COL5A1 Variants Research Articles

A diagnosis of vascular ehlers-danlos syndrome in childhood: clinical and molecular features of 63 individuals

Abstract Background Vascular Ehlers-Danlos syndrome (vEDS) is a rare inherited connective tissue disorder characterised by generalised severe tissue friability and predominantly caused by pathogenic COL3A1 variants. Diagnosis of vEDS in childhood primarily occurs due to a positive family history and/or the occurrence of an early arterial or intestinal event. However, little has been reported on additional clinical characteristics and management in this population. Purpose This study aims to identify critical drivers of a childhood vEDS diagnosis in individuals diagnosed through a national EDS service. Methods 63 individuals (24 index, 31 females) with a clinical and genetic diagnosis of vEDS in childhood (< 18 years) were identified through a national EDS service, and childhood cardiovascular surveillance was conducted through joint inherited cardiovascular disease clinics. Results Diagnosis of vEDS in childhood occurred at a median age of 7 years (IQR 3-12) and was predominantly driven by positive family history of vEDS (55.5%). Clinical investigations were directly initiated by occurrence of a major clinical event in n=13 (20.6%), and a total of 17 major clinical events in childhood were recorded in 13 individuals. First events occurred at a median age of 11 years (IQR 0-13) and vascular events, though infrequent, occurred as early as the first year of life. In individuals who did not have a positive family history or major event, easy and/or excessive bruising was the most frequent cause for referral (60%). Conclusions This cohort provides further details of the clinical and genetic characteristics of children with vEDS and contributes new insight into drivers of vEDS diagnosis in childhood and the importance of long-term management, including paediatric cardiovascular surveillance.

Clinical and Genetic characteristics of patients with Peripheral Retinal Fleck in Koreans.

To describe the clinical and genetic features of Korean patients with peripheral retinal flecks unrelated to aging. A retrospective analysis was conducted on the clinical characteristics of patients with symmetric peripheral retinal flecks. Age-related deposits such as reticular pseudodrusen were excluded, as well as secondary deposits related to intraocular inflammation, tumor, and drug toxicity. Multimodal imaging, electrophysiological examinations, and genetic testing were analyzed. A total of 10 patients (2 males and 8 females) with bilateral peripheral flecks were enrolled in this study. A mean age at diagnosis was 30.5±19.6 years (4-59 years). Within the 10 patients, 6 were genetically confirmed with monogenic retinal disorders. Biallelic pathogenic variants in RDH5 were found in 5 patients, and 1 patient was diagnosed with retinopathy related to Alport syndrome due to a pathogenic variant in COL4A5. Although not genetically confirmed, one case associated with nanophthalmos and another case showing chorioretinal mottling in a carrier of ocular albinism have been identified. In one patient, genetic testing also revealed unknown causes. The mean logMAR initial visual acuity was 0.12±0.18 and 0.07±0.18 in right and left eyes, respectively. Night blindness was reported by 4 patients (40%), with 3 showing decreased or delayed rod response in electroretinogram, particularly those with RDH5 mutations. Differences in the deposit layers and the patterns of flecks were observed on multimodal imaging. In the study population, we observed various causes and clinical differences in the retinal fleck patterns among Koreans, including RDH5-related fundus albipunctatus and Alport syndrome. Despite reports of night blindness symptoms in some cases, all patients demonstrated satisfactory corrected visual acuity.

Detection of Alport gene variants in children and young people with persistent haematuria.

Genetic kidney disease is an important cause of persistent microscopic haematuria in children and young people. We aimed to determine the frequency of variants in the Alport syndrome genes (COL4A3, COL4A4 or COL4A5) in individuals under 18 years of age presenting with persistent microscopic haematuria to a single specialist centre in the UK over a 10-year period. We conducted a retrospective longitudinal study of individuals referred to a tertiary paediatric nephrology service with persistent microscopic haematuria between April 2012 to 2022. A total of 224 individuals (female 51.8%) were evaluated with persistent microscopic haematuria of greater than 6 months duration. The age at presentation was 7.5 ± 4.3 years (mean ± SD) with a duration of follow-up of 6.8 ± 4.6 years (mean ± SD). Targeted exome sequencing was performed in 134 individuals and 91 (68%) had a pathogenic or likely pathogenic variant in COL4A3, COL4A4 or COL4A5. Only 49.5% of individuals with identified variants had a family history of microscopic haematuria documented and 37.4% (34/91) had additional proteinuria at presentation. COL4A5 was the most common gene affected and missense variants affecting glycine residues were the most common variant type. Over two-thirds of children and young people who underwent genetic testing had an identifiable genetic basis for their microscopic haematuria and over half did not have a documented family history. Genetic testing should be part of the evaluation of persistent microscopic haematuria despite a negative family history.

Whole genome sequencing in adults with clinical hallmarks of hypophosphatasia negative for ALPL variants.

