Incorrect Centromere Splitting Causes Genetic Disorders

The importance of centromere lies in its specialty of holding the chromosome arms together. Imagine two threads of 2cm each aligned in a cross-shape. Suppose you wish to align them on a pin-up board. How would you align them? The answer is very simple. Just place a pin on the intersecting point of the two threads such that they resemble a cross. Once you pin the threads, they remain intact. Similarly, consider another example of a paper cut out in a cross shape. The four arms of the cross-connect each other through a central point.

During the cell division, the spindle fibers attach to the centromere via kinetochore. Centromere splitting leads to separation of the chromatids. The centromere is also known as a primary constriction. Any error in the centromere proves catastrophic for the cell. During mitosis, centromere assists in proper segregation of chromosomes. The cell undergoes a cyclical pattern to grow and divide. The somatic cell cycle divides into interphase and a mitosis phase. Meiosis occurs in the gametes. During metaphase, the centromeres help in attachment with the kinetochores. Hence chromosomes align themselves on the equatorial plane. It occurs during normal centromere splitting. Later on, during anaphase, the centromeres separate the sister chromatids, each forming individual daughter chromosomes. These events have chances of centromere misdivision. The term centromere misdivision describes a condition in which a centromere or a near centromere region fails to split properly. In humans, errors in centromere separation result in various disorders. The chromosome segregation gets affected depending on the types of errors such as non-disjunction, out of phase separation, premature splitting, centromere puffing, and Isochromosome formation.

Centromere separation in humans:

The cells undergo a typical cyclical pattern consisting of interphase and mitosis. The division phase or mitosis involves an important phase dependent on the centromere splitting or separation. This phase is known as anaphase. It involves splitting of the centromere to move the daughter chromatids towards the opposite poles. Certain factors play a crucial role in the splitting of the centromeres. The initiation of the centromere splitting first requires an anaphase-promoting complex. This complex involves an inhibitory chaperone known as securin. The APC/C cyclosome degrades three main factors such as securin, S and M cyclins. The degradation of securin releases a protease known as separase which in turn cleaves the cohesins. The cohesins bond the sister chromatids intact and allow non-homologous centromere coupling. However, the cleavage of the cohesin happens during anaphase. It further accelerates the centromere splitting and sister chromatid separation. The centromere splitting may be either horizontal or longitudinal (vertical). Normally the centromere divides longitudinally. However, the centromere splitting may accompany errors. A common error occurs when the centromere divides horizontally. Two isochromosomes arise due to incorrect centromere splitting. These isochromosomes have genetically identical arms. The isochromosomes may have duplication on one arm and deletion on the other arm.

Image: Isochromosomes

More about isochromosomes:

An incorrect centromere splitting gives rise to a kind of metacentric chromosomes known as isochromosomes. A product of incorrect Centromeric division is due to a complete absence of the centromere. Another product of an incorrect division involves a centromere ready to duplicate. Thus an Isochromosome consists of an identical long arm or short arm depending on the Centromeric division product. An Isochromosome involving a long arm of X chromosome leads to a structural abnormality associated with gonadal dysgenesis. Isochromosome studies relate to the horizontal misdivision of the centromere. Misdivision occurs either in the maternal or a paternal chromosome. It is also known as centromeric fission. This type of horizontal splitting gives rise to i(Ap) and i(Aq) arms of the chromosome. The letter “i” indicates an Isochromosome. A U-type exchange results in a loop formation. This structure forms during mitosis and meiosis. Mosaicism arises due to a subsequent mitotic loss of an Isochromosome.

Two main types of isochromosomes reported so far include monocentric or dicentric chromosome. Monocentric Isochromosome consists of one centromere. A dicentric centromere consists of two centromeres. Some of the references mention the occurrence of the misdivisions in the pericentric regions consisting of homologous sequence sites. The early anaphase involves breakage and fusion of the sister chromatids with a U-type strand exchange. There is a repair mechanism for a double strand break. Fusion of the sister chromatids containing centromere repair a double-strand break.

