Systemic AL amyloidosis with unusual cutaneous presentation unmasked by carotenoderma.

We present a case study of an elderly woman with systemic lambda-type AL amyloidosis that featured unusually extensive cutaneous involvement. The case initially presented with a sudden hyper ß-carotenemia with carotenoderma that instigated the clinical examination including skin biopsy. A diagnosis of systemic amyloidosis was made. Immunohistochemistry and Western-blot analysis indicated the presence of lambda light chain proteins in skin amyloid deposits. However, notable co-deposition of wild-type apoA-I and transthyretin was observed which caused initial diagnostic confusion. Proteomic analysis of microdissected skin amyloid deposits by mass spectrometry confirmed lambda light chain proteins in amyloid deposits and co-deposition of apolipoprotein A-IV and serum amyloid P-component. The patient died from renal failure caused by amyloid nephropathy combined with analgesic nephropathy. The autopsy disclosed vascular, cardiac, renal and pulmonary amyloid deposition. While all amyloid deposits were positive for lambda light chain proteins, the immunodetection of apoA-I and transthyretin varied significantly among the visceral amyloid deposits. Although the patient exhibited a 1000-fold increase in serum ß-carotene levels, only a mild increase in retinol and lutein concentrations was observed. Increased ß-carotene values were also found in the liver and the skin. The mechanisms underlying this hyper ß-carotenemia remain undetermined.

Dubin-Johnson syndrome coinciding with colon cancer and atherosclerosis.

Hyperbilirubinemia has been presumed to prevent the process of atherogenesis and cancerogenesis mainly by decreasing oxidative stress. Dubin-Johnson syndrome is a rare, autosomal recessive, inherited disorder characterized by biphasic, predominantly conjugated hyperbilirubinemia with no progression to end-stage liver disease. The molecular basis in Dubin-Johnson syndrome is absence or deficiency of human canalicular multispecific organic anion transporter MRP2/cMOAT caused by homozygous or compound heterozygous mutation(s) in ABCC2 located on chromosome 10q24. Clinical onset of the syndrome is most often seen in the late teens or early adulthood. In this report, we describe a case of previously unrecognized Dubin-Johnson syndrome caused by two novel pathogenic mutations (c.2360_2366delCCCTGTC and c.3258+1G>A), coinciding with cholestatic liver disease in an 82-year-old male patient. The patient, suffering from advanced atherosclerosis with serious involvement of coronary arteries, developed colorectal cancer with nodal metastases. The subsequent findings do not support the protective role of Dubin-Johnson type hyperbilirubinemia.

Hypertrophic cardiomyopathy due to the mitochondrial DNA mutation m.3303C>T diagnosed in an adult male.

Mitochondrial disorders comprise a heterogeneous group of diseases with multisystem involvement including myocardium. Most cases of mitochondrial cardiomyopathy are associated with myopathy and encephalopathy and are generally present in infancy or childhood. The disease often exhibits a rapid downward course with death frequently occuring within the first year of life. We describe a unique case of hypertrophic cardiomyopathy due to mitochondrial DNA mutation m.3303C >T in the MT-TL1 gene, diagnosed accidentally in a 35-year-old male. The patient initially presented with stroke of assumed cardioembolic origin due to the presence of two interatrial communications associated with mobile aneurysm of the interatrial septum. No other extracardiac manifestations of mitochondrial disorder were observed.

Clinical picture of S-adenosylhomocysteine hydrolase deficiency resembles phosphomannomutase 2 deficiency.

We report on the seventh known patient with S-adenosylhomocysteine hydrolase (SAHH) deficiency presenting at birth with features resembling phosphomannomutase 2 (PMM2-CDG Ia) deficiency. Plasma methionine and total homocysteine levels were normal at 2 months and increased only after the 8th month of age. SAHH deficiency was confirmed at 4.5 years of age by showing decreased SAHH activity (11% in both erythrocytes and fibroblasts), and compound heterozygosity for a known mutation c.145C>T (p.R49C) and a novel variant c.211G>A (p.G71S) in the AHCY gene. Retrospective analysis of clinical features revealed striking similarities between SAHH deficiency and the PMM2-CDG Ia.

Distinctive histopathological features that support a diagnosis of cholesterol ester storage disease in liver biopsy specimens.

