Germ line mosaicism

Mosaicism in germ cells has been recognized, over the past few years, as an important and relatively frequent mechanism at the origin of genetic disorders. There are two possibilities for the existence of such a mosaicism: one is that the mutation occurs in a germ cell that continues to divide. The other possibility is that the mutation occurs very early in a somatic cell before the separation to germinal cells and is therefore present both in somatic and germinal cells. Depending on various factors, such as the gene involved and/or the degree of mosaicism, the carrier of a somatic and germline mosaicism may be asymptomatic or may present with various symptoms of the disease. There are still relatively few reports in the literature in which the origin of germ-line mosaicism has been analyzed; nevertheless, they allow for a better insight into the mechanisms involved. In some diseases, such as osteogenesis imperfecta, new mutations are often present as asymptomatic somatic and germline mosaicism in one of the parents of the propositus. In other disorders, such as neurofibromatosis, somatic mosaicism is very rare in the parents of the propositus, perhaps since such mosaicism causes clinical symptoms. These differences are particularly important for genetic counseling in order to evaluate the risk for another affected child after the birth of the propositus. Received: 15 September 1997 / Accepted: 12 January 1998     

MSH2-dependent germinal CTG repeat expansions are produced continuously in spermatogonia from DM1 transgenic mice

Myotonic dystrophy type 1 is a neuromuscular affection associated with the expansion of an unstable CTG repeat in the DM protein kinase gene. The disease is characterized by somatic tissue-specific mosaicism and very high intergenerational instability with a strong bias towards expansions. We used transgenic mice carrying more than 300 unstable CTG repeats within their large human genomic environment to investigate the dynamics of CTG repeat germinal mosaicism in males. Germinal mosaicism towards expansions was already present in spermatozoa at 7 weeks of age and continued to increase with age, suggesting that expansions are continuously produced throughout life. To determine the precise stage at which germinal expansions occur during spermatogenesis, we sorted and collected the different germ cell types produced during spermatogenesis from males of different ages and analyzed the CTG repeat mosaicism in each fraction. Strong mosaicisms towards expansions were already observed in spermatogonia before meiosis. In transgenic Msh2-deficient mice, germinal instability of the CTG repeats (only contractions) also occurs premeiotically. No significant difference in mosaicism was detected between spermatogonia and spermatozoa, arguing against continued expansions during postmeiotic stages. This indicates that germinal expansions are produced at the beginning of spermatogenesis, in spermatogonia, by a meiosis-independent mechanism involving MSH2.     

Recurrence risk of a new dominant mutation in children of unaffected parents.

Extensions to models originally described by Hartl for predicting the recurrence risk of new dominant mutations are developed in this paper. Additions to the models are (1) possible somatic mosaicism in a parent in some families, (2) the possibility that the parental origin of the mutation may or may not be known, and (3) mutation rates which change as a function of sex and/or time. The models indicate that recurrence risks are most critically affected by (a) the amount of somatic mosaicism which can be tolerated in the parent without manifesting disease and (b) knowledge of the parental origin of the mutation. In addition, there is a moderate effect on recurrence risks if mutation rates increase in the father as a function of time. Recurrence risks are at least as large as the risk of trisomy 21 in a child of a mother of age 35 years or older, probably much higher (5%-10%) when the mutation is known to be of maternal origin or if substantial somatic mosaicism in the parent is compatible with a normal phenotype. The recurrence risk of a new mutation is high because of a very high ascertainment bias of families with substantial germ-line mosaicism.     

Germinal and Somatic Trisomy 21 Mosaicism: How Common is it,What are the Implications for Individual Carriers and How Does it Come About?

