Perspective Chapter: Predictive Genomics

Abstract

Predictive genomics can support treatment decisions by giving people the chance to act in time to prevent serious illness. Tests based on single nucleotide polymorphism (SNP) can be analyzed by various methods. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry technology detects genetic variants based on their individual mass. Standardized workflow, automation, sensitivity, quick turnaround time, and reliability are the main advantages of the MALDI-TOF use in molecular analysis. Beside pharmacogenetics, SNP variation plays a role in various fields of medicine. In the present article importance of various SNPs for nutrigenetics is presented. Especially, various aspects of fat metabolism, vitamin metabolism, and intolerances were discussed.

1. Introduction

Predictive genetics includes various areas of genetics, including nutrigenetics, fitness genetics, and pharmacogenetics. Some authors use the word “lifestyle genetics” because it is different from medical genetics.

Genetic and nongenetic information has to be combined to understand diseases and include this information into personalized preventive medicine.

For the investigation of genetic polymorphisms, mass spectrometry seems to be a very reliable and cost-efficient method compared to next generation sequencing (NGS) technology when investigating not more than 250 SNPs.

We have developed several genetic test panels for various areas of genetic diagnostics and discuss some aspects of nutrigenetics as fat metabolism, vitamins, and intolerances in this report.

2. Mass spectrometry in medicine

Mass spectrometry represents a technology, which is increasingly applied in medical diagnostics. Recently, review articles were analyzed concerning mass spectrometry consisting of microbiological pathogens, diagnosis of diseases, DNA analysis, and small molecules [1].

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is used in everyday routine in clinical diagnostic of microorganism infections. MALDI-TOF technology has many advantages versus traditional techniques, especially fast turnaround time, low amount of hands-on time, and low cost [2]. Direct identification of viruses and bacteria is possible within minutes, allowing the administration of a targeted antimicrobial therapy [3]. Microorganisms were detected by mass spectrometry based on a mass spectrum identifying a characteristic spectrum, which is compared to a large database provided by the manufacturers of the mass spectrometers [4]. Furthermore, the technology may clarify microbial resistance mechanisms [5, 6]. Numerous reports have been published identifying bacteria, fungi [6], and various viruses [7, 8].

Mass spectrometry is able to identify drugs and other metabolites in various body fluids, tissues, and cells [9, 10]. This technique is not only able to identify molecular targets but also their spatial distribution providing a three-dimensional image of the targets. Spatial analysis of drug absorption, distribution, metabolism, and toxicology has been performed using mass spectrometry imaging (MSI) technique [11, 12]. One of the recent developments of MSI is the highly multiplexed immunohistochemistry (IHC) based on MALDI MSI (MALDI-IHC), where up to 30 different antibodies simultaneously can be detected and quantified within a tissue section [13].

Imaging technology was also used in tumor classification providing a tool to identify morphological features of a tissue combined with detection of proteins, glycans, or lipids directly without the limitations and expense of antibodies [14, 15].

The technique of mass spectrometry has been used for the detection of various molecules. The molecular targets for mass spectrometry include proteins [16], peptides [17], lipids [18], glycans [19, 20, 21, 22], and metabolites [23]. Application of mass spectrometry in nucleic acid analysis has been shown in various fields [24, 25, 26].

A new and growing class of medical tests, differing from conventional medical diagnostic tests, are tests in genetics [27, 28], including pharmacogenetics [29, 30].

3. Mass spectrometry in genetics

Predictive genetic tests represent a new and growing field in medicine that differs from conventional medical diagnostic tests. Unlike testing patients with a disease condition, predictive genetics is applied in asymptomatic people to predict the future risk of disease. Early identification of individuals at risk for a specific condition will lead to reduced morbidity and mortality. Unfortunately, predictive genetic tests carry a degree of uncertainty about whether a condition will develop, when it will develop, and its severity [31].

Various genetic tests were developed and integrated into medical diagnostics, especially in predictive medicine [32].

