A Review of Multi-dimensional Diagnosis of Alport Syndrome in 22 Children in Northeast China

Background Alport syndrome (AS) is progressive hereditary nephritis due to different gene mutations. Affected individuals usually develop hematuria during childhood with gradual deterioration of renal functions. We adopted multi-dimensional methods to diagnose Alport syndrome in order to decrease the misdiagnosis. Methods Twenty-two children were diagnosed and managed by the Department of Pediatric Nephrology of Jilin University First Hospital between January 2017 and January 2020 through multi-dimensional methods. Information collected included age of onset, age at diagnosis, clinical manifestations, family history (FH), renal pathology and their genotype. Results All patients presented with hematuria with various degrees of proteinuria in partial patients. While three children suffered from hearing loss, none of the children in the cohort had any visual problem or renal failure. Besides five patients estimated as Stage 2, the remain seventeen cases were at Stage 0. Renal biopsy were obtained in eighteen patients and fourteen of them showed glomerular basement membranes (GBM)-specific abnormalities. Thirteen children had mutations of the collagen IV genes. Conclusion Combined with the importance of early diagnosis and economic factors, we adopted multi-dimensional methods to diagnose Alport syndrome and estimate the risk of progression. We also reviewed the therapy progress.

in medical technology, in addition to electron microscopy, [5] other modes of investigations such as collagen IV analysis and genetic testing [6] have broadened the clinical and research repertoire that we can use to detect the changes in AS. The diagnostic criteria includes family history (FH) of hematuria, sensorineural hearing loss, characteristic eye signs, diffuse esophageal leiomyomatosi, ultrastructural changes and abnormal distributions of the α(IV) collagen chains by immunohistochemical staining of GBM, and genetic mutations of COL4A3, COL4A4 or COL4A5. [7] Clinicians may not be aware of this disease, either because of an incomplete evaluation or an atypical presentation. In this study, we adopted multi-dimensional methods to diagnose AS and estimated the risk of progression. In addition, we showed some atypical findings and possible differential diagnoses and reviewed the therapy progress.

Diagnostic criteria
In order to unify standardized diagnosis, we adopted the criteria established by the the Working Group for Alport Syndrome in the Japanese Society of Pediatric Nephrology (JSPN) in 2015 (Table1). [8] 1. Diagnostic criteria: In addition to the primary feature, patients should satisfy one or more secondary features or satisfy two or more of the accessory features. 2. If patients only have the primary feature and a family member diagnosed with Alport syndrome, the case is set as a "suspected case". 3. If patients have any one feature of type IV collagen (II-1 or II-2) among the secondary features, the case is set as "asymptomatic carriers". 4. Features caused by other diseases should be excluded, for example, a family history of kidney failure due to diabetes.

Clinical investigation
Clinical data collected included age of onset, diagnosis age, duration from onset of symptoms to diagnosis, hematuria, proteinuria, estimated glomerular filtration rate (eGFR), extrarenal symptoms and family history. Histological findings of light microscopy (LM) and electron microscopy (EM), immunofluorescence staining and immunohistochemical staining of type Ⅳ collagen were also collected for analysis. Gene data was obtained form partial children when AS is highly suspected.

Statistical analysis
SPSS 20.0 statistical software was used to process the data. The non-normal distribution data were expressed as median (range) and the counting data were expressed as percentage (%).

Results
According to clinical features, pathology features and gene detection, 22 children were diagnosed as AS from January 2017 to January 2020 in Department of Pediatric Nephrology of Jilin University First Hospital. All of them met the diagnostic criteria including the primary feature and at least one of the secondary features ( Table 5). The clinical characteristics, renal pathology characteristics and gene mutations were elaborated as follows.

Clinical characteristics
The diagnosis age of the 22 patients (12 boys and 10 girls) ranged from 34 to 170 months (median: 84 months). The interval time between onset and diagnosis ranged from 1 to 97 months (median: 5.5 months). All children (100%) had hematuria with dysmorphic red cells; eighteen of them (81.8%) had paroxysmal macroscopic hematuria during upper respiratory infection. Non-nephrotic range proteinuria was present in 10 children (45.5%), in which all of their proteinuria level were less than 30 mg albumin per g creatinine or per day (Stage 0). Five other children (22.7%) had nephrotic range proteinuria (P2, P9, P15, P19 and P20) who were at Stage 2. At diagnosis, while the eGFR and vision of all children was within the normal range, 3 children (13.6%) were confirmed to have mild to moderate sensorineural hearing loss (P2, P9 and P19). Positive FH was identified in 16 patients (72.7%) . Table 5 depicts the full details of the clinical findings.

Renal pathology characteristics
Renal biopsies were performed in 18 children. Sixteen (88.9%) of the examined biopsies showed minor glomerular abnormalities (MGA) in LM. In additional, one case of focal segmental glomerulosclerosis (FSGS) and one MsPGN (mesangial proliferative glomerulonephritis) was showed respectively. Immunoflourescence staining were negative in 6 (33.3%) while the others presenting with non-specific deposition of immune complex. GBM-specific abnormalities were observed in 14 cases (77.8%) in EM. Two cases presented as extensive thinning of the GBM (P8) and irregular thinning of the GBM (P13) respectively. Type Ⅳ collagen α 5 chain expression was tested for in 11 children and only 3 of them presented abnormal expression. Seeing inTable 5.