It is well known that varying degrees of mosaicism for Trisomy 21, primarily a combination of normal and Trisomy 21 cells within individual tissues, may exist in the human population. This involves both Trisomy 21 mosaicism occurring in the germ line and Trisomy 21 mosaicism documented in different somatic tissues, or indeed a combination of both in the same subjects. Information on the incidence of Trisomy 21 mosaicism in different tissue samples from people with clinical features of Down syndrome as well as in the general population is, however, still limited. One of the main reasons for this lack of detailed knowledge is the technological problem of its identification, where in particular low grade/cryptic Trisomy 21 mosaicism, i.e. occurring in less than 3-5% of the respective tissues, can only be ascertained by fluorescence in situ hybridization (FISH) methods on large cell populations from the different tissue samples.In this review we summarize current knowledge in this field with special reference to the question on the likely incidence of germinal and somatic Trisomy 21 mosaicism in the general population and its mechanisms of origin. We also highlight the reproductive and clinical implications of this type of aneuploidy mosaicism for individual carriers. We conclude that the risk of begetting a child with Trisomy 21 Down syndrome most likely is related to the incidence of Trisomy 21 cells in the germ line of any carrier parent. The clinical implications for individual carriers may likewise be dependent on the incidence of Trisomy 21 in the relevant somatic tissues. Remarkably, for example, there are indications that Trisomy 21 mosaicism will predispose carriers to conditions such as childhood leukemia and Alzheimer's Disease but there is on the other hand a possibility that the risk of solid cancers may be substantially reduced.     

Parental Somatic Mosaicism Is Underrecognized and Influences Recurrence Risk of Genomic Disorders

New human mutations are thought to originate in germ cells, thus making a recurrence of the same mutation in a sibling exceedingly rare. However, increasing sensitivity of genomic technologies has anecdotally revealed mosaicism for mutations in somatic tissues of apparently healthy parents. Such somatically mosaic parents might also have germline mosaicism that can potentially cause unexpected intergenerational recurrences. Here, we show that somatic mosaicism for transmitted mutations among parents of children with simplex genetic disease is more common than currently appreciated. Using the sensitivity of individual-specific breakpoint PCR, we prospectively screened 100 families with children affected by genomic disorders due to rare deletion copy-number variants (CNVs) determined to be de novo by clinical analysis of parental DNA. Surprisingly, we identified four cases of low-level somatic mosaicism for the transmitted CNV in DNA isolated from parental blood. Integrated probabilistic modeling of gametogenesis developed in response to our observations predicts that mutations in parental blood increase recurrence risk substantially more than parental mutations confined to the germline. Moreover, despite the fact that maternally transmitted mutations are the minority of alleles, our model suggests that sexual dimorphisms in gametogenesis result in a greater proportion of somatically mosaic transmitting mothers who are thus at increased risk of recurrence. Therefore, somatic mosaicism together with sexual differences in gametogenesis might explain a considerable fraction of unexpected recurrences of X-linked recessive disease. Overall, our results underscore an important role for somatic mosaicism and mitotic replicative mutational mechanisms in transmission genetics.     

Parent of Origin,Mosaicism, and Recurrence Risk: Probabilistic Modeling Explains the Broken Symmetry of Transmission Genetics

Most new mutations are observed to arise in fathers, and increasing paternal age positively correlates with the risk of new variants. Interestingly, new mutations in X-linked recessive disease show elevated familial recurrence rates. In male offspring, these mutations must be inherited from mothers. We previously developed a simulation model to consider parental mosaicism as a source of transmitted mutations. In this paper, we extend and formalize the model to provide analytical results and flexible formulas. The results implicate parent of origin and parental mosaicism as central variables in recurrence risk. Consistent with empirical data, our model predicts that more transmitted mutations arise in fathers and that this tendency increases as fathers age. Notably, the lack of expansion later in the male germline determines relatively lower variance in the proportion of mutants, which decreases with paternal age. Subsequently, observation of a transmitted mutation has less impact on the expected risk for future offspring. Conversely, for the female germline, which arrests after clonal expansion in early development, variance in the mutant proportion is higher, and observation of a transmitted mutation dramatically increases the expected risk of recurrence in another pregnancy. Parental somatic mosaicism considerably elevates risk for both parents. These findings have important implications for genetic counseling and for understanding patterns of recurrence in transmission genetics. We provide a convenient online tool and source code implementing our analytical results. These tools permit varying the underlying parameters that influence recurrence risk and could be useful for analyzing risk in diverse family structures.     

