POU6F2 mutation in humans with pubertal failure alters GnRH transcript expression

Idiopathic hypogonadotropic hypogonadism (IHH) is characterized by the absence of pubertal development and subsequent impaired fertility often due to gonadotropin-releasing hormone (GnRH) deficits. Exome sequencing of two independent cohorts of IHH patients identified 12 rare missense variants in POU6F2 in 15 patients. POU6F2 encodes two distinct isoforms. In the adult mouse, expression of both isoform1 and isoform2 was detected in the brain, pituitary, and gonads. However, only isoform1 was detected in mouse primary GnRH cells and three immortalized GnRH cell lines, two mouse and one human. To date, the function of isoform2 has been verified as a transcription factor, while the function of isoform1 has been unknown. In the present report, bioinformatics and cell assays on a human-derived GnRH cell line reveal a novel function for isoform1, demonstrating it can act as a transcriptional regulator, decreasing GNRH1 expression. In addition, the impact of the two most prevalent POU6F2 variants, identified in five IHH patients, that were located at/or close to the DNA-binding domain was examined. Notably, one of these mutations prevented the repression of GnRH transcripts by isoform1. Normally, GnRH transcription increases as GnRH cells mature as they near migrate into the brain. Augmentation earlier during development can disrupt normal GnRH cell migration, consistent with some POU6F2 variants contributing to the IHH pathogenesis

Silika metodu ile kemikten DNA ekstraksiyonu

TEZ7733Tez (Yüksek Lisans) -- Çukurova Üniversitesi, Adana, 2010.Kaynakça (s. 35-38) var.xi, 39 s. : rnk. res. ; 29 cm.Silica Based DNA Extraction From Bone. Determining the identity of a dead person is one of the most important steps in the practise of Forensic medicine. DNA can be retrieved from a variety of biological material for the correct identification. DNA is best preserved and least likely to be contaminated in the bone tissue after a range of physical and chemical impacts on the corpse. In this study, the feasibility of a silica-based method of DNA extraction from bone has been investigated. A total 15 costal bones obtained from autopsy cases at the Adana Forensic Medicine Institute has been used. Sufficient amount of good quality DNA to successfully run a 16 STR (D8S1179, D21S11, D7S820, CSFIPO, D3S1358, THO1, D13S317, D16S539, D2S1338, D19S433, VWA, TPOX, D18S51, AMEL, D5S818 and FGA) test has been extracted with a silica-based commercial kit, Qiagen DNA Blood Mini Kit) from six months old bones left in room temperature. It was also observed that decalcification was not necessary for the extraction of DNA from 15 fresh bones. In three old bones, the DNA extraction failed with or without decalcification. In one case, although there was not a recogniziable amount of DNA, we were able to determine D3S1358, THO1, D19S433, TPOX, AMEL and D5S818 in a 16 STR multiplex reaction, despite the relative fleurosence unit being below 50. This is explained by not using a system with a cooler during grinding of old samples in this study. These results demonstrate that a silica-based metod of DNA extraction from bone can be employed for routine diagnostics at the Forensic Serology and Genetics Laboratory of the Çukurova University, Faculty of Medicine, department of Forensic Medicine.Silika Metodu ile Kemikten DNA Ekstraksiyonu. Adli uygulamalarda ölüm olaylarının incelenmesinde en önemli aşamalardan birisi kişinin kimliğinin tespitidir. Kimliklendirme için çeşitli biyolojik delillerden DNA'ya ulaşmak mümkündür. Cesetlerin maruz kaldığı farklı fiziksel ve kimyasal faktörler sonrası DNA'nın en iyi korunabildiği ve kontaminasyondan en az etkilenen doku kemik dokusudur. Bu çalışmada kemik dokudan silika esaslı DNA ekstraksiyon metodu ile DNA elde edilebilirliğinin araştırılması amaçlanmıştır. Çalışmada Adana Adli Tıp Kurumunda otopsisi yapılan olgulardan alınan 15 adet kosta kemiği kullanılmıştır. 6 ay oda sıcaklığında bekletilen kemik örneklerinde silika temelli ticari kit olan Qiagen DNA Mini Kit ile 16 STR (D8S1179, D21S11, D7S820, CSFIPO, D3S1358, THO1, D13S317, D16S539, D2S1338, D19S433, VWA, TPOX, D18S51, AMEL, D5S818 ve FGA) sisteminin tanımlanabilmesi için yeterli kalite ve kantitede DNA elde edilebilmiştir. Çalışmada kullanılan 15 taze kemik örneğinden DNA eldesinde dekalsifikasyona gerek olmadığı görülmüştür. Çalışmada kullanılan üç eski kemik örneğinin ikisinde dekalsifikasyonlu ve dekalsifikasyonsuz başarılı DNA ekstraksiyonu yapılamamıştır. Birinde ekstraksiyonda tanımlanabilir miktarda DNA bulunmamasına karşın 16 STR sisteminin çalışıldığı multiplex reaksiyonda relatif floresans ünitesi 50'nin altında olmakla birlikte D3S1358, THO1, D19S433, TPOX, AMEL ve D5S818 STR sistemleri tanımlanabilmiştir. Bu durum eski örneklerin toz haline getirilme aşamasında soğutma sistemli bir düzeneğin olmamasına bağlandı. Elde edilen sonuçlar kemik dokudan silika temelli DNA ekstraksiyonunun Çukurova Üniversitesi Tıp Fakültesi Adli Tıp Anabilim Dalı Adli Seroloji ve Genetik Laboratuvarında rutin hizmette kullanılabilirliğini gösterilmiştir.Bu çalışma Ç.Ü. Bilimsel Araştırma Projeleri Birimi Tarafından Desteklenmiştir. Proje No: TF2009YL

