FINALS LEC: Molecular Detection of Inherited Diseases Flashcards
Caused by mutations (changes) in germ cells that are passed down from parent to child
Inherited Diseases
Mutations in Somatic Cell
- Cancer
- Congenital malformations (present at birth): due to factors upsetting the developmental process
Abnormal chromosome #
Genome Mutations
more than 2 sets
Polyploidy
gain (trisomy)/loss (monosomy)
Aneuploidy
Detection for genome mutations:
karyotyping, ploidy analysis, flow cytometry, & FISH
Flat facial profile, mental retardation, cardiac problems. risk of acute leukemia, eventual neuropathological disorders, abnormal immune system
down syndrome
Severe, clenched fist; survival less than 1 year
edward syndrome
Cleft palate, heart damage, mental retardation, survival usually less than 6 mo
patau syndrome
Male hypogonadism, long legs, gynecomastia (male breast enlargement), low testosterone level
klinefelter syndrome
Excessive height, acne, 1%-2% behavioral disorders
XYY SYNDROME
Bilateral neck webbing, heart disease, failure to develop secondary sex characteristics, hypothyroidism
turner syndrome
Mental retardation increases with increasing X
Multi X females
Trisomy 21, 47,XY+21
Down syndrome
Trisomy 18, 47,XY+18
Edward syndrome
Trisomy 13, 47,XY+13
Patau syndrome
47,XXY
Klinefelter syndrome
47,XYY
XYY SYNDROME
45X and variants
turner syndrome
47,XXX: 48,XXXX
Multi X females
Abnormalities in chromosome structure
Translocations, inversions, deletions, duplications, marker chromosomes, derivative chromosomes
caused by chemicals, radiation, chromosome breakage syndromes (Fanconi anemia, Bloom syndrome, ataxia telangiectasia)
Chromosome breakage
Chromosomal mutations detection
karyotyping, FISH, microarray technology (CGH)
CATCH 22 (cardiac abnormality/abnormal facies, T-cell deficit, cleft palate, hypercalcemia)
DiGeorge syndrome and velocardiofacial syndrome
Growth deficiency, catlike cry in infancy, small head, mental retardation
Cri du chat syndrome
Aniridia (absence of iris), hemihypertrophy (one side of the body seems to grow faster than the other), and other congenital anomalies.
Contiguous gene syndrome; Wilms tumor, aniridia, genitourinary anomalies, mental retardation syndrome
del(22q)
Cri du chat syndromeDiGeorge syndrome and velocardiofacial syndrome
del(5p)
Cri du chat syndrome
del (11p)
Contiguous gene syndrome; Wilms tumor, aniridia, genitourinary anomalies, mental retardation syndrome
Single-gene diseases affect structural proteins, cell surface receptor proteins, growth regulators, and enzymes
Patterns of Inheritance in Single-Gene Diseases
3 Dominance Relationships
Complete dominance
Partial/incomplete dominace
Codominance
Heterozygous phenotype (child=Tt) Homozygous phenotype (1 parent = TT)
Example: height
Complete dominance
Offspring phenotype is variably intermediate (combine) between the homozygous & heterozygous parentals
Example: gene affecting hair texture
Partial/incomplete dominance
Simultaneously demonstrate the phenotype of both parents
Example: ABO blood group
Codominance
Dominant allele will mask the effect of the recessive allele completely
Complete Dominance
Both the alleles are dominant
Codominance
A.K.A. transmission patterns/mode of inheritance
Patterns of Inheritance in Single-Gene Diseases
The manner in which a genetic trait, disorder, or risk of disorder is passed from one generation to the next
Determined by examination of family histories
Patterns of Inheritance in Single-Gene Diseases
diagram of family phenotype/genotype
Pedigree
Patterns of Inheritance in Single-Gene Disease 3 main patterns:
autosomal dominant, autosomal recessive, sex-linked (Xlinked)
Criteria:
1. Males and females can be affected. Male-to-male transmission may occur.
2. Males and females transmit the trait with equal frequency.
RECESSIVE DOMINANT
3. Successive generations are affected.
4. Transmission stops after a generation in which no one inherits the mutation.
Affected individual: has dominant allele
Parent 1 (affected) x Parent (2) (unaffected) = 50%-100% risk/likelihood of expressing the disease phenotype on the child
Autosomal Dominant Transmission
Criteria:
1. Males and females can be affected. 2. Affected males and females can transmit the gene, unless it causes death before reproductive age.
