Whole-exome sequencing reveals new potential genes and variants in patients with premature ovarian insufficiency.

PURPOSE: Premature ovarian insufficiency (POI) is a heterogeneous disorder characterized by the cessation of menstrual cycles before the age of 40 years due to the depletion or dysfunction of the ovarian follicles. POI is a highly heterogeneous disease in terms of etiology. The aim of this study is to reveal the genetic etiology in POI patients. METHODS: A total of 35 patients (mean age: 27.2 years) from 28 different families diagnosed with POI were included in the study. Karyotype, FMR1 premutation analysis, single nucleotide polymorphism (SNP) array, and whole-exome sequencing (WES) were conducted to determine the genetic etiology of patients. RESULTS: A total of 35 patients with POI were first evaluated by karyotype analysis, and chromosomal anomaly was detected in three (8.5%) and FMR1 premutation was detected in six patients (17%) from two different families. A total of 29 patients without FMR1 premutation were included in the SNP array analysis, and one patient had a 337-kb deletion in the chromosome 6q26 region including PARK2 gene, which was thought to be associated with POI. Twenty-nine cases included in SNP array analysis were evaluated simultaneously with WES analysis, and genetic variant was detected in 55.1% (16/29). CONCLUSION: In the present study, rare novel variants were identified in genes known to be associated with POI, which contribute to the mutation spectrum. The effects of detected novel genes and variations on different pathways such as gonadal development, meiosis and DNA repair, or metabolism need to be investigated by experimental studies. Molecular etiology allows accurate genetic counseling to the patient and family as well as fertility planning.

Secondary findings in 622 Turkish clinical exome sequencing data.

CES (Clinical Exome Sequencing) is a method that we use to diagnose rare diseases with nonspesific clinical features. Besides primary indication for testing genetic information may be detected about diseases which have not yet emerged. ACMG guidelines recommend to report pathogenic variations in medically actionable 59 genes. In this study we evaluated CES data of 622 cases which were tested for various indications. According to ACMG recommendations 59 genes were screened for reportable variations. The detected variations were reviewed using distinct databases and ACMG variation classification guidelines. Among 622 cases 13 (2.1%) had reportable variations including oncogenetic, cardiogenetic disorders, and malignant hyperthermia susceptibility-related genes. In 15 cases (2.4%) heterozygous pathogenic and likely pathogenic variations were detected in genes showing autosomal recessive inheritance. Ten novel variations causing truncated protein or splicing defect were reported. We detected 11 variations having conflicting interpretations in databases and 30 novel variations, predicted as likely pathogenic via insilico analysis tools which further evaluations are needed. As to our knowledge this is the first study investigating secondary findings in Turkish population. To extract the information that may lead to prevent severe morbidities and mortalities from big data is a valuable and lifesaving effort. Results of this study will contrbute to existing knowledge about secondary findings in exome sequencing and will be a pioneer for studies in Turkish population.

Effects of MC4R, FTO, and NMB gene variants to obesity, physical activity, and eating behavior phenotypes.

Obesity is a major contributory factor of morbidity and mortality. It has been suggested that biological systems may be involved in the tendency to be and to remain physically inactive also behaviors such as food and beverage preferences and nutrient intake may at least partially genetically determined. Consequently, besides environment, genetic factors may also contribute to the level of physical activity and eating behaviors thus effect obesity. Therefore the aim of this study is to investigate the effect of various gene mutations on obesity, physical activity levels and eating behavior phenotypes. One hundred patients and 100 controls were enrolled to the study. Physical activity levels were measured with an actical acceloremeter device. Eating behaviors were evaluated using Three-Factor Eating questionnaire (TFEQ). Associations between eating behavior scores and physical characteristics were also evaluated. The information about other obesity risk factors were also collected. Mutations were investigated with PCR, direct sequencing and Real-Time PCR. rs1051168, rs8050146 -2778C > T mutations were found statistically significant in patients, rs1121980 was found statistically significant in controls. 21 mutations were found in MC4R and near MC4R of which 18 of them are novel and 8 of them cause amino acid change. In addition, it was found that, some obesity related factors and questions of TFEQ are associated with various investigated gene mutations. Any relation between gene mutations and physical activity levels were not detected. It is thought that, due to the genotype data and eating behaviors, it may be possible to recommend patients for proper eating patterns to prevent obesity. © 2016 IUBMB Life, 68(10):806-816, 2016.

