SHROOM3 is a novel candidate for heterotaxy identified by whole exome sequencing
© Tariq et al.; licensee BioMed Central Ltd. 2011
Received: 19 July 2011
Accepted: 21 September 2011
Published: 21 September 2011
Heterotaxy-spectrum cardiovascular disorders are challenging for traditional genetic analyses because of clinical and genetic heterogeneity, variable expressivity, and non-penetrance. In this study, high-resolution SNP genotyping and exon-targeted array comparative genomic hybridization platforms were coupled to whole-exome sequencing to identify a novel disease candidate gene.
SNP genotyping identified absence-of-heterozygosity regions in the heterotaxy proband on chromosomes 1, 4, 7, 13, 15, 18, consistent with parental consanguinity. Subsequently, whole-exome sequencing of the proband identified 26,065 coding variants, including 18 non-synonymous homozygous changes not present in dbSNP132 or 1000 Genomes. Of these 18, only 4 - one each in CXCL2, SHROOM3, CTSO, RXFP1 - were mapped to the absence-of-heterozygosity regions, each of which was flanked by more than 50 homozygous SNPs, confirming recessive segregation of mutant alleles. Sanger sequencing confirmed the SHROOM3 homozygous missense mutation and it was predicted as pathogenic by four bioinformatic tools. SHROOM3 has been identified as a central regulator of morphogenetic cell shape changes necessary for organogenesis and can physically bind ROCK2, a rho kinase protein required for left-right patterning. Screening 96 sporadic heterotaxy patients identified four additional patients with rare variants in SHROOM3.
Using whole exome sequencing, we identify a recessive missense mutation in SHROOM3 associated with heterotaxy syndrome and identify rare variants in subsequent screening of a heterotaxy cohort, suggesting SHROOM3 as a novel target for the control of left-right patterning. This study reveals the value of SNP genotyping coupled with high-throughput sequencing for identification of high yield candidates for rare disorders with genetic and phenotypic heterogeneity.
Congenital heart disease (CHD) is the most common major birth defect, affecting an estimated 1 in 130 live births . However, the underlying genetic causes are not identified in the vast majority of cases [2, 3]. Of these, approximately 25% are syndromic while approximately 75% are isolated. Heterotaxy is a severe form of CHD, a multiple congenital anomaly syndrome resulting from abnormalities of the proper specification of left-right (LR) asymmetry during embryonic development, and can lead to malformation of any organ that is asymmetric along the LR axis. Heterotaxy is classically associated with heart malformations, anomalies of the visceral organs such as gut malrotation, abnormalities of spleen position or number, and situs anomalies of the liver and/or stomach. In addition, inappropriate retention of symmetric embryonic structures (for example, persistent left superior vena cava), or loss of normal asymmetry (for example, right atrial isomerism) are clues to an underlying disorder of laterality [4, 5].
Heterotaxy is the most highly heritable cardiovascular malformation . However, the majority of heterotaxy cases are considered idiopathic and their genetic basis remains unknown. To date, point mutations in more than 15 genes have been identified in humans with heterotaxy or heterotaxy-spectrum CHD. Although their prevalence is not known with certainty, they most likely account for approximately 15% of heterotaxy spectrum disorders [4, 7–9]. Human X-linked heterotaxy is caused by loss of function mutations in ZIC3, and accounts for less than 5% of sporadic heterotaxy cases . Thus, despite the strong genetic contribution to heterotaxy, the majority of cases remain unexplained and this indicates the need for utilization of novel genomic approaches to identify genetic causes of these heritable disorders.
LR patterning is a very important feature of early embryonic development. The blueprint for the left and right axes is established prior to organogenesis and is followed by transmission of positional information to the developing organs. Animal models have been critical for identifying key signaling pathways necessary for the initiation and maintenance of LR development. Asymmetric expression of Nodal, a transforming growth factor beta ligand, was identified as an early molecular marker of LR patterning that is conserved across species [10–12]. Genes in the Nodal signaling pathway account for the majority of genes currently known to cause human heterotaxy. However, the phenotypic variability of heterotaxy and frequent sporadic inheritance pattern have been challenging for studies using traditional genetic approaches. Although functional analyses of rare variants in the Nodal pathway have been performed that confirm their deleterious nature, in many cases these variants are inherited from unaffected parents, suggesting that they function as susceptibility alleles in the context of the whole pathway [7, 8].