Hypophosphatasia (HPP) is a rare disease caused by deficient activity of tissue-nonspecific alkaline phosphatase (ALP), encoded by the ALPL gene. The primary objective was to explore novel ALPL variants by whole genome sequencing (WGS) in patients with HPP who previously tested negative by standard methods for ALPL variants. The secondary objective was to search for genes beyond ALPL that may reduce ALP activity or contribute to HPP symptoms. WGS was performed in 16 patients clinically diagnosed with HPP who had ALP activity below the normal range and tested negative for ALPL variants. Genetic variants in ALPL and genes possibly associated with low ALP activity or phenotypic overlap with HPP were assessed. All 16 patients had ALP activity below the normal range. WGS did not identify any novel disease-causing ALPL variants. Positive findings for other gene variants were identified in 4 patients: 1 patient presented with variants in COL1A1, NLRP12, and SCN9A, coding for collagen, type, I alpha-1 chain, nod-like receptor pyrin domain containing 12, and sodium voltage-gated channel alpha subunit 9, respectively; 1 presented with a heterozygous, likely pathogenic variant in P3H1 coding for prolyl 3 hydroxylase 1; 1 presented with a heterozygous pathogenic variant in SGCE, coding for sarcoglycan epsilon; and 1 presented with a heterozygous variant of uncertain significance in VDR, encoding vitamin D receptor. Genomic analysis did not identify novel ALPL variants or a pattern of disease-causing variants in genes other than ALPL to explain the HPP phenotype in these patients. Clinicaltrials.gov identifier: NCT04925804.

Denovo glomerulonephritis associated with IgA anti-GBM alloantibodies after kidney transplantation in Alport syndrome: A case report with diagnostic implications

Alport syndrome (AS) is a hereditary disorder caused by pathogenic variants in COL4A3, COL4A4, or COL4A5 genes expressing α3, α4, and α5 chains of basement membrane type IV collagen (COL4). The triple-helical α3α4α5(IV) protomer is a major component of the mature glomerular basement membrane (GBM) whose defective formation in AS leads to structural GBM disruption and kidney dysfunction, often resulting in kidney replacement therapy. A genetically intact renal graft exposes the immune system to a non-tolerized α3α4α5(IV) component and an alloimmune response eventually ensues. So far, only IgG alloantibodies reacting against COL4 have been reported in AS alloimmune responses. Here, we report alloimmune glomerulonephritis mediated by IgA antibodies against the non-collagenous C-terminal domain 1 of the α5(IV) chain in a patient with autosomal recessive AS following a second kidney transplantation. The patient presented a not previously described biallelic variant in the COL4A4 gene. Immunological, diagnostic, and clinical implications are discussed.

Collagen IV deficiency causes hypertrophic remodeling and endothelium-dependent hyperpolarization in small vessel disease with intracerebral hemorrhage

Genetic variants in COL4A1 and COL4A2 (encoding collagen IV alpha chain 1/2) occur in genetic and sporadic forms of cerebral small vessel disease (CSVD), a leading cause of stroke, dementia and intracerebral haemorrhage (ICH). However, the molecular mechanisms of CSVD with ICH and COL4A1/COL4A2 variants remain obscure. Vascular function and molecular investigations in mice with a Col4a1 missense mutation and heterozygous Col4a2 knock-out mice were combined with analysis of human brain endothelial cells harboring COL4A1/COL4A2 mutations, and brain tissue of patients with sporadic CSVD with ICH. Col4a1 missense mutations cause early-onset CSVD independent of hypertension, with enhanced vasodilation of small arteries due to endothelial dysfunction, vascular wall thickening and reduced stiffness. Mechanistically, the early-onset dysregulated endothelium-dependent hyperpolarization (EDH) is due to reduced collagen IV levels with elevated activity and levels of endothelial Ca2+-sensitive K+ channels. This results in vasodilation via the Na/K pump in vascular smooth muscle cells. Our data support this endothelial dysfunction preceding development of CSVD-associated ICH is due to increased cytoplasmic Ca2+ levels in endothelial cells. Moreover, cerebral blood vessels of patients with sporadic CSVD show genotype-dependent mechanisms with wall thickening and lower collagen IV levels in those harboring common non-coding COL4A1/COL4A2 risk alleles. COL4A1/COL4A2 variants act in genetic and sporadic CSVD with ICH via dysregulated EDH, and altered vascular wall thickness and biomechanics due to lower collagen IV levels and/or mutant collagen IV secretion. These data highlight EDH and collagen IV levels as potential treatment targets. MRC, Wellcome Trust, BHF.

Genetic study of Alport syndrome in Tunisia.

