Fabry Disease Mutations Research Articles

Prevalence of CADASIL and Fabry Disease in a Cohort of MRI Defined Younger Onset Lacunar Stroke.

Background and PurposeCerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), caused by mutations in the NOTCH3 gene, is the most common monogenic disorder causing lacunar stroke and cerebral small vessel disease (SVD). Fabry disease (FD) due to mutations in the GLA gene has been suggested as an underdiagnosed cause of stroke, and one feature is SVD. Previous studies reported varying prevalence of CADASIL and FD in stroke, likely due to varying subtypes studied; no studies have looked at a large cohort of younger onset SVD. We determined the prevalence in a well-defined, MRI-verified cohort of apparently sporadic patients with lacunar infarct.MethodsCaucasian patients with lacunar infarction, aged ≤70 years (mean age 56.7 (SD8.6)), were recruited from 72 specialist stroke centres throughout the UK as part of the Young Lacunar Stroke DNA Resource. Patients with a previously confirmed monogenic cause of stroke were excluded. All MRI’s and clinical histories were reviewed centrally. Screening was performed for NOTCH3 and GLA mutations.ResultsOf 994 subjects five had pathogenic NOTCH3 mutations (R169C, R207C, R587C, C1222G and C323S) all resulting in loss or gain of a cysteine in the NOTCH3 protein. All five patients had confluent leukoaraiosis (Fazekas grade ≥2). CADASIL prevalence overall was 0.5% (95% CI 0.2%-1.1%) and among cases with confluent leukoaraiosis 1.5% (95% CI 0.6%-3.3%). No classic pathogenic FD mutations were found; one patient had a missense mutation (R118C), associated with late-onset FD.ConclusionCADASIL cases are rare and only detected in SVD patients with confluent leukoaraiosis. No definite FD cases were detected.

Identification of mutations in Colombian patients affected with Fabry disease

Fabry Disease (FD) is an X-linked inborn error of glycosphingolipid catabolism, caused by a deficiency of the lisosomal α-galactosidase A (AGAL). The disorder leads to a vascular disease secondary to the involvement of kidney, heart and the central nervous system. The mutation analysis is a valuable tool for diagnosis and genetic counseling. Although more than 600 mutations have been identified, most mutations are private. Our objective was to describe the analysis of nine Colombian patients with Fabry disease by automated sequencing of the seven exons of the GLA gene. Two novel mutations were identified in two patients affected with the classical subtype of FD, in addition to other 6 mutations previously reported. The present study confirms the heterogeneity of mutations in Fabry disease and the importance of molecular analysis for genetic counseling, female heterozygotes detection as well as therapeutic decisions.

Fabry disease in a male patient: De novo mutation, germline mosaicism or both?

Background: Fabry disease (FD) is an innate error of glycosphingolipid catabolism due to a genetic defect in the gene which codifies for alphagalactosidase A (α-Gal A) production (GLA gene). Mutations affecting the GLA gene result in deficiency of this lysosomal enzyme, which in turn leads to progressive accumulation of neutral glycosphingolipids, primarily globotriaosylceramide (GL-3), in different cell types throughout the body, including renal glomerular and tubular epithelial cells, myocardial cells and valvular fibrocytes, and nervous system. FD has an X linked inheritance. Currently, more than 600 disease-causing mutations in the GLA gene have been described. Most of these mutations are private, restricted to an individual family. De novo mutations are rare; population reports establish their frequency between 4 and 7%. We present the case of a pediatric male patient with a previously unreported de novomutation, which during family tree analysis, revealed a positive sibling, but nomutation found in the mother. Case presentation: A 17 year old male patient from Durango, Mexico with a negative family history presented at our ward. His initial symptoms began at 10 years of age and were primarily neurological: recurrent and progressive acroparesthesias several times a week, upper and lower extremity weaknesswithmovement limitations and fainting spells; he also complained of intermittent fever, hypohidrosis, abdominal pain and diarrhea. Clinical evaluation of the patient revealed tortuosity of retinal vessels, sinus bradycardia and hypotension and distal hyperesthesia in arms and legs. Fabry disease was suspected; αGal A enzyme activity, GLA gene analysis and lysoGL-3 measurement confirmed the diagnosis. Family tree evaluation was carried out; one six-year-old brother also had the mutation; however no mutation was found in the mother’s molecular analysis. FD specific evaluation of the index case revealed positive 24-hour proteinuria (6.5 mg/kg/h reference b4 ml/kg/h); CNSMRI was normal. Kidney biopsy was conducted; electron microscopy analysis of specimens revealed dense lamellar bodies corresponding to lipid material deposits in all cell groups, confirming the presence of a pathological mutation. Conclusions: De novo onset mutations in FD are rare; however, even more infrequent is the presence of germline mutations, which have been reported only periodically in case reports. Unfortunately, female germline cells are difficult to obtain to confirm this diagnosis. doi:10.1016/j.ymgme.2014.12.069

