Revealing the functions of novel mutations in RAB3GAP1 in Martsolf and Warburg micro syndromes.

PURPOSE: Martsolf (MS) and Warburg micro syndromes (WARBM) are rare autosomal recessive inherited allelic disorders, which share similar clinical features including microcephaly, intellectual disability, brain malformations, ocular abnormalities, and spasticity. Here, we revealed the functions of novel mutations in RAB3GAP1 in a Turkish female patient with MS and two siblings with WARBM. We also present a review of MS patients as well as all reported RAB3GAP1 pathogenic mutations in the literature. METHODS: We present a female with MS phenotype and two siblings with WARBM having more severe phenotypes. We utilized whole-exome sequencing to identify the molecular basis of these syndromes and confirmed suspected variants by Sanger sequencing. Quantitative (q) RT-PCR analysis was carried out to reveal the functions of novel splice site mutation detected in MS patient. RESULTS: We found a novel homozygous c.2607-1G>C splice site mutation in intron 22 of RAB3GAP1 in MS patient and a novel homozygous c.2187_2188delinsCT, p.(Met729_Lys730delinsIleTer) mutation in exon 19 of RAB3GAP1 in the WARBM patients. We showed exon skipping in MS patient by Sanger sequencing and gel electrophoresis. qRT-PCR analysis demonstrated the reduced expression of RAB3GAP1 in the patient with the c.2607-1G>C splice site mutation compared to a healthy control individual. CONCLUSION: Here, we have studied two novel RAB3GAP1 mutations in two different phenotypes; a MS associated novel splice site mutation, and a WARBM1 associated novel deletion-insertion mutation. Our findings suggest that this splice site mutation is responsible for milder phenotype and the deletion-insertion mutation presented here is associated with severe phenotype.

Novel POC1A mutation in primordial dwarfism reveals new insights for centriole biogenesis.

POC1A encodes a WD repeat protein localizing to centrioles and spindle poles and is associated with short stature, onychodysplasia, facial dysmorphism and hypotrichosis (SOFT) syndrome. These main features are related to the defect in cell proliferation of chondrocytes in growth plate. In the current study, we aimed at identifying the molecular basis of two patients with primordial dwarfism (PD) in a single family through utilization of whole-exome sequencing. A novel homozygous p.T120A missense mutation was detected in POC1A in both patients, a known causative gene of SOFT syndrome, and confirmed using Sanger sequencing. To test the pathogenicity of the detected mutation, primary fibroblast cultures obtained from the patients and a control individual were used. For evaluating the global gene expression profile of cells carrying p.T120A mutation in POC1A, we performed the gene expression array and compared their expression profiles to those of control fibroblast cells. The gene expression array analysis showed that 4800 transcript probes were significantly deregulated in cells with p.T120A mutation in comparison to the control. GO term association results showed that deregulated genes are mostly involved in the extracellular matrix and cytoskeleton. Furthermore, the p.T120A missense mutation in POC1A caused the formation of abnormal mitotic spindle structure, including supernumerary centrosomes, and changes in POC1A were accompanied by alterations in another centrosome-associated WD repeat protein p80-katanin. As a result, we identified a novel mutation in POC1A of patients with PD and showed that this mutation causes the formation of multiple numbers of centrioles and multipolar spindles with abnormal chromosome arrangement.

The role of miR-145 in stem cell characteristics of human laryngeal squamous cell carcinoma Hep-2 cells.

The cancer stem-like cells (CSLCs) are tumorigenic cells promoting initiation, progression, and spread of the tumor. Accumulating evidences suggested the presence of CSLCs in distinct tumors including laryngeal squamous cell carcinoma (LSCC). MicroRNAs have been proposed as significant regulators of carcinogenesis, and several of them have been demonstrated to have direct roles in survival of CSLCs. In this study, we aimed to explore the role of miR-145, which is downregulated in LSCC, on cancer stem cell potency of laryngeal cancer cells. We initially showed the downregulation of miR-145 expression in tumor tissue samples and in CD133-enriched CSLCs. Quantitative reverse-transcription PCR (qRT-PCR) analysis of miR-145-transfected Hep-2 cells demonstrated the inhibitory role of miR-145 on stem cell markers like SOX2, OCT4, KLF4, and ABCG2. We, then, investigated the stem cell features of miR-145-overexpressing Hep-2 cells by sphere formation assay, single-cell cloning assay, and aldehyde dehydrogenase (ALDH) assay, which all demonstrated the inhibition of stem cell potency upon miR-145 overexpression. Further qRT-PCR analysis demonstrated altered expression of epithelial to mesenchymal transition markers in miR-145-overexpressing Hep-2 cells. In conclusion, we demonstrated the regulatory role of miR-145 in stem cell characteristics of Hep-2 cells. Based on these results, we propose that miR-145 might carry crucial roles in LSCC tumorigenesis, prognosis, metastasis, chemoresistance, and recurrence through regulating stem cell properties of tumor cells.

