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.

An Efficient Muscle Segmentation Method Via Bayesian Fusion of Probabilistic Shape Modeling and Deep Edge Detection.

OBJECTIVE: Paraspinal muscle segmentation and reconstruction from MR images are critical to implement quantitative assessment of chronic and recurrent low back pains. Due to unclear muscle boundaries and shape variations, current segmentation methods demonstrate suboptimal performance with insufficient training samples. This work proposes a novel approach to modeling and inferring muscle shapes that enhances segmentation accuracy and efficiency with few training data. METHODS: Firstly, a probabilistic shape model (PSM) based on Fourier basis functions and Gaussian processes (GPs) is designed to encode 3D muscle shapes, where anatomical meanings are attributed to the model's geometric parameters. Muscle shape variations and correlations are described by the GPs of the geometric parameters, which allow a small size of parameters to model the distribution of muscle shapes. Secondly, a Bayesian framework is developed to achieve entire muscle segmentation by posterior estimations. The framework fuses the geometric prior of the PSM with observations of deep-learning-based edge detections (DED) and sparse manual annotations, by which issues of unclear boundaries and shape variations can be compensated. RESULTS AND CONCLUSION: Experiments on public and clinical datasets demonstrate that, with just three manually annotated slices, our method achieves a Dice similarity coefficient exceeding 90%, which outperforms other methods. Meanwhile, our method needs only a small training dataset and offers rapid inference speeds in clinical applications. SIGNIFICANCE: Our study enables precise assessment of paraspinal muscles in 2D and 3D, aiding clinicians and researchers in understanding muscle changes in various conditions, potentially enhancing treatment outcomes.

Rare variant association analyses reveal the significant contribution of carbohydrate metabolic disturbance in severe adolescent idiopathic scoliosis.

BACKGROUND: Adolescent idiopathic scoliosis (AIS), the predominant genetic-influenced scoliosis, results in spinal deformities without vertebral malformations. However, the molecular aetiology of AIS remains unclear. METHODS: Using genome/exome sequencing, we studied 368 patients with severe AIS (Cobb angle >40°) and 3794 controls from a Han Chinese cohort. We performed gene-based and pathway-based weighted rare variant association tests to assess the mutational burden of genes and established biological pathways. Differential expression analysis of muscle tissues from 14 patients with AIS and 15 controls was served for validation. RESULTS: SLC16A8, a lactate transporter linked to retinal glucose metabolism, was identified as a novel severe AIS-associated gene (p=3.08E-06, false discovery rate=0.009). Most AIS cases with deleterious SLC16A8 variants demonstrated early onset high myopia preceding scoliosis. Pathway-based burden test also revealed a significant enrichment in multiple carbohydrate metabolism pathways, especially galactose metabolism. Patients with deleterious variants in these genes demonstrated a significantly larger spinal curve. Genes related to catabolic processes and nutrient response showed divergent expression between AIS cases and controls, reinforcing our genomic findings. CONCLUSION: This study uncovers the pivotal role of genetic variants in carbohydrate metabolism in the development of AIS, unveiling new insights into its aetiology and potential 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.

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.

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.

Impaired glycine neurotransmission causes adolescent idiopathic scoliosis.

Adolescent idiopathic scoliosis (AIS) is the most common form of spinal deformity, affecting millions of adolescents worldwide, but it lacks a defined theory of etiopathogenesis. Because of this, treatment of AIS is limited to bracing and/or invasive surgery after onset. Preonset diagnosis or preventive treatment remains unavailable. Here, we performed a genetic analysis of a large multicenter AIS cohort and identified disease-causing and predisposing variants of SLC6A9 in multigeneration families, trios, and sporadic patients. Variants of SLC6A9, which encodes glycine transporter 1 (GLYT1), reduced glycine-uptake activity in cells, leading to increased extracellular glycine levels and aberrant glycinergic neurotransmission. Slc6a9 mutant zebrafish exhibited discoordination of spinal neural activities and pronounced lateral spinal curvature, a phenotype resembling human patients. The penetrance and severity of curvature were sensitive to the dosage of functional glyt1. Administration of a glycine receptor antagonist or a clinically used glycine neutralizer (sodium benzoate) partially rescued the phenotype. Our results indicate a neuropathic origin for "idiopathic" scoliosis, involving the dysfunction of synaptic neurotransmission and central pattern generators (CPGs), potentially a common cause of AIS. Our work further suggests avenues for early diagnosis and intervention of AIS in preadolescents.

