Screening for Complement System Abnormalities in Patients with Atypical Hemolytic Uremic Syndrome

Serum Measurement

When possible, blood should be taken for CFH or CFI measurement before plasma infusion/exchange is commenced. If blood is taken in a convalescent period, then it should be at least 2 wk after the last infusion of plasma to ensure that the levels measured are from natively synthesized proteins.

Serum C3 and C4 levels generally will form part of a basic complement screen. In almost all cases of aHUS, C4 levels are normal (9). Low C3 levels are commonly seen in patients with mutations in CFH, CFI, and MCP (9). However, this is not a sensitive screening test, and normal C3 levels do not exclude the presence of mutations in complement regulatory proteins (15). In those with mutations in CFH, approximately 50% have normal C3 levels. For CFI, this figure is 40% and for MCP approximately 70% (15). There is also a group with low C3 levels (approximately 30%) and no mutations in MCP, CFH, or CFI (9). This group likely represents a cohort with mutations in an as-yet-unidentified complement gene.

CFH and CFI Levels

Because most cases of CFH-associated HUS have a heterozygous mutation in CFH (15), patients in whom the mutant protein is not expressed would be expected to have a 50% level. Factor H serum levels, however, vary considerably, and the range changes with age (neonates 170 to 397 μg/ml; adults 242 to 759 μg/ml) (16). Most children reach adult levels by 1 yr of age. It therefore is essential that an age-matched control panel be established in a reference laboratory to give interpretable results. No significant difference in CFH levels has been shown between genders (16).

From the reports to date, serum measurement of CFH will detect approximately 25% of patients with mutations in CFH (15). As with CFH, there are differences in CFI concentration according to age (neonate 15 to 55 μg/ml; adults 39 to 100 μg/ml) (16). Again, most children reach adult levels by 1 yr of age. Serum measurement will be low in approximately 40% of mutations in CFI (15).

Commercially available radioimmunodiffusion assay kits for CFI and CFH are available and give reliable results on serum or plasma. Serum generally is preferred, and it should be collected and stored frozen as the laboratory does for C3 and C4 assays.

MCP Levels

FACS analysis of peripheral blood mononuclear cells provides a quick and relatively cheap screening option for MCP mutations. From currently available data, screening in this manner will detect approximately 75% of mutations (12). The mean fluorescence intensity in patients and family carriers of a heterozygous mutation should be approximately 50% of the normal range. Rare carriers of either compound heterozygous or homozygous MCP mutations show almost absent MCP signal by FACS (9,10,12).

There is a modest (approximately 20%) variation in MCP expression level on peripheral blood mononuclear cells using serial measurements up to 5 d after venipuncture (unpublished observations). When possible, blood should be examined at a standard number of days after collection and be compared with wild-type controls that were collected and stored under similar conditions. We recommend that blood be stored for no more than 5 d at 4°C before FACS analysis.

Genetic Analysis of the Genes for CFH, CFI, and MCP

Screening for mutations by serum levels or cell surface expression will fail to detect low protein levels in 25 to 75% of mutations. Genetic analysis therefore also is required.

Genetic screening for mutations has most frequently been performed using either denaturing HPLC or single-strand conformation polymorphism. Both of these options are relatively inexpensive but are not sensitive. In addition, these techniques do not reveal the position of a mutation, and further confirmatory direct sequencing is required (17).

Sequencing PCR amplicons is now becoming the method of choice for mutation screening. Although it can be expensive, it is the most sensitive method available (17).

The CFH gene consists of 23 exons. Primers for mutation screening of CFH using single-strand conformation polymorphism, denaturing HPLC, and direct sequencing have been reported (3–6,18). The primers chosen will reflect whichever technology is available and optimized in the laboratory of choice. Furthermore, it is vital that the primers chosen be able to differentiate between CFH and the factor H–related proteins, especially CFHL1 (19). Errors have been made as a result of use of nonspecific primers, and this is a vital component of the screening process because the majority of CFH mutants cluster in the region of maximal CFHL1 homology (19,20). For this reason, it may be prudent to adopt the primers that already are used and verified by one of the recognized groups in this area.

