Ambroxol as a novel disease-modifying treatment for Parkinson's disease dementia: protocol for a single-centre, randomized, double-blind, placebo-controlled trial.

BACKGROUND: Currently there are no disease-modifying treatments for Parkinson's disease dementia (PDD), a condition linked to aggregation of the protein α-synuclein in subcortical and cortical brain areas. One of the leading genetic risk factors for Parkinson's disease is being a carrier in the gene for ß-Glucocerebrosidase (GCase; gene name GBA1). Studies in cell culture and animal models have shown that raising the levels of GCase can decrease levels of α-synuclein. Ambroxol is a pharmacological chaperone for GCase and is able to raise the levels of GCase and could therefore be a disease-modifying treatment for PDD. The aims of this trial are to determine if Ambroxol is safe and well-tolerated by individuals with PDD and if Ambroxol affects cognitive, biochemical, and neuroimaging measures. METHODS: This is a phase II, single-centre, double-blind, randomized placebo-controlled trial involving 75 individuals with mild to moderate PDD. Participants will be randomized into Ambroxol high-dose (1050 mg/day), low-dose (525 mg/day), or placebo treatment arms. Assessments will be undertaken at baseline, 6-months, and 12-months follow up times. Primary outcome measures will be the Alzheimer's disease Assessment Scale-cognitive subscale (ADAS-Cog) and the ADCS Clinician's Global Impression of Change (CGIC). Secondary measures will include the Parkinson's disease Cognitive Rating Scale, Clinical Dementia Rating, Trail Making Test, Stroop Test, Unified Parkinson's disease Rating Scale, Purdue Pegboard, Timed Up and Go, and gait kinematics. Markers of neurodegeneration will include MRI and CSF measures. Pharmacokinetics and pharmacodynamics of Ambroxol will be examined through plasma levels during dose titration phase and evaluation of GCase activity in lymphocytes. DISCUSSION: If found effective and safe, Ambroxol will be one of the first disease-modifying treatments for PDD. TRIAL REGISTRATION: ClinicalTrials.gov NCT02914366, 26 Sep 2016/retrospectively registered.

Debunking Occam's razor: Diagnosing multiple genetic diseases in families by whole-exome sequencing.

BACKGROUND: Recent clinical whole exome sequencing (WES) cohorts have identified unanticipated multiple genetic diagnoses in single patients. However, the frequency of multiple genetic diagnoses in families is largely unknown. AIMS: We set out to identify the rate of multiple genetic diagnoses in probands and their families referred for analysis in two national research programs in Canada. MATERIALS & METHODS: We retrospectively analyzed WES results for 802 undiagnosed probands referred over the past 5 years in either the FORGE or Care4Rare Canada WES initiatives. RESULTS: Of the 802 probands, 226 (28.2%) were diagnosed based on mutations in known disease genes. Eight (3.5%) had two or more genetic diagnoses explaining their clinical phenotype, a rate in keeping with the large published studies (average 4.3%; 1.4 - 7.2%). Seven of the 8 probands had family members with one or more of the molecularly diagnosed diseases. Consanguinity and multisystem disease appeared to increase the likelihood of multiple genetic diagnoses in a family. CONCLUSION: Our findings highlight the importance of comprehensive clinical phenotyping of family members to ultimately provide accurate genetic counseling.

The expanding phenotype of MELAS caused by the m.3291T > C mutation in the MT-TL1 gene.

m.3291T > C mutation in the MT-TL1 gene has been infrequently encountered in association with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS), however remains poorly characterized from a clinical perspective. In the following report we describe in detail the phenotypic features, long term follow up (> 7 years) and management in a Caucasian family with MELAS due to the m.3291T > C mutation and review the literature on m.3291T > C mutation. The clinical phenotype in the proposita included overlapping features of MELAS, MERRF (Myoclonic epilepsy and ragged-red fiber syndrome), MNGIE (Mitochondrial neurogastrointestinal encephalopathy), KSS (Kearns-Sayre Syndrome) and CPEO (Chronic progressive external ophthalmoplegia).

