Storage Disease Type Research Articles

Recapture Lysosomal Enzyme Deficiency via Targeted Gene Disruption in the Human Near-Haploid Cell Line HAP1.

Background: Advancement in genome engineering enables rapid and targeted disruption of any coding sequences to study gene functions or establish human disease models. We explored whether this approach can be used to study Gaucher disease, one of the most common types of lysosomal storage diseases (LSDs) in a near-haploid human cell line (HAP1). Results: CRISPR-Cas9 targeting to coding sequences of β-glucocerebrosidase (GBA), the causative gene of Gaucher disease, resulted in an insertional mutation and premature termination of GBA. We confirmed the GBA knockout at both the gene and enzyme levels by genotyping and GBA enzymatic assay. Characterization of the knockout line showed no significant changes in cell morphology and growth. Lysosomal staining revealed more granular lysosomes in the cytosol of the GBA-knockout line compared to its parental control. Flow cytometry analysis further confirmed that more lysosomes accumulated in the cytosol of the knockout line, recapturing the disease phenotype. Finally, we showed that this knockout cell line could be used to evaluate a replacement therapy by recombinant human GBA. Conclusions: Targeted gene disruption in human HAP1 cells enables rapid establishment of the Gaucher model to capture the key pathology and to test replacement therapy. We expect that this streamlined method can be used to generate human disease models of other LSDs, most of which are still lacking both appropriate human disease models and specific treatments to date.

ВПЛИВ ОСНОВНИХ ФАКТОРІВ РИЗИКУ НА ПЕРЕБІГ ІНФАРКТУ МІОКАРДА У ХВОРИХ ІЗ НАДМІРНОЮ МАСОЮ ТІЛА

Основними факторами ризику інфаркту міокарда, що сприяють виникненню та погіршують перебіг захворювання, є артеріальна гіпертензія, ожиріння, куріння, малорухливий спосіб життя, порушення ліпідного обміну та цукровий діабет 2 типу, особливо при їх поєднанні.Мета дослідження – дослідити особливості поширення основних факторів ризику серцево-судинної патології, їх вплив на перебіг та частоту розвитку ускладнень інфаркту міокарда у хворих із надлишковою масою тіла жителів Тернопільської області.Матеріали та методи. Обстежено 76 хворих на інфаркт міокарда віком від 39 до 85 років, яких методом конвертів поділили на 3 дослідні групи: перша група – 33 пацієнтів із інфарктом міокарда з надмірною масою тіла, друга група – 13 пацієнтів з інфарктом міокарда та з ожирінням І ст., третя – 10 хворих на інфаркт міокарда та з ожирінням ІІ–ІІІ ст. Контрольну групу склали 20 пацієнтів із нормальною масою тіла.Результати досліджень та їх обговорення. Встановлено, що найбільш поширеними факторами ризику серед хворих виявились артеріальна гіпертензія (у 97 % пацієнтів із надмірною масою тіла та у 100 % хворих з ожирінням) та цукровий діабет 2 типу (у 24 % хворих із надмірною масою тіла та у 60 % пацієнтів з ожирінням). Одночасно у хворих на інфаркт міокарда з надмірною масою тіла частіше, ніж у пацієнтів із нормальною масою тіла, зустрічались підвищення найбільш атерогенного класу – холестерину ліпопротеїдів низької щільності та зниження антиатерогенного класу холестерину ліпопротеїдів високої щільності. У пацієнтів з ожирінням ІІ–ІІІ ст. достовірно частіше спостерігалась гіпертригліцеридемія (р=0,003).Висновки. Аналіз катамнезу засвідчив, що найчастішими ускладненнями у хворих на інфаркт міокарда з надлишковою масою тіла виявились серцева астма, набряк легень, гостра аневризма лівого шлуночка та епістенокардитичний перикардит.

The investigation and management of metabolic myopathies

Metabolic myopathies (MM) are rare inherited primary muscle disorders that are mainly due to abnormalities of muscle energy metabolism resulting in skeletal muscle dysfunction. These diseases include disorders of fatty...

