A bipartite DNA-binding domain in yeast Reb1p.

The REB1 gene encodes a DNA-binding protein (Reb1p) that is essential for growth of the yeast Saccharomyces cerevisiae. Reb1p binds to sites within transcriptional control regions of genes transcribed by either RNA polymerase I or RNA polymerase II. The sequence of REB1 predicts a protein of 809 amino acids. To define the DNA-binding domain of Reb1p, a series of 5' and 3' deletions within the coding region was constructed in a bacterial expression vector. Analysis of the truncated Reb1p proteins revealed that nearly 400 amino acids of the C-terminal portion of the protein are required for maximal DNA-binding activity. To further define the important structural features of Reb1p, the REB1 homolog from a related yeast, Kluyveromyces lactis, was cloned by genetic complementation. The K. lactis REB1 gene supports active growth of an S. cerevisiae strain whose REB1 gene has been deleted. The Reb1p proteins of the two organisms generate almost identical footprints on DNA, yet the K. lactis REB1 gene encodes a polypeptide of only 595 amino acids. Comparison of the two Reb1p sequences revealed that within the region necessary for the binding of Reb1p to DNA were two long regions of nearly perfect identity, separated in the S. cerevisiae Reb1p by nearly 150 amino acids but in the K. lactis Reb1p by only 40 amino acids. The first includes a 105-amino-acid region related to the DNA-binding domain of the myb oncoprotein; the second bears a faint resemblance to myb. The hypothesis that the DNA-binding domain of Reb1p is formed from these two conserved regions was confirmed by deletion of as many as 90 amino acids between them, with little effect on the DNA-binding ability of the resultant protein. We suggest that the DNA-binding domain of Reb1p is made up of two myb-like regions that, unlike myb itself, are separated by as many as 150 amino acids. Since Reb1p protects only 15 to 20 nucleotides in a chemical or enzymatic footprint assay, the protein must fold such that the two components of the binding site are adjacent.

The rRNA enhancer regulates rRNA transcription in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the rRNA genes are organized as a tandem array of head-to-tail repeats. An enhancer of rRNA transcription is present just at the end of each transcription unit, 2 kb away from the next one. This enhancer is unusual for S. cerevisiae in that it acts both upstream and downstream of, and even across, genes. The role of the enhancer in the nutritional regulation of rRNA transcription was studied by introducing a centromere plasmid carrying two rRNA minigenes in tandem, flanking a single enhancer, into cells. Analysis of the transcripts from the two minigenes showed that the enhancer was absolutely required for the stimulation of transcription of rRNA that occurs when cells are shifted from a poor carbon source to a good carbon source. While full enhancer function is provided by a 45-bp region at the 3' end of the 190-bp enhancer, some activity was also conferred by other elements, including both a T-rich stretch and a region containing the binding sites for the proteins Reb1p and Abf1p. We conclude that the enhancer is composed of redundant elements and that it is a major element in the regulation of rRNA transcription.

REB1, a yeast DNA-binding protein with many targets, is essential for growth and bears some resemblance to the oncogene myb.

REB1 is a DNA-binding protein that recognizes sites within both the enhancer and the promoter of rRNA transcription as well as upstream of many genes transcribed by RNA polymerase II. We report here the cloning of the gene for REB1 by screening a yeast genomic lambda gt11 library with specific oligonucleotides containing the REB1 binding site consensus sequence. The REB1 gene was sequenced, revealing an open reading frame encoding 809 amino acids. The predicted protein was highly hydrophilic, with numerous OH-containing amino acids and glutamines, features common to many of the general DNA-binding proteins of Saccharomyces cerevisiae, such as ABF1, RAP1, GCN4, and HSF1. There was some homology between a portion of REB1 and the DNA-binding domain of the oncogene myb. REB1 is an essential gene that maps on chromosome II. However, the physiological role that it plays in the cell has yet to be established.

Expression of Cdcrel-1 (Pnutl1), a gene frequently deleted in velo-cardio-facial syndrome/DiGeorge syndrome.

