Genotipificación del gen RHD en ADN fetal libre en plasma de embarazadas RhD negativo / Genotyping of the RHD gene in free fetal DNA in plasma of RhD negative pregnant women

Objetivo: Determinar el grupo RhD fetal a través del estudio del gen RHD en ADN fetal que se encuentra libre en plasma de embarazadas RhD negativo. Método: Se analizó la presencia de los genes RHD, SRY y BGLO en ADNfl obtenido de plasma de 51 embarazadas RhD negativo no sensibilizadas, utilizando una qPCR. Los resultados del estudio genético del gen RHD se compararon con el estudio del grupo sanguíneo RhD realizado por método serológico en muestras de sangre de cordón, y los resultados del estudio del gen SRY fueron cotejados con el sexo fetal determinado por ecografía. Se calcularon la sensibilidad, la especificidad, los valores predictivos y la capacidad discriminativa del método estandarizado. Resultados: El gen RHD estaba presente en el 72,5% de las muestras y el gen SRY en el 55,5%, coincidiendo en un 100% con los resultados del grupo RhD detectado en sangre de cordón y con el sexo fetal confirmado por ecografía, respectivamente. Conclusiones: Fue posible deducir el grupo sanguíneo RhD del feto mediante el estudio del ADN fetal que se encuentra libre en el plasma de embarazadas con un método molecular no invasivo desarrollado y validado para este fin. Este test no invasivo puede ser utilizado para tomar la decisión de administrar inmunoglobulina anti-D solo a embarazadas RhD negativo que portan un feto RhD positivo.

Objective: To determine the fetal RhD group through the study of the RHD gene in fetal DNA found free in plasma of RhD negative pregnant women. Method: The presence of the RHD, SRY and BGLO genes in fetal DNA obtained from plasma of 51 non-sensitized RhD negative pregnant women was analyzed using qPCR. The results of the genetic study of the RHD gene were compared with the RhD blood group study performed by serological method in cord blood samples, and the results of the SRY gene study were compared with the fetal sex determined by ultrasound. Sensitivity, specificity, predictive values and discriminative capacity of the standardized method were calculated. Results: The RHD gene was present in 72.5% of the samples and the SRY gene in 55.5%, coinciding 100% with the results of the RhD group detected in cord blood, and with the fetal sex confirmed by ultrasound, respectively. Conclusions: It was possible to deduce the RhD blood group of the fetus through the study of fetal DNA found free in the plasma of pregnant women with a non-invasive molecular method developed and validated for this purpose. This non-invasive test can be used to make the decision to administer anti-D immunoglobulin only to RhD-negative pregnant women carrying an RhD-positive fetus.

Sex Reversal Syndrome (SRS): A case of SRY-Positive 46,XX Testicular Disorder / Journal of the ASEAN Federation of Endocrine Societies

@#We report a case of an SRY-positive 46,XX Indian male who presented with small testis and phallus, poor beard and mustache development and gynecomastia at the age of 24 years. He was biochemically found to have hypergonadotropic hypogonadism. He had 46,XX karyotype and Quantitative Fluorescence-PCR (QF-PCR) identified the SRY gene on the X chromosome. SRY-positive 46 XX male SRS cases usually present as phenotypically male since birth but develop features of hypogonadism, poor testicular development, and infertility after puberty. Infertility, hypogonadism, external genital development, and psychological distress are the major concerns during the management of the patients. Testosterone therapy for hypogonadism, artificial reproductive technologies for fertility, surgical repair of hypospadias/cryptorchidism/under-virilized genitalia and psychological and genetic counseling are helpful for proper management of the patients.

Establishment of Multiplex Amplification System of STR Loci in Felis Catus and Its Forensic Application / 法医学杂志

OBJECTIVES@#To construct a Felis catus STR loci multiplex amplification system and to evaluate its application value by testing the technical performance.@*METHODS@#The published Felis catus STR loci data were reviewed and analyzed to select the STR loci and sex identification loci that could be used for Felis catus individual identification and genetic identification. The fluorescent labeling primers were designed to construct the multiplex amplification system. The system was validated for sensitivity, accuracy, balance, stability, species specificity, tissue identity and mixture analysis, and investigated the genetic polymorphisms in 145 unrelated Felis catus samples.@*RESULTS@#Sixteen Felis catus autosomal STR loci and one sex determining region of Y (SRY) were successfully selected, and constructed a multiplex amplification system containing the above loci. The complete profile of all alleles could still be obtained when the amount of DNA template was as low as 0.25 ng. There was no specific amplification peak in other common animal samples. Population genetic surveys showed that total discrimination power (TDP) of the 16 STR loci was 1-3.57×10-20, the cumulative probability of exclusion (CPE) was 1-6.35×10-5 and the cumulative probability of matching was 3.61×10-20.@*CONCLUSIONS@#The Felis catus STR multiplex amplification system constructed in this study is highly sensitive, species-specific, and accurate in typing results, which can provide an effective solution for Felis catus species identification, individual identification and kinship identification in the field of forensic science.

