COL7A1 Mutations Research Articles

#5782 AN EXCEPTIONAL CASE OF A PATIENT WITH TWO RARE GENETIC VARIANTS OF AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE AND ALPORT SYNDROME

Abstract Background Alport syndrome and autosomal dominant polycystic kidney disease (ADPKD) belong to the most prevalent monogenic causes of chronic kidney disease (CKD) and both progress to end stage renal disease (ESRD). Alport syndrome, is caused by mutations in genes encoding collagen IV protein family. Inheritance might be X-linked, autosomal recessive, autosomal dominant or digenic and several pathogenic variants have been identified. ADPKD is mostly caused by mutations in PKD1and PKD2 genes but recently, the GANAB gene, that encodes the glucosidase IIa subunit, has been identified has a cause of a milder form of ADPKD. We present a case report of a patients with CKD-4, bilateral sensorineural hearing loss and multiple renal cysts where two unrelated and distinct genetic variants of both diseases were concomitantly found. Case report A 63-year-old caucasian male with medical history of arterial hypertension, hematuria and bilateral sensorineural hearing loss since his 40 years-old, with cochlear implant surgery 13 years ago was referred to the Nephrology unit due to progressive CKD, currently stage 4. His mother had ESRD of unknown etiology, in peritoneal dialysis since the age of 78 years and he has two healthy sons (aged 16 and 20 years old). Physical examination was irremarkable. Laboratorial workout showed serum creatinine of 2.7 mg/dL, ratio albumin/creatinine of 105mg/g with 11 erythrocytes in urinary sediment. Renal ultrasound showed normal sized kidneys with more than 10 cysts in each kidney, the biggest with 44mm. Genetic testing via next generation sequencing was performed revealing heterozygous mutations in COL4A3 (c.4421T>C p.(Leu1474Pro)), reported in ClinVar, and described in literature as an autosomal dominant variant present in Alport families. A mutation in the GANAB (c.2281A>G p.(Thr761Ala)) gene, classified as variants of unknown significance in was also detected, but this mutations is not reported in gnomAD or ClinVar. The patient was referred for genetic counseling. Conclusion We describe a rare case of possible overlap of ADPKD and Alport syndrome. The COL4A3 mutation is described has having a risk ≥ 20% of developing ESRD, but the association of a mild variant of ADPKD might increase exponentially this risk.

#4879 PREDICTORS OF KIDNEY DISEASE PROGRESSION IN MALES WITH X-LINKED ALPORT SYNDROME

Abstract Background and Aims Alport's syndrome is the second most common monogenic cause of end-stage renal disease (ESRD). Males with X-linked Alport's syndrome (XLAS) have a high risk of early ESRD development. The aim of study was to determine the predictors of unfavorable renal prognosis in boys with XLAS. Method The children with genetically confirmed XLAS (n = 84, age 8.7±4.3 yrs, eGFR 102±16.2 ml/min/1.73 m2, 59 pts with missense COL4A5 mutations) were included in observation single center study (FU 6.9±2.4 yrs). Seventy three pts (q = 0.87) were treated with ACEi (age of start 7.6±3.3 yrs, dose for Ramipril 2.7±0.8 mg/m2/day). Arterial blood pressure (BP), proteinuria (Pr, mg/m2/day), eGFR (Schwartz equation, ml/min/1,73m2), gene mutation type and ACEi-treatment data (age of pts and disease stage at the therapy start, ACEi dosage, dynamics of Pr and eGFR) were obtained and updated for each patients. BP >90 perc for gender, age and height was defined as uncontrolled (uBP); Pr was categorised according to its level as a low (100-<250 mg/m2/day), moderate (≥250-500 mg/m2/day), high (≥500-1000 mg/m2/day) and nephrotic (≥1000 mg/m2/day). Gene mutations were divided into severe (nonsense, deletion, splicing) and less severe (missense) mutations. The eGFR<60 ml/min/1,73m2 was defined as primary outcome. Results Twenty pts (q = 0.24, age 13.5±2.96 yrs) reached the end point during observation period. Non-missense COL4A5 mutations (HR = 4.28, 95% CI 1.47-12.4, p = 0.007), uBP (HR = 20, 95% CI 4.68-29.6, p<0.001), persistent Pr >250 mg/m2/day (HR = 20, 95% CI 3.36-21, p<0.001), absence of ACEi treatment (HR = 10.7, 95% CI 2.45-14.7, p = 0.02) or start therapy at proteinuric stage of disease (HR = 20, 95% CI 2.2-13.8, p<0.001) were the risk factors of disease progression. Multiple regression analysis adjusted for age, initial eGFR revealed that late start of ACEi treatment (β = 0.23, p = 0.036), persistence of uBP (β = 0.26, p = 0.008) and Pr >250 mg/m2/day (β = 0.17, p = 0.04) had independent significance and predict unfavorable renal prognosis (R = 0.76, R2 = 0.57, p<0.000). Conclusion Persistent proteinuria and uncontrolled blood pressure, initiation of ACEi treatment at the proteinuric stage of glomerulopathy are the factors of unfavorable prognosis in male with XLAS.

