Chapter 13. Gene Diagnosis

To identify the cause of a disease or condition based on gene analysis or test.

Methods of diagnosing a genetic disease:

1) Direct diagnosis: detect disease-causing mutation

2) Indirect diagnosis: tracking the disease gene by linkage analysis

1. Direct diagnosis

Premise: for the known gene mutations 1.1 Point mutation (without size change)

Restriction site altered by mutation PCR-RFLP

No restriction site altered by mutation Allele specific oligonucleotide (ASO )

1.2 Insertion or deletion (with size change)large fragments – by Southernsmall fragments – by PCR

1.3 DNA Sequencing

1.1.1 Detect point mutation by PCR-RFLP

sickle cell anemia：又称Hb S病

1949 Pauling HbS

ＳＡ1956 Ingram :β-globin codon 6: Glu→Val

CCT GAG GAG … (Hb bA)

… CCT GTG GAG … (Hb bS)

Hb bA

Hb bS

MstII MstII MstII

Glu

Val

… CCT GAG GAG … (Hb bA)… CCT GTG GAG … (Hb bS)

CCT NAG G (MstII)

1.15 kb

1.35 kb

Southern blotting

Hb bA

Hb bS

MstII MstII MstII

Glu

Val

1.15 kb

1.35 kb

100 150

PCR product: 250bp

PCR-RFLP

PCR-RFLP Steps:

Amplify region of β-globin around codon 6

Cut with MstII

Run products on gel

Genotype analysis

Sickle Cell

Disease

(aa)

Heterozygote

(Aa)Normal

(AA)

250

150

100

DNA Diagnosis of HbS

250150100

AA

DNA Diagnosis of HbS

Aa

?

aa

Aa Aa

1.1.2 Detect point mutation by ASO

normal allele probe: 5′TCCATTAACAGTAAGTAATTT3′mutant allele probe: 5′TCCATTAACAATAAGTAATTTT3′

S1 S2 S3 S4 S5 S6

S1 S2 S3 S4 S5 S6

Probe a

Probe A

Aa Aa aa aaAA AA

1. Direct diagnosis

Premise: for the known gene mutations 1.1 Point mutation (without size change)

Restriction site altered by mutation PCR-RFLP

No restriction site altered by mutation Allele specific oligonucleotide (ASO )

1.2 Insertion or deletion (with size change)large fragments – by Southernsmall fragments – by PCR

1.2.1 Detect size change by Southern blot

a-thalassanemia (a-globin chains are deficient)

Hemoglobinopathies

Structurally abnormal hemoglobin chains

Thalassemias

Abnormal production of normal chains

Hemoglobinopathies

Qualitative structural abnormalities of globin polypeptide chains

Hemoglobin variants due to: single base pair substitution Stop codon muatation Framshift mutation Codon insertion or deletion Fusion gene

Hemoglobin Variants: Alpha Chain

Hemoglobin Variants: Beta Chain

Thalassemias

Decreased chain production

Decreased mRNA production

Beta thalassemia - decreased beta chains

Alpha thalassemia - decreased alpha chains

• Normal /

• Silent carrier - /

• Minor -/-

--/

• Hb H disease --/-

• Barts hydrops fetalis --/--

Alpha Thalassemia

Beta Thalassemia

Decreased Beta chain production

B+ - decreased synthesis

Bo - absent synthesis

Homozygous condition provides a wide range of disease and heterozygous condition can range from mild to moderately severe

1.2.1 Detect size change by Southern blot

5’ 3’16pz yz y 2 1

5’ 3’16pz yz y 2 1

a-thalassanemia (a-globin chains are deficient)

a2 a1

hydrops fetalis (fetal or early neonatal death)

Hb H disease (mild to severe anemia)

a1-thalassemia (no significant anemia)

a2-thalassemia (silent carrier state)

normal

Alpha Thalassemia

aA/a+

aA/a0 or

a+/a+

a+/a0

a0/a0

10kb

14kbnormal allele

mutant allele

BamHⅠ BamHⅠ

Genomic DNA Bam HI DNA fragmentselectrophoresis

Size fraction transfer DNA fragments on membrane Hybridizing

14kb

10kb

AA Aa aa

1 2

1 2

Ⅰ

Ⅱ

Results of Southern blot：

Ⅱ1 Ⅰ1 Ⅰ2 Ⅱ2

10kb

14kb

normal

Duchenne Muscular Dystrophy

17 18 19 44 45 46 47 48 49

17

19

44

45

48

C P1 P2 P3 P4Patient1: exon 44 deleted

Patient2: exon 17deleted

Patient4: exon 19 deleted

Patient3: exon 45 deleted

1.2.2 Detect size change by PCR

1 2

1 2

Ⅰ

Ⅱ

A DMD family

normal

2. Indirect diagnosis

Demonstration of a mutation in known gene

Demonstration of a disease allele of unknown genes by using tightly linked markers to the disease gene

2.1 detect a mutation in a known gene

Hemophilia A

1 1/2

2 1/1 2 1/2 2 1

XAXa

XaY XaY

XAY

XAY XAYXAXA XAXa

Ⅰ

Ⅱ1

2

2

1

1

3 4 5 6

Ⅲ 2/2

1/2

1

2

XAY

XaY

XAXA

XAXa

Disease gene

Marker 1 Marker 2

2/5

P

Ⅰ

Ⅱ1

2

2

1

1

3 4 5 6

Ⅲ

2/4 3/5

2/3 4/5 2/5 3/4 4/52/5

2

2/4

P

3/5

N

3/4

N

2/2 P

2/5 ？

5/5 N

2.2 detect a mutation in an unknown gene (mapped disease gene)

Aa aa

Aa Aa aa aa

Indirect Gene Diagnosis

M1 1 23 2 2 4 4 12 2

XAY XAXa

XaY

XaY XAY XaY

? ?

Disease gene

Marker 1

Recombination ?

M1 1 23 12 2

XAY XAXa XaY XaY

? ?

Indirect Gene Diagnosis

Premise

Family data

Tightly linked polymorphic marker

Heterozygote

Location known

Exercise

Ⅰ

Ⅱ

Ⅲ1

1

1 2

2

2 3

3 4

4

5 6

3/5 2/6

5/6 2/5 2/5 2/3 1/61/4

1/5 1/6 2/5 1/2

Aa aa

aa Aa Aa aa aa aa

aaaaAaAa aa

Ⅰ

Ⅱ

Ⅲ1

1 2

1

2

2 3

2.5/2.5 2.5/4

2.5/4 2.5/4 4/4

2.5/4 4/4

Aa

aa

Aa

Aa AA

AA Aa