Alport syndrome and thin glomerular basement membrane nephropathy: a practical approach to diagnosis.
Abstract: * Context.--Alport syndrome and thin glomerular basement membrane nephropathy (TBMN) are genetically heterogenous conditions characterized by structural abnormalities in the glomerular basement membrane and an initial presentation that usually involves hematuria. Approximately 40% of patients with TBMN are heterozygous carriers for autosomal recessive Alport syndrome, with mutations at the genetic locus encoding type IV collagen [[alpha].sub.3] [[[alpha].sub.3](IV)] and [[alpha].sub.4] chains. However, although the clinical course of TBMN is usually benign, Alport syndrome, particularly the X-linked form with mutations in the locus encoding the [[alpha].sub.5] chain of type IV collagen [[[alpha].sub.5](IV)], typically results in end-stage renal disease. Electron microscopy is essential to diagnosis of TBMN and Alport syndrome on renal biopsy, although electron microscopy alone is of limited value in distinguishing between TBMN, the heterozygous carrier state of X-linked Alport syndrome, autosomal recessive Alport syndrome, and even early stages of X-linked Alport syndrome.

Objectives.--To review diagnostic pathologic features of each of the above conditions, emphasizing the need for immunohistology for [[alpha].sub.3](IV) and [[alpha].sub.5](IV) in addition to electron microscopy to resolve this differential diagnosis on a renal biopsy. The diagnostic value of immunofluorescence studies for [[alpha].sub.5](IV) on a skin biopsy in family members of patients with Alport syndrome also is reviewed.

Data Sources.--Original and comprehensive review articles on the diagnosis of Alport syndrome and TBMN from the past 35 years, primarily the past 2 decades, and experience in our own renal pathology laboratory.

Conclusions.--Although Alport syndrome variants and TBMN do not show characteristic light microscopic findings and can be difficult to differentiate from each other even by electron microscopy, using a combination of electron microscopy and immunohistology for [[alpha].sub.3](IV) and [[alpha].sub.5](IV) enables pathologists to definitively diagnose these disorders on renal biopsy in most cases.
Article Type: Report
Subject: Alport's syndrome (Diagnosis)
Alport's syndrome (Genetic aspects)
Gene mutations (Research)
Gene mutations (Physiological aspects)
Fluorescent antibody technique (Usage)
Immunofluorescence (Usage)
Pathology (Practice)
Pathologists (Practice)
Author: Haas, Mark
Pub Date: 02/01/2009
Publication: Name: Archives of Pathology & Laboratory Medicine Publisher: College of American Pathologists Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2009 College of American Pathologists ISSN: 1543-2165
Issue: Date: Feb, 2009 Source Volume: 133 Source Issue: 2
Topic: Event Code: 310 Science & research; 200 Management dynamics
Accession Number: 230151958
Full Text: Alport syndrome and thin glomerular basement membrane nephropathy (TBMN) are genetically heterogenous conditions characterized by structural abnormalities in the glomerular basement membrane (GBM). Both conditions typically present with hematuria, with the course of Alport syndrome usually manifested by increasing proteinuria and progressive renal insufficiency developing at varying ages, ranging from childhood to early or even middle-aged adulthood, although this age is fairly constant within affected members of the same family. (1-3) Patients with Alport syndrome often, although not always, exhibit systemic manifestations, the most common of which are hearing impairments (1,4,5) and ocular defects, most typically anterior lenticonus. (1,4,6,7)

Approximately 85% of cases of Alport syndrome are due to various mutations in the COL4A5 gene, located on the X chromosome, which encodes the [[alpha].sub.5] chain of type IV collagen [[[alpha].sub.5](IV)]. (8) Inheritance of this mutation is X linked, with progressive and ultimately end-stage renal disease in affected males but only urinary abnormalities (hematuria with or without mild proteinuria, which may increase with age) in heterozygous females. (4,9,10) Although most patients with X-linked Alport syndrome will have a known family history of renal disease, 10% to 15% of cases appear to represent de novo mutations in the COLA5 gene. (11)

