Cécily Lucas

PhD Kay-Sara Sauter

MSc Michael Steigert

MD Delphine Mallet

PhD James Wilmouth

MS Julie Olabe

MD Ingrid Plotton

PhD Yves Morel

PhD Daniel Aeberli

MD Franca Wagner

MD Hans Clevers

PhD Amit V Pandey

PhD Pierre Val

# Phd

Florence Roucher-Boulez

PhD # Pharmd

Christa E Flück
email: christa.flueck@dbmr.unibe.ch

Loss of human LGR4/GPR48 causes a novel syndromic form of severe neonatal saltwasting due to disrupted WNT signaling altering adrenal zonation

Keywords: mineralocorticoid deficiency, aldosterone synthesis, salt wasting, adrenal zona glomerulosa

ABSTRACT

BACKGROUND

Isolated mineralocorticoid deficiency causing potentially life-threatening salt-wasting has been associated with gene variants of aldosterone biosynthesis or resistance, but in some patients no variant is found. WNT/β-catenin signaling is crucial for differentiation and maintenance of the aldosterone producing adrenal zona glomerulosa (zG).

METHODS

Whole exome sequencing in a girl presenting with adrenal crisis and born into a consanguineous family with multiple perinatal deaths identified a splice variant in the WNT signaling regulator

LGR4. The variant was characterized by structural analysis, protein expression, binding studies, and confocal microscopy.

LGR4 inactivation was also analyzed by adrenal cortex specific ablation of Lgr4 in mice.

FINDINGS

The proband presented with failure to thrive, salt-wasting crises associated with isolated hypoaldosteronism, nail anomalies, short stature, and deafness. We identified a homozygous splice variant in the R-SPONDIN receptor LGR4 gene (c.618-1G>C). The resulting transcripts affected protein function and stability and resulted in loss of Wnt/β-catenin signaling. Inactivation of Lgr4 within the adrenal cortex in the mouse model caused decreased WNT signaling, aberrant zonation and reduced aldosterone production.

INTERPRETATION

LGR4 mutations establish a direct link between LGR4 inactivation and decreased canonical WNT signaling with abnormal zG differentiation and endocrine function. Thus genetic variants in WNT signaling and its regulators should systematically be considered in familial hyperreninemic hypoaldosteronism.

FUNDING

RESEARCH IN CONTEXT Evidence before this study

Human disorders of isolated mineralocorticoid deficiency with elevated renin are either caused by genetic defects of aldosterone synthesis (CYP11B2 variants) and aldosterone resistance, or remain genetically unsolved. Mineralocorticoids are produced in the zona glomerulosa of the adrenal cortex, which is controlled in its differentiation by WNT signaling. WNT4 and R-SPONDIN3 (RSPO3) are important drivers of WNT activation and zG differentiation, through stabilization of β-catenin, which stimulates expression of CYP11B2 and angiotensin II receptor AGTR1. Mice with Ctnnb1, Wnt4, or Rspo3 deficiency have reduced zG differentiation. However, mutations in the WNT signaling pathway in relation with mineralocorticoid deficiency, were not found in patients.

Added value of this study

Here, we show that homozygous human splice variants of LGR4 (the R-SPONDIN receptor) are associated with potentially lethal neonatal salt wasting in the context of a syndrome with isolated aldosterone deficiency, short stature, deafness, and nail anomalies. We further show that consistent with our patient's phenotype, conditional Lgr4 knockout in mice leads to aberrant adrenal cortex zonation and reduced aldosterone production. Our study demonstrates the central role of Wnt/b-catenin signaling for proper adrenal cortex formation and function in humans.

Implications of all the available evidence

Our data suggest that LGR4 variants and potential variants in other genes involved in the complex network of LGR4-Wnt/b-catenin signaling should be considered in patients presenting with a saltwasting crisis at birth, especially when manifesting with other syndromic features. Conversely, patients with known genetic alterations in WNT signalling should be carefully monitored for adrenal functions.

INTRODUCTION

Disorders of isolated mineralocorticoid (MC) deficiency are potentially life-threatening 1 . So far, they have been described in humans with primary defects in aldosterone biosynthesis or with MC resistance due to failure of aldosterone action. Patients mostly manifest in neonatal life with a salt-wasting crisis, e.g. dehydration, vomiting, and failure to thrive, due to high potassium, low sodium, metabolic acidosis, and high renin. The disorder becomes usually less severe with age.

In humans, aldosterone is the principal MC produced in the zona glomerulosa (zG) of the adrenal cortex where the CYP11B2 gene for aldosterone synthesis is expressed 2 . Aldosterone synthesis regulated by the renin-angiotensin-aldosterone (RAA) feedback loop, controls salt and water homeostasis and blood pressure.

