Ionizing radiation and genetic risks VII. The concept of mutation component and its use in risk estimation for Mendelian diseases

The responsiveness of Mendelian diseases to an increase in the mutation rate is studied by using the concept of the mutation component (MC) of genetic diseases. Algebraic expressions to evaluate MC at any specific generation following either a one-time or a permanent increase in mutation rate are derived and are illustrated with numerical examples. For a one-time increase in mutation rate, the analysis shows that the first generation MC for autosomal dominant diseases is equal to the selection coefficient; this is also true for X-linked diseases (adjusted for the proportion of X-chromosomes in males). For autosomal recessive diseases the first generation MC is substantially smaller than that for autosomal dominants. In subsequent generations MC gradually decays to zero. Under conditions of a permanent increase in the mutation rate, the MC for autosomal dominant, X-linked and completely recessive autosomal disorders progressively increases to reach a value of one at the new equilibrium. For incompletely recessive autosomal disorders, however, the MC at equilibrium can be larger than one. The rates of approach to the new equilibrium are different for the different classes of diseases, dictated by selection and time (in generations) following radiation exposure. The effects of increases in mutation rate on MC are more pronounced for autosomal dominants, followed by X-linked and are far less for autosomal recessives. Even for autosomal dominants, the early generation effects of radiation exposures would not be appreciable unless the heterozygotes have a severely reduced fitness.     

Ionizing radiation and genetic risks. XIII. Summary and synthesis of papers VI to XII and estimates of genetic risks in the year 2000

This paper recapitulates the advances in the field of genetic risk estimation that have occurred during the past decade and using them as a basis, presents revised estimates of genetic risks of exposure to radiation. The advances include: (i) an upward revision of the estimates of incidence for Mendelian diseases (2.4% now versus 1.25% in 1993); (ii) the introduction of a conceptual change for calculating doubling doses; (iii) the elaboration of methods to estimate the mutation component (i.e. the relative increase in disease frequency per unit relative increase in mutation rate) and the use of the estimates obtained through these methods for assessing the impact of induced mutations on the incidence of Mendelian and chronic multifactorial diseases; (iv) the introduction of an additional factor called the "potential recoverability correction factor" in the risk equation to bridge the gap between radiation-induced mutations that have been recovered in mice and the risk of radiation-inducible genetic disease in human live births and (v) the introduction of the concept that the adverse effects of radiation-induced genetic damage are likely to be manifest predominantly as multi-system developmental abnormalities in the progeny.For all classes of genetic disease (except congenital abnormalities), the estimates of risk have been obtained using a doubling dose of 1 Gy. For a population exposed to low LET, chronic/ low dose irradiation, the current estimates for the first generation progeny are the following (all estimates per million live born progeny per Gy of parental irradiation): autosomal dominant and X-linked diseases, approximately 750-1500 cases; autosomal recessive, nearly zero and chronic multifactorial diseases, approximately 250-1200 cases. For congenital abnormalities, the estimate is approximately 2000 cases and is based on mouse data on developmental abnormalities. The total risk per Gy is of the order of approximately 3000-4700 cases which represent approximately 0.4-0.6% of the baseline frequency of these diseases (738,000 per million) in the population.     

Estimation of genetic risks of exposure to ionizing radiation: status in the year 2000

This paper provides an overview of the advances in the estimation of genetic risks of exposure of human populations to ionizing radiation with particular emphasis on the advances during the last decade. Among the latter are: (a) an upward revision of the estimates of the baseline frequencies of Mendelian diseases (from 1.25 to 2.4%); (b) the conceptual change to the use of a doubling dose based on human data on spontaneous mutation rates and mouse data on induced mutation rates (from the one based entirely on mouse data on spontaneous and induced mutation rates, which was the case thus far); (c) the fuller development of the concept of mutation component (MC) and its application to predict the responsiveness of Mendelian and chronic multifactorial diseases to induced mutations; (d) the concept that the major adverse effects of radiation exposure of human germ cells are likely to be manifest as multi-system developmental abnormalities and (e) the concept of potential recoverability correction factor (PRCF) to bridge the gap between induced mutations studied in mice and the risk of genetic disease in humans. For a population exposed to low LET, chronic/low dose-rate irradiation, the current estimates of risk for the first generation progeny are the following (all estimates per million live born progeny per Gy of parental irradiation): autosomal dominant and X-linked diseases, approximately 750 to 1,500 cases; autosomal recessive, nearly zero; chronic multifactorial diseases, approximately 250 to 1,200 cases and congenital abnormalities, approximately 2,000 cases. The total risk per Gy is of the order of approximately 3,000 to 4,700 cases which represent approximately 0.4 to 0.6% of the baseline frequency of these diseases. The main message is that at low doses of radiation of interest in risk estimation, the risk of adverse hereditary effects is small.     

