Molecular Genetics Testing

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Continuing Education Activity

The field of molecular genetic and genomic testing is undergoing rapid change due to improvements in our understanding of the molecular causes of both uncommon and common illnesses, as well as in DNA analysis technologies. Molecular diagnostics (MDx) deals with human, viral, and microbial genomes as well as the products they encode. Molecular genetics utilizes the laboratory tools of molecular biology to relate changes in the structure and sequence of human genes to functional changes in protein function and, ultimately, to health and disease. This activity reviews various methods for molecular diagnosis, specimen collection, procedures, Indications, potential diagnosis, normal and critical findings, interfering factors, and complications.


  • Describe the types of genetic molecular testing.
  • Explain the procedures and indications of genetic molecular testing.
  • Review the normal and critical findings of genetic molecular testing.
  • Summarize the interfering factors and complications of genetic molecular testing.


Genetic molecular testing has become a fundamental method for evaluating a growing number of inherited disorders, somatic or acquired diseases with genetic associations, and pharmacogenetic responses. Genotyping can provide valuable disease diagnosis, prognosis, and progression indicators, guide treatment selection, and response, and interrogate targets for gene-specific therapies.[1] The majority of genetic material is DNA, composed of two strands of a sugar-phosphate backbone bound together by hydrogen bonds between two purines and two pyrimidines attached to the sugar molecule, deoxyribose, in a double helix.[2][3]

DNA in human cells is wrapped around histone proteins and packaged into nucleosome units, which are compacted further to form chromosomes.[4] There are 23 pairs of chromosomes, two of which are the sex chromosomes X and Y. Each chromosome is a single length of DNA with a stretch of short repeats at the ends called telomeres and additional repeats in the centromere region.[5] In humans, there are two sets of 23 chromosomes, a mixture of DNA from the mother's egg and the father's sperm. Therefore, each egg and sperm is a single or haploid set of 23 chromosomes. The combination of the two creates a diploid set of human DNA, allowing each individual to possess two different sequences, genes, and alleles on each chromosome, one from each parent.[6] Each child has a unique combination of alleles because of homologous recombination between homologous chromosomes during meiosis in the development of gametes (egg and sperm cells). This creates genetic diversity within the human population.[7]

The completion of the full human genome sequence, the identification, and cloning of numerous genes associated with inherited and acquired conditions and diseases, plus the advent of powerful methods for molecular analysis of these genes in clinical specimens, have revolutionized the practice of molecular genetics and molecular pathology.[8] With the aid of these techniques, it is now possible to identify illness risk in individuals who are not yet showing symptoms, identify asymptomatic carriers of recessive traits, and make prenatal diagnoses for diseases that might not develop in pregnancy.[9] Molecular genetic, nucleic acid-based techniques are often the only approaches available for these applications. As such, they offer a powerful tool for diagnosis, genetic consultation, and prevention of heritable diseases.[10]

Many types of genetic tests are available to analyze changes in genes, chromosomes, or proteins. A healthcare provider will consider several factors when selecting the appropriate test, including what condition or conditions are suspected and the genetic variations typically associated with those conditions.[8] A test that looks at many genes or chromosomes may be used if a diagnosis is unclear. However, a more focused test may be done if a specific condition is suspected.[11]

Molecular tests look for changes in one or more genes. These types of tests determine the order of DNA, building blocks (nucleotides) in an individual's genetic code, and a process called DNA sequencing. These tests can vary in scope.[8] The targeted single variant is a single variant test that looks for a specific variant in one gene. The selected variant is known to cause a disorder (for example, the specific variant in the HBB gene that causes sickle cell disease). This type of test is often used to test family members of someone known to have a particular variant, to determine whether they have a familial condition.[12] Single-gene tests look for any genetic changes in one gene. These tests are typically used to confirm (or rule out) a specific diagnosis, particularly when many variants in the gene can cause the suspected condition. Gene panel tests look for variants in more than one gene.[13] This type of test is often used to pinpoint a diagnosis when a person has symptoms that may fit a wide array of conditions or when variants in many genes can cause the suspected condition.[14]

