Expression levels and DNA methylation profiles of the growth gene SHOX in cartilage tissues and chondrocytes.

All attempts to identify male-specific growth genes in humans have failed. This study aimed to clarify why men are taller than women. Microarray-based transcriptome analysis of the cartilage tissues of four adults and chondrocytes of 12 children showed that the median expression levels of SHOX, a growth gene in the pseudoautosomal region (PAR), were higher in male samples than in female samples. Male-dominant SHOX expression was confirmed by quantitative RT-PCR for 36 cartilage samples. Reduced representation bisulfite sequencing of four cartilage samples revealed sex-biased DNA methylation in the SHOX-flanking regions, and pyrosequencing of 22 cartilage samples confirmed male-dominant DNA methylation at the CpG sites in the SHOX upstream region and exon 6a. DNA methylation indexes of these regions were positively correlated with SHOX expression levels. These results, together with prior findings that PAR genes often exhibit male-dominant expression, imply that the relatively low SHOX expression in female cartilage tissues reflects the partial spread of X chromosome inactivation into PAR. Altogether, this study provides the first indication that sex differences in height are ascribed, at least in part, to the sex-dependent epigenetic regulation of SHOX. Our findings deserve further validation.

Gene-specific somatic epigenetic mosaicism of FDFT1 underlies a non-hereditary localized form of porokeratosis.

Porokeratosis is a clonal keratinization disorder characterized by solitary, linearly arranged, or generally distributed multiple skin lesions. Previous studies showed that genetic alterations in MVK, PMVK, MVD, or FDPS-genes in the mevalonate pathway-cause hereditary porokeratosis, with skin lesions harboring germline and lesion-specific somatic variants on opposite alleles. Here, we identified non-hereditary porokeratosis associated with epigenetic silencing of FDFT1, another gene in the mevalonate pathway. Skin lesions of the generalized form had germline and lesion-specific somatic variants on opposite alleles in FDFT1, representing FDFT1-associated hereditary porokeratosis identified in this study. Conversely, lesions of the solitary or linearly arranged localized form had somatic bi-allelic promoter hypermethylation or mono-allelic promoter hypermethylation with somatic genetic alterations on opposite alleles in FDFT1, indicating non-hereditary porokeratosis. FDFT1 localization was uniformly diminished within the lesions, and lesion-derived keratinocytes showed cholesterol dependence for cell growth and altered expression of genes related to cell-cycle and epidermal development, confirming that lesions form by clonal expansion of FDFT1-deficient keratinocytes. In some individuals with the localized form, gene-specific promoter hypermethylation of FDFT1 was detected in morphologically normal epidermis adjacent to methylation-related lesions but not distal to these lesions, suggesting that asymptomatic somatic epigenetic mosaicism of FDFT1 predisposes certain skin areas to the disease. Finally, consistent with its genetic etiology, topical statin treatment ameliorated lesions in FDFT1-deficient porokeratosis. In conclusion, we identified bi-allelic genetic and/or epigenetic alterations of FDFT1 as a cause of porokeratosis and shed light on the pathogenesis of skin mosaicism involving clonal expansion of epigenetically altered cells.

Systematic molecular analyses for 115 karyotypically normal men with isolated non-obstructive azoospermia.

