Alessandra Zanetti

Gene therapy approaches for lysosomal storage disorders, a good model for the treatment of mendelian diseases

This review describes the different gene therapy technologies applied to approach lysosomal storage disorders, monogenic conditions, with known genetic and biochemical defects, for many of which animal models are available. Both viral and nonviral procedures are described, underlying the specific needs that the treatment of genetic disorders requires.

Segregation analysis in a family at risk for the Maroteaux-Lamy syndrome conclusively reveals c.1151G>A (p.S384N) as to be a polymorphism

Maroteaux–Lamy syndrome is an autosomal-recessive disorder due to the deficit of the lysosomal enzyme, arylsulfatase B (ARSB). Among the numerous genomic lesions reported till now, the sequence variant, c.1151G>A (p.S384N), has been associated with a severe phenotype in more than 10% of the patients. We now report the first in vivo demonstration of the polymorphic nature of p.S384N, revealed during the segregation analysis in a family at risk for Maroteaux–Lamy syndrome. The proband, compound heterozygous for c.[944G>A]+[245T>G] (p.[R315Q]+[L82R]), did not carry the p.S384N change, which was instead present in two healthy members of the family, in trans with the causative mutations, p.R315Q and p.L82R, respectively. The hypothesis that p.S384N was a polymorphism was further addressed by reverse dot-blot analysis of 400 control alleles, estimating an allele frequency of 4.5%. To predict the consequences of p.R315Q, p.L82R and p.S384N, we also modeled and compared the three amino-acid changes in the three-dimensional ARSB structure. The in silico analysis predicted a local protein misfolding in the presence of p.R315Q and p.L82R. On the contrary, no evident problem was predicted in the case of p.S384N, occurring on the protein surface, far from the active site. Overall, these findings strongly support the hypothesis that the non-synonymous change p.S384N is a polymorphism. Moreover, our results emphasize the need for caution in drawing conclusions from a novel variant allele before screening at least 50 healthy control subjects.

Gene therapy of Hunter syndrome: evaluation of the efficiency of muscle electro gene transfer for the production and release of recombinant iduronate-2-sulfatase (IDS).

Mucopolysaccharidosis type II (MPSII) is an inherited disorder due to a deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). The disease is characterized by a considerable deposition of heparan- and dermatan-sulfate, causing a general impairment of physiological functions. Most of the therapeutic protocols proposed so far are mainly based upon enzyme replacement therapy which is very expensive. There is a requirement for an alternative approach, and to this aim, we evaluated the feasibility of muscle electro gene transfer (EGT) performed in the IDS-knockout (IDS-ko) mouse model. EGT is a highly efficient method of delivering exogenous molecules into different tissues. More recently, pre-treatment with bovine hyaluronidase has shown to further improve transfection efficiency of muscle EGT. We here show that, by applying such procedure, IDS was very efficiently produced inside the muscle. However, no induced IDS activity was measured in the IDS-ko mice plasma, in contrast to matched healthy controls. In the same samples, an anticipated and rapidly increasing immune response against the recombinant protein was observed in the IDS-ko vs control mice, although reaching the same levels at 5 weeks post-injection. Additional experiments performed on healthy mice showed a significant contribution of hyaluronidase pre-treatment in increasing the immune response.

QueryOR: a comprehensive web platform for genetic variant analysis and prioritization

BackgroundWhole genome and exome sequencing are contributing to the extraordinary progress in the study of human genetic variants. In this fast developing field, appropriate and easily accessible tools are required to facilitate data analysis.ResultsHere we describe QueryOR, a web platform suitable for searching among known candidate genes as well as for finding novel gene-disease associations. QueryOR combines several innovative features that make it comprehensive, flexible and easy to use. Instead of being designed on specific datasets, it works on a general XML schema specifying formats and criteria of each data source. Thanks to this flexibility, new criteria can be easily added for future expansion. Currently, up to 70 user-selectable criteria are available, including a wide range of gene and variant features. Moreover, rather than progressively discarding variants taking one criterion at a time, the prioritization is achieved by a global positive selection process that considers all transcript isoforms, thus producing reliable results. QueryOR is easy to use and its intuitive interface allows to handle different kinds of inheritance as well as features related to sharing variants in different patients. QueryOR is suitable for investigating single patients, families or cohorts.ConclusionsQueryOR is a comprehensive and flexible web platform eligible for an easy user-driven variant prioritization. It is freely available for academic institutions at http://queryor.cribi.unipd.it/.

