Background
Alport syndrome (ATS) initially described as a likely autosomal dominant condition, with males being more severely affected than females, [
1‐
5] has been redefined as an X-linked semi-dominant condition [
6,
7] after the discovery in 1990 of the causative gene
COL4A5 on the X chromosome (Xq22.3). With time it has become evident that ATS is a genetically heterogeneous disorder for which all three main models of Mendelian inheritance, namely X-linked (XL), autosomal recessive (AR) and autosomal dominant (AD), are applicable [
8]. ATS is characterized by clinical heterogeneity and extreme intrafamilial phenotypic variability including the degree of proteinuria, the onset of Chronic Renal Failure (CRF), the progression-rate to End Stage Renal Disease (ESRD), as well as the ocular and hearing involvement. While X-linked semidominant ATS is a well-established entity associated with mutations in
COL4A5, AR inheritance pattern is associated with two mutations, in either
COL4A3 or
COL4A4 [
7,
9] located on chromosome 2(2q35-q37) [
8,
10].
The existence of an AD form of ATS (ADAS) distinct from the thin basement membrane nephropathy (TBMN) is sometime debated; however, already in 2000, van der Loop and colleagues provided convincing evidence in favor of the existence of ADAS, identifying a heterozygous mutation in the
COL4A3 gene in a large ATS family from Northern Ireland [
11]. Shortly afterwards, some ADAS pedigrees have also been reported by our group [
9] and we have then provided clear evidence of the existence of an AD form accounting for about a 30% of cases [
12]. Recently, we have reported a digenic pattern of inheritance for ATS demonstrating that the severity of renal involvement correlates with the number of the collagen type IV mutated molecules [
13,
14]. However, the digenic model based on the combination of collagen IV mutations partially explains the phenotypic variability. A recent report underlined the contribution of
LAMA5 gene mutations co-segregating with
COL4A5 mutations in patients with a phenotypic spectrum including hematuria, proteinuria, focal segmental glomerulosclerosis, loss of kidney function and renal cortical cysts [
15].
Here, we selected patients with a likely digenic pattern of inheritance who presented a phenotype more severe than what expected for a dominant ATS or for whom family history was suggestive of a biparental inheritance and, using a whole-exome sequencing approach, we investigated whether variations in other genes of the extracellular matrix (basement membrane and slit diaphragm) or podocyte cytoskeleton are implicated in the intrafamilial phenotypic variability. We found that hypomorphic heterozygous variants in LAMA5 encoding for the alpha 5 subunit of laminin 511 segregate with the severity of renal involvement sometimes along with hypomorphic heterozygous variants in other kidney disease-related genes and we concluded for more complex mechanisms of inheritance in which non-collagen genes may play a pivotal role in disease pathogenesis.
Discussion
ATS is one of the most common inherited form of glomerulopathy, often associated with deafness and ocular lesions. Four clinical criteria, which include positive family history of hematuria with or without chronic renal failure, unique ultrastructural changes in the GBM, and extrarenal manifestations for the diagnosis of ATS were proposed by Flinter in 1988 [
18]. Genetic testing is always recommended and is the only tool able to confirm the diagnosis and define the inheritance pattern. Employing a targeted next-generation multigene panel in a clinically heterogeneous nephropathy such as ATS, we have recently been able to define a new digenic pattern of inheritance with pathogenic mutations in two collagen IV genes. However, daily clinical practice prompt us to realize the existence of families in which a collagen-related genes digenic pattern of inheritance is not sufficient to justify the clinical severity, the intrafamilial variable expressivity and sometimes the histopathological findings. Recent works have suggested a role of other genes in exacerbating clinical phenotype in ATS patients with
COL4A5 mutations [
19]. Recently an association of
LAMA5 and
COL4A5 variants has been described in four family members presenting with microhematuria and proteinuria and progressive ESRD in the spectrum of ATS [
15].
