Comprehensive gene profiling by Next-Generation sequencing in a cohort of Egyptian pediatric Atypical HUS

Graphical abstract

Abstract

Background

Atypical hemolytic uremic syndrome (aHUS) is a rare, severe condition in children, resulting from uncontrolled complement activation caused by genetic factors or autoantibodies; it usually has a poor prognosis. Early identification of the underlying genetic variants is crucial for guiding management and predicting outcomes. This study aimed to identify gene mutations in Egyptian children with aHUS at an early stage. This would enable the selection of the most appropriate treatment options and help prevent adverse outcomes for the patients.

Methods

This is an observational cohort study, included 21 children with a clinical diagnosis of aHUS who presented to the Pediatric Nephrology Unit and general wards of Cairo University Children’s Hospital between June 2022 and January 2024 with a follow-up duration of 12 months (median). All patients underwent whole exome sequencing (WES). Clinical data, treatment regimens, and outcomes were recorded and analyzed.

Result

Among the 21 patients, around one third had negative WES results while 28.57 % showed CFHR 3/CFHR1 deletion. Most patients progressed to chronic kidney disease (52.4 %), while 28.6 % recovered their kidney functions following plasmapheresis. A significant association was observed between WES category and disease relapse (p = 0.021); patients with CFHR3 deletion, CFHR5 deletion, or MMUT variant developed at least one attack of relapse.

Conclusions

Egyptian children with aHUS demonstrate marked genetic heterogeneity. A substantial proportion lacked identifiable pathogenic variants, highlighting the complexity of the disease. CFHR3/CFHR1 deletion was the most frequent finding. Genetic profiling remains crucial for anticipating relapse risk and guiding therapeutic decisions, particularly in resource-limited settings.

1. Introduction

AHUS represents one of the rare, life-threatening, progressive diseases characterized by chronic, uncontrolled activation of the complement system, which results in systemic thrombotic microangiopathy (TMA) 1, 2. Globally, the incidence of aHUS is estimated to be approximately 1 to 2 cases per million people per year, translating to about 8,000 to 16,000 new cases annually [3]. Among children, aHUS accounts for about 5–10 % of all hemolytic uremic syndrome (HUS) cases, with the age of onset varying widely from the neonatal period to adulthood [4].

Clinical manifestations include haemolytic anemia, thrombocytopenia, and acute renal failure, often accompanied by extra-renal symptoms, affect approximately 20 % of patients. Gastrointestinal tract, altered awareness, seizures, or focal neurologic impairments are among the symptoms that accompany this condition. Furthermore, prodromic diarrhea occurs in nearly 30 % of cases [5].Complications of aHUS can be severe, as half of the cases may develop end-stage kidney disease (ESKD) or death in the first twelve months following diagnosis, despite treatment with plasma exchange or plasma infusion [6]. Unfortunately, 33 %-40 % of patients either progressed to chronic kidney disease (CKD) or succumbed during the initial attack. [7].Up to 2014, the exchange of therapeutic plasma was the cornerstone of therapy, when significant death related to plasma treatment among kids was highlighted, and eculizumab, a terminal complement inhibitor, received approval [8].The introduction of complement inhibitors like eculizumab has significantly improved outcomes, reducing the risk of ESKD and mortality. However, early diagnosis and timely intervention remain critical for optimal management [8].

The diagnosis is challenging and often requires exclusion of other thrombotic microangiopathies (TMAs), including thrombotic thrombocytopenic purpura (TTP) and Shiga toxin-producing Escherichia coli (STEC)-HUS [9]. Genetic mutations in complement regulatory proteins, such as Factor H, Factor I, and membrane cofactor protein (MCP), are commonly implicated in aHUS, leading to uncontrolled complement activation and endothelial damage. The etiology of aHUS is multifactorial, including the genetic predisposition and environmental triggers like infections or medications [10]. Early and accurate diagnosis, followed by prompt initiation of complement blockade therapy, is crucial for improving outcomes and preventing irreversible kidney damage [11]. Approximately 30–40 % of patients with aHUS have identifiable genetic mutations [9]. Next-generation sequencing (NGS) has become the most updated methodology for analyzing these genetic mutations, providing a comprehensive genetic assessment that informs diagnosis, prognosis, and treatment decisions. Studies have shown that CFH, CFI, and CD46 are among the most frequently mutated genes in aHUS patients. The genetic information is crucial for predicting disease progression, guiding therapeutic interventions, and assessing the risk of recurrence in kidney transplant patients [10].

The study primarily aimed to identify gene mutations in Egyptian children with aHUS at an early stage. This would enable the selection of the most appropriate treatment options and help prevent adverse outcomes for the patients.

2. Patients and methods

The study is an observational cohort with a follow-up duration of 12 months (median), which included 21 patients diagnosed with aHUS, who presented to the Pediatric Nephrology Unit and general wards of Cairo University Children’s Hospital, during the study period between June 2022 and January 2024 and referred to Clinical Genetics Clinic and Medical Molecular units at the Human Genetics and Genome Institute, National Research Centre for genetic analysis and genetic counseling.

