GEN E S TR UCTU RE, EXPRESSI ON AN D MUTATI ON

SIGNIFICANCE
• GENETIC ISSUES ARISE BEFORE, DURING AND AFTER PREGNANCY • POSITIVE FAMILY HISTORY OF DISEASES • AMNIOCENTESIS OR CHORIONIC VILLUS SAMPLING • WORK-UP FOR INFERTILITY • DISORDERS OF SEXUAL DEVELOPMENT • GYNECOLOGIC MALIGNANCIES

Chromosome analysis can be performed on

• Peripheral blood – for analysis of fetal, neonatal, juvenile, or adult blood • Bone marrow - for malignant studies • Tissue – for malignant studies

Chromosome analysis can be performed on

• Fetal tissue – for identification of reasons for fetal demise and for tissue culture
• Amniotic Fluid • Chorionic Villi • Umbilical Cord Blood

Chromosome analysis is indicated in the following cases

Individuals with • suspected classic chromosomal syndrome • multiple congenital anomalies • dysmorphic features • failure to thrive • ambiguous genitalia • abnormalities of sexual development • amenorrhea • short stature • mental retardation of undetermined etiology • developmental delay

Couples with

• two or more miscarriages • Infertility • Advance age

Family members

• both parents of a child with structural chromosome rearrangement, deletion, or duplication • at risk of having a chromosome rearrangement

Pregnancy

• abortuses • stillbirth

Sample Collection

Peripheral Blood

Sterile sodium or lithium heparinized tube (green top vacutainer) or sterile tube with 0.05ml preservative free heparin. Ensure that sample is no clotted

Specimen Culture

Blood:
Children/adults - 8 drops of whole blood/ 5 ml culture media Infants - 4-5 drops/ 5 ml culture media

Bone marrow:
4 –5 drops into 8 ml T/C flask of media 2 flasks Per sample

Specimen Incubation

Incubate (37oC)
Blood Bone marrow = 72 Hours = overnight

Harvest
Cell cycle

• • • •

Metaphase arrest Hypotonic swelling Lysis by acetic acid Fixation

Metaphase Arrest

Metaphase arrest (Colchicine/ Colcemid)

Increasing Cell Volume by KCl
Hypotonic shock
The hypotonic solutions:
.075M KCL dilute balance salt solution, sodium citrate, 20% diluted serum in H2O

Lysis by Acetic Acid

Fixation
Fixation Methanol & Glacial acetic acid (3:1)

Slide Preparation

Slides

Chromosome Banding
C-banding stains centromeres. R-banding is the reverse of C-banding and stains noncentromeric regions in preference to centromeres. G-banding is obtained with Giemsa stain. It yields a series of lightly and darkly stained bands (right). Q-banding is a fluorescent pattern obtained using quinacrine for staining. The pattern of bands is very similar to that seen in G-banding. T-banding produce specific staining of the telometric regions of the chromosomes

GTG-banding

Chromosomes are G-banded to facilitate the identification of structural abnormalities. Slides are dehydrated, treated with the enzyme trypsin, and then stained.

Screening and Analysis

Chromosomes are described with the following categories:

Metacentric: centromere is median or near median chromosome has two well defined arms with a length ratio varying from 1:1 to 2.5:1 Acrocentric: centromere is close to one end of the chromosome one arm is substantially smaller than the other and the arm
ratio ranges from 3:1 to 10:1

Telocentric: centromere is a strictly terminal entity and the chromosome is one armed

Capturing and Analysis

Normal Male Karyotype

Normal Female Karyotype

DNA
• A molecule made up of linear sequence of nucleotides intertwined as a double helix. • Within cells, DNA is organized into structures called chromosomes. • -The main role of DNA molecules is the longterm storage of information

• What are Chromosomes? Chromosomes are tiny string-like structures in cells of the body. They contain the estimated 30,000 to 35,000 human gene pairs that determine traits like eye and hair color, as well as direct the growth and development of every part of our physical and biochemical systems.

