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 novel method for isolating lymphatic endothelial cells from lymphatic malformations and detecting PIK3CA somatic mutation in these isolated cells.

PURPOSE: Tissue disaggregation and the cell sorting technique by surface markers has played an important role in isolating lymphatic endothelial cells (LECs) from lymphatic malformation (LM). However, this technique may have the drawback of impurities or result in isolation failure because it is dependent on surface marker expressions, the heterogeneity of which has been found in the lymphatic system. We developed a novel method for isolating LM-LECs without using whole tissue disaggregation. METHODS: Seven LM surgical specimens were collected from seven patients with LMs. LM-LECs were detached from the LM cyst wall by "lumen digestion" and irrigating the cystic cavity with trypsin, and maintained in culture. RESULTS: The cells formed a monolayer with a cobblestone-like appearance. Immunohistochemistry and quantitative RT-PCR of these cells revealed high expression of lymphatic-specific genes, confirming their identity as LM-LECs. The whole-exome sequencing and PIK3CA sequencing of these cells revealed somatic mutations in PIK3CA in all cases. CONCLUSIONS: We established a novel technique for isolating LM-LECs from LM tissue by "lumen digestion" without whole-tissue disaggregation. The limited incorporation of non-LM LECs in the isolate in our method could make it an important tool for investigating the heterogeneity of gene expression as well as mutations in LM-LECs.

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.

An efficient genetic test flow for multiple congenital anomalies and intellectual disability.

BACKGROUND: Genetic testing has enabled the diagnosis of multiple congenital anomalies and/or intellectual disabilities. However, because of the phenotypic variability in these disorders, selection of an appropriate genetic test can be difficult and complex. For clinical examination, particularly in clinical facilities, a simple and standardized system is needed. METHODS: We compared microarray comparative genomic hybridization and clinical exome sequencing with regard to diagnostic yield, cost, and time required to reach a definitive diagnosis. After first performing G-banding for 200 patients with multiple congenital anomalies and/or intellectual disability, as a subsequent genetic test, microarray and clinical exome sequencing were compared with regard to diagnostic yield, cost, and time required. RESULTS: There was no obvious difference in the diagnostic rate between the two methods; however, clinical exome sequencing was superior in terms of cost and time. In addition, clinical exome sequencing could sufficiently identify copy number variants, and even smaller copy number variants could be identified. CONCLUSIONS: Clinical exome sequencing should be implemented earlier as a genetic test for undiagnosed patients with multiple congenital anomalies and/or intellectual disabilities. Our results can be used to establish inspection methods in clinical facilities.

Running suture versus interrupted suture for vesicourethral anastomosis in retropubic radical prostatectomy: a randomized study.

OBJECTIVES: To determine the feasibility of vesicourethral anastomosis using running suture during retropubic radical prostatectomy and to compare the surgical outcomes of vesicourethral anastomosis using running suture with those of the standard interrupted suture technique. METHODS: A total of 60 patients undergoing radical prostatectomy from 2010 to 2012 at the Yamaguchi University Hospital, Japan were included in the present study, and were randomly assigned to vesicourethral anastomosis using running suture (n = 30 patients) or a standard interrupted suture technique group (n = 30 patients). Vesicourethral anastomosis using running suture was carried out with 12-bite sutures using 3-0 poliglecaprone. The primary end-point was the time to catheter removal. Patients' health-related quality of life was assessed using the Expanded Prostate Cancer Index Composite in 56 patients (28 patients in each group). RESULTS: No significant difference was found in the median suturing time between the two study groups (both 19 min, P = 0.449). The time to catheter removal was significantly better in the vesicourethral anastomosis using running suture group (hazard ratio 5.23, 95% confidence interval 1.73-17.65, P = 0.003). The pad-free rate was significantly higher in the vesicourethral anastomosis using running suture group at 1 month after surgery (20.7% vs 3.3%, P = 0.0463); however, there was no significant difference at 3 months and beyond. The Expanded Prostate Cancer Index Composite urinary and bowel summary scores at 1 month were significantly better in the vesicourethral anastomosis using running suture patients (both P < 0.01), though no significant difference was observed thereafter. A vesicourethral anastomosis stricture was noted in three patients (10%) in the standard interrupted suture technique group, and none in the vesicourethral anastomosis using running suture group. CONCLUSION: Running suture for vesicourethral anastomosis is feasible during retropubic radical prostatectomy. Furthermore, it offers better outcomes than the conventional standard interrupted suture technique, with a higher likelihood of improvement in patients' health-related quality of life.

Novel COL2A1 variants in Japanese patients with spondyloepiphyseal dysplasia congenita.