Spatial distribution of brain ceramides in an acid ceramidase deficient murine model: Subsequent histological manifestations and functional deficits

Background: Fabry disease (FD) is an innate error of glycosphingolipid catabolism due to a genetic defect in the gene which codifies for alphagalactosidase A (α-Gal A) production (GLA gene). Mutations affecting the GLA gene result in deficiency of this lysosomal enzyme, which in turn leads to progressive accumulation of neutral glycosphingolipids, primarily globotriaosylceramide (GL-3), in different cell types throughout the body, including renal glomerular and tubular epithelial cells, myocardial cells and valvular fibrocytes, and nervous system. FD has an X linked inheritance. Currently, more than 600 disease-causing mutations in the GLA gene have been described. Most of these mutations are private, restricted to an individual family. De novo mutations are rare; population reports establish their frequency between 4 and 7%. We present the case of a pediatric male patient with a previously unreported de novomutation, which during family tree analysis, revealed a positive sibling, but nomutation found in the mother. Case presentation: A 17 year old male patient from Durango, Mexico with a negative family history presented at our ward. His initial symptoms began at 10 years of age and were primarily neurological: recurrent and progressive acroparesthesias several times a week, upper and lower extremity weaknesswithmovement limitations and fainting spells; he also complained of intermittent fever, hypohidrosis, abdominal pain and diarrhea. Clinical evaluation of the patient revealed tortuosity of retinal vessels, sinus bradycardia and hypotension and distal hyperesthesia in arms and legs. Fabry disease was suspected; αGal A enzyme activity, GLA gene analysis and lysoGL-3 measurement confirmed the diagnosis. Family tree evaluation was carried out; one six-year-old brother also had the mutation; however no mutation was found in the mother’s molecular analysis. FD specific evaluation of the index case revealed positive 24-hour proteinuria (6.5 mg/kg/h reference b4 ml/kg/h); CNSMRI was normal. Kidney biopsy was conducted; electron microscopy analysis of specimens revealed dense lamellar bodies corresponding to lipid material deposits in all cell groups, confirming the presence of a pathological mutation. Conclusions: De novo onset mutations in FD are rare; however, even more infrequent is the presence of germline mutations, which have been reported only periodically in case reports. Unfortunately, female germline cells are difficult to obtain to confirm this diagnosis. doi:10.1016/j.ymgme.2014.12.069

Plasma globotriaosylsphingosine (lysoGb3) could be a biomarker for Fabry disease with a Chinese hotspot late-onset mutation (IVS4 + 919G>A)

BackgroundPrevious studies revealed a high incidence of late-onset Fabry disease mutation, IVS4+919G>A, in Taiwan. However, the natural course is largely unclear and suitable biomarkers for monitoring disease progress are unavailable. Methods and resultsPatients carrying IVS4+919G>A or classical Fabry mutations were enrolled in this study. The subjects ranged from newborn to eighty year old adults. Plasma globotriaosylceramide (Gb3) and globotriaosylsphingosine (lysoGb3) were measured by LC-MS/MS in subjects to evaluate the sensitivity of these two biomarkers. All adult males and symptomatic females could be distinguished from healthy controls by an elevated plasma lysoGb3 level. The lysoGb3 level was also related to the left ventricular mass considering gender and age (p<0.01). Moreover, approximately 70% of male and 45% of female newborns already had an elevated plasma lysoGb3 level which increased gradually as the subjects got older (p<0.01). ConclusionsPlasma lysoGb3 is a more sensitive and reliable biomarker than plasma Gb3. LysoGb3 also correlated with age and left ventricular mass index in Fabry patients with IVS4+919G>A mutation. Because lots of infants with the IVS4+919G>A mutation already had elevated lysoGb3 levels at birth, that indicates that the development of hypertrophic cardiomyopathy may require a long and insidious course after lysoGb3 accumulation.