The remaining cases of Alport syndrome result from mutations at the COL4A3/COL4A4 locus on chromosome 2, encoding the [[alpha].sub.3] and [[alpha].sub.4] chains of type IV collagen [[[alpha].sub.3](IV) and [[alpha].sub.4](IV)], with autosomal recessive (12-14) or, much less commonly, autosomal dominant (15-17) inheritance. Unlike the case with X-linked Alport syndrome, which in affected males is nearly always a progressive disease leading to end-stage renal disease, the phenotypic expression of autosomally transmitted Alport syndrome is quite variable, ranging from benign urinary abnormalities to progressive nephropathy leading to end-stage renal disease. (14,17) Likewise, ultrastructural abnormalities in the GBM may be less severe in autosomal recessive than in X-linked Alport syndrome, with a predominance of GBM thinning (resembling changes seen in TBMN or heterozygous carriers of X-linked Alport syndrome) rather than extensive splitting and lamellation. (18,19) It is thus possible that some patients reported to have TBMN who developed hypertension, proteinuria, and late-onset renal insufficiency (20) may have instead had autosomal recessive Alport syndrome with homozygous or compound heterozygous COL4A3/COL4A4 mutations.

Approximately 40% of patients with TBMN have heterozygous mutations at the COL4A3/COL4A4 locus, and thus can be considered carriers for autosomal recessive Alport syndrome. (21-25) In addition, 2 such mutations (G1334E and G871C) were identified recently as being associated with a form of TBMN characterized by development of focal segmental glomerulosclerosis and proteinuria after age 30 years and renal insufficiency after age 50 years.25 This further blurs the once-distinct line between TBMN, a disease often (although not necessarily correctly) equated with the clinical syndrome of benign familial hematuria, and Alport syndrome, which carries a far more ominous prognosis. Thin glomerular basement membrane nephropathy appears to be far more common than Alport syndrome, occurring in approximately 1% of the population compared with 0.02% for Alport syndrome. (11,26) Still, as noted above, there is considerable phenotypic overlap between individuals, particularly children and young adults, with TBMN, the heterozygous carrier state of X-linked Alport syndrome, autosomally transmitted Alport syndrome, and even some children with X-linked Alport syndrome. This article reviews the diagnostic pathologic features of each, emphasizing the need for immunohistology for [[alpha].sub.3](IV) and [[alpha].sub.5](IV) in addition to electron microscopy (EM) to resolve this differential diagnosis.

DETERMINATION OF GBM THICKNESS

The diagnosis of TBMN first and foremost requires the demonstration of diffusely thin GBMs by EM. If these are found, it is then necessary to rule out Alport syndrome (including heterozygous X linked) by immunofluorescence studies for [[alpha].sub.3](IV) and [[alpha].sub.5](IV).

There are no standard criteria for defining the lower limit for normal GBM thickness below which the GBMcan be considered thin, and there is considerable variability between the values established as this lower limit at different centers. Much, although not all, of this variability is methodologically related. Dische, (27) using the orthogonal intercept/mean harmonic thickness method of GBM measurement, (28) established a normal range of GBM thickness in adults of 330 to 460 nm, and they defined thin GBM nephropathy as an average GBM thickness of less than 330 nm. Using similar methodology, Tiebosch et al (29) determined the lower limit of the normal range of GBM thickness to be 264 nm. This method of GBM thickness measurement is illustrated in Figure 1. Electron micrographs of random capillaries are first taken around the periphery of glomeruli. Electron microscopy prints (made at a final magnification of X12 000 in the study of Dische (27)) are then overlaid with a clear plastic 4-[cm.sup.2] grid, and GBM width (distance between endothelial and podocyte plasma membranes) is recorded in "classes." The latter are whole integers (1-9) based on measurements made using a logarithmic ruler, as shown in Figure 1, wherever grid lines intersect the endothelial cell plasma membrane, not including mesangial areas. In the study of Dische, (27) 9 prints were examined per biopsy, and the total number of such intersections per biopsy ranged from 164 to 315 (mean, 247). Notably, all intersections are included (Figure 1, arrows), including those made at oblique angles, thus making GBM thickness measurements by this method totally random. Once all measurements are made, the mean harmonic thickness of the GBM is calculated using a set of equations (27,28) that makes allowances for oblique sectioning. The advantages of this method, in addition to truly random sampling of the GBM, are that it yields a normal distribution of thicknesses and that highly repeatable results are obtained when the same glomerulus is rephotographed. (27) Until recently, a major disadvantage to the method is that it was extremely time consuming, probably too much so to be used in most diagnostic pathology laboratories. However, with today's digital cameras and appropriate software, this is much less of an issue. Still, a true understanding of the method requires a considerable background in mathematics, which many if not most pathologists (including the author of this review) do not have.