More recent studies have focused on pathways upstream of Nodal signaling, including ion channels and electrochemical gradients [13–15], ciliogenesis and intraflagellar transport , planar cell polarity (Dvl2/3, Nkd1) [17, 18] and convergence extension (Vangl1/2, Rock2) [19, 20], and non-transforming growth factor beta pathway members that interact with the Nodal signaling pathway (for example, Ttrap, Geminin, Cited2) [21–23]. Relevant to the current study, we recently identified a rare copy number variant containing ROCK2 in a patient with heterotaxy and showed that its knockdown in Xenopus causes laterality defects . Similar laterality defects were identified separately with knockdown of Rock2b in zebrafish . The emergence of additional pathways regulating LR development has led to new candidates for further evaluation. Given the mutational spectrum of heterotaxy, we hypothesize that whole-exome approaches will be useful for the identification of novel candidates and essential for understanding the contribution of susceptibility alleles to disease penetrance.
Very recently, whole-exome analysis has been used successfully to identify the causative genes for many rare disorders in affected families with small pedigrees and even in singlet inherited cases or unrelated sporadic cases [25–29]. Nevertheless, one of the challenges of whole-exome sequencing is the interpretation of the large number of variants identified. Homozygosity mapping is one approach that is useful for delineating regions of interest. A combined approach of homozygosity mapping coupled with partial or whole-exome analysis has been used successfully in identification of disease-causing genes in recessive conditions focusing on variants within specific homozygous regions of the genome [30–32]. Here we use SNP genotyping coupled to a whole-exome sequencing strategy to identify a novel candidate for heterotaxy in a patient with a complex heterotaxy syndrome phenotype. We further evaluate SHROOM3 in an additional 96 patients from our heterotaxy cohort and identify four rare variants, two of which are predicted to be pathogenic.
Clinical findings in LAT1180
Clinical findings in LAT1180
L-Transposition of the great arteries
Abdominal situs inversus
Sensorineural hearing loss
Chromosome microarray analysis
Major absence-of-heterozygosity regions identified in LAT1180 using SNP array
Number of markers
Genes in region
Exome statistics for LAT1180
Total amount of raw data generated (Gb)
Sequencing read length (bp)
Total reads generated (million pairs)
Reads aligning to human reference genome hg19 (million pairs)
Usable data for alignment (Gb)
Reads aligned to human reference genome hg19
Bases aligning to human exome (targets)
Total bases aligning to exome (Gb)
Mean depth of coverage of targets
Maximum depth of coverage of targets
Minimum depth of coverage of targets
Average depth of coverage
Bases covered at depth of ≥ 1×
Bases covered at depth of ≥ 5×
Bases covered at depth of ≥ 10×
Exome sequencing and filtering strategy in LAT1180¶
Total variants identified
Total coding variants identified
Total dbSNP132 variants
Total changes not present in dbSNP132 database
Homozygous missense changes
Homozygous missense changes not present in 1000 Genomes data
Homozygous missense changes on chromosomes 1, 4, 7, 13, 15, 18
Homozygous missense changes within absence-of-heterozygosity
Previously, we developed an approach for prioritization of candidate genes for heterotaxy spectrum cardiovascular malformations and laterality disorders based on developmental expression and gene function . In addition, we have developed a network biology analysis appropriate for evaluation of candidates relative to potential interactions with known genetic pathways for heterotaxy, LR patterning, and ciliopathies in animal models and humans (manuscript in preparation). Using these approaches, three of the genes, CXCL2, CTSO, and RXFP1, are considered unlikely candidates. CXCL2 is an inducible chemokine important for chemotaxis, immune response, and inflammatory response. Targeted deletion of Cxcl2 in mice does not cause congenital anomalies but does result in poor wound healing and increased susceptibility to infection . CTSO, a cysteine proteinase, is a proteolytic enzyme that is a member of the papain superfamily involved in cellular protein degradation and turnover. It is expressed ubiquitously postnatally and in the brain prenatally. RFXP1 (also known as LRG7) is a G-protein coupled receptor to which the ligand relaxin binds. It is expressed ubiquitously with the exception of the spleen. Mouse Genome Informatics shows that homozygous deletion of Rfxp1 leads to males with reduced fertility and females unable to nurse due to impaired nipple development. In contrast, SHROOM3 is considered a very strong candidate based on its known expression and function, including its known role in gut looping and its ability to bind ROCK2.
