Guidelines for the genetic diagnosis of hereditary recurrent fevers

Hereditary recurrent fevers (HRFs) are a group of monogenic autoinfl ammatory diseases characterised by recurrent bouts of fever and serosal infl ammation that are caused by pathogenic variants in genes important for the regulation of innate immunity.

# Introduction
Patients with hereditary recurrent fevers (HRFs) present with recurrent bouts of fever and infl ammatory symptoms involving, in particular, the abdomen, joints and skin. The causative genes for HRFs encode proteins involved in the regulation of innate immunity, mainly by affecting proinfl ammatory cytokines and apoptosis pathways. While familial Mediterranean fever (FMF) is relatively common in several Mediterranean and Middle Eastern populations, [bib_ref] Familial Mediterranean fever, Ben-Chetrit [/bib_ref] most HRFs are rare diseases. The best-characterised HRFs are two recessively inherited diseases: (FMF, gene MEFV, MIM 608107) and mevalonate kinase defi ciency (MKD, gene MVK, MIM 251170) and two dominantly inherited diseases: tumour necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS, gene TNFRSF1A, MIM 191190) and cryopyrin-associated periodic syndrome (CAPS, gene NLRP3, MIM 606416). Patients with HRF often display similar infl ammatory symptoms with variable intensity and localisation of symptoms, making their clinical diagnosis diffi cult.
Since the discovery of these four HRF genes, almost 700 nucleotide variants have been identifi ed and recorded in Infevers, a database dedicated to autoinfl ammatory sequence variants (http:// fmf.igh.cnrs.fr/ISSAID/infevers/). [bib_ref] INFEVERS: the Registry for FMF and hereditary infl ammatory disorders mutations, Sarrauste De Menthiere [/bib_ref] [bib_ref] Infevers: an evolving mutation database for auto-infl ammatory syndromes, Touitou [/bib_ref] [bib_ref] The infevers autoinfl ammatory mutation online registry: update with new genes and..., Milhavet [/bib_ref] Some of these variants are clearly pathogenic, but most are unconfi rmed or seemingly non-pathogenic variants. A signifi cant number of patients clinically diagnosed with recessive HRFs have been found to carry only one disease-associated mutation in the respective genes 7 despite extensive searching for a second pathogenic mutation in the coding region, and continuing search for mutations affecting regulatory sequences or transcript splicing that would affect gene expression. Genetic testing for HRF is a logical and feasible way to corroborate clinical diagnosis. Five-year experience of external quality assessment and profi ciency testing (PT) (external quality assurance/PT) conducted between 2006 and 2010 showed that although there has been an impressive improvement in the quality of HRF testing and reporting, many issues still remain to be addressed and standardised. [bib_ref] An international external quality assessment for molecular diagnosis of hereditary recurrent fevers:..., Touitou [/bib_ref] Guidelines using the standard defi nition by Field and Lohrare now proposed to provide a framework for best laboratory practice and reporting on the genetic diagnosis of HRFs as agreed by an international consortium of experts in the fi eld. They are intended to be used primarily by molecular geneticists and by other health professionals involved in the care of these patients.

# Methodology
A draft report was written by the organisers and assessors of the European Molecular Genetics Quality Network for HRFs with reference to relevant literature, reviews of reports issued during the fi ve previous international HRF meetings, web-based resources relating to the subject and examples of guidelines for other hereditary diseases (eg, haemochromatosis, 16 cystic fi brosis 17 and von Willebrand 18 diseases). The draft was disseminated to molecular geneticists and clinicians working in the fi eld of HRFs and discussed with them during a best practice workshop held in Bruges (Belgium) on 18 and 19 September 2011. In the light of feedback of the participants, amendments were made and a second draft was disseminated by email, after which, the fi nal document was ratifi ed.

## Indications for hrf testing minimal requirements for the genetic test
We suggest that the following minimum set of requirements should be obtained: patient name, date of birth, gender, ethnicity/origin, written informed consent (depending on countryspecifi c law), referring doctor's name and contact details of the person who will receive the results.

