Navigating the Labyrinth of Genetic Testing : Journal of Marine Medical Society

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Navigating the Labyrinth of Genetic Testing

Gulati, Deepika Col1; Gopinath, Manoj Col2; Singhal, Anuj Surgeon Captain3,

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Journal of Marine Medical Society 25(1):p 1-3, Jan–Jun 2023. | DOI: 10.4103/jmms.jmms_166_22
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Genetic testing includes tests which are used to detect changes in a DNA sequence or chromosome numerically or structurally. Increasingly, newer genetic testing technologies such as karyotyping, fluorescence in situ hybridization (FISH), Sanger sequencing, and next-generation sequencing (NGS) have reached the diagnostic laboratory; out of the realms of research laboratories, bringing them closer to the clinician and giving a huge push to the diagnosis of genetic disorders. However, the availability of all these tests has also left a dilemma among the untrained and the unexposed clinicians in the periphery, who are the first responders for the majority of these patients as to the ordering protocol of these tests. The correct ordering protocol is very important as most of these tests are very expensive with long turnaround times. Some of the tests require detailed clinical history, clinical examination, and high degree of suspicion for the generation of correct and relevant results.


A genetic disorder is a disease caused by a change in the normal DNA sequence in whole or in part. Genetic disorders can be caused by a mutation in a single gene (monogenic disorder), by mutations in multiple genes (polygenic disorder) by a combination of gene mutations and environmental factors (multifactorial inheritance disorder), or due to chromosomal abnormalities (changes in the number or structure of entire chromosomes or part thereof). Mutations can be inherited or de novo. The diagnosis of patients with de novo mutations is important for the treatment. The diagnosis of patients with inherited mutations becomes doubly important as it shall help not only in developing a treatment strategy but in developing a preventive protocol, especially if the parents are thinking of increasing the family size. It is also important to remember that many genetic disorders do not have a cure. Hence, the diagnosis of a genetic disorder in the majority of cases would be a preventive strategy.


“plus ça change, plus c’est la même chose” or “the more things change, the more they stay the same.”-Jean-Baptiste Alphonse Karr.

Any diagnosis always boils down to history and history. The suspicion of a genetic disorder rests on a detailed family and clinical history, pedigree analysis, detailed physical examination, and routine laboratory investigations. A USA National Faculty Development Initiative has come up with Genetics in Primary Care (GPC) project with the aim of developing primary care faculty expertise in genetics by enhancing the training of medical students and primary care residents. It has developed the concept of the red flag for genetic thinking. For this, they came up with practical categories with the mnemonic as follows.

  • F – Family history. Multiple affected siblings or individuals in multiple generations. It is important to note that a lack of a family history does not rule out genetic causes
  • G – Groups of congenital anomalies. Two or more anomalies are much more likely to indicate the presence of a syndrome with genetic implications
  • E – Extreme or exceptional presentation of common conditions. Like the early onset of disease or unusually severe reaction to infectious or metabolic stress
  • N – Neurodevelopmental delay or degeneration. Like developmental delay, developmental regression in children, or early-onset neurologic deterioration in adults
  • E – Extreme or exceptional pathology. Like rare tumors or other pathology or multiple primary cancers in one or different tissues
  • S – Surprising laboratory values. Abnormal laboratory values in an otherwise healthy individual; extreme laboratory values for a typical clinical situation.[1]

Other relevant histories would include multiple miscarriages, stillbirths, and childhood deaths. Dysmorphologies generally affecting the heart and face as well as growth issues are suggestive of a genetic disorder caused by an inherited mutation, spontaneous mutation, teratogen exposure, or unknown factors. Some physical attributes may be quite distinctive or are different than the norm such as wide-set or droopy eyes, flat face, short fingers, and tall stature. These rare and subtle features may often be missed and not ring a bell to a primary care provider regarding suspicion of a genetic disease. In such cases, it is imperative that an evaluation by a genetics specialist is done to rule out the presence of a genetic disease.


The testing strategy will depend on matching the phenotype with a suspected genotype and to determine whether it is due to a numerical chromosomal abnormality as in Downs syndrome or a structural abnormality of a whole chromosome or a part thereof or due to different mutations of a specific known gene or an unknown gene.