STUDY QUESTION: Do copy-number variations (CNVs) in the azoospermia factor (AZF) regions and monogenic mutations play a major role in the development of isolated (non-syndromic) non-obstructive azoospermia (NOA) in Japanese men with a normal 46, XY karyotype? SUMMARY ANSWER: Deleterious CNVs in the AZF regions and damaging sequence variants in eight genes likely constitute at least 8% and approximately 8% of the genetic causes, respectively, while variants in other genes play only a minor role. WHAT IS KNOWN ALREADY: Sex chromosomal abnormalities, AZF-linked microdeletions, and monogenic mutations have been implicated in isolated NOA. More than 160 genes have been reported as causative/susceptibility/candidate genes for NOA. STUDY DESIGN, SIZE, DURATION: Systematic molecular analyses were conducted for 115 patients with isolated NOA and a normal 46, XY karyotype, who visited our hospital between 2017 and 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: We studied 115 unrelated Japanese patients. AZF-linked CNVs were examined using sequence-tagged PCR and multiplex ligation-dependent probe amplification, and nucleotide variants were screened using whole exome sequencing (WES). An optimized sequence kernel association test (SKAT-O), a gene-based association study using WES data, was performed to identify novel disease-associated genes in the genome. The results were compared to those of previous studies and our in-house control data. MAIN RESULTS AND THE ROLE OF CHANCE: Thirteen types of AZF-linked CNVs, including the hitherto unreported gr/gr triplication and partial AZFb deletion, were identified in 63 (54.8%) cases. When the gr/gr deletion, a common polymorphism in Japan, was excluded from data analyses, the total frequency of CNVs was 23/75 (30.7%). This frequency is higher than that of the reference data in Japan and China (11.1% and 14.7%, respectively). Known NOA-causative AZF-linked CNVs were found in nine (7.8%) cases. Rare damaging variants in known causative genes (DMRT1, PLK4, SYCP2, TEX11, and USP26) and hemizygous/multiple-heterozygous damaging variants in known spermatogenesis-associated genes (TAF7L, DNAH2, and DNAH17) were identified in nine cases (7.8% in total). Some patients carried rare damaging variants in multiple genes. SKAT-O detected no genes whose rare damaging variants were significantly accumulated in the patient group. LIMITATIONS, REASONS FOR CAUTION: The number of participants was relatively small, and the clinical information of each patient was fragmentary. Moreover, the pathogenicity of identified variants was assessed only by in silico analyses. WIDER IMPLICATIONS OF THE FINDINGS: This study showed that various AZF-linked CNVs are present in more than half of Japanese NOA patients. These results broadened the structural variations of AZF-linked CNVs, which should be considered for the molecular diagnosis of spermatogenic failure. Furthermore, the results of this study highlight the etiological heterogeneity and possible oligogenicity of isolated NOA. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Grants from the Japan Society for the Promotion of Science (21K19283 and 21H0246), the Japan Agency for Medical Research and Development (22ek0109464h0003), the National Center for Child Health and Development, the Canon Foundation, the Japan Endocrine Society, and the Takeda Science Foundation. The results of this study were based on samples and patient data obtained from the International Center for Reproductive Medicine, Dokkyo Medical University Saitama Medical Center, Koshigaya, Japan. The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

Upregulated expression of lamin B receptor increases cell proliferation and suppresses genomic instability: implications for cellular immortalization.

Mammalian somatic cells undergo terminal proliferation arrest after a limited number of cell divisions, a phenomenon termed cellular senescence. However, cells acquire the ability to proliferate infinitely (cellular immortalization) through multiple genetic alterations. Inactivation of tumor suppressor genes such as p53, RB and p16 is important for cellular immortalization, although additional molecular alterations are required for cellular immortalization to occur. Here, we aimed to gain insights into these molecular alterations. Given that cellular immortalization is the escape of cells from cellular senescence, genes that regulate cellular senescence are likely to be involved in cellular immortalization. Because senescent cells show altered heterochromatin organization, we investigated the implications of lamin A/C, lamin B1 and lamin B receptor (LBR), which regulate heterochromatin organization, in cellular immortalization. We employed human immortalized cell lines, KMST-6 and SUSM-1, and found that expression of LBR was upregulated upon cellular immortalization and downregulated upon cellular senescence. In addition, knockdown of LBR induced cellular senescence with altered chromatin configuration. Additionally, enforced expression of LBR increased cell proliferation likely through suppression of genome instability in human primary fibroblasts that expressed the simian virus 40 large T antigen (TAg), which inactivates p53 and RB. Furthermore, expression of TAg or knockdown of p53 led to upregulated LBR expression. These observations suggested that expression of LBR might be upregulated to suppress genome instability in TAg-expressing cells, and, consequently, its upregulated expression assisted the proliferation of TAg-expressing cells (i.e. p53/RB-defective cells). Our findings suggest a crucial role for LBR in the process of cellular immortalization.

