Genetic testing in four Indian families with suspected Stickler syndrome

Purpose: Stickler syndrome is associated with the development of rhegmatogenous retinal detachment (RRD), and often presents with ocular, auditory, skeletal, and orofacial abnormalities. Molecular analysis has proven effective in diagnosis, confirmation and classification of the disease. We aimed to describe the utility of next?generation sequencing (NGS) in genetic analysis of four Indian families with suspected Stickler syndrome. Methods: The index cases presented with retinal detachment with family history. Genetic analysis in the index case was performed by next?generation sequencing of inherited retinal degeneration genes, and validated by Sanger sequencing followed by co?segregation analysis in the other family members. Results: Twenty patients were included for the genetic analysis (15 males and 5 females from four families). Clinical details were available for 15 patients (30 eyes). Fourteen eyes (11 patients) developed RRD. In the 16 eyes without RRD, 8 underwent barrage laser to lattice degeneration and 8 were under observation. Disease segregating heterozygous mutations with pathogenic/likely pathogenic effect was identified in COL2A1 (c.4318?1G>A, c.141G>A, c.1221+1G>A for 3 families) and COL11A1 (c.1737+1 G>A for 1 family) gene. In addition to the mutation in the COL2A1 gene, a pathogenic heterozygous variant associated with risk for arrhythmogenic right ventricular cardiomyopathy (ARVC) was identified in one member. Conclusion: NGS testing confirmed the presence of the causative gene for Stickler syndrome in the index case followed by evaluation of family members and confirmation of genetic and ocular findings. We believe that this may be the first such report of families with RRD from India

Identification of a COL2A1 mutation in a Chinese family with Stickler syndrome type 1 via whole exome sequencing / 中华实验眼科杂志

Objective:To identify the disease-causing mutation in a Chinese family with Stickler syndrome type 1.Methods:The pedigree investigation was conducted.A Chinese family with Stickler syndrome type 1 was enrolled in the Shantou International Eye Center in June 2012.Medical history collection and clinical examinations, such as vision, intraocular pressure, slit lamp microscopy and fundus, were carried out in all the included family members and the diagnosis was made by clinical experts.Total genomic DNAs were extracted from the peripheral blood samples (5 ml) obtained from 5 patients and 4 healthy members.The potential variant of the proband's father Ⅲ-5 were screened by whole exome sequencing (WES) and stepwise bioinformatic analysis.The segregation and mutation conformation of the variant was verified by Sanger sequencing.The pathogenicity of the variant was predicted by SIFT, Polyphen2, and MutationTaster.Conservation and three-dimensional structure of amino acid mutation were analyzed by multiple sequence alignment and UniProt.This study adhered to the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of Joint Shantou International Eye Center (No.EC20110310[2]-P02).Written informed consent was obtained from each subject or the guardian.Results:An autosomal dominant inherence in 39 members of 4 generations including 15 patients and 24 phenotypically normal members was found in the family.The proband (Ⅳ-4) showed high myopia, retinal detachment and strabismus in the right eye, and the left eye was blind.A patient (Ⅲ-5) showed high myopia and cataract in the right eye, atrophy in the left eye.A patient (Ⅳ-9) showed binocular high myopia.A heterozygous variation, c.1693C>T: p.Arg565Cys, within the exon 26 of COL2A1 gene was revealed in patient Ⅲ-5, which was only found in the patients and not in phenotypically normal members, indiacating co-separation in this family.The variant was predicted to be a severe damage by SIFT, Polyphen2 and MutationTaster.The amino acid mutation at position 565 was highly conservative among human, mouse, rat, bovine and Xenopus laevis, which caused the arginine to cysteine substitution at the X position in triple helix repeat region Gly-X-Y, affecting the function of fibrous protein and becoming pathogenic. Conclusions:Variant c.1693C>T: p.Arg565Cys in COL2A1 gene is disease-causing in this family and this is the first report about the variant in China.

