Mutations in KIF7 implicated in idiopathic scoliosis in humans and axial curvatures in zebrafish.

Idiopathic scoliosis (IS) is a spinal disorder affecting up to 3% of otherwise healthy children. IS has a strong familial genetic component and is believed to be genetically complex due to significant variability in phenotype and heritability. Previous studies identified putative loci and variants possibly contributing to IS susceptibility, including within extracellular matrix, cilia, and actin networks, but the genetic architecture and underlying mechanisms remain unresolved. Here, we used whole-exome sequencing from three affected individuals in a multigenerational family with IS and identified 19 uncommon variants (minor allele frequency < 0.05). Genotyping of additional family members identified a candidate heterozygous variant (H1115Q, G>C, rs142032413) within the ciliary gene KIF7, a regulator within the hedgehog (Hh) signaling pathway. Resequencing of the second cohort of unrelated IS individuals and controls identified several severe mutations in KIF7 in affected individuals only. Subsequently, we generated a mutant zebrafish model of kif7 using CRISPR-Cas9. kif7co63/co63 zebrafish displayed severe scoliosis, presenting in juveniles and progressing through adulthood. We observed no deformities in the brain, Reissner fiber, or central canal cilia in kif7co63/co63 embryos, although alterations were seen in Hh pathway gene expression. This study suggests defects in KIF7-dependent Hh signaling, which may drive pathogenesis in a subset of individuals with IS.

A polygenic burden of rare variants across extracellular matrix genes among individuals with adolescent idiopathic scoliosis.

Adolescent idiopathic scoliosis (AIS) is a complex inherited spinal deformity whose etiology has been elusive. While common genetic variants are associated with AIS, they explain only a small portion of disease risk. To explore the role of rare variants in AIS susceptibility, exome sequence data of 391 severe AIS cases and 843 controls of European ancestry were analyzed using a pathway burden analysis in which variants are first collapsed at the gene level then by Gene Ontology terms. Novel non-synonymous/splice-site variants in extracellular matrix genes were significantly enriched in AIS cases compared with controls (P = 6 × 10(-9), OR = 1.7, CI = 1.4-2.0). Specifically, novel variants in musculoskeletal collagen genes were present in 32% (126/391) of AIS cases compared with 17% (146/843) of in-house controls and 18% (780/4300) of EVS controls (P = 1 × 10(-9), OR = 1.9, CI = 1.6-2.4). Targeted resequencing of six collagen genes replicated this association in combined 919 AIS cases (P = 3 × 10(-12), OR = 2.2, CI = 1.8-2.7) and revealed a highly significant single-gene association with COL11A2 (P = 6 × 10(-9), OR = 3.8, CI = 2.6-7.2). Importantly, AIS cases harbor mainly non-glycine missense mutations and lack the clinical features of monogenic musculoskeletal collagenopathies. Overall, our study reveals a complex genetic architecture of AIS in which a polygenic burden of rare variants across extracellular matrix genes contributes strongly to risk.

Tracking DNA-based antigen-specific T cell receptors during progression to type 1 diabetes.

T cells targeting self-proteins are important mediators in autoimmune diseases. T cells express unique cell-surface receptors (TCRs) that recognize peptides presented by major histocompatibility molecules. TCRs have been identified from blood and pancreatic islets of individuals with type 1 diabetes (T1D). Here, we tracked ~1700 known antigen-specific TCR sequences, islet antigen or viral reactive, in bulk TCRß sequencing from longitudinal blood DNA samples in at-risk cases who progressed to T1D, age/sex/human leukocyte antigen-matched controls, and a new-onset T1D cohort. Shared and frequent antigen-specific TCRß sequences were identified in all three cohorts, and viral sequences were present across all ages. Islet sequences had different patterns of accumulation based upon antigen specificity in the at-risk cases. Furthermore, 73 islet-antigen TCRß sequences were present in higher frequencies and numbers in T1D samples relative to controls. The total number of these disease-associated TCRß sequences inversely correlated with age at clinical diagnosis, indicating the potential to use disease-relevant TCR sequences as biomarkers in autoimmune disorders.

Congruence as a measurement of extended haplotype structure across the genome.