Isolated hypoaldosteronism is mostly associated with autosomal recessive variants of the CYP11B2 gene, catalyzing aldosterone synthesis. However, a subset of typical cases of aldosterone deficiency, grouped under Familial Hyperreninemic Hypoaldosteronism (FHHA2) remains genetically unsolved [START_REF] Miller | Adrenal cortex and its disorders[END_REF] . Mutations in genes that regulate aldosterone biosynthesis, downstream of renin, including genes encoding angiotensin (I), the angiotensin-converting enzyme (ACE) or the angiotensin II receptor AGTR1 are associated with arterial hypotension in mice. In humans, these mutations are associated with renal tubular dysgenesis [START_REF] Krege | Malefemale differences in fertility and blood pressure in ACE-deficient mice[END_REF][START_REF] Tanimoto | Angiotensinogendeficient mice with hypotension[END_REF][START_REF] Sugaya | Angiotensin II type 1a receptor-deficient mice with hypotension and hyperreninemia[END_REF][START_REF] Gribouval | Mutations in genes in the renin-angiotensin system are associated with autosomal recessive renal tubular dysgenesis[END_REF] . However, they have not been linked with hypoaldosteronism. Other potential candidates comprise genes involved in the development and differentiation of the adrenal cortex.

Adrenal cortex physiology relies on functional zonation, essential for the production of aldosterone by the outer zG and glucocorticoids by the inner zona fasciculata (zF). The cortex undergoes constant cell renewal during postnatal life [START_REF] Pignatti | Adrenal cortex development and related disorders leading to adrenal insufficiency[END_REF] . This involves recruitment of subcapsular progenitor cells to zG fate and subsequent conversion to zF identity. This differentiation occurs in a centripetal manner, under the control of the WNT signaling pathway in zG and the PKA pathway in zF [START_REF] Drelon | PKA inhibits WNT signalling in adrenal cortex zonation and prevents malignant tumour development[END_REF] . WNT4 and R-SPONDIN3 (RSPO3) are important drivers of WNT activation and zG differentiation, through stabilization of β-catenin, which stimulates expression of CYP11B2 and angiotensin II receptor AGTR1 [START_REF] Berthon | WNT/beta-catenin signalling is activated in aldosterone-producing adenomas and controls aldosterone production[END_REF] . Consequently, mouse models with Ctnnb1, Wnt4, or Rspo3 deficiency have reduced zG differentiation [START_REF] Drelon | PKA inhibits WNT signalling in adrenal cortex zonation and prevents malignant tumour development[END_REF][START_REF] Heikkila | Wnt-4 deficiency alters mouse adrenal cortex function, reducing aldosterone production[END_REF][START_REF] Vidal | The adrenal capsule is a signaling center controlling cell renewal and zonation through Rspo3[END_REF][START_REF] Leng | beta-Catenin and FGFR2 regulate postnatal rosette-based adrenocortical morphogenesis[END_REF] . Conversely, constitutive WNT pathway activation resulting from activating CTNNB1 mutations or downregulation of negative WNT regulators is associated with the development of aldosterone-producing adenomas [START_REF] Boulkroun | Aldosterone-producing adenoma formation in the adrenal cortex involves expression of stem/progenitor cell markers[END_REF][START_REF] Berthon | Constitutive beta-catenin activation induces adrenal hyperplasia and promotes adrenal cancer development[END_REF] . Despite the central role of canonical WNT signaling in zG differentiation, mutations in this pathway have not been associated with hypoaldosteronism in humans so far. Here, we identified a loss of function splice variant of the R-SPONDIN receptor coding LGR4 gene (c.618-1G>C) in a girl born into a highly consanguineous family with a history of multiple perinatal deaths. The proband presented with failure to thrive, severe salt-wasting crises associated with isolated hypoaldosteronism, nail anomalies, short stature, and deafness. Our in silico, in vitro and in vivo studies establish a causal link between LGR4 inactivation, decreased canonical WNT signaling, abnormal zG differentiation and endocrine function. This suggests that anomalies in WNT signaling pathway regulators should systematically be evaluated in familial hyperreninemic hypoaldosteronism.