#3271 EXPRESSION OF THE COLLAGEN IV α345 MOLECULE ALONG THE RENAL TUBULE IN HEALTH AND DISEASE

Abstract Background and Aims Patients suffering from Alport Syndrome (AS) have a high life time risk of kidney failure and hearing loss. The underlying molecular cause is a germline mutation in one of the COL4A3/4/5 genes, whose gene products collectively form an important component of the glomerular basement membrane and the inner ear. Recent genetic studies have shown that the AS is much more prevalent than clinically recognized, suggesting that atypical clinical cases are frequent. Thus, patients with AS may phenocopy other kidney diseases. To date, pathomechanistic studies of the AS have focused exclusively on the glomerular membrane, yet equally strong expression of collagen IV can be found along the distal renal tubule. Structural disintegration of the distal tubular membrane may induce inflammation and fibrosis, thus progression of kidney disease. We hypothesize that genetic loss of the tubular collagen IV molecule contributes to kidney failure and may drive atypical phenotypes. Method Immunohistochemistry (IHC) and in situ hybridization (ISH, RNAscope) for COL IV α5, where IHC was performed on healthy human tissues as well as biopsies from patients with AS. Tubular marker staining for localization of COL IV α5 expression as well as spatial changes on consecutive sections. Human urinary primary tubular cells (huPTC) from healthy probands and patients with AS were generated and cultivated in cell culture, with analysis of COL IV mRNA by qPCR and detection of specifically expressed proteins by immunoblotting. Results COL IV α5 was detected within the tubular basement membrane (TBM) of the distal segments of renal tubules by IHC, including the thick ascending limb (TAL) of Henle (Tamm-Horsfall Protein), the distal convoluted tubule (11ß-Hydroxysteroid-Dehydrogenase) and the collecting duct (aquaporin 2). Spacial vicinity of these areas showed a predominance of interstitial fibrosis and inflammation in patient samples with AS, but not control samples with primary proteinuric diseases. Collagen IV protein is lost along the TBM of patients with AS, even in the hemizygous male and heterozygous female carriers of the hypomorphic but frequent COL4A5 mutation G624D. By ISH, COL4A5 expression was mainly observed in the glomerulus (most likely podocytes) and broadly in tubular epithelial cells, with only few tubulointerstitial cells showing mRNA expression. Likewise, the huPTC showed COL IV expression in qPCR, as well as COL IV α5 protein in immunoblotting. Conclusion Our studies show that the COL IV molecule is situated within the TBM of the complete distal tubule, starting from the TAL throughout the distant collecting duct. Although the epithelial cells have no detectable protein, in situ hybridization and primary cell cultures clearly suggest that tubular cells are a major source of the COL IV α345 molecule within the TBM. Renal biopsies of AS patients with diagnostic genetic COL IV variants indicate that tubulointerstitial fibrosis and inflammation starts in the vicinity of the distal tubule. Therefore, we postulate that the pathogenesis of AS may in part stem from the (distal) tubular apparatus, which is already known from other diseases such as ADTKD and nephronophthisis.