A first description of chronic arthritis and uveitis in an adolescent girl with glycogen storage disease

Purpose Glycogen storage disease (GSD) type 1a is an autosomal recessive inborn error of carbohydrate metabolism which affects glycogenolysis and gluconeogenesis. Severe fasting hypoglycemia and secondary biochemical abnormalities such as hyperuricemia occur. Molecular analysis differentiates GSD type 1 into different clinical phenotypes, of which Type 1b but not Type 1a, is associated with inflammatory bowel disease (IBD). While gout has been described in GSD, chronic arthritis and uveitis have not.

When Clinicians and a Parent Disagree on the Extent of Medical Care

TG is a 32-month-old girl with a rare lysosomal storage disease. The diagnosis was confirmed at 8 months of age; she underwent bone marrow transplant at 14 months. TG's father remained at home with her siblings whereas her mother lived at the transplant hospital with TG for 1 year. Significant respiratory infections led to dependency on a tracheotomy and ventilator. She was transferred to the current hospital 10 months ago for respiratory rehabilitation, which has been complicated by pulmonary hemorrhages. On examination, TG was in a wheelchair with truncal support and leg splints. She is a small child with coarse facial features, generalized hypotonia, and significant joint restriction. She exhibited intermittent repetitive arm movements. A tracheotomy was in place, and she did not vocalize. Variable levels of alertness were observed although she rarely signaled to others for social engagement or to express her needs. Eye contact was limited and she responded inconsistently to her name. TG's mother states that her daughter interacts well with her and uses a number of signs for communication. The rehabilitation staff reported limited progress and recommends withdrawal of services. TG's mother expressed frustration with her daughter's poor developmental improvement and believes she needs more intensive therapy, not less. Staff members are now frustrated with TG's mother's level of involvement with her daughter's care. Although TG's mother spends full weekdays at her daughter's bedside, she is often working on her computer managing her business. TG's father works full time and visits on the weekends, when her mother returns home. TG's mother has expressed frustration with her daughter's level of improvement; she believes TG needs more intensive therapy that the hospital is not providing. She informed the staff that other children with this type of storage disease may have delays, but are often higher functioning. TG's mother made the point to the staff that the family's reason for pursuing a bone marrow transplant was to attempt to correct the enzyme deficiency and improve her daughter's outcome.

When Clinicians and a Parent Disagree on the Extent of Medical Care

TG is a 32-month-old girl with a rare lysosomal storage disease. The diagnosis was confirmed at 8 months of age; she underwent bone marrow transplant at 14 months. TG's father remained at home with her siblings whereas her mother lived at the transplant hospital with TG for 1 year. Significant respiratory infections led to dependency on a tracheotomy and ventilator. She was transferred to the current hospital 10 months ago for respiratory rehabilitation, which has been complicated by pulmonary hemorrhages. On examination, TG was in a wheelchair with truncal support and leg splints. She is a small child with coarse facial features, generalized hypotonia, and significant joint restriction. She exhibited intermittent repetitive arm movements. A tracheotomy was in place, and she did not vocalize. Variable levels of alertness were observed although she rarely signaled to others for social engagement or to express her needs. Eye contact was limited and she responded inconsistently to her name. TG's mother states that her daughter interacts well with her and uses a number of signs for communication. The rehabilitation staff reported limited progress and recommends withdrawal of services. TG's mother expressed frustration with her daughter's poor developmental improvement and believes she needs more intensive therapy, not less. Staff members are now frustrated with TG's mother's level of involvement with her daughter's care. Although TG's mother spends full weekdays at her daughter's bedside, she is often working on her computer managing her business. TG's father works full time and visits on the weekends, when her mother returns home. TG's mother has expressed frustration with her daughter's level of improvement; she believes TG needs more intensive therapy that the hospital is not providing. She informed the staff that other children with this type of storage disease may have delays, but are often higher functioning. TG's mother made the point to the staff that the family's reason for pursuing a bone marrow transplant was to attempt to correct the enzyme deficiency and improve her daughter's outcome.

