P < 0.0001, Kruskal-Wallis test). Moreover, nucleotide changes were found in APC, KRAS, P53, PIK3CA, FBXW7 and SRC genes. Among those genes, KRAS exon 2 mutations were validated in another cohort of 70 CRC and 72 CAD specimens. Results showed that the difference in percentage of KRAS exon 2 mutations between CD166 positive and CD166 negative CRC specimens was significant . Long term follow-up of the CRC patients showed that CD166-positive KRAS exon 2 mutations was useful in discriminating CRC patients with worse outcome. This study has provided evidence that KRAS exon 2 mutations are concentrated in CD166-positive cancer cells, with prognostic significance in CRC, and those mutations are also detected in CAD.Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer deaths worldwide. Recent studies have shown that cancer stem cells (CSCs) are an important cause of tumor recurrence and metastasis. We hypothesized that CSCs marker CD166-positive CRC and colorectal adenoma (CAD) cells consist of more hotspot mutations than CD166-negative CRC and colorectal adenoma cells. To verify this, formalin fixed paraffin embedded tissue specimens from 42 patients each with CRC and CAD were recruited and CD166 immunohistochemical (IHC) staining followed by macrodissection was performed. DNA extracted was used for quantitative polymerase chain reaction detection on a somatic mutation array. Results showed that the immunoreactivity of CD166 protein had significant difference among CRC, CAD, and normal colorectal epithelial tissues (NCET) ( Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer deaths in the world . The disCarcinoembryonic antigen (CEA) is a widely used serum marker for CRC, but is unreliable in detecting postoperative recurrence . Other bCRC is a heterogeneous disease. Twenty-five percent and 30% of CRC patients, present respectively with tumour-node-metastasis (TNM) stages II and III , have a In the majority of cases, CRC develops over a long period of time through the adenoma-carcinoma sequence . TherefoCancer stem cells (CSCs) are a small subpopulation of tumor cells that are capable of initiating and maintaining tumor growth, as well as having the ability of self-renewal , 18. CSCOne strategy to identify CSCs in colorectal tissues is to detect the surface markers on colorectal CSCs, such as CD133, CD44 and CD166 . CD166, Kirsten rat sarcoma viral oncogene homolog (KRAS) is one of the key mutations involving CRC progression and metastasis [KRAS mutations have also been suggested to be an adverse prognostic marker of metastasis [KRAS mutations metastasized to the liver more rapidly than tumors carried wild-type KRAS [KRAS mutations were also associated with higher mortality when compared to their wild-type counterpart [One alternative approach of prognosis evaluation is to make use of CRC-associated DNA mutations. For example, the gain-of-function mutation in the oncogene tastasis , and hastastasis . Recentltastasis . CRC carype KRAS . Liver mnterpart . Hence, KRAS mutation and detected the hotspot exon 2 mutations of KRAS genes in another cohort of CD166 stained CRC and CAD specimens using Sanger sequencing. Our objective was to examine the clinical significance of hotspot exon 2 KRAS mutations in CD166-pcc and CD166-pac.Based on these findings, we hypothesize that by detecting tumor-associated mutations that present in CD166-positive cancer cells (CD166-pcc), a more accurate prediction of tumor recurrence and metastasis may be achieved. We reason that such potentially enhanced accuracy may due to a combined advantage that 1) CD166-pcc may be more relevant to recurrence and metastasis than those found in other parts of the tumor, and 2) tumor-specific mutations could act as a discriminating marker such that only tumor-derived cells, but not normal cells, could be specifically detected. Hence, we first identified CD166-pcc in CRC and CD166-positive adenoma cells (CD166-pac) in CAD followed by detecting their mutations after macrodissection of those cells. Among those mutations found, we selected The results generated from this study will lay down a solid foundation in using CD166-pcc and CD166-pac associated mutations in screening, diagnosis and prognosis of CRC patients.P < 0.0001, Kruskal-Wallis test).CD166 protein was expressed in 76% 32/42) of CRC specimens, 64% (27/42) of CAD specimens and 35% (7/20) of normal colorectal epithelial tissues adjacent to CRC tissues  whereas the number of CD166 negative specimens with nucleotide change is 2 (2/6 = 33.3%). In CAD, the number of CD166 positive specimens with nucleotide change is 24 (24/30 = 80%). Detailed analysis showed that 16 CD166 positive CAD specimens with 1 nucleotide change (16/30 = 53.3%) and 8 CD166 positive CAD specimens with 2 nucleotide change (8/30 = 26.7%) whereas the number of CD166 negative CAD specimens with nucleotide change is 4 (4/8 = 50%). Overall, there are a total of 44 nucleotide changes because 8 of the CD166 positive CAD specimens have 2 nucleotide change  CRC specimens were CD166 positive and 21 of 70 (30%) CRC specimens were CD166 negative. Similarly, 49 of 72 (68%) CAD specimens were CD166 positive and 23 of 72 (32%) CAD specimens were CD166 negative.Mutation array results showed that nucleotide changes were found in es Table . Out of KRAS exon 2 mutations were detected in 29 out of 49 CD166 positive (29/49 = 59%) CRC specimens and 7 out of 21 (7/21 = 33%) CD166 negative CRC specimens. On the other hand, KRAS exon 2 mutations were detected in 11 out of 49 CD166 positive (11/49 = 22%) CAD specimens and 4 out of 23 CD166 negative (4/23 = 17%) CAD specimens. The difference in percentage of KRAS exon 2 mutations between CD166 positive and CD166 negative CRC specimens is significant  whereas whereas the difference of it between CD166 positive and CD166 negative CAD specimens is not significant . Neglecting the status of CD166, the overall percentage of KRAS exon 2 mutations is 51% (36/70) in CRC and 21% (15/72) in CAD. Detailed analysis showed that the KRAS exon 2 mutations in CRC were composed of i) c.35G>A (G12D), ii) c.35G>T (G12V) and iii) c.34G>A (G12S). Moreover, c.35G>A (G12D) was detected in 18/49 (37%) CD166 positive CRC and 5/21 (28%) CD166 negative CRC; c.35G>T (G12V) was detected in 8/49 (16%) CD166 positive CRC and 2/21 (10%) CD166 negative CRC; c.34G>A (G12S) was detected in 3/49 (6%) CD166 positive CRC (Table KRAS exon 2 mutations were composed of i) c.35G>A (G12D), ii) c.35G>T (G12V), iii) c.38G>A (G13D) and iv) c.34G>A (G12S). Similarly, c.35G>A (G12D) was detected in 6/49 (12%) CD166 positive CAD and 1/23 (4%) CD166 negative CAD; c.35G>T (G12V) was detected in 5/49 (10%) CD166 positive CAD; c.38G>A was detected in 2/23 (9%) CD166 negative CAD and c.34G>A (G12S) was detected in 1/23 (4%) CD166 negative CAD . When considering CD166 IHC staining alone, the marker showed a sensitivity of positive staining for 22/25 (88%) of the poor-treatment outcome patients. As there were a total of 49 patients with positive staining, hence 27/45 (60%) of the good-treatment outcome patients with positive staining was detected. In line with this logistic, negative CD166 staining was found in 3/25 (12%) poor-treatment outcome patients and 18/45 (40%) good-treatment outcome patients. Similarly, KRAS exon 2 mutations without macrodissection were detected in 9/25 (36%) poor-treatment outcome patients and 16/45 (36%) good-treatment outcome patients whereas KRAS exon 2 mutations without macrodissection were not detected in 16/25 (64%) poor-treatment outcome patients and 29/45 (64%) good-treatment outcome patients. When detecting KRAS exon 2 mutations in CD166 positive cells, positive KRAS exon 2 mutations in CD166-positive cells were detected in 19/25 (76%) poor-treatment outcome and 10/45 (22%) good-treatment outcome patients whereas negative KRAS exon 2 mutations in CD166-positive cells were detected in 4/25 (16%) poor-treatment outcome and 16/45 (36%) good-treatment outcome patients  when compared to CD166-ncc in CRC (33%: 7/21). Detailed analysis demonstrated that the mutations c.35G>A (G12D) (37% vs 24%), c.35G>T (G12V) (16% vs 10%) and c.34G>A (G12S) (6% vs 0%) have a much higher prevalence in CD166-pcc than CD166-ncc. In contrast, a slightly higher percentage of KRAS exon 2 mutations was detected in CD166-pac (22%: 11/49) than CD166-negative adenoma cells (CD166-nac) (17%: 4/23) in CAD. More detailed analysis showed a higher detection rate of c.35G>A (G12D) (12% vs 4%) in CD166-pac than CD166-nac in CAD. In addition, c.35G>T (G12V) was only found in CD166-pac (10%) whereas c.38G>A  and c.34G>A  were only found in CD166-nac.The aim of this study was to examine whether CD166-pcc would consist of more hotspot mutations when compared to those CD166-ncc. If so, those hotspot mutations within the CD166-pcc may be used for diagnosis, prognosis as well as treatment benefit to CRC patients. The reason why we used this strategy because there is approximately only 36–40% of patients with CRC have tumor-associated utations . On the AD cells . It is ning CRCs . In thisKRAS mutations in CRC patients occur at codons 12, 13, and 61 of the KRAS gene. The result of these mutations is constitutive activation of KRAS signaling pathways [KRAS are unlikely to benefit from anti-EGFR antibody therapy, either as monotherapy [KRAS exon 2, codon 12 and 13 mutations have important diagnostic and prognostic values in CRC [The majority of the tumor-associated pathways . It is notherapy  or in cootherapy . On the s in CRC –33. As CKRAS exon 2 mutations in predicting poor treatment outcome. The first aspect can show that c.35 G>A (G12D), c.35 G>T (G12V) and c.34 G>A (G12S) are present in early and late TNM stages of CRC specimens. As there are nearly double the percentage of KRAS exon 2 mutations in those CD166-pcc in CRC specimens, we suggest that CD166 immunohistochemical staining followed by KRAS exon 2 sequencing can be performed on those specimens without KRAS exon 2 mutations for patient benefit in targeted therapy. The second aspect can show that KRAS exon 2 mutations have high potential to be an early CRC bio-marker. Although there was no significant