#5124 GENETIC GLOMERULAR DISEASES IN THE ADULT PATIENT: A WORK IN PROGRESS – SINGLE CENTRE COHORT

Abstract Background and Aims Recent advances in genetic molecular techniques have allowed the expansion of knowledge in glomerular diseases of genetic cause, including the identification of genes involved in focal and segmental glomerular sclerosis (FSGS). The prevalence of adult-onset genetic FSGS varies widely depending on the population studied. In central Europe, NPHS2 and COL4A3, COL4A4 e COL4A5 genes are postulated to be the most frequent genes implicated in genetic causes of adult FSGS. This study aims to characterize patients submitted to genetic study of glomerular diseases in the Nephrogenetics Clinic of our centre. Method We conducted a single-centre retrospective analysis of the patients submitted to genetic study of glomerular diseases in the Nephrogenetics Clinic of our centre from 2016 to 2022. Adult patients with a glomerular disease phenotype suggestive of a genetic cause were studied, using a predetermined phenotype-targeted panel of genes, through massive parallel sequencing. The number of genes included in this panel has increased (from 21 genes in 2016 to 49 genes since 2018); given this change, patients with a negative result on the first approach repeated screening with the broader panel. Additionally, patients presenting with advanced chronic kidney disease (CKD) without an identifiable phenotype, studied with a larger panel for CKD causing genes, are reported. In patients with a negative result but highly suggestive presentation, whole exome sequencing (WES) was considered, after multidisciplinary discussion with a Geneticist's evaluation. Patients with variants of unknown significance (VUS) were referred to a Genetics’ consultation for genetic counselling and segregation studies. Results Of the 80 patients studied (comprising 74 families), 52.5% were female (n=42) and 79% (n=63) of Caucasian ascent. Mean age was 45.4 ± 16.0 years. Nine patients were studied with the larger gene panel for CKD. 85% (n=68) had CKD (all stages) at presentation, with proteinuria (ranging from sub-nephrotic to nephrotic range) and 15% had asymptomatic urinary changes. 50% (n=40) of patients had positive family history, and 31% (n=25) had a syndromic presentation and/or extra renal manifestations. 27.5% (n=22) had a renal biopsy, and the most prevalent result was FSGS (11%; n=9). Genetic variants were identified in 70% (n=56) of the patients; but pathogenicity is not fully established in all: genotype-phenotype correlations, including inheritance pattern and histological correlation, and segregation studies (when indicated and possible) are ongoing. In 5 patients results are pending. Pathogenic / likely pathogenic variants were identified in at least 47.5% (n=38) patients. The most frequently implicated genes were: COL4A3, COL4A4 e COL4A5 (n=21), high risk APOL1 (n=8); NPSH2 (n=5), IFN2 (n=6, 3 pathogenic, 1 variant of unknown significance (VUS), 2 under segregation studies) and MYH9 (n=3). COL4A3, COL4A4 e COL4A5 mutations occur with Alport syndrome, but also with histologically documented FSGS, sub-nephrotic and nephrotic proteinuria, nephrotic syndrome and asymptomatic urinary changes. In our cohort, NPHS2 p.R229Q was identified in 5 patients (6%), but only with pathogenic significance in 3 patients, and APOL1 high risk haplotype was co-identified with COL4 variants in 3 patients. Conclusion The genetic study of a cohort of patients suspected of genetic FSGS allowed the identification of genetic variants in the genes of interest in a high percentage of cases. The most frequently implicated genes were COL4A3, COL4A4 e COL4A5, NPHS2, INF2 and APOL1 high risk haplotype. The high percentage of gene variants identified in our cohort is probably due to the selection of a population with a high likelihood of genetic FSGS. Further studies will clarify the causal and/or modifier effect of some genes, namely COL4A3, COL4A4 e COL4A5. The future use of WES in cases of high likelihood of genetic cause will probably allow the identification on novel candidate genes and increase the clinical benefits of a genetic diagnosis of FSGS.

