Mis-splicing of the GALNS gene resulting from deep intronic mutations as a cause of Morquio a disease.

BACKGROUND: Mucopolysaccharidosis-IVA (Morquio A disease) is a lysosomal disorder in which the abnormal accumulation of keratan sulfate and chondroitin-6-sulfate is consequent to mutations in the galactosamine-6-sulfatase (GALNS) gene. Since standard DNA sequencing analysis fails to detect about 16% of GALNS mutant alleles, gross DNA rearrangement screening and uniparental disomy evaluation are required to complete the molecular diagnosis. Despite this, the second pathogenic GALNS allele generally remains unidentified in ~ 5% of Morquio-A disease patients. METHODS: In an attempt to bridge the residual gap between clinical and molecular diagnosis, we performed an mRNA-based evaluation of three Morquio-A disease patients in whom the second mutant GALNS allele had not been identified. We also performed sequence analysis of the entire GALNS gene in two patients. RESULTS: Different aberrant GALNS mRNA transcripts were characterized in each patient. Analysis of these transcripts then allowed the identification, in one patient, of a disease-causing deep intronic GALNS mutation. The aberrant mRNA products identified in the other two individuals resulted in partial exon loss. Despite sequencing the entire GALNS gene region in these patients, the identity of a single underlying pathological lesion could not be unequivocally determined. We postulate that a combination of multiple variants, acting in cis, may synergise in terms of their impact on the splicing machinery. CONCLUSIONS: We have identified GALNS variants located within deep intronic regions that have the potential to impact splicing. These findings have prompted us to incorporate mRNA analysis into our diagnostic flow procedure for the molecular analysis of Morquio A disease.

Left ventricular diastolic dysfunction in type I Gaucher disease: an echo Doppler study.

Type I Gaucher disease (GD1) is an autosomal recessive lysosomal storage disease characterized by multiorgan damage. Left ventricular (LV) involvement has been rarely reported. Accordingly, the aim of the study was to evaluate LV geometry and function in a series of patients with GD1. Eighteen patients with GD1, 18 age- and sex-matched normal controls, and 18 age- and sex-matched hypertensive patients (HTN) were compared by standard echo Doppler examination. LV mass index, relative wall thickness and ejection fraction, transmitral E/A ratio, E velocity deceleration time (DT), atrial filling fraction (AFF = time-velocity integral of A velocity/time-velocity integral of total diastole × 100), E/e' ratio, and left atrial volume index were determined. Nine GD1 patients also exhibited arterial hypertension. The intergroup difference of LV mass index and relative wall thickness was not significant. Transmitral E/A ratio was lower in HTN than in normal controls and GD1 (P < 0.05). GD1 exhibited longer DT than NC and HTN (P = 0.009). AFF was higher in GD1 and HTN compared to NC (P = 0.034). After adjustment for heart rate, GD1 was associated with longer DT (P < 0.001) and greater AFF (P = 0.036), while HTN was associated only with AFF (P = 0.013). No interaction was found between GD1 and HTN. In conclusion, GD1 is associated with subclinical LV diastolic dysfunction, which is independent of the coexistence of arterial hypertension. Subclinical LV impaired relaxation in the context of myocardial infiltrative damage could be the mechanism underlying these alterations.

Pediatric poisoning management: How clinical practice can benefit from forensic approach.

Pediatric population represents the most vulnerable and at risk for unintentional poisoning, with children younger than 6 years old accounting for nearly half of poison exposures. Poisoning is a time-dependent emergency. The need to reach a scientific agreement on diagnostic protocol and treatment seems to be crucial to reduce morbidity and mortality. Starting from a buprenorphine pediatric intoxication case, this article highlights the limits and pitfalls of the traditional diagnostic approach. Diagnosis of drug intoxication was achieved after several days when an in-depth diagnostic investigation became necessary and complete forensic toxicological analyses were performed. Results evidenced an alarming lack of an unequivocal diagnostic protocol in case of suspect intoxication in structures not provided with a forensic toxicological service/unit. Collection of biological specimens according to forensic protocols at hospitalization plays a paramount role in the definitive diagnosis of intoxication. A diagnostic algorithm that focuses on medical history and biological specimen collection timing is herein proposed, in order to unify emergency approaches to the suspected poisoned child.

