Lithium treatment rescues dysfunctional autophagy in the cell models of Tay-Sachs disease.

Tay-Sachs disease is a rare lysosomal storage disorder (LSD) caused by a mutation in the HexA gene coding ß-hexosaminidase A enzyme. The disruption of the HexA gene causes the accumulation of GM2 ganglioside resulting in progressive neurodegeneration in humans. Surprisingly, Hexa-/- mice did not show neurological phenotypes. Our group recently generated a murine model of Tay-Sachs disease exhibiting excessive GM2 accumulation and severe neuropathological abnormalities mimicking Tay-Sachs patients. Previously, we reported impaired autophagic flux in the brain of Hexa/-Neu3-/- mice. However, regulation of autophagic flux using inducers has not been clarified in Tay-Sachs disease cells. Here, we evaluated the effects of lithium treatment on dysfunctional autophagic flux using LC3 and p62 in the fibroblast and neuroglia of Hexa-/-Neu3-/- mice and Tay-Sachs patients. We discovered the clearance of accumulating autophagosomes, aggregate-prone metabolites, and GM2 ganglioside under lithium-induced conditions. Our data suggest that targeting autophagic flux with an autophagy inducer might be a rational therapeutic strategy for the treatment of Tay-Sachs disease.

Clinical outcome assessments of disease burden and progression in late-onset GM2 gangliosidoses.

The late-onset GM2 gangliosidoses, comprising late-onset Tay-Sachs and Sandhoff diseases, are rare, slowly progressive, neurogenetic disorders primarily characterized by neurogenic weakness, ataxia, and dysarthria. The aim of this longitudinal study was to characterize the natural history of late-onset GM2 gangliosidoses using a number of clinical outcome assessments to measure different aspects of disease burden and progression over time, including neurological, functional, and quality of life, to inform the design of future clinical interventional trials. Patients attending the United States National Tay-Sachs & Allied Diseases Family Conference between 2015 and 2019 underwent annual clinical outcome assessments. Currently, there are no clinical outcome assessments validated to assess late-onset GM2 gangliosidoses; therefore, instruments used or designed for diseases with similar features, or to address various aspects of the clinical presentations, were used. Clinical outcome assessments included the Friedreich's Ataxia Rating Scale, the 9-Hole Peg Test, and the Assessment of Intelligibility of Dysarthric Speech. Twenty-three patients participated in at least one meeting visit (late-onset Tay-Sachs, n = 19; late-onset Sandhoff, n = 4). Patients had high disease burden at baseline, and scores for the different clinical outcome assessments were generally lower than would be expected for the general population. Longitudinal analyses showed slow, but statistically significant, neurological progression as evidenced by worsening scores on the 9-Hole Peg Test (2.68%/year, 95% CI: 0.13-5.29; p = 0.04) and the Friedreich's Ataxia Rating Scale neurological examination (1.31 points/year, 95% CI: 0.26-2.35; p = 0.02). Time since diagnosis to study entry correlated with worsening scores on the 9-Hole Peg Test (r = 0.728; p < 0.001), Friedreich's Ataxia Rating Scale neurological examination (r = 0.727; p < 0.001), and Assessment of Intelligibility of Dysarthric Speech intelligibility (r = -0.654; p = 0.001). In summary, patients with late-onset GM2 gangliosidoses had high disease burden and slow disease progression. Several clinical outcome assessments suitable for clinical trials showed only small changes and standardized effect sizes (change/standard deviation of change) over 4 years. These longitudinal natural history study results illustrate the challenge of identifying responsive endpoints for clinical trials in rare, slowly progressive, neurogenerative disorders where arguably the treatment goal is to halt or decrease the rate of decline rather than improve clinical status. Furthermore, powering such a study would require a large sample size and/or a long study duration, neither of which is an attractive option for an ultra-rare disease with no available treatment. These findings support the development of potentially more sensitive late-onset GM2 gangliosidoses-specific rating instruments and/or surrogate endpoints for use in future clinical trials.