Type of isochromosome	Condition
i(5p)	Refractory cytopenia pulmonary atresia
i(8p)	Mosaicism
i(18p); i(18q)	Mosaic tetrasomy
Robertsonion isochromosome	Down’s syndrome
i(17q)	Neoplasia
i(Xq)	Turner’s syndrome
i(11q), i(17q), i(21q)	AML
i(7q), i(9q), i(17q)	ALL
i(9q), i(17q), i(22q)	CML
i(X)(q13), i(17q), i(21q)	MDS
I(1q), i(16p), i(7q), i(18q), i(9q)	Recurrent isochromosomes in lymphoproliferative disorder.

Table: Types of isochromosomes and the conditions associated with the same.

Centromere separation and non-disjunction:

Failure of separation of chromosomes during anaphase leads to non-disjunction. It leads to an improper separation of the chromosome. Non-disjunction leads to an imbalance of chromosomes. It happens during mitosis or meiosis. Non-disjunction gives rise to conditions known as monosomy, trisomy or mosaicism. Pericentric exchanges contribute to non-disjunction. Premature separation of the sister chromatids arises due to degradation of the centromere. Non-disjunction in the first meiotic division arises due to a reduced exchange near a centromere. Non-disjunction in the second meiotic division arises as a result of an increased exchange near the centromere. In a tetrad, non-disjunction associates with the distance between the centromere and closest exchange.

Premature centromere division:

Non-disjunction arises due to premature centromere division. It occurs due to the absence of pairing proteins such as INCENPs (inner centromere proteins) or CLIPs (chromatid linkage protein). It may lead to failure of chromatid pairing and chromosome alignment. Premature centromere division is an age-dependent phenomenon and is more common in females. It arises mostly in the X chromosomes.

Out of phase centromere separation:

It involves centromere spreading and common in tumor cells. The chances of aberrations are very high. Out of phase separation involves very early or very late separating centromeres. The Centromeric division in human mitotic chromosomes is non-random. It occurs in a genetically controlled way. The timing of separation depends on the timing of the repetitive DNA replication. Pericentric heterochromatin mainly consists of such DNA. Out of phase separation of the centromere arises maximally in the X chromosome. Out of phase centromere separation in X chromosome results in pregnancy complications, repeated abortions, Klinefelter syndrome, and Ataxia telangiectasia. A few inactive centromeres result into myelomas and dicentrics.

Centromere puffing:

It involves a localized swelling of a Centromeric region. Centromere puffing is nothing but an excessive DNA replication in the centromere region. When centromere puffing occurs in an acrocentric chromosome, it increases the risk of Robert’s syndrome. Centromere puffing is common in acute myelogenous leukemia, acute non-lymphocytic leukemia, non-lymphocytic leukemia, and acute lymphocytic leukemia.

Robert’s syndrome accompanies deformities in the body, prenatal growth retardation, limb malformation, and craniofacial abnormalities. Hence, centromere misdivision and premature centromere splitting result in centromere instability syndromes.

References:
[1] Essentials Of Human Genetics Fifth Edition,  Manu L. Kothari, Lopa A. Mehta
[2] Vogel and Motulsky's Human Genetics: Problems and Approaches, Friedrich Vogel, Gunter Vogel, Arno G. Motulsky
[3] Mechanisms of Environmental Mutagenesis-Carcinogenesis, A. Kappas
[4] The Principles of Clinical Cytogenetics, Steven L. Gersen, Martha B. Keagle
[5] Centromeres and Kinetochores: Discovering the Molecular Mechanisms,  Ben E. Black

© Copyright, 2018 All Rights Reserved.

A Review on Microdeletion Syndromes

Microdeletions involve sub-microscopic or minute loss of the genetic material. Microdeletions either arise spontaneously during pregnancy or get inherited. A microdeletion syndrome or a contiguous gene syndrome arises due to chromosomal deletions spanning several genes. The genes are too small to be detected under a microscope. Autosomal microdeletions involve microdeletions in the autosomes or non-sex chromosomes. Microdeletions also affect the sex chromosomal genes. Microdeletions range from deletion of a small region in a gene or several genes. Microdeletions in Y chromosome lead to missed genes. The condition is known as YCM or Y chromosome microdeletion. Although men with YCM do not exhibit symptoms, their fertility gets reduced with low sperm count. Specified partial deletions known as AzFc-gr/gr deletions cause infertility. Microdeletions known as X chromosome microdeletion affect both males and females.

