Trimerization profile of type IV collagen COL4A5 exon deletion in X-linked Alport syndrome.

BACKGROUND: Alport syndrome (AS) is a genetic kidney disease caused by a mutation in type IV collagen α3, α4, and α5, which are normally secreted as heterotrimer α345(IV). Nonsense mutation in these genes causes severe AS phenotype. We previously revealed that the exon-skipping approach to remove a nonsense mutation in α5(IV) ameliorated the AS pathology. However, the effect of removing an exon on trimerization is unknown. Here, we assessed the impact of exon deletion on trimerization to evaluate their possible therapeutic applicability and to predict the severity of mutations associated with exon-skipping. METHODS: We produced exon deletion constructs (ΔExon), nonsense, and missense mutants by mutagenesis and evaluated their trimer formation and secretion activities using a nanoluciferase-based assay that we previously developed. RESULTS: Exon-skipping had differential effects on the trimer secretion of α345(IV). Some ΔExons could form and secrete α345(IV) trimers and had higher activity compared with nonsense mutants. Other ΔExons had low secretion activity, especially for those with exon deletion near the C-terminal end although the intracellular trimerization was normal. No difference was noted in the secretion of missense mutants and their ΔExon counterpart. CONCLUSION: Exon skipping is advantageous for nonsense mutants in AS with severe phenotypes and early onset of renal failure but applications may be limited to ΔExons capable of normal trimerization and secretion. This study provides information on α5(IV) exon-skipping for possible therapeutic application and the prediction of the trimer behavior associated with exon-skipping in Alport syndrome.

Clinical characteristics and outcomes of immune-complex membranoproliferative glomerulonephritis and C3 glomerulopathy in Japanese children.

BACKGROUND: Membranoproliferative glomerulonephritis (MPGN) can be divided into immune-complex MPGN (IC-MPGN) and C3 glomerulopathy (C3G), which includes dense deposit disease (DDD) and C3 glomerulonephritis (C3GN). These conditions result from abnormalities in different complement pathways and may lead to different prognoses. However, there are limited studies describing the respective clinical courses. METHODS: In this study, Japanese pediatric patients diagnosed with MPGN based on kidney biopsies conducted between February 2002 and December 2022 were reclassified as having IC-MPGN or C3G (DDD or C3GN). We retrospectively analyzed the clinical characteristics and outcomes of these patients. RESULTS: Out of 25 patients with MPGN, three (12.0%) were diagnosed with DDD, 20 (80.0%) with C3GN, and two (8.0%) with IC-MPGN. There were 13 (65.0%) patients and one (33.3%) patient in remission after treatment for C3GN and DDD, respectively, and no patients with IC-MPGN achieved remission. The median follow-up period was 5.3 (2.5-8.9) years, and none of the patients in either group progressed to an estimated glomerular filtration rate < 15 ml/min/1.73 m2. Patients with C3GN presenting mild to moderate proteinuria (n = 8) received a renin-angiotensin system inhibitor (RAS-I) alone, and these patients exhibited a significant decrease in the urinary protein creatinine ratio and a notable increase in serum C3 levels at the last follow-up. CONCLUSIONS: Most patients with MPGN were diagnosed with C3GN. The remission rate for C3GN was high, and no patients developed kidney failure during the approximately 5-year follow-up. Additionally, patients with C3GN with mild to moderate proteinuria had good outcomes with RAS-I alone, but continued vigilance is necessary to determine long-term prognosis.

Kidney resident macrophages have distinct subsets and multifunctional roles.