BACKGROUND: Historically, extended haplotypes have been defined using only a few data points, such as alleles for several HLA genes in the MHC. High-density SNP data, and the increasing affordability of whole genome SNP typing, creates the opportunity to define higher resolution extended haplotypes. This drives the need for new tools that support quantification and visualization of extended haplotypes as defined by as many as 2000 SNPs. Confronted with high-density SNP data across the major histocompatibility complex (MHC) for 2,300 complete families, compiled by the Type 1 Diabetes Genetics Consortium (T1DGC), we developed software for studying extended haplotypes. METHODS: The software, called ExHap (Extended Haplotype), uses a similarity measurement we term congruence to identify and quantify long-range allele identity. Using ExHap, we analyzed congruence in both the T1DGC data and family-phased data from the International HapMap Project. RESULTS: Congruent chromosomes from the T1DGC data have between 96.5% and 99.9% allele identity over 1,818 SNPs spanning 2.64 megabases of the MHC (HLA-DRB1 to HLA-A). Thirty-three of 132 DQ-DR-B-A defined haplotype groups have > 50% congruent chromosomes in this region. For example, 92% of chromosomes within the DR3-B8-A1 haplotype are congruent from HLA-DRB1 to HLA-A (99.8% allele identity). We also applied ExHap to all 22 autosomes for both CEU and YRI cohorts from the International HapMap Project, identifying multiple candidate extended haplotypes. CONCLUSIONS: Long-range congruence is not unique to the MHC region. Patterns of allele identity on phased chromosomes provide a simple, straightforward approach to visually and quantitatively inspect complex long-range structural patterns in the genome. Such patterns aid the biologist in appreciating genetic similarities and differences across cohorts, and can lead to hypothesis generation for subsequent studies.

Analysis of Human Leukocyte Antigen DR Alleles, Immune-Related Adverse Events, and Survival Associated With Immune Checkpoint Inhibitor Use Among Patients With Advanced Malignant Melanoma.

Importance: Treatment with immune checkpoint inhibitors (ICIs) has increased survival in patients with advanced malignant melanoma but can be associated with a wide range of immune-related adverse events (irAEs). The role of human leukocyte antigen (HLA)-DR alleles in conferring irAE risk has not been well studied. Objective: To evaluate the association between irAEs and treatment response, survival, and the presence of HLA-DR alleles after ICI therapy in advanced melanoma. Design, Setting, and Participants: This case-control study used the patient registry and biobanked samples from the tertiary referral University of Colorado Cancer Center. Specimens and clinical data were collected between January 1, 2010, and December 31, 2021. Patients with advanced (stage III unresectable and stage IV) melanoma who received ICI therapy (n = 132) were included in the analysis. Exposures: Immune checkpoint inhibitors (anti-cytotoxic T-lymphocyte antigen 4, anti-programmed cell death protein 1 or its ligand, or the combination) for the treatment of advanced melanoma. Main Outcomes and Measures: The association between irAEs and response to therapy, survival, and HLA-DR alleles. Results: Among the cohort of 132 patients with advanced melanoma (mean [SD] age, 63.4 [7.2] years; 85 men [64%] and 47 women [36%]) treated with ICIs, 73 patients had at least 1 irAE and 59 did not have an irAE. Compared with patients without an irAE, patients with an irAE had higher treatment response rates (50 of 72 [69%] vs 28 of 57 [49%]; P = .02) and increased survival (median, 4.8 [IQR, 0.2-9.6] vs 3.2 [IQR, 0.1-9.2] years; P = .02). Specific HLA-DR alleles were associated with the type of irAE that developed: 7 of 10 patients (70%) who developed type 1 diabetes had DR4; 6 of 12 (50%) who developed hypothyroidism had DR8; 5 of 8 (63%) who developed hypophysitis had DR15; 3 of 5 (60%) who developed pneumonitis had DR1; and 8 of 15 (53%) who developed hepatitis had DR4. Conclusions and Relevance: These findings suggest that IrAEs are associated with treatment response rates and increased survival after ICI therapy for advanced melanoma. Because distinct HLA-DR alleles are associated with given adverse events, HLA genotyping before ICI therapy may aid in identifying risk for specific irAEs that could develop with such treatment.