difference in KRAS exon 2 mutations between CD166-pac and CD166-nac in CAD, an overall percentage of 21% (15/72) KRAS exon 2 mutations are still encouraging for us to detect other KRAS mutations in CAD. As the mutation c.34G>A (G12S) was found in both CRC and CAD specimens from the same patient, the clinical significance of it will be examined thoroughly in order to explore its potential in screening, detection and monitoring of CRC. Our preliminary findings on the prognostic significance of CD166 IHC staining and KRAS exon 2 mutations provide evidence that KRAS exon 2 mutations in CD166 positive cells would be useful in discriminating CRC patients with poor post-treatment outcome such as metastasis, relapse and death. Our results are different from a recent study which has shown that KRAS mutation increased the risk of lymph node involvement by 8 times in CD166-negative patients [KRAS exon 2 mutations detection whereas that study detected KRAS mutations using a tissue microarray block to represent the whole specimen. Therefore, the immunoreactivity of CD166 in the specimens in that study may be under-estimated. Nevertheless, the limitations of this study are 1) small sample size especially in colon cancer array and 2) only CD166 was examined and 3) only KRAS exon 2 mutations were examined. Therefore, a larger cohort of study is now being prepared to validate these results so as to determine the diagnostic, prognostic and therapeutic significance of these mutations. Moreover, more CSC markers and more tumor-associated mutations in genes such as APC, p53 and BRAF will be examined. On the other hand, we are also preparing to detect those KRAS exon 2 mutations in the plasma of CRC patients by digital PCR or targeted re-sequencing. To study the functional relationship between CD166 and KRAS exon 2 mutations, we propose to use RNA interference–mediated knockdown of CD166 to compare the status of KRAS exon 2 mutations in CD166-positive and negative cells and examine the invasive potential of the CRC cells. This study has successfully provided evidence that 1) CD166-pcc has more KRAS exon 2 mutations than CD166-ncc in CRC and hotspot KRAS exon 2 mutations were detected in CAD and 2) these KRAS exon 2 mutations in CD166 positive cells may have prognostic significance in patients with CRC. We hope that these results can be verified in a large scale study and translated into clinical applications in future for CRC detection, monitoring and chemotherapeutic treatment response.This study produces significant results in four aspects: 1) c.35 G>A (G12D), c.35 G>T (G12V) and c.34 G>A (G12S) were detected in TNM stage II to IV CD166-pcc CRC specimens, 2) c.35 G>A (G12D), c.35 G>T (G12V), c.38G>A (G13D) and c.34G>A (G12S) were also detected in CAD, 3) a mutation c.34G>A (G12S) was found in both CRC and CAD specimens from the same patient and 4) prognostic significance of CD166-pcc The first cohort of formalin fixed paraffin embedded (FFPE) specimens from 42 CRC patients and 42 CAD patients were recruited. Besides, 20 normal colorectal epithelial tissues were also included for comparison. CD166-pcc and CD166-pac were selected for human colon cancer somatic mutation polymerase chain reaction (PCR) array.KRAS exon 2 mutations using Sanger sequencing. The study was approved by the Clinical Research Ethics Committee of the Queen Elizabeth Hospital, Kowloon Central Cluster, Hospital Authority, Hong Kong Special Administrative Region.The second cohort of FFPE specimens from 70 CRC patients and 72 CAD patients were recruited. CD166-pcc and CD166-pac were selected for CD166 (ALCAM)-specific antibodies  was used.Serial tissue sections (4 μm thick) were cut and antigen retrieval was performed using Bond Epitope Retrieval Solution 2 on the Bond-max automated immunostainer  at 100° C for 25 minutes. Staining was performed according to a standard protocol in the immunostainer. Polymer detection system was selected to avoid the problem of nonspecific endogenous biotin staining. Breast carcinoma was used as a positive control which was mounted on every test slide and negative controls were performed by replacing the antibody with tris buffered saline.The stained slides were completely evaluated under light microscope at ×400 magnification by 2 independent observers without knowledge of clinical outcomes and in the case of disagreement, consensus was reached after thorough discussion and slides examination using a multi-headed microscope. All slides were scored semi-quantitatively and expressed as an IHC score by multiplying the “percentage of positive cells” and the “staining intensity”. Staining intensity was scored as follows: 0 = negative; 1 = weak; 2 = moderate; 3 = strong; and 4 = very strong. The IHC score ranged from 0 to 400. The scoring of percentage and staining intensity was targeted to 1) CRC cells, 2) CAD cells and 3) adjacent normal epithelial cells. Membranous staining was the expected result.Thirty sections of each CD166 positive FFPE specimens were cut with 5 µm thickness per section. Each section was mounted on a superfrost slide. Microtome was cleaned with xylene before sectioning of each specimen in order to avoid any tissue carryover. The unstained sections of each specimen were deparaffinized with xylene followed by absolute alcohol. Selected areas on each slide were circled by comparing with a reference IHC stained slide of the same tissue section. Circled areas on each slide were filled with buffer ATL  followed by scrapping using a new scalpel for each tissue specimen. The scrapped tissues were then transferred into an RNase-free microcentrifuge tube and the final volume was made up to 180 µl using buffer ATL. DNA extraction was performed according to instructions of a QIAamp DNA FFPE tissue kit (Qiagen).™ dsDNA system .Genomic DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen) according to the manufacturer’s instructions with modifications. Briefly, macrodissected sections were deparaffinzed with xylene, followed by overnight proteinase K digestion. The mixture was then being loaded into the extraction column. DNA was eluted in 20 µl of water, and quantified by the QuantiFluorAPC, BRAF, CTNNB1/beta-catenin, FBXW7, KRAS, PIK3CA, SRC, and P53 . These mutations warrant extensive investigation to enhance the understanding of carcinogenesis and identify potential drug targets. This array includes 86 DNA sequence mutation assays designed to detect the most frequent, functionally verified, and biologically significant mutations in human colon cancer. These mutations are chosen from curated, comprehensive somatic mutation databases and peer-reviewed scientific literature, and represent the most frequently recurring somatic mutations compiled from over 29,000 colon cancer samples. The array layout and the gene table (Qiagen) were shown in This array is a translational research tool that allows rapid, accurate, and comprehensive profiling of the top somatic mutations in human colon cancer specimens for the following genes: The procedures involved DNA extraction (QIAGEN QIAamp DNA Mini Kit or FFPE Tissue Kit is recommended), quantitative PCR detection on qBiomarker Somatic Mutation PCR Array, and data analysis using the qBiomarker Somatic Mutation Data Analysis Template.Four specimens were tested in one qBiomarker Somatic Mutation PCR Array plate in each run. The protocol of qBiomarker Somatic Mutation PCR Array was followed. In brief, 700 ng genomic DNA extracted by QIAamp DNA FFPE Kit (Qiagen) was added to the master mix. Ten microliters of the reaction mix were used per reaction. The plate was then tightly sealed and centrifuged at 2000 rpm for 2 minutes and was put on ice until use. Real-time PCR was set up and run according to the protocol in a LightCycler 480 system .https://www.qiagen.com/hk/shop/genes-and-pathways/data-analysis-center-overview-page/. Free data analysis software for qBiomarker Somatic Mutation PCR Array available at the above website was utilised for further investigation. The mutation array consists of the following genes:Threshold cycle of each sample reaction was calculated using the cycle software. Linear view of the amplification plots was used to define the baseline value while log view of the amplification plots was used to define the threshold value. The threshold value was placed above the background signal but within the lower half to one-third of the linear phase of the amplification plot. The resulting threshold cycle values for all wells were exported to a blank excel spreadsheet and were formatted according to the excel-based PCR array Data Analysis Template which was available at The most commonly detected APC inactivation mutations are mainly composed of truncation mutations (due to nonsense mutations and frameshift mutations) and point mutations between codons 1250 and 1578.In colon cancer, the BRAF mutation that leads to increased kinase activity, p. V600E mutation, is the most important to test.The most frequently detected CTNNB1 mutations result in abnormal signaling in the WNT signaling pathway. The mutated codons are mainly several serine/threonine residues targeted for phosphorylation by GSK-3 beta.The mutations queried by these assays lay in either the third or fourth repeat of the protein’s WD40 domain, typically involved in protein-protein interactions.KRAS codons 12, 13, and 61. Mutations at these positions result in reduced intrinsic GTPase activity and/or cause KRAS to become unresponsive to RasGAP.The mutation assays include the most frequently occurring mutations in The most frequently occurring PIK3CA mutations mainly belong to two classes: gain-of-function kinase domain activating mutations and helical domain mutations that mimic activation by growth factors.SRC is a proto-oncogene and a tyrosine-protein kinase that plays a role in the regulation of embryonic development and cell growth. Mutations in this gene could be involved in the malignant progression of colon cancer.The most frequently detected somatic mutations in TP53 are largely composed of DNA-binding domain mutations which disrupt either DNA binding or protein structure.KRAS exon 2 was designed