied to confirm the final variant. The clinical and molecular genetic features of the three siblings from the new CTX family and other patients with the same mutations, as previously reported, were analyzed. The CYP27A1 gene was also studied for the number of pathogenic variants and their location. Results: We found a homozygous splicing mutation, NM_000784: exon6: c.1184+1G>A, in CYP27A1 gene, which was confirmed by Sanger sequencing. Among the detected pathogenic variants, the splice site mutation had the highest prevalence, and the mutations were mostly found in exon 4. Conclusion: This study is the first to report the c.1184+1G>A mutation in Iran. Our findings highlight the other feature of the disease, which is the lack of relationship between phenotype and genotype. Due to nonspecific symptoms and delay in diagnosis, CYP27A1 genetic analysis should be the definitive method for CTX diagnosis.

Comprehensive genotype-phenotype correlation in AP-4 deficiency syndrome; Adding data from a large cohort of Iranian patients.

Mutations in adaptor protein complex-4 (AP-4) genes have first been identified in 2009, causing a phenotype termed as AP-4 deficiency syndrome. Since then several patients with overlapping phenotypes, comprised of intellectual disability (ID) and spastic tetraplegia have been reported. To delineate the genotype-phenotype correlation of the AP-4 deficiency syndrome, we add the data from 30 affected individuals from 12 out of 640 Iranian families with ID in whom we detected disease-causing variants in AP-4 complex subunits, using next-generation sequencing. Furthermore, by comparing genotype-phenotype findings of those affected individuals with previously reported patients, we further refine the genotype-phenotype correlation in this syndrome. The most frequent reported clinical findings in the 101 cases consist of ID and/or global developmental delay (97%), speech disorders (92.1%), inability to walk (90.1%), spasticity (77.2%), and microcephaly (75.2%). Spastic tetraplegia has been reported in 72.3% of the investigated patients. The major brain imaging findings are abnormal corpus callosum morphology (63.4%) followed by ventriculomegaly (44.5%). Our result might suggest the AP-4 deficiency syndrome as a major differential diagnostic for unknown hereditary neurodegenerative disorders.

Natural Selection at the NHLH2 Core Promoter Exceptionally Long CA-Repeat in Human and Disease-Only Genotypes in Late-Onset Neurocognitive Disorder.

BACKGROUND: Approximately 2% of the human core promoter short tandem repeats (STRs) reach lengths of ≥6 repeats, which may in part be a result of adaptive evolutionary processes and natural selection. A single-exon transcript of the human nescient helix loop helix 2 (NHLH2) gene is flanked by the longest CA-repeat detected in a human protein-coding gene core promoter (Ensembl transcript ID: ENST00000369506.1). NHLH2 is involved in several biological and pathological pathways, such as motivated exercise, obesity, and diabetes. METHODS: The allele and genotype distribution of the NHLH2 CA-repeat were investigated by sequencing in 655 Iranian subjects, consisting of late-onset neurocognitive disorder (NCD) as a clinical entity (n = 290) and matched controls (n = 365). The evolutionary trend of the CA-repeat was also studied across vertebrates. RESULTS: The allele range was between 9 and 25 repeats in the NCD cases, and 12 and 24 repeats in the controls. At the frequency of 0.56, the 21-repeat allele was the predominant allele in the controls. While the 21-repeat was also the predominant allele in the NCD patients, we detected significant decline of the frequency (p < 0.0001) and homozygosity (p < 0.006) of this allele in this group. Furthermore, 12 genotypes were detected across 16 patients (5.5% of the entire NCD sample) and not in the controls (disease-only genotypes; p < 0.0003), consisting of at least one extreme allele. The extreme alleles were at 9, 12, 13, 18, and 19 repeats (extreme short end), and 23, 24, and 25 repeats (extreme long end), and their frequencies ranged between 0.001 and 0.04. The frequency of the 21-repeat allele significantly dropped to 0.09 in the disease-only genotype compartment (p < 0.0001). Evolutionarily, while the maximum length of the NHLH2 CA-repeat was 11 repeats in non-primates, this CA-repeat was ≥14 repeats in primates and reached maximum length in human. CONCLUSION: We propose a novel locus for late-onset NCD at the NHLH2 core promoter exceptionally long CA-STR and natural selection at this locus. Furthermore, there was indication of genotypes at this locus that unambiguously linked to late-onset NCD. This is the first instance of natural selection in favor of a predominantly abundant STR allele in human and its differential distribution in late-onset NCD.

