Dr. Joseph de Nanassy
Associate Professor, PALM, uOttawa
Chief of Anatomical Pathology, CHEO
Site Chief of Laboratory Medicine, CHEO jdenanassy@cheo.on.ca
613-737-7600 x 2897

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Develop a basic understanding of the genetic apparatus
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Comprehend definitions of major genetic abnormalities
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Correlate molecular abnormalities and genetic defects

I. Definitions
Genetic code
Chromosomes, Genes, Cell Division
Molecular mechanisms
II. Abnormal fetal development
Malformations, deformations, dysplasias, disruptions
III. Perinatal pathology
Birth defects
Metabolic disorders

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DNA : arranged in chromosomes
(network of granules = nuclear chromatin)
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RNA : spherical intranuclear structure(s)
- nucleolus / nucleoli

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A series of messages contained in the chromosomes
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This code regulates cell functions by way of directing the synthesis of cell proteins
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The code corresponds to the structure of the DNA
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The code is transmitted to new cells during cell division

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Exist in pairs – homologous: 22a + 1s
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Composed of double coils of DNA
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Basic unit: nucleotide phosphate group deoxyribose sugar base: purine (A, G) pyrimidine (T, C)

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A locatable region of genomic sequence, corresponding to a unit of inheritance
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A union of genomic sequences encoding a coherent set of potentially overlapping functional products; i.e. genes are one long continuum (2007)
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Determine cell properties, both structure and functions unique to the cell

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Sum total of all genes contained in a cell’s chromosomes
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Identical in all cells
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Not all genes are expressed in all cells
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Not all genes are active all the time
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May code for enzymes or other functional proteins, structural proteins, regulators of other genes

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A protein or RNA specified by a gene
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Transcribed into mRNA in the nucleus
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Translated through tRNA and cytoplasmic ribosomes into protein

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

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Genetic sex = composition of X and Y
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Large X: many genes, many activities
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Small Y: almost entirely male sexual diff.
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Female: XX, male XY
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One X randomly inactivated and nonfunctional after first week of embryonic development
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Same inactivated X in descendant cells

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Stains with some fluorescent dyes
- bright fluorescent spot in the nucleus
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Normal female: sex chromatin body but no fluorescent spot
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Normal male: fluorescent spot but no sex chromatin body

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Mitosis: somatic cells (PMAT)
Daughter cells have the same number of chromosomes as the parent cell.
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Meiosis: gametogenesis (1 st and 2 nd div)
Number of chromosomes reduced by half.

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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.

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Interphase: DNA duplication to form chromatids just before mitosis
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Prophase: centriole migration, mitotic spindle
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Metaphase: chromosomes line up in centre, chromatids still joined at centromere
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Anaphase: chromosomes separate into sister chromatids
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Telophase: sister chromatids form new chromosomes, new nuclear membranes form, cytoplasm divides

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

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Metaphase 1: paired homologous chromosomes align at the equatorial plate
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Anaphase 1: homologous chromosome pairs migrate to opposite poles of the cell; each chromosome is composed of two chromatids, the chromatids are not separated
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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

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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)

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Gonads: testes, ovaries; contain
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Precursor cells or germ cells; mature into
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Gametes: sperm, ova; in gametogenesis
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Spermatogenesis, oogenesis

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

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Locus: specific site of a gene on the chromosome. Since the chromosomes exist in pairs, genes are also paired.
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Alleles: alternate forms of a gene can occupy the same locus (homozygous, heterozygous)
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Recessive gene: expressed only when homozygous
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Dominant gene: expressed whether homozygous or heterozygous, both expressed when co-dominant
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Sex-linked gene: only X-linked in males, most are recessive, hemizygous (no allele on Y)

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Genes occur in pairs on homologous chromosomes, one from each parent
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Different effects of gene whether ♀ or ♂
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Genes modified during gametogenesis
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Gene imprinting: additional methyl groups added to DNA molecules
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Basic structure unchanged; in some diseases different expression
(behaviour) depending on parent of origin: hereditary disease as a result of imprinting

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Insertion of a gene encoding a desired product (e.g. insulin) into a bacterium
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Bacterial gene spliced enzymatically, recombinant DNA inserted into plasmid
(circular DNA segment in bacterium), dividing bacterial population produces desired protein

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Normal gene inserted into defective cell
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Compensates for the missing or dysfunctional gene, in somatic cells only
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Can be inserted into mature cell (ly)
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Can be inserted into stem cell (bone marrow)
☺ Used to treat e.g. ADA deficiency, CF, …

Congenital / Hereditary Diseases
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Congenital: present at birth
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Hereditary (genetic): result of chromosome abnormality or defective gene

1. Chromosomal abnormalities
2. Gene abnormalities
3. Intrauterine injury (e.g. drugs, radiation, infection, environmental, etc)
4. Environmental effect on genetically predisposed embryo

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Nondisjunction: failure of homologous chromosomes in germ cells to separate from one another during 1 st or 2 nd meiotic division
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Sex chromosomes or autosomes
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Extra chromosome: trisomy (24 or 47)
Absent chromosome: monosomy (22 or
45)

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Chromosome Deletion : Broken piece of chromosome is lost from cell
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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

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Loss of genetic material: aborted embryo
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Deletion of gene: congenital anomalies
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Trisomy: syndromic, e.g. 21, 18, 13

