Application of MALDI-TOF Mass Spectrometry for Non-invasive Diagnostics of Mucopolysaccharidosis IIIA

Abstract Mucopolysaccharidosis IIIA (MPS IIIA) is a lysosomal storage disorder (LSD) caused by deficiency of lysosomal N-sulphoglucosamine sulphohydrolase, which is one of four enzymes involved in heparan sulfate degradation. Traditional methods used for MPS IIIA diagnostics usually constitute of selective screening, based on the analysis of urinary glycosaminoglycans, further enzymatic assays in leukocytes, and mutation analysis. Nowadays, some LSDs, including mucopolysaccharidoses, can be precisely diagnosed by mass spectrometry-based techniques. Up to this date, there are no comprehensive studies of MPS IIIA diagnostics by MALDI-TOF analysis of free oligosaccharides in urine published. In the presented work, MALDI-TOF/TOF analysis of permethylated oligosaccharides was performed to obtain the set of glyco-biomarkers that together form the specific fingerprint of this disease. Early and accurate diagnostics of MPS IIIA is crucial to stabilize the progressive cellular damage and improve the overall well-being of patients.

Mutación novel en el Síndrome Sanfilippo B. Presentación de un caso / A novel mutation in Sanfilippo Syndrome type B. Case report

Introducción: El Síndrome Sanfilippo B es un error innato en el metabolismo lisosomal, con herencia autosómica recesiva. Se caracteriza por facie ligeramente tosca, deterioro neurológico progresivo y poca repercusión somática, provocado por mutaciones en el gen NAGLU, cuyo locus es 17q21.2. La incidencia internacionalmente es muy baja y en Cuba solo se han diagnosticado siete pacientes desde 1985. Objetivo: Describir las manifestaciones clínicas, bioquímicas y moleculares de un paciente cubano diagnosticado con Síndrome Sanfilippo B. Presentación de Caso: Se describió un paciente de 13 años, cuyas principales manifestaciones clínicas fueron: facie ligeramente tosca, sinofris, alteraciones de conducta y deterioro neurológico progresivo. El trastorno del sueño fue ocasional y frecuente las infecciones respiratorias. Se demostró la presencia de colitis ulcerativa y pólipo intestinal. Se confirmó excreción aumentada de heparán sulfato y disminución de la actividad enzimática N-acetil αD-glucosaminidasa. Se identificó la mutación c.640dupC en el gen NAGLU en homocigosis en el paciente y ambos padres resultaron ser portadores. Conclusiones: Predominaron las alteraciones de conducta, deterioro neurológico progresivo e infecciones respiratorias en el caso reportado; siendo la colitis ulcerativa y el pólipo intestinal un hallazgo no descrito anteriormente para esta enfermedad. Los estudios cromatográficos y enzimáticos resultaron positivos para Sanfilippo B. El genotipo de este paciente resultó ser homocigótico para una nueva variante alélica patogénica en el gen NAGLU. Se demostró la segregación mendeliana de la mutación en la familia(AU)

Introduction: Sanfilippo syndrome type B is an autosomal recessive lysosomal storage disease. The frequent clinical manifestations include slightly coarse facial features, progressive neurodegeneration and mild somatic repercussion caused by mutations in the NAGLU gene, whose locus is 17q21.2. The worldwide incidence is very low and only seven patients have been diagnosed in Cuba since 1985. Objective: To describe clinical, biochemical and molecular characteristics of a Cuban patient with the diagnosis of Sanfilippo Syndrome type B. Case presentation: A 13 years old patient was described. The main clinical manifestations included mild coarse facie, synophrys, behavior disturbances, and progressive neurologic deterioration. Intermittent sleep disturbance and frequent upper respiratory infections were identified. Ulcerative colitis and intestinal polyp were demonstrated. Increased excretion of heparan sulfate and very low N-acetyl α-Dglucosaminidase activity were confirmed. In addition, the presence of mutation c.640dupC in NAGLU gene was identified. The patient had homozygous genotype and both parents were heterozygous. Conclusions: Behavioral alterations, progressive neurological deterioration and respiratory infections predominated in the reported case. Other findings such as ulcerative colitis and intestinal polyps were not previously described in this disease. The chromatographic and enzymatic studies were positive for Sanfilippo type B. This patient's genotype was found to be homozygous for a novel pathogenic allelic variant in the NAGLU gene. Mendelian segregation of the mutation in the family was demonstrated(AU)

Determination of Reference Values for Alpha-N-Acetylglucosaminidase Activities in Patients with Sanfilippo Type B Disease and Control Population in Colombia

