Can a heterozygous allele show non-heterozygous expression in a family?

Can a heterozygous allele show non-heterozygous expression in a family?

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I'm doing a family study looking for novel cancer-associated variants in germ-line samples; the goal is to find candidate biomarkers which might be used for early detection. At an earlier step our analysis, we eliminated a family that showed a heterozygous mutation known to be associated with our cancer under the premise that this known mutation better explained the presence of the disease than any novel variant we may have found.

I've now found another mutation, strongly associated with out cancer, which we does not appear to be heterozygous. 2 out of 3 family members share this mutation with a within-family allele frequency of 0.5.

Trying to decide whether this means I need to discard this family from further consideration, adjust my thinking, or not worry about it. My current thinking is that it'd be safest to eliminate the family from consideration because the mutation pathogenic and found in the germline but I can't wrap my head around how the lack of a heterozygous pattern affects things. Does it absolutely mean the variant was not inherited? Can anyone help me get my thoughts straight?


Antonio Blanco , Gustavo Blanco , in Medical Biochemistry , 2017

Proteins are macromolecules formed by amino acids

Proteins are large size molecules (macromolecules), polymers of structural units called amino acids. A total of 20 different amino acids exist in proteins and hundreds to thousands of these amino acids are attached to each other in long chains to form a protein. Amino acids can be released from proteins by hydrolysis. (Hydrolysis is the cleavage of a covalent bond by addition of water in adequate conditions.)

Due to their large size, proteins obligatorily form colloids when they are dispersed in a suitable solvent. This property characteristically distinguishes proteins from solutions containing small size molecules.

Since amino acids are the “building blocks” for proteins, their structure and properties will be considered first.

Mendelian inheritance patterns

Within a population, there may be a number of alleles for a given gene. Individuals that have two copies of the same allele are referred to as homozygous for that allele individuals that have copies of different alleles are known as heterozygous for that allele. The inheritance patterns observed will depend on whether the allele is found on an autosomal chromosome or a sex chromosome, and on whether the allele is dominant or recessive.

Autosomal dominant

If the phenotype associated with a given version of a gene is observed when an individual has only one copy, the allele is said to be autosomal dominant. The phenotype will be observed whether the individual has one copy of the allele (is heterozygous) or has two copies of the allele (is homozygous).

Autosomal recessive

If the phenotype associated with a given version of a gene is observed only when an individual has two copies, the allele is said to be autosomal recessive. The phenotype will be observed only when the individual is homozygous for the allele concerned. An individual with only one copy of the allele will not show the phenotype, but will be able to pass the allele on to subsequent generations. As a result, an individual heterozygous for an autosomal recessive allele is known as a carrier.

Sex-linked or X-linked inheritance

In many organisms, the determination of sex involves a pair of chromosomes that differ in length and genetic content - for example, the XY system used in human beings and other mammals.

The X chromosome carries hundreds of genes, and many of these are not connected with the determination of sex. The smaller Y chromosome contains a number of genes responsible for the initiation and maintenance of maleness, but it lacks copies of most of the genes that are found on the X chromosome. As a result, the genes located on the X chromosome display a characteristic pattern of inheritance referred to as sex-linkage or X-linkage.

Females (XX) have two copies of each gene on the X chromosome, so they can be heterozygous or homozygous for a given allele. However, males (XY) will express all the alleles present on the single X chromosome that they receive from their mother, and concepts such as 'dominant' or 'recessive' are irrelevant.

A number of medical conditions in humans are associated with genes on the X chromosome, including haemophilia, muscular dystrophy and some forms of colour blindness.

What Are Heterozygous Traits?

Any time an individual has two different alleles for a gene, the individual's genotype is heterozygous. For genes with simple dominance, like the pea example, the dominant allele is always expressed, so heterozgyous individuals have the dominant trait. Not all genes exhibit simple dominance, though, and sometimes heterozygous individuals have a distinct phenotype. In incomplete dominance, the heterozygous phenotype is intermediate between the homozygous phenotypes. In codominance, both homozygous phenotypes are expressed in heterozygous individuals.


