Slide 1

Thank you very much. I would like to thank the organisers for their invitation to this very nice symposium. In the next few minutes I will present recent advances in the field of basement membrane diseases related to type IV collagen mutation.

Slide 2

Briefly, I would like to remind you that basement membranes are very specialised extracellular matrices and they are found in normal organs. Besides their structural role they also play a very important role in some physiological and pathophysiological processes. In the kidney the glomerular basement membrane plays a major role in the filtration barrier but extracellular basement membrane contributes to the activity and the function of tubules, vessels and Bowman’s capsules.

Slide 3

Basement membranes are composed of 4 main molecules type IV collagen IV, laminins, proteoglycans and entactin/nidogen. Other forms of type IV collagen and lamins display a tissue and development restricting this distribution that explain the heterogeneity of the basement membrane composition.

Slide 4

Type IV collagen is the main component of the basement membrane and 6 different alpha chains have been described. They share the same constitution with a large collagenous domain containing the classical Gly-X-Y aminoacid repeat, a small 7S domain and a non-collagenous domain at the NC1 terminal.

Slide 5

After synthesis specific interaction between the NC1 domain initiates the formation of three different trimers the α1α1α2 trimer, the α3α4α5 and the α5α5α6. Glycine residues play a very important role in the stabilisation of the collagenous-triple helix. In the extracellular space the type IV collagen networks result in the formation of NC1 hexamers, 7S domain heterotrimers and lateral interaction between helical domains.

Slide 6

The α1α1α2 trimer display is widely expressed, whereas the two other trimers display a more tissue-specific expression. In the kidney the α3α4α5 replaces the α1α1α2 in the glomerular basement membrane during development but in the basement membrane of tubules, vessels and Bowman’s capsule are mainly composed by the α1α1α2 trimers.
Each alpha chain is encoded by 6 different genes that are located head wards in three different chromosomes. As you know, many familial hematuria syndromes are related to type IV collagen mutations. The most common form of Alport syndrome is due to COL4A5 mutations while about 15% of Alport syndrome is due to an autosomal dominant or recessive mutation affecting either COL4A3 or COL4A4.
In addition about 40% of familial benign hematuria is related to defects in those genes.
To date no human diseases have been linked to COL4A defects and in the next part of my talk I will speak about human diseases related to the Col4a1 gene.

Slide 7

We know that the combined invalidation of Col4a1 and Col4a2 genes in mice used – mainly due to placental and systemic haemorrhages. However heterozygous mice display a normal phenotype thus suggesting that the lack of deleterious effect of Alport insufficiency.

Slide 8

However, heterozygous mice bearing mutations that lead to the deletion of Exon 15 display a high rate of neonatal lethality mainly due to cerebral haemorrhages.

Slide 9

In adults several animals, however develop porencephaly which is a brain cavity severe eye defect and with basement membrane abnormalities as you can see here with – thickening and focal rupture. Because of the brain phenotype in these animals authors lead to describe the first human mutation in human disease, the autosomal dominant porencephaly. This disease is characterised by the presence of brain cavities associated with brain white matter lesions and neurological signs of various severity.

Slide 10

After these first reports two French teams reported two additional mutations in families presenting with small vessel disease of the brain with leukoencephalopathy and cerebral haemorrhages but without porencephaly. In addition in this family these brain abnormalities were associated with retinal arteriolar --- which were responsible for --- visual loss due to retinal haemorrhages.

Slide 11

Interestingly in one patient an unexplained hematuria was reported. The spectrum of eye disease associated with Col4a1 mutation when extended with an additional family in which patients presented with several eye abnormalities affecting the anterior segment of the eyes and which were typical of Axenfeld-Rieger disease associating corneal abnormalities, optic nerve excavation and cataract. In this family however, patients presented with ischemic stroke and leukoencephalopathy. Interestingly, these high abnormalities were reported in Col4a1 mutant mice. All but one mutation in this family affected the glycine residue in the collagenous domain of the protein.

Slide 12

However, a wide spread excretion of the alpha 1 chain suggested that a COL4A1 mutation may lead to a more systemic phenotype than a brain-eye disease. We have reported in 3 families that presented with a systemic disease that we called HANAC for hereditary angiopathy, nephropathy, aneurysm and cramps. In this family there was also a brain disease with a leukoencephalopathy but this neurological disease was asymptomatic and only detected by brain imaging.
In addition all affected patients presenting with retinal arteriolar tortuosities and typical Raynaud phenomenon which was suggested for a systemic small vessel involvement.

Slide 13

More interestingly, unique or multiple aneurysm affecting the internal carotid artery were recorded in many patients thus suggesting that the molecular defect also affects large vessels. The systemic phenotype in HANAC syndrome associates a renal and muscular disease.

Slide 14

In the first family presented with isolated hematuria that secreted with HANAC symptoms and a renal biopsy performed in these 2 patients showed no glomerular abnormalities at light microscopy, normal distribution of the α3 and α5 type IV collagen chains and ultrastructural examination disclosed normal GBM thickness and structure.

