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.

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.

Identification and in silico structural analysis for the first de novo mutation in the cystic fibrosis transmembrane conductance regulator protein in Iran: case report and developmental insight using microsatellite markers.

Cystic fibrosis (CF) is an autosomal recessive disease caused by the inheritance of two mutant cystic fibrosis transmembrane conductance regulator (CFTR) alleles, one from each parent. Autosomal recessive disorders are rarely associated with germline mutations or mosaicism. Here, we propose a case of paternal germline mutation causing CF. The subject also had an identifiable maternal mutant allele. We identified the compound heterozygous variants in the proband through Sanger sequencing, and in silico studies predicted functional effects on the protein. Also, short tandem repeat markers revealed the de novo nature of the mutation. The maternal mutation in the CFTR gene was c.1000C > T. The de novo mutation was c.178G > A, p.Glu60Lys. This mutation is located in the lasso motif of the CFTR protein and, according to in silico structural analysis, disrupts the interaction of the lasso motif and R-domain, thus influencing protein function. This first reported case of de novo mutation in Asia has notable implications for molecular diagnostics, genetic counseling, and understanding the genetic etiology of recessive disorders in the Iranian population.

Identifying the first de novo mutation in the cystic fibrosis transmembrane conductance regulator protein in Iran: a case report with insights from microsatellite markersA child can develop Cystic Fibrosis (CF) if both parents pass on mutated genes. In some rare cases, new genetic mutations occur spontaneously, causing CF. This report discusses a unique case where a child has one gene with a spontaneous mutation and inherits another gene mutation from the mother. We used a method called Sanger sequencing to find the two different gene changes in the affected person. We also used computer analysis to predict how these changes might affect the protein responsible for this genetic disease. To confirm that the child's new change is not inherited, we used a type of genetic marker called microsatellite markers. The mutation inherited from the mother and the new spontaneous mutation resulted in a unique change in the responsible protein. This mutation is located in a specific part of the protein called the lasso motif. Our computer simulations show that this mutation disrupts the interaction between the lasso motif and another part of the protein called the R-domain, which ultimately affects the protein's function. This case is significant because it is the first reported instance of a de novo mutation causing CF in Asia. It has important implications for genetic testing, counseling, and understanding how recessive genetic disorders like CF occur within the Iranian population.

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 as an effective agent in the LIV-1-targeted treatment of cancers due to its longer half-life and stronger cytotoxicity.

Whole-Transcriptome Sequencing-Based Profiling of the Cutaneous Virome in Patients with Secondary Immunodeficiency.

Most viral infections can be self-limited, with no requirement for medical intervention. However, the same viruses can cause severe diseases in patients with compromised immunity due to single-gene diseases, acquired immune deficiency syndrome, or hematologic malignancies or those receiving immunosuppressive drugs. Occasionally, these immunocompromised patients harbor >1 infectious agent, requiring several concomitant diagnostic tests. We have developed, to our knowledge, a previously unreported whole-transcriptome sequencing-based pipeline that allows virome profiling, quantitation, and expression pattern analysis of 926 distinct viruses by sequencing of RNA isolated from a single lesional skin biopsy. This pipeline can also explore host genetics if there is a Mendelian predisposition to infection. We applied this pipeline to 6 Iranian patients with viral-induced skin lesions associated with immune deficiency secondary to HIV, human T-lymphotropic virus 1, chronic lymphocytic leukemia, and post transplant immunosuppression. In 5 cases, definitive human papillomavirus infections were identified, some caused by multiple viral types. In addition to human papillomavirus, coinfection with other viruses (Merkle cell polyomavirus, cytomegalovirus, and human herpesvirus 4) was detected in some lesions. In 1 case, whole-transcriptome sequencing validated the clinical diagnosis of adult T-cell leukemia/lymphoma in a patient with an initial diagnosis of mycosis fungoides/Sézary syndrome. These findings attest to the power of whole-transcriptome sequencing in profiling the cutaneous virome in the context of compromised immunity.

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.

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.

Isolation of stem cells from adult rat kidneys

The kidney has an inherent ability for recovery and regeneration following acute damage. However, there has been much contention as to the source of regenerating renal cells. The aim of this study was to isolate and characterize these cells. Normal rat kidneys were minced and cells were isolated with collagenase I and were cultured in an expansion medium. Adherent cells were isolated and expanded for more than 120 days in vitro. These cells had the potential of trans-lineage differentiation into neural cells, adipocytes and osteocytes. These cells also expressed Nucleostemin, Cyclin D1, Notch1 and Survivin which are commonly expressed in stem cells. The results of the current work show that the adult kidney contains a population of multipotent stem cells.

Isolation of stem cells from adult rat kidneys

The kidney has an inherent ability for recovery and regeneration following acute damage. However, there has been much contention as to the source of regenerating renal cells. The aim of this study was to isolate and characterize these cells. Normal rat kidneys were minced and cells were isolated with collagenase I and were cultured in an expansion medium. Adherent cells were isolated and expanded for more than 120 days in vitro. These cells had the potential of trans-lineage differentiation into neural cells, adipocytes and osteocytes. These cells also expressed Nucleostemin, Cyclin D1, Notch1 and Survivin which are commonly expressed in stem cells. The results of the current work show that the adult kidney contains a population of multipotent stem cells.(AU)