Alport syndrome is a genetic disorder affecting the kidneys, ears, and eyes, causing chronic kidney disease, sensorineural hearing loss, and ocular abnormalities. It results from pathogenic variants in the COL4A3, COL4A4, or COL4A5 genes, with different inheritance patterns: X-linked from COL4A5 variants, autosomal recessive from homozygous variants in COL4A3 or COL4A4, digenic from variants in both COL4A3 and COL4A4, and autosomal dominant from heterozygous variants in COL4A3 or COL4A4. We analyzed 45 patients with Alport syndrome from 11 Tunisian families to determine their clinical and genetic characteristics. Clinical data were collected retrospectively, and whole-exome sequencing was conducted on one patient from each family. Sanger sequencing validated pathogenic variants, and cascade screening extended the analysis to 53 individuals. We identified nine likely pathogenic variants among 11 index cases: six novel and three known variations. Of these, five were in COL4A3, and four were in COL4A5, with variants including frameshift, nonsense, missense, and alternative splicing. Most variations affected the Gly-XY codon. Among the 45 clinically identified siblings, 30 tested positive for Alport syndrome. The cascade screening identified 3 additional affected individuals, 10 unaffected siblings, and 10 unaffected parents. The mode of inheritance was autosomal recessive in six families and X-linked in four families. This study is the first to screen the mutational spectrum of Alport syndrome in Tunisia. It reveals novel pathogenic variants and suggests that autosomal recessive inheritance may be more common in the Tunisian population than X-linked inheritance, contrary to existing literature.

Clinical, Pathological, and Genetic Characteristics of Patients with Digenic Alport Syndrome.

Digenic Alport syndrome could be associated with poor renal prognosis. However, the characteristics of patients with digenic Alport syndrome remain ambiguous. We retrospectively investigated the clinical symptoms, pathological findings, genetic variants, and proportions of patients with digenic Alport syndrome. The ages at detection of proteinuria and development of end-stage kidney disease (ESKD) were compared between patients with digenic Alport syndrome with disease-causing variants in COL4A3 and COL4A4 and those with autosomal dominant Alport syndrome previously analyzed by our group. Eighteen patients from nine families with digenic variants in COL4A3 and COL4A4 and four male and five female patients with digenic variants in COL4A5 and COL4A3 or COL4A4 were enrolled in this study. Next-generation sequencing revealed that the proportion of patients with digenic Alport syndrome was 1.7% among all patients with Alport syndrome. In patients with digenic variants in COL4A3 and COL4A4, the median ages at detection of proteinuria and ESKD were 10.0 and 57.0 years, respectively. Compared to the patients with autosomal dominant Alport syndrome, the age at detection of proteinuria tended to be earlier (10.0 vs. 20.0 years old; P = 0.073) and that at development of ESKD was significantly earlier (57.0 vs. 72.0 years old; P = 0.045) in patients with digenic Alport syndrome. Overall, patients with digenic Alport syndrome harboring COL4A3 and COL4A4 variants exhibited poor renal compared to the patients with autosomal dominant Alport syndrome. Therefore, timely identification of the two disease-causing variants is critical for the renal prognostic assessment and early treatment of patients with digenic Alport syndrome.

Genotype-first analysis in an unselected health system-based population reveals variable phenotypic severity of COL4A5 variants.

Our knowledge of X-linked Alport Syndrome [AS] comes mostly from selected cohorts with more severe disease. We examined the phenotypic spectrum of X-linked AS in males and females with a genotype-based approach using data from the Geisinger MyCode DiscovEHR study, an unselected health system-based cohort with exome sequencing and electronic health records. Patients with COL4A5 variants reported as pathogenic (P) or likely pathogenic (LP) in ClinVar, or protein-truncating variants (PTVs), were each matched with up to 5 controls without COL4A3/4/5 variants by sociodemographics, diabetes diagnosis, and year of first outpatient encounter. AS-related phenotypes included dipstick hematuria, bilateral sensorineural hearing loss (BSHL), proteinuria, decreased eGFR, and ESKD. Out of 170,856 patients, there were 29 hemizygous males (mean age 52.0 y [SD 20.0]) and 55 heterozygous females (mean age 59.3 y [SD 18.8]) with a COL4A5 P/LP variant, including 48 with the hypomorphic variant p.Gly624Asp. Overall, penetrance (having any AS phenotypic feature) was highest for non-p.Gly624Asp P/LP variants (males: 94%, females: 85%), intermediate for p.Gly624Asp (males: 77%, females: 69%), compared to controls (males: 32%; females: 50%). The proportion with ESKD was highest for males with P/LP variants (44%), intermediate for males with p.Gly624Asp (15%) and females with P/LP variants (10%), compared to controls (males: 3%, females 2%). Only 47% of individuals with COL4A5 had completed albuminuria screening, and a minority were taking renin-angiotensin aldosterone system (RAAS) inhibitors. Only 38% of males and 16% of females had a known diagnosis of Alport syndrome or thin basement membrane disease. In an unselected cohort, we show increased risks of AS-related phenotypes in men and women compared to matched controls, while showing a wider spectrum of severity than has been described previously and variability by genotype. Future studies are needed to determine whether early genetic diagnosis can improve outcomes in Alport Syndrome.

Alport Syndrome: Clinical Utility of Early Genetic Diagnosis in Children.