Clinical significance of NRSN2-AS1 expression in ovarian cancer and its biological effect in vitro / 中华内分泌外科杂志

Objective:To investigate the clinical significance and biological effect of long non-coding RNA (lncRNA) NRSN2-AS1 in ovarian cancer.Methods:The expression of NRSN2-AS1 was detected by real-time quantitative PCR (RT-qPCR) in 84 cases of ovarian cancer tissues and corresponding normal adjacent tissues, and the relationship between NRSN2-AS1 expression and clinical data of patients was analyzed. Human ovarian epithelial cells HOSEpiC and human ovarian cancer cells A2780 and OVCAR3 were cultured in vitro, and overexpression or interference of NRSN2-AS1 and control plasmids were respectively transfected into A2780 and OVCAR3 cells as the blank group, overexpression group and control group, interference group. CCK-8 assay was used to detect cell proliferation, while transwell invasion and migration assay were used to detect the change of cell metastasis ability, RT-qPCR and Western blot were used to detect the expressions of SRY related HMG box transcription factor 12 (SOX12), a potential downstream gene of NRSN2-AS1.Results:The expressions of NRSN2-AS1 in ovarian cancer tissues and cells were significantly higher than normal adjacent tissues, and the high expression of NRSN2-AS1 was significantly correlated with poor prognosis, lymph node metastasis and high clinical stage ( P<0.05). In the blank and overexpression group, the expressions of NRSN2-AS1 were 1.00±0.08 and 5.78±0.41, the expressions of SOX12 mRNA were 1.00±0.10 and 3.08±0.23, the expression of SOX12 protein were 1.00±0.08 and 7.26±0.39, the invasion cells per field were 22.7±4.9 and 79.0±6.2, and the migration cells per field were 26.5±4.1 and 43.5±4.5. In the control and interference group, the expression of NRSN2-AS1 were 1.00±0.11 and 0.37±0.04, the expressions of SOX12 mRNA were 1.00±0.07 and 0.59±0.05, the expression of SOX12 protein were 1.00±0.07 and 0.36±0.03, the invasion cells per field were 68.3±6.1 and 30.6±5.5, and the migration cells per field were 85.2±7.0 and 22.7±4.2. Compared with the blank group, the expression of SOX12 mRNA and protein in the overexpression group was significantly increased ( P<0.05), and the cell proliferation and metastasis ability were significantly enhanced ( P<0.05). Compared with the control group, the expression of SOX12 mRNA and protein in the interference group was significantly decreased, and the ability of cell proliferation and metastasis was suppressed significantly ( P<0.05) . Conclusion:The expression of NRSN2-AS1 is up-regulated in ovarian cancer, which is closely related to poor prognosis and progression of ovarian cancer. NRSN2-AS1 can promote the expression of SOX12 and the malignant behavior of ovarian cancer cells in vitro.

Prenatal diagnosis and genetic analysis of a female fetus with 46, XX and positive SRY gene / 中华围产医学杂志

We report a case of a pregnant woman with 46,XX karyotype and positive sex-determining region on the Y chromosome ( SRY) gene and her female fetus. Ultrasound examination at 12 +6 gestational weeks indicated a thickened fetal nuchal translucency, and 46, XX with a positive SRY gene was detected in the fetus through quantitative fluorescent-polymerase chain reaction and amniotic fluid karyotype. However, the ultrasound showed that the gender of the fetus was female, which was inconsistent with the phenotype of male syndrome with 46, XX combining positive SRY gene. The fluorescent in situ hybridization (FISH) revealed that the short arm of the Y chromosome translocated to the long arm of one of the X chromosomes, namely Yp11.3-Xq28. In addition, a copy number variation at Yp11.31p11.2 copy (about 1 MB) was found by chromosomal microarray analysis, which validated the result of FISH and was consistent with the mother. After genetic counseling, the parents chose to continue the pregnancy to full term, and no abnormalities were found in the infant during the follow-up.

