Synergistic use of glycomics and single-molecule molecular inversion probes for identification of congenital disorders of glycosylation type-1.

Congenital disorders of glycosylation type 1 (CDG-I) comprise a group of 27 genetic defects with heterogeneous multisystem phenotype, mostly presenting with nonspecific neurological symptoms. The biochemical hallmark of CDG-I is a partial absence of complete N-glycans on transferrin. However, recent findings of a diagnostic N-tetrasaccharide for ALG1-CDG and increased high-mannose N-glycans for a few other CDG suggested the potential of glycan structural analysis for CDG-I gene discovery. We analyzed the relative abundance of total plasma N-glycans by high resolution quadrupole time-of-flight mass spectrometry in a large cohort of 111 CDG-I patients with known (n = 75) or unsolved (n = 36) genetic cause. We designed single-molecule molecular inversion probes (smMIPs) for sequencing of CDG-I candidate genes on the basis of specific N-glycan signatures. Glycomics profiling in patients with known defects revealed novel features such as the N-tetrasaccharide in ALG2-CDG patients and a novel fucosylated N-pentasaccharide as specific glycomarker for ALG1-CDG. Moreover, group-specific high-mannose N-glycan signatures were found in ALG3-, ALG9-, ALG11-, ALG12-, RFT1-, SRD5A3-, DOLK-, DPM1-, DPM3-, MPDU1-, ALG13-CDG, and hereditary fructose intolerance. Further differential analysis revealed high-mannose profiles, characteristic for ALG12- and ALG9-CDG. Prediction of candidate genes by glycomics profiling in 36 patients with thus far unsolved CDG-I and subsequent smMIPs sequencing led to a yield of solved cases of 78% (28/36). Combined plasma glycomics profiling and targeted smMIPs sequencing of candidate genes is a powerful approach to identify causative mutations in CDG-I patient cohorts.

Diagnostic serum glycosylation profile in patients with intellectual disability as a result of MAN1B1 deficiency.

Congenital disorders of glycosylation comprise a group of genetic defects with a high frequency of intellectual disability, caused by deficient glycosylation of proteins and lipids. The molecular basis of the majority of the congenital disorders of glycosylation type I subtypes, localized in the cytosol and endoplasmic reticulum, has been solved. However, elucidation of causative genes for defective Golgi glycosylation (congenital disorders of glycosylation type II) remains challenging because of a lack of sufficiently specific diagnostic serum methods. In a single patient with intellectual disability, whole-exome sequencing revealed MAN1B1 as congenital disorder of glycosylation type II candidate gene. A novel mass spectrometry method was applied for high-resolution glycoprofiling of intact plasma transferrin. A highly characteristic glycosylation signature was observed with hybrid type N-glycans, in agreement with deficient mannosidase activity. The speed and robustness of the method allowed subsequent screening in a cohort of 100 patients with congenital disorder of glycosylation type II, which revealed the characteristic glycosylation profile of MAN1B1-congenital disorder of glycosylation in 11 additional patients. Abnormal hybrid type N-glycans were also observed in the glycoprofiles of total serum proteins, of enriched immunoglobulins and of alpha1-antitrypsin in variable amounts. Sanger sequencing revealed MAN1B1 mutations in all patients, including severe truncating mutations and amino acid substitutions in the alpha-mannosidase catalytic site. Clinically, this group of patients was characterized by intellectual disability and delayed motor and speech development. In addition, variable dysmorphic features were noted, with truncal obesity and macrocephaly in ∼65% of patients. In summary, MAN1B1 deficiency appeared to be a frequent cause in our cohort of patients with unsolved congenital disorder of glycosylation type II. Our method for analysis of intact transferrin provides a rapid test to detect MAN1B1-deficient patients within congenital disorder of glycosylation type II cohorts and can be used as efficient diagnostic method to identify MAN1B1-deficient patients in intellectual disability cohorts. In addition, it provides a functional confirmation of MAN1B1 mutations as identified by next-generation sequencing in individuals with intellectual disability.

Genotype-phenotype spectrum of PYCR1-related autosomal recessive cutis laxa.