## Symptomatic patients
The main indication for genetic testing of HRFs is in the case of a patient with a clinical symptom pattern consistent with one or more of the syndromes. Thus, clinical data that justify the choice of one or more HRFs genetic tests are required. It is not unusual that when overlapping, partial or atypical clinical symptoms impede an accurate clinical diagnosis, screening of several HRF-responsible genes gives the correct diagnosis.
The clinical HRF referral usually includes the frequency of attacks, duration, sites affected, acute phase reactants levels, biomarkers for mevalonic aciduria or amyloidosis and a letter by an expert clinician. An example of a clinical chart is provided in online supplementary fi gure S1. It has been established in France by GenMAI, the national network for genetic diagnosis of autoinfl ammatory diseases, in conjunction with the clinical reference centres. Decision trees for genetic diagnosis in atypical patients and patients with sporadic disease have been proposed. In addition, a diagnostic score for children with periodic fever has been elaborated in Italy. 20

## Presymptomatic diagnosis and carrier status
In general, presymptomatic diagnosis is not advisable, as its interpretation is inconclusive, may be complicated for mutations with incomplete penetrance and it usually does not call for medical intervention. Presymptomatic testing may be recommended after genetic counselling for asymptomatic family members when a severe genotype has been found in relatives with an overt disease, or if there is a family history of amyloidosis. Follow-up of people at risk may avoid occurrence of this life-threatening complication. However, whether such cases should be given prophylactic treatment remains controversial. Evaluation of carrier status could be recommended in healthy relatives to phase two known disease-associated or new mutations, when identifi ed in an affected patient.

## Prenatal diagnosis and preimplantation genetic diagnosis
Generally prenatal diagnosis (PND) and preimplantation genetic diagnosis (PGD) are not considered appropriate for HRF as most of these conditions are treatable and symptoms usually decrease over time. However, it may be appropriate to discuss PND or PGD in families affected by a particularly severe form of MKD or chronic infantile neurological, cutaneous and articular syndrome (CINCA) as these disorders can be associated with debilitating complications: blindness, deafness, mental retardation, ataxia and bone deformation. However, most severe CINCA-associated mutations occur de novo and this should be considered before offering PND and PGD. If PND is planned, it should be performed after genetic counselling.

## Diagnostic strategy diagnostic laboratories and expert structures
Genetic diagnosis for HRFs is now widely available. There are 99 laboratories providing FMF testing in Europe (source Orphanet at www.orpha.net). [bib_ref] Orphanet: a European database for rare diseases), Weinreich [/bib_ref] A registry of sequence variants was developed online at http://fmf.igh.cnrs.fr/ISSAID/infevers to assist the molecular geneticist. It provides a comprehensive and updated list of gene variants and a reference database for the mutation nomenclature, but an accurate phenotype-genotype correlation is not available. In addition, clinical reference centres were formally nominated or are recognised in several countries (France, Italy, Spain, UK, Germany, Turkey, Israel, USA...). As HRFs are rare diseases mostly caused by single-nucleotide substitutions, genetic testing should be referred to specialised laboratories to ensure that pertinent tests are performed and proper information is reported to clinicians, particularly those inexperienced with HRFs. These laboratories should work within a comprehensive quality management system (accreditation), use validated methods, participate annually in interlaboratory comparisons such as external quality assessment and profi ciency testing for HRF and/ or the relevant techniques (eg, DNA sequencing) and defi ne a typical turn-around time.

## Testing strategy
Most laboratories focus the molecular analysis on mutational hot-spot regions in various genes. Recommendations on the reference sequence to be used for analysis and for the extent of the initial mutation screening are provided in table [bib_ref] Autoinfl ammatory diseases: the hereditary periodic fever syndromes, Fietta [/bib_ref]. The minimum diagnostic screen should include variants that are clearly shown to be pathogenic and that are frequently identifi ed in patients. Although this screening recommendation is valid worldwide, the ethnic background of the patient needs to be considered. For instance, the four clearly pathogenic MEFV variants are almost exclusively found in Mediterranean populations, while the frequency of the debated p.Glu148Gln (NM_000243.2:c.442GC) variant is as high as 20% in Asiatic countries. [bib_ref] The spectrum of Familial Mediterranean Fever (FMF) mutations, Touitou [/bib_ref] The p.Phe479Leu (NM_000243.2:c. 1437CG) is especially relevant in Greek and Iranian patients. [bib_ref] The spectrum of Familial Mediterranean Fever (FMF) mutations, Touitou [/bib_ref] The p.Pro75Leu (NM_001065.3:c.224CT) (usual name P46L) of TNFRSF1A is commonly found in Arabic and African populations. [bib_ref] Unexpected high frequency of P46L TNFRSF1A allele in sub-Saharan West African populations, Tchernitchko [/bib_ref] Accordingly, many laboratories have adopted a two-step strategy-that is, an initial search for the most common pathogenic variants followed, if necessary, by an extended search spanning the complete coding sequence of the various genes. For the MEFV gene, there is limited utility in searching for rare variants for patients with clinically established FMF and no mutations in exons 2, 3, 5 and 10.

