Chapter 4: Pathomechanisms of Genetic Diseases


From Genotype to Phenotype

It is unlikely that a patient will seek medical advice by saying, “Good morning, doctor. I have a problem with my compound heterozygosity. On one allele of my CFTR gene is a classical F508del mutation, on the other the 5T-intron variant—this worries me. Can you please help me?” Medical practice deals with real patients and not with variable copies of pathological gene sequences. So, when does a molecular alteration turn into a “real patient”? How does the genotype become a phenotype? And how can the understanding of this correlation help the doctor in diagnosis and therapy?

The term genotype denotes the entire genetic information of an organism or a cell. More specifically, especially with regard to a certain phenotype, the term relates to the genetic information at a specific locus (i.e., the combined DNA sequence information on the two copies of a particular gene). For example, the genotype of an autosomal gene tells whether a genetic variant occurs on both copies of the gene (homozygous) or only on one (heterozygous).

It considers the fact that two different variants of a gene can occur on the two homologous chromosomes (in trans) or just on one chromosome (in cis), the latter having occurred through successive mutations or after recombination. The genotype also makes a distinction between homozygosity and hemizygosity. If there is only one copy of a gene, such as most X-chromosomal genes in males, variants in this gene are hemizygous even though in standard molecular genetic tests they look like homozygous variants in autosomal genes.

The total genetic information available to an organism or a cell, either as a whole or with regard to a specific function.
The physical manifestation of the genetic information.
A genetic variant/sequence on both copies of a gene.
A genetic variant/sequence on only one of the two copies of a gene.
A genetic variant on a gene of which there is only a single copy.
In trans:
Two different genetic variants of a gene that occur on the two homologous chromosomal strands.
In cis:
Two different variants of a gene that occur beside each other on the same chromosomal strand and not on the homologous chromosomal strand.

A phenotype is “what we see,” meaning the physical manifestation of the genetic information. It describes what the cell or the organism creates from the genotype. It is important to note that the phenotype results from multiple factors, both genetic as well as nongenetic. The phenotype can be defined in different ways, depending on the focus that we choose to take (Fig. 4.1).

Figure 4.1.From Genotype to Phenotype.

From Genotype to Phenotype.

Patients and physicians usually think and work primarily at the level of the clinical phenotype. The patient normally consults a physician because of symptoms that may be perceived as serious and stressful. The physician examines the clinical phenotype, both physically and through diagnostic imaging techniques, and tries to change it through therapeutic intervention. Indeed, nongenetic factors (i.e., “environmental factors”), including treatment, exert their biggest impact on the clinical phenotype.

On a functional level, the metabolic or clinical chemical phenotype, meaning the concentration of certain key substances in body fluids, is of special diagnostic relevance. Not every clinical phenotype has a measurable clinical chemical correlate; for many genetic disorders there are no measurable biochemical abnormalities.

On the other hand, there are genetically caused conditions that have a distinct clinical chemical phenotype yet lack any clinical correlate. Autosomal recessive–inherited histidinemia is a good example of such a “nondisease.” There are clear metabolic abnormalities associated with increased histidine concentration in plasma and urine but no clinical symptoms whatsoever.

The same is true for the molecular phenotype, which represents the direct gene product and can be examined, for instance, through laboratory methods such as enzyme studies. Protein structure and function is more directly determined by the genotype, and its analysis, if possible, allows a more direct assessment of gene function. Nevertheless, measurable abnormalities in protein structure or function do not automatically imply clinical relevance. Histidinemia, caused by a loss of histidase enzyme activity, once again serves as an example.

Ultimately, the genotype is the etiological basis of many human conditions. However, it is not always easy to determine what a particular genetic variant really means for the patient. Geneticists may be specialists for diagnostic tests at the genotype level, but they must also be familiar with the various ways of identifying a genetic disorder on the phenotype level (e.g., clinical, functional, structural, cellular, and molecular levels). Determining which diagnostic approach to choose depends on many factors, and quite often a phenotype-based assay is superior to a gene test.

Nevertheless, for many disorders described in this book, it is not possible to measure the activity of enzymes, amounts of protein, substrate accumulation, or other phenotypic parameters in order to reach a firm diagnosis. Only if we understand the relationship between genetic variants and the different levels of phenotypic presentation will we be able to understand the patient’s disorder and to interpret the results of genetic tests correctly.