Rare variants in SHROOM3
- - -
+ + +
+ + +
- - -
+ + +
In the present study, we investigated a proband, LAT1180, from a consanguineous pedigree with a novel form of heterotaxy syndrome using microarray-based CNV analysis and whole-exome sequencing. Our initial genetic analysis using two microarray-based platforms (Illumina SNP genotyping and exon-targeted Agilent aCGH) failed to identify any potential structural mutation. However, we observed homozygous regions (absence-of-heterozygosity) from SNP genotyping data, suggesting that homozygous point mutations or small insertion/deletion events within these regions could be disease associated. Subsequently, whole-exome analysis resulted in the identification of a novel homozygous missense mutation in the SHROOM3 gene on chromosome 4. Additional sequencing in a cohort of 96 heterotaxy patients identified two additional patients with homozygous variants and two patients with heterozygous variants. Although in vivo loss of function analyses have demonstrated the importance of SHROOM3 for proper cardiac and gut patterning, specific testing of the variants identified herein will be useful to further establish pathogenicity and the most common mode of inheritance. This study demonstrates the usefulness of high-throughput sequencing and SNP genotyping to identify important candidates in disorders characterized by genetic and phenotypic heterogeneity.
SHROOM3 encodes a cytoskeletal protein of 1,996 residues that is composed of 3 main domains with distinct functions (Figure 5). SHROOM3, an actin binding protein, is responsible for early cell shape during morphogenesis through a myosin II-dependent pathway. It is essential for neural tube closure in mouse, Xenopus, and chick [40–42]. Early studies in model species showed that Shroom3 plays an important role in the morphogenesis of epithelial sheets, such as gut epithelium, lens placode invagination, and also cardiac development [43, 44]. Recent data indicate an important role for Shroom3 in proper gut rotation . Interestingly, gut malrotation is a common feature of heterotaxy and is consistent with a laterality disorder. In Xenopus, Shroom3 is expressed in the myocardium and is necessary for cellular morphogenesis in the early heart as well as normal cardiac tube formation with disruption of cardiac looping (Thomas Drysdale, personal communication, manuscript in revision). Downstream effector proteins of Shroom3 include Mena, myosin II, Rap1 GTPase and Rho Kinases [40–42, 44, 46].
Shroom3 may play an important role in LR development acting downstream of Pitx2. Pitx2 is an important transcription factor in the generation of LR patterning in Xenopus, zebrafish, and mice [47–49]. Recently it was shown that Pitx2 can directly activate expression of Shroom3 and ultimately chiral gut looping in Xenopus . Gut looping morphogenesis in Xenopus is most likely driven by cell shape changes in gut epithelium . The identification of Shroom3 as a downstream effector fills an important gap in understanding how positional information is transferred into morphogenetic movements during organogenesis. The presence of a Pitx2 binding-sites upstream of mouse Shroom3 combined with the similar gut looping phenotypes of mouse Pitx2 and Shroom3 mutants supports the interactive mechanism for these two proteins [41, 43, 51].
SHROOM3 is a novel candidate for heterotaxy-spectrum cardiovascular malformations. This study highlights the importance of microarray-based SNP/CNV genotyping followed by exome sequencing for identification of novel candidates. This approach can be useful for rare disorders that have been challenging to analyze with traditional genetic approaches due to small numbers, significant clinical and genetic heterogeneity, and/or multifactorial inheritance.
Materials and methods
DNA of proband LAT1180 was extracted from whole peripheral blood leukocytes following a standard protocol. Screening of SHROOM3 was performed using DNA samples from 96 additional sporadic heterotaxy patients. The heterotaxy cohort has been reported previously [7, 9]. DNA samples with previous positive genetic testing results were not used in the current study. This study was approved by the Institutional Review Boards at the Baylor College of Medicine and Cincinnati Children's Hospital Medical Center (CCHMC). Written informed consent for participation in this study as well as publication of clinical data of the proband was obtained. All the methods applied in this study conformed to the Declaration of Helsinki (1964) of the World Medical Association concerning human material/data and experimentation  and ethical approval was granted by the ethics committee of the Baylor College of Medicine and CCHMC.
Genome-wide SNP genotyping was performed using an Illumina HumanOmni-Quad Infinium HD BeadChip. The chip contains 1,140,419 SNP markers with an average call frequency of > 99% and is unbiased to coding and noncoding regions of the genome. CNV analysis was performed using KaryoStudio Software (Illumina Inc.).