3. The trait can skip generations. 4. Parents of an affected individual are heterozygous or have the traits
Affected individual: homozygous recessive genotypes
Carriers: heterozygotes/ asymptomatic
Autosomal Recessive Transmission
Sex-Linked Transmission
X-linked recessive transmission
X-linked dominant transmission
Common
Always expressed in males
Inherit the trait from heterozygote/homozygote mother
Females are carriers and can only be expressed if the causative allele is present in 2 copies
Inherit the trait from affected father & affected heterozygote mother
Ichthyosis, colorblindness, hemophilia
X-linked recessive transmission
Rare
Always expressed in females
Passed from male to all daughters but to no sons
Expressed also in males, with more severe effects
Rickets, Rett syndrome, incontinentia pigmenti, congenital hypertrichosis
X-linked dominant transmission
Freq. of expression of disease phenotype in individuals with a gene lesion
Penetrance
homozygous recessive
Complete penetrance
Range of phenotypes in individuals with the same gene lesion
Example: polydactyly
Variable expressivity
Detection: Molecular methods, morphological studies, clinical chemistry Final diagnosis: physiological, morphological, & laboratory results
Single-Gene Disorders
Cause: incompletely digested macromolecules due to loss of enzymatic degradation (acid hydrolases)
Defects in proteins required for normal lysosomal function physical abnormalities
Screening: gene product testing Molecular testing: genes that code for the enzymes & their subunits
Detection of mutation: direct sequencing
Lysosomal Storage Disease
Cause: Single point mutation in the coagulation factor V gene F5 (1q23) at exon 10 (1691G>A, R506Q)
Genotype: heterozygous form (4%8% of the general population) & homozygous (0.06%-0.25%)
Thrombophilia: inherited blood clotting disorder
Treatment for blood clot/deep venous thrombosis: anticoagulants
Molecular methods: PCR-RFLP, SSP-PCR
Other methods: Invader technology, clot-based methods, family history
Factor V Leiden
Precursor to thrombin in the coagulation cascade
Autosomal-dominant increased risk of thrombosis: mutation in the 3’ untranslated region of the gene that codes for prothrombin or coagulation factor II, F2 (11p11-q12)x
Laboratory tests: F2 & F5 mutations Molecular methods: multiplex PCRRFLP
Phenotypic methods: thrombin time, prothrombin time, platelet count, CBC
Automated systems: measure changes in light transmittance during clot formation generating a curve
Other: sequencing of factors XI & XIII
Prothrombin
autosomal recessive disorder caused by deficiency of the 5,10methylenetetrahydrofolate reductase (MTHFR) gene product
Hyperhomocysteinemia
Genetic alterations: 677C>T
(p.A222V) & 1298A>C (p.E429A) deficiencies in folate metabolism
Detection: standard/multiplex PCR with RFLP (HinfI & MboII) or sequencing, multiplex qPCR, HR-MCA
Methylenetetrahydrofolate Reductase
Autosomal recessive condition, overabsorption of iron from food pancreas, liver, & skin damage; heart disease; diabetes
Diagnosis: measurement of blood iron levels, transferrin saturation, liver biopsy
Molecular cause: dysfunction of the hemochromatosis type I HFE or HLAH gene product (C282Y, H63D, S65C)
Indications for mutation testing: clinical symptoms & increased serum ferritin & transferrin-iron saturation
C282Y mutation detection: PCRRFLP
Hemochromatosis
Life-threatening autosomal recessive disorder that causes severe lung damage & nutritional deficiencies
Affects cells that produces mucus, sweat, saliva, & digestive juices secretions become thick & sticky
