MutationHuman – Essaylink

Data Analysis Part 1 – MutationHuman cystic fibrosis transmembrane conductance regulator (CFTR) mRNA

 

1. The cDNA sequence representing the complete mRNA of the normal or wild-type human CFTR gene is given in Appendix I. Use this to determine the amino acid sequence of the protein.

There are several DNA translation tools that you can use for this:

https://web.expasy.org/translate/

https://www.ebi.ac.uk/Tools/st/emboss_transeq/

Which reading frame gives you the correct protein sequence (remember you only need to translate the top strand)?

 

1. Identify the protein coding sequence (that is, the region which is translated into protein), and the 3’ and 5’ untranslated regions and complete Table 1. How many amino acids are encoded by this region?

 

Table 1.  CFTR mRNA features

Feature	Nucleotide positions
3’ UTR
Coding sequence (CDS)
5’ UTR

 

 

 

 

 

2. Over 1700 mutations have been identified in the CFTR gene that are associated with cystic fibrosis. Four of the most common ones are listed in Table 2.

 

For each of the mutations determine (i) what kind of mutation it is (missense, nonsense) and (ii) what is the effect of the mutation on the protein sequence.

To do this you will need to identity and change the relevant nucleotide(s) in the wild-type cDNA and then use a translation tool to determine the effect on the protein. You can then use programmes designed to align multiple sequences to compare the mutant proteins to the wild-type. The most commonly used is Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/).

 

Table 2. Common CFTR mutations

Mutation	Nucleotide position(s)*	Mutation type	Amino acid change	Protein domain affected
M1:  G → A	420	Missense	R117H	TMD 1
M2: del CTT	1591 -1593	3 bp deletion	Deletion of F508	ATP binding domain 1
M3: G → T	1694	Nonsense	G542X	Premature termination
M4: G → A	1722	Nonsense	G551D	ATP binding domain 1

^* Refers to the nucleotide position in the cDNA

 

1. Using the information in Table 3, draw a cartoon of the CFTR protein showing the domain organisation and indicate on this the positions of the amino acid changes identified in the mutants.

 

Table 3. CFTR proteins domain regions

Domain	Amino acids
Transmembrane domain 1	85 – 303
ATP binding domain 1	389 – 670
Regulatory (R) domain	639 – 849
Transmembrane domain 2	866 – 1147
ATP binding domain 2	1208 – 1480

 

 

11. A patient was diagnosed with CF; neither parent had CF. Routine analysis identified a known mutant allele on one chromosome and an unknown mutant allele on the other chromosome. The novel mutation was found to be located between exons 8-11.  RT-PCR analysis was carried out on RNA samples from the patient and both parents, using primers designed to amplify this region (Figure 1B).  The PCR products were analysed by agarose gel electrophoreses (Figure 1A) and then sequenced (Appendix II).

 

 

 

 

 

 

 

 

 

Figure 1: (A) Analysis of RT-PCR products of RNA isolated from blood samples of the patient (J28), and his mother (Mo) and father (Fa).  The position of the 500 bp band in the 100 bp marker lane is indicated. (B) Sequences of the forward (F) and reverse (R) primers used in the PCR.

 

 

• Locate the position of the primers on the complete cDNA. Remember you will first need to determine the reverse complement of the R primer in order to do this.
• What size product is expected for amplification of the wild-type cDNA?
• How do you interpret the results of the RT-PCR?
• Compare the wild-type and mutant DNA sequences to determine the nature of the mutation using programmes Clustal Omega https://www.ebi.ac.uk/Tools/msa/clustalo/).
• Can you speculate how this mutation arose (hint: look at the positions of the exon junctions in the cDNA; Appendix III)?
• What protein domain is affected by the mutation and in what way?

 

 

Appendix I. CFTR cDNA sequence

 

Note the sequence below is presented in FASTA format, which is a text-based format used in bioinformatics to represent nucleotide or protein sequences using their single-letter abbreviations. The first line denoted by ‘>’ is a comment line and is used to identify or describe the sequence.

