VSIG10L is a major determinant of esophageal homeostasis and inherited predisposition to Barrett’s esophagus

Domain: neuroscience
Published: 2026-01-29
Editorial outcome: minor_revision

Abstract
The molecular underpinnings contributing to the onset of Barrett’s esophagus (BE) remain elusive. By studying familial clusters of the disease, here we identify a significant association between genetic variants in the V-set and Immunoglobulin Domain Containing 10 Like (VSIG10L) gene and BE predisposition. Using mammalian tissues and patient-derived organoids, we show VSIG10L is selectively expressed in the suprabasal squamous cells of the esophageal mucosa and is essential for epithelial maturation and homeostasis. Mice carrying human-orthologous germline mutations in Vsig10l exhibit loss of desmosomes, concomitant with disrupted epithelial differentiation programs, in the squamous mucosa. Upon long-term exposure to a bile acid (deoxycholate) supplemented diet, Vsig10l-mutant mice develop overt BE-like lesions in the forestomach. Furthermore, loss of esophageal VSIG10L expression is observed frequently in patients with chronic gastroesophageal reflux disease, a known risk factor for BE. Collectively, our study uncovers a fundamental link between VSIG10L, esophageal homeostasis, and BE predisposition.

Introduction
Barrett’s esophagus (BE) is characterized by intestinal-type mucinous metaplasia of squamous (SQ) epithelial cells in the distal esophagus1. Patients with BE in particular are at a high risk of developing esophageal adenocarcinoma (EAC), an increasingly prevalent and refractory malignancy, often diagnosed at advanced stages where treatments become ineffective2,3. While risk factors for BE/EAC such as age, male gender, Caucasian ethnicity, obesity, gastroesophageal reflux disease (GERD), and diet have been previously suggested, they lack sufficient predictive power to guide effective clinical management of this disease4–6. Moreover, the co-opting genetic / molecular aberrations contributing to the onset and progression of BE remain elusive and complex7.

Prior studies from our group identified that up to 10% of patients with BE/EAC report a family history, indicating a genetic predisposition to the disease8–12. Our segregation analysis in familial clusters of the disease strongly suggested that familial BE (FBE) is consistent with dominant transmission of one or more incompletely penetrant major Mendelian alleles13. Moreover, we have identified genetic linkage of familial BE to distinct chromosomes and genomic loci8, suggesting that FBE is genetically complex. Using familial association and high-throughput genomic approaches in a large multi-generational index family, we subsequently discovered the first inherited autosomal dominant BE susceptibility variant (S631G) mapped to a highly-conserved Serine residue within the Ig-like domain in the V-set And Immunoglobulin Domain Containing 10 Like (VSIG10L) gene14.

Here we sought to determine the extent of VSIG10L susceptibility variants in a large cohort of BE/EAC familial kindreds. Furthermore, we characterized the expression of VSIG10L in esophageal mucosa, developed human-orthologous Vsig10l-mutant mice, and performed molecular and phenotypic studies to interrogate the pathogenicity of VSIG10L variants. Overall, our study identifies VSIG10L as a key molecule involved in maintaining esophageal homeostasis; defects in which enhance susceptibility to BE.

Results
Identification of familial BE/EAC-associated susceptibility variants in VSIG10L
In follow-up to our previous report14, here we determined the extent of VSIG10L susceptibility variants associated with the disease by targeted sequencing of VSIG10L coding regions in 684 blood DNA samples, banked from BE/EAC affected and unaffected relatives from 302 families, accrued through an active multi-center familial BE registry8,14,15. We found additional germline VSIG10L susceptibility variants including: a rare deleterious frameshift deletion (dbSNP: rs1185522503, Minor Allele Frequency16: 0.0001) in a 50 year old female diagnosed with BE; a private missense variant (GRCh37/hg19/chr19:51842243: A>A/G, p.V543A) in a male diagnosed with BE at age 61; and a rare in-frame deletion (dbSNP: rs370588522, Minor allele frequency16: 0.0027) in a male diagnosed with EAC at age 82. Together, our studies in familial clusters of the disease indicated a pathogenetic link between VSIG10L and BE/EAC predisposition.

VSIG10L expression is localized to suprabasal squamous cells of the esophagus
VSIG10L is predominantly expressed in SQ cells, and its gene product encodes a membrane-bound protein with immunoglobulin (Ig)-like domains14. Nonetheless, its function in the esophagus or in other SQ tissues is hitherto unknown. We accordingly first sought to determine its expression within the stratified esophageal SQ mucosa. Given the lack of optimized VSIG10L antibodies, we developed custom RNAscope-in situ hybridization probes and subsequently assessed its expression in representative human and porcine esophagus, as well as in human SQ epithelial cells (EPC2) grown as 3D-organotypes17,18. As shown in Fig. 1, we found VSIG10L RNA to be selectively and consistently expressed in suprabasal SQ cells, with no detectable expression in basal squamous cells, suggesting a possible role of VSIG10L in epithelial maturation.

Esophageal organoids from VSIG10L-mutant patient show disrupted squamous maturation
We previously reported that EPC2 3D-organotypes engineered to express the VSIG10L-S631G FBE variant exhibit dysmaturation and loss of epithelial integrity, while ultrastructural assessments of esophageal mucosa from the VSIG10L S631G-mutant patient revealed loss of desmosomes and dilated intercellular spaces particularly within the suprabasal SQ cells14. To further explore the impact of VSIG10L on the developmental dynamics of esophageal SQ cells, we generated and assessed esophageal progenitor SQ organoids derived from pluripotent stem cells (hPSC) from the VSIG10L-S631G patient. Morphologically, organoids from the VSIG10L-S631G patient showed frequent disruption of SQ epithelial stratification and maturation, in stark contrast to the normal (anticipated) growth and differentiation of SQ organoids observed in the control subject grown under the same experimental conditions. Immunohistochemistry (IHC) assessments revealed that the single layer of cells in the abnormal glandular-appearing organoids in the mutant patient to be positive for p63 (basal SQ cell marker), albeit to varying degree, suggesting these may be predominantly basal-like SQ cells (Fig. 2). Additionally, unlike in the control subject, the spherical-appearing organoids from the mutant patient often showed loss of inward epithelial differentiation gradient, with strong p63-positivity in all the cells (Fig. 2), further suggesting a perturbation in SQ maturation. Together, these findings point to the pathogenicity of familial BE-associated VSIG10L variants, in vivo.

Accordingly, we generated the following human-orthologous germline Vsig10l-mutant murine models on a C57Bl/6J background: 1) Based on the human FBE variant (VSIG10L-S631G14), we generated mice carrying the orthologous Vsig10l-S516G missense mutation, mapping to the highly conserved Serine residue within the Ig-like domain14; and 2) Based on the deleterious frameshift variant (rs1185522503) identified in one of our familial BE kindreds mentioned above, we additionally generated mice that were null/knockout (KO) for Vsig10l.

First, similar to our observations in human and porcine tissues (Fig. 1), we found murine Vsig10l to be also selectively expressed in the subprabasal cells of the SQ mucosa (Fig. 3a). As BE typically develops in the distal portion of the esophagus contiguous with the gastro-esophageal (SQ-columnar) junction1, we next assessed for potential ultrastructural changes within the SQ mucosa of the forestomach in the Vsig10l-mutant mice, using Transmission Electron Microscopy (TEM). Both S516G and KO mice revealed a marked reduction in desmosomes in the suprabasal SQ cell layer where VSIG10L is expressed (Fig. 3b). Importantly, our findings in the Vsig10l-mutant mice are remarkably consistent with the ultrastructural changes we observed in the esophagus of familial BE patient carrying the orthologous germline VSIG10L S631G variant14, suggesting a potential role of VSIG10L in maintaining epithelial integrity that is conserved across species. To further understand the molecular programs impacted by Vsig10l disruption, we performed Spatial Transcriptomic Profiling (STP) using formalin-fixed and paraffin–embedded (FFPE) tissues derived from the SQ forestomach of representative wild type and Vsig10l-mutant mice (Fig. 3c). Subsequent gene set enrichment analysis of STP profiles revealed a significant disruption of cell structure and differentiation programs in the SQ mucosa of both S516G and KO mice (Fig. 3c). To orthogonally evaluate our TEM and STP findings, we assessed the expression of Desmogleins (Dsg1/3), members of the cadherin gene family and key components of desmosomes involved in cell-cell adhesion and epidermal cell differentiation19,20, in an independent set of wild type and Vsig10l-mutant mice. Immunohistochemistry (IHC) analyses of FFPE SQ tissues indeed showed a reduction of Dsg1 protein in S516G-mutant mice, with a more pronounced repression of both Dsg1 and Dsg3 proteins in the KO mice, compared to the wild type (Fig. 3d). Collectively, these findings add further support to the role of VSIG10L in maintaining SQ epithelial integrity and maturation.

Of note, in analyzing the STP data, we explored whether Vsig10l disruption additionally leads to perturbations in downstream effectors/signaling pathways, using our in-house developed computational framework, InFlo18,21,22. While no significant changes in canonical signaling pathways or large signaling networks were observed, few individual sub-networks showed altered activities in the Vsig10l-mutant mice compared to wild type (Supplementary Fig. 1). While some of these differentially-activated components such as Notch, Bmp, Smad have been implicated in BE/EAC pathobiology23, characterizing their functional relationship with VSIG10L (direct or indirect) and the broader significance of such changes in these specific nodes/components require further in-depth investigations.