Susceptibility genes related to non-traumatic femoral head necrosis: Improving detection accuracy and developing new treatment strategy / 中国组织工程研究

BACKGROUND: Although the mechanism of femoral head necrosis has a series of explanations such as glucocorticoids, ethanol, decompression sickness, sickle cell anemia, and genetic susceptibility, the specific pathogenesis is still unclear. OBJECTIVE: To highlight the genetics of non-traumatic femoral head necrosis by combining recent genetic studies, thus providing new treatments for the repair and reconstruction of femoral head. METHODS: A computer-based online retrieval of CNKI, WanFang, PubMed and Web of Science databases was performed to search the related articles published from January 2000 to April 2019. The keywords were “non-traumatic femoral head necrosis, susceptibility genes, pathogenesis” in Chinese and English, respectively. According to the inclusion and exclusion criteria, the irrelevant and repetitive articles were excluded, and finally 49 eligible articles were included for analysis. RESULTS AND CONCLUSION: Non-traumatic femoral head necrosis is an extremely complex disease whose pathogenesis is caused by a combination of factors rather than a single genetic gene. COL2A1 genetic mutation is the high susceptibility gene to non-traumatic femoral head necrosis. High-coagulation and low-fibrinolytic state-related loci in the blood, interleukin-related gene loci in immune system and lipid-related loci become more and more important in inducing non-traumatic femoral head necrosis. With the deep understanding of human genetic technology, the accuracy of detecting susceptible genes will be continuously improved, which is conducive to the prevention, diagnosis and precise individualized treatment of non-traumatic femoral head necrosis.

A Case of Platyspondylic Lethal Skeletal Dysplasia Torrance Type

Platyspondylic lethal skeletal dysplasia, Torrance type (PLSD-T), is one of the phenotypes of type II collagenopathy and is characteristic of severe bone growth disorder. This phenotype may limit the growth and expansion of the lungs, which is known to cause death from respiratory failure during or shortly after birth, but in few less severe cases, patients have been reported to have survived to adulthood. We have experienced a case of PLSD-T in a preterm infant who was delivered via cesarean section at the gestational age of 29 weeks 3 days, with a birth weight of 1.15 kg. Physical examination of the infant revealed characteristic findings of short arms and legs, small thorax, distended abdomen, and cleft palate. On the basis of the subsequent genetic testing, the patient had a heterozygous mutation in the encoded c-propeptide region of collagen, type II, alpha 1 (COL2A1), c.4335G>A (p.Trp1445*) in exon 52. This is the first case of PLSD-T diagnosed in Korea, and we hereby report the case.

Comparative Study of Longbie Capsules and Bone Marrow Mesenchymal Stem Cell Transplantation in Repairing Damaged Cartilage of Knee Osteoarthritis Rats / 广州中医药大学学报

Objective To compare the actions of Longbie Capsules and bone marrow mesenchymal stem cell (BMSC)transplantation in repairing the damaged cartilage of knee osteoarthritis(KOA)rats. Methods Thirty-six rats aged 4-6 weeks old were induced into KOA model(bilateral knees)by collagenase injection method. All of the modeled rats were randomly divided into model group(intragastric administration of normal saline), BMSC transplantation group(giving tail vein injection of 1 ×106 of BMSCs per time, 2 times every week), and Chinese medicine group (intragastric administration of Longbie Capsules of 7.5 g·kg-1·d-1),12 rats in each group. Six weeks later,the cartilage of rat bilateral knees was taken out. The pathological changes of cartilage were observed by hematoxylin-eosin(HE) staining method, and the protein and mRNA expression levels of Col2a1, X-linkedinhibitor of apoptosis protein (XIAP), HuR in rat knee cartilage were detected by immunohistochemistry and real-time quantitative polymerase chain reaction (qPCR), respectively. Results The HE staining results showed that the cartilage tissue surface was rough with more cracks, and the cartilage cells gathered with the cytoplasm collapsed and arranging disorderly in the model group. The number of chondrocytes was increased and cell surface was flat,and the cracks of the cartilage were decreased with the chondrocytes arranging uniformly in Chinese medicine group and BMSC transplantation group compared with the model group. The results of immunohistochemistry and qPCR detection showed that in Chinese medicine dosage group and BMSC transplantation group, the protein and mRNA expression levels of Col2a1,XIAP and HuR were significantly higher than those in the model group (P<0.05 or P<0.0001), but there was no significant difference between the two medication groups(P>0.05). Conclusion Longbie Capsules and BMSC transplantation can promote the secretion of Col2a1 in the cartilage tissue of KOA rats,improve cartilage, and their mechanism may be related with up-regulating apoptosis-related proteins HuR and XIAP.

