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+---+------+-------+----------------+----------------+----------------+
| * |      |       | **Phase of     |                |                |
| * |      |       | Testing --     |                |                |
| T |      |       | Information    |                |                |
| e |      |       | Needs**        |                |                |
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+---+------+-------+----------------+----------------+----------------+
|   | **   | *     | **             | **Analytic**   | **P            |
|   | Type | *Type | Pre-analytic** |                | ost-Analytic** |
|   | of   | of    |                |                |                |
|   | Outp | Inf   |                |                |                |
|   | ut** | ormat |                |                |                |
|   |      | ion** |                |                |                |
+---+------+-------+----------------+----------------+----------------+
| 1 | Full | DNA   | Pr             | Date processed | **Catego       |
|   | Ge   |       | imary clinical |                | ries/Subtypes/ |
|   | nome |       | phenotype      | Batch number   | Stages/Class** |
|   | Scan |       |                |                |                |
|   |      |       | Co-morbid      | Sample quality | **Probability  |
|   |      |       | conditions     | (score?)       | score**        |
|   |      |       |                |                |                |
|   |      |       | Test context   | **Whole genome |                |
|   |      |       |                | or partial**   |                |
|   |      |       | Ethnic         |                |                |
|   |      |       | origin/s (self | Data quality   |                |
|   |      |       | report)        | (score?)       |                |
|   |      |       |                |                |                |
|   |      |       | Age            | Duplicate test |                |
|   |      |       |                | (?)            |                |
|   |      |       | Gender         | Independent    |                |
|   |      |       |                | method?        |                |
|   |      |       | Date of        |                |                |
|   |      |       | sampling       | Bank Raw data? |                |
|   |      |       |                | Images?        |                |
|   |      |       | Sample type    |                |                |
|   |      |       |                |                |                |
|   |      |       | Germline or    |                |                |
|   |      |       | somatic sample |                |                |
|   |      |       | (              |                |                |
|   |      |       | interpretation |                |                |
|   |      |       | or always      |                |                |
|   |      |       | clear to lab?) |                |                |
+---+------+-------+----------------+----------------+----------------+
| 2 | Full | DNA   | Individual     | Sample         | Secure,        |
|   | Ge   |       | identifier     | tracking       | structured     |
|   | nome |       |                |                | electronic     |
|   | Se   |       | Source of DNA  | Sample         | reporting of   |
|   | quen |       |                | processing     | data to        |
|   | ce\* |       | Date of sample |                | EHR/PHR/Other  |
|   |      |       |                | Error checking | data           |
|   |      |       | Data sharing   |                | repository     |
|   |      |       | consent --     | Instrument     |                |
|   |      |       | research,      | reporting of   | a.             |
|   |      |       | relationships, | sequence (and  | **Pre-analytic |
|   |      |       | donor          | epigenetic?)   |     modifier   |
|   |      |       | registries     | data in        |     data**     |
|   |      |       |                | standardized,  |                |
|   |      |       | Contextual     | structured     | b.  **Raw      |
|   |      |       | family history | format         |     sequence   |
|   |      |       | -- appended to |                |     data**     |
|   |      |       | sequence       | **Major        |                |
|   |      |       |                | abnormality    | c              |
|   |      |       | **Contextual   | detection**    | .  **Annotated |
|   |      |       | DNA sequence   |                |     sequence   |
|   |      |       | information    | Secure         |     data       |
|   |      |       | (known         | temporary data |     (quality,  |
|   |      |       | sequence       | storage in     |     major      |
|   |      |       | variations in  | standardized,  |                |
|   |      |       | family for     | structured     |  abnormalities |
|   |      |       | example)--     | format         |     flagged)** |
|   |      |       | appended to    |                |                |
|   |      |       | sequence**     |                | Secure,        |
|   |      |       |                |                | structured     |
|   |      |       | Indication for |                | data storage   |
|   |      |       | testing        |                |                |
|   |      |       |                |                | d.  Data       |
|   |      |       |                |                |     integrity  |
|   |      |       |                |                |                |
|   |      |       |                |                |    maintenance |
|   |      |       |                |                |                |
|   |      |       |                |                | e.  Data       |
|   |      |       |                |                |     access     |
|   |      |       |                |                |     rules      |
|   |      |       |                |                |                |
|   |      |       |                |                | f.  Data       |
|   |      |       |                |                |     sharing    |
|   |      |       |                |                |     rules      |
|   |      |       |                |                |                |
|   |      |       |                |                |    (modifiable |
|   |      |       |                |                |     with time) |
|   |      |       |                |                |                |
|   |      |       |                |                | Structured     |
|   |      |       |                |                | indica         |
|   |      |       |                |                | tion-dependent |
|   |      |       |                |                | data analysis  |
|   |      |       |                |                |                |
|   |      |       |                |                | g.  Algorithms |
|   |      |       |                |                |     for        |
|   |      |       |                |                |     variation  |
|   |      |       |                |                |     detection  |
|   |      |       |                |                |     and        |
|   |      |       |                |                |     comparison |
|   |      |       |                |                |     to         |
|   |      |       |                |                |     reference  |
|   |      |       |                |                |     databases  |
|   |      |       |                |                |                |
|   |      |       |                |                | h.             |
|   |      |       |                |                | Interpretation |
|   |      |       |                |                |     of         |
|   |      |       |                |                |     variations |
|   |      |       |                |                |     in context |
|   |      |       |                |                |     of         |
|   |      |       |                |                |     available  |
|   |      |       |                |                |     clinical   |
|   |      |       |                |                |     data       |
|   |      |       |                |                |                |
|   |      |       |                |                | i.  Generation |
|   |      |       |                |                |     of         |
|   |      |       |                |                |                |
|   |      |       |                |                |   interpretive |
|   |      |       |                |                |     report for |
|   |      |       |                |                |     ordering   |
|   |      |       |                |                |     provider   |
|   |      |       |                |                |                |
|   |      |       |                |                | j.  Periodic   |
|   |      |       |                |                |     updates    |
|   |      |       |                |                |     based on   |
|   |      |       |                |                |     new        |
|   |      |       |                |                |     knowledge  |
|   |      |       |                |                |     applied to |
|   |      |       |                |                |     past data  |
|   |      |       |                |                |     queries    |
|   |      |       |                |                |                |
|   |      |       |                |                | Mechanisms for |
|   |      |       |                |                | secure         |
|   |      |       |                |                | sequence data  |
|   |      |       |                |                | transfer       |
|   |      |       |                |                | between        |
|   |      |       |                |                | individuals    |
|   |      |       |                |                | and healthcare |
|   |      |       |                |                | entities       |
|   |      |       |                |                | across a       |
|   |      |       |                |                | lifespan.      |
+---+------+-------+----------------+----------------+----------------+
| 3 | Full | DNA   | Test ordering  | Lab            | Reporting and  |
|   | Gene |       | guidance       | Information    | Interpretation |
|   | Se   |       |                | System         |                |
|   | quen |       | -   Validation |                | -   Software   |
|   | ce\* |       |     of         | -   extraction |     to support |
|   |      |       |                |     of DNA     |     creation   |
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|   |      |       |     reasons    |     specimen   |     report and |
|   |      |       |     for order  |                |     structured |
|   |      |       |                | -   sample     |     results,   |
|   |      |       | -   Testing    |     generation |     interface  |
|   |      |       |     r          |     (to        |     for        |
|   |      |       | ecommendations |     prepare    |     messaging  |
|   |      |       |     given a    |     material   |     of these   |
|   |      |       |     particular |     for        |     results    |
|   |      |       |     set of     |                |                |
|   |      |       |     s          |    sequencing) |     -          |
|   |      |       | igns/symptoms, |                |    non-patient |
|   |      |       |     clinic     |                |                |
|   |      |       | al/preliminary | -   **On-going |       specific |
|   |      |       |     diagnosis, |                |         data   |
|   |      |       |     and/or     |     monitoring |         to be  |
|   |      |       |     family     |         of     |                |
|   |      |       |     history.   |         primer |     maintained |
|   |      |       |                |                |         in a   |
|   |      |       | ```{=html}     |     quality.** |                |
|   |      |       | <!-- -->       |                |  knowledgebase |
|   |      |       | ```            | -   Instrument |                |
|   |      |       | -   laboratory |                |                |
|   |      |       |                |    Integration |       -   test |
|   |      |       |                |                |                |
|   |      |       |  -   reference |     -          |     dictionary |
|   |      |       |                |  Configuration |                |
|   |      |       |    information |         of     |                |
|   |      |       |         for    |                |     -   unique |
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|   |      |       |    association |         --     |     identifier |
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|   |      |       |                |         data   |                |
|   |      |       |     electronic |         upload |       specific |
|   |      |       |                |         to     |                |
|   |      |       |        medical |         case/  |      materials |
|   |      |       |         record | sample/aliquot |                |
|   |      |       |         and    |                |                |
|   |      |       |                | -   Raw Data   |       -   test |
|   |      |       |       ordering |     Review     |                |
|   |      |       |                |                |       specific |
|   |      |       |       provider |     -   QA/QC  |                |
|   |      |       |                |         of     |     literature |
|   |      |       |         -      |         batch  |                |
|   |      |       |    physician's |                |     references |
|   |      |       |                |       (samples |                |
|   |      |       |          name, |                |                |
|   |      |       |                |       prepared |    -   textual |
