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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">An Overview of Mutation Detection Methods in Genetic Disorders</title><author><name sortKey="Mahdieh, Nejat" sort="Mahdieh, Nejat" uniqKey="Mahdieh N" first="Nejat" last="Mahdieh">Nejat Mahdieh</name><affiliation><nlm:aff id="AF0001">Medical Genetic Group, Faculty of Medicine, Ilam University of Medical Sciences, Ilam, Iran</nlm:aff></affiliation></author><author><name sortKey="Rabbani, Bahareh" sort="Rabbani, Bahareh" uniqKey="Rabbani B" first="Bahareh" last="Rabbani">Bahareh Rabbani</name><affiliation><nlm:aff id="AF0002">Medical Genetic Group, Faculty of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran</nlm:aff></affiliation><affiliation><nlm:aff id="AF0003">Growth and Development Research Center, Tehran University of Medical Sciences, Tehran, Iran</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">24427490</idno><idno type="pmc">3883366</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3883366</idno><idno type="RBID">PMC:3883366</idno><date when="2013">2013</date><idno type="wicri:Area/Pmc/Corpus">004A89</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">004A89</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">An Overview of Mutation Detection Methods in Genetic Disorders</title><author><name sortKey="Mahdieh, Nejat" sort="Mahdieh, Nejat" uniqKey="Mahdieh N" first="Nejat" last="Mahdieh">Nejat Mahdieh</name><affiliation><nlm:aff id="AF0001">Medical Genetic Group, Faculty of Medicine, Ilam University of Medical Sciences, Ilam, Iran</nlm:aff></affiliation></author><author><name sortKey="Rabbani, Bahareh" sort="Rabbani, Bahareh" uniqKey="Rabbani B" first="Bahareh" last="Rabbani">Bahareh Rabbani</name><affiliation><nlm:aff id="AF0002">Medical Genetic Group, Faculty of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran</nlm:aff></affiliation><affiliation><nlm:aff id="AF0003">Growth and Development Research Center, Tehran University of Medical Sciences, Tehran, Iran</nlm:aff></affiliation></author></analytic><series><title level="j">Iranian Journal of Pediatrics</title><idno type="ISSN">2008-2142</idno><idno type="eISSN">2008-2150</idno><imprint><date when="2013">2013</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Genetic disorders are traditionally categorized into three main groups: single-gene, chromosomal, and multifactorial disorders. Single gene or Mendelian disorders result from errors in DNA sequence of a gene and include autosomal dominant (AD), autosomal recessive (AR), X-linked recessive (XR), X-linked dominant and Y-linked (holandric) disorders. Chromosomal disorders are due to chromosomal aberrations including numerical and structural damages. Molecular and cytogenetic techniques have been applied to identify genetic mutations leading to diseases. Accurate diagnosis of diseases is essential for appropriate treatment of patients, genetic counseling and prevention strategies. Characteristic features of patterns of inheritance are briefly reviewed and a short description of chromosomal disorders is also presented. In addition, applications of cytogenetic and molecular techniques and different types of mutations are discussed for genetic diagnosis of the pediatric genetic diseases. The purpose is to make pediatricians familiar with the applications of cytogenetic and molecular techniques and tools used for genetic diagnosis.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Mahdieh, N" uniqKey="Mahdieh N">N Mahdieh</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mahdieh, N" uniqKey="Mahdieh N">N Mahdieh</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Richette, P" uniqKey="Richette P">P Richette</name></author><author><name sortKey="Bardin, T" uniqKey="Bardin T">T Bardin</name></author><author><name sortKey="Stheneur, C" uniqKey="Stheneur C">C Stheneur</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Su, N" uniqKey="Su N">N Su</name></author><author><name sortKey="Sun, Q" uniqKey="Sun Q">Q Sun</name></author><author><name sortKey="Li, C" uniqKey="Li C">C 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Iran J Pediatr</journal-id><journal-id journal-id-type="iso-abbrev">Iran J Pediatr</journal-id><journal-id journal-id-type="publisher-id">IJPD</journal-id><journal-title-group><journal-title>Iranian Journal of Pediatrics</journal-title></journal-title-group><issn pub-type="ppub">2008-2142</issn><issn pub-type="epub">2008-2150</issn><publisher><publisher-name>Tehran University of Medical Sciences</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">24427490</article-id><article-id pub-id-type="pmc">3883366</article-id><article-id pub-id-type="publisher-id">IJPD-23-375</article-id><article-categories><subj-group subj-group-type="heading"><subject>Review Article</subject></subj-group></article-categories><title-group><article-title>An Overview of Mutation Detection Methods in Genetic Disorders</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Mahdieh</surname><given-names>Nejat</given-names></name><degrees>PhD</degrees><xref ref-type="aff" rid="AF0001">1</xref></contrib><contrib contrib-type="author"><name><surname>Rabbani</surname><given-names>Bahareh</given-names></name><degrees>PhD</degrees><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="aff" rid="AF0002">2</xref><xref ref-type="aff" rid="AF0003">3</xref></contrib></contrib-group><aff id="AF0001"><label>1</label>Medical Genetic Group, Faculty of Medicine, Ilam University of Medical Sciences, Ilam, Iran</aff><aff id="AF0002"><label>2</label>Medical Genetic Group, Faculty of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran</aff><aff id="AF0003"><label>3</label>Growth and Development Research Center, Tehran University of Medical Sciences, Tehran, Iran</aff><author-notes><corresp id="cor1"><label>*</label><bold>Corresponding Author:</bold><bold>Address:</bold> Medical Genetic Group, Faculty of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran. <bold>E-mail:</bold><email xlink:href="baharehrabbani@yahoo.com">baharehrabbani@yahoo.com</email></corresp></author-notes><pub-date pub-type="ppub"><month>8</month><year>2013</year></pub-date><volume>23</volume><issue>4</issue><fpage>375</fpage><lpage>388</lpage><history><date date-type="received"><day>04</day><month>7</month><year>2012</year></date><date date-type="accepted"><day>06</day><month>4</month><year>2013</year></date></history><permissions><copyright-statement>© 2013 Iranian Journal of Pediatrics & Tehran University of Medical Sciences</copyright-statement><copyright-year>2013</copyright-year><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by-nc/3.0/"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0), which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.