Core planar cell polarity genes VANGL1 and VANGL2 in predisposition to congenital vertebral malformations.

Congenital scoliosis (CS), affecting approximately 0.5 to 1 in 1,000 live births, is commonly caused by congenital vertebral malformations (CVMs) arising from aberrant somitogenesis or somite differentiation. While Wnt/ß-catenin signaling has been implicated in somite development, the function of Wnt/planar cell polarity (Wnt/PCP) signaling in this process remains unclear. Here, we investigated the role of Vangl1 and Vangl2 in vertebral development and found that their deletion causes vertebral anomalies resembling human CVMs. Analysis of exome sequencing data from multiethnic CS patients revealed a number of rare and deleterious variants in VANGL1 and VANGL2, many of which exhibited loss-of-function and dominant-negative effects. Zebrafish models confirmed the pathogenicity of these variants. Furthermore, we found that Vangl1 knock-in (p.R258H) mice exhibited vertebral malformations in a Vangl gene dose- and environment-dependent manner. Our findings highlight critical roles for PCP signaling in vertebral development and predisposition to CVMs in CS patients, providing insights into the molecular mechanisms underlying this disorder.

COL11A2 as a candidate gene for vertebral malformations and congenital scoliosis.

Human vertebral malformations (VMs) have an estimated incidence of 1/2000 and are associated with significant health problems including congenital scoliosis (CS) and recurrent organ system malformation syndromes such as VACTERL (vertebral anomalies; anal abnormalities; cardiac abnormalities; tracheo-esophageal fistula; renal anomalies; limb anomalies). The genetic cause for the vast majority of VMs are unknown. In a CS/VM patient cohort, three COL11A2 variants (R130W, R1407L and R1413H) were identified in two patients with cervical VM. A third patient with a T9 hemivertebra and the R130W variant was identified from a separate study. These substitutions are predicted to be damaging to protein function, and R130 and R1407 residues are conserved in zebrafish Col11a2. To determine the role for COL11A2 in vertebral development, CRISPR/Cas9 was used to create a nonsense mutation (col11a2L642*) as well as a full gene locus deletion (col11a2del) in zebrafish. Both col11a2L642*/L642* and col11a2del/del mutant zebrafish exhibit vertebral fusions in the caudal spine, which form due to mineralization across intervertebral segments. To determine the functional consequence of VM-associated variants, we assayed their ability to suppress col11a2del VM phenotypes following transgenic expression within the developing spine. While wildtype col11a2 expression suppresses fusions in col11a2del/+ and col11a2del/del backgrounds, patient missense variant-bearing col11a2 failed to rescue the loss-of-function phenotype in these animals. These results highlight an essential role for COL11A2 in vertebral development and support a pathogenic role for two missense variants in CS.

First copy number variant in trans with single nucleotide variant in CCN6 causing progressive pseudorheumatoid dysplasia revealed by genome sequencing and deep phenotyping in monozygotic twins.

Biallelic pathogenic variants in CCN6 cause progressive pseudorheumatoid dysplasia (PPD), a rare skeletal dysplasia. The predominant features include noninflammatory progressive joint stiffness and enlargement, which are not unique to this condition. Nearly 100% of the reported variants are single nucleotide variants or small indels, and missing of a second variant has been reported. Genome sequencing (GS) covers various types of variants and deep phenotyping (DP) provides detailed and precise information facilitating genetic data interpretation. The combination of GS and DP improves diagnostic yield, especially in rare and undiagnosed diseases. We identified a novel compound heterozygote involving a disease-causing copy number variant (g.112057664_112064205del) in trans with a single nucleotide variant (c.624dup(p.Cys209MetfsTer21)) in CCN6 in a pair of monozygotic twins, through the methods of GS and DP. The twins had received three nondiagnostic results before. The g.112057664_112064205del variant was missed by all the tests, and the recorded phenotypes were inaccurate or even misleading. The twins were diagnosed with PPD, ending a 13-year diagnostic odyssey. There may be other patients with PPD experiencing underdiagnosis and misdiagnosis due to inadequate genetic testing or phenotyping methods. This case highlights the critical role of GS and DP in facilitating an accurate and timely diagnosis.

Haplotype-specific MAPK3 expression in 16p11.2 deletion contributes to variable neurodevelopment.

