P.24 - A possible new phenotype associated with variants in COL6A2 gene

To explore the genetic basis of two fetuses with an osteogenesis imperfecta (OI) phenotype. Two fetuses diagnosed at the Affiliated Hospital of Weifang Medical College respectively on June 11, 2021 and October 16, 2021 were selected as the study subjects. Clinical data of the fetuses were collected. Amniotic fluid samples of the fetuses and peripheral blood samples of their pedigree members were collected for the extraction of genomic DNA. Whole exome sequencing (WES) and Sanger sequencing were carried out to identify the candidate variants. Minigene splicing reporter analysis was used to validate the variant which may affect the pre-mRNA splicing. For fetus 1, ultrasonography at 17+6 weeks of gestation had revealed shortening of bilateral humerus and femurs by more than two weeks, in addition with multiple fractures and angular deformities of long bones. WES revealed that fetus 1 had harbored a heterozygous c.3949_3950insGGCATGT (p.N1317Rfs*114) variant in exon 49 of the COL1A1 gene (NM_000088.4). Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), it was classified as a pathogenic variant (PVS1+PS2+PM2_Supporting) for disrupting the downstream open reading frame resulting in premature translational termination, being de novo in origin, and lacking records in the population and disease databases.For fetus 2, ultrasonography at 23 weeks of gestation also revealed shortening of bilateral humerus and femurs by one and four weeks, respectively, in addition with bending of bilateral femurs, tibias and fibulas. Fetus 2 had harbored a heterozygous c.1557+3A>G variant in intron 26 of the COL1A2 gene (NM_000089.4). Minigene experiment showed that it has induced skipping of exon 26 from the COL1A2 mRNA transcript, resulting in an in-frame deletion (c.1504_1557del) of the COL1A2 mRNA transcript. The variant was inherited from its father and had been previously reported in a family with OI type 4. It was therefore classified as a pathogenic variant (PS3+PM1+PM2_Supporting+PP3+PP5). The c.3949_3950insGGCATGT (p.N1317Rfs*114) variant in the COL1A1 gene and c.1557+3A>G variant in the COL1A2 gene probably underlay the disease in the two fetuses. Above findings not only have enriched the mutational spectrum of OI, but also shed light on the correlation between its genotype and phenotype and provided a basis for genetic counseling and prenatal diagnosis for the affected pedigrees.

BackgroundStickler syndrome is a group of connective tissue disorders that can affect eye (myopia, cataract, and retinal detachment), skeleton (spondyloepiphyseal dysplasia and precocious arthritis), craniofacies (midfacial under development and cleft palate), and inner ear (conductive and sensorineural); with the degree of symptoms varying among patients. Mutations in the COL2A1, COL11A1, COL11A2, COL9A1, COL9A2, and COL9A3 procollagen genes cause Stickler syndrome.Case presentationA 16‐year‐old Mongolian girl approached our clinics with retinal detachment. The proband had vitreous degeneration in both eyes, rhegmatogenous retinal detachment in her right eye, a large area of retina degeneration in her left eye, and coupled with severe myopia. No obvious hearing disorder was found, no abnormalities in bones and joints, and her communication and learning capability were also normal. Further clinical examination showed that the patient's other five family members across three generations had vitreous and retina degenerations. Exome sequencing showed a heterozygous splicing variant in COL2A1 in all patients.ConclusionsIn this case report, a pathogenic splicing variant in the COL2A1 gene was identified in a Mongolian family affected with Stickler syndrome type I by exome sequencing. This heterozygous splicing variant in COL2A1 (NM_001844.4:C.2518‐1G>A) that may impair splicing, which was suggested by in silico prediction. Next‐generation sequencing is helpful for the differential diagnosis of this clinically variable and genetically heterogeneous disorder.