To date, the medical genetic tests offered are mainly for BRCA1/2 (59, 40%), Lynch syndrome (23, 16%), and newborn screening (18, 12%).

3.1 SNP and GWAS

It is well-known that the DNA sequence at each locus may contain nucleotide bases: A, C, G, and T, which can be similar (homozygous) or different (heterozygous) at each DNA strength.

Single-nucleotide polymorphisms (SNPs) are DNA polymorphisms caused by a single-nucleotide substitution mutation. SNPs are caused by mutation and are present one SNP per thousand bases [33].

SNPs may influence various disease conditions and may alter metabolism of various drugs. The difference between SNP and SNV (single nucleotide variation) is that for the first more than 1% of a population has carry a variant nucleotide at a specific position of the DNA. SNPs can be present in coding (exons) or noncoding regions of DNA (introns). SNPs may cause change in the encoded amino acid or not and may be, therefore, of utmost importance or does not have any effect [34]. Although a particular SNP may not cause a disorder, some SNPs are associated with a disease. Improving knowledge may provide useful SNP markers for medical testing and a safer individualized medication to treat the most common disorders [34].

SNPs may help to provide information to prevent diseases or to give the opportunity of personalized medicine to patients.

In pharmacogenetics specific, SNPs can be used for treatment decisions or to choose the appropriate dosage of a drug. This could save time and could prevent adverse drug effects in patients.

Improved knowledge of the meaning of SNPs comes from genome-wide association studies (GWASs). The principle of GWAS is to compare genetics of two or more different groups of individuals [35, 36]. In the last years, the number of GWAS meta-analysis increased to study various traits in different populations [35].

Genetic analysis of SNPs can be done using various body materials as a source of human DNA such as saliva/buccal smears [37], blood samples [27, 38, 39], bone marrow cell lines [40], cytological liquid samples [41], formalin-fixed paraffin-embedded (FFPE) tissue [24, 26, 42, 43, 44].

3.1.1 Mass spectrometry for the detection of SNP (technical considerations)

MALDI-TOF mass spectrometry allows high efficiency in gentying. An efficient analysis of SNPs is offered by the Agena Bioscience iPLEX® procedure. Automatic extraction of DNA can be performed using Chemagic 360 Instrument (Perkin Elmer). DNA from a variety of biospecimen types (blood, saliva, cells collected by cheek brush, and even from FFPE) can be used for genotyping. Extracted DNA is then processed following manufacturer’s instruction for SNP genotyping by Agena Bioscience, described in the multiplex (iPLEX®) assay procedure.

The multiplex (iPLEX®) assay procedure and MassARRAY based on MALDI-TOF mass spectrometry includes several steps [45, 46]. The various steps of this analysis include amplification of targeted DNA sequence by PCR. Then, PCR products are neutralized with shrimp alkaline phosphatase (SAP) for unbound nucleotides. PCR products are then extended by one base. The mass is then measured using a mass spectrometer to produce/calculate a specific mass spectrum of targeted SNPs.

The MassARRAY Analyzer System is built to detect DNA fragments within a mass range of approximately 4500–9000 Da and can easily distinguish between analytes separated by 16 Da. The assay design process is assisted by an online suite of programs that allow for design using “default” settings, as well as “user-defined” settings (more advanced manipulation). Up to two plates of 96 or 384 samples can be genotyped for about 40-plex assay in around 8–12 h, resulting in the generation of more than 30,000 genotypes. Data analysis is performed using MassARRAY Typer Analyzer software from Agena Bioscience. MassARRAY, iPLEX® and SpectroCHIP are registered trademarks of Agena Bioscience, Inc.

3.2 Nutrigenetics

Nutrigenetics attempts to characterize and integrate the relationship between food constituents and gene expression. These approaches for precision nutrition and their relation to disease risk help to identify genetic variants that could modify the effects of dietary intake, affect food metabolism, and influence food preferences [47].

The aim combining genomics and nutrigenomics with clinical data is to get information about genetic variants, which are the basis for personalized nutritional supplementation. The substances of interest for nutritional genetics include lipids, proteins, vitamins, glycose, and iron or calcium [48].

