Genetic abnormalities can occur in constitutional or mosaic form All genetic abnormalities—everything from a gross chromosomal abnormality to a single nucleotide change—can be constitutional or mosaic.
A constitutional abnormality is present in all cells of the body. It was inherited in the egg or sperm from a parent who carried the abnormality (or just possibly it could have arisen very early in embryonic development, so that the one abnormal cell gave rise to the whole person).
Mosaicism is when an individual has two or more genetically different cell lines, all derived from one original zygote (Figure 1A). It is the result of a post-zygotic genetic change, probably in just a single cell of a developing embryo. The event might have occurred early in embryonic development, resulting in large-scale multitissue mosaicism, or much later, producing limited tissue-restricted mosaicism. Conditions that would be lethal in constitutional form may be seen in mosaic form in patients. Intriguingly, when an individual has a constitutional mutation that impairs the proliferation of a certain type of cell, one sometimes sees revertant mosaicism, where by chance a cell back-mutates to normal and so acquires a growth advantage over the mutant cells.
Fig1. Mosaics and chimeras. (A) Mosaics have two or more genetically different cell lines derived from a single zygote. The genetic change indicated may be a gene mutation, a numerical or structural chromosomal change, or the special case of X-inactivation (see Section 10.4). (B) A chimera is derived from two zygotes, which are usually both normal but genetically distinct.
Mosaicism is somatic if it involves only somatic cells, gonadal or germinal if it is in the germ line, and gonosomal if both somatic and germ-line cells are involved. Somatic mosaicism has rather different phenotypic consequences depending whether the gene product involved is diffusible or cell-autonomous. Mosaicism for lack of a circulating protein would show as a reduced level of the protein. Depending on the protein, this might or might not cause clinical symptoms. It might cause a mild version of the phenotype produced by the same mutation when it is in constitutional form. Mosaicism for a cell-autonomous product would create patches of tissue with the mutant phenotype. This would be particularly noticeable in skin, where various nevi and marks are the result of genetic mosaicism. Somatic mosaicism should be suspected in any condition that shows a patchy or variegated phenotype. Gonadal mosaicism, where a phenotypically normal person has a clone of mutant cells in his or her gonads, is a serious problem when estimating recurrence risks, as discussed below.
Note that mosaicism is different from chimerism (Figure 1B). Mosaics start life as a single fertilized egg. Chimeras, in contrast, are the result of fusion of two zygotes to form a single embryo (the reverse of twinning), or chimerism can be the result of intra uterine transfusion between dizygotic twins that share a placenta. Chimerism is rare. It is proved by the presence of too many parental alleles at several loci in a sample that is prepared from a large number of cells. If just one locus were involved, one would suspect mosaicism for a single mutation, rather than the much rarer phenomenon of chimerism.
Blood-grouping centers occasionally discover chimeras among normal donors, and some intersex patients turn out to be XX/XY chimeras. A fascinating example was described by Strain et al. (1998) (PMID 9428825; see Further Reading). They showed that a 46,XY/46,XX boy was the result of two embryos amalgamating after an in vitro fertilization in which three embryos had been transferred into the mother’s uterus.
Most mosaicism goes unnoticed
If we look carefully enough we are all mosaic. Most mutations arise through errors in DNA replication or cell division, or mistakes in repairing DNA damage. Given the num ber of mitoses involved as a fertilized egg develops into an adult human, and given the finite risk of error whenever a cell divides or repairs damage, it must follow that each one of us is mosaic many times over for a great variety of abnormalities. Additionally, all females are mosaic by virtue of X-inactivation. However, mosaicism is only noticed and commented on when a person has a relevant phenotypic abnormality or if they have multiple children affected by a dominant condition that occurs de novo in the family. This can happen in three ways:
• If the mutation occurred in an early embryo, affecting a cell that was the progenitor of a significant fraction of the whole person, they might show phenotypic signs of the mutation;
• If the abnormality conferred a growth advantage on cells they might multiply dis proportionately. Most obviously this happens in cancer, but there are also a number of congenital syndromes such as Proteus syndrome (OMIM #176920) where there is overgrowth of some part of the body caused by mosaicism;
• If the mutation occurred in a germ-line cell early in development, it could result in a phenotypically normal person harboring a clone of mutant germ-line cells. As a result, a normal couple with no previous family history may produce one or more children with the same serious disease. The possibility of germ-line mosaicism must be considered whenever there is a new mutant case of a condition. As described below, with serious dominant or X-linked conditions such cases are frequent and cause difficulties with pedigree interpretation and estimation of recurrence risks.
New mutations are often originally present in mosaic form
When an individual carries a new mutation, a common assumption is that an entirely normal parent produced a single mutant gamete. However, this is not necessarily what happened. Unless there is something special about the mutational process, such that it can happen only during gametogenesis, a transmitted mutation could have arisen in the parent at any time during post-zygotic life.
New mutations are frequent with serious dominant or X-linked recessive conditions
New mutations are individually rare. In Chapter 11 we describe the various ways they can arise and ways of estimating their frequency. However, in the context of serious dominant or X-linked diseases, they may appear to be very far from rare: they may account for a significant proportion of all cases. If a serious dominant or X-linked condition persists in a population over many generations despite selection removing disease alleles, there must be a compensating production of new mutant alleles.
• A fully penetrant, lethal dominant condition would necessarily always occur by fresh mutation, because the parents could never be affected—an example is thanatophoric dysplasia (severe shortening of long bones and abnormal fusion of cranial sutures; OMIM #187600).
