Unravelling complex mosaicism of sex chromosomes in a patient with primary amenorrhea through cytogenetic analysis on urothelial cells.

OBJECTIVE: Sex chromosome mosaicism remains challenging in the study of disorders of sex development (DSD). Aneuploid cells in the developing gonad play a major role in sex determination. Therefore, it is necessary acknowledge their presence by different methods. Our aim was to stand out the utility of urothelial cells for unravelling complex and hidden cell lines in DSD patients. CASE REPORT: Herein we report on a 19-year-old female with primary amenorrhea, short stature without ambiguous external genitalia. She had a 45,X/46, XY karyotype in leukocytes. Interphase FISH revealed hidden 45,X/47,XYY/47,XXY/46,XY/46, XX mosaicism in leukocytes and urothelial cells. CONCLUSION: These findings highlight the importance of investigating sex chromosome mosaicism in other tissues. Of particular interest in cases of DSD are the cells from the urinary epithelium, which may reflect the cell composition of the urogenital ridge, the analysis of these cells should be considered within the clinical assessment of DSD patients.

Psychometric properties of the 12-item Knee injury and Osteoarthritis Outcome Score (KOOS-12) Spanish version for people with knee osteoarthritis.

To evaluate the validity, reliability, and responsiveness to change of the 12-item Knee injury and Osteoarthritis Outcome Score (KOOS) Spanish version questionnaire. This study was based on a questionnaire validation design. A cross-sectional survey of 199 patients with knee osteoarthritis (KOA) and ten healthy controls was studied to evaluate the validity and reliability of KOOS-12. One hundred and sixteen patients were assessed for test-retest reliability, and 38 patients were included for a responsiveness assessment. Structural validity was assessed by the confirmatory factor analysis (CFA). Item response theory-based methods were used to determine the performance of the items. Internal consistency reliability was appropriate for all scales (Cronbach's alpha = 0.85-0.94). The intra-class correlation coefficient of KOOS-12 scales ranged from 0.60 to 0.71. The CFA and generalized partial credit model showed that KOOS-12 scales presented a good overall model fit. No differential item functioning was found. Convergent validity was demonstrated by strong correlations (Spearman's rho ≥ 0.70) with KOOS, International Knee Documentation Committee subjective knee evaluation form (IKDC), and Knee Intermittent and Constant Osteoarthritis Pain (ICOAP). Known-groups validity showed that KOOS-12 well discriminated subgroups of patients (radiographic severity and nutritional status). Standardized response means for KOOS-12 scales were ≥ 0.75. Changes in KOOS-12 scales had a moderate to strong correlation (Pearson's r ≥ 0.40) with the changes in the KOOS, ICOAP, and IKDC scales. The KOOS-12 Spanish version is a valid, reliable, and responsiveness to change questionnaire to measure patients' opinions about their knee and associated problems in Mexican subjects with KOA. Key Points • KOOS-12 is a short self-reported measure that assesses patient's opinions about the difficulties they experience due to problems with their knee and also covers aspects of pain, functional limitations, and knee-related quality of life. • The Spanish version of KOOS-12 questionnaire is a valid instrument for measuring the patients' opinions about their knee and associated problems, and is both reliable and responsiveness to change.

Interleukin 8 Is Overexpressed in Acute Rejection in Kidney Transplant Patients.

The main complication associated with renal graft loss is immune rejection. The gold standard for the diagnosis of renal graft rejection is percutaneous renal biopsy, which is expensive and can lead to complications. Inflammation is one of the main pathogenic pathways in allograft rejection, and urine samples seem to be efficient windows to explore the allograft condition with a high cost-benefit ratio. This study aimed to evaluate the messenger ribonucleic acid (mRNA) profile expression pattern for interleukin (IL) 2, IL-4, IL-6, IL-8, and IL-10; tumor necrosis factor alfa; gamma interferon; and transforming growth factor ß1 in the urine renal cells of patients with a diagnosis of humoral rejection and patients with a diagnosis of normal biopsy. METHODS: An observational, cross-sectional analytical study was performed. All kidney transplants were performed at the Organ Transplant Department between 2018 and 2019. Also, a healthy control with a normal blood test and no apparent infection was included. mRNA from urine samples and biopsies was isolated, and the expression of interleukins was analyzed in PCR real time. Data were analyzed by Shapiro-Wilk and Kruskal-Wallis tests. RESULTS: The proinflammatory IL expression pattern in urine samples of kidney rejection group showed overexpression for IL-8 (P = .0001). No differences were observed in the rest of the interleukins analyzed. When we compared the results in the rejected versus not rejected transplanted patients with a group of apparently healthy subjects, the difference remains consistent. Thus, mRNA of IL-8 could function as a diagnostic tool in cases of chronic damage secondary to fibrosis.

