For there even to be a chapter titled “Atypical Inheritance” in a book that focuses on human genetics, we are actually highlighting an important fact. On one hand, in spite of our developmental and functional complexity, the number of genetic rules is surprisingly small. The mechanisms of inheritance are generally so direct that most examples are “typical.” But that is not always the case. The path from an inherited DNA sequence to an expressed phenotype can sometimes be a convoluted and complex one. Most of the time this complexity is seen in the way genetic processes and their products interact with each other and with the environment. But not always.
A couple of examples will illustrate how our normal assumptions can lead to surprises. After fertilization, the regular nuclear divisions of mitosis yield a population of genetically identical cells that differentiate into the tissues of the adult. True? Yes, but not necessarily. Somatic mutation or other genetic events can yield genetic mosaics (Figure 12-1) in which subpopulations of cells within the individual differ from each other. But in other situations the genome itself does not change. Only the ability of the genes to function is affected. This is called genetic imprinting and is a normal phenomenon. It is similar to inactivation of one X chromosome in females in that some portions of the chromosome are turned off.
Figure 12-1. Mosaicism can occur in any multicellular specimen. Specific mutations may cause phenotypic differences that correlate with the distribution the affected cells. (a) Segmental mosaicism in an orange showing hypertrophy (overgrowth) of one section. (b) Clonal mosaicism seen in the feathers of a duck. The small patch of abnormal feathers on the head is actually a collection of abdominal feathers.
One example is a type of dwarfism in mice caused by imprinting of the insulin-like growth factor 2 (Igf2) gene. Let Igf2+ represent the normal allele and Igf2− represent the dwarf allele. Normally imprinting results in the inactivation of the Igf2 allele the offspring inherits from the mother. If a heterozygous offspring (Igf2+/Igf2−) inherits the normal allele from the father, it will be average sized. But, if it inherits the normal allele from the mother, the normal allele will be inactivated (Igf2+/Igf2−) and the offspring will be dwarf. Sometimes the genome is not even involved. Drosophila can inherit carbon dioxide sensitivity from their mother due to a rhabdovirus passed cytoplasmically just like the maternally inherited mitochondria.
The more we learn about the genome, the more we realize how diverse the body’s information pathways can be. Influences on development go well beyond the traditional role of genes controlling the synthesis of enzymes and structural proteins. Some special cases have been mentioned in other contexts. Here we will look at a sample of these mechanisms to explore their medical significance in a more applied fashion.
Part 1: Background and Systems Integration
Mosaicism
It is common to assume that cells within the same individual share the same genotype. But that is not necessarily the case. Mosaicism refers to the situation in which different cells within the same organism have different genotypes. Given the lower rate of genetic repair found in somatic cells compared to the germline, it is likely that all individuals are mosaics for some genetic differences. The earlier in development a somatic mutation occurs, the larger the number of cells that carry and potentially show it. Usually this is not noticed, because the mutated genes are either inactive in the affected tissue or the pathogenic defect can be covered by normal cells nearby. So, an individual can be genetically, but not phenotypically, mosaic. However, depending upon the nature of a particular mutation, the level of mosaicism and the distribution of the cells carrying the mutation, phenotypic differences may be observed.
X-chromosome inactivation in females represents a unique mechanism of mosaicism. As a means for dosage compensation in mammals, one of the two female X chromosomes is inactivated very early in embryogenesis, resulting in functional mosaicism for any X-linked heterozygous gene. Although inactivation is random most of the time with about half the cells inactivated for each allele, there can be skewed inactivation toward one chromosome so that one allele is phenotypically expressed more often than the other. This is usually a signal that there is a deleterious X-linked mutation: the cells preferentially inactivate the X with the mutation. Another example of genetic mosaicism is seen in most cancers, in which the tumor is genetically different from the normal cells around it.
Uniparental Disomy
In the usual situation, an individual typically inherits one copy of each chromosome from each parent. As the name suggests, uniparental disomy (“one parent-both bodies”—UPD) is an exceptional condition in which both copies of a given gene, gene region, or chromosome originate from the same parent. In the case of isodisomy, both alleles are the same, while in heterodisomy the alleles differ from each other but originate from the same parent (Figure 12-2). Several mechanisms have been proposed to explain this phenomenon. Most proposed mechanisms involve a genetic error with a second error that by chance corrects the first error. For example, consider events affecting a trisomic individual. Two of the chromosomes come from one parent and the third comes from the other parent. But if the latter copy is subsequently lost, the result will be a return to normal disomy, but with both copies originating from the same parent. The reverse situation would be an initial monosomic conception with a second division error resulting in a subsequent gain of two chromosomes from the original. Yet a third possible mechanism has been termed gamete complementation. In this situation, two abnormal gametes with reciprocal errors (one missing a chromosome with the other having two copies of the same one) correct each other at conception.
Figure 12-2. Schematic demonstrating UPD. The normal segregation pattern is in the middle. The panel on the left shows maternal isodisomy. The panel on the right shows paternal heterodisomy.
Imprinting
Imprinting was discussed briefly in Chapter 5 (Cytogenetics). This parent-of-origin epigenetic phenomenon is reintroduced here to put it into context with other examples of atypical gene expression. Before transmission, genes can be marked by differential methylation or histone alterations that affect their later levels of gene expression. These epigenetic changes are maintained throughout all somatic cells and are only erased when germline cells for the next generation are formed. Low levels of gene expression generally result from methylated alleles, while higher gene expression is generally found for alleles that are unmethylated. But methylation does not always mean inactivation; the effect depends on the gene. For loci that undergo imprinting, therefore, the phenotype of the offspring is determined both by the specific allele(s) it inherits and by which parent transmitted each one. The result is essentially monoallelic expression for the gene. Since some genes are imprinted in the parental female and others in the male, it is necessary that the imprinting be erased and then re-established each generation.
In mammals only a small proportion of genes (perhaps only 1% or less) undergo imprinting. In humans thus far fewer than 100 imprinted genes have been identified, with most working during the embryonic and placental phases of development. Because of these imprinted genes, naturally occurring cases of parthenogenesis cannot occur in humans.