AIMS: To identify reliable criteria with which to improve the diagnosis of lysosomal acid lipase (LAL) deficiency of the cholesterol ester storage disease (CESD) type in liver biopsies. METHODS AND RESULTS: We analysed a series of 16 liver biopsies of LAL deficiency of the CESD type confirmed by enzyme testing and DNA sequencing. The biopsy appearances were compared with those of biopsies of other causes of fatty liver. A predominantly microvesicular steatosis in CESD patients could not be reliably distinguished from other causes of fatty liver with cytosolic lipid accumulation in fixed paraffin-embedded tissues routinely stained with haematoxylin and eosin. The presence of luminal (cathepsin D) and membrane lysosomal markers [lysosomal-associated membrane protein (LAMP)1, LAMP2, and lysosomal integral membrane protein 2] around the lipid vacuoles facilitated the diagnosis of CESD in fixed paraffin-embedded material. Additional diagnostic clues included autofluorescent detection of ceroid induction in storage macrophages and the absence of lipopigment in hepatocytes. Stored liquid crystals of cholesteryl esters, which are associated with Maltese cross-type birefringence, were best appreciated in unfixed biopsy samples. CONCLUSIONS: The pathological diagnosis of CESD requires a high index of suspicion, and can be rapidly and effectively appreciated at the light microscopy level, even in routine fixed paraffin-embedded liver samples with immuohistochemical staining for lysosomal markers.

Glycosphingolipid profile of the apical pole of human placental capillaries: the relevancy of the observed data to Fabry disease.

A series of six full-term placentas and umbilical cords were examined using the in situ detection of globotriaosylceramide (Gb3Cer), GM1 ganglioside (GM1), GM3 ganglioside (GM3), cholesterol and caveolin 1. Immunohistochemical study showed uniform distinct staining of the apical membrane of villous capillary endothelial cells for Gb3Cer, GM1, GM3 and cholesterol. There was also a strong signal for caveolin 1. The immunophenotype suggests the presence of caveola-associated raft microdomains. The immunophenotype was almost completely shared with the extravillous intravascular trophoblast in the basal plate. It was absent in the endothelial cells of umbilical vessels and in the capillaries of somatic structures (heart, lung, skeletal muscle and skin) in neonates as well as in adults, including capillaries of the proliferative endometrium. Results of in situ analyses were confirmed by lipid chromatographic analysis of tissue homogenates and by tandem mass spectrometry. Lysosomal Gb3Cer turnover was followed in three placentas including umbilical cords from Fabry disease (α-galactosidase A deficiency). Lysosomal storage was restricted to vascular smooth muscle cells and to endothelial cells of umbilical vessels. Placental villous capillary endothelial cells displaying a strong non-lysosomal staining for Gb3Cer were free of lysosomal storage.

Mutations in DNAJC5, encoding cysteine-string protein alpha, cause autosomal-dominant adult-onset neuronal ceroid lipofuscinosis.

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.

Adipocytes participate in storage in α-galactosidase deficiency (Fabry disease).

Ultrastructural and histochemical studies of bioptic and postmortem tissue samples from ten Fabry hemizygotes showed lysosomal storage in adipocytes as a constant feature of the classic phenotype of α-galactosidase (GLA) deficiency. The storage was represented by a crescent-shaped line of storage lysosomes of varying thicknesses restricted to the perinuclear subplasmalemmal area. The ultrastructure of the storage lysosomes was dominated by concentric lipid membranes modified by simultaneous deposition of autofluorescent ceroid. Storage was widely expressed in adipose tissue. The number of storage lysosomes was increased, and the lysosomes were more clustered in adipocytes with less voluminous lipid content. The findings should attract interest to studies of adipose tissue biology in Fabry disease, a topic that has not been studied so far. In terms of cell biology, the observations represent indirect evidence of significant lysosomal turnover of α-galactose lipid conjugates in adipocytes demasked by GLA deficiency. The results extend the thus far limited information on the adipocyte lysosomal system and its participation in lysosomal storage disorders.

Cystathionine beta-synthase null homocystinuric mice fail to exhibit altered hemostasis or lowering of plasma homocysteine in response to betaine treatment.

