Search

Currently, there are no treatments for Alport syndrome, which is the second most commonly inherited kidney disease. Here we report the development of an exon-skipping therapy using an antisense-oligonucleotide (ASO) for severe male X-linked Alport syndrome (XLAS). We targeted truncating variants in exon 21 of the COL4A5 gene and conducted a type IV collagen α3/α4/α5 chain triple helix formation assay, and in vitro and in vivo treatment efficacy evaluation. We show that exon skipping enabled trimer formation, leading to remarkable clinical and pathological improvements including expression of the α5 chain on glomerular and the tubular basement membrane. In addition, the survival period was clearly prolonged in the ASO treated mice group. This data suggests that exon skipping may represent a promising therapeutic approach for treating severe male XLAS cases., Alport syndrome is a progressive inherited nephritis accompanied by sensorineural loss of hearing and ocular abnormalities, for which there is currently no effective therapy. Here, the authors develop an exon-skipping therapy using an antisense-oligonucleotide and show it is effective in mouse models.

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.

Background Nephrotic syndrome is the most common cause of chronic glomerular disease in children. Most of these patients develop steroid-sensitive nephrotic syndrome (SSNS), but the loci conferring susceptibility to childhood SSNS are mainly unknown.Methods We conducted a genome-wide association study (GWAS) in the Japanese population; 224 patients with childhood SSNS and 419 adult healthy controls were genotyped using the Affymetrix Japonica Array in the discovery stage. Imputation for six HLA genes (HLA-A, -C, -B, -DRB1, -DQB1, and -DPB1) was conducted on the basis of Japanese-specific references. We performed genotyping for HLA-DRB1/-DQB1 using a sequence-specific oligonucleotide-probing method on a Luminex platform. Whole-genome imputation was conducted using a phased reference panel of 2049 healthy Japanese individuals. Replication was performed in an independent Japanese sample set including 216 patients and 719 healthy controls. We genotyped candidate single-nucleotide polymorphisms using the DigiTag2 assay.Results The most significant association was detected in the HLA-DR/DQ region and replicated (rs4642516 [minor allele G], combined Pallelic=7.84×10-23; odds ratio [OR], 0.33; 95% confidence interval [95% CI], 0.26 to 0.41; rs3134996 [minor allele A], combined Pallelic=1.72×10-25; OR, 0.29; 95% CI, 0.23 to 0.37). HLA-DRB1*08:02 (Pc=1.82×10-9; OR, 2.62; 95% CI, 1.94 to 3.54) and HLA-DQB1*06:04 (Pc=2.09×10-12; OR, 0.10; 95% CI, 0.05 to 0.21) were considered primary HLA alleles associated with childhood SSNS. HLA-DRB1*08:02-DQB1*03:02 (Pc=7.01×10-11; OR, 3.60; 95% CI, 2.46 to 5.29) was identified as the most significant genetic susceptibility factor.Conclusions The most significant association with childhood SSNS was detected in the HLA-DR/DQ region. Further HLA allele/haplotype analyses should enhance our understanding of molecular mechanisms underlying SSNS.

Recent advances in high-throughput sequencing for clinical genetic testing have revealed novel disease-causing genes, such as Crumbs homolog 2 (CRB2) for early-onset steroid-resistant nephrotic syndrome (SRNS). We report the long-term clinicopathologic observation of a Japanese female patient with SRNS caused by a newly identified compound heterozygous mutation of CRB2 (p.Arg628Cys and p.Gly839Trp located in the 10th and 11th epidermal growth factor-like domains, respectively). She was initially examined during a mass urinary screening for 3.5-year-old children in Japan. Although she developed long-standing SRNS without any extrarenal clinical signs thereafter, her renal function was well-preserved over the next 17 years. In total, six sequential renal biopsy specimens revealed histologic alterations ranging from minor glomerular abnormalities to advanced focal segmental glomerulosclerosis (FSGS). A genetic analysis for SRNS performed at 19 years of age revealed a newly identified compound heterozygous mutation in CRB2. Glomerular CRB2 immunoreactivity in biopsy specimens from the patient was scanty, whereas intense expression was observed in those from patients with idiopathic FSGS or in controls. To our knowledge, this is the first report regarding a long-term outcome in a case of SRNS due to an identified CRB2 mutation. Although the phenotype of CRB2 mutation-related syndrome is now expanding, we believe that this case might provide a novel clinicopathologic aspect of this syndrome.