Temporal development of T cell receptor repertoires during childhood in health and disease.

T cell receptor (TCR) sequences are exceptionally diverse and can now be comprehensively measured with next-generation sequencing technologies. However, a thorough investigation of longitudinal TCR repertoires throughout childhood in health and during development of a common childhood disease, type 1 diabetes (T1D), has not been undertaken. Here, we deep sequenced the TCR-ß chain repertoires from longitudinal peripheral blood DNA samples at 4 time points beginning early in life (median age of 1.4 years) from children who progressed to T1D (n = 29) and age/sex-matched islet autoantibody-negative controls (n = 25). From 53 million TCR-ß sequences, we show that the repertoire is extraordinarily diverse early in life and narrows with age independently of disease. We demonstrate the ability to identify specific TCR sequences, including those known to recognize influenza A and, separately, those specific for insulin and its precursor, preproinsulin. Insulin-reactive TCR-ß sequences were more common and frequent in number as the disease progressed in those who developed T1D compared with genetically at risk nondiabetic children, and this was not the case for influenza-reactive sequences. As an independent validation, we sequenced and analyzed TCR-ß repertoires from a cohort of new-onset T1D patients (n = 143), identifying the same preproinsulin-reactive TCRs. These results demonstrate an enrichment of preproinsulin-reactive TCR sequences during the progression to T1D, highlighting the importance of using disease-relevant TCR sequences as powerful biomarkers in autoimmune disorders.

Whole Exome Sequencing of 23 Multigeneration Idiopathic Scoliosis Families Reveals Enrichments in Cytoskeletal Variants, Suggests Highly Polygenic Disease.

Adolescent idiopathic scoliosis (AIS) is a lateral spinal curvature >10° with rotation that affects 2-3% of healthy children across populations. AIS is known to have a significant genetic component, and despite a handful of risk loci identified in unrelated individuals by GWAS and next-generation sequencing methods, the underlying etiology of the condition remains largely unknown. In this study, we performed exome sequencing of affected individuals within 23 multigenerational families, with the hypothesis that the occurrence of rare, low frequency, disease-causing variants will co-occur in distantly related, affected individuals. Bioinformatic filtering of uncommon, potentially damaging variants shared by all sequenced family members revealed 1448 variants in 1160 genes across the 23 families, with 132 genes shared by two or more families. Ten genes were shared by >4 families, and no genes were shared by all. Gene enrichment analysis showed an enrichment of variants in cytoskeletal and extracellular matrix related processes. These data support a model that AIS is a highly polygenic disease, with few variant-containing genes shared between affected individuals across different family lineages. This work presents a novel resource for further exploration in familial AIS genetic research.

The role of CDHR3 in susceptibility to otitis media.

Otitis media (OM) is common in young children and can cause hearing loss and speech, language, and developmental delays. OM has high heritability; however, little is known about OM-related molecular and genetic processes. CDHR3 was previously identified as a locus for OM susceptibility, but to date, studies have focused on how the CDHR3 p.Cys529Tyr variant increases epithelial binding of rhinovirus-C and risk for lung or sinus pathology. In order to further delineate a role for CDHR3 in OM, we performed the following: exome sequencing using DNA samples from OM-affected individuals from 257 multi-ethnic families; Sanger sequencing, logistic regression and transmission disequilibrium tests for 407 US trios or probands with OM; 16S rRNA sequencing and analysis for middle ear and nasopharyngeal samples; and single-cell RNA sequencing and differential expression analyses for mouse middle ear. From exome sequence data, we identified a novel pathogenic CDHR3 splice variant that co-segregates with OM in US and Finnish families. Additionally, a frameshift and six missense rare or low-frequency variants were identified in Finnish probands. In US probands, the CDHR3 p.Cys529Tyr variant was associated with the absence of middle ear fluid at surgery and also with increased relative abundance of Lysobacter in the nasopharynx and Streptomyces in the middle ear. Consistent with published data on airway epithelial cells and our RNA-sequence data from human middle ear tissues, Cdhr3 expression is restricted to ciliated epithelial cells of the middle ear and is downregulated after acute OM. Overall, these findings suggest a critical role for CDHR3 in OM susceptibility. KEY MESSAGES: • Novel rare or low-frequency CDHR3 variants putatively confer risk for otitis media. • Pathogenic variant CDHR3 c.1653 + 3G > A was found in nine families with otitis media. • CDHR3 p.Cys529Tyr was associated with lack of effusion and bacterial otopathogens. • Cdhr3 expression was limited to ciliated epithelial cells in mouse middle ear. • Cdhr3 was downregulated 3 h after infection of mouse middle ear.