|   |      |       |           UPIN |         and    |                |
|   |      |       |             #, |         run    |    description |
|   |      |       |                |                |                |
|   |      |       |         etc... |       together |             of |
|   |      |       |                |         on the |                |
|   |      |       |                |                |      materials |
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|   |      |       |                |                |            and |
|   |      |       |          name, |     -   QA/QC  |                |
|   |      |       |                |         of     |        methods |
|   |      |       |           date |                |                |
|   |      |       |             of |     individual |                |
|   |      |       |                |         trace  |      -   **DNA |
|   |      |       |         birth, |         files, |                |
|   |      |       |                |                |       variants |
|   |      |       |         gender | identification |                |
|   |      |       |                |         of     |          known |
|   |      |       |         etc... |         those  |                |
|   |      |       |                |                |            for |
|   |      |       |         -   if |        needing |                |
|   |      |       |                |         to be  |            the |
|   |      |       |            the |         re-run |                |
|   |      |       |                |                |     particular |
|   |      |       |        patient |                |                |
|   |      |       |             is |    -   Support |           gene |
|   |      |       |                |         for    |                |
|   |      |       |          alive |         manual |       tested** |
|   |      |       |             or |         review |                |
|   |      |       |                |                |                |
|   |      |       |           dead |        process |      -   **DNA |
|   |      |       |                |                |                |
|   |      |       |          (also |                |        variant |
|   |      |       |                |       -   note |                |
|   |      |       |       relevant |                |          name, |
|   |      |       |                |        quality |                |
|   |      |       |            for |             of |           gene |
|   |      |       |             i  |                |                |
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|   |      |       |                |                |                |
|   |      |       |                |                |       location |
|   |      |       |        -   hos |   -   **record |                |
|   |      |       | pital/provider |                |             on |
|   |      |       |                |            DNA |                |
|   |      |       |          name, |                |            the |
|   |      |       |                |       variants |                |
|   |      |       |        medical |                |         gene** |
|   |      |       |                |    identified, |                |
|   |      |       |         record |             in |                |
|   |      |       |                |                |    -   **other |
|   |      |       |         number |     structured |                |
|   |      |       |                |                |         names, |
|   |      |       |                |         form** |                |
|   |      |       |    -   patient |                |            for |
|   |      |       |                |                |                |
|   |      |       |    information |  -   **Support |            the |
|   |      |       |         to be  |                |                |
|   |      |       |                |            for |       variant, |
|   |      |       |     considered |                |                |
|   |      |       |         in     |      automated |       commonly |
|   |      |       |                |                |                |
|   |      |       |       analysis |       sequence |           used |
|   |      |       |         and    |                |                |
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|   |      |       | interpretation |                |                |
|   |      |       |         of     |                |            the |
|   |      |       |                |    -   Support |                |
|   |      |       |        results |         for    |   literature** |
|   |      |       |                |                |                |
|   |      |       |         -      |       repeated |                |
|   |      |       | Race/Ethnicity |                |   -   clinical |
|   |      |       |                |        testing |                |
|   |      |       |                |         of the |   significance |
|   |      |       |     -   clinic |         same   |                |
|   |      |       | al/preliminary |         gene   |             -  |
|   |      |       |                |         region |    **frequency |
|   |      |       |      diagnosis |                |                |
|   |      |       |                |     -   **For  |       reported |
|   |      |       |                |         tests  |                |
|   |      |       |        -   age |                |             by |
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|   |      |       |                |         on     |            the |
|   |      |       |      diagnosis |         tumor  |                |
|   |      |       |                |                |            lab |
|   |      |       |                |     specimens, |                |
|   |      |       |    -   results |         if DNA |            and |
|   |      |       |             of |                |                |
|   |      |       |                |       variants |        linkage |
|   |      |       |       clinical |         are    |                |
|   |      |       |                |                |             to |
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|   |      |       |                |         the    |        reports |
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|   |      |       | igns/symptoms, |                |                |
|   |      |       |                |       specimen |        similar |
|   |      |       |            and |         needs  |                |
|   |      |       |                |         to be  |           test |
|   |      |       |      treatment |                |                |
|   |      |       |                |       examined |      results** |
|   |      |       |            key |         for    |                |
|   |      |       |             to |         the    |                |
|   |      |       |                |                | -   literature |
|   |      |       |         clinic |       presents |                |
|   |      |       | al/preliminary |         or     |     references |
|   |      |       |                |                |                |
|   |      |       |     diagnosis. |        absence |            for |
|   |      |       |                |         of the |                |
|   |      |       |                |         DNA    |            the |
|   |      |       |   -   relevant |                |                |
|   |      |       |                |        variant |        variant |
|   |      |       |         social |         (i.e.  |                |
|   |      |       |                |                |                |
|   |      |       |        history |  determination |    -   patient |
|   |      |       |                |         if it  |                |
|   |      |       |                |         was    |       specific |
|   |      |       |    -   patient |                |         data   |
|   |      |       |                |       acquired |                |
|   |      |       |         family |         or     |                |
|   |      |       |                |                |       -   data |
|   |      |       |        history |    inherited), |                |
|   |      |       |                |         as     |           from |
|   |      |       |                |         this   |                |
|   |      |       | -   indication |         will   | pre-analytical |
|   |      |       |             if |         affect |                |
|   |      |       |                |         the    |          phase |
|   |      |       |          other |         int    |                |
|   |      |       |                | erpretation.** |            for |
|   |      |       |         family |                |                |
|   |      |       |                | General        |        linkage |
|   |      |       |        members | laboratory     |             to |
|   |      |       |                | management     |                |
|   |      |       |           have | software for   |       hospital |
|   |      |       |                | genetic        |                |
|   |      |       |       received | testing        |       provider |
|   |      |       |                | laboratories   |                |
|   |      |       |        genetic |                |     electronic |
|   |      |       |                | -   reagen     |                |
|   |      |       |        testing | ts/instruments |       systems, |
|   |      |       |                |                |                |
|   |      |       |            for |                |       ordering |
|   |      |       |                |    -   reagent |                |
|   |      |       |           this |                |     physician, |
|   |      |       |                |     inventory, |                |
|   |      |       |        disease |                |            and |
|   |      |       |                |    re-ordering |                |
|   |      |       |         (gene) |                |        patient |
|   |      |       |                |       details, |                |
|   |      |       |                |         and    |                |
|   |      |       | -   high-level |                |    -   testing |
|   |      |       |                |        quality |                |
|   |      |       |     indication |                |        context |
|   |      |       |                |        testing |                |
|   |      |       |            for |                |                |
|   |      |       |                |      according | -   high-level |
|   |      |       |        testing |         to     |                |
|   |      |       |             to |                |     indication |
|   |      |       |             be |     guidelines |                |
|   |      |       |                |                |                |
|   |      |       |     considered |                |   -   specific |
|   |      |       |             in | -   instrument |                |
|   |      |       |                |                |     indication |
|   |      |       |   interpreting |     inventory, |                |
|   |      |       |                |                |                |
|   |      |       |            the |    maintenance |      -   **DNA |
|   |      |       |                |                |                |
|   |      |       |        results |        history |       variants |
|   |      |       |                |         and    |                |
|   |      |       |            and |                |   identified** |
|   |      |       |                |        quality |                |
|   |      |       |      providing |                |             -  |
|   |      |       |                |        testing |   **zygosity** |
|   |      |       |    appropriate |                |                |
|   |      |       |             r  |      according |                |
|   |      |       | ecommendations |         to     |     -   tissue |
|   |      |       |                |                |                |
|   |      |       |                |     guidelines |         origin |
|   |      |       | -   diagnosis, |                |                |
|   |      |       |                | -              |                |
|   |      |       |        carrier |    **primers** |    -   overall |
|   |      |       |                |                |                |
|   |      |       |       testing, |                | interpretation |
|   |      |       |                |  -   **primers |                |
|   |      |       |              p |         should |                |
|   |      |       | harmacogenomic |         be     |    -   textual |
|   |      |       |                |                |                |
|   |      |       |       guidance |       analyzed |            for |
|   |      |       |                |         on a   |                |
|   |      |       |         etc... |                |            the |
|   |      |       |                |       periodic |                |
|   |      |       |                |         basis  |         report |
|   |      |       |   -   specimen |         for    |                |
|   |      |       |                |         the    |                |
|   |      |       |    information |                |      -   coded |
|   |      |       |                |       presents |                |
|   |      |       |                |         of     |     vocabulary |
|   |      |       |         -   if |         SNP's  |                |
|   |      |       |                |         (i.e.  |            for |
|   |      |       |         cancer |                |                |
|   |      |       |                |       compared |            the |
|   |      |       |        tissue, |                |                |