Extension of the molecular analysis to the promoter region of the iduronate 2-sulfatase gene reveals genomic alterations in mucopolysaccharidosis type II patients with normal coding sequence

Hunter disease or mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal disorder caused by the deficit of the enzyme iduronate-2-sulfatase (IDS), involved in the catabolism of the glycosaminoglycans heparan and dermatan sulfate. Our aim was to search for molecular defects in the promoter region of the IDS gene in patients with previous biochemical diagnosis of MPS II and after we sequenced the whole IDS coding region and the exon/intron boundaries without detecting any pathogenic mutations. Screening of the promoter region of four patients detected in two of them a 178 bp deletion and in the other two a single nucleotide substitution 818 bp upstream of the coding region. The latter had never been described before in MPS II patients and it turned out to be a polymorphism. Our experience suggests that MPS II patients with no mutations detected in the IDS coding region should be screened in the promoter region of the gene. Findings will hopefully help to clarify the relationship between genotype and phenotype and will be useful for the correct molecular diagnosis of Hunter patients and the identification of female carriers, the latter particularly important for genetic counseling.

Chiari 1 Malformation and Holocord Syringomyelia in Hunter Syndrome

Compressive cervical myelopathy is a well-known life-threatening complication in mucopolysaccharidosis (MPS) patients. Glycosaminoglycan accumulation in the growing cartilage results in dens dysplasia, atlanto-axial instability, and subsequent periodontoid fibrocartilaginous tissue deposition with upper cervical stenosis.Chiari malformation type 1 (CM1) is a congenital downward cerebellar tonsil ectopia determined by clivus and posterior cranial fossa underdevelopment, possibly leading to progressive spinal cord cavitation (syringomyelia) and severe neurological impairment.We present a boy affected with Hunter syndrome (MPS II) and cerebellar tonsil ectopia who developed a holocord syringomyelia at the age of 6 years. The child underwent atlanto-occipital decompressive surgery with rapid clinical and neuroimaging improvement.Sharing a primary mesenchymal involvement of the cervical-occipital region, the coexistence of CM1 in MPS might be not unexpected and complicate further the disease course. In these patients, strict monitoring and prompt treatment might be of foremost importance for preventing major neurological complications.

Molecular Analysis of Turkish Maroteaux-Lamy Patients and Identification of One Novel Mutation in the Arylsulfatase B (ARSB) Gene

Mucopolysaccharidosis type VI (MPS VI, Maroteaux-Lamy syndrome) is an autosomal recessive disorder caused by the deficit of the arylsulfatase B (ARSB) enzyme, which leads to dermatan sulfate pathological storage, resulting in a wide spectrum of clinical phenotypes. To date more than 130 different mutations were reported, most of them being restricted to individual families. We here report the first study on the ARSB gene mutations in MPS VI patients of Turkish ethnogeographic origin. On the whole we analyzed 13 unrelated families recruited from 3 different Turkish clinical centers, for a total of 52 subjects, including patients, parents, and siblings. The molecular characterization of ARSB gene in these subjects lead to the identification of eight different mutations (6 missense mutations and two single-nucleotide deletions) one of which novel: c.532C>G (p.H178D). We characterized seven different genotypes, all homozygous except one. The analysis highlighted c.962T>C (p.L321P) as the most frequently detected mutation in the group of patients examined and the c.1072G>A (p.V358M) as the most frequent polymorphism. All parents and 50% of the healthy siblings analyzed carried in a heterozygous condition the mutation identified in the affected relative. The high number of homozygotes reported in this study reflects the high degree of consanguinity of the Turkish population, being the parents of most of the patients here examined, first-degree cousins. As consanguineous marriages are an integral part of the Turkish society, carriers identification accompanied by genetic counseling in families at risk is the eligible approach to minimize the effects of consanguinity in this population.

