Analysis of Globotriaosylceramide (Gb3) in Liquid Urine: A Straightforward Assay Using Tandem Mass Spectrometry.

Fabry disease (FD) is a lysosomal storage disorder caused by variants in the GLA gene encoding α-galactosidase A, an enzyme required for catabolism of globotriaosylceramide (Gb3). Accumulation of Gb3 in patients' cells, tissues, and biological fluids causes clinical manifestations including ventricular hypertrophy, renal insufficiency, and strokes. This protocol describes a methodology to analyze urinary Gb3 and creatinine. Samples are diluted with an internal standard solution containing Gb3(C17:0) and creatinine-D3, centrifuged, and directly analyzed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) using an 8.7-min method. Eight Gb3 isoforms [C16:0, C18:0, C20:0, C22:1, C22:0, C24:1, C24:0, and (C24:0)OH] are analyzed and the total is normalized to creatinine. Confirmation ions are monitored to detect potential interferences. The Gb3 limit of quantification is 0.023 µg/ml. Its interday coefficients of variation (3 concentrations measured) are ≤15.4%. This method minimizes matrix effects (≤6.5%) and prevents adsorption or precipitation of Gb3. Urine samples are stable (bias <15%) for 2 days at 21°C, 7 days at 4°C, and 4 freeze/thaw cycles, whereas prepared samples are stable for 5 days at 21°C, and 14 days at 4°C. The Gb3/creatinine age-related upper reference limits (mean + 2 standard deviations) are 29 mg/mol creatinine (<7 years) and 14 mg/mol creatinine (≥7 years). This simple, robust protocol has been fully validated (ISO 15189) and provides a valuable tool for diagnosis and monitoring of FD patients. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Analysis of urinary globotriaosylceramide (Gb3) and creatinine by UHPLC-MS/MS Support Protocol 1: Preparation of the urinary quality controls Support Protocol 2: Preparation of the urine matrix used for the Gb3 calibration curve Support Protocol 3: Preparation of the Gb3 calibrators Support Protocol 4: Preparation of the working solution containing the internal standards Support Protocol 5: Preparation of the creatinine calibrators Support Protocol 6: Preparation of the UHPLC solutions and mobile phases.

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9.

Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with unique protospacer adjacent motif (PAM) compatibilities. To expand this repertoire, we engineered nucleases, cytosine base editors, and adenine base editors from the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) system. We found that St1Cas9 strain variants enable targeting to five distinct A-rich PAMs and provide a structural basis for their specificities. The small size of this ortholog enables expression of the holoenzyme from a single adeno-associated viral vector for in vivo editing applications. Delivery of St1Cas9 to the neonatal liver efficiently rewired metabolic pathways, leading to phenotypic rescue in a mouse model of hereditary tyrosinemia. These robust enzymes expand and complement current editing platforms available for tailoring mammalian genomes.

Vitamin B12 deficiency in inflammatory bowel disease: a prospective observational pilot study.

BACKGROUND AND AIM: Diagnostic and management guidelines for vitamin B12 (cobalamin, Cbl) deficiency in inflammatory bowel disease (IBD) are lacking. True deficiency is defined as Cbl concentrations below reference range combined with elevated methylmalonic acid (MMA) concentrations. Studies analyzing Cbl status in IBD use only Cbl concentrations without confirmatory MMA. This study aims to determine the proportion of IBD patients with Cbl concentrations below reference range and their predisposing clinical and genetic characteristics. We then compared this to the proportion with true deficiency. PATIENTS AND METHODS: In a prospective observational pilot study of adult IBD outpatients, Cbl concentrations, MMA levels, and fucosyltransferase 2 mutations were measured at clinic visits. RESULTS: A total of 66 Crohn's disease (CD) and 30 ulcerative colitis (UC) patients were recruited. Mean Cbl concentrations (pmol/l) in CD (253.7) were not significantly lower than UC (320.5, P=0.24). Serum Cbl below reference range (<148) was observed in 7.6 and 10% of CD and UC patients, respectively (P=0.70). True deficiency in CD and UC was 3 and 3.3%, respectively (P=1.0). Patients with ileal resections more than 30 cm had lower mean Cbl concentrations (177, P=0.02) and a trend toward higher proportions with Cbl levels below reference range (40%, P=0.06), but not increased deficiency rates (0%, P=1.0). Disease location, severity, and fucosyltransferase 2 mutations were not associated with altered Cbl status. CONCLUSION: True Cbl deficiency was rare in IBD patients in this study. A disparity in Cbl status exists when confirmatory MMA levels are used compared with Cbl concentrations alone. Asymptomatic IBD patients with low serum Cbl require confirmatory tests to guide management and avoid unnecessary treatment.

Hypersuccinylacetonaemia and normal liver function in maleylacetoacetate isomerase deficiency.