Recurrence of lethal osteogenesis imperfecta due to parental mosaicism for a dominant mutation in a human type I collagen gene (COL1A1).

We have determined that two infants with perinatal lethal osteogenesis imperfecta in one family had the same new dominant point mutation. Although not detected in his dermal fibroblast DNA, the mutation was detected in somatic DNA from the father's hair root bulbs and lymphocytes. The mutation was also detected in the father's sperm, demonstrating that mosaicism in the father's germ line explains recurrence. The presence of both germ-line and somatic mosaicism indicates that the mutation occurred prior to segregation of the germ-line and somatic cell progenitors. About one in eight sperm carry the mutation, which implies that at least four progenitor cells populate the germ line in human males. The observation that the mosaic individual is clinically normal suggests that genetic diseases can have both qualitative and quantitative components.     

Mosaike bei monogenen Erkrankungen

Mosaicism is defined as the simultaneous presence of cells with different genotypes that originate from a common zygote. Mutations can either be present in germline or somatic cells. Monogenic disorders apparently caused by a de novo mutation may show a recurrence risk due to germline mosaicism in a parent. Duchenne muscular dystrophy is a well investigated example with a high frequency of germline mosaicism and the estimation for the risk of recurrence is based on theoretical models and empirical data. Recently, somatic mutations have been uncovered in various syndromic disorders, such as Proteus syndrome or hemimegalencephaly and respective mutations often show gain-of-function properties. Genetic testing is mainly based on next generation sequencing technologies but still remains challenging; however, detection of somatic mosaicism is expected to be of increasing relevance in the diagnosis of monogenic disorders. Somatic mosaicism may also play a hitherto underestimated role in common disorders.     

The use of Bayes theorum in genetic counseling in cases of Duchenne muscular dystrophy

Bayes' law or theorem (1763) allows the expression of a posterior probability of heterozygosity for an X-linked gene, from two different kinds of information, namely: 1. the prior probability for the mother of an isolated case of Duchenne muscular dystrophy: --to be a carrier by mutation of the gene in one of her parents, or by segregation from earlier generations; --to be herself the origin of the mutation; 2. conditional probabilities, taking into consideration the existence of this woman's normal brothers, sons or maternal uncles and the serum creatine-kinase levels in the possible carrier(s) of the mutant gene. In some situations, these calculations give a recurrence risk which is lower than expected at first and allows sometimes to reassure anxious consultants on their genetic risk.     

Characterization of a spontaneous mutation to a beta-thalassemia allele.

We have studied a nuclear family containing a single child with severe beta-thalassemia intermedia, a Greek-Cypriot mother with hematological findings of beta-thalassemia trait, and a Polish father who is hematologically normal. Since both the child and her father were heterozygous for a DNA polymorphism within the beta-globin gene, it was possible to clone and sequence the beta-globin gene identical by descent from both the child and her father. A nonsense mutation in codon 121 (GAA----TAA) was found in the beta-globin gene of the child, while the same gene from her father lacked this mutation and was normal. This mutation has not been previously observed among over 200 beta-thalassemia genes characterized in Caucasians. Since the mutation eliminates an EcoRI site in the beta-globin gene, we could show that the mutation is not present in genomic DNA of the father. To rule out germinal mosaicism, sperm DNA of the father was also digested with EcoRI, and the mutant EcoRI fragment was not observed under conditions that would detect the mutation if it were present in at least 2% of sperm cells. Routine HLA and blood group testing supported stated paternity. In addition, studies with 17 DNA probes that detect multiple allele polymorphisms increased the probability of stated paternity to at least 10(8):1. These data provide evidence that the G----T change in codon 121 of the beta-globin gene in the child is the result of a spontaneous mutation that occurred during spermatogenesis in a paternal germ cell.     