BACKGROUND: The kidney contains distinct glomerular and tubulointerstitial compartments with diverse cell types and extracellular matrix components. The role of immune cells in glomerular environment is crucial for dampening inflammation and maintaining homeostasis. Macrophages are innate immune cells that are influenced by their tissue microenvironment. However, the multifunctional role of kidney macrophages remains unclear. METHODS: Flow and imaging cytometry were used to determine the relative expression of CD81 and CX3CR1 (C-X3-C motif chemokine receptor 1) in kidney macrophages. Monocyte replenishment was assessed in Cx3cr1CreER X R26-yfp-reporter and shielded chimeric mice. Bulk RNA-sequencing and mass spectrometry-based proteomics were performed on isolated kidney macrophages from wild type and Col4a5-/- (Alport) mice. RNAscope was used to visualize transcripts and macrophage purity in bulk RNA assessed by CIBERSORTx analyses. RESULTS: In wild type mice we identified three distinct kidney macrophage subsets using CD81 and CX3CR1 and these subsets showed dependence on monocyte replenishment. In addition to their immune function, bulk RNA-sequencing of macrophages showed enrichment of biological processes associated with extracellular matrix. Proteomics identified collagen IV and laminins in kidney macrophages from wild type mice whilst other extracellular matrix proteins including cathepsins, ANXA2 and LAMP2 were enriched in Col4a5-/- (Alport) mice. A subset of kidney macrophages co-expressed matrix and macrophage transcripts. CONCLUSIONS: We identified CD81 and CX3CR1 positive kidney macrophage subsets with distinct dependence for monocyte replenishment. Multiomic analysis demonstrated that these cells have diverse functions that underscore the importance of macrophages in kidney health and disease.

iPSC-derived type IV collagen α5-expressing kidney organoids model Alport syndrome.

Alport syndrome (AS) is a hereditary glomerulonephritis caused by COL4A3, COL4A4 or COL4A5 gene mutations and characterized by abnormalities of glomerular basement membranes (GBMs). Due to a lack of curative treatments, the condition proceeds to end-stage renal disease even in adolescents. Hampering drug discovery is the absence of effective in vitro methods for testing the restoration of normal GBMs. Here, we aimed to develop kidney organoid models from AS patient iPSCs for this purpose. We established iPSC-derived collagen α5(IV)-expressing kidney organoids and confirmed that kidney organoids from COL4A5 mutation-corrected iPSCs restore collagen α5(IV) protein expression. Importantly, our model recapitulates the differences in collagen composition between iPSC-derived kidney organoids from mild and severe AS cases. Furthermore, we demonstrate that a chemical chaperone, 4-phenyl butyric acid, has the potential to correct GBM abnormalities in kidney organoids showing mild AS phenotypes. This iPSC-derived kidney organoid model will contribute to drug discovery for AS.

Systematic Review of Clinical Characteristics and Genotype-Phenotype Correlation in LAMB2-Associated Disease.

Introduction: Laminin subunit beta-2 (LAMB2)-associated disease, termed Pierson syndrome, presents with congenital nephrotic syndrome, ocular symptoms, and neuromuscular symptoms. In recent years, however, the widespread use of next-generation sequencing (NGS) has helped to discover a variety of phenotypes associated with this disease. Therefore, we conducted this systematic review. Methods: A literature search of patients with LAMB2 variants was conducted, and 110 patients were investigated, including 12 of our patients. For genotype-phenotype correlation analyses, the extracted data were investigated for pathogenic variant types, the severity of nephropathy, and extrarenal symptoms. Survival analyses were also performed for the onset age of end-stage kidney disease (ESKD). Results: Among all patients, 81 (78%) presented with congenital nephrotic syndrome, and 52 (55%) developed ESKD within 12 months. The median age at ESKD onset was 6.0 months. Kidney survival analysis showed that patients with biallelic truncating variants had a significantly earlier progression to ESKD than those with other variants (median age 1.2 months vs. 60.0 months, P < 0.05). Although the laminin N-terminal domain is functionally important in laminin proteins, and variants in the laminin N-terminal domain are said to result in a severe kidney phenotype such as earlier onset age and worse prognosis, there were no significant differences in onset age of nephropathy and progression to ESKD between patients with nontruncating variants located in the laminin N-terminal domain and those with variants located outside this domain. Conclusion: This study revealed a diversity of LAMB2-associated diseases, characteristics of LAMB2 nephropathy, and genotype-phenotype correlations.

Matrikines in kidney ageing and age-related disease.

PURPOSE OF REVIEW: Matrikines are cell-signalling extracellular matrix fragments and they have attracted recent attention from basic and translational scientists, due to their diverse roles in age-related disease and their potential as therapeutic agents. In kidney, the matrix undergoes remodelling by proteolytic fragmentation, so matrikines are likely to play a substantial, yet understudied, role in ageing and pathogenesis of age-related diseases. RECENT FINDINGS: This review presents an up-to-date description of known matrikines with either a confirmed or highly anticipated role in kidney ageing and disease, including their point of origin, mechanism of cleavage, a summary of known biological actions and the current knowledge which links them to kidney health. We also highlight areas of interest, such as the prospect of matrikine cross-tissue communication, and gaps in knowledge, such as the unexplored signalling potential of many kidney disease-specific matrix fragments. SUMMARY: We anticipate that knowledge of specific matrikines, and their roles in controlling processes of kidney pathology, could be leveraged for the development of exciting new future therapies through inhibition or even with their supplementation.