Introduction X-linked Alport syndrome (XLAS) is a hereditary disease characterized by progressive nephritis, hearing loss, and ocular abnormalities. Affected male patients usually progress to end-stage renal disease in early or middle adulthood, and disease severity is strongly correlated with genotype. However, the clinical course in female patients has rarely been reported. Methods We conducted a retrospective analysis of females with genetically proven XLAS (n = 275) and their affected female family members (n = 61) from 179 Japanese families. Patients suspected to have Alport syndrome from pathologic findings or a family history who were referred from anywhere in Japan for genetic diagnosis between 2006–2015 were included in this study. Clinical and laboratory data were collected from medical records at the time of registration for genetic analysis. Results Proteinuria was detected in 175 genetically proven patients (72.6%), and the median age for developing proteinuria was 7.0 years. Fifty-two of 336 patients developed end-stage renal disease with a median renal survival age of 65.0 years. No obvious genotype–phenotype correlation was observed. Additionally, targeted sequencing for podocyte-related genes in patients with severe phenotypes revealed no obvious variants considered to be modifier genes except for 1 patient with a COL4A3 gene variant. Discussion This study revealed that phenotypes in female XLAS patients may be severe, but genotype does not help to predict the disease severity. Clinicians must therefore pay careful attention to the clinical course and appropriate treatment in females with XLAS.

X-linked Alport syndrome (XLAS) is a progressive, hereditary nephropathy. Although males with XLAS usually develop end-stage renal disease before 30 years of age, some men show a milder phenotype and possess somatic mosaic variants of the type IV collagen α5 gene (COL4A5), with severity depending on variant frequencies. In females, somatic mosaic variants are rarely reported in XLAS, and it is not clear what determines severity. Two females with somatic mosaic mutations in COL4A5 with variant frequencies of 17.9 and 22.1% were detected using the next-generation sequencing. One patient only had hematuria. The other, however, had moderate proteinuria, which is a severe phenotype for a female XLAS patient of her age. The molecular mechanisms for the severe phenotype were investigated by examining variant frequencies in urinary sediment cells and X chromosome inactivation patterns, and by looking for modifier variants in podocyte-related genes using the next-generation sequencing. The severe phenotype patient had a variant frequency of 36.6% in urinary sediment cells, which is not markedly high, nor did she show skewed X chromosome inactivation. However, she did have the heterozygous variant in COL4A3, which can affect severity. Factors determining severity in female XLAS patients remain unclear. One studied patient with the somatic variant in COL4A5 showed a severe phenotype without skewed X chromosome inactivation, which might be derived from digenic variants in COL4A3 and COL4A5. Further studies are required to determine molecular mechanisms behind female XLAS resulting in the severe phenotype.

