Patient 1 (Registry code 193)
The male patient was born in Kosovo at week 39 of an uncomplicated pregnancy, with a birth weight of 3400 g. At the age of 7 months, he was taken to the Children’s Hospital in Prishtina due to sudden onset of pallor, weakness, and anemia. There was neither fever, nor diarrhea. His initial laboratory findings (see Table 1) showed severe anemia and thrombocytopenia with normal renal function and diuresis. Direct antiglobulin test was negative.
He was initially considered to have sepsis and was given antibiotic treatment. He received packed red blood cells and fresh frozen plasma transfusions on three occasions. His hematological parameters improved, but on day 10 of hospitalization, his renal function deteriorated rapidly. He became anuric and decompensated dilated cardiopathy developed, which required hospitalization at the University Children’s Hospital Skopje, for peritoneal dialysis.
Atypical HUS was suspected, but (after plasma therapy) his hematological parameters were inconclusive (haemoglobin level and platelet count were normal) on admission. Fragmented red blood cells could not be demonstrated even by repeated blood smear analysis, and complement C3 level was normal at 0.90 g/L. Peritoneal dialysis improved the patient’s clinical condition and cardiac function. His creatinine level peaked at 316 μmol/L.
On day 19 of hospitalization, his overall condition worsened, severe anemia and seizures occurred. This time, blood smear analysis revealed many schistocytes. Follow-up laboratory tests confirmed the relapse of severe anemia and thrombocytopenia. At this point, the clinical diagnosis of atypical HUS was certain and appropriate blood samples were sent to Hungary for the comprehensive analysis of complement factors. These blood samples, received 24 days after disease onset and after plasma therapy, were tested on the day of arrival of sample for C3, alternative pathway and ADAMTS13 activity, and the results indicated severe complement dysregulation.
The patient was intubated and mechanically ventilated, transfused on three occasions with packed red blood cells and fresh frozen plasma. Despite intensive therapy and the administration of plasma, the baby fell into coma. Respiratory failure accompanied by symptomatic multiorgan failure led to the patient’s death on day 29 of hospitalization. Autopsy was not performed.
Patient 2 (Registry code 229)
The male neonate was born in Hungary, from an uneventful pregnancy and delivered by the vaginal route, at 36 weeks of gestation, with a birth weight of 2150 g.
On the second day of life, poor feeding, abdominal distension, hepatomegaly, respiratory distress were observed along with reduced skin perfusion and oliguria despite normal blood pressure. Bacterial sepsis was suspected and antibiotic treatment was introduced.
The laboratory tests showed anemia, low platelet count, and metabolic acidosis, as well as bleeding disorder (increased INR; fibrinogen: under detection limit), and a marked elevation of LDH. However, no hyperbilirubinemia was present. On the 4th day of life, uremia, low platelet count, and metabolic acidosis persisted, and moderate increase of alanine aminotransferase and aspartate aminotransferase levels were seen. Direct antiglobulin test was negative.
Progressive circulatory failure required mechanical ventilation and catecholamine treatment from the 4th day of life. Echocardiography depicted a haemodynamically significant, patent ductus arteriosus. The differential diagnosis excluded bacterial sepsis, renal vein thrombosis, and metabolic disorder. The treatment was supplemented with regular, periodic administration of fresh frozen plasma, based on the clinical signs and laboratory results indicative of aHUS.
Progressive circulatory failure, oliguria and uremia culminated in extreme volume overload and pulmonary edema. Therefore, hemofiltration was started on the 9th day of life and repeated altogether 7 times. Despite all these efforts, the patient did not improve and died on the 19th day of life of circulatory failure unresponsive to the treatment.
The serum complement profile obtained on day 14 showed alternative pathway dysregulation and supported the diagnosis of aHUS. Investigations during autopsy could not identify any alternative (infective or malignant) causes of HUS. The histological examination of the kidney and the lung revealed signs of small vessel microangiopathy (thickening of capillary walls, lamellation, detachment of endothelial cells, narrowing of the capillary lumen).
Patient 3 (Registry code 304)
The female infant was born in Austria, at 38 weeks of gestation with a birth weight of 3750 g. The antenatal and the perinatal periods were uneventful.
At the age of 2 months, she developed acute abdominal distension and cyanosis. There was no history of infection, vomiting, or diarrhea in the previous days.
Initial laboratory findings included high LDH, CK, and serum transaminase levels. Serum albumin concentration was greatly reduced. INR, PTT, and thrombin time were prolonged, C-reactive protein was normal; interleukin-8 level and the leukocyte count were elevated. Renal function parameters, platelet count, and hemoglobin were normal on day 1.