Autosomal recessive cutis laxa type 2B (ARCL2B; OMIM # 612940) is a segmental progeroid disorder caused by mutations in PYCR1 encoding pyrroline-5-carboxylate reductase 1, which is part of the conserved proline de novo synthesis pathway. Here we describe 33 patients with PYCR1-related ARCL from 27 families with initial diagnoses varying between wrinkly skin syndrome, gerodermia osteodysplastica, De Barsy syndrome or more severe progeria syndromes. Given the difficult differential diagnosis of ARCL syndromes we performed a systematic comparison of clinical features of PYCR1-related ARCL. Intrauterine growth retardation, a characteristic triangular facial gestalt, psychomotor retardation, and hypotonia were the most relevant distinctive hallmarks of ARCL due to proline de novo synthesis defects. Corneal clouding or cataracts, athetoid movements, and finger contractures were rather rare features, but had a high predictive value. In our cohort we identified 20 different PYCR1 mutations of which seven were novel. Most of the mutations accumulated in exons 4 to 6. Missense alterations of highly conserved residues were most frequent followed by splice site changes and a single nonsense mutation. Analysis of genotype-phenotype correlation revealed that patients with mutations in the first two exons had lower average clinical scores and absent or only mild intellectual disability. Structural analyses predicted interference with PYCR1 multimerization for a subset of missense mutations. These findings have implications for the clinics as well as the pathomechanism of PYCR1-related ARCL.

Diagnosing lysosomal storage diseases in a Brazilian non-newborn population by tandem mass spectrometry.

OBJECTIVES: High-throughput mass spectrometry methods have been developed to screen newborns for lysosomal storage disorders, allowing the implementation of newborn screening pilot studies in North America and Europe. It is currently feasible to diagnose Pompe, Fabry, Gaucher, Krabbe, and Niemann-Pick A/B diseases, as well as mucopolysaccharidosis I, by tandem mass spectrometry in dried blood spots, which offers considerable technical advantages compared with standard methodologies. We aimed to investigate whether the mass spectrometry methodology for lysosomal storage disease screening, originally developed for newborns, can also discriminate between affected patients and controls of various ages. METHODS: A total of 205 control individuals were grouped according to age and subjected to mass spectrometry quantification of lysosomal α-glucosidase, ß-glucocerebrosidase, α-galactosidase, acid sphingomyelinase, galactocerebrosidase, and α-L-iduronidase activities. Additionally, 13 affected patients were analyzed. RESULTS: The median activities for each enzyme and each age group were determined. Enzyme activities were significantly lower in individuals aged older than 18 years compared with those in newborns. Affected patients presented enzymatic activities corresponding to less than 20% of the age-matched controls. CONCLUSIONS: Our data indicate that the mass spectrometry methodology can be used for the screening of lysosomal storage diseases in non-newborn patients. However, for some diseases, such as Fabry and mucopolysaccharidosis I, a combination of biochemical and clinical data may be necessary to achieve accurate diagnoses.

Analysis of novel ARG1 mutations causing hyperargininemia and correlation with arginase I activity in erythrocytes.

Hyperargininemia (HA) is an autosomal recessive disease that typically has a clinical presentation that is distinct from other urea cycle disorders. It is caused by the deficient activity of the enzyme arginase I, encoded by the gene ARG1. We screened for ARG1 mutations and measured erythrocyte enzyme activity in a series of 16 Brazilian HA patients. Novel mutations, in addition to previously described missense mutations, were analysed for their effect on the structure, stability and/or function of arginase I (ARG1) using bioinformatics tools. Three previously reported mutations were found (p.R21X; p.I11T and p.W122X), and five novel mutations were identified (p.G27D; p.G74V; p.T134I; p.R308Q; p.I174fs179). The p.T134I mutation was the most frequent in the Brazilian population. Patients carrying the p.R308Q mutation had higher residual ARG1 decreased activity, but presented no distinguishable phenotype compared to the other patients. Bioinformatics analyses revealed that missense mutations (1) affect the ARG1 active site, (2) interfere with the stability of the ARG1 folded conformation or (3) alter the quaternary structure of the ARG1. Our study reinforced the role of Arg308 residue for assembly of the ARG1 homotrimer. The panel of heterogeneous ARG1 mutations that cause HA was expanded, nevertheless a clear genotype-phenotype correlation was not observed in our series.

