Niederwanger et al. Eur J Med Res (2018) 23:13 https://doi.org/10.1186/s40001-018-0312-2 European Journal of Medical Research

RESEARCH Open Access Isolated choanal and gut atresias: pathogenetic role of serine protease inhibitor type 2 (SPINT2) unlikely Christian Niederwanger1, Silvia Lechner2, Lisa König3, Andreas R. Janecke3, Claus Pototschnig4, Beatrice Häussler5, Sabine Scholl‑Bürgi3, Thomas Müller3 and Peter Heinz‑Erian3*

Abstract Background: Choanal (CA) and gastrointestinal atresias (GA) are an important feature of syndromic congenital sodium diarrhea (sCSD), a disorder recently associated with mutations in the gene for serine protease inhibitor type 2 (SPINT2). It is, however, not known whether isolated non-syndromic CA and GA themselves might result from SPINT2 mutations. Methods: We performed a prospective cohort study to investigate 19 CA and/or GA patients without diarrhea (“non- sCSD”) for potential sCSD characteristic clinical features and SPINT2 mutations. Results: We found a heterozygous SPINT2 splice (c.593-1G>A), previously demonstrated in sCSD in homozygous form, in only 1 of the 19 patients of the “non-sCSD” cohort. This patient presented with isolated anal atresia and borderline low laboratory parameters of sodium balance. In the remaining 18 non-sCSD CA/GA patients investigated, SPINT2 sequence analysis and clinical markers of sodium homeostasis were normal. None of the 188 healthy controls tested in a regional Tyrolean population harbored the c.593-1G>A mutation, which is also not listed in the ExAc and gnomAD databases. Conclusions: The fnding of only one heterozygous SPINT2 mutation in 19 patients with isolated CA/GA was not statistically signifcant. Therefore, SPINT2 mutations are an unlikely cause of non-sCSD atresia. Trial registration ISRCTN73824458. Retrospectively registered 28 September 2014 Keywords: Isolated atresia, Gut, Choanae, Serine protease inhibitor type 2, SPINT2 splice mutation

Background SPINT2 encodes for Kunitz-type serine protease inhibi- Atresias of the choanae (CA) [1] and the gut (GA) have tor type two, also reported as placental bikunin [8] and been shown to be due to a large variety of pathomecha- activator type 2 [9], which has nisms [2–6]. One disorder also commonly exhibiting CA been shown to inhibit a number of serine proteases such and/or GA is autosomal recessive syndromic congeni- as matriptase, prostasin and furin [10–12]. Te latter tal sodium diarrhea (sCSD) [7]. Te phenotype of sCSD have been postulated to regulate the activity of the epi- involves voluminous watery stools and enormous fecal thelial sodium channel (ENaC) which allows uptake of losses of sodium, leading to hyponatremia and metabolic sodium by many cell types, including the intestinal epi- acidosis. Te disease is caused by homozygous and com- thelium [12]. SPINT2 mutations in sCSD patients may pound heterozygous mutations in the SPINT2 gene [7]. interfere with the normal process of regulated proteolytic activity and thus lead to defective function of ENaC. Tis

*Correspondence: Peter.Heinz‑Erian@i‑med.ac.at could result in enhanced diarrheal sodium loss via the gut 3 Department of Pediatrics I, Medical University of Innsbruck, Anichstrasse epithelium [7] as well as frequent occurrence of atretic 35, 6020 Innsbruck, Austria malformations in sCSD, although the pathomechanism Full list of author information is available at the end of the article

© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Niederwanger et al. Eur J Med Res (2018) 23:13 Page 2 of 7

is currently incompletely understood. We investigated two heterozygous SPINT2 nucleotide alterations were patients with isolated forms of CA and GA, who did not identifed in exon seven of two non-sCSD patients with sufer from sodium-losing diarrhea (non-sCSD atresia), anal atresia. c.598G>C het, demonstrated in patient #10 with regard to SPINT2 mutations and clinical picture, (Table 1), was found to be a common polymorphism with and compared the results with those from sCSD patients. an allele frequency of 2.8% in 61.000 individuals (ExAC database). c.593-1G>A het, detected in a second non- Patients and methods sCSD atresia patient (patient #14, Table 1 upper panel) Te study was approved by the ethics committee of the of our study is a splice mutation that had been identifed Medical University of Innsbruck. 84 patients diagnosed in homozygous state in fve previously reported Austrian with CA and/or GA between January 1, 1980 and Decem- sCSD patients [7]. Tis mutation afects the canoni- ber 31, 2011 were identifed from the patient registries cal acceptor splice site in intron 6 of the SPINT2 gene. of the University Hospitals of Innsbruck and invited for It is not listed in public databases of population-based a clinical examination. 19 patients (9 males, 10 females, exome and genome data, i.e., neither in ExAC (http:// aged 2–40 years) were included in the study after giving exac.broadinstitute.org/gene/ENSG00000167642) nor written informed consent. A careful history and physical in gnomAD (http://gnomad.broadinstitute.org/gene/ examination by an ear nose and throat (ENT) specialist, a ENSG00000167642). It emerged that all fve homozygous pediatric surgeon and a pediatrician was performed. Type patients and the heterozygous patient #14 of the present and localization of the respective atresia was determined study originated from the same rural area, suggesting from previous history and physical examination data, diag- that patient #14 of the non-sCSD group most likely was a nostic imaging results and surgery reports. Laboratory heterozygous sCSD carrier. workup consisted of electrolytes, blood gases, osmolality Te individual clinical data of the non-sCSD atre- and creatinine in venous plasma as well as of electrolytes, sia and sCSD patients are summarized in Table 1. osmolality and creatinine in urine. Te concentrations Te average age of the non-sCSD atresia group was of sodium and creatinine in plasma and urine were used 139 ± 101 months (range 25–480 months) and thus for the calculation of the fractional excretion of sodium signifcantly higher (p < 0.05) than the age of the sCSD (FENa) using the formula FENa = urine Na × plasma cre- patients (60 ± 70 months, range 4–192 months). Te atinine × 100/(plasma Na × urine creatinine). Concentra- gestational age of non-sCSD atresia patients was also sig- tions of uNa (urine sodium) and uCrea (urine creatinine) nifcantly higher than that of the sCSD group (38.8 ± 1.9 were used for the calculation of the urine sodium to urine vs. 36.7 ± 2.6 weeks, p < 0.05). Birth weight which creatinine (uNa/uCrea) ratio. Furthermore, stool samples was 3136 ± 554 g for non-sCSD atresia patients and were analyzed for electrolytes, pH and osmolality. 