Identification of a novel gene taking role in puberty.

TEZ9269Tez (Doktora) -- Çukurova Üniversitesi, Adana, 2014.Kaynakça (s. 55-63) var.xv, 79 s. : res. (bzs. rnk.), tablo ; 29 cm.Bu çalışma, insanlarda çocukluktan erişkinliğe geçişi sağlayan püberte sürecinin nasıl başladığının aydınlatılmasına katkıda bulunmak amacı ile süreçte yer alan yeni bir genin belirlenmesi için yapılmıştır. Buna yönelik olarak, koku alma duyusunun olmaması ve İdiopatik Hipogonadotropik Hipogonadizme bağlı püberte gelişmemesiyle karakterize olan Kallmann sendromu insan hastalık modeli olarak kullanılmıştır. Çukurova Üniversitesi Tıp Fakültesi Çocuk Endokrinoloji Polikliniğinde izlenen familyal Kallmann sendromlu 20 aile çalışmaya seçilmiştir. Genom boyu SNP genotiplemesine dayalı otozigosite haritalaması ve tüm eksom sekanslaması sonucu ailelerden ikisinin ortak olarak FEZF1 geninde olası zararlı varyantlar barındırdığı saptanmıştır. Fezf1 knockout edilmiş farelerde GnRH nöronlarının olfaktor plakoddan hipotalamusa doğru olan embriyonik göçlerinin kesintiye uğradığı literatürde daha önce gösterilmiştir. Birinci ailedeki varyantın (p.H278Y) çinko parmak özelliğindeki FEZF1 genin işlevini bozduğu HEK293T hücrelerinde yapılan fonksiyonel çalışmalarla kanıtlanmıştır. İkinci ailedeki çerçeve kayması varyantının (c.651delT; A217fs13X) ise Nonsense-mediated decay mekanizmasını etkinleştirip kodlanan proteinin hiç üretilememesine neden olduğu öngörülmüştür. Bu iki ailedeki hastalarda GnRH nöronları çok yüksek olasılıkla embriyonik göçlerini tamamlayıp hipotalamustaki nihai lokasyonlarına ulaşamamış böylelikle yaşamın ikinci on yılında aktiflenerek püberte sürecini başlatan hipotalamo-hipofizer-gonadal eksen hiç kurulamamıştır. Böylelikle, insanda FEZF1 geni fonksiyonunun püberte süreci için zorunlu olduğu ve bu gendeki zararlı mutasyonların bir sonucu olarak Kallmann sendromu fenotipinin geliştiği literatürde ilk kez gösterilmiştir.It is currently unknown how transition from childhood to adulthood, namely puberty, is started. This study was undertaken to identify a novel gene which has a role in pubertal process. To this end, familial anosmic hypogonadotropic hypogonadism (Kallmann syndrome), which is characterized by absence of pubertal development and sense of smell, was utilized as a human disease model. Patients from 20 multiplex families who were diagnosed with Kallmann Syndrome (KS) in the department of Pediatric Endocrinology of the Cukurova University, Faculty of Medicine were recruited. After screening out the genes known to be associated with KS, 10 of these families were further investigated for responsible gene mutations with SNPbased autozygosity mapping and whole exome sequencing. Two of these families harbored potentially harmful mutations in FEZF1. Knockout mice models for this gene were previously shown to suffer from a defective embryonic GnRH neuron migration from the olfactory placode to the hypothalamus. Mutation in the first family (p.H278Y), which involves a zinc finger motif, was demonstrated to be deleterious in a heterologous expression study in HEK298 cell line. Mutation in the second family (c.651delT; A217fs13X) is predicted to result in the absence of a protein product due to Nonsense-mediated decay. These functional deficits most probably led to unsuccessful embriyonic migration of GnRH neurons, thus resulting in failure to establish hypothalamo-pituitarygonadal axis (HPG), activation of which in the second decade of life is the basis of puberty. In conclusion, it has been shown for the first time in this study that FEZF1 is required for normal pubertal development and deleterious mutations in FEZF1 result in KS in humans.Bu çalışma Ç.Ü. Bilimsel Araştırma Projeleri Birimi tarafından desteklenmiştir. Proje No: FBE2013D1