</license-p></license></permissions><abstract><p>Genetic disorders are traditionally categorized into three main groups: single-gene, chromosomal, and multifactorial disorders. Single gene or Mendelian disorders result from errors in DNA sequence of a gene and include autosomal dominant (AD), autosomal recessive (AR), X-linked recessive (XR), X-linked dominant and Y-linked (holandric) disorders. Chromosomal disorders are due to chromosomal aberrations including numerical and structural damages. Molecular and cytogenetic techniques have been applied to identify genetic mutations leading to diseases. Accurate diagnosis of diseases is essential for appropriate treatment of patients, genetic counseling and prevention strategies. Characteristic features of patterns of inheritance are briefly reviewed and a short description of chromosomal disorders is also presented. In addition, applications of cytogenetic and molecular techniques and different types of mutations are discussed for genetic diagnosis of the pediatric genetic diseases. The purpose is to make pediatricians familiar with the applications of cytogenetic and molecular techniques and tools used for genetic diagnosis.</p></abstract><kwd-group><kwd>Cytogenetics</kwd><kwd>Chromosomal Aberration</kwd><kwd>Molecular Diagnostic Technique</kwd><kwd>Carrier Detection</kwd><kwd>Mutation</kwd></kwd-group></article-meta></front><body><sec id="S0001"><title>Introduction</title><p>The diploid human genome including twenty-three pairs of chromosomes is composed of 20–25 thousand genes; haploid set is estimated to be 3.2*10<sup>9</sup> base pairs<sup>[<xref ref-type="bibr" rid="CIT0001">1</xref>]</sup>. One member of each chromosome is received from the father, and the other member of the pair is transmitted through maternal lineage. DNA is made up of four base pairs adenine, thymine, cytosine and guanine abbreviated as A, T, C, and G, respectively. Genes consisting of DNA base pairs are located on chromosomes. A gene is a sequence of base pairs that produces a functional product including a RNA molecule or subsequently a peptide (<xref ref-type="fig" rid="F0001">Fig. 1A</xref>). An allele is positioned on a locus, the specific location of a gene or DNA sequence on a chromosome; so the diploid genome contains two alleles of each gene. Chromosomes 1 to 22 are called autosomes and the twenty-third pair is the sex chromosomes, i.e. X and Y<sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>]</sup>.</p><fig id="F0001" position="float"><label>Fig. 1</label><caption><p>A) Nucleus, chromosomes, gene, exon, intron and base pairs. B) Central dogma. C1) Common symbols for drawing a pedigree. C2) Autosomal recessive mode of inheritance; C3) X-linked recessive inheritance; C4) Autosomal dominant inheritance</p></caption><graphic xlink:href="IJPD-23-375-g001"></graphic></fig><p>Steps in the transmission of genetic information include replication (DNA makes DNA), transcription (DNA makes RNA), RNA processing (capping, splicing, tailing and RNA translocation to cytoplasm), translation (RNA makes protein), and protein processing, folding, transport and incorporation (<xref ref-type="fig" rid="F0001">Fig. 1B</xref>). If the DNA sequence is mutated and the alteration is not repaired by the cell, subsequent replications reproduce the mutation. A variety of mechanisms can cause mutations ranging from a single nucleotide alteration to the loss, duplication or rearrangement of chromosomes<sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>]</sup>. Genetic diseases are usually categorized into three major classes: single-gene, chromosomal, and multi-factorial disorders. A combination of genes and environmental factors is involved in multifactorial disorders such as congenital heart disease, most types of cleft lip/palate, club foot, and neural tube defects<sup>[<xref ref-type="bibr" rid="CIT0003">3</xref>]</sup>.</p><p>Here, Mendelian patterns of inheritance and chromosomal disorders are reviewed and a brief summary of genetic methods in genetic disorders is presented to make the pediatricians familiar with the basics of the cytogenetics and molecular methods of mutation detection as well.</p></sec><sec id="S0002"><title>Mendelian Disorders</title><p>Gregor Mendel discovered a set of principles of heredity in the mid-19th century; characteristic patterns of inheritance are determined on the basis of these principles. Single gene disorders arising from errors in DNA sequence of a gene are categorized into autosomal dominant (AD), autosomal recessive (AR), X-linked recessive (XR), X-linked dominant and Y-linked (holandric) disorders<sup>[<xref ref-type="bibr" rid="CIT0003">3</xref>]</sup>.</p><p>In autosomal dominant disorders (<xref ref-type="fig" rid="F0001">Fig. 1C1</xref> and <xref ref-type="fig" rid="F0004">4</xref>), damage in one allele of a pair of the gene leads to the deficiency (<xref ref-type="table" rid="T0001">Table 1</xref>)<sup>[<xref ref-type="bibr" rid="CIT0003">3</xref>]</sup>, e.g. a mutation in FGFR3 gene can cause achondroplasia<sup>[<xref ref-type="bibr" rid="CIT0004">4</xref>, <xref ref-type="bibr" rid="CIT0005">5</xref>]</sup>. A parent with an autosomal dominant disorder has a 50% chance of transmitting the disease to her/his child<sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>]</sup>. The range of signs and symptoms of some diseases in different people vary widely (variable expressivity), e.g. some people with Marfan syndrome (due to mutation in FBN1) have only mild symptoms (such as being tall and thin with long, slender fingers), while others have life-threatening complications involving the heart and blood vessels as well<sup>[<xref ref-type="bibr" rid="CIT0006">6</xref>]</sup>. Furthermore, some individuals exhibit signs and symptoms of a given disorder while others do not, even though they have the disease-causing mutation (i.e. a proportion of people with a particular mutation show the condition in this type of disorders), e.g. many people having mutation of the BRCA1 gene will develop breast cancer during their lifetime, while some people will not<sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>, <xref ref-type="bibr" rid="CIT0007">7</xref>]</sup>. In other words, in a pedigree a healthy individual has at least one affected parent and one affected child (skipped generation)<sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>]</sup>.
