Clinical Genetics

Juan Wang
13816721634 juanwang@tongji.edu.cn

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Module Objectives

-Diagnosis Gene
diagnosis
Prenatal Diagnosis

-Treatment
Regular treatment
gene therapy

- Prevention of genetic disorders

Genetic Screening
Genetic counseling

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Question

A couple, father 39 and mother 36, come to the

clinic for genetic counsel. They have a hemophilia
son, and would like to have another child.

What will you recommend if you are the physician?

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Clinical Genetics

A cross subject of medical genetics and

genetic medicine, which focuses on medical
practice of genetics

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 Structure of chromatin and chromosomes

Changing chromosomes, as duplications, deletions or mutation

have profound consequences on normal gene expression, leading
to severe developmental and physiologic abnormalities

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Clinical Genetics
Disorders addressed by clinical genetists include all age
groups and types of conditions, including
- Rare single-gene disorders
- Birth defects
- Familial cancer
- Metabolic disorders
- Neurologic disorders
- Multisystem disorders

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 Examples of H Monogenic Diseases, Modes of Inheritance, and Associated Genes
Disease Type of Inheritance Gene Responsible

Phenylketonuria (PKU) Autosomal recessive Phenylalanine hydroxylase (PAH)

Cystic fibrosis Autosomal recessive Cystic fibrosis conductance
transmembrane regulator (CFTR)
Sickle-cell anemia Autosomal recessive Beta hemoglobin (HBB)
Albinism, oculocutaneous, type II Autosomal recessive Oculocutaneous albinism II (OCA2)

Huntington's disease Autosomal dominant Huntingtin (HTT)

Myotonic dystrophy type 1 Autosomal dominant Dystrophia myotonica-protein

kinase (DMPK)

Hypercholesterolemia, Autosomal dominant Low-density lipoprotein receptor

autosomal dominant, type B (LDLR); apolipoprotein B (APOB)

Neurofibromatosis, type 1 Autosomal dominant Neurofibromin 1 (NF1)

Polycystic kidney disease 1 and 2 Autosomal dominant Polycystic kidney disease 1 (PKD1)
and polycystic kidney disease 2
(PKD2), respectively
Hemophilia A X-linked recessive Coagulation factor VIII (F8)
Muscular dystrophy, Duchenne type X-linked recessive Dystrophin (DMD)
Hypophosphatemic rickets, X-linked X-linked dominant Phosphate-regulating endopeptidase
dominant homologue, X-linked (PHEX)
Rett's syndrome X-linked dominant Methyl-CpG-binding protein 2 (MECP2)
Spermatogenic failure, Y-linked Ubiquitin-specific peptidase 9Y, Y-linked
nonobstructive, (USP9Y)
Y-linked

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Clinical Genetics

- Diagnosis

- Treatment

- Prevention of genetic disorders

(patient counseling)

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Clinical Genetics

- Diagnosis

- Treatment

- Prevention of genetic disorders

(patient counseling)

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Diagnosis of genetic disorders

Symptomatic diagnosis

History -family history as pedigree analysis

Cytogenetic detection

Biochemical detection

Gene diagnosis

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Family history is important
➢ It can be critical in diagnosis
➢Can provide info about natural history
of the disease & variation in its
expression
➢ Can clarify pattern of inheritance
◼ Note:
Diagnosis of a hereditary condition allows risk
estimation in other family members so that
proper management, prevention, & counseling
can be offered to patient & family

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Standard pedigree symbols

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Standard pedigree symbols
Male, Male, heterozygous for
affected autosomal recessive trait

Female, Female, heterozygous for

unaffected Autosomal or X-linked
recessive trait
Male,
deceased Dizygotic
(non-identical)
twins
Mating
Monozygotic
(identical)
Consanguineous twins
mating
Spontaneous abortion
Pregnancy or still birth

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 Dominant, recessive, X-linked,
and mitochondrial (matrilinear) inheritance

Pedigree
analysis

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Cytogenetic detection of chromosome abnormalites

Indications for Cytogenetic Analysis

(Cytogenetic Testing in Prenatal Diagnosis)

