Nephrol Dial Transplant (2018) 1–8

doi: 10.1093/ndt/gfy028

Genetic variants in the LAMA5 gene in pediatric

ORIGINAL ARTICLE
nephrotic syndrome

Daniela A. Braun1,*, Jillian K. Warejko1,*, Shazia Ashraf1, Weizhen Tan1, Ankana Daga1, Ronen Schneider1,
Tobias Hermle1, Tilman Jobst-Schwan1, Eugen Widmeier1 Amar J. Majmundar1, Makiko Nakayama1,
David Schapiro1, Jia Rao1, Johanna Magdalena Schmidt1, Charlotte A. Hoogstraten1, Hannah Hugo1,
Sevcan A. Bakkaloglu2, Jameela A. Kari3, Sherif El Desoky3, Ghaleb Daouk1, Shrikant Mane4,
Richard P. Lifton4,5, Shirlee Shril1 and Friedhelm Hildebrandt1
1
Department of Medicine, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA, 2Department of Pediatric Nephrology, Gazi
University, Ankara, Turkey, 3Pediatric Nephrology Center of Excellence and Pediatrics Department, Faculty of Medicine, King Abdulaziz
University, Jeddah, Saudi Arabia, 4Department of Genetics, Yale University School of Medicine, New Haven, CT, USA and 5Laboratory of
Human Genetics and Genomics, Rockefeller University, New York, NY, USA

Correspondence and offprint requests to: Friedhelm Hildebrandt; Email: [email protected]

*These authors contributed equally to this work.

ABSTRACT disease, data from genetic mapping and a mouse model that
Background. Nephrotic syndrome (NS), a chronic kidney dis- recapitulates the NS phenotypes suggest that these genetic var-
ease, is characterized by significant loss of protein in the urine iants may be inherited factors that contribute to the develop-
causing hypoalbuminemia and edema. In general, 15% of ment of NS in pediatric patients.
childhood-onset cases do not respond to steroid therapy and Keywords: genetic variants, inherited diseases, nephrotic syn-
are classified as steroid-resistant NS (SRNS). In 30% of cases drome, treatment response in pediatric nephrotic syndrome,
with SRNS, a causative mutation can be detected in one of 44 whole exome sequencing
monogenic SRNS genes. The gene LAMA5 encodes laminin-a5,
an essential component of the glomerular basement membrane.
Mice with a hypomorphic mutation in the orthologous gene INTRODUCTION
Lama5 develop proteinuria and hematuria. Nephrotic syndrome (NS), a chronic kidney disease, is
Methods. To identify additional monogenic causes of NS, we characterized by proteinuria (>40 mg/m2/h) that causes hypo-
performed whole exome sequencing in 300 families with pedia- albuminemia, edema and hyperlipidemia. While most affected
tric NS. In consanguineous families we applied homozygosity children and young adults show a clinical response to steroid
mapping to identify genomic candidate loci for the underlying therapy, 10–15% of cases exhibit steroid-resistant NS (SRNS;
recessive mutation. defined as persistent proteinuria after 6 weeks of oral steroid
Results. In three families, in whom mutations in known NS treatment) [1, 2]. Patients with steroid-sensitive NS (SSNS) can
genes were excluded, but in whom a recessive, monogenic cause have a single episode of NS, responding promptly to therapy, or
of NS was strongly suspected based on pedigree information, show a complicated clinical course with frequent relapses of NS
we identified homozygous variants of unknown significance after termination of steroid therapy. Some patients who are
(VUS) in the gene LAMA5. While all affected individuals had resistant to steroid therapy may still respond to a more intensi-
nonsyndromic NS with an early onset of disease, their clinical fied drug regimen with different immunosuppressive drugs,
outcome and response to immunosuppressive therapy differed whereas others are completely resistant to therapy [3]. This
notably. observation suggests that in some cases SSNS and SRNS can
Conclusion. We here identify recessive VUS in the gene represent a continuum of the same disease rather than being
LAMA5 in patients with partially treatment-responsive NS. two distinct disease entities. Early response to therapy is a
More data will be needed to determine the impact of these strong predictor of long-term renal outcome [4], and patients
VUS in disease management. However, familial occurrence of with SRNS progress almost inevitably to end-stage renal disease

