Mouse teratocarcinoma mutant clones deficient in adenine phosphoribosyltransferase and developmentally pluripotent

Mouse teratocarcinoma stem cells deficient in activity of adenine phosphoribosyltransferase (APRT; EC 2.4.2.7) were obtained in order to have this marker in developmentally versatile cells. Mutagenized stem-cell cultures were selected for resistance to 8-azaadenine and four clonal cell lines were isolated. Three had severe deficiencies of APRT activity (7% or less of wild type) and one had a moderate reduction (73%). The enzyme in the latter clone was found to be an electrophoretic variant with slightly less anodal migration than the wild-type enzyme. Each clone remained stably APRT-deficient for at least 3% weeks, after subcutaneous inoculation, in the absence of the selective agent. The tumors formed from the inocula comprised a variety of differentiated tissues and thus showed persistence of stem-cell developmental pluripotency despite mutagenesis and selection. All mutants also retained the quasinormal karyotype (X/0 sex chromosomal constitution, trisomy-19) of the parent line. These lines are appropriate for such uses as production (by blastocyst injection) of mouse models of the human genetic deficiency and for foreign-gene transfer, via teratocarcinoma cells, into mice.     

Asymptomatic and late-onset ornithine transcarbamylase (OTC) deficiency in males of a five-generation family, caused by an A208T mutation

In a large five-generation Polish family, late-onset ornithine transcarbamylase (OTC) deficiency in males segregated with the missense mutation Ala208Thr (A208T), and all heterozygous females were asymptomatic. No other mutations were found in the coding sequences and intron-exon boundaries of the OTC gene. Surprisingly, the mutation originated from the great-grandfather of the index patient who died at age 59 of liver carcinoma. He never had dietary restrictions or hyperammonemic spells throughout life and appears to be the oldest male reported with OTC deficiency. The index patient had a severe OTC deficiency (3% of normal). Eight males died suddenly at ages 4 months to 23 years (average 14 years) after a foudroyant episode triggered by a common infection. The patients remained undiagnosed for 28 years because a metabolic defect was not considered to be the cause of the acute episodes. Recognition of the familial pattern of inheritance was initially unnoticed since the patients were admitted to eight different hospitals. DNA analysis predicted that two 'healthy' boys also had OTC deficiency, which was confirmed by abnormal results of allopurinol challenge tests. Initial suspicion of OTC deficiency in such families is complicated, since symptoms can develop at any age, or even remain absent. This obscures the typical pattern of X-linked inheritance in small families.     

Generalized glycogen storage and cardiomegaly in a knockout mouse model of Pompe disease

Glycogen storage disease type II (GSDII; Pompe disease), caused by inherited deficiency of acid alpha-glucosidase, is a lysosomal disorder affecting heart and skeletal muscles. A mouse model of this disease was obtained by targeted disruption of the murine acid alpha-glucosidase gene (Gaa) in embryonic stem cells. Homozygous knockout mice (Gaa -/-) lack Gaa mRNA and have a virtually complete acid alpha-glucosidase deficiency. Glycogen-containing lysosomes are detected soon after birth in liver, heart and skeletal muscle cells. By 13 weeks of age, large focal deposits of glycogen have formed. Vacuolar spaces stain positive for acid phosphatase as a sign of lysosomal pathology. Both male and female knockout mice are fertile and can be intercrossed to produce progeny. The first born knockout mice are at present 9 months old. Overt clinical symptoms are still absent, but the heart is typically enlarged and the electrocardiogram is abnormal. The mouse model will help greatly to understand the pathogenic mechanism of GSDII and is a valuable instrument to explore the efficacy of different therapeutic interventions.      

Atypical expression of ß-galactosidase deficiency in a child with Hurler-like features but without neurological abnormalities

A 28-month-old child was found to have several clinical features of lysosomal storage diseases, including: coarse facies, hepatosplenomegaly, lumbar kyphosis due to hypoplastic beaked L1 and L2 vertebral bodies, vacuolated lymphocytes in blood smears and rare foamy hystiocytes in bone marrow. However, no signs of neurological or ocular abnormalities were detected. A beta-galactosidase deficiency was demonstrated in leukocytes and cultured skin fibroblasts, with a residual activity toward 4-methylumbelliferyl-beta-galactopyranoside ranging between 5 and 15% of the normal mean. Normal activities were found for several other lysosomal acid hydrolases. beta-Galactosidase activities in leukocytes and cultured skin fibroblasts from both parents were within the normal ranges. The patient seems to represent an atypical expression of acid beta-galactosidase deficiency, since his clinical picture does not exaclty correspond to that of either the two classical types of GM1-gangliosidosis or other atypical patients reported in the literature havining beta-galactosidase deficiency.     

