Divergent variant patterns among 19 patients with Rubinstein-Taybi syndrome uncovered by comprehensive genetic analysis including whole genome sequencing.

Rubinstein-Taybi syndrome (RSTS) is characterized by dysmorphic facial features, broad thumbs, and intellectual disability. CREB-binding protein (CREBBP) or E1A-binding protein P300 (EP300) are causative genes. To elucidate the underlying genetic and genomic architecture related to the RSTS phenotype, we performed comprehensive genetic analysis targeting CREBBP and/or EP300 in 22 clinically diagnosed patients. During the 11-year study period, we used several analysis methods including high-resolution melting, array-based comparative genomic hybridization, panel-based exome sequencing, whole exome sequencing, and whole genome sequencing (WGS). We identified the causative variants in 19 patients (86.3%), but they were variable and complex, so we must combine multiple analysis methods. Notably, we found genetic alterations in the non-coding regions of two patients (10.5%, 2/19): scattered deletions including a partial 5'-untranslated region of CREBBP in one patient (all coding exons were intact), and a deep 229-bp intronic deletion in another patient, resulting in a splicing error. Furthermore, we identified rare clinical findings: two patients with an EP300 variant showed abnormal development of the neural tube, and one patient with a CREBBP variant had anorectal atresia with a cloaca. Our findings expand the allelic heterogeneity of RSTS, underscore the utility of comprehensive genetic analysis, and suggest that WGS may be a practical diagnostic strategy.

A Recurrent Variant in POLR1B, c.3007C>T; p.Arg1003Cys, Associated with Atresia of the External Canal and Microtia in Treacher Collins Syndrome Type 4.

Treacher Collins syndrome (TCS) is a heterogenous malformation syndrome characterized by a distinct facial appearance including downslanting palpebral fissures, malar hypoplasia, conductive hearing loss, and mandibular hypoplasia. Recently, a new causative gene, POLR1B, encoding DNA-directed RNA polymerase I subunit RPA2, was identified as a fourth type of TCS (TCS4). We describe another patient with TCS4 caused by a recurrent POLR1B variant, c.3007C>T; p.Arg1003Cys. Including our patient, all 4 patients with p.(Arg1003Cys) had atresia of the external auditory canal and microtia. All of the reported pathogenic variants in POLR1B were clustered at only 2 residues. Our patient highlights the genotype-phenotype correlation in TCS4 associated with POLR1B.

Update of the genotype and phenotype of KMT2D and KDM6A by genetic screening of 100 patients with clinically suspected Kabuki syndrome.

Kabuki syndrome is characterized by a variable degree of intellectual disability, characteristic facial features, and complications in various organs. Many variants have been identified in two causative genes, that is, lysine methyltransferase 2D (KMT2D) and lysine demethylase 6A (KDM6A). In this study, we present the results of genetic screening of 100 patients with a suspected diagnosis of Kabuki syndrome in our center from July 2010 to June 2018. We identified 76 variants (43 novel) in KMT2D and 4 variants (3 novel) in KDM6A as pathogenic or likely pathogenic. Rare variants included a deep splicing variant (c.14000-8C>G) confirmed by RNA sequencing and an 18% mosaicism level for a KMT2D mutation. We also characterized a case with a blended phenotype consisting of Kabuki syndrome, osteogenesis imperfecta, and 16p13.11 microdeletion. We summarized the clinical phenotypes of 44 patients including a patient who developed cervical cancer of unknown origin at 16 years of age. This study presents important details of patients with Kabuki syndrome including rare clinical cases and expands our genetic understanding of this syndrome, which will help clinicians and researchers better manage and understand patients with Kabuki syndrome they may encounter.

Diamond-Blackfan anemia caused by chromosome 1p22 deletion encompassing RPL5.