Two Likely Pathogenic Variants of COL2A1 in Unrelated Korean Patients With Ocular-Only Variants of Stickler Syndrome: The First Molecular Diagnosis in Korea

Stickler syndrome is a genetically heterogeneous disorder that affects the ocular, auditory, and musculoskeletal systems. Ocular-only variant of Stickler syndrome type 1 (OSTL1) is characterized by high risk of retinal detachment without systemic involvement and is caused by alternatively spliced exon 2 mutation of COL2A1. We report the cases of two Korean families with OSTL1 carrying likely pathogenic variants of COL2A1. All patients presented with membranous vitreous anomaly, peripheral retinal degeneration, and/or rhegmatogenous retinal detachment, but no systemic manifestations. By genetic analysis, two likely pathogenic non-exon 2 variants, c.2678dupC (p.Ala895Serfs*49) and c.3327+ 1G>C, were identified in COL2A1. Our results demonstrate that COL2A1 defects in OSTL1 are not confined to mutations in exon 2. Together with molecular data, ophthalmologists should consider genetic diagnosis of Stickler syndrome in patients with vitreous anomaly to prevent blindness from retinal detachment. To our knowledge, this is the first report of genetically confirmed OSTL1 in Korea.

A Case of Familial Stickler Syndrome in a Newborn with COL2A1 Gene Mutation / 대한주산의학회잡지

Stickler syndrome is a progressive, hereditary disorder of connective tissue caused by mutations in different collagen genes. It is characterized by distinctive ocular, auditory, skeletal and oro-facial abnormalities and associated with long-term respiratory, nutritional, and developmental difficulties. Therefore, early detection and early treatment of Stickler syndrome is very important. We reported a case of Stickler syndrome in a newborn with family history and COL2A1 gene mutation.

A Case of Type 1 Stickler Syndrome Caused by a Novel Mutation in COL2A1 / 대한내과학회지

Stickler syndrome is a genetic disorder characterized by ophthalmologic, craniofacial, audiologic, and joint problems. In this report, we describe a 10-year-old boy presenting with a flat face, high myopia, retinal detachment, arthropathy, short stature, and mental retardation. Sequencing of the COL2A1 gene revealed a novel mutation, c.3055C > T (p.Pro1019Ser), consistent with a diagnosis of Type 1 Stickler Syndrome.

Association of COL2A1 Gene Polymorphism with Degenerative Lumbar Scoliosis

BACKGROUND: Degenerative lumbar scoliosis (DLS) progresses with aging after 50-60 years, and the genetic association of DLS remains largely unclear. In this study, the genetic association between collagen type II alpha 1 (COL2A1) gene and DLS was investigated. METHODS: COL2A1 gene polymorphism was investigated in DLS subjects compared to healthy controls to investigate the possibility of its association with COL2A1 gene. Based on a single nucleotide polymorphism (SNP) database, SNP (rs2276454) in COL2A1 were selected and genotyped using direct sequencing in 51 patients with DLS and 235 healthy controls. The SNP effects were analyzed using three models of codominant, dominant, and recessive. Logistic regression models were calculated for odds ratios (ORs) with 95% confidence intervals (CIs) and corresponding p-values, controlling age and gender as co-variables. RESULTS: SNP (rs2276454) in COL2A1 was significantly associated with the degenerative lumbar scoliosis in the codominant (OR, 1.90; 95% CI, 1.17 to 3.10; p = 0.008) and dominant models (OR, 3.58; 95% CI, 1.59 to 9.29; p = 0.001). CONCLUSIONS: The results suggest that COL2A1 is associated with the risk of DLS in Korean population.

