IN NONALCOHOLIC FATTY LIVER DISEASE (NAFLD)

The present invention relates to the use of the SNP rs17618244 as a predictive marker in NAFLD. In greater detail, the invention relates to the use of the single nucleotide polymorphism (SNP) rs17618244, wherein an adenine (A) is present in place of a guanine (G) in the gene encoding the beta-Klotho (KLB) coreceptor, located on chromosome 4 in position 39446909, as a biomarker in in vitro diagnosis for detecting a genetic predisposition to or the risk of developing non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASFI) and the tissue damage associated with NASFI.

As is well known, non-alcoholic fatty liver disease or NAFLD is a pathology characterized by an ample spectrum of histological damages to the liver that range from simple steatosis to non-alcoholic steatohepatitis (NASH), i.e. a combination of steatosis and inflammatory processes with structural lesions to the hepatocyte (such as for example ballooning). In some cases, the NASH can evolve into more serious forms such as fibrosis and cirrhosis [1 ].

Until today, NAFLD is considered to be a multifactor pathology where environmental factors (excessive consumption of food rich in fat and sugars and a sedentary lifestyle), and genetic factors together play a supplementary part in the development and progression of the disease [2]

With regard to the diagnosis of the disease, while NAFLD can be diagnosed for example with a liver ultrasound, NASH and the various types of tissue damage associated therewith, like ballooning, inflammation and fibrosis, can be diagnosed only following a liver biopsy, which, as is known, constitutes an invasive method. Further, in the particular case of paediatric patients, carrying out a biopsy brings further risks that have to be evaluated on a case by case basis [3].

In recent years, genome wide association studies (GWAS) have identified in genes involved in important paths of lipid and glucidic metabolism and of oxidative stress (in PNPLA3, TM6SF2, KLF6, LPIN1 , SOD2), different variations associated with the risk of NAFLD in adults. Different studies have shown that some of these single nucleotide polymorphisms (SNPs), like PNPLA3 and TM6SF2, constitute NAFLD risk factors also in paediatric patients [4-6]. Owing to their frequency, these SNPs can explain genetic susceptibilityto the development and progress of NAFLD in most individuals, but they are not sufficient for discriminating the type of histological damage associated with NASFI. On the other hand, further SNPs, although they are rarer, could be associated with a proportion of individuals who do not have the allelic variants of PNPLA3 and TM6SF2 or could be associated with specific histological traits that characterize the liver damage associated with NASFI.

In the light of what has been set out above, it is therefore apparent the need to be able to dispose of new biomarkers that are able to overcome the drawbacks and limits of known biomarkers.

In this regard, various studies are known relating to the role of fibroblast growth factors 19 and 21 (FGF19 and FGF21 ) and of their receptor complex (KLB/FGFR) in the development and progression of NAFLD [7,8]

FGF19 and FGF21 , unlike the canonical FGFs, act as hormones and are involved respectively in regulating the metabolisms of the bile acids and in regulating the metabolism of the fats. The interaction with the beta-Klotho (KLB) coreceptor is able to facilitate the FGF molecule bond with their receptors (FGFR), thus enabling activation of the entire receptor complex [9].

As already known [10], KLB is a glycoprotein consisting of a short intracellular domain, a transmembrane portion and a large extracellular domain containing the two sites bKL1 and bKL2, which are devoid of glycosidasic activity. KLB displays high sequence homology with the Klotho coreceptor (KL), which has both a transmembrane form and a trunk form that can act as a humoral factor. Although it has not been demonstrated, it is plausible that also KLB has both transmembrane and extracellular activity.

KLB is abundantly expressed at the level of the liver, pancreas and adipose tissue. At the liver level, it permits the interaction between the fibroblast growth factor 19 (FGF19) and its FGFR4 receptor, expressed on the membrane of the hepatocytes, permitting full activation of the receptor complex by acting on the synthesis of bile acids and the metabolism of carbohydrates [9].

Recently, a close association has been reported between the decrease of the plasma levels of the fibroblast growth factors 19 and 21 (FGF19 and FGF21 ), the reduced hepatocellular expression of their Klotho coreceptor (KL) and the gravity of the histopathological picture of NAFLD in paediatric patients [1 1 ].

It is further known that the variant rs17618244 G>A in the KLB gene, that entails the replacement of a guanine (G) with an adenine (A), is associated with a pathology known as irritable bowel syndrome (IBS)[12].

According to the present invention, it was found in obese children suffering from NAFLD that a reduced hepatocellular expression of KL was mainly ascribable to its homologue protein KLB, which is in turn linked to the presence of a specific SNP in its encoding gene.

The results obtained are very significant because they demonstrate for the first time a relationship between the rs17618244 variant in KLB and the severity of the liver damage in paediatric patients suffering from NAFLD.

In the population examined, it was observed that the rs17618244 variant in KLB was more present, i.e. significantly more frequent, in the patients with NAFLD than in the healthy controls. It is also clear that the presence of the homozygous variant (gene type AA) is correlated with an increased risk of developing NASH and the related tissue damage.

A further interesting datum is provided by the confocal microscope analysis, which shows a reduction in the levels of KLB protein in the liver tissue of patients with NASH compared with Not-NASH patients and this reduction is even more marked in patients carrying the mutation.

