Annalisa Sechi

Low molecular weight proteins in urines from healthy subjects as well as diabetic, nephropathic and diabetic-nephropathic patients : a MALDI study

Urine samples from healthy subjects as well as diabetic, nephropathic and diabetic-nephropathic patients were analyzed by matrix assisted laser desorption/ionization (MALDI) mass spectrometry in order to establish evidence of some possible differences in the peptide profile related to the pathological states. Multivariate analysis suggested the possibility of a distinction among the considered groups of patients. Some differences have been found, in particular, in the relative abundances of three ions at m/z 1912, 1219 and 2049. For these reasons, further investigation was carried out by MALDI/TOF/TOF to determine the sequence of these peptides and, consequently, to individuate their possible origin. By this approach, the peptide at m/z 1912 was found to originate from uromodulin, and its lower expression in the case of nephropathy can be well related to the pathological condition. Ions at m/z 2049 and 1219 originate from the collagen alpha-1(I) chain precursor and from the collagen alpha-5 (IV) chain precursor, respectively, and, also in this case, their different expressions can be related to the pathologies under investigation. The obtained data seem to indicate that urine is an interesting biological fluid to investigate on the peptide profile and to obtain, consequently, information on the dismetabolism activated by specific pathologies.

A further investigation on a MALDI-based method for evaluation of markers of renal damage.

The validity of the urinary protein profile to characterize the pathological states of diabetic, nephropathic and diabetic-nephropathic patients was considered on the basis of previously obtained results by MALDI/MS, showing a different abundance ratio of the collagen alpha1 and alpha5 chain precursor fragments at m/z 1219 and 2049 and of the uromodulin precursor fragment at m/z 1912 observed in healthy subjects and patients; a larger number of subjects was examined and the obtained results were statistically evaluated. The p values related to the observed differences indicate that they are statistically significant when comparing all patients versus healthy controls, diabetic with normo or microalbuminuria versus nephropathic with advanced renal disease patients and diabetic with normo or microalbuminuria versus diabetic with advanced nephropathy patients. The scatter plot matrix gives evidence of the strict inverse relationship between the abundances of ions at m/z 1912 and 1219, the correlation coefficient being particularly high (r = 0.921, p < 0.001). The relationship between the true positive rate (sensitivity) and false positive rate (1-specificity) for every possible cutoff value in abundance of the considered ionic species was investigated through the receiver-operating characteristic (ROC) curve. The obtained data indicate that a good differentiation of nephropathic patients with advanced renal disease and diabetic patients with advanced nephropathy versus healthy subjects can be easily obtained by this approach.

Did the Temporary Shortage in Supply of Imiglucerase Have Clinical Consequences? Retrospective Observational Study on 34 Italian Gaucher Type I Patients

Background. Enzyme Replacement Therapy (ERT) is the standard of care in Gaucher disease. The effects of withdrawal or reduced doses are debated, thus a retrospective cohort study was conducted to investigate clinical and laboratory differences in 34 Gaucher type 1 patients experiencing an ERT dosage reduction after the forced temporary imiglucerase shortage in 2009. Methods. Haemoglobin concentration, leukocytes and platelets counts, and chitotriosidase activity were assessed at baseline and after 6 and 12 months (t0, t6, t12), while bone pain, energy, work or school performance, concentration, memory and social life every 3 months. Results. The cohort was made up of 18 males and 16 females (medians: age 41.8 years, therapy duration 14.1 years, dosage reduction 35.5%). Haemoglobin, leukocytes and platelets remained substantially stable, while chitotriosidase activity showed an increase, especially after t6. Age, splenectomy or genotype were not associated with laboratory parameters changes, except for a significant median increase of chitotriosidase activity in non-splenectomised patients after 12 months (p = 0.01). At 3, 6, 9 and 12 months, more than 50% patients reported at least one problem in subjective well-being (56%, 65%, 70%, 58%, respectively), while bone pain occurred or worsened in 13/33, 13/32, 7/28 and 5/26 patients, respectively. No bone crises were reported. Conclusions. Drug reduction did not induce substantial modification in the laboratory values but seems to have influenced the well-being perception of some Gaucher patients. Thus, bone pain, general health and quality of life should be carefully monitored during ERT reductions.

Long term effects of enzyme replacement therapy in an Italian cohort of type 3 Gaucher patients.

