Shirley Soukup

Rapid Prenatal Diagnosis of Glycogen-Storage Disease Type II by Electron Microscopy of Uncultured Amniotic-Fluid Cells

Glycogen-storage disease Type IIa is a fatal, genetically determined disease of infancy or early childhood that is characterized by deficient activity of acid alpha-glucosidase and by the presence of intracellular vacuoles full of glycogen, which are found in most tissues, including skin and liver. On electron microscopy these specific vacuoles are tightly packed accumulations of glycogen particles surrounded by a single membrane. We did electron-microscopical examinations on uncultured amniotic-fluid cells from 26 women whose fetuses were at risk for glycogen-storage disease Type IIa and from 8 normal control pregnant women. We found specific vacuoles in cells from 6 of the 26 high-risk patients. At delivery, glycogen-storage disease Type IIa was present in the infants of these 6 women and absent in those of the other 20 according to results of clinical, biochemical, and electron-microscopical studies of gestational products. After amniocentesis at 15 to 18 weeks of gestation, the prenatal diagnosis made by electron microscopy of uncultured amniotic-fluid cells was available in three to six days, whereas it took from three to six weeks to make the diagnosis by enzymatic analysis of the cultured amniotic-fluid cells. We conclude that the electron-microscopical prenatal diagnosis of glycogen-storage disease Type IIa is rapid, safe, and reliable. It should facilitate earlier diagnosis and thereby help to preserve parental options.

Biologic characteristics of four Ewing's sarcomas

Four Ewings sarcomas were examined for chromosomal characteristics, growth in cell culture, tumorigenicity in nude mice, and presence of beta-adrenergic receptors. Three tumors were from untreated patients (one obtained directly from the patient and two after growth in nude mice) and one was a metastatic lesion obtained after treatment. All four tumors were diploid or near-diploid, with one or more structural rearrangements. In the metastatic lesion, 21 abnormalities were seen. No specific chromosome aberration was found to be common to all four tumors, although a t(1,16) was observed in two and a t(11;22) in two. Abnormalities involving chromosomes #1, #3, #11, #13, #16, and #22 were each found in two of four tumors. All four tumors were tumorigenic in nude mice; two grew well in cell culture, one of which became an established line, and all four expressed high concentrations of beta-adrenergic receptors.

Ultrastructure and Enzymatic Deficiency of Fibroblast Cultures in Type II Glycogenosis

Extract: Fibroblast cultures from skin biopsy specimens of a girl with type II glycogenosis showed less than 5% of control activity of lysosomal acid α-glucosidase. These cells showed numerous intracellular vacuoles that resembled the abnormal lysosomes found in liver and other tissues of the patient. Their characteristic ultrastructural abnormality was seen in fibroblast and epithelial cells of the primary cultures and thereafter in all the subcultures of which to date eight passages have been examined. Control fibroblast cultures did not contain such inclusions.Speculation: Demonstration of the disease-specific ultrastructural abnormality indicates the usefulness of fibroblast cultures to study pathophysiology and potential treatment in type II glycogenosis. In addition, fibroblast cultures of other lysosomal diseases may serve a similar purpose.

Chromosome analyses in a rhabdoid tumor of the brain.

A malignant rhabdoid tumor of the brain from a 19-month-old child was studied. Two related clones, 46,XX,-8,+der(8)t(8;22)(p11;q?12)x2,-22,del(22)(q12q?13) and 46,XX-8,+der(8)t(8;22) (p11;q?12) x2,-22,r(22) were found after chromosome analyses of primary and recurrent tumor, and multiple nude mouse passages of the tumor. Breakpoints were studied using FISH.

Further observations of ocular pathology in trisomy 9.

The ocular pathology in a new patient with mosaic trisomy 9 comprised major anomalies and contrasted sharply with the findings in a previous case reported by us. The ocular changes in this case were, in essence, indistinguishable from those encountered in the most severe form of trisomy 13. Similarities to trisomy 18 and 21 were further evidence of the overlap of ocular findings in autosomal trisomies. There is increasing evidence that most, if not all, chromosomes have some role in regulating ocular embryogenesis.

