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.     

Frequency of somatic and germ-line mosaicism in retinoblastoma: implications for genetic counseling.

Although mosaicism can have important implications for genetic counseling of families with hereditary disorders, information regarding the incidence of mosaicism is available for only a few genetic diseases. Here we describe an evaluation of 156 families with retinoblastoma; the initial oncogenic mutation in the retinoblastoma gene had been identified in these families. In 15 ( approximately 10%) families, we were able to document mosaicism for the initial mutation in the retinoblastoma gene, either in the proband or in one of the proband's parents. The true incidence of mosaicism in this group of 156 families is probably higher than our findings indicate; in some additional families beyond the 15 we identified, mosaicism was likely but could not be proven, because somatic or germ-line DNA from key family members was unavailable. Germ-line DNA from two mosaic fathers was analyzed: in one of these, the mutation was detected in both sperm and leukocyte DNA; in the other, the mutation was detected only in sperm DNA. Our data suggest that mosaicism is more common than is generally appreciated, especially in disorders such as retinoblastoma, in which a high proportion of cases represent new mutations. The possibility of mosaicism should always be considered during the genetic counseling of newly identified families with retinoblastoma. As demonstrated here, genetic tests of germ-line DNA can provide valuable information that is not available through analysis of somatic (leukocyte) DNA.     

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.     

Characterization of Fs(2)1, a germ-line dependent dominant female sterile mutation of Drosophila

Fs(2)1 is a germ-line dependent dominant female sterile mutation of Drosophila melanogaster. Fs(2)1 heterozygous females deposit very few abnormal eggs (collapsed, with malformed chorion). The degeneration of egg primorida starts around the end of egg maturation. Mitotic recombination mapping locates Fs(2)1 in a distal region of the left arm of the 2nd chromosome. Fs(2)1 is a good tool for studying germ-line functions (by the dominant female sterile technique) because the frequency of germ-line mosaicism exceeds 20% upon irradiation of adult females. Salivary gland polytene chromosomes of Fs(2)1 and the revertant heterozygous larvae appear normal.     

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.     

Somatic gene mutation and human disease other than cancer

While the focus of much mutation research is on germ-line mutation, somatic mutation is being found to be important in human disease. Neurofibromatosis 1 and McCune-Albright are disorders which are detected in the skin and other systems. The skin manifestations were essential for the demonstration of somatic mosaicism in neurofibromatosis 1, while analysis of blood DNA demonstrated somatic mutation in neurofibromatosis 2. Incontinentia pigmenti is also a disorder seen in skin and other tissues, but here it is the rare variant of the disorder in males, where it is usually lethal, that involves somatic mosaicism. Paroxysmal nocturnal hemoglobinuria is a disorder of the blood and cell separation of blood elements allows the demonstration of the somatic mosaicism. This review also discusses disorders in which somatic mosaicism, for mutations probably incompatible with life if the mutation had been germ-line, are likely to be involved. These include the Proteus syndrome, which involves both vascular tissues and bones, and two disorders which might be thought of as representing two subtypes of Proteus: Klippel-Trenaunay, which involves vascular tissues, and Maffuci, which involves bones. Embryonic mutations, which create mosaicism for both the soma and germ-line, are being increasingly found in a number of disorders and are discussed more briefly. Finally, reverse mutations involving the soma have been recently found in several disorders and such revertant mutations are also examined. While the review focuses on the clinical importance of somatic mutations, many of the mutations found to date are tabulated. It is too early to see if there is a different pattern of somatic mutation as compared to germ-line mutation. Although the parameters to allow careful quantitation are not yet available, it seems that the frequency of gene mutation in embryonic cells is not markedly different than that in the germ-line.     

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     

Multiple Molecular Mechanisms Underlying Subdiagnostic Variants of Marfan Syndrome

Mutations in the FBN1 gene, which encodes fibrillin-1, cause Marfan syndrome (MFS) and have been associated with a wide range of milder, overlap phenotypes. The factors that modulate phenotypic severity, both between and within families, remain to be determined. This study examines the relationship between the FBN1 genotype and phenotype in families with extremely mild phenotypes and in those that show striking clinical variation among apparently affected individuals. In one family, clinically similar but etiologically distinct disorders are segregating independently. In another, somatic mosaicism for a mutant FBN1 allele is associated with subdiagnostic manifestations, whereas germ-line transmission of the identical mutation causes severe and rapidly progressive disease. A third family cosegregates mild mitral valve prolapse syndrome with a mutation in FBN1 that can be functionally distinguished from those associated with the classic MFS phenotype. These data have immediate relevance for the diagnostic and prognostic counseling of patients and their family members.     

