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1.
Kidney Int ; 104(2): 367-377, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37230224

RESUMO

X-linked Alport syndrome (XLAS) is an inherited kidney disease caused exclusively by pathogenic variants in the COL4A5 gene. In 10-20% of cases, DNA sequencing of COL4A5 exons or flanking regions cannot identify molecular causes. Here, our objective was to use a transcriptomic approach to identify causative events in a group of 19 patients with XLAS without identified mutation by Alport gene panel sequencing. Bulk RNAseq and/or targeted RNAseq using a capture panel of kidney genes was performed. Alternative splicing events were compared to those of 15 controls by a developed bioinformatic score. When using targeted RNAseq, COL4A5 coverage was found to be 23-fold higher than with bulk RNASeq and revealed 30 significant alternative splicing events in 17 of the 19 patients. After computational scoring, a pathogenic transcript was found in all patients. A causative variant affecting COL4A5 splicing and absent in the general population was identified in all cases. Altogether, we developed a simple and robust method for identification of aberrant transcripts due to pathogenic deep-intronic COL4A5 variants. Thus, these variants, potentially targetable by specific antisense oligonucleotide therapies, were found in a high percentage of patients with XLAS in whom pathogenic variants were missed by conventional DNA sequencing.


Assuntos
Nefrite Hereditária , Humanos , Nefrite Hereditária/diagnóstico , Nefrite Hereditária/genética , Nefrite Hereditária/patologia , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Mutação , Éxons , Splicing de RNA
2.
Clin Genet ; 103(6): 693-698, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36705481

RESUMO

Whole-genome sequencing (WGS) now allows identification of multiple variants in non-coding regions. The large number of variants identified by WGS however complicates their interpretation. Through identification of the first deep intronic variant in NPHS2, which encodes podocin, a protein implicated in autosomal recessive steroid resistant nephrotic syndrome (SRNS), we compare herein three different tools including a newly developed targeted NGS-based RNA-sequencing to explore the splicing effect of intronic variations. WGS identified two different variants in NPHS2 eventually involved in the disease. Through RT-PCR, exon-trapping Minigene assay and targeted RNA sequencing, we were able to identify the splicing defect in NPHS2 mRNA from patient kidney tissue. Only targeted RNA-seq simultaneously analyzed the effect of multiple variants and offered the opportunity to quantify consequences on splicing. Identifying deep intronic variants and their role in disease is of utmost importance. Alternative splicing can be predicted by in silico tools but always requires confirmation through functional testing with RNA analysis from the implicated tissue remaining the gold standard. When several variants with potential effects on splicing are identified by WGS, a targeted RNA sequencing panel could be of great value.


Assuntos
Síndrome Nefrótica , Humanos , Mutação , Sequenciamento Completo do Genoma , Síndrome Nefrótica/genética , RNA Mensageiro/genética
3.
Proc Natl Acad Sci U S A ; 117(26): 15137-15147, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32554502

RESUMO

RNA modifications play a fundamental role in cellular function. Pseudouridylation, the most abundant RNA modification, is catalyzed by the H/ACA small ribonucleoprotein (snoRNP) complex that shares four core proteins, dyskerin (DKC1), NOP10, NHP2, and GAR1. Mutations in DKC1, NOP10, or NHP2 cause dyskeratosis congenita (DC), a disorder characterized by telomere attrition. Here, we report a phenotype comprising nephrotic syndrome, cataracts, sensorineural deafness, enterocolitis, and early lethality in two pedigrees: males with DKC1 p.Glu206Lys and two children with homozygous NOP10 p.Thr16Met. Females with heterozygous DKC1 p.Glu206Lys developed cataracts and sensorineural deafness, but nephrotic syndrome in only one case of skewed X-inactivation. We found telomere attrition in both pedigrees, but no mucocutaneous abnormalities suggestive of DC. Both mutations fall at the dyskerin-NOP10 binding interface in a region distinct from those implicated in DC, impair the dyskerin-NOP10 interaction, and disrupt the catalytic pseudouridylation site. Accordingly, we found reduced pseudouridine levels in the ribosomal RNA (rRNA) of the patients. Zebrafish dkc1 mutants recapitulate the human phenotype and show reduced 18S pseudouridylation, ribosomal dysregulation, and a cell-cycle defect in the absence of telomere attrition. We therefore propose that this human disorder is the consequence of defective snoRNP pseudouridylation and ribosomal dysfunction.