Additionally, in the future, nutrigenetics and nutrigenomics will be combined with data of other omics technologies, such as proteomics and metabolomics, as well as microbiome and data technology [49].

The basic knowledge of emerging nutrigenomics and nutrigenetics can be applied to optimize health, prevention, and treatment of diseases [50]. The increasing number of patients with diabetes and obesity has led focus to these diseases, including genetic risk factors in the last years [51].

Since, these diseases have at least in part a genetic background to explore gene-diet interactions on obesity and diabetes is of utmost interest [52].

Personalized nutrition seems to be necessary because of the substantial variation in the genetic pattern of various human subjects [53].

3.2.1 Fat mass and obesity (FTO) associated gene

FTO gene variants (fat mass and obesity-associated) gene were detected in a GWAS search for genes predisposing for diabetes [36].

This gene is involved in the expression of fat deposition and metabolism-related hormones and genes [54]. For these reasons, investigation of the polymorphism of this gene is included in nearly all specific nutrigenetics and nutrigenomics tests.

Various studies have shown that polymorphisms in this gene lead to a higher body mass index [55, 56].

It has been reported that polymorphisms in the FTO gene are associated with other genes involved in adipogenesis. Furthermore, their impact is not solely dependent on the expression of the polymorphisms themselves [57, 58].

The FTO rs9939609 has been found to relate to the hormone ghrelin, which is associated with digestive behavior [59].

Childhood metabolic syndrome is prevalent around the world and is associated with increased disease risk, especially of cardiovascular diseases, including hypertension and acute coronary syndrome. Some variants of the human FTO gene contribute to the early onset of childhood metabolic syndrome [60].

It must be emphasized that epigenetic influence on the FTO gene is possible as a new approach in the treatment and management of obesity depending on the genetic variant [61].

Figure 1 shows a representative genotyping result of the SNP FTO_ rs9939609 using the MassARRAY System.

Figure 1.

Representative genotyping result of the SNP FTO_ rs9939609 with the MassARRAY system. (A) Spectrum showing a representative multiplex assay of a patient with heterozygous genotype for the SNP FTO_ rs9939609 (AT). A zoomed view in the mass range 6700–6860 shows two peaks indicated with blue dotted lines representing the heterozygous alleles (AT). (B) Representative spectrum of multiplex assay of a patient with homozygous genotype for the SNP FTO_ rs9939609 (AA). A zoomed view in the mass range 6700–6860 shows the detection of one single peak of interest (blue dotted line).

FTO polymorphisms were reviewed in a minireview, which provides thorough inside into these genetic variants [61].

3.2.2 Lipids

Dyslipidemias are known risk factors, which could require precision nutrition designed according to characteristics, such as diet, phenotype, and genotype [62]. Increased intake of triglycerides and cholesterol is associated with an increased risk of metabolic diseases.

One of the best-studied genetic polymorphisms is that of the Apolipoprotein E (ApoE)-gene.

ApoE plays a key role in the transport of cholesterol. ApoE variants are associated with early-onset Alzheimer’s disease. Specifically, ApoE4 isoform may be responsible for alterations in insulin- and lipid metabolism, altered gray matter volume, and impaired cerebrovascular functions [63].

The ApoE2 isoform is generally the most favorable and ApoE4 the least favorable for cardiovascular and neurological health. Under metabolic stress, homozygosity for ApoE2 may result in dysbetalipoproteinemia [64].

It is of special interest that omega-3 fatty acid intake and physical activity may modify the impact of ApoE4 on Alzheimer’s disease and cardiovascular disease risk [65].

Genotyping for ApoE may help develop a targeted approach to disease prevention. Adherence to Mediterranean diet may lower Alzheimer’s disease-related anatomical or clinical symptoms in individuals without ApoE4 genotype [66].

The association of diet rich in saturated fatty acids may increase Alzheimer’s risks in ApoE4 carriers [67].

