Pathogenic synonymous variation of the COL4A3 gene causing Alport syndrome comorbid with IgA deposition in a toddler: a case report

Abstract

Background

Alport syndrome (AS) is a hereditary kidney disorder caused by pathogenic variations in COL4 genes and is clinically characterized by hematuria, proteinuria, and progressive renal impairment. IgA nephropathy (IgAN) is a clinicopathological syndrome characterized by the deposition of IgA or IgA-dominant in the glomerular mesangial areas.

Case presentation

This article reports a case of a 2-year-and−3-month-old female toddler who presented with hematuria and proteinuria. Renal biopsy revealed IgA deposition, and a few segments displayed atypical tearing and layering changes in the dense layer. Family screening revealed that the father and grandmother of the patient had been diagnosed with thin basement membrane disease. Genetic testing revealed compound heterozygous variations c.4793T > G (p.Leu1598Arg) and c.765G > A (p.Thr255Thr) in the COL4A3 gene. Both hematuria and proteinuria improved significantly with treatment involving steroids, mycophenolate mofetil, tacrolimus, and angiotensin-converting enzyme inhibitors (ACEIs), but both recurred and slowly increased under ACEIs monotherapy. The toddler was ultimately diagnosed with AS comorbid with IgAN, and the variant c.765G > A (p.Thr255Thr) from the father is suspected to be pathogenic based on familial segregation and predictive evidence.

Conclusion

Younger children with AS exhibit milder clinical manifestations or are asymptomatic. Biallelic pathogenic variations and IgA deposition may accelerate AS disease progression. Synonymous variations can also be pathogenic.

Introduction

Alport syndrome (AS) is a hereditary kidney disorder characterized by hematuria, proteinuria, and progressive renal impairment. AS results from pathogenic variations in COL4A3, COL4A4, and COL4A5, which encode the α3α4α5 chains of type IV collagen. The typical electron microscopy findings of AS include diffuse thinning or irregular thickening of the glomerular basement membrane (GBM), with splitting and lamellation of the lamina densa, resulting in a basket-weave appearance. IgA nephropathy (IgAN) is a clinicopathological syndrome characterized by the deposition of IgA or IgA-dominant in the glomerular mesangial areas. The central pathogenesis of IgAN involves the formation of circulating immune complexes between aberrantly glycosylated IgA1 molecules and specific IgG/IgA antibodies, which are subsequently deposited in the glomerular mesangium. IgAN is pathologically characterized by mesangial IgA-dominant immune deposits and mesangial cell proliferation. Here, we report a 2-year-and-3-month-old female toddler who was diagnosed with IgAN comorbid with AS caused by compound heterozygous pathogenic variations in the COL4A3 gene. The variant c.765G > A (p.Thr255Thr) from the father is suspected to be pathogenic based on familial segregation and predictive evidence.

Case presentation

General information

A 2-year-and-3-month-old female toddler first presented with an abnormal urine color at the age of eight months. Hematologic examination revealed mild anemia with a red blood cell (RBC) count of 3.42 *10¹²/L (reference range: 4.10–5.30 *10¹² /L) and a hemoglobin level of 99.0 g/L (reference range: 110–147 g/L). Urine analysis revealed hematuria 3+, proteinuria 1+, RBC count 5034.8 /µL, and dysmorphic RBCs 30.0%. The urine albumin-to-creatinine ratio was 149.4 µg/mg. However, renal function was normal, with a creatinine concentration of 28 µmol/L and an estimated glomerular filtration rate (eGFR) > 100 mL/min/1.73m² (calculated using the Schwartz equation).

Renal biopsy and family history

The pathological results of the renal biopsy (Fig. 1) revealed that the patient had IgAN (WHO Grade III, Oxford Classification E0M0S1T1-C1) with basement membrane abnormalities. Immunofluorescence staining was positive for IgA 3+, IgG 3+, WT1 3+, and lambda light chains 4+. Electron microscopy revealed GBM thicknesses ranging from 102 nm to 232 nm, with some segments showing curling. A few segments showed atypical tearing and layering changes in the dense layers. Some mesangial areas had small amounts of electron-dense deposits.

Fig. 1.

Renal pathology of the proband. (A) The GBM was uneven in thickness, with double-track signs visible in some segments. Immune deposits can be observed under the endothelium and epithelium (PAS stain: ×400). (B) The renal interstitium shows focal widening, edema, and fibrosis, with evident inflammatory cell infiltration (MASSON stain: ×400). (C) Positive reaction (3+) in the glomeruli (immunofluorescence IgA: ×400). (D) Positive reaction (3+) in the glomeruli (immunofluorescence IgG: ×400). (E)A few segments of the GBM are curled in the dense layer (EM: ×10000, pointed by green arrow). (F) A few segments show atypical tearing and layering changes in the dense layer (EM: ×5000, pointed by red arrow)

Notably, family screening (Fig. 2) revealed a history of hematuria in the patient’s father and grandmother, both of whom were diagnosed with thin basement membrane disease through renal biopsy.

