Novel MSX1 frameshift mutation in a Japanese family with nonsyndromic oligodontia

Congenital tooth agenesis is a common anomaly in humans. We investigated the etiology of human tooth agenesis by exome analysis in Japanese patients, and found a previously undescribed heterozygous deletion (NM_002448.3(MSX1_v001):c.433_449del) in the first exon of the MSX1 gene. The deletion leads to a frameshift and generates a premature termination codon. The truncated form of MSX1, namely, p.(Trp145Leufs*24) lacks the homeodomain, which is crucial for transcription factor function.

Here, we analyze Japanese patients with oligodontia diagnosed on the basis of clinical and radiographic examinations. Saliva samples were obtained from patients after they provided informed consent to participate in the study, which was approved by the Institutional Review Board of Aichi-Gakuin University, TOYOTA Memorial Hospital, and the Institute for Developmental Research, Aichi Developmental Disability Center. In the studied family, the proband (II-1) and her father (I-1) showed symptoms, i.e., loss of eight and six teeth, respectively ( Fig. 1A-C). Nevertheless, we did not analyze the nucleotide sequence of the MSX1 gene of the father because of disagreement with informed consent. Orofacial cleft, craniofacial abnormalities, or other health problems, including those related to ectodermal organs, such as the hair, nails, and sweat glands, were not noted in any of the affected family members.
Genomic DNA was extracted from saliva using Oragene DNA (OG-500; DNA Genotek, Ontario, Canada) according to the manufacturer's instructions. Briefly, each saliva sample was mixed with prepIT L2P (PT-2LP; DNA Genotek), incubated on ice, and centrifuged for 5 min at 15,000 × g. The supernatant was mixed with EtOH to precipitate DNA. After centrifugation, the DNA pellet was dissolved in elution buffer. Mutational analysis by wholeexome sequencing was subsequently performed according to our previously reported method 12 .
The 17-bp deletion generates an MSX1 protein with a Cterminus lacking the homeodomain. It has previously been demonstrated that the homeodomain (amino acids 175-229) plays a pivotal role in molecular interactions with DNA 13 and other transcription factors related to tooth development, such as PAX9, TATA-binding protein, and DLX family members 14 . The homeodomain is also associated with nuclear transport 15 , which is crucial for the biological function of transcription factors. We expressed mutant MSX1 in HEK293 cells to confirm the nuclear transport defect. Although immunoreactivity of wild-type MSX1 was detected in nuclei, as previously reported 16 , truncated MSX1 was mainly found in the cytoplasm (Fig. 2C).
In addition to its nonsyndromic form, haploinsufficiency of MSX1 causes the syndromic form of tooth agenesis that includes cleft lip and/or palate 17,18 . To date, 50 MSX1 gene mutations, including four truncated variants, have been identified in patients with syndromic and nonsyndromic tooth agenesis. Although amino acid substitutions in patients with tooth agenesis cluster in the homeodomain (70%), no variants associated with cleft lip and/or palate without tooth malformation have been identified in it 19 . Previous biochemical analyses have shown that some single-amino acid substitutions in the homeodomain of MSX1 can affect the transcriptional suppression activity of the MyoD promoter, which is one of the targets of MSX1 20 . Because MSX1 strongly suppresses target gene expression and represses cell differentiation both in vitro and in vivo 21 , it sustains cellular proliferation in the tooth germ during odontogenesis for robust tooth development in terms of size, number, and shape.
In summary, previous reports and the current results indicate that the p.(Trp145Leufs*24) mutation impairs the molecular function of MSX1; thus, the identified nucleotide deletion is the cause of the tooth agenesis in the studied family. Because of the pathogenic relationship between particular MSX1 mutations and facial clefts, we believe that clarifying MSX1 gene variations will help to improve the precision of genetic counseling to patients with odontogenic malformations and/or facial clefts.

HGV DATABASE
The relevant data from this Data Report are hosted at the Human Genome Variation Database at https://doi.org/10.6084/m9. figshare.hgv.3039