13.1 Single-Gene Mendelian Disorders & CFTR Testing
Key Takeaways
- Mendelian disorders follow classical inheritance patterns (autosomal dominant, autosomal recessive, X-linked recessive/dominant, mitochondrial); understanding penetrance, variable expressivity, and mosaicism is essential for diagnostic interpretation.
- Cystic fibrosis is caused by pathogenic variants in CFTR (7q31.2); the p.Phe508del (Delta-F508) mutation is a Class II trafficking defect accounting for ~70% of mutant alleles in Caucasian populations.
- CFTR mutations are categorized into six functional classes (I-VI) based on synthesis, processing, gating, conductance, abundance, and membrane stability, which dictate response to CFTR modulators (potentiators like ivacaftor vs. correctors like elexacaftor/tezacaftor).
- The intron 9 poly-T tract (5T/7T/9T) and adjacent TG repeats modulate exon 10 splicing efficiency; a 5T variant in cis with the R117H missense mutation substantially reduces full-length CFTR mRNA, causing classic cystic fibrosis or congenital bilateral absence of the vas deferens (CBAVD).
- Common Mendelian molecular targets on the ASCP MB exam include HBB point mutations and alpha-globin deletions in hemoglobinopathies, HFE C282Y/H63D in hereditary hemochromatosis, and Factor V Leiden (F5 c.1691G>A) in hereditary thrombophilia.
Last updated: August 2026
13.1 Single-Gene Mendelian Disorders & CFTR Testing
Quick Summary: Single-gene Mendelian disorders result from pathogenic DNA variants within specific genomic loci following predictable inheritance patterns: autosomal dominant (AD), autosomal recessive (AR), X-linked recessive (XLR), X-linked dominant (XLD), and maternal mitochondrial transmission. A premier clinical model is Cystic Fibrosis (CF), caused by mutations in the CFTR gene on chromosome 7q31.2. The most prevalent mutation, $\Delta$F508 (p.Phe508del), causes protein misfolding and premature endoplasmic reticulum-associated degradation (ERAD). Molecular diagnosis utilizes targeted mutation panels (e.g., ACMG core panels), Allele-Specific Oligonucleotide (ASO) hybridization, Amplification Refractory Mutation System (ARMS-PCR), Multiplex Ligation-dependent Probe Amplification (MLPA), and Next-Generation Sequencing (NGS). Testing often requires interrogating polymorphic modifier loci, such as the CFTR intron 9 poly-T (5T/7T/9T) and TG repeats, which modulate the clinical penetrance of variants like p.Arg117His (R117H).
1. Classical Mendelian Inheritance Patterns & Non-Mendelian Modifiers
Understanding the biochemical transmission of monogenic traits forms the foundation of clinical molecular genetics.
+----------------------------------------------------------------------------------------------------+
| SUMMARY OF MENDELIAN INHERITANCE MODES |
+-------------------+-------------------+-------------------+----------------------------------------+
| Inheritance Mode | Transmission Risk | Sex Distribution | Hallmark Genetic Disorders |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Autosomal | 50% risk per | Males and females | Huntington disease (*HTT*), Marfan |
| Dominant (AD)** | pregnancy from an | affected equally; | syndrome (*FBN1*), Achondroplasia |
| | affected parent | vertical lineage | (*FGFR3*), Lynch syndrome (*MMR*) |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Autosomal | 25% affected, | Males and females | Cystic Fibrosis (*CFTR*), Sickle Cell |
| Recessive (AR)** | 50% carrier risk | affected equally; | (*HBB*), Hemochromatosis (*HFE*), |
| | from carrier pair | horizontal pedigree| Tay-Sachs (*HEXA*), PKU (*PAH*) |
+-------------------+-------------------+-------------------+----------------------------------------+
| **X-Linked | Carrier mother -> | Predominantly | Duchenne / Becker Muscular Dystrophy |
| Recessive (XLR)** | 50% affected sons,| males; no male- | (*DMD*), Hemophilia A (*F8*) / B (*F9*),|
| | 50% carrier dtrs | to-male trans. | Fragile X syndrome (*FMR1*) |
+-------------------+-------------------+-------------------+----------------------------------------+
| **X-Linked | Affected father ->| Females affected | Rett syndrome (*MECP2*), Hypophos- |
| Dominant (XLD)** | 100% dtrs, 0% sons| 2x more than males| phatemic Rickets (*PHEX*), Incontinentia|
| | Affected mother->50%| often embryonic lethal| pigmenti (*IKBKG*) |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Mitochondrial | 100% of offspring | Transmitted ONLY | MELAS (m.3243A>G *MT-TL1*), MERRF |
| (Maternal)** | of affected female| by females to all | (m.8344A>G *MT-TK*), Leber Hereditary |
| | inherit mutation | children | Optic Neuropathy (LHON, *MT-ND4*) |
+-------------------+-------------------+-------------------+----------------------------------------+
Non-Mendelian Phenomena & Molecular Modifiers
- Incomplete Penetrance: An individual inherits a disease-causing pathogenic genotype but does not manifest the clinical phenotype (e.g., HFE C282Y homozygotes where only a fraction develop overt clinical hemochromatosis).
