HUMAN GENOME DATABASES

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Background

Transmembrane protein (TMEM132D) has an alternative symbol of KIAA1944 and an alternative name of Mature OligodendrocyteTransmembrane Protein (MOLT). Its approved gene symbol is TMEM132D with cytogenetic location of 12q24.33 to chromosome 12q24.3. It is moderately expressed in adult and fetal brain only with highest expression in caudate neucleus and lowest expression in amygdala, corpus callosum, hippocampus, substantianigra, subthalamicneucleus and thalamus. As expressed by Nomoto et al (2003) in OMIM webpage, transmembrane protein was found to have molecular mass of about 130kD and N-terminal hydrophobic signal peptide, 7 predicted N-glycosylation sites, 2 predicted O-glycosylation sites, phosphorylation sites and C-terminal transmembrane domain. It shares in rat homologs 83.4% and 83.2% amino acid identities in mouse homologs. The TMEM132D gene contains 9exons spanning 850kb.

TMEM132D is a polytopicprotein that aggregate and aggregate and precipitate in water. They can be extracted by use of non-polar solvents or detergents or better denaturing agents for some. Transmembrane protein spans from one side of a membrane to the other side where it is permanently attached by special membrane lipids known as annular lipid shells. They are capable of permitting or denying transport of specific substances across the biological membrane getting in and out of the cells by a folding up or bending technique.To explore the effects of TMEM132D gene, it is important to trace its associations and traits due to its fundamental roles. Mala cards website revealed 5 hits associated with this gene which includes leukemia, panic disorder, asthma and obesity. NCBI and Ensembl were used to gather information about the gene such as location, mutations and genetic disorders associated with it.

Chromosomal Map Position of TMEM132D

The chromosomal map position of Transmembrane Protein (TMEM132D) from NCBI website can be accessed through selecting option “Genomes and Maps” followed by “Tools” then “Map Viewer” options. Alternatively, from the web interface, straight away select option “Tools” then “Map Viewer”. On the “Map Viewer” interface, on search field on the top left corner, select “Homo Sapiens” option then search for the gene code by entering on the box labeled “for”. An interface with chromosomes will then appear. At the filter on top, “Search for” TMEM132D and on the “Assembly” field select Primary Assembly to establish the chromosome the Transmembrane Protein is located. The search highlights chromosome 12 with a red line with 33 hits. Select the highlighted chromosome (12). “Map Viewer” interface opens and below appears the map summary. (NCBI)

On the Ensemble browser, search for TMEM132D is entered in the search engine. Restrict the search to “human” and “gene” this can be done by directly selecting “TMEM132D (Human Gene). And the summary is as follows:

Summary

Name:TMEM132D (HGNC Symbol)

CCDS:This gene is a member of the Human CCDS set: CCDS9266

UniprotKB:This gene has proteins that correspond to the following Uniprot identifiers: Q14C87

RefSeq:Overlapping RefSeq Gene ID 121256 matches and has similar biotype of protein coding. (4)

Ensembl version:ENSG00000151952.11

GRCh37 assembly:This gene maps to 129,556,270-130,388,211 in GRCh37 coordinates.View this locus in the GRCh37 archive: ENSG00000151952

Gene type:Known protein coding

Prediction Method:Annotation for this gene includes both automatic annotation from Ensembland Havana manual curation.(4)

Alternative genes:This gene corresponds to the following database identifiers:Havana gene: OTTHUMG00000168400

These browsers evidently have different ways of displaying information on chromosomal map position with different characteristics. Though the information displayed is quite similar, there are notable differences. The NCBI browser display information about the gene in terms mapping and positioning as compared to Ensembl browser that gives information on transcript variants. NCBI is capable of displaying the variants but through engaging other procedures. Getting approximate size of the gene can be done easily through NCBI website and getting the transcript variants is easier through Ensembl browser which organizes the information in more specific and organized way.It is more possible to get useful links when using Ensembl through the selection of every transcript variant independently which brings expanded information that leads to anothe. All of the browsers are integrated to each other in gathering information about the genes. Moreover, the browsers are characterised by their different way in gathering information for example, to find out the approximate size of the gene can be easy found from NCBI easily compared with Ensembl, though they are integrated to each other in gathering information about the gene.

