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. Author manuscript; available in PMC: 2023 Sep 1.
Published in final edited form as: Am J Med Genet A. 2022 Sep 26;188(12):3469–3481. doi: 10.1002/ajmg.a.62972

TBX6 as a cause of a combined skeletal-kidney dysplasia syndrome

Guozhuang Li 1,2,3,*, Alanna Strong 4,5,*, Haojun Wang 6, Ji-Sun Kim 7, Deborah Watson 5, Sen Zhao 1,2,3, Courtney Vaccaro 5, Erum Hartung 8,9, Hakon Hakonarson 4,5,9,10, Terry Jianguo Zhang 1,2,3, Philip F Giampietro 7,11, Nan Wu 1,2,3
PMCID: PMC10473889  NIHMSID: NIHMS1920307  PMID: 36161696

Abstract

TBX6 encodes transcription-factor box 6, a transcription factor critical to paraxial mesoderm segmentation and somitogenesis during embryonic development. TBX6 haploinsufficiency is believed to drive the skeletal and kidney phenotypes associated with the 16p11.2 deletion syndrome. Heterozygous and biallelic variants in TBX6 are associated with vertebral and rib malformations (TBX6-associated congenital scoliosis, TACS) and spondylocostal dysostosis, and heterozygous TBX6 variants are associated with increased risk of genitourinary tract malformations. Combined skeletal and kidney phenotypes in individuals harboring heterozygous or biallelic TBX6 variants are rare. Here we present 7 individuals with vertebral and rib malformations and structural kidney differences associated with heterozygous TBX6 gene deletion in trans with a hypomorphic TBX6 allele or biallelic TBX6 variants. Our case series highlights the association between TBX6 and both skeletal and kidney disease.

Keywords: Congenital scoliosis, TBX6, Congenital anomalies of the kidney and urinary tract (CAKUT), Kidney dysplasia, Vertebral segmentation defects, Rib anomalies

Introduction:

TBX6 encodes transcription-factor box 6, a transcription factor that plays a critical role in paraxial mesoderm development and somitogenesis (Chapman et al., 1998). Knockout studies in zebrafish and mice support a role for TBX6 in skeletal development (Nacke et al., 2000; Watabe-Rudolph et al., 2002; Windner et al., 2015; Ren et al., 2020). Pathogenic variants in TBX6 are associated with congenital scoliosis in diverse populations (Wu et al., 2015; Liu et al., 2019; Otomo et al., 2019; Yang et al., 2019). Cohort-based studies suggest a dosage-dependent model where the combination of a pathogenic TBX6 variant in trans with a hypomorphic TBX6 variant is sufficient to cause disease, defined as TBX6-associated congenital scoliosis (TACS) (Wu et al., 2015; Nakamora et al., 2017; Takeda et al., 2017; Liu et al., 2019; Yang et al., 2019; Chen et al., 2020). The most commonly-reported hypomorphic allele is the T-C-A haplotype, defined by single-nucleotide polymorphisms (SNPs) at the positions of rs2289292, rs3809624 and rs38090627 (Sparrow et al., 2013; Wu et al., 2015; Lefebvre et al., 2017; Takeda et al., 2017; Otomo et al., 2019; Chen et al., 2020; Errichiello et al., 2020). Heterozygous nonsense and frameshift variants and biallelic missense variants in TBX6 have also been identified in individuals with the more severe skeletal dysplasia spondylocostal dysostosis, characterized by developmental vertebral and rib defects (Sparrow et al, 2013; Lefebvre et al., 2017; Otomo et al., 2019; Errichiello et al., 2020).