The term haploinsufficiency describes microdeletion. In simple terms, haploinsufficiency means insufficiency of a single copy of a normal gene for producing protein, thereby affecting the function. Two situations arise in the case of haploinsufficiency. An individual, heterozygous for the gene mutation gets affected with deletion or microdeletion for a gene segment, a gene or a corresponding allele. Other situation arises when the individual is hemizygous for a particular locus. Deletion syndromes arise due to copy number losses. Submicroscopic differences (losses) in few sections of the DNA result into copy number variations. Microdeletions involve two types such as a terminal or interstitial deletions. Independent of a location of a gene, microdeletions occur anywhere such as Centromeric regions, telomeric regions or any other regions of the chromosomes. The deletions in the interstitial regions involve the regions between the centromere and the site of rearrangement. The microdeletions involving the chromosomal ends are known as terminal microdeletions. The inheritance of microdeletion syndromes follows autosomal or sex-linked inheritance. Few references site novel cases of telomeric microdeletions.

Two main classes of copy number variants (CNVs) include recurrent and non-recurrent copy number variants. Non-allelic homologous recombination (NAHR) gives way to recurrent copy number variants with breakpoints in the large duplicated sequences. Breakpoints in the unique sequences mark the non-recurrent CNVs.

Following examples include microdeletion syndromes:

1.     Prader-Willi syndrome:

This genetic disorder affects the muscles and the feeding abilities of children. It leads to obesity and diabetes accompanying intellectual impairment. It arises due to a loss of function mutation. A part of the 15^th chromosome of the father gets deleted leading to loss of the gene function. Hence, the genetic changes occur due to microdeletions. Prader-Willi syndrome involves a phenomenon known as genomic imprinting. The expression of genes involves a parent of origin-specific manner. Genes are known as snRNPs and necdin genes accompanying a few snoRNA genes get deleted. A part of the q arm of the 15^th chromosome consists of the above genes. Prader-Willi syndrome also includes cases with snoRNA-HBII-52 microdeletions.

Image 1: Gene mutations in Prader Willi syndrome

2.     Angelman’s syndrome

Nervous system impairment arises due to gene defects in Angelman’s syndrome. New mutations arise due to microdeletions. The patient’s mother exhibits a microdeletion on the 15^th chromosome. The patients or the proband inherit a mutated UBE3A gene on the 15^th chromosome. Angelman’s syndrome accompanies an inheritance of a loss of function mutation from the mother. However, very rare cases involve inheritance from the father.

Image 2: Gene mutations in Angelman's syndrome

3.     Wilm’s tumor:

11^th chromosome microdeletions increase the risk of Wilm’s tumor. The malignant tumor mainly affects the kidneys. Wilm’s syndrome involves alterations in the WT1 gene. Wilm’s tumor includes a group of disorders known as Wilm’s tumor Aniridia- Genitourinary malformations (WAGR). It involves intellectual disability and anxiety related problems.

4.     William’s syndrome:

It arises due to a microdeletion in the 7^th chromosome. The genes such as CLIP2, GTF 21, GTF21RD1, LIMK1, and other genes help in the detection. Individuals show affected neurological and behavioral characteristics. The children with William’s syndrome require interaction, counseling, and motivation. The condition arises either sporadically or due to inheritance.

5.     Langer-Giedion syndrome:

This syndrome is a rare autosomal dominant one. It involves a microdeletion in chromosome 8. The missed regions include TRPS1 and EXT1 genes. It occurs sporadically. However, father to son and mother to daughter transmission is possible. These individuals exhibit physical and dental anomalies.

6.     Miller-Dieker syndrome:

Microdeletions involve small arm of the 17^th chromosome and accompany congenital malformations. Miller-Dieker syndrome follows an autosomal dominant inheritance. Microdeletions in the 17^th chromosome result into loss of multiple genes. The parent of the proband shows balanced translocations. These translocated genes become unbalanced while getting passed on from generation to generation. Hence, it results in either a loss of genes or gain of the extra material. Miller-Dieker syndrome is a contiguous gene syndrome. Submicroscopic deletion includes LIS 1 gene.

7.     Di-George syndrome:

It involves a deletion in a small segment of the 22^nd chromosome. Prevalence of the microdeletion involves the middle region of the 22^nd chromosome. Di-George syndrome is an autosomal dominant inheritance. The syndrome involves heterozygous microdeletions and TBX1 gene haploinsufficiency.