Spondyloepiphyseal dysplasia congenita (SEDC) is a multisystemic skeletal disorder caused by pathogenic variants in COL2A1. Here, we report the genotype-phenotype correlations in five Japanese patients with SEDC based on their clinical and radiological findings. All five patients had novel missense variants resulting in glycine substitutions (G474V, G543E, G567S, G594R, and G1170R). Genetic testing is important for early intervention for the extraskeletal complications of SEDC. Spondyloepiphyseal dysplasia congenita (SEDC) (OMIM#183900) is an autosomal dominant chondrodysplasia characterized by disproportionate short stature, abnormal epiphyses, flattened vertebral bodies (skeletal abnormalities), and extraskeletal features, including myopia, retinal degeneration with retinal detachment, and cleft palate. SEDC is caused by a heterozygous variant in the collagen II alpha 1 (COL2A1) gene.

Diagnostic performance of cold provocation test with hands immersion in water at 10°C for 5 min evaluated in vibration-induced white finger patients and matched controls.

PURPOSE: This study aimed to explore the diagnostic ability of the cold provocation test with hands immersion in water at 10°C for 5 min in diagnosing vibration-induced white finger (VWF). METHODS: Finger skin temperature (FST) was measured in 20 VWF patients and 20 matched healthy controls, at palmar side of the distal phalanges of fingers from both hands before, during, and after hands immersion in water at 10°C (for 5 min with waterproof coverings put on both hands). Data from 4 fingers (except thumb) were evaluated at five time points: just before immersion, last minute during immersion, and at 5th, 10th, and 15th min during the post-immersion or recovery period. RESULTS: A positive group difference between patients and controls was revealed during the recovery period. During recovery at 95 and 70% specificity, the sensitivity ranged from 20 to 30% and 50 to 70% for evaluation with average FST for 4 fingers and 15-35% and 60-65% for evaluation with minimum FST among 4 fingers, respectively. Overall, evaluation of absolute FST at 15th min of recovery offered better diagnostic ability. CONCLUSIONS: The cold provocation test with hands immersion in water at 10°C for 5 min could discriminate VWF patients from healthy controls; however, this test has a limited diagnostic value in diagnosing patients with VWF.

Acute Effects of Whole-Body Vibration on Peripheral Blood Flow, Vibrotactile Perception and Balance in Older Adults.

BACKGROUND: Non-invasive application of whole-body vibration (WBV) has the potential for inducing improvements in impaired peripheral circulation, cutaneous sensation and balance among older adults. However, relevant studies have frequently applied high magnitudes of vibration and show conflicting and inconclusive results. Therefore, we attempted to ascertain the acute responses in those parameters from exposure of thirty older subjects to WBV of three different magnitudes, defined according to ISO 2631-1 (1997). METHODS: Each subject randomly underwent four sessions of intervention (three bouts of 1 min exposure with 1 min between-bout rests): WBV at 15, 20, or 25 Hz with a peak-to-peak displacement of 4 mm, or control condition. RESULTS: Both during and after intervention, dorsal foot skin blood flow increased significantly under 20 and 25 Hz exposure conditions with greater responses under the latter condition, the magnitude of which slightly exceeded the recommended value. Plantar vibrotactile perception showed significant increases after WBV exposure with overall greater responses under higher frequencies of vibration. In contrast, no WBV-induced change in balance was observed. CONCLUSIONS: WBV at 20 Hz with a magnitude within the recommended limit can be effective in inducing enhancements in peripheral blood flow; however, the same magnitude of vibration seems insufficient in improving balance among older adults.

A systematic review of studies investigating the effects of controlled whole-body vibration intervention on peripheral circulation.

The findings of the published studies investigating the changes in peripheral circulation induced by exposure to whole-body vibration (WBV) are not consensual or conclusive. Also, those studies did not consider the role of vibration magnitude on the peripheral circulatory responses making the interpretation of the observed findings difficult. We aimed to review the published literature investigating the effects of controlled WBV intervention on peripheral circulation by characterizing the relevant exposure conditions including vibration magnitudes, and ascertain the specific patterns of responses in peripheral circulation of the lower extremity from such exposure. A computerized search was performed in PubMed and Scopus using selected key search terms, and the relevant data were extracted. The vibration magnitude in the included studies frequently exceeded the limit specified in the International Standard ISO 2631-1 (1997) for such exposure. As observed, exposure to WBV with frequencies ≤30 Hz caused an improvement in peripheral blood flow (BF) and/or skin temperature (ST) of the lower extremity. In contrast, the responses in BF and ST from exposure to WBV of higher frequencies (31 Hz-50 Hz) produced conflicting results. The findings of this review indicate that WBV exposure with relatively lower frequency and magnitude can be safely and effectively used to induce improvements in peripheral circulation. For this purpose, the limits recommended by the ISO 2631-1 (1997) should be considered till safe and effective vibration-related parameters are established.