Imiglucerase, cholecalciferol, and bone-diet in skeletal health management of type I Gaucher disease patients: a pilot study and systematic review.

Skeletal anomalies represent a characteristic feature of type 1 Gaucher disease (GD1). Here we evaluated the impact of an integrated therapy comprising enzyme-replacement therapy (ERT), cholecalciferol, and a normocalcemic-normocaloric-hyposodic diet (bone diet) on bone health in GD1 patients. We also performed a systematic review to compare our results with available data. From January 1, 2015 to February 28, 2019, all GD1 patients referred to Federico II University were enrolled and treated with the integrated therapy. Bone turnover markers and bone mineral density (BMD) were evaluated at baseline (T0) and after 24 months (T24). We enrolled 25 GD1 patients, all showing 25-hydroxy vitamin D (25OHD) levels < 50 nmol/l (hypovitaminosis D) at T0. Response to cholecalciferol treatment was effective, showing a direct relationship between 25OHD levels before and after treatment. At T0, 2 GD1 patients showed fragility fractures, 5 the Erlenmeyer flask deformity, 3 osteonecrosis, and 7 a BMD Z-score ≤ -2. Overall, GD1 patients with bone anomalies showed higher C-terminal telopeptide levels compared with those without bone anomalies. No new bone anomalies occurred during 2 years of follow-up. At T24, BMD remained stable across the entire study cohort, including in patients with bone anomalies. The systematic review showed that our study is the first that evaluated all bone health parameters. Hypovitaminosis D is prevalent in GD1 patients. The response to cholecalciferol treatment was effective but different to healthy subjects and in patients with metabolic bone disorders. Integrated therapy including ERT, cholecalciferol, and bone diet guarantees bone health.

Minimal disease activity in Gaucher disease: criteria for definition.

Gaucher disease type I is a metabolic disorder caused by a genetic deficiency of lysosomal ß-glucocerebrosidase that leads to accumulation of glucocerebroside in macrophages, thus causing damage in different organ systems. Enzyme replacement therapy with imiglucerase improves organ impairment and clinical manifestations, but patients differ in response to treatment. While clinical remission is the most desirable therapeutic outcome, a more realistic goal in patients with high disease burden is reasonably good clinical status despite persistence of residual biochemical or imaging abnormalities. Therefore, the concept of minimal disease activity--used in certain haematological or rheumatologic conditions--needs to be introduced in Gaucher disease, with a level of disease activity that patients and physicians consider a useful treatment target. In this paper, we propose specific parameters and criteria for defining minimal disease activity in Gaucher disease and its stability over time, based on three major systemic domains typically involved: haematological, visceral, and skeletal. Biomarker parameters were not included as criteria, because currently they do not adequately reflect disease evolution in individual patients. Neurological and respiratory domains were also excluded, as their involvement per se indicates severe disease unlikely to respond to enzyme replacement therapy and achieve minimal disease status. Our goal in defining minimal disease activity and stability is to identify a tool to facilitate treatment decisions in clinical practice.

Severe Lymphatic Disorder and Multifocal Atrial Tachycardia Treated with Trametinib in a Patient with Noonan Syndrome and SOS1 Mutation.

Noonan syndrome (NS) is a multisystemic disorder caused by germline mutations in the Ras/MAPK cascade, causing a broad spectrum of phenotypical abnormalities, including abnormal facies, developmental delay, bleeding diathesis, congenital heart disease (mainly pulmonary stenosis and hypertrophic cardiomyopathy), lymphatic disorders, and uro-genital abnormalities. Multifocal atrial tachycardia has been associated with NS, where it may occur independently of hypertrophic cardiomyopathy. Trametinib, a highly selective MEK1/2 inhibitor currently approved for the treatment of cancer, has been shown to reverse left ventricular hypertrophy in two RIT1-mutated newborns with NS and severe hypertrophic cardiomyopathy. Severe lymphatic abnormalities may contribute to decreased pulmonary compliance in NS, and pulmonary lymphangiectasias should be included in the differential diagnosis of a newborn requiring prolonged oxygen administration. Herein we report the case of a pre-term newborn who was admitted to our unit for the occurrence of severe respiratory distress and subentrant MAT treated with trametinib.