C The
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 (ESRD) [2]. In patients with SRNS, the most Michigan and Boston Children’s Hospital. Patients were enrolled
frequently observed histological diagnosis on renal biopsy is between 1998 and 2016. We performed WES in 335 individuals
focal segmental glomerulosclerosis (FSGS) [5]. Patients display- from 300 families. Patients were recruited in collaborating cen-
ing minimal change nephropathy (MCN) on renal biopsy when ters worldwide and were ethnically diverse. Individuals of con-
first diagnosed tend to have better outcomes [4]. However, fre- sanguineous kindred were preferentially sent for WES.
quently MCN evolves into FSGS over time [5]. Despite inten- Treatment response to immunosuppressive therapy in NS
sive research, no effective treatment for FSGS/SRNS exists and was defined as follows: complete response—remission of protei-
FSGS constitutes the third most frequent cause of ESRD in nuria, with a urine protein:creatinine ratio of <0.2 mg/mg, nor-
childhood and adolescence [6]. malization of albumin; partial response—a decrease in
The discovery of monogenic causes of SRNS has identified proteinuria by 50%; resistance—persistent, fixed proteinuria.
glomerular podocytes as the primary site of damage in this dis- In cases with complete response, the absence of proteinuria
ease, because many genes that, if mutated, cause SRNS are pre- was confirmed in laboratory values of negative proteinuria either
dominantly expressed in this cell type [7, 8]. Mutations in four in a 24-h urine specimen or in the first morning urine specimen.
genes, namely NPHS1, NPHS2, LAMB2 and WT1, are most fre- A total of 258 patients with renal ciliopathies based on
quently observed, and together explain up to 66% of cases with increased echogenicity and/or cysts on renal ultrasound who
an onset of SRNS within the first year of life [9]. Advances in did not have proteinuria were used as a control cohort.
next-generation sequencing have facilitated the identification of Informed consent, sample processing and WES evaluation were
more than 44 genes that cause SRNS if mutated [10, 11]. It was performed as for NS samples.
recently shown in a worldwide study in 1873 families that in
29.5% of cases with an onset of SRNS before age 25 years, a WES
causative single-gene mutation could be identified [12]. This Genomic material deoxyribonucleic acid (DNA) for WES
finding was confirmed in two additional studies that demon- was extracted from saliva or blood lymphocytes. Following
strated monogenic disease causation in 26.2% and 32.3% of library preparation and exome capture using the Agilent
patients with SRNS, respectively [13, 14]. SureSelect human exome capture arrays (Life Technologies,
The glomerular filter, which is functionally impaired in NS, Carlsbad, CA, USA), next-generation sequencing was per-
consists of podocytes, fenestrated endothelial cells, mesangial formed on a HighSeq platform (Illumina, San Diego, CA,
cells and the glomerular basement membrane (GBM). One USA). Sequence reads were aligned to the human reference
essential component of the GBM is laminin, which forms genome (National Center for Biotechnology Information build
obligate heterotrimers composed of a, b and c chains [15]. 37/hg19) using CLC Genomics Workbench (version 6.5.2)
Laminins and integrin receptors are important for cross talk (CLC bio, Aarhus, Denmark). Subsequent bioinformatics anal-
between podocytes and the GBM [16]. Laminin-a5, the ysis excluded all variants with a minor allele frequency >1% in
predominant a-chain in the mature GBM [17], is encoded by the Short Genetic Variations database (dbSNP, version 142) or
the gene LAMA5. Knockout of Lama5 in mice causes embryonic in the 1000 Genomes Project database (1094 subjects of various
lethality and severe defects of glomerular development [18]. In ethnicities; May 2011 data release). Noncoding variants outside
contrast, mice with a hypomorphic Lama5 allele in the homozy- canonical splice regions as well as synonymous variants were
gous state demonstrated proteinuria, cystic kidney disease and excluded, and variants were annotated using the Exome
death from progressive renal failure at 3–4 weeks of age [19]. Aggregation Consortium (ExAC) database. Variants that were
Interestingly, Lama5 mutations in mice have a strictly recessive reported more than 10 times in the homozygous state in the
mode of inheritance and heterozygous mice were indistinguish- ExAC database were excluded as likely nonpathogenic poly-
able from wild-type litter mates [18]. Two previous publications morphisms. Remaining variants were ranked based on estab-
suggested an association between heterozygous mutations of lished criteria [10, 11, 22] and were evaluated for likely
LAMA5 and adult-onset FSGS [20, 21]. pathogenicity under consideration of phenotypic as well as ped-
We here performed whole exome sequencing (WES) in 335 igree information.
individuals of 300 families with NS. After excluding likely
disease-causing mutations in known monogenic causes of Homozygosity mapping
SRNS, we used homozygosity mapping in consanguineous
families and identified recessive mutations in the gene LAMA5 Homozygosity mapping was calculated based on WES data.
in three unrelated families with childhood-onset NS that In brief, aligned Binary Alignment Map files were processed
responded to immunosuppressive therapy. using Picard and SAMtools4 as described by other groups [23].
Single-nucleotide variant calling was performed using Genome
Analysis Tool Kit [24]. The resulting variant call format files
MATERIALS AND METHODS were used to generate homozygosity mapping data and visual
outputs using the program HomozygosityMapper [25].
Study participants
Following informed consent, we obtained pedigree informa- Screening for mutations in known monogenic causes of
tion, clinical data and blood samples from individuals with pro- SRNS
teinuria or NS (onset <25 years of age). Study approval was We concatenated the genomic loci of 34 genes that are known
obtained from the institutional review boards of the University of to cause SRNS if mutated (Supplementary data, Table S1).