• Each person normally has 23 pairs of chromosomes, or 46 in all. We normally inherit one chromosome per pair from our mother and one from our father.

Backbone:
• - Phosphate – Pentose sugar (deoxyribose) – Nitrogen bases

Bases in a DNA molecule:
• Purines
Adenine and Guanine

• Pyrimidines
Thymine & Cytosine

Gen e
• Biologic unit of heredity • Located at a definite position (locus) on a particular chromosome. • consist of a long strand of DNA that contains a promoter, coding and non-coding sequence. • promoter- controls the activity of a gene, • . Coding sequence - determines what the gene produces, • non-coding sequence - regulate the conditions of gene expression.

Gene • When a gene is active, the coding and non-coding sequence is copied in a process called transcription, producing an RNA copy of the gene's information

• is a molecule of RNA encoding a chemical "blueprint" for a protein product.mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes. • In mRNA as in DNA, genetic information is encoded in the sequence of four nucleotides arranged into codons of three bases each. Each codon encodes for a specific amino acid, except the stop codons that terminate protein synthesis. This process requires two other types of RNA: • Transfer RNA (tRNA) mediates recognition of the codon and provides the corresponding amino acid, • while Ribosomal RNA (rRNA) is the central component of the ribosome's protein manufacturing machinery.

Messenger Ribonucl ei c Ac id (mRN A)

Tr anscri ptio n
Transcription is the synthesis of mRNA from a DNA template. During transcription, RNA polymerase makes a copy of a gene from the DNA to mRNA as needed.

Ste ps i n tra nsc rip tion • DNA unwinds. • RNA polymerase recognizes a specific base sequence in the DNA called a promoter and binds to it. The promoter identifies the start of a gene, which strand is to be copied, and the direction that it is to be copied. • Complementary bases are assembled (U instead of T). • A termination code in the DNA indicates where transcription will stop. • The mRNA produced is called a mRNA transcript.

Pr ocessin g th e mRNA Tr anscrip t • the newly-formed mRNA transcript (also called heterogenous nuclear RNA or hnRNA) must be further modified before it can be used. • A cap is added to the 5’ end and a poly-A tail (150 to 200 Adenines) is added to the 3’end of the molecule. • The newly-formed mRNA has regions that do not contain a genetic message. These regions are called introns and must be removed. Their function is unknown. • The remaining portions of mRNA are called exons. They are spliced together to form a mature mRNA transcript.

Processing the mRNA Transcript

Translatio n • Translation is the process where ribosomes synthesize proteins using the mature mRNA transcript produced during transcription. • During translation, the mRNA transported to the cytoplasm is "de-coded" or "translated" to produce the correct order of amino acids in a protein. • tRNA = transfer RNA; small RNA molecules that carry a specific amino acid at one end and an anticodon region that recognizes and binds mRNA at the other end. The tRNA that binds to that mRNA codon determines what amino acid is added to a protein chain.

The steps of transl ati on:
• 1. Initiation: mRNA enters the cytoplasm and becomes associated with ribosomes (rRNA + proteins). • tRNAs, each carrying a specific amino acid, pair up with the mRNA codons inside the ribosomes. Base pairing (A-U, G-C) between mRNA codons and tRNA anticodons determines the order of amino acids in a protein. • 2. Elongation: addition of amino acids one-by-one: As the ribosome moves along the mRNA, the tRNA transfers its amino acid to the growing protein chain, producing the protein codon by codon! 3. Termination: when the ribosomes hits a stop codon - UAA, UGA, or UAG - the ribosome falls apart! The same mRNA may be used hundreds of times during translation by many ribosomes before it is degraded (broken down) by the cell.