The frequency and the effects of 21-hydroxylase gene defects in congenital adrenal hyperplasia patients.

Congenital adrenal hyperplasia (CAH) is a group of genetic endocrine disorders, caused by enzyme deficiencies in the conversion of cholesterol to cortisol. More than 90% of the cases have 21-hydroxylase deficiency (21-OHD). The clinical phenotype of the disease is classified as classic, the severe form, and nonclassic, the mild form. In this study, it was planned to characterize the mutations that cause 21-OHD in Turkish CAH patients by direct sequencing and multiplex ligation-dependent probe amplification (MLPA) analysis and to investigate the type of CAH (classic or nonclassic type) that these mutations cause. A total of 124 CAH patients with 21-OHD and 100 healthy volunteers were recruited to the study. Most of the mutations were detected by direct sequencing. Large gene deletions/duplications/conversions were investigated with MLPA analysis. Results were evaluated statistically. At the end of our study, 66 different variations were detected including SNPs and deletions/duplications/conversions. Of these variations, 18 are novel, of which three cause amino acid substitutions. In addition, 15 SNPs which cause amino acid changes were identified among these variations. If similar results are obtained in different populations, these mutations, in particular the novel mutation 711 G>A, may be used as markers for prenatal diagnosis.

BRCA Mutations and MicroRNA Expression Patterns in the Peripheral Blood of Breast Cancer Patients.

Breast cancer (BC) persists as the predominant malignancy globally, standing as the foremost cause of cancer-related mortality among women. Despite notable advancements in prevention and treatment, encompassing the incorporation of targeted immunotherapies, a continued imperative exists for the development of innovative methodologies. These methodologies would facilitate the identification of women at heightened risk, enhance the optimization of therapeutic approaches, and enable the vigilant monitoring of emergent treatment resistance. Circulating microRNAs (miRNAs), found either freely circulating in the bloodstream or encapsulated within extracellular vesicles, have exhibited substantial promise for diverse clinical applications. These applications range from diagnostic and prognostic assessments to predictive purposes. This study aimed to explore the potential associations between BRCA mutations and specific miRNAs (miR-21, miR-155, miR-126, and miR-200c) expression that are known to be dysregulated in BC patient samples. Our findings indicate a robust correlation between miRNA expression status and disease subtypes. We found a correlation between the expression status of miRNAs and distinct disease subtypes. Intriguingly, however, no significant associations were discerned between disease status, subtypes, or miRNA expression levels and the presence of BRCA mutations. To advance the validation of miRNAs as clinically relevant biomarkers, additional investigations within larger and meticulously selected patient cohorts are deemed imperative. These microRNA entities hold the potential to emerge as groundbreaking and readily accessible tools, poised for seamless integration into the landscape of clinical practice.

Multigene Panel Testing in Turkish Hereditary Cancer Syndrome Patients.