The study included pediatric patients' aged 2 months to 14 years of both sexes, who presented with acute kidney injury (AKI), as defined by KDIGO guidelines. AKI was characterized by serum creatinine levels exceeding the higher than the standard limit for age or hindered urine output necessitating renal replacement therapy [12]. Additionally, included patients exhibited non-immune microangiopathic hemolytic anemia and/or thrombocytopenia (<150/μL), irrespective of preceding diarrheal episodes.

Exclusion criteria encompassed children with typical HUS associated with STEC infection or other known infectious causes, alternative etiologies of kidney injury unrelated to aHUS, drug-induced HUS (e.g., Cyclosporine and Tacrolimus), and secondary HUS linked to systemic lupus erythematosus, antiphospholipid syndrome, or bone marrow transplantation.

In this study, the term aHUS applied to non-complement mediated and complement-mediated aHUS. Patient outcomes were classified as regarding complete recovery, determined as eGFR > 90 mL/min/1.73 m² or CKD after 3 months from the onset of the first attack as stage 1, stages 2–4, or stage 5, according to calculating eGFR (ml/min/1.73 m²) making use of the Schwartz formula as following: GFR = 0.413 x (height/ serum creatinine) with the height expressed in centimeters and serum creatinine in mg/dl [13], following KDIGO guidelines [14].

Prior to data collection, the study's structure and purpose were thoroughly explained to the patients’ parents or guardians, and informed consents were obtained. Subsequent investigations were conducted as outlined below.

2.1. History taking and clinical examination

Pedigree construction up to three generations, personal history, including sex, age, consanguinity, family history of aHUS, preceding symptoms, clinical manifestations at admission, management details including renal replacement therapy and plasmapharesis or transfusion, clinical outcomes and response to therapy.

General examination includes weight, height, extrarenal manifestations involving neurological and cardiac examination including arterial blood pressure measuring. According to the guidelines established by the European Society of Hypertension for children and teenagers, the diagnosis of high blood pressure obtained an average of 3 blood pressure measurements that were ≥ the 95th percentile for age, gender, and height in children aged 0 to 15 years. This was accomplished by utilizing a cuff of an appropriate size and a sphygmomanometer that had been validated [15].

2.2. Pre-transfusion laboratory assessment

Before administrating any blood products, all samples were collected to ensure a comprehensive evaluation of hematologic and serologic parameters.

Complete Hematologic Profile: Hemoglobin (Hb), platelet count (PLT), white blood cell count (WBCs), reticulocyte count, lactate dehydrogenase (LDH), haptoglobin levels, and renal function markers (urea, creatinine).

To exclude STEC-HUS, all patients underwent stool culture and Shiga toxin assays when clinically indicated. TTP was ruled out based on ADAMTS13 activity (A Disintegrin and Metalloproteinase with Thrombospondin Type 1 Motif, Member 13). Secondary TMAs (autoimmune, drug-induced) were excluded through autoimmune serologies (ANA, dsDNA) beside clinical assessment. Serological assessment of complement system components—C3 (reference range: 900–1800 mg/L) and C4 (reference range: 150–400 mg/L) were done to all patients but Factor H, anti-Factor H antibodies, and ADAMTS13 assays were available for a subset of patients: Factor H levels in 7 patients, anti–Factor H autoantibodies in 6 patients, and ADAMTS13 activity in 6 patients with deficiency (<10 %) excluded in all tested cases.

2.3. Kidney Biopsy Consideration

In cases where the diagnosis remained inconclusive, particularly in patients who did not develop thrombocytopenia or presented with an ambiguous hemolytic profile, a kidney biopsy was performed to facilitate definitive diagnostic assessment.

2.4. Molecular Methods

The methodology involved in the present study begins with genomic DNA extraction from peripheral blood samples of all cases utilizing standard measures. The DNA samples are then quantified making use of a Qubit fluorometer with the dsDNA BR Assay Kit (ThermoFisher, Waltham, Massachusetts, United States of America).

2.5. WES Analysis

Whole–exome sequencing was performed to comprehensively interrogate the coding regions of the genome and identify variants relevant to atypical hemolytic uremic syndrome such as ADAMTS13, C2, C3, C3AR1, CD46 (MCP), CFB, CFD, CFH, CFHR1, CFHR2, CFHR3, CFHR4, CFHR5, CFI, DGKE, MASP2, MMACHC, THBD, PLG, WT1, and C5 p.Arg885. Raw sequence data were processed through a standardized pipeline beginning with quality assessment and adapter trimming, followed by alignment to the human reference genome. Variant calling was conducted across all exons, with particular attention to genes implicated in complement regulation and metabolic pathways. Depth of coverage and read distribution were evaluated to ensure reliable detection of single–nucleotide variants, small insertions or deletions, and copy–number changes. Exon–level coverage profiles were generated to highlight potential structural alterations, including partial deletions and hybrid events in paralogous regions such as CFH and CFHR1. Candidate variants were annotated using multiple databases to assess pathogenicity, population frequency, and functional relevance. The final dataset was curated to distinguish primary pathogenic mutations from secondary findings, with integration of clinical and biochemical data to support interpretation. This approach provided a robust framework for identifying disease–associated variants while minimizing the risk of misclassification due to technical artifacts or common polymorphisms.