- Had chromosomally abnormal child in the past

- 3 or more of past pregnancies ended in miscarriages
- She or her spouse is known to have a chromosomal abnormality
- There is a history of chromosomal abnormalities in the family
- Had male relative with severe X- link chromosomal abnormalities
- fetus is at increase risk for some hereditary error metabolism

and a neural tube defect

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FISH: fluorescence in situ hybridization

X Y 13 18 21

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FISH: fluorescence in situ hybridization

13 21

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 Biochemical detection

Various biochemical detect methods to different defects and

different types of hereditary diseases

Hemoglobinopathies and Thalassemia

Hemophila
Enzyme protein disease
Receptor protein disease

Membrane Transport apoprotein disease

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 -A2/C S F A+
Normal
Hb SS
Hb AS
Hb SC
Hb CC

Cellulose acetate Hemoglobin electrophoresis

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Gene diagnosis

Example

LHON leber hereditary optic neuropathy

One-shot mutation takes 90%

MTND1*LHON3460A
MTND4*LHON11778A 50% cases
MTND6*LHON14484C

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 SfaNⅠ MaeⅢ
normal abnormal
11778 G MTND4*LHON11778A

PCR

SfaNⅠ MaeⅢ

normal abnormal normal abnormal

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22
Sequencing result for MTND4*LHON11778A

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Gene diagnosis

Genetic tests search for mutation

- Restriction Fragment Length Polymorphism, RFLP

Endonuclease
EcoR1 5 -G↓AATTC-3 Hind III 5 -A↓AGCTT-3'
3 -CTTAA↑G-5 3 -TTCGA↑A-5

- DNA sequencing
- Polymerase Chain Reaction, PCR and related technique
- DNA Chip
- Western Blot

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 Restriction Fragment Length Polymorphism, RFLP
These are different (polymorphic) pieces of DNA (length) that are produced
by cutting the DNA with restriction enzymes and probing them with
radioactive labeled DNA. Scientists use Southern Blotting to detect RFLPs,
they now serve as genetic markers

Gene diagnosis of sickle cell anemia

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Cystic fibrosis (mucoviscidosis)

Most common lethal genetic disorder in Caucasians

Defect: mutation of the chloride channel protein, cystic fibrosis transmembrane

conductance regulator (CFTR) - deletion in amino acid position 508 (AF508)

Pathogenesis: defective chloride channel protein -thick viscous mucous

- Lungs: recurrent pulmonary infections, bronchiectasis
- Pancreas: Pancreatic insufficiency-fat malabsorption
- Male reproductive system: Male infertility
- Liver: biliary cirrhosis
- GI tract: meconium ileus

Diagnosis: Sweat test (elevated NaCl), DNA probes

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 Phenylketonuria (PKU)

Pathogenesis: Enzyme defect, deficiency of phenylalanine hydroxylase,

resulting in toxic levels of phenylalanine

Clinical findings:
- Normal at birth but develop profound mental retardation by 6 months of age
- Lack of tyrosine: light-colored skin and hair
- Have a mousy or musty odor to the sweat and urine
(secondary to metabolite phenyl-acetate accumulation)

Diagnosis: screened at birth

Treatment: dietary restriction of phenylalanine

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 Down syndrome (Trisomy 21)
Karyotype: 47XXor XY+21, Most common chromosomal disorders
Most common cause of inherited mental retardation

Pathogenesis: Meiotic nondisjunction (95%), robertsonian translocation (4%),

mosaicism due to mitotic nondisjunction during embryogenesis (1%)

Clinical findings:
- Severe mental retardation
- Apperance: flat face, low-bridged nose, epicanthal folds
Broad short neck, Palmar (simian) crease Brushfield
spots of iris
- Muscular hypotonia
- Congenital heart defects. Endocardial cushion defect, Atrioventricular canal
- GI: Duodenal atresia ("double-bubble" sign), Hirschsprung disease
- Increased risk (IS-20x) of acute lymphoblastic leukemia (ALL)
- Alzheimer disease (by age 40 virtually all will develop Alzheimer disease)

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 Cytogenetic detection
(Chromosome or Karaotype analysis)

A. An idealized human chromosome, showing the centromere (cen), long (q) and short (p) arms, and telomeres.
B. A G-banded human karyotype from a normal (46,XX) female.

