CHROMOSOMAL BASIS OF HUMAN DISEASES History of human cytogenetics The “Trisomy Period” The “Banding Era Historically, cytogenetics was developed before molecular genetics.Cytogenetics is Introduction of different banding a brench of genetics concerned with the techniques enabled the study of the structure and function of identification each chromosome chromosomes. The visualization of and the detection of chromosomes and correct model human chromosomal abnormalities, chromosome number was determined by such as translocations, Тjio and Levan in 1956. inversions, deletions and They serendipitously used hypotonic insertions. solution for releasing chromosomes from cells. History of human cytogenetics The “Molecular Era” Molecular karyotyping

FISH Аrray-based CGH Chomosome-based CGH

The major technique in molecular cytogenetics is fluorescent in situ hybridization (FISH), which is an approach that allows nucleic acid sequence to be examined inside cells or on chromosomes. In 2003 the “so-called” array techniques were introduced in genetic diagnostics, providing high resolution for the determination of chromosomal gain or loss. Formal cytogenetics – group of chromosomes

Human chromosomes are classified by position of centromere into: metacentric, submetacentric, acrocentric. There are 22 paires of autosomes and a pair of sex chromosomes – XX or XY. Formal cytogenetics – nomenclature An ideogram represents the typical chromosome pattern and the relation of the p and q arm. Databases that can help to identify a syndrome

1. Elements of Morphology: helps to find the right terminology for subsequent literature and database search Free: http://elementsofmorphology.nih.gov/index.cgi 2. Phenomizer: postnatal, to find a syndrome based on combination of several malformations/dysmorphic signs Free: http://compbio.charite.de/phenomizer/

3. Phenotip: prenatal, find a syndrome from the combination of several malformations/dysmorphic signs Free: http://www.phenotip.com/ 4. Orphanet: postnatal, find a syndrome, get information on diagnostics and other support, in diff. languages Free: http://www.orpha.net/consor/cgi-bin/index.php?lng=EN

5. Other commercial databases Face2Gene: http://www.fdna.com/ London Dysmorphology Database: http://www.lmdatabases.com POSSUM: http://www.possum.net.au/ Indications for karyotype study

• Diagnose of constitutional disorders – disorders present at birth (birth defects, ambiguous genitalia) – intellectual disability and physical development delay – adults with reproductive failure – fetal chromosome diagnosis (preimplantaion/prenatal diagnostics) • Diagnose of an acquired disorders – most commonly haematological malignancies and solid tumor – evaluation of chromosome aberrations in mutagenesis (in vitro) Mutation types Genome mutation (chromosome number) Chromosome mutation (chromosome structure) Gene mutation (DNA sequence)

Genome mutation is total number of chromosomes is changed due to errors during gametogenesis in meiotic nondisjunction

Paternal % Maternal % Trisomy 13 5 85 Trisomy 18 10 90 Trisomy 21 5 95 45,X 80 20 47,XXX 5 95 47,XXY 45 55 47,XYY 100 0

Non-disjunction more frequent in (older) women genome mutations Point mutations more frequent in (older) male gene mutations Chromosome Abnormalities

Numerical Structural Chromosome changes result from errors occurring during meiotic or mitotic segregation. Two classes of Aneuploidy numerical chromosomal Chromosome loss or gain abnormalities can be distinguished. • Trisomy – having three copies of a particular chromosome • Monosomy - corresponds to lack Triploidy of a chromosome Three haploid set (23xn) very seldom survive to term, and the condition is not compatible with life. AUTOSOMAL TRISOMIES Trisomy 21: Down syndrome- Trisomy 13: Patau Trisomy 18: Edwards 47,XX+21 syndrome - 47,XX,+13 syndrome – 47,XX +18