Steps in Translocation

Mutation
Gene Mutation• Occurs as a result of environmental damage to DNA, through errors during DNA replication or repair. • Reserved for new changes in the genetic code that lead to altered function and clinical consequences. Point Mutations- results in amino acid substitution, leading to different products with altered functions. E.g. sickle cell anemia

Mol ecul ar Tool s and Di agnosi s in Human Geneti cs

1. PCR 2. RE/ RFLP

Mol ecul ar Tool s and Di agnosi s in Human Geneti cs

Polymerase Chain Reaction- a common method of creating copies of specific fragments of DNA. PCR rapidly amplifies a single DNA molecule into many billions of molecules.

Rest ric tio n En do nuc lea ses an d R estric tion F ragm en t Le ngt h Po lymo rph isms

• are enzymes that recognize and cut specific nucleotide sequences in the double stranded DNA molecule • the sites of their action are known as restriction sites • The restriction fragment length polymorphisms can be used to follow the transmission of a gene in a family.

Mi cro arra y DN A An alysi s • Permits the expression and analysis of thousands of genes simultaneously. • It was used to understand the molecular basis of cancer and the biological behavior of tumors. • This powerful tool can provide a molecular fingerprint of an individuals disease and is referred to as gene expression profiling(GEP)

Genetic Testing: Direct and In dir ect Me thods

Direct Method • Direct testing for the actual mutation in an affected individual to confirm the clinical diagnosis or prenatal diagnosis would be possible by obtaining DNA from the subject.

Indi rect Method • when direct testing is not possible • - DNA markers located to the presumptive disease-causing gene/mutation are used as road maps to identify the travel or passage of the gene from an affected parent to an at-risk offspring.

Indi rect Method • This requires that the affected individual has markers that are informative, in other words, unique or distinctive from markers of the non affected individual. • Multiple family members, both affected and unaffected must have DNA available for analysis in order for this approach to be informative. • The markers are often RFLPS.

Mol ecul ar Cyt oge net ics
• powerful tool to analyze chromosome abnormalities that are not visible using traditional karyotyping and microscopy. • most widely used procedure is fluorescent in situ hybridization(FISH) • FISH takes advantage of the complementary nature of DNA • Denatured DNA sequences labeled with a fluorescent dye are hybridized onto denatured chromosomes that have been immobilized onto a slide. The chromosomes are then viewed with a wavelength of light that excites the fluorescent dye.

Mol ecul ar Cytogenetics
• FISH is commonly used to screen for chromosome anueploidy in amniotic fluid cells in prenatal diagnosis • Powerful tool to confirm or diagnose syndromes that are due to microdeletions of segments of chromosomal material • Used to identify the actual physical location of a gene or to order a serias of DNA sequences or genes on a chromosome

Comparative Genome Hybridization- used to measure differences in copy number or dosage of a particular chromosome segment. • its widest application is in the study of a gene dosage in normal and cancer cell lines. Spectral Karyotyping- uses the FISH principle to visualize all 24 chromosomes by “painting” with chromosome-specific probes in different colors simultaneously.

Patterns of Inheritance
Pedigree = graphic representation of family history data that assists in mining the transmission pattern of the gene = the pattern of transmission and the constellation of the characteristics of the affected individuals in the pedigree confirms the diagnosis

As a review….
• A recessive trait is one that is "obscured" by dominant traits, but is expressed when two recessive genes are present. When symbolizing recessive traits, lower-case letters are used (aa). • A dominant trait is one that is expressed even in the presence of other genes for the same trait. When symbolizing dominant traits, a capital letter is used (AA or Aa).

AUTOSOMAL DOMINANT

• Only one of the mutated gene is required for expression of the trait • Individual is said to be heterozygous for the trait

General Characteristics
– Every affected individual has an affected parent. – If reproductively fit, the affected person has a 50% chance in transmitting the gene with each pregnancy. – The sexes are affected equally. – There is a father to son transmission. – An individual who does not carry the mutation will have no risk of transmission to his or her offspring.

