Altered subcortical and cortical brain morphology in adult women with 47,XXX: a 7-Tesla magnetic resonance imaging study.

BACKGROUND: Triple X syndrome (47,XXX) is a relatively common sex chromosomal aneuploidy characterized by the presence of a supernumerary X chromosome in females and has been associated with a variable cognitive, behavioural and psychiatric phenotype. 47,XXX may serve as a suitable model for studying the effect of genetic architecture on brain morphology. Previous studies have shown alterations in brain structure in 47,XXX particularly in childhood and adolescence. In this study, we examined subcortical and cortical brain morphology in adult women with 47,XXX using ultra-high field 7T MRI. Given previous evidence of impaired social functioning and emotion recognition in adults with 47,XXX, we also investigated the relationship of these functions with brain morphology. METHODS: Twenty-one adult women with 47,XXX and 22 age- and sex-matched healthy controls were included. Structural T1-weighted images were acquired using a 7-Tesla magnetic resonance scanner. Measures of subcortical brain volumes, cortical surface area and thickness, and cortical folding were obtained and compared between the groups using general linear models. Additionally, we examined potential relationships between brain outcome measures and social functioning and social cognition in 47,XXX using correlation analyses. RESULTS: Adults with 47,XXX showed lower volumes of the thalamus, caudate, putamen, hippocampus, nucleus accumbens and pallidum, and larger lateral ventricle volumes. Lower surface area was found in the superior frontal gyrus and superior temporal gyrus in 47,XXX participants compared to healthy controls. Altered cortical thickness and cortical folding were not present in 47,XXX. Cortical thickness was associated with social cognition in 47,XXX. CONCLUSIONS: Results suggest that a supernumerary X chromosome in females affects subcortical and lateral ventricle volumes, and cortical surface area in adulthood. 47,XXX may serve as a suitable model for studying genetic influences on structural brain morphology across developmental stages in order to understand neurobiological mechanisms underlying cognitive and behavioural impairments.

Prevalence of Y chromosome microdeletion in north Indian infertile males with spermatogenesis defect.

Deletion of specific genes present in the long arm of Y chromosome has been identified as the most common genetic cause of defective spermatogenesis. Studies have shown that frequency of Y chromosome microdeletion varies in different geographical location and is related to genetic and environmental influence preponderance. Therefore, the present study was carried out to identify the frequency of Y chromosome microdeletion in the northern region of India and to define subgroup of infertile patients who are critically under more risk of having microdeletion. A total of 292 north Indian infertile males with nonobstructive azoospermia and oligozoospermia were selected for screening the Y chromosome microdeletion. Healthy fertile males (n=100) were also enrolled as control subjects. Frequency of Y chromosome microdeletion in north Indian infertile males was found to be about 8.5%, with azoospermia factor (AZFc) region as the most susceptible region for microdeletion. Comparatively microdeletion is more common in patients with nonobstructive azoospermia than oligozoospermia (9.2% versus 7.1%). Statistical analysis also revealed that patients with hormonal FSH level between 20 and 40 mIU/mL have more chances of harbouring microdeletion. Hence, the present study highlights the importance of screening AZFc region among infertile patients with very high serum FSH value.

Y chromosome structural variation in infertile men detected by targeted next-generation sequencing.

PURPOSE: To provide a validated method to identify copy number variation (CNV) in regions of the Y chromosome of infertile men by next-generation sequencing (NGS). METHODS: Semen analysis was used to determine the quality of semen and diagnose infertility. Deletion of the azoospermia factor (AZF) region in the Y chromosome was detected by a routine sequence-tagged-site PCR (STS-PCR) method. We then used the NGS method to detect CNV in the AZF region, including deletions and duplications. RESULTS: A total of 326 samples from male infertility patients, family members, and sperm donors were studied between January 2011 and May 2017. AZF microdeletions were detected in 120 patients by STS-PCR, and these results were consistent with the results from NGS. In addition, of the 160 patients and male family members who had no microdeletions detected by STS-PCR, 51 cases were found to exhibit Y chromosome structural variations by the NGS method (31.88%, 51/160). No microdeletions were found in 46 donors by STS-PCR, but the NGS method revealed 11 of these donors (23.91%, 11/46) carried structural variations, which were mainly in the AZFc region, including partial deletions and duplications. CONCLUSION: The established NGS method can replace the conventional STS-PCR method to detect Y chromosome microdeletions. The NGS method can detect CNV, such as partial deletion or duplication, and provide details of the abnormal range and size of variations.

