Globotriaosylceramide accumulation in the Fabry kidney is cleared from multiple cell types after enzyme replacement therapy.
Fabry disease, a lysosomal storage disease caused by deficient lysosomal ...α-galactosidase A activity, is characterized by globotriaosylceramide (GL-3) accumulation in multiple cell types, particularly the vasculature, leading to end organ failure. Accumulation in the kidney is responsible for progressive decline in renal function in male patients with the classical phenotype, resulting in renal failure in their third to fifth decades of life. With the advent of recombinant protein synthesis technology, enzyme replacement therapy has become a viable alternative to dialysis or renal transplantation, previously the only available treatment options for end-stage renal disease.
The pre- and post-treatment renal biopsies were analyzed from fifty-eight Fabry patients enrolled in a Phase 3 double-blind, randomized, placebo-controlled trial followed by a six-month open label extension study of the recombinant human enzyme, α-galactosidase A (r-hαGalA), administered IV at 1mg/kg biweekly. The purpose of this investigation was to detail the pathologic changes in glycosphingolipid distribution and the pattern of post-treatment clearance in the kidney.
Baseline evaluations revealed GL-3 accumulations in nearly all renal cell types including vascular endothelial cells, vascular smooth muscle cells, mesangial cells and interstitial cells, with particularly dense accumulations in podocytes and distal tubular epithelial cells. After 11 months of r-hαGalA treatment there was complete clearance of glycolipid from the endothelium of all vasculature as well as from the mesangial cells of the glomerulus and interstitial cells of the cortex. Moderate clearance was noted from the smooth muscle cells of arterioles and small arteries. Podocytes and distal tubular epithelium also demonstrated evidence for decreased GL-3, although this clearance was more limited than that observed in other cell types. No evidence of immune complex disease was found by immunofluorescence despite circulating anti-r-hαGalA IgG antibodies.
These findings indicate a striking reversal of renal glycosphingolipid accumulation in the vasculature and in other renal cell types, and suggest that long-term treatment with r-hαGalA may halt the progression of pathology and prevent renal failure in patients with Fabry disease.
The inborn errors of heme biosynthesis, the porphyrias, are 8 genetically distinct metabolic disorders that can be classified as "acute hepatic," "hepatic cutaneous," and "erythropoietic cutaneous" ...diseases. Recent advances in understanding their pathogenesis and molecular genetic heterogeneity have led to improved diagnosis and treatment. These advances include DNA-based diagnoses for all the porphyrias, new understanding of the pathogenesis of the acute hepatic porphyrias, identification of the iron overload-induced inhibitor of hepatic uroporphyrin decarboxylase activity that causes the most common porphyria, porphyria cutanea tarda, the identification of an X-linked form of erythropoietic protoporphyria due to gain-of-function mutations in erythroid-specific 5-aminolevulinate synthase (ALAS2), and new and experimental treatments for the erythropoietic prophyrias. Knowledge of these advances is relevant for hematologists because they administer the hematin infusions to treat the acute attacks in patients with the acute hepatic porphyrias, perform the chronic phlebotomies to reduce the iron overload and clear the dermatologic lesions in porphyria cutanea tarda, and diagnose and treat the erythropoietic porphyrias, including chronic erythrocyte transfusions, bone marrow or hematopoietic stem cell transplants, and experimental pharmacologic chaperone and stem cell gene therapies for congenital erythropoietic protoporphyria. These developments are reviewed to update hematologists on the latest advances in these diverse disorders.
Human hydroxymethylbilane synthase Bung, Navneet; Roy, Arijit; Chen, Brenden ...
Proceedings of the National Academy of Sciences - PNAS,
04/2018, Letnik:
115, Številka:
17
Journal Article
Recenzirano
Odprti dostop
Hydroxymethylbilane synthase (HMBS), the third enzyme in the heme biosynthetic pathway, catalyzes the head-to-tail condensation of four molecules of porphobilinogen (PBG) to form the linear ...tetrapyrrole 1-hydroxymethylbilane (HMB). Mutations in human HMBS (hHMBS) cause acute intermittent porphyria (AIP), an autosomal-dominant disorder characterized by life-threatening neurovisceral attacks. Although the 3D structure of hHMBS has been reported, the mechanism of the stepwise polymerization of four PBG molecules to form HMB remains unknown. Moreover, the specific roles of each of the critical active-site residues in the stepwise enzymatic mechanism and the dynamic behavior of hHMBS during catalysis have not been investigated. Here, we report atomistic studies of HMB stepwise synthesis by using molecular dynamics (MD) simulations, mutagenesis, and in vitro expression analyses. These studies revealed that the hHMBS active-site loop movement and cofactor turn created space for the elongating pyrrole chain. Twenty-seven residues around the active site and water molecules interacted to stabilize the large, negatively charged, elongating polypyrrole. Mutagenesis of these active-site residues altered the binding site, hindered cofactor binding, decreased catalysis, impaired ligand exit, and/or destabilized the enzyme. Based on intermediate stages of chain elongation, R26 and R167 were the strongest candidates for proton transfer to deaminate the incoming PBG molecules. Unbiased random acceleration MD simulations identified R167 as a gatekeeper and facilitator of HMB egress through the space between the enzyme’s domains and the active-site loop. These studies identified the specific active-site residues involved in each step of pyrrole elongation, thereby providing the molecular bases of the active-site mutations causing AIP.
