IDENTIFICATION OF MESODERM DEVELOPMENT (MESD) CANDIDATE GENES BY COMPARATIVE MAPPING AND GENOME SEQUENCE ANALYSIS

Mary E. Wines^1,2*, Lance Lee^1,3*, Manpreet S. Katari², Ying Shi1, Liqun Zhang¹, Signa Perkins¹,
Michael Feldman1, W. Richard McCombie⁴, and Bernadette C. Holdener^1#
1Institute for Cell and Developmental Biology, Department of Biochemistry and Cell Biology,
State University of New York at Stony Brook, Stony Brook, NY 11794-5215
² Graduate Program in Genetics, SUNY at Stony Brook
³ Graduate Program in Molecular and Cellular Biology, SUNY at Stony Brook
⁴ Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724

^* authors contributed equally

The proximal albino deletions identify several functional regions on mouse chromosome 7, including mesoderm development (mesd), neuroendocrine lineage development (nelg), and hepatocyte-specific developmental regulator-1 (hsdr1). Through comparative mapping and sequence analysis, we have identified a conserved region of human synteny homologous to the mesd, nelg, and hsdr-1 functional regions. Three human diseases cosegregate with microsatellite markers used in construction of the human BAC/YAC physical map, including autosomal dominant frontal lobe epilepsy (ADNFLE), a syndrome of mental retardation, spasticity, and tapetoretinal degeneration (MR/S/TD); and a pyogenic arthritis, pyoderma gangrenosum, and acne syndrome (PAPA). Using comparative mapping and genomic sequence comparison, we have identified two mesd candidate genes and three novel genes located outside the mesd critical region. The function of these transcription units was examined by northern analysis and TgN171M12 complemention of mesd defects. These results demonstrate that genes located outside the mesd critical region are not essential for fetal development.

FIG. 1: Comparison of mouse chromosome 7 and human chromosome 15q

FIG. 2: Three human disorders segregate with markers in the mesd homologous region.

CONCLUSIONS:

FIG. 1: COMPARISON OF MOUSE CHROMOSOME 7 AND HUMAN CHROMOSOME 15q

• The mouse mesd, nelg, and hsdr deletion intervals are homologous to a region of human synteny located on chromosome 15q in bin 60 (G3 radiation hybrid panel).
• Using comparative mapping and genome sequence analysis we identified two novel candidate genes mesd-cg1 and mesd-cg2 in the 75 kb mesd critical region defined by the rescuing BAC 171M12.
• The mouse and human Mesd-cg1 genes are highly conserved and encode predicted proteins of 362 aa (98% identical) similar to a central domain of Talin (45% similarity).
• A nuclear localization signal within the Mesd-cg1 protein suggests a distinct role from Talin.
• The mouse and human Mesd-cg2 (KIAA0081) genes are conserved and encode predicted proteins of 224 and 234 aa (82% identical).
• A hydrophobic stretch at the N-terminus suggests that Mesd-cg2 is a transmembrane protein.

FIG. 2: THREE HUMAN DISORDERS SEGREGATE WITH DNA MARKERS LOCATED IN THE MESD HOMOLOGOUS REGION

• Three human disorders, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), a syndrome of mental retardation, spasticitiy, and tapetoretinal degeneration (MR/S/TD), and pyogenic arthritis, pyoderma gangrenosum, and acne syndrome (PAPA) segregate with markers located within the mesd homologous interval.
• ADNFLE is located between two markers that flank the mesd homologous interval. Both MESD-CG1 and MESD-CG2 as well as AL137657 (encoding a novel StAR related protein) and IL-16 are located within the ADNFLE critical region.
• MR/S/TD and PAPA critical regions extend beyond the mesd homology region and include a plethora of genes.

CONCLUSIONS:

The 1.3 Mb BAC contig encompassing the mouse mesd, nelg, and hsdr1 functional intervals covers 7 genes, including known genes: Fah, Arnt2, and Il-16 as well as 4 novel genes 18B7-T7(GS), Mesd-cg1, Mesd-cg2, and 12H19.01.T7. Despite the expression of 18B7-T7(GS) and 12H19.01.T7 in the early embryo, we have utilized the TgN171M12 BAC transgenic line to demonstrate that the only genes essential for fetal development are located within the 75 kb BAC 171M12. We have completely sequenced the 75 kb BAC clone. Comparison with the human genome draft sequence confirms the location of Mesd-cg1 and Mesd-cg2. Both genes are expressed in the embryo during gastrulation and both encode predicted proteins that are consistent with the cell autonomous mesd phenotype. Current efforts focus on the characterization of early expression of the two candidate genes and generation of transgenics and targetted mutations.

We have identified the homologous region of human chromosome 15q Bin 60-61 (Stanford G3). Construction of a human BAC contig demonstrates that the order of loci is conserved between mouse and human. The human disorder ADNFLE was localized to the mesd homology region. Both MESD-CG1 and MESD-CG2 as well as AL137657 (encoding a novel StAR related protein) and IL-16 are located within the ADNFLE critical region.