Abstract Sanfilippo B is a lysosomal disorder characterized by the pathological accumulation of heparan sulfate. It is caused by mutations in the NAGLU gene that codes for the alpha-N-acetylglucosaminidase enzyme. The objective of this study was to determine the reference values and frequency of Sanfilippo B in Colombia through an enzyme analysis of leukocytes extracts. We aim to inform the community and the health system so that they can work in a preventive way, providing an early diagnosis of patients and thus providing an appropriate management of the symptoms. We carried out an endpoint assay that indirectly quantifies NAGLU activity through the cleavage of 4-methylumbelliferone from the 4-methylumbelliferyl-2-acetamido-2-deoxy-α-D-glucopyranoside substrate. The activity of 463 healthy volunteers (Range: 0.6 - 4 nmol/mg/h, Median: 1.69 +/- 0.73) as well as 462 patients referred for clinical suspicion, was calculated. From the last group, 7 cases turned out to be positive (Range: 0 - 0.24 nmol/mg/h, Median: 0.13 +/- 0.09). The cut-off point according to ROC analysis between affected patients and controls was 0.42 nmol/mg/h. To our knowledge, this study is the first in Colombia where an estimated frequency of Sanfilippo type B is calculated by providing enzyme activity ranges and a cut-off point.

A Novel Mutation (p.Met1?) of a Cuban Patient in the NAGLU Gene with Mucopolysaccharidosis IIIB

Abstract Mucopolysaccharidosis III (MPSIII) or Sanfilippo syndrome is an autosomal recessive disorder of lysosomal metabolism. MPS III is caused by mutations in genes that encode for the enzymes involved in the degradation of heparan sulfate. It is classified into 4 subtypes (MPSIII A-D). MPS IIIB is induced by mutations in the gene encoding the alpha-N-acetylglucosaminidase enzyme. We report a 6-year-old boy with phenotypic findings of Sanfilippo syndrome type B, such as mild coarse facie, clear corneas, hirsutism, hepatomegaly, mild joint stiffness and mild dysostosis multiplex. He also presents frequent upper respiratory infections, bilateral hearing loss, sleep disturbances, progressive neurologic deterioration and behavioral problems. He is compound heterozygous for the NAGLU gene (c.503G˃A; p.Trp168Ter/ c.3G˃A; p.met1?). One of the mutation was described in two patients before. A novel pathogenic variant was detected.

A New Fluorescent Method to Detect Sulfamidase Activity in Blood, Tissue Extracts and Dried Blood Spots

Abstract Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder due to the deficient activity of sulfamidase (SGSH). Traditionally, measurement of this enzymatic activity has been performed using a fluorescently (4-MU) labeled glycoside substrate. While this substrate is inexpensive and readily available, the current method requires a 2-step procedure that is performed over 2 days. Here we report a new and simplified procedure using the 4-MU substrate. Major advantages of this assay method over the existing fluorescent method include a single step vs. 2-step procedure, an incubation time of 1 hour, and high sensitivity. The reaction is also run on UPLC equipment, which is available in most research labs and permits separation of the endogenous, autofluorescent material from the 4-MU signal. This assay method was developed using the MPS IIIA mouse model, and was validated using mouse plasma, liver and brain extracts, and dried blood spots. Human MPS IIIA skin fibroblasts and dried blood spots also were used to validate the method.

Mucopolysacaridosis type iiib misdiagnosed as an autistic spectrum disorder: a case report and literature review / Mucopolissacaridose tipo iii b mal diagnosticada como transtorno de espectro autista: relato de caso e revisão de literatura

ABSTRACT Objective: To report a rare case of mucopolysaccharidosis IIIB in a pediatric patient, with emphasis on the description of the clinical manifestations and the early diagnosis. Case description: A 14-year-old male patient, who presented regression of neuropsychomotor development since his three years and six months old, with speech loss and frequent falls, evolving with behavioral changes, with agitation and aggressiveness. Although being diagnosed with autism, there was no response to the established treatment; he was subsequently submitted to metabolic investigation, which lead to the diagnosis of Mucopolysaccharidosis IIIB. Comments: Identifying a metabolic disorder requires connecting multiple signs and symptoms, as well as eliminating other apparent causes. MPS IIIB is a diagnostic challenge, particularly in the early stages and in the absence of a family history of the disease.

RESUMO Objetivo: Relatar o caso raro de um paciente pediátrico com mucopolissacaridose III B, com ênfase na descrição de manifestações clínicas. Descrição do caso: Paciente masculino de 14 anos que, a partir dos 3 anos e 6 meses de idade, apresentou regressão do desenvolvimento neuropsicomotor, com perda da fala e quedas frequentes, evoluindo com alterações comportamentais, agitação e agressividade. Diagnosticado como autista, não obteve resposta ao tratamento estabelecido, sendo posteriormente submetido à investigação metabólica, que evidenciou o diagnóstico de mucopolissacaridose III B. Comentários: A identificação de um distúrbio metabólico exige conectar vários sinais e sintomas, além de eliminar outras causas aparentes. A mucopolissacaridose III B é um desafio diagnóstico, particularmente nos estágios iniciais e na ausência de história familiar da doença.