Early cardiovascular remodelling in Fabry disease

Fabry disease (FD) is a rare X-linked genetic disorder caused by the deficiency or absent activity of lysosomal α-galactosidase A. Cardiovascular remodelling is a hallmark of FD. The present study aimed to comprehensively evaluate the cardiac, vascular and microvascular status in a population of patients with genetic mutations for FD without left ventricular hypertrophy (LVH). This study includes subjects carrying genetic mutations for FD (Fabry disease mutation-carrier, FDMC) without LVH (n = 19). A group of control subjects (n = 19) matched for age, sex, body mass index and cardiovascular risk factors were also included. All subjects underwent echocardiography, carotid ultrasound scan, endothelial flow-mediated dilatation (FMD) and nailfold capillaroscopy (NFC) assessment. When compared to the subjects in the control group, FDMC patients showed significantly lower mean values of systolic myocardial velocity (7.33 ± 1.28 vs. 10.08 ± 1.63 cm/s, p < 0.0001), longitudinal systolic strain (-18.07 ± 1.72 vs. -21.15 ± 2.22%, p < 0.0001), significantly higher E/E' mean values (7.15 ± 1.54 vs. 5.98 ± 1.27, p = 0.016) and intima-media thickness mean values (0.80 ± 0.20 vs. 0.61 ± 0.19 mm, p = 0.005), significantly lower FMD (8.3 ± 4.6 vs. 12.2 ± 5.0%, p = 0.02), more atypical capillaries and irregular NFC architecture in FDMC than control subjects (52.6 vs. 0%, p < 0.0001; 78.9 vs. 36.8%, p = 0.02 respectively). FD progressively involves cardiac, macrovascular and microvascular systems in an early stage. These features are present even in asymptomatic mutation carriers without LVH.

Fabry disease in unselected patients with TIA or stroke: population‐based study

Fabry disease (FD) is an X-linked lysosomal storage disorder frequently associated with cerebrovascular disease. In recent years, the prevalence of FD has been reported to be up to 4% in cryptogenic young stroke patients. However, there have been no population-based studies in unselected patients with transient ischaemic attack (TIA) or stroke across the full range of ages. We determined the prevalence of FD mutations in consecutive patients from a population-based study of acute TIA or ischaemic stroke (Oxford Vascular Study). Analysis included amplifying of the α-galactosidase A gene by polymerase chain reaction, denaturing high-performance liquid chromatography (dHPLC) analysis and sequencing using standard automated sequencing protocols [Mutation Surveyor software (Softgenetics)] where the dHPLC indicated a possible mutation. Samples of 1046 consecutive patients (52% women; mean age 73.2years; 15% age <60 years; 572 stroke; 474 TIA) were tested. No patient had a known gene mutation causing FD, giving an upper 95% confidence interval around the estimated frequency of 0.35% overall and 2.37% in the 154 patients aged under 60years. However, in 5 (0.48%) samples, a known polymorphism or sequence variation in the gene was identified that can be associated with lower than normal enzyme activity in plasma without causing the full clinical manifestation of FD. Fabry disease is rare in an unselected group of UK patients with TIA or stroke. Larger studies in unselected younger patients with cryptogenic stroke are required to determine whether routine screening is justified in this group.

Fabry Disease Mutations Addressable with Migalastat HCl, an Investigational Chaperone Therapy. Screening Results from FACETS, a Phase 3 Study in Male and Female Patients

for HSCT evaluation. It was determined that there might also be a maternal family history of Fragile X syndrome. EB, and his mother, were tested and the mother was found to be a carrier of a premutation allele (82 repeats) and EB was found to be mosaic for a premutation allele and a full mutation (107 and >200 repeats). Since Fragile X syndrome, even in a mosaic state, is expected to cause cognitive disability in males, it was determined that even if HSCT was performed, this child may see only minimal neurological benefit due to the underlying Fragile X syndrome. This abstract will discuss the ethical dilemmas presented by these results and how the dilemma was eventually resolved with emphasis on ethical principles.

