Alport syndrome–like basement membrane changes in frasier syndrome: an electron microscopy study

Alport syndrome–like basement membrane changes in frasier syndrome: an electron microscopy study

Alport Syndrome–Like Basement Membrane Changes in Frasier Syndrome: An Electron Microscopy Study Shu-ichi Ito, MD, Hiroshi Hataya, MD, Masahiro Ikeda,...

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Alport Syndrome–Like Basement Membrane Changes in Frasier Syndrome: An Electron Microscopy Study Shu-ichi Ito, MD, Hiroshi Hataya, MD, Masahiro Ikeda, MD, Ayako Takata, MD, Haruhito Kikuchi, MD, Jun-ichi Hata, MD, Yukihiko Morikawa, MD, Sadao Kawamura, MD, and Masataka Honda, MD ● Frasier syndrome (FS) is a rare disease characterized by male pseudohermaphroditism and slowly progressing nephropathy. FS originates from heterozygous mutation in the intron 9 splicing donor site of Wilms’ tumor suppressor gene (WT1). Focal segmental glomerular sclerosis is common in FS, but there have not been so many detailed pathologic investigations. The authors examined the kidneys of 3 patients with FS. The results showed that nephropathy started as mesangial proliferative glomerulonephritis, and later a concomitant focal segmental lesion developed. In all cases, electron microscopy results showed widespread thinning, splitting, and lamellation of the glomerular basement membrane, which mimicked hereditary nephritis. Throughout adulthood, WT1 protein expresses on glomerular podocytes. Recent reports described that podocytes expressing WT1 play an important role in maintaining the glomerular basement membrane. Hereditary nephritis-like glomerular basement membrane findings in FS suggest that one of the important functions of podocytes is to form and maintain the glomerular basement membrane. Am J Kidney Dis 41:1110-1115. © 2003 by the National Kidney Foundation, Inc. INDEX WORDS: Frasier syndrome; Wilms’ tumor suppressor gene (WT1); Alport syndrome; electron microscopy; basement membrane; thinning; splitting.

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ILMS’ TUMOR suppressor gene (WT1) encodes 4 zinc finger lesions and associates with the transcriptional factor during the development of urogenital organs.1,2 Congenital mutation of WT1 causes some type of nephropathy. Exonal point mutation of WT1 causes Denys-Drush syndrome, which is characterized by diffuse mesangial sclerosis, Wilms’ tumor, and male pseudohermaphroditism.4,5 Mutations at the intron 9 splicing donor site of WT1 are found in patients with Frasier syndrome (FS),6,7 which is characterized by male pseudohermaphroditism and slowly progressive nephropathy.8 The renal manifestations usually observed in FS develop in childhood and slowly progress to

From the Yokomama City University Medical Center, Yokomama, Japan; Departments of Nephrology and Pathology, Tokyo Metropolitan Kiyose Children’s Hospital, Tokyo, Japan; Department of Pathology, Keio University School of Medicine, Keio, Japan; National Research Institute for Child Heath and Development, Tokyo, Japan; and the Department of Pathology, Toho University, School of Medicine, Tokyo, Japan. Received June 24, 2002; accepted in revised form October 10, 2002. Address reprint requests to Shu-ichi Ito, MD, Department of Pediatrics, Yokohama City University Medical Center 4-57, Urafune-cho, Minami-ku, Yokohama 232-0024, Japan. E-mail: [email protected] © 2003 by the National Kidney Foundation, Inc. 0272-6386/03/4105-0022$30.00/0 PII: S0272-6386(03)00209-9 1110

renal failure in adolescent or adult age. Some of the previous reports described that focal segmental glomerular sclerosis (FSGS) was a characteristic finding in FS. We examined the renal pathologic findings of 3 patients with FS. Electron microscopy results showed thinning, splitting, and wrinkling of glomerular basement membrane (GBM) mimicking hereditary nephritis, as in Alport syndrome (AS), in all these patients. These interesting findings suggested the unique pathogenesis of renal involvement in FS. CASE REPORT

Patient 1 Patient 1, a girl, was found to have proteinuria at the age of 3 years but had not received any medication until the age of 10. She was admitted to our hospital because of nephrotic syndrome at the age of 10 and was treated with prednisolone and cyclophosphamide, but nephrotic syndrome did not resolve. The chromosomal analysis performed at the age of 12 because of delayed puberty, found a male karyotype (46,XY). This patient had female external genitalia, but the internal genitalia were streak gonads. The results of renal biopsy performed when she was 13 years old are summarized in Table 1. Light microscopic findings showed mild mesangial proliferative glomerulonephritis but without focal segmental sclerotic lesion (Fig 1A). Many foam cells were observed in the interstitium. Mild mesan-

American Journal of Kidney Diseases, Vol 41, No 5 (May), 2003: pp 1110-1115

BASEMENT MEMBRANE CHANGES IN FRASIER SYNDROME Table 1.