Assuntos
Catarata/genética , Proteínas de Ciclo Celular/genética , Enterocolite/genética , Perda Auditiva Neurossensorial/genética , Síndrome Nefrótica/genética , Proteínas Nucleares/genética , Ribonucleoproteínas Nucleolares Pequenas/genética , Animais , Criança , Feminino , Predisposição Genética para Doença , Humanos , Longevidade , Masculino , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Linhagem , Conformação Proteica , RNA Ribossômico/genética , Peixe-Zebra
4.
Am J Hum Genet ; 104(2): 348-355, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30661770

RESUMO

Steroid-resistant nephrotic syndrome (SRNS) is characterized by high-range proteinuria and most often focal and segmental glomerulosclerosis (FSGS). Identification of mutations in genes causing SRNS has improved our understanding of disease mechanisms and highlighted defects in the podocyte, a highly specialized glomerular epithelial cell, as major factors in disease pathogenesis. By exome sequencing, we identified missense mutations in TBC1D8B in two families with an X-linked early-onset SRNS with FSGS. TBC1D8B is an uncharacterized Rab-GTPase-activating protein likely involved in endocytic and recycling pathways. Immunofluorescence studies revealed TBC1D8B presence in human glomeruli, and affected individual podocytes displayed architectural changes associated with migration defects commonly found in FSGS. In zebrafish we demonstrated that both knockdown and knockout of the unique TBC1D8B ortholog-induced proteinuria and that this phenotype was rescued by human TBC1D8B mRNA injection, but not by either of the two mutated mRNAs. We also showed an interaction between TBC1D8B and Rab11b, a key protein in vesicular recycling in cells. Interestingly, both internalization and recycling processes were dramatically decreased in affected individuals' podocytes and fibroblasts, confirming the crucial role of TBC1D8B in the cellular recycling processes, probably as a Rab11b GTPase-activating protein. Altogether, these results confirmed that pathogenic variations in TBC1D8B are involved in X-linked podocytopathy and points to alterations in recycling processes as a mechanism of SRNS.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Mutação com Perda de Função , Síndrome Nefrótica/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Peixe-Zebra/genética , Animais , Transporte Biológico/genética , Proteínas de Ligação ao Cálcio/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Glomérulos Renais/metabolismo , Masculino , Podócitos/citologia , Podócitos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Sequenciamento do Exoma , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
6.
PLoS Genet ; 14(5): e1007386, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29768408

RESUMO

Recent evidence suggests that the presence of more than one pathogenic mutation in a single patient is more common than previously anticipated. One of the challenges hereby is to dissect the contribution of each gene mutation, for which animal models such as Drosophila can provide a valuable aid. Here, we identified three families with mutations in ADD3, encoding for adducin-γ, with intellectual disability, microcephaly, cataracts and skeletal defects. In one of the families with additional cardiomyopathy and steroid-resistant nephrotic syndrome (SRNS), we found a homozygous variant in KAT2B, encoding the lysine acetyltransferase 2B, with impact on KAT2B protein levels in patient fibroblasts, suggesting that this second mutation might contribute to the increased disease spectrum. In order to define the contribution of ADD3 and KAT2B mutations for the patient phenotype, we performed functional experiments in the Drosophila model. We found that both mutations were unable to fully rescue the viability of the respective null mutants of the Drosophila homologs, hts and Gcn5, suggesting that they are indeed pathogenic in flies. While the KAT2B/Gcn5 mutation additionally showed a significantly reduced ability to rescue morphological and functional defects of cardiomyocytes and nephrocytes (podocyte-like cells), this was not the case for the ADD3 mutant rescue. Yet, the simultaneous knockdown of KAT2B and ADD3 synergistically impaired kidney and heart function in flies as well as the adhesion and migration capacity of cultured human podocytes, indicating that mutations in both genes may be required for the full clinical manifestation. Altogether, our studies describe the expansion of the phenotypic spectrum in ADD3 deficiency associated with a homozygous likely pathogenic KAT2B variant and thereby identify KAT2B as a susceptibility gene for kidney and heart disease in ADD3-associated disorders.