Three additional properties • variable expressivity= severity of phenotype in individuals who have the mutation • penetrance= probability that a gene will have any clinical manifestation at all in a person known to have the mutation • new mutations

AUTOSOMAL RECESSIVE
– rare; require the affected individual to have two copies of the mutant allele (homozygous) in order to manifest the condition

GENERAL CHARACTERISTICS
• The characteristic will occur equally in both sexes. • for an offspring to be at risk, both parents must have at least one copy of the mutation • If both parents are heterozygous (carrier) for the condition, 25% of the offspring will be homozygous for the mutation and manifest the condition, 50% will be carriers and unaffected. The remaining 25% will not have inherited the mutation at all, will be unaffected, and will not be at risk of transmitting the mutation to any offspring. • consanguinity is often present in families demonstrating rare autosomal recessive conditions. • If the disease is relatively rare, it will be clustered among the siblings, and will not be seen among other family members such as ancestors, cousins, aunts and uncles.

X-LINKED TRAIT
chromosome – Most are recessive – Expression of genes located on the x chromosome demonstrate a unique characteristic known as DOSAGE COMPENSATION – Achievement of dosage compensation is through the principles of X inactivation, also known as the LYON HYPOTHESIS

-diseases caused by genes on the x

LYON HYPOTHESIS
• One x chromosome in each cell is randomly inactivated in the early female embryo (soon after fertilization). • The inactivation process is random; either the paternally or maternally derived x chromosome is chosen. The female is thus a mosaic for genes located on the x chromosome. • All descendants of the cell will have the same inactive X chromosome.

X-LINKED DOMINANT INHERITANCE – All heterozygotes, both male and female, manifest the condition – Affected males never have affected sons, and all daughters of affected males are affected.

Atypical Patterns of Inheritance

• TRINUCLEOTIDE-REPEAT DISORDERS: UNSTABLE MUTATIONS

• A new class of genetic conditions in which the gene mutation was dynamic and would change with different affected individuals within a family • Most common group of disorders • Unstable in that they tend to expand as the gene is passed on from generation to generation---progressively earlier onset or more severe manifestations of disease with each successive generation---- anticipation (generally characteristic of genetic conditions caused by unstable repeats) • Molecular mechanism: misalignment at the time of meiosis

• FRAGILE X SYNDROME • Most common heritable form of moderate mental retardation • Gene is located on X chromosome Xq27.3 • FMR1 (fragile X mental retardation 1) --- triplet expansion blocks normal function of the FMR1 gene • Individuals with an intermediate number of copies (52-200)= PREMUTATION CARRIERS= unaffected

• GENOMIC IMPRINTING AND UNIPARENTAL DISOTOMY

• Differential activation or expression of genes depending on the parent of origin • A group of diseases in which the parent of origin of a gene or chromosome plays a role in the phenotype of the affected individual

EXAMPLES
PRADER-WILLI SYNDROME • Characterized by obesity, hyperphagia, small hands and feet, hypogonadism, and mental retardation ANGELMAN SYNDROME • Due to the absence of a paternal contribution of the genes located at 15q11q13 • Flattened back of the head. Deep-set eyes. Wide, ever-smiling mouth. Prominent jaw and widely spaced teeth. Lightly pigmented hair, skin and eyes.

• GERMLINE MOSAICISM • Presence of two or more genetically different cell lines in the same individual or tissue derived from a single zygote • Mutation is present in only one parent and arose from embryogenesis in all or some of the germ cells but few or none of the somatic cells of the embryo

• OSTEOGENESIS IMPERFECTA TYPE II (LETHAL FORM)

• At the molecular level, the mutation causing the condition is dominant, that is, only one copy of the abnormal gene is necessary to cause this perinatal lethal condition

