C-terminal truncations in IQSEC2: implications for synaptic localization, guanine nucleotide exchange factor activity, and neurological manifestations.

IQSEC2 gene on chromosome Xq11.22 encodes a member of guanine nucleotide exchange factor (GEF) protein that is implicated in the activation of ADP-ribosylation factors (Arfs) at the postsynaptic density (PSD), and plays a crucial role in synaptic transmission and dendritic spine formation. Alterations in IQSEC2 have been linked to X-linked intellectual developmental disorders including epilepsy and behavioral abnormalities. Of interest, truncating variants at the C-terminus of IQSEC2 can cause severe phenotypes, akin to truncating variants located in other regions. Here, we present a 5-year-old boy with severe intellectual disability and progressive epilepsy. The individual carried a nonsense variant p.Q1227* in the last exon of the IQSEC2 gene that was supposed to escape nonsense-mediated mRNA decay, thereby leading to a translation of C-terminus truncated IQSEC2 protein with residual activity. The functional analyses showed that the GEF activity of IQSEC2 Q1227* was compromised, and that the IQSEC2 Q1227* lacked preferential synaptic localization due to the absence of functional domains for binding to scaffolding proteins in the PSD. The impaired GEF activity and disrupted synaptic localization of the mutant IQSEC2 protein could impact dendritic and spine development in neurons, potentially explaining the patient's severe neurological manifestations. Our findings indicate that C-terminal truncations in IQSEC2, previously not well-characterized, may have significant pathogenic implications.

The pathogenic role of the BRCA2 c.7847C>T (p.Ser2616Phe) variant in breast and ovarian cancer predisposition.

Substantial numbers of variants of unknown significance (VUSs) have been identified in BRCA1/2 through genetic testing, which poses a significant clinical challenge because the contribution of these VUSs to cancer predisposition has not yet been determined. Here, we report 10 Japanese patients from seven families with breast or ovarian cancer harboring the BRCA2 c.7847C>T (p.Ser2616Phe) variant that was interpreted as a VUS. This variant recurs only in families from Japan and has not been reported in the global general population databases. A Japanese patient with Fanconi anemia with compound heterozygous variants c.7847C>T (p.Ser2616Phe) and c.475+1G>A in BRCA2 was reported. In silico predictions and quantitative cosegregation analysis suggest a high probability of pathogenicity. The clinical features of the variant carriers were not specific to, but were consistent with, those of patients with hereditary breast and ovarian cancer. A validated functional assay, called the mixed-all-nominated-in-one-BRCA (MANO-B) method and the accurate BRCA companion diagnostic (ABCD) test, demonstrated the deleterious effects of the variant. Altogether, following the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines, this variant satisfied the "PS3," "PM2," "PM3," and "PP3" criteria. We thus conclude that the BRCA2 c.7847C>T (p.Ser2616Phe) variant is a "likely pathogenic" variant that is specifically observed in the Japanese population, leading to a breast and ovarian cancer predisposition.

A novel pathogenic variant of the FH gene in a family with hereditary leiomyomatosis and renal cell carcinoma.

Hereditary leiomyomatosis and renal cell carcinoma caused by loss-of-function germline variants of the FH gene can develop into aggressive renal cell carcinoma (RCC). We report the case of a 27-year-old man who died of RCC. Genetic testing revealed a novel pathogenic variant of FH, NM_000143.3:c.1013_1014del (p.Ile338Serfs*3), that was also identified in healthy siblings. Identification of genetic causes in the proband helped us to provide relatives with precise genetic counseling and appropriate surveillance programs.

Loss of imprinting of the human-specific imprinted gene ZNF597 causes prenatal growth retardation and dysmorphic features: implications for phenotypic overlap with Silver-Russell syndrome.

BACKGROUND: ZNF597, encoding a zinc-finger protein, is the human-specific maternally expressed imprinted gene located on 16p13.3. The parent-of-origin expression of ZNF597 is regulated by the ZNF597:TSS-DMR, of which only the paternal allele acquires methylation during postimplantation period. Overexpression of ZNF597 may contribute to some of the phenotypes associated with maternal uniparental disomy of chromosome 16 (UPD(16)mat), and some patients with UPD(16)mat presenting with Silver-Russell syndrome (SRS) phenotype have recently been reported. METHODS: A 6-year-old boy presented with prenatal growth restriction, macrocephaly at birth, forehead protrusion in infancy and clinodactyly of the fifth finger. Methylation, expression, microsatellite marker, single nucleotide polymorphism array and trio whole-exome sequencing analyses were conducted. RESULTS: Isolated hypomethylation of the ZNF597:TSS-DMR and subsequent loss of imprinting and overexpression of ZNF597 were confirmed in the patient. Epigenetic alterations, such as UPD including UPD(16)mat and other methylation defects, were excluded. Pathogenic sequence or copy number variants affecting his phenotypes were not identified, indicating that primary epimutation occurred postzygotically. CONCLUSION: We report the first case of isolated ZNF597 imprinting defect, showing phenotypic overlap with SRS despite not satisfying the clinical SRS criteria. A novel imprinting disorder entity involving the ZNF597 imprinted domain can be speculated.

