Monday, December 30, 2013

Article Review (2008) - Three German fibrinogen Alpha-chain amyloidosis patients with the p.Glu526Val mutation

We will wrap up the 2013 article reviews with one from 2008, which is the first report of fibrinogen amyloidosis from Germany. Or is it?

Title: Three German fibrinogen Alpha-chain amyloidosis patients with the p.Glu526Val mutation (1)

Authors: Magdalena Eriksson, Stefan Schonland, Raoul Bergner, Ute Hegenbart, Peter Lohse, Hartmut Schmidt, Christoph Rocken (Charite University Hospital, Berlin, Germany; University of Heidelberg, Heidelberg, Germany; Klinikum der Stadt Ludwigshafen, Ludwigshafen, Germany; University of Munich, Munich, Germany; University of Munster, Munster, Germany)

Journal: Virchows Archiv: The European Journal of Pathology (2008)

Abstract:
Plasma protein fibrinogen variants cause fibrinogen Aα-chain (AFib) amyloidosis, which presents with hypertension, proteinuria, and azotemia. Six AFib mutations have been reported thus far. We identified three patients who presented with marked proteinuria and serum creatinine elevations. Their kidney biopsies revealed destruction of the glomerular architecture by amyloid deposits with typical, apple-green birefringence in polarized light after Congo red staining. We found immunoreactivity against fibrinogen, which is typical for this type of amyloidosis. We sequenced the FGA exon 5 and demonstrated heterozygosity for the p.Glu526Val mutation in all three cases. This amino acid substitution is the most common fibrinogen Aα-chain variant causing AFib amyloidosis. The mutation has been reported in individuals of European and American descent but not yet in German patients. AFib amyloidosis should therefore be considered an important differential diagnosis in German patients with renal amyloidosis. In the cases described here, the use of antibodies directed against fibrinogen, followed by direct gene sequencing, revealed the underlying cause.
The introduction of this article has an overview of hereditary amyloidosis, which narrows to hereditary renal amyloidosis, which then narrows to what was known at the time about fibrinogen amyloidosis, such as the known mutations, typical symptoms, and treatment options. It then describes the three patients who were diagnosed with the Glu526Val mutation for fibrinogen amyloidosis.

Case 1: A 62-yer-old woman developed hypertension around the age of 52, and then at age 60 she was observed to have proteinuria and elevated creatinine clearance. A kidney biopsy showed amyloidosis of unknown type. No other organs were affected and a bone marrow biopsy was normal. She did show early signs of polyneuropathy in her fingers and feet. She had no family history of hereditary amyloidosis or kidney disease.

Case 2: A 49-year-old man with hypertension, hyperlipidemia (elevated lipid levels) and coronary artery disease was admitted to the hospital with severe nephrotic syndrome. Upon admission his proteinuria was 15 grams per day. A kidney biopsy showed amyloidosis, and no other organs were involved. The patient's mother had an unknown renal disease and died of a heart attack at the age of 61.

Case 3: A 64-year-old man with hypertension for 20 years and shortness of breath for four years developed progressive proteinuria and renal failure, eventually ending up on dialysis. A kidney biopsy showed amyloidosis.

All three of these patients were referred to the Amyloidosis Clinic at The University of Heidelberg in Germany. The article then describes the additional analysis done on the biopsy samples and the genetic testing on the individual patients. The biopsies of all three patients showed extensive amyloid deposits concentrated in the glomeruli. They performed immunohistochemistry on the biopsies, meaning they were stained with different antibodies that would react to the various precursor proteins known to cause amyloidosis. Some of the deposits did intensely stain in reaction to the fibrinogen antibody, which raised the suspicion of fibrinogen amyloidosis.

The genetic testing showed all three patients were heterozygous for the Glu526Val fibrinogen amyloidosis mutation. They also established that the three patients were not related (as far as the available geneaology records indicated.) Additional genetic testing on family members who consented showed the following:

Case 1: This patient has a son and a daughter. The daughter underwent genetic testing and she has the mutation but is asymptomatic.

Case 2: This patient has no siblings or children. His mother had an unknown kidney disease before she died, so her DNA was never tested for the mutation. But his mother's sister does have the mutation and is asymptomatic. She (the patient's aunt) has a daughter and two grandchildren who have not been tested.

Case 3: This patient's father and aunt developed kidney disease but were never genetically tested for the mutation. The patient has three healthy children who have not been tested.

The final section of the article gives some technical information about the fibrinogen molecule itself, and the molecular impact of the various mutations known to cause fibrinogen amyloidosis. It then discusses the importance of immunohistochemistry in the analysis of these biopsy samples, and recommends that fibrinogen amyloidosis be included in the differential diagnosis of any German patient presenting with renal amyloidosis.

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This article describes three patients with the typical symptoms of fibrinogen amyloidosis, whose kidney biopsies showed the usual pattern of amyloid deposits concentrated in the glomeruli. Fortunately they were not misdiagnosed with AL amyloidosis, but were instead referred to an amyloidosis clinic and were correctly diagnosed. We also have one of these patients having no family history of kidney disease, which is not unusual.

One thing that got my attention regarding these cases was how many years two of these patients had hypertension before developing any of the other usual symptoms of fibrinogen amyloidosis, such as proteinuria or elevated serum creatinine levels. I do not recall such long time periods in previous articles. Perhaps hypertension could be a very early warning sign of the kidneys being affected by fibrinogen amyloidosis in patients who are known to have the mutation. Hypertension alone would certainly not lead a doctor to suspect any type of amyloidosis, given all the other possible causes of hypertension. But hypertension in a patient with a known genetic mutation for fibrinogen amyloidosis should cause a doctor (and a knowledgeable patient) to suspect amyloidosis.