|   |      |       |                |        against |     structured |
|   |      |       |      pathology |                |                |
|   |      |       |                |        dbSNP). |        results |
|   |      |       |        results |         If a   |                |
|   |      |       |                |         primer |                |
|   |      |       |                |         is     |  -   reporting |
|   |      |       |         -   if |         found  |                |
|   |      |       |                |         to be  |        process |
|   |      |       |            the |         in the |                |
|   |      |       |                |         region |                |
|   |      |       |           test |         of a   |    -   patient |
|   |      |       |                |         SNP,   |                |
|   |      |       |       requires |         it     |           test |
|   |      |       |                |         should |                |
|   |      |       |            two |         be     |          order |
|   |      |       |                |                |                |
|   |      |       |          types |     redesigned |                |
|   |      |       |                |         to     |                |
|   |      |       |             of |                |                |
|   |      |       |                |        prevent |                |
|   |      |       |      specimens |                |                |
|   |      |       |                |        allelic |                |
|   |      |       |        (sample |                |                |
|   |      |       |                |        dropout |                |
|   |      |       |             in |         (i.e.  |                |
|   |      |       |                |         the    |                |
|   |      |       |       question |                |                |
|   |      |       |                |        failure |                |
|   |      |       |            and |         of one |                |
|   |      |       |                |         or     |                |
|   |      |       |              a |         more   |                |
|   |      |       |                |         of the |                |
|   |      |       |      reference |                |                |
|   |      |       |                |        patient |                |
|   |      |       |        sample) |                |                |
|   |      |       |                |        alleles |                |
|   |      |       |             as |         to be  |                |
|   |      |       |                |                |                |
|   |      |       |             in |      amplified |                |
|   |      |       |                |         which  |                |
|   |      |       |            the |         would  |                |
|   |      |       |                |         result |                |
|   |      |       |           case |         in a   |                |
|   |      |       |                |         false  |                |
|   |      |       |           with |         test   |                |
|   |      |       |                |                |                |
|   |      |       |       prenatal |      result)** |                |
|   |      |       |                |                |                |
|   |      |       |            and | -   personnel  |                |
|   |      |       |                |                |                |
|   |      |       |         cancer |                |                |
|   |      |       |                |   -   training |                |
|   |      |       |         tissue |         etc..  |                |
|   |      |       |                |                |                |
|   |      |       |       testing, | -   inspection |                |
|   |      |       |                |     records    |                |
|   |      |       |           than |                |                |
|   |      |       |                |                |                |
|   |      |       |           each |                |                |
|   |      |       |                |                |                |
|   |      |       |       specimen |                |                |
|   |      |       |                |                |                |
|   |      |       |          needs |                |                |
|   |      |       |                |                |                |
|   |      |       |       separate |                |                |
|   |      |       |                |                |                |
|   |      |       |     identifier |                |                |
|   |      |       |                |                |                |
|   |      |       |          along |                |                |
|   |      |       |                |                |                |
|   |      |       |           with |                |                |
|   |      |       |                |                |                |
|   |      |       |              a |                |                |
|   |      |       |                |                |                |
|   |      |       |      mechanism |                |                |
|   |      |       |                |                |                |
|   |      |       |            for |                |                |
|   |      |       |                |                |                |
|   |      |       |          cross |                |                |
|   |      |       |                |                |                |
|   |      |       |      reference |                |                |
|   |      |       |                |                |                |
|   |      |       |                |                |                |
|   |      |       |    -   general |                |                |
|   |      |       |                |                |                |
|   |      |       |       specimen |                |                |
|   |      |       |                |                |                |
|   |      |       |    information |                |                |
|   |      |       |                |                |                |
|   |      |       |                |                |                |
|   |      |       |   -   specimen |                |                |
|   |      |       |                |                |                |
|   |      |       |           type |                |                |
|   |      |       |                |                |                |
|   |      |       |                |                |                |
|   |      |       |    -   Billing |                |                |
|   |      |       |                |                |                |
|   |      |       |    information |                |                |
|   |      |       |                |                |                |
|   |      |       |                |                |                |
|   |      |       |  -   Physician |                |                |
|   |      |       |         and    |                |                |
|   |      |       |                |                |                |
|   |      |       |        patient |                |                |
|   |      |       |                |                |                |
|   |      |       |        contact |                |                |
|   |      |       |                |                |                |
|   |      |       |    information |                |                |
+---+------+-------+----------------+----------------+----------------+
| 4 | SNP  | DNA   | Information    | IT system that | Electronic     |
|   | anal |       | suggested for  | supports work  | report         |
|   | ysis |       | test order     | process        |                |
|   |      |       | decision       | regarding      | Flag           |
|   |      |       |                | workflow,      | clinicians     |
|   |      |       | -              | quality        | that results   |
|   |      |       | Recommendation | control, etc.  | for genetic    |
|   |      |       | regarding      |                | tests are      |
|   |      |       | a              | Medical Device | available      |
|   |      |       | ppropriateness | Interface      |                |
|   |      |       | of test in     | to/from        | Decision       |
|   |      |       | light of       | instruments to | support:       |
|   |      |       | clinical       | LIS            |                |
|   |      |       | presentation   |                | Provide        |
|   |      |       |                | Automate       | warning when   |
|   |      |       | -Summaries     | analytical     | action (ex.    |
|   |      |       | clinical       | result         | ordering a     |
|   |      |       | utility for    | interpretation | specific drug) |
|   |      |       | current and    |                | is             |
|   |      |       | new genetic    | Integrating    | c              |
|   |      |       | test or        | LIS with       | ontraindicated |
|   |      |       | variant        | existing       | due to         |
|   |      |       |                | databases of   | patient's      |
|   |      |       | Rules engine   | known variants | genotype       |
|   |      |       | for warning    | and other      |                |
|   |      |       | message        | relevant       | Integrating    |
|   |      |       | regarding need | genetic        | EMR with       |
|   |      |       | for a specific | information    | existing       |
|   |      |       | genotyping     |                | databases of   |
|   |      |       | test ex. when  |                | known variants |
|   |      |       | prescribing    |                | and other      |
|   |      |       | certain drugs  |                | relevant       |
|   |      |       |                |                | clinical       |
|   |      |       | When ordering  |                | genetic        |
|   |      |       | the test       |                | information    |
|   |      |       |                |                |                |
|   |      |       | Rules engine   |                |                |
|   |      |       | for            |                |                |
|   |      |       | identification |                |                |
|   |      |       | of test        |                |                |
|   |      |       | already        |                |                |
|   |      |       | performed-show |                |                |
|   |      |       | results        |                |                |
|   |      |       |                |                |                |
|   |      |       | Information    |                |                |
|   |      |       | required for   |                |                |
|   |      |       | performing the |                |                |
|   |      |       | test           |                |                |
|   |      |       |                |                |                |
|   |      |       | -Family        |                |                |
|   |      |       | history        |                |                |
|   |      |       |                |                |                |
|   |      |       | -Clinical      |                |                |
|   |      |       | history        |                |                |
|   |      |       |                |                |                |
|   |      |       | -Presume Dx    |                |                |
|   |      |       |                |                |                |
|   |      |       | -Documentation |                |                |
|   |      |       | of Informed    |                |                |
|   |      |       | Consent        |                |                |
+---+------+-------+----------------+----------------+----------------+
| 5 | Chr  | -     | -Diagnosis     | -Complete      | -patients      |
|   | omos | Polyp |                | protocol       | clinical       |
|   | omal | loidy | -Tissue type   |                | history        |
|   | A    |       |                | -Reagents used |                |
|   | bnor | -     | -Method of     |                | -              |
|   | mali | Aneup | extraction     | -Reagent       | interpretation |
|   | ty\* | loidy |                | concentration  |                |
|   |      |       | -Amount        |                |                |
|   |      | -Del  | obtained       | -CLSpCG        |                |
|   |      | etion |                |                |                |
|   |      |       |                | -result        |                |
|   |      | -D    |                | reporting      |                |
|   |      | uplic |                |                |                |
|   |      | ation |                |                |                |
|   |      |       |                |                |                |
|   |      | -Tra  |                |                |                |
|   |      | nsloc |                |                |                |
|   |      | ation |                |                |                |
|   |      |       |                |                |                |
|   |      | -Inve |                |                |                |
|   |      | rsion |                |                |                |
|   |      |       |                |                |                |
|   |      | -Iso  |                |                |                |
|   |      | chrom |                |                |                |
|   |      | osome |                |                |                |
|   |      |       |                |                |                |
|   |      | -Ring |                |                |                |