RMAE is of interest as an epigenetic phenomenon as it requires unequal regulation of two alleles, even if they are identical. Moreover, since parental imprinting mechanisms are not at play, there is a requirement for the RMAE of individual genes to be established independently in individual cells during development, much like X-inactivation in females, except that the decision occurs at the level of the individual autosomal gene as opposed to the entire X-chromosome. DNA rearrangement-mediated RMAE of immunoglobulin and T-cell receptor genes were the only known examples of autosomal RMAE until the report of monoallelic expression in the odorant receptor gene family and subsequent reports of a handful of other genes, most involved in the immune and the immune chemosensory systems.

Using SNP genotyping arrays capable of genome-scale query, we have recently established that RMAE of autosomal genes is widespread in the human genome, affecting approximately 10% of assessed genes [10]. In order to understand the extent of this phenomenon and its evolutionary conservation, in this work we examined RMAE in mouse cells, using a genome-scale approach in clonal cell lines. We observe widespread monoallelic expression in the mouse, comprising over 10% of genes, as evidenced by allele-specific expression observed in one or more mouse lymphoblast clonal cell lines. A smaller but still sizeable number of genes were found to be RMAE in fibroblasts (Figure S1 and Note 1 in Additional file 1). As we found in analyzing human genes, for most RMAE mouse genes, the allelic expression state varies from clone to clone: sometimes maternal monoallelic, sometimes paternal monoallelic, sometimes biallelic (98.1% of genes have at least one BAE clone). We did not repeat the extensive in vivo validation of RMAE that was performed for the earlier study of RMAE of human genes. As such, it is formally possible some of the RMAE observed in mouse cell lines differs from the expression patterns in vivo. However, given the conclusiveness with which RMAE detected in human cell lines was validated in vivo taken together with the overall similarity of gene regulation mechanisms in human and mouse, it is reasonable to expect that the RMAE found in mouse cell lines generally reflects the situation in vivo.

As for humans, the choice of active allele is not coordinated chromosome-wide in a given clone, the expression state of each monoallelic gene is independent of the others. As a result, each clone has a unique signature of allele-specific expression, creating extensive epigenetic heterogeneity in otherwise identical cells. The diversity of patterns observed within a population of cell lines could be explained by an initial period of plasticity (or random choice) followed by a fixation of each allele's allelic expression state. The properties of RMAE described above for the mouse are similar to what was observed in human clonal lymphocytes [10].

Autosomal RMAE has the potential to impact biological function by creating three distinct cell states for each gene in instances when both alleles encode functional gene products. For each given gene, these states would be defined by expression of the maternal allele, the paternal allele, or both alleles. The observed stability of the allele-specific choice in a given clone [9, 10], together with in vivo clonal expansion, can lead to growth of macroscopic patches of tissue with subtly distinct properties. In studies of human RMAE, such patches were shown in the normal placenta [10]. In general, the size of these patches would be dependent on the stage in development at which the allelic choice is made for each developing tissue. Given the large number of autosomal genes involved, there is a clear potential for RMAE to contribute to phenotypic differences among individual organisms.

Considering orthologous genes assessed for RMAE in similar cell types in mouse and human, we find that the number of genes subject to RMAE in both species is five-fold greater than would be expected by chance (Figures 3a, b). We can thus conclude that regulatory features allowing a gene to be RMAE were present in the last common ancestor of rodents and primates, and that these features have been maintained in the intervening 65 to 85 million years [17]. Under one interpretation, such evolutionary conservation could be due to the selective advantage of these genes' RMAE. Indeed, for some previously known examples of autosomal monoallelic expression, such as olfactory receptors and immunoglobulins, the adaptive advantage of monoallelic expression is clear: it confers a unique specificity to otherwise identical cells. However, it is also formally possible that for at least some of the newly identified mouse RMAE genes, RMAE is not in itself adaptive, but is rather a consequence of other regulatory features being acted upon by selective pressures. Another possibility supported by these data would be that RMAE of some genes adversely affects the fitness of the organism to the point that these genes are excluded from RMAE, resulting in obligate, and thus conserved, BAE. This would thus limit the pool of orthologs that have the potential to display RMAE.

