Biallelic ERBB3 loss-of-function variants are associated with a novel multisystem syndrome without congenital contracture

The Erb-B2 receptor tyrosine kinase (ERBB)/HER family of receptor tyrosine kinases includes four transmembrane glycoprotein epidermal growth factor receptors (EGFR/HER1, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4) that play essential roles in the regulation of cell survival, proliferation, adhesion, and differentiation through activation of phosphatidylinositol 3-kinase (PI3K)/AKT and extracellular signal-regulated kinase (ERK) signaling pathways [1, 2]. The ERBB3 gene (NM_001982.3) is located on 12q13.2 and encodes a 1342-amino acid protein that is widely expressed in various tissues in humans and mice. The ERBB3 protein has a heregulin (HRG)-binding domain and can bind to and is phosphorylated by neuregulin (NRG)1 depending on interaction with integrins [3]. However, phosphorylated ERBB3 cannot directly transmit intracellular signals since it lacks an active kinase domain; instead, it activates downstream pathways via heterotypic interactions with other members of the EGFR family, with the ERBB2/ERBB3 heterodimer eliciting the strongest downstream response [4]. Somatic heterozygous activating mutations of the ERBB3 gene have been linked to the occurrence of numerous solid tumors including those of the prostate, bladder, gall bladder, colon, gastric, and breast [5‐7]. In addition, a heterozygous germline activating variant of the ERBB3 gene has been implicated in myelodysplastic syndromes [8].

In 2003, 23 cases of autosomal recessive congenital contractural syndrome (OMIM#607598) were reported in a large Israeli Bedouin kindred pedigree; the phenotype included multiple joint contractures, anterior horn atrophy in the spinal cord, and an unusual distended urinary bladder [9]. Subsequent work identified a homozygous germline splice variant (c.1184-9A > G) of the ERBB3 gene in two pedigrees that was predicted to produce a frameshift variant (p.G395Afs*4) [10, 11]. This was the first report of a human phenotype that might result from loss of function of the ERBB3 gene. Recently, a homozygous missense variant (c.3425C > T; p.P1142L) of ERBB3 was reported in a patient with knee dislocation and bilateral hip dislocation [12]. However, due to the limitation of the number of cases and the lack of definite evidence from functional studies, there is still insufficient evidence of a link between germline loss-of-function variants of the ERBB3 gene and Mendelian phenotypes.

ERBB2 is activated through heterodimerization with other EGFR family members; however, it fails to bind to HRG; instead, it depends on HRG to bind to ERBB3 or ERBB4. The ERBB2/ERBB3 heterodimer functions as an oncogenic unit that drives tumorigenesis; for instance, ERBB3 phosphorylation is increased in many ERBB2-overexpressing breast tumors [18‐20]. Additionally, ERBB2 and ERBB3 gain-of-function mutations have been shown to contribute to the occurrence and development of a variety of cancers [17]. However, there is limited knowledge of the loss-of-function phenotype of ERBB3 in humans. In this study, we report for the first time that germline loss-of-function variants of the ERBB3 gene are associated with the development of multiple congenital deformities. We identified compound heterozygous variants (c.1253 T > C;p.I418T and c.3182dupA;p.N1061Kfs*16) in the ERBB3 gene; the former had no impact on ERBB3 expression or interaction with ERBB2 protein, but impaired ERBB2 and ERBB3 phosphorylation and blocked the activation of AKT and ERK pathways, whereas the latter truncated protein that was also unable to activate downstream signaling. The following two points may explain why the ERBB3 (p.I418T)/ERBB2 heterodimer fails to induce signaling. Firstly, although the missense variant has no significant impact on the secondary structure of the protein (Fig. 3), it might alter the asymmetrical interaction of the heterodimeric complex, which is crucial for downstream signaling activation. It has been observed that the catalytically inactive ERBB3 can still be an ‘active’ participant in a heterodimeric complex [21, 22]. Secondly, ERBB3 possesses direct binding sites for the p85 subunit of PI3K, allowing activation of PI3K and its downstream signaling components independent of interaction with ERBB2 [23]. Thus, inactive ERBB3 may directly impair downstream signaling activation.