Whole exome sequencing/whole genome sequencing tests analyze the bulk of an individual's DNA to find genetic variations. This test is typically used when a single gene or panel testing has not provided a diagnosis or when the suspected condition or genetic cause is unclear.[15] This sequencing method is often more cost- and time-effective than performing multiple single gene or panel tests.[16] Chromosomal tests analyze whole chromosomes or long lengths of DNA to identify large-scale changes. Changes that can be found include an extra or missing copy of a chromosome (trisomy or monosomy, respectively), a large piece of a chromosome that is added (duplicated) or missing (deleted), or rearrangements (translocations) of segments of chromosomes.[17] Certain genetic conditions are associated with specific chromosomal changes, and a chromosomal test can be used when one of these conditions is suspected. (For example, Williams syndrome is caused by a deletion of a section of chromosome 7).

Gene expression tests look at which genes are turned on or off (expressed) in different types of cells. When a gene is turned on (active), the cell produces a molecule called mRNA from the instructions in the genes, and the mRNA molecule is used as a blueprint to make proteins.[18] Gene expression tests study the mRNA in cells to determine which genes are active. Too much activity (overexpression) or too little activity (underexpression) of certain genes can be suggestive of particular genetic disorders, such as many types of cancer.[19] Biochemical tests do not directly analyze DNA, but they study the amount or activity level of proteins or enzymes that are produced from genes.[20] Abnormalities in these substances can indicate that there are changes in the DNA that underlie a genetic disorder.

Mutations associated with heritable disorders are detectable in all nucleated cells and thus are considered germline or constitutional genetic changes. Somatic genetic changes are characteristic of acquired or sporadic diseases, such as cancer.[21] The molecular biology methods applied to investigate these two scenarios are very similar and focus on detecting DNA and RNA variations. However, the interpretation and utility of the laboratory results may be quite distinct.[22]

Fluorescent in situ hybridization (FISH), chromosomal microarray analysis (CMA), and cytogenetic analysis (karyotyping) can be used to detect gross mutations like whole and large-scale gene deletions, duplications or rearrangements. Conventional karyotyping is limited to detecting rearrangements involving more than 5 Mb of DNA.[23] The resolution of the FISH technique, using fluorescent probes, is about 100kb-1Mb in size. The minor changes as single-base substitutions, insertions, and deletions can be detected with single-strand conformation polymorphism (SSCP) and sequence analysis through next-generation sequencing (NGS), a procedure that can use genomic DNA or complementary DNA (cDNA) with three modalities that include whole genomic DNA,[11][24] targeted or exome sequencing; the denaturing high-performance liquid chromatography (DHPLC) is valid for detection of small deletions and duplications too; the extension of the deletions and duplications detected by multiplex ligation-dependent probe amplification (MLPA)is in some way in between of those identified by FISH or cytogenetic analysis and HPLC, i.e.,[25] MLPA  is useful for detection of complete or single and multiexon deletions or duplications.[26]

Specimen Collection

The specimen required for the FISH, MLPA, DHPLC, and sequencing is peripheral blood; cells from amniotic fluid and, more recently, cell-free fetal DNA have been used as the sample for non-invasive prenatal testing.[27] Ethylenediaminetetraacetic acid (EDTA) is the most commonly used anticoagulant for molecular-based testing, but acid–citrate–dextrose (ACD) is an acceptable alternative in cases where preservation of both form and the function of cellular components is required. There are two ACD tube designations: ACD A and ACD B. These differ only by the concentrations of the additives.[28] Both enhance the viability and recovery of white blood cells for several days after collection of the specimen; thus, they are suitable for both molecular diagnostic testing and cytogenetic testing.


