Exon-Trapping Assay Improves Clinical Interpretation of COL11A1 and COL11A2 Intronic Variants in Stickler Syndrome Type 2 and Otospondylomegaepiphyseal Dysplasia

Stickler syndrome (SS) is a hereditary connective tissue disorder affecting bones, eyes, and hearing. Type 2 SS and the SS variant otospondylomegaepiphyseal dysplasia (OSMED) are caused by deleterious variants in COL11A1 and COL11A2, respectively. In both genes, available database information indicates a high rate of potentially deleterious intronic variants, but published evidence of their biological effect is usually insufficient for a definite clinical interpretation. We report four previously unpublished intronic variants in COL11A1 (c.2241 + 5G>T, c.2809 − 2A>G, c.3168 + 5G>C) and COL11A2 (c.4392 + 1G>A) identified in type 2 SS/OSMED individuals. The pathogenic effect of these variants was first predicted in silico and then investigated by an exon-trapping assay. We demonstrated that all variants can induce exon in-frame deletions, which lead to the synthesis of shorter collagen XI α1 or 2 chains. Lacking residues are located in the α-triple helical region, which has a crucial role in regulating collagen fibrillogenesis. In conclusion, this study suggests that these alternative COL11A1 and COL11A2 transcripts might result in aberrant triple helix collagen. Our approach may help to improve the diagnostic molecular pathway of COL11-related disorders.

Variants in the 5′UTR reduce SHOX expression and contribute to SHOX haploinsufficiency

SHOX haploinsufficiency causes 70–90% of Léri-Weill dyschondrosteosis (LWD) and 2–10% of idiopathic short stature (ISS). Deletions removing the entire gene or enhancers and point mutations in the coding region represent a well-established cause of haploinsufficiency. During diagnostic genetic testing on ISS/LWD patients, in addition to classic SHOX defects, five 5′UTR variants (c.-58G > T, c.-55C > T, c.-51G > A, c.-19G > A, and c.-9del), were detected whose pathogenetic role was unclear and were thus classified as VUS (Variants of Uncertain Significance). The purpose of the present study was to investigate the role of these noncoding variations in SHOX haploinsufficiency. The variants were tested for their ability to interfere with correct gene expression of a regulated reporter gene (luciferase assay). The negative effect on the mRNA splicing predicted in silico for c.-19G > A was assayed in vitro through a minigene splicing assay. The luciferase assay showed that c.-51G > A, c.-19G > A, and c.-9del significantly reduce luciferase activity by 60, 35, and 40% at the homozygous state. Quantification of the luciferase mRNA showed that c.-51G > A and c.-9del might interfere with the correct SHOX expression mainly at the post-transcriptional level. The exon trapping assay demonstrated that c.-19G > A determines the creation of a new branch site causing an aberrant mRNA splicing. In conclusion, this study allowed us to reclassify two of the 5′UTR variants identified during SHOX diagnostic screening as likely pathogenic, one remains as a VUS, and two as likely benign variants. This analysis for the first time expands the spectrum of the genetic causes of SHOX haploinsufficiency to noncoding variations in the 5′UTR.

TGFβ3, MSX1, and MMP3 as Candidates for NSCL±P in an Indian Population

Objective: To evaluate the association of transforming growth factor β3 ( TGFβ3), muscle segment homeobox 1 ( MSX1), Metalloproteinases 3 ( MMP3), and MMP9 genes as candidates for nonsyndromic cleft lip and/or palate in an Indian population. Design: Case–control association study, mutational screening, and functional evaluation of obtained mutations. Setting: Mutational screening of the developmental genes, TGFβ3 and MSX1, along with functional evaluation and association of promoter region SNPs—one each in MMP3 and MMP9. Patients, Participants: Two hundred forty five NSCL±P cases from G. S. Memorial Plastic Surgery Hospital and Trauma Center, Varanasi and 201 healthy controls without a family history of congenital malformations from nearby schools, primary health centers, and the university hospital. Main Outcome Measure(s): Sequencing, SSCP, and PCR-RFLP were used for candidate gene screening. MatInspector and electrophoretic mobility shift assay (EMSA) were used to check the differential transcription factor binding of the variants at promoter region. Luciferase assay was used to test the transcriptional potential of the variant, and evaluation of the alternative splice site was carried out using exon-trapping experiment. Results: Metalloproteinases3 −1171 5A/6A was associated with NSCL±P, whereas MMP9 −1562 C/T did not show association. A rare variant in the promoter region of TGFβ3 (rs117462711) creates a differential binding site, confirmed by EMSA. Luciferase assay showed 3.7-fold increased expression level in mutant construct. A synonymous change in MSX1 (rs34165410) showed association with NSCL±P, which may create an alternative splice site or lead to low codon usage. Exon-trapping experiment failed to confirm alternative splicing, indicating low codon usage frequency of the mutant affecting the gene function. Conclusions: TGFβ3, MSX1, and MMP3 are candidates for NSCL±P.

