Advances in Molecular Biology Involving Recombinant DNA Technology

GENETIC DISORDERS (Dr. Dotollo)

Advances in Molecular Biology involving Recombinant DNA Technology

molecular basis of human disease

- to study a variety of inborn errors of metabolism

-insertion of molecularly cloned human DNA into the germ line of mice

production of human biologically active agent

- by inserting the requisite gene into bacteria or other suitable cells in tissue culture

- e.g. tissue plasminogen activator (tPA)

- for treatment of thrombotic states

growth hormone

- treatment of deficiency states

erythropoietin

- to reverse the anemia of renal disease

myeloid growth & differentiation factors (GM-CSF, G-CSF)

- to enhance production of monocytes & neutrophils in states of poor marrow function

gene therapy

-by transfer of somatic cells transfected with the normal gene

disease diagnosis

- molecular probes are proven to be useful in the diagnosis of both genetic & non-genetic disease

classification of human diseases (cause)

environmentally determined (microbiologic infections which are conditioned by the genotype)
genetically determined
both genetic & environmental factors are involved (nature-nurture interplay)

terminologies:

hereditary disorders (familial)

- are derived from one's parents & are transmitted in the germ line thru generations

congenital disorders ("born with")

- some congenital diseases are not genetic (congenital syphilis)

- not all genetic disease are congenital (Huntington's disease)

MUTATIONS

- a permanent change in the DNA

in germ cells - are transmitted to progeny & may give rise to inherited diseases
in somatic cells - do not cause hereditary disease (but important in genesis of

cancers & some congenital malformation)

categories of mutation (based on genetic change)

genome mutations

- involve loss or gain of whole chromosomes (monosomy/trisomy)

chromosome mutations

- result from rearrangement of genetic material & give rise to visible structural changes in the chromosome

gene mutations

- result in partial or complete deletion of a gene or more often affect a single base

e.g.point mutation

- a single nucleotide base may be substituted by a different base

missense mutation
nonsense mutation

frameshift mutation

- one or 2 base pairs may be inserted or deleted from the DNA

leading to alteration of the reading frame of DNA strand

predispositions to mutation:

radiation

chemicals inc. the rate of spontaneous mutation because of their role in carcinogenesis

viruses

Major Categories of Genetic Disorders

those related to mutant genes of large effect

- includes many relatively uncommon conditions all resulting from single-gene mutations of large effect (e.g. storage disease)

- referred to as Mendelian disorders

single gene disorders with non-classic inheritance

- includes disorders resulting from triplet repeat mutations, those arising from

mutations in mitochondrial DNA & those in which the transmission is influenced

by genomic imprinting

disease with multifactorial (polygenic inheritance)

- includes some of the most common diseases of humans (hypertension & DM)

- influenced by both genetic & environmental factors

chromosomal disorders

- includes diseases that result from genomic or chromosomal & are associated

with numerical & structural changes in chromosomes

MENDELIAN or SINGLE GENE DISORDERS

result of expressed mutations in single genes of large effect transmitted by autosomal recessive, or X-linked modes of inheritance
some autosomal mutations produce partial expression in the heterozygote & full expression in
the homozygote
80-85% are familial & the remainder represent new mutations acquired de novo by an affected
individual

codominance - both alleles of a gene pair may be fully expressed in the heterozygote

pleiotropism - single mutant gene may lead to many end effects

Transmission Patterns of Single-Gene Disorders

autosomal dominant disorders

are manifested in the heterozygous state

both males & females are affected, & both can transmit the condition

2 non-enzyme proteins which are affected:

those involved in regulation of complex metabolic pathways

(membrane receptors & transport proteins)

key structural proteins such as collagen & cytoskeletal component of RBC

( specrin)

characteristic conditions:

with every autosomal dominant disorder, some patients do not have affected
parents ( due to new mutations involving the egg & the sperm)
"reduced penetrance"

- some inherit the genes mutant gene but are phenotypically normal

"variable expressivity"

- if a trait is seen in all individuals carrying the mutant gene but is

expressed differently among individuals

in many conditions, the age at onset is delayed:

- symptoms & signs do not appear until adulthood (Huntington's disease)

autosomal recessive

result only when both alleles at a given gene locus are mutants

include almost all inborn errors of metabolism

features:

the trait does not usually affect the parents, but siblings may show the disease
siblings have one chance in four of being affected
if a mutant gene occurs with a low frequency in the population, there is a strong likelihood that the proband is the product of a consanguineous marriage

autosomal recessive vs. autosomal dominant

The expression of the defect tend to be more uniform than in autosomal dominant disorders.
Complete penetrance is common
Onset is frequently early in life.
Although new mutations for recessive disorders do occur, they are rarely detected clinically.
In many cases, enzyme proteins are affected by the mutation.

X-linked disorders

are X-linked, almost all X-linked recessive

X-linked recessive inheritance

hemizygous males
An affected male does not transmit the disease to his sons, but all daughters are carriers.
Son of heterozygous women have one chance in two of receiving the mutant gene.

 X-linked dominant disorders

transmitted by an affected heterozygous female to half her sons & half her daughters, & by an affected male parent to all his daughters but none of his sons, if the female parent is unaffected.

Biochemical & Molecular Basis of Single-Gene Disorders (mechanisms)

enzyme defects & their consequences
accumulation of substrate

- depends on: site of block & accumulation of one of both intermediates

- tissue injury may result if if the precursor, the intermediates, or the products of the alternative minor pathways are toxic in high concentrations

e.g.  phenylalanine hydroxylase - accumulation of phenylalanine

 lysozymes - lysosomal storage diseases

presence of metabolic block & decrease amount of end product

- if the end product is a feedback inhibitor of the enzymes involved in the early reactions, the deficiency of the end product may permit overproduction of intermediates & their catabolic products, some of which may be injurious at high concentrations.

e.g. block in tyrosinase   melanin  albinism

failure to inactivate a tissue damaging substrate

e.g.alpha1-antitrypsin deficiency - unable to inactivate neutrophil elastase

in their lungs

defect in membrane receptors & transport systems
defect in initial binding to specific receptor site

e.g.familial hypercholesterolemia

defect in "carrier" of transport system

e.g.cystic fibrosis (transport of Cl in lungs, sweat glands & pancreas)

defect in protein that regulates cell growth

e.g. neurofibromatosis

alterations in structure, function & quantity of non-enzyme proteins

structure - e.g. hemoglobinopathies (defect in structure of globin molecule)

Marfan's/Ehlers-Danlos (defect in collagen synthesis)

quantity - e.g. thalassemia ( amounts of  or  globin chains)

unusual reaction to drugs

e.g. G6PD deficiency - in normal conditions - no disease

- on administration - of primaquine  severe hemolytic anemia

SINGLE-GENE DISORDERS with NON-CLASSIC INHERITANCE

diseases caused by triplet-repeat mutations

fragile X syndrome

- is the prototype of diseases in which the mutation is characterized by a long repeating sequence of 3 nucleotides

- one of the most common causes of familial mental retardation

- a fragile site because it is particularly liable to chromatid breaks when cells are cultured in folate-deficient media

Huntington disease - a neurodegenerative disorder & myotonic dystrophy

disorders caused by mutations in mitochondrial genes

Leber hereditary optic neuropathy

- a neurodegenerative disease that manifests itself as progressive bilateral loss

of central vision

disorders associated with genomic imprinting

Prader-Willi Syndrome

- characterized by mental retardation, short stature, hypotonia, obesity, small

hands & feet, & hypogonadism

- the deletion affects the paternally derived chromosome 15

Angelman Syndromes

- deletion of chromosome 15 from their mothers

- are also mentally retarded, with ataxic gait, seizures & inappropriate laughter

"genomic imprinting"

- an epigenetic process where there are functional differences between the paternal & maternal gene

MULTIFACTORIAL DISORDERS

result from the combined actions of environmental influences & 2 or more mutanbt genes having additive effects

"the greater the inherited deleterious genes, the more severe the expression of the disease"

environmental influences significantly modify the risk of expressing the disease

CHROMOSOMAL MUTATON (cytogenetic)