Late-onset Tay-Sachs disease presenting with a neuromuscular phenotype-a case series.

BACKGROUND AND PURPOSE: Tay-Sachs disease is a rare and often fatal, autosomal recessive, lysosomal storage disease. Deficiency in ß-hexosaminidase leads to accumulation of GM2 ganglioside resulting in neuronal swelling and degeneration. Typical onset is in infancy with developmental regression and early death. Late-onset Tay-Sachs disease (LOTS) is extremely rare, especially in the non-Ashkenazi Jewish population, and is characterized by a more indolent presentation typically encompassing features of cerebellar and anterior horn cell dysfunction in addition to extrapyramidal and neuropsychiatric symptoms. CASES: A case series of four unrelated patients of non-Ashkenazi Jewish origin with a predominantly, and in some cases pure, neuromuscular phenotype with evidence of a motor neuronopathy on electromyography is presented. Cerebellar atrophy, reported to be a ubiquitous feature in LOTS, was absent in all patients. CONCLUSION: This case series provides evidence to support a pure neuromuscular phenotype in LOTS, which should be considered in the differential diagnosis of anterior horn cell disorders.

Plasma GM2 ganglioside potential biomarker for diagnosis, prognosis and disease monitoring of GM2-Gangliosidosis.

GM2-Gangliosidosis are a group of inherited lysosomal storage pathologies characterized by a large accumulation of GM2 ganglioside in the lysosome. They are caused by mutation in HEXA or HEXB causing reduced or absent activity of a lysosomal ß-hexosaminidase A, or mutation in GM2A causing defect in GM2 activator protein (GM2AP), an essential protein for the activity of the enzyme. Biochemical diagnosis relies on the measurement of ß-hexosaminidases A and B activities, which is able to detect lysosomal enzyme deficiency but fails to identify defects in GM2AP. We developed a rapid, specific and sensitive liquid chromatography-mass spectrometry-based method to measure simultaneously GM1, GM2, GM3 and GD3 molecular species. Gangliosides were analysed in plasma from 19 patients with GM2-Gangliosidosis: Tay-Sachs (n = 9), Sandhoff (n = 9) and AB variant of GM2-Gangliosidosis (n = 1) and compared to 20 age-matched controls. Among patients, 12 have a late adult-juvenile-onset and 7 have an infantile early-onset of the disease. Plasma GM2 molecular species were increased in all GM2-Gangliosidosis patients (19/19), including the patient with GM2A mutation, compared to control individuals and compared to patients with different other lysosomal storage diseases. GM234:1 and GM234:1/GM334:1 ratio discriminated patients from controls with 100% sensitivity and specificity. GM234:1 and GM234:1/GM334:1 were higher in patients with early-onset compared to those with late-onset of the disease, suggesting a relationship with severity. Longitudinal analysis in one adult with Tay-Sachs disease over 9 years showed a positive correlation of GM234:1 and GM234:1/GM334:1 ratio with age at sampling. We propose that plasma GM2 34:1 and its ratio to GM3 34:1 could be sensitive and specific biochemical diagnostic biomarkers for GM2-Gangliosidosis including AB variant and could be useful as a first line diagnostic test and potential biomarkers for monitoring upcoming therapeutic efficacy.

Gene expression changes in Tay-Sachs disease begin early in fetal brain development.