One other item of note, which I was not aware of until I recently read this article again, is that these patients were not the first reported German patients with fibrinogen amyloidosis. They were the first reported with the Glu526Val mutation, but there was a previous German patient with a different mutation reported in an abstract presented at the Xth International Symposium on Amyloid and Amyloidosis in April of 2004. That abstract can be found in the book Amyloid and Amyloidosis, published in November of 2004, edited by Gilles Grateau, Robert Kyle and Martha Skinner. I have not gotten my hands on a copy of that book yet, but I did find this complete one-page abstract online at the publisher's web site at this link: http://www.crcnetbase.com/doi/abs/10.1201/9781420037494.ch126. This particular mutation is discussed a bit more in a 2009 article, but I want to go ahead and review the abstract from the 2004 symposium in this blog post.

Title: Hereditary Renal Amyloidosis In A German Family Associated With Fibrinogen A Alpha Chain Glu540Val (2)

Authors: A. Bybee, M. Hollenbeck, E. R. Debusmann, D. Gopau, J. A. Gilbertson, H. J. Lachmann, M. B. Pepys, P. N. Hawkins (Nephrologische Klinik, Knappschaftskrankenhaus, Bottrop, Germany; National Amyloidosis Centre, London, UK)

Book: Amyloid and Amyloidosis (2004)


A German woman presented with proteinuria and haematuria (blood in the urine) at the age of 49. A kidney biopsy showed amyloid deposits in the glomeruli, which suggested fibrinogen amyloidosis. DNA analysis determined she was heterozygous for a newly discovered mutation, Glu540Val. (This is the exact same substitution that occurs in the Glu526Val mutation, but it occurs just 14 amino acids later in the sequence.)


This patient's father had died of renal failure at the age of 47. The patient's sister was found to also have the Glu540Val mutation, and her only symptom is proteinuria. She had a kidney biopsy that was similar to her sister's. Two of her three children also have the mutation and are currently asymptomatic.

There was no evidence of amyloid involvement in any other organs, although SAP scintigraphy on both sisters showed some amyloid desposits in the spleen. Based on these two patients, the Glu540Val mutation is indistinguishable from the Glu526Val mutation.

The next articles up for review will also come from Europe, this time from France. We have already had fibrinogen amyloidosis reported in France, but this next report includes something we have not seen before.

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Citations:

(1) Eriksson M, Schönland S, Bergner R, et al. Three German fibrinogen Aalpha-chain amyloidosis patients with the p.Glu526Val mutation. Virchows Arch. 2008;453(1):25-31.

(2) Grateau G, Kyle RA, Skinner M. Amyloid and Amyloidosis: Taylor & Francis; 2004.

Tuesday, December 24, 2013

Article Review (2007) - Structural analyses of fibrinogen amyloid fibrils

Today's article under review actually covers two articles with essentially the same title and the same authors. The 2007 article published in the journal Amyloid is actually a slightly longer and more detailed version of a paper presented at the XIth International Symposium on Amyloidosis in November of 2006. I have previously reviewed three papers from the 2006 symposium that were published in the book XIth International Symposium on Amyloidosis, edited by Drs. Skinner, Berk, Connors and Seldin. The first of those reviews was in the November 25, 2013 post.

Since these two articles cover the same material, I will give the information for both articles first, and then review the second one. In contrast to the previous article review, which showed how fibrinogen amyloidosis is different than other types of amyloidosis, these articles show how fibrinogen amyloidosis is similar to the other types, including AL.

Title: Structural Analyses of Fibrinogen A Alpha-Chain Amyloid Fibrils (1)

Authors: L. C. Serpell, M. D. Benson, J. J. Liepneiks, and P. E. Fraser (University of Sussex, Brighton, UK; Indiana University School of Medicine, Indianaposis, IN, USA; Richard L. Roudebush Veterans Affairs Medical Center, Indianapolis, IN, USA; Centre for Research into Neurodegenerative Diseases, Toronto, Ontario, Canada)

BookXIth International Symposium on Amyloidosis (2007)

Abstract: None




Title: Structural analyses of fibrinogen amyloid fibrils (2)

Authors
L. C. Serpell, M. D. Benson, J. J. Liepneiks, and P. E. Fraser (University of Sussex, Brighton, UK; Indiana University School of Medicine, Indianaposis, IN, USA; Richard L. Roudebush Veterans Affairs Medical Center, Indianapolis, IN, USA; Centre for Research into Neurodegenerative Diseases, Toronto, Ontario, Canada)

Journal: Amyloid (2007)

Abstract

Hereditary fibrinogen amyloidosis is characterized by deposition of amyloid fibrils in renal glomeruli. The subunit protein of the amyloid fibrils is a proteolytic fragment of the fibrinogen Aα-chain. To investigate the structure of fibrinogen amyloid, fibrils were extracted from the tissues of a patient and studied by X-ray fiber diffraction and electron microscopy. We have carried out a full structural characterization of amyloid fibrils taken from disease tissue. These studies revealed that ex vivo fibrinogen amyloid fibrils have a cross-β structure similar to other chemical types of amyloid fibrils.

As I mentioned at the end of the previous post, these articles have a lot of technical information. The topic is the amyloid fibrils themselves, specifically their molecular structure. Most of it is beyond my understanding, and although it does not have any direct impact currently in terms of diagnosis or treatment, it may be important as amyloidosis treatments are developed in the future.

If you read much about amyloidosis in general, you will likely encounter the terms "beta sheets," "beta-pleated sheets," or "cross-beta structure." (Instead of writing out the word "beta", the symbol "β" will often be used, as it is in the abstract above.) In reviewing these articles today we learn what a beta sheet is, and whether or not fibrinogen amyloidosis forms beta sheets like other forms of amyloidosis. So, what is a beta sheet? Quoting from Wikipedia, beta sheet is the second form of regular secondary structure in proteins, and it is less common than the alpha helix. Huh? Let's see if I can explain that a little better, given my ten minutes of Wikipedia research. (For a more thorough explanation than the following, see the Wikipedia articles on "biomolecular structure" and "beta sheet.")