ERBB3 plays a critical role in early embryonic development in mice. Erbb3^+/− mice were healthy and fertile, while Erbb3^−/− mice were embryonic lethal at embryonic day (E)11.5 to E13.5 due to a lack of connective tissue in the atrioventricular valves, leading to blood reflux [24, 25]. These embryos also showed severe anomalies in brain development including stagnation of cerebellar development, an absence of Schwann cell precursors, mandibular division of the trigeminal nerve, and abnormal development of stomach, pancreas, and adrenal glands. In the Israeli Bedouin kindred pedigree, 17 patients died from soon after birth to 1 year old, possibly due to a complete lack of functional ERBB3 expression. In contrast, the I418T variant in our patient showed partial functional expression, which ensured a certain degree of growth and development.

To date, only two homozygous variants of the ERBB3 gene have been reported to cause Mendelian phenotypes in humans (Fig. 2c) [11, 12]. Due to a lack of detailed clinical information in the second report [12], we mainly compared the phenotypes of our patient to those observed in the Israeli Bedouin kindred pedigree [11]. Although there were some shared features such as cardiac malformation and ophthalmologic problems, several differences were observed (Table 1). Firstly, all 23 patients in the Israeli Bedouin kindred pedigree had multiple joint contracture and two had hip dislocation, while the patient reported by Alfares et al. also had hip and knee dislocation [12], suggesting that joint deformities arise from ERBB3 variation. However, our patient did not show any joint deformity. Secondly, half of the patients (12/23) in the earlier report had an enlarged urinary bladder and five exhibited kidney malformation, which were also absent in our patient. Thirdly, our patient showed more serious facial malformations along with developmental delay, asymmetrical limbs, immunodeficiency, liver damage, feeding difficulties, and anemia, which have not been previously described. The definite molecular mechanism by which ERBB3 maintains the normal growth and development of different organ tissues and cells remains unclear. Previous studies on the two ERBB3-related contractures as well as the murine knockout model also did not reveal any such mechanism. Considering the widespread expression of ERBB3 and the importance of AKT and ERK signaling in development, different mechanisms are very likely to exist in different organ tissues and cells. ERBB3 is known to primarily function as an oncogene for the activation of PI3K/AKT and ERK signaling to promote tumor cell proliferation or differentiation; hence, we believe that the inactivation of these pathways might limit cell proliferation, leading to dysplasia of multiple organs. Meanwhile, prolonged activation of PI3K/AKT and ERK signaling disrupts the regulation of cell growth and division, leading to the characteristic features of Noonan syndrome (OMIM# 163950) that is caused by germline variation in KRAS genes (PTPN11, SOS1, RAF1, LZTR1, etc.) [26]. Therefore, we assume that organ development requires the appropriate expression of these two pathways. Both over-activation and inactivation of these pathways will result in a complex phenotype. Moreover, the 23 patients reported by Landau et al. harbored the same homozygous variant, but showed variable phenotypes. Differences in the proportion of the mutation in different tissues might also be a cause of the phenotypic differences in these patients.

Our patient was suspected of having SRS at 7 months of age mainly based on lower limb asymmetry, as well as postnatal growth failure, protruding forehead, feeding difficulties, and a low BMI [27]. However, the lower limb asymmetry had almost completely recovered at 24 months of age. Moreover, she was not small-for gestational age (birth weight at − 1.9 SD), which is a primary feature of SRS; however, we could not obtain birth head circumference data for the patient, making it very difficult to assess the patient using the SRS criteria. Therefore, we prefer to define the patient’s condition as novel multisystem syndrome without congenital contracture.

Taking into consideration the substantial phenotype differences between our patient and the previously reported cases, we performed a trio-whole genome sequencing for the patient in an attempt to identify possibly detrimental intronic variants implicated in development disorders. However, no unexpected intronic variants were found (data not shown). Identification of more cases with ERBB3 loss-of-function variants and similar phenotypes will be helpful to establish the genotype-phenotype relationship in the future studies. Moreover, further studies should consider using animal models with the specific variant (I418T) identified in the patient and try to rescue the phenotype with wild type ERBB3 cDNA, rather than the mutant cDNA, in ERBB3-deficient cells derived from patients or animal models. Generation of an I418T knock-in cell line, for example in U2OS cells, is also necessary to study the molecular mechanism.

In summary, we reported the first patient with a novel multisystem syndrome associated with novel compound heterozygous variants of the ERBB3 gene. The in vitro functional results suggested that loss of function of ERBB3 is related to this human phenotype. It implies that the spectrum of features associated with ERBB3 variation is broader than previously thought, and provides new evidence that biallelic loss of function variants of ERBB3 may contribute to a developmental disorder involving multiple organ systems.