In a striking difference from human RMAE, in 129S1/SvImJ × Cast/EiJ murine cells we often observe statistically significant 'skewed RMAE', wherein, for a given gene, there is preferential expression of one allele for cells with monoallelic expression (Figure 4). In extreme cases that still lie within the boundaries of our definition of RMAE, all instances of monoallelic expression originate from the same allele, while the other allele is active only in clones that show BAE. This skewed RMAE resembles the skewed X-chromosome inactivation that has been observed in mouse F1 hybrids, and that has been traced to distinct properties of the sequence elements known as X-inactivation centers [15]. In the case of skewed X-chromosome inactivation, each cell still has chosen one or the other copy of the X-chromosome to be active. What is skewed is the relative abundance of these cells with one X active versus cells with the other X active.

The notion of skewed RMAE has important implications for the interpretation of data measuring allele-specific expression of autosomal genes, since such skewing could underlie some instances in which 'incomplete imprinting' has been observed, as well as instances where allelic imbalance tracks with strain of origin (and cis-regulatory polymorphisms have been presumed to be the sole regulatory mechanism). For example, a gene that is equally expressed from both alleles in half of the cells, and from only one allele in the other half of the cells, would appear to show significant allelic imbalance when assessed in a mixed cell population (for example, 2:1 if the level of expression is fixed per allele Figure 5). However, the underlying mechanisms and functional consequences (especially for genes with cell autonomous functions, such as tumor suppressors) would be quite different than in a 'classical' allelic imbalance (that is, one due to the interaction of cis- and trans-factors [18].

Clone-specific monoallelic expression and tissue-scale allelic imbalance. Allelic imbalance has been noted for a variety of genes in different tissues and is typically attributed to either parent-of-origin imprinting or to cis-regulatory variants. Skewed random monoallelic expression (RMAE), when one allele is preferentially chosen for expression, could also result in a tissue-wide allelic imbalance. In a traditional view (left) the expression level varies between the two alleles, with the paternal (pat) allele contributing more. This is uniformly true among the cells in the tissue. By contrast, in the scenario depicted at right, a difference in the relative abundance of cells with different RMAE states results in a tissue-scale allelic imbalance. Mat, maternal Pat, paternal.

Finally, we compared the RMAE seen in immortalized lymphoblasts to that seen in fibroblast lines. Similar to results reported for lymphoblasts, fibroblasts demonstrate RMAE across a large number of genes throughout the genome (Figure S1 in Additional file 1). By contrast, a lower overall level of RMAE (Note 1 in Additional file 1) was seen in fibroblasts, a finding that is consistent with the idea that RMAE is cell type specific [9, 10].

Practical Work for Learning

Class practical

Reebops are imaginary animals, made from marshmallows (or foam packing pieces). Their features are determined by the characteristics encoded in their chromosomes. Both parents are heterozygous for all their characteristics (except their sex). Making models in this way has proved very popular with post-16 students – many of whom keep their baby Reebops at home and talk of them with some affection!

Inheritance and variation are two key observations on which the theory of evolution by natural selection is based. You can develop this model of inheritance to show how a population of Reebops could change over successive generations – how it could evolve – if you generate rules to model the advantage that could be conferred on individuals by different characteristics.

Lesson organisation

Prepare for this lesson by explaining what your course requires students to know about the structure of DNA, what genes are, and the fact that chromosomes exist in homologous pairs. Make sure students understand the idea of gametes including one chromosome from each homologous pair, and the offspring having homologous pairs that include one chromosome from the male and one from the female parent.

Present models of adult male and adult female Reebops to the students and draw attention to their characteristics.

Explain that the students have been given instructions for breeding a male and a female Reebop in order to make a baby Reebop, and find out what it might be like.

Provide students working in pairs with the student procedure sheet, envelopes of chromosomes, a decoder key and the materials to build their baby Reebops.

