CHARACTERIZATION and STATISTICAL ANALYSIS of GENES on HUMAN X-CHROMOSOME Xq22.1

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www.jatit.org CHARACTERIZATION AND STATISTICAL ANALYSIS OF GENES ON HUMAN X-CHROMOSOME Xq22.1

VIDYA NIRANJAN1, RIAZ MAHMOOD1 , PARESH KUMAR SINGH2, KONTHOUJAM DEEPIKA2, RAMESHWAR.G.K2, JAYARAMA REDDY3

St. Joseph’s Post Graduate and Research Centre 1Department of Bioinformatics and Biotechnology, Kuvempu University, Sankarghatta ,India 2Oxford College of Science, Bangalore, India. 3St. Joseph’s Post Graduate and Research Centre E-mail: [email protected]

ABSTRACT:

Human Xq22.1 is a genomic region, which has 20 mammalian rapidly evolving genes and involved in numerous genetic diseases such as XLMR, Fabry disease, pre mature ovarian failure, cancer etc. Many complex diseases may arise from quantitative, rather than qualitative differences in the gene products. Review, characterization and statistical analysis is performed on protein coding genes in human genomic region, Xq22.1. The analysis is based on literature review, molecular weight, molecular class, biological process, gene localization, gene expression, splicing events and mutational study. Role in biological processes, cellular component and molecular class of most of the genes in human genome Xq22.1 is unknown. Tracing the specific genes will help us in understanding the genetics of human disease associated with Xq22.1. Study on splicing events and SNP studies revealed interesting results on APG4A, the gene responsible for autophagy.

1. INTRODUCTION: cytogenetic expression of gene activity (4). The analysis of protein coding genes were done using 20 mammalian rapidly-evolving genes on human a combination of bioinformatics and chromosome Xq22.1 in various protein families experimental verification using HPRD BEX, WEX and GASP were identified(1). (2) (www.hprd.org) on molecular weight, molecular identified a novel human homeobox gene class, molecular function, biological process, ESXR1 with that of mouse Esx1 homeobox localization , expression, isoforms, Proteolytic gene. ESXR1 has been localized to human Cleavage and Single Nucleotide Polymorphisms Xq22.1-q22.3, and their expressions are (SNPs) of 40 genes reported. restricted to placenta and testis. The findings that there are similarities between ESXR1 and Esx1, Review on disease association on Xq22.1 yet differences between their encoded products are consistent with the idea that placental genes Pandita A eta.al gave insight on the pathogenesis evolve rapidly between mammalian species. of gangliogliomas and associated rare malignant (3) discovered a new member of the RAB family progression(5). The gene encoding the human was identified through a public database search, protein is located on chromosome Xq22.1, which revealed that the gene is divided into three involved in premature ovarian failure. Merhi ZO exons and spans approximately 7.2kb of the et.al revealed that premature ovarian failure due genome DNA of chromosome Xq22.1-q22.3 to Xp duplication and Xq deletion(6). Clarke NF region. eta.al described an unreported form of congenital Xq22.1 plays a major role in chromosomal fiber-type disproportion that follows an X-linked fragile site expression. The expression of inheritance pattern and demonstrated linkage to common chromosomal fragile sites on human two regions of the X chromosome, Xp22.13 to chromosomes has been proposed to be a Xp11.4 and Xq13.1 to Xq22.1.

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www.jatit.org the genotype and the phenotype. Studies of Chen CP identified antenatal diagnosis of de additional Fabry families will provide novo partial trisomy Xq (Xq22.1-->qter) and information on the nature and frequency of the terminal Xp deletion accompanying sonographic mutations causing this disease as well as detection of uterine growth restriction (7). A potential insights into the structure/function novel NXF gene cluster on Xq22.1, a good relationships of this lysosomal hydrolase. candidate gene X-linked Mental Retardation (XLMR)(8.9) and can be classified into (Autophagy-related cysteine endopeptidase A syndromal and non syndromal and the (APG4A), Ras related protein Rab-40A observation confirmed the suspicion of a gene (RAB40A), Translocase of inner mitochondrial involved in growth hormone regulation being membrane 8 homolog A (TIMM8A) is located in localized in Xq22.1. Xq22.1. These genes are reported as possibly implicated in cancer.