Lastly, further results obtained according to the present invention have shown, by in vitro experiments on cells, that the over-expression of the mutated KLB gene (KLB rs17618244) causes an increase in intracellular lipid content and lipotoxicity, for example by increased expression of genes like p62, increased expression of genes involved in the regulation of the metabolism of fatty acids and increased expression of genes encoding pro-inflammatory cytokines. The results obtained according to the present invention suggest that the presence of the rs17618244 variant in KLB is able to cause a change in a codon of the messenger RNA causing the replacement of an amino acid that is different from the original one and consequently the alteration of the structure, synthesis and function of the protein. In fact, the reduction of the KLB protein at the tissue level could be due to the decrease of the levels of synthesised protein and could follow the decrease of the plasma levels of its cleaved form.

The rs17618244 variant in KLB can then be advantageously used as biomarker to identify individuals who are genetically susceptible to NAFLD, or to NAFLD progression into NASH and to the histological NASH-related damage. This would then enables the patient’s clinical picture to be clarified further and the actual need for an invasive technique like liver biopsy to be confirmed.

It is therefore a specific object of the present invention an in vitro diagnosis method for detecting the risk, or the genetic predisposition, of a person of developing non-alcoholic fatty liver disease (NAFLD) or of developing its more serious form, i.e. non-alcoholic steatohepatitis (NASH) and the tissue damage associated with non-alcoholic steatohepatitis (NASH), said method consisting of or comprising detecting the single nucleotide polymorphism (SNP) rs17618244, wherein an adenine (A) is present in place of a guanine (G) in the gene encoding the beta-Klotho (KLB) coreceptor, located in the chromosome 4 in position 39446909, in a biological sample, wherein the presence of said single nucleotide polymorphism rs17618244, in homozygous form or in heterozygous form, indicates the risk of developing non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH) and the tissue damages associated with non-alcoholic steatohepatitis.

Therefore, the presence of said single nucleotide polymorphism rs17618244, both in homozygous form and in heterozygous form, in a subject indicates a greater predisposition or a higher risk of developing non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH) and the tissue damages associated with non-alcoholic steatohepatitis (NASH) compared with a subject in which the gene encoding the beta-Klotho (KLB) coreceptor is present in wild type form.

According to the method of the present invention, there is a greater risk of developing non-alcoholic steatohepatitis (NASH) and the tissue damage associated with non-alcoholic steatohepatitis (NASH) when said single nucleotide polymorphism rs17618244 is detected in homozygous form with respect to when said single nucleotide polymorphism rs17618244 is detected in heterozygous form.

In particular, the tissue damages associated with non-alcoholic steatohepatitis (NASH) can be ballooning and/or inflammation and/or fibrosis.

According to the method of the present invention, the biological sample can be a peripheral blood sample or a saliva sample or a urine sample.

According to one embodiment of the present invention, the method can further comprise detecting one or more of the following single nucleotide polymorphisms: PNPLA3 rs738409, SOD2 rs4880, LPIN1 rs13412852, KLF6 rs3750861 , TM6SF2 rs58542926, ZNF624 rs12603226.

In particular, the techniques that can be used for detecting the single nucleotide polymorphism rs17618244 and further polymorphisms mentioned above can be: direct sequencing (Sanger method), pyrosequencing, Next Generation Sequencing (NGS: lllumina; Life Technologies; Roche), Restriction Fragment Length Polymorphism (RFLP), Flap endonuclease (FEN), primer extension, Oligonucleotide Ligation Assay (OLA), Denaturing Gradient Gel Electrophoresis (DGGE), Single Stranded Conformation Polymorphism (SSCP), Strand Displacement Amplification (SDA), isothermic in vitro amplification of DNA and its variants (Loop Amplification, LAMP), Isothermal Amplified Detection of DNA and RNA, Linear Amplification (LLA), denaturing High Performance Liquid Chromatography (dHPLC), 5’nuclease assay (TaqMan Assay), PCR with reverse hybridization (Reverse Dot Blot), Allele-Specific Oligonucleotide (ASO), Dynamic Allele-Specific Hybridization ( DASH), molecular beacons and other variants;“scorpions” probes; Fluorescence Resonance Energy Transfer (FRET); High Melt Resolution Analysis (HRMA), SNParray, Nucleic Acid Arrays, Multiplex Ligation Dependent Probe Amplification (MLPA), genetic analyses mediated by ligase/polimerase and related variants and methods, Southern Blot, Transcription Mediated Amplification (TMA); NASBA (Nucleic Acid Sequence Based Amplification); self-sustained sequence replication.

According to the method of the present invention the following forward primer CGAGCCTCTGTTGCATGC (SEQ ID NO:1 ) and the following reverse primer TTGAGCAGCCTCCTTTCGG (SEQ ID NO:2) can be used for detecting the single nucleotide polymorphism (SNP) rs17618244, for example by the Sanger method.