Assuntos
Proteínas de Ligação a Calmodulina/genética , Drosophila/genética , Mutação , Fatores de Transcrição de p300-CBP/genética , Anormalidades Múltiplas/genética , Adolescente , Adulto , Animais , Proteínas de Ligação a Calmodulina/deficiência , Linhagem Celular , Células Cultivadas , Análise Mutacional de DNA , Proteínas de Drosophila/genética , Feminino , Cardiopatias/genética , Homozigoto , Humanos , Falência Renal Crônica/genética , Masculino , Linhagem , Fenótipo
7.
Nephrol Dial Transplant ; 34(11): 1885-1893, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-29992269

RESUMO

BACKGROUND: Apolipoprotein L1 (APOL1) risk variants are strongly associated with sporadic focal segmental glomerulosclerosis (FSGS) in populations with African ancestry. We determined the frequency of G1/G2 variants in steroid-resistant nephrotic syndrome (SRNS)/FSGS patients with African or French West Indies ancestry in France and its relationships with other SRNS genes. METHODS: In a cohort of 152 patients (139 families), the APOL1 risk variants were genotyped by direct Sanger sequencing and pathogenic mutations were screened by next-generation sequencing with a panel including 35 SRNS genes. RESULTS: The two risk allele [high-risk (HR)] genotypes were found in 43.1% (66/152) of subjects compared with 18.9% (106/562) in a control population (P < 0.0001): 33 patients homozygous for APOL1 G1 alleles, 4 homozygous for G2 and 29 compound heterozygous for G1 and G2. Compared with patients in the low-risk (LR) group, patients in the HR group were more likely to originate from the French West Indies than from Africa [45/66 (68.2%) versus 30/86 (34.9%); P < 0.0001]. There were more familial cases in the HR group [27 (41.5%) versus 8 (11.4%); P < 0.0001]. However, causative mutations in monogenic SRNS genes were found in only 1 patient in the HR group compared with 16 patients (14 families) in the LR group (P = 0.0006). At diagnosis, patients in the HR group without other mutations were more often adults [35 (53.8%) versus 19 (27.1%); P = 0.003] and had a lower estimated glomerular filtration rate (78.9 versus 98.8 mL/min/1.73 m2; P = 0.02). CONCLUSIONS: The HR genotype is frequent in FSGS patients with African ancestry in our cohort, especially in those originating from the West Indies, and confer a poor renal prognosis. It is usually not associated with other causative mutations in monogenic SRNS genes.


Assuntos
Apolipoproteína L1/genética , População Negra/genética , Resistência a Medicamentos , Predisposição Genética para Doença , Glomerulosclerose Segmentar e Focal/genética , Mutação , Síndrome Nefrótica/genética , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Estudos de Coortes , Feminino , França/epidemiologia , Genótipo , Glomerulosclerose Segmentar e Focal/diagnóstico , Glomerulosclerose Segmentar e Focal/etnologia , Homozigoto , Humanos , Masculino , Síndrome Nefrótica/diagnóstico , Síndrome Nefrótica/etnologia , Linhagem , Prognóstico , Fatores de Risco , Esteroides/farmacologia , Taxa de Sobrevida , Adulto Jovem
8.
Nephrol Dial Transplant ; 34(3): 458-467, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29474669

RESUMO

BACKGROUND: Recommendations for management of Finnish-type congenital nephrotic syndrome (CNS) followed by many teams include daily albumin infusions, early bilateral nephrectomy, dialysis and transplantation. We aimed to assess the treatment and outcome of patients with CNS in France. METHODS: We conducted a nationwide retrospective study on 55 consecutive children born between 2000 and 2014 treated for non-infectious CNS. RESULTS: The estimated cumulative incidence of CNS was 0.5/100 000 live births. The underlying defect was biallelic mutations in NPHS1 (36/55, 65%), NPHS2 (5/55, 7%), PLCE1 (1/55, 2%), heterozygous mutation in WT1 (4/55, 7%) and not identified in nine children (16%). Fifty-three patients (96%) received daily albumin infusions from diagnosis (median age 14 days), which were spaced and withdrawn in 10 patients. Twenty children (35%) were managed as outpatients. Thirty-nine patients reached end-stage kidney disease (ESKD) at a median age of 11 months. The overall renal survival was 64% and 45% at 1 and 2 years of age, respectively. Thirteen children died during the study period including four at diagnosis, two of nosocomial catheter-related septic shock, six on dialysis and one after transplantation. The remaining 13 patients were alive with normal renal function at last follow-up [median 32 months (range 9-52)]. Renal and patient survivals were longer in patients with NPHS1 mutations than in other patients. The invasive infection rate was 2.41/patient/year. CONCLUSIONS: Our study shows: (i) a survival free from ESKD in two-thirds of patients at 1 year and in one-half at 2 years and (ii) a significant reduction or even a discontinuation of albumin infusions allowing ambulatory care in a subset of patients. These results highlight the need for new therapeutic guidelines for CNS patients.