Recurrent proximal 16p11.2 deletion (16p11.2del) is a risk factor for diverse neurodevelopmental disorders with incomplete penetrance and variable expressivity. Although investigation with human induced pluripotent stem cell models has confirmed disruption of neuronal development in 16p11.2del neuronal cells, which genes are responsible for abnormal cellular phenotypes and what determines the penetrance of neurodevelopmental abnormalities are unknown. We performed haplotype phasing of the 16p11.2 region in a 16p11.2del neurodevelopmental disorders cohort and generated human induced pluripotent stem cells for two 16p11.2del families with distinct residual haplotypes and variable neurodevelopmental disorder phenotypes. Using transcriptomic profiles and cellular phenotypes of the human induced pluripotent stem cell-differentiated cortex neuronal cells, we revealed MAPK3 to be a contributor to dysfunction in multiple pathways related to early neuronal development, with altered soma and electrophysiological properties in mature neuronal cells. Notably, MAPK3 expression in 16p11.2del neuronal cells varied on the basis of a 132 kb 58 single nucleotide polymorphism (SNP) residual haplotype, with the version composed entirely of minor alleles associated with reduced MAPK3 expression. Ten SNPs on the residual haplotype were mapped to enhancers of MAPK3. We functionally validated six of these SNPs by luciferase assay, implicating them in the residual haplotype-specific differences in MAPK3 expression via cis-regulation. Finally, the analysis of three different cohorts of 16p11.2del subjects showed that this minor residual haplotype is associated with neurodevelopmental disorder phenotypes in 16p11.2del carriers.

TBX6 as a cause of a combined skeletal-kidney dysplasia syndrome.

TBX6 encodes transcription-factor box 6, a transcription factor critical to paraxial mesoderm segmentation and somitogenesis during embryonic development. TBX6 haploinsufficiency is believed to drive the skeletal and kidney phenotypes associated with the 16p11.2 deletion syndrome. Heterozygous and biallelic variants in TBX6 are associated with vertebral and rib malformations (TBX6-associated congenital scoliosis) and spondylocostal dysostosis, and heterozygous TBX6 variants are associated with increased risk of genitourinary tract malformations. Combined skeletal and kidney phenotypes in individuals harboring heterozygous or biallelic TBX6 variants are rare. Here, we present seven individuals with vertebral and rib malformations and structural kidney differences associated with heterozygous TBX6 gene deletion in trans with a hypomorphic TBX6 allele or biallelic TBX6 variants. Our case series highlights the association between TBX6 and both skeletal and kidney disease.

Identification of chemical constituents in vitro and in vivo of Er Shen Zhenwu Decoction by utilizing ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectrometry.

Er Shen Zhenwu Decoction is a prescription for treating chronic heart failure of heart and kidney yang deficiency, while its active ingredients remain unclear and difficult to identify. This paper aims to apply a rapid assay strategy of ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry to collect the mass spectrometry data of Er Shen Zhenwu Decoction and its decomposed recipes (monarch, minister, and assist). By comparing with retention time and MSE fragmentation patterns, 67 and 34 components in vitro and in vivo were identified, respectively, the main ingredients include saponins, terpenes, alkaloids, phenolic acids, tanshinone, urea, steroids, aromatics, organic acids, carbohydrates, and so forth, of which the monarch medicine > minister medicine > assist medicine. By comparison with reference standards, paeoniflorin, rosmarinic acid, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1 and atractylenolide III were identified in vitro and paeoniflorin, ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 were identified in vivo. In this study, the chemical ingredients of Er Shen Zhenwu Decoction were analyzed by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry technology and each compound was grouped into the decomposed recipes. The identified substances can be used as references for Er Shen Zhenwu Decoction quality control and potential medicinal substances in chronic heart failure of heart and kidney yang deficiency treatment.

Diagnosis and treatment of the Ehlers-Danlos syndromes in China: synopsis of the first guidelines.

BACKGROUND: The Ehlers-Danlos syndromes (EDS) are a group of rare hereditary connective tissue disorders. EDS is clinically and genetically heterogeneous and usually involves multiple systems. There are 14 subtypes of EDS with hallmark features including joint hypermobility, skin hyperextensibility, and tissue fragility. The clinical manifestations and their severity differ among the subtypes, encompassing recurrent joint dislocations, scoliosis, arterial aneurysm and dissection, and organ rupture. Challenges in diagnosis and management arise from the complexity of the disease, which is further complicated by its rarity. The development of clinical guidelines and implementation of coordinated multi-disciplinary team (MDT) approaches have emerged as global priorities. MAIN BODY: Chinese Multi-Disciplinary Working Group on the Ehlers-Danlos Syndromes was therefore established. Healthcare professionals were recruited from 25 top hospitals across China. The experts are specialized in 24 fields, including genetics, vascular surgery, dermatology, and orthopedics, as well as nursing care, rehabilitation, psychology, and nutrition. Based on GRADE methodology, the Guidelines were written by the Group supervised by methodologists, following a systemic review of all 4453 articles in PubMed published before August 9, 2023, using the search term "Ehlers Danlos". A coordinated MDT approach for the diagnosis and management of EDS is highly recommended by the Group, along with 29 specific recommendations addressing key clinical questions. In addition to the treatment plan, the Guidelines also emphasize integrating support from nursing care, rehabilitation, psychology, and nutrition. This integration not only facilitates recovery in hospital settings, but most importantly, the transition from an illness-defined life to a more "normalized" life. CONCLUSION: The first guidelines on EDS will shorten the diagnostic odyssey and solve the unmet medical needs of the patients. This article is a synopsis of the full guidelines.

