15.3 Clinical Calculations, ACMG Variant Classification & HGVS

Key Takeaways

Spectrophotometric quantification utilizes specific absorption constants ($1.0\ OD_{260} = 50\ \mu\text{g/mL}$ dsDNA, $40\ \mu\text{g/mL}$ RNA, $33\ \mu\text{g/mL}$ ssDNA), with pure dsDNA exhibiting an $A_{260}/A_{280}$ ratio of 1.7–1.9 and $A_{260}/A_{230}$ ratio of 2.0–2.2.
Diagnostic performance metrics evaluate clinical utility: Clinical Sensitivity ($TP / [TP + FN]$), Clinical Specificity ($TN / [TN + FP]$), and Positive Predictive Value ($TP / [TP + FP]$), where PPV increases with higher disease prevalence.
The ACMG/AMP 5-tier classification system categorizes sequence variants (Pathogenic, Likely Pathogenic, VUS, Likely Benign, Benign) based on weighted evidence criteria (PVS1, PS1–4, PM1–6, PP1–5, BA1, BS1–4, BP1–7).
PVS1 (Very Strong Pathogenicity) applies strictly to null variants (nonsense, frameshift, canonical $\pm 1, 2$ splice sites, initiation codon loss, whole-exon deletion) in genes where loss-of-function is an established disease mechanism.
HGVS nomenclature strictly designates coding DNA variants relative to `c.1` (the A of the ATG translation initiation codon, with no nucleotide zero), using `+` or `-` for intronic coordinates and standardized protein three-letter notation (`Ter` or `*` for stop codons).

Last updated: August 2026

15.3 Clinical Calculations, ACMG Variant Classification & HGVS

Quick Summary: Clinical molecular laboratory practice requires mastery of quantitative laboratory calculations, diagnostic assay validation metrics, standardized ACMG/AMP sequence variant classification, and HGVS nomenclature. Technologists must calculate nucleic acid yields from UV spectrophotometry ($1.0\ OD_{260} = 50\ \mu\text{g/mL}$ for dsDNA, $40\ \mu\text{g/mL}$ for RNA), interpret purity ratios ($A_{260}/A_{280} \approx 1.8$; $A_{260}/A_{230} \ge 2.0$), compute copy numbers from mass and fragment size, and determine clinical sensitivity, specificity, and predictive values. In clinical variant reporting, variants are categorized into a 5-tier system (Pathogenic, Likely Pathogenic, VUS, Likely Benign, Benign) using rule-based criteria, notably PVS1 for loss-of-function null mutations, and described using standardized HGVS coding (c.) and protein (p.) notation anchored at c.1 of the ATG start codon.

1. Molecular Laboratory Calculations & Spectrophotometric Quantification

UV Spectrophotometric Quantification of Nucleic Acids

Nucleic acid purines and pyrimidines exhibit resonance peak absorbance at $\lambda = 260\text{ nm}$, while aromatic amino acids in proteins (tryptophan, tyrosine, phenylalanine) absorb maximally at $\lambda = 280\text{ nm}$. Organic contaminants, chaotropic salts, and carbohydrates absorb at $\lambda = 230\text{ nm}$.