Failed Genetic Protection: Type 1 Diabetes in the Presence of HLA-DQB1*06:02.

Certain HLA class II genes increase the risk for type 1 diabetes (T1D) development while others provide protection from disease development. HLA class II alleles encode MHC proteins on antigen-presenting cells, which function to present peptides and activate CD4 T cells. The DRB1*15:01 (DR15)-DQA1*01:02-DQB1*06:02 (DQ6) haplotype provides dominant protection across all stages of T1D and is a common haplotype found in Caucasians. However, it is present in <1% of people with T1D. Knowing which metabolic, immunologic, and genetic features are unique to individuals who fail genetic protection and develop T1D is important for defining the underlying mechanisms of DQB1*06:02-mediated protection. We describe a T1D cohort with DQB1*06:02 (n = 50) and compare them to individuals with T1D and without DQB1*06:02 (n = 2,759) who were identified over the last 26 years at the Barbara Davis Center for Diabetes. The age at diagnosis was similar between the cohorts and normally distributed throughout childhood and early adulthood. The average hemoglobin A1c was 10.8 ± 2.8% (95 ± 7 mmol/mol) at diagnosis in those DQB1*06:02 positive. The majority of T1D DQB1*06:02 + individuals were positive for one or more islet autoantibodies; however, there was a greater proportion who were islet autoantibody negative compared with those T1D DQB1*06:02 - individuals. Interestingly, DQB1*03:02, which confers significant T1D risk, was present in only those DQB1*06:02 + individuals with islet autoantibodies. This is one of the largest studies examining patients presenting with clinical T1D in the presence of DQB1*06:02, which provides a population to study the mechanisms of failed genetic protection against T1D.

Multi-omic studies on missense PLG variants in families with otitis media.

Otitis media (OM), a very common disease in young children, can result in hearing loss. In order to potentially replicate previously reported associations between OM and PLG, exome and Sanger sequencing, RNA-sequencing of saliva and middle ear samples, 16S rRNA sequencing, molecular modeling, and statistical analyses including transmission disequilibrium tests (TDT) were performed in a multi-ethnic cohort of 718 families and simplex cases with OM. We identified four rare PLG variants c.112A > G (p.Lys38Glu), c.782G > A (p.Arg261His), c.1481C > T (p.Ala494Val) and c.2045 T > A (p.Ile682Asn), and one common variant c.1414G > A (p.Asp472Asn). However TDT analyses for these PLG variants did not demonstrate association with OM in 314 families. Additionally PLG expression is very low or absent in normal or diseased middle ear in mouse and human, and salivary expression and microbial α-diversity were non-significant in c.1414G > A (p.Asp472Asn) carriers. Based on molecular modeling, the novel rare variants particularly c.782G > A (p.Arg261His) and c.2045 T > A (p.Ile682Asn) were predicted to affect protein structure. Exploration of other potential disease mechanisms will help elucidate how PLG contributes to OM susceptibility in humans. Our results underline the importance of following up findings from genome-wide association through replication studies, preferably using multi-omic datasets.

Defining multiple common "completely" conserved major histocompatibility complex SNP haplotypes.

The availability of both HLA data and genotypes for thousands of SNPs across the major histocompatibility complex (MHC) in 1240 complete families of the Type 1 Diabetes Genetics Consortium allowed us to analyze the occurrence and extent of megabase contiguous identity for founder chromosomes from unrelated individuals. We identified 82 HLA-defined haplotype groups, and within these groups, megabase regions of SNP identity were readily apparent. The conserved chromosomes within the 82 haplotype groups comprise approximately one third of the founder chromosomes. It is currently unknown whether such frequent conservation for groups of unrelated individuals is specific to the MHC, or if initial binning by highly polymorphic HLA alleles facilitated detection of a more general phenomenon within the MHC. Such common identity, specifically across the MHC, impacts type 1 diabetes susceptibility and may impact transplantation between unrelated individuals.