To explore the phenomenon experimentally, a mouse embryo series was produced that carried small chromosomal regions from either the mother alone or the father alone. This series of UPDs, explored in more detail in the next section, was used to define an imprinting map. Many chromosome sections were found to contain several imprinted genes. In fact, about 80% of the known imprinted genes are located in clusters, called imprinted domains, suggesting that they might be regulated as a group.
It is important to note that imprinting is a normal phenomenon. For the approximately 80 human genes known to be imprinted, monoallelic expression is what is expected for that locus. As with most features in biological systems, any deviation (over or under expression) from the expected norm usually results in an abnormal phenotype. Figure 12-3 depicts a hypothetical imprinted gene and several possible scenarios of how specific genetic changes can alter the normal level of gene expression. Knowing that imprinting is a normal occurrence, it is interesting to speculate why such a mechanism is needed. Possible reasons that have been suggested include regulation of placentation, avoidance of parthenogenesis, providing flexibility during development, playing a role in the immunological escape of the fetus, and “dominance modification.”
Figure 12-3. Graphic representation of a hypothetical imprinted gene. (a) Right panel depicts the maternal and paternal alleles of a segment of DNA. All 10 genes show bi-allelic expression. Left panel shows 2 imprinted genes. Gene 3 on the paternal strand is imprinted. Gene 7 on the maternal strand is imprinted. (b) In the event of paternal UPD of this region, two abnormalities result: bi-allelic expression of gene 7 and no expression of gene 3. (c) A deletion of the paternal gene 3 will have no effect as this gene is normally imprinted. (d) A deletion of the maternal gene 3 will produce an abnormality—no expression of gene 3. (e) “Imprinting genes” are responsible for actually altering the gene expression. A mutation in the imprinting gene responsible for imprinting gene 3 would prevent gene 3 from being “turned off.” This too would be abnormal as there would now be bi-allelic expression of gene 3. Any deviation from the normal configuration has the potential to produce an abnormal phenotype.
Other Types of Gene × Gene Interactions
In Chapter 6, we described genetic heterogeneity, in which a large number of different gene mutations can give rise to the same phenotype. One example was retinitis pigmentosa, a phenotype that can be produced by defects in a range of contributing gene processes. One of these is actually digenic inheritance, i.e., the condition is produced when mutations occur together in two different genes.
A classic example of digenic inheritance is comb shape in roosters (Figure 12-4). Here the rose comb (R–) is dominant to rr and the pea comb (P–) is dominant to pp. R and P are co-dominant and produce a walnut comb. The double recessive (rrpp) yields a single comb. In these examples, the phenotype produced by the combined action of the two genes is not simply predictable from knowing the effect of each gene alone.
Figure 12-4. The comb appearance in a rooster depends on two genes, rose (R) and pea (P). Different combinations of dominant and recessive genotypes at these two loci determine four common comb types. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
Phenotypes can usually be explained in terms of simple diploid genotypes. But occasionally it is necessary to postulate the involvement of a third allele, i.e., to postulate triallelic inheritance. Although it is generally difficult to identify these situations, some estimates put them at less than 10% of the known cases. In those few instances, however, traditional Mendelian risk estimates used by genetic counselors may not be completely accurate.
By using the term multi-locus inheritance we mean that the observed phenotype is influenced by genes at more than one locus. This should not come as a surprise to the reader at this point. We have already discussed several examples of multi-locus inheritance. In Chapter 10 (Multifactorial Inheritance and Gene × Environment Interactions) we discussed polygenic inheritance. Clearly polygenic inheritance falls within the definition of “multi-locus” as it represents the cumulative (often additive) effects of multiple loci on a quantitative trait. Likewise the digenic and triallelic inheritance patterns described earlier are also examples of multiple loci influencing a specific phenotype.
Another example of multi-loci interactions is that of modifier genes. A study of modifier genes reflects more a specific perspective than a unique type of gene interaction. Modifier genes are simply genes that influence the expression of other genes. Generally the context refers to the variable expression of the gene or mutation of primary interest. Modifier genes are loci of secondary importance or more minor effect that influence the degree of severity of the primary gene.
An example from Drosophila will illustrate the phenomenon. The fly’s wings are supported by longitudinal and cross veins that stabilize it during flight (Figure 12-5a). A number of mutations have been isolated that either reduce the length of the vein or increase the amount of venation formed on the wing (Figure 12-5b, c). But as for other quantifiable traits like this, all sampled populations carry genes that either lengthen or shorten a vein gap or that add to or remove extra venation. In other words, all natural populations appear to be segregating for alleles that modify the expression of the mutations that affect Drosophila wing vein length. This may not seem very surprising until you step back and consider: in a normal fly the veins extend all the way to the edge of the wing, and they have done so for millions of years. Why would all tested populations of Drosophila carry modifiers of a trait that has not varied for thousands of generations?
Figure 12-5. Drosophila wings provide a model system for studying major genes and quantitative variation affecting expression. (a) Normal wing. (b) Veinlet mutation reduces venation. (c) Plexus increases wing vein fragments. Polygenic modifiers of veinlet and of plexusexpression can make the mutant wings indistinguishable from the normal.
The answer is subtle, but important. Just because we detect a gene’s action by the way it modifies something we choose to measure does not mean we are looking at the actual role of that modifier gene in development. For Drosophila wing vein lengths, the modifier genes are not there to influence the expression of a shortened vein. In nature the veins are never short. Instead, the modifier genes appear to be part of a developmental buffering system for wing blade organization. The lesson from this example is that a phenotypic association does not always indicate the primary functional relationship between a gene and its role in development.
Opposite (Anti-Sense) Transcripts
The traditional understanding of the process of transcription in humans is that the coding strand of DNA is copied as messenger RNA. In this process, one strand, the coding strand, is read in a 3′ to 5′ direction. However, anywhere along the genome either strand has the potential to act as the template. In other words some genes run one direction, some the other. In a few remarkable cases, at one locus, the same segment of double-stranded DNA contains genetic information on both strands. The products of such a “double read” have been termed opposite (or anti-sense) transcripts. It is important to note, however, that in either situation the RNA polymerase still transcribes the DNA strand in its 3′ to 5′ direction. Natural antisense transcripts (NATs) have been identified from most eukaryotes, including humans, and the RNAs generated include both protein-coding and noncoding examples.