Pathologic Findings

Light Microscopy

Patient, Age at the Biopsy

Patient 1, 13 y Patient 2, 10 y Patient 3 1st, 7 y 2nd, 11 y 3rd, 14 y

Mesangial Cell Proliferation

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Electron Microscopy

Immunofluorescent Staining

Thin GBM Splitting of GBM

Dense Deposit in Glomeruli

Many Many

Fibrinogen (⫾⬃⫹; mesangial) C3 (⫹⬃2⫹; GBM), IgA (2⫹; GBM)

P P

N N

N Slight Many

IgM (2⫹; GBM), IgG (⫾⬃⫹; GBM) IgM (2⫹; GBM), IgA (⫹; GBM) IgM (2⫹; GBM), C3 (⫾; GBM), Fibrinogen (2⫹; mesangial)

Not done Not done

Not done Not done

P

P

FSGS

Foam Cell in Interstitium

Mild Mild

N N

None Mild Mild to moderate

N N P

Abbreviations: P, positive; N, negative.

gial hypercellularity, widespread thinning and splicing of GBM, like in AS, were characteristic (Fig 1B and C). The width of the thin lesion of GBM was under 150 nm. Especially, the width of the lamina densa was markedly thin. Dense deposits were not observed under electron microscopy. Immunohistochemical staining for type IV collagen ␣-5 was positive. Her serum level of albumin had been between 2.0 g/dL (20 g/L) and 2.5 g/dL (25 g/L), but she never showed hematuria throughout the clinical course. This patient had FS diagnosed at the age of 19 based on the presence of a splicing donor site mutation on intron 9 of WT1(⫹4 c3t). The serum level of creatinine was about 5 mg/dL (381␮mol/L) at the age of 23. Clinical manifestations are summarized in Table 2.

Widespread thinning and splitting of the GBM like in AS, dense deposits were observed by electron microscopy. The width of the thin lesion of GBM was under 150 nm. The lamina densa was markedly thin. These findings were almost the same as those of her sister (patient 1). Immunohistochemical staining for type IV collagen ␣-5 was positive. Her serum albumin level was persistently between 2.0 g/dL (20 g/L) and 2.5 g/dL (25 g/L), but she never showed hematuria or occult blood in urine until end-stage renal failure. This patient had FS diagnosed at the age of 19 based on the presence of the same splicing donor site mutation of intron 9 of WT1 (⫹4 c3t) detected in her sister. Hemodialysis was started at the age of 20. Clinical manifestations are summarized in Table 2.

Patient 2 Patient 2 was monozygotic twin of patient 1. She was found to have proteinuria at the age of 3 years but had not been treated until she became 10 years old. She was transferred to our hospital because of nephritic syndrome at the age of 10, and a renal biopsy was performed. Chromosomal analysis was performed because of delayed puberty, the same as for patient 1, and the results showed a 46,XY karyotype. This patient had female external genitalia, but the internal genitalia were streak gonads. The results of renal pathologic findings are summarized in Table 1. Light microscopy showed mild mesangial proliferative glomerulonephritis with increased foam cells in the interstitium, but without focal segmental sclerotic lesions (data not shown). These findings were very similar to those of her sister.

Patient 3 Patient 3 was found to have proteinuria at the age of 5 years and had been placed under observation for chronic glomerulonephritis without medication. The first renal biopsy performed at the age of 7 showed abnormal changes. Nephrotic syndrome developed and was prescribed oral prednisolone. However, she showed no improvement. The second biopsy performed at the age of 11, showed mild mesangial cell proliferation and mild increase of the mesangial matrix. The first and second biopsies were not examined under an electron microscope. The third biopsy was performed when she was 14 years old. Light microscopic findings showed mild to moderate mesangial proliferation, mildly increased mesangial matrix, and many foam cells in the interstitium. Some of the glomeruli showed focal seg-

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Fig 1. (A) Photomicrograph shows mild mesangial proliferation in the glomeruli (Periodic acid methanamine [PAM] stain, original magnification ⴛ 300). (B) Electron micrograph shows widespread thinning (arrow). (Original magnification ⴛ 8,000.) (C) Electron micrograph shows splitting and lamellation (arrowheads) of the glomerular basement membrane. (Original magnification ⴛ 10,000.)

BASEMENT MEMBRANE CHANGES IN FRASIER SYNDROME Table 2.

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Clinical Manifestations

Hematuria

External and Internal Genital Status

Genotype

Mutation at the Splicing Donor Site of WT1

— — —

female, streak gonads female, streak gonads female, streak gonads

46,XY 46,XY 46,XY

intron 9 (⫹4 c 3 t) intron 9 (⫹4 c 3 t) intron 9 (⫹2 t 3 c)

Age (y) Patient No.

