Jean H. Priest

Incidence and significance of chromosome mosaicism involving an autosomal structural abnormality diagnosed prenatally through amniocentesis: A collaborative study

Among 179 663 prenatal diagnosis cases collected from ten institutions and two publications, 555 (0·3 per cent) were diagnosed as having chromosome mosaicism. Of these, 57 (10·3 per cent) were mosaic for an autosomal structural abnormality, 28 (5 per cent) for a sex chromosome structural abnormality, and 85 (15·3 per cent) were mosaic for a marker chromosome. Ninety‐five cases of prenatally diagnosed mosaicism with a structural abnormality in an autosome and a normal cell line, and with a known phenotypic outcome, were collected for karyotype–phenotype correlations through our collaboration (40 cases), a prior survey (26 cases), and published reports (29 cases). They included 13 balanced reciprocal translocations, one unbalanced reciprocal translocation, four balanced Robertsonian translocations, four unbalanced Robertsonian translocations, four inversions, 17 deletions, three ring chromosomes, 19 i(20q), seven +i(12p), six other isochromosomes, and 17 partial trisomies resulting from a duplication or other rearrangement. All cases mosaic for a balanced structural rearrangement resulted in a normal phenotype. All cases of 46/46,i(20q) resulted in normal liveborns. Five of seven cases with 46/47,+i(12p) had an abnormal phenotype compatible with Killian–Pallister syndrome. The overall risk for an abnormal outcome for a mosaic case with an unbalanced structural abnormality, excluding 46/46,i(20q) and 46/47,+i(12p), is 40·4 per cent. In the same category, the study also suggested a correlation between the percentage of abnormal cells and an abnormal phenotype. For mosaicism involving a terminal deletion, the possibility of a familial fragile site should be considered.

Maple Syrup Urine Disease: Coenzyme function and prenatal monitoring

Abstract A pregnancy at high risk for “cofactor resistant” Maple Syrup Urine Disease (MSUD) was monitored by quantitating valine, leucine, and isoleucine concentrations in maternal 24-hr urines, maternal plasma, and amniotic fluid. The fetal genotype was determined by assaying the conversion of radiolabeled branched-chain amino acids to 14 CO 2 by intact cultured amniotic fluid cells. Although branched-chain amino acid concentrations in maternal fluids were similar to control values, cells cultured from the high-risk pregnancy produced 14 CO 2 at one-half the rate of control cells. This finding suggested that the unborn 46 XY fetus was heterozygous for the MSUD gene. To test this hypothesis and to study normal and mutant branched-chain α-keto-acid dehydrogenase and co-factor interaction, a broken-cell system was developed that decarboxylated branched-chain α-keto-acids only when reconstituted with required cofactors. In control systems reduced coenzyme A (CoASH), β-nitotinamide adenine dinucleotide (NAD), thiamine pyrophosphate (TPP), and magnesium chloride (Mg 2+ ) stimulated 14 CO 2 production from α-ketoisocaproic acid-I- 14 C (KIC), α-ketoisovaleric acid-I- 14 C (KIV), and L-α-keto-β-methylvaleric acid-I- 14 C (KMV) by five to fifteen times base line over a 2-hr time course. TPP and Mg 2+ alone failed to increase either KIC or KIV decarboxylase, but reconstituted 30% of KMV decarboxylase. This “TPP-reconstituted” KMV decarboxylase was also present in mutant cells homozygous for this MSUD gene. KIV decarboxylase activity was essentially absent in the homozygous-affected line and partially impaired in heterozygous lines. Cells from the newborn male off-spring had partial impairment of KIV and KMV decarboxylase. These studies indicated that the MSUD mutation in this family resulted in the production of a defective subunit of the branched-chain α-keto-acid dehydrogenase complex that was common to all three branched-chain α-keto-acids, that a “TPP-reconstituted” KMV decarboxylase was present and under separate genetic control, and that a heterozygote for this MSUD gene was predicted before birth.