Alport syndrome (AS) is a hereditary glomerulopathy due to pathogenic variants in COL4A3, COL4A4, and COL4A5. Treatment with Renin-Angiotensin-Aldosterone System (RAAS) inhibitors can delay progression to end stage renal disease (ESRD). From 2018 until today, we performed Whole Exome Sequencing (WES) in 19 patients with AS phenotype with or without positive family history. Fourteen of these patients were children. Genetic testing was extended to family members at risk. All patients received a genetic diagnosis of AS: five X-linked AS (XLAS) males, five X-linked AS (XLAS) females, six autosomal dominant AS (ADAS), and one autosomal recessive AS (ARAS). After cascade screening four XLAS males and eight XLAS females, six ADAS and three ARAS heterozygotes were added to our initial results. Fifteen patients were eligible to start treatment with RAAS inhibitors after their diagnosis. All XLAS female patients, ARAS heterozygotes, and ADAS have been advised to be followed up, so that therapeutic intervention can begin in the presence of microalbuminuria. Genetic diagnosis of AS ensures early therapeutic intervention and appropriate follow up to delay progression to chronic kidney disease, especially in thet pediatric population.

A pilot study of childhood-onset Takayasu arteritis using whole exome sequencing suggests oligogenic inheritance involving classical complement, collagen, and autoinflammatory pathways.

Takayasu arteritis (TA) is a chronic granulomatous inflammatory disease affecting the aorta and its branches. Paediatric TA (pTA) may present from 6months after birth till the adolescent age group. Genetics and pathogenesis of pTA are not fully understood. Earlier studies reported monogenic mutation in NOD2, XIAP, and STAT1 genes in patients with pTA. TA, a relatively rare disease, is more common in geographical pockets, including India. We hypothesized that South Asian patients with pTA, namely, those of Indian subcontinent origin, may have clinically relevant and unique pathogenic variants involving one or more genes, especially those linked to genetically driven vasculitic illnesses, including autoinflammatory pathologies. Children with pTA fulfilling EULAR/PRINTO/PReS classification criteria and presenting with clinical symptoms to the Paediatric Rheumatology clinic of Christian Medical College, Vellore, were included. Blood samples were collected after getting informed consent from parents or guardians and assent forms from children. DNA was extracted from whole blood using the Qiagen DNA extraction kit. Initially, the common variant in Indian population, namely, ADA2 c.139G > A; p.Gly47Arg, was screened, followed by whole exome sequencing. Fourteen children were recruited for the study. Median age of patients was 11years (4months-14years) with a male-to-female ratio of 4:10. Distribution of angiographic subsets by Numano's classification of included children were as follows: type 5 (n = 7), type 4 (n = 5), and type 3 (n = 2). We identified novel variants in ten different genes. This include variants in genes of classical complement pathway, namely, C2, C3, C6, C7, and C9, and other genes, namely, CYBA, SH3BP2, GUCY2C, CTC1, COL5A1, and NLPR3. Two of 14 patients have heterozygous pathogenic variants; this implies that combination of heterozygous variants in C3 and COL5A1 might lead to disease development, suggesting digenic inheritance. One patient has a homozygous variant in CYBA. None of the patients were identified to have ADA2 variants. Whole exome sequencing reveals combination of rare variants in genes C3, COL5A1, and CYBA associated with disease development in children with Takayasu Arteritis. Key Points • We identified novel variants in genes of classical complement pathway, namely, C2, C3, C6, C7, and C9, and other genes, namely, CYBA, SH3BP2, GUCY2C, CTC1, COL5A1, and NLPR3. • Two of 14 patients have heterozygous pathogenic variants in C3 and COL5A1; this may have implications in disease development, suggesting digenic inheritance. • One patient has homozygous variant in CYBA. • None of the patients were identified to have ADA2 variants.

Molecular Genetic Diagnosis with Targeted Next Generation Sequencing in a Cohort of Turkish Osteogenesis Imperfecta Patients and Their Genotype-phenotype Correlation.

Osteogenesis imperfecta (OI) is a group of phenotypically and genetically heterogeneous connective tissue disorders that share similar skeletal anomalies causing bone fragility and deformation. This study aimed to investigate the molecular genetic etiology and determine the relationship between genotype and phenotype in OI patients with targeted next-generation sequencing (NGS). In patients with OI, a targeted NGS analysis panel (Illumina TruSight One) containing genes involved in collagen/bone synthesis was performed on the Illumina Nextseq550 platform. Fifty-six patients (female/male: 25/31) from 46 different families were enrolled in the study. Consanguinity between parents was noted in 15 (32.6%) families. Clinically according to Sillence classification; 18(33.1%) patients were considered to type I, 1(1.7%) type II, 26(46.4%) type III and 11(19.6%) type IV. Median body weight was -1.1 (-6.8, - 2.5) SDS, and height was -2.3 (-7.6, - 1.2) SDS. Bone deformity was detected in 30 (53.5%) of the patients, while 31 (55.4%) were evaluated as mobile. Thirty-six (60.7%) patients had blue sclera, 13 (23.2%) had scoliosis, 12 (21.4%) had dentinogenesis imperfecta (DI), and 2 (3.6%) had hearing loss. Disease-causing variants in COL1A1 and COL1A2 genes were found in 24 (52.1%) and 6 (13%) families, respectively. In 8 (17.3%) of the remaining 16 (34.7%) families, the NGS panel revealed disease-causing variants in three different genes (FKBP10, SERPINF1, and P3H1). Nine (23.6%) of the variants detected in all investigated genes were not previously reported in the literature and were classified to be pathogenic according to ACMG guidelines pathogenity scores. In ten (21.7%) families, a disease-related variant was not found in a total of 13 OI genes included in the panel. Genetic etiology was found in 38 (82.6%) of 46 families by targeted NGS analysis. In addition, 9 new variants were assessed in known OI genes which is a significant contribution to the literature.