Identification of Novel Genes and Biological Pathways That Overlap in Infectious and Nonallergic Diseases of the Upper and Lower Airways Using Network Analyses.

Previous genetic studies on susceptibility to otitis media and airway infections have focused on immune pathways acting within the local mucosal epithelium, and outside of allergic rhinitis and asthma, limited studies exist on the overlaps at the gene, pathway or network level between the upper and lower airways. In this report, we compared [1] pathways identified from network analysis using genes derived from published genome-wide family-based and association studies for otitis media, sinusitis, and lung phenotypes, to [2] pathways identified using differentially expressed genes from RNA-sequence data from lower airway, sinus, and middle ear tissues, in particular cholesteatoma tissue compared to middle ear mucosa. For otitis media, a large number of genes (n = 1,806) were identified as differentially expressed between cholesteatoma and middle ear mucosa, which in turn led to the identification of 68 pathways that are enriched in cholesteatoma. Two differentially expressed genes CR1 and SAA1 overlap in middle ear, sinus, and lower airway samples and are potentially novel genes for otitis media susceptibility. In addition, 56 genes were differentially expressed in both tissues from the middle ear and either sinus or lower airways. Pathways that are common in upper and lower airway diseases, whether from published DNA studies or from our RNA-sequencing analyses, include chromatin organization/remodeling, endocytosis, immune system process, protein folding, and viral process. Taken together, our findings from genetic susceptibility and differential tissue expression studies support the hypothesis that the unified airway theory wherein the upper and lower respiratory tracts act as an integrated unit also applies to infectious and nonallergic airway epithelial disease. Our results may be used as reference for identification of genes or pathways that are relevant to upper and lower airways, whether common across sites, or unique to each disease.

Type 1 diabetes: chronic progressive autoimmune disease.

A wealth of data in animal models indicates that type 1A diabetes results from T cell-mediated specific destruction of islet beta cells. There is evidence for the NOD mouse model that insulin is the primary autoantigen and a specific insulin peptide B:9-23 is central to pathogenesis. It is also now possible to predict the development of type 1A (immune mediated) diabetes for the great majority of individuals with a combination of genetic, immunological and metabolic parameters. Such prediction is possible because of the chronic nature of the autoimmunity and loss of beta cell function that precedes the disease. Given the ability to predict type 1A diabetes trials at all stages of the disorder to prevent beta cell destruction are now possible.

Methyldopa blocks MHC class II binding to disease-specific antigens in autoimmune diabetes.

Major histocompatibility (MHC) class II molecules are strongly associated with many autoimmune disorders. In type 1 diabetes (T1D), the DQ8 molecule is common, confers significant disease risk, and is involved in disease pathogenesis. We hypothesized that blocking DQ8 antigen presentation would provide therapeutic benefit by preventing recognition of self-peptides by pathogenic T cells. We used the crystal structure of DQ8 to select drug-like small molecules predicted to bind structural pockets in the MHC antigen-binding cleft. A limited number of the predicted compounds inhibited DQ8 antigen presentation in vitro, with 1 compound preventing insulin autoantibody production and delaying diabetes onset in an animal model of spontaneous autoimmune diabetes. An existing drug with a similar structure, methyldopa, specifically blocked DQ8 in patients with recent-onset T1D and reduced inflammatory T cell responses to insulin, highlighting the relevance of blocking disease-specific MHC class II antigen presentation to treat autoimmunity.

A missense variant in SLC39A8 is associated with severe idiopathic scoliosis.