[FIGURE 1 OMITTED]

An alternative to the above method involves direct measurement of GBM thickness (distance from endothelial to podocyte plasma membrane) and determination of the arithmetic mean of such measurements. Such a method is easily applied in diagnostic laboratories without a specialized camera or software, although it excludes obliquely sectioned areas of the GBM from measurement and tends to yield lower normal ranges of GBM thickness than the orthogonal intercept/mean harmonic thickness method. (26,30-32) Das et al (31) found that if 16 measurements from each of 2 glomeruli were made using this direct measurement/ arithmetic mean method, the results were equivalently reproducible to those obtained using the orthogonal intercept/mean harmonic thickness method, although the by approximately 40% using the latter method. (31)

[FIGURE 2 OMITTED]

We have applied a modification of the direct measurement/ arithmetic mean method to measure GBMthickness in our laboratory, and in addition we used this method to establish normal ranges of GBM thickness for adult males and females and to estimate the frequency of incidental TBMN in our renal biopsy population. Basically, the method is as follows (26):

1. Electron micrographs with print magnifications of X20 000 to X32 500 (original magnifications of X8000 to X13 000) are taken of multiple capillaries from 1 to 2 glomeruli.

2. A total of 30 GBM measurements are made on 10 randomly selected capillaries to determine an average (arithmetic mean) GBM thickness for that biopsy. For capillary loops with the full circumference represented (Figure 2, A), the mesangium is taken to represent 6 o'clock, and measurements of GBM thickness (see below) are made at the 3 points of nonoblique sectioning of the GBM closest to 3 o'clock, 9 o'clock, and 12 o'clock, similar to the procedure described by Meleg-Smith et al. (10) Areas of GBM notching are avoided. For other capillaries, measurements are made at the 2 points of nonoblique GBM sectioning closest to the edges of the photograph or to the interface with the mesagium (but not including the actual mesangial portion of the GBM) and farthest from each other (Figure 2, B). A third measurement then is made at the point of nonoblique GBM sectioning closest to the midpoint (with respect to GBM length) between the above 2 points.

Direct measurements of GBM thickness are made using a X10 eyepiece magnifier with gradations of 0.1 mm to measure the distance between the endothelial and podocyte plasma membranes. This value then is divided by the print magnification to obtain the GBM thickness.

Using this method we obtained mean [+ or -] SD values for the normal GBM thickness of males and females aged 9 years or older of 330 [+ or -] 50 nm and 305 [+ or -] 45 nm, respectively. These values were based on average GBM thicknesses for each of 50 males and 50 females, aged 9 to 80 years, with minimal change nephropathy or acute interstitial nephritis without hematuria.26 Normal ranges for each sex (230-430 nm for males, 215-395 nm for females) were defined as being within 2 SD of these means. These normal ranges are similar to those determined by others using a similar methodology. (30-33) Excluding biopsies performed specifically because of hematuria and those with immunoglobulin (Ig) A nephropathy (which is known to be frequently associated with thin GBMs (34,35)), diabetic nephropathy (which is associated with GBM thickening (36)), and Alport syndrome, we found the frequency of incidentally discovered TBMN in our biopsy population to be 0.9%. (26)

Based on the data of Morita et al, (37) who found that GBM thickness in children increases almost linearly with increasing age between 1 and 9 years and then plateaus, we did not make any adjustments of GBM thickness for age in patients 9 years or older. To determine a lower limit for normal GBM thickness at ages younger than 9 years, the following calculation was made, based on the assumptions that: (1) the rate of increase in GBM thickness is linear up to the age of 9 years; (2) the ratio of the lower limit of normal GBM thickness to mean normal GBM thickness remains constant from birth; and (3) mean GBM thickness at birth is 194 nm for both males and females (extrapolated from the data of Morita et al (37)). Thus, the estimated lower limit for GBM thickness for males at birth is (194/ 330 X 230) nm = 135 nm, based on a mean normal GBM thickness of 194 nm at birth and of 330 nm at 9 years or older, and a lower limit of normal GBM thickness of 230 nm in males 9 years or older. The estimated lower limit for GBM thickness for males aged Y years then would be (Y/9 X [230 - 135]) nm + 135 nm. The estimated lower limit for GBM thickness for females at birth is (194/305 X 215) nm = 137 nm, based on a mean normal GBM thickness of 194 nm at birth and of 305 nm at 9 years or older, and a lower limit of normal GBM thickness of 215 nm in females 9 years or older. The estimated lower limit for GBM thickness for females aged Z years then would be (Z/9 X [215 - 137]) nm + 137 nm. (26) Table 1 shows estimated normal ranges for GBM thickness in males and females aged 1 to 9 years based on these calculations and similar calculations for upper limits of normal GBM thickness (Table 1 footnote). The ranges listed in Table 1 and their midpoints are in good agreement with the results of direct measurements of GBM thickness in children made using similar methodologies. (37,38)