Fig. 2.

Family pedigree chart of the patient. Blackened circles and squares indicate individuals with clinical manifestations of hematuria, proteinuria, or renal dysfunction (kidney disease)

Gene tests

Integrated analysis of whole-exome sequencing (Fig. 3) data incorporating bioinformatics predictions, clinical relevance, and inheritance patterns revealed compound heterozygous variations c.4793T > G (p.Leu1598Arg) and c.765G > A (p.Thr255Thr) in the COL4A3 gene. The variation c.4793T > G is inherited from the mother and is associated with autosomal recessive Alport syndrome (ARAS), as reported in the HGMD database [1]. However, the patient and her mother were only heterozygous carriers, and the patient’s mother had normal urine test results, leaving the impact of this variation on the patient’s phenotype open for further discussion. The pathogenic variation c.765G > A was a synonymous change from the father and was predicted by SplicingPre software to possibly affect gene splicing. RNA sequencing (Fig. 4) demonstrated that the pathogenic variant c.765G > A disrupted splicing, resulting in complete skipping of exon 13 and an in-frame deletion. This aberrant splicing event significantly compromises gene function and is likely to cause for autosomal dominant Alport syndrome (ADAS), as previously reported in the literature [2]. The synonymous mutation was classified as likely pathogenic (PM2, PP1, PS3) based on the American College of Medical Genetics and Genomics (ACMG) guidelines. The patient might been diagnosed with ADAS, with the paternally inherited c.765G > A variant identified as the primary pathogenic determinant. The maternally inherited c.4793T > G variant (in heterozygous state) currently demonstrates no significant clinical contribution.

Fig. 3.

Whole-exome sequencing results. Abbreviations: ACMG, American College of Medical Genetics and Genomics; Het, Heterozygous; LP, Likely pathogenic; VUS, Variant of uncertain significance; Wt, Wild type

Fig. 4.

RNA sequencing results. Abbreviations: HGVS, Human Genome Variation Society

Diagnosis

The patient was diagnosed with IgAN comorbid with AS caused by compound heterozygous variations in the COL4A3 gene.

Treatment and follow-up

The patient was initially treated with steroids (70 mg/kg/day for 3 days as pulse therapy, followed by 1.75 mg/kg every other day), mycophenolate mofetil at 30 mg/kg/day, and angiotensin-converting enzyme inhibitors (ACEIs) at 1.75 mg/kg/day for nearly one month. Owing to a suboptimal response to the initial treatment regimen, tacrolimus was added to the therapy at 0.13 mg/kg/day. After more than three months of treatment, a follow-up urinalysis revealed negative proteinuria, trace hematuria, and a RBC count of 12.5 /µL (Fig. 5). Subsequently, most oral medications are gradually discontinued, but hematuria and proteinuria recurred under ACEIs treatment alone. After more than three years of follow-up, there was a slow increase in hematuria and proteinuria under ACEIs monotherapy. Recent urinalysis of the patient (5-year-and-8-month-old) revealed hematuria 3+, a RBC count of 885.72 /µL, and a urinary microalbumin concentration of 151 mg/L. Regrettably, the patient did not complete the required audiological and ophthalmic screenings during the clinical evaluation process. The parents of the patient did not undergo genetic counseling for future family planning.

Fig. 5.

Changes in the urine RBC count with age in the patient

Recent urinalysis of the patient’s father (35-year-old) and grandmother (56-year- old) also revealed proteinuria 2+, and the eGFR of the patient’s grandmother was 50 mL/min/1.73m² (calculated by the CKD-EPI Formula).

The urine RBC count decreased significantly with treatment involving steroids, mycophenolate mofetil, tacrolimus, and ACEIs but recurred and slowly increased under ACEIs monotherapy.

Discussion

The main components of GBM are laminin-521 (α5β2γ1), type IV collagen (α3α4α5), nidogen, and heparan sulfate proteoglycans [3]. During glomerulogenesis, there is a shift in the composition of collagen IV α-chains, involving a transition from the α1α1α2(IV) network to the α3α4α5(IV) network, which defines the mature GBM [4]. The absence or reduction of the α3α4α5(IV) chains leads to thin basement membranes and AS. However, there is substitution or compensation by the α1α1α2(IV) and/or α5α5α6(IV) network, which may slow the progression of the disease for several years [5, 6]. A study found that patients with ARAS progressively developed clinical manifestations at distinct median ages: hematuria at 4 years, proteinuria at 7 years, hearing loss at 10 years, and ocular lesions at 11 years [7]. These findings suggest that younger children with AS exhibit milder clinical manifestations and are even asymptomatic. In this case, both hematuria and proteinuria improved significantly after treatment, which initially indicated the impact of IgAN. However, proteinuria and hematuria recurred with ACEIs monotherapy, suggesting a possible contribution from AS, while not excluding the potential recurrence of IgAN. Thus, long-term follow-up and screening for younger patients with AS with less severe clinical presentations are advisable. If necessary, a second kidney biopsy was performed to assess the progression of the patients’ kidney disease.

