Joe T.R. Clarke

Identification and Characterization of Ambroxol as an Enzyme Enhancement Agent for Gaucher Disease

Gaucher disease (GD), the most prevalent lysosomal storage disease, is caused by a deficiency of glucocerebrosidase (GCase). The identification of small molecules acting as agents for enzyme enhancement therapy is an attractive approach for treating different forms of GD. A thermal denaturation assay utilizing wild type GCase was developed to screen a library of 1,040 Food and Drug Administration-approved drugs. Ambroxol (ABX), a drug used to treat airway mucus hypersecretion and hyaline membrane disease in newborns, was identified and found to be a pH-dependent, mixed-type inhibitor of GCase. Its inhibitory activity was maximal at neutral pH, found in the endoplasmic reticulum, and undetectable at the acidic pH of lysosomes. The pH dependence of ABX to bind and stabilize the enzyme was confirmed by monitoring the rate of hydrogen/deuterium exchange at increasing guanidine hydrochloride concentrations. ABX treatment significantly increased N370S and F213I mutant GCase activity and protein levels in GD fibroblasts. These increases were primarily confined to the lysosome-enriched fraction of treated cells, a finding confirmed by confocal immunofluorescence microscopy. Additionally, enhancement of GCase activity and a reduction in glucosylceramide storage was verified in ABX-treated GD lymphoblasts (N370S/N370S). Hydrogen/deuterium exchange mass spectrometry revealed that upon binding of ABX, amino acid segments 243–249, 310–312, and 386–400 near the active site of GCase are stabilized. Consistent with its mixed-type inhibition of GCase, modeling studies indicated that ABX interacts with both active and non-active site residues. Thus, ABX has the biochemical characteristics of a safe and effective enzyme enhancement therapy agent for the treatment of patients with the most common GD genotypes.

Enzyme-Replacement Therapy With Agalsidase Alfa in Children With Fabry Disease

CONTEXT. Fabry disease is an X-linked multisystem disorder. Enzyme-replacement therapy in adults has limited efficacy in treating major sequelae of advanced Fabry disease, such as kidney failure or stroke. This prompted a study of the safety and efficacy of enzyme replacement at an earlier stage of Fabry disease. OBJECTIVES. Our purpose with this work was to evaluate safety and to explore efficacy of enzyme treatment with agalsidase alfa in pediatric patients with Fabry disease. METHODS. We conducted a 6-month open-label study at 3 tertiary care centers with 24 children (19 boys and 5 girls) with a mean age of 11.8 (range: 6.5–18) years, to examine safety parameters, including infusion reactions and antiagalsidase alfa antibodies. RESULTS. Agalsidase alfa was well tolerated, and all of the patients completed the study. Six boys and 1 girl had mild-to-moderate infusion reactions. One boy developed transient immunoglobulin G antibodies against agalsidase alfa. The boys showed a significant reduction in plasma globotriaosylceramide on treatment. Mean estimated glomerular filtration rate, cardiac structure, and function were normal and did not change over 26 weeks. Heart rate variability, as determined by 2-hour ambulatory monitoring, was decreased in the boys compared with the girls at baseline. All indices of heart rate variability improved significantly in the boys. Three patients with anhidrosis, as determined by quantitative sudomotor axon reflex testing, developed sweating. Six of 11 patients could reduce or cease their use of antineuropathic analgesics. CONCLUSIONS. Enzyme replacement with agalsidase alfa was safe in this study. The exploratory efficacy analysis documented increased clearance of globotriaosylceramide and improvement of autonomic function. Prospective long-term studies are needed to assess whether enzyme replacement initiated early in patients with Fabry disease is able to prevent major organ failure in adulthood.

Genomic rearrangements resulting in PLP1 deletion occur by nonhomologous end joining and cause different dysmyelinating phenotypes in males and females.

