Diagnostic issues faced by a rare disease healthcare network during Covid-19 outbreak: data from the Campania Rare Disease Registry.

BACKGROUND: The aims of this study were: to investigate the capacity of the rare disease healthcare network in Campania to diagnose patients with rare diseases during the outbreak of Covid-19; and to shed light on problematic diagnoses during this period. METHODS: To describe the impact of the Covid-19 pandemic on the diagnosis of patients with rare diseases, a retrospective analysis of the Campania Region Rare Disease Registry was performed. A tailored questionnaire was sent to rare disease experts to investigate major issues during the emergency period. RESULTS: Prevalence of new diagnoses of rare disease in March and April 2020 was significantly lower than in 2019 (117 versus 317, P < 0.001 and 37 versus 349, P < 0.001, respectively) and 2018 (117 versus 389, P < 0.001 and 37 versus 282, P < 0.001, respectively). Eighty-two among 98 rare disease experts completed the questionnaire. Diagnostic success (95%), access to diagnosis (80%) and follow-up (72%), lack of Personal Protective Equipment (60%), lack of Covid-19 guidelines (50%) and the need for home therapy (78%) were the most important issues raised during Covid-19 outbreak. CONCLUSIONS: This study describes the effects of the Covid-19 outbreak on the diagnosis of rare disease in a single Italian region and investigates potential issues of diagnosis and management during this period.

Cutting Edge: Increased Autoimmunity Risk in Glycogen Storage Disease Type 1b Is Associated with a Reduced Engagement of Glycolysis in T Cells and an Impaired Regulatory T Cell Function.

Glycogen storage disease type 1b (GSD-1b) is an autosomal-recessive disease caused by mutation of glucose-6-phosphate transporter and characterized by altered glycogen/glucose homeostasis. A higher frequency of autoimmune diseases has been observed in GSD-1b patients, but the molecular determinants leading to this phenomenon remain unknown. To address this question, we investigated the effect of glucose-6-phosphate transporter mutation on immune cell homeostasis and CD4+ T cell functions. In GSD-1b subjects, we found lymphopenia and a reduced capacity of T cells to engage glycolysis upon TCR stimulation. These phenomena associated with reduced expression of the FOXP3 transcription factor, lower suppressive function in peripheral CD4+CD25+FOXP3+ regulatory T cells, and an impaired capacity of CD4+CD25- conventional T cells to induce expression of FOXP3 after suboptimal TCR stimulation. These data unveil the metabolic determinant leading to an increased autoimmunity risk in GSD-1b patients.

Lysosomal storage diseases: from pathophysiology to therapy.

Lysosomal storage diseases are a group of rare, inborn, metabolic errors characterized by deficiencies in normal lysosomal function and by intralysosomal accumulation of undegraded substrates. The past 25 years have been characterized by remarkable progress in the treatment of these diseases and by the development of multiple therapeutic approaches. These approaches include strategies aimed at increasing the residual activity of a missing enzyme (enzyme replacement therapy, hematopoietic stem cell transplantation, pharmacological chaperone therapy and gene therapy) and approaches based on reducing the flux of substrates to lysosomes. As knowledge has improved about the pathophysiology of lysosomal storage diseases, novel targets for therapy have been identified, and innovative treatment approaches are being developed.

Guidelines for the diagnosis and management of cystathionine beta-synthase deficiency.

Cystathionine beta-synthase (CBS) deficiency is a rare inherited disorder in the methionine catabolic pathway, in which the impaired synthesis of cystathionine leads to accumulation of homocysteine. Patients can present to many different specialists and diagnosis is often delayed. Severely affected patients usually present in childhood with ectopia lentis, learning difficulties and skeletal abnormalities. These patients generally require treatment with a low-methionine diet and/or betaine. In contrast, mildly affected patients are likely to present as adults with thromboembolism and to respond to treatment with pyridoxine. In this article, we present recommendations for the diagnosis and management of CBS deficiency, based on a systematic review of the literature. Unfortunately, the quality of the evidence is poor, as it often is for rare diseases. We strongly recommend measuring the plasma total homocysteine concentrations in any patient whose clinical features suggest the diagnosis. Our recommendations may help to standardise testing for pyridoxine responsiveness. Current evidence suggests that patients are unlikely to develop complications if the plasma total homocysteine concentration is maintained below 120 µmol/L. Nevertheless, we recommend keeping the concentration below 100 µmol/L because levels fluctuate and the complications associated with high levels are so serious.

The contribution of the citrate pathway to oxidative stress in Down syndrome.