Cause: loss of function of the CFTR gene (3-bp deletion F508del & 1,900 other mutations such as G542X, G551D, N1301K, R117H, W1282X, 1717-1G>A) (Human Genome Variation Society www.genet.sickkids.on.ca)
Molecular tests for mutation detection: RFLP, PCR-RFLP, HA, temporal-gradient gel electrophoresis, SSCP, SSP-PCR, cleavase, bead array technology, & direct sequencing
Cystic Fibrosis
Group of mono-oxygenase enzymes localized to the ER
Present in high concentrations in the liver & small intestine enzymes metabolize & detoxify compounds (drugs)
Polymorphisms affect the metabolism of hormones, caffeine, chemotherapeutic drugs, antidepressants, & oral contraceptives: Tests are used to predict the response to drugs
Detection of polymorphisms: allele -specific PCR
Screening tests: microarray, bead array, sequencing
Cytochrome P-450
Do not follow Mendelian rules of inheritance:
Mitochondrial gene mutations
Genomic imprinting
Gonadal mosaicism
Nucleotide-repeat expansion disorders
Multifactorial inheritance
Maternally inherited
mtDNA
Circular, 16,569 bp, with 37 genes, 1000-nt control region
Database of mt genes & mutations:
http://www.MITOMAP.org
Mutations affect energy production muscles & nervous system
Heteroplasmy: mutated mt & normal mt in the same cell
Molecular methods:
Large deletions: Southern blot
Point mutations: PCR-RFLP
Mitochondrial (mt) Gene Mutations
Only 1 copy of a gene in an individual (either from mother or father) is expressed, while the other copy is suppressed
Example: mules (male donkey x female horse) & hinnies (male horse x female donkey)
Cause: transcriptionally silencing through histone/DNA modification
Genetic disorders: 1 or other allele of a gene is lost (uniparental disomy)
Examples:
1. Prader-Willi syndrome: paternal del(15)(q11q13)
2. Angelman syndrome: same region, maternal
Genomic Imprinting
Cytogenetic methods:
Translocations & some deletions: standard karyotyping
Microdeletions: HR-karyotyping
FISH with labeled probes
Molecular methods:
PCR-RFLP/STR analysis
Methylation-specific PCR
Southern blot using methylation-specific restriction enzymes
Assays developed for CNV detection: FISH, array-based CGH, NGS
Generation of new mutations in germline cells giving rise to eggs/sperm carrying the mutation which then becomes a heritable phenotype
Expected when phenotypically normal parents have more than 1 affected child
Example: osteogenesis imperfecta
Gonadal Mosaicism
Nucleotide repeats, such as STRs (1-10 bp repeating units) can expand in length during DNA replication & meiosis
Triplet-repeat mutations: expansions of STR w/ 3-bp repeating units in the gene sequences
Fragile X syndrome
Huntington disease Idiopathic congenital central hypoventilation syndrome (CCHS)
Nucleotide-Repeat Expansion Disorder
CGG expansion (up to >2,000 repeats) in the noncoding region 5’ to the FMR-1 gene
Symptoms (increase in severity with each generation): learning disorders & mental retardation (IQ~20), long face, large ears, macroorchidism
Detection:
Karyotyping
PCR
Southern blot
Capillary electrophoresis
Fragile X Syndrome
CAG expansion (9-37 repeats to 3886 repeats) in the huntingtin structural gene (4p16.3)
Symptoms: impaired judgment, slurred speech, difficulty in swallowing, chorea, personality changes, depression, mood swings, unsteady gait, intoxicated appearance
Detection: standard PCR methods, capillary electrophoresis
Huntington Disease
Gene mutations in PHOX2b gene in chromosome 4: insertion of multiple alanine residues
Inadequate breathing while asleep, hypoventilation while awake
Occurs in association with an intestinal disorder (Hirschsprung disease) & symptoms of ANS