Transposable Elements
Transposable elements (“jumping genes”) are mobile segments of genetic material that are present in all eukaryotes. They are nonrandomly distributed throughout the genome. By a series of mechanisms, they can move themselves or a copy from one chromosomal locus to another. In the process of moving about, they may increase (or decrease) the amount of DNA in the genome of the cell. If such changes occur in gamete precursor cells, the change is potentially heritable. Potentially one-third of the entire human genome is made up of repetitive sequences that represent degenerate copies of transposable elements.
Transposable elements (also called transposons) are classified by the type and size of nucleic acids involved and the mechanism of movement. Class I transposons (retrotransposons) transcribe DNA into RNA and then utilize reverse transcriptase to remake a DNA copy of the RNA to insert into a new location. There are several different types of retrotransposons. The types and characteristics vary among species. In humans, two types should be mentioned. Long interspersed elements (LINEs or L1) are found in large numbers in most eukaryotic genomes. They are transcribed to an RNA using an RNA polymerase II promoter that is within the LINE itself. Short interspersed elements (SINEs) are shorter DNA segments. SINEs do not have their own reverse transcriptase enzyme and rely on other mobile elements to move about. The most common SINEs in humans and other primates are called Alu sequences. Class II transposons consist of only DNA that moves directly from locus to locus. Class III transposons are also known as miniature inverted-repeats transposable elements or MITEs.
The major clinical implications of transposons are that they act as mechanisms for the spontaneous generation of mutations (i.e., they are mutagens). This then represents a “natural” source of genetic variation. In migratory populations, such as Drosophila, there is a well-described phenomenon of hybrid dysgenesis. When two different strains of Drosophila meet, intermingle, and mate, the spontaneous mutation rate increases phenomenally. This sudden increase in mutation rate has been shown to be due to a transposable element in the flies known as a P element.
The most common mechanism of transposon generated mutations is insertional mutagenesis. The movement of the element into a normal gene may simply disrupt the gene. Alternatively, the element may exert its influence on nearby genes by affecting the promoter or enhancer.
Genetic Anticipation
Anticipation as a genetic term refers to a phenotype of a condition that becomes more severe (phenotypically worse) as it passes from one generation to the next. As clinicians have observed families through time, genetic anticipation has often been postulated. Until relatively recently there have been significant disagreements as to whether or not genetic anticipation was an actual phenomenon. Skeptics held the opinion that what was being observed was simply a bias of ascertainment: when a genetic condition was diagnosed in a single family member, it then became easier to identify the condition in subsequent (or previous) generations. Thus, the condition would appear to be getting worse through the generations, although it actually was not.
The debate was ultimately resolved when genetic anticipation was identified in association-specific genetic markers—expanding trinucleotide repeats. Trinucleotide repeats, also known as microsatellites, can cause disease by expanding the number of repeated copies at a locus. An individual with a low number of repeated units is typically normal, but the number of repeats has the potential to change each generation. If the size of the repeat increases, it can ultimately disrupt gene function by a variety of mechanisms to the point where the individual becomes symptomatic.
Epigenetics
Epigenetic inheritance is the transmission of information from a cell or multicellular organism to its descendants without that information being encoded in the nucleotide sequence of the genes. It occurs through interactions among developmental processes above the level of primary gene action. Epigenetic variation does not follow the rules of Mendelian inheritance, is often the result of changed gene expression, and may be reversible. It may be somatically inherited, but it is not transmitted through meiosis.
All of these examples show how much information potential the genome contains beyond its traditionally-recognized role in gene regulation and protein coding. The interpretation of genetic causality must, therefore, always be done with an open mind. Most patients may fit into well-understood patterns of inheritance and expression. But one should always be open to the significance of the unexpected or the complex.
Part 2: Medical Genetics
The mechanisms of “atypical inheritance” described in the first section of this chapter are fascinating indeed. Simply knowing that such mechanisms exist is worthwhile just for their intellectual curiosity. However, the importance for the health care professional is that all of these mechanisms have “real life” clinical applicability. For this section of the chapter, we will not describe mechanisms again. Instead we will provide clinical examples of each and discuss how they might appear in patient encounters.
Mosaicism
Somatic mosaicism
Mosaicism can become clinically recognizable depending upon the nature of the mutation and what cells harbor the mutation. Somatic mutations can occur in the embryo very early after conception. If so, a large number of cells may be “affected” and clinical features may be recognized that reflect the distribution of the cells harboring the mutation. Marfan syndrome is a connective tissue disorder characterized by exceptionally long limbs and digits, hyper-flexible joints, fragile stretchy skin, ocular abnormalities, and cardiac abnormalities such as dissecting aortic aneurysms and valvular prolapse. Most cases of Marfan syndrome have been shown to be due to mutations in a connective tissue protein called fibrillin. The young lady shown in Figure 12-6 has a distinct body asymmetry. From a clinical standpoint, her doctors felt that she had the features of Marfan syndrome on one side of her body and not the other (i.e., she had “hemi-Marfan syndrome”). Skin biopsies performed on each side of her body confirmed that one side of her body had deficient fibrillin, while the other side had normal amounts (Figure 12-7). The explanation for this would be somatic mosaicism with a fibrillin mutation in the cells of the left side of her body. Of course somatic mutations can occur at any point in embryogenesis throughout all of postnatal life. Small clonal patches of cells harboring a mutation might be completely asymptomatic or evident as something as subtle as a freckle.
Figure 12-6. Adolescent female with clinical features of Marfan syndrome on the left side of her body. (a) Note the hip tilt due to leg length discrepancy. (b, c) Note excessive length of left hand and foot. (Reprinted with permission from Am J Hum Genet. 1990;46:661-671.)
Figure 12-7. Two panels of immunohistochemical staining of the protein fibrillin from skin biopsies on the left and right side of the patient in Fig. 12-6. Almost complete absence of fibrillin is seen in the panel from the left side of the patient which corresponds with this patient’s clinical picture of “hemi-Marfan syndrome.”
