Spontaneous mutations can cause either loss or gain of function of the normal gene through different mechanisms. For example, loss-of-function mutations in genes that code for structural or regulatory proteins may result in dominant phenotypes through haploinsufficiency (i.e., a single normal gene is not sufficient for normal functioning) or through dominant negative effects (i.e., the mutant product interferes with the function of the normal gene in the heterozygote). While loss of function of a gene can result from a variety of molecular alterations including deletions, gain-of-function mutations are likely only when specific changes in the gene cause a given disease phenotype. Radiation-induced mutations, because they are often multigene deletions, cause loss of function through haploinsufficiency.

Despite the existence of a number of differences between spontaneous and radiation-induced mutations as outlined above, radiation mutagenesis studies with a variety of experimental systems have been very successful. The possible reasons for this are now becoming evident: although the choices of marker genes in early studies of induced mutations were dictated more by practical considerations (e.g., obtaining sufficient numbers of mutants, unambiguous identification through their respective phenotypes) than by their relevance to human genetic diseases, in retrospect it is clear that the “successful” mutation test systems have been those in which most of these marker genes, and the genomic regions in which they are located, are nonessential for the viability of heterozygotes (in vivo) or of the cell carrying the induced genetic change (in vitro). Consequently, induced mutations—predominantly deletions—could be recovered and studied. Most human genes, however, do not appear to be of this type.

ANNEX 4E: CRITERIA USED TO ASSIGN HUMAN GENES TO ONE OF THREE GROUPS FROM THE STANDPOINT OF THE RECOVERABILITY OF INDUCED MUTATIONS IN LIVE BIRTHS

The genes included in the analysis are a subset of those in which mutations cause autosomal dominant and X-linked diseases, which have provided the basis for the overall incidence estimates for these diseases discussed earlier (Sankaranarayanan 1998). Since not all of them fulfilled the requirements for inclusion (because of insufficient information about one or more of the following: gene size, structure, function, genomic context, etc.), only a subset could be used. The “gene-richness” or “gene poorness” of given genomic regions was assessed using the MIM (Medelian Inheritance in Man) gene maps that present the cytogenetic location of “disease genes” and other expressed genes in given cytogenetic bands (McKusick 2000.).

A gene is assigned to group 1 (induced deletions unlikely to be recovered and/or unlikely to cause the phenotype of the disease under study) when the phenotype of the naturally occurring disease is due to specific (1) gain-of-function mutations (e.g., the FGFR3 gene involved in achondroplasia); (2) trinucleotide repeat expansions (e.g., Huntington’s disease); (3) dominant negative mutations (e.g., the COL1A1 gene involved in osteogenesis imperfecta); and (4) restricted array of point mutations (e.g., mutations in the APOB gene involved in one form of familial hypercholesterolemia). Also included in this group are genes that are relatively small in size and located in putative gene-rich regions (e.g., the VMD2 gene in Best’s macular dystrophy).

The gene is assigned to group 2 (uncertain recoverability) when (1) it is large, it codes for an essential structural protein, and the known genetic changes are missense or nonsense mutations; (2) whole-gene deletions are rare; (3) whole-gene deletions are not rare, but the gene is located in a putative gene-rich region; and (4) information on these other genes and their function is insufficient (e.g., BRCA2; VHL [von Hippel-Lindau syndrome]).

Group 3 (potentially recoverable) includes genes that are generally large and constitutional deletions, some extending beyond the confines of genes, and translocations or inversions with breakpoints in the gene causing the disease phenotype are known despite the putative gene-rich nature of the genomic region (e.g., EXT1 [multiple exotoses]; RB1 [retinoblastoma]).

For X-linked genes, the assessment is based on whether the induced deletion will be compatible with viability in males and cause disease (since the loss of the whole X chromosome is compatible with viability but results in 45,X females).

ANNEX 4F: RADIATION STUDIES WITH EXPANDED SIMPLE TANDEM REPEAT LOCI IN THE MOUSE AND MINISATELLITE LOCI IN HUMAN GERM CELLS

Introduction

The mouse and human nuclear genomes, like those of other complex eukaryotes, contain a large amount of highly repeated DNA sequence families most of which are transcriptionally inactive (Singer 1982). Among these are the simple sequence repeats that are perfect or slightly imperfect tandem repeats of one or a few base pairs (bp). In the mouse genome, the tandem repeat loci are represented by (1) relatively short microsatellites (<500 bp) with a repeat size of 1 to 4 bp; (2) long expanded simple tandem repeats (0.5 to 16 kilobases, repeat size 4 to 6 bp); and (3) true minisatellites (0.5 to 10 kb) with repeat size of 14 to 47 bp (Gibbs and others 1993; Bois and others 1998a, 1998b; Blake and others 2000).

Mouse ESTRs

The ESTRs were originally called minisatellites but have recently been renamed to distinguish them from the much



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