Diamond-Blackfan anemia (DBA) is an inherited anemia with multiple congenital malformations, and mutations in ribosomal protein genes have been identified as the underlying cause. We describe a female patient with mild DBA due to 1p22 deletion, encompassing the gene encoding 60S ribosomal protein L5 (RPL5). Considering previously reported cases together with our patient, we suggest that RPL5 haploinsufficiency might cause a less severe form of DBA than loss-of-function mutations.

Expression of RSOsPR10 in rice roots is antagonistically regulated by jasmonate/ethylene and salicylic acid via the activator OsERF87 and the repressor OsWRKY76, respectively.

Plant roots play important roles in absorbing water and nutrients, and in tolerance against environmental stresses. Previously, we identified a rice root-specific pathogenesis-related protein (RSOsPR10) induced by drought, salt, and wounding. RSOsPR10 expression is strongly induced by jasmonate (JA)/ethylene (ET), but suppressed by salicylic acid (SA). Here, we analyzed the promoter activity of RSOsPR10. Analyses of transgenic rice lines harboring different-length promoter::ß-glucuronidase (GUS) constructs showed that the 3-kb promoter region is indispensable for JA/ET induction, SA repression, and root-specific expression. In the JA-treated 3K-promoter::GUS line, GUS activity was mainly observed at lateral root primordia. Transient expression in roots using a dual luciferase (LUC) assay with different-length promoter::LUC constructs demonstrated that the novel transcription factor OsERF87 induced 3K-promoter::LUC expression through binding to GCC-cis elements. In contrast, the SA-inducible OsWRKY76 transcription factor strongly repressed the JA-inducible and OsERF87-dependent expression of RSOsPR10. RSOsPR10 was expressed at lower levels in OsWRKY76-overexpressing rice, but at higher levels in OsWRKY76-knockout rice, compared with wild type. These results show that two transcription factors, OsERF87 and OsWRKY76, antagonistically regulate RSOsPR10 expression through binding to the same promoter. This mechanism represents a fine-tuning system to sense the balance between JA/ET and SA signaling in plants under environmental stress.

De novo duplication of 17p13.1-p13.2 in a patient with intellectual disability and obesity.

17p13.1 Deletion encompassing TP53 has been described as a syndrome characterized by intellectual disability and dysmorphic features. Only one case with a 17p13.1 duplication encompassing TP53 has been reported in a patient with intellectual disability, seizures, obesity, and diabetes mellitus. Here, we present a patient with a 17p13.1 duplication who exhibited obesity and intellectual disability, similar to the previous report. The 9-year-old proposita was referred for the evaluation of intellectual disability and obesity. She also exhibited insulin resistance and liver dysfunction. She had wide palpebral fissures, upturned nostrils, a long mandible, short and slender fingers, and skin hyperpigmentation. Array comparative genomic hybridization (array CGH) detected a 3.2 Mb duplication of 17p13.1-p13.2 encompassing TP53, FXR2, NLGN2, and SLC2A4, which encodes the insulin-responsive glucose transporter 4 (GLUT4) associated with insulin-stimulated glucose uptake in adipocytes and muscle. We suggest that 17p13.1 duplication may represent a clinically recognizable condition characterized partially by a characteristic facial phenotype, developmental delay, and obesity.

Pure duplication of 19p13.3.

Chromosomal abnormalities involving 19p13.3 have rarely been described in the published literature. Here, we report on a girl with a pure terminal duplication of 6.1 Mb on 19p13.3, caused by an unbalanced translocation der(19)t(10;19)(qter;p13.3)dn. Her phenotype included severe psychomotor developmental delay, skeletal malformations, and a distinctive facial appearance, similar to that of a patient previously reported by Lybaek et al. [Lybaek et al. (2009); Eur J Hum Genet 17:904-910]. These results suggest that a duplication of >3 Mb at the terminus of 19p13.3 might represent a distinct chromosomal syndrome.

Expression analysis of a 17p terminal deletion, including YWHAE, but not PAFAH1B1, associated with normal brain structure on MRI in a young girl.

Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide (YWHAE), on chromosome 17p13.3, has been shown to play a crucial role in neuronal development. The deletion of YWHAE, but not platelet-activating factor acetylhydrolase, isoform 1b, subunit 1 (PAFAH1B1), underlies a newly recognized neurodevelopmental disorder, characterized by significant growth retardation, developmental delay/intellectual disability (DD/ID), distinctive facial appearance, and brain abnormalities. Here, we report on a girl with a terminal deletion of 17p13.3, including YWHAE but not PAFAH1B1, showing normal brain structure on MRI. She had mild developmental delay, a distinctive facial appearance, and severe growth retardation despite normal growth hormone levels, which was improved by growth hormone therapy. Expression analysis of YWHAE and PAFAH1B1 yielded results consistent with array CGH and FISH results. These results indicate that the dosage effect of YWHAE varies from severe to very mild structural brain abnormalities, and suggest that the expression of YWHAE is associated with a complex mechanism of neuronal development.

Spastic quadriplegia in Down syndrome with congenital duodenal stenosis/atresia.

Down syndrome is an autosomal chromosome disorder, characterized by intellectual disability and muscle hypotonia. Muscle hypotonia is observed from neonates to adulthood in Down syndrome patients, but muscle hypertonicity is extremely unusual in this syndrome. During a study period of nine years, we found three patients with severe spastic quadriplegia among 20 cases with Down syndrome and congenital duodenal stenosis/atresia (3/20). However, we could find no patient with spastic quadriplegia among 644 cases with Down syndrome without congenital duodenal stenosis/atresia during the same period (0/644, P < 0.05). Further, we did not find any cases with spastic quadriplegia among 17 patients with congenital duodenal stenosis/atresia without Down syndrome admitted during the same period to use as a control group (0/17, P < 0.05). Our results suggest that congenital duodenal stenosis/atresia is a potential risk factor for spastic quadriplegia in patients with Down syndrome. Long-term survival is improving, and the large majority of people with Down syndrome are expected to live well into adult life. Management and further study for the various problems, representing a low prevalence but serious and specific to patients with Down syndrome, are required to improve their quality of life.

RSOsPR10 expression in response to environmental stresses is regulated antagonistically by jasmonate/ethylene and salicylic acid signaling pathways in rice roots.

Plant roots play important roles not only in the absorption of water and nutrients, but also in stress tolerance. Previously, we identified RSOsPR10 as a root-specific pathogenesis-related (PR) protein induced by drought and salt treatments in rice. Transcripts and proteins of RSOsPR10 were strongly induced by jasmonate (JA) and the ethylene (ET) precursor 1-aminocyclopropane-1-carboxylic acid (ACC), while salicylic acid (SA) almost completely suppressed these inductions. Immunohistochemical analyses showed that RSOsPR10 strongly accumulated in cortex cells surrounding the vascular system of roots, and this accumulation was also suppressed when SA was applied simultaneously with stress or hormone treatments. In the JA-deficient mutant hebiba, RSOsPR10 expression was up-regulated by NaCl, wounding, drought and exogenous application of JA. This suggested the involvement of a signal transduction pathway that integrates JA and ET signals in plant defense responses. Expression of OsERF1, a transcription factor in the JA/ET pathway, was induced earlier than that of RSOsPR10 after salt, JA and ACC treatments. Simultaneous SA treatment strongly inhibited the induction of RSOsPR10 expression and, to a lesser extent, induction of OsERF1 expression. These results suggest that JA/ET and SA pathways function in the stress-responsive induction of RSOsPR10, and that OsERF1 may be one of the transcriptional factors in the JA/ET pathway.

Flow cytometric analysis of peripheral blood and tumor-infiltrating regulatory T cells in dogs with oral malignant melanoma.