(Scientist identified that Fabry disease which is a Autophagy is the process by which endogenous lysosomal disease caused as the result of the proteins and damaged organelles are destroyed gradual accumulation of Glycosphingolipid in intracellularly. Autophagy is postulated to be the alpha-galactosidase A enzyme in Xq22- essential for cell homeostatis and cell remodeling 1(10,11,12,13). Eng CM eta.al (14, 15, 16) found during differentiation, metamorphosis, Non- an X-linked inborn error of glycosphingolipid apoptotic cell death and aging. Reduced levels of catabolism revealed that most mutations in the autophagy have been described in some alpha-galactosidase A were private, and that malignant tumors, and a role for autophagy in different substitutions of the same codon resulted controlling the unregulated cell growth linked to in markedly different disease phenotypes. cancer. Hence our detailed analysis is Though several mutations of the alpha accentuated in a 4.2 Mb region of the human X- galactosidase A gene have been spotted but to chromosome, Xq22.1 and in detail on APG4A date no correlation has been established between

RESULTS: STATISTICAL ANALYSIS:

2. MOLECULAR WEIGHT ANALYSIS:

200000MOLECULAR WEIGHT ANALYSIS

150000 T H IG100000 E W Series1 R A 50000 L U C E 0 L O M GENE SYMBOL

Statistical analysis is performed Molecular weight Vs Gene Symbol.

It shows GPRASP1 (G Protein-coupled receptor encode large proteins, A full-length cDNA associated sorting protein 1) has a highest encoding KIAA0443 called GASP. The molecular weight (156845 Da). By screening a predicted protein contains 1,395 amino acids. cDNA library for cDNAs with the potential to RT-PCR analysis revealed ubiquitous expression

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www.jatit.org of KIAA043, with highest levels in brain, and abundantly expressed in heart and skeletal kidney, and ovary. muscle. SMPX (Small muscular protein) has a lowest molecular weight protein-encoding gene in 3. MOLECULAR CLASS: Xq22.1 (9560 Da). The gene consists of five exons (> or =172, 57, 84, 148, > or =422 bp) and These following molecular classes are with four introns (3639, 10410, 6052, 31134 bp) percentage identified in Xq22.1 are discussed comprising together 52.1 kb and is preferentially below.

Molecular Class

Transcription factor Cysteine Protease Transcription Regulatory Protein 0% Ribosomal subunit RNA binding protein Adaptor molecule Enzyme : methyltransferase GTPase Integral membrane protein Transport/Cargo protein Enzyme:Translocase Unknown 7% 7% 38% 12%

2% 10%

8% 5% 3% 3% 3% 2%

In Xq22.1, molecular classes of most of the NXF2) and 8% of the molecular class identified genes are unknown (37%). Now at present, as GTPase (RAB9B, RAB40A). Transcription transcription regulatory protein (TCEAL1, factor (TAF7L, ESX1L) and cysteine protease TCEAL2) is a major molecular class (12%) (APG4A) are the molecular class identified as identified in Xq22.1. Pillutla RC eta.al1999 (17) 7%. Transport / Cargo protein (GPRASP1, revealed that TCEAL1 is the gene which SYTL4) is one of the molecular classes as 5%. consists of three exons and two introns that Ribosomal subunit (RPL36A), Adaptor molecule codes for p21/SIIR and their expression was (NGFRAP1), Enzyme: Methyltransferase lowest in hematopoietic cells of both normal and (CXorf34), Integral membrane protein tumor origin. It was mapped to human (TMEM35), Enzyme: Translocase, these are the chromosome Xq22.1 by fluorescence in situ molecular classes’ shows similar percentage hybridization. 9% of the molecular class (3%) in human genome Xq22.1. identified as RNA binding protein (CSTF2,

