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1.
Kidney Int ; 98(6): 1605-1614, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32712167

RESUMO

Early kidney failure in the hereditary type IV collagen disease, Alport syndrome, can be delayed by renin-angiotensin inhibitors. However, whether all patients and all different genotypes respond equally well to this kidney-protective therapy remains unclear. Here, we performed a retrospective study on 430 patients with male X-linked Alport syndrome to examine the relationships among kidney prognosis, genotype, and treatment effect in a large cohort of Japanese patients. We analyzed the clinical features, genotype-phenotype correlation, and kidney survival period for patients treated with or without renin-angiotensin inhibitors. As a result, the median kidney survival period of patients in this cohort was found to be at 35 years with a strong genotype-phenotype correlation. The median age at the onset of end stage kidney disease (ESKD) significantly differed between patients treated with and without renin-angiotensin inhibitors (over 50 years versus 28 years, respectively). Moreover, these drugs delayed the onset of ESKD in patients with truncating variants for 12 years, extending the median age from 16 years to 28 years. Thus, our results confirmed a strong genotype-phenotype correlation in patients with male X-linked Alport syndrome. Additionally, it was suggested that renin-angiotensin inhibitors could significantly delay ESKD progression. Despite these therapies, patients with truncating variants developed ESKD at the median age of 28 years.


Assuntos
Angiotensinas , Nefrite Hereditária , Preparações Farmacêuticas , Colágeno Tipo IV/genética , Estudos de Associação Genética , Humanos , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Nefrite Hereditária/tratamento farmacológico , Nefrite Hereditária/genética , Estudos Retrospectivos
2.
J Hum Genet ; 64(1): 3-9, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30401917

RESUMO

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.


Assuntos
Mutação , Síndromes Orofaciodigitais/genética , Síndromes Orofaciodigitais/patologia , Proteínas/genética , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Linhagem , Prognóstico
3.
J Am Soc Nephrol ; 29(8): 2244-2254, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29959198

RESUMO

BACKGROUND: X-linked Alport syndrome (XLAS) is a progressive hereditary nephropathy caused by mutations in the COL4A5 gene. Genotype-phenotype correlation in male XLAS is relatively well established; relative to truncating mutations, nontruncating mutations exhibit milder phenotypes. However, transcript comparison between XLAS cases with splicing abnormalities that result in a premature stop codon and those with nontruncating splicing abnormalities has not been reported, mainly because transcript analysis is not routinely conducted in patients with XLAS. METHODS: We examined transcript expression for all patients with suspected splicing abnormalities who were treated at one hospital between January of 2006 and July of 2017. Additionally, we recruited 46 males from 29 families with splicing abnormalities to examine genotype-phenotype correlation in patients with truncating (n=21, from 14 families) and nontruncating (n=25, from 15 families) mutations at the transcript level. RESULTS: We detected 41 XLAS families with abnormal splicing patterns and described novel XLAS atypical splicing patterns (n=14) other than exon skipping caused by point mutations in the splice consensus sequence. The median age for developing ESRD was 20 years (95% confidence interval, 14 to 23 years) among patients with truncating mutations and 29 years (95% confidence interval, 25 to 40 years) among patients with nontruncating mutations (P=0.001). CONCLUSIONS: We report unpredictable atypical splicing in the COL4A5 gene in male patients with XLAS and reveal that renal prognosis differs significantly for patients with truncating versus nontruncating splicing abnormalities. Our results suggest that splicing modulation should be explored as a therapy for XLAS with truncating mutations.


Assuntos
Colágeno Tipo IV/genética , Estudos de Associação Genética/métodos , Predisposição Genética para Doença/epidemiologia , Nefrite Hereditária/genética , Mutação Puntual/genética , Adulto , Estudos de Coortes , Análise Mutacional de DNA , Humanos , Japão , Masculino , Nefrite Hereditária/diagnóstico , Nefrite Hereditária/epidemiologia , Linhagem , Estudos Retrospectivos
4.
J Hum Genet ; 63(5): 589-595, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29459630

RESUMO

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.