Population genetic studies of retinitis pigmentosa.

A questionnaire survey characterized a sample of 670 probands with retinitis pigmentosa (RP) and allied disorders. Segregation analysis provided some evidence for a small proportion of sporadic cases and for decreased segregation ratios of the dominant and recessive genotypes, which could be attributed to delayed age of onset in some cases. The overall incidence of RP was indirectly calculated to be approximately 1 in 3,700, while the incidence of autosomal recessive RP, including at least two genocopies, was estimated to be about 1 in 4,450. Family data analysis included the calculation of the likelihood that each family represented autosomal recessive, autosomal dominant, and X-linked inheritance patterns. These likelihoods were then converted to relative probabilities and summed over the sample population to yield estimates of the proportions of the three Mendelian types. This large, heterogeneous sample indicated that approximately 84% of the cases in the United States may be autosomal recessive, while about 10% are dominant and 6% X-linked recessive.     

Ionizing radiation and genetic risks. III. Nature of spontaneous and radiation-induced mutations in mammalian in vitro systems and mechanisms of induction of mutations by radiation

This paper (1) presents an analysis of published data on the molecular nature of spontaneously arising and radiation-induced mutations in mammalian somatic cell systems and (2) examines whether the molecular nature and mechanisms of origin of radiation-induced mutations, in mammalian in vivo and in vitro systems, as currently understood, are consistent with expectations based on the biophysical and microdosimetric properties of ionizing radiation. Depending on the test system (CHO cells, human T lymphocytes and human lymphoid cell line TK6), 80-97% of spontaneous HPRT mutations show normal Southern patterns; the remainder is due to gross changes, predominantly partial (intragenic) deletions. Total gene deletions at the HPRT locus are rare except in the TK6 cell line. At the APRT locus in CHO cells, 80-97% of spontaneous mutations are due to base-pair changes, the remainder being, mostly, partial deletions. The latter can extend upstream in the 5' direction but not beyond the APRT gene in the 3' direction. At the human HLA-A locus (T lymphocytes), the percentage of mutations with normal Southern patterns is lower than that for HPRT, and in the range of 50-60%. At the HLA-A locus, mitotic recombination contributes substantially to the mutation spectrum (approximately 30% of mutations recovered) and this is likely to be true of the TK locus in the TK6 cell line as well. With a few exceptions, most of the radiation-induced mutations show altered Southern patterns and are consistent with their being deletions and/or other gross changes (HPRT, 70-90% (CHO); 50-85% (TK6); 50-75% (T lymphocytes); TK, 60-80% (TK6); HLA-A, 80% (T lymphocytes); DHFR, 100% (CHO]. The exceptions are APRT mutations in CHO cells (16-20% of mutants with deletions or other changes) and HPRT mutations in T lymphocytes from A-bomb survivors (15-25%); the latter finding is consistent with the occurrence of in vivo selection against HPRT mutant cells. In cases of HPRT intragenic deletions analyzed (CHO cells and V79 Chinese hamster cells), there is evidence for a non-random distribution of breakpoints. The spontaneous mutation frequencies vary widely, from about 0.04/10(6) cells (sickle cell mutations at the human HBB locus) to 30.8/10(6) cells (HLA-A mutations in T lymphocytes) and are dependent on the locus, the system employed and a number of other factors. Those for the other loci fall between these limits.(ABSTRACT TRUNCATED AT 400 WORDS)     

Estimation of the incidence of a rare genetic disease through a two-tier mutation survey.

