Genetic Diseases

Dr. Joseph de NanassyAssociate Professor, PALM, uOttawaChief of Anatomical Pathology, CHEO

Site Chief of Laboratory Medicine, CHEO

jdenanassy@cheo.on.ca613-737-7600 x 2897

Objectives

☺ Develop a basic understanding of the genetic apparatus

☺ Comprehend definitions of major genetic abnormalities

☺ Correlate molecular abnormalities and genetic defects

Outline

I. DefinitionsGenetic codeChromosomes, Genes, Cell DivisionMolecular mechanisms

II. Abnormal fetal developmentMalformations, deformations, dysplasias, disruptions

III. Perinatal pathologyBirth defectsMetabolic disorders

The Cell

Nucleus

☺ DNA: arranged in chromosomes

(network of granules = nuclear chromatin)

☺ RNA: spherical intranuclear structure(s)

- nucleolus / nucleoli

Genetic Code

☺ A series of messages contained in the chromosomes

☺ This code regulates cell functions by way of directing the synthesis of cell proteins

☺ The code corresponds to the structure of the DNA

☺ The code is transmitted to new cells during cell division

DNA structure

DNA replication

mRNA and tRNA

Chromosomes

☺ Exist in pairs – homologous: 22a + 1s

☺ Composed of double coils of DNA

☺ Basic unit: nucleotide

phosphate group

deoxyribose sugar

base: purine (A, G)

pyrimidine (T, C)

Genes

☺ A locatable region of genomic sequence, corresponding to a unit of inheritance

☺ A union of genomic sequences encoding a coherent set of potentially overlapping functional products; i.e. genes are one long continuum (2007)

☺ Determine cell properties, both structure and functions unique to the cell

Genome

☺ Sum total of all genes contained in a cell’s chromosomes

☺ Identical in all cells

☺ Not all genes are expressed in all cells

☺ Not all genes are active all the time

☺ May code for enzymes or other functional proteins, structural proteins, regulators of other genes

Gene Product

☺ A protein or RNA specified by a gene

☺ Transcribed into mRNA in the nucleus

☺ Translated through tRNA and cytoplasmic ribosomes into protein

Human Genome

☺ 3 billion+ pairs of DNA nucleotides☺ ~ 50,000 – 100,000 genes☺ Protein-coding Genes = <10% (2%) of human genome☺ Exons: parts of the DNA chain that code for specific

proteins☺ Introns: the parts in-between the exons☺ Both exons and introns are transcribed but only the

exons are translated (introns are removed from mRNA before leaving nucleus)

☺”Junk DNA”: no obvious function but 80% expressed

Sex chromosomes

☺ Genetic sex = composition of X and Y

☺ Large X: many genes, many activities

☺ Small Y: almost entirely male sexual diff.

☺ Female: XX, male XY

☺ One X randomly inactivated and nonfunctional after first week of embryonic development

☺ Same inactivated X in descendant cells

(Mary) Lyon Law

(Murray) Barr body

Y chromosome

☺ Stains with some fluorescent dyes

- bright fluorescent spot in the nucleus

☺ Normal female: sex chromatin body

but no fluorescent spot

☺ Normal male: fluorescent spot

but no sex chromatin body

Cell Division

☺ Mitosis: somatic cells (PMAT)

Daughter cells have the same number of chromosomes as the parent cell.

☺ Meiosis: gametogenesis (1st and 2nd div)

Number of chromosomes reduced by half.

Chromatids

☺ Before mitosis, the DNA chains duplicate to form new chromosome material.

The duplicated chromosome material lies side by side = two sister chromatids.

Mitosis = the process by which conjoined chromatids separate into sister chromatids and move into new daughter cells.

Mitosis

☺ Interphase: DNA duplication to form chromatids just before mitosis

☺ Prophase: centriole migration, mitotic spindle☺ Metaphase: chromosomes line up in centre,

chromatids still joined at centromere☺ Anaphase: chromosomes separate into sister

chromatids☺ Telophase: sister chromatids form new

chromosomes, new nuclear membranes form, cytoplasm divides

Mitosis

Meiosis

☺ First meiotic division interphase: duplication of chromosomes to form paired chromatids

☺ Prophase 1 of meiosis: homologous chromosomes lie side by side over entire length = synapse.