• microcephaly impairment of cognitive ability • polyductily • severe psychomotor and • stunted growth • cleft palate, cleft lip growth retardation, • redundant neck skin • holoprosencephaly • growth deficiency • flat face (failure of the forebrain to divide • structural heart defects • epicantus properly) • micrognatia (small jaw) • flat nasal bridge • low vitality • narrow eyelid folds • flat neck • incidence 1:11000 palbebral fissures • heart defect • webbing of the 2-nd and 3-d • semian crease fingers • incidence 1:700 The critical region has • microcephaly been reported to include • feeding, breathing difficulties Molecular analysis has revealed that 13q14-13q32 with • low vitality the 21q22.1-q22.3 region appears to variable expression, contain the gene(s) responsible for the • incidence 1:15000 congenital heart disease observed in gene interactions, or Down syndrome. interchromosomal 95% of the cases of trisomy 21 the effects. extra chromosome comes from the mother and only in 5% from the father. SEX-CHROMOSOME ANEUPLOIDIES • Turner syndrome: inherit only one X chromosome; their genotype is X0. • Triple-X females: inherit three X chromosomes; their genotype is XXX or more rarely XXXX. Some of them are fertile. • Klinefelter syndrome: males inherit one or more extra X chromosomes; their genotype is XXY or more rarely XXXY, XXXXY, or XY/XXY mosaic (hypogonadism, increased stature, infertility, gynecomastia, physical and behavioral differences) • Jacobs syndrome:males inherit an extra Y chromosome; their genotype is XYY (increased growth velocity, some of them are fertile, half of 47,XYY boys have learning difficulties)

The increased gene dosage of three X/Y chromosome pseudoautosomal region (PAR1) SHOX genes has been postulated as a cause of the increased stature seen in all three sex chromosome trisomies: 47,XXX, 47,XXY, and 47,XYY.

10 X-Y related syndromes Turner syndrome is the only monosomy in liveborn. However Klinefelter’s syndrome – 47.XXY the syndrome presents with different karyotypes 45,X; 46,XX/45,X; 46,Xi(Xq)

• Wide or weblike neck • Receding or small lower jaw • High, narrow roof of the mouth (palate) • Low-set ears • Low hairline at the back of the head • hypogonadism • Broad chest with widely spaced nipples • increased stature • Short fingers and toes • infertility • Arms that turn outward at the elbows • physical and behavioral • Fingernails and toenails that are narrow differences and turned upward • gynecomastia • Swelling of the hands and feet, especially at birth • Slightly smaller than average height at birth • Delayed growth Frequency of cytogenetic aberrations • ~50% of abortions show chromosomal abnormalities • 1 in 120 newborn have chromosomal aberrations, ~50% of them are of clinical relevance.

trisomy 13 0.08% sSMC 0.4% trisomy 18 0.15% 45,X 0.3% trisomy 21 1.2% XXX 1.1%

triploidy 0.02% unbalanced structural aberr. 0.3% XXY 1.2% balanced rec. translocation 2.5% XYY 1.2% balanced Robertsonian transl. 1.0% other aneupl. 0.15% I nversions 0.8%

• cause for early sudden infant death (5-7%, most frequently trisomy 18 and 21) • mentally retarded individuals (without FRA-X-syndrome): IQ < 20 3-10% most frequent: IQ = 20-49 12-35% Down-syndrome, IQ = 50-69 3% deletions/ microdeletions, unbalanced rtanslocations…

• in infertile individuals, hermaphroditism, recurrent abortions etc. Structural Chromosome Rearrangements

Balanced Unbalanced is a structural chromosomal there is a gain or loss of abnormalities if there is no gain or chromosome material loss of chromosome material

translocations inversions • insertion • deletion • duplication reciprocal robertsonian • ring • dicentric • isochromosome TYPES OF CHROMOSOME ABERRATIONS

arised from gamets and somatic cells

Constitutional chromosomal Acquired chromosomal aberrations anomalies

They result from chromosome On the other hand, any breakage with subsequent changes in the chromosomes reunion in a different which arise during configuration. Chromosomal development or during the life rearrangements originating in of an organism are referred as the germ line, whether inherited acquired chromosomal from the parents or from a de rearrangements novo mutation in the gametes, are referred to as constitutional INVERSION PARACENTRIC INVERSION PERICENTRIC INVERSION If the inversion is outside An inversion involves two breaks the centromere, it is in a chromosome and the termed a paracentric segment is reversed or inverted inversion whereas inversion in the position spanning the centromere, involving both the chromosome arms, is known as pericentric inversion.