The erythropoietic porphyrias are inborn errors of heme biosynthesis with prominent cutaneous manifestations. They include autosomal recessive Congenital Erythropoietic Porphyria (CEP) due to ...loss-of-function (LOF) mutations in the Uroporphyrinogen III Synthase (UROS) gene, Erythropoietic Protoporphyria (EPP) due to LOF mutations in the ferrochelatase (FECH) gene, and X-Linked Protoporphyria (XLP) due to gain-of-function mutations in the terminal exon of the Aminolevulinic Acid Synthase 2 (ALAS2) gene. During the 11-year period from 01/01/2007 through 12/31/2017, the Mount Sinai Porphyrias Diagnostic Laboratory provided molecular diagnostic testing for one or more of these disorders in 628 individuals, including 413 unrelated individuals. Of these 628, 120 patients were tested for CEP, 483 for EPP, and 331 for XLP, for a total of 934 tests. For CEP, 24 of 78 (31%) unrelated individuals tested had UROS mutations, including seven novel mutations. For EPP, 239 of 362 (66%) unrelated individuals tested had pathogenic FECH mutations, including twenty novel mutations. The IVS3-48 T > C low-expression allele was present in 231 (97%) of 239 mutation-positive EPP probands with a pathogenic FECH mutation. In the remaining 3%, three patients with two different FECH mutations in trans were identified. For XLP, 24 of 250 (10%) unrelated individuals tested had ALAS2 exon 11 mutations. No novel ALAS2 mutations were identified. Among family members referred for testing, 33 of 42 (79%) CEP, 62 of 121 (51%) EPP, and 31 of 81 (38%) XLP family members had the respective family mutation. Mutation-positive CEP, EPP, and XLP patients who had been biochemically tested had marked elevations of the disease-appropriate porphyrin intermediates. These results expand the molecular heterogeneity of the erythropoietic porphyrias by adding a total of 27 novel mutations. The results document the usefulness of molecular testing to confirm the positive biochemical findings in these patients and to identify heterozygous family members.
Congenital erythropoietic porphyria (CEP) is a rare autosomal recessive disease due to the deficient, but not absent, activity of uroporphyrinogen III synthase (UROS), the fourth enzyme in the heme ...biosynthesis pathway. Biallelic variants in the UROS gene result in decreased UROS enzymatic activity and the accumulation of non-physiologic Type I porphyrins in cells and fluids. Overproduced uroporphyrins in haematopoietic cells are released into the circulation and distributed to tissues, inducing primarily hematologic and dermatologic symptoms. The clinical manifestations vary in severity ranging from non-immune hydrops fetalis in utero to mild dermatologic manifestations in adults. Here, the biochemical, molecular and clinical features of CEP as well as current and new treatment options, including the rescue of UROS enzyme activity by chaperones, are presented.
The success of prenatal carrier screening as a disease prevention strategy in the Ashkenazi Jewish (AJ) population has driven the expansion of screening panels as disease‐causing founder mutations ...have been identified. However, the carrier frequencies of many of these mutations have not been reported in large AJ cohorts. We determined the carrier frequencies of over 100 mutations for 16 recessive disorders in the New York metropolitan area AJ population. Among the 100% AJ‐descended individuals, screening for 16 disorders resulted in ∼1 in 3.3 being a carrier for one disease and ∼1 in 24 for two diseases. The carrier frequencies ranged from 0.066 (1 in 15.2; Gaucher disease) to 0.006 (1 in 168; nemaline myopathy), which averaged ∼15% higher than those for all screenees. Importantly, over 95% of screenees chose to be screened for all possible AJ diseases, including disorders with lower carrier frequencies and/or detectability. Carrier screening also identified rare individuals homozygous for disease‐causing mutations who had previously unrecognized clinical manifestations. Additionally, prenatal testing results and experience for all 16 disorders (n = 574) are reported. Together, these data indicate the general acceptance, carrier frequencies, and prenatal testing results for an expanded panel of 16 diseases in the AJ population. Hum Mutat 31:1-11, 2010.