Identification of novel L2HGDH gene mutations and update of the pathological spectrum.

L-2-hydroxyglutaric aciduria (L-2-HGA, MIM 236792) is a neurometabolic disorder caused by the toxic accumulation of high concentration of L-2-hydroxyglutaric acid in plasma and cerebrospinal fluid. Distinct mutations on the L2HGDH gene have been associated with the clinical and biochemical phenotype. Here we present three novel mutations (Gln197X, Gly211Val and c.540+1 G>A), which increase the present deleterious collection of L2HGDH gene up to 35 mutations that we have compiled in this study. In addition, we used the haplotypic information based on polymorphic markers to demonstrate the common origin of Gly57Arg harboring chromosomes.

Molecular markers for the follow-up of enzyme-replacement therapy in mucopolysaccharidosis type VI disease.

MPS VI (mucopolysaccharidosis type VI) is a lysosomal storage disease in which deficient activity of the enzyme N-acetylgalactosamine 4-sulfatase [ASB (arylsulfatase B)] impairs the stepwise degradation of the GAG (glycosaminoglycan) dermatan sulfate. Clinical studies of ERT (enzyme replacement therapy) by using rhASB (recombinant human ASB) have been reported with promising results. The release of GAG into the urine is currently used as a biomarker of disease, reflecting in some cases disease severity and in all cases therapeutic responsiveness. Using RNA studies in four Italian patients undergoing ERT, we observed that TNFalpha (tumour necrosis factor alpha) might be a biomarker for MPS VI responsive to therapy. In addition to its role as a potential biomarker, TNFalpha expression could provide insights into the possible pathophysiological mechanisms underlying the mucopolysaccharidoses.

Impaired gait kinematics in type 1 Gaucher's Disease.

Type 1 Gaucher's disease (GD1) is traditionally regarded as "non-neurological". Spatiotemporal and kinematic analysis of gait was carried on thirteen GD1 patients and thirteen healthy controls. We identified a previously unknown subclinical reduction of amplitude of movements in GD1. Articular excursion of ankle, knee and hip was reduced during the swing phase of gait (p <  0.0001). Furthermore, the excursion of the knee appeared also significantly more asymmetric in GD1 patients (p = 0.02). Correction for age, BMI and basal walking speed did not modify the significance. Accordingly to the recent observations that GD1 predisposes to Parkinson's disease, the impaired and asymmetric gait kinematics that we observed might be interpreted as a form of extrapyramidal involvement.

Hypoglycemia, polycythemia and hyponatremia in a newborn exposed to nebivolol during pregnancy.

Nebivolol is a third-generation beta blocker that exerts selective antagonistic activity on ß1 receptors. It has vasodilating properties that result from direct stimulation of endothelial nitric oxide synthase. Nebivolol is indicated for the treatment of hypertension and heart failure, and is generally well tolerated. In this article, we report a case of an infant who was admitted to the Pediatrics and Neonatology Unit of the Moscati Hospital (Aversa, Italy) about 24 hours after birth. The reason for hospitalization was persistent severe hypoglycemia (blood glucose = 30 mg/dL) and jaundice (total bilirubin = 12.5 mg/dL, indirect bilirubin 11.75 mg/dL). He was born by spontaneous delivery after a normal term pregnancy. Birth weight was 3040 g and the Apgar score was 6-9. The mother reported taking nebivolol 5 mg/day for unspecified tachycardia in the last 4 months of pregnancy. Clinical and instrumental investigations carried out during hospitalization did not reveal any congenital or perinatal abnormalities. After treatment for metabolic and electrolyte imbalance, he was discharged on the 10(th) day of hospitalization, in good clinical condition and with normalization of clinical and laboratory parameters. Currently, there are no specific studies on nebivolol tolerability during pregnancy. Our data suggest that the risk profile of nebivolol during pregnancy is the same as that of other ß-blockers. Therefore, further studies are required to determine the safety of ß-blockers during pregnancy and the risks to the unborn child.