ՌԵՑԻՊՐՈԿ ՏՐԱՆՍԼՈԿԱՑԻԱ ՔՐՈՄՈՍՈՄՆԵՐԻ ՏԱՐԱԲԱՇԽՈՒՄԸ ԳԱՄԵՏՆԵՐՈՒՄ ԿՐԵԼԻՈՒԹՅԱՆ ԴԵՊՔՈՒՄ

A carrier of a balanced reciprocal translocation can produce gametes that give rise to an entirely normal child, a phenotypically normal balanced carrier, or various unbalanced karyotypes ISCN - International System for Human Cytogenetic Nomenclature • most recent edition pub. 2016 • provides standardized “grammatical” rules for the designation of cytogenetic findings

A uniform system of chromosomal classification is internationally accepted for identification.The pattern of bands on each chromosome is numbered on each arm. By this numbering, location of any band, DNA sequences and genes within it, and its involvement in a chromosome abnormality can be precisely described.

Example: 46,XY,der(9)(9qter→9p23:: ::4q26→4qter),pat CLINICAL CASE: Partial trisomy 4q26-qter as a result of a reciprocal translocation in parent Various ocular abnormalities are presented. Ophthalmologic assessment revealed: divergent strabismus, microphtalmiа, optic nerve subatrophy,retina atrophy, locus oculosum with searching look. Others include receding jaw, pointed chin highly arched palate, short neck, nipples asymmetric set with hypertelorism Unbalanced translocation (proband) Balanced translocation (carrier) 46,XY,der(9)(9qter→9p23:: ::4q26→4qter),pat 46,XY,t(4;9)(q26;p23),mat Robertsonian translocation (centric fusion) with acrocentric chromosomes

Proband Carrier 46,XX,t(14;21)(q10;q10),+21 45,XX,t(14;21)(q10;q10) DOWN SYNDROME KARYOTYPE VARIANTS a )47, XX,+21 Carriers of Robertsonian translocation have 45 b) 46,XY,t (13,21),+21 chromosome where long arm chr.21 joins with the long arm c) 46,XX,t (14;21),+21 of chr. 14 or 13,15, Depends on what chromosomes are d) 46,XX,t (15;21),+21 involved in the translocation different outcomes might be e) 46,XY,t(21;21)+21 detected in the family history:misscarigies, trisomy 21(Down syndrome), trisomy 13(Patau syndrome). Gamets of a carrier of 21/21 translocation have anomalies: monosomy 21 or trisomy 21. All of the viable offspring of an isochromosome 21 individual will have Down syndrome. Case

A phenotypically normal woman with a 45, XX, -14, -21, +t(14q, 21q) 47, XY, +21 karyotype has a karyotypically normal 46, XY, der(14;21),+21 (46, XY) husband. Among their 46, XY, der(14;21),+14 liveborn offspring, the most likely 45, XY, -14, -21, +t(14q, 21q) karyotypes are 46, XY and 46, XX. 46, XY, -14, -21, +16 Which of the following karyotypes is the next most likely among liveborn offspring Types of unbalanced chromosomal aberrations

RING CHROMOSOME

DUPLICATION ISOCHROMOSOMES A ring chromosome is a chromosome whose arms have fused together to form a ring Isochromosome is abnormal chromosome that has two identical arms due to duplication of one arm and loss of the other. Isochromosomes are found in tumors and in some girls with Turner syndrome. Gene duplication or chromosomal duplication or gene amplification is a major mechanism through which new genetic material is generated during molecular evolution. It can be defined as any duplication of a region of DNA that contains a gene. Chromosomal deletion syndromes typically involve larger deletions, that are typically visible on karyotyping. Syndromes involving smaller deletions (and additions) that affect one or more contiguous genes on a chromosome and are not visible on karyotyping are considered microdeletion and duplication syndromes. Advantages and Disadvantages of conventional cytogenetic technique

Advantages 1- Enable the entire genome to be viewed at one time.

