We describe a child of Middle Eastern descent by first-cousin mating with idiopathic neurogenic bladder and high grade vesicoureteral reflux at 1 year of age, whose characteristic facial grimace led to the diagnosis of Ochoa (Urofacial) syndrome at age 5 years. We used homozygosity mapping, exome capture and paired end sequencing to identify the disease causing mutation in the proband. We reviewed the literature with respect to the urologic manifestations of Ochoa syndrome. A large region of marker homozygosity was observed at 10q24, consistent with known autosomal recessive inheritance, family consanguinity and previous genetic mapping in other families with Ochoa syndrome. A homozygous mutation was identified in the proband in HPSE2: c.1374_1378delTGTGC, a deletion of 5 nucleotides in exon 10 that is predicted to lead to a frameshift followed by replacement of 132 C-terminal amino acids with 153 novel amino acids (p.Ala458Alafsdel132ins153). This mutation is novel relative to very recently published mutations in HPSE2 in other families. Early intervention and recognition of Ochoa syndrome with control of risk factors and close surveillance will decrease complications and renal failure.

The N-terminal region of human HOXA13 has seven discrete polyalanine tracts. Our previous analysis of these tracts in multiple major vertebrate clades suggested that three are mammal-specific. We now report the N-terminal HOXA13 repetitive tract structures in the monotreme Tachyglossus aculeatus (echidna). Contrary to our expectations, echidna HOXA13 possesses a unique set of polyalanine tracts and an unprecedented polyglycine tract. The data support the conclusion that the emergence of expanded polyalanine tracts in proteins occurred very early in the stem lineage that gave rise to mammals, between 162 and 315 MYA.

We previously demonstrated that a ~1 Mb domain of genes upstream of and including Hoxa13 is co-expressed in the developing mouse limbs and genitalia. A very highly conserved non-coding sequence, mmA13CNS, located ~350 kb upstream of the Hoxa13 gene, was shown to be insufficient in transgenic mice to direct precise Hoxa13-like expression in the limb buds or genital bud, although some LacZ expression from the transgene was found in those tissues. In this report, we used overlapping β-globin minimal promoter LacZ recombinant BAC mouse transgenes encompassing mmA13CNS to localize genital bud and distal limb enhancers. Hoxa13-like embryonic genital bud expression was observed with both BACs, suggesting that a genital bud enhancer lies within the region of BAC overlap. In contrast, at least two separate regions of sequence remote to the HoxA cluster are required to drive Hoxa13-like expression in developing distal limbs. Given that the paralogous posterior HoxD and neighboring genes have been shown to be under the influence of long-range distal limb and genital bud enhancers, we hypothesize that both HoxA and HoxD long-range enhancers have one ancestral origin, which diverged in both sequence and function after the HoxA/D cluster duplication.

BMP4 loss-of-function mutations and deletions have been shown to be associated with ocular, digital, and brain anomalies, but due to the paucity of these reports, the full phenotypic spectrum of human BMP4 mutations is not clear. We screened 133 patients with a variety of ocular disorders for BMP4 coding region mutations or genomic deletions. BMP4 deletions were detected in two patients: a patient affected with SHORT syndrome and a patient with anterior segment anomalies along with craniofacial dysmorphism and cognitive impairment. In addition to this, three intragenic BMP4 mutations were identified. A patient with anophthalmia, microphthalmia with sclerocornea, right-sided diaphragmatic hernia, and hydrocephalus was found to have a c.592C>T (p.R198X) nonsense mutation in BMP4. A frameshift mutation, c.171dupC (p.E58RfsX17), was identified in two half-siblings with anophthalmia/microphthalmia, discordant developmental delay/postaxial polydactyly, and poor growth as well as their unaffected mother; one affected sibling carried an additional BMP4 mutation in the second allele, c.362A>G (p.H121R). This is the first report indicating a role for BMP4 in SHORT syndrome, Axenfeld–Rieger malformation, growth delay, macrocephaly, and diaphragmatic hernia. These results significantly expand the number of reported loss-of-function mutations, further support the critical role of BMP4 in ocular development, and provide additional evidence of variable expression/non-penetrance of BMP4 mutations.

HOX proteins are important transcriptional regulators in mammalian embryonic development and are dysregulated in human cancers. However, there are few known direct HOX target genes and their mechanisms of regulation are incompletely understood. To isolate and characterize gene segments through which HOX proteins regulate transcription we used cesium chloride centrifugation-based chromatin purification and immunoprecipitation (ChIP). From NIH 3T3-derived HOXA13-FLAG expressing cells, 33% of randomly selected, ChIP clones were reproducibly enriched. Hox-enriched fragments (HEFs) were more AT-rich compared with cloned fragments that failed reproducible ChIP. All HEFs augmented transcription of a heterologous promoter upon coexpression with HOXA13. One HEF was from intron 2 of Enpp2, a gene highly upregulated in these cells and has been implicated in cell motility. Using Enpp2 as a candidate direct target, we identified three additional HEFs upstream of the transcription start site. HOXA13 upregulated transcription from an Enpp2 promoter construct containing these sites, and each site was necessary for full HOXA13-induced expression. Lastly, given that HOX proteins have been demonstrated to interact with histone deacetylases and/or CBP, we explored whether histone acetylation changed at Enpp2 upon HOXA13-induced activation. No change in the general histone acetylation state was observed. Our results support models in which occupation of multiple HOX binding sites is associated with highly activated genes.