Treatment of monogenic disorders has historically relied on symptomatic management with limited ability to target primary molecular deficits. However, recent advances in gene therapy and related technologies aim to correct these underlying deficiencies, raising the possibility of disease management or even prevention for diseases that can be treated pre-symptomatically. Tay-Sachs disease (TSD) would be one such candidate, however very little is known about the presymptomatic stage of TSD. To better understand the effects of TSD on brain development, we evaluated the transcriptomes of human fetal brain samples with biallelic pathogenic variants in HEXA. We identified dramatic changes in the transcriptome, suggesting a perturbation of normal development. We also observed a shift in the expression of the sphingolipid metabolic pathway away from production of the HEXA substrate, GM2 ganglioside, presumptively to compensate for dysfunction of the enzyme. However, we do not observe transcriptomic signatures of end-stage disease, suggesting that developmental perturbations precede neurodegeneration. To our knowledge, this is the first report of the relationship between fetal disease pathology in juvenile onset TSD and the analysis of gene expression in fetal TSD tissues. This study highlights the need to better understand the "pre-symptomatic" stage of disease to set realistic expectations for patients receiving early therapeutic intervention.

Quantitative longitudinal natural history of 8 gangliosidoses-conceptual framework and baseline data of the German 8-in-1 disease registry. A cross-sectional analysis.

PURPOSE: Gangliosidoses are a group of inherited neurogenetic autosomal recessive lysosomal storage disorders usually presenting with progressive macrocephaly, developmental delay, and regression, leading to significant morbidity and premature death. A quantitative definition of the natural history would support and enable clinical development of specific therapies. METHODS: Single disease registry of 8 gangliosidoses (NCT04624789). Cross-sectional analysis of baseline data in N = 26 patients. Primary end point: disease severity assessed by the 8-in-1 score. Secondary end points: first neurologic sign or symptom observed (1) by parents and (2) by physicians, diagnostic delay, as well as phenotypical characterization. Tertiary end points: neurologic outcomes (development, ataxia, dexterity) and disability. RESULTS: The 8-in-1 score quantitatively captured severity of disease. Parents recognized initial manifestations (startle reactions) earlier than physicians (motor developmental delay and hypotonia). Median diagnostic delay was 3.16 (interquartile range 0.69-6.25) years. In total, 8 patients presented with late-infantile phenotypes. CONCLUSION: Data in this registry raise awareness of these rare and fatal conditions to accelerate diagnosis, inform counseling of afflicted families, define quantitative end points for clinical trials, and can serve as historical controls for future therapeutic studies. We provide further insight into the rare late-infantile phenotype for GM2-gangliosidosis. Longitudinal follow up is planned.

Pontocerebellar atrophy is the hallmark neuroradiological finding in late-onset Tay-Sachs disease.

PURPOSE: Late-onset Tay-Sachs disease (LOTS) is a form of GM2 gangliosidosis, an autosomal recessive neurodegenerative disorder characterized by slowly progressive cerebellar ataxia, lower motor neuron disease, and psychiatric impairment due to mutations in the HEXA gene. The aim of our work was to identify the characteristic brain MRI findings in this presumably underdiagnosed disease. METHODS: Clinical data and MRI findings from 16 patients (10F/6 M) with LOTS from two centers were independently assessed by two readers and compared to 16 age- and sex-related controls. RESULTS: Lower motor neuron disease (94%), psychiatric symptoms-psychosis (31%), cognitive impairment (38%) and depression (25%)-and symptoms of cerebellar impairment including dysarthria (94%), ataxia (81%) and tremor (69%), were the most common clinical features. On MRI, pontocerebellar atrophy was a constant finding. Compared to controls, LOTS patients had smaller mean middle cerebellar peduncle diameter (p < 0.0001), mean superior cerebellar peduncle diameter (p = 0.0002), mesencephalon sagittal area (p = 0.0002), pons sagittal area (p < 0.0001), and larger 4th ventricle transversal diameter (p < 0.0001). Mild corpus callosum thinning (37.5%), mild cortical atrophy (18.8%), and white matter T2 hyperintensities (12.5%) were also present. CONCLUSION: Given the characteristic clinical course and MRI findings of the pontocerebellar atrophy, late-onset Tay-Sachs disease should be considered in the differential diagnosis of adult-onset cerebellar ataxias.

sp2-Iminosugars targeting human lysosomal ß-hexosaminidase as pharmacological chaperone candidates for late-onset Tay-Sachs disease.

