Complement System Disorder Panel

The complement system disorders are a group of primary immunodeficiencies resulting in absent or suboptimal function of complement system proteins. In general, deficiencies of the classical and alternative complement pathways are rather rare, while deficiencies of the proteins in the mannose-binding lectin (MBL) pathway are more common. C2-deficiency is the most common classical pathway complement deficiency in many populations and its prevalence is estimated to be 1:10,000. There are two main categories of complement system disorders, originating from mutations in genes encoding proteins either inhibiting or activating the complement system and thus resulting in overactive or underactive responses, respectively. Complement system disorders predispose patients for example to (Neisserial) infections, atypical hemolytic uremic syndrome, age-related macular degeneration, systemic lupus erythematosus SLE and preeclampsia. This panel covers genes associated with autosomal recessive, autosomal dominant as well as X-linked forms of complement deficiencies. In addition to complement system disorders, this panel has the ability to diagnose other conditions, such as primary ciliary dyskinesia, that is characterized by recurrent respiratory infections. OTHER INFORMATION ON CFH AND CFHR1-4 GENES

CFH gene have multiple exons that are pseudogenic (exons 8-9, 11, 21-23). Moreover, the function of CFHR1, CFHR2, CFHR3 and CFHR4 has not been established and they are highly homologues (see below chapter ‘CFHR1-4 genes‘).
Genetics of atypic hemolytic uremic syndrome (aHUS)
Mutations in CFH account for approximately 30% of the cases, CD46 (also known as MCP) 12%, CFI 5%-10%, C3 5%, THBD 3%-5%. In early onset aHUS, disease manifesting before age 1 year, mutations in DGKE explain 27% of the cases. Inheritance mode is difficult to determine for most of the genes related to aHUS due to low penetrance but the predisposition to disease is commonly autosomal dominant. In the ClinVar mutation database, vast majority of the novel disease associated variants in major aHUS genes such as CFH, CD46, CFI and C3 are classified as risk factors but not pathogenic or likely pathogenic. In most of the families where probands has novel variant in aHUS genes, some of the unaffected parents or other family members carry the same variant. However, one study showed fully penetrant recessive aHUS relating to homozygous CFH mutations in a large Bedouin pedigree with 10 aHUS cases (PubMed: 9811382). In addition, deletions in CFHR1 to CFHR5 genes have shown to increase slightly a risk for aHUS. The Newcastle cohort of 66 aHUS patients showed deletions in CFHR1 were more frequent in aHUS patients compared to controls (zero copies 10% vs. 2%; one copy 35%vs 9% and two copies 55% vs.89%) indicating odds ratios (OR) 6.3 for homozygous deletion and 3.8 for heterozygous. Absence of CFHR1 and/or CFHR3 was shown to contribute to the defective regulation of complement activation on cell and tissue surfaces (PubMed: 17367211). Hofer et al evaluated 116 aHUS patients and 118 control. Homozygous deletion in CFHR1 was detected in 32% of the patients with aHUS tested and in 2.5% of controls. CFH antibodies were present in 25% of the patients and none of the controls. CFH antibodies were detected in 82% of patients with homozygous CFHR1 deletion and in 6% of patients without. CFH antibody-positive patients with aHUS showed a significantly lower platelet nadir at disease onset and significantly less frequent involvement of the central nervous system than did antibody-negative patients. Antibody-positive patients also received plasma therapy more often (PubMed: 23243267). It is noteworthy that disease activity appears to correlate better with immune complex titers than FHAA titers (PubMed: 22922817). In 2016, Challis et al described novel CFH/CFHR3 hybrid gene in a patient with aHUS secondary to a de novo 6.3-kb deletion that arose through microhomology-mediated end joining rather than nonallelic homologous recombination. Secreted protein product lacked the recognition domain of factor H and exhibits impaired cell surface complement regulation. The fact that the formation of this hybrid gene arose as a de novo event suggests that this cluster is a dynamic area of the genome in which additional genomic disorders may arise (PubMed: 26490391). CFHR1-4 genes
In August 25 2017, Blueprint Genetics excluded CFHR1, CFHR2, CFHR3, CFHR4 genes from three diagnostic NGS panels including Primary Immunodeficiency Panel, Complement System Disorder Panel and Hemolytic Uremic Syndrome Panel. This was done due to extensive homology between these genes making it difficult or even impossible to determine copy number reliably from these genes with short read length NGS methods. Moreover, homozygous or heterozygous deletions involving these gene are common in population even though enriched in patients with aHUS. By relying on three estimates: 1) higher end of aHUS prevalence (9 per 1,000,000), 2) frequency of homozygous CFHR1 deletion (2%) and 3) assuming that all aHUS cases would be caused by this defect (over estimating the effect), we are left with the fact that 99.95% of the individuals with homozygous CFHR1 deletion will never get aHUS. Thus, we consider releasing copy number from CFHR1-4 genes may be misleading, and is not considered helpful in clinical practice. We believe that fusion genes between CFH and CFHR1-4 may be the mechanism that explain the association between CFHR1-4 gene deletions and aHUS. However, this kind of alterations are not reliably detected by targeted sequencing approaches.