In the majority of patients with Pelizaeus-Merzbacher disease, duplication of the proteolipid protein gene PLP1 is responsible, whereas deletion of PLP1 is infrequent. Genomic mechanisms for these submicroscopic chromosomal rearrangements remain unknown. We identified three families with PLP1 deletions (including one family described elsewhere) that arose by three distinct processes. In one family, PLP1 deletion resulted from a maternal balanced submicroscopic insertional translocation of the entire PLP1 gene to the telomere of chromosome 19. PLP1 on the 19qtel is probably inactive by virtue of a position effect, because a healthy male sibling carries the same der(19) chromosome along with a normal X chromosome. Genomic mapping of the deleted segments revealed that the deletions are smaller than most of the PLP1 duplications and involve only two other genes. We hypothesize that the deletion is infrequent, because only the smaller deletions can avoid causing either infertility or lethality. Analyses of the DNA sequence flanking the deletion breakpoints revealed Alu-Alu recombination in the family with translocation. In the other two families, no homologous sequence flanking the breakpoints was found, but the distal breakpoints were embedded in novel low-copy repeats, suggesting the potential involvement of genome architecture in stimulating these rearrangements. In one family, junction sequences revealed a complex recombination event. Our data suggest that PLP1 deletions are likely caused by nonhomologous end joining.