</p><table-wrap id="T0001" position="float"><label>Table 1</label><caption><p>Modes of inheritance and their properties</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" style="background-color: #000080; color:white" rowspan="1" colspan="1">Inheritance pattern</th><th align="left" style="background-color: #000080; color:white" rowspan="1" colspan="1">Description</th><th align="center" style="background-color: #000080; color:white" rowspan="1" colspan="1">Examples</th></tr></thead><tbody><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>Autosomal Dominant</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">One mutated allele causes the disease Each affected person usually has one affected parent Appears in every generation of an affected family (Vertical)</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">Marfan syndrome; Achondroplasia; Huntington disease; Myotonic dystrophy</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Autosomal Recessive</bold></td><td align="left" rowspan="1" colspan="1">Two mutated alleles needed to cause the disease Parents are usually unaffected heterozygotes Not typically seen in every generation (Horizontal).</td><td align="center" rowspan="1" colspan="1">Beta thalassemia; Cystic fibrosis; Homocystinuria</td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>X-linked Dominant</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">Females are more frequently affected than males no male-to-male transmission</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">Rett syndrome; Hypophosphatemia</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>X-linked Recessive</bold></td><td align="left" rowspan="1" colspan="1">Males are more frequently affected than females Both parents of an affected daughter must be carriers Fathers cannot pass X-linked traits to their sons</td><td align="center" rowspan="1" colspan="1">Hemophilia; Duchenne Muscular Dystrophy</td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>Mitochondrial</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">Only females can pass on mitochondrial conditions to their children (maternal inheritance) Both males and females can be affected Can appear in every generation of a family</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">LHON: Leber's hereditary optic neuropathy</td></tr></tbody></table></table-wrap><p>Mutations in both alleles (loss of function) of a gene are required to cause the defect to appear in an autosomal recessive disorder (<xref ref-type="table" rid="T0001">Table 1</xref> and <xref ref-type="fig" rid="F0001">Fig. 1C2</xref>), i.e. an affected person has got one abnormal allele from one heterozygous parent. In this type of disorders, there is a 25% chance of having an affected offspring for heterozygous parents. In case of common autosomal recessive disorders or traits (sickle cell anemia due to a specific mutation in HBB gene encoding beta globin<sup>[<xref ref-type="bibr" rid="CIT0008">8</xref>]</sup> or nonsyndromic autosomal recessive hearing loss due to mutations in GJB2 gene encoding connexin 26<sup>[<xref ref-type="bibr" rid="CIT0009">9</xref>–<xref ref-type="bibr" rid="CIT0014">14</xref>]</sup>), sometimes a homozygous affected person marries a heterozygous carrier; such an example, in which apparently dominant transmission of this disorder occurs, is called pseudodominant inheritance<sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>]</sup>.</p><p>In an X-linked disorder (<xref ref-type="fig" rid="F0001">Fig. 1C3</xref> and <xref ref-type="table" rid="T0001">Table 1</xref>), the mutated gene is located on the X chromosome. A recessive mutation can lead to the disease. The gene in chromosome X should be mutated to cause the condition; hence, an X-linked recessive disorder is carried by females, while usally affects males.</p><p>Some of genetic conditions follow none of the mentioned patterns of inheritance; mitochondrial diseases, trinucleotide expansion disorders and genomic imprinting defects have non-Mendelian or nontraditional pattern of inheritance<sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>, <xref ref-type="bibr" rid="CIT0015">15</xref>]</sup>.</p></sec><sec id="S0003"><title>Chromosomal Disorders</title><p>Typically, somatic cells proliferate via division called mitosis while germ cells are produced through meiosis division. Meiosis involves a reductional division followed by an equational division, Meiosis I and II, respectively.</p><p>Oogenesis begins in the female fetus at 12 weeks, but it is stopped in a stage of meiosis I (when the homologous chromosomes have replicated and paired as bivalents or tetrads) at about 20 weeks<sup>[<xref ref-type="bibr" rid="CIT0016">16</xref>]</sup>. At puberty usually only one oocyte is released per month; a primary oocyte completes meiosis I and produces one secondary oocyte and one polar body. Chromosomal aberrations including numerical (due to errors at chromosome pairing and crossing-over) and structural damages lead to chromosomal disorders (<xref ref-type="table" rid="T0002">Table 2</xref>, <xref ref-type="table" rid="T0003">3</xref> and <xref ref-type="table" rid="T0004">4</xref>; <xref ref-type="fig" rid="F0002">Fig. 2A</xref> and <xref ref-type="fig" rid="F0002">B</xref>). Aneuploidy is usually due to failure of segregation of chromosomes in meiosis I or meiosis II (non-disjunction, premature disjunction or anaphase lag) <sup>[<xref ref-type="bibr" rid="CIT0017">17</xref>]</sup>; examples of numerical aberrations include Down syndrome (trisomy 21), Edwards syndrome (trisomy 18), Patau syndrome (trisomy 13), Klinefelter syndrome (XXY syndrome), Turner syndrome (monosomy X) and trisomy X. Chromosomal errors in oocytes are increased dramatically with maternal age. Non-disjunction or chromosome lag during mitosis can lead to mosaicism<sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>]</sup>.
</p><fig id="F0002" position="float"><label>Fig. 2</label><caption><p>A) Chromosome segregation during meiosis, nondisjunction and its consequences. Nondisjunction (NDJ) at meiosis I and II leads to uniparental heterodisomy and uniparental isodisomy, respectively. B) Structural aberrations of human chromosomes. C) Human karyotype. Chromosomes in a typical male. D) Fluorescence in situ hybridization (FISH).</p></caption><graphic xlink:href="IJPD-23-375-g002"></graphic></fig><table-wrap id="T0002" position="float"><label>Table 2</label><caption><p>Examples of numerical aberrations (aneuploidies)</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" style="background-color: #000080; color:white" rowspan="1" colspan="1">Aneuploidy</th><th align="center" style="background-color: #000080; color:white" rowspan="1" colspan="1">Karyotype</th><th align="center" style="background-color: #000080; color:white" rowspan="1" colspan="1">Incidence</th><th align="center" style="background-color: #000080; color:white" rowspan="1" colspan="1">Main features</th></tr></thead><tbody><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>Down syn. Trisomy 21</bold></td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">47, XX or XY, +21</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">1/700 live births</td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">Epicanthal folds, hypotonia, flat occiput, Brushfield spots in irides, single transverse crease, clinodactyly, etc.</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Edward syn. Trisomy 18</bold></td><td align="center" rowspan="1" colspan="1">47, XX or XY, +18</td><td align="center" rowspan="1" colspan="1">1/3000 live births</td><td align="left" rowspan="1" colspan="1">Clenched fists, rocker bottom feet, low-set, malformed ears, micrognathia, cardiac and renal abnormalities, etc.</td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>Patau syn. Trisomy 13</bold></td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">47, XX or XY, +13</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">1/5000 live births</td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">Microcephaly, holoprosencephaly, rocker-bottom feet, microphthalmia, anophthalmia, cyclopia, cryptorchidism, heart defects; cleft lip and palate, etc.</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Klinefelter syn</bold>.
</td><td align="center" rowspan="1" colspan="1">47, XXY ( 48, XXXY; 49, XXXXY)</td><td align="center" rowspan="1" colspan="1">1/500 male births</td><td align="left" rowspan="1" colspan="1">Gynecomastia, small genitalia and infertility</td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>Turner syn</bold>.
</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">45, X</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">1/5000 female births</td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">Fail to mature sexually, lymphedema, webbed neck, low posterior hairline, cubitus valgus, etc.</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>XYY syn</bold>.
</td><td align="center" rowspan="1" colspan="1">47, XYY</td><td align="center" rowspan="1" colspan="1">1/1000 male births</td><td align="left" rowspan="1" colspan="1">Tall stature, large teeth; fertility is normal</td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>Triple X syn</bold>.
</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">47, XXX</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">1/1000 female births</td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">Some learning problems</td></tr></tbody></table><table-wrap-foot><p>Syn: syndrome</p></table-wrap-foot></table-wrap><table-wrap id="T0003" position="float"><label>Table 3</label><caption><p>Chromosomal aberrations in human disorders</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" style="background-color: #000080; color:white" rowspan="1" colspan="1">Mutation</th><th align="center" style="background-color: #000080; color:white" rowspan="1" colspan="1">Definition</th><th align="center" style="background-color: #000080; color:white" rowspan="1" colspan="1">Example (Gene)</th><th align="center" style="background-color: #000080; color:white" rowspan="1" colspan="1">Disease/condition</th><th align="center" style="background-color: #000080; color:white" rowspan="1" colspan="1">Ref</th></tr></thead><tbody><tr><td colspan="5" align="left" rowspan="1"><bold>Structural aberrations</bold></td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>Deletion</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">A part of a chromosome is deleted</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">46,XX,del (5p15.2-pter)</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">Cat Cry Syn.</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1"><sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>]</sup></td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Duplication</bold></td><td align="left" rowspan="1" colspan="1">A portion of a chromosome is duplicated</td><td align="center" rowspan="1" colspan="1">46, XX, dup (22q11.2)</td><td align="center" rowspan="1" colspan="1">Cat Eye Syn.</td><td align="center" rowspan="1" colspan="1"><sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>]</sup></td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>Translocation</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">An interchange of genetic material between nonhomologous chromosomes</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">46, XX, t (9; 22) (q34; q11)</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">CML</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1"><sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>]</sup></td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Reciprocal T</bold>.