Cystathionine beta-synthase (CBS) deficient homocystinuria is an inherited metabolic defect that if untreated typically results in mental retardation, thromboembolism and a range of connective tissue disturbances. A knockout mouse model has previously been used to investigate pathogenic mechanisms in classical homocystinuria (Watanabe et al., PNAS 92 (1995) 1585-1589). This mouse model exhibits a semi-lethal phenotype and the majority of mice do not survive the early neonatal period. We report here that the birth incidence of cbs (-/-) mice produced from heterozygous crosses is non-Mendelian and not significantly improved by treatment with either the Hcy lowering compound betaine or the cysteine donor N-acetylcysteine. Betaine treatment did improve survival of cbs (-/-) mice and restored fertility to female cbs (-/-) mice but did so without significantly lowering Hcy levels. Surviving cbs (-/-) mice failed to show any alteration in coagulation parameters compared to wild-type controls. Moribund cbs (-/-) mice exhibited severe liver injury and hepatic fibrosis while surviving cbs (-/-) mice although less severely affected, still exhibited a level of severe liver injury that is not found in the human disease. The hepatopathy observed in this model may offer an explanation for the failure of cbs (-/-) mice to respond to betaine or exhibit a hypercoagulative phenotype. We conclude that although this model provides useful data on the biochemical sequelae of classical homocystinuria, it does not successfully recapitulate a number of important features of the human disease and its use for studying mechanisms in homocystinuria should be treated with caution as the hepatopathy produces changes which could influence the results.

A novel transgenic mouse model of CBS-deficient homocystinuria does not incur hepatic steatosis or fibrosis and exhibits a hypercoagulative phenotype that is ameliorated by betaine treatment.

Cystathionine beta-synthase (CBS) catalyzes the condensation of homocysteine (Hcy) and serine to cystathionine, which is then hydrolyzed to cysteine by cystathionine gamma-lyase. Inactivation of CBS results in CBS-deficient homocystinuria more commonly referred to as classical homocystinuria, which, if untreated, results in mental retardation, thromboembolic complications, and a range of connective tissue disorders. The molecular mechanisms that underlie the pathology of this disease are poorly understood. We report here the generation of a new mouse model of classical homocystinuria in which the mouse cbs gene is inactivated and that exhibits low-level expression of the human CBS transgene under the control of the human CBS promoter. This mouse model, designated "human only" (HO), exhibits severe elevations in both plasma and tissue levels of Hcy, methionine, S-adenosylmethionine, and S-adenosylhomocysteine and a concomitant decrease in plasma and hepatic levels of cysteine. HO mice exhibit mild hepatopathy but, in contrast to previous models of classical homocystinuria, do not incur hepatic steatosis, fibrosis, or neonatal death with approximately 90% of HO mice living for at least 6months. Tail bleeding determinations indicate that HO mice are in a hypercoagulative state that is significantly ameliorated by betaine treatment in a manner that recapitulates the disease as it occurs in humans. Our findings indicate that this mouse model will be a valuable tool in the study of pathogenesis in classical homocystinuria and the rational design of novel treatments.

Abnormal nonstoring capillary endothelium: a novel feature of Gaucher disease. Ultrastructural study of dermal capillaries.

Ultrastructural study of skin biopsies in two cases of Gaucher disease (GD) patients (types II and III) revealed hitherto unknown alteration of the blood capillary endothelial cells (ECs) featured by hypertrophy and numerous subplasmalemmal microvesicles underneath both the apical and basal membranes. There was also prominent apical membrane folding with formation of filiform and large cytoplasmic projections, with occasional transcapillary cytoplasmic bridges. Similar, though less frequently expressed, changes were manifested at the basal membrane by numerous cytoplasmic projections into the subendothelial space. Regressive changes with EC breakdown were rare. Lysosomal storage was always absent. Besides EC hypertrophy, there was also increased EC density in the capillary lumen, leading to pronounced changes in capillary architecture with loose or incomplete EC anchoring. There were also signs of EC sprouting. Some pericytes displayed an increase in size and number of cytoplasmic processes, which often extended into distant pericapillary regions. The spectrum of changes suggests that a significant positive growth effect on EC occurs in GD. The putative mechanisms triggered by GBA1 deficiency leading to EC involvement are discussed. The authors are well aware of the fact the results, based on a nontraditional type of bioptic samples, are preliminary, but they are worth following, as further ultrastructural and functional studies of blood endothelium in GD may open a novel field in molecular cell pathophysiology of the disorder: endothelial dysfunction.

Association between cardiac energy metabolism and gain of left ventricular mass in Fabry disease.

