Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 34
Filter
Add more filters

Country/Region as subject
Publication year range
1.
Cell ; 178(3): 521-535.e23, 2019 07 25.
Article in English | MEDLINE | ID: mdl-31348885

ABSTRACT

Intracellular accumulation of misfolded proteins causes toxic proteinopathies, diseases without targeted therapies. Mucin 1 kidney disease (MKD) results from a frameshift mutation in the MUC1 gene (MUC1-fs). Here, we show that MKD is a toxic proteinopathy. Intracellular MUC1-fs accumulation activated the ATF6 unfolded protein response (UPR) branch. We identified BRD4780, a small molecule that clears MUC1-fs from patient cells, from kidneys of knockin mice and from patient kidney organoids. MUC1-fs is trapped in TMED9 cargo receptor-containing vesicles of the early secretory pathway. BRD4780 binds TMED9, releases MUC1-fs, and re-routes it for lysosomal degradation, an effect phenocopied by TMED9 deletion. Our findings reveal BRD4780 as a promising lead for the treatment of MKD and other toxic proteinopathies. Generally, we elucidate a novel mechanism for the entrapment of misfolded proteins by cargo receptors and a strategy for their release and anterograde trafficking to the lysosome.


Subject(s)
Benzamides/metabolism , Bridged Bicyclo Compounds/pharmacology , Heptanes/pharmacology , Lysosomes/drug effects , Vesicular Transport Proteins/metabolism , Activating Transcription Factor 6/metabolism , Animals , Benzamides/chemistry , Benzamides/pharmacology , Bridged Bicyclo Compounds/therapeutic use , Epithelial Cells/cytology , Epithelial Cells/metabolism , Female , Frameshift Mutation , Heptanes/therapeutic use , Humans , Imidazoline Receptors/antagonists & inhibitors , Imidazoline Receptors/genetics , Imidazoline Receptors/metabolism , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Kidney/cytology , Kidney/metabolism , Kidney/pathology , Kidney Diseases/metabolism , Kidney Diseases/pathology , Lysosomes/metabolism , Male , Mice , Mice, Transgenic , Mucin-1/chemistry , Mucin-1/genetics , Mucin-1/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Unfolded Protein Response/drug effects , Vesicular Transport Proteins/chemistry
2.
Kidney Int ; 105(4): 799-811, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38096951

ABSTRACT

Sporadic cases of apolipoprotein A-IV medullary amyloidosis have been reported. Here we describe five families found to have autosomal dominant medullary amyloidosis due to two different pathogenic APOA4 variants. A large family with autosomal dominant chronic kidney disease (CKD) and bland urinary sediment underwent whole genome sequencing with identification of a chr11:116692578 G>C (hg19) variant encoding the missense mutation p.L66V of the ApoA4 protein. We identified two other distantly related families from our registry with the same variant and two other distantly related families with a chr11:116693454 C>T (hg19) variant encoding the missense mutation p.D33N. Both mutations are unique to affected families, evolutionarily conserved and predicted to expand the amyloidogenic hotspot in the ApoA4 structure. Clinically affected individuals suffered from CKD with a bland urinary sediment and a mean age for kidney failure of 64.5 years. Genotyping identified 48 genetically affected individuals; 44 individuals had an estimated glomerular filtration rate (eGFR) under 60 ml/min/1.73 m2, including all 25 individuals with kidney failure. Significantly, 11 of 14 genetically unaffected individuals had an eGFR over 60 ml/min/1.73 m2. Fifteen genetically affected individuals presented with higher plasma ApoA4 concentrations. Kidney pathologic specimens from four individuals revealed amyloid deposits limited to the medulla, with the mutated ApoA4 identified by mass-spectrometry as the predominant amyloid constituent in all three available biopsies. Thus, ApoA4 mutations can cause autosomal dominant medullary amyloidosis, with marked amyloid deposition limited to the kidney medulla and presenting with autosomal dominant CKD with a bland urinary sediment. Diagnosis relies on a careful family history, APOA4 sequencing and pathologic studies.


Subject(s)
Amyloidosis , Apolipoproteins A , Nephritis, Interstitial , Renal Insufficiency, Chronic , Humans , Middle Aged , Nephritis, Interstitial/diagnosis , Nephritis, Interstitial/genetics , Nephritis, Interstitial/complications , Mutation , Renal Insufficiency, Chronic/diagnosis , Renal Insufficiency, Chronic/genetics , Renal Insufficiency, Chronic/complications
3.
Am J Med Genet C Semin Med Genet ; 190(3): 309-324, 2022 09.
Article in English | MEDLINE | ID: mdl-36250282

ABSTRACT

The clinical characteristics of autosomal dominant tubulointerstitial kidney disease (ADTKD) include bland urinary sediment, slowly progressive chronic kidney disease (CKD) with many patients reaching end stage renal disease (ESRD) between age 20 and 70 years, and autosomal dominant inheritance. Due to advances in genetic diagnosis, ADTKD is becoming increasingly recognized as a cause of CKD. Pathogenic variants in UMOD, MUC1, and REN are the most common causes of ADTKD. ADTKD-UMOD is also associated with hyperuricemia and gout. ADTKD-REN often presents in childhood with mild hypotension, CKD, hyperkalemia, acidosis, and anemia. ADTKD-MUC1 patients present only with CKD. This review describes the pathophysiology, genetics, clinical manifestation, and diagnosis for ADTKD, with an emphasis on genetic testing and genetic counseling suggestions for patients.