2- Suitable for mosaicism detection

Disadvantages 1- Low resolution. Detect major structural abnormalities ~ 10Mb 2- Labor intensive, time consuming and highly dependent upon operator experience and skills. Molecular era in cytogenetics Fluorescent in situ Hybridization (FISH) Principles and Applications The major advance in human cytogenetics has been the development and implementation different molecular genetics technologies to detect specific DNA sequences and to locate them to specific chromosomal site. This approaches extended the limitation of conventional cytogenetics and contributed to bridging the gap between the microscope and the molecule. Today chromosome analysis is an increasingly important diagnostic procedure in numerous areas of clinical medicine. The advanced development of the karyotype study is the technique called fluorescent in situ hybridization (FISH), because the DNA in metaphase chromosomes fixed on the slides is denatured in place to expose the two strands of DNA, thus allowing a labeled probe to hybridize to the chromosomal DNA. FLUORESCENT IN SITU HYBRIDIZATION (FISH) FISH (fluorescent in situ hybridization) features Uses оf a fluorescent probe to hybridize to specific areas of the chromosomes

•The least of chromosomal region disorder that can be detected on karyotype even by the high resolution chromosome analysis comes to 10 Mb from at least 200 genes. •FISH allows to detect changes in chromosome structure, if it consists of 5Mb and less. •FISH can be used in metaphase cells to detect specific microdeletions beyond the resolution of routine cytogenetics or identify extra material of unknown origin. •FISH can be used in interphase cells Types of FISH probes

Whole chromosome paint probes to rule out complex rearrangements within chromosomes

Locus specific probes to rule out deletions or gains of specific loci Telomere probes to rule out deletions in telomere regions FISH APPLICATIONS Interphase-FISH

Preimplantation genetic diagnosis - PGD

Myc break point translocation between chromosomes 8 and 14 detected in lymphoblastic Prenatal diagnosis - FISH on uncultured leukemia amniocytes Philadelphian chromosome in chronic myeloid leukemia (Ph-chromosome)

Translocation lead to the BCR-ABL "fusion" gene creation. This abnormal "fusion" gene generates a protein of p210 Microdeletion Syndromes diagnosed with FISH technique

 Cri-du-chat (5p-).  Miller-Dieker syndrome (7q11.23).  Smith-Magenis syndrome (17p13.3).  Steroid Sulfatase Deficiency (Xp22.3).  DiGeorge/Velo-cardio-facial/CATCH-22/ Shprintzen Syndrome (22q11.2).  Kallman Syndrome (Xp22.3).  Williams Syndrome (7q11.23).  Wolf-Hirsch horn (4p-).  Prader-Willi/Angelman Syndrome (15q11.2-13).  X-Linked Icthyosis (xp22.3).  Retinoblastoma (13q14). Mechanism underlying the majority of chromosome rearrangements

Low copy repeats Non-allelic homologue Deletion (LCRs) or segmental recombination (NAHR). Duplication duplication This is mechanism of Inversion translocation formation

Certain regions of the human genome are especially prone to structural rearrangements due to the presence of repetitive sequence elements, which are the hotpots susceptible to genome instability. Genome architecture has been implicated in the formation of increasing numbers of genomic disorders.1 The best-studied example is the association between segmental duplications or low-copy repeats (LCRs) and microdeletion and microduplication syndromes . Interaction between chromosome-specific LCRs leads to gain, loss or inversion of the intervening sequence. Where a particular region contains dosage sensitive or imprinted genes this can lead to a specific genetic disease: loss of 7q11.23 results in Williams syndrome (MIM 194050), loss of 22q11 results in Di George syndrome/VCFS and loss of 15q11–q13 results in either Prader–Willi syndrome or Angelman syndrome Examples of microdeletion syndrome Williams-Beuren Syndrome - del 7q11.23, ELN gene deletion

