Novel Gross Deletion Mutations in NTRK1 Gene Associated With Congenital Insensitivity to Pain With Anhidrosis

Abstract

Background: Congenital insensitivity to pain with anhidrosis (CIPA) is a rare inherited autosomal recessive disorder characterized by insensitivity to noxious stimuli, anhidrosis, recurrent fever, and intellectual disability. CIPA is mainly caused by mutations in the neurotrophic tyrosine kinase receptor type 1 gene (NTRK1). This study aims to identify pathogenic mutations underlying CIPA in two unrelated Chinese families. Methods: DNA was extracted from blood samples of patients and their available family members and subjected to whole exome sequencing (WES). Real-time PCR (qPCR), Gap-PCR, and Sanger sequencing were applied to verify the identified variants. Result: We found novel compound gross deletion mutations [exon1-6 del (g.1-1258_10169del); exon5-7 del (g.6995_11999del)] of NTRK1 (MIM 191315) gene in family 1 and the compound heterozygous mutations [c.851-33T>A; exon5-7 del (g.6995_11999del)] in family 2. Interestingly, we discovered the intragenic novel gross deletion [exon5-7 del (g.6995_11999del)] mediated by recombination between Alu elements. Conclusions: The present study highlights two rare gross deletion mutations in the NTRK1 gene associated with CIPA in two unrelated Chinese families. The deletion of exon1-6 (g.1-1258_10169del) is thought to be the largest NTRK1 deletion reported to date. Our findings expand the mutation spectrum of NTRK1 mutations in the Chinese and could be useful for prenatal interventions and more precise pharmacological treatments to patients. WES conducted in our study is a convenient and useful tool for clinical diagnosis of CIPA and other associated disorders.

Keywords: CIPA; Chinese families; HSAN; NTRK1; mutations; whole exome sequencing.

Figure 1

Identification of compound heterozygous gross deletion mutations of neurotrophic tyrosine kinase receptor type 1 (NTRK1) in family 1. (A) Pedigree chart of family1, black arrow indicates the proband. (B) Current situation of patient 1. (C) The result of whole exome sequencing (WES), the blue bar chart Means coverage of each exon of NTRK1 gene. Bar chart of readcount showing a suspicious area of CNVs (red squares) of NTRK1. It indicates the mutation conclude deletion of exon1-6 and the deletion of exon6-7. (D) The real-time quantitative PCR result (right) verified the compound heterozygous deletion. Deletion of exon1-6 derived from the father and deletion of exon6-7 derived from the mother. (E) Identification of the gross deletion E6-E7del (g.6995_11999del): (a) Gap-PCR products about 2,388 bp, the forward primer in exon4 and the reverse primer in exon8, proband and his mother amplified the products. Except for the patient 1, the other three people could amplify the internal reference fragment (112 bp); (b) DNA sequencing of the Gap-PCR products unveiled a deletion of 5,004 bp, and the breakpoint junction was located within two Alu repetitive elements with 14 bp common fusion; (c) The schematic map of gross deletion: E5-E7del (g.6995_11999del), gross deletion covering exons5, exon6, exon7, and introns in NTRK1. (F) Identification of the gross deletion E1-E6del (g.1-1258_10169del): (a) Gap-PCR products about 864 bp, the forward primer in upstream of the NTRK1 gene, and the reverse primer in exon7, proband and his father amplified the products. The internal reference fragment (112 bp) only found between the control and the parents; (b) DNA sequencing of the Gap-PCR products unveiled a deletion of 11,427 bp, and the breakpoint junction with 2 bp common fusion; (c) The schematic map of gross deletion:E1-E6del (g.1-1258_10169del) gross deletion covering exon1-6, upstream of the NTRK1 gene, and introns in NTRK1.

Figure 2

Identification of compound heterozygous mutations of NTRK1 in family 2. (A) Pedigree chart of family 2, black arrow indicates the proband. (B) The clinical manifestations of the proband: (a) current situation of patient 2 (b) damaged hand; (c) X-ray image showing a humeral fracture. (C) The result of whole exome sequencing (WES), the blue bar chart means coverage of each exon of NTRK1 gene. Bar chart of readcount showing a suspicious area of CNVs (red squares) of NTRK1. It indicates the heterozygous gross deletion mutation from exon5 to exon7. (D) The real-time quantitative PCR result verified the gross deletion, and the heterozygous mutation of deletion derived from the mother. (E) Identification of the gross deletion: E6-E7del (g.6995_11999del): (a) Gap-PCR products about 2,388 bp, proband, and his mother amplified the products; the internal reference fragments (112 bp) can be amplified in all samples; (b) DNA sequencing of the Gap-PCR products, the breakpoint junction was located within intron4 and intron7. (F) Gene sequencing results: the proband carries heterozygous splicing mutation of NTRK1 gene c.851-33T>A, and the mutation derived from the father.