Testing Frequently Asked Questions

This section is a work in progress. If you have a question that is not answered herein, ask us. If your question is brief and general (not regarding a specific patient or person), our Director (Richard G. Boles, M.D.) will provide a brief generalized response. If your question is thought to be of interest to many, you might see it converted by into a future FAQ. We cannot answer specific medical questions, unless you are an existing patient. Note that the answers are summaries designed for physicians/patients without a detailed understanding of genetics and genetic testing. As such, the text will be basic for some, yet complicated for others. This is not a “course” in genetics, and does not replace expert consultation, but is meant to be helpful to physicians. If you are a patient or family member of one, feel free to forward the link to your physician.

 

 

 

 

 

 

Questions and Answers

Yes, the Genetics Home Reference is an excellent source at https://ghr.nlm.nih.gov/. See the
menu on the top right “Help Me Understand Genetics”.

Genetic sequencing, or DNA sequencing, refers to testing that reads the genetic code,
nucleotide by nucleotide. The purpose of genetic testing, like all diagnostic testing, is to identify
the cause or disease or factors leading to disease. The primary reason for this identification is to
assist in treatment and otherwise improve the medical care rendered. In addition, the knowledge
gained may be helpful even if it does not alter patient care, such as to inform school placement
and reproductive choices.

There are many levels to genetic sequencing that developed at different phases in the
evolution of testing. While recent advances have greatly expanded the scope of how much of the
DNA can be sequenced, older and smaller sequencing tests are still in use in limited

circumstances. Below is a list in order from the smallest to largest in terms of the amount of
DNA sequenced.

Single variant sequencing

  • What it is?
    This test looks at a specific change in the DNA code (variant), generally one that is known or
    highly suspected to be related to disease. These tests may look at a single variant in a single
    gene, more than one variant in a single gene, or more than one variant in more than one gene.
  • When to consider?
    This test is generally done today when a specific variant(s) was found in an individual, to see if it
    is present in certain family members.
    Additionally, this type of test can be performed when only one or a few genes are of interest, and
    within those genes only one or a few variants cause most disease.
  • Examples
    Testing of parents for a variant identified in their child to determine if it was inherited or new (de
    novo)
    Testing of an affected sibling for a variant identified in their child to determine if it is present.
    Common MTHFR variant testing
    Sequencing for the common MELAS variant in the mtDNA of patients presenting with classical
    MELAS syndrome

Single gene sequencing

  • What it is?
    This test looks at all changes (variants) in the coding sequences (exons) of a single gene.
  • When to consider?
    This test is generally ordered when only one or a few genes are of interest, yet more than one
    variant within the gene(s) is of interest.
  • Examples
    Sequencing of the POLG gene in an infant with neuromuscular conditions and valproate-induced
    liver disease, an excellent fit for disease related to this specific gene.
    Sequencing of the ACADM gene in patients with newborn screening results that are highly
    suggestive of disease related to this specific gene.

Small sequencing panels

  • What it is?

This test looks at all changes (variants) in the coding sequences (exons) of about 5 to 100 genes.
Note, this is no accepted definition as to what constitutes a “small” versus a “large” panel.

  • When to consider?
    This test is generally ordered when the patient has a clear diagnosis of a condition that is
    generally caused by variants in a relatively small number of genes, for which an existing small
    panel has a relatively high likelihood of identifying causes or factors related to that condition.
  • Examples
    Ordering a mtDNA depletion syndrome sequencing panel in patients with biopsy findings of
    mtDNA depletion.
    Ordering a periodic fever sequencing panel in patients with periodic fever.

Large sequencing panels

  • What it is?
    This test looks at all changes (variants) in the coding sequences (exons) of about 100 to a few
    thousand genes. Note, this is no accepted definition as to what constitutes a “small” versus a
    “large” panel.
  • When to consider?
    This test is generally ordered when the patient has a clear diagnosis of a condition that is
    generally caused by variants in a large number of genes, for which an existing large panel has a
    relatively high likelihood of identifying causes or factors related to that condition.
  • Examples
    Ordering an epilepsy sequencing panel in patients with epilepsy.
    Ordering an ataxia sequencing panel in patients with ataxia.
    Ordering a hearing loss sequencing panel in patients with hearing loss.