## Techniques
A variety of techniques are used to identify HRFs gene sequence variations but direct mutation analysis by DNA sequencing is the method employed by most laboratories. Other methods include PCR with restriction enzyme digest, allele-specifi c PCR, PCRsingle-strand conformation polymorphism and primer extension and reverse hybridisation-based kits. Commercially available kits should specify whether they are CE-IVD (in vitro diagnostics) marked or Food and Drug Administration approved. General guidelines 18 should be followed. No standard primer set is recommended for amplifi cation of the essential regions in HRF genes, but:
1. Primer sequences should be regularly checked for underlying single-nucleotide polymorphisms (SNPs) particularly for highly polymorphic exons of the HRF genes (eg, MEFV exons 2 or 5).
2. PCR primer design should avoid possible amplifi cation of sequences from homologous genes (eg, NLRP genes).

## Interpretation classifi cation and validation of hrf sequence variants
The Human Genome Variation Society (HGVS) has recommended avoiding the use of 'mutation' and polymorphism' as terms with opposite meanings (pathogenic vs non-pathogenic), since functional studies are scarce or unavailable for most of the sequence variants to evaluate their pathogenicity. HRF sequence variants are associated with a broad range of phenotypes, but only a small proportion of them have been clearly shown to be the direct cause of the disease. The most common variants seen in the HRF genes are non-synonymous nucleotide changes and with the exception of MKD, no large structural mutations (deletions, duplications, rearrangement) have been reported. This is probably because such deleterious pathogenic variants would not be tolerated in genes that regulate host defence pathways.
We have established a classifi cation of gene variants based on the expertise of HRF diagnostic laboratories and on the review of current publications . Interpretation should differentiate the following:
Clearly pathogenic variants. Genetic confi rmation of 1.
HRFs is more straightforward in cases of sequence variants that associate with a well-recognised HRF phenotype (eg, p. [bib_ref] The clinical continuum of cryopyrinopathies: novel CIAS1 mutations in North American patients..., Aksentijevich [/bib_ref] We believe that reporting these SNPs in the context of genetic diagnosis of HRF might mislead the report recipient.

## Genetic confi rmation of the clinical diagnosis
As for any Mendelian conditions, the defi nitive genetic diagnosis of HRFs is based on the fi nding of unambiguous mutations in the causative genes. Theoretically, identifi cation of one single pathogenic variant in dominant diseases, homozygosity or compound heterozygosity (confi rmed by studying the parental alleles) in recessive disorders, should be enough to confi rm the diagnosis. Of note, fi nding a single MEFV pathogenic variant in patients of Mediterranean origin does not exclude the possibility of disease-causing mutations in other HRF genes. [bib_ref] Web resources for rare auto-infl ammatory diseases: towards a common patient registry, Touitou [/bib_ref] However, interpretation of a result should always take into account the sensitivity of the molecular screening strategy. Failure to identify a causal mutation in a given gene can almost never exclude the diagnosis. Indeed, the entire gene is not sequenced in most routine settings and even complete gene sequencing based on PCR technology could miss a pathogenic variant (primer variants, inversion of exons, duplication of exons...) Likewise mosaicism will be most likely missed by standard sequencing.

## Results: two [type of the variants] were found
Mutations and genotype to be given using HGVS nomenclature
[formula] Mutations DNA level Protein level 1. c.--- p.--- 2. c.--- p.--- [/formula]

## Genotype: p.[---];[---] if phased not allelic p.[---;---] if phased allelic p.[---(;)---]
if not phased Interpretation:
[To be reported here as suggested in table 2]
Recommendations: Genetic counseling is advised.

## Date of report name of the molecular geneticist(s)

## Reporting
We recommend that genetic test results should be sent to the doctor(s) designated by the patient and not directly to the patient. The referring doctor should be invited to contact the laboratory if they have not fully understood the clinical significance of the test result. In the case of family studies, genetic results should not be communicated to other relatives without their consent. We recommend that a patient's information on genetic testing results should be given by a doctor skilled in the fi eld of HRFs or by an expert geneticist, if available. We strongly suggest that the reports include all items recommended by the OECD quality assurance in genetic testing (http://www.oecd.org/dataoecd/43/6/38839788.pdf) and match the international standard ISO-15189. The laboratory report should be limited to a single page, with the genetic results and interpretation highlighted and the rest in smaller characters or presented in footnotes. A model report is proposed (fi gure 1).

# Genetic results
1. Variants should be described at both the protein and nucleotide level and should comply with the latest version of HGVS nomenclature (http://www.hgvs.org/mutnomen/). It is preferable to use the three-letter amino acid code. 2. Wherever the sequence variant name has changed owing to renumbering of the start codon, both the standard and HGVS nomenclature should be reported, to allow the referring clinician access to the relevant literature. Examples are p.Arg121Gln (NM_001065.3:c.362GA) (common name R92Q) of TNFRSF1A and p.Val198Met It is preferable to recommend referral to genetic counselling and/or to a clinical reference centre rather than to comment directly on treatment options or the predicted risk for the offspring or other family members. The clinical reference centres are better placed to fully discuss these pertinent issues. Testing should be offered to other symptomatic family members and to the parents where this can help with interpretation of the proband's results.