+----------------------------------------------------------------------------------------------------+
|                         SPECTROPHOTOMETRIC ABSORBANCE CONVERSION FACTORS                           |
+-------------------+-------------------+-------------------+----------------------------------------+
| Nucleic Acid Type | Standard Optical  | Formula for Concentration ($C$)                            |
|                   | Density ($1.0\ OD_{260}$) | (where $DF = \text{Dilution Factor}$)                 |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Double-Stranded | **$50\ \mu\text{g/mL}$**| $C\ (\mu\text{g/mL}) = A_{260} \times 50\ \mu\text{g/mL} \times DF$   |
| DNA (dsDNA)**     | ($50\ \text{ng/}\mu\text{L}$)| $C\ (\text{ng/}\mu\text{L}) = A_{260} \times 50 \times DF$            |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Single-Stranded | **$40\ \mu\text{g/mL}$**| $C\ (\mu\text{g/mL}) = A_{260} \times 40\ \mu\text{g/mL} \times DF$   |
| RNA (ssRNA)**     | ($40\ \text{ng/}\mu\text{L}$)| $C\ (\text{ng/}\mu\text{L}) = A_{260} \times 40 \times DF$            |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Single-Stranded | **$33\ \mu\text{g/mL}$**| $C\ (\mu\text{g/mL}) = A_{260} \times 33\ \mu\text{g/mL} \times DF$   |
| Oligos (ssDNA)**  | ($33\ \text{ng/}\mu\text{L}$)| $C\ (\text{ng/}\mu\text{L}) = A_{260} \times 33 \times DF$            |
+-------------------+-------------------+-------------------+----------------------------------------+

$\text{Total Yield (}\mu\text{g)} = \text{Concentration (}\mu\text{g/mL)} \times \text{Total Elution Volume (mL)}$

Spectrophotometric Purity Ratios & Troubleshooting

$A_{260}/A_{280}$ Ratio (Protein & Phenol Assessment):
- Pure dsDNA: Ideal ratio is $1.8$ (acceptable range: $1.7 - 1.9$).
- Pure ssRNA: Ideal ratio is $2.0$ (acceptable range: $1.9 - 2.1$).
- Low Ratio ($<1.6$): Indicates residual protein contamination, phenol carryover, or cellular debris.
- High Ratio ($>2.0$ for DNA): Indicates RNA contamination in a DNA extraction.
$A_{260}/A_{230}$ Ratio (Salt & Organic Solvent Assessment):
- Pure Nucleic Acids: Expected ratio is $2.0 - 2.2$.
- Low Ratio ($<1.5$): Indicates carryover of chaotropic salts (guanidinium thiocyanate / guanidine HCl), EDTA, carbohydrates, glycogen carrier, or phenol/chloroform.

Copy Number Calculations from Mass and Length

To determine the absolute number of DNA molecules (copy number) in a given mass of double-stranded DNA (e.g., standard curve for RT-qPCR or viral load assay):

$\text{Number of Copies} = \frac{\text{Mass of dsDNA (ng)} \times 6.022 \times 10^{23}\text{ molecules/mol}}{\text{Length of DNA (bp)} \times 1 \times 10^9\text{ ng/g} \times 660\text{ g/(mol}\cdot\text{bp)}}$

Constants to Remember:

Average molecular mass of 1 base pair of dsDNA $\approx 660\text{ Da (g/mol)}$.

Average molecular mass of 1 nucleotide of ssDNA $\approx 330\text{ Da (g/mol)}$.

Average molecular mass of 1 nucleotide of ssRNA $\approx 340\text{ Da (g/mol)}$.

Avogadro's constant $= 6.022 \times 10^{23}\text{ molecules/mol}$.

Laboratory Dilutions & Reagent Preparation ($C_1 V_1 = C_2 V_2$)

Working Stock Formula: $C_1 V_1 = C_2 V_2$, where $C_1$ is initial concentration, $V_1$ is initial volume, $C_2$ is final desired concentration, and $V_2$ is total final volume.
Serial Dilutions & Dilution Factor ($DF$): $DF = \frac{\text{Total Volume}}{\text{Aliquot Volume}} = \frac{\text{Sample Volume} + \text{Diluent Volume}}{\text{Sample Volume}}$ A $1:10$ dilution equals $1\text{ part sample} + 9\text{ parts diluent}$ (total 10 parts, $DF = 10$).

Population Genetics: Hardy-Weinberg Equilibrium

For a gene with two alleles ($p = \text{dominant allele frequency}$, $q = \text{recessive allele frequency}$):

$p + q = 1$ $p^2 + 2pq + q^2 = 1$

$p^2 =$ frequency of homozygous dominant individuals.
$2pq =$ frequency of heterozygous carriers.
$q^2 =$ frequency of homozygous recessive affected individuals (disease incidence).