</td><td align="left" rowspan="1" colspan="1">An interchange of genetic material between two nonhomologous chromosomes</td><td align="center" rowspan="1" colspan="1">46, XX, rcp (9; 22) (q34; q11)</td><td align="center" rowspan="1" colspan="1">CML</td><td align="center" rowspan="1" colspan="1"><sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>]</sup></td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>Robertsonian T</bold>.
</td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">The fusion of the long arms of two acrocentric chromosomes and loss of their short arms</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">45, XX, rob (14q21q)</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">Normal</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1"><sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>]</sup></td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Inversion</bold></td><td align="left" rowspan="1" colspan="1">A portion of a chromosome is inverted</td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>Pericentric Inv</bold>.
</td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">The inverted segment includes the centromere</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">46, XX, Inv (9) (p11q13)</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">Normal?</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1"><sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>]</sup></td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Paracentric Inv</bold>.
</td><td align="left" rowspan="1" colspan="1">The inverted segment is located on one arm of the chromosome</td><td align="center" rowspan="1" colspan="1">inv(14)(q13q24)</td><td align="center" rowspan="1" colspan="1">Microcephaly</td><td align="center" rowspan="1" colspan="1"><sup>[<xref ref-type="bibr" rid="CIT0051">51</xref>]</sup></td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>Ring</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">Both arms of a chromosome have fused together as a ring</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">46, Xr (X)</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">Turner syn.</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1"><sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>]</sup></td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Isochromosome</bold></td><td align="left" rowspan="1" colspan="1">A chromosome that has two identical arms because of duplication of one arm of the chromosome; a mirror-image of one arm of a chromosome</td><td align="center" rowspan="1" colspan="1">46, Xi (Xq)</td><td align="center" rowspan="1" colspan="1">Turner syn.</td><td align="center" rowspan="1" colspan="1"><sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>]</sup></td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>Dicentric chromosome</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">An abnormal chromosome that has two centromeres</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">46, X, psu dic (Y) (pter→q11.2::q11.2→pter)</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">Azoospermia</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1"><sup>[<xref ref-type="bibr" rid="CIT0052">52</xref>]</sup></td></tr><tr><td colspan="5" align="left" rowspan="1"><bold>Numerical aberrations</bold></td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>Aneuploidy</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">An abnormal number of chromosomes</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1"></td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1"></td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Monosomy</bold></td><td align="left" rowspan="1" colspan="1">The presence of only one of two homologous chromosome in a diploid organism (e.g. Human)</td><td align="center" rowspan="1" colspan="1">45, X</td><td align="center" rowspan="1" colspan="1">Turner Syn.</td><td align="center" rowspan="1" colspan="1"><sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>]</sup></td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>Uniparental disomy</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">Inheritance of two pairs of a homologous chromosome from one parent and no copy from the other parent (Fig 2)</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">46, XX, upd (15) mat</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">Prader-Willi syndrome</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1"><sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>]</sup></td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Trisomy</bold></td><td align="left" rowspan="1" colspan="1">Existence of three copies of a homologous chromosome</td><td align="center" rowspan="1" colspan="1">47, XX, +21</td><td align="center" rowspan="1" colspan="1">Down syn.</td><td align="center" rowspan="1" colspan="1"><sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>]</sup></td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>Tetrasomy</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">Existence of four copies of a homologous chromosome</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">48, XXXX</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">X tetrasomy</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1"><sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>]</sup></td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Monoploidy</bold></td><td align="left" rowspan="1" colspan="1">The state of having a single (non-homologous) set of chromosomes</td><td align="center" rowspan="1" colspan="1">23X</td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>Triploidy</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">Having three sets of chromosomes instead of two</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">69, XXX</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">Abortion, Hydatidiform mole</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1"><sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>]</sup></td></tr></tbody></table><table-wrap-foot><fn><p>Syn: Syndrome; Ref: Reference; T: Translocation; Inv: Inversion; psu dic: Psuedodicentric; rcp: Reciprocal; rob: Robertsonian; upd: Uniparental Disomy</p></fn></table-wrap-foot></table-wrap><table-wrap id="T0004" position="float"><label>Table 4</label><caption><p>Gene-based Mutations in human disorders</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" style="background-color: #000080; color:white" rowspan="1" colspan="1">Mutation</th><th align="center" style="background-color: #000080; color:white" rowspan="1" colspan="1">Definition</th><th align="center" style="background-color: #000080; color:white" rowspan="1" colspan="1">Example (Gene)</th><th align="center" style="background-color: #000080; color:white" rowspan="1" colspan="1">Disease/ condition</th><th align="center" style="background-color: #000080; color:white" rowspan="1" colspan="1">Ref</th></tr></thead><tbody><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>Point Mutation</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">A single base pair alteration; it includes transition: purine (A,G) to purine (G,A) or pyrimidine (C,T) to pyrimidine (T,C), and transversion: purine (A,G) to pyrimidine (T,C) or pyrimidine (T,C) to purine (A,G)</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">A > G, A > T</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1"></td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1"><sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>, <xref ref-type="bibr" rid="CIT0045">45</xref>]</sup></td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>a. Missense (Nonsynonymous)</bold></td><td align="left" rowspan="1" colspan="1">A single nucleotide resulting in a codon that codes for a different amino acid</td><td align="center" rowspan="1" colspan="1">A82P (HSD3B2)</td><td align="center" rowspan="1" colspan="1">3βHSD deficiency</td><td align="center" rowspan="1" colspan="1"><sup>[<xref ref-type="bibr" rid="CIT0046">46</xref>]</sup></td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>b. Nonsense</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">A single nucleotide resulting in a premature stop codon</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">G23X (HBB)</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">Beta Thalassaemia</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1"><sup>[<xref ref-type="bibr" rid="CIT0047">47</xref>]</sup></td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>c. Synonymous</bold></td><td align="left" rowspan="1" colspan="1">A single nucleotide that changes a codon to an amino acid with similar properties e.g. Lysine to Arginine</td><td align="center" rowspan="1" colspan="1">V153I (GJB2)</td><td align="center" rowspan="1" colspan="1">Hearing