Case Report: A prenatal diagnosis of osteogenesis imperfecta in a patient with a novel pathogenic variant in COL1A2

Osteogenesis imperfecta is considered a rare genetic condition which is characterized by bone fragility. In 85% of cases, it is caused by mutations in COL1A1 and COL1A2 genes which are essential to produce type I collagen. We report the case of a female neonate delivered to a 27-year-old women at San Bartolomé Teaching Hospital with a family history of clavicle fracture. A prenatal control with ultrasound was performed to the mother at 29 weeks. A fetus with altered morphology and multiple fractures was found. Therefore, a prenatal diagnosis of osteogenesis imperfecta was performed. The neonate was born with a respiratory distress syndrome and an acyanotic congenital heart disease. Therefore, she remained in NICU until her death. We highlight the importance of prenatal diagnosis, genetic counseling and a multidisciplinary evaluation in this type of pathologies and report a new probably pathogenic variant in the COL1A2 gene detected by exomic sequencing in amniotic fluid.

Genotype–phenotype relationship and comparison between eastern and western patients with osteogenesis imperfecta

PurposeTo evaluate the genotypic and phenotypic relationship in a large cohort of OI patients and to compare the differences between eastern and western OI cohorts.MethodsA total of 671 OI patients were included. Pathogenic mutations were identified, phenotypic information was collected, and relationships between genotypes and phenotypes were analyzed. Literature about western OI cohorts was searched, and differences were compared between eastern and western OI cohorts.ResultsA total of 560 OI patients were identified as carrying OI pathogenic mutations, and the positive detection rate of disease-causing gene mutations was 83.5%. Mutations in 15 OI candidate genes were identified, with COL1A1 (n = 308, 55%) and COL1A2 (n = 164, 29%) being the most common mutations, and SERPINF1 and WNT1 being the most common biallelic variants. Of the 414 probands, 48.8, 16.9, 29.2 and 5.1% had OI types I, III, IV and V, respectively. Peripheral fracture was the most common phenotype (96.6%), and femurs (34.7%) were most commonly affected. Vertebral compression fracture was observed in 43.5% of OI patients. Biallelic or COL1A2 mutation led to more bone deformities and poorer mobility than COL1A1 mutation (all P < 0.05). Glycine substitution of COL1A1 or COL1A2 or biallelic variants led to more severe phenotypes than haploinsufficiency of collagen type I α chains, which induced the mildest phenotypes. Although the gene mutation spectrum varied among countries, the fracture incidence was similar between eastern and western OI cohorts.ConclusionThe findings are valuable for accurate diagnosis and treatment of OI, mechanism exploration and prognosis judgment. Genetic profiles of OI may vary among races, but the mechanism needs to be explored.

The same heterozygous Col4A4 mutation triggered different renal pathological changes in Chinese family members.

Background: Mutations in the collagen components of the glomerular basement membrane (GBM) often lead to hereditary glomerulonephritis. Previous studies have identified that autosomal dominant mutations of Col4A3, Col4A4 or Col4A5 are associated with thin basement membrane nephropathy (TBMN), Alport syndrome and other hereditary kidney diseases. However, the genetic mutations underlying other glomerulonephritis types have not been elucidated. Methods: In this study, we investigated a Chinese family with hereditary nephritis using the methods of genetic sequencing and renal biopsy. Genomic DNA was extracted from peripheral blood of the proband and her sister, and subsequently was performed genetic sequencing. They were found to have the similar mutation sites. Other family members were then validated using Sanger sequencing. The proband and her sister underwent renal puncture biopsies, and experienced pathologists performed PAS, Masson, immunofluorescence, and immunoelectron microscopic staining of the kidney tissue sections. Results: Through genetic sequencing analysis, we detected a novel heterozygous frameshift mutation c.1826delC in the COL4A4 (NM_000092.4) gene coding region, and 1 hybrid missense variation c.86G>A (p. R29Q) was also detected in the TNXB (NM_019105.6) gene coding region in several members of this Chinese family. Interestingly, we found that the same mutations caused different clinical features and distinct pathological changes in individual family members, which confirmed that pathological and genetic testing are crucial for the diagnosis and treatment of hereditary kidney diseases. Conclusion: In this study, we found a novel heterozygous mutation in Col4A4 and co-mutations of the TNXB gene in this Chinese family. Our study indicated that the same Col4A4 mutated variants produced different pathological and clinical changes in different family members. This discovery may provide novel insights into the study of hereditary kidney disease. In addition, new genetic biology techniques and renal biopsy of individual family members are essential.