Interchange of segments of homologous chromosomes = crossover.

2 Xs side by side just like the autosomes.

X and Y end-to-end: no crossover.

Meiosis

☺ Metaphase 1: paired homologous chromosomes align at the equatorial plate

☺ Anaphase 1: homologous chromosome pairs migrate to opposite poles of the cell;

each chromosome is composed of two chromatids, the chromatids are not separated

☺ Telophase 1: two new daughter cells form;

each contains half the chromosome number = reduction of chromosomes by half; interchange of genetic material occurred during synapse

Meiosis

☺ Second meiotic division = mitotic division

Prophase 2: DNA does not replicate

Metaphase 2: chromosomes align at the equatorial plate

Anaphase 2: sister chromatids migrate separately

Telophase 2: four haploid cells (half the normal number of chromosomes)

Meiosis

Gametogenesis

☺ Gonads: testes, ovaries; contain

☺ Precursor cells or germ cells; mature into

☺ Gametes: sperm, ova; in gametogenesis

☺ Spermatogenesis, oogenesis

Gametogenesis

Primary follicles

Oogenesis vs. spermatogenesis

☺ One ovum (+ 3 polar bodies) vs. four spermatozoa

☺ Oocytes formed before birth vs. continuous spermatogenesis (‘fresh’ sperm)

Prolonged Prophase 1 until ovulation –

more frequent congenital abnormalities in ova of older women (longer exposure to potentially harmful environmental influences until meiotic division resumes at ovulation)

Chromosome Analysis

Karyotype

Genes and Inheritance

☺ Locus: specific site of a gene on the chromosome. Since the chromosomes exist in pairs, genes are also paired.

☺ Alleles: alternate forms of a gene can occupy the same locus (homozygous, heterozygous)

☺ Recessive gene: expressed only when homozygous

☺ Dominant gene: expressed whether homozygous or heterozygous, both expressed when co-dominant

☺ Sex-linked gene: only X-linked in males, most are recessive, hemizygous (no allele on Y)

Gene Imprinting

☺ Genes occur in pairs on homologous chromosomes, one from each parent

☺ Different effects of gene whether ♀ or ♂☺ Genes modified during gametogenesis☺ Gene imprinting: additional methyl

groups added to DNA molecules☺ Basic structure unchanged; in some diseases different expression

(behaviour) depending on parent of origin: hereditary disease as a result of imprinting

Genetic Engineering

☺ Insertion of a gene encoding a desired product (e.g. insulin) into a bacterium

☺ Bacterial gene spliced enzymatically, recombinant DNA inserted into plasmid (circular DNA segment in bacterium), dividing bacterial population produces desired protein

Gene Therapy

☺ Normal gene inserted into defective cell

☺ Compensates for the missing or dysfunctional gene, in somatic cells only

☺ Can be inserted into mature cell (ly)

☺ Can be inserted into stem cell (bone marrow)

☺ Used to treat e.g. ADA deficiency, CF, …

Congenital / Hereditary Diseases

☺ Congenital: present at birth

☺ Hereditary (genetic): result of chromosome abnormality or

defective gene

Causes of malformations

1. Chromosomal abnormalities

2. Gene abnormalities

3. Intrauterine injury (e.g. drugs, radiation, infection, environmental, etc)

4. Environmental effect on genetically predisposed embryo

Chromosomal abnormalities

☺ Nondisjunction: failure of homologous chromosomes in germ cells to separate from one another during 1st or 2nd meiotic division

☺ Sex chromosomes or autosomes

☺ Extra chromosome: trisomy (24 or 47)

Absent chromosome: monosomy (22 or 45)

Nondisjunction in meiosis

☺ Chromosome Deletion: Broken piece of chromosome is lost from cell

☺ Translocation: Not lost, just misplaced and attached to another chromosome- reciprocal: between two nonhomologous chromosomes (no loss or gain of genetic material - no loss of cell function)- in germ cells: deficient or excess chromosome material – abnormal zygote