The murine Cdcrel-1 (Pnutl1) gene belongs to the family of septins, which are thought to be involved in cytokinesis in yeast, Drosophila and vertebrates. Recent studies implicate Cdcrel-1 in the regulation of vesicle transport in neurons of the adult brain. The human homologue, hCDCREL-1 maps to chromosome 22q11.2, a region commonly deleted in patients displaying velo-cardio-facial syndrome (VCFS) or DiGeorge syndrome (DGS). During development, Cdcrel-1 transcripts are expressed from E10.5 on in the nervous system such as the dorsal root ganglia and the cranial ganglia as well as the lateral layer of the neural tube, the area where terminally differentiated neurons are located. Low level expression is found in the mesenchyme of the frontonasal mass and the limb bud mesenchyme of E11.5 and E13.5 murine embryos. At E15.5, expression is detected in the nervous tissue and in the neural layer of the eye. Based on the expression pattern as well as clinical data, Cdcrel-1 may be involved in the etiology of VCFS/DGS.

The 22q11 deletion syndrome candidate gene Tbx1 determines thyroid size and positioning.

Thyroid dysgenesis is the major cause of congenital hypothyroidism in humans. The underlying molecular mechanism is in most cases unknown, but the frequent co-incidence of cardiac anomalies suggests that the thyroid morphogenetic process may depend on proper cardiovascular development. The T-box transcription factor TBX1, which is the most probable gene for the 22q11 deletion syndrome (22q11DS/DiGeorge syndrome/velo-cardio-facial syndrome), has emerged as a central player in the coordinated formation of organs and tissues derived from the pharyngeal apparatus and the adjacent secondary heart field from which the cardiac outflow tract derives. Here, we show that Tbx1 impacts greatly on the developing thyroid gland, although it cannot be detected in the thyroid primordium at any embryonic stage. Specifically, in Tbx1-/- mice, the downward translocation of Titf1/Nkx2.1-expressing thyroid progenitor cells is much delayed. In late mutant embryos, the thyroid fails to form symmetric lobes but persists as a single mass approximately one-fourth of the normal size. The hypoplastic gland mostly attains a unilateral position resembling thyroid hemiagenesis. The data further suggest that failure of the thyroid primordium to re-establish contact with the aortic sac is a key abnormality preventing normal growth of the midline anlage along the third pharyngeal arch arteries. In normal development, this interaction may be facilitated by Tbx1-expressing mesenchyme filling the gap between the pharyngeal endoderm and the detached thyroid primordium. The findings indicate that Tbx1 regulates intermediate steps of thyroid development by a non-cell-autonomous mechanism. Thyroid dysgenesis related to Tbx1 inactivation may explain an overrepresentation of hypothyroidism occurring in patients with the 22q11DS.

Low-copy repeats mediate the common 3-Mb deletion in patients with velo-cardio-facial syndrome.

Velo-cardio-facial syndrome (VCFS) is the most common microdeletion syndrome in humans. It occurs with an estimated frequency of 1 in 4, 000 live births. Most cases occur sporadically, indicating that the deletion is recurrent in the population. More than 90% of patients with VCFS and a 22q11 deletion have a similar 3-Mb hemizygous deletion, suggesting that sequences at the breakpoints confer susceptibility to rearrangements. To define the region containing the chromosome breakpoints, we constructed an 8-kb-resolution physical map. We identified a low-copy repeat in the vicinity of both breakpoints. A set of genetic markers were integrated into the physical map to determine whether the deletions occur within the repeat. Haplotype analysis with genetic markers that flank the repeats showed that most patients with VCFS had deletion breakpoints in the repeat. Within the repeat is a 200-kb duplication of sequences, including a tandem repeat of genes/pseudogenes, surrounding the breakpoints. The genes in the repeat are GGT, BCRL, V7-rel, POM121-like, and GGT-rel. Physical mapping and genomic fingerprint analysis showed that the repeats are virtually identical in the 200-kb region, suggesting that the deletion is mediated by homologous recombination. Examination of two three-generation families showed that meiotic intrachromosomal recombination mediated the deletion.

Isolation and characterization of a novel gene containing WD40 repeats from the region deleted in velo-cardio-facial/DiGeorge syndrome on chromosome 22q11.

Three congenital disorders, cat-eye syndrome (CES), der(22) syndrome, and velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS), result from tetrasomy, trisomy, and monosomy, respectively, of part of 22q11. They share a 1.5-Mb region of overlap, which contains 24 known genes. Although the region has been sequenced and extensively analyzed, it is expected to contain additional genes, which have thus far escaped identification. To understand completely the molecular etiology of VCFS/DGS, der(22) syndrome, and CES, it is essential to isolate all genes in the interval. We have identified and characterized a novel human gene, located within the 1.5-Mb region deleted in VCFS/DGS, trisomic in der(22) syndrome and tetrasomic in CES. The deduced amino acid sequence of the human gene and its mouse homologue contain several WD40 repeats, but lack homology to known proteins. We termed this gene WDR14 (WD40 repeat-containing gene deleted in VCFS). It is expressed in a variety of human and mouse adult and fetal tissues with substantial expression levels in the adult thymus, an organ hypoplastic in VCFS/DGS.