|   |      | chrom |                |                |                |
|   |      | osome |                |                |                |
+---+------+-------+----------------+----------------+----------------+
| 6 | Gene | Cur   | Patient        | #              | Result         |
|   | copy | rent: | demographics   |  *Gene Dosage* |                |
|   | numb |       | and unique     |                | Method         |
|   | ers/ | N     | identification | **Multiplex    |                |
|   |      | umber |                | Sem            | Interpretation |
|   | ex   | of    | Diagno         | i-quantitative |                |
|   | pres | funct | sis/indication | fluorescent    | Clinical       |
|   | sion | ional |                | PCR**          | Correlation    |
|   | le   | genes | Family History |                |                |
|   | vels | expr  |                | **Restriction  | Family History |
|   |      | essed | Tissue type    | enzyme         |                |
|   | \*   |       |                | digestion,     | Genetic        |
|   |      | Vari  | (Blood, AF,    | probe          | Counseling     |
|   |      | ation | CVS)           | detection and  |                |
|   |      | in    |                | Southern       | Identification |
|   |      | copy  | Sample         | Blot**         | of treatment   |
|   |      | n     | handling       |                | protocols      |
|   |      | umber |                | **Gapped-PCR** |                |
|   |      | w     | Sample         |                | Clinical       |
|   |      | ithin | preparation    | **Automated    | follow up      |
|   |      | re    |                | DNA analysis   |                |
|   |      | gions | Amount/quality | (ABI3100)**    |                |
|   |      | of    | DNA            |                |                |
|   |      | the   |                | **Data         |                |
|   |      | g     | Clinic         | analysis       |                |
|   |      | enome | o-pathological | software       |                |
|   |      | assoc | correlation    | (Genemapper)** |                |
|   |      | iated |                |                |                |
|   |      | with  | Patient        | ## *Array CGH* |                |
|   |      | solid | history,       |                |                |
|   |      | t     | including the  | **Microarray   |                |
|   |      | umors | results of     | (BAC clones,   |                |
|   |      | and   | prior testing, | cDNA or        |                |
|   |      | ge    | treatment      | olig           |                |
|   |      | netic | protocols and  | onucleotides** |                |
|   |      | dis   | pre-existing   |                |                |
|   |      | eases | conditions     | **Dual color   |                |
|   |      |       |                | hybridization  |                |
|   |      |       |                | using          |                |
|   |      |       |                | fluorescent    |                |
|   |      |       |                | probes of      |                |
|   |      |       |                | different      |                |
|   |      |       |                | wavelength**   |                |
|   |      |       |                |                |                |
|   |      |       |                | **Digital      |                |
|   |      |       |                | image          |                |
|   |      |       |                | capture**      |                |
|   |      |       |                |                |                |
|   |      |       |                | **Analysis     |                |
|   |      |       |                | software to    |                |
|   |      |       |                | quantitate the |                |
|   |      |       |                | fluorescent    |                |
|   |      |       |                | signal         |                |
|   |      |       |                | intensities**  |                |
|   |      |       |                |                |                |
|   |      |       |                | *              |                |
|   |      |       |                | *Normalization |                |
|   |      |       |                | and            |                |
|   |      |       |                | statistical    |                |
|   |      |       |                | analysis       |                |
|   |      |       |                | software to    |                |
|   |      |       |                | calculate      |                |
|   |      |       |                | signal ratios  |                |
|   |      |       |                | between test   |                |
|   |      |       |                | and reference  |                |
|   |      |       |                | samples for    |                |
|   |      |       |                | all            |                |
|   |      |       |                | representative |                |
|   |      |       |                | regions of the |                |
|   |      |       |                | genome**       |                |
|   |      |       |                |                |                |
|   |      |       |                | *mRNA          |                |
|   |      |       |                | expression     |                |
|   |      |       |                | profiles for   |                |
|   |      |       |                | identification |                |
|   |      |       |                | of the origin  |                |
|   |      |       |                | of metastatic  |                |
|   |      |       |                | tumors*        |                |
|   |      |       |                |                |                |
|   |      |       |                | **O            |                |
|   |      |       |                | ligonucleotide |                |
|   |      |       |                | microarray**   |                |
|   |      |       |                |                |                |
|   |      |       |                | **Real-time    |                |
|   |      |       |                | Quantitative   |                |
|   |      |       |                | PCR**          |                |
|   |      |       |                |                |                |
|   |      |       |                | **Genetic      |                |
|   |      |       |                | al             |                |
|   |      |       |                | gorithm/Neural |                |
|   |      |       |                | Net "training" |                |
|   |      |       |                | algorithms**   |                |
|   |      |       |                |                |                |
|   |      |       |                | **Immuno       |                |
|   |      |       |                | histochemistry |                |
|   |      |       |                | staining of    |                |
|   |      |       |                | tumor tissue   |                |
|   |      |       |                | samples,       |                |
|   |      |       |                | microscopic    |                |
|   |      |       |                | interpretive   |                |
|   |      |       |                | diagnosis of   |                |
|   |      |       |                | the            |                |
|   |      |       |                | histology**    |                |
+---+------+-------+----------------+----------------+----------------+
| 7 | A    | RN    |                |                |                |
|   | lter | A/DNA |                |                |                |
|   | nate |       |                |                |                |
|   | RNA  | (see  |                |                |                |
|   | sp   | gene  |                |                |                |
|   | lici | seque |                |                |                |
|   | ng\* | ncing |                |                |                |
|   |      | exa   |                |                |                |
|   |      | mple) |                |                |                |
+---+------+-------+----------------+----------------+----------------+
| 8 | E    | Cur   | Ger            | **Methyl       | Ger            |
|   | pige | rent: | mline/Somatic: | ation-specific | mline/Somatic: |
|   | neti |       |                | PCR**          |                |
|   | cs\* | DNA   | Diagno         |                | Result         |
|   |      | m     | sis/indication | **Melting      |                |
|   |      | ethyl |                | curve          | Method         |
|   |      | ation | Tissue type    | analysis**     |                |
|   |      |       |                |                | Interpretation |
|   |      | Fu    | Sample         | **Real-time    |                |
|   |      | ture: | handling       | quantitative   | Limitations    |
|   |      |       |                | PCR**          | (doesn't       |
|   |      | Hi    | Sample prep    |                |                |
|   |      | stone |                | **PCR          | define         |
|   |      | m     | Amount/quality | single-strand  | underlying     |
|   |      | ethyl | DNA            | conformation   |                |
|   |      | ation |                | polymorphism** | abnormality)   |
|   |      |       |                |                |                |
|   |      | Hi    |                | **Methyl       | R              |
|   |      | stone |                | ation-specific | ecommendations |
|   |      | a     |                | restriction    |                |
|   |      | cetyl |                | enzymes**      | next steps     |
|   |      | ation |                |                |                |
|   |      |       |                | **Southern     |                |
|   |      | Hi    |                | blot**         |                |
|   |      | stone |                |                |                |
|   |      | ubiq  |                | **DNA          |                |
|   |      | uitin |                | microarray**   |                |
|   |      | ation |                |                |                |
|   |      |       |                | **Sanger       |                |
|   |      | Small |                | sequencing**   |                |
|   |      | RNA   |                |                |                |
|   |      | sp    |                | **Py           |                |
|   |      | ecies |                | rosequencing** |                |
|   |      |       |                |                |                |
|   |      | mic   |                | **Chromatin    |                |
|   |      | roRNA |                | immunop        |                |
|   |      |       |                | recipitation** |                |
|   |      | Anti- |                |                |                |
|   |      | sense |                |                |                |
|   |      |       |                |                |                |
|   |      | RNA   |                |                |                |
|   |      |       |                |                |                |
|   |      | Other |                |                |                |
|   |      | non-c |                |                |                |
|   |      | oding |                |                |                |
|   |      | RNAs  |                |                |                |
+---+------+-------+----------------+----------------+----------------+
| 9 | Pro  | -pre  | -State         | -Test type     | -Confirmation  |
|   | tein | sence | screening laws |                | of results     |
|   | d    | of    |                | -Complete      | with           |
|   | ysfu | ce    | -Symptoms      | protocol       | additional     |
|   | ncti | rtain |                |                | tests          |
|   | on\* | metab | -Diagnosis     | -Reagents used |                |
|   |      | olite |                |                | -              |
|   |      |       | -Tissue type   | -Reagent       | Interpretation |
|   |      | -ove  |                | concentration  |                |
|   |      | rabun | -Amount        |                | []{.mark}      |
|   |      | dance | obtained       | -Result        |                |
|   |      | of    |                | reporting      |                |
|   |      | ce    |                |                |                |
|   |      | rtain |                |                |                |
|   |      | metab |                |                |                |
|   |      | olite |                |                |                |
|   |      |       |                |                |                |
|   |      | -lack |                |                |                |
|   |      | of    |                |                |                |
|   |      | ce    |                |                |                |
|   |      | rtain |                |                |                |
|   |      | metab |                |                |                |
|   |      | olite |                |                |                |
|   |      |       |                |                |                |
|   |      | -e    |                |                |                |
|   |      | nzyme |                |                |                |
|   |      | fun   |                |                |                |
|   |      | ction |                |                |                |
|   |      |       |                |                |                |
|   |      | -misf |                |                |                |
|   |      | olded |                |                |                |
|   |      | pr    |                |                |                |
|   |      | otein |                |                |                |
+---+------+-------+----------------+----------------+----------------+

