**2. Single-nucleotide polymorphisms in innate and adaptive immunity genes in IBD**

The latest research on genetics and immunology has confirmed that the innate immune system is of great importance in inducing intestinal inflammation [13], and the publication of Dudzińska et al. [14], "Single nucleotide polymorphisms in selected genes in inflammatory bowel disease," is dedicated to this issue. This paper attempts to demonstrate polymorphisms *NOD2/CARD15* (*nucleotide-binding oligomerization domain containing 2/caspase recruitment domain family number 15*) and the *DLG5* gene (*discs large homolog 5*) in patients with IBD hospitalized at the Department of Gastroenterology, Regional Specialist Hospital, SPZOZ im. Stefan Cardinal Wyszynski in Lublin.

*NOD2/CARD15* is located on chromosome 16q12.1 and was the first diseasesusceptibility gene discovered for CD. *NOD2/CARD15* is a pattern-recognition receptor that is involved in the homeostasis of intestinal immunity [15].

*CARD15/NOD2* plays an important role in immune function. In response to bacterial infection, *CARD15/NOD2* acts as an intracellular bacterial receptor and activates the kappa B nuclear factor (NF-κB), particularly after recognizing the bacterial wall component muramyl dipeptide (MDP) [16].

*NOD2/CARD15* mutations lead to dysregulation of host-microbe interactions, contributing to the development of inflammation in the ileum, which is characteristic of CD [17].

Today more than 60 polymorphisms of this gene have been identified; however, three common mutations Leu1007fsinsC, Arg702Trp, and Gly908Arg have been specifically associated with ileal involvement, stricturing complications, and earlier age of onset [18].

While the gene *DLG5* encodes scaffold proteins belonging to the MAGUK family, which participate in the formation of cellular connections, maintenance of cell shape, and intracellular signal transduction [19]. Expression of this gene is widely expressed in the tissues of the small and large intestines. *DLG5* gene polymorphisms have been shown to increase susceptibility to IBD, including both CD and UC [20]. *DLG5* has been shown to be localized at cell-cell contact sites and is involved in maintaining epithelial integrity. Different variants of *DLG5* may contribute to the loss of cell polarization complexes and adhesion complexes, so that epithelial cell polarity is not maintained, and epithelialmesenchymal transition (EMT) is induced [20]. EMT is a process involving the transformation of immobile, polarized cells with an epithelial phenotype into cells with a mesenchymal phenotype. The characteristic features of EMT include lack of polarity and cell adhesion, reduced expression of E-cadherin, and increased mobility and invasion capacity [21].

Thus, it can be assumed that *DLG5* polymorphisms may impair the epithelial barrier in the gastrointestinal tract and lead to abnormal epithelial structure, making it more susceptible to IBD (CD and UC). Furthermore, the *DLG5* scaffold protein also belongs to the CARD family of proteins (like *CARD15*/*NOD2*). Thus, *DLG5* is probably involved in the regulation of NF-κB activation or caspase activation within the host defense mechanisms [22]. Therefore, both the *NOD2* and *DLG5* genes may interact functionally to contribute to the risk of developing CD.

Our research in the group of patients diagnosed with CD and UC is related to the occurrence of polymorphisms in the *NOD2* and *DLG5* genes.

The test material was blood collected from patients on an empty stomach after 12 hours of rest. In addition, medical history was taken with regard to the occurrence of extraintestinal symptoms and autoimmune diseases in the family. The family history of all subjects was negative. DNA was isolated using a QIAamp DNA Blood Mini Kit (QIAgen), followed by quantitative and qualitative evaluation of the isolated DNA samples.

The following primers were used for PCR: (F) GACTCTTTTGGCCTTTTCAGATT and (R) CCAATGGTCTTTTTTCCTTACTCC for *CARD15/NOD2* and (F) TTATTCCCCTTCCACAGGCACTAC and (R) GCCGCAGCTGAATGGAGA for *DLG5*.