Biochemical and Hematologic Manifestations of Gastric Intrinsic Factor (GIF) Deficiency: A Treatable Cause of B12 Deficiency in the Old Order Mennonite Population of Southwestern Ontario.

Intrinsic factor deficiency (OMIM #261000, IFD) is a rare inherited disorder of vitamin B12 metabolism due to mutations in the gastric intrinsic factor (GIF) gene.We report three individuals from an Old Order Mennonite community who presented with B12 deficiency. Two cases are siblings born to consanguineous parents and the third case is not known to be closely related. The older male sib presented at 4 years with gastrointestinal symptoms, listlessness, and pallor. He had pancytopenia with megaloblastic anemia. Serum B12 was 61 (198-615 pmol/L). Methylmalonic aciduria was present. C3 was elevated on acylcarnitine profile. Homocysteine was high at 16.7 (5.0-12.0 umol/L). His asymptomatic female sibling was also found to have B12 deficiency. Genetic testing for methylmalonic aciduria (MMAA), transcobalamin deficiency (TCN2), and Imerslund-Gräsbeck syndrome (AMN) showed no mutation in both siblings. The third patient, a 34-year-old woman, had presented in infancy with a diagnosis of pernicious anemia. Mutation analysis of GIF revealed compound heterozygosity for a c.79+1G>A substitution and a c.973delG deletion in all three individuals. Oral or parenteral vitamin B12 has led to complete recovery of clinical parameters and vitamin B12 levels. Newborn screening samples on the siblings revealed normal methylcitrate, C3, and C3/C2 ratios thus indicating no disruption of propionic or methylmalonic acid metabolism.A high index of suspicion should be maintained if children present with megaloblastic anemia since GIF deficiency is a treatable disorder and newborn screening may not be able to detect this condition.

MELAS: A Multigenerational Impact of the MTTL1 A3243G MELAS Mutation.

BACKGROUND: the maternally inherited MTTL1 A3243G mutation in the mitochondrial genome causes MelaS (Mitochondrial encephalopathy lactic acidosis with Stroke-like episodes), a condition that is multisystemic but affects primarily the nervous system. Significant intra-familial variation in phenotype and severity of disease is well recognized. METHODS: retrospective and ongoing study of an extended family carrying the MTTL1 A3243G mutation with multiple symptomatic individuals. tissue heteroplasmy is reviewed based on the clinical presentations, imaging studies, laboratory findings in affected individuals and pathological material obtained at autopsy in two of the family members. RESULTS: there were seven affected individuals out of thirteen members in this three generation family who each carried the MTTL1 A3243G mutation. the clinical presentations were varied with symptoms ranging from hearing loss, migraines, dementia, seizures, diabetes, visual manifestations, and stroke like episodes. three of the family members are deceased from MelaS or to complications related to MelaS. CONCLUSIONS: the results of the clinical, pathological and radiological findings in this family provide strong support to the current concepts of maternal inheritance, tissue heteroplasmy and molecular pathogenesis in MelaS. neurologists (both adult and paediatric) are the most likely to encounter patients with MelaS in their practice. genetic counselling is complex in view of maternal inheritance and heteroplasmy. newer therapeutic options such as arginine are being used for acute and preventative management of stroke like episodes.

Update on transcobalamin deficiency: clinical presentation, treatment and outcome.

Transcobalamin (TC) transports cobalamin from blood into cells. TC deficiency is a rare autosomal recessive disorder usually presenting in early infancy with failure to thrive, weakness, diarrhoea, pallor, anemia, and pancytopenia or agammaglobulinemia. It can sometimes resemble neonatal leukemia or severe combined immunodeficiency disease. Diagnosis of TC deficiency is suspected based on megaloblastic anemia, elevation of total plasma homocysteine, and blood or urine methylmalonic acid. It is confirmed by studying the synthesis of TC in cultured fibroblasts, or by molecular analysis of the TCN2 gene. TC deficiency is treatable with supplemental cobalamin, but the optimal type, route and frequency of cobalamin administration and long term patient outcomes are unknown. Here we present a series of 30 patients with TC deficiency, including an update on multiple previously published patients, in order to evaluate the different treatment strategies and provide information about long term outcome. Based on the data presented, current practice appears to favour treatment of individuals with TC deficiency by intramuscular injections of hydroxy- or cyanocobalamin. In most cases presented, at least weekly injections (1 mg IM) were necessary to ensure optimal treatment. Most centres adjusted the treatment regimen based on monitoring CBC, total plasma homocysteine, plasma and urine methylmalonic acid, as well as, clinical status. Finally, continuing IM treatment into adulthood appears to be beneficial.