Vsig10l-mutant mice develop BE-like mucinous metaplasia lesions upon chronic exposure to reflux-associated injury
Since factors disrupting epithelial homeostasis can render the esophagus more susceptible to GERD injury and BE pathogenesis7,24,25, we posited Vsig10l-mutant mice may exhibit an inherent propensity to developing BE-like pathologies upon exposure to deoxycholate (DCA), a key bile acid involved in reflux injury and a risk factor for BE26–29. Accordingly, wild type and Vsig10l-mutant mice were fed either standard diet or diet supplemented with 0.2% DCA, and followed longitudinally for a period of up to 24 months. Mice were then sacrificed, and esophageal, forestomach contiguous with the SQ-columnar junction, and gastric tissues were harvested. Respective FFPE tissue sections were subsequently subjected to histopathological examination. Tissues were primarily assessed for the presence of BE-like lesions, hereafter referred to as Barrett’s-like mucinous metaplasia (BEMM), by a gastrointestinal pathologist (J.E.W) who was blinded to the sample genotypes. Subsequently, the incidences of BEMM lesions in mice were enumerated across respective heterozygous and homozygous Vsig10l genotypes. While no noticeable pathologies were observed in the standard diet group, the vast majority (70%–100%) of Vsig10l-mutant mice on DCA diet developed BEMM lesions at the SQ-columnar junction in the forestomach, significantly different from the infrequent diminutive lesions developing in ~20% of wild type mice (Fig. 4). Importantly, BEMM lesions developing in Vsig10l-mutant mice were also qualitatively quite distinct than those developing in wild type; i.e, BEMM lesions in the Vsig10l-mutant mice were more exacerbated and extensive, unlike the occasional and minor/restricted glandular changes observed in the wild type mice (Fig. 5). Beyond histomorphometric similarities, BEMM lesions in Vsig10l-mutant mice exhibited molecular features that are characteristic of human BE, including: induction of intestinal-type acidic mucins30–32; expression of BE-associated mucin, Mucin 6 (Muc6)33–35; induction of Trefoil Factor 3 (Tff3), a BE-associated trefoil protein and a marker of established intestinal columnar metaplasia and true goblet cells36,37; as well as SRY-Box Transcription Factor 9 (Sox9), a key transcription factor implicated in BE pathogenesis that drives columnar differentiation of esophageal SQ epithelium38 (Fig. 5). Also, similar to human BE, BEMM lesions in these mice were negative for the SQ marker, p63 (Fig. 5). We further note that no such abnormal lesions or gross histologic changes were apparent elsewhere in the esophagus proper in the Vsig10l-mutant mice. Collectively, these findings underscore the pathogenicity of FBE-associated VSIG10L susceptibility variants in facilitating BE development.

Esophageal SQ mucosa from GERD patients reveal a loss of VSIG10L expression
As injury from chronic reflux is a key risk factor for the onset of BE4–6, we further sought to conversely determine whether GERD-associated injury to esophageal SQ mucosa can impact VSIG10L expression. Accordingly, we accrued a random set of snap-frozen distal esophageal SQ tissue biopsies from non-familial patients with GERD, and subsequently assessed VSIG10L RNA expression using real-time qPCR. Of note, erosive or ulcerative esophagitis cases were excluded from this analysis. Compared to a random cohort of non-GERD subjects, esophageal tissues from GERD patients showed a significantly lower VSIG10L expression (~3-fold, P=0.008), while no such differences were observed in TP63 (SQ marker) RNA between the two cohorts (Fig. 6a). To further orthogonally assess VSIG10L expression in the context of GERD, we performed RNAscope-in situ hybridization in distal esophageal FFPE tissue sections, derived from an independent set of five GERD patients and two control subjects that were available for testing. In stark contrast to healthy/normal SQ epithelia, we found a profound reduction of VSIG10L expression or a near-all depletion of VSIG10L-positive differentiation gradient in the stratified SQ layer of GERD-associated reactive esophageal mucosa (Fig. 6b). As anticipated, we observed an expansion of basal-like immature SQ cells expressing p63 particularly in these reactive mucosal regions from GERD patients (Fig. 6b). Taken together, these findings provocatively suggest that GERD-associated disruption of VSIG10L dynamics in the esophageal mucosa may be one of the early molecular changes by which reflux injury impairs SQ homeostasis and potentially enhances the risk of further BE development.

Methods
Our research study complies with relevant ethical regulations. Written informed consent was obtained from all donors for use of their samples and clinical data in the present study. All human protocols were approved by University Hospitals Cleveland Medical Center Institutional Review Board (IRB) for human investigations (CASE3207CC301) and Duke GI Tissue Repository (protocol number; 00001662), adherent to the principles outlined in the Declaration of Helsinki. PBMC-derived human pluripotent stem cells from the VSIG10L S631G mutant patient and control subject were obtained under an IRB-approved protocol (CASE3207CC301) with written consent, and included MTA agreements where appropriate. Archived Porcine tissues were accessed through North Carolina State University (NCSU), approved by NCSU Institutional Animal Care and Use Committee-approved protocol (NCSU 13-116-B). All mouse studies were approved by Case Western Reserve University Institutional Animal care and Use Committee (IACUC), and conform to the ARRIVE 2.049 guidelines. A flow diagram illustrating all the experiments performed and reported in the current study is provided in Supplementary Fig. 2.

Familial patients and VSIG10L DNA sequencing
Probands with BE or EAC as well as unaffected controls were recruited and gave written informed consent for collection of biopsy tissues and blood for research as part of the Barrett’s Esophagus Translational Network (BETRNet) Consortium using a consent and biobanking protocol (protocol number, CASE3207CC30) approved by the Institutional Review Board for Human Investigation at University Hospitals Cleveland Medical Center and collaborating institutions8,12–15, adherent to the principles outlined in the Declaration of Helsinki. The observational study is registered at clinicaltrials.gov, NCT00288119. Study subjects were given a questionnaire that collects information about BE and EAC risk factors along with family history. Study subjects provide written consent to use banked biospecimens for genetic studies and other future research. Permission is also obtained from probands to contact their relatives. Histologic diagnosis for family members reported to have BE or EAC is confirmed by review of records whenever available. Family members who had no prior esophagogastroduodenoscopy (EGD) were offered screening. Blood or saliva is banked from probands and family members who are alive and consent to the study. Study subjects gave written consent and blood lymphocytes were collected, immortalized, and banked at the Rutgers University DNA Repository (RUCDR) as a source of germline DNA from all family members who had screening endoscopy. For deceased family members, permission is obtained from nearest living relatives to obtain formalin fixed paraffin embedded (FFPE) biopsy tissues from treating hospitals. Germline DNA was extracted from blood, immortalized lymphocytes, saliva, or FFPE tissues for subsequent sequencing.

Targeted capture and DNA resequencing of VSIG10L exons in 684 samples, belonging to 302 families, was performed by Novogene (Sacramento, CA). Of the 684 subjects, 428 were men and 256 women (self-reported), mean age was 61.5 years (range; 14–100 yrs), and all familial BE subjects were of European ancestry. Only samples passing internal quality controls were included in the analysis. Raw 100-bp FASTQ reads were aligned to the human reference genome (hg19) using the Burrows-Wheeler Aligner (BWA-v0.7.17)50. Alignment quality was assessed with GATK Picard tools (v2.18.12), followed by pre-processing of BAM files with a custom script utilizing GATK51 to mark duplicates, recalibrate base quality scores, and realign indels. For variant calling, we used two independent callers; Unified Genotyper52 and HaplotypeCaller53 from the GATK Suite. Variant calls were filtered to exclude those with coverage below 30 reads or an alternate base frequency under 0.1. Filtered variants were annotated using Oncotator (v1.9.1.0)54, and were further analyzed for population-level allele frequencies using the ExAC55 and gnomAD (v4.1.0)56 databases. All rare/private variants were further manually reviewed using Integrative Genomics Viewer57. Lastly, selected variants were orthogonally confirmed in fresh DNA aliquots from respective samples using direct Sanger sequencing.

Porcine tissue procurement
Archival porcine esophageal tissue blocks of normal squamous epithelium were accessed for the VSIG10L localization assays. All porcine tissues were originally derived from Yorkshire crossbred pigs (Sus scrofa) of either sex, weighing approximately 35kg, and cared for according to North Carolina State University Institutional Animal Care and Use Committee-approved protocol (NCSU 13-116-B) and as set forth in the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health. Porcine tissues were collected by North Carolina State University Large Animal Models Core of the Center for Gastrointestinal Biology and Disease (CGIBD) under veterinary supervision58. For euthanasia, initial sedation was provided with xylazine-ketamine injection, and then animals were euthanized with pentobarbital (60 mg/kg IV) and clinically assessed by veterinary staff to confirm death prior to collection of esophageal tissues that were subsequently used for histologic analyses.