The data processing and bioinformatic analysis began with converting the sequence data from enriched libraries into FASTQ files using MiSeq software. The Burrows Wheeler Aligner (BWA) was employed to match the FASTQ reads to the GRCh37/hg19 reference genome, resulting in BAM files. The Genome Analysis Toolkit (GATK) is utilized in order to analyze variants in the targeted regions of the specified genes. Variant filtration and analysis are performed using Illumina Variant Studio software, taking into account sequencing depth, quality, and allele frequency in various databases, including dbSNP [16], Genome Aggregation Database (GnomAD) [17], 1000 Genomes Project (1000G), and EXAC. Variants are named following the Human Genome Variation Society guidelines, and their pathogenicity is discussed in line with the standards of the American College of Medical Genetics and Genomics (ACMG).

2.6. Ethical approval

The study received approval from the Research Ethical Committee, Faculty of Medicine, Cairo University (MD-89–2022). Every procedure carried out complied with the 1964 Helsinki Declaration.

2.7. Statistical analysis

The analysis of data employed SPSS version 25 (IBM Corp). Descriptive statistics represented qualitative data in the form of percentages and numbers, whereas the quantitative were represented in the form of mean ± standard deviation (SD). Analytical statistics included the Chi-square test (χ²) for comparing and associating two qualitative variables, and ANOVA (f) test for comparing three or more groups with quantitative variables. A p-value of < 0.05 was set to denote statistically significant values. The p-value of < 0.001 indicated a highly significant result for two-tailed tests. But given the small sample size in each genetic subgroup, all p-values should be interpreted as exploratory and hypothesis-generating rather than confirmatory.

3. Results

3.1. Patient characteristics at presentation

The age of our cases at presentation ranged from 2 months to 12 years, with body weights varying from 5 to 40 kg. The study included a nearly equal number of males and females (11 males and 10 females). Two patients had a family history of renal disease, while 10 patients (47.6 %) came from consanguineous families.

We summarized all patients' clinical data in Table 1. as following, in all cases; the onset of aHUS was linked to a triggering event. Gastrointestinal symptoms such as diarrhea were observed in 10 patients (47.6 %), and vomiting occurred in 17 patients (81 %). Upper respiratory tract infection (URTI) was documented in 7 patients (33.3 %), and combinations of triggers were also noted—for example, vomiting and URTI co-occurred in 3 patients (14.3 %). During the initial episode, 11 patients (52.4 %) presented with uremic manifestations. Oliguria was reported in 17 patients (81 %), while neurological symptoms, including convulsions, temporary vision loss, or altered consciousness, were found in 8 patients (38.1 %). Cardiomyopathy developed in 1 patient (4.8 %), and hypertension was observed in 17 patients (81 %), with left ventricular hypertrophy (LVH) occurring in 4 patients (19 %) as a complication. Kidney biopsies were performed on 13 patients (63.9 %), all of which revealed thrombotic microangiopathy (TMA).

Table 1.

Clinical data of studied cohort:

Clinical Findings	Number of Patients	Percentage (%)
Triggering Events
Gastrointestinal symptoms (diarrhea)	10	47.6
Vomiting	17	81.0
URTI	7	33.3
Vomiting + URTI (co-occurrence)	3	14.3
Initial Episode Manifestations
Uremic manifestations	11	52.4
Oliguria	17	81.0
Neurological symptoms (convulsions, vision loss, altered consciousness)	8	38.1
Cardiomyopathy	1	4.8
Hypertension	17	81.0
LVH	4	19.0
Renal Biopsy Findings
Renal biopsies performed	13	63.9
TMA detected	13	100.0

LVH: Left ventricular hypertrophy, TMA: Thrombotic microangiopathy, URTI: Upper respiratory tract infection

The initial laboratory assessment revealed a mean hemoglobin level of 6.36 ± 0.847 g/dl and a mean platelet count of 89.43 ± 42.25 × 10³cells/mm³. Reticulocyte counts and LDH levels were elevated in all patients. The mean urea level was 196.24 ± 51.77 mg/dl, and creatinine averaged 6.08 ± 3.09 mg/dl. Proteinuria was observed in 71.4 % of patients, categorized as 1 + in 6 patients (28.6 %), 2 + in another 6 patients (28.6 %), and 3 + in 3 patients (14.3 %), with a mean albumin-to-creatinine ratio in urine of 1567 ± 1216 mg/g.