Apparatus and Chemicals

For each group of students:

Envelope of Mum Reebop chromosomes
(Note 1 and see Resources sheet)

Envelope of Dad Reebop chromosomes
(Note 1 and see Resources sheet)

Large marshmallows, white, 4 (Note 2)

Small marshmallows, in three other colours, up to 6 (Note 2)

Pins, with coloured plastic covers – map pins, thumb tacks or other pins, 8 (Note 2)

Decoder key, 1 (see Resources sheet)

For the class – set up by technician/ teacher:

Adult male and adult female Reebop (Note 1)

Health & Safety and Technical notes

Make sure students don’t eat the marshmallows if you are working in a laboratory.

1 Chromosomes: Photocopy the chromosome templates onto card. Copy the Mum Reebop onto pink or red card and the Dad Reebop onto blue. Laminating the card increases the number of times you can use them. Cut up the chromosomes and secure in bundles with an elastic band.

2 Making adult Reebops: Marshmallows that have dried out slightly work better. You could make the Reebop pieces in any colour, but remember to update the genotype/ phenotype decoder sheet accordingly.

  • Join three large white marshmallows with cocktail sticks as ‘ligaments’ to hold them together.
  • Attach a head (another large white marshmallow) above the first segment with a cocktail stick.
  • Make a tail from a piece of pipe cleaner. Mum and Dad both have curly tails.
  • Make four legs with blue push pins.
  • Add two antennae – pins of the same colour. Glass-headed dressmaking pins would be ideal here.
  • Add eyes – coloured thumb tacks (or small marshmallows).
  • Add a nose – a small marshmallow or another kind of pin.
  • Add humps to the body segments with small marshmallows, held in place with short pieces of cocktail stick.
  • Indicate that your Reebop is male or female. Use your imagination!

Ethical issues

There are no ethical issues associated with this procedure. However, our understanding of this model for allele reassortment is at the centre of applications such as genetic disorder screening and other selective reproduction techniques. Be prepared for discussions around this model that introduce these ethical issues.


SAFETY: Do not allow students to eat the marshmallows if you are working in a laboratory. Even in a classroom, discourage eating the marshmallows after they have been handled and stuck with pins!


a Make up the adult Reebops, the decoder key and the chromosome cards.

b Collect enough material to make one baby Reebop for every working pair.


c Discuss the adult Reebops and their features.

d Allow students to read and follow instructions on sorting and re-assorting the chromosomes.

e Allow students to decode the genotype of their baby Reebop and build a model.

f Discuss the outcomes.

Teaching notes

The whole exercise is a model for the processes of meiosis, fertilisation and development. You can draw attention to what happens when working with the model, and highlight how they reflect (or differ from) our ideas about the living process. Here are some examples.

  • An elastic band keeping the chromosomes together can be thought of as representing histone proteins or the nuclear envelope.
  • If students ‘get it wrong’, consider any mistakes carefully and try to draw parallels with natural errors such as non-disjunction or mutations.
  • Extend the exercise further by choosing two babies allow them to ‘mature’ rapidly, and use them as parents for the next generation.
  • Draw up family trees to follow the inheritance of selected features.
  • If your new adults are both heterozygous for a dominant feature and starting a new population, think about the outcome of introducing a recessive mutation for some feature and see how it could spread through a population.
  • Consider how natural selection might act on this group of animals. Which features could confer an advantage? or a disadvantage in certain situations? How could a characteristic be eliminated from a population? If you split the Reebops into two groups, how might they develop differently in different situations?

Eight chromosomes should give a good range of phenotype expression (384 possible outcomes). If you have a large class you might want to introduce extra features, such as wings or gills, to increase the variation between the offspring. If so, include an extra chromosome, extra materials and extra information on the decoder key.


Download the student sheet Making Reebops: a model for meiosis (70 KB) with questions and answers.

Adapted with permission from Salters-Nuffield Advanced Biology. Pearson Education 2008. Original source material is available as pdfs attached to this document.

© 2019, Royal Society of Biology, 1 Naoroji Street, London WC1X 0GB Registered Charity No. 277981, Incorporated by Royal Charter


Some genes affect more than one phenotypic trait. This is called pleiotropy. There are numerous examples of pleiotropy in humans. They generally involve important proteins that are needed for the normal development or functioning of more than one organ system. An example of pleiotropy in humans occurs with the gene that codes for the main protein in collagen, a substance that helps form bones. This protein is also important in the ears and eyes. Mutations in the gene result in problems not only in bones but also in these sensory organs, which is how the gene's pleiotropic effects were discovered.