The PCR product was sequenced, and the sequences obtained were recorded in FASTA format. The nucleotide sequences of the *CARD15/NOD2* and *DLG5* gene fragments were compared using DNA Baser software.

The size of the analyzed *CARD15/NOD2* gene fragment was 243 bp. No SNPs were observed in this fragment in patients with CD or UC.

Although numerous reports confirm that *CARD15/NOD2* gene polymorphisms are associated with a predisposition to IBD [23], our research did not show this relationship. This may be linked to the absence of parenteral symptoms in the subjects.

One of the most common single-nucleotide polymorphisms of the *CARD15/ NOD2* gene is P268S (SNP5), where the cytosine residue at position 802 is replaced by thymine. In the Polish population, P268S polymorphism has been found in 49.5% of patients with CD, and its presence in both alleles is associated with earlier

**45**

IBD [25].

**Figure 3.**

*Single-Nucleotide Polymorphisms in Inflammatory Bowel Disease*

abnormalities, iritis, and erythema nodosum [24].

IBD, in particular Crohn's disease.

in patients with IBD [27].

onset of disease symptoms and increased risk of parenteral symptoms such as joint

*SNP at position rs1248696, T > C substitution in a patient with Crohn's disease (source: Dudzi*ń*ska et al. 2018).*

Despite numerous reports indicating the presence of polymorphisms of the *NOD2/CARD15* gene, our studies did not show the position of SNP in patients with IBD from the Lublin region. The study suggests that SNPs (T > C substitution) affect the function of the *DLG5* protein and thus play a role in the development of

Autophagy is a conserved lysosome-dependent catabolic process, degrading and recycling protein aggregates or damaged organelle. Autophagy affects the pathogenesis of IBD in multiple ways, including secretion of antimicrobial materials from Paneth cells, clearance of invading pathogens, presentation of antigen, or

SNPs of autophagy genes such as autophagy-related gene 16 like 1 (*ATG16L1)* and immunity-related GTPase family M (*IRGM*) have been identified to be associated with CD. *ATG16L1* and *IRGM* genes affect cellular autophagy processes and bacterial clearance in immune cells and may affect bacterial composition of the gut

There is some overlap in these autophagy-related genetic variants in both CD and UC, but majority of the identified variants are more associated with ileal CD. Despite this association, the positive predictive value for disease development in individuals carrying autophagy variants is low because the *ATG16L1* T300A polymorphism is linked with CD susceptibility, which is also present in a large

**3. The role of autophagy genes in the pathogenesis of IBD**

proinflammatory cytokine production by macrophages [26].

proportion of healthy individuals who do not develop IBD [28].

was found in the gene fragment. A T > C substitution occurred in one sample (Patient 3) from the group of patients with Crohn's disease (**Figure 3**). Other authors note that the R30Q (Rs1248696) variant of *DLG5*, where amino acid 30 in exon 3 changes from arginine to glutamine, is associated with the development of

The size of the *DLG5* gene fragment was 107 bp. One SNP at position 1,248,696

*DOI: http://dx.doi.org/10.5772/intechopen.92051*

*Single-Nucleotide Polymorphisms in Inflammatory Bowel Disease DOI: http://dx.doi.org/10.5772/intechopen.92051*

#### **Figure 3.**

*The Recent Topics in Genetic Polymorphisms*

tic of CD [17].

age of onset [18].

isolated DNA samples.