Pharmacological chaperone therapy for Fabry disease

Fabry disease is an inherited lysosomal storage disorder caused by deficient α-galactosidase A activity. Many missense mutations in Fabry disease often cause misfolded gene products, which leads to their retention in the endoplasmic reticulum by the quality control system; they are then removed by endoplasmic reticulum-associated degradation. We discovered that a potent α-galactosidase A inhibitor, 1-deoxygalactonojirimycin, acts as a pharmacological chaperone to facilitate the proper folding of the mutant enzyme by binding to its active site, thereby improving its stability and trafficking to the lysosomes in mammalian cells. The oral administration of 1-deoxygalactonojirimycin to transgenic mice expressing human mutant α-galactosidase A resulted in significant increases in α-galactosidase A activity in various organs, with concomitant reductions in globotriaosylceramide, which contributes to the pathology of Fabry disease. Seventy-eight missense mutations were found to be responsive to 1-deoxygalactonojirimycin. These data indicate that many patients with Fabry disease could potentially benefit from pharmacological chaperone therapy.

Elevation of urinary globotriaosylceramide (GL3) in infants with Fabry disease

Background Fabry disease is caused by a deficiency of α-galactosidase A (α-Gal A), which results in the accumulation of globotriaosylceramide (GL3) and related glycosphingolipids in different organs. Urinary GL3 levels increase in symptomatic Fabry disease patients, but it is not clear whether urinary GL3 excretion also increases in young or pre-symptomatic patients. Subjects and methods Eighty-nine newborns with leukocyte α-Gal A activities of less than 30% of the normal mean were discovered by newborn screening. Urine samples were collected on filter paper, and GL3 levels were measured using liquid chromatography–tandem mass spectrometry. Results Five newborns with classic Fabry disease mutations all had elevated urinary GL3 levels (mean = 5.2 mg/mmol creatinine (creat.), range = 0.80–14.39, normal < 0.6). Among the 84 newborns with later-onset mutations, 45 (54%) had a mild elevation of urinary GL3 levels (mean = 1.1 mg/mmol creat., range = 0.60–3.07, normal < 0.6). The urinary GL3 levels decreased in all newborns over the course of a three-year follow-up period. However, four children with classic mutations and seven with IVS4+919G>A mutations still had elevated GL3 levels at the end of the study. Conclusion Elevated urinary GL3 levels can be present at birth in Fabry disease patients, suggesting an early involvement of the kidneys in this disease. The increased urinary GL3 excretion in those with later-onset mutations supports a pathogenic role for these mutations.

Novel sequence variants of the α-galactosidase A gene in patients with Fabry disease

We carried out molecular studies of 15 unrelated Hungarian families diagnosed with Fabry disease (FD). Genetic analysis of the α-galactosidase A gene was performed in 22 hemizygous males and 34 females. One of the female patients with severe disease phenotype showed homozygosity for the recurrent c.644A > G mutation due to parental consanguinity. The c.644A > G mutation that has previously been found mostly in patients with the cardiac variant of FD, was associated with renal but not cardiac involvement in this female and in two other family members. In nine families, eight novel sequence variants such as small deletions (c.363delT, c.477delT, c.746delAC) and single nucleotide changes (c.107T > C, c.493G > C, c.796G > T, c.866T > G, c.871G > A) were found in addition to six previously described private mutations. This report contributes to the identification of novel disease-causing mutations in FD, and increases our knowledge on demographics and molecular characteristics of this rare lysosomal storage disorder. This is the first comprehensive overview of molecular genetic features of Hungarian patients with FD.