Whole exome sequencing (WES)

  • What it is?
    This test looks at all changes (variants) in the coding sequences (exons) of almost all of the
    known 23,000 genes.
  • When to consider?
    This test historically has been ordered when the patient has a condition that is unclear or not well
    defined (e.g. an unclassified neurodevelopmental disorder), the patient has more than one
    condition (e.g. epilepsy and ataxia), a very-large number of genes are associated with the
    condition (e.g. autism), or no good panel exists (e.g. functional disease). Due to recent cost
    considerations, WES is now oftentimes the preferred test to order in most patients.
  • ExamplesSince essentially all known genes are sequenced, WES can be ordered for just about every
    clinical scenario.
    WES is of particular importance in phenotypes associated with several hundreds to thousands of
    genes, including:

    • Congenital anomalies (birth defects)
    • Intellectual disability (mental retardation)
    • Autism
    • Epilepsy

Whole genome sequencing (WGS)

  • What it is?
    This test looks at all changes (variants) in the entire genetic code, including all genes and regions
    that do not contain genes.
  • When to consider?
    This test has been ordered predominately in research setting, but lowered pricing and increased
    utility has provided a recent place for WGS in clinical diagnostics. The exome that is sequenced
    by WES only constitutes about 2% of the entire DNA. The other 98% is predominately
    regulatory, and not well understood. Most known disease-related variants are located in the
    exome; however, this is likely to change over the coming years. Thus, WGS represents an
    investment for the future. What is causing WGS to be seriously considered for clinical testing
    today is that this single test covers small mutations (sequencing) as well as large mutations
    (historically identified my chromosomal microarray, CMA, testing). WGS is sometimes
    comparable or less expensive than ordering WES and CMA separately.
  • Examples
    Same as WES above, especially since both WES/WGS and CMA are indicated in most patients
    with congenital anomalies, intellectual disability, autism, and epilepsy.
    Of note, results for SNPs and pharmacogenetics can be deduced from WGS with the appropriate
    software.

There are several important genetic tests beyond the sequencing of nuclear DNA. These tests
ae generally ordered separately from sequencing testing, in the appropriate settings. Every test in
this list provide unique information, and the tests do not overlap (exception: some tests overlap
with WGS in some laboratories).

Chromosomal microarray (CMA)

  • What it is?
    This test looks at the entire genetic code (all DNA) for copy number variants. Copy number
    variants (CNVs) refers to variants that result in too much of too few copies of each region of the
    DNA. This testing is designed to look for disease-associated CNVs of a few hundreds to millions
    of nucleotide pairs in size.
  • When to consider?
    For any condition, disease-associated variants can be large or small. Large variants are detected
    by CMA, while small variants are detected by sequencing. Thus, CMA and sequencing are
    complementary, and non-overlapping, methodologies to identify disease-associated variants.
    Thus, CMA is generally ordered when the patient has virtually any condition for which genetic
    testing is performed.
  • Examples
    In practice, CMA is generally ordered in patients with higher likelihood of a positive test result,
    including in patients with:

    • Congenital anomalies (birth defects)
    • Intellectual disability (mental retardation)
    • Autism
    • Epilepsy
  • Special Issues
    While CMA is generally performed as a singleton (patient only), most abnormal results will
    require parental testing to determine if it is de novo or inherited. In general, a de novo variant is
    likely disease causal, while an inherited variant may be benign or a risk factor, especially if
    inherited from an unaffected parent. Some laboratories will perform parental testing for no extra
    charge. It is very difficult to get insurance to pay for parental testing.