Clinical Carrier Frequency Calculation: For Cystic Fibrosis (autosomal recessive) with a disease incidence of $1\text{ in }2,500$ live births: $q^2 = \frac{1}{2500} = 0.0004 \implies q = \sqrt{0.0004} = 0.02$ $p = 1 - q = 1 - 0.02 = 0.98$ $\text{Carrier Frequency } (2pq) = 2 \times (0.98) \times (0.02) \approx 0.0392 = \mathbf{1\text{ in }25.5}$

2. Clinical Diagnostic Performance Metrics

Validation of a molecular assay (under CLIA/CAP guidelines) requires establishing analytical and clinical performance characteristics using a $2 \times 2$ contingency matrix.

                           THE 2x2 CLINICAL CONTINGENCY TABLE
                           
                                          [ True Disease State (Gold Standard) ]
                                          +-------------------+-------------------+
                                          | Disease Positive  | Disease Negative  |
    +-----------------+-------------------+-------------------+-------------------+
    | Molecular Test  | Positive Result   | True Positive (TP)| False Positive(FP)| -> PPV = TP/(TP+FP)
    | Result          | Negative Result   | False Negative(FN)| True Negative (TN)| -> NPV = TN/(TN+FN)
    +-----------------+-------------------+-------------------+-------------------+
                                            |                   |
                                            v                   v
                                      Sensitivity =       Specificity =
                                      TP / (TP + FN)      TN / (TN + FP)

+----------------------------------------------------------------------------------------------------+
|                         DIAGNOSTIC PERFORMANCE METRICS FORMULAS                                    |
+-------------------+-------------------------------+------------------------------------------------+
| Metric            | Mathematical Formula          | Clinical Definition & Utility                  |
+-------------------+-------------------------------+------------------------------------------------+
| **Clinical        | $\text{Sensitivity} =          | Ability of the assay to correctly identify     |
| Sensitivity**     | \frac{TP}{TP + FN} \times 100$| individuals **with the disease** (Low FN rate) |
+-------------------+-------------------------------+------------------------------------------------+
| **Clinical        | $\text{Specificity} =          | Ability of the assay to correctly identify     |
| Specificity**     | \frac{TN}{TN + FP} \times 100$| individuals **without the disease** (Low FP)   |
+-------------------+-------------------------------+------------------------------------------------+
| **Positive        | $\text{PPV} =                  | Probability that a patient with a **positive   |
| Predictive Value**| \frac{TP}{TP + FP} \times 100$| test result** truly has the disease            |
+-------------------+-------------------------------+------------------------------------------------+
| **Negative        | $\text{NPV} =                  | Probability that a patient with a **negative   |
| Predictive Value**| \frac{TN}{TN + FN} \times 100$| test result** is truly free of the disease     |
+-------------------+-------------------------------+------------------------------------------------+
| **Diagnostic      | $\text{Accuracy} =             | Proportion of all test results (both positive  |
| Accuracy**        | \frac{TP + TN}{TP+TN+FP+FN}$   | and negative) that are correct                 |
+-------------------+-------------------------------+------------------------------------------------+

Impact of Disease Prevalence on PPV and NPV

Positive Predictive Value (PPV) is highly dependent on disease prevalence in the tested population. As disease prevalence decreases, PPV decreases drastically (more false positives relative to true positives), while NPV increases toward $100%$.
Analytical Sensitivity (Limit of Detection / LoD): The lowest concentration of target analyte that can be consistently detected with $\ge 95%$ certainty.
Analytical Specificity: The ability of the assay to detect only the target sequence without cross-reacting with homologous non-target sequences or interfering substances.

