Usefulness of combined NGS and QF-PCR analysis for product of conception karyotyping.

Purpose: Since chromosomal abnormalities can be detected in more than half of miscarriages, cytogenetic testing of the product of conception (POC) can provide important information when preparing for a subsequent pregnancy. Conventional karyotyping is the common diagnostic method for a POC but can be problematic due to the need for cell culture. Methods: We here conducted shallow whole-genome sequencing (sWGS) using next-generation sequencing (NGS) for alternative POC cytogenomic analysis. Since female euploidy samples can include 69,XXX triploidy, additional QF-PCR was performed in these cases. Results: We here analyzed POC samples from miscarriages in 300 assisted reproductive technology (ART) pregnancies and detected chromosomal abnormalities in 201 instances (67.0%). Autosomal aneuploidy (151 cases, 50.3%) was the most frequent abnormality, consistent with prior conventional karyotyping data. Mosaic aneuploidy was detected in seven cases (2.0%). Notably, the frequency of triploidy was 2.3%, 10-fold lower than the reported frequency in non-ART pregnancies. Structural rearrangements were identified in nine samples (3%), but there was no case of segmental mosaicism. Conclusions: These data suggest that NGS-based sWGS, with the aid of QF-PCR, is a viable alternative karyotyping procedure that does not require cell culture. This method could also assist with genetic counseling for couples who undergoes embryo selection based on PGT-A data.

Analysis of the Origin of Double Mosaic Aneuploidy in Two Cases.

We present 2 cases of double mosaic aneuploidy harboring 2 or more different aneuploid cell lines, but no line with a normal chromosome constitution. One of these cases presented mosaicism of sex chromosome aneuploid cell lines (47,XXX/45,X) along with another line containing an autosomal trisomy (47,XX,+8), while the other case showed mosaicism of 2 different autosomal trisomy cell lines (47,XY,+5 and 47,XY,+8). To elucidate the mechanisms underlying these mosaicisms, we conducted molecular cytogenetic analyses. Genotyping data from the SNP microarray indicated that 2 sequential meiotic or early postzygotic segregation errors likely had occurred followed by natural selection. These cases suggest that frequent segregation errors and selection events in the meiotic and early postzygotic stages lead to this condition.

A case of a parthenogenetic 46,XX/46,XY chimera presenting ambiguous genitalia.

Sex-chromosome discordant chimerism (XX/XY chimerism) is a rare chromosomal disorder in humans. We report a boy with ambiguous genitalia and hypospadias, showing 46,XY[26]/46,XX[4] in peripheral blood cells. To clarify the mechanism of how this chimerism took place, we carried out whole-genome genotyping using a SNP array and microsatellite analysis. The B-allele frequency of the SNP array showed a mixture of three and five allele combinations, which excluded mosaicism but not chimerism, and suggested the fusion of two embryos or a shared parental haplotype between the two parental cells. All microsatellite markers showed a single maternal allele. From these results, we concluded that this XX/XY chimera is composed of two different paternal alleles and a single duplicated maternal genome. This XX/XY chimera likely arose from a diploid maternal cell that was formed via endoduplication of the maternal genome just before fertilization, being fertilized with both X and Y sperm.

The effect of early irregular cell division of human embryos on blastocyst euploidy: considerations from the subsequent development of the blastomeres by direct or reverse cleavage.

OBJECTIVE: To investigate whether blastocysts that divide irregularly reduce subsequent blastocyst euploidy. DESIGN: Retrospective study. SETTING: Private clinic. PATIENT(S): A total of 122 blastocysts for which consent for disposal and research use was obtained. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Results of next-generation sequencing analysis of the blastocysts and whether blastomeres by normal or irregular divisions subsequently participated in blastocyst formation or not. RESULT(S): The embryos were classified according to their dynamics until the second cleavage. The blastocyst euploidy rates were 33.3% (19/57) in the normal cleavage (NC) group, 38.3% (18/47) in the direct cleavage (embryos with one cell dividing into 3 cells) (DC) group, and 72.2% (13/18) in the reverse cleavage (RC) (embryos with fused cells once divided) group. The rate of the RC group was significantly higher than that of the NC group. The blastocyst participation rate of the blastomeres were 95.6% in the NC group and 56.5% in that derived from DC of the first cleavage, and 91.7% in that of blastomeres derived from normal division of the second cleavage and 53.6% in that derived from DC of the second cleavage, both of which were significantly lower in the latter. In the RC group, the rates of fused and nonfused blastomeres were 62.1% and 87.5%, respectively, with no significant difference. CONCLUSION(S): The blastomeres generated by DC were often excluded from blastocyst formation, and we speculate that this is one reason why their division does not reduce blastocyst euploidy. The association between RC and euploidy of blastocysts merits further study.