Alport syndrome comprises a group of inherited heterogeneous disorders involving CKD, hearing loss, and ocular abnormalities. Autosomal dominant Alport syndrome caused by heterozygous mutations in collagen 4A3 and/or collagen 4A4 accounts for5% of patients. However, the clinical, genetic, and pathologic backgrounds of patients with autosomal dominant Alport syndrome remain unclear.We conducted a retrospective analysis of 25 patients with genetically proven autosomal dominant Alport syndrome and their family members (a total of 72 patients) from 16 unrelated families. Patients with suspected Alport syndrome after pathologic examination who were referred from anywhere in Japan for genetic analysis from 2006 to 2015 were included in this study. Clinical, laboratory, and pathologic data were collected from medical records at the point of registration for genetic diagnosis. Genetic analysis was performed by targeted resequencing of 27 podocyte-related genes, including Alport-related collagen genes, to make a diagnosis of autosomal dominant Alport syndrome and identify modifier genes or double mutations. Clinical data were obtained from medical records.The median renal survival time was 70 years, and the median age at first detection of proteinuria was 17 years old. There was one patient with hearing loss and one patient with ocular lesion. Among 16 patients who underwent kidney biopsy, three showed FSGS, and seven showed thinning without lamellation of the glomerular basement membrane. Five of 13 detected mutations were reported to be causative mutations for autosomal recessive Alport syndrome in previous studies. Two families possessed double mutations in both collagen 4A3 and collagen 4A4, but no modifier genes were detected among the other podocyte-related genes.The renal phenotype of autosomal dominant Alport syndrome was much milder than that of autosomal recessive Alport syndrome or X-linked Alport syndrome in men. It may, thus, be difficult to make an accurate diagnosis of autosomal dominant Alport syndrome on the basis of clinical or pathologic findings. No modifier genes were identified among the known podocyte-related genes.

Glomerulopathy with fibronectin deposits (GFND) is a rare autosomal dominant disease characterized by massive fibronectin deposits, leading to end-stage renal failure. Although mutations within the heparin-binding domains of the fibronectin 1 gene (FN1) have been associated with GFND, no mutations have been reported within the integrin-binding domains. In this study, FN1 mutational analysis was conducted in 12 families with GFND. Biochemical and functional features of mutated proteins were examined using recombinant fibronectin fragments encompassing both the integrin- and heparin-binding domains. We report six FN1 mutations from 12 families with GFND, including five that are novel (p.Pro969Leu, p.Pro1472del, p.Trp1925Cys, p.Lys1953_Ile1961del, and p.Leu1974Pro). p.Pro1472del is localized in the integrin-binding domain of fibronectin, while the others are in heparin-binding domains. We detected p.Tyr973Cys, p.Pro1472del, and p.Leu1974Pro mutations in multiple families, and haplotype analysis implied that p.Pro1472del and p.Leu1974Pro are founder mutations. The protein encoded by the novel integrin-binding domain mutation p.Pro1472del showed decreased cell binding ability via the integrin-binding site. Most affected patients developed urine abnormalities during the first or second decade of life, and some mutation carriers were completely asymptomatic. This is the second large-scale analysis of GFND families and the first report of an integrin-binding domain mutation. These findings may help determine the pathogenesis of GFND.

Phenotypic overlap exists among type III Bartter syndrome (BS), Gitelman syndrome (GS), and pseudo-BS/GS (p-BS/GS), which are clinically difficult to distinguish. We aimed to clarify the differences between these diseases, allowing accurate diagnosis based on their clinical features.A total of 163 patients with genetically defined type III BS (n = 30), GS (n = 90), and p-BS/GS (n = 43) were included. Age at diagnosis, sex, body mass index, estimated glomerular filtration rate, and serum and urine electrolyte concentrations were determined.Patients with p-BS/GS were significantly older at diagnosis than those with type III BS and GS. Patients with p-BS/GS included a significantly higher percentage of women and had a lower body mass index and estimated glomerular filtration rate than did patients with GS. Although hypomagnesemia and hypocalciuria were predominant biochemical findings in patients with GS, 17 and 23% of patients with type III BS and p-BS/GS, respectively, also showed these abnormalities. Of patients with type III BS, GS, and p-BS/GS, 40, 12, and 63%, respectively, presented with chronic kidney disease.This study clarified the clinical differences between BS, GS, and p-BS/GS for the first time, which will help clinicians establish differential diagnoses for these three conditions.