3. ACMG/AMP Sequence Variant Interpretation Guidelines

The American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology (AMP) established a standardized 5-tier classification system (Richards et al., 2015) for interpreting germline sequence variants:

                            THE ACMG 5-TIER CLASSIFICATION SYSTEM
                            
           [ Pathogenic (P) ]  -------- (Certainty > 99% Disease-Causing)
                   |
        [ Likely Pathogenic (LP) ] ---- (>90% Certainty Disease-Causing)
                   |
      [ Variant of Uncertain ] -------- (Conflicting / Insufficient Evidence)
      [   Significance (VUS) ]
                   |
         [ Likely Benign (LB) ] ------- (>90% Certainty Non-Pathogenic)
                   |
            [ Benign (B) ] ------------ (Certainty > 99% Non-Pathogenic)

+----------------------------------------------------------------------------------------------------+
|                         ACMG / AMP EVIDENCE CODE HIERARCHY                                         |
+-------+-------------------+------------------------------------------------------------------------+
| Level | Evidence Codes    | Criteria Definitions & Clinical Application Examples                   |
+-------+-------------------+------------------------------------------------------------------------+
| **Very| **PVS1**          | **Null Variant**: Nonsense, frameshift, canonical $\pm 1$ or $2$      |
| Strong| (Pathogenic Very  | splice site, initiation codon loss, or whole-exon deletion in a gene   |
| **    | Strong)           | where **Loss of Function (LoF) is an established disease mechanism**  |
+-------+-------------------+------------------------------------------------------------------------+
|**Strong| **PS1**           | Same amino acid change as a previously established pathogenic variant  |
| **    | **PS2**           | *De novo* variant with confirmed paternity and maternity               |
|       | **PS3**           | Well-established *in vitro* or *in vivo* functional assay shows damage |
|       | **PS4**           | Variant prevalence in affected cases significantly higher than controls|
+-------+-------------------+------------------------------------------------------------------------+
|**Mod- | **PM1**           | Located in a mutational hotspot or critical functional domain          |
| erate | **PM2**           | Absent or extremely low frequency in population databases (gnomAD)     |
| **    | **PM3**           | Detected in trans with a pathogenic variant for recessive disorder    |
|       | **PM4**           | Protein length change (in-frame insertion/deletion or stop-loss)       |
|       | **PM5**           | Novel missense at an amino acid where different missense is pathogenic |
|       | **PM6**           | Assumed *de novo* without confirmed maternity and paternity            |
+-------+-------------------+------------------------------------------------------------------------+
|**Sup- | **PP1**           | Co-segregation with disease in multiple affected family members        |
| port- | **PP2**           | Missense variant in gene with low rate of benign missense variation    |
| ing** | **PP3**           | Multiple computational in silico tools predict deleterious effect      |
|       | **PP4**           | Patient phenotype/family history highly specific for single gene defect|
|       | **PP5**           | Reputable source reports as pathogenic without individual evidence     |
+-------+-------------------+------------------------------------------------------------------------+
|**Stand| **BA1**           | Allele frequency **$>5\%$ in population databases (gnomAD / ExAC)**     |
| Alone | (Benign Standalone)| (Stand-alone evidence for benign classification in Mendelian disease)  |
+-------+-------------------+------------------------------------------------------------------------+
|**Benign| **BS1 - BS4**     | BS1 (Frequency higher than expected); BS2 (Observed in healthy adult); |
| Strong| (Benign Strong)   | BS3 (Functional studies show no effect); BS4 (Lack of segregation)     |
+-------+-------------------+------------------------------------------------------------------------+
|**Benign| **BP1 - BP7**     | BP1 (Missense in LoF gene); BP3 (In-frame indel in non-conserved loop);|
| Supp**| (Benign Supporting| BP4 (In silico tools predict benign); BP7 (Synonymous variant, no splice)|
+-------+-------------------+------------------------------------------------------------------------+

Combining Evidence to Reach a Classification

Pathogenic (P):
- $1\text{ PVS1} + \ge 1\text{ Strong (PS1–PS4)}$
- $1\text{ PVS1} + \ge 2\text{ Moderate (PM1–PM6)}$
- $1\text{ PVS1} + 1\text{ Moderate} + 1\text{ Supporting (PP1–PP5)}$
- $1\text{ PVS1} + \ge 2\text{ Supporting}$
- $\ge 2\text{ Strong (PS)}$
- $1\text{ Strong} + \ge 3\text{ Moderate}$
Benign (B):
- $1\text{ BA1}$ (Stand-alone: allele frequency $>5%$)
- $\ge 2\text{ Benign Strong (BS1–BS4)}$
Variant of Uncertain Significance (VUS):
- Criteria for pathogenic and benign are not met, or conflicting evidence exists between pathogenic and benign criteria.