METHODS

Patients

The index patient was referred at the age of 17 years for adrenal insufficiency, short stature, deafness, developmental delay, and dysplastic nails (Figure 1 and Suppl Figure S1). She was born at term and manifested within days, with failure to thrive and signs of an adrenal salt-wasting crisis. Corticosteroid treatment was successfully installed without further investigations. Parents are first-degree cousins from Syria. The presence of the same phenotype in 3 siblings deceased in the neonatal period, and two cousins (with consanguineous parents) was indicative of an autosomal recessive disorder. Laboratory workup revealed normal cortisol response to ACTH stimulation, but hyperreninemic hypoaldosteronism (Table 1). Thus the diagnosis was revised to isolated mineralocorticoid deficiency and therapy continued with fludrocortisone, while genetic workup was initiated. Additional important findings were short stature, microcephaly, structural S1-4 and Table S1). Pubertal development was late with menarche at 16 years.

Of note, two cousins with the same clinical phenotype (one female and one male) are alive under steroid replacement therapy in Syria but are not available for investigations. A detailed description of the clinical findings is given in the Supplementary Appendix.

Laboratory studies

Studies in humans or on human material were conducted in accordance with Swissethics (KEK Bern ID 04/07). Written informed consent was obtained from all subjects. Animal experiments were approved by the Auvergne ethics committee (CEMEAA) APAFIS #39127.

We sequenced the exome of the affected child, her parents, and her two unaffected brothers (details in Supplementary Appendix 1). Primary fibroblasts of skin biopsies from the proband and healthy controls permitted LGR4 transcript analysis and studies of protein expression. The putative impact of the specific LGR4 variants was analyzed in silico using the three-dimensional structure of the human LGR4 extracellular domain in complex with a part of R-SPONDIN (PDB # 4KT1). The function of identified LGR4 variants was analyzed in vitro, using the TOP-Flash WNT signaling luciferase reporter assay in the presence or absence of RSPO1. Localization of mutant

LGR4 and interaction with RSPO1 was investigated by confocal microscopy in HEK293 cells expressing HA-tagged LGR4 and GFP-tagged RSPO1. The impact of LGR4 inactivation in vivo was analyzed by adrenal cortex specific ablation of Lgr4, using Lgr4 Flox/Flox mice mated with Sf1:Cre mice. Full experimental details are provided in the Supplementary Appendix.

Role of the funding sources

Authors confirm that the study sponsors played no role in study design, data collection, analysis, and interpretation of data; they were also not involved in the writing of the report and in the decision to submit the paper for publication.

RESULTS

In a highly consanguineous family from Syria, newborns were found to suffer from salt-wasting crises soon after birth due to isolated aldosterone deficiency. In addition, they revealed a common syndromic phenotype of nail dysplasia, deafness, growth restriction, and mental disability (Figure 1A).

Identification of a human LGR4 variant

After exclusion of CYP11B2 mutations, filtering of the exome sequences identified a homozygous mutation in the proband LGR4 gene: NM_018490:c.618-1G>C. Both parents and one brother were heterozygous for the same LGR4 mutation but showed no overt phenotype (Figure 1B; Suppl Table S2). The variant affects a splicing acceptor site, predicted to result in exon 6 skipping, and deletion r.618_689del or p.(His207_Leu230del). mRNA transcript analysis of patient's fibroblasts confirmed exon 6 skipping, but also identified a second transcript with an alternative acceptor site within exon 6, leading to a shorter deletion of -24 bp, r.618_641del or p.(His207_Arg214) (Figure 1C).

In silico analysis of LGR4 variants for prediction of pathogenicity

Leucine-rich repeat-containing G-protein-coupled receptors (LGR) are characterized by Leucine-Rich Repeats (LRRs) that provide the rigid structure of their large extracellular domain. LGR4 is a receptor for R-SPONDINS (RSPOs). The binding of RSPOs to LGR4 stimulates Wnt/β-catenin signaling pathway by inhibiting the E3-ubiquitin ligases ZNRF3/RNF43 [START_REF] Ordaz-Ramos | The Role of LGR4 (GPR48) in Normal and Cancer Processes[END_REF] . RSPOs bind to the first LRR and LRR3-LRR9 [START_REF] Petrie | In a Class of Their Own -RXFP1 and RXFP2 are Unique Members of the LGR Family[END_REF] . The amino acid deletions caused by the loss of 8 and 24 amino acids in

LGR4 are located in LRR7 and 8, in the extracellular domain of LGR4 (Figure 1D). A protein sequence alignment of LGR4 across species revealed that sections of LGR4 that comprise LRR7 and LRR8 are highly conserved (Suppl Figure S5). Regions of RSPOs that interact with LGR4 are conserved among RSPOs isoforms (Suppl Figure S6) and across species (Suppl Figure S7).