Proteinuria

Nephrosis

Dialysis

1 2 3

3 3 5

10 10 10

CRF 20 23

Abbreviation: CRF, chronic renal failure, but did not need dialysis.

mental sclerotic lesions (Fig 2A). Widespread thinning, splitting, and wrinkling of the GBM, like in AS, were characteristic (Fig 2B). Immuno-

Fig 2. (A) Photomicrograph shows mild mesangial proliferation and focal segmental sclerotic lesions in the glomeruli. (PAM stain, original magnification ⴛ 300.) (B) Electron micrograph shows widespread thinning of the basement membrane and effacement of foot processus of podocytes (open arrow), wrinkling (arrowhead), splitting, and lamellation (arrow) of the basement membrane (original magnification ⴛ 6,000.)

histochemical staining for type IV collagen ␣-5 was positive. The width of the thin lesion of the GBM was under 150 nm. There were a few

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subepithelial, subendothelial, and mesangial deposits, but they seemed to accumulate later. Electron microscopic findings were significantly different from the typical findings of FSGS. These renal pathologic findings are summarized in Table 1. Chromosomal analysis was performed when the patient was 18 years old because of absence of puberty and menstruation; the result was a 46,XY karyotype. A donor-splice site point mutation at the intron 9 of WT1 (⫹2 t3c) was detected at the age of 19, and FS was diagnosed. The external genitalia of this patient were those of a female, but her internal genitalia were streak gonads. At the age of 19 she underwent gonadectomy to prevent the development of a gonadoblastoma. Immunohistochemical staining for type IV collagen ␣-5 was positive. She was started on hemodialysis at the age of 23. Serum level of albumin was between 2.0 g/dL (20 g/L) and 2.5 g/dL (25 g/L), until end-stage renal failure, but she had never showed hematuria or occult blood in urine. Clinical manifestations are summarized in Table 2. DISCUSSION

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Moorthy et were the first to report FS as a syndrome characterized by male pseudohermaphroditism and nephropathy in 1987. WT1 is the gene responsible for Wilms’ tumor and plays a crucial role in the development of kidneys and gonads.1,2 Barbaux et al6 and Kikuchi et al7 reported that this syndrome originated from an intronic donor splice site mutation of WT1. The intron 9 alternative splice donor site mutation seen in FS leads to loss of 3 amino acids (⫹KTS isoform), which disrupts the normal ratio of ⫹KTS/-KTS isoforms critical for a proper gonadal and renal development.9 However, exonal point mutations of WT1 cause Denys-Drash syndrome (DDS) characterized by male pseudohermaphroditism, Wilms’ tumor and nephropathy.3 Additionally, these exonal and intronal mutations are found also in some patients with isolated diffuse mesangial sclerosis.10,11 The diffuse mesangial sclerosis observed in Denys-Drash syndrome has been well described, but no pathologic studies regarding the renal involvement of FS have been done except for those involving light microscopic examination. In some previous cases, renal involvement of this syndrome was reported as FSGS lesions.6-9 However, in our

study, pathologic examination of FS showed unique findings that differed from those of typical FSGS, as the primary nephrotic syndrome. As for light microscopic findings, mild to moderate mesangial cell proliferation and many clusters of foam cells in the interstitium were characteristic in all cases. Except for the third biopsy in patient 3, no focal segmental sclerotic lesion was observed. In patients 1 and 2, renal biopsies were performed 7 and 10 years after the onset, respectively. This fact may suggest that focal segmental sclerotic lesions developed later in the clinical course of FS. FSGS may be the consequence of persistent proteinuria. Their serum level of albumin was kept between 2.0 g/dL (20 g/L) and 2.5 g/dL (25 g/L), until end-stage renal failure. The slowly progressive clinical course and persistent mild nephrotic syndrome also differed from the typical clinical course of FSGS developed as primary nephrotic syndrome. We could not find common and characteristic immunofluorescent findings in FS. However, widespread thinning, splitting, or wrinkling of the GBM were found in our 3 patients. The width of the thin lesion of GBM was under 150 nm in all of them. There have been no previous reports of electron microscopy findings in FS. All concerned pathologists thought that the changes were similar to those of hereditary nephritis, like in AS or thin GBM disease,12 but immunohistological staining of type IV collagen ␣-5 was positive in all cases. It is interesting that all of our patients showed no hematuria, because most of the patients with AS or thin GBM disease usually develop hematuria from the early stage of the diseases. From the result of immunohistologic staining and its clinical course, there are some structural differences of GBM between FS and AS or thin GBM disease. Subepithelial deposits were focally seen in patient 3, but it should be noticed that those deposits accumulated after the change of the basement membrane. Some double track formations were observed under light microscopy in all patients; this finding might be compatible with the splitting of the GBM. After birth, WT1 protein expresses only in glomerular podocytes13 and plays an important role in maintaining the differentiation of adult podocytes.14 Additionally, some animal models of glomerulitis showed that damage of podocytes induces glomerulosclerosis.15,16 All our patients