Generation of Vsig10l mutant mice
Vsig10l S516G missense mutant (orthologous to the human VSIG10L variant S631G14) and Vsig10l knockout (KO) (targeted deletion of Exon 1-Exon 10) mice were generated on a C57Bl/6J (The Jackson Laboratory, Bar Harbor, ME) genetic background (Strain no: 000664), using standard CRISPR-Cas9 methodology. Briefly, single guide RNAs (sgRNAs) for generating murine Vsig10l (Gene ID: 75690) S516G and KO were selected using the CRISPR design tool (http://crispr.mit.edu/)59. In the end, the following sgRNA sequences (5’-3’) were selected for the missense S516G mutant (CGACTACAGCTTAGCCAGGA on exon 6), and for the KO (upstream-ACCTGGGACAATGCAAGCTG; downstream-AGGACTGGACCCTAAGAGTG). Cleavage efficiency of the selected sgRNAs were confirmed using the Guide-it Complete sgRNA Screening System (Takara Bio USA Inc, San Jose, CA; cat. no. 632636), with C57Bl/6J mouse genomic DNA as the template. PAGE-purified sgRNAs along with Cas9 protein (PNABio, CA) were microinjected into fertilized eggs and implanted into pseudopregnant females to generate mice with precise genomic edits at specific loci. For the S516G mutant, the following PAGE-purified donor ultramer was co-injected: 5’-CTCGGCAAGGACGGCCCCTGGCTCCAGGTGGTGGGGGCCGACTACAGCTTGGCCAAGATGGCCGAAAGCTCCTCATCAACAACTTCAGCCTGGATTGGGA. Tail DNA extracted from the pups were confirmed for genomic editing using the Illumina Miseq platform (San Diego, CA) for founder mice. F1 founder mice were selected following confirmation of germline transmission by backcrossing with wild type C57Bl/6J mice. The above CRISPR-based Vsig10l mutant mice were generated in collaboration with the Transgenic and Targeting facility at Case Western Reserve University (CWRU). All animal studies were approved by Case Western Reserve University Institutional Animal care and Use Committee (IACUC), under the protocol number 2019-0082, and were handled in compliance with the 3Rs (Replacement, Reduction and Refinement) ethics guidelines. All wild type and Vsig10l mutant mice were housed in the CWRU animal core facility and maintained on a standard 12h:12h light-dark cycle, 70–74°F temperature, and ~50% humidity. The Vsig10l-mutant mouse models developed here will be shared with other investigators upon request and following review by the corresponding author to ensure that distribution complies with ethical, institutional, and material-transfer guidelines.

Breeding and genotyping of Vsig10l mutant mice
Homozygous Vsig10l mutant mice (S516G and KO) were viable and exhibited fair breeding performances. Genotyping was performed using DNA isolated from tail digests. Briefly, mutant mice for S516G were genotyped using PCR-Sanger sequencing with primer sequences on Exon 6 (forward; TGCTGCTCCAGCCCATAGT, reverse; TGGACCTGAGAGAGGGTGATT, amplicon size of 361 bp). Vsig10l KO mice were genotyped using conventional PCR amplification with primer sequences upstream of Exon 1 (forward; TGGGCTTCTCCATACAACCTC) and downstream of Exon 10 (reverse; TGCCAACCACATTCATCCGT) resulting in an amplicon size of 401 bp for the deleted region. Genotypes of heterozygous mice (-/+) were confirmed using the above primers as well as PCR amplification of the wild type Exon 9 region (forward; GCTCACCCCACCAGCAAAAA, reverse; ACAGTGGGAGGCAACGACAC), resulting in 401bp and 139bp products. PCR conditions included initial denaturation at 95°C for 4 min, followed by 40 cycles of 95°C for 30 sec, 66°C (S516G) or 58°C (KO) annealing for 45s, and 72 °C extension for 45sec. All animal procedures were approved by CWRU in accordance with Institutional Animal care and Use Committee (IACUC) and followed the National Institutes of Health guidelines.

Animal diet and tissue harvest
Animals were fed either standard irradiated diet containing 12 kcal% fat with Palm Oil (Research Diet Inc, NJ; cat. no. 19102101), or standard irradiated diet containing 12 kcal% fat with Palm Oil supplemented with 0.2% Deoxycholic Acid (Millipore Sigma-Aldrich, cat no. D2510; Research Diet Inc; cat. no. 19102102) starting at 5 weeks of age and for a period of 24 months. Animals within each genotype were randomly assigned to the respective standard or standard plus DCA diet groups. The diets were stored in cold dry place. Mice were allowed to have ad libitum access to respective diets (replenished weekly), and drinking water was supplied through standard sipper tubes during the entire duration of the study. At the end of the study, esophagus and forestomach from each mouse were isolated, formalin-fixed (10% neutral buffer) and paraffin–embedded (FFPE) for subsequent histopathologic and molecular assessments. The overall sex distribution of the mice available for final analyses (N=46) were 50% male and 50% female; wild type (50% male), Vsig10l S516G/+ (58% male), Vsig10l S516G/S516G (44% male), Vsig10l -/+ (57% male), Vsig10l -/- (38% male). As genetic predisposition can enhance BE/EAC risk in both men and women8,15,60, data from both male and female mice in respective genotypes were pooled for all analysis. All animal experiments were conducted in accordance with the ARRIVE 2.0 guidelines49.

EPC2 squamous 3D-Organotypic culture model
EPC2 human esophageal squamous esophageal cell line (EPC2), originally derived from a male non-BE patient14,18,61, was gifted to us by Dr. John P. Lynch MD, Ph.D at University of Pennsylvania. Briefly, EPC2 cells were grown in serum free keratinocyte-SFM media (Thermo Fisher scientific, Waltham, MA; cat no. 17005042), supplemented with Bovine Pituitary Extract (BPE, Thermo Fisher scientific; cat no. 13028-014) and human recombinant epidermal growth factor (rEGF, Thermo Fisher scientific; cat no. 10450-013), and maintained at 37°C and 5% CO2. Cells were tested for authenticity using short tandem repeat genotyping and were screened periodically for mycoplasma contamination. EPC2 3D-organotypes were generated as follows: Transwell inserts were added to deep-well six-well plates. 3D collagen/Matrigel matrices (bottom and top layer in the transwell insert) were prepared on Day 1. The bottom layer was prepared using 10X EMEM (BioWhittaker, Walkersville, MD; cat no. 12-684F), Fetal Bovine serum (Hyclone, Logan, UT; cat no. SH30088.3), L-glutamine (Cellgro, Corning, NY; cat no. 25-005-CI), sodium bicarbonate (BioWhittaker; cat no. 17-613E) and high concentration (2.2 mg/ml) collagen rat tail (Corning; cat no. 354249). The reagents were mixed well and incubated at 37°C for 20 min to let the matrix solidify. The top layer of the insert was prepared by mixing 10X EMEM, FBS, L-glutamine, Sodium bicarbonate, collagen, matrigel (Corning; cat no. 354234), and 7.5×104 human esophageal fibroblasts (ScienCell, Carlsbad, CA; cat. no. 2730), and then added to the bottom of the transwell. On Day 7, media was changed to Dulbecco’s Modified Eagles Medium (Sigma-Aldrich, St. Louis, MO; cat no. 51441C) and Ham’s F-12 (Gibco, Grand Island, NY; cat no. 11765-054) in a 3:1 ratio and added to the bottom and top of the transwell and incubated for 1 h at 37°C. Media was then removed from the top and approximately 1.5×106 EPC2 cells were added as droplets. After a 2-hour incubation at 37 °C, Epidermalization I media was added to the plate supplemented with the following: DMEM, F-12, L-glutamine, Hydrocortisone (Sigma-Aldrich, cat no. H0888), ITES (500x) (Biowhittaker; cat no. 17839Z), O-phoshory-ethanolamine (Sigma-Aldrich; cat no. P0503), adenine (Sigma-Aldrich; cat no. A9795), progesterone (Sigma-Aldrich; cat no. P8783), triiodothyronine (Sigma-Aldrich; cat no. T5516), newborn calf serum chelated (Hyclone; cat no. SH3011802). On Day 11, media was removed and the Epidermalization II medium was added to the bottom of the plate only, allowing the epithelial cells to differentiate at the air-liquid interface. Epidermalization I and II media have similar components, except that no progesterone is added to Epidermalization II media. On day 17 organotype cultures were harvested by fixing in 10% formaldehyde (Thermo Fisher Scientific; cat. no. SF100-4), and later were paraffin-embedded for further histopathological analysis.

RNAscope In Situ Hybridization
RNAscope ISH was performed on 5 μM FFPE sections62 derived from human, porcine and mouse tissues, using the RNAscope 2.5 HD Singleplex Chromogenic Assay kit as per the manufacturer’s instructions (Advanced Cell Diagnostics, Newark, CA; cat. no. 322360). Briefly, 5 μM tissue/organoid sections were baked for 60 min at 60°C, deparaffinized in xylene for 5 min twice and rehydrated in 100% ethanol for 3 minutes and air dried for 5 min. The sections were then quenched with hydrogen peroxide for 10 minutes at room temperature and heat-induced RNAScope antigen retrieval was carried out at 100-104°C for 15 minutes. Following antigen-retrieval, slides were rinsed quickly in distilled water at room temperature and dipped in 100% ethanol for 2 min and air dried for 5 min. The sections were covered with RNAscope protease plus, followed by incubation in a humidity-controlled oven for 25 min at 40°C and rinsed in distilled water. Sections were then subjected to VSIG10L ISH with custom RNAscope-ISH probes, designed using Advanced Cell Diagnostics RNAscope probe design pipeline, against respective human (cat. no. 1086661-C1), pig (cat. no. 1161521-C1) or mouse (cat. no. 1088591-C1) species. ISH probe hybridization was carried out in a humidity-controlled oven for 120 min at 40°C. Following hybridization, sections were washed in RNAscope 1X wash buffer at least twice for 2 min at room temperature. Sections were subsequently processed for signal amplification using RNAscope amplification reagent 1 in a humidity-controlled oven for 30 min at 40°C. Following amplification, sections were rinsed with RNAscope 1X wash buffer at least twice for 2 minutes at room temperature. The above step was repeated with amplification reagents 2, 3, and 4, followed by rinsing with RNAscope 1X wash buffer for each section. Sections were then incubated with amplification reagent 5 and 6 for 15-30 min at room temperature with intermittent rinsing using RNAscope 1X wash buffer at least twice for 2 min at room temperature. Sections were then covered with Fast Red Chromagen (Advanced Cell Diagnostics; cat. no. 322360) by using a 60:1 dilution of Buffer Fast Red A: Chromagen Fast Red B for 3-5 minutes. After decanting the chromagen, 120 μl of 3,3’-Diaminobenzidine chromogen (DAB) mixture was added to each section and incubated at room temperature for 10 minutes. Sections were then rinsed in distilled water and stained with CAT Hematoxylin counter stain (Biocare Medical, Pacheco, CA; cat. no. CATHE-M) for 30 s, rinsed under running tap water for 30 s, followed by additional rinses in distilled water for 1 min at least three times. Cell nuclei were stained with bluing reagent for one minute with TBST wash buffer and rinsed in running tap water for 30 s and dehydrated in Xylene, and coverslipped with Ecomount medium (Biocare Medical; cat. no. EM897L). Tissue/organoid sections were also stained for standard Hematoxylin and eosin (H&E) for histologic characterization in respective serial sections. Stained sections were visualized using EVOS M7000 imaging system (Thermo Fisher Scientific). All images were captured using identical settings on the EVOS M7000 imaging system, and each tissue section on a slide was imaged in its entirety using the M7000 image tile-stitching software function.

