Regan Veith

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.

Altered TGFβ signaling and cardiovascular manifestations in patients with autosomal recessive cutis laxa type I caused by fibulin-4 deficiency

Fibulin-4 is a member of the fibulin family, a group of extracellular matrix proteins prominently expressed in medial layers of large veins and arteries. Involvement of the FBLN4 gene in cardiovascular pathology was shown in a murine model and in three patients affected with cutis laxa in association with systemic involvement. To elucidate the contribution of FBLN4 in human disease, we investigated two cohorts of patients. Direct sequencing of 17 patients with cutis laxa revealed no FBLN4 mutations. In a second group of 22 patients presenting with arterial tortuosity, stenosis and aneurysms, FBLN4 mutations were identified in three patients, two homozygous missense mutations (p.Glu126Lys and p.Ala397Thr) and compound heterozygosity for missense mutation p.Glu126Val and frameshift mutation c.577delC. Immunoblotting analysis showed a decreased amount of fibulin-4 protein in the fibroblast culture media of two patients, a finding sustained by diminished fibulin-4 in the extracellular matrix of the aortic wall on immunohistochemistry. pSmad2 and CTGF immunostaining of aortic and lung tissue revealed an increase in transforming growth factor (TGF)β signaling. This was confirmed by pSmad2 immunoblotting of fibroblast cultures. In conclusion, patients with recessive FBLN4 mutations are predominantly characterized by aortic aneurysms, arterial tortuosity and stenosis. This confirms the important role of fibulin-4 in vascular elastic fiber assembly. Furthermore, we provide the first evidence for the involvement of altered TGFβ signaling in the pathogenesis of FBLN4 mutations in humans.

Genomics in Clinical Practice: Lessons from the Front Lines

This Commentary explores the challenges in launching a medical genomics clinic for whole genome sequencing and analysis of patient samples. The price of whole-genome and -exome sequencing has fallen to the point where these methods can be applied to clinical medicine. Here, we outline the lessons we have learned in converting a sequencing laboratory designed for research into a fully functional clinical program.

Characterization of a New DGKE Intronic Mutation in Genetically Unsolved Cases of Familial Atypical Hemolytic Uremic Syndrome

BACKGROUND AND OBJECTIVES Genetic and acquired abnormalities causing dysregulation of the complement alternative pathway contribute to atypical hemolytic uremic syndrome (aHUS), a rare disorder characterized by thrombocytopenia, nonimmune microangiopathic hemolytic anemia, and acute kidney failure. However, in a substantial proportion of patients the disease-associated alterations are still unknown. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Whole-exome and whole-genome sequencing were performed in two unrelated families with infantile recessive aHUS. Sequencing of cDNA from affected individuals was used to test for the presence of aberrant mRNA species. Expression of mutant diacylglycerol kinase epsilon (DGKE) protein was evaluated with western blotting. RESULTS Whole-exome sequencing analysis with conventional variant filtering parameters did not reveal any obvious candidate mutation in the first family. The report of aHUS-associated mutations in DGKE, encoding DGKE, led to re-examination of the noncoding DGKE variants obtained from next-generation sequencing, allowing identification of a novel intronic DGKE mutation (c.888+40A>G) that segregated with disease. Sequencing of cDNA from affected individuals revealed aberrant forms of DGKE mRNA predicted to cause profound abnormalities in the protein catalytic site. By whole-genome sequencing, the same mutation was found in compound heterozygosity with a second nonsense DGKE mutation in all affected siblings of another unrelated family. Homozygous and compound heterozygous patients presented similar clinical features, including aHUS presentation in the first year of life, multiple relapsing episodes, and proteinuria, which are prototypical of DGKE-associated aHUS. CONCLUSIONS This is the first report of a mutation located beyond the exon-intron boundaries in aHUS. Intronic mutations such as these are underreported because conventional filtering parameters used to process next-generation sequencing data routinely exclude these regions from downstream analyses in both research and clinical settings. The results suggest that analysis of noncoding regions of aHUS-associated genes coupled with mRNA sequencing might provide a tool to explain genetically unsolved aHUS cases.

