Analysis of rearrangements of the CFTR gene in patients from Turkey with CFTR-related disorders: frequent exon 2 deletion.

Cystic fibrosis is a hereditary disease that mostly affects the sweat glands, respiratory system, digestive system, and reproductive system. Many and various types of mutations have been reported in CFTR in different ethnicities and countries/regions. Analysis of CFTR gene rearrangements is recommended in patients with unidentified mutated alleles in CFTR sequencing analysis. We collected MLPA analyses of 527 patients from Turkey who had at least one unidentified mutation in CFTR sequence analysis. Heterozygous/homozygous deletions were detected in the CFTR gene in 49 individuals (9.2%) from 35 families. Twelve different single/multi exon deletions were demonstrated, two of which were not previously reported in the literature. Mutations have previously reported in patients from various regions including Asia, Europe, and Africa, and Turkey is located at a crossroads between them. The most frequent mutation was the exon 2 deletion, accounting for 60%. Moreover, patients with exon 2 deletions, were especially originated from northern Turkey. This finding is valuable in leading and shaping planned screening programs in Turkey. Our study, the most comprehensive study for rearrangement analysis in patients from Tukey, revealed a candidate hotspot region of patients suspected of having CFTR-related disorders from Turkey.

Differential expression of ABCB1, ABCG2, and KLF4 as putative indicators for paclitaxel resistance in human epithelial type 2 cells.

Laryngeal squamous cell carcinoma (LSCC) is the second most common malignancy of the head and neck region in the USA with a declining 5-year survival rate. Paclitaxel resistance of tumors including LSCC still stands as a vital cause for poor clinical outcome in patients. In the current study, our aim was to explore the expressions of ATP-binding cassette transporters and stemness associated genes in human epithelial type 2 (Hep-2) cells with paclitaxel resistance. Resistant cells were developed via treatment with increasing doses of paclitaxel to acquire four sub-lines resistant to one-, two-, four-, and eightfold concentrations of paclitaxel (1×, 2×, 4×, 8×). Then, we profiled the expressions of ten selected ABC transporters (ABCA5, ABCB1, ABCB6, ABCC1, ABCC2, ABCC3, ABCC5, ABCC10, ABCF2, and ABCG2) and four stem cell markers (SOX2, OCT4, KLF, and CXCR4) using quantitative real time polymerase chain reaction in paclitaxel resistant cells to look for a link between these markers and chemoresistance. We demonstrated that ABCB1 and ABCG2 expressions gradually elevated and reached a maximum level in Taxol 8× cells. Considering stem cell markers, KLF4 expression elevated significantly, as soon as parental cells acquired resistance to the lowest dose of paclitaxel and its expression elevated stepwise. Expression levels of other tested ATP-binding cassette transporters and stem cell markers also elevated, although at different steps of paclitaxel resistance acquisition. Our findings suggest that higher expressions of ABCB1, ABCG2, and KLF4 might be considered as putative indicators for paclitaxel resistance in LSCC patients.

A novel CLN5 mutation in Turkish patient with variant late-onset neuronal ceroid lipofuscinosis and recurrent fractures that causes severe morbidity.

Neuronal ceroid lipofuscinosis (NCL) is characterized by ataxia, epilepsy, mental and motor deterioration, and visual loss. The phenotype of patients is highly heterogeneous. We report a patient with late-infantile-onset psychomotor retardation, visual loss, seizure, movement disorder, and recurrent bone fractures. Clinical exome sequencing revealed a novel homozygous c.1113_1116del, p.Y371fs mutation in CLN5. No variant was detected associated with simple bone cyst. While NCL disease is difficult disease in itself, recurrent fractures significantly increased morbidity. This case report contributes to genotypic spectrum of CLN5 and emphasizes clinical importance of Turkish patients with CLN5 mutations, and non-NCL factors/diseases can adversely affect morbidity.

Expression profile of stem cell markers and ABC transporters in 5-fluorouracil resistant Hep-2 cells.