Assuntos
Doença de Dent/diagnóstico , Doença de Dent/genética , Sequenciamento de Nucleotídeos em Larga Escala , Inativação do Cromossomo X , Biomarcadores , Biópsia , Canais de Cloreto/genética , Cromossomos Humanos X , Feminino , Perfilação da Expressão Gênica , Variação Genética , Humanos , Leucócitos/metabolismo , Linhagem , Análise de Sequência de DNA , Transcriptoma
5.
Clin Exp Nephrol ; 22(4): 881-888, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29372472

RESUMO

BACKGROUND: Comprehensive genetic approaches for diagnosing inherited kidney diseases using next-generation sequencing (NGS) have recently been established. However, even with these approaches, we are still failing to detect gene defects in some patients who appear to suffer from genetic diseases. One of the reasons for this is the difficulty of detecting copy number variations (CNVs) using our current approaches. For such cases, we can apply methods of array-based comparative genomic hybridization (aCGH) or multiplex ligation and probe amplification (MLPA); however, these are expensive and laborious and also often fail to identify CNVs. Here, we report seven cases with CNVs in various inherited kidney diseases screened by NGS pair analysis. METHODS: Targeted sequencing analysis for causative genes was conducted for cases with suspected inherited kidney diseases, for some of which a definitive genetic diagnosis had not been achieved. We conducted pair analysis using NGS data for those cases. When CNVs were detected by pair analysis, they were confirmed by aCGH and/or MLPA. RESULTS: In seven cases, CNVs in various causative genes of inherited kidney diseases were detected by pair analysis. With aCGH and/or MLPA, pathogenic CNV variants were confirmed: COL4A5 or HNF1B in two cases each, and EYA1, CLCNKB, or PAX2 in one each. CONCLUSION: We presented seven cases with CNVs in various genes that were screened by pair analysis. The NGS-based CNV detection method is useful for comprehensive screening of CNVs, and our results revealed that, for a certain proportion of cases, CNV analysis is necessary for accurate genetic diagnosis.


Assuntos
Variações do Número de Cópias de DNA , Sequenciamento de Nucleotídeos em Larga Escala , Nefropatias/genética , Adulto , Pré-Escolar , Hibridização Genômica Comparativa , Feminino , Humanos , Lactente , Nefropatias/diagnóstico , Masculino , Pessoa de Meia-Idade
6.
J Hum Genet ; 62(2): 335-337, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27784896

RESUMO

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.


Assuntos
Síndrome de Gitelman/diagnóstico , Síndrome de Gitelman/genética , Túbulos Renais Distais/patologia , Sequência de Bases , Pré-Escolar , Éxons/genética , Feminino , Síndrome de Gitelman/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Íntrons/genética , Mutação/genética , Análise de Sequência de DNA , Membro 3 da Família 12 de Carreador de Soluto/genética , Membro 3 da Família 12 de Carreador de Soluto/metabolismo
7.
Clin Exp Nephrol ; 21(5): 877-883, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27796712

RESUMO

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.


Assuntos
Cromossomos Humanos X , Colágeno Tipo IV/genética , Mosaicismo , Mutação , Nefrite Hereditária/genética , Adulto , Criança , Análise Mutacional de DNA , Feminino , Genes Modificadores , Predisposição Genética para Doença , Hematúria/genética , Hereditariedade , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Taxa de Mutação , Nefrite Hereditária/diagnóstico , Linhagem , Fenótipo , Proteinúria/genética , Índice de Gravidade de Doença , Inativação do Cromossomo X
8.
Genet Med ; 18(2): 180-8, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25880437

RESUMO

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.


Assuntos
Síndrome de Bartter/diagnóstico , Síndrome de Gitelman/diagnóstico , Adolescente , Adulto , Síndrome de Bartter/genética , Síndrome de Bartter/fisiopatologia , Pré-Escolar , Cloretos/urina , Análise Mutacional de DNA , Diagnóstico Diferencial , Feminino , Síndrome de Gitelman/genética , Síndrome de Gitelman/fisiopatologia , Humanos , Laxantes/efeitos adversos , Masculino , Sódio/urina
9.
Clin Exp Nephrol ; 20(5): 699-702, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26581810

RESUMO

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.


Assuntos
Colágeno Tipo IV/genética , Nefrite Hereditária/genética , Mutação Puntual , Sítios de Splice de RNA , Biópsia , Cápsula Glomerular/química , Colágeno Tipo IV/metabolismo , Análise Mutacional de DNA , Progressão da Doença , Éxons , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Membrana Basal Glomerular/química , Humanos , Imuno-Histoquímica , Falência Renal Crônica/diagnóstico , Falência Renal Crônica/genética , Falência Renal Crônica/metabolismo , Masculino , Pessoa de Meia-Idade , Nefrite Hereditária/diagnóstico , Nefrite Hereditária/metabolismo , Linhagem , Fenótipo , Adulto Jovem
10.
Clin Nephrol ; 79(6): 484-7, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23725647

