Page 1346 - Clinical Immunology_ Principles and Practice ( PDFDrive )

CHaPTEr 96 Molecular Methods 1307

sift/SIFT.html), PolyPhen (http://coot.embl.de/PolyPhen/), and
Tertiary Data Analysis—Variant Annotation, several others. CADD represents a class of software that creates
Interpretation, and Reporting a joint model based on integration of many of these methods.
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The next steps in the analysis involve annotation, filtering, and All these criteria are put together in a process called gene and
classification of variants as to their likely role in the disease or variant curation. Recommendations for scoring variants into
phenotype for which the test was submitted. The laboratory must five categories—pathogenic, likely pathogenic, variant of unknown
ensure that the final report is accurate and complete without significance, likely benign, and benign—have been given by the
overinterpretation. Annotation of each variant detected in the American College of Medical Genetics and Association for
sequencing means attaching information about population Molecular Pathology. 34
frequency (or lack of previous observation), differences in The next step in the process is assembly of filtered and classified
frequency between various ethnic groups, effect of the variant on variants into the laboratory report. The report must be simple
protein coding sequence, effect on splicing, and so on. Diagnostic enough for physicians who are not experts in genomics to
laboratories must actively use information contained in the public understand but, at the same time, must document the intermediate
sequence databases to interpret the biological consequences of results required to demonstrate analytical accuracy and clinical
mutations and polymorphisms. There are databases that have validity. Clinical reports must contain clear nomenclature for
an important role: (i) GenBank, which contains the reference the position and consequence of relevant variants along with
human genome sequences (http://www.ncbi.nlm.nih.gov/entrez); appropriate indication of test and interpretive limitations. The
(ii) dbSNP, which contains more than 150 000 000 common final steps of interpretation and reporting may be facilitated by
single-nucleotide polymorphisms; (iii) Exome Sequencing Project communication with the referring clinician so that there is active
(https://esp.gs.washington.edu/drupal/), which has exome data collaboration in arriving at a clinical diagnosis.
on about 6500 individuals from several research projects; (iv) the The final steps in bioinformatics management of NGS tests
1000 Genomes Project (http://www.1000genomes.org/), which has are aggregation and data sharing. Submission of deidentified
exome and genome data from about 2500 multiethnic controls; and cases to public-knowledge databases, such as ClinVar, will allow
the (v) Exome Aggregation Consortium (http://exac.broadinstitute aggregation of variant data accompanied by sufficient clinical
.org/), which has assembled variant data on more than 60 000 information to support variant classification. Once this informa-
subjects from various disease and populations studies. tion is accumulated in many clinical contexts, the value for patient
Interpretation of sequence and genotype information is the care will be magnified beyond what could be inferred from the
next step in carrying out a molecular diagnostic test. The labora- case data alone. Data sharing that is consistent, standardized,
tory must do the following: take into consideration the patient’s and collected with appropriate protections for confidentiality
presenting symptoms and other clinical data; examine the strength will be an invaluable resource for the future.
of the evidence that specific gene mutations can cause the
suspected disease; look for variants in genes that might provide Clinical Performance of Genomics Assays
a causal explanation; determine whether a candidate variant has The development of specialized training programs in molecular
previously been reported to cause a disease; determine whether genetics and molecular genetic pathology by the American
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variants that appear in previous databases have a low frequency Boards of Medical Genetics and Pathology highlights the recogni-
(i.e., compatible with the observed frequency of the rare disease); tion by professional groups that this area of clinical testing
determine whether loss-of-function, gain-of-function, dominant is exceptionally complex. Although inferences made about
negative, haploinsufficiency, and so on are known underlying disease diagnosis and carrier status based on direct detection of
mechanisms of disease; determine whether variant occurs in an mutations are inherently categorical, data interpretation should
exon and/or protein domain compatible with alteration of incorporate known genotype–phenotype correlations, variable
function; and summarize whether the candidate variants occur disease expression/severity, incomplete penetrance, gender-specific
in similarly affected family members (pattern of inheritance risk, and other available data. In the future, as DNA testing is
and cosegregation). If a variant has been observed in the employed in the assessment of more complex traits such as
proband and other family members are tested, then the expecta- autoimmune and neoplastic diseases, genotype data will perhaps
tion is that it will exactly segregate with the disease. An example be expressed as “relative risk” and incorporate gene–gene and
would be testing for a mutation in X-linked SCID in two brothers. gene–environment interactions. The reliability of these estimates
The causal variant will be present in both. When the proband will require standard data acquisition algorithms and constant
is the only affected individual, then one may look to the literature; updating of population-based data.
ClinVar (www.clinicalgenome.org/data-sharing/clinvar/), a
database of variants with curated interpretations; and locus- RECOMMENDATIONS FOR USE
specific public databases (bioinf.uta.fi/base_root/mutationdata
bases.php) to determine whether the specific variant has previ-
ously been observed in another affected person. However, it is KEY CONCEPTS
routine to find variants that have not previously been observed.
Assessment of the functional consequence of a newly observed Principles of Molecular Diagnostics
sequence variant is problematic, as there is no guarantee that it • DNA diagnostics can play an important role in the diagnosis of specific
is anything more than a rare, but neutral, polymorphism. Loss- diseases in affected individuals and in genetic risk assessment for
of-function mutations (e.g., nonsense, frameshift, and conserved their family members.
splice site mutations) have clear functional consequences. Missense • Genotyping can be used to conduct prenatal diagnoses.
substitutions are more difficult. Several methods based on • Prenatal diagnosis may be used not only for elective termination of
sequence conservation and the chemical properties of amino pregnancy but also for treatment planning.
acids have been developed. These are available in software, such • Provision of genetic testing results to patients and their families carries
responsibilities for sensitivity and confidentiality.
as Sorting Intolerant From Tolerant (SIFT) (http://blocks.fhcrc.org/