Beta sheet is one of two standard ways of describing the general three-dimensional form of a protein. The other one is alpha helix. These are not intended to be accurate three-dimensional models of the protein molecules, but instead have to do with how they bond together. Just think of alpha helix as a spiral, and beta sheet as a curved sheet or a ribbon, and that gets you close enough for our purposes.

Amyloidosis is often referred to as a protein misfolding disorder. In over-simplified terms, the shape of a protein, or how it is folded, is very critical to ensure it does its job in the body, whatever that may be. When a protein is misfolded, sometimes the body just gets rid of it. But at other times, such as with amyloidosis, these misfolded proteins build up and form deposits in various places in the body. In the case of AL amyloidosis, it has been known for some time that the misfolded proteins form beta sheets. The purpose of this article was to determine if amyloid deposits due to fibrinogen amyloidosis also form beta sheets.

In order to analyze the structure of fibrinogen amyloid material they isolated some tissue from a spleen that was removed from a fibrinogen amyloidosis patient. This patient was the propositus of one of the kindreds described in the first report of the Glu526Val mutation, which I reviewed in the March 20, 2013 blog post. The current article states this patient had a kidney transplant that failed, so he was on dialysis for several years and eventually developed an enlarged spleen that had to be removed.

The article goes into great detail on the laboratory procedures that were used to isolate and then analyze the amyloid fibrils from the spleen. They did stain some amyloid fibrils with Congo red at one point, to confirm they were indeed amyloid. And when viewed under an electron microscope, the fibrils were long, straight and unbranching, which resembles amyloid fibrils due to other types of amyloidosis. Using a technique called X-ray diffraction they were able to determine that the amyloid fibrils formed a cross-beta structure, which is also common to other types of amyloidosis. The article then goes into some more detail on the actual dimensions and angles of the beta sheet, and it states that the amyloid fibrils are composed of a fragment of the fibrinogen A alpha chain that is approximately 80 amino acids long. (A complete fibrinogen A alpha chain is composed of 610 amino acids.)

So this article is definitely on the technical side and does not give us any new information regarding the diagnosis or treatment of fibrinogen amyloidosis. But perhaps this information will some day be useful in the development of drugs that can either prevent the amyloid fibrils from initially forming, or cause the amyloid deposits to be dissolved.

In the next article up for review, we have a report of fibrinogen amyloidosis from yet another country in Europe.


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Citations:

(1) Skinner M, Berk JL, Connors LH, Seldin DC. XIth International Symposium on Amyloidosis: Taylor & Francis; 2010.

(2) Serpell LC, Benson M, Liepnieks JJ, Fraser PE. Structural analyses of fibrinogen amyloid fibrils. Amyloid. 2007;14(3):199-203.

Wednesday, December 18, 2013

Article Review (2007) - Human plasma fibrinogen is synthesized in the liver

Before getting to today's article review, I would like to mention a milestone the blog recently reached. On the evening of December 12, less than 30 minutes after the previous post was published, the pageview counter for the blog went from four digits to five. Yes, it hit 10,000. Woohoo!!  <Tonight we're gonna party like we passed nine nine nine nine!>  No, 10,000 is not a huge number of page views, but it is a power of ten, so it must be important. We'll see how long it takes to get to 100,000. I doubt it will take nine more years, given all the pageviews that probably come from spammers and bots crawling the interwebs. Enough blog news for now. Back to business . . .

Today's article under review, from 2007, comes to us from the National Amyloidosis Centre in London. (You may notice some amyloidosis superstars among the list of authors.) The findings in this article are what make fibrinogen amyloidosis different from all other types of amyloidosis (as far as I am aware.)


Authors: Glenys A. Tennent, Stephen O. Brenna, Arie J. Stangou, John O'Grady, Philip N. Hawkins, and Mark B. Pepys (National Amyloidosis Centre, London, UK; Christchurch Hospital, Christchurch, New Zealand; King's College Hospital, Londin, UK)

Journal: Blood (2007)

Abstract:
Hereditary systemic amyloidosis caused by fibrinogen Aα-chain gene mutations is an autosomal dominant condition with variable penetrance, usually of late onset, and typically presents with nephropathy leading to renal failure. Amyloid deposits often develop rapidly in transplanted kidneys, and concomitant orthotopic liver transplantation has lately been performed in several patients with the hope of halting amyloid deposition. Fibrinogen is produced in vitro by hepatocytes but also by other human cell types, and although the liver is the source of plasma fibrinogen in vivo in rats, this is not known in humans. Transplantation of livers expressing wild-type fibrinogen into patients with variant fibrinogen amyloidosis provides a unique opportunity to establish the source of human plasma fibrinogen. We therefore characterized plasma fibrinogen Aα-chain allotypes by electrospray ionization mass spectrometry mapping of tryptic digests before and after liver transplantation. Before liver transplantation, fibrinogen amyloidosis patients with the Glu526Val Aα-chain variant had approximately equal proportions of peptide with the wild-type sequence TFPGFFSPMLGEFVSETESR, and with the amyloidogenic variant sequence TFPGFFSPMLGEFVSVTESR, as expected for individuals heterozygous for the mutation. After transplantation, only the wild-type sequence was detected, and the liver is thus the source of at least 98% of the circulation fibrinogen.
Here is a link to the PDF if you would like to follow along: http://bloodjournal.hematologylibrary.org/content/109/5/1971.full.pdf

The article starts with some basic info about fibrinogen. You may already know that fibrinogen is a protein that is important in the formation blood clots, and there are many fibrinogen mutations known to cause blood clotting disorders. But here are some bits of information to keep in the back of your mind when someone asks you "what is fibrinogen?"