8.     Smith-Magenis Syndrome:

This type of microdeletion leads to a deletion in the short arm of the 15^th chromosome. Mainly the RAI1 gene of the 17^th chromosome gets affected. The patients with the condition show abnormalities in the jaw, eyes, nasal bridge and the teeth. Such an individual has a short stature and hearing problems.

9.     Rubinstein Taybi syndrome:

It involves physical and facial deformities such as short stature, broad thumbs, and toes. These individuals show susceptibility to cancer. The condition is an autosomal dominant one. A microdeletion in the 16^th chromosome involves CREBBP gene deletion. The gene CREBBP encodes for CREB binding protein that regulates the cell cycle and development.

10. Neurofibromatosis:

Two types of neurofibromatosis involve NF-1 and NF-2 respectively. NF-1 or Neurofibromatosis type 1 involves a mutation in a gene present on the 17^th chromosome. The gene encodes a protein known as neurofibromin, needed for normal functioning of human cell types. NF-1 is an autosomal dominant disorder. Neurofibromatosis type 2 is a genetic disorder involving NF-2 gene mutation on the 22^nd chromosome. It is also an autosomal dominant disorder.

11.Wolf-Hirschhorn syndrome:

It involves a partial deletion in the short arm of chromosome 4. Most of the cases exhibit de novo deletions. These patients exhibit craniofacial anomalies and intellectual disability.

12.Cri-du-chat syndrome:

It results in deletion in the short arm of the 5^th chromosome. The syndrome also arises due to microdeletions. Individuals with this condition have a high pitched voice resembling that of a cat.

Microdeletions in the mitochondrial DNA:

Few cases of infertility in males involved microdeletions in the mitochondrial DNA in the spermatozoa. In girls, a microdeletion in the cytochrome c oxidase (COX) subunit II in the mitochondrial DNA passes on exclusively from the mother, since the mtDNA inheritance follows maternal inheritance.

Detection of the microdeletion syndromes:

Various new methods help to detect microdeletions. Detection plays an important role in therapeutics. Following examples include detection tests:

1.     Prenatal diagnosis:

The invasive ways of the prenatal diagnosis involve a collection of fetal cells through amniocentesis or chorionic villus sampling. However, with the advancement in technology, the development of non-invasive techniques came into existence. There is an expansion in the global market for non-invasive prenatal techniques. NIPT or non-invasive prenatal testing helps in detecting aneuploidies, microdeletions and many other conditions. NIPT uses ultrasonography and serum screening. Unlike amniocentesis and chorionic villus sampling, NIPT uses cell-free DNA floating in the maternal plasma. These tests involve cell-free fetal DNA (cfDNA) screening along with different algorithms. The cfDNA test easily detects microdeletions.

2.     Next-generation sequencing and array CGH:

Screening of microdeletions also involves microarray and NGS technologies. These techniques detect small deletions. More and more advances are happening in the whole genome sequencing and exome sequencing techniques. Microarrays measure gain or loss of genes or portion of the genes throughout the genome. It involves detection of the copy number variants and single nucleotide polymorphisms. The whole genome sequencing analyses the entire genome including the introns, exons and other sequences. Exome sequencing involves the study of only the exons since introns are non-coding sequences.

3.     FISH:

The FISH technique involves detection of microdeletions and deletions less than five megabases. The technique identifies specific chromosomes, regions, genes and gene segments through hybridization. The fluorescently labeled probes attach to specific regions or DNA segments. The examination of the sample slides under a fluorescent microscope reveals striking results. The fluorescent lighting detects the presence of hybridized DNA through a fluorescent signal. The flashing of the fluorescent signal indicates the presence of the chromosomal material under study. No fluorescent signal indicates the absence of the material under study. The FISH technique detects microdeletions in the chromosomes, interphase nuclei, and the sperms. Metaphase FISH involves analyzing the chromosomes in the metaphase.

4.     Multiplex ligation-dependent probe amplification (MLPA):

This technique spans 50 different DNA sequences in one go. Thus, it is very helpful in detecting the copy number variations, microdeletions, microduplications, and sub-telomeric deletions and duplications. It also uses the PCR technique. MLPA is a variation of multiplex-PCR and allows amplification of multiple targets with only a primer pair.