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 47 XX(XY) +21

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46 XX(XY) +21 ?
46 XX(XY) t(14;21)

Robertsonian
translocation
(t)

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32
 Tay-Sachs disease

Common lysosomal storage disorder in Ashkenazi Jews(1 in 30 carrier rate)

Pathogenesis: Enzyme defect: deficiency of hexosaminidase A, leads to the

accumulation of GM2 ganglioside in the lysosomes of the CNS and retina.

Clinical findings: Normal at birth with onset of symptoms by 6 months

Progressive mental deterioration and motor incoordination
Death by age 2-3.
Retina: cherry-red spot (accentuation of the macula).
CNS:dilated neurons with cytoplasmic vacuoles.
EM: distended lysosomes with whirled membranes

Diagnosis: enzyme assays and DNA probes

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 Familial hypercholesterolemia
Most common inherited disorder (1 in 500)

Pathogenesis: low density lipoprotein (LDL) receptor gene mutation

Five major classes of mutations
- Class I: no LDL receptor synthesis
- Class II: defect in transportation out of the endoplasmic reticulum
- Class III: defect in LDL receptor binding
- Class IV: defect in ability to internalize bound LDL
- Class V: defect in the recycling of the LDL receptor

Clinical findings:
- Elevated serum cholesterol.
- Skin xanthomas and anthelasma around the eyes
- Premature atherosclerosis (homozygotes MIs in late teens and twenties)

Biochemical detection: Receptor protein

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 Type 1 Neurofibromatosis (von
Recklinghausen disease)

Accounts for 90% of cases of neurofibromatosis

Genetics: Chromosome 17 (17q 11.2)Tumor suppressor gene: NF-l.

. Normal gene product (neurofibromin) inhibits p21 ras oncoprotein

Multiple neurofibromas:
- Benign tumor of peripheral nerves often numerous and may be disfiguring.
Plexiform neurofibromas are diagnostic, rare (3%) malignant transformation.
- Pigmented skin lesions ("cafe-au-lait spots").
- Light brown macules usually located over nerves .
- Pigmented iris hamartomas.
- Increased risk of meningiomas and pheochromocytoma.

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 Huntington's disease

Genetics: triplet repeat mutation (CAG) of the Huntington gene produces an

abnormal protein (Huntington), which is neurotoxic. Atrophy of caudate nucleus

➢Unstable expansion of a CAG repeat

in males and females with full mutation or mosaicism (full
mutation+premutation).

Clinical presentation:
- Early onset (age range: 20-50) of progressive dementia.
- Choreiform movements

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Dynamic mutation

In some conditions, the phenotype seemed to

become progressively worse in later generations.

➢It is caused by a novel mutational mechanism called

dynamic mutation involving expansion of a tandem repeat
of three nucleotides (CAG, CGG or CTG).
➢Such trinucleotide is tandemly repeated in everyone to
some degree.
➢However, marked expansion of the number of repeats may
lead to disease as repeated sequences of certain length are
unstable during meiosis and likely contain a mutant gene with a
repeat longer than that of parent’s gene.

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 Disorders caused by dynamic mutation
Disorde Location Repeat Normal range Disease range
r of gene sequence repeat no repeat no
FXS Xq27.3 CCG** 6~54 pre-mutation 60~200
full mutation 200~2000
HD 4p16.3 CAG* 9~34 pre-mutation 36~121

MD 19q13.3 CTG*** 5~35 pre-mutation 50~80

full mutation
80~2000
* encode region ** 5’untranslate region ***3’UTR

FXS: Fragile X syndrome

HD: Huntington disease
MD: Myotonic dystrophy

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Dynamic mutation ( )
Unstable repeat expansion( )

Anticipation( )

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 Presymptomatic Diagnosis of delayed onset
inherited diseases such as Huntington diseases

DNA analysis
Genomic DNA from family
is digested with HindIII
and hybridized to the
linkage G8 probe

Molecular haplotype of the Huntington disease linked G8 probe

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 β-Thalassemia

Hemoglobin protein has two alpha subunits and two beta subunits. Each
alpha globin gene produces only about half the quantity of protein of a single
beta globin gene. This keeps the production of protein subunits equal.
Thalassemia occurs when a globin gene fails, and the production of globin
protein subunits is thrown out of balance.

