Content

Alexei's Case Study

Welcome to Your Patient!

Alexei, 8 year old boy, recently arrived at the emergency room with a severe hematoma on his left thigh...

Attending's Instruction sticky-note: Have this done by Friday and be ready to present.

Previous Diagnosis: Hemophilia

Key Symptoms: Repeated episodes of profuse bleeding, bruising and intermittent severe pain

Due to the history of bleeding issues, Alexei (and his father) was referred to you for a second opinion and genetic analysis. Laboratory tests from the ER visit will be added to his EHR for your review.

Researching the Referral

Here's a link to the Referral that Alexei's doctor sent. Read it over to learn more about Alexei and his case, then do the following steps to learn more about the preliminary diagnosis.

Search MedGen (https://www.ncbi.nlm.nih.gov/medgen/) with:

hemophilia[exacttitle]

If you need it, you can click here to get to a link for the Medgen record page.

Try this!

Based on the results of that search, what is a possible diagnosis for Alexei and how does this correspond to the preliminary diagnosis listed in the referral?

In the “Term Hierarchy” section of this record, you can see you can see more specific sub-types of “Hemophilia” - two major forms of hereditary disprder are displayed. Click the names of the disorders to open the MedGen records and read about each sub-type.

What is/are the major differences in the sub-types of Hemophilia? Identify the gene or genes associated with each subtype.

The referral mentions Hemophilia B (Factor IX Deficiency). What do you think you should try to test for to rule in this diagnosis and rule out the other possible one?

GeneReviews® is a project run by the University of Washington producing expert-authored, point-of-care information with clinically relevant and medically actionable information for inherited conditions. It is an incredible review article-type of resource, and is thus featured in it's own section on relevant MedGen records - as an abstract with links to key sections.

Let's focus on the preliminary diagnosis of Hemophilia B (Factor IX Deficiency).

- To learn more about this disorder, in the "Disease Characteristics" section scan the summary "Excerpted from the GeneReview" and consider accessing and reading the whole article.

Which features within this summary seem to confirm that this is a possible disorder to be considered for the diagnosis?

- To begin your case work-up, note the links to specific GeneReview sections below and click on the link "Differential Diagnosis"

Which other conditions you should consider during your work-up?

- To learn about which test(s) could be ordered to diagnose this condition, click the link to the "Diagnosis" section.

The referring physician ordered standard Lab tests and the Lab Test Results have come in! Read them over to learn more about Alexei's results. (Note: This report is in an "old" format of report that used to be sent as a print-out or electronic PDF. Now-a-days these results are generally available as part of the patient's Electronic Health Record (EHR) - which may be formatted differently.)

Which analytes are "flagged" as outside normal ranges?

How does the information in the lab test correspond to the preliminary diagnosis? Does this support the preliminary diagnosis?

- GeneReview articles contain information on genetic testing when relevant for that disorder or condition. Continue reading the "Diagnosis" section through the "Establishing the Diagnosis" sub-section.

For this disorder, is a genetic test(s) recommended for validation?

To assist you further in learning about this disorder and for preparation for discussions with your patient, there are links to:
- many relevant publications available in PubMed - you can look on this record for these as well as on other closely-related disorder records that you saw linked in the "Term Hierarchy" section
- Clinical Trials, when available, linked in the "Clinical Resources" section on the right-hand side of the page.
- often there are a few "Consumer Resources" listed on the right-hand side of the page - which can helpful for patient education

You discuss the diagnosis and hand Alexei's father some Patient Education materials.
He is worried about Alexei and the implications of this genetic condition for him and the family. Also, if the bleeding is likely to be a problem for him throughout his life, he wonders what can be done to help Alexei on a more permanent basis.

Finding a Genetic Test and Understanding the Results

From the MedGen record, you can quickly link (on the right-hand side of the page) to relevant Genetic Tests that are available for this disorder or condition.

Click on any of the Genetic Testing Registry or GTR links on the right-hand side of the page to retrieve a list of relevant tests. Then use the left-hand sidebar filters to narrow down those shown to those relevant for the case - in this particular case, you should look for one from a CLIA-certified clinical lab (so that "Insurance" is more likely to pay for it) and in the country he is currently visiting - Finland. Click on the lab test title to learn more about the genetic test including "How to Order" it and often the "Methodology" employed for the test.

Please note The information shown in the NIH Genetic Testing Registry (GTR) is submitted by the testing laboratory. You should look at the "Laboratory Contact" tab for ways to contact them directly for any question you have about the test.

If you need it, you can click here to get to a link for the Medgen record page.