Development of sexual dimorphism of skeletal muscles through the adrenal cortex, caused by androgen-induced global gene suppression.

The zona fasciculata (zF) in the adrenal cortex contributes to multiple physiological actions through glucocorticoid synthesis. The size, proliferation, and glucocorticoid synthesis characteristics are all female biased, and sexual dimorphism is established by androgen. In this study, transcriptomes were obtained to unveil the sex differentiation mechanism. Interestingly, both the amount of mRNA and the expressions of nearly all genes were higher in females. The expression of Nr5a1, which is essential for steroidogenic cell differentiation, was also female biased. Whole-genome studies demonstrated that NR5A1 regulates nearly all gene expression directly or indirectly. This suggests that androgen-induced global gene suppression is potentially mediated by NR5A1. Using Nr5a1 heterozygous mice, whose adrenal cortex is smaller than the wild type, we demonstrated that the size of skeletal muscles is possibly regulated by glucocorticoid synthesized by zF. Taken together, considering the ubiquitous presence of glucocorticoid receptors, our findings provide a pathway for sex differentiation through glucocorticoid synthesis.

Biallelic non-productive enhancer-promoter interactions precede imprinted expression of Kcnk9 during mouse neural commitment.

It is only partially understood how constitutive allelic methylation at imprinting control regions (ICRs) interacts with other regulation levels to drive timely parental allele-specific expression along large imprinted domains. The Peg13-Kcnk9 domain is an imprinted domain with important brain functions. To gain insights into its regulation during neural commitment, we performed an integrative analysis of its allele-specific epigenetic, transcriptomic, and cis-spatial organization using a mouse stem cell-based corticogenesis model that recapitulates the control of imprinted gene expression during neurodevelopment. We found that, despite an allelic higher-order chromatin structure associated with the paternally CTCF-bound Peg13 ICR, enhancer-Kcnk9 promoter contacts occurred on both alleles, although they were productive only on the maternal allele. This observation challenges the canonical model in which CTCF binding isolates the enhancer and its target gene on either side and suggests a more nuanced role for allelic CTCF binding at some ICRs.

Loss of NSD2 causes dysregulation of synaptic genes and altered H3K36 dimethylation in mice.

Background: Epigenetic disruptions have been implicated in neurodevelopmental disorders. NSD2 is associated with developmental delay/intellectual disability; however, its role in brain development and function remains unclear. Methods: We performed transcriptomic and epigenetic analyses using Nsd2 knockout mice to better understand the role of NSD2 in the brain. Results and discussion: Transcriptomic analysis revealed that the loss of NSD2 caused dysregulation of genes related to synaptic transmission and formation. By analyzing changes in H3 lysine 36 dimethylation (H3K36me2), NSD2-mediated H3K36me2 mainly marked quiescent state regions and the redistribution of H3K36me2 occurred at transcribed genes and enhancers. By integrating transcriptomic and epigenetic data, we observed that H3K36me2 changes in a subset of dysregulated genes related to synaptic transmission and formation. These results suggest that NSD2 is involved in the regulation of genes important for neural function through H3K36me2. Our findings provide insights into the role of NSD2 and improve our understanding of epigenetic regulation in the brain.

Variations in gender identity and sexual orientation of university students.

