Dexamethasone protects against asthma via regulating Hif-1α-glycolysis-lactate axis and protein lactylation.

PURPOSE: Asthma can not be eradicated till now and its control primarily relies on the application of corticosteroids. Recently, glycolytic reprogramming has been reportedly contributed to asthma, this study aimed to reveal whether the effect of corticosteroids on asthma control is related to their regulation of glycolysis and glycolysis-dependent protein lactylation. METHODS: Ovalbumin (OVA) aeroallergen was used to challenge mice and stimulate human macrophage cell line THP-1 following dexamethasone (DEX) treatment. Airway hyperresponsiveness, airway inflammation, the expressions of key glycolytic enzymes and pyroptosis markers, the level of lactic acid, real-time glycolysis and oxidative phosphorylation (OXPHOS), and protein lactylation were analyzed. RESULTS: DEX significantly attenuated OVA-induced eosinophilic airway inflammation, including airway hyperresponsiveness, leukocyte infiltration, goblet cell hyperplasia, Th2 cytokines production and pyroptosis markers expression. Meanwhile, OVA-induced Hif-1α-glycolysis axis was substantially downregulated by DEX, which resulted in low level of lactic acid. Besides, key glycolytic enzymes in the lungs of asthmatic mice were notably co-localized with F4/80-positive macrophages, indicating metabolic shift to glycolysis in lung macrophages during asthma. This was confirmed in OVA-stimulated THP-1 cells that DEX treatment resulted in reductions in pyroptosis, glycolysis and lactic acid level. Finally, protein lactylation was found significantly increased in the lungs of asthmatic mice and OVA-stimulated THP-1 cells, which were both inhibited by DEX. CONCLUSION: Our present study revealed that the effect of DEX on asthma control was associated with its suppressing of Hif-1α-glycolysis-lactateaxis and subsequent protein lactylation, which may open new avenues for the therapy of eosinophilic asthma.

Melatonin regulates circadian clock proteins expression in allergic airway inflammation.

Asthma demonstrates a strong circadian rhythm with disrupted molecular clock. Melatonin which can directly regulate circadian rhythm has been reported to alleviate asthma, but whether this effect is related to its regulation on circadian clock has not yet been known. Here, female C57BL/6 mice were challenged with ovalbumin (OVA) to establish allergic airway inflammation, and were treated with melatonin or Luzindole to investigate whether the expressions of circadian clock proteins were changed in response to OVA and were affected by exogenous/endogenous melatonin. Airway inflammation, mucus secretion, protein expressions of circadian proteins (Bmal1, Per1, Clock, Timeless, Cry1 and Cry2), melatonin biosynthetase (ASMT, AANAT) and melatonin receptor (Mel-1A/B-R) were analyzed accordingly. The results showed that in the successfully established allergic airway inflammation model, inflammatory cells infiltration, expressions of circadian clock proteins in the lung tissues of OVA-challenged mice were all notably up-regulated as compared to that of the vehicle mice. Meanwhile, the protein expression of ASMT and the level of melatonin in the lung tissues were reduced in allergic mice, while the expression of melatonin receptor Mel-1A/B-R was markedly increased. After addition of exogenous melatonin, the OVA-induced airway inflammation was pronouncedly ameliorated, while simultaneously the OVA-induced expressions of Per1 and Clock were further increased. However, a melatonin receptor antagonist Luzindole further augmented the OVA-induced airway inflammation, accompanied with remarkably decreased expressions of Per1, Bmal1, Cry1 and Cry2 but notably increased expression of Timeless. Collectively, our results demonstrated that the expression of circadian clock proteins was increased in the lungs during allergic airway inflammation, and Per1 was a clock protein that can be regulated by both exogenous and endogenous melatonin, suggesting Per1 may be an important potential circadian clock target for melatonin as a negative regulatory factor against Th2-type airway inflammation.

Genetic and clinical characterization of familial renal glucosuria.