X-linked Alport syndrome (XLAS) is a progressive hereditary nephropathy caused by mutations in the type IV collagen alpha chain 5 gene (COL4A5). Although many COL4A5 mutations have previously been identified, pathogenic synonymous mutations have not yet been described. A family with XLAS underwent mutational analyses of COL4A5 by PCR and direct sequencing, as well as transcript analysis of potential splice site mutations. In silico analysis was also conducted to predict the disruption of splicing factor binding sites. Immunohistochemistry (IHC) of kidney biopsies was used to detect α2 and α5 chain expression. We identified a hemizygous point mutation, c.876A>T, in exon 15 of COL4A5 in the proband and his brother, which is predicted to result in a synonymous amino acid change, p.(Gly292Gly). Transcript analysis showed that this mutation potentially altered splicing because it disrupted the splicing factor binding site. The kidney biopsy of the proband showed lamellation of the glomerular basement membrane (GBM), while IHC revealed negative α5(IV) staining in the GBM and Bowman’s capsule, which is typical of XLAS. This is the first report of a synonymous COL4A5 substitution being responsible for XLAS. Our findings suggest that transcript analysis should be conducted for the future correct assessment of silent mutations.

The nuclear pore complex (NPC) is a huge protein complex embedded in the nuclear envelope. It has central functions in nucleocytoplasmic transport, nuclear framework, and gene regulation. Nucleoporin 107 kDa (NUP107) is a component of the NPC central scaffold and is an essential protein in all eukaryotic cells. Here, we report on biallelic NUP107 mutations in nine affected individuals who are from five unrelated families and show early-onset steroid-resistant nephrotic syndrome (SRNS). These individuals have pathologically focal segmental glomerulosclerosis, a condition that leads to end-stage renal disease with high frequency. NUP107 is ubiquitously expressed, including in glomerular podocytes. Three of four NUP107 mutations detected in the affected individuals hamper NUP107 binding to NUP133 (nucleoporin 133 kDa) and NUP107 incorporation into NPCs in vitro. Zebrafish with nup107 knockdown generated by morpholino oligonucleotides displayed hypoplastic glomerulus structures and abnormal podocyte foot processes, thereby mimicking the pathological changes seen in the kidneys of the SRNS individuals with NUP107 mutations. Considering the unique properties of the podocyte (highly differentiated foot-process architecture and slit membrane and the inability to regenerate), we propose a “podocyte-injury model” as the pathomechanism for SRNS due to biallelic NUP107 mutations.

Gitelman syndrome (GS) is an autosomal recessive renal tubulopathy characterized by hypokalemic metabolic alkalosis with hypocalciuria and hypomagnesemia. GS clinical symptoms range from mild weakness to muscular cramps, paralysis or even sudden death as a result of cardiac arrhythmia. GS is caused by loss-of-function mutations in the solute carrier family 12 member 3 (SLC12A3) gene, but molecular mechanisms underlying such a wide range of symptoms are poorly understood. Here we report cryptic exon activation in SLC12A3 intron 12 in a clinically asymptomatic GS, resulting from an intronic mutation c.1669+297?T>G that created a new acceptor splice site. The cryptic exon was sandwiched between the L3 transposon upstream and a mammalian interspersed repeat downstream, possibly contributing to inclusion of the cryptic exon in mature transcripts. The mutation was identified by targeted next-generation sequencing of candidate genes in GS patients with missing pathogenic SLC12A3 alleles. Taken together, this work illustrates the power of next-generation sequencing to identify causal mutations in intronic regions in asymptomatic individuals at risk of developing potentially fatal disease complications, improving clinical management of these cases

Recent advances in high-throughput sequencing for clinical genetic testing have revealed novel disease-causing genes, such as Crumbs homolog 2 (CRB2) for early-onset steroid-resistant nephrotic syndrome (SRNS). We report the long-term clinicopathologic observation of a Japanese female patient with SRNS caused by a newly identified compound heterozygous mutation of CRB2 (p.Arg628Cys and p.Gly839Trp located in the 10th and 11th epidermal growth factor-like domains, respectively). She was initially examined during a mass urinary screening for 3.5-year-old children in Japan. Although she developed long-standing SRNS without any extrarenal clinical signs thereafter, her renal function was well-preserved over the next 17 years. In total, six sequential renal biopsy specimens revealed histologic alterations ranging from minor glomerular abnormalities to advanced focal segmental glomerulosclerosis (FSGS). A genetic analysis for SRNS performed at 19 years of age revealed a newly identified compound heterozygous mutation in CRB2. Glomerular CRB2 immunoreactivity in biopsy specimens from the patient was scanty, whereas intense expression was observed in those from patients with idiopathic FSGS or in controls. To our knowledge, this is the first report regarding a long-term outcome in a case of SRNS due to an identified CRB2 mutation. Although the phenotype of CRB2 mutation-related syndrome is now expanding, we believe that this case might provide a novel clinicopathologic aspect of this syndrome.

