Role of Iron in Children With Immunoglobulin A Nephropathy and Macrohematuria-Induced Acute Kidney Injury.

Introduction: The role of iron in, and the prognosis of, pediatric Immunoglobulin A nephropathy (IgAN) with macrohematuria (MH)-induced acute kidney injury (AKI) (MH-AKI) have not been evaluated. Thirty percent of adults with MH-AKI, and especially those who are older, show progression to chronic kidney disease. Methods: We evaluated the immunohistopathologic characteristics of renal biopsy samples from pediatric patients with MH-AKI IgAN and controls, using Berlin Blue to identify iron, CD163 (a hemoglobin-scavenging receptor), and CD68 (a total macrophage marker), then compared the findings against the clinical characteristics of the patients. Results: We enrolled 44 children as follows: 19 with IgAN but no MH or AKI; 5 with IgAN and MH but no AKI (MH(+)AKI(-) IgAN); 11 with MH-AKI IgAN; and 9 with no IgAN, MH, or AKI, according to a renal biopsy. Berlin Blue staining was detected predominantly at the injured tubulointerstitium, and the areas of staining in children with MH(+)AKI(-) and MH-AKI IgAN were significantly more extensive. The areas of Berlin Blue and CD163 staining did not perfectly match; however, areas of Berlin Blue were surrounded by immunopositivity for CD163. No children with MH-AKI IgAN showed decreased renal function at their last visit. Conclusion: Children with IgAN and MH, with or without AKI, showed considerable iron deposition in their renal tubules. CD163-positive cells might scavenge hemoglobin in patients with MH-AKI IgAN, but not their roles as macrophages. The renal prognosis of pediatric MH-AKI IgAN is good.

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.

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.

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.

BCS1L mutations produce Fanconi syndrome with developmental disability.

Fanconi syndrome is a functional disorder of the proximal tubule, characterized by pan-aminoaciduria, glucosuria, hypophosphatemia, and metabolic acidosis. With the advancements in gene analysis technologies, several causative genes are identified for Fanconi syndrome. Several mitochondrial diseases cause Fanconi syndrome and various systemic symptoms; however, it is rare that the main clinical symptoms in such disorders are Fanconi syndrome without systematic active diseases like encephalomyopathy or cardiomyopathy. In this study, we analyzed two families exhibiting Fanconi syndrome, developmental disability and mildly elevated liver enzyme levels. Whole-exome sequencing (WES) detected compound heterozygous known and novel BCS1L mutations, which affect the assembly of mitochondrial respiratory chain complex III, in both cases. The pathogenicity of these mutations has been established in several mitochondria-related functional analyses in this study. Mitochondrial diseases with isolated renal symptoms are uncommon; however, this study indicates that mitochondrial respiratory chain complex III deficiency due to BCS1L mutations cause Fanconi syndrome with developmental disability as the primary indications.

A disease-causing variant of COL4A5 in a Chinese family with Alport syndrome: a case series.

BACKGROUND: Alport syndrome (AS), which is a rare hereditary disease caused by mutations of genes including COL4A3, COL4A4 and COL4A5, has a wide spectrum of phenotypes. Most disease-causing variants of AS are located in the exons or the conservative splicing sites of these genes, while little is known about the intronic disease-causing variants. METHODS: A Chinese AS family was recruited in this study. All the clinical data of AS patient were collected from medical records. After pedigree analysis, the pathogenic variants were studied by the whole exome sequencing (WES). Minigene assay and in vivo RT-PCR analysis were performed to validate the functions of the variants. RESULTS: Renal biopsy showed a typical histopathology changes of AS. WES revealed compound heterozygous substitution, NM_033380 c.991-14(IVS17) A > G, in the intron 17 of the COL4A5 gene, which were confirmed by Sanger sequencing. Moreover, the variant was co-segregated with the phenotype in this family. Minigene assay in cultured cell lines showed that a splicing error was induced by this intronic variant, which further confirmed by in vivo RT-PCR analysis. CONCLUSION: A novel intronic disease-causing variant in COL4A5 gene was identified by WES, which was the molecular pathogenic basis of AS.

Genotype-Phenotype Correlation in WT1 Exon 8 to 9 Missense Variants.