Views of primary care providers regarding the return of genome sequencing incidental findings

Sequencing of the entire exome or genome is increasingly used in clinical practice. Debate continues, however, regarding which incidental findings (IFs) should be returned and who should be involved in those decisions. Previous empirical research regarding stakeholder attitudes to the return of IFs has primarily involved genetics professionals; non‐genetics health professionals have not been widely surveyed. Given this, a survey regarding return of IFs was administered at the Best Practices in Pediatrics Conference following an educational presentation on genetics terminology and genetic condition examples. A total of 258 participants completed the survey. Of particular note, respondents who were positively disposed to sequencing did not always report wanting to learn about IFs, even if actionable. This is noteworthy given recent American College of Medical Genetics and Genomics guidelines recommending particular actionable IF be returned ‘without reference to patient preference’. This studys findings are important because they provide insight regarding the attitudes to the return of genome sequencing results for an important professional group, primary care providers. Ultimately, as likely gatekeepers to referrals for this technology, their opinions about the test will be key to its successful deployment.

Successful Application of Whole Genome Sequencing in a Medical Genetics Clinic

A pilot program was initiated using whole genome sequencing (WGS) to diagnose suspected genetic disorders in the Genetics Clinic at Childrens Hospital of Wisconsin. Twenty-two patients underwent WGS between 2010 and 2013. Initially, we obtained a 14% (3/22) diagnosis rate over 2 years; with subsequent reanalysis, this increased to 36% (8/22). Disease causing variants were identified in SKIV2L, CECR1, DGKE, PYCR2, RYR1, PDGFRB, EFTUD2, and BCS1L. In 75% (6/8) of diagnosed cases, the diagnosis affected treatment and/or medical surveillance. Additionally, one case demonstrated a homozygous A18V variant in VLDLR that appears to be associated with a previously undescribed phenotype.

Clinical diagnostic whole genome sequencing in a paediatric population: experience from our WGS genetics clinic

Background We are in an unprecedented era where the genome of an individual can be sequenced in a day for low cost in a single sequencing run. The availability this information is changing and challenging the practice of medicine. At The Medical College of Wisconsin (MCW) we have been running a whole genome sequencing (WGS) based genetics clinic for over a year. WGS together with the processes and tools developed for analysis and interpretation are being used to identify disease-causing mutations in rare pediatric disorders in children being treated at our affiliated Children’s Hospital of Wisconsin.

Pancreatic insufficiency in Toriello-Carey syndrome: report of a second patient.

Toriello–Carey syndrome is characterized by multiple congenital anomalies. Pancreatic insufficiency is suspected when patients present with poor weight gain, diarrhea, or maldigestion. The diagnosis is confirmed by low stool elastase and pancreatic stimulation testing. To our knowledge, only one patient with Toriello–Carey syndrome has been reported to have pancreatic insufficiency. We report on a second patient with Toriello–Carey syndrome and pancreatic insufficiency, and describe the management of pancreatic insufficiency in patients with this syndrome.

Hypertrophic Cardiomyopathy: A New Mutation Illustrates the Need for Family-Centered Care

This is a case series of a family positive for a previously undescribed mutation in the myofilament gene MYH7, causing hypertrophic cardiomyopathy (HCM), a potentially lethal cardiac disease with strong hereditability. The family’s significant disease became strikingly apparent with the unanticipated diagnosis of their newborn infant shortly after her birth. This led to the discovery of the MYH7 mutation in the infant, as well as her father and two siblings, all of whom had varying degrees of disease severity. Despite prior diagnosis of HCM for the paternal grandmother and great uncles, this family’s situation points to the need for continued education of healthcare providers, when heritable diseases are encountered. Genetics consult should occur early and has been shown to be helpful in making an accurate diagnosis and identifying relatives at risk of developing the condition. It may, as in this case series, lead to the discovery of a novel mutation and contribute to the growing genetic database for familial HCM.