Background: Previous studies have shown that a small percentage of people in the general population have atypical gender identity and/or sexual orientation. Aim: This study aimed to explore variations in gender identity and sexual orientation in university students and determine genetic factors associated with these variations. Methods: Deviations from complete gender congruence and exclusive heterosexual orientation in 736 Japanese university students were quantitatively assessed with self-assessment questionnaires. Next, we conducted genetic tests for 80 participants who showed relatively low gender identity scores and/or atypical sexual orientation. These genetic tests consisted of repeat number analysis of the androgen receptor gene (AR) and a SKAT-O: an optimal unified sequence kernel association test, which is an exome-based rare variant association study. The results of the genetic tests were compared with the Japanese reference data and the results of our 637 control samples. Outcomes: We calculated the gender identity and sexual orientation scores of all participants and analyzed the molecular data of 80 selected participants. Results: The gender identity scores of 736 participants were broadly distributed: only ~15% of natal males and ~5% of natal females had the maximum score that corresponds to complete gender congruence. The sexual orientation scores also varied: ~80% of natal males and ~60% of natal females showed exclusive heterosexual orientation. We found no association between gender characteristics and AR repeat numbers. The SKAT-O showed that rare damaging variants of TDRP and 3 other genes were more common in the 80 participants than in the control group. Clinical Implications: Our data support the view that gender is a phenotypic continuum rather than a binary trait. Strength and Limitations: This study quantitatively assessed the gender characteristics of a large cohort of university students. Moreover, we conducted systematic screening for genetic factors associated with gender variations. The weaknesses of the study were the limited analytic power of the questionnaires, the relatively small sample for molecular analyses, and incomplete clinical information and relatively advanced ages of the control group. Conclusion: This study revealed significant variations in gender identity and sexual orientation in university students, which may be partly associated with variants in TDRP or other genes.

Physical Properties of Ultrafine Bubbles Generated Using a Generator System.

BACKGROUND/AIM: Ultrafine bubbles (UFBs) have been extensively researched owing to their promising physical and biological properties. However, determining the lifespan or ideal concentration of UFBs for various biological events is challenging. This study aimed to determine the maximum concentration and longest lifespan of UFBs and to verify the validity of UFBs for assessing cell properties. MATERIALS AND METHODS: A generator system (HMB-H0150+P001, TOSSLEC Corporation Limited, Kyoto, Japan) generated UFBs using various gases. The size and concentration of UFBs in ultrapure water and cell culture medium were measured through a nanoparticle tracking analysis method. RESULTS: The UFB concentration increased when the generator operated in a time dependent manner. The mean size of UFBs was approximately 120 nm. In the UFB lifespan, the concentration decreased by approximately 30% within the first two weeks of generation and was stable for up to 6 months. The UFB size increased by approximately 20% within the first two weeks of generation and demonstrated minor changes until the 6th month. The number of cells differed significantly with various concentrations of nitrogen gas UFBs. CONCLUSION: The generator system can generate UFBs with multiple concentrations within a suitable temperature. Consequently, the solution containing UFBs could be widely acceptable in cell culture systems.

Myelodysplasia after clonal hematopoiesis with APOBEC3-mediated CYBB inactivation in retroviral gene therapy for X-CGD.

Stem cell gene therapy using the MFGS-gp91phox retroviral vector was performed on a 27-year-old patient with X-linked chronic granulomatous disease (X-CGD) in 2014. The patient's refractory infections were resolved, whereas the oxidase-positive neutrophils disappeared within 6 months. Thirty-two months after gene therapy, the patient developed myelodysplastic syndrome (MDS), and vector integration into the MECOM locus was identified in blast cells. The vector integration into MECOM was detectable in most myeloid cells at 12 months after gene therapy. However, the patient exhibited normal hematopoiesis until the onset of MDS, suggesting that MECOM transactivation contributed to clonal hematopoiesis, and the blast transformation likely arose after the acquisition of additional genetic lesions. In whole-genome sequencing, the biallelic loss of the WT1 tumor suppressor gene, which occurred immediately before tumorigenesis, was identified as a potential candidate genetic alteration. The provirus CYBB cDNA in the blasts contained 108 G-to-A mutations exclusively in the coding strand, suggesting the occurrence of APOBEC3-mediated hypermutations during the transduction of CD34-positive cells. A hypermutation-mediated loss of oxidase activity may have facilitated the survival and proliferation of the clone with MECOM transactivation. Our data provide valuable insights into the complex mechanisms underlying the development of leukemia in X-CGD gene therapy.

Gene expression signatures associated with chronic endometritis revealed by RNA sequencing.