loss</td><td align="center" rowspan="1" colspan="1"><sup>[<xref ref-type="bibr" rid="CIT0010">10</xref>]</sup></td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>d. Silent</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">A single nucleotide which does not alter amino acid sequences e.g. GCT, GCC, GCA and GCG all code for alanine; any change in the third position of the codon (e.g. GCA > GCG), does not alter the amino acid sequence</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">I69I (GJB2)</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">Hearing loss</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1"><sup>[<xref ref-type="bibr" rid="CIT0010">10</xref>]</sup></td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>e. Neutral</bold></td><td align="left" rowspan="1" colspan="1">A single nucleotide which does not have any harmful or beneficial effect on the organism, it usually occurs at noncoding DNA regions</td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>Duplication</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">A region of a sequence is duplicated; if the number of nucleotides is not evenly divisible by three from DNA sequence it is called a frameshift mutation</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">920dupTCAG (LDLR)</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">Familial hypercholesterolemia</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1"><sup>[<xref ref-type="bibr" rid="CIT0048">48</xref>]</sup></td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Deletion</bold></td><td align="left" rowspan="1" colspan="1">A portion of a sequence is deleted; if the number of nucleotides is not evenly divisible by three from DNA sequence it is called a frameshift mutation</td><td align="center" rowspan="1" colspan="1">delE120 (GJB2)</td><td align="center" rowspan="1" colspan="1">Hearing loss</td><td align="center" rowspan="1" colspan="1"><sup>[<xref ref-type="bibr" rid="CIT0011">11</xref>]</sup></td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>Insertion</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">Addition of one or more nucleotide base pairs into a DNA sequence; if the number of nucleotides is not evenly divisible by three from DNA sequence it is called a frameshift mutation</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">3524insA (FBN1)</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">Marfan syndrome</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1"><sup>[<xref ref-type="bibr" rid="CIT0049">49</xref>]</sup></td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Splice mutation</bold></td><td align="left" rowspan="1" colspan="1">A sequence change in the site splicing of an intron; it may result in one or more introns remaining in mature mRNA.</td><td align="center" rowspan="1" colspan="1">IVS1 + 1G > A (GJB2)</td><td align="center" rowspan="1" colspan="1">Hearing loss</td><td align="center" rowspan="1" colspan="1"><sup>[<xref ref-type="bibr" rid="CIT0010">10</xref>]</sup></td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>Dynamic mutation</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">An unstable mutation in which the number of copies of a sequence is changed during meiosis division e.g. trinucleotide expansions</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">(CGG)n > 200 (FMR1)</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">Fragile X syndrome</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1"><sup>[<xref ref-type="bibr" rid="CIT0050">50</xref>]</sup></td></tr></tbody></table><table-wrap-foot><fn><p>Syn: Syndrome; Ref: Reference</p></fn></table-wrap-foot></table-wrap><p>Most of structural aberrations, including translocations, deletions, inversions, duplications, ring chromosomes and isochromosomes (<xref ref-type="table" rid="T0003">Table 3</xref>, <xref ref-type="table" rid="T0004">4</xref> and <xref ref-type="fig" rid="F0002">Fig. 2B</xref>) result from unequal exchange between chromosomes or enzymatic misrepairing of two chromosome breakages; examples of structural aberration are cat cry syndrome (5p-), Williams syndrome (7q11.2 deletion), DiGeorge syndrome (22q11.2 deletion), etc.</p></sec><sec id="S0004"><title>Types of Mutations</title><p>A mutation is a change in the nucleotide sequence in coding portions of the DNA which may alter the amino acid sequences of proteins, or a change in noncoding regions of DNA which has the potential for changing expression of the gene, for example by altering the strength of a promoter. There are many mutations which are classified to chromosomal and DNA-based mutations (<xref ref-type="table" rid="T0003">Table 3</xref> and <xref ref-type="table" rid="T0004">4</xref>). Mutations can also be categorized on the basis of the function: 1) The loss-of-function mutations cause a decrease or a loss of the gene product or the activity of the gene product; 2) The gain-of-function mutations cause an increase in the amount of gene product or its activity, and sometimes create a new property, leading to a toxic product responsible for a pathological effect.</p><p>Three types of mutations usually observed in dominant disorders are gain-of-function, haploinsuficiency and dominant negative. Mutations may act as dominant or recessive when the amount of product from one allele is not sufficient for a complete function (Haplo- insufficiency), e.g. mutations in LDLR leading to haploinsufficiency in familial hypercholesterolemia<sup>[<xref ref-type="bibr" rid="CIT0018">18</xref>]</sup>. If the product of the defective allele interferes with the product of normal allele (Dominant negative) it affects the function of normal protein; basically collagen mutations are dominant negative ones<sup>[<xref ref-type="bibr" rid="CIT0019">19</xref>]</sup>. A mutated allele may gain a new or excessive activity (Gain of function) e.g. mutations of FGFR3 in achondroplasia<sup>[<xref ref-type="bibr" rid="CIT0004">4</xref>, <xref ref-type="bibr" rid="CIT0005">5</xref>]</sup>.</p></sec><sec id="S0005"><title>Mutation Detection</title><p>With the development of new technologies for more accurate understanding of the genome and potential gene therapies, the detection of mutations has an increasingly central role in various areas of genetic diagnosis including preimplantation genetic diagnosis (PGD), prenatal diagnosis (PND), presymptomatic testing, confirmational diagnosis and forensic/identity testing. Two groups of tests, molecular and cytogenetic, are used in genetic syndromes. In general, single base pair mutations are identified by direct sequencing, DNA hybridization and/or restriction enzyme digestion methods. However, there are two approaches for genetic diagnosis; indirect approach depends on the results from a genetic linkage analysis using DNA markers such as STR(short tandem repeat) or VNTR (variable number tandem repeat) markers flanking or within the gene<sup>[<xref ref-type="bibr" rid="CIT0020">20</xref>–<xref ref-type="bibr" rid="CIT0022">22</xref>]</sup>. The direct approach for diagnosis essentially depends on the detection of the genetic variations responsible for the disease.</p></sec><sec id="S0006"><title>Cytogenetics and Molecular Cytogenetics</title><p><bold>Conventional Karyotyping</bold>: Chromosome studies are advised in the following situations: suspected chromosome abnormality, sexual disorders, multiple congenital anomalies and/or developmental retardation, undiagnosed learning disabilities, infertility or multiple miscarriage, stillbirth and malignancies<sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>]</sup>. Traditionally, the microscopic study of chromosomes is performed on compacted chromosomes at a magnification of about 1000 at metaphase.</p><p>Preparation of a visual karyotype (<xref ref-type="fig" rid="F0002">Fig 2C</xref>) is done by arresting dividing cells at metaphase stage with a microtubule polymerization inhibitor such as colchicine; the cells, then, are spread on a glass slide and stained with Giemsa stain (G-banding). Chromosomes are studied by making a photograph or digital imaging and subsequent assembling of chromosomes. Human chromos-omes are categorized based on position of centromere; in metacentric chromosomes, centromere is located in the middle (chromosomes 1, 3, 16, 19 and 20), chromosomes 13, 14, 15, 21, 22 and Y are acrocentric (the centromere near one end), and other chromosomes are sub-metacentric. Chromosome arms are defined by region number (from centromere), band, sub-band and sub-sub-band numbers, e.g. 12q13.12 refers to chromosome 12, long arm, region 1, band 3, sub-band 1, sub-sub-band 2 (read chromosome 12, q, 1, 3, point, 1, 2). High resolution banding needs fixation before the chromosomes are fully compacted. The convenient methods of chromosome banding are G-(Giemsa), R-(reverse), C-(centromere) and Q-(quinacrine) banding.