Mass spectrometry glycophenotype characterization of ALG2-CDG in Argentinean patients with a new genetic variant in homozygosis.

Human ALG2 encodes an α 1,3mannosyltransferase that catalyzes the first steps in the synthesis of N-glycans in the endoplasmic reticulum. Variants in ALG2cause a congenital disorder of glycosylation (CDG) known as ALG2-CDG. Up to date, nine ALG2-CDG patients have been reported worldwide. ALG2-CDG is a rare autosomal recessive inherited disorder characterized by neurological involvement, convulsive syndrome of unknown origin, axial hypotonia, and mental and motor regression. In this study, we used MALDI-TOF MS to define both total serum protein and transferrin (Tf) N-glycan phenotypes in three ALG2-CDG patients carrying a c.752G > T, p.Arg251Leu ALG2 missense variant in homozygous state, as determined by exome sequencing. Comparing it to control samples, we have observed Tf under-occupancy of glycosylation site(s) typical of a defective N-glycan assembly and the occurrence of oligomannose and hybrid type N-glycans. Moreover, we have observed a slight oligomannose accumulation in total serum glyco-profiles. The increased heterogeneity of serum N-glycome in the studied patients suggests a marginal disarrangement of the glycan processing in ALG2-CDG. Previous studies reported on slightly increased concentrations of abnormal serum N-glycans in CDG-I due to defects in the mannosylation steps of dolichol-linked oligosaccharide biosynthesis. This preliminary work aims at considering serum N-glycan accumulation of high mannosylated glycoforms, such as oligomannose and hybrid type N-glycans, as potential diagnostic signals for ALG2-CDG patients.

Ten years of screening for congenital disorders of glycosylation in Argentina: case studies and pitfalls.

BACKGROUND: Congenital Disorders of Glycosylation (CDG) are genetic diseases caused by hypoglycosylation of glycoproteins and glycolipids. Most CDG are multisystem disorders with mild to severe involvement. METHODS: We studied 554 patients (2007-2017) with a clinical phenotype compatible with a CDG. Screening was performed by serum transferrin isoelectric focusing. The diagnosis was confirmed by genetic testing (Sanger or exome sequencing). RESULTS: A confirmed abnormal pattern was found in nine patients. Seven patients showed a type 1 pattern: four with PMM2-CDG, two with ALG2-CDG, and one with classical galactosemia. A type 2 pattern was found in two patients: one with a CDG-IIx and one with a transferrin protein variant. Abnormal transferrin pattern were observed in a patient with a myopathy due to a COL6A2 gene variant. CONCLUSIONS: CDG screening in Argentina from 2007 to 2017 revealed 4 PMM2-CDG patients, 2 ALG2-CDG patients with a novel homozygous gene variant and 1 CDG-IIx.

Identification and functional analyses of CBS alleles in Spanish and Argentinian homocystinuric patients.

Homocystinuria due to CBS deficiency is a rare autosomal recessive disorder characterized by elevated plasma levels of homocysteine (Hcy) and methionine (Met). Here we present the analysis of 22 unrelated patients of different geographical origins, mainly Spanish and Argentinian. Twenty-two different mutations were found, 10 of which were novel. Five new mutations were missense and five were deletions of different sizes, including a 794-bp deletion (c.532-37_736 + 438del794) detected by Southern blot analysis. To assess the pathogenicity of these mutations, seven were expressed heterologously in Escherichia coli and their enzyme activities were assayed in vitro, in the absence and presence of the CBS activators PLP and SAM. The presence of the mutant proteins was confirmed by Western blotting. Mutations p.M173del, p.I278S, p.D281N, and p.D321V showed null activity in all conditions tested, whereas mutations p.49L, p.P200L and p.A446S retained different degrees of activity and response to stimulation. Finally, a minigene strategy allowed us to demonstrate the pathogenicity of an 8-bp intronic deletion, which led to the skipping of exon 6. In general, frameshifting deletions correlated with a more severe phenotype, consistent with the concept that missense mutations may recover enzymatic activity under certain conditions.

