Temple syndrome and Kagami-Ogata syndrome: clinical presentations, genotypes, models and mechanisms

Abstract

Temple syndrome (TS) and Kagami-Ogata syndrome (KOS) are imprinting disorders caused by absence or overexpression of genes within a single imprinted cluster on human chromosome 14q32. TS most frequently arises from maternal UPD14 or epimutations/deletions on the paternal chromosome, whereas KOS most frequently arises from paternal UPD14 or epimutations/deletions on the maternal chromosome. In this review, we describe the clinical symptoms and genetic/epigenetic features of this imprinted region. The locus encompasses paternally expressed protein-coding genes (DLK1, RTL1 and DIO3) and maternally expressed lncRNAs (MEG3/GTL2, RTL1as and MEG8), as well as numerous miRNAs and snoRNAs. Control of expression is complex, with three differentially methylated regions regulating germline, placental and tissue-specific transcription. The strong conserved synteny between mouse chromosome 12aF1 and human chromosome 14q32 has enabled the use of mouse models to elucidate imprinting mechanisms and decipher the contribution of genes to the symptoms of TS and KOS. In this review, we describe relevant mouse models and highlight their value to better inform treatment options for long-term management of TS and KOS patients.

Introduction

Genomic imprinting is a mammalian-specific phenomenon that results in the parental-specific expression of a small number of genes (1). Imprinted genes are generally found in clusters and regulated by imprinting control regions (ICRs), which exhibit parental-specific DNA methylation that is acquired during germline development. Critically, gamete-specific DNA methylation at ICRs is maintained after fertilization despite the large amount of epigenetic reprogramming that occurs at this time. This unique maintenance of DNA methylation can be explained by ICR-specific recognition of the Krüppel-associated box-containing zinc finger proteins (KZFP) ZFP57 and ZFP445 (2,3), as well the maintenance DNA methyltransferase DNMT1. Deletions or abnormal DNA methylation of ICRs results in perturbed imprinting of multiple genes in a cluster.

Much of what has been learned about imprinting in the past four decades is conserved between mouse and humans, enabling the use of both human genetics and mouse models to elucidate genes and mechanisms. Though numbering only in the hundreds, imprinted genes can explain the block to complete uniparental development in mammals. Additionally, because imprinted genes are expressed from one parental allele, heterozygous mutations can result in abnormal phenotypes or even lethality. Further, given the central role for epigenetic gene regulation of imprinted genes, defects in DNA methylation, or epimutations, trigger abnormal expression and phenotypes. Consistently, human imprinting disorders have been described that result from heterozygous mutations, large or small deletions, uniparental disomy (UPD) and ICR epimutations (biallelic loss or gain of DNA methylation). Examples of human imprinting disorders include the growth disorders, Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS), and the neurobehavioral disorders Prader-Willi syndrome (PWS) and Angelman syndrome (AS) (4,5). Importantly, because of human and mouse imprinting conservation, mouse models have been instrumental to understanding mechanisms, genotype-phenotype correlations and pursuing therapeutic options (6,7).

Temple syndrome (TS: OMIM #616222; ORPHA: #254516) and Kagami-Ogata syndrome (KOS: OMIM #608149; ORPHA: #254534) are two additional genomic imprinting disorders that result from genetic and epigenetic alterations of a large imprinted gene cluster in the Chromosome 14q32 region. As with the imprinting disorders above, this region is conserved in mouse. TS was first described in individuals who exhibited short stature and premature puberty (8), whereas KOS was first described in individuals with multiple congenital anomalies (9). Although the initial cases were classified as maternal or paternal UPDs, respectively, and were considered rare (<1 in 1,000,000), there is growing realization that the disorders are more common and have a variety of etiologies. In some cases, molecular genetic and epigenetic testing is required to confirm a diagnosis, as patients with either KOS (10) or TS (11) have been misdiagnosed as having more common imprinting disorders such as PWS, AS, BWS or SRS.

This review is designed to (1) provide a clinical overview of symptoms and (2) describe the genetic and epigenetic alterations underlying TS and KOS, with the goal of appropriately identifying infants, children and adults with these disorders. Moreover, we describe how mouse models are used to provide insight into the mechanisms of imprinting and the contribution of genes in the region to phenotypes. Ultimately, the diagnosis of TS or KOS is essential for infants to receive appropriate medical treatment early in life and to provide families with expectations for their children’s clinical course, recommendations for support services and appropriate counseling for recurrence risks.