Newborn transient patterned hyperpigmentation and anophthalmia / Patrón de hiperpigmentación transitoria del recién nacido y anoftalmia

Abstract Background: Transient pigmentary lines of the newborn are uncommon cutaneous lesions of unknown etiology. To date, only a few cases have been described. Case report: A patient with a combination of transient pigmentary lines and ocular malformation is described. Molecular analysis of the SRY-box 2 (SOX2) and MIFT genes was conducted to rule out any monogenetic etiology. Conclusions: Worldwide, this is the eighth case of transient pigmentary lines of the newborn reported, and the first associated with anophthalmia. No mutations in the analyzed genes (SOX2 and MIFT) were identified. Therefore, somatic mutations could be responsible for this anomaly.

Resumen Introducción: Las líneas transitorias pigmentarias del recién nacido son lesiones cutáneas poco comunes. A la fecha, pocos casos se han descrito. Caso clínico: Paciente neonato con la combinación de líneas transitorias hiperpigmentadas y una malformación ocular. Se realizó secuenciación molecular de los genes SOX2 y MIFT para descartar una etiología monogénica. Conclusiones: En todo el mundo, este es el octavo caso reportado de líneas transitorias hiperpigmentadas del recién nacido, y el primero asociado con anoftalmia. No se identificaron mutaciones en los genes estudiados (SOX2 y MIFT). Por lo tanto, las mutaciones somáticas pueden ser la causa de la afección.

Effect of bone morphogenetic protein-7 combined with porous tantalum on chondrogenic differentiation and function of bone marrow mesenchymal stem cells / 中国组织工程研究

BACKGROUND: The establishment of coculture system combined with physical factors and scaffold materials and the Induction of cytokines have become the focus of chondrogenlc differentiation of bone marrow mesenchymal stem cells. OBJECTIVE: To observe the effect of bone morphogenetic protein 7 combined with porous tantalum on chondrogenlc differentiation of bone marrow mesenchymal stem cells. METHODS: Bone marrow mesenchymal stem cells of Sprague-Dawley rats (provided by Beijing Huafukang Biology) were Isolated and cultured. Group Intervention: (1) in the experimental group, porous tantalum tablet was added, while In the control group, porous tantalum tablet was not added. At 5 days after culture, cell growth on the surface of porous tantalum tablet was observed by phalloidin staining. At 1, 3,5, and 7 days after culture, CCK-8 method was used to detect cell proliferation. (2) Group A was added with chondrocyte Inducer; group B with chondrocyte Inducer and bone morphogenetic protein 7; group C with domestic porous tantalum material and chondrocyte Inducer; group D with domestic porous tantalum material and chondrocyte Inducer and bone morphogenetic protein 7. At 7,14 and 21 days after culture, the levels of type II collagen, SRY type high mobility group protein and matrix metalloproteinase-13 secreted by cells In each group was detected by ELISA. Western blot assay was used to detect the expression of type II collagen, SRY type high mobility group protein and matrix metalloproteinase-13. This study was approved by the Animal Experimental Ethics Committee of North China University of Science and Technology. RESULTS AND CONCLUSION: (1) The phalloidin staining results showed that bone marrow mesenchymal stem cells grew well on and around the porous tantalum surface. (2) At 3 and 5 days after culture, the proliferation of bone marrow mesenchymal stem cells was slower In the experimental group than in the control group (P 0.05). (3) At 7,14 and 21 days, the expression of type II collagen and SRY high mobility group protein Increased gradually among groups A, B, C and D (P 0.05). At 21 days, there was no significant difference among groups A, B, C and D (P > 0.05). (4) Western blot assay showed that at 7,14 and 21 days after culture, the expression level of type II collagen and SRY high mobility group protein Increased gradually In groups A, B, C and D (P 0.05). (5) The results showed that bone morphogenetic proteln-7 combined with domestic porous tantalum could Induce cartilage differentiation of bone marrow mesenchymal stem cells, facilitate the expression of type II collagen and SRY high mobility group protein, and Inhibit the expression of matrix metalloproteinase-13.

Paciente masculino con cariotipo 46 XX negativo para el gen SRY y sin ambigüedad genital: reporte de un caso / Male patient 46, XX SRY -negative and unambiguous genitalia: A case report

En la mayoría de los casos, la diferenciación sexual masculina ocurre con la participación del gen SRY. Sin embargo, se pueden presentar otros genotipos excepcionales, como en el caso que se presenta en este reporte. Se trata de un paciente adulto de sexo masculino atendido en el Servicio de Paternidades del Instituto de Genética de la Universidad Nacional de Colombia. Se le hicieron los análisis del gen de la amelogenina y de repeticiones cortas en tándem (Short Tandem Repeat, STR) específicas para el gen SRY con estuches comerciales de identificación humana, así como los de cariotipo convencional e hibridación in situ fluorescente del SRY, y el estudio de microdeleciones del cromosoma Y mediante reacción en cadena de la polimerasa (PCR). Se le hizo la evaluación clínica y se le brindó asesoramiento genético. El paciente no presentaba ambigüedad genital, su cariotipo era 46 XX, y el perfil molecular era negativo para el gen SRY y positivo para el ZFY. Se le diagnosticó un trastorno de diferenciación sexual 46 XX testicular no sindrómico, una rara condición genética. Solo el 20 % de los pacientes con este diagnóstico son negativos para SRY y exhiben perfiles moleculares diversos. La información disponible parece indicar que el ZFY está relacionado con la diferenciación sexual masculina, aún en ausencia del gen SRY.