Array comparative genomic hybridization
The custom exon-targeted aCGH array was designed by Baylor Medical Genetics Laboratories  and manufactured by Agilent Technology (Santa Clara, CA, USA). The array contains 180,000 oligos covering 24,319 exons (4.2/exon). Data (105 k) were normalized using the Agilent Feature Extraction software. CNVs were detected by intensities of differentially labeled test DNA samples and LAT1180 DNA samples hybridized to Agilent array containing probes (probe-based). Results were interpreted by an experienced cytogeneticist at the Baylor College of Medicine. The Database of Genomic Variants  and in-house cytogenetic databases from the Baylor College of Medicine and CCHMC were used as control datasets for CNV analysis.
Genomic DNA (3 μg) from proband LAT1180 was fragmented and enriched for human exonic sequences with the NimbleGen SeqCap EZ Human Exome v2.0 Library (2.1 million DNA probes). A total of approximately 30,000 consensus coding sequence genes (approximately 300,000 exons, total size 36.5 Mb) are targeted by this capture, which contains probes covering a total of 44.1 Mb. The resulting exome library of the proband was sequenced with 50 bp paired-end reads using Illumina GAII (v2 Chemistry). Data are archived at the NCBI Sequence Read Archive (SRA) under an NCBI accession number [NCBI: SRP007801] . All sequence reads were mapped to the reference human genome (UCSC hg 19) using the Illumina Pipeline software version 1.5 featuring a gapped aligner (ELAND v2). Variant identification was performed using locally developed software 'SeqMate' (submitted for publication). The tool combines the aligned reads with the reference sequence and computes a distribution of call quality at each aligned base position, which serves as the basis for variant calling. Variants are reported based on a configurable formula using the following additional parameters: depth of coverage, proportion of each base at a given position and number of different reads showing a sequence variation. The minimum number of high quality bases to establish coverage at any position was arbitrarily set at 10. Any sequence position with a non-reference base observed more than 75% of the time was called a homozygous variant. Any sequence position with a non-reference base observed between 25% and 75% of the time was called a heterozygous variant. Amino acid changes were identified by comparison to the UCSC RefSeq database track. A local realignment tool was used to minimize the errors in SNP calling due to indels. A series of filtering strategies (dbSNP132, 1000 Genomes project (May 2010)) were applied to reduce the number of variants and to identify the potential pathogenic mutations causing the disease phenotype.
Mutation screening and validation
Primers were designed to cover exonic regions containing potential variants of SHROOM3 and UGT2A1 genes in LAT1180. For screening additional heterotaxy patients, primers were designed to include all exons and splice junctions of SHROOM3 (primer sequences are available upon request). A homozygous nonsense variant (p.Y192X) was confirmed in the UGT2A1 gene within the same homozygous region on chromosome 4 but was later excluded because of its presence in the 1000 Genomes project data. PCR products were sequenced using BigDye Terminator and an ABI 3730XL DNA Analyzer. Sequence analysis was performed via Bioedit Sequence Alignment Editor, version 6.0.7 . All positive findings were confirmed in a separate experiment using the original genomic DNA sample as template for new amplification and bi-directional sequencing reactions.
array comparative genomic hybridization
congenital heart disease
copy number variation
single nucleotide polymorphism.
We thank the heterotaxy patients and families for their cooperation. We thank Dr Thomas Drysdale for discussions and sharing data on Shroom3 in cardiac morphogenesis. We also thank the Genetic Variation and Gene Discovery Core (GVGDC) at CCHMC for providing genotyping and high-throughput sequencing facilities. This project was supported by a Burroughs Wellcome Fund Clinical Scientist Award in Translational Research #1008496 (SMW).
