Biology-Driven Candidate Gene Strategy to Identify Modifier Genes
Strategies used to show the role of genetic factors in phenotypic expression can be classified into two categories: (i) a systematic approach in which the whole genome is scanned, and (ii) an approach focusing on candidate genes or pathways. The candidate-gene approach can be defined as the study of genetic determinants of a complex trait based on: (i) generating hypotheses and identifying candidate genes that might have a pathogenic role; (ii) identifying variants (SNPs) in or near these genes; and (iii) genotyping of variants in a population, followed by statistical methods (linkage or association) to identify correlations with the phenotype. Testing of variants in carefully selected candidates is attractive for several reasons: the number of variants is generally small, thereby avoiding severe penalties for multiple comparisons during the statistical analysis. A detailed understanding of the candidate gene product and its variants provides mechanistic insight and facilitates experimental studies to evaluate the modifier effects.
Several approaches can be used to select candidate genes. A deeper understanding of the biochemical functions of neurofibromin may lead to the discovery of interacting proteins and of upstream and downstream effectors that are critical for the development of particular phenotypic features. In both humans and mice, NF1 tumour development results from a combination of ubiquitous NF1 heterozygosity and unpredictable NF1 loss of heterozygosity in different cell lineages. Neurofibroma-derived Schwann cells harbouring two mutated NF1 alleles (NF1) have been isolated from several neurofibromas. Mitotic recombination is the likely mechanism underlying this loss of heterozygosity. As mitotic recombination shows inter-individual variation, genes that control this phenomenon may partly explain the variable number of neurofibromas in NF1 patients, by influencing the somatic mutation rate.
The variable number of NF1-associated neurofibromas could also be due to variable accumulation of somatic NF1 mutations. Two research groups have described the role of MMR genes in neurofibroma development in NF1. Both provided evidence that a reduction in MMR capacity can result in NF1 mutations in a high percentage of neurofibromas. It has been postulated that constitutional or early alterations of MMR genes in NF1 patients may lead to an accumulation of second hits in NF1, a human gene with one of the highest mutation rates. However, apart from one report, no constitutional mutations in MMR genes have been detected in NF1 patients. A recent study examined whether increased tumour load in NF1 (higher number of cutaneous neurofibromas) was associated with methylation of MMR genes. Titze et al performed methylation-specific PCR and pyrosequencing of MMR gene promoters most frequently involved in human cancers (MLH1, MSH6, PMS2, and MSH2) in leukocytes of NF1 patients. They found that in NF1 patients with a high number of cutaneous neurofibromas versus those with a low, methylation of two (out of six) CpG dinucleotides in MSH2 promoter was enhanced.
Biology-driven modifier genes have also been suggested to play a role in dermal neurofibromas. Dermal neurofibromas occur in virtually all individuals with NF1. Recent elegant studies have pointed to skin-derived neural progenitors (SKPs), or their derivatives, as the cell of origin of NF1-associated dermal neurofibromas. When Nf1-homozygous SKPs were autologously implanted intradermally in mice, they only gave rise to dermal neurofibromas in female mice that were pregnant at the time of implantation, and not in males or non pregnant females. This suggested that the hormonal environment may be a critical factor in the onset of dermal neurofibromas. NF1 patients typically begin to develop dermal neurofibromas around puberty, and the number and size of neurofibromas increases during pregnancy. McLaughlin et al reported that 5% of human neurofibromas express the oestrogen receptor (ER), while 75% express the progesterone receptor. Studies have confirmed steroid hormone receptor expression and ligand-mediated cell growth and survival in both normal human Schwann cells and neurofibroma-derived Schwann cell cultures. Moreover, an increased potential for malignant transformation of plexiform neurofibromas has been reported during pregnancy. The selective ER modulator tamoxifen has been tested for its ability to inhibit MPNST tumourigenesis. Tamoxifen showed potent antitumor activity in mice orthotopically xenografted with human MPNST cells, providing a rationale for clinical trials. Thus, steroid hormones may directly influence neurofibroma initiation or progression by acting through their cognate receptors, but these effects may only apply to a subset of tumours.
Because of their co-localisation with neurofibromin, mitochondria are also attractive NF1 modifier candidates. In Drosophila, neurofibromin regulates longevity and stress resistance through cAMP regulation of mitochondrial respiration and ROS production. Recently, the role of mitochondria in tumour development has gained much attention, with reports of somatic mitochondrial DNA (mtDNA) mutations in several human cancers. Somatic mtDNA mutations have also been described in NF1-associated neurofibromas. Moreover, variations in mtDNA copy numbers are increasingly reported in a range of primary human cancers, suggesting that they may be critical for cancer pathogenesis and progression. Mitochondria contain multiple copies of circular double-stranded DNA molecules that exhibit a high degree of sequence variation across individuals. Detjen et al studied nucleated blood cells from four pairs of NF1 discordant MZ twins and from cutaneous neurofibromas of one twin pair, but failed to find evidence of mtDNA sequence differences or different degrees of heteroplasmy.