To understand the genetic heterogeneity underlying developmental delay, we compare copy-number variants (CNVs) in 15,767 children with intellectual disability and various congenital defects to 8,329 adult controls. We estimate that ~14.2% of disease in these individuals is due to large CNVs > 400 kbp. We find greater CNV enrichment in patients with craniofacial anomalies and cardiovascular defects than epilepsy or autism. We identify 59 pathogenic CNVs including 14 novel or previously weakly supported candidates. We refine the critical interval for several genomic disorders such as the 17q21.31 microdeletion syndrome and identify 940 candidate dosage-sensitive genes. We also develop methods to opportunistically discover small, disruptive CNVs within the large and growing diagnostic array datasets. This evolving CNV morbidity map combined with exome/genome sequencing will be critical for deciphering the genetic basis of developmental delay, intellectual disability, and autism spectrum disorders.

We report the identification of a recurrent 520-kbp 16p12.1 microdeletion significantly associated with childhood developmental delay. The microdeletion was detected in 20/11,873 cases vs. 2/8,540 controls (p=0.0009, OR=7.2) and replicated in a second series of 22/9,254 cases vs. 6/6,299 controls (p=0.028, OR=2.5). Most deletions were inherited with carrier parents likely to manifest neuropsychiatric phenotypes (p=0.037, OR=6). Probands were more likely to carry an additional large CNV when compared to matched controls (10/42 cases, p=5.7×10-5, OR=6.65). Clinical features of cases with two mutations were distinct from and/or more severe than clinical features of patients carrying only the co-occurring mutation. Our data suggest a two-hit model in which the 16p12.1 microdeletion both predisposes to neuropsychiatric phenotypes as a single event and exacerbates neurodevelopmental phenotypes in association with other large deletions or duplications. Analysis of other microdeletions with variable expressivity suggests that this two-hit model may be more generally applicable to neuropsychiatric disease.

Lymphedema-distichiasis (LD) is an autosomal dominant disorder that classically presents as lymphedema of the limbs, with variable age at onset, and double rows of eyelashes (distichiasis). Other complications may include cardiac defects, cleft palate, extradural cysts, and photophobia, suggesting a defect in a gene with pleiotrophic effects acting during development. We previously reported neonatal lymphedema, similar to that in Turner syndrome, associated with a t(Y;16)(q12;q24.3) translocation. A candidate gene was not found on the Y chromosome, and we directed our efforts toward the chromosome 16 breakpoint. Subsequently, a gene for LD was mapped, by linkage studies, to a 16-cM region at 16q24.3. By FISH, we determined that the translocation breakpoint was within this critical region and further narrowed the breakpoint to a 20-kb interval. Because the translocation did not appear to interrupt a gene, we considered candidate genes in the immediate region that might be inactivated by position effect. In two additional unrelated families with LD, we identified inactivating mutations-a nonsense mutation and a frameshift mutation-in the FOXC2 (MFH-1) gene. FOXC2 is a member of the forkhead/winged-helix family of transcription factors, whose members are involved in diverse developmental pathways. FOXC2 knockout mice display cardiovascular, craniofacial, and vertebral abnormalities similar to those seen in LD syndrome. Our findings show that FOXC2 haploinsufficiency results in LD. FOXC2 represents the second known gene to result in hereditary lymphedema, and LD is only the second hereditary disorder known to be caused by a mutation in a forkhead-family gene.

Nager syndrome, first described more than 60 years ago, is the archetype of a class of disorders called the acrofacial dysostoses, which are characterized by craniofacial and limb malformations. Despite intensive efforts, no gene for Nager syndrome has yet been identified. In an international collaboration, FORGE Canada and the National Institutes of Health Centers for Mendelian Genomics used exome sequencing as a discovery tool and found that mutations in SF3B4, a component of the U2 pre-mRNA spliceosomal complex, cause Nager syndrome. After Sanger sequencing of SF3B4 in a validation cohort, 20 of 35 (57%) families affected by Nager syndrome had 1 of 18 different mutations, nearly all of which were frameshifts. These results suggest that most cases of Nager syndrome are caused by haploinsufficiency of SF3B4. Our findings add Nager syndrome to a growing list of disorders caused by mutations in genes that encode major components of the spliceosome and also highlight the synergistic potential of international collaboration when exome sequencing is applied in the search for genes responsible for rare Mendelian phenotypes.

We conclude that FGD1 and Vav are regulators of the Rho GTPase family. Along with their target proteins Cdc42, Rac and Rho, FGD1 and Vav control essential signals required during embryonic development.