DIAGNOSIS OF TBMN

Most experienced renal pathologists are able to discriminate between GBMs of normal thickness (Figure 3, A) and those that are clearly thin (Figure 3, B), so in most cases it is not necessary to perform GBM measurements to rule out a diagnosis of TBMN, particularly when this is not suspected clinically. However, to make a diagnosis of TBMN, we require that each of the following 4 criteria are met (25):

1. The average of the 30 measurements of GBM thickness is below the lower limit of the normal range for the patient's sex and age, as specified above.

2. Fifty percent or more of the individual GBM measurements are below the lower limit of the normal range for the patient's sex and age. Although GBM thinning may be somewhat segmental in some individuals, (39,40) there is presently general agreement among renal pathologists that thinning should involve at least 50% of the GBM for a diagnosis of TBMN to be made. (33)

3. Widespread splitting or lamellation of the GBM is not present, although very localized splitting or lamellation (Figure 3, C) does not rule out TBMN. (19) The maximum amount of GBM splitting or lamellation that can be observed in TBMN has not been systematically studied, although in our laboratory we have not observed any biopsies showing splitting/lamellation involving an estimated 5% or more of the total GBM length examined without clinical, familial, and/or immunohistologic evidence of Alport syndrome.

4. Indirect imunofluorescence (IF) studies for [[alpha].sub.3](IV) and [[alpha].sub.5](IV) (see below) show no evidence for Alport syndrome.

Because TBMN occurs in approximately 1% of the general population, (26) one occasionally encounters cases of other glomerular diseases superimposed on TBMN. (26,41,42) Patients with minimal-change nephropathy or primary focal segmental glomerulosclerosis superimposed on TBMN present with heavy proteinuria as well as persistent hematuria. (42) Distinguishing TBMN with superimposed primary focal segmental glomerulosclerosis from secondary focal segmental glomerulosclerosis developing in the context of certain forms of TBMN (eg, with some specific heterozygous COL4A3/COL4A4 mutations (25)) represents a major diagnostic challenge and may not be possible in some cases, (43) although with the latter, significant proteinuria does not appear to occur before age 30 years, and renal insufficiency is uncommon before age 50 years. (25)

It may also be extremely difficult if not impossible to perform GBM measurements in some cases where there is an immune complex-related glomerular disease, particularly membranous nephropathy, membranoproliferative glomerulonephritis, or fibrillary glomerulonephritis. We do not attempt to diagnose TBMN in biopsies showing the latter 2 lesions, advanced membranous nephropathy, or diabetic nephropathy. However, with relatively early membranous lesions we select for measurement the points of nonoblique GBM sectioning not containing or directly adjacent to a deposit that are closest to those sites that would be designated for measurement by the method detailed above. (26) We and others (43) also do not make a diagnosis of TBMN in cases of IgA nephropathy. Our rationale for this is that GBM abnormalities, most often thinning, are seen in as many as 35% to 50% of cases of IgA nephropathy. (34,35) Furthermore, although patients with IgA nephropathy and thin GBMs were found to have a significantly higher incidence of gross hematuria than patients with IgA nephropathy and normal GBM thickness, other parameters more closely associated with clinical outcomes, including proteinuria, hypertension, and renal insufficiency at the time of biopsy, were not significantly different between the 2 groups. As such, we do not feel that patients with IgA nephropathy and thin GBMs warrant a separate diagnosis or imunofluorescence studies for [[alpha].sub.3](IV) and [[alpha].sub.5](IV) to rule out Alport syndrome. More severe GBM changes in IgA nephropathy,

including lamellation, irregular thickening and thinning, disruption, and partial lysis are associated with subendothelial and/or subepithelial immune complex deposits, and also tend to be associated with more severe histologic glomerular lesions (crescents, diffuse mesangial hypercellularity). (44,45) If a biopsy with IgA nephropathy shows true GBM splitting/ lamellation that is not clearly associated with subepithelial and/or subendothelial deposits, we will perform imunofluorescence studies for [[alpha].sub.3](IV) and [[alpha].sub.5](IV) to rule out Alport syndrome.