dysregulation/dysfunction
Detection: PCR w/ 32P-labeled primer & polyacrylamide gel electrophoresis
Standard PCR & agarose gel electrophoresis
Idiophatic Congenital Central Hypoventilation Syndrome (CCHS)
Disorders (& normal conditions) controlled by multiple genetic & environmental factors (nutritional/chemical exposures)
Phenotypes: conditioned by the no. of controlling genes inherited
Detection: HR-array methods NGS
Interpretation: Databases (ClinVar & dbSNP)
Prognostic & diagnostic value of gene mutation analysis: Annotation of demographics (ethnicity/gender, lifestyles)
Multifactorial Inheritance
Phenotypic methods: treatment is directed to the phenotype
Genes with variable expressivity
Gene mutation may not predict the severity of the phenotype
Clotting time & transferrin saturation
Better guides for anticoagulant treatment
Molecular testing
May discover genetic lesions in the absence of symptoms
Offer only a diagnosis, not a cure
Limitations of Molecular Testing
Lysosomal Storage Disease screening:
gene product testing
Lysosomal Storage Disease molecular testing:
genes that code for the enzymes & their subunits
Lysosomal Storage Disease Detection of mutation:
direct sequencing
inherited blood clotting disorder
Thrombophilia
Treatment for blood clot/deep venous thrombosis:
anticoagulants
Molecular methods of Factor V Leiden:
PCR-RFLP, SSP-PCR
Autosomal-dominant increased risk of thrombosis:
mutation in the 3’ untranslated region of the gene that codes for prothrombin or coagulation factor II, F2 (11p11-q12)x
Laboratory tests for prothrombin:
F2 & F5 mutations
Molecular methods of prothrombin:
multiplex PCR-RFLP
Phenotypic methods of prothrombin:
thrombin time, prothrombin time, platelet count, CBC
Automated systems of prothrombin:
measure changes in light transmittance during clot formation generating a curve
Detection of Methylenetetrahydrofolate Reductase:
standard/multiplex PCR with RFLP (HinfI & MboII) or sequencing, multiplex qPCR, HR-MCA
C282Y mutation detection:
PCR-RFLP
Molecular tests for mutation detection of cystic fibrosis:
RFLP, PCR-RFLP, HA, temporal-gradient gel electrophoresis, SSCP, SSP-PCR, cleavase, bead array technology, & direct sequencing
Detection of polymorphisms for Cytochrome P-450:
allele -specific PCR
Screening tests for Cytochrome P-450:
microarray, bead array, sequencing
Mutations affect energy production
muscles & nervous system
Mutations affect energy production
muscles & nervous system
mutated mt & normal mt in the same cell
Heteroplasmy
Molecular methods for mitochondrial gene mutations:
Large deletions:
Point mutations:
Southern blot , PCR-RFLP
Cause: transcriptionally silencing through histone/DNA modification
Genomic Imprinting
Examples:
1. Prader-Willi syndrome: paternal del(15)(q11q13)
2. Angelman syndrome: same region, maternal
Genomic Imprinting
Expected when phenotypically normal parents have more than 1 affected child
Gonadal Mosaicism
expansions of STR w/ 3-bp repeating units in the gene sequences
Triplet-repeat mutations
Symptoms (increase in severity with each generation): learning disorders & mental retardation (IQ~20), long face, large ears, macroorchidism
Fragile X Syndrome
Symptoms: impaired judgment, slurred speech, difficulty in swallowing, chorea, personality changes, depression, mood swings, unsteady gait, intoxicated appearance
Huntington Disease
Inadequate breathing while asleep, hypoventilation while awake
Occurs in association with an intestinal disorder (Hirschsprung disease) & symptoms of ANS
dysregulation/dysfunction
Idiophatic Congenital Central Hypoventilation Syndrome (CCHS)