The target region for each gene includes coding exons and ±20 base pairs from the exon-intron boundary. In addition, the panel includes non-coding and regulatory variants if listed above (Non-coding variants covered by the panel). Some regions of the gene(s) may be removed from the panel if specifically mentioned in the ‘Test limitations” section above. If the test includes the mitochondrial genome the target region gene list contains the mitochondrial genes. The sequencing data generated in our laboratory is analyzed with our proprietary data analysis and annotation pipeline, integrating state-of-the art algorithms and industry-standard software solutions. Incorporation of rigorous quality control steps throughout the workflow of the pipeline ensures the consistency, validity and accuracy of results. Our pipeline is streamlined to maximize sensitivity without sacrificing specificity. We have incorporated a number of reference population databases and mutation databases including, but not limited, to 1000 Genomes Project, gnomAD, ClinVar and HGMD into our clinical interpretation software to make the process effective and efficient. For missense variants, in silico variant prediction tools such as  SIFT, PolyPhen,MutationTaster are used to assist with variant classification. Through our online ordering and statement reporting system, Nucleus, ordering providers have access to the details of the analysis, including patient specific sequencing metrics, a gene level coverage plot and a list of regions with suboptimal coverage (<20X for nuclear genes and <1000X for mtDNA) if applicable. This reflects our mission to build fully transparent diagnostics where ordering providers can easily visualize the crucial details of the analysis process.

We provide customers with the most comprehensive clinical report available on the market. Clinical interpretation requires a fundamental understanding of clinical genetics and genetic principles. At Blueprint Genetics, our PhD molecular geneticists, medical geneticists, and clinical consultants prepare the clinical statement together by evaluating the identified variants in the context of the phenotypic information provided in the requisition form. Our goal is to provide clinically meaningful statements that are understandable for all medical professionals regardless of whether they have formal training in genetics.

Variant classification is the cornerstone of clinical interpretation and resulting patient management decisions. Our classifications follow the ACMG guideline 2015.

The final step in the analysis is orthogonal confirmation. Sequence and copy number variants classified as pathogenic, likely pathogenic, and variants of uncertain significance (VUS) are confirmed using bi-directional Sanger sequencing or by orthogonal methods such as qPCR/ddPCR when they do not meet our stringent NGS quality metrics for a true positive call.

Our clinical statement includes tables for sequencing and copy number variants that include basic variant information (genomic coordinates, HGVS nomenclature, zygosity, allele frequencies, in silico predictions, OMIM phenotypes, and classification of the variant). In addition, the statement includes detailed descriptions of the variant, gene, and phenotype(s) including the role of the specific gene in human disease, the mutation profile, information about the gene’s variation in population cohorts, and detailed information about related phenotypes. We also provide links to the references, abstracts, and variant databases used to help ordering providers further evaluate the reported findings if desired. The conclusion summarizes all of the existing information and provides our rationale for the classification of the variant.

Identification of pathogenic or likely pathogenic variants in dominant disorders or their combinations in different alleles in recessive disorders are considered molecular confirmation of the clinical diagnosis. In these cases, family member testing can be used for risk stratification. We do not recommend using variants of uncertain significance (VUS) for family member risk stratification or patient management. Genetic counseling is recommended.

Our interpretation team analyzes millions of variants from thousands of individuals with rare diseases. Our internal database and our understanding of variants and related phenotypes increases with every case analyzed. Our laboratory is therefore well-positioned to re-classify previously reported variants as new information becomes available. If a variant previously reported by Blueprint Genetics is re-classified, our laboratory will issue a follow-up statement to the original ordering healthcare provider at no additional cost, according to our latest follow-up reporting policy.