3.2.3 Nonalcoholic fatty liver disease NFLD

Nonalcoholic fatty liver disease (NAFLD) is a chronic condition associated with genetic and environmental factors, obesity, type 2 diabetes, and dyslipidemia in which fat abnormally accumulates in the liver. Different genetic polymorphisms seem to be involved in this context [68].

3.2.4 Vitamins

3.2.4.1 Vitamin A

Retinol (Vitamin A) plays a crucial role in the anti-aging industry, primarily due to its ability to neutralize free radicals in tissues, which subsequently leads to a reduced appearance of wrinkles.

β-carotene 15,15′-monooxygenase 1 (BCMO1) is the most critical enzyme involved in retinoid metabolism [69].

Especially, A379V TT variant was inversely related to vitamin A status [69]. Assessment of the responsiveness to beta-carotene confirmed that carriers of variant alleles had a reduced ability to convert beta-carotene [70].

Individual responsiveness was associated with genetic variants in SNP rs7501331 of the carotenoid metabolizing enzyme BCMO1, resulting of single nucleotide variation (SNV) from C to T [71]. Carriers of T nucleotide have lower ability to convert beta-carotene. Studies shown that only 5% of the population have TT genotypes, while 56% have CC. Having this on mined and knowing that lacking of normal retinol metabolism is responsible for several diseases, supplementation/treatment of vitamin A should be completely personalized in the future in regards to genetic variations in the BCO1 gene [72, 73].

3.2.4.2 Vitamin B9 (folic acid)

Vitamin B9 is molecule responsible for normal cell growth and development. It is crucial supplement in the prevention of pregnancy complication. Enzyme, which plays a key role in vitamin D metabolisms, is 5,10-methylenetetrahydrofolate reductase (MTHFR), and it regulates around 60% of folic acid metabolism [48].

Mutations in the MTHFR gene can result in abnormal folate metabolism, which is associated with and may contribute to various pathological conditions, including stroke, depression, and reduced cognitive function etc.

Two mutations in SNPs, rs1801131 (SNV, T > G) and rs1801133 (SNV, G > A) have been reported that affect enzymatic activity of MTHFR [74].

Depending in which SNP is the mutation and the state of mutation (is it homozygous or not) several possible phenotypes can be detected. Each phenotype is associated with specific enzyme the activity and function.

Individuals with MTHFR (rs1801133) genotype CC has normal homocysteine levels, on the other hand, patients with TT genotype have high level of homocysteine and low folate levels. So, autosomal recessive MTHFR polymorphism led to wide range of vascular and neurological unfunctionally [75, 76].

The risk genotype of rs1801133 has been related with various pathological conditions such as deep vein thrombosis, various cardiovascular diseases (CVDs), then cancer, diabetes, etc. [76, 77, 78].

TT genotype is also known as C677T MTHFR polymorphism. Supposition of C with T will lead to amino acid change from alanine to valine. If a patient has two defective alleles of the MTHFR gene, enzyme activity can be reduced by 80 to 90%. Recent clinical studies have demonstrated that carriers of these alleles are at a significantly higher risk of ischemic stroke [79].

Studies on MTHFR have been successfully used to develop disease prevention strategies [80]. And therefore, future health education has to be based on personalized nutritional recommendations and prevention strategies in the field of vitamins supplementation.

3.2.4.3 Vitamin D

Vitamin D has been highlighted as a prime example of nutrigenomics. It is a molecule with multiple roles in the human body, including important functions in metabolism and various clinical applications [81, 82].

This vitamin plays a role in numerous system, such as in the immune system [83], skeletal system [84], reproductive system [85], insulin secretion [86], and intestinal system [87].

Vitamin D deficiency is very frequent, with almost 40% of the Europeans presenting levels below 50 nmol/L [88].

Vitamin D receptor (VDR) regulates several target gene transcription processes necessary for various biological functions of vitamin D. VDR will make hormone-receptor complex with active form of vitamin D (1,25(OH)2D3) in target cells. This complex interfere with specific DNA sequences of target genes to control the expression of numerous genes [89, 90].