All reported non-canonical splice site variants in GLA cause aberrant splicing.

BACKGROUND: Fabry disease is an X-linked lysosomal storage disorder caused by insufficient α-galactosidase A (GLA) activity resulting from variants in the GLA gene, which leads to glycosphingolipid accumulation and life-threatening, multi-organ complications. Approximately 50 variants have been reported that cause splicing abnormalities in GLA. Most were found within canonical splice sites, which are highly conserved GT and AG splice acceptor and donor dinucleotides, whereas one-third were located outside canonical splice sites, making it difficult to interpret their pathogenicity. In this study, we aimed to investigate the genetic pathogenicity of variants located in non-canonical splice sites within the GLA gene. METHODS: 13 variants, including four deep intronic variants, were selected from the Human Gene Variant Database Professional. We performed an in vitro splicing assay to identify splicing abnormalities in the variants. RESULTS: All candidate non-canonical splice site variants in GLA caused aberrant splicing. Additionally, all but one variant was protein-truncating. The four deep intronic variants generated abnormal transcripts, including a cryptic exon, as well as normal transcripts, with the proportion of each differing in a cell-specific manner. CONCLUSIONS: Validation of splicing effects using an in vitro splicing assay is useful for confirming pathogenicity and determining associations with clinical phenotypes.

Corrigendum to "Detecting MUC1 Variants in Patients Clinicopathologically Diagnosed With Having Autosomal Dominant Tubulointerstitial Kidney Disease"Kidney International Reports, Volume 7, Issue 4, April 2022, Pages 857-866.

[This corrects the article DOI: 10.1016/j.ekir.2021.12.037.].

Aberrant splicing caused by exonic single nucleotide variants positioned 2nd or 3rd to the last nucleotide in the COL4A5 gene.

BACKGROUND AND OBJECTIVES: The evident genotype-phenotype correlation shown by the X-linked Alport syndrome warrants the assessment of the impact of identified gene variants on aberrant splicing. We previously reported that single nucleotide variants (SNVs) in the last nucleotide of exons in COL4A5 cause aberrant splicing. It is known that the nucleotides located 2nd and 3rd to the last nucleotides of exons can also play an essential role in the first step of the splicing process. In this study, we aimed to investigate whether SNVs positioned 2nd or 3rd to the last nucleotide of exons in COL4A5 resulted in aberrant splicing. METHODS: We selected eight candidate variants: six from the Human Gene Variant Database Professional and two from our cohort. We performed an in-vitro splicing assay and reverse transcription-polymerase chain reaction (RT-PCR) for messenger RNA obtained from patients, if available. RESULTS: The candidate variants were initially classified into the following groups: three nonsense, two missense, and three synonymous variants. Splicing assays and RT-PCR for messenger RNA revealed that six of the eight variants caused aberrant splicing. Four variants, initially classified as non-truncating variants, were found to be truncating ones, which usually show relatively more severe phenotypes. CONCLUSION: We revealed that exonic SNVs positioned 2nd or 3rd to the last nucleotide of exons in the COL4A5 were responsible for aberrant splicing. The results of our study suggest that attention should be paid when interpreting the pathogenicity of exonic SNVs near the 5' splice site.

Is influenza vaccination associated with nephrotic syndrome relapse in children? A multicenter prospective study.

BACKGROUND: Prospective research of children receiving heterogeneous vaccines has shown that immunization is not associated with pediatric idiopathic nephrotic syndrome (NS) relapses. However, prospective data concentrating only on influenza (flu) virus vaccines are not available. METHODS: This multicenter prospective study was conducted in children with NS who received inactivated flu vaccines from June 2017 to July 2018. The day of flu vaccination was defined as day 0, and the period between prevaccination and postvaccination days was defined as - X to + Y (period from day - 180 to 0 as the precontrolled period). The primary outcome was the NS relapse rate from day 0 to + 30 as a direct association with vaccination compared with those in the precontrolled period. Exacerbation was defined as children experiencing more NS relapses after vaccination compared with those in the precontrolled period, or children starting any new immunosuppressants due to NS relapse after vaccination. RESULTS: Sixty-three children were included. Relapse rates were not significantly different between the precontrolled period and 0 to + 30 periods (0.38 vs. 0.19 times/person-year, p = 0.95). Although the exacerbation rate during the 0 to + 180 period in children without NS relapse in the precontrolled period was very low (4/54 [7.4 %]), children with at least one NS relapse in the precontrolled period showed a remarkable increase in the rate (4/9 [44.4%]; p = 0.01). CONCLUSIONS: Flu vaccination did not significantly precipitate the direct relapse of NS in children. However, it might increase the disease activity in children with at least one NS relapse within a half year before vaccination. A higher resolution version of the Graphical abstract is available as Supplementary information.

