Fabry Disease and the GLA Gene

Sunday, December 7, 2014

IPA Pathways

This week, we use IPA to "model, analyze and understand complex biological and chemical systems." (IPA)

In IPA, there are no drugs associated with the product of the GLA gene.

Growing a Pathway
The following is the direct pathway grown using limited to humans as species and deselecting chemicals and biological drugs. Two relationships with GLA are identified. The HNF1A molecule is located in the nucleus and is a transcription regulator. It has an reaction relationship with the GLA product as Protein-DNA binding (PD). The TFEB molecule is also located in the nucleus and is also a transcription regulator. It also has a reaction relationship with the GLA product involving expression (E).

Autolayout view

As depicted below, the GLA gene product is located in the cytoplasm.

Canonical Pathway

IPA does not have a canonical pathway for GLA. As such, a pathway involving HNF1A was identified. HNF1A is involved with the Maturity Onset Diabetes of Young (MODY) Signaling canonical pathway. This is a disease-specific pathway involving the endocrine system. "MODY, the monogenic form of diabetes, results from defects that affect the functioning of islet beta-cells where the pancreas does not produce enough insulin. This in turn leads to hyperglycemia...The impaired secretion of insulin seen in MODY is similar to the deficiency found in Type-1 diabetes. Yet, unlike Type 1 diabetes, MODY develops slowly and does not completely destroy the ability of the pancreas to produce insulin. Rather, it impairs insulin secretion so that the body cannot adequately control blood glucose levels from one moment to the next." Molecular defects in 6 different genes including the HNF1A gene have been identified in MODY patients. "A mutation in one of the alleles encoding HNF1A leads to a reduction in beta-cell glucokinase activity resulting in decreased glucose phosphorilation in the beta-cell and glucose stimulated insulin release at any blood glucose concentration."

Friday, November 28, 2014

GENOME ANALYSIS

Commentary:
The decision of whether an individual should pursue genetic testing when he or she presents with unusual variants of a condition or does not respond to the typical treatments is plagued with challenges. Will the information gained from whole genome sequencing (WGS) or whole exome sequencing (WES) be beneficial clinically? Is the testing affordable? Is there insurance reimbursement for the testing? Will the clinician treating the patient be able to interpret the results of the testing? How should the clinician and patient respond to incidental findings?

Consider Mary who has various renal, cardiac and cerebrovascular manifestations of Fabry disease that have developed despite the typical enzyme replacement therapy. Her brother also has Fabry disease but has responded to enzyme replacement therapy.

Briefly, Fabry disease is a lysosomal storage disorder caused by GLA gene mutations. It is inherited in an X-liked pattern. As males have only one X chromosome, one altered copy of the GLA gene is sufficient to cause the condition. However, unlike other X-linked disorders, Fabry disease causes significant medical problems in many females with just one variant of the GLA gene. More information about Fabry disease can be found here NIH Fabry Disease.
As Mary has been confirmed as being homozygous for a disease causing variant of the GLA gene, she has presumably undergone clinical genome or exome sequencing for Fabry disease. But her lack of response to typical treatment may indicate the presence of other disorders with renal, cardiac and cerebrovascular manifestations. As such one option is for Mary to participate in a clinical trial offering full exome analysis at no cost to her and her parents. However, her ability to participate will depend on the inclusion/exclusion criteria of the trial. In addition, this trial likely requires the participation of her parents and Mary's ability to enroll in the study will also depend on her parents' willingness to give consent. As a small portion of disease-causing mutations are found in introns, the sequencing offered in this trial may not provide Mary with a comprehensive explanation of her lack of response to traditional therapy. Another option is for Mary to seek insurance provider approval for full genome analysis. Given, however, that this likely involves a 4 to 6 month negotiation with her insurance carrier, this may not be an option if her condition is deteriorating quickly. Full genome analysis, although decreasing in price, still remains out of reach of many individuals given the $5,000 to $10,000 cost. Mary may consider direct-to-consumer genome analysis from a commercial lab without the involvement of her health care providers. Recently, however, the American College of Medicine Genetics Board (ACGM) has issued a statement against this practice due to potentials harms including misinterpretation of test results and lack of necessary follow up ( ACMG statement). The American Society of Human Genetics (ASHG) has also issued a statement regarding the genetic testing done by laboratories not of high quality whose claims about the testing may be misleading to the consumer (ASHG statement). Mary will also need to have an in-depth discussion with her provider as to how to handle "incidental" findings during sequencing. The ACMG has recommended that laboratories search for specific types of mutations for which "preventive measures and/or treatment are available and disorders in which individuals with pathogenic mutations might be asymptomatic for long periods of time" (ACMG policy statement, see table 1).
In my opinion, Mary should enroll in the clinical trial if her parents agree as this option provides her with the most information possible with the lowest out-of-pocket cost. She should have a very detailed discussion with the investigators about the types of conditions, genes and variants that can be expected as per ACMG guidelines.

Conversion of GLA variant to Variant Call Format (VCF):
Per OMIM, the variant of the GLA gene which results in arginine substitution by tryptophan at position 356 of the protein (enzyme) is represented by dbSNP:rs104894827.

To following screen shot illustrates that the beginning position of the variant relative to position 1 of the X chromosome (on which the GLA gene is located) is position 101398033.

Exon 7 of the GLA gene contains this variant (as screen in line entitled CCDS).