Electron microscopy
Esophageal tissues from euthanized mice were prepared for electron microscopy using the protocol previously described by us14,63,64. Briefly, tissues were fixed with triple aldehyde-DMSO fixative for 2 h at room temperature. The specimens were thoroughly rinsed in 0.1M phosphate buffer, pH 7.4, then post-fixed for 2 h in an unbuffered 1:1 mixture of 2% osmium tetroxide and 3% potassium ferricyanide. After rinsing with distilled water, the specimens were soaked overnight in an acidified solution of 0.25% uranyl acetate. After another rinse in distilled water, they were dehydrated in ascending concentrations of ethanol, passed through propylene oxide, and embedded in Poly/Bed resin mixture. Thin sections were sequentially stained with acidified uranyl acetate followed by a modification of Sato’s triple lead stain and examined in a FEI Tecnai Spirit (T12) transmission electron microscope with a 4k × 4k Gatan US4000 CCD camera.

Spatial Transcriptomics
The Visium Spatial Gene Expression platform from 10x Genomics was used for spatial transcriptomic profiling (STP). Briefly, esophageal FFPE tissue sections (5 μM) from wild type and Vsig10l mutant mice (with DV200 ≥ 80%) were placed within a 6.5 mm2 field on a 10x Genomics gene expression slide that contains 5000 barcoded probes. Sections were deparaffinized via xylene immersion, ethanol to remove the solubilized paraffin, and then immersion in water to remove residual ethanol. The deparaffinized tissues were then stained with hematoxylin and eosin (H&E) to visualize tissue architecture. RNA sequestered by formalin fixation within the tissue is de-crosslinked by serial treatment with HCl at room temperature followed by heating at 70°C in TE buffer pH 9.0 for 1 h. Next, cells within the tissue were permeabilized prior to RNA hybridization with whole transcriptome mouse probe panels (21k), consisting of RNA-templated ligation (RTL) probes that target the protein-coding transcriptome. Following targeted gene probe ligation, the poly-A tail from probes were then used as template for the on-surface probe priming. Probes were extended by the addition of a UMI, Spatial Barcode and partial Read 1, then released from the slide and collected for qPCR to optimize sample index PCR cycle number. Targeted gene products then underwent index PCR to ligate Illumina sequencing adapters and sample dual indices followed by final library cleanup by SPRIselect size selection. Final libraries were assessed on a Tapestation 4200 and quantified by qPCR. Libraries were sequenced on Illumina NGS paired end sequencers at 25k reads/spot on an Illumina paired end, dual indexed flow cell in the format of 28x10x10x50. Data were then uploaded to analyses packages for visualization.

Gene Set Enrichment analyses (GSEA)
Spatial transcriptomics gene expression data from wild type and Vsig10l mutant mice were subjected to Gene Set Enrichment analyses (GSEA) using the fgsea package in R. This analyses aimed to pinpoint pathways and biological processes significantly enriched among the differentially expressed genes (DEGs) between the experimental groups: Wild type vs. S516G; Wild type vs. KO. Gene sets were sourced from multiple databases, including the Molecular Signatures Database (MSigDB), Gene Ontology (GO), KEGG, and Reactome, encompassing curated pathways and gene ontologies. The input for GSEA consisted of ranked gene lists based on log2 fold change values for differential expression between the aforementioned comparisons. For each analyses, GSEA calculated an enrichment score (ES) for each gene set, indicating the extent to which the genes within that set were over-represented at the top or bottom of the ranked gene list. The significance of the ES was determined through permutation testing (1000 permutations), which yielded a normalized enrichment score (NES). We considered gene sets with a false discovery rate (FDR) q-value of < 0.05 to be significantly enriched.

Immunohistochemistry
Immunohistochemical evaluation was performed using standard protocols and methods as previously described by our group18,22. Briefly, 5 μM mouse and human tissue/organoid sections were baked for 75 min at 60°C followed by de-paraffinization in xylene and subsequent rehydration in series of graded ethanols (100%, 95%, 70%, respectively) and rinsed in distilled water. Heat-induced antigen retrieval was carried out in a pressure cooker in citrate solution (pH 6.0) at 123°C for 30 s and endogenous peroxidase was quenched with 3% hydrogen peroxide for 8 min. Charged proteins and endogenous IgG was blocked in background sniper solution for 20 min. Slides were incubated with the primary Dsg1 (Abcam; cat. no. ab124798, 1:1000 dilution), Dsg3 (Thermo Fisher; cat. no. BS-6585R, 1:400 dilution), Muc6 (Thermo Fisher; cat. no. PA5-79706, 1:1500 dilution), Tff3 (Thermo Fisher; cat. no. 14-4758-82, 1:5000 dilution), Sox9 (Abcam; cat. no. ab185230, 1:1000 dilution), p63 (GeneTex, cat. no. GTX102425, 1:100 dilution), or CK7 (Abcam, cat. no. ab181598, 1:50,000) overnight at 4°C. Detection of antibodies was achieved by incubating the tissue sections with anti-mouse or anti-rabbit horseradish peroxidase (HRP) conjugated polymers for 30 minutes. Sections were stained with 3,3’-Diaminobenzidine chromogen for 5 min to visualize the antigen-antibody binding, and hematoxylin was used as a nuclear counterstain. Stained sections were visualized using the EVOS M7000 imaging system (Thermo Fisher Scientific). In parallel, FFPE sections were subjected to standard Alcian blue/Periodic Acid Schiff’s stain procedure to evaluate the presence of mucins. Briefly, 5 μM esophageal and forestomach tissue sections were deparaffinized and hydrated with distilled water. Sections were then placed in Alcian Blue (AB) pH 2.5 stain solution for 30 min at room temperature, and rinsed with deionized water. Subsequently, sections were placed in Periodic acid solution (PAS) for 5 min at room temperature, rinsed with deionized water, followed by staining with Schiff’s reagent for 15 min and rinsed with deionized water. All sections were counter stained with hematoxylin 7211 for one minute, rinsed, and followed by staining with bluing reagent for one minute. Sections were then dehydrated in anhydrous alcohol followed by clearing and mounting. The acidic mucosubstances appear blue in color and the neutral polysaccharides appear purple/magenta in color. All images were captured using identical settings on the EVOS M7000 imaging system, and each tissue section on a slide was imaged in its entirety using the M7000 image tile-stitching software function. As appropriate, IHC findings were assessed on a qualitative scale for the presence or absence of signal (yes or no) in a blinded fashion.