Analyses of Programmed Cell Death Protein 1 in High Immunologic-Risk Transplant Patients.

Kidney transplant (KT) is the first therapeutic option for most patients with chronic renal failure that requires renal function replacement. The main complication associated with renal graft loss is immune rejection. The T regulatory pathways play a key role in this process, and abnormalities in some of these molecules could participate in the graft rejection. In this paper, our group performed an exploratory analysis of the behavior of the coinducible molecules (CD28, CTLA-4, ICOS, PD-1) in patients with KT rejection and control KT patients without rejection. The Mann-Whitney U test, used for 2 groups, showed significant differences (P = .0005), indicating that PD-1 is underexpressed in patients with allograft rejection. No differences were found in CD28+, regulatory T cells (T reg), CTLA-4, and ICOS, so we are proposing that PD-1 is a key player in the immunotolerance phenomenon and its underexpression participates in the rejection process. More research needs to be performed on this topic.

Alexithymia is associated with mood disorders, impairment in quality of life and disability in women with fibromyalgia.

OBJECTIVES: Alexithymia is a personality trait related to the quality of life of women with fibromyalgia (FM). It is still unknown whether alexithymia is associated with the clinical manifestations of FM. The present study describes the relationship between alexithymia and the domains included in the core set recommended by the Outcome Measures in Rheumatology (OMERACT) for FM evaluation. METHODS: One hundred two women with FM were enrolled in the cross-sectional study. The domains evaluated were alexithymia, pain, fatigue, health-related quality of life, sleep quality, depression, anxiety, and disability. Univariate and multivariate (Kernel Regularized Least Squares method) analyses were performed to assess the relationship between alexithymia and the domains included in the core set recommended by the OMERACT. RESULTS: Alexithymia prevalence was 64.5% (95% Confidence Interval [CI], 54.6%-73.9%) and higher in women with depression (76.1%; 95%CI, 63.8%-86%). Female patients with FM and alexithymia showed higher pain intensity, anxiety and depression levels, and disability perception and lower quality of life, as compared to those with FM without alexithymia. Size effect differences ranged from medium to large and all of them were statistically significant (p<0.05). Using multivariate analysis, alexithymia was significantly associated with worse perceptions of quality of life (except physical health domain) and more disability perception, independently of other variables. However, alexithymia was not significantly associated with pain intensity. CONCLUSIONS: Alexithymia plays an important role in clinical manifestations of FM, mainly in the psychological and social dimensions of quality of life and the degree of perceived disability.

Gender-specific differences in clinical and metabolic variables associated with NAFLD in a Mexican pediatric population.

INTRODUCTION AND OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in children and it is more prevalent in Hispanic males. The gender differences can be explained by body fat distribution, lifestyle, or sex hormone metabolism. We evaluated anthropometric and metabolic differences by gender in children with and without NAFLD. METHODS: We included 194 participants (eutrophic, overweight, and individuals with obesity). The presence of NAFLD was determined using ultrasonography, and we evaluated the association between this disease with metabolic and anthropometric variables by gender. RESULTS: The mean age was 10.64±2.54 years. The frequency of NAFLD in boys was 24.51% and in girls was 11.96% (OR=2.39; 95%CI=1.10-5.19; p=0.025). For girls, NAFLD was significantly associated with triglycerides (p=0.012), homeostatic model assessment of insulin resistance (HOMA-IR) (p=0.048), and the visceral adiposity index (VAI) (p=0.024). The variables related to NAFLD in a gender-specific manner were body mass index (BMI) (p=0.001), waist circumference (WC) (p<0.001), HDL cholesterol (p=0.021), alanine aminotransferase (ALT) (p<0.001), and aspartate aminotransferase (AST) (p=0.002). CONCLUSIONS: In our study NAFLD is more frequent in boys, only ALT, and no other clinical or metabolic variables, were associated with NAFLD in these patients. HOMA-IR, VAI, triglyceride levels, and ALT were associated with NAFLD only in girls. The ALT cut-off points for the development of NAFLD in our study were 28.5U/L in females and 27.5U/L in males. Our findings showed that NAFLD should be intentionally screened in patients with obesity, particularly in boys.