References:
[1] Medical Genetics, Ian D Young
[2] Clinical Cytogenetics, An Issue of Clinics in Laboratory Medicine, Caroline Astbury
[3] Management of Genetic Syndromes, Suzanne B. Cassidy, Judith E. Allanson
[4] Molecular Cytogenetics: Protocols and Applications, Yao-Shan Fan

© Copyright, 2018 All Rights Reserved.

A review on human chromosomes

An Introduction to chromosomes:

The ability of the chromosomes in getting stained reveals the true nature of the chromosome (derived from the Greek work word. The word chroma indicates color). These stainable bodies appear like threads under a microscope. They contain the genetic material, mainly the DNA coiled around the proteins. Human chromosomes are visible with when the cell is undergoing mitotic or meiotic cell division. There are total 46 chromosomes in each human cell.

·    Autosomes: There are total 44 autosomes or 22 pairs. Each pair consists of homologous chromosomes. In a pair, one chromosome comes from the father and the other chromosome comes from the mother.

·        Sex Chromosomes: There are two different types of sex chromosomes, X and Y respectively.

The average size of the human metaphase chromosome is 5 millimeters. Chromosomes tightly coil and get condensed during metaphase. Chromosomes appear in different shapes during each phase of the cell cycle. They appear thread-like during interphase. During metaphase, chromosomes look like rod-shaped. They look like V, J or rod-shaped during anaphase. Von Hartz coined the term chromosome. The scientists who first discovered the structure of chromosomes were Schleiden, Virchow, and Bütschli. Walter Sutton and Theodor Boveri independently developed the chromosome theory of inheritance in 1902.

An interphase nucleus contains strands of a material called chromatin. There are two regions in the chromatin, mainly coiled and extended regions.

·    Heterochromatin: It is the dark staining area of the chromatin. There are two types of heterochromatin. Constitutive heterochromatin contains repetitive sequences. It is present near the centromere. Constitutive heterochromatin never expresses itself. There is one more type of heterochromatin, known as facultative heterochromatin, which expresses itself.

·        Euchromatin: It is the light staining area of the chromatin.

The chief constituent of chromatin is DNA, the blueprint of life. At the time of cell division, chromatin strands coil into compact structures, so that they easily fit into the cell nucleus. Chromosomes appear as thick rods only during the cell division. They uncoil and form chromatin at the end of cell division.

Image: Human chromosomes revealed through karyotyping

Structure of the chromosome:

Metaphase chromosome appears clearly under the microscope. Following are the principal point to be discussed:

·        Chromatids: Each metaphase chromosome consists of two symmetrical halves parallel to each other. They are called chromatids. These chromatids are present in the form of chromonema during prophase. There are two types of chromatids mainly, sister and non-sister chromatids. Two chromatids are present on a single chromosome. Thus they are called sister chromatids. The concept of dyad describes a pair of sister chromatids. These structures join with the help of a centromere.  Non-sister chromatids are either of the two chromatids of a chromosome pair.

·        Centromere: A centromere is a light staining constricted area to which both the chromatids are attached. A centromere divides the chromatids into short and long arms respectively. The short arm is known as “p” arm. The long arm is known as “q” arm. Centromere produces a primary constriction. It is the position of the centromere. It is different for different chromosomes. Secondary constriction is also known as nucleolar organizer region. It is involved in the formation of the nucleolus. Human centromere consists of several hundred kilobases of repetitive DNA. Centromeres are the sites where spindle fibers are attached. Thus, centromere helps in the movement of the chromosome.

·     Satellite: A satellite is a region that is attached to the chromosome by a thread of chromatin. It is present at the distal region of the arm of the chromosome.

·        Telomere: A special DNA-protein complex is present at the ends of the chromosomes. This complex is known as a telomere, with tandem repeats of TTAGGG-3’ sequences between 3-20 kilobases in length. Telomeres are not genes since they do not code for any functional molecule. Telomeres provide structural stability to the chromosomes by sealing their ends. Telomeres protect the chromosomes from damage. They also protect the chromosome from fusing into a ring or binding to other DNA.