FISH is a technique where the fixed genetic material of cells on interphase or metaphase hybridizes with a fluorescent DNA probe designed to target several specific sequences of the gene of interest; probes against sequences of housekeeping genes always serve as positive internal controls.[24] The probe must be large enough to hybridize specifically with its target but not so large as to impede the hybridization process. A conventional FISH reaction is typically performed as a bench-top assay by pipetting the FISH hybridization mix, containing the probes, onto the cytological sample and incubating it.FISH can be performed on suspended cells followed by cell sorting procedures for separating the fluorescent signal, as well as on cultured cells and frozen or formalin-fixed paraffin-embedded tissue sections.[29] During the fixation of the sample, it is important to preserve nucleic acid integrity and cell morphology. The actual experimental FISH procedure includes several preparatory steps, the hybridization reaction itself, and the removal of unbound probes.[30] 

The probe can be tagged directly with fluorophores or with targets for fluorescently labeled antibodies or other substrates. Different types of tags can be used; therefore, different targets can be detected in the same sample simultaneously (multi-color FISH). Tagging can be done in various ways, such as nick translation or PCR using tagged nucleotides. Probes can vary in length from 20 to 30 nucleotides to much longer sequences. Locus-specific probes target a specific gene sequence of interest.[31] These probes can be used to determine whether a gene is amplified, deleted, or present in a normal copy number. Dual Fusion probes are used to detect common translocations involving gene regions that, when rearranged and joined, result in cancer. They are designed to bind to regions spanning the breakpoint of both translocation partners. If the green and red signals are closer than the width of one signal, they are said to be intact.[32] When a break in the gene sequence occurs, the green and red signals will not be close together anymore and will thus appear as separate green and red signals.[31]

Break-apart probes target two areas of a specific gene sequence. Usually, a green fluorescent label is used on one end of a gene sequence, and a red fluorescent label is used on the other end of the gene sequence. When the gene sequences are intact, the green and red signals will usually fluoresce as a yellow signal, known as a fusion signal. Whole chromosome probes are collections of smaller probes, each of which binds to a different sequence along the length of a given chromosome.[33] Using multiple probes labeled with a mixture of different fluorescent dyes, scientists are able to label each chromosome in its own unique color. The resulting full-color map of the chromosome is known as a spectral karyotype which makes it possible to identify all 24 chromosomes by single hybridization.[32] Whole chromosome probes are particularly useful for examining chromosomal abnormalities, for example, when a part of one chromosome is translocated to another chromosome.[33]

The chromosomal microarray (CMA) is like a grid covered with thousands of tiny probes consisting of small pieces of DNA from known locations on each of the 46 chromosomes. CMA looks for imbalances of chromosomal material between DNA from the control and the patient’s DNA.[34] When a patient’s sample and the control sample are labeled and added to the microarray, one can determine if there are any differences in copy number, also known as gains (duplications) or losses (deletions) in specific segments of DNA. If a difference is found, the location and type of change (gain or loss) will often determine the cause of the patient’s health condition.[35]

Denaturing high-performance liquid chromatography (DHPLC) is based on differential chromatography retention of DNA heteroduplexes after denaturation and renaturation; the migration of DNA heteroduplexes is determined not only by the length of the molecule but also by its melting temperature, which is a crucial point for the sensitivity of the test.[25] Denaturing HPLC typically compares two PCR products amplified from two genes, wild type and mutated; PCR products can become amplified from RNA (cDNA) or genomic DNA; amplified PCR products are denatured at 95 C and gradually reannealed by cooling from 95 C to 65 C prior to chromatography. A major advantage of this technology is that multiple samples can be pooled together for variant detection with increased throughput.[36] It can detect in minutes with close to 100% sensitivity and specificity single-base substitutions and small deletions and insertions in DNA fragments ranging from 80 to 1500 base pairs in size.[37]