COMPARISON OF CHROMOSOME ANALYSIS TO DNA CONTENT BY FLOW CYTOMETRY FOR PEDIATRIC TUMORS

Chromosome analyses in a series of 50 pediatric tumor samples showed abnormal chromosome number, ranging from hypodiploid to tetraploid. Aliquots of the same solid tumor samples were analyzed by flow cytometry (FCM). Material from spontaneous abortions and 12 tumor samples with normal chromosomes were also compared in a control series of 83 samples. Tumors or abortuses with 44-48 chromosomes could not be differentiated from material with normal diploid complement by FCM. However, greater than or equal to 3-4 extra chromosomes produced detectable differences in mean DNA index. Triploidy and tetraploidy were readily identified by FCM. It is concluded that FCM could identify an important group of hyperdiploid pediatric tumors, as well as 3N and 4N tumor complements.

Electron microscopy of uncultured amniotic fluid cells: In utero diagnosis of type II glycogenosis

Electron microscopy (EM) of uncultured amniotic fluid cells has been performed on 14 specimens. Of these, 10 specimens served as “normal controls” while the other 4 were from women who had previous babies with type II glycogenosis (type II GSD). EM of “normal controls” indicated the presence of two cell types: (1) frequent squamous epithelial cells with varying amounts of cytoplasmic glycogen but without membrane bounded accumulations of glycogen (i.e., without lysosomal glycogen); and (2) rare ciliated cells that may derive from the fetal trachea. Of the 4 high risk pregnancies, two had amniotic fluid cells indistinguishable from “normal controls” and produced clinically and biochemically healthy children. Amniotic fluid cells of the remaining two pregnancies contained glycogen accumulations surrounded by membrances (lysosomal glycogen) that are the hallmark of type II GSD. Upon termination of one of these pregnancies at 21 weeks of gestation, the fetus had type II GSD by biochemical and EM criteria. The other pregnancy resulted in the delivery at term of a boy who apparently was healthy clinically but who at birth had the abnormal lysosomes in skin, liver and muscle and who died of type II GSD at age 4 months. Eight obligatory heterozygotes for type II GSD did not have abnormal lysosomes in hepatic biopsy specimens. We conclude that direct EM of uncultured amniotic fluid cells may help with the in utero diagnosis of type II GSD. A major advantage of this method is diagnosis within three days of amniocentesis.

148 THREE FORMS OF CARNITINE PALMITOYL TRANSFERASE (CPT) II DEFICIENCY: (1) FATAL AT BIRTH OR (2) AT AGE 20 YEARS WITH CARDIAC FAILURE, OR (3) LIFE-COMPATIBLE WITH MODERATE MUSCLE SYMPTOMS

Three unrelated patients had Vmax normal for CPT I but reduced for CPT II, and normal Km for both. Palmitoyl CoA synthase, carnitine acetyl transferase, 4 acyl CoA dehydrogenases were normal. The patients: (1) A girl died at 5 days of encephalo-cardiomyopathy. Lipid was high in heart, liver, muscle; in these and in fibroblasts. CPT II was < 6% or not detectable. Total Carnitine was low, acyl carnitine high (Hug el al.Pediatr Res 25:115A;1989). (2) A male without muscle symptoms died at 20 years of dilated cardiomyopathy, as did his half-brother (of a different father). In heart, lipid was not excessive, carnitine was 50% and CPT II was 19%. (3) A male at 20 years had symptoms as in muscle CPT deficiency (DiMauro et al.Science 182:929;1973). Liver and heart were normal clinically. CPT II was 6% in muscle and fibroblasts. - Deficient CPT II and normal CPT I in the same patient indicate thai the 2 enzymes differ. Whether different phenotypes in CPT deficiency reflect differences in tissue distribution and in extent of the defect must be studied in multiple tissues obtained at biopsy or autopsy.