Missense variants in homeobox domain of PBX1 cause coracoclavicular ankylosis

doi:10.21203/rs.3.rs-5211072/v1

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Missense variants in homeobox domain of PBX1 cause coracoclavicular ankylosis

https://doi.org/10.21203/rs.3.rs-5211072/v1

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There have been several reports on heterozygous loss of function variants in PBX1 associated with congenital anomalies of the kidney and urinary tract (CAKUT). We report three patients harboring de novo heterozygous missense variants in PBX1, who did not have CAKUT, but instead presented with respiratory failure, developmental delay, and, the most important, a unique skeletal phenotype characterized by broad and short clavicles with coracoclavicular ankylosis and broad ischia with premature fusion of the ischiopubic synchondrosis. All the variants are clustered at the last portion of the homeobox domain. These phenotypes are consistent with mouse models with functional dysregulation in Pbx1 or its interacting factor, Emx2. This study reports a novel phenotype affecting the clavicle and ischia due to PBX1 variants and expands the clinical spectrum of PBX1-related disorder.

Health sciences/Medical research/Genetics research

Biological sciences/Developmental biology/Disease model

PBX1

broad clavicles

coracoclavicular ankylosis

homeobox domain

EMX2

CAKUT

PBX1 is a global transcriptional regulator, involved in the PBC family of TALE superclass homeobox genes, with a PBC domain and a TALE homeodomain [1]. Pbx1 interacts with several factors, including Emx2, and has a pleiotropic effect in embryonic development [2]. While Pbx1^+/− mice are viable, fertile, and display no apparent abnormalities, Pbx1^−/− mice are embryonic lethal and are associated with multiple organ abnormalities, including severe hypoplasia in the lungs, liver, stomach, gut, pancreas, and kidneys. Ectopic thymus, kidneys, and spleen are also demonstrated. The axial and appendicular skeleton of Pbx1 deficient mice have multiple malformations, likely to be caused by premature ossification and perturbation in chondrocyte proliferation [3].

In humans, truncating variants in PBX1 result in congenital anomalies affecting the kidney and urinary tract (CAKUT) [4, 5]. Additional malformations include external ear anomalies, abnormal branchial arch derivatives, heart malformations, diaphragmatic hernia, and developmental delay [6]. While skeletal abnormalities are prominent in Pbx1 deficient mice, little attention has been paid to the skeletal malformations in humans with PBX1 variants, except for the patient with hypoplastic clavicles by Riedhammer et al. [7].

Here, we report three children harboring de novo missense variants in the homeobox domain of PBX1, who showed a novel PBX1-related phenotype that is characterized by dysostosis of the clavicles and ischia, but not CAKUT phenotypes. They all showed respiratory failure due to laryngotracheomalacia in infancy.

Genetic analysis was performed using trio (Patient 1, 3) and targeted (Patient 2) full exome sequencing [8, 9]. The variants classification was according to the guidelines for the interpretation of sequence variants by American College of Medical Genetics and Genomics [10]. Clinical details of the three patients are included in the Supplementary materials and summarized in Table 1.

All three patients shared common skeletal features, including broad medial clavicles and lateral clavicular tapering with coracoclavicular ankylosis and broad ischia with premature fusion of the ischiopubic synchondrosis (Fig. 1). The coracoclavicular ankylosis represents a congenital fusion between the coracoid process of the scapula and the coracoid tubercle of the clavicle. The scapular body may be mildly hypoplastic as compared with the scapular spine. The thoracic cage was mildly narrow and slim. Limb abnormalities were absent for the patients (Supplementary Figure). Respiratory failure at birth was also common but the severity was variable from dependence of mechanical ventilation (Patient 3) to a transient neonatal tachypnea (Patient 2). The respiratory failure may be caused by a narrow thoracic cage, neonatal hypotonia, and laryngomalacia with or without tracheomalacia. The patent ductus arteriosus (PDA) common to all patients might be due to the lung immaturity caused by the thoracic hypoplasia. Developmental delay was also common to the patients with variable degrees of delay ranging from mild to profound. Brain magnetic resonance image (MRI) was normal for Patient 1 and 2, and showed some non-specific abnormalities for Patient 3 likely due to long mechanical ventilation. Notably, there was no manifestation of CAKUT phenotype, except for hyper-retractile testes requiring orchidopexy in patient 1. Dysmorphic facial appearance was noted in patient 3, but overlapping facial features were not observed (Supplementary Figure).

We identified novel and de novo missense variants in PBX1 (Fig. 2). The variants were absent in the gnomAD (v2.1.3, v3.1.2) and were likely pathogenic as with high scores of several predictive values including > 25 of CADD [11, 12] or > 0.9 of REVEL [13]. Other candidate variants were unlikely to be the cause.

We describe three individuals with a unique skeletal phenotype, associated with novel heterozygous missense variants of the PBX1 homeobox domain. PBX1 shows high scores of being loss-of-function intolerant (pLI = 1), and missense Z (3.83) in the constraint metrics. Given the different clinical phenotype of previously reported individuals with PBX1 haploinsufficiency, the molecular pathogenesis of these missense variants would be hypomorphic effects with or without dominant-negative mechanism.