Subject(s)
Genetic Testing , Renal Insufficiency, Chronic , Humans , Young Adult , Adult , Middle Aged , Aged , Uromodulin/genetics , Mutation
4.
Hum Mol Genet ; 28(22): 3805-3814, 2019 11 15.
Article in English | MEDLINE | ID: mdl-31600779

ABSTRACT

We report for the first time an autosomal recessive inborn error of de novo purine synthesis (DNPS)-PAICS deficiency. We investigated two siblings from the Faroe Islands born with multiple malformations resulting in early neonatal death. Genetic analysis of affected individuals revealed a homozygous missense mutation in PAICS (c.158A>G; p.Lys53Arg) that affects the structure of the catalytic site of the bifunctional enzyme phosphoribosylaminoimidazole carboxylase (AIRC, EC 4.1.1.21)/phosphoribosylaminoimidazole succinocarboxamide synthetase (SAICARS, EC 6.3.2.6) (PAICS). The mutation reduced the catalytic activity of PAICS in heterozygous carrier and patient skin fibroblasts to approximately 50 and 10% of control levels, respectively. The catalytic activity of the corresponding recombinant enzyme protein carrying the mutation p.Lys53Arg expressed and purified from E. coli was reduced to approximately 25% of the wild-type enzyme. Similar to other two known DNPS defects-adenylosuccinate lyase deficiency and AICA-ribosiduria-the PAICS mutation prevented purinosome formation in the patient's skin fibroblasts, and this phenotype was corrected by transfection with the wild-type but not the mutated PAICS. Although aminoimidazole ribotide (AIR) and aminoimidazole riboside (AIr), the enzyme substrates that are predicted to accumulate in PAICS deficiency, were not detected in patient's fibroblasts, the cytotoxic effect of AIr on various cell lines was demonstrated. PAICS deficiency is a newly described disease that enhances our understanding of the DNPS pathway and should be considered in the diagnosis of families with recurrent spontaneous abortion or early neonatal death.


Subject(s)
Carboxy-Lyases/genetics , Peptide Synthases/genetics , Purines/metabolism , Abnormalities, Multiple/genetics , Adenylosuccinate Lyase/deficiency , Autistic Disorder , Carboxy-Lyases/metabolism , Denmark , Fatal Outcome , Humans , Infant, Newborn , Male , Mutation , Peptide Synthases/metabolism , Perinatal Death , Phenotype , Purine-Pyrimidine Metabolism, Inborn Errors , Purines/biosynthesis
5.
Am J Nephrol ; 52(5): 378-387, 2021.
Article in English | MEDLINE | ID: mdl-34098564

ABSTRACT

INTRODUCTION: Patients with ADTKD-MUC1 have one allele producing normal mucin-1 (MUC1) and one allele producing mutant MUC1, which remains intracellular. We hypothesized that ADTKD-MUC1 patients, who have only 1 secretory-competent wild-type MUC1 allele, should exhibit decreased plasma mucin-1 (MUC1) levels. To test this hypothesis, we repurposed the serum CA15-3 assay used to measure MUC1 in breast cancer to measure plasma MUC1 levels in ADTKD-MUC1. METHODS: This cross-sectional study analyzed CA15-3 levels in a reference population of 6,850 individuals, in 85 individuals with ADTKD-MUC1, and in a control population including 135 individuals with ADTKD-UMOD and 114 healthy individuals. RESULTS: Plasma CA15-3 levels (mean ± standard deviation) were 8.6 ± 4.3 U/mL in individuals with ADTKD-MUC1 and 14.6 ± 5.6 U/mL in controls (p < 0.001). While there was a significant difference in mean CA15-3 levels, there was substantial overlap between the 2 groups. Plasma CA15-3 levels were <5 U/mL in 22% of ADTKD-MUC1 patients, in 0/249 controls, and in 1% of the reference population. Plasma CA15-3 levels were >20 U/mL in 1/85 ADTKD-MUC1 patients, in 18% of control individuals, and in 25% of the reference population. Segregation of plasma CA15-3 levels by the rs4072037 genotype did not significantly improve differentiation between affected and unaffected individuals. CA15-3 levels were minimally affected by gender and estimated glomerular filtration rate. DISCUSSION/CONCLUSIONS: Plasma CA15-3 levels in ADTKD-MUC1 patients are approximately 40% lower than levels in healthy individuals, though there is significant overlap between groups. Further investigations need to be performed to see if plasma CA15-3 levels would be useful in diagnosis, prognosis, or assessing response to new therapies in this disorder.


