Hi there,So basically, I need help with the 'results' section of the article. And I am doing...

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Hi there,So basically, I need help with the 'results' section of the article. And I am doing material and methods, results (3.1 and 3.2) from the article. The questions that need to be answered are;• Describe (communicate your understanding of) methods used in figures• What do individual figures show?• Include enough of methods so data can be understood• Why use a particular technique, its limitations?• Report major finding of a figure/table but don’t discuss implicationsDisease‐causing mutations in the promoter and enhancer of the ornithine transcarbamylase gene Received: 15 September 2017 Revised: 19 December 2017 Accepted: 21 December 2017 DOI: 10.1002/humu.23394 R E S E A RCH ART I C L E Disease-causingmutations in the promoter and enhancer of the ornithine transcarbamylase gene Yoon J. Jang1 Abigail L. LaBella2 Timothy P. Feeney3 Nancy Braverman4 Mendel Tuchman1 HirokiMorizono1 Nicholas AhMew5 Ljubica Caldovic1 1Center forGeneticMedicine Research, Chil- dren'sNational Health System,Washington, District of Columbia 2Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 3Harvard T.H. Chan School of PublicHealth, HarvardUniversity, Cambridge,Massachusetts 4McGill UniversityHealthCentre,McGill Uni- versity,Montreal, Quebec, Canada 5Center for Translational Sciences, Children's National Health System,Washington, District of Columbia Correspondence Dr. LjubicaCaldovic,Center forGenetic MedicineResearch,Children'sNationalMed- icalCenter, 111MichiganAveNW,Washington DC,20010. Email: lcaldovic@childrensnational.org Funding information MarylandHHMIUndergraduateResearch Fellowship;RashidFamilyFund Contract grant sponsor:National Institute ofDiabetesDigestive andKidneyDiseases (R01DK047870,R01DK064913). Communicatedby JohannesZschocke Abstract The ornithine transcarbamylase (OTC) gene is on the X chromosome and its product catalyzes the formation of citrulline from ornithine and carbamylphosphate in the urea cycle. About 10%– 15% of patients, clinically diagnosed with OTC deficiency (OTCD), lack identifiable mutations in the coding region or splice junctions of the OTC gene on routine molecular testing. We collected DNA from such patients via retrospective review and by prospective enrollment. In nine of 38 subjects (24%), we identified a sequence variant in theOTC regulatory regions. Eight subjects had unique sequence variants in theOTCpromoter andone subject had a novel sequence variant in the OTC enhancer. All sequence variants affect positions that are highly conserved inmammalianOTC genes. Functional studies revealed reduced reporter gene expression with all sequence variants. Two sequence variants caused decreased binding of the HNF4 transcription factor to its mutated binding site. Bioinformatic analyses combined with functional assays can be used to identify and authenticate pathogenic sequence variants in regulatory regions of the OTC gene, in other urea cycle disorders or other inborn errors of metabolism. K EYWORDS enhancer mutation, gene expression gene regulation, hyperammonemia, ornithine transcarbamy- lase, ornithine transcarbamylase deficiency, promoter mutation, urea cycle 1 INTRODUCTION The urea cycle functions in the liver where it converts ammonia, a neu- rotoxic product of protein catabolism, into urea, which is excreted in urine (Brusilow & Horwich, 2001). Ornithine transcarbamylase (OTC; EC 2.1.3.3, MIM# 300461) is a mitochondrial enzyme that catalyzes the second reaction of the urea cycle: the formation of L-citrulline from L-ornithine and carbamylphosphate. The human OTC gene, located on the short arm of the X chromosome (Xp11.4), is 70 kb long and has 10 exons that contain a 1,062 bp coding sequence (Hata et al., 1986). This gene codes for a protein comprised of 354 amino acids, includ- ing a 32 amino acid mitochondrial targeting peptide at its N-terminus (Horwich et al., 1984; Horwich, Kalousek, Fenton, Pollock, & Rosen- berg, 1986). Primary OTC deficiency (OTCD;MIM# 311250) is caused by mutations in the OTC gene that lead to either reduced or absent functional OTC enzyme, thus limiting ammonia flux through the urea cycle. This results in the accumulation of blood ammonia, which may manifest as