The altered promoter methylation of oxytocin receptor gene in autism.

Autism spectrum disorder (ASD) is one of the lifelong existing disorders. Abnormal methylation status of gene promoters of oxytonergic system has been implicated as among the etiologic factors of ASDs. We, therefore, investigated the methylation frequency of oxytocin receptor gene (OXTR) promoter from peripheral blood samples of children with autistic features. Our sample includes 66 children in total (22-94 months); 27 children with ASDs according to the DSM-IV-TR and the Childhood Autism Rating Scale (CARS) and 39 children who do not have any autistic like symptoms as the healthy control group. We investigated the DNA methylation status of OXTR promoter by methylation specific enzymatic digestion of genomic DNA and polymerase chain reaction. A significant relationship has been found between ASDs and healthy controls for the reduction of methylation frequency of the regions MT1 and MT3 of OXTR. We could not find any association in the methylation frequency of MT2 and MT4 regions of OXTR. Although our findings indicate high frequency of OXTR promoter hypomethylation in ASDs, there is need for independent replication of the results for a bigger sample set. We expect that future studies with the inclusion of larger, more homogeneous samples will attempt to disentangle the causes of ASDs.

Identification of microRNA profile specific to cancer stem-like cells directly isolated from human larynx cancer specimens.

BACKGROUND: Emerging evidences proposed that microRNAs are associated with regulation of distinct physio-pathological processes including development of normal stem cells and carcinogenesis. In this study we aimed to investigate microRNA profile of cancer stem-like cells (CSLCs) isolated form freshly resected larynx cancer (LCa) tissue samples. METHODS: CD133 positive (CD133+) stem-like cells were isolated from freshly resected LCa tumor specimens. MicroRNA profile of 12 pair of CD133+ and CD133- cells was determined using microRNA microarray and differential expressions of selvected microRNAs were validated by quantitative real time PCR (qRT-PCR). RESULTS: MicroRNA profiling of CD133+ and CD133- LCa samples with microarray revealed that miR-26b, miR-203, miR-200c, and miR-363-3p were significantly downregulated and miR-1825 was upregulated in CD133+ larynx CSLCs. qRT-PCR analysis in a total of 25 CD133+/CD133- sample pairs confirmed the altered expressions of these five microRNAs. Expressions of miR-26b, miR-200c, and miR-203 were significantly correlated with miR-363-3p, miR-203, and miR-363-3p expressions, respectively. Furthermore, in silico analysis revealed that these microRNAs target both cancer and stem-cell associated signaling pathways. CONCLUSIONS: Our results showed that certain microRNAs in CD133+ cells could be used as cancer stem cell markers. Based on these results, we propose that this panel of microRNAs might carry crucial roles in LCa pathogenesis through regulating stem cell properties of tumor cells.

Hereditary spastic paraplegia with recessive trait caused by mutation in KLC4 gene.

We report an association between a new causative gene and spastic paraplegia, which is a genetically heterogeneous disorder. Clinical phenotyping of one consanguineous family followed by combined homozygosity mapping and whole-exome sequencing analysis. Three patients from the same family shared common features of progressive complicated spastic paraplegia. They shared a single homozygous stretch area on chromosome 6. Whole-exome sequencing revealed a homozygous mutation (c.853_871del19) in the gene coding the kinesin light chain 4 protein (KLC4). Meanwhile, the unaffected parents and two siblings were heterozygous and one sibling was homozygous wild type. The 19 bp deletion in exon 6 generates a stop codon and thus a truncated messenger RNA and protein. The association of a KLC4 mutation with spastic paraplegia identifies a new locus for the disease.

Role of miR-145 in human laryngeal squamous cell carcinoma.