as in Table The sequence of primers against KRAS gene. Each PCR reaction was consisted of i) 2.5 μL BigDye Direct PCR Master Mix, ii) 1 μL of 0.8 μM M13-tailed forward and reverse primer mix, iii) 1.5 μL genomic DNA (<2 ng/μL), or 1 μL of 2.0 ng/μL genomic DNA and iv) 0.5 μL deionized water to add up the total volume to 5 μL. Amplification and cycle sequencing of KRAS were conducted under the same condition. The PCR condition was shown as follows: 95° C for 10 minutes; 35 cycles each of i) 96° C for 3 seconds, ii) 62° C for 15 seconds and iii) 68° C for 30 seconds; and finally hold at 72° C for 2 minutes. Each PCR product was then diluted with 5 μL of water.BigDye Direct Cycle Sequencing Kit {Applied Biosystems (ABI), Thermo Fisher Scientific, Foster City, CA 94404, USA} and ABI Veriti thermal cyclers were used for amplification and cycle sequencing of Each sequencing reaction consisted of 5 μL diluted PCR product, 1 μL of BigDye Direct Sequencing Master Mix (ABI) and 0.5 μL of BigDye Direct M13 Forward or Reverse Primer. Condition of cycle sequencing was shown as follows: 37° C for 15 minutes; 80° C for 2 minutes; 96° C for 1 minute; then 25 cycles each of i) 96° C for 10 seconds, ii) 50° C for 5 seconds and iii) 60° C for 75 seconds.BigDye XTerminator Purification Kit (ABI) was used to purify the sequencing products. Each sequencing product was mixed with 36 μL SAM Solution and 8 μL XTermination solution on a spin plate and vortexed on IKA MS3 digital vortexer at 2000 rpm for 20 minutes. It was then centrifuged in a swinging-bucket centrifuge at 1000 g for 2 minutes. Sequencing was performed on Applied Biosystems 3500 Genetic Analyzer.KRAS exon 2 mutations was studied using chi-square test. Finally, multivariate regression  was used to analyze whether CD166 status, KRAS exon 2 mutations were correlated with the clinico- histopathological factors of the patients . A P value < 0.05 is considered to be statistically significant in all analyses. All P values are two-tailed.The difference in IHC scores of CD166 protein among CRC, CAD and adjacent normal colorectal epithelial cells was studied using non-parametric Kruskal-Wallis test. Moreover, the association between CD166 status and


ABCB1 (C3435T) and ABCC1 (G2012T) polymorphisms with clinical response to atorvastatin in Iranian primary hyperlipidemic patients.Atorvastatin is prescribed for the primary and the secondary prevention of coronary artery diseases. A wide variation in inter-individual statin response suggests that genetic differences may contribute to this variation. This study investigated the association of Individuals (n=179) with primary hypercholesterolemia were enrolled, and peripheral blood samples were collected. Genotyping of two polymorphisms were performed by amplification refractory mutation system PCR.ABCB1 (P=0.018) and wild-type group (GG) of ABCC1 genes (P=0.029). Logistic regression analysis revealed a significant difference between male and female responses to 10 mg/day atorvastatin .Following four weeks of treatment, a significant reduction of LDL-C was observed in variant groups (CT+TT) of Our finding indicated that these polymorphisms may be attributed to LDL-C serum levels in the primary hypercholesterolemia patients receiving atorvastatin. Coronary artery disease (CAD) was responsible for approximately 300,000 deaths in 2010. Serum lABCC1) play an important role in drug transport through the cell membrane, which may contribute to drug disposition and response[ABCB1), located on chromosome 7. Until now, more than 20 polymorphisms have been recognized; some of which have been associated with altered P-gp expression and activity[P-glycoprotein (P-gp) and multidrug resistance protein 1  is located in the exon 26 and has a relationship with differences in total serum cholesterol, LDL-C, and HDL-C in response to atorvastatin in American populations[ABCC1 appears to be ubiquitously expressed in many human tissues and transports a wide spectrum of substrates and toxicants[G2012T (rs 45511401) polymorphism, located in exon 16, is common among Caucasian populations[ABCB1 and ABCC1 genes and differences in serum LDL-C and HDL-C levels, which indicates the potential role of these polymorphisms in the pharmacogenetics of the atorvastatin[ABCB1 (C3435T) and ABCC1 (G2012T) polymorphisms with para-clinical and clinical responses to atorvastatin in Iranian patients with hyperlipidimia.Several investigations have focused on a synonymous polymorphism in the nd safety-11. The pulations. ABCC1 atoxicants. A non-sT rs 455101 polymorvastatin. RegardiPatients with primary hypercholesterolemia were selected from the outpatients, who were evaluated for the presence of CAD, at the cardiovascular disease clinics affiliated with Shiraz University of medical sciences  between January 2012 and October 2013. The study protocol was approved by the Ethics Committees of Shiraz University of Medical Sciences (code number 91-01-36-5018). Pregnant women as well as individuals with serum triglycerides above 400 mg/dl or with thyroid disease (TSH≥5 or ≤0.4 µu/dl), renal failure , liver dOne week before and four weeks after atorvastatin administration, peripheral blood samples were collected from the individuals after an overnight fasting (8-12 h). Total sC3435T polymorphism on ABCB1 and G2012T on ABCC1 was carried out using amplification refractory mutation system PCR method. Briefly, PCR assays were performed with 30-40 ng genomic DNA, 10 µl Master mix 2SO4, 5.5 mM MgCl2, 130 mM Tris-HCl, pH 8.8, 0.4 mM of each dNTP, 0.02% Tween-20), 0.6 µM ABCB1 reveres primer (5’-GGA GAC CCC CTT ATA AATC-3’), 1 µM ABCB1 forward primer (5’-TGG TGT CAC AGG AAG ATA TC-3’), 1 µM ABCC1 reveres primer (5’-CCA CCA CGG CCA CCA AAT CAA-3’) and 0.7 µM ABCC1 forward primer (5’-CTG CCT CAC TTC AAG GGA CAC-3’). The thermal cycle  protocol for ABCB1 and ABCC1 consisted of an initial denaturation at 95ºC for 5 min, followed by 30 and 35 cycles of denaturation at 94ºC for 25 s and 40 s, annealing at 59 ºC for 35 s and 62ºC for 45 s and extension at 72ºC for 25 s and 40 s. PCR products were analyzed by 2% agarose gel electrophoresis and stained by SYBR Green dye.Genomic DNA was extracted from buffy coat of EDTA-treated peripheral blood samples by the DNP kit  according to the manufacturer’s instruction. Genotyping of the ABCB1 and ABCC1 polymorphisms and serum lipid concentration, individuals carrying the homozygous form of the less common allele were grouped with the heterozygous carriers (variant group), and those carrying the homozygous form of the more common allele were put in the wild-type group. General linear regression model was employed to evaluate the association of C3435T and G2012T genotypes with LDL-C reduction. Logistic regression analysis was carried out to compare the effect of demographic factors and C3435T and G2012T polymorphisms on clinical response to atorvastatin . The allele frequency of these variants was assessed for deviation from the Hardy-Weinberg equilibrium using Chi-square test. A P<0.05 was considered statistically significant. SPSS 17 software  was used for statistical analysis.Continuous variables were presented as mean±SD. Categorical variables were reported as counts (percentage). To find any potential association between genotypes in candidate genes and demographic phenotypes and response to atorvastatin, we first divided the patients into two different dosage groups, individuals treated with 10 mg atorvastatin per day and those who were treated with 20 or 40 mg. To evaluate the association between P>0.05). The genotype and allele distribution of two polymorphisms are shown in Patients (n=179) with primary hypercholesterolemia were enrolled in this study. Based on the laboratory data, the mean±SD of LDL-C, triglyceride, total cholesterol and HDL-C were139.8±27.2, 163.4±60.8, 217.7±37.9 and 42.6±8.6 mg/dl, respectively. As shown in P=0.004, odds ratio=0.2, CI 95%= 0.06-0.6). The result indicated that women are better responders to the therapy by five-fold. Furthermore, no significant association was detected between the ABCB1 (C3435T) and ABCC1 (G2012T) polymorphisms and clinical response with different dosages of atorvastatin in Iranian hyperlipidemic patients. Linkage disequilibrium between C3435T and G2012T polymorphisms was not observed in our study population (P=0.56).The percentage change in lipid parameters by genotype is shown in ABCB1 genotype  were not similar to those found in Iranian population in previous studies (P>0.05)[et al.[ABCB1 wild-type genotype (3435CC) in 19% of Iranian renal transplant patients, whereas 51% and 30% were heterozygous (3435CT) and homozygous (3435 TT), respectively. The genetic risk variants such as APOE, PCSK9, CDKN2A and CDKN2B are associated with conventional risk factors for CAD and very commonly occur in 50% of the population[ABCB1 polymorphism might be considered as a genetic risk factor for CAD development, and it may affect the genotype profile of these patients. However, no data were found regarding ABCB1 genotype distribution in healthy control group to compare with our patient group.The present investigation showed that the frequencies of the  (P>0.05)-19. Azar5)[et al. have fouopulation. In thisDe novo sequencing at ABCC1 was performed on 142 healthy individuals from four different populations  to identify polymorphisms at multidrug resistance protein 1/ABCC1[G2012T polymorphism was common among Caucasians but it was not detected in Asian populations[n 1/ABCC1. T alleln 1/ABCC1. Furtherpulations.P=0.004, odds ratio=0.2, CI95%=0.06-0.6). Women in the first group showed better response than men. One study indicated lower mean area under the concentration-time curve and shorter half-life of atorvastatin in women than in men[ABCB1 gene polymorphism (CT+TT) and wild-type group of ABCC1 gene (GG). The LDL-C reduction demonstrated that the effect of these polymorphisms on atorvastatin efficacy was more clear in low-dose atorvastatin. Taking dose dependency of atorvastatin into consideration[In the present study, logistic regression analysis revealed significant differences between males and females’ response to atorvastatin regarding the achievement of LDL-C goal after four weeks of treatment with 10 mg/day atorvastatin . Furthermore, our study confirmed the investigation by Hoenig et al.