Gonadal mosaicism
Somatic mosaicism localized to the germ cells (gonadal mosaicism) has special implications as germ cell mutations are potentially heritable. If the mutation is only in the germ cells, there will likely be no physical manifestations in the parents as the germ cells are not performing a specific “function” for the body other than reproduction. However, such a mutation may produce problems in the next generation. For example, osteogenesis imperfecta (OI) is an autosomal dominant skeletal disorder characterized by the tendency to have fractures easily. Like most conditions there is a marked variability ranging from a severe form that is lethal at birth to milder forms that are associated with the tendency to have a fracture with only mild trauma. Figure 12-8a, b shows two siblings with a moderately severe form of OI. Both children had over 20 fractures each simply as a result of the birthing process. As noted in Figure 12-8c, these are the only two children to this couple. Neither parent has OI. Given this information, one would be tempted to predict that in this family OI was an autosomal recessive disorder. However, molecular testing revealed that this was not the situation. Most cases of osteogenesis imperfecta are caused by abnormalities in type I collagen. Type I collagen is a multimeric protein composed of two alpha-1 polypeptide chains (COL1A1) and one alpha-2 chain (COL1A2). Mutation testing in these two siblings showed that each one of them carried an identical mutation of only one copy of their COL1A1 gene! Since only one allele of the COL1A1 gene had a mutation, this means that for these children the disorder was autosomal dominant (COL1A1 is on chromosome 17).
Figure 12-8. (a, b) Male and female siblings with osteogenesis imperfecta. Both children have multiple abnormally healed fractures. (c) Pedigree of this family. Initial inspection might suggest autosomal recessive inheritance. See text for explanation.
So then, what is the explanation for both children having an identical mutation? The odds of two spontaneous identical mutations are astronomically small. The answer then is in the title of this section—gonadal mosaicism. One of the parents has this mutation in some of their germ cells. Because the mutation is not present in other tissues, the parent is unaffected. Gonadal mosaicism can be present in either parent, but in this particular family, genetic analysis of individual sperm confirmed that it was the father who harbored the same mutation that was found in his children. Additional testing demonstrated that this mutation was present in about 40% of his sperm. Thus, a plausible presumption of autosomal recessive inheritance would be wrong. For this family it is an autosomal dominant condition. As such the recurrence risk for future children would not be 25%, but actually 40%, reflective of the degree of mosaicism in the father’s spermatozoa.
Uniparental Disomy
As noted earlier, UPD is a unique situation in which an individual has inherited both copies of an allele or chromosome from one parent, rather than the typical situation of receiving one copy from each parent. The first question that might arise could be: “so what? What harm could there be in inheriting both copies from the same parent?” As it turns out, there are several fascinating consequences of such events. Take for example cystic fibrosis (CF) (Figure 4-21). Cystic fibrosis is an autosomal recessive disorder caused by mutations in a gene called CFTR—a membrane transporter of chloride ions. Once DNA testing became available, children could be tested to identify the particular mutations causing their disorder. Likewise their parents can be tested to identify which parent is carrying which mutation–information that can be useful for others in the family as they try and ascertain their particular risks. As families were tested, an interesting observation was sometimes encountered. A child with CF would be tested, and two identical mutations would be identified on their two alleles. When the parents were tested, one parent would be identified as having one of the two mutations, but the other would have no identifiable mutation. Further studies were conducted to verify paternity and to rule out new mutations. Ultimately, these studies identified UPD as the explanation! Figure 12-2 provides a schematic demonstrating UPD. Population studies have suggested that maternal UPD may be present in 1 in 500 children with CF. (Note that in order for the child to be affected with CF, that there would have to be uniparental isodisomy). A very important aspect of this occurrence would be in recurrence risk counseling. In the typical situation of autosomal recessive inheritance, the recurrence risk for a couple who has had a child with CF would be 25%. In the event of UPD, the same rare abnormal event would have to happen a second time making recurrence essentially zero.
Imprinting
As noted earlier, fewer than 100 human genes are currently known to be imprinted. Specifically then, for this handful of alleles, the normal situation is for monoallelic expression. Disruption of the normal imprinting pattern can result in an abnormal phenotype. Several fascinating human disorders are now known to occur as a result of abnormal imprinting. We encourage you to review the section above on imprinting and to understand Figure 12-3completely before reading any further.
The first described and probably best understood “imprinting disorders” are Prader-Willi syndrome (PWS) and Angelman syndrome. Prader-Willi syndrome (Figure 12-9) is characterized by hypotonia, cognitive deficits, typical facial changes, obesity, and small hands and feet. Patients with PWS exhibit many features and behaviors that are due to hypothalamic dysfunction (poor satiety control, hyperefficient metabolic rate, hypogonadotrophic hypogonadism). Patients with Angelman syndrome also have a characteristic appearance (Figure 12-10) that is distinctly different from that of PWS. These patients often have seizures and an abnormal gait that is somewhat spastic/hypertonic in nature. In addition they often show “happy” behavior under circumstances not expected to evoke a happy response.
Figure 12-9. Young woman with Prader-Willi syndrome (a) Typical facial changes and body habitus. (b, c) Small hands and feet.
Figure 12-10. Young girl with Angelman syndrome showing a positive “tuning fork sign”.
High resolution cytogenetic studies and cytogenetic microarrays have shown that a specific deletion of chromosome region 15p11-13 is found in 70% to 80% of patients with either disorder (Figure 12-11). The obvious question arising from this observation, of course, is how can the same deletion produce two very different phenotypes? Investigations revealed a parent-of-origin answer. For those patients in which the deletion was on the paternally inherited chromosome 15, the phenotype was PWS. Conversely, if it was the maternally inherited chromosome 15 with the deletion, the phenotype was Angelman syndrome. Further studies have shown that for the 20% of patients with PWS who do not have a chromosome 15 deletion, maternal UPD of chromosome 15 is present. If one considers these two observations, a quick conclusion becomes apparent. The common theme is that PWS occurs if the paternal component of this region of chromosome 15 is missing—either because it is deleted or because the person has inherited both copies of chromosome 15 from the mother. Ultimately these differences have been shown to be due to imprinted genes in this region.
Figure 12-11. (a) Idiogram of chromosome 15. The arrow points to the region 15q11. (b) Idiograms and photomicrographs of two different chromosome 15s demonstrating a 15q11 deletion.