Background: Cardiological complications are a leading cause of adverse outcomes in patients with systemic diseases. Genetic factors, particularly those influencing myocardial structure and vascular regulation, play a significant role in individual susceptibility. Polymorphisms in the COL1A1 gene are associated with myocardial fibrosis and remodeling, while variations in the EDN1 gene are linked to endothelial dysfunction and vasoconstriction. However, the combined effect of these polymorphisms on cardiovascular risk remains underexplored. Objective: To assess the association of COL1A1 and EDN1 gene polymorphisms with the risk of developing cardiological complications and to evaluate their combined effect on structural and vascular abnormalities. Materials and Methods: An observational case-control study included 199 participants: 102 patients with an underlying disease (subdivided into 64 with and 38 without cardiological complications) and 97 healthy controls. Cardiological complications were diagnosed based on clinical, ECG, echocardiographic, and laboratory criteria. Genotyping of COL1A1 and EDN1 polymorphisms was performed using PCR. Statistical analysis included allele frequency comparison, odds ratios (OR), and risk stratification. Results: Carriers of the minor A allele of COL1A1 and the Asn allele of EDN1 showed a trend toward increased risk of complications (OR=1.22, 95% CI: 0.67–2.22 and OR=1.36, 95% CI: 0.70–2.64, respectively). Combined carriage of both unfavorable alleles was associated with a higher frequency of complications and demonstrated an additive risk increase compared to isolated variants. Conclusion: Polymorphisms in COL1A1 and EDN1 are associated with an elevated risk of cardiological complications, with combined carriage resulting in additive risk. These findings support an integrated genetic approach to cardiovascular risk prediction and may inform personalized prevention strategies.

Developmental dysplasia of the hip (DDH) is one of the most common hip deformities in children. Since the COL1A1 gene is located in the DDH relating region, we investigated the COL1A1 promoter variations in the development of DDH. We collected 154 unrelated female patients and 180 matched healthy female children, and then sequenced the COL1A1 gene promoter for detection of variations. Three variations in the COL1A1 gene promoter were detected in ten patients (T-139C, C-106T, and C-35T [rs113647555]), but none of the 180 health controls. The chi-square test showed that the rate of total variations in COL1A1 gene contributed to DDH (p=0.0016). We detected three variations in the COL1A1 gene promoter in patients and first demonstrated that the higher rate of total variations of COL1A1 gene contributed to DDH in Chinese female children; thus, the COL1A1 gene is a new candidate gene for DDH disease.

Osteogenesis imperfecta (OI) is a rare genetic disorder. Clinical severity is heterogeneous. The purpose of this study was to investigate the genetic characteristics of a fetus with OI by whole exome sequencing (WES) and identify the cause of the disease. In this study, a fetus with osteogenic dysplasia was referred to our hospital. DNA was extracted from the aborted fetal tissue and peripheral blood of the parents. To identify the pathogenic genes, we conducted the trio-WES using DNA. A de novo variant in the COL1A1 gene is suspected to be the cause of the OI phenotype. We used Sanger sequencing for validation and various bioinformatics methods (such as SIFT, PolyPhen2, Mutation Taster, conservative analysis, SWISS Model, glycosylation site prediction, and I-Mutant 2.0) for analysis. Both WES and Sanger sequencing identified a novel de novo variant of COL1A1 (c. 1309G>A, p. Gly437Ser) in a fetus with OI. Bioinformatic analysis showed that the affected residue, p. Gly437, was highly conserved in multiple species and predicted that the variant was deleterious and may have an impact on protein function. This variant is present in highly conserved glycine residues of Gly-X-Y sequence repeats of the triple helical region of the collagen type I α chain, which may be the cause of OI. This study revealed that the c.1309G>A (p. Gly437Ser) variant in the COL1A1 gene may be the genetic cause of fetal OI in this case. The discovery of this variant enriched the variation spectrum of OI. WES improves the accurate diagnosis of fetal OI, and doctors can provide patients with appropriate geneticcounseling.