In addition to its role in skeletogenesis, TBX6 is expressed in the nephric mesenchyme and plays a role in kidney development (Concepcion et al., 2017; Yang et al., 2020; Hayashi et al., 2021). Loss-of-function variants in TBX6 cause kidney malformations and urogenital anomalies in mice (Nacke et al., 2000; Tewes et al., 2015; Chu et al., 2019; Verbitsky et al., 2019; Yang et al., 2020; Hayashi et al., 2021), and TBX6 haploinsufficiency is believed to drive the urogenital phenotype associated with the 16p11.2 deletion syndrome (Sampson et al., 2010; Verbitsky et al., 2019; Yang et al., 2020).

Costovertebral and urogenital malformations have both been described in association with TBX6 pathogenic variants; however, the combined phenotype is rare (Nacke et al., 2000; Tewes et al., 2015; Yang et al., 2020; Chen et al., 2021). Here we report 7 individuals with skeletal and kidney malformations found to have TBX6 insufficiency either caused by a heterozygous deletion of 16p11.2 including the TBX6 gene in trans with the T-C-A haplotype or by biallelic TBX6 variants. Our cases highlight the association between TBX6 and both skeletal and kidney malformations.

Methods:

Editorial Policies and Ethical Considerations:

Consent for participation was obtained from each family, and appropriate consent forms were signed. For the individual evaluated at The Children’s Hospital of Philadelphia (Patient 1), the study was approved by the Institutional Review Board (IRB, Protocol number 16–013278). For individuals evaluated in China (Patients 2 – 7), the study was approved by the IRB (JS-2364) of Peking Union Medical College Hospital (PUMCH) under the framework of the Deciphering Disorders Involving Scoliosis and COmorbidities (DISCO, http://www.discostudy.org/) study.

Genetic Testing Methodology:

Clinical duo exome sequencing (proband + mother) was performed on Patient 1 at GeneDx. After negative clinical exome testing, Patient 1 was recruited into the Center for Applied Genomics (CAG) at CHOP for exome reanalysis. Variant call format (VCF) was obtained from Gene Dx and reanalyzed using an in-house variant annotation, filtration and prioritization platform developed within the CAG. Variants were initially filtered at 0.5% gnomAD MAF and annotated with a combination of multiple tools and databases, including Variant Effect Predictor, HGMD, ClinVar, dbSNP, OMIM, HPO, PolyPhen-2 and SIFT, and a custom-built splice-site annotator. Variants were assigned a priority score of likelihood as the causal variant for the patient’s disease, ranked using a weighted combination of a) overlap with HPO terms, b) patient and family genotypes, c) predicted functional impact, d) inheritance modeling, e) presence in mutation databases such as HGMD and ClinVar; and other factors. The identified TBX6 variants were validated clinically by Invitae laboratories. For Patients 3 – 7, research exome sequencing was performed as part of the DISCO study (Zhao et al., 2021). Patients 2 was newly enrolled and were not reported in the previous studies. Raw data were processed using the Peking Union Medical college hospital Pipeline (PUMP) (Chen et al., 2021; Zhao et al., 2021). Gene Matcher was used to match these cases (Sobreira et al., 2015). The genomic coordinates of the 16p11.2 deletion are 29,638,676 – 30,188,531 on the Genomic Reference Consortium Human Build 38 platform.

Diagnostic Criteria for Congenital Scoliosis and Congenital Anomalies of the Kidney and Urinary Tract:

Congenital scoliosis (CS) was defined as individuals with idiopathic structural malformations of the vertebrae revealed by X-ray or CT resulting in lateral curvature of the spine greater than or equal to ten degrees. Congenital anomalies of the kidney and urinary tract (CAKUT) were detected via ultrasound (Patients 1) or MRI imaging (Patients 2 – 7) with independent interrogation of the left and right kidneys. Differences in kidney volumes of >2SDs below the means in age and ethnically matched controls were considered renal hypoplasia (Cain et al., 2010). Kidney volume was calculated using the equation 0.5233×length×width×thickness (Han et al., 1985). The reference values for kidney volume were obtained from a study of healthy Chinese children without a history of renal disease or congenital anomalies (Shi et al., 2015).