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 Aligned reads from WES data for all 335 individuals were first families with NS that shared a homozygous peak region on chro-
filtered using this file and evaluated for potentially disease- mosome 20q13 (Figure 1), we identified three different homozy-
causing variants in these known monogenic genes of SRNS. gous variants of unknown significance (VUS) in the gene
Further analysis seeking potential novel candidate genes was LAMA5 (NM_005560.4) encoding laminin-a5. In family A4389
only performed if no likely pathogenic mutation was identified. from Turkey we detected the variant c.2239 C >T, p.Arg747Trp,
conserved to Drosophila melanogaster; in family B150 from
Variant filtering to identify novel monogenic causes of NS Egypt we detected the variant c.3002 A>G, p.Glu1001Gly, con-
We used homozygosity mapping data in consanguineous served to Ciona intestinalis; and in family B1284 from Saudi
families (>100 Mb of cumulative homozygosity according to Arabia we identified the variant c.8842 G>A, p.Gly2948Ser, con-
mapping) to identify genomic candidate loci for the underlying served to D. melanogaster (Table 1, Figure 2).
recessive mutation. Within these areas we restricted our analysis Each family had two affected children with NS, strongly sug-
to homozygous mutations. Remaining calls were ranked for their gesting an inherited cause of disease. The parents, who were
predicted pathogenicity based on the following criteria [10, 11, heterozygous carriers of the VUS, were phenotypically healthy
22]: (i) protein truncating or obligatory splice site mutation and did not have proteinuria. In family B1284, a DNA sample
versus missense mutation or in-frame deletion/insertion, for the younger sibling was not available. In the other two fami-
(ii) evolutionary conservation, (iii) minor allele frequency in lies (A4389 and B150) we demonstrated the presence of the
control databases [Genome Aggregation Database (gnomAD), homozygous VUS in both affected children. Segregation analy-
Exome Variant Server (EVS)], (iv) chemical difference between sis in families B150 and B1284 showed that, as expected in a
wild-type and altered amino acid residue and (v) web-based recessive mode of inheritance, the parents were healthy hetero-
mutation analysis prediction tools [Sorting Intolerant From zygous carriers of the VUS (Figure 2D). All three VUS were
Tolerant (SIFT), PolyPhen-2, MutationTaster]. missense alleles affecting amino acid residues of strong evolu-
tionary conservation among orthologue proteins of laminin-a5
Implementation of structural data and evolutionary (Figure 2C, Table 1). Bioinformatics scoring programs (SIFT,
conservation for variant evaluation PolyPhen-2 and MutationTaster) predicted the impact of the
Protein domain structure cartoons and evaluation were identified variants as likely pathogenic (Table 1). Furthermore,
based on the uniprot (Universal Protein Resource) database. none of the three variants has been reported in the homozygous
Orthologue proteins used to evaluate evolutionary conservation state in databases of healthy control populations (EVS and
were obtained from the Ensemble Genome Browser and were gnomAD [27]) (Table 1). The protein laminin-a5 carries an N-
aligned using the Clustal Omega multiple sequence alignment terminal LN (laminin N-terminal) domain, followed by multi-
tool [European Molecular Biology Laboratory (EMBL) - ple epidermal growth factor (EGF) domains and 5 C-terminal
European Bioinformatics Institute (EBI)] [26]. laminin G-like (LamG) domains. Interestingly, two of the var-
iants affected amino acid residues that are located within one of
Web resources the functional domains of the protein, namely the ninth EGF-
The following web resources were used: UCSC Genome like domain (p.Arg747Trp, A4389) and the second laminin G-
Browser, http://genome.ucsc.edu/cgi-bin/hgGateway; 1000 like domain (p.Gly2948Ser, B1284) (Figure 2B, Table 1).
Genomes Browser, http://browser.1000genomes.org; Clustal Because individuals of Middle Eastern descent are poorly
Omega, https://www.ebi.ac.uk/Tools/msa/clustalo/; Ensembl represented in publicly available control databases, we used 258
Genome Browser, http://www.ensembl.org; Uniprot individuals with renal ciliopathies, a different entity of inherited
Consortium, http://www.uniprot.org/; Exome Variant Server, kidney disease in which proteinuria is characteristically absent,
http://evs.gs.washington.edu/EVS ExAC, http://exac.broadinsti as a control cohort. However, we did not identify recessive
tute.org; gnomAD, http://gnomad.broadinstitute.org; HGMD mutations in the LAMA5 gene in these individuals.
Professional 2016.3, https://portal.biobase-international.com/
Treatment response and renal outcome differ in three
hgmd; Online Mendelian Inheritance in Man (OMIM), http://
families with VUS in LAMA5 and NS
www.omim.org; Polyphen2, http://genetics.bwh.harvard.edu/
pph2; SIFT, http://sift.jcvi.org; MutationTaster, http://www. All five patients in whom we detected homozygous VUS in
mutationtaster.org and HomozygosityMapper, http://www. LAMA5 shared the phenotype of NS with onset before 4 years
homozygositymapper.org/. of age. Extrarenal manifestations or syndromic features were
All websites were last accessed on 2 February 2018. not present in any of the affected children. However, the five
children differed notably in their response to immunosuppres-
RESULTS sive treatment, their clinical presentation and regarding pro-
gression to ESRD.
Recessive variants of LAMA5 in patients with NS
3In family A4389, the two children presented with NS at ages
We performed WES in 335 individuals of 300 families with 22 months and 3 years, respectively. Both siblings responded
NS. As a first step, we excluded likely disease-causing mutations promptly to steroids but had frequent relapses within the first year
in 34 genes that are known to cause monogenic NS if mutated of diagnosis. Subsequently, stable remission was achieved and
(Supplementary data, Table S1). We subsequently used homo- they have been maintained off steroids without further relapse for
zygosity mapping in consanguineous families to identify candi- an interval of 3 years (at the time of this publication). The younger
date loci for the underlying recessive mutation. In three unrelated sibling received cyclophosphamide due to steroid dependency. No