Clinical, Pathological, and Genetic Characteristics in Patients with Focal Segmental Glomerulosclerosis.

Background: Approximately 30% of children with steroid-resistant nephrotic syndrome (SRNS) have causative monogenic variants. SRNS represents glomerular disease resulting from various etiologies, which lead to similar patterns of glomerular damage. Patients with SRNS mainly exhibit focal segmental glomerulosclerosis (FSGS). There is limited information regarding associations between histologic variants of FSGS (diagnosed using on the Columbia classification) and monogenic variant detection rates or clinical characteristics. Here, we report FSGS characteristics in a large population of affected patients. Methods: This retrospective study included 119 patients with FSGS, diagnosed using the Columbia classification; all had been referred to our hospital for genetic testing from 2016 to 2021. We conducted comprehensive gene screening of all patients using a targeted next-generation sequencing panel that included 62 podocyte-related genes. Data regarding patients' clinical characteristics and pathologic findings were obtained from referring clinicians. We analyzed the associations of histologic variants with clinical characteristics, kidney survival, and gene variant detection rates. Results: The distribution of histologic variants according to the Columbia classification was 45% (n=53) FSGS not otherwise specified, 21% (n=25) cellular, 15% (n=18) perihilar, 13% (n=16) collapsing, and 6% (n=7) tip. The median age at end stage kidney disease onset was 37 years; there were no differences in onset age among variants. We detected monogenic disease-causing variants involving 12 of the screened podocyte-related genes in 34% (40 of 119) of patients. The most common genes were WT1 (23%), INF2 (20%), TRPC6 (20%), and ACTN4 (10%). The perihilar and tip variants had the strongest and weakest associations with detection of monogenic variants (83% and 0%, respectively; P<0.001). Conclusions: We revealed the distributions of histologic variants of genetic FSGS and nongenetic FSGS in a large patient population. Detailed data concerning gene variants and pathologic findings are important for understanding the etiology of FSGS.

Detecting pathogenic deep intronic variants in Gitelman syndrome.

Gitelman syndrome (GS) is a rare, autosomal recessive, salt-losing tubulopathy caused by loss of function in the SLC12A3 gene (NM_000339.2), which encodes the natrium chloride cotransporter. The detection of homozygous or compound heterozygous SLC12A3 variants is expected in GS, but 18%-40% of patients with clinical GS carry only one mutant allele. Previous reports identified some pathogenic deep intronic variants in SLC12A3. Here, we report the screening of SLC12A3 deep intronic variants in 13 patients with suspected GS carrying one mutated SLC12A3 allele. Variant screening used the HaloPlex Target Enrichment System Kit capturing whole introns and the promotor region of SLC12A3, followed by SureCall variant analysis. Rare intronic variants (<1% frequency) were identified, and pathogenicity evaluated by the minigene system. Deep intronic variant screening detected seven rare SLC12A3 variants from six patients. Only one variant showed pathogenicity in the minigene system (c.602-16G>A, intron 4) through activation of a cryptic acceptor site. No variants were detected in the promotor region. Deep intronic screening identified only one pathogenic variant in patients with suspected GS carrying monoallelic SLC12A3 variants. Our results suggest that deep intronic variants partially explain the cause of monoallelic variants in patients with GS.

Detecting MUC1 Variants in Patients Clinicopathologically Diagnosed With Having Autosomal Dominant Tubulointerstitial Kidney Disease.