- abnormal number of chromosomes or alterations in the structure of 1 or more chromosomes

a. cytogenetic disorders involving autosomes

trisomy 21(Down's Syndrome)

most common

1:1000

90% have trisomy 21 so their chromosome is 47

maternal age - 1:1550 under 20 years

- 1: 25 over 45 years of age

diagnostic clinical features:

1)Flat facial profile

2)Oblique palpebral fissures

3)Epicanthic folds - "mongolism" and "mongolian idiocy"

4)Gentle, shy manner, hypotonia

5)Easily directed

40% die by age 10 (cardiac problems)

30% - congenital heart disease (ventricular septal defects)

Increase frequency of acute leukemia (myoblastic)

leukemoid reactions ( transient marked elevation in WBC count)

Male - always infertile or lack of sexual potency

trisomy 18(Edward's Syndrome)

1:5000 - 10000

Mental retardation is severe

95% cardiac anomalies (ventricular septal defect)

10% mosaicism

trisomy 13(Patau's Syndrome)

Most severe of all chromosomal abnormalities

Microcephaly and mental retardation are marked

Arrhinencephaly (congenital absence of those regions of forebrain derived from olfactory system)

Virtual absence of eyes

Cyclopia (single eyes with the nose absent)

Cri du Chat Syndrome

deletion of the short arm of chromosome 5 (5p-)

affected infants up to the age of one year have the characteristic cry of a cat

severe mental retardation, microcephaly & round faces

thrive better than those with trisomies & some survive in adult life

b. cytogenetic disorders involving sex chromosomes

In general, they induce subtle, chronic problems relating to sexual development & fertility.
They are often difficult to diagnose at birth, & many are first recognized at the time of puberty.
In general, the higher the number of X chromosomes, in both male & female, the greater the likelihood of mental retardation.

monosomy:

(45,X) Turner's Syndrome

results from complete or partial monosomy of the X chromosome & is characterized primarily by hypogonadism in phenotypic females

gonadal dysgenesis

45, X, karyotype

clinical manifestations vary with age

3% survive to birth

edema - (due to lymph stasis) of dorsum hand and foot
swelling of the nape of the neck

- cystio-hygroma

congenital heart disease ( preductal coarctation of the aorta)
aortic stenosis with endocardial fibroelastosis
failure to develop normally secondary sexual characteristics

genitalia is infantile

breast development inadequate

little pubic hair

normal mental status
shortness of stature - rarely exceeding 150 cm in height

amenorrhea - important in establishing diagnosis in adult

trisomies:

(47,XXY) Klinefelter Syndrome

male hypogonadism that occurs when there are 2 or more X chromosomes & one or more Y chromosomes

Testicular dysgenesis

Most common cause of hypogonadism in male

1:850 live male births

Rarely diagnosed before puberty since testicular abnormalities does not develop before early puberty

characteristics: (key word SALES)

Small, atrophic testes
Abnormally long legs
Lack of secondary sex characteristics
Eunochoid bodily habitus
Small penis

confirmatory lab. Finding (key word PAIL)

Positive X chromatin
Absence of/or striking reduction of sperm in seminal fluid
Increased urinary excretion of FSH
Lower than normal testosterone levels

2 Important Clinical Significances:

1)Important cause of sterility in male

2)Often associated with decreased intelligence

testicular lobules are totally atrophied and are replaced by pink, hyaline,

collagenous ghosts

47, XXY karyotype (+ sex chromatin test)

advance maternal age and irradiation

(47,XYY) XYY Syndrome

phenotypic males, no symptoms

excessively tall, severe acne, normal intelligence

Supernumerary Y's, maybe found in male

47, XYY or even greater Y polysomy

dx - more than one chromatin body in interphase nuclei

- chromosome analysis

phenotypically normal

excessively tall

aggressive behavior

susceptibility to acne

increased frequency among inmates of penal institution

antisocial (not violent)

delinquent

impulsive acting - out disorders

(47,XXX) Multi-X Females

female with no apparent symptoms

the greatest number of X, the greater tendency to mental retardation

1:1200 newborn females

diagnosis is by demonstration of 2 or more X chromatin bodies most women are entirely small

