We characterized the mutational landscape of melanoma, the form of skin cancer with the highest mortality rate, by sequencing the exomes of 147 melanomas. Sun-exposed melanomas had markedly more ultraviolet (UV)-like C>T somatic mutations compared to sun-shielded acral, mucosal and uveal melanomas. Among the newly identified cancer genes was PPP6C, encoding a serine/threonine phosphatase, which harbored mutations that clustered in the active site in 12% of sun-exposed melanomas, exclusively in tumors with mutations in BRAF or NRAS. Notably, we identified a recurrent UV-signature, an activating mutation in RAC1 in 9.2% of sun-exposed melanomas. This activating mutation, the third most frequent in our cohort of sun-exposed melanoma after those of BRAF and NRAS, changes Pro29 to serine (RAC1P29S) in the highly conserved switch I domain. Crystal structures, and biochemical and functional studies of RAC1P29S showed that the alteration releases the conformational restraint conferred by the conserved proline, causes an increased binding of the protein to downstream effectors, and promotes melanocyte proliferation and migration. These findings raise the possibility that pharmacological inhibition of downstream effectors of RAC1 signaling could be of therapeutic benefit.

Objective

Myoclonus is characterized by sudden, brief involuntary movements and its presence is debilitating. We identified a family suffering from adult-onset, cortical myoclonus without associated seizures. We performed clinical, electrophysiological, and genetic studies to define this phenotype.

Methods

A large, four-generation family with history of myoclonus underwent careful questioning, examination, and electrophysiological testing. Thirty-five family members donated blood samples for genetic analysis, which included SNP mapping, microsatellite linkage, targeted massively parallel sequencing, and Sanger sequencing. In silico and in vitro experiments were performed to investigate functional significance of the mutation.

Results

We identified 11 members of a Canadian Mennonite family suffering from adult-onset, slowly progressive, disabling, multifocal myoclonus. Somatosensory evoked potentials indicated a cortical origin of the myoclonus. There were no associated seizures. Some severely affected individuals developed signs of progressive cerebellar ataxia of variable severity late in the course of their illness. The phenotype was inherited in an autosomal dominant fashion. We demonstrated linkage to chromosome 16q21-22.1. We then sequenced all coding sequence in the critical region, identifying only a single co-segregating, novel, nonsynonymous mutation, which resides in the gene NOL3. Furthermore, this mutation was found to alter post-translational modification of NOL3 protein in vitro.

Interpretation

We propose that Familial Cortical Myoclonus (FCM) is a novel movement disorder that may be caused by mutation in NOL3. Further investigation of the role of NOL3 in neuronal physiology may shed light on neuronal membrane hyperexcitability and pathophysiology of myoclonus and related disorders.

The advent of next-generation sequencing technologies has facilitated the detection of rare variants. Despite the significant cost reduction, sequencing cost is still high for large-scale studies. In this article, we examine DNA pooling as a cost-effective strategy for rare variant detection. We consider the optimal number of individuals in a DNA pool to detect an allele with a specific minor allele frequency (MAF) under a given coverage depth and detection threshold. We found that the optimal number of individuals in a pool is indifferent to the MAF at the same coverage depth and detection threshold. In addition, when the individual contributions to each pool are equal, the total number of individuals across different pools required in an optimal design to detect a variant with a desired power is similar at different coverage depths. When the contributions are more variable, more individuals tend to be needed for higher coverage depths. Our study provides general guidelines on using DNA pooling for more cost-effective identifications of rare variants. Genet. Epidemiol. 35:139-147, 2011. © 2011 Wiley-Liss, Inc.

The recent advent of next-generation sequencing technologies has dramatically changed the nature of biomedical research. Human genetics is no exception-it has never been easier to interrogate human patient genomes at the nucleotide level to identify disease-associated variants. To further facilitate the efficiency of this approach, whole exome sequencing (WES) was first developed in 2009. Over the past three years, multiple groups have demonstrated the power of WES through robust disease-associated variant discoveries across a diverse spectrum of human diseases. Here, we review the application of WES to different types of inherited human diseases and discuss analytical challenges and possible solutions, with the aim of providing a practical guide for the effective use of this technology.

