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.

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.

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.

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.

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.

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.

Differentiation of skeletal muscle from pituitary folliculo-stellate cells and endocrine progenitor cells.

We previously reported the ectopic differentiation of skeletal muscle cells in a pituitary gland transplanted beneath a kidney capsule. Morphological observation suggested that the skeletal muscle cells may have differentiated from folliculo-stellate (FS) cells in the anterior pituitary gland. However, at that time, we did not confirm this directly with an in vitro system. To obtain direct evidence, we used the Tpit/F1 cell line. The Tpit/F1 cell line was recently established from the pituitary gland of a temperature-sensitive T antigen transgenic mouse and has the characters of pituitary FS cells. Using Tpit/F1 cells, we have found that FS cells of the pituitary are able to differentiate into muscle cells in vitro. Additionally, we showed that the cells have some characteristics of pituitary FS cells and also express pituitary endocrine cell-specific transcription factor (pit-1) and prolactin genes, and can differentiate into striated muscle cells. The anterior pituitary gland is known to be of ectodermal origin, so the differentiation of its cells into striated muscle is completely unexpected. This is the first report of direct evidence of ectopic differentiation of skeletal muscle cells from pituitary cells.