Clinical Symptoms in TS

The common signs and symptoms of TS are shown in Table 1. The cardinal features of children with TS are low birth weight, feeding problems, hypotonia and motor delay, mild facial dysmorphism, short stature and premature puberty (OMIM #616222; ORPHA: #254516). Hypotonia is associated with poor feeding and limited suck reflex early in life (11–13). Low birth weight is due to intrauterine growth restriction (11–13). In some cases, these characteristics contribute to a formal diagnosis of failure to thrive (14–16).

Table 1

Clinical characteristics of Temple syndrome and Kagami-Ogata syndrome

Features	Temple syndrome	Kagami-Ogata syndrome
Prenatal	Oligohydramniosa	Polyhydramniosa
	Preterm deliverya	Preterm deliverya
	Intrauterine growth restriction/small for gestational age (SGA)a	Omphalocelea
	Small placenta	Placentomegalya
	Reduced fetal movement	Macrosomiaa
	Delivery requiring cesarean section	Delivery requiring cesarean section
Perinatal/Postnatal	Low birth weight and lengtha	Respiratory distressa
	Feeding problemsa	Feeding problemsa
	Postnatal short staturea	Postnatal growth retardation
Developmental/Neurological	Hypotoniaa	Hypotoniaa
	Motor delaya	Speech delaya
	Speech delaya	Motor delaya
	Global developmental delaya	Mild intellectual disability
	Intellectual disabilitya
Craniofacial	Abnormal head circumferencea	Frontal bossinga
	Trigonocephalya	Depressed nasal bridgea
	Depressed nasal bridgea	Anteverted naresa
	Broad nosea	Prominent philtruma
	Short philtruma	Full cheeksa
	Micrognathiaa	Micrognathiaa
	Epicanthus	Short necka
	Anteverted nares	Hairy forehead
	High-arched palate
Thoracic	Uncommon	Small bell-shaped thoraxa
		Coat-hanger ribsa
		Narrow chest walla
		Heart anomaly
Abdominal	Uncommon	Omphalocelea
		Diastasis rectia
		Inguinal hernia
Musculo-skeletal	Small hands and/or feeta	Joint contracturesa
	Clinodactylya	Kyphoscoliosis
	Joint hypermobilitya
	Body asymmetry
	Kyphoscoliosis
Endocrine	Obesitya	Uncommon
	Early onset pubertya
	Advanced bone age
	Hypercholesterolemia
	Type II diabetes

Features listed from most common to least common in each category; ^adenotes a very common feature. Symptoms above were noted in 5 or more patients with TS or KOS. References are listed in Supplementary Material, Supplementary File #1; articles with patient photographs are indicated.

Facial features range from mild to moderate dysmorphia and may not be evident in neonates, but become distinguishable with age. Frontal bossing and micrognathia are common in infants (11–13,17). Face shape may be long or triangular, due to prominent forehead and small jaw (12,15,18). Many patients have been described clinically with ‘flat facial features’ (11,12,15,17,18). Skeletal features that help with diagnosis include small hands and/or feet, disproportionate to the rest of the body, as well as clinodactyly and joint hypermobility (11–13,15,17,18). Growth hormone has been used as a treatment for short stature (11,13,19).

Development ranges from normal to severely delayed. Motor delay often presents with delay in walking (13,17,18). Children may experience speech delay and/or intellectual disability, and require special accommodations in school settings (11,12). Children with TS should be evaluated for developmental delays and provided with early intervention services.

Endocrine anomalies are prevalent, with truncal obesity developing as early as 4–6 years (11,12,18). Obesity has been associated with compulsive excessive eating habits as well as normal eating habits (12). Diabetes and hypercholesterolemia have been reported (12,20,21). Most TS patients experience precocious puberty (11–13) along with advanced bone age (11,17,18); treatment with gonadotropin-releasing hormone agonists can delay the early signs of puberty (22).

Clinical Symptoms in KOS

Common signs and symptoms of KOS are shown in Table 1. The cardinal features of KOS infants are small bell-shaped thorax, coat-hanger ribs and narrow chest wall, which lead to significant respiratory distress upon delivery (OMIM #608149; ORPHA: #254534). Neonates often require intubation and high level care in the neonatal intensive care unit, and are discharged with oxygen and respiratory monitoring systems (23,24). Abnormal formation of the thoracic cavity can complicate feeding and contribute to failure to thrive and postnatal growth failure in infancy (10,25). Some KOS infants are born with heart anomalies (26,27).