49, XXXXX karyotype - (+) mental retardation

47, XXX - unaffected

karyotype

- composed of:

22 homologous pairs of autosomes 46 chromosomes

2 sex chromosomes (XX & XY)

- are described using a shorthand system of notation

total number of chromosomes
sex chromosome complement
description of any abnormality

- e.g. a male with trisomy 21

47,XY,+21

euploid - any exact multiple of the haploid number

aneuploidy

- if an error occurs in mitosis or meiosis & a cell acquires a chromosome complement that is

not exact multiple of 23

- usual causes are nondisjunction (when a homologous pair of chromosomes fail to disjoin at the 1st meiotic division) & anaphase lag (2 chromatids fail to separate resulting in 2 aneuploid cells)

- one homologous chromosome in meiosis or one chromatid in mitosis lags & is left out in nucleus

monosomy

- involving an autosome generally represents loss of too much genetic information to permit live birth or even embryogenesis

trisomy

- some do permit survival

- with the exception of trisomy 21, all yield severely handicapped infants who almost die invariably at an early age

mosaicism

- a condition which refers to mitotic errors in early development which give rise to 2 or more populations of cells in the same individual

- result from mitotic errors during cleavage of fertilized ovum or in somatic cells

types of chromosomal rearrangements

deletion

- refers to loss of a portion of chromosome

a. terminal - result from a single break from the arm of a chromosome, producing a fragment with no centromere, which is then lost in the next cell division

b. interstitial - when there are 2 breaks in the chromosome followed by loss of the of the regions between the breaks

translocation

- a segment of one chromosome is transferred to another

balanced reciprocal translocation

- there are single breaks in each of two chromosomes, with exchange of material

- no loss of genetic material

- phenotypically normal

- increased risk of producing abnormal gametes

Robertsonian translocation (centric fusion)

- a reciprocal translocation between 2 acrocentric chromosomes

- one very large chromosome & one extremely small

inversion

- refers to a rearrangement that involves two breaks within a single chromosome with reincorporation of the inverted segment

paracentric - involving only one arm of the chromosome
pericentric - if the breaks are on opposite sides of the centromere

- perfectly compatible with normal development

isochromosome formation

- results when arm of a chromosome is lost & the remaining arm is duplicated, resulting in a chromosome consisting of 2 short arms only or of 2 long arms

ring chromosome

- a special form of deletion which is produced when a deletion occurs at both ends of a chromosome with fusion of the damaged ends

SEXUAL AMBIGUITIES

genetic sex

determined by the presence or absence of a Y chromosome

no matter how many X chromosomes are present, a single Y chromosome dictates testicular development & the genetic male gender

gonadal sex

is based on the histologic characteristics of the gonads

ductal sex

depends on the presence of the derivatives of the mullerian or wolffian ducts

phenotypic of genital sex

is based on the appearance of the external genitalia

HERMAPHRODISM & PSEUDOHERMAPHRODITISM

"true hermaphrodite"

- implies both the presence of both ovarian & testicular tissue

"pseudohermaphrodite"

- represents a disagreement between the phenotypic & gonadal sex

e.g. female pseudohermaphrodite

- has ovaries but male external genitalia

male pseudohermaphrodite

- has testicular tissue but female-type genitalia

"true hermaphroditism"

- implying the presence of both ovarian & testicular tissues

testes on one side & ovary on the other side
combined ovarian &testicular tissue (ovotestes)

- are heterogenous group, having in common the presence of 2 X chromosomes as well as a

complete or partial Y chromosome

female pseudohermaphroditism

- the genetic sex in all cases is XX, & the development of the gonads & internal genitalia is

normal

- only the external genitalia are ambiguous or virilized

- exposure to androgenic steroids during the early part of gestation

male pseudohermaphroditism

- represents the most complex of all disorders

- possess a Y chromosome, & their gonads are exclusively testes, but the genital ducts or the external genitalia are incompletely differentiated along the male phenotype

- the external genitalia are either ambiguous or completely female