Resistance of laryngeal squamous cell carcinoma cells to traditional therapeutic regimens still remains to be a major reason for therapeutic failure in patients. In this study, we aimed at investigating the expression profiles of ATP-binding cassette (ABC) transporters and stem cell markers in 5-fluorouracil (5-FU) resistant laryngeal Hep-2 cells. We treated parental Hep-2 cells, with stepwise increased doses of 5-FU for almost 1 year to develop 5-FU resistant sub-lines with resistance against varying levels of 5-FU concentrations (4 sub-lines resistant to 1, 2, 4, and eightfold of 5-FU). Then, we measured the expression levels of 10 genes from ABC transporters family and 4 stem cell associated markers using quantitative reverse transcription polymerase chain reaction (qRT-PCR) to find out a potential relationship between these markers and chemoresistance. We found that stemness-associated markers had elevated expressions from the beginning of 5-FU resistance acquisition. Their expressions elevated stepwise while parental Hep-2 cells got resistance to higher doses of 5-FU. Expressions of tested ABC transporters (ABCA5, ABCB1, ABCB6, ABCC1, ABCC2, ABCC3, ABCC5, ABCC10 and ABCF2, and ABCG2) were also deregulated in 5-FU resistant Hep-2 cells. Although their expressions remained unaltered at the beginning of acquisition of resistance, expressions of ABC transporters except from ABCB6 increased significantly when cells became resistant to higher doses of 5-FU. Our results suggest that enrichment of cells with stemness characteristics and upregulation of ABC transporters might be amongst the crucial contributors of chemoresistance in laryngeal cancer cells.

Three novel mutations in 20 patients with hereditary spastic paraparesis.

Hereditary spastic paraparesis (HSP) constitutes both genetic and clinically heterogeneous group of upper motor neuron diseases. Half of the individuals with autosomal dominant (AD) HSP have mutations in SPAST, ATL1, and REEP1 genes. This study was conducted to elucidate the genetic etiology of patients with the pure type AD-HSP diagnosis. The patient group consisted of 23 individuals from 6 families in Turkey. In the first step of work, Sanger sequencing (SS) was performed in ATL1, SPAST, and REEP1 genes and the second phase whole-exome sequencing (WES) was performed following SS analysis for the patients with no detected mutations in these genes. The results of this study revealed that in ATL1, 6 patients have previously reported c.776C > A mutation and 6 patients have novel c.470 T > C mutation. In SPAST, 3 patients have novel c.1072G > C mutation and 2 patients have novel c.1099-1G > C mutation. WES was performed in three patients, who had no detected mutation in these genes with SS analysis. In this approach, as previously reported c.1859 T > C mutation in KIAA0196 was detected, and it was confirmed with the patient's relatives by SS. In three of patients, no HSP-associated variant could be identified in SS and WES. With this study, the molecular genetic etiology in 20 of 23 (87%) individuals that were included in this study with the utilization of SS and WES was elucidated. Utilization of SS and WES methods have enabled the identification of genetic etiology of HSP further with appropriate genetic counseling that was provided to the patients.

The role of miRNAs in cancer: from pathogenesis to therapeutic implications.

Cancer is still one of the dominating causes of deaths worldwide, although there have been important enhancements for detection and diagnosis of cancer recently. miRNAs are shown to participate in carcinogenesis of several types of tumors and their aberrant expression of miRNAs has been detected in cell lines, xenografts and clinical samples. miRNAs are thought to target and modulate the expression of more than 60% of human genes, which makes the expressional regulation by miRNAs the most abundant post-transcriptional regulation mode. Here, we have reviewed the most current literature to shed a light on the functions of miRNAs on human carcinogenesis. Possible roles of miRNAs in oncogenesis through both genetic and epigenetic changes occurring during cancer initiation, progression, invasion or metastasis are summarized.

Comparison of microRNA expression levels in patients with schizophrenia before and after electroconvulsive therapy.