Neonates with KOS may present with abdominal wall defects (25,26). Omphalocele is most prevalent, often detected on ultrasound before delivery and requiring surgical repair after delivery (10). Diastasis recti is highly reported in neonates as well. Inguinal hernia often presents later in infancy or during childhood (10,26). Hepatoblastoma has been reported in three children with KOS and monitoring has been suggested, but no formal guidelines have been developed (10).

Individuals with KOS often exhibit mildly dysmorphic craniofacial features, which may include a depressed nasal bridge, frontal bossing and a prominent philtrum (10,27,28). Other noteworthy traits include short neck, short palpebral fissures, anteverted nares and micrognathia (10,26). As they develop, children with KOS may experience speech and/or motor delays (10). Some children have mild intellectual disability, while others have normal intellectual ability (10). Children with KOS should be closely monitored for signs of developmental delay and provided with early intervention services.

Clinical Symptoms During the Prenatal Period

While TS and KOS are often not diagnosed until birth, several nonspecific symptoms may indicate a fetus carries one of these genetic abnormalities. Both syndromes are associated with preterm labor and often require cesarean section for delivery (10,12). In TS, oligohydramnios and small placenta contribute to intrauterine growth restriction (12,18). TS babies are almost always small for gestational age (SGA) (11–13). Occasionally, reduced fetal movement may be detected on ultrasound during pregnancy (11,18). In KOS, polyhydramnios is a cardinal feature, often causing dyspnea in pregnant women and requiring amnioreduction (10,26). Omphaloceles are commonly detected on ultrasound, necessitating cesarean delivery and surgical repair (10). In contrast to babies with TS, KOS newborns often exhibit macrosomia (10,28).

Structure and Genes of the Chr 14q32 Imprinted Region

The imprinted region responsible for TS and KOS is shown in Figure 1. The centromeric end is proximal to the DLK1 gene and the telomeric end is distal to the DIO3 gene. The locus contains three differentially methylated regions (IG-DMR, MEG3-DMR, MEG8-DMR) (29–31). Protein-coding genes (DLK1, RTL1, DIO3), long ncRNAs (MEG3, MEG8, RTL1as, DIO3OS) and short ncRNAs (SNORDs and miRNAs) are transcribed based on parent-of-origin (Table 2). TS and KOS are most commonly caused by UPD followed by epimutations and deletions (Fig. 1). Rare cases involve inherited or de novo Robertsonian translocations (15,24) or mosaicism (32–34).

Figure 1

Genetic and epigenetic alterations on Chromosome 14 lead to Temple syndrome or Kagami-Ogata syndrome. The human Chr 14q32 region extending from 100.7 to 101.6 Mb is shown (Ensembl: Human GRCh38.p13). Hashmarks (//) indicate the largest interval of 513 kb which lies between the 3′ end of the MEG8 gene and the 3′ end of the DIO3OS gene. The methylation pattern of the IG-DMR is established in the germline (29). The MEG3-DMR lies in the promoter region of the MEG3 gene; its methylation pattern is set postfertilization and is dependent on the methylation pattern of the IG-DMR (30). The MEG8-DMR lies in intron 2 of the MEG8 gene (72). Methylation of the MEG8-DMR occurs at or after week 17 of fetal development and may be dependent on the IG-DMR and/or MEG3-DMR (31), as well as transcription starting at the upstream MEG3 promoter and extending through the MEG8-DMR (11). Most postnatal tissues show differential methylation of the MEG8-DMR, consistent with imprinting (11). The MEG3, RTL1as, MEG8 and SNORD genes may be transcribed as polycistronic transcripts (65,73). The CpG-rich 1.2 kb promoter of the DIO3OS and DIO3 genes lies between them, although additional promoters outside this region have been suggested (74). Although the direction of transcription of DIO3OS is known (74), the parental origin of transcription in humans is unknown. Alterations leading to TS and KOS are diagrammed in the left and right columns, respectively. The % represents an estimate of the frequency of cases that are due either to UPD, epimutations or deletions, based on reports describing multiple cases (11,12,22,65,75). (A) Normal imprinting, methylation and gene expression at the Chr 14q32 region. The imprinted region begins proximal to the DLK1 gene and ends distal to the DIO3 gene. (B) TS can be caused by maternal UPD leading to expression of only maternally expressed genes from both chromosomes, whereas (C) KOS can be caused by paternal UPD leading to expression of only paternally expressed genes from both chromosomes. UPD may be segmental or involve the whole chromosome. (D) TS may be caused by hypomethylation of the IG-DMR and MEG3-DMR on the paternal chromosome, leading to a maternal chromosome-like expression pattern. (E) Conversely, KOS may be caused by hypermethylation of the IG-DMR and MEG3-DMR on the maternal chromosome, leading to a paternal chromosome-like expression pattern. (F) TS may be caused by deletions in the paternally inherited chromosome, leading to absence of paternally expressed genes, (G) whereas KOS may be caused by deletions in the maternal chromosome, leading to absence of maternally expressed genes. Microdeletions involving the IG-DMR and/or the MEG3-DMR may also cause TS or KOS. Not drawn to scale.