Purification and characterization of the yeast rDNA binding protein REB1.

In the yeast Saccharomyces cerevisiae, the ribosomal RNA genes are present in a single tandem array. A transcriptional enhancer element lies within the spacer region between each rRNA gene, 2.2 kilobases upstream from the transcription initiation site. We have identified previously two proteins, REB1 and REB2, that bind to specific sites within the enhancer (Morrow, B. E., Johnson, S. P., and Warner, J. R. (1989) J. Biol. Chem. 264, 9061-9068). REB1 binds also to a second, higher affinity site near the promoter, 210 base pairs upstream from the initiation site. This report describes the purification and further characterization of REB1. REB1 is a single polypeptide with an apparent molecular mass of 125,000 Da that binds to the sequence CCGGGTAA. It has been found to bind also within transcriptional control regions of several genes transcribed by RNA polymerase II, such as the UASG of the GAL1-GAL10 spacer. Immunoprecipitation analysis demonstrated that REB1 is phosphorylated.

Proteins that bind to the yeast rDNA enhancer.

The transcription of ribosomal RNA genes differs from that of other genes in several respects: the use of a specialized polymerase, the generally high level of transcription, and the tandem arrangement of the genes. In the yeast Saccharomyces cerevisiae, we identified a nucleotide sequence in the "nontranscribed" spacer region that had many characteristics of an enhancer of transcription (8). More recently, it has become apparent that transcription of this sequence occurs (9) and that it may also be involved in some aspect of termination of 35 S rRNA transcription. The likelihood that there are protein factors involved in termination and activation of transcription and that these may participate in the coupling of the transcription of adjacent rRNA genes led us to search for proteins that might bind to the enhancer. We have identified two such proteins, termed REB1 and REB2, that bind to the enhancer and protect specific sequences from attack by chemical and enzymatic reagents. It is noteworthy that there is a second REB1 binding site approximately 210 base pairs upstream of the origin of transcription of rRNA and that binding of REB1 to this site alters the conformation of DNA adjacent to the site of initiation.

A model for transcription termination by RNA polymerase I.

The transcription termination site for yeast RNA polymerase I requires not only an 11 bp binding site for Reb1p, but also about 46 bp of 5' flanking sequence. We propose that Reb1p bound to its site is part of a pause element, while the 5' flanking sequence contains a release element. Pausing requires little other than the DNA-binding domain of Reb1p and is not specific for polymerase I. The release element, however, can be polymerase specific. We propose a general model for eukaryotic transcription terminators in which termination occurs when a relatively nonspecific signal induces polymerase to pause in the context of a release element.

AT-rich palindromes mediate the constitutional t(11;22) translocation.

The constitutional t(11;22) translocation is the only known recurrent non-Robertsonian translocation in humans. Offspring are susceptible to der(22) syndrome, a severe congenital anomaly disorder caused by 3&rcolon;1 meiotic nondisjunction events. We previously localized the t(11;22) translocation breakpoint to a region on 22q11 within a low-copy repeat termed "LCR22" and within an AT-rich repeat on 11q23. The LCR22s are implicated in mediating different rearrangements on 22q11, leading to velocardiofacial syndrome/DiGeorge syndrome and cat-eye syndrome by homologous recombination mechanisms. The LCR22s contain AT-rich repetitive sequences, suggesting that such repeats may mediate the t(11;22) translocation. To determine the molecular basis of the translocation, we cloned and sequenced the t(11;22) breakpoint in the derivative 11 and 22 chromosomes in 13 unrelated carriers, including two de novo cases and der(22) syndrome offspring. We found that, in all cases examined, the reciprocal exchange occurred between similar AT-rich repeats on both chromosomes 11q23 and 22q11. To understand the mechanism, we examined the sequence of the breakpoint intervals in the derivative chromosomes and compared this with the deduced normal chromosomal sequence. A palindromic AT-rich sequence with a near-perfect hairpin could form, by intrastrand base-pairing, on the parental chromosomes. The sequence of the breakpoint junction in both derivatives indicates that the exchange events occurred at the center of symmetry of the palindromes, and this resulted in small, overlapping staggered deletions in this region among the different carriers. On the basis of previous studies performed in diverse organisms, we hypothesize that double-strand breaks may occur in the center of the palindrome, the tip of the putative hairpin, leading to illegitimate recombination events between similar AT-rich sequences on chromosomes 11 and 22, resulting in deletions and loss of the palindrome, which then could stabilize the DNA structure.