Background and Aims Variants in COL4A3, COL4A4 and COL4A5 genes were the most common variants associated with focal segmental glomerulosclerosis (FSGS). Split-luciferase-based trimer formation assay of α345 collagen is a technique that test collagen structure formation by transfect COL4A3/4/5 plasmids into cells. This method was recently used to successfully determine the pathogenicity of variants in COL4A5 gene in Alport syndrome patients. We used the trimer formation assay to evaluate the variants found in our FSGS patients and correlate with genomic variants of COL4A gene. Method We transfected wild-type COL4A3-SmBiT, COL4A4-LgBiT and COL4A5 plasmids into human embryonic kidney cells. In wild-type plasmids, α345 collagen will be formed, then SmBiT and LgBiT will properly fuse, and luminescence will be measured (Figure 1A). We selected the pathogenic and likely pathogenic variants in three FSGS patients from our FSGS cohort, which are two missense variants in COL4A4 (c.1805G>A, p.Gly602Glu and c.2752G>A, p.Gly918Arg), and a deletion in COL4A4, leading to frameshift and premature stop codon (c.905delG, p.Gly302ValfsTer32). We also used COL4A4, c.2906C>G, p.Ser969Ter and COL4A4, c.1396G>A, p.Gly466Arg as nonsense and missense mutation negative controls, respectively. We did the mutagenesis in COL4A4-LgBiT plasmid and repeat the steps as in wild-type plasmids. If the variants caused abnormal protein structure, SmBiT and LgBiT will not properly fuse, and luminescence will be measured less than wild-type (Figure 1B). Results The luminescence of nonsense control and missense control were at 22.50% and 23.58% of relative light unit (RLU) from cells expressing wild-type, respectively. The luminescences of cells expressing mutated plasmid were at 33.68%, 30.94% and 19.74% of RLU from cells expressing wide type for c.905delG, c.1805G>A and c.2752G>A, respectively. (Figure 1C) The lower luminescence means cells with mutated plasmids produced less normal α345 collagen than cells with wild-type plasmids. The results confirmed that these three variants could really cause abnormal α345 collagen in cell model. Conclusion This is the first study to demonstrate that split-luciferase-based trimer formation assay of α345 collagen can be used as a functional study to evaluate the effect of COL4A3/4/5 variants in FSGS.

Alport's syndrome (AS) is a rare disorder characterized by a triad of deafness, progressive renal dysfunction, and ocular abnormalities. We presented a patient of early onset AS with a novel frameshift pathogenic variant in the COL4A5 gene and discuss the utility of genetic testing in the family as well as for the transplant recipient. The patient was a 17-year-old adolescent male with end-stage renal disease (ESRD) and hearing loss. In the setting of ESRD, since hearing loss and anterior lenticonus was detected on an ophthalmologic exam, AS was suspected. On genetic testing, a novel hemizygous frameshift variant was identified in the COL4A5 gene (c.1392del (p.Asp464GlufsTer10)), which was also segregated in the family. In this report, we discussed the early severe presentation, typical ocular findings, genotype-phenotype correlation, and implications of genetic testing for renal transplant. We also explored the challenges of genetic testing in developing countries and the potential of pharmacogenomics.