Diagnosing lysosomal storage diseases in a Brazilian non-newborn population by tandem mass spectrometry

OBJECTIVES: High-throughput mass spectrometry methods have been developed to screen newborns for lysosomal storage disorders, allowing the implementation of newborn screening pilot studies in North America and Europe. It is currently feasible to diagnose Pompe, Fabry, Gaucher, Krabbe, and Niemann-Pick A/B diseases, as well as mucopolysaccharidosis I, by tandem mass spectrometry in dried blood spots, which offers considerable technical advantages compared with standard methodologies. We aimed to investigate whether the mass spectrometry methodology for lysosomal storage disease screening, originally developed for newborns, can also discriminate between affected patients and controls of various ages. METHODS: A total of 205 control individuals were grouped according to age and subjected to mass spectrometry quantification of lysosomal α-glucosidase, β-glucocerebrosidase, α-galactosidase, acid sphingomyelinase, galactocerebrosidase, and α−L-iduronidase activities. Additionally, 13 affected patients were analyzed. RESULTS: The median activities for each enzyme and each age group were determined. Enzyme activities were significantly lower in individuals aged older than 18 years compared with those in newborns. Affected patients presented enzymatic activities corresponding to less than 20% of the age-matched controls. CONCLUSIONS: Our data indicate that the mass spectrometry methodology can be used for the screening of lysosomal storage diseases in non-newborn patients. However, for some diseases, such as Fabry and mucopolysaccharidosis I, a combination of biochemical and clinical data may be necessary to achieve accurate diagnoses. .

Estudo nosólogico, clínico e etiológico de pacientes com atraso do desenvolvimento psico-motor, retardo mental e/ou regressão neurológica em hospital de doenças do aparelho locomotor

Foram selecionados, para o presente estudo, 490 pacientes atendidos no Ambulatório da Rede Sarah, num período de sete anos, que apresentavam quadros de atraso do desenvolvimento psico-motor, retardo mental e/ou regressão neurológica e que foram referenciados ao laboratório de bioquímica genética para investigação como parte de rotina estabelecida para estudo. Etiologia genética foi encontrada em 29,79 por cento dos pacientes, distribuídos da seguinte forma: doenças monogênicas em 22,65 por cento dos casos, cromossômicas em 5,51 por cento, poligênica esporádica em 1,02 por cento e monogênica esporádica em 0,61 por cento. Etiologia ambiental respondeu por 27,35 por cento dos pacientes e casos cuja etiologia não pode ser determinada por representarem distúrbios sem diagnóstico específico contribuíram com 42,86 por cento dos casos. Retardo mental foi o grupo mais numeroso (57,96 por cento dos indivíduos) e participações iguais foram encontradas nos probandos com quadros de regressão neurológica e atraso no desenvolvimento psico-motor. Os propósitos foram agrupados em seis classes [...] Dentre as patologias monogênicas, as autossômicas ocorreram na maioria dos diagnósticos (75,67 por cento dos casos de herança monogênica), patologias de herança autossômica dominante em 7,21 por cento desses, heranças ligadas ao cromossomo X em 9,91 por cento, e herança inconclusiva (autossômica recessiva ou ligadas ao cromossomo X) em 7,21 por cento dos casos. As anormalidades do sistema nervoso central tiveram alta frequência em nossa amostra (63,76 por cento), justificando a realização de tomografia computadorizada e/ou ressonância nuclear magnética nos casos de RM, RGN e ADPM, o que poderá indicar ao profissional de saúde o processo responsável por esses quadros, permitindo a elucidação diagnóstica, aconselhamento genético e orientando o tratamento. Patologias de etiologia ou ambiental, responsável por parcela expressiva dos pacientes estudados, podem ser prevenidas ou tratadas de forma adequada, através de melhoria nos cuidados pré-, peri- e pós-natais, disponibilização de exames laboratoriais e radiológicos necessários à investigação, além de ampliação do rastreamento neo-natal de doenças metabólicas, bem como a viabilização de tratamento para os casos identificados