Mechanisms for phenotypic variation in Lesch-Nyhan disease and its variants.

Lesch-Nyhan disease is a neurogenetic disorder caused by mutation of the HPRT1 gene on the X chromosome. There is significant variation in the clinical phenotype, with more than 300 different known mutations. There are few studies that have addressed whether similar mutations result in similar phenotypes across different patients because hypoxanthine-guanine phosphoribosyltransferase (HGprt) deficiency is rare, and most mutations are unique or limited to individual families. However, recent studies have revealed multiple unrelated patients with similar mutations, providing an opportunity to examine genotype-phenotype correlations. We found significant variation among the clinical features of 10 patients from 8 unrelated families all carrying a mutation replacing guanine with adenine at base position 143 (c.143G>A) in the HPRT1 gene. This mutation results in replacement of arginine by histidine at amino acid position 48 (p.arg48his) in the HGprt enzyme. Biochemically, the enzyme exhibits reduced thermal integrity, a mechanism that may explain clinical variation. The literature reveals similar clinical variation among other patients with similar mutations, although the variation is relatively minor across the whole population of patients. Identifiable sources of clinical variation include known limitations of clinical ascertainment and mechanisms that affect residual enzyme activity and stability. These results are helpful for understanding genotype-phenotype correlations and discordance and likely are applicable to other neurogenetic disorders where similar variation occurs.

Attenuated variants of Lesch-Nyhan disease.

Lesch-Nyhan disease is a neurogenetic disorder caused by deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase. The classic form of the disease is described by a characteristic syndrome that includes overproduction of uric acid, severe generalized dystonia, cognitive disability and self-injurious behaviour. In addition to the classic disease, variant forms of the disease occur wherein some clinical features are absent or unusually mild. The current studies provide the results of a prospective and multi-centre international study focusing on neurological manifestations of the largest cohort of Lesch-Nyhan disease variants evaluated to date, with 46 patients from 3 to 65 years of age coming from 34 families. All had evidence for overproduction of uric acid. Motor abnormalities were evident in 42 (91%), ranging from subtle clumsiness to severely disabling generalized dystonia. Cognitive function was affected in 31 (67%) but it was never severe. Though none exhibited self-injurious behaviours, many exhibited behaviours that were maladaptive. Only three patients had no evidence of neurological dysfunction. Our results were compared with a comprehensive review of 78 prior reports describing a total of 127 Lesch-Nyhan disease variants. Together these results define the spectrum of clinical features associated with hypoxanthine-guanine phosphoribosyltransferase deficiency. At one end of the spectrum are patients with classic Lesch-Nyhan disease and the full clinical phenotype. At the other end of the spectrum are patients with overproduction of uric acid but no apparent neurological or behavioural deficits. Inbetween are patients with varying degrees of motor, cognitive, or behavioural abnormalities. Recognition of this spectrum is valuable for understanding the pathogenesis and diagnosis of all forms of hypoxanthine-guanine phosphoribosyltransferase deficiency.

Evidence for genetic heterogeneity in D-2-hydroxyglutaric aciduria.

We performed molecular, enzyme, and metabolic studies in 50 patients with D-2-hydroxyglutaric aciduria (D-2-HGA) who accumulated D-2-hydroxyglutarate (D-2-HG) in physiological fluids. Presumed pathogenic mutations were detected in 24 of 50 patients in the D-2-hydroxyglutarate dehydrogenase (D2HGDH) gene, which encodes D-2-hydroxyglutarate dehydrogenase (D-2-HGDH). Enzyme assay of D-2-HGDH confirmed that all patients with mutations had impaired enzyme activity, whereas patients with D-2-HGA whose enzyme activity was normal did not have mutations. Significantly lower D-2-HG concentrations in body fluids were observed in mutation-positive D-2-HGA patients than in mutation-negative patients. These results imply that multiple genetic loci may be associated with hyperexcretion of D-2-HG. Accordingly, we suggest a new classification: D-2-HGA Type I associates with D-2-HGDH deficiency, whereas idiopathic D-2-HGA manifests with normal D-2-HGDH activity and higher D-2-HG levels in body fluids compared with Type I patients. It remains possible that several classifications for idiopathic D-2-HGA patients with diverse genetic loci will be revealed in future studies.

