Targeted Next-Generation Sequencing in the Diagnosis of Facial Dysostoses

Abstract

Background: Defects in the development of the first and second pharyngeal arches and their derivatives result in abnormal formation of the craniofacial complex, consequently giving rise to facial dysostoses (FDs). FDs represent a group of rare and highly heterogeneous disease entities that encompass mandibulofacial dysostoses (MFDs) with normal extremities and acrofacial dysostoses (AFDs) with limb anomalies in addition to craniofacial defects.

Methods: We examined 11 families with variable clinical symptoms of FDs, in most of which only one member was affected. We applied two custom gene panels-first comprising 37 genes related to the genetic disorders of craniofacial development such as FDs (On-Demand AmpliSeq Thermo Fisher Scientific gene panel with two primer pools) and second composed of 61 genes and 11 single nucleotide variants (SNVs) known to be involved in the development of skull malformations, mainly in the form of craniosynostoses (SureSelect Agilent Technologies). Targeted next-generation sequencing (NGS) was performed using the Ion Torrent S5 platform. To confirm the presence of each detected variant, we have analyzed a genomic region of interest using Sanger sequencing.

Results: In this paper, we summarized the results of custom targeted gene panel sequencing in the cohort of sixteen patients from 11 consecutive families affected by distinct forms of FDs. We have found three novel pathogenic variants in the TCOF1 gene-c.2145_2148dupAAAG p.(Ser717Lysfs ^∗42), c.4370delA p.(Lys1457Argfs ^∗118), c.83G>C p.(Arg28Pro) causing Treacher Collins syndrome type 1, two novel missense variants in the EFTUD2 gene-c.491A>G p.(Asp164Gly) and c.779T>A p.(Ile260Asn) in two female patients affected by acrofacial dysostosis Guion-Almeida type, one previously reported-c.403C>T (p.Arg135Cys), as well as one novel missense variant-c.128C>T p.(Pro43Leu) in the DHODH gene in the male patient with Miller syndrome and finally one known pathogenic variant c.574G>T p.(Glu192^∗) in the SF3B4 gene in the patient with Nager syndrome.

Conclusion: Our study confirms the efficiency and clinical utility of the targeted gene panel sequencing and shows that this strategy is suitable and efficient in the molecular screening of variable forms of FDs.

Keywords: acrofacial dysostoses; craniofacial development; facial dysostoses; mandibulofacial dysostoses; pharyngeal arch; rare diseases; targeted next-generation sequencing.

FIGURE 1

Clinical characteristics of family 1–patient 1.1 (A,B), patient 1.2 (C,D), patient 1.3 (E,F), patient 1.4 (G,H), patient 1.5 (I,J), patient 1.6 (K), The pedigree (L).

FIGURE 2

Sanger sequencing results of family 1 showing the absence of the pathogenic variant c.2145_2148dupAAAG p.(Ser717Lysfs*42) within the TCOF1 gene in a healthy family member (A) and its presence in patients affected by Treacher Collins syndrome (B–G).

FIGURE 3

Clinical characteristics and segregation results of patient 2 (A–C) and 3 (D–F) affected by Treacher Collins syndrome. Patient 2 carries a heterozygous variant c.83G>C p.(Arg28Pro) within the TCOF1 gene, which occurred de novo (C) whereas patient 3 carries a heterozygous variant c.4370delA p.(Lys1457Argfs*118) within the TCOF1 gene, which also occurred de novo.

FIGURE 4

Clinical characteristics of patient 4 affected by Nager syndrome (A,B). Patient 4 carries a heterozygous variant c.574G>T p.(Glu192*) within the SF3B4 gene, which was excluded in her healthy mother (C).

FIGURE 5

Clinical characteristics and segregation results of patient 5 (A–D) and 6 (E–H) affected by acrofacial dysostosis Guion-Almeida type. Patient 5 carries a heterozygous variant c.491A>G p.(Asp164Gly) within the EFTUD2 gene, which occurred de novo (D) whereas patient 6 carries a heterozygous variant c.779T>A p.(Ile260Asn) within the EFTUD2 gene, which also occurred de novo (H).

FIGURE 6

Clinical characteristics (A–D) and segregation results (E) of patient 7 affected by Miller syndrome. Patient 7 carries c.403C>T (p.Arg135Cys) and c.128C>T p.(Pro43Leu) missense variants, both located in trans orientation in the DHODH gene (E).