Mitochondrial DNA (mtDNA) sequencing

  • What it is?
    While WGS refers to the nuclear DNA (contained in the chromosomes, within the nucleus),
    mtDNA sequencing is a whole genome sequencing test for the DNA located inside the
    mitochondria. The mtDNA includes 37 genes and small non-coding regions. Unlike most of the rest of the genome that is inherited from both parents, mtDNA is inherited only from the mother.
    In addition, mtDNA is present at high copy number within cells, and the genetic code is
    somewhat different from that of nuclear DNA. Thus, sequencing of this small genome is often
    considered to be a separate test (a small panel) and may or may not be included as part of
    WES/WGS, depending on the laboratory.
  • When to consider?
    Since the mtDNA is involved in energy metabolism, and all cells need energy to conduct most
    cellular processes, variants in the mtDNA can be associated with a very wide range of clinical
    conditions.
  • Examples
    In practice, mtDNA sequencing is generally ordered in patients with higher likelihood of a
    positive test result, including in patients with:

    • Functional disease (pain, fatigue, gastrointestinal symptoms, dysautonomia, mood
      disorders, etc.)
    • Any neurological or neurodevelopmental condition
    • Any multisystem condition

Trinucleotide repeat testing

  • What it is?
    Some specific areas of the genome have trinucleotide sequences (e.g. CAG) that normally repeat
    a number of times. These trinucleotide repeats are unstable in cell division, and different
    individuals have different numbers of repeats. Particularly large numbers of repeats are highly
    unstable, and at risk for extreme expansion in the number of repeats in meiosis, when the egg
    and sperm nuclei are created. Trinucleotide repeat testing counts the number of repeats to
    determine if the number is consistent with disease or reproductive risk of disease.
  • When to consider?
    Expansion of specific trinucleotide repeats results in specific disorders, most of which are
    neurological, especially intellectual disability (including autism), ataxia, and movement
    disorders.
  • Examples
    Fragile X is a single variant test that is often ordered in patients with intellectual disability or
    autism.
    Other trinucleotide repeat testing is important in hereditary or early-onset ataxias.
    Some laboratories include trinucleotide repeat testing (fragile X and others) in WGS, while
    others do not.

Imprinting/methylation testing

  • What it is?                                                                                                                                                                                                                                                                Usually, the gene inherited from the mother is treated identically to the same gene inherited from
    the father, but this is not always the case. A minority of regions on the chromosomes are
    “imprinted” such that the gene inherited from one of the parent is methylated, and thus turned
    off, while the gene inherited from the opposite parent is unmethylated, and thus turned on. In
    certain situations, both copies of a gene are methylated, which leads to disease. This can be
    detected by imprinting or methylation testing.
  • Examples
    Angelman syndrome testing is an imprinting or methylation test that is often ordered in patients
    with intellectual disability, autism, and/or epilepsy. Many cases of this condition are not detected
    by CMA and sequencing.
    Prader-Willi syndrome testing is an imprinting or methylation test that is often ordered in
    patients with overeating and hypotonia. Most cases of this condition are not detected by CMA
    and sequencing.

Pharmacogenetics

Information to come.

Single nucleotide polymorphism (SNP) testing

Information to come.

The answer to your question revolves around what are the added advantages of “triome” over
“singleton” sequencing.

Singleton: This term refers herein to when only DNA from the patient is sequenced. While not
perfect, most of the advantages of sequencing can be gained from testing the patient alone, at
least in many patients (see below).

Triome: This term refers to when DNA from the patientand both parentsare sequenced. Triome
sequencing offers certain advantages over singleton sequencing, including:

  • The primary advantage of triome is the identification ofde novovariants, which are
    “new mutations” that are present in the patient but absent in both parents. Triome
    sequencing is frequently often used in patients with neurodevelopmental disorders
    (intellectual disability, autism, etc.), epilepsy, “syndromes”, and/or birth defects,
    whereasde novo variants are fairly common and oftentimes the main cause of the
    patient’s disease.
  • Most metabolic disorders are autosomal recessive, in which both parents are (usually
    unaffected) carriers of one disease-causing variant each, and the patient inherited both
    of these variants, resulting in disease. When two different variants that might be
    disease-related are identified in a patient, recessive disease is likely. To be disease
    causing, each variant should be on a different chromosome, inherited one each from
    both parents (in which the “phase” is termed as being “in trans”). The alternative is that
    both variants are located on the same chromosome, inherited from only one parent (in
    which the “phase” is termed as being “in cis”). In the latter case, the variants are likely
    not disease causal, but could be a risk factor. The easiest, and often the only, way to
    determine the phase of the variants (in cis versusin trans) is to sequence at least one
    parent.
  • In the case that one parent is clinically affected, sequencing can determine which of
    several “variants of uncertain significance” were inherited from the affected parent.
    Obviously, these variants would be more important to consider than are the variants
    inherited from the unaffected parent.