Introduction: Autosomal dominant tubulointerstitial kidney disease (ADTKD)-MUC1 is predominantly caused by frameshift mutations owing to a single-base insertion into the variable number tandem repeat (VNTR) region in MUC1. Because of the complexity of the variant hotspot, identification using short-read sequencers (SRSs) is challenging. Although recent studies have revealed the usefulness of long-read sequencers (LRSs), the prevalence of MUC1 variants in patients with clinically suspected ADTKD remains unknown. We aimed to clarify this prevalence and the genetic characteristics and clinical manifestations of ADTKD-MUC1 in a Japanese population using an SRS and an LRS. Methods: From January 2015 to December 2019, genetic analysis was performed using an SRS in 48 patients with clinically suspected ADTKD. Additional analyses were conducted using an LRS in patients with negative SRS results. Results: Short-read sequencing results revealed MUC1 variants in 1 patient harboring a cytosine insertion in the second repeat unit of the VNTR region; however, deeper VNTR regions could not be read by the SRS. Therefore, we conducted long-read sequencing analysis of 39 cases and detected MUC1 VNTR variants in 8 patients (in total, 9 patients from unrelated families). With the inclusion of family-affected patients (n = 31), the median age at the development of end-stage kidney disease (ESKD) was 45 years (95% CI: 40-40 years). Conclusion: In Japan, the detection rate of MUC1 variants in patients with clinically suspected ADTKD was 18.8%. More than 20% of patients with negative SRS results had MUC1 variants detected by an LRS.

Evaluation of Suspected Autosomal Alport Syndrome Synonymous Variants.

Background: Alport syndrome is an inherited disorder characterized by progressive renal disease, variable sensorineural hearing loss, and ocular abnormalities. Although many pathogenic variants in COL4A3 and COL4A4 have been identified in patients with autosomal Alport syndrome, synonymous mutations in these genes have rarely been identified. Methods: We conducted in silico splicing analysis using Human Splicing Finder (HSF) and Alamut to predict splicing domain strength and disruption of the sites. Furthermore, we performed in vitro splicing assays using minigene constructs and mRNA analysis of patient samples to determine the pathogenicity of four synonymous variants detected in four patients with suspected autosomal dominant Alport syndrome (COL4A3 [c.693G>A (p.Val231=)] and COL4A4 [c.1353C>T (p.Gly451=), c.735G>A (p.Pro245=), and c.870G>A (p.Lys290=)]). Results: Both in vivo and in vitro splicing assays showed exon skipping in two out of the four synonymous variants identified (c.735G>A and c.870G>A in COL4A4). Prediction analysis of wild-type and mutated COL4A4 sequences using HSF and Alamut suggested these two variants may lead to the loss of binding sites for several splicing factors, e.g., in acceptor sites and exonic splicing enhancers. The other two variants did not induce aberrant splicing. Conclusions: This study highlights the pitfalls of classifying the functional consequences of variants by a simple approach. Certain synonymous variants, although they do not alter the amino acid sequence of the encoded protein, can dramatically affect pre-mRNA splicing, as shown in two of our patients. Our findings indicate that transcript analysis should be carried out to evaluate synonymous variants detected in patients with autosomal dominant Alport syndrome.

The Contribution of COL4A5 Splicing Variants to the Pathogenesis of X-Linked Alport Syndrome.

X-linked Alport syndrome (XLAS) is caused by pathogenic variants in COL4A5 and is characterized by progressive kidney disease, hearing loss, and ocular abnormalities. Recent advances in genetic analysis and further understanding of genotype-phenotype correlations in affected male patients raises the importance of detecting splicing variants in COL4A5. Aberrant splicing of COL4A5 is caused not only by canonical splice site variants but also non-canonical splice site variants such as deep intronic changes or even substitutions in exons. Patients with splicing variants account for ~15% of all cases in XLAS. In addition, it has been shown that there is a significant difference in kidney survival depending on the aberrant splicing patterns of transcripts- in particular in-frame or out-of-frame nucleotide changes in transcripts. Therefore, cDNA analysis of patient mRNA is necessary to determine the impact of splice site variants and to confirm a diagnosis of XLAS and to predict the kidney prognosis. However, it is usually difficult to amplify COL4A5 transcripts extracted from peripheral blood leukocytes. For these cases, in vitro minigene assays or RNA sequence extracted from urine derived cells can confirm aberrant splicing patterns. Moreover, controlling aberrant splicing by nucleic acids or small molecular compounds in genetic diseases are attracting attention as a potential therapeutic strategy. Here, we review the frequency of splicing variants in COL4A5, the latest diagnostic strategies, and the prospects for new therapeutic approaches.