BACKGROUND The chronic neuropathic form of Gaucher disease (GD3) is characterised by hepatosplenomegaly, anaemia, thrombocytopenia, bone alterations and central neurological involvement. Enzyme replacement therapy (ERT) has been demonstrated to be effective in non neuropathic Gaucher disease, but long term results in patients with GD3 are still limited and contrasting. A possible role of genotype in determining the response to ERT has been hypothesised. PATIENTS AND METHODS All patients affected by GD3, treated with ERT, and followed-up in 4 different Italian centres (Udine, Catanzaro, Sassari and Florence) were included. Data on clinical conditions, laboratory values, neurological and neuropsychological examinations, radiological and electrophysiological features were collected retrospectively from clinical records. RESULTS Ten patients (6 females, 4 males) with four different genotypes (L444P/L444P, L444P/F231I, P159T/unknown, C.115+1G>A/N188S) were identified. They received ERT infusions from 3 to 21years. Haematological parameters and organomegaly improved/normalised in all patients. Three patients showed severe progressive skeletal deformities. 6/10 patients were neurologically asymptomatic when they started ERT for systemic symptoms. During the follow-up, 2/6 developed an important central nervous system disease; 2/6 developed mild central symptoms; and 2/6 did not show any neurological symptom after 5, and 20years of treatment respectively, despite the presence of epileptiform abnormalities at the electroencephalogram. Overall, neurological involvement worsened over time in 6/10 patients, 3 of whom developed progressive myoclonic encephalopathy and died. CONCLUSIONS ERT improved the systemic manifestations in patients with GD3, but was not able to counteract the progression of neurological symptoms in the long term.

Urinary peptides as a diagnostic tool for renal failure detected by matrix-assisted laser desorption/ionisation mass spectrometry: an evaluation of their clinical significance

The development of new analytical methodologies related to the proteome for the evaluation of renal physiology and pathology is surely of wide interest for physicians, giving them new tools for monitoring complications associated with diabetes, such as end-stage renal disease. In the present study, the clinical significance of the urinary abundance of two peptides, SGSVIDQSRVLNLGPITR (the uromodulin precursor, m/z 1912) and IGPHypGPHypGLMGPP [present in the collagen-α-5(IV) chain precursor, m/z 1219], detected by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI/MS) in microalbuminuric or nephropathic diabetic patients and in non-diabetic nephropathic patients was evaluated. A progressive increase in the abundance of the ion at m/z 1219 and a decrease in the abundance of the ion at m/z 1912 have been found in diabetic microalbuminuric, diabetic–nephropathic and nephropathic patients. Linear correlations are present between serum creatinine values and the abundances of the ions at m/z 1219 (positive correlation, r = 0.3645, P < 0.0001) and at m/z 1912 (negative correlation, r = −0.3053, P < 0.0005). Correlations between the MALDI data and the estimated glomerular filtration rate were also found, while relationships with urinary albumin excretion were found only in sub-sets of patients. Analysis of receiver operating characteristic curves showed a sensitivity up to 96% and a specificity of up to 84% for the two ionic species, or their ratio, for distinguishing diabetic patients with different degrees of nephropathy from healthy subjects, proving that the urinary abundance of the two peptides at m/z 1219 and m/z 1912, determined with MALDI/MS, may be considered as a possible diagnostic tool for the determination of progression toward renal failure, also with the aim of monitoring kidney function, in diabetic patients.

Profile of eliglustat tartrate in the management of Gaucher disease.

Gaucher disease (GD) is a lysosomal storage disorder caused by the deficient activity of acid beta glucosidase, with consequent accumulation of glucosylceramide in the spleen, liver, bone marrow, and various organs and tissues. Currently, the gold standard for GD treatment is enzyme replacement therapy (ERT). The efficacy of ERT in improving or stabilizing the visceral and hematological symptoms of GD is well-proven. However, since ERT has to be administered by frequent intravenous infusions, this therapeutic approach has an important impact on the patient’s quality of life. Eliglustat tartrate is a new substrate reduction therapy for GD, which acts as a specific and potent inhibitor of glucosylceramide synthase and can be administered orally. This review summarizes the results of the preclinical and clinical trials, which experimented with eliglustat, and discusses its possible role in the management of GD, when compared to the currently available treatments and the new experimental approaches.

Effects of miglustat treatment in a patient affected by an atypical form of Tangier disease.

BackgroundTangier disease (TD) is a rare autosomal recessive disorder, resulting from mutations in the ATP binding cassette transporter (ABCA1) gene. The deficiency of ABCA1 protein impairs high density lipoprotein (HDL) synthesis and cholesterol esters trafficking.Case ReportA 58 year-old female, presenting with complex clinical signs (splenomegaly, dysarthria, dysphagia, ataxia, tongue enlargement, prurigo nodularis, legs lymphedema, pancytopenia and bone marrow foam cells), was misdiagnosed as Niemann-Pick C (NPC) and treated with miglustat (300 mg/day), normalizing neurological symptoms and improving skin lesions and legs lymphedema. Subsequently filipin-staining and molecular analysis for NPC genes were negative. Lipid profiling showed severe deficiency of HDL, 2 mg/dl (n.v. 45-65) and apoAI, 5.19 mg/dl (n.v. 110-170), suggesting TD as a probable diagnosis. Molecular analysis of ABCA1 gene showed the presence of a novel homozygous deletion (c.4464-486_4698 + 382 Del). Miglustat treatment was then interrupted with worsening of some neurological signs (memory defects, slowing of thought processes) and skin lesions. Treatment was restarted after 7 months with neurological normalization and improvement of skin involvement.ConclusionsThese results suggest miglustat as a possible therapeutic approach in this untreatable disease. The mechanisms by which miglustat ameliorates at least some clinical manifestations of TD needs to be further investigated.