</p><p><bold>Fluorescence in situ hybridization (FISH)</bold>: FISH is applied to provide specific localization of genes on chromosomes. Rapid diagnosis of trisomies and microdeletions is acquired using specific probes. Usually a denatured probe is added to a metaphase chromosome spread and incubated overnight to allow sequence-specific hybridization. After washing off the unbound probe, the bound probe is visualized by its fluorescence under UV light; thus, the site of the gene of interest is observed as in situ (<xref ref-type="fig" rid="F0002">Fig 2D</xref>)<sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>, <xref ref-type="bibr" rid="CIT0023">23</xref>]</sup>. This technique is used to check the cause of trisomies, microdeletion syndromes, etc.</p><p><bold>Comparative genomic hybridization (CGH)</bold>: CGH, a special FISH technique (dual probes), is applied for detecting all genomic imbalances. The basics of technique is comparison of total genomic DNA of the given sample DNA (e.g. tumor DNA) with total genomic DNA of normal cells. Typically, an identical amount of both tumor and normal DNAs is labeled with two different fluorescent dyes; the mixture is added and hybridized to a normal lymphocyte metaphase slide. A fluorescent microscope equipped with a CCD camera and an image analysis system are used for evaluation<sup>[<xref ref-type="bibr" rid="CIT0024">24</xref>]</sup>. Technical details have been described in numerous CGH publications<sup>[<xref ref-type="bibr" rid="CIT0025">25</xref>, <xref ref-type="bibr" rid="CIT0026">26</xref>]</sup>. Copy number of genetic material (gains and losses) is calculated by evaluation software<sup>[<xref ref-type="bibr" rid="CIT0027">27</xref>]</sup>. CGH is used to determine copy number alterations of genome in cancer and those cells whose karyotype is hard or impossible to prepare or analyze. In array-CGH, metaphase slide is replaced by specific DNA sequences, spotted in arrays on glass slides<sup>[<xref ref-type="bibr" rid="CIT0028">28</xref>]</sup>; so its resolution is increased.</p></sec><sec id="S0007"><title>Molecular Diagnostics</title><p>In addition to genetic causes of disorders, predisposition to a disease or treatment options could be revealed by determining DNA variations. Molecular diagnostics provide a way for assessment of the genetic makeup of human; it combines laboratory medicine with molecular genetics to develop DNA/RNA-based analytical methods for monitoring human pathologies. A wide range of methods has been used for mutation detection. Molecular methods for identification of the disease-causing mutations could be classified as methods for known and methods for unknown mutations. Several criteria, however, have to be met for choosing a suitable method; for example the following points should be considered: type of nucleic acid (DNA or RNA), kind of specimen (e.g., blood, tissues, etc.), the number of mutations, and reliability of the method. The pediatricians need to be noticed when prescribing these tests to provide an accurate diagnosis for the patients.</p></sec><sec id="S0008"><title>A) Known Mutations</title><p>Many different approaches have been used for identifying known mutations. Usually starting with the polymerase chain reaction (PCR), additional assay steps are performed based on the type of mutation. <xref ref-type="table" rid="T0005">Table 5</xref> shows examples of some <italic>of the frequently used techniques as well as their advantages and disadvantages. Here, a brief view of some of these techniques is presented with focus on their applications</italic>.
</p><table-wrap id="T0005" position="float"><label>Table 5</label><caption><p>Cytogenetics and molecular methods for mutation detection</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" style="background-color: #000080; color:white" rowspan="1" colspan="1">Method</th><th align="left" style="background-color: #000080; color:white" rowspan="1" colspan="1"></th><th align="left" style="background-color: #000080; color:white" rowspan="1" colspan="1">Application</th><th align="center" style="background-color: #000080; color:white" rowspan="1" colspan="1">Advantage/disadvantage</th><th align="center" style="background-color: #000080; color:white" rowspan="1" colspan="1">Known mutation</th><th align="center" style="background-color: #000080; color:white" rowspan="1" colspan="1">Unknown mutation</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"><bold>Cytogenetics</bold></td><td align="left" rowspan="1" colspan="1"><bold>Karyotype</bold></td><td align="left" rowspan="1" colspan="1">Detecting numerical and gross structural aberrations</td><td align="center" rowspan="1" colspan="1">Low resolution Time consuming and labor requirements</td><td align="center" rowspan="1" colspan="1">+</td><td align="center" rowspan="1" colspan="1">+</td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>FISH</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">Detecting trisomies, monosomies and microdeletions</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">Detects mosaicism</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">+</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">−</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><bold>CGH</bold></td><td align="left" rowspan="1" colspan="1">Detects copy number variations of genetic material</td><td align="center" rowspan="1" colspan="1">Used only for losses and gains</td><td align="center" rowspan="1" colspan="1">+</td><td align="center" rowspan="1" colspan="1">+</td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>Molecular</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>RFLP</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">Restriction fragments are separated by electrophoresis</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">Requires mutation in restriction site</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">+</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">−</td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>ARMS PCR</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">Allele-specific amplification of mutant and normal allele, determination of the genotype of an individual</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">Highly sensitive Possible to detect any known mutation May increase time and costs</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">+</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">−</td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>Multiplex PCR</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">Amplification of more than one target simultaneously</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">Reduces time and labor requirements Lower sensitivity and specificity</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">+</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">−</td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>Nested PCR</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">Amplification using external and internal primer sets</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">More sensitive Decreases nonspecific amplification</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">+</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">−</td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>RT-PCR</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">Amplification of RNA</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">Amplification of all RNA types May increase time and costs</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">+</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">+</td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>Real-time PCR</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">Amplification, detection, and quantification of target</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">Increased specificity Usually eliminates postamplification analyses More expensive</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">+</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">−</td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>MLPA</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">Deletions and duplications</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">A multiplex technique Identifies very small single gene aberrations (50-70 nt)</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">+</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">+</td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>DGGE</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">Based on migration within gradient gel electrophoresis</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">Detects close to 100% of point