Left ventricular (LV) hypertrophy is the hallmark of cardiac involvement in Fabry disease (FD). However, its pathogenesis is not clearly understood as pathologic substrate accumulation represents only 1-2% of the total cardiac mass. Abnormal myocardial energy metabolism has been previously demonstrated in different forms of cardiomyopathies. We hypothesized that myocardial energy status at the time of diagnosis could have a relationship to gain of LV mass in FD. In the group of 16 affected subjects, the indicators of energetic state of cardiac muscle determined by magnetic resonance spectroscopy showed significant negative correlation with annual increase in LV mass, evaluated during long-term follow-up (8 ± 3 years). Myocardial energy metabolism may therefore represent one of the mechanisms contributing to development of FD-related cardiomyopathy.

Dominant renin gene mutations associated with early-onset hyperuricemia, anemia, and chronic kidney failure.

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.

Right ventricular involvement in Fabry disease.

The aim of the study was to describe right ventricular (RV) structural and functional changes in Fabry disease (FD). A detailed echocardiographic examination was performed in 58 patients with proven FD (mean age 40 +/- 16 years, 24 men). RV hypertrophy (RVH) was present in 40% of affected subjects with similar prevalence in both genders. Approximately two thirds of patients with left ventricular hypertrophy (LVH) also exhibited RVH. RV dilatation was not present in any subject. RV systolic dysfunction was noted in only 1 female subject. RV diastolic dysfunction was present in 47% of 45 subjects in whom RV filling was assessed. RV diastolic dysfunction was associated with the presence of RVH. A significant correlation between RV wall thickness and age (r = 0.52, P < .001) and left ventricular mass index (r = 0.70, P < .001) was noted. RVH with normal chamber size and preserved systolic but impaired diastolic function represents a typical RV structural change in FD. Its prevalence and degree are related to the prevalence and degree of LVH and the age of the patient.

Replacement of alpha-galactosidase A in Fabry disease: effect on fibroblast cultures compared with biopsied tissues of treated patients.

The function and intracellular delivery of enzyme therapeutics for Fabry disease were studied in cultured fibroblasts and in the biopsied tissues of two male patients to show diversity of affected cells in response to treatment. In the mutant fibroblasts cultures, the final cellular level of endocytosed recombinant alpha-galactosidases A (agalsidases, Fabrazyme, and Replagal) exceeded, by several fold, the amount in control fibroblasts and led to efficient direct intra-lysosomal hydrolysis of ((3)H)Gb3Cer. In contrast, in the samples from the heart and some other tissues biopsied after several months of enzyme replacement therapy (ERT) with Fabrazyme, only the endothelial cells were free of storage. Persistent Gb3Cer storage was found in cardiocytes (accompanied by increase of lipopigment), smooth muscle cells, fibroblasts, sweat glands, and skeletal muscle. Immunohistochemistry of cardiocytes demonstrated, for the first time, the presence of a considerable amount of the active enzyme in intimate contact with the storage compartment. Factors responsible for the limited ERT effectiveness are discussed, namely post-mitotic status of storage cells preventing their replacement by enzyme supplied precursors, modification of the lysosomal system by longstanding storage, and possible relative lack of Sap B. These observations support the strategy of early treatment for prevention of lysosomal storage.

Proteomic analysis of hepatic iron overload in mice suggests dysregulation of urea cycle, impairment of fatty acid oxidation, and changes in the methylation cycle.

Liver iron overload can be found in hereditary hemochromatosis, chronic liver diseases such as alcoholic liver disease, and chronic viral hepatitis or secondary to repeated blood transfusions. The excess iron promotes liver damage, including fibrosis, cirrhosis, and hepatocellular carcinoma. Despite significant research effort, we remain largely ignorant of the cellular consequences of liver iron overload and the cellular processes that result in the observed pathological changes. In addition, the variability in outcome and the compensatory response that likely modulates the effect of increased iron levels are not understood. To provide insight into these critical questions, we undertook a study to determine the consequences of iron overload on protein levels in liver using a proteomic approach. Using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) combined with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), we studied hepatic iron overload induced by carbonyl iron-rich diet in mice and identified 30 liver proteins whose quantity changes in condition of excess liver iron. Among the identified proteins were enzymes involved in several important metabolic pathways, namely the urea cycle, fatty acid oxidation, and the methylation cycle. This pattern of changes likely reflects compensatory and pathological changes associated with liver iron overload and provides a window into these processes.