Segregation analysis of 1885 DMD families: significant departure from the expected proportion of sporadic cases

Summary The proportion of sporadic cases of Duchenne muscular dystrophy has been estimated by classical segregation analysis in a pooled sample of 1885 sibships from 7 different countries. A significant departure from the theoretical expectations based on mutation-selection equilibrium is observed (segregation frequency = 0.439 ± 0.017; frequency of sporadic cases = 0.229 ± 0.026, at the maximum likelihood). The occurrence of germinal mosaicism in some of the mothers of Duchenne cases may account for this peculiar finding, although a possible role of inequality of mutation rates in the two sexes cannot be ruled out.     

Rett syndrome: exclusion mapping following the hypothesis of germinal mosaicism for new X-linked mutations

Summary The hypothesis of germinal mosaicism in the unaffected mother of two half-sisters affected with Rett syndrome is postulated to explain the unusual recurrence of this genetic disorder affecting only females (1/10000); it might be caused by new X-linked mutations with lethality in male fetuses. The analysis of 34 X-linked restriction fragment length polymorphisms (RFLPs) in these two affected females and in their unaffected mother and half-brother, together with the reconstruction of phase for 15 informative RFLPs in somatic cell hybrids retaining a single X chromosome from each female, has made it possible to exclude some regions of the X chromosome as possible sites of the mutation(s) causing Rett syndrome.     

A growth and division model for retinoblastoma

A stochastic growth and division model for studying a two hit cancer is developed and applied to retinoblastoma. Retinoblastoma occurs if both genes coding for a tumor suppressor protein on homologous chromosomes become defective. Germinal cases occur when a patient or carrier, born with one defective gene, suffers a second insult to any progeny retinal cell. Somatic cases are far less likely as two hits to the same cell during development are required. Details of the disease, germinal or somatic, unilateral or bilateral, in combination with case data allow for the estimation of the two parameters of the model: mutation rate, estimated at p=7x10(-7) per chromosome per cell division, and carrier frequency, estimated at f=40 per million. The model indicates that carriers of the disease arise from similar mutations to germ cells; in particular, heridary transmission can occur for only a generation or two before dying out. The results show that a stochastic simulation of a multi-hit cancer is feasible and may predict tumor growth dynamics. A simulation run will have to consist of a few million cells in order to observe even a small number of mutations. And several dozens such runs will have to be simulated.     

Sex-specific mutation rates for x-linked disorders: estimation and application

Emerging human molecular data are adding to our knowledge about the frequency and pattern of genetic mutations. This not only gives important insight into the biological processes underlying mutation, but also provides data which must be incorporated in the clinical setting. An example is the assumption of equal mutation probability in the male and female germ lines. This is a key assumption in Bayesian risk calculation for families segregating an X-linked recessive disorder. For some disorders, data are now available that demonstrate that the mutation probability in males differs from that in females. In this paper, we review the estimation of the male-female mutation rate ratio, including the construction of confidence intervals, and apply sex-specific mutation rates to carrier risk calculation in a variety of pedigree structures. In several instances, the difference in risk is substantial.     

Description of first germinal mosaic mutation identified in dominant skeletal mutation experiments and considerations about how to deal with this kind of spontaneous mutation in analyses

Germinal mosaicism is a well-established mechanism by which new spontaneous mutations enter the human population, but it is only rather recently that clusters of mutations arising in that way have been acknowledged and dealt with in specific-locus experiments on male mice. This paper reports the first cluster of germinal mosaic mutations to have been identified in experiments on the induction of dominant skeletal mutations. The mutation was detected in six offspring of a control male from the radiation part of an Assessment-of-Dominant-Damage (ADD) experiment. Reasons are provided to explain why this one litter of six mutants was excluded from the analysis of induction of dominant mutations causing the more common skeletal anomalies, which is reported in another paper. The effects of excluding this litter from that analysis are fully described. There is discussion of why such clusters should be included in some analyses but omitted in others. They should certainly always be reported because, in some cases, they can have a major impact on conclusions. Details on this one cluster of FCGM mutations provide numerous examples of how a dominant skeletal mutation that causes rare effects can also cause many of the more common anomalies.     