The purpose of triome sequencing is to better understand the sequence results in the patient.
Triome sequencing is NOT intended to identify genetic disease or risks is the parents, most of
which will not be identified. Of course, triome sequencing will identify a parent as having a
disease-causing variant if both the patient and a parent share the variant.
Of note, triome sequencing can be used for large panels, whole exome sequencing (WES),
and whole genome sequencing (WGS).

As a practical matter, triome sequencing is recommended forpatients with neurodevelopmental
disorders (intellectual disability, autism, etc.), epilepsy, “syndromes”, and/or birth defects,
whereas in many other settings, singleton sequencing is appropriate.

Before reading on, read the section entitled “Triome versus singleton testing”. Certainly, all
of the advantages of singleton testing are available if one parent is unavailable, or even if both
parent are unavailable (e.g. the child is adopted).
However, without both parents, de novo variants cannot be identified. If only one parent is
available, sequencing of that parent can be helpful in terms of phasing if two variants of interest
in the same gene are found (see #2 in “Triome versus singleton testing”). Furthermore, the parent
of origin can generally be determined from a single parent (see #3), as a de novo variant is very
unlikely if the variant identified is not very rare.

Choosing the correct test(s) is complicated, is based on the specifics of each available case,
and is best determined by a genetics professional or highly-knowledgeable physician. The
information herein is simplified into general principles in order to assist physicians to order the
correct testing in many less-complicated situations. To some degree, this is an opinion piece
written by Dr. Boles, although the opinions herein are shared by many geneticists. Before
reading on, please read the sections entitled “The different levels of genetic sequencing”,
“Additional genetic testing to consider beyond nuclear sequencing”, and “Triome versus
singleton testing”.

  1. Choose the appropriate level of sequencing:
    • Lower levels of testing such as single variant sequencing, single gene sequencing,
      and small sequencing panels are only indicated if the exact diagnosis is highly
      suspected, and an inexpensive test is desired for confirmation. If the test is
      negative, then testing should be reflexed to a higher level, generally WES.
    • Large panels are appropriate for certain conditions, but are generally being
      replaced by WES as the difference in the costs narrows, especially considering the
      added price of reflux to WES should panel testing be negative or inconclusive.

      • Note that all testing below the level of WES will become outdated very
        rapidly as additional variants and genes are rapidly being associated with
        disease.
    • WES is the standard level of testing for most patients undergoing sequencing for
      metabolic, morphological (syndromes), neurological, and neurodevelopmental
      conditions.