Inflammatory conditions and oxidative stress have a crucial role in Down syndrome (DS). Emerging studies have also reported an altered lipid profile in the early stages of DS. Our previous works demonstrate that citrate pathway activation is required for oxygen radical production during inflammation. Here, we find up-regulation of the citrate pathway and down-regulation of carnitine/acylcarnitine carrier and carnitine palmitoyl-transferase 1 genes in cells from children with DS. Interestingly, when the citrate pathway is inhibited, we observe a reduction in oxygen radicals as well as in lipid peroxidation levels. Our preliminary findings provide evidence for a citrate pathway dysregulation, which could be related to some phenotypic traits of people with DS.

Pharmacological Chaperone Therapy: Preclinical Development, Clinical Translation, and Prospects for the Treatment of Lysosomal Storage Disorders.

Lysosomal storage disorders (LSDs) are a group of inborn metabolic diseases caused by mutations in genes that encode proteins involved in different lysosomal functions, in most instances acidic hydrolases. Different therapeutic approaches have been developed to treat these disorders. Pharmacological chaperone therapy (PCT) is an emerging approach based on small-molecule ligands that selectively bind and stabilize mutant enzymes, increase their cellular levels, and improve lysosomal trafficking and activity. Compared to other approaches, PCT shows advantages, particularly in terms of oral administration, broad biodistribution, and positive impact on patients' quality of life. After preclinical in vitro and in vivo studies, PCT is now being translated in the first clinical trials, either as monotherapy or in combination with enzyme replacement therapy, for some of the most prevalent LSDs. For some LSDs, the results of the first clinical trials are encouraging and warrant further development. Future research in the field of PCT will be directed toward the identification of novel chaperones, including new allosteric drugs, and the exploitation of synergies between chaperone treatment and other therapeutic approaches.

A restricted spectrum of mutations in the SMAD4 tumor-suppressor gene underlies Myhre syndrome.

Myhre syndrome is a developmental disorder characterized by reduced growth, generalized muscular hypertrophy, facial dysmorphism, deafness, cognitive deficits, joint stiffness, and skeletal anomalies. Here, by performing exome sequencing of a single affected individual and coupling the results to a hypothesis-driven filtering strategy, we establish that heterozygous mutations in SMAD4, which encodes for a transducer mediating transforming growth factor ß and bone morphogenetic protein signaling branches, underlie this rare Mendelian trait. Two recurrent de novo SMAD4 mutations were identified in eight unrelated subjects. Both mutations were missense changes altering Ile500 within the evolutionary conserved MAD homology 2 domain, a well known mutational hot spot in malignancies. Structural analyses suggest that the substituted residues are likely to perturb the binding properties of the mutant protein to signaling partners. Although SMAD4 has been established as a tumor suppressor gene somatically mutated in pancreatic, gastrointestinal, and skin cancers, and germline loss-of-function lesions and deletions of this gene have been documented to cause disorders that predispose individuals to gastrointestinal cancer and vascular dysplasias, the present report identifies a previously unrecognized class of mutations in the gene with profound impact on development and growth.

Progression of renal damage in glycogen storage disease type I is associated to hyperlipidemia: a multicenter prospective Italian study.

Angiotensin converting enzyme (ACE)-inhibitors decrease glomerular hyperfiltration but not microalbuminuria and proteinuria in glycogen storage disease type I. In the current study, we demonstrated that severe hyperlipidemia is associated with ACE-inhibitor ineffectiveness. We underline the importance of adequate metabolic control in glycogen storage disease type I. A combination therapy with ACE-inhibitors and lipid lowering drugs might be considered.

A chaperone enhances blood α-glucosidase activity in Pompe disease patients treated with enzyme replacement therapy.

Enzyme replacement therapy is currently the only approved treatment for Pompe disease, due to acid α-glucosidase deficiency. Clinical efficacy of this approach is variable, and more effective therapies are needed. We showed in preclinical studies that chaperones stabilize the recombinant enzyme used for enzyme replacement therapy. Here, we evaluated the effects of a combination of enzyme therapy and a chaperone on α-glucosidase activity in Pompe disease patients. α-Glucosidase activity was analyzed by tandem-mass spectrometry in dried blood spots from patients treated with enzyme replacement therapy, either alone or in combination with the chaperone N-butyldeoxynojirimycin given at the time of the enzyme infusion. Thirteen patients with different presentations (3 infantile-onset, 10 late-onset) were enrolled. In 11 patients, the combination treatment resulted in α-glucosidase activities greater than 1.85-fold the activities with enzyme replacement therapy alone. In the whole patient population, α-glucosidase activity was significantly increased at 12 hours (2.19-fold, P = 0.002), 24 hours (6.07-fold, P = 0.001), and 36 hours (3.95-fold, P = 0.003). The areas under the curve were also significantly increased (6.78-fold, P = 0.002). These results suggest improved stability of recombinant α-glucosidase in blood in the presence of the chaperone.