Epigenetic and transcriptomic consequences of excess X-chromosome material in 47,XXX syndrome-A comparison with Turner syndrome and 46,XX females.

47,XXX (triple X) and Turner syndrome (45,X) are sex chromosomal abnormalities with detrimental effects on health with increased mortality and morbidity. In karyotypical normal females, X-chromosome inactivation balances gene expression between sexes and upregulation of the X chromosome in both sexes maintain stoichiometry with the autosomes. In 47,XXX and Turner syndrome a gene dosage imbalance may ensue from increased or decreased expression from the genes that escape X inactivation, as well as from incomplete X chromosome inactivation in 47,XXX. We aim to study genome-wide DNA-methylation and RNA-expression changes can explain phenotypic traits in 47,XXX syndrome. We compare DNA-methylation and RNA-expression data derived from white blood cells of seven women with 47,XXX syndrome, with data from seven female controls, as well as with seven women with Turner syndrome (45,X). To address these questions, we explored genome-wide DNA-methylation and transcriptome data in blood from seven females with 47,XXX syndrome, seven females with Turner syndrome, and seven karyotypically normal females (46,XX). Based on promoter methylation, we describe a demethylation of six X-chromosomal genes (AMOT, HTR2C, IL1RAPL2, STAG2, TCEANC, ZNF673), increased methylation for GEMIN8, and four differentially methylated autosomal regions related to four genes (SPEG, MUC4, SP6, and ZNF492). We illustrate how these changes seem compensated at the transcriptome level although several genes show differential exon usage. In conclusion, our results suggest an impact of the supernumerary X chromosome in 47,XXX syndrome on the methylation status of selected genes despite an overall comparable expression profile.

A rare case of NIPT discrepancy caused by the placental mosaicism of three different karyotypes, 47,XXX, 47,XX,+21, and 48,XXX,+21.

BACKGROUND: Placental mosaicism is one of the major reasons for noninvasive prenatal testing (NIPT) discrepancy. Herein, we discovered a rare case of placenta with complex karyotypes that caused false-positive and false-negative results in noninvasive prenatal testing. METHODS: Next-generation sequencing (NGS) and Quantitative fluorescent polymerase chain reaction (QF-PCR) were performed on the cord blood sample, fetal tissues, and eight placental biopsies. Fluorescent In Situ Hybridization (FISH) and karyotyping were also carried to confirm the fetal genome status. RESULTS: The results suggested that the fetal chromosome was 47,XXX and the placenta had three karyotypes of 48,XXX,+21, 47,XX,+21, and 47,XXX. QF-PCR indicated that the extra chromosome 21 and chromosome X were all from the father. It is speculated that the zygote may have 48,XXX,+21 karyotype and trisomy rescue could be the main mechanism for the development of the homogeneous fetus and complex mosaic placenta. CONCLUSION: Overall, the complicated nature of our case underlines the importance of discussing with parents the possibility of both atypical and discordant results during preconfirmatory amniocentesis counseling and consent.

Efficacy of MLPA for detection of Y-chromosome microdeletions in infertile Brazilian patients.

PURPOSE: Worldwide publications follow the gold standard method-the polymerase chain reaction (PCR)-for detecting Y-chromosome microdeletions; however, markers are frequently variable between the studies. Can we detect the deletions by another molecular method with more genomic coverage? The Y chromosome harbors several different genes responsible for testicular development and spermatogenesis, and its repetitive conformation predisposes it to complex rearrangements that have clinical impact. Our aim was to evaluate a molecular diagnostic method, the Multiplex Ligand Probe-dependent Amplification (MLPA), which is also a valuable ancillary method for the identification of deletions, duplications, and rearrangements in a single and faster reaction, leading to a better comprehension of patients' phenotypes, and should be considered a useful tool for detection of Y chromosome deletions. METHODS: This is a study of diagnostic accuracy (transversal prospective study) conducted to investigate Y-chromosome deletions in 84 individuals through PCR and MLPA methods. Forty-three infertile men (azoospermic and oligozoospermic) and 41 controls (40 fertile men and 1 normal karyotyped woman) were analyzed by PCR and MLPA techniques. RESULTS: We diagnosed seven (7) deletions (16.2%) by PCR and 9 with MLPA (21%). In addition, we found five (5) duplications and a suggestive mosaic. CONCLUSION: Our results demonstrate that MLPA technique is valuable in the investigation of microdeletions and microduplications. Besides deletions, duplications can cause instability of chromosome genes, possibly leading to infertility. Both studied techniques provide an advantageous diagnostic strategy, thus enabling a better genetic counseling.