Der(22) syndrome and velo-cardio-facial syndrome/DiGeorge syndrome share a 1.5-Mb region of overlap on chromosome 22q11.

Derivative 22 (der[22]) syndrome is a rare disorder associated with multiple congenital anomalies, including profound mental retardation, preauricular skin tags or pits, and conotruncal heart defects. It can occur in offspring of carriers of the constitutional t(11;22)(q23;q11) translocation, owing to a 3:1 meiotic malsegregation event resulting in partial trisomy of chromosomes 11 and 22. The trisomic region on chromosome 22 overlaps the region hemizygously deleted in another congenital anomaly disorder, velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS). Most patients with VCFS/DGS have a similar 3-Mb deletion, whereas some have a nested distal deletion endpoint resulting in a 1.5-Mb deletion, and a few rare patients have unique deletions. To define the interval on 22q11 containing the t(11;22) breakpoint, haplotype analysis and FISH mapping were performed for five patients with der(22) syndrome. Analysis of all the patients was consistent with 3:1 meiotic malsegregation in the t(11;22) carrier parent. FISH-mapping studies showed that the t(11;22) breakpoint occurred in the same interval as the 1.5-Mb distal deletion breakpoint for VCFS. The deletion breakpoint of one VCFS patient with an unbalanced t(18;22) translocation also occurred in the same region. Hamster-human somatic hybrid cell lines from a patient with der(22) syndrome and a patient with VCFS showed that the breakpoints occurred in an interval containing low-copy repeats, distal to RANBP1 and proximal to ZNF74. The presence of low-copy repetitive sequences may confer susceptibility to chromosome rearrangements. A 1.5-Mb region of overlap on 22q11 in both syndromes suggests the presence of dosage-dependent genes in this interval.

Two functional copies of the DGCR6 gene are present on human chromosome 22q11 due to a duplication of an ancestral locus.

The DGCR6 (DiGeorge critical region) gene encodes a putative protein with sequence similarity to gonadal (gdl), a Drosophila melanogaster gene of unknown function. We mapped the DGCR6 gene to chromosome 22q11 within a low copy repeat, termed sc11.1a, and identified a second copy of the gene, DGCR6L, within the duplicate locus, termed sc11.1b. Both sc11.1 repeats are deleted in most persons with velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS), and they map immediately adjacent and internal to the low copy repeats, termed LCR22, that mediate the deletions associated with VCFS/DGS. We sequenced genomic clones from both loci and determined that the putative initiator methionine is located further upstream than originally described, but in a position similar to the mouse and chicken orthologs. DGCR6L encodes a highly homologous, functional copy of DGCR6, with some base changes rendering amino acid differences. Expression studies of the two genes indicate that both genes are widely expressed in fetal and adult tissues. Evolutionary studies using FISH mapping in several different species of ape combined with sequence analysis of DGCR6 in a number of different primate species indicate that the duplication is at least 12 million years old and may date back to before the divergence of Catarrhines from Platyrrhines, 35 mya. These data suggest that there has been selective evolutionary pressure toward the functional maintenance of both paralogs. Interestingly, a full-length HERV-K provirus integrated into the sc11.1a locus after the divergence of chimpanzees and humans.

Characterization and mutation analysis of goosecoid-like (GSCL), a homeodomain-containing gene that maps to the critical region for VCFS/DGS on 22q11.