#3084 Familial nephropathy associated with a combined mutations of Alport and Nail Patella syndrome genes in the same family

Abstract Introduction Alport syndrome is a genetic condition stemming from mutation in Type IV collagen and is characterized by kidney disease and frequently hearing loss and ocular abnormalities. Renal involvement is from an abnormality of the glomerular basement membrane with hematuria and proteinuria progressing eventually to renal failure. In the vast majority, the inheritance is X- linked affecting the alpha 5 chain of type IV collagen while in about 15 percent of the patients the inheritance is autosomal recessive or dominant with mutations affecting alpha 3 or 4 chains of type IV collagen. Sensorineural deafness is common and ocular abnormalities may include lenticonus, corneal opacities, fleck retinopathy and temporal retinal thinning. Nail Patella syndrome (NPS) is a rare genetic condition from mutation in the LMX1B gene on chromosome 9 with an autosomal dominant inheritance. Glomerular disease is from characteristic glomerular basement membrane changes and is associated with nail, pelvic girdle and limb abnormalities. A forme fruste with glomerular lesions but lacking extrarenal features is recognized but uncommon. We present a family with mutations in both COL4A5 and LMX1B presenting with hematuria and proteinuria Case Presentation A 23 year old female was evaluated for hematuria and proteinuria. Her history was notable for kidney biopsy at the age thirteen showing focal and segmental glomerulosclerosis (FSGS). She was treated with tacrolimus for four years from age thirteen through seventeen. Patient was reportedly diagnosed with Nail Patella Syndrome after genetic testing. Her family history was notable for hematuria in her brother and hematuria and proteinuria in her mother. On physical examination she was noted to have no nail or skeletal abnormalities associated with Nail Patella Syndrome. Her labs were notable for a serum creatinine of 0.6 mg/dl and proteinuria of 155 mg/g creatinine of proteinuria. Urine microscopy revealed dysmorphic hematuria. Radiological imaging did not show skeletal abnormalities observed in Nail Patella Syndrome but genetic testing identified a pathogenic variant in COL4A5 c.919G>A (p.Gly307Ser) in addition to a variant of uncertain significance in LMX1B c.349G>A (p.Gly117Ser). Her brother aged 20 had a creatinine of 0.87 mg/dl with dysmorphic hematuria and no proteinuria. Genetic testing showed the same pathogenic variant in COL4A5 as in his sibling. His mother aged 55 was subsequently evaluated and had a creatinine 0.73 mg/dl with dysmorphic hematuria and 152 mg/g of proteinuria. Genetic testing was with the same variant in COL4A5 noted in her two children. Renal biopsy was extensive thinning and segmental lamellation of the glomerular basement membrane with eight percent FSGS. The typical “moth-eaten” appearance of the glomerular basement membrane characteristic of NPS was not evident. Segmental staining was observed with alpha 5 unit of Type IV collagen. Audiology and retinal optical coherence optometry studies are pending. Discussion Alport syndrome and Nail Patella Syndrome are rare genetic disorders that cause variable degrees of renal insufficiency from glomerular base membrane defects and are associated with characteristic extra renal manifestations. This is the first report of an inherited nephritis with combined mutations in COL4A5 and LMX1B. The clinical features and renal biopsy are compatible with Alport Syndrome and the significance of the mutation in LMX1B is uncertain. Conclusion This is the first case report of a combined mutation in COL4A5 and LMX1B gene presenting with hematuria and minimal proteinuria with preserved kidney function.

#817 Genetic analysis in a group of patients with persistent isolated microscopic hematuria