Results

Individuals with pathogenic variants in TBX6

Patient 1:

Patient 1 is of Guatemalan ancestry. He was born full-term via vaginal delivery to a 16-year-old G1P0→1 mother. There was no known family history of kidney disease or skeletal malformations. Birth weight was 2.95 kg (75%); birth length was 45 cm (25 – 50%). He was diagnosed with bilateral pupillary colobomas, cleft soft palate, sensorineural hearing loss, intestinal malrotation, feeding intolerance, hindgut duplication cyst, anemia, stage V chronic kidney disease, and short and bowed limbs. Kidney ultrasound demonstrated right to left cross fused renal ectopia and bilateral cystic dysplasia. Skeletal survey showed multiple vertebral segmentation defects and rib fusions. Follow up skeletal survey at 5 months of age was notable for wormian bones in the coronal sutures, widened coronal sutures, fusion of the right fourth and fifth ribs, seventh and eighth ribs, and the ninth through twelfth ribs, paired fusions throughout the left rib cage, multiple thoracic vertebral segmentation defects without scoliosis, a hemivertebrae at L4 with an associated scoliosis, sacral dysplasia, delayed ossification of the humeral head, femoral head, and hand ossification centers, metatarsal brachydactyly, and small phalanges, consistent with spondylothoracic dysostosis (Figure 1). Brain MRI demonstrated thin corpus callosum, prominent extra-axial spaces and overall immature brain parenchyma. Growth parameters at 5 months were notable for a weight of 4.88 kg (<3%: 50% for 5 weeks), a length of 52 cm (<3%; 50% for 2 weeks), and a head circumference of 40 cm (<3%; 50% for 2 months). Physical examination was notable for relative macrocephaly, mild plagiocephaly, coarse facial features with prominent cheeks, left esotropia, low-set ears with lower lobe hypoplasia, preauricular skin tags bilaterally, high palate, short arms, brachydactyly with spatula-shaped fingers and toes, broad thumbs, sandal gap, single palmar crease, dysplastic nails, joint hypermobility, doughy skin, and global hypotonia (Figures 2A–F). A chromosomal microarray (Integrated Genetics) was notable for regions of homozygosity on chromosomes 2, 5 and 16 encompassing 41 Mb (1.5% of the autosomal genome). These regions include 861 genes, 178 OMIM genes, and 58 OMIM genes with associated disorders. A skeletal dysplasia panel (Invitae Laboratories) was notable for heterozygous variants of uncertain significance in DVL1 (NM_004421: c.1379A>G; p.Asn460Ser), IHH (NM_002181: c.1222G>A; p.Gly408Arg), and SGSH (NM_000199: c.67C>T; p.Arg23Trp), and 1 homozygous variant of uncertain significance in TBX6 (NM_004608: c.699G>C; p.Trp233Cys). Duo exome sequencing was notable for a heterozygous variant of uncertain significance in PKD1 (NM_001009944.2: c.7906 C>T (p.Arg2636Trp)), which was not maternally-inherited. Research-based exome reanalysis was performed within the Center for Applied Genomics with a particular focus on ciliopathy genes and was notable only for the previously-reported variants. Patient 1 is currently 15 months of age. His clinical course has been complicated by kidney failure requiring hemodialysis, hypertension, anemia, and developmental delay.

Figure 1:

Figure 1:

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A) Chest view demonstrating 12 ribs bilaterally with multiple rib fusions, including right ribs 4–5, 7–8 and 9–12. There are multiple paired fusions of the left ribs. B) More detailed view of the left chest demonstrating multiple paired rib fusions C) Spine view demonstrating multiple thoracic vertebral segmentation anomalies without scoliosis and 6 lumbar hemivertebrae with scoliosis D) Left hand demonstrating brachydactyly, small phalanges, and diminished ossification of several ossification centers E) Right foot demonstrating brachydactyly and small phalanges.

