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Clear evidence of LAMA5 gene biallelic truncating variants causing infantile nephrotic syndrome

identified in only 7 patients far and analysis conducted for all but report 3 patients with LAMA5 biallelic truncating variants manifesting infantile nephrotic syndrome and in vitro heterotrimer assays report one case with biallelic missense Abstract: Background: Pathogenic variants in single genes encoding podocyte-associated proteins have been implicated in about 30% of steroid resistant nephrotic syndrome (SRNS) patients in children. However, LAMA5 gene biallelic variants have been identified in only 7 patients so far, and most are missense variants of unknown significance. Furthermore, no functional analysis had been conducted for all but one of these variants. Here, we report three patients with LAMA5 gene biallelic truncating variants manifesting infantile nephrotic syndrome and one SRNS case with biallelic LAMA5 missense variants. Methods: We conducted comprehensive gene screening of Japanese patients with severe proteinuria. Using targeted next-generation sequencing, 62 podocyte-related genes were screened in 407 unrelated patients with proteinuria. For the newly discovered LAMA5 variants, we conducted in vitro heterotrimer formation assays. Results: Biallelic truncating variants in the LAMA5 gene (NM_005560) were detected in 3 patients from 2 families. All patients presented with proteinuria within 6 months of age. Patients 1 and 2 were siblings possessing a nonsense variant (c.9232C>T, p.(Arg3078*)) and a splice site variant (c.1282+1G>A) that led to exon 9 skipping and a frameshift. Patient 3 had a remarkable irregular contour of the glomerular basement membrane. She was subsequently found to have a nonsense variant (c.8185C>T, p.(Arg2720*)) and the same splice site variant in patients 1 and 2. By in vitro heterotrimer formation assays, both truncating variants produced smaller laminin α5 proteins that nevertheless formed trimers with laminin β1 and γ1 chains. Patient 4 showed SRNS at the age of eight and carried compound heterozygous missense variants (c.1493C>T, p.(Ala498Val) and c.8399G>A, p.(Arg2800His)). Conclusions: Our patients showed clear evidence of biallelic LAMA5 truncating variants causing infantile nephrotic syndrome. We also discerned the clinical and pathological characteristics observed in LAMA5-related nephropathy. LAMA5 variant screening should be performed in congenital/infantile nephrotic syndrome patients. Abstract Background Pathogenic variants in single genes encoding podocyte-associated proteins have been implicated in about 30% of steroid resistant nephrotic syndrome (SRNS) patients in children. However, LAMA5 gene biallelic variants have been identified in only 7 patients so far, and most are missense variants of unknown significance. Furthermore, no functional analysis had been conducted for all but one of these variants. Here, we report three patients with LAMA5 gene biallelic truncating variants manifesting infantile nephrotic syndrome and one SRNS case with biallelic LAMA5 missense variants. We conducted comprehensive gene screening of Japanese patients with severe proteinuria. Using targeted next-generation sequencing, 62 podocyte-related genes were screened in 407 unrelated patients with proteinuria. For the newly discovered LAMA5 variants, we conducted in vitro heterotrimer formation assays. Results This presents three infantile nephrotic syndrome patients with LAMA5 gene biallelic truncating variants and one SRNS case with LAMA5 biallelic missense variants. For our patients, we performed in vitro heterotrimer formation assays and demonstrated defects in the function of truncating variants. This is the first report to show clear evidence that LAMA5 pathogenic variants can lead to nephrotic syndrome in the absence of major developmental anomalies. Our patients suggest the need for awareness of LAMA5 gene screening in patients with early-onset proteinuria.

The glomerular basement membrane (GBM) is an important component of the kidney's glomerular filtration barrier. The GBM contains members of the four major classes of basement membrane proteins: type IV collagen, laminin, heparan sulfate proteoglycan, and nidogen 5 . The GBM's major laminin isoform is laminin-521 (LM-521), a cross-shaped heterotrimeric glycoprotein composed of the laminin α5, β2, and γ1 chains 6 . The LAMA5 gene encodes laminin α5, which is a widely expressed chain 7 found in many embryonic and adult basement membranes 8 .
We have characterized in detail three infantile nephrotic syndrome patients with biallelic LAMA5 truncating variants and one SRNS case with a LAMA5 missense variant.
These patients provided clear evidence for the first time that biallelic truncating variants in LAMA5 can cause early-onset nephrotic syndrome.