LAMA5
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 FIGURE 1: Homozygosity mapping in families with NS identifies recessive candidate loci. Homozygosity mapping was calculated from WES
data using HomozygosityMapper. A homozygous peak on chromosome 20q13 (arrow head) that includes the LAMA5 locus was present in all
three families. (A, B) Homozygosity mapping for the two affected siblings (-21 and -22) of family A4389. (C, D) The two affected siblings (-21
and -22) of family B150. (E) Homozygosity mapping data of patient B1284-21.

further immunosuppressive therapy was required and both sib- DISCUSSION

lings showed normal renal function and persistent remission of We here identify recessive VUS in the gene LAMA5 in three
NS at ages 11 and 9 years, respectively (Table 1). unrelated families with two children each with childhood-onset
The two siblings of family B150 had their first episode of NS. The familial occurrence and presence of homozygosity in
NS at ages 1.5 and 4 years, respectively. Both siblings devel- all three families strongly suggested a recessive cause of disease.
oped SRNS. However, while the older sibling (B150-21) ini- We excluded mutations in 34 genes that are known to cause
tially responded to steroids and only later developed SRNS, NS, if mutated. By homozygosity mapping, we identified the
the younger sibling (B150-22) had SRNS at her initial pre- LAMA5 locus as a candidate region common to all three fami-
sentation. The renal histology in both siblings demonstrated lies. We then used WES and identified recessive VUS in the
FSGS and the younger sister progressed to ESRD by the age LAMA5 gene in all three families. However, because experimen-
of 6 years. Unfortunately, this patient died prior to renal tal evidence is lacking, the impact of these genetic variants on
transplantation so data on disease recurrence after trans- protein function and disease pathogenesis cannot be judged with
plantation are not available. certainty. Therefore, more data are needed to evaluate the func-
Individual B1284-21 first presented with NS at 4 years of tional relevance of these VUS in NS and to determine their
age. He initially responded to steroid therapy but had frequent potential impact on prognosis and disease management.
relapses and ultimately developed steroid-dependent NS, While ~80% of pediatric patients with NS promptly respond
requiring additional immunosuppressive agents to achieve to therapy with steroids, inherited forms of the disease typically
remission. He has a younger brother with a similar clinical pre- present with the phenotype of SRNS. We and others have previ-
sentation. However, his genotype is not known because a DNA ously shown that, depending on age of onset, consanguinity
sample for genetic analysis could not be obtained. and familial occurrence of disease, in ~30% of SRNS cases a

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LAMA5
Table 1. Genetic variants of LAMA5 in three families with nephrotic syndrome.

variants in pediatric NS
Family- Nucleotide Amino acid Exon (zygo- PPH2 SIFT Mut Amino acid gnomAD allele EVS allele Gender Ethnic Parental Age of Extrarenal Biopsy Therapy and
indivi- change change protein sity, score Taster conservation frequencies frequenciesa origin consan- onset manifestations (at age) response

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dual domain segregation) to species (hom/het/ (hom/het/ guinity (ESRD)
wild-type) wild-type)
A4389 c.2239C>T p.Arg747Trp 18 1.0 del DC Drosophila 0/34/275, 534 0/1/4,297 Turkey Yes None SSNS
melanogasterb
-21 EGF-like (hom, ND) E: 0/4/125, 468 Male 3 yrs (no) n.p. 3 relapses (no IS
domain 9 A: 0/9/23, 894 beyond steroids)
SA: 0/20/30, 752
-22 (hom, ND) EA: 0/0/18, 826 Male 22 mo (no) n.p. 4 relapses (IS with
steroids and
cyclophosphamide)
Normal renal function
in both siblings (11
and 9 yrs.)
B150 c.3002A>G p.Glu1001Gly 24 0.92 del DC Ciona Not reported Not reported Egypt Yes None
intestinalis
-21 n/a (hom) Male 4 yrs (no) FSGS (2 yrs) SRNS
-22 (hom ) Female 1.5 yrs (6 yrs) FSGS (2 yrs) SRNS (no
(het, m; het, p) response to CSA)

