Elevation of glycosaminoglycans in the amniotic fluid of a fetus with mucopolysaccharidosis VII.

OBJECTIVE: The aim of this study was to quantify glycosaminoglycans (GAGs) in amniotic fluid (AF) from an MPS VII fetus compared with age-matched fetuses obtained from normal pregnancies. METHOD: Disaccharides were measured by liquid chromatography tandem mass spectrometry, compared to age-matched controls. Enzyme assay was performed in AF supernatant or cultured amniocytes. GUSB was analyzed by next generation sequencing using Ion Torrent Personal Genome Machine with a customized panel. RESULTS: No activity of ß-glucuronidase was detected in fetal cells. The pregnancy was spontaneously terminated in the third trimester. Genetic studies identified a homozygous mutation of p.N379D (c.1135A > G) in the GUSB gene. Liquid chromatography tandem mass spectrometry showed that chondroitin sulfate, dermatan sulfate, heparan sulfate, and keratan sulfate levels were markedly increased in the MPS VII AF, compared to those in age-matched control AF (dermatan sulfate, heparan sulfate, and chondroitin-6-sulfate more than 10 × than age-matched controls; chondroitin-4-sulfate and keratan sulfate more than 3 times higher). CONCLUSION: This is the first report of specific GAG analysis in AF from an MPS VII fetus, indicating that GAG elevation in AF occurs by 21 weeks of gestation and could be an additional tool for prenatal diagnosis of MPS VII and potentially other MPS types. © 2017 John Wiley & Sons, Ltd.

[Prenatal diagnosis by amniocentesis. Clinical and cytogenetic experience in 1,500 cases]. / Diagnóstico prenatal por amniocentesis. Experiencia clínica y citogenética en 1,500 casos.

BACKGROUND: Genetic amniocentesis is performed in México 25 years ago but only few works have been published. OBJECTIVE: To analyze clinical and cytogenetic findings in consecutive patients submitted to genetic amniocentesis. MATERIAL AND METHOD: An analysis was made of the clinical features, amniocentesis results and pregnancy outcome in 1500 consecutive cases of genetic amniocentesis. RESULTS: Sixty-eight fetuses with chromosomopathy (4.5%) were detected and two, with an inborn error of metabolism. The most frequent abnormalities were trisomy 21 (32 cases), trisomy 18 (10 cases), trisomy 13(6 cases), 45,X (6 cases), 47,XXY (4 cases). Pregnancy outcome is known in 474 patients (32%). There were five fetal losses (1%). Of the 68 cases with chromosomopathy, the outcome is known in 45, of which, 29 (64%) decided to have an abortion while 16 (35%) continued the pregnancy, six had a spontaneous abortion or perinatal death and ten had an alive new born. Among fetuses with normal or balanced karyotype and normal ultrasound, 11 out of 419 (2.6%) had congenital anomalies. Two of them had a condition known to be related with epigenetic regulation, (Russell Silver and Angelman syndrome). CONCLUSIONS: Amniocentesis is a reliable and low risk method. Cytogenetic findings in this series are similar to those reported in the literature. Most patients with fetal disease decided to have an abortion. The finding of two patients with a condition related with abnormal epigenetic regulation suggests that the magnitude of this risk remains to be defined.

Widespread gene transduction to the central nervous system by adenovirus in utero: implication for prenatal gene therapy to brain involvement of lysosomal storage disease.