Chromosomal abnormalities/cytogenetic abnormalities cause genetic conditions generally diagnosed in the perinatal period, infancy, and childhood. The cytogenetic diagnosis is rare in the adult population except for those whose diagnosis is missed in childhood, especially those presenting with infertility issues. The standard cytogenetic tool is karyotyping which apart from picking up aneuploidy also gives information on large loss (deletion), gain (duplication), or rearrangements (translocations and inversions) of genetic material. Patients with dysmorphic features and an intellectual handicap are generally investigated by this test. In investigating couples with recurrent pregnancy loss, about 4% of one partner may be found to have a translocation or inversions. However, this technique has a long turnaround time and is dependent on the quality of the chromosome preparation and the skill and experience of the cytogeneticist. Newer sophisticated cytogenetic diagnostic techniques like FISH which can pick up deletions, duplications, or rearrangements of genetic material not visible by usual techniques have also been developed and are being used routinely.[2] This technique detects chromosomal DNA with the help of short sequences of single-stranded DNA (probes) which carry fluorescent tags and are complementary. Gene-specific probes, also called “locus-specific,” bind to single areas of a chromosome, whether a gene or a repetitive sequence such as a centromere or telomere. FISH does not need cells to be in the metaphase before analysis, as it relies upon the presence or absence of a fluorescent signal to identify chromosomes or parts of chromosomes, rather than a specific banding pattern. This technique requires a very small quantity of samples. Hence, this test is used generally when specimens are in limited quantity (prenatal diagnosis) or negative results (microdeletion syndromes) on karyotyping.[3]

Comparative genomic hybridization also known as chromosomal microarray analysis (CMA) is the best available test to detect copy number variations in chromosomal makeup. CMA provides similar information as G-banded karyotyping but at a much higher resolution for genomic imbalances and therefore has a higher sensitivity for submicroscopic deletions and duplications. This technique is ideal for imbalanced rearrangements. However, balanced rearrangements and low-level mosaicism are not detected and hence G-banded karyotyping remains the investigation of choice. CMA provides the sensitivity of high-resolution genome-wide detection of clinically significant copy number variants (CNVs), but it comes with the challenge of interpreting variants of uncertain clinical significance, which can impose a burden and adds on to the dilemma of clinicians and laboratories. Studies in the evaluation of reporting of CNVs among clinical laboratories show the variability of interpretation.[4]

Single-gene disorders have been cataloged by Mckusick since 1966 and since 1988, this catalog has become electronic and called Online Mendelian Inheritance in Man. Various methods of direct mutation analysis exist, each with their advantages and disadvantages, including varying levels of ability to detect different types of mutations. As a result of this, the type of mutation analysis may need to be tailored to the type of mutation to be detected. If the gene and the type of mutation associated with the suspected condition are known, then direct analyses by specific polymerase chain reaction protocols would be the simplest, cheapest, and fastest method of detection and will provide the most definitive genetic diagnosis. For the detection of point mutations and small variants, bidirectional Sanger sequencing has been considered the “gold standard” in clinical genetic diagnosis.

Testing for unknown gene involves whole-exome analysis (WES) or whole-genome analysis (WGS), wherein the entire exonic (coding) genetic component or both exonic and intronic components are analyzed, respectively. The technique used is called NGS.

NGS uses powerful massively parallel sequencing assays to sequence many genes of interest, the whole exome, or the whole genome for variants in a variety of patients with rare and complex disorders. WGS data over WES data creates a challenge at a whole new level and adds on to the dilemma of interpreting noncoding variants that may contribute to the genetic load of a patient’s phenotype. A targeted NGS approach is sometimes used, based on a suspected syndrome to minimize costs and to maximize variant identification. Targeted approaches, especially specific gene panels and whole exomes may be of greater analytical sensitivity (target coverage is better to detect heterozygous changes) but limits the clinical sensitivity vis a vis to WGS, which might restrict the scope of interpretation to coding regions. For efficient interpretation of sequencing data, WES and WGS often include sequencing of the probands and both unaffected parents as a trio to ascertain efficiently de novo and inherited mutations under limited information about the mode of inheritance. Confirmation testing by Sanger sequencing in probands and family members is typical.[5] The interpretation of NGS testing results is challenging as it identifies many variants. The clinical significance of these variants may vary from a benign, likely benign, variant of uncertain significance, likely pathogenic, and pathogenic. For some variants, the clinical significance is well known. However, for some other variants, the clinical significance is not known or not certain since these variants might have not been reported before or have only been reported rarely.


In view of the plethora of tests available and the variability of results and interpretation, it is important to note that genetic testing should always be offered along with a pretest and posttest genetic counseling by a trained geneticist/genetic counselor.

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Conflicts of interest

There are no conflicts of interest.


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2. Gilchrist DM. Medical genetics:3. An approach to the adult with a genetic disorder. CMAJ 2002;167:1021–9.
3. Sinclair A. Genetics 101:Cytogenetics and FISH. CMAJ 2002;167:373–4.
4. Miller DT, Adam MP, Aradhya S, Biesecker LG, Brothman AR, Carter NP, et al. Consensus statement:Chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies. Am J Hum Genet 2010;86:749–64.
5. Katsanis SH, Katsanis N. Molecular genetic testing and the future of clinical genomics. Nat Rev Genet 2013;14:415–26.
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