As noted earlier, imprinted genes are often found in clusters called imprinting domains, which likely have linked functions of the genes in the region. Clearly this region of chromosome 15 is such a region with several known imprinted genes. Two of these genes are particularly notable. The small nuclear ribonucleoprotein polypeptide N (SNRPN) gene seems to be highly associated with PWS and the ubiquitin protein ligase E3A (UBE3A) gene with Angelman syndrome.
The genetic etiology of Angelman syndrome is slightly more complicated than PWS. Like PWS, around 70% of patients with Angelman syndrome will have a 15q11-13 deletion, which will be of maternalorigin. However, only about 5% of patients with Angelman syndrome will have UPD. But as predicted, when this is the case, it will be paternal uniparental disomy. The remaining cases of Angelman syndrome are largely due to mutations in the imprinted gene UBE3A.
Besides these two conditions, there are several other human disorders that are known to be due to disorders of imprinting. A list of some of these is in Table 12-1. It is interesting to note that many of these are inverse disorders like Prader-Willi and Angelman syndromes. While the conditions listed are largely recognizable syndromes and conditions, it is likely that imprinting errors play a role in more complex conditions. Studies have suggested a role of imprinting defects in conditions like Alzheimer disease, autism, schizophrenia, and even some cancers.
Table 12-1 Disorders Caused by Abnormalities of Imprinting
Albright hereditary osteodystrophy/McCune-Albright syndrome
Beckwith-Wiedemann syndrome/Russel-Silver syndrome
Familial nonchromaffin paraganglioma
Maternal/paternal uniparental disomy14 syndromes
Transient neonatal diabetes mellitus
More Examples of Gene-Gene Interactions
The traditional definition of compound heterozygosity is the presence of two different mutant alleles at a particular gene locus, one on each allele of the pair (Figure 12-12). In a monogenic, autosomal recessive disorder, it simply means that each allele carries its own unique mutation. This is in fact quite common given the degree of genetic heterogeneity typically present in human genetic disorders.
Figure 12-12. Graphic representation of compound heterozygosity for a simple autosomal recessive disorder at a single locus.
With better access to molecular diagnostic information, more complex mechanisms of inheritance have been identified. Digenic inheritance as noted before occurs when mutations occur at two different loci. For example, most states in the United States currently perform newborn screening for hearing loss. Newborn hearing loss is relatively common, occurring in about 1 newborn per 1000 births. If a congenital hearing loss is identified, a genetic evaluation to identify the cause is indicated. The two most common causes of hearing loss identified in infants are teratogenic (congenital cytomegalovirus) and mutations in a gene called connexin 26 or GJB2. Connexin 26 is one of the so-called “gap junction proteins” that allows for rapid ion transport that circumvents osmosis by directly connecting the cytoplasmic regions of contacting cells. This gene is located on chromosome 13. In fact, connexin 26 mutations account for almost 15% of all hearing loss in newborns.
Typically infant hearing loss due to connexin 26 mutations is inherited as a straightforward autosomal recessive condition (Figure 12-13a). Given the high frequency of connexin 26 related hearing loss, it is not surprising that the carrier frequency of connexin 26 mutations is relatively high at about 1 in 30 individuals of Northern European descent. Connexin 30 is another gap junction protein. It also lies on chromosome 13 just upstream and close to connexin 26. About 1 in 100,000 people in the general population carry a connexin 30 gene deletion. Given the low carrier frequency it is rare, but occasionally there is an individual who is homozygous for this deletion (Figure 12-13b). These individuals, then, also have an autosomal recessive hearing loss. When genetic testing for the genes became available, an interesting phenomenon was noted. A significant number of newborn were seen in which only a single connexin 26 mutation was identified. Early assumptions were that this was simply chance and unlikely to be the cause of the hearing loss. However, this occurrence was soon recognized as occurring significantly more often than could be predicted by chance and carrier frequency.
Figure 12-13. Compound heterozygosity at two different loci in hereditary hearing loss. (a) Autosomal recessive hearing loss due to mutations in the gene connexin 26. (b) Autosomal recessive hearing loss due to deletions in the gene connexin 30. (c) Compound heterozygosity for a connexin 26 mutation and a connexin 30 deletion also produces hearing loss. This could be termed “digenic” inheritance.
Ultimately it was shown that 20% of connexin 26 heterozygotes with neurosensory hearing loss also have a connexin 30 deletion (Figure 12-13c). This compound heterozygosity at two different loci is thus the cause of the hearing loss. The inheritance can be described as digenic inheritance.
Bardet-Biedel syndrome (BBS) is a recognizable condition with the key clinical features being mental retardation, pigmentary retinopathy, polydactyly and other digital abnormalities, central obesity, and hypogenitalism (Figure 12-14). As originally described, BBS was felt to be an autosomal recessive disorder. Research into the genetics of BBS has revealed genetic heterogeneity with at least 15 genes thus far described that are associated with this condition. The majority of cases of BBS typically show simple autosomal recessive inheritance due to mutations in the BBS 1 gene (Figure 12-15a). Notably, mutations on both alleles of the BBS 2 gene do not cause any apparent disease (Figure 12-15b). However, patients who are homozygous for BBS2 mutations who also harbor a single BBS6 mutation will be affected with Bardet-Biedel syndrome (Figure 12-15c). This type of compound heterozygosity involving three alleles at two different loci has been termed “triallelic” inheritance.
Figure 12-14. Young girl with Bardet-Biedel syndrome. This child has cognitive deficits, short stature, mild obesity, and pigmentary retinal abnormalities.
Figure 12-15. Compound heterozygosity involving 3 alleles at 2 different loci for Bardet-Biedel syndrome. This could be termed “triallelic inheritance.” See text for detailed descriptions.
Multi-locus Inheritance
Multi-locus inheritance as defined in the first section of this chapter means that the observed phenotype is influenced by genes at more than one locus. For many of the most common human medical conditions (if not all) this is the case. The examples above of digenic and triallelic inheritance patterns clearly meet this definition.