AZF deletions in Indian populations: original study and meta-analyses.

PURPOSE: To identify the frequency of Y chromosome microdeletions in Indian populations and to quantitatively estimate the significance of association between these deletions and male infertility. METHODS: A total of 379 infertile males (302 azoospermic and 77 oligozoospermic infertile males) and 265 normozoospermic fertile males were evaluated for Y chromosome microdeletions (YCD) using PCR amplification and gel electrophoresis. Meta-analyses were performed on AZFa (2079 cases and 1217 controls), AZFb (2212 cases and 1267 controls), AZFc (4131 cases and 2008 controls), and AZFb+c (1573 cases and 942 controls) deletions data to quantitatively estimate the significance of association between these deletions and male infertility in Indian populations. RESULTS: The results revealed that out of 379 infertile azoospermic and oligozoospermic males, 38 (10.02%) had AZF deletions. No deletion was found in control samples. The highest percentage of deletions was observed in the AZFc region, followed by AZFa and AZFb. Qualitative analysis showed that AZF deletions were present in 0.59 to 32.62% (average 13.48%) of infertile cases in Indian populations. Meta-analysis revealed a significant association of AZFa (OR = 6.74, p value = 0.001), AZFb (OR = 4.694, p value = 0.004), AZFc (OR = 13.575, p value = 0.000), and AZFb+c (OR = 5.946, p value = 0.018) deletions with male infertility. CONCLUSION: AZF deletions were seen in 10.02% of azoospermic and oligozoospermic cases with the highest frequency of AZFc deletions. Pooled analysis for all studies showed deletion frequency from 0.59 to 32.62% (average = 13.48%). Meta-analysis showed significant association of AZFa, AZFb, and AZFb+c deletions with male infertility. Analysis of Y chromosome microdeletions should be reckoned as an essential testing for diagnostic and therapeutic purposes.

Atypical Rett syndrome in a girl with mosaic triple X and MECP2 variant.

BACKGROUND: Rett syndrome (RTT) is a neurodevelopmental disorder that predominantly affects girls, resulting from a loss-of-function variant in X-linked MECP2. Here, we report a rare case of a girl with RTT with an X chromosome mosaic karyotype (46,XX/47,XXX). METHODS: Fluorescent in situ hybridization (FISH) was carried out to confirm the mosaic karyotype. Sanger sequencing was carried out to genetically diagnose RTT. Furthermore, we assessed the X chromosome inactivation (XCI) pattern. MECP2 expression levels were examined via RT-PCR. RESULTS: The patient presented with preserved speech variant, the milder form of RTT. Genetic examination revealed a de novo, heterozygous, truncating variant of MECP2. FISH revealed mosaicism in the 47,XXX karyotype in 6% of her cells. The XCI assay revealed unbalanced inactivation with skewing in favor of the paternal X chromosome. MECP2 was downregulated to only 84% of the control, indicating that the patient's variant was probably of paternal origin. Unbalanced XCI in this patient might have contributed to the alleviation of the phenotype. However, her supernumerary X chromosome was derived from maternal X chromosome harboring the wild-type allele and might have had no preferential effect on her RTT-related phenotype. CONCLUSION: The present results indicate that phenotypic effects of X chromosome aneuploidy depend on the nature of the supernumerary X chromosome, the pattern of mosaicism, and XCI status.

Chromosomal microarray analysis of infertile men with azoospermia factor microdeletions.