Novel SYNGAP1 variant in a patient with intellectual disability and distinctive dysmorphisms.

We describe a novel de novo heterozygous variant in SYNGAP1 (c.1741C>T, p.R581W), identified through targeted resequencing in an 8-year-old boy with intellectual disability, autism spectrum disorder, distinctive dysmorphic features, and no seizures. Our data strongly suggest that the SYNGAP1 variant is causative of intellectual disability in this patient.

Clinical Evaluation of the Immunochromatographic System Using Silver Amplification for the Rapid Detection of Mycoplasma pneumoniae.

Mycoplasma pneumoniae infection is conventionally diagnosed using serum antibody testing, microbial culture, and genetic testing. Recently, immunochromatography-based rapid mycoplasma antigen test kits have been developed and commercialised for rapid diagnosis of M. pneumoniae infection. However, as these kits do not provide sufficient sensitivity and specificity, a rapid test kit with improved accuracy is desired. The present prospective study evaluated a rapid M. pneumoniae diagnostic system utilizing a newly developed silver amplification immunochromatography (SAI) system. We performed dilution sensitivity test and the prospective clinical study evaluating the SAI system. The subjects of the clinical study included both children and adults. All patients suspected to have mycoplasma pneumonia (169 patients) were sequentially enrolled. Twelve patients did not agree to participate and 157 patients were enrolled in the study. The results demonstrate excellent performance of this system with 90.4% sensitivity and 100.0% specificity compared with real-time polymerase chain reaction. When compared with loop-mediated isothermal amplification (LAMP) methods, the results also demonstrate a high performance of this system with 93.0% sensitivity and 100.0% specificity. The SAI system uses a dedicated device for automatic analysis and reading, making it highly objective, and requires less human power, supporting its usefulness in clinical settings.

Evaluation of the effect of heat exposure on the autonomic nervous system by heart rate variability and urinary catecholamines.

The aim of this study was to investigate the usefulness of heart rate variability (HRV) and urinary catecholamines (CA) as objective indices of heat stress effect. We examined physiological responses, subjective symptoms, HRV and urinary CA to evaluate the effect of heat exposure on the autonomic nervous system. Six healthy male students volunteered for this study. They were exposed on different days to either a thermoneutral condition at wet bulb globe temperature (WBGT) 21 degrees C, or a heated condition at WBGT 35 degrees C for 30 min, while seated on a chair. In the thermoneutral condition, differences of all parameters between the values before and after 30 min exposure were not statistically significant. In the heated condition, heart rate, body temperature and scores for subjective symptoms (feverishness, sweating, mood, and face flushing) significantly increased after 30 min exposure (p<0.05). Also, the high frequency component (HF%) of HRV significantly decreased and the low frequency/high frequency (LF/HF) ratio of HRV significantly increased after 30 min exposure to the heated condition (p<0.05). There were no significant differences between the amounts of urinary CA before and after the 30 min exposures; however, the norepinephrine amount after 30 min exposure to the heated condition was significantly greater than that of the thermoneutral condition (p<0.05). The heat exposure (WBGT 35 degrees C) induced activation of the sympathetic nervous system and a withdrawal of the parasympathetic nervous system. These findings coincide with observed changes of heart rate, body temperature and subjective symptoms. It is suggested that HRV (HF% and LF/HF ratio) and urinary norepinephrine may be useful objective indices of heat stress; HRV seems to be more sensitive to heat stress than urinary CA.

Assessing the influence of antivibration glove on digital vascular responses to acute hand-arm vibration.

This study was designed to assess the influence of an antivibration glove on digital vascular responses in healthy subjects exposed to short-term grasping of a vibrating handle. To measure finger blood flow (FBF) and finger skin temperature (FST) once at the end of every min, a blood flowmeter sensor was attached to the dorsum and a thermistor sensor was attached to the medial surface of the subject's middle phalanx of the third finger of the right hand. After 5 min of baseline measurements without or with an antivibration glove meeting ISO standard 10819, worn on the right hand, subjects gripped a vibrating handle with the same hand for a period of 5 min. Vibration was generated at two frequencies of 31.5 Hz and 250 Hz with a frequency weighted rms acceleration of 5.5 m/s(2). FBF and FST continued to be recorded for a further 5 min after release of the vibrating handle. Statistical analysis showed no significant change after vibration exposure in either FST or FBF at 250 Hz, compared to baseline (control) measurements while using the antivibration glove. At 31.5 Hz, FBF data exhibited a significant difference between before and after grasping of vibrating handle, which was less under the condition of wearing the antivibration glove than under the condition of bare hand. The results provide evidence that the antivibration glove considerably influenced finger vascular changes in healthy subjects induced by vibration exposure, especially against high frequency vibration. Further studies are required to assess finger vascular responses to hand-transmitted vibration with antivibration gloves of different manufacturers.