Figure 2:

Figure 2:

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A) Frontal view demonstrating coarse facial features and prominent cheeks B) Right ear demonstrating lower lobe hypoplasia and preauricular skin tags C) Left hand demonstrating brachydactyly, broad thumbs, and spatula-shaped fingers D) Right hand demonstrating brachydactyly and single palmar creases E) Right foot with brachydactyly, sandal gap, and dysplastic nails F) Underside of right foot demonstrating brachydactyly and sandal gap

Patient 2:

Patient 2 is a 7-year-old male of Han Chinese ancestry. Family history is notable for a father and paternal grandfather with scoliosis. He was born full term via vaginal delivery to a 26-year-old mother after an uncomplicated pregnancy. Birth weight was 3.6 kg (75%). Current weight is 20 kg (10–25%) and length is 120 cm (50%). He has demonstrated normal development. He has been diagnosed with left inguinal hernia, kidney dysplasia with preserved kidney function, and multiple skeletal malformations, including multiple rib fusions and bifurcations, hemivertebrae, butterfly vertebrae and scoliosis. Exome sequencing and Massarray (Gabriel et al., 2009) showed a frameshift variant in TBX6 (NM_004608: c.1121_1122del; p.Pro374Argfs*112) in trans with the hypomorphic TBX6 T-C-A haplotype. Parental samples were not available.

Patients with the 16p11.2 deletion in trans with the T-C-A haplotype

Patient 3:

Patient 3 is a 9-year-old male of Han Chinese ancestry. Family history is notable for a paternal grandmother with scoliosis. He was born full term via caesarian delivery to a 30-year-old mother after an uncomplicated pregnancy. Birth weight was 3.45 kg (50%). Current weight is 40 kg (90–95%) and length is 139 cm (75%). He has speech delay and spoke his first words at 4 years of age. Other diagnoses include bilateral maxillary and ethmoid sinusitis, kidney dysplasia with preserved function, L4/5 hemivertebrae, L3/4 segmentation defects, and scoliosis. Exome sequencing and long-range PCR (Zhao et al., 2021) with subsequent Sanger sequencing validation showed a heterozygous 16p11.2 deletion in trans with the hypomorphic TBX6 T-C-A haplotype. Parental samples were not available.

Patient 4:

Patient 4 is a 15-year-old female of Han Chinese ancestry. She was born full term to a 32-year-old mother after an uncomplicated pregnancy. Current weight is 52 kg (50%) and length is 150 cm (7%). She has speech delay, kidney dysplasia with preserved function, absent left rib at T12, segmented right hemivertebrae at T12, lumbar sacralization, hypoplastic scapula, and congenital scoliosis. Exome sequencing and long-range PCR with subsequent Sanger sequencing validation showed a heterozygous 16p11.2 deletion in trans with the hypomorphic TBX6 T-C-A haplotype.

Patient 5:

Patient 5 is a 10-year-old female of Han Chinese ancestry. She has kidney dysplasia with preserved kidney function, butterfly vertebra at L1, congenital kyphosis, and scoliosis. Development has been normal. Growth parameters are not available. Exome sequencing and long-range PCR with subsequent Sanger sequencing validation showed a heterozygous 16p11.2 deletion in trans with the hypomorphic TBX6 T-C-A haplotype. Parental samples were not available.

Patient 6:

Patient 6 is a 7-year-old male of Miao ancestry. He was born full term via caesarian delivery to a 24-year-old mother after an uncomplicated pregnancy. Current weight is 30 kg (95%) and length is 130 cm (95%). He has demonstrated normal development, but carries a diagnosis of sinus tachycardia, kidney dysplasia with normal function, absent ninth and twelfth right ribs, segmented butterfly vertebra at T7, left hemivertebrae at T9 and T12, and hypoplastic scapulae. Exome sequencing and long-range PCR with subsequent Sanger sequencing validation showed a heterozygous 16p11.2 deletion in trans with the hypomorphic TBX6 T-C-A haplotype. Parental samples were not available.