Mucopolysaccharidosis type VI (MPS VI) and molecular analysis: Review and classification of published variants in the ARSB gene

Maroteaux–Lamy syndrome (MPS VI) is an autosomal recessive lysosomal storage disorder caused by pathogenic ARSB gene variants, commonly diagnosed through clinical findings and deficiency of the arylsulfatase B (ASB) enzyme. Detection of ARSB pathogenic variants can independently confirm diagnosis and render genetic counseling possible. In this review, we collect and summarize 908 alleles (201 distinct variants, including 3 polymorphisms previously considered as disease‐causing variants) from 478 individuals diagnosed with MPS VI, identified from literature and public databases. Each variant is further analyzed for clinical classification according to American College of Medical Genetics and Genomics (ACMG) guidelines. Results highlight the heterogeneity of ARSB alleles, with most unique variants (59.5%) identified as missense and 31.7% of unique alleles appearing once. Only 18% of distinct variants were previously recorded in public databases with supporting evidence and clinical significance. ACMG recommends publishing clinical and biochemical data that accurately characterize pathogenicity of new variants in association with reporting specific alleles. Variants analyzed were sent to ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), and MPS VI locus‐specific database (http://mps6-database.org) where they will be available. High clinical suspicion coupled with diagnostic testing for deficient ASB activity and timely submission and classification of ARSB variants with biochemical and clinical data in public databases is essential for timely diagnosis of MPS VI.

Brain RNA-seq profiling of the mucopolysaccharidosis type II mouse model

Lysosomal storage disorders (LSDs) are a group of about 50 genetic metabolic disorders, mainly affecting children, sharing the inability to degrade specific endolysosomal substrates. This results in failure of cellular functions in many organs, including brain that in most patients may go through progressive neurodegeneration. In this study, we analyzed the brain of the mouse model for Hunter syndrome, a LSD mostly presenting with neurological involvement. Whole transcriptome analysis of the cerebral cortex and midbrain/diencephalon/hippocampus areas was performed through RNA-seq. Genes known to be involved in several neurological functions showed a significant differential expression in the animal model for the disease compared to wild type. Among the pathways altered in both areas, axon guidance, calcium homeostasis, synapse and neuroactive ligand–receptor interaction, circadian rhythm, neuroinflammation and Wnt signaling were the most significant. Application of RNA sequencing to dissect pathogenic alterations of complex syndromes allows to photograph perturbations, both determining and determined by these disorders, which could simultaneously occur in several metabolic and biochemical pathways. Results also emphasize the common, altered pathways between neurodegenerative disorders affecting elderly and those associated with pediatric diseases of genetic origin, perhaps pointing out a general common course for neurodegeneration, independent from the primary triggering cause.

Personalized Medicine in Rare Pediatric Neurometabolic Diseases

Enormous advances in life sciences and molecular biology mean that research today generates massive amounts of data that can potentially serve to advance the development of individualized therapies. Such treatments are based on the principles of disease prevention, prediction, early diagnosis and reliable monitoring of therapeutic efficacy with the goal of providing personalized treatments that improve the health of individuals. The development of personalized medicine is of considerable importance for pediatric patient populations, and represents a move away from the use of treatment dosages based on experience with the same compounds in adults. Currently, however, we know little about developmental pharmacogenomics and, although many biomarkers are available for use in clinical research, there have been few applications in the management of pediatric diseases. This chapter reviews the requirements for pediatric personalized medicine and describes a group of diseases that require such an approach. Personalized medicine is particularly relevant for the treatment of rare childhood diseases, and the group of life-threatening neurological diseases known as lysosomal storage diseases (LSDs) represents a potential study population. The genetic bases of these disorders are generally well defined, there is the potential for diagnosis at birth or prenatally, and there are several therapeutic options available or under development.