Genetic factors predictive of severe adolescent idiopathic scoliosis (AIS) are largely unknown. To identify genetic variation associated with severe AIS, we performed an exome-wide association study of 457 severe AIS cases and 987 controls. We find a missense SNP in SLC39A8 (p.Ala391Thr, rs13107325) associated with severe AIS (P = 1.60 × 10-7, OR = 2.01, CI = 1.54-2.62). This pleiotropic SNP was previously associated with BMI, blood pressure, cholesterol, and blood manganese level. We replicate the association in a second cohort (841 cases and 1095 controls) resulting in a combined P = 7.02 × 10-14, OR = 1.94, CI = 1.63-2.34. Clinically, the minor allele of rs13107325 is associated with greater spinal curvature, decreased height, increased BMI and lower plasma manganese in our AIS cohort. Functional studies demonstrate reduced manganese influx mediated by the SLC39A8 p.Ala391Thr variant and vertebral abnormalities, impaired growth, and decreased motor activity in slc39a8 mutant zebrafish. Our results suggest the possibility that scoliosis may be amenable to dietary intervention.

Idiopathic Scoliosis Families Highlight Actin-Based and Microtubule-Based Cellular Projections and Extracellular Matrix in Disease Etiology.

Idiopathic scoliosis (IS) is a structural lateral spinal curvature of ≥10° that affects up to 3% of otherwise healthy children and can lead to life-long problems in severe cases. It is well-established that IS is a genetic disorder. Previous studies have identified genes that may contribute to the IS phenotype, but the overall genetic etiology of IS is not well understood. We used exome sequencing to study five multigenerational families with IS. Bioinformatic analyses identified unique and low frequency variants (minor allele frequency ≤5%) that were present in all sequenced members of the family. Across the five families, we identified a total of 270 variants with predicted functional consequences in 246 genes, and found that eight genes were shared by two families. We performed GO term enrichment analyses, with the hypothesis that certain functional annotations or pathways would be enriched in the 246 genes identified in our IS families. Using three complementary programs to complete these analyses, we identified enriched categories that include stereocilia and other actin-based cellular projections, cilia and other microtubule-based cellular projections, and the extracellular matrix (ECM). Our results suggest that there are multiple paths to IS and provide a foundation for future studies of IS pathogenesis.

The fission yeast DNA structure checkpoint protein Rad26ATRIP/LCD1/UVSD accumulates in the cytoplasm following microtubule destabilization.

BACKGROUND: DNA structure checkpoints are conserved eukaryotic signal transduction pathways that help preserve genomic integrity. Upon detecting checkpoint signals such as stalled replication forks or double-stranded DNA breaks, these pathways coordinate appropriate stress responses. Members of the PI-3 kinase related kinase (PIKK) family are essential elements of DNA structure checkpoints. In fission yeast, the Rad3 PIKK and its regulatory subunit Rad26 coordinate the detection of checkpoint signals with pathway outputs. RESULTS: We found that untreated rad26Delta cells were defective for two microtubule-dependent processes: chromosome segregation and morphogenesis. Interestingly, cytoplasmic accumulation of Rad26-GFP occurred following treatment with microtubule destabilizing drugs, but not during treatment with the genotoxic agent Phleomycin. Cytoplasmic accumulation of Rad26-GFP depended on Rad24, a 14-3-3 protein also required for DNA structure checkpoints and morphogenesis. Results of over expression and epistasis experiments confirm that Rad26 and Rad24 define a response to microtubule destabilizing conditions. CONCLUSION: Two DNA structure checkpoint proteins with roles in morphogenesis define a response to microtubule destabilizing conditions.

Sequencing of the TBX6 Gene in Families with Familial Idiopathic Scoliosis.