Patient 7:

Patient 7 is a 12-year-old male of Han Chinese ancestry. He was born full term via caesarian delivery to a 21-year-old mother after an uncomplicated pregnancy. Current weight is 55 kg (25%) and length is 150 cm (75%). Development has been normal. He additionally has kidney dysplasia with preserved function, incomplete paralysis of the lower limbs possibly related to scoliosis surgery, missing ribs, T9 and T11 segmentation defects, left T10 hemivertebrae, kyphosis, and scoliosis. Exome sequencing and long-range PCR with subsequent Sanger sequencing validation showed a paternally-inherited heterozygous 16p11.2 deletion in trans with a maternally-inherited hypomorphic TBX6 T-C-A haplotype.

Discussion:

TBX6 encodes the protein transcription-factor box 6, a critical coordinator of skeletal and genitourinary tract development. TBX6 variants are associated with costovertebral malformations (Takeda et al., 2000; Watabe et al., 2002; Sparrow et al., 2013; Wu et al., 2015; Lefebvre et al., 2017; Nakamura et al., 2017; Otomo et al., 2019; Chen et al., 2019) and congenital anomalies of the kidney and genitourinary tract in animal models and humans (Takeda et al., 2000; Watabe et al., 2002; Sampson et al., 2019; Yang et al., 2020). Combined kidney-skeletal phenotypes associated with TBX6 loss-of-function have been reported in animal models of TBX6 insufficiency, whereas this association in TBX6-related human disease is rare (Tewes et al., 2015; Nacke et al., 2000; Chen et al., 2021). We report 7 individuals with combined skeletal and kidney phenotypes associated with biallelic TBX6 variants or TBX6 gene deletion in trans with the hypomorphic T-C-A haplotype, further supporting the role of TBX6 in combined skeletal-kidney disease (Table 1).

TABLE 1.

Clinical features and variant information of seven patients.

Patient - 4 Patient - 5 Patient - 6 Patient - 7
SCO1907P0219 SCO1908P0101 SCO1909P0008 SCO2003P1900
15 10 7 12
F F M M
Han NA Miao NA
N NA N N
16p11.2deletion 16p11.2deletion 16p11.2deletion 16p11.2deletion
NA NA NA NA
Deletion Deletion Deletion Deletion
Present Present Present Present
32 NA 24 21
Normal NA Normal Normal
N NA N N
NA NA Cesarean Cesarean
NA NA 3.0kg 3.3kg
NA NA NA NA
NA NA NA NA
52Kg NA 30kg 55kg
NA NA NA NA
150cm NA 130cm 150cm
N N N N
N N N N
N N N N
N N N N
N N N N
N N N N
N N N N
N N N N
N N N N
N N Sinus tachycardia N
N N N N
N N N N
N N N N
N N N N
N N N N
N N N N
N N N N
N N N N
Yes Yes Yes Yes
N N N N
N N N N
N N N N
N N N N
N N N N
N N N N
N N N N
N N N N
N N N N
delay without exat age NA NA NA
Language retardation Normal Normal Normal
N N N N
Normal Normal Normal Normal
Absent left rib of T12 N Missing rib Missing rib
Completely segmented right hemivertebrae of T12 L1 posterior butterfly vertebrae Butterfly vertebrae, Hemivertebrae Hemivertebrae
Y Y Y Y
N N N N
Completely segmented right hemivertebrae of T12 L1 posterior butterfly vertebrae Butterfly vertebrae, Hemivertebrae Hemivertebrae
N N N N
Lumbar sacralization, Hypoplasia of the scapula Kyphosis (hunchback) Hypoplasia of the scapula Kyphosis (hunchback)
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Combined kidney and skeletal phenotypes have been reported in the 16p11.2 deletion syndrome, a recurrent ~600 kB deletion that includes TBX6 and is characterized by intellectual disability, autism, obesity, vertebral anomalies, and structural kidney differences of variable severity (Sampson et al., 2010; Al-Kateb et al., 2014; Ching et al., 2021). SH2B1, a signaling adaptor protein, and MAZ, a zinc-finger transcription factor, were hypothesized to contribute to the skeletal and kidney phenotypes of the 16p11.2 deletion syndrome; however, TBX6 is believed to be the dominant phenotype driver (Sampson et al., 2010; Nik-Zainal et al., 2011; Haller et al., 2018; Sadler et al., 2019; Sampson et al., 2019; Chung et al., 2021). It is unclear why some individuals with the 16p11.2 deletion syndrome have skeletal and kidney phenotypes while others do not. One explanation is a hypomorphic TBX6 allele in trans to the deletion, such as the T-C-A haplotype, causing a further decrease in overall TBX6 expression. This phenomenon may explain the combined phenotypes seen in Patients 3 – 7.

