The diagnosis of susceptibility concerns diseases that are widespread in adults, and, in general, the molecular test can predict an increased or reduced risk of developing the dis ease. This susceptibility, but not the disease, can be transferred to descendants in a non-Mendelian and unpredictable way. The development of the various conditions associated with this risk is also affected by lifestyle and environmental factors and epigenetic modifications.

In recent years, the number of diseases for which susceptibility markers are known has increased significantly and continues to grow exponentially due to the availability of massive human genome sequencing technologies. Indeed, a complete genome sequencing analysis can be performed quickly and cost-effectively by selecting patients with a dis ease and healthy controls. Then, the myriad of data obtained is analyzed by appropriate bioinformatics systems, and the gene loci (usually single nucleotide polymorphisms [SNPs]) statistically associated with the disease are defined. The polymorphisms identified do not necessarily have to fall within a disease-gene and in themselves may also be devoid of a direct pathogenetic effect; in many cases, they are close to a disease-causing gene (or even present within the gene itself in non-coding intronic regions) that is not yet known and that contributes to the pathogenesis of the disease. The strict observance of the selection criteria of patients and controls and the stringency of the statistical criteria of data processing make the marker usable in routine, even without identifying the disease-gene. Indeed, for many multifactorial diseases (Table 1), panels for the routine analysis of susceptibility loci are already on the market.

Table1. Multifactorial diseases for which panels for the routine analysis of predisposition loci are commercially available

Thrombophilia

In some cases, susceptibility genes are defined by a bio chemical approach, i.e., by studying the proteins (and therefore the genes) involved in a biological process. For example, alterations in specific genes encoding proteins that inhibit the coagulation process may predispose to thrombotic episodes. Even these gene variants do not give rise to disease, but they predispose to developing thrombotic episodes that may be triggered in the presence of concomitant causes, or in patients immobilized for long periods, etc. The most known prothrombotic genetic factors are:

• Antithrombin III (ATIII) deficiency

• Protein C (PC) deficiency

• Protein S (PS) deficiency

 • Factor V of Leiden (FV Leiden)

 • G20210A prothrombin mutation (FII G20210A)

Hundreds of different mutations have been described for each of the three deficiency conditions (ATIII, PC, or PS deficiency) that can cause a more or less severe reduction in the functional activity of the protein. However, the search for mutations is carried out only for research purposes since, for diagnostic purposes, the functional activity of the three proteins can be assessed. The search for FV Leiden and FII G20210A mutations is, instead, part of the screening proto col for thrombophilia.

Pharmacogenomics

Clinical molecular biology has a key role in studying mutations of genes encoding for enzymes involved in drug metabolism. Along these lines, a new area of clinical molecular biology defined as pharmacogenomics has developed recently. It is well known that about 40% of the general population is resistant to one or more drugs and, at the same time, about 15–20% of hospital admissions are due to adverse drug reactions which, in the United States, represent the fourth cause of death (after cardiovascular disease, cancer, and stroke). All drugs (antibiotics, anti neoplastics, etc.) are inactivated or, in some cases, activated through enzymatic reactions that occur mainly in the liver and constitute the metabolism of xenobiotics. The efficiency of the activation or inactivation reactions (and thus the more or less effective response to the drug) varies from individual to individual depending on the efficiency of the enzymes of xenobiotic metabolism, which in turn depends on mutations in the corresponding genes. One example, shown in Table 2, concerns cytochrome P450 2D6, which is involved in the metabolism of at least a quarter of all currently used drugs. Today, at least 70 variants of the gene encoding for cytochrome are known and, depending on the type of variant, individuals are divided into three groups:

 1. Rapid metabolizers (about 90% of the population)

2. Slow metabolizers (about 5% of the population)

3. Ultrarapid metabolizers (who inactivate the drug early: 1% of the population)

Table2. Drugs metabolized by cytochrome P450 2D6

The analysis of these variants can therefore allow to predict the individual response to the drug and modulate the drug’s type (or dosage) to be administered. In other cases, the alterations mentioned above may concern the gene encoding for the protein targeted by the drug, as in the case of the β-adrenergic receptor 2, the target of anti asthmatic drugs. For many last-generation chemotherapies and antibiotics, it is now possible to predict the patient’s response by analyzing genes encoding for proteins involved in their metabolism, adopting more targeted therapeutic choices.