Mutation analysis and prenatal diagnosis in a Lesch-Nyhan family showing non-random X-inactivation interfering with carrier detection tests

Summary A nonsense mutation at the CpG-site in the codon for Arg(169) in the gene for hypoxanthine phosphoribosyltransferase (hprt) was identified by genomic polymerase chain reaction (PCR) and DNA sequencing in cultured fibroblasts from two brothers with Lesch Nyhan's syndrome. The recurrence of mutation at this CpG-site in several unrelated Lesch-Nyhan families suggests that deamination of 5-methylcytosine is a possible mechanism for mutagenesis. The level of hprt-mRNA in the fibroblasts of the patients was similar to that in healthy controls, whereas hprt-enzyme activity was not detectable. The mutation in this family was also identified in five female relatives and prenatally in a male fetus. Unexpectedly, results from hair follicle analyses and fibroblast selection studies in 8-azaguanine and 6-thioguanine medium showed a non-carrier phenotype in three of the female heterozygotes, whereas X-inactivation mosaicism was demonstrated in one heterozygote. A possible explanation for the apparent non-random X-inactivation in this family is the co-existence of the hprt mutation with an undefined X-linked lethal mutation. This observation is of practical relevance for carrier detection in other Lesch-Nyhan families.     

Germ line origins of de novo mutations in hemophilia B families

The germ line of origin for 13 of 14 de novo hemophilia B mutations has been determined. When added to previous reports, the origin, assuming no mosaicism, occurred in 43 female and 33 male gametes. Mutation rate estimates are twofold higher in males than in females. The pooled data also indicate that male and female germ lines have different mutation rates depending upon the type of mutation.     

Recurrence of Marfan syndrome as a result of parental germ-line mosaicism for an FBN1 mutation

Mutations in the FBN1 gene cause Marfan syndrome (MFS), a dominantly inherited connective tissue disease. Almost all the identified FBN1mutations have been family specific, and the rate of new mutations is high. We report here a de novo FBN1mutation that was identified in two sisters with MFS born to clinically unaffected parents. The paternity and maternity were unequivocally confirmed by genotyping. Although one of the parents had to be an obligatory carrier for the mutation, we could not detect the mutation in the leukocyte DNA of either parent. To identify which parent was a mosaic for the mutation we analyzed several tissues from both parents, with a quantitative and sensitive solid-phase minisequencing method. The mutation was not, however, detectable in any of the analyzed tissues. Although the mutation could not be identified in a sperm sample from the father or in samples of multiple tissue from the mother, we concluded that the mother was the likely mosaic parent and that the mutation must have occurred during the early development of her germ-line cells. Mosaicism confined to germ-line cells has rarely been reported, and this report of mosaicism for the FBN1 mutation in MFS represents an important case, in light of the evaluation of the recurrence risk in genetic counseling of families with MFS.     

Maturation of the immune response in germinal centers.

Germinal centers develop in peripheral lymphatic tissue during the primary immune response and may play a crucial role in affinity maturation. We have compared the diversification of the antigen-specific repertoire of B cells, both from within and from outside the germinal centers, during the murine response to 2-phenyloxazolone (phOx). By sequencing V kappa Ox1 L-chains characteristic of phOx-specific antibodies, we show that somatic mutations accumulate in germinal center B cells and that a mutation conferring high affinity binding is found with increasing frequency. An analysis of V/D/J rearrangements suggests that this mutation occurred independently in many B cells, which were then preferentially expanded. We conclude that, although the hypermutation mechanism may be activated before germinal centers develop, affinity maturation by hypermutation and selection takes place in the germinal centers.