Mucolipidosis IV: report of a case with ocular restricted phenotype caused by leaky splice mutation.

PURPOSE: To confirm and define a molecular basis for a case of mucolipidosis type IV (ML IV) with an extremely atypical phenotype pattern. DESIGN: Observational case report of a patient with ML IV with disease progression restricted to ocular symptoms. METHODS: Complete ophthalmologic and neurologic examination. Ultrastructural examination of white blood cells, skin, conjunctiva, and corneal epithelium. The MCOLN1 gene was sequenced from cDNA and the proportion of splicing variants were assessed by quantitative allele-specific polymerase chain reaction. RESULTS: Absence of any neurological abnormalities. Retinal pathologic features were the main cause of visual disability: low visual acuity and cloudy corneas since 2 years of age, progressive decrease in visual acuity since the age of 9 years. Ultrastructural examination showed storage lysosomes filled with either concentric membranes or lucent precipitate in corneal and conjunctive epithelia and in vascular endothelium. Cultured fibroblasts were free of any autofluorescence. Sequencing of the MCOLN1 gene identified compound heterozygosity for D362Y and A-->T transition leading to the creation of a novel donor splicing site and a 4-bp deletion from exon 13 at the mRNA level. Both normal and pathologic splice forms were detected in skin fibroblasts and leukocytes, with the normal form being more abundant. CONCLUSIONS: The case of this patient with ML IV is unique and is characterized by a curious lack of generalized symptoms. In this patient, the disorder was limited to the eyes and appeared without the usual psychomotor deterioration. The resulting phenotype is the mildest seen to date.

Relationship between X-inactivation and clinical involvement in Fabry heterozygotes. Eleven novel mutations in the alpha-galactosidase A gene in the Czech and Slovak population.

We have identified 21 different alpha-galactosidase A gene (GLA) mutations in 22 unrelated Czech and Slovak families with Fabry disease. Eleven of these mutations were novel (point mutations D93N, A135V, D155H, G171R, Q280K, G360S, Q330X, splicing errors c.194ins14, c.801ins36 and deletions c.674_732del59, g.3405_6021del2617). Genotyping of family members for family-specific mutations revealed 55 heterozygotes that manifested clinical symptoms of different severity. To examine the contribution of X-inactivation skewing to disease manifestation in Fabry heterozygotes, we have adopted the Mainz severity scoring scheme and compared the score values with the X-inactivation status in 39 carriers in an age-dependent manner. The age-score trendline of Fabry females who had a predominantly inactivated X-chromosome bearing a wild-type GLA allele (10 of 38 females) was markedly steeper than in the rest of the cohort. One female carrier with an inactivated mutated allele had a low score value when compared to the other heterozygotes of the same age. These data suggest that X-inactivation is indeed a major factor determining the severity of clinical involvement in Fabry heterozygotes. There was a statistically significant difference between the severity score values of heterozygotes with random and non-random X-chromosome inactivation at the 5% level of significance. Further studies will show if the degree of the wildtype allele inactivation will be useful as a predictive marker of severity of phenotype in Fabry heterozygotes. Although the correlation between X-inactivation skewing and presentation of the disease in Fabry heterozygotes has previously been suggested in the literature, this report is among the first attempts to examine this relationship systematically.

Recurrence of Fabry disease as a result of paternal germline mosaicism for alpha-galactosidase a gene mutation.

We present two sisters with a severe form of Fabry disease, who both carry the same mutation in the alpha-galactosidase A (alpha-gal A) gene (Q330X). Each of the sisters developed renal failure in the third decade of life; the older sibling underwent renal transplantation at 40 years of age. The severe phenotype of the siblings correlates with results of the X-inactivation study: examination of methylation status in human androgene receptor (HUMARA) gene suggests preferential inactivation of the wild-type allele in both patients. Patients' parents had no symptoms of Fabry disease and were tested negative for the mutation Q330X in DNA isolated from peripheral leukocytes, mouth wash cells, and urinary sediment cells. Genotype analysis using DXS7424 marker showed paternal origin of the mutation. The father's sperm was then tested for presence of the mutation to examine the possibility of the germline mosaicism. Both mutant and wild-type alleles were found in DNA isolated from father's sperm. The apparent explanation of these findings is germline mosaicism due to mutation event during the embryonic development of sperm producing cells (spermatogonia). This is the first case of germline mosaicism in Fabry disease reported in the literature.