SIMULACIÓN Y ASIGNACIÓN DE CARGAS

As few H E SXl mutations were identified other candidate genes were considered which could account for the SOD phenotype. However, this produced a large number o f potential SOD candidate genes. Therefore the portion o f the patient cohort with additional features was examined for phenotypes strongly suggestive o f particular candidate genes. Two such phenotypes were readily identifiable. The first consisted o f 13 patients with cleft lip and/or palate in addition to components o f SOPD, in particular pituitary defects. This group were screened for mutations in the gene P T X l. The second consisted o f 8 patients with

components o f SOPD, in particular pituitary defects, in addition to defects o f left-right asymmetry. This group was sequenced for mutations in PTX2.

4.1

PTXL

4.1.1 Introduction.

P txl {PITXl,POTXl,BFT) is a bicoid-like homeodomain transcription factor. Originally cloned in the mouse as a gene expressed in the pituitary that is able to activate the POMC gene (Lamonerie et a l, 1996), it is the founder member o f the pituitary OZY'-related factors. In addition to its ability to activate POMC, it has been shown to have a role in limb

development, craniofacial development, and in the expression o f interferon a genes.

The murine P tx l gene is first expressed during gastrulation at E6-7 within the newly formed mesoderm at the posterior margin o f the embryonic endoderm, where the primitive streak will initially appear. Expression is maintained in derivatives o f this tissue, the allantois and posterior lateral mesoderm (E7.5). By E8, expression is observable in the somatic and splanchnic mesoderm. Later, by E9-9.5, expression is additionally detectable in the umbilical vessels. Expression within the posterior is limited to the lateral plate mesoderm. Following this pattern o f expression, P txl is expressed within the hind limb mesenchyme by E l 1.5 (Lanctot et a l, 1997). P txl is only expressed in the latter stages o f fore limb development (Szetao et a l, 1999). At E8, an additional zone o f expression within the anterior occurs within the oral ectoderm, oral membrane, and rostal foregut, extending into a v-shaped segment within the facial ectoderm from underneath the future lens placode to the first branchial arch. At E9, P txl expression appears in the first branchial arch mesenchyme. Expression is maintained until at least E l 0 in a number o f tissues derived from these regions

(Lanctot et a l, 1997). Crucially, expression is maintained throughout anterior pituitary development and P txl is expressed in the adult post-natal pituitary. Expression o f P tx l initially occurs throughout the pituitary, but after E l 6 expression is limited to the POMC expressing cells (Lamonerie et a l, 1996). Expression o f the chick gene, cPtxl, is very similar to that seen in the mouse (Lamonerie et a l, 1996).

At the transcription level, P txl has been shown to interact with a number o f other transcription factors, SF-1 and P itl (Tremblay et a l, 1998), Tpit (Lamolet et a l, 2001), N euroD l/^2 (Poulin et a l, 1997), and H esxl (Quirk and Brown 2002). The role o f P txl in expression o f aG SU and PLH is well characterised, and two target binding sites 5 ’-CTTA-3’ (aG SU ) and 5 '-ATTA (PLH) have been identified (Tremblay et a l 1998 and Quirk et a l, 2001). H esxl is also able to use these two sites to repress transcription. However, P tx l is able to bypass H esxl monomer mediated repression at the aG SU site, whilst H esxl cooperative dimérisation is able to block P txl access to the |3LH site (Quirk and Brown 2002). The significance o f these data remains unclear, given that H E SX l expression is extinguished before gonadotrope differention occurs. It might be that H E SXl acts to limit PTX l activation o f these Gonadotrope specific genes during early pituitary development. P txl -/- mice show a striking phenotype where the distal hind limbs resemble the fore limbs (Szetao er a/., 1999). In addition, defects of the anterior pituitary are also apparent. (3LH, PPSH, pTSH and aG SU levels are diminished in P txl -/- mice indicating that the number o f gonadotropes and thyrotropes is reduced and/or defective. There appears to be no reduction in POMC transcripts, indeed ACTH levels are increased. It is a variable phenotype and P txl -/- mice can also manifest midline abnormalities, such as cleft palate.

P TX l maps to 5q31 (Crawford et a l, 1997) and based upon the expression and function of P tx l, as well as, the phenotype o f P txl -/- mice P T X l is considered to be a candidate gene for anterior pituitary defects associated with facial clefting.

A sub-population o f the patient cohort (n=31) was screened for mutations in P T X l. 13 patients with cleft lip and/or palate in addition to components o f SOPD and 1 patient with facial dysplasia in addition to SOPD were screened for P T X l mutations given the cranio­ facial phenotype o f the P txl null mutant mouse. Similarly, 3 patients with multiple midline abnormalities were screened. As P txl plays an important role in pituitary development 7 patients with isolated pituitary disease were screened, as were 7 patients with SOPD. The sporadic cases (n=28) were screened by dHPLC-heteroduplex detection with all samples “spiked” with WT DNA, whilst familial cases (n=3) were directly sequenced.

4.1.2 Methods.

Oligonucleotides were derived from genomic contig gi:7708834 using identical procedures as in section 2.3.1 (Table 4.1.2a).

Oligonucleotide

Sequence