A novel NTRK1 splice site variant causing congenital insensitivity to pain with anhidrosis in a Chinese family.

Background: Congenital insensitivity to pain with anhidrosis (CIPA, OMIM #256800), also known as hereditary sensory and autonomic neuropathy type Ⅳ (HSAN-IV), is a rare autosomal recessive disorder characterized by recurrent episodic fevers, anhidrosis, insensitivity to noxious stimuli, self-mutilating behavior and intellectual disability. CIPA can be caused by the variants in NTRK1 gene, which encodes a high-affinity tyrosine kinase receptor for nerve growth factor. To ascertain the hereditary cause of a patient with CIPA accompanied by the additional symptoms of mild growth retardation, prone to fracture, underdeveloped nails of fingers and toes, irregular tooth alignment, enamel hypoplasia, postoperative wound healing difficulty, hand and limb deformity, and dislocation of hip joint, whole exome sequencing was used and revealed a compound heterozygous variant in NTRK1. Methods: DNA was extracted from peripheral blood samples of pediatric patients and their parents, and subjected to comprehensive analysis using whole-exome sequencing (WES), followed by verification of variant sites in the NTRK1 gene through Sanger sequencing. To elucidate the functional impact of the newly discovered variants, an in vitro experimental system was established. Splicing analysis was conducted using PCR and Sanger sequencing, while expression levels were assessed through qPCR and Western blot techniques. Results: One hotspot variant c.851-33T>A(ClinVar ID: 21308) and a novel variant c.850 + 5G>A(ClinVar ID:3069176) was inherited from her father and mother, respectively, identified in the affected individuals. The c.850 + 5G>A variant in NTRK1 resulted in two forms of aberrant mRNA splicing: 13bp deletion (c.838_850del13, p. Val280Ser fs180) and 25bp deletion (826_850del25, p. Val276Ser fs180) in exon 7, both leading to a translational termination at a premature stop codon and forming a C-terminal truncated protein. The expression of two abnormal splicing isoforms was decreased both in the level of mRNA and protein. Conclusion: In conclusion, this study elucidated the genetic cause of a patient with CIPA and identified a novel variant c.850 + 5G>A in NTRK1, which broadened the and enriched the NTRK1 mutation spectrum.

Assessment of serum vitamin D levels in children aged 0-17 years old in a Chinese population: a comprehensive study.

Vitamin D deficiency and insufficiency pose global public health challenges, yet research on serum vitamin D levels in the 0-17-year-old age group in southeastern China remains limited. This study aimed to fill this gap by investigating serum 25(OH)D levels in children in the region aged 0-17 years, contributing crucial data for understanding vitamin D nutritional status. Liquid chromatography‒mass spectrometry/mass spectrometry (LC‒MS/MS) technology was used. Vitamin D testing was integrated into routine diagnostic procedures for 11,116 children in Wujiang District, Suzhou City. Among the 0-17-year age group, comprising 6348 boys and 4768 girls, the prevalence of serum 25(OH)D deficiency and insufficiency was 21.4% and 31.0%, respectively. The median serum 25(OH)D concentration was 29.72 ng/mL (21.84-39.84 ng/mL) in boys compared to 28.48 ng/mL (20.65-39.23 ng/mL) in girls. Seasonal variations were observed, with median serum 25(OH)D concentrations of 29.02 ng/mL (20.73-39.72 ng/mL) in spring, 28.79 ng/mL (21.53-39.37 ng/mL) in summer, 30.12 ng/mL (22.00-39.70 ng/mL) in autumn, and 28.58 ng/mL (19.97-39.46 ng/mL) in winter. Statistically significant differences were noted in the serum 25(OH)D levels during autumn and winter. In conclusion, the rate of adequate vitamin D levels in local children was 47.5%, revealing a relatively high prevalence of vitamin D deficiency (21.4%) and insufficiency (31.0%), especially during the post-preschool period. Advocating for vitamin D supplementation in children is crucial for ensuring adequate vitamin D support.

T-cell infiltration in the central nervous system and their association with brain calcification in Slc20a2-deficient mice.