Sanfilippo Syndrome: The Tale of a Challenging Diagnosis

Abstract Sanfilippo syndrome or mucopolysaccharidosis III (MPS III), includes a group of four autosomal recessive lysosomal storage disorders caused by deficient activity of enzymes involved in the catabolism of heparan sulfate. The four types of MPS III are recognized in accordance with the deficient enzyme, resulting in the accumulation of heparan sulfate with particularly deleterious effects in the central nervous system. The incidence of MPS III remains to be established in Latin American countries. We describe the journey of a patient with MPS IIIB whom, even in the presence of speech delay and deterioration, behavioral problems and motor incoordination, showed unaltered urinary glycosaminoglycans (GAGs) levels. An investigation for MPS was undertaken and enzyme analysis indicated a deficiency of alpha-N-acetylglucosaminidase, leading to the diagnosis of MPS IIIB. With the correct diagnosis, the patient's symptoms could be properly managed, and the parents received appropriate genetic counseling. The present case report reinforces the need of investigating MPS III in patients with language delay and/or regression, neurological impairment and behavioral alterations, even when urinary GAGs are within normal range. A definitive diagnosis ends the diagnostic journey and enables the medical team and family to provide a better care for the child.

Development of a Clinical Algorithm for the Early Diagnosis of Mucopolysaccharidosis III

Abstract Mucopolysaccharidosis III (MPS III) is a rare inherited metabolic disease primarily affecting the central nervous system, leading to developmental and/or speech regression. Early diagnosis of the disease is important to introduce appropriate management measures and to optimize therapeutic outcomes. The diagnosis of MPS III is often significantly delayed due to the rarity of the disease, the more attenuated somatic presentation compared to other MPS types, and the symptom overlap with other developmental disorders. To shorten the time to diagnosis, a list of eight early signs and symptoms was identified through an expert system approach by a global, multidisciplinary working group of 13 specialists with expertise in various aspects of MPS and developmental disorders and three parents of MPS III patients. Coarse facial features and persistent hirsutism or prominent, thick eyebrows were identified as the most important MPS III early signs. The list of eight early MPS III signs and symptoms is the first step towards the development of a clinical algorithm aiming to identify neonates and infants with MPS III before the onset of neurocognitive damage, ultimately shortening the diagnostic journey of MPS III patients.

Genética clínica comunitaria: exploración de patología genética en Boyacá, Colombia / Clinical community genetics: exploring genetic disorders in Boyacá, Colombia

RESUMEN Objetivo Explorar la presencia de patología genética sindrómica en el Departamento de Boyacá, mediante un acercamiento de medicina genética comunitaria. Materiales y Métodos Un grupo conformado por genetistas, neurólogo pediátrico y genetista bioquímico, llevó a cabo jornadas clínicas en las cuales se evaluaron pacientes con sospecha de enfermedad genética. Se obtuvieron datos demográficos, epidemiológicos y clínicos y se realizó el cálculo de frecuencias de los mismos. En los centros de referencia visitados se realizaron actividades de capacitación al personal médico. Resultados Se encontraron dos agrupamientos genéticos: MPSIII y Síndrome de Ellis Van Creveld, con incidencias mayores a lo reportado en la literatura, además una alta frecuencia de patologías de herencia autosómica recesiva, así como sospecha de síndromes de microdeleción-microduplicación. Conclusiones Se deben establecer mecanismos no convencionales de atención médica para facilitar el acceso a las comunidades a un diagnóstico y tratamiento adecuados en genética. Se espera que el apoyo brindado a los pacientes, familias y personal asistencial de los hospitales a través de las jornadas clínicas y la capacitación, permitan alcanzar este objetivo y a la vez sea un punto de inicio de procesos de prevención primaria y secundaria.(AU)

