Ingenuity Pathways

Ingenuity Pathways

IPA ingenuity is a program that helps to unlock the relationships between genes and relevant pathways. Utilizing IPA pathways, I was able to locate my gene, FBN1. Upon the initial search, there does not appear to be any drugs that are associated with the product of FBN1 (as depicted below).

IPA Pathway: FBN 1. Absence of drug interactions. 

When I grew a pathway using my gene and constraining the relationships to direct relationships using all molecules, human species data, and removal of chemical and biological drugs, the following image was obtained with identification of 3 molecules and 3 relationship. The 3 molecules are TP63, MMP25, VCAN.

FBN1 pathway with listed constraints : 3 pathways identified

Since only 3 relationships were observed, I did not need to apply the trim instructions. The following is the screen shot of the pathway after applying “autolayout”.  

Autolayout of selected FBN1 pathways

There are 3 different types of relationship types for the 3 molecules identified. For example, TP63 has an "expression" relationship, MMP25 has a "molecular cleavage" relationship, and VCAN has a protein - protein interaction.  Binding of human FBN1 protein and a protein fragment containing a C-type lectin domain from human VERSICAN (VCAN) protein occurs in a system of purified components from A204 cells. Therefore, there is one protein-protein interaction and no activation or inhibition relationships. 

Protein-protein interaction between VCAN and FBN1

Molecular cleavage relationship of MMP25 to  FBN1

Expression relationship of TP63 to FBN1

Using the "Toggle SubcellularLayout" it is evident that the FBN1 gene is located in the extracellular space along with VCAN. MMP25 is located in the plasma membrane. TP63 increases the expression of human FBN1 mRNA and is appropriately located in the nucleus of cells.

Subcellular layout of FBN1 and related pathways

Overlay Canonical Pathway

Using the canonical pathway function, there was no single pathway that included FBN1 (as seen below). So I chose to proceed with assessing TP63  for the p53 signaling pathway.

Canonical pathways

The p53 signaling pathway is depicted below with a close up of where TP63 is in relation to the pathway. According to the pathway report, the p53 (TP53)  tumor suppressor protein is a key transcriptional regulator that responds to a variety of cellular stresses such as DNA damage, UV irradiation, and hypoxia. the p53 gene regulates key cellular processes such as DNA repair, cell-cycle progression, angiogenesis, and apoptosis. Loss of p53 is thought to be a contributing factor to cancer. 

p53 signaling pathway

Location of p63 in the p53 signaling pathway

Genome Analysis

Part 1: Commentary

Mary has been confirmed as being homozygous for the Fibrillin gene (FBN1) for Marfan's Syndrome which is typically inherited in a autosomal dominant pattern. She has demonstrated complex response to multiple medications and has multiple clinical conditions that are not explained by the disease variant or by 1 year of traditional diagnostic testing. Mary and her family have several options: a) to participat in a clinical trial offering full exome analysis to her and her parents at no cost, b) seek full gemone analysis through their insurance carrier which may take 4-6 months, c) pay out of pocket for full genome analysis (can run between $5-10 K), or d) use direct-to-consumer services and perform independent analysis of raw results. 

Marfan's syndrome is autosomal dominant where approximately 75% of individuals with Marfan syndrome have an affected parent and approximately 25% of probands with Marfan syndrome have a de novo mutation. There are 68 allelic variants that are responsible for a wide array of phenotypical features associated with Marfan's. For example, CYS1409SER is responsible for a mild variant where clinical features are not apparent and diagnosis was only made by genetic testing. However, the more typical variant is ARG1137PRO, in which the classic severe features are noted such as aortic root dilatation. The primitive genetic tests only searched for known defects and if not found, the patients were deemed clear of the disease. However, with advancements in genetic testing, we now know that variants are also responsible for clinical disease. Even tho the typically the same variants are passed on from generation to generation, there can be other defects that occur de novo and can cause other symptoms. So in Mary's case, it is appropriate to proceed with a diagnostic full genome sequence analysis. 

Out of her options, the most valid one appears to be participating in a clinical trial for testing. Not only will the hefty costs of the test be eliminated for her and her family, there may be a possible treatment they could offer if a known variant is found on her analysis. She would have access to health care professionals that are involved with genetic tests and possibly be the best people to interpret her analysis.  It is also feasible to go through her insurance company, however, if waiting 4-6 months would be detrimental to her life span and obtaining life saving medications, it may not be worth the wait.

The American College of Medicine Genetics released several statements on direct-to-consumer testing which I agree with. ( ACMG statement on direct-to-consumer testing and ACMG statement on direct-to-consumer testing in the USA.) Consumers who are searching for answers to their medical conditions, are vulnerable to being misled by the results of unproven and invalid genetic tests. Guidance from a health care professional can help guide the consumers in the appropriate direction for their test results. The consumer may be concerned about only one or two results that they know about and are interested in, however, if other results are found that can affect their health and the health of their family members in a different way, this should be addressed by a health care professional and not left to the companies performing the tests. Also, genetic tests provide only one piece of information about a person's health -- other issues such as environmental factors, life style choices, family history, and reproduction history, may also need to be addressed in order to obtain maximal benefit in their treatment plan. These factors cannot be addressed by at -home kits or the testing companies. 

With regards to incidental findings, their provider should address, inform and discuss  these findings with the patient. The patient has a right to know the results of any test that is performed on them, especially since they have provided consent for testing with the anticipation that they will find some answers. Even though we may pick up incidental findings that are not deemed to have clinical significance (yet), the patients should be informed of this and continued to be monitored if necessary. It is absolutely pertinent to discuss any results that have known treatment or prevention strategies that could possibly save the patient's or her family's life. 