Acceptance of amniocentesis by low-income patients in an urban hospital☆☆☆

A study was made of increased accessibility of genetic services to low-income obstetric patients in Atlanta, Georgia. The proportion of black patients averaged 83%. Of 522 patients counseled from August, 1976, through 1978, 157 were offered amniocentesis, and 95 (61%) elected the procedure. For most of the patients (120, or 76%) who were eligible for amniocentesis, age (greater than or equal to 35 years at delivery) was an indication; and of these, only six (5%) had any prior knowledge of genetic risk. During the same time interval, 188 patients over 35 years of age who initiated prenatal care too late for prenatal diagnosis were counseled in the hospital after delivery; 101 (54%) indicated that they would have accepted amniocentesis. The conclusion was that (1) genetic services are acceptable to this socioeconomic group, and (2) accessibility and publicity are needed to promote utilization in this population.

Differentiation in human amniotic fluid cell cultures: I: Collagen production.

The collagen produced by differentiated cells cultured from human amniotic fluid was characterized in two ways. By chain composition and by 4-hydroxyproline:3-hydroxyproline isomer ratio, the collagen synthesized by F-type (fibroblast) cells was indistinguishable from that made by cultured fetal dermal fibroblasts. The predominant cells in young amniotic fluid cultures, termed AF-type, produced collagen with a lower isomer ratio, resembling that of basement membrane collage. The chain composition, as determined by chromatography on carboxymethyl cellulose, varied for different cultures of the AF-type, but the major pattern was consistent with that of basement membrane collagen. On the basis of these characteristics, F cells are of fibroblast origin, whereas most AF cells are of a different origin either endothelial or epithelial. Other evidence (Megaw et al., 1977) suggests an epithelial origin for AF cells.

Dicentric chromosome 13 and centromere inactivation.

SummaryThe karyotype of a child with dysmorphic findings suggestive of both trisomy 13 and the 13q-syndrome was found to have cells with one of two different dicentric chromosomes: one bearing a duplication of chromosome 13q [46,XX,-13, + psu dic(13)t(13;13)(pter→ce→q34::q34→pter)] and the other a deletion of 13q [46,XX,-13,+psu dic(13)t(13;13)(pter→cen→q22::q11→pter). Longitudinal cytogenetic studies in leukocytes demonstrated a loss of those cells possessing the small dicentric [psu dic(13)(q22;q11)], whereas fibroblasts from two separate skin biopsies contained only this marker. Q-band polymorphisms indicated that both dicentrics were of paternal origin, with the smaller dicentric derived from the larger via the bridge-breakage-fusion cycle. The presence of two active centromeres could not be confirmed in either dicentric.

Differentiation in human amniotic fluid cell cultures: Chorionic gonadotropin production

SummaryTwo of the distinguishable cell classes subcultured from human amniotic fluid were examined for their capability to produce human chorionic gonadotropin (hCG) as determined by radioimmunoassay. The class that predominates in most cultures used for prenatal genetic diagnosis, previously termed AF (for amniotic fluid), secretes hCG into the culture medium. Dermal fibroblasts do not, nor does another type of cultured cell from amniotic fluid, previously termed F because of a resemblance to fibroblasts. Primary AF cultures produce more hCG than do subcultures. Evidence that this hormone is intact hCG is provided by its immunoreactivity with antisera raised against the β-subunit and against the intact molecule of hCG. Furthermore, a dose-response curve for hormone in culture medium is parallel to that of highly purified intact hCG. It is postulated that AF cultures are derived from fetal membranes and retain properties of trophoblast.

Cell kinetic disturbances induced by treatment of human diploid fibroblasts with 5-azacytidine indicate a major role for DNA methylation in the regulation of the chromosome cycle

SummaryBrdU-Hoechst flow cytometry was used to investigate the effects of DNA hypomethylation, induced by treatment with 5-azacytidine (5AC), on cell proliferation. When human fibroblast-like cells derived from skin and amniotic fluid were exposed to 5AC during three successive cell cycles, their clone-forming ability was diminished after removal of the drug. Treated cells were rendered quiescent by culture with low serum in the absence of the drug. Upon serum stimulation, they showed a diminished fraction of proliferating cells, which exhibited a prolonged transit through the S and G2 phase of the cell cycle, and a permanent arrest within the G2 compartment. This pattern of disturbed cell proliferation may in part explain the changes in replication banding pattern reported in the literature. Cytogenetic analysis of 5AC-treated cells revealed numerous endomitoses and tetraploid metaphases indicating a disturbed chromosome cycle in association with these cell kinetic perturbations.