\* indicates more detailed description/use case after table

***2. Full Genome Sequencing***

Within the next one to two decades full genome sequencing will likely
become available at a cost comparable to current common radiology
diagnostics such as CT scans and MRIs. If clinical applications of full
genome sequencing become a reality, current approaches to clinical
genetic testing will almost certainly change dramatically and
unpredictably.

One could envision a number of points in an individual's lifespan where
the data could be of value. A potential approach would be to take a DNA
sample at the time of birth. Currently most states perform biochemical
testing at the time of delivery to detect inborn errors of metabolism;
one could imagine that genome sequencing might be an attractive adjunct
to the biochemical approach. Given that the genetic code of an
individual is reasonably immutable, sequencing an individual once in
infancy then storing the data *in silica* for future data mining on an
as-needed basis would seem a reasonable strategy. As a child, the data
might be used to inform drug selection or predict to risk for, and
potentially mitigate against the development of childhood disorders. At
the age of majority, the tested individual might choose to mine the data
set for information regarding risk of adult onset disorders and choose
to act upon those risks. During reproductive planning the data set could
be mined for potential variations that might affect reproductive
decisions. This approach would require a radical change in the way many
currently think about clinical laboratory testing and healthcare
delivery.

A clear barrier to any future effective clinical use of full genome
sequence data is the current lack of health information technology (HIT)
capable of securely storing, accessing, and interpreting the huge amount
of data that would be generated by such testing. Speculation on the data
needs that might drive HIT development for full genome sequence data is
challenging in that there are not yet agreed upon indications for this
type of testing.

***3. Gene Sequencing Tests***

Germline Usecase:

-   patient x communicates that his/her father died at 30, due to a
    heart problem

-   results from clinical tests support a preliminary diagnosis of
    Hypertrophic Cardiomyopathy (HCM) and is interested in genetic
    testing for the disease

-   physician calls the molecular diagnostic laboratory that performs
    HCM testing and receives test ordering guidance (sequence test HCM
    panel A \[covering 80% of known mutations\] with a reflex to another
    sequencing test HCM panel B \[which covers another 20% of known
    mutations\])

-   specimen is collected and sent with completed requisition and
    consent form

-   laboratory accessions specimen into LIS (laboratory information
    system) software, that will track workflow throughout the testing
    process, as well as, configure analytical instruments, collect raw
    data output, run QA/QC algorithms against the raw output, and
    support the data review process.

-   Integrated with the LIS software is a knowledgebase and reporting
    software. This software is used for creating both the textual and
    structured results and interpretation, which are then inserted into
    the information flow leading to the electronic medical record
    systems (typically sent via HL7).

-   Mechanisms exist within the clinical environment to either update
    the interpretation, should the clinical significance of a mutation
    identified in patient x change, or alert the physician to the change
    and need for re-interpretation or re-testing.

Somatic Usecase:

-   A pathologist diagnoses patient y with non-small cell lung cancer

-   patient y's physician would like pharmacogenomic guidance to
    determine course of therapy and orders EGFR sequencing test to be
    performed on patient y's biopsy

-   a blood sample is also taken from patient y and sent along with
    their cancer tissue and completed requisition/consent form to the
    molecular diagnostic lab for testing

-   both of patient y's specimens are accessioned into the laboratory
    information management system

-   laboratory accessions specimen into LIS (laboratory information
    system) software, that will track workflow throughout the testing
    process, as well as, configure analytical instruments, collect raw
    data output, run QA/QC algorithms against the raw output, and
    support the data review process.