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4. GENE LOCALISATION:

GENE LOCALISATION ANALYSIS

Series1

30% 18% 45% 3% 3% 3%

In Xq22.1, most of the gene localization is are localized in mitochondria (3%).3% genes unknown (37%). 29% genes are localized in (SYTL4) are localized in secretory granule. In nucleus (TCEAL1, ARMCX5, TCEAL2, some genes (TAF7L) primary localization is CSTF2, ESX1L, NXF2 and SMPX). 18% genes nucleus and alternative localization is cytoplasm are localized in cytoplasm (APG4A, RPL36A, (3%). NGFRAP1 and MID2). Some genes (TIMM8A)

5. BIOLOGICAL PROCESS:

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www.jatit.org BIOLOGICAL PROCESS ANALYSIS

3% 3% Nucleic acid metabolism

5% Unknown 10% 32% Protein metabolism

10% Signal transduction

Transport

Metabolic Pathways 37% Cell growth factor maintenance

Most of the biological process in human genome biological processes observed in genome Xq22.1 Xq22.1 about 37% is unknown. 32% of the as 10%. 5% of biological process observed as biological process is nucleic acid metabolism transport (GPRASP1, SYTL4). Metabolic (TAF7L, TCEAL1, TCEAL2, CSTF2, CXorf34, pathways (TIMM8A) and cell growth factor ESX1L and NXF2). Protein metabolism maintenance (MID2) are the biological processes (APG4A, RPL36A) and Signal transduction observed as least (3%) in Xq22.1. (NGFRAP1, RAB9B and RAB40A) are the

6. GENE EXPRESSION:

In gene expression analysis maximum gene (TAF7L, FSHTRH1, APG4A, TCEAL3, expression about 30% is observed in Testis NGFRAP1 and ESX1L).

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www.jatit.org Next to that, about 9% genes are expressed in includes Lung (FSHTRH1, TCEAL1), Placenta Heart (APG4A, TCEAL1, TIMM8A and SMPX) (TCEAL1, ESX1L), Liver (TCEAL1, TIMM8A) and Prostate (APG4A, TCEAL1 and MID2). 8% and Kidney (TCEAL1, TIMM8A). In Fetus gene expression is observed in Spleen (APG4A, MID2), Colon (TCEAL1) and Thymus (FSHPRH1, APG4A and TCEAL1) and skeletal (TCEAL1) the gene expression is about 4%. muscle (APG4A, TCEAL1, TIMM8A and About 2% genes are expressed in the Ubiquitous SMPX). Brain (FSHPRH1, TCEAL1, TIMM8A (RAB9B). Finally the least expression of gene and SMPX) and Ovary (FSHPRH1, TCEAL1, i.e. about 1% is observed in B cell (CSTF2) and NGFRAP1 and MID2) shows similar gene small intestine (MID2). expression about 7%. 5% gene expression is observed in most parts of the body. The list

7. ISOFORM ANALYSIS IN Xq22.1:

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DIFFERENT FORMS OF GENE IN Xq22.1

80.00%

60.00% Series1

40.00% Others, 68% 20.00% Transcript variant, Isoform, 8%25% 0.00%

Statistical analysis of the genes present on protein (APG4A). APG4A is has a three Xq22.1 indicated that 8% isoform genes were isoforms denoted as Isoform a, Isoform b and present in this region. This percentage included Isoform C. an Autophagy-related cysteine endopeptidase-2

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www.jatit.org 8. GENETIC MAPPING by CLUSTALW Results:

9. PROTEOLYTIC CLEAVAGE:

We now turn to a different mechanism of precursors that are subsequently activated by enzyme regulation. Many enzymes acquire full cleavage of one or a few specific peptide bonds. enzymatic activity as they spontaneously fold The inactive precursor is called a zymogen (or a into their characteristic 3D forms. In contrast, proenzyme). An energy source (ATP) is not other enzymes are synthesized as inactive needed for cleavage. Therefore, in contrast to the

