Novel GJA1 mutations in patients with oculo-dento-digital dysplasia (ODDD).

Oculo-dento-digital dysplasia (ODDD) is an autosomal dominant disorder characterized by developmental anomalies of the face, the eyes, the limbs and the teeth. Patients with ODDD usually present with complete syndactyly of the fourth and fifth fingers (type III syndactyly), ocular changes, abnormalities of primary and permanent dentition and specific craniofacial malformations. Mutations in GJA1, a gene that encodes the gap junction protein connexin 43, are responsible for ODDD. Gap junctions are assemblies of intercellular channels that allow exchange of various ions and signaling molecules between cells. In this way, gap junctions play an important regulatory role in a variety of physiologic and developmental processes. We identified three novel and one previously described GJA1 mutation in two large ODDD families and two sporadic ODDD cases.

PA26 is a candidate gene for heterotaxia in humans: identification of a novel PA26-related gene family in human and mouse.

Heterotaxia is an aetiologically heterogeneous condition caused by an abnormal left-right axis formation, resulting in reversed left-right polarity of one or more organ systems. In a patient with heterotaxia and a de novo reciprocal translocation t(6;18)(q21;q21), we found that the PA26 gene was disrupted by the 6q21 breakpoint. Northern blot analysis showed decreased expression of the PA26 gene in an Epstein-Barr virus-transformed cell line of this patient. During early embryogenesis of Xenopus, the orthologue of PA26, XPA26 is exclusively expressed in the notochord, a midline structure. This further supports a possible role of PA26 in human situs determination. Mutation analysis of human PA26 gene in 40 unrelated individuals with unexplained heterotaxia failed to identify mutations, indicating that PA26 mutations are not a frequent cause of heterotaxia in humans. Analysis of the PA26 gene structure resulted in the identification of a novel PA26-related gene family, which we have named the sestrin family, and which comprises three closely related genes in human and in mouse.

Involvement of a palindromic chromosome 22-specific low-copy repeat in a constitutional t(X; 22)(q27;q11).

Segmental duplications or low-copy repeats (LCRs) on chromosome 22q11 have been implicated in several chromosomal rearrangements. The presence of AT-rich regions in these duplications may lead to the formation of hairpin structures, which facilitate chromosomal rearrangement. Here we report the involvement of such a low-copy repeat in a t(X;22) associated with a neural tube defect. Molecular analysis of the chromosomal breakpoints revealed that the chromosome 22 breakpoint maps in the palindromic non-AT-rich NF1-like region of low-copy repeat B (LCR-B). No palindromic region was encountered near the breakpoint on chromosome X. Our findings confirm that there is no single mechanism leading to translocations with chromosome 22q11 involvement. Because LCR-B does not contain genes involved in neural tube development, we believe that the gene responsible for the observed phenotype is most likely localized on chromosome X.

LHFP, a novel translocation partner gene of HMGIC in a lipoma, is a member of a new family of LHFP-like genes.

A major cytogenetic subgroup among human lipomas is characterized by translocations involving the HMGIC gene at 12q15. In the context of an ongoing research program aiming at the elucidation of the functional consequences of HMGIC translocations in the etiology of lipomas, we have isolated a novel human gene, LHFP (lipoma HMGIC fusion partner), that acts as a translocation partner of HMGIC in a lipoma with t(12;13). The LHFP gene was mapped to the long arm of chromosome 13, a region recurrently targeted by chromosomal aberrations in lipomas. By Northern blot analysis, a transcript of 2. 4 kb was detected in a variety of human tissues. We assembled a cDNA contig containing the entire coding region of LHFP. Nucleotide sequence analysis of the composite LHFP cDNA revealed an open reading frame encoding a protein of 200 amino acids. The predicted human LHFP protein is almost identical to a translated mouse EST that covers almost the entire LHFP coding region. In addition, BLAST searches revealed that the LHFP protein belongs to a new protein family consisting of at least four or five members. In the lipoma studied, the expressed HMGIC/LHFP fusion transcript encodes the three DNA binding domains of HMGIC followed by 69 amino acids encoded by frame-shifted LHFP sequences. LHFP is the second translocation partner of HMGIC identified in lipomas and represents a candidate target gene for lipomas with 13q aberrations.

Allelic knockout of novel splice variants of human recombination repair gene RAD51B in t(12;14) uterine leiomyomas.