INTRODUCTION: WT1 missense mutation in exon 8 or 9 causes infantile nephrotic syndrome with early progression to end-stage kidney disease (ESKD), Wilms tumor, and 46,XY female. However, some patients with missense mutations in exon 8 or 9 progress to ESKD in their teens or later. Therefore, we conducted a systematic review and functional analysis of WT1 transcriptional activity. METHODS: We conducted a systematic review of 174 cases with WT1 exon 8 or 9 missense variants from our cohort (n=13) and previous reports (n=161). Of these cases, mild and severe genotypes were selected for further in vitro functional analysis using luciferase assay. RESULTS: The median age of developing ESKD was 1.17 years. A comparative study was conducted among three WT1 genotype classes: mutations of the DNA-binding site (DBS group), mutations outside the DNA-binding site but at sites important for zinc finger structure formation by 2 cysteines and 2 histidines (C2H2 group), and mutations leading to other amino acid changes (Others group). The DBS group showed the severest phenotype and the C2H2 group was intermediate, whereas the Others group showed the mildest phenotype (developing ESKD at 0.90, 2.00, and 3.92 years, respectively, with significant differences). In vitro functional analysis showed dominant-negative effects for all variants; in addition, the DBS and C2H2 mutations were associated with significantly lower WT1 transcriptional activity than the other mutations. CONCLUSION: Not only the DNA-binding site but also C2H2 zinc finger structure sites are important for maintaining WT1 transcriptional activity, and their mutation causes severe clinical symptoms.

X-chromosome inactivation patterns in females with Fabry disease examined by both ultra-deep RNA sequencing and methylation-dependent assay.

BACKGROUND: Fabry disease is an X-linked inherited lysosomal storage disorder caused by mutations in the gene encoding α-galactosidase A. Males are usually severely affected, while females have a wide range of disease severity. This variability has been assumed to be derived from organ-dependent skewed X-chromosome inactivation (XCI) patterns in each female patient. Previous studies examined this correlation using the classical methylation-dependent method; however, conflicting results were obtained. This study was established to ascertain the existence of skewed XCI in nine females with heterozygous pathogenic variants in the GLA gene and its relationship to the phenotypes. METHODS: We present five female patients from one family and four individual female patients with Fabry disease. In all cases, heterozygous pathogenic variants in the GLA gene were detected. The X-chromosome inactivation patterns in peripheral blood leukocytes and cells of urine sediment were determined by both classical methylation-dependent HUMARA assay and ultra-deep RNA sequencing. Fabry Stabilization Index was used to determine the clinical severity. RESULTS: Skewed XCI resulting in predominant inactivation of the normal allele was observed only in one individual case with low ⍺-galactosidase A activity. In the remaining cases, no skewing was observed, even in the case with the highest total severity score (99.2%). CONCLUSION: We conclude that skewed XCI could not explain the severity of female Fabry disease and is not the main factor in the onset of various clinical symptoms in females with Fabry disease.

Clinical and histological features in pediatric and adolescent/young adult patients with renal disease: a cross-sectional analysis of the Japan Renal Biopsy Registry (J-RBR).

BACKGROUND: Only a few studies have investigated epidemiological and clinicopathological information regarding pediatric and adolescent and young adult (AYA) patients with renal disease. The purpose of this study was to clarify the differences and relationship of clinicopathological findings between pediatric and AYA patients using the Japan Renal Biopsy Registry (J-RBR). METHODS: This cross-sectional study analyzed data from patients registered in the J-RBR between 2007 and 2017. Clinicopathological findings at diagnosis were analyzed for 3,463 pediatric (age < 15 years) and 6,532 AYA (age 15-30 years) patients. RESULTS: Although chronic nephritic syndrome was the most common clinical diagnosis at age > 5 years, nephrotic syndrome was the most frequent diagnosis at age < 4 years. The most common pathological diagnosis as classified by pathogenesis in pediatric patients was primary glomerular disease (except IgA nephropathy), whereas IgA nephropathy was increased in AYA patients. Mesangial proliferative glomerulonephritis was the most common pathological diagnosis as classified by histopathology in both pediatric and AYA patients. Minor glomerular abnormalities were the most frequent histopathologic diagnoses of nephrotic syndrome in childhood, but their frequency decreased with age. CONCLUSION: To the best of our knowledge, this is the first report of clinicopathological features of pediatric and AYA patients in a large nationwide registry of renal biopsy. There were differences of clinical, pathological and histopathologic findings between pediatric and AYA patients.

Usefulness of functional splicing analysis to confirm precise disease pathogenesis in Diamond-Blackfan anemia caused by intronic variants in RPS19.