Objective: Hereditary cancer syndromes (HCSs) are a heterogenous group of disorders caused by germline pathogenic variations in various genes that function in cell growth and proliferation. This study aimed to describe the germline variations in patients with hereditary cancer using multigene panels. Methods: The molecular and clinical findings of 218 patients with HCS were evaluated. In addition, 25 HCS-related genes were sequenced using a multigene panel, and variations were classified according to the American College of Medical Genetics and Genomics (ACMG) criteria. In total, 218 HCS patients predominantly with breast, colorectal, ovarian, gastric, and endometrium cancers were included. Results: Pathogenic variations in 12 distinct genes were detected in 36 of 218 (16.5%) cases. In this study, the most affected gene was the ATM gene, in which pathogenic variations were detected in 8 of 218 cases, followed by CHEK2 (3.2%), MUTYH (3.2%), BRIP1 (1.8%), BARD1 (0.9%), TP53 (0.9%), PALB2 (0.4%), MLH1 (0.4%), MSH2 (0.4%), PMS2 (0.4%), RAD50 (0.4%), and RAD51C (0.4%). Conclusions: This study contributes to genotype-phenotype correlation in HCSs and expands the variation spectrum by introducing three novel pathogenic variations. The wide spectrum of the gene pathogenic variations detected and the presence of multiple gene defects in the same patient make the multigene panel testing a valuable tool in detecting the hereditary forms of cancer and providing effective genetic counseling and family specific screening strategies. Amaç: Herediter kanser sendromlari (HCS) hücre büyümesi ve proliferasyonunda görevli genlerde saptanan germline mutasyonlardan kaynaklanan heterojen bir grup hastaliktir. Bu çalismada kalitimsal kanser sendrom ön tanisiyla degerlendirilen olgularda çoklu gen paneli ile germ hatti varyasyonlarinin degerlendirilmesi planlanmistir. Yöntemler: Kalitimsal kanser sendromu düsünülen 218 olgudan periferik kandan DNA izolasyonu sonrasi HCS ile iliskili 25 gen multigen panel kullanilarak dizilendi ve varyasyonlar American College of Medical Genetics and Genomics (ACMG) kriterlerine göre degerlendirildi. Bulgular: Meme, kolorektal, over, gastrik ve endometriyum kanseri basta olmak üzere toplam 218 herediter kanser sendromlu olgu degerlendirildi. Tüm çalisma grubu incelendiginde en sik ATM gen varyasyonlari (8/218, %3,6) tespit edildi ve bunu siklik sirasina göre CHEK2 (%3,2), MUTYH (%3,2), BRIP1 (%1,8), BARD1 (%0,9), TP53 (%0,9), PALB2 (%0,4), MLH1 (%0,4), MSH2 (%0,4), PMS2 (%0,4), RAD50 (%0,4), RAD51C (%0,4) varyasyonlari takip etmekteydi. Sonuçlar: Bu çalismada farkli kanser türlerinde kalitimsal kansere yol açan genler analiz edilmis ve fenotiple iliskisi degerlendirilmistir. Ayrica bu çalismada ilk kez saptanan üç yeni varyasyon ile literatüre katki saglanmaktadir. Patojenik varyasyon tespit edilen genlerin genis dagilimi ve ayni hastada birden fazla genetik varyasyonun varligi düsünüldügünde, uygun genetik danisma ve aileye özgü tarama planlamasi yapmak için çoklu gen taramasi kalitimsal kanser hastalarinin degerlendirilmesinde hizli ve etkin bir yöntem olarak görünmektedir.

A Novel ATM Gene Mutation Affecting Splicing in an Ataxia-Telangiectasia Patient.

Ataxia-telangiectasia (AT) is an autosomal recessive disorder characterized by progressive ataxia, choreoathetosis and immunodeficiency beginning in early childhood. An 8-year-old girl was referred with a diagnosis of AT. She had gait disturbance and dysarthria for 3years. Multiple cutaneous telangiectases were observed on her face, trunk and limbs. Sequence analysis of the ATM gene revealed a homozygous c.7308-15A>G mutation in IVS49. Human Splicing Finder predicted that the mutation could activate an intronic cryptic acceptor site. We designed primers for amplification of related exons (48-50) from cDNA for evaluating splicing pattern. Sequencing of ATM exons 48-50 revealed a 14-nucleotide insertion from intron 49, between exons 49 and 50, resulting in premature termination of translation at codon 2439. To conclude, we report a novel mutation in a classical AT case, which resulted in an alternatively spliced transcript and was predicted to form a truncated protein or null protein due to nonsense-mediated decay.