mutaions</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">−</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">+</td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>SSCP</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">Based on migration within gel electrophoresis</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">Detects about 80–90% of point mutations</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">−</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">+</td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>Hetero-duplex analysis</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">Based on homoduplices and heteroduplices motilities in gel electrophoresis</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">Detects nearly 80% of mutations</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">−</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">+</td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>CCM</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">DNA: DNA or DNA:RNA heteroduplices are cleaved by piperidine</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">All possible mutations are detectable Uses toxic substances</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">+</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">+</td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>PTT</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">It is based on a combination of PCR, transcription, and translation</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">Detects translation-terminating mutations Missense mutations are not detected.</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">−</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">+</td></tr><tr><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1"><bold>OLA</bold></td><td align="left" style="background-color: #dcdcff" rowspan="1" colspan="1">It is based on ligation of two flanked primers annealed with target sequences</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">Detects all base exchanges</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">+</td><td align="center" style="background-color: #dcdcff" rowspan="1" colspan="1">+</td></tr></tbody></table><table-wrap-foot><fn><p><italic>FISH: Fluorescence in situ hybridization; CGH: Comparative genomic hybridization; RELP: Restriction fragment length polymorphism;</italic> ARMS; Amplification refractory mutation system; PCR: Polymerase chain reaction; RT: Reverse transcriptase; MLPA; Multiplex ligation-dependent probe amplification; DGGE: Denaturing Gradient Gel Electrophoresis; SSCP: Single Strand Conformational Polymorphism; CCM: Chemical cleavage of mismatch; PTT: Protein truncation test; OLA: Oligonucleotide ligation assay</p></fn></table-wrap-foot></table-wrap><p><italic><bold>Polymerase chain reaction (PCR) and its versions:</bold></italic> In 1980s, Dr Mullis introduced a method for amplifying DNA fragment to a large number of fragments in only a few hours; this method, named polymerase chain reaction (PCR), was a critical point in molecular biology<sup>[<xref ref-type="bibr" rid="CIT0029">29</xref>, <xref ref-type="bibr" rid="CIT0030">30</xref>]</sup>. Essential components of polymerase chain reaction are template DNA, primers (a pair of synthetic oligonucleotides complementary to the two strands of target DNA), thermostable DNA polymerase enzyme (e.g. Taq), divalent cations (usually Mg<sup>2+</sup> ), deoxynucleoside triphosphates (dNTPs) and buffer solution. PCR, consisting of 25-40 repeated cycles, has three discrete steps of temperature changes (<xref ref-type="fig" rid="F0003">Fig. 3A</xref>); after a single temperature step at a high temperature (>90°C), a series of cycles of denaturation, annealing of primers and extension are performed and followed by a single temperature step called final product extension or brief storage. These steps are as follow:</p><fig id="F0003" position="float"><label>Fig. 3</label><caption><p>A) Polymerase chain reaction. B) DNA sequencing</p></caption><graphic xlink:href="IJPD-23-375-g003"></graphic></fig><p>Initial denaturation step includes heating the reaction to a temperature of 92–96°C for 1–9 minutes.<list list-type="order"><list-item><p>Denaturation step includes heating the reaction to 92–98°C for 20–30 seconds. The hydrogen bonds between complementary bases are disrupted and DNA molecules are denatured, yielding single-stranded DNA molecules (DNA melting).</p></list-item><list-item><p>Annealing step is performed by decreasing temperature to 50–65°C for 25–40 seconds; so the primers are annealed to their targets on single stranded DNAs by hydrogen bonds and a polymerase can bind to the primer-template hybrid and begin DNA polymerization in next step.</p></list-item><list-item><p>Extension step includes polymerization of the bases to the primers; a thermostable such as Taq polymerase extends a new strand complementary to the DNA template strand by adding matched dNTPs in 5’ to 3’ direction at a temperature of 72°C. A series of 25-40 repeated cycles of denaturation, annealing of primers and extension is performed to amplify the template fragment (<xref ref-type="fig" rid="F0003">Fig. 3A</xref>). Subsequently, a final elongation is sometimes done at 70–74°C for 5–15 minutes after the last PCR cycle to ensure full extension of any remaining single-stranded DNA<sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>]</sup>.
</p></list-item></list></p><p>Checking the PCR products, electrophoresis (agarose or polyacrylamide gel electrophoresis) is employed for sizing the PCR products by comparison with a DNA ladder (a molecular weight marker). Here, applications of some PCR versions are mentioned.<list list-type="order"><list-item><p><italic>Reverse transcriptase PCR (RT-PCR):</italic> In this version, a strand of RNA molecule is transcribed reversely into its complementary DNA (cDNA) using the reverse transcriptase enzyme. This cDNA is then amplified by PCR. RT-PCR is applied to study the mutations at RNA level.</p></list-item><list-item><p><italic>Multiplex PCR:</italic> In this technique, multiple selected target regions in a sample are amplified simultaneously using different pairs of primers.</p></list-item><list-item><p><italic>Nested PCR:</italic> It includes two successive PCRs; the product of the first PCR reaction is used as a template for the second PCR. This type of PCR is employed to amplify templates in low copy numbers in specimens. It has the benefits of increased sensitivity and specificity.</p></list-item><list-item><p><italic>Amplification refractory mutation system (ARMS) PCR:</italic> Allele-specific amplification (AS-PCR) or ARMS-PCR is a general technique for the detection of any point mutation or small deletion<sup>[<xref ref-type="bibr" rid="CIT0031">31</xref>]</sup>. The genotype (normal, heterozygous and homozygous states) of a sample could be determined using two complementary reactions: one containing a specific primer for the amplification of normal DNA sequence at a given locus and the other one containing a mutantspecific primer for amplification of mutant DNA. ARMS-PCR has been used to check the most common mutation in GJB2 gene, 35delG mutation among deaf children.</p></list-item><list-item><p><italic>Real time PCR:</italic> In this technique, the amplified DNA is detected as the PCR progresses. It is commonly used in gene expression studies and quantification of initial copy number of the target<sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>]</sup>.</p></list-item></list></p><p><italic><bold>DNA microarray:</bold></italic> DNA “chips” or microarrays have been used as a possible testing for multiple mutations. In this technology, single DNA strands including sequences of different targets are fixed to a solid support in an array format. On the other hand, the sample DNA or cDNA labeled with fluorescent dyes is hybridized to the chip (<xref ref-type="fig" rid="F0004">Fig. 4E</xref>)<sup>[<xref ref-type="bibr" rid="CIT0032">32</xref>]</sup>. Then using a laser system, the presence of fluorescence is checked; the sequences and their quantities in the sample are determined.</p><fig id="F0004" position="float"><label>Fig. 4</label><caption><p>A) Multiplex ligation-dependent probe amplification (MLPA). B) Single-strand conformation polymorphism (SSCP). C) Denaturing gradient gel electrophoresis (DGGE). D) Restriction fragment length polymorphism (RFLP). E) Microarray.