The late-onset form of Tay-Sachs disease displays when the activity levels of human ß-hexosaminidase A (HexA) fall below 10% of normal, due to mutations that destabilise the native folded form of the enzyme and impair its trafficking to the lysosome. Competitive inhibitors of HexA can rescue disease-causative mutant HexA, bearing potential as pharmacological chaperones, but often also inhibit the enzyme O-glucosaminidase (GlcNAcase; OGA), a serious drawback for translation into the clinic. We have designed sp2-iminosugar glycomimetics related to GalNAc that feature a neutral piperidine-derived thiourea or a basic piperidine-thiazolidine bicyclic core and behave as selective nanomolar competitive inhibitors of human Hex A at pH 7 with a ten-fold lower inhibitory potency at pH 5, a good indication for pharmacological chaperoning. They increased the levels of lysosomal HexA activity in Tay-Sachs patient fibroblasts having the G269S mutation, the highest prevalent in late-onset Tay-Sachs disease.

CRISPR/nCas9-Based Genome Editing on GM2 Gangliosidoses Fibroblasts via Non-Viral Vectors.

The gangliosidoses GM2 are a group of pathologies mainly affecting the central nervous system due to the impaired GM2 ganglioside degradation inside the lysosome. Under physiological conditions, GM2 ganglioside is catabolized by the ß-hexosaminidase A in a GM2 activator protein-dependent mechanism. In contrast, uncharged substrates such as globosides and some glycosaminoglycans can be hydrolyzed by the ß-hexosaminidase B. Monogenic mutations on HEXA, HEXB, or GM2A genes arise in the Tay-Sachs (TSD), Sandhoff (SD), and AB variant diseases, respectively. In this work, we validated a CRISPR/Cas9-based gene editing strategy that relies on a Cas9 nickase (nCas9) as a potential approach for treating GM2 gangliosidoses using in vitro models for TSD and SD. The nCas9 contains a mutation in the catalytic RuvC domain but maintains the active HNH domain, which reduces potential off-target effects. Liposomes (LPs)- and novel magnetoliposomes (MLPs)-based vectors were used to deliver the CRISPR/nCas9 system. When LPs were used as a vector, positive outcomes were observed for the ß-hexosaminidase activity, glycosaminoglycans levels, lysosome mass, and oxidative stress. In the case of MLPs, a high cytocompatibility and transfection ratio was observed, with a slight increase in the ß-hexosaminidase activity and significant oxidative stress recovery in both TSD and SD cells. These results show the remarkable potential of CRISPR/nCas9 as a new alternative for treating GM2 gangliosidoses, as well as the superior performance of non-viral vectors in enhancing the potency of this therapeutic approach.

[Progressive psychomotor regression for 2.5 years in a boy aged 5 years]. / 5岁男童进行性智力运动功能倒退2.5年.

A boy, aged 5 years, attended the hospital due to progressive psychomotor regression for 2.5 years. Motor function regression was the main manifestation in the early stage, and brain MRI and whole-exome sequencing (WES) of the family showed no abnormalities. After the age of 4 years and 9 months, the boy developed cognitive function regression, and brain MRI showed cerebellar atrophy. The reanalysis of WES results revealed a compound heterozygous mutation, [NM_000520, c.784C>T(p.His262Tyr]), c.1412C>T(p.Pro471Leu)], in the HEXA gene. The enzyme activity detection showed a significant reduction in the level of ß-hexosaminidase encoded by this gene. The boy was diagnosed with juvenile Tay-Sachs disease (TSD). TSD has strong clinical heterogeneity, and cerebellar atrophy may be an important clue for the diagnosis of juvenile TSD. The reanalysis of genetic data when appropriate based on disease evolution may improve the positive rate of WES.

In-silico screening and microsecond molecular dynamics simulations to identify single point mutations that destabilize ß-hexosaminidase A causing Tay-Sachs disease.