BASEMENT MEMBRANE CHANGES IN FRASIER SYNDROME

showed the GBM change like AS, but AS rarely developed to FSGS. WT1 mutation on podocytes may relate to the structural change in GBM, but persistent proteinuria and nephritic condition rather than the GBM change itself may induce the slowly progressive segmental glomerular sclerosis. The pathologic change of GBM similar to that found in hereditary nephritis in FS suggests that one of the important functions of podocytes is to form and maintain the GBM. More cases of FS should be examined for renal involvement from the early stage to clarify the pathogenesis of FS and the role of podocytes and WT1 in forming and maintaining the GBM. REFERENCES 1. Gessler M, Poutska A, Cavanee W, Neve RL, Orkin SH, Bruns GAP: Homozygous deletion in Wilms’ tumors of a zinc finger gene identified by chromosome jumping. Nature 343:774-778, 1990 2. Call KM, Glaser T, Ito CY, Buckler AJ, Pelletier J, Haber DA: Isolation and characterization of zinc finger polypeptide gene at the human chromosome 11 Wilms’ tumor locus. Cell 60:509-520, 1990 3. Pelletier J, Bruening W, Kashtan CE, et al: Germline mutations in the Wilms’ tumor suppressor gene are associated with abnormal urogenital development in Denys-Drash syndrome. Cell 67:437-447, 1992 4. Denys P, Malvaux P, Berghe HVD, Tanghe W, Proesmans W: Association d’un syndrome anatomopathologique de pseudohermaphrodisme masculin, d’une tumeur de Wilm’s, d’une nephropahtie parenchymateuse et d’un mosaicisme XX/XY. Arch Fr Pediatr 24:729-739, 1967 5. Drash A, Sherman F, Hartman WH, Blizzard RM: A syndrome of pseudohermaphroditism, Wilm’s tumor, hypertension and degenerative renal disease. J Pediatr 76:585593, 1970

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6. Barbaux S, Niaudet P, Gubler MC, et al: Donor splicesite mutations in WT1 are responsible for Frasier syndrome. Nat Genet 17:467-701, 1997 7. Kikuchi H, Takata A, Akasaka Y, et al: Do intronic mutations affecting splicing of WT1 exon 9 cause Frasier syndrome. J Med Genet 35:45-48, 1998 8. Moorthy AV, Chesney RW, Lubinsky M: Chronic renal failure and XV gonadal dysgenesis; “Frasier” syndrome — A commentary on reported cases. Am J Med Genet 3:397302, 1987 (suppl) 9. Klamt B, Koziell A, Poulat F, et al: Frasier syndrome is caused by defective alternative splicing of WT1 leading to an altered ratio of WT1⫹/KTS splice isoforms. Hum Mol Genet 7:709-714, 1998 10. Jeanpierre C, Denamur E, Henry I, et al: Identification of constitutional WT1 mutations, in patients with isolated diffuse mesangial sclerosis, and analysis of genotype/ phenotype correlations by use of a computerized mutation database. Am J Hum Genet 62:824-33, 1998 11. Ito S, Takata A, Hataya H, et al: Isolated diffuse mesangial sclerosis and Wilms tumor suppressor gene. J Pediatr 138:425-427, 2001 12. Rumpelt HJ: Alport’s syndrome: Specificity and pathogenesis of glomerular basement membrane alterations. Pediatr Nephrol 1:422-7, 1987 13. Mundlos S, Pelletier J, Darveau A, Bachmann M, Winterpacht A, Zabel B: Nuclear localization of the protein encoded by the Wilms’ tumor gene WT1 in embryonic and adult tissues. Development 119:1329-1341, 1993 14. Mundel P, Reiser J, Kriz W: Induction of differentiation in cultured rat and human podocytes. J Am Soc Nephrol 8:697-705, 1997 15. Kretzler M, Koeppen-Hagemann I, Kriz W: Podocyte damage is a critical step in the development of glomerulosclerosis in the uninephrectomised-desoxycorticosterone hypertensive rat. Virchows Arch 425:181-193, 1994 16. Shirato I, Hosser H, Kimura K, Sakai T, Tomino Y, Kriz W: The development of focal segmental glomerulosclerosis in masugi nephritis is based on progressive podocyte damage. Virchows Arch 429:255-273, 1996