RECOMENDACIONES

Exclusion o f coding region mutations does not necessarily suggest that a candidate gene is not involved in the pathogenesis of a particular disorder. In addition to an intact coding sequence, with absence of deleterious mutations, normal gene function requires correctly functioning regulatory control. Position effects can give rise to disease as a result of a disruption of the normal chromatin environment

surrounding an intact gene known to be responsible for disease (reviewed by Kleinjan and van Heyningen 1998). There are a number of examples of cases with cytogenetic rearrangements situated outside the causative gene which lead to disease as a result of disrupted gene expression. This phenomenon has been previously described in

craniofacial disorders including holoprosencephaly as a result o f loss of SHH

expression (Roessler et al. 1997), and Saethre-Chotzen syndrome in affected

individuals with translocations downstream of an intact TWIST gene (Rose et al.

1994; Krebs et al. 1997). Such position effects may arise as a result of a change in the position o f a gene relative to its normal environment. Therefore cw-acting regulatory elements, such as the promoter region or distant enhancer/repressor elements, may be unable to regulate gene transcription. Such an effect may also arise in the absence of

transcribed portion of a gene. This has been dramatically demonstrated in the mouse

aphakia mutant, characterized by hyploplasia of the eyes with absence of the lens.

The mutant phenotype arises from deregulated expression of an intact Pitx3 gene as a

result o f a 652 bp deletion situated approximately 2.5 Kb upstream o f the gene (Semina et al. 2000).

None of the genes screened for mutations in family P in this study can be excluded from having a causative role in the development of HFM as a result of a

possible position effect. With respect to GSC, the most suitable candidate in the

region, although no deletions were detected within a 9 Kb genomic fragment encompassing the gene, this effect still cannot be ruled out. Long ranging position effects over several hundred kilobases in some cases have been identified in humans. Patients with X-linked deafness mapping to the DFN3 locus, characterized by fixation of the stapes and mixed hearing loss, have been shown to result from

duplications/inversions and deletions located up to 900 Kb proximal to the POU3F4

gene, in which mutations had previously been identified in some affected individuals mapping to DFN3 (de Kok et al. 1995a; de Kok et al. 1995b; de Kok et al. 1996). Furthermore, transgenic deletion mapping experiments in the mouse have revealed that some genes may have complex mechanisms of regulation through a number of distinct and discrete enhancer elements dispersed over several kilobases of the gene locus. For certain genes, individual enhancers may be responsible for regulating temperospatial gene expression in specific domains o f the developing embryo

(Summerbell et al. 2000). This suggests that loss or alteration of a specific regulatory element may result in aberrant gene expression in a particular tissue at a certain time, which may give rise to localised defects.

Given the diverse distribution and general lack of knowledge o f regions involved in the regulation of expression of human genes, a search for potentially pathogenic regulatory mutations is likely to be extremely time consuming and minimally productive. Techniques such as Southern blotting, pulsed field gel

electrophoresis, or FISH could be used to look for potential pathogenic microdeletions on 14q32 in affected members of family P. Potential sites likely to be involved in gene regulation could be targeted by means of comparative sequence analysis

between closely related species to identify conserved non-coding regions, particularly those containing putative transcription factor binding sites (Wasserman et al. 2000).

The strategy undertaken so far, in this work, o f screening for exonic mutations by direct sequencing in family P may be ineffectual in identifying certain pathogenic mutations. Whole exon deletions may not be detected using this approach. If the extent o f the deletion encompasses the primer sets used in the analysis, then only the

intact normal copy of the gene would be amplified. With the exception of GSC in this

study, no attempt was made to identify intragenic deletions in any o f the other genes studied. However, in most cases where single nucleotide polymorphic variation was detected, heterozygous affected individuals were observed. The application of genotyping SNPs in affected individuals to detect possible deletions provides a rapid method to exclude the possibility o f complete loss of a particular exon. This has the advantage o f being less time consuming than Southern blotting, as in most cases heterozygous SNPs should be detected by sequencing or direct restriction enzyme digestion o f PCR products. However, in the absence of intragenic SNPs other methods for detection of deletions, or duplications, may be required.

Additionally, the approach of screening exonic sequence for mutations will fail to detect the possibility o f pathogenic intronic variation. Intronic regions may

contain regulatory sequences critical for normal gene expression. BDKRBl, for

example, on chromosome 14q32 contains a putative promoter element in intron 2 of the gene which has been suggested to be involved in tissue specific expression (Yang and Polgar 1996). Furthermore, intronic mutations may result in aberrant splicing of the mRNA transcript, for example by disrupting the branch site, which plays an important role in the splicing mechanism (Burrows et al. 1998; Janssen et al. 2000).

More recently, an intronic mutation has been reported in the CDKN2A gene in

melanoma families resulting in the creation of a splice donor site deep within the second intron of the gene. This leads to the aberrant splicing of the transcript with some mRNA species ignoring the newly created splice donor, but retaining the entire intronic sequence (Harland et al. 2001). Incidentally, mutations in the 5’ untranslated

region o f the CDKN2A gene had previously been identified in a different subset of

melanoma patients leading to the creation of a novel translation initiation codon and a truncated gene product, with decreased translation from the wild-type start codon (Liu et al. 1999). Such observations present another mechanism for mutations in non­ coding regions o f a gene having a role in disease causation. Directly sequencing cDNA derived from affected individuals is likely to detect mutations resulting in an altered transcript, however access to this material may not always be possible.

Lymphoblastoid cell lines are available for affected members of family P which could be used to extract RNA, however not all genes will be expressed in lymphoblasts, particularly those with critical functions in early embryogenesis. This is demonstrated

by the failure to detect GSC expression in a normal lymphoblast cell line by RT-PCR

Additionally it must be considered that unidentified exons located upstream of the known gene sequence may exist, that could be involved in the production of alternative transcripts. This is a particular concern for genes predicted by computer analysis whose exonic structure has not been confirmed by experimental methods.

In addition to those screened as part o f this study, other potential candidate genes exist within the candidate HFM region on 14q32, particularly genes o f as yet undetermined function. Therefore, it may be a more practical compromise to continue the strategy o f prioritising and screening these genes prior to undertaking a time consuming and somewhat blind search for potential regulatory mutations. However, extension of the mutation screening approach to include intronic regions of genes in some cases, and the identification of intragenic SNPs to rule out potential deletions should be considered.