Subject(s)
Mucin-1/blood , Nephritis, Interstitial/blood , Uromodulin/genetics , Adult , Aged , Alleles , Biomarkers/blood , Case-Control Studies , Cross-Sectional Studies , Female , Healthy Volunteers , Humans , Male , Middle Aged , Mucin-1/genetics , Mutation , Nephritis, Interstitial/genetics , Prognosis
6.
Kidney Int ; 98(6): 1589-1604, 2020 12.
Article in English | MEDLINE | ID: mdl-32750457

ABSTRACT

There have been few clinical or scientific reports of autosomal dominant tubulointerstitial kidney disease due to REN mutations (ADTKD-REN), limiting characterization. To further study this, we formed an international cohort characterizing 111 individuals from 30 families with both clinical and laboratory findings. Sixty-nine individuals had a REN mutation in the signal peptide region (signal group), 27 in the prosegment (prosegment group), and 15 in the mature renin peptide (mature group). Signal group patients were most severely affected, presenting at a mean age of 19.7 years, with the prosegment group presenting at 22.4 years, and the mature group at 37 years. Anemia was present in childhood in 91% in the signal group, 69% prosegment, and none of the mature group. REN signal peptide mutations reduced hydrophobicity of the signal peptide, which is necessary for recognition and translocation across the endoplasmic reticulum, leading to aberrant delivery of preprorenin into the cytoplasm. REN mutations in the prosegment led to deposition of prorenin and renin in the endoplasmic reticulum-Golgi intermediate compartment and decreased prorenin secretion. Mutations in mature renin led to deposition of the mutant prorenin in the endoplasmic reticulum, similar to patients with ADTKD-UMOD, with a rate of progression to end stage kidney disease (63.6 years) that was significantly slower vs. the signal (53.1 years) and prosegment groups (50.8 years) (significant hazard ratio 0.367). Thus, clinical and laboratory studies revealed subtypes of ADTKD-REN that are pathophysiologically, diagnostically, and clinically distinct.


Subject(s)
Anemia , Polycystic Kidney Diseases , Adult , Child , Cohort Studies , Female , Humans , Male , Mutation , Polycystic Kidney Diseases/genetics , Renin/genetics , Young Adult
7.
Am J Hum Genet ; 99(1): 174-87, 2016 Jul 07.
Article in English | MEDLINE | ID: mdl-27392076

ABSTRACT

Autosomal-dominant tubulo-interstitial kidney disease (ADTKD) encompasses a group of disorders characterized by renal tubular and interstitial abnormalities, leading to slow progressive loss of kidney function requiring dialysis and kidney transplantation. Mutations in UMOD, MUC1, and REN are responsible for many, but not all, cases of ADTKD. We report on two families with ADTKD and congenital anemia accompanied by either intrauterine growth retardation or neutropenia. Ultrasound and kidney biopsy revealed small dysplastic kidneys with cysts and tubular atrophy with secondary glomerular sclerosis, respectively. Exclusion of known ADTKD genes coupled with linkage analysis, whole-exome sequencing, and targeted re-sequencing identified heterozygous missense variants in SEC61A1-c.553A>G (p.Thr185Ala) and c.200T>G (p.Val67Gly)-both affecting functionally important and conserved residues in SEC61. Both transiently expressed SEC6A1A variants are delocalized to the Golgi, a finding confirmed in a renal biopsy from an affected individual. Suppression or CRISPR-mediated deletions of sec61al2 in zebrafish embryos induced convolution defects of the pronephric tubules but not the pronephric ducts, consistent with the tubular atrophy observed in the affected individuals. Human mRNA encoding either of the two pathogenic alleles failed to rescue this phenotype as opposed to a complete rescue by human wild-type mRNA. Taken together, these findings provide a mechanism by which mutations in SEC61A1 lead to an autosomal-dominant syndromic form of progressive chronic kidney disease. We highlight protein translocation defects across the endoplasmic reticulum membrane, the principal role of the SEC61 complex, as a contributory pathogenic mechanism for ADTKD.


Subject(s)
Anemia/genetics , Heterozygote , Kidney Diseases/genetics , Mutation , SEC Translocation Channels/genetics , Adult , Aged , Alleles , Amino Acid Sequence , Animals , Biopsy , Child , Chronic Disease , Disease Progression , Endoplasmic Reticulum/metabolism , Exome/genetics , Female , Fetal Growth Retardation/genetics , Genes, Dominant , Golgi Apparatus/metabolism , Humans , Infant, Newborn , Kidney Diseases/pathology , Male , Middle Aged , Models, Molecular , Mutation, Missense/genetics , Neutropenia/genetics , Pedigree , Phenotype , RNA, Messenger/analysis , RNA, Messenger/genetics , SEC Translocation Channels/chemistry , Syndrome , Young Adult , Zebrafish/embryology , Zebrafish/genetics
8.
J Am Soc Nephrol ; 29(9): 2418-2431, 2018 09.
Article in English | MEDLINE | ID: mdl-29967284

ABSTRACT

BACKGROUND: Autosomal dominant tubulointerstitial kidney disease caused by mucin-1 gene (MUC1) mutations (ADTKD-MUC1) is characterized by progressive kidney failure. Genetic evaluation for ADTKD-MUC1 specifically tests for a cytosine duplication that creates a unique frameshift protein (MUC1fs). Our goal was to develop immunohistochemical methods to detect the MUC1fs created by the cytosine duplication and, possibly, by other similar frameshift mutations and to identify novel MUC1 mutations in individuals with positive immunohistochemical staining for the MUC1fs protein. METHODS: We performed MUC1fs immunostaining on urinary cell smears and various tissues from ADTKD-MUC1-positive and -negative controls as well as in individuals from 37 ADTKD families that were negative for mutations in known ADTKD genes. We used novel analytic methods to identify MUC1 frameshift mutations. RESULTS: After technique refinement, the sensitivity and specificity for MUC1fs immunostaining of urinary cell smears were 94.2% and 88.6%, respectively. Further genetic testing on 17 families with positive MUC1fs immunostaining revealed six families with five novel MUC1 frameshift mutations that all predict production of the identical MUC1fs protein. CONCLUSIONS: We developed a noninvasive immunohistochemical method to detect MUC1fs that, after further validation, may be useful in the future for diagnostic testing. Production of the MUC1fs protein may be central to the pathogenesis of ADTKD-MUC1.