Table 2

Genes in the Human Chr 14q32 and Mouse Chr 12 imprinted regions

Human symbola	Human gene name	Mouse symbol	Expression	Function	References
DIO3	Deiodinase, Iodothyronine Type III	Dio3	High biallelic expression in placenta and pregnant uterus; high expression in fetus, fetal liver, testis, bladder; preferential paternal expression in neonatal foreskin; preferential maternal expression in adult skin; region-specific preferential maternal expression of a larger DIO3 transcript in brain, similar to DIO3OS	Maintains low serum T3 concentrations in placenta; the DIO3 selenoenzyme catalyzes the conversion of T4 and T3 to the inactive metabolites rT3 and T2, respectively; may prevent premature exposure of developing fetal tissues to adult levels of thyroid hormones; regulates circulating fetal thyroid hormone concentrations; dysregulation may contribute to neurological symptoms	Hernandez et al. (74); Martinez et al. (77); Hernandez and Stohn (78)
DIO3OS (DIO3-AS1)	Deiodinase, Iodothyronine Type III, Opposite Strand	Dio3os (Dio3as)	LncRNA with 12 isoforms; expressed in most tissues with highest expression in testis, adrenal cortex, prostate, bladder, uterus, placenta, and fetal lung; region-specific imprinted expression in brain, similar to DIO3	Suggested role in maintaining paternal-only expression of DIO3; the 5′ end of two DIO3OS transcripts encode hsa-mir-1247	Hernandez et al. (74)
DLK1 (PREF1)	Delta-like non-canonical notch ligand 1	Dlk1	Paternal expression in fetus, hypothalamus, preadipocytes	An epidermal growth factor repeat-containing transmembrane protein; the protein is cleaved by TACE to generate an active soluble form; soluble DLK1 interacts with FN1 to activate integrin downstream MEK/ERK signaling, upregulate SOX9, and inhibit adipocyte differentiation; loss of DLK1 results in precocious puberty and increased adiposity	Dauber et al. (69,85)
MEG3 (GTL2)	Maternally expressed gene 3	Meg3 (Gtl2)	LncRNA—maternal expression with multiple transcripts; highly expressed in brain and pituitary	Unclear in humans; potential role in angiogenesis, brain development and function, and tumorigenesis from mouse studies	Gordon et al. (79)
MEG8	Maternally expressed gene 8	Rian (Meg8)	LncRNA—maternal expression with multiple transcripts that include the SNORDs; highly expressed in brain, uterus, and heart	Unknown	Charlier et al. (80)
RTL1 (PEG11)	Retrotransposon-like gene 1	Rtl1	Highly expressed protein at the late fetal stage in fetus and placenta; expression is 5-fold higher in the absence of RTL1as	Essential for normal development of placenta and fetus; maintenance of capillaries in the fetus	Ito et al. (67)
RTL1as (anti-PEG11)	Retrotransposon-like gene 1 antisense	Rtl1as	LncRNA encoding miRNAs − 431, −433, −127, −432, and − 136	Contains miRNAs targeting the RTL1 transcript in trans through an RNA interference (RNAi) mechanism	Seitz et al. (45); Davis et al. (81)
miRNAs (hsa-mir-#)	microRNAs	Mirg and miRNAs (Meg9)	Maternal expression of miRNA-154, miRNA-379, miRNA-544, miRNA-654 family members and other miRNAs; expressed throughout human vasculature	Translational repression and/or gene silencing of target mRNAs; potential role in vascular remodeling and Type II Diabetes; summary of individual miRNA’s involvement in disease pathogenesis in Benetatos et al. 2013 (73)	Benetatos et al. (73); Goossens et al. (82); Kameswaran et al. (83)
SNORDs (orphan snoRNAs)	C/D box small nucleolar RNAs	Snord(s)	Multiple tandem copies of SNORD-112, −113, −114 are highly expressed ncRNAs from maternal chromosome; differential expression in brain and throughout human vasculature	General role in posttranslational rRNA modification, pre-mRNA splicing, and polyadenylation of genes; Chr 14q32 SNORDs direct 2’O-ribose-methylation via Fibrillarin of non-canonical RNA targets; potential roles in vascular remodeling and cardiovascular disease	Hakansson et al. (84)

