NGS-Based Identification of Two Novel PCDH19 Mutations in Female Patients with Early-Onset Epilepsy

Abstract

Developmental and epileptic encephalopathy-9 (DEE9) is characterized by seizure onset in infancy, mild to severe intellectual impairment, and psychiatric features and is caused by a mutation in the PCDH19 gene on chromosome Xq22. The rare, unusual X-linked type of disorder affects heterozygous females and mosaic males; transmitting males are unaffected. In our study, 165 patients with epilepsy were tested by Next Generation Sequencing (NGS)-based panel and exome sequencing using Illumina technology. PCDH19 screening identified three point mutations, one indel, and one 29 bp-long deletion in five unrelated female probands. Two novel mutations, c.1152_1180del (p.Gln385Serfs*6) and c.830_831delinsAA (p.Phe277*), were identified and found to be de novo pathogenic. Moreover, among the three inherited mutations, two originated from asymptomatic mothers and one from an affected father. The PCDH19 c.1682C>T and c.1711G>T mutations were present in the DNA samples of asymptomatic mothers. After targeted parental testing, X chromosome inactivation tests and Sanger sequencing were carried out for mosaicism examination on maternal saliva samples in the two asymptomatic PCDH19 mutation carrier subjects. Tissue mosaicism and X-inactivation tests were negative. Our results support the opportunity for reduced penetrance in DEE9 and contribute to expanding the genotype-phenotype spectrum of PCDH19-related epilepsy.

Keywords: NGS; PCDH19 mutation; WES; epilepsy; protocadherin.

Figure 1

Schematic presentation of the cellular interference mechanism with PCDH19 mutation and in normal individuals. The figure was created using BioRender.com (accessed on 16 April 2024).

Figure 2

(A) Schematic illustration of the identified pathogenic PCDH19 c.830_831delTCinsAA (p.Phe277*) mutation in Patient 4; The burgundy coloured TC nucleotides were replaced by a pale pink coloured AA nucleotides. (B) Reads obtained from the exome sequencing analyzed using the Integrative Genomics Viewer (2.12.0) show the two affected bases located on the same allele of PCDH19. Light blue arrows represent forward reads, green arrows show reverse reads. The figure was created using BioRender.com (accessed on 16 April 2024).

Figure 3

Pedigree of Patient 5. Dotted line diamond represents miscarriage. I, II, III numbers show different generations.

Figure 4

Sanger sequencing electropherogram of heterozygous PCDH19 (A) c.1682C>T and (B) c.1711G>T mutations. (A/1) Blood sample of Patient 1. (A/2) Blood sample of Patient 1’s mother. (A/3) Saliva sample of Patient 1’s mother. (B/1) Blood sample of Patient 3. (B/2) Blood sample of Patient 3’s mother. (B/3) Saliva sample of Patient 3’s mother.

Figure 5

Electropherogram of X-inactivation tests in Patient 1, her mother, and a female control in undigested and predigested samples using methylation-specific restriction endonuclease HpaII. A slightly skewed ratio in Patient 1 (proband) (33:67), in her mother (60:40), and a random (54:46) X-inactivation pattern in a healthy female control.

Figure 6

Illustration of the identified 29 bp-long c.1152_1180del (p.Gln385Serfs*6) deletion in PCDH19 gene in Patient 2. The red coloured C and A nucleotides are the first and last nucleotide involved in the 29 bp-long deletion. The figure was created using BioRender.com (accessed on 16 April 2024).