Human PSC-derived esophageal organoids
PBMC-derived human pluripotent stem cells (hPSC) were generated from the VSIG10L S631G mutant patient who had provided written consent under the protocol approved by the Institutional Review Board for Human Investigation at the University Hospitals Cleveland Medical Center14. The control BU3 hPSC line65, derived from a healthy male Caucasian, was purchased from Boston university stem cell core under an MTA agreement and approved informed consent. Esophageal progenitor cells (EPCs) were subsequently derived from the hPSCs as previously described66. Briefly, hPSCs were first cultured in mTeSR plus medium (Stemcell technologies, Vancouver, BC; cat. no. 85850) on 6-well plates coated with 100x diluted Matrigel. After 24 hours, the medium was replaced with Definitive Endoderm Medium plus supplements MR and CJ (StemCell Technologies; cat. no. 05110). After additional 24 hours of culture (day 2), the media was replaced and cells incubated in Definitive Endoderm (DE) medium plus supplement CJ. At day 3, the media was replaced again with fresh DE media plus supplement CJ. At day 4, Serum-Free Differentiation (SFD) Media was prepared as following: 375 ml of reconstituted Iscove’s DMEM (Corning, Corning, NY; cat. no. 15-016-CV), 125 ml of Ham’s F-12 (Corning; cat. no. 10-080-CV), 5 ml of N2 (Thermo Fisher Scientific; cat. no. 17502048), 10 ml of B27 (Thermo Fisher Scientific; cat. no. 12587010), 3.3 ml of 7.5% Bovine Albumin Fraction V Solution (Thermo Fisher Scientific; cat. no. 15260037), 19.6 μl of 11.5M monothioglycerol (Sigma-Aldrich, St. Louis, MO; cat. no. M6145), 500 μl of L-Ascorbic acid (Sigma-Aldrich; cat. no. A92902) and 1 ml of 50 mg/ml primocin (Invitrogen, Waltham, MA; cat. no. NC9141851). At day 4, the cells were passaged into new Matrigel-coated 6-well plates as clusters and cells were further induced by culturing in the SFD medium plus 10 μM SB431542 (Selleckchem, Houston, TX; cat. no. S1067), 200 ng/ml Noggin (R&D Systems, Minneapolis, MN; cat. no. 6057-NG), 100 ng/ml EGF (Peprotech, Cranbury, NJ; cat. no. AF-100-15) and 10 μM Y-2763210 (Selleckchem; cat. no. S1049) for 24 hours and the SFD medium plus 10 μM SB431542, 200 ng/ml Noggin and 100 ng/ml EGF for another 48 hours (day 5-6). Cells were maintained at 5% O2/90% N2/5% CO2 from day 1–6. To induce esophageal progenitor cell differentiation, anterior cells were cultured from day 7 to day 16 in the SFD medium plus 10 μM SB431542, 200 ng/ml Noggin and 100 ng/ml EGF. From day 16 to day 24, cells were maintained in the SFD medium plus 100 ng/ml EGF. Cells were cultured at 95% air/5% CO2 from day 7 onwards. On day 24, the cells were sorted using FACS to obtain EPCs. To perform cell surface marker staining, cells were disassociated with Accutase added DNase (Thermo Fisher Scientific) and 10 μM Y-2763210 and stained with PE/Cyanine7 anti-human CD326 (EpCAM) antibody (BioLegend, San Diego, CA; cat. no. 324201) in FACS buffer (1× PBS, 2% FBS, added DNase and 10 μM Y-2763210) for 1 hour with live/dead staining dye SYTOX (Thermo Fisher Scientific; cat. no. S7020) to exclude dead cells. Stained cells were analyzed using BD FACS CariaII (BD Biosciences, Franklin Lakes, NJ) and sorted EPCAM-positive cells. Sorted hPSC-derived EPCs were counted and seeded at 20,000 cells per 50 μL Geltrex (Thermofisher Scientific; cat. no. A1413202) per one well of a 24-well plate, and the medium was added to after Geltrex solidified. The organoid culture medium included the SFD medium, supplemented with 10 μM Y27632, 10 μM SB431542, 10 μM GSK3 inhibitor (Stem cell technologies; cat. no. CHIR99021), 20 ng/ml FGF2 and 200 ng/ml EGF. hPSC-derived EPC 3D-organoids were subsequently FFPE embedded, sectioned, and used for analysis.

Semi-quantitative PCR analysis of VSIG10L in GERD vs. non-GERD biopsy tissues
Endoscopically-normal appearing mucosal biopsies were collected from the distal 5 cm of the squamous esophagus from control patients without BE or EAC during upper endoscopy who consented under the observational study biobanking protocol approved by the Institutional Review Board for Human Investigation of the University Hospitals Cleveland Medical Center9. Esophageal samples from Duke University hospitals were obtained from participants who enrolled in the GI Tissue Repository, and longitudinal observational study of patients presenting for endoscopic procedures who agreed to use of excess clinical samples approved by the Institutional Review Board for Human Investigation at Duke Universtiy Medical Center (protocol number; 00001662), adherent to the principles outlined in the Declaration of Helsinki. Archival samples from research participants were accessed through CWRU and Duke BioRepository & Precision Pathology Center (BRPC). Clinical data were collected and updated, including demographics, gastrointestinal symptoms, endoscopic and pathologic findings as previously described67. Chronic GERD was defined as the presence of at least weekly heartburn or regurgitation for at least five years as assessed by the Mayo gastroesophageal reflux questionnaire (GERQ)68. Erosive or ulcerative esophagitis cases were not included. Non-GERD was defined as either none or less than once a month heartburn or regurgitation for less than five years. All biopsy tissues were snap-frozen at the site of collection. Among the GERD subjects (n=23), 77% were men and 23% were women (self-reported), and the mean age of the sampled cohort at the time of diagnosis was 54.7 years (range; 28-77 yrs). 92% of GERD subjects were of European ancestry and 8% Asians. All but 4 of the subjects with GERD were on proton pump inhibitors or histamine-2 blockers. For the non-GERD control cohort (n=24), 73% were men and 27% were women (self-reported), and the mean age of the sampled cohort was 60.1 years (range; 41-80 yrs). 96% of non-GERD control subjects were of European ancestry and 4% African Americans. Total RNA was extracted from respective distal esophageal squamous biopsies using RNeasy Plus Mini kit (Qiagen, Germantown, MD; cat. no. 74134,). Subsequently, 0.5-1 μg of total RNA was reverse-transcribed using Superscript IV First-Strand Synthesis (Thermo Fisher Scientific; cat. no. 18091) according to the manufacturer’s protocol. qPCR of VSIG10L (Qiagen; cat. no. QT02451393), TP63 (Qiagen; cat. no. QT02424051), and beta-2-microglobulin (B2M) (Qiagen; cat. no. QT00088935) were performed using iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA; cat. no. 170-8887) on the CFX96 system (Bio-Rad). TP63 was used as a control for SQ epithelial content in the biopsy tissues and B2M was used as an endogenous control for RNA. Each qPCR reaction was carried out in triplicates and the mean expression levels across the triplicates were normalized to the mean expression values of B2M using the ΔCt method. A two-tailed non-parametric Mann-Whitney U test was used to determine significant differences in expression between GERD vs non-GERD tissues.

