Towards cloning the gene for holoprosencephaly on chromosome 2
- Yale Univ., New Haven, CT (United States); and others
Holoprosencephaly (HPE) is a developmental defect of the forebrain and mid face in humans. Because of the non-random association of HPE with chromosome anomalies in 2p21, 7q36, 18p, and 21q22.3, we hypothesized that these regions contain putative HPE genes. As a first step towards isolating the H P E 2 gene in 2p21 we have established six cell lines from patients with HPE and cytogenetically visible 2p21 deletions, the smallest of which (LCL002) is located between D2S174 and D2S123. Both maternal and paternal origin of the del(2) chromosome could be confirmed by PCR analysis using 7 markers in this region. By dosage blot analysis, we have identified 12 of over 50 microdissected microclones from the smallest deletion [del(2)(p2101-p2109)]. The inserts of the 12 microclones were sequenced and primers for sequence-tagged sites (STSs) generated. Two somatic cell hybrid clones with the del(2) but not the normal chromosome 2 of LCL002 were generated and used to confirm the location of the 12 STSs within 2p2101-p2109. Yeast artificial chromosomes (YACs) from the CEPH library were mapped by fluorecent in situ hybridization (FISH) to the smallest 2p21 deletion. YACs from within this deletion were used to screen the Genethon data base for additional overlapping YACs. With the available FISH, YAC, and STS data, we were able to construct a contig consisting of 18 overlapping YACs which span the smallest deletion. We used these YACs for FISH analysis of two crucial HPE cell lines. One carries a de novo balanced translocation, another one an insertion with breakpoints in 2p21, both of which appear to disrupt the putative H P E 2 gene. Both breakpoints are located in the smallest deletion. Furthermore, we were able to identify 6 YACs which cross these breakpoints. Subcloning of the 6 YACs into cosmids is now in progress in an attempt to isolate H P E 2 gene-specific sequences in 2p21.
- OSTI ID:
- 133389
- Report Number(s):
- CONF-941009-; ISSN 0002-9297; TRN: 95:005313-0117
- Journal Information:
- American Journal of Human Genetics, Vol. 55, Issue Suppl.3; Conference: 44. annual meeting of the American Society of Human Genetics, Montreal (Canada), 18-22 Oct 1994; Other Information: PBD: Sep 1994
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
BASIC STUDIES
GENES
GENE MUTATIONS
DNA SEQUENCING
HUMAN CHROMOSOME 2
GENETIC MAPPING
CHROMOSOMAL ABERRATIONS
PATIENTS
NERVOUS SYSTEM DISEASES
SOMATIC CELLS
DNA-CLONING
DNA HYBRIDIZATION
YEASTS
HUMAN CHROMOSOME 7
HUMAN CHROMOSOME 18
HUMAN CHROMOSOME 21
FLUORESCENCE
CONTIGS
POLYMERASE CHAIN REACTION