Azoospermia factors, located in the long arm of the Y chromosome, are critical for spermatogenesis, the microdeletions of AZF are considered to be associated with male infertility. In addition to complete deletion, several AZFc partial deletions were also detected in infertile men with wide phenotypic heterogeneity. In this study, we investigated the relevance of Y chromosome deletions, Y-linked CNVs and variable phenotypes in infertile men. To clarify the relationship between phenotypic heterogeneity and Y chromosome deletion in male infertility, we performed chromosomal microarray analysis (CMA) capable of analyzing thousands of loci simultaneously to investigate Y-linked copy number variations (CNVs). Firstly, we reviewed the results of Y chromosome screening in 554 infertile patients and then compared the results of CMA to routine Y chromosome screening in 29 patients with Y chromosomal microdeletions. Then, the Y-linked CNVs associated with oligoasthenospermia were identified according to ACMG standards and guidelines. The results indicated that the prevalence of Yq microdeletions was 5.23% (29/554), with 93% (27/29) of the deletions in the AZFc region among 554 infertile men recruited in this study. The results of CMA and multiplex-PCR-based AZFc deletion analysis were generally concordant, but CMA provided more details about location, size and OMIM genes involved in deletion fragments of the AZF region. Of 29 clinically infertile phenotype-related CNVs detected by CMA, nine were pathogenic and the remaining 20 CNVs were OVUS. Except for a 15.69 Mb loss CNV in AZFa + b + c and an 8.25 Mb loss CNV in AZFb + c, others were located in the AZFc region. Based on a combination of the clinical symptoms and loss CNVs, we concluded that the CNV size and the involvement of spermatogenesis critical genes are two important factors that determine the relevance of a CNV in the AZFc region to the presence or absence of a clinically infertile phenotype.

Efficient generation of functional haploid spermatids from human germline stem cells by three-dimensional-induced system.

Generation of functional spermatids from human spermatogonial stem cells (SSCs) in vitro is of utmost importance for uncovering mechanisms underlying human germ cell development and treating infertility. Here we report a three-dimensional-induced (3D-I) system by which human SSCs were efficiently differentiated into functional haploid spermatids. Human SSCs were isolated and identified phenotypically. Meiotic chromatin spreads and DNA content assays revealed that spermatocytes and haploid cells were effectively generated from human SSCs by 3D-I system. Haploid cells derived from human SSCs harbored normal chromosomes and excluded Y chromosome microdeletions. RNA sequencing and bisulfite sequencing analyses reflected similarities in global gene profiles and DNA methylation in human SSCs-derived spermatids and normal round spermatids. Significantly, haploid spermatids generated from human SSCs via 3D-I system were capable of fertilizing mouse oocytes, which subsequently enabled the development of hybrid embryos. This study thus provides invaluable human male gametes for treating male infertility.

Generation of integration-free induced pluripotent stem cells (GZHMUi001-A) by reprogramming peripheral blood mononuclear cells from a 47, XXX syndrome patient.

47, XXX syndrome is one of several sex-chromosomal aneuploidies, and it has an incidence of approximately 1/1000 in newborn females. Because of heterogeneity in X-inactivation, these patients may exhibit a variety of clinical symptoms. Here, we report the generation of an integration-free human induced pluripotent stem cell line (GZHMUi001-A) by using Sendai virus to reprogram peripheral blood mononuclear cells from a 47, XXX syndrome patient with premature ovarian failure. This 47, XXX iPS cell line has characteristics of pluripotent stem cells and is a useful tool for the investigation of this X chromosome aneuploid disease.

Noninvasive prenatal screening for fetal common sex chromosome aneuploidies from maternal blood.

Objective To explore the feasibility of high-throughput massively parallel genomic DNA sequencing technology for the noninvasive prenatal detection of fetal sex chromosome aneuploidies (SCAs). Methods The study enrolled pregnant women who were prepared to undergo noninvasive prenatal testing (NIPT) in the second trimester. Cell-free fetal DNA (cffDNA) was extracted from the mother's peripheral venous blood and a high-throughput sequencing procedure was undertaken. Patients identified as having pregnancies associated with SCAs were offered prenatal fetal chromosomal karyotyping. Results The study enrolled 10 275 pregnant women who were prepared to undergo NIPT. Of these, 57 pregnant women (0.55%) showed fetal SCA, including 27 with Turner syndrome (45,X), eight with Triple X syndrome (47,XXX), 12 with Klinefelter syndrome (47,XXY) and three with 47,XYY. Thirty-three pregnant women agreed to undergo fetal karyotyping and 18 had results consistent with NIPT, while 15 patients received a normal karyotype result. The overall positive predictive value of NIPT for detecting SCAs was 54.54% (18/33) and for detecting Turner syndrome (45,X) was 29.41% (5/17). Conclusion NIPT can be used to identify fetal SCAs by analysing cffDNA using massively parallel genomic sequencing, although the accuracy needs to be improved particularly for Turner syndrome (45,X).