Background: Familial renal glucosuria (FRG) is a hereditary disorder caused by variants in SLC5A2 encoding sodium-glucose cotransporter 2 (SGLT2). In this study, we aimed to characterize proximal tubule solute transport, glucagon secretion and the genotype-phenotype relationship in FRG patients. Methods: We sequenced SLC5A2 and PDZK1IP1 in 21 FRG patients and measured the renal threshold of glucose (RTG) in 15 patients. We built an open-source online calculator of RTG, evaluated the proximal tubule transport of amino acid, uric acid and phosphate, and explored glucagon secretion after glucose ingestion in FRG patients. Results: We identified 12 novel SLC5A2 variants (G484D, R564W, A212S, c.574+1G>C, W649*, S592Cfs*6, Q579*, Y339*, V39F, G491E, A464E and G360D) in our cohort and yielded 111 SLC5A2 variants from literature review. RTG in our cohort ranged from 1.0 to 9.2 mmol/L. Patients with two SLC5A2 variants had lower RTG (3.9 vs 6.2 mmol/L) and higher 24-h urinary glucose excretion (24hUG) than single-variant carriers (291.0 vs 40.0 mmol/1.73 m2). Patients with homozygous missense or in-frame indels had mean 24hUG of 457.2 mmol/1.73 m2, comparable to those with homozygous truncating variants (445.0 mmol/1.73 m2) and significantly more than those with homozygous splicing variants (196.6 mmol/1.73 m2). Patients with homozygous missense variants involving conservative residues (582.0 mmol/1.73 m2) had more 24hUG than those with variants at non-conservative residues (257.6 mmol/1.73 m2). Four out of 14 tested patients had mild aminoaciduria. The RTG of FRG patients had no significant correlation to phosphate reabsorption but a potential negative correlation to the fractional excretion of uric acid. Postprandial suppression of glucagon secretion was absent in most FRG patients. Conclusions: We built a comprehensive map showing the impact of SLC5A2 variant type and variant location on glucosuria severity. Our results highlighted the role of key residues in maintaining the transport function of SGLT2 and the functional link between glucosuria and reabsorption of amino acid and uric acid in FRG patients.

Model development and validation of noninvasive parameters based on coronary computed tomography angiography to predict culprit lesions in acute coronary syndromes within 3 years: value of plaque characteristics, hemodynamics and pericoronary adipose tissue.

Background: Machine learning (ML) is combined with noninvasive parameters from coronary computed tomography angiography (CTA) to construct predictive models to identify culprit lesions that may lead to acute coronary syndrome (ACS). Methods: We retrospectively analyzed 132 patients with ACS at the Fourth Affiliated Hospital of Harbin Medical University who had coronary CTA between 3 months and 3 years before the ACS event, with a total of 240 lesions. Lesions from 2020 (n=154) were included in the training set, and lesions from 2021 (n=86) were included in the test set for internal validation. We evaluated the role of plaque characteristics, hemodynamic parameters and pericoronary adipose tissue (PCAT) attenuation from CTA in identifying culprit ACS lesions. In the training set, logistic regression was used to screen CTA-derived parameters with P values <0.05 for the model construction. Logistic regression, random forest, Bayesian and K-nearest neighbor algorithms were used to build classification models, and their performance was assessed using the test set. The following models were established to evaluate the effectiveness of different combinations of models to identify culprit lesions: Model 1 was established for plaque characteristics; Model 2 was established for hemodynamic parameters; Model 3 was established for PCAT attenuation; Model 4 was established for plaque characteristics and hemodynamic parameters; and Model 5 was established for plaque characteristics, hemodynamic parameters and PCAT attenuation. Results: A total of ten high-risk factors were screened for the ML model construction, and the ML model based on the logistic regression algorithm had the best performance among the four algorithms (accuracy =0.721; sensitivity =0.892; specificity =0.592; positive prediction =0.623; and negative prediction =0.879). In this model, the minimum lumen area, positive remodeling and lesion-specific fat attenuation index (FAI) were the risk factors significantly associated with the culprit lesion. Analysis of the effect of different combinations of models to identify culprit lesions showed that Model 5 had the best predictive effect (AUC =0.819 and 95% CI: 0.722-0.916). Conclusions: ACS can be predicted using ML based on CTA parameters. Compared to other models, the model combining plaque characteristics, hemodynamic parameters and PCAT attenuation performed best in predicting the culprit lesion.

Identification of patients with acute coronary syndrome and representation of their degree of inflammation: application of pericoronary adipose tissue within different radial distances of the proximal coronary arteries.