X-linked Menkes disease: first documented report of germ-line mosaicism

This work investigated a three-generation Menkes disease family, where germ-line mosaicism was suspected in the maternal grandmother of the index patient. She had given birth to 2 boys who died of suspected Menkes disease on the basis of clinical and photographic evidence. Biochemical analysis of the index patient confirmed the diagnosis of Menkes disease, and DNA analysis established a partial gene deletion (EX11_EX23del), involving exons 11-23 and the 3'-untranslated region (UTR) of ATP7A. A junction fragment was detectable by Southern blot analysis, which enabled carrier analysis. The mother was demonstrated to be a carrier, whereas analysis of lymphoblasts and skin fibroblasts from the maternal grandmother gave no indication of a partial gene deletion. No materials were available from the possibly affected maternal uncles. Further genetic analyses, including biochemical testing of the grandmother and haplotype analysis using four intragenic markers on DNA from selected members of the family, corroborated this finding. The combined results from DNA analyses showed that the grandmother had transmitted three different ATP7A haplotypes to her offspring: (1) the at-risk allele (CA(B))-1 and the deletion; (2) the at-risk allele (CA(B))-1 without deletion; and (3) the second allele (CAB)-2 without deletion. In conclusion, our study demonstrated segregation of Menkes disease within the family investigated that can best be explained by extensive germ-line mosaicism in the maternal grandmother. The finding of germ-line mosaicism has obvious implications for genetic counseling of Menkes disease families.     

Absence of somatic mosaicism in 17 families with hemophilia B: an analysis with a sensitivity 10- to 1000-fold greater than that of sequencing gels

Most estimates of germ-line mosaicism have been derived from families in which there has been transmission of a mutated allele to two or more children by an unaffected individual. Previously, analyses for somatic mosaicism detected five such individuals by PCR-based sequencing and haplotype analysis at a sensitivity of approximately 1 mutant per 10 wild-type alleles. To determine whether mutations that occur later in embryogenesis also give rise to somatic mosaicism, we analyzed leukocyte DNA from 17 individuals in whom a mutation in the factor IX gene was known to have originated. Methods capable of detecting 1 mutant allele in 100–10 000 were utilized, and no further examples of somatic mosaicism were detected. If confirmed by future studies, the paucity of somatic mosaicism with mutant:wild-type allele frequencies ranging from 1:10 to 1:1000 (relative to the 11% of somatic mosaicism detected with mutant:wild-type allele frequencies of 1:1 to 1:10) may reflect a higher mutation rate and/or germ-line lineage allocation very early in embryogenesis. Received: 14 July 1995 / Revised: 1 April 1996     

A new Rett syndrome family consistent with X-linked inheritance expands the X chromosome exclusion map.

Although familial recurrences of Rett syndrome (RTT) comprise only approximately 1% of the reported cases, it is these cases that hold the key for the understanding of the genetic basis of the disorder. Families in which RTT occurs in mother and daughter, aunt and niece, and half sisters are consistent with dominant inheritance and variable expressivity of the phenotype. Recurrence of RTT in sisters is likely due to germ-line mosaicism in one of the parents, rather than to recessive inheritance. The exclusive occurrence of classic RTT in females led to the hypothesis that it is X-linked and may be lethal in males. In an X-linked dominant disorder, unaffected obligate-carrier females would be expected to show nonrandom or skewed inactivation of the X chromosome bearing the mutant allele. We investigated the X chromosome inactivation (XCI) patterns in the female members of a newly identified family with recurrence of RTT in a maternal aunt and a niece. Skewing of XCI is present in the obligate carrier in this family, supporting the hypothesis that RTT is an X-linked disorder. However, evaluation of the XCI pattern in the mother of affected half sisters shows random XCI, suggesting germ-line mosaicism as the cause of repeated transmission in this family. To determine which regions of the X chromosome were inherited concordantly/discordantly by the probands, we genotyped the individuals in the aunt-niece family and two previously reported pairs of half sisters. These combined exclusion-mapping data allow us to exclude the RTT locus from the interval between DXS1053 in Xp22.2 and DXS1222 in Xq22.3. This represents an extension of the previous exclusion map.     

Germ-line mosaicism in tuberous sclerosis: how common?