Translocation in gametes

Sex chromosome abnormalities

Turner syndrome

Klinefelter syndrome

Autosomal abnormalities

☺ Loss of genetic material: aborted embryo

☺ Deletion of gene: congenital anomalies

☺ Trisomy: syndromic, e.g. 21, 18, 13

Trisomy 21 (Down)

T21 causes

1. Nondisjunction during gametogenesis (95%)

2. Translocation (few)

3. Nondisjunction in zygote (rare)

Translocation T21

Zygote nondisjunction T21- Mosaic

Abnormal gene diseases

☺ Individual gene abnormalities☺ Hereditary diseases transmitted mostly

on autosomes, only a few on sex chromosomes.

☺ Gene mutation: spontaneous environmental

☺ Minor structural change may result in major functional abnormality (e.g. SCD: HgbSA, co-dominant, Hgb beta gene)

Modes of Inheritance

☺ Autosomal dominant (a dominant gene expressed in the heterozygous state)

☺ Autosomal recessive (expressed only in homozygous individual, disease only if both alleles are abnormal, carrier if only one abN)

☺ Codominant (full expression of both alleles in heterozygous state)

☺ X-linked (usually affects male offspring; the abnormal X-linked gene acts as dominant gene when paired with the Y chromosome)

Intrauterine Injury

1. Drugs: thalidomide (phocomelia), DES (cervical cancer), street drugs (IUFD), smoking (IUGR), alcohol (FAS), etc

2. Radiation: x-rays3. Maternal infections:

- Rubella virus (CVS, CNS, chr. infection)- CMV (microcephaly, chronic infection)- Toxoplasma gondii (hydrocephalus,

systemic infection)

Thalidomide baby

Prenatal CMV infection

Multifactorial Inheritance

☺ Combined effect of multiple genes interacting with environmental agents,

e.g. cleft palate, cardiac malformations, club foot, hip dislocation, spina bifida, etc

☺ Cause: developmental sequence fails to reach a certain point at an appropriate time (threshold)

Genetically determined variationin rate of development

Effect of harmful environmental agents on susceptibility for

congenital malformations

Interaction of genetic predisposition and environmental factors

in cleft palate

Prenatal Diagnosis of Congenital Abnormalities

1. Examination of fetal cells for chromosomal, genetic or biochemical abnormalities

2. Examination of amniotic fluid for products secreted by the fetus

3. Ultrasound of the fetus to detect malformations (NTD, hydrocephalus, PCKD, etc)

Prenatal Diagnosis of Congenital Abnormalities

Main indications for amniocentesis

1. Maternal age (>35)2. Previous infant with T21 or other

chromosomal abnormality3. Known translocation T21 carrier4. Other chromosomal abnormality in either

parent, e.g. t(7;21)5. Risk of genetic disease in the fetus that

can be detected prenatally (thalassemia)6. Previous infant born with neural tube

defect (multifactorial inheritance, ~5%)

Methods of fetal DNA analysis

1. Enzyme analysis of DNA: resultant

DNA fragments different in health and disease, e.g. sickle cell anemia

2. DNA probes: same complementary nucleotide arrangement as in defective DNA gene – binds to mutant gene

Molecular Genetics of Solid Pediatric Tumors

☺ Mechanisms for tumor development

1. Creation of novel fusion proteins

2. Loss of tumor suppressor genes

3. Activation of proto-oncogenes

Translocations, Oncogenes, Tumor suppressor genes

NB: MYCN amplification and 1p deletion by FISH

NB: Double-minute chromosomes by FISH

RB: MYCN probe to detect homogeneously staining region in

metaphase spread and interphase nuclei

Ewing sarcoma: t(11;22) EWS green, FLI-1 pink, t yellow

E-RMS: Spectral karyotypet(1;3), t(1;15), t(1;21)

Abnormal Fetal Development

☺ Malformation

☺ Deformation

☺ Dysplasia

☺ Disruption

Prenatal development, pre-embryonic

Prenatal development, early embryonic

Prenatal development, late embryonic

Fetal development

Normal gametogenesis

Meiosis

Abnormal gametogenesis

♂ & ♀ gametes

Sperm penetrating oocyte

Fertilization

Causes of human congenital anomalies

Malformations

☺ Intrinsic abnormalities of blastogenesis and organogenesis affecting the morphogenetically reactive fields of the embryo = developmental field defects