Diamond-Blackfan anemia (DBA) is mainly caused by pathogenic variants in ribosomal proteins and 22 responsible genes have been identified to date. The most common causative gene of DBA is RPS19 [NM_001022.4]. Nearly 180 RPS19 variants have been reported, including three deep intronic variants outside the splicing consensus sequence (c.72-92A > G, c.356 + 18G > C, and c.411 + 6G > C). We also identified one case with a c.412-3C > G intronic variant. Without conducting transcript analysis, the pathogenicity of these variants is unknown. However, it is difficult to assess transcripts because of their fragility. In such cases, in vitro functional splicing assays can be used to assess pathogenicity. Here, we report functional splicing analysis results of four RPS19 deep intronic variants identified in our case and in previously reported cases. One splicing consensus variant (c.411 + 1G > A) was also examined as a positive control. Aberrant splicing with a 2-bp insertion between exons 5 and 6 was identified in the patient samples and minigene assay results also identified exon 6 skipping in our case. The exon 6 skipping transcript was confirmed by further evaluation using quantitative RT-PCR. Additionally, minigene assay analysis of three reported deep intronic variants revealed that none of them showed aberrant splicing and that these variants were not considered to be pathogenic. In conclusion, the minigene assay is a useful method for functional splicing analysis of inherited disease.

Common risk variants in NPHS1 and TNFSF15 are associated with childhood steroid-sensitive nephrotic syndrome.

To understand the genetics of steroid-sensitive nephrotic syndrome (SSNS), we conducted a genome-wide association study in 987 childhood SSNS patients and 3,206 healthy controls with Japanese ancestry. Beyond known associations in the HLA-DR/DQ region, common variants in NPHS1-KIRREL2 (rs56117924, P=4.94E-20, odds ratio (OR) =1.90) and TNFSF15 (rs6478109, P=2.54E-8, OR=0.72) regions achieved genome-wide significance and were replicated in Korean, South Asian and African populations. Trans-ethnic meta-analyses including Japanese, Korean, South Asian, African, European, Hispanic and Maghrebian populations confirmed the significant associations of variants in NPHS1-KIRREL2 (Pmeta=6.71E-28, OR=1.88) and TNFSF15 (Pmeta=5.40E-11, OR=1.33) loci. Analysis of the NPHS1 risk alleles with glomerular NPHS1 mRNA expression from the same person revealed allele specific expression with significantly lower expression of the transcript derived from the risk haplotype (Wilcox test p=9.3E-4). Because rare pathogenic variants in NPHS1 cause congenital nephrotic syndrome of the Finnish type (CNSF), the present study provides further evidence that variation along the allele frequency spectrum in the same gene can cause or contribute to both a rare monogenic disease (CNSF) and a more complex, polygenic disease (SSNS).

Clinical and genetic variability of PAX2-related disorder in the Japanese population.

Pathogenic variants of paired box gene 2 (PAX2) cause autosomal-dominant PAX2-related disorder, which includes renal coloboma syndrome (RCS). Patients with PAX2-related disorder present with renal and ophthalmological pathologies, as well as with other abnormalities, including developmental problems and hearing loss. We sequenced PAX2 in 457 patients with congenital anomalies of the kidney and urinary tract or with renal dysfunction of unknown cause and identified 19 different pathogenic variants in 38 patients from 30 families (6.5%). Thirty-four patients had renal hypodysplasia or chronic kidney disease of unknown cause, and three had focal segmental glomerulosclerosis. Although no obvious genotype-phenotype correlation was observed, six of the seven patients who developed end-stage renal disease in childhood had truncating variants. Twenty-three patients had ocular disabilities, mostly optic disc coloboma. Non-renal and non-ophthalmological manifestations included developmental disorder, electrolyte abnormality, and gonadal abnormalities. Two unrelated patients had congenital cystic adenomatoid malformations in their lungs. Six of ten probands with PAX2 mutation identified by next-generation sequencing did not show typical RCS manifestations. We conclude that PAX2-related disorder has a variable clinical presentation and can be diagnosed by next-generation sequencing even in the absence of typical RCS manifestations.

Clinical characteristics of HNF1B-related disorders in a Japanese population.

