PRKG1 mutation identified by whole-exome sequencing: a potential genetic etiology for He-Zhao deficiency : Journal of Bio-X Research

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PRKG1 mutation identified by whole-exome sequencing: a potential genetic etiology for He-Zhao deficiency

Hu, Xiaowena; Wang, Dandana; Yang, Xuhana,b; Song, Zhongchenc,d; Wang, Zuoline; Zhang, Juana; Wan, Chunlinga,*; He, Lina,*

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doi: 10.1097/JBR.0000000000000128



Tooth development involves highly programmed bone turnover events, which requires the complex and elaborate coordination of several types of bone cells.[1] Odontoblasts and odontoclasts, which are primarily involved in tooth root formation and resorption, respectively, coordinate in cycles during the bone turnover.[2,3] Dysfunctions in either phase of the cycle can lead to abnormalities of tooth development.[4,5] Hypodontia can manifest differently in terms of both the amount of missing teeth and the specific teeth that are missing,[6] implying that there is considerable genetic heterogeneity underlying this condition. Mutations in several genes have been identified as the etiological agents of nonsyndromic tooth agenesis in recent decades, increasing the rate of genetic diagnosis in patients with this condition.[7–14] However, the molecular basis of some forms of nonsyndromic tooth agenesis remains elusive.

Several pathogenic mechanisms are involved in hypodontia, such as Wnt/β-catenin signaling,[15] BMP signaling,[15] TNF-α signaling[16] in root formation, and RANKL signaling[17] in osteoclast differentiation. Hypodontia often affects a limited amount of teeth but seldom leads to severe impact on patient’s appearance. In our previous study, we described an autosomal dominant inherited form of hypodontia in a large pedigree and named it He-Zhao deficiency (OMIM entry %610926).[18] Distinctive symptoms observed in the affected family members included the persistence of deciduous teeth and the agenesis of permanent teeth. The amount of missing permanent teeth in the pedigree ranges from a few teeth to the entire set of teeth. The most frequently affected teeth in this He-Zhao deficiency pedigree are the second maxillary and mandibular premolars, followed by the maxillary lateral incisors. Panoramic radiographs revealed the absence of permanent tooth germs in the position of permanent teeth.[18] The affected members showed no clinical features other than tooth agenesis, including other missing ectodermal appendages or decreased intelligence, suggesting that He-Zhao deficiency is a form of nonsyndromic hypodontia. Linkage analysis was performed among the family members, and chromosome 10q11.2 was linked with He-Zhao deficiency.[19] In the current study, we employed next generation sequencing to analyze the whole exomes of members of this pedigree and detect genetic mutations that may cause He-Zhao deficiency.

Materials and methods

Study participants

The He-Zhao deficiency pedigree involved in this study was from Xunyi County, Shaanxi Province, China during 1997 to 1999 as previously described.[19] Briefly, tooth developments were recorded by self-report method and checked by visual examination. Representative family members with tooth agenesis were verified by dental X-ray photography. The demographic information is shown in Additional Table 1, Peripheral blood samples from individuals in the He-Zhao deficiency pedigree were obtained during the investigation. Whole-exome sequencing was performed in five affected family members and two unaffected members. Subsequently, 141 family members, excluding spouses, were genotyped according to their genomic DNAs extracted from peripheral blood, and 93 sequenced members older than 12 years were analyzed with the guidance of cosegregation.

To investigate the phenotypes of sporadic carriers of the PRKG1 mutation identified in the pedigree with He-Zhao deficiency, an unrelated cohort of Chinese Han individuals was recruited from Jining Medical University, Shandong Province, China. Self-reports involving characteristics of ectodermal appendages, potential symptoms referring to the functions of PRKG1 and the family histories were collected from individuals who were eligible for re-investigation. Dentition was assessed by clinical examination and panoramic radiograph at the Department of Stomatology of the Affiliated Hospital of Jining Medical University. College students under the age of 18 were excluded. The study was approved by the Research Ethics Committee of Bio-X Institutes, Shanghai Jiao Tong University (M2011004) and conducted in accordance with the 1964 Declaration of Helsinki, as revised in 2013. Informed consent was obtained from all participants or their legal guardians.

Whole-exome sequencing

Genomic DNA of all study participants was extracted from peripheral blood cells of all study participants using a QIAamp DNA mini kit (QIAGEN, Hilden, Germany). The DNA was fragmented, and the exomes were captured using an Agilent SureSelect Human All Exon V6 kit (Agilent, Santa Clara, CA) according to the manufacturer’s instructions. The enriched libraries were sequenced on an Illumina Novaseq 6000 system (Illumina, San Diego, CA) using 150-bp paired-end reads.