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B12843 c.8842G>A p.Gly2948Ser 65 1.0 del DC D. melanogaster 0/33/195, 366 Not reported Arabic Yes
-21 Laminin G-like (hom) E: 0/0/85, 398 Male 4 yrs (no) None n.p. SSNS/SDNS with
domain 2 (het, m; het, p) A: 0/0/17, 686 freq. relapses (IS
SA: 0/1/24, 934 with Tac, MMF)
EA: 0/31/12, 864
LAMA5, NM_005560.4.
a
Allele frequencies in European Americans.
b
Chemically conserved as arginine or lysine.
c
Younger brother presented with SSNS at age 4 years, frequent relapses of NS, no DNA sample available.
A, African (allele frequencies, gnomAD); DC, predicted to be disease-causing (SIFT); del, predicted to be deleterious (MutationTaster); E, European (allele frequencies, gnomAD); EA, East Asian (allele frequencies, gnomAD); het, heterozygous;
hom, homozygous; IS, immunosuppressive therapy; m, maternal; mo, months; n/a; not applicable; ND, no data; n.p., no biopsy performed; m, maternal; MMF, mycophenolate mofetil; p, paternal; PPH2 score humvar PolyPhen2 prediction score;
SA, South Asian (allele frequencies, gnomAD); SDNS, steroid-dependent nephrotic syndrome; Tac, tacrolimus; tol, tolerated; yrs, years.

5
 A
LAMA5 (NM_005560.4)

11,088 bp
B
LAMA5 (NP_005551.3)
EEEEEEEEEEE EEEE EEEEEE
LAM LAM LAM LAM LAM LAM
LamNT GGGGGGGGGGG GGGG GGGGGG
FFFFFFFFFFF F F F F IV-A F F F F F F G G G G G

3695 aa

A4389 B150 B1284

c.2239C>T c.3002A>G c.8842G>A
p.Arg747Trp (Hom) p.Glu1001Gly (Hom) p.Gly2948Ser (Hom)
C

Homo sapiens
Mus musculus
Gallus gallus
Xenopus tropicalis
Danio rerio
Ciona intestinalis
C. elegans
D. melanogaster

D A
G
G
A

Control Father Gly Father Ser

(wildtype) (het) (het)

A G
G A

A4389-21 Mother
Gly Ser
Mother
(Hom) (het) (het)

B150-21 B1284-21
A4389-22
(Hom) (Hom)
(Hom)

B150-22
(Hom)

FIGURE 2: WES identifies recessive variants in the gene LAMA5 in three families with nephrotic syndrome. (A) Exon structure of LAMA5
cDNA (NM_005560.4). The positions of start codons, stop codons and mutated nucleotides are indicated. (B) Domain structure of the
encoded protein laminin-a5. Arrows indicate the positions of the altered amino acid residues in families A4389, B150 and B1284. (C)
Evolutionary conservation among orthologous proteins of laminin-a5. Altered amino acid residues in families A4389, B150 and B1284 are
indicated with arrowheads and a yellow box. (D) Sanger sequencing of the respective regions of LAMA5 in genomic DNA of affected patients,
parental DNA and control samples as indicated. aa, amino acid; bp, base pairs; C. elegans, Caenorhabditis elegans; D. melanogaster, Drosophila
melanogaster; EGF, laminin EGF-like domain; het, heterozygous; Hom, homozygous; LAM G, laminin G-like domain; LAM IV-A, laminin IV
type A; LAM NT, laminin N-terminal domain.

