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Familial microscopic hematuria (FMH) is associated with a genetically heterogeneous group of conditions including the collagen-IV nephropathies, the heritable C3/CFHR5 nephropathy and the glomerulopathy with fibronectin deposits. The clinical course varies widely, ranging from isolated benign familial hematuria to end-stage renal disease (ESRD) later in life. We investigated 24 families using next generation sequencing (NGS) for 5 genes: COL4A3, COL4A4, COL4A5, CFHR5 and FN1. In 17 families (71%), we found 15 pathogenic mutations in COL4A3/A4/A5, 9 of them novel. In 5 families patients inherited classical AS with hemizygous X-linked COL4A5 mutations. Even more patients developed later-onset Alport-related nephropathy having inherited heterozygous COL4A3/A4 mutations that cause thin basement membranes. Amongst 62 heterozygous or hemizygous patients, 8 (13%) reached ESRD, while 25% of patients with heterozygous COL4A3/A4 mutations, aged >50-years, reached ESRD. In conclusion, COL4A mutations comprise a frequent cause of FMH. Heterozygous COL4A3/A4 mutations predispose to renal function impairment, supporting that thin basement membrane nephropathy is not always benign. The molecular diagnosis is essential for differentiating the X-linked from the autosomal recessive and dominant inheritance. Finally, NGS technology is established as the gold standard for the diagnosis of FMH and associated collagen-IV glomerulopathies, frequently averting the need for invasive renal biopsies.

Most Alport cases, 85%, are caused by mutations in the X-linked gene, COL4A5 that encodes the α5 chain of type IV collagen, the most abundant structural protein in the glomerular basement membrane (GBM). The remaining 15% of cases are caused by autosomal recessive mutations in the genes that encode the α3 and α4 chains of the type IV collagen, COL4A3/COL4A4 1,2. Thin basement membrane nephropathy (TBMN) is reportedly also a genetically heterogeneous condition, caused by heterozygous COL4A3/COL4A4 mutations in about 40-50% of the cases. No other responsible genes have been identified as yet. Collagen type IV, as all collagens, is a trimer formed by combinations of three of the six alpha chains, α1-α6. Genes COL4A1 and COL4A2 map to chromosome 13q34, COL4A3 and COL4A4 map to chromosome 2q36–q37 and COL4A5 and COL4A6 map to Xq22-23. All six genes are encoded in nearly 50 exons and close to 1600 aminoacids, and consist of an Nterminal 7S domain, a C-terminal non-collagenous (NC1) domain and a large collagenous domain in between, containing the characteristic Gly-X-Y repeat, common to all collagens. All six alpha chains contain 22-26 natural interruptions of the Gly-X-Y repeats, spread throughout their central collagenous domain. These are presumably regions of specific function and/or ligand binding sequences, of significance to its structural role in the basement membrane. Although there are many possible combinations among the six alpha chains, only three are biologically compatible and found in basement membranes. These are composed of α1α1α2, α3α4α5 and α5α5α6. Type IV collagen participates in forming networks interacting with additional important components of the basement membranes, such as laminin and nidogen 3,4. As a component of the glomerular filtration barrier, along