Classification of chromosomes:

1.     Classification based on the position of the centromere:  

·     Metacentric: The two arms are almost equal in their lengths. The location of the centromere is at the center of the chromosome.

·   Submetacentric: The two arms of the submetacentric chromosomes are unequal in length. The location of the centromere is slightly away from the center.

·   Acrocentric: One arm of an acrocentric chromosome is short. Whereas, the other arm is long.

·      Telocentric: A telocentric chromosome has only one arm.

2.     Standard classification: It is also known as Denver classification. It classifies chromosomes into seven groups, depending on the length of the chromosomes.

·  Group A: This group consists of pairs of chromosomes 1, 2 and 3. Chromosome 1 is the largest human chromosome. It represents 8% of the total DNA content.  Chromosome 2 is the second one. The third chromosome represents 6.5 % of the total DNA content.

·       Group B: This group consists of pairs of chromosome 4 and 5.

·    Group C: This group consists of pairs of chromosome 6, 7, 8, 9, 10, 11 and 12.

·        Group D: This group consists of pairs of chromosomes 13, 14 and 15.

·        Group E: This group consists of pairs of chromosomes 16, 17 and 18.

·        Group F: This group consists of pairs of chromosomes 19 and 20.

·        Group G: This group consists of pairs of chromosomes 21 and 22.

·  Sex Chromosomes: There are two types of sex chromosomes. X chromosome and Y chromosome are called sex chromosomes.

3.     Paris nomenclature: According to this method, the long and short arms of the chromosomes have specific regions that get stained. These regions are further stained using banding techniques. Such techniques may not only help to identify specific chromosomes, but also find out the location within the chromosome. Banding techniques may help to detect minor structural abnormalities.

What is sex chromatin?

The nucleus in the interphase is in the resting phase. An interphase nucleus shows a dark stain chromatin mass attached on one side of the nuclear membrane. Sex chromatin is also known as the Barr body. It is observed only in females. However, the chromatin determination using the Barr body is not as accurate as the Karyotyping technique.

What are chromosomal aberrations?

Chromosomal mutations or aberrations are variation in the normal chromosome structure or chromosome number.

A deletion is a chromosomal mutation in which a part of a chromosome is missing. Chromosomal breaks result in deletions. Sometimes an entire chromosome may get deleted. Duplication may lead to doubling of a chromosomal segment. Excision of a chromosomal segment follows reinsertion leading to an inversion. A translocation is a chromosomal mutation in which a chromosome segment gets positioned in a different location in the genome.

Chromosome Analysis:

Chromosome analysis indicates a proper diagnosis of many clinical conditions. It is a microscopic analysis of chromosomes in the dividing cells. Chromosomal analysis can detect chromosome number and structure.

Uses of chromosome analysis are as follows:

·        Detection of congenital malformations, mental retardation, and repeated abortions.

·        Prenatal diagnosis

·        Diagnosis of malignancies

Karyotyping:

Karyotyping is a test to evaluate the number and the structure of the chromosomes. In this procedure, the metaphase chromosomes are obtained and photographed. The procedure of karyotyping is specialized. Peripheral blood lymphocytes, bone marrow cells or amniotic fluid samples are collected and analyzed for chromosomes. A photo-micrograph reveals chromosomes scattered randomly. These chromosomes are arranged into groups, using the software. A karyotype can be sued to detect chromosomal abnormalities.

Chromosome Banding:

Analysis of chromosome becomes precise with the help of banding techniques. There are four types of banding techniques such as G-banding, Q-banding, R-banding, and C-banding. A unique pattern of light and dark bands are obtained using the G-banding technique.

Fluorescence in-situ hybridization (FISH):

FISH is a new diagnostic technique that involves a single-stranded probe annealing to its complementary sequence. FISH can be used to detect minute chromosomal aberrations, malignancies, and study of chromosomes.

References:
[1] Medical genetics, G.P. Pal
[2] Human Genetics, 3/e, Gangane
[3] Vogel and Motulsky's Human Genetics: Problems and Approaches, Friedrich Vogel, Gunter Vogel, Arno G. Motulsky
[4] Biology for the IB Diploma: Standard and Higher Level, Andrew Allott
[5] Principles of Medical Genetics, Thomas D. Gelehrter

© Copyright, 2018  All Rights Reserved