Clinical, biochemical and genetic characteristics of MOGS-CDG: a rare congenital disorder of glycosylation.

PURPOSE: To summarise the clinical, molecular and biochemical phenotype of mannosyl-oligosaccharide glucosidase-related congenital disorders of glycosylation (MOGS-CDG), which presents with variable clinical manifestations, and to analyse which clinical biochemical assay consistently supports diagnosis in individuals with bi-allelic variants in MOGS. METHODS: Phenotypic characterisation was performed through an international and multicentre collaboration. Genetic testing was done by exome sequencing and targeted arrays. Biochemical assays on serum and urine were performed to delineate the biochemical signature of MOGS-CDG. RESULTS: Clinical phenotyping revealed heterogeneity in MOGS-CDG, including neurological, immunological and skeletal phenotypes. Bi-allelic variants in MOGS were identified in 12 individuals from 11 families. The severity in each organ system was variable, without definite genotype correlation. Urine oligosaccharide analysis was consistently abnormal for all affected probands, whereas other biochemical analyses such as serum transferrin analysis was not consistently abnormal. CONCLUSION: The clinical phenotype of MOGS-CDG includes multisystemic involvement with variable severity. Molecular analysis, combined with biochemical testing, is important for diagnosis. In MOGS-CDG, urine oligosaccharide analysis via matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry can be used as a reliable biochemical test for screening and confirmation of disease.

The critical role of psychosine in screening, diagnosis, and monitoring of Krabbe disease.

PURPOSE: Newborn screening (NBS) for Krabbe disease (KD) is performed by measurement of galactocerebrosidase (GALC) activity as the primary test. This revealed that GALC activity has poor specificity for KD. Psychosine (PSY) was proposed as a disease marker useful to reduce the false positive rate for NBS and for disease monitoring. We report a highly sensitive PSY assay that allows identification of KD patients with minimal PSY elevations. METHODS: PSY was extracted from dried blood spots or erythrocytes with methanol containing d5-PSY as internal standard, and measured by liquid chromatography-tandem mass spectrometry. RESULTS: Analysis of PSY in samples from controls (N = 209), GALC pseudodeficiency carriers (N = 55), GALC pathogenic variant carriers (N = 27), patients with infantile KD (N = 26), and patients with late-onset KD (N = 11) allowed for the development of an effective laboratory screening and diagnostic algorithm. Additional longitudinal measurements were used to track therapeutic efficacy of hematopoietic stem cell transplantion (HSCT). CONCLUSION: This study supports PSY quantitation as a critical component of NBS for KD. It helps to differentiate infantile from later onset KD variants, as well as from GALC variant and pseudodeficiency carriers. Additionally, this study provides further data that PSY measurement can be useful to monitor KD progression before and after treatment.

Urine oligosaccharide screening by MALDI-TOF for the identification of NGLY1 deficiency.

N-glycanase deficiency (NGLY1 deficiency, NGLY1-CDDG), the first autosomal recessive congenital disorder of N-linked deglycosylation (CDDG), is caused by pathogenic variants in NGLY1. The majority of affected individuals have been identified using exome or genome sequencing. To date, no reliable, clinically available biomarkers have been identified. Urine oligosaccharide analysis was included as part of a routine evaluation for possible biomarkers in patients with confirmed NGLY1-CDDG. During the qualitative review of oligosaccharide profiles by an experienced laboratory director an abnormal analyte with a proposed structure of Neu5Ac1Hex1GlcNAc1-Asn was identified in NGLY1-CDDG patient urine samples. The same species has been observed in profiles from individuals affected with aspartylglucosaminuria, although the complete spectra are not identical. Additional studies using tandem mass spectrometry confirmed the analyte's structure. In addition to the known NGLY1-CDDG patients identified by this analysis, a single case was identified in a population referred for clinical testing who subsequently had a diagnosis of NGLY1-CDDG confirmed by molecular testing. Urine oligosaccharide screening by MALDI-TOF MS can identify individuals with NGLY1-CDDG. In addition, this potential biomarker might also be used to monitor the effectiveness of therapeutic options as they become available.

Acetylcholinesterase inhibition in patients with orthostatic intolerance.