CFI has 13 exons, and MCP has 14 exons. Primers for mutation screening for CFI and for MCP using direct sequencing again have been reported by several groups who are experienced in this field (9,10,13,14).

Genomic Rearrangements

The genes for CFH and MCP, as well as many other complement regulatory genes, all are tightly linked in a region that is known as the regulators of complement activation (RCA) cluster on chromosome 1q32. The RCA cluster arose by several large genomic duplications. These duplications have resulted in a high degree of sequence identity between the gene for CFH and the genes for the five factor H–related proteins (CFHL1 through 5) (21,22). These large genomic duplications predispose to gene conversion events and genomic rearrangements (23).

Unambiguous evidence of a role for gene conversion in generation of CFH mutants in aHUS was provided by Heinen et al. (24). In that study, the Goodship group described two patients with conclusive evidence of gene conversion and another nine patients in whom a CFH mutation could have arisen by gene conversion. This type of mutation can be detected by genomic sequencing.

The Goodship group also described a large genomic rearrangement in CFH that resulted in a hybrid gene in which exons 1 to 21 are derived from CFH and exons 22 and 23 are derived from CFHL1 (25). Subsequent analysis of a large panel of patients with aHUS revealed similar deletions of exons 22 and 23 with formation of a hybrid gene in an additional three of 80 patients. Such a change accounts for approximately 5% of mutations in aHUS in this series. This is of particular importance when screening patients for mutations in CFH, because large genomic rearrangements of this type would not be detected by genomic sequencing using CFH-specific primers. Because these individuals lacked CFH exons 22 and 23 on one allele, the specific primers would amplify only exons 22 and 23 of CFH on the normal allele. The sequencing therefore would seem normal.

Given the high frequency of genomic rearrangements in CFH, it is recommended that all patients be screened with multiplex ligation–dependent probe amplification. No data yet exist for genomic rearrangements in MCP and CFI, however, multiplex ligation–dependent probe amplification of these genes should be considered in those in whom a mutation has not been discovered (17).

Combined Mutations

When screening for mutations in complement regulators, it also should be noted that in several cases, individuals have had mutations in two complement regulators (9,11,26). This raises the question of whether after the identification of a mutation in one complement regulator, efforts should be made to seek out another predisposing gene. The small number of cases with multiple mutations discovered so far makes genotype–phenotype correlations difficult to derive. We believe that when a mutation has been identified in either CFH or CFI, which are poor prognostic indicators for successful renal transplantation, there is no clinical utility in searching for another mutation. In those in whom an MCP mutation has been identified, which has a better prognosis for renal transplantation, there may be an argument for screening CFH and/or CFI to exclude mutations in these genes that would adversely affect transplant outcome.

Autoantibodies

In those without detectable mutations in CFH, CFI, or MCP, autoantibodies to CFH have been identified in a small percentage of cases (approximately 6%) (27). An ELISA with purified human factor H–coated plates to capture anti-CFH antibodies is used.

Interpreting Results

Conclusion

We propose a method for screening for mutations in complement regulatory proteins in patients with aHUS. We believe that the evidence suggests that all patients who have aHUS and are being considered for renal transplantation should undergo screening for mutations in complement regulators. This will allow patients and clinicians to make informed decisions regarding listing for transplantation, on the basis of the risk for recurrence and its not inconsiderable consequent morbidity and mortality. Screening of unaffected family members has limited clinical utility unless they are being considered as living-related kidney donors. A useful source of guidance for genetic screening in aHUS is GeneTests, an National Institutes of Health–funded Web site that provides information on commercial and research-based testing for various genetic disorders (http://www.genetests.org).

Disclosures

None.