Genetic Alterations in Gorlin Syndrome

Gorlin syndrome (GS) is an autosomal dominant disorder that predisposes affected individuals to developmental defects and tumorigenesis, and caused mainly by heterozygous germline PTCH1 mutations. Despite exhaustive analysis, PTCH1 mutations are often unidentifiable in some patients; the failure to detect mutations is presumably because of mutations occurred in other causative genes or outside of analyzed regions of PTCH1, or copy number alterations (CNAs). In this study, we subjected a cohort of GS-affected individuals from six unrelated families to next-generation sequencing (NGS) analysis for the combined screening of causative alterations in Hedgehog signaling pathway-related genes. Specific single nucleotide variations (SNVs) of PTCH1 causing inferred amino acid changes were identified in four families (seven affected individuals), whereas CNAs within or around PTCH1 were found in two families in whom possible causative SNVs were not detected. Through a targeted resequencing of all coding exons, as well as simultaneous evaluation of copy number status using the alignment map files obtained via NGS, we found that GS phenotypes could be explained by PTCH1 mutations or deletions in all affected patients. Because it is advisable to evaluate CNAs of candidate causative genes in point mutation-negative cases, NGS methodology appears to be useful for improving molecular diagnosis through the simultaneous detection of both SNVs and CNAs in the targeted genes/regions

Stable lines and clones of long-term proliferating normal, genetically unmodified murine common lymphoid progenitors.

Common lymphoid progenitors (CLPs) differentiate to T and B lymphocytes, dendritic cells, natural killer cells, and innate lymphoid cells. Here, we describe culture conditions that, for the first time, allow the establishment of lymphoid-restricted, but uncommitted, long-term proliferating CLP cell lines and clones from a small pool of these cells from normal mouse bone marrow, without any genetic manipulation. Cells from more than half of the cultured CLP clones could be induced to differentiate to T, B, natural killer, dendritic, and myeloid cells in vitro. Cultured, transplanted CLPs transiently populate the host and differentiate to all lymphoid subsets, and to myeloid cells in vivo. This simple method to obtain robust numbers of cultured noncommitted CLPs will allow studies of cell-intrinsic and environmentally controlled lymphoid differentiation programs. If this method can be applied to human CLPs, it will provide new opportunities for cell therapy of patients in need of myeloid-lymphoid reconstitution

Role of PERK in mitochondrial function

Mitochondria play a central role in the function of brown adipocytes (BAs). Although mitochondrial biogenesis, which is indispensable for thermogenesis, is regulated by coordination between nuclear DNA transcription and mitochondrial DNA transcription, the molecular mechanisms of mitochondrial development during BA differentiation are largely unknown. Here, we show the importance of the ER-resident sensor PKR-like ER kinase (PERK) in the mitochondrial thermogenesis of brown adipose tissue. During BA differentiation, PERK is physiologically phosphorylated independently of the ER stress. This PERK phosphorylation induces transcriptional activation by GA-binding protein transcription factor α subunit (GABPα), which is required for mitochondrial inner membrane protein biogenesis, and this novel role of PERK is involved in maintaining the body temperatures of mice during cold exposure. Our findings demonstrate that mitochondrial development regulated by the PERK–GABPα axis is indispensable for thermogenesis in brown adipose tissue

Dysregulation of the DNA Damage Response and <i>KMT2A</i> Rearrangement in Fetal Liver Hematopoietic Cells

<p>(A) Dose-dependence of γH2AX positivity in fetal liver hematopoietic stem cells (FL-HSCs), analyzed 4 h after IP injection of etoposide at the indicated doses. Etoposide was IP injected into E13.5 pregnant mice. (B) DNA double-strand breaks were detected according to γH2AX positivity, and cell-cycle distribution was monitored by propidium iodide (PI) incorporation. Etoposide (10 mg/kg) was IP injected into E13.5 pregnant mice, and samples were analyzed at the indicated time points. A two-dimensional dot blot is shown. FL-HSC: fetal liver hematopoietic stem cells; mBM: maternal bone marrow mono nuclear cells. (C) The kinetics of γH2AX positivity in the samples shown in B are expressed as a line graph. Bold line indicates FL-HSC, and broken line indicates mBM. (D) Percentage of apoptotic cells in the samples shown in B.</p

FL-HSCs of <i>Atm</i>-knockout mice were pulse-treated with etoposide for 4 h <i>in vitro</i>; metaphase spreads were generated 24 h after pulse treatment.

<p>(A) Representative metaphase spreads. Arrows indicate chromosomal or chromatid breaks. (B) Number of chromosomal breaks and chromatid breaks, shown as a table. (C) Sequence electropherogram of <i>Kmt2a</i>-<i>Ptp4a2</i> fusion mRNA.</p

Fetal etoposide concentration after intraperitoneal (IP) etoposide injection.

<p>Etoposide (10 mg/kg) was IP injected into E13.5 pregnant mice, and fetal livers were collected at the indicated time points.</p

Simultaneous Detection of Both Single Nucleotide Variations and Copy Number Alterations by Next-Generation Sequencing in Gorlin Syndrome.

Gorlin syndrome (GS) is an autosomal dominant disorder that predisposes affected individuals to developmental defects and tumorigenesis, and caused mainly by heterozygous germline PTCH1 mutations. Despite exhaustive analysis, PTCH1 mutations are often unidentifiable in some patients; the failure to detect mutations is presumably because of mutations occurred in other causative genes or outside of analyzed regions of PTCH1, or copy number alterations (CNAs). In this study, we subjected a cohort of GS-affected individuals from six unrelated families to next-generation sequencing (NGS) analysis for the combined screening of causative alterations in Hedgehog signaling pathway-related genes. Specific single nucleotide variations (SNVs) of PTCH1 causing inferred amino acid changes were identified in four families (seven affected individuals), whereas CNAs within or around PTCH1 were found in two families in whom possible causative SNVs were not detected. Through a targeted resequencing of all coding exons, as well as simultaneous evaluation of copy number status using the alignment map files obtained via NGS, we found that GS phenotypes could be explained by PTCH1 mutations or deletions in all affected patients. Because it is advisable to evaluate CNAs of candidate causative genes in point mutation-negative cases, NGS methodology appears to be useful for improving molecular diagnosis through the simultaneous detection of both SNVs and CNAs in the targeted genes/regions