causative single-gene mutation can be identified [12–14]. known monogenic causes of SRNS and better availability of
Having a conclusive, monogenic diagnosis provides affected genetic testing, it becomes increasing important to determine
patients and their families with an unequivocal diagnosis, can standardized criteria that help distinguish polymorphisms from
help guide therapy and predicts a lower risk for disease recur- true pathogenic variants [28]. This is particularly important if
rence after renal transplantation [5]. Therefore, genetic testing functional data for the identified variants are lacking and if eth-
should be implemented as the standard of care in all patients nically matched control populations are unrepresented in avail-
with SRNS [10]. However, considering the growing number of able large datasets [27].

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 Familial cases of SSNS have been described [29] and genetic targeted deletion of Lama5 in the kidney resulted in severely
causation may not be limited to patients with NS that is strictly disrupted glomerulogenesis, defects in branching of the ureteric
resistant to drug treatment. This was first observed for the gene bud and sporadic renal agenesis [18]. Both findings suggest an
EMP2, which, if mutated, caused a steroid-sensitive form of NS important function of Laminin-a5 during renal development
in some patients [30]. Subsequently, recessive mutations of and glomerular maturation. Interestingly, a hypomorphic
TNS2, DLC1, CDK20, ITSN1 and ITSN2 were found to cause Lama5 allele (Lama5neo) allowed normal formation of glomeru-
partially treatment-sensitive NS [31]. Our findings suggest that lar architecture but caused delayed onset of proteinuria with
VUS of LAMA5 may contribute to the development of a form thickening of the GBM and secondary glomerulosclerosis [15,
of NS that in some patients responds to drug treatment. 19]. This finding is consistent with the phenotype that we
Traditionally, SSNS and SRNS are regarded as two distinct dis- observed in three families with recessive, hypomorphic VUS of
ease entities, and it has been proposed that the pathogenesis of LAMA5. Furthermore, Lama5 hypomorphic mice developed
SSNS is likely immune mediated [32]. Interestingly, the three polycystic kidney disease [19]. In contrast, the renal ultrasound
families with VUS in LAMA5 differed significantly in their in all five patients with VUS in LAMA5 was unremarkable. In
response to drug therapy, ranging from SSNS to SRNS, includ- particular, no cystic lesions were detected and there were no
ing one family with an intermediate phenotype (B1284, radiological signs of congenital malformations of the kidney or
Table 1). The observation that the same molecular defect, an urinary tract.
alteration of the laminin-a5 protein, can act as a contributing In summary, we here identify recessive VUS in the LAMA5
factor in the pathogenesis of both phenotypes (SSNS and gene as genetic variants that may contribute to the development
SRNS) may suggest that steroid resistance and steroid of NS in pediatric patients. Interestingly, some of the described
sensitivity in NS occupy a phenotypic spectrum. patients responded partially to steroids or other immunosup-
Despite their long-term therapeutic use, the exact mode of pressive drug regimens, whereas others were resistant to ther-
action of steroids is unknown. Growing evidence suggests that in apy. Additional data are required to determine the impact of