We performed a genome-wide association study of IgA nephropathy (IgAN), a major cause of kidney failure worldwide. Discovery was in 1,194 cases and 902 controls of Chinese Han ancestry, with targeted follow-up in Chinese and European cohorts comprising 1,950 cases and 1,920 controls. We identified three independent loci in the major histocompatibility complex (MHC), a common deletion of CFHR1 and CFHR3 at Chr. 1q32 and a locus at Chr. 22q12 that each surpassed genome-wide significance (p-values for association between 1.59 × 10−26 and 4.84 × 10−9 and minor allele odds ratios of 0.63–0.80). These five loci explain 4–7% of the disease variance and up to a 10-fold variation in interindividual risk. Many of the IgAN–protective alleles impart increased risk of other autoimmune or infectious diseases, and IgAN risk allele frequencies closely parallel the variation in disease prevalence among Asian, European and African populations, suggesting complex selective pressures.

Hypertension affects one billion people and is a principal reversible risk factor for cardiovascular disease. A rare Mendelian syndrome, pseudohypoaldosteronism type II (PHAII), featuring hypertension, hyperkalemia, and metabolic acidosis, has revealed previously unrecognized physiology orchestrating the balance between renal salt reabsorption versus K+ and H+ excretion1. We used exome sequencing to identify mutations in Kelch-like 3 (KLHL3) or Cullin 3 (CUL3) in 41 PHAII kindreds. KLHL3 mutations are either recessive or dominant, while CUL3 mutations are dominant and predominantly de novo. CUL3 and BTB-Kelch proteins such as KLHL3 are components of Cullin/RING E3 ligase complexes (CRLs) that ubiquitinate substrates bound to Kelch propeller domains2–8. Dominant KLHL3 mutations are clustered in short segments within the Kelch propeller and BTB domains implicated in substrate9 and Cullin5 binding, respectively. Diverse CUL3 mutations all result in skipping of exon 9, producing an in-frame deletion. Because dominant KLHL3 and CUL3 mutations both phenocopy recessive loss-of-function KLHL3 mutations, they may abrogate ubiquitination of KLHL3 substrates. Disease features are reversed by thiazide diuretics, which inhibit the Na-Cl cotransporter (NCC) in the distal nephron of the kidney; KLHL3 and CUL3 are expressed in this location, suggesting a mechanistic link between KLHL3/CUL3 mutations, increased Na-Cl reabsorption, and disease pathogenesis. These findings demonstrate the utility of exome sequencing in disease gene identification despite combined complexities of locus heterogeneity, mixed models of transmission, and frequent de novo mutation, and establish a fundamental role for KLHL3/CUL3 in blood pressure, K+, and pH homeostasis.

Background

Aldosterone producing lesions are a common cause of hypertension, but genetic alterations for tumorigenesis have been unclear. Recently, either of two recurrent somatic missense mutations (G151R or L168R) was found in the potassium channel KCNJ5 gene in aldosterone producing adenomas. These mutations alter the channel selectivity filter and result in Na+ conductance and cell depolarization, stimulating aldosterone production and cell proliferation. Because a similar mutation occurs in a Mendelian form of primary aldosteronism, these mutations appear to be sufficient for cell proliferation and aldosterone production. The prevalence and spectrum of KCNJ5 mutations in different entities of adrenocortical lesions remain to be defined.

Materials and Methods

The coding region and flanking intronic segments of KCNJ5 were subjected to Sanger DNA sequencing in 351 aldosterone producing lesions, from patients with primary aldosteronism and 130 other adrenocortical lesions. The specimens had been collected from 10 different worldwide referral centers.

Results

G151R or L168R somatic mutations were identified in 47% of aldosterone producing adenomas, each with similar frequency. A previously unreported somatic mutation near the selectivity filter, E145Q, was observed twice. Somatic G151R or L168R mutations were also found in 40% of aldosterone producing adenomas associated with marked hyperplasia, but not in specimens with merely unilateral hyperplasia. Mutations were absent in 130 non-aldosterone secreting lesions.

KCNJ5 mutations were overrepresented in aldosterone producing adenomas from female compared to male patients (63 vs. 24%). Males with KCNJ5 mutations were significantly younger than those without (45 vs. 54, respectively; p<0.005) and their APAs with KCNJ5 mutations were larger than those without (27.1 mm vs. 17.1 mm; p<0.005).

Discussion

Either of two somatic KCNJ5 mutations are highly prevalent and specific for aldosterone producing lesions. These findings provide new insight into the pathogenesis of primary aldosteronism.