In terms of immunological findings, Coombs test results were negative for all patients. Low C3 levels were identified in 4 patients (19 %), while low C4 levels were found in 2 patients (9.5 %). Factor H assessments were conducted in 7 patients (33.3 %), revealing low levels in 3 of them (14.3 %). Anti-factor H analysis was performed in 6 patients (28.6 %), with positive results in 3 (14.3 %). ADAMTS13 activity was tested in 6 patients (28.6 %), and all showed normal results.

3.2. Genetic testing (WES results)

Regarding the WES results, 33.3 % of patients showed negative findings. CFHR3/CFHR1 deletion was identified in 6 patients (28.57 %). Other deletions included CFHR1/CFHR3/CFHR4, CFHR2/CFHR1, CFHR3, and CFHR5. Additionally, homozygous and heterozygous pathogenic variants were detected in DGKE, C8B, MMACHC, and MMUT genes, with each variant found in only one patient (4.8 %), as shown in Table 2.. Given the few cases for each mutated complement protein, data analysis was conducted using the descriptive method rather than through statistics-based comparison.

Table 2.

Molecular analysis of our patients (N = 21).

WES results	Zygosity	Pathogenicity	ACMG	N = 21	%
CFHR 1/CFHR3 Deletion	Homozygous or Heterozygous	−	−	6	28.57
CFHR 1/CFHR 3/CFHR 4 Deletion	Homozygous	−	−	1	4.8
CFHR 2/CFHR 1 Deletion	Homozygous	−	−	1	4.8
CFHR 3 Deletion	Heterozygous	−	−	1	4.8
CFHR 5 Deletion	Heterozygous	−	−	1	4.8
DGKE gene mutation (NM_003647.3: c.127C > T, p.Gln43*) (NM_003647:c.1632 T > G,p.Tyr544*)	Compound Heterozygous	Pathogenic VUS	PM2/PVS1 PM2/PVS1	1	4.8
MMACHC gene mutation (NM_001330540: c.269G > A, p.Gly90Asp)	Heterozygous	Pathogenic	PM3/PS3	1	4.8
MMUT gene mutation (NM_000255.4:c.643G > A, p.Gly215Ser)	Homozygous	Pathogenic	PM3/PP2	1	4.8
C8B gene mutation (NM_000066:c.1282C > T, p.Arg428*)	Heterozygous	Pathogenic	PVS1/PM3	1	4.8
Negative result	−	−	−	7	33.3

Whole exome sequencing (WES), The American College of Medical Genetics and Genomics (ACMG), Variants of uncertain significance (VUR).

Among the WES results, a patient with a homozygous MMUT gene mutation showed consumption of both C3 and C4. Another patient, who had a homozygous deletion of CFHR1, CFHR3, and CFHR4 genes, exhibited consumption of C3 only. Additionally, two patients with negative WES results also demonstrated C3 consumption. Furthermore, one out of six patients with a heterozygous deletion of the entire CFHR1 and CFHR3 genes displayed consumption of C4 only Table 3.. ADAMTS13 activity was assessed in six patients to exclude TTP as a differential diagnosis but all results were normal Table 3. In addition to the functional assay, genetic screening of ADAMTS13 was performed. No pathogenic or likely pathogenic variants were identified in these patients. Only benign polymorphisms and variants of uncertain significance (VUS) were detected, none of which are known to impair ADAMTS13 function.

Table 3.

Summarizes age at presentation, immunological and adams13 findings in different genotypes among our patients (n = 21).

WES results (n = 21)	The number of copies	Age at presentation (years, months)	C3	C4	Factor H	Antifactor H antibodies	ADAMTS 13
C8B gene mutation	one	9	normal	normal	low	positive	Normal
CFHR 1/CFHR 3/CFHR 4 Deletion	two	9 year and 6 months	low	normal	low	positive	NA
CFHR 2/CFHR 1 Deletion	two	11 months	normal	normal	NA	NA	NA
CFHR 3 Deletion	one	1 year and 2 months	normal	normal	NA	NA	NA
CFHR 3/CFHR 1 Deletions
CFHR 3/CFHR 1 Deletion (a)	one	1 year and 6 months	normal	low	low	positive	Normal
CFHR 3/CFHR 1 Deletion (b)	two	9 years	normal	normal	normal	negative	Normal
CFHR 3/CFHR 1 Deletion (c)	two	6 years	normal	normal	NA	NA	NA
CFHR 3/CFHR 1 Deletion (d)	one	7 years	normal	normal	NA	NA	NA
CFHR 3/CFHR 1 Deletion (e)	two	4 years	normal	normal	NA	NA	NA
CFHR 3/CFHR 1 Deletion (f)	one	2 years	normal	normal	NA	NA	NA
CFHR 5 Deletion	one	5 years	normal	normal	NA	NA	NA
DGKE gene mutation	two	2 months	normal	normal	NA	NA	NA
MMACHC	one	4 years and 6 months	normal	normal	NA	NA	NA
MMUT gene mutation	two	8 months	low	low	NA	NA	NA
Negative results
Negative result (a)	−	4 years	low	normal	NA	NA	NA
Negative result (b)	−	1 year and 7 months	low	normal	normal	negative	Normal
Negative result (c)	−	6 months	normal	normal	normal	negative	Normal
Negative result (d)	−	1 year	normal	normal	normal	NA	Normal
Negative result (e)	−	4 months	normal	normal	NA	NA	NA
Negative result (f)	−	7 years	normal	normal	NA	NA	NA
Negative result (g)	−	12 years	normal	normal	NA	NA	NA

Letters (a–f) indicate individual patients carrying CFHR3/CFHR1 deletions or different patients with negative results. One copy (heterozygous), two copies (homozygous). NA: Not Applicable. WES: whole exome sequencing, ADAMTS 13: A DisintegrinAnd Metalloproteinase with ThromboSpondin type 1 motif, member 13.