Correlation of genetic results with testicular histology, hormones and sperm retrieval in nonobstructive azoospermia patients with testis biopsy.

To investigate the frequency and types of genetic results in different testicular histology of patients with nonobstructive azoospermia (NOA), and correlated with hormones and sperm retrieval (SR), a retrospective study was conducted in 286 Chinese NOA patients who underwent testis biopsy and 100 age-matched fertile men as the control group. Chromosome karyotype analyses were performed by the peripheral blood chromosome G-band detection method. Screening of Y chromosome microdeletions of azoospermia factor (AZF) region was performed by polymerase chain reaction (PCR) amplification of 11 sequence-tagged sites (STS). The serum levels of follicle-stimulating hormone, luteinising hormone and testosterone (T) and the appearance of scrotal ultrasound were also obtained. In 286 cases of NOA, 14.3% were found to have chromosomal alterations. The incidence of chromosomal abnormality was 2.8%. Sex chromosomal abnormalities were seen in six cases (four cases of Klinefelter's syndrome (47, XXY) and two cases of mosaics). The incidence of polymorphic chromosomal variants was 3% in the normal group and 11.5% in the NOA group. In total, 15.7% of NOA patients were found to have AZF microdeletions and AZF (c + d) was the most frequent one. The results of hormone and SR were found to be significantly different among all testicular histological types, whereas no significant differences were found when it comes to genetic alterations. It is concluded that the rate of cytogenetic alterations was high in NOA patients. So screening for chromosomal alterations and AZF microdeletions would add useful information for genetic counselling in NOA patients with testis biopsy and avoid vertical transmission of genetic defects by assisted reproductive technology.

Correlation between Y chromosome microdeletion and male infertility.

Dyszoospermia due to genetic factors is the leading cause of male infertility. To explore the correlation between azoospermia factor (AZF) microdeletion of the Y chromosome and male infertility, we evaluated AZF microdeletion on the long arm of the Y chromosome in 166 infertile males and 50 fertile males using multiplex polymerase chain reactions amplification and gel electrophoresis. The results demonstrated that 28 individuals had varying degrees of microdeletion in the AZF region (16.90%); 12 out of the 76 males with azoospermia and 16 out of the 90 males with oligospermia had AZF microdeletion. AZF microdeletion was not observed in any of the healthy controls. In addition, 53.60% of the AZF microdeletions occurred in the AZFc region. It can be concluded that AZF microdeletion on the long arm of the Y chromosome can result in male spermatogenesis dysfunction. Detection of AZF microdeletion can provide a theoretical basis for genetic counseling, as well as improve the diagnosis and treatment of this disease.

Aplastic Anemia in Two Patients with Sex Chromosome Aneuploidies.

Sex chromosome aneuploidies range in incidence from rather common to exceedingly rare and have a variable phenotype. We report 2 patients with sex chromosome aneuploidies who developed severe aplastic anemia requiring treatment. The first patient had tetrasomy X (48,XXXX) and presented at 9 years of age, and the second patient had trisomy X (47,XXX) and presented at 5 years of age. Although aplastic anemia has been associated with other chromosomal abnormalities, sex chromosome abnormalities have not been traditionally considered a risk factor for this condition. A review of the literature reveals that at least one other patient with a sex chromosome aneuploidy (45,X) has suffered from aplastic anemia and that other autosomal chromosomal anomalies have been described. Despite the uncommon nature of each condition, it is possible that the apparent association is coincidental. A better understanding of the genetic causes of aplastic anemia remains important.

Identification of the origin of marker chromosomes by two-color fluorescence in situ hybridization and polymerase chain reaction in azoospermic patients.