IgA nephropathy (IgAN), major cause of kidney failure worldwide, is common in Asians, moderately prevalent in Europeans, and rare in Africans. It is not known if these differences represent variation in genes, environment, or ascertainment. In a recent GWAS, we localized five IgAN susceptibility loci on Chr.6p21 (HLA-DQB1/DRB1, PSMB9/TAP1, and DPA1/DPB2 loci), Chr.1q32 (CFHR3/R1 locus), and Chr.22q12 (HORMAD2 locus). These IgAN loci are associated with risk of other immune-mediated disorders such as type I diabetes, multiple sclerosis, or inflammatory bowel disease. We tested association of these loci in eight new independent cohorts of Asian, European, and African-American ancestry (N = 4,789), followed by meta-analysis with risk-score modeling in 12 cohorts (N = 10,755) and geospatial analysis in 85 world populations. Four susceptibility loci robustly replicated and all five loci were genome-wide significant in the combined cohort (P = 5×10−32–3×10−10), with heterogeneity detected only at the PSMB9/TAP1 locus (I2 = 0.60). Conditional analyses identified two new independent risk alleles within the HLA-DQB1/DRB1 locus, defining multiple risk and protective haplotypes within this interval. We also detected a significant genetic interaction, whereby the odds ratio for the HORMAD2 protective allele was reversed in homozygotes for a CFHR3/R1 deletion (P = 2.5×10−4). A seven–SNP genetic risk score, which explained 4.7% of overall IgAN risk, increased sharply with Eastward and Northward distance from Africa (r = 0.30, P = 3×10−128). This model paralleled the known East–West gradient in disease risk. Moreover, the prediction of a South–North axis was confirmed by registry data showing that the prevalence of IgAN–attributable kidney failure is increased in Northern Europe, similar to multiple sclerosis and type I diabetes. Variation at IgAN susceptibility loci correlates with differences in disease prevalence among world populations. These findings inform genetic, biological, and epidemiological investigations of IgAN and permit cross-comparison with other complex traits that share genetic risk loci and geographic patterns with IgAN.

Author Summary

IgA nephropathy (IgAN) is the most common cause of kidney failure in Asia, has lower prevalence in Europe, and is very infrequent among populations of African ancestry. A long-standing question in the field is whether these differences represent variation in genes, environment, or ascertainment. In a recent genome-wide association study of 5,966 individuals, we identified five susceptibility loci for this trait. In this paper, we study the largest IgAN case-control cohort reported to date, composed of 10,775 individuals of European, Asian, and African-American ancestry. We confirm that all five loci are significant contributors to disease risk across this multi-ethnic cohort. In addition, we identify two novel independent susceptibility alleles within the HLA-DQB1/DRB1 locus and a new genetic interaction between loci on Chr.1p36 and Chr.22q22. We develop a seven–SNP genetic risk score that explains nearly 5% of variation in disease risk. In geospatial analysis of 85 world populations, the genetic risk score closely parallels worldwide patterns of disease prevalence. The genetic risk score also predicts an unsuspected Northward risk gradient in Europe. This genetic prediction is verified by examination of registry data demonstrating, similarly to other immune-mediated diseases such as multiple sclerosis and type I diabetes, a previously unrecognized increase in IgAN–attributable kidney failure in Northern European countries.

Endocrine tumors such as aldosterone-producing adrenal adenomas (APAs), a cause of severe hypertension, feature constitutive hormone production and unrestrained cell proliferation; the mechanisms linking these events are unknown. We identify two recurrent somatic mutations in and near the selectivity filter of the potassium (K+) channel KCNJ5 that are present in 8 of 22 human APAs studied. Both produce increased sodium (Na+) conductance and cell depolarization, which in adrenal glomerulosa cells produces calcium (Ca2+) entry, the signal for aldosterone production and cell proliferation. Similarly, we identify an inherited KCNJ5 mutation that produces increased Na+ conductance in a Mendelian form of severe aldosteronism and massive bilateral adrenal hyperplasia. These findings explain pathogenesis in a subset of patients with severe hypertension and implicate loss of K+ channel selectivity in constitutive cell proliferation and hormone production.

