Sporadic Case of Heterozygous X-Linked Alport Syndrome

A 3-year-old girl with no significant past medical history was referred to UCLA Children’s Health Center for further evaluation of tea color urine for the prior 2 months. She was initially evaluated at her pediatrician office, and urinalysis revealed 3+ hematuria and proteinuria (random urine protein creatinine ratio around 0.6 mg/mg creatinine). Vital signs included normal blood pressure of 97/65 mm Hg, pulse of 110 beats/min, BMI of 14.94 kg/m² (63rd percentile for age). A physical examination was unremarkable. Her laboratory workup demonstrated normal kidney function (estimated glomerular filtration rate 113 mL/min/1.73 m²) [1]. Urinalysis showed occult blood (3+), >210 RBCs/high-power field and a protein/creatinine ratio of 0.7 mg/mg creatinine. An extensive serological workup was unrevealing (Table 1). Due to concern for possible nutcracker syndrome, renal ultrasound with Doppler was obtained and it demonstrated no significant abnormalities. She had a febrile illness during the time of her work up with development of transient gross hematuria. No other history of fevers, cough, sore throat, rashes, urinary tract infections, flank pain, extremity, or facial swelling. The family history was significant for history of microscopic hematuria and gross hematuria in maternal grandmother and maternal great aunt but negative for end-stage kidney disease, hearing loss/deafness, autoimmune disease, or hypertension other than that in older age. Given concern for an underlying glomerular disease, a kidney biopsy was obtained.

The kidney biopsy (shown in Fig. 1) was composed of cortex, corticomedullary junction, and medulla and contained 87 non-sclerotic glomeruli. Focally immature-appearing glomeruli were present, and glomeruli appeared small, consistent with the patient’s age. There was mild mesangial hypercellularity, and glomerular capillary loops showed slightly weak silver staining. Rare red blood cell casts were present. There was no significant interstitial fibrosis, tubular atrophy, or arterio/arteriolosclerosis. There was no significant immunofluorescence staining. Electron microscopy demonstrated diffuse glomerular basement membrane structural abnormalities including segmental subepithelial scalloping, lamina dense splitting, and basket weave type remodeling. In areas without significant remodeling, glomerular basement membranes were diffusely thin (156 nm in thickness, standard deviation 58 nm, direct measurement, and arithmetic mean [2]). Some tubular basement membranes exhibited multi laminations. There was segmental mild podocyte foot process effacement (∼20%). There was increased mesangial matrix. There were no tubuloreticular inclusions or any electron dense deposits.

A diagnosis of atypical glomerular basement membranes suggestive of Alport syndrome was rendered. Subsequent molecular testing was pursued via 385 gene panel next-generation sequencing assay which included evaluation of collagen IV alpha chains 3, 4, and 5 genes (COL4A5, COL4A4, COL4A5). This molecular assay was positive for a heterozygous likely pathogenic variant in the COL4A5 gene (c. 485del(p.Gly1618Valfs*35) diagnostic of X-linked Alport syndrome. No mutations in other COL4A genes were identified. A repeat single-gene analysis of the COL4A5 gene was confirmatory. This sequence change creates a premature translational stop signal (p.Gly1618Valfs*35) in the COL4A5 gene that is expected to disrupt the last 68 amino acids of the COL4A5 protein. This variant is not present in the gnomAD population database and has not been reported in the literature in individual affected with COL4A5-related conditions. This variant disrupts a region of the COL4A5 protein in which other variant(s) (p.Cys1678Tyr) have been determined to be pathogenic [3]. Thus, the variant present in this patient was likely to be disease causing.

The patient was evaluated in audiology and found to have normal hearing screen. She was also evaluated by pediatric ophthalmologist and had normal examination except for hyperopia. Both parents underwent targeted variant testing of the COL4A5 gene, and the results of this test showed that the pathogenic variant in the COL4A5 gene carried by the patient was de novo and not inherited from either parent. The patient continues to follow-up in pediatric nephrology every 6 months. Her kidney function and blood pressure have remained normal with a stable urine protein/creatinine ratio. She is not currently on angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker therapy. She will continue to be followed annually in audiology and ophthalmology clinics. Her eGFR has remained stable around 124 mL/min/1.73 m² based on CKiD U25 GFR equation [1].

Alport syndrome is a genetically and phenotypically heterogeneous disorder that can be transmitted as an X-linked, autosomal recessive, or autosomal dominant fashion and can affect glomerular, cochlear, and ocular basement membranes. The disorder results from mutations in the collagen IV genes COL4A5 (X chromosome), COL4A3, and COL4A4. Alport patients are at lifetime risk for kidney failure, sensorineural deafness, and ocular abnormalities [4].

The diagnosis and classification of Alport syndrome has undergone a recent paradigm shifts that recognize the heterogeneity in the genetics and presentation of this disorder [5]. This new paradigm emphasizes the role of genetically defined disease rather than specific kidney or extra-renal signs/symptoms. Important conceptual paradigm shifts include the concept that females with heterozygous variants in COL4A5 are at risk for progressive kidney disease [6‒8]. Random X inactivation (the process known as lyonization) accounts for the variable clinical course of the disease in female patients [6]. The risk of developing ESRD before the age of 40 years is 12% in girls and women versus 90% in boys and men, but the risk of progression in women appears to increase after the age of 60 years [7]. This case also illustrates that in some cases, heterozygous X-linked Alport syndrome may have a severe and early phenotype.