Velocardiofacial syndrome (VCFS) is a developmental disorder characterized by conotruncal heart defects, craniofacial anomalies, and learning disabilities. VCFS is phenotypically related to DiGeorge syndrome (DGS) and both syndromes are associated with hemizygous 22q11 deletions. Because many of the tissues and structures affected in VCFS/DGS derive from the pharyngeal arches of the developing embryo, it is believed that haploinsufficiency of a gene(s) involved in embryonic development may be responsible for its etiology. A homeodomain-containing gene, Goosecoidlike (GSCL), has been recently described, and it resides in the critical region for VCFS/DGS on 22q11. GSCL is related to the Goosecoid gene (GSC) in both sequence of the homeodomain and genomic organization. Gsc in the mouse is expressed during early and midembryogenesis and is required for craniofacial rib, and limb development. The chick homolog of GSCL, termed GSX, is expressed during early chick embryogenesis. We detected GSCL expression in human embryos and biphasic expression in mouse embryos. It is possible that the vertebrate GSCL gene is also required for embryonic development. Due to its location in the critical region on 22q11, GSCL is an excellent candidate gene for VCFS/DGS. The vertebrate GSC protein has the same DNA binding specificity as the Drosophila morphogen, bicoid. Upon examination of the putative GSCL promoter, we found three sequence elements with an exact match to the reverse complement of the bicoid DNA recognition motif, suggesting that GSC, or possibly GSCL itself, regulates the transcription of GSCL. Sequence analysis of the putative promoter and the coding region of GSCL was performed on the DNA template from 17 VCFS patients who did not have a detectable 22q11 deletion to identify mutations. We did not detect a mutation in this set of VCFS patients. A polymorphism was detected in codon 47 of exon 1.

A common molecular basis for rearrangement disorders on chromosome 22q11.

The chromosome 22q11 region is susceptible to rearrangements that are associated with congenital anomaly disorders and malignant tumors. Three congenital anomaly disorders, cat-eye syndrome, der() syndrome and velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS) are associated with tetrasomy, trisomy or monosomy, respectively, for part of chromosome 22q11. VCFS/DGS is the most common syndrome associated with 22q11 rearrangements. In order to determine whether there are particular regions on 22q11 that are prone to rearrangements, the deletion end-points in a large number of VCFS/DGS patients were defined by haplotype analysis. Most VCFS/DGS patients have a similar 3 Mb deletion, some have a nested distal deletion breakpoint resulting in a 1.5 Mb deletion and a few rare patients have unique deletions or translocations. The high prevalence of the disorder in the population and the fact that most cases occur sporadically suggest that sequences at or near the breakpoints confer susceptibility to chromosome rearrangements. To investigate this hypothesis, we developed hamster-human somatic hybrid cell lines from VCFS/DGS patients with all three classes of deletions and we now show that the breakpoints occur within similar low copy repeats, termed LCR22s. To support this idea further, we identified a family that carries an interstitial duplication of the same 3 Mb region that is deleted in VCFS/DGS patients. We present models to explain how the LCR22s can mediate different homologous recombination events, thereby generating a number of rearrangements that are associated with congenital anomaly disorders. We identified five additional copies of the LCR22 on 22q11 that may mediate other rearrangements leading to disease.

A common breakpoint on 11q23 in carriers of the constitutional t(11;22) translocation.

Structural chromosomal rearrangements occur commonly in the general population. Individuals that carry a balanced translocation are at risk of having unbalanced offspring; therefore, the frequency of translocations in couples with recurrent spontaneous abortions is higher than that in the general population. The constitutional t(11;22) translocation is the most common recurrent non-Robertsonian translocation in humans and may serve as a model to determine the mechanism that causes recurrent meiotic translocations. We previously localized the t(11;22) translocation breakpoint to a region on 22q11 within a low-copy repeat, termed "LCR22." To define the breakpoint on 11q23 and to ascertain whether this region shares homology with LCR22 sequences, we performed haplotype analysis on patients with der(22) syndrome. We found that the breakpoint on 11q23 occurred between two genetic markers, D11S1340 and APOC3-tetra, both being present within a single bacterial-artificial-chromosome clone. To determine whether the breakpoint occurred within the same region among a larger set of carriers, we performed FISH mapping studies. The breakpoints were all within the same clone, suggesting that this region may harbor sequences that are prone to breakage. We narrowed the breakpoint interval, in both derivative chromosomes from two unrelated carriers, to a 190-bp, AT-rich repeat, which indicates that this repeat may mediate recombination events on chromosome 11. Interestingly, the LCR22s harbor AT-rich repeats, suggesting that this sequence motif may mediate recombination events in nonhomologous chromosomes during meiosis.

Molecular definition of 22q11 deletions in 151 velo-cardio-facial syndrome patients.