^aPrevious gene symbols are shown in () below the current gene symbol.

Genetic and Epigenetic Testing

A diagnosis of TS or KOS based on clinical characteristics alone may sometimes be difficult, especially if symptoms are mild. The rarity of these disorders along with features that resemble those in more common imprinting syndromes may contribute to misdiagnosis. For example, TS can be considered an undergrowth disorder, with some features (e.g. SGA, short stature, hypotonia and speech delay) resembling those found in PWS or SRS (11,22). Although a hallmark feature of TS is precocious puberty, this diagnosis cannot be made in infants. Similarly, KOS can be considered a disorder of overgrowth, with some prenatal features (e.g. placentomegaly, omphalocele and fetal macrosomia) resembling those found in BWS (10,27).

Genetic and epigenetic platforms that could collectively analyze and distinguish among genomic imprinting disorders (such as PWS, AS, BWS, SRS and now TS and KOS) are needed to aid clinical diagnoses. Although single nucleotide polymorphism microarrays detect isodisomy and large deletions; next-generation sequencing can define deletion breakpoints and other alterations. Platforms to assess the entire methylome exist, but regional analysis of DNA methylation status must be ordered individually for each locus using methylation-specific (MS) multiplex ligation-dependent probe amplification (MS-MLPA) or MS-PCR (35). Recently, a DNA methylation platform was specifically designed to detect multiple methylation abnormalities across a set of imprinting conditions and genes (EpiSign Complete) (36,37). These assays can confirm a clinical diagnosis as well as detail the genetic/epigenetic anomalies that underlie individual cases of TS and KOS. This determination is necessary to appropriately counsel families regarding recurrence risk. Although mosaicism has been reported in only a few cases of TS and KOS (32–34), the true number of mosaic individuals may be underestimated. Current tests have a high sensitivity to detect mosaicism, especially if multiple tissues are evaluated, as demonstrated in related imprinting disorders, such as BWS (38,39).

Imprinting at mouse distal chromosome 12

Imprinted genes at mouse chromosome 12aF1 were first suggested when embryonic lethality was observed for both maternal and paternal disomies or duplications of the distal portion of chromosome 12 in historical genetic studies using Robertsonian or reciprocal translocation heterozygote intercrosses (40). Indeed, this region contains the ~1 Mb Dlk1-Dio3 imprinted region syntenic with human chromosome 14 where TS and KOS map (41–48).

The largely conserved regulatory mechanisms within the Dlk1-Dio3 locus between mouse and human (see Figs 1 and 2A) (49) substantiate the use of genetic mouse models to explore the molecular basis for KOS and TS. The characteristic bell-shaped thorax, abdominal wall defects and placentomegaly characteristic of KOS is recapitulated in several mouse models with loss of maternally expressed genes (Megs) or overexpression of paternally expressed genes (Pegs). In contrast, while several mouse models with overexpression of Megs or loss of Pegs display the growth restriction and hypotonia observed in TS, these perturbations to the Dlk1-Dio3 locus do not cause the precocious puberty prevalent in TS patients. Subsequent discussion will focus on relating changes in gene expression within the locus to the phenotypes observed in these mouse models (summarized in Fig. 2B and C).

Figure 2

Mouse Dlk1-Dio3 Imprinted locus. (A) Located in the distal portion of chromosome 12, the mouse Dlk1-Dio3 spans ~1 Mb and shares similar regulatory mechanisms as the human DLK1-DIO3 locus (detailed in Fig. 1). The germline ICR, IG-DMR, is located ~15 Kb upstream of Meg3 and 70 Kb downstream of Dlk1. In mouse embryonic stem cells, CTCF binding to the unmethylated Meg3-DMR is important for monoallelic expression of Dlk1 (76). (B and C) Summary of KOS and TS mouse models described in this review. Gene deletions or insertions are aligned to the native locus illustrated above in (A). Striped boxes indicate gene deletions, while a filled triangle indicates gene insertion. Color filling designates the parental inheritance of the mutated allele.