The biological basis for regional and inter-species differences in cerebral cortical morphology is poorly understood. We focused on consanguineous Turkish families with a single affected member with complex bilateral occipital cortical gyration abnormalities. By using whole-exome sequencing, we initially identified a homozygous 2-bp deletion in LAMC3, the laminin γ3 gene, leading to an immediate premature termination codon. In two other affected individuals with nearly identical phenotypes, we identified a homozygous nonsense mutation and a compound heterozygous mutation. In human but not mouse fetal brain, LAMC3 is enriched in postmitotic cortical plate neurons, localizing primarily to the somatodendritic compartment. LAMC3 expression peaks between late gestation and late infancy, paralleling the expression of molecules that are important in dendritogenesis and synapse formation. The discovery of the molecular basis of this unusual occipital malformation furthers our understanding of the complex biology underlying the formation of cortical gyrations.

Genetic studies of midgut carcinoid cancer have exclusively focused on genomic changes of the tumor cells. We investigated the role of constitutional genetic polymorphisms in predisposing individuals to ileal carcinoids. In all, 239 cases and 110 controls were collected from three institutions: the Uppsala University Hospital; the Dana-Farber Cancer Institute; and the MD Anderson Cancer Center, and were genotyped using microarrays assaying >300 000 single nucleotide polymorphisms. Association with rs2208059 in KIF16B approached statistical significance (Mantel-Haenszel odds ratio=2.42, P=4.16×10−7) at a Bonferroni-corrected level (<1.62×10−7). Using two computational algorithms, four copy-number variants (CNVs) were identified in multiple cases that were absent in study controls and markedly less frequent in ~1500 population-based controls. Of these four constitutional CNVs identified in blood-derived DNA, a 40 kb heterozygous deletion in Chr18q22.1 corresponded with a region frequently showing loss of heterozygosity (LOH) in ileal carcinoid tumor cells based on our meta-analysis of previously published cytogenetic studies (69.7% LOH, 95% confidence interval=60.0–77.9%). We analyzed the constitutional 40 kb deletion on chr18 in our study samples with a real-time quantitative PCR assay; 14/226 cases (6.19%) and 2/97 controls (2.06%) carried the CNV, although the exact boundaries of each deletion have not been determined. Given the small sample size, our findings warrant an independent cohort for a replication study. Owing to the rarity of this disease, we believe these results will provide a valuable resource for future work on this serious condition by allowing others to make efficient use of their samples in targeted studies.

Expression of astrocyte elevated gene-1 (AEG-1) is elevated in multiple human cancers including brain tumors, neuroblastomas, melanomas, breast cancers, non-small cell lung cancers, liver cancers, prostate cancers, and esophageal cancers. This gene plays crucial roles in tumor cell growth, invasion, angiogenesis and progression to metastasis. In addition, over-expression of AEG-1 protects primary and transformed cells from apoptosis-inducing signals by activating PI3K-Akt signaling pathways. These results suggest that AEG-1 is intimately involved in tumorigenesis and may serve as a potential therapeutic target for various human cancers. However, the normal physiological functions of AEG-1 require clarification. We presently analyzed the expression pattern of AEG-1 during mouse development. AEG-1 was expressed in mid-to-hindbrain, fronto-nasal processes, limbs, and pharyngeal arches in the early developmental period from E8.5 to E9.5. In addition, at stages of E12.5-E18.5 AEG-1 was localized in the brain, and olfactory and skeletal systems suggesting a role in neurogenesis, as well as in skin, including hair follicles, and in the liver, which are organ sites in which AEG-1 has been implicated in tumor development and progression. AEG-1 co-localized with Ki-67, indicating a role in cell proliferation, as previously revealed in tumorigenesis. Taken together, these results suggest that AEG-1 may play a prominent role during normal mouse development in the context of cell proliferation as well as differentiation, and that temporal regulation of AEG-1 expression may be required during specific stages and in specific tissues during development.

The advent of next-generation sequencing technologies has facilitated the detection of rare variants. Despite the significant cost reduction, sequencing cost is still high for large-scale studies. In this article, we examine DNA pooling as a cost-effective strategy for rare variant detection. We consider the optimal number of individuals in a DNA pool to detect an allele with a specific minor allele frequency (MAF) under a given coverage depth and detection threshold. We found that the optimal number of individuals in a pool is indifferent to the MAF at the same coverage depth and detection threshold. In addition, when the individual contributions to each pool are equal, the total number of individuals across different pools required in an optimal design to detect a variant with a desired power is similar at different coverage depths. When the contributions are more variable, more individuals tend to be needed for higher coverage depths. Our study provides general guidelines on using DNA pooling for more cost-effective identifications of rare variants.

