Genetic evaluation using next-generation sequencing of children with short stature: a single tertiary-center experience.

PURPOSE: We used next-generation sequencing (NGS) to investigate the genetic causes of suspected genetic short stature in 37 patients, and we describe their phenotypes and various genetic spectra. METHODS: We reviewed the medical records of 50 patients who underwent genetic testing using NGS for suspected genetic short stature from June 2019 to December 2022. Patients with short stature caused by nongenetic factors or common chromosomal abnormalities were excluded. Thirty-seven patients from 35 families were enrolled in this study. We administered one of three genetic tests (2 targeted panel tests or whole exome sequencing) to patients according to their phenotypes. RESULTS: Clinical and molecular diagnoses were confirmed in 15 of the 37 patients, for an overall diagnostic yield of 40.5%. Fifteen pathogenic/likely pathogenic variants were identified in 13 genes (ACAN, ANKRD11, ARID1B, CEP152, COL10A1, COL1A2, EXT1, FGFR3, NIPBL, NRAS, PTPN11, SHOX, SLC16A2). The diagnostic rate was highest in patients who were small for their gestational age (7 of 11, 63.6%). CONCLUSION: Genetic evaluation using NGS can be helpful in patients with suspected genetic short stature who have clinical and genetic heterogeneity. Further studies are needed to develop patient selection algorithms and panels containing growth-related genes.

Novel In-Frame Deletion CNOT3 Variant in a Family With Intellectual Developmental Disorder With Speech Delay and Dysmorphic Facies.

Objectives: Intellectual developmental disorder with speech delay, autism, and dysmorphic facies (IDDSADF) is caused by heterozygous CNOT3 (MIM# 604910) variants on chromosome 19q13. This study aimed to identify and describe the clinical features of a Korean family with maternally inherited speech delay and intellectual and developmental disability to elucidate the underlying genetic mechanism. Methods: We conducted whole-exome sequencing and confirmatory Sanger sequencing on the proband, the mother, and unaffected grandparents with wild-type genotypes. Results: The phenotypes of the mother and 2 daughters presented muscular hypotonia, global developmental delay, speech delay, intellectual disability, macrocephaly, facial dysmorphic features, and focal corpus callosum hypoplasia. Whole-exome sequencing identified a novel in-frame deletion, c.2017_2019del (p.Phe673del) in CNOT3, located in the C-terminal negative on the TATA-less-box domain. Discussion: This report presents a new possible mechanism underlying IDDSADF caused by CNOT3 variants-an in-frame deletion. The findings enhance our understanding of early-life neurodevelopment and the genotype-phenotype relationships of IDDSADF caused by CNOT3 variants. In addition, this report could assist in early diagnosis and facilitate genetic counseling.

Genetic Profile and Associated Characteristics of 150 Korean Patients with Retinitis Pigmentosa.

PURPOSE: Retinitis pigmentosa (RP) shows great diversity between genotypes and phenotypes, and it is important to identify the causative genes. This study aimed to analyze the molecular profiles, associated ocular characteristics, and progression of RP in Korean patients. METHODS: All the genetic variants in patients with RP, identified using targeted next-generation sequencing (NGS) with a panel of 88 RP-related genes between November 2018 and November 2019, were retrospectively reviewed. All the patients underwent comprehensive ophthalmological evaluations, and their clinical and family histories were recorded. The best-corrected visual acuity (BCVA) deterioration and photoreceptor disruption progression rates were determined based on the major causative mutational genes using nonlinear mixed models, and the differences among them were investigated using the interaction effect. RESULTS: Among the 144 probands, 82 variants in 24 causative genes were identified in 77 families (53.5%). Most of the RP cases were associated with autosomal recessive variants (N = 64 (44.4%)), followed by autosomal dominant (N = 10 (6.9%)) and X-linked variants (N = 3 (2.1%)). The four most frequently affected genes were EYS (N = 15 (10.4%)), USH2A (N = 12 (8.3%)), PDE6B (N = 9 (6.3%)), and RP1 (N = 8 (5.6%)). Epiretinal membranes and cystoid macular edema were frequently noted in the patients with USH2A (75.0%) and PDE6B (50.0%) variants, respectively. During the follow-up period, the BCVA and photoreceptor disruption changes were significantly different among the patients carrying the four common causative genes (P=0.014 and 0.034, resp.). Patients with PDE6B variants showed faster BCVA changes (0.2 LogMAR/10 years), and those with USH2A variants showed the fastest ellipsoid zone disruptions (-170.4 µm/year). CONCLUSION: In conclusion, our genetic analysis using targeted NGS provides information about the prevalence of RP-associated mutations in Korean patients. Delineating clinical characteristics according to genetic variations may help clinicians identify subtype features and predict the clinical course of RP.

