Lysosomal gene Hexb displays haploinsufficiency in a knock-in mouse model of Alzheimer's disease.

Lysosomal network abnormalities are an increasingly recognised feature of Alzheimer's disease (AD), which appear early and are progressive in nature. Sandhoff disease and Tay-Sachs disease (neurological lysosomal storage diseases caused by mutations in genes that code for critical subunits of ß-hexosaminidase) result in accumulation of amyloid-ß (Aß) and related proteolytic fragments in the brain. However, experiments that determine whether mutations in genes that code for ß-hexosaminidase are risk factors for AD are currently lacking. To determine the relationship between ß-hexosaminidase and AD, we investigated whether a heterozygous deletion of Hexb, the gene that encodes the beta subunit of ß-hexosaminidase, modifies the behavioural phenotype and appearance of disease lesions in App NL-G-F/NL-G-F (App KI/KI ) mice. App KI/KI and Hexb +/- mice were crossed and evaluated in a behavioural test battery. Neuropathological hallmarks of AD and ganglioside levels in the brain were also examined. Heterozygosity of Hexb in App KI/KI mice reduced learning flexibility during the Reversal Phase of the Morris water maze. Contrary to expectation, heterozygosity of Hexb caused a small but significant decrease in amyloid beta deposition and an increase in the microglial marker IBA1 that was region- and age-specific. Hexb heterozygosity caused detectable changes in the brain and in the behaviour of an AD model mouse, consistent with previous reports that described a biochemical relationship between HEXB and AD. This study reveals that the lysosomal enzyme gene Hexb is not haplosufficient in the mouse AD brain.

Oxidative profile exhibited by Mucopolysaccharidosis type IVA patients at diagnosis: Increased keratan urinary levels.

Morquio A disease (Mucopolysaccharidosis type IVA, MPS IVA) is one of the 11 mucopolysaccharidoses (MPSs), a heterogeneous group of inherited lysosomal storage disorders (LSDs) caused by deficiency in enzymes need to degrade glycosaminoglycans (GAGs). Morquio A is characterized by a decrease in N-acetylgalactosamine-6-sulfatase activity and subsequent accumulation of keratan sulfate and chondroitin 6-sulfate in cells and body fluids. As the pathophysiology of this LSD is not completely understood and considering the previous results of our group concerning oxidative stress in Morquio A patients receiving enzyme replacement therapy (ERT), the aim of this study was to investigate oxidative stress parameters in Morquio A patients at diagnosis. It was studied 15 untreated Morquio A patients, compared with healthy individuals. The affected individuals presented higher lipid peroxidation, assessed by urinary 15-F2t-isoprostane levels and no protein damage, determined by sulfhydryl groups in plasma and di-tyrosine levels in urine. Furthermore, Morquio A patients showed DNA oxidative damage in both pyrimidines and purines bases, being the DNA damage positively correlated with lipid peroxidation. In relation to antioxidant defenses, affected patients presented higher levels of reduced glutathione (GSH) and increased activity of glutathione peroxidase (GPx), while superoxide dismutase (SOD) and glutathione reductase (GR) activities were similar to controls. Our findings indicate that Morquio A patients present at diagnosis redox imbalance and oxidative damage to lipids and DNA, reinforcing the idea about the importance of antioxidant therapy as adjuvant to ERT, in this disorder.

Lysoplex: An efficient toolkit to detect DNA sequence variations in the autophagy-lysosomal pathway.

The autophagy-lysosomal pathway (ALP) regulates cell homeostasis and plays a crucial role in human diseases, such as lysosomal storage disorders (LSDs) and common neurodegenerative diseases. Therefore, the identification of DNA sequence variations in genes involved in this pathway and their association with human diseases would have a significant impact on health. To this aim, we developed Lysoplex, a targeted next-generation sequencing (NGS) approach, which allowed us to obtain a uniform and accurate coding sequence coverage of a comprehensive set of 891 genes involved in lysosomal, endocytic, and autophagic pathways. Lysoplex was successfully validated on 14 different types of LSDs and then used to analyze 48 mutation-unknown patients with a clinical phenotype of neuronal ceroid lipofuscinosis (NCL), a genetically heterogeneous subtype of LSD. Lysoplex allowed us to identify pathogenic mutations in 67% of patients, most of whom had been unsuccessfully analyzed by several sequencing approaches. In addition, in 3 patients, we found potential disease-causing variants in novel NCL candidate genes. We then compared the variant detection power of Lysoplex with data derived from public whole exome sequencing (WES) efforts. On average, a 50% higher number of validated amino acid changes and truncating variations per gene were identified. Overall, we identified 61 truncating sequence variations and 488 missense variations with a high probability to cause loss of function in a total of 316 genes. Interestingly, some loss-of-function variations of genes involved in the ALP pathway were found in homozygosity in the normal population, suggesting that their role is not essential. Thus, Lysoplex provided a comprehensive catalog of sequence variants in ALP genes and allows the assessment of their relevance in cell biology as well as their contribution to human disease.

Expanding the spectrum of HEXA mutations in Indian patients with Tay-Sachs disease.

Tay-Sachs disease is an autosomal recessive neurodegenerative disorder occurring due to impaired activity of ß-hexosaminidase-A (EC 3.2.1.52), resulting from the mutation in HEXA gene. Very little is known about the molecular pathology of TSD in Indian children except for a few mutations identified by us. The present study is aimed to determine additional mutations leading to Tay-Sachs disease in nine patients confirmed by the deficiency of ß-hexosaminidase-A (< 2% of total hexosaminidase activity for infantile patients) in leucocytes. The enzyme activity was assessed by using substrates 4-methylumbelliferyl-N-acetyl-ß-d-glucosamine and 4-methylumbelliferyl-N-acetyl-ß-d-glucosamine-6-sulfate for total-hexosaminidase and hexosaminidase-A respectively, and heat inactivation method for carrier detection. The exons and exon-intron boundaries of the HEXA gene were bi-directionally sequenced on an automated sequencer. 'In silico' analyses for novel mutations were carried out using SIFT, Polyphen2 and MutationT@ster software programs. The structural study was carried out by UCSF Chimera software using the crystallographic structure of ß-hexosaminidase-A (PDB-ID: 2GJX) as the template. Our study identified four novel mutations in three cases. These include a compound heterozygous missense mutation c.524A>C (D175A) and c.805G>C (p.G269R) in one case; and one small 1 bp deletion c.426delT (p.F142LfsX57) and one splice site mutation c.459+4A>C in the other two cases respectively. None of these mutations were detected in 100 chromosomes from healthy individuals of the same ethnic group. Three previously reported missense mutations, (i) c.532C>T (p.R178C), (ii) c.964G>T (p.D322Y), and (iii) c.1385A>T (p.E462V); two nonsense mutations (i) c.709C>T (p.Q237X) and (ii) c.1528C>T (p.R510X), one 4 bp insertion c.1277_1278insTATC (p.Y427IfsX5) and one splice site mutation c.459+5G>A were also identified in six cases. We observe from this study that novel mutations are more frequently observed in Indian patients with Tay-Sachs disease with clustering of ~ 73% of disease causing mutations in exons 5 to 12. This database can be used for a carrier rate screening in the larger population of the country.