Background
The type IV collagen α3α4α5 chain is a major component of the glomerular basement membrane (GBM) and is a heterotrimer that is encoded by three genes:
COL4A3,
COL4A4 and
COL4A5. While
COL4A3 and
COL4A4 are located on chromosome 2,
COL4A5 lies on the X chromosome. Mutations in any of these genes can lead to type IV collagen-related nephropathy in which there is disruption of the normal glomerular basement membrane architecture and kidney disease [
1]. In general, heterozygous mutations result in mild disease characterized by focal or diffuse thinning of the GBM, with or without isolated microscopic hematuria (MH) and termed thin basement membrane nephropathy (TBMN). In a minority (estimated at 10-20%) of patients with TBMN and heterozygous mutation of
COL4A3,
4 or
5 there is progressive renal dysfunction with end stage renal disease (ESRD) in later life, usually after the fifth decade [
2]. Males hemizygous for a
COL4A5 mutation, and individuals of either sex with homozygous or compound heterozygous
COL4A3/4 mutations, are at risk of X-linked and autosomal recessive Alport Syndrome (AS) respectively, in which there is a high likely of ESRD within the first three decades of life, associated with sensorineural deafness and ocular abnormalities, including asymptomatic dot and fleck retinopathy and lenticonus.
Genetic testing is the gold standard in diagnosing collagen IV-related nephropathies. In AS (a rare disease with 1:5000 prevalence), X-linked (XL) inheritance is reported in 80-85% of patients autosomal recessive AS (ARAS) accounts for 15% of cases. TBMN reported to affect at least 1% of the worldwide population is frequently associated with heterozygous mutations of
COL4A3,
COL4A4 or
COL4A5. Although more than 1000 mutations (nearly 300 in
COL4A3 and
COL4A4, and 756 in
COL4A5) have been identified in AS and TBMN [
3], the challenges in the diagnosis of collagen IV nephropathies remain. The large size of
COL4A3,
COL4A4 and
COL4A5 genes, comprising 48–53 exons each, and the lack of mutational hot spots make individual screening of multiple genes by Sanger sequencing difficult and expensive. While historically a
COL4A5 mutation has been identified in 90% of XLAS [
4], in suspected autosomal disease the detection rate of detection of
COL4A3/COL4A4 mutations has been reported as low as 20% [
5]. The clinical and light microscopic (LM) kidney biopsy findings of some collagen IV-related nephropathy patients are difficult to distinguish from other conditions such as focal segmental glomerulosclerosis (FSGS) and glomerulonephritis, which may lead to incorrect diagnosis [
6]. In families presenting with isolated microscopic hematuria only, kidney biopsy may not be clinically indicated and genetic testing is frequently not performed, presumably owing to the cost.
Recent availability of next-generation sequencing (NGS) technologies including whole exome sequencing (WES) make it possible to simultaneously test thousands of genes in an unbiased, high-throughput and cost-efficient manner. WES has recently corrected the diagnosis of AS patients mistaken for familial focal segmental glomerulosclerosis (FSGS) [
7,
8]. Targeted exome capture of
COL4A3/
COL4A4/
COL4A5 followed by NGS has also been used clinically to screen clinically suspected AS patients [
9,
10]. In this study using WES, we identified novel and rare type IV collagen gene mutations in 3 unrelated Chinese families with glomerulonephritis and no extra-renal involvement, in whom diagnosis had been difficult to make because of atypical clinical presentation and complex or incomplete histological findings. Since affected members of all 3 families had progressed to ESRD, precise molecular diagnosis was clinically valuable because it allowed genetic counseling and predictive testing to be offered to relatives, aided therapeutic decision-making and allowed more accurate prognostic advice to be given.
Discussion
Current options for genetic testing include Sanger sequencing of candidate gene(s), next generation sequencing of a selected panel of candidate genes, WES, and whole genome sequencing. In the clinical setting, targeted panels are gaining favor because of the relatively modest cost and the detail with which the targeted regions are analyzed which can allow reliable copy number estimation, detecting or excluding within-gene duplications and deletions. Two recent studies using targeted panels of
COL4A3/A4/A5 for the genetic diagnosis in patients suspected of having a Type IV collagen-related nephropathy identified a likely pathogenic mutation in 55% and 83.2% patients tested [
9,
10]. A major limitation of this approach is that if the panel used does not include the gene responsible in a patient, the mutation will not be detected. This is a significant problem in the current era when new genes are being implicated in Mendelian kidney diseases each year. WES yields data on a far greater number of genes, including those not initially considered candidates for the phenotype being investigated. This allows a diagnosis to be made where detailed phenotype data (such as a kidney biopsy) are misleading or lacking, but may detect variants that predict disease unrelated to the indication for testing in an individual patient, and which they would rather not know about. These considerations must be taken into account when consenting for and analyzing WES data, and are of course avoided when only genes known to be related to the trait under investigation are sequenced. A further advantage is that unbiased screening of large numbers of genes may allow epistatic effects to be detected, such as that of
NPHS2 variant p.R229Q which, although not detected in any of the patients in this study, is known to be associated with proteinuria and renal impairment in TBMN [
21,
22]. Whole genome sequencing is currently less widely applied outside a research setting – the amount of sequencing, data storage and analysis required are significantly greater than for WES and it is not clear if the associated costs are justified when the vast majority of detectable disease-causing variants lie within coding regions that are covered by WES.