Assuntos
Proteínas de Membrana/genética , Mutação , Nefrectomia/mortalidade , Síndrome Nefrótica/mortalidade , Progressão da Doença , Feminino , França/epidemiologia , Humanos , Incidência , Lactente , Recém-Nascido , Masculino , Síndrome Nefrótica/epidemiologia , Síndrome Nefrótica/genética , Síndrome Nefrótica/terapia , Estudos Retrospectivos , Taxa de Sobrevida , Resultado do Tratamento
9.
Kidney Int ; 94(5): 1013-1022, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30348286

RESUMO

Monogenic forms of Steroid-Resistant Nephrotic Syndrome (SRNS) have been widely characterized, but genetic screening paradigms preferentially address congenital, infantile onset, and familial cases. Our aim was to characterize the distribution of disease-causing gene mutations in adults with sporadic SRNS or focal segmental glomerulosclerosis (FSGS). We selected adult patients with non-syndromic, biopsy-proven FSGS or SRNS in the absence of known family history. Strict clinical criteria included lack of response to glucocorticoids and cyclosporine, and no recurrence after kidney transplantation. Mutations in SRNS genes were detected using a targeted gene panel. Sixteen of 135 tested participants (11.8%) carried pathogenic mutations in monogenic SRNS genes, and 14 others (10.4%) carried two APOL1 high-risk alleles. Autosomal recessive disease was diagnosed in 5 participants, autosomal dominant disease in 9, and X-linked disease in 2. Four participants carried a de novo heterozygous mutation. Among the 16 participants with identified mutations in monogenic SNRS genes, 7 (43.7%) had type IV collagen mutations. Mutations in monogenic SNRS genes were identified primarily in participants with proteinuria onset before 25 years of age, while the age at disease onset was variable in those with APOL1 high-risk genotype. Mean age at diagnosis was lower and renal survival was worse in participants with identified mutations in SNRS genes than in those without mutations. We found a significant rate of pathogenic mutations in adults with SRNS, with Type IV collagen mutations being the most frequent. These findings may have immediate impact on clinical practice.


Assuntos
Glucocorticoides/uso terapêutico , Mutação , Síndrome Nefrótica/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Apolipoproteína L1/genética , Autoantígenos/genética , Colágeno Tipo IV/genética , Proteínas do Citoesqueleto/genética , Resistência a Medicamentos , Feminino , Humanos , Rim/patologia , Masculino , Pessoa de Meia-Idade , Síndrome Nefrótica/tratamento farmacológico , Adulto Jovem
10.
Pediatr Nephrol ; 33(3): 473-483, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29058154

RESUMO

BACKGROUND: Familial steroid-sensitive nephrotic syndrome (SSNS) is a rare condition. The disease pathophysiology remains elusive. However, bi-allelic mutations in the EMP2 gene were identified, and specific variations in HLA-DQA1 were linked to a high risk of developing the disease. METHODS: Clinical data were analyzed in 59 SSNS families. EMP2 gene was sequenced in families with a potential autosomal recessive (AR) inheritance. Exome sequencing was performed in a subset of 13 families with potential AR inheritance. Two variations in HLA-DQA1 were genotyped in the whole cohort. RESULTS: Transmission was compatible with an AR (n = 33) or autosomal dominant (AD, n = 26) inheritance, assuming that familial SSNS is a monogenic trait. Clinical features did not differ between AR and AD groups. All patients, including primary (n = 7) and secondary steroid resistant nephrotic syndrone (SRNS), (n = 13) were sensitive to additional immunosuppressive therapy. Both HLA-DQA1 variations were found to be highly linked to the disease (OR = 4.34 and OR = 4.89; p < 0.001). Exome sequencing did not reveal any pathogenic mutation, neither did EMP2 sequencing. CONCLUSIONS: Taken together, these results highlight the clinical and genetic heterogeneity in familial SSNS. Clinical findings sustain an immune origin in all patients, whatever the initial steroid-sensitivity. The absence of a variant shared by two families and the HLA-DQA1 variation enrichments suggest a complex mode of inheritance.


Assuntos
Glucocorticoides/uso terapêutico , Cadeias alfa de HLA-DQ/genética , Glicoproteínas de Membrana/genética , Síndrome Nefrótica/genética , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Heterogeneidade Genética , Predisposição Genética para Doença , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Mutação , Síndrome Nefrótica/tratamento farmacológico , Análise de Sequência de DNA/métodos , Adulto Jovem
11.
Am J Hum Genet ; 94(2): 288-94, 2014 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-24439109

RESUMO

Renal hypodysplasia (RHD) is a heterogeneous condition encompassing a spectrum of kidney development defects including renal agenesis, hypoplasia, and (cystic) dysplasia. Heterozygous mutations of several genes have been identified as genetic causes of RHD with various severity. However, these genes and mutations are not associated with bilateral renal agenesis, except for RET mutations, which could be involved in a few cases. The pathophysiological mechanisms leading to total absence of kidney development thus remain largely elusive. By using a whole-exome sequencing approach in families with several fetuses with bilateral renal agenesis, we identified recessive mutations in the integrin α8-encoding gene ITGA8 in two families. Itga8 homozygous knockout in mice is known to result in absence of kidney development. We provide evidence of a damaging effect of the human ITGA8 mutations. These results demonstrate that mutations of ITGA8 are a genetic cause of bilateral renal agenesis and that, at least in some cases, bilateral renal agenesis is an autosomal-recessive disease.