Background: Pericoronary adipose tissue (PCAT) around the proximal right coronary artery (RCA) is considered a marker of coronary inflammation. We aimed to explore the segments of PCAT that represent coronary inflammation in patients with acute coronary syndrome (ACS) and to identify patients with ACS and stable coronary artery disease (CAD) prior to intervention. Methods: We retrospectively enrolled consecutive patients with ACS and stable CAD who underwent invasive coronary angiography (ICA) after coronary computed tomography angiography (CCTA) from November 2020 to October 2021 at the Fourth Affiliated Hospital of Harbin Medical University. The fat attenuation index (FAI) was obtained using PCAT quantitative measurement software, and the coronary Gensini score was also calculated to indicate the severity of CAD. The differences and correlations between FAI within different radial distances of proximal coronary arteries were evaluated, and the recognition ability of FAI for patients with ACS and stable CAD was evaluated by establishing receiver operator characteristic (ROC) curves. Results: A total of 267 patients were included in the cross-sectional study, including 173 patients with ACS. With the increase of radial distance from the outer wall of proximal coronary vessels, the FAI decreased (P<0.001). The FAI around the proximal left anterior descending artery (LAD) within the reference diameter from the outer wall of the vessel (LADref) had the highest correlation with the FAI around culprit lesions [r=0.587; 95% confidence interval (CI): 0.489-0.671; P<0.001]. The model based on clinical features, Gensini score, and LADref had the highest recognition performance for patients with ACS and stable CAD [area under the curve (AUC): 0.663; 95% CI: 0.540-0.785]. Conclusions: LADref is most correlated with FAI around culprit lesions in patients with ACS and has higher value in the preintervention differentiation of patients with ACS and stable CAD compared to the use of clinical features alone.

Genetic Screening of Targeted Region on the Chromosome 22q11.2 in Patients with Microtia and Congenital Heart Defect.

Microtia is a congenital malformation characterized by a small, abnormally shaped auricle (pinna) ranging in severity. Congenital heart defect (CHD) is one of the comorbid anomalies with microtia. However, the genetic basis of the co-existence of microtia and CHD remains unclear. Copy number variations (CNVs) of 22q11.2 contribute significantly to microtia and CHD, respectively, thus suggesting a possible shared genetic cause embedded in this genomic region. In this study, 19 sporadic patients with microtia and CHD, as well as a nuclear family, were enrolled for genetic screening of single nucleotide variations (SNVs) and CNVs in 22q11.2 by target capture sequencing. We detected a total of 105 potential deleterious variations, which were enriched in ear- or heart-development-related genes, including TBX1 and DGCR8. The gene burden analysis also suggested that these genes carry more deleterious mutations in the patients, as well as several other genes associated with cardiac development, such as CLTCL1. Additionally, a microduplication harboring SUSD2 was validated in an independent cohort. This study provides new insights into the underlying mechanisms for the comorbidity of microtia and CHD focusing on chromosome 22q11.2, and suggests that a combination of genetic variations, including SNVs and CNVs, may play a crucial role instead of single gene mutation.

Functional Pathway and Process Enrichment Analysis of Genes Associated With Morphological Abnormalities of the Outer Ear.

Congenital anomalies of the outer ear are common birth defects, including a variety of congenital deformities or malformations ranging from mild structural anomalies to total absence of the ear. Despite its high incidence and detrimental impact on patients, the etiology of outer ear abnormalities remains poorly understood. The goal of this study was to summarize the related genes and improve our understanding of the genetic etiology of morphological abnormalities of the outer ear. Human Phenotype Ontology (HPO) database, Mouse Genome Informatics (MGI) database, and PubMed search engine were used to acquire the genes associated with abnormal human or mouse outer ear. Metascape was employed on the genes above to conduct functional annotation, pathway and process enrichment analysis, protein-protein interaction network analysis, and MCODE component analysis. After a comprehensive review of the databases and literature, we identified 394 human genes and 148 mouse genes that have been associated with abnormal phenotypes of the outer ear, and we identified several biological pathways for human and mouse respectively. Especially, the analysis of common genes shared by human and mouse emphasized the importance of certain genes ( PAX6 , PBX1 , HOXA1 , HOXA2 , TBX1 , TBX15 , PRRX1 , and HMX1 ) in the embryonic development of the external ear. Through our analysis of genes associated with morphological abnormalities of the outer ear, the authors have shown that embryonic development pathways take important roles in the morphogenesis of abnormal external ear and highlighted some potential genetic drivers.