• For a nonlethal but deleterious dominant condition a similar argument applies, but to a lesser degree. Achondroplastic dwarfism is an example, as described in Chapter 12.
• Serious X-linked recessive diseases also show a significant proportion of fresh mutations, because the disease allele is exposed to natural selection whenever it is in a male.
• Autosomal recessive pedigrees, by contrast, are not significantly affected. Ultimately there must have been a mutational event, but the mutant allele can propagate for many generations in asymptomatic carriers, and so it is reasonable to assume that the parents of an affected child are both carriers.
The relation between intensity of selection, mutation rate, and population frequency of a condition is explored further in Box 1.
Box1. MUTATION–SELECTION EQUILIBRIUM
New mutations complicate pedigree interpretation
When a normal couple with no relevant family history have a child with severe abnormalities (Figure 2), deciding the mode of inheritance can be very difficult—the problem might be autosomal recessive, autosomal dominant with a new mutation, X-linked recessive (if the child is male), or nongenetic. In the absence of a direct molecular test, this leads to uncertainties in interpreting the pedigree and still greater uncertainties in estimating recurrence risks.
Fig2. A problem in pedigree interpretation. Is this an inherited autosomal or X-linked recessive condition, a new autosomal dominant or X-linked mutation, or maybe a nongenetic condition?
In every pedigree with a new mutation the possibility of germ-line mosaicism must be considered. This complicates estimation of the recurrence risk. For the pedigree in Figure 2, even if it is proven that the condition affecting the child is caused by a new dominant mutation, it is very difficult to calculate a recurrence risk to use in counseling the parents. Figure 3 shows an example of this uncertainty with an X-linked disease. The problem is discussed by van der Meulen et al. (1995) (PMID 7760316; see Further Reading). Usually an empiric risk (see below) is quoted.
Fig3. An X-linked pedigree with a new mutation. III1 has a serious X-linked disease. The three grandparental X chromosomes were distinguished by using genetic markers; here we distinguish them with three different colors (ignoring recombination). III1 has the grandpaternal X, which must have acquired a mutation at some point in the pedigree. See text for discussion.
In the family shown in Figure 3 there has been a new mutation. It could have occurred at any one of four possible points in the pedigree—each with very different implications for genetic counseling:
• If III1 carries a new mutation that was not present in any form in II1 , the recurrence risk for all family members is very low;
• If the mutation happened post-zygotically in II1 so that she is a germinal mosaic, there is a significant (but hard to quantify) risk for her future children, but not for those of her three sisters;
• If II1 was the result of a single mutant sperm, her own future offspring have the standard recurrence risk for an X-linked recessive trait, but her sisters are free of risk;
• If I1 was a germinal mosaic, all four sisters in generation II have a significant (but hard to quantify) risk of being carriers of the condition.
Detecting mosaicism
Molecular studies can be a great help where there is a suspicion of mosaicism. Sometimes it is possible to demonstrate directly that a normal father is producing a proportion of mutant sperm, in which case the proportion can be used to give an accurate recurrence risk. Direct testing of the germ line is not feasible in women, but other accessible tissues such as fibroblasts or hair roots can be examined for evidence of mosaicism. A negative result on somatic tissues does not rule out germ-line mosaicism, but a positive result, in conjunction with an affected child, proves it.
If individual cells are being examined for the presence of a mosaic variant—for example, if karyotyping or fluorescence in situ hybridization is being used to check for a chromosomal variant—the ability to detect mosaicism depends simply on the number of cells examined. Mosaicism can never be totally excluded, but an upper limit can be placed on its possible extent. If the actual proportion of mutant cells is x, then the chance of failing to see any mutant cell when a sample of N cells is tested is (1 − x)N. So, for example, if 300 cells are checked and no variant found, might the variant actually be present in 1% of the general cell population? The chance of this can be calculated to be 0.05 since (0.99)300 = 0.05.
A similar argument holds for next-generation sequencing. A very high read depth will allow detection of low-level mosaicism. By contrast, Sanger sequencing is unlikely to detect mosaicism present in less than around 20% of molecules, and other techniques that produce a single answer from bulk DNA will similarly be poor at picking up low-level mosaicism.
Often the question is whether a particular known sequence variant is present in low level mosaic form. Provided PCR primers can be designed that amplify the variant but not the wild-type sequence, the variant sequences can be detected and quantitative real-time PCR can be used to estimate their frequency. Often one is interested in very-low-level mosaicism—for example, an oncologist might want to check for the low-level presence of a pathogenic variant to attack it before it can progress, or a virologist might want to check whether a treatment has eliminated a patient’s HIV infection. For detecting and quantifying a variant present in a few copies per million cells, droplet digital PCR (Figure4) is a useful technique. Target sequences can be directly counted without the need for calibration with standard samples, and for very-low-level targets the results are more reliable than those obtained with real-time quantitative PCR.
Fig4. Droplet digital polymerase chain reaction (PCR). To detect a very few mutant sequences among a vast preponderance of wild-type sequences, the test sample is mixed with a reaction mix that will specifically PCR-amplify the mutant sequence, then emulsified. The PCR is run on the emulsion, which is then partitioned into individual droplets and the proportion of positive droplets measured. Ideally the initial concentration should be such that most droplets contain either 0 or 1 mutant molecule. Knowing the total concentration of the wild-type sequence, the proportion of mutant sequences can be directly calculated.