Introduction: Chronic endometritis (CE) is a persistent inflammatory condition of the endometrium characterized by the infiltration of plasma cells in the endometrial stroma. CD138 immunohistochemistry is considered to improve the CE diagnosis rate. Methods: Using the number of CD138-positive cells equal or greater than five as a diagnostic criterion for CE, we identified 24 CE and 33 non-CE cases among women with infertility. We conducted RNA-sequencing analysis for these 57 cases in total as an attempt to elucidate the molecular pathogenesis of CE and to search for new biomarkers for CE. Results and Discussion: By comparing CE and non-CE groups, we identified 20 genes upregulated in the endometria of CE patients, including 12 immunoglobulin-related genes and eight non-immunoglobulin genes as differentially expressed genes. The eight genes were MUC5AC, LTF, CAPN9, MESP1, ACSM1, TVP23A, ALOX15, and MZB1. By analyzing samples in the proliferative and secretory phases of the menstrual cycle separately, we also identified four additional non-immunoglobulin genes upregulated in CE endometria: CCDC13 by comparing the samples in the proliferative phase, and OVGP1, MTUS2, and CLIC6 by comparing the samples in the secretory phase. Although the genes upregulated in CE may serve as novel diagnostic markers of CE, many of them were upregulated only in a limited number of CE cases showing an extremely high number of CD138-positive cells near or over one hundred. Exceptionally, TVP23A was upregulated in the majority of CE cases regardless of the number of CD138-positive cells. The upregulation of TVP23A in the endometria of CE cases may reflect the pathophysiology of a cell-type or cell-types intrinsic to the endometrium rather than the accumulation of plasma cells. Our data, consisting of clinical and transcriptomic information for CE and non-CE cases, helped us identify gene expression signatures associated with CE.

Rare sequence variants associated with the risk of non-syndromic biliary atresia.

AIM: The etiology of non-syndromic biliary atresia (BA) remains largely unknown. In this study, we performed genome-wide screening of genes associated with the risk of non-syndromic BA. METHODS: We analyzed exome data of 15 Japanese patients with non-syndromic BA and 509 control individuals using an optimal sequence kernel association test (SKAT-O), a gene-based association study optimized for small-number subjects. Furthermore, we examined the frequencies of known BA-related single-nucleotide polymorphisms in the BA and control groups. RESULTS: SKAT-O showed that rare damaging variants of MFHAS1, a ubiquitously expressed gene encoding a Toll-like receptor-associated protein, were more common in the BA group than in the control group (Bonferroni corrected p-value = 0.0097). Specifically, p.Val106Gly and p.Arg556Cys significantly accumulated in the patient group. These variants resided within functionally important domains. SKAT-O excluded the presence of other genes significantly associated with the disease risk. Of 60 known BA-associated single-nucleotide polymorphisms, only eight were identified in the BA group. In particular, p.Ile3421Met of MYO15A and p.Ala421Thr of THOC2 were more common in the BA group than in the control group. However, the significance of these two variants is questionable, because MYO15A has been linked to deafness, but not to BA, and the p.Ala421Thr of THOC2 represents a relatively common single-nucleotide polymorphism in Asia. CONCLUSIONS: The results of this study indicate that rare damaging variants in MFHAS1 may constitute a risk factor for non-syndromic BA, whereas the contribution of other monogenic variants to the disease predisposition is limited.

Inhibitory Effect of Pyra-Metho-Carnil on Cancer Spheroid Growth Through Decrease in Glycolysis-associated Molecules.

BACKGROUND/AIM: Pyra-Metho-Carnil (PMC) has been identified as a novel candidate compound for treating numerous malignancies; however, its mechanism of action remains unknown. In this study, we conducted RNA-sequencing (RNA-seq) analyses to elucidate the mechanism of PMC against human colorectal cancer cells harboring mutant KRAS (mtKRAS). MATERIALS AND METHODS: RNA-seq analyses of the HKe3-wild-type KRAS and HKe3-mtKRAS spheroids treated with DMSO or PMC for 6 days were performed. RESULTS: RNA-seq data suggested that PMC treatment suppresses the aerobic glycolysis pathway in HKe3-mtKRAS spheroids through the down-regulation of the HIF1 pathway. Indeed, treatment with PMC markedly suppresses the absorption of glucose by spheroids and the secretion of lactate from them. CONCLUSION: PMC suppresses growth of cancer spheroid through down-regulation of cancer-specific glucose metabolism.

