We are happy to accommodate requests for single genes or a subset of these genes. The price will remain the list price. If desired, free reflex testing to remaining genes on panel is available. Alternatively, a single gene or subset of genes can also be ordered on our PGxome Custom Panel.

The great majority of tests are completed within 20 days.

The great majority of tests are completed within 20 days.

Periodic Fever Syndromes (PFS) (also known as monogenic autoinflammatory syndromes) is a collective group of disorders highlighted by recurrent fever and inflammatory episodes. Fever episodes can range from hours up to ~ 2 weeks with recurrences ranging from several bouts per month to a few per year. PFS frequently involve inflammation of the skin, serous membranes, joints, lymph nodes, gastrointestinal tract, and nervous system. Onset of PFS occurs during the first year of life or early childhood with only familial mediterranean fever (FMF) and tumor necrosis factor receptor-associated periodic syndrome having cases of adult onset described. In severe cases, development of amyloidosis may occur. PFS are caused by aberrant inflammasome activation leading to heightened interleukin 1 levels and innate immune dysregulation. Differential diagnosis of individual PFS is important for employing appropriate therapeutics such as colchicine to treat FMF or antibiotics for cyclic neutropenia. PFS may mirror other immune dysfunction disorders such as cyclic neutropenia and SCID due to recurrent fever bouts, but unlike those disorders, bouts are not a result of recurrent infections (Caso et al. 2013; Touitou et al. 2013).

Disorders tested in this PFS panel include familial mediterranean fever/FMF, familial cold autoinflammatory syndrome/FCAS, muckle-wells syndrome/MWS, chronic infantile neurological cutaneous articular syndrome/CINCA, tumor necrosis factor receptor-associated periodic syndrome/TRAPS, mevalonate kinase deficiency/MKD, NLRP12 associated autoinflammatory disorder, pyogenic arthritis pyoderma gangrenosum and cystic acne syndrome/PAPA, majeed syndrome, cyclic neutropenia, pediatric granulomatous arthritis/PGA, CANDLE syndrome, DITRA syndrome, CARD14 mediated psoriasis/CAMPS, and TNFAIP3-mediated early onset autoinflammatory disease (Sanchez et al. 2013).

Autosomal dominant PFS disorders include TRAPS (TNFRSF1A), FACS (NLRP3), MWS (NLRP3), CINCA (NLRP3), NLRP12 associated autoinflammatory disorder (NLRP12), PAPA (PSTPIP1), CAMPS (CARD14), TNFAIP3-mediated early onset inflammatory disease, PGA (NOD2), and cyclic neutropenia (ELANE). Autosomal recessive PFS disorders include FMF (MEFV), MKD (MVK), CANDLE (PSMB8), DITRA (IL36RN) and majeed syndrome (LPIN2) (Caso et al. 2013; Sanchez et al. 2013).

See individual test descriptions for additional information on the molecular biology of each gene product.

For this NGS test, the full coding regions plus ~10bp of non-coding DNA flanking each exon are sequenced for each of the genes listed below.  Sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization method, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for any regions not captured or with insufficient number of sequence reads. All pathogenic, undocumented and questionable variant calls are confirmed by Sanger sequencing. 

This panel provides 100% coverage of the aforementioned regions of the indicated genes. We define coverage as > 20X NGS reads for exons and 0-10 bases of flanking DNA, > 10X NGS reads for 11-20 bases of flanking DNA, or Sanger sequencing.

Candidates present with periodic/episodic fevers without an underlying infection typically within the first few years of life. Attacks are short and recurrent with spontaneous remission. Cyclic Neutropenia mirrors many features of other PFS, but is triggered by underlying infection. Fever episodes often present with elevated serum amyloid A, elevated erythrocyte sedimentation rate, and leukocytosis (Caso et al. 2013; Touitou et al. 2013).

Diseases

We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~10 bases of non-coding DNA flanking each exon.  As required, genomic DNA is extracted from the patient specimen.  For NGS, patient DNA corresponding to these regions is captured using an optimized set of DNA hybridization probes.  Captured DNA is sequenced using Illumina’s Reversible Dye Terminator (RDT) platform (Illumina, San Diego, CA, USA).  Regions with insufficient coverage by NGS are often covered by Sanger sequencing.

For Sanger sequencing, Polymerase Chain Reaction (PCR) is used to amplify targeted regions.  After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Patient DNA sequence is aligned to the genomic reference sequence for the indicated gene region(s). All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories, listed below, per ACMG Guidelines (Richards et al. 2015).

(1) Pathogenic Variants
(2) Likely Pathogenic Variants
(3) Variants of Uncertain Significance
(4) Likely Benign Variants
(5) Benign Variants

Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://www.hgvs.org).  Rare variants and undocumented variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing.

As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.

In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.   

Interpretation of the test results is limited by the information that is currently available.  Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.

When Sanger sequencing does not reveal any difference from the reference sequence, or when a sequence variant is homozygous, we cannot be certain that we were able to detect both patient alleles.  Occasionally, a patient may carry an allele which does not amplify, due to a large deletion or insertion.   In these cases, the report will contain no information about the second allele.  Our Sanger and NGS Sequencing tests are generally not capable of detecting Copy Number Variants (CNVs).

We sequence all coding exons for each given transcript, plus ~10 bp of flanking non-coding DNA for each exon.  Test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions or any currently uncharacterized alternative exons.

In most cases, we are unable to determine the phase of sequence variants.  In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants due to somatic mosaicism is limited.  Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes from whole blood).   Test reports contain no information about the DNA sequence in other cell-types.

We cannot be certain that the reference sequences are correct.

Rare, low probability interpretations of sequencing results, such as for example the occurrence of de novo mutations in recessive disorders, are generally not included in the reports.

We have confidence in our ability to track a specimen once it has been received by PreventionGenetics.  However, we take no responsibility for any specimen labeling errors that occur before the sample arrives at PreventionGenetics.

Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are analyzed.

PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene.

Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.

This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.

aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.

In the case of duplications, aCGH will not determine the chromosomal location of the duplicated DNA. Most duplications will be tandem, but in some cases the duplicated DNA will be inserted at a different locus. This method will also not determine the orientation of the duplicated segment (direct or inverted).

Breakpoints, if occurring outside the targeted gene, may be hard to define.

The sensitivity of this assay is dependent upon the quality of the input DNA. In particular, highly degraded DNA will yield poor results.