BACKGROUND: Laryngeal squamous cell carcinoma (SCC), being an aggressive malignancy, is one of the most commonly diagnosed malignant types of head and neck SCC worldwide. Incidences of laryngeal SCC have been reported to increase recently. In this study, we aimed to explore the biological effects of miR-145 on laryngeal cancer cells. METHODS: The relative miR-145 level in laryngeal SCC tumor tissues and normal samples was investigated. Then, Hep-2 cells were utilized for functional analysis of miR-145. The proliferation abilities of transfected cells were measured using MTS assay. Scratch assay and single colony migration assay were performed to observe the alterations in migration behavior of transfected cells. Caspase assay and cell cycle analysis were used to investigate the underlying reasons of proliferative inhibition in cells in which miR-145 is overexpressed. Moreover, expression of SOX2 was analyzed using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analysis in Hep-2 cells upon miR-145 transfection and its expression was evaluated in tumor and normal tissue sample of the larynx. RESULTS: The miR-145 expression in laryngeal SCC tumor samples has been shown to be downregulated. The miR-145 overexpression caused inhibition of proliferation and migration in Hep-2 cells through induction of apoptosis and cell cycle arrest. The SOX2 level was demonstrated to be overexpressed in tumor samples and its expression was significantly decreased in miR-145 overexpressed Hep-2 cells. CONCLUSION: We have demonstrated the deregulation of miR-145 and SOX2 in laryngeal SCC. Based on these results, we propose that miR-145, as an important regulator of SOX2, carries crucial roles in laryngeal SCC tumorigenesis.

Draft Genome Sequences of Two Heat-Resistant Mutant Strains (A52 and B41) of the Photosynthetic Hydrogen-Producing Bacterium Rhodobacter capsulatus.

The draft genome sequences of two heat-resistant mutant strains, A52 and B41, derived from Rhodobacter capsulatus DSM 1710, and with different hydrogen production levels, are reported here. These sequences may help understand the molecular basis of heat resistance and hydrogen production in R. capsulatus.

Alpha-B-crystallin expression in human laryngeal squamous cell carcinoma tissues.

BACKGROUND: Laryngeal squamous cell carcinoma (SCC), being an aggressive malignancy, is one of the most commonly diagnosed malignant types of head and neck SCC worldwide. The recent studies suggested that αB-crystallin might play an important role in tumorigenesis. The purpose of this study was to investigate the αB-crystallin expression level in metastatic and nonmetastatic laryngeal SCC tissues and to determine its prognostic significance. METHODS: Alpha-B-crystallin expression status in metastatic, nonmetastatic laryngeal SCC, and normal tissue samples was investigated by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: We demonstrated that the expression of αB-crystallin was significantly upregulated in laryngeal SCC tumor tissue samples in comparison with the corresponding normal tissues (p < .001), although no significant association has been found between αB-crystallin expression and either the metastatic potential or the T classification of the specimens. CONCLUSION: Although expression of αB-crystallin is not statistically correlated with neck metastases, we demonstrated that αB-crystallin is significantly overexpressed in laryngeal cancer.

A clinical variant in SCN1A inherited from a mosaic father cosegregates with a novel variant to cause Dravet syndrome in a consanguineous family.

A consanguineous family from Turkey having two children with intellectual disability exhibiting myoclonic, febrile and other generalized seizures was recruited to identify the genetic origin of these phenotypes. A combined approach of SNP genotyping and exome sequencing was employed both to screen genes associated with Dravet syndrome and to detect homozygous variants. Analysis of exome data was extended further to identify compound heterozygosity. Herein, we report identification of two paternally inherited genetic variants in SCN1A (rs121917918; p.R101Q and p.I1576T), one of which was previously implicated in Dravet syndrome. Interestingly, the previously reported clinical variant (rs121917918; p.R101Q) displayed mosaicism in the blood and saliva of the father. The study supported the genetic diagnosis of affected children as Dravet syndrome possibly due to the combined effect of one clinically associated (rs121917918; p.R101Q) and one novel (p.I1576T) variants in SCN1A gene. This finding is important given that heterozygous variants may be overlooked in standard exome scans of consanguineous families. Thus, we are presenting an interesting example, where the inheritance of the condition may be misinterpreted as recessive and identical by descent due to consanguinity and mosaicism in one of the parents.

Characterization of stem-like cells directly isolated from freshly resected laryngeal squamous cell carcinoma specimens.

Larynx cancer (LCa) is an aggressive malignancy, which is the second most common malignant neoplasm of head and neck squamous cell carcinoma. Its incidences have been reported to increase and therapeutic options mostly fail to give positive clinical response especially for the advanced LCa cases. In this study we aimed to isolate stem-like cells from freshly resected LCa tumor specimens and characterize them by quantitative real time PCR (qRT-PCR) for expression of cancer stem cell markers including SOX2, OCT4, KLF4, ABCG2, CXCR4 and CD44. Our results showed that CD133(high) cells directly isolated from freshly resected tumor specimens exhibit elevated levels of SOX2, OCT4 and KLF4, and have increased expression levels of ABCG2 and CXCR4, which were associated with resistance of tumors to regular chemotherapeutic reagents. In conclusion, this study offers a useful approach utilizing CD133 to isolate stem cells directly from fresh tissues, which gives the opportunity to develop novel therapeutic tools specifically targeting these cells through their further characterization.