Integrating deep phenotyping with genetic analysis: a comprehensive workflow for diagnosis and management of rare bone diseases.

Phenotypes play a fundamental role in medical genetics, serving as external manifestations of underlying genotypes. Deep phenotyping, a cornerstone of precision medicine, involves precise multi-system phenotype assessments, facilitating disease subtyping and genetic understanding. Despite their significance, the field lacks standardized protocols for accurate phenotype evaluation, hindering clinical comprehension and research comparability. We present a comprehensive workflow of deep phenotyping for rare bone diseases from the Genetics Clinic of Skeletal Deformity at Peking Union Medical College Hospital. Our workflow integrates referral, informed consent, and detailed phenotype evaluation through HPO standards, capturing nuanced phenotypic characteristics using clinical examinations, questionnaires, and multimedia documentation. Genetic testing and counseling follow, based on deep phenotyping results, ensuring personalized interventions. Multidisciplinary team consultations facilitate comprehensive patient care and clinical guideline development. Regular follow-up visits emphasize dynamic phenotype reassessment, ensuring treatment strategies remain responsive to evolving patient needs. In conclusion, this study highlights the importance of deep phenotyping in rare bone diseases, offering a standardized framework for phenotype evaluation, genetic analysis, and multidisciplinary intervention. By enhancing clinical care and research outcomes, this approach contributes to the advancement of precision medicine in the field of medical genetics.

SIGMA leverages protein structural information to predict the pathogenicity of missense variants.

Leveraging protein structural information to evaluate pathogenicity has been hindered by the scarcity of experimentally determined 3D protein. With the aid of AlphaFold2 predictions, we developed the structure-informed genetic missense mutation assessor (SIGMA) to predict missense variant pathogenicity. In comparison with existing predictors across labeled variant datasets and experimental datasets, SIGMA demonstrates superior performance in predicting missense variant pathogenicity (AUC = 0.933). We found that the relative solvent accessibility of the mutated residue contributed greatly to the predictive ability of SIGMA. We further explored combining SIGMA with other top-tier predictors to create SIGMA+, proving highly effective for variant pathogenicity prediction (AUC = 0.966). To facilitate the application of SIGMA, we pre-computed SIGMA scores for over 48 million possible missense variants across 3,454 disease-associated genes and developed an interactive online platform (https://www.sigma-pred.org/). Overall, by leveraging protein structure information, SIGMA offers an accurate structure-based approach to evaluating the pathogenicity of missense variants.

Unraveling the genetic architecture of congenital vertebral malformation with reference to the developing spine.

Congenital vertebral malformation, affecting 0.13-0.50 per 1000 live births, has an immense locus heterogeneity and complex genetic architecture. In this study, we analyze exome/genome sequencing data from 873 probands with congenital vertebral malformation and 3794 control individuals. Clinical interpretation identifies Mendelian etiologies in 12.0% of the probands and reveals a muscle-related disease mechanism. Gene-based burden test of ultra-rare variants identifies risk genes with large effect sizes (ITPR2, TBX6, TPO, H6PD, and SEC24B). To further investigate the biological relevance of the genetic association signals, we perform single-nucleus RNAseq on human embryonic spines. The burden test signals are enriched in the notochord at early developmental stages and myoblast/myocytes at late stages, highlighting their critical roles in the developing spine. Our work provides insights into the developmental biology of the human spine and the pathogenesis of spine malformation.

Retrospective Analysis of Associated Anomalies in 636 Patients with Operatively Treated Congenital Scoliosis.