Some genetic variants of the gene encoding VDR modify either its expression or function, with the consequent disruption of the vitamin D signaling pathway. Recent publications on the relationship between VDR genetic variants and the risk of type 2 diabetes, metabolic syndrome, overweight, and obesity were reviewed and give only partial answers to this question [91].

Vitamin D analogs bind to vitamin D receptors in tumor cells and activate downstream pathways to inhibit tumor growth. VDR expression is a prognostic indicator for digestive system tumors. That the intake of vitamin D analogs should be determined according to vitamin D receptor expression was stated in a comprehensive review on tumors of the digestive tract [92].

Findings considering gene polymorphisms in the VDR gene, which are based on the role of VDR SNPs in gene regulation and protein expression, will help to understand the detected role of VDR in various diseases [93].

For personalized medicine and pharmacogenomics new studies of VDR polymorphisms and vitamin D-VDR signaling are necessary for better understanding of role of this complex in various diseases [94].

Individuals who are genetically predisposed to low vitamin D benefit from foods rich in this vitamin [95, 96].

Individuals with genetic changes in the VDR gene may benefit from foods rich in vitamin D and from calcium and/or vitamin D supplementation [97, 98].

3.3 Intolerances

3.3.1 Gluten

Celiac disease (CD) is an autoimmune disorder, affecting about 1% of the population, where individuals are genetically predisposed to gluten intolerance [99, 100]. Gluten is a protein complex present in some cereals such as wheat, barley, and rye, in which gliadins and glutenin proteins are considered to be responsible for the inappropriate immune response of the genetically predisposed individuals. This condition can cause a localized complication in the mucosa of the intestine with toxic effects, leading to villous atrophy and lymphocyte infiltration in the small intestinal mucosa [101]. Typical symptoms include diarrhea, digestive tract pain and discomfort, weight loss, and malabsorption of nutrients [102].

There is a genetic basis at the origin of CD that determines susceptibility to the disease, which is correlated with genes in the human leukocyte antigen (HLA) system. More specifically, genetic testing for CD consists of determining the presence of the HLA DQ2 and DQ8 alleles [103].

Most CD cases (90%) are associated with the presence of the HLA-DQ2 haplotype encoded by (HLA-DQA1*05-DQB1*02). Some patients (5%) carry a second HLA DQ8 heterodimer encoded by (DQA1*03-DQB1*0302), and the remaining 5% of patients hold at least one of the two genes [104]. Six SNPs in HLA-DQ genes are responsible for CD, and in 75% patients CD has hereditary pattern. Sporadic, non -HLA related CD will occur in 68% patients [105].

Screening of single nucleotide polymorphisms by mass spectrometry within HLA region is an efficient method to accurately analyze multiple SNPs at the same time [106]. For example, individuals with CC (around 1% in Caucasian population) genotype in HLA-DQ8 gene (rs7454108) have high risk of gluten intolerance, while CT genotype indicates moderate risk of gluten intolerance However, the most frequent genotype for this polymorphism in Caucasian population is TT (no risk), around 80% (Figure 2), indicating that other genes. SNPs from HLA-DQ system plays key role in gluten intolerance.

Figure 2.

(A) Representative cluster plot displaying the SNP HLADQ8_rs7454108 genotypes of 70 samples assayed. Heterozygous region is close to the 45°-line (green squares represent heterozygous (TC) samples. Homozygous region for the low mass allele is close to the X coordinate (blue triangles represent homozygous (TT) samples for the same SNP. (B) Representative spectrum of homozygous genotype (TT) for the SNP HLADQ8_rs7454108 where one single peak of interest (blue dotted line) is indicated for the allele T. (C) Example of a spectrum of heterozygous genotype (TC) for the same SNP HLADQ8_rs7454108 where two peaks of interest are indicated with blue dotted lines representing the heterozygous alleles (AT).