The development of the human cerebral cortex is an orchestrated process involving the birth of neural progenitors in the peri-ventricular germinal zones, cell proliferation characterized by both symmetric and asymmetric mitoses, followed by migration of post-mitotic neurons to their final destinations in 6 highly ordered, functionally-specialized layers1,2. An understanding of the molecular mechanisms guiding these intricate processes is in its infancy, substantially driven by the discovery of rare mutations that cause malformations of cortical development (MCD)3-6. Mapping of disease loci in putative Mendelian forms of MCD has been hindered by marked locus heterogeneity, small kindred sizes and diagnostic classifications that may not reflect molecular pathogenesis. Here we demonstrate the use of whole-exome sequencing to overcome these obstacles by identifying recessive mutations in WDR62 as the cause of a wide spectrum of severe cerebral cortical malformations including microcephaly, pachygria with cortical thickening as well as hypoplasia of the corpus callosum. Some patients with WDR62 mutations had evidence of additional abnormalities including lissencephaly, schizencephaly, polymicrogyria and, in one instance, cerebellar hypoplasia, all traits traditionally regarded as distinct entities. In mouse and humans, WDR62 transcripts and protein are enriched in neural progenitors within the ventricular and subventricular zones. WDR62 expression in the neocortex is transient, spanning the period of embryonic neurogenesis. Unlike other known microcephaly genes, WDR62 does not apparently associate with centrosomes and is predominantly nuclear in localization. These findings unify previously disparate aspects of cerebral cortical development and highlight the utility of whole-exome sequencing to identify disease loci in settings in which traditional methods have proved challenging.

Somatic loss of wild-type alleles can produce disease traits such as neoplasia. Conversely, somatic loss of disease-causing mutations can revert phenotypes, however these events are infrequently observed. We demonstrate that ichthyosis with confetti, a severe, sporadic skin disease, is associated with thousands of revertant clones of normal skin that arise from loss of heterozygosity on chromosome 17q via mitotic recombination. This enabled mapping and identification of disease-causing mutations in keratin 10 (KRT10); all result in frameshifts into the same alternative reading frame, producing an arginine-rich C-terminal peptide that redirects keratin 10 from the cytokeratin filament network to the nucleolus. The general rarity of spontaneous reversion and the specific absence of reversion of other dominant mutations in KRT10 implicate the frameshift peptide in the appearance of revertants. These results may have ramifications for reversion of other mutations.

Summary

The neural crest is a multipotent, migratory cell population arising from the border of the neural and surface ectoderm. In mouse, the initial migratory neural crest cells occur at the five-somite stage. Bone morphogenetic proteins (BMPs), particularly BMP2 and BMP4, have been implicated as regulators of neural crest cell induction, maintenance, migration, differentiation and survival. Mouse has three known BMP2/4 type I receptors, of which Bmpr1a is expressed in the neural tube sufficiently early to be involved in neural crest development from the outset; however, earlier roles in other domains obscure its requirement in the neural crest. We have ablated Bmpr1a specifically in the neural crest, beginning at the five-somite stage. We find that most aspects of neural crest development occur normally; suggesting that BMPRIA is unnecessary for many aspects of early neural crest biology. However, mutant embryos display a shortened cardiac outflow tract with defective septation, a process known to require neural crest cells and to be essential for perinatal viability. Surprisingly, these embryos die in mid-gestation from acute heart failure, with reduced proliferation of ventricular myocardium. The myocardial defect may involve reduced BMP signaling in a novel, minor population of neural crest derivatives in the epicardium, a known source of ventricular myocardial proliferation signals. These results demonstrate that BMP2/4 signaling in mammalian neural crest derivatives is essential for outflow tract development and may regulate a crucial proliferation signal for the ventricular myocardium.