Comprehensive genetic analysis using next-generation sequencing for the diagnosis of nephronophthisis-related ciliopathies in the Japanese population.

Nephronophthisis is an autosomal-recessive kidney disease that is caused by abnormalities in primary cilia. Nephronophthisis-related ciliopathies (NPHP-RCs) are a common cause of end-stage kidney disease (ESKD) in children and adolescents. NPHP-RCs are often accompanied by extrarenal manifestations, including intellectual disability, retinitis pigmentosa, or polydactyly. Although more than 100 causative genes have been identified, its diagnosis is difficult because the clinical features of each mutation often overlap. From September 2010 to August 2021, we performed genetic analysis, including next-generation sequencing (NGS), in 574 probands with kidney dysfunction and retrospectively studied cases genetically diagnosed with NPHP-RCs. RESULTS: We detected mutations related to NPHP-RCs in 93 patients from 83 families. Members of 60 families were diagnosed using NGS, and the mutations and the corresponding number of families are as follows: NPHP1 (24), NPHP3 (10), OFD1 (7), WDR35 (5), SDCCAG8 (4), BBS10 (3), TMEM67 (3), WDR19 (3), BBS1 (2), BBS2 (2), IFT122 (2), IFT140 (2), IQCB1 (2), MKKS (2), SCLT1 (2), TTC21B (2), ALMS1 (1), ANKS6 (1), BBS4 (1), BBS12 (1), CC2D2A (1), DYNC2H1 (1), IFT172 (1), and MAPKBP1 (1). A total of 39 cases (41.9%) progressed to ESKD at the time of genetic analysis, whereas 58 cases (62.3%) showed extrarenal manifestations, the most common being developmental delay, intellectual disability, and autism spectrum disorder in 44 patients. Comprehensive genetic analysis using NGS is useful for diagnosing patients with NPHP-RCs.

Efficacy of combination therapy for childhood complicated focal IgA nephropathy.

BACKGROUND: Patients with immunoglobulin A nephropathy who present with focal mesangial proliferation (focal IgAN) can have a relatively good prognosis, and renin-angiotensin system inhibitor (RAS-i) is commonly used as the initial treatment. However, there are some complicated focal IgAN cases with resistance to RAS-i treatment or nephrotic-range proteinuria. Thus, combination therapy including corticosteroids is often used. This study aimed to evaluate the efficacy of combination therapy for complicated focal IgAN cases by comparing to diffuse mesangial proliferation (diffuse IgAN). METHODS: We conducted a multicenter retrospective study on 88 children who received 2-year combination therapy. The participants were classified based on pathological severity: focal IgAN (n = 26) and diffuse IgAN (n = 62). RESULTS: In total, 26 patients with focal IgAN and 52 with diffuse IgAN achieved proteinuria disappearance within 2 years (100 vs. 83.9%, P = 0.03). Moreover, the time to proteinuria disappearance was significantly shorter in the focal IgAN group than in the diffuse IgAN group (2.9 vs. 4.2 months, P < 0.01) and all patients with focal IgAN achieved proteinuria disappearance within 8 months. At the last observation (8.6 vs. 10.4 years, P = 0.13), only patients with diffuse IgAN (n = 12) had greater than stage 2 chronic kidney disease. In terms of irreversible adverse events, one patient exhibited cataracts. CONCLUSION: Combination therapy was significantly effective in patients with complicated focal IgAN. Moreover, the long-term prognosis was good, and the duration of combination therapy for complicated focal IgAN can be decreased to reduce adverse events.

Use of renin-angiotensin system inhibitors as initial therapy in children with Henoch-Schönlein purpura nephritis of moderate severity.

