CARRIER DETECTION

NASSER A. ELHAWARY

Professor of Medical Genetics

Some Definitions Genetic locus: is a specific position or location

on a chromosome. Locus usually refers to a specific gene.

Alleles are alternative forms of a gene at a given locus.

Homozygous: a subject in which both alleles on a locus are identical.

Heterozygous: a subject in which both alleles on a locus are different.

Compound heterozygote: a subject having 2 different mutant alleles on a given locus.

Screening for genetic disease

Screening those at high risk

Carrier testing for AR & XL disorders

Pre-symptomatic diagnosis of AD

How to Detect Carriers?.... through

1. Clinical manifestations in carriers.

2. Biochemical abnormalities in carriers.

3. Linkage between a disease locus and a

polymorphic marker.

1. Clinical manifestations in carriers…

Carriers for some disorders have mild clinical manifestations.

e.g., In X-linked ocular albinism, retinal pigmentation may be seen in a manifesting female carrier.

Sometimes, either there is no sign at all in AR- or XL-carriers or overlap with general variations (like aging).

2. Biochemical abnormalities in carriers…

The biochemical abnormality seen is a direct

product of the gene, and then carrier detection

ensures.

e.g. In carriers of Tay-Sachs disease, the range in

enzyme activity ‘hexosaminidase’ is intermediate

between the normal and patient.

Sometimes, the biochemical abnormality is not a

direct result of action of the gene product, in DMD

(CPK).

Creatine phosphokinase (CPK) in obligate carrier females of DMD

• DNA polymorphic markers: e.g. STR

• Potential pitfall with linked

polymorphic DNA markers:

- Recombination

- Polymorphic variation: e.g. (CGG)n

- Locus heterogeneity: A disorder inherited in

the same manner can be due to mutations in

more than one gene (sensori-neural hearing

impairment/deafness. e.g. 1ry AR microcephaly

have 6 distinct loci.

3. Linkage between a disease locus & polymorphic marker…

Elhawary et al., 2006Clin Biochem

STR44STR45STR48STR50

A pedigree of X-linked DMD showing STRs Elhawary et al., 2006Clin Biochem

STR44STR45STR48STR50

Presymptomatic diagnosis of AD

Due to either delayed age of onset or

reduced penetrance, the following may

help to diagnose the AD disorders:

Clinical examination

Specialist investigation

Biochemical testing

Linkage DNA markers

Examples of Autosomal dominant

Fam. Hypercholestrolemia (1 in 500): High serum

levels of total cholesterol and LDL-cholesterol. Polycyctic kideny disease (1 in 1,250): Large

number of fluid filled cysts form within the kidneys and cause renal failure.

Marfan syndrome (1 in 4,000): Abnormal patient's eyes, cardiovascular system, musculoskeletal system (Spider-like fingers.

Huntington disease (1 in 15,000): neurodegenerative mutations in (CAG)n

Maternal serum screening Neural tube defects (NTDs, 1/1000) (↑ in AFP).

Down syndrome & Chromosomal abnormalities

‘Triple test’ at 16 wks:1. Maternal serum AFP (↓ in DS pregnant)

2. unconjugated estriol (μE3) (↓ in DS pregnant).

3. hCG (human chorionic gonadotropin) (↑).

Ultrasonography

NTDs (open, closed)

Neural tube defects (NTDs) are one of the most common

birth defects.

An NTD is an opening in the spinal cord or brain that occurs

very early in human development.

The 3rd or 4th wk of pregnancy, specialized cells on the dorsal

side of the fetus begin to fuse and form the neural tube (NT).

When the NT does not close completely, an NTD develops.

- ttt by Folic acid & vit B12

The hormone hCG is produced during pregnancy. It is made by cells that form the placenta, which

nourishes the Egg after it has been fertilized and becomes attached to the uterine wall.

hCG Levels can first be detected by a blood test about 11 days after conception and about 12-14 days after conception by a urine test. In general, the hCG levels will double every 72 h.

The level will reach its peak in the first 8-11 wk of pregnancy and then will decline and level off for the remainder of the pregnancy.

hCG (human chorionic gonadotropin)

μE3 (unconjugated estriol)

Estriol is one of the three main estrogens produced by the human body (estrone E1 ‘menopause’, estradiol E2’during reproductive’, estriol E3 ‘during pregnancy’).

Estriol can be measured in maternal blood or urine and can be used as a marker of fetal health.

If levels of "unconjugated estriol" are abnormally low in a pregnant woman, this may indicate chromosomal or congenital anomalies like Down syndrome (47,+21) or Edward's syndrome (XX,47,+18).

It is included as part of the triple test & quadruple test for antenatal screening for fetal anomalies.

Because many pathological conditions in a pregnant woman can cause deviations in estriol levels, these screenings are often seen as less definitive of fetal-placental health. Conditions which can create false positives and false negatives in estriol testing for fetal distress include preeclampsia, anemia and impaired kidney function.