Radiographic features of the skeleton in disorders of post-squalene cholesterol biosynthesis.

Disorders of post-squalene cholesterol biosynthesis are inborn errors of metabolism characterised by multiple congenital abnormalities, including significant skeletal involvement. The most frequent and best-characterised example is the Smith-Lemli-Opitz syndrome. Nine other disorders are known, namely autosomal-recessive Antley-Bixler syndrome, Greenberg dysplasia, X-linked dominant chondrodysplasia punctata, X-linked recessive male emopamil-binding protein deficiency, CHILD syndrome, CK syndrome, sterol C4 methyloxidase-like deficiency, desmosterolosis and lathosterolosis. This study provides an overview of the radiologic features observed in these diseases. A common pattern of limb abnormalities is recognisable, including polydactyly, which is typically post-axial and rarely interdigital and can involve all four limbs, and syndactyly of the toes. Chondrodysplasia punctata is specifically associated with a subgroup of disorders of cholesterol biosynthesis (Greenberg dysplasia, CHILD syndrome, X-linked dominant chondrodysplasia punctata, male emopamil-binding protein deficiency). The possible occurrence of epiphyseal stippling in the Smith-Lemli-Opitz syndrome, initially reported, does not appear to be confirmed. Stippling is also associated with other congenital disorders such as chromosomal abnormalities, brachytelephalangic chondrodysplasia punctata (X-linked recessive chondrodysplasia punctata, disruptions of vitamin K metabolism, maternal autoimmune diseases), rhizomelic chondrodysplasia punctata (peroxisomal disorders) and lysosomal storage disorders. In the differential diagnosis of epiphyseal stippling, a moth-eaten appearance of bones, asymmetry, or presence of a common pattern of limb abnormalities indicate inborn errors of cholesterol biosynthesis. We highlight the specific differentiating radiologic features of disorders of post-squalene cholesterol biosynthesis.

Recurrent dominant mutations affecting two adjacent residues in the motor domain of the monomeric kinesin KIF22 result in skeletal dysplasia and joint laxity.

Spondyloepimetaphyseal dysplasia with joint laxity, leptodactylic type (lepto-SEMDJL, aka SEMDJL, Hall type), is an autosomal dominant skeletal disorder that, in spite of being relatively common among skeletal dysplasias, has eluded molecular elucidation so far. We used whole-exome sequencing of five unrelated individuals with lepto-SEMDJL to identify mutations in KIF22 as the cause of this skeletal condition. Missense mutations affecting one of two adjacent amino acids in the motor domain of KIF22 were present in 20 familial cases from eight families and in 12 other sporadic cases. The skeletal and connective tissue phenotype produced by these specific mutations point to functions of KIF22 beyond those previously ascribed functions involving chromosome segregation. Although we have found Kif22 to be strongly upregulated at the growth plate, the precise pathogenetic mechanisms remain to be elucidated.

Hyperhomocysteinemia: related genetic diseases and congenital defects, abnormal DNA methylation and newborn screening issues.

Homocysteine, a sulfur-containing amino acid derived from the methionine metabolism, is located at the branch point of two pathways of the methionine cycle, i.e. remethylation and transsulfuration. Gene abnormalities in the enzymes catalyzing reactions in both pathways lead to hyperhomocysteinemia. Hyperhomocysteinemia is associated with increased risk for congenital disorders, including neural tube closure defects, heart defects, cleft lip/palate, Down syndrome, and multi-system abnormalities in adults. Since hyperhomocysteinemia is known to affect the extent of DNA methylation, it is likely that abnormal DNA methylation during embryogenesis, may be a pathogenic factor for these congenital disorders. In this review we highlight the importance of homocysteinemia by describing the genes encoding for enzymes of homocysteine metabolism relevant to the clinical practice, especially cystathionine-ß-synthase and methylenetetrahydrofolate reductase mutations, and the impairment of related metabolites levels. Moreover, a possible correlation between hyperhomocysteine and congenital disorders through the involvement of abnormal DNA methylation during embryogenesis is discussed. Finally, the relevance of present and future diagnostic tools such as tandem mass spectrometry and next generation sequencing in newborn screening is highlighted.

Bronchial isomerism in a Kabuki syndrome patient with a novel mutation in MLL2 gene.