• "elfin" facial appearance with hypercalcemia • long philtrum • unusually cheerful demeanor • wide mouth • flattened nasal bridge • cardiovascular problems (supravalvular aortic stenosis) • caused by a deletion of about 26 genes from the long arm of chromosome 7 • prevalence - 10,000-20,000 births Williams syndrome, also known as Williams-Beuren syndrome, is a rare genetic disorder characterized by growth delays before and after birth (prenatal and postnatal growth retardation), short stature, a varying degree of mental deficiency, and distinctive facial features that typically become more pronounced with age. Such characteristic facial features may include a round face, full cheeks, thick lips, a large mouth that is usually held open, and a broad nasal bridge with nostrils that flare forward (anteverted nares). Williams syndrome results from deletions of genetic material from adjacent genes (contiguous genes) located on the long arm (q) of chromosome 7 (7q11.23).

According to investigators, 28 genes within the 7q11.23 chromosomal region may play a causative role in Williams syndrome including those known as the ELN (elastin) gene, the LIMK1 (or LIM kinase-1) gene, and the RFC2 (replication factor C, subunit 2) gene. The LIMK1 gene is believed to be involved with visual-spatial problems associated with Williams syndrome. Examples of microdeletion syndrome

Cri du chat syndrome – del 5p

• feeding problems because of difficulty swallowing and sucking; • low birth weight and poor growth; • severe cognitive, speech, and motor delays; • behavioral problems such as hyperactivity, aggression, repetitive movements; • unusual facial features which may change over time; • excessive drooling; • small head and jaw; • wide eyes; • skin tags in front of eyes. The syndrome gets its name from the characteristic cry of affected infants, which is similar to that of a meowing kitten, due to problems with the larynx and nervous system. About 1/3 of children lose the cry by age 2.

Cri du chat syndrome is due to a partial deletion of the short arm of chromosome number 5, also called "5p monosomy" or "partial monosomy." Approximately 90% of cases result from a sporadic, or randomly occurring, de novo deletion. The remaining 10-15% are due to unequal segregation of a parental balanced translocationwhere the 5p monosomy is often accompanied by a trisomic portion of the genome. These individuals may have more severe disease than those with isolated monosomy of 5p DiGeorge syndrome

Children with DiGeorge syndrome tend to have the following features:

• a long, narrow face • wide-set, almond-shaped eyes • a broad nasal bridge and bulbous nose tip • a small mouth • small, low-set ears that are folded over at the top • a cleft lip • a cleft palate • an irregular skull shape

Catch -22 gene deletion DiGeorge syndrome is a genetic disorder that's usually noticeable at birth. DiGeorge syndrome is the most common microdeletion syndrome characterized by low copy repeats and the deletion occurs near the middle of the chromosome at a location designated 22q11.2—signifying its location on the long arm of one of the pair of chromosomes 22, on region 1, band 1, sub- band 2 The inheritance pattern is autosomal dominant and it has a prevalence estimated at 1:4000. The syndrome was described in 1968 by the pediatric endocrinologist Angelo DiGeorge 22q11 deletion is also associated with truncus arteriosus and tetralogy of Fallot. The inheritance pattern is autosomal dominant and it has a prevalence estimated at 1:4000 Parent-of-Origin Effects - For some disorders, expression of disease phenotypes depends on parental origin of mutant allele or abnormal chromosome

- Differences in gene expression b/w allele inherited from mother and allele inherited from father are the result of genomic imprinting.