Statistics & Reproducibility
Data analyses and pertinent statistical tests are outlined in respective Methods sub-sections above. Sample size estimations or randomization were not applicable for human genetic studies, as all individuals that were accrued were sequenced. The study focuses on identifying BE susceptibility variants in VSIG10L in affected families. Unaffected/healthy family relatives were sequenced where available. To assess the minor allele frequencies of the detected VSIG10L variants in families, large-scale population databases like gnomAD and ExAC were used. Results from gene expression studies in human samples were estimated using non-parametric tests. For animal studies, assuming 80% power and a significance level of 0.05, 6 mice in each group will enable detecting ≥50% metaplasia incidence (qualitative estimates) with a 95% CI of ±28.1. For diet studies, respective age-matched genotype mice were randomly assigned to the standard or DCA diet groups, and dietary interventions were initiated at the same age across genotypes. Vsig10l-mutant genotypes were compared to respective wild type littermates in all instances. Results from animal expression studies were also estimated using non-parametric tests, and additional multiple testing corrections were performed for STP datasets where appropriate. Where appropriate, biologic/technical replicates were included in the experiments to confirm reproducibility of the results. As stated before, mouse genotypes were blinded for all subsequent histopathologic and electron microscopy assessments. No data were excluded for either human or animal studies.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The study ‘VSIG10L is a Major Determinant of Esophageal Homeostasis and Inherited Predisposition to Barrett’s Esophagus’ by Durgadevi Ravillah et al. explore the functional significance of V-set and Immunoglobulin Domain Containing 10 Like (VSIG10L) in development of BE. This study builds upon the previous work identifying a significant association between germline variants in the previously uncharacterized gene VSIG10L and genetic predisposition to BE. The current study provides insight into the localization of VSIG10L in esophageal epithelium using tissues from humans as well as organotypic and organoids cultures, the latter developed from iPSCs derived from human subjects with or without VSIG10L mutation. Then, the authors generate genetically engineered mouse lines with Vsig10l S516G mutation or Vsig10l knockout, both of which reveal defects in esophageal epithelium suggesting impaired epithelial integrity. Upon challenge with deoxycholate (DCA) these mice develop lesions BE-like lesions at the squamo-columnar junction. Finally, the authors demonstrate decreased expression of VSIG10L in esophageal biopsies from patients with GERD. There is a paucity of knowledge related to the genetic factors that predispose one to BE, supporting the significance of this study. Below I provide suggestions to improve the rigor of this study:\nPlease provide H&E images of organoids from Figure 1B as the current images make it difficult to assess architecture. It appears that the highlighted organoids from the Vsig10l S516G patient consist of a single layer of cells and lack evidence of squamous differentiation. Are the cells in this later basal or do they express columnar markers?\nIn Vsig10l S516G and KO mice:\n• The presence of dilated intracellular spaces in esophageal epithelium is not convincing. Please refer to PMID: 8898633.\n• It appears that all phenotypes described in Figure 2 occur in the esophagus. It is important to validate these in the squamous forestomach as this is where BE-like lesions are detected.\n• It would be interest for the authors to comment on any phenotype (or lack thereof) observed in esophageal tissues of Vsig10l S516G and KO mice. It is implied that there may be no phenoptye (lines 233-235), but this should be directly addressed.\n• Assays to confirm impaired epithelial barrier integrity are needed as this is essential to the premise of the model proposed in Figure 6.\n3-The human subjects classified as GERD should be more clearly defined, including status of VSIG10L as well as PPI use at the time of biopsy. It is unclear if these GERD subjects had active inflammation at the time of biopsy. It is surprising that no difference in p63 was shown as GERD is associated with basal zone hyperplasia. Using ISH to visualize VSIG10L in biopsies from these subjects should be performed.", "reviewer_2": "In the submitted manuscript, Dr. Kishore Guda's group based on the familial clusters studies of the onset of Barrett's esophagus, focus on a germline variants in the VSIG10L, inherited predisposition to Barrett's esophagus. Although this work has certain merits on mice model by validating a key gene variant in BE pathogenesis, it is unfortunately pure descriptive without novel mechanistic insights. Many of the analyses are at most speculations and many are well known facts in BE-EAC studies.\nHowever, their analysis and validation is not comprehensive, and the proposal in VSIG10L as a key determinant in BE pathogenesis is not innovate (2016, JAMA Oncol), either. I have some concerns and comments regarding the contents of this study listed below.\n1.Strengthen Human Functional Validation and add human Cohort Support for VSIG10L Pathogenicity.\nWhile the authors have identified several rare VSIG10L variants in BE/EAC families and established mouse models carrying key mutations, the current evidence primarily centers on genetic associations and animal phenotypes. Incorporating direct functional validation in human cells or tissues—such as knock-in of the corresponding mutations—would help to more definitively demonstrate the pathogenicity of these mutations. In addition, verifying these findings using a more human tissue samples would further support the association between VSIG10L variants and disease risk. These additional studies would enhance the causal inference and translational value of the work.\n2.Further Elucidation of the Gene-Environment Interaction Mechanisms and Downstream Molecular Pathways.\nThe manuscript presents evidence that VSIG10L mutations, together with environmental factors such as bile acid exposure, contribute to the development of Barrett’s-like metaplasia, and spatial transcriptomics have revealed perturbations in epithelial homeostasis pathways. To further enrich the mechanistic understanding, the authors could consider deeper analyses of their spatial transcriptomics and differential gene expression data to pinpoint key downstream effectors and signaling pathways. Clarifying how VSIG10L deficiency influences epithelial repair, differentiation, and barrier integrity by studying the specific downstream molecules or pathways—as well as detailing the interplay between genetic and environmental factors at the molecular level—would provide a more comprehensive mechanistic framework.\n3. Enhancing the Statistical Generalizability and Population-Level Validation of Rare Pathogenic Variants.\nThe study’s identification of pathogenic VSIG10L variants is largely based on familial screening, with most variants appearing to be extremely rare or private to individual families. To further establish the broader relevance of these findings, the authors might consider integrating analyses from population-based whole-genome or exome sequencing datasets relevant to BE. Such efforts would clarify the population frequency, penetrance, and risk effect of these variants, thereby increasing the statistical power and clinical significance of the study’s conclusions.\nMinor:\nNo subtitles in section of Results. Needs more data supports the working model (figure 6).", "reviewer_4": "Authors have shown that the gene VSIG10L is associated with increased BE / EAC in clusters of Familial BE , mice model and organoids.\nthey must be applauded for the extensive and thorough work.\nThey have shown that gene missense and frame shift mutation was noted in human BE families and when incorporated in murine knock out models predisposed them to develop abnormal squamous mucosa which further developed into BEMM on exposure to diet rich in DCA (bile).\nthey noted areas of BEMM near SQ-C junction ie near the esophago- gastric junction as opposed to none noted in mutant gene mice not exposed to DCA.\ngiven that the DCA was given in diet and that the mutation would have affected the entire esophageal epithelium is there a reason that the BEMM changes were noted only at the native GEJ and not along the length of the esophagus. Some discussion to this affect would help shed light on this.\nAnother question which arises- do authors believe that the mutation enabled unstable mucosa simply provides a 'fertile ground; for DCA to work as a BEMM inducing agent or does this specific mutation is somehow needed for the BE genesis- though may not be able to be answered based on current study but should be discussed.\nMinor comments:\n• In Figure 1a, the framed picture of the SQ 3D-organoid is not providing any additional magnification to the ISH slide in the background.\n• Could the authors provide details on how the IHC assessment was conducted (qualitative, semi-quantitative, or quantitative)? Was it based on rounded percentages, cell count, area-based analysis? Was a computer-based system or AI used for staining detection? Was a quantitative comparison performed or was it observation-based?\n• For comparisons presented in Figure 3, the authors report a p-value estimated using a one-tailed Fisher’s exact test. Should corrections or post-hoc tests for multiple group comparisons be applied to reduce the risk of false positives, particularly given the small number of animals in each group (n=7–12) and the number of groups (5)?\n• Similarly, when comparing expression of VSIG10L and TP63 in GERD vs. non-GERD patients, the authors assume a normal distribution of expression levels. As this is rarely the case, was a data normality assessment performed upfront? If the data were nonparametric, I suggest using the Mann–Whitney U test instead of a t-test.\n• I recommend that the authors include a study flow diagram representing all the experiments performed and reported in the current manuscript.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We thank the editors and reviewers for their valuable time and volunteering to review our manuscript, and for their overall enthusiasm and endorsement of the study. Also, we very much appreciate the reviewers in recognizing the arduous efforts involved in pursuing human genetic studies and generating animal models, and the high clinical-translational potential of such studies, especially in complex disorders like Barrett’s esophagus (BE).\nIn brief, we originally made the observation of inherited predisposition in BE/EAC, and have been accruing families across the U.S and Europe for over two decades. Using genetic association, high-throughput genomics, and subsequently developing orthologous animal models, we discovered VSIG10L to be a key determinant in BE pathobiology. Our findings, in toto, underscore the many routes to BE genesis, in addition to identifying the genes and processes involved in BE predisposition. Also, the Vsig10l mutant mouse models we present here are the first of its kind in this disorder that is directly based on human disease and predisposition. We believe our study will stimulate new areas of investigations into VSIG family of proteins and their connection to esophageal SQ biology and BE pathogenesis. From a clinical-translational standpoint, our continued efforts in identifying such genetic determinants of BE/EAC should enable developing effective screening and surveillance strategies for patients, and in formulating improved prevention approaches for this complex disorder. Below we have provided our responses to the reviewers’ thoughtful comments to the best of our ability.\nReviewer #1:\n1. Please provide H&E images of organoids from Figure 1B as the current images make it difficult\nto assess architecture. It appears that the highlighted organoids from the Vsig10l S516G patient\nconsist of a single layer of cells and lack evidence of squamous differentiation. Are the cells in\nthis later basal or do they express columnar markers?\n• We apologize for this, and have now included the H&E and also p63 staining of the organoids\n(performed on new serial sections) in revised Figure 2 (originally Figure 1b). In regard to the\nabnormal glandular-appearing organoids in the mutant patient, the single layer of cells do\nshow p63 positivity, albeit to a different degree (we also tried CK7 columnar marker but it\nshowed no reactivity in these single layer of cells). Additionally, unlike in the control subject,\nmajority of the normal-appearing SQ spherical organoids from the mutant patient also showed\nloss of inward epithelial differentiation gradient, with near-all p63-positivity in the cells, further\nsuggesting a perturbation in SQ differentiation. Nonetheless, given the conceptual/technical\nlimitations associated with organoids (in general), assessing/gauging further metaplasia/BE\ntransformation may not be feasible in these models. Out of an overabundance of caution,\nwhat we can say at this point is that hPSC-derived esophageal organoids from the VSIG10L-\nmutant patient exhibit aberrant SQ maturation/stratification.