Mutations in the patient resulted in the deletion of parts of the LGR4 protein in its extracellular domain within LRR7 (for -8AA variant) and LRR7 and LRR8 (for -24AA variant). Structural analysis of contacts between LGR4 and RSPO1/RSPO3 showed that multiple contact points between the two proteins in the complex are located in LRR7 and LRR8 (Val204, His207, Asn226, Thr229, Tyr234 and Glu252) (Suppl Figure S8). A loss of multiple contact points due to the deletions would result in significantly weaker interaction between LGR4 and RSPOs and impact the overall structures of complexes involving other interaction partners (ZNRF3, RNF43, UBB, UBC etc). In addition, the deletions were predicted to result in loss of protein stability and decreased half-life, which could alter LGR4 protein expression levels in the patients. Together with weaker complex formation, lower protein levels should result in an overall loss of interaction of the -8AA and -24AA variants of LGR4 found in patients, with the -24AA variant predicted to have a higher impact.

Analysis of LGR4 protein expression in human fibroblasts

Western blot analysis showed that patient fibroblasts expressed LGR4 protein minimally compared to controls (Figure 2A), but the expression was also low in control fibroblasts from healthy individuals. Unfortunately, biomaterial from heterozygote family members was not available.

Functional analysis of LGR4 variants in cell models

To assess the function of identified LGR4 variants on RSPO1 activated Wnt signaling, we used an established TOP-Flash luciferase reporter assay [START_REF] Carmon | R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/beta-catenin signaling[END_REF][START_REF] Glinka | LGR4 and LGR5 are R-spondin receptors mediating Wnt/beta-catenin and Wnt/PCP signalling[END_REF][START_REF] Korinek | Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/-colon carcinoma[END_REF][START_REF] Ruffner | R-Spondin potentiates Wnt/beta-catenin signaling through orphan receptors LGR4 and LGR5[END_REF][START_REF] Tomaselli | Human RSPO1/Rspondin1 is expressed during early ovary development and augments beta-catenin signaling[END_REF][START_REF] De Lau | Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling[END_REF] . When using fibroblasts, RSPO1 stimulation of endogenous LGR4 -Wnt/ β-catenin signaling was not strong enough for luciferase readout. Therefore, we performed the studies in HEK293T cells that were transiently transfected with wild-type and variants of LGR4. While the basal activity of the Wnt/β-catenin signaling was low and did not differ between wild-type and LGR4 variants, RSPO1 activated signaling was increased 6.1-fold with WT-LGR4 (p<0.0001, Figure 2B). By contrast, the LGR4 nt-24 variant increased the Wnt/β-catenin signaling only 3.3.-fold (p<0.0001), and the LGR4 nt-72 variant completely failed to activate Wnt/β-catenin signaling (Figure 2B). Thus, compared to WT, the LGR4 nt-72 showed loss of function, while the LGR4 variant nt -24 had 54% activity. In line with these results, loss of interaction by deletion of 24 amino acids in the nt-72 variant was predicted to have a higher impact on the binding as well as protein stability (Suppl Figure S8), which explains the very low level of LGR4 protein detected in western blots of patient fibroblasts.

Analysis of localization of LGR4 and binding to RSPO1 in HEK293 cells

LGR4 localizes to the cell membrane and reveals its signal-transducing functionality upon binding to R-SPONDINS [START_REF] Carmon | R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/beta-catenin signaling[END_REF][START_REF] Glinka | LGR4 and LGR5 are R-spondin receptors mediating Wnt/beta-catenin and Wnt/PCP signalling[END_REF][START_REF] Korinek | Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/-colon carcinoma[END_REF][START_REF] Ruffner | R-Spondin potentiates Wnt/beta-catenin signaling through orphan receptors LGR4 and LGR5[END_REF][START_REF] Tomaselli | Human RSPO1/Rspondin1 is expressed during early ovary development and augments beta-catenin signaling[END_REF][START_REF] De Lau | Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling[END_REF][START_REF] Kudo | The nematode leucine-rich repeatcontaining, G protein-coupled receptor (LGR) protein homologous to vertebrate gonadotropin and thyrotropin receptors is constitutively active in mammalian cells[END_REF] . To assess localization and RSPO1 binding characteristics of wild-type and mutant LGR4 proteins, we expressed HA-tagged LGR4 in HEK293 cells and studied its binding to GFP-tagged RSPO1 by confocal microscopy. As depicted in Figure 2C, LGR4 localized to the cell surface. nt-24-LGR4 was expressed at a lower level than WT-LGR4, and cells carrying the nt-72-LGR4 expressed the lowest level of LGR4 (Figure 2D), in line with LGR4 protein expression in patient fibroblasts (Figure 2A). RSPO1 binding with LGR4 protein was also altered with nt-24-

LGR4 compared to wild-type, and almost absent with nt-72 (Figure 2 C,D). Altogether, these in vitro data show that the mutant LGR4 proteins identified in the proband are deficient in their ability to bind R-SPONDINS and stimulate downstream WNT signaling.