CHD7 mutations in patients with anosmic or normosmic idiopathic hypogonadotropic hypogonadism

WOS: 000485922401023

Kallmann Syndrome Due to a Homozygous Missense c.217C > T (p.R73C) Mutation Detected in the Exon-2 of the PROK2 Gene

WOS: 000384166801412

DLG2 Mutations in the Etiology of Pubertal Delay and Idiopathic Hypogonadotropic Hypogonadism

Introduction: Idiopathic hypogonadotropic hypogonadism (IHH) is caused by dysfunction of the hypothalamic-pituitary-gonadal axis. DLG2 was recently implicated as a gene associated with delayed puberty and which may also contribute to IHH. The confirmation of the candidate puberty genes in independent IHH cohorts has become crucial due to the lack of proper genotype-phenotype segregations in reported pedigrees. Therefore, we aimed to screen DLG2 in patient variants in a large cohort of IHH patients. Methods: The present study included a total of 336 IHH patients from 290 independent families. The coding and flanking regions of DLG2 were screened for potentially important variants in the WES data. Candidate variants were evaluated in the -gnomAD and GME databases according to their allele frequencies, and only those with a frequency Results: We found 1 homozygous and 2 heterozygous missense variants in 3 independent pedigrees. Identified variants were found extremely rare or not reported in gnomAD. Two variants were categorized as "uncertain significance," and the other one was "likely pathogenic" according to the ACMG criteria. All patients were normosmic, and in 2 of the 3 families, there were no causal variants in other IHH-related genes. Conclusion: We detected 3 rare sequencing variants in DLG2 in 5 patients with IHH or delayed puberty in a large IHH cohort. Our results support the contention that the DLG2 mutations are associated with IHH in human puberty

BMP4 mutations as a novel cause of normosmic hypogonadotropic hypogonadism

WOS: 000485922401062

A nonsense variant in FGFR1: a rare cause of combined pituitary hormone deficiency

Objectives: Variants in fibroblast growth factor receptor-1 (FGFR1) may either cause isolated hypogonadotropic hypogonadism (IHH) or Kallmann syndrome (KS). Although the relationship of genes classically involved in IHH with combined pituitary hormone deficiency (CPHD) is well established, variants in FGFR1 have been presented as a rare cause of this phenotype recently

Idiopathic Hypogonadotropic Hypogonadism Caused by Inactivating Mutations in SRA1

Objective: What initiates the pubertal process in humans and other mammals is still unknown. We hypothesized that gene(s) taking roles in triggering human puberty may be identified by studying a cohort of idiopathic hypogonadotropic hypogonadism (IHH). Methods: A cohort of IHH cases was studied based on autozygosity mapping coupled with whole exome sequencing. Results: Our studies revealed three independent families in which IHH/delayed puberty is associated with inactivating SRA1 variants. SRA1 was the first gene to be identified to function through its protein as well as noncoding functional ribonucleic acid products. These products act as co-regulators of nuclear receptors including sex steroid receptors as well as SF-1 and LRH-1, the master regulators of steroidogenesis. Functional studies with a mutant SRA1 construct showed a reduced co-activation of ligand-dependent activity of the estrogen receptor alpha, as assessed by luciferase reporter assay in HeLa cells. Conclusion: Our findings strongly suggest that SRA1 gene function is required for initiation of puberty in humans. Furthermore, SRA1 with its alternative products and functionality may provide a potential explanation for the versatility and complexity of the pubertal process