lethargy, vomiting, behavioral and neurological abnormal- ities, and, in severe cases, coma and death (Breningstall, 1986). In addition to high plasma ammonia, biochemical abnormalities associ- ated with OTCD include elevated plasma glutamine, low or absent cit- rulline, and increased excretion of orotic acid and orotidine in the urine (Brusilow&Horwich, 2001). Because the human OTC gene is located on the X-chromosome, severe OTCD primarily affects hemizygous males and accounts for approximately one-half of all urea cycle disorders (Lindgren, de Mar- tinville, Horwich, Rosenberg, & Francke, 1984). Male hemizygotes with mutations that abrogate or severely impair OTC function invari- ably exhibit hyperammonemia within the first week of life (Ah Mew et al., 2013; McCullough et al., 2000). Males with hypomorphic OTC alleles that retain residual enzyme activity, or female heterozygotes with skewed lyonization (Caldovic, Abdikarim, Narain, Tuchman, & Morizono, 2015; McCullough et al., 2000) typically present symp- tomatically after the first week of life (Caldovic et al., 2015; Numata HumanMutation. 2018;39:527–536. c© 2017Wiley Periodicals, Inc. 527wileyonlinelibrary.com/journal/humu http://orcid.org/0000-0002-9140-5585 528 JANG ET AL. et al., 2010). The true prevalence of OTCD is unknown, but it has been estimated to be between one in 14,000 and one in 76,000 (Balasubramaniam et al., 2010; Brusilow & Maestri, 1996; Dionisi- Vici et al., 2002; Nettesheim et al., 2017; Summar et al., 2013). In 85%–90% of patients with the biochemical phenotype of OTCD, a mutation can be identified through commercially available sequencing or deletion/duplication testing. In the remaining 10%–15% of affected individuals, a molecular cause of OTCD cannot be ascertained through clinical testing. In a few such instances, mutations were ultimately identified in intronic or regulatory regions (Caldovic, Abdikarim, Narain, Tuchman, & Morizono, 2015; Engel et al., 2008; Luksan et al., 2010). Mutations in these regions may, in fact, be responsible for a large proportion of OTCD, in patients with no identified variants via current clinical methods. However, sequencing of “deep” intronic or regulatory regions is currently neither routine nor clinically available. OTC is expressed in the liver and intestine of humans and other mammals (Brusilow&Horwich, 2001). Transcription of the humanOTC gene appears to initiate at multiple transcription start sites (Luksan et al., 2010),whereas transcriptionof themouseand ratOtcgenes initi- ate 136 and 98 bp upstreamof the translation initiation codon, respec- tively (Takiguchi, Murakami, Miura, & Mori, 1987; Veres, Craigen, & Caskey, 1986). In the rat Otc promoter, four regions, A–D, bind tran- scription factors that regulate expression of the Otc gene (Figure 1; (Murakami, Nishiyori, Takiguchi, & Mori, 1990)). The rat Otc promoter was sufficient to direct expression of transgenes in the liver and intes- tine of transgenic mice, but expression was higher in the intestine than in the liver (Jones et al., 1990; Murakami, Takiguchi, Inomoto, Yamamura, & Mori, 1989). Expression studies in cultured cells and transgenic animals revealed that an enhancer, located approximately 11 kb upstream of the first exon of the rat Otc gene, is essential for a high level of expression of the Otc gene in the liver (Murakami et al., 1990). In vitro binding studies revealed four sites, designated I–IV, that are important for the function of the rat −11 kb enhancer (Figure 2 (Murakami et al., 1990)). In this study, we screened conserved upstream regulatory regions of the OTC gene in 38 subjects with clinically diagnosed OTCD, but in whom no deleteriousOTCmutation was identified by clinical sequenc- ing of the coding regionor splice junctions. Eight of these subjectswere found to harbor one of six unique sequence variants in the OTC pro- moter. One subject had a novel sequence variant in the OTC enhancer. Five sequence variants were within either known or predicted tran- scription factor binding sites and the remaining two affected base pairs that are highly conserved in vertebrates. Six sequence variants were not found in any of the databases of single nucleotide polymor- phisms. Functional testing confirmed that all seven sequence variants described herein cause either reduced expression of reporter gene in cultured cells or reduced binding of the hepatic nuclear factor 4 (HNF4) transcription factor. These results highlight the importance of seeking sequence variants in regulatory regions of genes of patients with OTCD without identifiable mutations as well as the need for bet- ter and simpler functional testing of regulatory mutations in disease- causing genes. 