RESUMO

Multicystic dysplastic kidney (MCDK) is one of the most common congenital abnormalities of the kidney and urinary tract (CAKUT), although its pathophysiology remains unknown. Familial occurrence of MCDK suggests that mutations in genes associated with nephrogenesis are involved in the pathogenesis in at least some cases. Hepatocyte nuclear factor 1ß (HNF1ß) is a member of the homeodomain-containing super family of transcription factors, and is known to regulate tissue-specific gene expression in a number of organs including the kidneys, pancreas and liver. It has been recently postulated to be associated with CAKUT, including MCDK. We recently encountered a family with a deletion mutant of HNF1ß, of which the 2nd son, the proband, developed bilateral MCDK resulting in renal loss of function in infancy while the 1st son developed unilateral MCDK. Their father has two normal kidneys. This family confirmed that mutations in the HNF1ß gene are strongly associated with the development of MCDK. Furthermore, the fact that no clear phenotype-genotype correlation exists suggests that gene(s) other than HNF1ß are also involved in nephrogenesis and the development of MCDK.


Assuntos
Fator 1-beta Nuclear de Hepatócito/genética , Rim Displásico Multicístico/diagnóstico , Rim Displásico Multicístico/genética , Deleção de Sequência , Irmãos , Adulto , Biomarcadores/metabolismo , Criança , Pai , Humanos , Lactente , Masculino , Rim Displásico Multicístico/diagnóstico por imagem , Fenótipo , Ultrassonografia Pré-Natal
11.
Nat Commun ; 11(1): 2777, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32488001

RESUMO

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.


Assuntos
Colágeno Tipo IV/metabolismo , Éxons/fisiologia , Nefrite Hereditária/metabolismo , Nefrite Hereditária/terapia , Animais , Colágeno Tipo IV/química , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Células HEK293 , Humanos , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Masculino , Camundongos , Modelos Moleculares , Nefrite Hereditária/genética , Nefrite Hereditária/patologia , Insuficiência Renal Crônica
12.
Hum Genet ; 126(4): 533-8, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19513753

RESUMO

Type I Bartter syndrome (BS), an inherited salt-losing tubulopathy, is caused by mutations of the SLC12A1 gene. While several intronic nucleotide changes in this gene have been detected, transcriptional analysis had not been conducted because mRNA analysis is possible only when renal biopsy specimens can be obtained or occasionally when mRNA is expressed in the leukocytes. This report concerns a type I BS patient due to compound heterozygosity for the SLC12A1 gene. Genomic DNA sequencing disclosed the presence of two novel heterozygous mutations of c.724 + 4A > G in intron 5 and c.2095delG in intron 16, but it remains to be determined whether the former would be likely to influence the transcription. In this report, we conducted both in vivo assay of RT-PCR analysis using RNA extracted from the proband's urinary sediments and in vitro functional splicing study by minigene construction, and obtained evidence that this intronic mutation leads to complete exon 5 skipping. To the best of our knowledge, this is the first study to use non-invasive methods for both an in vivo assay and an in vitro functional splicing assay of inherited kidney disease. These analytical assays could be adapted for all inherited kidney diseases.


Assuntos
Síndrome de Bartter/genética , Íntrons/genética , Mutação/genética , Splicing de RNA/genética , Cloreto de Sódio/metabolismo , Simportadores de Cloreto de Sódio-Potássio/genética , Síndrome de Bartter/patologia , Criança , Primers do DNA/química , Primers do DNA/genética , Éxons/genética , Feminino , Humanos , Técnicas In Vitro , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Membro 1 da Família 12 de Carreador de Soluto
13.
Pediatr Res ; 66(5): 590-3, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19668106

RESUMO

Many mutations have been detected in the SLC12A3 gene of Gitelman syndrome (GS, OMIM 263800) patients. In previous studies, only one mutant allele was detected in approximately 20 to 41% of patients with GS; however, the exact reason for the nonidentification has not been established. In this study, we used RT-PCR using mRNA to investigate for the first time transcript abnormalities caused by deep intronic mutation. Direct sequencing analysis of leukocyte DNA identified one base insertion in exon 6 (c.818_819insG), but no mutation was detected in another allele. We analyzed RNA extracted from leukocytes and urine sediments and detected unknown sequence containing 238bp between exons 13 and 14. The genomic DNA analysis of intron 13 revealed a single-base substitution (c.1670-191C>T) that creates a new donor splice site within the intron resulting in the inclusion of a novel cryptic exon in mRNA. This is the first report of creation of a splice site by a deep intronic single-nucleotide change in GS and the first report to detect the onset mechanism in a patient with GS and missing mutation in one allele. This molecular onset mechanism may partly explain the poor success rate of mutation detection in both alleles of patients with GS.


