Taad Case Study

1. How was the LOX mutation found in TAAD?
The LOX mutation was detected via whole-genome sequencing on the patient with TAAD family history who was experiencing TAAD symptoms. A missense substitution was found at position 893 in the DNA sequence, causing the thymine replaced by guanine, resulting in the replacement of methionine by arginine at the position 298. LOX is a copper-dependent enzyme that cross-links the two structural proteins, collagen and elastin, on the blood vessel membrane. Methionine is the amino acid that is essential for blood vessels growing and is located within the copper binding domain of LOX. Thus, the substitution of methionine suggests potential structural alteration on the blood vessel membrane, which could lead
to TAAD. The study used the mouse model to investigate the association between the LOX mutation and the TAAD. Their data showed that the LOX mutant disturbs the structure of the membrane proteins. In mice with heterozygous LOX mutant, longer ascending aortas with discontinuous elastic lamellae were observed. The homozygous mice died early due to severe internal hemorrhages in multiple areas. Thus, the findings confirmed that the LOX mutation is correlated to TAAD.
2. Which experimental methodologies were used to address whether the specific mutation M298R, is causative for human TAAD?
The CRISPR/Cas9 genome editing was used to introduce the human mutation M298R into the orthologous position of the mouse genome and created mice that were heterozygous and homozygous for the mutant.
CRISPR is a genome engineering tool that cleaves the desired DNA sequence by a programmable RNA-guided DNA endonucleases. The single-guided RNA is made of crRNA (a 20nt-guided sequence) and tracrRNA (trans-activating crRNA that binds to Cas9 and activate the cleavage). In other words, the sgRNA with 5’-NGG PAM cleaves the DNA sequence at the specific site and Cas9 binds to it and program dsDNA at the site. Mice that were heterozygous of the mutant showed aberrant aorta wall arrangement and homozygous mice died shortly after birth due to ruptured aortic aneurysms in multiple areas. Hence, the mutation M298R could potentially causative for the human TAAD due to weakening vessel wall formation.
3. Describe the similarities and differences between the mouse model and human patients having heterozygous mutation of LOX gene.
Impaired aortic wall structure was observed in both heterozygous mice and the patient with heterozygous mutant LOX gene. Therefore, the mutation of LOX gene plays a role in alternating the formation of vessel walls in both mice and human. The heterozygous mice had increased ascending aortic length and discontinued elastic lamellae, which suggested that they might be predisposed to vascular diseases. However, no aneurysm or any arterial diseases were detected in heterozygous mice, whereas the human patient experienced multiple heart diseases and was diagnosed with the Marfan syndrome.
4. What is the phenotype of mice having homozygous LOX gene
mutation?
Homozygous mice were born alive but died few hours after birth. The body size is similar to the wild-typed and heterozygous mice, but severe internal bleedings in the cranial, thoracic, and abdominal area were observed. Severe kyphosis was also seen in some homozygous mice.