  • The typical concentration of fibrinogen in the blood is 2 to 4 grams per liter. Assuming an average adult has a total blood volume of approximately 5 liters, that average adult would have roughly 10 to 20 grams of fibrinogen circulating in their blood under normal conditions.
  • The half-life of fibrinogen is approximately four days. That means that if you could track all of the fibrinogen molecules that your body manufactured on a specific day, half of that fibrinogen would be gone four days later. (In case you are curious, the half life of white blood cells is about 7 hours, and the half life of red blood cells is about 50 days.)
  • A fibrinogen molecule consists of three chains, usually written in the medical literature as Aα, Bβ, and γ. Those symbols, if they appeared correctly wherever you are reading this, are alpha, beta, and gamma, the first three letters of the Greek alphabet. (That will likely be the last time I use the symbol for alpha instead of just writing "alpha," since I have to copy it over from Word.) The mutations that cause amyloidosis affect the A alpha chain, which is why the journal articles often refer to it as "fibrinogen A alpha chain" or "fibrinogen alpha chain" amyloidosis. Those are the same thing as "fibrinogen amyloidosis." There are mutations that affect the fibrinogen beta or gamma chains, but none of those are known to cause amyloidosis. In the future, if a mutation of the beta or gamma chain is discovered to cause amyloidosis, then there will be a need to differentiate between fibrinogen alpha chain amyloidosis and fibrinogen beta or gamma chain amyloidosis.

At the time this article was written, fibrinogen was known to be produced by liver cells in vitro (outside the body, such as in a test tube or petri dish), but it was unknown to what extent fibrinogen is produced by other types of cells in vivo (within the body). Other proteins are known to be produced as needed by other types of cells, and the concentration of fibrinogen is known to increase in response to injury, infection or inflammation, so the authors of this article set out to determine whether or not a significant amount of fibrinogen was produced other than in the liver.

The article then gives an overview of current treatments for fibrinogen amyloidosis, which is kidney or combined liver and kidney transplants. When just a kidney transplant is done, the transplanted kidney is usually affected by amyloid deposits, whereas when a combined liver and kidney transplant is done, patients have shown improvement due to a lack of buildup of additional amyloid deposits and regression of the existing deposits. The article states that the first liver transplants for fibrinogen amyloidosis were done on the presumption that the liver is the source of the variant (mutant) fibrinogen, but that has never been investigated until now.

Before continuing I need to define some terms. When a person has the mutation for fibrinogen amyloidosis (and they are heterozygous, meaning they have one normal copy of the gene and one mutated copy), half of the fibrinogen produced will be normal, and the other half will be mutated instead of normal. The medical term most often used to refer to the mutated fibrinogen (or any other protein) is "variant," and the term most often used to refer to the normal fibrinogen (or any other protein) is "wild-type." I had no idea what the term "wild-type" meant the first time I heard it, and I was really surprised when I first learned that it simply means normal. But if you think of "wild-type" as the type most often occurring in nature, or "in the wild," it starts to make some sense. So this article review will frequently refer to variant fibrinogen and wild-type fibrinogen.

The last item discussed in the article before describing the actual analysis that was performed was the use of liver transplantation to treat transthyretin amyloidosis (ATTR). Those patients may improve after a liver transplant, but with many of the ATTR mutations, amyloid deposits can still build up for two reasons. First, transthyretin is also produced outside the liver, and second, wild-type transthyretin can still deposit as amyloid. (That was discussed at the familial amyloidosis meeting in October. They believe that the amyloid deposits initially formed by the variant transthyretin act as sort of a scaffold that wild-type transthyretin continues to build on, even without additional variant transthyretin.) The good news for those of us with fibrinogen amyloidosis is that there is no evidence that wild-type fibrinogen can form amyloid deposits.

Ok, now for the data analysis in the article. They analyzed blood taken at various times from five unrelated patients, all of whom had the Glu526Val mutation for fibrinogen amyloidosis. They also analyzed blood taken from healthy subjects of the same ages for comparison. Here is a description of each patient and when blood was taken from the ones who had received transplants:

Case 1: Male. Combined liver and kidney transplant in 2004. Blood was drawn 29 days before transplant, 46 days after, and 137 days after

Case 2: Male. Combined liver and kidney transplant in 2004. Blood was drawn immediately before the transplant and 92 days after.

Case 3: Female. Combined liver and kidney transplant approximately 1999. Blood was drawn 7 years after transplant.

Case 4: Male, waiting on transplant.

Case 5: Female, waiting on transplant.


The next few paragraphs of the article describe in detail the laboratory procedures used to isolate fibrinogen and do other analysis of the various blood samples. I will not pretend to come close to understanding any of that, so we will move on to the results.

And here are the results, summarized in one simple chart:


Figure 2 from article
Representative ESIMS tryptic maps of purified A alpha chains recorded in the m/z range 1060-1150.

Don't worry if you do not have a clue what that chart means. I didn't either the first time I saw it, and it took a little digging into the article and a few other places to understand it enough to know what is important for this discussion. But I think I can explain it without getting too technical.

First, this chart displays something called a tryptic map for each of six different blood samples, represented by the letters A through F. Think of a tryptic map as a way of indicating the various components a substance is made of. In this case the substance is the fibrinogen A alpha chains extracted from a blood sample. The horizontal scale along the bottom of each tryptic map, as noted in the not-very-helpful caption, runs from 1060 to 1150. Those numbers have something to do with atomic weights (m/z is mass-to-charge ratio), and we care about what shows up at 1118 and 1133. Wild-type fibrinogen shows up at 1133, and variant fibrinogen (for the Glu526Val mutation) shows up at 1118. So we see in the map for row A there is a large spike at 1133, and there is basically nothing at 1118. Now compare that to row A, which has shorter spikes at both 1133 and 1118, both of which seem to be at about the 50% level on the vertical scale.

