The patient (patient 1) was a 2-year-old Japanese girl who was the first child of healthy non-consanguineous parents. Her younger brother had no proteinuria or hematuria. Her maternal aunt had an episode of nephritis, and a renal biopsy was performed during childhood, but the findings were uncertain. At 14 months of age, patient 1 was referred to our hospital because of microscopic hematuria that had persisted from the neonatal period. She had no clinically detectable hearing loss or ocular abnormalities. A urine culture revealed no infection. At 15 months of age, enalapril malate was initiated for proteinuria, with a urinary protein/creatinine ratio (P/Cr) of 1.3-3.6 g/gCr. At 21 months of age, she was admitted to our hospital for a renal biopsy. On admission, a urinalysis showed 2+ proteinuria (P/Cr, 1.3 g/gCr) and 3+ hematuria, with the urine sediment containing >100 red cells per high-power field. Her blood urea nitrogen (BUN) level was 17.7 mg/dL, serum creatinine level was 0.22 mg/dL, serum total protein level was 5.9 g/dL, and albumin level was 3.7 g/dL. Serum C3 and C4 levels were 118.2 mg/dL and 24.8 mg/dL, respectively. Antinuclear antibodies were negative. Renal ultrasonography was unremarkable.
Her mother (patient 2) also had a history of proteinuria and hematuria without renal dysfunction, deafness, or ocular abnormalities. The pedigree of her family is shown in Fig. 1a. At 34 years of age, she was admitted to our hospital along with her daughter (patient 1) for a renal biopsy. A urinalysis showed 3+ proteinuria (P/Cr, 1.7 g/gCr) and 2+ hematuria, with the sediment containing 10-19 red cells per high-power field. Her BUN level was 20.0 mg/dL, serum creatinine level was 0.59 mg/dL, serum total protein level was 6.8 g/dL, and albumin level was 4.0 g/dL. Serum C3 and C4 levels were 134.4 mg/dL and 34.2 mg/dL, respectively. Antinuclear antibodies were negative. Renal ultrasonography showed a cystic lesion in the right kidney.
The renal biopsy findings of the two patients are shown in Fig. 2. In patient 1, 29 glomeruli were observed on light microscopy; the glomerulus, tubules, and interstitium showed no significant alterations. Immunofluorescence (IF) staining for alpha 5 chains of type IV collagen showed segmental and mosaic patterns in the glomerular basement membrane (GBM). Electron microscopy (EM) demonstrated diffusely thinned-out GBMs (139-143 nm) with focal lamellation and splitting. In patient 2, 40 glomeruli were observed on light microscopy, two of which were globally sclerotic. IF staining for alpha 5 chains of type IV collagen showed segmental and mosaic patterns in the GBM, Bowman's capsule, and distal tubular basement membrane (TBM). EM demonstrated diffusely thinned-out GBMs (149-166 nm) with dense granules and splitting. In both patients, the merged IF staining images for alpha 2 and 5 chains of type IV collagen clarified the findings of segmental and mosaic patterns in the GBM.
A sequence analysis of COL4A5 in the index patient and her mother was performed. The study was approved by the Institutional Review Board of Kobe University School of Medicine, and written informed consent was obtained. Genomic DNA was isolated from each patient's peripheral blood leukocytes using the Quick Gene Mini 80 System (Kurabo Industries, Tokyo, Japan), according to the manufacturer's instructions. Mutational analyses of COL4A5 were performed using polymerase chain reaction (PCR) and direct sequencing of genomic DNA of all exons and exon-intron boundaries. All 51 specific exons of COL4A5 were amplified by PCR. The PCR-amplified products were then purified and subjected to direct sequencing using a Dye Terminator Cycle Sequencing Kit (Amersham Biosciences, Piscataway, USA) with an automatic DNA sequencer (ABI Prism 3130; Perkin Elmer Applied Biosystems, Foster City, USA). The analysis revealed that both patients had a heterozygous mutation (c.2767G>C) in exon 32 (Fig. 1a). To investigate X chromosome inactivation, the human androgen receptor (HUMARA) assay was performed in both patients. Genomic DNA was digested by a methylation-sensitive enzyme, HpaII, at 37 C for 18 hours followed by PCR using DNA with HUMARA primers, as described previously. A DNA fragment analysis was performed on a 310 Genetic Analyzer (Thermo Fisher Scientific, Waltham, USA). Fragment data analyses were performed using the Gene Mapper Software program (Thermo Fisher Scientific). The HUMARA assay for patients 1 and 2 revealed that the X chromosome inactivation pattern was 77:23 and 31:69, respectively (Fig. 1b).