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.

Perturbations of genes essential for Müllerian duct and Wölffian duct development in Mayer-Rokitansky-Küster-Hauser syndrome.

Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is associated with congenital absence of the uterus, cervix, and the upper part of the vagina; it is a sex-limited trait. Disrupted development of the Müllerian ducts (MD)/Wölffian ducts (WD) through multifactorial mechanisms has been proposed to underlie MRKHS. In this study, exome sequencing (ES) was performed on a Chinese discovery cohort (442 affected subjects and 941 female control subjects) and a replication MRKHS cohort (150 affected subjects of mixed ethnicity from North America, South America, and Europe). Phenotypic follow-up of the female reproductive system was performed on an additional cohort of PAX8-associated congenital hypothyroidism (CH) (n = 5, Chinese). By analyzing 19 candidate genes essential for MD/WD development, we identified 12 likely gene-disrupting (LGD) variants in 7 genes: PAX8 (n = 4), BMP4 (n = 2), BMP7 (n = 2), TBX6 (n = 1), HOXA10 (n = 1), EMX2 (n = 1), and WNT9B (n = 1), while LGD variants in these genes were not detected in control samples (p = 1.27E-06). Interestingly, a sex-limited penetrance with paternal inheritance was observed in multiple families. One additional PAX8 LGD variant from the replication cohort and two missense variants from both cohorts were revealed to cause loss-of-function of the protein. From the PAX8-associated CH cohort, we identified one individual presenting a syndromic condition characterized by CH and MRKHS (CH-MRKHS). Our study demonstrates the comprehensive utilization of knowledge from developmental biology toward elucidating genetic perturbations, i.e., rare pathogenic alleles involving the same loci, contributing to human birth defects.

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.

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.

Data Mining Suggests That CXCL14 Gene Silencing in Colon Cancer Is Due to Promoter Methylation.

CXCL14 is one of the most evolutionarily conserved members of the chemokine family and is constitutionally expressed in multiple organs, suggesting that it is involved in the homeostasis maintenance of the system. CXCL14 is highly expressed in colon epithelial cells and shows obvious gene silencing in clinical colon cancer samples, suggesting that its silencing is related to the immune escape of cancer cells. In this paper, we analyzed the expression profiles of multiple human clinical colon cancer datasets and mouse colon cancer models to reveal the variation trend of CXCL14 expression during colitis, colon polyps, primary colon cancer, and liver metastases. The relationship between CXCL14 gene silencing and promoter hypermethylation was revealed through the colorectal carcinoma methylation database. The results suggest that CXCL14 is a tumor suppressor gene in colorectal carcinoma which is activated first and then silenced during the process of tumor occurrence and deterioration. Promoter hypermethylation is the main cause of CXCL14 silencing. The methylation level of CXCL14 is correlated with the anatomic site of tumor occurrence, positively correlated with patient age, and associated with prognosis. Reversing the hypermethylation of CXCL14 may be an epigenetic therapy for colon cancer.

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.

A genotype-first analysis in a cohort of Mullerian anomaly.

Müllerian anomaly (M.A.) is a group of congenital anatomic abnormalities caused by aberrations of the development process of the Müllerian duct. M.A. can either be isolated or be involved in Mendelian syndromes, such as Dandy-Walker syndrome, Holt-Oram syndrome and Bardet-Biedl syndrome, which are often associated with both uterus and kidney malformations. In this study, we applied a genotype-first approach to analyze the whole-exome sequencing data of 492 patients with M.A. Six potential pathogenic variants were found in five genes previously related to female urogenital deformities (PKD1, SON, SALL1, BMPR1B, ITGA8), which are partially overlapping with our patients' phenotypes. We further identified eight incidental findings in seven genes related to Mendelian syndromes without known association with reproductive anomalies (TEK, COL11A1, ANKRD11, LEMD3, DLG5, SPTB, BMP2), which represent potential phenotype expansions of these genes.

Diagnostic yield and clinical impact of exome sequencing in early-onset scoliosis (EOS).