Abdominal ultrasound showed massive edema of the bowels, particularly of the colon and the duodenum. A plain abdominal film did not depict any sign of free air. The progressive decline of oxygen saturation and worsening respiratory insufficiency necessitated respiratory support and then, intubation and mechanical ventilation.
During the next hours, oliguria progressed to anuria, and generalised edema developed. Simultaneously, platelet count and hemoglobin level decreased, whereas renal function parameters increased. Peritoneal dialysis was initiated on day 2 due to progressive edema and renal insufficiency. The follow-up laboratory tests showed further elevation of the LDH level. Complement dysregulation was detected on day 2 (Table 2). The direct antiglobulin test was negative; haptoglobin level was decreased, and free hemoglobin concentration was increased in the plasma. These findings indicated intravasal hemolysis; however, fragmentocytes were absent from the blood smear. Irreversible multiorgan failure developed during the next hours; the CT scan confirmed generalized brain edema, while further testing excluded underlying infectious and/or metabolic causes of the multiorgan failure. The patient died on day 3.
Investigations during autopsy could not identify any alternative (infective or malignant) causes of HUS. The histological examination of the kidney, spleen, lung, bowel, and myocardium confirmed thrombotic microangiopathy.
The results of complement- and genetic testing
The detailed results of complement profiling are presented in Table 2. Alternative complement pathway activity was decreased in all three patients. The consumption of C3, as well as decreased levels of complement factor B and I were found in all cases. Factor H level was below the detection limit in patient 1 and 3, and it was decreased in patient 2. The level of C4 and the activity of the classical pathway were decreased in patients 2 and 3. Since we observed unusual concomitant decrease in these factors in patients 2 and 3, we have measured the levels of potassium, magnesium, calcium and phosphate in these serum samples and none of them was decreased excluding the possibility that samples were diluted with infusion.
ADAMTS13 activity was decreased but not deficient in all cases, and the presence of the ADAMTS13 inhibitor was excluded in two cases (Table 2). None of the family members had low (<67%) ADAMTS13 level indicative of ADAMTS13 deficiency. We suspect secondary ADAMTS13 consumption by the ultra-large von Willebrand factor due to extensive endothelial activation.
All patients were negative for anti-factor H autoantibody. Regarding the family members of patients, no signs of complement deficiency or dysregulation was detected in the family of patient 1. The father of patient 2 had low levels of factor B, I and C3, the father of patient 3 had below normal level of factor B, while other family members in these families had normal complement parameters.
To investigate the genetic alterations in our patients, we sequenced the coding regions of complement genes CFH, CFI, CD46, THBD, CFB, C3 and DGKE. Patient 1 was heterozygous for a cytosine to adenine substitution (c.2165C>A) in exon 15 of CFH (encoding short consensus repeat 12) that causes a serine to STOP change at p.722 (Ser722Stop) in the protein. Mutation at this site has not been reported previously. Based on the analysis of inheritance in the patient’s family, this mutation was located on an allele coding histidine at p.402. As the patient was heterozygous for the Tyr402His (Y402H) polymorphism, we applied an ELISA test system using two different monoclonal antibodies, specific for the His402- and Tyr402- containing factor H proteins (HK353 ELISA kit, Hycult Biotech). The level of factor H protein from the Ser722Stop mutation-containing allele (p.402His-containing factor H) was below the lower limit of quantification, while the level of factor H from the other allele (p.402Tyr-containing factor H) was low, but still measurable in the serum sample of the patient. The mutation-carrying father of the patient was not heterozygous for the Tyr402His polymorphism, therefore there are no comparable alleles in his serum, so we did not apply this method in this case. Based on these results we concluded that the mutation p.Ser722Stop by causing the premature termination of translation presumably leads to deficient synthesis and/or secretion of the mutated protein. No disease-causing mutation was detected in the other two patients.
Sequencing of the selected complement genes revealed the presence of multiple polymorphic variants. As inferred from genotype data, one patient (patient 1) carried the H3 risk haplotype of the CFH gene, and two patients (patient 1 and patient 3) carried risk alleles of MCP polymorphisms (MCPggaac). The results of the pedigree analysis are presented in Table 2.
To reveal deletions or duplications that may influence disease development, CFI, CD46 (MCP) as well as CFH and its related genes were studied by applying MLPA probemixes of MRC-Holland. None of the patients showed copy number alterations in the selected genes.