Lgr4 ablation results in disrupted Wnt/b-catenin signaling pathway in mice

To study the role of LGR4 in adrenal function in vivo, we conditionally inactivated Lgr4 within steroidogenic cells in the adrenal cortex of Lgr4cKO mice. RTqPCR showed a reduction in Lgr4 mRNA, confirming efficient deletion of the floxed allele in Lgr4cKO mice (Suppl Figure S9A).

Consistent with our in vitro data, conditional inactivation of Lgr4 resulted in a significant decrease in expression of WNT target genes in the adrenals of Lgr4cKO mice (Apcdd1, Axin2, and Lef1) (Figure 3A) with decreased accumulation of both β-Catenin and LEF1 proteins in the presumptive zG of mutant mice, where they normally accumulate in control mice (Figure 3B-C).

Lgr4 ablation causes adrenal hypoplasia and aberrant zonal differentiation

The reduced canonical WNT signaling in Lgr4cKO mice was associated with decreased adrenal weight at 5 weeks (Figure 3D), massive cortical thinning, off-center localization of the adrenal medulla, steroidogenic cell cytomegaly, and a significant decrease in cortical cells numbers (Figure 3E-F). Interestingly, adrenal cortex thinning was not associated with decreased proliferation or increased apoptosis, suggesting that it relied on altered development/maintenance of the gland (Suppl Figure S9B-C). Analysis of adrenal cortex differentiation by immunohistochemistry showed a marked decrease in the number of cells expressing the zG marker DAB2 and expansion of the expression domain of zF marker AKR1B7 up to the capsule, where the zG normally resides (Figure 3G). Lgr4 deficiency also resulted in the accumulation of cells with both zG (DAB2+) and zF (AKR1B7+) identity that were not found in control mice (Figure 3G, arrowheads), demonstrating a marked impairment of adrenal cortex differentiation. Aberrant cortical differentiation was further confirmed by RTqPCR showing increased Akr1b7 (zF) and decreased expression of the zG markers Dab2 and Hsd3b6 (Figure 3H), consistent with previous data showing decreased zG differentiation and expansion of zF in mice with decreased adrenal WNT signaling [START_REF] Drelon | PKA inhibits WNT signalling in adrenal cortex zonation and prevents malignant tumour development[END_REF][START_REF] Vidal | The adrenal capsule is a signaling center controlling cell renewal and zonation through Rspo3[END_REF] .

Lgr4 ablation inhibits zG zonation resulting in primary hypoaldosteronism

Consistent with observations in our patient, zG differentiation anomalies in Lgr4cKO mice resulted in a significant decrease in plasma aldosterone (Figure 4A) and an increase in hematocrit, suggestive of dehydration (Figure S9C). The observation of normal plasma renin activity (Figure 4B) suggested that hypoaldosteronism in Lgr4cKO mice was of primary adrenal origin. This was further supported by the almost complete extinction of CYP11B2 protein expression in the Lgr4cKO zG (Figure 4C-D). Interestingly, plasma corticosterone concentration was significantly decreased (Figure 4E), which was associated with a significant decrease in Cyp11a1 expression, which is essential for the first step of both aldosterone and corticosterone synthesis (Figure 4G).

However, there was a concomitant increase in Cyp21 and Cyp11b1 expression, which was not associated with altered plasma ACTH concentration (Figure 4F-G). This may reflect the aberrant expansion of zF at the expense of zG, and hence an increased ratio of zF to zG cells, rather than a direct effect of ACTH on steroidogenic gene expression.

Altogether, these data show that Lgr4 inactivation is sufficient to significantly reduce WNT signaling in the adrenal cortex, which results in early-onset primary hypoaldosteronism.

DISCUSSION

Although adrenal insufficiency with MC and GC deficiencies has been reported for several complex syndromes where genetic variants lead to structural and/or functional defects of the adrenals and other organ systems (e.g. IMAGe, MIRAGE syndromes) [START_REF] Pignatti | Adrenal cortex development and related disorders leading to adrenal insufficiency[END_REF] , an inherited syndrome with isolated MC deficiency at birth, has not been described so far. In this study, we identified a novel syndromic form of severe neonatal salt-wasting in a highly consanguineous family. In the index patient, isolated mineralocorticoid deficiency was diagnosed and treated successfully with mineralocorticoid replacement therapy, while cortisol production remained normal in the first two decades of life. Associated defects included nail anomalies, hearing loss, short stature, and mental disability in the index patient and both affected cousins (Figure 1A). We were aided by the consanguinity in this family to reveal the underlying genetic cause, involving a homozygous splice site variation in the LGR4 gene (ch11p14.1) producing two shorter splice variants. LGR4, also named GPR48, is a leucine-rich repeat-containing G-protein coupled receptor, widely expressed in multiple tissues from early embryogenesis to adulthood 15, 24 . LGR4 potentiates canonical WNT signaling, through inhibition of the ZNRF3/RNF43-mediated degradation of Frizzled receptors, after binding to R-SPONDINS [START_REF] Ordaz-Ramos | The Role of LGR4 (GPR48) in Normal and Cancer Processes[END_REF] . Consistent with this, our in vitro studies showed that the two aberrant LGR4 transcripts found in the proband, coded for proteins with significantly reduced activity on WNT/β catenin signaling in vitro. We further showed that genetic inactivation of Lgr4 within steroidogenic cells of the adrenal cortex of transgenic mice resulted in decreased canonical WNT signaling, deficient zG differentiation, and reduced aldosterone production. Even though previous reports had shown adrenal dysgenesis in patients with inactivating mutations of WNT4 in the context of SERKAL syndrome, profound developmental defects resulted in embryonic lethality, precluding evaluation of adrenal differentiation and endocrine activity [START_REF] Mandel | SERKAL syndrome: an autosomal-recessive disorder caused by a loss-of-function mutation in WNT4[END_REF] . Therefore, to the best of our knowledge, our study is the first to demonstrate a key role of LGR4 and more broadly of deficient canonical WNT signaling in adrenal differentiation and zG hypofunction in patients.

Beyond primary hypoaldosteronism, the index case also presented with a spectrum of defects including nail anomalies, hearing loss, and short stature, which were also associated with in utero death of presumably affected siblings. Our model of conditional Lgr4 ablation within steroidogenic cells did not allow evaluation of LGR4 in these phenomena. However, studies of LGR4 variants in patients and of whole-body Lgr4 knockout mice demonstrated the association between LGR4 alterations and fetal/ perinatal death, short stature, deafness, and dysplastic nails (Supplementary Table S3). This strongly suggests that the broad defects observed in our proband are the result of the identified LGR4 mutation. So far only individuals who carry heterozygous LGR4 variants were described. Heterozygous human LGR4 variants are associated with low bone mineral density, electrolyte imbalance, reduced testosterone production, and increased risk of cancers of the biliary system and skin [START_REF] Styrkarsdottir | Nonsense mutation in the LGR4 gene is associated with several human diseases and other traits[END_REF] . More recently, 3 rare heterozygous missense variants in LGR4 were associated with delayed puberty, resulting from alterations in the development of hypothalamic GnRH neurons [START_REF] Mancini | LGR4 deficiency results in delayed puberty through impaired Wnt/beta-catenin signaling[END_REF] . Loss of LGR4 is also potentially implicated in the rare aniridiagenitourinary anomalies-mental retardation (AGR) syndrome, where a contiguous gene deletion of the 11p13-14 region has been identified comprising the LGR4 gene [START_REF] Yi | LGR4/GPR48 inactivation leads to aniridiagenitourinary anomalies-mental retardation syndrome defects[END_REF] . Similar to the phenotype of AGR syndrome, whole-body deletion of Lgr4 in mouse led to aniridia, polycystic kidney disease, genitourinary anomalies, and mental retardation. Although there were no reports of adrenal dysfunction in these cases, our results suggest that patients presenting with homozygous LGR4associated genetic variations should be carefully evaluated for adrenal function.

Whereas Lgr4cKO mice show defects in both aldosterone and corticosterone secretion as early as 5 weeks, our index case presented with isolated mineralocorticoid deficiency in the first two decades of life. This phenotypic discrepancy could be accounted for by the residual activity of mutant LGR4 proteins in our patient, compared with the complete inactivation of LGR4 in the adrenal cortex of our transgenic model. However, ACTH stimulation testing of our patient at 21 years, showed subclinical glucocorticoid deficiency. This suggests that the endocrine phenotype may progress towards full-fledged adrenal deficiency over time. Lineage tracing studies in mice have shown that adrenal cortex cell renewal requires initial differentiation of progenitors into zG cells that subsequently differentiate into zF cells [START_REF] Freedman | Adrenocortical zonation results from lineage conversion of differentiated zona glomerulosa cells[END_REF][START_REF] King | Shh signaling regulates adrenocortical development and identifies progenitors of steroidogenic lineages[END_REF] . It is thus tempting to speculate that aberrant zG differentiation in our patient hampered cortical cell renewal, resulting in progressive exhaustion of the zF, associated with progressive glucocorticoid insufficiency. This warrants careful monitoring of patients initially presenting with primary hypoaldosteronism, without CYP11B2 inactivating mutations.