Narrative Review: Fabry Disease

The patient was a thin, unhealthy-looking 29-year-old man with a 15-month history of increasing general malaise and fatigability, polyuria, and swelling of the ankles. He reported frequent episodes of tingling in his fingers and toes associated with severe, lancinating pain in the legs (especially during febrile illnesses) dating back to middle childhood. The pain was generally unresponsive to treatment with acetaminophen or ibuprofen. The patient also reported almost daily attacks of diffuse central and upper abdominal pain, which were aggravated by eating fatty or spicy foods and were accompanied by frequent, loose, small-volume stools, often associated with urgency and tenesmus. He also reported marked heat intolerance and almost complete lack of sweating. The patient did not have a history of drug or alcohol abuse. The patients mother reported that her maternal grandfather and her only brother had died of end-stage renal disease, the former at age 55 years and the latter at age 49 years. Her other son, the patients brother, was apparently healthy at age 14 years, and a sister, age 17 years, was also apparently well. On physical examination, the patient appeared chronically ill and had mild, diffuse, upper abdominal tenderness on palpation. Examination of the external genitalia revealed numerous discrete skin lesions on the scrotum and penis. The lesions were 1 to 2 mm in diameter, slightly raised, dark red to black, and lacked inflammation. They did not blanch with pressure. Results on neurologic examination were normal aside from patchy impairment of temperature sensation in the lower legs and feet. Urinalysis revealed a specific gravity of 1.010 and moderate proteinuria (2.4 g/d). The hemoglobin level was 110 g/L (11.0 g/dL). The leukocyte count and differential were normal, but the erythrocyte sedimentation rate was increased at 16 mm/h. Electrocardiography showed sinus bradycardia, first-degree heart block, and voltage evidence of left ventricular hypertrophy (Figure 1). The plasma creatinine concentration was elevated (267 mol/L [3.02 mg/dL]). Figure 1. Electrocardiogram showing sinus bradycardia and evidence of left ventricular hypertrophy. Measurement of -galactosidase A in the plasma revealed less than 2% normal enzyme activity, which was consistent with Fabry disease. Mutation analysis subsequently showed that the patient was hemizygous for a single base sequence change causing substitution of a proline for an alanine residue in codon 143 (A143P). Renal biopsy was done. Microscopic examination showed an irregular pattern of glomerular sclerosis and vacuolation of glomerular and tubular epithelial cells (Figure 2, A). Electron microscopic examination revealed masses of membrane-bound, concentric lamellar, membranous inclusions within glomerular epithelial cells (Figure 2, B). Enzyme replacement therapy (ERT) was started with biweekly intravenous infusions of agalsidase-, a recombinant form of -galactosidase A, which is produced in cultured cells. One week before the first treatment, the patient experienced a sudden onset of double vision, associated with the appearance on magnetic resonance imaging of a small discrete lesion posteromedial to the right red nucleus in the upper midbrain (Figure 3). Figure 2. Histopathologic changes in the kidney. A. Two glomeruli, 1 with marked vacuolation of glomerular epithelial cells (left) and 1 with moderately advanced sclerosis (right) (periodic acidSchiff stain). B. Electron photomicrograph of glomerular epithelial cells showing intralysosomal concentric lamellar membranous inclusions typical of Fabry disease. Figure 3. Axial fluid-attenuated inversion recovery magnetic resonance image (TR9002. 0/TE170.5) of the brain showing a lesion in the right posteromedial midbrain ( arrow ). Clinicopathologic Correlations Fabry disease is an X-linked lysosomal storage disease caused by deficiency of the enzyme -galactosidase A. This deficiency results in accumulation of 2 neutral glycosphingolipids (GSLs), globotriaosylceramide (Gb3) and digalactosylceramide, in various tissues throughout the body. Although the enzyme is also required for degradation of blood group substances B and P1, these GSLs do not seem to play a substantial role in the pathogenesis of the disease. The incidence of the disease has been estimated to be approximately 1 in 55000 male births (1), although this almost certainly is a substantial underestimate of the true frequency, particularly of milder variants of the disease (2). Our understanding of the natural history of this rare disease is incomplete. Although death from Fabry diseaserelated complications before adulthood is probably very rare, most affected males die by the end of the sixth decade of life (3, 4). As many as 70% of carrier women experience symptoms of the disease, although the clinical manifestations of the disease are generally later in onset and milder in female carriers than in affected males (57). Some women eventually develop the same severe complications as affected males. The decreased overall severity and variability of the disease in women is almost certainly the result, in part, of the effects of X-chromosome inactivation (8, 9). Pain Although the patient had had burning pain and acroparesthesias in his hands and feet for 20 years, the diagnosis of Fabry disease was not made until he developed clinical and pathologic evidence of advancing renal disease. The first clinical manifestation of Fabry disease is generally episodes of burning pain or acroparesthesia, usually involving the hands and feet (3, 1015). Acroparesthesia may be transient or may persist for several hours and is often precipitated by febrile illness, exercise, emotional stress, or exposure to heat or sometimes to cold. Other forms of pain are also common, sometimes involving the large joints. However, the bizarre, intermittent and transient nature of the acroparesthesia; the absence of substantial physical findings; and a general lack of awareness of Fabry disease can cause diagnosis to be delayed for decades (16). In fact, in the absence of a known family history, very few affected males are diagnosed before adulthood. Table 1 shows some of the features that should alert the clinician to the possibility of Fabry disease. Table 1. Clinical Problems That Should Raise Suspicion of Fabry Disease The pain and acroparesthesia in Fabry disease are believed to be caused by lysosomal accumulation of GSL in peripheral nerves, dorsal root ganglia, and the spinal cord and atrophy of the small, unmyelinated nerves involved in pain and temperature sensation (1727). The acroparesthesia is typically resistant to treatment with conventional analgesics, such as acetaminophen or ibuprofen. Acute painful crises may require treatment with narcotic analgesics, such as codeine, meperidine, or morphine. Carbamazepine is also effective (28). Chronic or frequently recurrent pain often responds, at least partially, to treatment with phenytoin, amitriptyline, or gabapentin (28, 29) or combinations of these drugs. By the fourth decade of life, the pain and acroparesthesia often become less severe, at about the same age that temperature sensation becomes noticeably blunted. Acroparesthesia in women is also an early symptom, but it is rarely as severe as that experienced by men (57). Gastrointestinal Symptoms Chronic abdominal pain is also common in Fabry disease. The pain often mimics the discomfort of gastroesophageal reflux, hiatal hernia, or spastic colitis and frequently is associated with nausea and vomiting (30, 31). The gastrointestinal symptoms do not respond well to treatment with conventional antispasmodic agents, antacids, or antidiarrheal drugs. However, many patients report that diarrhea improves with treatment with loperamide or bismuth subsalicylate. Angiokeratomata The patient had angiokeratomata of the skin of his scrotum and penis. The lesions are caused by accumulation of GSL in the walls of the small blood vessels, with secondary ectasia and hyperkeratosis. Biopsy specimens of the lesions typically show vacuolation of the capillary endothelial cells and smooth-muscle cells of the media (Figure 4). Figure 4. Histopathologic changes in a small cutaneous blood vessel showing vacuolation of endothelial and smooth-muscle cells (periodic acidSchiff stain). Impaired Renal Function Impairment of renal function is particularly common in males with Fabry disease (3, 3235). The first indication is often isosthenuria, which generally occurs in the third or fourth decade of life. Proteinuria generally follows and may be severe. In a review of the natural history of renal involvement in 105 males with the disease, Branton and coworkers (4) reported that 50% of patients had proteinuria by age 35 years and had end-stage renal disease by age 47 years. Increased awareness and improvements in diagnosis have led to the identification of some important subgroups of patients with organ-specific variants of Fabry disease. A subgroup of patients with serious renal impairment has been described, but with relatively few nonrenal manifestations of the disease (3641). Surveys of males receiving dialysis to treat end-stage renal disease of obscure cause have revealed that as many as 2% in some series have Fabry disease (40, 42, 43). Cardiac Arrhythmias and Left Ventricular Hypertrophy Cardiac arrhythmias and conduction defects are common and early manifestations of Fabry disease and often produce symptoms as early as the second decade of life (4449). The disease is also associated with progressive left ventricular hypertrophy, which may be aggravated by arterial hypertension (44, 45, 5052). As the condition advances, progressive impairment of diastolic filling causes decreased cardiac output and early death (5355). In a subgroup of patients with severe cardiomyopathy, noncardiac manifestations of the disease may be trivial (56, 57). The pathophysiology of cardiomyopathy in Fabry disease is