Alport syndrome (AS) is a rare inherited kidney disease associated with progressive renal failure and visual and hearing disorders. The purpose of this study was to find genetic variants in patients with suspected Alport syndrome from Central and Southwestern Poland and their association with the clinical course of the disease, and to evaluate the impact of Alport syndrome on pregnancy. Initially, 90 patients with suspected Alport syndrome were evaluated by molecular-based testing. Clinical analyses, including urinalysis, evaluation of serum parameters, ultrasound, ophthalmologic, cardiovascular and audiology examination, and genetic testing were performedusing next-generation sequencing and the Sanger method. Seventy-seven patients (40.26% male; 59.74% female) with a median age of 6years were included in the study group, after receivinga diagnosis of Alport syndrome. Twenty pathogenic/potentially pathogenic variants within the COL4A3, COL4A4 and COL4A5 genes were identified in these patients. The c.1871G > A variant in the COL4A5 gene was the most common (53.25%). Isolated hematuria was the most common initial sign of Alport syndrome (70.8%). Genetic testing confirmed Alport syndrome in 85% of symptomatic patients and in 15% of asymptomatic patients. Sensorineural hearing loss (17%) and ocular abnormalities (6%) were also detected in patients in the study group. Isolated hematuria showed a significant association with COL4A5 gene variants (p < 0.001). Genetic variants showed an association with initial clinical symptoms and age at Alport syndrome manifestation. Regular urinalysis and genetic testing should be considered in suspected cases of Alport syndrome for rapid diagnosis and effective patient management.

Lattice degeneration of the retina is a vitreoretinal disorder characterized by a visible fundus lesion predisposing the patient to retinal tears and detachment. The etiology of this degeneration is still uncertain, but it is likely that both genetic and environmental factors play important roles in its development. To identify genetic susceptibility regions for lattice degeneration of the retina, we performed a genome-wide association study (GWAS) using a dense panel of 23,465 microsatellite markers covering the entire human genome. This GWAS in a Japanese cohort (294 patients with lattice degeneration and 294 controls) led to the identification of one microsatellite locus, D2S0276i, in the collagen type IV alpha 4 (COL4A4) gene on chromosome 2q36.3. To validate the significance of this observation, we evaluated the D2S0276i region in the GWAS cohort and in an independent Japanese cohort (280 patients and 314 controls) using D2S0276i and 47 single nucleotide polymorphisms covering the region. The strong associations were observed in D2S0276i and rs7558081 in the COL4A4 gene (Pc = 5.8×10−6, OR = 0.63 and Pc = 1.0×10−5, OR = 0.69 in a total of 574 patients and 608 controls, respectively). Our findings suggest that variants in the COL4A4 gene may contribute to the development of lattice degeneration of the retina.

Mutations in one of the two genes encoding type I procollagen (COL1A1 and COL1A2) are frequently the cause of osteogenesis imperfecta (OI), a disorder characterized by brittle bones. Here we tested whether patients with low bone density also have mutations in these genes. The 26 patients studied had no apparent metabolic bone disease, but most had a positive family history of osteopenia or osteoporosis. Although a diagnosis of OI was considered by the clinician in some cases, the clinical criteria for OI were not satisfied. Our strategy for mutation analysis consisted of PCR amplification of cDNA made to fibroblast mRNA using primers specific for the coding regions of COL1A1 and COL1A2. The PCR products were then sequenced directly with primers located within each PCR product. We found that 3 of 26 patients had mutations that altered the encoded amino acid. One mutation, at position alpha 2(I)-661 has been reported (Spotila et al. 1991 Proc Natl Acad Sci USA PNAS 88:5423). The other 2 patients, who were not related to each other, had a mutation that altered the proline codon at alpha 1(I)-27 to alanine. This mutation was not found in 81 normal individuals or in 37 additional osteopenic individuals. However, its effect on the biologic function of type I collagen, as well as its role in osteopenia, is uncertain. In addition to the two mutations, we found a polymorphism in codon alpha 2(I)-459. Although this polymorphism involved an amino acid substitution, it was present with equal frequency in the patient and the normal population. By analyzing this and previously reported neutral sequence variants in the COL1A2 gene, we determined that all patients expressed both alleles of the COL1A2 gene. The 12 patients who were heterozygous for a COL1A1 neutral sequence variant also expressed both alleles. Here we present all PCR primer and sequencing primer information. The results suggest that surveying a larger group of similarly selected individuals may reveal additional mutations in the COL1A1 or COL1A2 genes.