BACKGROUND: Early-onset scoliosis (EOS), defined by an onset age of scoliosis less than 10 years, conveys significant health risk to affected children. Identification of the molecular aetiology underlying patients with EOS could provide valuable information for both clinical management and prenatal screening. METHODS: In this study, we consecutively recruited a cohort of 447 Chinese patients with operative EOS. We performed exome sequencing (ES) screening on these individuals and their available family members (totaling 670 subjects). Another cohort of 13 patients with idiopathic early-onset scoliosis (IEOS) from the USA who underwent ES was also recruited. RESULTS: After ES data processing and variant interpretation, we detected molecular diagnostic variants in 92 out of 447 (20.6%) Chinese patients with EOS, including 8 patients with molecular confirmation of their clinical diagnosis and 84 patients with molecular diagnoses of previously unrecognised diseases underlying scoliosis. One out of 13 patients with IEOS from the US cohort was molecularly diagnosed. The age at presentation, the number of organ systems involved and the Cobb angle were the three top features predictive of a molecular diagnosis. CONCLUSION: ES enabled the molecular diagnosis/classification of patients with EOS. Specific clinical features/feature pairs are able to indicate the likelihood of gaining a molecular diagnosis through ES.

A Hybrid Rule-Based and Data-Driven Approach to Illegal Transshipment Identification with Interpretable Behavior Features.

Illegal transshipment of maritime ships is usually closely related to illegal activities such as smuggling, human trafficking, piracy plunder, and illegal fishing. Intelligent identification of illegal transshipment has become an important technical means to ensure the safety of maritime transport. However, due to different geographical environments, legal policies and regulatory requirements in each sea area, there are differences in the movement characteristics and geographical distribution of illegal transshipment behavior in different time and space. Moreover, in areas with dense traffic flow, normal navigation behavior can easily be identified as illegal transshipment, resulting in a high rate of misidentification. This paper proposes a hybrid rule-based and data-driven approach to solve the problem of missing identification in fixed threshold methods and introduces a traffic density feature to reduce the misidentification rate in dense traffic areas. The method is both interpretable and adaptable through unsupervised clustering to get suitable threshold distribution combination for regulatory sea areas. The evaluation results in two different sea areas show that the proposed method is applicable. Compared with other widely used identification methods, this method identifies more illegal transshipment events, which are highly suspicious, and gives warning much earlier. The proposed method can even filter out misidentification events from compared methods' results, which account for more than half of the total number.

Mutational landscape and genetic signatures of cell-free DNA in tumour-induced osteomalacia.

Tumour-induced osteomalacia (TIO) is a very rare paraneoplastic syndrome with bone pain, fractures and muscle weakness, which is mostly caused by phosphaturic mesenchymal tumours (PMTs). Cell-free DNA (cfDNA) has been regarded as a non-invasive liquid biopsy for many malignant tumours. However, it has not been studied in benign tumours, which prompted us to adopt the targeted next-generation sequencing approach to compare cfDNAs of 4 TIO patients, four patients with bone metastasis (BM) and 10 healthy controls. The mutational landscapes of cfDNA in TIO and BM groups were similar in the spectrum of allele frequencies and mutation types. Markedly, deleterious missense mutations in FGFR1 and loss-of-function mutations in MED12 were found in 3/4 TIO patients but none of BM patients. The gene ontology analysis strongly supported that these mutated genes found in TIOs would play a potential role in PMTs' process. The genetic signatures and corresponding change in expression of FGFR1 and FGF23 were further validated in PMT tissues from a test cohort of another three TIO patients. In summary, we reported the first study of the mutational landscape and genetic signatures of cfDNA in TIO/PMTs.

A novel COMP mutation in a Chinese family with multiple epiphyseal dysplasia.

BACKGROUND: Multiple epiphyseal dysplasia (MED) is a skeletal disorder characterized by delayed and irregular ossification of the epiphyses and early-onset osteoarthritis. At least 66% of the reported autosomal dominant MED (AD-MED) cases are caused by COMP mutations. METHODS: We recruited a four-generation Chinese family with early-onset hip osteoarthritis, flatfoot, brachydactyly, and mild short stature. An assessment of the family history, detailed physical examinations, and radiographic evaluations were performed on the proband and other family members, followed by the performance of whole-exome sequencing (WES). The pathogenicity of the candidate mutation was also analyzed. RESULTS: An AD-MED family with 10 affected members and 17 unaffected members was recruited. The main radiographic findings were symmetrical changes in the dysplastic acetabulum and femoral heads, irregular contours of the epiphyses, a shortened femoral neck, and flatfoot. Lower bone density was also observed in the ankle joints, wrist joints, and knees, as well as irregular vertebral end plates. In the proband, we identified the missense mutation c.1153G > T (p. Asp385Tyr), located in exon 11 of the COMP gene. This mutation was assessed as 'pathogenic' because of its low allele frequency and its high likelihood of co-segregation with disease in the reported family. Sanger sequencing validated the novel heterozygous mutation c.1153G > T (p. Asp385Tyr) in exon 11 of COMP in all affected individuals in the family. CONCLUSIONS: Our results underlined a key role of the Asp385 amino acid in the protein function of COMP and confirmed the pathogenicity of the COMP (c.1153G > T; p. Asp385Tyr) mutation in AD-MED disease. We have therefore expanded the known mutational spectrum of COMP and revealed new phenotypic information for AD-MED.