Clinical characteristics and disease progression of retinitis pigmentosa associated with PDE6B mutations in Korean patients.

Due to the genotype-phenotype heterogeneity in retinitis pigmentosa (RP), molecular diagnoses and prediction of disease progression is difficult. This study aimed to report ocular and genetic data from Korean patients with PDE6B-associated RP (PDE6B-RP), and establish genotype-phenotype correlations to predict the clinical course. We retrospectively reviewed targeted next-generation sequencing or whole exome sequencing data for 305 patients with RP, and identified PDE6B-RP in 15 patients (median age, 40.0 years). Amongst these patients, ten previously reported PDE6B variants (c.1280G > A, c.1488del, c.1547T > C, c.1604T > A, c.1669C > T, c.1712C > T, c.2395C > T, c.2492C > T, c.592G > A, and c.815G > A) and one novel variant (c.712del) were identified. Thirteen patients (86.7%) experienced night blindness as the first symptom at a median age of 10.0 years. Median age at diagnosis was 21.0 years and median visual acuity (VA) was 0.20 LogMAR at the time of genetic analysis. Nonlinear mixed models were developed and analysis revealed that VA exponentially decreased over time, while optical coherence tomography parameters linearly decreased, and this was related with visual field constriction. A high proportion of patients with the c.1669C > T variant (7/9, 77.8%) had cystoid macular edema; despite this, patients with this variant did not show a higher rate of functional or structural progression. This study will help clinicians predict functional and structural progression in patients with PDE6B-RP.

The first familial case of inherited intellectual developmental disorder with dysmorphic facies and behavioral abnormalities (IDDFBA) with a novel FBXO11 variant.

Intellectual developmental disorder with dysmorphic facies and behavioral abnormalities (IDDFBA) caused by germline de novo variants in FBXO11 was recently recognized as a novel intellectual disability (ID) syndrome through reverse phenotyping after whole-exome sequencing (WES). Fewer than 50 disease-causing de novo FBXO11 variants in IDDFBA are reported thus far. Here, we present the first report of a family showing autosomal dominantly inherited IDDFBA, harboring a novel heterozygous variant in FBXO11 (c.2401_2405dup;p. Gly803Leufs*6) identified by WES. In this family, the mother and two daughters showed mild ID and mild facial dysmorphism. This finding is expected to increase our understanding of the genotype-phenotype of IDDFBA and to facilitate genetic counseling for the disorder caused by FBXO11.

Differences in PD-1 expression on CD8+ T-cells in chronic myeloid leukemia patients according to disease phase and TKI medication.

Malignant cells can increase in number using immune escape mechanisms such as immune checkpoints. In this study, we evaluated the expression of an immune checkpoint programmed death 1 (PD-1) on T-cell subsets in chronic myeloid leukemia (CML). We obtained bone marrow aspirate samples from CML patients and from individuals without evidence of hematologic malignancies (controls). PD-1 expression on T-cell subsets was measured using flow cytometric analysis. PD-1 expression levels on CD8+ T-cells were significantly lower in complete hematologic response (CHR) than in controls, chronic phase, and blast phase (BP). In CML patients receiving imatinib and dasatinib, PD-1 expression levels on CD8+ T-cells were lower than that at diagnosis. PD-1 expression levels on CD8+ T-cells were positively correlated with quantitative levels of the BCR/ABL fusion gene. PD-1 expression levels on CD4+ T-cells were higher in BP than in CHR. PD-1 expression levels on CD4+ T-cells did not differ significantly according to different medications or quantitative BCR/ABL1 fusion gene levels. Low PD-1 expression on CD8+ T-cells might play a role in maintaining CHR in CML patients. Immune monitoring of PD-1 expression on CD8+ T-cells may predict the disease course. In cases of refractory disease or resistance to imatinib or dasatinib, the use of PD-1 inhibitors would be helpful.

POEMS Syndrome: Bone Marrow, Laboratory, and Clinical Findings in 24 Korean Patients.