In most cases, male sexual differentiation occurs with SRY gene mediation. However, exceptional genotypes have been identified, as shown in this paper. This was a male adult patient seen at the Servicio de Paternidades, Instituto de Genética, Universidad Nacional de Colombia. The following procedures were carried out: Amelogenin gene and short tandem repeat analyses using human identification commercial kits, conventional karyotype, SRY fluorescent in situ hybridization, PCR analysis for Y chromosome microdeletions, clinical evaluation, and genetic counseling. We present an adult male with unambiguous genitalia, karyotype 46,XX, and an SRY negative and ZFY positive molecular profile. The diagnosis of nonsyndromic 46,XX testicular disorder of sex development (DSD) -a rare genetic condition- was established. Only 20 % of similarly diagnosed patients are SRY negative and exhibit diverse molecular profiles. Until now, available evidence seems to indicate that, even in the absence of SRY, the ZFY factor is involved in male sexual differentiation.

MiR-195 inhibits migration, invasion and epithelial-mesenchymal transition (EMT) of endometrial carcinoma cells by targeting SOX4

MicroRNAs (miRNAs) have been identified as potential biomarkers for endometrial carcinoma (EC) diagnosis, prognosisand therapy. The purpose of the present study was to investigate the detailed role and molecular mechanism of miR-195 inEC metastasis. qRT-PCR assay was performed to assess the expression of miR-195 and SRY-related high-mobility groupbox 4 (SOX4) mRNA in EC tissues and cells. The levels of N-cadherin, Vimentin, E-cadherin and SOX4 protein weredetermined by western blot. SOX4 protein expression in EC tissues was also determined by Immunohistochemistry (IHC)experiment. Transwell assay was used to analyze cell migration and invasion abilities. Dual-luciferase reporter assay andRNA Immunoprecipitation (RIP) assay were performed to confirm the targeted interaction between miR-195 and SOX4.Our data supported that miR-195 was downregulated and SOX4 was upregulated in EC tissues and cell lines. Upregulationof miR-195 inhibited migration, invasion and epithelial-mesenchymal transition (EMT) of EC cells. Moreover, SOX4 was adirect target of miR-195. MiR-195 overexpression-mediated anti-migration, anti-invasion and anti-EMT effects wereantagonized by SOX4 restoration in EC cells. In conclusion, our study suggested that miR-195 inhibited the migration,invasion and epithelial mesenchymal transition (EMT) of EC cells at least partly by targeting SOX4. Our study provided anovel underlying mechanism for EC metastasis and a promising therapeutic target for EC management.

Análisis clínico y citogenético de un caso de trastorno del desarrollo sexual testicular XX con SRY negativo / Clinical and Cytogenetic Analysis of a Case of SRY-Negative XX Testicular Disorder of Sex Development / Análise clínica e citogenética de um caso de transtorno do desenvolvimento sexual testicular XX com SRY-negativo

Resumen: El trastorno del desarrollo sexual (TDS) testicular XX es una patología que se presenta en un individuo con cariotipo 46,XX con un fenotipo anatómico de genitales externos masculinos, que pueden variar desde la normalidad hasta la ambigüedad genital. Clínicamente se han descrito dos subgrupos de hombres 46,XX con SRY-negativos y SRY-positivos, dependiendo de la presencia o no del gen SRY que normalmente se encuentra en el cromosoma Y participando en la determinación testicular. En este artículo se describen los antecedentes personales y los hallazgos clínicos de un infante con anomalías del meato urinario en el cual se identificó un complemento cromosómico 46,XX. También, se realizó hibridación in situ fluorescente en linfocitos de sangre periférica que demostró la ausencia del gen SRY y confirmó la presencia de dos cromosomas X.

Abstract XX testicular disorder of sex development (DSD) is a pathology that occurs in an individual with a 46,XX karyotype and an anatomical phenotype of male external genitalia, which may vary from normal to ambiguous. Clinically, two subgroups of SRY-negative and SRY-positive, 46, XX men have been described, depending on the presence of the SRY gene that is normally found on the Y chromosome participating in testicular determination. This article describes the personal history and clinical findings of an infant with urethral meatus abnormalities in whom a 46,XX chromosome set was identified. Also, fluorescent in situ hybridization was performed in peripheral blood lymphocytes which demonstrated the absence of the SRY gene and confirmed the presence of two X chromosomes.