[Does alpha1-blocker provide additional improvement of quality-of-life in patients with overactive bladder?--a multi-center prospective randomized trial between anti-cholinergic (propiverine chloride) with and without alpha1-blocker (urapidil)].

AIM: Storage/filling symptoms caused by overactive bladder (OAB) are bothersome to patients. The aim of this study is to clarify if alpha1-blocker provides additional benefit in combination with anticholinergic treatment in patients with OAB. METHODS: In total, 100 patients (men/women: 43/57, mean age: 71.3 years) who had frequency (more than eight times a day) and urgency (more than three times a week) were prospectively randomized, and allocated to two groups (monotherapy group [n = 52]: propiverine alone or combination group [n = 48]: propiverine plus urapidil). The primary end point was to compare the improvement of storage symptoms (numbers of frequency, urgency, disappearance of urge incontinence) as well as patients' quality of life (QOL) assessed by King's Health Questionnaires (KHQ) at baseline, 2 weeks, and 6 weeks after the start of treatment in both groups. The second end point was to evaluate the safety of these agents. RESULTS: Statistically significant improvements in terms of urgency and frequency were observed in both groups at two-weeks after the start of treatment as compared with baseline (p < 0.01 and < 0.05, respectively), while no inter-group difference was observed between the two groups. Significant improvement of QOL was observed after six weeks treatment in overall mean score, general health perception, incontinence impact, sleep/energy domains in both groups as compared with baseline. No significant difference was observed in terms of toxic events between the two groups. CONCLUSIONS: Although both groups showed identical improvement of storage symptoms and tolerability, no additional benefit of alpha1-blocker was observed.

Clinical significance of serum complement factor 3 in patients with type 2 diabetes mellitus.

AIMS: Although serum complement factor 3 (C3) is an acute phase reactant mainly synthesized in the liver, several recent studies have shown high C3 gene expression in adipose tissue (AT). However, the relationship between C3 and AT levels has not been fully clarified in type 2 diabetes mellitus (T2DM) patients. METHODS: A total of 164 T2DM patients (109men and 55 women) participated in this cross-sectional study. A computed tomography scan was performed to measure visceral, subcutaneous, and total AT. The correlation between these factors and C3 levels was examined using Pearson's correlation analysis. A multivariate regression model was used to assess an independent determinant associated with C3 levels after adjusting the explanatory variables (i.e., all ATs [visceral, subcutaneous, and total], and clinical features [sex, age, body mass index, waist circumference, glycated hemoglobin, duration of diabetes, systolic blood pressure, diastolic blood pressure, aspartate aminotransferase levels, alanine aminotransferase levels, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, log(triglyceride levels), estimated glomerular filtration rate, and log(high-sensitivity C-reactive protein levels)]). RESULTS: Serum C3 levels were correlated with visceral, subcutaneous, and total AT among both men (r=0.505, p<0.001; r=0.545, p<0.001; r=0.617, p<0.001, respectively) and women (r=0.396, p=0.003; r=0.517, p<0.001; r=0.548, p<0.001, respectively). In the multivariate regression model, the association between total AT and C3 levels remained significantly positive (ß=0.490, p<0.001). CONCLUSIONS: Serum C3 levels are associated with visceral, subcutaneous, and total AT in T2DM patients. Furthermore, C3 levels seem to be a marker for overall adiposity rather than regional adiposity.

Prevalence of cervical spondylosis and musculoskeletal symptoms among coolies in a city of Bangladesh.