ß-hexosaminidase A (HexA) protein is responsible for the degradation of GM2 gangliosides in the central and peripheral nervous systems. Tay-Sachs disease occurs when HexA within Hexosaminidase does not properly function and harmful GM2 gangliosides begin to build up within the neurons. In this study, in silico methods such as SIFT, PolyPhen-2, PhD-SNP, and MutPred were utilized to analyze the effects of nonsynonymous single nucleotide polymorphisms (nsSNPs) on HexA in order to identify possible pathogenetic and deleterious variants. Molecular dynamics (MD) simulations showed that two mutants, P25S and W485R, experienced an increase in structural flexibility compared to the native protein. Particularly, there was a decrease in the overall number and frequencies of hydrogen bonds for the mutants compared to the wildtype. MM/GBSA calculations were performed to help assess the change in binding affinity between the wildtype and mutant structures and a mechanism-based inhibitor, NGT, which is known to help increase the residual activity of HexA. Both of the mutants experienced a decrease in the binding affinity from -23.8 kcal/mol in wildtype to -20.9 and -18.7 kcal/mol for the P25S and W485R variants of HexA, respectively.

Screening for autosomal recessive and X-linked conditions during pregnancy and preconception: a practice resource of the American College of Medical Genetics and Genomics (ACMG).

Carrier screening began 50 years ago with screening for conditions that have a high prevalence in defined racial/ethnic groups (e.g., Tay-Sachs disease in the Ashkenazi Jewish population; sickle cell disease in Black individuals). Cystic fibrosis was the first medical condition for which panethnic screening was recommended, followed by spinal muscular atrophy. Next-generation sequencing allows low cost and high throughput identification of sequence variants across many genes simultaneously. Since the phrase "expanded carrier screening" is nonspecific, there is a need to define carrier screening processes in a way that will allow equitable opportunity for patients to learn their reproductive risks using next-generation sequencing technology. An improved understanding of this risk allows patients to make informed reproductive decisions. Reproductive decision making is the established metric for clinical utility of population-based carrier screening. Furthermore, standardization of the screening approach will facilitate testing consistency. This practice resource reviews the current status of carrier screening, provides answers to some of the emerging questions, and recommends a consistent and equitable approach for offering carrier screening to all individuals during pregnancy or preconception.

Natural history of Tay-Sachs disease in sheep.

Tay-Sachs disease (TSD) is a fatal neurodegenerative disease caused by a deficiency of the enzyme ß-N-acetylhexosaminidase A (HexA). TSD naturally occurs in Jacob sheep is the only experimental model of TSD. TSD in sheep recapitulates neurologic features similar to juvenile onset and late onset TSD patients. Due to the paucity of human literature on pathology of TSD, a better natural history in the sheep TSD brain, which is on the same order of magnitude as a child's, is necessary for evaluating therapy and characterizing the pathological events that occur. To provide clinicians and researchers with a clearer understanding of longitudinal pathology in patients, we compare spectrum of clinical signs and brain pathology in mildly symptomatic (3-months), moderately symptomatic (6-months), or severely affected TSD sheep (humane endpoint at ~9-months of age). Increased GM2 ganglioside in the CSF of TSD sheep and a TSD specific biomarker on MRS (taurine) correlate with disease severity. Microglial activation and reactive astrocytes were observed globally on histopathology in TSD sheep with a widespread reduction in oligodendrocyte density. Myelination is reduced primarily in the forebrain illustrated by loss of white matter on MRI. GM2 and GM3 ganglioside were increased and distributed differently in various tissues. The study of TSD in the sheep model provides a natural history to shed light on the pathophysiology of TSD, which is of utmost importance due to novel therapeutics being assessed in human patients.

A pathogenic HEXA missense variant in wild boars with Tay-Sachs disease.