Evaluation of the biotinidase activity in hepatic glycogen storage disease patients. Undescribed genetic finding associated with atypical enzymatic behavior: an outlook.

Repeated evaluation of biotinidase (BTD) activity was carried out for a long-term follow-up in patients with hepatic glycogen storage diseases (GSDs). The results indicated inter-intra variability among the GSD-Ia, GSD-III and GSD-IX patients. In addition, a c.1330G>C transversion in the BTD gene, resulting in a p.Asp444His substitution was detected in one allele of a GSD-Ia patient with sustained normal enzyme activity. Thus far, it is necessary to be cautious in the interpretation of the results of BTD activity as a presumptive GSD diagnostic element. It is not known why plasma BTD activity increases in GSDs patients, or the clinical importance of the increment. When viewed from a global perspective, there are some lines of biotin biology that could indicate a relationship between BTD´s behavior and GSDs.

The molecular landscape of propionic acidemia and methylmalonic aciduria in Latin America.

In this work, we review the clinical and genetic data in 14 Latin American propionic acidemia (PA) and 15 methylmalonic aciduria (MMAuria) patients. In the PA patients, we have identified four different changes in the PCCA gene, including one novel one (c.414+5G>A) affecting the splicing process. The PCCB mutational spectrum included two prevalent changes accounting for close to 60% of the mutant alleles studied and one novel change (c.494G>C) which by functional analysis is clearly pathogenic. We have also identified the deep intronic change c.654+462A>G, and the results of the antisense treatment in the patient's cell line confirmed the functional recovery of PCC activity. All PA patients bearing out-of-frame mutations presented the disease earlier while patients bearing in hemizygous fashion p.E168K and p.R165W presented the disease later. Regarding the MMAuria patients, we have found three novel mutations in the MUT gene (c.1068G>A, c.1587_1594del8 and c.593delA) and one in the MMAB gene (c.349-1 G>C). Two patients with MMAuria with homocystinuria cblC type are carriers of the frequent c.271dupA mutation. All mut(0), cblB and cblC patients presented the symptoms early and in general had more neurological complications, while cblA and mut(-) patients exhibited a late-onset presentation, and in general the long-term outcome was better. The results presented in this work emphasize the importance of the genetic analysis of the patients not only for diagnostic purposes but also to research into novel therapies based on the genotype.

Efficacy of citrulline supplementation to decrease the risk of pulmonary hypertension after congenital heart disease surgery. A local experience.

Introduction: Pulmonary hypertension (PH) is a major cause of morbi-mortality among patients with congenital heart disease (CHD) and also a potentially severe complication after surgical repair. Oral citrulline, a precursor to NO synthesis, is safe and efficacious for decreasing the risk of postoperative PH. Objective: Objetive: The aim of the present study was to investigate in pediatric patients the changes of plasma citrulline, arginine, homocysteine and nitric oxide (NO) metabolites and pulmonary artery pressures (PAP) pre-post cardiac surgery in order to describe our population status with regard to the risk of pulmonary hypertension and look for potential biomarkers for early detection and treatment. Main results/Discussion: 16 Argentine pediatric patients with CHD undergoing cardiopulmonary bypass were randomized in two groups: (A) with and (B) without perioperative citrulline supplementation. We found that plasma citrulline median levels before surgery were lower in both groups respect to referential values, probably due to the poor nutritional status of our patients; only group A surpassed post-surgery the minimum recommended level to avoid PH. Furthermore, none of the patients in group A showed mean PAP higher than 20 mmHg, whereas in group B, 67% of the measurements were ≥ than the reference level. Conclusions: We reaffirm that citrulline supplementation it is effective in reducing postoperative pulmonary hypertension and biomarkers could evidence patient status as a translational medicine application.

