Clinical Genetics

CLINICAL GENETICS

The anatomy of human genome
The term "genome" refers to "gene" and "chromosome".
The Human Genome Project provided highly accurate information on a sequence of 3x109 base pairs (3000 million base or 3000 megabases) of DNA that constitute the human genome.
This sequence contains all the genes that determine development of human body.
The estimated number of genes was 30 000 which was much less than the 100 000 that was expected.
The 30 000 genes produce 30 000 different proteins.
An average gene has 1400 base pairs but only 1.5% of the genome represents primary coding sequence.
The proteins can be modified by glycolysation (i.e. the addition of sugar side chains to aminoacids) to produce 100 000s different proteins.
DNA, genes and chromosomes
DNA molecule is a double-stranded helical structure with hydrogen bonding.
Each strand is composed of a sequence of nucleotides.
Each nucleotide is composed of:
A nitrogen base; guanine (G), cytocine (C), adenenin (A) and thymine (T).
A sugar molecule called deoxyribose
A Phosphate group.
The two strands are opposed together so that the bases in each strand are bound to the bases of other strands. Each G only binds to C (G-C) and each A only binds to T (A-T).
Each pair of opposing bases in the two strands is called a base-pair (bp).
If fully opened, the length of DNA molecule of chromosomes ranges between 1-3 centimeter.
This is very long in relation to the size of the nucleus (6-8 micrometers) that is why the double-stranded DNA molecule is thrown into a complex coiling to reduce its length. This coiling is accomplished by successive twisting around long particles of a nuclear protein called histone, to form spool-like structures called nucleosome.
The chromosomes in the resting phase of the cells, that is called the interphase, are thread like structures called chromatin fibres, and are thrown in the nuclear space irregularly to form what is called the chromatin network.
At the time of cell division, the chromatin fibers replicate itself into two copies called chromatids.
The chromatids are fully separated except at the point of centromere.
The chromatids, in order to complete cell division, undergo further shortening by a process of folding to form the small short and thick chromosomes with upper limbs (p) and lower limbs (q) and a centromere.
The short chromosomes, then, arrange in a line at the equator of the nucleus to start the anaphase (i.e. the phase of separation to form two nuclei).
All human cells contain 46 chromosomes, 22 pairs of autosomes and one pair of sex chromosome (XX in the female or XY in the male).
Each member of the chromosome pair is derived from each parent.
The somatic cells contains a diploid number (i.e. double set) of chromosomes.
But the germ cell in the gonads contain haploid number (single set) of chromosomes (i.e. 23 chromosomes; 22 autosomal chromosome and one sex chromosome either X or Y).
Sex chromosomes determine the sex of the individual.
Each cell of female's body contains two X chromosomes, but one of them is inactivated (lyonised- lyonisation) and became stuck to nuclear membrane as a mass of DNA called Barr body.
The normal female number of chromosomes is symbolized as: 2n (46, XX)
The normal male's number of chromosomes is symbolized as: 2n (46, XY)
The genetic code, genes and loci
Each three successive nucleotides (triplet) in the DNA strand form one codon.
A series of codons (triplets) running from site 5' toward 3' along the strand of DNA forms the genetic code.
Protein coding regions of the DNA are referred to as genes.
Each codon in the gene specifies one amino acid, e.g ATG codon specifies methionine.
There are 64 different codons (triplets); 61 code for the twenty aminoacids and 3 codons; TAA, TAG and TGA, do not code for anything, so are called nonsense codons.
The nonsense codons terminate the growing polypeptide chain on the dorsum of ribosomes.
Some of the amino acids are specified by more than one codon.
Genes constitute very small fraction of human genome.
In the gene itself, not all the sequence codes for specific protein, the coding regions within the gene are called exons and the noncoding are introns.
Any area of the genome is referred to as locus.
To code for proteins, an RNA transcript of the gene is first made inside the nucleus by a process called transcription.
The RNA transcript is then processed by excising and removing the introns and splicing the remaining exons at their ends to form what is called messenger RNA (mRNA)
mRNA, (which is composed of spliced exons), leaves the nucleus to the cytoplasm, through pores in the nuclear membrane.
In the cytoplasm, the mRNA rests on the dorsae of rounded structures called the ribosomes.
Specific amino acids in the cytoplasm stuck to their codons in the mRNA sequentially. The ultimate result is the formation of a chain of amino acids linked to the underlying sequence of codons of the mRNA.
The chain formation stops when a non-coding codon in the mRNA comes.
When the amino acid fully forms, it is released from the m RNA to the cytoplasm.
The formed protein can be a hormone e.g. insulin, or enzyme e.g. renin, or a cell membrane protein like protein kinase C, or a coagulation factor, or a digestive enzyme, or hemoglobin, or collagen or intercellular matrix …etc.
The genotype of individual means his genetic make-up i.e. the sequence of the genes.
The phenotype means the shape, structure, development and pathophysiology of an individual.