[C3435T polymorphism in ABCB1 was associated with reduced atorvastatin efficacy regardless of the cholesterol metabolism following a six-week administration of atorvastatin. In Alzoubi et al.[G2677T (ABCB1) and the TT genotype of the C3435T (ABCB1) polymorphisms were associated with lower levels of LDL-C after atorvastatin treatment in Jordanians hypercholesterolemic patients. However, the effects of atorvastatin on the levels of total cholesterol, triglyceride and HDL-C had no relationship with any of the genotypes in both polymorphisms. In Rebecchi et al.[ABCC1 (G2012T) polymorphism, but ABCB1 and ABCC1 mRNA levels were modulated by atorvastatin and ABCB1G2677T/A polymorphism. Also, ABCB1 baseline expression was related to the differences in the serum LDL-C and ApoB in response to atorvastatin. Unequal group sizes and the risk of selection bias with the groups are the main limitations of this study. A short-term follow-up and the lack of control group for comparison may be considered as other important limitations. However, we concluded that in a low dose of atorvastatin, ABCB1 and ABCC1 polymorphisms may have an effective impact on atorvastatin efficacy in reduction of LDL-C serum level in Iranian patients with primary hypercholesterolemia.In a recent study, Kajinami et al. found noig et al. on Austrbi et al. study, bhi et al. study on


KDM5C cause Mental Retardation, X-linked, Syndromic, Claes-Jensen type  and are one of the most common causes of X-linked ID. KDM5C encodes a histone demethylase for di- and tri-methylated histone H3 lysine 4 (H3K4me2/3), which are enriched in transcriptionally engaged promoter regions. KDM5C regulates gene transcription; however, it remains unknown whether removal of H3K4me is fully responsible for KDM5C-mediated gene regulation. Most mutations functionally tested to date result in reduced enzymatic activity of KDM5C, indicating loss of demethylase function as the primary mechanism underlying MRXSCJ. Here, we report a novel KDM5C mutation, R1115H, identified in an individual displaying MRXSCJ-like symptoms. The carrier mother’s cells exhibited a highly skewed X-inactivation pattern. The KDM5C-R1115H substitution does not have an impact on enzymatic activity nor protein stability. However, when overexpressed in post-mitotic neurons, KDM5C-R1115H failed to fully suppress expression of target genes, while the mutant also affected expression of a distinct set of genes compared to KDM5C-wildtype. These results suggest that KDM5C may have non-enzymatic roles in gene regulation, and alteration of these roles contributes to MRXSCJ in this patient.Intellectual disability (ID) affects up to 2% of the population world-wide and often coincides with other neurological conditions such as autism spectrum disorders. Mutations in  KDM5C is one of the most frequently mutated genes in XLID and estimated to explain approximately 0.7–2.8% of all XLID cases. KDM5C-deficiency is characterized by frequent autistic and aggressive behaviors   affect 1.5–2% of the population world-wide . Clinicaehaviors  and is cH3K4me3) .Kdm5c led to defective development of dendrites and dendritic spines  on genomic DNA from the proband and both parents. The Agilent SureSelect XT2 All Exon V4 kit was used to target the exon regions of the genomes. The targeted regions were sequenced using the Illumina HiSeq 2000 sequencing system with 100 bp paired-end reads. The DNA sequence was mapped to and analyzed in comparison with the published human genome build UCSC hg19 reference sequence. The targeted coding exons and splice junctions of the known protein-coding RefSeq genes were assessed for the average depth of coverage of 64X and data quality threshold of 95.9%. The XomeAnalyzer was used to evaluate sequence changes in the proband compared to other sequenced family members. All sequence variants in the proband and parental samples were confirmed by Sanger sequencing analysis.6 lymphoblastoid cells from the proband and his father. A roughly 2 kb region surrounding the residue was amplified using the Q5 High-Fidelity Polymerase (NEB) with the following primers: 5′-AGAGGTTGTAGAGGAGGCCG-3′ and 5′-CTGTCATGCGAGGACTGTTGGTC-3′. The PCR reaction was purified (Qiagen) and exon 22 was Sanger sequenced using the following primers: 5′-gtgaggcctgggaccttg-3′ inside intron 21–22, and 5′-ccccatctgtgtcgaagc-3′ inside intron 22–23. Pedigree was made using pedigreedraw.com, from Genial Genetics. Multiple species sequence alignments were made using Clustal Omega.For Sanger sequencing validation, genomic DNA was isolated (Promega) from 3 × 10X-inactivation (XI) analysis using the well-characterized CpG methylation site and polymorphic CAG repeats within the Androgen Receptor (AR) locus was performed using standard protocol . In thisThe R1115H substitution was introduced into pENTR-KDM5C (human)  using a Wild-type and mutant KDM5C cDNAs were cloned into a baculovirus expression vector, pFastBac (Life Technologies), and expressed in Sf9 (for H3K4me3 assay) or Hi5  insect cells using the Bac-to-bac baculoviral expression system (Life Technologies). Cells were lysed in Buffer A  with 0.2 mM PMSF and protease inhibitor cocktail (Sigma). Recombinant proteins were immobilized on Strep-Tactin affinity resin (Qiagen), washed with Buffer A, and eluted in Buffer A containing 2.5–25 mM desthiobiotin. Enzymatic activity was assessed using the Histone Demethylase Fluorescent Activity Kit . 4–5 mM (for H3K4me3 assay) or 1 mM  purified KDM5C protein and 4 mM peptides were incubated at 30°C with freshly prepared 4 mM alpha ketoglutarate, 2 mM ascorbate, 100 mM iron ammonium sulfate. The following synthetic histone N-terminal peptides were purchased from Anaspec: H3K4 me3: H2N-ART(Kme3)QTARKSTGGKAPRKQL-amide, and H3K4me3/K9me3: H2N-ART(Kme3)QTAR(Kme3)STGGKAPRKQL-amide. Reactions were quenched with 4 mM deferoxamine mesylate at 10, 20, or 30 min, and detected with formaldehyde detection reagent according to the kit instructions. Fluorescence end point measurement was performed using the Tecan Safire 2 plate reader and XFluro4 V4.62 software.2. Cycloheximide (Sigma) was resuspended in DMSO and used at a final concentration of 100 μg/ml. For in situ demethylation assays in U2OS cells, Strep-tagged KDM5C expression plasmids (pHAGE) were transfected into U2OS cells with Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. For in situ demethylation assays in neurons, cells were transduced after 1 day in vitro (DIV1) cells with lentivirus of equal titer containing either human KDM5C cDNA  or vector alone.Lymphoblastoid cell lines from the proband and his father were isolated and cultured under identical conditions as described previously . Briefly5 cells/well, and transfected as described above for 48 h. Mouse forebrain tissue was dissected from embryonic day 16 (E16) CD1 mouse embryos. Cells were dissociated, plated, and cultured as described previously (in vitro 1 (DIV1) as described above, and harvested at DIV3. Cells were fixed with 4% paraformaldehyde, permeabilized with Triton X-100, and blocked with 10% fetal bovine serum. Coverslips were incubated with appropriate primary antibodies overnight at 4°C, washed, and incubated with the corresponding secondary antibodies and DAPI stain for 1 h at room temperature. Coverslips were then washed, mounted with ProLong Gold Antifade Mountant (Invitrogen). U2OS cells were analyzed on an Olympus BX61 microscope using a 60x oil objective, and images were acquired with cellSens Dimension (1.14) software and processed with ImageJ (1.48) and Adobe Photoshop (CS6). Neurons were analyzed on a Nikon A-1 confocal microscope using a 60x oil objective, and images were acquired with Nikon’s Elements software. Primary antibodies were used at the following concentrations: 1:1,000 anti-H3K4me1 (Abcam ab8895), 1:20,000 anti-H3K4me2 (Abcam ab7766), 1:1,000 anti-H3K4me3 (Abcam ab8580), 1:500 (for U2OS) or 1:1,000 (for neurons) anti-Strep (Genscript A01732), 1:1,000 anti-Map2 (Millipore AB5543). Alexa 647-donkey-anti-mouse (for neurons), 594-donkey-anti-rabbit (for U2OS) or -anti-chicken (for neurons), and 488-donkey-anti-mouse (for U2OS) or -anti-rabbit (for neurons) secondary antibodies were all used at 1:1,000. DAPI was used to stain the nucleus at a 1:1,000 dilution.U2OS cells were plated on PDL-coated coverslips in 24-well dishes at 1 × 10eviously , transduCell were lysed in SDS-PAGE sample buffer. Infrared fluorescence-based Western blot was performed using the LI-COR Odyssey Western Blotting RD system according to standard protocol. Primary antibodies were used at the following dilutions: anti-KDM5C  at 1:250in vitro (DIV1) cells were infected with lentivirus of equal titer containing either human KDM5C cDNA  or vector alone. Puromycin selection occurred at DIV4 with 0.2 μg/ml puromycin (Sigma). Control puromycin selection with untransduced cultures eliminated nearly all cells, indicating that majority of survived cells carry the KDM5C transgene. Cells were harvested at DIV10 by direct addition of TRI Reagent (Sigma). Samples were subject to total RNA isolation, and subsequent purification was performed using RNEasy Mini Kit (Qiagen). Ribosomal RNA was depleted using RiboMinus Eukaryotic Kit v2 (Life Technologies). Libraries were prepared using Direct Ligation of Adapters to First-strand cDNA as described previously (Mouse forebrain tissue was dissected from embryonic day 16 (E16) CD1 mouse embryos. Cells were dissociated, plated, and cultured as described previously . After 1eviously . Multiplp-value < 0.01. Cuffdiff (Cufflinks 2.2.1) was used to calculate FPKM. Differentially expressed genes were examined for functional annotation clustering using DAVID (6.8). To validate overexpression conditions, reads were mapped to a custom genome using STAR, allowing no mismatches, and only uniquely mapped reads were analyzed further. The custom genome contained human KDM5C cDNA (NM_001146702.1), where bases were masked with “N” at the H514 (c.1540-1541CA > NN) and R1115 (c.3344G > N) loci to allow for specific mapping under strict no-mismatch conditions.Reads were trimmed to 60 bp using BBDuk (35.51) and mapped to the mm9 mouse genome using STAR (2.5.3a) allowing zero mismatches, and only uniquely mapped reads were analyzed further. Due to low mapability of read2, only read1 was used for further analysis. BAM files were indexed and converted to BigWig files in a strand specific manner. BigWigs were normalized to 10 million non-rRNA and non-mitochondrial reads. DESeq2 (1.14.1.) was used to call differential gene expression between conditions, using a cutoff of RNA-seq files can be found at Gene Expression Omnibus GEO:GSE104319.Figures 2A,B, error bars represent standard error of the mean (SEM) of a technical triplicate. For analysis of H3K4me levels in Figure 2D, H3K4me signals were normalized to pan-H3 signal and error bars represent SEM of technical triplicate. For RNA-seq data in Figures 5 and Supplementary Figure S3, differentially expressed genes were determined by DESeq2 using a cutoff of p < 0.01. For GO term analysis in Figure 5, the modified Fisher Exact p-value is represented as calculated by the DAVID functional annotation tool . This variant results in an arginine-to-histidine substitution at amino acid position 1115 (R1115H), and was inherited from the maternal grandmother. The carrier mother and grandmother were phenotypically normal, and the variant was not present in the unaffected siblings or maternal uncle. We isolated genomic DNA from lymphoblastoid cell lines from father (UM1 II-1) and proband (UM1 III-3), and confirmed by Sanger sequencing that the variant is present specifically in the proband .The proband of family UM1 was first clinically examined at 26 months for developmental delay and microcephaly, after observing delays in developmental milestones from 1 year of age. He was diagnosed with ASD due to cognitive impairment and behavioral concerns, including severe tantrums, aggression, and anxiety Figure 1C). KDM5C belongs to a family of histone H3K4 demethylases, in which KDM5A, KDM5B, and KDM5D are the additional members . Consistently, multiple genetic variant assessment algorithms predict this variant to be pathogenic (Supplementary Table S1). We did not find any homozygous or hemizygous substitutions of R1115 in the Genome Aggregation Database (gnomAD)2, indicating that variation in this amino acid residue is intolerant in the population. To date, 25 KDM5C mutations have been identified in MRXSCJ patients . The R1115H variant lies within the C-terminal segment of the KDM5C protein, making it the most distal missense mutation in KDM5C reported to date.The R1115H variant lies in a region of high conservation and is itself highly conserved among vertebrates ( members . The R11patients . Most ofTable 1]. When we tested the X-inactivation status of the carrier mother (UM1 II-2), we observed highly skewed inactivation , and moderate skewing in the grandmother (UM1 I-2). In the carrier mother, the X-chromosome passed on to the proband is dominantly CpG-methylated at the androgen receptor (AR) locus. AR and KDM5C are located nearby (∼14.5 Mb), flanking the centromere, and no meiotic recombination site is known between the two loci .In female mammals, one of the two X chromosomes is randomly chosen to be silenced during early embryogenesis; therefore, females are chimeras of cells that silenced either the paternally- or the maternally-inherited X . Female two loci ; therefoKDM5C-MRXSCJ mutations that have been tested for enzymatic activity exhibit a decrease in histone demethylase activity, suggesting a loss-of-function pathogenic mechanism . We performed in vitro demethylation assays using purified KDM5C and two synthetic histone H3 N-terminal tail peptides carrying the following modifications: K4me3, the direct substrate for the KDM5C catalytic subunit; or K4me3 and K9me3, which is recognized by the first PHD domain of KDM5C . These data indicate intrinsic demethylation ability of the KDM5C R1115H protein is largely retained.A majority of echanism . We set of KDM5C . Our resKDM5C cDNA in U2OS cells and examined H3K4me3 levels using immunofluorescence . Cells expressing KDM5C WT showed dramatically reduced H3K4me3 staining. Similar to WT, KDM5C R1115H-expressing cells exhibited a marked reduction in H3K4me3 signal. In contrast, cells expressing the catalytically-inactive KDM5C H514A mutant . Cells expressing KDM5C WT often showed reduced H3K4me2 and H3K4me3 staining, and no effect on H3K4me1, as predicted. Similar to WT, KDM5C R1115H-expressing cells frequently exhibited a marked reduction in H3K4me2/3 signal. In contrast, cells expressing the catalytically inactive KDM5C H514A mutant . Taken together, these data strongly suggest that KDM5C R1115H substitution does not lead to substantial reduction in histone demethylation activity.We next tested demethylation activities in cells. We over-expressed Strep-tagged human A mutant  retain hA mutant  retain hKDM5C-MRXSCJ mutations have been shown to destabilize KDM5C protein, representing another potential loss-of-function effect. To test the stability of KDM5C R1115H, we treated the lympoblastoid cells from the father and the proband with cycloheximide, an inhibitor of protein synthesis, for 0–28 h. Reduction kinetics of endogenous KDM5C levels were not dramatically different in proband compared to father throughout the time course, suggesting protein stability is largely unaffected by the R1115H mutation .Some Kdm5c regulates neurodevelopment genes in cultured mouse forebrain neurons and multiple brain regions in vivo including the cortex and the amygdala . Mapping the sequencing reads to the human KDM5C cDNA also allowed us to validate similar levels of human KDM5C WT and R1115H mutant cDNAs, while we noted moderately higher level of H514A mutant expression compare to the other two KDM5C cDNAs . We compared the sequencing reads mapped to human KDM5C cDNA and mouse Kdm5c gene without allowing any mismatches, and estimated that each KDM5C cDNA was expressed approximately 15- to 30-fold higher than endogenous Kdm5c mRNA . The expression of endogenous Kdm5c does not appear to be largely affected by human KDM5C overexpression .We previously reported that amygdala . To testamygdala . As outlp < 0.01; Figure 4B). Consistent with its role as a transcriptional repressor in primary neuron culture . Both KDM5C R1115H and H514A failed to fully repress this group of genes, suggesting that the two mutations interfere with KDM5C’s gene-regulatory function . The partial deficiency of H514A mutant despite the higher level of KDM5C-H514A mRNA implicates that demethylation activity is not the sole mechanism for KDM5C-mediated gene control. It is noteworthy that KDM5C R1115H mutant showed a similar level of deficiency compared to H514A mutant in regulating the 42 genes.Compared to the vector control, overexpression of KDM5C-WT resulted in 42 differentially-expressed (DE) genes . KDM5C R1115H expression resulted in 64 DE genes  compared to WT, while KDM5C H514A gave 99 DE genes  . Representative genes for the following four expression patterns across conditions are shown in Supplementary Figures S3D–G: (1) genes repressed by KDM5C WT but not by mutants include PWWP domain containing 2b (Pwwp2b) and Zinc finger protein 198 (Zfp189); (2) genes that show unique response to KDM5C-H514A include Protein kinase C, theta (Prkcq) and Trichoplein, keratin filament binding (Tchp); (3) genes strongly repressed by KDM5C-R1115H include TATA-box binding protein associated factor 5 like (Taf5l) and coordinator of PRMT5, differentiation stimulator (Coprs); and finally (4) genes uniquely up-regulated by KDM5C-R1115H including Transmembrane protein 251 (Tmem251) and Keratin associated protein 4-8 (Krtap4-8).We then compared the identity of DE genes upon overexpression of KDM5C-WT, R1115H, and H514A. Interestingly, overlap between DE genes in each condition were limited, suggesting that R1115H and H514A mutants can regulate different sets of genes compared to KDM5C-WT and the two mutants are functionally distinct from each other . These GO terms are absent from both R1115H  and H514A  down-regulated genes, pointing to the inability of the mutant KDM5Cs to regulate the relevant genes as WT (see Supplementary Table S2 for complete list of GO terms). Meanwhile, down-regulated genes by R1115H and H514A were enriched in largely distinct sets of GO terms compared to WT, and these GO terms are also different between the two mutants. For example, KDM5C-R1115H mutant down-regulated and “membrane-bound organelle” and “muscle structure development,” while KDM5C-H514A down-regulated “gliogenesis” and “protein binding.” Distinct sets of genes appear to contribute the diverse GO term enrichments between the conditions (Supplementary Table S2). Taken together, these data suggest that KDM5C R1115H is less potent in suppressing WT-regulated genes, yet the mutant gains unique gene regulatory roles, which may lead to deleterious and distinct consequences in neuronal development and functions.Having observed both common and unique impacts of R1115H and H514A substitutions, we sought to gain biological insights associated with the mutations by using the database for annotation, visualization, and integrated discovery (DAVID) ,b. GivenMRXSCJ has been primarily attributed to either reduced enzymatic activity and/or stability of the KDM5C protein by associated mutations. In the present study, we describe a novel KDM5C missense mutation, R1115H, which was identified in an individual with a typical MRXSCJ phenotype. While the R1115H substitution does not result in appreciable changes in enzymatic activity or protein stability, this substitution appears to alter the transcriptional regulatory function of KDM5C. Another KDM5C mutation, D87G, was shown to not interfere with the demethylation activity . HoweverFigure 1). Third, KDM5C-R1115 is highly conserved among vertebrates and the R1115H substitution is predicted to be functionally damaging by multiple prediction algorithms . Forth, the carrier mother showed highly skewed X-inactivation (Table 1). KDM5C was originally discovered as an X-linked gene that escapes X-inactivation both in human and mouse, although escaping is not complete — KDM5C is expressed at a lower level from the inactive X compared to that of the active X . The N-terminal half of KDM5 family demethylases, encompassing JmjN, Bright, PHD1, JmjC, and zinc finger domains, was shown to be sufficient for its catalysis . Given the intact H3K4 demethylation activity of KDM5C-R1115H and low frequency of its cytoplasmic presence, the mislocalized protein may not have a major impact. However, we cannot rule out the possibility that KMD5C-R1115H could demethylate ectopic target(s) at the cytoplasm. Future studies are needed to determine the roles of the C-terminus in KDM5C-mediated transcriptional regulation, impact of disease mutations in this segment, and the roles of enzymatically active mutants that can be present outside nuclei.The previously reported missense mutations in KDM5C all fall within the N-terminal half of KDM5C, which harbors a JmjC domain, the catalytic core for histone H3K4 demethylation (atalysis . By contatalysis , it is patalysis . These obona fide KDM5C target-genes. Nonetheless, KDM5C-R1115H illuminates a novel gene regulatory function of KDM5C, which is independent of its enzymatic activity, and potentially represents a novel molecular etiology contributing to MRXSCJ.Our RNA-seq data suggest that the R1115H mutation may not only lead to loss of function of KDM5C but also acquisition of a new gene-regulatory function. It is tempting to speculate that KDM5C neofunctionalization might be involved in the pathophysiology of this specific MRXSCJ-like patient. However, limitations of interpreting the RNA-seq results should be noted. The RNA-seq was carried out upon overexpression of WT- and mutant-KDM5C, 10 days after introducing KDM5C transgenes into neurons. Gene expression changes may therefore involve both direct impact of on KDM5C’s target genes as well as indirect consequences, such as alteration in neuronal maturation processes. In addition, gene expression changes upon KDM5C overexpression may not simply reflect the dysregulation of CNV, CEK, and SI conceived the study and designed the experiments. CEK performed the clinical evaluation of the proband and coordinated genetic testing of the proband and family members. MJF oversaw the X-chromosome inactivation analysis. MB oversaw the generation of lymphoblastoid cell lines. CNV performed all experiments and analysis. All authors contributed to the writing and editing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer YB and handling Editor declared their shared affiliation.


Oxysterols are oxidised derivatives of cholesterol, formed by the enzymatic activity of several cytochrome P450 enzymes and tumour-derived oxysterols have been implicated in tumour growth and survival. The aim of this study was to profile the expression of oxysterol metabolising enzymes in primary colorectal cancer and assess the association between expression and prognosis.Immunohistochemistry was performed on a colorectal cancer tissue microarray containing 650 primary colorectal cancers using monoclonal antibodies to CYP2R1, CYP7B1, CYP8B1, CYP27A1, CYP39A1, CYP46A1 and CYP51A1, which we have developed. Unsupervised hierarchical cluster analysis was used to examine the overall relationship of oxysterol metabolising enzyme expression with outcome and based on this identify an oxysterol metabolising enzyme signature associated with prognosis.2=12.984, p<0.001, HR=1.983, 95% CI 1.341-2.799) than the poor prognosis group . For the mismatch repair proficient cohort, the good prognosis group had a significantly better survival  than the poor prognosis group. Multi-variate analysis showed that cluster group was independently prognostically significant in both the whole patient cohort  and the mismatch repair proficient group .Cluster analysis of the whole patient cohort identified a good prognosis group  that had a significantly better survival (δIndividual oxysterol metabolising enzymes are overexpressed in colorectal cancer and an oxysterol metabolising enzyme expression profile associated with prognosis has been identified in the whole patient cohort and in mismatch repair proficient colorectal cancers. Colorectal cancer is one of the most common types of malignancy affecting both men and women, with a worldwide annual incidence of greater than 1.2 million new cases [Currently, colorectal cancer is commonly staged using the tumour, node, metastasis (TNM) staging system to guide treatment decisions and indicate prognosis. However, patients with the same stage of tumour often experience a wide range of different clinical outcomes. Despite the unequivocal value of current staging systems, there is a still need to develop reliable biomarkers to more accurately predict prognosis and risk stratify patients with colorectal cancer. Biomarkers can have a variety of roles in colorectal cancer including early detection, predicting prognosis, predicting response to therapy and aiding post-operative monitoring .Oxysterols are oxidised derivatives of cholesterol, formed by the enzymatic activity of several cytochrome P450 enzymes –12. In aDespite the recognition of the role of oxysterol signalling in tumourigenesis, the key cytochrome P450s involved in the oxysterol pathway have received very limited study in existing research with regard to their expression in tumours , 18. ThiThe specificity of the monoclonal antibodies to CYP2R1, CYP7B1, CYP8B1, CYP27A1, CYP39A1, CYP46A1 and CYP51A1 was determined by ELISA using the immunogenic peptides and also by immunoblotting using whole cell lysates from cells overexpressing of each protein , CYP7B1 (p<0.001), CYP8B1 (p<0.001), CYP46A1 (p<0.001) and CYP51A1 (p=0.001). CYP27A1 and CYP39A1 showed no statistically significant difference in expression between primary tumour and normal colonic mucosa , CYP39A1 (p=0.001), CYP46A1 (p<0.001) and CYP51A1 (p<0.001) each showed a significant decrease in immunoreactivity in lymph node metastasis compared to primary tumour. There was no statistically significant difference in expression of CYP7B1 or CYP27A1 between the paired cases of Dukes C (stage 3) colorectal cancer and their corresponding lymph node metastasis. However, CYP2R1 (p=0.034), CYP8B1 (p=0.002), CYP39A1 (p<0.001), CYP46A1 (p<0.001) and CYP51A1 (p=0.001) each demonstrated significantly reduced expression in paired lymph node metastasis compared to Dukes C (stage 3) colorectal cancer.The relationship between expression of each protein and relevant clinico-pathological parameters are summarised in Table Unsupervised hierarchical cluster analysis was used as an unbiased exploratory statistical tool to examine the overall relationship of oxysterol metabolising enzyme expression with outcome and based on this identify an oxysterol metabolising enzyme signature associated with prognosis. A range of cluster solutions (number of clusters) was investigated to determine the optimum number of clusters that produced groups with different outcomes. Clustering the data into five clusters was identified as the optimum number of clusters for analysis in relation to the most prognostically significant groups Figure . These f2=12.984, p<0.001, HR=1.983, 95% CI 1.341-2.799) than the poor prognosis group   had a significantly better survival  than the poor prognosis group .For the mismatch repair proficient cohort good prognosis group  had a significantly better survival  than the poor prognosis group .For the mismatch repair deficient cohort good prognosis group  had a significantly better survival  and the mismatch repair proficient group  in a model that included information about Dukes stage Table , Model BThe relationship between expression of each protein and overall survival was investigated using different cut-off points of immunostaining intensity to allow a total of four comparisons to be made . 2=14.97, p=0.002). The mean survival in patients with tumours that did not express CYP8B1 (n=201) was 128 months (95% CI 113-141), declining to 119 months (95% CI 104-134) for tumours with weak CYP8B1 immunostaining (n=171). The mean survival for CYP8B1 moderately expressing tumours (n=133) was 101 months (95% CI 89-113) and the mean survival for CYP8B1 strongly expressing tumours (n=118) was 79 months (95% CI 67-91). When negative/weak staining for CYP8B1 was compared to moderate/strong staining, there was a statistically significant relationship between CYP8B1 expression and survival . The mean survival in patients with tumours showing negative/weak CYP8B1 immunoreactivity (n=371) was 126 months (95% CI 115-136) compared to 91 months (95% CI 82-99) in patients with tumours showing moderate/strong immunoreactivity (n=251). Comparing CYP8B1 negative/weak/moderate tumours with CYP8B1 strongly expressing tumours demonstrated a highly significant relationship with survival . Patients with strongly staining tumours for CYP8B1 (n=118) survived a mean of 79 months (95% CI 67-91) whereas negative/weak/moderate CYP8B1 immunostaining was associated with a better prognosis and a mean survival of 123 months (95% CI 114-132).Expression of CYP8B1 was consistently associated with patient survival. Considering each CYP8B1 intensity as a distinct group, increasing intensity was related to poorer prognosis . Patients with strongly scoring tumours for CYP27A1 (n=16) had decreased survival, with a mean survival of 59 months (95% CI 35-84) in this group. For CYP27A1 negative tumours (n=367) the mean survival was 118 months (95% CI 108-128), for CYP27A1 weakly stained tumours (n=195) the mean survival was 108 months (95% CI 96-120) and for CYP27A1 moderately stained (n=44) tumours the mean survival was 91 months (95% CI 71-111). When negative/weak CYP27A1 immunostaining was compared to moderate/strong immunostaining, higher intensities of staining were significantly associated with poorer survival . The mean survival in patients with tumours showing moderate/strong staining for CYP27A1 (n=60) was 82 months (95% CI 65-98) whereas for negative/weak tumours (n=562) the mean survival was 119 