- Pierpont ME, Basson CT, Benson DW, Gelb BD, Giglia TM, Goldmuntz E, McGee G, Sable CA, Srivastava D, Webb CL: Genetic basis for congenital heart defects: current knowledge: a scientific statement from the American Heart Association Congenital Cardiac Defects Committee, Council on Cardiovascular Disease in the Young: endorsed by the American Academy of Pediatrics. Circulation. 2007, 115: 3015-3038. 10.1161/CIRCULATIONAHA.106.183056.PubMedView ArticleGoogle Scholar
- Ransom J, Srivastava D: The genetics of cardiac birth defects. Semin Cell Dev Biol. 2007, 18: 132-139. 10.1016/j.semcdb.2006.12.005.PubMedView ArticleGoogle Scholar
- Weismann CG, Gelb BD: The genetics of congenital heart disease: a review of recent developments. Curr Opin Cardiol. 2007, 22: 200-206. 10.1097/HCO.0b013e3280f629c7.PubMedView ArticleGoogle Scholar
- Sutherland MJ, Ware SM: Disorders of left-right asymmetry: heterotaxy and situs inversus. Am J Med Genet C Semin Med Genet. 2009, 151C: 307-317. 10.1002/ajmg.c.30228.PubMedView ArticleGoogle Scholar
- Zhu L, Belmont JW, Ware SM: Genetics of human heterotaxias. Eur J Hum Genet. 2006, 14: 17-25.PubMedGoogle Scholar
- Oyen N, Poulsen G, Boyd HA, Wohlfahrt J, Jensen PK, Melbye M: Recurrence of congenital heart defects in families. Circulation. 2009, 120: 295-301. 10.1161/CIRCULATIONAHA.109.857987.PubMedView ArticleGoogle Scholar
- Mohapatra B, Casey B, Li H, Ho-Dawson T, Smith L, Fernbach SD, Molinari L, Niesh SR, Jefferies JL, Craigen WJ, Towbin JA, Belmont JW, Ware SM: Identification and functional characterization of NODAL rare variants in heterotaxy and isolated cardiovascular malformations. Hum Mol Genet. 2009, 18: 861-871.PubMedPubMed CentralGoogle Scholar
- Roessler E, Ouspenskaia MV, Karkera JD, Velez JI, Kantipong A, Lacbawan F, Bowers P, Belmont JW, Towbin JA, Goldmuntz E, Feldman B, Muenke M: Reduced NODAL signaling strength via mutation of several pathway members including FOXH1 is linked to human heart defects and holoprosencephaly. Am J Hum Genet. 2008, 83: 18-29. 10.1016/j.ajhg.2008.05.012.PubMedPubMed CentralView ArticleGoogle Scholar
- Ware SM, Peng J, Zhu L, Fernbach S, Colicos S, Casey B, Towbin J, Belmont JW: Identification and functional analysis of ZIC3 mutations in heterotaxy and related congenital heart defects. Am J Hum Genet. 2004, 74: 93-105. 10.1086/380998.PubMedPubMed CentralView ArticleGoogle Scholar
- Shiratori H, Hamada H: The left-right axis in the mouse: from origin to morphology. Development. 2006, 133: 2095-2104. 10.1242/dev.02384.PubMedView ArticleGoogle Scholar
- Hamada H, Meno C, Watanabe D, Saijoh Y: Establishment of vertebrate left-right asymmetry. Nat Rev Genet. 2002, 3: 103-113.PubMedView ArticleGoogle Scholar
- Essner JJ, Vogan KJ, Wagner MK, Tabin CJ, Yost HJ, Brueckner M: Conserved function for embryonic nodal cilia. Nature. 2002, 418: 37-38. 10.1038/418037a.PubMedView ArticleGoogle Scholar
- Vandenberg LN, Levin M: Perspectives and open problems in the early phases of left-right patterning. Semin Cell Dev Biol. 2009, 20: 456-463. 10.1016/j.semcdb.2008.11.010.PubMedPubMed CentralView ArticleGoogle Scholar
- Vandenberg LN, Levin M: Far from solved: a perspective on what we know about early mechanisms of left-right asymmetry. Dev Dyn. 2010, 239: 3131-3146. 10.1002/dvdy.22450.PubMedView ArticleGoogle Scholar
- Aw S, Levin M: What's left in asymmetry?. Dev Dyn. 2008, 237: 3453-3463. 10.1002/dvdy.21560.PubMedPubMed CentralView ArticleGoogle Scholar
- Cardenas-Rodriguez M, Badano JL: Ciliary biology: understanding the cellular and genetic basis of human ciliopathies. Am J Med Genet C Semin Med Genet. 2009, 151C: 263-280. 10.1002/ajmg.c.30227.PubMedView ArticleGoogle Scholar