Identification of novel FBN1 variations implicated in congenital scoliosis.

Congenital scoliosis (CS) is a form of scoliosis caused by congenital vertebral malformations. Genetic predisposition has been demonstrated in CS. We previously reported that TBX6 loss-of-function causes CS in a compound heterozygous model; however, this model can explain only 10% of CS. Many monogenic and polygenic CS genes remain to be elucidated. In this study, we analyzed exome sequencing (ES) data of 615 Chinese CS from the Deciphering Disorders Involving Scoliosis and COmorbidities (DISCO) project. Cosegregation studies for 103 familial CS identified a novel heterozygous nonsense variant, c.2649G>A (p.Trp883Ter) in FBN1. The association between FBN1 and CS was then analyzed by extracting FBN1 variants from ES data of 574 sporadic CS and 828 controls; 30 novel variants were identified and prioritized for further analyses. A mutational burden test showed that the deleterious FBN1 variants were significantly enriched in CS subjects (OR = 3.9, P = 0.03 by Fisher's exact test). One missense variant, c.2613A>C (p.Leu871Phe) was recurrent in two unrelated CS subjects, and in vitro functional experiments for the variant suggest that FBN1 may contribute to CS by upregulating the transforming growth factor beta (TGF-ß) signaling. Our study expanded the phenotypic spectrum of FBN1, and provided nove insights into the genetic etiology of CS.

Molecular characterization of carp edema virus disease: An emerging threat to koi Cyprinus carpio in China.

Carp edema virus disease (CEVD) has resulted in great economic losses in koi (Cyprinus carpio koi) and common carp (Cyprinus carpio carpio) populations in the world. In this study, the diseased koi were diagnosed as CEV infection based on 5' untranslated region (5'UTR) and 4a protein genes by the conventional PCR, nested PCR and quantitative PCR (qPCR) analyses. Phylogenetic tree analysis showed that the TJ201708 strain was classified into the genogroup IIa. Furthermore, qPCR of 5'UTR gene revealed that the lowest detection limit was 4.0 fg/µL. The pathogenicity of CEV for koi was demonstrated in the infection experiments. Histopathological examination revealed the petechial hemorrhages of liver and spleen, vacuolization of lamina propria of intestine and swelling and necrosis of respiratory epithelial cells of gills. To our knowledge, this is the first report the qPCR of 5'UTR gene in the detection of carp edema virus.

Skin proteome profiling of tongue sole (Cynoglossus semilaevis) challenged with Vibrio vulnificus.

Vibrio vulnificus is a major pathogen of cultured Cynoglossus semilaevis and results in skin ulceration and haemorrhage, but the proteomic mechanism of skin immunity against V. vulnificus remains unclear. In this study, we investigated the histopathology and skin immune response in C. semilaevis with V. vulnificus infection at the protein levels, the differential proteomic profiling of its skin was examined by using iTRAQ and LC-MS/MS analyses. A total of 951 proteins were identified in skin, in which 134 and 102 DEPs were screened at 12 and 36 hpi, respectively. Selected eleven immune-related DEPs (pvß, Hsp71, MLC1, F2, α2ML, HCII, C3, C5, C8ß, C9 and CD59) were verified for their immune roles in the V. vulnificus infection via using qRT-PCR assay. KEGG enrichment analysis revealed that most of the identified immune proteins were significantly associated with complement and coagulation cascades, antigen processing and presentation, salivary secretion and phagosome pathways. To our knowledge, this study is the first to describe the proteome response of C. semilaevis skin against V. vulnificus infection. The outcome of this study contributed to provide a new perspective for understanding the molecular mechanism of local skin mucosal immunity, and facilitating the development of novel mucosal vaccination strategies in fish.

The mutational burden and oligogenic inheritance in Klippel-Feil syndrome.