MITOCHONDRIAL INHERITANCE- MATERNAL INHERITANCE
• Growing number of conditions due to abnormalities of mitochondria • Do not follow the typical mendelian pattern of inheritance MITOCHONDRIAL DNA (mtDNA) • MITOCHONDRIAL DISEASES CAN BE CAUSED BY MUTATIONS IN THE mtDNAOR IN THE NUCLEAR GENES THAT CODE FOR COMPONENTS OF THE OXPHOS SYSTEM. LEBER’S HEREDITARY OPTIC NEUROPATHY • Rapid, bilateral loss of central vision occurs

MULTIFACTORIAL INHERITANCE • traits or characteristics produced by the action of several genes, with or without the interplay of environmental factors. -Cleft lip with or without cleft palate -Open neural tube defect -Cardiac defects

CHROMOSOME ABNORMALITIES
• Numerical chromosomal abnormalities

1. aneuploidy- refers to an extra chromosome, such as in Trisomy 21 (Down Syndrome), Monosomy X (Turner Syndrome) 2. Polyploidy- refers to numerical chromosome abnormalities in which there is addition of an entire complement of haploid chromosome such as triploidy, in which three haploid sets occur (XXY or XYY).

• Numerical or aneuploid chromosome abnormalities invade either autosomes or sex chromosomes. Most occur as the result of nondisjunction during meiosis or mitosis in which homologous chromosomes fails to disjoin

• Molecular structures for the parent of origin have identified that the majority of autosomal aneuploidies result from non disjunctional error in maternal meiosis I.

• The majority of trisomic conceptions are nonviable, autosomal trisomies have been seen in abortus material in all 21, 18, 13, and 22 result in live chromosomes 1 and 17. However, trisomies births and are associated with advance maternal age

Tris omy 1 3 (P atau Syndrome

• occurs in approximately 1/10,000 live births. • Characterized by gross multiple structural defecits: procencephaly, cleft lip/palate, cardiac defects, and polydactyly.

Tris omy 1 8 (Ed ward Syndrome)

• 1/6,000 of live births • associated with prenatal growth restriction, rocker bottom feet, cardiac and renal defects.

Tri so my 21 (Do wn Sy ndrome )

• most common viable autosomal trisomy and has an incidence of 1/800 of live births. *The majority (95 %) of individuals with Down syndrome have trisomy 21 because of maternal disjunction.

• However, about 2% to 3% of individuals with clinical Down syndrome have structural rearrangement (robertsonian Translocation) and another 1 % to 3 % is mosaic for trisomy 21.

Tri so my 22

• has been seen in a few live born individuals and is associated with severe neurologic impairment. *monosomic states involving autosomes are extremely rare and generally lethal.

Sex Chromosome aneuploidy usually occurs in the trisomic states.

Monosom y Y

• has never been seen in clinical situation or even in abortus

Monosom y X (45 X, Tur ner Sy ndrome )

• typical finding, However, because most 45 X conceptions are lethal, the actual incidence of live births is about 1/5,000. • characterized by lymphedema, hypotonia and webbed neck.

• girls with Turner syndrome have short stature, broad chest with wide spaced nipples, cubitus valgus ( widened carrying angle of the arms), gonodal dysgenesis resulting in lack of secondary characteristics, amenorrhea, and infertility. • others features include congenital heart disease (COA, most common), kidney disease, and hypertension in later life.

• Intelligence is normal although spatial perception abnormalities are common.

• * Hormonal supplementation during puberty allows girls to develop secondary Characteristics.

• the 45 X karyotype occurs paternal non disjunction • and is not associated with advanced maternal or paternal age.

• There is no recurrence for 45 X • Another 30 to 40 % of individuals are mosaic for the 45 X cell line and another cell line (usually 46 XX) because of postzygotic nondisjunction during mitosis.

• Females who are mosaic with a 45 X or 46 XY karyotype are at an increased risk for gonadoblastoma.

• Other trisomies involving the sex chromosomes are seen in 47 XXX, 47 XXY (Klinefelter Syndrome ), 47 XYY karyotypes.