According to one embodiment of the present invention, the method can provide for the use of two probes targeting the following sequences: GCCCTGGCCTGGCGCCT CT ACGACCAGCAGTT CAGGCCCT CACAGC GCGGG (SEQ ID NO:3),

GCCCTGGCCTGGCGCCT CT ACGACCGGCAGTT CAGGCCCT CACAGC GCGGG (SEQ ID NO:4), for detecting the single nucleotide polymorphism (SNP) rs17618244, for example when SNP rs17618244 is detected by Real Time PCR.

In particular, the method of the present invention can be used for paediatric subjects.

It is a further object of the present invention the use of single nucleotide polymorphism (SNP) rs17618244, wherein an adenine (A) is present in place of a guanine (G) in the gene encoding the beta-Klotho (KLB) coreceptor, located in the chromosome 4 in position 39446909, as a biomarker in in vitro diagnosis for detecting the risk, or the genetic predisposition, of a subject of developing non-alcoholic fatty liver disease (NAFLD) or of developing its more serious form, i.e. non-alcoholic steatohepatitis (NASH) and the tissue damages associated with non alcoholic steatohepatitis (NASH).

In particular, as mentioned above, the risk of developing non alcoholic steatohepatitis (NASH) and the tissue damages associated with non-alcoholic steatohepatitis (NASH) is higher when said single nucleotide polymorphism rs17618244 is present in homozygous form than when said single nucleotide polymorphism rs17618244 is present in heterozygous form .

The tissue damages associated with non-alcoholic steatohepatitis (NASH) can be ballooning and/or inflammation and/or fibrosis.

According to the present invention, the SNP rs17618244 biomarker can be used in combination with one or more of the following single nucleotide polymorphisms: PNPLA3 rs738409, SOD2 rs4880, LPIN1 rs13412852, KLF6 rs3750861 , TM6SF2 rs58542926, ZNF624 rs12603226.

The biomarker according to the present invention can be advantageously used in paediatric subjects.

It is a further object of the present invention a kit for in vitro diagnosis for detecting the risk, or the genetic predisposition, of a subject of developing non-alcoholic fatty liver disease (NAFLD) or of developing its more serious form, i.e. non-alcoholic steatohepatitis (NASH) and the tissue damage associated with non-alcoholic steatohepatitis (NASH), said kit comprising primers or probes for detecting the single nucleotide polymorphism (SNP) rs17618244, wherein an adenine (A) is present in place of a guanine (G) in the gene encoding the beta-Klotho (KLB) coreceptor, located in the chromosome 4 in position 39446909, in combination with primers or probes for detecting one or more of the following single nucleotide polymorphisms: PNPLA3 rs738409, SOD2 rs4880, LPIN1 rs13412852, KLF6 rs3750861 , TM6SF2 rs58542926, ZNF624 rs12603226.

According to one embodiment of the present invention, the kit can comprise the following primers for detecting the single nucleotide polymorphism (SNP) rs17618244: forward primer with CGAGCCTCTGTTGCATGC (SEQ ID NO:1 ) sequence and reverse primer with TT GAGCAGCCT CCTTT CGG (SEQ ID N0:2) sequence.

Further, the probes for detecting the single nucleotide polymorphism (SNP) rs17618244 can be the probes constructed using the following target sequences:

GCCCTGGCCTGGCGCCT CT ACGACCAGCAGTT CAGGCCCT CACAGC GCGGG (SEQ ID NO:3),

GCCCTGGCCTGGCGCCT CT ACGACCGGCAGTT CAGGCCCT CACAGC GCGGG (SEQ ID NO:4).

The present invention will now be described, in an illustrative, but non-limiting, way, according to preferred embodiments thereof, with particular reference to the figures of the attached drawings, wherein:

Figure 1 shows the Distribution of minor allele frequency (MAF). Minor allele frequency (MAF) of the KLB rs17618244 variant in 128 healthy paediatric controls(CTRL) and in 210 paediatric patients suffering from NAFLD (NAFLD). ^* p < 0.05 calculated by Cochran-Armitage trend test.

Figure 2 shows the Distribution of patients with NAFLD on the basis of the presence of NASH and of the genotype. Distribution of the genotypes on the basis of the presence of NASFI in the 210 paediatric patients suffering from NAFLD (NAFLD). ^*** p < 0.001 calculated by D'Agostino-Pearson omnibus test.

Figure 3 shows the Tissue expression of KLB on the basis of genotype in NAFLD patients. An image representative (40x) of the liver expression of the KLB and of its intracellular distribution in NAFLD patients with or without NASFI. The immunofluorescence was carried out on 2 pm thick sections of liver tissue fixed in formalin incorporated in paraffin. The KLB is shown in green whereas the nuclei are shown in blue.

Figure 4 shows the expression of the gene and the KLB protein in HepG2 cells over-expressing the wild type KLB or the mutated KLB. (A) Relative expression of the mRNA of the KLB, evaluated by Real Time PCR, (B) immunoelectrophoretic profile and (C-D) densitometric analysis of the KLB protein in FlepG2 cells over-expressing the wild type KLB (indicated as KLB WT), or the mutated KLB (indicated as KLB rs17618244), with respect to the control cells transfected with an empty plasmid (indicated as pCMV6). The histograms show the average ±SD of three independent experiments repeated in triplicate. ^** P <0.01 , ^*** P <0.001 with respect to the control.

