Introduction:Alport syndrome (AS) is an inherited disorder characterized by hematuria, proteinuria, and kidney function impairment, and frequently associated with extrarenal manifestations. Pathogenic variants in COL4A5 usually cause X-linked Alport syndrome (XLAS), whereas those in the COL4A3 or COL4A4 genes are associated with autosomal dominant (AD) or recessive (AR) inheritance. To date, more than 3000 different disease-causing variants in COL4A5, COL4A3, and COL4A4 have been identified. The aim of this study was to evaluate the clinical and genetic spectrum of individuals with novel, pathogenic or likely pathogenic variants in the COL4A3-A5 genes in a previously unstudied cohort.MethodsIn this study molecular analysis by next generation sequencing (NGS) was performed on individuals from a Lithuanian cohort, with suspected AS. The presence of AS was assessed by reviewing clinical evidence of hematuria, proteinuria, chronic kidney disease (CKD), kidney failure (KF), a family history of AS or persistent hematuria, and specific histological lesions in the kidney biopsy such as thinning or lamellation of the glomerular basement membrane (GBM). Clinical, genetic, laboratory, and pathology data were reviewed. The novelty of the COL4A3-A5 variants was confirmed in the genetic variant databases (Centogene, Franklin, ClinVar, Varsome, InterVar). Only undescribed variants were included in this study.ResultsMolecular testing of 171 suspected individuals led to the detection of 99 individuals with 44 disease causing variants including 27, previously undescribed changes, with the frequency of 9/27 (33,3%) in genes COL4A5, COL4A3 and COL4A4 equally. Three individuals were determined as having digenic AS causing variants: one in COL4A3 and COL4A4, two in COL4A4 and COL4A5. The most prevalent alterations in genes COL4A3-5 were missense variants (n = 19), while splice site, frameshift, unknown variant and stop codon changes were detected more in genes COL4A4 and COL4A5 and accounted for 3, 3, 1 and 1 of all novel variants, respectively.ConclusionGenotype-phenotype correlation analysis suggested that some variants demonstrated intra-familial phenotypic variability. These novel variants represented more than half of all the variants found in a cohort of 171 individuals from 109 unrelated families who underwent testing. Our study expands the knowledge of the genetic and phenotypic spectrum for AS.

BACKGROUND AND AIMS Alport syndrome (AS) is a hereditary renal disease characterized by progressive loss of kidney function and often accompanied by sensorineural hearing loss and ocular abnormalities [1]. The variety of symptoms and their severity is closely associated with mode of inheritance, type of mutation, as well as the sex of the patient [2]. In cases of twins with Alport syndrome, genotype–phenotype manifestation shows a large intrafamilial heterogeneity, although only few reports have been published. In this case series, we describe three different manifestations of three unrelated families with AS with twin pattern. METHOD Molecular analysis by next-generation sequencing was performed on individuals with suspected AS in Vilnius University Hospital Santaros Klinikos. Only patients with confirmed pathogenic AS variants and presence of the twin sibling were included in this study. Clinical, genetic, and laboratory data were reviewed and compared among the siblings and their family members. RESULTS Case 1: Two heterozygous twin sisters with two compound heterozygous pathogenic variants c.4702C > T and c.3247G > C in COL4A3 gene led to autosomal recessive AS. The first sister with a history of nephritis from a childhood reached kidney failure at the age of 33. Further investigations revealed mild bilateral sensorineural hearing loss and no ocular lesions. Her 33-year-old heterozygous twin sister had a similar past history consisting of hematuria since the age of 5 and preeclampsia during her first pregnancy at the age of 30. However, at the age of 33, this patient still had preserved kidney function with eGFR >45 mL/min/1.73 m3. She also suffered by mild bilateral hearing loss while no ocular abnormalities were found. Case 2: Heterozygous pathogenic variant c.2623G > A in COL4A5 gene in female heterozygous twin and her relatives (her mother, son and granddaughter) led to X-linked AS while her twin brother was born healthy. The 51-year-old female’s medical history revealed hematuria since 7 years of age. Her kidney function gradually declined and the patient reached kidney failure at the age of 43 years. Family screening showed a history of kidney disease. Her mother developed hematuria and proteinuria at the age of 20 years and kidney failure at 60 years. The patient's son reached kidney failure at his 20s and he also had bilateral sensorineural hearing loss and typical ocular lesions. Her granddaughter has experienced persistent hematuria and proteinuria since 2 months old, but no extrarenal manifestations were discovered. Case 3: Two heterozygous twin brothers with heterozygous pathogenic variant c.593G > T in COL4A3 gene led to autosomal dominant AS. A 53-year-old male has experienced persistent hematuria and proteinuria since the age of 26. At the age of 53, kidney biopsy was performed where FSGS, thin and split glomerular basement membrane were observed. Further examination showed mild bilateral sensorineural hearing loss and no ocular abnormalities. Family screening revealed that multiple family members had a history of kidney disease. Patient's 55-year-old twin brother experienced persistent hematuria since early adulthood; however, he did not show any signs of hearing loss or ocular abnormalities. Elder brother demonstrated hematuria as well. The patient's deceased father reached kidney failure at the age of 69; however, no hearing abnormalities were found. CONCLUSION Observing genotype–phenotype manifestations suggest that some pathogenic variants demonstrate intrafamilial phenotypic variability. Therefore, despite having identical mutations, twins can manifest AS differently.