Re: MDR3 Defects...I think I am answering my own question...

[Guest guest]

This is from the Fall 2006 PFIC.org newsletter... I think I

understand things a bit more...and I am posting it in case anyone

else was having trouble deciphering. I know it doesn't cover it

all...but I am closer

PFIC 3 " MDR3 deficiency " , or " ABCB4 disease

By Knisely, MD

Consultant Histopathologist

Institute of Liver Studies

King's College Hospital

Some persons will have come to this website to learn more about a

disorder called " PFIC-3. " A more precise, and less confusing, name

for the

disorder is " MDR3 deficiency " , or " ABCB4 disease. " Just different

spoonsful of alphabet soup, I can hear people saying – but let me try

to set out

why I think the distinctions matter.

The shorthand name for one of the clinical-laboratory studies,

or " blood tests " , done to monitor children with liver disease

is " GGT " . GGT

stands for " gamma-glutamyl transpeptidase. " This is an enzyme, a

protein that speeds up a chemical reaction. Samples of blood serum

(what is

left of blood after clotting has occurred and the clot is separated

out) contain some GGT. The concentrations of GGT activity in serum

can be

measured in different ways; the values obtained are used to track the

severity of cholestasis. In all but a few forms of cholestasis, GGT

values rise

in parallel with bilirubin values. (Bilirubin is the yellow substance

that gives jaundice its color.)

" PFIC " was originally defined as the class of rare inherited

childhood liver disorders in which GGT values did not rise in

parallel with bilirubin

values. " PFIC-1 " was the first member of that class to be described,

the original " Byler disease " , and the first member for which a

responsible

gene was identified. " PFIC-2 " was the second member to be

distinguished, and the second member for which a responsible gene was

identified.

The search continues for others.

In " PFIC-3 " , however, GGT values are not low. They are high.

So confusion has been great. Doctors and nurses are puzzled – can

there be a " high-GGT " form of PFIC, when the definition of PFIC

requires

that GGT values be low? (In my opinion: No; logically there can't.)

Many other inherited childhood liver disorders, as well as even more

acquired

childhood liver disorders, are characterized by high GGT values. More

puzzlement – my patient or my family member, who has a small

child, has " high-GGT " liver disease; does (s)he have " PFIC-3 " ? (Not

necessarily.)

Those puzzled questions, I think, are the result of a misleading

name, one that has got even medical professionals muddled. I'd rather

that the

terms used to describe any disorder be clear and specific, and that's

why I prefer the designations " MDR3 deficiency " and " ABCB4 disease " to

" PFIC-3. "

What, then, are " MDR3 " and " ABCB4 " ?

MDR3 is an officially approved abbreviation for an enzyme

called " multidrug resistance protein 3. " (The third identified member

of a set, that is,

of proteins that in structure resemble " multidrug resistance protein

1 " , which acts to push various substances out of liver cells.) Yes,

there are

committees of scientists that track, evaluate, and assign names to

newly discovered substances, to ensure that everyone sings from the

same page

of the hymnbook. The officially approved abbreviation for the gene

encoding MDR3 (providing the cell with instructions on how to make

MDR3)

is ABCB4, short for " ATP-binding cassette gene class B, member 4. "

Defects in ABCB4 – gene designations are put in italics, if you were

wondering

– lead to complete or partial lack of working MDR3. This lack then

causes disease of the liver, including the bile ducts.

How does that happen? To explain that, I have to describe what MDR3

does in the healthy liver.

MDR3 contributes to forming bile. Bile is a mix of water, and salts,

and bilirubin pigment, and wastes or toxins that the liver has partly

broken

down, and fatty substances called lipids, and detergents called bile

acids. Without MDR3, the bile is deficient in one of those fatty

substances, a

lipid called phosphatidylcholine. The tiniest branches of the biliary

tract (the bile canaliculi) have walls made of parts of liver cells

(hepatocytes).

Those walls, or membranes, are two layers thick. MDR3 in the

hepatocyte spans the inner and outer layer of the canalicular

membrane and

moves phosphatidylcholine from the inner layer to the outer layer.

A quick digression about " detergents. " That oil and water don't mix

is proverbial. Detergents, however, let such substances mingle with

one

another. Laundry detergent breaks up grease and lets it disperse in

water. Bile acids inside the small intestine break up the fat in what

we've

eaten and let it disperse in the mix (mostly water) of partly

digested food. Once the fat is dispersed, the cells that line the

intestinal wall can take

it up. Without bile acids, we can't absorb fat, or fat-soluble

substances like some vitamins, from our diet. This is why cholestatic

children require

vitamin supplements: Bile acids in such children aren't making the

journey from hepatocytes to small intestine.

