Alport syndrome is characterized by hematuria, renal failure, and extra-renal alterations, such as: hearing loss, lenticonus, and retinal flecks [4–7]. The disease is caused by changes in the collagen type IV chains, resulting the damage to the base membrane of several organs. Approximately 85% of families have X-linked inheritance with mutations in COL4A5 gene [3, 15–17], and most of the others have autosomal recessive disease with alterations in both copies of COL4A3 or COL4A4 [18–20]. Autosomal dominant inheritance is very rare and results from heterozygous COL4A3 or COL4A4 variants.
In our study, we took a comprehensive three-generation family history. The proband (III:1) was presented a course of progressive deterioration of renal function with persistent hematuria and proteinuria, and underwent a kidney transplant at aged 12. His family history is significant for progressing to ESRD in her mother, over a 10-year follow-up period of hematuria and proteinuria. Besides, His maternal grandma (I:1), maternal aunt (II:4) and aunt’s daughter (III:2) had asymptomatic microscopic hematuria (30/HP) and proteinuria (0.5 g/d) with normal renal function. Characteristic pathological changes were observed in renal biopsy specimens from the proband (III:1) and his maternal aunt’s daughter (III:2), including segmental uneven thickness with lamellation and splitting of the dense layer in GBM under electron microscopy. In addition, immunostaining showed nearly complete loss for a5 (IV) collagen chain in GBM, complete absence in Bowman’s capsule and distal tubule basement membrane in the praband, and his mother (II:1) exhibited segmental distribution of a5 (IV) expression in the skin BM. According to the diagnostic algorithm published in 2013 [7], the diagnosis of Alport syndrome is established in the proband (III:1), his mother (II:1) and his maternal aunt’s daughter (III:2) based on pedigree study, clinical manifestation and skin/renal biopsy.
The COL4A3 and COL4A4 genes reside on chromosome 2 while the COL4A5 gene is located on the X chromosome, which encode the collagen IV a3, a4 and a5 chain, respectively. In normal individuals, the collagen IV a3a4a5 chains are highly expressed and co-distributed within the mature kidney (GBM and distal tubular BM), cochlea, and eye, and the collagen IV a5a5a6 network occurs in the skin BM and kidney (Bowman’s capsule) [5]. X-linked Alport syndrome accounts for of the majority of Alport syndrome cases, arising from mutations in the COL4A5 gene. In the proband and his mother, morphologic phenotype demonstrated reduced and depleted expression of the collagen IV a5 chain in kidney and/or skin. Further clinical and genealogical study indicated that the affected proband (III:1) are much more severe than females (his mother (II:1), maternal grandma (I:1), maternal aunt (II:4) and aunt’s daughter (III:2) ) in the pedigree, and male-to-male transmission is absent. Hence, the mode of inheritance was suspected to be X-linked.
Molecular genetic testing is one of the criteria for the diagnosis of Alport syndrome with a high sensitivity and specificity (༞90%) [7, 21, 22]. High-throughput next generation sequencing (NGS) technology can improve the diagnosis of Alport syndrome by providing molecular confirmation of COL4A3, COL4A4, or COL4A5 mutations. Then we performed whole-exome sequencing for 4 individuals (II:1, II:2, III:1, III:2), however, we did not identify any variation in thought to be possibly disease-causing in the three type IV collagen genes. It is possible that a small proportion of variants may have been missed. Alternatively, a rare deep intronic variant affecting splicing and only detectable by RNA analysis. In addition, pathogenic genes in families with Alport syndrome may not be confined to a few regions. Known COL4A3, COL4A4, and COL4A5 genes are scattered throughout many exons, making it difficult to develop predictive genetic tests.
Noteworthily, a marked C3 staining with the corresponding electron-dense deposits along the mesangium was detected by immunofluorescence and electron microscope analysis of the kidney biopsy in the proband (III:1). Serum C3 was reduced in his mother (II:1), while normal in the proband and other affected family members. Excessive glomerular C3 fragment deposition, with scanty C1q and immunoglobulin, showed some morphological features of C3GN. In addition, the proband and his mother were affected more severely than other members. In the pedigree, familial C3GN is suspicious. The findings above prompted us to take a further investigation of the complement system in the index family. A broad range of genetic contributors definitely implicate in the pathogenesis of C3GN [23–25], then we screened a set of complement genes.
We identified a rare heterozygous missense variant c.508G > A (p.V170M; rs201073457) in the complement regulatory gene CFHR5 in 3 members in this family (I:1, II:1 and III:1). CFHR5 colocalizes with complement-containing glomerular immune deposits in a variety of glomerular pathologic states [26]. Based on genetic studies, copy number variations in CFHR5 is implicated in C3GN [8, 27–28], but significant enrichment of disease-associated rare variants is less [29]. The c.508G > A in the gene CFHR5 generates a nonsynonymous alteration at amino acid position 170 (Val in wild-type and Met in mutant), which is strictly conserved among organisms. In silico programs (SIFT, SNAP, PolyPhen-2 and Mutation Taster) independently indicated the CFHR5 c.508G > A to be potential functional variant. Then we simulated 3D homology-modeled structures, which indicated mutant-type CFHR5 increased conformational stability and induced an increase in the C3b-binding affinity. Additionally, the rare alteration has been previously reported in a patient with aHUS [30] and verified to exhibit significantly higher C3b binding capacity compared with wild-type CFHR5 in vitro functional assays [31]. The above computational and functional assessments indicated that CFHR5 c.508G > A may be responsible for the C3GN phenotype in the proband.