Background Nephrotic syndrome is the most common cause of chronic glomerular disease in children. Most of these patients develop steroid-sensitive nephrotic syndrome (SSNS), but the loci conferring susceptibility to childhood SSNS are mainly unknown. Methods We conducted a genome-wide association study (GWAS) in the Japanese population; 224 patients with childhood SSNS and 419 adult healthy controls were genotyped using the Affymetrix Japonica Array in the discovery stage. Imputation for six HLA genes (HLA-A, -C, -B, -DRB1, -DQB1, and -DPB1) was conducted on the basis of Japanese-specific references. We performed genotyping for HLA-DRB1/-DQB1 using a sequence-specific oligonucleotide-probing method on a Luminex platform. Whole-genome imputation was conducted using a phased reference panel of 2049 healthy Japanese individuals. Replication was performed in an independent Japanese sample set including 216 patients and 719 healthy controls. We genotyped candidate single-nucleotide polymorphisms using the DigiTag2 assay. Results The most significant association was detected in the HLA-DR/DQ region and replicated (rs4642516 [minor allele G], combined P(allelic)=7.84×10(−23); odds ratio [OR], 0.33; 95% confidence interval [95% CI], 0.26 to 0.41; rs3134996 [minor allele A], combined P(allelic)=1.72×10(−25); OR, 0.29; 95% CI, 0.23 to 0.37). HLA-DRB1*08:02 (Pc=1.82×10(−9); OR, 2.62; 95% CI, 1.94 to 3.54) and HLA-DQB1*06:04 (Pc=2.09×10(−12); OR, 0.10; 95% CI, 0.05 to 0.21) were considered primary HLA alleles associated with childhood SSNS. HLA-DRB1*08:02-DQB1*03:02 (Pc=7.01×10(−11); OR, 3.60; 95% CI, 2.46 to 5.29) was identified as the most significant genetic susceptibility factor. Conclusions The most significant association with childhood SSNS was detected in the HLA-DR/DQ region. Further HLA allele/haplotype analyses should enhance our understanding of molecular mechanisms underlying SSNS.

ETV6–ABL1 fusion is a rare but recurrent oncogenic lesion found in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL), without an established chromosomal abnormality, and is associated with poor outcome. In ETV6–ABL1-positive cases, an in-frame fusion produced by a complex rearrangement results in constitutive chimeric tyrosine kinase activity. Monosomy 7 is also a rare and unfavorable chromosomal abnormality in childhood BCP-ALL. Here, we report a 14-year-old female BCP-ALL patient with ETV6–ABL1 fusion combined with monosomy 7. She was admitted to our hospital because of persistent fever. Bone marrow nuclear cell count on admission was 855,000/µL with 90.0% blastic cells of lymphoid morphology. Blasts were positive for CD10, CD19, CD20, CD34, cyCD79a, cyTdT, HLA-DR, and CD66c, had a karyotype of 45, XX, − 7 [18/20] and a split signal for ABL1 FISH probe (92.7%), and were sensitive to tyrosine kinase inhibitors, imatinib and dasatinib, in vitro. ETV6–ABL1 fusion transcript was identified by whole transcriptome sequencing and confirmed by RT-PCR. She was treated with the high-risk protocol based on ALL-BFM 95, achieved complete remission (CR) after induction chemotherapy, and maintained CR for 4 months. To our knowledge, this is the first report of ETV6–ABL1 fusion combined with monosomy 7 in childhood BCP-ALL.