In the presence of a mismatch, not only the original homoduplexes, formed upon reannealing of the perfectly matching sense and antisense strands (25% each) are produced again but, simultaneously, two heteroduplexes form upon reannealing of the sense strand of either homoduplex with the antisense strand of the other homoduplex (also 25% each). As the heteroduplexes denature more extensively than the homoduplexes, they elute earlier than the homoduplexes that undergo less denaturation.[25] All four species separate according to their differences in stacking interactions with the chromatography column (solid phase); this and a more detailed explanation of the theoretical basis of DHPLC exists in the literature.[37]

The sample for MLPA is genomic DNA; specific MLPA probes become hybridized with denatured genomic DNA; MLPA probes are designed with two peculiarities; the first one is that each pair of probes can hybridize next to each other on the target DNA region where they are ligated; the second peculiarity of the probes is that their design confers a unique length to each pair of amplified MLPA probe; thus they are detectable and quantifiable by capillary electrophoresis.[38] All the MLPA probes are amplified with the same pair of primers. The abundance of each amplified PCR fragment is proportional to the number of copies of its target in the sample.[26]

NGS amplifies DNA with random priming generating millions of reads and a genome-wide view of the genetic background of the patients. The first step for NGS is the generation of a library of fragments that are representative of the entire genome or transcriptome of the individual; the library generation starts with the fragmentation of the nucleic acid; in the modality of whole-exome sequencing, the fragments come from cDNA, while the whole-genome modality includes the complete genomic DNA.[39] Fragments join using sequence adaptors and are enriched; for targeted libraries where only a part of the genome or some genes are analyzed (gene panel), fragments of the library hybridize with DNA fragments that are complementary to the sequence of the region or genes of interest and then, specifically enriched.[40] In the sequencing step, every nucleotide addition is detected by a nucleotide-specific fluorescent dye or by pH changes originating from the release of hydrogen ions during the DNA polymerization.[39]

Sanger sequencing is a multistep process that begins with PCR-based amplification of target DNA, followed by the removal of excess deoxynucleotide triphosphates (dNTPs) and PCR primers.[41] Sanger sequencing with 99.99% base accuracy is considered the “gold standard” for validating DNA sequences, including those already sequenced through next-generation sequencing (NGS). The Sanger sequencing method consists of 6 steps, the double-stranded DNA (dsDNA) is denatured into two single-stranded DNA (ssDNA), a primer that corresponds to one end of the sequence is attached, and four polymerase solutions with four types of dNTPs but only one type of ddNTP is added, the DNA synthesis reaction initiates and the chain extends until a termination nucleotide is randomly incorporated, the resulting DNA fragments are denatured into ssDNA, the denatured fragments are separated by gel electrophoresis, and the sequence is determined.[42] Because DNA polymerase only synthesizes DNA in the 5’ to 3’ direction starting at a provided primer, each terminal ddNTP will correspond to a specific nucleotide in the original sequence (e.g., the shortest fragment must terminate at the first nucleotide from the 5’ end, the second-shortest fragment must terminate at the second nucleotide from the 5’ end, etc.) Therefore, by reading the gel bands from smallest to largest, we can determine the 5’ to 3’ sequence of the original DNA strand.[43][42]

In manual Sanger sequencing, the user reads all four lanes of the gel at once, moving from bottom to top, using the lane to determine the identity of the terminal ddNTP for each band.[42] For example, if the bottom band is found in the column corresponding to ddGTP, then the smallest PCR fragment terminates with ddGTP, and the first nucleotide from the 5’ end of the original sequence has a guanine (G) base.[41][42] In automated Sanger sequencing, a computer reads each band of the capillary gel in order, using fluorescence to call the identity of each terminal ddNTP. In short, a laser excites the fluorescent tags in each band, and a computer detects the resulting light emitted. Because each of the four ddNTPs is tagged with a different fluorescent label, the light emitted can be directly tied to the identity of the terminal ddNTP. The output is called a chromatogram, which shows the fluorescent peak of each nucleotide along the length of the template DNA.[42]