Bioinformatics analysis

Paired-end reads were mapped to the human reference genome (UCSC hg19) using the Burrows–Wheeler Aligner[20] (BWA, version 0.7.8-r455, UK). Single nucleotide polymorphisms (SNPs) and insertion and/or deletion (InDel) variants were called using SAMtools[21] (version 1.0, and annotated using ANNOVAR[22] (version 2017June01, The filtering criteria were as follows: nonsynonymous variants in exons or variants located in splice sites or in untranslated regions (UTRs) with a minor allele frequency (MAF) lower than 0.05 in the East Asian population as reported by the 1000 Genomes Project[23] and the Genome Aggregation Database (gnomAD, version 3.1.1,[24]

Sanger sequencing

Primers flanking the -144 C>A variant in PRKG1 were designed using the online tool Primer-BLAST ([25]: forward primer: 5′-AAGATAATCACCTACTCCCCAG-3′; reverse primer: 5′-GGCATTTGTGGAGTTTCCTC-3′. PCR amplification was carried out using Pfu PCR Master Mix (Tiangen, Beijing, China) and a GeneAmp PCR System 9700 (Applied Biosystems, Pleasanton, CA). Sanger sequencing was performed using an ABI 3730xl DNA Analyzer (Life Technologies, Carlsbad, CA) in accordance with the manufacturer’s instructions.

Plasmid construction and luciferase assay

Fragments containing the promoter region of PRKG1 transcript variant 1 (NM_001098512) and the 5′ end of either the wide-type allele or the mutant (-144 C>A) allele (−2000/+228 bp) were cloned into the luciferase reporter pGL3-basic vector (Promega, Madison, WI), denoted as PGL3-PRKG1α WT and PGL3-PRKG1α Mut, respectively. The DNA sequences of all constructs were verified by Sanger sequencing.[26,27] A pGL3-basic plasmid and a pGL3-promoter plasmid were used as negative controls and positive controls, respectively. Human embryonic kidney cells (HEK 293T, Cat# CRL-3216, ATCC, Manassas, VA, USA) were used for functional analyses. The cells were maintained in Dulbecco’s modified Eagle’s medium (HyClone, Logan, UT) with 10% fetal bovine serum (Gibco, Carlsbad, CA), 100 U/mL penicillin, 100 mg/mL streptomycin, and 250 ng/mL amphotericin B and incubated at 5% CO2 at 37°C. HEK 293T cells were seeded in 24-well culture plates 24 hours prior to transfection with 2 × 104 plasmid copies per well. Cells were co-transfected with 1μg of the PRKG1 reporter plasmid and 100 ng of the pRL plasmid as a normalizing control using X-tremegene HP (Roche, Basel, Switzerland), according to the manufacturer’s instructions. Luciferase activity was measured after 24 hours of culture using the Dual-Luciferase® Reporter Assay System (E1910, Promega, Madison, WI) and measured on a BioTek Synergy microplate reader (BioTek, Santa Clara, CA). The experiment was performed in triplicate. Promoter activity is expressed as relative Firefly luciferase activity normalized to Renilla luciferase activity.

Statistical analysis

In this He-Zhao deficiency pedigree, 93 sequenced family members older than 12 years were analyzed with the guidance of cosegregation between genotype and disease phenotype. The difference of transcription between PGL3-PRKG1α WT and PGL3-PRKG1α Mut was analyzed using t-test. Statistical significance was determined by the P value less than 0.05. Statistical analyses were performed using R environment (version 3.4.3).


Cosegregation of PRKG1 variant c.-144 C>A with disease phenotypes in a pedigree with He-Zhao deficiency

Analysis of cosegregation between phenotype and genotype identified the heterozygous variant rs114234235 (c.-144 C>A, NM_001098512) in the 5′-UTR region of PRKG1α as a candidate causative mutation (Fig. 1). One hundred forty-one available samples from the members in the pedigree with spouses excluded were subjected to Sanger sequencing. Generally, deciduous teeth should be completely replaced by permanent teeth before the age of 12, at which time permanent tooth agenesis can be easily detected without clinical dental examination. The c.-144 C>A variant of PRKG1α cosegregated with tooth agenesis in 93 sequenced family members who were older than 12, suggesting that it is the causative factor of He-Zhao deficiency (Fig. 2 and Additional Table 1, PRKG1 is located on chromosome 10q11.2, which is consistent with our previous linkage work.[19] In addition, PRKG1 has an extremely high haploinsufficiency rank (HI%=1.27%), indicating that the function of its gene product would be sensitive to changes at the transcriptional level, which is consistent with the fact that He-Zhao deficiency is inherited in an autosomal dominant manner.