SummaryTriple screen of maternal serum

AFP hCG uE3 Associated conditions

Low High Low Down Syndrome

Low Low Low Trisomy 18 (Edward’s)

High n/a n/a NTDs, e.g. spina bifida

- Triple test measures with a 70% sensitivity and means high risk of chrom. abnormalities and NTDs. Direct testing of fetal DNA in maternal blood simplify current screening programs.

- Quad test (81% sensitivity by adding inhibin A ‘inhibits FSH secretion’

Quad screen of maternal serum:

Quantification at gestational age 16-18 wks of

Inhibin A (increased) Beta-hCG (increased) AFP (decreased) Unconjugated estriol (uE3) (decreased) suggest of a fetus with DS. Also, inhibin A is used as a marker of

ovarian cancer.

Preimplantation genetic diagnosis (PGD)

Prenatal diagnosis is so difficult on an established pregnancy to view a possible termination. So,

The female is given hormones to induce hyper-ovulation.

Oocytes are harvested transcervically, under sedation, with US guidance.

Motile sperm from a semen sample are added to the oocytes in culture (in-vitro fertilization, IVF).

Incubation to allow fertilization

Preimplantation genetic diagnosis…

At the early embryo (blastocyst) at the eight-cell stage on the 3rd day, fertilization is achieved using intracytoplasmic sperm injection (ICSI).

At the 8-cell stage, the early embryo is biopsied and one or 2 cells are removed for analysis. The IVF is strictly regulated by Human Fertilization & Embryology Authority (HFEA).

PGD is used in CF, DMD, HD, β-thal, SMA, FraX & chromosomal abnormalities by FISH is performed.

Genetic Counseling

Dr. NASSER A. ELHAWARY

Professor of Medical Genetics

Genetic Counseling

The counselor should ensure that the consultand understands that:

1. The medical diagnosis, prognosis, and treatment.

2. Mode of inheritance of the disorder and Risk of developing and transmitting it.

3. Choices or options available for dealing with the risk.

Steps in genetic counseling…

Accurate Diagnosis based on..

1) Take personal-family-medical history

(pedigree, checklist),

2) Carry out an accurate examination,

3) Undertake appropriate investigations

(include chromosome, molecular studies

or referral to neurologist… etc).

Problems arise with firm diagnosis…- If the disorder shows etiological heterogeneity

(e.g., hearing loss & non-specific mental retardation both have genetic and environ-mental factors).

- The GC can be extremely difficult when the heterogeneity extends to different modes of inheritance.

- e.g., Ichthyosis.. AD, AR, XR

- e.g., Ehlers-Danlos syndrome.. AD, AR, XL

Steps in genetic counseling…

Calculating & representing the risk Discussing the options. After the last 2 steps,

(e.g. prenatal diagnosis should be discussed with details of techniques, limitations, risks associated with various

applied methods). Communication and support adoption (between

the counselor and the consultand due to psychlogical and emotional factors in infertile cases, AD)

Special problems in GC

1- Consanguinity: Kuwait (54%), KSA (54%), Pakistan (40-50%), Egypt (28%), Algeria (23%), Japan (2-4%), UK or USA (2%).

2- Incest (relationships occur betn. 1st degree relatives;

3- Adoption 4- Disputed paternity

Steps in genetic counseling…

Treatment of Genetic Diseases

Treatment of Genetic

Diseases

Conventional Approaches

TherapeuticApplications

of RecombinantDNA tech.

GeneTherapy

Conventional Approaches treatment…

1- Protein/enzyme Replacement

If a genetic disorder is found to be the result of

a deficiency or an abnormality of an enzyme or

a protein, ttt may involve replacement of the

deficient enzyme or protein (e.g. use of factor

VIII concentrate in ttt of hemophilia).

2- Drug Treatment

Statins can help to lower the cholesterol levels in

FHC.

Avoiding some drugs or foods to prevent

manifestations of some genetic diseases

(Sulfonamides & fava beans in G6PD).

Gentamicin (aminoglycoside antibiotics) was used

as nasal drops to treat patients with CF.

Conventional Approaches treatment…

3- Tissue Transplantation

- Replacement of diseased tissue has been

replaced.

e.g. Renal transplantation in adult poly-

cyctic kidney (PCK) disease.

or Lung transplantation in CF.

Conventional Approaches treatment…

Therapeutic Applications of Recombinant DNA tech...

Biosynthesis of Gene products- Insulin extracted from pig pancreas.

- Using recDNA technology, insulin can be obtained from human insulin gene by introducing cDNA of insulin into vectors.

- Other examples: HGH (dwarfism), Factor VIII (hemophilia A), Factor IX (hemophilia B), b-interferon (multiple sclerosis).

GENE THERAPY

Gene therapy: defined by GTAC as the deliberate introduction of genetic materials into human somatic cells for therapeutics, prophylactic or diagnostic purposes.

Regulatory requirements:

- All programs are focusing only on somatic

cell gene therapy but not on germline gene

therapy.