 

>human_CFTR_wildtype_cDNA_6070 bp

gtagtaggtctttggcattaggagcttgagcccagacggccctagcagggaccccagcgcccgagagaccatgcagaggtcgcctctggaaaaggccagcgttgtctccaaactttttttcagctggaccagaccaattttgaggaaaggatacagacagcgcctggaattgtcagacatataccaaatcccttctgttgattctgctgacaatctatctgaaaaattggaaagagaatgggatagagagctggcttcaaagaaaaatcctaaactcattaatgcccttcggcgatgttttttctggagatttatgttctatggaatctttttatatttaggggaagtcaccaaagcagtacagcctctcttactgggaagaatcatagcttcctatgacccggataacaaggaggaacgctctatcgcgatttatctaggcataggcttatgccttctctttattgtgaggacactgctcctacacccagccatttttggccttcatcacattggaatgcagatgagaatagctatgtttagtttgatttataagaagactttaaagctgtcaagccgtgttctagataaaataagtattggacaacttgttagtctcctttccaacaacctgaacaaatttgatgaaggacttgcattggcacatttcgtgtggatcgctcctttgcaagtggcactcctcatggggctaatctgggagttgttacaggcgtctgccttctgtggacttggtttcctgatagtccttgccctttttcaggctgggctagggagaatgatgatgaagtacagagatcagagagctgggaagatcagtgaaagacttgtgattacctcagaaatgattgaaaatatccaatctgttaaggcatactgctgggaagaagcaatggaaaaaatgattgaaaacttaagacaaacagaactgaaactgactcggaaggcagcctatgtgagatacttcaatagctcagccttcttcttctcagggttctttgtggtgtttttatctgtgcttccctatgcactaatcaaaggaatcatcctccggaaaatattcaccaccatctcattctgcattgttctgcgcatggcggtcactcggcaatttccctgggctgtacaaacatggtatgactctcttggagcaataaacaaaatacaggatttcttacaaaagcaagaatataagacattggaatataacttaacgactacagaagtagtgatggagaatgtaacagccttctgggaggagggatttggggaattatttgagaaagcaaaacaaaacaataacaatagaaaaacttctaatggtgatgacagcctcttcttcagtaatttctcacttcttggtactcctgtcctgaaagatattaatttcaagatagaaagaggacagttgttggcggttgctggatccactggagcaggcaagacttcacttctaatggtgattatgggagaactggagccttcagagggtaaaattaagcacagtggaagaatttcattctgttctcagttttcctggattatgcctggcaccattaaagaaaatatcatctttggtgtttcctatgatgaatatagatacagaagcgtcatcaaagcatgccaactagaagaggacatctccaagtttgcagagaaagacaatatagttcttggagaaggtggaatcacactgagtggaggtcaacgagcaagaatttctttagcaagagcagtatacaaagatgctgatttgtatttattagactctccttttggatacctagatgttttaacagaaaaagaaatatttgaaagctgtgtctgtaaactgatggctaacaaaactaggattttggtcacttctaaaatggaacatttaaagaaagctgacaaaatattaattttgcatgaaggtagcagctatttttatgggacattttcagaactccaaaatctacagccagactttagctcaaaactcatgggatgtgattctttcgaccaatttagtgcagaaagaagaaattcaatcctaactgagaccttacaccgtttctcattagaaggagatgctcctgtctcctggacagaaacaaaaaaacaatcttttaaacagactggagagtttggggaaaaaaggaagaattctattctcaatccaatcaactctatacgaaaattttccattgtgcaaaagactcccttacaaatgaatggcatcgaagaggattctgatgagcctttagagagaaggctgtccttagtaccagattctgagcagggagaggcgatactgcctcgcatcagcgtgatcagcactggccccacgcttcaggcacgaaggaggcagtctgtcctgaacctgatgacacactcagttaaccaaggtcagaacattcaccgaaagacaacagcatccacacgaaaagtgtcactggcccctcaggcaaacttgactgaactggatatatattcaagaaggttatctcaagaaactggcttggaaataagtgaagaaattaacgaagaagacttaaaggagtgcttttttgatgatatggagagcataccagcagtgactacatggaacacataccttcgatatattactgtccacaagagcttaatttttgtgctaatttggtgcttagtaatttttctggcagaggtggctgcttctttggttgtgctgtggctccttggaaacactcctcttcaagacaaagggaatagtactcatagtagaaataacagctatgcagtgattatcaccagcaccagttcgtattatgtgttttacatttacgtgggagtagccgacactttgcttgctatgggattcttcagaggtctaccactggtgcatactctaatcacagtgtcgaaaattttacaccacaaaatgttacattctgttcttcaagcacctatgtcaaccctcaacacgttgaaagcaggtgggattcttaatagattctccaaagatatagcaattttggatgaccttctgcctcttaccatatttgacttcatccagttgttattaattgtgattggagctatagcagttgtcgcagttttacaaccctacatctttgttgcaacagtgccagtgatagtggcttttattatgttgagagcatatttcctccaaacctcacagcaactcaaacaactggaatctgaaggcaggagtccaattttcactcatcttgttacaagcttaaaaggactatggacacttcgtgccttcggacggcagccttactttgaaactctgttccacaaagctctgaatttacatactgccaactggttcttgtacctgtcaacactgcgctggttccaaatgagaatagaaatgatttttgtcatcttcttcattgctgttaccttcatttccattttaacaacaggagaaggagaaggaagagttggtattatcctgactttagccatgaatatcatgagtacattgcagtgggctgtaaactccagcatagatgtggatagcttgatgcgatctgtgagccgagtctttaagttcattgacatgccaacagaaggtaaacctaccaagtcaaccaaaccatacaagaatggccaactctcgaaagttatgattattgagaattcacacgtgaagaaagatgacatctggccctcagggggccaaatgactgtcaaagatctcacagcaaaatacacagaaggtggaaatgccatattagagaacatttccttctcaataagtcctggccagagggtgggcctcttgggaagaactggatcagggaagagtactttgttatcagcttttttgagactactgaacactgaaggagaaatccagatcgatggtgtgtcttgggattcaataactttgcaacagtggaggaaagcctttggagtgataccacagaaagtatttattttttctggaacatttagaaaaaacttggatccctatgaacagtggagtgatcaagaaatatggaaagttgcagatgaggttgggctcagatctgtgatagaacagtttcctgggaagcttgactttgtccttgtggatgggggctgtgtcctaagccatggccacaagcagttgatgtgcttggctagatctgttctcagtaaggcgaagatcttgctgcttgatgaacccagtgctcatttggatccagtaacataccaaataattagaagaactctaaaacaagcatttgctgattgcacagtaattctctgtgaacacaggatagaagcaatgctggaatgccaacaatttttggtcatagaagagaacaaagtgcggcagtacgattccatccagaaactgctgaacgagaggagcctcttccggcaagccatcagcccctccgacagggtgaagctctttccccaccggaactcaagcaagtgcaagtctaagccccagattgctgctctgaaagaggagacagaagaagaggtgcaagatacaaggctttagagagcagcataaatgttgacatgggacatttgctcatggaattggagctcgtgggacagtcacctcatggaattggagctcgtggaacagttacctctgcctcagaaaacaaggatgaattaagtttttttttaaaaaagaaacatttggtaaggggaattgaggacactgatatgggtcttgataaatggcttcctggcaatagtcaaattgtgtgaaaggtacttcaaatccttgaagatttaccacttgtgttttgcaagccagattttcctgaaaacccttgccatgtgctagtaattggaaaggcagctctaaatgtcaatcagcctagttgatcagcttattgtctagtgaaactcgttaatttgtagtgttggagaagaactgaaatcatacttcttagggttatgattaagtaatgataactggaaacttcagcggtttatataagcttgtattcctttttctctcctctccccatgatgtttagaaacacaactatattgtttgctaagcattccaactatctcatttccaagcaagtattagaataccacaggaaccacaagactgcacatcaaaatatgccccattcaacatctagtgagcagtcaggaaagagaacttccagatcctggaaatcagggttagtattgtccaggtctaccaaaaatctcaatatttcagataatcacaatacatcccttacctgggaaagggctgttataatctttcacaggggacaggatggttcccttgatgaagaagttgatatgccttttcccaactccagaaagtgacaagctcacagacctttgaactagagtttagctggaaaagtatgttagtgcaaattgtcacaggacagcccttctttccacagaagctccaggtagagggtgtgtaagtagataggccatgggcactgtgggtagacacacatgaagtccaagcatttagatgtataggttgatggtggtatgttttcaggctagatgtatgtacttcatgctgtctacactaagagagaatgagagacacactgaagaagcaccaatcatgaattagttttatatgcttctgttttataattttgtgaagcaaaattttttctctaggaaatatttattttaataatgtttcaaacatatataacaatgctgtattttaaaagaatgattatgaattacatttgtataaaataatttttatatttgaaatattgactttttatggcactagtatttctatgaaatattatgttaaaactgggacaggggagaacctagggtgatattaaccaggggccatgaatcaccttttggtctggagggaagccttggggctgatgcagttgttgcccacagctgtatgattcccagccagcacagcctcttagatgcagttctgaagaagatggtaccaccagtctgactgtttccatcaagggtacactgccttctcaactccaaactgactcttaagaagactgcattatatttattactgtaagaaaatatcacttgtcaataaaatccatacatttgtgtgaaa