</p></caption><graphic xlink:href="IJPD-23-375-g004"></graphic></fig><p><italic><bold>DNA Sequencing:</bold></italic> As a powerful technique in molecular genetics, DNA sequencing provides analysis of genes at the nucleotide level. The main aim of DNA sequencing is to determine the sequence of small regions of interest (∼1 kilobase) using a PCR product as a template. Dideoxynucleotide sequencing or Sanger sequencing represents the most widely used technique for sequencing DNA<sup>[<xref ref-type="bibr" rid="CIT0033">33</xref>]</sup>. In this method, double stranded DNA is denatured into single stranded DNA with NaOH. A Sanger reaction consists of a single strand DNA, primer, a mixture of a particular ddNTP with normal dNTPs (e.g. ddATP with dATP, dCTP, dGTP, and dTTP). A fluorescent dye molecule is covalently attached to the dideoxynucleotide. ddNTPs cannot form a phosphodiester bond with the next deoxynucleotide so that they terminate DNA chain elongation. This step is done in four separate reactions using a different ddNTP for each reaction (<xref ref-type="fig" rid="F0003">Fig. 3B</xref>)<sup>[<xref ref-type="bibr" rid="CIT0002">2</xref>]</sup>. DNA sequencing could be used to check all small known and unknown DNA variations.</p><p><italic><bold>Multiplex ligation-dependent probe amplification</bold></italic><bold>(MLPA):</bold> MLPA is commonly applied to screen deletions and duplications of up to 50 different genomic DNA or RNA sequences. Altogether gene deletions and duplications account up to 10%, and in many disorders up to 30% of disease-causing mutations<sup>[<xref ref-type="bibr" rid="CIT0034">34</xref>, <xref ref-type="bibr" rid="CIT0035">35</xref>]</sup>. In this technique, briefly, the probe set is hybridized to genomic DNA in solution. Each probe consists of two halves; one half is composed of a target specific sequence and a universal primer sequence, and other half has other more sequences, a variable length random fragment to provide the size differences for electrophoretic resolution. A pair of probes is hybridized on the target region adjacently so that they can then be joined by use of a ligase; the contiguous probe can be amplified by PCR<sup>[<xref ref-type="bibr" rid="CIT0036">36</xref>]</sup>. After PCR amplification, the copy number of target sequence i.e. deletion or duplication of target sequence can be determined and quantified using the relative peak heights (<xref ref-type="fig" rid="F0004">Fig. 4A</xref>).</p></sec><sec id="S0009"><title>B) Unknown Mutations</title><p><italic><bold>Single Strand Conformational Polymorphism (SSCP):</bold></italic> SSCP is one of the simplest screening techniques for detecting unknown mutations (microlesions) such as unknown single-base substitutions, small deletions, small insertions, or microinversions. A DNA variation causes alterations in the conformation of denatured DNA fragments during migration within gel electrophoresis. The logic is comparison of the altered migration of denatured wild-type and mutant fragments during gel electrophoresis<sup>[<xref ref-type="bibr" rid="CIT0037">37</xref>]</sup>. In this technique, briefly, DNA fragments are denatured, and renatured under special conditions preventing the formation of double-stranded DNA and allowing conformational structures to form in single-stranded fragments (<xref ref-type="fig" rid="F0004">Fig. 4B</xref>). The conformation is unique and resulted from the primary nucleotide sequence. Mobility of these fragments is differed through nondenaturing polyacrylamide gels; detection of variations is based on these conformational structures. PCR is used to amplify the fragments, called PCR-SSCP, because the optimal fragment size can be 150 to 200 bp. About 80–90% of potential point mutations are detected by SSCP<sup>[<xref ref-type="bibr" rid="CIT0037">37</xref>, <xref ref-type="bibr" rid="CIT0038">38</xref>]</sup>.</p><p><italic><bold>Denaturing Gradient Gel Electrophoresis (DGGE):</bold></italic> DGGE has been used for screening of unknown point mutations. It is based on differences in the melting behavior of small DNA fragments (200-700 bp); even a single base substitution can cause such a difference. In this technique, DNA is first extracted and subjected to denaturing gradient gel electrophoresis. As the denaturing condition increases, the fragment completely melts to single strands. The rate of mobility in acrylamide gels depends on the physical shape of the fragment (<xref ref-type="fig" rid="F0004">Fig. 4C</xref>). Detection of mutated fragments would be possible by comparing the melting behavior of DNA fragments on denaturing gradient gels. Approximately less than 100% of point mutations can be detected using DGGE. Maximum of a nearly 1000 bp fragment can be investigated by this technique<sup>[<xref ref-type="bibr" rid="CIT0039">39</xref>]</sup>.</p><p><italic><bold>Heteroduplex analysis:</bold></italic> A mixture of wild-type and mutant DNA molecules is denatured and renatured to produce heteroduplices. Homoduplices and heteroduplices show different electrophoretic mobilities through nondenaturing polyacrylamide gels. In this technique, fragment size ranges between 200 and 600 bp. Nearly 80% of point mutations have been estimated to be detected by heteroduplex analysis<sup>[<xref ref-type="bibr" rid="CIT0040">40</xref>]</sup>.</p><p><italic><bold>Restriction fragment length polymorphism (RFLP):</bold></italic> Point mutations can change restriction sites in DNA causing alteration in cleavage by restriction endonucleases which produce fragments with various sizes (<xref ref-type="fig" rid="F0004">Fig. 4D</xref>). RFLP is used to detect mutations occurring in restriction sites<sup>[<xref ref-type="bibr" rid="CIT0041">41</xref>]</sup>.</p></sec><sec id="S0010"><title>Next Generation Sequencing</title><p>In recent years, newer technologies for DNA sequencing in a massive scale have been emerged that are referred to as next-generation sequencing (NGS). High speed and throughput, both qualitative and quantitative sequence data are allowed by means of NGS technologies so that genome sequencing projects can be completed in a few days<sup>[<xref ref-type="bibr" rid="CIT0042">42</xref>, <xref ref-type="bibr" rid="CIT0043">43</xref>]</sup>. NGS systems provide several sequencing approaches including whole-genome sequencing (WGS), whole exome sequencing (WES), transcriptome sequencing, methylome, etc. The coding sequences compromises about 1% (30Mb) of the genome. More than 95% of the exons are covered by WES; on the other hand, 85% of disease-causing mutations in Mendelian disorders are located in coding regions. Sequencing of the complete coding regions (exome)<sup>[<xref ref-type="bibr" rid="CIT0044">44</xref>]</sup>, therefore, could potentially uncover the mutations causing rare, mostly monogenic, genetic disorders as well as predisposing variants in common diseases and cancer.</p></sec><sec sec-type="conclusion" id="S0011"><title>Conclusion</title><p>Any change in DNA sequence could be pathogenic if it has abnormal effect on biologic pathways within the cell. Characterization of the genetic basis of the disease is required for an accurate diagnosis. PCR as a powerful and sensitive technique can amplify very small amounts of DNA. This technique has many applications in various areas of biology and it has been used for diagnosis of inherited diseases on the DNA level. Examining DNA would demonstrate the changes in the genes that may cause disease. Molecular diagnosis of genetic disorders is noticed as the detection of the pathogenic mutations in DNA and/or RNA samples. It could facilitate fine subclassification, prognosis, and therapy of disorders. Since most hereditary disorders affect people at childhood ages, it is important for pediatricians to be familiar with genetic testing methodology as well as applications of these tests in clinic to get an accurate diagnosis. The clinicians should be able to recognize and categorize genetic disorders and affected patients on the basis of symptoms and signs to a subtype of chromosomal or single gene disorders, so that they could offer an appropriate genetic test for diagnosing the disease. They could also discuss and consult with a medical geneticist.</p></sec></body><back><ack><title>Acknowledgment</title><p>We would like to appreciate Dr Gh-R Walizadeh for critical reading of the manuscript.</p></ack><sec id="S0012"><title>Conflict of Interest</title><p>None</p></sec><ref-list><title>References</title><ref id="CIT0001"><label>1</label><element-citation publication-type="journal"><article-title>International human genome sequencing consortium:Finishing the euchromatic sequence of the human genome</article-title><source>Nature</source><year>2004</year><volume>431</volume><issue>7011</issue><fpage>931</fpage><lpage>45</lpage><pub-id pub-id-type="pmid">15496913</pub-id></element-citation></ref><ref id="CIT0002"><label>2</label><element-citation publication-type="book"><person-group person-group-type="author"><name><surname>Mahdieh</surname><given-names>N</given-names></name></person-group><article-title>A Comprehensive Review on 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