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www.jatit.org reversible regulation by phosphorylation, even a cell proliferation, differentiation and migration, protein located outside cells can be activated by morphogenesis and tissue remodeling, and this means. angiogenesis and apoptosis. An additional Proteolytic enzymes through their process in which proteolytic enzymes have also ability to catalyze irreversible hydrolytic been recently implicated is autophagy. reactions play crucial roles in the development and maintenance of all living organisms. Proteolytic cleavage in APG4A: Proteases were initially characterized as Autophagy is a biological process involved in nonspecific degradative enzymes associated with the intracellular destruction of endogenous protein catabolism, but recent studies have protein and the removal of damaged organelles demonstrated that they influence a wide range of and has been suggested to be essential for cell cellular functions by processing multiple homeostasis as well as for cell remodeling bioactive molecules. These essential processes during differentiation, metamorphosis, non- initiated, regulated, or terminated by proteases apoptotic cell death, and ageing. include DNA replication, cell cycle progression,

Through analysis, it was shown that in Xq22.1 variations that occur when a single nucleotide genome, 8% of APG4A isoform genes were (A, T, C, or G) in the genome sequence is formed by proteolytic cleavage. This was caused altered. For example a SNP might change the by a proteolytic enzyme called HsApg4A. DNA sequence AAGGCTAA to ATGGCTAA. Therefore, 8% proteolytic cleavage occurs in the For a variation to be considered an SNP, it must Xq22.1 of X-chromosome genome. This was occur in at least 1% of the population. SNPs, also confirmed by the statistical studies of which make up about 90% of all human genetic proteolytic cleavage in Xq22.1. GATE-16 variation occur every 100 to 300 bases along the (Golgi-associated ATPase enhancer of 60kDa) is 3-billion-base human genome. Two of every an essential component of intra- Golgi transport three SNPs involve the replacement of cytosine and post-mitotic Golgi reassembly. The COOH (C) by thymine (T). SNPs can occur in both terminus of GATE-16 undergoes post- coding (gene) and noncoding regions of the translational cleavage by a human cysteine genome. Many SNPs have no effect on cell protease HsApg4A, which exposes the glycine function, but scientists believe others could 116 residues. predispose people to disease or influence their response to a drug. Although more than 99% of Single nucleotide polymorphisms (SNPs): human DNA sequences are the same across the Single nucleotide polymorphisms or SNPs population, variations in DNA sequence can (pronounced "snips") are DNA sequence have a major impact on how humans respond to

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www.jatit.org a disease; environmental insults such as bacteria, endopeptidase 2 or APG4A in Xq22.1 is a good viruses, toxins, and chemicals; and drugs and example of how SNPs affect disease other therapies. This makes SNPs of great value development. This gene contains 65 SNPs .A for biomedical research and for developing change of one amino acid in the APG4A protein pharmaceutical products or medical diagnostics. alters its structure and function enough to make SNPs are also evolutionarily stable --not disease development more likely. SNPs are not changing much from generation to generation -- absolute indicators of disease development. making them easier to follow in population studies. Scientists believe SNP maps will help SNPs Databases: them identify the multiple genes associated with The single nucleotide polymorphism database such complex diseases as cancer, diabetes, (dbSNP) has been designed to support vascular disease, and some forms of mental submissions and research into a broad range of illness. biological problems. These include physical mapping, functional analysis and SNPs do not cause disease, but they can help pharmacogenomics, association studies and determine the likelihood that someone might evolutionary studies. develop a particular disease. One of the genes associated with Autophagy-related cysteine

In Xq22.1, it was discovered that APG4A had insertion polymorphisms (or) DIPs) and much more single nucleotide polymorphisms retroposable element insertion and microsatellite (SNPs) than any other gene. This collection of repeat variations (also called short tandem polymorphisms includes single-base nucleotide repeats (or) STRs). substitutions (also known as single nucleotide polymorphism (or) SNPs), small-scale multi- . base deletion or insertion (also called deletion