ABSTRACT Objectives To explore the incidence of syndromic genetic pathologies in Boyacá, Colombia, through a community genetics approach. Materials and Methods A group made up by different medical specialists (geneticists, a pediatric neurologist, and a biochemical geneticist) developed clinical campaigns, in which patients with clinical suspicion of genetic diseases were involved. Demographic, epidemiological and clinical data were collected, and frequency calculations were made based on the collected data. Several training workshops for health personnel were done in each center visited. Results Two genetic clusters were found: mucopolysaccharidosis type III, and Ellis-Van Creveld Syndrome, both of them with higher incidences than those found in the literature. Also, a high frequency of autosomal recessive diseases was found, as well as microdeletion/microduplication syndromes. Conclusions Conventional mechanisms of medical attention must be established, in order to facilitate the access to an appropriate diagnosis and treatment. This work intended to provide support to patients, families and health care services personnel through the workshops and clinical campaigns, and to become a starting point to develop primary and secondary prevention processes.(AU)

Mucopolisacaridosis: características clínicas, diagnóstico y de manejo / Mucopolysaccharidosis: clinical features, diagnosis and management

Las mucopolisacaridosis (MPS) son un grupo de enfermedades raras (huérfanas), de baja prevalencia, caracterizadas por la deficiencia de enzimas que participan en el metabolismo de glucosaminglucanos (GAG) a nivel lisosomal. Se caracteriza por acumulación de GAG intracelular, produciendo alteraciones de múltiples órganos y sistemas. Su diagnóstico se basa en el conocimiento de las manifestaciones clínicas, realizar el análisis bioquímico para identificar el tipo de GAG que se está acumulando y confirmar el tipo de enfermedad con la determinación enzimática correspondiente. Su identificación es fundamental para iniciar un tratamiento oportuno, teniendo en cuenta que actualmente existe manejo transdisciplinario y tratamiento de reemplazo enzimático para MPS I (síndrome de Hurler), MPS II (síndrome de Hunter), MPS IV (síndrome de Morquio) y MPS VI (síndrome de Maroteaux-Lamy). En esta revisión se analizan cada uno de estos síndromes, su diagnóstico y tratamiento.

The mucopolysaccharidoses (MPS) are a group of rare (orphan) diseases, characterised by a deficiency of enzymes involved in the metabolism of glycosaminoglycans (GAGs) at lysosomal level. When there is a deficiency of a particular enzyme there is an accumulation of GAGs in the cells resulting in progressive cellular damage, which can affect multiple organ systems and lead to organ failure. Diagnosis is based on knowledge of the clinical manifestations, performing biochemical analyses to identify the type of GAG that is accumulating, and confirm the type of disorder with the corresponding enzymatic determination. Their identification is essential to initiate early treatment, taking into account that multidisciplinary management and enzyme replacement therapy is available for MPS I (Hurler syndrome), MPS II (Hunter syndrome), MPS IV (Morquio syndrome), and MPS VI (Maroteaux-Lamy syndrome. In this review, an analysis is made of each of these syndromes, as well as their diagnosis and treatment.

Mucopolisacaridosis (síndrome de Hunter) / Mucopolysaccharidosis (Hunter syndrome)

Se realiza una presentación de un caso interesante, no comúnmente visto en la práctica médica, de unos de los tipos de mucopolisacaridosis, específicamente de un síndrome de Hunter. Se hace esta presentación con el objetivo de dar a conocer a estudiantes y profesionales de la salud, mediante fotos, las características físicas del paciente con dicho sídrome, quien llegó desnutrido al hospital; se le operó de la hernia umbilical y se mejoró su estado nutricional al compensarse su hepatopatía. Se le da el alta médica en mejores condiciones(AU)

We present here an interesting case of mucopolysaccharidoses, which is not commonly seen in medical practice, specifically a Hunter syndrome. This presentation is done in order to make known to students and health professionals, through photos, the physical characteristics of the patient with such syndrome. This patient arrived malnourished at the hospital, he was operated on the umbilical hernia and improved his nutritional status by compensating for his liver disease. This patient had medical discharge in better conditions(AU)

The First Korean Case of Mucopolysaccharidosis IIIC (Sanfilippo Syndrome Type C) Confirmed by Biochemical and Molecular Investigation

Mucopolysaccharidosis (MPS) III has 4 enzymatically distinct forms (A, B, C, and D), and MPS IIIC, also known as Sanfilippo C syndrome, is an autosomal recessive lysosomal storage disease caused by a deficiency of heparan acetyl-CoA:alpha-glucosaminide N-acetyltransferase (HGSNAT). Here, we report a case of MPS IIIC that was confirmed by molecular genetic analysis. The patient was a 2-yr-old girl presenting with skeletal deformity, hepatomegaly, and delayed motor development. Urinary excretion of glycosaminoglycan (GAG) was markedly elevated (984.4 mg GAG/g creatinine) compared with the age-specific reference range (A (IVS2+1G>A) and c.1150C>T (p.Arg384*). To the best of our knowledge, this is the first case of MPS IIIC to be confirmed by clinical, biochemical, and molecular genetic findings in Korea.