Assuntos
Anormalidades Congênitas/genética , Genes Recessivos , Cadeias alfa de Integrinas/genética , Nefropatias/congênito , Rim/anormalidades , Anormalidades Urogenitais/genética , Anormalidades Congênitas/patologia , Feminino , Feto/anormalidades , Homozigoto , Humanos , Cadeias alfa de Integrinas/metabolismo , Rim/patologia , Nefropatias/genética , Nefropatias/patologia , Masculino , Mutação , Linhagem , Anormalidades Urogenitais/patologia
12.
Am J Hum Genet ; 95(6): 637-48, 2014 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-25466283

RESUMO

Galloway-Mowat syndrome is a rare autosomal-recessive condition characterized by nephrotic syndrome associated with microcephaly and neurological impairment. Through a combination of autozygosity mapping and whole-exome sequencing, we identified WDR73 as a gene in which mutations cause Galloway-Mowat syndrome in two unrelated families. WDR73 encodes a WD40-repeat-containing protein of unknown function. Here, we show that WDR73 was present in the brain and kidney and was located diffusely in the cytoplasm during interphase but relocalized to spindle poles and astral microtubules during mitosis. Fibroblasts from one affected child and WDR73-depleted podocytes displayed abnormal nuclear morphology, low cell viability, and alterations of the microtubule network. These data suggest that WDR73 plays a crucial role in the maintenance of cell architecture and cell survival. Altogether, WDR73 mutations cause Galloway-Mowat syndrome in a particular subset of individuals presenting with late-onset nephrotic syndrome, postnatal microcephaly, severe intellectual disability, and homogenous brain MRI features. WDR73 is another example of a gene involved in a disease affecting both the kidney glomerulus and the CNS.


Assuntos
Hérnia Hiatal/genética , Deficiência Intelectual/genética , Microcefalia/genética , Nefrose/genética , Síndrome Nefrótica/genética , Proteínas/genética , Adolescente , Encéfalo/fisiopatologia , Linhagem Celular , Sobrevivência Celular , Criança , Pré-Escolar , Citosol/metabolismo , Exoma/genética , Hérnia Hiatal/fisiopatologia , Homozigoto , Humanos , Glomérulos Renais/fisiopatologia , Masculino , Microcefalia/fisiopatologia , Microtúbulos/metabolismo , Mitose , Modelos Moleculares , Mutação , Nefrose/fisiopatologia , Síndrome Nefrótica/fisiopatologia , Podócitos , Transporte Proteico , Proteínas/metabolismo , Polos do Fuso/metabolismo
13.
J Am Soc Nephrol ; 27(1): 63-8, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25967120

RESUMO

Hereditary defects of coenzyme Q10 biosynthesis cause steroid-resistant nephrotic syndrome (SRNS) as part of multiorgan involvement but may also contribute to isolated SRNS. Here, we report 26 patients from 12 families with recessive mutations in ADCK4. Mutation detection rate was 1.9% among 534 consecutively screened cases. Patients with ADCK4 mutations showed a largely renal-limited phenotype, with three subjects exhibiting occasional seizures, one subject exhibiting mild mental retardation, and one subject exhibiting retinitis pigmentosa. ADCK4 nephropathy presented during adolescence (median age, 14.1 years) with nephrotic-range proteinuria in 44% of patients and advanced CKD in 46% of patients at time of diagnosis. Renal biopsy specimens uniformly showed FSGS. Whereas 47% and 36% of patients with mutations in WT1 and NPHS2, respectively, progressed to ESRD before 10 years of age, ESRD occurred almost exclusively in the second decade of life in ADCK4 nephropathy. However, CKD progressed much faster during adolescence in ADCK4 than in WT1 and NPHS2 nephropathy, resulting in similar cumulative ESRD rates (>85% for each disorder) in the third decade of life. In conclusion, ADCK4-related glomerulopathy is an important novel differential diagnosis in adolescents with SRNS/FSGS and/or CKD of unknown origin.