Combined sialidase (neuraminidase) and β-galactosidase deficiency. Clinical, morphological and enzymological observations in a patient

A patient with combined deficiency of sialidase and beta-galactosidase is described. This now 39-year-old man, who is of Japanese origin, showed gradually progressive clinical features from the age of six years. Many of these features are commonly found in sialidosis type 2 or in GM1-gangliosidosis. Both sialidase and beta-galactosidase activities were deficient in leucocytes and cultured fibroblasts. Leucocytes of his mother showed activities of both enzymes in the lower limit of the control range. Morphologically, the pattern of storage products in a skin biopsy resembled in many respects that seen in GM1-gangliosidosis. Moreover, storage products which could be typical of sialidosis were also observed. Since the patient showed angiokeratomata, the morphological findings were compared with those specific to Fabry's disease, but no similarities were found. An enzymological diagnosis of the disease is most reliable on cultured fibroblasts, discriminating it from sialidosis type 2 and GM1-gangliosidosis. In view of recent findings, leucocytes seem to be less suitable for the establishment of the diagnosis galactosialidosis.     

Improved assay for differential diagnosis between Pompe disease and acid α-glucosidase pseudodeficiency on dried blood spots

The high frequency (3.3-3.9%) of acid α-glucosidase pseudodeficiency, c.[1726G>A; 2065G>A] homozygote (AA homozygote), in Asian populations complicates newborn screening for Pompe disease (glycogen storage disease type II or acid maltase deficiency) on dried blood spots, since AA homozygotes have a considerably low enzyme activity. We observed that hemoglobin in the enzyme reaction solution strongly interferes with the fluorescence of 4-methylumbelliferone released from 4-methylumbelliferyl α-D-glucopyranoside (4MU-αGlc) by acid α-glucosidase. Therefore, we have searched for a method to effectively eliminate hemoglobin in the reaction solution. Hemoglobin precipitation with barium hydroxide and zinc sulfate (Ba/Zn method) carried out after the enzyme reaction considerably enhances the fluorescence intensity while it does not reduce the intensity to any extent as can occur with conventional deproteinization agents like trichloroacetic acid. The Ba/Zn method greatly improved the separation between 18 Japanese patients with Pompe disease and 70 unaffected AA homozygotes in a population of Japanese newborns in the assay with 4MU-αGlc on dried blood spots. No overlap was observed between both groups. We further examined acid α-glucosidase activity in fibroblasts from 11 Japanese patients and 57 Japanese unaffected individuals including 31 c.[1726G; 2065G] homozygotes, 18 c.[1726G; 2065G]/[1726A; 2065A] heterozygotes and 8 AA homozygotes to confirm that fibroblasts can be used for definitive diagnosis. The patients were reliably distinguished from three control groups. These data provide advanced information for the development of a simple and reliable newborn screening program with dried blood spots for Pompe disease in Asian populations.     

A case of adult Pompe disease presenting with severe fatigue and selective involvement of type 1 muscle fibers

We present a case of adult Pompe disease (acid maltase deficiency) with an uncommon clinical presentation characterized by severe fatigue and myalgia prior to the onset of limb girdle weakness. Remarkably, the muscle biopsy demonstrated selective involvement of type 1 muscle fibers. The cause and clinical effects of fiber type specific involvement are currently unknown, but the phenomenon might contribute to the clinical heterogeneity in Pompe disease and the variable response to enzyme replacement therapy.     

Fatigue: an important feature of late-onset Pompe disease

Abstract Objective To investigate the prevalence and severity of fatigue in adult patients with Pompe disease. Methods The Fatigue Severity Scale (FSS) was assessed in an international population of 225 adults with Pompe disease, a metabolic disorder presenting as a slowly progressive proximal myopathy. The FSS scores were compared to those of healthy controls and the relationship between the level of fatigue and other patient characteristics was investigated. Results The mean age of the participants was 47 (SD 13) years and the mean disease duration 11 (SD 8) years. 43% used a wheelchair and 46% had respiratory support, 29% needed both. 67% of the participants had a FSS score ≥5, indicating severe fatigue. The mean FSS score was 5.2 (SD 1.5), which was significantly higher than that of healthy controls (p < 0.001). Fatigue was not related to age, sex or disease duration. Patients who used a wheelchair or respiratory support were on average more fatigued than those who did not (p = 0.01). However, of the patients who did not use these aids, 59% also had a FSS score ≥5. FSS scores were highest among patients who reported a high frequency of sleep disorders, but patients who never experienced sleep difficulties were also fatigued (mean FSS score = 4.8). Conclusion Fatigue is highly prevalent among both mildly and severely affected adult patients with Pompe disease. The FSS appears a useful tool in assessing fatigue in Pompe disease.