Assuntos
Síndrome de Gitelman/genética , Íntrons , Mutação , Receptores de Droga/genética , Simportadores/genética , Alelos , Sequência de Bases , Criança , Análise Mutacional de DNA , Éxons , Feminino , Humanos , Masculino , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Membro 3 da Família 12 de Carreador de Soluto
14.
Kidney Int Rep ; 4(1): 119-125, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30596175

RESUMO

INTRODUCTION: Gitelman syndrome (GS) is a tubulopathy exhibited by salt loss. GS cases are most often diagnosed by chance blood test. Aside from that, some cases are also diagnosed from tetanic symptoms associated with hypokalemia and/or hypomagnesemia or short stature. As for complications, thyroid dysfunction and short stature are known, but the incidence rates for these complications have not yet been elucidated. In addition, no genotype-phenotype correlation has been identified in GS. METHODS: We examined the clinical characteristics and genotype-phenotype correlation in genetically proven GS cases with homozygous or compound heterozygous variants in SLC12A3 (n = 185). RESULTS: In our cohort, diagnostic opportunities were by chance blood tests (54.7%), tetany (32.6%), or short stature (7.2%). Regarding complications, 16.3% had short stature, 13.7% had experienced febrile convulsion, 4.3% had thyroid dysfunction, and 2.5% were diagnosed with epilepsy. In one case, QT prolongation was detected. Among 29 cases with short stature, 10 were diagnosed with growth hormone (GH) deficiency and GH replacement therapy started. Interestingly, there was a strong correlation in serum magnesium levels between cases with p.Arg642Cys and/or p.Leu858His and cases without these variants, which are mutational hotspots in the Japanese population (1.76 mg/dl vs. 1.43 mg/dl, P < 0.001). CONCLUSION: This study has revealed, for the first time, clinical characteristics in genetically proven GS cases in the Japanese population, including prevalence of complications. Patients with hypokalemia detected by chance blood test should have gene tests performed. Patients with GS need attention for developing extrarenal complications, such as short stature, febrile convulsion, thyroid dysfunction, epilepsy, or QT prolongation. It was also revealed for the first time that hypomagnesemia was not severe in some variants in SLC12A3.

15.
Mol Genet Genomic Med ; 7(9): e883, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31364286

RESUMO

BACKGROUND: Alport syndrome (AS) is a hereditary disease caused by mutations in COL4A3-5 genes. Recently, comprehensive genetic analysis has become the first-line diagnostic tool for AS. However, no reports comparing mutation identification rates between conventional sequencing and comprehensive screening have been published. METHODS: In this study, 441 patients clinically suspected of having AS were divided into two groups and compared. The initial mutational analysis method involved targeted exome sequencing using next-generation sequencing (NGS) (n = 147, NGS group) or Sanger sequencing for COL4A3/COL4A4/COL4A5 (n = 294, Sanger group). RESULTS: In the NGS group, 126 patients (86%) were diagnosed with AS by NGS, while two had pathogenic mutations in other genes, NPHS1 and EYA1. Further, 239 patients (81%) were diagnosed with AS by initial analysis in the Sanger group. Thirteen patients who were negative for mutation detection in the Sanger group were analyzed by NGS; three were diagnosed with AS. Two had mutations in CLCN5 or LAMB2. The final variant detection rate was 90%. DISCUSSION: Our results reveal that Sanger sequencing and targeted exome sequencing have high diagnostic ability. NGS also has the advantage of detecting other inherited kidney diseases and pathogenic mutations missed by Sanger sequencing.


Assuntos
Nefrite Hereditária , Análise de Sequência de DNA , Adulto , Canais de Cloreto/genética , Colágeno/genética , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Laminina/genética , Masculino , Proteínas de Membrana/genética , Nefrite Hereditária/diagnóstico , Nefrite Hereditária/genética , Proteínas Nucleares/genética , Proteínas Tirosina Fosfatases/genética
16.
Nephrol Dial Transplant ; 23(8): 2525-30, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18332068