2900 ± 448 g for the sCSD group was not signifcantly SPINT2 mutational analysis was performed as described diferent (p = 0.19). Diarrhea was not reported in any of previously [7]: briefy, genomic DNA was prepared from the non-CSD atresia subjects, while it was the predomi- leukocytes using a robot (GenoM 48, Qiagen, Vienna, Aus- nant feature in all sCSD patients. Moreover, average val- tria). Te complete coding region of the SPINT2 gene and ues of laboratory indicators of sodium balance (fecal Na, all exon–intron-boundaries were amplifed using intronic plasma Na, urinary Na, ­FENa and uNa/uCrea ratio) and of primers. Te 190–504-bp amplicons were sequenced and metabolic acidosis were normal in the non-sCSD atresia the sequencing reactions analyzed on an ABI 3100 DNA group and were thus signifcantly diferent from the sCSD sequencer. A panel of 188 DNA samples from anony- cohort where these parameters were generally pathologi- mous healthy subjects was investigated for the presence cal (Table 2). Tere was also no diarrhea reported in the of sequence variants [7]. Clinical and mutational analysis non-sCSD atresia patient with the heterozygous splice data of the 19 non-CSD atresia patients were compared mutation c.593-1G>A (patient #14). However, the FENa with published results from 16 patients with sCSD [7]. and uNa/uCrea in that patient showed borderline low Data of clinical parameters are presented as mean ± SD. values for these two sodium balance parameters and also Te statistical signifcance of diferences between both a rather low uNa concentration (Table 1). We speculate patient groups was determined using the two-tailed Stu- that these results refect an efect of the c.593-1G>A car- dent’s t test. Allele frequencies for SPINT2 variants were rier state on sodium status, because we had previously taken from the ExAC and gnomAD databases. found low urinary sodium concentrations in two obligate heterozygous sCSD parents [13]. Tis resembles fndings Results in other autosomal recessive disorders, where clinically Among non-sCSD CA or GA patients, no homozy- healthy patients have been shown to exhibit pathologi- gous or compound heterozygous mutations were found cal laboratory markers as in, e.g., alpha-1-antitrypsin in the present study (Table 1 upper panel). However, defciency [14, 15]. Apart from patient #14, however, Niederwanger et al. Eur J Med Res (2018) 23:13 Page 3 of 7 n.i. fpH (6.5–7.5) 6.0 5.0 5.5 7.5 6.0 7.5 5.5 8.0 7.0 5.5 9.0 6.0 5.5 6.5 6.0 n.i. 6.0 5.5 7.5 6.5 6.0 6.0 8.1 8.6 n.i. 7.5 7.8 8.0 n.i. 106 fNa (mmol/L) (20–50) 90 28 86 17 90 29 32 38 24 24 39 14 30 n.i. 19 n.i. 21 31 30 30 49 37 152 190 n.i. 105 103 133 100 n.i. uNa/uCrea uNa/uCrea (mmol/ mmol) (17–53) n.i. 26.6 n.i. 18.4 n.i. 15.4 40.0 20.9 16.6 21.6 n.i. 9.3 25.0 9.8 26.3 40.7 16.5 47.7 24.1 27.6 31.8 39.5 0.6 0.7 n.i. 1.6 1.1 0.9 n.i. n.i. FENa (%) (0.5–1.5) n.i. 1.7 n.i. 1.3 n.i. 0.7 2.8 0.8 0.6 0.8 n.i. 0.4 1.1 0.4 1.0 1.8 0.6 1.7 0.9 1.0 2.5 1.4 < 0.1 < 0.1 n.i. < 0.1 < 0.1 < 0.1 n.i. n.i. uCrea uCrea (mmol/L) (3.5–24.6) n.i. 3.8 n.i. 7.0 n.i. 3.5 6.2 9.2 9.7 9.2 n.i. 9.3 4.0 18.1 0.8 4.5 1.7 3.0 7.4 2.4 3.9 5.7 17.7 14.2 n.i. 10.0 9.0 11.2 n.i 0.07 pCrea pCrea (mmol/L) (0.05–0.1) 0.09 0.09 1.1 0.10 0.09 0.06 0.10 0.05 0.05 0.06 0.05 0.04 0.06 0.05 0.05 0.06 0.05 0.05 0.05 0.05 0.06 0.05 0.19 0.16 n.i. 0.08 0.11 0.12 0.06 n.i. uNa (mmol/L) (30–250) n.i. 101 n.i. 