Two-thirds of cases of tuberous sclerosis complex (TSC) are sporadic and usually are attributed to new mutations, but unaffected parents sometimes have more than one affected child. We sought to determine how many of these cases represent germ-line mosaicism, as has been reported for other genetic diseases. In our sample of 120 families with TSC, 7 families had two affected children and clinically unaffected parents. These families were tested for mutations in the TSC1 and TSC2 genes, by Southern blotting and by single-strand conformational analysis. Unique variants were detected in six families. Each variant was present and identical in both affected children of a family but was absent in both parents and the unaffected siblings. Sequencing of the variants yielded two frameshift mutations, one missense mutation, and two nonsense mutations in TSC2 and one nonsense mutation in TSC1. To determine which parent contributed the affected gametes, the families were analyzed for linkage to TSC1 and TSC2, by construction of haplotypes with markers flanking the two genes. Linkage analysis and loss-of-heterozygosity studies indicated maternal origin in three families, paternal origin in one family, and either being possible in two families. To evaluate the possibility of low-level somatic mosaicism for TSC, DNA from lymphocytes of members of the six families were tested by allele-specific PCR. In all the families, the mutant allele was detected only in the known affected individuals. We conclude that germ-line mosaicism was present in five families with mutations in the TSC2 gene and in one family with the causative mutation in the TSC1 gene. The results have implications for genetic counseling of families with seemingly sporadic TSC.     

Germ-line mosaicism simulates genetic heterogeneity in Wiskott-Aldrich syndrome.

The Wiskott-Aldrich syndrome (IMD2) is an X-linked recessive immunodeficiency. Initial linkage studies mapped the disease locus on the proximal short arm of the X chromosome, a localization which was further refined to the interval framed by DXS7 and DXS14. We have recently shown that a novel hypervariable locus, DXS255, is very closely linked to the disease gene and is likely to be, at present, the marker closest to the disease gene. The analysis of one family, however, displayed conflicting linkage results, as all of the informative markers situated in the Xp11-q22 region appeared to recombine with the disease locus in two "phase-known" meioses. We have shown by X-inactivation studies that the segregation of the disease through three obligate carrier females in this family originates from a grandpaternal mosaicism, which accounts for the apparent recombinations. This shows that germ-line mosaicism can simulate genetic heterogeneity in linkage studies.     

Somatic mosaicism for a newly identified splice-site mutation in a patient with adenosine deaminase-deficient immunodeficiency and spontaneous clinical recovery.

Absent or severely reduced adenosine deaminase (ADA) activity produces inherited immunodeficiency of varying severity, with defects of both cellular and humoral immunity. We report somatic mosaicism as the basis for a delayed presentation and unusual course of a currently healthy young adult receiving no therapy. He was diagnosed at age 2 1/2 years because of life-threatening pneumonia, recurrent infections, failure of normal growth, and lymphopenia, but he retained significant cellular immune function. A fibroblast cell line and a B cell line, established at diagnosis, lacked ADA activity and were heteroallelic for splice-donor-site mutation in IVS 1 (+1GT-->CT) and a missense mutation (Arg101Gln). All clones (17/17) isolated from the B cell mRNA carried the missense mutation, indicating that the allele with the splice-site mutation produced unstable mRNA. In striking contrast, a B cell line established at age 16 years expressed 50% of normal ADA; 50% of ADA mRNA had normal sequence, and 50% had the missense mutation. Genomic DNA contained the missense mutation but not the splice-site mutation. All three cell lines were identical for multiple polymorphic markers and the presence of a Y chromosome. In vivo somatic mosaicism was demonstrated in genomic DNA from peripheral blood cells obtained at 16 years of age, in that less than half the DNA carried the splice-site mutation (P < .002, vs. original B cell line). Consistent with mosaicism, erythrocyte content of the toxic metabolite deoxyATP was only minimally elevated. Somatic mosaicism could have arisen either by somatic mutation or by reversion at the site of mutation. Selection in vivo for ADA normal hematopoietic cells may have played a role in the return to normal health, in the absence of therapy.     

Evidence of autosomal dominant mutations in childhood-onset proximal spinal muscular atrophy.

Autosomal recessive and dominant inheritance of proximal spinal muscular atrophy (SMA) are well documented. Several genetic studies found a significant deviation from the assumption of recessive inheritance in SMA, with affected children in one generation. The existence of new autosomal dominant mutations has been assumed as the most suitable explantation, which is supported by three observations of this study: (1) The segregation ratio calculated in 333 families showed a significant deviation from autosomal recessive inheritance in the milder forms of SMA (P = .09 +/- .06 for onset at 10-36 mo and .13 +/- .07 for onset at > 36 mo; and P = .09 +/- .07 for SMA IIIa and .12 +/- .07 for SMA IIIb). (2) Three families with affected subjects in two generations are reported, in whom the disease could have started as an autosomal dominant mutation. (3) Linkage studies with chromosome 5q markers showed that in 5 (5.4%) of 93 informative families the patient shared identical haplotypes with at least one healthy sib. Other mechanisms, such as the existence of phenocopies, pseudodominance, or a second autosomal recessive gene locus, cannot be excluded in single families. The postulation of spontaneous mutations, however, is a suitable explanation for all three observations. Estimated risk figures for genetic counseling are given.     

Chromosome mosaicism in a population sample.