Gangliosidoses are inherited lysosomal storage disorders caused by reduced or absent activity of either a lysosomal enzyme involved in ganglioside catabolism, or an activator protein required for the proper activity of a ganglioside hydrolase, which results in the intra-lysosomal accumulation of undegraded metabolites. We hereby describe morphological, ultrastructural, biochemical and genetic features of GM2 gangliosidosis in three captive bred wild boar littermates. The piglets were kept in a partially-free range farm and presented progressive neurological signs, starting at 6 months of age. Animals were euthanized at approximately one year of age due to their poor conditions. Neuropathogens were excluded as a possible cause of the signs. Gross examination showed a reduction of cerebral and cerebellar consistency. Central (CNS) and peripheral (PNS) nervous system neurons were enlarged and foamy, with severe and diffuse cytoplasmic vacuolization. Transmission electron microscopy (TEM) of CNS neurons demonstrated numerous lysosomes, filled by parallel or concentric layers of membranous electron-dense material, defined as membranous cytoplasmic bodies (MCB). Biochemical composition of gangliosides analysis from CNS revealed accumulation of GM2 ganglioside; furthermore, Hex A enzyme activity was less than 1% compared to control animals. These data confirmed the diagnosis of GM2 gangliosidosis. Genetic analysis identified, at a homozygous level, the presence of a missense nucleotide variant c.1495C > T (p Arg499Cys) in the hexosaminidase subunit alpha gene (HEXA), located within the GH20 hexosaminidase superfamily domain of the encoded protein. This specific HEXA variant is known to be pathogenic and associated with Tay-Sachs disease in humans, but has never been identified in other animal species. This is the first report of a HEXA gene associated Tay-Sachs disease in wild boars and provides a comprehensive description of a novel spontaneous animal model for this lysosomal storage disease.

Pronounced Therapeutic Benefit of a Single Bidirectional AAV Vector Administered Systemically in Sandhoff Mice.

The GM2 gangliosidoses, Tay-Sachs disease (TSD) and Sandhoff disease (SD), are fatal lysosomal storage disorders caused by mutations in the HEXA and HEXB genes, respectively. These mutations cause dysfunction of the lysosomal enzyme ß-N-acetylhexosaminidase A (HexA) and accumulation of GM2 ganglioside (GM2) with ensuing neurodegeneration, and death by 5 years of age. Until recently, the most successful therapy was achieved by intracranial co-delivery of monocistronic adeno-associated viral (AAV) vectors encoding Hex alpha and beta-subunits in animal models of SD. The blood-brain barrier crossing properties of AAV9 enables systemic gene therapy; however, the requirement of co-delivery of two monocistronic AAV vectors to overexpress the heterodimeric HexA protein has prevented the use of this approach. To address this need, we developed multiple AAV constructs encoding simultaneously HEXA and HEXB using AAV9 and AAV-PHP.B and tested their therapeutic efficacy in 4- to 6-week-old SD mice after systemic administration. Survival and biochemical outcomes revealed superiority of the AAV vector design using a bidirectional CBA promoter with equivalent dose-dependent outcomes for both capsids. AAV-treated mice performed normally in tests of motor function, CNS GM2 ganglioside levels were significantly reduced, and survival increased by >4-fold with some animals surviving past 2 years of age.

Preconception risk assessment for thalassaemia, sickle cell disease, cystic fibrosis and Tay-Sachs disease.