Abstract Background and Aims Autosomal dominant Alport syndrome (ADAS) is a genetic disease caused by pathogenic variants in COL4A3 and COL4A4 genes. ADAS is the main cause of isolated persistent microscopic hematuria (PMH). However, other diseases like IgA nephropathy (IgAN) can present with PMH. Establishing a precise genetic diagnosis in this type of patients would avoid invasive studies like kidney biopsies. In this study we performed a comprehensive genetic test in a cohort of patients with isolated PMH to evaluate the frequency of ADAS or other genetic abnormalities and compare the clinical characteristics of patients with or without ADAS Method Fifty-two patients with PMH were included in the study. PMH was defined by the presence of 3 or more dysmorphic red blood cells per field in at least 5 consecutive urine sediments and a time-averaged proteinuria <1 gr/day. Average proteinuria was determined for every 6 months of follow-up and time-averaged (TA) proteinuria represented an average of the mean proteinuria measurements in each 6-month period. A comprehensive genetic study using whole exome next-generation sequencing was performed. One hundred and one genes related to familial hematuria, focal segmental glomerulosclerosis and cystic kidney diseases were analyzed. Patients with known secondary causes of hematuria were excluded. The baseline clinical characteristics of patients with and without ADAS were compared. Patients with pathogenic variants in COL4A5 gene were excluded of this analysis. Results Thirty-one (60%) patients were diagnosed with ADAS (16 patients with pathogenic variants in COL4A3, 15 in COL4A4), 3 (6%) patients (2 women, 1 man) presented pathogenic variant in COL4A5 gene, and the genetic study was negative in the remaining 18 patients (variants of unknown significance in COL4A3 or COL4A4 were found in 3 of the latter). No other genetic abnormalities were detected in the panel of 101 genes. Table 1 shows the baseline clinical characteristics of patients with and without ADAS. Patients with ADAS presented a higher TA proteinuria [0.23 (0.11-0.6) vs 0.08 (0.05-0.16), p = 0.01], and a more frequent familial history of isolated microscopic hematuria (76% vs 40%, p = 0.01) chronic kidney disease (CKD) (44% vs 0%, p = 0.01) or end stage kidney disease (32% vs 0%, p = 0.02). In the multivariate analysis, only a family history of microscopic hematuria was associated with ADAS: OR 5.62 (1.25-25.49, p = 0.02). Conclusion ADAS is the most common cause of PMH. A genetic study should be the first diagnostic step in order to obtain a precise cause of the disease and avoid invasive procedures. A family history of microscopic hematuria and of CKD increases the likelihood of ADAS prior to the performance of genetic test.

#2438 ADPKD and collagen genes (COL4A3, COL4A4, COL4A5)

Abstract Background and Aims Familial hematuria diseases are a heterogeneous group of monogenic conditions caused by mutations in one of the collagen IV genes: COL4A3 (2q36.3), COL4A4 (2q36.3), and COL4A5 (Xq22.3) that are expressed in the glomerular basement membranes (GBM) and are responsible for the most frequent forms of microscopic hematuria (MH), Alport syndrome, and thin basement membrane nephropathy (TBMN). Recent data suggest that about 1% (1 in 106 individuals) of the world population may have heterozygous predicted pathogenic COL4A3 or COL4A4 variants, a frequency that leads to the occasional superimposition of TBMN with other glomerulopathies. Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a major genetic disorder affecting up to 12.5 million individuals worldwide and it is the fourth most common global cause of renal replacement therapy. Two are the principal causative genes: PKD1(16p13.3) and PKD2 (4q22.1). The aim of the study was to describe a cohort of clinically ADPKD patients studying collagen IV genes COL4A3, COL4A4, and COL4A5. Method We performed NGS with Sophia Genetic “Nephropathies Solution” Panel on clinically ADPKD patients (pts). This panel includes 44 genes (target region 105.8 kb) involved in different types of nephropathies. We considered variants that are categorized as Variant of Uncertain Significance (Vus), likely pathogenic, or pathogenic (Class 3,4, or 5 of American College of Medical Genetics (ACMG)) in COL4A3, COL4A4, and COL4A5 genes. Results We analyzed 250 consecutive clinically ADPKD pts (consecutive in the outpatient clinic). We found 13 (6.3%) clinically potentially interesting (Class 3,4 or 5 of ACMG) variants in collagen genes. We found 2 pts that were negative for PKD1, PKD2, PKHD1 analysis: 1 pt carrier COL4A4 NM_000092.5: c.2908C>T (p.Gln970Ter), Pathogenic and c.2756A>G (p.Glu919Gly) Vus; 1 male pt carrier COL4A3 NM_000091.5: c.609+3_609+6del, Vus and COL4A5 NM_000495:c.169G>A p.(Gly57Arg), likely pathogenic. We also found 4 pts with 4 Vus variants in collagens genes combined with at least 1 pathogenic or likely pathogenic variant in PKD1 or PKD2 genes. We found 3 pts with likely pathogenic variants in COL4A4 or COL4A3 genes combined with Vus or likely benign variants in PKD1 or PKD2 genes and 1 patient with 1 likely pathogenic variant in COL4A3 and 1 likely pathogenic variant in PKD1. Conclusion Variants in collagen genes are very frequent and the associated phenotype is varied, sometimes very mild such as microhematuria. Furthermore, penetrance has to be considered. The study outlines the importance of considering also collagen genes, even if we have a clear clinical manifestation of ADPKD. An additional variant in a kidney already compromised can have an impact on the phenotype. Analysis of genes involved in these 2 pathologies could lead to a better understanding of the phenotype-genotype correlation at least in patients with both conditions.