Recently, the high mobility group protein gene HMGIC was identified as the chromosome 12q15 target gene in a variety of benign solid tumors. Here, we report that the recombinational repair gene RAD51B on chromosome 14q23-24 is the preferential translocation partner of HMGIC in uterine leiomyomas. The pathogenetically critical sequences seem to reside in the last coding exon of a novel RAD51B isoform, which encode a domain containing a putative transmembrane anchor and are expressed in the uterus but not in a wide variety of other tissues tested. By fluorescence in situ hybridization, rapid amplification of 3' cDNA ends, and reverse transcription-PCR analysis, we demonstrated consistent chromosomal rearrangements within RAD51B and expression of fusion transcripts, structurally resulting in an allelic knockout of the uterine isoform of RAD51B and confirming a pleiotropic function of this gene.

Mapping of the translocation breakpoints of primary pleomorphic adenomas and lipomas within a common region of chromosome 12.

Recent molecular cytogenetic analysis of uterine leiomyoma cell lines with chromosomal aberrations of 12q14-q15 have indicated that the chromosome 12 breakpoints cluster in a 445-kb region designated ULCR12 (uterine leiomyoma cluster region of the chromosome 12 breakpoints). Here we report the results of FISH studies of five primary pleomorphic adenomas and six primary lipomas and established cell lines of these tumor types characterized by translocations involving the chromosomal segment 12q13-q15. The results reveal that for nearly all tumors and cell lines analyzed, the chromosome 12 breakpoints map within a 350-kb region included in ULCR12, despite the previous cytogenetic assignment of the breakpoints to different bands of that region. In some cases the primary material and additionally analyzed cell lines allowed an even more precise localization of the breakpoints to less than 100 kb. Furthermore, a previously hidden translocation of ULCR12 in one primary tumor could be detected by FISH.

A 6-Mb yeast artificial chromosome contig and long-range physical map encompassing the region on chromosome 12q15 frequently rearranged in a variety of benign solid tumors.

Cytogenetic analysis of a variety of benign solid tumors, among which uterine leiomyoma, lipoma, pleomorphic salivary gland adenoma, and pulmonary chondroid hamartoma, has indicated that these tumors often display chromosome breakpoints in region q13-q15 of chromosome 12. In previous studies, we have reported that these breakpoints map between locus D12S8 and the CHOP gene, the latter of which has been shown to be consistently rearranged in myxoid liposarcomas with t(12;16)(q13;p11). Here, we report directional chromosome walking studies starting from D12S8 and resulting in the construction of a YAC contig of about 6 Mb. This YAC contig, whose orientation on chromosome 12 was determined by double-color fluorescence in situ hybridization (FISH) analysis, has at least double coverage and consists of 75 overlapping YAC clones, all isolated from CEPH YAC libraries. Their insert sizes were estimated by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Chromosomal localization and chimerism of the YACs were investigated by FISH analysis. Chimerism of YAC clones was independently determined by restriction mapping. On the basis of YAC end-derived DNA markers and sequence-tagged sites (STSs), with an average spacing of approximately 70 kb, as well as restriction enzyme analysis, a long-range physical map was established for the 6-Mb DNA region of chromosome 12 covered by the YAC contig. Within the YAC contig, the relative positions of various known genes, an expressed sequence-tagged site, and a number of CEPH/Généthon polymorphic markers were determined. The latter data allow full integration of our mapping data with those obtained by CEPH/Généthon as well as those reported at the Second International Workshop on Human Chromosome 12 Mapping. Finally, this YAC contig constitutes the basis for the contstruction of a transcriptional map of this region and is likely to facilitate identification of genes involved in the formation of various benign solid tumor types.

Regulation of squalene synthetase activity in rat liver: elevation by cholestyramine, but no diurnal variation.

Squalene synthetase activity in liver microsomes from rats sacrificed at three different times of the diurnal cycle showed no significant differences. Addition of 4% cholestyramine to the food resulted in a marked increase in activity (280% of control), independent of the time of killing. 3-Hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7 alpha-hydroxylase activity, determined as positive controls, were also found to be elevated by cholestyramine and additionally showed a diurnal variation. On the other hand, five control enzyme activities, not directly related to cholesterol metabolism, i.e. glutamate dehydrogenase, NADPH cytochrome-c reductase, beta-hexosaminidase, catalase and acyl coenzyme A oxidase, showed neither an influence of cholestyramine feeding nor a time of sacrifice dependent variation.