Molecular genetic testing has a unique range of indications, most of which are quite different from the uses of traditional clinical laboratory testing and even molecular biologic testing in other disease classes (e.g., infectious disease, cancer).[44] The uses of genetic testing include newborn screening, diagnostic testing to identify or rule out a specific genetic or chromosomal condition, carrier testing to identify people who carry one copy of a gene mutation that, when present in two copies, causes a genetic disorder, prenatal testing to detect changes in a fetus's genes or chromosomes before birth, predictive and presymptomatic types of testing to detect gene mutations associated with disorders that appear after birth, often later in life, forensic testing to identify an individual for legal purposes.[45]

FISH is indicated in patients with a family history of disease with a known deletion and has recently been used to detect deletions in single blastomeres on a preimplantation genetic diagnosis.[30] FISH tests using panels of gene-specific probes for deletions, amplification, and translocations are used to study hematologic and solid tumors and detect intracellular microorganisms and parasites.[32] CMA should be considered for individuals who lack a sufficient specific history or features on physical examination to suggest a specific genetic (or non-genetic) cause for intellectual disability, developmental delay, autism spectrum disorder, or multiple congenital anomalies.[46] Chromosomal microarray analysis may be beneficial when prenatally detected structural anomalies are associated with specific microdeletions and microduplications or to assess for copy number variants when a de novo balanced rearrangement or marker chromosome is diagnosed.[47]

MLPA has a variety of applications, including the detection of mutations and single nucleotide polymorphisms analysis of DNA methylation, relative mRNA quantification, chromosomal characterization of cell lines and tissue samples, detection of gene copy number, detection of duplications and deletions in human cancer predisposition genes such as BRCA1, BRCA2, hMLH1, and hMSH2 and aneuploidy determination.[26] MLPA has potential applications in prenatal diagnosis, both invasive and noninvasive.[48] Denaturing high-performance liquid chromatography (DHPLC) is ideal for scanning genes for novel mutations and analyzing large sample sizes at a low cost.DHPLC can also be used for genotyping specific mutations or polymorphisms.[25] These systems can also be used for several applications besides the detection of genetic variants, such as size-based double-strand DNA separation analysis, single-strand DNA separation, and analysis of DNA purification.[49]  

NGS has been used for rapidly sequencing whole genomes, deeply sequencing target regions, utilizing RNA sequencing (RNA-Seq) to discover novel RNA variants and splice sites, quantifying mRNAs for gene expression analysis, analyzing epigenetic factors such as genome-wide DNA methylation and DNA-protein interactions, Sequence cancer samples to study rare somatic variants, tumor subclones and Identify novel pathogens.[39] Sanger sequencing, also known as the "chain termination method," is a method for determining DNA nucleotide sequences.[42] 

Potential Diagnosis

FISH provides a swift means to diagnose common fetal aneuploidies; its diagnostic scope has reduced sensitivity compared with conventional cytogenetic analysis; this technique will not identify cases with cytogenetic abnormalities other than the most frequent ones (e.g., translocations, inversions, markers).[33] Denaturing high-performance liquid chromatography (DHPLC) results in detecting a single nucleotide of small deletions or insertions that have to be subsequently confirmed by sequencing; this methodology allows the discovery of unknown mutations, an advantage for diseases like NF1 with 50% of de Novo mutations.[25]

Chromosomal microarrays are considered a first-tier test for individuals with developmental delays, intellectual disabilities, autism spectrum disorders, or multiple congenital disabilities and are recommended in lieu of a karyotype.[35]

Detection of abnormalities by multiplex ligation-dependent probe amplification analysis is dependent on the probes employed, which are typically designed to identify specific small gene deletions (e.g., MEN1 partial or complete deletion).[26] In addition, MLPA may be used in specific circumstances to evaluate for alterations in methylation [e.g., pseudohypoparathyroidism 1b (PHP1b) in which deletion of one or more of four differentially methylated regions is typically observed].[48]