The VCF format of the rs104894827 variant in a patient who is homozygous for the disease associated variant is represented as follows:

#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT CB00001

X 101398033 rs104894827 C T 25 PASS NS=1;DP=35;AF=0.5;DB GT:GQ 1|1:52

Sunday, November 9, 2014

Molecular Diagnostics

Per ClinVar there are 150 recognized pathogenic variants of the GLA gene. None are recognized by a professional society and none are identified by an expert panel.

According to Genetests, 51 laboratories in the US offer diagnostic testing related to the GLA gene. A variety of different methodologies are offered including Micro-array, Deletion/Duplication/Copy Number, Quantitative Biochemical Analysis, Sequencing, and Genotyping.

According to NCBI Genetic Test Registry (GTR), 46 labs in the U.S. offer testing related to GLA variants which are associated with Fabry’s Disease. Testing methods include Biochemical Genetics (Analyte, Enzyme Assay) and Molecular Genetics (Sequence analysis of the entire coding region, Deletion/duplication analysis, Sequence analysis of select exons, Targeted variant analysis).

Identification of a potential RFLP test for the 1066C>T variant of the GLA sequence was not possible as there were no changes in enzyme sites of the codon associated with this variant. Therefore, ClinVar was used to identify the 677G>A variant which did result in a new restriction enzyme (Bpu101) site at position 675 of the ORF of the variant that was not present in the ORF of the normal GLA gene sequence. (The Bpu101 enzyme is an infrequent cutter with 3 sites in the native GLA sequence as compared to 4 sites in 677G>A variant sequence.)

Agarose gel simulations for the Bpu101 enzyme of the native GLA sequence and the 677G>A sequence are depicted below. The additional Bpu101 enzyme site in the 677G>A variant resulted in creating 4 bands (602bp, 322bp, 259bp,235bp) as compared to 3 bands (602bp,494bp,322bp) in the native sequence of the GLA gene.

Native GLA sequence

677G>A variant

Sunday, October 12, 2014

The GLA gene encodes alpha-galactosidase (GLA; EC 3.2.1.22), a lysosomal hydrolase. In a patient with Fabry disease, a 1066C>T transition in the GLA gene results in a substitution from Arginine to Tryptophan at position 356 of the protein. The substitution results in an enzymatic defect that results in the accumulation of globotriaoslyceramide and related glycosphingolipids in the plasma and cellular lysosomes of vessels, nerves, tissues and other organs throughout the body. The result is a systemic disease manifested as progressive renal failure, cardiac disease, cerebrovascular disease, and skin lesions.

This week, we analyze the 3D Structure of the GLA protein. From NCBI, "Structure of Human Alpha-galactosidase [Hydrolase EC:3.2.1.22] 2013/2/12" was chosen as this was the most recently updated representative human model of the GLA enzyme. The PDB (Protein Data Bank) ID for this structure is 1R46.

Using CN3D, the GLA enzyme, a homodimeric glycoprotein, is represented below.

One of the 2 molecules is then removed and the following is a space filling view of the alpha-galactosidase molecule with position 356 highlighted in yellow. In the variant protein, Arginine is substituted by Tryptophan at position 356 of the protein structure resulting in deficient activity of the enzyme.

By zooming in within the Worms View of CN3D, the following representation of position 356 (highlighted in yellow) is illustrated. It is apparent that position 356 is located in a turn region within the molecule.

Last week, we evaluated the secondary structure and hydropathy of the GLA protein. The Garnier-Robson method predicted that in the normal GLA protein, position 356 would be located in a Beta sheet whereas the Chou-Fasman method predicted that position 356 would be located in an alpha helix. With regards to the evaluation of hydropathy, both the Kyte-Doolittle and the Hopp-Woods methods predicted that position 356 would be hydrophilic. By creating the 3D model via CN3D, we see that neither the Garnier-Robson nor the Chou-Fasman alogorithm of the secondary structure of the GLA protein correctly predicted location of position 356 as the above illustration (worms view) depicts position 356 in a turn region. [The illustration below from Protean 3D also depicts position 356 (highlighted in blue) in a turn region.] However, the Kyte-Doolittle and the Hopp-Woods algorithms for hydropathy created in DNASTAR's Protean 3D were correct in predicting the 3 dimensional location of position 356 of the GLA protein as hydrophilic as predicted in the space filling view above.

Sunday, October 5, 2014

GLA protein 350 to 359 helical wheel

OMIM allele associated with Fabry Disease: 300644.0001

Amino Acid variant: arg356-to-trp substitution (R356W)

R356W variant protein 350 to 359 helical wheel

Clinical impact of variant: This substitution alters the lysosomal enzyme alpha-galactosidase A’s kinetic properties and stability and alters the glycosphingolipid metabolic pathway. The substitution of Arginine to Tryptophan at position 356 results in deficient activity of the α-GAL enzyme. Partial or complete deficiency of α-GAL results in an inability to catabolize lipids with terminal α-galactosyl residues. These lipids, particularly globotriaosylceramide (GL-3; Gb3, ceramide trihexoside, or CTH), accumulate progressively in lysosomes in multiple cell types throughout the body resulting primarily in progressive multisystemic damage to the kidney, heart, and cerebrovascular system.

Protein Analysis: The substitution from amino acid Arginine(R) to Tryptophan (W) at position 356 of the protein changes the secondary structure as well as the hydropathy of the protein. In the normal protein Arginine (R) is hydrophilic and is in a beta region of the protein. In the R356W variant, Tryptophan (W) is hydrophobic and is in an alpha region of the protein. The R356W substitution, however, causes no changes in the transmembrane regions of the protein.