      • WES has the most favorable current-clinically-relevant-information to
        cost ratio among all levels of testing, baring cases in which the physician
        is confirming a highly-suspected diagnosis or variant identified in a
        relative.
      • Note that even WES will become outdated as additional non-
        exonic variants are associated with disease.
    • WGS is of clinical relevance in cases whereas multiple testing is considered. In
      particular, WGS in some laboratories can provide all of the following testing, for
      one cost: whole genome (including WES), CMA, mtDNA sequencing, fragile X
      and other trinucleotide repeats. In some cases, WGS is less expensive than
      individual testing.
  2. Choose singleton or triome for sequencing:
    • Triome testing is recommended for early-onset cases with neurodevelopmental
      disorders (intellectual disability, autism, etc.), epilepsy, and morphological
      conditions (“syndromes”, birth defects).
    • Singleton testing is recommended for most other conditions, including late-onset
      neurological and functional disorders
  3. Choose additional testing:
    • CMA as appropriate, particularly in cases with congenital anomalies, intellectual
      disability, autism, and/or epilepsy.
    • mtDNA sequencing as appropriate, particularly in cases with multiple functional
      symptomatology, any neurological or neurodevelopmental condition, and/or a
      multisystem condition.
    • Trinucleotide repeat testing as appropriate, particularly fragile X testing in cases
      with intellectual disability or autism. Testing for other repeats should be
      conditioned for ataxias or certain movement disorders.
    • Imprinting/methylation testing as appropriate, particularly Angelman syndrome
      testing in cases with intellectual disability, autism, and/or epilepsy. Consider
      Prader-Willi syndrome testing as appropriate, particularly in cases with overeating
      and hypotonia.
  4. Choose a laboratory, based on the following criteria:
    • Testing: Does the laboratory do all of the testing requested. However, sometimes
      it is appropriate to send testing to two or more laboratories.
    • Methodology: Does the laboratory use best practices? Does it “miss” diagnoses
      found elsewhere?
    • Reporting: Does the laboratory report contain the information desired for a
      clinical interpretation?

      • Laboratory reporting vary tremendously in terms of the reporting of
        variants of uncertain significance, how narrow is considered “on target”
        versus “incidental”, and in regards to polygenic and integrative modeling.
    • Pricing: Certainly, lower is better. However, in determining price, remember to
      consider parental testing/confirmation (e.g. triome pricing, does the laboratory
      confirm positive CMA variants for no extra charge), bundling (e.g. some labs
      include mtDNA in WES/WGS, most testing can be bundled in WGS), insurance
      coverage, and out-of-pocket pricing and liability for families (especially, will the
      family receive an unexpected bill if insurance fails to pay?).
    • Invasiveness: Blood, saliva, or buccal swabs?
    • Process: Does the laboratory provide assistance with ordering and insurance
      coverage? Is full sequence data transferred easily upon request? Are results sent
      promptly? Are questions answered promptly and professionally?

In many cases, a patient already has a diagnosis. This diagnosis may be based on a variety of
different criteria. There are several situations in which additional genetic testing can be helpful
and is indicated:

  • The current diagnosis is only a description: Most patients carry a diagnosis that is simply
    a description of the patient’s disease, and the diagnosis is not an underlying physiological
    or genetic cause or factor. Examples include epilepsy, autism, intellectual disability,
    migraine, cyclic vomiting syndrome, chronic fatigue syndrome, fibromyalgia, and many
    others. In these cases, genetic testing is often indicated in order to identify underlying
    disease-related factors or causes, some of which may be treatable.
  • The current diagnosis is in doubt: In many cases, the diagnosis is not definitive, and may
    or may not be correct. In these cases, genetic testing is often indicated in order to look for
    alternative potential diagnoses, some of which may be treatable.
  • The primary diagnosis is likely correct, but does not account for all of the clinical
    manifestations present in the patient: In these cases, genetic testing is often indicated in
    order to identify additional underlying disease-related factors or causes, some of which
    may be treatable. A common example is a patient who has a copy number variant (CNV)
    identified on chromosomal microarray (CMA) that fits the patient’s phenotype, but was
    inherited from a minimally affected or unaffected parent. Obviously, the CNV alone
    cannot cause the patient’s disease, and the CNV is assumed to be a risk factor, for which
    additional risk factors remain to be identified. Most patients with complex disease,
    including all of the conditions listed in the first bullet above, are thought to have multiple
    factors, both genetic and environmental, that together lead to the development of disease.
  • A phenotype is of significant distress to patient/family, and is refractory to the usual
    therapies: Never assume that all of the factors leading to disease have been identified, as
    even siblings with the same “monogenic” disorder generally demonstrate wide
    phenotypic heterogeneity. In treatment-refractory cases, genetic testing is often indicated
    in order to identify additional underlying disease-related factors or causes, some of which
    may be treatable. An example is a patient with a known epilepsy-predisposition gene in
    which seizure are severe and refractory despite standard and gene-specific therapies.