The 3 families in this study posed well-recognized diagnostic challenges in kidney disease, including atypical or late clinical presentation with unavailable or inconclusive pathology findings: patients from family 1 and family 2 in this study were initially diagnosed with MsPGN, IgAN and FSGS. Some individuals were treated with extended courses of corticosteroids that very likely had no beneficial effect on their renal disease and would not have been used had the correct underlying diagnosis been known to the treating clinician at the time.
In family 1, although we identified compound heterozygous
COL4A4 mutations in the index patient, his reprocessed EM findings and clinical course of disease was more suggestive of heterozygous
COL4A-related disease than ARAS. Interestingly, the index patient’s grandmother was initially diagnosed with IgAN and the unexpected identification of the co-segregating
COL4A4 [c.G2636A (p.Gly879Glu)] mutation in her suggested co-existence of a collagen IV-related nephropathy. Since she was the only family member who developed ESRD in this family, her poor renal outcome may be attributable to the coincidence of these two kidney diseases, although it is well recognized that a single heterozygous
COL4A3/COL4A4 mutation is associated with late-onset ESRD in up to 20% of patients [
2]. Superimposition of other comorbidities, such as IgAN, in collagen IV-related nephropathy (especially in TBMN) is not rare. A previous study has shown that 30% of familial IgAN cases had GBM abnormalities and TBMN could be a significant contributor to disease in these families [
23].
LM findings of AS often resemble those of FSGS or MsPGN. Data retrieved from the Alport Syndrome database in South China recently showed that the misdiagnosis rate of AS is 13% (52 out of 398), with MsPGN (26.9%) and FSGS (19.2%) being the most common misdiagnoses [
24]. In family 2, WES identified no known or likely FSGS-associated variants, but a novel heterozygous
COL4A3 [c.G2290A (p.Gly997Glu)] mutation that is predicted to have a functional impact on the collagen IV heterotrimer structure. This strongly suggests an underlying diagnosis of collagen IV-related nephropathy rather than an inherited or acquired podocytopathy.
Apart from pathological heterogeneity, collagen IV-related nephropathy is clinically heterogeneous within and between families. In male XLAS patients, the mutation location and nature predict clinical severity. Large deletions and insertions, rearrangements, splicing and nonsense mutations, and mutations within the NC1 domain of
COL4A5 are associated with severe consequences with ESRD occurred before age 20, whereas missense mutations involving the collagenous domain are responsible for a less severe type of AS [
25]. In female XLAS patients, the explanation for the wide variability in outcomes is uncertain, as genotype-phenotype correlation has not been observed. The largest study, of 288 female patients from 195 families, showed that 18% patients progressed to ESRD over the course of the follow-up and only 12% of them reached ESRD by age 40 [
26]. In family 3, in which we detected a
COL4A5 donor splice site mutation (c.687 + 1G > A) known to cause XLAS, the disease was unusually severe (for X-linked disease) in the female patients I-2, II-2 and II-5 and unusually mild in the male patient III-2. The intrafamilial heterogeneity in female patients of family 3 may be due to variable X-chromosomal inactivation [
27]. Other factors such as unknown modifying genes and environmental factors may also affect the natural history of disease in different affected family members.
Recently, the first clinical application of WES reported that the success rate of mutation identification in patients with genetic disorder is approximately 25% [
28]. In our study, sequencing one affected family member using WES, combined with stepwise variant filtering strategy, we identified mutations of collagen IV genes in 3 undiagnosed glomerulonephritis families with no extra-renal involvement. This result shows that WES is a powerful diagnostic tool that can complement renal biopsy and can non-invasively provide a diagnosis in patients with familial kidney disease, particularly when clinical information is limited or non-specific.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
LFJ analyzed data and performed experiments supervised by DPG. LFJ and DPG obtained funding. LFJ, BF, ZJ, SJP, LW, JG and YY obtained the samples and clinical data. WXR and ZJ performed renal pathological analysis. JGR directed the project. All authors read and approved the final manuscript.