BACKGROUND: Hepatocyte nuclear factor 1ß (HNF1B), located on chromosome 17q12, causes renal cysts and diabetes syndrome (RCAD). Moreover, various phenotypes related to congenital anomalies of the kidney and urinary tract (CAKUT) or Bartter-like electrolyte abnormalities can be caused by HNF1B variants. In addition, 17q12 deletion syndrome presents with multi-system disorders, as well as RCAD. As HNF1B mutations are associated with different phenotypes and genotype-phenotype relationships remain unclear, here, we extensively studied these mutations in Japan. METHODS: We performed genetic screening of RCAD, CAKUT, and Bartter-like syndrome cases. Heterozygous variants or whole-gene deletions in HNF1B were detected in 33 cases (19 and 14, respectively). All deletion cases were diagnosed as 17q12 deletion syndrome, confirmed by multiplex ligation probe amplification and/or array comparative genomic hybridization. A retrospective review of clinical data was also conducted. RESULTS: Most cases had morphological abnormalities in the renal-urinary tract system. Diabetes developed in 12 cases (38.7%). Hyperuricemia and hypomagnesemia were associated with six (19.3%) and 13 cases (41.9%), respectively. Pancreatic malformations were detected in seven cases (22.6%). Ten patients (32.3%) had liver abnormalities. Estimated glomerular filtration rates were significantly lower in the patients with heterozygous variants compared to those in patients harboring the deletion (median 37.6 vs 58.8 ml/min/1.73 m2; p = 0.0091). CONCLUSION: We present the clinical characteristics of HNF1B-related disorders. To predict renal prognosis and complications, accurate genetic diagnosis is important. Genetic testing for HNF1B mutations should be considered for patients with renal malformations, especially when associated with other organ involvement.

TGFBI-associated corneal dystrophy and nephropathy: a novel syndrome?

Transforming growth factor beta-induced (TGFBI)-associated corneal dystrophies are a group of inherited progressive corneal diseases. One of these TGFBI-associated corneal dystrophies is Avellino corneal dystrophy, an autosomal dominant corneal dystrophy characterized by multiple asymmetric stromal opacities that potentially impair vision. Recently, a case with corneal dystrophy complicated by nephropathy possessing a pathogenic variant of the TGFBI gene was reported for the first time. Here, we report the second case with the same condition and the same mutation in the TGFBI gene. The patient was an 18-year-old male. He and his father had already been diagnosed with corneal dystrophy. Proteinuria was revealed in the patient during urine screening at school. Since his serum creatinine level was raised, a percutaneous renal biopsy was performed. Light microscopy demonstrated oligomeganephronia. Electron microscopy demonstrated an irregular basement membrane. TGFBI was analyzed by direct sequencing. A heterozygous mutation c.371G > A in exon 4, which caused an amino acid substitution from arginine to histidine at codon 124, was identified in the patient and his father. Although only one case of TGFBI-associated corneal dystrophy and nephropathy has been reported, our case's clinical and pathological findings were almost identical to those in that reported case. Further investigations of this new disease entity should be reported to all nephrologists and ophthalmologists.

Clinical spectrum of male patients with OFD1 mutations.

Oral-facial-digital syndrome type 1 (OFD1) is a ciliopathy characterized by oral, facial, and digital malformations that are often accompanied by polycystic lesion of the kidney and central nervous involvement. OFD1 shows an X-linked recessive inheritance caused by mutation in the OFD1 gene (Xp22.2). The disease is generally considered embryonic lethal for hemizygous males. However, males with OFD1 mutations were recently reported. Here, we report four additional Japanese male patients with OFD1 variants and describe the variable clinical manifestation and disease severity among the four patients. Patient 1 with pathogenic indels including a 19-bp deletion and 4-bp insertion (c.2600-18_2600delinsACCT) had end-stage renal disease (ESRD) with bilateral cystic kidneys and sensory hearing loss. He showed neither intellectual disability nor facial or digital dysmorphism. Patient 2 with a missense variant in exon 7 (c.539 A > T, p.Asp180Val) presented head circumference enlargement, brachydactyly, high-arched palate, micropenis, severe global developmental delay, and ESRD. Patient 3 had a single base substitution at the splice donor site of intron 16 (c.2260 + 2 T > G) causing a 513-bp deletion at the transcript level. The patient had chronic kidney disease and speech delay, but no oral, facial, or digital dysmorphism. His uncle (patient 4) carried the same OFD1 variant and showed ESRD with extra-renal malformations including obesity and micropenis, which was previously diagnosed as Bardet-Biedl syndrome. The OFD1 mutations were not lethal in these four male patients, likely because the three mutations were in-frame or missense. This report provided insights into the onset mechanism and phenotype-genotype association in patients with OFD1 mutations.

Three Severe Cases of Viral Infections with Post-Kidney Transplantation Successfully Confirmed by Polymerase Chain Reaction and Flow Cytometry.

Viral infections in patients with post-kidney transplantation are often difficult to diagnose as well as treat. We herein report three cases with severe viral infections after kidney transplantation. All their causative pathogens could be detected promptly by polymerase chain reaction and flow cytometry during the early stages of infection. These examinations would also be of great use to monitor therapeutic responses and disease activity. It is indeed true that no specific treatment is available for most of the viral infections, but we should be aware that some infections, such as Epstein-Barr virus infection, can be treatable with prompt and specific treatment, such as rituximab.