Gene detection
Thirteen children and their parents were tested for genetic mutations of the collagen IV genes using high throughput sequencing; with positive results confirmed by Sanger sequencing or real-time PCR

Discussion
Mutations in the collagen IV genes leading to AS is well documented. Novel mutations and genotype and phenotype correlation has been under study in China. [11][12] However, as a developing country, there are still many economic less-developed regions in China, especially in Northeast China. WES can not be accepted in partial families due to economic reason, while renal biopsy not be accepted for conservative ideas. Combined with the above reasons, we adopted the Japanese diagnostic criteria [8] for multi-dimensional diagnosis of this disease. We hope to reduce misdiagnosis and improper treatment, estimate the risk of the progressive renal disease, provide timely intervention, and minimize economic costs.
Through the diagnostic criteria above, 22 children were diagnosed as AS. Although some of them didn't have a renal biopsy and some didn't have a genetic test, the diagnosis was precise and welldocumented. Similar to those published articles [13] , patients in our cohort generally presented with hematuria, partially with proteinuria. Though five of them have manifeted with nephrotic-range proteinuria which made them be estimated as Stage 2 of AS, three of them have displayed hearing loss, fourteen cases presented with GBM-specific abnormalities in electron microscope, none of them showed renal failure. That may due to they were diagnosed within their first or second decades of lifetime.
A total of ten COL4A5 mutation, one compound heterozygous mutation at COL4A4, one autosomal dominant (AD) mutation of COL4A3, and a Digenic AS with mutation of COL4A3 and COL4A5 were identified in this study. In XL-AS, hemizygous male patients have a 100% risk of progression to ESRD, although rate of progression and timing of extrarenal manifestations are related to COL4A5 genotype. [9] Heterozygous female patients have a lifetime risk of progression to ESRD of approximately 25%.
But that depends on many risk factors including a history of gross hematuria in childhood, sensorineural deafness, proteinuria, and extensive GBM thickening and lamellation. [14] Since all of our patients with COL4A5 mutation were estimated as Type MS or Type M, with risk factors in majority, renal function should be monitored closely within the next decade.
Autosomal Alport syndrome associated with biallelic mutations (homozygous or compound heterozygous) in COL4A3 or COL4A4 exhibits a recessive inheritance pattern and is associated with a 100% risk of ESRD, with rate of progression and timing of extrarenal manifestations influenced by genotype. [9] P3 in our study got the compound heterozygous mutations of COL4A4 with GBM-specitic renal pathology. According to this, he is estimated with a 100% risk of ESRD. Patients with heterozygous mutations in COL4A3 or COL4A4 are considered affected if they exhibit hematuria or proteinuria and include patients who would have previously been diagnosed with thin basement membrane nephropathy (TBMN). In these individuals the risk of ESRD is up to 20% among those with risk factors for progression, which include proteinuria, sensorineural deafness, family history of progression to ESRD, and renal biopsy findings of focal segmental glomerulosclerosis, or GBM thickening and lamellation, or all of these. P8 who has been diagnosed with TBMN was identified to have the COL4A3 dominant mutation. Since her mother presented isolated hematuria without ESRD, it is hopeful to look forward to the benign progression. P13 was a bit different. He got glycine-XY substitutions involving exons 1-20 in COL4A5 and irregular thinning of the GBM, but he also got a COL4A3 mutation, which contributed him high risk of renal progression. We are actively following these children and closely monitoring their renal progression.
Not all of our patients were correctly diagnosed to have AS at presentation. The median of interval time between onset and diagnosis was 5.5months, ranging from 1 to 97 months. That means it took nearly half of a year to get diagnosed since onset. For partial patients who were willing to accept necessary examminations, we just spent one month to identify the etiology. However, more patients may need much longer time, even 4 to 8 years. In that case, many patients accepted some improper treatments under a ambiguous diagnosis.
P2 was initially diagnosed to have glomerulonephritis at the age of 3 years at local clinic due to hematuria and nephrotic range of proteinuria. Subsequent renal biopsy, which was done at his 9-yearold, was compatible with minimal change disease (MCD). Despite further history did reveal his mother had persistent hematuria and proteinuria of unknown etiologyand his grandfather died of uremia, his parents refused to undergo further investigation, therefore, the diagnosis of AS was not entertained at that time (Seeing in Fig.1A). The child was initially managed by using corticosteroids, followed by cyclophosphamide and mycophenolate mofetil, due to steroid resistance. After accepted the treatment of Tacrolimus , he obtained partial remission with urine protein being controlled below 1 gram per day. He was later confirmed to have XL-AS (a missense mutation of COL4A5 inherited from her mother) at the age of 11 by genetic testing (Seeing in Fig.1B). He is currently treated with tacrolimus and ACE inhibitors. Therefore, it is important to choise the right time for both renal biopsy or re-biopsy and genetic testing for those with positive family history. Likewise, P9, P15, P19 and P20 presented with heavy proteinuria who would have been managed as nephrotic syndrome if further eaxminations were not performed. Interestingly, P9 and P15 were finally treated with tacrolimus after genetic confirmation of AS, and their proteinuria reduced to below 1 gram per day, this phenomenon corroborates with the previous studies that showed therapeutic benefits of calcineurin inhibitors in AS patients. [15] The presentation of AS can occasionally mimic other clinical entities. P4 of our cohort, who had a missense mutation of COL4A5 inherited from her mother (Seeing in Fig.2B), presented with macroscopic hematuria during infection, and she did not suffer from any hearing loss or visual problem. Her renal biopsy revealed minor glomerular abnormalities with mild IgA deposition that was compatible with IgA nephroparthgy. If not for the presence of family history (Seeing in Fig.2A), she would have been managed as IgA nephropathy and genetic testing would not be offered.