The variant p.Arg290Leu of patient 3 occurs at the same amino acid position p.Arg290Trp reported by Ruscitti et al.[14]. The patient described by Ruscitti et al. had cleidosternal dysostosis, pulmonary hypoplasia, complex cardiovascular anomalies, and intestinal malrotation, suggesting PBX1 missense variants may cause a spectrum of abnormalities in the shoulder girdle. In a previous study by Mathiasen et al., it was observed that the substitution of p.Arg290 into Alanine in the highly conserved homeodomain of PBX1 resulted in a marked reduction in DNA binding activity [15]. Based on the Mathiasen’s results combining their own results, Ruscitti et al. suggested that the structural change of homeobox domain could lead to the loss of function of PBX1 in their patient with p.Arg290Trp [14]. In line with these results, it is assumed that our patients’ phenotypes, with common skeletal manifestations and tightly clustered variants, may be caused by a functional hypomorphs of PBX1.

The skeletal features were unique, and the hallmarks included clavicular dysplasia with coracoclavicular fusion and dysplastic ischia with premature fusion of the ischiopubic synchondrosis. The scapular wing and thorax were mildly hypoplastic. Other common phenotypes include a variable degree of developmental delay, respiratory failure associated with laryngomalacia or tracheomalacia, and minor facial dysmorphism. Unlike previously reported patients with PBX1 pathogenic variants, the three patients did not have CAKUT.

PBX1 has a crucial role in embryonic development. Pbx1-deficient mice (Pbx1-knockout mice) show aplasia of multiple organs and defects of the axial and appendicular skeleton resulting from abnormalities in chondrocyte proliferation and differentiation [3]. The skeletal abnormalities include severely hypoplastic sternum, vertebral and rib malformations, hypoplastic scapulae and clavicles, and a malformed pelvic girdle. Notably, the distal fore- and hindlimbs were intact [3]. The present individuals had malformation of the shoulder and pelvic girdles. However, the skeletal phenotype of our patients was much milder than that of Pbx1-knockout mice. The major abnormalities were confined to the clavicle and ischium. However, the distal clavicular tapering and coracoclavicular ankylosis in the present patients may recapitulate “the attenuated clavicle” [3] and “the coracoid expansion” [2] in Pbx1-knockout mice, respectively. In Pbx1-knockout mice, the ischium was described to be rudimentary [3]. In the present individuals, the ischium appeared thick, and may have shown early ossification as compared with that of Pbx1-knockout mice. The genetic interaction between Pbx1 and Emx2 has a crucial role in shoulder girdle development in interacting with homeodomain proteins [2]. The alteration in the interaction of Pbx1 and Emx2 resulting the expression reduction of Sox9, leading to pelvic girdle malformation [16]. In line with these results, the missense variants of the homeobox domain in PBX1 could alter the function of the PBX1-EMX2 complex, leading to the unique phenotype of our patients.

However, several questions remain. The first is why variants associated with scapuloclavicular and ischial dysplasia are concentrated in the last portion of the homeobox domain of PBX1 (Fig. 2). Skeletal evaluation of patients with homeobox domain variants is limited, and additional patients would need to be added to confirm the relationship between skeletal features and missense variants. Second is why patients with haploinsufficiency of PBX1 do not show overt skeletal abnormalities, including scapulae [4, 7]. There are a few reports that brought attention to alterations of the skeleton including the shoulder girdle in PBX1 haploinsufficiency. However, the skeletal changes of the shoulder girdle are so mild that they are essentially different from what we reported in this report. The correlation between the skeletal abnormalities and variant effects is likely to be incomplete. Finally, pathogenic variants of EMX2 have never been reported to cause skeletal dysplasia in humans [17]. Comprehensive skeletal evaluation in PBX1-related diseases is needed to determine the functional impact of PBX1 and its partners. Further study with Pbx1 knock-in mice with missense variants will be required.

In summary, we described three patients characterized by broad clavicles and ischia, scapuloclavicular fusion and respiratory failure due to laryngomalacia, but without CAKUT phenotype, associated with the missense variants in the homeobox domain of PBX1. Further radiographic analysis of patients with PBX1-related disorder and the generation of animal models with missense variants in the PBX1 homeobox domain will provide insights into the underlying mechanism of cleidoischial dysostosis.

DATA AVAILABILITY

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

ACKNOWLEDGEMENTS

We thank the patients and their families for participating in this research.

AUTHOR CONTRIBUTIONS

MI, KES, KM, LAL, TK, YE, HS, TK, K Kosaki, MSP, K Kurosawa, and GN analyzed and interpreted the data; MI, KES, KM, LAL, TS, SM, NA, MSP, and K Kurosawa, and GN interpreted and described clinical findings; MI, KES, KM, LAL, MSP, K Kurosawa, and GN wrote the manuscript; all authors reviewed and discussed the manuscript during preparation and approved the final manuscript.

FUNDING

This work was supported by the Initiative on Rare and Undiagnosed Diseases (IRUD) (20ek0109301) from the Japan Agency for Medical Research and Development (AMED).

COMPETING INTERESTS

The authors declare no competing interests.

ETHICAL APPROVAL AND CONSENT TO PARTICIPATE

The procedures employed were reviewed and approved by the central ethics committee at Tohoku University on November 27, 2018 (CEC No. 201-2-216). Informed consents were provided by the UBC, CCAOC, and KCMC patients to publish this report.

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Table 1 is available in the Supplementary Files section.

There is no duality of interest

Table12024.10.4.xlsx
Table 1
Supplementarymaterials241014b.pdf
Supplementary materials

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Missense variants in homeobox domain of PBX1 cause coracoclavicular ankylosis

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INTRODUCTION

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