Patient 2 presented with left inguinal hernia, kidney dysplasia, multiple rib fusions and bifurcations, hemivertebrae, butterfly vertebrae and scoliosis and was found to have a heterozygous frameshift variant in TBX6 in trans with the T-C-A haplotype. Heterozygous TBX6 frameshift, splice and extension variants have been associated with autosomal dominant spondylocostal dysostosis, distal vaginal atresia, and Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome (Sparrow et al., 2013; Chu et al., 2019; Errichiello et al., 2020; Chen et al., 2021). It is unclear why the Pro374Argfs*112 TBX6 variant identified in Patient 2 caused such severe disease. Possible explanations include a gain-of-function/dominant-negative mechanism, stochastic epigenetic modifications that resulted in preferential silencing of the wild-type allele, or influences of genetic background such as the T-C-A haplotype.

Patient 1 presented with severe, multiorgan disease including not only skeletal and kidney malformations but also colobomas, cleft palate, hearing loss, intestinal malrotation, brain malformations, and developmental delay. Panel testing, exome sequencing, and research-based exome reanalysis were notable for the previously reported TBX6 missense variants (Trp233Cys) and the heterozygous DVL1 variant (Asn460Ser). The Trp233Cys TBX6 variant resides within the T-box domain, which is the portion of TBX6 that binds DNA. This variant has previously been identified in trans with a different missense variant in a child with kyphosis, short stature, low weight, multiple vertebral segmentation defects and rib anomalies (Lefebvre et al., 2017). In vitro studies support pathogenicity, with the variant causing TBX6 mislocalization in cells (Otomo et al., 2019). Multiple missense variants have been reported as causal for TBX6-related disease and many disrupt TBX6 localization; however, this degree of multi-organ involvement has not been described previously.

Patient 1 was initially suspected to have a ciliopathy-spectrum disease. We performed region of homozygosity analysis and research-based exome reanalysis within The Center for Applied Genomics with a focus on genes related to the cilium to identify a potential comorbid ciliopathy; however, no variants of interest were detected. Of note, Tbx6 deficiency has been reported to interfere with the motility, morphology and calcium signaling ability of the nodal cilium in mouse embryos and cause heterotaxy-spectrum disease (Hadjantonakis et al., 2008; Conception et al., 2018). Though not specifically attributed to TBX6 haploinsufficiency, ciliary dysfunction is believed to contribute to the 16p11.2 deletion phenotype (Migliavacca et al., 2015). We hypothesize that homozygosity for a missense variant that localizes within the T-box DNA binding domain interferes with the nodal and primary cilium causing the severe, ciliopathy-spectrum disease seen in this individual.