1. Nondisjunction during gametogenesis
(95%)
2. Translocation (few)
3. Nondisjunction in zygote (rare)

Zygote nondisjunction T21- Mosaic

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Individual gene abnormalities
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Hereditary diseases transmitted mostly on autosomes, only a few on sex chromosomes.
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Gene mutation: spontaneous environmental
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Minor structural change may result in major functional abnormality (e.g. SCD:
HgbSA, co-dominant, Hgb beta gene)

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Autosomal dominant (a dominant gene expressed in the heterozygous state)
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Autosomal recessive (expressed only in homozygous individual, disease only if both alleles are abnormal, carrier if only one abN)
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Codominant (full expression of both alleles in heterozygous state)
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X-linked (usually affects male offspring; the abnormal X-linked gene acts as dominant gene when paired with the Y chromosome)

1. Drugs: thalidomide (phocomelia), DES
(cervical cancer), street drugs (IUFD), smoking (IUGR), alcohol (FAS), etc
2. Radiation: x-rays
3. Maternal infections:
- Rubella virus (CVS, CNS, chr. infection)
- CMV (microcephaly, chronic infection)
- Toxoplasma gondii (hydrocephalus, systemic infection)

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Combined effect of multiple genes interacting with environmental agents, e.g. cleft palate, cardiac malformations, club foot, hip dislocation, spina bifida, etc
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Cause: developmental sequence fails to reach a certain point at an appropriate time (threshold)

Genetically determined variation in 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 abnormality
3. Known translocation T21 carrier
4. 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%)

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
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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 karyotype t(1;3), t(1;15), t(1;21)

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Malformation
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Deformation
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Dysplasia
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Disruption

Prenatal development, pre-embryonic

Prenatal development, early embryonic

Prenatal development, late embryonic

Normal gametogenesis

Meiosis

Abnormal gametogenesis

Sperm penetrating oocyte

Fertilization

Causes of human congenital anomalies

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Intrinsic abnormalities of blastogenesis and organogenesis affecting the morphogenetically reactive fields of the embryo = developmental field defects
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Occur alone or in combination
(syndromes or associations)
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Severe (spina bifida aperta) or mild (spina bifida occulta)

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Causally heterogeneous
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Intrinsic causes: mendelian mutations, chromosome abnormalities, environmental interactions (multifactorial), mitochondrial mutations

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Environmental (exogenous) causes producing abnormalities of morphogenetic field dynamics
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E.g. rubella, thalidomide, isotretinoin, alcohol, etc

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Disturbances of histogenesis, occurring later and somewhat independently of morphogenesis
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Morphogenesis is prenatal, histogenesis continues postnatally in all tissues that have not undergone end differentiation
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Dysplasias may predispose to cancer

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Secondary changes in form or shape of previously normally formed organs or body parts
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Caused by extrinsic forces (e.g. Potter syndrome) or intrinsic defects (e.g. fetal akinesia syndrome with congenital arthrogryposis)

Oligohydramnios (Potter) sequence

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Secondary consequences of malformations, disruptions, dysplasias, or deformities
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E.g. renal adysplasia leads to Potter oligohydramnios sequence
DiGeorge anomaly leads to tetany, hypoparathyroidism, heart failure, conotruncal congenital heart defect

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Disturbance of phenogenesis in fetal life
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Phenogenesis: the process of attaining final quantitative anthropometric traits of the race and family (variant familial developmental pattern)
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Causes: intrinsic (chromosome imbalance) extrinsic (teratogens)

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Patterns of anomalies proven or presumed causally related
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Causes:
- chromosome mutations
- imprinting defects
- aneuploidy
- multifactorial disorders
- teratogenic sequences

Treacher-Collins syndrome
(mandibulofacial dysostosis) AD

Leprechaunism
(defective insulin binding) AR

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Idiopathic multiple congenital anomalies of blastogenesis
Vertebral anomalies
Anorectal anomalies
V
A
TracheoEsophageal defects TE
Radial and Renal defects R
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Single hit during gastrulation affecting multiple, morphogenetically closely related structural primordia

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Most are inherited as AR, some are
X-linked, a few are AD.
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Great variability in presentation
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Some present with dysmorphic features
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Storage material in RES and other tissues

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Lysosomal Lipid Storage Diseases
Nieman-Pick: sphyngomyelin
Gaucher disease: glucocerebrosidase
Tay-Sachs disease: Gangliosidoses
Metachromatic leukodystrophy
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Mucopolysaccharidoses (I, II, III, VII) glycosaminoglycans and glycolipids

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Glycogen Storage Diseases
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Galactosemia

Glycogen storage disease type II

Amino Acid Disorders

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Fatty Acid Beta-Oxidation Defects
(LCAD, MCAD, SCAD)
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Organic Acidemias
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Defects in Purine Metabolism
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Carnitine Deficiency
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Peroxisomal Disorders
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Disorders in Metal Metabolism
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Defects in Copper Metabolism

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Wigglesworth: Textbook of Fetal and
Neonatal Pathology
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Moore, Persaud: The Developing Human
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Perspectives in Pediatric Pathology,
Volume 21, Society for Pediatric Pathology
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GilbertBarness: Potter’s Atlas of Fetal and Infant Pathology
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Crowley: An Introduction to Human
Disease, Pathology and Pathophysiology