4. HGVS Sequence Variant Nomenclature Standards

The Human Genome Variation Society (HGVS) publishes strict international standards for describing DNA, RNA, and protein sequence variants.

                           HGVS REFERENCE SEQUENCE PREFIXES
                           
    - g.  = Genomic DNA reference sequence (e.g., NC_000007.14:g.117559590G>A)
    - c.  = Coding DNA reference sequence (e.g., NM_000492.4:c.1521_1523delCTT)
    - n.  = Non-coding RNA reference sequence
    - r.  = RNA reference sequence (e.g., r.1521_1523delcuu)
    - p.  = Protein reference sequence (e.g., NP_000483.3:p.Phe508del)
    - m.  = Mitochondrial DNA reference sequence (e.g., m.3243A>G)

                          CODING DNA (c.) NUMBERING SYSTEM
                          
          5' UTR                 Coding Exons (1 to N)                  3' UTR
     ... c.-3  c.-2  c.-1    c.1  c.2  c.3 ... c.1584           c.*1  c.*2  c.*3 ...
     ---------------------[ A  T  G ... ... ... T  A  A ]------------------------
                            |                   |
                            | (Start Codon A)   | (Stop Codon End)
                            v                   v
                    *** THERE IS NO NUCLEOTIDE ZERO (c.0 DOES NOT EXIST!) ***
                    Base preceding c.1 is c.-1
                    Base following stop codon is c.*1

+----------------------------------------------------------------------------------------------------+
|                         HGVS CODING DNA (c.) SYNTAX RULES & EXAMPLES                               |
+-------------------+-------------------+-------------------+----------------------------------------+
| Mutation Type     | HGVS Format       | Clinical Example  | Exact Molecular Description            |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Substitution**  | `c.positionRef>Alt`| `c.1799T>A`       | Single nucleotide change at pos 1799   |
|                   |                   | (*BRAF* V600E)    | from Thymine to Adenine                |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Deletion**      | `c.start_enddel`  | `c.1521_1523delCTT`| Deletion of 3 nucleotides (CTT) from   |
|                   |                   | (*CFTR* F508del)  | position 1521 to 1523                  |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Insertion**     | `c.flank_flankins`| `c.1521_1522insATG`| Insertion of ATG between adjacent      |
|                   |                   |                   | nucleotides 1521 and 1522              |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Duplication**   | `c.start_enddup`  | `c.775_776dup`    | Tandem duplication of 2 nucleotides    |
|                   |                   | (*HER2* exon 20)  | at positions 775 to 776                |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Deletion /      | `c.start_end`     | `c.112_117delinsTG`| Deletion of 6 nucleotides (112 to 117) |
| Insertion (Delins)| `delinsSequence`  |                   | and insertion of TG                    |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Intronic Donor  | `c.exon_end+pos`  | `c.1584+1G>A`     | G to A transition at the first base    |
| Splice Site (+)** | `Ref>Alt`         | (Splice donor)    | of the intron following exon pos 1584  |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Intronic Acceptor| `c.exon_start-pos`| `c.489-2A>G`      | A to G transition at the -2 base of the|
| Splice Site (-)** | `Ref>Alt`         | (Splice acceptor) | intron preceding coding exon pos 489   |
+-------------------+-------------------+-------------------+----------------------------------------+

The HGVS 3' Rule (Right-Shift Rule): For insertions, deletions, or duplications occurring in tandem repetitive sequences (e.g., homopolymer runs or microsatellites), the variant must always be described at the most $3'$ position relative to the reference sequence.