It is also possible that Patient 1’s severe phenotype is related to multiple hits on developmental pathways. Specifically, Patient 1 has a missense variant in DVL1 (c.1379A>G; p.Asn460Ser). This variant was not detected in his mother; paternal sample was not provided. Heterozygous pathogenic variants in DVL1 are associated with autosomal dominant Robinow syndrome. Patient 1 shares many features with Robinow syndrome, including coarse facial features, ear malformations, cleft palate, brachydactyly, kidney differences and developmental delay. The Asn460Ser missense variant is predicted to have a deleterious effect on protein function by Polyphen and SIFT and though it is reported in gnomAD in the heterozygous state in 2 individuals of Latino-Admixed American descent, it is present at a low allele frequency (2/250372 alleles). Importantly, heterozygous DVL1 variants reported in Robinow syndrome are typically truncating and not missense variants (Bunn et al., 2015; White et al., 2015). DVL1 is a component of the Wnt signaling pathway. TBX6 is involved in the NOTCH (Hofmann et al., 2004; White et al., 2005; Hadjantonakis et al., 2008), Wnt (Wittler et al., 2007; Dunty et al., 2008) and BMP (Szeto et al., 2004; Chen et al., 2009) signaling pathways. It is possible that the combination of a mild DVL1 variant with biallelic TBX6 variants sufficiently disrupted developmental signaling to cause a Robinow-like phenotype. Exacerbation of DVL1-associated disease by second variants in related morphogen genes has been previously reported (Mishra et al., 2020).

We have presented 7 cases that highlight an association between TBX6 variants and combined skeletal and kidney phenotypes. Limitations of our study include incomplete clinical records for Patients 2 – 7 and a small sample size, which make it difficult to establish genotype-phenotype correlations. Specifically, the causal variants identified in our cohort are diverse, including biallelic missense variants (Patient 1), a heterozygous frameshift variant in trans with the T-C-A haplotype (Patient 2), and a 16p11.2 deletion including the TBX6 gene in trans with the hypomorphic T-C-A haplotype (Patients 3 – 7). It remains unclear why some individuals with TBX6 variants have severe disease. One possible explanation is incomplete phenotyping of individuals with TBX6-related disease, including the 16p11.2 deletion syndrome. Without clinical signs of kidney disease or skeletal dysplasia/scoliosis, imaging may not be obtained leading to a falsely low estimate of the true prevalence of skeletal and kidney phenotypes in TBX6-related disease. We propose that individuals with the 16p11.2 deletion have designated spine films/skeletal survey, kidney imaging, and kidney function testing to identify potential subclinical or mild disease. Another explanation is the presence of other hypomorphic TBX6 variants in trans with the 16p11.2 deletion or additional variants in related genes that encode components of developmental pathways, similar to Patient 1. Based on this model, we would recommend that individuals with significant skeletal and kidney comorbidities undergo additional genetic testing for hypomorphic TBX6 variants or possible modifier genes.

In summary, we propose that TBX6-related disease be considered in the differential of costovertebral and kidney disease and that individuals with TBX6 variants be screened for vertebral and kidney phenotypes.

Acknowledgements:

The authors would like to thank the families for their participation in this study. The authors would also like to thank their funding sources: the CAMS Innovation Fund for Medical Sciences (CIFMS, 2021-I2M-1-051 to J.Z. and N.W.), National Natural Science Foundation of China (82072391 to N.W., 81972037 and 82172382 to J.Z.), Beijing Natural Science Foundation (JQ20032 to N.W.), the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (No. 2019PT320025), K08 Mentored Career Development Award K08DK128606 (A.S.), the Institutional Development Fund (H.H.), and the R03 HD099516-01A1 (P.F.G). We thank GeneSeeq Inc. for exome sequencing technical support. We also thank Beijing Ekitech Co. Ltd. for support in bioinformatic analyses and multimodal data management.

Footnotes

Conflict of Interest Disclosure: None

Editorial Policies And Ethical Considerations: This study was approved by the institutional review board (IRB, protocol #16-013278) at The Children’s Hospital of Philadelphia and the IRB (JS-2364) at the Peking Union Medical College Hospital (PUMCH). The patients’ families provided informed consent to participate in this study as well as for publication.

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