Figure 1.:
Monoallelic rs114234235 identified in the pedigree with He-Zhao deficiency. (A) Schematic representation of the location of the rs114234235 mutation within PRKG1α (c.-144 C>A) in comparison with PRKG1β. (B) Sanger sequencing results. The specific location of the candidate pathogenic mutation is highlighted in yellow.
Figure 2.:
A general introduction of the pedigree with He-Zhao deficiency. (A) Genogram of the pedigree with He-Zhao deficiency. Filled shapes indicate affected individuals, and empty shapes indicate unaffected individuals. Half-filled shapes indicate individuals under the age of 12, whose deciduous teeth may not yet have been completely replaced by permanent teeth, so that tooth agenesis could not be easily distinguished without clinical dental examination. Squares indicate males, and circles indicate females. Blue arrows indicate individuals from whom samples were taken for whole-exome sequencing. Red shading indicates individuals for whom clinical samples were available, and black shading indicates individuals for whom clinical samples were unavailable. The icon with a slash on it indicates deceased individuals. (B–E) Dental panoramic radiographs of 4 family members: (B) an 18-year-old man with normal permanent teeth (V52 in the genogram); (C) a 21-year-old woman with three teeth left (V50, sister of V52); (D) a 66- year-old man (IV13, father of V50 and V52), the proband of this pedigree. He had normal deciduous teeth when he was a child but his permanent teeth did not erupt. He had only eight molars left at the age of 40 according to his self-report; (E) a 35-year-old woman with several permanent teeth and deciduous teeth left (V102). Detailed information can be seen in our previous study.[ 18 ]

Verification study in an unrelated population

We then investigated the prevalence of the variant rs114234235 in an unrelated Chinese Han cohort of 466 adults. Forty of them harbored the c.-144 C>A heterozygous mutation, indicating an allele frequency of 4.29%. This is consistent with the reported frequency of this mutation in the East Asian population (MAF=3.98%),[24] and is much higher than the reported frequency in other populations (eg, MAF=1.49% in Latino/Admixed American population), as indicated by the Genome Aggregation Database (gnomAD v3.1.1).[24] Twenty-two variant carriers from the sporadic cohort and 23 controls who were negative for the mutation underwent subsequent clinical investigation. Panoramic radiograph analysis showed that four of the 22 mutation carriers were missing permanent teeth, indicating a penetrance of 18.18%. Each of these four individuals was missing one or two teeth, either lateral incisors or canines, and none were missing third molars (Fig. 3). In contrast, none of the 23 individuals from the control group who did not have the variant were missing any teeth. Collectively, these findings indicate that tooth agenesis is common in carriers of the c.-144 C>A PRKG1 variant. However, the number of missing teeth in variant carriers from the unrelated control group differed from that seen in variant carriers in the pedigree, suggesting a high degree of heterogeneity among mutation carriers.

Figure 3.:
Panoramic radiographs of 4 sporadic carriers of the c.-144 C>A PRKG1 variant with tooth agenesis in the unrelated cohort. The missing teeth were: (A) the left mandibular lateral incisor, (B) the left maxillary lateral incisor, (C) the right maxillary canine, (D) the left and right mandibular lateral incisor. Red arrows in the panoramic radiographs and red shading in the top-right teeth position table indicate the missing teeth of each variant carrier. The permanent teeth positions were numbered as: 1-central incisor, 2-lateral incisor, 3-canine, 4-first premolar, 5-second premolar, 6-first molar, 7-second molar, and 8-third molar.

Effects of the c.-144 C>A variant on PRKG1α expression in vitro

To determine the impact of the identified PRKG1 mutation, we sought to elucidate the functional consequence of the mutant allele. PRKG1 encodes a cyclic GMP (cGMP)-dependent protein kinase with two isoforms, α and β, that differ in their N-termini, which results in interaction with different sets of proteins.[28–30] The -144 C/A mutation is located in the 5′-UTR of PRKG1 transcript variant 1, which would potentially affect transcription and translation of PRKG1α but have no effect on the β isoform (Fig. 1A). We performed in vitro luciferase reporter assays to investigate the effects of the mutation on the expression of PRKG1α. The luciferase constructs contained the entire 2228-bp promoter region of PRKG1 transcript variant 1 from the wild-type or mutant allele, followed by the firefly luciferase gene (pGL3-basic reporter vectors). Transcription of the reporter gene from the PRKG1α promoter harboring the c.-144 C>A variant was significantly higher than that from the wild-type promoter (fold change=1.23, P<0.001; Fig. 4). This finding suggests that the c.-144 C>A mutation increases promoter activity, and hence PRKG1α transcription.

Figure 4.:
Results from the luciferase assay performed in HEK293T cells to assess the impact of the PRKG1α c.-144 C>A variant. The construct containing the mutant promoter (PGL3-PRKG1α Mut) showed a significantly (1.23-fold) higher transcription level than that containing the wild-type promoter (PGL3-PRKG1α WT). The difference of transcription between PGL3-PRKG1α Mut and PGL3-PRKG1α WT was analyzed using t-tests. ***P<0.001. The experiment was performed in triplicate. Data are expressed as the mean±SD.