3.3. Treatment and Clinical outcomes

According to modality of treatment, nineteen patients (90.50 %) needed renal replacement therapy, 12 patients required hemodialysis with mean frequency of sessions needed 7.83 ± 7.396 (2 to 30 sessions), and 11 patients were treated by acute peritoneal dialysis with mean of 3.09 ± 1.514 (1 to 6) sessions. 4 of these patients required both hemodialysis and peritoneal dialysis.

The two patients, who did not need renal replacement therapy, had the DGKE gene mutation and heterozygous pathogenic variant within the MMACHC which is defective in patients with cobalamine disorders belonging to cb1C complementation group.

Fourteen patients (66.6 %) were treated by plasmapheresis, with a mean number of sessions of 17.86 ± 9.945, and 9(42.9 %) patients received plasma transfusions. Only one patient could be treated by Eculizumab, this patient carried heterozygous pathogenic variant in the C8B gene cause complement C8 deficiency, Eculizumab was initiated due to poor response to plasmapheresis in the first attack of relapse (he was improved on plasmapheresis at presentation) but unfortunately the patient showed no response and died.

After treatment most patients progressed to chronic kidney disease (11 patients) (52.4 %). Six (28.6 %) patients recovered their kidney functions, all of them were treated with plasmapheresis; and 4(19 %) passed away. There was no significant relation between all outcomes (recovered, CKD, deceased) and all modalities of treatment except peritoneal dialysis (p = 0.035).

As regards correlation between WES results and the outcome, it showed no significant difference (p = 0.443). The 6 patients who recovered their kidney functions harbored the following mutations: CFHR2/CFHR 1 deletion in one patient, CFHR 3/CFHR 1 deletion in three patients, MMACHC in one patient, and in one patient no mutation could be detected. While the eleven patients who progressed to chronic kidney disease exhibited the following mutations: CFHR 1/CFHR 3, CFHR 4 deletion in one patient, CFHR 3 deletion in one patient, CFHR 3/CFHR 1 deletion in three patients, CFHR 5 deletion in one patient, DGKE gene in one patient, MMUT gene variation in one patient, and no mutation could be detected in three patients. As regards the four patients who passed away, only one had mutation in C8B gene who received Eculizumab, while in the other three patients no mutation could be detected.

Relapse is a feature of aHUS, this study shows significant correlation between WES results and occurrence of relapse (p = 0.021). Patients with CFHR 3 deletion, CFHR 5 deletion, and MMUT gene mutation developed at least on attack of relapse.

4. Discussion

This study represents a pioneering examination of the clinical presentation and genetic findings using WES of pediatric aHUS cases in Egypt, where only one study highlighted the role of complement dysregulation in aHUS and the prevalence of anti-factor H antibodies in Egyptian children [18]. While no gender-based differences in disease incidence were observed, consanguinity was notable among parents, affecting 47.6 % of cases reflecting regional population genetics and cultural practices in Egypt, however, a familial trend for aHUS was absent, as no patient demonstrated a documented family history of the disease, also there were two cases reported a family history of renal disorders, none had a documented family history of aHUS specifically or thrombotic microangiopathy.

WES has significantly advanced the understanding of aHUS by identifying pathogenic variants within the aHUS disease gene panel. Pathogenic variants, such as those in complement regulatory genes like CFH, CFI, and MCP (CD46), have been directly linked to the dysregulation of the complement system, a hallmark of aHUS. These findings have informed targeted therapeutic strategies, such as the use of complement inhibitors, such as ravulizumab and eculizumab, which have demonstrated efficacy in mitigating disease progression [19].

Previous reports underscored the importance of genetic insights in aHUS management as studies by Shawky et al., 2021 and Fakhouri et al., 2023 10, 18, These illustrate how WES results, whether pathogenic or variant of unknown significant are pivotal in shaping personalized treatment approaches for aHUS.

AHUS relates to genetic variations and autoimmune forms, nonallelic homologous recombination events causing hetero- or homozygous deletions of CFHR1 and CFHR3 of anapproximately 24-kbchromosomal segment that encompasses the CFHR3-CFHR1 genes is noticed in approximately 15 % of predominantly young cases with aHUS. For these patients with DEAP-HUS (Deficiency of CFHR plasma proteins and factor H- HUS), the genetic scenario is frequently related to the presence of autoantibodies that bind to the C-terminal region of Factor H (SCRs 19–20) and block surface binding [20].