Study participants
Following informed consent, we obtained clinical data and blood samples from individuals with severe proteinuria in Japan. Study approval was obtained from the Institutional Review Board of Kobe University Graduate School of Medicine (IRB approval number 301). Patients were enrolled between January 2016 and January 2021.
We performed targeted next generation sequencing in 407 families.

Genetic analysis
Genomic DNA was extracted from peripheral blood using the Quick Gene Mini 80 system (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) and used for targeted sequencing and Sanger sequencing. Targeted sequencing using next-generation sequencing (NGS) was conducted for genes responsible for inherited renal disease (listed in Supplementary Table 1). The sample library for NGS analysis was prepared using SureSelect XT2 custom capture library 0.5-2.9Mb (Agilent Technologies, Santa Clara, CA, USA), in accordance with the manufacturer's workflow. Briefly, 150 ng of genomic DNA was used for a restricted reaction and hybridized using SureSelect XT Low Input reagents. All indexed DNA samples were amplified by polymerase chain reaction and sequenced using the Miseq platform (Illumina, San Diego, CA, USA). The results were analyzed using SureCall 3.0 (Agilent Technologies).

Transcript analysis
Total RNA was isolated from urine-derived cells using RNeasy Mini Kit (QIAGEN Hilden, Germany). Urine-derived cells were cultured in accordance with a previously reported protocol 9 . Total RNA was reverse-transcribed into cDNA using Ecodry Premix (Double Primed) (Takara Bio Inc., Kusatsu, Japan), and PCR was performed. The primers used are shown in Supplementary Table 2.

Immunofluorescence analysis of laminin α5
Double immunostaining for laminin α5 and laminin β2 or type IV collagen α5 chain were performed using frozen kidney tissues. Each sample was fixed with acetone for 10 min, followed by heat-induced epitope retrieval. After blocking with 10% goat serum (Vector Laboratories, Burlingame, CA, USA), samples were incubated with primary monoclonal antibodies (mAbs) against the laminin α5 chain (AMAb91124) (Atlas Antibodies AB, Stockholm, Sweden), laminin β2 chain (sc-20777, H-300) (Santa Cruz Biotechnology, Dallas, CA, USA) or type IV collagen α5 chain (H53) (Shigei Medical Research Institute, Okayama, Japan) overnight. Bound mAbs were detected using specific secondary antibodies, Alexa Fluor 546 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-rat IgG, for 90 min. Finally, samples were incubated with DAPI for 5 min to stain nuclei.
The resultant cDNA fragment was inserted into the HindIII and SacII sites of LM5-pcDNA3.1 using GeneArt™ Seamless Cloning and Assembly Kit (Thermo SCIENTIFIC). Expression vectors for two human LAMA5 truncation mutants, Arg2720* and Arg3078*, were prepared by extension PCR using a LM5His-pcDNA3.1 as a template. Resultant cDNA fragments were inserted into the BbrCI and AscI sites of LM5His-pcDNA3.1 using GeneArt™ Seamless Cloning and Assembly Kit. The DNA sequence of the resulting vectors were confirmed using an ABI PRISM 3130 Genetic Analyzer.

In Vitro Secretion of wild-type LM511 and its Chain-termination Mutants
Wild-type LM511 and its mutants were transiently expressed in the To confirm the heterotrimerization of LM5 chain-termination mutants in LM511, the conditioned media were incubated with anti-human laminin 5 mAb 5D6 14