\n2. In Vsig10l S516G and KO mice: The presence of dilated intracellular spaces in esophageal\nepithelium is not convincing. Please refer to PMID: 8898633.\n• We agree that DIS is not marked and as readily apparent in our review. To avoid over\ninterpretation, we have removed DIS when mentioning the results in mice in the revised\nmanuscript and stated only the clearly apparent reduction in desmosomes.\n3. It appears that all phenotypes described in Figure 2 occur in the esophagus. It is important to\nvalidate these in the squamous forestomach as this is where BE-like lesions are detected.\n• We apologize for not appropriately mentioning the anatomical location of the tissues used for\nthe studies in Figure 2. As we (us) are used to referring “distal esophagus” routinely during\nour conversations regarding BE in humans, we inadvertently used the esophagus terminology\nfor the mice as well. In fact, samples for Figure 2 experiments were obtained from the\nsquamous forestomach region for the exact reason the reviewer alludes to. We have now\nclarified this in the Figure legend, and in the text (where appropriate) of the revised\nmanuscript. Please note that original Figure 2 is now Figure 3 in the revised manuscript.\n4. It would be interest for the authors to comment on any phenotype (or lack thereof) observed in\nesophageal tissues of Vsig10l S516G and KO mice. It is implied that there may be no phenotype\n(lines 233-235), but this should be directly addressed.\n• We did not observe any apparent changes in the esophagus proper of S516G and KO mice.\nWe have now included an explicit statement in this regard in the appropriate Results section\nof the revised manuscript.\n5. Assays to confirm impaired epithelial barrier integrity are needed as this is essential to the\npremise of the model proposed in Figure 6.\n• VSIG10L expression is selective to squamous epithelia. We previously showed that\nintroducing the S631G mutation into SQ 3D-organotypes disrupts squamous architecture,\nmaturation and polarity, while electron microscopy studies in the VSIG10L-mutant patient\nrevealed dilated intercellular spaces and loss of desmosomes in the SQ esophagus. This loss\nof desmosomes was particularly recapitulated in Vsig10l-mutant mice. These complementary\nfindings (from human tissues, 3D models, animals), when taken together, suggested a\nplausible hypothesis where such changes could compromise epithelial integrity and\npresumably barrier function, thus enhancing risk of BE. Based on the above, we conceived\nan illustration that was meant as hypothesis-generating rather than an established paradigm;\n(i.e.) suggesting one of the potential mechanisms by which mutant VSIG10L could promote\nBE pathobiology. Nonetheless, we have now updated the illustration (based on the\nsuggestions and new data generated as part of comment 6 below), and have additionally\nclarified the text in the Figure legend to reflect this. We further note that our prior and current\n(long-term) translational studies on VSIG10L in FBE families, model systems, and orthologous\nanimals were predominantly geared towards identifying VSIG10L susceptibility variants and\ndetermining their pathogenicity and phenotypic consequences, a critically-important step in\nascertaining causality. VSIG10L remains largely uncharacterized, and it is highly likely that\nthere could be additional (yet to be determined) regulatory and functional aspects for VSIG10L\npotentially involved in BE pathogenesis. We certainly plan to pursue these additional lines of\ninvestigations moving forward now that we have a strong genetic and functional evidence\nlinking VSIG10L to BE pathogenesis/predisposition (a first in this complex disorder).\n6. The human subjects classified as GERD should be more clearly defined, including status\nof VSIG10L as well as PPI use at the time of biopsy. It is unclear if these GERD subjects had\nactive inflammation at the time of biopsy. It is surprising that no difference in p63 was shown as\nGERD is associated with basal zone hyperplasia. Using ISH to visualize VSIG10L in biopsies\nfrom these subjects should be performed.\n• We have now included this information in the respective Methods section of the revised\nmanuscript. We note that we bank only endoscopically normal-appearing biopsies and not\nerosive or ulcerative esophagitis samples. Secondly, in regard to P63, the normalized qPCR\nassessments from whole tissue (snap-frozen) biopsies showed no significant changes in P63\nexpression between non-GERD vs. GERD patients. One prior study in cell culture model also\nnoted no change in P63 RNA, albeit reduced protein levels, when squamous epithelial cells\nwere exposed to acid/bile (PMID: 17615180). Also, to our knowledge, we came across only\none article on this topic in the literature which investigated highly expressed hub\ngenes/networks in non-erosive reflux patients (N=10) vs. healthy controls (N=6) (PMID:\n34848992), using RNA sequencing. Here again, the study did not identify P63 RNA or related\nnetwork as being altered in samples from reflux patients. It is possible that cellular-admixture\nin whole tissue biopsies, non-homogeneous or varying degree of P63 expression pattern, PPI\nusage, and/or differences in RNA vs protein stability/estimation etc. may in part influence\nthese measurements. Accrual of well-defined / clinically-annotated non-GERD/GERD\nsamples is quite challenging (even at a high volume hospital like ours). And, unfortunately,\nwe do not have sister (FFPE) biopsies matching the snap-frozen tissues used for our qPCR\nassessments. Nonetheless, in considering the reviewer’s comment, we tried hard to procure\nprocure an independent set of FFPE specimens by pooling resources from Case and Duke University\nhospitals patient path core repositories, and in the end were able to identify a few clinically-\nannotated GERD patient samples, as well a couple of non-GERD subjects. These subjects\nwere classified as GERD or non-GERD based not only on clinical diagnosis but also on\nhistopathologic changes well associated with GERD injury or absence thereof. We\nsubsequently performed VSIG10L ISH (as well as P63 IHC) on these FFPE sections. This\nnew data is included in Figure 6 of the revised manuscript. In stark contrast to the healthy\nmucosa, GERD-associated reactive SQ mucosa showed a profound reduction/depletion of\nVSIG10L-positive differentiated cells across all GERD patients tested, often concomitant with\nan expansion of basal-like, p63-positive, immature SQ cells. We have now included the\nupdated results and text in the appropriate results section and Figure legend of the revised\nmanuscript. These findings offer new insights into the pathobiologic effects of chronic injury\non esophageal VSIG10L-mucosal dynamics. We have accordingly also updated our\nhypothetical illustration in Figure 7 taking into account some of the above findings. Despite\nthe time and effort it took us to procure these FFPE specimens, we are glad we did this\nanalysis as it added further support and insight into the impact of reflux injury on VSIG10L\nstatus in situ and we thank the reviewer for this particular suggestion.\nReviewer #2:\n1.Strengthen Human Functional Validation and add human Cohort Support\nfor VSIG10L Pathogenicity. While the authors have identified several rare VSIG10L variants in\nBE/EAC families and established mouse models carrying key mutations, the current evidence\nprimarily centers on genetic associations and animal phenotypes. Incorporating direct functional\nvalidation in human cells or tissues—such as knock-in of the corresponding mutations—would\nhelp to more definitively demonstrate the pathogenicity of these mutations. In addition, verifying\nthese findings using a more human tissue samples would further support the association\nbetween VSIG10L variants and disease risk. These additional studies would enhance the causal\ninference and translational value of the work.\n• We have shown that introducing the VSIG10L S631G mutant in EPC2 3D-organoids disrupts\nSQ maturation and integrity (PMID: 27467440). These findings are also conceptually in line\nwith our observations in the hPSC-derived esophageal SQ organoids in the patient (Fig. 2 of\nthe revised manuscript). Human culture/organoid models have significant conceptual and\ntechnical limitations; more so for a complex disorder like BE. Moreover, such culture systems\nare not suited for studying long-term BE predisposition/risk. That’s why we opted to test the\nconsequence of these mutations in vivo by painstakingly generating the VSIG10L mutant\nanimals and following them for up to 2 years under different dietary exposures. The similarities\nin ultrastructural and phenotypic manifestations between VSIG10L-mutant mice and human\nBE provide further strong evidence for a causal link between identified VSIG10L mutations\nand BE pathobiology.\n2.Further Elucidation of the Gene-Environment Interaction Mechanisms and Downstream\nMolecular Pathways. The manuscript presents evidence that VSIG10L mutations, together with\nenvironmental factors such as bile acid exposure, contribute to the development of Barrett’s-like\nmetaplasia, and spatial transcriptomics have revealed perturbations in epithelial homeostasis\npathways. To further enrich the mechanistic understanding, the authors could consider deeper\nanalyses of their spatial transcriptomics and differential gene expression data to pinpoint key\ndownstream effectors and signaling pathways. Clarifying how VSIG10L deficiency influences\nepithelial repair, differentiation, and barrier integrity by studying the specific downstream\nmolecules or pathways—as well as detailing the interplay between genetic and environmental\nfactors at the molecular level—would provide a more comprehensive mechanistic framework.\n• In considering the reviewer’s comment, we further explored whether Vsig10l disruption\nadditionally leads to perturbations in downstream effectors / signaling pathways, using our in-\nhouse developed computational framework, InFlo. InFlo uses RNAseq-based expression and\nother omics data to estimate signaling pathway activities (based on established pathway\nnetwork maps), including activities of components and intermediary complexes that are not\ndirectly measured (example references to InFlo; PMID: 27819677; PMID: 30768984; PMID:\n35870513). While assessment of STP expression profiles using InFlo showed no significant\nperturbations in major signaling pathway networks in the Vsig10l-mutant mice, there were a\nfew (distinct) small nodes with alterations in estimated activities in the Vsig10l-mutant mice.\nSome of these small sub-networks, including Notch, Bmp, Smad etc have been implicated in\nthe BE/EAC disease. We have now included this exploratory analysis as a new\nSupplementary Fig. 1 and outlined in the appropriate Results section of the revised\nmanuscript. While this analysis provides us with several hypotheses and directions to pursue,\ndetermining their functional relationship with VSIG10L (direct or indirect) and the broader\nsignificance of such changes in these specific nodes / sub-networks require further\ncomprehensive investigations, which we are planning to tackle in future studies.\n3. Enhancing the Statistical Generalizability and Population-Level Validation of Rare Pathogenic\nVariants. The study’s identification of pathogenic VSIG10L variants is largely based on familial\nscreening, with most variants appearing to be extremely rare or private to individual families. To\nfurther establish the broader relevance of these findings, the authors might consider integrating\nanalyses from population-based whole-genome or exome sequencing datasets relevant to BE.\nSuch efforts would clarify the population frequency, penetrance, and risk effect of these variants,\nthereby increasing the statistical power and clinical significance of the study’s conclusions.\n• Familial Barrett’s Esophagus, similar to other complex genetic diseases (e.g., Familial\nPancreatic / Colorectal / Breast Cancer etc.), is genetically quite heterogeneous. We\ndescribed Familial BE 25 years ago. Since then we have accrued more than 300 families\nacross U.S and Europe. Our prior linkage study (PMID: 27468417) indeed found multiple loci\nconfirming heterogeneity. We have spent considerable expense and effort in whole genome\nand whole exome sequencing the germline DNA from these families. Like with other complex\ngenetic diseases, identification of susceptibility genes has proven to be challenging.