Resumo: O transtorno do desenvolvimento sexual (TDS) testicular XX é uma patologia apresentada em um indivíduo com cariótipo 46,XX com um fenótipo anatômico de genitais externos masculinos, que podem variar da normalidade à ambiguidade genital. Clinicamente, são descritos dois subgrupos de homens 46,XX com SRY-negativos e SRY-positivos, dependendo da presença ou não do gene SRY que normalmente se encontra em Y cromossomo participando da determinação testicular. Neste artigo, são descritos os antecedentes pessoais e os achados clínicos de uma criança com anomalias de meato urinário em que foi identificado um complemento cromossômico 46,XX. Além disso, foi rea -lizada hibridação in situ fluorescente em linfócitos de sangue periférico que demonstrou a ausência do gene SRY e confirmou a presença de dois cromossomos X.

Varones 46, XX: a propósito de dos casos genéticamente distintos / Males 46, XX: Presentation of Two Genetically Different Cases

Introducción Los desórdenes de diferenciación sexual son condiciones clínicas en las que existe una discrepancia entre el sexo cromosómico y el sexo fenotípico de un individuo. Esas condiciones suelen resultar angustiantes para los pacientes y sus familias e incluso para el equipo médico tratante debido a la dificultad en diagnosticarlas. Objetivo Presentar las características clínicas, genéticas y hormonales de dos varones con desórdenes de diferenciación sexual. Método Se realizó un estudio descriptivo basado en la revisión y análisis de datos de la historia clínica y la confrontación de los resultados con reportes similares. Resultados Se observaron dos individuos con fenotipo masculino y diagnóstico de hipogonadismo hipergonadotrófico con cariotipo 46, XX. El primer caso presentó testes pequeños y azoospermia, mientras que el segundo caso presentó baja talla, criptorquidea bilateral congénita y escrotos hipoplásicos. En ambos pacientes se exploró la presencia del gen SRY, confirmando su presencia en el primer caso y ausencia en el segundo caso. Conclusiones El diagnóstico genético-molecular actual apela a la combinación de técnicas tradicionales junto a técnicas modernas, como secuenciación por paneles genéticos a fin de identificar etiológicamente los desórdenes de diferenciación sexual. La presentación de esos casos aún se considera rara debido a su baja tasa de frecuencia poblacional, por lo que su reporte siempre resultará útil a la comunidad científica ya que muestran las distintas formas de presentación clínica y el manejo multidisciplinario de esos casos en diferentes contextos clínicos

Introduction Disorders of Sexual Development are clinical conditions in which a discrepancy between the chromosomal sex and the phenotypic sex occurs in an individual. These conditions are often distressing for patients and their families and even for the medical team due to the difficulty of diagnosing them. Objective The aim of this study was to present the clinical, genetic and hormonal characteristics of two males with sexual differentiation disorders. Method A descriptive study was performed based on the review and analysis of the clinical history data and the comparison of the results with similar cases reported. Results Two individuals with a male phenotype and a diagnosis of hypogonadotropic hypogonadism with 46, XX karyotype were observed. The first case presented small testes and azoospermia, while the second case presented low height, congenital bilateral cryptorchid and hypoplastic scrotums. The SRY gene was explored in both patients, and it was confirmed its presence in the first case and its absence in the second case. Conclusions The current molecular-genetic diagnosis calls for the combination of traditional techniques combined with modern techniques, such as the genetic panel sequencing, to identify etiologically the Disorders of Sexual Development. The presentation of these cases is even considered rare because of their low population frequency rate, so their report is always useful to the scientific community, for they show the different ways of the clinical disease presentation and the multidisciplinary management of these cases in different clinical contexts.