Recurrent encephalopathy: NAGS (N-acetylglutamate synthase) deficiency in adults.

N-acetyl-glutamate synthase (NAGS) deficiency is a rare autosomal recessive urea cycle disorder (UCD) that uncommonly presents in adulthood. Adult presentations of UCDs include; confusional episodes, neuropsychiatric symptoms and encephalopathy. To date, there have been no detailed neurological descriptions of an adult onset presentation of NAGS deficiency. In this review we examine the clinical presentation and management of UCDs with an emphasis on NAGS deficiency. An illustrative case is provided. Plasma ammonia levels should be measured in all adult patients with unexplained encephalopathy, as treatment can be potentially life-saving. Availability of N-carbamylglutamate (NCG; carglumic acid) has made protein restriction largely unnecessary in treatment regimens currently employed. Genetic counselling remains an essential component of management of NAGS.

Severe phenotypic spectrum of mevalonate kinase deficiency with minimal mevalonic aciduria.

Mevalonate kinase deficiency is a rare autosomal recessively inherited organic aciduria with a complex multi-systemic phenotype. We describe two deceased patients with clinically severe mevalonate kinase (MK) deficiency confirmed by MK mutation analysis. The phenotype in our patients ranged from neonatal hydrops in the first patient to severe failure to thrive, hepatosplenomegaly, recurrent febrile episodes and lymphadenopathy in the second. Both infants excreted relatively low amounts of mevalonic acid intermittently.

Transcobalamin (TC) deficiency--potential cause of bone marrow failure in childhood.

It is unusual for inborn errors of metabolism to be considered in the investigative work-up of pancytopenia. We report a family in which the proband presented with failure to thrive at 2 months of age and subsequent bone marrow failure. A previous sibling had died at 7 months of age with suspected leukaemia. Haematological findings in the proband were significant for pancytopenia, and bone marrow aspiration showed dysplastic changes in all cell lineages. Urinary organic acid analysis revealed elevated methylmalonic acid. The synthesis of transcobalamin II (transcobalamin, TC) by cultured fibroblasts was markedly reduced, confirming the diagnosis of TC deficiency. The proband and his younger asymptomatic sister (also found to have TC deficiency) were homozygous for R399X (c.1195C>T), a novel mutation resulting in the loss of the C- terminal 29 amino acids of TC, a highly conserved region. Response to parenteral vitamin B(12) in the proband was dramatic. At 6 years 3 months of age, physical examination is normal and developmental level is age appropriate. His sister is clinically asymptomatic and is also developing normally. Propionylcarnitine concentrations were not elevated in the newborn screening cards from the proband and sister, but that was for specimens retrieved from storage after 7 years and 5 years, respectively. Inherited and acquired cobalamin disorders should both be considered in the differential diagnosis of bone marrow failure syndromes in young children. Early detection of the metabolic causes of bone marrow failure can ensure prompt recovery in some cases involving the vitamin B(12) pathway.

Development of a clinical assay for detection of GAA mutations and characterization of the GAA mutation spectrum in a Canadian cohort of individuals with glycogen storage disease, type II.