Familial Whole Exome Sequencing Study of 30 Families With Early-Onset High Myopia.

This study was conducted to investigate potential candidate pathogenic genes in early-onset high myopia (eoHM) in families with eoHM. Whole-exome sequencing was performed on probands with eoHM to identify potential pathogenic genes. Sanger sequencing was used to verify the identified gene mutations causing eoHM in first-degree relatives of the proband. The identified mutations were screened out by bioinformatics analysis combined with segregation analysis. A total of 131 variant loci, involving 97 genes, were detected in the 30 families. A total of 28 genes (37 variants), which were carried by 24 families, were verified and analyzed by Sanger sequencing. We identified five genes and 10 loci associated with eoHM, which have not been reported in previous research. Hemizygous mutations in COL4A5, NYX, and CACNA1F were detected in this study. Inherited retinal disease-associated genes were found in 76.67% (23/30) of families. Genes that can be expressed in the retina in the Online Mendelian Inheritance in Man database were found in 33.33% (10/30) of families. Mutations in the genes associated with eoHM, including CCDC111, SLC39A5, P4HA2, CPSF1, P4HA2, and GRM6, were detected. The mutual correlation between candidate genes and phenotype of fundus photography was revealed in our study. The eoHM candidate gene mutation types contain five categories: missense mutations (78.38%), nonsense (8.11%), frameshift mutation (5.41%), classical splice site mutation (5.41%), and initiation codon mutation (2.70%). Candidate genes carried by patients with eoHM are closely related to inherited retinal diseases. Genetic screening in children with eoHM facilitates the early identification and intervention of syndromic hereditary ocular disorders and certain hereditary ophthalmopathies.

Cervical Instability and Quadriparesis Requiring Stabilization in Pediatric Patient Caused by a Mutation in COL2A1

A 3-year-old male with no past medical history presented with flaccid plegia of his upper extremities and significant weakness in his lower extremities after wrestling with his brother. Cervical spine magnetic resonance imaging was consistent with cord edema and intraparenchymal hemorrhage at C1-C2. A nonossified tissue mass at the expected location of the upper dens created narrowing of the canal at the C1-2 level and mass effect on the cord. Head computed tomography showed periventricular leukomalacia. Initial findings favored dysplasia of the odontoid with associated soft tissue mass/pannus caused by a possible underlying genetic or metabolic bone dyscrasia. The patient underwent suboccipital craniotomy/C1 laminectomy and occiput to C4 fusion, for decompression and stabilization. Genetic testing showed a COL2A1 collagen disorder, with the child harboring a de novo mutation for c.3455 G>T (p.G1152V). The patient was discharged to inpatient acute rehabilitation, with gradual improvement in strength in all 4 extremities.

Clinical, histological and molecular characteristics of Alport syndrome in Chinese children.

Alport syndrome is caused by COL4A3, COL4A4, or COL4A5 gene mutations. The present study aims to compare the clinicopathological features, gene mutations, and outcome of Chinese children with different forms ofAlport syndrome. One hundred twenty-eight children from 126 families diagnosed with Alport syndrome through pathological and genetic examination between 2003 and 2021 were included in this single-center retrospective study. The laboratory and clinicopathological features of the patients with different inheritance patterns were analyzed. The patients were followed-up for diseaseprogression and phenotype-genotype correlation. Of the 126 Alport syndrome families, X-linked formsaccounted for 77.0%, autosomal recessive for 11.9%, autosomal dominant for 7.1%, and digenic for 4.0%. Among the patients, 59.4% were males and 40.6% were females. Altogether, 114 different mutations were identified in 101 patients from 99 families by whole-exome sequencing, of which 68 have not been previously reported. The most prevalent type of mutation was glycine substitution, which was identified in 52.1%, 36.7%, and 60% of the patients with X-linkedAlport syndrome, autosomal recessiveand autosomal dominantAlport syndrome, respectively. At the end of a median follow up of 3.3 (1.8-6.3) years, Kaplan-Meier curves showed kidney survival was significantly lower in autosomal recessive compared to X-linkedAlport syndrome (P = 0.004).Pediatric patients with Alport syndromeseldom presented extrarenal involvement. X-linkedAlport syndrome is the most frequent form found in this cohort. Progression was more rapid in autosmal recessivethan in X-linked Alport syndrome.