Transport and Golgi organization 2 deficiency with a prominent elevation of C14:1 during a metabolic crisis: A case report.

Mutations in transport and Golgi organization 2 homolog (TANGO2) have recently been described as a cause of an autosomal recessive syndrome characterized by episodes of metabolic crisis associated with rhabdomyolysis, cardiac arrhythmias, and neurodegeneration. Herein, we report a case of a one-and-a-half-year-old Japanese girl, born to nonconsanguineous parents, who presented with metabolic crisis characterized by hypoglycemia with hypoketonemia, rhabdomyolysis, lactic acidosis, and prolonged corrected QT interval (QTc) at the age of 6 months. Acylcarnitine analysis during the episode of crisis showed prominent elevation of C14:1, suggesting very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. In addition, worsening rhabdomyolysis was observed after intravenous administration of L-carnitine. VLCAD deficiency was initially suspected; however, the enzyme activity in lymphocytes was only mildly decreased at the gene carrier level, and no mutation in the VLCAD gene (ADADVL) was detected. Subsequently, acylcarnitine analysis was nonspecific at 17-h fasting and almost normal during the stable phase. Eventually, a trio whole-exome sequencing revealed a compound heterozygous variant of two novel variants in the TANGO2 gene, a missense variant, and a deletion of exon 7. This is the first case of TANGO2 deficiency in Asians. Our case suggests that elevated C14:1 may be seen in severe metabolic crises and that the use of L-carnitine should be avoided during metabolic crises.

Novel compound heterozygous mutations in the PLEC gene in a neonate with epidermolysis bullosa simplex with pyloric atresia.

Epidermolysis bullosa (EB) is a heterogeneous group of inherited disorders characterized by the blistering of the skin and mucous membranes. Although the molecular basis of EB has been significantly elucidated, the precise phenotypes of the lethal types of EB have not been completely characterized. Herein, we report a severe case of EB with pyloric atresia (PA). The patient was a Japanese boy who not only had skin lesions but also various complications such as PA, dysphagia, hypotonia, infectious keratitis with corneal ulcer, obstructive uropathy and protein-losing enteropathy. Genetic analysis led to the identification of two novel compound heterozygous mutations in the last exon of the plectin (PLEC) gene. Based on this finding, EB simplex with PA was diagnosed. Immunostaining with anti-plectin antibodies revealed truncated plectin proteins lacking the C-terminus in the patient's skin. We also conducted a prenatal diagnosis in subsequent pregnancy. Our report further highlights the crucial role of plectin in many organs and provides valuable information regarding the phenotypes resulting from mutations in the PLEC gene.

Mitochondrial DNA mutations can influence the post-implantation development of human mosaic embryos.

Introduction: Several healthy euploid births have been reported following the transfer of mosaic embryos, including both euploid and aneuploid blastomeres. This has been attributed to a reduced number of aneuploid cells, as previously reported in mice, but remains poorly explored in humans. We hypothesized that mitochondrial function, one of the most critical factors for embryonic development, can influence human post-implantation embryonic development, including a decrease of aneuploid cells in mosaic embryos. Methods: To clarify the role of mitochondrial function, we biopsied multiple parts of each human embryo and observed the remaining embryos under in vitro culture as a model of post-implantation development (n = 27 embryos). Karyotyping, whole mitochondrial DNA (mtDNA) sequencing, and mtDNA copy number assays were performed on all pre- and post-culture samples. Results: The ratio of euploid embryos was significantly enhanced during in vitro culture, whereas the ratio of mosaic embryos was significantly reduced. Furthermore, post-culture euploid and culturable embryos had significantly few mtDNA mutations, although mtDNA copy numbers did not differ. Discussion: Our results indicate that aneuploid cells decrease in human embryos post-implantation, and mtDNA mutations might induce low mitochondrial function and influence the development of post-implantation embryos with not only aneuploidy but also euploidy. Analyzing the whole mtDNA mutation number may be a novel method for selecting a better mosaic embryo for transfer.