A capture methyl-seq protocol with improved efficiency and cost-effectiveness using pre-pooling and enzymatic conversion.

OBJECTIVE: The opportunities for sequencing-based methylome analysis of clinical samples are increasing. To reduce its cost and the amount of genomic DNA required for library preparation, we aimed to establish a capture methyl-seq protocol, which adopts pre-pooling of multiple libraries before hybridization capture and TET2/APOBEC-mediated conversion of unmethylated cytosine to thymine. RESULTS: We compared a publicly available dataset generated by the standard Agilent protocol of SureSelect XT Human Methyl-Seq Kit and our dataset obtained by our modified protocol, EMCap, that adopted sample pre-pooling and enzymatic conversion. We confirmed that the quality of DNA methylation data was comparable between the two datasets. As our protocol, EMCap, is more cost-effective and reduces the amount of input genomic DNA, it would serve as a better choice for clinical methylome sequencing.

Peripheral immune system modulates Purkinje cell degeneration in Niemann-Pick disease type C1.

Niemann-Pick disease type C1 (NPC1) is a fatal lysosomal storage disorder characterized by progressive neuronal degeneration. Its key pathogenic events remain largely unknown. We have, herein, found that neonatal BM-derived cell transplantation can ameliorate Purkinje cell degeneration in NPC1 mice. We subsequently addressed the impact of the peripheral immune system on the neuropathogenesis observed in NPC1 mice. The depletion of mature lymphocytes promoted NPC1 phenotypes, thereby suggesting a neuroprotective effect of lymphocytes. Moreover, the peripheral infusion of CD4-positive cells (specifically, of regulatory T cells) from normal healthy donor ameliorated the cerebellar ataxic phenotype and enhanced the survival of Purkinje cells. Conversely, the depletion of regulatory T cells enhanced the onset of the neurological phenotype. On the other hand, circulating inflammatory monocytes were found to be involved in the progression of Purkinje cell degeneration, whereas the depletion of resident microglia had little effect. Our findings reveal a novel role of the adaptive and the innate immune systems in NPC1 neuropathology.

Identification of a KDM6A somatic mutation responsible for Kabuki syndrome by excluding a conflicting KMT2D germline variant through episignature analysis.

Kabuki syndrome (KS) is a congenital disorder caused by mutations in either KMT2D on chromosome 12 or KDM6A on chromosome X, encoding a lysine methyltransferase and a lysine demethylase, respectively. A 9-year-4-month-old male patient with a normal karyotype presented with KS and autism spectrum disorder. Genetic testing for KS was conducted by Sanger sequencing and episignature analysis using DNA methylation array data. The patient had a mosaic stop-gain variant in KDM6A and a heterozygous missense variant (rs201078160) in KMT2D. The KDM6A variant is expected to be deleterious. The KMT2D variant pathogenicity has been inconsistently reported in the ClinVar database. Using biobanking resources, we identified two heterozygous individuals possessing the rs201078160 variant. In a subsequent episignature analysis, the KS patient showed the KS episignature, but two control individuals with the rs201078160 variant did not. Our results indicate that the mosaic stop-gained variant in KDM6A, but not the rs201078160 variant in KMT2D, is responsible for the KS phenotype in the patient. This study further demonstrated the utility of DNA methylation information in diagnosing rare genetic diseases and emphasized the importance of a reference dataset containing both genotype and DNA methylation information.

Genome-wide association study of preterm birth and gestational age in a Japanese population.

Preterm birth (PTB), defined as the birth of a baby at <37 weeks of gestation, is known to be the main cause of neonatal morbidity and mortality. Here, we report genetic associations between preterm birth and gestational age in a Japanese population. We conducted a genome-wide association study (GWAS) of 384 cases who delivered prematurely and 644 controls and considered gestational age as a quantitative trait in 1028 Japanese women. Unfortunately, we were unable to identify any significant variants associated with PTB or gestational age using the current sample. We also examined genetic associations previously reported in European populations and identified no associations, even with the genome-wide subthreshold (p value < 10-6). This data report aims to provide summary statistics of current GWASs on PTB in a Japanese population for future meta-analyses of genetics and PTB with larger sample sizes.