BACKGROUND: Kabuki syndrome (KS) is a rare, multiple congenital anomalies/intellectual disability syndrome caused by mutations of MLL2 gene, which codifies for a histone methyltrasferase that regulates the embryogenesis and the tissue development. Left-bronchial isomerism is a rare congenital abnormality that can be defined as the absence of the normal lateralizing features which distinguish right and left-sides in the lungs. To date, this is the first report of left-bronchial isomerism in association with KS. CASE PRESENTATION: A one-month-old Caucasian male patient underwent our attention for microcephaly, dysmorphic features (long palpebral fissures, eyebrows with sparse lateral third, everted lower eyelids, blue sclerae, large dysplastic ears, lower lip pits), persistent fetal fingertip pads, short stature, heart defects (interventricular defect and aortic coarctation), unilateral cryptorchidism, hypotonia and delay in gross motor skills. These features suggested a diagnosis of KS and a molecular analysis confirmed a novel frame-shift mutation in the exon 11 of MLL2 gene. Subsequently, given recurrent respiratory infections with a normal immunological status, he underwent a chest CT scan that showed a left bronchial isomerism. CONCLUSION: We report a patient affected by KS, with a novel MLL2 mutation and an atypical phenotype characterized by left-side bronchial isomerism. Interestingly, genes involved in the heterotaxia/isomerism such as ROCK2 and SHROOM3 are known to interact with MLL2 gene. In order to achieve a correct diagnosis and an appropriate therapy, the presence of pulmonary anatomical variations should be investigated in KS patients with respiratory signs not associated to immunological deficiency. Finally, our findings support the hypothesis that the mutations leading to a complete loss of function of MLL2 gene is often associated with complex visceral malformations.

Complex chromosomal rearrangements causing Langer-Giedion syndrome atypical phenotype: genotype-phenotype correlation and literature review.

Langer-Giedion syndrome (LGS) is caused by a deletion of chromosome 8q23.3-q24.11. The LGS clinical spectrum includes intellectual disability (ID), short stature, microcephaly, facial dysmorphisms, exostoses. We describe a 4-year-old girl with ID, short stature, microcephaly, distinctive facial phenotype, skeletal signs (exostoses on the left fibula, coccyx agenesis, stubby and dysmorphic sphenoid bone, osteoporosis), central nervous system malformations (hypoplastic and dysmorphic corpus callosum and septum pellucidum), pituitary gland hypoplasia and hyperreninemia. Array-CGH revealed complex chromosomal rearrangements. A diagnosis of LGS was confirmed by the detection of a 8q23.3-q24.1 deletion. Associated chromosomal abnormalities were a 21q22.1 deletion and a balanced reciprocal translocation t(2;11)(p24;p15) de novo, confirmed by FISH analysis. We document the patient's atypical findings, never described in LGS patients, in order to update the genotype-phenotype correlation. We speculate that the disruption of regulatory elements mapping upstream CYP11B2 involved in the deleted region could cause hyperreninemia.

Early diagnosis of Gaucher disease in pediatric patients: proposal for a diagnostic algorithm.

Gaucher disease (GD) is caused by an enzyme deficiency that leads to the accumulation of glycolipids in various organs. Although the signs and symptoms of GD emerge in childhood in the majority of patients, the disease often remains unrecognized for many years with delay of benefits of therapy or development of irreversible complications. Based on published data and data from the International Collaborative Gaucher Group Registry, an algorithm has been drafted for early diagnosis of GD in pediatric patients. It will help hematologists in promoting a timely diagnosis and early access to therapy for pediatric patients with GD.

Mitochondrial DNA methylation as a next-generation biomarker and diagnostic tool.

Recent expansion of our knowledge on epigenetic changes strongly suggests that not only nuclear DNA (nDNA), but also mitochondrial DNA (mtDNA) may be subjected to epigenetic modifications related to disease development, environmental exposure, drug treatment and aging. Thus, mtDNA methylation is attracting increasing attention as a potential biomarker for the detection and diagnosis of diseases and the understanding of cellular behavior in particular conditions. In this paper we review the current advances in mtDNA methylation studies with particular attention to the evidences of mtDNA methylation changes in diseases and physiological conditions so far investigated. Technological advances for the analysis of epigenetic variations are promising tools to provide insights into methylation of mtDNA with similar resolution levels as those reached for nDNA. However, many aspects related to mtDNA methylation are still unclear. More studies are needed to understand whether and how changes in mtDNA methylation patterns, global and gene specific, are associated to diseases or risk factors.