Novel large deletion involving EVC and EVC2 in Ellis-van Creveld syndrome.

Ellis-van Creveld syndrome is an autosomal recessive skeletal dysplasia that is characterized by thoracic hypoplasia, polydactyly, oral abnormalities, and congenital heart disease. It is caused by pathogenic variants in the EVC or EVC2 genes. We report a case of a newborn with a compound heterozygous variant comprising NM_147127.5: c.1991dup:[p.Lys665Glufs*10] in the EVC2 gene and a novel large deletion involving exon 1 in EVC and exons 1-7 in EVC2.

Genotype-phenotype correlation of renal lesions in the tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant disease caused by loss-of-function mutations in either of two tumor suppressor genes, TSC1 and TSC2. These mutations lead to the growth of benign tumors and hamartomas in many organs, including those of the central nervous system, the skin, and the kidneys. To investigate the genotype-phenotype correlation, we performed sequence analysis of the TSC1/2 genes using next-generation sequencing. We classified 30 patients with TSC whose pathogenic variants were identified into two groups: those with mutations producing premature termination codons (PTCs) and those with missense mutations. Then, we compared the phenotypes between the two groups. Patients with a PTC were significantly more likely to manifest the major symptoms of the diagnostic criteria than those without a PTC (P = 0.035). The frequencies of subependymal nodules (P = 0.026), cortical tubers (P = 0.026), and renal cysts (P = 0.026) were significantly higher in PTC-containing variants than in cases without a PTC. When the analyses were limited to renal angiomyolipoma (AML) cases with TSC2 mutations, there was no difference in tumor size between cases with and without a PTC. However, the cases with a PTC showed a trend toward disease onset at a younger age and multiple tumors, and bilateral disease was observed in their AML lesions. TSC patients with PTC-producing mutations might potentially manifest more severe TSC phenotypes than those with missense mutations. A larger-scale study with appropriate samples deserves further investigation.

Streamlined human antibody generation and optimization by exploiting designed immunoglobulin loci in a B cell line.

Monoclonal antibodies (mAbs) are widely utilized as therapeutic drugs for various diseases, such as cancer, autoimmune diseases, and infectious diseases. Using the avian-derived B cell line DT40, we previously developed an antibody display technology, namely, the ADLib system, which rapidly generates antigen-specific mAbs. Here, we report the development of a human version of the ADLib system and showcase the streamlined generation and optimization of functional human mAbs. Tailored libraries were first constructed by replacing endogenous immunoglobulin genes with designed human counterparts. From these libraries, clones producing full-length human IgGs against distinct antigens can be isolated, as exemplified by the selection of antagonistic mAbs. Taking advantage of avian biology, effective affinity maturation was achieved in a straightforward manner by seamless diversification of the parental clones into secondary libraries followed by single-cell sorting, quickly affording mAbs with improved affinities and functionalities. Collectively, we demonstrate that the human ADLib system could serve as an integrative platform with unique diversity for rapid de novo generation and optimization of therapeutic or diagnostic antibody leads. Furthermore, our results suggest that libraries can be constructed by introducing exogenous genes into DT40 cells, indicating that the ADLib system has the potential to be applied for the rapid and effective directed evolution and optimization of proteins in various fields beyond biomedicine.

Exome-First Approach in Fetal Akinesia Reveals Chromosome 1p36 Deletion Syndrome.

BACKGROUND: Fetal akinesia refers to a broad spectrum of disorders with reduced or absent fetal movements. There is no established approach for prenatal diagnosis of the cause of fetal akinesia. Chromosome 1p36 deletion syndrome is the most common subtelomeric terminal deletion syndrome, recognized postnatally from typical craniofacial features. However, the influence of chromosome 1p36 deletion on fetal movements remains unknown. CASE REPORT: A 32-week-old fetus with akinesia showed multiple abnormalities, including fetal growth restriction, congenital cardiac defects, and ventriculomegaly. G-banding analysis using cultured amniocytes revealed 46,XY,22pstk+. Postnatal whole exome sequencing and subsequent chromosomal microarray identified a 3 Mb deletion of chromosomal region 1p36.33-p36.32. These results of molecular cytogenetic analyses were consistent with the fetal sonographic findings. CONCLUSION: Using the exome-first approach, we identified a case with fetal akinesia associated with chromosome 1p36 deletion. Chromosome 1p36 deletion syndrome may be considered for differential diagnosis in cases of fetal akinesia with multiple abnormalities.

