Limits...
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.

Show MeSH

Related in: MedlinePlus

Characterization of the effect of IVS 5 (c.574+1G>C) and IVS16 (c.2206-2A>G) point mutations on NTRK1 splicing. A. Schematic partial representation of NTRK1 exon/IVS structure, indicating the position of the primer sets (P1-F/P1-R and P2-F/P2-R) designed for RT-PCR analysis. B. Left, agarose gel showing the two differently sized amplicons obtained by PCR analysis of cloned P1-F/P1-R RT-PCR products (see "Methods" section for details). As illustrated on the right, lane 1 fragment corresponds to normally spliced mRNA, encoding the wild-type (wt) NTRK1 protein, whose structural domains are schematically depicted in the figure (LRM: leucine-rich motif; IGL: immunoglobulin-like; TM: transmembrane; TK: tyrosine kinase). Lane 2 fragment corresponds to an abnormally spliced mRNA lacking exon 5. The encoded NTRK1 protein, depicted below, is predicted to bear a premature stop codon following a novel 64 aminoacid sequence after the L100 residue. C. Left, agarose gel showing the three differently sized amplicons obtained by PCR analysis of cloned P2-F/P2-R RT-PCR products. Lane 1 corresponds to normally spliced mRNA. Lane 2 corresponds to an abnormally spliced mRNA retaining a 336 bp fragment of IVS16. The predicted NTRK1 protein, depicted below, would have a premature stop codon following a novel 5 aminoacid sequence after the E375 residue. Lane 3 corresponds to an abnormally spliced mRNA lacking a 21 bp fragment of exon 17. As indicated, this in frame deletion is predicted to encode an NTRK1 protein bearing a 7 aminoacid interstitial (A736_Q742del) deletion in its TK domain.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3141629&req=5

Figure 2: Characterization of the effect of IVS 5 (c.574+1G>C) and IVS16 (c.2206-2A>G) point mutations on NTRK1 splicing. A. Schematic partial representation of NTRK1 exon/IVS structure, indicating the position of the primer sets (P1-F/P1-R and P2-F/P2-R) designed for RT-PCR analysis. B. Left, agarose gel showing the two differently sized amplicons obtained by PCR analysis of cloned P1-F/P1-R RT-PCR products (see "Methods" section for details). As illustrated on the right, lane 1 fragment corresponds to normally spliced mRNA, encoding the wild-type (wt) NTRK1 protein, whose structural domains are schematically depicted in the figure (LRM: leucine-rich motif; IGL: immunoglobulin-like; TM: transmembrane; TK: tyrosine kinase). Lane 2 fragment corresponds to an abnormally spliced mRNA lacking exon 5. The encoded NTRK1 protein, depicted below, is predicted to bear a premature stop codon following a novel 64 aminoacid sequence after the L100 residue. C. Left, agarose gel showing the three differently sized amplicons obtained by PCR analysis of cloned P2-F/P2-R RT-PCR products. Lane 1 corresponds to normally spliced mRNA. Lane 2 corresponds to an abnormally spliced mRNA retaining a 336 bp fragment of IVS16. The predicted NTRK1 protein, depicted below, would have a premature stop codon following a novel 5 aminoacid sequence after the E375 residue. Lane 3 corresponds to an abnormally spliced mRNA lacking a 21 bp fragment of exon 17. As indicated, this in frame deletion is predicted to encode an NTRK1 protein bearing a 7 aminoacid interstitial (A736_Q742del) deletion in its TK domain.

Mentions: One μg total RNA was reverse-transcribed using the SuperScript VILO cDNA Synthesis kit (Invitrogen), to generate complementary DNA (cDNA). Two sets of specific primers (P1-F/P1-R and P2-F/P2-R), whose location is indicated in Figure 2, were designed as described above. PCR reactions with each of the primer sets were carried out using 2 μl of cDNA. PCR products were inserted into the pCR2.1-TOPO vector using the TOPO TA Cloning kit (Invitrogen). DNA was extracted from 20 randomly selected bacterial clones. The size of the inserted PCR fragment in each clone was determined using PCR with a set of primers directed against vector sequences flanking the cloning site. Plasmid DNA from bacterial clones containing inserted PCR products of different size was purified using the QIAprep Spin Miniprep Kit (Qiagen) and sequenced as described above.