Table 1 . Patient characteristics and laboratory findings at initial presentation and during 4 years follow-up

 1 Footnotes: nd, not detected; numbers in bold mark findings outside the normative range brain anomalies, and mental disability; deafness with functional but without structural anomalies of the cochlea and hearing nerve; low bone mineral density, and small kidneys with cortical microlesions (Suppl Figures

	Age	years		17	17 1/3	17 1/3	18	20	21	21
	Height	cm			141		142	142	142
	Weight	kg			33.5		36.1	34.7	36.4
	BMI	kg/m2					17.9	17.2	18.05
	Blood pressure	mmHg			103/74		113/76	127/85	116/80
	Pubertal stage	Tanner			4-5		5	5	5
	Bone age	years GP			16-17		adult
	Hydrocortisone	mg/m2/d		23.9	24h off	0	0	0	0
					treatment
	Florinef	ug/d		150	24h off	0	100	100	24h off
					treatment				treatment
			Normal	basal	basal	ACTH	basal	basal	basal	ACTH
			range			stimulated				stimulated
			basal
	Na	mmol/l			136		138	137	142
	K				4.2		4.5	3.8	3.7
	Cl				106		108	106	110
	Creat	umol/l			79		72	69	64
	ACTH	ng/l	7.2-63.3	9.3	47.8		8	6.1	44.3
	Renin	ng/l (*mU/L) 1.7-23.9	38	133		*187	20.8	9.1
			(*4.4-46)
	Corticosterone	nmol/l	1.69-63.8						1.08	9.91
	Aldosterone	pmol/l	87-662	41	nd		49.2		nd	nd
	17OHProg	nmol/l	0.24-6.84 4.2	16.6	20.2	3	1.5	8.26	15.7
	Progesterone	nmol/l			20.5	15.6	3		42.1
	Cortisol	nmol/l	133-537		400	564	288.7	205	206	303
	DHEA-S	umol/l	1.77-9.99 3.7	4.35	4.55	2.3	2.4	3.79	3.29
	DHEA	nmol/l	1.7-38.3		8.2	9			7.93	11.3
	Androstendione	nmol/l	1.06-7.72 8.5	13	15.9	3.4	3.3	6.89	7.45
	Testosteron	nmol/l	0.31-2.29						1.33	1.54
	E2	pmol/l	45-854	151	903			475	1264
	LH	U/l		11.8	17.7			12.2	5.7
	FSH	U/l		4.7	3.2			4.6	1.3
	AMH	pmol/l	7.14-57.1		13.3				22.4
	IGF-1	ng/ml		170	205			207
	IGF-BP3	mg/l		4.26	5.16			3.5
	iPTH	pg/ml	15-65						29.3
	25OHVitD3	nmol/l	50-135						60
	Osteocalcin	ng/ml	11-43						34.3
	b-Cross-Laps	pg/ml	<573						357
	totP1NP	ng/ml	15.1-58.6						84.3
	FGF23	pg/ml	10-50						50.7

Acknowledgments

We thank the patient and family for participating in the study. We acknowledge the help of Christelle Damon-Soubeyrand and the Anipath histopathology facility (iGReD), Clermont-Ferrand, for mouse tissue analyses. We also thank Khirredine Ouchen, Sandrine Plantade and Philippe Mazuel for animal care.

Ligue Nationale Contre le Cancer 2021-2025 and ANR-21-CE14-0044-ADREMAC to PV. grant by the Lyon University Hospital to FRB. SNF grant ID 320030_146127 to CEF.

Funding

Pierre Val is supported by a program grant (Labellisation) by the "Ligue Nationale Contre le Cancer" 2021-2025. Florence Roucher-Boulez is supported by the Endocrinology Research Grant RECORDATI Rare Diseases of the French Society of Endocrinology (SFE) 2020 and the Young Researchers 2021 grant by the Lyon University Hospital (HCL). This work has also been supported by the Swiss National Science Foundation grant ID 320030_146127 to Christa E. Flück.

Data sharing

All data and materials presented in the manuscript will be made available upon written request to the corresponding author.