-   A DNA variant is identified in patient y's tumor tissue; therefore,
    a known mutation test is performed on patient y's DNA extracted from
    their blood sample, in order to determine if the DNA variant was
    acquired or inherited.

-   Integrated with the LIS software is a knowledgebase and reporting
    software. This software is used for creating both the textual and
    structured results and interpretation, which are then inserted into
    the information flow leading to the electronic medical record
    systems (typically sent via HL7).

-   Mechanisms exist within the clinical environment to either update
    the interpretation, should the clinical significance of a mutation
    identified in patient y change, or alert the physician to the change
    and need for re-interpretation or re-testing.

Gene Sequence Test - Definition: Sequence one or more genes (or gene
regions) to test for pathogenic or causal variants. Often these tests
are preformed reflexively (e.g. perform this test; if it is negative,
perform this test...and so on). (Note, sequencing tests may also be
performed to confirm results from a DNA Microarray test.)

1.  Pre-analytic

    a.  Clinicians would benefit from test ordering guidance in the
        electronic medical record.

        i.  Validation of clinically appropriate reasons for ordering a
            test

        ii. Testing recommendations given a particular set of
            signs/symptoms, clinical/preliminary diagnosis, and/or
            family history. This would include recommendations on the
            order in which to perform the tests, for a cost effective
            approach to diagnosis.

> *Needs: A publicly available knowledgebase containing all commercially
> available genetic tests, containing an interface that can be queried
> by electronic record systems to provide robust ordering guidance for a
> clinician.*
>
> *Known activities in this area: GeneTests.org is incomplete and not
> programmatically accessible by electronic systems.*

b.  laboratory would benefit from the following information:

    i.  reference information for association to patients electronic
        medical record and ordering provider

        1.  physician's name, UPIN #, etc...

        2.  patient name, date of birth, gender etc...

        3.  if the patient is alive or dead (also relevant for
            interpretation)

        4.  hospital/provider name, medical record number

    ii. patient information to be considered in analysis and
        interpretation of results

        1.  Race/Ethnicity (standards need to be developed)

        2.  clinical/preliminary diagnosis (ICD9 codes, SNOMED codes,
            free text)

        3.  age at diagnosis

        4.  results of clinical tests/studies, noted signs/symptoms, and
            treatment key to clinical/preliminary diagnosis. (not all of
            these should be accompanied by age of the patient.

        5.  relevant social history (e.g. smoker/non-smoker)

        6.  patient family history

            a.  ethnicity of paternal side and maternal side

            b.  pedigree with affected and non-affected members, as well
                as, carriers (for recessively inherited conditions)

            c.  if members of the family are deceased, note age and
                cause of death, if known

        7.  indication if other family members have received genetic
            testing for this disease (gene) and attachment of their
            genetic test report. If performed to determine the presence
            or absence of a known mutation, that the mutation should be
            noted.

        8.  high-level indication for testing to be considered in
            interpreting the results and providing appropriate
            recommendations

            a.  diagnosis, carrier testing, pharmacogenomic guidance
                etc...

        9.  specimen information

            a.  if cancer tissue, pathology results

            b.  if the test requires two types of specimens (sample in
                question and a reference sample) as in the case with
                prenatal and cancer tissue testing, than each specimen
                needs separate identifier along with a mechanism for
                cross reference

*Needs: Increased compliance of physicians to send requested phenotypic
and family history information when ordering a genetic test. OR an
electronic test ordering system that is able to pre-populate this
information from the patient medical record.*

*Activities in this area: HL7 Clinical Genomics model is working on
modeling the genetic test order.*

iii. general specimen information

     1.  specimen

         a.  specimen type

         b.  if DNA, DNA concentration

         c.  if tissue, origin (adenocarcinoma of the lung), preparation
             method (e.g. formalin-fixed, paraffin embedded), and note
             if primary tumor or metastases etc...

iv. Billing information

v.  Physician and patient contact information

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2.  Analytic (precondition: start with a system that captures
    pre-analytical data from section 1.b above)

    a.  Lab Information System, ensuring data flow integrity through
        testing process (i.e. the right results, delivered for the right
        patient, in the right format)

        i.  extraction of DNA from the specimen

        ii. sample generation (to prepare material for sequencing). For
            example, in our lab, a complete Hypertrophic Cardiomyopathy
            sequencing test, across 8 genes, requires the DNA to be
            added to \~ 200 PCR reactions to amplify the \~200 regions
            of interest across the 8 genes. These regions of
            amplification are called amplicons. Each of these regions of
            interest will be sequenced in both the forward and reverse
            directions, resulting in \~ 400 sequencing reactions with a
            sequence trace file generated for each. Trace files not
            meeting quality standards will need to be re-run.
            Additionally, it is standard practice to re-run or confirm
            identified mutations; therefore, sequencing is an iterative
            process taking place over a period of days, weeks, or a
            month, depending on the complexity of the test. Instrument
            configuration, trace file management an organized approach
            to QA/QC and mutation analysis (i.e. review of the traces)
            are of high importance. Software to manage this process also
            needs to support the iterative workflow natural to this
            testing process.

            1.  On-going monitoring of primer quality.

                a.  primers should be analyzed on a periodic basis for
                    the presents of SNP's (i.e. compared against dbSNP).
                    If a primer is found to be in the region of a SNP,
                    it should be redesigned to prevent allelic dropout
                    (i.e. the failure of one or more of the patient
                    alleles to be amplified which would result in a
                    false test result)

        iii. Instrument Integration

             1.  Configuration of instrument --

             2.  Automatic data upload to case/sample/aliquot

*Needs: 1) Laboratory Information Systems that contain a mechanism to
catalog primers used in clinical testing and on a periodic basics checks
for the presence of newly identified SNP's 2) Formal published
instrument API's*

iv. Raw Data Review

    1.  QA/QC

        a.  QA/QC of batch (samples prepared and run together on the
            instrument)

        b.  QA/QC of individual trace files, identification of those
            needing to be re-run

    2.  Raw data review

        a.  Support for manual review process, using third party
            mutation analysis software

            i.  note quality of trace

            ii. record DNA variants identified, in structured form (to
                be sent on for interpretation and reporting)

        b.  Support for automated sequence analysis

    3.  Support for repeated testing of the same gene region, either to
        confirm the presence of a DNA variant or to rerun a low quality
        trace

    4.  For tests preformed on tumor specimens, if DNA variants are
        identified, the reference specimen needs to be examined for the
        presents or absence of the DNA variant (i.e. determination if it
        was acquired or inherited), as this will affect the
        interpretation.

*Needs: 1) Robust automated (or semi-automated) sequence analysis
software for mutation identification and QA/QC of raw data.*

*2) Structured knowledgebase containing all known DNA variant for a gene
and their clinical significance. If particular variants, need to be
present in combination (or homozygous form) to be causal for a
condition, this would also be represented. This knowledgebase can be
leveraged by medical record systems to monitor the accuracy of
interpretations of a patient's genetic tests over time, as well as,
potentially provide interpretive services in the medical record.*

3.  Post-Analytic (precondition: start with a system that captures
    pre-analytic and analytic data from sections 1&2 above)

    a.  Reporting and Interpretation

        i.  Software to support the simultaneous creation of both the
            textual report and structured results, as well as, having an
            interface for messaging of these results to receiving record
            systems (according to healthcare guidelines)

            1.  non-patient specific data to be maintained in a
                knowledgebase

                a.  test dictionary

                    i.  unique identifier (either a national standard id
                        or a lab id that can be mapped to a national
                        standard, so that the same test performed by
                        different laboratories can e treated in a
                        similar manner within the EHR)

                    ii. test specific materials - primers, reference
                        sequence, region of interest -- captured for
                        each region of amplification

                    iii. test specific literature references

                    iv. textual description of materials and methods
                        (for report)

                b.  DNA variants known for the particular gene tested

                    i.  DNA variant name, gene and location on the gene

                    ii. other names, for the variant, commonly used in
                        the literature (e.g. amino acid change)

                    iii. clinical significance

                    iv. frequency reported by the lab and linkage to
                        reports for similar test results

                    v.  literature references for the variant (e.g.
                        publication on pathogenicity of the variant)