Now that we know what these charts are showing, and we know to focus on what appears at 1118 and 1133, we can see the big picture a little better once we know who these blood samples were taken from, and when. Here is that info:

A: Healthy person without the Glu526Val mutation

B: Case 1, 29 days before transplant

C: Case 1, 46 days after transplant

D: Case 1, 137 days after transplant

E: Case 2, immediately before transplant

F: Case 2, 92 days after transplant


Now we can see what these charts are telling us. As we would expect, the person without the mutation (Row A) has no variant fibrinogen. There is only a spike at 1133. Rows B and E, on the other hand, both show patients with the mutation before transplant. They each have approximately equal amounts of variant and wild-type fibrinogen (spikes at 1118 and 1133.)

When we look at the rows representing blood drawn after transplant (Rows C, D and F), we only see spikes at 1133 again, indicating those patients have no circulating variant fibrinogen. Although it is not shown here, the article also mentions that no variant fibrinogen was detected in Case 3, the patient who had a liver transplant 7 years prior.

So that is really the main point of this article, that fibrinogen appears to be produced solely in the liver. If there is any variant fibrinogen produced outside the liver, it is essentially undetectable. The article concludes with this: ". . . our present results thus formally validate the rationale for use of liver transplantation in treatment of hereditary fibrinogen amyloidosis."

I want to discuss one more thing about the chart from the article, which sheds some light on the terminology sometimes used when referring to this mutation. At the bottom of the figure above, below chart A, there are two lines, each starting with "T-62." T-62 is a peptide, and peptides are building blocks of proteins. In this case, T-62 is a building block of the fibrinogen protein molecule. The building blocks of peptides are amino acids, and what is shown on those two rows are the amino acid sequences for the variant and wild-type versions of this particular peptide, which is the one affected by the Glu526Val mutation. You will notice there is only one letter different between the two, and it is underlined. In wild-type fibrinogen it is "E" and in variant fibrinogen it is "V." So, what do E and V represent? Actually, E and V are the same thing as Glu and Val in Glu526Val. In case you do not remember from my basic DNA lesson in the post from March 20, 2013, Glu is the three-letter abbreviation for the amino acid Glutamic Acid, and Val is the three-letter abbreviation for the amino acid Valine. So E is the single-letter abbreviation for Glutamic Acid (the letter G is assigned to Glycine), and V is the single-letter abbreviation for Valine. That is why this mutation is sometimes written as E526V.


This article gives us some good, solid data to explain why liver transplantation is so successful in the treatment of fibrinogen amyloidosis. It's a little on the technical side, but science has to be that way sometimes. And if you thought this article was technical, just wait until the next one.

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Citation:

(1) Tennent GA, Brennan SO, Stangou AJ, O'Grady J, Hawkins PN, Pepys MB. Human plasma fibrinogen is synthesized in the liver. Blood 2007; 109: 1971-1974.

Thursday, December 12, 2013

Article Review (2006) - Amyloidosis-associated kidney disease

In the previous post I discussed a couple of papers that give us an indication of how rare fibrinogen amyloidosis actually is, at least in the US. Oddly enough, the day after I published that post I found some additional data that would have been nice to add to that discussion. I have updated the previous post with the new info, but for the benefit of you email subscribers I will go ahead and include the update in this post, and then we'll move on.

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[Start of update to previous post.]

As luck would have it, I found some more data the day after I published this blog post. This additional info is from an abstract presented December 7, 2013, at the 2013 Meeting of the American Society of Hematology. Here is the title and a link to the abstract:

Title: Proteome of Amyloidosis: Mayo Clinic Experience In 4139 Cases

Link: https://ash.confex.com/ash/2013/webprogram/Paper62434.html

First of all, what is a proteome? The dictionary definition is that a proteome is the entire complement of proteins found within a cell, tissue or organism. In this context, the proteome of amyloidosis is the complete list of proteins that are known to cause amyloidosis. (These are often referred to as precursor proteins.) This paper gives the breakdown of amyloid subtypes found in 4139 cases where a tissue biopsy was evaluated at Mayo Clinic since 2008, when they started using a newly developed method for subtyping amyloid deposits. (It's called liquid chromatography electrospray tandem mass spectrometry, or LC-MS/MS. This is probably the technique people refer to as mass spec.) I assume there will be some overlap with the previous article that covered the years 2007 to 2011 in the Renal Pathology Lab at Mayo Rochester.

Here is the table showing the distribution of amyloid subtypes in these 4139 cases.


Frequency of amyloid subtypes observed in the Mayo Clinic Amyloid Cohort (n=4139).
Asterisk denotes non-canonical subtypes.

As shown in the table, there were 26 fibrinogen amyloidosis (AFib) diagnoses among these 4139 cases (0.63%), over a period of five to six years (2008 to 2013 inclusive). So that is still only 4 or 5 cases per year diagnosed at Mayo Clinic. But what if we compare the data in this article to the previous one, which had data from the Mayo Clinic Renal Pathology Laboratory from 2007 to 2011? The earlier article reported six cases of Afib, whereas this more recent article reported 26 cases. Does that mean there have been at least 20 cases diagnosed at Mayo Clinic since the data was gathered for the earlier paper? It looks that way based solely on the data we have, but we cannot say for certain without more information, such as the date of each fibrinogen amyloidosis diagnosis at Mayo Clinic since 2007.

Whatever the true numbers may be, the data clearly shows that fibrinogen amyloidosis is among the rare types of familial amyloidosis, and familial amyloidosis only accounts for about 20% of the total amyloidosis cases, and amyloidosis itself is a rare disease.

[End of update to previous post.]
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Today's article under review is the last of the articles from 2006. Although this is a rather long article (ten pages plus four more to list the 107 references), there is only a small amount of information specifically related to fibrinogen amyloidosis. My review here will not go into detail on most of this article, so you do not have to worry about this review being longer than the article. I also detour a bit and go over Mom's biopsy report again due to some information in this article.

