Mechanisms of human genetic disease

The goal of medical evaluation is to make a diagnosis. To achieve this we need to explore medical histories, evaluate the symptoms of a condition and perform relevant diagnostic tests. As you are aware, all human diseases are caused by environmental or genetic factors, or a combination (i.e. multifactorial). Earlier in the step Genetic variations, we discussed how several genomic alterations can result in human diseases.

Generally, several factors determine how a genetic variant contributes to the phenotype. Benign variants do not affect the phenotype, whereas pathogenic variants can either have a ‘small influence’ on the phenotype (as in multifactorial disease) or a ‘large influence’ on the phenotype (as in highly penetrant monogenic diseases). Environmental (lifestyle, pollution, toxicant exposures) and epigenetic (a change in gene function by the addition of epigenetic marks, e.g. methylation) factors can also influence the phenotype. In addition, some genetic variations cause a phenotype at the cellular level (e.g. microcytosis in carriers of beta thalassemia), but do not have a clinically detectable external phenotype.

Click to expand

Figure 1. Factors influencing human genetic diseases

Genetic alterations can affect the expression of a gene or the function of its encoded protein leading to an alteration in cellular metabolism/homeostasis or other important functions in human development or the maintenance of health. The resultant phenotype can be monogenic (due to variants in a single gene), oligogenic (variants in a few genes) or polygenic (variants in many genes, often with additional environmental influence). Chromosomal variants (including structural and numerical abnormalities) cause human phenotypes by altering the function/dosage of one or several genes or their regulatory elements. Genetic and environmental factors together contribute to common diseases (multifactorial). It is important to understand that any variant operates in a background of other factors, explaining the difference in phenotypes seen in individuals with the same genotype.

In this course, we will focus on monogenic disorders, which account for the majority of rare genetic disorders. These are caused by a pathogenic alteration in one or both alleles of a gene. The main molecular mechanisms underlying monogenic disorders are loss of function, haploinsufficiency, and dominant negative or gain of function (described in detail below). Note, the allelic requirement (e.g. autosomal monoallelic, autosomal biallelic, or hemizygous) should always be considered in addition to the disease mechanism.

Loss of function

Loss of function (LoF) can occur when the variant reduces or ablates the function of the gene. Variants that cause a loss of function can be:

• Chromosomal deletions: resulting in the absence of one or more exons or a complete gene.
• Chromosomal rearrangements: disrupting a gene sequence.
• Nonsense variants: introduce stop codons UAA, UAG or UGA, leading to truncated mRNAs that finally undergo Nonsense Mediated Decay (NMD). Variants in the last exon, in the first ~100bp of coding nucleotides and up to 50-bases upstream of the last exon-exon junction usually do not lead to NMD of mRNA and need to be interpreted with great caution.
• Frameshift variants: cause loss of mRNA-reading frame, which leads to premature truncation or elongation of a protein.
• Splicing variants: affect canonical splice sites that are critical for removing introns from primary mRNA, leading to the retention of intron or excision of exon, which results in an abnormal protein. Some sequence variations can also create new splice sites in the non-canonical (within exonic or intronic) regions.
• Missense variants and in-frame deletion/insertions: these result in substitution, deletion or replacement of an essential amino acid at a specific position and can lead to loss of function of the protein. Although they can cause a LoF, these variants can be difficult to interpret as they can also be functionally silent (benign), or can perturb protein function by other mechanisms (e.g. gain of function or dominant negative effects (described below).
• Alteration in regulatory elements: a change in the promoter, transcription initiation site or an enhancer hinders or blocks transcription/translation initiation or elongation and can affect the expression of a gene product at an appropriate time and location.

Nearly all autosomal recessive and X-linked recessive disorders are caused by a LoF mechanism. However, the degree of disease severity depends on the combined functional effect of variants present in both alleles at a locus (gene) or in the case of 46, XY males at a locus (gene) on their single X chromosome. Hypomorphic variants may diminish protein function but not entirely eliminate the gene product.

Haploinsufficiency (HI)

HI refers to insufficient monoallelic expression of a gene resulting in a disease. Due to the presence of loss-of-function variants in an allele of a gene, the amount of gene product is reduced to half (50%). This reduction in gene product may either go undetected as gene product produced by a single wild allele is sufficient to ensure normal metabolic function (as in autosomal recessive diseases) or manifest in disease.