Slide 15

In the second and the third family the renal phenotype was characterised by large bilateral cysts but without kidney enlargement. In addition, a mild GFR decrease was observed in affected patients. However, no hematuria or proteinuria was detected in these patients and because of the cystic phenotype no renal biopsy was performed in these patients.

Slide 16

The muscular defect affects all patients and was characterised by muscular cramps with persistent elevation of CPK while no muscle abnormalities were observed by light microscopy or ultrastructural examination. Because the molecular defect in HANAC affects basement membrane we have extensively analysed skin and kidney biopsies.

Slide 17

This slide shows BM abnormalities in a kidney of the two patients which were biopsied in the third family. You can see that BM showed an abnormal thickening with a multi-lamination aspect with an electron lucent area.

In addition by immunoelectron microscopy performed by – we show normal expression of the α 1 and α 2 chains except in the abnormal electron lucent area, as you can see here in the tubular basement membrane and in the interstitial capillary basement membrane.

Slide 18

In the skin the same BM abnormalities were seen with abnormal duplication at the dermal epidermal junction and in dermal vessels you can see that smooth muscle cells were dissociated by abnormal spreading of the basement membrane.

Slide 19

In HANAC families COL4A1 mutations also were responsible for glycine substitution in the collagenous domain of the protein and this glycine residue closely localised in the 30 aminoacid segments. In addition you can see for the first and the third mutation that glycine has an identical 7 aminoacid segment.

Slide 20

So many questions are raised by COL4A1 associated diseases mainly because of the phenotypic heterogeneity of the disease.

Slide 21

In the next few slides I will show you some animal data where I’ll try to explain such discrepancies. To date many Col4a1 mutant mice have been reported with different mutations and this table shows the eye phenotype and the brain phenotype. Concerning the kidney phenotype there are only mild abnormalities reported in Bru mice with Bowman’s capsule defect and in Col mice with only focal GBM duplication but without significant functional abnormalities.

Slide 22

Most interesting is the potential role of the genetic background and modifier gene in the severity of the phenotype and Gould nicely demonstrated that when crossing BL6 mice who have the severe eye phenotype with mice on different background F1 mice display only weak phenotypes and they identify the modifier locus for this strain.

Slide 23

In order to explore the role of the mutant alpha chains in the formation and the secretion of the α1α1α2 trimer α 1 and α 2 chain expression have been analysed in Col4a1 mutant mice and in C. elegans. It has been demonstrated that there was an abnormal retention of the α 1 and α 2 chains in the cells and at the same time a decreased expression in the basement membrane. Additional data suggests that the mutant alpha chain is retained in the endoplasmic reticulum and that this accumulations leads to abnormal expression of ER markers suggesting the occurrence of ER stress.

Slide 24

To conclude the Col4a1 mutation induced brain or eye restricted disease or could induce a more systemic phenotype, the HANAC syndrome which associates large and small vessel diseases, renal and muscular disease and several ultrastructural defects of the basement membrane.

Slide 25

When should nephrology search for Col4a1 mutations? In case of familial hematuria with normal GBM thickness and no deafness and in cases of multicystic kidney disease with bilateral large cysts, and normal kidney size. You then have to search for a muscular disease, a brain disease and to perform a fundoscopic examination to search for retinal arteriolar tortuosities. Because of the potential severe complications associated with cerebral aneurysms and the risk of anticoagulant therapy or trauma, we think that in suspected cases you have to search for Col4a1 mutations in patients and relatives.

Slide 26

So this HANAC story was made in Pierre Ronco’s nephrology unit and I would like to thank the patients and all the collaborators in this work. Thank you very much.

Slide 27

Chairman: Now we have time for a few questions.

Questions: A very interesting work and somehow there is only one, the α 2 mutations have not been found in any diseases yet, that’s what you said, α 2 chain mutations.

Prof. Plaisier: To date we have many families with a similar phenotype and no Col4a1 mutation and we have found in some of them Col4a2 mutation and we have not yet detected mutation but we can expect that Col4a2 mutation could produce a similar phenotype. There are most strains with Col4a2 mutation who have many a – phenotype and a – phenotype.

Question: Of course the basement membranes are everywhere and the α 1 chain is in almost all basement membranes. Did you see any changes in the intestine basement membranes?

Prof. Plaisier: We didn’t look at anything other than the skin and renal basement membrane.

Question: I was wondering with the HANAC disease syndrome what is the most severe defect in the patients, is it the kidney or is it the vasculature? Do they get insults? What limits their life?

Prof. Plaisier: The most symptomatic signs are muscular cramps but as you see, there are potential severe complications associated with cerebral aneurysm even if we haven’t observed it in our families.

Question: Can you do anything for the muscular cramps?

Prof. Plaisier: No unfortunately no.

Chairman: Thank you.