Glycogen storage disease, type II (GSDII; Pompe disease; acid maltase deficiency) is an autosomal recessive disease caused by mutations of the GAA gene that lead to deficient acid alpha-glucosidase enzyme activity and accumulation of lysosomal glycogen. Although measurement of acid alpha-glucosidase enzyme activity in fibroblasts remains the gold standard for the diagnosis of GSDII, analysis of the GAA gene allows confirmation of clinical or biochemical diagnoses and permits predictive and prenatal testing of individuals at risk of developing GSDII. We have developed a clinical molecular test for the detection of GAA mutations based on cycle sequencing of the complete coding region. GAA exons 2-20 are amplified in six independent PCR using intronic primers. The resulting products were purified and sequenced. Preliminary studies using this protocol were conducted with DNA from 21 GSDII-affected individuals from five centers across Canada. In total, 41 of 42 mutations were detected (96.7% detection rate). Mutations spanned intron 1 through exon 19 and included nine novel mutations. Haplotype analysis of recurrent mutations further suggested that three of these mutations are likely to have occurred independently at least twice. Additionally, we report the identification of the c.-32-13T>G GAA mutation in an individual with infantile variant GSDII, despite reports of this mutation being associated almost exclusively with late-onset forms of the disease. The development of a clinical molecular test provides an important tool for the management and counseling of families and individuals with GSDII, and has provided useful information about the GAA mutation spectrum in Canada.

Aspartylglucosaminuria in a Canadian family.

Aspartylglucosaminuria (McKusick 208400) is a lysosomopathy associated with aspartylglucosaminidase (L-aspartamido-beta-N-acetylglucosamine amidohydrolase, EC 3.5.1.26) deficiency. It has been most frequently encountered in Finland, where the regional incidence may be as high as 1 in 3600 births. In North America it is very rare, having been reported in only 8 patients. We encountered 4 patients with aspartylglucosaminuria in a Canadian family of 12 siblings. The 4 siblings affected--2 brothers and 2 sisters--were apparently normal at birth; however, their developmental milestones, particularly speech, were slow, and they acquired only a simple vocabulary. Throughout life, there was a progressive coarsening of facial features; 3 had inguinal hernia and recurrent diarrhea; all became severely retarded and by the 4th decade showed evident deterioration of both cognitive and motor skills; 2 exhibited cyclical behavioural changes. Three of the siblings have died, at 33, 39 and 44 years of age. Two died of bronchopneumonia and 1 of asphyxiation following aspiration. In the urine of all 4 siblings, and in the 1 liver examined, we found 2-acetamido-1-N-(4-L-aspartyl)-2-deoxy-beta-D-glucosamine (GlcNAc-Asn) and alpha-D-mannose-(1,6)-beta-D-mannose-(1,4)-2-acetamido- 2-deoxy-beta-D-glucose-(1,4)-2-acetamido-1-N-(4-L-aspartyl)-2-deoxy-beta - D-glucosamine (Man2-GlcNAc2-Asn). Compared with the level of activity in controls, aspartylglucosaminidase activity was less than 2% in fibroblasts from 3 of the siblings, less than 0.5% in leukocytes from 1 sibling, and less than 1% in the liver of 1 sibling, whereas other acid hydrolase activities in these tissues were normal. Ultrastructural studies of skin showed that fibroblasts, endothelial cells and pericytes contained vacuoles with fine reticulo-floccular material. Glial and neuronal cells of the central nervous system showed similar inclusions as well as others composed of concentric or parallel membranous arrays intermingled with lipid droplets.

Consequences of aging on mitochondrial respiratory chain enzymes in cultured human fibroblasts treated with ascorbate.

The activities of mitochondrial respiratory chain enzymes with and without ascorbate pretreatment were assayed in 10- to 20-week-old cultures of human fibroblasts. Aging was associated with a significant loss of respiratory chain enzyme activities. The presence of ascorbate in the medium reduced the rate of loss of these enzymes. Free radical-mediated injuries may also contribute to aging since the changes seen in respiratory chain enzyme activities are similar to those seen in oxidatively stressed cells. This study demonstrates an age-related decline in mitochondrial respiratory chain activity as well as a protective role for ascorbate in aging.

Isolated sulfite oxidase deficiency.