2. Exon / intron structure of TMEM132D gene.

Exon and intron structure of TMEM132D gene from NCBI website is found through selecting Gene box followed by filter button to restrict the search to the gene TMEM132D, which brings hits. After that the first option with gene ID 121256 is selected.Genomic regions, transcripts, and products are selected and then GenBank that reveals the displayed region of the chromosome. From NCBI Reference Sequences “mRNA and proteins” is chosen and that shows the transcript variants of the gene. From the transcript it is possible to get the following:Location: 12q24.33 Exon count: 9 Intron : 8

In Ensembl browser, same procedure of locating chromosomal mapping position is followed. Of the three revealed transcript variants, the first one which is the longestis selected and the following is obtained.

StatisticsExons: 9 Coding exons: 9 Transcript length: 5,776 bps Translation length: 1,099 residues

Comparing between Ensembl and NCBI gene browsers each of them is characterised and specified in the way of searching and displaying information. NCBI browser provides less information with regards to exon and intron structure as compared to Enseml browser. The arrangement of the exon and intron structures is different between both browsers.Ensembl browser gives more specific and independent and gives more access to information. There is also integration between Ensembl and NCBI web browser so that they are complementary to each other’s in handling information.

3. The Rare homozygous SNPs for the patient.

NCBI and Ensembl browsers are used to investigate the rare homozygous Single Nucleotide Polymorphism for the patient. NCBIbrowser straight forward shows the location of thehits of the rarehomozygous, which are allocated in the intron region as follows

 rs12819591is located in the intron region of NM_133448.2.

rs7960253 is located in the intron region of NM_133448.2.

The gene SNPs were identified by clicking on the variation table under “Genetic variations” in the Gene based display on the left column in the Map viewer page. An output table showing the short nucleotide polymorphism of the gene was generated.

Ensembl browser is able to provide more information represented as follows

For rs12819591 SNP Original sourceVariants (including SNPs and indels) AllelesC/T | Ancestral: T | Ambiguity code: Y | MAF: 0.10 (C)LocationChromosome 12:129535078 (forward strand) 

For rs7960253Original sourceVariants (including SNPs and indels) AllelesC/T | Ancestral: C | Ambiguity code: Y | MAF: 0.05 (T)LocationChromosome 12:129597445 (forward strand)

The SNPs are located in the intron regions of the gene therefor they don’t have a major effect in the gene protein products. They are not changing the coding region for the gene and have no potential effect of the final product of the gene. Ensembl and NCBI as browsers differently display the number of the SNPs in the rare homozygous. NCBI browser is specific on the number of SNPs in the coding region and the whole gene, whileEnsembl page configuration which dictates the size of the region displayed which affectthe number of the SNPs. Ensembl browser displays the SNPs in organised format with their source.

4. Single nucleotide polymorphisms (SNPs) within the gene

A search run through the NCBI website obtained the SNPs within the gene. The information was filtered for human gene.

NCBI browser display the information straight forward, and the number of the SNPs can be seen in both coding region and whole gene area. There are different numbers of variations for each rare homozygosis and also the number of the assay chips is different in the browsers used.

5. PCR primers for the rare homozygous SNPs using a primer design program

The designing of PCR is different in both of NCBI and Ensembl browsers. The principle of designing of the primers is mainly based on copying the area of the sequence round the SNPs( 300-600 bases in long )  and pasted in the primer. PCR designing performed for the three rare homozygous using both NCBI and Ensembl browsers, showed differences between NCBI and Ensembl with regards to the PCR designing, in NCBI browser the designing result shows left primer, right primer, the product size and statistics.On the other hand, when using Ensembl browser SNPs highlighted in red letter as it was visualized through the flanking sequence. It is possible to configure the page that gives an option for expanding or limiting the displayed regions of the SNPs. Ensembl browser is different from NCBI browser in the page configuration option. The result of designing the PCR from the surrounding sequence in Ensembl browser depends on the size of selected region according to the page configuration.

