MOLECULAR GENETICS REPORT: Glycogen Storage Disease NextGen Sequencing Panel

Transcription

1 Patient LAST, First ID#: DOB: Sex: CLIA #: 52D CAP #: S. Business Park Ave. Marshfield, WI Sample Information Type: Collected: Received: PG ID: Ordering Provider(s) MOLECULAR GENETICS REPORT: Glycogen Storage Disease NextGen Sequencing Panel SUMMARY OF RESULTS: NEGATIVE RESULTS AND INTERPRETATIONS: In this patient, for the AGL, G6PC, GAA, GBE1, GYS2, PFKM, PHKA1, PHKA2, PHKB, PHKG2, PYGL, PYGM, SLC2A2 and SLC37A4 genes, we found no sequence variants that are likely to be a primary cause of disease. This patient is heterozygous in the PFKM gene for a likely benign missense which bears further mention. This variant is defined as c.2087g>a, and is predicted to result in the amino acid substitution p.arg696his. This variant was reported in a patient with GSD Type VII (Raben et al Am. J. Hum. Genet. 56: ). However, a second clearly causative variant was not found in this patient, and in vitro expression of the Arg696His protein in yeast showed no reduction in enzyme activity. Furthermore, the programs PolyPhen-2 and SIFT (Adzhubei et al. Nat Methods 7: , 2010; Ng and Henikoff Genome Res 11: , 2001) predict the p.arg696his change to be benign and tolerated, respectively. It should be noted that this variant has a minor allele frequency greater than 1.0% in several different populations ( and has been observed in several presumably unaffected individuals in the homozygous state ( evs.gs.washington.edu/evs/; In addition, we have observed this variant in several patients at PreventionGenetics in the absence of a second causative variant in the PFKM gene. In summary, we classify this variant as likely benign. These results should be interpreted in context of clinical findings, family history and other laboratory data. All genetic tests have limitations. Please see limitations and other information for this test on pages 3-5. RECOMMENDATION: Genetic counseling is recommended. Electronically signed on [date] by: Electronically signed on [date] by: Page 1 of 5

2 3800 S. Business Park Ave. Marshfield, WI Patient Name: LAST, First PG ID Number: APPROACH: Using patient genomic DNA, we sequenced the indicated gene region(s). We then aligned and compared the patient s sequences with the reference sequences. All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories per ACMG Guidelines (Richards et al Genet Med 17: ). All sequence variants are reported below or made available. Pathogenic Variants: None found Likely Pathogenic Variants: None found Uncertain Variants: None found Likely Benign Variants: Gene Transcript DNA Variation Predicted Effect Reference PFKM NM_ c.2087g>a, Heterozygous p.arg696his rs PHKB NM_ c g>t, Heterozygous Intronic rs Benign Variants: We also found many benign sequence variants. For the sake of brevity, these variants are not listed here but are available upon request. The benign variants are often useful in interpretation of the patient s test results, and we recommend that they be retained. GENES SEQUENCED (Transcript Numbers): AGL (NM_ ), G6PC (NM_ ), GAA (NM_ ), GBE1 (NM_ ), GYS2 (NM_ ), PFKM (NM_ ), PHKA1 (NM_ ), PHKA2 (NM_ ), PHKB (NM_ , NM_ ), PHKG2 (NM_ ), PYGL (NM_ ), PYGM (NM_ ), SLC2A2 (NM_ ), SLC37A4 (NM_ ) DATA TRANSFER: PreventionGenetics recommends that DNA sequence from this test be stored in the patient s electronic medical record. This will permit automatic reinterpretation of the sequence in the future, and will best benefit the patient and family members. Upon request, we will be pleased to transfer the patient s sequence. Page 2 of 5

3 Test Methods SUPPLEMENTAL INFORMATION September 15, 2014 NEXTGEN SEQUENCING We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~20 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 covered by Sanger sequencing. All pathogenic, undocumented and suspect NGS variant calls are confirmed 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. Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants ( 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. Limitations and Other Test Notes 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 Next Gen or 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 capture or 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 tests are generally not capable of detecting Copy Number Variants (CNVs). We sequence all coding exons for each given transcript, plus ~20 bp of flanking non-coding DNA for each exon. Unless specifically indicated, 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 amplification. Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes if taken 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. 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. Genetic counseling to help to explain test results to the patients and to discuss reproductive options is recommended. FDA Notes These results should be used in the context of available clinical findings, and should not be used as the sole basis for treatment. This test was developed and its performance characteristics determined by PreventionGenetics. US Food and Drug Administration (FDA) does not require this test to go through premarket FDA review. This test is used for clinical purposes. It should not be regarded as investigational or for research. This laboratory is certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA) as qualified to perform high complexity clinical laboratory testing. Page 3 of 5

4 SUPPLEMENTAL INFORMATION June 19, 2014 GLYCOGEN STORAGE DISEASE PANEL NEXTGEN SEQUENCING Clinical Features: The Glycogen Storage Diseases (GSDs) are a group of inherited metabolic disorders that result from a defect in any one of several enzymes required for either glycogen synthesis or glycogen degradation (see Table below). Broadly speaking, the GSDs can be divided into those with hepatic involvement, which present as hypoglycemia, and those which are associated with neuromuscular disease and weakness. The severity of the GSDs range from those that are fatal in infancy if untreated to mild disorders with a normal lifespan. While some forms of GSD affect a single tissue type (for example, skeletal muscle in McArdle disease), other GSDs affect multiple systems. GSD Type (Disease Name) Enzyme/Protein Deficiency Hepatic vs. Neuromuscular GSD 0 Glycogen synthase Hepatic GSD Ia (von Gierke) Glucose-6-phosphatase-α catalytic subunit Hepatic GSD Ib and Ic Glucose-6-phosphate transporter Hepatic GSD II (Pompe) α-1,4-glucosidase Neuromuscular GSD III (Cori Disease) Glycogen debranching enzyme Both GSD IV (Andersen Disease and APBD) Glycogen branching enzyme Both GSD V (McArdle) Glycogen Phosphorylase (muscle) Neuromuscular GSD VI (Hers) Glycogen Phosphorylase (liver) Hepatic GSD VII (Tarui) Phosphofructokinase (muscle) Neuromuscular GSD IXa1 Phosphorylase kinase α subunit (liver) Hepatic GSD Ixb Phosphorylase kinase β subunit Both GSD IXc Phosphorylase kinase y subunit Hepatic GSD Ixd Phosphorylase kinase α subunit (muscle) Neuromuscular GSD XI (Fanconi-Bickel) GLUT2 transporter Hepatic Many of the glycogen storage diseases are reviewed individually in GeneReviews. In addition, Wolfsdorf and Weinstein (2003) provide a comprehensive review of the hepatic forms of GSD, and DiMauro and Spiegel (2011) provide a comprehensive review of the GSD myopathies. For comprehensive review of all glycogen storage diseases, see Chen et al. (2014) and Hicks et al. (2011). The GSDs have traditionally been diagnosed using a combination of clinical symptoms, biochemical results, and pathology findings. Within the last decade, DNA mutation analysis has become the primary method for diagnosing glycogen storage disease. While such testing was initially performed to complement enzymatic activity studies and clarify ambiguous results, such testing is now becoming the gold standard to confirm a suspected diagnosis. The benefits of such testing are numerous and include the following: Molecular analysis obviates the need for invasive biopsy. Unlike enzyme studies which may require a significant amount of fresh frozen tissue, DNA-based testing does not usually require extremely careful handling of sensitive specimens. Molecular analysis may help differentiate affected patients with higher levels of residual enzyme activity from heterozygous carriers not predicted to develop disease. Page 4 of 5

5 Genetics: The estimated disease incidence for all forms of glycogen storage disease in the United States is approximately 1 in 20,000 to 1 in 25,000 births (Roth 2009). These disorders are found in all ethnic groups, but founder mutations may put certain populations at higher risk (for example, GSD Type III in the Faroe Islands and GSD Type VI in the U.S. Mennonite community) (Santer el al. 2001; Chang et al. 1988). Although the majority of Glycogen Storage Diseases show autosomal recessive inheritance, two forms of GSD Type IX (due to mutations in the PHKA1 and PHKA2 genes) show X-linked recessive inheritance. Massively parallel sequencing plus Sanger confirmation will detect the vast majority of mutations known to cause GSD. One exception, however, is GSD Type II (Pompe Disease), where at least 11 different gross deletions and one gross insertion have been reported in the GAA gene (Human Gene Mutation Database). Please see individual test descriptions for additional information on the molecular biology of each gene. Testing Strategy: For this NGS panel, the full coding regions plus ~20bp 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. Indications for Test: Genetic testing for Glycogen Storage Disease is available for patients suspected to have this condition. Molecular testing is useful to confirm a clinical diagnosis of GSD and to aid in disease treatment and management. Analytical Sensitivity: Greater than 99% of single nucleotide substitutions, small insertions and deletions. This test does not detect large deletions or duplications spanning one or more exons. Clinical Sensitivity: The vast majority of mutations known to cause GSD are detectable using massively parallel sequencing plus Sanger confirmation. It is estimated that gene sequencing will detect at least one causative allele in 98% of tested patients. In some cases, GSD caused by rare gross deletions of a homozygous or hemizygous nature may initially be suspected using sequencing methods, but diagnostic confirmation requires gene-specific deletion analysis. References: Please see the test description for this test on our website () for a full list of complete citations. Page 5 of 5