*Needs: 1) Method for uniquely identifying a genetic test (e.g. EGFR
Sequencing Test)*

*2) Ideally, a publicly available knowledgebase to record primers and
precise region examined for each commercially available test. This
information can be used to monitor the reliability of the test result
overtime, as well as, add in follow-up on negative results.*

*3) public repository of reference sequences for clinical genetic
testing use (similar to RefSeq)*

*Known Activities: 1) LOINC is currently working to expand their
guidelines to support coding for the modern, commercially available,
genetic tests. 2) Perhaps GeneTests.org integrated with NCBI's probe
database could fill this gap 3)NCBI is in the process for creating a
repository of reference sequences for clinical use*

2.  patient specific data

    a.  data from pre-analytical phase for linkage to hospital provider
        electronic systems, ordering physician, and patient

    b.  testing context

        i.  high-level indication (from section 1.b.ii.8.a above;
            diagnostic, carrier testing, pharmacogenomic guidance etc...
            )

        ii. specific indication (clinical/preliminary diagnosis)

    c.  DNA variants identified

        i.  zygosity

        ii. tissue origin (i.e. somatic/germline/prenatal)

    d.  overall interpretation

        i.  textual for the report

        ii. coded vocabulary for the structured results

3.  reporting process

    a.  patient test order (e.g. test(s) performed, indication for
        testing...) and result information (e.g. DNA variants
        identified, tissue source and zygosity) are queried against the
        knowledgebase and a default report is generated. If the DNA
        variant is not found in the knowledgebase, the geneticist adds
        this variant and its clinical significance (to the
        knowledgebase) and continues reporting. The draft report is
        reviewed, altered as needed and signed-out by a geneticist. The
        report and structured results then flow from the laboratory
        system into the electronic medical record. If not reporting into
        a medical record system, results are faxed or mailed to the
        physician.

*Needs: 1) Standard for reporting structured genetic test results (and
textual report) to an electronic medical record 2) Mechanism for
ensuring the accuracy/reliability of the genetic test
results/interpretations, within the medical record, over time*

*Known Activities: 1) HL7 Clinical Genomics Special Interest Group is
working on this model. The model to support sequencing based test
results in draft standard status and has been successfully piloted by
Partners Healthcare; it is scheduled to go to normative standard in the
next 6-12 months. 2) Partners Healthcare has developed a knowledgebase
that can be leveraged in the clinical environment within a limited
setting and is interested in working with others to see how this could
be integrated with (or share/import data from) other knowledgebases to
scale for broad support of genetic testing.*

4.  General laboratory management software for genetic testing
    laboratories

    a.  reagents/instruments

        i.  reagent inventory, re-ordering details, and quality testing
            according to guidelines

        ii. instrument inventory, maintenance history and quality
            testing according to guidelines

    b.  primers

        i.  primers should be analyzed on a periodic basis for the
            presents of SNP's (i.e. compared against dbSNP). If a primer
            is found to be in the region of a SNP, it should be
            redesigned to prevent allelic dropout (i.e. the failure of
            one or more of the patient alleles to be amplified which
            would result in a false test result)

    c.  personnel

        i.  training etc..

    d.  inspection records

    e.  .....

*Needs: Development of genetics laboratory management systems.*

***5. Chromosomal Abnormalities***

Disease's caused by chromosomal abnormalities such as autosomal and
sex-chromosome aneuploids, duplications, deletions, translocations, and
inversions can be diagnosed using a karyotype of the affected
individual's cells.

*Karyotype*

\[Pre-Analytic\] Cells can be obtained from any tissue of an affected
individual if mutation is germline. In the case of some cancers, tissue
can be taken directly from the tumor. Chromosome tests are most commonly
performed on cells from fetuses to detect and prepare for disease *in
utero.* Fetal tissue is extracted using amniocentesis, chrionic villi
sampling, or fetal-maternal cell sorting. Tissue is sent to CLIA
certified diagnostic lab.

\[Analytic\] Cells are arrested during mitosis then placed in a
hypotonic salt solution which lyses the cell and separates the
chromosomes. The chromosomes are fixed, rinsed, and stained for AT-rich
or GC-rich stretches of DNA, or for heterochromatin then prepared for
microscopy. Alternatively, fluorescent *in situ* hybridization (FISH) is
used to probe a specific region of the chromosome. The sample DNA and
the fluorescently labeled probe are hybridized using
co-denature/re-anneal process. Using a microscope (fluorescent in the
case of FISH), a clinical laboratory specialist in cytogenetics
(CLSp(CG)) analyzes and summarizes the results.

\[Post-Analytical\] A board-certified medical geneticist and a
pathologist review the summary then write an interpretation taking into
account the patient's clinical history. The results are reported using
*International System for Human Cytogenetic Nomenclature 2005.*

***6. Gene copy number/expression level***

*Example 1 Determination of alpha globin "dosage" (number of genes
expressed)*

Several diseases are caused by gene deletions within the germline. For
example, over 95% of alpha thalassemia alleles are caused by large gene
deletions. Normal individuals express 4 copies of alpha globin genes;
two highly homologous genes, HBA1 and HBA2 are present in a cluster on
chromosome 16p13.3. The clinical phenotype of the alpha thalassemia
carrier depends on the number of functioning genes. Carriers of 3 normal
genes are usually asymptomatic, while carriers of 2 functioning genes
have mild anemia. Individuals with only one functioning gene present
with hemoglobin H disease and severe anemia. Inheritance of no normal
alpha globin gene in most instances leads to Hb Barts hydrops fetalis
and fetal death.

# *Example 2 Array CGH for the detection of deletions and/or duplications within the genome*

Genome alterations resulting in changes in copy number are found in
somatic cells in solid tumors and also in germline cells in association
with developmental abnormalities and/or mental retardation. Microarray
based comparative genomic hybridization employs an array of BAC clones,
cDNA or oligonucleotides with representation from the entire genome.
Differentially labeled test and reference DNA are hybridized to the
clones and the ratios of the fluorescent intensities of the labels is
calculated in order to derive a quantitative measure of the variation in
copy number between the test and reference DNA samples.

Within solid tumors, there are reports of correlation between histology,
degree of differentiation and copy number profiles for tumors such as
liposarcoma and adenocarcinoma of the colon. There are also reports of
correlations between copy number changes and alterations in gene
expression in breast cancers and head and neck tumors, leading to a
"targeted" approach to identify possible oncogenes by whole genome array
CGH.

Within germline cells, chromosomal abnormalities with variation in copy
number are associated with various dysmorphologies and/or mental
retardation. Whole genome array CGH may be utilized as a screening
procedure to target and quantify regions in a test sample and
verification of specific regional abnormalities may be obtained by
fluorescent in-situ hybridization (FISH). A specific example may be seen
in diGeorge syndrome in which a deletion on chromosome 22q11.2 is
associated with cardiac abnormalities, dysmorpic facies and other
abnormalities (Albertson and Pinkel Human Molecular Genetics 2003 12
145-152)

*Example 3 mRNA expression profile for prediction of the origin of a
metastatic tumor in "carcinoma of unknown primary" (CUP)*

Identification of the likely site of origin of a metastatic tumor of
unknown primary is approximately 25% accurate using immunohistochemistry
of specific tissue markers. Oligonucleotide microarrays containing, for
example 22000 genes, are used in dual color mRNA hybridization assays in
which reference and test RNA are differentially labeled and hybridized
with the microarray. The fluorescent intensity ratios are then used to
calculate the expression profiles of the gene products. One CUP assay
used a genetic algorithm analysis of the microarray data to search for
gene combinations optimal for multitumor classification. From the
analysis, 92 genes were chosen which were then developed into a real
time quantitative PCR assay (Ma et al Arch Pathol Lab Med 130 April 2006
465-473). The assay model is then trained using known tumor types to
identify expression profiles which may be used to predict the likely
site of origin of metastatic tumors. The model demonstrated an overall
82% accuracy in cross-validation studies, though some tumor types (for
example adenocarcinoma of the breast) demonstrated a more accurate
prediction than others (for example adenocarcinoma of the stomach).

***7. mRNA Splicing Defect Disease***

mRNA splicing is a common occurrence in mRNA processing and is
responsible for multiple protein variations arising from a single gene.
Mutations in the DNA can cause introns to be included in the mature
transcript or essential exons skipped over during processing. These
types of situations can render a protein functionless thus causing
certain diseases like spinal muscular atrophy (SMA) and lactic acidosis.
It has been reported that 15% of point mutants that result in human
genetic disease disrupted splicing (Krawczak et al. 1992).

\[Currently\] Clinically, genetic diagnosis tests for these alternative
splice diseases rely on DNA sequencing of gene. *Refer to DNA sequencing
for details.*

\[Future\] However, additional methods used in research for detecting
mRNA splice disorders can potentially be used in the clinical setting.
These methods have yet to be used for disease diagnostics in the
clinical setting, but the potential for clinical use is present.

> -mRNA microarray could be used to detect alternatively spliced mRNA
> deviants that include introns or exclude essential exons.
>
> -Extracting aberrant mature mRNA and using reverse transcriptase PCR
> to convert mature mRNA sequence back into cDNA for comparison with
> cDNA of an mRNA with the normal splice pattern.

***8. Epigenetics***

Epigenetics is defined as "the altering of gene expression by any means
that is not related to the sequence of bases along the DNA molecule."
\[from Encyclopedia of Genetics, Genomics, Proteomics and
Bioinformatics, 2005 p. 3676\] Alterations in epigenetic modification
are known to cause disease, including multiple congenital anomaly
syndromes (e.g. Angelman and Prader-Willi syndromes), cancer (e.g. Wilms
tumors, paragangliomas, and a subset of colorectal cancers) and are
responsible, in part, for differences in prevalence of common disease in
monozygotic twins. Epigenetic changes can be inherited (i.e. germ-line)
or acquired (i.e. somatic).

Critical components of the epigenetic modification machinery include
histone acetyltransferases and deacetylases, histone
methyl-transferases, methyl cytosine binding proteins, DNA
methyl-transferases and small RNA species including microRNAs,
anti-sense RNAs and other non-coding RNAs.

Epigenetic modification can involve nearly whole chromosomes
(X-inactivation), chromosomal regions (15q11.2-15q13), individual genes
(*GNAS*) and single base pairs. Alteration in DNA methylation is the
only known human disease-causing mechanism at the present time, although
there is, for instance, evidence in the mouse that histone methylation
is important in regulation of *Igf2* and that non-coding RNAs may play a
role in regulation of *G~S~α* (*GNAS* gene), so other types of
epigenetic alterations could influence the phenotypes of disorders
associated with these loci. Altered histone acetylation may play a role
in Oculo-Auriculo-Vertebral (OAV) spectrum. (Fischer et al. (2006)
Histone acetylation dependent allelic expression imbalance of *BAPX1* in
patients with the oculo-auriculo-vertebral spectrum. Hum Mol Genet.
15:581-587).

*Clinical Examples:*

Germline: A 5 yo female is identified to have mental retardation, lack
of expressive language and seizures. **\[Preanalytic**\] Angelman
syndrome is suspected clinically (Diagnosis/indication). Blood (tissue
type) is drawn and sent to the laboratory in compliance with the
laboratory requirements for sample handling. The laboratory extracts the
DNA and prepares the DNA using the bisulfite conversion method (sample
preparation). The converted sample meets the laboratory's standard for
sample adequacy. **\[Analytic\]** The sample is analyzed by
Methylation-specific PCR with appropriate controls.
**\[Post-analytic\]** The result shows a methylation pattern consistent
with absence of the maternal imprint of chromosome 15q11.2-q13 (result)
consistent with the clinical diagnosis of Angelman syndrome
(interpretation). The method used to determine this result is given
(method) and the ordering physician is told that this method is unable
to determine whether the abnormal methylation is due to a chromosomal
deletion, uniparental disomy, or a mutation in the imprinting center
(limitations). Additional tests to determine the etiology are provided
(recommendations). Alternatively, the test shows a normal methylation
pattern (result). The clinician is told that that this does not rule out
Angelman syndrome, as mutations in the *UBE3A* gene account for 11% of
cases (limitation) and methylation analysis (method) will not detect
these cases. *UBE3A* gene sequencing is recommended if clinically
appropriate (recommendation).

Somatic: A 45 yo male has a resection of a colorectal cancer (CRC).
Because of the early age of presentation, microsatellite instability
(MSI) analysis is performed on the tumor and is positive.
**\[Pre-analytic\]** This finding can be attributed to one of two
mechanisms: a somatic or germline mutation in one of the mismatch repair
(MMR) genes, or somatic hypermethylation of the *hMLH1* promoter
(diagnosis/indication). A tumor specimen is sent to the laboratory in
compliance with the laboratory requirements for sample handling. The
laboratory extracts the DNA and prepares the DNA using the bisulfite
conversion method (sample preparation). The converted sample meets the
laboratory's standard for sample adequacy. **\[Analytic\]** The sample
is analyzed using the PCR single-strand conformation polymorphism
technique with appropriate controls. **\[Post-analytic\]** The test
shows hypermethylation of the *hMLH1* promoter (result). The ordering
physician is informed that this result is consistent with a sporadic
CRC, as hypermethylation of the *hMLH1* promoter is rarely seen in
association with a mutation in a MMR gene (interpretation) although this
study would not detect such a mutation (limitation). No further testing
is recommended, unless there are clinical indications that warrant
(recommendation). Alternatively, the test does not show hypermethylation
of the *hMLH1* promoter (result). The observation of MSI in the tumor
specimen could be due to a germline or somatic mutation in one of the
MMR genes (interpretation). This test will not detect such mutations
(limitation). Suggest immunohistochemical staining (IHC), testing for
the V600E mutation in *BRAF*, or sequencing of the MMR genes if
indicated (recommendation).

***9. Protein dysfunction***

Protein dysfunction can be illustrated by a broad array of tests
depending on the protein of interest. The tests mainly detect an
overabundance of a certain metabolite (i.e. phenylalanine) caused by
genetically induced dysfunctional or sub-functional protein (i.e.
phenylalanine hydrolase). Tandem mass spectrometry allows for the
detection of a large number of congenital metabolic diseases that
affects blood levels of organic acids. Enzyme assay tests can detect a
genetic disorder by determining the level of functionality of a specific
enzyme of interest (i.e. hexosaminidase activity in Tay Sachs).

There is a broad array of diseases caused by protein dysfunction that
can be tested. They include: endocrine disorders (hyperthyroidism),
blood cell disorders (sickle cell anemia), amino acid metabolism (PKU),
organic acid metabolism (biotinidase metabolism), fatty acid metabolism
(Tay-Sachs), carbohydrate metabolism (galactosemia), congenital
infections (HIV), and others (cystic fibrosis).

*A newborn screening example- PKU*

\[Pre-Analytic\] State regulations require a drop blood to be taken from
newborn between 2-3 days old, placed on filter paper and sent to a CLIA
certified testing laboratory.

\[Analytic\] The test involves placing the blood sample on an agar plate
with active cultures of bacteria and B-2-thienylalanine (bacterial
growth inhibitor). Bacterial growth is inhibited on the agar plate
except in the location of excess phenylalanine leached from blood
samples of elevated phenylalanine levels characteristic of PKU. Colony
growth is proportional to phenylalanine levels in blood.

\[Post-Analytic\] Newborn screening test results must be confirmed be
additional tests before a specific diagnosis can be made, however
specific responses to positive results vary between states. Results are
reported to physician, PKU positive results prompt the doctor to put
newborn on strict diet low in phenylalanine.