Mutation Spectrum of Familial Adenomatous Polyposis Patients in Turkish Population: Identification of 3 Novel APC Mutations.

BACKGROUND: Familial adenomatous polyposis (OMIM #175100) and MUTYH-associated polyposis (OMIM #608456) are rare cancerprone disorders characterized by hundreds of adenomatous polyps in the colon and rectum, which have a high probability of malignant transformation. Attenuated familial adenomatous polyposis is a variant of familial adenomatous polyposis, which is a term used for the condition in which patients have less than 100 colorectal polyps. Germline heterozygous Adenomatous polyposis coli (APC) and biallelic MUTYH (mutY DNA glycosylase) pathogenic variations are responsible for familial adenomatous polyposis and MUTYH-associated polyposis respectively. The aim of this study is to discuss the clinical manifestations of patients having pathogenic APC and MUTYH variations. METHODS: We included 27 probands who have more than 10 colonic polyps in this study. After evaluation of their clinical and family histories, the probands were screened for APC and MUTYH variations via next generation sequencing. The family members of the probands carrying pathogenic variations were screened via Sanger sequencing. RESULTS: Among 27 probands, pathogenic APC and MUTYH variations were detected in 3 and 6 probands respectively. In the APC gene, 3 novel truncating variations (p.Leu360*, p.Leu1489Phefs*23, and p.Leu912*) were detected in 3 unrelated probands. In the MUTYH gene, only 2 distinct pathogenic variations were detected (p.Pro295Leu and p.Glu480del) in the homozygous or compound heterozygous state. CONCLUSION: In this study, molecular etiology was clarified in 9 familial polyposis patients. The p.Pro295Leu and p.Glu480del variations seem to be common in the Turkish population and may be considered as a first-step genetic test in Turkish familial polyposis patients showing autosomal recessive inheritance. However more studies are needed to reveal the exact frequency of these variations.

When do we need to suspect maturity onset diabetes of the young in patients with type 2 diabetes mellitus?

OBJECTIVE: Maturity onset diabetes of the young (MODY) patients have clinical heterogeneity as shown by many studies. Thus, often it is misdiagnosed to type 1 or type 2 diabetes(T2DM). The aim of this study is to evaluate MODY mutations in adult T2DM patients suspicious in terms of MODY, and to show clinical and laboratory differences between these two situations. METHODS: In this study, we analyzed 72 type 2 diabetic patients and their relatives (35F/37M) who had been suspected for MODY and referred to genetic department for mutation analysis. The gene mutations for MODY have been assessed in the laboratory of Marmara University genetics. Totally 67 (32F/35M; median age 36.1) diabetic patients were analyzed for 7 MODY mutations. Twelve patients who have uncertain mutation (VUS) were excluded from study for further evaluation. MODY(+) (n:30) patients and T2DM patients (n:25) were compared for clinical and laboratory parameters. RESULTS: In MODY(+) subjects, mutations in GCK (MODY 2) (n:12; 40%) were the most common followed by HNF4A (MODY 1) (n:4; 13.3%). Diabetes diagnosis age was younger in MODY(+) group but not statistically significant. Sixty-six percent of MODY(+) subjects had diabetes history at 3-consecutive generations in their family compared with 28% of T2DM patients statistically significant (p:0.006). Gender, BMI, C-peptide, HbA1c, lipid parameters, creatinine, GFR, microalbuminuria, vitamin D and calcium were not statistically different between the groups. CONCLUSION: According to present study results, MODY mutation positivity is most probable in young autoantibody (-) diabetic patients diagnosed before 30 years of age, who have first degree family history of diabetes.

Genetic and Clinical Characterization of Patients with Maturity-Onset of Diabetes of the Young (MODY): Identification of Novel Variations.