Recent attempts to detect mutations involving single base changes or small deletions that are specific to genetic diseases provide an opportunity to develop a two-tier mutation-screening program through which incidence of rare genetic disorders and gene carriers may be precisely estimated. A two-tier survey consists of mutation screening in a sample of patients with specific genetic disorders and in a second sample of newborns from the same population in which mutation frequency is evaluated. We provide the statistical basis for evaluating the incidence of affected and gene carriers in such two-tier mutation-screening surveys, from which the precision of the estimates is derived. Sample-size requirements of such two-tier mutation-screening surveys are evaluated. Considering examples of cystic fibrosis (CF) and medium-chain acyl-CoA dehydrogenase deficiency (MCAD), the two most frequent autosomal recessive disease in Caucasian populations and the two most frequent mutations (delta F508 and G985) that occur on these disease allele-bearing chromosomes, we show that, with 50-100 patients and a 20-fold larger sample of newborns screened for these mutations, the incidence of such diseases and their gene carriers in a population may be quite reliably estimated. The theory developed here is also applicable to rare autosomal dominant diseases for which disease-specific mutations are found.     

Ionizing radiation and genetic risks. IV. Current methods, estimates of risk of Mendelian disease, human data and lessons from biochemical and molecular studies of mutations

This paper is aimed at a synthesis of conclusions and concepts from the first three papers of this series and an inquiry of their relevance to the estimation of the risk of autosomal dominant and X-linked diseases in man, due to exposure to ionizing radiation. For a population under conditions of continuous irradiation, the doubling-dose method (DD method) enables the prediction of the excess risk of dominant and X-linked diseases at equilibrium. Per unit dose, this quantity is the product of the natural prevalence of these diseases (assumed to be 10,000/10(6) livebirths) and the reciprocal of the DD. The DD currently used is 1 Gy and is based primarily on data on the induction of recessive specific-locus mutations in male mice. The estimate of risk to the first generation is derived from that at equilibrium; the figure is about 15% of the equilibrium value (i.e., 15 cases/10(6) livebirths/cGy). With the direct method, the first-generation risk of dominant disease is estimated using data on the induction of dominant skeletal and cataract mutations in male mice and a number of correction factors. The estimates are about 10-20 cases and 0-9 cases, respectively, for irradiation of males and females, per 10(6) livebirths/cGy. In the Japanese studies, no significant adverse genetic effects, attributable to exposure of the parents to the atomic bombs, could be demonstrated with respect to any of the endpoints used. Most of the latter are clinically and socially relevant but mutationally insensitive. On the basis of these data, Neel and colleagues have estimated that the gametic DD for genetic effects of radiation in man is at least about 4-5 times the 1 Gy value thus far used. The concepts, assumptions, and the data-base used with the DD method have been re-examined. Arguments are advanced to support the thesis that ionizing radiation is probably not very efficient in inducing the very specific molecular changes that are known to underlie spontaneous mutations which cause naturally occurring dominant genetic diseases. It is suggested that (i) the DD estimate of 1 Gy that is used to estimate risk for autosomal dominant and X-linked diseases is conservative and (ii) the 1% prevalence figure for these diseases that is used for this purpose may be too high. If these suggestions are correct, then the estimate of risk for the dominant and X-linked diseases may need to be revised downwards.(ABSTRACT TRUNCATED AT 400 WORDS)     

Clonal analysis of delayed karyotypic abnormalities and gene mutations in radiation-induced genetic instability.

Many tumors exhibit extensive chromosomal instability, but karyotypic alterations will be significant in carcinogenesis only by influencing specific oncogenes or tumor suppressor loci within the affected chromosomal segments. In this investigation, the specificity of chromosomal rearrangements attributable to radiation-induced genomic instability is detailed, and a qualitative and quantitative correspondence with mutagenesis is demonstrated. Chromosomal abnormalities preferentially occurred near the site of prior rearrangements, resulting in complex abnormalities, or near the centromere, resulting in deletion or translocation of the entire chromosome arm, but no case of an interstitial chromosomal deletion was observed. Evidence for chromosomal instability in the progeny of irradiated cells also included clonal karyotypic heterogeneity. The persistence of instability was demonstrated for at least 80 generations by elevated mutation rates at the heterozygous, autosomal marker locus tk. Among those TK- mutants that showed a loss of heterozygosity, a statistically significant increase in mutation rate was observed only for those in which the loss of heterozygosity encompasses the telomeric region. This mutational specificity corresponds with the prevalence of terminal deletions, additions, and translocations, and the absence of interstitial deletions, in karyotypic analysis. Surprisingly, the elevated rate of TK- mutations is also partially attributable to intragenic base substitutions and small deletions, and DNA sequence analysis of some of these mutations is presented. Complex chromosomal abnormalities appear to be the most significant indicators of a high rate of persistent genetic instability which correlates with increased rates of both intragenic and chromosomal-scale mutations at tk.     

