Cell-type-resolved mosaicism reveals clonal dynamics of the human forebrain.

Debate remains around the anatomical origins of specific brain cell subtypes and lineage relationships within the human forebrain1-7. Thus, direct observation in the mature human brain is critical for a complete understanding of its structural organization and cellular origins. Here we utilize brain mosaic variation within specific cell types as distinct indicators for clonal dynamics, denoted as cell-type-specific mosaic variant barcode analysis. From four hemispheres and two different human neurotypical donors, we identified 287 and 780 mosaic variants, respectively, that were used to deconvolve clonal dynamics. Clonal spread and allele fractions within the brain reveal that local hippocampal excitatory neurons are more lineage-restricted than resident neocortical excitatory neurons or resident basal ganglia GABAergic inhibitory neurons. Furthermore, simultaneous genome transcriptome analysis at both a cell-type-specific and a single-cell level suggests a dorsal neocortical origin for a subgroup of DLX1+ inhibitory neurons that disperse radially from an origin shared with excitatory neurons. Finally, the distribution of mosaic variants across 17 locations within one parietal lobe reveals that restriction of clonal spread in the anterior-posterior axis precedes restriction in the dorsal-ventral axis for both excitatory and inhibitory neurons. Thus, cell-type-resolved somatic mosaicism can uncover lineage relationships governing the development of the human forebrain.

Research progress on the roles of dopamine and dopamine receptors in digestive system diseases.

Dopamine (DA) is a neurotransmitter synthesized in the human body that acts on multiple organs throughout the body, reaching them through the blood circulation. Neurotransmitters are special molecules that act as messengers by binding to receptors at chemical synapses between neurons. As ligands, they mainly bind to corresponding receptors on central or peripheral tissue cells. Signalling through chemical synapses is involved in regulating the activities of various body systems. Lack of DA or a decrease in DA levels in the brain can lead to serious diseases such as Parkinson's disease, schizophrenia, addiction and attention deficit disorder. It is widely recognized that DA is closely related to neurological diseases. As research on the roles of brain-gut peptides in human physiology and pathology has deepened in recent years, the regulatory role of neurotransmitters in digestive system diseases has gradually attracted researchers' attention, and research on DA has expanded to the field of digestive system diseases. This review mainly elaborates on the research progress on the roles of DA and DRs related to digestive system diseases. Starting from the biochemical and pharmacological properties of DA and DRs, it discusses the therapeutic value of DA- and DR-related drugs for digestive system diseases.

The role of ncRNAs-mediated pyroptosis in diabetes and its vascular complications.

Over the past decade, the prevalence of diabetes has increased significantly worldwide, leading to an increase in vascular complications of diabetes (VCD), such as diabetic cardiomyopathy (DCM), diabetic nephropathy (DN), and diabetic retinopathy (DR). Noncoding RNAs (ncRNAs), such as microRNAs (miRNAs), long Noncoding RNAs (lncRNAs), and circular RNAs (circRNAs), play a key role in cellular processes, including the pathophysiology of diabetes and VCD via pyroptosis. ncRNAs (e.g., miR-17, lnc-MEG3, and lnc-KCNQ1OT1) can regulate pyroptosis in pancreatic ß cells. Some ncRNAs are involved in VCD progression. For example, miR-21, lnc-KCNQ1OT1, lnc-GAS5, and lnc-MALAT1 were reported in DN and DCM, and lnc-MIAT was identified in DCM and DR. Herein, this review aimed to summarize recent research findings related to ncRNAs-mediated pyroptosis at the onset and progression of diabetes and VCD.

Application and configuration analysis of electric muck transfer equipment in plateau railway tunnel: a case study in southwest China.

