Mutation screening of the LPP gene in patients with esophageal atresia, tracheoesophageal fistula, and VACTERL association Andrés Hernández-García1, Erwin Brosens5,6, Hitisha Zaveri1, Zhiyin Yu1, Caraciolo Fernandes2, Anthony Johnson1,3,4, Maria Blazo7, Seema Lalani1, Dick Tibboel6, Annelies de Klein5, Daryl A. Scott1 1) Department of Molecular and Human Genetics, 2) Pediatrics, 3) Obstetrics and Gynecology, 4) Pediatric Surgery, Baylor College of Medicine, Houston, Texas, USA; 5) Department of Clinical Genetics, 6) Department of Pediatric Surgery, Erasmus Medical Center, Rotterdam, the Netherlands; 7) Texas A&M Health Science Center College of Medicine, Temple, Texas, USA

Summary Esophageal Atresia (EA) and Tracheoesophageal Fistula (TEF) are common, life-threatening birth defects. Approximately 10% of individuals with EA/TEF also meet criteria for VACTERL association. The genetic factors that contribute to most cases of EA/TEF and VACTERL association have yet to be identified. To determine if copy number changes in LPP are a common cause of EA/TEF or VACTERL association, we screened 195 individuals with these diagnoses for chromosomal deletions/duplications. A deletion affecting the coding region of LPP was identified in one individual with VACTERL association. We are now working to determine if this change was de novo or inherited. LPP sequence analysis of 75 patients revealed two relatively rare, non-synonymous changes—S246L and R388C—in three patients. These changes may deleteriously affect protein function, but each was inherited from an unaffected mother. Our results suggest that clearly deleterious alterations in the LPP gene are not a common cause of EA/TEF or VACTERL association. If the non-synonymous LPP sequence changes indentified in this study contribute to these phenotypes, they are likely to have a relatively mild effect and to do so in combination with other genetic, environmental and/or stochastic factors.

Background Esophageal atresia (EA) and Tracheoesophageal fistula (TEF) Incidence: 1:3,500 EA/TEF plus other abnormalities: 50% VACTERL association Vertebral, Anal, Cardiac, TracheoEsophageal fistula, Renal, and Limb 10% of EA/TEF cases also meet criteria for VACTERL

EA/TEF, VACTERL Association

Schematic representation of LPP gene > 400 kb Genomic Region Exons 1 2 3 4 5 6 7 8 9 10 11 LPP Gene LPP Protein α-actinin binding site VASP binding site Nuclear export signal LPP/TRIP-6 similar region Zyxin/LPP/TRIP-6/LIMD1 similar region Tumor breakpoints Pre-LIM region LIM domains Schematic representation of LPP gene

Genetic Factors of EA/TEF Multifactorial etiology Low twin concordance rate Evidence for genetic factors in EA/TEF Up to 25% in children with trisomy 18 Recurrent duplications: 3p25-pter and 5q34-qter Recurrent deletions: 2q37.2-qter, 4q31-qter, 5p14-pter, 6q13-q15, 14q32.2-qter, 17q22-q23 Syndromic EA/TEF: Feingold, CHARGE, anophthalmia-esophageal-genital syndrome, Fanconi anemia

LIM domain containing preferred translocation partner in lipoma (LPP) Arrington et al. 2010 Individual with VACTERL Tetralogy of Fallot (TOF), right aortic arch EA/TEF Small kidneys 451 kb interstitial deletion of (LPP) Small nuclear family with TOF Intronic deletion in LPP segregated with TOF Reduced LPP expression Arrington et al. Am J Med Genet A 152A(11):2919-2923

LPP deletions in EA/TEF, VACTERL RESULTS LPP deletions in EA/TEF, VACTERL Cohort of 195 patients screened 48 Isolated EA/TEF 77 EA/TEF with other anomalies 64 VACTERL with EA/TEF 6 VACTERL without EA/TEF 1 Deletion (1/195; 0.51%; inheritance?) Vertebral anomalies Cardiac anomalies EA/TEF

Table 1. Clinical and molecular synopsis of individuals with non-synonymous changes in LPP. Het = individuals were heterozygous for the change; Homo = individuals were homozygous for the change 9

Table 2. Evaluation of changes in the LPP gene identified in patients with EA/TEF and VACTERL.

DISCUSSION EA/TEF can occur as an isolated finding, in association with other birth defects, or as part of a genetic syndrome. For individuals with isolated EA/TEF without a clear etiology and patients with VACTERL association the recurrence risk in their siblings is low—approximately 1%. Although Arrington et al. have described a 451 kb interstitial deletion involving only the LPP in an individual diagnosed with EA/TEF and VACTERL association, the inheritance pattern could not be confirmed. In our study, a single deletion was found in a cohort of 195 individuals with EA/TEF or VACTERL association. This observation suggests that the deletion is present in 0.51% of the patients. Several non-synonymous changes in LPP were identified in our cohort—S144T, S246L, Y346H and R388C. Although the S144T change is rare, its unlikely to contribute to the phenotype of the patient in which it was found since not only was it predicted to be benign by all three of the prediction programs used in this study but also inherited from an unaffected father. The Y346H allele was predicted to be possibly damaging by one prediction programs used in this study but benign by the other two calling into question its effect on LPP function. The c.1036T>C change associated with the Y346H allele is also a relatively common variant with a reported minor allele frequency of 0.2099 in dbSNP. Based on Hardy-Weinberg equilibrium, the Y346H allele would be found in the homozygous state in 4.4% of the population. In contrast to the Y346H change, the S246L change that was seen in two patients and the R388C change seen in one patient are relatively rare. The S246L change was predicted to be deleterious by only one of the three prediction programs used in this study and the R388C change was predicted to be deleterious by all three prediction programs. However, in all cases, these changes were inherited from unaffected parents. The R388C change was also seen in 1 out of 78 African American controls suggesting it may be seen more commonly in this ethnic group.

CONCLUSION Taken together, these results suggest that clearly deleterious alterations in the LPP gene are not a common cause of EA/TEF or VACTERL association. This makes it unlikely that clinical screening of the LPP gene would prove to be cost effective in individuals with EA/TEF or VACTERL association. Our data also suggests that if the non-synonymous LPP sequence changes indentified in this study contribute to the development of these phenotypes, they are likely to have a relatively mild effect and do so in combination with other genetic, environmental and/or stochastic factors. This study was funded by the Doris Duke Charitable foundation grand 2007053 and the Sophia Stichting Wetenschappelijk Onderzoek (SWOO, project 493), Rotterdam, The Netherlands.