Primary familial brain calcification (PFBC) is a rare neurodegenerative and neuropsychiatric disorder characterized by bilateral symmetric intracranial calcification along the microvessels or inside neuronal cells in the basal ganglia, thalamus, and cerebellum. Slc20a2 homozygous (HO) knockout mice are the most commonly used model to simulate the brain calcification phenotype observed in human patients. However, the cellular and molecular mechanisms related to brain calcification, particularly at the early stage much prior to the emergence of brain calcification, remain largely unknown. In this study, we quantified the central nervous system (CNS)-infiltrating T-cells of different age groups of Slc20a2-HO and matched wild type mice and found CD45+CD3+ T-cells to be significantly increased in the brain parenchyma, even in the pre-calcification stage of 1-month-old -HO mice. The accumulation of the CD3+ T-cells appeared to be associated with the severity of brain calcification. Further immunophenotyping revealed that the two main subtypes that had increased in the brain were CD3+ CD4- CD8- and CD3+ CD4+ T-cells. The expression of endothelial cell (EC) adhesion molecules increased, while that of tight and adherents junction proteins decreased, providing the molecular precondition for T-cell recruitment to ECs and paracellular migration into the brain. The fusion of lymphocytes and EC membranes and transcellular migration of CD3-related gold particles were captured, suggesting enhancement of transcytosis in the brain ECs. Exogenous fluorescent tracers and endogenous IgG and albumin leakage also revealed an impairment of transcellular pathway in the ECs. FTY720 significantly alleviated brain calcification, probably by reducing T-cell infiltration, modulating neuroinflammation and ossification process, and enhancing the autophagy and phagocytosis of CNS-resident immune cells. This study clearly demonstrated CNS-infiltrating T-cells to be associated with the progression of brain calcification. Impairment of blood-brain barrier (BBB) permeability, which was closely related to T-cell invasion into the CNS, could be explained by the BBB alterations of an increase in the paracellular and transcellular pathways of brain ECs. FTY720 was found to be a potential drug to protect patients from PFBC-related lesions in the future.

High-Dose Versus Standard-Dose Intensity-Modulated Radiotherapy With Concurrent Paclitaxel Plus Carboplatin for Patients With Thoracic Esophageal Squamous Cell Carcinoma: A Randomized, Multicenter, Open-Label, Phase 3 Superiority Trial.

PURPOSE: The standard dose (SD) of definitive concurrent chemoradiotherapy (dCRT) remains 50.4 Gy in patients with esophageal cancer; a higher dose, when applied with conventional radiation therapy techniques, increases toxicities without improving survival. We investigated whether a high dose of 59.4 Gy using intensity-modulated radiation therapy (IMRT) would improve survival without increasing toxicities. METHODS: Patients with inoperable thoracic esophageal squamous cell carcinoma (SCC) referred for dCRT were randomly assigned (1:1) to high-dose (HD) IMRT (59.4 Gy) or SD IMRT (50.4 Gy). Chemotherapy consisted of 6 cycles of concurrent weekly paclitaxel and carboplatin and a maximum of 2 cycles of consolidation chemotherapy. Nutritional intervention was implemented for patients with malnutrition on the basis of nutritional screening. The primary endpoint was median overall survival (mOS). Analyses were by modified intention to treat. RESULTS: Between April 30, 2016, and April 30, 2019, 167 patients were enrolled at 9 participating centers in China. Seventy-one patients in the HD and 73 patients in the SD groups were included in the analysis; 86.8% of the patients completed radiation therapy and 70.1% received 5 or 6 cycles of concurrent chemotherapy. The median follow-up was 36.0 months. The mOS was 28.1 and 26.0 months in the HD and SD arms, respectively (P = .54). A total of 7 treatment-related deaths were observed. Grade 3 or worse treatment-related toxicities were observed in 62% and 68.5% of the patients in the HD and SD arms, respectively (P = .675). CONCLUSIONS: For patients with inoperable thoracic esophageal SCC, a dose of 59.4 Gy did not improve survival compared with the SD of dCRT using IMRT.

Case report: Kidney perivascular epithelioid cell tumor treated with anti-VEGFR tyrosine kinase inhibitor and MTOR inhibitor.

Perivascular epithelioid cell tumors (PEComas) are rare mesenchymal tumors arising from perivascular epithelial cells. There was no standard treatment for unresectable PEComa before 2021. For a low incidence and a rarely curable disease, development of new therapy is essential. A 45-year-old female was diagnosed with malignant renal PEComa (likely with TFE3 rearrangement) that underwent rapid progression after 10 months of surgery. The patient then received the tyrosine kinase inhibitor (TKI) Apatinib, and the tumor remained stable for 15 months before another progression. The patient then received the MTOR inhibitor everolimus that alleviated her symptoms but the tumor went into remission again after another 15 months. This result suggests that antagonizing the vascular endothelial growth factor receptor (VEGFR) pathway be a useful strategy for malignant PEComas, along with the MTOR pathway inhibition that had recently been approved for the rare tumor.

