Doctoral Dissertations

Date of Award

12-1994

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Eugene Rinchik

Committee Members

Luis J. Stubbs, Liam B. Russell

Abstract

Analyses of 20 radiation-induced deletion mutations of the pinkeyed ip) locus in mouse chromosome 7 have identified a specific genetic interval associated with a recessive, neonatally lethal mutation resulting in isolated cleft palate. This interval extends from the distal breakpoint of the p83FBFo deletion to Gabrb3, the gene encoding the β33 subunit of the type A γ-aminobutyric acid receptor (GABAA), defining a gene(s) designated cp1, which controls secondary palate development. Complete concordance between alterations at the Gabrb3 transcription unit and the clefting phenotype was found in these 20 mutations. When combined with earlier teratological studies, this concordance suggested that Gabrb3 is cp1 and that a particular GABAA receptor that includes the β3 subunit may be necessary for normal palate development. The extents of the distal breakpoints of the deletions used to localize cp1 were further differentiated by determining whether any deleted a third GABAA receptor subunit gene encoding the γ3 subunit (Gabrg3). The data indicated the following gene order: p-Gabrg3-Gabra5-Gabrb3-Znf127. A mutant mouse line that fails to express the γ3 transcript and another that expresses neither the γ3 and α5 transcripts were identified. Mice from both mutant lines are phenotypically normal and do not exhibit any of the neurological symptoms characteristic of the rare survivors that are deleted for all three (γ3, α5, and β3) subunits but do not have a cleft palate. These two mutant lines provide a whole-organism GABAA-receptor background that is devoid of any receptor subtypes that normally contain the γ3 and/or α5 subunits. Neither Gabra5 nor Gabrg3, like Gabrb3, is functionally imprinted in mouse brain. The absence of an overt neurological phenotype in mice lacking the γ3 and/or α5 subunits, along with the lack of imprinting of all three GABAA-receptor genes in mouse brain, suggest that GABRG3 and GABRA5 (and quite possibly GABRB3) are unlikely to contribute to Angelman syndrome in humans as previously proposed. Analysis of mouse genomic DNA by Pulsed-Field Gel Electrophoresis (PFGE), and molecular characterization of two overlapping mouse fragments covering the cp1 interval cloned as yeast artifical chromosomes (YAC), established a physical map around cp1 and estimated the interval to be ~100 kb. The restriction map of rare-cutting enzymes within the interval do not show the presence of any other strong CpG islands that could indicate the presence of alternate candidate genes. A comparison of this mouse physical map with the established map in human 15q11-q13 reveals a high degree of conservation between these homologous chromosomal regions. Based on our genetic studies, on earlier in vitro and in vivo studies showing a teratological effect of GABA and its agonists in palate development, and on our RNA analysis showing expression of the gene in isolated palate shelves during the critical stage of palate shelf elevation and fusion, we hypothesized that cp1 is Gabrb3 and that a deficiency in a GABAA-receptor containing β3 as a subunit can cause clefting in mouse. This hypothesis was rigorously tested by a phenotype-rescue experiment. Neonatally lethal cleft palate in mice homozygous for a chromosome-7 deletion (p4THO-11) that deletes Gabrb3 has been corrected by the introduction of a rat Gabrb3 cDNA transgene under the control of the human β-actin promoter. p4THO-11 homozygotes rescued from the lethal cleft palate by the expression of the Gabrb3 transgene were observed from two independently derived lines. The rescued deletion homozygotes are typically smaller than their littermates, but this decrease in size correlates with homozygosity for the deletion rather than with expression of the transgene. The Gabrb3 transgene is imprinted in at least one line, such that rescue occurs only when the transgene is inherited from the mother. RNA analysis showed that the transgene is expressed equally well when passed through either germline; hence, this imprinting may be a tissue specific difference. Since the Gabrb3 gene is not imprinted endogenously, this imprinting of the transgene is probably not related to its native function. Regardless of the imprinting of the transgene in this one line, the phenotype-rescue experiment does confirm the hypothesis that Gabrb3 is cp1 Moreover, the extraordinarily conserved Gabrb3 gene has been placed in the homologous human chromosomal region 15q11-q13, which is associated with syndromes that manifest craniofacial anomalies. Thus, mutations in the human homologue GABRB3 may contribute to inherited or sporadic craniofacial disorders in humans.

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