Modifier genes are simply genes that influence the expression of other genes. Usually these are genes that have small quantitative effects on the level of expression of another gene. One might appropriately ask: “isn’t this what was defined as polygenic inheritance?” In many ways this is correct. The distinction here is subtle, and maybe not always completely delineated. One can model polygenic inheritance as having multiple genes each more or less equally influencing a single quantitative trait. Modifiers genes, on the other hand, exert influence on a major gene’s effects. Take the example of van der Woude syndrome. Van der Woude syndrome is an autosomal dominant condition caused by mutations in IRF6 on chromosome 1q32. The major phenotypic feature that can be tracked through a family is that of bilateral lower lip pits (Figure 12-16). Cleft lip and/or cleft palate are variably expressed in association with the lip pits (Figure 12-17). It is obviously the same gene with the same mutation within a family, so why then do only some have clefting? Linkage studies have identified a locus at 17p11.2 that contains a gene (yet to be identified) that influences the expression of clefts in patients with van der Woude syndrome.
Figure 12-16. Van der Woude syndrome. The child has a repaired cleft lip and palate. Note the paired lower lip pits.
Figure 12-17. Pedigree of a family with van der Woude syndrome.
Multi-locus interactions are particularly notable in cancer. It is now known that all cancers are genetic (the reader is reminded that this is not synonymous with heritable, since mutations can occur in somatic tissue alone). The current understanding of the etiologic pathogenesis of cancer is a model of sequentially accumulated mutations (Figure 12-18). That is, multiple genes are involved in neoplasia initiation and progression. All evidence points to the clonal nature of cancers. This means that all of the daughter cancerous cells share a common origin from a cell line that has accumulated mutations in all of the genes necessary to change the growth characteristics of the cells from benign to malignant.
Figure 12-18. Conceptual model of multi-locus etiology of colorectal cancer.
Opposite (Anti-Sense) Transcripts
The use of a section of DNA for more than one transcript is not uncommon in viruses, in which compaction of information is at a premium. But when only a tiny percent of the whole human genome is actually used to code for all of the required protein products, it seemed improbable for portions to overlap in coding function. Improbable, i.e., until it was discovered. In reality, the generation of products is a function of their usefulness, not some probability of their spatial distribution. Current estimates suggest there are at least 1600 such transcript pairs scattered across the human genome.
Interleukin-14 (ILI4) is one such example in humans. Two distinctly different transcripts are created from the opposite strands of the ILI4 gene: IL14α and IL14β. Another example is the Lit1 gene in Beckwith-Wiedeman syndrome (see the clinical correlation in Part 3 of this chapter). Abnormal processing of natural antisense transcripts has been implicated in diverse group of human conditions, such as cancer, Alzheimer disease, and hemoglobinopathies.
Transposable Elements
As noted in Part 1 of this chapter, transposable elements (transposons) are migratory segments of the genome. There are several different types of transposons that differ by their size, nucleic acid type, and the mechanism of movement. The major clinical implication of transposons lies in their muta-genic potential. In some situations, this is actually a normal and desired effect. Transposons play a key role in the generation of antibodies in a normal immune response. In the processes of recombination and “somatic hypermutation” that are necessary to generate a wide diversity of antibodies, the presence of a natural source of “shuffling the deck” is advantageous. Transposons have also been shown to play a role in the origin and perpetuation of several genetic disorders. In Chapter 7 (Mutations) it was noted that some loci have a higher than average mutation rate. For many of these “mutation hotspots,” the higher mutation rate has been shown to be related to the presence of nearby or internal transposons. A list of a few of these is provided in Table 12-2. Transposons are implicated in the pathogenesis of cancer and aging. It is interesting to note that the HIV-1 virus and other similar human retroviruses demonstrate replication patterns that are strikingly similar to retrotransposons.
Table 12-2. Disorders Shown to Be Related to Transposon Generated Mutations
Acute intermittent porphyria (hydroxymethylbilane synthase gene)
Duchenne muscular dystrophy (dystrophin gene)
Familial adenomatosis polyposis (APC gene)
Hemophilia A (factor VIII gene)
Hemophilia B (factor IX gene)
X-linked severe combined immunodeficiency (IL-2 receptor gene)
Genetic Anticipation
Genetic anticipation is defined as the apparent worsening of a disorder with subsequent generations. For decades the issue was debated. Was anticipation real or simply a bias of ascertainment? If it were real, what possible mechanism could explain it? The issue was finally resolved with the identification of the underlying pathogenesis of fragile X syndrome. (Before the genetic basis of fragile X syndrome was discovered, the associated phenotype had been described as “Martin-Bell” syndrome.) Fragile X syndrome is now known to be an X-linked condition associated with mental retardation and autistic like behaviors. Boys with fragile X have mildly dysmorphic facies (somewhat thin and elongated with large ears), macrocephaly, hypotonia, joint laxity, and postpubertal macroorchidism (Figure 4-27). In the mid-1970s several laboratories were studying families with X-linked mental retardation. As part of these studies, a marker was found in some families. In these families, a “fragile site” could be expressed on the X chromosome at location Xq28 if the cells were cultured in folate-deficient media (Figure 12-19).
Figure 12-19. Karyotype showing the “fragile site” on the X chromosome at location Xq28.
Using this marker, investigators were able to look at this subgroup of families with X-linked mental retardation separately. In doing so, they identified a novel inheritance in these families. The transmission of fragile X syndrome within families showed X-linked semi-dominant inheritance with anticipation—but only when transmitted through the mother. This unique pattern was designated the “Sherman paradox” named after Stephanie Sherman who described it. Figure 4-28 shows a hypothetical pedigree of a family with fragile X syndrome. In this drawing, all of the mentioned features can be seen. The condition is X-linked; hence no male-to-male transmission is seen. The condition is semi-dominant with partial (milder) expression in females. The observed anticipation is represented by the statistical recurrence risks. Note the increasing recurrence risk with each generation—but only if transmission is maternal. Herein was the first objective evidence of genetic anticipation!