Background: Spinal muscular atrophy (SMA) is the most common genetic neurological disease leading to infant death. It is caused by loss of survival motor neuron (SMN) 1 gene and subsequent reduction of SMN protein in motor neurons. Because SMN is ubiquitously expressed and functionally linked to general RNA metabolism pathway, fibroblasts are most widely used for the assessment of SMN expression in SMA patients but usually isolated from skin biopsy samples after the onset of overt symptoms. Although recent translational studies of SMN-targeted therapies have revealed the very limited time-window for effective SMA therapies during perinatal period, the exact time-point when SMN shortage became evident is unknown in human samples. In the present study, we analyzed SMN mRNA and protein expression during perinatal period by using umbilical cord-derived mesenchymal stem cells (UC-MSCs) obtained from preterm and term infants. Methods: UC-MSCs were isolated from 16 control infants delivered at 22-40 weeks of gestation and SMA fetus aborted at 19 weeks of gestation (UC-MSC-Control and UC-MSC-SMA). Fibroblasts were isolated from control volunteer and SMA patient (FB-Control and FB-SMA). SMN mRNA and protein expression in UC-MSCs and fibroblasts was determined by RT-qPCR and Western blot. Results: UC-MSC-Control and UC-MSC-SMA expressed the comparable level of MSC markers on their cell surface, and were able to differentiate into adipocytes, osteocytes, and chondrocytes. At steady state, SMN mRNA and protein expression was decreased in UC-MSC-SMA compared to UC-MSC-Control, as observed in FB-SMA and FB-Control. In response to histone deacetylase inhibitor valproic acid, SMN mRNA and protein expression in UC-MSC-SMA as well as FB-SMA was increased. During perinatal development from 22 to 40 weeks of gestation, SMN mRNA and protein expression in UC-MSC-Control was positively correlated with gestational age. Conclusions: UC-MSCs isolated from 17 fetus/infant of 19-40 weeks of gestation are expressed functional SMN mRNA and protein. SMN mRNA and protein expression in UC-MSCs is increased with gestational age during perinatal development.

Introduction: ETV6-ABL1 fusion represents a rare subgroup of pediatric acute lymphoblastic leukemia (ALL) with unfavorable outcomes. ETV6-ABL1-positive ALL is recently identified in Philadelphia chromosome (Ph)-like ALL and exhibits a gene expression profile similar to BCR-ABL1-positive ALL. Analogous to BCR-ABL1 fusion, ETV6-ABL1 fusion results in the formation of constitutively active non-receptor tyrosine kinases that can also be targeted by selective ATP-competitive tyrosine kinase inhibitors (TKIs). Since TKIs are currently incorporated into the standard treatment of BCR-ABL1-positive ALL, they will be a promising option also for the treatment ETV6-ABL1-positive ALL. However, TKI resistance becomes a common problem in TKI-treated patients, where it is frequently caused by BCR-ABL1-dependent alterations including mutations, genomic amplification, and enhanced expression of BCR-ABL1-fusion kinase. In addition, BCR-ABL1-independent alterations have also been reported to cause TKI resistance. It includes a variety of activating and/or inactivating alterations in RAS, NF-kB, PI3K-AKT, and JAK-STAT signaling pathways that mediate the oncogenic activity of BCR-ABL1 fusion kinase. In contrast, the molecular mechanisms of TKI resistance have been poorly described in ETV6-ABL1-positive ALL, except for T315I mutation of ETV6-ABL1 fusion gene in a single patient and K89M mutation of GNB1 gene in a cell line model. Patient and Results: A previously healthy 14-year-old girl was admitted to our hospital because of persistent fever. Laboratory data showed white blood cell count of 417,800 /µL and increased LDH level and uric acid level. Bone marrow examination showed nuclear cell count of 855,000 /µL with 90.0% blastic cells of lymphoid morphology. Bone marrow blasts at initial diagnosis were positive for CD10, CD19, CD20, CD34, cyCD79a, cyTdT, HLA-DR, and CD66c; had a karyotype of 45, XX, -7; and were sensitive to TKIs (imatinib and dasatinib) in vitro. A split signal analyzed by ABL1 FISH probe was positive (92.7%), while major and minor BCR-ABL1 fusion transcripts were not detected by RT-qPCR. She was treated with the high-risk protocol based on BFM 95 protocol because of prednisolone poor response. After induction chemotherapy, she achieved complete remission (CR) without ABL1 split signal and IgH gene rearrangement. However, she relapsed 19 months after initial diagnosis, and failed to achieve second CR by alternating administration of dasatinib and antileukemic drugs. Bone marrow blasts at initial diagnosis and after relapse were subjected to whole-transcriptome sequencing. ETV6-ABL1 fusion transcripts were identified in both initial diagnostic and relapsed samples, and their level of expression was not significantly changed. No known alteration in ETV6-ABL1 fusion and GNB1 genes was detected. These results suggested a novel mechanism of TKI resistance in ETV6-ABL1-positive ALL. Conclusion: To our knowledge, this is a first case of ETV6-ABL1-positive ALL who acquired ETV6-ABL1-independent TKI resistance. It will provide a foundation for the treatment of TKI-resistant ETV6-ABL1-positive ALL. Citation Format: Suguru Uemura, Daiichiro Hasegawa, Akemi Shono, Khin Kyae Mon Thwin, Nanako Nino, Satoru Takafuji, Takeshi Mori, Akihiro Tamura, Nobuyuki Yamamoto, Atsuro Saito, Kenji Kishimoto, Toshiaki Ishida, Yoshiyuki Kosaka, Kazumoto Iijima, Noriyuki Nishimura. A pediatric ETV6-ABL1-positive acute lymphoblastic leukemia case with ETV6-ABL1-independent resistance to tyrosine kinase inhibitor [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A11.