The efficacy of current therapeutic measures in orthostatic intolerance (OI) varies among patients and is oftentimes unsatisfactory. New approaches to alleviate symptoms of OI are therefore clearly needed. Recent reports have demonstrated that acetylcholinesterase inhibition is effective in the treatment of orthostatic hypotension with the presumed mechanism of enhancing sympathetic ganglionic transmission. Based on the hypothesis that acetylcholinesterase inhibition, by improving the safety factor of cholinergic transmission, will result in enhanced vascular adrenergic tone and a vagal shift in cardiac sympathovagal balance, we evaluated the role of acetylcholinesterase inhibition in the treatment of patients with OI. We monitored heart rate (HR), blood pressure, and indexes for cardiac output, end-diastolic volume, and systemic resistance continuously in 18 patients with OI during supine rest and during 5 minutes of 70 degrees head-up tilt before and 1 hour after oral administration of 60 mg pyridostigmine. Plasma catecholamines and baroreflex sensitivity were determined for the supine and upright position before and after medication. Patients scored orthostatic symptoms for both tilt studies. The excessive HR response to orthostatic stress was significantly blunted after pyridostigmine administration. HR was significantly lower in the supine and more so in the upright position. Baroreflex sensitivity in the upright position was significantly higher after pyridostigmine. Norepinephrine was increased in both supine and upright position. These changes were associated with significant improvement of orthostatic symptoms. We conclude that pyridostigmine improves orthostatic tolerance in patients with OI. Our findings support the suggested mechanisms of enhanced sympathetic ganglionic neurotransmission and a vagal shift in cardiac sympathovagal balance. Acetylcholinesterase inhibition could be a new useful concept in the treatment of OI.

Stability of acetylcholine chloride solution in autonomic testing.

Acetylcholine (ACh) is the neurotransmitter used as an agent to evoke a sudomotor axon reflex response in autonomic testing. Adequate stimulus of postganglionic axons requires ACh solutions to be stable, but its stability in the clinical laboratory is uncertain. We evaluated the stability of standard (0.55 M) ACh solutions stored at temperatures of -20 degrees C, 4 degrees C, 25 degrees C, and 50 degrees C for 10 time points between 0 and 84 days. ACh and choline (Ch) were measured by reverse-phase HPLC with electrochemical detection using an Acetylcholine/Choline Assay Kit. Linear regressions of ACh and Ch standards were used to calculate the levels in the stored samples. The inherent levels of Ch were used as the internal standard. Regression analyses were used to examine the effects of length of storage and temperature. The samples of ACh stored at -20 degrees C and 4 degrees C showed an extremely small breakdown over the 84-day period and had no evidence to show the regression lines differed. ACh solution stored at 25 degrees C was stable for about 28 days, after such time, modest breakdown occurs. At a temperature of 50 degrees C, ACh showed a rapid breakdown after 1 day. We conclude ACh solution should not be stored at room temperature for more than 28 days and should not be exposed to higher temperatures to assure an adequate axon stimulus.

A screen of candidate genes and influence of beta2-adrenergic receptor genotypes in postural tachycardia syndrome.

OBJECTIVE: To screen candidate genes, encoding beta2-adrenergic receptor (beta2AR), alpha2C-adrenergic receptor (alpha(2C)AR), norepinephrine transporter (NET), and mitochondrial complex I (COI), for common single nucleotide polymorphisms (SNPs) in patients with postural tachycardia syndrome (POTS); alterations could potentially cause or aggravate orthostatic tachycardia and to relate beta2AR SNPs, known to effect venomotor tone, to heart rate (HR) and blood pressure measurements during 10-min head-up tilt. METHODS: (a) DNA extraction from leukocytes of 29 patients with POTS; (b) Denaturing high performance liquid chromatography analysis to screen for the 12-bp deletion (Del322-325) in alpha(2C)AR and for the alanine to proline mutation at amino acid 457 (Ala457Pro) in NET; (c) Systematic direct sequence analysis to screen for SNPs in beta2AR, NET, and COI. RESULTS: Three common polymorphisms were abundant in at least one allele in beta2AR resulting in a cysteine to arginine in the 5' promoter region (72% of patients), an arginine to glycine at amino acid-16 (Gly16; 86%), and a glutamine to glutamic acid at amino acid-27 (Glu27; 66%), a frequency that was no different to the normal Caucasian population. Orthostatic HR was significantly greater in patients with Glu27. Diastolic blood pressure (DBP) was significantly lower in a subset of patients with Gly16 whose HR were > or =120 beats/min with head-up tilt. All patients did not show the Ala457Pro mutation of NET; all sequence variants detected in alpha(2C)AR, NET, and COI were not considered causally related to POTS. CONCLUSIONS: Of the candidate genes screened, none harbored a SNP considered to be causally related to POTS. There was significant association of HR and DBP with SNPs of the gene encoding beta2AR; Gly16 or Glu27 could aggravate orthostatic tachycardia by excessive venous pooling.