Different mutations in the same gene can be responsible for the development of different disorders. For example, mutations in the lamin A/C gene can result in several different disorders, including mandibuloacral dysostosis, Charcot-Marie-Tooth disease, familial lipodystrophy, and progeria.
Genetic heterogeneity:
The same disorder can be triggered by mutations in different genes. For example, Lynch syndrome can result from mutations in MLH1, MSH2, PMS1, PMS2, or MSH6.
The variable phenotypic manifestation caused by a genetic variant.
Variable expressivity:
The same mutation can result in variable clinical phenotypes.
The likelihood with which a genetic variant causes any kind of phenotypic manifestation.
The phenomenon in which the effects of one gene are modified by one or several other genes.
The phenomenon in which the effects of one gene are modified by external factors.
A disease that is caused by mutations in a single gene.
A disease that is caused by the combined effect of mutations in two different genes.
A disease that is caused by the combined effect of mutations in multiple genes.
A disease that is caused by the combined effect of multiple genetic and nongenetic factors.

The identification of genotype–phenotype correlations is one of the main tasks for human genetics, both in research and in clinical practice. Quite often mutations in a gene are associated with different clinical manifestations even within the same family (i.e., variable expressivity) or in some individuals may have no functional effect whatsoever (i.e., reduced penetrance). This may be due to the influence of other genetic factors within the organism (i.e., epistasis), due to chance effects in dominant disorders caused by clonal loss of the second (i.e., wild-type) allele (e.g., loss of heterozygosity as seen in cancer predisposition syndromes), or due to exogenous factors (i.e., polyphenism).

Interestingly, successful treatment that changes the phenotype, such as dietary treatment that prevents intellectual disability in phenylketonuria (PKU) (as discussed in Chapter 24.1.3), could be regarded as a form of polyphenism, but the term is mostly used for the adaptation of animals and plants to different environments. For the development of some clinical phenotypes, the mutation(s) of one single gene is not sufficient. Only if mutations in two or several specific genes interact does the clinical phenotype develop (i.e., digenic or polygenic inheritance). An example is Bardet-Biedl syndrome (see Section 29.3).

Dominant and Recessive

The founder of modern genetics was the Augustinian monk Johann Gregor Mendel (1822–1884), who in 1865 published “Experiments on Plant Hybridization” after cultivating and studying varieties of garden peas at his monastery in Bru?nn (now the Slovakian town of Brno). He showed that inheritance is based on distinct and separate factors. Today these “factors” are called genes. Mendel also coined the terms dominant and recessive by writing, “. . . those characters which are transmitted entire, or almost unchanged in the hybridization, and therefore in themselves constitute the characters of the hybrid, are termed the dominant, and those which become latent in the process, recessive.” Understanding the molecular basis of dominance and recessiveness is the basis for understanding the molecular basis of inherited disorders.

There are two different approaches to the terms dominant and recessive. The traditional approach is to look at inheritance patterns in a family and to check whether a phenotype (i.e., a disease) is found, for example, in successive generations, siblings, or male individuals (see Chapter 5). Although this works in many instances, it has major limitations and does not really help in understanding the pathophysiology. Much more enlightening is the question of what happens clinically (i.e., phenotypically) when an individual is heterozygous, a “hybrid” in Mendel’s terms.

The terms dominant and recessive can only be understood properly as a description of the functional relationship of two different alleles of the same gene in a (compound) heterozygous organism. Mendel demonstrated this in the case of pea plants, which bloomed either red or white. If a genetically pure red-blooming plant is pollinated by a genetically pure white-blooming plant (or vice versa), the resulting seed produces only red-blooming plants in the F1 generation.

The “red” allele dominates over the “white” allele in the hybrid. The same is true with regard to disease-causing mutations in humans. A pathogenic mutation on one allele (with the wild-type allele on the homologous chromosome) is enough to cause a dominant disorder, while heterozygous carriers of recessive disorder are phenotypically healthy. Recessive disorders only arise if there are mutations on both copies of the respective gene.