Generation and characterization of induced pluripotent stem cell lines from two patients with recessive dystrophic epidermolysis Bullosa

Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare and severe genetic disease responsible for blistering of the skin and mucosa caused by a wide variety of mutations in COL7A1 encoding type VII collagen. We have generated Induced Pluripotent Stem Cells (iPSCs) from two RDEB patients’ fibroblasts harboring homozygous recurrent mutations in COL7A1. Their pluripotent state was confirmed by gene and protein expression of stem cell markers OCT4, SOX2, TRA1/60 and SSEA4. Embryoid body formation followed by immunostaining and TaqMan scorecard analysis confirmed the capacity of RDEB iPSCs to differentiate into cell types from the three germ layers in vitro.

872 One-step correction through CRISPR/Cas9-mediated HDR and reprograming in induced pluripotent stem cells from a patient with a highly recurrent mutation in COL7A1

872 One-step correction through CRISPR/Cas9-mediated HDR and reprograming in induced pluripotent stem cells from a patient with a highly recurrent mutation in COL7A1

816 A novel COL7A1 mutation c.3570G>A resulted in relatively mild clinical phenotype of recessive dystrophic epidermolysis bullosa

816 A novel COL7A1 mutation c.3570G>A resulted in relatively mild clinical phenotype of recessive dystrophic epidermolysis bullosa

A novel compound heterozygous mutation of COL6A3 in Chinese patients with isolated cervical dystonia.

The etiology and pathogenesis of idiopathic dystonia remain obscure. Recent studies revealed that compound heterozygous mutations in collagen type VI alpha-3 gene COL6A3 may cause recessive isolated dystonia (DYT)-27. However, whether COL6A3 mutations are associated with Chinese patients with isolated dystonia is not yet reported. In this study, 45 Chinese patients with isolated cervical dystonia were recruited, and their blood DNA samples were subjected to whole-exome sequencing. The potential causal variants of COL6A3 were identified based on the criteria of the American College of Medical Genetics and Genomics and by prediction software. Among 45 isolated cervical dystonia patients, 18 patients (10 female patients and eight male patients) were found to have seven potential causal variants in the COL6A3 gene. Among these variants, a compound heterozygous mutation was found in one patient. One allele had a c.1264G>A mutation in exon 4 that resulted in an amino acid substitution of methionine for valine at codon 422 (p.Val422Met) and the other a c.8965+9G>A mutation involving a splicing change in exon 40. In addition, other five missense variants, including c.958G>A (p.Ala320Thr), c.1478T>C (p.Val493Ala), c.1597C>T (p.Arg533Cys), c.1762G>A (p.Asp588Asn), and c.4912G>A (p.Ala1638Thr), were identified as well. We identified a novel deleterious compound heterozygous mutation as well as five missense variants in the COL6A3 gene of Chinese patients with cervical dystonia. These findings may expand the spectrum of the COL6A3 genotype in isolated dystonia.