- Hashimoto M, Shinohara K, Wang J, Ikeuchi S, Yoshiba S, Meno C, Nonaka S, Takada S, Hatta K, Wynshaw-Boris A, Hamada H: Planar polarization of node cells determines the rotational axis of node cilia. Nat Cell Biol. 2010, 12: 170-176. 10.1038/ncb2020.PubMedView ArticleGoogle Scholar
- Schneider I, Schneider PN, Derry SW, Lin S, Barton LJ, Westfall T, Slusarski DC: Zebrafish Nkd1 promotes Dvl degradation and is required for left-right patterning. Dev Biol. 2010, 348: 22-33. 10.1016/j.ydbio.2010.08.040.PubMedPubMed CentralView ArticleGoogle Scholar
- Antic D, Stubbs JL, Suyama K, Kintner C, Scott MP, Axelrod JD: Planar cell polarity enables posterior localization of nodal cilia and left-right axis determination during mouse and Xenopus embryogenesis. PLoS One. 2010, 5: e8999-10.1371/journal.pone.0008999.PubMedPubMed CentralView ArticleGoogle Scholar
- Wang G, Cadwallader AB, Jang DS, Tsang M, Yost HJ, Amack JD: The Rho kinase Rock2b establishes anteroposterior asymmetry of the ciliated Kupffer's vesicle in zebrafish. Development. 2011, 138: 45-54. 10.1242/dev.052985.PubMedPubMed CentralView ArticleGoogle Scholar
- Esguerra CV, Nelles L, Vermeire L, Ibrahimi A, Crawford AD, Derua R, Janssens E, Waelkens E, Carmeliet P, Collen D, Huylebroeck D: Ttrap is an essential modulator of Smad3-dependent Nodal signaling during zebrafish gastrulation and left-right axis determination. Development. 2007, 134: 4381-4393. 10.1242/dev.000026.PubMedView ArticleGoogle Scholar
- Lopes Floro K, Artap ST, Preis JI, Fatkin D, Chapman G, Furtado MB, Harvey RP, Hamada H, Sparrow DB, Dunwoodie SL: Loss of Cited2 causes congenital heart disease by perturbing left-right patterning of the body axis. Hum Mol Genet. 2011, 20: 1097-1110. 10.1093/hmg/ddq554.PubMedView ArticleGoogle Scholar
- Huang S, Ma J, Liu X, Zhang Y, Luo L: Geminin is required for left-right patterning through regulating Kupffer's vesicle formation and ciliogenesis in zebrafish. Biochem Biophys Res Commun. 2011, 410: 164-169. 10.1016/j.bbrc.2011.04.085.PubMedView ArticleGoogle Scholar
- Fakhro KA, Choi M, Ware SM, Belmont JW, Towbin JA, Lifton RP, Khokha MK, Brueckner M: Rare copy number variations in congenital heart disease patients identify unique genes in left-right patterning. Proc Natl Acad Sci USA. 2011, 108: 2915-2920. 10.1073/pnas.1019645108.PubMedPubMed CentralView ArticleGoogle Scholar
- Krawitz PM, Schweiger MR, Rodelsperger C, Marcelis C, Kolsch U, Meisel C, Stephani F, Kinoshita T, Murakami Y, Bauer S, Isau M, Fischer A, Dahl A, Kerick M, Hecht J, Kohler S, Jager M, Grunhagen J, de Condor BJ, Doelken S, Brunner HG, Meinecke P, Passarge E, Thompson MD, Cole DE, Horn D, Roscioli T, Mundlos S, Robinson PN: Identity-by-descent filtering of exome sequence data identifies PIGV mutations in hyperphosphatasia mental retardation syndrome. Nat Genet. 2010, 42: 827-829. 10.1038/ng.653.PubMedView ArticleGoogle Scholar
- Ng SB, Bigham AW, Buckingham KJ, Hannibal MC, McMillin MJ, Gildersleeve HI, Beck AE, Tabor HK, Cooper GM, Mefford HC, Lee C, Turner EH, Smith JD, Rieder MJ, Yoshiura K, Matsumoto N, Ohta T, Niikawa N, Nickerson DA, Bamshad MJ, Shendure J: Exome sequencing identifies MLL2 mutations as a cause of Kabuki syndrome. Nat Genet. 2010, 42: 790-793. 10.1038/ng.646.PubMedPubMed CentralView ArticleGoogle Scholar
- Ng SB, Buckingham KJ, Lee C, Bigham AW, Tabor HK, Dent KM, Huff CD, Shannon PT, Jabs EW, Nickerson DA, Shendure J, Bamshad MJ: Exome sequencing identifies the cause of a mendelian disorder. Nat Genet. 2010, 42: 30-35. 10.1038/ng.499.PubMedPubMed CentralView ArticleGoogle Scholar