Molecular Diagnostic Yield of Exome Sequencing and Chromosomal Microarray in Short Stature: A Systematic Review and Meta-Analysis.

Importance: Currently, the diagnostic yield of exome sequencing (ES) and chromosomal microarray analysis (CMA) for short stature cohorts is uncertain. Despite previous studies reporting the widespread use of ES and CMA, a definitive diagnostic yield has not been established. Objective: To investigate the diagnostic yield of ES and CMA in short stature. Data Sources: A systematic literature search was conducted using relevant keywords in 3 databases (PubMed, Embase, and Web of Science) in February 2023. Study Selection: Eligible studies for meta-analysis were those that had at least 10 participants with short stature who were diagnosed using either ES or CMA and the number of diagnosed patients was reported. Of 5222 identified studies, 20 were eventually included in the study. Data Extraction and Synthesis: Two independent investigators extracted relevant information from each study, which was then synthesized using proportional meta-analysis to obtain the overall diagnostic yield of ES and CMA. Main Outcomes and Measures: The primary outcome measure was to determine the overall diagnostic yield of ES and CMA. A subgroup meta-analysis was also performed to assess if the diagnostic yield varied depending on whether ES was used as a first-tier or last-resort test. Additionally, a meta-regression was carried out to investigate how the diagnostic yield varied over time. Results: Twenty studies were included, comprising 1350 patients with short stature who underwent ES and 1070 patients who completed CMA. The overall diagnostic yield of ES among the cohorts and CMA among the cohorts was found to be 27.1% (95% CI, 18.1%-37.2%) and 13.6% (95% CI, 9.2%-18.7%), respectively. No statistically significant difference was observed between the first-tier (27.8%; 95% CI, 15.7%-41.8%) and last-resort groups (25.6%; 95% CI, 13.6%-39.6%) (P = .83) or in the percentage of positively diagnosed patients over time. No statistically significant difference was observed between the first-tier (27.8%; 95% CI, 15.7%-41.8%) and last-resort groups (25.6%; 95% CI, 13.6%-39.6%) (P = .83) or in the percentage of positively diagnosed patients over time. Conclusion and Relevance: This systematic review and meta-analysis provides high-level evidence supporting the diagnostic efficacy of ES and CMA in patients with short stature. The findings serve as a solid reference for clinicians when making informed decisions about recommending these genetic tests.

Exploring the association between congenital vertebral malformations and neural tube defects.

Congenital vertebral malformations (CVMs) and neural tube defects (NTDs) are common birth defects affecting the spine and nervous system, respectively, due to defects in somitogenesis and neurulation. Somitogenesis and neurulation rely on factors secreted from neighbouring tissues and the integrity of the axial structure. Crucial signalling pathways like Wnt, Notch and planar cell polarity regulate somitogenesis and neurulation with significant crosstalk. While previous studies suggest an association between CVMs and NTDs, the exact mechanism underlying this relationship remains unclear. In this review, we explore embryonic development, signalling pathways and clinical phenotypes involved in the association between CVMs and NTDs. Moreover, we provide a summary of syndromes that exhibit occurrences of both CVMs and NTDs. We aim to provide insights into the potential mechanisms underlying the association between CVMs and NTDs, thereby facilitating clinical diagnosis and management of these anomalies.

Variants in the SOX9 transactivation middle domain induce axial skeleton dysplasia and scoliosis.

SOX9 is an essential transcriptional regulator of cartilage development and homeostasis. In humans, dysregulation of SOX9 is associated with a wide spectrum of skeletal disorders, including campomelic and acampomelic dysplasia, and scoliosis. The mechanism of how SOX9 variants contribute to the spectrum of axial skeletal disorders is not well understood. Here, we report four novel pathogenic variants of SOX9 identified in a large cohort of patients with congenital vertebral malformations. Three of these heterozygous variants are in the HMG and DIM domains, and for the first time, we report a pathogenic variant within the transactivation middle (TAM) domain of SOX9 . Probands with these variants exhibit variable skeletal dysplasia, ranging from isolated vertebral malformation to acampomelic dysplasia. We also generated a Sox9 hypomorphic mutant mouse model bearing a microdeletion within the TAM domain ( Sox9 Asp272del ). We demonstrated that disturbance of the TAM domain with missense mutation or microdeletion results in reduced protein stability but does not affect the transcriptional activity of SOX9. Homozygous Sox9 Asp272del mice exhibited axial skeletal dysplasia including kinked tails, ribcage anomalies, and scoliosis, recapitulating phenotypes observed in human, while heterozygous mutants display a milder phenotype. Analysis of primary chondrocytes and the intervertebral discs in Sox9 Asp272del mutant mice revealed dysregulation of a panel of genes with major contributions of the extracellular matrix, angiogenesis, and ossification-related processes. In summary, our work identified the first pathologic variant of SOX9 within the TAM domain and demonstrated that this variant is associated with reduced SOX9 protein stability. Our finding suggests that reduced SOX9 stability caused by variants in the TAM domain may be responsible for the milder forms of axial skeleton dysplasia in humans.