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 Sickle cell disease
Genetics: single nucleotide change in codon causes valine (neutral) to replace
normal glutamic acid (acidic) at the sixth position of the β-globin chain.

Factors affecting formation of irreversibly sickled red blood cells

- Increased concentration (dehydration) makes symptoms worse; decreased
concentration (with thalassemia) makes symptoms better
- Decreased pH decreases oxygen affinity and makes symptoms worse
- Increased hemoglobin F makes symptoms better (rationale for therapy with
hydroxyurea, which increased blood hemoglobin F levels)
- Presence of hemoglobin C (SC: double-heterozygote individual) makes
symptoms better

Increased RBC destruction causes a severe hemolytic anemia.

Capillary thrombi result from sickle cells blocking small vessels: Vaso-occlusive
(painful) crisis, Hand-foot syndrome (swelling) in children, Autosplenectomy.

Lab tests for hemoglobin S: Hemoglobin electrophoresis.

Prenatal diagnosis: genetic testing (Mst II endonuclease)

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Hemophilia A

Genetics: Deficiency of factor VIII, X-linked recessive

Clinical features:
-Predominately affects males
- Symptoms are variable dependent on the degree of deficiency
- Spontaneous hemorrhages into joints (hemarthrosis)
- Easy bruising and hematoma formation after minor trauma
- Severe prolonged bleeding after surgery or lacerations
- No petechiae or ecchymoses

Lab test:
- Normal platelet count and bleeding time
- Normal PT and prolonged PTT

Treatment: factor VIII concentrate

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Gene diagnosis of hemophilia caused
by factor VIII partial deletion

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Prenatal Diagnosis

Genetic analysis of the cells from

- Amniocentesis: amniotic fluid amniocyte

- Chorionic villlus sampling, CVS: chorion

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Indication for Prenatal Diagnosis

- Either parent has chromosomal abnormalities

- Pregnant women above 35
- prior birth of a child with neural tube defect
- Pregnant woman with X-linked hereditary diseases
- recurrent spontaneous abbortion
- polyhydramnios
- The couple is consanguineous

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Method and Application of Prenatal Diagnosis

Non-invasive Method
- Type B ultrasound
- X rays

Invasive Method
- Amniocentesis
- Chorionic villlus sampling, CVS
- Cordocentesis
- Fetoscopy

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Diagnose chromosomal disease, hereditary metabolic
diseases, sex, NTD etc.
17-21 weeks pregnancy
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 Cordocentesis

Detect hematological diseases, congenital immune deficiency,

single gene inheritance disease, and so on.
26-30 weeks pregnancy
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 Fetoscopy

Fetal skin or liver biopsy

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Non-Invasive Prenatal Testing
(NIPT)

is a blood test used to screen

for Down syndrome and a few
other chromosomal conditions.
You can have NIPT at 10 weeks
of pregnancy or later.

vs. Amniocentesis?

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https://v.qq.com/x/page/v077762vudi.html

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Case Study

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An 8-year-old boy is brought to his pediatrician by his mother
because she was concerned that he was having language-
speech problems, was hyperactive, and was told by teachers
that he may have mental retardation. The mother reports a
strong family history of mental retardation in males. The boy on
exam is found to have a large jaw, prominent ears, and
enlarged testes (macroorchidism). The mother was told her
family had a genetic problem causing the mental retardation.

The patient underwent a series of blood tests and was

scheduled to see a genetic counselor, who expressed that the
etiology of the genetic defect was likely transmitted from his
mother. The genetic counselor states that his mother likely has
a silent mutation.
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•What is the most likely diagnosis?

•Which chromosome is likely to be affected?

•What are some types of biochemical mutations?

•What is the biochemical basis of the different
types of mutations?

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Clinical Genetics

- Diagnosis

- Treatment

- Prevention of genetic disorders

(patient counseling)

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Therapy of Hereditary Disease

Thalassemia

Thalassemia is a treatable disorder that

can be well-managed with blood
transfusions and chelation therapy. It is
important for people with thalassemia to
learn how to stay healthy.

Treatment depends on the type and severity

of thalassemia.