He-Zhao deficiency is a rare type of hypodontia that in some cases involves agenesis of the entire set of permanent teeth, which is much more severe than more commonly reported forms of hypodontia.[18] We previously mapped the causative gene locus to a 5.5-cM region of chromosome 10q11.2 using linkage analysis with pooled DNA samples.[19] The development of next generation sequencing has enabled comprehensive scanning of the whole exome, and thus breakthroughs in determining the genetic basis of this and other hereditary diseases. In the current study, we identified variant rs114234235 in PRKG1 as the pathogenetic mutation causing He-Zhao deficiency, which is strongly supported by cosegregation with permanent tooth agenesis in 23 affected family members from three generations of the pedigree and the location of this mutation in the previously identified 10q11.2 region.[19]

Tooth development involves highly programmed bone turnover events, which involve elaborately coordinated cycles of odontoblast and odontoclast activity. Odontoclasts function during the absorption of deciduous tooth roots, which is critical for their exfoliation and the subsequent eruption of permanent teeth. The absorption process initiates with the formation of a sealing zone between odontoclasts and the degradative surface,[31] followed by the subsequent local acidification of the bone matrix.[1,32] Focal adhesion depends on the integrin αvβ3[33,34] and involves integrin-associated cytoskeleton arrangement for spatial colonization of the peripheral ring in odontoclasts.[30,35] PRKG1α is activated by NO/cGMP signaling and then phosphorylates Ser239 of the vasodilator-stimulated phosphoprotein (VASP) in odontoclasts.[30] The -144 C>A variant of PRKG1α identified in our study resulted in overexpression of its gene product, which could be speculated to increase phosphorylation of VASP.[30] VASP binds to the integrin αvβ3, while phosphorylation of Ser239 of VASP causes it to detach from integrin αvβ3 and bind to migfilin instead,[30,36,37] which would result in the redistribution of integrin along the peripheral ring of osteoclasts.[30,35] This cytoskeletal rearrangement could impede the integrin-based adhesion of osteoclasts to the extracellular matrix and block formation of the resorptive pit and subsequent local acidification, resulting in a decrease in odontoclast activity during resorption of primary tooth roots.


Expression of the reporter gene from the PRKG1α promoter in vitro suggests that the c.-144 C>A mutation increases its transcription. However, it would be more informative using odontoclasts or osteoclasts in consideration of the pathogenic mechanism that we proposed. Animal experiments are also needed to illustrate the effect of the PRKG1α mutation on tooth development. In addition, the high phenotypic heterogeneity among both the affected family members of the He-Zhao deficiency pedigree and the unrelated mutant carriers in the control group suggests that other factors are also involved in the etiology of tooth agenesis, indicating the need for further research to investigate the functional alterations that the variant induces.


In this study, we identified mutations in PRKG1α as the potential genetic etiology of He-Zhao deficiency. The -144 C>A variant of PRKG1α cosegregated with tooth agenesis in the He-Zhao deficiency pedigree, and the variant allele was confirmed to induce overexpression of PRKG1α, which could attenuate osteoclast adhesion. Our study identified a novel genetic basis for hypodontia, as well as a distinct disease mechanism involving osteoclasts, which provides new insight into tooth development and may increase the yield of clinical molecular diagnosis for tooth agenesis.


We are grateful to all individuals that participated in this study for their contributions.

Author contributions

LH and CW designed the study. XH, DW, and XY wrote the manuscript and contributed to the acquisition of data. XH and DW contributed to statistical analysis. All authors contributed to the critical revision of the manuscript for important intellectual content and approved the final version of the manuscript for publication.

Financial support

This work was supported by the National Natural Science Foundation of China (Nos. 81971254 and 81771440) and Shanghai Municipal Science and Technology Major Project (No. 2017SHZDZX01). The funders had no role in manuscript design, data collection and analysis, preparation, or decision to publish.

Institutional review board statement

The study was approved by the Research Ethics Committee of Bio-X Institutes, Shanghai Jiao Tong University (approval No. M2011004).

Declaration of participant consent

The authors certify that they have obtained the participant consent forms. In the forms, participants have given their consent for their images and other clinical information to be reported in the journal. The participants understand that their names and initials will not be published and due efforts will be made to conceal their identity.

Conflicts of interest

There are no conflicts of interest.

Editor note: LH is an Editorial Board member of Journal of Bio-X Research. He was blinded from reviewing or making decisions on the manuscript. The article was subject to the journal’s standard procedures, with peer review handled independently of this Editorial Board member and their research groups.


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genetic etiology; He-Zhao deficiency; novel gene; PRKG1; whole-exome sequencing

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