BACKGROUND: Cases of Henoch-Schönlein purpura nephritis (HSPN) with moderate severity were demonstrated to achieve good prognosis after treatment with renin-angiotensin system (RAS) inhibitors. However, some patients required additional treatment for recurrence after remission. This study aimed to clarify the effect of RAS inhibitors in HSPN cases with moderate severity, including the proportion of cases with recurrence and their response to additional treatment. METHODS: Among 126 patients diagnosed with HSPN between 1996 and 2019, 71 patients with clinicopathologically diagnosed HSPN of moderate severity, defined as ISKDC grade II-IIIa and serum albumin ≥ 2.5 g/dL, were investigated. RESULTS: Proteinuria became negative after RAS inhibitor treatment alone in all 71 cases. However, 16 (22.5%) had recurrence. Eleven recurrent cases achieved negative proteinuria again following additional treatment. At the last follow-up (median 46.5 months; IQR, 23.2-98.2), 5 patients had persistent mild proteinuria; no patients had estimated glomerular filtration rate < 90 mL/min/1.73 m2. The pathological findings in all recurrent cases were ISKDC grade IIIa. The 16 recurrent cases had significantly higher proportions of glomeruli with global/segmental sclerosis (25.0 vs. 0%, P < 0.001) and tubular atrophy/interstitial fibrosis (37.5 vs. 12.7%, P =0.0 24) than 55 cases without recurrence. CONCLUSIONS: Japanese childhood HSPN cases with moderate severity had good outcomes without need for corticosteroids or immunosuppressants, when prescribed RAS inhibitor treatment. Even in recurrent cases, abnormal proteinuria was transient, and prognosis was excellent. A higher resolution version of the Graphical abstract is available as Supplementary information.

Last Nucleotide Substitutions of COL4A5 Exons Cause Aberrant Splicing.

INTRODUCTION: COL4A5 is a causative gene of X-linked Alport syndrome (XLAS). Male patients with XLAS with nonsense variants have the most severe phenotypes of early onset end-stage kidney disease (ESKD); those with splicing variants have middle phenotypes and those with missense variants have the mildest phenotypes. Therefore, genotyping for male patients with XLAS can be used to predict kidney prognosis. Single-base substitutions at the last nucleotide position in each exon are known to affect splicing patterns and could be splicing variants. Nevertheless, in XLAS, these variants are generally considered to be missense variants, without conducting a transcript analysis, which underestimates some patients as having mild phenotypes. This study aimed to investigate whether single-base substitutions at the last nucleotide position of COL4A5 exons cause aberrant splicing. METHODS: In total, 20 variants were found in the Human Gene Mutation Database (n = 14) and our cohort (n = 6). We performed functional splicing assays using a hybrid minigene analysis and in vivo transcript analyses of patients' samples when available. Then, we investigated genotype-phenotype correlations for patients with splicing variants detected in this study by comparing data from our previous studies. RESULTS: Among the 20 variants, 17 (85%) caused aberrant splicing. Male patients with splicing variants had more severe phenotypes when compared with those with missense variants. Findings from the in vivo analyses for 3 variants were identical to those from the minigene assay. CONCLUSION: Our study revealed that most single-base substitutions at the last nucleotide position of COL4A5 exons result in splicing variants, rather than missense variants, thereby leading to more severe phenotypes.

Identification of novel OCRL isoforms associated with phenotypic differences between Dent disease-2 and Lowe syndrome.

BACKGROUND: Although Lowe syndrome and Dent disease-2 are caused by Oculocerebrorenal syndrome of Lowe (OCRL) mutations, their clinical severities differ substantially and their molecular mechanisms remain unclear. Truncating mutations in OCRL exons 1-7 lead to Dent disease-2, whereas those in exons 8-24 lead to Lowe syndrome. Herein we identified the mechanism underlying the action of novel OCRL protein isoforms. METHODS: Messenger RNA samples extracted from cultured urine-derived cells from a healthy control and a Dent disease-2 patient were examined to detect the 5' end of the OCRL isoform. For protein expression and functional analysis, vectors containing the full-length OCRL transcripts, the isoform transcripts and transcripts with truncating mutations detected in Lowe syndrome and Dent disease-2 patients were transfected into HeLa cells. RESULTS: We successfully cloned the novel isoform transcripts from OCRL exons 6-24, including the translation-initiation codons present in exon 8. In vitro protein-expression analysis detected proteins of two different sizes (105 and 80 kDa) translated from full-length OCRL, whereas only one protein (80 kDa) was found from the isoform and Dent disease-2 variants. No protein expression was observed for the Lowe syndrome variants. The isoform enzyme activity was equivalent to that of full-length OCRL; the Dent disease-2 variants retained >50% enzyme activity, whereas the Lowe syndrome variants retained <20% activity. CONCLUSIONS: We elucidated the molecular mechanism underlying the two different phenotypes in OCRL-related diseases; the functional OCRL isoform translated starting at exon 8 was associated with this mechanism.