Title: Amyloidosis-associated Kidney Disease (1)

Author: Laura M. Dember (Boston University, Boston, MA)

Journal: Journal of the American Society of Nephrology

Abstract:
The amyloidoses are a group of disorders in which soluble proteins aggregate and deposit extracellularly in tissues as insoluble fibrils, causing progressive organ dysfunction. The kidney is one of the most frequent sites of amyloid deposition in AL, AA, and several of the hereditary amyloidoses. Amyloid fibril formation begins with the misfolding of an amyloidogenic precursor protein. The misfolded variants self-aggregate in a highly ordered manner, generating protofilaments that interact to form fibrils. The fibrils have a characteristic appearance by electron microscopy and generate birefringence under polarized light when stained with Congo red dye. Advances in elucidating the mechanisms of amyloid fibril formation, tissue deposition, and tissue injury have led to new and more aggressive treatment approaches for these disorders. This article reviews the pathogenesis, diagnosis, clinical manifestations, and treatment of the amyloidoses, focusing heavily on the renal aspects of each of these areas.

Here is a link to the article if you would like to follow along:  http://jasn.asnjournals.org/content/17/12/3458.full

This article is a very thorough discussion of how all types of amyloidosis affect the kidneys. It was written by Dr. Laura Dember, who was a nephrologist at Boston University at the time and is now with The University of Pennsylvania. The article starts with a general overview of amyloidosis, including the various types and subtypes. It then discusses detection of amyloid deposits in tissue biopsies using Congo red dye. It also mentions something I was unaware of, which is amyloid fibrils have a characteristic look when viewed with an electron microscope. Whereas Congo red staining is not always a routine part of a biopsy evaluation, electron microscopy is. If amyloid fibrils are detected with an electron microscope, then the tissue should be stained with Congo red to confirm the presence of amyloid.

The next section of the article is "Determination of the Type of Amyloidosis." We know how tricky that can be, so I will skip right to the end of that section and just copy the last sentence here: "Isolated glomerular involvement on kidney biopsy with no amyloid in the tubules, interstitium, or vessels has been found to be characteristic of fibrinogen A alpha amyloidosis, and this histologic pattern should raise suspicion for fibrinogen A alpha disease." No real surprise there, as we know from previous articles that kidney biopsies of fibrinogen amyloidosis patients show amyloid deposits predominantly in the glomeruli.

The next section of the article is "Renal Pathology." This section seems to be written for pathologists, as it goes into detail on what amyloid looks like in a kidney biopsy and exactly where in the kidney the deposits typically form. There are the usual pictures of biopsy slides, with the Congo red stained amyloid deposits under nonpolarized and polarized light, plus some other stains for AL amyloidosis. There is also a picture of what amyloid fibrils look like under an electron microscope, which I don't think I have seen before. The pictures (with captions) are available at this link: http://jasn.asnjournals.org/content/17/12/3458/F2.expansion.html. If you click on the pictures you get an even larger view. (In my opinion, the one with Congo red stain under polarized light (photo C), is not very impressive. There are much better examples out there.)

Sometimes when I read about techniques for analyzing kidney biopsies I go back and read over Mom's biopsy report to see if I understand it any better. I did that again after reading over this article, and a few things stood out this time. (Feel free to skip the rest of this paragraph if biopsy findings do not excite you.) If you look at Mom's biopsy report from the October 1, 2012 blog post, you will see three sections under Microscopic Description. They are Light Microscopy, Immunofluorescence, and Electron Microscopy. The Light Microscopy section details the findings from just looking at the tissue under a microscope with very high magnification. I believe that section says 12 of 28 glomeruli did have amyloid deposits. The Immunofluorescence section has the results of applying the various stains in an attempt to determine the type of amyloid deposits. One of the stains used is intended to detect fibrinogen, but it came up negative as did most of the others. That is not unusual, as these immunofluorescence techniques are far from 100 percent accurate. The Electron Microscopy section has the results of examining material under an electron microscope. I believe the part about the glomerular sections having "crosshatched fibrilla material having size characteristics of amyloid" is exactly what this article is talking about.

Ok, back to the article. It gets even more technical in the next few sections, and I did not highlight much of anything worth discussing here. The section headings are Clinicopathologic Correlates, Determinants of Renal Deposition of Amyloid, and How Does Amyloidosis Cause Renal Disease? The next section, Treatment Approaches and Impact on the Kidney, primarily deals with the treatment of AL and AA amyloidosis, but there is a subsection on hereditary amyloidosis. It begins by discussing the use of liver transplantation to treat TTR amyloidosis. Then it states that there is considerably less experience in using liver transplantation to treat fibrinogen amyloidosis, and it refers to three of the articles we have previously reviewed which contain case reports of combined liver and kidney transplantation, with satisfactory outcomes. It then gives some reasons why liver transplantation is not as successful, or not appropriate at all, for some of the other types of hereditary amyloidosis.

The remainder of the article discusses in more general terms end stage renal disease, regression of amyloid deposits and improvement of kidney function after treatment to eliminate the precursor protein, and then it closes with a short section on emerging treatment strategies. (It does mention diflunisal, a drug which is now very far along in clinical trials and is showing good outcomes in the treatment of TTR amyloidosis.)

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So overall, we do not learn much new information about fibrinogen amyloidosis from this article, but we do see how it compares with other types of amyloidosis. And if you want some very detailed information about what is happening to kidneys that are affected by amyloidosis and how amyloidosis appears to a pathologist (with the tools available in 2006), this article is a very good reference.

Next up: Is fibrinogen only produced in the liver? How do we know?

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Citation:

(1) Dember, L. M. Amyloidosis-associated kidney disease. J. Am Soc Nephrol 2006; 17: 3458-3471.

Friday, December 6, 2013

Article Reviews (2006 and 2013) - How rare is fibrinogen amyloidosis in the US?

Before getting to today's article reviews, I have a quick update on Mom and a note about the monthly blog stats that appear at the end of this post.