SOX9 promotes epithelial-mesenchymal transition in non-small cell lung cancer A549 cells via Wnt/β-catenin pathway / 中国肿瘤生物治疗杂志

@#Objective:To explore the mechanism by which SRY-related high mobility group-box 9 (SOX9) promotes the epithelial mesenchymal transition (EMT) of non-small cell lung cancer (NSCLC) A549 cells via the Wnt/β-catenin pathway. Methods: The human NSCLCA549 cell line was divided into three groups: OE-NC group, OE-SOX9 group and OE-SOX9+XAV-939 group. The cells in OESOX9 group were transfected with SOX9 pcDNA plasmid to up-regulate the expression level of SOX9; The cells in OE-SOX9+XAV939 group were transfected with SOX9 pcDNA plasmid while the β-catenin inhibitor XAV-939 (1.0 μmol/L) was added to the medium. qPCR was used to detect SOX9 mRNA levels; CCK-8 was used to examine the proliferation of A549 cells; Wound-healing assay and Transwell chamber assay were used to detect the migration and invasion ofA549 cells, respectively; and WB was used to detect protein expressions of SOX9, β-catenin, E-cadherin, γ-catenin, N-cadherin and vimentin. Results: The mRNA and protein levels of SOX9 in OE-SOX9 group and OE-SOX9+XAV-939 group were significantly higher than those in the OE-NC group after transfection (all P< 0.05), while there was no significant difference between the OE-SOX9 group and the OE-SOX9+XAV-939 group (P>0.05). The proliferation, migration and invasion of cells in OE-SOX9 group were significantly higher than those in OE-NC group; however, those abilities in OE-SOX9+XAV-939 group were significantly lower than those in OE-SOX9 group (all P<0.05). The level of β-catenin protein in OE-SOX9 group was significantly higher than that in the OE-NC group, while the level of β-catenin protein in OE-SOX9+XAV-939 group was lower than that in OE-SOX9 group (all P<0.05). Compared with the OE-NC group, the levels of phenotypic markers of epithelial cells, E-cadherin and γ-catenin, were down-regulated, and the phenotypic markers of mesenchymal cells, N-cadherin and vimentin, were up-regulated in cells of OE-SOX9 group; however, E-cadherin and γ-catenin were higher, and N-cadherin and vimentin were lower in OE-SOX9+XAV-939 group than those in OE-SOX9 group (all P<0.05). Conclusion: SOX9 could promote proliferation, migration and EMT of NSCLCA549 cells by activating the Wnt/β-catenin pathway. ··

A rapid and useful method for gender identification of grey red-backed voles / 中国比较医学杂志

Objective Grey red-backed voles (Myodes rufocanus) are agile, fierce and hard to catch, thus, it is difficult to judge their gender by external appearance, especially for the juvenile voles. Therefore, it may cause difficulties to their allocation and later breeding in laboratories. The aim of this paper is to establish a rapid, simple and accurate method for gender identification of grey red-backed voles. Methods Fresh hair follicles were taken from 6 adult male voles, 3 adult females and 14 4-week-old juvenile voles, 5 male and 5 female 9-week-old Wistar rats, and 5 male and 3 female 6-week-old BALB/c mice. The genomic DNA was extracted using Chelex-100 resin and the zinc-finger Y/X gene (ZFY/ZFX) and the gene of sex-determining region of the Y (SRY) chromosome were amplified by PCR, and a double PCR amplification method was established. Results The ZFY/ZFX gene and SRY gene were simultaneously amplified from the male voles, while only the ZFY/ZFX gene was amplified from the females. The gender of all 23 voles, 10 Wistar rats and 8 BALB/c mice were correctly identified with this method, and the PCR results were consistent with the phenotypic and autopsy results. Conclusions Using fresh hair follicles as experimental materials for gender identification of grey redbacked voles can alleviate shock and damage to the animals. The established double PCR amplification method is accurate, simple, rapid, and deserves to be used for gender identification of grey red-backed voles.

Three cases of novel SRY mutations causing 46,XY disorder of sex development / 基础医学与临床

Objective To detect SRY mutation in 46,XY disorder of sex development (46,XY DSD), analyze SRY mutation frequency , and to define the clinical features of the patients with the mutation .Methods A total of sixty-three 46,XY DSD patients admitted to department of endocrinology of Peking Union Medical College Hospital from 2009 to 2014 were enrolled and detailed clinical data were collected .Genomic DNA was extracted from peripheral blood, and SRY was amplified and sequenced .The mutation was identified by comparing with the online database , and the clinical features were analyzed .Results Three novel mutations of SRY gene were detected in 3 of 63 pa-tients (5%).The 3 patients' social genders were all female and their karyotypes are 46, XY.Vaginal and uterine structures were present .Sex hormone profiles were consistent with hypergonadotropic hypogonadism .The 3 novel mutations were Pro131His, R76C and L35Afs*25.The former two were mutations in the nuclear localization signal regions of HMG box and highly-conservative amino acids were affected .The latter one was a frameshift mutation re-sulting in deletion of the entire HMG box .All these were presumably affecting the functional domain of SRY protein severely.Conclusions This study identified three novel mutations of SRY gene causing 46,XY DSD.The detection rate of SRY mutation was about 5%.It is recommended that SRY testing be performed to identify the etiology of the disease .