Natural course of Fabry disease: changing pattern of causes of death in FOS – Fabry Outcome Survey

Background: Fabry disease is a rare X-linked lysosomal storage disorder characterised by severe multisystemic involvement that leads to major organ failure and premature death in affected men and women. Over the past 7 years, the Fabry Outcome Survey (FOS) has collected data on the natural history of Fabry disease, and the long-term efficacy and safety of enzyme-replacement therapy. This paper provides an update on the first analysis of FOS data. Design: Baseline data on clinical manifestations and causes of death in a cohort of 1453 patients (699 male, 754 female) from 19 countries worldwide were analysed. Causes of death of affected relatives were analysed separately. Results: The most frequently reported signs and symptoms of Fabry disease were neurological. Cardiac, ocular, gastrointestinal, dermatological, auditory and renal manifestations were also common. The principal causes of death among 181 affected relatives of patients in FOS (most of whom had died before 2001) were renal failure in males (42%) and cerebrovascular disease in females (25%). In contrast, of the 42 patients enrolled in FOS whose deaths were reported between 2001 and 2007, cardiac disease was the main cause of death in both male (34%) and female (57%) patients. Conclusion: These data suggest that the importance of renal disease as a cause of death in patients with Fabry disease is decreasing while the importance of cardiac disease is increasing. This pattern probably reflects improvements in the management of renal disease in patients with Fabry disease.