Let's head back to the membranes that line the biliary tract. If MDR3

is present in the canalicular wall, and if the MDR3 present is

working normally,

the outer layer of the canalicular membrane will contain

phosphatidylcholine. Under the influence of bile acids, then,

phosphatidylcholine

can float out of the membrane and into the bile, like a grease spot

out of a work shirt and into the wash water. In the bile the

phosphatidylcholine

forms a complex with the bile acids and acts as a " chaperone. "

The action of phosphatidylcholine as a chaperone – sticking close to

the bile acids and keeping them from causing trouble, like teachers

watching

rowdy teenagers on Prom Night – is essential because bile acids are

very corrosive substances. The canalicular membranes, and the

membranes

of the cells that line the bile ducts, contain large quantities of

lipids. If bile acids in the bile are not chaperoned by

phosphatidylcholine, they will

attack the membranes and injure them by leaching lipids away. This

sort of injury can lead to cell death or dysfunction. Cell death and

dysfunction

in the hepatobiliary system lead to jaundice, and itching, and

inflammation, and scarring, which eventually may be fatal without a

liver transplant.

And that is the sequence proceeding from mutation in ABCB4 (or ABCB4

disease) to absence or malfunction of MDR3 (or MDR3 deficiency) to

unhappy, undergrown, scratching, yellowish, miserable children on the

transplant waiting list.

Why are GGT values high in MDR3 deficiency? Because GGT is present in

large quantities in the canalicular and bile-duct membranes (mostly

canalicular). When those are damaged, GGT is released into the bile.

GGT then leaks across the wall of the biliary tract into the blood

and the

concentrations of GGT activity in the serum rise.

Why are GGT values low in PFIC-1? Because GGT is lacking in the

canalicular membranes in that disorder – if it isn't there, it can't

be released.

Why are GGT values low in PFIC-2? Because bile acids are not normally

pumped into bile in that disorder – although GGT is present in the

canalicular

membranes, without the detergent action of bile acids the GGT can't

be released.

MDR3 deficiency has a broad clinical spectrum. In total deficiency,

the effects are quite severe, often are evident in early childhood,

and can require

liver transplantation for relief. In partial deficiency, the effects

are milder. They may include gallstones, or off-and-on itching, and

may show

up only in adulthood or even middle age. When MDR3 deficiency is

suspected in adults, two principal approaches to diagnosis exist.

When it is

suspected in young children, a third approach also can be useful.

Most definitive, of course, is analysis of ABCb4, looking for gene

changes that can be predicted to disrupt gene function. This is

expensive and

takes a long time. In some cases, the abnormalities found are not

clear-cut, and additional studies must be done. It is most useful for

families

who want to take advantage of prenatal diagnosis.

A reasonable tack to take is to analyze bile itself to learn if

phosphatidylcholine is deficient. Such deficiency can be inferred, if

other things fit the

picture, to be the result of lack of MDR3, or of subnormal MDR3

function. (Sampling bile, however, is not an easy business,

particularly in the

very young.) Bile analysis can permit the diagnosis of functional

deficiency of MDR3.

PFIC 3 " MDR3 deficiency " , or " ABCB4 disease –Continued

Genetic analysis and bile analysis can be used in diagnosis of both

severe and mild forms of MDR3 deficiency. For severe forms,

immunohistochemical

studies of liver biopsy materials also can be conducted. Antibodies

that tag MDR3 protein are used in this approach. If no MDR3 protein

can be demonstrated in liver tissue, as shown in Figure 1, actual

(rather than functional) MDR3 deficiency can be diagnosed. If the

antibodies

react, and if the pattern of reaction is appropriate, then MDR3 is

present, as shown in Figure 2. This does not give any information,

however, on

how well the MDR3 actually functions. Persons with disease manifest

after infancy likely have some MDR3 protein – protein that works, but

that

that does not work very well. Immunohistochemical studies are not

conclusive in such cases. Persons with disease manifest in infancy,

that is,

with severe clinical signs and symptoms, are more likely to lack all

MDR3 protein, and are better candidates for immunohistochemical study

of

liver biopsy materials.

So far, to my knowledge, no one has been found who has functional

severe MDR3 deficiency, as demonstrated by analyses of bile that show

phosphatidylcholine

to be almost totally absent – and who has documented ABCB4 mutations –

and who has no lack, on immunohistochemical study,

of MDR3 protein along canaliculi in a liver biopsy specimen. But such

a person will almost certainly be found, because some mutations in

other

genes like ABCB4 lead cells to make forms of proteins that are

normally handled by the cell, going to the right places and reacting

properly with

antibodies, but do not perform their usual function at all. In my

opinion, it is just a matter of time till a person with this sort of

ABCB4 disease

shows up. When immunohistochemical studies demonstrate no MDR3

protein, the test has given a clear answer: " Severe MDR3 deficiency is

present. " When they demonstrate MDR3 protein, the answer can only

be: " This study does not support the idea that severe MDR3 deficiency

is

causing this patient's disease. However, it also does not disprove

that idea. "

Figure 1. Antibodies against MDR3 did not react in this liver biopsy

specimen from a boy with itching,

mild jaundice, high serum GGT activity values, and a family history

of liver disease. The diagnosis of severe

MDR3 deficiency can be made. Compare with Figure 2.