Quality of life in adult patients with glycogen storage disease type I: results of a multicenter italian study.

BACKGROUND Glycogen storage disease type I (GSD I) is a chronic metabolic disease that requires a lifelong strict dietetic treatment to avoid hypoglycemia and can lead to severe complications during adult age. Impaired quality of life (QoL) has been reported in affected children, but this aspect has not been previously investigated in adults. OBJECTIVE To assess QoL in adult patients with GSD I. PATIENTS AND METHODS Italian patients with GSD type Ia and Ib, who were 16 years or older, were asked to complete the SF-36 questionnaire, assessing their QoL. Data on demographic characteristics and clinical history were collected from clinical records and interviews. RESULTS Thirty-eight patients (22 females, 16 males; 27 with GSD Ia, 11 with GSD Ib, median age 26.5 years) completed the SF-36 questionnaire. Overall, when compared to normal values, patients with GSD I had lower median scores in general health perception and social functioning, but better median scores for bodily pain and mental health. Patients with GSD Ib had a lower Z-score than GSD Ia patients for emotional health problems. Male patients showed better Z-scores in physical functioning, general health perception, and social functioning when compared to females. Emotional health problems Z-score was lower in nephropathic patients. CONCLUSION QoL can be impaired in adult patients with GSD I. The results of this study show that patients with GSD type Ib, women, and those with renal complications are more likely to experience a poorer QoL.

Myocardial fibrosis as the first sign of cardiac involvement in a male patient with Fabry disease: report of a clinical case and discussion on the utility of the magnetic resonance in Fabry pathology.

BackgroundCardiovascular magnetic resonance (CMR) with late gadolinium enhancement (LGE) imaging is increasingly used to assess myocardial involvement in patients with Fabry disease, an X linked lipid storage disorder. However, it is often proposed as an optional tool. A different cardiomyopathic disease progression between male and female patients was hypothesised in previous studies, as in female myocardial fibrosis was found without left ventricular (LV) hypertrophy, while myocardial fibrosis was always detected in association to LV hypertrophy in men.Case presentationA male Caucasian patient, 19 years old, diagnosed through a family-based molecular screening, presented with LGE of the LV inferolateral wall evidenced at the CMR, without LV hypertrophy, or other clinical signs of the disease.ConclusionThis is the first report of cardiac fibrosis as the first sign of organ involvement in a male patient with Fabry disease. This finding stresses the importance of performing CMR with LGE imaging for the initial staging and monitoring of Fabry patients of both genders.

Role of LIMP-2 in the intracellular trafficking of β-Glucosidase in different human cellular models

Acid β‐glucosidase (GCase), the enzyme deficient in Gaucher disease (GD), is transported to lysosomes by the lysosomal integral membrane protein (LIMP)‐2. In humans, LIMP‐2 deficiency leads to action myoclonus‐renal failure (AMRF) syndrome. GD and AMRF syndrome share some clinical features. However, (hey are different from clinical and biochemical points of view, suggesting that the role of LLMP‐2 in the targeting of GCase would be different in different tissues. Besides, the role of LLMP‐2 in the uptake and trafficking of the human recombinant (hr) GCase used in the treatment of GD is unknown. Thus, we compared GCase activity and intracellular localization in immortalized lymphocytes, fibroblasts, and a neuronal model derived from multi‐potent adult stem cells, from a patient with AMRF syndrome, patients with GD, and control subjects. In fibroblasts and neuronlike cells, GCase targeting to the lysosomes is completely dependent on LLMP‐2, whereas in blood cells, GCase is partially targeted to lysosomes by a LLMP‐2‐independent mechanism. Although hrGCase cellular uptake is independent of LLMP‐2, its trafficking to the lysosomes is mediated by (his receptor. These data provide new insights into the mechanisms involved in the intracellular trafficking of GCase and in the pathogeneses of GD and AMRF syndrome.—Malini, E., Zampieri, S., Deganuto, M., Romanello, M., Sechi, A., Bembi, B., Dardis, A Role of LIMP‐2 in the intracellular trafficking of β‐glucosidase in different human cellular models. FASEB J. 29, 3839‐3852 (2015). www.fasebj.org