Here we characterize the consequences of elevated bone morphogenetic protein (BMP) signaling on neural tube morphogenesis by analyzing mice lacking the BMP antagonist, Noggin. Noggin is expressed dorsally in the closing neural folds and ventrally in the notochord and somites. All Noggin−/− pups are born with lumbar spina bifida; depending on genetic background, they may also have exencephaly. The exencephaly is due to a primary failure of neurulation, resulting from a lack of mid/hindbrain dorsolateral hinge point (DLHP) formation. Thus, as previously shown for Shh signaling at spinal levels, BMP activity may inhibit cranial DLHP morphogenesis. However, the increased BMP signaling observed in the Noggin−/− dorsal neural tube is not sufficient to cause exencephaly; it appears to also depend on the action of a genetic modifier, which may act to increase dorsal Shh signaling. The spinal neural tube defect results from a different mechanism: increased BMP signaling in the mesoderm between the limb buds leads to abnormal somite differentiation and axial skeletal malformation. The resulting lack of mechanical support for the neural tube causes spina bifida. We show that this defect is due to elevated BMP4 signaling. Thus, Noggin is required for mammalian neurulation in two contexts, dependent on position along the rostrocaudal axis.

Stroke is the world’s third leading cause of death. One cause of stroke, intracranial aneurysm, affects ~2% of the population and accounts for 500,000 hemorrhagic strokes annually in midlife (median age 50), most often resulting in death or severe neurological impairment1. The pathogenesis of intracranial aneurysm is unknown, and because catastrophic hemorrhage is commonly the first sign of disease, early identification is essential. We carried out a multistage genome-wide association study (GWAS) of Finnish, Dutch and Japanese cohorts including over 2,100 intracranial aneurysm cases and 8,000 controls. Genome-wide genotyping of the European cohorts and replication studies in the Japanese cohort identified common SNPs on chromosomes 2q, 8q and 9p that show significant association with intracranial aneurysm with odds ratios 1.24-1.36. The loci on 2q and 8q are new, whereas the 9p locus was previously found to be associated with arterial diseases, including intracranial aneurysm2-5. Associated SNPs on 8q likely act via SOX17, which is required for formation and maintenance of endothelial cells6-8, suggesting a role in development and repair of the vasculature; CDKN2A at 9p may have a similar role9. These findings have implications for the pathophysiology, diagnosis and therapy of intracranial aneurysm.

Point mutations in TBX1 can recapitulate many of the structural defects of 22q11 deletion syndromes (22q11DS), usually associated with a chromosomal deletion at 22q1.2. 22q11DS often includes specific cardiac and pharyngeal organ anomalies, but the presence of characteristic craniofacial defects is highly variable. Even among family members with a single TBX1 point mutation but no cytological deletion, cleft palate and low-set ears may or may not be present. In theory, such differences could depend on an unidentified, second-site lesion that modifies the craniofacial consequences of TBX1 deficiency. We present evidence for such a locus in a mouse model. Null mutations of chordin have been reported to cause severe defects recapitulating 22q11DS, which we show are highly dependent on genetic background. In an inbred strain in which chordin−/− is fully penetrant, we found a closely linked, strong modifier—a mutation in a Tbx1 intron causing severe splicing defects. Without it, lack of chordin results in a low penetrance of mandibular hypoplasia but no cardiac or thoracic organ malformations. This hypomorphic Tbx1 allele per se results in defects resembling 22q11DS but with a low penetrance of hallmark craniofacial malformations, unless chordin is mutant. Thus, chordin is a modifier for the craniofacial anomalies of Tbx1 mutations, demonstrating the existence of a second-site modifier for a specific subset of the phenotypes associated with 22q11DS.

Author Summary

A range of structural malformations is associated with 22q11 deletion syndrome (22q11DS), which is usually associated with microdeletions at chromosome 22q11.2. Variable defects in cardiovascular, pharyngeal, and craniofacial structures occur, but the basis for such variability is unknown. Mutations in TBX1, a gene within the region typically deleted, can recapitulate the structural anomalies of 22q11.2 deletions. However, even among family members with a single TBX1 point mutation, craniofacial defects are variable. In theory, such differences could depend on an unknown, second-site lesion that modifies the craniofacial consequences of TBX1 deficiency. We identify such a locus in mouse. In certain strains, lack of the chordin gene results in a phenotype resembling severe 22q11DS, also seen in mice lacking Tbx1. We find that the chordin phenotype depends on a closely linked, strong modifier—a cryptic, partial-function mutation in Tbx1. Without it, lack of chordin sometimes results in mandibular truncations but no cardiac or thoracic organ malformations. This novel Tbx1 allele per se results in defects resembling 22q11DS, but with a low frequency of hallmark craniofacial malformations, unless chordin is mutant. Thus chordin is a modifier for the craniofacial anomalies of Tbx1 mutations, demonstrating the existence of a second-site modifier for a specific subset of phenotypes associated with 22q11DS.