I know it has been awhile since I mentioned anything about a kidney transplant for Mom, and that is because there has not been much of anything to report. The two people who did offer to be living donors had both been ruled out as of earlier this year, and there have been no other inquiries since then. In June of this year Mom was notified that she would be removed from the list of transplant candidates within 90 days if she did not have a potential living donor, so we knew where things were likely headed. Her case was presented to the transplant committee in October, and she did receive an official letter dated November 1 that she has been removed from the transplant waiting list. Although it was not really a surprise, it's still a bit depressing, to say the least.

Regarding the monthly blog stats, I have some bad news for you loyal readers who anxiously await the first post each month so you can jump to the end and see what new countries have visited the blog. It is with great sadness that I must report that no new countries viewed the blog in November. After a streak of 13 consecutive months of adding a minimum of two countries to the list, we came up empty in November. Hopefully this will be just a short pause in my quest for world domination. (FYI, the three most populous countries that have not yet visited the blog are Ethiopia, Democratic Republic of the Congo, and Burma.)

The two articles being reviewed today, along with some information I received in 2010, attempt to answer the question posed in the title of this post, which is: How rare is fibrinogen amyloidosis in the US? (It is more common in the UK than in the US, but still rare.) We all know it is rare, and Dr. Skinner's presentation at the 2013 familial amyloidosis meeting listed it as making up less than one percent of the total systemic amyloidosis cases. I have heard the number of amyloidosis cases diagnosed annually in the US is around 3000. That would include types that are not systemic, such as localized, but let's just use 3000 for the approximate number of systemic amyloidosis cases each year. If fibrinogen amyloidosis is less than one percent of that, then all we know is there are fewer than 30 cases of fibrinogen amyloidosis diagnosed in the US each year. By the end of this post it will be obvious that the number of new US cases each year is likely much less than 30.

The first article under review today is the third of three papers I will be reviewing that were presented at the eleventh annual International Symposium on Amyloidosis, which was held in Woods Hole, Massachusetts in November of 2006. The papers presented at this symposium can be found in the book XIth International Symposium on Amyloidosis, edited by Drs. Skinner, Berk, Connors and Seldin, all of the Amyloidosis Center at Boston University.

Title: Recent novel and rare mutations in a clinic population of patients with amyloidosis (1)

Authors: B. Spencer, L. H. Connors, T. Prokaeva, P. Soohoo, C'Hara, and M. Skinner (Boston University, Boston, MA)

Book: XIth International Symposium on Amyloidosis (2007)

Abstract: None


This paper presents some data about the types of familial amyloidosis diagnosed by the team at Boston University between the Xth and XIth International Symposiums on Amyloidosis. The Xth symposium was held in April of 2004, and the XIth symposium was held in November of 2006, so this paper covers a little more than two years. Although there are a number of reasons why you cannot reach any hard conclusions about the general population based on this set of data alone, this paper does give us a good idea of how rare a diagnosis of fibrinogen amyloidosis really is.

The article begins by stating that familial amyloidosis is diagnosed in approximately 10% of the patients evaluated at the Boston University Amyloid Program. It states that the total number of familial patients is 253, which has to be since the program began in 1980. (Currently they see about 200 new patients per year.) Out of those 253 familial amyloidosis patients, 224 (89%) had transthyretin mutations (ATTR). That means through 2006, out of about 2500 patients, Boston had only diagnosed 29 patients with a familial form of amyloidosis other than ATTR.

The paper goes into detail on the mutations that were diagnosed (two for the first time) during this two-year period, but I won't go into all of those here. They did happen to have three cases of fibrinogen amyloidosis, all with the Glu526Val mutation. All three patients had kidney disease, and biopsies showed amyloid deposits predominantly in the glomeruli of the kidneys.

Although this paper does not give us any new information in terms of the diagnosis or treatment of fibrinogen amyloidosis, it does give us some idea of how rare the diagnosis really is. Over the course of two years, Boston diagnosed three patients (out of about 400) with fibrinogen amyloidosis. Adding some more data to the Boston numbers, when I was corresponding with Dr. Skinner shortly after Mom's diagnose in 2010, she said Boston had only seen a total of 10 to 15 patients with fibrinogen amyloidosis. So that means since the discovery of fibrinogen amyloidosis in the early 1990s, Boston has averaged just under one case per year out of the 200 new patients they see annually.

So that is the picture from Boston. How about Mayo Clinic? It just so happens there was an article published this year that gives us some data from the Mayo Clinic.

Title: Renal Amyloidosis: Origin and Clinicopathologic Correlations of 474 Recent Cases (2)

Authors: S. M. Said, S. Sethi, A. M. Valeri, N. Leung, L. D. Cornell, M. E. Fidler, L. H. Hernandez, J. A. Vrana, J. D. Theis, P. S. Quint, A. Dogan, S. H. Nasr (Mayo Clinic, Rochester, MN; Columbia University, New York, NY)

Journal: Clinical Journal of the American Society of Nephrology (2013)