The burgeoning development of railway construction in plateau regions of southwest China necessitates innovative and environmentally sustainable approaches, particularly in the realm of tunnel construction, where the transfer of muck poses significant operational and environmental challenges. This research, pivoting around the application and configuration of electric muck transfer equipment in plateau railway tunnels, seeks to dissect the potentialities and impediments of transitioning from conventional diesel-powered machinery to electric alternatives, with a spotlight on mitigating environmental impacts and enhancing operational efficiency. Through an analytical lens, the study employs a case study methodology, leveraging data and insights from existing electric equipment models and their applications, provided by major manufacturers in China, to weave a comprehensive narrative around the practicalities, specifications, and challenges embedded in the adoption of electric machinery in plateau environments. The findings unveil a nuanced landscape, where the environmental and operational advantages of electric equipment are juxtaposed against a backdrop of technological, financial, and infrastructural hurdles, thereby crafting a complex tapestry of opportunities and challenges. The research further extrapolates policy recommendations and practical guidelines, advocating for a harmonized amalgamation of governmental policies, technological advancements, and strategic planning to navigate through the identified challenges and optimize the integration of electric equipment in tunnel construction practices. Envisaging future research pathways, the study underscores the criticality of perpetuating technological innovations, policy adaptations, and interdisciplinary research to further refine and enhance the application of electric muck transfer equipment in plateau railway tunnel projects, thereby contributing to the broader narrative of sustainable construction practices in challenging terrains.

Possible melatonin-induced salt stress tolerance pathway in Phaseolus vulgaris L. using transcriptomic and metabolomic analyses.

Melatonin plays important roles in multiple stress responses; however, the downstream signaling pathway and molecular mechanism remain unclear. This study aimed to elucidate the transcriptional regulation of melatonin-induced salt stress tolerance in Phaseolus vulgaris L. and identify the key downstream transcription factors of melatonin through transcriptomic and metabolomic analyses. The melatonin-induced transcriptional network of hormones, transcription factors, and functional genes was established under both control and stress conditions. Among these, eight candidate transcription factors were identified via gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, one gene related to transmembrane transport of salts (Phvul.004G177300). These genes may play a role in maintaining the cell structure and excreting sodium ions outside the cell or transporting them to the vacuoles for storage. Melatonin regulates the Phvul.009G210332 gene and metabolites C05642 (N-acetyl-N-2-formyl-5-methoxycanurine), C05643 (6-hydroxymelatonin), C05660 (5-methoxyindoleacetic acid) involved in tryptophan metabolism. The metabolites C05642 and C05643 were identified as decomposition products of tryptophan, indicating that exogenous melatonin entered the P. vulgaris tissue and was metabolized. Melatonin promotes the synthesis and metabolism of tryptophan, which is crucial to plant metabolism, growth, maintenance, and repair.

[Clinical study of modified suspension reduction method combined with percutaneous vertebroplasty in the treatment of thoracolumbar osteoporotic compression fracture].

OBJECTIVE: To investigate the clinical effect of modified suspension reduction method combined with percutaneous vertebroplasty in the treatment of osteoporotic thoracolumbar compression fractures. METHODS: From February 2020 to October 2021, 92 patients with thoracolumbar osteoporotic compression fracture were treated by percutaneous vertebroplasty. According to different treatment methods, they were divided into the observation group and the control group. The observation group was treated with modified suspension reduction and then percutaneous vertebroplasty, while the control group was treated with percutaneous vertebroplasty alone. The observation group (47 cases), including 20 males and 27 females, the age ranged from 59 to 76 years old with an average of (69.74±4.50) years old, fractured vertebral bodies:T10(2 cases), T11(7 cases), T12(19 cases), L1(14 cases), L2(5 cases);the control group(45 cases), including 21 males and 24 females, the age ranged from 61 to 78 years old with an average of (71.02±3.58) years old, fractured vertebral bodies:T10(3 cases), T11(8 cases), T12(17 cases), L1(12 cases), L2(5 cases);The leakage of bone cement were observed, the visual analogue scale (VAS), Oswestry lumbar dysfunction index (ODI), anterior vertebrae height (AVH), Cobb angle of kyphosis and the amount of bone cement injected before and after operation were recorded and compared between the two groups. RESULTS: All patients were followed up, ranged from 6 to10 with an average of (8.45±1.73) months. Two patients ocurred bone cement leakage in observation group and 3 patients in control group. AVH of observation group increased (P<0.05) and Cobb angle of injured vertebrae decreased (P<0.05). Cobb angle of injured vertebrae and AVH of the control group were not significantly changed (P>0.05). Cobb angle of injured vertebrae of the observation group was lower than that of control group (P<0.05) and AVH was higher than that of the control group (P<0.05). In the observation group, VAS before operation and 1 week, 3 and 6 months after operation respectively were(7.32±1.05) scores, (3.56±1.18) scores, (1.83±0.67) scores, (1.27±0.34) scores, and ODI were(40.12±14.69) scores, (23.76±10.19) scores, (20.15±6.39) scores, (13.45±3.46) scores. In the control group, VAS before operation and 1 week, 3 and 6 months after operation respectively were(7.11±5.26) scores, (3.82±0.68) scores, (1.94±0.88) scores, (1.36±0.52) scores, and ODI were(41.38±10.23) scores, (25.13±14.22) scores , (20.61±5.82) scores, (14.55±5.27) scores . The scores of VAS and ODI after operation were lower than those before operation (P<0.05), but there was no significant difference between the two groups (P<0.05). CONCLUSION: Modified suspension reduction method combined with PVP surgery for osteoporotic thoracolumbar compression fractures has achieved good clinical results, which can effectively relieve lumbar back pain, restore vertebral height, correct kyphosis, improve lumbar function and patients' quality of life.