Try this!

Okay, so you ordered the test, it was performed, and the Genetic Test Results have come back from the lab! Read them over to learn more about Alexei's results. (Note: There is currently no established standard format for the results. Each laboratory creates it's own report - some are very detailed, some pretty terse.)

What is/are the specific gene(s) and variation(s) identified in Alexei?

What does the Genetic Testing Lab's report assert for Alexei's’ diagnosis? Based on the information in the GeneReviews article, does this make sense?

To validate what is asserted by this clinical testing laboratory, search NCBI’s ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/) with: F9 c.278-3A>G

If you need it, you can click here to get to a link for the ClinVar record page.

Try this!

In which position in the Gene is this variant located?
(Write down the HGVS information for the NG_ g. Note: You may have to expant the table to find it!)

What does the ClinVar record indicate in the “Interpretation” field for this gene and variation?

In the middle section, click on "Conditions" to see a summary of interpretations per disorder or condition. This variation may have been submitted with information about several different disorders, conditions or even risk factors.
You can click on any relevant "Variation/condition record" links to learn more about what information was provided for that particular one from the different sources.

What does the aggregate table and/or Variant/condition record(s) indicate about the Alexei's specific preliminary diagnosis?

What do you conclude about the assertion for this genetic variation with regard to Alexei's diagnosed disorder?

You discuss the results of the genetic test and diagnosis with Alexei's father and write a referral to a hematologist to discuss treatment.
He wants to understand more about how this genetic variation causes issues in his son. Helping him to understand the specific mechanism causing his disorder can help her feel more able to participate in his care plan.

Learning About the Implicated Gene

On the MedGen record, click the link for the Gene identified as having a variant in Alexei.

If you need it, you can click here to get to a link for the Medgen record page.

Try this!

Or click here to get a link to go straight to the Gene record page.

Try this!

Read the Summary section and identify what does this gene normally does.

Here's some additional information which may also help you - I've pulled it together from an old molecular biology textbook available on the NCBI Bookshelf!

The Blood Clotting Cascade

Blood is a vital fluid containing critical oxygen transport cells and cells necessary for immune surveillance and protection from infection.
Damage to the blood transport vasculature can trigger clotting to prevent loss of the vital fluid and it's components.
Activation of a series of tightly regulated "clotting factor" protein activation steps through intrinsic or extrinsic pathway induces blood clotting, or coagulation to stop bleeding.
Regulation of clotting is critical, because too little clotting results in continued bleeding and too much clotting can occlude vital vessels and block the flow of vital blood.
Some key proteins in the intrinsic pathway have been associated with significant bleeding disregulation - either too much clotting, such as Factor V or too little, such as Factor VIII or Factor IX.

The Gene Ontology (GO) Consortium's mission is to build a comprehensive, human-readable and computable, hierarchical knowledgebase for the molecular functions, cellular locations, and processes that gene products (usually proteins) carry out.

In the Gene record's “Gene Ontology” section, learn more about the gene's product (protein) with GO-assigned standardized terms for where this gene product is likely to be found within a cell (Component), what processes it is often involved in (Process), and what it does (Function).

What type(s) of process(es) is/are this protein normally involved with?

What specific function(s) does this protein have?

In which component(s) (sub-cellular location) is this protein normally found?

Does this make sense based on the Gene Summary of the Gene that you found above?

Scroll down to the Gene record's “Expression” section to see in which tissues this gene has been observed to be expressed.

In which tissues has this gene been found to be expressed?

Do any of these tissues correlate with those associated with Alexei's symptoms?

Mapping and Understanding the Impact of the Variant

So, you now know information about what the "wild-type" or non-variant-containing gene product does and where. How might your patient's variant impact ths gene and it's product? First, it depends upon where the variant is located and then it depends upon it's impact in that location.

Click here to see a graphic that I made based on the central dogma of molecular biology and how to systematically step through an assessment of variant location and possible physiological impact.

A Review of the Central Dogma of Molecular Biology & How to Assess and Predict Variant Impact
Figure showing "The Central Dogma of Molecular Biology and Possible Sites for Variant Impact"

Figure showing "The Central Dogma of Molecular Biology and Possible Sites for Variant Impact"

Genome/Chromosome: First, let's map where that particular gene is within the chromosome.

In the "Genomic Context" section of the Gene record it will tell you exactly where the gene is located, on which chromosome, in which cytogenetic band and even down to the basepair position.

On which chromosome is the implicated gene (and, presumably, the variant)?

How many of these chromosomes does Alexei have? What importance is this information when thinking about the severity of Alexei's condition?

