A mild form of Alport syndrome: Hereditary nephropathy in the absence of extra-renal features Yoon H-S, Wilson JC & Eccles MR Pathology, University of Otago, Dunedin, New Zealand

Alport syndrome (AS) A hereditary disorder resulting from abnormal type IV collagen Nephropathy with considerable genetic and clinical heterogeneity characterized by haematuria, proteinuria and progressive renal failure, first reported by Alport in 1927 Frequently associated with: Eye abnormalities High tone sensorineural deafness Rarely associated with: Mental retardation Leiomyomatois

Alport syndrome: genetics 85% of AS patients: X-linked inheritance of mutations in the COL4A5 gene on Xq22 encoding the a5(IV) collagen chain COL4A5 is a large gene comprising 51 exons As many as 609 mutations have been described to date and are spread throughout the gene without any hot spots (Arup Laboratory 2011) 15%: Autosomal inheritance of mutations of the COL4A3 or COL4A4 gene encoding the a3(IV) or a4(IV) on 2q36-37: 14%: autosomal recessive 1%: autosomal dominant

Type IV collagen formation 7S Collagenous NC1 a1 a1a1a2 a2 a3 a3a4a5 a4 a5 a5a5a6 a6 a(VI) chain Protomer Meshwork formation Hudson et al, NEJM 348:2543, 2003

Type IV collagen distribution a1.a1.a2(IV): Ubiquitously present in basement membrane (BM) in many tissue a3.a4.a5(IV) and a5.a5.a6(IV): Restricted tissue distribution In the kidney a1.a1.a2(IV) network predominates during early nephrogenesis in GBM. During the 2nd trimester of foetal development, a3.a4.a5(IV) network gradually becomes dominant a3.a4.a5(IV) is also expressed in the eye, cochlea, lung and testis while a5.a5.a6(IV) network is present in skin, oesophagus and smooth muscle.

Initial presentation of the NZ family Two sisters (IV6:58 yo and IV8:54 yo) presented to the clinic to be considered as potential live kidney donors for their sons (V29:27 yo and V35:33 yo, respectively) who had ESRF. Both women were found to have significant proteinuria (1.8, 1.4g/d, respectively) and hypertension and they proceeded to a renal biopsy (mild mesangial proliferation and hypertensive arteriosclerosis). Routine questioning revealed a strong familial history of hypertension and the possibility of a familial form of renal disease. Subsequent clinical review of the family identified a number of additional family members with renal disease. Negative for hearing loss or eye abnormalities in all individuals tested.

1. Subjects with renal disease identified before DNA tests Identif-ication number Age (yrs old) Gender Presentation Renal Function and Blood Pressure Biopsy Inheritance III2 F   Died on dialysis Not done Affected/ Carrier IV3 57 M ESRF. Dialysis Renal Transplant at 41 Affected IV5 46 M Proteinuria Hypertension Chronic kidney disease V24 39 Haematuria Normal renal function Mild mesangial matrix expansion V29 27 Acute nephritic syndrome 2nd renal transplant at 26 Chronic glomerulo-nephritis V31 36 BP 136/86 Mesangial cell proliferation V35 33 ESRF and renal transplant at 28 IV39 72 Proteinuria 1.6g/24 hr No haematuria Mild chronic kidney disease BP 144/76 V42 Proteinuria 1.1g/24 hr BP 126/80 Identif-ication number Age(yrs old) Gender Presentation Renal Function and Blood Pressure Biopsy Inheritance IV26 58 F Proteinuria 1.8g/24 hr Hypertension BP 152/76 Mesangial cell proliferation Hypertensive arteriosclerosis Carrier IV28 54 Proteinuria 1.4g/24 hr Hypertension Normal renal function Mesangial cell proliferation Hypertensive arteriosclerosis Dead:1, ESRF:3, Chronic disease:3

Histology of V42

IHC for a3, a4 and a5(IV) a3 a4 a5 Abs gift from Dr Sado

Diagnostic dilemma Is this Alport syndrome? No hearing or eye abnormalities Mild form of kidney disease and late onset 11 Glomerulonephritis 4 ESRF (3 males and 1 female) Low penetrance!! A new entity of hereditary kidney disease?