Another example of pleiotropy occurs with sickle cell anemia. This recessive genetic disorder occurs when there is a mutation in the gene that normally encodes the red blood cell protein called hemoglobin. People with the disorder have two alleles for sickle-cell hemoglobin, so named for the sickle shape (Figure (PageIndex<4>)) that their red blood cells take on under certain conditions such as physical exertion. The sickle-shaped red blood cells clog small blood vessels, causing multiple phenotypic effects, including stunting of physical growth, certain bone deformities, kidney failure, and strokes.

Figure (PageIndex<4>): The sickle-shaped red blood cell on the left is shown next to several normal red blood cells for comparison.


Probably the biggest challenge for accurate estimates of ASE comes from the fact that reads from both alleles are mapped against a common reference. If one of the alleles is more similar to the reference than the other one, this results in an unequal success rate of read mapping (mapping bias) (Degner et al. 2009 Kofler et al. 2011 ).

Several studies exist that propose frameworks to identify allele-specific gene expression from RNA-seq data (Rozowsky et al. 2011 Skelly et al. 2011 Turro et al. 2011 Graze et al. 2012 Satya et al. 2012 Shen et al. 2012 ). However, only two studies, AlleleSeq (Rozowsky et al. 2011 ) and MMSEQ (Turro et al. 2011 ) provide a software pipeline, which can be used by researchers to conduct similar analysis. In accordance with the proposed frameworks for ASE identification, both software tools generate a polymorphism-aware diploid genome as a reference for read mapping to reduce the mapping bias. However, their usage is limited to specific data sets. AlleleSeq does not infer polymorphisms between parental genomes, but requires these polymorphisms as input (Rozowsky et al. 2011 ). MMSEQ allows polymorphism detection on the RNA-seq data directly, but requires phasing of genotype calls prior to reconstruction of the parental haplotypes (Turro et al. 2011 ). Both software tools use Bowtie (Langmead et al. 2009b ) for short read mapping, which does not support gapped alignments, split mapping and SNP aware mapping. Furthermore, the statistical framework of AlleleSeq does not account for replicate data, and neither tool considers a residual mapping bias.

Here, we introduce a new comprehensive and user-friendly software tool, Allim, for measuring allele-specific gene expression in F1 individuals, which accounts for the inevitable mapping bias by combining two strategies. First, a polymorphism-aware diploid reference genome is constructed from parental RNA or genomic short read data. Second, a sequence-specific simulation tool estimates the residual mapping bias. Furthermore, within Allim, a statistical framework is provided, which includes a correction of the residual mapping bias and can take advantage of replicate data. For optimal short read mapping, Allim uses GSNAP, which is capable of SNP tolerant mapping, split mapping and allows gapped alignments (Wu & Nacu 2010 ).

Tt x Tt

Indeed, we can now see why 1/4 of the individuals were short since only one in four (lower right) possess the tt genotype i.e. no big T is present in 25% of the offspring. Thus, the genotype of the F2 population is 25% TT, 50% Tt, and 25% tt (a ratio of 1:2:1). The phenotype is different, however. Since 3/4 possess at least one big T, they will be tall. So, the phenotypic ratio is 3:1 (tall vs short).

The totally recessive individuals are highly useful in genetics. Whenever you see one, you automatically know the entire genotype (i.e. both alleles) for that gene. For instance, if you have a tall individual, you know that at least one big T is present but you don't know if the second allele is "T" or "t." Not so for the short pea plants as they can only be "tt." These recessive offspring are extremely valuable in genetics. You not only know the genotype of the individual in question, but you also know that each of the parents carries at least one recessive allele whether you can see it or not (i.e., the offspring HAD to get a little "t" from EACH parent). These recessive individuals can be used to "test" an unknown plant and probe for any hidden, recessive alleles. This is called a testcross.