The Rho family of small GTP-binding proteins plays important roles in the regulation of actin cytoskeleton organization and cell growth. Activation of these GTPases involves the replacement of bound GDP with GTP, a process catalyzed by the Dbl-like guanine-nucleotide exchange factors, all of which seem to share a putative catalytic motif termed the Dbl homology (DH) domain, followed by a pleckstrin homology (PH) domain. Here we have examined the role of a Dbl-like molecule, the faciogenital dysplasia gene product (FGD1), which when mutated in its Dbl homology domain, cosegregates with the developmental disease Aarskog-Scott syndrome. We report that a polypeptide of FGD1 encompassing the DH and PH domains can bind specifically to the Rho family GTPase Cdc42Hs and stimulates the GDP-GTP exchange of the isoprenylated form of Cdc42Hs. Microinjection of this FGD1 polypeptide into Swiss 3T3 fibroblast cells induces the formation of peripheral actin microspikes, similar to that previously observed when cells were injected with a constitutively active form of Cdc42Hs. This effect of FGD1 on actin organization is readily inhibited by coinjection of a dominant-negative mutant of Cdc42Hs. Examination of NIH 3T3 cells expressing the FGD1 fragment revealed that similar to cells expressing Dbl, two independent elements downstream of Cdc42Hs, the Jun NH 2 -terminal kinase and the p70 S6 kinase, became activated. Hence, our results indicate that FGD1, through its DH and PH domains, acts as a Cdc42Hs-specific guanine-nucleotide exchange factor and suggest that the Cdc42Hs GTPase may have a role in mammalian development.The Rho family of small GTP-binding proteins have been implicated in multiple signaling pathways leading to cytoskeleton reorganization (1) and cell growth (2). RhoA has been shown to be involved in actin stress fiber and focal adhesion formation (3); Rac1 is required for membrane ruffling and lamellipodia formation induced by growth factors (4); and Cdc42Hs has been demonstrated to mediate the induction of peripheral actin microspikes (PAM) 1 and filopodia by bradykinin (5). In fibroblast cells, Cdc42Hs/Rac1/RhoA have been suggested to work in a hierarchical cascade mediating these changes in the actin cytoskeleton (6). In addition, Rho, Rac, and Cdc42Hs all seem to be involved in the transcriptional regulation by serum response factor (7) and in the regulation of cell cycle progression (8). Particularly, Cdc42Hs and Rac participate in the p21-activated kinase-mediated Jun N-terminal kinase (JNK) activation (9, 10) and, through an independent pathway, in the regulation of p70 S6 kinase (S6K) (11).The activation of Rho proteins requires the exchange of bound GDP for GTP (12), a process catalyzed by a growing family of guanine-nucleotide exchange factors (GEFs) for which the Dbl oncoprotein is a prototype (13). These putative GEFs share the structural arrangement of an ϳ200-amino acid motif, the Dbl homology (DH) domain, followed by a second putative signaling motif, the pleckstrin homology (PH) domain (13). The ...

We report the complete 5025-base sequence of the human 28S rRNA gene. Variability within the species has been demonstrated by sequencing a variable region from six separately cloned genes. This region is one of three large subunit rRNA regions that show extreme sequence and size variation among species. The interspecies differences suggest species-specific functions for these sections, while the intraspecies heterogeneity indicates differences among ribosomes.Comparison of the human gene with a partial sequence from the chimpanzee 28S gene yields divergence rates for the two species: 0.8% for conserved regions of the gene and 3.7% for a variable region. The rapid divergence rates of variable regions in the ribosomal gene may permit answers to the question of time of separation of closely related species.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder which affects approximately 1 in 3,300 males, making it the most common of the neuromuscular dystrophies. The biochemical basis of the disease is unknown and as yet no effective treatment is available. A small number of females are also affected with the disease, and these have been found to carry X; autosome translocations involving variable autosomal sites but always with a breakpoint within band Xp21 of the X chromosome (implicated by other kinds of genetic evidence as the site of the DMD lesion). In these female patients the normal X chromosome is preferentially inactivated, which it is assumed silences their one normal DMD gene, leading to expression of the disease. In one such affected female the autosomal breakpoint lies in the middle of the short arm of chromosome 21, within a cluster of ribosomal RNA genes. Here we have used rRNA sequences as probes to clone the region spanning the translocation breakpoint. A sequence derived from the X-chromosomal portion of the clone detects a restriction fragment length polymorphism (RFLP) which is closely linked to the DMD gene and uncovers chromosomal deletions in some male DMD patients.