Variability of thymidylate synthase activity in whole blood cultures treated with FUdR.

Induction of some fragile sites including fragile X [fra(X)] depends on the depletion of thymidine monophosphate (TMP) from the culture medium. This can be accomplished by use of inhibitors such as 5-fluorodeoxyuridine (FUdR) and by culturing cells in medium deficient in folate and TMP. FUdR inhibits the activity of thymidylate synthase (TS), thereby depleting cells of TMP. To determine the degree of FUdR inhibition of TS under routine cytogenetic culture conditions, we modified the tritiated dUMP TS method for use in short-term whole blood cultures stimulated with phytohemagglutinin. TS inhibition was highly variable across whole blood cultures from 30 individuals exposed to FUdR during the last 24 hours of a 4 day culture. If an additional dose of FUdR was added 12 hours before harvest, TS inhibition usually increased. These findings have a potential impact on the use of FUdR for the diagnosis of the fra(X) syndrome.

Acid alpha-glucosidase deficiency in cultured fibroblasts with phenotype 2 of acid alpha-glucosidase.

The enzyme acid alpha-glucosidase shows a polymorphism determined by two alleles, namely phenotypes 1, 2-1 and 2. We report that the activity of acid alpha-glucosidase, measured in fibroblasts homozygous for isozyme 2 is significantly reduced. In fibroblasts harvested at early confluency the activity in the strain with phenotype 2 of acid alpha-glucosidase was 23% and 5% compared to the levels in normal phenotype 1 strains using maltose and glycogen as substrate, respectively. At late confluency, the same fibroblasts showed an increase in activity to 60% and 15% of normal, respectively. At both early and late confluency, two fibroblast strains with the adult form of acid alpha-glucosidase deficiency had activity of approximately 7% of the activity in normal phenotype 1 fibroblasts with both substrates. In general, an increased amount of glycogen was measured in deficient fibroblasts, including the strain with phenotype 2, but overlapping was observed with normal cells. These findings show that fibroblasts homozygous for the isozyme 2 allele have a reduced ability to hydrolyse the natural substrate, glycogen.

Farber lipogranulomatosis: an unusual presentation in a black child.

Farber disease, a rare, inherited condition of lipid metabolism usually appears within the first two months of life. The patients may die in the first few years of life or may live into the second decade. We believe this patient to be the first black American reported with Farber disease. Additionally, the characteristics of the disease in this patient were at variance with previously reported cases.

Angiokeratoma corporis diffusum in GM1 gangliosidosis, type 1.

A patient with severe deficiency of beta-galactosidase, who developed skin lesions of angiokeratoma corporis diffusum between the 3rd and 10th month of life, is described. The activity of other lysosomal enzymes, including alpha-neuraminidase, was normal. The first signs of the disease were noticed during the first month of life. By 3 months coarseness of the face and psychomotor retardation were present. In addition to angiokeratoma, he had large mongolian spots and several scattered slate-blue spots of pigmentation over his body. With the exception of the skin lesions, the other clinical signs and the course of the psychomotor deterioration were within the clinical picture of GM1 gangliosidosis, Type 1. Angiokeratoma, a manifestation of several lysosomal disorders, may appear in GM1 gangliosidosis during the first year of life.

Oligosaccharyl diphosphodolichols in the ceroid-lipofuscinoses.

Autopsy brain samples from patients with late-infantile, juvenile and adult forms of ceroid-lipofuscinosis (CL) and cultured skin fibroblasts from juvenile CL were analyzed for the content of phosphodolichol (P-Dol) related compounds. The levels of P-Dol obtained on treatment with hot dilute acid of the chloroform-methanol (CM 2:1) extract and the chloroform-methanol-water (CMW 1:1:3) extract of the residue were estimated by high performance liquid chromatography. Compared to age-matched control individuals, the levels of P-Dol obtained in both the extracts were increased more than 6.6 times in all the patient samples. Further analysis of the CMW extract indicates that the increased P-Dol is primarily due to oligosaccharyl diphosphodolichol. Cultured skin fibroblasts from the juvenile form of CL show normal level of free dolichol and elevated level of phosphorylated dolichols. Glycoprotein synthesis measured by incorporation of labeled glucosamine show no deficit in the transfer of oligosaccharides from lipids to proteins. A hypothesis is presented to explain the accumulation of oligosaccharyl diphosphodolichol and deficiency of lysosomal proteases in ceroid-lipofuscinosis.

Sanfilippo disease in Greece.

In a series of cases collected in most parts of the world, Sanfilippo disease type A is more frequent than type B. Skin biopsies were obtained from Greek patients suspected for Sanfilippo disease and cultured fibroblasts were assayed for both N-acetyl-a-glucosaminidase and sulfamidase activity. Eleven patients with Sanfilippo disease were identified. Ten of them were type B and one type A. The 10 patients with type B came from East-Central Greece and the neighboring areas of Thessaly and Macedonia. Both parents of the type A patient were from the Greek ethnic community of Turkey. It remains unknown whether or not the higher frequency of type B than type A appears only in Greece or if it occurs in other Mediterranean countries as well.