The effect of fecal microbiota transplantation on psychiatric symptoms among patients with irritable bowel syndrome, functional diarrhea and functional constipation: An open-label observational study.

BACKGROUNDS: The intestinal microbiota is considered as a potential common underpinning pathophysiology of Functional Gastrointestinal Disorders (FGIDs) and psychiatric disorders such as depression and anxiety. Fecal Microbiota Transplantation (FMT) has been reported to have therapeutic effects on diseases related to dysbiosis, but few studies have evaluated its effect on psychiatric symptoms. METHODS: We followed 17 patients with either Irritable Bowel Syndrome (IBS), Functional Diarrhea (FDr) or Functional Constipation (FC) who underwent FMT for the treatment of gastrointestinal symptoms and observation of psychiatric symptoms. Changes in Hamilton Rating Scale for Depression (HAM-D) and subscale of sleep-related items, Hamilton Rating Scale for Anxiety (HAM-A) and Quick Inventory for Depressive Symptoms (QIDS) between baseline and 4 weeks after FMT, and relationship with the intestinal microbiota were measured. RESULTS: At baseline, 12 out of 17 patients were rated with HAM-D ≥ 8. Significant improvement in HAM-D total and sleep subscale score, HAM-A and QIDS were observed (p = 0.007, p = 0.007, p = 0.01, p = 0.007, respectively). Baseline Shannon index indicated that microbiota showed lower diversity in patients with HAM-D ≥ 8 compared to those of healthy donors and patients with HAM-D < 8. There was a significant correlation between baseline Shannon index and HAM-D score, and a correlation between Shannon index change and HAM-D improvement after FMT. LIMITATIONS: The small sample size with no control group. CONCLUSIONS: Our results suggest that depression and anxiety symptoms may be improved by FMT regardless of gastrointestinal symptom change in patients with IBS, FDr and FC, and the increase of microbiota diversity may help to improve patient's mood.

Fecal microbiota transplantation for recurrent Clostridium difficile infection in a patient with ulcerative colitis.

Fecal microbiota transplantation (FMT) has been reported as a safe and effective therapy in patients with refractory and recurrent Clostridium difficile infection (CDI). FMT has also been reported as a promising therapy in patients with ulcerative colitis (UC). Both, CDI and UC, are believed to be caused by dysbiosis, such as altered compositions or decreased diversity of the intestinal microbiota. This report describes a patient with UC in remission with a second recurrent episode of CDI, who was treated with FMT. A single FMT performed via colonoscopy completely resolved the patient's diarrhea and eradicated C. difficile bacteriologically without any severe complications. Molecular biological analysis of the patient's fecal microbiota showed that FMT could dramatically change the altered composition of intestinal microbiota and restore its diversity. Despite the restoration of the intestinal microbiota, FMT could not prevent a relapse of UC in this patient. However, it improved the intestinal symptoms of CDI and could prevent further recurrences of CDI.

Bifidobacterium-Rich Fecal Donor May Be a Positive Predictor for Successful Fecal Microbiota Transplantation in Patients with Irritable Bowel Syndrome.

BACKGROUND/AIMS: Dysbiosis is associated with various systemic disorders including irritable bowel syndrome (IBS). Fecal microbiota transplantation (FMT) might restore intestinal microbial balance. The study aimed to determine the safety and efficacy of FMT in IBS patients, as well as also positive predictors for FMT. METHODS: This was a single-arm, open-label study. Eligible patients were diagnosed based on Rome III Diagnostic Criteria. Fecal materials were administered to the patient via colonoscopy. The primary end point was a change in the Bristol stool form scale at 4 weeks after FMT. Recovery to types 3-4 was considered a clinical response. The secondary end point was a change in intestinal microbiota and psychological status using the Hamilton Rating Scale. RESULTS: Ten patients were enrolled. Six patients achieved a clinical response. The diversity of patients 4 weeks after FMT increased significantly compared with patients before FMT, and that of responding patients was significantly higher than non-responder patients. The abundance of Bifidobacterium in effective donors was significantly higher than in ineffective donors and patients. Psychological status of all patients was significantly improved after FMT. CONCLUSIONS: FMT for patients with IBS is safe, and relatively effective. Bifidobacterium-rich fecal donor may be a positive predictor for successful FMT. Key Summary: (1) Dysbiosis is associated with various gastrointestinal disorders including IBS. (2) FMT has potential to restore intestinal microbial balance. (3) We showed that FMT improved stool form and psychological status of IBS patients. (4) Bifidobacterium-rich donor efficiently induced symbiosis in IBS patients.

Single fecal microbiota transplantation failed to change intestinal microbiota and had limited effectiveness against ulcerative colitis in Japanese patients.