The load of genetic and partially genetic diseases in man. III. Mental retardation

This paper summarizes estimates of detriment associated with different etiologic categories of mental retardation (MR) in Hungary. The basic data derive from an earlier study carried out in Budapest on 1276 school-age mentally retarded children (with some etiologic reclassification based on recent studies). Detriment associated with these different categories of MR is expressed in terms of years of lost and impaired life. About 30 per 10(3) school-age children in Hungary are mentally retarded (mild + severe MR), one-tenth of whom have severe MR (IQ less than or equal to 50); 50% of the latter are institutionalized. The breakdown on the basis of etiology is as follows: gene mutations and chromosomal abnormalities, about 4 per 10(3); 'familial' (multifactorial) causes, 12 per 10(3); adverse pre-, peri- and post-natal causes, 11 per 10(3); and 'causes as yet unknown', the remainder. The estimates of mean number of years of lost life range from 42 to 68 (depending on the etiologic category), with an overall mean of 58. The total number of years of lost life is about 36,000 per 10(4) live births of which over 70% is due to pre-, peri- and post-natal causes, 18% due to 'familial' causes and the remainder due to Mendelian and chromosomal diseases. The total number of years of impaired life is about 7300 per 10(4) livebirths, 50% of which is due to 'familial' causes. While admittedly approximate, these estimates suggest that detriment associated with MR-related causes is not inconsiderable. Additionally, they provide some indication of causes of MR which are minimizable.     

Ionizing radiation and genetic risks. II. Nature of radiation-induced mutations in experimental mammalian in vivo systems

This paper reviews data on the nature of spontaneous and radiation-induced mutations in the mouse. The data are from studies using a variety of endpoints scorable at the morphological or the biochemical level and include pre-selected as well as unselected loci at which mutations can lead to recessive or dominant phenotypes. The loci used in the morphological recessive specific-locus tests permit the recovery of a wide spectrum of induced changes. Important variables that affect the nature of radiation-induced mutations (assessed primarily using tests for viability of homozygotes) include: germ cell stage, type of irradiation and the locus. Most of the results pertain to irradiated stem cell spermatogonia. The data on morphological specific-locus mutations show that overall, more than two-thirds of the X- or gamma-ray-induced mutations are lethal when homozygous. This proportion may be lower for those that occur spontaneously, but the numbers of tested mutants are small. For spontaneous mutations, there is evidence for the occurrence of mosaics and for proviral insertions. Most or all tested induced enzyme activity variants, dominant visibles (recovered in specific-locus experiments) and dominant skeletal mutations are lethal when homozygous and this is true of 50% of dominant cataract mutations, but again, the numbers of tested mutants are small. Electrophoretic mobility variants, which are known to be due to base-pair changes, are seldom induced by irradiation. At the histocompatibility loci, no radiation-induced mutations have been recovered, presumably because deletions are incompatible with survival even in heterozygotes. All these findings are consistent with the view that in mouse germ cells, most radiation-induced mutations are DNA deletions. Some mutations (in the morphological specific-locus tests) which had previously been inferred to be deletions on the basis of genetic analyses have now been shown to be DNA deletions by molecular methods. However, the possibility cannot be excluded that at least a small proportion of induced mutations may be intragenic changes. The data on the rates of induction of recessive lethals and of dominant skeletal and dominant cataract mutations (and proportions of the latter two which are homozygous lethal) can be used to estimate the proportions of recessive lethals which are expressed as skeletal abnormalities or cataracts. These calculations show that about 10% of recessive lethals manifest themselves as skeletal and less than 0.2% as cataract mutations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ionizing radiation and genetic risks. XII. The concept of "potential recoverability correction factor" (PRCF) and its use for predicting the risk of radiation-inducible genetic disease in human live births