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.

The effect of pedicle screw instrumentation at a young age on upper thoracic vertebra and canal development.

BACKGROUND CONTEXT: Pedicle screws are widely used in spinal surgeries. Pedicle screw fixation has shown better clinical effects than other techniques by providing steady fixation from the posterior arch to the vertebral body. However, there are several concerns about the impact of pedicle screw instrumentation insertion on vertebral development in young children, including early closure of the neurocentral cartilage (NCC). The effect of pedicle screw insertion in an early age on further growth of the upper thoracic spine is still unclear. PURPOSE: This study aimed to evaluate the impact of pedicle screw insertion on further growth of the upper thoracic vertebra and spinal canal. STUDY DESIGN: A retrospective case study. PATIENT SAMPLE: Twenty-eight patients. OUTCOME MEASUREMENTS: X-ray and CT parameters including length, height and area of the vertebrae and spinal canal were manually measured. METHODS: Twenty-eight patients who underwent pedicle screw fixation (T1-T6) before the age of 5 years from March 2005 to August 2019 at Peking Union Medical College Hospital were recruited, and records were retrospectively reviewed. Vertebral body and spinal canal parameters were measured at instrumented and adjacent noninstrumented levels and compared using statistical methods. RESULTS: Ninety-seven segments met the inclusion criteria (average age at instrumentation 44.57 months, range from 23-60 months). Thirty-nine segments had no screws, and 58 had at least one screw. There was no significant difference between the preoperative and final follow-up values of the measurement of vertebral body parameters. No significant difference was observed between the growth rates in levels with or without screws in pedicle length, vertebral body diameter, or spinal canal parameters. CONCLUSION: Pedicle screw instrumentation in the upper thoracic spine does not cause a negative effect on the development of the vertebral body and spinal canal in children younger than 5 years old.

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.

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.

Clinical outcomes of the traditional dual growing rod technique combined with apical pedicle screws in the treatment of early-onset scoliosis: preliminary results from a single center.

OBJECTIVE: Limited control of an apical deformity is a major disadvantage in the traditional dual growing rod (TDGR) technique. Previous literature has reported the results of apical pedicle screw placement (APS) as an apical control technique in patients with early-onset scoliosis (EOS). However, the clinical outcomes, indications, and complications of the TDGR technique combined with APSs have not been well described. The purpose of this study was to evaluate the preliminary clinical outcomes of the TDGR technique combined with APSs in EOS patients. METHODS: Clinical data of 12 patients with EOS who were treated with the TDGR technique combined with APSs at the index surgery at the authors' center from January 2010 to January 2020, with a minimum 2-year follow-up, were retrospectively reviewed. Indications for the use of APSs included 1) no vertebral segmentation failure, fused ribs, or multiple hemivertebrae at the apex; 2) at least 2 normal discs around the apex; and 3) proper development of apical pedicles on the convex side. Etiology, age at index surgery, number of lengthening procedures, follow-up duration, and complications were recorded. Radiographic measurements included Cobb angle, apical vertebral translation (AVT), apical vertebral rotation (AVR), thoracic kyphosis, lumbar lordosis, spine height, and space available for the lung (SAL). RESULTS: The mean follow-up period was 4.0 ± 1.4 years, with a mean of 4.8 lengthening procedures per patient. The mean Cobb angle improved from 61.7° ± 10.4° to 19.9° ± 9.0° after the index surgery (19.6° ± 9.4° at the latest follow-up). The mean postindex AVT decreased to 16.8 ± 8.9 mm from a preindex AVT of 56.3 ± 9.7 mm and further improved to 13.6 ± 10.0 mm at the latest follow-up. The mean annual increases in T1-12 and T1-S1 height were 9.0 ± 4.7 mm and 13.9 ± 6.5 mm, respectively. The SAL improved from 0.91 to 1.04 at the latest follow-up. AVR improved significantly after the index surgery (p = 0.013), while minor deterioration was observed after repeat lengthening procedures. Five complications (2 implant related and 3 alignment related) occurred in 4 patients. CONCLUSIONS: For EOS patients with good flexibility (without segmentation failure or multiple hemivertebrae at the apex), the TDGR technique combined with APSs can improve primary curve correction, maintain good correction results, and allow continuous spine growth, which may reduce the risks of complications during lengthening treatment. More multicenter prospective studies with larger samples are needed to further validate the findings of this study.

