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  <head>
    <title>092-03 Cytogenetics, sequencing, genomic methods, variant interpretation, and clinical limits</title>
    <ownerName>Integrated Medical Foundations</ownerName>
  </head>
  <body>
    <outline text="Genomic methods, variant interpretation, and limits">
      <outline text="Choosing a test">
        <outline text="Genomic testing is not one technology"/>
        <outline text="Each method has blind spots"/>
        <outline text="Select by phenotype and suspected mechanism"/>
        <outline text="Integrate quality, frequency, function, context"/>
      </outline>
      <outline text="Cytogenetic methods">
        <outline text="Karyotype: aneuploidy and large rearrangements">
          <outline text="Detects balanced translocations and inversions"/>
          <outline text="Misses small copy and nucleotide changes"/>
          <outline text="Culture can select against cell populations"/>
        </outline>
        <outline text="FISH: labelled probes on selected regions">
          <outline text="Break-apart shows disruption, fusion juxtaposition"/>
          <outline text="Normal result does not exclude other abnormalities"/>
        </outline>
        <outline text="Microarray measures genome-wide copy number">
          <outline text="Submicroscopic deletions and duplications"/>
          <outline text="Long regions of homozygosity"/>
          <outline text="Misses balanced changes, repeats, small variants"/>
        </outline>
      </outline>
      <outline text="Arrays and PCR">
        <outline text="SNP arrays assay predefined markers">
          <outline text="Imputation depends on LD and ancestry"/>
          <outline text="Rare and structural variants imputed poorly"/>
        </outline>
        <outline text="PCR amplifies a selected region">
          <outline text="Quantitative, reverse-transcription, digital"/>
          <outline text="Allele dropout, contamination, primer-site variants"/>
        </outline>
      </outline>
      <outline text="Sequencing technologies">
        <outline text="Sanger: accurate reading of a targeted fragment">
          <outline text="Confirms variants and familial sites"/>
          <outline text="Misses copy number and low-level mosaicism"/>
        </outline>
        <outline text="Short-read sequencing reads millions of molecules">
          <outline text="Panel, exome, genome breadth"/>
          <outline text="Align, call, filter, annotate, review"/>
        </outline>
        <outline text="Coverage is uneven">
          <outline text="Repeats, pseudogenes, high GC are difficult"/>
          <outline text="Mean depth does not prove every base"/>
          <outline text="Orthogonal confirmation for hard findings"/>
        </outline>
        <outline text="Short reads struggle with phasing and long repeats"/>
        <outline text="Long reads span structure, repeats, haplotypes"/>
        <outline text="No platform is comprehensive alone"/>
      </outline>
      <outline text="RNA and methylation">
        <outline text="RNA sequencing: expression, splicing, fusion">
          <outline text="Depends on tissue expression and stability"/>
          <outline text="Nonsense-mediated decay may erase transcript"/>
          <outline text="Complements, not replaces, DNA analysis"/>
        </outline>
        <outline text="Methylation: imprinting, silencing, episignatures">
          <outline text="Bisulfite is indirect and damages DNA"/>
          <outline text="Varies by tissue, age, cell composition"/>
        </outline>
      </outline>
      <outline text="Nomenclature and classification">
        <outline text="Specify reference, coordinate, alleles, protein">
          <outline text="Transcripts change exon numbers and effects"/>
        </outline>
        <outline text="Five germline classification categories">
          <outline text="Evidence of association, not personal certainty"/>
        </outline>
        <outline text="Loss of function needs an established mechanism">
          <outline text="Late stop, exon skipping, in-frame splice"/>
          <outline text="Gene may tolerate haploinsufficiency"/>
        </outline>
        <outline text="Missense predictions are supporting evidence"/>
        <outline text="Segregation strengthens evidence">
          <outline text="Unaffected carrier weakens, not refutes"/>
        </outline>
        <outline text="De novo strong when parentage confirmed"/>
      </outline>
      <outline text="Uncertain and somatic results">
        <outline text="Uncertain variants should not drive irreversible acts">
          <outline text="Reclassification as evidence grows"/>
          <outline text="Patients need a plan for review"/>
        </outline>
        <outline text="Negative result does not exclude genetic disease"/>
        <outline text="Tumour allele fraction reflects purity, clonality"/>
        <outline text="Tumour variant may reveal germline predisposition"/>
        <outline text="Clonal haematopoiesis mimics findings"/>
      </outline>
      <outline text="Reproductive and population testing">
        <outline text="Cell-free DNA is screening, not diagnosis">
          <outline text="Placental mosaicism, vanished twin, low fraction"/>
          <outline text="Predictive value depends on prior probability"/>
        </outline>
        <outline text="Newborn screening targets treatable conditions">
          <outline text="Screen-positive result needs confirmation"/>
        </outline>
        <outline text="Preimplantation testing of trophectoderm cells">
          <outline text="Not an absolute guarantee"/>
        </outline>
      </outline>
      <outline text="Validity, equity, iteration">
        <outline text="Secondary findings affect relatives"/>
        <outline text="Analytical, clinical validity and utility distinct"/>
        <outline text="Underrepresented groups get more uncertainty"/>
        <outline text="Avoid race as a shortcut for genotype"/>
        <outline text="Negative result reflects current knowledge"/>
      </outline>
    </outline>
  </body>
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