From NCBI web browser the surrounded sequence of rs12819591rare homozygous is obtained. The sequence was copied and pasted in the primer3. Appendix 1 shows the designresult.

From NCBI web browser the surrounded sequence of rs7960253 rare homozygous is obtained. The sequence was copied and pasted to primer3. Appendix 2 shows the design result.

Reference.

1. "OMIM http://www.omim.org/120252 10 Sep. 2014..
2. Mala Cards The Human Malady Compendium 2013.
3. Committee HHGN. Gene Symbol Report 2013 [31/07/2013]. Available from: http://www.genenames.org/data/hgnc_data.php?hgnc_id=221510 Sep. 2014.
4. Enbeslhttp://asia.ensembl.org/index.html
5. NCBI http://www.ncbi.nlm.nih.gov/

Appendix 1

3-     No mispriming library specified

4-     Using 1-based sequence positions

5-     WARNING: Unrecognized base in input sequence

6-      

7-     OLIGO            startlentmgc%  any  3'seq

8-     LEFT PRIMER        208   21   59.25   42.86  7.00  3.00 CAGGCCCTGAAGTGTTAAAAA

9-     RIGHT PRIMER       368   20   60.18   45.00  3.00  0.00 TTTGCTTCTGGGGAATCTTG

10-  SEQUENCE SIZE: 601

11-  INCLUDED REGION SIZE: 601

12-   

13-  PRODUCT SIZE: 161, PAIR ANY COMPL: 6.00, PAIR 3' COMPL: 1.00

14-  TARGETS (start, len)*: 301,1

15-   

16-      1 TTTTGTTTCCAAATATTTTCTATCCACAGTTGGTTGGATCCACAGACACAGAACCCACAG

17-   

18-   

19-     61 ACACAGAGGGCTGACTGTATACAATCAAGATAGGAGAGGACATTTTTGCTGATCTCAGTG

20-   

21-   

22-    121 GGAGAGGTACTTAAACAAACAACAGTGATATACTCCTGTTTACTTTATTTGTACACATTG

23-   

24-   

25-    181 GTAACCAGAATGTCATAAATTATTCTTCAGGCCCTGAAGTGTTAAAAAAGTATTTATAAA

26-  >>>>>>>>>>>>>>>>>>>>> 

27-   

28-    241 TAAAATAAAAACAACTTTCCTATAATGAACACCCCATACTTACATCAAACAGAGCTGTAG

29-   

30-   

31-    301 YGCTTACCAGCATGGTAAGTAAAATGATTATAAAGGTAAGTGTTCCATCAAGATTCCCCA

32-        *                                               <<<<<<<<<<<<

33-   

34-    361 GAAGCAAATAACATTCACGGAGTACAAGTAGGACAGTCCCCTGAGACAGTAATATTGTCT

35-  <<<<<<<< 

36-   

37-    421 CTGAATCCATCATATAAGCATGTTCCCTCAGGCAGTCCAAGGACTAAAATATCATTCCCC

38-   

39-   

40-    481 TAAGTGAAACATGAAAGAAATCCCTTTAAAAACAAAAATGTATAGTCAGGGAACCAAAAC

41-   

42-   

43-    541 ATTGTATTCACACTCTCACCTGCGTTTCAACCATAACATAGCATTTAGAAAGTGTAGGTA

44-   

45-   

46-    601 C

47-   

48-   

49-  KEYS (in order of precedence):

