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A short in-frame deletion in NTRK1 tyrosine kinase domain caused by a novel splice site mutation in a patient with congenital insensitivity to pain with anhidrosis.

Sarasola E, Rodríguez JA, Garrote E, Arístegui J, García-Barcina MJ - BMC Med. Genet. (2011)

Bottom Line: We also found a second mutation, c.2206-2 A>G, not previously reported in the literature, which is located at the splice acceptor site of intron 16.As a consequence, this mutation would result in the production of a mutant NTRK1 protein with a seven aminoacid in-frame deletion in its tyrosine kinase domain.We present the first description of a CIPA-associated NTRK1 mutation causing a short interstitial deletion in the tyrosine kinase domain of the receptor.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Basurto University Hospital (OSAKIDETZA/Servicio Vasco de Salud), Bilbao, Spain.

ABSTRACT

Background: Congenital insensitivity to pain with anhidrosis (CIPA) is a rare autosomal recessive genetic disease characterized by the lack of reaction to noxious stimuli and anhidrosis. It is caused by mutations in the NTRK1 gene, which encodes the high affinity tyrosine kinase receptor I for Neurotrophic Growth Factor (NGF).

Case presentation: We present the case of a female patient diagnosed with CIPA at the age of 8 months. The patient is currently 6 years old and her psychomotor development conforms to her age (RMN, SPECT and psychological study are in the range of normality). PCR amplification of DNA, followed by direct sequencing, was used to investigate the presence of NTRK1 gene mutations. Reverse transcriptase (RT)-PCR amplification of RNA, followed by cloning and sequencing of isolated RT-PCR products was used to characterize the effect of the mutations on NTRK1 mRNA splicing. The clinical diagnosis of CIPA was confirmed by the detection of two splice-site mutations in NTRK1, revealing that the patient was a compound heterozygote at this gene. One of these alterations, c.574+1G>A, is located at the splice donor site of intron 5. We also found a second mutation, c.2206-2 A>G, not previously reported in the literature, which is located at the splice acceptor site of intron 16. Each parent was confirmed to be a carrier for one of the mutations by DNA sequencing analysis. It has been proposed that the c.574+1G>A mutation would cause exon 5 skipping during NTRK1 mRNA splicing. We could confirm this prediction and, more importantly, we provide evidence that the novel c.2206-2A>G mutation also disrupts normal NTRK1 splicing, leading to the use of an alternative splice acceptor site within exon 17. As a consequence, this mutation would result in the production of a mutant NTRK1 protein with a seven aminoacid in-frame deletion in its tyrosine kinase domain.

Conclusions: We present the first description of a CIPA-associated NTRK1 mutation causing a short interstitial deletion in the tyrosine kinase domain of the receptor. The possible phenotypical implications of this mutation are discussed.

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Pedigree of the family and results of the NTRK1 genetic analysis in CIPA patients' DNA. A. As indicated by the symbols in the pedigree, individual II-1 is affected by CIPA, whereas her parents and sister are all carriers. The NTRK1 mutation detected in each individual is indicated under the corresponding symbol. B. Electropherograms demonstrating the presence of two NTRK1 point mutations (in IVS5 and IVS16) in DNA from individual II-1. The position of each mutation is indicated by an asterisk.
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Figure 1: Pedigree of the family and results of the NTRK1 genetic analysis in CIPA patients' DNA. A. As indicated by the symbols in the pedigree, individual II-1 is affected by CIPA, whereas her parents and sister are all carriers. The NTRK1 mutation detected in each individual is indicated under the corresponding symbol. B. Electropherograms demonstrating the presence of two NTRK1 point mutations (in IVS5 and IVS16) in DNA from individual II-1. The position of each mutation is indicated by an asterisk.

Mentions: In addition to the patient, her parents and sister were also available for molecular analysis. The pedigree of the family is shown in Figure 1. Blood samples were collected from all the family members. Genomic DNA was purified using a standard "salting-out" purification protocol. Total RNA was obtained using the Puregene RNA isolation kit (Gentra). The concentration of both DNA and RNA was measured using a spectrophotometer. All the 17 exons and intron-exon boundaries of the longest NTRK1 isoform (NM_002529) were analysed using PCR amplification of genomic DNA from the patient followed by direct DNA sequencing. The presence of the identified NTRK1 mutations was subsequently investigated in her relatives using the same methods. Primers for PCR amplification were designed using the Primer3 on-line application (http://frodo.wi.mit.edu/primer3/input.htm). Primer sequences and optimal annealing temperature for each primer pair are available upon request. All PCR reactions were carried out using TaqGold DNA polymerase (Applied Biosystems) with 5% DMSO (Sigma). Direct sequencing of both strands of the amplified DNA fragments was performed using the BigDye Terminator v3.1 Sequencing kit (Applied Biosystems). Sequencing reactions were analysed on an ABI PRISM 3130 Genetic Analyzer using the Sequence Scanner software (Applied Biosystems). The mutations described in this report are named following the recommendations from the Human Genome Variation Society (http://www.hgvs.org/mutnomen/).