In conclusion, we describe the first patients harboring biallelic LGR4 variants and offer the mechanistic explanation for their life-threatening salt loss at birth, due to primary adrenal hypoaldosteronism. Our study confirms the important role of Wnt/b-catenin signaling for proper adrenal cortex zG and zF formation and function. Thus LGR4 variants and potential variants in other genes involved in the complex network of LGR4-Wnt/b-catenin signaling should be considered in patients presenting with a salt-wasting crisis at birth, especially when manifesting with other syndromic features.

Declaration of interests

The authors declare no conflict of interest


Paul Moulaire

MSc Pierre Emmanuel Poulet

MSc Emilien Petit

MSc Thomas Klockgether

MD Alexandra Durr

PhD Tetsuo Ashisawa

MD Sophie Tezenas Du Montcel

Introduction Methods

Results Conclusion

In 2006, the Scale for the Assessment and Rating of Ataxia (SARA) was developed to assess the presence and the severity of ataxia. SARA was validated for participants with spinocerebellar ataxia, translated in several languages, and validated in related diseases such as Friedreich ataxia. It is nowadays the reference scale for the clinical evaluation of ataxia.

In this study we aimed to: 1) Assess the temporal dynamic of each of the eight items composing the scale. 2) Study the global progression of the sum score. 3) Define the most reliable part of the scale 4) Asses the number of required participants for therapeutic trials with different scenarios Data from four international cohorts (EUROSCA, RISCA, CRC-SCA, SPATAX) were pooled together for a total of 1210 SCA1, SCA2, SCA3 and SCA6 participants and 4092 visits from 2006 to 2020.

A Bayesian ordinal mixed effect model (Leaspy) was used to estimate:

1) The time (in years) spent at each level of each of the eight items composing SARA scale. 2) The average time for a one-point increase of each items.

3) The average time for a one point-increase of each of the SARA score and the associated variability. 4) The linearity of progression of different items and SARA scale. Sample size calculations for therapeutics trials was done with different scenarios.

Seven of the eight different items had a non-linear progression (Figure 1 and Figure 3B). The speed of progression was different between most of the items with an average time for a onepoint increase from 3.5 years [3.4;3.6] (median, 95% credible interval) for the fastest item to 11.4 [10.9;12.0] years (Figure 3A). The total SARA score had a linear progression with an average time for a one-point increase of 0.95 [0.92; 0.98] years (Figure 2). The most reliable part of the scale, (with high overlap between the different steps) was between SARA score 4 and 36 (figure 2).

Selecting this range as an inclusion criterion reduced the required sample size from 234 to 218 in an heterogeneous group of SCA participants. (Figure 4). Despite a heterogeneous temporal dynamic at the item level, the global progression of the SARA scale was linear. This important result suggests that all items are complementary and that they each provide specific information at different stages of the disease. Selecting patients with a baseline SARA score between 4 and 36 reduced the required sample size, reflecting a better performance of the scale on this range. This new information about the temporal dynamic of the scale should help to design outcome of future clinical trials.

As the SARA scale is used in all types of SCAs, and to reach more power, the different SCAs and cohorts were pooled together. This information must be taken into account, and the individual progression at the SCA level could be not linear.

Figure 2 :

 2 Figure 2: Global temporal dynamic of the SARA scale. A: Average score progression as a function of years from start of progression B: Posterior distribution of the time spent in each level of the SARA scale.

Figure 1 :

 1 Figure 1: Average score progression as a function of years from start of progression Average trajectory of progression of each of the eight item composing the SARA scale.

Figure 3 :

 3 Figure 3: Score progression at the item level A: Posterior distribution of the average time for a one-point increase of the corresponding item. For instance, the blue distribution is the average time for a one point-increase of the item 1-Gait of the SARA scale. B: Each panel corresponds to the progression of one item of the SARA scale. In one panel, each distribution represents the time spent in each level of this item. For instance, for panel B-1-Gait, the red distribution, Score1, is the average time spent at Gait score 1.

Figure 4 :

 4 Figure 4: Total number of participants required for a therapeutic trial Sample size for a two-group interventional trial (1:1 ratio per group) of 12 months duration with a treatment effect of 50% on the disease progression, a power of 90% and an alpha of 5% SCA = Spinocerebellar ataxia Total = Heterogeneous group of SCA SARA 4-36 = Inclusion criterion: participants between SARA score 4 and 36.

  Acknowledgments: READISCA consortium, grant : U01 NS104326 EUROSCA, CRC-SCA and SPATAX consortiums Temporal dynamics of the Scale for the Assessment and Rating of Ataxia in autosomal dominant cerebellar ataxias patients