Subject(s)
Genetic Predisposition to Disease/epidemiology , Mucin-1/genetics , Polycystic Kidney, Autosomal Dominant/genetics , Polycystic Kidney, Autosomal Dominant/pathology , Biopsy, Needle , Case-Control Studies , Female , Humans , Immunohistochemistry , Incidence , Male , Mutation/genetics , Pedigree , Polycystic Kidney, Autosomal Dominant/mortality , Prognosis , Registries , Retrospective Studies , Risk Assessment
9.
Hum Mol Genet ; 25(18): 4062-4079, 2016 09 15.
Article in English | MEDLINE | ID: mdl-27466185

ABSTRACT

The Acadian variant of Fanconi Syndrome refers to a specific condition characterized by generalized proximal tubular dysfunction from birth, slowly progressive chronic kidney disease and pulmonary interstitial fibrosis. This condition occurs only in Acadians, a founder population in Nova Scotia, Canada. The genetic and molecular basis of this disease is unknown. We carried out whole exome and genome sequencing and found that nine affected individuals were homozygous for the ultra-rare non-coding variant chr8:96046914 T > C; rs575462405, whereas 13 healthy siblings were either heterozygotes or lacked the mutant allele. This variant is located in intron 2 of NDUFAF6 (NM_152416.3; c.298-768 T > C), 37 base pairs upstream from an alternative splicing variant in NDUFAF6 chr8:96046951 A > G; rs74395342 (c.298-731 A > G). NDUFAF6 encodes NADH:ubiquinone oxidoreductase complex assembly factor 6, also known as C8ORF38. We found that rs575462405-either alone or in combination with rs74395342-affects splicing and synthesis of NDUFAF6 isoforms. Affected kidney and lung showed specific loss of the mitochondria-located NDUFAF6 isoform and ultrastructural characteristics of mitochondrial dysfunction. Accordingly, affected tissues had defects in mitochondrial respiration and complex I biogenesis that were corrected with NDUFAF6 cDNA transfection. Our results demonstrate that the Acadian variant of Fanconi Syndrome results from mitochondrial respiratory chain complex I deficiency. This information may be used in the diagnosis and prevention of this disease in individuals and families of Acadian descent and broadens the spectrum of the clinical presentation of mitochondrial diseases, respiratory chain defects and defects of complex I specifically.


Subject(s)
Electron Transport Complex I/genetics , Fanconi Syndrome/genetics , Mitochondria/metabolism , Mitochondrial Diseases/genetics , Mitochondrial Proteins/genetics , Adult , Alleles , Canada , Chromosome Mapping , Exome/genetics , Fanconi Syndrome/pathology , Female , Genetic Predisposition to Disease , Heterozygote , Homozygote , Humans , Kidney/metabolism , Kidney/pathology , Lung/metabolism , Lung/pathology , Male , Middle Aged , Mitochondria/pathology , Mitochondrial Diseases/metabolism , Mitochondrial Diseases/pathology , Mutation
10.
Rheumatology (Oxford) ; 57(7): 1180-1185, 2018 07 01.
Article in English | MEDLINE | ID: mdl-30423175

ABSTRACT

Objectives: Phosphoribosylpyrophosphate synthetase (PRPS1) superactivity is an X-linked disorder characterized by urate overproduction Online Mendelian Inheritance in Man (OMIM) gene reference 300661. This condition is thought to rarely affect women, and when it does, the clinical presentation is mild. We describe a 16-year-old African American female who developed progressive tophi, nephrolithiasis and acute kidney failure due to urate overproduction. Family history included a mother with tophaceous gout who developed end-stage kidney disease due to nephrolithiasis and an affected sister with polyarticular gout. The main aim of this study was to describe the clinical manifestations of PRPS1 superactivity in women. Methods: Whole exome sequencing was performed in affected females and their fathers. Results: Mutational analysis revealed a new c.520 G > A (p.G174R) mutation in the PRPS1 gene. The mutation resulted in decreased PRPS1 inhibition by ADP. Conclusion: Clinical findings in previously reported females with PRPS1 superactivity showed a high clinical penetrance of this disorder with a mean serum urate level of 8.5 (4.1) mg/dl [506 (247) µmol/l] and a high prevalence of gout. These findings indicate that all women in families with PRPS1 superactivity should be genetically screened for a mutation (for clinical management and genetic counselling). In addition, women with tophaceous gout, gout presenting in childhood, or a strong family history of severe gout should be considered for PRPS1 mutational analysis.