BACKGROUND: A high level of succinylacetone (SA) in blood is a sensitive, specific newborn screening marker for hepatorenal tyrosinemia type 1 (HT1, MIM 276700) caused by deficiency of fumarylacetoacetate hydrolase (FAH). Newborns with HT1 are usually clinically asymptomatic but show liver dysfunction with coagulation abnormalities (prolonged prothrombin time and/or high international normalised ratio). Early treatment with nitisinone (NTBC) plus dietary restriction of tyrosine and phenylalanine prevents the complications of severe liver disease and neurological crises. METHODS AND RESULTS: Six newborns referred for hypersuccinylacetonaemia but who had normal coagulation testing on initial evaluation had sequence variants in the GSTZ1 gene, encoding maleylacetoacetate isomerase (MAAI), the enzyme preceding FAH in tyrosine degradation. Initial plasma SA levels ranged from 233 to 1282 nmol/L, greater than normal (<24 nmol/L) but less than the initial values of patients with HT1 (16 944-74 377 nmol/L, n=15). Four individuals were homozygous for c.449C>T (p.Ala150Val). One was compound heterozygous for c.259C>T (p.Arg87Ter) and an intronic sequence variant. In one, a single heterozygous GSTZ1 sequence variant was identified, c.295G>A (p.Val99Met). Bacterial expression of p.Ala150Val and p.Val99Met revealed low MAAI activity. The six individuals with mild hypersuccinylacetonaemia (MHSA) were not treated with diet or nitisinone. Their clinical course has been normal for up to 13 years. CONCLUSIONS: MHSA can be caused by sequence variants in GSTZ1. Such individuals have thus far remained asymptomatic despite receiving no specific treatment.

A Metabolic Signature of Mitochondrial Dysfunction Revealed through a Monogenic Form of Leigh Syndrome

A decline in mitochondrial respiration represents the root cause of a large number of inborn errors of metabolism. It is also associated with common age-associated diseases and the aging process. To gain insight into the systemic, biochemical consequences of respiratory chain dysfunction, we performed a case-control, prospective metabolic profiling study in a genetically homogenous cohort of patients with Leigh syndrome French Canadian variant, a mitochondrial respiratory chain disease due to loss-of-function mutations in LRPPRC. We discovered 45 plasma and urinary analytes discriminating patients from controls, including classic markers of mitochondrial metabolic dysfunction (lactate and acylcarnitines), as well as unexpected markers of cardiometabolic risk (insulin and adiponectin), amino acid catabolism linked to NADH status (α-hydroxybutyrate), and NAD(+) biosynthesis (kynurenine and 3-hydroxyanthranilic acid). Our study identifies systemic, metabolic pathway derangements that can lie downstream of primary mitochondrial lesions, with implications for understanding how the organelle contributes to rare and common diseases.

Orbicularis oculi muscle biopsies for mitochondrial DNA analysis in suspected mitochondrial myopathy.

AIMS: We wished to demonstrate the feasibility of performing diagnostic mitochondrial DNA (mtDNA) analysis on biopsies of the orbicularis oculi muscle in patients with a chronic progressive external ophthalmoplegia (CPEO) phenotype and suspicion of an underlying mitochondrial disorder. METHOD: Case series of three patients who underwent ptosis surgery and had simultaneous biopsy of the orbicularis oculi muscle because of a suspicion of a mitochondrial disorder. Orbicularis muscle samples were divided into two pieces at the time of biopsy. The first was snap-frozen in liquid nitrogen, DNA was extracted and mtDNA deletion analysis was performed by two complementary methods (long PCR and Southern blot analysis). The second piece of muscle was assessed using routine histopathology, electron microscopy and immuno-histochemical analysis. RESULTS: Three patients with clinical features of CPEO, without any positive family history, underwent orbicularis muscle biopsies at time of eyelid ptosis surgery. All biopsies were adequate to conduct histopathological and immuno-histochemical analysis, which showed evidence of abnormal muscle structure and function. mtDNA was successfully extracted from all biopsies, and long PCR and Southern blot analysis confirmed diagnostic large single mtDNA deletions in all three cases. CONCLUSIONS: Orbicularis oculi muscle biopsies are useful in patients with CPEO to perform mtDNA analysis, thus avoiding a separate biopsy of skeletal muscle elsewhere.

Levator palpebrae biopsy and diagnosis of progressive external ophthalmoplegia.

BACKGROUND: Progressive external ophthalmoplegia (PEO) is a mitochondrial myopathy of ocular muscles. Diagnostic investigation usually involves limb skeletal muscle biopsy and molecular genetic studies, although diagnostic yield tends to be low. The purpose of this study was to evaluate the diagnostic yield obtained by analysis of levator palpebrae (LP) muscle tissue. METHODS: This is a clinicopathologic study of 8 patients with a diagnosis of PEO, who had LP muscle biopsies as part of oculoplastic procedures. Six of these patients also had limb muscle biopsies. Histopathology, electron microscopy and genetic studies were performed. RESULTS: Diagnostic histopathologic findings were present in 4/6 quadriceps biopsies, and 7/8 LP biopsies. Genetic testing on DNA extracted from LP muscle revealed abnormalities in 4 patients. CONCLUSION: In patients whose LP. muscle demonstrate both genetic defects and histopathological abnormalities, the diagnosis of PEO can be confirmed without limb muscle biopsy. Patients having LP resection during oculoplastics procedures for treatment of ptosis may therefore be able to avoid a separate procedure for limb muscle biopsy. Further study is required to determine the specificity of these findings.