 

 

 

Appendix II. Sequence of RT-PCR products

 

>Wild-type_RT-PCR product

ctgcgcatggcggtcactcggcaatttccctgggctgtacaaacatggtatgactctcttggagcaataaacaaaatacaggatttcttacaaaagcaagaatataagacattggaatataacttaacgactacagaagtagtgatggagaatgtaacagccttctgggaggagggatttggggaattatttgagaaagcaaaacaaaacaataacaatagaaaaacttctaatggtgatgacagcctcttcttcagtaatttctcacttcttggtactcctgtcctgaaagatattaatttcaagatagaaagaggacagttgttggcggttgctggatccactggagcaggcaagacttcacttctaatggtgattatgggagaactggagccttcagagggtaaaattaagcacagtggaagaatttcattctgttctcagttttcctggattatgcctggcaccattaaagaaaatatcatctttggtgtttcctatgatg

 

 

>Mutant_RT-PCR product

ctgcgcatggcggtcactcggcaatttccctgggctgtacaaacatggtatgactctcttggagcaataaacaaaatacaggatttcttacaaaagcaagaatataagacattggaatataacttaacgactacagaagtagtgatggagaatgtaacagccttctgggaggagacttcacttctaatggtgattatgggagaactggagccttcagagggtaaaattaagcacagtggaagaatttcattctgttctcagttttcctggattatgcctggcaccattaaagaaaatatcatctttggtgtttcctatgatg

 

 

 

 

 

Appendix III. Exons positions

 

exon	nucleotides		exon	nucleotides		exon	nucleotides
exon 1	1 – 123		exon 10	1280 – 1462		exon 19	3059 – 3209
exon 2	124 – 234		exon 11	1463 – 1654		exon 20	3210 – 3437
exon 3	235 – 343		exon 12	1655 – 1749		exon 21	3438 – 3538
exon 4	344 – 559		exon 13	1750 – 1836		exon 22	3539 – 3787
exon 5	560 – 649		exon 14	1837 – 2560		exon 23	3788 – 3943
exon 6	650 – 813		exon 15	2561 – 2689		exon 24	3944 – 4033
exon 7	814 – 939		exon 16	2690 – 2727		exon 25	4034 – 4206
exon 8	940 – 1186		exon 17	2728 – 2978		exon 26	4207 – 4312
exon 9	1187 – 1279		exon 18	2979 – 3058		exon 27	4313 – 6070

 

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