Figure 5 shows the analysis of the lipid content in the HepG2 over-expressing the wild type and mutated KLB. The histograms show the lipid content, measured by ORO assay, like O.D./mg proteins in the HepG2 over-expressing the wild type KLB (indicated as KLB WT) or the mutated KLB (indicated as KLB rs17618244), with respect to the control cells transfected with the empty plasmid (indicated as pCMV6).

The data show the average ±SD of three independent experiments repeated in triplicate. ^*** p < 0.001 with respect to the control cells.

Figure 6 shows the Gene expression analysis in HepG2 cells over-expressing the mutated KLB. The histograms show the expression of the messenger RNA, evaluated by Real Time PCR of p62, ACOX1 and of ACSL1 in the HepG2 over-expressing the mutated KLB (KLB rs17618244). The data show the average ±SD of three independent experiments repeated in triplicate. ^* p < 0.05, ^*** p < 0.001 with respect to the control cells.

Figure 7 shows the Gene expression analysis in cells HepG2 over-expressing the mutated KLB. The histograms show the expression of the messenger RNA, evaluated by Real Time PCR of IL-1 beta and TNF-alpha in the HepG2 over-expressing the mutated KLB (KLB rs17618244). The data show the average ±SD of three independent experiments repeated in triplicate. ^** p < 0.01 , ^*** p < 0.001 with respect to the control cells.

Example 1 : Study of the rs17618244 variant in the KLB gene in paediatric patients suffering from NAFLD.

Materials and methods

Case-control Study

Our case-control study included children of Italian nationality in paediatric age between 2 and 18 years, enrolled in the Liver Pathologies Unit of Rome’s Ospedale Pediatrico Bambino Gesu children’s hospital between the months of September 201 1 and November 2015. This population is divided as follows: a group of 210 patients who were not related to one another who were suffering from NAFLD diagnosed by liver biopsy, and a group of 128 healthy children who joined special liver pathology screening programmes run at Rome’s Ospedale Pediatrico Bambino Gesu hospital between 2013 and 2015. Other causes of hepatic disease, including alcohol abuse (>30/20 grams/day), viral hepatitis and autoimmune hepatitis, hereditary haemochromatosis, alpha-1 -antitripsyn deficiency and hepatitis B and C infections, were excluded from the study.

The study was approved by a competent Ethical Committee and all the enrolled subjects and their parents gave their informed written consent to the gathering of the anthropometric and biochemical data and samples on which to conduct the genetic analysis.

For healthy children (CTRL) age, gender, weight, height and body mass index (BMI) were reported, BMI being calculated by the formula weight (expressed in Kg) / height (expressed in cm) ² . (Table 1 ).

The characteristics of the healthy children (CTRL) are set out in Table 1 , in which the displayed values show the average ± standard deviation or the percentage (%) and BMI indicates the Body Mass Index.

Table 1

On the other hand, for children with NAFLD, in addition to age, gender, weight, height and BMIthe waist circumference was reported and, by standard laboratory procedures, the following clinical biochemical parameters were measured: total cholesterol, high-density lipoproteins (HDL) and low-density lipoproteins (LDL), triglycerides, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma- glutamyltranspeptidase (GGT) (Table 2).

The characteristics of the children with NAFLD are shown in Table 2, in which the reported values indicate the average ± standard deviation or the percentage (%) and BMI indicates Body Mass Index.

Table 2

The histological examination of the liver was evaluated by an expert pathologist who was unaware of the clinical and genetic data.

As a further control group, 502 healthy Europeans from the 1000 Genomes Project were used whose gene type of interest is available (http://www.internationalgenome.org). Genomic DNA extraction

Peripheral blood samples in EDTA (from the patients) and mouth swabs (from healthy children) were collected and processed for genomic DNA extraction, carried out by QIAamp DNA Blood Mini Kit(QIAGEN) following the instructions supplied by the kit. The thus obtained DNA was quantified by using a Nanodrop spectrophotometer (ThermoFisher).

Sanger method sequencing

The patients were genotyped for the variant rs17618244 G>A in KLB by direct sequencing of Sanger type.

The sequences of the (forward and reverse) primers were then used to amplify the DNA region under study by PCR (MyTaq DNA Polymerase; Bioline) are reported below.

PRIMERS:

Forward: CG AGCCT CT GTTGCATGC (SEQ ID NO:1 )

Reverse: TT G AGCAGCCT CCTTT CGG (SEQ ID NO:2)

PCR REACTION SET UP:

• 5x MyTaq Reaction Buffer: 4 mI_

• DNA: 100 ng

• 10mM primers: 1 .5 mI_

• MyTaq DNA Polymerase: 0.2 mI_

• Double distilled water: bring up to the final volume of 20mI_

The PCR was performed on an Eppendorf Mastercycler Pro S thermocycler in the following conditions: 3 minutes at 95 °C, 40 cycles at 95 °C for 20 seconds, at 66 °C for 30 seconds and at 72 °C for 30 seconds, lastly 7 minutes at 72 °C.

