Genetics, Molecular Testing


Molecular testing, as part of genetic counseling, is strongly recommended to offer a guided diagnosis and management of genetic diseases to the patients and their families. For example, NF-1. Other indications for molecular testing in genetics include patients who wish to confirm a clinical diagnosis or with an atypical presentation of the syndrome and patients with the need for prenatal or preimplantation diagnosis.

Neurofibromatosis 1 (NF1) is an autosomal dominant disorder caused by mutations of NF1 gene with a broad spectrum of clinical characteristics, the disease is suggested by the presence of multiple café-au-lait spots, neurofibromas, inguinal freckling, iris hamartomas, tumors or skeletal abnormalities, learning disabilities are present in 50% of the patients.[1] Individuals are variably affected, not only between families but also inside the same family, and even the same person during his or her lifetime can have very different clinical evolution.[2] Genetically, the disease is also heterogenic, with observed mutations in the NF1 gene include stop mutations, amino acid substitutions, insertions, deletions (partial or whole), and gross chromosomal rearrangements; besides, 50% of the patients have de novo mutations.[1] Diagnosis of the disease is based on clinical criteria specified by NIH that are specific and sensitive in adults or children with a family history of the disease, but only 50% of children with no family history of NF1 meet clinical criteria for diagnosis by the age of 1 year [3]; thus, NF1 is the most common syndrome associated with café au lait spots in children and although the presence of them on children without a family history of NF1 is highly suggestive of Neurofibromatosis.[1] Molecular diagnosis of NF1 has existed for a long time.[4] 

Recently next-generation sequencing (NGS) technologies have been incorporated for the diagnosis of this and other diseases to provide less expensive and faster identification of mutations.[5][6] Detecting mutations on NF1 is challenging, and current protocols combine complementary methods for detection of minor changes as single-base substitutions, insertions and deletions, exon deletions or duplications, and microdeletions or complete gene deletions.

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, however, these methodologies detect only the 2 to 5%, 5% and less than 1% of all mutations on NF1, respectively. Other methodologies can also detect some of these mutations.[7][8][9] 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, targeted or exome sequencing; the Denaturing high-performance liquid chromatography (DHPLC) is useful for detection of small deletions and duplications too; the extension of the deletions and duplications detected by MLPA (Multiplex ligation-dependent probe amplification) is in some way in between of those identified by FISH or cytogenetic analysis and HPLC, i.e., MLPA  is useful for detection of complete or single and multiexon deletions or duplications.[10] 

As has been explained before, any of these methodologies cannot efficiently detect all NF1 gene mutations, but there are reports of multistep mutation detection protocols with a sensitivity of up to 95% have been reported.[10][1] In the protocol reported by Valero et al., the first step consisted on the identification of complete or partial NF1 gene deletions and duplications using MLPA, when samples were negative for this test, a second-tier applied HPLC to cDNA to detect splice mutations, small deletion/duplications and; as part of the second tier, a region of the NF1 gene was analyzed from genomic DNA, since the intrinsic characteristics of the mRNA sequence, a high GC content, make difficult the amplification of cDNA. The third step on this analysis focuses on identifying, through a haplotype analysis, which of the found mutations were potentially pathogenic, since mutations can also be non-pathogenic variants; all mutations identified at every step had confirmation with Sanger sequencing.

Molecular testing of NF1 constitutes a good example to explain the theoretical basis on a tier-based protocol, in the next sections, this article will discuss FISH, MLPA, DHPLC as components of a tiered-protocol and briefly explain the differences between gene-targeted, exome and whole NGS that have used isolated or as part of tiered protocols in the diagnosis of NF1 and other diseases.[5]

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 has been used as the sample for non-invasive prenatal testing.[11]


FISH is an assay 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.

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, that is a crucial point for the sensitivity of the test.[12] Denaturing HPLC compares typically 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 degrees C and gradually reannealed by cooling from 95 degrees C to 65 degrees C prior to chromatography.

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 homoduplexes, they elute earlier than the homoduplexes that undergo less denaturation. All four species separate according to their differences in stacking interactions with the chromatography column (solid phase); this and a more detailed explanation about the theoretical basis of DHPLC exists in the literature.[13]

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. 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.[14]

NGS amplifies DNA with random priming generating millions of reads and a genome-wide view of the genetic background of the patients.[15] 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. Fragments join using sequence adaptors and 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. In the sequencing step, every nucleotide addition is detected by a nucleotide-specific fluorescent dye or by pH changes originated by the release of hydrogen ions during the DNA polymerization.[16]


FISH is indicated in patients with a family history of disease with a known deletion, recently has been used to detect deletions in single blastomeres on a preimplantation genetic diagnosis.[17] This methodology was not included on the tiered protocol of Valero et al., however, it has been the first tier for other protocols.