- Variable Expressivity: Individuals harboring the identical pathogenic genotype display wide variations in clinical severity and organ involvement (e.g., Neurofibromatosis type 1, NF1).
- Pleiotropy: A single mutant gene causes multiple, seemingly unrelated phenotypic traits across diverse organ systems (e.g., FBN1 mutations causing lens dislocation, aortic root aneurysm, and skeletal arachnodactyly in Marfan syndrome).
- Mosaicism: Presence of two or more genetically distinct cell lines within a single individual arising from post-zygotic somatic mutations. Germline (gonadal) mosaicism occurs when the mutation is restricted to gametes, explaining why unaffected parents can have multiple children with an autosomal dominant disorder without showing somatic symptoms (frequent in DMD and COL1A1/COL1A2 osteogenesis imperfecta).
- Mitochondrial Heteroplasmy: Cells contain a mixture of mutant and wild-type mitochondrial DNA (mtDNA) molecules. Clinical symptoms manifest only when the proportion of mutant mtDNA exceeds a critical biochemical threshold (typically 60–80%), which segregates randomly into daughter cells during replicative segregation.
2. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Molecular Diagnostics
Cystic fibrosis is the most common life-limiting autosomal recessive genetic disorder in individuals of European descent (carrier frequency $\sim 1$ in $25–29$). The disease is caused by mutations in the CFTR gene located on chromosome 7q31.2, spanning $27\text{ exons}$ and encoding a $1,480\text{ amino acid}$ ATP-binding cassette (ABC) transporter functioning as a cAMP-regulated epithelial chloride and bicarbonate channel.
CFTR PROTEIN ARCHITECTURE & MEMBRANE TOPOLOGY
Extracellular Space
====================[ Apical Membrane ]====================
|| || || || || || || || || || || ||
|| || || || || || || || || || || ||
--++---++---++---++---++---++---------++---++---++---++---++---++---
Cytoplasm [ TMD1 ] [ TMD2 ]
12 Membrane-Spanning Helices (Chloride Pore)
/ \ / \
/ \ / \
+---------------+ +---------------+ +---------------+
| NBD1 | | R-Domain | | NBD2 | --> ATP Binding
| Nucleotide | | (Regulatory; | | Nucleotide | & Hydrolysis
| Binding | | PKA Phosphor- | | Binding | Drive Gating
| Domain 1 | | ylation Sites)| | Domain 2 | Open / Close
+---------------+ +---------------+ +---------------+
^
| (Position 508: Phenylalanine Deletion Site!)
[ ΔF508 / p.Phe508del ]
The $\Delta$F508 (p.Phe508del) Mutation
- Molecular Mechanism: A 3-base-pair in-frame deletion (c.1521_1523delCTT or c.1522_1524delTTT) in exon 11 (legacy exon 10) that deletes the codon for phenylalanine at position 508 within Nucleotide-Binding Domain 1 (NBD1).
- Pathobiology: The absence of Phe508 causes improper protein folding, triggering the endoplasmic reticulum quality-control machinery to target the nascent protein for ubiquitination and degradation via the Endoplasmic Reticulum-Associated Degradation (ERAD) pathway. Consequently, virtually zero CFTR reaches the apical plasma membrane (Class II mutation).