Similarly, patients with thin basement membrane lesion alone and any variant in COL4A3, 4, or 5 genes may also be at risk for disease progression. Thus, the terms “thin basement membrane nephropathy” and “benign familial hematuria” are best avoided [9‒12]. For an initial pathology report, the term “thin basement membrane lesion” or a descriptive diagnosis of “diffusely or segmentally thin glomerular basement membranes” should be used. Subsequent genetic evaluation and clinical/pathologic correlation can then be used to risk stratify and appropriate classify patients with such biopsy findings. There is controversy regarding the consensus nomenclature and classification of Alport syndrome and thin basement membrane lesions which is beyond the scope of this brief review [13].

It is also important to be aware of the limitations of genetic studies as up to 10% of cases’ clinical and pathological findings’ essential diagnosis for Alport syndrome may have genetic changes not identified by next-generation sequencing panels or even whole-exome sequencing [14]. Additionally, some gene variants identified will be of uncertain significance, especially in patients whose family geographic background is not well represented in reference gene data sets [15]. Genetic counseling is also an important aspect of this work-up, especially as nephrologist may have limited experience or training in the interpretation of molecular pathology reports. Recommendations for genetic testing are beyond the scope of this manuscript; we refer the reader to this comprehensive work by Savige et al. [16] for recommendations.

This case also highlights how family history of kidney disease (or lack thereof) may be misleading in the diagnosis of Alport syndrome or other genetic kidney diseases. In this case, despite some history of familial hematuria, the patient’s mutation was sporadic and not shared by either parent. Such disease-causing sporadic mutations have been characterized to occur; however, there are limited data on the frequency of such events [17, 18].

Kidney biopsy can be extremely helpful in the diagnosis of Alport syndrome, especially when the disease is renal limited (or initially renal limited) and there is no definitive family history. Kidney biopsies should include a full light, routine immunofluorescence, and electron microscopic workup. The light microscopic findings are typically non-specific (see Fig. 2) and can range from near normal morphology to extensive interstitial fibrosis/tubular atrophy and global glomerulosclerosis with or without segmental glomerulosclerosis. Mesangial widening and hypercellularity has been noted in some cases [19, 20]. Some cases show prominent interstitial foam cells. The routine immunofluorescence panel typically shows no immune complex deposits. Electron microscopic studies are heterogenous and can show thin glomerular basement membrane lesion alone or variable amounts of atypical glomerular basement membrane remodeling (e.g., lamina densa splitting, basket weave pattern lamina densa remolding, and subepithelial scalloping) [21]. The presence of any of these electron microscopic findings should trigger considerations for genetic studies.

Measurements of glomerular basement membranes should also be interpreted in the context of the age and sex of the patient and normal reference range for the renal pathology laboratory. We recommend using direct measurement of glomerular basement membrane thickness – distance from endothelial to podocyte plasma membrane; 10 peripheral capillary loops and at least 30 measurements – and determination of the arithmetic mean of such measurements as it is straightforward and convenient with modern digital cameras [2]. Furthermore, it avoids pitfalls of measuring tangential-sectioned areas or other processing artifacts which may skew the orthogonal intercept/mean harmonic thickness method. Measurement using simple direct measurement produces thinner measurements than the orthogonal intercept/mean harmonic thickness method.

Immunofluorescence studies to demonstrate loss of the normal collagen IV alpha 5 and alpha 3 staining pattern can also be helpful in diagnosis and provide prognostic information, however, may be equivocal or normal in a high percentage of cases [22]. Typically, we defer directly to genetic studies with any suspicious biopsy findings as this will generally provide a definitive diagnosis with the most prognostic information. Furthermore, genetic studies also are important to rule out the rare biopsy mimics of Alport syndrome including, LMX1B, LAMB2, and WT-1 mutations [21].

The progression of Alport nephropathy follows a consistent pattern including isolated hematuria with subsequent mild then severe proteinuria and decline in glomerular filtration rate [23]. There is marked heterogeneity of progression rate among patients influenced predominantly by COL4A genotype and sex [7]. Affected male patients with X-linked COL4A5 mutation have a very high probability of developing end-stage kidney disease by 30 years of age [7]. The risk of developing hearing defects and ocular changes like lenticonus is also dependent of COL4A genotype and deletion of gene. Hearing and ocular defects are more common with deletion and missense mutations in COL4A5 genes in males with X-linked Alport syndrome [24].

The current goals of therapy are to halt or decrease the rate of disease progression. The mainstay of therapy is ACE inhibitor treatment, optimally initiated prior to any GFR decline and in all patients with overt proteinuria [14, 25, 26]. Initiation of therapy can be tailored to patient sex and severity of mutation in cases of mild to moderate proteinuria. There are currently no KDIGO guidelines for Alport syndrome treatment. Clinical trials are underway to understand the role of ACE inhibitor therapy with mild to moderate proteinuria or hematuria alone. A variety of clinical trials of novel therapies, therapeutic strategies, and biomarker discover for Alport disease are underway (Fig. 3). Kidney transplant is a treatment often pursued in patients who develop end-stage kidney disease from Alport syndrome. Unfortunately, it is not often definitive, especially in young people given the high risk of allograft loss over decades.

Written informed consent was obtained from the parent/legal guardian of the patient for publication of the details of their medical case and any accompanying images. IRB approval is not required for this study based on UCLA guidelines.

J.E.Z. has been a paid consultant for Leica Biosystem and PathAI as well as an Editorial Board Member of PathologyOutlines.com.

No funding was received for this work.

J.E.Z. and R.S. wrote the manuscript.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.