Disruption of a long-range cis-acting regulator for Shh causes preaxial polydactyly

Identification of a PPD Translocation Breakpoint.

A 3-year-old girl presented with bilateral duplication of the triphalangeal thumb and triplication of the great toe without any associated abnormality (Fig. 1A). She was found to carry a de novo reciprocal translocation t(5,7)(q11,q36). Fine mapping of the translocation breakpoint by use of FISH analysis in the lymphoblastoid cell line derived from the patient (Fig. 1C) identified a BAC clone Rp11-332E22 (GenBank accession no. AC007097) that spanned the breakpoint (Fig. 1B). Using fragments from the BAC, a cosmid clone 171g2 was identified that spanned the breakpoint (Fig. 1B). Sequencing the ends of the cosmid insert identified the genomic fragment as lying inside one of the genes (originally given the designation C7orf2 (5) within the genetically defined critical region for PPD. This gene is the orthologue of the mouse Lmbr1 gene (14). Southern blot analysis of DNA from a lymphoblastoid cell line further refined the site of the breakpoint (Fig. 1D). A number of repeat-free STSs including 13–293 and 13–479 were PCR-amplified and used as probes. This analysis showed that the translocation breakpoint is located in a 714-bp BamHI/XbaI fragment residing between exons 5 and 6 of LMBR1 without an accompanying large deletion (Fig. 1E). This DNA fragment consists of 95% LINES and 5% simple repeated sequence, and seems to neither encode an ORF nor contain known regulatory sequences.

• Download figure
• Open in new tab
• Download powerpoint

Figure 1

Phenotype and identification of the breakpoint in the t(5 7)(q11,q36)translocation patient. (A) Left hand (Left, ventral view) of the PPD translocation patient showing duplication of the triphalangeal thumb. Radiogram of the right hand (Center) showing bilateral duplications and of the right foot (Right) showing triplication of the great toe. (B) The YACs, PACs, BACs, and a cosmid used to define the translocation breakpoint. (C) FISH analysis of metaphase chromosomes from a lymphoblastoid cell line from the patient, using cosmid clone171G2 as a probe. Hybridization signals are marked by arrows on the wild-type chromosome 7 and on the products of the translocation, the derived chromosome 5 (der 5) and derived chromosome 7 (der 7). (D) Southern blot analysis with genomic DNA from the lymphoblastoid cell line from the patient (P) and a normal control (N). DNA was digested with restriction endonucleases BamHI and XbaI. STS probe 13–293 (Left) and STS probe 13–479 (Right) were used. The relative positions of the STS probes, restriction sites, and the translocation breakpoint are indicated (E).

Subsequently, we performed mutation analysis on the LMBR1 gene in patients. Analysis of the exons and the intron/exon boundaries of the LMBR1 gene in five unrelated PPD families (1, 4) uncovered no pathogenic mutations (data not shown). Therefore, in the PPD families mutations interrupting either the coding region or the structure of the transcript are not apparent.

Isolation of the Ssq Insertion Site.

To isolate the transgenic insertion site from the Ssq mutant mouse, a λ genomic library was made from Ssq/Ssq mice. Clones were isolated using the transgene as a probe, one of which contained a junction of the transgene insertion site (Fig. 2B). The genomic end fragment of this λ clone (Fig. 2B) was used to identify mouse PAC clones which, by chromosomal FISH analysis, were shown to co-localize with the transgene (Fig. 2A). Subsequent exon trapping (12) of the PAC RPCI21-542N10 identified a surrounding gene. The complete sequence of the corresponding mouse cDNA was determined and is identical to mouse Lmbr1 (14).