Assuntos
Glomerulosclerose Segmentar e Focal/genética , Mutação , Proteínas Quinases/genética , Adolescente , Idade de Início , Criança , Pré-Escolar , Humanos , Lactente
14.
Hum Mol Genet ; 23(6): 1479-91, 2014 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-24163131

RESUMO

Renal tubular dysgenesis (RTD) is a recessive autosomal disease characterized most often by perinatal death. It is due to the inactivation of any of the major genes of the renin-angiotensin system (RAS), one of which is the angiotensin I-converting enzyme (ACE). ACE is present as a tissue-bound enzyme and circulates in plasma after its solubilization. In this report, we present the effect of different ACE mutations associated with RTD on ACE intracellular trafficking, secretion and enzymatic activity. One truncated mutant, R762X, responsible for neonatal death was found to be an enzymatically active, secreted form, not inserted in the plasma membrane. In contrast, another mutant, R1180P, was compatible with life after transient neonatal renal insufficiency. This mutant was located at the plasma membrane and rapidly secreted. These results highlight the importance of tissue-bound ACE versus circulating ACE and show that the total absence of cell surface expression of ACE is incompatible with life. In addition, two missense mutants (W594R and R828H) and two truncated mutants (Q1136X and G1145AX) were also studied. These mutants were neither inserted in the plasma membrane nor secreted. Finally, the structural implications of these ACE mutations were examined by molecular modelling, which suggested some important structural alterations such as disruption of intra-molecular non-covalent interactions (e.g. salt bridges).


Assuntos
Morte Fetal/genética , Túbulos Renais Proximais/anormalidades , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Anormalidades Urogenitais/genética , Animais , Células CHO , Cricetulus , Cristalografia por Raios X , Feminino , Células HEK293 , Humanos , Recém-Nascido , Masculino , Modelos Moleculares , Mutação de Sentido Incorreto , Peptidil Dipeptidase A/sangue , Peptidil Dipeptidase A/química , Conformação Proteica , Estrutura Secundária de Proteína , Transporte Proteico
15.
J Am Soc Nephrol ; 25(2): 407-19, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24309185

RESUMO

Since the recent publication of data showing favorable outcomes for patients with HIV-1 and ESRD, kidney transplantation has become a therapeutic option in this population. However, reports have documented unexplained reduced allograft survival in these patients. We hypothesized that the unrecognized infection of the transplanted kidney by HIV-1 can compromise long-term allograft function. Using electron microscopy and molecular biology, we examined protocol renal transplant biopsies from 19 recipients with HIV-1 who did not have detectable levels of plasma HIV-1 RNA at transplantation. We found that HIV-1 infected the kidney allograft in 68% of these patients. Notably, HIV-1 infection was detected in either podocytes predominately (38% of recipients) or tubular cells only (62% of recipients). Podocyte infection associated with podocyte apoptosis and loss of differentiation markers as well as a faster decline in allograft function compared with tubular cell infection. In allografts with tubular cell infection, epithelial cells of the proximal convoluted tubules frequently contained abnormal mitochondria, and both patients who developed features of subclinical acute cellular rejection had allografts with tubular cell infection. Finally, we provide a novel noninvasive test for determining HIV-1 infection of the kidney allograft by measuring HIV-1 DNA and RNA levels in patients' urine. In conclusion, HIV-1 can infect kidney allografts after transplantation despite undetectable viremia, and this infection might influence graft outcome.


Assuntos
Infecções por HIV/virologia , HIV-1/isolamento & purificação , Falência Renal Crônica/cirurgia , Transplante de Rim , Rim/virologia , Transplantes/virologia , Adulto , Aloenxertos , Apoptose , Biópsia , DNA Viral/urina , Feminino , Sobrevivência de Enxerto , Infecções por HIV/complicações , Infecções por HIV/urina , Hepatite C Crônica/complicações , Humanos , Hibridização In Situ , Rim/patologia , Falência Renal Crônica/complicações , Túbulos Renais/virologia , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Podócitos/virologia , Reação em Cadeia da Polimerase , Proteinúria/etiologia , RNA Viral/urina , Transplantes/patologia , Carga Viral
16.
J Am Soc Nephrol ; 25(11): 2435-43, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24876116