It is well known that tumor-infiltrating lymphocytes (TILs) and peripheral blood lymphocytes (PBLs) from patients with advanced-stage cancer have a poor immune response. Regulatory T cells (Tregs), characterized by the expression of a cluster of differentiation 4 and intracellular FoxP3 markers, can inhibit antitumor immunoresponse. In the present study, the prevalence of Tregs in peripheral blood and tumor tissue from dogs with oral malignant melanoma was evaluated by triple-color flow cytometry. The percentage of Tregs in the peripheral blood of the dogs with malignancy was significantly increased compared with healthy control dogs, and the percentage of Tregs within tumors was significantly increased compared with Tregs in peripheral blood of dogs with oral malignant melanoma. This finding suggests that the presence of tumor cells induced either local proliferation or selective migration of Tregs to tumor-infiltrated sites. A better understanding of the underlying mechanisms of Treg regulation in patients with cancer may lead to an effective anticancer immunotherapy against canine malignant melanoma and possibly other tumors.

Relationship between regulatory and type 1 T cells in dogs with oral malignant melanoma.

Recent data suggest a decreased prevalence of IFN-gamma-producing T lymphocytes (Type 1 T cells) in tumor-bearing hosts. Moreover, it has been reported that Treg have a strong impact on the activation and proliferation of CD4 (+) and CD8 (+) lymphocytes; however, no previous reports have described the relationship between Treg and the progression of tumor, or Type 1 T cell populations in dogs with malignant tumor. In this study, the percentage of Treg, Th1, and Tc1 in the peripheral blood of dogs with oral malignant melanoma and healthy dogs was measured and compared. Although the percentages of Th1 and Tc1 in dogs with oral malignant melanoma were less than those in healthy dogs (Th1: P < 0.01, Tc1: P < 0.05), the percentage of Treg was increased (P < 0.01). A significant inverse correlation between the percentage of Tc1 and the clinical tumor stage (P < 0.01), and a significant correlation between that of Treg and the clinical tumor stage (P < 0.001) was found. Moreover, there was a significant inverse correlation between the percentages of Treg and Th1 (P < 0.05) or Tc1 (P < 0.001). In conclusion, the percentage of Treg increases with the tumor stage in the peripheral blood of dogs with oral malignant melanoma. In dogs, Treg appears to suppress Type 1 immunity, which may be responsible for anti-tumor responses.

Increase of regulatory T cells in the peripheral blood of dogs with metastatic tumors.

It is well known that lymphocytes from patients with advanced-stage cancer have impaired immune responsiveness and that type1 T lymphocyte subsets in tumor bearing hosts are suppressed. Treg have been reported to comprise a subgroup which inhibits T cell mediated immune responses. In the present study, the percentage of Treg, Th1 and Tc1 in the peripheral blood of tumor bearing dogs with or without metastases was evaluated. The percentages of Th1 and Tc1 in dogs with metastatic tumor were significantly less, and that of Treg was significantly greater, than those of dogs without metastatic tumor. The percentage of Treg showed an inverse correlation with that of Th1 and Tc1 in tumor bearing dogs. It was concluded that an increase in Treg in the peripheral blood of dogs with metastatic tumor may induce suppression of tumor surveillance by the Type1 immune response and lead to metastasis of tumor[0][0].[0].

Systemic candidiasis and mesenteric mast cell tumor with multiple metastases in a dog.

A 5-year-old female miniature dachshund presenting with persistent vomiting and diarrhea had two concurrent rare pathological conditions: systemic candidiasis and mesenteric mast cell tumor with multiorgan metastases. Neoplastic mast cells formed mass in the mesentery of the cecal-colonic region and were also found in the liver, spleen, kidneys, lungs, adrenal grands, ovaries, bone marrow and other tissues. The cells had intracytoplasmic granules with metachromasia and were immunohistochemically positive for c-kit and histamine. Granulomatous lesions with fungal organisms were present in the heart, lungs, kidneys, pancreas, subserosal and surrounding adipose tissue of the duodenum, thyroid glands and mesenteric mass, and phagocytosed organisms were detected in the liver and bone marrow. Bacteriologically and immunohistochemically, the fungi were consistent with Candida albicans.