BACKGROUND: Globally, about 6% of children are born with a serious birth defect of genetic or partially genetic origin. Carrier screening or testing is one way to identify couples at increased risk of having a child with an autosomal recessive condition. The most common autosomal recessive conditions are thalassaemia, sickle cell disease, cystic fibrosis and Tay-Sachs disease, with higher carrier rates in high-risk populations of specific ancestral backgrounds. Identifying and counselling couples at genetic risk of the conditions before pregnancy enables them to make fully informed reproductive decisions, with some of these choices not being available if testing is only offered in an antenatal setting. This is an update of a previously published review. OBJECTIVES: To assess the effectiveness of systematic preconception genetic risk assessment to enable autonomous reproductive choice and to improve reproductive outcomes  in women and their partners who are both identified as carriers of thalassaemia, sickle cell disease, cystic fibrosis and Tay-Sachs disease in healthcare settings when compared to usual care. SEARCH METHODS: We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group's Trials Registers. Date of latest search of the registers: 04 August 2021. In addition, we searched for all relevant trials from 1970 (or the date at which the database was first available if after 1970) to date using electronic databases (MEDLINE, Embase, CINAHL, PsycINFO), clinical trial databases (National Institutes of Health, Clinical Trials Search portal of the World Health Organization, metaRegister of controlled clinical trials), and hand searching of key journals and conference abstract books from 1998 to date (European Journal of Human Genetics, Genetics in Medicine, Journal of Community Genetics). We also searched the reference lists of relevant articles, reviews and guidelines and also contacted subject experts in the field to request any unpublished or other published trials. Date of latest search of all these sources: 25 June 2021.  SELECTION CRITERIA: Any randomised controlled trials (RCTs) or quasi-RCTs (published or unpublished) comparing reproductive outcomes of systematic preconception genetic risk assessment for thalassaemia, sickle cell disease, cystic fibrosis and Tay-Sachs disease when compared to usual care. DATA COLLECTION AND ANALYSIS: We identified 37 papers, describing 22 unique trials which were potentially eligible for inclusion in the review. However, after assessment, we found no RCTs of preconception genetic risk assessment for thalassaemia, sickle cell disease, cystic fibrosis and Tay-Sachs disease. MAIN RESULTS: No RCTs of preconception genetic risk assessment for thalassaemia, sickle cell disease, cystic fibrosis and Tay-Sachs disease are included. A trial identified earlier has published its results and has subsequently been listed as excluded in this review. AUTHORS' CONCLUSIONS: As there are no RCTs of preconception genetic risk assessment for thalassaemia, sickle cell disease, cystic fibrosis, or Tay-Sachs disease included in either the earlier or current versions of this review, we recommend considering potential non-RCTs studies (for example prospective cohorts or before-and-after studies) for future reviews. While RCTs are desirable to inform evidence-based practice and robust recommendations, the ethical, legal and social implications associated with using this trial design to evaluate the implementation of preconception genetic risk assessment involving carrier testing and reproductive autonomy must also be considered.  In addition, rather than focusing on single gene-by-gene carrier testing for specific autosomal-recessive conditions as the intervention being evaluated, preconception expanded genetic screening should also be included in future searches as this has received much attention in recent years as a more pragmatic strategy. The research evidence for current international policy recommendations is limited to non-randomised studies.

Tay-Sachs Disease: Two Novel Rare HEXA Mutations from Pakistan and Morocco.

BACKGROUND: Tay-Sachs disease (TSD) is a rare autosomalrecessive genetic disorder characterized by progressive destruction of nerve cells in the brain and spinal cord. It is caused by genetic variations in the HEXA gene leading to a deficiency of ß hexosaminidase A (HEXA) isoenzyme activity. This study aimed to identify causative gene variants in 3 unrelated consanguineous families presented with TSD from Pakistan and Morocco. METHODS: Detailed clinical investigations were carried out on probands in 3 unrelated consanguineous families of Pakistani and Moroccan origin. Targeted gene sequencing and Whole Exome Sequencing (WES) were performed for variant identification. Candidate variants were checked for co-segregation with the phenotype using Sanger sequencing. Public databases including ExAC, GnomAD, dbSNP and the 1,000 Genome Project were searched to determine frequencies of the alleles. Conservation of the missense variants was ensured by aligning orthologous protein sequences from diverse vertebrate species. RESULTS: We report on 3 children presented with Tay-Sachs Disease. The ß hexosaminidaseA enzyme activity was reduced in the Pakistani patient in one of the pedigrees. Genetic testing revealed 2 novel homozygous variants (p.Asp386Alafs*13 and p.Trp266Gly) in the gene HEXA in Pakistani and Moroccan patients respectively.The third family of Pakistani origin revealed a previously reported variant (p.Tyr427Ilefs*5) in HEXA. p.Tyr427Ilefs*5 is the most commonly occurring pathogenic variationin Ashkenazi but was not reported in Pakistani population. CONCLUSION: Our study further expands the ethnic and mutational spectrum of Tay-Sachs disease emphasizing the usefulness of WES as a powerful diagnostic tool where enzymatic activity is not performed for Tay-Sachs disease. The study recommends targeted screening for these mutations (p.Tyr427Ilefs5) for cost effective testing of TSD patients. Further, the study would assist in carrier testing and prenatal diagnosis of the affected families.