Genetic risks of radiation exposure of humans are generally expressed as expected increases in the frequencies of genetic diseases over those that occur naturally in the population as a result of spontaneous mutations. Since human data on radiation-induced germ cell mutations and genetic diseases remain scanty, the rates derived from the induced frequencies of mutations in mouse genes are used for this purpose. Such an extrapolation from mouse data to the risk of genetic diseases will be valid only if the average rates of inducible mutations in human genes of interest and the average rates of induced mutations in mice are similar. Advances in knowledge of human genetic diseases and in molecular studies of radiation-induced mutations in experimental systems now question the validity of the above extrapolation. In fact, they (i) support the view that only in a limited number of genes in the human genome, induced mutations may be compatible with viability and hence recoverable in live births and (ii) suggest that the average rate of induced mutations in human genes of interest from the disease point of view will be lower than that assumed from mouse results. Since, at present, there is no alternative to the use of mouse data on induced mutation rates, there is a need to bridge the gap between these and the risk of potentially inducible genetic diseases in human live births.In this paper, we advance the concept of what we refer to here as "the potential recoverability correction factor" (PRCF) to bridge the above gap in risk estimation and present a method to estimate PRCF. In developing the concept of PRCF, we first used the available information on radiation-induced mutations recovered in experimental studies to define some criteria for assessing potential recoverability of induced mutations and then applied these to human genes on a gene-by-gene basis. The analysis permitted us to estimate unweighted PRCFs (i.e. the fraction of genes among the total studied that might contribute to recoverable induced mutations) and weighted PRCFs (i.e. PRCFs weighted by the incidences of the respective diseases). The estimates are: 0.15 (weighted) to 0.30 (unweighted) for autosomal dominant and X-linked diseases and 0.02 (weighted) to 0.09 (unweighted) for chronic multifactorial diseases. The PRCF calculations are unnecessary for autosomal recessive diseases since the risks projected for the first few generations even without using PRCFs are already very small. For congenital abnormalities, PRCFs cannot be reliably estimated.With the incorporation of PRCF into the equation used for predicting risk, the risk per unit dose becomes the product of four quantities (risk per unit dose=Px(1/DD)xMCxPRCF) where P is the baseline frequency of the genetic disease, 1/DD is the relative mutation risk per unit dose, MC is the mutation component and PRCF is the disease-class-specific potential recoverability correction factor instead of the first three (as has been the case thus far). Since PRCF is a fraction, it is obvious that the estimate of risk obtained with the revised risk equation will be smaller than previously calculated values.     

Ionizing radiation and genetic risks. X. The potential "disease phenotypes" of radiation-induced genetic damage in humans: perspectives from human molecular biology and radiation genetics.

Estimates of genetic risks of radiation exposure of humans are traditionally expressed as expected increases in the frequencies of genetic diseases (single-gene, chromosomal and multifactorial) over and above those of naturally-occurring ones in the population. An important assumption in expressing risks in this manner is that gonadal radiation exposures can cause an increase in the frequency of mutations and that this would result in an increase in the frequency of genetic diseases under study. However, despite compelling evidence for radiation-induced mutations in experimental systems, no increases in the frequencies of genetic diseases of concern or other adverse effects (i.e., those which are not formally classified as genetic diseases), have been found in human studies involving parents who have sustained radiation exposures. The known differences between spontaneous mutations that underlie naturally-occurring single-gene diseases and radiation-induced mutations studied in experimental systems now permit us to address and resolve these issues to some extent. The fact that spontaneous mutations (among which are point mutations and DNA deletions generally restricted to the gene) originate through a number of different mechanisms and that the latter are intimately related to the DNA organization of the genes, are now well-documented. Further, spontaneous mutations include those that cause diseases through loss of function as well as gain of function of genes. In contrast, most radiation-induced mutations studied in experimental systems (although identified through the phenotypes of the marker genes) are predominantly multigene deletions which cause loss of function; the recoverability of an induced deletion in a livebirth seems dependent on whether the gene and the genomic region in which it is located can tolerate heterozygosity for the deletion and yet be compatible with viability. In retrospect, the successful mutation test systems (such as the mouse specific locus test) used in radiation studies have involved genes which are non-essential for survival and are also located in genomic regions, likewise non-essential for survival. In contrast, most of the human genes at which induced mutations have been looked for, do not seem to have these attributes. The inference therefore is that the failure to find induced germline mutations in humans is not due to the resistance of human genes to induced mutations but due to the structural and functional constraints associated with their recoverability in livebirths. Since the risk of inducible genetic diseases in humans is estimated using rates of "recovered" mutations in mice, there is a need to introduce appropriate correction factors to bridge the gap between these rates and the rates at which mutations causing diseases are potentially recoverable in humans. Since the whole genome is the "target" for radiation-induced genetic damage, the failure to find increases in the frequencies of specific single-gene diseases of societal concern does not imply that there are no genetic risks of radiation exposures: the problem lies in delineating the phenotypes of recoverable genetic damage that are recognizable in livebirths. Data from studies of naturally-occurring microdeletion syndromes in humans and those from mouse radiation studies are instructive in this regard. They (i) support the view that growth retardation, mental retardation and multisystem developmental abnormalities are likely to be among the quantitatively more important adverse effects of radiation-induced genetic damage than mutations in a few selected genes and (ii) underscore the need to expand the focus in risk estimation from known genetic diseases (as has been the case thus far) to include these induced adverse developmental effects although most of these are not formally classified as "genetic diseases". (ABSTRACT TRUNCATED)     