Clinical and genetic findings in patients with congenital cataract and heart diseases.

BACKGROUND: Congenital cataract (CC) and congenital heart disease (CHD) are significant birth defects. In clinical practice, the concurrence of CC and CHD is frequently observed in patients. Additionally, some monogenic diseases, copy number variation (CNV) syndromes, and diseases associated with intrauterine infection involve both cataract and heart defects. However, little is known about the association between CC and CHD. Here, we characterised the demographic, clinical, and genetic features of patients with CC and heart defects. METHODS: Medical records for 334 hospitalised patients diagnosed with CC were reviewed. Demographic and clinical features of patients with CC with and without CHD were compared. Clinical and genomic information for patients with 'cataract' and 'cardiac defects' were reviewed from Database of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources (DECIPHER). Microarray-based comparative genomic hybridisation and whole-exome sequencing were performed in 10 trio families with CC and CHD to detect de novo genomic alterations, including copy number variants and single nucleotide changes. RESULTS: In a retrospective analysis of 334 patients with CC over the past 10 years at our hospital, we observed a high proportion of patients (41.13%) with CHD (including innocent CHD, which reported as left-to-right shunt in echocardiography test). The CC with CHD group had higher incidences of preterm birth and Down's syndrome than the CC without CHD group. Atrial septal defect was the most frequent heart defect. A total of 44 cases with cataracts and heart diseases were retrieved from Database of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources (DECIPHER). In total, 52 genomic alterations were reported, 44% of which were de novo germline variants. In the 10 trio families with CC and CHD, we found de novo CNVs responsible for two well-known chromosomal disorders and identified a novel pathogenic mutation in GJA8 responsible for CC. CONCLUSIONS: We observed significant associations between CHD and CC in our 10-year patient cohort. Based on the cohort and data from DECIPHER, developmental syndromes in some patients were due to genetic defects, thus explaining the concurrence of CC and CHD. Additionally, we detected de novo mutations as an independent cause of cataracts. Our findings suggest that developmental syndromes in patients with CC deserve more attention in clinical practice by ophthalmologists.

Case Report: Two Novel Frameshift Mutations in SLC20A2 and One Novel Splice Donor Mutation in PDGFB Associated With Primary Familial Brain Calcification.

Primary familial brain calcification (PFBC, OMIM#213600), also known as Fahr's disease, is characterized by bilateral and symmetric brain calcification in the basal ganglia (globus pallidus, caudate nucleus, and putamen), thalamus, subcortical white matter, and cerebellum. PFBC can be caused by loss-of-function mutations in any of the six known causative genes. The most common clinical manifestations include movement disorders, cognitive impairment, and neuropsychiatric signs that gradually emerge in middle-aged patients. To broaden the PFBC mutation spectrum, we examined nine members of a family with PFBC and two sporadic cases from clinical departments, and sequenced all PFBC-causative genes in the index case. Two novel frameshift mutations in SLC20A2 [NM_001257180.2; c.806delC, p.(Pro269Glnfs*49) and c.1154delG, p.(Ser385Ilefs*70)] and one novel splice donor site mutation (NM_002608.4, c.456+1G>C, r.436_456del) in PDGFB were identified in the patient cohort. c.806delC co-segregated with brain calcification and led to SLC20A2 haploinsufficiency among the affected family members. The c.456+1G>C mutation in PDGFB resulted in aberrant mRNA splicing, thereby forming mature transcripts containing an in-frame 21 base pair (bp) deletion, which might create a stably truncated protein [p.(Val146_Gln152del)] and exert a dominant negative effect on wild-type PDGFB. All three mutations were located in highly conserved regions among multiple species and predicted to be pathogenic, as evaluated by at least eight common genetic variation scoring systems. This study identified three novel mutations in SLC20A2 and PDGFB, which broadened and enriched the PFBC mutation spectrum.

Slc20a2-Deficient Mice Exhibit Multisystem Abnormalities and Impaired Spatial Learning Memory and Sensorimotor Gating but Normal Motor Coordination Abilities.