Proper localization of nephrin determines integrity of the glomerular slit diaphragm. Slit diaphragm proteins assemble into functional signaling complexes on a raft-based platform, but how the trafficking of these proteins coordinates with their signaling function is unknown. Here, we demonstrate that a raft-mediated endocytic (RME) pathway internalizes nephrin. Nephrin internalization was slower with raft-mediated endocytosis than with classic clathrin-mediated endocytosis. Ultrastructurally, the RME pathway consisted of noncoated invaginations and was dependent on cholesterol and dynamin. Nephrin constituted a stable, signaling-competent microdomain through interaction with Fyn, a Src kinase, and podocin, a scaffold protein. Tyrosine phosphorylation of nephrin triggered its own RME-mediated internalization. Protamine-induced hyperphosphorylation of nephrin led to noncoated invaginations predominating over coated pits. These results demonstrate that an RME pathway couples nephrin internalization to its own signaling, suggesting that RME promotes proper spatiotemporal assembly of slit diaphragms during podocyte development or injury.

Mesenchymal stem cells (MSCs) are a heterogeneous cell population that is isolated initially from the bone marrow (BM) and subsequently almost all tissues including umbilical cord (UC). UC-derived MSCs (UC-MSCs) have attracted an increasing attention as a source for cell therapy against various degenerative diseases due to their vigorous proliferation and differentiation. Although the cell proliferation and differentiation of BM-derived MSCs is known to decline with age, the functional difference between preterm and term UC-MSCs is poorly characterized. In the present study, we isolated UC-MSCs from 23 infants delivered at 22–40 weeks of gestation and analyzed their gene expression and cell proliferation. Microarray analysis revealed that global gene expression in preterm UC-MSCs was distinct from term UC-MSCs. WNT signaling impacts on a variety of tissue stem cell proliferation and differentiation, and its pathway genes were enriched in differentially expressed genes between preterm and term UC-MSCs. Cell proliferation of preterm UC-MSCs was significantly enhanced compared to term UC-MSCs and counteracted by WNT signaling inhibitor XAV939. Furthermore, WNT2B expression in UC-MSCs showed a significant negative correlation with gestational age (GA). These results suggest that WNT signaling is involved in the regulation of GA-dependent UC-MSC proliferation.