Urea cycle disorders in Argentine patients: clinical presentation, biochemical and genetic findings.

BACKGROUND: The incidence, prevalence, and molecular epidemiology of urea cycle disorders (UCDs) in Argentina remain underexplored. The present study is the first to thoroughly assess the clinical and molecular profiles of UCD patients examined at a single reference center in Argentina. RESULTS: Forty-nine UCD cases were collected. About half (26/49, 53%) manifested neonatally with classical presentation and had a high mortality (25/26, 96%). Ornithine transcarbamylase deficiency (OTCD) was the most common UCD (26 patients). Argininosuccinate synthetase deficiency (ASSD) was detected in 19 cases, while argininosuccinate lyase deficiency (ASLD) was diagnosed in 4 cases. Molecular genetic analysis revealed 8 private OTC mutations and two large deletion/duplication events in the OTC gene. Most mutations in the ASS1 and ASL genes were recurrent missense changes, and four alterations were novel. The clinical outcome of our UCD cohort was poor, with an overall mortality of 57% (28/49 cases), and a 28% (6/21) disability rate among the survivors. CONCLUSIONS: Most patients in our case series showed severe neonatal onset, with high morbidity/mortality. We detected in total 19 mutations, most of them recurrent and of high frequency worldwide. Noteworthy, we highlight the presence of a geographic cluster with high prevalence of a point mutation in the ASS1 gene. This study suggests that these disorders may be more frequent than commonly assumed, and stresses the need for increased awareness amongst health professionals and greater availability of diagnostic tools for accurate identification, early diagnosis, and timely treatment.

Erratum to: The p.T191M mutation of the CBS gene is highly prevalent among homocystinuric patients from Spain, Portugal and South America.

In this article, one of the novel mutations, c.208_209+ 8del10, was incorrectly given as c.69_70+8del10. It corresponds to patient 64 in Table 4.

Delineation of the motor disorder of Lesch-Nyhan disease.

Lesch-Nyhan disease (LND) is caused by deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). Affected individuals exhibit over-production of uric acid, along with a characteristic neurobehavioural syndrome that includes mental retardation, recurrent self-injurious behaviour and motor disability. Prior studies involving relatively small numbers of patients have provided different conclusions on the nature of the motor disorder. The current study includes the results of a multi-centre international prospective study of the motor disorder in the largest cohort of patients studied to date. A total of 44 patients ranging from 2 to 38 years presented a characteristic motor syndrome that involved severe action dystonia superimposed on baseline hypotonia. Although some patients also displayed other extrapyramidal or pyramidal signs, these were always less prominent than dystonia. These results are compared with a comprehensive review of 122 prior reports that included a total of 254 patients. Explanations for the differing observations available in the literature are provided, along with a summary of how the motor disorder of LND relates to current understanding of its pathophysiology involving the basal ganglia.

[Juvenile form of Sandhoff disease: first case reported in Argentina]. / Variante juvenil de la enfermedad de Sandhoff: presentación del primer caso descrito en Argentina.

Sandhoff disease is a neurodegenerative, lysosomal and autosomal recessive disease caused by mutations in the HEXB gene. Three forms are recognized: infantile, juvenile and adult. Previously, an endogamous population in Córdoba, Argentina, was identified with a high incidence of Sandhoff disease, all reported cases were of the infantile type. In this work, we describe a child with the juvenile form of Sandhoff disease, the first case reported in Argentina. The patient is a 7-year-old boy presenting with ataxia, speech disturbances and global developmental delay, symptoms starting at the age of 2 years. Diagnosis was based on the hexosaminidase deficiency. Sequencing of genomic DNA revealed compound heterozygosity for two HEXB gene mutations: c.796T>G (p.Y266D) and c.1615C>T (p.R539C), both already reported.