Large deletion involving exon 5 of the arylsulfatase B gene caused apparent homozygosity in a mucopolysaccharidosis type VI patient.

Apparent homozygosity for the mutation p.R315X present on exon 5 of the arylsulfatase B (ARSB) gene in a mucopolysaccharidosis type VI patient was solved in this study by further testing for a second mutation. Patient cDNA analysis revealed that the entire exon 5 of the ARSB gene was lacking; this new mutation was identified as c.899-1142del. As the genomic DNA sequencing excluded the presence of splicing mutations, polymerase chain reaction analysis was performed for polymorphisms listed in the NCBI SNP database for the ARSB gene. This allowed the mutation at the genomic DNA level to be identified as g.99367-102002del; this gross deletion, involving the entire exon 5 of the gene and parts of introns 4 and 5 led to a frameshift starting at amino acid 300 and resulting in a protein with 39% amino acids different from the normal enzyme. We stress that extensive DNA analysis needs to be performed in case of apparent homozygosity to avoid potential errors in genetic counseling.

Functional and structural characterization of novel mutations and genotype-phenotype correlation in 51 phenylalanine hydroxylase deficient families from Southern Italy.

Hyperphenylalaninemia (Online Mendelian Inheritance in Man database: 261600) is an autosomal recessive disorder mainly due to mutations in the gene for phenylalanine hydroxylase; the most severe form of hyperphenylalaninemia is classic phenylketonuria. We sequenced the entire gene for phenylalanine hydroxylase in 51 unrelated hyperphenylalaninemia patients from Southern Italy. The entire locus was genotyped in 46 out of 51 hyperphenylalaninemia patients, and 32 different disease-causing mutations were identified. The pathologic nature of two novel gene variants, namely, c.707-2delA and p.Q301P, was demonstrated by in vitro studies. c.707-2delA is a splicing mutation that involves the accepting site of exon 7; it causes the complete skipping of exon 7 and results in the truncated p.T236MfsX60 protein. The second gene variant, p.Q301P, has very low residual enzymatic activity (approximately 4.4%), which may be ascribed, in part, to a low expression level (8-10%). Both the decreased enzyme activity and the low expression level are supported by analysis of the 3D structure of the molecule. The putative structural alterations induced by p.Q301P are compatible with protein instability and perturbance of monomer interactions within dimers and tetramers, although they do not affect the catalytic site. In vivo studies showed tetrahydrobiopterin responsiveness in the p.Q301P carrier but not in the c.707-2delA carrier. We next investigated genotype-phenotype correlations and found that genotype was a good predictor of phenotype in 76% of patients. However, genotype-phenotype discordance occurred in approximately 25% of our patients, mainly those bearing mutations p.L48S, p.R158Q, p.R261Q and p.P281L.

Brain damage in glycogen storage disease type I.

OBJECTIVE: To investigate brain morphology and function in patients with glycogen storage disease type I (GSDI). STUDY DESIGN: Nineteen patients (13 females and 6 males, aged 0.9-22.6 years) and 38 sex- and age-matched controls entered the study. Neurological examinations, psychometric tests (IQ, tests of performance and verbal abilities), standard electroencephalogram (EEG), somatosensory (SEPs), visual (VEPs), and brain-stem auditory evoked potentials (BAEPs), and brain magnetic resonance imaging (MRI) were performed. RESULTS: The results of tests of performance ability were lower in patients than in controls (P <.05). The prevalence of abnormal EEG findings (26.3% versus 2.6%), VEPs (38.4% versus 7.7%), SEPs (23.0% versus 0%), and BAEPs abnormalities (15.7% versus 0%) was higher in patients than in controls (P <.05). MRI pattern was altered in 57.1% of patients and was normal in all controls (P <.05). Both results of tests of performance ability and BAEPs abnormalities significantly correlated with the frequency of admissions for hypoglycemia, whereas EEG abnormalities correlated with dietary compliance (P <.05). CONCLUSIONS: Brain damage, probably caused by recurrent severe hypoglycemia, may be present in patients with GSDI.