A Case of Suspected Lysosomal Storage Disease in a Neonatal Japanese Black Calf

A 5-day-old Japanese black calf was necropsied and intracytoplasmic vacuolations were histologically observed in many tissues. In the central nervous system, intracytoplasmic inclusions and vacuoles were found in neuronal cells. Intracytoplasmic inclusions were more conspicuous in the nuclei containing large nerve cells, especially in the brain stem and spinal cord. These inclusions were stained weak positive to positive with alcian blue, Giemsa, Luxol fast blue and periodic acid-Schiff stains but not with oil red O. Ultrastructurally, neuronal inclusions were observed in lysosomes and consisted of an amorphous electron-dense substance and occasional membranous structures. These findings seem to differ from the cases of bovine lysosomal diseases that have been reported, and this case may be another type of lysosomal storage disease.

Dietary management of inborn errors of metabolism.

Inborn errors of metabolism are individually rare but are an important cause of mortality and morbidity in infants and children. Dietary therapy is the mainstay of treatment in phenylketonuria, maple syrup urine disease, homocystinuria, galactosemia and glycogen storage disease (Type I/III). Some disorders like urea cycle disorders and organic acidurias require dietary modification in addition to other modalities. Certain basic principles of dietary management should be clearly understood for proper management of these disorders. Commercially available diets are very expensive and modification in routine Indian diet may be tried based on content of different nutrients but the desirable fine control is not achieved.

The mannosidoses and ceroid-lipofuscinoses: Experimental studies on two types of storage disease

alpha-Mannosidosis of Angus calves was studied both for its veterinary importance and as a model of analogous human lysosomal storage diseases. This study facilitated a similar study in Australia on Swainsona spp. intoxication of livestock in which the toxic principle was shown to be an indolizidine alkaloid, Swainsonine. These genetic and acquired alpha-mannosidoses are compared with beta-mannosidosis. Collectively the study has helped the understanding of the processes of glycosylation and catabolism of glycoproteins. An experiment of nature involving an alpha-mannosidosis chimeric calf born co-twin to a normal calf helped to define the expectations and limitations of bone marrow transplants in this type of storage disease in humans. The inherited ceroid-lipofuscinoses (Batten disease) were studied in an ovine model. Isolation and analyses of the fluorescent accumulated lipopigment denied the dogma of lipid peroxidation current in the 1970s and 1980s. It was shown that in this, and analogous diseases in humans, the dominantly accumulated species was the very hydrophobic protein, subunit c of mitochondrial ATP synthase. Contrary to the adage that this should reflect a disorder of lysosomal proteolysis, there is accumulating evidence that the primary defect resides in mitochondria. Because of its hydrophobic nature, subunit c forms paracrystaline complexes which appear resistant to proteolysis within the lysosomal apparatus.

First Trimester Prenatal Diagnosis of Glycogen Storage Disease Type III

Glycogen storage disease (GSD) type III (McKusick 23240) is an autosomal recessive disorder due to a deficiency of glycogen debranching enzyme (Illingworth et al., 1956). This enzyme presents a transferase activity: oligo a-1,4-glucan-a-1,4 glucan-4-glycosyltransferase (EC 2.4.1.25) and a hydrolytic activity: amylo-1,6-glucosidase (EC 3.2.1.33); both activities have been located on the same macromolecule. Debranching enzyme deficiencies have been reported in muscle, liver, leukocytes, erythrocytes and fibroblasts. Generally, the course of the disease seems rather mild, in spite of some difficulties in long-term outcome (De Parscau et al., 1988); but, in some cases, an unusually severe course in a previous child leads the parents to request a prenatal diagnosis for a further pregnancy. We have previously reported the possibility of prenatal diagnosis of GSD type III using cultured amniotic fluid (AF) cells (Maire and Mathieu, 1986) and the feasibility of the diagnosis using chorionic villi (CV) has been suggested by Van Diggelen et al. (1985). We report here the first trimester prenatal diagnosis of an affected case.

Finnish type of sialic acid storage disease with sialuria (Salla disease): the occurrence and diagnostic significance of cytoplasmic vacuoles in blood lymphocytes.