Expression and Methylation Pattern of hsa-miR-34 Family in Sperm Samples of Infertile Men.

Production of high-quality spermatozoa is necessary for male fertility. In this regard, post-mitotic stage in spermatogenesis is very important which posttranscriptional microRNAs (miRNAs) playing a key role at this stage. In this research, we evaluated the expression and methylation of hsa-miR-34 family in sperm samples of infertile men. We recruited 102 infertile men (asthenozoospermia, teratozoospermia, asthenoteratozoospermia, and oligoasthenoteratozoospermia) and 52 fertile men as control. The expression of hsa-miR-34a,b,c was determined by quantitative real-time PCR (qRT-PCR) technique. Methylation of hsa-miR-34b,c promoter was evaluated by methylation-specific PCR (MS-PCR) method. Our data indicated under-expression of three miRNAs (hsa-miR-34a,b,c) in the sperm samples of infertile men in compared to their fertile counterparts. The highest rate of expression reduction was observed in hsa-miR-34c-5p and in oligoasthenoteratospermic patients (P = 0.011, F = 4.01). The results revealed that the frequency of methylation of the promoter region of hsa-miR-34b,c in infertile men was higher than that of fertile men (82.4% versus 23.3%), and the highest frequency of methylation was observed in patients with asthenoteratospermia (92.9%) and oligoasthenoteratospermia (93.8%). In conclusion, our results indicated lower expression of hsa-miR-34a,b,c and hypermethylation of hsa-miR-34b,c promoter in sperm samples of infertile men. Aberrant under-expression of these miRNAs could be duo to the hypermethylation of the promoter region and indicative of a defect in spermatogenesis.

A novel PTC mutation in the BTB domain of KLHL7 gene in two patients with Bohring-Opitz syndrome-like features.

The bric-a-brac, tramtrack and broad complex (BTB) superfamily of conserved proteins are involved in ubiquitin-proteasome system that contains the Kelch-like (KLHL) gene family. Kelch-like family member 7 (KLHL7), one of the KLHL gene family, consists of one BTB/POZ domain, one BACK domain and five or six Kelch motifs. Numerous variants in KLHL7 gene domains have been reported with Crisponi syndrome/cold-induced sweating syndrome type 1 (CS/CISS1)-like features and retinitis pigmentosa 42, and have recently been identified as causing Bohring-Opitz syndrome (BOS)-like features. We report two siblings with BOS-like phenotype with healthy parents and living in Qazvin province (Central Iran). We performed whole-exome sequencing (WES) on the older patient and Sanger sequencing was carried out for validation of potential causative variants in the close family. A novel homozygous frameshift mutation, p.(Phe83Leufs*3), was identified in the BTB domain of KLHL7 that caused a premature translation-termination codon (PTC) in the two siblings with severe developmental delay, microcephaly, facial dysmorphism, peripheral retinal and optic disc atrophy and cardiac septal defects. Our findings are in agreement with the clinical spectrum of KLHL7 mutations, which are associated with BOS-like features that reports for first time in our population.

Distinct genetic variation and heterogeneity of the Iranian population.

Iran, despite its size, geographic location and past cultural influence, has largely been a blind spot for human population genetic studies. With only sparse genetic information on the Iranian population available, we pursued its genome-wide and geographic characterization based on 1021 samples from eleven ethnic groups. We show that Iranians, while close to neighboring populations, present distinct genetic variation consistent with long-standing genetic continuity, harbor high heterogeneity and different levels of consanguinity, fall apart into a cluster of similar groups and several admixed ones and have experienced numerous language adoption events in the past. Our findings render Iran an important source for human genetic variation in Western and Central Asia, will guide adequate study sampling and assist the interpretation of putative disease-implicated genetic variation. Given Iran's internal genetic heterogeneity, future studies will have to consider ethnic affiliations and possible admixture.