\nSequencing of rare, large multi-generational families such as the family in which VSIG10L\nwas originally discovered, and where heterogeneity is not a concern, has been uniquely\nsuccessful. The variants in VSIG10L we have discovered are either private or have extremely\nrare population allele frequencies (based on Genome aggregation database). The sample\nsize that would be required to statistically study these rare variants would be prohibitive. Also,\nprior sequencing studies and available public datasets pertain mostly to somatic alterations in\nBE and/or EAC, and not focused on genetic predisposition. On the other hand, GWAS-based\nlinkage datasets are also not suitable for studying rare, disease-associated/causal variants.\nThe above points apply more broadly to other disease contexts where rare susceptibility\nvariants in genes account for much of the inherited predisposition, as we have previously\ndemonstrated for colorectal cancer (as example; PMID: 19617566, PMID: 20551049, PMID:\n29749045, PMID: 24838973). Our discovery of such susceptibility variants in VSIG10L in\nfamilial BE and subsequent demonstration of disease induction in relevant animal models in\nparticular are of high translational impact, and underscore the pathogenicity of identified\nVSIG10L susceptibility variants and their involvement in BE predisposition. In regard to clinical\nsignificance, we respectfully submit that the discovery of such rare causal variants in families\nenables robust screening/surveillance in patients for prognosticating risk and/or early\ndetection, in implementing improvised strategies for disease prevention and intervention, as\nwell as provide critical/novel insights into genes and processes involved in disease\npathogenesis.\nMinor:\n1. No subtitles in section of Results. Needs more data supports the working model (figure 6).\n• Our manuscript was originally written as a letter format article to better convey the major points\nof the study by combining the results and discussion section, while minimizing tangential\ninterpretations and discussions. However, in the revised manuscript, we have adhered to the\nformatting guidelines of Nature Communications and have included subtitles for the Results\nsection for better clarity. Secondly, as outlined above under comment 5 (Reviewer 1), the\nillustration in Figure 6 is a not an established model, rather meant to be hypothesis generating.\nFindings from our electron microscopy studies in humans and mice, prior VSIG10L S631G\nmutant studies in squamous 3D organotype, hPSC-derived esophageal organoids from\nVSIG10L mutant patient, and the animal models developing Barrett’s esophagus like\nmucinous metaplasia (BEMM) phenotypes all suggested a plausible hypothesis of how\nVSIG10L could be linked to BE pathobiology by disrupting epithelial homeostasis and creating\na risky field. Over time, such changes can co-opt with other risk factors such as GERD to\npromote BE pathogenesis. We have further clarified and updated Figure 7 (originally Figure\n6) based on new data, speculating on the likely routes of BE development. We also note that\nthis is also the first study providing direct evidence connecting a squamous gene mutation\n(molecular etiology) to disrupted squamous programs (process) and BE\npredisposition/pathogenesis (disease).\nReviewer #4:\n1. given that the DCA was given in diet and that the mutation would have affected the entire\nesophageal epithelium is there a reason that the BEMM changes were noted only at the native\nGEJ and not along the length of the esophagus. Some discussion to this affect would help shed\nlight on this.\n• We agree with the reviewer that it is intriguing that mucinous metaplasia develops at the\nsquamo-columnar (Gastro-esophageal) junction (SCJ/GEJ). Much effort has gone into\nunderstanding why transitional squamo-columnar zones in organs such as esophagus,\nrectum, and cervix are predisposed to the development of metaplasia and cancer. Indeed,\nstudies (PMID: 29019984) have proposed transitional cells as one putative mechanism for the\ndevelopment of Barrett’s esophagus. SCJ/GEJ may remain the primary site for reflux injury\nallowing the backflow of stomach acid resulting in metaplastic changes when compared to the\nlength of esophagus. Another possibility could be esophageal epithelium may show cellular\nheterogeneity and hence exposure to DCA show differential response to the insult along the\nlength of esophagus compared to SCJ/GEJ. We have now added some text outlining the\nabove in the discussion section of the revised manuscript.\n2. Another question which arises- do authors believe that the mutation enabled unstable mucosa\nsimply provides a 'fertile ground; for DCA to work as a BEMM inducing agent or does this specific\nmutation is somehow needed for the BE genesis- though may not be able to be answered based\non current study but should be discussed.\n• This is an interesting question and our thought is also that these susceptibility variants are\nlikely creating a risky field for subsequent development of the disease. Considering the\nfindings from our in vitro and in vivo studies thus far, it is reasonable to presume that these\nsusceptibility variants are likely pathogenic and disrupting SQ architecture and maturation.\nThat being said, both our missense and KO animals showed development of BEMM only in\nthe presence of a DCA (mimicking reflux injury). It is therefore likely that disruption in\nsquamous homeostasis caused by these mutations provide a “risky field”, and over time, can\nco-opt with additional insults promoting BE pathogenesis (either by rendering the mucosa\nmore prone to injury and/or impaired healing following injury). Taken together, our findings\nsuggest a plausible hypothesis (revised Figure 7) where a compromise in SQ homeostasis,\nmuch like that observed in GERD, may be one of the key initial processes in the development\nof BE. Screening additional families prospectively and identifying potentially new genetic\nmutations in VSIG10L might help us in further determining the relative-pathogenicity and\nfunctional impact of a given mutation. Another approach could be to generate animal models\ncarrying orthologous common missense polymorphisms in Vsig10l to compare and contrast\ndisease predisposition between the familial susceptibility variants vs. common\npolymorphisms. We further note that VSIG10L remains largely uncharacterized and much of\nits function is yet to be determined. Given the now strong genetic and functional evidence\nlinking VSIG10L to BE pathogenesis, we hope to tackle these lines of investigation moving\nforward. We have included some of the above points in the discussion section of the revised\nmanuscript.\nMinor:\n1. In Figure 1a, the framed picture of the SQ 3D-organoid is not providing any additional\nmagnification to the ISH slide in the background.\n• We have now removed the framed inset from Fig. 1 for the SQ 3D-organoid in the revised\nmanuscript.\n2. Could the authors provide details on how the IHC assessment was conducted (qualitative,\nsemi-quantitative, or quantitative)? Was it based on rounded percentages, cell count, area-based\nanalysis? Was a computer-based system or AI used for staining detection? Was a quantitative\ncomparison performed or was it observation-based?\n• Our main goal for the follow-up IHC studies was to assess whether the BEMM lesions exhibit\nsome of the key markers associated with human BE, on a qualitative scale (yes or no), and\nwanted to minimize any over interpretation by further assessing on a quantitative level.\nMoreover, the profound differences in the type and extent of the lesions between WT vs.\nKI/KO mice also precluded us to perform any meaningful quantitative differences by IHC\nbetween the genotypes, in addition to the limitations associated with quantitating IHC signals\nfor such analysis. We have now included this note in the appropriate Methods section of the\nrevised manuscript. We further note that all the images were acquired using M7000 scope\nwith similar intensity settings across the genotypes. Moreover, we captured the tissue section\non a slide using the automated image tile-stitching software function on the M7000, and\naccordingly was able to assess the whole section in a single image. All histopathologic\nanalysis was conducted by our GI pathologist (J.E.W.) who was blinded to the genotypes.\n3. For comparisons presented in Figure 3, the authors report a p-value estimated using a one-\ntailed Fisher’s exact test. Should corrections or post-hoc tests for multiple group comparisons be\napplied to reduce the risk of false positives, particularly given the small number of animals in each\ngroup (n=7–12) and the number of groups (5)?\n• We contemplated this when analyzing the data. Basically, we enumerated the number of\nanimals containing any changes at the squamo-columnar junction (even single glandular-\nappearing lesions) occurring in each mouse exposed to DCA diet (genotypes blinded to our\npathologist). As mentioned and shown in the revised manuscript (updated Fig. 5), the lesions\ndeveloping in the KI/KO mice were profoundly exacerbated, compared to the infrequent and\nminor glandular changes we saw in the WT, underscoring a fundamental difference. Given\nthe pronounced phenotypic differences between WT vs. KI/KO, we strongly believe the\nlikelihood of such qualitatively exacerbated lesions to occur by random chance in an otherwise\nnormal/wild-type mouse (even exposed to DCA) would be extremely rare. That being said,\nfrom a statistical perspective, given that we conducted 4 hypothesis tests, we can adjust\nsignificance at P = 0.0125 (0.05/4) using Bonferroni. We have now added this Bonferroni\ncorrected P value for additional consideration in the Figure 4 legend (originally Figure 3) of\nthe revised manuscript.\n4. Similarly, when comparing expression of VSIG10L and TP63 in GERD vs. non-GERD patients,\nthe authors assume a normal distribution of expression levels. As this is rarely the case, was a\ndata normality assessment performed upfront? If the data were nonparametric, I suggest using\nthe Mann–Whitney U test instead of a t-test.\n• We appreciate the comment and correctly pointing this out (as the Shapiro-Wilk showed\ndeviation from normality). We re-ran the analysis using the non-parametric Mann Whitney U\ntest. We have accordingly provided the updated P-value in the revised manuscript (showing\nsignificant differences in VSIG10L expression, but not P63, between non-GERD vs. GERD\nsamples).\n5. I recommend that the authors include a study flow diagram representing all the experiments\nperformed and reported in the current manuscript.\n• We thank the reviewer for making this suggestion and have now included a study flow diagram\nas a new supplementary Fig. 2 in the revised manuscript.\n• We again thank all the reviewers for their time and effort and enthusiastically endorsing\nour translational efforts in this complex disorder."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have appropriately addressed each of my comments.", "reviewer_2": "The revised manuscript has been improved, I think this paper is appropriate for Nat. Commun. now. The authors have solved almost all my concerns. This work is significance to the field in Esophageal related diseases.", "reviewer_6": "This study is a valuable extension of the authors’ prior work mapping the BE susceptibility variant S631G to VSIG10L. Their efforts to characterize variability in VSIG10L expression and its potential contribution to BE susceptibility are commendable and advance the early understanding of VSIG10L’s role in this disease. The study methodology are sound, and following the first round of edits are well-described and comprehensive.\nKey strengths include the demonstration that VSIG10L RNA is selectively and consistently expressed in suprabasal squamous cells; the finding that organoids harboring the VSIG10L-S631G variant exhibit disrupted squamous stratification and maturation compared with controls; and mouse models showing reduced desmosomes in the suprabasal squamous layer in S516G and knockout variants. These findings support ongoing work to define the pathogenic and phenotypic impact of VSIG10L variants, while recognizing that further studies are needed to clarify the mechanisms of BE pathogenesis and causality.\nPlease note that I served as a co-reviewer after Reviewer #4 became unavailable to evaluate the authors’ responses to their earlier comments. The authors’ responses to Reviewer #4’s comments are satisfactory. I recommend that this manuscript be accepted for publication."}, "author_response": ""}]

Decision letter