129 n.i. 54 248 192 161 199 n.i. 133 100 178 21 183 28 143 178 106 124 225 < 10 < 10 n.i. 16 < 10 < 10 n.i. 129 pNa (mmol/L) (136–145) 119 140 119 140 119 138 141 139 142 145 139 140 138 136 138 139 141 143 139 140 139 141 119 123 n.i. 119 118 116 120 ppH n.i. 7.38 n.i. 7.37 7.22 7.38 7.39 7.29 7.20 7.14 7.30 7.35 7.36 7.40 7.38 7.36 7.33 7.36 7.39 7.38 7.35 7.37 7.04 7.11 n.i. 6.99 7.13 7.09 7.33 7.25 BW (g) BW 3635 3270 3620 3360 2540 2510 2500 1835 3650 3350 2700 3270 2760 3200 4010 3880 3440 3185 3820 2510 3030 3305 2850 2910 2190 2900 2190 2670 2995 3345 GA (months) 40 39 39 40 35 37 n.i. 33 38 39 39 n.i. 39 39 41 41 40 38 41 37 40 39 35 38 32 36 32 37 40 39 Sex (m/f)Sex M M F M F M F M F M M F F F F F M F F M F M M F M F F F F F 7 6 4 6 84 96 49 60 86 88 25 85 74 13 10 98 14 Actual age (months) 192 180 180 156 180 480 108 184 158 144 192 146 252 molecular data of non-CSDClinical and molecular data and sCSD patients 9 1 10 2 11 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 1 2 3 4 5 6 7 8 1 Table Patient # Patient Non - sCSD sCSD Niederwanger et al. Eur J Med Res (2018) 23:13 Page 4 of 7 Diagnosis CA CA EA IIIb EA EA IIIb EA IIIb EA AA AA AA AA AA AA AA AA AA AA AA AA sCSD sCSD sCSD sCSD, ON sCSD, fpH (6.5–7.5) 7.0 n.i. 8.2 n.i. n.i. fNa (mmol/L) (20–50) 56 66 92 95 119 uNa/uCrea uNa/uCrea (mmol/ mmol) (17–53) n.i. n.i. n.i. 0.5 n.i. SPINT2 nucleotide alteration None None None None None None None None None c.598G>C het c.598G>C None None None - 1G>A het c.593 None None None None None - 1G>A homozygousc.593 - 1G>A homozygousc.593 - 1G>A homozygousc.593 No DNA, sister of pat. 3 No DNA, sister - 1G>A homozygousc.593 - 1G>A homozygousc.593 FENa (%) (0.5–1.5) n.i. n.i. n.i. < 0.1 n.i. uCrea uCrea (mmol/L) (3.5–24.6) n.i. n.i. n.i. 6.0 7.0 Atresia Choanal Choanal Esophageal Esophageal Esophageal Esophageal Esophageal Anal Anal Anal Anal Anal Anal Anal Anal Anal Anal Anal Anal Corneal erosions Choanal, corneal erosions Choanal, n.i. Corneal erosions Choanal None pCrea pCrea (mmol/L) (0.05–0.1) 0.09 0.08 0.08 0.15 n.i. uNa (mmol/L) (30–250) n.i. n.i. n.i. 3 10 Diarrhea No No No No No No No No No No No No No No No No No No No Yes Yes Yes Yes Yes Yes pNa (mmol/L) (136–145) 123 116 124 125 n.i. ppH 7.27 7.21 7.30 7.19 n.i. Sex (m/f)Sex M M M F M F M M F F F F F M F F M F M F M M F F F BW (g) BW 3130 3050 3075 2400 n.i. GA (months) 36 40 36 36 n.i. Sex (m/f)Sex M F F M F 7 6 4 84 96 49 60 86 88 25 85 74 13 10 98 Actual age (months) 180 156 480 108 184 158 144 192 146 252 31 11 22 72 Actual age (months) 108 continued 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 1 2 4 3 5 6 12 13 14 15 16 1 Table Patient # Patient # Patient Non - sCSD sCSD Niederwanger et al. Eur J Med Res (2018) 23:13 Page 5 of 7 Diagnosis sCSD sCSD sCSD, PPN sCSD, sCSD, PPN sCSD, sCSD, PPN sCSD, sCSD sCSD sCSD sCSD sCSD, PPN sCSD, gestational age; GIA gestational SPINT2 nucleotide alteration c.488A>G homozygousc.488A>G c.488A>G homozygousc.488A>G c.488A>G homozygousc.488A>G c.488A>G homozygousc.488A>G + 2T>C/c.488A>G c.337 c.488A>G homozygousc.488A>G No DNA, sister of pat. 12 No DNA, sister + 2T>A c.488A>G/c.553 c.488 G/c.1A>T c.488 c.488A>G homozygousc.488A>G Atresia Choanal Choanal None? Choanal Choanal Choanal Choanal, anal Choanal, Choanal, corneal erosions Choanal, Choanal Choanal, anal Choanal, Diarrhea Yes Yes Yes Yes Yes yes Yes Yes Yes Yes Sex (m/f)Sex F F M F F M F F M F fractional excretion of sodium; GA FENa fractional excretion pH; f female; Na; fpH fecal fNa fecal EA esophageal atresia; BW birth CA choanal atresia; anal atresia; weight; 6 14 31 11 22 72 Actual age (months) 192 180 180 108 continued 7 8 9 10 11 12 13 14 15 16 1 Table # Patient AA in alphabetical order: Abbreviations oral nutrition; nutrition; pNa plasma Na; ppH pH; PPN partial ON oral uNa urinary n.a . not applicable; n.i no information; m male; mo month; parenteral urinary Na; uNa/uCrea atresia; gastrointestinal Na:urinary ratio creatinine Niederwanger et al. Eur J Med Res (2018) 23:13 Page 6 of 7

Table 2 Clinical parameters refecting diferences factor inhibitor 2 (HAI2) has been shown to be essen- between non-sCSD and sCSD patients tial for organogenesis [17]. Furthermore, loss of SPINT2 Parameter non-sCSD sCSD p value protein has been implicated in severe clefting of the embryonic ectoderm in mice [18]. Tis also raises the Actual age (months) 139 101 60 70 < 0.05 ± ± possibility of an infuence of SPINT2 on the develop- Gestational age (weeks) 39 2 37 3 < 0.05 ± ± ment of CA and GA during prenatal organ formation. Birth weight (g) 3136 554 2900 448 n.s. ± ± Another mechanism by which SPINT2 mutations may Plasma Na (mmol/L) 140 2 120 4 < 0.001 ± ± lead to atretic malformations could be related to the Plasma pH 7.34 0.07 7.18 0.11 < 0.001 ± ± function of the epithelial sodium channel (ENaC). Tis Urinary Na (mmol/L) 139 64 10 4 < 0.001 ± ± channel has been shown to be regulated by alterations FE (%) 1.2 0.7 0.1 0.0 < 0.001 Na ± ± of the serine protease–protease inhibitor balance which uNa/uCrea (mmol/mmol) 25.0 11.1 0.9 0.4 < 0.001 ± ± may, if defective, disturb the volume of the epithelial sur- Fecal Na (mmol/L) 29 9 106 33.2 < 0.001 ± ± face liquid layer [19]. Such a mechanism has been shown Fecal pH 6.5 1.1 7.3 1.1 n.s. ± ± to be the case for the airway epithelium of cystic fbrosis Abbreviations in alphabetical order: FENa fractional excretion of sodium; mo (CF) patients in which hyperabsorption of sodium (and month; n.s. not signifcant; uNa/uCrea urinary Na/urinary creatinine ratio; wk week fuid) causes increased viscosity of the epithelial sur- face liquid. An analogous pathomechanism of epithelial fuid–electrolyte imbalance due to malfunction of the cystic fbrosis transmembrane regulator (CFTR) could the results in all other 18 non-sCSD patients were nor- be imagined to cause adhesion of epithelial layers induc- mal. Besides the fndings in the non-sCSD cohort, the ing the commonly encountered aplasia of the vas defer- absence of the c.593-1G>A mutation among 188 ethni- ens in CF [20]. cally matched controls argued against a role for SPINT2 in isolated CA/GA. Conclusion Discussion Our study aimed at investigating whether a similar mechanism of epithelial adhesion potentially related to It has been previously shown that mutations in SPINT2 SPINT2 mutations could be responsible for the develop- are associated with sCSD [7], a disease featuring a ment of isolated CA and/or GA. However, our fnding sodium-losing diarrhea, recurrent corneal erosions and that no homozygous mutations were identifed in the frequently atretic malformations of the choanae (CA) non-sCSD group argues against the possibility of SPINT2 and the gastrointestinal tract (GA). Furthermore, intesti- mutations being a cause of isolated