GENETIC DISEASE
>99.9% of human genome sequence is identical in humans.
The variation mainly involves the non-coding regions that have no direct relevance to development and function.
Very little variation in millions of base-pairs of the coding sequence produces polymorphism between individuals.
If a variation in the coding sequence alters a protein to the degree that alters its function a genetic disease may results.
Genetic disease may present at any age from early development to old age.
Any system or more than one system may be affected.
TYPES OF GENETIC DISEASE
Chromosomal disorders:
Numerical abnormalities (i.e. incorrect number of chromosomes)
Tetraploidy (4n), leads to→ spontaneous abortion
Triploidy (3n)→ spontaneous abortion
Diploidy (2n from same parent) called parthenogenesis (Parthenos G, means virgin)→ can be compatible with life in chimera.
Aneuploidy of autosomal chromosome (2n + specific chromosome):
Trisomy 21 (47, XY, +21)→Down's syndrome (characteristic facies, IQ usually <50, congenital heart disease, reduced life expectancy).
Trisomy 18 (47, XY, +18)→Edwards' syndrome (characteristic skull and facies, frequent malformation of heart, kidney and other organs)
Aneuploidy of sex chromosome (2n with one added or lost sex chromosome):
47, XXY→Klinefilter's syndrome (infertility, gynecomastia and small testes)- phenotypically male.
47, XYY→ asymptomatic, often tall, may be psychopath.
47, XXX→ Trisome X (usually asymptomatic, 20% mentally handicapped)
45, X0→ Turner's syndrome (short stature, webbed neck, primary amenorrhea)
Structural abnormalities of chromosomes (due to alteration of structure of one or more chromosomes. They may be inherited or acquired).
Inherited aberration e.g. 46, XY, del(5p)→Cri du chat syndrome with deletion (del) of short arm (p) of chromosome 5.
Acquired abberation- occur in 50% of hematologic malignancies e.g.
46, XY,t(9,22) which forms an abnormal chromosome seen in WBCs of chronic myeloid leukemia called Philadelphia chromosome. It's due to translocation (t) of a segment from chromosome 9 to chromosome 22.
46,XY,t(8,22), (8,2) or (8,14)- seen in Burkitt's lymphoma


Gene disorders (Mutations of genes)
Mutation is a disease-causing DNA alteration.
A sequence change that does not cause disease is called polymorphism.
Alteration of DNA sequence in mutation causes alteration in protein product that may cause a disease.
Mutations in nuclear and mitochondrial DNA are of many types:
Point mutations: it is a change of a single nucleotide, also called substitution. It is the most common form of mutation e.g. familial polyposis coli and autosomal dominant polycystic kidney disease (ADPKD) and achndroplasia.
Insertions: insertion of one or more nucleotide.
Deletions: deletion of one or more nucleotide
Both, deletion and insertion, when occur in a gene may result in abnormal splicing of exons or alteration of the reading frame (frameshift mutation).
The insertion or deletion of a single nucleotide will alter the coding sequence next to it.
This will cause abnormal protein synthesis e.g. cystic fibrosis.
Duplication: duplication of a region of DNA due to the presence of repeated DNA sequence resulting in misalignment during DNA replication. An entire gene can be duplicated e.g hereditary motor and sensory neuropathy (HMSN type1).
Triplet (codon) repeat mutations: usually results in inherited neurologic disorders like Huntington's disease, Friedreich's ataxia, myotonic dystrophy ….etc.
Imprinted genes: the genes that manifest differently when they are inherited from the fathers or from the mothers are called imprinted genes. For example, a critical region on chromosome 15q contains several genes in which only the paternal or maternal allel is transcriptionally active. Deletion of these genes on the paternal chromosome causes Prader-willi syndrome, whereas deletion of maternal chromosome causes Angelman's syndrome. This is called genomic imprinting.
C. Polymorphism:
We all share genome sequences that are 99.9% identical. The remaining 0.1% is responsible for all the genetic diversity between individuals.
Polymorphism is a change in DNA that does not result in an overt disease.
The role of genetic factors in common diseases
Common diseases like bronchial asthma, atopy, hypertension, diabetes, ischemic heart disease and many infectious diseases show increased incidence in first degree relatives of affected individuals.
This sort of inheritance is not in the form of single gene (monogenic) inheritance.
It is usually polygenic and multifactorial.
Multifactorial means that genes work together with environmental influences giving rise to susceptibility to disease.