  

Part 2: 

Using OMIM for FBN1, and .0001 MARFAN SYNDROME, SEVERE CLASSIC, the rs ID is

rs137854456. Below is the identification of rs137854456 within the sequence:

The beginning position of the variant site appears to be at 48487365.

Beginning position of variant site

My variant appears to be on exon 28 as depicted below.

The VCF formatted data is noted below:

CHROM  POS       ID          REF ALT QUAL FILTER  INFO                  FORMAT  CB00001

15     48487365  rs137854456 G   C   25   PASS    NS=1;DP=35;AF=0.5;DB  GT:GQ  1│1:52

Molecular Genetics - part II

Restriction Enzyme Sites:

There did not appear to be a modification in RFLP sites when the codon 1137 was changed from CGC to CCC (Arginine to Proline) as previously described. Utilizing ClinVar,  a variant was identified as 2055C>G (#95 on ClinVar). This variant lead to a change in the frequency of the BsRDI cutting enzyme from 6 to 5 with a loss of the enzyme near the site of the variant.

Agarose gel simulations for this change are depicted below. The native sequence had 6 bands (6079bp, 2595bp, 1395bp, 891 bp, 402bp, and 333 bp). The variant sequence has 5 bands (8674 bp, 1395 bp, 891 bp, 402 bp, and 333 bp).  

RFLP summary of original sequence

Agarose gel simulation of original sequence

RFLP summary of variant sequence

Agarose gel simulation of variant sequence

Molecular Genetics

ClinVar

Utilizing ClinVar, 106 pathogenic variants are described for the FBN1 gene. Unfortunately, there are no professional societies or expert panels that recognize these variants.

Pathogenic variants: 106

Professional society: 0

Expert panel: 0

Genetests.org

Utilizing genetest.org, I searched for FBN1 in the list of genes they have on their website. It appears that 70 labs across the USA offer testing for Marfan’s syndrome. All the testing is molecular testing, however, several tests methods are used. The majority of them use array testing (deletion/duplication/copy number) or next gen sequencing, however a few are using capillary sequencing, mutation scanning of the entire coding region, mutation scanning of select exons, and genotyping.

NCBI Genetic Test Reference

Using the NCBI genetic test reference website, there appears to be 54 labs in the USA that test for FBN1 to identify Marfan’s Syndrome. The also solely use molecular genetic testing, however the majority of the testing appears to be via sequence analysis of the entire coding region. Other test methods include: deletion/duplication analysis, sequence analysis of select exons, mutation scanning of the entire coding region, and target variant analysis.

More to follow.... :)

3D Analysis (Week6)

3D Analysis of FBN1:

The PBD ID for this structure : 1LMJ.

Using NCBI "structure" and the protein FBN1, 9 results were obtained. I chose to use PBD ID: 1LMJ because it is a human protein structure, recently updated (in 2013), and a structural protein that contained the region of substitution (codon 1137: Arginine (CGC) is replaced with Proline (CCC).)

Using Cn3D, the FBN1 protein is depicted below:

Utilizing Cn3D, a space filling view of the protein is depicted below with position 1137 highlighted in yellow. The site is externally placed on the protein.

Space filling view of Position 1137

Upon zooming in and utilizing the "worms" view in Cn3D, the following image was obtained. Position 1137 is highlighted in yellow (and circled in red). It appears that position 1137 is located on a beta sheet within the molecule.

Worms view of position 1137

Upon review of the secondary structures from last week, the Garnier Robson and Chou Fasman methods predicted position 1137 to be located on a turn region, not on an alpha helix or beta sheet. Utilizing the file obtained fron NCBI "structure" and reviewing it in Protean 3D (as illustrated below), the secondary structure reveals position 1137 to be on a beta sheet. Hence, both algorithms from last week incorrectly predicted the position of 1137.

With regards to prediction of hydropathy, Kyte Doolittle algorithm predicted a hydrophilic region which was correctly identified. This is portrayed on the space filling view as noted above.

Overall view of position 1137 in Protean 3D

Position 1137 located on a beta sheet

Protein Structure Prediction, human variation (Week 5)

.0001 MARFAN SYNDROME, SEVERE CLASSIC

FBN1, ARG1137PRO [dbSNP:rs137854456] ClinVar

According to OMIM, a G to C transversion at nucleotide 3410 converted codon 1137 from CGC (arginine) to CCC (Proline) is associated with this gene. This mutation was previously known as ARG239PRO. This is a nonconservative change where a basic amnio acid is replaced with a nonpolar alpha-amino acid proline. There appeared to be no clinical impact of this mutation as the two original patients this was identified in did not show any abnormalities. 

Protein Analysis:

The substitution of Arginine with Proline on Codon 1137 did not have any significant impact on the secondary structure, hydropathy, or transmembrane region of the protein. The alpha and  beta regions of the secondary structure are hydrophilic and the same was noted on the variant mutation. Upon magnification of the Kyte Doolittle regions, all areas remained hydrophobic in both sequences. There was no change on the transmembrane regions of the protein. 

Original Sequence: Overall view of secondary structure

Original sequence: Magnified view of secondary structure

Variant Sequence: Overall view of secondary structure

Variant sequence: Magnified view of secondary structure

Orignial sequence: Overall view of Kyte Doolittle Regions

Originial sequence: Magnified view of Kyte Doolittle Region

Variant sequence: Overall view of Kyte Doolittle Region

Variant Sequence: Magnified view of Kyte Doolittle Region

Originial sequence: Overall view of transmembrane regions

Originial sequence: Magnified view of transmembrane regions

Variant sequence: Overall view of transmembrane regions

Variant sequence: Magnified view of transmembrane regions

Originial Sequence Helical Wheel

Variant Sequence Helical Wheel