Alan N. Mayer

Making a definitive diagnosis: Successful clinical application of whole exome sequencing in a child with intractable inflammatory bowel disease

Purpose: We report a male child who presented at 15 months with perianal abscesses and proctitis, progressing to transmural pancolitis with colocutaneous fistulae, consistent with a Crohn disease-like illness. The age and severity of the presentation suggested an underlying immune defect; however, despite comprehensive clinical evaluation, we were unable to arrive at a definitive diagnosis, thereby restricting clinical management.Methods: We sought to identify the causative mutation(s) through exome sequencing to provide the necessary additional information required for clinical management.Results: After sequencing, we identified 16,124 variants. Subsequent analysis identified a novel, hemizygous missense mutation in the X-linked inhibitor of apoptosis gene, substituting a tyrosine for a highly conserved and functionally important cysteine. X-linked inhibitor of apoptosis was not previously associated with Crohn disease but has a central role in the proinflammatory response and bacterial sensing through the NOD signaling pathway. The mutation was confirmed by Sanger sequencing in a licensed clinical laboratory. Functional assays demonstrated an increased susceptibility to activation-induced cell death and defective responsiveness to NOD2 ligands, consistent with loss of normal X-linked inhibitor of apoptosis protein function in apoptosis and NOD2 signaling.Conclusions: Based on this medical history, genetic and functional data, the child was diagnosed as having an X-linked inhibitor of apoptosis deficiency. Based on this finding, an allogeneic hematopoietic progenitor cell transplant was performed to prevent the development of life-threatening hemophagocytic lymphohistiocytosis, in concordance with the recommended treatment for X-linked inhibitor of apoptosis deficiency. At >42 days posttransplant, the child was able to eat and drink, and there has been no recurrence of gastrointestinal disease, suggesting this mutation also drove the gastrointestinal disease. This report describes the identification of a novel cause of inflammatory bowel disease. Equally importantly, it demonstrates the power of exome sequencing to render a molecular diagnosis in an individual patient in the setting of a novel disease, after all standard diagnoses were exhausted, and illustrates how this technology can be used in a clinical setting.