Pericarotid adipose tissue computed tomography attenuation distinguishes different stages of carotid atherosclerotic disease: a cross-sectional study.

Background: Carotid atherosclerotic plaque inflammation plays a critical role in guiding the prevention of secondary stroke. Increased perivascular adipose tissue attenuation observed on computed tomography angiography (CTA) may indicate local inflammation. Our objective was to investigate whether pericarotid adipose tissue (PCAT), as a local inflammation biomarker, could distinguish between different stages of carotid atherosclerotic disease plaques. Methods: We prospectively enrolled 45 consecutive acute stroke patients with carotid artery stenosis from September 2019 to September 2021. We then matched them to non-stroke patients (n=67) and no carotid atherosclerotic disease controls (n=65) based on gender, age, and cardiovascular risk factors. We compared PCAT attenuation, carotid plaque features on CTA, clinical risk factors, and serum inflammatory factors across the different groups. To detect the association of PCAT attenuation with stage of carotid atherosclerotic disease, we used multivariable logistic regression analysis. Results: Patients with acute stroke had a higher PCAT attenuation (-78.80±11.62 HU) than patients with non-stroke (-89.01±10.81 HU, P<0.001) and no carotid atherosclerotic disease controls (-95.24±10.81 HU, P<0.001). PCAT attenuation was significantly increased in non-stroke patients compared to non-stroke patients over no carotid atherosclerotic disease controls (P=0.004). The association between PCAT attenuation and the stage of carotid atherosclerotic disease was independent of age, gender, cardiovascular risk factors, and CTA plaque characteristics. No interaction was observed between clinical features and CTA plaque characteristics on PCAT attenuation. Conclusions: PCAT attenuation, which is an imaging biomarker of local inflammation, independently distinguishes patients with different stages of carotid atherosclerotic disease. Quantitative evaluation of PCAT attenuation in carotid atherosclerotic disease is expected to guide targeted surgical treatment of carotid plaque.

Cell-type-resolved somatic mosaicism reveals clonal dynamics of the human forebrain.

Debate remains around anatomic origins of specific brain cell subtypes and lineage relationships within the human forebrain. Thus, direct observation in the mature human brain is critical for a complete understanding of the structural organization and cellular origins. Here, we utilize brain mosaic variation within specific cell types as distinct indicators for clonal dynamics, denoted as cell-type-specific Mosaic Variant Barcode Analysis. From four hemispheres from two different human neurotypical donors, we identified 287 and 780 mosaic variants (MVs), respectively that were used to deconvolve clonal dynamics. Clonal spread and allelic fractions within the brain reveal that local hippocampal excitatory neurons are more lineage-restricted compared with resident neocortical excitatory neurons or resident basal ganglia GABAergic inhibitory neurons. Furthermore, simultaneous genome-transcriptome analysis at both a cell-type-specific and single-cell level suggests a dorsal neocortical origin for a subgroup of DLX1+ inhibitory neurons that disperse radially from an origin shared with excitatory neurons. Finally, the distribution of MVs across 17 locations within one parietal lobe reveals restrictions of clonal spread in the anterior-posterior axis precedes that of the dorsal-ventral axis for both excitatory and inhibitory neurons. Thus cell-type resolved somatic mosaicism can uncover lineage relationships governing the development of the human forebrain.

Genomic data resources of the Brain Somatic Mosaicism Network for neuropsychiatric diseases.