Disorders associated with imprinting

• Prader-Willi / Angelman

• Beckwith–Wiedemann syndrome / Silver–Russell dwarfism Imprinting affects expression but not DNA sequence. It is a reversible form of gene inactivation but not a mutation, i.e., epigenetic effect. Epigenetics are with significant influences on gene expression and phenotype both in normal individuals and a variety of disorders (cytogenetic, single gene and cancer) Imprinting takes place during gametogenesis, before fertilization, and marks certain genes as having come from mother or father. Imprint controls gene expression in some or all somatic tissues of embryo. Imprinted state persists postnatally into adulthood. Control over conversion appears to be governed by DNA elements called “imprinting centers” located within imprinted regions, their precise mechanism is unknown, they must initiate epigenetic change in chromatin, which then spreads outward over imprinted region • It is likely that several dozen or ~ 100 genes show imprinting effects.

• Only one allele (either maternal or paternal) is expressed. Non-imprinted loci (majority of loci) are expressed from both alleles.

Map of imprinted regions in human genome (gray: expressed only from maternal; blue: expressed from paternal) Syndrome Prader-Willi - del 15q11-q13 Angelman syndrome del 15q11-q13

Interstitial deletion 15q11- q13 region on the maternal chromosome

Interstitial deletion 15q11-q13 region on the paternal chromosome • severe mental retardation • jerky movements (especially • obesity, • hand flapping) •hypogonadism • seizures •small hands and feet • frequent laughter or smiling, •short stature •developmental delay. and usually a happy demeanor PW is a relatively common dysmorphic syndrome: obesity, excessive and indiscriminate eating habits, small hands short stature, hypogonadism, and mental retardation. In ~ 70% of cases, there is a interstitial deletion involving (15q11- q13), occurring only on the chromosome 15 inherited from the patient's father.Thus, patients have genetic info in 15q11-q13 derived from their mothers. In contrast, in ~ 70% of patients with the rare Angelman syndrome: unusual facial appearance, short stature, severe mental retardation, spasticity, and seizures, there is a deletion of ~ the same chr. region but on chr 15 inherited from mother. Patients with AS, therefore, have genetic info. in 15q11-q13 derived from their fathers. This demonstrates that parental origin of genetic material (15q11- q13) can have a profound effect on the clinical expression of a defect. Prader-Willy/Angelman Syndromes region

D15S13 D15S63 SNRPN UBE3A D15S10 D15S113 GABRB3 Another cause of AS is uniparental disomy (UPD)c where the child inherits both copies o chromosome 15 from the father with no copy inhereted from the mother. In this case there is no mutation or deletion, but the child is still missing the active UBE3A gene, because the paternal-derived chromosomes only have brain-inactivated UBE3A genes. Genetic Mechanisms of Leading to Angelman Syndrome 15q11-q13 region

Prader-Willi syndrome Angelman syndrome

• 70% - del 15q11-q13 in the • 70% - del 15q11-q13 in the paternal chromosome maternal chromosome • 28% – UPD of the maternal • 28% –UPD of the paternal chromosome chromosome • 2% - imprinting error • 2% - imprinting error FISH Advantages • Targeted analysis • Higher resolution (2Mb-100kb) Disadvantages • Cannot detect small mutations. • Cannot detect whole genome • Probes are not yet commercially available for all chromosomal regions. Further progress in cytogenetics - Comparative Genomic Hybridization (CGH) • Comparative genomic hybridization (CGH) is developed for the analysis of cryptic genetic imbalances in whole genome or chromosome set. • Assess the relative copy number of genomic DNA sequences in a comprehensive manner • Complements karyotyping and provides very sensitive, high resolution genome assessment. • Balanced translocations and rearrangements can not be resolved CGH. Comparative genomic hybridization (CGH) was developed to survey DNA copy-number variations across a whole genome.

With CGH, differentially labeled test (i.e. tumor) and reference (i.e. normal individual) genomic DNAs are cohybridized to normal metaphase chromosomes, and fluorescence ratios along the length of chromosomes provide a cytogenetic representation of the relative DNA copy-number variation.

ArrayCGH resolution is limited to 1Mb