Multiplex ligation-dependent probe amplification analysis constitutes the first tier on protocols to identify NF1 mutations allowing to identify multiple exon deletions and duplications simultaneously in the same reaction; these mutations correspond to around the 15% of the alterations on NF1 gene [10]; known mutations are also identifiable using this method when using specific probes for these mutations. MLPA specific kits for NF1 are commercially available.[6] 

Denaturing high-performance liquid chromatography is a methodology included in most of the tiered genetic testing protocols for NF1, and it is performed after discarding single or multiple exon deletions or duplications.[10] DHPLC can detect small mutations like polymorphisms, and small deletions and mutations that are not detected by FISH or MLPA with 1-bp resolution. It also works to detect alternatively spliced transcripts that are hard to detect by other methods because of their scarcity and discrepancy of just a few nucleotides with the genomic sequence.[12]

NGS has been used for the detection of mutations on NF1, followed by functional characterization of the detected mutations. alone or as part of tiered protocols.[5] The percentage of NF1 mutations detected by NGS ranges from 60 to 90%, depending on the study and the modality of NGS used.[7] Gene panel sequencing is indicated when a diagnosis is suggested by the clinical hypothesis as would be the case in NF1, whole-exome sequencing (WES) expands the results to all the coding sequences but avoids the interpretation of the 99% of the genome. Whole-genome sequencing (WGS) is an option mainly when there is a high suspicion of a genetic syndrome, but results from exome sequencing are negative.[15]

Potential Diagnosis

FISH can detect partial or complete deletions of the NF1 gene but cannot detect small deletions of around 50-70 nucleotides on length. A negative FISH test cannot discard the presence of deletions from DNA regions that are not covered by the used probe. FISH provides a swift means by which to diagnose common fetal aneuploidies; its diagnostic scope has reduced sensitivity compared with conventional cytogenetic analysis; that is, this technique will not identify cases with cytogenetic abnormalities other than the most frequent ones (e.g., translocations, inversions, markers).[11]

MLPA analysis can detect known point mutations and single and multiple exon deletions/duplications. It constitutes the first tier on some protocols for NF1, and it can detect 15% of all the NF1 mutations. 

DHPLC results in the detection of a single nucleotide of small deletions or insertions that have to be subsequently confirmed by sequencing, this methodology allows discovering unknown mutations, an advantage for diseases like NF1 with 50% of de Novo mutations.

NGS generates millions of sequences that are processed, analyzed, and interpreted to identify the variants. The bioinformatic analysis starts with raw data generated by the signal detection of nucleotide incorporation. The quality of the reads undergoes evaluation during the primary analysis of the data. 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 tolerating some mismatches to allow the detection of the genetic variants.[16]

Normal and Critical Findings

Since the autosomal dominant character of NF1, the finding on FISH of only one fluorescent spot for the NFI gene indicates a positive result for NFI deletion.

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.

The presence of heteroduplexes on a DHPLC indicates the presence of a mutation relative to the reference genome used in the same sample.

NGS sequencing detects genetic variants ranging from single nucleotide, small insertions or deletions, and some structural variants. The detection of a genetic variant does not imply that it has a role in the disease. The detected variants require analysis in a clinical context, and its pathological potential requites assessment using different approaches.

Interfering Factors

The specificity of the probe used in FISH is critical to avoid its hybridization with other genes 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.

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.

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.

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.


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.[18] 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.

Patient Safety and Education

Besides the potential complications mentioned before about sampling procedure, other legal, medical, psychological, and there are ethical issues associated with molecular testing. 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.

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 requires explanation to the patient. Legal issues as informed consent, especially in children that cannot give it, must be covered. Patient education is an essential part of molecular testing and genetic counseling.[19] 

Finally, with NGS technologies applied to genetic counseling. The complexity of the results is higher than the results obtained with traditional genetic tests. Considerable information is generated and available for sharing, and there are ethical issues implied in divulging it.[20] All these aspects need to be considered, informed, and explained to every patient subjected to genetic testing.

Clinical Significance

Any permanent difference in the nucleotide sequence of a gene concerning a reference genome merits consideration as a genetic change or mutation.[21] All changes identified following the tiered protocol have to be confirmed by sequencing, and their pathogenic role in disease has to be determined. A variant found during genetic testing can be benign, likely benign, pathogenic, likely pathogenic, or of uncertain significance. Each variant is rigorously classified based on different kinds of evidence (population, computational, functional, or segregation data) to describe its clinical significance.

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. 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.[22]

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. 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.

Article Details

Article Author

Cecilia Ishida

Article Editor:

Vikas Gupta


7/21/2021 11:43:52 PM



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