Differentiation in human amniotic fluid cell cultures: II: Secretion of an epithelial basement membrane glycoprotein.

Cells obtained by amniocentesis for prenatal diagnosis were grown in vitro and examined for the presence of a glycoprotein component epithelial basement membrane. Isolated colonies or clones of amniotic fluid-type cells secrete the glycoprotein, which was identified in association with the cells using indirect immunofluorescent antibody techniques. In addition, the glycoprotein was isolated from tissue culture medium and identified as a component of epithelial basement membranes by passive haemagglutination (PHA) and immunodiffusion assays. Fibroblast-type cells do not secrete the glycoprotein. These results correlate well with the synthesis of type IV collagen by amniotic fluid cells reported in the accompanying paper (Priest et al., 1977) and indicate that amniotic fluid cells are epithelial in origin.

Ascorbate regulation of collagen biosynthesis in Ehlers-Danlos syndrome, type VI

We studied two unrelated individuals with Ehlers-Danlos syndrome type VI, which is characterized by congenital hypotonia, lax joints, severe kyphoscoliosis, friable skin, and hemorrhagic hypotrophic scars. The diagnosis was confirmed by decreased hydroxylysine residues in dermal collagen and decreased collagen lysyl hydroxylase activities in their cultured skin fibroblasts. Despite the diminished hydroxylysine residues in dermal collagen from the probands, we found no differences in hydroxylysyl residues of collagen synthesized by fibroblasts in culture. When patient 1 was given oral sodium ascorbate (5 g/d) for 3 weeks, ascorbate concentrations increased two-fold in plasma and 300-fold in urine. Urinary excretion of hydroxylysine and hydroxyproline increased during ascorbate administration. After a 1-year interval, bleeding time, wound healing, and muscle strength improved. Ascorbate supplementation (50 micrograms/mL) to confluent fibroblasts cultured from the two patients and controls increased hydroxyprolyl and hydroxylysyl residues of fibroblasts four to seven and three to four-fold respectively. Total protein associated with the cell layer increased 14% to 32% without concomitant change in cellular DNA. Total soluble collagenous material recovered from culture media increased 61% to 103% with ascorbate supplementation. These studies demonstrate that ascorbate improves the clinical status of patients with impaired collagen lysyl hydroxylase activity by enhancing lysyl and prolyl hydroxylation and total collagen production.

Acceptance of amniocentesis by women in the state of Montana (USA) who are screen positive for Down's syndrome.

Objective To assess factors influencing uptake of amniocentesis after a positive Downs syndrome screening result. Methods Interviews of 53 Montana women with screening risks ≥1 in 300 after delivery. Results Thirty had accepted amniocentesis (“yes” group) and 23 had declined (“no” group) (57% uptake). Age at delivery was significantly higher (p=0.02) for the “no” than the “yes” group (mean 35.3 v 31.7 years). The mean risk of Downs syndrome ascertained by screening was 1 in 190 for the “no” group and 1 in 115 for the “yes” group (p=0.05). Statistically significant differences (p≤0.05) between opinions in the two groups included: (a) desire to know if the fetus had Downs syndrome; (b) perception of the burden of care for an affected child; (c) support of doctor, spouse, and relatives for choice about amniocentesis; (d) attitudes toward abortion; (e) importance of religion; and (f) concerns about the amniocentesis procedure. The most important factor for those choosing amniocentesis was knowing if the fetus had Downs syndrome, and for those not choosing amniocentesis, attitude about abortion. Conclusion Our results show the need for prescreening education to enable pregnant women to make informed decisions about screening for Downs syndrome and diagnostic testing.