Subject(s)
Genetic Diseases, X-Linked/diagnosis , Genetic Diseases, X-Linked/genetics , Purine-Pyrimidine Metabolism, Inborn Errors/diagnosis , Purine-Pyrimidine Metabolism, Inborn Errors/genetics , Ribose-Phosphate Pyrophosphokinase/metabolism , Adolescent , Adult , Arthritis, Gouty/etiology , Arthritis, Gouty/genetics , Female , Humans , Male , Molecular Structure , Mutation , Nephrolithiasis/etiology , Nephrolithiasis/genetics , Pedigree , Purine-Pyrimidine Metabolism, Inborn Errors/complications , Ribose-Phosphate Pyrophosphokinase/genetics , Whole Genome Sequencing/methods
11.
Am J Hum Genet ; 92(5): 792-9, 2013 May 02.
Article in English | MEDLINE | ID: mdl-23602711

ABSTRACT

The genetic cause of GAPO syndrome, a condition characterized by growth retardation, alopecia, pseudoanodontia, and progressive visual impairment, has not previously been identified. We studied four ethnically unrelated affected individuals and identified homozygous nonsense mutations (c.262C>T [p.Arg88*] and c.505C>T [p.Arg169*]) or splicing mutations (c.1435-12A>G [p.Gly479Phefs*119]) in ANTXR1, which encodes anthrax toxin receptor 1. The nonsense mutations predictably trigger nonsense-mediated mRNA decay, resulting in the loss of ANTXR1. The transcript with the splicing mutation theoretically encodes a truncated ANTXR1 containing a neopeptide composed of 118 unique amino acids in its C terminus. GAPO syndrome's major phenotypic features, which include dental abnormalities and the accumulation of extracellular matrix, recapitulate those found in Antxr1-mutant mice and point toward an underlying defect in extracellular-matrix regulation. Thus, we propose that mutations affecting ANTXR1 function are responsible for this disease's characteristic generalized defect in extracellular-matrix homeostasis.


Subject(s)
Alopecia/genetics , Anodontia/genetics , Chromosomes, Human, Pair 2/genetics , Extracellular Matrix/genetics , Genetic Predisposition to Disease/genetics , Growth Disorders/genetics , Homeostasis/genetics , Neoplasm Proteins/genetics , Optic Atrophies, Hereditary/genetics , Receptors, Cell Surface/genetics , Alopecia/pathology , Alternative Splicing/genetics , Anodontia/pathology , Base Sequence , Codon, Nonsense/genetics , DNA Primers/genetics , Extracellular Matrix/metabolism , Fibroblasts , Fluorescent Antibody Technique , Gene Frequency , Growth Disorders/pathology , Humans , Male , Microfilament Proteins , Molecular Sequence Data , Optic Atrophies, Hereditary/pathology , Pedigree , RNA Splice Sites/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA
12.
Mol Genet Metab ; 119(3): 270-277, 2016 11.
Article in English | MEDLINE | ID: mdl-27590927

ABSTRACT

Purines are essential molecules for nucleic acid synthesis and are the most common carriers of chemical energy in all living organisms. The cellular pool of purines is maintained by the balance between their de novo synthesis (DNPS), recycling and degradation. DNPS includes ten reactions catalysed by six enzymes. To date, two genetically determined disorders of DNPS enzymes have been described, and the existence of other defects manifested by neurological symptoms and the accumulation of DNPS intermediates in bodily fluids is highly presumable. In the current study, we prepared specific recombinant DNPS enzymes and used them for the biochemical preparation of their commercially unavailable substrates. These compounds were used as standards for the development and validation of quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). To simulate manifestations of known and putative defects of DNPS we prepared CRISPR-Cas9 genome-edited HeLa cells deficient for the individual steps of DNPS (CR-cells), assessed the substrates accumulation in cell lysates and growth media and tested how the mutations affect assembly of the purinosome, the multi-enzyme complex of DNPS enzymes. In all model cell lines with the exception of one, an accumulation of the substrate(s) for the knocked out enzyme was identified. The ability to form the purinosome was reduced. We conclude that LC-MS/MS analysis of the dephosphorylated substrates of DNPS enzymes in bodily fluids is applicable in the selective screening of the known and putative DNPS disorders. This approach should be considered in affected individuals with neurological and neuromuscular manifestations of unknown aetiology. Prepared in vitro human model systems can serve in various studies that aim to provide a better characterization and understanding of physiology and pathology of DNPS, to study the role of each DNPS protein in the purinosome formation and represent an interesting way for the screening of potential therapeutic agents.


Subject(s)
CRISPR-Cas Systems , Multienzyme Complexes/metabolism , Purines/biosynthesis , Chromatography, Liquid , HeLa Cells , Humans , Multienzyme Complexes/chemistry , Multienzyme Complexes/genetics , Mutation , Purines/metabolism , Substrate Specificity , Tandem Mass Spectrometry
13.
Hum Mol Genet ; 21(7): 1534-43, 2012 Apr 01.
Article in English | MEDLINE | ID: mdl-22180458