The Six Functional Classes of CFTR Mutations
+----------------------------------------------------------------------------------------------------+
| CFTR MUTATION FUNCTIONAL CLASSES |
+-------+-------------------+-------------------+-------------------+--------------------------------+
| Class | Functional Defect | Molecular Basis | Example Mutations | Pharmacological Strategy |
+-------+-------------------+-------------------+-------------------+--------------------------------+
| **I** | **No functional | Nonsense, frame- | **G542X, W1282X, | Premature stop codon read- |
| | protein made** | shift, or canonical| **R553X, 621+1G>T**| through agents or mRNA therapy |
| | | splice variants | | |
+-------+-------------------+-------------------+-------------------+--------------------------------+
| **II**| **Defective | Misfolding and | **$\Delta$F508 | **CFTR Correctors**: |
| | processing / | premature ERAD | (p.Phe508del), | Elexacaftor, Tezacaftor, |
| | trafficking** | degradation | N1303K, I507del** | Lumacaftor (rescue folding) |
+-------+-------------------+-------------------+-------------------+--------------------------------+
| **III**| **Defective gating| Protein reaches | **G551D, G1349D, | **CFTR Potentiator**: |
| | / regulation** | membrane but channel| S549N** | Ivacaftor (Kalydeco - locks |
| | | fails to open | | channel in open conformation) |
+-------+-------------------+-------------------+-------------------+--------------------------------+
| **IV**| **Decreased | Channel opens but | **R117H, R334W, | **CFTR Potentiator**: |
| | conductance** | ion transport rate| R347P** | Ivacaftor (amplifies open-state|
| | | is reduced | | ion flow) |
+-------+-------------------+-------------------+-------------------+--------------------------------+
| **V** | **Reduced | Aberrant intronic | **3849+10kbC>T, | Splicing modulators or |
| | synthesis** | splicing generates| 2789+5G>A, | potentiators / correctors |
| | | low normal mRNA | A455E** | |
+-------+-------------------+-------------------+-------------------+--------------------------------+
| **VI**| **Accelerated | Destabilized | **4326delTC, | Protein membrane stabilizers |
| | membrane turnover**| apical residence | rP205S** | |
+-------+-------------------+-------------------+-------------------+--------------------------------+
The ACMG 23-Mutation Core Screening Panel
In 2001 (updated 2004), the American College of Medical Genetics and Genomics (ACMG) and American College of Obstetricians and Gynecologists (ACOG) established a standardized 23-mutation core panel for pan-ethnic cystic fibrosis carrier screening. The panel includes pan-ethnic variants with allele frequencies $\ge 0.1%$ in CF patients, including $\Delta$F508, $\Delta$I507, G542X, G551D, W1282X, N1303K, R553X, 621+1G>T, 3849+10kbC>T, and R117H.
The CFTR Intron 9 Poly-T and TG Tract Modifiers
The polymorphic polythymidine (poly-T) tract located at the splice acceptor site of CFTR intron 9 (legacy intron 8) directly dictates the splicing efficiency of exon 10.
CFTR INTRON 9 SPLICING MODIFICATION
Intron 9 Exon 10
5'--[ (TG)m ]---[ (T)n Tract ]-----[ Splice Acceptor AG ]=====[ Exon 10 Coding ]--3'
m = 11, 12, 13 n = 5T, 7T, 9T
[ 9T Allele ]: Robust U2AF65 binding -> 100% normal Exon 10 inclusion -> Full functional CFTR
[ 7T Allele ]: Moderate U2AF65 binding -> ~90% Exon 10 inclusion -> Normal phenotype
[ 5T Allele ]: Weak U2AF65 binding -> Severe Exon 10 skipping -> Non-functional CFTR
(Exon 10 skipping eliminates essential transmembrane domains!)
+----------------------------------------------------------------------------------------------------+
| R117H AND POLY-T / TG TRACT INTRON 9 COMPOUND INTERACTIONS |
+-------------------+-------------------+------------------------------------------------------------+
| Genotype Pattern | Phase (Linkage) | Clinical Phenotype & Outcome |
+-------------------+-------------------+------------------------------------------------------------+
| **R117H + 5T** | **In cis** | **Classic Cystic Fibrosis** (severe pulmonary & pancreatic |
| | (same chromosome) | disease when paired with a severe trans CFTR mutation) |
+-------------------+-------------------+------------------------------------------------------------+
| **R117H + 7T** | **In cis** | **CBAVD / Mild CF** (Congenital Bilateral Absence of the |
| | (same chromosome) | Vas Deferens causing male infertility; mild late pulmonary)|
+-------------------+-------------------+------------------------------------------------------------+
| **R117H + 9T** | **In cis** | **Benign / Non-Penetrant** (Normal respiratory function and|
| | (same chromosome) | typically normal fertility) |
+-------------------+-------------------+------------------------------------------------------------+
| **TG12 or TG13** | **In cis with 5T**| **Increased Exon Skipping**: Longer TG repeats combined with|
| | | 5T further destabilize splicing, exacerbating severity |
+-------------------+-------------------+------------------------------------------------------------+
Critical Laboratory Reporting Rule: Reflex testing for the intron 9 poly-T tract (5T/7T/9T) is indicated only when the R117H variant is detected, or during diagnostic workup for isolated male infertility due to Congenital Bilateral Absence of the Vas Deferens (CBAVD). Reporting 5T status in the absence of R117H in general carrier screening causes unwarranted patient anxiety.