[FIGURE 3 OMITTED]

At our center, it is standard procedure to perform indirect IF staining for [[alpha].sub.1](IV) (as a general control to assure that staining is working), [[alpha].sub.3](IV), and [[alpha].sub.5](IV) on all renal biopsies with clinical and/or EM findings suggestive of TBMN or Alport syndrome. Staining is performed on crysostat sections using the primary mouse monoclonal antibodies fromWieslab AB (Lund, Sweden) and fluorescein isothiocyanate-conjugated goat anti-mouse IgG (ICN/Cappel, Aurora, Ohio), according to directions supplied by Wieslab. The 3 primary antibodies are sold together as a kit, and use of all 3 antibodies allows the pathologist to discriminate TBMN and the different forms of Alport syndrome in most cases. A positive control (a portion of normal renal cortex from a nephrectomy specimen with carcinoma that is stored at -70[degrees]C) is included with all cases. Although the instructions provided by Wieslab with their antibody kit describe a method for performing immunoperoxidase staining of paraffin sections of formalin-fixed tissue using these same primary antibodies, we have not been able to obtain satisfactory results with these antibodies on such sections, despite trying several antigen retrieval methods. Therefore, our practice when triaging renal biopsies from patients with unexplained hematuria is to, whenever possible, freeze a generous sample of cortical tissue for IF studies.

Figure 4, A, illustrates the normal staining pattern of renal cortex for [[alpha].sub.5](IV) with diffuse, linear staining of the GBM and the basement membrane of Bowman capsule, and focal linear tubular basement membrane staining, representing staining of the distal tubular basement membrane. Although not shown, an identical staining pattern is seen for [[alpha].sub.3](IV). This staining pattern for both [[alpha].sub.3](IV) and [[alpha].sub.5](IV) is required for a diagnosis of TBMN in our laboratory. The rationale for performing such staining on all cases of suspected TBMN is that heterozygous carriers of X-linked Alport syndrome (Figure 4, B), individuals with autosomal recessive Alport syndrome (Figure 4, C), and even some children with X-linked Alport syndrome (Figure 4, D) may show predominantly thin GBMs with little or no splitting or lamellation by EM. (1,18,19)

If faced with a biopsy from a patient with unexplained hematuria containing renal cortical tissue with no glomeruli or only globally sclerotic glomeruli on the tissue collected for IF, we will proceed with indirect IF staining of this tissue for [[alpha].sub.3](IV) and [[alpha].sub.5](IV) in the event that (1) EM studies show GBM abnormalities suggestive of TBMN or Alport syndrome or they cannot be done due to an inadequate sample, and (2) IF studies on paraffin sections do not show findings of IgA nephropathy. Assessment of staining of cortical tubular basement membranes for [[alpha].sub.3](IV) and [[alpha].sub.5](IV) may be diagnostically useful even in the absence of glomeruli, and in addition it has been our limited experience that sclerotic glomeruli from non-Alport syndrome patients often retain staining for [[alpha].sub.3](IV) and [[alpha].sub.5](IV), at least to an extent sufficient to rule out homozygous X-linked Alport syndrome (see below).

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

Our laboratory presently does not perform staining for [[alpha].sub.4](IV), and overall experience with staining for [[alpha].sub.4](IV) is much less than with [[alpha].sub.3](IV) and [[alpha].sub.5](IV). Still, there are uncommon forms of Alport syndrome linked to mutations in the COL4A4 gene encoding [[alpha].sub.4](IV),15,16 and as such in a patient whose renal biopsy shows EM findings suspicious for Alport syndrome but normal IF staining for [[alpha].sub.3](IV) and [[alpha].sub.5](IV), testing for a COL4A4 mutation may be indicated.