For the first time, here we describe three families, two with an apparent autosomal dominant ATS and one with an X-linked pattern of transmission, in which a pathogenic mutation in
COL4A4 or
COL4A5 cosegregate with a likely damaging variant in
LAMA5. Notably, in family 1, a very large informative family, the combination of
LAMA5/COL4A4 variants was observed in all severely affected family members, while the single
COL4A4 variant was detected in one individual with episodic microhematuria and in two asymptomatic older cousins, suggesting, a digenic inheritance rather than a modifier effect, according to which the coexistence of both variants is necessary in order to display all the clinical criteria for ATS diagnosis. We have previously provided clear evidence of the existence of an AD form of Alport syndrome [
12]. Here we like to introduce the concept of a mutation type-dependent pattern of transmission based on which severely damaging COL4a3 and a4 chains mutations in heterozygous state are sufficient to determine an autosomal dominant ATS while hypomorphic mutations need to cosegregate together or in association with variants in other genes of the extracellular matrix or podocyte cytoskeleton in order for a fully penetrant phenotype to develop.
Previous works have shown that the glomerular filtration barrier integrity is ensured by laminin alpha5. Podocytes specific inactivation of Lama5 in mice, results in varying degrees of proteinuria and progression to nephrotic syndrome likely related to a thickening of the GBM associated with podocyte foot processes disruption [
20]. This is in line with the histopathological findings of the probands in family 2 and 3 who displayed thickening and thinning of the GBM associated with podocytes foot processes retraction or fusion which is rarely observed in collagen 4 genes-related ATS and that should be regarded as a pathognomonic sign of
LAMA5 variants coinheritance. Proband’s father in family 2, harboring the single
LAMA5 mutation displays constant microhematuria suggesting that damaging
LAMA5 mutations in a heterozygous state could alone explain familial hematuria. In line with previous reports [
15] renal cysts were also observed in family 1 affected members who harbor the combination of
LAMA5/COL4A4 mutations. These findings urge the need to investigate for a
LAMA5 mutation in all ATS individuals in which kidney biopsy is positive for the presence of renal cysts and podocytes foot processes fusion or retraction.
In family 3 affected individuals also harbored a
NPHS2 variant. The
NPHS2 variant c.686G > A (p.(Arg229Gln)) (rs61747728), is reported as one of the most important predictive factors for steroid-resistant nephrotic syndrome and FSGS [
21]. Some studies have suggested that a recessive inheritance of the variant is enough to determine the phenotype [
22], while others have suggested that the p.(Arg229Gln) is pathogenic only when it is inherited in a compound heterozygote state with another
NPHS2 mutation or in combination with other variants [
23]. Our data suggests that in a heterozygote state, this allele contributes to an oligogenic form of disease in which any single gene plays a different role sometimes distinct among the affected individuals. Due to the young age of the proband at the moment of the clinical evaluation we cannot exclude the future onset of proteinuria as reported in the paternal uncle with whom he likely shares the combination of
COL4A4 mutation with at least one of the other two variants. However, it is also plausible to think that in each family member the existence of different trans-splicing factors would determine a variable effect of the
LAMA5 splicing variant on its own transcript leading to a distinct clinical expressivity.
The mean age of onset of ESRD in our group of patients harboring a combination of
COL4/
LAMA5 variants (II;2, II;3, II;4; II;5 in family 1 and II;2 in family 2) is 45 years, in line with previously reported data about collagen 4 chains-related digenic inheritance [
13,
14], suggesting to suspect a digenic inheritance when the age of the renal failure onset is intermediate between an autosomal dominant and a recessive form. Ultimately, these data highlight the need for a next generation sequencing approach broadened to include non- collagen genes in order to better define clinical severity and recurrence risk for other family members. Definitely, a larger study which compares patients with only a collagen 4-gene mutation and patients harboring a combination of collagen 4/non-collagen genes variant is needed in order to prove an additive effect of the identified alterations.
Acknowledgements
We would like first to thank ATS patients and their families. The ‘Cell lines and DNA bank of Rett Syndrome, X-linked mental retardation and other genetic diseases’ and the ‘Cell line and DNA Bank of Genetic Movement Disorders and Mitochondial Diseases (GMD-MDbank)’, members of the Telethon Network of Genetic Biobanks (project no. GTB12001), funded by Telethon Italy, and of the EuroBioBank network provided us with specimens.We also thank a donation in favor of ‘Graziano and Marco Laurini’ to AR.