NGS generates millions of sequences that are processed, analyzed, and interpreted to identify the variants.[40] The bioinformatics analysis starts with raw data generated by the signal detection of nucleotide incorporation. The quality of the reads undergoes evaluation during the primary data analysis.[41] The sequences are subsequently aligned or mapped against a reference genome; during this step, computational algorithms will try to find the best match for each read with the reference genome but tolerate some mismatches to detect the genetic variants.[50] Sanger sequencing is a robust testing strategy able to determine whether a point mutation or small deletion/duplication is present. It has been widely used for several decades in many settings, including defining the mutational spectrum of a tumor as well as identifying a constitutional variant in diagnostic testing.[41] Primers can be created to cover several regions (amplicons) to cover any size region of interest.[42]

Normal and Critical Findings

In fluorescence in situ hybridization (FISH), when a probe binds to a chromosome, its fluorescent tag provides information on chromosomal abnormalities.[33] On MLPA, the intensity of peaks of PCR amplicons during capillary electrophoresis is proportional to the copy number in the sample. The amplification of an MLPA probe that includes a mutation indicates the presence of it in the sample.[48] Two possible results can be obtained from an MLPA test:

  • Positive (Pathogenic and likely pathogenic): A positive result indicates that a gene deletion or duplication has been associated with the disease phenotype under study. 
  • Negative: A negative result indicates that no disease-causing deletions or duplications are identified in the test performed. It does not guarantee that the person will be healthy or free from other genetic disorders or medical conditions. Additionally, a negative result does not rule out a genetic cause of the disease nor eliminates the risk for future offspring. However, if a negative test result is obtained and the variant in question is known to be present in affected family members, this then rules out a diagnosis of that genetic disorder in the proband. Several causes may explain a negative result, including limited genetic knowledge and limitations associated with the methodology.[51]

The presence of heteroduplexes on a DHPLC indicates the presence of a mutation relative to the reference genome used in the same sample.[25] NGS sequencing detects genetic variants ranging from single nucleotides, small insertions or deletions, and some structural variants. Seeing a genetic variant does not imply its role in the disease.[40] The detected variants require analysis in a clinical context, and their pathological potential requires assessment using different approaches.[52] Reading the Sanger sequencing results properly will depend on which of the two complementary DNA strands is of interest and what primer is available. If the two strands of DNA are A and B and strand A is of interest, but the primer is better for strand B, the output fragments will be identical to strand A. On the other hand, if strand A is of interest and the primer is better for strand A, then the output will be identical to strand B. Accordingly, the output must be converted back to strand A.[42]

Interfering Factors

The specificity of the probe used in FISH is critical to avoid its hybridization with genes other than the target. Some FISH preparations have autofluorescence; thus, an adequate wash of cells to remove any fluorescent buffer or medium is recommended to eliminate the chances of background fluorescence.[32]

MPLA has some general limitations. The mutations to be detected have to be known to include them in the design of the probes, and gene rearrangements like inversions and translocations are not detectable by the method. The samples have to be free of impurities. The presence of some contaminants in the sample, like phenol, can interfere with the ligation step. False positive or negative results may occur due to rare sequence variants in target regions detected by MLPA probes.[51] A point mutation or polymorphism in the sequence detected by a probe, which results in reduced probe binding efficiency, can also cause a reduction in the relative peak area. Therefore, single exon deletions detected by MLPA should always be confirmed by other methods like multiplex PCR or sequencing.[53] 

Melting temperature is critical for the sensitivity of DHPLC. Computational algorithms can predict this temperature, and usually, the experimental procedure is made with at least two different melting temperatures to increase the sensitivity of the test.[25] Chromosomal microarray (CMA) does not detect small changes in the sequence of single genes (point mutations), tiny duplications and deletions of DNA segments within a single gene (Fragile X syndrome, for example), and balanced chromosomal rearrangements (balanced translocations, inversions).[35]