3. Hemoglobinopathies: Sickle Cell Disease & Thalassemia
Hemoglobin consists of a heterotetramer of two $\alpha$-like globin chains (HBA1, HBA2 on chromosome 16p13.3) and two $\beta$-like globin chains (HBB on chromosome 11p15.4).
HEMOGLOBIN VARIANT ARCHITECTURE
Normal Adult HbA (α2β2): Codon 6 of HBB = GAG (Glutamate, Acidic/Hydrophilic)
Sickle Cell HbS (α2βS2): Codon 6 of HBB = GTG (Valine, Nonpolar/Hydrophobic) [c.20A>T]
Hemoglobin C HbC (α2βC2): Codon 6 of HBB = AAG (Lysine, Basic/Positively Charged) [c.19G>A]
+----------------------------------------------------------------------------------------------------+
| MOLECULAR BASIS OF HEMOGLOBINOPATHIES |
+-------------------+-------------------+-------------------+----------------------------------------+
| Disorder | Gene & Chromosome | Mutational Type | Diagnostic Characteristics & Phenotype |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Sickle Cell | *HBB* (11p15.4) | Point Mutation | Deoxy-HbS polymerizes into rigid fibers|
| Anemia (HbSS)** | | c.20A>T | causing vaso-occlusive crisis; |
| | | (p.Glu6Val) | abolishes *DdeI* and *MstII* cut sites |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Hemoglobin C | *HBB* (11p15.4) | Point Mutation | Hexagonal intracellular crystals; mild |
| Disease (HbCC)** | | c.19G>A | hemolytic anemia and target cells |
| | | (p.Glu6Lys) | |
+-------------------+-------------------+-------------------+----------------------------------------+
| **$\beta$-Thalassemia| *HBB* (11p15.4) | Point mutations | $\beta^0$ (complete absence of HbA) vs |
| Major / Minor** | | (Promoter, splice,| $\beta^+$ (reduced synthesis); severe micro-|
| | | nonsense, frameshift)| cytic hypochromic anemia in Major |
+-------------------+-------------------+-------------------+----------------------------------------+
| **$\alpha$-Thalassemia| *HBA1* & *HBA2*| **Large Genomic | Caused by unequal homologous crossing |
| Syndromes** | (16p13.3) | **Deletions** | over; 4 functional $\alpha$-globin genes|
| | (4 alleles total) | ($-\alpha^{3.7}$, | per diploid genome |
| | | $-\alpha^{4.2}$, $--^{MED}$,| |
| | | $--^{SEA}$) | |
+-------------------+-------------------+-------------------+----------------------------------------+
$\alpha$-Thalassemia Gene Dosage Spectrum
- Silent Carrier ($-\alpha / \alpha\alpha$): 3 functional genes; clinically asymptomatic, normal hematologic indices.
- $\alpha$-Thalassemia Trait / Minor: 2 functional genes.
- Trans configuration ($-\alpha / -\alpha$): Prevalent in African populations; cannot pass two deleted genes on one chromosome.
- Cis configuration ($-- / \alpha\alpha$): Prevalent in Southeast Asian and Mediterranean populations; carriers can transmit $--$, putting offspring at risk for lethal Hydrops Fetalis.
- Hemoglobin H Disease ($-- / -\alpha$): 1 functional gene; excess $\beta$-chains form unstable $\beta_4$ tetramers (HbH), leading to inclusion bodies and moderate to severe hemolytic anemia.
- Hydrops Fetalis / Hb Bart's ($-- / --$): 0 functional genes; excess fetal $\gamma$-chains form $\gamma_4$ tetramers (Hb Bart's) with extreme oxygen affinity, causing intrauterine death or fatal hydrops.