Expression of SOX9 and COL2A1 gene in human cervical intervertebral disc nucleus pulpusus and its significance / 第二军医大学学报

Objective: To observe the expression of SRYCsex determining region Y)-box 9 (SOX9) and collagen type E alpha 1 (COL2A1) in human cervical intervertebral disc nucleus pulposus (NP) tissues of various degeneration degrees, so as to discuss the role of their expression during cervical intervertebral disc degeneration(CIDD). Methods: Totally 36 human cervical intervertebral disc specimens and their pre-operation MRI findings were obtained from surgical patients or patients with cervical trauma, and they were classified into five grades according to the Miyazaki grading system. The expression of SOX9 and COL2A1 was examined by fluorescence quantitative RT-PCR, Western blotting analysis and immunohistochemistry staining. The relationship between SOX9, COL2A1 expression and the degrees of CIDD was analyzed, and the relationship between SOX9 and COL2A1 was also discussed. Results: Both SOX9 and COL2A1 were expressed in NP tissues, with SOX9 seen in the nucleus and COL2A1 in the extracellular matrix. The expression of SOX9 and COL2A1 mRNA and protein gradually decreased as Miyazaki I progressed to Miyazaki V. We also found a linear relationship between COL2A1 (Y) and SOX9 (X) expression: Y=0. 923X-0. 059 (P<0. 001). Conclusion: The expression of SOX9 and COL2A1 mRNA decrease with the increase of CIDD severity, with SOX9 and COL2A1 mRNA having a similar decreasing tendency. SOX9 positively regulate COL2A1 expression, indicating that changes of SOX9 and COL2A1 gene might be one of the molecular mechanisms of CIDD.

Clinical and genetic research in a Chinese family with Stickler syndrome type 1 / 中华实验眼科杂志

Background Stickler syndrome is a genetic connective tissue disorder that affects the ocular,skeletal,orofacial and auditory systems.To determine the gene mutation loci can offer a basis for genetic diagnosis and management of Stickler syndrome.Objective The aim of this study was to research the clinical characteristics of a pedigree with Stickler syndrome and identify the disease-causing gene mutation.Methods This study was approved by Ethic Committee of Peking Union Medical College Hospital.The clinical study and pedigree analysis were performed in one family with Stickler syndrome type Ⅰ (STL Ⅰ).Nine family members were examined with informed consent.The entire coding regions of COL2A1 gene with flanking intronic regions were amplified by PCR and directly sequenced.The detected sequence change was confirmed to be mutationloci by examining whether they existed in normal control individuals.Mutant proteins were predicted with online software.Results There were 4 generations and 11 members in this family,and 2 members died,including 1 patient.Three patients were found in 9living families.Inheritance of this family complicd with an autosomal dominant inheritance mode.All affected individuals showed the consistent phenotypes with STL Ⅰ,including high myopia,membranous vitreous anomaly and surface central flat,short nose,palatoschisis,etc.Mutation screening of COL2A1 gene revealed that the first base of intron 12 was deleted(IVS12+1G del).Nucleotide sequence analysis showed that this mutation led to the functional abnormal of this gene by forming termination cordon in advance.This mutation occurred in all affected individuals,however,no mutation was observed in any unaffected member or 100 normal unrelated individuals.Conclusions This study identifies a novel splice-site mutation(IVS12+ 1G del)in COL2A1 gene in a Chinese STL Ⅰ pedigree.This is the first report on a mutation in a Chinese STL Ⅰ family.