Within the scope of this research, 10 out of 21 participants (47.6 %) exhibited deletion mutations in the CFHR gene. Among them, only 2 individuals (22.2 %) tested positive for factor H autoantibodies. Notably, one patient possessed a homozygous deletion encompassing CFHR1, CFHR3, and CFHR4, whereas another displayed a heterozygous deletion of CFHR1 and CFHR3 Table 3. The precise mechanism through which CFHR deletions contribute to the emergence of autoantibodies remains enigmatic. Nonetheless, approximately 87 % of HUS patients presenting with factor H autoantibodies demonstrate CFHR3/CFHR1 deletions [21], although verification is hindered by the constrained accessibility of anti-factor H analysis. Pediatric cases involving anti-factor H antibodies predominantly manifest between the ages of 6 and 17 years [22], with occurrences among infants being exceptionally rare. These observations are consistent with the findings of this study, which include two patients aged 9–10 years however one patient was 1.5 years old, CHO et al., 2007 study, reported aHUS cases with factor H autoantibodies presenting in infancy often showing severe symptoms like renal failure, anemia, and thrombocytopenia, sometimes mimicking lupus, with diagnosis relying on low complement (C3/C4), high factor H autoantibodies, and sometimes genetic factors (like CFH mutations) causing severe complement dysregulation [23]. In Table 3, normal C3 plasma concentration does not eliminate the presence of a mutation in the complement system or of factor H autoantibodies. Conversely, decreased C3 level signs the presence of a complement abnormality [24].

Different studies have contributed comparative insights into the prevalence and clinical implications of CFHR1/CFHR3 deletions, it is a common genetic variation associated with aHUS, with its prevalence varying by ethnicity, and it was found in 5–10 % of studied populations [25]. This deletion is significantly related to anti-factor H antibodies, detected in 5–13 % of European aHUS patients [26] and up to 56 % in Indian populations [27].While the mechanism underpinning anti-factor H antibody development remains elusive, Moore et al., 2010 study has established a robust association between CFHR gene deletions and the presence of these autoantibodies after screening of 142 aHUS patients. A substantial proportion of aHUS patients exhibit complete CFHR1 deficiency, predominantly driven by homozygous CFHR3-CFHR1 deletions, with occasional cases of compound heterozygous CFHR3-CFHR1 and CFHR1-CFHR4 deletions [25]. This genetic variability contributes to a spectrum of clinical outcomes, including severe extra-renal manifestations like cardiomyopathy and neurological symptoms [28]. Table 3 showed that one out of 6 patients with CFHR1/CFHR3 deletions had complement C3 consumption that is in accordance with Bhandari et al., 2023 study who reported that the same pathogenic variant in aHUS might result in variable effects at the systemic complement levels (that might be normal in some cases) due to several factors, primarily the “two-hit” hypothesis, the nature of the affected protein, and individual genetic variability [29].

Additionally, among our studied cohort group, in only one patient WES identified deletion in the CFHR5 gene. The deletion or mutation in the CFHR5 gene has been linked to aHUS and other complement-mediated disorders, as highlighted in several studies 30, 31. CFHR5, a protein involved in complement regulation, serves a twofold purpose as both a regulator and activator of the complement system, depending on the microenvironment [32]. In aHUS patients, CFHR5 mutations can lead to dysregulated complement activation, resulting in hemolysis and thrombotic microangiopathy (TMA) [31]. Clinical findings from case reports and studies revealed that patients with CFHR5 mutations often present with severe renal impairment, including acute kidney injury and progression to ESKD [28]. Extra-renal manifestations, such as cardiomyopathy, pulmonary involvement, and neurological symptoms, have also been observed, particularly in cases resistant to standard therapies like eculizumab. Genetic studies have identified structural variants in the CFHR5 gene, including hybrid genes and copy number variations, which exacerbate complement dysregulation [30]. The results highlight the significance of timely diagnosis and targeted treatment to mitigate organ damage and improve outcomes in affected patients. The clinical variability and complexity of CFHR5-related aHUS highlight the need for further research to unravel its precise role in complement-mediated diseases.

DGKE gene was reported to cause autosomal recessive Nephrotic syndrome, type 7 and susceptibility to aHUS. The age of presentation for aHUS associated with DGKE gene mutations of compound heterozygous pathogenic stop codon variants (NM_003647.3: c.127C > T, p.Gln43*) and (NM_003647.3: c.1632 T > G, p.Tyr 544*) is notably early, as evidenced by our study, where a patient exhibited symptoms at just two months of age, and who passed into CKD. This aligns with findings from Ozaltin et al., 2013 study that identified a subgroup of aHUS patients with mutations in the DGKE gene manifesting before one year of age, DGKE encodes diacylglycerol kinase-e, an intracellular protein crucial for maintaining podocyte homeostasis and regulating protein kinase C activity in endothelial cells and platelets. Its dysfunction disrupts cellular signaling pathways, contributing to the pathogenesis of aHUS [33]. The role of DGKE in aHUS outcomes is significant, as mutations in this gene are associated with severe disease progression- as in our patient- and resistance to complement-targeted therapies like eculizumab. Recent studies emphasize the need for alternative therapeutic approaches tailored to DGKE-related aHUS. For instance, a case reported by Dai et al., 2023[34] highlighted the poor prognosis and high mortality rate in DGKE-aHUS patients, underscoring the importance of early diagnosis and intervention. Similarly, Shin et al., 2022 [35] demonstrated the challenges in managing recurrent aHUS caused by DGKE mutations, advocating for personalized treatment strategies. These findings reinforce the critical impact of DGKE mutations on disease outcomes and the necessity for innovative management protocols.