BACKGROUND: Klippel-Feil syndrome (KFS) represents a rare anomaly characterized by congenital fusion of the cervical vertebrae. The underlying molecular etiology remains largely unknown because of the genetic and phenotypic heterogeneity. METHODS: We consecutively recruited a Chinese cohort of 37 patients with KFS. The clinical manifestations and radiological assessments were analyzed and whole-exome sequencing (WES) was performed. Additionally, rare variants in KFS cases and controls were compared using genetic burden analysis. RESULTS: We primarily examined rare variants in five reported genes (GDF6, MEOX1, GDF3, MYO18B and RIPPLY2) associated with KFS and detected three variants of uncertain significance in MYO18B. Based on rare variant burden analysis of 96 candidate genes related to vertebral segmentation defects, we identified BAZ1B as having the highest probability of association with KFS, followed by FREM2, SUFU, VANGL1 and KMT2D. In addition, seven patients were proposed to show potential oligogenic inheritance involving more than one variants in candidate genes, the frequency of which was significantly higher than that in the in-house controls. CONCLUSIONS: Our study presents an exome-sequenced cohort and identifies five novel genes potentially associated with KFS, extending the spectrum of known mutations contributing to this syndrome. Furthermore, the genetic burden analysis provides further evidence for potential oligogenic inheritance of KFS.

Skin transcriptome, tissue distribution of mucin genes and discovery of simple sequence repeats in crucian carp (Carassius auratus).

Crucian carp (Carassius auratus) is one of the major freshwater species and important food fish in China. Fish skin acts as the first line of defence against pathogens, yet its molecular and immune mechanism remains unclear. In this study, a de novo transcriptome assembly of C. auratus skin was performed with the Illumina Hiseq 2000 platform. A total of 49,154,776 clean reads were assembled, among which 60,824 (46.86%), 37,103 (28.59%), 43,269 (33.33%) unigenes were annotated against National Center for Biotechnology Information, Gene Onotology and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, respectively. KEGG Orthology categories were significantly involved in immune system (20.50%), signal transduction (18.04%) and mucosal mucin genes (e.g., muc2, muc5AC, muc5B, muc17, muc18). The high expression of muc18 gene was observed in brain; that of muc2 in intestine; and that of muc5AC in skin, liver, spleen, intestine and muscle. Moreover, the potential 28,928 simple sequence repeats with the three most abundant dinucleotide repeat motifs (AC/GT, AG/CT, AT/AT) were detected in C. auratus. To authors' knowledge, this is the first report to describe the transcriptome analysis of C. auratus skin, and the outcome of this study contributed to the understanding of mucosal immune response of the skin and molecular markers in cyprinid species.

TBX6-associated congenital scoliosis (TACS) as a clinically distinguishable subtype of congenital scoliosis: further evidence supporting the compound inheritance and TBX6 gene dosage model.

PURPOSE: To characterize clinically measurable endophenotypes, implicating the TBX6 compound inheritance model. METHODS: Patients with congenital scoliosis (CS) from China(N = 345, cohort 1), Japan (N = 142, cohort 2), and the United States (N = 10, cohort 3) were studied. Clinically measurable endophenotypes were compared according to the TBX6 genotypes. A mouse model for Tbx6 compound inheritance (N = 52) was investigated by micro computed tomography (micro-CT). A clinical diagnostic algorithm (TACScore) was developed to assist in clinical recognition of TBX6-associated CS (TACS). RESULTS: In cohort 1, TACS patients (N = 33) were significantly younger at onset than the remaining CS patients (P = 0.02), presented with one or more hemivertebrae/butterfly vertebrae (P = 4.9 × 10‒8), and exhibited vertebral malformations involving the lower part of the spine (T8-S5, P = 4.4 × 10‒3); observations were confirmed in two replication cohorts. Simple rib anomalies were prevalent in TACS patients (P = 3.1 × 10‒7), while intraspinal anomalies were uncommon (P = 7.0 × 10‒7). A clinically usable TACScore was developed with an area under the curve (AUC) of 0.9 (P = 1.6 × 10‒15). A Tbx6-/mh (mild-hypomorphic) mouse model supported that a gene dosage effect underlies the TACS phenotype. CONCLUSION: TACS is a clinically distinguishable entity with consistent clinically measurable endophenotypes. The type and distribution of vertebral column abnormalities in TBX6/Tbx6 compound inheritance implicate subtle perturbations in gene dosage as a cause of spine developmental birth defects responsible for about 10% of CS.

Perturbations of BMP/TGF-ß and VEGF/VEGFR signalling pathways in non-syndromic sporadic brain arteriovenous malformations (BAVM).