[Elevational Pattern and Control Factors of Soil Microbial Carbon Use Efficiency in the Daiyun Mountain].

Microbial carbon use efficiency (CUE) refers to the C transformation to microbial biomass from C uptake. The study of soil microbial CUE is very important for understanding the soil C cycle. Here, CUE, Cgrowth, and Crespiration were measured using the 18O-H2O-DNA labeling method at six elevational sites (980-1765 m) in Daiyun Mountain, a subtropical montane forest, to understand the variation characteristics and influencing mechanisms. The results showed that:CUE varied from 0.1 to 0.4 and increased linearly with elevation; CUE was positively correlated with Cgrowth, Crespiration, and qgrowth but negatively correlated with qCO2, indicating that CUE increased with elevation by increasing microbial growth and inhibiting respiration; and temperature was the first controlling factor for the elevation variation in microbial CUE in the subtropical forest ecosystem.

Effect of 320-row CT reconstruction technology on fractional flow reserve derived from coronary CT angiography based on machine learning: single- versus multiple-cardiac periodic images.

Background: Fractional flow reserve derived from computed tomography (CT-FFR) can be used to noninvasively evaluate the functions of coronary arteries and has been widely welcomed in the field of cardiovascular research. However, whether different image reconstruction schemes have an effect on CT-FFR analysis through single- and multiple-cardiac periodic images in the same patient has not been investigated. Methods: This study retrospectively enrolled 122 patients who underwent 320-row computed tomography (CT) examination with both single- and multiple-cardiac periodic reconstruction schemes; a total of 366 coronary arteries were analyzed. The lowest CT-FFR values of each vessel and the poststenosis CT-FFR values of the lesion-specific coronary artery were measured using the two reconstruction techniques. The Wilcoxon signed-rank test was used to compare differences in CT-FFR values between the two reconstruction techniques. Spearman correlation analysis was performed to determine the relationship between CT-FFR values derived using the two methods. Bland-Altman and intraclass correlation coefficient (ICC) analyses were performed to evaluate the consistency of CT-FFR values. Results: In all blood vessels, the lowest CT-FFR values showed no significant differences between the two reconstruction techniques in the left anterior descending artery (LAD; P=0.65), left circumflex artery (LCx; P=0.46), or right coronary artery (RCA; P=0.22). In blood vessels with atherosclerotic plaques, the poststenosis CT-FFR values (2 cm distal to the maximum stenosis) exhibited no significant differences between the two reconstruction techniques in the LAD (P=0.78), LCx (P=1.00), or RCA (P=1.00). The mean CT-FFR values of single- and multiple-cardiac periodic images showed excellent correlation and minimal bias in all groups. Conclusions: CT-FFR analysis based on an artificial intelligence deep learning neural network is stable and not affected by the type of 320-row CT reconstruction technology.

Identification of patients with acute myocardial infarction based on coronary CT angiography: the value of pericoronary adipose tissue radiomics.

OBJECTIVE: To determine whether radiomics analysis of pericoronary adipose tissue (PCAT) captured by coronary computed tomography angiography (CCTA) could discriminate acute myocardial infarction (MI) from unstable angina (UA). METHODS: In a single-center retrospective case-control study, patients with acute MI (n = 105) were matched to patients with UA (n = 105) and all patients were randomly divided into training and validation cohorts with a ratio of 7:3. Fat attenuation index (FAI) and PCAT radiomics features selected by Max-Relevance and Min-Redundancy (mRMR) and least absolute shrinkage and selection operator (LASSO) around the proximal three major epicardial coronary vessels (LAD [left anterior descending artery], LCx [left circumflex artery], and RCA [right coronary artery]) were used to build logistic regression models. Finally, a FAI model, three radiomics models of PCAT (LAD, LCx, and RCA), and a combined model that used the scores of these independent models were constructed. The performance of the models was evaluated by identification, calibration, and clinical application. RESULTS: In training and validation cohorts, compared with the FAI model (AUC = 0.53, 0.50), the combined model achieved superior performance (AUC = 0.97, 0.95) while there was a significant difference of AUC between two models (p < 0.05). The calibration curves of the combined model demonstrated the smallest Brier score loss. Decision curve analysis suggested that the combined model provided higher clinical benefit than the FAI model. CONCLUSIONS: The CCTA-based radiomics phenotype of PCAT outperforms the FAI model in discriminating acute MI from UA. The combination of PCAT radiomics and FAI could further enhance the performance of acute MI identification. KEY POINTS: • Fat attenuation index based on CCTA can detect inflammation-induced changes in the ratio of lipid to aqueous phase in pericoronary adipose tissue. • Fat attenuation index cannot distinguish acute MI patients from UA patients, suggesting that the two groups have the same degree of ratio of lipid to aqueous phase in pericoronary adipose tissue. • Radiomics features of PCAT have the potential to distinguish acute MI patients from UA patients.