Somatic mosaicism is defined as an occurrence of two or more populations of cells having genomic sequences differing at given loci in an individual who is derived from a single zygote. It is a characteristic of multicellular organisms that plays a crucial role in normal development and disease. To study the nature and extent of somatic mosaicism in autism spectrum disorder, bipolar disorder, focal cortical dysplasia, schizophrenia, and Tourette syndrome, a multi-institutional consortium called the Brain Somatic Mosaicism Network (BSMN) was formed through the National Institute of Mental Health (NIMH). In addition to genomic data of affected and neurotypical brains, the BSMN also developed and validated a best practices somatic single nucleotide variant calling workflow through the analysis of reference brain tissue. These resources, which include >400 terabytes of data from 1087 subjects, are now available to the research community via the NIMH Data Archive (NDA) and are described here.

Genomic Mosaicism of the Brain: Origin, Impact, and Utility.

Genomic mosaicism describes the phenomenon where some but not all cells within a tissue harbor unique genetic mutations. Traditionally, research focused on the impact of genomic mosaicism on clinical phenotype-motivated by its involvement in cancers and overgrowth syndromes. More recently, we increasingly shifted towards the plethora of neutral mosaic variants that can act as recorders of cellular lineage and environmental exposures. Here, we summarize the current state of the field of genomic mosaicism research with a special emphasis on our current understanding of this phenomenon in brain development and homeostasis. Although the field of genomic mosaicism has a rich history, technological advances in the last decade have changed our approaches and greatly improved our knowledge. We will provide current definitions and an overview of contemporary detection approaches for genomic mosaicism. Finally, we will discuss the impact and utility of genomic mosaicism.

Detection of Salmonella DNA and drug-resistance mutation by PCR-based CRISPR-lbCas12a system.

Salmonella is an important foodborne pathogen, which can cause serious public health problems. Rapid and accurate detection of Salmonella infection and drug resistance mutations in patients will provide timely guidance for clinical treatment and avoid disease progression and other related clinical problems. Here, we established a highly sensitive and quick method for Salmonella and drug resistance mutation detection based on polymerase chain reaction (PCR) and CRISPR-lbCas12a system and evaluated its practicability with clinical samples.Specific CRISPR RNAs (crRNAs) and primers are designed for Salmonella DNA and parC gene S80I mutation diagnosis. CrRNAs with and without phosphorylated modification and different crRNA preparation methods are used to assess the effect on the detection system. After optimization, we detected as low as one copy of Salmonella DNA and drug resistance mutation parC S80I with the Salmonella DNA standard. For 94 clinical samples, this method also showed high sensitivity (100%, 95% CI: 84.98-100%) and specificity (98.48%, 95% CI: 90.73-99.92%) with less time (3 h) than plate culture (16 h) and conventional antimicrobial susceptibility testing (over 16 h). Besides, one parC S80I mutant strain was detected, which is consistent with the result of DNA sequencing. Taken together, we established a highly sensitive and specific method for Salmonella infection and parC S80I drug resistance mutation detection with fewer reagents and ordinary instruments. This assay has wide application prospects for fast detection of pathogen (bacterium and virus) infection, drug resistance determination, and proper treatment guidance.

Estrogen regulates duodenal glucose absorption by affecting estrogen receptor-α on glucose transporters.

The mechanisms of estrogen in glucose metabolism are well established; however, its role in glucose absorption remains unclear. In this study, we investigated the effects of estrogen on glucose absorption in humans, mice, and SCBN intestinal epithelial cells. We first observed a correlation between estrogen and blood glucose in young women and found that glucose tolerance was significantly less in the premenstrual phase than in the preovulatory phase. Similarly, with decreased serum estradiol levels in ovariectomized mice, estrogen receptors alpha (ERα) and beta (ERß) in the duodenum were reduced, and weight and abdominal fat increased significantly. The expression of sodium/glucose cotransporter 1 (SGLT1) and glucose transporter 2 (GLUT2) and glucose absorption in the duodenum decreased significantly. Estrogen significantly upregulated SGLT1 and GLUT2 expression in SCBN cells. Silencing of ERα, but not ERß, reversed this trend, suggesting that ERα may be key to estrogen-regulating glucose transporters. A mechanistic study revealed that downstream, estrogen regulates the protein kinase C (PKC) pathway. Overall, our findings indicate that estrogen promotes glucose absorption, and estrogen and ERα deficiency can inhibit SGLT1 and GLUT2 expression through the PKC signaling pathway, thereby reducing glucose absorption.