Cytogenetics and Molecular Genetic Analysis of Chimerism in Marmosets (Callithrix: Primates)

ABSTRACT The birth of fraternal twins is a characteristic frequently observed in callitrichids. Cytogenetic studies have demonstrated hematopoietic chimerism in marmosets with the occurrence of two cell lines 2n=46,XX/46,XY in females and males co-twins, without phenotypic changes. Amplification by PCR have also been used to verify the presence of the SRY gene in female chimaeras. Our aim was to verify the occurrence of chimerism in Callithrix sp. individuals considered as hybrids according to their intermediate phenotypes between C. jacchus and C. penicillata. Blood samples from 37 Callithrix sp. individuals were collected. Hematopoietic chimerism 2n=46,XX/46,XY was detected by cytogenetic analysis in five individuals, three males and two females. A fragment of approximately 200bp of the SRY gene was amplified in seven females with normal external genitalia. The percentage of 32% of chimeric individuals detected in the present study is similar to that observed for pure specimens of Callithrix. These data suggests that hybridization probably does not interfere with the occurrence of twin gestation, nor of chimerism. Although cytogenetics is the main tool to identify the two cell lineages present in cases of chimerism, the amplification of the SRY gene by PCR has proved to be more efficient to identify the Y chromosome in cases of chimeric female marmoset.

Highly Sensitive and Specific Detection of SRY Gene for Non-invasive Prenatal Diagnosis / 分析化学

By detecting SRY gene of cell-free fetal DNA ( cffDNA) in maternal peripheral blood, the sex of fetuses was determined, the risks of sex-linked genetic disorders were assessed and the birth rate of sick fetuses was decreased. A method of real-time polymerase chain reaction ( PCR) coupled with invader assay was established to detect SRY gene. This method possessed the advantages such as high sensitivity, high specificity and non-contaminated with closed tube detection. Under the optimized reaction conditions such as 250 nmol/L detection probes, 7. 5 U FEN1 enzyme, 0. 5 U Taq polymerase and 67℃ of annealing temperature in pre-amplification, the simulated samples as low as 4% ( 4 copies/μL ) were detected and two clinical samples with the gestation age of 9 weeks and 10 weeks were successfully detected. The detection results showed that this method could be used to detect SRY gene of cffDNA in maternal peripheral blood, providing an effective technique for clinical non-invasive prenatal diagnosis based on SRY gene.

Identification of Cervus nippon, C.elaphus and their hybridize samples based on COI and SRY gene / 中国中药杂志

For rapid identification of Cervus nippon, C. elaphus and their hybridize samples, the specific PCR for mutual authentication of them was established based on the SNPs in COI and SRY sequence. C. nippon, C. elaphus and their hybridize samples were collected from different origins, total DNA of 24 identified samples were extracted, and the COI and SRY gene was seqenced. SNPs in the COI and SRY sequences of the samples were found by Clustul X 2.1 program. Primers for identifying C. nippon and C. elaphus were designed according to the SNP site, two multi-PCR reaction system were established to identify them. In addition, 24 samples which were randomly collected in different herbal medicine market were identified. The band special for C. nippon (232 bp)and band special for C. elaphus (518 bp) based on COI sequence,and the band special for C. nippon (803 bp)and band special for C. elaphus (425 bp) based on SRY sequence, were found using multi-PCR reaction, and three of the twenty-four samples were identified as the hybridize samples. The multi-PCR reaction system could be used to identify C. nippon, C. elaphus and their hybridize samples.

Highly Sensitive and Specific Detection of SRY Gene for Non-invasive Prenatal Diagnosis / 分析化学

By detecting SRY gene of cell-free fetal DNA ( cffDNA) in maternal peripheral blood, the sex of fetuses was determined, the risks of sex-linked genetic disorders were assessed and the birth rate of sick fetuses was decreased. A method of real-time polymerase chain reaction ( PCR) coupled with invader assay was established to detect SRY gene. This method possessed the advantages such as high sensitivity, high specificity and non-contaminated with closed tube detection. Under the optimized reaction conditions such as 250 nmol/L detection probes, 7. 5 U FEN1 enzyme, 0. 5 U Taq polymerase and 67℃ of annealing temperature in pre-amplification, the simulated samples as low as 4% ( 4 copies/μL ) were detected and two clinical samples with the gestation age of 9 weeks and 10 weeks were successfully detected. The detection results showed that this method could be used to detect SRY gene of cffDNA in maternal peripheral blood, providing an effective technique for clinical non-invasive prenatal diagnosis based on SRY gene.