Moreover, in our study, WES identified a heterozygous pathogenic variation in the C8B gene (NM_000066:c.1282C > T,p.Arg428*) leading to complement C8 deficiency type II in one patient (4.8 %). Tragically, this patient succumbed within the first year, likely due to severe infections episodes of gastroenteritis consistent with the role of complement pathway variants in infection–related disease onset. This finding emphasizes the critical role of the C8B protein in immune defense, particularly in forming the membrane attack complex (MAC) essential for combating bacterial infections [36]. C8 deficiency, an autosomal recessive condition, manifests in two forms, with C8 beta subunit deficiency being one. Patients with this deficiency are highly susceptible to recurrent Neisseria infections, such as meningitis. Comparatively, studies have reported varying prevalence rates of complement deficiencies across populations. For instance, the Primary Immunodeficiency Network documented a prevalence of 0.0027 % for C8 deficiency in Japan and 70 reported cases in the UK. Additionally, deficiencies in terminal complement components, including C5, C6, C7, and C8, are consistently linked to heightened vulnerability to Neisseria infections. Interestingly, individuals with C9 deficiency retain some hemolytic activity, as C5b-C8 can mediate erythrocyte lysis [37]. In another study [38] highlighted the clinical significance of C8B mutations;the studyemphasized the importance of early diagnosis and prophylactic measures, such as vaccination and antibiotic therapy, to prevent severe infections in patients with C8 deficiency. Similarly, findings from the Immune Deficiency Foundation underscore the necessity of complement screening in at-risk individuals to mitigate infection-related complications [39]. These insights reinforce the pivotal function of C8B in immune function and the need for timely intervention in affected patients.

In our study, we examined a unique case of a single patient who presented with a heterozygous variant in MMACHC (NM_001330540: c.269G > A, p.Gly90Asp). Mutations in the MMACHC gene are well-documented as causative of autosomal recessive Methylmalonic aciduria and homocystinuria, cblC type. Our patient exhibited symptoms at 4.5 years of age, which is an older age compared to the French cohort (mean age of 1.5 years) and the Dutch cohort (typically under 1 year of age) 40, 41. Unlike these aforementioned cohorts, where severe kidney dysfunction frequently necessitated renal replacement therapy upon presentation, our patient experienced a remarkable recovery of renal function within the first year without requiring such intervention. This divergence could potentially be attributed to variations in genetic expression, possibly influenced by the inheritance of a monoallelic pathogenic variant. Furthermore, the probability of deep intronic mutationsparticularly as pathogenic variants in the MMACHC deep intron gene have been documented [42] alongside environmental factors. Recent studies support these findings, such as the Fakhouri et al., 2021 study that analyzed 187 patients with aHUS and identified genetic variants, including MMACHC, correlating them with clinical outcomes. The study highlighted the variability in disease presentation and the importance of genetic testing in guiding treatment [43]. Similarly, a cohort study by Connaughton et al, 2023 emphasized the challenges in interpreting genetic variants like MMACHC and their impact on disease progression [44]. These studies underscore the need for individualized approaches to managing aHUS based on genetic and clinical profiles.

The homozygous missense variant in the MMUT gene (NM_000255.4: c.643G > A, p.Gly215Ser) identified in one patient (4.8 %) has not been previously reported in aHUS cases; nevertheless, other mutations in MMUT gene have been well-documented in connection with methylmalonic acidemia (MMA) [45], a disorder linked to defects in methylmalonyl-CoA mutase [46]. We therefore interpreted MMUT variants as causative for MMA, with aHUS occurring as a secondary complication in selected case, rather than attributing aHUS directly to homozygous MMUT variants alone. This clarification ensures accurate gene nomenclature and a precise distinction between MMA–related aHUS and other genetic forms of aHUS. For instance, the c.278G > A p.Arg93His variant has been identified as pathogenic, impairing enzyme activity and contributing to MMA. The c.322C > T p.Arg108Cys mutation, predominantly observed in the Mexican population, induces significant structural alterations in the catalytic domain, leading to severe clinical manifestations such as neurodevelopmental delays and high mortality rates [47]. Additionally, the p.Thr230Arg (c.689C > G) mutation was reported in a consanguineous Pakistani family and was associated to MMA mmut type, disrupting enzyme function and causing metabolic acidosis alongside neurological complications [46]. Also, high frequency of N219Y in Caucasians, French, Turkish descent were reported [47]. Collectively, these mutations compromise the conversion of methylmalonyl-CoA to succinyl-CoA, which results in accumulating toxic metabolites that precipitate variable clinical outcomes, including developmental delays, organ dysfunction, and metabolic crises 28, 48.