Transcriptomic analysis of the upper lip and primary palate development in mice.

Background: Normal fusion of the upper lip and primary palate is a complex process involving a series of characteristic and orderly regulated cellular events. Cleft lip with or without palate (CL/P), one of the most common congenital malformations, may be induced by abnormalities in any of these events. However, less is known about the precise regulatory process in the fusion of the upper lip and primary palate. Methods: Lambdoidal junction tissues of mice from embryonic days 10.5, 11.5, and 12.5- three key fusion stages-were acquired for RNA sequencing. Results: Gene expression profiles in distinct fusion stages of mice were identified. Some of the differentially expressed genes (DEGs) have been reported to affect upper lip and primary palate development. However, other DEGs, such as Krt5, Pax1, Ambn, Hey2, and Tnmd, have not previously been investigated. Gene set enrichment analysis (GSEA) of these DEGs revealed the sequential intensification of Wnt, PI3K-Akt, MAPK, Hippo, and TGF-beta signaling pathways and identified relatively highly expressed genes including Tnn, Wnt3a, and Wnt16. We also observed substantial alternative splicing events during the fusion of the upper lip and primary palate and identified potentially important genes including Gtpbp8, Armcx1, Tle3, and Numa1. Protein-protein interaction (PPI) network analysis identified a series of hub genes, including Col1a2, Fos, Bmp2, Shh, Col1a1, Wnt3a, Anxa1, Gem, etc. Conclusion: Overall, the results of this study provided a comprehensive analysis of the development of the upper lip and primary palate. Our work provides insight into future studies of normal upper lip and primary palate development and the etiology of CL/P.

De novo 22q11.2 deletions and auricular findings in two Chinese patients with microtia.

BACKGROUND: Congenital microtia is a common craniofacial malformation resulting from both environmental and genetic factors. Recurrent chromosomal imbalances were observed in patients with microtia. The 22q11.2 deletion is one of the most common microdeletions in human beings. The cell division cycle 45 gene (CDC45) embedded in the proximal 22q11.2 deleted region is involved in craniofacial development. However, only a few studies have focused on the 22q11.2 deletion as genetic etiology in microtia patients and studied its associated external ear deformity characteristics in detail. METHODS: In this research, a total of 65 patients from north China with sporadic microtia were studied. Copy number variations of CDC45 were screened using AccuCopy assay. The 22q11.2 deletion harboring CDC45 was identified by whole-genome sequencing and targeted next-generation sequencing. A parental test was carried out to determine the origin of the deletion. RESULTS: CDC45 copy number loss was identified in two patients with microtia. A set of qPCR assays demonstrated two patients carried a typical proximal 22q11.2 deletion between the low-copy repeats on chromosome 22q11.2 (LCR22A and LCR22D), encompassing CDC45. The 22q11.2 deletions were de novo in each patient. In-depth auricular phenotype assessment showed these two patients have a distinct concha-type ear malformation while other microtia patients have lobule-type microtia among the 65 microtia patient cohort in this study. CONCLUSION: Here we present two additional Chinese microtia patients with de novo 22q11.2 proximal deletion harboring CDC45 and further report these patients' distinct ear malformation.

Heterogeneity of Accompanying Phenotypes and Genomic Variants Involved in Microtia.