OBJECTIVE: Exploring the role of microRNAs in the antipsychotic efficacy of electroconvulsive therapy (ECT) will contribute to understanding the underlying mechanism through which ECT exerts its therapeutic effects. The primary objective of this study was to identify microRNA alterations before and after ECT in patients with schizophrenia. METHODS: We compared microarray-based microRNA profiles in peripheral blood from eight patients with schizophrenia before and after ECT and eight healthy controls. Then, we aimed to validate selected differentially expressed microRNAs in 30 patients with schizophrenia following a course of ECT, alongside 30 healthy controls by using quantitative reverse-transcription PCR. RESULTS: Microarray-based expression profiling revealed alterations in 681 microRNAs when comparing pre- and post-ECT samples. Subsequent quantitative reverse-transcription PCR analysis of the selected microRNAs (miR-20a-5p and miR-598) did not reveal any statistical differences between pre- and post-ECT samples nor between pre-ECT samples and those of healthy controls. CONCLUSION: As neuroepigenetic studies on ECT are still in their infancy, the results reported in this study are best interpreted as exploratory outcomes. Additional studies are required to explore the potential epigenetic mechanisms underlying the therapeutic efficacy of ECT.

Rare Findings in Cleidocranial Dysplasia Caused by RUNX Mutation.

Background Cleidocranial dysplasia (CCD, #MIM119600) is an autosomal-dominant skeletal dysplasia characterized by delayed closure of the cranial sutures, aplasia, or hypoplasia of the clavicles and dental abnormalities. These findings were accompanied by mobile and drooping shoulders, frontal and parietal bossing, hypertelorism, brachycephaly, short stature, supernumerary, and late erupting teeth. Radiographic studies can reveal involvement of multiple bones including skull, chest, pelvis, and limbs. CCD can be diagnosed with clinical and radiological evaluation and validated by molecular studies. Heterozygous loss of function RUNX2 gene, which plays an important role in osteogenesis and differentiation of precursor cells, causes CCD phenotype. Methods In this article, we reported five cases from three unrelated families with CCD phenotype. All exons and exonic-intronic boundary regions of RUNX2 gene from five patients were analyzed by polymerase chain reaction amplification and direct Sanger-sequencing. Results Our patients had classical CCD phenotype and we detected three different previously described mutations including c.1171C > T, IVS4 + 4delAAGT and c.676G > A. However, nail dysplasia has never been associated with these mutations. Our patients had varying degrees of nail dysplasia. Two of three mutations are related with Runt DNA-binding domain of RUNX2 protein in Wnt signaling and c.1171C > T had effect on proline/serine/threonine-rich (PST) domain. Recently, Wnt signaling pathway was presented as a key regulator of digit and nail differentiation. Our data suggest that RUNX2 gene may have an essential role on embryogenesis of nails, probably by protecting their integrity.

Acrocapitofemoral dysplasia: Novel mutation in IHH in two adult patients from the third family in the literature and progression of the disease.

Acrocapitofemoral dysplasia (ACFD) is a rare autosomal recessive skeletal dysplasia characterized by short stature with short limb dwarfism, brachydactyly, and a narrow thorax. Major radiographic features are egg-shaped capital femoral epiphyses with a short femoral neck and cone-shaped epiphyses, mainly in the hands and hips. To date, only four child patients from two families have been reported. We describe two adult patients with ACFD with a novel homozygous c.478C>T (p.Arg160Cys) mutation in IHH in the third family of the literature. The reported cases showed a middle phalanges which fused with distal phalanges in the fifth toes, the typical configuration of metacarpals, radial angulation and extremely short femoral neck. These findings could help the diagnosis of ACFD in adult patients. We hope that this new family will be a helpful guide for predicting and managing the prognosis of diagnosed children.

Recurrent c.776T>C mutation in CHST3 with four other novel mutations and a literature review.

The chondrodysplasia with congenital joint dislocations, CHST3 type, which was distinguished by predominantly contractures, marked vertebral changes, and normal facial appearance. Although, some clinical clues can be used for differential diagnosis, it is mostly too difficult to discriminate one type from another on basis of clinical findings only. Eight patients with multiple dislocations from five unrelated families were included in this study to elucidate molecular diagnoses. Clinical exome sequencing (CES) was performed on one patient from each family. Variable degree vertebral changes, pes equinovarus, and kyphoscoliosis accompanied multiple dislocations and short stature. In CES analyses, all mutations showed in CHST3. Previously reported c.776T>C homozygous mutations were detected in two families, compound heterozygous novel c.740G>C and c.881T>C mutations were found in one family, and homozygous novel c.564C>A and c.963G>A mutations were also determined in remaining two families, separately. Biallelic CHST3 c.776T>C mutations are most frequent mutation in CHST3 and have been reported predominantly in Turkish patients which may be remarkable for genotype-ethnicity correlation in chondrodysplasia with congenital joint dislocations, CHST3 type. It is suggested that c.776T>C mutation can be accepted as a recurrent mutation in CHST3 for Turkish patients who are suspected of having chondrodysplasia with congenital joint dislocations, CHST3 type.