In this case, the patient developed symptoms in infancy, indicating a severe AS phenotype compared to that of the father. Genetic testing revealed that the patient had compound heterozygous variations in the COL4A3 gene. Similarly, studies in the literature have reported that individuals with a monoallelic disease-causing variant develop microscopic hematuria and proteinuria significantly later than individuals with biallelic disease-causing variants [8]. This suggests that biallelic pathogenic variations may lead to more severe clinical manifestations.

In addition, these findings may suggest that IgA deposition accelerates disease progression in children with AS. A prospective study revealed that pediatric IgAN patients with COL4A3 pathogenic variations presented more severe manifestations than non‒COL4A3-IgAN patients [9]. The precise mechanisms underlying this process remain unclear. However, IgA deposition can activate the complement system, initiate inflammatory cascades, and exacerbate GBM damage [10]. Furthermore, structural defects in the GBM may increase the permeability and deposition of IgA immune complexes [11]. Over time, persistent IgA deposition and GBM compromise can drive chronic inflammation and fibrotic remodeling [12]. Nevertheless, due to the high frequency of heterozygous COL4A3/4 variants in the general population (approximately 1 in 107), the co-occurrence of AS (particularly autosomal dominant AS) and IgAN may be coincidental, especially in Asia. Chen [13] and Vischini [14] have also reported cases diagnosed with COL4A3-mediated AS coexisting with IgAN. However, our case is the youngest reported in the literature. Its uniqueness lies in the extremely early onset and the application of RNA sequencing, underscoring the necessity of early genetic testing and renal biopsy in children presenting with hematuria to uncover underlying syndromic diagnoses. Therefore, attention should be paid to the coexistence and progression of multiple diseases in toddlers with kidney disease.

Early studies suggested that synonymous variations can be pathogenic, similar to nonsynonymous pathogenic variations, and are associated with 1% of human diseases. They can disrupt transcription, splicing, cotranslational folding, and mRNA stability, causing various function-related changes that lead to disease [15]. RNA sequencing revealed that the pathogenic variant c.765G > A may affect RNA splicing, as reported in the literature [2]. This variant is suspected to be likely pathogenic based on familial segregation and predictive evidence. Another study also reported the identical COL4A3 synonymous variant c.888G > A (p.Gln296Gln), SpliceAI, dbscSNV_RF, and dbscSNV_ADA, indicating that it may affect splicing [13]. Therefore, synonymous variations can also be pathogenic, and RNA sequencing is highly important when whole-exome sequencing results are inconclusive regarding the pathogenicity of variations.

Conclusion

Younger children with Alport syndrome exhibit milder clinical manifestations or are asymptomatic but still require renal biopsy. Biallelic pathogenic variations and IgA deposition may accelerate the progression of Alport syndrome. Synonymous variations can also be pathogenic.

Abbreviations

AS

Alport syndrome

IgAN

IgA nephropathy

ACEIs

Angiotensin-converting enzyme inhibitors

RBC

Red blood cell

eGFR

Estimated glomerular filtration rate

GBM

Glomerular basement membrane

PAS

Periodic acid-Schiff

MASSON

Masson’s trichrome

EM

Electron microscope

ARAS

Autosomal recessive Alport syndrome

ADAS

Autosomal dominant Alport syndrome

ACMG

American College of Medical Genetics and Genomics

Het

Heterozygous

VUS

Variant of uncertain significance

HGVS

Human Genome Variation Society

LP

Likely pathogenic

Wt

Wild type

Author contributions

PpS: conception and design. JbX, JhZ and HqY: acquisition of data. WpL, RrX, YhH, XqM and JyP: analysis and interpretation of the data. PpS and LrQ: writing and reviewing the manuscript. All authors contributed to the article and approved the submitted version.

Funding

Funding was provided by the Hubei Province Health Commission General Project (grant number: WJ2023M004) and the Beijing Medical Award Foundation (grant number: YXJL-2023-0866-0329).

Data availability

The datasets generated and/or analyzed during the current study are available in the National Genomics Data Center repository [HRA011415].

Declarations

Ethics and consent to participate declarations

This study was conducted according to the guidelines laid down in the Declaration of Helsinki. This research protocol was reviewed and approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (No. TJ-IRB20231280).

Disclosure

No potential conflict of interest was reported by the author(s).

Consent for publication

Written informed consent was obtained from the patient’s parents for publication of this case report and any accompanying images. A copy of the written consent is available for review by the editor of this journal.

Competing interests

The authors declare no competing interests.