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)

Characterization of the effect of IVS 5 (c.574+1G>C) and IVS16 (c.2206-2A>G) point mutations on NTRK1 splicing. A. Schematic partial representation of NTRK1 exon/IVS structure, indicating the position of the primer sets (P1-F/P1-R and P2-F/P2-R) designed for RT-PCR analysis. B. Left, agarose gel showing the two differently sized amplicons obtained by PCR analysis of cloned P1-F/P1-R RT-PCR products (see "Methods" section for details). As illustrated on the right, lane 1 fragment corresponds to normally spliced mRNA, encoding the wild-type (wt) NTRK1 protein, whose structural domains are schematically depicted in the figure (LRM: leucine-rich motif; IGL: immunoglobulin-like; TM: transmembrane; TK: tyrosine kinase). Lane 2 fragment corresponds to an abnormally spliced mRNA lacking exon 5. The encoded NTRK1 protein, depicted below, is predicted to bear a premature stop codon following a novel 64 aminoacid sequence after the L100 residue. C. Left, agarose gel showing the three differently sized amplicons obtained by PCR analysis of cloned P2-F/P2-R RT-PCR products. Lane 1 corresponds to normally spliced mRNA. Lane 2 corresponds to an abnormally spliced mRNA retaining a 336 bp fragment of IVS16. The predicted NTRK1 protein, depicted below, would have a premature stop codon following a novel 5 aminoacid sequence after the E375 residue. Lane 3 corresponds to an abnormally spliced mRNA lacking a 21 bp fragment of exon 17. As indicated, this in frame deletion is predicted to encode an NTRK1 protein bearing a 7 aminoacid interstitial (A736_Q742del) deletion in its TK domain.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Characterization of the effect of IVS 5 (c.574+1G>C) and IVS16 (c.2206-2A>G) point mutations on NTRK1 splicing. A. Schematic partial representation of NTRK1 exon/IVS structure, indicating the position of the primer sets (P1-F/P1-R and P2-F/P2-R) designed for RT-PCR analysis. B. Left, agarose gel showing the two differently sized amplicons obtained by PCR analysis of cloned P1-F/P1-R RT-PCR products (see "Methods" section for details). As illustrated on the right, lane 1 fragment corresponds to normally spliced mRNA, encoding the wild-type (wt) NTRK1 protein, whose structural domains are schematically depicted in the figure (LRM: leucine-rich motif; IGL: immunoglobulin-like; TM: transmembrane; TK: tyrosine kinase). Lane 2 fragment corresponds to an abnormally spliced mRNA lacking exon 5. The encoded NTRK1 protein, depicted below, is predicted to bear a premature stop codon following a novel 64 aminoacid sequence after the L100 residue. C. Left, agarose gel showing the three differently sized amplicons obtained by PCR analysis of cloned P2-F/P2-R RT-PCR products. Lane 1 corresponds to normally spliced mRNA. Lane 2 corresponds to an abnormally spliced mRNA retaining a 336 bp fragment of IVS16. The predicted NTRK1 protein, depicted below, would have a premature stop codon following a novel 5 aminoacid sequence after the E375 residue. Lane 3 corresponds to an abnormally spliced mRNA lacking a 21 bp fragment of exon 17. As indicated, this in frame deletion is predicted to encode an NTRK1 protein bearing a 7 aminoacid interstitial (A736_Q742del) deletion in its TK domain.
Mentions: One μg total RNA was reverse-transcribed using the SuperScript VILO cDNA Synthesis kit (Invitrogen), to generate complementary DNA (cDNA). Two sets of specific primers (P1-F/P1-R and P2-F/P2-R), whose location is indicated in Figure 2, were designed as described above. PCR reactions with each of the primer sets were carried out using 2 μl of cDNA. PCR products were inserted into the pCR2.1-TOPO vector using the TOPO TA Cloning kit (Invitrogen). DNA was extracted from 20 randomly selected bacterial clones. The size of the inserted PCR fragment in each clone was determined using PCR with a set of primers directed against vector sequences flanking the cloning site. Plasmid DNA from bacterial clones containing inserted PCR products of different size was purified using the QIAprep Spin Miniprep Kit (Qiagen) and sequenced as described above.

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