Muscular dystrophy is an inherited disease with clinical and genetic heterogeneity. Muscle weakness is the primary symptom of these disorders that often leads to disability and death. The overall prevalence for all types of muscular dystrophies worldwide is 19.8-25.1 per 100,000 population. Autosomal recessive types of muscular dystrophies are more common in Iran, likely due to the high rate of consanguineous marriage. We aimed at deciphering molecular defects in three unrelated families with muscular dystrophies not related to Duchene muscular dystrophy (MD) or limb girdle muscular dystrophies. We are reporting families having affected children with MD owing to the mutations in three genes related to the COL6A (collagen type VI, alpha subunit) gene family. Three unrelated families, who had at least one member affected with MD and for whom a definite molecular diagnosis was not provided by routine methods, were investigated by WES and confirmed by Sanger sequencing. In the first family, a homozygous variant was found in the COL6A3 gene (NM_004369.4:c.4390C>T:p.Arg1464Ter), which explains the clinical symptoms observed in this family. In the second family, two homozygote missense variants with possible relevance to the patient’s phenotype were identified in COL6A1 and COL6A2 genes (NM_001848.2:c.803A>G: p.Glu268Gly and NM_001849.3:c.2489G>A:p.Arg830Gln). Also, a heterozygous pathogenic variant in the COL6A2 gene (NM_001849.3: c.1053+1G>T) was detected in the third family. WES can serve as an effective method for detecting the causative mutations in families with unresolved cases of MD. The data provided herein broadens the spectrum of mutations causing MD in Iran.

An estimated 30%–50% of dilated cardiomyopathy (DCM) cases are attributable to genetic factors, with titin (TTN) mutations constituting the most prevalent genetic etiology, accounting for 20%–25% of hereditary DCM cases. The majority of pathogenic TTN variants are heterozygous truncating mutations (TTNtv), including frameshift, nonsense, and canonical splice-site variants. Alport syndrome (AS) represents the second most common monogenic cause of end-stage kidney disease (ESKD). While COL4A5 gene variants cause X-linked AS, mutations in COL4A3 or COL4A4 genes (both located on chromosome 2) are associated with autosomal recessive AS, autosomal dominant AS, and thin basement membrane nephropathy. We present a unique case featuring both a novel TTN variant and a rare COL4A4 mutation in a DCM patient. This dual rare variant presentation is clinically exceptional and may contribute to expanding the genetic landscape of DCM and informing future investigations into genotype-phenotype correlations between TTN mutations and DCM pathogenesis.

Génétique des lombalgies

Abnormal mRNA Splicing Effect of COL4A3 to COL4A5 Unclassified Variants

Variants in collagen genes can cause diverse growth plate disorders frequently associated with short stature. This study aimed to evaluate clinical phenotypes in two autosomal dominant familial short stature (AD-FSS), along with the responses to recombinant human growth hormone (rhGH). Two AD-FSS children treated with rhGH from two families were included. Next-generation sequencing (NGS) was performed to screen the gene variants that may be related to short stature. The genetic test results were evaluated using the guidelines set by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP). The response of the children to rhGH was evaluated. The first case (child 1) was a girl aged 8 years and 7 months with a height of 118.8 cm. Her mother had a height of 145 cm. The child's maternal aunt, grandmother, grandmother's sisters, and great-grandmother were also under 150 cm in height, sharing the characteristic of short limbs. NGS revealed a c.688G>T heterozygous variant in exon 5 of the COL11A2 gene for the girl and her mother. The second case (child 2) was a boy aged 4 years and 8 months with a height of 96 cm. A heterozygous variant c.2458G>A in exon 32 of the COL11A2 gene was identified in the boy and his father. After 18 and 19 months of rhGH treatment, their heights increased by 15 and 20 cm, respectively, with no adverse events. We presented two AD-FSS cases carrying the c.688G>T variant in exon 5 and the c.2458G>A variant in exon 32 of the COL11A2 gene, respectively. The short-term response to rhGH treatment is promising for AD-FSS children.