A nonsense mutation causing decreased levels of insulin receptor mRNA: detection by a simplified technique for direct sequencing of genomic DNA amplified by the polymerase chain reaction.

Abstract

Mutations in the insulin receptor gene can render the cell resistant to the biological action of insulin. We have studied a patient with leprechaunism (leprechaun/Minn-1), a genetic syndrome associated with intrauterine growth retardation and extreme insulin resistance. Genomic DNA from the patient was amplified by the polymerase chain reaction catalyzed by Thermus aquaticus (Taq) DNA polymerase, and the amplified DNA was directly sequenced. A nonsense mutation was identified at codon 897 in exon 14 in the paternal allele of the patient's insulin receptor gene. Levels of insulin receptor mRNA are decreased to <10% of normal in EpsteinBarr virus-transformed lymphoblasts and cultured skin fibroblasts from this patient. Thus, this nonsense mutation appears to cause a decrease in the levels of insulin receptor mRNA. In addition, we have obtained indirect evidence that the patient's maternal allele of the insulin receptor gene contains a cis-acting dominant mutation that also decreases the level of mRNA, but by a different mechanism. The nucleotide sequence ofthe entire protein-coding domain and the sequences of the intron-exon boundaries for all 22 exons of the maternal allele were normal. Presumably, the mutation in the maternal allele maps elsewhere in the insulin receptor gene. Thus, we conclude that the patient is a compound heterozygote for two cis-acting dominant mutations in the insulin receptor gene: (i) a nonsense mutation in the paternal allele that reduces the level Qf insulin receptor mRNA and (ii) an as yet unidentified mutation in the maternal allele that either decreases the rate of transcription or decreases the stability of the mRNA.

The molecular defect of ferrochelatase in a patient with erythropoietic protoporphyria.

“…pEPP1 also exhibited a frameshift mutation that would result in a truncated protein. Since the existence of such a premature termination codon in a mRNA is known to significantly reduce its stability (23)(24)(25)(26), it is conceivable that the aberrant ferrochelatase transcript in the proband's cells also has a decreased stability. Thus it is likely that the observed decrease in ferrochelatase mRNA content in the proband's cells is the result of decreased stability of the abnormal transcript.…”

Mutations of the Gs alpha-subunit gene in Albright hereditary osteodystrophy detected by denaturing gradient gel electrophoresis.

“…PCR-amplified genomic fragments were sequenced using multiple oligonucleotide primers (17). Genomic DNA was amplified by PCR using the same oligonucleotides as for DGGE and was directly sequenced (17). Sequence shown is that of the complementary strand, obtained by using an oligonucleotide primer complementary to sequence within intron 10 (5'-CTTGCACGGGGTTCTTCTCTA-3').…”

“…PCR-amplified genomic fragments were sequenced using multiple oligonucleotide primers (17). Genomic DNA was amplified by PCR using the same oligonucleotides as for DGGE and was directly sequenced (17).…”

Functional properties of a heterozygous mutation (Arg1174 → Gln) in the tyrosine kinase domain of the insulin receptor from a Type A insulin resistant patient

“…Indeed, many mutations of the insulin receptor gene have been identified over the past few years in patients with insulin resistance. Inherited defects of the insulin receptor gene may cause diminished receptor expression [1][2][3][4][5][6] or impaired receptor function, i.e. insulin binding [7,8] or tyrosine kinase activity [9][10][11].…”