Isolated sulfite oxidase (SO) deficiency is an autosomal recessively inherited inborn error of sulfur metabolism. In this report of a ninth patient the clinical history, laboratory results, neuropathological findings and a mutation in the sulfite oxidase gene are described. The data from this patient and previously published patients with isolated sulfite oxidase deficiency and molybdenum cofactor deficiency are summarized to characterize this rare disorder. The patient presented neonatally with intractable seizures and did not progress developmentally beyond the neonatal stage. Dislocated lenses were apparent at 2 months. There was increased urine excretion of sulfite and S-sulfocysteine and a decreased concentration of plasma cystine. A lactic acidemia was present for 6 months. Liver sulfite oxidase activity was not detectable but xanthine dehydrogenase activity was normal. The boy died of respiratory failure at 32 months. Neuropathological findings of cortical necrosis and extensive cavitating leukoencephalopathy were reminiscent of those seen in severe perinatal asphyxia suggesting an etiology of energy deficiency. A point mutation that resulted in a truncated protein missing the molybdenum-binding site has been identified.

Heteroallelic missense mutations of the galactosamine-6-sulfate sulfatase (GALNS) gene in a mild form of Morquio disease (MPS IVA).

Morquio disease (MPS IVA) is an autosomal recessive disorder caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) activity. Patients commonly present in early infancy with growth failure, spondyloepiphyseal dysplasia, corneal opacification, and keratan sulfaturia, but milder forms have been described. We report on a patient who grew normally until age 5 years. Her keratan sulfaturia was not detected until adolescence, and she now has changes restricted largely to the axial skeleton. She has experienced only mildly impaired vision. At age 22, thin-layer chromatography of purified glycosaminoglycans showed some keratan sulfaturia. GALNS activity in fibroblast homogenate supernatants was 20 +/- 5% of controls (as compared to 5 +/- 3% of controls in severe MPS IVA, P < .003). Kinetic analysis of residual fibroblast GALNS activity in patient and parents revealed decreased K(m) and increased Vmax in the mother and daughter, but not in the father, compatible with compound heterozygosity. GALNS exons were amplified from patient genomic DNA and screened by SSCP. Two missense mutations, a C to T transition at position 335 (predicting R94C) and a T to G transversion at position 344 (predicting F97V), were found on sequencing an abnormally migrating exon 3 amplicon. Digestion of the amplicon with FokI and AccI restriction enzymes (specific for the R94C and F97V mutations, respectively) confirmed heterozygosity. In fibroblast transfection experiments, heterozygous R94C and F97V mutants independently expressed as severe and mild GALNS deficiency, respectively. We interpret these findings to indicate that our patient bears heteroallelic GALNS missense mutations, leading to GALNS deficiency and mild MPS IVA. Our findings expand the clinical and biochemical phenotype of MPS IVA, but full delineation of the genotype-phenotype relationship requires further study of native and transfected mutant cell lines.

Nerve biopsy findings in Niemann-Pick type II (NPC)

The severe infantile form of Niemann-Pick disease type II was diagnosed in a 4-year-old girl and confirmed by demonstrating in cultured skin fibroblasts a deficiency of low-density lipoprotein-stimulated cholesterol ester synthesis of < 5% of normal. Electrodiagnostic studies revealed changes of a predominantly demyelinating motor and sensory polyneuropathy. Light microscope and ultrastructural examination of a peroneal nerve biopsy showed unique changes. Compacted myelin sheaths were disproportionately thin with marked globular irregularities in single teased nerve fibres and evidence of chronic demyelination. The majority of axons were preserved but axonal spheroids and cytoskeletal abnormalities akin to neuroaxonal dystrophy were noted. Membrane-bound multi-lobulated lysosomal inclusions of floccular and electron-dense material were present in Schwann cells (SC), endoneurial fibroblasts, macrophages, pericytes and endothelial cells. SC of myelinated fibres were stuffed with whorls of concentric osmiophilic membraneous profiles and electron-lucent material. The findings are diagnostic and differ from those of classical Niemann-Pick disease.

Retrospective diagnosis of medium chain acyl-CoA dehydrogenase deficiency.

A male infant is reported who died suddenly and who at post-mortem had pathological evidence suggestive of a genetic defect of fatty acid beta-oxidation. A specific diagnosis could not be made enzymatically because of unavailability of suitable tissue for assay. The diagnosis of medium chain acyl-CoA dehydrogenase (MCAD) deficiency was made by specific mutation analysis using the polymerase chain reaction and DNA extracted from the newborn screening card of this infant. This powerful new molecular diagnostic technique should prove to be of use in similar circumstances.