Genetic susceptibility to heart disease in Canada: lessons from patients with familial hypercholesterolemia.

Much of the recent progress in treating patients with heart disease due to narrowed coronary arteries has resulted from studying disease evolution in patients with rare monogenic forms of disease. For instance, autosomal dominant familial hypercholesterolemia (FH, MIM (Mendelian Inheritance in Man) 143890) typically results from heterozygous mutations in LDLR encoding the low-density lipoprotein (LDL) receptor. Deficient LDLR activity results in elevated circulating LDL cholesterol, which accumulates within blood vessel walls, forming arterial plaques that can grow and eventually occlude the arterial lumen. Heterozygous LDLR mutations are usually detected using exon-by-exon sequence analysis (EBESA) of genomic DNA, a technology that has identified approximately 50 mutations in heterozygous FH (HeFH) subjects in Ontario. However, approximately 35% of Ontario HeFH patients had no EBESA-identified LDLR mutation. The diagnostic gap relates both to the genetic heterogeneity of FH and also to inadequate sensitivity of EBESA to detect certain mutation types, such as large deletions or insertions in LDLR. By means of a dedicated method to detect copy number variations (CNVs), additional heterozygous mutations in LDLR ranging from approximately 500 to >15 000 bases were uncovered, accounting for most of the remainder of Ontario HeFH patients. The appreciation of the key role of genomic CNVs in disease coincides with recent genome-wide mapping studies demonstrating that CNVs are common in apparently healthy people. CNVs thus represent a new level of genomic variation that is both an important mechanism of monogenic disease and a contributor to genomic variation in the general population; as well, it may have implications for evolution, biology, and possibly susceptibility to common complex diseases.     

Gaucher's and Fabry's diseases: biochemical and genetic aspects

Gaucher and Fabry's diseases are lysosomal storage disorders. They are due to glucocerebrosidase or alpha galactosidase deficiency, respectively. Gaucher disease, transmitted as an autosomal recessive trait, is frequent among Ashkenazi Jews. Cloning of the gene has allowed the characterization of few common mutations. Some of them have a prognosis value, in favour of either a non neurological form (type 1) or more severe forms (types 2 and 3). There mutations were found in 70% of the alleles, the other alleles carrying private mutations. Fabry disease is transmitted as an X-linked recessive trait. Genetic counselling in at-risk families relies on the detection of carrier females. As the alpha galactosidase gene shows various mutations, the establishment of phenotype-genotype correlations is limited. These two diseases, well defined at the biochemical and genetic level, are good models of inherited diseases for the development of specific therapies.     

Diverse prevalence of large deletions within the OA1 gene in ocular albinism type 1 patients from Europe and North America

Ocular albinism type 1 (OA1) is an X-linked disorder mainly characterized by congenital nystagmus and photodysphoria, moderate to severe reduction of visual acuity, hypopigmentation of the retina, and the presence of macromelanosomes in the skin and eyes. We have previously isolated the gene for OA1 and characterized its protein product as melanosomal membrane glycoprotein displaying structural and functional features of G protein-coupled receptors. We and others have identified mutations of various types within the OA1 gene in patients with this disorder, including deletions and splice site, frameshift, nonsense, and missense mutations. However, different prevalences of large intragenic deletions have been reported, ranging from 10% to 50% in independent studies. To determine whether these differences might be related to the geographic origin of the OA1 families tested, we performed a further extensive mutation analysis study leading to the identification of pathogenic mutations in 30 unrelated OA1 patients mainly from Europe and North America. These results, together with our earlier mutation reports on OA1, allow us to resolve the apparent discrepancies between previous studies and point to a substantial difference in the frequency of large intragenic deletions in European (<10%) compared with North American (>50%) OA1 families. These observations and our overall refinement of point mutation distribution within the OA1 gene have important implications for the molecular diagnosis of OA1 and for the establishment of any mutation detection program for this disorder.     