Abstract:
BACKGROUND AND OBJECTIVES:  The kidney is the organ most commonly involved in systemic amyloidosis. This study reports the largest clinicopathologic series of renal amyloidosis.
DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS:  This study provides characteristics of 474 renal amyloidosis cases evaluated at the Mayo Clinic Renal Pathology Laboratory from 2007 to 2011, including age, sex, serum creatinine, proteinuria, type of amyloid, and tissue distribution according to type.
RESULTS:  The type of amyloid was Ig amyloidosis in 407 patients (85.9%), AA amyloidosis in 33 (7.0%), leukocyte chemotactic factor 2 amyloidosis in 13 (2.7%), fibrinogen A α chain amyloidosis in 6 (1.3%), Apo AI, Apo AII, or Apo AIV amyloidosis in 3 (0.6%), combined AA amyloidosis/Ig heavy and light chain amyloidosis in 1 (0.2%), and unclassified in 11 (2.3%). Laser microdissection/mass spectrometry, performed in 147 cases, was needed to determine the origin of amyloid in 74 of the 474 cases (16%), whereas immunofluorescence failed to diagnose 28 of 384 light chain amyloidosis cases (7.3%). Leukocyte chemotactic factor 2 amyloidosis and Apo AI, Apo AII, or Apo AIV amyloidosis were characterized by diffuse interstitial deposition, whereas fibrinogen A α chain amyloidosis showed obliterative glomerular involvement. Compared with other types, Ig amyloidosis was associated with lower serum creatinine, higher degree of proteinuria, and amyloid spicules.
CONCLUSIONS:  In the authors' experience, the vast majority of renal amyloidosis cases are Ig derived. The newly identified leukocyte chemotactic factor 2 amyloidosis form was the most common of the rarer causes of renal amyloidosis. With the advent of laser microdissection/mass spectrometry for amyloid typing, the origin of renal amyloidosis can be determined in >97% of cases.
Here is a link to the article (not freely available):  http://cjasn.asnjournals.org/content/8/9/1515.abstract

I do not have this article, so I will just be discussing the abstract. This article describes the types of amyloidosis that were diagnosed in 474 renal amyloidosis cases evaluated at the Mayo Clinic Renal Pathology Laboratory from 2007 to 2011. So these were instances where a diagnosis was made from a kidney biopsy. Out of these 474 cases over five years, only six were diagnosed with fibrinogen amyloidosis. Keep in mind that this may not be the total number of fibrinogen cases diagnosed at Mayo during this time period, since sometimes a diagnosis cannot be determined from a kidney biopsy and genetic testing is required. (That was the case with Mom's diagnosis from Boston.) The abstract does state that there were 11 cases where the type of amyloidosis could not be determined from the biopsy, so it is possible some of those cases were eventually diagnosed with fibrinogen amyloidosis.

So now we have rough estimates from Boston (just under one) and from Mayo Clinic (a little over one) on the number of new fibrinogen amyloidosis cases diagnosed per year. Those two centers certainly see the vast majority of new amyloidosis patients each year in the US, and the only other location that might diagnosis nearly as many (or perhaps more) fibrinogen amyloidosis cases would be Dr. Benson in Indianapolis. But even if we estimate one from Boston, two from Mayo Clinic, and two from Dr. Benson annually, that's only five new cases per year in the US. Keep in mind that these are new cases where a patient has symptoms and gets a diagnosis, so there will be additional cases where people test positive for the mutation but are asymptomatic. So let's double the number to account for the asymptomatic patients, and now we're up to ten per year, which is probably on the high side.

So whenever someone in the US learns they have a genetic mutation for fibrinogen amyloidosis, I think it is safe to say they can use their fingers to count all the other people in the US who learned the same thing that year, and they may not even need both hands.

Aren't we special?

[The following update, regarding an abstract presented at the 2013 Annual Meeting of the American Society of Hematology, was added December 7, 2013.]

As luck would have it, I found some more data the day after I published this blog post. This additional info is from an abstract presented December 7, 2013, at the 2013 Meeting of the American Society of Hematology. Here is the title and a link to the abstract:

Title: Proteome of Amyloidosis: Mayo Clinic Experience In 4139 Cases

Link: https://ash.confex.com/ash/2013/webprogram/Paper62434.html

First of all, what is a proteome? The dictionary definition is that a proteome is the entire complement of proteins found within a cell, tissue or organism. In this context, the proteome of amyloidosis is the complete list of proteins that are known to cause amyloidosis. (These are often referred to as precursor proteins.) This paper gives the breakdown of amyloid subtypes found in 4139 cases where a tissue biopsy was evaluated at Mayo Clinic since 2008, when they started using a newly developed method for subtyping amyloid deposits. (It's called liquid chromatography electrospray tandem mass spectrometry, or LC-MS/MS. This is probably the technique people refer to as mass spec.) I assume there will be some overlap with the previous article that covered the years 2007 to 2011 in the Renal Pathology Lab at Mayo Rochester.

Here is the table showing the distribution of amyloid subtypes in these 4139 cases.

Frequency of amyloid subtypes observed in the Mayo Clinic Amyloid Cohort (n=4139).
Asterisk denotes non-canonical subtypes.

As shown in the table, there were 26 fibrinogen amyloidosis (AFib) diagnoses among these 4139 cases (0.63%), over a period of five to six years (2008 to 2013 inclusive). So that is still only 4 or 5 per year diagnosed at Mayo Clinic. But what if we compare the data in this article to the previous one, which had data from the Mayo Clinic Renal Pathology Laboratory from 2007 to 2011? The earlier article reported six cases of Afib, whereas this more recent article reported 26 cases. Does that mean there have been at least 20 cases diagnosed at Mayo Clinic since the data was gathered for the earlier paper? It looks that way based solely on the data we have, but we cannot say for certain without more information, such as the date of each fibrinogen amyloidosis diagnosis at Mayo Clinic since 2007.

Whatever the true numbers may be, the data clearly shows that fibrinogen amyloidosis is among the rare types of familial amyloidosis, and familial amyloidosis only accounts for about 20% of the total amyloidosis cases, and amyloidosis itself is a rare disease.


=====Monthly Blog Status Update=====

Total posts: 113 (7 in November)

Total pageviews: 9700 (~1000 in November)

Email subscribers: 6

Total number of countries that have viewed the blog: 79

No new countries viewed the blog in November: :-(
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Citations:

(1) Skinner M, Berk JL, Connors LH, Seldin DC. XIth International Symposium on Amyloidosis: Taylor & Francis; 2010.

(2) Said SM, Sethi S, Valeri AM, et al. Renal amyloidosis: origin and clinicopathologic correlations of 474 recent cases. Clin J Am Soc Nephrol. 2013;8(9):1515-1523.