Further research ultimately identified a fascinating mechanism of pathogenesis in fragile X syndrome. The condition was shown to be due to an “expanding gene.” Specifically, the normal gene, designated FMR1, was found at a locus that was better defined as Xq27.3. A series of trinucleotide (CGG) repeats was identified in the 5′ untranslated region of the gene. Normal individuals typically have 35 to 40 CGG repeats in this region. Fragile X syndrome is now known to be caused expansion of the CGG repeat number in the FMR1 gene. When the number of repeats exceeds 200, abnormal methylation occurs with resulting suppression of FMR1 transcription and decreased production of the normal protein. The normal protein product of this gene is designated the fragile X mental retardation protein (FMRP). This protein is found in most tissues, but has its highest concentrations within the brain and testicles. The protein is critical in the formation and organization of synapses. Thus, fragile X syndrome is ultimately caused by deficiency of the FMRP. Additional studies have shown that the clinical spectrum of problems seen in families with fragile X syndrome correlate with the number of repeats, which expand as the mutation is transmitted to the next generation (Table 12-3). Thus, the argument could be definitively settled. Genetic anticipation is a real phenomenon, and a specific mechanism that can explain how it can occur had been discovered. Current technology then allows for ready diagnosis of fragile X syndrome. Using any one of several techniques, the number of trinucleotide repeats can be quantified to establish or rule out the diagnosis (Figure 12-20).
Table 12-3. Correlation of Trinucleotide Repeat Number and Phenotype in Fragile X Syndrome
Figure 12-20. Molecular testing for the trinucleotide repeats in fragile X syndrome. Shown are 3 chromatograms depicting repeat number. (a) Normal female. Note the two peaks, one for each X chromosome. (b) Normal male. One peak, one X chromosome. (c) Male with full expansion. This individual has over 200 CGG repeats noted by the large displaced peak. (Chromatograms courtesy of Dr. Jennifer Wei, Ambry Genetics.)
Myotonic dystrophy (Figure 12-21) is a muscle disease characterized by myotonia (the inability to relax muscles adequately after a sustained contraction). Affected individuals may show a variety of other medical complications including cataracts, heart arrhythmias, hypogonadism, and male pattern baldness. Clinicians had long suspected that myotonic dystrophy showed genetic anticipation. Thus, shortly after the discovery of the expanding trinucleotide repeats in fragile X syndrome, a similar mechanism was discovered in myotonic dystrophy. In a similar fashion, myotonic dystrophy was shown to have genetic anticipation due to an expanding trinucleotide repeat. There are, however, several significant differences. The trinucleotide repeat in fragile X syndrome is CGG, but in myotonic dystrophy it is GAA. The location of the repeats in fragile X is the 5′ untranslated region. In myotonic dystrophy, it is in the 3′ untranslated region of the gene dystrophia myotonica protein kinase (DMPK). In fragile X syndrome, the repeat only enlarges as it passes through maternal meiosis. In myotonic dystrophy the repeat can enlarge as it is transmitted by either sex; however, the expansion tends to be larger as it is transmitted by mothers.
Figure 12-21. Mother and child with myotonic dystrophy. The mother is more mildly affected having only mild myotonia. Note the hypotonia in the child.
Since the discovery of the trinucleotide expansions in fragile X syndrome and myotonic dystrophy, several more trinucleotide repeat disorders have been described (Table 12-4). Interestingly, most of these conditions are neuromotor disorders. As with fragile X and myotonic dystrophy, these disorders differ in the trinucleotide repeat, its size and stability, where in the gene it is located, and whether or not the repeat is translated or transcribed (Figure 12-22). Categories have been established to group the conditions that have similar properties of the repeats. Category I includes conditions due to repeats affecting the coding region of the gene. Two well-known examples are Huntington disease and the spinocerebellar ataxias (due to CAG repeats). Category II expansions are also found in the exons of genes but tend to be phenotypically diverse and generally small. Category III expansions tend to be the largest and are located outside the protein-coding region of a gene. Fragile X and myotonic dystrophy are included in this third group.
Table 12-4. Disorders Caused By Expanding Trinucleotide Repeats
Dentatorubral-pallidoluysian atrophy (DRPLA)
Fragile-X syndrome (FRAXA) Other “fragile” mental retardation syndromes Fragile X type E (FRAXE), and so forth
Friedreich ataxia
Huntington disease
Kennedy disease (X-linked spinal-bulbar muscular atrophy, SBMA)
Myotonic dystrophy
Oculopharyngeal dystrophy
Spinocerebellar ataxias
Figure 12-22 Schematic showing the different types of trinucleotide abnormalities that may be seen in different disorders.
While we will discuss pathogenesis in more detail in Chapter 16, it is worth brief discussion here. The specific question is: “how do expanding trinucleotide repeats cause disease?” To date at least three mechanisms have been identified:
1. Loss of function. As already described before, fragile X syndrome is caused by a loss of functional protein due to abnormal methylation. Friedreich ataxia, the only known trinucleotide repeat disorder that shows autosomal recessive inheritance (not surprisingly) also is due to protein insufficiency.
2. Gain of function. In some conditions, the mechanism of disease is not a deficiency of a protein product, but rather a direct toxicity from excess metabolites, which inhibit other enzyme and/or regulatory systems. Huntington disease is one such example. Normally the enzyme GADPH binds to stretches of glutamine. Excess glutamine (>760 CAG repeats) will inhibit the enzyme, which results in cumulative cell death. As is the case in Huntington disease, most of these conditions show an adult onset degenerative phenotype.
3. Dominant negative effect. Sometimes the trinucleotide change results in an abnormal product that interferes with its normal physiological function. This is the proposed mechanism in myotonic dystrophy.
One way of looking at these three mechanisms is to consider a race. Always remember that the normal physiological state is balance. Think about two people in racing lanes with the goal being to run at the same pace. A loss of function mutation would make one racer run slower than the other. A gain of function mutation would make one run faster than the other. A dominant negative effect would be if one had a leg injury that caused them to weave out of their lane and run into the second runner disabling both of them.
Epigenetics
A very important and rapidly emerging field of genetics concerns epigenetics. One of the many reasons that humans have a relatively small number of genes compared with much simpler organisms is the fact that we can do a lot more with the genes we have. As discussed in earlier chapters, this would include mechanisms such as multiply cleaved transcripts or posttranslational modification of proteins. Another mechanism of getting varied responses from a single specific genetic code is epigenetic modification.