ABSTRACT

The purinosome is a multienzyme complex composed by the enzymes active in de novo purine synthesis (DNPS) that cells transiently assemble in their cytosol upon depletion or increased demand of purines. The process of purinosome formation has thus far been demonstrated and studied only in human epithelial cervical cancer cells (HeLa) and human liver carcinoma cells (C3A) transiently expressing recombinant fluorescently labeled DNPS proteins. Using parallel immunolabeling of various DNPS enzymes and confocal fluorescent microscopy, we proved purinosome assembly in HeLa, human hepatocellular liver carcinoma cell line (HepG2), sarcoma osteogenic cells (Saos-2), human embryonic kidney cells (HEK293), human skin fibroblasts (SF) and primary human keratinocytes (KC) cultured in purine-depleted media. Using the identical approach, we proved in cultured skin fibroblasts from patients with AICA-ribosiduria and ADSL deficiency that various mutations of ATIC and ADSL destabilize to various degrees of purinosome assembly and found that the ability to form purinosomes correlates with clinical phenotypes of individual ADSL patients. Our results thus shown that the assembly of functional purinosomes is fully dependent on the presence of structurally unaffected ATIC and ADSL complexes and presumably also on the presence of all the other DNPS proteins. The results also corroborate the hypothesis that the phenotypic severity of ADSL deficiency is mainly determined by structural stability and residual catalytic capacity of the corresponding mutant ADSL protein complexes, as this is prerequisite for the formation and stability of the purinosome and at least partial channeling of succinylaminoimidazolecarboxamide riboside-ADSL enzyme substrates-through the DNPS pathway.


Subject(s)
Adenylosuccinate Lyase/genetics , Hydroxymethyl and Formyl Transferases/genetics , Multienzyme Complexes/genetics , Nucleotide Deaminases/genetics , Purine-Pyrimidine Metabolism, Inborn Errors/enzymology , Purine-Pyrimidine Metabolism, Inborn Errors/genetics , Adenylosuccinate Lyase/deficiency , Autistic Disorder , Cell Line, Tumor , Cells, Cultured , Fibroblasts/enzymology , HeLa Cells , Humans , Hydroxymethyl and Formyl Transferases/analysis , Keratinocytes/enzymology , Multienzyme Complexes/analysis , Mutation , Nucleotide Deaminases/analysis , Purines/metabolism , Skin/cytology
14.
Am J Hum Genet ; 89(2): 241-52, 2011 Aug 12.
Article in English | MEDLINE | ID: mdl-21820099

ABSTRACT

Autosomal-dominant adult-onset neuronal ceroid lipofuscinosis (ANCL) is characterized by accumulation of autofluorescent storage material in neural tissues and neurodegeneration and has an age of onset in the third decade of life or later. The genetic and molecular basis of the disease has remained unknown for many years. We carried out linkage mapping, gene-expression analysis, exome sequencing, and candidate-gene sequencing in affected individuals from 20 families and/or individuals with simplex cases; we identified in five individuals one of two disease-causing mutations, c.346_348delCTC and c.344T>G, in DNAJC5 encoding cysteine-string protein alpha (CSPα). These mutations-causing a deletion, p.Leu116del, and an amino acid exchange, p.Leu115Arg, respectively-are located within the cysteine-string domain of the protein and affect both palmitoylation-dependent sorting and the amount of CSPα in neuronal cells. The resulting depletion of functional CSPα might cause in parallel the presynaptic dysfunction and the progressive neurodegeneration observed in affected individuals and lysosomal accumulation of misfolded and proteolysis-resistant proteins in the form of characteristic ceroid deposits in neurons. Our work represents an important step in the genetic dissection of a genetically heterogeneous group of ANCLs. It also confirms a neuroprotective role for CSPα in humans and demonstrates the need for detailed investigation of CSPα in the neuronal ceroid lipofuscinoses and other neurodegenerative diseases presenting with neuronal protein aggregation.


Subject(s)
Genes, Dominant/genetics , HSP40 Heat-Shock Proteins/genetics , Membrane Proteins/genetics , Mutation/genetics , Neuronal Ceroid-Lipofuscinoses/epidemiology , Neuronal Ceroid-Lipofuscinoses/genetics , Adult , Age of Onset , Base Sequence , Brain/metabolism , Brain/pathology , Brain/ultrastructure , Chromosome Segregation/genetics , Exons/genetics , Family , Female , Gene Dosage/genetics , Gene Expression Regulation , Genetic Linkage , Humans , Lipoylation , Lysosomes/metabolism , Lysosomes/ultrastructure , Male , Molecular Sequence Data , Neuronal Ceroid-Lipofuscinoses/pathology , Neurons/metabolism , Neurons/pathology , Neurons/ultrastructure , Pedigree , Protein Transport , Sequence Analysis, DNA
15.
Virchows Arch ; 484(1): 135-140, 2024 Jan.
Article in English | MEDLINE | ID: mdl-37787787

ABSTRACT

Despite the adenoids are regularly removed in patients with mucopolysaccharidoses (MPS), the underlying tissue and cellular pathologies remain understudied. We characterized an (immuno)histopathologic and ultrastructural phenotype dominated by lysosomal storage changes in a specific subset of adenotonsillar paracortical cells in 8 MPS patients (3 MPS I, 3 MPS II, and 2 MPS IIIA). These abnormal cells were effectively detected by an antibody targeting the lysosomal membrane tetraspanin CD63. Important, CD63+ storage vacuoles in these cells lacked the monocytes/macrophages lysosomal marker CD68. Such a distinct patterning of CD63 and CD68 was not present in a patient with infantile neurovisceral variant of acid sphingomyelinase deficiency. The CD63+ storage pathology was absent in two MPS I patients who either received enzyme-replacement therapy or underwent hematopoietic stem cells transplantation prior the adenoidectomy. Our study demonstrates novel features of lysosomal storage patterning and suggests diagnostic utility of CD63 detection in adenotonsillar lymphoid tissue of MPS patients.