Prevalence of pathogenic variants in cancer-predisposing genes in second cancer after childhood solid cancers.

BACKGROUND: Second malignant neoplasms (SMNs) are one of the most severe late complications after pediatric cancer treatment. However, the effect of genetic variation on SMNs remains unclear. In this study, we revealed germline genetic factors that contribute to the development of SMNs after treatment of pediatric solid tumors. METHODS: We performed whole-exome sequencing in 14 pediatric patients with SMNs, including three brain tumors. RESULTS: Our analysis revealed that five of 14 (35.7%) patients had pathogenic germline variants in cancer-predisposing genes (CPGs), which was significantly higher than in the control cohort (p < 0.01). The identified genes with variants were TP53 (n = 2), DICER1 (n = 1), PMS2 (n = 1), and PTCH1 (n = 1). In terms of the type of subsequent cancer, leukemia and multiple episodes of SMN had an exceptionally high rate of CPG pathogenic variants. None of the patients with germline variants had a family history of SMN development. Mutational signature analysis showed that platinum drugs contributed to the development of SMN in three cases, which suggests the role of platinum agents in SMN development. CONCLUSIONS: We highlight that overlapping effects of genetic background and primary cancer treatment contribute to the development of second cancers after treatment of pediatric solid tumors. A comprehensive analysis of germline and tumor samples may be useful to predict the risk of secondary cancers.

Chromosomal microdeletion leading to pituitary gigantism through hormone-gene overexpression.

Pituitary gigantism is a rare endocrinopathy characterized by tall stature due to growth hormone (GH) hypersecretion. This condition is generally linked to a genetic predisposition to tumors that produce GH or GH-releasing hormone (GHRH). Here, we report a Japanese woman who exhibited prominent body growth from infancy to reach an adult height of 197.4 cm (+7.4 standard deviation). Her blood GH levels were markedly elevated. She carried no pathogenic variants in known growth-controlling genes but had a hitherto unreported 752 kb heterozygous deletion at 20q11.23. The microdeletion was located 8.9 kb upstream of GHRH and encompassed exons 2-9 of a ubiquitously expressed gene TTI1 together with 12 other genes, pseudogenes and non-coding RNAs. Transcript analyses of the patient's leukocytes showed that the microdeletion produced chimeric mRNAs consisting of exon 1 of TTI1 and all coding exons of GHRH. In silico analysis detected promoter-associated genomic features around TTI1 exon 1. Genome-edited mice carrying the same microdeletion recapitulated accelerated body growth from a few weeks after birth. The mutant mice developed pituitary hyperplasia and exhibited ectopic Ghrh expression in all tissues examined. Thus, the extreme phenotype of pituitary gigantism in the patient likely reflects GHRH overexpression driven by an acquired promoter. The results of this study indicate that germline submicroscopic deletions have the potential to cause conspicuous developmental abnormalities due to gene overexpression. Furthermore, this study provides evidence that constitutive expression of a hormone-encoding gene can result in congenital disease.

Two sporadic cases of childhood-onset Hailey-Hailey disease with superimposed mosaicism.

A prenatal second-hit genetic change that occurs on the wild-type allele in an embryo with a congenital pathogenic variant allele results in mosaicism of monoallelic and biallelic defect of the gene, which is called superimposed mosaicism. Superimposed mosaicism of Hailey-Hailey disease (HHD) has been demonstrated in one familial case. Here, we report two unrelated HHD cases with superimposed mosaicism: a congenital monoallelic pathogenic variant of ATP2C1, followed by a postzygotic copy-neutral loss of heterozygosity. Uniquely, neither patient had a family history of HHD at the time of presentation. In the first case, the congenital pathogenic variant had occurred de novo. In the second case, the father had the pathogenic variant but had not yet developed skin symptoms. Our cases showed that superimposed mosaicism in HHD can lack a family history and that genetic analysis is crucial to classify the type of mosaicism and evaluate the risk of familial occurrence.