2 MATERIALS AND METHODS 2.1 Study subjects This project, which included retrospective and prospective compo- nents, was conducted with the approval of the Institutional Review Board of the Children's National Health System. Inclusion criteria were: suspected urea cycle disorder with reduced or absent OTC enzymatic activity in the liver biopsy and/or urinary orotate above 30 ?mol/mmol of urinary creatinine, but absence of a disease-causing mutation in the exons and intron/exon boundaries of the OTC gene. When available, gender, age of disease onset, enzymatic activity of CPS1 in the liver biopsy, and concentrations of ammonia and citrulline in the plasma were collected for eligible patients. All participants were probands. Participants and their families were also prospectively enrolled into the study after referral by their metabolic physicians. In the retrospective study component, participants were selected from a database of de-identified patients who were screened for mutations in the coding region and intron/exon junctions of the OTC gene at the Children's National Health SystemMolecular Genetics Laboratory. Of 1,035 patients in the database, 106 were eligible for this study, 43 were diagnosed with OTCD based on reduced or absent OTC activity in the liver, and the remaining 63 had a documented history of hyper- ammonemia and elevated urinary orotate. DNAwas available from 24 subjects. Subjects 1–4 were ascertained retrospectively and subjects 5–9were participants in the prospective study. 2.2 Subjects with sequence changes in the regulatory regions of theOTC gene Subject 1 was a male diagnosed with late-onset OTCD based on OTC activity of 6.5 ?mol min−1 g−1, which was approximately one-tenth of the control value (56.7 ?molmin−1 g−1 of liver tissue). TheCPS1 activ- ity in the same liver sample was normal (2.9 ?mol min−1 g−1). Values of urinary orotate, plasma ammonia, glutamine, and citrullinewere not available. Subject 2was amale diagnosedwith late onsetOTCDbased onOTC activity of 10.3 ?mol min−1 g−1, which was approximately one-tenth of the control value (110.6 ?mol min−1 g−1 of liver tissue). The CPS1 activity in the same liver biopsy was normal (6.1 ?mol min−1 g−1). His urinary orotate concentration was 77 ?mol/mmol creatinine (normal

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DISEASE-CAUSING MUTATIONS IN THE PROMOTOR AND ENHANCER OF THE ORNITHINE TRANSCARBAMYLASE GENE
Describe (communicate your understanding of) methods used in figures.
The authors used bioinformatic instruments to recognize locales of preservation inside the 5′ - flanking groupings of 8 mammalian ornithine transcarbamylase (OTC) genes thinking that arrangement variations in the relating districts of the human OTC gene are probably going to influence its demeanor. Bioinformatics is a subdiscipline of science and software engineering dealing with the obtaining, stockpiling, examination, and dispersal of natural information, regularly DNA and amino acid arrangements. Pair-wise correlations distinguished 3 saved areas upstream of the main exon of OTC. The primary area is roughly 600 bp long and is found promptly upstream of the starting ATG codon. This region includes the promotor of OTC and the 5′ - untranslated locales of the OTC mRNA. The subsequent area, the human OTC enhancer, is roughly 900 bp long and compares to the −11 kb enhancer in the rodent OTC gene.
What do individual figures show?
Figure 1 shows the promotor regions present in the human OTC gene and the figure 2 represents the liver-specific enhancer (LSE) that are present in the human OTC gene. These figures again are divided into section A and B which illustrates the gene sequence and LOGO representations respectively for each of them.

Disease‐causing mutations in the promoter and enhancer of the ornithine transcarbamylase gene Received: 15 September 2017 Revised: 19 December 2017 Accepted: 21 December 2017 DOI:...

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