Supplementary Table. Complement Genetics, Auto Antibodies and Levels in Patients With

Supplementary Table. Complement genetics, auto antibodies and levels in patients with secondary aHUS Variants of uncertain Anti FH auto Patient Genes analyzed (1) Pathogenic variants FH* MCP* C3* C4* significance (2) antibodies (3) NGS panel 1 None CFHR2: Y264C No 183 92 130 100 (Ion Torrent) CFH, MCP, CFI 2 None - No 166 49 69 53 (Sanger) NGS panel 3 None - No ND 70 96 106 (Illuminia) CFH, MCP, CFI 4 None - No 198 87 104 100 (Sanger) NGS panel 5 None - No 148 76 110 108 (Ion Torrent) NGS panel 6 None - No 206 99 116 94 (Illuminia) NGS panel 7 None CFHR2: Y264C No 113 89 73 94 (Ion Torrent) NGS panel 8 None - No 182 78 58 86 (Ion Torrent) MCP: c.-325A>C NGS panel 10 None THBD: c.*36G>A No 140 101 125 104 (Ion Torrent) ADMTS13: W470L NGS panel 357-1094 11 None THBD: c.*23_*40del 135 83 143 110 (Illuminia) AU/ml NGS panel 12 None - 157 AU/ml 162 100 97 85 (Illuminia) NGS panel 14 None - No 231 92 106 97 (Ion Torrent) NGS panel 15 None - No 186 86 126 115 (Ion Torrent) CFH, MCP, CFI 16 None - No ND 79 17 100 (Sanger) NGS panel 17 None - No 148 87 87 120 (Illuminia) NGS panel 21 None - No 177 137 90 110 (Illuminia) NGS panel CFHR1: p.L290S 22 - No 72 118 76 ND (Illuminia) CFHR1: p.A296V NGS panel 24 None - No 174 98 98 93 (Ion Torrent) NGS panel 25 None CR1: p.A495V No 138 145 116 99 (Illuminia) NGS panel 26 None - No ND 117 119 111 (Illuminia) NGS panel 27 None - No 170 87 108 100 (Ion Torrent) NGS panel CFHR5: p.C568* 29 CFH: p.C597* No 58 81 73 103 (Ion Torrent) CFHR5: G145E (1) NGS panel (Ion Torrent) includes the CFH, CFI, MCP, C3, CFB, THBD, ADAMTS13, DGKE, CFHR1, CFHR2, CFHR3, CFHR4, CFHR5 and CFP genes. NGS panel (Illumina) interrogates as many as 48 genes and includes all known complement genes. (2) We consider variants of uncertain significance those with unknown functional consequences or unknown relationship with aHUS. (3) Auto antibodies are expressed in arbitrary units (AU). Lower limit is 135 AU/ml. Abbreviations: ND: not done. *Normal values: FH: 90-302 µg/ml, MCP: 91-109%, C3: 75-135 mg/dl, FI: 71-115% SupplementaryMethods

Data collection

Data were compiled from the medical records of the participating centers using a uniform protocol and included age, gender, blood pressure and cause of aHUS. Analytical variables included hemoglobin, platelet count, haptoglobin, lactate dehydrogenase, presence of schystocytes in peripheral blood smear, Coombs test, ADAMTS-13 determination, Shiga

Toxin detection, SCr, eGFR, (which was calculated using the 4-variable Modification of Diet in Renal Disease equation), serum albumin, and urinalysis. Results of the genetic analysis, performed in 22 patients were registered following a common protocol. Renal biopsies were performed in 18 patients and were reviewed for this study at every participating center.

Treatments received before eculizumab, number of plasmapheresis sessions and the time interval between aHUS diagnosis and onset of eculizumab were recorded, as well as the duration of eculizumab therapy and the cumulative dose administered to every patient.

Complications occurring during plasmapheresis and eculizumab treatment, side effects, the occurrence of end-stage renal disease (ESRD) and deaths were also recorded.

Genetic analysis and other complement studies

Complement studies were performed to identify anomalous FH and FHRs bands by western blot analyses of plasma samples. Plasma C3, FH and FI levels were measured in serum or plasma using a sandwich ELISA method. MCP levels in peripheral blood lymphocytes were determined by flow cytometry. Auto antibodies to FH were analysed by ELISA using purified

FH-coated plates.

For genetic analysis, genomic DNA was prepared from peripheral blood cells according to standard procedures. In three patients, the CFH, CFI, MCP and CFHR1 genes were analyzed by Sanger sequencing. In these cases all gene exons were amplified from genomic DNA by using specific primers derived from the 5’and 3’intronic sequences and automatic sequencing was performed in an ABI3730 sequencer using a dye terminator cycle sequencing kit

(Applied Biosystems). Nineteen patients were analyzed for mutations in the complement genes using two different next generation sequencing (NGS) panels that we developed to screen the complement genes and other genes associated with aHUS and C3G. The Ion proton

NGS panel includes the CFH, MCP, CFI, C3, CFB, DGKE, THBD, CFHR1, CFHR2,

CFHR3, CFHR4, CFHR5, CFP and ADAMTS13 genes. In this protocol the targeted genomic enrichment is performed using the multiplex PCR Ampliseq platform and the libraries generated using the Ion Xpress Library kit 4471269. Sequencing was performed on a 318v2 chip and the Ion PGM Sequencing 200 kit v2 4482006 using an IonProton sequencer. The

Illumina NGS panel was designed to include all the complement genes and some additional genes that have been described relevant to the pathologies under consideration; 48 genes in total. Targeted genomic capture in this case is performed using Nextera rapid capture custom

Enrichment Kit from Illumina and sequencing data generated in a Miseq equipment using

Miseq reagent kit v2 (300 cycles). Sequence data from both NGS panels was analyzed using the Burrows–Wheeler Alignment and Picard software with additional filtering using customs tools. Variant calling was performed both with bcftools and VarScan and the variant calling files generated merged in one single file using customs tools. Variant annotation was carried out with ANNOVAR. Common variants with a minor allele frequency value >1% in any population were excluded. We identified novel and/or pathogenic variants we used different databases (Exome Variant Server, 1000 Genomes, NCBI dbSNP, aHUS mutation database

(www.fh-hus.org) or our in-house database), combined multiple functional prediction methods (SIFT, PolyPhen2, etc) and performed functional determinations in the plasma of the carriers. The analysis of copy number variations within the CFH-CFHRs gene region was performed by multiplex ligation-dependent probe amplification (MLPA) with the P236 A1

ARMD mix 1 (MRC-Holland, Amsterdam, Netherlands).