- Ostergaard P, Simpson MA, Brice G, Mansour S, Connell FC, Onoufriadis A, Child AH, Hwang J, Kalidas K, Mortimer PS, Trembath R, Jeffery S: Rapid identification of mutations in GJC2 in primary lymphoedema using whole exome sequencing combined with linkage analysis with delineation of the phenotype. J Med Genet. 2011, 48: 251-255. 10.1136/jmg.2010.085563.PubMedView ArticleGoogle Scholar
- Walsh T, Shahin H, Elkan-Miller T, Lee MK, Thornton AM, Roeb W, Abu Rayyan A, Loulus S, Avraham KB, King MC, Kanaan M: Whole exome sequencing and homozygosity mapping identify mutation in the cell polarity protein GPSM2 as the cause of nonsyndromic hearing loss DFNB82. Am J Hum Genet. 2010, 87: 90-94. 10.1016/j.ajhg.2010.05.010.PubMedPubMed CentralView ArticleGoogle Scholar
- Becker J, Semler O, Gilissen C, Li Y, Bolz HJ, Giunta C, Bergmann C, Rohrbach M, Koerber F, Zimmermann K, de Vries P, Wirth B, Schoenau E, Wollnik B, Veltman JA, Hoischen A, Netzer C: Exome sequencing identifies truncating mutations in human SERPINF1 in autosomal-recessive osteogenesis imperfecta. Am J Hum Genet. 2011, 88: 362-371. 10.1016/j.ajhg.2011.01.015.PubMedPubMed CentralView ArticleGoogle Scholar
- Caliskan M, Chong JX, Uricchio L, Anderson R, Chen P, Sougnez C, Garimella K, Gabriel SB, dePristo MA, Shakir K, Matern D, Das S, Waggoner D, Nicolae DL, Ober C: Exome sequencing reveals a novel mutation for autosomal recessive non-syndromic mental retardation in the TECR gene on chromosome 19p13. Hum Mol Genet. 2011, 20: 1285-1289. 10.1093/hmg/ddq569.PubMedPubMed CentralView ArticleGoogle Scholar
- Otto EA, Hurd TW, Airik R, Chaki M, Zhou W, Stoetzel C, Patil SB, Levy S, Ghosh AK, Murga-Zamalloa CA, van Reeuwijk J, Letteboer SJ, Sang L, Giles RH, Liu Q, Coene KL, Estrada-Cuzcano A, Collin RW, McLaughlin HM, Held S, Kasanuki JM, Ramaswami G, Conte J, Lopez I, Washburn J, Macdonald J, Hu J, Yamashita Y, Maher ER, Guay-Woodford LM, et al: Candidate exome capture identifies mutation of SDCCAG8 as the cause of a retinal-renal ciliopathy. Nat Genet. 2010, 42: 840-850. 10.1038/ng.662.PubMedPubMed CentralView ArticleGoogle Scholar
- Lander ES, Botstein D: Homozygosity mapping: a way to map human recessive traits with the DNA of inbred children. Science. 1987, 236: 1567-1570. 10.1126/science.2884728.PubMedView ArticleGoogle Scholar
- NCBI Sequence Read Archive: SHROOM3 is a novel candidate for heterotaxy identified by whole exome sequencing. [http://www.ncbi.nlm.nih.gov/sra?term=SRP007801]
- Luan J, Furuta Y, Du J, Richmond A: Developmental expression of two CXC chemokines, MIP-2 and KC, and their receptors. Cytokine. 2001, 14: 253-263. 10.1006/cyto.2001.0882.PubMedView ArticleGoogle Scholar
- PolyPhen-2. [http://genetics.bwh.harvard.edu/pph/]
- PANTHER. [http://www.pantherdb.org/tools/csnpScoreForm.jsp]
- Mutation Taster. [http://www.mutationtaster.org/]
- SIFT. [http://sift.jcvi.org/www/SIFT_BLink_submit.html]
- Haigo SL, Hildebrand JD, Harland RM, Wallingford JB: Shroom induces apical constriction and is required for hingepoint formation during neural tube closure. Curr Biol. 2003, 13: 2125-2137. 10.1016/j.cub.2003.11.054.PubMedView ArticleGoogle Scholar
- Hildebrand JD, Soriano P: Shroom, a PDZ domain-containing actin-binding protein, is required for neural tube morphogenesis in mice. Cell. 1999, 99: 485-497. 10.1016/S0092-8674(00)81537-8.PubMedView ArticleGoogle Scholar
- Nishimura T, Takeichi M: Shroom3-mediated recruitment of Rho kinases to the apical cell junctions regulates epithelial and neuroepithelial planar remodeling. Development. 2008, 135: 1493-1502. 10.1242/dev.019646.PubMedView ArticleGoogle Scholar