BACKGROUND: Primary familial brain calcification (PFBC, OMIM#213600), also known as Fahr's disease, is a rare autosomal dominant or recessive neurodegenerative disorder characterized by bilateral and symmetrical microvascular calcifications affecting multiple brain regions, particularly the basal ganglia (globus pallidus, caudate nucleus, and putamen) and thalamus. The most common clinical manifestations include cognitive impairment, neuropsychiatric signs, and movement disorders. Loss-of-function mutations in SLC20A2 are the major genetic causes of PFBC. OBJECTIVE: This study aimed to investigate whether Slc20a2 knockout mice could recapitulate the dynamic processes and patterns of brain calcification and neurological symptoms in patients with PFBC. We comprehensively evaluated brain calcifications and PFBC-related behavioral abnormalities in Slc20a2-deficient mice. METHODS: Brain calcifications were analyzed using classic calcium-phosphate staining methods. The Morris water maze, Y-maze, and fear conditioning paradigms were used to evaluate long-term spatial learning memory, working memory, and episodic memory, respectively. Sensorimotor gating was mainly assessed using the prepulse inhibition of the startle reflex program. Spontaneous locomotor activity and motor coordination abilities were evaluated using the spontaneous activity chamber, cylinder test, accelerating rotor-rod, and narrowing balance beam tests. RESULTS: Slc20a2 homozygous knockout (Slc20a2-HO) mice showed congenital and global developmental delay, lean body mass, skeletal malformation, and a high proportion of unilateral or bilateral eye defects. Brain calcifications were detected in the hypothalamus, ventral thalamus, and midbrain early at postnatal day 80 in Slc20a2-HO mice, but were seldom found in Slc20a2 heterozygous knockout (Slc20a2-HE) mice, even at extremely old age. Slc20a2-HO mice exhibited spatial learning memory impairments and sensorimotor gating deficits while exhibiting normal working and episodic memories. The general locomotor activity, motor balance, and coordination abilities were not statistically different between Slc20a2-HO and wild-type mice after adjusting for body weight, which was a major confounding factor in our motor function evaluations. CONCLUSION: The human PFBC-related phenotypes were highly similar to those in Slc20a2-HO mice. Therefore, Slc20a2-HO mice might be suitable for the future evaluation of neuropharmacological intervention strategies targeting cognitive and neuropsychiatric impairments.

LRG1 Suppresses Migration and Invasion of Esophageal Squamous Cell Carcinoma by Modulating Epithelial to Mesenchymal Transition.

Background: Esophageal squamous cell carcinoma (ESCC) is a common cancer with poor prognosis. The molecular pathogenesis underlying ESCC remains to be explored. Leucine-rich ɑ-2-glycoprotein 1 (LRG1) has been implicated in the pathogenesis of various cancer types, however its role in ESCC is unknown. Materials and Methods: Data from the public database was analyzed to address the expression of LRG1 in ESCC. Gain-of-function studies were performed in select ESCC cell lines by over-expression or addition of recombinant LRG1, while loss-of-function studies achieved by small interfering RNA mediated knockdown. Wound healing and transwell assays were conducted to investigate ESCC cell migration and invasion upon manipulating LRG1 levels. Western blot and Immunofluorescence staining were used to examine the changes in epithelial to mesenchymal transition (EMT) and TGFß signaling pathway. Results: LRG1 mRNA levels were found to be significantly down-regulated in patients with ESCC as well as in several ESCC cell lines. Silencing of LRG1 promoted, while overexpression of LRG1 inhibited ESCC cell migration and invasion. In line with this, Silencing of LRG1 enhanced, while overexpression of LRG1 reduced TGFß signaling and EMT of ESCC cells. Conclusion/Significance: LRG1 suppresses ESCC cell migration and invasion via negative modulation of TGFß signaling and EMT. Down-regulation of LRG1 in ESCC patients may favor tumor metastasis and disease progression.

[Experimental study on co-culture of early embryo and cancer cells in vitro].

The co-culture system of early embryos and cancer cells is an important means to observe the biological behavior changes of embryos and cancer cells in vitro. In this study, we co-cultured the 3.5 dpc mouse embryo with malignant tumor cells, investigated the development of blastocyst by observing the hatchment, attachment and outgrowth, observed the biological behavior changes of cancer cells in the embryonic circumstances, and detected the proliferation and apoptosis of cancer cells. Compared with the control, the embryos developed normally in the tumor environments, and the rate of hatchment, attachment and outgrowth increased significantly (P<0.05). However, there was no significant change of cancer cells in morphology, proliferation and apoptosis in the co-culture system (P>0.05). Under the co-culture system, the early embryo developed normally, and the cancer cells also grew well. There may be similarities between the embryos and cancer cell's choice for living. Moreover, the growth of embryos could be promoted by cancer cells in the co-culture system. This might be related to the similarities of gene expression, growth factors and signal transduction mechanisms between embryos and cancer cells.