months (95% CI 110-127). Investigating strong CYP27A1 immunostaining compared to all other stain intensities was also significantly associated with survival . Strong CYP27A1 immunostaining (n=16) resulted in a mean survival of 59 months (95% CI 35-84) compared to 118 months (95% CI 109-126) for negative/weak/moderate staining for CYP27A1 (n=606).CYP27A1 expression was associated with patient outcome in three out of four comparisons. Considering each CYP27A1 stain intensity separately, expression was significantly associated with survival . Tumours showing negativity for CYP39A1 (n=453) were associated with a mean survival of 125 months (95% CI 115-134), for CYP39A1 weakly stained tumours (n=164) the mean survival was 89 months (95% CI 79-99) and for CYP39A1 moderately stained tumours (n=12) the mean survival was 33 months (95% CI 16-50). Comparing CYP39A1 negatively staining tumours versus CYP39A1 positively staining tumours, CYP39A1 expression demonstrated a statistically significant relationship with survival . Patients whose tumours did not express CYP39A1 (n=453) had a mean survival of 125 months (95% CI 115-135) whereas patients with weak/moderate staining for CYP39A1 (n=176) had a mean survival of 86 months (95% CI 76-96). Comparing negative/weakly stained tumours for CYP39A1 with moderately stained tumours revealed a highly significant relationship between expression and survival . The mean survival for CYP39A1 negative/weak staining tumours (n=617) was 117 months (95% CI 109-126), declining to 33 months (95% CI 17-50) for patients whose tumours demonstrated moderate immunostaining for CYP39A1 (n=12).Immunohistochemistry for CYP39A1 did not reveal any strong staining. In a comparison of negative versus weak versus moderate staining for CYP39A1, a highly statistically significant association with survival was identified . Tumours that were negative for CYP46A1 (n=74) showed a mean patient survival of 117 months (95% CI 95-138), weakly stained tumours (n=173) had a mean survival of 128 months (95% CI 113-144) and moderately stained tumours (n=205) had a mean survival of 113 months (95% CI 100-126). Strong staining reflected a poorer outcome, with patients who had strongly staining tumours for CYP46A1 (n=173) surviving a mean of 87 months (95% CI 76-98). A comparison of strong CYP46A1 immunostaining versus all other stain intensities was also significantly associated with survival . In common with all other associations noted, higher CYP46A1 expression was linked to poorer prognosis. Tumours demonstrating strong CYP46A1 immunostaining (n=173) had a mean patient survival of 87 months (95% CI 76-98) whereas negative/weak/moderate CYP46A1 immunostaining (n=452) was associated with a mean patient survival of 122 months (95% CI 112-131).Overall expression of CYP46A1 considering negative, weak, moderate and string staining as separate groups showed a statistically significant relationship with survival  were compared to the paired lymph node metastasis, expression of CYP2R1, CYP8B1, CYP39A1, CYP46A1 and CYP51A1 were each significantly reduced in the lymph node metastasis. This highlights the role of the tumour microenvironment in influencing the expression of the target proteins, a concept increasingly recognised in studies of the metastatic spread of malignancy –46. The In conclusion this study has defined the expression of oxysterol metabolising P450s in a well-characterised cohort of colorectal cancers. An oxysterol metabolizing enzyme signature has been identified which is associated with prognosis in the whole patient cohort and the mismatch repair proficient cohort. This good prognosis group showed tumours with low expression of CYP2R1, CYP8B1, CYP27A1 and CYP46A1 relative to the poor prognosis group and a schematic model of the relationship of oxysterol metabolizing enzymes in good and poor prognosis colorectal cancers is outlined in Figure Monoclonal antibodies to individual oxysterol metabolising enzymes  were developed in collaboration with Vertebrate Antibodies Ltd  using synthetic peptides. Peptides within the protein sequences were selected from regions which were antigenic, exposed on the surface and unique to the target protein , 49–51. Whole cell lysates from cells (human embryonic kidney cells-HEK 293) overexpressing CYP2R1, CYP7B1, CYP8B1, CYP27A1, CYP39A1, CYP46A1 and CYP51A1 respectively were used as positive controls for immunoblotting while lysates from cells containing vector only were used as negative controls. The cell lysates and their corresponding controls were obtained from . Cell lysates (5 μg protein/lane) were resolved by electrophoresis on NuPAGE 4-12% Bis-Tris gels . Following protein transfer to nitrocellulose membrane the membranes were washed for 45 minutes at room temperature in phosphate buffered saline-Tween-20 (PBST) containing 3 % (w/v) skimmed milk powder to block non-specific protein binding. Membranes were incubated overnight at 4°C with individual monoclonal antibodies diluted in PBST (1/2 dilution) and then washed 6 times for a total of 60 minutes in 1% skimmed milk. The membranes were subsequently probed for 60 minutes with a secondary antibody conjugated horseradish-peroxidase-conjugated anti-mouse IgG . Membranes were then washed (6 times) for a total of 60 minutes in 1% skimmed milk and protein bands visualized using the enhanced chemiluminescence detection system (Fisher Scientific) , 49.www.biorepository.nhsgrampian.org), Aberdeen, UK provides governance for this tissue bank, see ethics statement). Survival information, in the form of all-cause mortality , was available for each patient. At the time of censoring patient outcome data there had been 309 (47.5%) deaths. The mean patient survival was 115 months (95% CI 108-123 months). The study was conducted according to REMARK criteria and clinico-pathological characteristics of the patients and their tumours and relationship with survival are detailed in Table The patient cohorts of mismatch repair proficient and deficient colorectal cancers included 650 patients with a histologically confirmed diagnosis of primary colorectal cancer. All patients had undergone elective surgery for primary colorectal cancer, at Aberdeen Royal Infirmary , between the years of 1994 and 2009. Any patients who had received neoadjuvant chemotherapy and/or radiotherapy were excluded. Tissue was obtained retrospectively from the Aberdeen Colorectal Tumour Bank the Grampian Biorepository  using a Dako autostainer as previously described , 49, 54.The initial step in the automated staining protocol was a wash buffer rinse (Dako). Next, slides were incubated with the primary antibody for 60 minutes then washed with buffer. Each primary antibody was applied as undiluted tissue culture supernatant. Slides were then washed in buffer and peroxidase enzyme block was applied for a period of 7 minutes and slides were then again rinsed with wash buffer. Subsequently, the pre-diluted peroxidase labelled polymer coupled to goat anti-mouse/rabbit secondary antibody was applied for 30 minutes then rinsed with buffer to remove any unbound antibody. The diaminobenzidine (DAB) substrate was then applied for 7 minutes to demonstrate sites of peroxidase activity before a final wash with buffer then water. Slides were immersed in 0.5% copper sulphate for 2 minutes to intensify the DAB stain then washed with running water. Finally, slides were immersed in filtered Harris haematoxylin to lightly counterstain the nuclei before being dehydrated in alcohol and xylene and mounted. Omitting the primary monoclonal antibody from the immunohistochemical procedure and replacing it with antibody diluent (Dako) acted as a negative control. Normal liver was used as a positive control for CYP2R1, CYP7B1, CYP8B1, CYP39A1, CYP46A1 and CYP51A1. Grade 3 breast cancer of no special type was used a positive control for CYP27A1.Immunohistochemistry was also performed on whole sections of a sub-set of tumours to investigate possible intra-tumour heterogeneity. Sections from the same tissue blocks from which tissue cores were obtained were used.Following the completion of the immunohistochemistry protocol, the slides were examined by light microscopy using an Olympus BX 51 light microscope  equipped with an Olympus C4040 camera (Olympus). The intensity of immunostaining was quantified using a semi-quantitative scoring method as previously described , 49, 54.Mismatch repair protein status had previously been assessed by immunohistochemistry using antibodies to MLH1 and MSH2 . MismatcStatistical analysis of the data including the Mann-Whitney U test, Wilcoxon signed rank test, chi-squared test, Kaplan-Meier survival analysis, log-rank test and Cox multi-variate analysis  including the calculation of hazard ratios and 95% confidence intervals was performed using IBM SPSS version 22 for Windows 7™ . The log rank test was used to determine survival differences between individual groups. A probability value of p≤0.05 was regarded as significant. The influence of different cut-off points in relation to survival was investigated by dichotomising the immunohistochemistry intensity score for each marker. The groups that were analysed were negative staining versus any positive staining, negative and weak staining versus moderate and strong staining and negative, weak and moderate staining versus strong staining.Unsupervised hierarchical cluster analysis was carried out using the within-group average linkage method with Pearson correlation as the cluster measure and cluster analysis was performed without any transformation of the data or imputation of missing values.The colorectal cancer tissue microarray is held under the auspices of the Grampian Biorepository which has delegated research ethics authority (11/NS/0015) from The North of Scotland research ethics committee to approve research projects involving human tissue and data. This project was approved by the Grampian Biorepository scientific access group committee (Tissue request No. 0002). Written consent for the formalin fixed wax embedded tissue samples included in the colorectal cancer tissue microarray was not required.