AHUS cases are highly risked to progress to CKD stages 3–5 [49]. About half of the patients of aHUS have progression to ESKD, which a high mortality rate of up to a quarter of the cases [50]. The rates fluctuate according to the specific mutation; for instance, the rates of ESKD are 60–80 % for mutations involving C3, FH, FB, and FI, although they only reach 30–40 % in instances with MCP mutations [51]. But regarding our study group, 52.4 % of patients progressed to CKD while mortality rate was 19 % and there is no significant association between WES results and this outcome (p = 0.443).

About 28.6 % of our patients recovered their kidney functions, all of them after plasmapheresis which is considered low percentage if we compare it with a recent study by Zagożdżon et al., in 2024 as 55 % of cases with aHUS recovered their kidney functions initially, and a total of 50 % still were fully recovered when followed up for two years. Forty five percent developed CKD and 5.2 % passed away [52]. In our cohort patients recovered their kidney functions within the first year after initial presentation, which may be related to early initiation of plasmapheresis.

Relapse is a common occurrence for those who have genetic aHUS, even after they have made a full recovery from the current episode [53]. So as relapse is a feature of aHUS, this study shows 14.3 % (three patients) developed at least one attack of relapse with significant correlation between WES results and occurrence of relapse (p = 0.021). We found that patients with CFHR 3 deletion, CFHR 5 deletion, and MMUT gene mutation developed at least one relapse.

This contrast of findings among our patients and other studied cohorts may be attributed to potential variations in genetic expression, environmental factors, and the availability of early interventions across different populations. Furthermore, the variability in renal outcomes accentuates the importance of genetic and clinical profiling for individualizing treatment approaches and predicting disease trajectories.

In cases where only a single heterozygous variant was identified in a recessive gene, possible susceptibility factors to TMA phenotype, rather than established causes. This distinction acknowledges the multifactorial nature of aHUS/TMA pathogenesis.

Limitations: Unfortunately, lack of access to complement inhibitors, limited diagnostic capabilities, in addition to the economic burden of the disease, hinder the implementation of international guidelines for management of aHUS in developing countries as Egypt including genetic analysis. This justifies the development of local practice guidelines that can be implemented in developing countries according to the availability of different diagnostic tools and treatment options. The restricted testing for anti–factor H autoantibody may underestimate the frequency of anti–FH autoantibody positivity among our patients, and future studies with expanded screening are warranted to more accurately define the role of anti–FH autoantibodies in Egyptian aHUS cases.

5. Conclusion

The vast spectrum and varied manifestations of aHUS make it difficult to identify, which may result in a postponement in the initiation of targeted therapy. In our Egyptian patient group; negative genetic analysis can be found in large proportion while CFHR 3/CFHR 1 deletion was the most common gene mutation that is often associated with relapse of the disease. Further studies on a larger cohort and longer periods of follow-up are needed for further characterization of laboratory and clinical features and long-term results of our cohort, and to be able to correlate these data with WES results, and also to verify the prevalent genetic mutations in our population.

6. Ethical approval and consent to participate

The study received approval from the Research Ethical Committee, Faculty of Medicine, Cairo University (MD-89–2022). Every procedure carried out complied with the 1964 Helsinki Declaration. Before being included in the study, the care givers of the cases provided informed consent.

7. Consent for publication

Not applicable (no identifying information about participants is available in the article.

8. Clinical trial number

not applicable.

9. Data availability

The datasets used and/or analysed during the current study are available from the corresponding authors on reasonable request.

Author contribution

All authors contributed to the study conception and design. Material preparation done by Fatina I. Fadel and Ghada El-kamah. Data collection and Statistical analysis were performed by Rasha EssamEldin Galal, Khalda Amr, Mohamed A Abdel Mawla, Amr Mohamed Salem, Mohamed S. Thabet and Shorouk A.Othman. The first draft of the manuscript was written by Shorouk A.Othman and Khalda Amr and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

CRediT authorship contribution statement

Fatina I. Fadel: Supervision, Methodology, Conceptualization. Khalda Amr: Writing – original draft, Data curation, Conceptualization. Rasha EssamEldin Galal: Supervision, Methodology. Ghada El-kamah: Supervision, Formal analysis. Mohamed A Abdel Mawla: Formal analysis, Data curation, Conceptualization. Amr Mohamed Salem: Formal analysis, Data curation. Mohamed S. Thabet: Data curation, Conceptualization. Shorouk A.Othman: Writing – review & editing, Writing – original draft, Methodology, Investigation, Data curation, Conceptualization.

Funding

No funding resources were received for conducting this study.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.