A timely arrival for genomic medicine

In this issue of GIM,1 we describe a young boy who presented with unusually aggressive inflammatory bowel disease refractory to medical and surgical treatment. To reach a diagnosis, we were compelled to use genomic technology that, at the time, was not a clinically validated test. This case stimulated many discussions within our group and institution on the boundary between research and clinical care. Many of the same issues were raised again during review of the manuscript, further shaping our thinking about how this case speaks to the broader issue of genomics in medical practice. The purpose of this commentary is to expound on these issues, hopefully to stimulate discussion within the wider medical community. CLINICAL COURSE AND THE DECISION TO SEQUENCE Our article1 provides an abbreviated history of this patient’s unusual clinical course. In this limited space, it is difficult to convey the profound disability and suffering the child endured through numerous long hospital stays, and the resulting frustration that we experienced as we struggled to control the disease. Over a 3-year period, there were more than 100 surgical procedures, clinical consultations with physicians from around the world, innumerable informal discussions, weekly clinical care meetings, and informal e-mail consultations with world-leading experts. Despite these measures, we enjoyed little strategic success. Allogeneic hematopoietic progenitor cell transplant was regularly brought up as a potential therapy, but two main barriers prevented us from moving forward. First, for the majority of the clinical course, the child was judged to be too ill to have a reasonable chance of surviving the first 100 days of the transplant process. Second, we lacked a firm diagnosis; hence, we could not predict whether bone marrow transplant would be likely to help. However, the risks of morbidity and mortality were high. Although the disease could be intermittently brought into remission, it was felt that eventually the child would succumb to drug toxicity, total parenteral nutrition liver disease, or recurrence. Thus, from a therapeutic standpoint, we were left with no viable long-term options. The disease shared some similarities with Crohn disease, but the severity and tempo of disease progression was highly atypical. Exhaustive efforts to reach a diagnosis revealed numerous abnormalities in this child’s immune system, but none of these were pathognomonic for a specific disease. Similarly, conventional genetic testing of numerous candidate genes had failed to reach an answer. Therefore, we decided the next logical step was to sequence the patient’s exome (all known exons within the patient’s genome). FROM DATA TO DIAGNOSIS Initially, we formulated the following hypothesis: the disease was likely to be a single gene disorder with a recessive mode of inheritance. As we were looking for a recessive disease with a population frequency of 1:10,000, we could exclude genetic variants found in more than 1% of the general population as being causal of the child’s disease. Initial analysis was limited to a set of 2006 target genes to reduce the risk of discovering off target information. After it was clear that none of the candidate genes harbored a pathogenic mutation, we broadened the analysis to include all known genes, eventually leading to the identification of a mutation in the XIAP gene. Because XIAP deficiency was not previously known to cause a severe Crohn-like phenotype, we then confirmed the loss of XIAP protein function in the patient’s cells. Having diagnosed the patient with XIAP deficiency, we needed to reorient the clinical approach to address the attendant risks of hemophagocytic lymphohistiocytosis (HLH), regardless of its role in the etiology of the patients inflammatory bowel disease. 2 Accordingly, we evaluated for Epstein-Barr Virus infection (negative to date) and considered approaches to chemoprophylaxis. We reviewed the intestinal pathology and bone marrow specimens, performed a liver biopsy, and established that there was no evidence of active HLH. Nevertheless, the data regarding the natural history of XLP2 suggest that this child had a high probability of death due to HLH in the future, an outcome that could be prevented by hematopoietic stem cell transplant. Therefore, this was the singular basis for the decision to perform a transplant. Furthermore, the link between XIAP and a loss of NOD2 signaling, a pathway implicated in Crohn disease, gave us hope that a transplant could improve the gastrointestinal condition as well.

PhotoMorphs: a novel light-activated reagent for controlling gene expression in zebrafish.

Manipulating gene expression in zebrafish is critical for exploiting the full potential of this vertebrate model organism. Morpholino oligos are the most commonly used antisense technology for knocking down gene expression. However, morpholinos suffer from a lack of control over the timing and location of knockdown. In this report, we describe a novel light‐ activatable knockdown reagent called PhotoMorph™. PhotoMorphs can be generated from existing morpholinos by hybridization with a complementary caging strand containing a photocleavable linkage. The caging strand neutralizes the morpholino activity until irradiation of the PhotoMorph with UV light releases the morpholino. We generated PhotoMorphs to target genes encoding enhanced green fluorescent protein, No tail, and E‐cadherin to illustrate the utility of this approach. Temporal control of gene expression with PhotoMorphs permitted us to circumvent the early lethal phenotype of E‐cadherin knockdown. A splice‐blocking PhotoMorph directed to the rheb gene showed light‐dependent gene knockdown up to 72 hpf. PhotoMorphs thus offer a new class of laboratory reagents suitable for the spatiotemporal control of gene expression in the zebrafish. genesis 47:736–743, 2009.

RBM19 is essential for preimplantation development in the mouse

BackgroundRNA-binding motif protein 19 (RBM19, NCBI Accession # NP_083038) is a conserved nucleolar protein containing 6 conserved RNA recognition motifs. Its biochemical function is to process rRNA for ribosome biogenesis, and it has been shown to play a role in digestive organ development in zebrafish. Here we analyzed the role of RBM19 during mouse embryonic development by generating mice containing a mutation in the Rbm19 locus via gene-trap insertion.ResultsHomozygous mutant embryos failed to develop beyond the morula stage, showing defective nucleologenesis, activation of apoptosis, and upregulation of P53 target genes. A unique feature of RBM19 is its localization to the cytoplasm in morula stage-embryos, whereas most other nucleolar proteins are localized to the nucleolar precursor body (NPB). The nucleoli in the Rbm19 mutant embryos remain immature, yet they can carry out rRNA synthesis. The timing of developmental arrest occurs after expression of the inner cell mass markers OCT3/4 and NANOG, but prior to the specification of trophectoderm as reflected by CDX2 expression.ConclusionThe data indicate that RBM19 is essential for preimplantation development, highlighting the importance of de novo nucleologenesis during this critical developmental stage.