4. Hereditary Hemochromatosis (HFE) & Inherited Thrombophilia
+----------------------------------------------------------------------------------------------------+
| HEREDITARY HEMOCHROMATOSIS & THROMBOPHILIA GENOTYPES |
+-------------------+-------------------+-------------------+----------------------------------------+
| Gene & Variant | DNA / Protein Alt.| Restriction Enzyme| Clinical Pathology & Laboratory Impact |
+-------------------+-------------------+-------------------+----------------------------------------+
| **HFE C282Y** | c.845G>A | **Gains *RsaI*** | Disrupts Cys282-Cys277 disulfide bond, |
| (6p22.2) | (p.Cys282Tyr) | restriction site | preventing association with $\beta_2$- |
| | | | microglobulin; excessive iron absorption|
+-------------------+-------------------+-------------------+----------------------------------------+
| **HFE H63D** | c.187C>G | Loses *BclI* site | Mild variant; compound heterozygotes |
| (6p22.2) | (p.His63Asp) | | (C282Y/H63D) have modest iron overload |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Factor V Leiden**| c.1691G>A | **Loses *MnlI*** | Replaces arginine cleavage site with |
| (*F5*, 1q24.2) | (p.Arg506Gln) | restriction site | glutamine; factor Va resistant to |
| | | | Activated Protein C (APC) degradation |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Prothrombin** | c.*97G>A | Allele-specific | Located in 3' UTR; increases mRNA |
| (*F2*, 11p11.2) | (Legacy G20210A) | qPCR / Sanger | stability, elevating circulating pro- |
| | | | thrombin levels (2-3x VTE risk) |
+-------------------+-------------------+-------------------+----------------------------------------+
RESTRICTION FRAGMENT ANALYSIS: FACTOR V LEIDEN
Wild-Type F5 Allele (1691G): ---[ MnlI Site 1 ]------[ MnlI Site 2 (Arg506) ]---[ MnlI Site 3 ]---
Digestion yields: 37 bp, 116 bp, 67 bp (Normal fragments)
Mutant Leiden Allele (1691A): ---[ MnlI Site 1 ]------[ MUTATION (Site Lost!) ]---[ MnlI Site 3 ]---
Digestion yields: 37 bp, 183 bp (116 bp + 67 bp Fuse into 183 bp!)
5. Clinical Molecular Methodologies for Mendelian Variants
+----------------------------------------------------------------------------------------------------+
| MENDELIAN MOLECULAR TESTING METHODOLOGIES COMPARISON |
+-------------------+-------------------+-------------------+----------------------------------------+
| Method | Primary Principle | Common Applications| Key Strengths & Technical Limitations |
+-------------------+-------------------+-------------------+----------------------------------------+
| **ARMS-PCR** | Allele-specific | *CFTR*, *HFE*, | Rapid, inexpensive; requires precise |
| (Allele-Specific) | 3'-end primer | *F5* Leiden | primer mismatch design to avoid false |
| | extension | genotyping | amplification |
+-------------------+-------------------+-------------------+----------------------------------------+
| **PCR-RFLP** | Restriction enzyme| *F5* Leiden, | Straightforward; risk of false mutant |
| | cleavage of PCR | *HFE* C282Y | calls if restriction digestion is |
| | products | | incomplete (partial digest artifact) |
+-------------------+-------------------+-------------------+----------------------------------------+
| **MLPA** | Multiplex probe | *DMD*, *HBA1/2*, | Gold standard for large single/multi- |
| (Ligation-Based) | hybridization & | *SMN1* copy number| exon deletions and duplications; does |
| | ligation | variations | not detect balanced point mutations |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Gap-PCR** | Primers flank | $\alpha$-Thalassemia| Directly sizes large common deletions; |
| | deletion junction | ($-\alpha^{3.7}$, | Cannot detect novel or uncharacterized |
| | breakpoints | $--^{MED}$) | deletion breakpoints |
+-------------------+-------------------+-------------------+----------------------------------------+
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Test Your Knowledge
A molecular diagnostic laboratory receives a blood sample for CFTR carrier screening. The core panel detects the p.Arg117His (R117H) missense mutation. What reflex testing must be performed to accurately determine the clinical significance and penetrance of this variant, and how does the modifier allele alter phenotype?
A
B
C
D
Test Your Knowledge
A molecular technologist performs Factor V Leiden genotyping using PCR followed by restriction fragment length polymorphism (PCR-RFLP) with the MnlI endonuclease. The assay produces digestion fragments of 37 bp, 116 bp, and 67 bp in a wild-type sample. How does the presence of the Factor V Leiden pathogenic variant (c.1691G>A) alter the restriction pattern in a homozygous mutant patient?
A
B
C
D
Test Your Knowledge
Which clinical scenario illustrates the genetic concept of germline (gonadal) mosaicism?
A
B
C
D