Congenital chloride diarrhea needs to be distinguished from Bartter and Gitelman syndrome.

Pseudo-Bartter/Gitelman syndrome (p-BS/GS) encompasses a clinically heterogeneous group of inherited or acquired disorders similar to Bartter syndrome (BS) or Gitelman syndrome (GS), both renal salt-losing tubulopathies. Phenotypic overlap frequently occurs between p-BS/GS and BS/GS, which are difficult to diagnose based on their clinical presentation and require genetic tests for accurate diagnosis. In addition, p-BS/GS can occur as a result of other inherited diseases such as cystic fibrosis, autosomal dominant hypocalcemia, Dent disease, or congenital chloride diarrhea (CCD). However, the detection of the variants in genes other than known BS/GS-causing genes by conventional Sanger sequencing requires substantial time and resources. We studied 27 cases clinically diagnosed with BS/GS, but with negative genetic tests for known BS/GS genes. We conducted targeted sequencing for 22 genes including genes responsible for tubulopathies and other inherited diseases manifesting with p-BS/GS symptoms. We detected the SLC26A3 gene variants responsible for CCD in two patients. In Patient 1, we found the SLC26A3 compound heterozygous variants: c.354delC and c.1008insT. In Patient 2, we identified the compound heterozygous variants: c.877G > A, p.(Glu293Lys), and c.1008insT. Our results suggest that a comprehensive genetic screening system using targeted sequencing is useful for the diagnosis of patients with p-BS/GS with alternative genetic origins.

Development of ultra-deep targeted RNA sequencing for analyzing X-chromosome inactivation in female Dent disease.

The pattern of X-chromosome inactivation (XCI) can affect the clinical severity of X-linked disorders in females. XCI pattern analysis has been conducted mainly by HUMARA assay, a polymerase chain reaction-based assay using a methylation-sensitive restriction enzyme. However, this assay examines the XCI ratio of the androgen receptor gene at the genomic DNA level and does not reflect the ratio of either targeted gene directly or at the mRNA level. Here, we report four females with Dent disease, and we clarified the correlation between XCI and female cases of Dent disease using not only HUMARA assay but also a novel analytical method by RNA sequencing. We constructed genetic analysis for 4 female cases showing high level of urinary low-molecular-weight proteinuria and their parents. Their XCI pattern was analyzed by both HUMARA assay and an ultra-deep targeted RNA sequencing of the CLCN5 gene using genomic DNA and mRNA extracted from both leukocytes and urine sediment. All four cases possessed pathogenic variants of the CLCN5 gene. XCI analysis revealed skewed XCI in only two cases, while the other two showed random XCI. All assay results of HUMARA and targeted RNA sequencing in both leukocytes and urinary sediment were clearly identical in all four cases. We developed a novel XCI analytical assay of ultra-deep targeted RNA sequencing and revealed that skewed XCI explains the mechanism of onset of female Dent disease in only half of such cases.

A birth of bipartite exon by intragenic deletion.

BACKGROUND: Disease-causing mutations that activate transposon-derived exons without creating a new splice-site consensus have been reported rarely, but they provided unique insights into our understanding of structural motifs required for inclusion of intronic sequences in mature transcripts. METHODS: We employ a combination of experimental and computational techniques to characterize the first de novo bipartite exon activation in genetic disease. RESULTS: The exon originated from two separate introns as a result of an in-frame COL4A5 deletion associated with a typical Alport syndrome. The deletion encompassed exons 38 through 41 and activated a cryptic 3' and 5' splice site that were derived from intron 37 and intron 41, respectively. The deletion breakpoint was in the middle of the new exon, with considerable complementarity between the two exonic parts, potentially bringing the cryptic 3' and 5' splice site into proximity. The 3' splice site, polypyrimidine tract and the branch site of the new exon were derived from an inactive, 5' truncated LINE-1 retrotransposon. This ancient LINE-1 copy sustained a series of mutations that created the highly conserved AG dinucleotide at the 3' splice site early in primate development. The exon was fully included in mature transcripts and introduced a stop codon in the shortened COL4A5 mRNA, illustrating pitfalls of inferring disease severity from DNA mutation alone. CONCLUSION: These results expand the repertoire of mutational mechanisms that alter RNA processing in genetic disease and illustrate the extraordinary versatility of transposed elements in shaping the new exon-intron structure and the phenotypic variability.