DIAGNOSIS OF DIFFERENT FORMS OF ALPORT SYNDROME

The most distinctive light microscopic finding in Alport syndrome is the presence of lipid-laden interstitial foam cells. (11,46-48) These cells are most often clustered in the deep cortex, and when abundant may be seen in linear rows and nests that correspond to yellow streaks seen grossly in autopsy specimens. (46) Still, such cells are absent in a considerable fraction of biopsies, including most early biopsy specimens. In a total of 97 renal specimens (84 biopsies, 13 autopsies or nephrectomies) from Alport syndrome patients aged 2 to 41 years and pooled from 3 separate studies, (46-48) interstitial foam cells were seen in only 39 (40%). Foam cells were most commonly seen in specimens from older children and adults that also showed chronic changes such as glomerular sclerosis, tubular atrophy, and interstitial fibrosis. (46-48) Furthermore, in 1 pediatric study proteinuria of 40 mg/h/[m.sup.2] or higher was seen in 9 (75%) of 12 Alport patients whose biopsies showed interstitial foam cells, but only 6 (24%) of 25 such patients whose biopsies lacked these cells. (48) Interstitial foam cells are certainly not specific for Alport syndrome, because these may be observed in other glomerular diseases characterized by persistent heavy proteinuria,11 although they can be a helpful feature in the diagnosis of Alport syndrome in those patients with little or no proteinuria.

The classic ultrastructural picture of Alport syndrome is characterized by alternating zones of thinning and thickening of the GBM (the former tending to predominate earlier, and the latter in more advanced lesions, particularly in males with X-linked disease), splitting and lamellation of the GBM with loss of the normal lamina densa, small granules within the GBM, and an irregular outer contour of the GBM (4,9,11,48-50) (Figure 5, A through C). Still, as noted above, not all individuals with Alport syndrome will show all of these characteristic findings, and even among affected individuals of the same family there can be considerable ultrastructural variability. (51) Furthermore, GBM splitting with variable thickness and an irregular outer contour is not specific for Alport syndrome, as similar changes may be seen with hyperfiltration injury,52 chronic lesions of membranous nephropathy, postinfectious glomerulonephritis where the immune complex deposits have been extensively resorbed, (53) and as noted above, IgA nephropathy. (35) As such, the combined use of EM and immunofluorescence studies for [[alpha].sub.3](IV) and [[alpha].sub.5](IV) adds both sensitivity and specificity to the diagnosis of Alport syndrome, and by using both diagnostic tools it is possible to diagnose Alport syndrome in more than 90% of individuals with COL4A5 mutations. (51)

[FIGURE 6 OMITTED]

Figure 4 illustrates staining patterns for [[alpha].sub.5](IV) in different forms of Alport syndrome, which are also summarized in Table 2. X-linked heterozygotes show a discontinuous or mosaic pattern, with alternating positive and negative staining areas of the GBM and of Bowman capsule (Figure 4, B). In X-linked Alport syndrome there is complete absence of staining for [[alpha].sub.5](IV) in the GBM, Bowman capsule, and tubular basement membranes (Figure 4, D). In each of these latter 2 instances, staining for [[alpha].sub.3](IV) mirrors that for [[alpha].sub.5](IV).9 In patients with autosomal recessive Alport syndrome (homozygous or compound heterozygous mutations), there is a complete absence of staining for [[alpha].sub.3](IV) as well as an absence of GBM staining for [[alpha].sub.5](IV), although [[alpha].sub.5](IV) staining in Bowman capsule and distal tubular basement membranes is preserved (Figure 4, C). There are, however, a small number of Alport syndrome kindreds where staining for [[alpha].sub.3](IV) and [[alpha].sub.5](IV) remains normal, and as such, a normal immunofluorescence result cannot completely rule out either X-linked or autosomal recessive Alport syndrome. (9)