Interaction of Breast Cancer and Insulin Resistance on PD1 and TIM3 Expression in Peripheral Blood CD8 T Cells.

Epidemiological evidence points to a link between insulin resistance (IR) and breast cancer (BrCA). Insulin plays a role in CD8+ T cells (CD8T) differentiation and function and affects adipocytokines levels. CD8T activity in BrCA is associated with favorable outcome; while PD1 and TIM3 are markers of CD8T exhaustion and play critical roles in the negative regulation of T cell responses. Patients with (BrCA) have high expression levels of PD1 on circulating. Therefore, we hypothesized that BrCA and IR could affect PD1 and/or TIM3 expression on circulating CD8T. We determine PD1 and TIM3 expression on CD8T and analyze the relationship of CD8T phenotype with serum insulin and plasma adipocytokines levels in the different groups. We enrolled four groups of treatment-naive patients: women without neoplasms (Neo-)/without IR (IR-), Neo-/with IR (IR+), BrCa/IR- and BrCa/IR+. We found interactions between BrCA and IR with respect to TIM3 on naïve and central memory (CM) CD8T subsets. Furthermore, BrCA had a greater PD1 + TIM3- CD8T frequency in CD8T subsets than Neo-. IR+ presented a significantly lower PD1 + TIM3- frequency in CD8T subsets compare to Non-IR. In addition, we found a negative correlation between insulin levels, HOMA and frequency of PD1 + TIM3- in CD8T and a positive correlation between adiponectin levels and the frequency PD1 + TIM3- in CD8T. The increased expression of PD1 on different subsets of CD8T from BrCa patients is consistent with immunological tolerance, whereas IR has a contrary effect. IR could have a deleterious role in the activation of CD8T that can be relevant to new BrCa immunotherapy.

Physical Activity Protects Men but Not Women for Sarcopenia Development.

Objective: Sarcopenia is among the most deleterious effects of aging. The objective of this study was to analyze the relationship between performance tests and muscular volume over the life span of male and female participants. Method: A correlation study was conducted with healthy individuals (50 males and 47 females) between the ages of 20 and 94; the study group included active older people, sedentary younger people, and young athletes. Muscular volume was determined by tomography and muscular performance (4-meter speed tests [4 MSTs], chair test, and handgrip test), and a correlation analysis between the groups was performed. Results: Sex-related differences were observed between the variables; in males, muscle volume and functional parameters were closely related with age and physical activity, whereas in females, they were not related at all. Conclusion: Male and female muscle volume and performance demonstrate strong differences, which should be considered during clinical evaluations of sarcopenia.

Aneusomy detection with Karyolite-Bac on Beads® is a cost-efficient and high throughput strategy in the molecular analyses of the early pregnancy conception losses.

BACKGROUND: Approximately 10-15 % of all clinically recognized pregnancies end in miscarriage, the majority of them occur during the first trimester, underlying the cause of the loss. Genetic analysis of fetal tissues has the potential to provide valuable information and is highly recommended in some cases. Around 3-4 years ago, the gold standard for the analysis was the GTG Kayrotype, is well known that around 50 % of the tissue samples received failed to grow in culture. Different molecular techniques are used to improve the quality and the specificity of the study, intending to circumvent the limits of the Karyotype. RESULTS: Karyolite-BoBs™ (KL-BoB™) assay is a recent bead-based suspension, low density array technology with consistent results, probed that is an efficient molecular method to detect aneusomies in early pregnancy losses. Fifty samples from abortions were analyzed in order to probe and give more information about the methodology and analyze if KL-BoBs™ is a good and cost-efficient strategy. We detected 32 % of chromosomal abnormalities, in some of the cases more than one aberration was identified, the array CGH validate the observations. CONCLUSIONS: This molecular strategy is a cost-effective sensitive tool in the early pregnancy loss study.

