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.

Methylmalonic acidemia (MMA) is typically a severe disease with onset in infancy. Patients may present with lethargy, vomiting, hepatomegaly, acidosis, hypoglycemia and neutropenia. Many patients die in childhood; those that survive often experience neurological and renal complications. Milder forms of the disease are also known. Today, many cases are detected through routine neonatal screening with tandem mass spectrometry (Fenton et al. 2014; Watkins and Rosenblatt 2014; Manoli et al. 2016).

The MMADHC, MMAA, MMAB, MCEE, MUT and CD320 genes encode proteins directly related to cobalamin transport or metabolism, or methylmalonyl-CoA metabolism. Defects in these genes have been shown to lead to the development of isolated MMA. Defects in each gene are associated with a specific type of MMA disorder (cblD, cblA, cblB, MCEE, MUT or CD320 deficiency, respectively). The majority of MMA patients have defects in the MUT gene, followed by the MMAA and then MMAB genes (Manoli et al. 2016). Defects in the MMADHC, MCEE and CD320 genes are rare, having only been reported in a small number of families (Bikker et al. 2006; Dobson et al 2006; Carrillo et al. 2013; Manoli et al. 2016). It should be noted that pathogenic variants in the CD320 gene lead to transient methylmalonic aciduria and/or homocystinuria, which has not been associated with the neurological or hematological symptoms of the cblD, cblA, cblB, mcee or mut deficiency disorders. However, CD320 deficient individuals may be detected via newborn screening (Quadros et al. 2010; Karth et al. 2012; Watkins and Rosenblatt 2014).

This sequencing panel also includes coverage for several genes that do not encode proteins directly involved in cobalamin or methylmalonyl-CoA metabolism. Defects in the ALDH6A1 gene lead to methylmalonate semialdehyde dehydrogenase deficiency (MMSDH), which is an inborn error of valine and thymine metabolism (Chambliss et al. 2000; Marcadier et al. 2013). Defects in the ACSF3 and MLYCD genes lead to combined malonic and methylmalonic aciduria (CMAMMA) (FitzPatrick et al. 1999; Alfares et al. 2011; Sloan et al. 2011; Baertling et al. 2014). Defects in the SUCLA2 and SUCLG1 genes lead to mitochondrial DNA depletion syndromes (MDSs) (Elpeleg et al. 2005; Carrozzo et al. 2007; Ostergaard et al. 2007; Carrozzo et al. 2016). These five genes are included in this panel as defects in all of them have been shown to result in methylmalonic acidemia in at least some patients. Phenotypically, these disorders may be similar or quite different from the cblD, cblA, cblB, mcee or mut types of methylmalonic acidemia.

More details regarding each disorder are available on individual gene test pages.

Isolated methylmalonic acidemia (MMA) can result from pathogenic variants in at least six different genes directly associated with inborn errors of cobalamin metabolism or transport, or methylmalonyl-CoA metabolism (MMADHC, MMAA, MMAB, MCEE, MUT and CD320). In addition, defects in an additional five genes (ACSF3, ALDH6A1, MLYCD, SUCLA2 and SUCLG1) may also lead to methylmalonic acidemia, although these genes are not directly related to cobalamin metabolism or transport or methylmalonyl-CoA metabolism. All of these disorders are inherited in an autosomal recessive manner.

Massively parallel sequencing plus Sanger confirmation will detect the vast majority of sequence variants in the MMADHC, MMAA, MMAB, MCEE, MUT, CD320, ACSF3, ALDH6A1, MLYCD, SUCLA2 and SUCLG1 genes. It should be noted that large deletions that may not be detectable via sequencing have been reported in the MMAA, MUT, ACSF3 and SUCLA2 genes (Human Gene Mutation Database).

See individual gene test descriptions for information on molecular biology of gene products.

For this NextGen test, the full coding regions plus ~10 bp of non-coding DNA flanking each exon are sequenced for the genes listed. Sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization kit, 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.

In addition to the regions described above, this testing includes coverage of the following deep intronic variant: MCEE c.379-644A>G.

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.

Patients with biochemical and/or clinical symptoms suggestive of isolated methylmalonic acidemia (MMA) are good candidates for this test.

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.