Expression of proliferation markers in human pituitary incidentalomas.

This study aimed to immunohistochemically assess the proliferation activity of pituitary incidentalomas. A series of 52 incidentalomas studied included 22 gonadotroph cell adenomas, 21 null cell adenomas, and 9 clinically silent adenomas (identified as functioning by immunohistochemistry). We also analyzed the differences in proliferation activity between 43 non-functioning pituitary incidentalomas (not including 9 silent adenomas), and 43 symptomatic non-functioning adenomas (NFAs) that caused visual disturbance. Cell proliferation markers were immunostained using monoclonal Ki-67 (MIB-1) antibody and monoclonal anti-topoisomerase II alpha (Topo-II alpha) antibody. The average of MIB-1 labeling indices in pituitary incidentalomas was 0.61% +/- 0.06%. Overall, both MIB-1 and Topo-II alpha labeling indices of the incidentalomas were significantly lower than those of symptomatic NFAs. There were no significant differences in immunopositivity between the two groups based on gender, age, or subtype. The MIB-1 index of the smallest adenoma group in pituitary incidentalomas was significantly lower than in symptomatic NFAs, while the Topo-II alpha incidentaloma was significantly lower than in symptomatic NFAs. Our findings suggest that small or less invasive pituitary incidentalomas should be observed with follow-up MRI. Large or invasive incidentalomas should be surgically treated if the patients show visual disturbances, hypopituitarism, or pituitary apoplexy during the follow-up period.

Immunonegative "null cell" adenomas and gonadotropin (Gn) subunit (SUs) immunopositive adenomas share frequent expression of multiple transcription factors.

The differentiation of pituitary cells and human pituitary adenomas follow three cell lineages: GH-PRL-TSH, ACTH, and FSH/LH, which are regulated by a combination of various transcription factors and co-factors. We have used RT-PCR and immunohistochemistry to show that immunonegative, "null cell" adenomas are equipped with multiple transcription factors and co-factors. The "null cell" adenomas showed similar frequencies of transcription factors as did the gonadotropin subunit (GnSU)-positive adenomas, with the exception that there were fewer instances of SF1 in the former. We speculate, therefore, that null cell adenomas and GnSU-positive adenomas share common molecular mechanisms in functional differentiation, even though the former do not produce hormones. From the high frequency of various transcription factors, we also speculate that both null cell adenomas and GnSU-positive adenomas are derived from "committed" pituitary progenitor stem cells. The questions, why a certain proportion of these pituitary tumor groups lack hormone production and why they are molecularly more committed to Gn transcription, remain to be further investigated.

Dietary soybean enhances Pit-1 dependent pituitary hormone production in iodine deficient rats.

Reports have shown that soybeans are goitrogenic. In the present study, we investigated the effects of a high soybean diet in rats that were fed normal or iodine-deficient chow on the regulation of anterior pituitary hormone production. Iodine deficiency alone resulted in thyroid hyperplasia, reduced serum thyroxine levels, and a tendency towards an increase in serum thyroid stimulating hormone (TSH). The combination of a high soybean and low iodine diet (ID + DS) acted synergistically to induce thyroid hypertrophy, reduce serum thyroxine and tri-iodothyronine, and markedly increase serum TSH. Immunohistochemical analysis revealed that rats fed the ID + DS diet exhibited a marked increase in their number of pituitary TSH, prolactin (PRL), and growth hormone (GH) producing cells. Pituitary transcription factor-1 (Pit-1) which is involved in the expression of the TSH, PRL, and GH genes was also increased in ID + DS fed rats. These results suggest that a diet high in soybean products modulates anterior pituitary hormone production by regulating Pit-1 induction, in iodine-deficient animals.

Molecular pathology of the pituitary. Development and functional differentiation of pituitary adenomas.