47 XXX
• -1/1000 live births • - due to maternal nondisjunctiuon associated with increasing maternal age. • - most women are phenotypically normal with exception of possible mild developmental delay: fertility is normal and there may be a slightly increased risk for offspring with aneuploidy involving the sex chromosomes and autosomes.

47 XXY ( kl inef elter Sy ndrome )

• -1/1000 live male births • -is a common sex chromosome abnormality associated with advanced maternal age.

• Clinical features include tall gynecoid stature, gynecomastia (with an increased risk for breast cancer) and testicular atrophy. • -mental retardation is not a typical feature, but affected individuals may have IQ scores that are lower than those of their siblings.

47 XYY
• - nondisjunction during spermatogenesis involving the Y chromosome. • taller than average,but are otherwise phenotypically normal • contrary to previous and outdated observational studies, this sex chromosomes aneuploidy is not associated with violent crime. However, behavioral problems such as attention deficit disorder may be observed.

B . Str uct ur al chr omosomal abnor mal ities

• Chromosomes breaks and rearrangements may lead to no obvious phenotypic consequences (genetically balanced), loss or gain of chromosomal material (genetically imbalanced) that produce abnormalities, or abnormalities due to interruption of a critical gene at

STRUCTURAL CHROMOSOMAL ABNORMALITIES

DELETION

RING CHROMOSOME

INVERSION

ISOCHROMOSOM E

DUPLICATION

TRANSLOCATION

INSERTION

Ty pes:

• translocations (reciprocal and robertsonian) • insertions • inversions • isochromosomes • duplications • deletions

Balanced recip rocal tr anslo cations

• translocations occur as a result of a mutual and physical exchange of chromosomes material between non homologous chromosomes. • found in about 1/11000 newborns.

* The carrier of reciprocal balanced translocations is usually phenotypically normal. However, the carrier is at an increased risk for producing offspring who are chromosomally abnormal.

In meiosis, the two pairs of nonhomologous chromosomes involved in the translocation resolve their pairing difficulties by forming a quadriradial.

• Most reciprocal translocations are unique to a family, and, consequently, the reproductive fitness of the carrier depends on the carrier’s sex and nature of the translocations. • In general, the recurrence for unbalanced conception is 3% to 5% for male carriers and 10% to 15% for female carriers.

Ro bertsoni an tr ansl ocati on • structural rearrangement between the acrocentric chromosomes: chromosome pairs 13, 14, 15, and 22. • in this structural rearrangement, the short arms (p arm) of two nonhomologous chromosomes are lost, and the long arms fuse at the centromere, forming a single chromosome structure.

• The phenotypically normal carrier of a robertsonian translocation has a 45 chromosomes in each cell because the two acrocentric chromosomes involved in the translocation have formed into one chromosome structure.

• This person is genetically balanced, that is he or she has two copies of each chromosome. However, the gametes are at risk to be unbalanced. • As in reciprocal translocations, the chromosomes involved in the rearrangement resolve the pairing of homologous segments at meiosis by forming

Inversions
-occur when two breaks occur on a chromosomes followed by 180o turn of the segment and reinsertion at its original breakpoints. • Pericentric inversion-if the centromere is included in the inverted segment • Paracentric inversion- if the centromere is not involved.

Isochromoso mes
• occur as a result of the chromosome dividing along the horizontal axis rather than the longitudinal axis at the centromere. • the result is a chromosome that has two copies of one arm and no copies of the other.

* isochromosomes involving the autosomes are generally lethal because the resultant conception will be both trisomic and monosomic for genetic information. However ischromosomes involving the long arm of the X chromosomes (iso Xq ) is compatible with life.

Deletions and Duplications

• arise from unequal crossing over at meiosis, or from crossing over during pairing of inversion or translocations.

• Terminal deletions- break resulting in loss of chromosomes material at the tip. • Interstitial deletions- a loss of chromosome material between two breaks within a chromosome.