The S100 calcium-binding protein A6 plays a crucial role in hepatic steatosis by mediating lipophagy.

BACKGROUND: S100 calcium-binding protein A6 (S100A6) is a calcium-binding protein that is involved in a variety of cellular processes, such as proliferation, apoptosis, and the cellular response to various stress stimuli. However, its role in NAFLD and associated metabolic diseases remains uncertain. METHODS AND RESULTS: In this study, we revealed a new function and mechanism of S100A6 in NAFLD. S100A6 expression was upregulated in human and mouse livers with hepatic steatosis, and the depletion of hepatic S100A6 remarkably inhibited lipid accumulation, insulin resistance, inflammation, and obesity in a high-fat, high-cholesterol (HFHC) diet-induced murine hepatic steatosis model. In vitro mechanistic investigations showed that the depletion of S100A6 in hepatocytes restored lipophagy, suggesting S100A6 inhibition could alleviate HFHC-induced NAFLD. Moreover, S100A6 liver-specific ablation mediated by AAV9 alleviated NAFLD in obese mice. CONCLUSIONS: Our study demonstrates that S100A6 functions as a positive regulator of NAFLD, targeting the S100A6-lipophagy axis may be a promising treatment option for NAFLD and associated metabolic diseases.

Ion channels and transporters regulate nutrient absorption in health and disease.

Ion channels and transporters are ubiquitously expressed on cell membrane, which involve in a plethora of physiological process such as contraction, neurotransmission, secretion and so on. Ion channels and transporters is of great importance to maintaining membrane potential homeostasis, which is essential to absorption of nutrients in gastrointestinal tract. Most of nutrients are electrogenic and require ion channels and transporters to absorb. This review summarizes the latest research on the role of ion channels and transporters in regulating nutrient uptake such as K+ channels, Ca2+ channels and ion exchangers. Revealing the mechanism of ion channels and transporters associated with nutrient uptake will be helpful to provide new methods to diagnosis and find potential targets for diseases like diabetes, inflammatory bowel diseases, etc. Even though some of study still remain ambiguous and in early stage, we believe that ion channels and transporters will be novel therapeutic targets in the future.

Nanoscale reorganisation of synaptic proteins in Alzheimer's disease.

AIMS: Synaptic strength depends strongly on the subsynaptic organisation of presynaptic transmitter release and postsynaptic receptor densities, and their alterations are expected to underlie pathologies. Although synaptic dysfunctions are common pathogenic traits of Alzheimer's disease (AD), it remains unknown whether synaptic protein nano-organisation is altered in AD. Here, we systematically characterised the alterations in the subsynaptic organisation in cellular and mouse models of AD. METHODS: We used immunostaining and super-resolution stochastic optical reconstruction microscopy imaging to quantitatively examine the synaptic protein nano-organisation in both Aß1-42-treated neuronal cultures and cortical sections from a mouse model of AD, APP23 mice. RESULTS: We found that Aß1-42-treatment of cultured hippocampal neurons decreased the synaptic retention of postsynaptic scaffolds and receptors and disrupted their nanoscale alignment to presynaptic transmitter release sites. In cortical sections, we found that while GluA1 receptors in wild-type mice were organised in subsynaptic nanoclusters with high local densities, receptors in APP23 mice distributed more homogeneously within synapses. This reorganisation, together with the reduced overall receptor density, led to reduced glutamatergic synaptic transmission. Meanwhile, the transsynaptic alignment between presynaptic release-guiding RIM1/2 and postsynaptic scaffolding protein PSD-95 was reduced in APP23 mice. Importantly, these reorganisations were progressive with age and were more pronounced in synapses in close vicinity of Aß plaques with dense cores. CONCLUSIONS: Our study revealed a spatiotemporal-specific reorganisation of synaptic nanostructures in AD and identifies dense-core amyloid plaques as the major local inductor in APP23 mice.

The impact of medical insurance on family financial support: evidence from CHARLS in China.