PDK2 promotes chondrogenic differentiation of mesenchymal stem cells by upregulation of Sox6 and activation of JNK/MAPK/ERK pathway

This study was undertaken to clarify the role and mechanism of pyruvate dehydrogenase kinase isoform 2 (PDK2) in chondrogenic differentiation of mesenchymal stem cells (MSCs). MSCs were isolated from femurs and tibias of Sprague-Dawley rats, weighing 300-400 g (5 females and 5 males). Overexpression and knockdown of PDK2 were transfected into MSCs and then cell viability, adhesion and migration were assessed. Additionally, the roles of aberrant PDK2 in chondrogenesis markers SRY-related high mobility group-box 6 (Sox6), type ΙΙ procollagen gene (COL2A1), cartilage oligomeric matrix protein (COMP), aggrecan (AGC1), type ΙX procollagen gene (COL9A2) and collagen type 1 alpha 1 (COL1A1) were measured by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The expressions of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK) and extracellular regulated protein kinase (ERK) were measured. Overexpressing PDK2 promoted cell viability, adhesion and inhibited cell migration in MSCs (all P<0.05). qRT-PCR assay showed a potent increase in the mRNA expressions of all chondrogenesis markers in response to overexpressing PDK2 (P<0.01 or P<0.05). PDK2 overexpression also induced a significant accumulation in mRNA and protein expressions of JNK, p38MAPK and ERK in MSCs compared to the control (P<0.01 or P<0.05). Meanwhile, silencing PDK2 exerted the opposite effects on MSCs. This study shows a preliminary positive role and potential mechanisms of PDK2 in chondrogenic differentiation of MSCs. It lays the theoretical groundwork for uncovering the functions of PDK2 and provides a promising basis for repairing cartilage lesions in osteoarthritis.

Diagnóstico molecular, citogenético y anatomohistopatológico del Síndrome Freemartin en hembras bovinas en Colombia / Molecular, cytogenetic and anatomohistopatologic diagnostic of freemartin syndrome on females cattle in

El síndrome Freemartin es un estado de intersexualidad de muchas de las hembras bovinas provenientes de parto múltiple heterosexual (macho - hembra). Éste se origina en la vida fetal entre los 30 y 40 días de gestación producto del intercambio transplacentario de células mediante anastomosis vasculares, presentándose fenómenos de quimerismo 60XX/XY en varios tejidos, y esterilidad consecuente. En el presente trabajo se tomaron 106 muestras de sangre de terneras provenientes de parto múltiple heterosexual, se realizó extracción de ADN de leucocitos y se buscó la amplificación del gen SRY asociado al cromosoma "Y" mediante PCR y lectura en gel de agarosa. 90 terneras (84.9%) de las 106 amplificaron SRY, verificando el quimerismo 60XX/XY, y 16 terneras (15.1%) que no amplificaron el gen, libres del síndrome quimérico y por lo tanto, aptas reproductivamente. El análisis citogenético realizado mediante cultivo de linfocitos demostró la presencia del cromosoma "Y" en linfocitos de hembras positivas a SRY y la ausencia del quimerismo en hembras SRY negativas. El análisis anatómico post mortem de tractos reproductivos de hembras positivas a SRY detectó anormalidades características del síndrome tales como, clítoris hipertrofiados y atresias ductales cervicales. El análisis histopatológico de placas de gónadas de estos animales evidenció la presencia de ovotestículos. El presente estudio confirma la utilidad de las técnicas de biología molecular como herramientas diagnósticas del síndrome, para el aprovechamiento de hembras de reemplazo al servicio del hato bovino.

Freemartin syndrome is a intersexuality condition developed in many of the female calfs born from heterosexual multiple calvings (male female). This originates in the fetal development between 30 and 40 days of gestation with transplacental exchange of cells through vascular anastomosis, occurring 60XX/XY chimerism in various tissues, and consequent sterility. In this paper was took blood samples from 106 female calves born from a multiple heterosexual calving. DNA was extracted from leucocytes and searched the SRY gene amplification associated with the Y chromosome by PCR and reading in agarose gel. 90 of 106 samples (84.9%) was amplified the SRY gene, verifying the 60XX/XY chimerism, and 16 samples (15.1%) that did not amplify the gene. Cytogenetic analysis using lymphocytes cell cultures showed the presence of Y chromosome in samples positive for SRY and absence of chimerism in SRY negative samples. The postmortem analysis of female reproductive tracts of SRY positive calves, shows anatomical abnormalities such as clitoris hypertrophia and cervical atresia. Histological examination of gonads of these animals confirmed the presence of ovotestis. This study confirms the usefulness of molecular biology techniques as diagnostic tools of the syndrome, for the use of replacement females in cattle.