Patients
A total of 407 unrelated patients (216 men/boys and 191 women/girls) were included for genetic analysis by targeted sequencing using NGS. LAMA5 (NM_005560.4) gene variants were detected in four patients in three families. The genetic and concise clinical information for patients 1 and 2 were already published in our previous report 15 , but no precise clinical and pathological findings were described. Table 1 summarizes the characteristics of our four patients and the seven patients from previous reports with LAMA5 variants.
Patient 1 was a 13-year-old boy. At 3 months of age, his edema was detected by chance when he had a fever. Laboratory findings were as follows: serum total protein, 3.9 g/dl; serum albumin, 1.8 g/dl; serum Cr, 0.55 mg/dl and cholesterol, 940 mg/dl. Urinalysis showed heavy proteinuria. He was diagnosed with nephrotic syndrome. As his renal function was gradually deteriorating, peritoneal dialysis was started at the age of one-year. We conducted genetic testing using targeted sequencing of the genes known to be responsible for inherited kidney disease and detected compound heterozygous variants in LAMA5. The variants include a heterozygous G to A substitution at base position 1282+1 in intron 9 (IVS9+1G>A) and a heterozygous C to T substitution at base position 9232 in exon 68, which replaces the amino acid arginine with a stop codon at codon 3078 (p.Arg3078*) (Fig 1A, 1B). IVS9+1G>A led to the entire exon 9 (91bp) being skipped at the transcript level (Fig 1C), meaning that this is also a truncating variant. The patient's parents each carried a different variant in heterozygous form. The patient's mother was phenotypically healthy and did not have proteinuria. The patient's father had hypoplastic kidney, but he did not have proteinuria and did not reach ESKD.
Patient 2 was a 5-year-old girl. She is patient 1's younger sister. At 4 months of age, edema and hypoalbuminemia were detected. Urinalysis showed heavy proteinuria, and she was diagnosed with nephrotic syndrome. She did not have any extra-renal abnormalities. Peritoneal dialysis was started at the age of three and half months. She carried the same variants as Patient 1.
Patient 3 was a 6-year-old girl in whom hypoalbuminemia was detected by chance when she had been infected with the virus at 6 months of age. She had congenital cataract and hypoplastic kidney. There were no family history of kidney diseases. She was diagnosed with infantile nephrotic syndrome and started steroid therapy. However, she did not respond to immunosuppressive therapy. At 11 months of age, she underwent a renal biopsy. Pathologically, light microscopy demonstrated diffuse mesangial sclerosis, and electron microscopy demonstrated thinning and irregular structure of the GBM (Fig 1D).
She reached end stage kidney disease at three years and three months of age. We conducted targeted sequencing analysis using NGS and found compound heterozygous variants in LAMA5. The variants include a heterozygous G to A substitution at base position 1282+1 in intron 9 (the same as Patients 1 and 2), and a heterozygous C to T substitution at base position 8158 in exon 60, which replaces the amino acid arginine with a stop codon at codon 2720 (p.Arg2720*) (Fig 1E, 1F). We conducted immunostaining analysis of laminin α5. In the control, laminin α5 showed the characteristic linear GBM pattern; however, Patient 3 showed weakly positive staining for laminin α5, although laminin β2 and collagen α5 (IV) showed normal patterns (Fig 2A, 2B). This suggests that mutant laminin α5 could form laminin-521 heterotrimers, though levels in the GBM appear reduced.
Patient 4 was a 9-year-old girl. At 8 years of age, she was referred to the hospital because of proteinuria, which had been identified in a school-based urine screening program. Urinalysis showed heavy proteinuria. She did not have any extra-renal abnormalities. She was diagnosed with nephrotic syndrome and started steroid therapy.
She underwent a renal biopsy because of SRNS. Pathologically, light microscopy demonstrated focal segmental glomerulosclerosis cellular variant, and electron microscopy demonstrated thinning and irregular structure of the GBM (Fig 3A). We conducted targeted sequencing analysis using NGS. She had compound heterozygous missense variants in LAMA5. The variants include a heterozygous C to T substitution at base position 1493 in exon 12, which replaces the amino acid alanine with valine at codon 498 (p.Ala498Val) and a heterozygous G to A substitution at base position 8399 in exon 62, which replaces the amino acid arginine with histidine at codon 2800 (p.Arg2800His) ( Fig 3B, 3C). We conducted immunostaining analysis of laminin α5. In the control, laminin α5 shows a characteristic linear GBM pattern; Patient 4 showed the same degree of expression of laminin α5 as control (Fig 3D, 3E). The patient's parents each carried a different variant in heterozygous form. They were phenotypically healthy and did not have proteinuria.