5. Protein HGVS Syntax & Common Diagnostic Pitfalls

Protein alterations are described using standard three-letter amino acid abbreviations (preferred) or single-letter codes.

+----------------------------------------------------------------------------------------------------+
|                         HGVS PROTEIN (p.) SYNTAX RULES & EXAMPLES                                  |
+-------------------+-------------------+-------------------+----------------------------------------+
| Protein Change    | Standard Format   | Clinical Example  | Detailed Description                   |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Missense**      | `p.OrigPosNew`    | `p.Val600Glu`     | Valine replaced by Glutamic Acid at    |
|                   |                   | (or `p.V600E`)    | amino acid position 600                |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Nonsense / Stop | `p.OrigPosTer`    | `p.Trp1282Ter`    | Tryptophan replaced by premature stop  |
| Gained**          | (or `p.Trp1282*`) | (or `p.W1282*`)   | codon (Ter/*) at position 1282         |
+-------------------+-------------------+-------------------+----------------------------------------+
| **In-Frame Del**  | `p.OrigPosdel`    | `p.Phe508del`     | Phenylalanine deleted at position 508; |
|                   |                   | (*CFTR* $\Delta$F508)| reading frame remains intact           |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Frameshift**    | `p.OrigPosfsTer#` | `p.Lys179fsTer21` | Lysine 179 is first shifted codon; new |
|                   | (or `p.Lys179fs`) |                   | reading frame stops 21 codons downstream|
+-------------------+-------------------+-------------------+----------------------------------------+
| **Silent /        | `p.OrigPos=`      | `p.Gly670=`       | Nucleotide change does not alter amino |
| Synonymous**      |                   | (or `p.(=)`)      | acid sequence (Glycine remains Glycine)|
+-------------------+-------------------+-------------------+----------------------------------------+
| **Initiation      | `p.Met1?`         | `p.Met1?`         | Mutation in methionine start codon;    |
| Codon Defect**    | (or `p.0?`)       |                   | translation initiation compromised     |
+-------------------+-------------------+-------------------+----------------------------------------+

Critical Exam Pitfalls to Avoid in HGVS Nomenclature

Never use "X" for stop codons in HGVS: Ter or * is required (e.g., p.Trp1282Ter, NOT p.W1282X).
Never use c.0: Numbering transitions directly from c.-1 (5' UTR) to c.1 (coding start).
Never use slash (/) or hyphen (-) for substitutions: Write c.1799T>A, NOT c.1799T/A or c.1799T-A.
Coordinate Order for Insertions: Coordinates must reflect the two adjacent flanking bases (e.g., c.1521_1522insATG, NOT c.1521insATG).

Loading diagram...

ACMG Variant Classification Decision Tree

Test Your Knowledge

A molecular technologist performs UV spectrophotometry on a 1:50 dilution of an extracted genomic DNA sample. The spectrophotometer yields an A260 reading of 0.400 and an A280 reading of 0.222. What is the DNA concentration of the original undiluted sample, and what does the A260/A280 purity ratio indicate?

Concentration is 200 µg/mL; purity ratio of 0.55 indicates severe phenol contamination.

Concentration is 500 µg/mL; purity ratio of 2.22 indicates heavy RNA contamination.

Concentration is 20 µg/mL; purity ratio of 1.45 indicates high salt carryover.

Concentration is 1000 µg/mL (1.0 mg/mL); purity ratio of 1.80 indicates highly pure double-stranded DNA.

Test Your Knowledge

Under the ACMG/AMP sequence variant interpretation guidelines, under which specific condition can the 'Very Strong' evidence code PVS1 be applied to a novel frameshift or nonsense sequence variant?

The variant is identified in at least three unrelated individuals in the gnomAD database at a frequency greater than 1%.