ANTECEDENTES: Aproximadamente entre el 10­15 % de los embarazos reconocidos clínicamente terminan en aborto involuntario. El análisis genético de los tejidos de aborto proporciona información para el seguimiento médico de algunos casos. El estándar de oro hasta hace pocos años para el análisis de restos embrionarios era el Cariotipo GTG; sin embargo, alrededor del 50 % de las muestras no logran crecer en cultivo, por lo que recientemente se ha implementado el uso de las técnicas moleculares. La plataforma Karyolite-BoBs™ es un microarreglo de baja densidad que analiza con sondas específicas los brazos p, q y centrómeros de los 24 cromosomas los que permite mejorar la calidad y la especificidad del diagnóstico. RESULTADOS: Se analizaron 50 muestras de abortos espontáneos procedentes de pacientes con pérdidas gestacionales recurrentes provenientes de clínicas de reproducción, mediante Karyolite-BoBsTM, con el objetivo de obtener información del método de análisis y determinar si es una estrategia eficiente con un buen costo-beneficio. Los resultados se validaron mediante array CGH Constitutional Chip R 4.0 microarray de PerkinElmer Resultados: En todos los casos se pudo realizar el estudio molecular. Se detectaron 32 % de anomalías cromosómicas, se observaron alteraciones frecuentes en abortos espontáneos como las trisomías de los cromosomas 18, 21 y la monosomía del cromosoma X. También se identificaron alteraciones poco frecuentes como la monosomía de los cromosomas 11 y 19. En un par de casos se observó más de una aberración. Los resultados se validaron mediante el array CGH y con ambas técnicas se obtuvieron los mismos resultados. CONCLUSIONES: El análisis molecular mediante Karyolite-BoBsTM es una herramienta sensible y eficaz en costos para el estudio de tejidos embrionarios de pérdida temprana del embarazo.

Prevalence of recurrent pathogenic microdeletions and microduplications in over 9500 pregnancies.

OBJECTIVES: The implementation of chromosomal microarray analysis (CMA) in prenatal testing for all patients has not achieved a consensus. Technical alternatives such as Prenatal BACs-on-Beads(TM) (PNBoBs(TM) ) have thus been applied. The aim of this study was to provide the frequencies of the submicroscopic defects detectable by PNBoBs(TM) under different prenatal indications. METHODS: A total of 9648 prenatal samples were prospectively analyzed by karyotyping plus PNBoBs(TM) and classified by prenatal indication. The frequencies of the genomic defects and their 95%CIs were calculated for each indication. RESULTS: The overall incidence of cryptic imbalances was 0.7%. The majority involved the DiGeorge syndrome critical region (DGS). The additional diagnostic yield of PNBoBs(TM) in the population with a low a priori risk was 1/298. The prevalences of DGS microdeletion and microduplication in the low-risk population were 1/992 and 1/850, respectively. CONCLUSIONS: The constant a priori risk for common pathogenic cryptic imbalances detected by this technology is estimated to be ~0.3%. A prevalence higher than that previously estimated was found for the 22q11.2 microdeletion. Their frequencies were independent of maternal age. These data have implications for cell-free DNA screening tests design and justify prenatal screening for 22q11 deletion, as early recognition of DGS improves its prognosis.

The high frequency of genetic diseases in hypotonic infants referred by neuropediatrics.

Neonatal hypotonia is a relatively common cause of consultation in daily pediatric practice. It is part of the clinical presentation of a large group of heterogeneous diseases, many of which have an important and classifiable genetic background. Identification of the specific disorder can help optimize the management and treatment of the patient and inform genetic counseling for the family, and therefore input from clinical geneticists is critical at the earliest stages of medical management. Here we present 30 patients with hypotonia of unknown etiology referred by a neuropediatrician to clinical genetics. Clinical, genetic, and molecular evaluation of each patient was performed. Sixty-nine percent of the patients included in the study had a genetic disease, including eight with Prader-Willi syndrome, three with spinal muscular atrophy, one with Rett syndrome, and one with Sotos syndrome harboring a previously undescribed mutation. Our data demonstrate that a multidisciplinary approach used from the outset that includes molecular analysis can help improve diagnosis and management of hypotonic infants.

