Identification of mutations in the c-mpl gene in congenital amegakaryocytic thrombocytopenia

Abstract

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare disorder expressed in infancy and characterized by isolated thrombocytopenia and megakaryocytopenia with no physical anomalies. Our previous hematological analysis indicated similarities between human CAMT and murine c-mpl (thrombopoietin receptor) deficiency. Because the c-mpl gene was considered as one of the candidate genes for this disorder, we analyzed the genomic sequence of the c-mpl gene of a 10-year-old Japanese girl with CAMT. We detected two heterozygous point mutations: a C-to-T transition at the cDNA nucleotide position 556 (Q186X) in exon 4 and a single nucleotide deletion of thymine at position 1,499 (1,499 delT) in exon 10. Both mutations were predicted to result in a prematurely terminated c-Mpl protein, which, if translated, lacks all intracellular domains essential for signal transduction. Each of the mutations was segregated from the patient's parents. Accordingly, the patient was a compound heterozygote for two mutations of the c-mpl gene, each derived from one of the parents. The present study suggests that at least a certain type of CAMT is caused by the c-mpl mutation, which disrupts the function of thrombopoietin receptor.

Figure 1

T1-weighted brain MRI of the patient at 10 months of age. The MRI showed a hypoplastic cerebellar vermis with a communication between the fourth ventricle and cisterna magna. (A) Sagittal section. (B) Axial section.

Figure 2

The two truncating c-mpl mutations in the CAMT patient. (A) Sequences of c-mpl exon 4 in the CAMT patient and a control. A heterozygous C-to-T transition at cDNA position 556 (arrow), which is the first nucleotide of codon 156, changes a glutamine residue for a premature stop codon in the patient. (B) Sequences of c-mpl exon 10 in the patient and a control. A heterozygous deletion of nucleotide thymine at position 1,499 in the patient (arrow) results in a frameshift, leading to a premature truncation. For A and B, the nucleotide numbers are based on the predicted cDNA sequence (GenBank accession no. M90102). (C) The schematic structure of human c-Mpl and two point mutations found in the CAMT patient.

Figure 3

c-mpl gene mutations in the family with CAMT. (A) A pedigree of the family. Circles represent females and squares males. Half-shaded (left side of the symbol) symbols represent carriers of the Q186X mutation, and half-hatched (right side of the symbol) symbols represent carriers of the 1,499 delT mutation. The proband is II-1. (B) Verification of the C-to-T mutation at cDNA position 556 by PCR amplification and restriction-enzyme analysis. PCR products of exon 4 were digested with PvuII and checked by agarose gel electrophoresis. The 380-bp PCR product of exon 4 is cleaved to 175-bp, 105-bp, and 100-bp fragments by PvuII. The Q186X mutation abolishes one of two PvuII sites, resulting in 275-bp and 105-bp fragments. The patient (lane 2), her mother (lane 4), and her brother (lane 5) possess this mutation in one allele, whereas her father (lane 3) and her sister (lane 6) do not have this mutation. Lane 1 shows the molecular size marker. (C) Restriction-enzyme analysis of the 1,499 delT. The 265-bp PCR product of exon 10 is cleaved to 162-bp, 70-bp, and 33-bp fragments by Bfa I. The 1,499 delT abolishes one of two Bfa I sites, resulting in 195-bp and 70-bp fragments (70-bp and 33-bp bands are not shown). Although the patient (lane 2) and her father (lane 3) have this mutation in one allele, her mother (lane 4), her brother (lane 5), and her sister (lane 6) do not have this mutation. Lane 1 shows the molecular size marker.