BACKGROUND: Maturity-onset diabetes of the young (MODY) is a rare monogenic type of diabetes, and accounts for 2-5% of all diabetes cases. An early age of onset, a family history supporting autosomaldominant inheritance, insulin resistance, and the absence of autoimmunity are the major characteristics of MODY. However, genetic testing is crucial for diagnosis. AIMS: To investigate the 7 MODY-related genes and clinical findings of patients with a preliminary clinical diagnosis of MODY. STUDY DESIGN: Retrospective cross-sectional study. METHODS: In this study, 7 genes (KCNJ11, ABCC8, INS, GCK, HNF4A, HNF1A, and HNF1B) related to MODY were screened via targeted sequencing in 182 cases with a confirmed pre-diagnosis of MODY. The clinical characteristics of the patients were evaluated retrospectively. RESULTS: A total of 182 patients, 48% of whom were women, between the ages of 18-62 were included in the study. In 30 cases (16.4%), 28 different pathogenic variations were found, of which 20 were previously reported and 8 were novel variations segregated by disease within the family. Pathogenic variations were detected in the following genes in order of mutation frequency; GCK, HNF1A, ABCC8, HNF4A, HNF1B and KCNJ11. Interestingly, six of the 30 cases (20%) carried a pathogenic variation in the ABCC8 gene. No mutation was detected in the INS gene. A family history of vertically transmitted diabetes and elevated HbA1C at the time of diagnosis were found in 20 (66%) and 16 (52%) cases, respectively. CONCLUSION: In this series, 28 different pathogenic variations are identified, 8 of which are novel. The rate of pathogenic variation in the ABCC8 gene is unexpectedly high. Two-thirds of cases have a family history of vertically transmitted diabetes.

Genetic diagnosis in infertile men with numerical and constitutional sperm abnormalities.

Infertile men having numerical or structural sperm defects may carry several genetic abnormalities (karyotype abnormalities, Y chromosome microdeletions, cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations, androgen receptor gene mutations, and abnormalities seen in sperm cells) leading to this situation. First we aimed to investigate the relationship between the numerical and constitutional (morphological) sperm anomalies and the genetic disorders that can be seen in infertile males. Our other aim was to compare two different kinds of kits that we use for the detection of Y chromosome microdeletions. Sixty-three infertile males [44 nonobstructive azoospermic, 8 severe oligozoospermic, and 11 oligoasthenoteratozoospermic] were investigated in terms of somatic chromosomal constitutions and microdeletions of the Y chromosome. Sperm aneuploidy levels were analyzed by fluorescence in situ hybridization (FISH) in sperm cells obtained from the semen of six OAT patients. Microdeletion and sex chromosome aneuploidy (47,XXY) rates in somatic cells were found to be approximately 3.2% and 4.7%, respectively. Sperm aneuploidy rates were determined as 9%, 22%, and 47% in three patients out of six. Two of these three patients also had high rates of head anomalies in semen samples. High correlation was found between sperm aneuploidy rates and sperm head anomalies. Since the introduction of the assisted reproductive techniques for the treatment of severe male infertility, genetic tests and genetic counseling became very important due to the transmission of genetic abnormalities to the next generation. Thus in a very near future, for a comprehensive male infertility panel, it will be essential to include additional genetic tests, such as CFTR gene mutations, sperm mitochondrial DNA mutations, and androgen receptor gene mutations, besides the conventional chromosomal analyses, Y chromosome microdeletion detection, and sperm-FISH analyses.

Investigation of the association between mitochondrial DNA and p53 gene mutations in transitional cell carcinoma of the bladder.