• Download figure
• Open in new tab
• Download powerpoint

Figure 2

Identification of the mouse Ssq insertion site. PAC RPCI21-542N10 was isolated as containing the transgene integration site. (A) FISH analysis confirms the colocalization of the PAC with the transgene. PAC RPCI21-542N10 is labeled in green and the human placental alkaline phosphatase reporter gene (HPAP)-containing transgene is labeled in red. (Upper) Colocalization in an interphase nucleus; (Lower) colocalization in a metaphase spread. (B) The regions of the Lmbr1 gene covered by the two PAC clones (RPCI21 542N10 and 576B18) isolated. The position of the intron in the Lmbr1 gene (exon/intron gene structure from human sequence) and the intronic repeat structure (boxes) derived from the complete sequence between exons 5 and 6 is shown. Unique sequence DNA was predicted and four probes were generated (positions shown below intronic structure numbered 1–4). The positions of transgene integration (arrows with asterisks) and the area of intron duplication (below asterisks) are indicated. The resolved structure after integration is shown (bottom line), indicating intronic duplication as defined by probes 1–4. The positions of the λ and cosmid genomic clones (lines underneath) that defined the integration sites are marked. (C) RT-PCR samples were removed after 25, 30, and 35 cycles. RNA is from 11.5-day embryos of wild-type (+) and Ssq/Ssq (S) genotypes; a no RNA control (0) is also included. Top and Middle make use of primer pairs specific to Lmbr1; the first is from exon 3 to exon 5 and examines expression levels 5′ of the transgene integration. Comparable levels are detected in wild-type and Ssq/Ssq embryos. Middle uses primers that cross the integration site spanning exons 3 and 6. With these, at 30 cycles a product is only detected in the wild-type sample. The primers used in this experiment cross-hybridize with the mouse glycosylphosphatidylinositol-anchored protein homologue (closed arrowhead), which serves as positive control for the experiment and to normalize the samples. (Bottom) primers from the HPRT locus were used as positive control for RNA integrity and controls for reverse transcription.

Subsequently, a cosmid library was made from Ssq genomic DNA and additional clones were isolated to more fully elucidate the integration site. The transgene/genomic junction from the other end of transgenic integration was identified (Fig. 3B). Comparison of sequence from this cosmid clone with the complete intron 5 sequence we additionally assembled defined the position of this integration. Multiple copies of the transgene have integrated, lying wholly within intron 5 of the Lmbr1 gene (genomic sites of insertion are marked by asterisks in Fig. 2B). The process of integration has resulted in a duplication of approximately 20 kb of the intron with the transgene residing between the duplication endpoints (lower line in Fig. 2B). The duplication was confirmed by Southern blot analysis using unique sequence probes (probes numbered 1–4 in Fig. 2B). The surrounding DNA including exons 5 and 6 was apparently unaffected by the integration.

• Download figure
• Open in new tab
• Download powerpoint

Figure 3

Cis–trans genetic test. A shows the genetic cross used. A homozygous Ssq mouse was crossed to a Shh^null heterozygote. The F₁ mice that carry both mutations on opposite chromosomes, all of which exhibit preaxial polydactyly, were crossed to wild-type mice. The bottom row depicts the outcome of the cross. The majority of G₂ progeny were, as expected, found to be heterozygous for either the Ssq allele or the Shh^null (218 and 220 mice, respectively). The mice of interest were those identified as carrying a chromosome 5 that has recombined between the Shh locus and the region of the Ssq insertion. Three had inherited the wild-type alleles at both loci and five carried the Ssq allele and the Shh^null allele in cis. These mice show no additional digits and have wild-type paws. This cross results in a genetic distance of 1.8 cM between Shh and Ssq. B shows examples of the genotyping PCRs used to analyze the G₂ progeny. Lanes 1–3 are mice heterozygous for the Shh^null allele. Lanes 4–6 are wild type for Shh but carry the Ssq insertion. Mice in lanes 7–12 carry the recombinant chromosomes; 7–9 show G₂s with both Shh^null and Ssq, whereas 10–12 are wild type at both loci.

The consequence of the Ssq transgenic insertion on Lmbr1 transcription was analyzed. The transgene containing Lmbr1 gene encodes full-length transcripts, further supporting the proposal that arrangement of exons is unaffected by the insertional mutation. However, Lmbr1 transcription in Ssq embryos does show a high level of premature termination. The 5′ end of the mutant Lmbr1 transcript (Fig. 2C Top) is produced at wild-type levels, but transcription of exons 3′ of the insertion site is detectably lower, representing about 10–30% of wild-type levels (Fig. 2C Middle). The transgene insertion apparently leads to termination of transcription between exons 5 and 6.