Interactions with co-factors provide a means by which HOX proteins exert specificity. To identify candidate protein interactors of HOXA13, we created and screened an E11.5–E12.5, distal limb bud yeast two-hybrid prey library. Among the interactors, we isolated the BMP-signaling effector Smad5, which interacted with the paralogous HOXD13 but not with HOXA11 or HOXA9, revealing unique interaction capabilities of the AbdB-like HOX proteins. Using deletion mutants, we determined that the MH2 domain of Smad5 is necessary for HOXA13 interaction. This is the first report demonstrating an interaction between HOX proteins and the MH2 domain of Smad proteins. HOXA13 and HOXD13 also bind to other BMP and TGF-β/Activin-regulated Smad proteins including Smad1 and Smad2, but not Smad4. Furthermore, HOXD13 could be co-immunoprecipitated with Smad1 from cells. Expression of HOXA13, HOXD13 or a HOXD13 homeodomain mutant (HOXD13IQN>AAA) antagonized TGF-β-stimulated transcriptional activation of the pAdtrack-3TP-Lux reporter vector in Mv1Lu cells as well as the Smad3/Smad4-activated pTRS6-E1b promoter in Hep3B cells. Finally, using mammalian one-hybrid assay, we show that transcriptional activation by a GAL4/Smad3-C-terminus fusion protein is specifically inhibited by HOXA13. Our results identify a new co-factor for HOX group 13 proteins and suggest that HOX proteins may modulate Smad-mediated transcriptional activity through protein–protein interactions without the requirement for HOX monomeric DNA-binding capability.

Serial Analysis of Gene Expression (SAGE) is becoming a widely used gene expression profiling method for the study of development, cancer and other human diseases. Investigators using SAGE rely heavily on the quantitative aspect of this method for cataloging gene expression and comparing multiple SAGE libraries. We have developed additional computational and statistical tools to assess the quality and reproducibility of a SAGE library. Using these methods, a critical variable in the SAGE protocol was identified that has the potential to bias the Tag distribution relative to the GC content of the 10 bp SAGE Tag DNA sequence. We also detected this bias in a number of publicly available SAGE libraries. It is important to note that the GC content bias went undetected by quality control procedures in the current SAGE protocol and was only identified with the use of these statistical analyses on as few as 750 SAGE Tags. In addition to keeping any solution of free DiTags on ice, an analysis of the GC content should be performed before sequencing large numbers of SAGE Tags to be confident that SAGE libraries are free from experimental bias.

Individual cancers harbor a set of genetic aberrations that can be informative for identifying rational therapies currently available or in clinical trials. We implemented a pilot study to explore the practical challenges of applying high-throughput sequencing in clinical oncology. We enrolled patients with advanced or refractory cancer who were eligible for clinical trials. For each patient, we performed whole-genome sequencing of the tumor, targeted whole-exome sequencing of tumor and normal DNA, and transcriptome sequencing (RNA-Seq) of the tumor to identify potentially informative mutations in a clinically relevant time frame of 3 to 4 weeks. With this approach, we detected several classes of cancer mutations including structural rearrangements, copy number alterations, point mutations, and gene expression alterations. A multidisciplinary Sequencing Tumor Board (STB) deliberated on the clinical interpretation of the sequencing results obtained. We tested our sequencing strategy on human prostate cancer xenografts. Next, we enrolled two patients into the clinical protocol and were able to review the results at our STB within 24 days of biopsy. The first patient had metastatic colorectal cancer in which we identified somatic point mutations in NRAS, TP53, AURKA, FAS, and MYH11, plus amplification and overexpression of cyclin-dependent kinase 8 (CDK8). The second patient had malignant melanoma, in which we identified a somatic point mutation in HRAS and a structural rearrangement affecting CDKN2C. The STB identified the CDK8 amplification and Ras mutation as providing a rationale for clinical trials with CDK inhibitors or MEK (mitogenactivated or extracellular signal–regulated protein kinase kinase) and PI3K (phosphatidylinositol 3-kinase) inhibitors, respectively. Integrative high-throughput sequencing of patients with advanced cancer generates a comprehensive, individual mutational landscape to facilitate biomarker-driven clinical trials in oncology.

Chromosome region 1q21.1 contains extensive and complex low-copy repeats, and copy number variants (CNVs) in this region have recently been reported in association with congenital heart defects1, developmental delay2,3, schizophrenia and related psychoses4,5. We describe 21 probands with the 1q21.1 microdeletion and 15 probands with the 1q21.1 microduplication. These CNVs were inherited in most of the cases in which parental studies were available. Consistent and statistically significant features of microcephaly and macrocephaly were found in individuals with micro-deletion and microduplication, respectively. Notably, a paralog of the HYDIN gene located on 16q22.2 and implicated in autosomal recessive hydrocephalus6 was inserted into the 1q21.1 region during the evolution of Homo sapiens7; we found this locus to be deleted or duplicated in the individuals we studied, making it a probable candidate for the head size abnormalities observed. We propose that recurrent reciprocal microdeletions and microduplications within 1q21.1 represent previously unknown genomic disorders characterized by abnormal head size along with a spectrum of developmental delay, neuropsychiatric abnormalities, dysmorphic features and congenital anomalies. These phenotypes are subject to incomplete penetrance and variable expressivity.