CA or GA. nal biopsies have shown tufts, consisting of focal crowds of enterocytes within the epithelium [16]. Mutational Authors’ contributions CM organized the study, supervised the patient management, collected analysis of SPINT2 had previously identifed fve distinct the data and wrote the frst draft of the manuscript. SL performed mutation homozygous or compound heterozygous mutations [7] analysis of SPINT2. LK assisted in data collection and patient management. in each of the investigated sCSD patients (Table 1, lower AJ devised and supervised genetic studies and wrote the genetic part of the paper. CP performed ENT investigations. BH provided surgical investigations. panel). Our study was undertaken to investigate whether SSB provided laboratory investigations. TM provided general support of the the SPINT2 mutations identifed in sCSD would also be study and proof-read the manuscript. PH-E devised and organized the study, present in patients with non-syndromic forms of isolated wrote the fnal manuscript and submitted the manuscript as the correspond‑ ing author. All authors read and approved the fnal manuscript. atresias, who did not have diarrhea (non-sCSD). No homozygous or compound heterozygous SPINT2 Author details 1 mutations were identifed in isolated non-sCSD atre- Department of Pediatrics III, Medical University of Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria. 2 VAMED, 1230 Vienna, Austria. 3 Department sia subjects. However, in the non-sCSD patient #14 we of Pediatrics I, Medical University of Innsbruck, Anichstrasse 35, 6020 Inns‑ found a heterozygous splice mutation, c.593-1G>A, bruck, Austria. 4 Department of Ear, Nose and Throat Diseases, Medical 5 which is identical to a SPINT2 mutation shown previ- University of Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria. Department of General Surgery, Pediatric Surgery Unit, Medical University of Innsbruck, ously in fve Austrian sCSD patients [7]. Tis patient also Anichstrasse 35, 6020 Innsbruck, Austria. had borderline laboratory values (Table 1) for sodium balance, indicating potential sodium depletion. Tis sug- Acknowledgements Not applicable. gests that carriers of the c.593-1G>A SPINT2 mutation might be recognizable by testing for phenotypical labo- Competing interests ratory markers of sodium status (e.g., FENa, uNa/uCrea). The authors declare that they have no competing interests. Regulation of the cell surface serine protease Availability of data and materials matriptase by the SPINT2-encoded hepatocyte growth Trial registration: ISRCTN ISRCTN73824458. Registered 28 September 2014. [email protected] Niederwanger et al. Eur J Med Res (2018) 23:13 Page 7 of 7

Ethics approval and consent to participate 9. Kawaguchi T, Qin L, Shimomura T, et al. Purifcation and cloning of The study was approved by the ethics committee of the Medical University of hepatocyte growth factor activator inhibitor type 2, a Kunitz-type serine Innsbruck (UN3987 session 288/4.10). protease inhibitor. J Biol Chem. 1997;272(44):27558–64. 10. Planes C, Leyvraz C, Uchida T, et al. In vitro and in vivo regulation of Funding transepithelial lung alveolar sodium transport by serine proteases. Am J This study was supported by grants from the Jubiläumsfonds der Österreichis‑ Physiol Lung Cell Mol Physiol. 