The variant is a null mutation (nonsense, frameshift, canonical +/- 1,2 splice site) in a gene where loss of function (LoF) is an established mechanism of human disease.

Multiple computational in silico algorithms (SIFT, PolyPhen-2, CADD) uniformly predict that the missense change alters protein tertiary structure.

The variant co-segregates with disease in two affected second-degree relatives.

Test Your Knowledge

According to official HGVS sequence variant nomenclature rules, which statement correctly describes coding DNA coordinate numbering and intronic splice site notation?

Coordinate c.1 is the A of the ATG translation initiation start codon, nucleotide zero does not exist (the base preceding c.1 is c.-1), and intronic variants are designated relative to the nearest coding exon boundary using + or - offsets (e.g., c.1584+1G>A).

Coordinate c.0 represents the first nucleotide of the 5' untranslated region, and intronic variants are numbered consecutively from 1 to 10,000 using the lowercase letter 'i'.

Coding coordinates begin at the transcription start cap site as c.1, while stop codons are represented exclusively with the letter 'X'.

Intronic donor splice site mutations must be written as slash-separated genomic coordinates (e.g., g.1584/1585del).

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ASCP Molecular Biology Technologist

ASCP Molecular Biology Technologist

15.3 Clinical Calculations, ACMG Variant Classification & HGVS

Key Takeaways

15.3 Clinical Calculations, ACMG Variant Classification & HGVS

1. Molecular Laboratory Calculations & Spectrophotometric Quantification

UV Spectrophotometric Quantification of Nucleic Acids

Spectrophotometric Purity Ratios & Troubleshooting

Copy Number Calculations from Mass and Length

Laboratory Dilutions & Reagent Preparation ($C_1 V_1 = C_2 V_2$)

Population Genetics: Hardy-Weinberg Equilibrium

2. Clinical Diagnostic Performance Metrics

Impact of Disease Prevalence on PPV and NPV

3. ACMG/AMP Sequence Variant Interpretation Guidelines

Combining Evidence to Reach a Classification

4. HGVS Sequence Variant Nomenclature Standards

5. Protein HGVS Syntax & Common Diagnostic Pitfalls

Critical Exam Pitfalls to Avoid in HGVS Nomenclature

ASCP Molecular Biology Technologist

1Introduction & Exam Strategy

2Nucleic Acid Chemistry & Molecular Structure

3Central Dogma, Genome Organization & Cytogenetics

4Biochemical Reagents & Enzymes

5Nucleic Acid Isolation & Purification

6Nucleic Acid Quantification & Electrophoresis

7PCR Principles & Primer Design Optimization

8Advanced Amplification Technologies

9DNA Sequencing Technologies

10Hybridization, Blotting & Cytogenetic Assays

11Molecular Infectious Disease Diagnostics

12Molecular Oncology & Hematopathology

13Medical Genetics, Pharmacogenomics & Identity Testing

14Quality Assurance, Controls & Method Validation

15Laboratory Operations, Safety & Clinical Variant Reporting

ASCP Molecular Biology Technologist

15.3 Clinical Calculations, ACMG Variant Classification & HGVS

Key Takeaways

15.3 Clinical Calculations, ACMG Variant Classification & HGVS

1. Molecular Laboratory Calculations & Spectrophotometric Quantification

UV Spectrophotometric Quantification of Nucleic Acids

Spectrophotometric Purity Ratios & Troubleshooting

Copy Number Calculations from Mass and Length

Laboratory Dilutions & Reagent Preparation ($C_1 V_1 = C_2 V_2$)

Population Genetics: Hardy-Weinberg Equilibrium

2. Clinical Diagnostic Performance Metrics

Impact of Disease Prevalence on PPV and NPV

3. ACMG/AMP Sequence Variant Interpretation Guidelines

Combining Evidence to Reach a Classification

4. HGVS Sequence Variant Nomenclature Standards

5. Protein HGVS Syntax & Common Diagnostic Pitfalls

Critical Exam Pitfalls to Avoid in HGVS Nomenclature