INHERITANCE PATTERNS
Dominant inheritance
There is an abnormal gene in one chromosome but the second copy of the gene on the homologous chromosome cannot compensate for the mutated copy.
If there is full penetrance, half of the offspring are affected, male=female.
e. g. neurofibromatosis, hypertrophic cardiomyopathy, benign familial hematuria, Marfan's syndrome and familial Alzheimer's disease

Recessive inheritance

There is an abnormal gene on one of the two chromosomes, but the second copy of the gene on the homologous chromosome compensate for the mutated copy. This is called a carrier state.
If both parents are carriers, then one quarter of their offspring are affected, and one half are carriers.
In the affected, both chromosomes (one from the father and one from the mother) carry abnormal genes.
Unaffected carrier individuals transmit disease.
Usually only one generation is affected.
e.g. α1 antitrypsin deficiency, cystic fibrosis, renal tubular acidosis and Wilson's disease.

X-linked inheritance

The abnormal gene is on the X chromosome.
In the females where there are two X chromosome, the normal one will compensate for the abnormal.
In the males, where there is one X chromosome, the abnormality on this chromosome will manifest frankly.
So only males are affected.
Females are always carriers but they transmit the disease.
Half of the carrier female's offspring inherit the abnormal gene, but only the males of them show the disease.
Affected males cannot transmit the disease to their sons, but all their daughters are carriers.
X-linked diseases are occasionally dominant i.e. the females show the disease because the second copy on the normal X cannot compensate for the abnormal.
e. g. color blindness, hemophilia A and B


Non-germ line cytoplasmic inheritance (e.g. gene on mitochondrial DNA).
Males and females are affected.
No male transmit the disease like sex linked disease.
Variable proportion of offspring from female are affected.
e. g. Leber's optic atrophy.

INVESTIGATION OF GENETIC DISEASE

Accurate history
Thorough clinical examination
Diagnostic tests:
Hematology
Biochemistry
Radiology
Histopathology
Electrophysiology in certain neurological diseases
Chromosome and DNA analysis- to confirm or exclude the diagnosis:
Polymerase chain reaction (PCR)- this test can amplify any gene sequence for analysis by gel electrophoresis or DNA sequencing. By it, it is possible to identify the gene or genes responsible for the disorder. Some diseases are easily found due to single mutation in large gene like cystic fibrosis, while other, due to multiple mutations, require more complex investigation of all the exons and adjacent sequences.
GENETIC COUNCELLING AND TESTING
This is the effective provision of information to individuals or families concerning the diagnosis of, or the risk of inheriting, a genetic disease.
It is an essential part of the management of individuals and families with genetic disease.
Collecting information is the most fundamental part of genetic counseling. This is usually achieved by constructing a pedigree or family tree.
Constructing a pedigree
Inheritance of a mutant gene can be depicted (drawn) using conventional symbol designation:
male is depicted as square
female is depicted as circle
unknown sex is depicted as a diamond
unaffected subject is depicted as white
affected subject is depicted as black
unaffected carrier is stippled
deceased (dead) is strike-through
Genetic testing
Genetic testing includes;
identification of markers of genetic diseases whether clinical, biochemical, hematological or radiological e.g. renal cysts in PCKD, raised creatine kinase in muscular dystrophy,
karyotyping e.g. trisomy 21 or Down's syndrome
direct DNA testing e.g. CAG expansion in Huntington's disease.
Genetic tests are used for many purposes mainly genetic screening and prenatal testing.


Genetic screening المسح الوراثي
Genetic screening is carried out on population or at risk groups. Examples are:
At risk groups screening for carriers of hemoglobinopathies and Tay-Sachs disease.
Newborn screening for phenylketonuria and cystic fibrosis.
Pregnant women screening (prenatal) e.g. for Down's syndrome at 15 weeks of gestation, by measuring serum alpha fetoprotein for neural tube defects (NTD) and unconjugated estradiol and human chorionic gonadotrophin (the triple test). Also screening for fetal defects by ultrasound.

Indications of prenatal testing:

Prenatal testing has a lot of ethical considerations.
Prenatal testing is indicated when:
Advanced maternal age
High risk maternal serum screening result.
A previous child with chromosomal abnormality
A child with genetic disease
A parent with chromosomal abnormality
A parent with a genetic disease

Types of prenatal testing are:

Ultrasound at 1st trimester onwards, may show increased nuchal translucency (edema at base of neck) for trisomies and for Turner's. it also shows NTDs and congenital heart disease.
Chorionic villous biopsy, from 11th week, used for early chromosomal, DNA and biochemical analysis.
Aminocentesis, from 14th weeks, for chromosomal and some biological tests such as alphafetoprotein for NTD.
Cordocentesis, from 19th week, for chromosomal and DNA analysis.