3.3.2 Lactose

The most common carbohydrate, which is the main component of milk and milk products, is disaccharide lactose. Necessary enzymes in lactose metabolism (located at LCT/MCM6 gene) have role to cut lactose in glucose and galactose [107]. The ability to digest and have a normal metabolism of lactose in adults is called lactase persistence/persistent (LP). LP is regulated by five genetic polymorphisms, which have dominant distribution in humans [108].

Lactase non-persistence/intolerance is a worldwide phenomenon, but it affects people with varying degrees of severity. Completely inactive lactose gene is very rare and present in patients with alactasia [109].

Lactose intolerance can manifest as secondary lactase deficiency, which can be either temporary or chronic, depending on the duration and nature of the harmful mediator affecting the small intestinal mucosa cells. Alternatively, it can appear as primary lactase deficiency, typically emerging in adolescence or early adulthood [110].

In the natural condition lactase non-persistence (LPN) in primary lactase deficiency, the activity of the enzyme LCT decreases with age. Various studies have been done and shown that genetic alterations are responsible for type of reduced lactase enzyme activity [111].

In the European population polymorphism in gene MCM6 (rs4988235, C > T), which is placed in the promoter region of MCM6 gene is mainly responsible for tolerance or intolerance for lactose.

Lactase intolerance in adults is triggered by a recessively congenital polymorphism of the MCM6 gene, and this phenotype is inherited as an autosomal dominant characteristic. Individuals who carry C alle are likely that will develop lactose intolerance during lifetime, meaning that CT individuals have more chance that they can digest milk in older ages, while TT individuals can digest lactose during all lifetime [112].

The decreased ability of the body to hydrolyze lactose is due to a programmed regulatory phenomenon involving the MCM6 gene intron, 13,14 kb upstream of the MCM6 gene. This gene has several single nucleotide base polymorphisms including the (rs4988235) for which the Thymine (T) allele forms an haplotype that is commonly evaluated in LP studies [113].

The −13,910∗T allele is commonly distributed in the European population with an average frequency of 50.8%. However, genetic distribution of T allele is most prevalent in northern Europe, especially England and Scotland (72.0% of the population), while it progressively decreases in the southern Europe with a frequency of 8.9% in Tuscany, Italy [114].

Mutations in the SNP rs4988235 cause intolerance problems [115]. Specifically, a genotype that has two (TT) at position –13,910 results in a lactase persistence (LP), while a homozygous CC genotype in the same position results in a lactase non-persistent (LNP) phenotype [116, 117].

The undigested lactose that remains in the intestine is metabolized by intestinal bacteria with the generation of an osmotic effect causing a recall of water, resulting in symptoms such as diarrhea, cramping, meteorism, intestinal discomfort, and sometimes nausea and vomiting [118, 119].

Twenty-nine percentage of individuals reported symptoms attributed to the ingestion of fresh milk, with abdominal pain, bloating, and flatulence being the most frequent [120].

Previous studies have shown that gut microbiota may be capable of adapting to lactose consumption in LNP individuals [121].

Mass spectrometry seems to be an ideal method to detect SNPs in the MCM-Gene. By excluding the genetic predisposition to lactose intolerance, people can avoid unnecessary dietary restrictions on dairy products [122].

4. Conclusions

This book chapter aimed to present the application of mass spectrometry for DNA analysis. After a small introduction of application of mass spectrometry in modern medicine methods for the detection of SNPs were discussed. Furthermore, an overview about studies using SNPs as genetic markers related to nutrigenetics, including fat metabolism, vitamins, and intolerances, were provided.

In the last years, MALDI-TOF mass spectrometry technique has been proven to be a versatile tool for the characterization of point mutations. This method allows the detection of SNPs in a rapid, precise, cost-effective, and high-throughput way. MALDI-TOF is a technique, which allows assessment of up to 250 SNPs. For the analysis of a larger amount of SNPs or genome-wide association studies, next-generation sequencing is the method of choice.

Conflict of interest

The authors declare no conflict of interest.