Absence of Cornea Verticillata in Hemizygotes of a Novel Mutation in Fabry Disease

To report a new pedigree of Fabry disease that does not display corneal involvement in hemizygotes. A 44-year-old man presented with proteinuria, chronic obstructive airway disease, hypoacusia, teleangiectasis, and hypohidrosis. A kidney biopsy, genetic study, and ophthalmological examination were conducted. The patient's 66-year-old mother and 45-year-old sister were also investigated. Glomerular changes associated with Fabry disease were found. The diagnosis was confirmed by genetic study of the alpha-galactosidase A gene. In exon 2, an adenine was changed to a guanine at codon 92. This mutation is called D92G or Asp92Gly. This mutation has not been reported previously. Systemic examination showed cardiac involvement and chronic obstructive airway disease in the mother of the patient. The same mutation was present in the mother and in the sister of the patient. Cornea verticillata was not present in the hemizygous male. The mother and the sister of the patient showed the features of cornea verticillata. The patient and his mother were treated with enzyme replacement therapy using recombinant alpha-galactosidase A. We report here a new mutation in Fabry disease. The hemizygote did not show corneal manifestations as opposed to heterozygotes in the described family. The severity of systemic disease was not correlated with the presence of corneal deposits.

Genomic analysis of Brazilian patients with Fabry disease

Fabry disease is an X-linked lysosomal disorder due to a-galactosidase A deficiency that causes storage of globotriaosylceramide. The gene coding for this lysosomal enzyme is located on the long arm of the X chromosome, in region Xq21.33-Xq22. Disease progression leads to vascular disease secondary to involvement of kidney, heart and the central nervous system. Detection of female carriers based solely on enzyme assays is often inconclusive. Therefore, mutation analysis is a valuable tool for diagnosis and genetic counseling. Many mutations of the a-galactosidase A gene have been reported with high genetic heterogeneity, being most mutations private found in only one family. The disease is panethnic, and estimates of incidence range from about 1 in 40,000 to 60,000 males. Our objective was to describe the analysis of 6 male and 7 female individuals belonging to 4 different Fabry disease families by automated sequencing of the seven exons of the alpha-galactosidase gene. Sequencing was performed using PCR fragments for each exon amplified from DNA extracted from peripheral blood. Three known mutations and one previously described in another Brazilian family were detected. Of 7 female relatives studied, 4 were carriers. Although the present study confirms the heterogeneity of mutations in Fabry disease, the finding of the same mutation previously detected in another Fabry family from our region raises the possibility of some founder effect, or genetic drift. Finally, the present study highlights the importance of molecular analysis for carrier detection and genetic counseling.

A sensitive mutation screening strategy for Fabry disease: detection of nine mutations in the alpha-galactosidase A gene.

Fabry disease is an X-linked recessive lysosomal storage disorder caused by a deficiency of alpha-galactosidase A (alpha-gal; EC 3.2.1.22). In the past, it has been difficult to give an unequivocal diagnosis of carrier status in Fabry disease because of the overlap between normal and heterozygote enzyme levels. To facilitate rapid and accurate carrier and hemizygote detection, a mutation detection strategy was devised to determine the lesion in our Fabry disease patients. The seven alpha-gal exons and adjacent intron boundaries from a representative member of each kindred were PCR amplified and analysed for the presence of sequence alterations by single-stranded conformation polymorphism (SSCP) analysis followed by PCR sequencing. Here we report the use of this strategy in the detection and analysis of the causative mutations in 9 patients with classic severe Fabry disease. Three deletions of 1-, 2-, and 3-bp (987delC, 717delAA, and delta E358), five amino acid substitutions (C52R, G128E, P205T, M284T, and N298K) and a mutation that affects the initiating methionine (M1I) were found in these patients. Counting a previously reported mutation, this strategy has now successfully detected all the Fabry disease mutations present in the 10 kindreds that have been analysed.

A sensitive mutation screening strategy for Fabry disease: Detection of nine mutations in the α‐galactosidase A gene

Fabry disease is an X-linked recessive lysosomal storage disorder caused by a deficiency of α-galactosidase A (α-gal; EC 3.2.1.22). In the past, it has been difficult to give an unequivocal diagnosis of carrier status in Fabry disease because of the overlap between normal and heterozygote enzyme levels. To facilitate rapid and accurate carrier and hemizygote detection, a mutation detection strategy was devised to determine the lesion in our Fabry disease patients. The seven α-gal exons and adjacent intron boundaries from a representative member of each kindred were PCR amplified and analysed for the presence of sequence alterations by single-stranded conformation polymorphism (SSCP) analysis followed by PCR sequencing. Here we report the use of this strategy in the detection and analysis of the causative mutations in 9 patients with classic severe Fabry disease. Three deletions of 1-, 2-, and 3-bp (987delC, 717delAA, and ΔE358), five amino acid substitutions (C52R, G128E, P205T, M284T, and N298K) and a mutation that affects the initiating methionine (M1I) were found in these patients. Counting a previously reported mutation, this strategy has now successfully detected all the Fabry disease mutations present in the 10 kindreds that have been analysed. © 1996 Wiley-Liss, Inc.