Evidence from randomised controlled trials suggests that vitamin D may reduce multimorbidity, but very few studies have investigated specific determinants of vitamin D2 and D3 (two isoforms of 25-hydroxyvitamin D). The aim of the study was to investigate the determinants of vitamin D2 and D3 and to identify the risk factors associated with hypovitaminosis D.Cross-sectional study.Northern Finland Birth Cohort 1966.2 and 25(OH)D3 concentrations measured at 31 years of age (1997), together with comprehensive measures of daylight, anthropometric, social, lifestyle and contraceptive cofactors.2374 male and 2384 female participants with data on serum 25(OH)D2, 25(OH)D3 and 25(OH)D concentrations were analysed by linear regression and risk factors for being in lower tertile of 25(OH)D concentration by ordinal logistic regression.We assessed a wide range of potential determinants prior to a nationwide fortification programme introduced in Finland. The determinants of 25(OH)D2 and negatively with 25(OH)D3 concentrations. Use of oral contraceptives (vs non-users) was associated with an increase of 0.17 nmol/L (95% CI 0.08 to 0.27) and 0.48 nmol/L (95% CI 0.41 to 0.56) in 25(OH)D2 and 25(OH)D3 concentrations. Sex, season, latitude, alcohol consumption and physical activity were the factors most strongly associated with 25(OH)D concentration. Risk factors for low vitamin D status were low sunlight exposure defined by time of sampling, residing in northern latitudes, obesity, higher waist circumference, low physical activity and unhealthy diet.At the time of sampling, 72% of the participants were vitamin D sufficient (≥50 nmol/L). Low sunlight exposure period (vs high) was associated positively with 25(OH)D2 and 25(OH)D3 raising important questions related to personalised healthcare. Future strategies could implement lifestyle modification and supplementation to improve vitamin D2 and D3 status, accounting for seasonal, lifestyle, metabolic and endocrine status.We demonstrate some differential associations of environmental and lifestyle factors with 25(OH)D This limits the availability of inferences that could help to identify people at risk of vitamin D deficiency, and improved fortification policies to meet the requirements of those living at northern latitudes.14There is limited knowledge about the factors associated with each isoform that may have differential environmental determinants.2, 25(OH)D3 and total 25(OH)D concentrations in Finnish adults aged 31 years prior to the implementation of a nationwide supplementation of vitamin D via fortification of milk products and margarine in 2002.14We examined here factors associated with 25(OH)DWe analysed data on participants from the Northern Finland Birth Cohort 1966 (NFBC1966) which has previously been described in detail.10.1136/bmjopen-2016-013161.supp1supplementary figure2 and 25(OH)D3 were measured by liquid chromatography tandem mass spectrometry and the detailed assay procedure is published elsewhere.2 values under the detectable limit were assigned a value of 1.25 nmol/L.2 low value assignment. Consequently, in the tables, total 25(OH)D may differ slightly from exact sum of D2 and D3. Vitamin D sufficiency criteria were defined according to the Institute of Medicine (IOM) guidelines as ≤30 nmol/L (risk/deficiency), 30–50 nmol/L (risk/insufficiency) and ≥50 nmol/L (sufficient).19Serum 25(OH)D2) was calculated and categorised according to the WHO 1998.22The season of participant attendance at the clinical assessment was categorised according to the Finnish Meteorological Institute standard as high sunlight (summer (1 June–30 August) autumn (1 September–31 October)) and low sunlight season (winter (1 November–31 March) and spring (1 April–31 May)).Categorisation of following lifestyle variables was based on the responses in the postal questionnaire. Current smoking was categorised as non-smoker, former/occasional or active smoker. Alcohol consumption during the 6 months prior to the questionnaire was calculated as grams per day (g/day) and has been described elsewhere.2 test for categorical variables. We performed univariable linear regression analysis to explore the association between explanatory variables and serum 25(OH)D2, 25(OH)D3 and total 25(OH)D concentrations. We log transformed 25(OH)D2, 25(OH)D3 and 25(OH)D, and expressed these on standardised scales (z-scores). To examine whether sex was an effect modifier of associations, an interaction term (sex × explanatory variable) was additionally included in univariable analyses. We conducted multivariable analyses aiming to examine mutually adjusted associations of different exposures with 25(OH)D2, 25(OH)D3 and 25(OH)D measures, namely season of blood sampling (low and high sunlight period), latitude, BMI, waist circumference, SEP, smoking status, alcohol consumption, leisure time computer use, physical activity, diet score and contraception status. In addition, we examined serum 25(OH)D2, 25(OH)D3 and 25(OH)D concentrations by excluding women using OCPs.All statistical analyses were performed using SAS V.9.4 . The variables were assessed for normality and log transformed where relevant. Mean differences between sexes for continuous variables were measured by independent samples t-test and analysis of variance; and Pearson χ2, 25(OH)D3 and 25(OH)D concentrations, we performed multinomial ordinal logistic regression analysis to assess the risk factors associated with being in the lower tertile (reference: tertile III) of vitamin D. Owing to equivocal definitions of cut-off values for vitamin D status in the general population, we categorised the analysis sample into tertiles of 25(OH)D. Statistical significance was set at global p<0.05 using two-tailed test.Following examination of the determinants associated with 25(OH)DThe characteristics of the study population at age 31 years are summarised in Characteristics of the study population across the tertiles of serum 25(OH)D concentration are summarised in 10.1136/bmjopen-2016-013161.supp2supplementary tables2, 25(OH)D3 and 25(OH)D in the total population are shown in 2 and 25(OH)D3 were sex, season of blood sampling, latitude, obesity, waist circumference and physical activity. Unhealthy diet and active smoking were univariably associated with lower 25(OH)D2 concentrations; and SEP was associated univariably with lower 25(OH)D3 concentrations.Univariable and multivariable associations of daylight, anthropometric, social and lifestyle factors with 25(OH)D2 and 25(OH)D3 concentrations. Men had 0.5 nmol/L lower 25(OH)D2 but 1.6 nmol/L higher 25(OH)D3 than women. When women using oral contraceptives were excluded from the analysis, the association between sex and 25(OH)D2 concentration was attenuated . Conversely, the sex difference still persisted for 25(OH)D3 concentrations , that is, women having lower concentrations. Low sunlight exposure period (vs high) at sampling associated with higher concentrations of 25(OH)D2 but lower concentrations of 25(OH)D3. Alcohol abstainers were associated with lower 25(OH)D3 concentrations than any other level of drinker. In addition, unhealthy diet score and leisure time computer use were associated with lower 25(OH)D3 concentrations.In multivariable analyses, sex was associated with serum 25(OH)D2 and 25(OH)D3 concentrations. Female OCP users (vs non-users) had greater serum 25(OH)D2 and 25(OH)D3 concentrations of 0.17 nmol/L and 0.48 nmol/L, respectively.In sex-stratified analyses, the associations were in the same direction and of similar magnitude with 25(OH)D3 concentrations, with the exception of waist circumference and leisure time computer use D associations with potential determinants reflect similar associations as reported for 25(OH)Duter use . OCP useAccording to the present data collected in 1997, 28% of young adults in Northern Finland were exposed to the risk of vitamin D insufficiency defined by IOM. The average vitamin D status observed in our study was higher than those reported by other studies from the same geographical location  of the circulating 25(OH)D concentrations in the present population. Our study and the study performed by Tolppanen et al2 and 25(OH)D3. The determinants associated with the vitamin D status also influenced the serum concentrations of 25(OH)D3, with the highest effect being exerted by the season. Importantly, we replicated the associations of the seasonal variation but not the SEP as first observed in children (mean age 9.8 years) of the Avon Longitudinal Studies of Parents and Children.3, known as the main contributor of vitamin D status obtained from sunlight, was positively associated with the season of blood sampling and latitude of residence. Interestingly, we observed a heightened vitamin 25(OH)D2 status during the winter months that has yet to be understood. However, we do not have information on supplement use which hinders the ability to assess the increased vitamin 25(OH)D2 status during winter. As suggested by Tolppanen and colleagues, if serum vitamin D2 is largely associated with dietary and some socioeconomic related factors, this may provide an indication of compensatory behaviour which can be adopted to correct the vitamin D status during the low sunlight months.31Public health recommendations and clinical diagnostics do not currently distinguish between vitamin D2 and D3.2 and 25(OH)D3) will help better understand the role of vitamin D in research, clinical and public health implications. The data also supported a differential association of 25(OH)D2 and 25(OH)D3 concentrations with sunlight which might have an impact on future strategy for supplementation. These differential results also question current strategies of vitamin D supplementation and IOM cutoffs for vitamin D sufficiency and warrant a personalised approach, accounting for individual and lifestyle characteristics. The fortification of fluid milk products (0.5 μg/100 g) was introduced in Finland in 2002 with limited efficiency in all age groups.Our results have provided information on the potential determinants associated with the vitamin D status prior to the implementation of a nationwide fortification policy. Understanding the associations between sex, season, latitude and multiple lifestyle factors with dual sources of vitamin D (25(OH)D