Gene/Transcript: Now that you know the chromosome the gene and variant are on, it will be imporant to focus in on the gene itself.

On the right-hand side of the Gene record in the "Related information" section, click the “RefSeqGene” link to see the graphical view of the gene structure defined on the chromosome on a RefSeqGene nucleotide page (or click on the image below to open a new web browser window with the "live" version on the web).

Image of the F9 Gene in NCBI's Sequence Viewer

Where is Alexei's genetic variant located in this gene and how does it relate to the start of the next exon in the mRNA? (Visualize a vertical line at the variant’s position to map it on the gene (green) and mRNA (purple). On the live site, type the variant’s gene position into the “Find” box to automatically zoom in)

Below is a zoomed-in picture showing the genome sequence (both strands) and the red “G” indicates the base change of the exact genetic variation ("-3" bases up stream from the beginning of the exon) next to the “-GA-” at position 15,340-15,341 which is the normal splice site acceptor indicated by the exon 4 purple bar with a “GAT” codon. The normal encoded amino acid sequence is shown below the red bar. Image of the F9 Gene in NCBI's Sequence Viewer with the intronic variation indicted

Image of the F9 Gene in NCBI's Sequence Viewer with the intronic variation indicted

Alexei’s genetic variation actually creates a new “-GA-” two letters up from the usual one….which causes a -2 base frameshift in splicing and the start of the encoded amino acid sequence. I could ask you to figure out what the new amino acid sequence would be…..starting with the new GAG codon, but instead…Scan each 3 letter codon and find where there a “TAA” stop codon shows up in the new frameshifted sequence.

Based on the position of the variant in the gene and it's impact on exon-processing of the transcript, what is the most likely mechanism for impacting the gene's product (such as altering gene expression, influencing transcript processing, or changing the encoded protein sequence)?

Protein: Since you've identified that the variant is located in the coding region of the gene and transcript, identifying the position and type of altered protein residue will be important to figuring out potential impact on the protein's structure and function.

On the right-hand side of the RefSeqGene page, click the “Protein” link OR go back to the Gene record and click the “RefSeq Proteins” link. Find "Isoform 1" (the longest and the most common expresed protein form), and click “Graphics” to see a graphical view of the annotated regions curated on the protein sequence (or click on the image below to open a new web browser window with the "live" version on the web). The information shown in in these “tracks” of this view can help you to learn more about this protein.

Based on the type (stop codon change) and it's position in the protein, can you predict what might be the mechanism for impacting the protein (such as altering protein folding, impacting the binding of a cofactor, ligand or substrate, changing a residue often targeted for modification, or truncating the production of the rest of the encoded protein)?

Okay, so let's bring this full circle now - Alexei's father wants answers.

If you haven't already, make sure to fill out that Summary sheet so that you can answer his quesitons. Some help in making sure all of your ducks-are-in-a-row - is below:

Click here to review some things to consider when formulating the answer to his questions.

- - Which gene is impacted by the genetic variation and what does the gene product normally "do"?
    - what is it's biomolecular function?
    - what is it's impact on cellular physiology?
    - in which cells/tissues is the gene product usually expressed?
  - Based on the patient's variation(s):
    - what do you think this would do to the gene product's structure and biomolecular function?
    - what would this do to cellular physiology?
    - what tissues or organs impact be impacted?
  - Based on the proposed impacted-tissues/organs, may some of the the patient's symptoms be explained by this? (validating his experience)

Click here for some more clinically-related considerations.

- - What is his specific disorder or condition? (Final diagnosis)
  - How do you know this is the correct diagnosis? (Take everything into account, - clinical features, lab and genetic test results, as well as response to any previous therapies.)
  - What can you do with the knowledge of the precise genetic lesion that causes this disorder? (Think about your next steps in case management planning.

What is wrong with Alexei?

Well? What do you think?

Alexei's father asks:

- "What is wrong with Alexei?"
- "How do you know for sure?"
- "What can we do now?"

Take-away Message!

We'll discuss this in class!

Think about what you would expect for this with regard to:

Case management for the disorder - why would understanding the genetic etiology help in understanding and also in developing your therapeutic approach?

Communications with the patient's family - why would understanding the genetic-based molecular mechanism help in answering questions and explaining "why and how"?

Your own your understanding of human biology and disease - as you prepare for your exam, can you integrate the genetics with the molecular biology/biochemistry with the physiology with the patient's symptoms...to understand the human disease etiology and how this can be exploited for both diagnosis and to target therapies?

Last Reviewed: August 23, 2022