1. 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, ) 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

2. 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.

3. 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

4. EA/TEF, VACTERL Association

5. Schematic representation of LPP gene
> 400 kb Genomic Region
Exons
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

6. 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

7. 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):

8. 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

9. 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

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

11. 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 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.

12. 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 and the Sophia Stichting Wetenschappelijk Onderzoek (SWOO, project 493), Rotterdam, The Netherlands.