Utility of OCT3/4, TSPY and ß-catenin as biological markers for gonadoblastoma formation and malignant germ cell tumor development in dysgenetic gonads.

BACKGROUND: Gonadoblastoma (GB) is regarded as an in situ form of germ cell tumor in dysgenetic gonads, and 30% of patients with GB develop a dysgerminoma/seminoma tumor. OBJECTIVE: Determine whether OCT3/4 and ß-catenin are expressed in dysgenetic gonads before GB development and whether TSPY participates in the OCT3/4-ß-catenin pathways in the malignant invasive behavior. METHODS: dysgenetic gonads of Disorders of sex differentiation (DSD) patients with mixed gonadal dysgenesis were analyzed by immunohistochemistry and immunofluorescence for comparison with GB and dysgerminoma/seminoma. RESULTS: Our results suggest that the development of GB is secondary to the interaction of OCT3/4 and TSPY, that ß-catenin does not participate in this process. CONCLUSIONS: The use of this biological markers detects the potential high risk gonads.

Small nuclear ribonucleoprotein polypeptide N quantitative methylation analysis in infants with central hypotonia.

Central hypotonic is one of the most difficult issues in neurology, ruling out neurogenetic syndromic causes is critical, Prader-Willi syndrome (PWS) it is the most frequent genetic syndrome, it is caused by the loss of expression of the paternal allele in a group of imprinted genes within 15q11-q13, and is characterized by severe prenatal and postnatal hypotonia. SNURF-SNRPN gene methylation detects 99% of the cases but fluorescent in situ hybridization (FISH) analysis is necessary to confirm chromosome microdeletions. The advantage of SNRP-quantitative strategy of methylated alleles is that it makes it possible to make the diagnosis and identify deletions and mosaicism in one reaction. In infants clinical diagnosis is difficult. It has been proposed that around 40% of hypotonic patients have PWS but an accurate percentage has not been established. Twenty-four central hypotonic infants were studied by this molecular strategy, showing 41.5% with the disease. This molecular approach also permitted calculation of gene dosage and detection of those cases with microdeletion.

Loss of cytochrome P450 17A1 protein expression in a 17alpha-hydroxylase/17,20-lyase-deficient 46,XY female caused by two novel mutations in the CYP17A1 gene.

17alpha-Hydroxylase deficiency (17OHD) is a rare form of congenital adrenal hyperplasia caused by mutations in the CYP17A1 gene. This condition shows considerable clinical and biochemical variation. Molecular characterization of novel mutations in the CYP17A1 gene and detailed study of their structural, enzymatic, and clinical consequences are required to fully understand enzyme behavior. Here, we present the first molecular characterization of two novel mutations in CYP17A1 in a 15-year-old female Mexican mestizo 46,XY female with primary amenorrhea and lack of pubertal development and severe hypertension that manifested only after surgery. A complete clinical and biochemical evaluation was compatible with 17OHD. Structural anomalies in the CYP17A1 gene were discovered by direct automated sequencing, which revealed a novel compound heterozygous K110X/R362H mutation that leads to a complete lack of enzyme activity. Immunohistochemical analyses performed to determine protein expression and localization showed that cytochrome P450 17A1 was completely absent in the patient's testicular tissue. Studies of novel mutations, such as those described here, provide important information that allows us to better understand the effect of a given mutation on enzyme function and to observe the impact of the mutation on clinical phenotype.

Participation of OCT3/4 and beta-catenin during dysgenetic gonadal malignant transformation.