Urinary AASA excretion is elevated in patients with molybdenum cofactor deficiency and isolated sulphite oxidase deficiency.

Analysis of α-aminoadipic semialdehyde is an important tool in the diagnosis of antiquitin deficiency (pyridoxine-dependent epilepsy). However continuing use of this test has revealed that elevated urinary excretion of α-aminoadipic semialdehyde is not only found in patients with pyridoxine-dependent epilepsy but is also seen in patients with molybdenum cofactor deficiency and isolated sulphite oxidase deficiency. This should be taken into account when interpreting the laboratory data. Sulphite was shown to inhibit α-aminoadipic semialdehyde dehydrogenase in vitro.

A variant of unknown significance in the GLA gene causing diagnostic uncertainty in a young female with isolated hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is genetically heterogeneous, and largely caused by mutations in genes encoding sarcomere proteins. However, GLA mutations causing Fabry disease, an X-linked lysosomal storage disorder, may also present with isolated HCM. As HCM genetic testing panels are increasingly being used clinically, variants of unknown significance (VUS) are encountered, leading to challenges in interpretation. We present an illustrative case: a 10-year-old girl with isolated HCM who, on testing with a HCM multi-gene panel, was found to carry a maternally inherited p.W24R variant in GLA. Attempts to evaluate the significance of this variant, by direct biochemical testing of patient specimens, gave inconclusive results. Subsequent in vitro protein expression studies suggested that the variant is unlikely to be pathogenic. This case highlights diagnostic dilemmas that can be provoked by VUS in general, and specifically raises a question whether GLA sequencing should be included in first-line diagnostic testing for female children with isolated hypertrophic cardiomyopathy.

Using next-generation sequencing for the diagnosis of rare disorders: a family with retinitis pigmentosa and skeletal abnormalities.

Linkage analysis with subsequent candidate gene sequencing is typically used to diagnose novel inherited syndromes. It is now possible to expedite diagnosis through the sequencing of all coding regions of the genome (the exome) or full genomes. We sequenced the exomes of four members of a family presenting with spondylo-epiphyseal dysplasia and retinitis pigmentosa and identified a six-base-pair (6-bp) deletion in GNPTG, the gene implicated in mucolipidosis type IIIγ. The diagnosis was confirmed by biochemical studies and both broadens the mucolipidosis type III phenotype and demonstrates the clinical utility of next-generation sequencing to diagnose rare genetic diseases.

Progression of organ manifestations upon enzyme replacement therapy in a patient with mucopolysaccharidosis type I/Hurler.

BACKGROUND: Enzyme replacement therapy (ERT) has been increasingly used as an interim treatment in severe mucopolysaccharidosis type I (MPSI)/Hurler patients prior to hematopoietic stem cell transplantation (HSCT). METHODS: We present the outcome of a patient with MPSI/Hurler after 14 months of ERT prior to HSCT. RESULTS: Urinary glucosaminoglycan excretion decreased by 70% after one month of ERT. Liver volume decreased by 14% of baseline after 12 months of ERT. Pre-existing thoracolumbar kyphosis progressed to thoracolumbar dislocation with complete displacement of facets after 12 months of ERT. New development of mitral valve thickening was found by echocardiography and mild hearing loss progressed to severe sensorineural hearing loss after 13 months of ERT. CONCLUSIONS: ERT over a period of 14 months did not prevent progression of organ manifestations in our patient. Patients should be monitored every 6 months for cardiac, skeletal and audiological involvement on ERT.

Screening for Fabry disease in patients with chronic kidney disease: limitations of plasma alpha-galactosidase assay as a screening test.

BACKGROUND AND OBJECTIVES: Fabry disease is a progressive X-linked disorder of glycosphingolipid metabolism that typically presents in childhood and progresses to heart failure and renal failure in adulthood. This study sought to determine the prevalence of Fabry disease in a multiethnic male chronic kidney disease population, involving dialysis-dependent, non-dialysis-dependent, and transplant patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: A total of 499 patients were screened with assay of alpha-galactosidase activity using fluorometric enzyme assay on plasma prepared from fresh heparinized blood, followed by leukocyte alpha-galactosidase activity in the subset of patients with plasma alpha-galactosidase activity below the second percentile (corresponding to a value <3.0 nmol/h per ml plasma). RESULTS: This study did not identify any new cases of Fabry disease; however, repeat testing of some of the study patients identified three limitations of the plasma enzyme assay that is commonly used as a high throughput screening method for Fabry disease: (1) False-negative results can occur; (2) these false-negative results are not prevented by use of inhibitors of alpha-galactosidase B activity; and (3) considerable intraindividual variation in plasma alpha-galactosidase levels reduces the discriminatory power of the screening test. CONCLUSION: Clinicians need to be aware that screening using plasma will fail to detect some patients with Fabry disease.