RESUMO

Several genes, mainly involved in podocyte cytoskeleton regulation, have been implicated in familial forms of primary FSGS. We identified a homozygous missense mutation (p.P209L) in the TTC21B gene in seven families with FSGS. Mutations in this ciliary gene were previously reported to cause nephronophthisis, a chronic tubulointerstitial nephropathy. Notably, tubular basement membrane thickening reminiscent of that observed in nephronophthisis was present in patients with FSGS and the p.P209L mutation. We demonstrated that the TTC21B gene product IFT139, an intraflagellar transport-A component, mainly localizes at the base of the primary cilium in developing podocytes from human fetal tissue and in undifferentiated cultured podocytes. In contrast, in nonciliated adult podocytes and differentiated cultured cells, IFT139 relocalized along the extended microtubule network. We further showed that knockdown of IFT139 in podocytes leads to primary cilia defects, abnormal cell migration, and cytoskeleton alterations, which can be partially rescued by p.P209L overexpression, indicating its hypomorphic effect. Our results demonstrate the involvement of a ciliary gene in a glomerular disorder and point to a critical function of IFT139 in podocytes. Altogether, these data suggest that this homozygous TTC21B p.P209L mutation leads to a novel hereditary kidney disorder with both glomerular and tubulointerstitial damages.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Cílios/fisiologia , Glomerulosclerose Segmentar e Focal/genética , Proteínas Associadas aos Microtúbulos/genética , Podócitos/fisiologia , Adolescente , Adulto , Animais , Linhagem Celular Transformada , Criança , Cílios/patologia , Saúde da Família , Feminino , Glomerulosclerose Segmentar e Focal/patologia , Haplótipos , Homozigoto , Humanos , Masculino , Camundongos , Mutação de Sentido Incorreto , Linhagem , Fenótipo , Podócitos/patologia , Fibras de Estresse/patologia , Fibras de Estresse/fisiologia , Adulto Jovem
17.
Hum Mutat ; 35(2): 178-86, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24227627

RESUMO

Mutations in the NPHS2 gene encoding podocin are implicated in an autosomal-recessive form of nonsyndromic steroid-resistant nephrotic syndrome in both pediatric and adult patients. Patients with homozygous or compound heterozygous mutations commonly present with steroid-resistant nephrotic syndrome before the age of 6 years and rapidly progress to end-stage kidney disease with a very low prevalence of recurrence after renal transplantation. Here, we reviewed all the NPHS2 mutations published between October 1999 and September 2013, and also all novel mutations identified in our personal cohort and in international genetic laboratories. We identified 25 novel pathogenic mutations in addition to the 101 already described. The mutations are distributed along the entire coding region and lead to all kinds of alterations including 53 missense, 17 nonsense, 11 small insertions, 26 small deletions, 16 splicing, two indel mutations, and one mutation in the stop codon. In addition, 43 variants were classified as variants of unknown significance, as these missense changes were exclusively described in the heterozygous state and/or considered benign by prediction software. Genotype-phenotype analyses established correlations between specific variants and age at onset, ethnicity, or clinical evolution. We created a Web database using the Leiden Open Variation Database (www.lovd.nl/NPHS2) software that will allow the inclusion of future reports.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Mutação , Síndrome Nefrótica/congênito , Adulto , Idade de Início , Animais , Pré-Escolar , Modelos Animais de Doenças , Variação Genética , Genótipo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Síndrome Nefrótica/genética , Síndrome Nefrótica/patologia , Fenótipo , Polimorfismo de Nucleotídeo Único , Software
18.
N Engl J Med ; 365(25): 2377-88, 2011 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-22187985

RESUMO

BACKGROUND: Charcot-Marie-Tooth neuropathy has been reported to be associated with renal diseases, mostly focal segmental glomerulosclerosis (FSGS). However, the common mechanisms underlying the neuropathy and FSGS remain unknown. Mutations in INF2 were recently identified in patients with autosomal dominant FSGS. INF2 encodes a formin protein that interacts with the Rho-GTPase CDC42 and myelin and lymphocyte protein (MAL) that are implicated in essential steps of myelination and myelin maintenance. We therefore hypothesized that INF2 may be responsible for cases of Charcot-Marie-Tooth neuropathy associated with FSGS. METHODS: We performed direct genotyping of INF2 in 16 index patients with Charcot-Marie-Tooth neuropathy and FSGS who did not have a mutation in PMP22 or MPZ, encoding peripheral myelin protein 22 and myelin protein zero, respectively. Histologic and functional studies were also conducted. RESULTS: We identified nine new heterozygous mutations in 12 of the 16 index patients (75%), all located in exons 2 and 3, encoding the diaphanous-inhibitory domain of INF2. Patients presented with an intermediate form of Charcot-Marie-Tooth neuropathy as well as a glomerulopathy with FSGS on kidney biopsy. Immunohistochemical analysis revealed strong INF2 expression in Schwann-cell cytoplasm and podocytes. Moreover, we demonstrated that INF2 colocalizes and interacts with MAL in Schwann cells. The INF2 mutants perturbed the INF2-MAL-CDC42 pathway, resulting in cytoskeleton disorganization, enhanced INF2 binding to CDC42 and mislocalization of INF2, MAL, and CDC42. CONCLUSIONS: INF2 mutations appear to cause many cases of FSGS-associated Charcot-Marie-Tooth neuropathy, showing that INF2 is involved in a disease affecting both the kidney glomerulus and the peripheral nervous system. These findings provide new insights into the pathophysiological mechanisms linking formin proteins to podocyte and Schwann-cell function. (Funded by the Agence Nationale de la Recherche and others.).