Bladder carcinoma is the most common malignancy of the urinary tract. The major aim of the present study is to investigate the association between mitochondrial DNA (mtDNA) and p53 gene mutations in bladder carcinoma. A total of 30 patients with transitional cell carcinoma and 27 controls were recruited for the study. Bladder cancer tissues were obtained by radical cystectomy or transurethral resection. Genomic DNA was extracted from peripheral blood. mtDNA and p53 genes were amplified by polymerase chain reaction and sequenced directly. A total of 37 polymorphisms were identified, among which, 2 mutations were significant in the patient group, and 1 mutation was significant in the control group. Additionally, 5 different moderate positive correlations between mtDNA mutations and 3 different positive correlations between p53 gene and mtDNA mutations were detected. The high incidence of mtDNA and p53 gene mutations in bladder cancer suggests that these genes could be important in carcinogenesis.

Transforming growth factor-ß3 intron 5 polymorphism as a screening marker for non-syndromic cleft lip with or without cleft palate.

In this study, we evaluated the effect of transforming growth factor ß3 intron 5 position +104 A➝G (TGF-ß3 IVS5+104AG) transition in patients with a non-syndromic cleft lip with or without cleft palate (NSCL/P). A total of 68 patients and 114 controls were recruited for the study. A genotyping procedure was carried out using the PCR-RFLP method. For statistical analysis, the Chi-square test was used to compare data between the patient and control groups. The frequencies of the AA, AG and GG genotypes were 24, 29 and 47%, respectively, for the patients and 54, 36 and 10%, respectively, for the control group. The GG genotype and G allele were significantly different in the patient group compared with the control (p=0.0001). We conclude that SfaN1 polymorphism in TGF-ß3 may be a good screening marker for the prediction of NSCL/P in patients. However, more studies with extended sample numbers should be carried out to clarify the effect of the examined gene region on NSCL/P.

Detection of mitochondrial DNA mutations in nonmuscle invasive bladder cancer.

BACKGROUND: Mitochondrial DNA (mtDNA) mutations have been recently described in various tumors; however, data focusing on bladder cancer are scarce. To understand the significance of mtDNA mutations in bladder cancer development, we investigated the mtDNA alterations in bladder cancer cases. METHODS: We studied the mtDNA in 38 bladder tumors and 21 microdissected normal bladder tissue samples. Mitochondrial genes ATPase6, CytB, ND1, and D310 region were amplified by polymerase chain reaction and then sequenced. RESULTS: We detected 40 mutations in our patient population. Our findings indicate that G8697A, G14905A, C15452A, and A15607G mutations are frequent in bladder cancers (p<0.05). In addition, the incidence of A3480G, T4216C, T14798C, and G9055A mutations were higher in patients with bladder tumors. CONCLUSIONS: In conclusion, the high incidence of mtDNA mutations in bladder cancer suggests that mitochondria could play an important role in carcinogenesis and mtDNA could be a valuable marker for early bladder cancer diagnosis.

Autosomal recessive idiopathic epilepsy in an inbred family from Turkey: identification of a putative locus on chromosome 9q32-33.

PURPOSE: The study describes the clinical features of an inbred family from Turkey with three members affected by seizures and tests possible autosomal recessive (AR) inheritance by means of linkage analysis. METHODS: Personal and family history was obtained from each subject, and general physical, neurologic, and EEG examinations were performed. A set of 382 fluorescence-labeled markers was used for the initial genome-wide search. A further set of 83 markers was used to map the locus precisely and to exclude the remaining genome. RESULTS: Twelve individuals from three generations were examined. Two subjects were affected by idiopathic epilepsy, whereas, their brother experienced a single unprovoked generalized seizure. Two siblings affected by idiopathic epilepsy and their unaffected sister showed a photoparoxysmal response to photic stimulation. Nine family members reported migraine. The genome-wide search led to the identification of a unique homozygous, 15.1-cM region shared by subjects with seizures on chromosome 9q32-33 and providing a lod score of 2.9. This locus, however, was not associated with migraine in this pedigree. CONCLUSIONS: The study suggests that idiopathic epileptic traits with AR inheritance might be underestimated in the general population and that inbred pedigrees may represent powerful tools for the identification of AR genes.