Fabry disease, an inherited deficiency of the lysosomal enzyme alpha-galactosidase A, causes progressive intralysosomal accumulation of globotriaosylceramide (GL-3) and premature death from renal, ...cardiac, and cerebrovascular manifestations. To determine the long-term safety and efficacy of recombinant human alpha-galactosidase A, an open-label, phase III extension study was conducted, involving 58 patients who had classic Fabry disease and completed a 20-wk, double-blind, randomized, placebo-controlled, phase III study of agalsidase beta and were transitioned to an extension trial to receive biweekly 1 mg/kg agalsidase beta for up to an additional 54 mo. GL-3 accumulation was evaluated in the capillary endothelia of the skin, kidney, and heart. Renal function was assessed. By month 54, all patients with optional kidney biopsies (n = 8) maintained complete GL-3 clearance in renal capillary endothelial cells and multiple cell types. Continued, complete clearance of skin (31 of 36) and heart (six of eight) capillary endothelium was demonstrated. Mean plasma GL-3 levels remained decreased in the normal range. Median serum creatinine and estimated GFR remained stable (normal) in patients with renal data at month 54 (n = 41). Six patients had renal disease progression; most (four of six) were older than 40 yr and had significant proteinuria at baseline and evidence of sclerotic glomeruli pretreatment. Adverse events were generally mild and unrelated to treatment. The most common treatment-related adverse events were infusion-associated reactions, which decreased over time. Long-term agalsidase beta therapy stabilizes renal function in patients without renal involvement at baseline, maintains reduction of plasma GL-3, and sustains GL-3 clearance in capillary endothelial cells and multiple renal cell types.
Dersimelagon in Erythropoietic Protoporphyrias Balwani, Manisha; Bonkovsky, Herbert L.; Levy, Cynthia ...
The New England journal of medicine,
04/2023, Letnik:
388, Številka:
15
Journal Article
Recenzirano
In a phase 2 trial, once-daily oral treatment with dersimelagon safely improved tolerance to sun exposure in patients with erythropoietic protoporphyria and X-linked protoporphyria.
Defects in hydroxymethylbilane synthase (HMBS) can cause acute intermittent porphyria (AIP), an acute neurological disease. Although sequencing-based diagnosis can be definitive, ∼⅓ of clinical HMBS ...variants are missense variants, and most clinically reported HMBS missense variants are designated as “variants of uncertain significance” (VUSs). Using saturation mutagenesis, en masse selection, and sequencing, we applied a multiplexed validated assay to both the erythroid-specific and ubiquitous isoforms of HMBS, obtaining confident functional impact scores for >84% of all possible amino acid substitutions. The resulting variant effect maps generally agreed with biochemical expectations and provide further evidence that HMBS can function as a monomer. Additionally, the maps implicated specific residues as having roles in active site dynamics, which was further supported by molecular dynamics simulations. Most importantly, these maps can help discriminate pathogenic from benign HMBS variants, proactively providing evidence even for yet-to-be-observed clinical missense variants.
van Loggerenberg et al. functionally assay ∼90% of all possible missense variants in HMBS, which is associated with acute intermittent porphyria. The resulting variant effect maps reveal sequence-structure-function relationships and provide evidence to distinguish pathogenic from benign variants.
Fabry disease results from deficient α-galactosidase A (α-Gal A) activity and the pathologic accumulation of the globotriaosylceramide (GL-3) and related glycosphingolipids, primarily in vascular ...endothelial lysosomes. Treatment is currently palliative, and affected patients generally die in their 40s or 50s. Preclinical studies of recombinant human α-Gal A (r-hαGalA) infusions in knockout mice demonstrated reduction of GL-3 in tissues and plasma, providing rationale for a phase 1/2 clinical trial. Here, we report a single-center, open-label, dose-ranging study of r-hαGalA treatment in 15 patients, each of whom received five infusions at one of five dose regimens. Intravenously administered r-hαGalA was cleared from the circulation in a dose-dependent manner, via both saturable and non-saturable pathways. Rapid and marked reductions in plasma and tissue GL-3 were observed biochemically, histologically, and/or ultrastructurally. Clearance of plasma GL-3 was dose-dependent. In patients with pre- and posttreatment biopsies, mean GL-3 content decreased 84% in liver (
n=13), was markedly reduced in kidney in four of five patients, and after five doses was modestly lowered in the endomyocardium of four of seven patients. GL-3 deposits were cleared to near normal or were markedly reduced in the vascular endothelium of liver, skin, heart, and kidney, on the basis of light- and electron-microscopic evaluation. In addition, patients reported less pain, increased ability to sweat, and improved quality-of-life measures. Infusions were well tolerated; four patients experienced mild-to-moderate reactions, suggestive of hypersensitivity, that were managed conservatively. Of 15 patients, 8 (53%) developed IgG antibodies to r-hαGalA; however, the antibodies were not neutralizing, as indicated by unchanged pharmacokinetic values for infusions 1 and 5. This study provides the basis for a phase 3 trial of enzyme-replacement therapy for Fabry disease.
PDF