Y chromosomal microdeletions at the azoospermia factor locus and chromosome abnormalities have been implicated as the major causes of idiopathic male infertility. A marker chromosome is a structurally abnormal chromosome in which no part can be identified by cytogenetics. In this study, to identify the origin of the marker chromosomes and to perform a genetic diagnosis of patients with azoospermia, two-color fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) techniques were carried out. The marker chromosomes for the two patients with azoospermia originated in the Y chromosome; it was ascertained that the karyotype of both patients was 46,X, ish del(Y)(q11)(DYZ3+, DXZ1-). The combination of two-color FISH and PCR techniques is an important method for the identification of the origin of marker chromosomes. Thus, genetic counseling and a clear genetic diagnosis of patients with azoospermia before intracytoplasmic sperm injection or other clinical managements are important.

Varicocele and male infertility in Northeast China: Y chromosome microdeletion as an underlying cause.

The prevalence of Y chromosome microdeletions among azoospermic, severe oligozoospermic, moderate oligozoospermic, and mild oligozoospermic patients with varicocele-related and idiopathic infertility shows conflicting data in Asian countries. We aimed to detect this frequency in Northeast China, and investigated spermatogenic defects whether associated with varicocele or Y chromosome microdeletions. All samples underwent a thorough physical examination, semen analysis, and PCR analyses for Y chromosome microdeletions. We randomly selected 150 infertile non-obstructive azoospermic patients with left varicocele (Group 1), 150 idiopathic non-obstructive azoospermic infertility patients (Group 2), 150 infertile severe oligozoospermic patients with left varicocele (Group 3), 150 idiopathic severe oligozoospermic infertility patients (Group 4), 150 infertile moderate oligozoospermic patients with left varicocele (Group 5), 150 idiopathic moderate oligozoospermic infertility patients (Group 6), 150 infertile mild oligozoospermic patients with left varicocele (Group 7), 150 idiopathic mild oligozoospermic infertility patients (Group 8), and 60 healthy unrelated men with proven fertility were recruited as control subjects (Group 9). We observed that our samples from Northeastern China had a higher frequency of microdeletions among the non-obstructive azoospermic individuals with varicocele, as compared with other Asian countries. Furthermore, the spermatogenic defect is due to the underlying Y chromosome microdeletion, and not the varicocele itself. Although varicocele is not the cause of male infertility, it may be associated with male infertility in the Northeastern Chinese population.

37,X/38,XY Mosaicism in a Cryptorchid Bengal Cat with Müllerian Duct Remnants.

A 23-month-old tomcat was referred to our clinic because of male behavioral problems, cryptorchidism, and an undefined intra-abdominal organ resembling a uterus. Ultrasonography and computed tomography showed 2 fluid-filled tubular structures dorsolaterally to the bladder and connected to the pelvic urethra. The cat was castrated, and the tubular structures were surgically removed. Histology identified them as Müllerian duct remnants. The testes were hypoplastic, the epididymes and deferent ducts were normal. Cytogenetic analyses revealed the presence of a mosaic 37,X/38,XY karyotype which explains the clinical findings.

A case-control study of brain structure and behavioral characteristics in 47,XXX syndrome.

Trisomy X, the presence of an extra X chromosome in females (47,XXX), is a relatively common but under-recognized chromosomal disorder associated with characteristic cognitive and behavioral features of varying severity. The objective of this study was to determine whether there were neuroanatomical differences in girls with Trisomy X that could relate to cognitive and behavioral differences characteristic of the disorder during childhood and adolescence. MRI scans were obtained on 35 girls with Trisomy X (mean age 11.4, SD 5.5) and 70 age- and sex-matched healthy controls. Cognitive and behavioral testing was also performed. Trisomy X girls underwent a semi-structured psychiatric interview. Regional brain volumes and cortical thickness were compared between the two groups. Total brain volume was significantly decreased in subjects with Trisomy X, as were all regional volumes with the exception of parietal gray matter. Differences in cortical thickness had a mixed pattern. The subjects with Trisomy X had thicker cortex in bilateral medial prefrontal cortex and right medial temporal lobe, but decreased cortical thickness in both lateral temporal lobes. The most common psychiatric disorders present in this sample of Trisomy X girls included anxiety disorders (40%), attention-deficit disorder (17%) and depressive disorders (11%). The most strongly affected brain regions are consistent with phenotypic characteristics such as language delay, poor executive function and heightened anxiety previously described in population-based studies of Trisomy X and also found in our sample.