Characteristics of a Three-Generation Family with Stickler Syndrome Type I Carrying Two Different COL2A1 Mutations

Stickler Syndrome is typically characterized by ophthalmic manifestations including vitreous degeneration and axial lengthening that predispose to retinal detachment. Systemic findings consist of micrognathia, cleft palate, sensorineural hearing loss, and joint abnormalities. COL2A1 mutations are the most common, however, there is a lack of genotype-phenotype correlations. Retrospective, single-center case series of a three-generation family. Clinical features, surgical requirements, systemic manifestations, and genetic evaluations were collected. Eight individuals clinically displayed Stickler Syndrome, seven of whom had genetic confirmation, and two different COL2A1 mutations (c.3641delC and c.3853G>T) were identified. Both mutations affect exon 51, but display distinct phenotypes. The c.3641delC frameshift mutation resulted in high myopia and associated vitreous and retinal findings. Individuals with the c.3853G>T missense mutation exhibited joint abnormalities, but mild ocular manifestations. One individual in the third generation was biallelic heterozygous for both COL2A1 mutations and showed ocular and joint findings in addition to autism and severe developmental delay. These COL2A1 mutations exhibited distinct eye vs. joint manifestations. The molecular basis for these phenotypic differences remains unknown and demonstrates the need for deep phenotyping in patients with Stickler syndrome to correlate COL2A1 gene function and expression with ocular and systemic findings.

Patient-derived podocyte spheroids reveal new insights into the etiopathogenesis of Alport syndrome

Alport syndrome (AS) is a rare disease characterized by defective glomerular basement membranes, caused by mutations in COL4A3, COL4A4, and COL4A5, which synthesize collagen type IV. Patients present with progressive proteinuria, hematuria and podocyte loss. There is currently no cure for Alport syndrome, and this is mainly due to its complex and variable pathogenesis, as well as the lack of models that can faithfully mimic the human phenotype. Here we have developed a novel human culture model of Alport syndrome and used it to study the effects of different mutations on podocyte development and biology. First, we established a differentiation protocol that allowed us to generate podocyte spheroids from patient-derived human induced pluripotent stem cells (hiPSCs). We have then carried out discovery proteomics and demonstrated that a total of 178 proteins were differentially expressed between Alport (AS1 and AS3) and control (LT) podocytes. GO analysis indicated alterations in several metabolic pathways, such as oxidative phosphorylation, RNA maturation, chromatin condensation, and proliferation. Although functional assays showed no changes in lactate production and mitochondrial potential compared to healthy controls, immunofluorescence and electron microscopy analysis showed key morphological changes related to the phenotypical maturation of Alport podocytes. Moreover, the studied mutations led to persistent proliferation, increased reactive oxygen species (ROS) production and the concomitant expression of peroxisome proliferator-activated receptors α and γ (PPARα and PPARγ) in podocytes. These data on patient-derived podocytes provide evidence that collagen mutations, in addition to playing a central role in the defective development of the glomerular filtration barrier, cause significant alterations in podocyte development and metabolism very early in development, even before the formation of the filtering apparatus. In conclusion, our study provides a new methodological platform for the differentiation of podocytes and to study human podocytopathies in a personalized manner, and reveals new insights into the etiopathogenesis and pathobiology of Alport syndrome.

Aortic dissection in a patient with novel frameshift COL5A1 variant of classical Ehlers-Danlos syndrome

Classical Ehlers-Danlos syndrome (cEDS) is one of the 13 subtypes of Ehlers-Danlos syndrome, which has the major clinical criteria of hyperextensibility skin, atrophic scars, and generalised joint hypermobility. The occurrence of aortic dissection has been described in some subtypes of Ehlers-Danlos, but it has a rare association with the cEDS subtype. This case report discusses a 39-year-old female with a past medical history of transposition of great arteries with a Senning repair at the age of 18 months and controlled hypertension with medication, who presents a spontaneous distal aortic dissection. The diagnosis of cEDS was made using the major criteria, and a novel frameshift mutation in COL5A1 was discovered. The reported case emphasises that in patients with cEDS, vascular fragility may be a complication.

