Computational protocol: Medical Sequencing of Candidate Genes for Nonsyndromic Cleft Lip and Palate

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Protocol publication

[…] Two collections of CL/P cases, 91 from the Philippines and 93 from Iowa, United States, were used to search for mutations in 20 candidate genes (). We selected the more-severe cases from those available to us, and the sequenced samples were enriched by bilateral cleft lip and palate cases with a positive family history for clefts (39/91 from the Philippines and 16/93 from Iowa). Two cases were later found to have associated features—one with Stickler syndrome in which family history was initially not available, and a second with polydactyly. Cases from the Philippines were studied under the auspices of Operation Smile International []. Patients were seen and examined by a board certified clinical geneticist (JCM or colleagues; see Acknowledgments) at one of four sites within the Philippines (Cavite, Kalibo, Cebu, and Negros). Iowa cases were collected through the Iowa Birth Defects Registry []. Signed consents were obtained from all participants before a blood sample was obtained. DNA was extracted according to published protocols.For each of the 20 candidate genes, all exons and 5′ and 3′ untranslated regions were sequenced in both directions. Primer sequences and PCR conditions are available on our Web site ( Primers for FOXE1, GLI2, MSX2, OSR2, and TGFBR1 were obtained from the literature [,–]. Cycle sequencing was performed in a 20-μl reaction using 4 μl of Applied Biosystems Big Dye Terminator sequencing reagent, 1 μl of 5 μM sequencing primer, 1 μl of DMSO, 4 μl of 2.5× Buffer, and 2.5 ng/100 base pair of DNA template. Following a denaturation step at 96 °C for 30 s, reactions were cycle sequenced at 96 °C for 10 s, 50 °C for 5 s, and 60 °C for 4 min for 40 cycles. Cleanup was performed using standard protocols. Samples were resuspended in 40 to 100 μl of ddH2O, and 2.5 μl were then injected on an Applied Biosystems 3700 sequencer. The Applied Biosystems sequence software (version 2.1.2) was used for lane tracking and first pass base calling. Chromatograms were transferred to a Unix workstation, base called with PHRED (version 0.961028), assembled with PHRAP (version 0.960731), scanned by POLYPHRED (version 0.970312), and the results viewed with CONSED (version 4.0) []. When the results indicated a possible new variant, the case sample was resequenced, as well as other available family members, and population controls. These data were analyzed using the same method.For any coding variant, we performed direct sequencing in 186 population-matched controls. Control populations were collected as described above for the cases and consisted of individuals with no CL/P or other recognized birth defect from adults at the same sites where cases were collected. If the variant was found in one or more controls, it was considered a polymorphism. To expand the number of controls tested, we developed allele-specific assays for the LHX8 E221A, SATB2 T190A, SKI A388V, SPRY2 D20A, and TBX10 R354Q mutations. We tested them in the CEPH Diversity Cell Line Panel [], which is comprised of 1,064 DNA samples from cultured lymphoblastoid cell lines derived from individuals representing 51 different human populations.We also developed an assay for the MSX1 P147Q missense mutation, recently reported in three Vietnamese cleft families []. Besides the 1,064 CEPH Diversity Cell Line Panel controls, we tested the MSX1 P147Q assay in an additional 607 Filipino controls and in a collection of 1,468 cleft cases from the Philippines as well.For the nine genes with potentially etiologic missense mutations, we identified orthologs through BLAST search of the non-redundant database using Homo sapiens FOXE1, GLI2, LHX8, JAG2, MSX2, SATB2, SKI, SPRY2, and TBX10, as reference sequences. We performed protein sequence comparisons with the available species. We also used the ESEfinder software available online to predict the presence of exonic splicing enhancers [], which appear to be prevalent, and may be present in most, if not all exons [,]. We screened the 141 exonic splicing silencer decamers that were identified by Wang et al. [] to check if any of those could be affected by the missense mutations we found. Finally, we used the PolyPhen software, also available online, to predict the impact of the amino acid substitutions identified on the structure and function of the human protein [–].Two single nucleotide polymorphisms in weak linkage disequilibrium with each other were selected for each population to perform linkage disequilibrium studies in the genes with missense mutations in cases but not in controls. Four single nucleotide polymorphisms were chosen for GLI2 based on the International HapMap Project's linkage disequilibrium pattern of the gene (data not shown). Frequency of the alleles can be found in the supplemental material ( TaqMan-based assays [] were performed on Applied Biosystems 7900 HT Sequence Detection System (Applied Biosystems, Foster City, California, United States). For one marker in SKI (rs2843159), we used a kinetic polymerase chain reaction assay previously reported []. These linkage disequilibrium studies were composed of 296 complete triads (mother/father/affected child) from the Philippines and 205 from Iowa. These samples were obtained as described above for cases and controls investigated by sequencing. The Family Based Association Test (FBAT) [–] program was used for this analysis. Significance figures were accounted for using Bonferroni correction taking into account the number of tests carried out []. With the Bonferroni correction, alpha is 0.0003 (0.05/192 comparisons) for the individual marker analysis and 0.0001 (0.05/384 comparisons) for the haplotype analysis. Linkage disequilibrium studies for FOXE1 were previously reported in Marazita et al. [].A third clefting population sample set of 434 case/mother pairs from South America was used to replicate any significant association. These population samples are derived from ECLAMC, which is a hospital-based birth defects registry study that includes sites in Argentina, Bolivia, Brazil, Chile, Ecuador, Paraguay, Uruguay, and Venezuela. This study population has previously been described in detail [,]. To analyze the ECLAMC samples, the likelihood ratio test (LRT) of Weinberg [] was applied under the assumption that the distribution of paternal alleles is the same as maternal. […]

Pipeline specifications

Software tools PolyPhred, ESEfinder, PolyPhen, FBAT
Databases International HapMap Project
Applications WGS analysis, Sanger sequencing, GWAS
Organisms Homo sapiens
Diseases Cleft Lip, Cleft Palate