In this study peripheral blood smears from 29 patients (17 males and 12 females; mean age 28 years, range 3-65 years) with a confirmed diagnosis of the Finnish type of sialic acid storage disease (FSASD) and 200 controls with mental retardation without any evidence of metabolic disease were examined for the presence of vacuolated lymphocytes. Urine samples were analysed by thin-layer chromatography for free sialic acid. Only 62% of the patients with FSASD had a clearly increased percentage of vacuolated lymphocytes (greater than normal mean + 2 s.d.). In thin-layer chromatography all the FSASD patients gave a positive test result. No false positive or negative results were obtained. Electronmicroscopical examination of peripheral blood lymphocytes demonstrated only non-specific changes in a few cells. Examination of peripheral lymphocytes for vacuoles is not a reliable screening test for FSASD. The screening method of choice is the analysis of free sialic acid by thin-layer chromatography.

A case of fructose-1-6-diphosphatase deficiency

We describe the case of a 5-yr-old boy first hospitalized at the age of 14 mth with hypoglycemia, ketonuria, severe metabolic acidosis and hepatomegaly. The provisional diagnosis of Reye's syndrome or glycogen storage disease (G.S.D.) Type I was made. In the following 12 mth he was readmitted on many occasions with mild intercurrent infections precipitating a metabolic crisis. He always responded well to glucose and bicarbonate. Open liver biopsy was performed. Histology showed moderate fatty changes, abundant glycogen but no cytoplasmic changes typical of G.S.D. Type I. Chemical pathology results on the specimen were: glycogen 4.6 g/100 g wet tissue (normal 5-7), glucose-6-phosphatase 6.4 U/g wet tissue (normal 4–13). In the non-fasting state the glucagon stimulation test showed a rise in blood glucose from 4.7 to 8.3 mmol/l in 30 minutes; a fasting glucagon stimulation test produced a drop in blood glucose from 1.4 to 0.8 mmol/l, with an increase in blood lactate from 7.1 mmol/l to 7.7 mmol/l. Subsequently we were able to detect no fructose-1-6-diphosphatase activity in the liver biopsy. A 2-deoxyglucose infusion test resulted in a fall in plasma glucose from 4.4 to 1.7 mmol/l and a lactate increase from 4.0 to 11.0 mmol/l. An oral fructose tolerance test caused a fall in plasma glucose from 2.7 mmol/l to 0.3 mmol/l and a lactate increase from 4.7 mmol/l to 9.2 mmol/l. All results obtained are consistent with a diagnosis of fructose-1-6-diphosphatase deficiency.

Mucopolysaccharidosis type 3 (Sanfilippo's syndrome).

In a clinically diagnosed case of Sanfilippo's syndrome (Mucopolysaccharidosis type III) histopathological examination of the eyes revealed accumulation of a granular material mainly in the sclera and to a lesser degree in the cornea and ciliary body. Similar scleral changes have previously been reported in various types of mucopolysaccharidosis in six cases only, and ciliary body changes in two cases only. It is emphasized that ocular involvement including corneal changes may be found histopathologically even if not clinically detected. The main histopathological findings in the cerebrum and cerebellum were ballooning of ganglion cells similar to the findings in other types of storage disease. By applying a battery of histochemical methods to the ocular and cerebral material, the findings indicated 1) a difference between the stored material in the brain and in the eyes, 2) that the stored material in the ganglion cells of the brain was probably of a ganglioside nature and 3) that the material in the eyes was consistent with an acid mucopolysaccharide, undoubtedly sul‐phated and probably rich in heparitin sulphate. These findings are compared with the suggested biochemical changes and the correlation between these and the morphology is discussed. The writer is in favour of the theory of a basic enzyme defect leading to accumulation of the granular material in the cells. Addition of normal and pathological cytoplasmic constituents are probably responsible for the difference in histochemical findings, especially in fibrocytes and ganglion cells, as revealed by the present study. The eyes have not previously been histopathologically or histochemically examined in Sanfilippo's syndrome.