Subject(s)
Mucopolysaccharidoses , Humans , Mucopolysaccharidoses/diagnosis , Mucopolysaccharidoses/drug therapy , Mucopolysaccharidoses/genetics , Lymphoid Tissue/pathology , Lysosomes , Enzyme Replacement Therapy , Tetraspanin 30
16.
Kidney Int Rep ; 9(7): 2209-2226, 2024 Jul.
Article in English | MEDLINE | ID: mdl-39081747

ABSTRACT

Introduction: Monoallelic variants in the ALG5 gene encoding asparagine-linked glycosylation protein 5 homolog (ALG5) have been recently shown to disrupt polycystin-1 (PC1) maturation and trafficking via underglycosylation, causing an autosomal dominant polycystic kidney disease-like (ADPKD-like) phenotype and interstitial fibrosis. In this report, we present clinical, genetic, histopathologic, and protein structure and functional correlates of a new ALG5 variant, p.R79W, that we identified in 2 distant genetically related Irish families displaying an atypical late-onset ADPKD phenotype combined with tubulointerstitial damage. Methods: Whole exome and targeted sequencing were used for segregation analysis of available relatives. This was followed by immunohistochemistry examinations of kidney biopsies, and targeted (UMOD, MUC1) and untargeted plasma proteome and N-glycomic studies. Results: We identified a monoallelic ALG5 variant [GRCh37 (NM_013338.5): g.37569565G>A, c.235C>T; p.R79W] that cosegregates in 23 individuals, of whom 18 were clinically affected. We detected abnormal localization of ALG5 in the Golgi apparatus of renal tubular cells in patients' kidney specimens. Further, we detected the pathological accumulation of uromodulin, an N-glycosylated glycosylphosphatidylinositol (GPI)-anchored protein, in the endoplasmic reticulum (ER), but not mucin-1, an O- and N-glycosylated protein. Biochemical investigation revealed decreased plasma and urinary uromodulin levels in clinically affected individuals. Proteomic and glycoproteomic profiling revealed the dysregulation of chronic kidney disease (CKD)-associated proteins. Conclusion: ALG5 dysfunction adversely affects maturation and trafficking of N-glycosylated and GPI anchored protein uromodulin, leading to structural and functional changes in the kidney. Our findings confirm ALG5 as a cause of late-onset ADPKD and provide additional insight into the molecular mechanisms of ADPKD-ALG5.

17.
Mol Genet Metab ; 108(3): 178-189, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23394948

ABSTRACT

Purines are molecules essential for many cell processes, including RNA and DNA synthesis, regulation of enzyme activity, protein synthesis and function, energy metabolism and transfer, essential coenzyme function, and cell signaling. Purines are produced via the de novo purine biosynthesis pathway. Mutations in purine biosynthetic genes, for example phosphoribosylaminoimidazole carboxylase/phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS, E.C. 6.3.2.6/E.C. 4.1.1.21), can lead to developmental anomalies in lower vertebrates. Alterations in PAICS expression in humans have been associated with various types of cancer. Mutations in adenylosuccinate lyase (ADSL, E.C. 4.3.2.2) or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC, E.C. 2.1.2.3/E.C. 3.5.4.10) lead to inborn errors of metabolism with a range of clinical symptoms, including developmental delay, severe neurological symptoms, and autistic features. The pathogenetic mechanism is unknown for these conditions, and no effective treatments exist. The study of cells carrying mutations in the various de novo purine biosynthesis pathway genes provides one approach to analysis of purine disorders. Here we report the characterization of AdeD Chinese hamster ovary (CHO) cells, which carry genetic mutations encoding p.E177K and p.W363* variants of PAICS. Both mutations impact PAICS structure and completely abolish its biosynthesis. Additionally, we describe a sensitive and rapid analytical method for detection of purine de novo biosynthesis intermediates based on high performance liquid chromatography with electrochemical detection. Using this technique we detected accumulation of AIR in AdeD cells. In AdeI cells, mutant for the ADSL gene, we detected accumulation of SAICAR and SAMP and, somewhat unexpectedly, accumulation of AIR. This method has great potential for metabolite profiling of de novo purine biosynthesis pathway mutants, identification of novel genetic defects of purine metabolism in humans, and elucidating the regulation of this critical metabolic pathway.


Subject(s)
Carboxy-Lyases/genetics , Metabolomics , Mutation , Peptide Synthases/genetics , Purines/biosynthesis , Animals , Base Sequence , CHO Cells , Carboxy-Lyases/metabolism , Chromatography, High Pressure Liquid , Cricetinae , Cricetulus , Electrochemical Techniques , Models, Biological , Models, Molecular , Molecular Sequence Data , Peptide Synthases/metabolism , Purine-Pyrimidine Metabolism, Inborn Errors/genetics , Purine-Pyrimidine Metabolism, Inborn Errors/metabolism , Sequence Alignment
18.
Am J Hum Genet ; 85(2): 204-13, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19664745