BACKGROUND: Congenital scoliosis is frequently associated with anomalies in multiple organ systems. However, the prevalence and distribution of associated anomalies remain unclear, and there is a large amount of variation in data among different studies. METHODS: Six hundred and thirty-six Chinese patients who had undergone scoliosis correction surgery at Peking Union Medical College Hospital from January 2012 to July 2019 were recruited, as a part of the Deciphering disorders Involving Scoliosis and COmorbidities (DISCO) study. The medical data for each subject were collected and analyzed. RESULTS: The mean age (and standard deviation) at the time of presentation for scoliosis was 6.4 ± 6.3 years, and the mean Cobb angle of the major curve was 60.8° ± 26.5°. Intraspinal abnormalities were found in 186 (30.3%) of 614 patients, with diastematomyelia being the most common anomaly (59.1%; 110 of 186). The prevalence of intraspinal abnormalities was remarkably higher in patients with failure of segmentation and mixed deformities than in patients with failure of formation (p < 0.001). Patients with intraspinal anomalies showed more severe deformities, including larger Cobb angles of the major curve (p < 0.001). We also demonstrated that cardiac anomalies were associated with remarkably worse pulmonary function, i.e., lower forced expiratory volume in the first second (FEV1), forced vital capacity (FVC), and peak expiratory flow (PEF). Additionally, we identified associations among different concomitant malformations. We found that patients with musculoskeletal anomalies of types other than intraspinal and maxillofacial were 9.2 times more likely to have additional maxillofacial anomalies. CONCLUSIONS: In our cohort, comorbidities associated with congenital scoliosis occurred at a rate of 55%. To our knowledge, our study is the first to show that patients with congenital scoliosis and cardiac anomalies have reduced pulmonary function, as demonstrated by lower FEV1, FVC, and PEF. Moreover, the potential associations among concomitant anomalies revealed the importance of a comprehensive preoperative evaluation scheme. LEVEL OF EVIDENCE: Diagnostic Level III. See Instructions for Authors for a complete description of levels of evidence.

[The changes and significance of T lymphocytes and subsets before and after receiving highly active anti-retroviral therapy in children infected with HIV or AIDS patients].

OBJECTIVE: To investigate the changes of the T lymphocytes and their subsets before and after receiving highly active anti-retroviral therapy (HAART) in children who were infected with HIV or AIDS patients. METHODS: Ninety-nine children met the criteria were recruited. All of them had received HAART in Guangxi Center for Disease Control and Prevention from May 2006 to April 2009. Peripheral blood of 2 milliliter was collected before treatment (D0) and after 3, 6, 12, 18, 24, 30, and 36 months (M3, M6, M12, M18, M24, M30 and M36), respectively. Four-color fluorescence flow cytometry was used for the detection of the absolute numbers of CD3(+), CD4(+), CD8(+) T lymphocytes in peripheral blood. And then, the percentages of CD3(+), CD4(+), CD8(+) T lymphocytes in the CD45(+) cells and the ratio of CD4/CD8 were calculated. RESULTS: Sixteen-five (65.66%) cases were treated with lamivudine (3TC)/zidovudine (AZT)/nevirapine (NVP), and 16 (16.16%), 8 (8.08%) and 10 (10.10%) cases were treated with 3TC/stavudine (D4T)/NVP, 3TC/AZT/efavirenz (EFV) and 3TC/AZT/lpv-rtv (LPV/r), respectively. The median of the ratio of CD4/CD8 were 0.39, 0.51, 0.61, 0.65, 0.70, 0.73 and 0.76 in M3, M6, M12, M18, M24, M30 and M36, respectively which were significantly higher than that in D0 (0.19) (Z values were -5.158, -7.375, -9.078, -8.853, -8.373, -5.845 and -5.844 respectively, P < 0.000). The median of CD4% were 16.92%, 22.70%, 25.54%, 26.66%, 27.99%, 30.36% and 29.30% respectively in M3, M6, M12, M18, M24, M30 and M36 respectively, which were also higher significantly than that in D0 (9.92%) (Z values were -5.268, -7.568, -9.496, -9.171, -8.760, -6.190 and -5.964 respectively, P < 0.000). In addition, the median of the absolute numbers of CD4(+)T lymphocytes in peripheral blood were 631, 813, 1050, 946, 1057, 1166 and 894 cells/mm(3) respectively in M3, M6, M12, M18, M24, M30 and M36, which were higher significantly than that of D0 (382 cells/mm(3)) (Z values were -3.318, -5.288, -6.661, -5.886, -5.801, -4.110 and -3.600 respectively, P < 0.000). However, the median of CD8% were 47.25%, 43.01%, 43.04%, 42.60%, 41.37%, 40.83% and 38.31% respectively in M3, M6, M12, M18, M24, M30 and M36, which were lower significantly than that of D0 (53.17%) (Z values were -3.082, -4.697, -5.282, -5.846, -5.757, -3.883 and -4.380 respectively, P < 0.001). CONCLUSION: There is certain rules for the changes of T lymphocytes and their subsets, which may play important roles in the evaluation of the therapeutic effect and the clinical application guidance of HAART.