50-  ****** target

51-  >>>>>> left primer

52-  <<<<<< right primer

53-   

54-  ADDITIONAL OLIGOS

55-  startlentmgc%  any  3'seq

56-   

57-   1 LEFT PRIMER        208   21   59.25   42.86  7.00  3.00 CAGGCCCTGAAGTGTTAAAAA

58-     RIGHT PRIMER       365   20   59.63   50.00  4.00  2.00 GCTTCTGGGGAATCTTGATG

59-     PRODUCT SIZE: 158, PAIR ANY COMPL: 6.00, PAIR 3' COMPL: 0.00

60-   

61-   2 LEFT PRIMER        208   21   59.25   42.86  7.00  3.00 CAGGCCCTGAAGTGTTAAAAA

62-     RIGHT PRIMER       362   20   60.39   45.00  4.00  0.00 TCTGGGGAATCTTGATGGAA

63-     PRODUCT SIZE: 155, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 0.00

64-   

65-   3 LEFT PRIMER        208   21   59.25   42.86  7.00  3.00 CAGGCCCTGAAGTGTTAAAAA

66-     RIGHT PRIMER       363   20   60.39   45.00  4.00  0.00 TTCTGGGGAATCTTGATGGA

67-     PRODUCT SIZE: 156, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 0.00

68-   

69-   4 LEFT PRIMER        208   21   59.25   42.86  7.00  3.00 CAGGCCCTGAAGTGTTAAAAA

70-     RIGHT PRIMER       364   20   59.48   50.00  4.00  0.00 CTTCTGGGGAATCTTGATGG

71-     PRODUCT SIZE: 157, PAIR ANY COMPL: 6.00, PAIR 3' COMPL: 0.00

72-   

73-  Statistics

74-           con   too    in    in          no    tm    tm  high  highhigh

75-  sid  many   tar  excl   bad    GC   too   too   any    3'  poly   end      

76-  ered    Ns   get   reg   GC% clamp   low  high complcompl     X  stab    ok

77-  Left    2599     0     0     0   302     0  1376   284    48     8    11    19   551

78-  Right   2632     0     0     0    73     0  1441   331     0     1     0    30   756

79-  Pair Stats:

80-  considered 2459, unacceptable product size 2449, ok 10

81-  primer3 release 1.1.4

82-   

83-   

84-  (primer3_results.cgi release 0.4.0)