## Clerical information
The other important items suggested by the OECD are summarised below:
- More than one identifi er that unequivocally links the report to the patient (name, date of birth, internal reference laboratory number). - The name of the referring healthcare professional and contact information. - The indication for testing and specifi c medical information where it is relevant. - The date of receipt of the sample and of report issuing. - The laboratory contact information and the identity of the individual approving the report.

# Conclusion
A consensus set of best practice guidelines has been developed for molecular genetic testing of HRFs based on feedback received from experts in this fi eld. The guidelines described here are aimed at improving the quality of HRF molecular diagnostics and promoting harmonisation and standardisation of laboratory test reports. Understanding the molecular pathology of these diseases, their heterogeneity and genotype-phenotype correlations is steadily evolving as more data become available from large population cohorts of patients and healthy controls. A particular challenge for the genetic diagnosis of HRF will be in the interpretation of clinical relevance of variants that are found at low, but >1%, frequency in various populations. These may function as susceptibility alleles to infl ammation rather than disease-associated mutations and as such may give rise to an infl ammatory phenotype when inherited through digenic inheritance (in the form of double heterozygous). In that context we feel that these guidelines may need to be regularly updated. Contributors IT led the project, organised the meeting, wrote the fi rst draft of the paper and was aided by YS, LO and IA. BB and HH edited the paper for English language. All authors participated in the development of the guidelines and contributed to the writing of the paper.
[fig] Figure 1: A proposed model for reporting the genetic diagnosis of hereditary recurrent fevers (HRFs). This is an example of a recessive disease, showing the minimal items to be reported. A report should ideally highlight the major fi ndings and not exceed one page. HGVS, Human Genome Variation Society.LABORATORY identifiers and contact information GENETIC DIAGNOSIS OF [ACRONYME HRF] (Extended name HRF) Name of referring health care professional : Date of receipt of the sample : Subject: [Family name, given name patient] Date of birth: --/--/----Patient ID: [internal ID] Type of sample: [DNA/blood] Indication for testing: Method: Method (sequencing, kit etc...) and extent of the screening (exons ---) of the [HRF] gene (ref seq: NM_XXXX.X). This allows identification of XX% of the known mutations. [/fig]
[table] Table 2: Guidelines for reporting and interpreting genetic results in the four main HRFs One clearly pathogenic variant One clearly pathogenic variant was identifi ed in the (MEFV/MVK) gene This individual is not likely to develop (MKD/FMF) CRP, C reactive protein; SAA, serum amyloid A; VUS, variant of uncertain clinical signifi cance (includes debated variants and rare and novel variants with no relevant information). The number for the reference sequence used should be provided. 4. A brief description of the testing technique and its clinical and analytical sensitivities for the specifi c population being screened (ie, extent of mutation screening and coverage of disease-associated mutations, region and limits of detection) should be provided.Typical cases together with suggested clinical interpretation are listed in table 2. Reports should state, at a minimum, if the genotype is consistent or not with a diagnosis of HRF. Where the diagnosis is not confi rmed genetically, the report should not state that a diagnosis of HRF is excluded. In this situation diagnosis relies on clinical judgement or criteria. Additional comments in the report may refer to the presumed effect of the sequence variant on protein function and suggest further genetic testing or clinical management. Comments related to genotype-phenotype correlations are appropriate if there is enough evidence in the literature to support them. They are critical when discussing the risk of amyloidosis in patients with FMF with mutations affecting codons 680 or 694 of the MEFV gene, in patients with TRAPS with cysteine pathogenic variants and in familial cases of HRF with a history of amyloidosis. A comment on mild disease outcome can be considered for genotypes with mild or low penetrance sequence variants. [/table]
[table] Useful links: Infevers: Registry of autoinfl ammatory mutations: http://fmf. igh.cnrs.fr/ISSAID/infevers/ ISSAID: Website of the International Society of Systemic Autoinfl ammatory Diseases: http://fmf.igh.cnrs.fr/ISSAID/ Eurofever: Registry of autoinfl ammatory patients: http://www. printo.it/eurofever/ Orphanet: Reference portal for information on rare diseases and orphan drugs: http://www.orpha.net/ HGVS: Reference for the nomenclature for the description of sequence variants: http://www.hgvs.org/mutnomen/ EMQN: European Molecular Genetics Quality Network: http:// www.emqn.org/emqn/ For FMF: The Centre of Arab Genomic Studies (http://www. cags.org.ae) and the Israeli National Genetic Database (http:// www.goldenhelix.org/server/israeli/) [/table]