Cri-du-chat (5p) syndrome

• microcephaly, profound mental retardation, growth retardation, a unique facial appearance, and a distinctive”cat like” cry.

*most cases occur as a de novo chromosome deletions of the tip of the short arm of chromosome 5, but approximately 10% to 15% arise as a consequence of parental reciprocal translocation chromosome 5 and another chromosome.

Mi crod eleti on/ dupl ication or Conti guous Gene Syndr ome • syndromes in which the involved chromosome region are submicroscopic and so small that molecular cytogenetic technique such as fluorescent in situ hybridization is necessary to localized the affected region. • The phenotypes of these conditions are due to absence ( or duplication) of multiple contiguous genes within the involved region.

Chromosome Abnormalities and Pregnancy Outcome

• incidence and types of chromosome abnormalities differ between spontaneous abortions, stillbirths, and live births.

• chromosome and lethal genetic abnormalities play a major role in early losses. 30 to 60 % of first trimester abortuses are found to have a chromosome abnormality of which approximately 50% are due to autosomal trisomies.

• certain chromosomes are more commonly involved than the others; for example, trisomy 16 accounts for one third of trisomic abortuses.autosomal monosomies are extremely rare,accounting for less than 1% of chromosomally abnormal fetus. Triploidy accounts for 16% of spontaneous abortions.

• Turner Syndrome due to 45 X by far the most common chromosome abnormality, accounting for 20% of spontaneous abortions that are chromosomally abnormal.

Molar Gestation

Partial moles (associated with triploidy)

• found in 16% of abortuses. • they occur as a consequence of errors in meiosis or from double fertilization of a single ovum.

• clinical features depend on the parent of origin of the extra set of chromosomes. In 2/3 of cases, there is one maternal set and two paternal set sets of chromosomes, resulting in poor embryonic development but a large hydropic placenta, the partial mole.

Conversely there is an extra maternal set, an anomalous and growthrestricted fetus and a small fibrotic placenta are seen.

Complete mole

• diploid with a 46 XX karyotype. • paternally derived • arise through the fertilization of an unucleated oocyte and subsequent duplication of haploid chromosome complement (23 X) of the sperm.

* the absence of maternal contribution results in development of abundant, hydropic trophoblastic tissue, and no recognizable embryonic tissue.

Down Syndr ome

47,XY,+21

Ed wa rd’s Syndrome

47,XY,+18

Translocation

46,XY,t(1;17)

BCR/ABL Metaphase
• A metaphase cell positive for the bcr/abl rearrangement using FISH. The chromosomes can be seen in blue. The chromosome that is labeled with green and red spots (up left) is the one where the wrong rearrangement is present

BCR/ABL Interphase
• Interphase cells positive for a chromosomal t(9;22) rearrangement.

NUMERICAL CHROMOSOMAL ABNORMALITIES

• Polyploidy is a state in which the number of sets of chromosomes exceeds the diploid number (2n) by a multiple of n • Triploidy - A complete extra set of chromosomes raises the total number to 69. This usually arises from: - Fertilization by two sperm (dispermy) - Failure of one of the maturation divisions of either the egg or the sperm, producing a diploid gamete

TRIPLOIDY
• A triploid fetus (which usually miscarries) would be 69,XXY (most common), 69,XXX or 69,XYY depending on the origin of the extra set of chromosomes

TETRAPLOIDY • Tetraploidy - Four times the haploid number is usually due to failure to complete the first zygotic division • Tetraploid cells are also a normal feature of regenerating liver and other tissues

POLYPLOID • A proportion of cells that occurs normally in human bone marrow, as megakaryocytes usually have 8-16 times the haploid number

BIOCHEMICAL GENETICS

BIOCHEMICAL GENETICS • RENDERS SERVICES FOR THE DIAGNOSIS AND CLINICAL MANAGEMENT OF PATIENTS WITH INBORN ERRORS OF METABOLISM (IEM) • PROVIDES KEY INVESTIGATIONS FOR VARIOUS METABOLIC DISORDERS