Dissection of mendelian predisposition and complex genetic architecture of craniovertebral junction malformation.

The craniovertebral junction (CVJ) is an anatomically complex region of the axial skeleton that provides protection of the brainstem and the upper cervical spinal cord. Structural malformation of the CVJ gives rise to life-threatening neurological deficits, such as quadriplegia and dyspnea. Unfortunately, genetic studies on human subjects with CVJ malformation are limited and the pathogenesis remains largely elusive. In this study, we recruited 93 individuals with CVJ malformation and performed exome sequencing. Manual interpretation of the data identified three pathogenic variants in genes associated with Mendelian diseases, including CSNK2A1, MSX2, and DDX3X. In addition, the contribution of copy number variations (CNVs) to CVJ malformation was investigated and three pathogenic CNVs were identified in three affected individuals. To further dissect the complex mutational architecture of CVJ malformation, we performed a gene-based rare variant association analysis utilizing 4371 in-house exomes as control. Rare variants in LGI4 (carrier rate = 3.26%, p = 3.3 × 10-5) and BEST1 (carrier rate = 5.43%, p = 5.77 × 10-6) were identified to be associated with CVJ malformation. Furthermore, gene set analyses revealed that extracellular matrix- and RHO GTPase-associated biological pathways were found to be involved in the etiology of CVJ malformation. Overall, we comprehensively dissected the genetic underpinnings of CVJ malformation and identified several novel disease-associated genes and biological pathways.

Functional characteristics of a broad spectrum of TBX6 variants in Mayer-Rokitansky-Küster-Hauser syndrome.

PURPOSE: Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is characterized by congenital absence of the uterus, cervix, and the upper part of the vagina in females. Whole-gene deletion and loss-of-function variants in TBX6 have been identified in association with MRKHS. We aimed to expand the spectrum of TBX6 variants in MRKHS and explore the biological effect of the variant alleles. METHODS: Rare variants in TBX6 were called from a combined multiethnic cohort of 622 probands with MRKHS who underwent exome sequencing or genome sequencing. Multiple in vitro functional experiments were performed, including messenger RNA analysis, western blotting, transcriptional activity assay, and immunofluorescence staining. RESULTS: We identified 16 rare variants in TBX6 from the combined cohort, including 1 protein-truncating variant reported in our previous study and 15 variants with unknown effects. By comparing the prevalence of TBX6 variants in the Chinese MRKHS cohort vs 1038 female controls, we observed a significant mutational burden of TBX6 in affected individuals (P = .0004, odds ratio = 5.25), suggesting a causal role of TBX6 variants in MRKHS. Of the 15 variants with uncertain effects, 7 were shown to induce a loss-of-function effect through various mechanisms. The c.423G>A (p.Leu141=) and c.839+5G>A variants impaired the normal splicing of TBX6 messenger RNA, c.422T>C (p.Leu141Pro) and c.745G>A (p.Val249Met) led to decreased protein expression, c.10C>T (p.Pro4Ser) and c.400G>A (p.Glu134Lys) resulted in perturbed transcriptional activity, and c.356G>A (p.Arg119His) caused protein mislocalization. We observed incomplete penetrance and variable expressivity in families carrying deleterious variants, which indicates a more complex genetic mechanism than classical Mendelian inheritance. CONCLUSION: Our study expands the mutational spectrum of TBX6 in MRKHS and delineates the molecular pathogenesis of TBX6 variants, supporting the association between deleterious variants in TBX6 and MRKHS.

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.