The PCR product was then sequenced using the 3500 Genetic Analyzer (Applied Biosystems) sequencer

Allelic discrimination by Real Time PCR

Subsequently, in the population under examination the rs17618244 G>A in KLB variant was genotyped by allelic discrimination assay of TaqMan type that is commercially available (ThermoFisher). This technology uses as a method Real Time PCR (RT-PCR). The assay consists of two TaqMan® probes, in which one provides a specific pairing for the sequence of an allele (for example G), and the other probe provides a specific pairing for the other allele (for example A). Each probe is marked with a distinctive fluorochrome at the respective end 5’, named the reporter (in the assay, VIC® or FAM™ fluorescences are used). Specifically, the probe marked in VIC recognizes the A mutated allele, whereas the probe marked in FAM recognizes the wild-type G allele. The fluorescent signal generated is detected at the end of the PCR (end-point) and immediately indicates the presence of the sought sequence or sequences. Specifically, the presence of the only fluorescence in VIC indicates the homozygosity condition for the A mutated allele (AA genotype); the presence of the only fluorescence in FAM indicates the homozygosity condition for the wild-type G allele (GG genotype); lastly, the presence of both VIC and FAM fluorescences indicate the heterozygosity condition (G/A genotype).

All the genotyping reactions were performed in 96-well plates

(MicroAmp Fast Optical 96-Well Reaction Plate Applied Biosystems), in a total volume of 25 pL containing 2X TaqMan Universal PCR Master Mix No Amperase UNG (Applied Biosystems), 20X SNP Genotyping Assay and 10 ng DNA.

The RT-PCRs were performed on the 7900 FIT Fast (Applied

Biosystem) thermocycler in the following conditions: 10 minutes at 95 ° C, then 40 cycles at 92 ° C for 15 seconds and 60 ° C per 1 minute.

The allelic discrimination was conducted on the post-PCR product and the SDS 2.1 software (Applied Biosystem) was used to analyze fluorescence.

The characteristics of SNP rs17618244 and the DNA sequence are shown in Table 3.

Table 3

Immunofluorescence

Sections that were 2 miti thick of liver tissue of patients without NASH (indicated as Not-NASH) and with NASH (NASH) with GG, GA and AA genotypes were used to evaluate the tissue distribution of the KLB by immunofluorescence. The sections were incubated all night at 4 °C with a specific antibody for KLB (rabbit, Abeam, ab106794 dilution 1 :300) and measured with the secondary antibody Alexa Fluor 488 goat antirabbit (dilutionl :500, Applied Biosystems, Life Technologies, Carlsbad, CA, USA). The nuclei were marked with 4',6-diamidino-2-phenylindole (DAPI). The analysis used an Olympus Fluoview FV1000 confocal microscope and FV10-ASW version 4.1 software.

Results

The rs17618244 variant In KLB is more frequent in children suffering from NAFLD

The SNP under examination was validated in the Caucasian population from the Single Nucleotide Database; http://www.ncbi.nlm.nih.gov/SNP/) of the website of the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/). As a supplementary control unit, 502 healthy Europeans, who were enrolled in the 1000 Genomes Project and for whom the genotype of interest is available (http://www.internationalgenome.org), were used.

The allele frequencies in the population studied are reported in Table 4. In particular, the frequencies of the wild type allele (containing the nitrogenous base guanine, G) and of the mutated allele (containing the nitrogenous base adenine, A) are shown in the healthy individuals belonging to the project 1000 genomes (N=502), in the healthy children (CTRL) (N=128) and in paediatric patients with NAFLD (N=210) recruited at Rome’s Ospedale Pediatrico Bambino Gesu hospital. The allele frequency values of the examined cohorts were calculated by direct count.

Table 4

Wild type allele (containing the nitrogenous base guanine, G) and of the mutated allele (containing the nitrogenous base adenine, A) frequencies in the healthy individuals belonging to the project 1000 genomes (N=502), in the healthy children (N=128) and paediatric patients with NAFLD (N=210) recruited at Rome’s Ospedale Pediatrico Bambino Gesu hospital.

The genotype frequencies of the SNP were shown to be consistent with the Hardy-Weinberg (H-W) equilibrium.

The A allele of the rs17618244 variant in KLB tends to be significantly more frequent in NAFLD patients than in healthy individuals (p=0.045) (Figure 1 ).

The rs17618244 variant in KLB increases the risk of NASH in children suffering from NAFLD

The distribution between the presence of the variant and NASH, evaluated by the D'Agostino-Pearson omnibus test, is shown in Figure 2. The A allele of the rs17618244 variant in KLB tends to be more frequent in NAFLD patients with NASH (NASH) than in subjects with NAFLD but without NASH (Not-NASH) (Figure 2). The analysis of the Pearson correlation shows that the presence of the mutated allele correlates significantly with the presence of fibrosis (r = 0.31 ; p<0.001 ) inflammation (r = 0.43; p<0.001 ) and ballooning (r = 0.35; p<0.001 ).

The KLB liver expression is reduced in paediatric patients suffering from NASH and carrying the variant.

The KLB liver expression is reduced in children with NASH compared with Not-NASH children, indicated in Figure 3. This reduction is greater in patients with the rs17618244 variant G>A KLB. Further, in our studies no homozygous NASH patient was found for the variant in question.

Example 2: Study of the effects of the rs17618244 variant in KLB gene in in vitro models.