Uniparental disomies of mouse chromosome 12

Paternal UPD of chromosome 12—PatUpd(12)—is 50% lethal by late gestation (E18.5) (50,51). In addition to recapitulating the skeletal and placental abnormalities of KOS, PatUpd(12) causes a mouse-specific phenotype of enlarged myofibers comprised of cells with centrally located nuclei resembling immature myotubules (50). Duplication of the paternal distal chromosome 12—PatDp(dist12)—phenocopies the whole chromosome duplication with increased severity of lethality by E16.5, implicating distal chromosome 12 as the location of the imprinted loci (52).

In agreement with human MatUPD(14) and TS, maternal UPD of chromosome 12—MatUpd(12)—is associated with a less severe phenotype than PatUpd(12) as pups can survive to term, but die perinatally due to severe respiratory distress (50). The consequence of overexpression of the maternally expressed non-coding transcripts within the Dlk1-Dio3 locus was further clarified using transgenic overexpression of Meg3 (also called Gtl2), Rtl1as and Meg8 (Meg3-Meg8 MatTg), which recapitulate the growth restriction and perinatal lethality observed in MatUpd(12) pups (53). In this model, expression of several downstream growth promoting genes are suppressed during embryonic development, suggesting that maternally expressed non-coding transcripts govern expression of growth regulators (53).

Deletions targeting IG-DMR

The germline ICR for the Dlk1-Dio3 locus is an 8 kb intergenic CpG island that is methylated on the paternal allele (Fig. 2) (44). Maternal transmission of IG-DMR deletion (IG-DMR^KO/+) leads to embryonic lethality by E16.5 and KOS-like phenotypes in the embryo (54). This deletion causes locus-wide loss of imprinting (LOI), including biallelic Dlk1 expression, overexpression of Rtl1 (4.5x), and Dio3 (2x), hypermethylation of the Meg3-DMR and silencing of downstream Megs expression (54,55). Placental defects are not observed in IG-DMR^KO/+ as explained by a less severe LOI in the IG-DMR^KO/+ placenta, downregulated expression of Megs and only modest upregulation of Dlk1, Rtl1 and Dio3 (54). This result suggests non-conserved placenta-specific imprinting regulation for the Dlk1-Dio3 locus. In contrast to the maternal deletion, paternal deletion of the methylated IG-DMR (IG-DMR^+/KO) is viable and exhibits no gene expression changes (55).

As described above, methylation at ICRs requires protection during postfertilization genome wide reprogramming (2,3). Recently, a model was developed to investigate the function of a tandem repeat array within the mouse IG-DMR (IG-DMR^+/ΔRep) (56). Paternal transmission of the tandem repeat deletion caused postfertilization loss of IG-DMR methylation, approximating a type of epimutation that is observed in TS cases (16,56–58). In this model, the paternal Meg3-DMR never acquired methylation, resulting in biallelic expression of maternal transcripts and the loss of Pegs expression (56). Both IG-DMR^+/ΔRep embryo and placenta become severely growth restricted beginning at E13.5, with pups dying shortly after birth. These results suggest that ZFP57 or ZFP445 binding motifs with the tandem repeats are required for ICR methylation protection (2,3,49,56).

Meg3-DMR and promoter mutations

The 5′ end of Meg3 gains methylation on the paternal allele proximal to implantation (E6.5) (59). Maternal deletion of the Meg3-DMR (Meg3DMR-Exon5^KO/+) eliminates the functional Meg3 promoter and is accompanied by decreased expression of Rtl1as, Meg8 (Rian) and miRNAs from the Mirg clusters (60,61). Two independent lines of Meg3DMR-Exon5^KO/+ were developed, with the mutant described by Zhou and colleagues resulting in overexpression of Pegs (60,61). Regardless of the expression status of Pegs, neither knockout line develops KOS-like skeletal defects. Instead, perinatal lethality occurred in both models due to severe hypoplastic pulmonary alveoli and diaphragm muscle defects (60,61).