A short in-frame deletion in NTRK1 tyrosine kinase domain caused by a novel splice site mutation in a patient with congenital insensitivity to pain with anhidrosis.

Sarasola E, Rodríguez JA, Garrote E, Arístegui J, García-Barcina MJ - BMC Med. Genet. (2011)

Pedigree of the family and results of the NTRK1 genetic analysis in CIPA patients' DNA. A. As indicated by the symbols in the pedigree, individual II-1 is affected by CIPA, whereas her parents and sister are all carriers. The NTRK1 mutation detected in each individual is indicated under the corresponding symbol. B. Electropherograms demonstrating the presence of two NTRK1 point mutations (in IVS5 and IVS16) in DNA from individual II-1. The position of each mutation is indicated by an asterisk.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3141629&req=5

Figure 1: Pedigree of the family and results of the NTRK1 genetic analysis in CIPA patients' DNA. A. As indicated by the symbols in the pedigree, individual II-1 is affected by CIPA, whereas her parents and sister are all carriers. The NTRK1 mutation detected in each individual is indicated under the corresponding symbol. B. Electropherograms demonstrating the presence of two NTRK1 point mutations (in IVS5 and IVS16) in DNA from individual II-1. The position of each mutation is indicated by an asterisk.
Mentions: In addition to the patient, her parents and sister were also available for molecular analysis. The pedigree of the family is shown in Figure 1. Blood samples were collected from all the family members. Genomic DNA was purified using a standard "salting-out" purification protocol. Total RNA was obtained using the Puregene RNA isolation kit (Gentra). The concentration of both DNA and RNA was measured using a spectrophotometer. All the 17 exons and intron-exon boundaries of the longest NTRK1 isoform (NM_002529) were analysed using PCR amplification of genomic DNA from the patient followed by direct DNA sequencing. The presence of the identified NTRK1 mutations was subsequently investigated in her relatives using the same methods. Primers for PCR amplification were designed using the Primer3 on-line application (http://frodo.wi.mit.edu/primer3/input.htm). Primer sequences and optimal annealing temperature for each primer pair are available upon request. All PCR reactions were carried out using TaqGold DNA polymerase (Applied Biosystems) with 5% DMSO (Sigma). Direct sequencing of both strands of the amplified DNA fragments was performed using the BigDye Terminator v3.1 Sequencing kit (Applied Biosystems). Sequencing reactions were analysed on an ABI PRISM 3130 Genetic Analyzer using the Sequence Scanner software (Applied Biosystems). The mutations described in this report are named following the recommendations from the Human Genome Variation Society (http://www.hgvs.org/mutnomen/).

Bottom Line: We also found a second mutation, c.2206-2 A>G, not previously reported in the literature, which is located at the splice acceptor site of intron 16.As a consequence, this mutation would result in the production of a mutant NTRK1 protein with a seven aminoacid in-frame deletion in its tyrosine kinase domain.We present the first description of a CIPA-associated NTRK1 mutation causing a short interstitial deletion in the tyrosine kinase domain of the receptor.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Basurto University Hospital (OSAKIDETZA/Servicio Vasco de Salud), Bilbao, Spain.

ABSTRACT

Background: Congenital insensitivity to pain with anhidrosis (CIPA) is a rare autosomal recessive genetic disease characterized by the lack of reaction to noxious stimuli and anhidrosis. It is caused by mutations in the NTRK1 gene, which encodes the high affinity tyrosine kinase receptor I for Neurotrophic Growth Factor (NGF).

Case presentation: We present the case of a female patient diagnosed with CIPA at the age of 8 months. The patient is currently 6 years old and her psychomotor development conforms to her age (RMN, SPECT and psychological study are in the range of normality). PCR amplification of DNA, followed by direct sequencing, was used to investigate the presence of NTRK1 gene mutations. Reverse transcriptase (RT)-PCR amplification of RNA, followed by cloning and sequencing of isolated RT-PCR products was used to characterize the effect of the mutations on NTRK1 mRNA splicing. The clinical diagnosis of CIPA was confirmed by the detection of two splice-site mutations in NTRK1, revealing that the patient was a compound heterozygote at this gene. One of these alterations, c.574+1G>A, is located at the splice donor site of intron 5. We also found a second mutation, c.2206-2 A>G, not previously reported in the literature, which is located at the splice acceptor site of intron 16. Each parent was confirmed to be a carrier for one of the mutations by DNA sequencing analysis. It has been proposed that the c.574+1G>A mutation would cause exon 5 skipping during NTRK1 mRNA splicing. We could confirm this prediction and, more importantly, we provide evidence that the novel c.2206-2A>G mutation also disrupts normal NTRK1 splicing, leading to the use of an alternative splice acceptor site within exon 17. As a consequence, this mutation would result in the production of a mutant NTRK1 protein with a seven aminoacid in-frame deletion in its tyrosine kinase domain.

Conclusions: We present the first description of a CIPA-associated NTRK1 mutation causing a short interstitial deletion in the tyrosine kinase domain of the receptor. The possible phenotypical implications of this mutation are discussed.

Show MeSH
Related in: MedlinePlus