RESUMO

BACKGROUND: Alport syndrome is the most common form of hereditary nephritis and is mainly caused by mutations in the COL4A5 gene, which shows the X-linked form. It is well known that some male Alport syndrome cases show a relatively mild phenotype, but few molecular investigations have been conducted to clarify the mechanism of this phenotype. Methods and results. This report concerns an 8-year-old male sporadic Alport syndrome patient. While electron microscopy of the glomerular basement membrane showed typical findings for Alport syndrome, however, the immunohistochemical analysis of the glomerulus showed mosaic staining of the type IV collagen alpha 5 chain. The mutational analysis of the COL4A5 gene unexpectedly disclosed two peaks at the intron 43 splicing acceptor site (c. 3998-2 a/t) with direct sequencing. Restriction enzyme analysis demonstrated that the presence of somatic mosaicism was responsible for this mutation. mRNA extracted from the urinary sediments was analysed by RT-PCR and two PCR fragments were amplified, one consisting of a normal sequence and one with skipping of exon 44. CONCLUSIONS: Our findings indicate that somatic mosaicism for COL4A5 is responsible for male X-linked Alport syndrome with an alpha 5 mosaic staining pattern. Several cases with somatic mosaicism have previously been reported, however, this is the first case where the presence of this mutation was proved with a comprehensive analysis of genomic DNA, mRNA and alpha 5 expression in the tissues. Somatic mosaicism may thus be one of the causes of the mild phenotype in Alport syndrome.


Assuntos
Colágeno Tipo IV/genética , Mosaicismo , Nefrite Hereditária/genética , Sequência de Bases , Criança , Cromossomos Humanos X/genética , Colágeno Tipo IV/metabolismo , DNA/genética , Análise Mutacional de DNA , Primers do DNA/genética , Éxons , Membrana Basal Glomerular/metabolismo , Membrana Basal Glomerular/patologia , Hematúria/genética , Humanos , Rim/metabolismo , Rim/patologia , Masculino , Mutação , Nefrite Hereditária/metabolismo , Nefrite Hereditária/patologia , Fenótipo , Proteinúria/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
17.
Nephron ; 138(2): 166-171, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29131116

RESUMO

BACKGROUND/AIMS: Glomerulopathy with fibronectin deposits (GFND; OMIM: 601894) is a very rare inherited kidney disease caused by pathogenic variants in the FN1 gene. Only 9 exonic pathogenic variants in FN1, 9 at the heparin-binding site, and 1 at the integrin-binding site have been reported. No intronic variants in FN1 have been detected. METHODS: We found a pathogenic intronic variant in intron 36 (c.5888-2A>G) located at the heparin-binding site. To determine whether this mutation influences splicing processes, we conducted RT-PCR analysis and an in vitro splicing assay using minigene construction. RESULTS: RT-PCR using RNA extracted from leukocytes of the proband failed because of the low expression of FN1 mRNA in leukocytes. We conducted in vitro functional splicing analysis using minigenes and found that c.5888-2A>G caused a 12 bp deletion at exon 37 by the activation of a novel splicing acceptor site within exon 37. We were able to detect the same abnormal transcript in mRNA extracted from the patient's urinary sediment and confirmed the pathogenicity of c.5888-2A>G by both RT-PCR using the patient sample and an in vitro splicing assay. CONCLUSION: Intronic variants can cause GFND. Minigene analysis is useful for determining the pathogenicity of the intronic variants and could be used for all inherited kidney diseases.


Assuntos
Fibronectinas/metabolismo , Glomerulonefrite Membranosa/genética , Isoformas de Proteínas/genética , Adulto , Sítios de Ligação/genética , Simulação por Computador , Análise Mutacional de DNA , Éxons/genética , Fibronectinas/genética , Glomerulonefrite Membranosa/patologia , Heparina/metabolismo , Humanos , Íntrons , Masculino , Mutação/genética , Reação em Cadeia da Polimerase , RNA/genética
18.
Mol Genet Genomic Med ; 5(3): 287-294, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28546999

RESUMO

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.

19.
Kidney Int Rep ; 2(5): 850-855, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29270492

RESUMO

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.

20.
Clin J Am Soc Nephrol ; 11(8): 1441-1449, 2016 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-27281700

RESUMO

BACKGROUND AND OBJECTIVES: 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 for <5% of patients. However, the clinical, genetic, and pathologic backgrounds of patients with autosomal dominant Alport syndrome remain unclear. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: 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. RESULTS: 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. CONCLUSIONS: 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.


Assuntos
Autoantígenos/genética , Colágeno Tipo IV/genética , Rim/patologia , Nefrite Hereditária/genética , Nefrite Hereditária/patologia , Adolescente , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Biópsia , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Testes Genéticos , Haplótipos , Humanos , Falência Renal Crônica/genética , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Nefrite Hereditária/complicações , Linhagem , Fenótipo , Proteinúria/etiologia , Estudos Retrospectivos , Adulto Jovem
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