Agalsidase Alfa and Kidney Dysfunction in Fabry Disease

In male patients with Fabry disease, an X-linked disorder of glycosphingolipid metabolism caused by deficient activity of the lysosomal enzyme alpha-galactosidase A, kidney dysfunction becomes apparent by the third decade of life and invariably progresses to ESRD without treatment. Here, we summarize the effects of agalsidase alfa on kidney function from three prospective, randomized, placebo-controlled trials and their open-label extension studies involving 108 adult male patients. The mean baseline GFR among 54 nonhyperfiltrating patients (measured GFR <135 ml/min per 1.73 m(2)) treated with placebo was 85.4 +/- 29.6 ml/min per 1.73 m(2); during 6 mo of placebo, the mean annualized rate of change in GFR was -7.0 +/- 32.9 ml/min per 1.73 m(2). Among 85 nonhyperfiltrating patients treated with agalsidase alfa, the annualized rate of change was -2.9 +/- 8.7 ml/min per 1.73 m(2). Treatment with agalsidase alfa did not affect proteinuria. Multivariate analysis revealed that GFR and proteinuria category (< 1 or > or = 1 g/d) at baseline significantly predicted the rate of decline of GFR during treatment. This summary represents the largest group of male patients who had Fabry disease and for whom the effects of enzyme replacement therapy on kidney function have been studied. These data suggest that agalsidase alfa may stabilize kidney function in these patients.

Pyrimethamine as a Potential Pharmacological Chaperone for Late-onset Forms of GM2 Gangliosidosis

Late-onset GM2 gangliosidosis is composed of two related, autosomal recessive, neurodegenerative diseases, both resulting from deficiency of lysosomal, heterodimeric β-hexosaminidase A (Hex A, αβ). Pharmacological chaperones (PC) are small molecules that can stabilize the conformation of a mutant protein, allowing it to pass the quality control system of the endoplasmic reticulum. To date all successful PCs have also been competitive inhibitors. Screening for Hex A inhibitors in a library of 1040 Food Drug Administration-approved compounds identified pyrimethamine (PYR (2,4-diamino 5-(4-chlorophenyl)-6-ethylpyrimidine)) as the most potent inhibitor. Cell lines from 10 late-onset Tay-Sachs (11 α-mutations, 2 novel) and 7 Sandhoff (9 β-mutations, 4 novel) disease patients, were cultured with PYR at concentrations corresponding to therapeutic doses. Cells carrying the most common late-onset mutation, αG269S, showed significant increases in residual Hex A activity, as did all 7 of the β-mutants tested. Cells responding to PC treatment included those carrying mutants resulting in reduced Hex heat stability and partial splice junction mutations of the inherently less stable α-subunit. PYR, which binds to the active site in domain II, was able to function as PC even to domain I β-mutants. We concluded that PYR functions as a mutation-specific PC, variably enhancing residual lysosomal Hex A levels in late-onset GM2 gangliosidosis patient cells.

Early treatment of Menkes disease with parenteral Cooper-Histidine: Long-term follow-up of four treated patients

We report on the long-term clinical course of 4 boys with Menkes disease, treated from early infancy with parenteral copper-histidine, with follow-up over 10-20 years. Three of the 4 had male relatives with a severe clinical course compatible with classical Menkes disease. As a consequence of early treatment, our patients have normal or near-normal intellectual development, but have developed many of the more severe somatic abnormalities of the related disorder, occipital horn syndrome, including severe orthostatic hypotension in 2. In addition, 1 boy developed a previously unreported anomaly, namely, massive splenomegaly and hypersplenism as a consequence of a splenic artery aneurysm. Previously reported molecular studies in 2 of these patients had shown gene defects which would have predicted a truncated and probably nonfunctional gene product. Despite the favorable effects on the neurological symptoms, parenteral copper treatment for Menkes disease should still be regarded as experimental. The development of more effective treatments must await a more precise delineation of the role which the Menkes protein plays in intracellular copper trafficking.

The Natural History of Juvenile or Subacute GM2 Gangliosidosis: 21 New Cases and Literature Review of 134 Previously Reported

