TRPV6 compound heterozygous variants result in impaired placental calcium transport and severe undermineralization and dysplasia of the fetal skeleton

Transient receptor potential vanilloid 6 (TRPV6) functions in tetramer form for calcium transport. Until now, TRPV6 has not been linked with skeletal development disorders. An infant with antenatal onset thoracic insufficiency required significant ventilatory support. Skeletal survey showed generalized marked undermineralization, hypoplastic fractured ribs, metaphyseal fractures, and extensive periosteal reaction along femoral, tibial, and humeral diaphyses. Parathyroid hormone (PTH) elevation (53.4–101 pmol/L) initially suggested PTH signaling disorders. Progressively, biochemical normalization with radiological mineralization suggested recovery from in utero pathophysiology. Genomic testing was undertaken and in silico protein modeling of variants. No abnormalities in antenatal CGH array or UPD14 testing. Postnatal molecular genetic analysis found no causative variants in CASR, GNA11, APS21, or a 336 gene skeletal dysplasia panel investigated by whole exome sequencing. Trio exome analysis identified compound heterozygous TRPV6 likely pathogenic variants: novel maternally inherited missense variant, c.1978G > C p.(Gly660Arg), and paternally inherited nonsense variant, c.1528C > T p.(Arg510Ter), confirming recessive inheritance. p.(Gly660Arg) generates a large side chain protruding from the C‐terminal hook into the interface with the adjacent TRPV6 subunit. In silico protein modeling suggests steric clashes between interface residues, decreased C‐terminal hook, and TRPV6 tetramer stability. The p.(Gly660Arg) variant is predicted to result in profound loss of TRPV6 activity. This first case of a novel dysplasia features severe but improving perinatal abnormalities. The TRPV6 compound heterozygous variants appear likely to interfere with fetoplacental calcium transfer crucial for in utero skeletal development. Astute clinical interpretation of evolving perinatal abnormalities remains valuable in complex calcium and bone pathophysiology and informs exome sequencing interpretation.

Progressively, biochemical normalization with radiological mineralization suggested recovery from in utero pathophysiology. Genomic testing was undertaken and in silico protein modeling  (Bonafe et al., 2015).
TRPV6 is relevant to calcium absorption (Fecher-Trost, Wissenbach, & Weissgerber, 2017). TRPV6 gene location and expression varies across species; in humans, TRPV6 is situated on chromosome 7q33-q34 and is expressed in prostate, placenta, epididymis, and exocrine pancreas (Wissenbach et al., 2001). Research has focused on the potential contribution of TRPV6 overexpression in prostate and breast cancer. Fewer reports address underexpression: the trpv6 −/− knockout mouse has hypofertility due to altered calcium homeostasis in the epididymis and in humans reduced placental expression is associated with preeclampsia (Haché et al., 2011). Until now, TRPV6 has not been linked with disorders of bone mineralization or skeletal dysplasia. We describe a patient with this phenotype and compound heterozygote TRPV6 variants occurring in trans, illustrating how astute clinical interpretation of evolving perinatal calcium and bone pathophysiology informed the interpretation of trio exome sequencing.

| CASE
A term female infant required significant and prolonged ventilatory support from birth due to severe thoracic insufficiency, accompanied by skeletal and biochemical abnormalities. Management included difficult discussions around survival. Therefore, identifying the underlying diagnosis in this neonate with rare bone disease was invaluable to better inform prognosis.
Antenatal abnormalities were noted early in pregnancy on the 20/40 gestation scan: small chest, unusual rib configuration, short but straight long bones, and no fractures identified. Pregnancy was complicated by severe and reaccumulating polyhydramnios which required three amniotic fluid drainage procedures from 27/40 gestation. There was no preeclampsia. Parents were counseled regarding the severe phenotype of the potentially lethal skeletal dysplasia, with no clear diagnosis.
Elective induction of labor at 39 1 /40, emergency cesarean section delivery due to fetal distress, birth weight 3.6 kg, Apgars Early biochemical abnormalities featured markedly elevated PTH 53.4 rising to 101 pmol/L (normal 1.1-6.9 pmol/L), normocalcemia FIGURE 1 Clinical and radiological findings. (a) The bell-shaped chest was associated with respiratory distress. (b-i) The skeletal survey at the age of 2 weeks showed generalized undermineralization, short, thin, and fractured ribs, absence of Wormian bones and normal vertebrae. The long bones showed a similar pattern of metaphyseal irregularities with corner fractures and periosteal reaction most obvious along the diaphyses of femora, tibiae and humeri. (j, k) Radiographs of chest and femur at the age of 10 weeks, showed broader, longer ribs, partial resolution of the metaphyseal lesions, and improved bone mineralization [Color figure can be viewed at wileyonlinelibrary.com] corrected calcium 2.43 mmol/L, normophosphatemia 1.4 mmol/L, normal Alkaline Phosphatase (ALP) 289 IU/L, normal urinary calcium/creatinine ratio 1.05, and vitamin D insufficiency 29 nmol/L. Parental biochemistry was normal.
PTH normalized, serum calcium did not rise, including after cinacalcet cessation. Transient mild hypocalcemia and hypophosphatemia occurred at weeks 3-4.
At the age of 6 weeks, X-rays showed improved rib mineralization and width and there was partial resolution of long bone periosteal abnormalities (Figure 1j,k). Ongoing metabolic bone strategy was oral calcium provision as substrate to facilitate bone mineralization and routine vitamin D supplementation (400 IU/day). The ventilatory requirement remained significant but sufficiently improved, with a decision to commence long-term ventilation through a tracheostomy from 8 weeks.