Extended family pedigree A total of 155 family members for 6 generations examined (81M and 74F). Black symbols: Biopsy confirmed GN (6M). Gray symbols: Clinically GN, biopsy not done (4M & 1F). Black dots: Obligate carriers (21F). White symbols: No clinical disease (71M & 73F). Cross: Confirmed by DNA testing. Predominance of GN in males and lack of male to male transmission consistent with X-linked inheritance

Family pedigree (simplified) X-chromosome microsatellite marker

Two point LOD scores between the GN locus and markers mapping to chromosome Xq21.33-23 Theta 0.01 0.05 0.1 0.2 0.3 0.4 DXS6809 -infinity -0.31 0.88 1.21 1.25 0.99 0.57 DXS6789 3.14 3.08 2.86 2.58 1.98 1.35 0.69 DXS8096 3.59 3.53 3.28 2.96 2.28 1.56 0.8 DXS1210 DXS6749 -0.61 -0.01 0.16 0.13 The linked region encompassing COL4A5

Nucleotide sequence alteration in COL4A5 in affected family members G>A substitution at nucleotide 4913 in Exon 50 (asterisk) Resulting in Cys1638Tyr Mae III digest of exon 50 PCR products A allele Present in all affected family members and not in 192 healthy control M NC Affected males Affected & carrier females

1.Renal disease identified before DNA tests 2.Renal disease/carriers identified after DNA tests Identif-ication number Age (yrs old) Gender Presentation Renal Function and Blood Pressure Biopsy Inheritance III2 F   Died on dialysis Not done Affected/ Carrier IV3 57 M ESRF. Dialysis Renal Transplant at 41 Affected IV5 46 M Proteinuria Hypertension Chronic kidney disease V24 39 Haematuria Normal renal function Mild mesangial matrix expansion V29 27 Acute nephritic syndrome 2nd renal transplant at 26 Chronic glomerulo-nephritis V31 36 BP 136/86 Mesangial cell proliferation V35 33 ESRF and renal transplant at 28 IV39 72 Proteinuria 1.6g/24 hr No haematuria Mild chronic kidney disease BP 144/76 V42 Proteinuria 1.1g/24 hr BP 126/80 Identif-ication number Age(yrs old) Gender Presentation Renal Function and Blood Pressure Biopsy Inheritance IV24 69 F Trace microscopic haematuria Normal renal function BP 168/86 Not done Carrier IV26 58 Proteinuria 1.8g/24 hr Hypertension BP 152/76 Mesangial cell proliferation Hypertensive arteriosclerosis IV28 54 Proteinuria 1.4g/24 hr Hypertension Mesangial cell proliferation Hypertensive arteriosclerosis IV31 Hypertension Negative urine IV34 65 IV36 61 Microscopic haematuria V40 38 Haematuria BP 118/60 Mild mesangial cell proliferation IV47 Negative urine BP 148/70 V44 36 Intermittent microscopic haematuria BP 120/76 V49 43 BP 120/70 V37 39

Depiction of NC1 domain showing locations of cysteine sequence alterations and clinical details of patients

Summary We report a unique cysteine to tyrosine substitution in the NC1 domain of COL4A5 in a New Zealand family who presented with a phenotypically mild form of Alport syndrome, suggesting that in this family substitution of Cys1638Tyr led to late onset renal failure without hearing loss or eye abnormalities.

Histology of female carriers (IV26) 6B

EM (carrier IV26) 150nm

EM (carriers IV26)

Summary Thin basement membrane nephropathy could be seen in some carrier women containing COL4A5 mutations.