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C/T APG4A GENE SNPs OBSERVED DATA G/T ANALYSIS A/G

3% 2% A/C

‐/T 7% 23% ‐/ATGTGTGC 2% 7% 2% ATGTGTGC 1% ‐/GT

8% C/G

20% A/T

‐/TTATT 25% ‐/G

Analysis of the nature of SNPs in APG4A 10 FUNCTIONAL ANALYSIS: revealed that more than 20% of the single nucleotide polymorphisms were caused by the Variations that occur in functional regions of three different alleles C/T, G/T and A/G. But all genes or in conserved non-coding regions can other alleles were less than 8% and some of the cause significant changes in the complement of APG4A alleles were insertion deletion transcribed sequences, leading to changes in polymorphisms, deletions represented by ‘-‘ in protein expression that can affect aspects of the allele string (in del) type. phenotype such as metabolism or cell signaling. We noted possible functional implications of DNA sequence variations in dbSNP in terms of how variation could alter mRNA transcripts.

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locus‐ coding‐ region,mrna‐ FUNCTION ANALYSIS synon,intron utr,intron 2% 5%

mRNA‐ intron UTR,intron 41% 52%

In APG4A gene SNPs’, a variant may have more 11 VALIDATION ANALYSIS: than one functional role. It was indicated in the SNPs assays validated directly by the statistical analysis that 52% of functional regions submitter through the validation section showed had variants in transcripts, but not in the coding the type of evidence used to confirm the region intervals. 41% of functional regions variation. showed variation in introns, but not in the first two or last two bases of introns. VALIDATION STATUS ANALYSIS

Series1 40%

15.38% 9.23% 7.69% 8% 4.62% 6.15% 3.08% 1.54% 3.08% 1.54%

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Validation status of APG4A gene variant may be 9% by genotype and 8% by frequency and by found by more than one method. 15% validation cluster. Maximum validation status in APG4A status of APG4A was found by-2hit-2allele. All was found to be unknown (or) no validation alleles have been observed in 2+ chromosomes could be reported for this ref SNP.

12. DISCUSSION: 13. CONCLUSION: The genome Xq22.1 contains 46 genes in that 40 genes are protein coding genes. The statistical The statistical analysis confirmed the analysis is performed on Protein coding genes in characterization of each protein coding gene human X-Chromosome, Xq22.1 based on present in human genome Xq22.1. Analysis molecular weight, molecular class, biological gives us insight identification and process, gene localization and gene expression. characterization of proteins in Xq22.1 will throw Molecular analysis shows GPRASP1 has a light on origin of various genetic diseases. Basic highest molecular weight (156845 Da) and research on Autophagy and SMPX has a lowest molecular weight (9560 Da). APG4A may reveal probable drug target for APG4A In genome Xq22.1, the molecular classes of most of the genes are unknown. Transcription 14. Acknowledgment: We acknowledge Kalai regulatory protein is one of the major molecular arasi and Prem kumar for their valuable help in classes observed in this genome segment. The data mining. statistical analysis of gene localization shows most of the gene localization is unknown. Many protein coding genes are localized in Nucleus and Cytoplasm. Some genes are localized in Mitochondria and Secretary Granule. Most of the biological process in the genome is unknown. Nucleic acid metabolism is played a major role in Xq22.1. Protein metabolism and Signal transduction also observed in this genome. The majority of the gene expression is observed in Testis. The protein coding genes are also expressed in Heart, Prostate, Spleen, Skeletal muscle, Brain and Ovary. The least gene expression is observed in B cell and Small intestine.

Phosphorylation is the only Post translational modification observed in genome Xq22.1. In 40 protein coding genes, SYTL4 is an only one plasma protein.

The statistical analysis reported localization, biological process and gene expression of the protein coding genes in the human genome Xq22.1. Isoform and SNP analysis of APG4A mutation at functional level can be a breakthrough in autophagy discovery.

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