In vitro analysis
To investigate the potential pathogenicity of the two nonsense variants, LM5-Arg2720* and -Arg3078* (Fig 4A), we assayed their synthesis and secretion in the context of the LM-511 trimer. We expressed either wild-type or mutant tagged human LM5 along with tagged LM1 and 1 to produce either wild-type or mutant LM-511 trimers by cotransfecting 293-F cells with three expression vectors: LMα5His-pcDNA3.1, LMβ1-pCEP4, and LMγ1FLAG-pcDNA3.1. Conditioned media were separated by SDS-PAGE, followed by immunoblotting using the mAb against the 5×His tag at the amino-terminus of LM5. This analysis showed that both truncating mutants,LM5-Arg2720* and -Arg3078*, exhibited a decreased trimer secretion level by about 40% and 30%, respectively, compared with wild-type ( Fig. 4B and C).
Consistent with these results, the signal intensities in cell lysates were increased by 4-5 fold for the mutants compared with wild-type ( Fig. 4B and C).
To further explore whether secreted LM5-Arg2720* and -Arg3078* mutants can trimerize with laminin 1 and 1, we performed immunoprecipitation using mAbs against the human laminin 5 chain (5D6) and the FLAG tag at the amino-terminus of the laminin 1 chain. Immunoblotting analyses showed that laminin 1 and 1 were detected in mAb 5D6 immunoprecipitates (Fig. 4D left panel). When the 1 chain was immunoprecipitated with anti-FLAG mAb, laminin 5 and 1 chains were detected in the precipitates (Fig.4D right panel). These results indicate that both 5-Arg2720* and -Arg3078* mutants are able to assemble with laminin 1 and 1 to yield a trimer, but compared with wild-type, secretion efficiencies of the mutant LM-511s were decreased by an unidentified intracellular quality control mechanism. This is in good agreement with our in vivo findings that the level of laminin 5 was decreased in glomeruli of Patient 3.

Discussion
This paper presents three infantile nephrotic syndrome patients with LAMA5 gene biallelic truncating variants and one SRNS case with LAMA5 biallelic missense variants. All five patients had nephrotic syndrome with onset before 4 years of age. One of them did not respond to immunosuppressive treatment and reached ESKD at age 6, but on the other hand, other patients responded to immunosuppressive treatment. This indicates significant phenotypic heterogeneity in this disease, which resembles somewhat the situation for LAMB2 gene variants 16 .
Our three patients with truncations (Patients 1, 2 and 3) showed more severe phenotypes than the patients with missense variants, but not lethality. Lama5 null mice die at late fetal stages with multiple development defects 17 8 . The GBM of proteinuric mice appeared thickened with a moth-eaten appearance 8 . These mouse pathological findings were in concordance with our patients. We also performed immunofluorescence analysis that showed weak deposition of laminin α5, but it was not negative in Patient 3.
This result was somewhat surprising to us, because we did not expect biallelic truncating variants to produce laminin α5 able to form laminin-521 trimers. But our in vitro trimer formation assay clearly proved that the two truncated laminin α5 variants can trimerize.         (C) Band densities in B were quantified using ImageJ software. Data was expressed as average ± standard deviation.
(D) 5-R2720* and -R3078* mutants heterotrimerize with laminin 1 and 1. 6×His tagged laminin 5 and the two chain-termination mutants were coexpressed with    Figure 1 Direct sequencing for transcript analysis ①The single band shown in the control (Co) and the upper band shown in patient (Pt) corresponded to exon 8, exon 9 and 10; the splicing pattern was normal.
② The lower band shown in Pt corresponded to exon 8 and exon 10; exon 9 skipping occurred.

Exon10
Exon8 Exon10 Galloway-Mowat Supplementary   Figure 1 Direct sequencing for transcript analysis ①The single band shown in the control (Co) and the upper band shown in patient (Pt) corresponded to exon 8, exon 9 and 10; the splicing pattern was normal.
② The lower band shown in Pt corresponded to exon 8 and exon 10; exon 9 skipping occurred.