BACKGROUND: Brain arteriovenous malformations (BAVM) represent a congenital anomaly of the cerebral vessels with a prevalence of 10-18/100 000. BAVM is the leading aetiology of intracranial haemorrhage in children. Our objective was to identify gene variants potentially contributing to disease and to better define the molecular aetiology underlying non-syndromic sporadic BAVM. METHODS: We performed whole-exome trio sequencing of 100 unrelated families with a clinically uniform BAVM phenotype. Pathogenic variants were then studied in vivo using a transgenic zebrafish model. RESULTS: We identified four pathogenic heterozygous variants in four patients, including one in the established BAVM-related gene, ENG, and three damaging variants in novel candidate genes: PITPNM3, SARS and LEMD3, which we then functionally validated in zebrafish. In addition, eight likely pathogenic heterozygous variants (TIMP3, SCUBE2, MAP4K4, CDH2, IL17RD, PREX2, ZFYVE16 and EGFR) were identified in eight patients, and 16 patients carried one or more variants of uncertain significance. Potential oligogenic inheritance (MAP4K4 with ENG, RASA1 with TIMP3 and SCUBE2 with ENG) was identified in three patients. Regulation of sma- and mad-related proteins (SMADs) (involved in bone morphogenic protein (BMP)/transforming growth factor beta (TGF-ß) signalling) and vascular endothelial growth factor (VEGF)/vascular endotheliual growth factor recepter 2 (VEGFR2) binding and activity (affecting the VEGF signalling pathway) were the most significantly affected biological process involved in the pathogenesis of BAVM. CONCLUSIONS: Our study highlights the specific role of BMP/TGF-ß and VEGF/VEGFR signalling in the aetiology of BAVM and the efficiency of intensive parallel sequencing in the challenging context of genetically heterogeneous paradigm.

Genetic polymorphisms of GPR126 are functionally associated with PUMC classifications of adolescent idiopathic scoliosis in a Northern Han population.

GPR126 has been identified to be associated with AIS (Adolescent Idiopathic Scoliosis) in different populations, but data on the northern Chinese population are unavailable. Additionally, it is important to know the exact clinical phenotypes associated with specific genetic polymorphisms. Fourteen SNP (single nucleotide polymorphism) loci in GPR126 were genotyped in 480 northern Chinese Han AIS patients and 841 controls. These patients were classified into three types based on the PUMC classification system. Luciferase assays were used to investigate their regulation of GPR126 transcription activity. Combined and stratified genotype-phenotype association analyses were conducted. The alleles rs225694, rs7774095 and rs2294773 were significantly associated with AIS (P = 0.021, 0.048 and 0.023, respectively). rs225694 and rs7774095 potentially have regulatory functions for the GRP126 gene. Correlation analysis revealed that allele A of rs225694 was a risk allele only for PUMC type II AIS (P = 0.036) and allele G of rs2294773 was a risk allele only for PUMC type I AIS (P = 0.018). In summary, rs225694, rs7774095 and rs2294773 are significantly associated with disease in northern Chinese Han AIS patients. The SNPs rs225694 and rs2294773 are associated with different AIS PUMC classifications.

The coexistence of copy number variations (CNVs) and single nucleotide polymorphisms (SNPs) at a locus can result in distorted calculations of the significance in associating SNPs to disease.

With the recent advance in genome-wide association studies (GWAS), disease-associated single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) have been extensively reported. Accordingly, the issue of incorrect identification of recombination events that can induce the distortion of multi-allelic or hemizygous variants has received more attention. However, the potential distorted calculation bias or significance of a detected association in a GWAS due to the coexistence of CNVs and SNPs in the same genomic region may remain under-recognized. Here we performed the association study within a congenital scoliosis (CS) cohort whose genetic etiology was recently elucidated as a compound inheritance model, including mostly one rare variant deletion CNV null allele and one common variant non-coding hypomorphic haplotype of the TBX6 gene. We demonstrated that the existence of a deletion in TBX6 led to an overestimation of the contribution of the SNPs on the hypomorphic allele. Furthermore, we generalized a model to explain the calculation bias, or distorted significance calculation for an association study, that can be 'induced' by CNVs at a locus. Meanwhile, overlapping between the disease-associated SNPs from published GWAS and common CNVs (overlap 10%) and pathogenic/likely pathogenic CNVs (overlap 99.69%) was significantly higher than the random distribution (p < 1 × 10-6 and p = 0.034, respectively), indicating that such co-existence of CNV and SNV alleles might generally influence data interpretation and potential outcomes of a GWAS. We also verified and assessed the influence of colocalizing CNVs to the detection sensitivity of disease-associated SNP variant alleles in another adolescent idiopathic scoliosis (AIS) genome-wide association study. We proposed that detecting co-existent CNVs when evaluating the association signals between SNPs and disease traits could improve genetic model analyses and better integrate GWAS with robust Mendelian principles.