For some genes, the genetic defect in a single allele (heterozygous state) cannot be tolerated and manifests in disease. These genes are haploinsufficient genes and are LoF intolerant. LoF variants in haploinsufficient genes are inherited in an autosomal dominant manner (particularly in diseases with late-onset or incomplete penetrance) or may arise as a result of de novo events and manifest in severe clinical phenotypes in children (with reduced reproductive fitness).

Dominant negative (DN)

Dominant-negative (DN) mechanisms are caused by monoallelic genetic variants that cause the mutant protein to directly or indirectly block the normal biological function of the wild-type protein. This most commonly occurs in proteins that assemble into homomeric complexes, made up of multiple copies of the same protein subunit. For example, a variant resulting in a glycine substitution in COL1A1 will distort the triple helix comprising two COL1A1 polypeptides and a COL1A2 polypeptide that make up a type 1 collagen fibril. This causes a disproportionate (>50%) loss of function, even though only half of the protein is mutated. Alternatively, variants that disrupt specific interactions (e.g. protein-protein or protein-DNA) can have DN effects by competing with wild-type proteins. Variants that cause disease by a DN mechanism usually cluster in specific regions of a protein or affect a specific amino acid and are often caused by missense variants.

Gain of Function (GoF)

Gain-of-function mechanisms have conventionally been explained by the monoallelic genetic variants that result in gene products with enhanced activity or increased dosage. However, a deeper understanding of phenotype-genotype correlation has revealed that GoF variants have several distinct functional effects, including triplet-repeat expansion, protein aggregation and changes in target-binding specificity. GoF mechanism has been implicated in:

• Gene duplication
• Uncontrolled gene expression
• Increased protein stability or reduced degradation
• Hyperactivity of the altered protein
• Chromosomal rearrangement in a regulatory region

GoF mechanisms are usually due to a restricted repertoire of variants in a gene, sometimes affecting a single codon or nucleotide. GoF variants tend to cluster together in specific sites in the gene and commonly increase the stabilisation of the altered protein. They are inherited in an autosomal dominant manner with pathogenic alteration in one allele of the gene. GoF variants are often missense changes or alterations in regulatory sequence.

Dominant variants occasionally result in novel protein functions, referred to as gain-of-new function. These are an important cause of rare diseases. For example, the enzyme Alpha-1 antitrypsin binds to elastin thereby protecting the body from its harmful effects. However, in the ‘Pittsburgh variant’ (p.Met358Arg) methionine is substituted with arginine, which causes the protein to bind to thrombin instead of elastin, causing severe bleeding disorder due to the decrease in thrombin levels.

Disease-associated variant consequences

To assist with the characterisation of disease-gene relationships, the Gene Curation Coalition (GenCC) has recently recommended consistent terminology for allelic requirements, inheritance modes and disease mechanisms. For a novel variant, the protein sequence consequence (e.g. amino acid sequence) is often understood, but the functional effect, which relates to the mechanism, is not. The ability to capture disease-associated variant consequences (i.e. predictable consequences), when the precise functional effect is unknown, is therefore beneficial.

The following high-level, disease-associated variant consequence terms have been recommended:

• Altered gene product level: a sequence variant that alters the level or amount of gene product produced. It can be used where the change in level is unknown or is not yet confirmed
• Increased gene product level: a variant that increases the level or amount of gene product produced.
• Decreased gene product level: a sequence variant that decreases the level or amount of gene product produced.
• Absent gene product: a sequence variant that results in no gene product.
• Altered gene product sequence: a sequence variant that alters the sequence of a gene product. Downstream mechanisms are diverse: functionally null – misfolded, mislocalized, inactive, hypomorphic; disruptive presence of abnormal protein (GoF, DN) etc.
• Functionally normal: a sequence variant that is not expected to alter gene product sequence or levels (eg, a synonymous variant).

Think outside the box

This article is not an exhaustive list of disease mechanisms. Do you know any other mechanism of disease causation? Discuss in the comments what is cellular interference.

Learn more about this topic by reading the articles below:

PCDH19-related epilepsy: understanding cellular interferenc

P141: X-linked inheritance and it’s exceptions: Lessons of X-inactivation and cellular interference