Appendix 2

No mispriming library specified

Using 1-based sequence positions

WARNING: Unrecognized base in input sequence

OLIGO            startlentmgc%  any  3'seq

LEFT PRIMER         48   20   60.21   55.00  3.00  2.00 CCCACCTCTCTGCCATTCTA

RIGHT PRIMER       223   20   60.03   45.00  5.00  1.00 GTGAATTTCCCCCAACCTTT

SEQUENCE SIZE: 1001

INCLUDED REGION SIZE: 1001

PRODUCT SIZE: 176, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 1.00

    1 ACCCAATCTCTTTCCTGGATAGCAGTATCTTCATTTTGCTTATATGACCCACCTCTCTGC

>>>>>>>>>>>>> 

   61 CATTCTATACCACCATGGTAGACTTGTAAGTCAAGAGTTTCTCCCTCCAACCCTGGACAA

>>>>>>> 

  121 AAGGTGGACACATGACCTAAGCCAGTCCAGCTGAAAGATTCTCTCTGGAATATAATCTTG

  181 AACAAAGAGACACAGCAAATAAAAAAGGTTGGGGGAAATTCACCCAGCCTTGTGGCCCTG

<<<<<<<<<<<<<<<<<<<< 

  241 CTACAACTGTCTATTGCTTCCTTCTCTCTGGTTTGCAGGAATTAACACAGCTAACATTTC

  301 TGAATCTGGTTCTCAGCCTTCCCTTTAGGCATACAAATATTCCCCATCCTCCCATCCAGT

  361 CCCTTCTTGTTTAACGTAGCCTGAGTTATTTATGATCATTTCCAATTATATAACAAGGGG

  421 AGGCTAAGATTCAGTGACTTCAAGAACAAGGAAGTGTATTTCCTCCTCCTGTGAACATCC

  481 ATTGCTGGTAACCCAGGTTGYTGGGTGTCTCTGTTCCACTCAGTCATTTGGGGATCAAGG

  541 CTGAAGGCATCTCTACTGTTTTTAATGTATAACTTCTAATGTCTCTGTGCTCATGACCAT

  601 CCCAGCCAAGAGGAAGAGGGAAGAGCAGTAGACCAGAACAAGGGATTTCCTCTTAAGCAG

  661 GTGAGGTGGGCACTGCATGCCCAAATTCACTCACCTCCCATTGGAAGGAGCCTAGTCTCA

  721 TGGCCACACCTGACTGCAAGAGCAGCGAGTGCTGGGAAGTCAGTCTTTACCTGGACAGCC

  781 GTATCCCCACTGTGACCCTGTCACTATAGTGGAAAGAGAGATGGATTTGGATGGAGGATT

  841 TGCATCTCCCTCCACCACAACCACCAGGGGACTCCAAATGATGGGATGTGTCAGCCTCCT

  901 AAAGTCTATAAATTTGAGTTCATTATCCAAAGCACTGTGTACAGAGACTTAGGCTGATGA

  961 CAACATTGCCCCAAAGAGAAACTCCCTACTTACGCAATCTT

KEYS (in order of precedence):

>>>>>> left primer

<<<<<<right primer

ADDITIONAL OLIGOS

startlentmgc%  any  3'seq

 1 LEFT PRIMER        461   20   60.05   55.00  4.00  0.00 TCCTCCTCCTGTGAACATCC

   RIGHT PRIMER       704   20   59.78   50.00  5.00  2.00 CCAATGGGAGGTGAGTGAAT

   PRODUCT SIZE: 244, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 2.00

 2 LEFT PRIMER        604   20   59.95   55.00  2.00  0.00 AGCCAAGAGGAAGAGGGAAG

   RIGHT PRIMER       801   20   60.24   55.00  5.00  2.00 ACAGGGTCACAGTGGGGATA

   PRODUCT SIZE: 198, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 0.00

 3 LEFT PRIMER        604   20   59.95   55.00  2.00  0.00 AGCCAAGAGGAAGAGGGAAG

   RIGHT PRIMER       800   20   60.24   60.00  5.00  2.00 CAGGGTCACAGTGGGGATAC

   PRODUCT SIZE: 197, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 1.00

 4 LEFT PRIMER        337   20   59.80   50.00  4.00  0.00 ATATTCCCCATCCTCCCATC

   RIGHT PRIMER       554   20   60.10   50.00  3.00  3.00 AGAGATGCCTTCAGCCTTGA

   PRODUCT SIZE: 218, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 2.00

Statistics

con   too    in    in          no    tm    tm  high  highhigh

sid  many   tar  excl   bad    GC   too   too   any    3'  poly   end      

ered    Ns   get   reg   GC% clamp   low  high complcompl     X  stab    ok

Left    7626    27     0     0   105     0  2835  2503     0    34    19   137  1965

Right   7677    27     0     0   115     0  2767  2602     0     4    22   134  2005

Pair Stats:

considered 4854, unacceptable product size 4827, high end compl 4, ok 23

primer3 release 1.1.4

(primer3_results.cgi release 0.4.0)

Appendix 3

Diagram 1

References

Barth L., Weinstein M., Jorgensen H. and Ferrao J. Antimicrobial Sensitivity Testing: A review of General Principles and Contemporary Practice. Clinical Infectious Diseases (2009) 49 (11) 1749-1755

Brock - Biology of Micro organismschp 26

Davis W. and Stout R. Disc Plate Method of Microbiological Antibiotic Assay - Factors Influencing Variability and Error. Applied Microbiology, Oct. 1971, p. 659-665 Vol. 22, No. 4.23

CLSI website http://clsi.org/edu/ accessed on (5^th September 2015)

University of Melbourne Laboratory Techniques Manual

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