La enfermedad de Sandhoff es una patología neurodegenerativa, de almacenamiento lisosomal, causada por mutaciones en el gen HEXB. Existen tres formas clínicas: infantil, juvenil y adulta. Previamente, fue identificada una población endogámica en la provincia de Córdoba, Argentina, que presentaba una alta incidencia de la enfermedad; todos los casos correspondieron a la forma infantil. En este trabajo, se presenta por primera vez el caso de un paciente argentino con la variante juvenil de la enfermedad de Sandhoff. El paciente es un niño de 7 años que, a partir de los 2, presentó ataxia, trastorno del habla y retraso global en el desarrollo. El diagnóstico se confirmó con la detección de valores residuales de enzima hexosaminidasa y con la identificación de dos mutaciones ya descritas en estado de heterocigosis: c.796T>G (p.Y266D) y c.1615C>T (p.R539C).

Creatine metabolism: detection of creatine and guanidinoacetate in saliva of healthy subjects.

Creatine (Cr) plays an important role in storage and transmission of phosphate-bound energy. Cerebral creatine deficiency syndromes comprise three inherited defects in Cr biosynthesis and transport. The aim of this study was to investigate whether Cr and Guanidinoacetate (GAA) can be detected in saliva of healthy subjects and to establish the relationship between salivary and plasma levels of these molecules. An adapted gas chromatography (GC) method is described for the quantification of Cr and GAA biomarkers in saliva. Reference values were established for GAA and Cr in saliva. These values were age dependent (p= 0.001). No difference between genders was observed. We detected a difference between GAA and Cr concentrations in saliva and in plasma. The GC method for simultaneous determination of GAA and Cr in human saliva is fast, reliable, sensitive, non-invasive and precise to use as a biochemical approach in early detection of cerebral creatine deficiency syndromes.

La creatina (Cr) juega un importante rol en el almacenamiento y el transporte de energía unida al fosfato. Los síndromes de deficiencia de creatina cerebral comprenden tres defectos genéticos en la biosíntesis y transporte de creatina. Es propósito de este estudio investigar si el guanidinoacetato (GAA) y la Cr pueden ser detectados en saliva de sujetos sanos e investigar la relación entre los valores de GAA y Cr en saliva con los niveles en plasma de estas moléculas. Se describe un método modificado de cromatografía gaseosa para la cuantificación de los biomarcadores, Cr y GAA en este biofluído. Se establecieron valores de referencia para GAA y Cr. Estos valores dependen de la edad (P=0.001). No se observaron diferencias entre género. Se detectó una diferencia entre la concentración de GAA y Cr en saliva con respecto al plasma. El método adaptado de cromatografía gaseosa para la determinación simultánea de GAA y Cr en saliva humana es fácil, seguro, sensible, no invasivo y preciso para utilizar como aproximación bioquímica en la detección temprana de los síndromes de deficiencia de creatina cerebral.

Neuronal ceroid lipofuscinosis type CLN2: a new rationale for the construction of phenotypic subgroups based on a survey of 25 cases in South America.