Assuntos
Doença de Charcot-Marie-Tooth/genética , Glomerulosclerose Segmentar e Focal/etiologia , Rim/metabolismo , Proteínas dos Microfilamentos/genética , Células de Schwann/metabolismo , Actinas/metabolismo , Adolescente , Adulto , Idade de Início , Animais , Doença de Charcot-Marie-Tooth/complicações , Criança , Feminino , Forminas , Heterozigoto , Humanos , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Mutação , Proteínas da Mielina/metabolismo , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina , Fenótipo , Proteolipídeos/metabolismo , Adulto Jovem
19.
Am J Kidney Dis ; 63(5): 816-9, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24518129

RESUMO

We report an occurrence of progressive loss of transplant function and ultimately transplant failure after living related kidney transplantation involving monozygotic twin brothers of Afro-Caribbean origin who were both heterozygous for the G1 and G2 kidney disease risk alleles in the APOL1 gene, which encodes apolipoprotein L-I. A 21-year-old man with end-stage kidney disease of unknown cause received a kidney from his brother, who was confirmed as a monozygotic twin by microsatellite analysis. Thirty months after transplantation, the patient presented with proteinuria and decreased estimated glomerular filtration rate; a biopsy of the transplant showed typical focal segmental glomerulosclerosis lesions. He received steroid therapy, but progressed to kidney failure 5 years later. The twin brother had normal kidney function without proteinuria at the time of transplantation; however, 7 years later, he was found to have decreased estimated glomerular filtration rate (40mL/min/1.73m(2)) and proteinuria (protein excretion of 2.5g/d). APOL1 genotyping revealed that both donor and recipient were heterozygous for the G1 and G2 alleles. This case is in stark contrast to the expected course of kidney transplantation in identical twins and suggests a role for APOL1 polymorphisms in both the donor and recipient.


Assuntos
Apolipoproteínas/genética , Doenças em Gêmeos , Transplante de Rim/efeitos adversos , Lipoproteínas HDL/genética , Doadores Vivos , Polimorfismo Genético , Insuficiência Renal Crônica/genética , Gêmeos Monozigóticos/genética , Apolipoproteína L1 , Apolipoproteínas/metabolismo , Biópsia , Seguimentos , Predisposição Genética para Doença , Genótipo , Humanos , Rim/patologia , Lipoproteínas HDL/metabolismo , Masculino , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/cirurgia , Adulto Jovem
20.
J Am Soc Nephrol ; 24(8): 1216-22, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23687361

RESUMO

LMX1B encodes a homeodomain-containing transcription factor that is essential during development. Mutations in LMX1B cause nail-patella syndrome, characterized by dysplasia of the patellae, nails, and elbows and FSGS with specific ultrastructural lesions of the glomerular basement membrane (GBM). By linkage analysis and exome sequencing, we unexpectedly identified an LMX1B mutation segregating with disease in a pedigree of five patients with autosomal dominant FSGS but without either extrarenal features or ultrastructural abnormalities of the GBM suggestive of nail-patella-like renal disease. Subsequently, we screened 73 additional unrelated families with FSGS and found mutations involving the same amino acid (R246) in 2 families. An LMX1B in silico homology model suggested that the mutated residue plays an important role in strengthening the interaction between the LMX1B homeodomain and DNA; both identified mutations would be expected to diminish such interactions. In summary, these results suggest that isolated FSGS could result from mutations in genes that are also involved in syndromic forms of FSGS. This highlights the need to include these genes in all diagnostic approaches to FSGS that involve next-generation sequencing.


Assuntos
Glomerulosclerose Segmentar e Focal/genética , Proteínas com Homeodomínio LIM/genética , Síndrome da Unha-Patela/genética , Fatores de Transcrição/genética , Adolescente , Adulto , Criança , Feminino , Genes Dominantes , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Linhagem , Análise de Sequência de DNA , Adulto Jovem
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