Gene Therapy…

Technical aspects: we have to address:

- Gene characterization

- Target cells, tissue & organ: e.g. b-thal-assemia ttt involves removing bone marrow from affected individuals, treating it in vitro, and then returning it to the patient by transfusion.

- Vector system

- Vector system: The vector by which the foreign gene is introduced, need to be both efficient and safe.

- The treated tissue or cells has a reasonable lifespan, and the body doesn’t react adversely to the gene product (produce antibodies product).

- Some vectors resulted in a malignancy or mutagenic effect on either the somatic cell or germ-cell lines through insertion of the gene or DNA sequence in the host DNA.

Gene Therapy…

Animal models: These should be present to assist

the suitability of gene therapy trials in humans (DMD,

FA*, CF, HD).

- In-utero fetal gene therapy: Risk to use adeno-

virus vector is overcome by using in-utero stem-cell

transplantation (Krabbe disease or Hurler syndrome).

Target organs: liver, CNS, muscle, bone marrow.

Gene Therapy…

*Friedreich’s ataxia (1860): Genetic disease (AR) due to progressive damage of nervous system resulting in symptoms ranging from gait disturbance to speech problems

Enrichment info…

Krabbe disease (globoid cell leukodystrophy) is a

rare AR (1/100,000 births)

Often fatal degenerative disorder that affects the

myelin sheath of the nervous system.

Mutation by GALC gene causes a deficiency of an

enzyme called galactocerebrosidase

Hurler syndrome, also known as

muco-polysaccharidosis type I (MPS I).

It is an AR that results in the buildup of MPS due to a

deficiency of α-L-iduronidase, an enzyme responsible for

the degradation of MPS in lysosomes.

Without this enzyme, a buildup of heparan sulfate and

dermatan sulfate occurs.

Symptoms appear during childhood and early death can

occur due to organ damage.

Enrichment info…

Gene Transfer: can be carried out either ex-vivo (ttt of cells or tissue from an affected individual in culture, and re-introduce into the affected one; or

- in-vivo (if cells can’t be cultured or replaced in the affected individual).

- Viral agents…

- Non-viral agents…

Gene Therapy…

In-vivo and ex-vivo gene therapy

1- Viral agents: used to transport foreign genetic

materials into cells.

- Oncoretrovirus: RNA viruses integrate into the host DNA

by making copy of their RNA molecule using RTase.

If these can be integrated to dividing stem cells, all progeny

cells will inherit a copy of the viral genome. It Introduces a

relatively small <7kb DNA sequences.

- Lentivirus: It includes HIV, they are complex viruses that

infect macrophages & lymphocytes, integrated to non-

dividing cells, hence useful in neurological conditions.

Viral agents, Gene Therapy…

- Adenovirus: used as vectors in gene therapy to infect a wide variety of cell types.

- Unlike retroviruses, they can infect non-dividing cells & carry up to 36 kb of foreign DNA.

- They don’t integrate into the host genome, thus avoiding possible insertional mutagenesis.

- Its potential effect in malignancy.- Expression of the introduced gene is unstable

and often transient.

Viral agents, Gene Therapy…

- Adeno-associated virus: are non-pathogenic

viruses in humans that require a co-infection with

helper adenoviruses or certain Herpes virus to

infect.

* In absence of helper, they integrates to chrom

DNA (chrom 19q13.3-qter).

* Carry and insert up to 5 kb into the cell.

Viral agents, Gene Therapy…

- Herpes-virus: They are neurotropic (infect

nervous tissue) and may target gene therapy

in Parkinson disease.

* Toxic effects on nerve cells, hence immune

response

* Don’t integrate to host genome and thus

expression of the gene product is temporarily

and unstable.

Viral agents, Gene Therapy…

2- Non-viral methods:

It is harmful to immune system, safer, simple to use, enable large scale production, but efficacy is limited.

- Naked DNA: via injection (mini-dystrophin). - Liposome-mediated DNA transfer:

Liposomes can facilitate introduction of foreign DNA, but not efficient in gene transfer.

- Receptor-mediated endocytosis:

Glycoprotein containing galactose will be recognized by receptors on the surface of liver cells.

Non-viral methods, Gene Therapy…

Diagrammatic representation of liposome-mediated gene therapy

Gene Therapy…

RNA modification targets mRNA either by

suppressing mRNA levels or by correcting/

adding function to the mRNA.

- Antisense oligonucleotides: The sequence-

specific binding of an antisense oligo-

nucleotide (18-30 mer) to a target mRNA to

inhibit the expression of protein.

RNA interference: Any gene may be a potential silencing by RNA interference.

- In contrast to antisense oligo (inhibited mRNA), mRNA is cleaved into 1000-fold more active.

- Hence, homologous sequences to synthetic double-stranded RNA molecules known as small interfering RNAs (siRNAs).

- siRNAs can be delivered in drug form to stabilize antisense oligonucleotides.

Gene Therapy…