Prospective differentiation of multiple system atrophy from Parkinson disease, with and without autonomic failure.

OBJECTIVE: To report preliminary results of a prospective ongoing study of multiple system atrophy (MSA) and Parkinson disease (PD), with a large subset of patients with PD with autonomic failure (25%), to evaluate autonomic indices that distinguish MSA from PD. METHODS: We used consensus criteria, detailed autonomic studies (Composite Autonomic Symptom Scale, Composite Autonomic Scoring Scale, thermoregulatory sweat test, and plasma catecholamines), and functional scales (Unified MSA Rating Scale [UMSARS] I-IV and Hoehn-Yahr grading) on a prospective, repeated, and ongoing basis. RESULTS: We report the results of a study on 52 patients with MSA (mean [SD], age, 61.1 [7.8] years; body mass index (calculated as weight in kilograms divided by height in meters squared), 27.2 [4.6]; Hoehn-Yahr grade, 3.2 [0.9]; UMSARS I score, 21.5 [7.4]; and UMSARS II score, 22.7 [9.0]) and 29 patients with PD, including PD with autonomic failure (mean [SD], age, 66.0 [8.1] years; body mass index, 26.6 [5.5]; Hoehn-Yahr grade, 2.2 [0.8]; UMSARS I score, 10.4 [6.1]; and UMSARS II score, 13.0 [5.9]). Autonomic indices were highly significantly more abnormal in MSA than PD (P < .001) for the Composite Autonomic Scoring Scale (5.9 [1.9] vs 3.3 [2.3], respectively), Composite Autonomic Symptom Scale (54.4 [21.8] vs 24.7 [20.5], respectively), and thermoregulatory sweat test (percentage anhidrosis, 57.4% [35.2%] vs 9.9% [17.7%], respectively). These differences were sustained and greater at 1-year follow-up, indicating a greater rate of progression of dysautonomia in MSA than PD. CONCLUSIONS: The severity, distribution, and pattern of autonomic deficits at study entry will distinguish MSA from PD, and MSA from PD with autonomic failure. These differences continue and are increased at follow-up. Our ongoing conclusion is that autonomic function tests can separate MSA from PD. Autonomic indices support the notion that the primary lesion in PD is ganglionic and postganglionic, while MSA is preganglionic.

Assessment of the "common" 4.8-kb mitochondrial DNA deletion and identification of several closely related deletions in the dorsal root ganglion of aging and streptozotocin rats.

The identification of several mitochondrial DNA (mtDNA) deletions and the accumulation of the "common" 4.8-kb mitochondrial DNA deletion (mtDNA(4834)) with aging and experimental streptozotocin-induced diabetes (STZ) were studied in the rat dorsal root ganglion (DRG). Twenty-one mtDNA deletions, including mtDNA(4834), were identified in rat L4-L6 DRG mtDNA of 15-month-old Spraque-Dawley rats with 13 months of STZ and age-matched controls. These deletions were flanked by breakpoints that ranged from 16-bp direct repeats to no direct repeats. The sciatic nerve contained undetectable levels of mtDNA deletions. Levels of mtDNA(4834) in rat DRG mtDNA significantly accumulated with age at a rate much higher than those reported in the brain, yet were not statistically different in STZ. Southern blot analysis demonstrated no significant accumulation of the total amount of mtDNA deletions in STZ over age-matched controls. The accumulation of mtDNA(4834) has not been studied in rat peripheral nerve tissue. Our identification of several mtDNA deletions with and without direct repeats at their breakpoint support the hypothesis that deletions can occur by both the slip-replication model and random recombination. Although there is a significant increase in accumulation of mtDNA(4834) associated with aging, the lack of significant accumulations of mtDNA deletions in STZ over age-matched controls indicates that this type of mtDNA damage is likely not a major alteration in STZ, although the changes could be confined to a small population of neurons that undergo apoptosis between 8 and 15 months.