POEMS syndrome is a rare paraneoplastic syndrome, which includes polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes due to plasma cell (PC) neoplasm. Diagnosis of this disease is challenging because of its rarity and complex clinical manifestations. We attempted to identify the key clinical features and characteristic bone marrow (BM) findings of POEMS syndrome, by reviewing the medical records and BM analyses of 24 Korean patients. Frequent clinical manifestations included polyneuropathy (100%), monoclonal gammopathy (100%), organomegaly (92%), extravascular volume overload (79%), and endocrinopathy (63%). The BM analyses revealed mild PC hyperplasia (median PCs: 5.5%) and frequent megakaryocytic hyperplasia (88%), megakaryocyte clusters (88%), and hyperlobation (100%). Flow cytometry of BM aspirates using CD138/CD38/CD45/CD19/CD56 showed normal (67%, 4/6) or neoplastic PC immunophenotypes (33%, 2/6). A diagnosis of POEMS syndrome must be considered when a patient suspected of having PC dyscrasia shows the above clinical presentation and BM findings.

Prognostic significance of recurrent additional chromosomal abnormalities in adult patients with Philadelphia chromosome-positive acute lymphoblastic leukemia.

In Philadelphia (Ph) chromosome-positive acute lymphoblastic leukemia (ALL), additional chromosomal abnormalities (ACAs) are frequently observed. We investigated the cytogenetic characteristics and prognostic significance of ACAs in Ph-positive ALL. We reviewed the clinical data and bone marrow cytogenetic findings of 122 adult Ph-positive ALL patients. The ACAs were examined for partial or whole chromosomal gains or losses, and structural aberrations. The overall survival (OS) and disease-free survival (DFS) of patients who received hematopoietic cell transplantation were compared between the isolated Ph group and ACA group. ACAs were present in 73.0% of all patients. The recurrent ACAs were extra Ph (24.7%), 9/9p loss (20.2%), and 7/7p loss (19.1%). Complex karyotype was found in 28.1% of patients in the ACA group. Younger patients (19-30 years) in the ACA group showed the highest frequency of extra Ph (54%) compared to other age groups. The OS in the ACA group was significantly shorter than in the isolated Ph group. The presence of an extra Ph chromosome or 9/9p loss was significantly associated with shorter OS and DFS, whereas 7/7p loss and complex karyotype were not associated with poorer prognosis. We suggest that subclassification of ACAs could be applied to prognostic investigation of Ph-positive ALL.

Co-colonization of vanA and vanB Enterococcus faecium of clonal complex 17 in a patient with bacteremia due to vanA E. faecium.

A 53-year-old Vietnamese man with liver cirrhosis was transferred from a Vietnamese hospital to our tertiary care hospital in Korea in order to undergo a liver transplantation. Bacteremia due to vanA Enterococcus faecium was diagnosed, and stool surveillance cultures for vancomycin-resistant enterococci (VRE) were positive for both vanA and vanB E. faecium. Pulsed-field gel electrophoresis analysis revealed that the 2 vanA VRE isolates from the blood and stool were clonal, but the vanB VRE was unrelated to the vanA VRE. vanA and vanB VRE were ST64 and ST18, single-allele variations of clonal complex 17, respectively. This is the first case report of vanA VRE bacteremia in a Vietnamese patient and demonstrates the reemergence of vanB VRE since a single outbreak occurred 15years ago in Korea. The reemergence of vanB VRE emphasizes the importance of VRE genotyping to prevent the spread of new VRE strains.

The first Korean case of disseminated mycetoma caused by Nocardia pseudobrasiliensis in a patient on long-term corticosteroid therapy for the treatment of microscopic polyangiitis.

Nocardia pseudobrasiliensis is predominantly associated with invasive infections in immunocompromised patients. We report a case of disseminated mycetoma caused by N. pseudobrasiliensis in a 57-yr-old woman with microscopic polyangiitis, who was treated for 3 months with corticosteroids. The same organism was isolated from mycetoma cultures on the patient's scalp, right arm, and right leg. The phenotypic characteristics of the isolate were consistent with both Nocardia brasiliensis and N. pseudobrasiliensis, i.e., catalase and urease positivity, hydrolysis of esculin, gelatin, casein, hypoxanthine, and tyrosine, but no hydrolysis of xanthine. The isolate was identified as N. pseudobrasiliensis based on 16S rRNA and hsp65 gene sequencing. The patient was treated for 5 days with intravenous ampicillin/sulbactam, at which time both the mycetomas and fever had subsided and discharged on amoxicillin/clavulanate. This case highlights a very rare presentation of mainly cutaneous mycetoma caused by N. pseudobrasiliensis. This is the first reported case of N. pseudobrasiliensis infection in Korea.

The role of neuronal nitric oxide synthase on hypobaric hypoxia-induced antinociception in writhing test.

