August 28, 2024

Aleksandra Gavrilovic  

10th Grade

Pine Crest School

A new recessive syndrome was discovered through researching the protein complex COPI. The endomembrane system which is made up of vital organelles like the endoplasmic reticulum (ER), the Golgi apparatus, and the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), is where the modification, packaging, and transport of proteins occurs. With each transport vesicle, protein coat complexes such as the COPI complex are also included, primarily involved in transporting proteins from the Golgi Apparatus back to the ER. This specific protein complex ensures the structure and function of the Golgi network along with recycling ER proteins. The actual protein structure itself is composed of seven different subunits, split into two subcomplexes ( α, β',  ε go under the B-subcomplex which is cage-like, and γ, δ, ζ, β go under the F-subcomplex which is adaptor-like). AFR1, a small GTPase allows for COPI recruitment to vesicles through the binding of the GTPase with the β-COP and γ-COP subunits. Since COPI brings membrane curvature any disruption to the protein complex can create defective cargo transport which interrupts the successful transportation. Certain disruptions, like mutations in COPI-related genes, are what can lead to different diseases such as microcephaly syndrome, cataracts, autoimmune disorders, developmental abnormalities, and even Alzheimer’s disease. Microcephaly is a condition where the baby’s head is smaller than expected due to abnormal brain development or when development abruptly ends. There are different severities linked to the condition that correspond to certain effects such as developmental delays, intellectual disabilities, and neurological issues. The main cause of microcephaly is genetic mutations but can also occur due to infections during pregnancy, malnutrition, and exposure to harmful substances. Cataracts are when the eye’s lens is clouded, causing blurry vision, scattering light, difficulty seeing during night time, and colors appearing faded. In the normal eye, the lens is clear and can focus light on the retina, this is unable to happen if you have cataracts. It normally develops slowly due to aging, but genetic disorders can also cause cataracts to occur. In order to identify how the protein complex is linked to such diseases, specifically focusing on microcephaly and cataracts, research was conducted on two families. In both families, severe intellectual disability, microcephaly, and cataracts were all present, as well as in the Xenopus tropicalis, confirming that there are biallelic variants in COPB1 that are linked to microcephaly and cataracts.

Multiple experiments were conducted across different areas. Family 1 consisted of people of Arab (Saudi) ethnicity. Experiments were also conducted on Xenopus tropicalis since the exon structure of the specific copb1 gene is identical in both Xenopus tropicalis and humans. Throughout the experiments, genetic analysis, structural modeling, and functional studies to explain the phenotypes observed were all included. 

Two different types of gene sequencing were performed. Whole exome sequencing (WES) was conducted on lymphocyte-extracted DNA using Twist Core Human Exome and Illumina NextSeq technologies from two of the affected sisters in Family 1. For Family 2, whole genome sequencing (WGS) was conducted on lymphocyte-extracted DNA as well and data was collected from electronic health records that were analyzed at King Abdulaziz Medical City, Riyadh, Saudi Arabia using HiSeq 4000. Once the gene is sequenced in to check for genetic mutation near the splice sites which are crucial in RNA splicing, silico analysis is used. Following this, reverse transcriptase PCR is then used to identify specific RNA molecules in a sample and focus on gene expression levels revealing the effect of Family 1’s variant on splicing. Sanger sequencing is later used to confirm it. Silico analysis was also used for Family 2 but instead of focusing on the splice sites, it was used as an analysis of the missense variants on β-COP protein. Plasmid preparation was needed to introduce the mutations into COPB1 and then they were purified, Sanger sequenced, and imaged using confocal microscopy. The proteins were separated by gel electrophoresis and detected with the usage of specific antibodies designed for the targeted gene. For the Xenopus Tropicalis experiments the frogs were housed in specific conditions and egg collection was performed. Using CRISPR-Cas9 the specific copb1 gene was identified then that DNA was cut at the exact identified spot. The sgRNA, which is the synthetic RNA molecule that combines the gRNA in CRISPR RNA and allows for a sequence to be created that matches the copb1 gene, is synthesized, performed, and used then injected into the embryos of the frogs. Imaging took place and gross morphological differences between un-injected tadpoles and crisp, genetically engineered, tadpoles were identified. 

With all these experiments the results gathered proved the link between the copb1 gene with certain diseases. In Family 1, the splice variant causes there to be a skipping of exon 8 which creates an in-frame deletion of 36 amino acids in the protein. Both the missense and splice variants in Family 1, along with the frameshift indels in exon 3 of the copb1 gene from the Xenopus experiment confirmed that microcephaly was a prominent phenotype and clearly reproduced in the Xenopus model. Not only this but the homozygous variant was also linked to a primary microcephaly which fit the characteristics of the individuals represented in Family 2; severe intellectual disability, language impairment, social impairment, cataracts, poor mobility, delayed motor skills, etc. COPI also has numerous roles in neuronal function, specifically in the transport of RNA neurons and in the transport of SNARE complex proteins associated with neurotransmitters. On top of this β-COP was identified as one of the two most connected proteins in the protein network of interferon-stimulated genes which is another cause of microcephaly. Moreover, the individuals in Family 1 with similar symptoms to those in Family 2 both have delayed motor skills allowing for there to be a link in COPI with it. Since β-COP interacts with another protein important in the transmission in motor circuit neurons, if something were to happen with COPI that could cause a negative effect on the transmission leading to delayed motor skills. On top of neurological defects, six out of six individuals developed cataracts which were able to become linked to a homozygous variation in the COPI complex, the COPII gene SEC23A. The patients, like the ones who developed microcephaly, had a missense mutation in the gene which created abnormal Golgi to ER trafficking. 

The common phenotypic changes specifically in regards to microcephaly and cataracts between the individuals demonstrate the link between the syndromes with biallelic variants in COPB1. The usage of frogs to express diseases is also proven to be successful because the exon skipping was able to be shown in the Xenopus studies which correlated to the ones found in the human studies. This overall study concludes the importance the COPI structure has not just in regards to microcephaly and cataracts, but brain development as a whole, along with human health. With this, and other research that can be conducted, who knows what links we can find between two things that will help both scientists and families in the future?