A method for stimulating the differentiation of human pluripotent stem cells into kidney lineages remains to be developed. Most cells in kidney are derived from an embryonic germ layer known as intermediate mesoderm. Here we show the establishment of an efficient system of homologous recombination in human pluripotent stem cells by means of bacterial artificial chromosome-based vectors and single-nucleotide polymorphism array-based detection. This system allowed us to generate human-induced pluripotent stem cell lines containing green fluorescence protein knocked into OSR1, a specific intermediate mesoderm marker. We have also established a robust induction protocol for intermediate mesoderm, which produces up to 90% OSR1(+) cells. These human intermediate mesoderm cells can differentiate into multiple cell types of intermediate mesoderm-derived organs in vitro and in vivo, thereby supplying a useful system to elucidate the mechanisms of intermediate mesoderm development and potentially providing a cell source for regenerative therapies of the kidney.

Minimal-change disease (MCD) is the most common cause of nephrotic syndrome (NS) and is characterized only by minor morphological alterations in podocytes. A subtype of MCD arises from mutations in nephrin, a major component of the slit diaphragm (SD). Idiopathic MCD is a complex trait where interactions of genetic and immunological factors are implicated. However, the pathogenic mechanisms remain unclear. Here we studied the molecular basis for familial NS characterized by frequent relapses and minimal-change histology. Our previous mutational analysis revealed that the two affected children were compound heterozygotes for nephrin variants C265R and V822M (Kidney Int., 2008). When heterologously expressed, these variants exhibited normal metabolic half-life and raft binding. C265R exhibited substantial ER retention, reflecting an intracellular trafficking defect. In contrast, V822M was able to reach the plasma membrane, but was restricted in lateral diffusion as well as trafficking at the cell surface. Clustering of V822M failed to evoke a maximum tyrosine-phosphorylation and actin reorganization, suggesting the inability to assemble into functioning membrane microdomains. Our results suggest that C265R and V822M compose a dysfunctional SD complex due to their mixed defects comprising reduced cell surface targeting and ineffective assembly of signaling microdomains. The defective SD likely confers a susceptibility to immunogenic stimuli and predisposes to a relapsing phenotype.

The predominant type of cellular junction between normal podocyte foot processes is the slit diaphragm. Under nephrotic conditions,however, foot process effacement leads to the loss of slit diaphragms and the new formationof tight junctions composed of the proteins coxsackievirus and adenovirus receptor (CAR) and zonula occludens 1 (ZO-1). Podocin, a protein that plays a key role in maintaining the integrity of the slit diaphragm, has also been localized to these tight junctions, but its function at this site is unknown. In this study, we confirmed that podocin colocalizes with CAR and ZO-1 at the tight junction between foot processes in nephrotic rats. Using primary cultures of rat podocytes, as well as cell lines that co-expressed podocin and CAR, we observed that podocin was recruited to sites of cell-cell contact and that it co-localized with CAR and ZO-1. Immunoprecipitation suggested that these three junctional proteins from a multi-protein complex. Consistent with this, we found that podociin facilitated the coalescence of preassembled lipid rafts containing CAR and restricted their lateral mobility, the latter likely a result of dynamic actin reorganization and subsequent tethering of CAR-podocin complexes to the cytoskeleton. In conclusion, in addition to serving as a structural protein of the slit diaphragm of normal podocytes, our data suggest that podocin may also serve as a scaffold that links tight junction proteins to the actin cytoskeleton in nephrotic foot processes.

Akiko Kitamura, Hiroyasu Tsukaguchi, Kenichi Maruyama, Akemi Shono, Kazumoto Iijima, Shoji Kagami and Toshio Doi Department of Pediatrics, University of Tokushima Graduate School, Tokushima, Japan; Department of Clinical Biology and Medicine, University of Tokushima Graduate School, Tokushima, Japan; Department of Pediatrics, Gunma Children’s Medical Center, Gunma, Japan and Department of Nephrology, National Center for Child Health and Development, Tokyo, Japan