#2327 Extent of proteinuria in autosomal dominant Alport syndrome compared to X linked Alport syndrome

Abstract Background and Aims Alport syndrome (AS), as first described by Arthur Cecil Alport in late XIX century, encompasses a clinical picture of neurosensorial hypoacusia, ocular abnormalities, hematuria and proteinuric kidney disease progressing to end-stage kidney disease in the young adulthood. Differently from X-linked and autosomal recessive AS, autosomal dominant AS (ADAS), a more recently recognized entity, whose prevalence based on populational studies may be near to 1%, generally presents with a milder kidney involvement and with negligible extrarenal features. Nevertheless, some of these patients progress to chronic kidney disease (CKD) category G5, with marked intra and interfamilial variability. Although hematuria is almost universally present all three inheritance patterns, the magnitude of proteinuria, a reliable prognostic marker of the X-linked form has not been thoroughly studied in ADAS. We aimed to compare the extent of proteinuria between patients with X-Linked and autosomal dominant AS. Method Retrospective registry data on type of Alport hereditary pattern and spot urine protein to creatinine ratio per CKD category were collected. The 2021 CKD-EPI formula for calculation of glomerular filtration rate (eGFR) was used. Non-parametric test was used to analyze variables that did not follow normal distribution. Results We analyzed proteinuria and eGFR of 127 patients with Alport Syndrome. Information on patient gender, hereditary pattern and gene involved is illustrated it Table 1. Increased proteinuria was observed with CKD progression in all categories (p < 0.05). Within each CKD category, X-linked males had greater median proteinuria followed by, X-linked females and autosomal dominant patients (Table 2 and Fig. 1). No difference in median proteinuria was observed between X-linked females and those with autosomal dominant inheritance throughout CKD categories G1, G2, G4 and G5 (p = 0.142, 0.878, 0.327, 0.909 respectively). Difference in median proteinuria between X-linked females and autosomal dominant inheritance revealed statistical significance in CKD category G3 (p = 0.034), however the small sample size in this group must be taken into account. Conclusion Pathogenic variants in COL4A3, COL4A4, and COL4A5 are all causes of CKD, and proteinuria serves as a hallmark of disease progression. However, the extent of proteinuria in ADAS is significantly lower than that observed in XLAS. Additionally, some ADAS patients develop CKD with only albuminuria. While there is still much to be understood about this common condition, the renal mechanism of damage in ADAS appears to differ definitively from that in XLAS or ARAS.

#1468 Alport syndrome family screening and management—experience of a tertiary center

Abstract Background and Aims Alport syndrome (AS) is the most common cause of inherited chronic kidney disease. This disorder is caused by deleterious variants on COL4A3, COL4A4 or COL4A5 genes. These genes codify the proteins that constitute collagen type IV of the glomerular basement membrane (GBM). Importantly, AS patients can develop chronic kidney disease (CKD) kidney disease that can be delayed or possibly prevented by timely initiation of therapy. The identification of at-risk relatives and their genetic testing allow to identify AS patients and initiate appropriate renal care and avoid unnecessary vigilance if the test is negative. Our aim was to assess the outcomes of Alport Syndrome cascade family screening in a tertiary hospital. Method In this study, we retrospectively assessed familial relatives at risk of AS index cases in our Nephrogenetics consult, from January 2018 to November 2023. After referral, routine tests to detect renal disease were done and AS gene panel was performed. Demographic and clinical data were also compiled. Results A total of 75 at-risk relatives were assessed, belonging to 26 families with a COL4A variant identified in the index case. Relatives at risk were mostly referred by primary care doctors (n = 52, 69.3%), with other 15 patients (20%) directly scheduled for consultation, and 8 (10.7%) referred by a nephrology clinic. In this cohort, an AS diagnosis was confirmed in 43 relatives (57.3%), with an additional 13 (17.3%) of the at-risk relatives with ongoing biochemical or molecular analyses. In 19 (25.4%) relatives the molecular study allowed the exclusion of AS. In the subgroup of patients diagnosed with AS (n = 43): median age at molecular diagnosis was 40.5 years (IQR 20.2-56.5years) and 26 (60.5%) were female. Thirty-five patients (81.4%) patients had autosomal dominant AS: 32 had a variant in COL4A3 (6 different variants were found: 4 were pathogenic or likely pathogenic and 2 were of uncertain significance) and 3 patients on COL4A4. COL4A5 variants were identified in 8 patients, 5 of them were female. Three (7%) patients presented with intermittent hematuria, 14 (32.6%) patients presented isolated hematuria, 10 (23.2%) patients had urinary protein/creatine (Up/cr) <0,3g/g, 7 (16.2%) presented Up/cr between 0.3g/g and 1g/g, 3 (7%) patients had Up/cr >1g/g; Six (14%) had CKD. Twenty-two (51.2%) patients were started on a renin angiotensin system inhibitor (RASi) and 5 (11.6%) were already under a RASi. At the last visit in this group of AS patients, the mean epiTFG was 104.2 ml/min (IQ 85.6-120-5 ml/min) and 6 (14%%) patients had epiTFG below 60 ml/min; mean Uprot/creat was 0.11g/g (IQR 0.06-0.32), 12 (28%) patients had no proteinuria. In the subgroup of relatives at risk were AS excluded (n = 19): median age was 38.5 years (IQR 20.2-56.0) and 11 (58%) were female. One patient had CKD, 7 (36.8%) had no signs of renal disease and 8 (42%) patients had intermittent hematuria. Conclusion Our retrospective review showed that the systematic screening of relatives at risk in our hospital was met with a high discovery rate of AS patients. Since there is clinical benefit in an early AS diagnosis, namely through improved renal care and prevention of disease progression, timely genetic/biochemical screening of at-risk relatives is advisable. Additionally, by discharging the relatives with a negative test, this is a more cost-effective approach than providing continuous vigilance to all.