Furthermore, we investigated for possible premature transcriptional termination in the familial PPD patients. We amplified LMBR1 transcripts by reverse transcription (RT)-PCR from RNA from lymphoblastoid cell lines of Dutch and Cuban patients, and sequenced the products. Polymorphisms in a Cuban (in exons 13 and 17) and two related Dutch (one in exon 5 and the other in exon 14) patients indicated that both LMBR1 alleles, at the 5′ end and the 3′ end, were capable of being expressed. Furthermore, an overlapping series of three PCR products covering the whole LMBR1 transcript showed no detectable difference in band size between patients and controls (data not shown), indicating that no exons are skipped in patients. These data argue that mutations disrupting LMBR1 transcriptional capacity are not commonly associated with the PPD phenotype.

Cis–Trans Genetic Analysis.

We previously showed (9) an intriguing relationship between the Ssq mutation and Shh expression. The reporter gene contained within the Ssq transgene insertion has acquired a limb-specific, Shh-like expression pattern; not only in the appropriate posterior ZPA (15), but also at an ectopic site at the anterior margin of the limb bud. It is unclear from the present data whether the mechanism for Shh misexpression involves disruption of the Lmbr1 gene and is therefore indirect or, as suggested (9), direct because of disruption of a long range Shh regulatory element. Long range regulation of Shh has been previously postulated for a translocation identified in a holoprosencephaly patient in which the breakpoint resides 250 kb upstream (16).

To examine the basis for preaxial polydactyly we devised a cis–trans genetic test. We predicted that an Shh regulator would function in a cis-acting manner—i.e., that the Ssq mutation would affect only chromosomally linked Shh. In contrast, disruption of a gene that secondarily affected Shh expression would operate on both Shh alleles and, therefore, in trans. A mouse cross was devised to derive a recombinant chromosome 5 in which the Ssq mutation was located in cis to an easily distinguishable Shh allele (Fig. 3A). Toward this end, we crossed Ssq/Ssq male mice with females carrying the mutant Shh null allele (Shh^null) (13) to produce the F₁ generation. The Shh^null allele provided a straightforward assay for both the genotype (Fig. 3B) and the phenotype of each offspring. In the analysis of the phenotype, mice carrying the recombinant chromosome would exhibit extra preaxial toes if Ssq is acting on Shh in trans and wild-type feet—i.e., suppression of the Ssq phenotype—if acting in cis.

The F₁ generation (n = 14) carrying Ssq and Shh^null on opposing chromosomes showed complete penetrance of the Ssq mutant phenotype. (Shh^null shows no phenotype in the heterozygous state.) The second generation (the G₂) was produced by mating the F₁ mice to wild type with the aim of generating the recombinant chromosome 5. Each G₂ mouse was assessed by PCR for the presence of the Ssq transgene insertion (Fig. 3B Upper) and the neo^r gene associated with Shh^null (Fig. 3B Lower). In this cross we produced 446 G₂ offspring of which eight were recombinants, representing a recombination frequency of 1.8 ± 0.6%. Three of the recombinants were uninformative because they contained the wild-type alleles (Fig. 3A). The other five recombinants carried both the Ssq insertion and Shh^null allele situated in cis. Analysis of the limb phenotype in these five recombinants showed no preaxial polydactyly or other detectable limb phenotypes. In addition, we bred two males carrying the recombinant chromosome 5 (Ssq and Shh^null) with wild-type females and showed that the next generation had no limb abnormalities (21 mice carrying the recombinant chromosome of 37 offspring). On some genetic backgrounds the Ssq heterozygous mutation is not fully penetrant. In this cross 7% of the Ssq/+ mice generated showed no detectable limb phenotype. Two of these Ssq/+ males, which displayed no limb phenotype, were bred further and were shown to transmit the phenotype (7 with additional preaxial digits of 19 offspring). Thus the data demonstrate that the Shh null allele inactivates the effects of the Ssq mutation when located in cis (but not in trans) on chromosome 5. It follows that the Ssq insertion is a dominant acting mutation that interferes with the limb-specific expression of Shh. The data are consistent with a long range limb-specific regulator of Shh residing within or near the Lmbr1 gene.