In this study, clinical, pathological and genetic information was provided for 24 patients from nine families. Ten pathogenic variants were identified in type IV collagen genes (COL4A3, COL4A4 and COL4A5) and included eight missense mutations, two nonsense mutations and one frameshift mutation. Five of the mutations were novel. A new missense mutation in UMOD was also identified.
Type IV collagen alpha chains form a network of α1α1α2, α3α4α5 and α5α5α6 helical heterotrimers that form the mature GBM. Abnormalities in α3α4α5 alters the expression of the other two chains, which affects trimer formation in the GBM [14, 15]. The molecular conformation of the collagen triple helix imposes stringent amino acid sequence constraints and requires a (GLY-X-Y) (n) repeat pattern and a high content of sub amino acids. The most common pathogenic variant is a single base substitution that leads to a glycine residue substitution, which breaks the GLY-X-Y repeat pattern. The single glycine substitution destabilizes the triple helix through a local break in the hydrogen bond and creates a discontinuity in the alignment of the helix [16]. In this study, nine of the 11 pathogenic variants occurred in the GLY-X-Y repeat sequence. The COL4A3 missense mutation (p.G1170A), the COL4A4 missense mutations (p.G223V, p.G466R) and the COL4A5 missense mutations (p.G869R, p.G787R) are all glycine substitutions in the GLY-X-Y repeat sequence. The COL4A3 nonsense mutation in Family 7 (E405*) occurs at the second position of the GLY-X-Y repeat and the COL4A4 frameshift mutation in Family 8 (p.P502Lfs*151) occurs at the third position of the repeat. The COL4A4 nonsense mutation in Family 7 (p.R1377*) causes a truncation that is not within the repeat. However, a large number of repetitive sequences are present downstream of this truncation. Overall, with the exception of the COL4A4 missense mutation in Family 8 (p.R1682W), all the pathogenic variants were associated with GLY-X-Y repeat sequences, which would cause a change in protein conformation, as verified by our 3D structure predictions.
With the development of genetic technology, more degenic mutations in the COL4A3 and COL4A4 genes have been identified and reported [17, 18]. In this study, there were two degenic mutant families. Family 2 had degenic missense mutations in the COL4A3 and UMOD genes, whilst Family 3 had a COL4A3 missense mutation and a heterozygous COL4A4 frameshift mutation. Degenic mutations are often considered to cause more severe phenotypes than single gene mutations. This phenomenon was also found in the two degenic mutant families in this study. Family 2, Ⅱ-3, had no hematuria or albuminuria but the renal function was abnormal, which was consistent with the clinical presentation of UMOD mutation-induced, autosomal dominant, tubulointerstitial kidney disease.
In AS caused by pathogenic variants in the COL4A3 and COL4A4 genes, there is a high variability in patient symptoms. Some patients with a mutation, as identified by genetic testing, are asymptomatic, have isolated hematuria or present with renal failure. This presents a major obstacle for the diagnosis of AS and is highly controversial for the definition of the disease [13, 19–22]. In previous reports of COL4A3 and COL4A4 mutations, the terms "familial benign hematuria (BFH)" and "thin GBM disease" were used [23, 24]. In this study, patients with COL4A3 or COL4A4 mutations showed thin GBM, by EM, and indicators of loss of renal function in the form of increased urinary protein and decreased eGFR, in addition to hematuria. This demonstrates that the disease is not benign, as previously defined, when it is caused by COL4A3 and COL4A4 mutations. In this study, BFH, TBMN and others were uniformly referred to as AS.
It is generally accepted that pathogenic variants contribute to AS and the severity and progression of clinical symptoms correlate with the type of mutation. The differential impact of various mutations may be an important predictor of disease severity and frameshift mutations are often considered to be the more severe type of mutation [25]. Family 3 had a COL4A4 frameshift mutation (p.P502Lfs*151) that appeared to have a variable phenotype. Two members of the family, I-1 (64 year old, male) and III-1 (seven year old, male), carried the frameshift mutation but their renal function was normal and they did not develop progressive hematuria. In contrast, II-2 (38 year old, female) developed severe hematuria. Similarly, in Family 6, Family 8 and Family 9, patients with the same pathogenic variant exhibited varying degrees of clinical symptoms. This suggested that there are differences in the severity of clinical symptoms in AS, which may be caused by differences in gender, epigenetic material, environmental factors, smoking and other lifestyle habits [26]. This is consistent with Savige J, Torra R et al [9, 21, 27, 28], who reported that AS, which included thin GBM nephropathy, has high clinical variability when caused by mutations in the COL4A3 and COL4A4 genes.
To date, multiple different COL4A5 mutations have been identified and include deletions, frameshift mutations, nonsense mutations, splice mutations and missense mutations. Mutation of COL4A5 can interfere with the synthesis of the type IV collagen α5 chain and result in the loss of expression of the type IV collagen α3α4α5 chain in the GBM. The deleteriousness of these mutations is mostly predicted by tools such as SIFT [13]. However, In vitro confirmation of COL4A5 gene mutations has been rarely reported. In vitro experiments on the COL4A5 missense mutations (p.G869R, p.G787R) have not been reported. In this study, we found that mRNA levels were significantly lower in the presence of the COL4A5 c.2359G > C or c.2605G > A mutation in HEK-239T cells, than in cells without the mutation. Subcellular localization was not altered by either missense mutation and both the wild-type and mutations were expressed in the cytoplasm. This is further evidence that the COL4A5 missense mutations, c.2359G > C, p.G787R and c.2605G > A, p.G869R, are deleterious. Unfortunately, due to the limitation of antibodies, we did not carry out quantitative analysis of the COL4A5 protein. The pathogenic mechanism of COL4A5 mutation needs further study.
In conclusion, this study broadens the scope of COL4A3, COL4A4 and COL4A5 mutations and also provides a better understanding of genotype and phenotype correlation, which has important implications for the diagnosis of AS.