BACKGROUND: In China, public medical insurance has expanded rapidly in the past 20 years. Many studies have discussed the benefits of medical insurance in improving residents' health and financial stability, and increasing the utilization of medical services. Less attention is paid to the effect of medical insurance on family support between parents and children. This study focuses on the effect of medical insurance on promoting family financial support in China. METHOD: Fifty-five thousand sixty-two individual samples were obtained from four waves of the China Health and Retirement Longitudinal Study (CHARLS): 2011, 2013, 2015 and 2018. Linear-regression model and propensity score matching are used to determine the relationship between medical insurance and family financial support. Then, mediation model is introduced to identify the mediation mechanisms. Also, moderation model is used to estimate the moderation effect of parental education and health. RESULTS: Medical insurance has significantly increased family financial support between the insured parents and their children. Moreover, this positive effect is heterogeneous since only families living in rural areas were affected, and the direction of family financial support changed with the aging of the parents. The welfare of medical insurance on financial status have also been proven in this paper. The results indicate that medical insurance reduces the out-of-pocket ratio of medical expenses and increases health investment, which can perform as as two mediation mechanisms to affect family financial support. Besides, the education and health status of the insured parents play a role in moderating the effect of medical insurance.

Fine mapping and characterisation of a PV-PUR mediating anthocyanin synthesis in snap bean (Phaseolus vulgaris L.).

Anthocyanin makes snap bean (Phaseolus vulgaris L.) pods purple, which helps seed dispersal and protects against environmental stress. In this study, we characterised the snap bean purple mutant pv-pur, which has purple cotyledon, hypocotyl, stem, leaf vein, flower and pod tissues. Total anthocyanin, delphinidin and malvidin levels in mutant pods were significantly higher than in wild-type plants. We constructed two populations for fine mapping of the PV-PUR purple mutation gene, located in the 243.9-kb region of chromosome 06. We identified Phvul.006g018800.3, encoding F3'5'H, as a candidate gene for PV-PUR. Six single-base mutations occurred in the coding region of this gene, altering protein structure. PV-PUR and pv-pur genes were transferred into Arabidopsis, respectively. Compared with the wild-type, the leaf base and internode of T-PV-PUR plant were purple, and the phenotype of T-pv-pur plant remained unchanged, which verified the function of the mutant gene. The results demonstrated that PV-PUR is a crucial gene for anthocyanin biosynthesis in snap bean, resulting in purple colouration. The findings lay a foundation for future breeding and improvement of snap bean. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01362-8.

MECP2 germline mosaicism plays an important part in the inheritance of Rett syndrome: a study of MECP2 germline mosaicism in males.

BACKGROUND: Germline mosaicisms could be inherited to offspring, which considered as "de novo" in most cases. Paternal germline MECP2 mosaicism has been reported in fathers of girls with Rett syndrome (RTT) previously. For further study, we focused on MECP2 germline mosaicism in males, not only RTT fathers. METHODS: Thirty-two fathers of RTT girls with MECP2 pathogenic mutations and twenty-five healthy adult males without history and family history of RTT or other genetic disorders were recruited. Sperm samples were collected and ten MECP2 hotspot mutations were detected by micro-droplet digital PCR (mDDPCR). And routine semen test was performed at the same time if the sample was sufficient. Additionally, blood samples were also detected for those with sperm MECP2 mosaicisms. RESULTS: Nine fathers with RTT daughters (28.1%, 9/32) were found to have MECP2 mosaicism in their sperm samples, with the mutant allele fractions (MAFs) ranging from 0.05% to 7.55%. Only one father with MECP2 c.806delG germline mosaicism (MAF 7.55%) was found to have mosaicism in the blood sample, with the MAF was 0.28%. In the group of healthy adult males, MECP2 mosaicism was found in 7 sperm samples (28.0%, 7/25), with the MAFs ranging from 0.05% to 0.18%. None of the healthy adult males with MECP2 germline mosaicisms were found with MECP2 mosaicism in blood samples. There were no statistical differences in age, or the incidence of asthenospermia between fathers with RTT daughters and healthy adult males with MECP2 germline mosaicisms. Additionally, there was no linear correlation between MAFs of MECP2 mosaicisms and the age of males with germline MECP2 mosaicisms. CONCLUSIONS: Germline MECP2 mosaicism could be found not only in fathers with RTT daughters but also in healthy adult males without family history of RTT. As germline mosaic mutations may be passed on to offspring which commonly known as "de novo", more attention should be paid to germline mosaicism, especially in families with a proband diagnosed with genetic disorders.