Alpha-galactosidase gene mutations in Fabry disease: heterogeneous expressions of mutant enzyme proteins.

Five point mutations were identified in unrelated Japanese Fabry disease hemizygotes: three new missense mutations, C142Y (425 G-->A), A156V (467 C-->T), and L166V (496 C-->G) in exon 3; one new splice site mutation at the 3' end of the consensus sequence in exon 4; one previously reported nonsense mutation, W44X (131 G-->A). C142Y expressed 50% of the normal enzyme protein in COS-1 cells, but catalytic activity was not detected. Both A156V and L166V expressed significant amounts of residual enzyme activity (6.7% and 9.8%) and enzyme proteins (10% each), the latter were more thermolabile at neutral pH than at acid pH, in vitro.

Synthesis and processing of alpha-galactosidase A in human fibroblasts. Evidence for different mutations in Fabry disease.

The synthesis and processing of the human lysosomal enzyme alpha-galactosidase A was examined in normal and Fabry fibroblasts. In normal cells, alpha-galactosidase A was synthesized as an Mr = 50,500 precursor, which contained phosphate groups in oligosaccharide chains cleavable by endoglucosaminidase H. The precursor was processed via ill-defined intermediates to a mature Mr 46,000 form. Processing was complete within 3-7 days after synthesis. In the presence of NH4Cl and in I-cell fibroblasts, the majority of newly synthesized alpha-galactosidase A was secreted as an Mr = 52,000 form. For comparison, the processing and stability of alpha-galactosidase A were examined in fibroblasts from five unrelated patients with Fabry disease, which is caused by deficient alpha-galactosidase A activity. In one cell line, synthesis of immunologically cross-reacting polypeptides was not detectable. In another, the synthesis, processing, and stability of alpha-galactosidase A was indistinguishable from that in normal fibroblasts. In a third Fabry cell line, the mutation retarded the maturation of alpha-galactosidase A. Finally, in two cell lines, alpha-galactosidase A polypeptides were synthesized that were rapidly degraded following delivery to lysosomes. These results clearly indicate that Fabry disease comprises a heterogeneous group of mutations affecting synthesis, processing, and stability of alpha-galactosidase A.

Fabry disease: isolation of a cDNA clone encoding human alpha-galactosidase A.

Fabry disease is an X-linked inborn error of metabolism resulting from the deficient activity of the lysosomal hydrolase, alpha-galactosidase A (alpha-Gal A; alpha-D-galactoside galactohydrolase, EC 3.2.1.22). To investigate the structure, organization, and expression of alpha-Gal A, as well as the nature of mutations in Fabry disease, a clone encoding human alpha-Gal A was isolated from a lambda gt11 human liver cDNA expression library. To facilitate screening, an improved affinity purification procedure was used to obtain sufficient homogeneous enzyme for production of monospecific antibodies and for amino-terminal and peptide microsequencing. On the basis of an amino-terminal sequence of 24 residues, two sets of oligonucleotide mixtures were synthesized corresponding to adjacent, but not overlapping, amino acid sequences. In addition, an oligonucleotide mixture was synthesized based on a sequence derived from an alpha-Gal A internal tryptic peptide isolated by reversed-phase HPLC. Four positive clones were initially identified by antibody screening of 1.4 X 10(7) plaques. Of these, only one clone (designated lambda AG18) demonstrated both antibody binding specificity by competition studies using homogeneous enzyme and specific hybridization to synthetic oligonucleotide mixtures corresponding to amino-terminal and internal amino acid sequences. Nucleotide sequencing of the 5' end of the 1250-base-pair EcoRI insert of clone lambda AG18 revealed an exact correspondence between the predicted and known amino-terminal amino acid sequence. The insert of clone lambda AG18 appears to contain the full-length coding region of the processed, enzymatically active alpha-Gal A, as well as sequences coding for five amino acids of the amino-terminal propeptide, which is posttranslationally cleaved during enzyme maturation.