Pharmacological enhancement of α-glucosidase by the allosteric chaperone N-acetylcysteine.

Pompe disease (PD) is a metabolic myopathy due to the deficiency of the lysosomal enzyme α-glucosidase (GAA). The only approved treatment for this disorder, enzyme replacement with recombinant human GAA (rhGAA), has shown limited therapeutic efficacy in some PD patients. Pharmacological chaperone therapy (PCT), either alone or in combination with enzyme replacement, has been proposed as an alternative therapeutic strategy. However, the chaperones identified so far also are active site-directed molecules and potential inhibitors of target enzymes. We demonstrated that N-acetylcysteine (NAC) is a novel allosteric chaperone for GAA. NAC improved the stability of rhGAA as a function of pH and temperature without disrupting its catalytic activity. A computational analysis of NAC-GAA interactions confirmed that NAC does not interact with GAA catalytic domain. NAC enhanced the residual activity of mutated GAA in cultured PD fibroblasts and in COS7 cells overexpressing mutated GAA. NAC also enhanced rhGAA efficacy in PD fibroblasts. In cells incubated with NAC and rhGAA, GAA activities were 3.7-8.7-fold higher than those obtained in cells treated with rhGAA alone. In a PD mouse model the combination of NAC and rhGAA resulted in better correction of enzyme activity in liver, heart, diaphragm and gastrocnemia, compared to rhGAA alone.

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.

Clinical description of a patient carrying the smallest reported deletion involving 10p14 region.

Haploinsufficiency of a region located distal to 10p14 designated HDR1, is responsible for hypoparathyroidism, sensorineural deafness, and renal anomalies (HDR syndrome). Haploinsufficiency of a more proximal region, located on 10p13-10p14, designated as DGCR2 is associated with congenital heart defects and thymus hypoplasia/aplasia or T cell defect. We describe a patient showing facial dysmorphisms, delayed psychomotor development and bilateral sensorineural hearing loss and carrying a 10p14 deletion, the smallest deletion found in the literature so far. Our patient, carrying a partial deletion of the DGCR2 region and of the HDR1 region, including the GATA3 gene, showed, unexpectedly, only few of the clinical features of DiGeorge 2 syndrome (psychomotor retardation, palpebral ptosis, epicanthic folds, anteverted nares, cryptorchidism, hand/foot abnormalities) and did not show other typical signs, such as cardiac defect, cleft palate, and abnormal T cell levels. Of the three characteristic features of the HDR syndrome, our patient had only sensorineural deafness. On the basis of the revision of the other cases reported in the literature with a deletion including the 10p14 region, we suggest that GATA3 haploinsufficiency, although not recorded for each patient, is responsible for deafness. The present case shows that even this small 10p deletion is responsible for a specific phenotype. We also underline the importance of CGH-array, in order to obtain a more precise physical mapping of the 10p deletions and an accurate genotype-phenotype correlation.

Synergy between the pharmacological chaperone 1-deoxygalactonojirimycin and the human recombinant alpha-galactosidase A in cultured fibroblasts from patients with Fabry disease.

Fabry disease (FD) is an X-linked inherited disease due to alpha-galactosidase A (alpha-Gal A) deficiency and characterized by lysosomal storage of globotriaosylceramide (Gb3) and related neutral glycosphingolipids. Storage of these substrates results in multisystem manifestations, including renal failure, cardiomyopathy, premature myocardial infarctions, stroke, chronic neuronopathic pain, gastrointestinal disturbances, and skin angiokeratoma. Enzyme replacement therapy (ERT) with recombinant human alpha-galactosidase A (rh-alpha-Gal A) is now available for the treatment of FD and in most patients results in clinical improvement or stabilization. However, ERT efficacy may vary in different tissues and its long-term effects remain to be defined. As a strategy to improve the efficacy of ERT, we tested the combination of rh-alpha-Gal A with the chaperone molecule 1-deoxynojirimycin (DGJ) in cultured FD fibroblasts with negligible residual enzyme activity. Compared to the effects of rh-alpha-Gal A alone, co-administration of DGJ and rh-alpha-Gal A resulted in better correction (4.8 to 16.9-fold) of intracellular alpha-Gal A activity, and increased amounts of the enzyme within the lysosomal compartment. The clearance of lyso-Gb3, one of the substrates stored in FD and a potent inhibitor of alpha-Gal A, was also significantly improved with the co-administration of DGJ and rh-alpha-Gal A. This study provides additional evidence for a synergistic effect between ERT and pharmacological chaperone therapy and supports the idea that the efficacy of combination protocols may be superior to ERT alone.