Paternal deletion of the Meg3-DMR activates downstream Megs expression from the normally silenced paternal allele and loss of expression of Dlk1, Rtl1 and Dio3 (60). These molecular phenotypes are also observed following gene insertion immediately upstream of the paternal Meg3-DMR, which is associated with hypomethylation of the DMR (Gtl2lacZPat, Gtl2Δ5’NeoPat) (62,63). These mutations cause severe growth restriction that is consistent with TS and the majority of mutant pups die neonatally. Proportional dwarfism remains in offspring that survive to adulthood, although precocious puberty is not observed in fertile adult mutants (60,63). Additional characterization of Gtl2lacZPat mutants shows disruption to the insulin-like growth factor-1 (Igf-1) signaling pathway and compromised glucose tolerance in the surviving animals (64). This finding may inform the underlying cause of endocrine and metabolic phenotypes observed in a subset of TS cases (11,12,18).

Single gene knockouts within the Dlk1-Dio3 locus

Because UPDs and large deletions or epimutations often cause misexpression of multiple genes in the KOS/TS critical region, it has been difficult to attribute phenotypes to specific genes. Single gene mutations, however, have provided more insight. A candidate etiology for KOS embryonic and placental phenotypes is the overexpression of RTL1 (65). In both mouse and human, miRNAs processed from the maternally expressed Rtl1as antagonize Rtl1 transcripts through an RNA interference mechanism (45). Maternal deletion of Rtl1as (Rtl1as-ko) (66) or miR-127 (ΔmiR127) (67) results in functional overexpression of Rtl1. Both deletions lead to placentomegaly with dilated fetal capillaries beginning at E16.5, consistent with KOS placentas. In contrast, embryos are unaffected by Rtl1 overexpression because pups develop normally (45,66), suggesting that Rtl1 protein is likely to be the most functional in the placenta.

Maternal deletion of miR-379/410 (miR-379/mir-410ΔMat) from the Mirg clusters causes poor neonatal survival due to inefficient activation of gluconeogenesis in the term fetus, a process that is required to survive parturition (68). This finding may better inform the developmental delay and feeding difficulties observed in a subset of KOS neonates (65).

In human, loss of function of the paternally expressed DLK1 gene is associated with familial cases of central precocious puberty, often unaccompanied by growth restriction that is characteristic of TS (69,70). In mouse, Dlk1 knockout (Dlk1^−/−, Dlk1^+/−) causes severe embryonic and postnatal growth retardation, typically resulting in perinatal death (71). Of Dlk1^−/− mice that survived to adulthood, no central precocious puberty was observed. Rather, Dlk1^−/− mice displayed metabolic phenotypes of hypercholesterolemia, hyperlipidemia and fatty liver disease when challenged on high fat diet, a feature reported in 10–20% of TS cases (12,22).

Conclusions

TS and KOS are complex imprinting disorders that largely manifest in physical defects necessitating obligatory supportive therapies early in the life of affected individuals. Improved understanding of the molecular mechanisms regulating the DLK1-DIO3 imprinted gene cluster have aided in the clinical diagnosis of TS and KOS, particularly in their differentiation from more common imprinting disorders. Clinical phenotypes associated with TS and KOS likely arise from complex interactions of multiple aberrantly expressed genes within the Dlk1-Dio3 cluster, as evidenced from the various single gene knockout or overexpression models that cannot precisely recapitulate TS (e.g. precocious puberty) or KOS (e.g. skeletal defects) phenotypes.

Importantly, alterations in gene-dosage within the Dlk1-Dio3 locus affect very early developmental processes including placentation, thus impacting fetal growth and musculoskeletal formation. Identification of targets of maternally expressed non-coding transcripts in this locus should be considered a priority for understanding TS and KOS. Emerging evidence shows that these non-coding transcripts can orchestrate expression of developmental growth regulators (53). Moving forward, knowledge gained from mouse models described here may inform treatment options for long-term management of TS and KOS patients through identification of druggable signaling pathways that are altered due to genetic and epigenetic mutations at the locus.

Acknowledgements

We thank Dr Jennifer Kalish and Dr Helio Pedro manuscript review. The authors acknowledge the astute observations and significant contributions of Dr Karen Temple, Dr Masayo Kagami, Dr Tsutomu Ogata and Dr Jin-Chen Wang, which led to the identification of TS and KOS as distinct genomic imprinting disorders.

Conflict of Interest statement. None declared.