Identification of miR-139-5p as a saliva biomarker for tongue squamous cell carcinoma: a pilot study.

BACKGROUND: Of all human oral carcinomas, 41 % are localized to the tongue. Despite considerable improvements in both diagnosis and treatment, tongue squamous cell carcinoma (TSCC) has remained one of the most lethal types of cancer. Here, we aimed at identifying a salivary microRNA (miRNA) expression signature specific for TSCC patients. METHODS: To identify putative diagnostic biomarkers, we compared the miRNA expression profiles of saliva samples from three TSCC patients and four healthy control individuals using an Agilent miRNA microarray platform (V19). Three of the differentially expressed miRNAs identified were selected for further validation using quantitative reverse-transcription PCR (qRT-PCR) in saliva samples from 25 TSCC patients and 25 healthy control individuals. RESULTS: Through microarray-based expression profiling, we found that 419 miRNAs were deregulated in the saliva samples from the TSCC patients compared to those from the healthy control individuals tested. Subsequent qRT-PCR analysis revealed that the expression level of miR-139-5p was significantly reduced in the TSCC validation samples compared to the controls. Further analysis of post-operative saliva samples derived from TSCC patients revealed that the miR-139-5p expression levels had turned back to normal again. In addition, we found that miR-139-5p exhibited enough power to discriminate pre-operative TSCC patients from both normal individuals (AUC: 0.805) and post-operative TSCC patients (AUC: 0.713), thereby underscoring its diagnostic potential. CONCLUSIONS: From our results we conclude that saliva can be used as a feasible source for routine TSCC diagnostics and that miR-139-5p may serve as a potential biomarker for early TSCC detection.

Revealing the function of a novel splice-site mutation of CHD7 in CHARGE syndrome.

Most cases of CHARGE syndrome are sporadic and autosomal dominant. CHD7 is a major causative gene of CHARGE syndrome. In this study, we screened CHD7 in two Turkish patients demonstrating symptoms of CHARGE syndrome such as coloboma, heart defect, choanal atresia, retarded growth, genital abnomalities and ear anomalies. Two mutations of CHD7 were identified including a novel splice-site mutation (c.2443-2A>G) and a previously known frameshift mutation (c.2504_2508delATCTT). We performed exon trapping analysis to determine the effect of the c.2443-2A>G mutation at the transcriptional level, and found that it caused a complete skip of exon 7 and splicing at a cryptic splice acceptor site. Our current study is the second study demonstrating an exon 7 deficit in CHD7. Results of previous studies suggest that the c.2443-2A>G mutation affects the formation of nasal tissues and the neural retina during early development, resulting in choanal atresia and coloboma, respectively. The findings of the present study will improve our understanding of the genetic causes of CHARGE syndrome.

Genes that Affect Brain Structure and Function Identified by Rare Variant Analyses of Mendelian Neurologic Disease.

Development of the human nervous system involves complex interactions among fundamental cellular processes and requires a multitude of genes, many of which remain to be associated with human disease. We applied whole exome sequencing to 128 mostly consanguineous families with neurogenetic disorders that often included brain malformations. Rare variant analyses for both single nucleotide variant (SNV) and copy number variant (CNV) alleles allowed for identification of 45 novel variants in 43 known disease genes, 41 candidate genes, and CNVs in 10 families, with an overall potential molecular cause identified in >85% of families studied. Among the candidate genes identified, we found PRUNE, VARS, and DHX37 in multiple families and homozygous loss-of-function variants in AGBL2, SLC18A2, SMARCA1, UBQLN1, and CPLX1. Neuroimaging and in silico analysis of functional and expression proximity between candidate and known disease genes allowed for further understanding of genetic networks underlying specific types of brain malformations.