☺ Occur alone or in combination (syndromes or associations)

☺ Severe (spina bifida aperta) or

mild (spina bifida occulta)

Malformations

☺ Causally heterogeneous

☺ Intrinsic causes: mendelian mutations, chromosome abnormalities,

environmental interactions (multifactorial), mitochondrial mutations

Disruptions

☺ Environmental (exogenous) causes producing abnormalities of morphogenetic field dynamics

☺ E.g. rubella, thalidomide, isotretinoin, alcohol, etc

Rubella embryopathy

Diabetic embryopathy

Dysplasias

☺ Disturbances of histogenesis, occurring later and somewhat independently of morphogenesis

☺ Morphogenesis is prenatal,

histogenesis continues postnatally in all tissues that have not undergone

end differentiation

☺ Dysplasias may predispose to cancer

Neurofibromatosis

Tuberous sclerosis

Deformities

☺ Secondary changes in form or shape of previously normally formed organs or body parts

☺ Caused by extrinsic forces (e.g. Potter syndrome) or intrinsic defects (e.g.

fetal akinesia syndrome with congenital arthrogryposis)

Oligohydramnios (Potter) sequence

Arthrogryposis

Sequences

☺ Secondary consequences of malformations, disruptions, dysplasias, or deformities

☺ E.g. renal adysplasia leads to Potter oligohydramnios sequence

DiGeorge anomaly leads to tetany, hypoparathyroidism, heart failure, conotruncal congenital heart defect

Minor Anomalies

☺ Disturbance of phenogenesis in fetal life

☺ Phenogenesis: the process of attaining final quantitative anthropometric traits of the race and family (variant familial developmental pattern)

☺ Causes:

intrinsic (chromosome imbalance)

extrinsic (teratogens)

Syndromes

☺ Patterns of anomalies proven or presumed causally related

☺ Causes: - chromosome mutations - imprinting defects- aneuploidy- multifactorial disorders - teratogenic sequences

Treacher-Collins syndrome(mandibulofacial dysostosis) AD

Leprechaunism(defective insulin binding) AR

Associations

☺ Idiopathic multiple congenital anomalies of blastogenesis

Vertebral anomalies VAnorectal anomalies ATracheoEsophageal defects TERadial and Renal defects R

☺ Single hit during gastrulation affecting multiple, morphogenetically closely related structural primordia

Metabolic Disorders

☺ Most are inherited as AR, some are

X-linked, a few are AD.

☺ Great variability in presentation

☺ Some present with dysmorphic features

☺ Storage material in RES and other tissues

Storage Diseases

☺ Lysosomal Lipid Storage Diseases

Nieman-Pick: sphyngomyelin

Gaucher disease: glucocerebrosidase

Tay-Sachs disease: Gangliosidoses

Metachromatic leukodystrophy

☺ Mucopolysaccharidoses (I, II, III, VII)

glycosaminoglycans and glycolipids

Hurler syndrome (MPS 1A) AR

COH Disorders

☺ Glycogen Storage Diseases

☺ Galactosemia

Glycogen storage disease type II

Amino Acid Disorders

Misc.

☺ Fatty Acid Beta-Oxidation Defects (LCAD, MCAD, SCAD)

☺ Organic Acidemias☺ Defects in Purine Metabolism☺ Carnitine Deficiency☺ Peroxisomal Disorders☺ Disorders in Metal Metabolism☺ Defects in Copper Metabolism

References

☺ Wigglesworth: Textbook of Fetal and Neonatal Pathology

☺ Moore, Persaud: The Developing Human☺ Perspectives in Pediatric Pathology,

Volume 21, Society for Pediatric Pathology☺ Gilbert-Barness: Potter’s Atlas of Fetal

and Infant Pathology☺ Crowley: An Introduction to Human

Disease, Pathology and Pathophysiology

Thank you