Molecular genetic analysis of recessive mutations at a heterozygous autosomal locus in human cells

We have investigated the genotypic changes that lead to expression of a recessive allele at a heterozygous autosomal locus in a human cell line. Mutant clones lacking thymidine kinase activity were derived from a B-cell lymphoblastoid line initially heterozygous at the tk locus, and restriction mapping was performed to detect intragenic structural alterations in the tk gene. In addition, informative molecular markers located elsewhere on chromosome 17 were analysed in order to detect large-scale (multilocus) events. We report that among 325 spontaneous and induced mutants, allele loss was more common than intragenic rearrangements or point mutations; in many cases, loss of heterozygosity appears to have extended well beyond the locus under selection. Cytogenetic analysis of a subset of these mutants showed that expression of the recessive TK-deficient phenotype and the associated loss of heterozygosity for chromosome 17 markers was not typically associated with detectable chromosomal changes.     

Developing genetic reagents to facilitate recovery, analysis, and maintenance of mouse mutations

Because the mouse has become the pre-eminent model system for functional genomics and analysis of complex-systems/pathways in mammals, there has been an escalation of interest in the generation and analysis of mouse mutations to use as tools in these analyses. I argue here for a parallel investment in continuing the development of appropriately marked chromosomal rearrangements to use as genetic reagents in mutation recovery, analysis, and maintenance crosses. Specifically, visibly marked interstitial chromosomal deletions can be valuable for regional mutagenesis screens for recessives based on hemizygosity, and they can also be used to simplify genetic fine-mapping as a prelude to gene identification based on positional cloning/candidacy strategies. Dominantly marked chromosomal inversions that also manifest some kind of recessive phenotype can be exploited in more extensive regional mutagenesis screens based on homozygosity, and are invaluable for simplified, low-cost and error-reduced mutant-stock maintenance. Also discussed are several issues concerning genetic background, particularly from the point of view of genetic-reagent resource development. Received: 16 December 1999 / Accepted: 16 December 1999     

Population-based risk estimates of Wilms tumor in sporadic aniridia. A comprehensive mutation screening procedure of PAX6 identifies 80% of mutations in aniridia

Aniridia is a severe eye disease characterized by iris hypoplasia; both sporadic cases and familial cases with an autosomal dominant inheritance exist. Mutations in the PAX6 gene have been shown to be the genetic cause of the disease. Some of the sporadic cases are caused by large chromosomal deletions, some of which also include the Wilms tumor gene (WAGR syndrome), resulting in an increased risk of developing Wilms tumor. Based on the unique registration of both cancer and aniridia cases in Denmark, we have made the most accurate risk estimate to date for Wilms tumor in sporadic aniridia. We have found that patients with sporadic aniridia have a relative risk of 67 (confidence interval: 8.1-241) of developing Wilms tumor. Among patients investigated for mutations, Wilms tumor developed in only two patients out of 5 with the Wilms tumor gene (WT1) deleted. None of the patients with smaller chromosomal deletions or intragenic mutations were found to develop Wilms tumor. Our observations suggest a smaller risk for Wilms tumor than previous estimates, and that tumor development requires deletion of WT1. We report a strategy for the mutational analysis of aniridia cases resulting in the detection of mutations in 68% of sporadic cases and 89% of familial cases. We also report four novel mutations in PAX6, and furthermore, we have discovered a new alternatively spliced form of PAX6.     

Evaluation of point mutations in dystrophin gene in Iranian Duchenne and Becker muscular dystrophy patients: introducing three novel variants

Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked neuromuscular diseases characterized by progressive muscular weakness and degeneration of skeletal muscles. Approximately two-thirds of the patients have large deletions or duplications in the dystrophin gene and the remaining one-third have point mutations. This study was performed to evaluate point mutations in Iranian DMD/BMD male patients. A total of 29 DNA samples from patients who did not show any large deletion/duplication mutations following multiplex polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA) screening were sequenced for detection of point mutations in exons 50–79. Also exon 44 was sequenced in one sample in which a false positive deletion was detected by MLPA method. Cycle sequencing revealed four nonsense, one frameshift and two splice site mutations as well as two missense variants.