Materials and methods

Cell lines and genotyping.

A human hepatocellular carcinoma cell line was used: HepG2 (ATCC; Manassas, VA, USA). The cells were grown in DMEM medium (Dulbecco’s Modified Eagles Medium, GIBCO) to which 1 % penicillin/streptomycin (Pen/Strep, Euroclone) and 10% foetal bovine serum (FBS South America, GIBCO) were added. The cells were incubated at 37 °C in a humid atmosphere with 5% (v/v) CO2.

The cells were gathered and centrifuged at 300 g for 5 minutes at ambient temperature in order to obtain a pellet from which the genomic DNA used per genotyping the HepG2 was extracted using the DNA Blood DNA Extraction Kit (Qiagen, Valencia, CA, USA).

Plasmid

The expression vector used in this work is a pCVM6-AC-GFP commercial vector containing the cDNA of the wild type KLB (KLB NM_175737 Human Tagged ORF Clone; Catalogue number: RG21 1 186; OriGene Technologies, Inc. Rockville, MD USA). A vector that expresses green fluorescent protein (GFP), that enables the expression to be detected with fluorescence microscope or cytofluorimetric methods, was chosen. The vector has a dimension of 9.7 Kb and contains: the site for insertion of the human cDNA of the KLB (3132 bp); a cytomegalovirus promoter (CMV promoter) that permits the constitutive expression of the insert cloned in many cell lines; the replication origin SV40 (SV40ori) for replication in mammalian cells; the gene for resistance to neomycin (Neo) for selection in mammalian cell lines; the ColE1 replication origin for replication in bacterial cells and the gene for ampicillin resistance (Amp) (Figure 4).

Transformation by thermal shock

For the transformation, 10 ng of plasmid were added to 50 pi of competent bacterial cells of E. coli, the DFI5-alpha (Invitrogen; Carlsbad, CA, USA), followed by incubation in ice for 20 minutes. Then, a thermal shock was induced by immersing the test tube for was immersed for 30 seconds in a 42 °C bath and then again in ice for 2 minutes. 450 mI of Luria-Bertani medium (LB; for one litre of medium, 20 g LB broth (Sigma- Aldrich, St. Louis, MO, USA) were weighed) were added to the test tube, which was left to incubate for an hour at 37 °C whilst being stirred.

Subsequently, the suspension was plated on an LB-agar culture medium (for one litre of medium, 35g LB broth with agar (Sigma-Aldrich, St. Louis, MO, USA) were weighed, to which ampicillin antibiotic was added (at a final concentration of 100 pg/ml). The plates were incubated at 37 °C for 16 hours.

Purification of plasmid DNA

In order to extract and purify the plasmid DNA of a bacterial colony, the HiSpeed Maxi Kit was used (Qiagen, Valencia, CA, USA).

It was necessary to inoculate a single colony of transformed bacteria in 5ml of LB medium containing the ampicillin antibiotic (at a final concentration of 100 pg/ml).

After an incubation of 8 hours, whilst being stirred at 37°, 1 mL of transformed bacteria were moved to a flask containing 250 mL of LB medium and ampicillin (at a final concentration of 100 pg/ml) and left to incubate whilst being stirred at 37 °C for 16 hours.

The next day, the bacteria were precipitated in a centrifuge at 6000 g for 15 minutes at 4°C. After the supernatant was eliminated, the obtained pellet was resuspended in 10 mL of P1 buffer and lysated by adding 10 mL of P2 buffer, overturning the bottle two or three times and incubating for 5 minutes. In this step, the alkaline solution causes cell lysis and denaturation and precipitatiion of the molecules of genomic and plasmid DNA. 10 mL of P3buffer were then added to return the cell lysate to neutral pH so as to enable the renaturation of only the plasmid DNA .

Lastly, the isolated plasmid DNA is precipitated in isopropanol, washed with 70% ethanol and eluted with 1 mL of sterile water. The concentration of plasmid DNA was measured by using a Nanodrop spectrophotometer ( Nanodrop ® Spectrophtometer 2000, Thermo Scientific).

Site-specific mutagenesis by PCR

The kit QuickChange II XL Site Directed Mutagenesis (Agilent Technologies, CA, USA) was used to introduce the genetic variant rs17618244 in KLB.

The protocol provides for the synthesis of the mutant strand starting from the relative template by PCR, digestion of the parental DNA and lastly the transformation of the mutant molecule into competent E. coli cells (XL1 - Gold strain supplied by the kit).

First of all, the experimental procedure provides for of the synthesis reaction of the mutant strand by PCR. The following mixture is prepared: 5 pi of 10x buffer, 10 ng of plasmid containing the wild type sequence of the KLB gene, 1 pi of dNTPS mix, 3 mI of QuickSolution, 125 ng of forward primer, 125 ng of reverse primer, 1 mI of PfuUltra HF DNA polymerase (2.5 U/ mI), H2O up to a final volume of 50 mI.

The following sequences of forward and reverse primers are used to introduce the genetic variant rs17618244 in KLB:

Forward: CTGGCGCCT CT ACG ACCAGCAGTT CAGGCCCT CAC (SEQ ID NO:5)

Reverse: GT G AGGGCCT G AACTGCTGGT CGT AGAGGCGCCAG (SEQ ID NO:6).