addition to their immunoregulatory function, immunosuppres- genetic variants in the LAMA5 gene on the pathogenesis,
sive drugs also act directly on podocytes [33, 34]. Via direct bind- prognosis and potentially on disease management in pediatric
ing to the mineralocorticoid receptor, which has been shown to patients with NS.
be expressed in podocytes, steroid therapy induced changes in
the podocyte transcriptome [35]. In addition, it has been shown
that cyclosporine A (CsA) acts directly on the podocyte actin SUPPLEMENTARY DATA
cytoskeleton, thereby influencing actin dynamics [36]. While Supplementary data are available at ndt online.
inherited forms of NS are characteristically regarded as non-
responsive to immunosuppressive therapy, there are also
some reports demonstrating that CsA can be effective in ACKNOWLEDGEMENTS
patients with Alport syndrome [37]. Alport syndrome is a We thank the study participants and their families for their
form of inherited glomerulopathy caused by mutations in contributions. We thank the Yale Center for Mendelian
genes that encode collagens of the GBM. Based on these Genomics (U54HG006504) for WES.
observations, one could speculate that steroids act directly or
indirectly on components of the GBM by changing the gene
expression profile of podocytes. FUNDING
Heterozygous mutations in the gene LAMA5 have been pre- This research was supported by grants from the National
viously reported by two other groups as being associated with Institutes of Health to F.H. (DK076683) and J.K.W. and
adult-onset FSGS in an autosomal dominant manner [20, 21]. A.J.M. (T32DK007726-31A1). A.J.M. was supported by the
However, the pathogenicity of the heterozygous mutations Harvard Stem Cell Institute, Kidney Group. WT was sup-
described in LAMA5 remains controversial [21]. In addition, a ported by the ASN Benjamin J. Lipps Research Fellowship
genetic mouse model of Lama5, in which heterozygous mice Award (FP01014311). T.H. was supported by the Deutsche
are phenotypically indistinguishable from wild-type littermates, Forschungsgemeinschaft, DFG fellowship (HE 7456/1-1).
demonstrates a strictly recessive mode of inheritance [18]. In T.J.S. was supported by grant Jo 1324/1-1 of the Deutsche
accordance with these findings, heterozygous parents in our Forschungsgemeinschaft. E.W. was supported by the German
study did not manifest with NS. However, it is possible that, as National Academy of Sciences Leopoldina (LPDS-2015-07).
described for other genes, such as COL4A3 and COL4A4 [38],
heterozygous carriers may manifest with adult-onset disease of
variable penetrance, whereas recessive mutations cause a fully AUTHORS’ CONTRIBUTIONS
penetrant, early-onset disease manifestation. Further studies D.A.B., J.K.W., S.A., S.S., W.T., A.D., R.S., T.H., T.J.S., E.W.,
will be required to determine the pathogenicity of heterozygous A.J.M., M.N., J.M.S., C.A.H., H.H., J.R., S.M., R.P.L. and F.H.
mutations of LAMA5. generated total genome linkage data, performed exome cap-
The mouse orthologue Lama5 encodes Laminin-a5, an ture with massively parallel sequencing and performed WES
essential component of basement membranes. A complete and mutation analysis. D.A.B., J.K.W., S.A., S.A.B., J.A.K.,
genetic knockout of Lama5 in mice caused various develop- S.E.D., G.D. and F.H. recruited patients and gathered detailed
mental defects and was embryonic lethal at e14–e17 [39]. A clinical information for the study. All authors critically