To assess the prevalence of cervical spondylosis and musculoskeletal symptoms among coolies a cross-sectional study was performed in Narayangonj City of Bangladesh on a random sample of 98 male coolies, using a questionnaire and the results of cervical spine X-rays. Statistical associations were investigated using the chi-square test. The results show a considerably higher prevalence of cervical spondylosis among the coolies (39.8%). More than half (51.3%) of the cases of cervical spondylosis were in subjects below the age of 40 yr, and a significant association was found between age group and prevalence of cervical spondylosis. The study also observed a significant association between duration of occupation and prevalence of cervical spondylosis. Coolies who had worked for 10 to 15 yr, or more than 15 yr, had higher rates of cervical spondylosis. In this study it was found that those who carried heavier loads suffered more from cervical spondylosis. Musculoskeletal symptoms in multiple body regions (two or more) were more prevalent (61.2%) than those in single body region (29.6%). Symptoms in the hands/fingers were the most frequent followed by the back and arms/forearms. In conclusion, the high prevalence of cervical spondylosis and musculoskeletal symptoms among professional coolies may be associated with the practice of carrying heavy loads on the head. Further study with a large sample of population is required to investigate this problem and to explore preventive measures.

A study on the relationship between subjective unpleasantness and body surface vibrations induced by high-level low-frequency pure tones.

Human body surface vibrations induced by high-level low-frequency pure tones were measured at the chest and the abdomen. At the same time, the subject rated the unpleasantness that he had just perceived during the exposure to low-frequency noise stimulus. Examining the relationship between the measured vibration and the rating score of the unpleasantness revealed that the unpleasantness was in close correlation with the vibration acceleration level (VAL) of the vibration measured. Taking previous results into account, this finding suggests that noise-induced vibrations primarily induce vibratory sensations and through the vibratory sensation or together with some other factors, secondarily contribute to the unpleasantness. The present results suggest that in evaluating high-level low-frequency noise, the effect of vibration should be taken into account.

A systematic review of diagnostic performance of quantitative tests to assess musculoskeletal disorders in hand-arm vibration syndrome.

The purpose was to systematically review the published reports for the clinical utility of quantitative objective tests commonly used for diagnosing musculoskeletal disorders in hand-arm vibration syndrome (HAVS). Two reviewers independently conducted a computerized literature search in PubMed and Scopus using predefined criteria, and relevant papers were identified. The articles were screened in several stages and considered for final inclusion. Quality of the selected papers was evaluated by a modified QUADAS tool. Relevant data were extracted as necessary. For this review, only 4 relevant studies could be identified for detailed examination. Grip strength, pinch strength, and Purdue pegboard tests were commonly used with their reported sensitivity and specificity ranging between 1.7 to 65.7% and 65.2 to 100%, 1.7 to 40% and 94 to 100%, and 44.8 to 85% and 78 to 95%, respectively. A considerable difference across the studies was observed with respect to patient and control populations, diagnostic performance and cut-off values of different tests. Overall, currently available English-language limited literature do not provide enough evidence in favour of the application of grip strength and pinch strength tests for diagnosing musculoskeletal injuries in HAVS; Purdue pegboard test seems to have some diagnostic value in evaluating impaired dexterity in HAVS.

Activation of restriction enzyme by electrochemically released magnesium ion.

Observation and cutting of DNA molecules at intended positions permit several new experimental methods that are completely different from conventional molecular biology methods; therefore several cutting methods have been proposed and studied. In this paper, a new cutting method for a DNA molecule by localizing the activity of a restriction enzyme is presented. Since most restriction enzymes require magnesium ions for their activation, local restriction enzyme activity can be controlled by the local concentration of magnesium ions. Applying a direct current (dc) voltage to a needle electrode of metallic magnesium made it possible to control the local magnesium ion concentration at the tip of the needle. The restriction enzyme was activated only when magnesium ions were electrochemically supplied.

Heart rate variation and urinary catecholamine excretion in response to acute psychological stress in hand-arm vibration syndrome patients.

The aim of this study was to evaluate heart rate variation and urinary catecholamines in response to acute psychological stress in hand-arm vibration syndrome (HAVS) patients and healthy controls. LF% (indicator of both the sympathetic and parasympathetic nervous activity), HF% (indicator of the parasympathetic nervous activity) and their ratio LF/HF (indicator of sympathovagal balance) were calculated from short-term electrocardiographic data from 16 HAVS patients and 14 healthy controls before and immediately after exposure to acute psychological stress. Urinary catecholamines (norepinephrine, epinephrine and dopamine) were analyzed from urine samples collected from every subject during rest and after exposure. The LF% and LF/HF of the patients significantly increased after exposure. The after exposure LF/HF of the patients was significantly greater than that of the controls. The amount of norepinephrine in the patients significantly increased after exposure. The after exposure amount of norepinephrine and epinephrine in the patients were significantly greater than the respective amount of the controls. The results of the present study indicated the predominance of sympathetic tone in the cardiac sympathovagal balance and greater sensitivity of the sympathoadrenal medullary system in response to acute psychological stress in the patients than in the healthy controls.