The thus prepared and mixed samples were subjected to the amplification cycles shown below in Table 5.

Table 5

1 mI of Dpnl restriction enzyme (10 U/ mI) was then added and the reaction mixture was incubated for an hour at 37 °C in order to digest the parental hemimethylated or methylated DNA.

The last step provided for the transformation of 1 mI of DNA treated with Dpnl in 50 mI of competent E. coli bacteria (XL1 - Gold strain provided with the kit), the mixture then being incubated in ice for 30 minutes. The bacteria were then subjected to thermal shock by exposure at 42 °C for 30 seconds, cooled in ice for 2 minutes and incubated at 37 °C for an hour in 450 mI of LB, thus permitting the expression and the synthesis of the protein that gives resistance to the selection antibiotic. Lastly, half of the suspension was sowed in Petri plates containing LB-broth with agar added with ampicillin (at a final concentration of 100 pg/ml) and incubated for 16 hours at 37 °C in order to select the transformed bacteria. The next day, the bacterial colonies obtained were incubated in 5 ml of liquid LB medium containing 100 pg/ml ampicillin and left to incubate at 37 °C for about 16 hours whilst being stirred. The mutated plasmid DNA was finally purified by using the HiSpeed Maxi Kit (Qiagen, Valencia, CA, USA) (see previous paragraph) and subsequently sequenced to evaluate the insertion of the desired mutation.

Transfection

HepG2 were sown in 6-well plates at the density of 2.5 c 10 ⁵ cells per plate. After 24 hours, the cells were transiently transfected with 2 micrograms of plasmid containing the wild type sequence of the KLB gene, or with 2 micrograms of plasmid into which the rs17618244 variant was introduced, or with 2 micrograms of empty pCMV6-AC-GFP plasmid used as a control (Catalogue number: PS100010; OriGene Technologies, Inc. Rockville, MD USA). Lipofectamin (Invitrogen, Carlsbad, CA, USA) was used as a transfecting agent for the transient transfection. The cells were collected at 48 hours to perform all the experiments.

Oil Red O Staining (ORO)

The HepG2s were sowed in 6-well plates at the density of 2.5 c 10 ⁵ cells per well. After 48 hours from the transfection, the cells were fixed in 10% formalin for 60 minutes, permeabilized for 5 minutes with 60% isopropanol and stained with an ORO solution (Sigma-Aldrich, St. Louis, MO, USA) for 10 minutes. After removal of the stain and after 5 washes with double distilled water, the intracellular ORO content, proportional to the accumulation of fatty acids, was then determined by measuring the absorption at 492 nm using a spectrophotometer for ELISA microplates, after eluting the ORO by adding isopropanol (100%) to each well. The results were then expressed as optical density (O.D.) / mg protein.

Extraction of the proteins, electrophoresis on acrylamide gel and Western Blotting

The total proteins extraction from the cell pellets was performed by collecting and centrifuging the cells for 5 minutes at 300 g and by removing the supernatant. The cells were lysated using a lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) to which protease inhibitors and phosphatase inhibitors were added (Thermo Scientific, Carlsbad, CA, USA). The lysate samples were kept in ice and then centrifuged at 20000 g for 10 minutes at 4°C. The supernatant containing the proteins was removed and stored at -20 °C. A quota of each sample of cell lysate was used for protein quantification by spectrophotometric quantitative analysis, with BCA TMProtein Assay Kit (Thermo Scientific, Carlsbad, CA, USA). The samples were then loaded onto polyacrylamide gel using a 4% and 8% concentration for stacking and resolving respectively. The run was carried out in a running buffer at a constant amperage (100 V) until the stain leakage from the gel. After separation on gel, the proteins were transferred onto nitrocellulose membrane, by the Western Blotting method for about 1 hour and 40 minutes at 4°C. Subsequently, the membranes were incubated with the following primary antibodies: polyclonal antibody directed against KLB (dilutionl :1000; Abeam, Cambridge, MA, USA), monoclonal antibody directed against alpha tubulin (dilution 1 :5000; Novus Biologicals, Littleton, Colorado, USA), monoclonal antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH; dilutionl :1000; Cell Signaling, Danvers, Massachusetts, USA), the last two being used as normalizers.

Hybridization was run for the whole night at 4°C. After three washes in PBS, the membranes were incubated with the secondary antibodies conjugated to horseradish peroxidase (dilution 1 :10000; Jackson ImmunoResearch Laboratories, Ely, Cambridgeshire, United Kingdom). Hybridization was run for 1 hour at ambient temperature. All the antibodies, both primary and secondary, were diluted in a 2.5% powdered milk solution dissolved in PBS. The specifically recognized proteins were detected by chemiluminescence by adding to the filter a solution containing luminol, horseradish peroxidase substrate (SuperSignal West Pico Plus ECL; Thermo Scientific, Waltham, Massachusetts, USA). The developed chemiluminescence was detected by exposure by contact of a photographic plate.