Figure 2. The fine network of canaliculi – little canals – among

hepatocytes is well demonstrated in this

liver specimen from an adult (who required liver surgery for a tumor

but was not jaundiced). Antibodies

against MDR3 were allowed to incubate with tissue sections; then

another antibody, bearing a pigmented

tag, was used to show where the first antibodies had united with the

tissue. The walls of the canaliculi

mark well for MDR3.

[Guest guest]

Hi ;

Sorry for not replying sooner. I'm glad that you found some

explanations for your questions in the www.PFIC.org newsletter

article. Thanks for posting it.

I'd just like to say that there certainly needs to be more research

on the bile transporter genes like MDR3 in PSC. Other genes should be

looked at as well ... such as CFTR. All of the nuclear receptors that

control the expression of these genes ... such as RXR, PXR, FXR, LXR,

CAR, and PPARs ... should also be looked at closely. I believe that

PPAR-alpha/RXR are the key receptors that control MDR3.

I think that we also need to change our thinking that mutations in

these genes that might lead to susceptibility to forming " toxic "

or " agressive " bile that is capabable of damaging bile ducts, must

always be recessive ... meaning that one would have to carry two

copies of the mutant gene in order to be susceptible. In the case of

CFTR it may be that only ONE copy of the mutant allele is sufficient

to result in susceptibility when combined with other genes or

environmental triggers that cause IBD or a " leaky gut " . Case in

point ... studies on MDR3 suggest that certain mutations can be

dominant .... meaning that only ONE copy of the mutant allele is

required to lead to susceptibility to intrahepatic cholestasis of

pregnancy.

Hepatology. 2007 Jan;45(1):150-8.

Linkage between a new splicing site mutation in the MDR3 alias ABCB4

gene and intrahepatic cholestasis of pregnancy.

Schneider G, Paus TC, Kullak-Ublick GA, Meier PJ, Wienker TF, Lang T,

van de Vondel P, Sauerbruch T, Reichel C

Department of Internal Medicine I, University of Bonn, Bonn, Germany.

Intrahepatic cholestasis of pregnancy (ICP) is defined as pruritus

and elevated bile acid serum concentrations in late pregnancy.

Splicing mutations have been described in the multidrug resistance p-

glycoprotein 3 (MDR3, ABCB4) gene in up to 20% of ICP women.

Pedigrees studied were not large enough for linkage analysis. Ninety-

seven family members of a woman with proven ICP were asked about

pruritus in earlier pregnancies, birth complications and symptomatic

gallstone disease. The familial cholestasis type 1 (FIC1, ATP8B1)

gene, bile salt export pump (BSEP, ABCB11) and MDR3 gene were

analyzed in 55 relatives. We identified a dominant mode of

inheritance with female restricted expression and a new intronic MDR3

mutation c.3486+5G>A resulting in a 54 bp (3465-3518) inframe

deletion via cryptic splicing site activation. Linkage analysis of

the ICP trait versus this intragenic MDR3 variant yielded a LOD score

of 2.48. A Bayesian analysis involving MDR3, BSEP, FIC1 and an

unknown locus gave a posterior probability of >0.9966 in favor of

MDR3 as causative ICP locus. During the episode of ICP the median

gamma-glutamyl transpeptidase (gamma-GT) activity was 10 U/l (95% CI,

6.9 to 14.7 U/l) in the index woman. Four stillbirths were reported

in seven heterozygous women (22 pregnancies) and none in five women

(14 pregnancies) without MDR3 mutation. Symptomatic gallstone disease

was more prevalent in heterozygous relatives (7/21) than in relatives

without the mutation (1/34), (P = 0.00341). CONCLUSION: This study

demonstrates that splicing mutations in the MDR3 gene can cause ICP

with normal gamma-GT and may be associated with stillbirths and

gallstone disease. PMID: 17187437.

I think that this is why Trauner is suggesting taking a

closer look at MDR3 in " selected PSC subgroups, such as those

with small duct PSC (making up only ~5% of PSC cases) and younger,

female PSC patients with gallstones. "

Best regards,

Dave

(father of (21); PSC 07/03; UC 08/03)