INBORN ERRORS OF METABOLISM

• DISORDERS THAT ARISE FROM A DEFECT IN A METABOLIC PATHWAY, WHICH COULD BE DUE TO EITHER AN ENZYME OR AN ABNORMALITY OF A TRANSPORT PROCESS

COMMON SIGNS AND SYMPTOMS

• ACUTE ENCEPHALOPATHY • CHRONIC ENCEPHALOPATHY
– MENTAL RETARDATION – DEVELOPMENTAL DELAY – SEIZURES, ETC

• MYOPATHY • DIFFUSE LIVER DISEASE • RENALTUBULAR DISEASE

TYPES OF IEM
• DISORDERS OF PROTEIN METABOLISM
– (AMINO ACIDOPATHIES, ORGANIC ACIDOPATHIES, UREA CYCLE DEFECTS)

• DISORDERS OF CARBOHYDRATE METABOLISM
– (CHO INTOLERANCE DISORDERS, GLYCOGEN STORAGE DISORDERS, DISORDERS OF GLUCONEOGENESIS AND GLYCOGENOLYSIS)

• • • •

LYSOSOMAL STORAGE DISORDERS FATTY ACID OXIDATION DEFECTS MITOCHONDRIAL DISORDERS PEROXISOMAL DISORDERS

LOCALLY AVAILABLE TEST • COMPREHENSIVE URINE METABOLIC PROFILE (URINE METABOLIC SCREEN AND ORGANIC ACID ANALYSIS) • URINE METABOLIC SCREEN (AMINO ACID PROFILE, METHYLMALONIC ACID SCREEN) • PLASMA AMINOACID QUANTITATIVE ANALYSIS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY(HPLC)

LOCALLY AVAILABLE TEST • CSF AMINOACID QUANTITATIVE ANALYSIS BY HPLC • URINE ORGANIC ACID ANALYSIS FOR GAS CHROMATOGRAPHY-MASS SPECTROPHOTOMETRY(GCMS) • EXPANDED NEWBORN SCREENING

PRIMARY REASONS FOR REQUESTING GENETIC METABOLIC TEST
• CHRONIC ENCEPHALOPATHY • ACUTE ENCEPHALOPATHY • PSYCHIATRIC SYMPTOMS • MOVEMENT DISODERS • STROKE • MYOPATHY • OCULAR SYMPTOMS • CARDIAC ARRYTHMIAS • HEPATIC SYMPTOMS

• HEMATOLOGIC SYMPTOMS • ENDOCRINE • METABOLIC ACIDOSIS • BIOCHEMICAL ABNORMALITIES • DYSMORPHIC FEATURES • OTHER VISCERAL SYMPTOMS

MOLECULAR GENETICS

FLUORESCENT IN SITU HYBRIDIZATION (FISH) • A technique that can be used to detect and localize the presence or absence of specific DNA sequences on chromosomes. It uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence similarity. Fluorescence microscopy can be used to find out where the fluorescent probe bound to the chromosome. FISH is often used for finding specific features in DNA. These features can be used in genetic counseling, medicine, and species identification.

FISH Principle

Medical appl ications
• In medicine, FISH can be used to form a diagnosis, to evaluate prognosis, or to evaluate remission of a disease, such as cancer. • Examples of diseases that are diagnosed using FISH include Prader-Willi syndrome, Angelman syndrome, chronic myelogenous leukemia, acute lymphoblastic leukemia, Cri-du-chat, Velocardiofacial syndrome, and Down syndrome

DNA/RNA ANALYSIS
DNA EXTRACTION DNA PURIFICATION DNA AMPLIFICATION DNA CLEAN-UP DNA ANALYSIS

POLYMERASE CHAIN REACTION

DUCHENNE MUSCLE DYSTROPHY

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