It is not quite that simple in most cases, however, and the analysis of what really happens on the molecular or cellular level in dominant and recessive disorders helps one understand the pathogenesis of human disorders.

A genetic variant that causes a recognizable phenotype in the heterozygous state, or the phenotype caused by this type of variant.
A genetic variant that causes a recognizable phenotype only in the homozygous state, or the phenotype caused by this type of variant.
Semidominance (incomplete dominance):
The phenomenon that a genetic variant causes a recognizable phenotype in the heterozygous state and another (usually more severe) phenotype in the homozygous state.
The phenomenon that two distinct phenotypes associated with two different genetic variants are both found in a compound heterozygous individual.

Dominant in Every Respect?
It is possible that heterozygous carriers of a recessive disease can have relevant phenotypic traits. Heterozygous carriers for PKU have slightly elevated phenylalanine concentrations in their blood and markedly reduced enzymatic activity of phenylalanine hydroxylase in the liver. This is not clinically relevant, but it shows that the normal allele does not completely dominate over the mutated one.

Heterozygote Advantage

Evolutionary pressure reduces the allele frequency of recessive disease mutations if affected (i.e., homozygous) individuals do not procreate. Why is it, then, that some recessive disorders are very common in various different populations? Why is cystic fibrosis so common in Europeans, with a carrier frequency of up to 5% or more? Why are 9% to 10% of African Americans heterozygous carriers for sickle cell anemia? It appears that, in certain situations, heterozygosity represents a selection advantage over homozygosity for the wild-type allele (i.e., heterozygote advantage). The best-known examples of this are the hemoglobinopathies.

In Equatorial Africa, where malaria is endemic, the mutation for sickle cell anemia (i.e., a mutation in the ?-chain of hemoglobin; see Chapter 21.5) has an extremely high allele frequency of 20% to 40%. Epidemiological studies have shown that heterozygous carriers for sickle cell anemia are more resistant toward Plasmodium falciparum, the pathogen that causes tropical malaria. Infected erythrocytes of heterozygous individuals are probably destroyed more rapidly, thereby reducing the ability of the plasmodium to multiply in the blood.

Homozygotes for normal hemoglobin are more likely to have serious complications of tropical malaria and to die early; in consequence, they are less fertile (have fewer offspring). Although homozygous patients with sickle cell anemia mutation have a serious life-threatening disease that, in the past, precluded them from having children, this effect was counterbalanced in areas with a high incidence of malaria, as reproduction of carriers of sickle cell mutation is increased by about 15%.

If exogenous factors are stable over many generations, the favorable and unfavorable effects of a genetic variant in a population achieve a balance, which is readjusted only when exogenous effects change. The sickle cell mutation, in this regard, represents a balanced polymorphism in populations with a high prevalence of malaria.

Semidominance or Incomplete Dominance

For most disorders inherited as dominant traits, homozygosity for a disease-causing mutation results in a much more severe clinical phenotype than heterozygosity. An example is familial hypercholesterolemia (Chapter 24.3), a genetic disorder resulting from mutations of the low-density lipoprotein (LDL) receptor gene. Individuals with a heterozygous loss-of-function mutation show elevated LDL cholesterol levels (greater than 7 to 10 mmol/L) and typically suffer their first myocardial infarction in midlife. Homozygous carriers have a much higher LDL cholesterol level (10 to 30 mmol/L), with the onset of symptoms in early childhood and coronary heart disease as early as school age.

Another example is achondroplasia (Chapter 26.2), in which homozygosity for the disease-causing FGFR3 mutation causes a severe skeletal dysplasia typically leading to death in utero or shortly after birth. In these examples, as indeed in most genetic traits, the phenotype of heterozygotes (Aa) is somewhere in between the phenotypes of wild-type and mutant homozygotes (AA and aa). The inheritance pattern is called semidominant or incompletely dominant, in contrast to complete dominance that is found in very few conditions, such as Huntington disease (Chapter 31.1.1), in which the phenotype of the heterozygous and homozygous mutation carriers is more or less identical.

It is worth thinking about reasons why a condition may show complete penetrance. For practical purposes, both types of conditions may be called dominant because the definition rests on the clinical phenotype in the heterozygote, irrespective of what is observed in the homozygote.