ABSTRACT

Through linkage analysis and candidate gene sequencing, we identified three unrelated families with the autosomal-dominant inheritance of early onset anemia, hypouricosuric hyperuricemia, progressive kidney failure, and mutations resulting either in the deletion (p.Leu16del) or the amino acid exchange (p.Leu16Arg) of a single leucine residue in the signal sequence of renin. Both mutations decrease signal sequence hydrophobicity and are predicted by bioinformatic analyses to damage targeting and cotranslational translocation of preprorenin into the endoplasmic reticulum (ER). Transfection and in vitro studies confirmed that both mutations affect ER translocation and processing of nascent preprorenin, resulting either in reduced (p.Leu16del) or abolished (p.Leu16Arg) prorenin and renin biosynthesis and secretion. Expression of renin and other components of the renin-angiotensin system was decreased accordingly in kidney biopsy specimens from affected individuals. Cells stably expressing the p.Leu16del protein showed activated ER stress, unfolded protein response, and reduced growth rate. It is likely that expression of the mutant proteins has a dominant toxic effect gradually reducing the viability of renin-expressing cells. This alters the intrarenal renin-angiotensin system and the juxtaglomerular apparatus functionality and leads to nephron dropout and progressive kidney failure. Our findings provide insight into the functionality of renin-angiotensin system and stress the importance of renin analysis in families and individuals with early onset hyperuricemia, anemia, and progressive kidney failure.


Subject(s)
Anemia/genetics , Genes, Dominant , Hyperuricemia/genetics , Kidney Failure, Chronic/genetics , Renin/genetics , Adolescent , Adult , Age of Onset , Anemia/metabolism , Cell Line , Child , Child, Preschool , Computer Simulation , Female , Genetic Linkage , Humans , Hyperuricemia/metabolism , Kidney/cytology , Kidney/ultrastructure , Kidney Failure, Chronic/metabolism , Male , Middle Aged , Mutation , Pedigree , Renin/metabolism , Sequence Analysis, DNA , Young Adult
19.
Metabolites ; 12(12)2022 Dec 02.
Article in English | MEDLINE | ID: mdl-36557247

ABSTRACT

Cytotoxicity of de novo purine synthesis (DNPS) metabolites is critical to the pathogenesis of three known and one putative autosomal recessive disorder affecting DNPS. These rare disorders are caused by biallelic mutations in the DNPS genes phosphoribosylformylglycineamidine synthase (PFAS), phosphoribosylaminoimidazolecarboxylase/phosphoribosylaminoimidazolesuccinocarboxamide synthase (PAICS), adenylosuccinate lyase (ADSL), and aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) and are clinically characterized by developmental abnormalities, psychomotor retardation, and nonspecific neurological impairment. At a biochemical level, loss of function of specific mutated enzymes results in elevated levels of DNPS ribosides in body fluids. The main pathogenic effect is attributed to the accumulation of DNPS ribosides, which are postulated to be toxic to the organism. Therefore, we decided to characterize the uptake and flux of several DNPS metabolites in HeLa cells and the impact of DNPS metabolites to viability of cancer cell lines and primary skin fibroblasts. We treated cells with DNPS metabolites and followed their flux in purine synthesis and degradation. In this study, we show for the first time the transport of formylglycinamide ribotide (FGAR), aminoimidazole ribotide (AIR), succinylaminoimidazolecarboxamide ribotide (SAICAR), and aminoimidazolecarboxamide ribotide (AICAR) into cells and their flux in DNPS and the degradation pathway. We found diminished cell viability mostly in the presence of FGAR and AIR. Our results suggest that direct cellular toxicity of DNPS metabolites may not be the primary pathogenetic mechanism in these disorders.

20.
Life Sci Alliance ; 5(4)2022 04.
Article in English | MEDLINE | ID: mdl-35064074

ABSTRACT

The human Sec61 complex is a widely distributed and abundant molecular machine. It resides in the membrane of the endoplasmic reticulum to channel two types of cargo: protein substrates and calcium ions. The SEC61A1 gene encodes for the pore-forming Sec61α subunit of the Sec61 complex. Despite their ubiquitous expression, the idiopathic SEC61A1 missense mutations p.V67G and p.T185A trigger a localized disease pattern diagnosed as autosomal dominant tubulointerstitial kidney disease (ADTKD-SEC61A1). Using cellular disease models for ADTKD-SEC61A1, we identified an impaired protein transport of the renal secretory protein renin and a reduced abundance of regulatory calcium transporters, including SERCA2. Treatment with the molecular chaperone phenylbutyrate reversed the defective protein transport of renin and the imbalanced calcium homeostasis. Signal peptide substitution experiments pointed at targeting sequences as the cause for the substrate-specific impairment of protein transport in the presence of the V67G or T185A mutations. Similarly, dominant mutations in the signal peptide of renin also cause ADTKD and point to impaired transport of this renal hormone as important pathogenic feature for ADTKD-SEC61A1 patients as well.


Subject(s)
Phenylbutyrates/pharmacology , Renin/metabolism , SEC Translocation Channels/genetics , Calcium/metabolism , Endoplasmic Reticulum/metabolism , HEK293 Cells , Humans , Kidney Diseases/physiopathology , Molecular Chaperones/metabolism , Mutation, Missense , Phenylbutyrates/metabolism , Polycystic Kidney Diseases , Protein Transport/genetics , Renin/genetics , SEC Translocation Channels/chemistry , SEC Translocation Channels/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL