CPT codes 81470 and 81471 can be used if at least 60 of the genes in the panel are analyzed. We are happy to accommodate requests for testing single genes in this panel 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 via our PGxome Custom Panel tool.

The great majority of tests are completed within 26 days.

Intellectual Disability (ID) is characterized by significant limitations in intellectual functioning and impairments in adaptive behavior, manifesting before 18 years of age (American Association of Intellectual and Developmental Disabilities, AAIDD). Intellectual functioning is assessed using an intelligence quotient (IQ) test, and a cutoff of ≤70 is considered intellectually impaired. Adaptive behavior is assessed across three domains - conceptual, social, and practical abilities (AAIDD). ID is diagnosed in ~1-3% of the total population, and X-linked genes account for ~5-10% of ID cases in males (Vissers et al. 2016. PubMed ID: 26503795; Kaufman et al. 2010. PubMed ID: 21124998; Lubs et al. 2012. PubMed ID: 22482801; Tzschach. 2015. PubMed ID: 25649377). X-linked intellectual disability (XLID) is phenotypically diverse, with conditions ranging from severe, early onset syndromic diseases involving multiple systems, to mild nonsyndromic forms (Neri et al. 2018. PubMed ID: 29696803).

While true treatments are often limited for XLID disorders, advantages of testing and molecular diagnosis are still numerous. These include prognostic information, early identification and treatment of symptoms (autism, seizures, comorbidities), and ability to join condition-specific support groups. When parental inheritance is observed, families can use this information for future family planning (including prenatal testing or pre-implantation genetic diagnosis). Alternatively, if a patient has a confirmed de novo variant (undetectable in parents), this may ease anxiety on the family, due to the dramatically reduced recurrence risk.

X-linked intellectual disability occurs in both X-linked dominant and X-linked recessive inheritance patterns. Males are disproportionately affected with X-linked recessive (XLR) forms of XLID due to hemizygosity of the X-chromosome, while several factors affect disease presentation in females. One of these factors is skewed X-chromosome inactivation patterns in females, which can impact disease severity or alter presenting symptoms (Plenge et al. 2002. PubMed ID: 12068376; Fieremans et al. 2016. PubMed ID: 27159028). In cases where inactivation is skewed completely toward the mutated allele, an XLR disorder may present similarly in a female to that seen in male. Alternatively, skewing toward the normal allele or tissue specific patterns of skewing may cause female carriers of an X-linked dominant disorder to present with significantly milder or variable presentations. The cellular mosaicism resulting from random X-inactivation in females is proposed as the mechanism for one female-limited form of XLID, caused by pathogenic variation in the PCDH19 gene (Dibbens et al. 2008. PubMed ID: 18469813). Another factor affecting gender-skewing in X-linked disorders is early lethality in males for some X-linked diseases, leading to these disorders appearing to disproportionately affect females (Franco et al. 2006. PubMed ID: 16650755).

This panel focuses on genes associated with established monogenic forms of XLID (Neri et al. 2018. PubMed ID: 29696803). A wide variety of causative variant types in these genes have been reported to cause XLID (including missense, nonsense, splicing, frameshift, large insertions and deletions, complex rearrangements, and trinucleotide repeat expansions)(Human Gene Mutation Database). Causative XLID variants in males are frequently inherited from an unaffected or mildly affected mother; yet, de novo mutations are another common cause of XLID in both males and females.

For this Next Generation Sequencing (NGS) test, sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization kit, followed by massively parallel sequencing of the captured DNA fragments.

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.

Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.

This panel typically provides 97.9% coverage of all coding exons of the genes plus 10 bases of flanking noncoding DNA in all available transcripts along with other non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. We define coverage as ≥20X NGS reads or Sanger sequencing.

Genes of this panel that are likely to get low coverage in an area of high pathogenic variation include (but are not necessarily limited to): ABCD1, IKBKG, and RPS6KA3. If a disorder associated with one of these genes is suspected, additional testing may be warranted.

Since this test is performed using exome capture probes, a reflex to any of our exome based tests is available (PGxome, PGxome Custom Panels).

This panel is recommended for patients with syndromic or nonsyndromic intellectual disability, and a family history suggestive of an X-linked pattern of inheritance. Fragile X repeat expansion testing is recommended in addition to this test.

Diseases

For PGxome® we use Next Generation Sequencing (NGS) technologies to cover the coding regions of targeted genes plus 10 bases of flanking non-coding DNA in all available transcripts along with other non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. As required, genomic DNA is extracted from patient specimens. Patient DNA corresponding to these regions is captured using Agilent Clinical Research Exome hybridization probes. Captured DNA is sequenced on the NovaSeq 6000 using 2x150 bp paired-end reads (Illumina, San Diego, CA, USA). The following quality control metrics are generally achieved: >97% of target bases are covered at >20x, and mean coverage of target bases >120x. Data analysis and interpretation is performed by the internally developed software Titanium-Exome. In brief, the output data from the NovaSeq 6000 is converted to fastqs by Illumina Bcl2Fastq, and mapped by BWA. Variant calls are made by the GATK Haplotype caller and annotated using in house software and SnpEff. Variants are filtered and annotated using VarSeq (www.goldenhelix.com).

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.

Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.

NextGen Sequencing: 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.

Copy Number Variant Analysis: The PGxome test detects most larger deletions and duplications including intragenic CNVs and large cytogenetic events; however aberrations in a small percentage of regions may not be accurately detected due to sequence paralogy (e.g., pseudogenes, segmental duplications), sequence properties, deletion/duplication size (e.g., 1-3 exons vs. 4 or more exons), and inadequate coverage. In general, sensitivity for single, double, or triple exon CNVs is ~70% and for CNVs of four exon size or larger is >95%, but may vary from gene-to-gene based on exon size, depth of coverage, and characteristics of the region.

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 sequencing does not reveal any heterozygous differences from the reference sequence, we cannot be certain that we were able to detect both patient alleles.

For technical reasons, the PGxome test is not 100% sensitive. Some exons cannot be efficiently captured, and some genes cannot be accurately sequenced because of the presence of multiple copies in the genome. Therefore, a small fraction of sequence variants will not be detected.

We sequence coding exons for all available transcripts plus 10 bp of flanking non-coding DNA for each exon. We also sequence other regions within or near genes in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere.  Unless specifically indicated, test reports contain no information about other portions of genes.

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.

Balanced translocations or inversions are only rarely detected.

Certain types of sex chromosome aneuploidy may not be detected.  

Our ability to detect CNVs due to somatic mosaicism is limited.

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.

A negative finding does not rule out a genetic diagnosis.

Genetic counseling to help to explain test results to the patients and to discuss reproductive options is recommended.