Interestingly, a child with similar clinical presentations to our patient was misdiagnosed to have IgA nephropathy. [16] His renal biopsy did not show features of AS until he had his second renal biopsy 4 years later. Similarly, in another recent Chinese report, [17] the proband who presented with hematuria and proteinuria was initially diagnosed as IgAN by renal biopsy and Immunofluorescence detection.
Because of the poor treatment outcome, he was identified with a novel mutation of COL4A5 under the gene detection. By the time he was diagnosed, he has been treated with prednisolone companying with mycophenolate mofetil and tacrolimus successively.
Different from the typical manifestations, P3 displayed as isolated hematuria with negative family history. He accidentally found microscopic hematuria during a health check. During the eight-month follow-up, the urine red blood cell count fluctuated from 10/HPF to 30/HPF. GBM-specific abnormalities were observed in renal biopsy. Gene detection revealed compound heterozygous mutaitons of COL4A4. The renal pathology, gene mutation and family pedigree of P3 are showed in Fig.3(A-G).
In addition, whether the characteristic changes in GBM can be present depend on multiple factors, such as the age of biopsy and different mutation. In our cohort, 14 of the 18 renal biopsies showed GBM-specific abnormalities, while two cases presented as extensive thinning of the GBM (P8) and irregular thinning of the GBM (P13) respectively. TBMN is a relatively common disease that has been reported in 1% of the general population. [18] According to the newest classification, [9] TBMN is now considered to be a lesion description rather than a diagnosis, and its likely that some of our patients previously diagnosed to have TBMN were actually patients suffered from AS. P8 had extensive thinning of GBM and would have been diagnosed to have TBMN if genetic testing was not performed.
Different from other cases, she got an autosomal dominant mutation of COL4A3. The renal pathology, gene mutation and the family pedigree are showed in Fig.4(A-F). P13 had irregular thinning of the GBM and digenic mutation of COL4A3 and COL4A5. We showed the detailed information in Fig.5(A-G).
There is no radical cure for the disease and attempts to use various stem cell therapies in animal models have been met with ambiguous success. It is reported that with the exception of cyclosporine, a calcineurin inhibitor, the use of which remains controversial due to its possible long term nephrotoxic effects, [15] Renin-Angiotensin-Aldosterone System (RAAS) inhibitors are efficient and well tolerated to retard chronic kidney disease (CKD) progression in AS. [19] Gross et al. [20] reported a double-blind, randomized, placebo-controlled, multicentre phase III trial in order to clarify the safety and efficacy of ramipril in pediatric patients with AS, in which they discussed about the efficacy of ramipril when they are presenting only with microhaematuria. So far, The Alport Syndrome Classifification Working Group recommanded to use ACEI when presenting hematuria and overt proteinruia. [9] In addition, future therapies including stem cells, chaperon therapy, collagen receptor blockade and anti-microRNA therapy will expand our perspective in protecting the kidneys of Alport patients from further damage. [21] Through different mechanism, therapies such as Bardoxolone, anti-miRNA-21, paricalcitol, lipid-lowering agents and epidermal growth factor receptor inhibition play role in decreasing renal fibrosis. [22] Meanwhile, Chaperone and Stem-cell based therapies are expected to be therapeutic at collagen chains and GBM level respectively. However, when kidney failure is inevitable, patients with Alport syndrome who undergo renal transplantation would have generally excellent outcomes. [23] Although genotype-phenotype correlation is prominent, severe mutations do not impact on patient and graft survival after transplantation. [24] Conclusion In conclusion, considering the importance of early diagnosis and economic factors, we adopted multidimensional methods to diagnose AS and estimate the risk of progression. In condition-limited settings, it is important to follow a pragmatic approach. RAAS inhibitors are testified to have safety and efficacy in delaying renal progression. Patients after renal transplantation and the graft survival rates are excellent. Future therapies are on the way to change the "inevitable" outcome of disease.

Ethics aproval and consent to participate
This study was approved by the Ethics Committee of the First Hospital of Jilin University. Permission was obtained from the hospital to access the mentioned data.

Consent for publication
Written informed consents were obtained from the parents of patients for publication of case history and clinical results. A copy of the written consent is available for review by the Editor of this journal.

Availability of data and materials
The datasets used analyzed during the current study are available from the corresponding author on reasonable request.