The results of Mendel's crosses allowed him to formulate his Law of Segregation, which states that each organism contains two factors (i.e. alleles) for each trait, and the factors segregate during the formation of gametes so that each gamete contains only one factor for each trait. What this means is that the alleles of an organism exist as pairs (we now know on separate, homologous chromosomes) and that one member of the pair enter different gametes (i.e., we now know that the homologous chromosomes separate during meiosis I).

OK. You now, hopefully, understand some of the basics of how alleles segregate into the gametes and how the gametes fuse to form a zygote. Using what we've learned above, lets see if you can work the following two monohybrid genetics problems.

Hypothetically, brown color (B) in naked mole rats is dominant to white color (b). Suppose you ran across a brown, male, naked mole rat in class and decided to find out if he was BB or Bb by using a testcross. You'd mate him to a white (totally recessive) female, and examine the offspring produced. Now, if only 2-3 offspring were born and they were all brown, you'd still be uncertain whether he was BB or Bb (for instance, even though the odds are 50:50 that you will produce a boy or girl, there are plenty of people that produce 4-5 girls and never a boy and vice versa). But, if the mole rats produce 50 offspring and all are brown, then it is likely that no hidden alleles are present and that the male is BB. But, what if white offspring are produced? You'd know that the brown parent had a hidden little "b" allele. So, what you need to do is perform a testcross on this brown, male, heterozygous, naked mole rat. What are the expected genotypic and phenotypic ratios of such a cross?

What if you bred some snap dragons and crossed a homozygous red plant (RR) with a homozygous white plant (rr)? In botony, "true breeding" means homozygous. In this case, 100% of the F1 individuals would be pink! This is an example of "incomplete dominance," where both alleles contribute to the outcome. In some cases of incomplete dominance, both alleles might contribute equally so one allele would produce red pigment and the other white thus, a pink plant appears. In another case, one allele may be non-functional. Although in many cases only a single allele is needed, perhaps in this case only one-half the amount of needed pigment is produced and so pink is due the low amount of red pigment in the petals. Who knows. Anyway, use a Punnett's square and set up a cross between a homozygous red plant and a homozygous white plant. Then, take the resulting offspring and cross these among themselves as well (i.e. F1 x F1). Then, determine the phenotypic and genotypic ratios.

The above examples involved a single gene only, and a single set of alleles. But, what if TWO different genes are involved. Well, that makes the crosses more complicated and we term this a dihybrid cross. As long as the genes are on separate chromosomes, things go pretty easily. Only the Punnett squares are larger (16 squares rather than 4). So, lets cross an individual who is homozygous dominant for gene A (AA) and homozygous dominant for gene B (BB) with someone who is homozygous recessive for gene A (aa) and homozygous recessive for gene B (bb). If you follow the movement of alleles into the gametes, for instance with the AABB, the alleles will assort as follows: the first A in the AABB goes with the first B (I've underlined this set) and is placed in column 1 (light blue) in the second pairing the first A goes with the second B (column 2) in the third pairing the second A goes with the first B (column 3) and in the fourth pairing the second A goes with the second B (column 4). Then, do the same for the aabb individual and place these pairs of alleles in the four rows (light green). As you will note below, all offspring are AaBb and will possess the same phenotype.

How can recessive traits skip generations?

Recessive traits can skip generations because a dominant phenotype can be produced by either a homozygous dominant genotype or a heterozygous genotype. So two heterozygous individuals would have the dominant phenotype for a trait, but since they each have a recessive allele, they both could pass a recessive allele to an offspring, producing a homozygous recessive offspring with the recessive phenotype. This would be a monohybrid cross.

In mice, black coat color is dominant, and white is recessive. The black allele is represented by the letter B, and the white allele is represented by the letter b.

Two black mice that are heterozygous (Bb) for the black phenotype produce offspring. The following Punnett square represents the possibilities for their offspring.

As you can see, there is a 1 in 4 (25%) chance that they will have a homozygous recessive (bb) offspring with the white phenotype.

So both of the heterozygous (Bb) parents have the dominant black coat color, but the next generation, their offspring, could have the homozygous recessive genotype (bb), producing the recessive white coat color.