THE DIAGNOSTIC VALUE OF SKIN BIOPSY IMMUNOFLUORESCENCE STUDIES

A number of investigators have demonstrated the usefulness of immunofluorescence for [[alpha].sub.5](IV) as a marker for X-linked Alport syndrome and its heterozygous carrier state. (9,54-57) As shown in Figure 6, A, there is normally diffuse, linear staining of the epidermal basement membrane (EBM) for [[alpha].sub.5](IV). Small areas of discontinuity of staining, mainly near the bottoms of dermal papillae, may be present normally.57 However, discontinuity of [[alpha].sub.5](IV) staining is far more pronounced in many heterozygous carriers of X-linked Alport syndrome (Figure 6, B), and [[alpha].sub.5](IV) staining along the EBM is completely absent in many male patients with X-linked Alport syndrome (Figure 6, C). Still, in different studies 30% to 55% of males and heterozygous females with COL4A5 mutations exhibited normal EBM staining for [[alpha].sub.5](IV). (56,57) Immunofluorescence studies of skin biopsies, which are far less invasive than renal biopsies, can therefore be a very useful diagnostic tool for testing individuals suspected of having COL4A5 mutations, such as family members of known X-linked Alport syndrome patients. However, although the value of skin biopsy in such testing is great when IF staining for [[alpha].sub.5](IV) is abnormal, it is quite limited when this staining is normal. Furthermore, as [[alpha].sub.3](IV) is normally absent from the EBM, skin biopsy is of no value for diagnosis of autosomal recessive Alport syndrome, with EBM staining for [[alpha].sub.5](IV) being normal in these individuals. (56,58)

SUMMARY

This article intends to summarize diagnostic methods available to the pathologist for the diagnosis of TBMN and different forms of Alport syndrome. For reviews of the genetic and clinical aspects of these conditions, the reader is directed elsewhere. (4,8-10,22) None of these conditions show diagnostic light microscopic findings and, as discussed, they can be difficult to differentiate from each other, even by EM. However, using a combination of EM and immunohistology for [[alpha].sub.3](IV) and [[alpha].sub.5](IV) enables us to definitively diagnose these disorders in most cases.

Accepted for publication December 18, 2007.

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Mark Haas, MD, PhD

From the Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Md.

The author has no relevant financial interest in the products or companies described in this article.

Reprints: Mark Haas, MD, PhD, Department of Pathology, Johns Hopkins University School of Medicine, 600 N Wolfe St, Pathology 712, Baltimore, MD 21287 (e-mail: mhaas@jhmi.edu).
Table 1. Estimated Normal Ranges of Glomerular
Basement Membrane (GBM) Thickness in Children *

              Normal Range of GBM Thickness, nm
Age,
[gamma]       Males     Females

Birth         135-253   137-251
1             146-273   146-267
2             156-292   154-283
3             167-312   163-299
4             177-332   172-315
5             188-351   180-331
6             198-371   189-347
7             209-391   198-363
8             219-410   206-379
9 and older   230-430   215-395

* These ranges of GBM thickness are based on measurements made
in our laboratory using the modification of the direct measurement/
arithmetic mean method described in the text, and estimated lower
limits of GBM thickness were calculated for each age using the
equations provided in the text. Similarly, the upper limits of GBM
thickness at birth were estimated to be 194/330 x 430 nm = 253 nm
for males, and 194/305 x 395 nm = 251 nm for females. Estimated upper
limits of GBM thickness for males of age Y and females of age Z were
calculated as (Y/9 x [430-253]) nm + 253 nm and (Z/9 x [395-251])
nm + 251 nm, respectively.

Table 2. Staining for [[alpha].sub.3](IV) and [[alpha].sub.5](IV) in
Thin Glomerular Basement Membrane Nephropathy (TBMN) and Alport
Syndrome Variants *

                        [[alpha].sub.3](IV)           [[alpha].sub.5]
                                                      (IV)

                        GBM       BC        TBM       GBM

Normal/TBMN             +         +         +         +
Alport variants
([dagger])
  X-linked carrier      Discont   Discont   Discont   Discont
  (heterozygote)
  X-linked male         -         -         -         -
  Autosomal recessive   -         -         -         -

                        [[alpha].sub.5](IV)

                        BC        TBM       EBM

Normal/TBMN             +         +         +
Alport variants
([dagger])
  X-linked carrier      Discont   Discont   Discont
  (heterozygote)                            ([double dagger])
  X-linked male         -         -         -
                                            ([double dagger])
  Autosomal recessive   -         -         -

* GBM indicates glomerular basement membrane; BC, Bowman capsule;
TBM, distal tubular basement membrane; EBM, epidermal basement
membrane; and Discont, discontinuous staining (mosaic pattern).

([dagger]) Infrequent exceptions to these patterns have been noted on
renal biopsies (Kashtan9; also see text).

([double dagger]) Up to approximately 50% of individuals in each of
these categories will show normal staining for [[alpha].sub.5](IV) on
skin biopsy (Patey-Mariaud de Serre et al (57); also see text).
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