NGS technologies remain evolving to circumvent every challenge; large insertions, duplications, and deletions are detected only by some massive sequencers that can support long reads. Long homopolymers are also problematic regions for sequencing.[40] The biggest drawback of NGS in a clinical environment is the need to build the necessary infrastructure, such as computer power and storage, and the personnel expertise required to comprehensively analyze and interpret the subsequent data.[50]

Despite automation, Sanger-based sequencing is still relatively labor-intensive, time-consuming, expensive, and requires specialized equipment.[42] It also has limited sensitivity for detecting point mutations, approximately 20% of mutant DNA within a wild-type background. Sanger sequencing is not precisely quantifiable. For example, one cannot conclude if a mutation is present in 25% versus 40% of cells based on peak sizes; additional testing strategies must be used for quantification.[43]


The complications during the peripheral blood collection by venipuncture are infrequent and of low risk; some patients can present hematomas, pain, and, especially in children, fear is common; other procedures like amniocentesis are more invasive and harbor more serious complications like infection, preterm delivery, respiratory distress, trauma, alloimmunization, among others though they are infrequent.[54] The genetic tests based on NGS of free-cell DNA obtained from the maternal peripherical blood sample are an alternative to diagnosis using amniocentesis fluid as a sample.[55] 

Patient Safety and Education

Besides the potential complications mentioned before about the sampling procedure, other legal, medical, psychological, and ethical issues are associated with molecular testing.[56] Although the direct objective of molecular testing is the molecular demonstration of the presence of a genetic trait that correlates with a disease, the recommendation is to provide this molecular diagnosis as part of genetic counseling.[57]

Genetic counseling is a service that involves a multidisciplinary team headed by the genetic counselor and several other professionals. As the first step, clinical identification of a suspected disease is necessary to narrow and direct molecular testing. Information about the potential disease, the procedure of the molecular testing, and its potential results require explanation to the patient. Legal issues such as informed consent, especially in children that cannot give it, must be covered.[58] Patient education is an essential part of molecular testing and genetic counseling.

Finally, NGS technologies are applied to genetic counseling. The results' complexity is higher than those obtained with traditional genetic tests. Considerable information is generated and available for sharing, and ethical issues are implied in divulging it. All these aspects must be considered, informed, and explained to every patient subjected to genetic testing.[59] When performing a molecular genetic test, laboratories should consider the preexamination, examination, and post-examination issues. In addition, the test methodology and result interpretation must be based on the test indication and application. Ethical issues associated with genetic testing and the privacy of genetic information should also be considered.[1]

Clinical Significance

Any permanent difference in the nucleotide sequence of a gene concerning a reference genome merits consideration as a genetic change or mutation. All changes identified following the tiered protocol must be confirmed by sequencing, and their pathogenic role in disease has to be determined.[60] A variant found during genetic testing can be benign, likely benign, pathogenic, likely pathogenic, or of uncertain significance.[61] Each variant is rigorously classified based on different kinds of evidence (population, computational, functional, or segregation data) to describe its clinical significance.[62]

The American College of Medical Genetics and Genomics recommends following this nomenclature and classification for the findings of genetics tests, including genotyping, single genes, panels, exomes, and genomes. The application of NGS has enhanced our comprehension of genetic diseases but also has driven the simultaneous identification of numerous genetic variants whose role in disease needs to be carefully studied.[63] Interprofessional approaches are necessary to rationally use the available genetic tests for the benefit of the patients, and the panel of experts strongly recommends the performance of genetic tests by certified laboratories and the results to be interpreted by a board-certified geneticist.[64]

Molecular genetic testing had accelerated progress with the incorporation of PCR on it, and this was followed by a giant step when next-generation sequencing allowed the generation of genome-wide data.[60] Multidisciplinary teams are working together to integrate the different methodologies of genetic testing with clinical, pathological, functional, computational, ethical, and social aspects of diseases to translate technological advances and knowledge for the benefit of patients.[65]

Article Details

Article Author

Cecilia Ishida

Article Author

Muhammad Zubair

Article Editor:

Vikas Gupta


4/7/2023 5:07:35 AM



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