Gonadoblastoma (GB) is an in situ tumor consisting of a heterogeneous population of mature and immature germ cells, other cells resembling immature Sertoli/granulosa cells, and Leydig/lutein-like cells, may also be present. GB almost exclusively affects a subset of patients with intersex disorders and in 30% of them overgrowth of the germinal component of the tumor is observed and the lesion is term dysgerminoma/seminoma. Several pathways have been proposed to explain the malignant process, and abnormal OCT3/4 expression is the most robust risk factor for malignant transformation. Some authors have suggested that OCT3/4 and beta-catenin might both be involved in the same oncogenic pathway, as both genes are master regulators of cell differentiation and, overexpression of either gene may result in cancer development. The mechanism by which beta-catenin participates in GB transformation is not completely clear and exploration of the E-cadherin pathway did not conclusively show that this pathway participated in the molecular pathogenesis of GB. Here we analyze seven patients with mixed gonadal dysgenesis and GB, in an effort to elucidate the participation of beta-catenin and E-cadherin, as well as OCT3/4, in the oncogenic pathways involved in the transformation of GB into seminoma/dysgerminoma. We conclude that the proliferation of immature germ cells in GB may be due to an interaction between OCT3/4 and accumulated beta-catenin in the nuclei of the immature germ cells.

Extragonadal germ cell tumors are often associated with Klinefelter syndrome.

Klinefelter syndrome is a well documented abnormality of sex differentiation, with an incidence of 1 in 600 newborn males. It is characterized by a 47,XXY or a mosaic karyotype and clinical findings of hypergonadotrophic hypogonadism, small testes, infertility, reduced body hair, gynecomastia, and tall stature. Other conditions like venous disease, autoimmune disorders, mild neurobehavioral deficit, diabetes mellitus, sexual precocity, and osteoporosis may also affect these patients. Different malignancies such as breast cancer, testicular tumors, leukemia, and lymphomas occur in 1%-2% of the cases. Klinefelter syndrome has been associated with other malignancies such as extragonadal germ cell tumors; however, some authors consider this association an unusual finding. We report the molecular cytogenetic studies performed in 4 young males with mediastinal germ cell tumors. In 2 cases, a 47,XXY karyotype was recognized in different tissues by fluorescent in situ hybridization, whereas the other 2 had a normal XY karyotype. We propose that in young patients with mediastinal teratoma, a cytogenetic analysis must always be performed.

A familial missense mutation in the hinge region of DAX1 associated with late-onset AHC in a prepubertal female.

Mutations in the DAX1 (Dosage-sensitive sex reversal-Adrenal hypoplasia congenita (AHC) critical region on the X chromosome gene 1; NR0B1) cause X-linked AHC, a disease characterized by primary adrenal failure in infancy and hypogonadotropic hypogonadism. All known missense mutations impair DAX1 repression of steroidogenic factor 1 (SF1) transactivation and have been localized to the putative ligand binding domain. Here, an asymptomatic father and his late-onset AHC daughter were both shown to share a novel DAX1 mutation (C200W), the first missense mutation identified in the hinge region of DAX1. Using immunohistochemistry we demonstrate that the sub-cellular localization of the C200W mutant DAX1 protein is shifted from the nucleus to the cytoplasm. The disturbed localization of the C200W mutant in transfected cells correlates with impaired transcriptional repression activity. The import defect is relatively mild, retaining 80% of wild-type activity, which may explain the unusually mild phenotype. Import of DAX1 into the nucleus involves a direct interaction with SF1. In vitro assays demonstrate that the C200W mutant retains the ability to functionally interact with SF1, which suggests that SF1-independent interactions of DAX1 could be responsible for the import defect.

Unusual mixed gonadal dysgenesis associated with Müllerian duct persistence, polygonadia, and a 45,X/46,X,idic(Y)(p) karyotype.

Mixed gonadal dysgenesis (MGD) is a developmental anomaly in which most of the patients have a dysgenetic testis, a contralateral streak and a 45,X/46,XY karyotype. This entity involves an heterogeneous group of gonadal and phenotypic abnormalities with a wide clinical spectrum. The phenotype depends on the ratio of testicular tissue which induces virilization. Although the karyotype in these patients is 45,X/46,XY, no genotype-phenotype correlation has been found to date. Müllerian ducts persistence (MDP) in MGD is rare; however, four patients with both entities and different karyotypes have been described. Here we present the data on a newborn patient with an atypical MGD associated with MDP, two left testes, a gonadal streak on the right, and absence of Wolffian derivatives. PCR analysis identified all the Y-derived sequence tested in the father, while the patient had them all except the AZF b,c regions which were lost. FISH analysis of the paternal Y chromosome documented Yq paracentric inversion while the patient's karyotype was 45,X/46,X,idic(Yp). No mutations were observed in MIS/MISRII genes.