STUDY DESIGN: A hypothesis-driven study was conducted in a familial cohort to determine the potential association between variants within the TBX6 gene and Familial Idiopathic Scoliosis (FIS). OBJECTIVE: To determine if variants within exons of the TBX6 gene segregate with the FIS phenotype within a sample of families with FIS. SUMMARY OF BACKGROUND DATA: Idiopathic Scoliosis (IS) is a structural curvature of the spine whose underlying genetic etiology has not been established. IS has been reported to occur at a higher rate than expected in family members of individuals with congenital scoliosis (CS), suggesting that the two diseases might have a shared etiology. The TBX6 gene on chromosome 16p, essential to somite development, has been associated with CS in a Chinese population. Previous studies have identified linkage to this locus in families with FIS, and specifically with rs8060511, located in an intron of the TBX6 gene. METHODS: Parent-offspring trios from 11 families (13 trios, 42 individuals) with FIS were selected for Sanger sequencing of the TBX6 gene. Trios were selected from a large population of families with FIS in which a genome-wide scan had resulted in linkage to 16p. RESULTS: Sequencing analyses of the subset of families resulted in the identification of five coding variants. Three of the five variants were novel; the remaining two variants were previously characterized and account for 90% of the observed variants in these trios. In all cases, there was no correlation between transmission of the TBX6 variant allele and FIS phenotype. However, an analysis of regulatory markers in osteoblasts showed that rs8060511 is in a putative enhancer element. CONCLUSIONS: Although this study did not identify any TBX6 coding variants that segregate with FIS, we identified a variant that is located in a potential TBX6 enhancer element. Therefore, further investigation of the region is needed.

Exome sequencing identifies a rare HSPG2 variant associated with familial idiopathic scoliosis.

Idiopathic scoliosis occurs in 3% of individuals and has an unknown etiology. The objective of this study was to identify rare variants that contribute to the etiology of idiopathic scoliosis by using exome sequencing in a multigenerational family with idiopathic scoliosis. Exome sequencing was completed for three members of this multigenerational family with idiopathic scoliosis, resulting in the identification of a variant in the HSPG2 gene as a potential contributor to the phenotype. The HSPG2 gene was sequenced in a separate cohort of 100 unrelated individuals affected with idiopathic scoliosis and also was examined in an independent idiopathic scoliosis population. The exome sequencing and subsequent bioinformatics filtering resulted in 16 potentially damaging and rare coding variants. One of these variants, p.Asn786Ser, is located in the HSPG2 gene. The variant p.Asn786Ser also is overrepresented in a larger cohort of idiopathic scoliosis cases compared with a control population (P = 0.024). Furthermore, we identified additional rare HSPG2 variants that are predicted to be damaging in two independent cohorts of individuals with idiopathic scoliosis. The HSPG2 gene encodes for a ubiquitous multifunctional protein within the extracellular matrix in which loss of function mutation are known to result in a musculoskeletal phenotype in both mouse and humans. Based on these results, we conclude that rare variants in the HSPG2 gene potentially contribute to the idiopathic scoliosis phenotype in a subset of patients with idiopathic scoliosis. Further studies must be completed to confirm the effect of the HSPG2 gene on the idiopathic scoliosis phenotype.

Replication and further characterization of a Type 1 diabetes-associated locus at the telomeric end of the major histocompatibility complex.

BACKGROUND: We recently reported an association between Type 1 diabetes and the telomeric major histocompatibility complex (MHC) single nucleotide polymorphism (SNP) rs1233478. As further families have been analyzed in the Type 1 Diabetes Genetics Consortium (T1DGC), we tested replication of the association and, with more data, analyzed haplotypic associations. METHODS: An additional 2717 case and 1315 control chromosomes have been analyzed from the T1DGC, with human leukocyte antigen (HLA) typing and data for 2837 SNPs across the MHC region. RESULTS: We confirmed the association of rs1233478 (new data only: P=2.2E-5, OR=1.4). We also found two additional SNPs nearby that were significantly associated with Type 1 diabetes (new data only rs3131020: P=8.3E-9, OR=0.65; rs1592410: P=2.2E-8, OR=1.5). For studies of Type 1 diabetes in the MHC region, it is critical to account for linkage disequilibrium with the HLA genes. Logistic regression analysis of these new data indicated that the effects of rs3131020 and rs1592410 on Type 1 diabetes risk are independent of HLA alleles (rs3131020: P=2.3E-3, OR=0.73; rs1592410: P=2.1E-3, OR=1.4). Haplotypes of 12 SNPs (including the three highly significant SNPs) stratify diabetes risk (high risk, protective, and neutral), with high-risk haplotypes limited to approximately 20,000 bp in length. The 20,000-bp region is telomeric of the UBD gene and contains LOC729653, a hypothetical gene. CONCLUSIONS: We believe that polymorphisms of the telomeric MHC locus LOC729653 may confer risk for Type 1 diabetes.