RNA Extraction

The extraction of the total RNA from the HepG2, following the transfection, was performed using the reagent Trizol® (Invitrogen, Carlsbad, CA, USA). The samples were collected with 1 ml trizol and left at ambient temperature for 5 minutes to enable the dissociation of the nucleus complexes. Subsequently, chloroform (0.2 ml for 1 ml of TRIZOL) was added; the sample was stirred vigorously for at least 15 seconds and left at room temperature for 3 minutes, then centrifuged for 15 minutes at 12000 g at 4°C to permit phase separation.

Following centrifugation, the mixture is separated into a lower organic phase containing proteins, an intermediate phase containing DNA, and an upper aqueous phase in which the RNA is located. The latter, equal to about 60% of the initial volume, was then removed carefully and transferred to a new tube. The RNA was precipitated by adding 0.5 ml of isopropanol per ml of TRIZOL for each sample so as to make the pellet visible; after about 10 minutes at room temperature, the samples were centrifuged for 10 minutes at 12000g at 4°C. The supernatant was aspirated and the pellet was washed with 1 ml of 75% ethanol (1 ml ETOH/ml of Trizol) centrifuged at 7500 g per 5 minutes at 4°C. At the end of washing, the ethanol was eliminated and the excess was evaporated under the flow of a chemical hood to avoid interference with the subsequent reverse transcription reaction. The RNA was then resuspended in a small volume of double distilled sterile H2O that was proportional to the RNA pellet and was brought to 55-60 ° C for 10 minutes to eliminate ethanol residues. The concentration of RNA was measured by using a ultraviolet-visible Nanodrop® Spectrophotometer 2000 (Thermo Scientific).

Reverse transcription

The total RNA extracted from the HepG2 was reverse transcripted in cDNA using the Superscript VILO cDNA Synthesis kit (Invitrogen, Carlsbad, CA, USA), using 2 pg of RNA per reaction. The protocol provides that a mixture for each sample consisting of the following is prepared: 4 mI of 5X VILO Reaction Mix, 2 mI of 10X Superscript Enzyme Mix and 4 mI of H2O to reach 10 pL volume. The reverse transcription reaction was performed by thermocycler Mastercycler@pro (Eppendorf) and had three steps: 25 °C for 10 minutes, 42 °C for 60 minutes and 85 °C for 5 minutes.

Real Time PCR

The cDNA obtained from the HepG2 was used to evaluate the expression of the KLB genes, p62, Acyl-CoA oxidase 1 (ACOX1 ), acyl- CoA synthetase long chain 1 (ACSL1 ), interleukin-1 beta (IL-1 beta) and tumour necrosis factor alpha (TNF-alpha). For the gene expression analysis, the following TaqMan® probes were used (Applied Biosystems, Carlsbad, CA, USA):

1) KLB (Hs01573147_m1 );

2) p62(Hs00177654_m1 );

3) ACOX1 (Hs01074241_m1 );

4) ACSL1 (Hs00960561_m1 ); 5) IL-1 beta (Hs00174097_m1 );

6) TNF-alpha (Hs00174128_m1 )

7) Glyceraldehyde 3-phosphate dehydrogenase (GAPDH;

Hs.PT.39a.22214836; Integrated DNA Technologies, IDT, Coralville, IA, USA) was used as an endogenous control.

The relative quantity of gene expression was calculated by the relative method through the formula 2 ^_DDa .

The Real Time PCR reaction was conducted using the SensiFast Probe Fli-ROX Kit (2X) (Bioline, London, UK) by 7900FIT Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA).

Statistical analysis

The values are expressed as average ± standard deviation (SD). The statistical analyses were conducted using Student’s t test.

Results

The over-expression of the KLB gene containing the rs17618244 variant influences lipid accumulation and the expression of genes involved in the regulation of lipid metabolism and in inflammation in HepG2 cells

In order to evaluate the effects of the rs17618244 variant in the KLB gene in NAFLD, an in vitro model of HepG2 cells was used that were genotyped for the rs17618244 variant in KLB. The cells were found to be wild type homozygotes (GG, thus devoid of the gene variant in question).

Subsequently, the HepG2 were transfected so that they would over express the wild type (WT) KLB or the mutated KLB gene (i.e. the gene into which the rs17618244 variant was introduced) to evaluate whether the presence of this variant would have an effect on the lipid accumulation that characterizes NAFLD.

Figure 4 shows control of the over-expression of the gene and the KLB protein both in wild type form and in mutated form. Subsequently, intracellular lipid accumulation was evaluated by ORO assay. As shown in Figure 5, the over-expression of the mutated KLB gene causes a statistically significant increase of the intracellular lipid content with respect to the control cells (indicated as pCMV6), whereas there is no variation in the cells over-expressing the wild type KLB .

Lastly, the over-expression of the mutated KLB gene causes increased expression of genes like p62, considered a lipotoxicity marker, increased expression of genes involved in the regulation of the metabolism of fatty acids like ACOX1 (is the first enzyme involved in the beta oxidation of fatty acids), and ACSL1 (an enzyme that converts free long-chain fatty acids into fatty acyl-CoA esters, and thus perform a key role in the biosynthesis of lipids and in degradation the fatty acids (Figure 6) and increased expression of genes encoding pro-inflammatory cytokines such as IL-1 beta and TNF-alpha (Figure 7).

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