Tripeptidyl-peptidase 1 (TPP1) null or residual activity occurs in neuronal ceroid lipofuscinosis (NCL) with underlying TPP1/CLN2 mutations. A survey of 25 South American CLN2 affected individuals enabled the differentiation of two phenotypes: classical late-infantile and variant juvenile, each in approximately 50% of patients, with residual TPP1 activity occurring in approximately 32%. Each individual was assigned to one of three subgroups: (I) n=11, null TPP1 activity in leukocytes; (II) n=8, residual TPP1 activity of 0.60-15.85 nmol/h/mg (nr 110-476); (III) n=6, activity not measured in leukocytes. Curvilinear bodies (CB) appeared in almost all studied CLN2 subjects; the only exceptions occurred in cases of subgroup II: two individuals had combined CBs/fingerprints (FPs), and one case had pure FPs. There were 15 mutations (4 first published in this paper, 3 previously observed in South America by our group, and 8 previously observed by others). In subgroup I, mutations were either missense or nonsense; in subgroups II and III, mutations prevailed at the non-conserved intronic site, c.887-10A>G (intron 7), and to a lesser extent at c.89+5G>C (intron 2), in heterozygous combinations. Grouping phenotypically and genetically known individuals on the basis of TPP1 activity supported the concept that residual enzyme activity underlies a protracted disease course. The prevalence of intronic mutations at non-conserved sites in subgroup II individuals indicates that some alternative splicing might allow some residual TPP1 activity.

Molecular epidemiology of citrullinemia type I in a risk region of Argentina: a first step to preconception heterozygote detection.

Classical citrullinemia type I (CTLN1) is an autosomal recessive disorder encoded by the ASS1 gene, which codes for argininosuccinate synthetase (ASS), the rate-limiting enzyme in the urea cycle. Previously, we identified the mutation p.G390R in patients with CTLN1 in the San Luis Province of Argentina. Here, we report the results of p.G390R analysis in a larger number of probands, relatives of involved families and additionally, a population study to identify carriers. Altogether, we analyzed 420 alleles, belonging to 12 probands, 26 relatives, and 172 healthy volunteers. All the probands were homozygous for the mutation, and 21 of 26 relatives were carriers. The occurrence of the disease in descendants of couples at risk was 57% showing a preferential transmission of the mutant allele compared to the normal allele. The carrier frequency in the general San Luis Province population was 4.1%, suggesting the incidence of CTLN1 to be 1:2,427, which is approximately 20 times higher than for the general population. This work suggests that there should be an increased awareness of preconceptual screening of CTNL1 among individuals/couples who are at risk in the San Luis Province in order to better inform them of their reproductive options.Cascade/family and population molecular screening for carrier identification were performed in an Argentinean province with high incidence of CTLN1, a first step to preconceptional screening.

Variante juvenil de la enfermedad de Sandhoff: presentación del primer caso descrito en Argentina / Juvenile form of Sandhoff disease: first case reported in Argentina

La enfermedad de Sandhoff es una patología neurodegenerativa, de almacenamiento lisosomal, causada por mutaciones en el gen HEXB. Existen tres formas clínicas: infantil, juvenil y adulta. Previamente, fue identificada una población endogámica en la provincia de Córdoba, Argentina, que presentaba una alta incidencia de la enfermedad; todos los casos correspondieron a la forma infantil. En este trabajo, se presenta por primera vez el caso de un paciente argentino con la variante juvenil de la enfermedad de Sandhoff. El paciente es un niño de 7 años que, a partir de los 2, presentó ataxia, trastorno del habla y retraso global en el desarrollo. El diagnóstico se confirmó con la detección de valores residuales de enzima hexosaminidasa y con la identificación de dos mutaciones ya descritas en estado de heterocigosis: c.796T>G (p.Y266D) y c.1615C>T (p.R539C).

Sandhoff disease is a neurodegenerative, lysosomal and autosomal recessive disease caused by mutations in the HEXB gene. Three forms are recognized: infantile, juvenile and adult. Previously, an endogamous population in Córdoba, Argentina, was identified with a high incidence of Sandhoff disease, all reported cases were of the infantile type. In this work, we describe a child with the juvenile form of Sandhoff disease, the first case reported in Argentina. The patient is a 7-year-old boy presenting with ataxia, speech disturbances and global developmental delay, symptoms starting at the age of 2 years. Diagnosis was based on the hexosaminidase deficiency. Sequencing of genomic DNA revealed compound heterozygosity for two HEXB gene mutations: c.796T>G (p.Y266D) and c.1615C>T (p.R539C), both already reported.