OBJECTIVE. Juvenile GM2 gangliosidosis is a group of inherited neurodegenerative diseases caused by deficiency of lysosomal β-hexosaminidase resulting in GM2 ganglioside accumulation in brain. The purpose of this study was to delineate the natural history of the condition and identify genotype-phenotype correlations that might be helpful in predicting the course of the disease in individual patients. METHODS. A cohort of 21 patients with juvenile GM2 gangliosidosis, 15 with the Tay-Sachs variant and 6 with the Sandhoff variant, was studied prospectively in 2 centers. Our experience was compared with previously published reports on 134 patients. Information about clinical features, β-hexosaminidase enzyme activity, and mutation analysis was collected. RESULTS. In our cohort of patients, the mean (±SD) age of onset of symptoms was 5.3 ± 4.1 years, with a mean follow-up time of 8.4 years. The most common symptoms at onset were gait disturbances (66.7%), incoordination (52.4%), speech problems (28.6%), and developmental delay (28.6%). The age of onset of gait disturbances was 7.1 ± 5.6 years. The mean time for progression to becoming wheelchair-bound was 6.2 ± 5.5 years. The mean age of onset of speech problems was 7.0 ± 5.6 years, with a mean time of progression to anarthria of 5.6 ± 5.3 years. Muscle wasting (10.6 ± 7.4 years), proximal weakness (11.1 ± 7.7 years), and incontinence of sphincters (14.6 ± 9.7 years) appeared later in the course of the disease. Psychiatric disturbances and neuropathy were more prevalent in patients with the Sandhoff variant than in those with the Tay-Sachs variant. However, dysphagia, sphincter incontinence, and sleep problems occurred earlier in those with the Tay-Sachs variant. Cerebellar atrophy was the most common finding on brain MRI (52.9%). The median survival time among the studied and reviewed patients was 14.5 years. The genotype-phenotype correlation revealed that in patients with the Tay-Sachs variant, the presence of R178H and R499H mutations was predictive of an early onset and rapidly progressive course. The presence of either G269S or W474C mutations was associated with a later onset of symptoms along with a more slowly progressive disease course. CONCLUSIONS. Juvenile GM2 gangliosidosis is clinically heterogeneous, not only in terms of age of onset and clinical features but also with regard to the course of the disease. In general, the earlier the onset of symptoms, the more rapidly the disease progresses. The Tay-Sachs and Sandhoff variants differed somewhat in the frequency of specific clinical characteristics. Speech deterioration progressed more rapidly than gait abnormalities in both the Tay-Sachs variant and Sandhoff variant groups. Among patients with the Tay-Sachs variant, the HEXA genotype showed a significant correlation with the clinical course.

Screening for carriers of Tay-Sachs disease among Ashkenazi Jews

BACKGROUND AND METHODS The prevention of Tay-Sachs disease (GM2 gangliosidosis, type 1) depends on the identification of carriers of the gene for this autosomal recessive disorder. We compared the enzyme-based test widely used in screening for Tay-Sachs disease with a test based on analysis of DNA. We developed methods to detect the three mutations in the HEXA gene that occur with high frequency among Ashkenazi Jews: two mutations cause infantile Tay-Sachs disease, and the third causes the adult-onset form of the disease. DNA segments containing these mutation sites were amplified with the polymerase chain reaction and analyzed for the presence of the mutations. RESULTS Among 62 Ashkenazi obligate carriers of Tay-Sachs disease, the three specific mutations accounted for all but one of the mutant alleles (98 percent). In 216 Ashkenazi carriers identified by the enzyme test, DNA analysis showed that 177 (82 percent) had one of the identified mutations. Of the 177, 79 percent had the exon 11 insertion mutation, 18 percent had the intron 12 splice-junction mutation, and 3 percent had the less severe exon 7 mutation associated with adult-onset disease. The results of the enzyme tests in the 39 subjects (18 percent) who were defined as carriers but in whom DNA analysis did not identify a mutant allele were probably false positive (although there remains some possibility of unidentified mutations). In addition, of 152 persons defined as noncarriers by the enzyme-based test, 1 was identified as a carrier by DNA analysis (i.e., a false negative enzyme-test result). CONCLUSIONS The increased specificity and predictive value of the DNA-based test make it a useful adjunct to the diagnostic tests currently used to screen for carriers of Tay-Sachs disease. Although some false positive results may be desirable on an enzyme-based test that is used in screening, the DNA test allows precise definition of the carrier state for the known mutations.