A whole-animal microplate assay for metabolic rate using zebrafish.

Regulation of whole-body metabolism and energy homeostasis has been shown to require signaling between multiple organs. To identify genetic programs that determine metabolic rate, and compounds that can modify it, a whole-animal assay amenable to large-scale screening was developed. The direct correlation of acid production with metabolic rate was exploited to use a noninvasive colorimetric assay for acid secretion by individual zebrafish larvae in a 96-well plate format. A 3-fold increase in metabolic rate was detected that accompanied development between 24 and 96 h postfertilization. Dynamic changes in metabolic rate were also detected in response to different conditions such as temperature and drug treatments, in general agreement with the rate of oxygen consumption measured concomitantly. This assay was used to measure metabolic rate in the progeny of fish known to carry a recessive mutation in a gene required for ribosome biogenesis ( npofW07-g ), which would be expected to reduce energy consumption. A strong correlation was found (p < 10—6 ) between reduced metabolic rate and genotype even before the developmental defect was visually evident. These studies support the conclusion that whole-animal acid secretion can be used as a readout for energy metabolism, thus enabling large-scale screening for genetic and chemical regulators of metabolic rate in a vertebrate. (Journal of Biomolecular Screening 2008;960-967)

Dynamic Lkb1-TORC1 signaling as a possible mechanism for regulating the endoderm-intestine transition

The intestinal epithelium arises from undifferentiated endoderm via a developmental program known as the endoderm‐intestine transition (EIT). Previously we found that the target of rapamycin complex 1 (TORC1) regulates intestinal growth and differentiation during the EIT in zebrafish. Here we address a possible role for the tumor‐suppressor kinase Lkb1 in regulating TORC1 in this context. We find that TORC1 activity is transiently upregulated during the EIT in both zebrafish and mouse. Concomitantly, Lkb1 becomes transiently localized to the nucleus, suggesting that these two phenomena may be linked. Morpholino‐mediated knockdown of lkb1 stimulated intestinal growth via upregulation of TORC1, and also induced precocious intestine‐specific gene expression in the zebrafish gut epithelium. Knockdown of tsc2, which acts downstream of lkb1, likewise induced early expression of intestine‐specific genes. These data suggest that programmed localization of Lkb1 could represent a novel mechanism for regulating the EIT during intestinal development in vertebrates. Developmental Dynamics 239:3000–3012, 2010.

Zebrafish Offers New Perspective on Developmental Role of TOR Signaling

Genetic studies on the molecular basis of growth control have converged on the target of rapamycin (TOR) pathway as a key regulator. When stimulated by nutrients (i.e. amino acids) or growth factors (i.e. insulin), TOR activates protein synthesis and other anabolic pathways to promote cell growth. Our knowledge of TORs function in vivo is still rudimentary, particularly in the setting of vertebrate development. An important question is whether TOR functions as a constitutive regulator of growth in all cell types, or as a stage- and organ-specific regulator. Recently we employed the zebrafish as a vertebrate model system to study the developmental role of TOR signaling. We found that TOR signaling was required for a discrete step prior to epithelial differentiation. The results support the view that different organs may be reliant on TOR activity to differing degrees. In the case of the zebrafish, the digestive tract exhibits the greatest sensitivity to rapamycin, which may reflect its reliance on TOR signaling for normal growth. We suggest the hypothesis that TOR signaling may regulate the size of the intestines absorptive surface area in response to systemic nutrient demand.