- Chung MI, Nascone-Yoder NM, Grover SA, Drysdale TA, Wallingford JB: Direct activation of Shroom3 transcription by Pitx proteins drives epithelial morphogenesis in the developing gut. Development. 2010, 137: 1339-1349. 10.1242/dev.044610.PubMedPubMed CentralView ArticleGoogle Scholar
- Plageman TF, Chung MI, Lou M, Smith AN, Hildebrand JD, Wallingford JB, Lang RA: Pax6-dependent Shroom3 expression regulates apical constriction during lens placode invagination. Development. 2010, 137: 405-415. 10.1242/dev.045369.PubMedPubMed CentralView ArticleGoogle Scholar
- Plageman TF, Zacharias AL, Gage PJ, Lang RA: Shroom3 and a Pitx2-N-cadherin pathway function cooperatively to generate asymmetric cell shape changes during gut morphogenesis. Dev Biol. 2011, 357: 227-234. 10.1016/j.ydbio.2011.06.027.PubMedPubMed CentralView ArticleGoogle Scholar
- Lee C, Scherr HM, Wallingford JB: Shroom family proteins regulate gamma-tubulin distribution and microtubule architecture during epithelial cell shape change. Development. 2007, 134: 1431-1441. 10.1242/dev.02828.PubMedView ArticleGoogle Scholar
- Capdevila J, Vogan KJ, Tabin CJ, Izpisua Belmonte JC: Mechanisms of left-right determination in vertebrates. Cell. 2000, 101: 9-21. 10.1016/S0092-8674(00)80619-4.PubMedView ArticleGoogle Scholar
- Davis NM, Kurpios NA, Sun X, Gros J, Martin JF, Tabin CJ: The chirality of gut rotation derives from left-right asymmetric changes in the architecture of the dorsal mesentery. Dev Cell. 2008, 15: 134-145. 10.1016/j.devcel.2008.05.001.PubMedPubMed CentralView ArticleGoogle Scholar
- Logan M, Pagan-Westphal SM, Smith DM, Paganessi L, Tabin CJ: The transcription factor Pitx2 mediates situs-specific morphogenesis in response to left-right asymmetric signals. Cell. 1998, 94: 307-317. 10.1016/S0092-8674(00)81474-9.PubMedView ArticleGoogle Scholar
- Muller JK, Prather DR, Nascone-Yoder NM: Left-right asymmetric morphogenesis in the Xenopus digestive system. Dev Dyn. 2003, 228: 672-682. 10.1002/dvdy.10415.PubMedView ArticleGoogle Scholar
- Kitamura K, Miura H, Miyagawa-Tomita S, Yanazawa M, Katoh-Fukui Y, Suzuki R, Ohuchi H, Suehiro A, Motegi Y, Nakahara Y, Kondo S, Yokoyama M: Mouse Pitx2 deficiency leads to anomalies of the ventral body wall, heart, extra- and periocular mesoderm and right pulmonary isomerism. Development. 1999, 126: 5749-5758.PubMedGoogle Scholar
- Aw S, Adams DS, Qiu D, Levin M: H,K-ATPase protein localization and Kir4.1 function reveal concordance of three axes during early determination of left-right asymmetry. Mech Dev. 2008, 125: 353-372. 10.1016/j.mod.2007.10.011.PubMedPubMed CentralView ArticleGoogle Scholar
- Danilchik MV, Brown EE, Riegert K: Intrinsic chiral properties of the Xenopus egg cortex: an early indicator of left-right asymmetry?. Development. 2006, 133: 4517-4526. 10.1242/dev.02642.PubMedView ArticleGoogle Scholar
- Gardner RL: Normal bias in the direction of fetal rotation depends on blastomere composition during early cleavage in the mouse. PLoS One. 2010, 5: e9610-10.1371/journal.pone.0009610.PubMedPubMed CentralView ArticleGoogle Scholar
- Kuroda R, Endo B, Abe M, Shimizu M: Chiral blastomere arrangement dictates zygotic left-right asymmetry pathway in snails. Nature. 2009, 462: 790-794. 10.1038/nature08597.PubMedView ArticleGoogle Scholar
- Declaration of Helsinki (1964) of the World Medical Association. [http://www.wma.net/en/30publications/10policies/b3/17c.pdf]
- Baylor Medical Genetics Laboratories, Baylor College of Medicine. [http://www.bcm.edu/geneticlabs/cma/tables.html]
- The Database of Genomic Variants (DGV). [http://projects.tcag.ca/variation]
- Bioedit Sequence Alignment Editor. [http://www.mbio.ncsu.edu/bioedit/bioedit.html]
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.