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 Treatment

•Blood transfusions: These can replenish hemoglobin and red

blood cell levels. Patients with thalassemia major will need
between eight and twelve transfusions a year. Those with less
severe thalassemia will need up to eight transfusions each year,
or more in times of stress, illness, or infection.
•Iron chelation: This involves removing excess iron from the
bloodstream. Sometimes blood transfusions can cause iron
overload. This can damage the heart and other organs. Patients
may be prescribed deferoxamine, a medication that is injected
under the skin, or deferasirox, taken by mouth.
•Patients who receive blood transfusions and chelation may also
need folic acid supplements. These help the red blood cells
develop.

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Regular blood transfusions may be
necessary for people with some types
of thalassemia.
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 Treatment

•Bone marrow, or stem cell, transplant: Bone marrow

cells produce red and white blood cells, hemoglobin, and
platelets. A transplant from a compatible donor may be an
effective treatment, in severe cases.
•Surgery: This may be necessary to correct bone
abnormalities.
•Gene therapy: Scientists are investigating genetic
techniques to treat thalassemia. Possibilities include
inserting a normal beta-globin gene into the patient’s bone
marrow, or using drugs to reactivate the genes that produce
fetal hemoglobin.

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Treatment for Genetic Diseases
Treatment strategies

Regular treatment
-Surgical Treatment
- Operation to correct congenital heart disease
- Liver transplant to treat a-antitrypsin defect

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Treatment for Genetic Diseases
Treatment strategies
(1) correct metabolic consequences of disease by
administration of missing product or limiting
availability of substrate
• e.g., dietary treatment of PKU
(2) replace absent enzyme or protein or to increase
its activity
• e.g., replacement therapy for hemophilia

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Treatment for Genetic Diseases
Treatment strategies
(3) remove excess of stored compound
•e.g., removal of iron by periodic bleeding in
hemochromatosis

(4) correct basic genetic abnormality

• e.g., gene therapy

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Gene Therapy

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A great discovery in the 20th century

- somatic cell therapy

- germ cell therapy

Only somatic gene therapy is permissible in

humans at present

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http://www.genetherapynet.com/clinical-trials.html

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http://www.genetherapynet.com/clinical-trials.html

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Gene Therapy

Three strategies of gene therapy

➢ Replacement - mutant gene removed and
replace with a normal gene

➢Correction - mutated area of affected gene

would be corrected and remainder left unchanged

➢Augmentation - introduction of foreign genetic

material into cell to compensate for defective product of
mutant gene (only gene therapy currently available)

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Methods of Gene Therapy

Physical method
- Microinjection
- Electroporation
- DNA Particle Bombardment
- Liposome

Biological Methods
- Retroviral/Lentiviral Vector
- Adenoviral Vector
- Antisense Nucleic Acid Method
- Plasmid-liposome complexes
- Gene editing

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http://www.genetherapynet.com/clinical-trials.html

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 Methods of Gene Therapy

(1) Adenoviruses
• replication-deficient
• does not integrate into host cell genome
disadvantage: expression of transgene
gradually declines requiring additional treatments
(may develop immune response to vector)
•treatment in vivo, vector can be introduced into upper
respiratory tract in aerosolized form

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 Methods of Gene Therapy

(2) Retroviruses
• Foreign gene integrates at random sites on
chromosomes, may interrupt (insertional
mutagenesis) the expression of host cell genes
• Replication-deficient
• Recipient cells must be actively growing for
integration into genome
• Usually performed ex vivo

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Figure. Nuclease-induced genome editing.
Nuclease-induced double strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ)
or homology-directed repair(HDR) pathways. Imprecise NHEJ mediated repair can produce mutations
at the site of the DSB. HDR-mediate repair can introduce precise point mutations or insertions from a
ss or dsDNA donor template.
From Internet.