Epigenetic mechanisms are those that can change gene expression without modifying the code itself. While the reader may not have specifically recognized it as such, epigenetic mechanisms have already been introduced in earlier parts of this book and were discussed under different topic headings. The inactivation of an X-chromosome in Lyonization is one such example. Changes in methylation as discussed earlier in this chapter in the role of imprinting genes is another. Other examples of epigenetic mechanisms are listed in Table 12-5. It is beyond the scope of this text to discuss this very complex and expansive topic in detail. The greatest importance in understanding the concept of epigenetic mechanisms lies in their therapeutic potential. The fact that many human diseases, including cancer, have an epigenetic etiology has encouraged the development of new therapeutic options that might be termed “epigenetic therapies.” Many agents have been discovered that alter methylation patterns on DNA or the modification of histones, and several of these agents are currently being tested in clinical trials.
Table 12-5. Types of Epigenetic Mechanisms
Chromatin modifications
DNA methylation
Histone modification
RNA-associated silencing
Imprinting X-inactivation
Part 3: Clinical Correlation
Beckwith-Wiedemann syndrome (BWS) is a recognizable multiple anomaly syndrome (Figure 12-23 a-c). It is characterized by macrosomia (large body size), macroglossia (large tongue), and omphalocoele. Other features include visceromegaly (enlarged internal organs)—especially the kidneys and pancreas—often severe neonatal hypoglycemia, abnormal earlobe creases, posterior helical pits, and an increased risk of embryonal tumors (Wilms tumor, hepatoblastoma, neuroblastoma, rhabdomyosarcoma). Clinical observations noted a clear familial nature to BWS without a Mendelian inheritance pattern. Other notable observations included an association of discordance of the phenotype in monozygotic twins (Figure 10-5) and an increased presence of BWS after certain types of in vitro fertilization methods.
Figure 12-23. (a) Newborn female with Beckwith-Wiedemann syndrome. (b) Close-up view of the child’s omphalocoele. (c) Abnormal helical pits.
Ultimately, molecular genetic studies of BWS identified an extremely complex inheritance pattern. The different mechanisms involved here tie together much of what has been discussed in this chapter. If after reading this clinical correlation, you understand it well, you have it down!
In short, the majority of cases of BWS are due to problems with genes within the locus 11p15. Of the identified cases:
1. For BWS 50% to 60% of cases are due to a problem with a gene in this region called Lit1. In the normal situation Lit1 is an imprinted gene. The maternal allele is usually methylated and thus “turned off.” The paternal allele is normally expressed. An error that causes loss of the methylation (hypo-methylation) of Lit1 (the normally imprinted maternal allele is thus activated) will result in increased Lit1expression and a person with BWS. It is also interesting to note that the gene Lit1 has within its coding sequence a gene that has an opposite transcript. The gene KCNQ1 is a gene that codes for a potassium channel protein. Its coding sequence is read in the opposite direction of Lit1. Thus, an alternative name for Lit1 is KCNQ1OT1 (KCNQ1 opposite transcript)!
2. In contrast, 2% to 7% of all BWS cases are due to a problem with a gene called H19 in this same region. In the normal situation H19 is an imprinted gene. The paternal allele is usually methylated and thus “turned off.” The maternal allele is normally expressed. An error that causes gain of methylation (hyper-methylation) of H19 (the normally non-imprinted maternal allele is turned off) will result in decreased H19 expression. Decreased H19expression leads to increased IgF2 expression (another gene in this region that is a growth factor) and likewise a person affected with BWS.
3. For BWS 10% to 20% of cases are due to paternal uniparental disomy of the 11p15 region.
4. For BWS 5% to 10% of cases have an identifiable mutation in CDKN1C (another imprinted gene in this region).
5. Finally, about 1% of patients with BWS will have a chromosome 11 rearrangement of the region 11p15 such as a maternal translocation or inversion or a paternal duplication.
Thus, when a patient is born with Beckwith-Wiedeman syndrome, any of these five etiologies are possible. From the information mentioned, it would seem obvious that the implications for things such as recurrence risks differ greatly depending on the cause. Therefore, targeted genetic testing must but performed—usually in a tiered (stepwise) set of tests. When an etiology is defined, this very complicated information needs to be communicated to the family in an understandable and usable manner. This is not an easy task—one that benefits from the skills and training of a genetic counselor.
Board-Format Practice Questions
1. Suppose that there is an important growth factor during fetal development (GF). Like most polypeptide hormones it has a receptor (GFR). It appears that during fetal life only the paternal GF gene is active and only the maternal GFR gene is active. Mutations that cause both copies of either gene to become active typically result in fetal overgrowth. This is an example of:
A. Uniparental disomy.
B. Genomic imprinting.
C. Unstable DNA.
D. Genetic anticipation.
E. Partial expression.
2. In regards to imprinting, which of the following statements is most likely true?
A. Imprinting represents a pathologic mechanism of gene expression.
B. Imprinting changes the DNA code.
C. Imprinting is erased during mitosis.
D. The most common mechanism of imprinting is by DNA glycosylation.
E. All human gene loci are normally imprinted.
3. In regards to X-inactivation:
A. Males will usually have one Barr body in their cells.
B. Because it is random and thus there is almost always a distribution of very close to a 50:50 proportion of one or the other X being inactivated.
C. A structurally abnormal X chromosome is preferentially inactivated, leaving the normal X active.
D. It is permanent in germ cells
E. Its clonal distribution suggests that it occurs late in embryonic development.
4. Which part of the Sherman paradox can be explained by the phenomenon of expanding trinucleotide repeats?
A. X-linked inheritance.
B. Semi-dominant pattern of transmission.
C. Genetic anticipation.
D. Females being more severely affected than males.
E. Mosaicism.
5. Which of the mechanisms below is an epigenetic mechanism?
A. X-autosome chromosome translocation.
B. Genomic imprinting.
C. Microsatellites.
D. Spontaneous mutation.
E. Expanding trinucleotide repeats.
6. Which is a mechanism whereby expanding trinucleotide repeats could cause disease (i.e., pathogenesis)?
A. Dominant negative effect.
B. Insertional mutations.
C. X-inactivation.
D. Reverse (opposite) transcription.
E. Modifier genes.