COL7A1 Editing via RNA Trans-Splicing in RDEB-Derived Skin Equivalents

Mutations in the COL7A1 gene lead to malfunction, reduction or complete absence of type VII collagen (C7) in the skin's basement membrane zone (BMZ), impairing skin integrity. In epidermolysis bullosa (EB), more than 800 mutations in COL7A1 have been reported, leading to the dystrophic form of EB (DEB), a severe and rare skin blistering disease associated with a high risk of developing an aggressive form of squamous cell carcinoma. Here, we leveraged a previously described 3'-RTMS6m repair molecule to develop a non-viral, non-invasive and efficient RNA therapy to correct mutations within COL7A1 via spliceosome-mediated RNA trans-splicing (SMaRT). RTM-S6m, cloned into a non-viral minicircle-GFP vector, is capable of correcting all mutations occurring between exon 65 and exon 118 of COL7A1 via SMaRT. Transfection of the RTM into recessive dystrophic EB (RDEB) keratinocytes resulted in a trans-splicing efficiency of ~1.5% in keratinocytes and ~0.6% in fibroblasts, as confirmed on mRNA level via next-generation sequencing (NGS). Full-length C7 protein expression was primarily confirmed in vitro via immunofluorescence (IF) staining and Western blot analysis of transfected cells. Additionally, we complexed 3'-RTMS6m with a DDC642 liposomal carrier to deliver the RTM topically onto RDEB skin equivalents and were subsequently able to detect an accumulation of restored C7 within the basement membrane zone (BMZ). In summary, we transiently corrected COL7A1 mutations in vitro in RDEB keratinocytes and skin equivalents derived from RDEB keratinocytes and fibroblasts using a non-viral 3'-RTMS6m repair molecule.

Cytosine Deaminase Base Editing to Restore COL7A1 in Dystrophic Epidermolysis Bullosa Human: Murine Skin Model

Recessive dystrophic epidermolysis bullosa is a debilitating blistering skin disorder caused by loss-of-function mutations in COL7A1, which encodes type VII collagen, the main component of anchoring fibrils at the dermal-epidermal junction. Although conventional gene therapy approaches through viral vectors have been tested in preclinical and clinical trials, they are limited by transgene size constraints and only support unregulated gene expression. Genome editing could potentially overcome some of these limitations, and CRISPR/Cas9 has already been applied in research studies to restore COL7A1 expression. The delivery of suitable repair templates for the repair of DNA cleaved by Cas9 is still a major challenge, and alternative base editing strategies may offer corrective solutions for certain mutations. We show highly targeted and efficient cytidine deamination and molecular correction of a defined recessive dystrophic epidermolysis bullosa mutation (c.425A>G), leading to restoration of full-length type VII collagen protein expression in primary human fibroblasts and induced pluripotent stem cells. Type VII collagen basement membrane expression and skin architecture were restored with de novo anchoring fibrils identified by electron microscopy in base-edited human recessive dystrophic epidermolysis bullosa grafts recovered from immunodeficient mice. The results show the potential and promise of emerging base editing technologies in tackling inherited disorders with well-defined single nucleotide mutations.

A mutational signature and significantly mutated driver genes associated with immune checkpoint inhibitor response across multiple cancers

Immune checkpoint inhibitor (ICI) treatments dramatically prolong the survival outcomes of several advanced cancers. However, as multiple studies reported, only a subset of patients could benefit from the ICI treatment. In this study, we aim to uncover novel molecular biomarkers predictive of immunotherapy efficacy across multiple cancers. Pre-treatment somatic mutational profiles and immunotherapy clinical information were obtained from 1097 samples of multiple cancers, including melanoma, non-small cell lung cancer (NSCLC), clear cell renal cell carcinoma (ccRCC), bladder carcinoma (BLCA), and head and neck squamous cell carcinoma (HNSCC). Mutational signatures, molecular subtypes, and significantly mutated genes (SMGs) were determined, and their connections with ICI response and outcome were also evaluated. We extracted a total of six mutational signatures across all samples. Among, a mutational signature featured by T > C substitutions was identified to associate with an ICI resistance. A molecular subtype determined based on mutational activities was connected with a significantly improved ICI response rate and outcome. Totaling 50 SMGs were identified, and we observed that patients with COL11A1 or COL4A6 mutations exhibited a superior ICI treatment efficacy than those without such mutations. In this study, we uncovered several novel molecular determinants of cancer immunotherapy response under a multiple-cancer setting, which provides clues for enrolling patients to receive immunotherapy and customizing personalized treatment strategies.