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TALEN transcription activator-like effector nuclease)
ZFN Zinc-finger nuclease
CRISPR clustered regulatory interspaced short palindromic repeat

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Clinical Application of Gene Therapy

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http://www.genetherapynet.com/clinical-trials.html

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http://www.genetherapynet.com/clinical-trials.html

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http://www.genetherapynet.com/clinical-trials.html

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http://www.genetherapynet.com/clinical-trials.html

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 Retrovirus, ADA gene

Retrovirus, TGFb gene, Osteoarthritis

Cervical cancer
http://www.abedia.com/wiley/
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Gene
Target cell
Delivery methods

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Problems of Gene Therapy

- Stability
- Safety
- Expression Efficiency
- Immunity
- Ethics

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Problems of Gene Therapy

Stability: Transgene expression maybe lost during

replication of target cells

- Gene transcription system

- Form the wrong mRNA
- Complicate control factor of gene expression
- Short lived target cells

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Problems of Gene Therapy

Safety: especially when use viral vectors

- viral infection
- disruption of endogenous gene
- induction of cancer

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 Gene Therapy

Conclusions based on recent gene therapy trials

•Gene therapy is feasible (i.e., evidence for expression
of transgene, and transient improvements in clinical
condition in some cases.
•So far it has proved safe (only inflammatory or
immune reactions directed toward vector or some
aspect of administration method rather than
toward transgene.
• No genetic disease cured by this method.
•Major problem is efficacy, levels of transgene
product expression often low or transient.

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Clinical Genetics

- Diagnosis

- Treatment

- Prevention of genetic disorders

(patient counseling)

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Prevention of Genetic Disorders

important
Measures

- Genetic Screening

- Genetic counseling
the incidence of frequent hereditary disease

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 Genetic Screening
- prenatal: >100 genetic diseases (genetic counsel)
- chromosomal disease
- hereditary metabolic diseases, DNA diagnosed hereditary diseases
- neural tube defect (NTD) and congenital malformation

- neonatal: PKU, congenital hypothyroidism, G-6-PD deficiency

- Carrier
- clinically significant and severe diseases
- high risk population
- inexpensive test (cost effective option)
- definitive test
- available reproductive option

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Genetic counsel

A process of providing individuals and families with information

on the nature, inheritance and implications of genetic disorders
to help them make medical and personal decisions

- comprehend medical factors

- understand how heredity contribute to disorders

- choose the course of action

- make the best possible adjust to the disorder

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People need genetic counsel

- 1 or more person in the family has a genetic disease

(reproductive guidance)

- A couple has an affect baby

(estimation of recurrent risk)

- A couple infertile or recurrent miscarriage

(underlying genetic reason)

- consanguineous couple

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Steps of genetic counsel

- careful detailed but confidential documentation

- thorough physical exam and key lab testing

- estimation of the recurrent risk

- discussion of a available options

- follow-up visit and counsel to relatives

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Estimation of the recurrent risk
of chromosomal disease

1/3: high
Mother > Father

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Estimation of the recurrent risk
of single gene disorder

1/2
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Estimation of the recurrent risk
of single gene disorder

1/4

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Estimation of the recurrent risk
of single gene disorder

Female ½ carrier
Male ½ affected

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 Estimation of recurrent risk example 1:

II-5 carrier: ½ x 1/16 = 1/32

not ½
Posterior probability carrier
1/32(1/2+1/32)=1/17

Risk of counseling carrier

1/17X1/2=1/34
First child risk 1/34x1/4=1/136
son 1/34x1/2=1/68

Table: the heterozygote probability of II-5 in the hemophilia family

Probability II-5 is Normal Homozygote(HH) II-5 is Heterzygote(Hh)
Proior probability 1/2 1/2
Conditional probability 1^4=1 (1/2)^4=1/16
Joint probability (1/2)(1)=1/2=16/32 (1/2)(1/16)=1/32

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98
Estimation of the recurrent risk
of single gene disorder

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Estimation of the recurrent risk of
polygenic diseases

Empirical risk: population risk

the number of relatives in the faimly
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Virtual Simulate Experiment

http://medicine.fudan.edu.cn/genetics/yichuan/index.html
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Genetic Register

- Observe the
natural history

- assess the effects

of treatment

- List patients

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Genetic Follow-up

- short-term
understand and remember the information

- long term
update the registration
identify risk early
bring new development
provide support
monitor the effect

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 The end.

Juan Wang
juanwang@tongji.edu.cn
Dept. of Regenerative Medicine
Division of Medical Genetics
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Clinical Application of Gene Therapy

- Adenosine Deaminase ADA Deficiency

- Hemophilia B
- Cystic Fibrosis

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