Velo-cardio-facial syndrome (VCFS) is a relatively common developmental disorder characterized by craniofacial anomalies and conotruncal heart defects. Many VCFS patients have hemizygous deletions for a part of 22q11, suggesting that haploinsufficiency in this region is responsible for its etiology. Because most cases of VCFS are sporadic, portions of 22q11 may be prone to rearrangement. To understand the molecular basis for chromosomal deletions, we defined the extent of the deletion, by genotyping 151 VCFS patients and performing haplotype analysis on 105, using 15 consecutive polymorphic markers in 22q11. We found that 83% had a deletion and >90% of these had a similar approximately 3 Mb deletion, suggesting that sequences flanking the common breakpoints are susceptible to rearrangement. We found no correlation between the presence or size of the deletion and the phenotype. To further define the chromosomal breakpoints among the VCFS patients, we developed somatic hybrid cell lines from a set of VCFS patients. An 11-kb resolution physical map of a 1,080-kb region that includes deletion breakpoints was constructed, incorporating genes and expressed sequence tags (ESTs) isolated by the hybridization selection method. The ordered markers were used to examine the two separated copies of chromosome 22 in the somatic hybrid cell lines. In some cases, we were able to map the chromosome breakpoints within a single cosmid. A 480-kb critical region for VCFS has been delineated, including the genes for GSCL, CTP, CLTD, HIRA, and TMVCF, as well as a number of novel ordered ESTs.

Molecular and clinical study of 183 patients with conotruncal anomaly face syndrome.

To investigate molecular and clinical aspects of conotruncal anomaly face (CAF), we studied the correlation between deletion size and phenotype and the mode of inheritance in 183 conotruncal anomaly face syndrome (CAFS) patients. Hemizygosity for a region of 22ql1.2 was found in 180 (98%) of the patients with CAFS by fluorescence in situ hybridization (FISH) using the N25(D22S75) DiGeorge critical region (DGCR) probe. No hemizygosity was found in three (2%) of the patients with CAFS by FISH using nine DiGeorge critical region probes and a SD1OP1 probe (DGA II locus). None of these three patients had mental retardation and just one had nasal intonation, which was observed in almost all of the 180 CAFS patients who carried deletions (mental retardation, 92%; nasal voice, 88%). Nineteen of 143 families (13%) had familial CAFS and 16 affected parents (84%) were mothers. Although only two of the affected parents had cardiovascular anomalies, the deletion size in the 16 affected parents and their affected family members, who were studied by FISH analysis, was the same. It indicates that extragenic factors may play a role in the genesis of phenotypic variability, especially in patients with cardiovascular anomalies. No familial cases were found among CAFS patients with absent thymus/DiGeorge anomaly (DGA). Also, in all 18 CAFS patients with completely absent thymus/DGA and all 6 CAFS patients with schizophrenia, it was revealed that the deletion was longer distally. A study of the origin of the deletion using microsatellite analyses in 48 de novo patients showed that in 65% of CAFS patients it was maternal, while in 64% of DGA patients it was paternal. The findings of this study indicated that CAF was almost always associated with the deletion of 22ql1.2. As well as the major features of the syndrome, other notable extracardiac anomalies were found to be susceptibility to infection, schizophrenia, atrophy or dysmorphism of the brain, thrombocytopenia, short stature, facial palsy, anal atresia, and mild limb abnormalities.

Mice overexpressing genes from the 22q11 region deleted in velo-cardio-facial syndrome/DiGeorge syndrome have middle and inner ear defects.

Velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS) is a congenital anomaly disorder associated with hemizygous 22q11 deletions. We previously showed that bacterial artificial chromosome (BAC) transgenic mice overexpressing four transgenes, PNUTL1, (CDCrel-1), GP1B beta, TBX1 and WDR14, had reduced viability, cardiovascular malformations and thymus gland hypoplasia. Since these are hallmark features of VCFS/DGS, we analyzed the mice for additional anomalies. We found that the mice have important defects in the middle and inner ear that are directly relevant to the disorder. The most striking defect was the presence of chronic otitis media, a common finding in VCFS/DGS patients. In addition, the mice had a hyperactive circling behavior and sensorineural hearing loss. This was associated with middle and inner ear malformations, analogous to Mondini dysplasia in humans reported to occur in VCFS/DGS patients. We propose that overexpression of one or more of the transgenes is responsible for the etiology of the ear defects in the mice. Based upon its pattern of expression in the ear and functional studies of the gene, TbX1 likely plays a central role. Haploinsufficiency of TBX1 may be responsible for ear disorders in VCFS/DGS patients.