An analysis has been made of mosaicism found in the different types of chromosome abnormalities among the 19000 persons examined at the Cytogenetic Laboratory, Risskov. The percentage with mosaicism was 36 in both triple-X and Turner's syndrome, it was 7 and 11% in XYY and Klinefelter's syndrome, respectively, and 2 in autosomal abnormalities. We found a mosaicism frequency of 11% in population studies with 5 cells analyzed primarily compared with 7% in other studies, in which 10-50 cells were analyzed primarily. (The difference is not significant.) The total frequency of mosaicism was 8%. The first cell with the chromosome aberration establishing the mosaicism was found among the first 5 cells in 40 of the 44 cases with mosaicism, and all but one of the 44 cases would have been established as mosaics, if the guidlines indicated by Bochkov et al. (1974) had been followed; that is 11 cells analyzed primarily, and if one of these cells has a chromosome aberration, the number of cells analyzed is increased to 17; if 2 cells have the same chromosome aberration, the number of cells analyzed is extended to 23, and if 3 cells with the same chromosome aberration is found among these 23 cells, the mosaicism is established. Aneuploid or structural chromosome abnormalities present in all cells may be detected by analysis of 2-3 cells of good quality. Mosaicism with 2 or more cell clones with different chromosome patterns are extremely difficult to detect, if the percentage of cell clones with chromosome aberration is low. The incidence of chromosome abnormalities found in all cells in newborn children in the different studies is very similar as shown in a recent survey of 6 different studies by Jacobs et al. (1974). The incidence of mosaicism varies according to the frequency of artefactual aneuploidy, the variety of tissue studied, number of cells analyzed from each tissue as well as the acuity of the observer and the checking procedures.     

Somatic mosaicism in a patient with bilateral retinoblastoma.

We describe two cell lines with different deletions of the retinoblastoma gene in a patient with bilateral retinoblastoma. This patient has transmitted the mutation less frequent in his lymphocytes to two affected children. We cloned, mapped, and sequenced the junction fragments of the two deletions and found that they share one breakpoint but extend into opposite directions. An insertion of 4 bp of unknown origin is present between the breakpoints in one of the deletions. The second deletion shows a more complex rearrangement, including an inversion at the 5' end. Short regions of homology were found at the breakpoints and flanking the inversion. These results support the notion that bilateral retinoblastoma may not only be due to a germ-line mutation but also to a postzygotic mutation leading to somatic mosaicism.     

Germinal mosaicism and risk calculation in X-linked diseases.

Germinal mosaicism is a major problem in risk estimation for an X-linked disease. A mutation can happen anytime in germ cell development, and the proportion of germ cells bearing the mutated gene is twice the probability of recurrence of the mutation. This proportion could be either very low in late mutations or very high in germinal and somatic mosaicism. When this heterogeneity is taken into consideration, the distribution of the recurrence risk is conveniently represented as a set of discrete classes that may be derived either from models of gametogenesis or from empirical data. A computer program taking into account germinal mosaicism has been devised to calculate the probability of a possible carrier belonging to any of these classes, in order to settle the origin of the mutation of a given family. Germinal mosaicism increases the probability of inheriting the mutation, but this effect is always lowered by the possibility of heterogeneity. When the mother of a possible carrier is not herself a carrier, the risk of her daughter being a carrier is approximately halved, even under the assumption of a high recurrence risk from mosaicism.     

Study of a single BRCA2 mutation with high carrier frequency in a small population.

Germ-line changes in the cancer-predisposition gene BRCA2 are found in a small proportion of breast cancers. Mutations in the BRCA2 gene have been studied mainly in families with high risk of breast cancer in females, and male breast cancer also has been associated with BRCA2 mutations. The importance of germ-line BRCA2 mutations in individuals without a family history of breast cancer is unknown. The same BRCA2 mutation has been found in 16/21 Icelandic breast cancer families, indicating a founder effect. We determined the frequency of this mutation, 999del5, in 1,182 Icelanders, comprising 520 randomly selected individuals from the population and a series of 632 female breast cancer patients (61.4% of patients diagnosed during the study period) and all male breast cancer patients diagnosed during the past 40 years. We detected the 999del5 germ-line mutation in 0.6% of the population, in 7.7% of female breast cancer patients, and in 40% of males with breast cancer. The mutation was strongly associated with onset of female breast cancer at age <50 years, but its penetrance and expression are varied. A number of cancers other than breast cancer were found to be increased in relatives of mutation carriers, including those with prostate and pancreatic cancer. Furthermore, germ-line BRCA2 mutation can be present without a strong family history of breast cancer. Comparison of the age at onset for mother/daughter pairs with the 999del5 mutation and breast cancer indicates that age at onset is decreasing in the younger generation. Increase in breast cancer incidence and lower age at onset suggest a possible contributing environmental factor.