Further evidence for genetic heterogeneity in the fragile X syndrome.

The X-linked fragile X [fra(X)] syndrome, associated with a fragile site at Xq27.3, is the most common Mendelian inherited form of mental deficiency. Approximately 1 in 1060 males and 1 in 677 females carry the fra(X) chromosome. However, diagnosis of carrier status can be difficult since about 20% of males and 44% of females are nonpenetrant for mental impairment and/or expression of fra(X). We analyzed DNA from 327 individuals in 23 families segregating fra(X) for linkage to three flanking polymorphic probes: 52A, F9, and ST14. This allowed probable nonpenetrant, transmitting males and carrier females to be identified. A combined linkage analysis was conducted using these families and published probe information on F9 in 27 other families, 52A in six families, and ST14 in five families. The two-point recombination fraction for 52A-F9 was 0.13 (90% confidence interval, 0.10-0.16), for F9-fra(X) was 0.21 (0.17-0.24), and for fra(X)-ST14 was 0.12 (0.07-0.17). Tight linkage between F9 and fra(X) was observed in some families; in others loose linkage was seen suggesting genetic linkage heterogeneity. Risk analysis of carrier status using flanking DNA probes showed that probable nonpenetrant transmitting males were included in families showing both tight and loose linkage. Thus, in contrast to our previous conclusions, it appears that the presence or absence of nonpenetrant, transmitting males in a family is not an indicator of heterogeneity. To determine if heterogeneity was present, we employed the admixture test. Evidence for linkage heterogeneity between F9 and fra(X) was found, significant at P less than 0.0005. Nonsignificant heterogeneity was seen for 52A-F9 linkage. No heterogeneity was found for fra(X)-ST14. The frequency of fra(X) expression was significantly lower in families with tight F9-fra(X) linkage than in families with loose linkage. Cognition appeared to relate to linkage type: affected males in tight linkage families had higher IQs than those in loose linkage families. These findings of genetic heterogeneity can account in part for the high prevalence and apparent high new mutation rate of fra(X). They will affect genetic counseling using RFLPs. An understanding of the basis for genetic heterogeneity in fra(X) will help to clarify the nature of the unusual pattern of inheritance seen in this syndrome.

Chromosomal abnormalities in amniotic fluid cell cultures: a comparison of apparent pseudomosaicism in Chang and RPMI-1640 media.

The finding of chromosome mosaicism is one of the most difficult problems in fetal chromosome analysis. Whether the finding indicates true mosaicism or pseudomosaicism must be investigated. Studies detailing the incidence of true mosaicism and pseudomosaicism have been reported (Hsu & Perlis 1984, Bui et al. 1984, Worton & Stern 1984) but do not correlate pseudomosaicism with any particular type of culture media. Benn & Hsu (1985) compared cell growth and chromosome abnormalities in amniotic fluid cell cultures grown in Chang medium and RPMI-1640 medium and found no statistically significant difference in the rate of abnormalities in the two media. We have previously shown that Chang medium exhibited more abnormalities which were not verified in second and third cultures (Masia et al. 1986). In the current study we examined 212 cases grown in both Chang and RPMI-1640 media, and compared apparent single and multiple cell pseudomosaic abnormalities to medium type. The number of observed abnormalities was 22, occurring in 19 of the cases studied. Apparent pseudomosaic chromosome anomalies were observed in 18 Chang cultures and in 4 RPMI-1640 cultures. Statistical analysis found significant correlation between medium type and the degree of observed pseudomosaic cells. We conclude that the rate at which pseudomosaic cells are observed is partly a function of medium type, and in our laboratory Chang medium caused apparent pseudomosaicism at a greater level than RPMI-1640 medium.

Distal duplication 14q: report of three cases and further delineation of the syndrome.

Three cases of distal duplication 14q are presented. The first two cases are cousins in a kindred segregating a balanced translocation t(14;18)(q31;q23). The third case resulted from a maternal translocation t(14;18)(q24;p11). By review of these cases and those previously reported, a distal duplication 14q syndrome is further delineated. Common features include postnatal growth retardation, mental retardation, hypotonia, microcephaly, slanted palpebral fissures, ocular hypertelorism, sparse eyelashes and eyebrows, nasal dysmorphism, tented lip, micrognathia, posteriorly rotated ears, and minor skeletal anomalies.

Atypical Down syndrome and partial trisomy 21.

A case of "atypical" Down Syndrome (DS), where the proposita did not exhibit all of the clinical features of DS and had de novo partial trisomy 21, was studied. Results from phenotypic, chromosome banding and superoxide dismutase (SOD) gene dosage studies suggest a karyotype of 46,XX,-12,+t(12pter to 12qter::21q21 to 21q22.?2). Additional studies of such atypical cases will provide more precise sublocalization for both gene and phenotypic mapping of the bands that are responsible for the DS phenotype.