Testing Frequently Asked Questions

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.

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).

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.