#1616 Pregnancy and fetal outcome in women with Alport syndrome

Abstract Background and Aims Pregnancy in patients with CKD is associated with maternal and fetal risks. The data about pregnancy outcome in Alport syndrome (AS) is limited. The aim was to evaluate the fetal and maternal pregnancy outcome in women with AS. Method One center retrospective study included 88 women with AS (86 with COL4A5, 2 with COL4A4 variants). Data at conception (age, arterial hypertension (HP), proteinuria (Pr), GFR), course of pregnancy (HP, Pr, GFR, pre-eclampsia (PE)), delivery (preterm (PD): < 37 weeks; early preterm (EPD): < 34 weeks), fetus characteristics (intrauterine fetal growth restriction (IUGR): L < 2 z-score for gestational age; small for gestational age (SGA): m < 2 z-score (SGA)) were obtained from medical charts and a cross-sectional survey. Results Information of 88 women, 133 term pregnancies (2 in 25 pts, 3 in 2 pts) were analyzed. Only 16 (q = 0,18) from 85 (q = 0,96) women with hematuria had diagnosis of AS before pregnancy; one of them had HP, 13 pts had Pr. HP, Pr, decreased GFR were revealed in 11 (q = 0,13), 27 (q = 0,2) and 9 (q = 0,06) pregnant, respectively. PE occurred in 8 cases (q = 0,06); PD was seen in 24 (q = 0,18) and EPD in 1 (q = 0,01) cases; cesarean section was performed in 8 cases (q = 0,07). IUGR and SGA were recorded in 8 (q = 0,06) and 11 (q = 0,08) neonates, respectively; 6 babies (q = 0,05) required a neonatal intensive care unit stay. Pr was the risk factor for PE (CI 14,86 ± 0,85, p < 0,001), PD (CI 6,267 ± 0,49; p < 0,001), IUGR (CI 14,86 ± 0,85, p < 0,001), SGA (CI 8,93 ± 0,67, p < 0,001). Conclusion The risk of pre-eclampsia and slowing of fetal growth in women with AS and normal kidney function appears comparable to that in the general population. But AS should be considered as a potential risk factor for preterm delivery. Proteinuria is associated with unfavorable pregnancy and fetal outcome in AS.

Identification of seven variants in the col4a1 gene that alter RNA splicing by minigene assay.

Type IV collagen is an integral component of basement membranes. Mutations in COL4A1, one of the key genes encoding Type IV collagen, can result in a variety of diseases. It is clear that a significant proportion of mutations that affect splicing can cause disease directly or contribute to the susceptibility or severity of disease. Here, we analyzed exonic mutations and intronic mutations described in the COL4A1 gene using bioinformatics programs and identified candidate mutations that may alter the normal splicing pattern through a minigene system. We identified seven variants that induce splicing alterations by disrupting normal splice sites, creating new ones, or altering splice regulatory elements. These mutations are predicted to impact protein function. Our results help in the correct molecular characterization of variants in COL4A1 and may help develop more personalized treatment options.

Genotype-Phenotype Correlations in Alport Syndrome-A Single-Center Experience.

Alport syndrome (AS) is a common and heterogeneous genetic kidney disease, that often leads to end-stage kidney disease (ESKD). This is a single-center, retrospective study that included 36 adults with type IV collagen (COL4) mutations. Our main scope was to describe how genetic features influence renal survival. A total of 24 different mutations were identified, of which eight had not been previously described. Mutations affecting each of the type IV collagen α chains were equally prevalent (33.3%). Most of the patients had pathogenic variants (61.1%). Most patients had a family history of kidney disease (71%). The most prevalent clinical picture was nephritic syndrome (64%). One-third of the subjects had extrarenal manifestations, 41.6% of patients had ESKD at referral, and another 8.3% developed ESKD during follow-up. The median renal survival was 42 years (95% CI, 29.98-54.01). The COL4A4 group displayed better renal survival than the COL4A3 group (p = 0.027). Patients with missense variants had higher renal survival (p = 0.023). Hearing loss was associated with lower renal survival (p < 0.001). Patients with COL4A4 variants and those with missense mutations had significantly better renal survival, whereas those with COL4A3 variants and those with hearing loss had worse prognoses.