OBJECTIVES: The symptoms associated with microtia are ever-changing and not to stick to 1 pattern. The symptoms associated with microtia are constantly changing and are not set in stone. The aim of this article was to describe the various phenotypes from multiple systems found in microtitis patients included in the DatabasE of genomiC varIation and Phenotype in Humans using Ensembl Resources database, and to analyze possible pathogenic mutations. METHODS: DatabasE of genomiC varIation and Phenotype in Humans using Ensembl Resources is an interactive web-based database, which incorporates a suite of tools designed to aid the interpretation of genomic variants. The term "microtia" was used as the search term, and the data extracted from the DatabasE of genomiC varIation and Phenotype in Humans using Ensembl Resources for this study was updated until October 2020. Pearson chi-squared test was used to test associations between types of genomic variants and the pathogenicity of variants. RESULTS: Of the 386 cases enrolled in the study, 99% (n = 382) had 1 or more associated abnormalities. The most frequently detected abnormalities were those of the face and neck (n = 362 [93.8% of all cases]); musculoskeletal system (n = 337 [87.3%]); and nervous system (n = 334 [86.5%]), followed by abnormalities of limbs (n = 252 [65.3%]); the eye (n = 212 [54.9%]); and the integument (n = 200 [51.8%]). Besides, a total of 479 genomic variants were determined, including sequence variants and copy number variants (loss and gain). The pathogenicity of loss-type variants was significantly higher among other types (P < 0.001). Twelve sharing variants had more than 5 repeats, and the repeated fragments were concentrated on chromosome 3, 7, 9, 10, 11, 15, 17, 18, and 22. CONCLUSIONS: Identification of the relation between phenotypes and genotypes will facilitate the uncovering of the mechanism of microtia and the study of potential therapeutic targets.

Generation of a human induced pluripotent stem cell line (PSHi002-A) from a Treacher-Collins syndrome patient carrying a TCOF1 gene mutation (c.1966_1969dup).

Mutations of the Treacle Ribosome Biogenesis Factor 1 (TCOF1) gene can lead to Treacher Collins syndrome (TCS). In present study, the peripheral blood mononuclear cells (PBMCs) of a 33-year-old male TCS patient with the heterozygous TCOF1 mutation c.1966_1969dup (p.Ser657Trpfs*25) were reprogrammed into induced pluripotent stem cells (iPSCs) named PSHi002-A through episomal plasmids encoding hOCT4, hSOX2, hNANOG, hLIN28, hKLF4, and hL-MYC. The established iPSC line expressed pluripotent markers, had a normal karyotype (46, XY), and can be differentiated into the three germ layers in vivo.

Generation of an induced pluripotent stem cell line from a congenital microtia patient with 4p16.1 microduplication involving the long-range enhancer of HMX1.

Congenital microtia is a malformation of the middle and external ear. Duplications involving the ECR, an ear-specific long-range enhancer of HMX1, lead to ear malformation in different species. Use of electroporation of episomal plasmids encodes OCT4, SOX2, NANOG, LIN28, KLF4, and LMYC into peripheral blood mononuclear cells (PBMCs), we generated an induced pluripotent stem cell (iPSCs) line of a microtia patient carrying the duplication involving ECR. The iPSCs express pluripotency markers, have the potential to differentiate into three germ layers, and show the normal karyotype. This patient-specific iPSC will be used for modeling the pathophysiology of ear malformation.

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.

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.

Identification of loss-of-function HOXA2 mutations in Chinese families with dominant bilateral microtia.

HOX genes are important regulatory genes patterning head formation, including development of the ear. Microtia is a congenital ear anomaly characterized by lacking all or part of the structures of the outer ear. To date, only four HOXA2 mutations were reported in families with autosomal-recessive or dominant microtia, with or without hearing impairment. More identified mutations are needed to confirm the correlation between genotype and phenotype. Here, we collect two Chinese families with non-syndromic bilateral microtia. Next generation sequencing identified two heterozygous nonsense HOXA2 mutations, one in each family. One mutation (c.637A > T, p.Lys213*) is newly reported, while the other one (c.703C > T,p.Gln235*) is consistent with a previous report. In mouse, Hoxa2 can bind to a long-range enhancer and regulate expression of the Hmx1 gene, which is a crucial transcription factor in eye and ear development. Using dual luciferase reporter assays, we showed that both HOXA2 mutations have impaired activation of the long-range enhancer of HMX1. In the present study, we report the first two bilateral non-syndromic microtia cases with HOXA2 mutations of Chinese origin and identify a novel mutation. Our results also provide molecular insights about how these nonsense HOXA2 mutations could affect the activation of its downstream target HMX1 by interacting with the long-range enhancer.