Guide Therapy in Oncology: The BRCA1/2 Test

The recent inter-societal recommendations of AIOM, SIGU, SIBIOC, and SIAPEC-IAP have indicated the diagnostic and therapeutic pathway for high-grade serous ovarian cancer (HGSOC) patients. For this neoplasm, the indications of the various international medical agencies (FDA, EMA, AIFA) provide for the execution of genetic testing BRCA1/2 to identify pathogenic variants (class 4 or 5) that give the patient access to treatment with PARP-1 inhibitors (Olaparib). The identification of a pathogenetic variant in one of the two BRCA genes has not only prognostic but also predictive importance. Also, it allows preventive planning strategies at a personal (e.g., assessing the risk of breast cancer) or family level (increased risk for breast, ovarian, prostate, and pancreatic cancer, mainly). Therefore, the BRCA test is essential for diagnosing and managing patients with ovarian cancer, mainly because about 22% (17–25% depending on the case series) of women with HGSOC are carriers of an alteration in one of the BRCA genes.

The test for BRCA1/2 is performed mainly using massively parallel sequencing technology (NGS), on the most common platforms, using CE-IVD certified kits coupled to bioinformatics software for data analysis. These methodologies allow the identification of most sequence variants, i.e., about 90% of those present in exonic regions and exon/intron junctions: from single nucleotide variants to deletions/insertions and truncating mutations.

NGS technology can fail in the detection of large rearrangements (partial or whole gene deletions), for which other analysis techniques, such as multiple ligation probe assay (MLPA) and multiplex amplicon quantification (MAQ), are required. The setting of these two methodologies requires using DNA with a high degree of integrity and a certain number of control samples; therefore, choosing reference samples is very important. Although apparently over lapping, these two technologies have peculiar characteristics making them complementary. Therefore, both should be used to thoroughly understand the CNV (copy number variation) status of BRCA1/2 genes.

The integration of NGS and MLPA/MAQ technologies allows an excellent coverage of all diagnostic needs related to the BRCA1/2 test. In some cases, however, it is necessary to resort to an analysis by CGH array (comparative genomic hybridization array) to characterize large rearrangements. The latter generally occurs in about 2% of patients analyzed and more frequently in BRCA1 due to the presence of the BRCA1 pseudogene, which has a homology of about 98%.

Finally, one of the significant problems for laboratory medicine professionals concerns the classification of variants of uncertain significance (VUS) that frequently occur in BRCA1/2. For a correct evaluation of such VUS, referring to the criteria of the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA; https://enigmaconsortium.org/) is necessary. It is important to follow these criteria because studies in mouse models have shown that variants in BRCA1/2 associated with increased risk of developing cancer are not necessarily indicators of better response to PARP-1 inhibitor therapy. It is, therefore, necessary for laboratories to develop very robust and sufficiently documented evaluation systems (in silico, in vitro, and/or in vivo) regarding the models of interpretation of BRCA1/2 variants, especially for a correct stratification of both the risk of disease and the possibility of access to targeted therapies. The laboratory should ensure a continuous update of the status of VUS to update the classification of these variants continuously.

Therefore, laboratories performing BRCA1/2 testing must have highly qualified personnel, complementary technologies (NGS, MLPA/MAQ, cGH array), and a bioinformatics infrastructure adequate to the test and workload. Finally, participation in recognized external quality assessment programs (EQA, EMQN) and rigorous internal testing controls guarantee the maintenance of the standards required by the significant accreditation systems (e.g., ISO15189, CPA).

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مواضيع ذات صلة

neutrophil antibody screen (Granulocyte antibodies, Polymorphonucleocyte antibodies [PMN ab], Antigranulocyte antibodies, Antineutrophil antibodies, Neutrophil antibodies, Leukoagglutinin)

Myoglobin

metal testing (lead, arsenic, mercury)

Laboratory diagnosis of SARS-CoV-2 : Point of Care Molecular Test

Laboratory diagnosis of SARS-CoV-2 : Real-time PCR

marijuana (THC) test (Cannabinoid or cannabis testing, Tetrahydrocannabinol [THC] testing)

lung cancer molecular testing (AKT1, ALK, BRAF, Epidermal growth factor [EGFR], HER2, KRAS, MEK1, MET, NRAS, PIK3CA, RET, and ROS1)

kidney stone analysis (Urinary stone analysis, Renal calculus analysis)

Variability and Standardization of CSF Examination

Lab Diagnosis of Yellow fever

hexosaminidase (Hexosaminidase A, Hex A, Total hexosaminidase, Hexosaminidase A and B)

hemoglobin electrophoresis (Hgb electrophoresis)