BACKGROUND/AIMS: Recent developments in analytical techniques including next-generation sequencing have clarified the correlation between intestinal microbiota and inflammatory bowel disease. Fecal microbiota transplantation (FMT) for patients with ulcerative colitis (UC) is proposed as a potential approach to resolving their dysbiosis; however, its safety and efficacy have not been confirmed. This single-arm, open-label, non-randomized study aimed to evaluate the safety and efficacy of FMT for Japanese patients with UC as the first registered clinical trial in Japan. METHODS: We enrolled 10 patients with active UC despite medical therapy. The donors were the patients' relatives and were carefully screened for infectious diseases. Fecal material was administered via colonoscopy, and the primary endpoint was the presence or absence of serious adverse events related to FMT. The secondary endpoint was a change in partial Mayo score at 12 weeks post-FMT. Scores ≤2 were considered a clinical response. Fecal samples were collected to follow changes in gut microbiota, while extracted complementary DNA were analyzed by a next-generation sequencer. We obtained written informed consent from all patients and donors. This study was approved by our Institutional Review Board and is registered in the University hospital Medical Information Network (UMIN) Clinical Trials Registry (UMIN 000012814). RESULTS: Five patients with moderate disease and five with severe disease were enrolled. No severe adverse effects were observed. One patient achieved clinical response; however, none of the patients' microbiota diversity recovered to the donor levels. CONCLUSIONS: The use of single FMT for UC was safe; however, we failed to show its clinical efficacy and potential to change the intestinal microbiota.

Phytochrome controls alternative splicing to mediate light responses in Arabidopsis.

Plants monitor the ambient light conditions using several informational photoreceptors, including red/far-red light absorbing phytochrome. Phytochrome is widely believed to regulate the transcription of light-responsive genes by modulating the activity of several transcription factors. Here we provide evidence that phytochrome significantly changes alternative splicing (AS) profiles at the genomic level in Arabidopsis, to approximately the same degree as it affects steady-state transcript levels. mRNA sequencing analysis revealed that 1,505 and 1,678 genes underwent changes in their AS and steady-state transcript level profiles, respectively, within 1 h of red light exposure in a phytochrome-dependent manner. Furthermore, we show that splicing factor genes were the main early targets of AS control by phytochrome, whereas transcription factor genes were the primary direct targets of phytochrome-mediated transcriptional regulation. We experimentally validated phytochrome-induced changes in the AS of genes that are involved in RNA splicing, phytochrome signaling, the circadian clock, and photosynthesis. Moreover, we show that phytochrome-induced AS changes of SPA1-RELATED 3, the negative regulator of light signaling, physiologically contributed to promoting photomorphogenesis. Finally, photophysiological experiments demonstrated that phytochrome transduces the signal from its photosensory domain to induce light-dependent AS alterations in the nucleus. Taking these data together, we show that phytochrome directly induces AS cascades in parallel with transcriptional cascades to mediate light responses in Arabidopsis.

Deletion of the RS domain of RRC1 impairs phytochrome B signaling in Arabidopsis.

Phytochrome B (phyB), a major photoreceptor in plants, interacts with transcription factors to regulate gene expression and induce various light responses. Recently, we identified an SR-like splicing factor, RRC1 (reduced red-light responses in cry1cry2 background 1), as a novel component of phyB signaling in Arabidopsis. RRC1 has a C-terminal arginine/serine-rich (RS) domain that is generally important for the regulation of alternative splicing. Whereas rrc1 hypomorphic mutant alleles produce truncated RRC1 proteins that lack the C-terminal region, including the RS domain, and exhibit splicing defects and reduced phyB signaling, the rrc1-4 null allele additionally displays pleiotropic developmental abnormalities with more severe splicing defects. Here, we show that transgenic Arabidopsis plants that express truncated RRC1 lacking the RS domain in the rrc1-4 null allele background exhibited the same phenotype as the hypomorphic alleles. Hence, we conclude that deletion of the RS domain of RRC1 reduces phyB signaling, probably due to aberrant regulation of alternative splicing of target genes.

The RS domain of Arabidopsis splicing factor RRC1 is required for phytochrome B signal transduction.

Plants monitor the light environment through informational photoreceptors that include phytochromes. In seedling de-etiolation, phytochrome B (phyB), which is the most important member of the phytochrome family, interacts with transcription factors to regulate gene expression and transduce light signals. In this study, we identified rrc1 (reduced red-light responses in cry1cry2 background 1), an Arabidopsis mutant that is impaired in phyB-mediated light responses. A genetic analysis demonstrated that RRC1 affected light signaling in a phyB-dependent manner. RRC1 encodes an ortholog of the human potential splicing factor SR140. The RRC1 polypeptide contains a C-terminal arginine/serine-rich (RS) domain that is important for the regulation of alternative splicing. Although the complete loss of RRC1 caused pleiotropic developmental abnormalities, the deletion of the RS domain specifically reduced phyB signaling and caused aberrant alternative splicing of several SR protein genes. Moreover, semi-quantitative RT-PCR analysis revealed that the alternative splicing patterns of some of the SR protein genes were altered in a red-light-dependent manner, and that these responses were reduced in both phyB and rrc1 mutants. These findings suggest that the regulation of alternative splicing by the RS domain of RRC1 plays an important role in phyB signal transduction.