Down syndrome: Gene dosage at the transcriptional level in skin fibroblasts

Abstract

Chromosome imbalance (aneusomy) is the leading known cause of both spontaneous abortion and mental retardation in human beings. The primary abnormality is thought to result from quantitative changes of transcription products from the unbalanced genetic material. To document this point, I compared chromosome 21-specific transcription in skin fibroblasts from subjects with monosomy 21, disomy 21 (normal), and trisomy 21 (Down syndrome (aneusomy) are shown by the lethality in human beings of monosomy for more than 2-3% and trisomy for more than 5-6% of the haploid autosomal genome (1).Smaller deficiencies and duplications of autosomal material are associated with altered phenotypes which can be recognized by specific patterns of malformation and are usually associated with mental retardation (2-4). Chromosome imbalance represents, in sum, the major known cause of both abortion (5) and mental retardation (6) in human beings. Down syndrome (trisomy 21) is the most common viable human aneusomy and comprises the majority of individuals who are retarded because of chromosomal imbalance (7).The actual mechanism whereby chromosomal imbalance results in major phenotypic effects remains poorly understood. However, several points are clear: (i) The defect is due to quantitative alteration of normal genetic material. This point is best illustrated by a case report of monozygotic twins who are discordant for Down syndrome (8 Given this background, elucidation of the mechanism(s) whereby aneusomy results in metabolic and phenotypic aberrations will require analyses at the primary level of gene action-i.e., transcription. This report describes some first steps toward such an analysis: RNA sequences transcribed from human chromosome 21 are quantitated in skin fibroblasts with one, two, and three copies per genome of chromosome 21. MATERIALS AND METHODSSynthesis of cDNAs. Human skin fibroblasts were grown to confluency in Dulbecco-Vogt medium containing 16% heat-inactivated fetal calf serum, fed, and harvested 4 days later. They were scraped off with a rubber policeman into ice-cold phosphate-buffered saline containing 1:200 (vol/vol) diethylpyrocarbonate (Sigma). The cells were pelleted and resuspended in 5 vol of 5 M guanidine thiocyanate (Tridom), 50 mM Tris-HCl (pH 7.6), 10 mM EDTA, and 5% (vol/vol) 2-mercaptoethanol. Sarkosyl was added to 4% (wt/vol), and 0.5 g of CsCl was added per ml of solution. The solution was layered over a 1-ml cushion of 5.7 M CsCl in 100 mM EDTA and centrifuged in a Spinco SW 50.1 rotor at 35,000 rpm for 12 hr at 20°C (16 2372The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Alterations in the relative amounts of specific mRNA species in the developing human brain in Down's syndrome

“…Although the genetic basis for the syndrome is known (Lejeune et al, 1959) the molecular mechanisms governing expression of the phenotype are poorly understood, although it is generally accepted that the syndrome arises from dosage-dependent transcription of qualitatively normal genetic material (Kurnit, 1979). Evidence for this proposal comes from the demonstration that the activity and synthesis of specific proteins with structural loci on chromosome 21 are dependent on gene dosage (Tan, 1975;Feaster et al, 1977;Bartley & 1980; Brown et al, 1981).…”

“…Evidence for this proposal comes from the demonstration that the activity and synthesis of specific proteins with structural loci on chromosome 21 are dependent on gene dosage (Tan, 1975;Feaster et al, 1977;Bartley & 1980; Brown et al, 1981). In addition, dosagedependent transcription of chromosome-21 sequences has been shown to occur in human fibroblast strains (Kurnit, 1979). However, the demonstration of changes in the activities of enzymes with structural loci outside chromosome 21 (Hsai et al, 1968) After removal of the CsCl cushion, the RNA pellet was washed briefly twice in sterile water and resuspended in 10mM-Tris/HCl (pH8.5)/0.1M-NaCl/0.5% (w/v) SDS/5mM-EDTA/5% (w/v) phenol with a glass Teflon homogenizer.…”

Human cytoplasmic superoxide dismutase cDNA clone: a probe for studying the molecular biology of Down syndrome.

“…Although trisomy 21 was identified as a human genetic disease over 20 years ago (5), little is known about the mechanisms by which the extra chromosome or the extra chromosomal segment 21q22 results in reduced viability and abnormalities of morphogenesis and mental function. It is generally assumed that the extra chromosome or chromosomal segment codes for normal products and that the abnormalities found in Down syndrome are produced by an imbalance due to changes in gene dosage (12). Namely, the presence of additional genetic material in the cell will result in the production of commensurately increased amounts of the gene products coded by the extra chromosomal segment.…”

Demonstration, by somatic cell genetics, of coordinate regulation of genes for two enzymes of purine synthesis assigned to human chromosome 21.

“…Third, we possessed a number of mutants of each complementation group. Fourth, the Ade-C gene in humans has been assigned to chromosome 21 (12 (13)(14)(15). Therefore, if genes can be excluded from this region of the chromosome, they must not be involved in the pathology of the disease.…”

“…The following 23 isozymes and 1 nutritional marker were assayed in the hybrid clones by using methods as described (19). Numbers in brackets correspond to human chromosome assignments (20): 6-phosphogluconate dehydrogenase [1]; malate dehydrogenase, soluble [2]; 1-galactosidase [3]; phosphoglucomutase 2 [4]; arylsulfatase B [5]; superoxide dismutase, mitochondrial [6]; f-glucuronidase [7]; glutathione reductase [8]; adenylate kinase [9]; proline requirement [10]; lactate dehydrogenase A [11]; lactate dehydrogenase B [12]; esterase D [13]; nucleoside phosphorylase [14]; hexosaminidase-A and pyruvate kinase [15]; phosphogluconate phosphatase [16]; galactokinase [17]; peptidase A [18]; glucose phosphate isomerase [19]; adenosine deaminase [20]; superoxide dismutase, soluble [21]; arylsulfatase-A [22]; and glucose-6-phosphate dehydrogenase [X].…”