A Case of Stickler Syndrome Type I Caused by a Novel Variant of COL2A1 Gene

Stickler syndrome is a very rare connective tissue disorder. The authors of the present study describe an 11-month-old girl with high myopia, retinal abnormalities, flat nose, cleft palate, retrognathia, micrognathia, short stature and arthrogryposis. Radiological evaluation also showed irregularity of the epiphysis of the femur and tibia and spondyloepiphyseal dysplasia. Genetic analysis using a peripheral blood sample revealed a novel variant c.787G>A (p.Gly246Asp) mutation of the COL2A1 gene. This is the first Korean case with Stickler syndrome confirmed by genetic testing.

COL2A1 gene mutation of a family with spondyloepiphyseal dysplasia conginita / 医学研究生学报

Objective:To investigate a large Chinese family in which 9 patients over 4 generations were diagnosed with a form of autosomal dominant spondyloepimphyseal dysplasia(SEMD).Mothods:X-Ray radiograph of proand at 18-month showed absence of secondary ossification centra of femoral heads.His father at 24-year presented severe spondyloepiphyseal changes that principally involved the vertebral bodies,the femoral necks and femoral heads and characterized by generalized platyspondyly with thoracolumbar scoliosis,irregular femoral necks,absent ossification of femoral heads,flat acetabular roofs and coxa vara.The other patients had similar clinical and radiological features.Haplotyping was performed with leukocyte DNA for 5 micosatellite repeat markers from chromosome 12 and the result showed COL2A1 gene as a candidate gene.A total of 54 exons and promoter of COL2A1 gene were amplified and sequenced from all patients and available normal relatives.In addition,exon 23 of COL2A1 gene was amplified and sequenced from 10 controls simultaneously.Results:All patients were identified a 1510(G→A) transition in exon 23 of COL2A1 gene that caused a change from a COL2A1 coding region in available glycine to serine at amino acid position 504.No mutation was found in the normal relatives and 10 controls. Conclusion:The mutation of COL2A1 gene is responsible for this form of SEDC of the family.This is the first familial report of SEDC relating to 1510G→A mutation of COL2A1 gene.The detailed clinical radiogram data will be useful for extending the phenotypic spectrum of type Ⅱcollagenopathies.

Prenatal molecular diagnosis of two pregnancies in familial G504S mutation of COL2A1 gene resulting spondylepiphyseal dysplasia congenita / 临床检验杂志

Objective To report the prenatal molecular diagnosis for two gravida in a family with spondylepiphyseal dysplasia congenita(SEDC)caused by G504S mutation of COL2A1 gene.Methods DNA of the two fetuses was extracted from amniotic fluid at the 19+3 and 18+6 weeks of gestation respectively.Direct sequencing of two samples were performed after amplifying exon 23 of COL2A1 containing the potential mutation.The femur length and biparietal diameter of the first fetus were measured by sonographic scans every two weeks from 17+3 weeks to 27+3 weeks of gestation,and for the second fetus these parameters were measured from 16+1 to 19+1 weeks of gestation.Results Sequncing analysis revealed the first fetus and his mother presented the same mutation which is specifically associated with SEDC,but the second fetus did not show the mutation of COL2A1 gene.Biparietal diameters of the both fetuses were appropriate for gestational age.Femur length of the second fetus was normal for gestational age but that of the first fetus was shortened evidently after the 23 week of gestation.The parents of the first fetus determined to terminate the pregnancy.A medical termination was carried out at 27+5 weeks of gestation and a male fetus with a relatively large head and short limbs was delivered.The radiological findings of the fetus were consistent with SEDC including generalized platy spondesand shortened long bones.Conclusions Prenatal molecular diagnosis is important for the fetus with risk of SEDC and useful for genetic counseling.Genotype of fetus with risk of SEDC can be identified before sonographic scan.Molecular genetic analysis in conjunction with sonographic monitoring was helpful in prenatal diagnosis of SEDC.