LAMA5
Downloaded from variants in pediatric NS
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on 12 March 2018
 reviewed the article. F.H. conceived of and directed the proj- 19. Shannon MB, Patton BL, Harvey SJ et al. A hypomorphic mutation in the
ect. D.A.B., J.K.W. and F.H. contributed by writing the article. mouse laminin alpha5 gene causes polycystic kidney disease. J Am Soc
Nephrol 2006; 17: 1913–1922
20. Chatterjee R, Hoffman M, Cliften P et al. Targeted exome sequencing inte-
grated with clinicopathological information reveals novel and rare muta-
CONFLICT OF INTEREST STATEMENT tions in atypical, suspected and unknown cases of Alport syndrome or
proteinuria. PLoS One 2013; 8: e76360
F.H. is a cofounder of Goldfinch Biopharma and receives roy- 21. Gast C, Pengelly RJ, Lyon M et al. Collagen (COL4A) mutations are the
alties from Claritas Genomics. The other authors declare that most frequent mutations underlying adult focal segmental glomerulosclero-
they have no competing financial interests. sis. Nephrol Dial Transplant 2016; 31: 961–970
22. MacArthur DG, Manolio TA, Dimmock DP et al. Guidelines for investigating
causality of sequence variants in human disease. Nature 2014; 508: 469–476
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