Investigating Immune Responses to the scAAV9-HEXM Gene Therapy Treatment in Tay-Sachs Disease and Sandhoff Disease Mouse Models.

GM2 gangliosidosis disorders are a group of neurodegenerative diseases that result from a functional deficiency of the enzyme ß-hexosaminidase A (HexA). HexA consists of an α- and ß-subunit; a deficiency in either subunit results in Tay-Sachs Disease (TSD) or Sandhoff Disease (SD), respectively. Viral vector gene transfer is viewed as a potential method of treating these diseases. A recently constructed isoenzyme to HexA, called HexM, has the ability to effectively catabolize GM2 gangliosides in vivo. Previous gene transfer studies have revealed that the scAAV9-HEXM treatment can improve survival in the murine SD model. However, it is speculated that this treatment could elicit an immune response to the carrier capsid and "non-self"-expressed transgene. This study was designed to assess the immunocompetence of TSD and SD mice, and test the immune response to the scAAV9-HEXM gene transfer. HexM vector-treated mice developed a significant anti-HexM T cell response and antibody response. This study confirms that TSD and SD mouse models are immunocompetent, and that gene transfer expression can create an immune response in these mice. These mouse models could be utilized for investigating methods of mitigating immune responses to gene transfer-expressed "non-self" proteins, and potentially improve treatment efficacy.

A novel gene editing system to treat both Tay-Sachs and Sandhoff diseases.

The GM2-gangliosidoses are neurological diseases causing premature death, thus developing effective treatment protocols is urgent. GM2-gangliosidoses result from deficiency of a lysosomal enzyme ß-hexosaminidase (Hex) and subsequent accumulation of GM2 gangliosides. Genetic changes in HEXA, encoding the Hex α subunit, or HEXB, encoding the Hex ß subunit, causes Tay-Sachs disease and Sandhoff disease, respectively. Previous studies have showed that a modified human Hex µ subunit (HEXM) can treat both Tay-Sachs and Sandhoff diseases by forming a homodimer to degrade GM2 gangliosides. To this end, we applied this HEXM subunit in our PS813 gene editing system to treat neonatal Sandhoff mice. Through AAV delivery of the CRISPR system, a promoterless HEXM cDNA will be integrated into the albumin safe harbor locus, and lysosomal enzyme will be expressed and secreted from edited hepatocytes. 4 months after the i.v. of AAV vectors, plasma MUGS and MUG activities reached up to 144- and 17-fold of wild-type levels (n = 10, p < 0.0001), respectively. More importantly, MUGS and MUG activities in the brain also increased significantly compared with untreated Sandhoff mice (p < 0.001). Further, HPLC-MS/MS analysis showed that GM2 gangliosides in multiple tissues, except the brain, of treated mice were reduced to normal levels. Rotarod analysis showed that coordination and motor memory of treated mice were improved (p < 0.05). Histological analysis of H&E stained tissues showed reduced cellular vacuolation in the brain and liver of treated Sandhoff mice. These results demonstrate the potential of developing a treatment of in vivo genome editing for Tay-Sachs and Sandhoff patients.