| METHODS
Editorial Policies and Ethical Considerations: written consent was obtained from the patient's parents for publication of clinical photographs and clinical information.
Whole exome sequencing was performed with DNA samples from the patient and her unaffected parents using the Agilent SureSelect All Exon v6 system, with sequencing on an Illumina NextSeq 500.
Likely causative variants were confirmed by Sanger sequencing.

| RESULTS
Antenatal genetic tests were normal including Array comparative genomic hybridization (aCGH) and investigation for paternal UPD14 (Kagami-Ogata syndrome), undertaken in view of the thoracic appearance.
Postnatally, careful clinical assessment suggested skeletal developmental pathology was the likely primary mechanism resulting in small lungs instead of primary lung pathology (pulmonary hypoplasia).
Biochemical abnormalities were not typical of Jeune asphyxiating thoracic dystrophy (in which constrained bone growth would continue postnatally). In contrast, correcting metabolic bone abnormalities could improve bone development aiding rib growth to facilitate lung function.
Initial postnatal genetic testing adopted a targeted single gene approach to explore NSHPT; the initial working diagnosis supported by periosteal changes and marked PTH elevation, although absent hypercalcemia was contradictory. Sanger sequencing of the CASR gene did not identify biallelic inactivating variants. We performed whole exome sequencing in the proband to test for variants in the

| DISCUSSION
This case adds significantly to understanding of TRPV6 function in humans. We hypothesize that structural/quantitative alterations in TRPV6 in our patient reduced placental calcium transfer and resulted in severely compromised in utero skeletal development and  6bo8); chains A-D are colored cyan, yellow, gray, and pink respectively; atoms of Gly660 are shown in all subunits as spheres colored by atom type (white, carbon; blue, nitrogen; red, oxygen), and are labeled for subunits A and D. The complex is oriented to show view in the plane of the membrane, represented by the gray bar, with the cytoplasmic region at the top. (b) As A, but rotated to show the view from the cytoplasm; the ion channel lies at the center of the tetramer. (c) Detail of the interface between the C-terminal hook of subunit A (cyan) and the N-terminal helix of subunit B (yellow); the position of the Gly660 backbone is indicated, and side chains shown in stick format, colored by atom type, for other relevant residues; residues from subunits A and B are labeled in black or blue font, respectively. Identification of compound heterozygous TRPV6 variants in this patient coincides with new information on how TRPV6 operates for calcium transfer. TRPV6 crystal structure has been recently described (McGoldrick et al., 2017;Saotome et al., 2016). The gene changes in our patient lie at the tetrameric interface crucial for calcium channel function (McGoldrick et al., 2017) and are predicted to destabilize the protein complex resulting in loss of calcium transport.
Although trpv6 is expressed in many mouse tissues, including intestine, expression in humans appears restricted to placenta, exocrine pancreas, and some exocrine glands (Fecher-Trost et al., 2017).
Our case showed effects of compromised placental calcium transfer, but did not demonstrate any exocrine pancreatic dysfunction features such as intestinal malabsorption. The absence of preeclampsia does not reinforce the hypothesis of TRPV6 dysfunction in preeclampsia (Haché et al., 2011). Our case showed normal intestinal calcium transfer during early infancy; although oral calcium supplementation was briefly required to compensate for skeletal calcium deficiency, ongoing normal milk calcium content achieved normocalcemia.
The TRPV gene family has previously been implicated in skeletal dysplasia: mutations in the TRPV4 gene are associated with skeletal dysplasia, arthritis-like, and neurological phenotypes including spondylometaphyseal dysplasia Kozlowski type, brachyolmia Type 3, metatropic dysplasia, spondyloepiphyseal dysplasia Maroteaux type, familial digital arthropathy-brachydactyly, Charcot-Marie-Tooth disease Type 2C, congenital distal spinal muscular atrophy, and scapuloperoneal spinal muscular atrophy (Krakow et al., 2009;Lamandé et al., 2014;Zimo n et al., 2010). Severe perinatal skeletal dysplasia has not been reported in association with TRPV6. Our case has intriguing similarities to severe antenatal-onset Caffey disease including polyhydramnios, hyperostosis, periosteal reaction, and progressive resolution of bony lesions over time (Schweiger et al., 2003), although we did not observe soft tissue swellings or fever. Antenatal Caffey disease is very rare and although heterozygote COL1A1 mutations have been identified in some cases (Gensure et al., 2005), genetic heterogeneity seems likely and TRPV6 may be a candidate worth investigation in unsolved cases.
Combined clinical and genetic expertise achieved diagnostic precision for this case and contributes to understanding of TRPV6 in human disease.

ACKNOWLEDGMENTS
The authors gratefully acknowledge the kind permission of the family to publish this case. The authors would like to thank clinical and radiological colleagues who contributed to case discussion. The trio exome sequence bioinformatics analysis pipeline was provided by Dr. Matthew Wakeling. The authors would also like to thank Andrew Parrish and Dr. Julia Baptista for their assistance with the exome sequencing data analysis. S.E. holds Wellcome Trust Senior Investigator Award 098395/Z/12/A.

CONFLICT OF INTEREST
The authors declared that they have no conflict of interest.

AUTHOR CONTRIBUTIONS
CB: Clinical care of patient including management of calcium homeostasis, recognition of candidate genes for phenotype, main author of this manuscript.