2005;288(6):L1099–109. chen Nationalbank Grant #14496 and the Tiroler Wissenschaftsfonds Grant 11. Donaldson SH, Hirsh A, Li DC, et al. Regulation of the epithelial #UNI-404/1286 to Andreas Janecke. sodium channel by serine proteases in human airways. J Biol Chem. 2002;277(10):8338–45. 12. Szabo R, Bugge TH. Membrane-anchored serine proteases in vertebral Publisher’s Note cell and developmental biology. Annu Rev Cell Dev Biol. 2011;27:213–35. Springer Nature remains neutral with regard to jurisdictional claims in pub‑ 13. Heinz-Erian P. Unpublished observation. 2010. lished maps and institutional afliations. 14. Dahl M, Tybjaerg-Hansen A, Lange P, Vestbo J, Nordestgaard BG. Change in lung function and morbidity from chronic obstructive pulmonary Received: 15 October 2017 Accepted: 23 February 2018 disease in alpha-1-antitrypsin MZ heterozygotes: a longitudinal study of the general population. Ann Intern Med. 2002;136(4):270–9. 15. Endres W. Inherited metabolic diseases afecting the carrier. J Inherit Metab Dis. 1997;20(1):9–20. 16. Salomon J, Goulet O, Canioni D, Brousse N, Lemale J, Tounian P, Coulomb A, Marinier E, Hugot JP, Ruemmele F, Dufer JL, Roche O, Bodemer C, References Colomb V, Talbotec C, Lacaille F, Campeotto F, Cerf-Bensussan N, Janecke 1. Kancherla V, Romitti PA, Sun L, et al. Descriptive and risk factor analysis for AR, Mueller T, Koletzko S, Bonnefont JP, Lyonnet S, Munnich A, Poirier F, choanal atresia: The National Birth Defects Prevention Study. Eur J Med Smahi A. Genetic characterization of congenital tufting enteropathy: Genet. 2014;57(5):220–9. epcam associated phenotype and involvement of SPINT2 in the syndro‑ 2. Spitz L. Oesophageal atresia. Orphanet J Rare Dis. 2007;2:24. mic form. Hum Genet. 2014;133(3):299–310. 3. Bednarczyk D, Sasiadek MM, Smigiel R. aberrations and 17. Szabo R, Hobson JP, Kristoph K, et al. Regulation of cell surface gene mutations in patients with esophageal atresia. J Pediatr Gastroen‑ protease matriptase by HAI2 is essential for placental development, terol Nutr. 2013;57(6):688–93. neural tube closure and embryonic survival in mice. Development. 4. Dalla Vecchia LK, Grosfeld JL, West KW, et al. Intestinal atresia and stenosis: 2009;136(15):2653–63. a 25 year experience with 277 cases. Arch Surg. 1998;133(5):490–6. 18. Mitchell KJ, Pinson KI, Kelly OG, et al. Functional analysis of secreted and 5. Boles ET Jr, Vassy LE, Ralston M. Atresia of the colon. J Pediatr Surg. transmembrane proteins critical to mouse development. Nat Genet. 1976;11(1):69–75. 2001;28(3):241–9. 6. Spouge D, Baird PA. Imperforate anus in 700,000 consecutive liveborn 19. Myerburg MM, Butterworth MB, McKenna EE, et al. Airway surface liquid newborns. Am J Med Genet. 1986;25(Suppl. 2):151–61. volume regulates ENaC by altering the serine protease-protease inhibitor 7. Heinz-Erian P, Müller T, Krabichler B, et al. Mutations in SPINT2 cause balance: a mechanism for sodium hyperabsorption in cystic fbrosis. J Biol a syndromic form of congenital sodium diarrhea. Am J Hum Genet. Chem. 2006;281(38):27942–9. 2009;84(2):188–96. 20. Shin D, Gilbert F, Goldstein M, et al. Congenital absence of the vas 8. Delaria KA, Muller DK, Marlor CW, et al. Characterization of placen‑ deferens: incomplete penetrance of cystic fbrosis gene mutations. J Urol. tal bikunin, a novel human serine protease inhibitor. J Biol Chem. 1997;158(5):1794–8. 1997;272(18):12209–14.

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