This review article describes functional differentiation of the pituitary cells and pituitary adenomas with special emphasis on transcription factors and co-factors. Human pituitary adenomas generally follow the combination of transcription factors and co-factors, which are similar to those of physiologic anterior pituitary cells. On very rare occasions, the single pituitary adenoma produces two hormones, which belong to different cell lineage 'trans-cell lineage'. Basic mechanism for this was considered to be 'aberrant expression' of transcription factors, i.e. NeuroD1 and Pit-1. This was experimentally supported by the induction of GH (mRNA and protein) in AtT-20 cells by transfecting Pit-1 gene. Various mechanisms have been reported for the experimental pituitary oncogenesis. Among these, GHRH has been emphasized as one of oncogenic factors for both human GHomas as well as in the transgenic animals.

Upregulation of plasma CCL8 in mouse model of graft-vs-host disease.

OBJECTIVE: Using a proteomic approach, we recently identified plasma CCL8 as a potential biomarker for diagnosis of graft-vs-host-disease (GVHD) in mice as well as humans. Because mass spectrometric analysis is only semi-quantitative, a quantitative method of measuring plasma CCL8 levels in mice is needed. MATERIALS AND METHODS: We established an enzyme-linked immunosorbent assay for the quantitative measurement of CCL8 concentrations in mouse plasma. RESULTS: Our newly established enzyme-linked immunosorbent assay revealed that the plasma CCL8 concentrations (mean +/- standard error; n=12) were 1287+/-55.7 ng/mL and 1604+/-110.8 ng/mL on days 7 and 14 after allogeneic bone marrow transplantation (BMT), respectively, while the plasma concentrations was 316.6+/-16.3 ng/mL on day 7 after syngeneic BMT. A Western blotting analysis also showed a difference in the plasma CCL8 levels between the allogeneic and syngeneic BMT groups, as did clinical GVHD scores. Neither lipopolysaccharide nor poly(I:C) elevated the plasma CCL8 concentrations, although a dramatic increase in interleukin-6 was detected after both treatments. CONCLUSION: An elevated plasma CCL8 concentration may be a promising plasma marker for GVHD in mouse models.

Induction of GH, PRL, and TSH beta mRNA by transfection of Pit-1 in a human pituitary adenoma-derived cell line.

The functional development of pituitary cells depends on the expression of a combination of transcription factors and co-factors. Pituitary-specific transcription factor-1 (Pit-1) is required for the expression of growth hormone (GH), prolactin (PRL), and the thyroid-stimulating hormone beta subunit (TSH beta) and acts synergistically with the estrogen receptor (ER) and GATA-binding protein 2 (GATA-2) to induce PRL and TSH beta expression, respectively. The glycoprotein hormone alpha subunit (alpha SU) is the first hormone to be expressed during pituitary development. In addition to being expressed in follicle-stimulating hormone, luteinizing hormone (LH), and TSH cells, alpha SU is reported to co-localize with GH in pituitary cells. These findings have led to the suggestion that the expression of Pit-1 in cells of the alpha SU-based gonadotropin cell lineage might also lead to the expression of GH. In this study, we transfected HP 75 cells (derived from a human non-functioning pituitary adenoma that expressed alpha SU and LH beta) with Pit-1 by using an adenovirus FLAG-Pit-1 construct. Most of the transfected cells expressed GH mRNA, with fewer cells expressing PRL and TSH beta mRNA. The HP 75 cells expressed the genes for ER and GATA-2, thus allowing their expression of GH, PRL, and TSH beta mRNA in response to Pit-1. These results support the hypothesis that GH can be induced in cells that possess an active alpha SU gene and shed light on the basic molecular mechanism that drives the development of GH, PRL, and TSH beta expression in the alpha SU-based gonadotroph lineage.

Recent progress in studies of pituitary tumor pathogenesis.

The mechanisms of tumorigenesis of the human pituitary have been elucidated to a limited extent. Classically, pituitary tumor formation was shown to be induced by thyroidectomy and estrogen administration. Molecular biological and immunohistochemical studies have revealed several aspects of pituitary tumorigenesis. Translineage cell differentiation has been shown to be induced by the aberrant expression of transcription factors and co-factors, such as Pit-1, Prop-1, and estrogen receptor. Defects or overexpression of cell cycle regulators, such as CDK inhibitors, PTTG, and GADD45gamma, result in the abnormal proliferation of pituitary cells. Recently, epigenetic regulation has been suggested to be related to pituitary tumor formation. This article presents a review and update of recent progress in studies of the development and differentiation of pituitary tumors.