Two Novel HOGA1 Splicing Mutations Identified in a Chinese Patient with Primary Hyperoxaluria Type 3

Background: Twenty-six HOGA1 mutations have been reported in primary hyperoxaluria (PH) type 3 (PH3) patients with c.700 + 5G>T accounting for about 50% of the total alleles. However, PH3 has never been described in Asians. Methods: A Chinese child with early-onset nephrolithiasis was suspected of having PH. We searched for AGXT, GRHPR and HOGA1 gene mutations in this patient and his parents. All coding regions, including intron-exon boundaries, were analyzed using PCR followed by direct sequence analysis. Results: Two heterozygous mutations not previously described in the literature about HOGA1 were identified (compound heterozygous). One mutation was a successive 2 bp substitution at the last nucleotide of exon 6 and at the first nucleotide of intron 6, respectively (c.834_834 + 1GG>TT), while the other one was a guanine to adenine substitution of the last nucleotide of exon 6 (c.834G>A). Direct sequencing analysis failed to find these mutations in 100 unrelated healthy subjects and the functional role on splicing of both variants found in this study was confirmed by a minigene assay based on the pSPL3 exon trapping vector. In addition, we found a SNP in this family (c.715G>A, p.V239I). There were no mutations detected in AGXT and GRHPR. Conclusion: Two novel HOGA1 mutations were identified in association with PH3. This is the first description and investigation on mutant gene analysis of PH3 in an Asian.

Abstract 332: Deletion of ADAMTS7 in Mice Reveals a Role in Vascular Smooth Muscle Cell Response to Injury

The zinc metalloprotease ADAMTS7 was recently shown by GWAS to be a locus on chr15 for human coronary artery disease (CAD). However, the molecular mechanisms tying ADAMTS7 function to atherosclerosis are unknown. We hypothesized that ADAMTS7 may promote atherosclerosis by facilitating vascular smooth muscle cell (VSMC) proliferation and migration in vivo. We obtained mice containing an exon-trapping cassette between exon 4 and 5 of Adamts7, leading to premature termination of the mRNA and a grossly truncated protein. These mice bred normally and did not display any obvious phenotype. The exon trapping cassette also contained a LacZ reporter construct, allowing for XGal staining to determine gene expression. We assessed Adamts7 gene expression via XGal staining in multiple tissues of interest, including the heart, lungs, liver, and spleen. We also confirmed KO of the gene message through TaqMan rtPCR in additional tissues. We performed wire injury and sham surgeries of femoral arteries in adult Adamts7+/+ and -/- mice (N=4 and 5, respectively) and investigated lesion formation at 28 days. Adamts7 -/- mice showed reduced neointima formation (64%), intima-to-media ratio (47%) and percent stenosis (61%) as compared to wild type. By immunohistochemistry and Trichrome staining we observed trends toward reduced VSMC proliferation (Ki67), reduced collagen IV and fibronectin matrix staining, and less disruption of laminin in ADAMTS7 -/- injured arteries. Finally, ADAMTS7 immunofluorescence of human primary aortic SMCs revealed colocalization to cortactin domains at the leading edge of migrating SMCs as well as to cytoskeletal filaments. In summary, Adamts7 deletion attenuates the VSMC and matrix responses to arterial injury suggesting a role for ADAMTS7 in VSMC migration in atherosclerotic lesion initiation and progression.