It has been reported that hypobaric hypoxia exposure by high altitude is responsible for neuropsychological impairment. In the present study, we examined an effect of hypobaric hypoxia on the writhing test. The ICR mice were exposed in hypobaric chamber with several altitudes (5000, 10,000 or 20,000 ft) for 1 or 2 h, and then immediately injected intraperitoneally (i.p.) with 1% acetic acid for writhing test. Our results show that both 10,000 ft and 20,000 ft exposure induce antinociceptive effect in writhing test, but 5,000 ft does not. In addition, this antinociceptive effect was abolished by L-NAME (nitric oxide synthase inhibitor) pre-treated intraperitoneally, but not naloxone (non-specific opioid receptor antagonist). Furthermore, we examined that neuronal NOS immunoreactivities in the hypothalamus (paraventricular nucleus and arcuate nucleus) were increased by hypobaric hypoxic exposure (10,000ft). These results suggest that hypobaric hypoxic-induced antinociception may be associated with neuronal NOS IR in the hypothalamus.

Stretch-activated currents in cardiomyocytes isolated from rabbit pulmonary veins.

Evidence is growing of a relationship between atrial dilation and atrial fibrillation (AF), the most prevalent type of arrhythmia. Pulmonary veins, which are important ectopic foci for provoking AF, are of increasing interest in relation to the early development of AF. Here, using single cardiomyocytes isolated from rabbit pulmonary veins, we characterised the stretch-activated currents induced by swelling and axial mechanical stretching. Swelling induced both a stretch-activated nonselective cationic current (NSC) and a Cl(-) current. The swelling-induced Cl(-) current (I Cl,swell) was inhibited by DIDS, whereas the swelling-induced NSC (I NSC,swell) was inhibited by Gd3+. The cationic selectivity of the I NSC,swell was K+ >Cs+ >Na+ >Li+, whilst the PK/PNa, PCs/PNa, and PLi/PNa permeability ratios were 2.84, 1.86, and 0.85, respectively. Activation of the I NSC,swell was faster than that of the I Cl,swell. Given a high K+ concentration in the bath solution, the I NSC,swell showed limited amplitude (<-70 mV). Mechanical stretching induced an immediate Gd3+- and streptomycin-sensitive NSC (I NSC,stretch) that was permeable to Na+, K+, Cs+ and NMDG. Persistent stretching activated a DIDS-sensitive current (I Cl,stretch). The I NSC,stretch, but not the I NSC,swell, was completely blocked by 400 microM streptomycin; therefore, the two currents may not be associated with the same channel. In addition, the type of current induced may depend on the type of stretching. Thus, stretch-induced anionic and cationic currents are functionally present in the cardiomyocytes of the main pulmonary veins of rabbits, and they may have pathophysiological roles in the development of AF under stretched conditions.

Simulation of spontaneous action potentials of cardiomyocytes in pulmonary veins of rabbits.

Atrial fibrillation is the most prevalent arrhythmia, but the mechanisms by which it develops are not clear. Recently, over 90% of paroxysmal atrial fibrillation was found to be located inside the main pulmonary veins (PVs). We found that single cardiac myocytes isolated from the main PVs of rabbits generate spontaneous action potentials (SAP). We therefore assayed the electrical characteristics of these cardiomyocytes. Among the diverse ionic currents identified were INa, ICa,L, IK1, IKr, IKs, Ito, IKsus, Incx, Ipump, IKH and ICl,Ca. In contrast, IK1 was minimal, IKs could be detected only in the presence of 10 microM forskolin, and we were unable to detect If and ICa,T, the most important currents for pacemaking activity in sinoatrial node cells. To identify the main cause of SAP, we developed a model that can explain the electrical properties of these cardiomyocytes. After reconstructing the ionic currents based on experimental observations, we were able to use our model to successfully reconstruct the characteristics of the SAP of PV cardiomyocytes. The simulation showed that the major currents contributing to pacemaking depolarization were ICaL, IKr, a background current and Na+-K+ pump current. Deactivation kinetics of IKr was one of the major determinants of the rate of pacemaking depolarization. The steady state inactivation of Ito was shifted to the negative voltage and the activity of Ito was minimal in the range of the SAP. The major currents for the repolarization were IKr and Ipump. The amplitude of most currents in these cardiac myocytes was small and no currents did not exceed 30 pA during the SAP, indicating that slight activation of other inward or outward currents will have profound effects on the SAP. To our knowledge, this report is the first to show the simulation of SAP of PV cardiomyocytes. This model may help to study on the electrophysiological basis of paroxysmal atrial fibrillation originating from PVs.