BACKGROUND: In some lysosomal storage diseases, considerable alterations of the central nervous system (CNS) occur prior to birth and neurodegeneration progresses rapidly soon after birth causing early death in patients. No effective treatment is available after birth. Treatment may need to be initiated before birth to prevent the onset or progression of neurological changes and thereby irreversible brain damage. The aim of this study is to investigate the feasibility and effectiveness of brain-directed prenatal gene therapy for lysosomal storage diseases. METHODS: Recombinant adenovirus encoding the lacZ gene was injected into the lateral ventricles of mouse embryos and the pattern of gene transduction to the CNS was investigated. In the therapeutic experiment, adenovirus expressing beta-glucuronidase was injected into the cerebral ventricles of the embryos of mucopolysaccharidosis VII mice and the therapeutic effects on the brain were evaluated. RESULTS: Injection of adenoviral vectors to the cerebral ventricles of mouse embryos led to widespread gene transduction throughout the brain and the spinal cord and transgene expression persisted over 10 months in those surviving the procedure. The prenatal transduction of the therapeutic gene to the brain of the mucopolysaccharidosis VII mouse efficiently prevented lysosomal storage in most brain cells before birth until 4 months after birth. CONCLUSIONS: Brain-directed in utero gene therapy through an intra-ventricular route would be an effective strategy to treat some lysosomal storage diseases with early and severe CNS alterations.

Therapeutic neonatal hepatic gene therapy in mucopolysaccharidosis VII dogs.

Dogs with mucopolysaccharidosis VII (MPS VII) were injected intravenously at 2-3 days of age with a retroviral vector (RV) expressing canine beta-glucuronidase (cGUSB). Five animals received RV alone, and two dogs received hepatocyte growth factor (HGF) before RV in an attempt to increase transduction efficiency. Transduced hepatocytes expanded clonally during normal liver growth and secreted enzyme with mannose 6-phosphate. Serum GUSB activity was stable for up to 14 months at normal levels for the RV-treated dogs, and for 17 months at 67-fold normal for the HGF/RV-treated dog. GUSB activity in other organs was 1.5-60% of normal at 6 months for two RV-treated dogs, which was likely because of uptake of enzyme from blood by the mannose 6-phosphate receptor. The body weights of untreated MPS VII dogs are 50% of normal at 6 months. MPS VII dogs cannot walk or stand after 6 months, and progressively develop eye and heart disease. RV- and HGF/RV-treated MPS VII dogs achieved 87% and 84% of normal body weight, respectively. Treated animals could run at all times of evaluation for 6-17 months because of improvements in bone and joint abnormalities, and had little or no corneal clouding and no mitral valve thickening. Despite higher GUSB expression, the clinical improvements in the HGF/RV-treated dog were similar to those in the RV-treated animals. This is the first successful application of gene therapy in preventing the clinical manifestations of a lysosomal storage disease in a large animal.

In utero fetal liver cell transplantation without toxic irradiation alleviates lysosomal storage in mice with mucopolysaccharidosis type VII.

Lysosomal storage diseases, such as Mucopolysaccharidosis type VII (MPS VII), cause progressive loss of mobility and intellect and result in early death. Treatment of progressive diseases must occur before the blood-brain barrier closes. In MPS VII mice, normal donor hematopoietic cells secrete the missing enzyme beta-glucuronidase (GUSB) that reverses disease manifestations. Correction of lysosomal storage is limited to the visceral organs unless transplantation is preceded by high-dose irradiation. We hypothesize that irradiation opens the blood-brain barrier allowing passage of corrective cells. Here we transplanted genetically myeloablated MPS VII fetuses to determine whether earlier treatment without toxic irradiation is systemically corrective. Cells with a selective advantage in utero were identified. Donor fetal liver cells (FLC), a substitute for difficult to obtain murine cord blood cells, were increased 10-fold in the host peripheral blood over equivalent numbers of adult marrow cells injected simultaneously and were stable long term in both primary and secondary hosts. GUSB- MPS VII fetuses injected with GUSB+ FLC were assessed longitudinally after birth. Donor FLC replaced host stem cell descendants, prolonged life dramatically, and reduced bone dysplasia and lysosomal storage in all tissues long term. GUSB, donor leptomeningeal cells, and microglia were present in the brain at 11 months postinjection. Lysosomal storage in cortical neurons and glia, although not completely corrected, was reduced. We conclude that in utero intervention without toxic pretreatment in this model reduces the storage disease long term and improves the length and quality of life despite exerting only minor effects on the brain.