*NOD2/CARD15* mutations lead to dysregulation of host-microbe interactions, contributing to the development of inflammation in the ileum, which is characteris-

Today more than 60 polymorphisms of this gene have been identified; however, three common mutations Leu1007fsinsC, Arg702Trp, and Gly908Arg have been specifically associated with ileal involvement, stricturing complications, and earlier

While the gene *DLG5* encodes scaffold proteins belonging to the MAGUK family, which participate in the formation of cellular connections, maintenance of cell shape, and intracellular signal transduction [19]. Expression of this gene is widely expressed in the tissues of the small and large intestines. *DLG5* gene polymorphisms have been shown to increase susceptibility to IBD, including both CD and UC [20]. *DLG5* has been shown to be localized at cell-cell contact sites and is involved in maintaining epithelial integrity. Different variants of *DLG5* may contribute to the loss of cell polarization complexes and adhesion complexes, so that epithelial cell polarity is not maintained, and epithelialmesenchymal transition (EMT) is induced [20]. EMT is a process involving the transformation of immobile, polarized cells with an epithelial phenotype into cells with a mesenchymal phenotype. The characteristic features of EMT include

lack of polarity and cell adhesion, reduced expression of E-cadherin, and

genes may interact functionally to contribute to the risk of developing CD.

occurrence of polymorphisms in the *NOD2* and *DLG5* genes.

The following primers were used for PCR: (F)

fragments were compared using DNA Baser software.

were observed in this fragment in patients with CD or UC.

GCCGCAGCTGAATGGAGA for *DLG5*.

Thus, it can be assumed that *DLG5* polymorphisms may impair the epithelial barrier in the gastrointestinal tract and lead to abnormal epithelial structure, making it more susceptible to IBD (CD and UC). Furthermore, the *DLG5* scaffold protein also belongs to the CARD family of proteins (like *CARD15*/*NOD2*). Thus, *DLG5* is probably involved in the regulation of NF-κB activation or caspase activation within the host defense mechanisms [22]. Therefore, both the *NOD2* and *DLG5*

Our research in the group of patients diagnosed with CD and UC is related to the

The test material was blood collected from patients on an empty stomach after 12 hours of rest. In addition, medical history was taken with regard to the occurrence of extraintestinal symptoms and autoimmune diseases in the family. The family history of all subjects was negative. DNA was isolated using a QIAamp DNA Blood Mini Kit (QIAgen), followed by quantitative and qualitative evaluation of the

GACTCTTTTGGCCTTTTCAGATT and (R) CCAATGGTCTTTTTTCCTTACTCC

The PCR product was sequenced, and the sequences obtained were recorded in FASTA format. The nucleotide sequences of the *CARD15/NOD2* and *DLG5* gene

The size of the analyzed *CARD15/NOD2* gene fragment was 243 bp. No SNPs

Although numerous reports confirm that *CARD15/NOD2* gene polymorphisms are associated with a predisposition to IBD [23], our research did not show this relationship. This may be linked to the absence of parenteral symptoms

One of the most common single-nucleotide polymorphisms of the *CARD15/ NOD2* gene is P268S (SNP5), where the cytosine residue at position 802 is replaced by thymine. In the Polish population, P268S polymorphism has been found in 49.5% of patients with CD, and its presence in both alleles is associated with earlier

for *CARD15/NOD2* and (F) TTATTCCCCTTCCACAGGCACTAC and (R)

increased mobility and invasion capacity [21].

**44**

in the subjects.

*SNP at position rs1248696, T > C substitution in a patient with Crohn's disease (source: Dudzi*ń*ska et al. 2018).*

onset of disease symptoms and increased risk of parenteral symptoms such as joint abnormalities, iritis, and erythema nodosum [24].

The size of the *DLG5* gene fragment was 107 bp. One SNP at position 1,248,696 was found in the gene fragment. A T > C substitution occurred in one sample (Patient 3) from the group of patients with Crohn's disease (**Figure 3**). Other authors note that the R30Q (Rs1248696) variant of *DLG5*, where amino acid 30 in exon 3 changes from arginine to glutamine, is associated with the development of IBD [25].

Despite numerous reports indicating the presence of polymorphisms of the *NOD2/CARD15* gene, our studies did not show the position of SNP in patients with IBD from the Lublin region. The study suggests that SNPs (T > C substitution) affect the function of the *DLG5* protein and thus play a role in the development of IBD, in particular Crohn's disease.
