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The change in the typical sequence of the gene by inserting a particular sequence or removing a specific segment is known as mutation. The location in which the mutation takes place determines the severity of the mutation.

The middle of the gene may contain the segment of an intron or an exon. Introns are the non-coding region, and exons are the coding region of DNA. The part removed from the middle of the gene does not affect the gene expression at a higher frequency.

Thus, removing three nucleotides from the middle of a gene has a lesser possibility of causing a harmful effect.

Therefore, the given option is false.

Removing nucleotide from a non-coding region does not result in a harmful mutation. This is because the introns are the non-coding region that does not code for the protein.

Thus, the mutation in the introns does not result in harmful mutation. The protein remains unaffected in this condition.

Therefore, the given option is false.

Mutation at the end of the coding sequence does not have much effect because the sequence present in the end can get sliced due to the post-transcriptional or post-translational modification.

The mutation occurs at the end of the coding sequences and does not majorly affect an organism.

Therefore, the given option is false.

The sequence inserted downstream of the coding region has a major impact on the protein product. Since the coding sequences are the region that codes for a protein product

Coding regions or exons are the sequences that contain the information which is encoded in the protein. A single nucleotide mutation will result in harmful mutations in an organism.

Therefore, the given option is true.