D. Hunter Best

EIF2AK4 Mutations in Pulmonary Capillary Hemangiomatosis

BACKGROUND Pulmonary capillary hemangiomatosis (PCH) is a rare disease of capillary proliferation of unknown cause and with a high mortality. Families with multiple affected individuals with PCH suggest a heritable cause although the genetic etiology remains unknown. METHODS We used exome sequencing to identify a candidate gene for PCH in a family with two affected brothers. We then screened 11 unrelated patients with familial (n = 1) or sporadic (n = 10) PCH for mutations. RESULTS Using exome sequencing, we identified compound mutations in eukaryotic translation initiation factor 2 α kinase 4 (EIF2AK4) (formerly known as GCN2) in both affected brothers. Both parents and an unaffected sister were heterozygous carriers. In addition, we identified two EIF2AK4 mutations in each of two of 10 unrelated individuals with sporadic PCH. EIF2AK4 belongs to a family of kinases that regulate angiogenesis in response to cellular stress. CONCLUSIONS Mutations in EIF2AK4 are likely to cause autosomal-recessive PCH in familial and some nonfamilial cases.

Pulmonary Arterial Hypertension: A Current Perspective on Established and Emerging Molecular Genetic Defects

Pulmonary arterial hypertension (PAH) is an often fatal disorder resulting from several causes including heterogeneous genetic defects. While mutations in the bone morphogenetic protein receptor type II (BMPR2) gene are the single most common causal factor for hereditary cases, pathogenic mutations have been observed in approximately 25% of idiopathic PAH patients without a prior family history of disease. Additional defects of the transforming growth factor beta pathway have been implicated in disease pathogenesis. Specifically, studies have confirmed activin A receptor type II‐like 1 (ACVRL1), endoglin (ENG), and members of the SMAD family as contributing to PAH both with and without associated clinical phenotypes. Most recently, next‐generation sequencing has identified novel, rare genetic variation implicated in the PAH disease spectrum. Of importance, several identified genetic factors converge on related pathways and provide significant insight into the development, maintenance, and pathogenetic transformation of the pulmonary vascular bed. Together, these analyses represent the largest comprehensive compilation of BMPR2 and associated genetic risk factors for PAH, comprising known and novel variation. Additionally, with the inclusion of an allelic series of locus‐specific variation in BMPR2, these data provide a key resource in data interpretation and development of contemporary therapeutic and diagnostic tools.

Mosaic ACVRL1 and ENG mutations in hereditary haemorrhagic telangiectasia patients

Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant disorder caused by mutations in the ACVRL1, ENG, and SMAD4 genes. HHT is commonly characterised by small arteriovenous malformations (AVMs) known as telangiectasias of the skin, oral or gastrointestinal mucosa, as well as larger AVMs of solid organs (lungs, liver, brain). However, the manifestations of HHT are extremely variable. Two patients with no family history of HHT and strikingly different clinical presentations, who are mosaic for mutations in the ACVRL1 or ENG gene, are reported here. These cases represent the first report of mosaicism in patients clinically affected with classical HHT and pulmonary arterial hypertension, and suggest the need for awareness of mosaicism when performing clinical testing for this disorder.

Molecular Pathology Methods

Molecular pathology is based on the principles, techniques, and tools of molecular biology as they are applied to diagnostic medicine in the clinical laboratory. These tools were developed in the research setting and perfected throughout the second half of the 20th century, long before the Human Genome Project was conceived. Molecular biology methods were used to elucidate the genetic and molecular basis of many diseases, and these discoveries ultimately led to the field of molecular diagnostics. Eventually the insights these tools provided for laboratory medicine were so valuable to the armamentarium of the pathologist that they were incorporated into pathology practice. Today, molecular diagnostics continues to grow rapidly as in vitro diagnostic companies develop new kits for the marketplace and as the insights into disease gained by the progress of the Human Genome Project develop into laboratory tests.

EIF2AK4 Mutations in Patients Diagnosed With Pulmonary Arterial Hypertension

Background Differentiating pulmonary venoocclusive disease (PVOD) and pulmonary capillary hemangiomatosis (PCH) from idiopathic pulmonary arterial hypertension (IPAH) or heritable pulmonary arterial hypertension (HPAH) is important clinically. Mutations in eukaryotic translation initiation factor 2 alpha kinase 4 (EIF2AK4) cause heritable PVOD and PCH, whereas mutations in other genes cause HPAH. The aim of this study was to describe the frequency of pathogenic EIF2AK4 mutations in patients diagnosed clinically with IPAH or HPAH. Methods Sanger sequencing and deletion/duplication analysis were performed to detect mutations in the bone morphogenetic protein receptor type II (BMPR2) gene in 81 patients diagnosed at 30 North American medical centers with IPAH (n = 72) or HPAH (n = 9). BMPR2 mutation‐negative patients (n = 67) were sequenced for mutations in four other genes (ACVRL1, ENG, CAV1, and KCNK3) known to cause HPAH. Patients negative for mutations in all known PAH genes (n = 66) were then sequenced for mutations in EIF2AK4. We assessed the pathogenicity of EIF2AK4 mutations and reviewed clinical characteristics of patients with pathogenic EIF2AK4 mutations. Results Pathogenic BMPR2 mutations were identified in 8 of 72 (11.1%) patients with IPAH and 6 of 9 (66.7%) patients with HPAH. A novel homozygous EIF2AK4 mutation (c.257+4A>C) was identified in 1 of 9 (11.1%) patients diagnosed with HPAH. The novel EIF2AK4 mutation (c.257+4A>C) was homozygous in two sisters with severe pulmonary hypertension. None of the 72 patients with IPAH had biallelic EIF2AK4 mutations. Conclusions Pathogenic biallelic EIF2AK4 mutations are rarely identified in patients diagnosed with HPAH. Identification of pathogenic biallelic EIF2AK4 mutations can aid clinicians in differentiating HPAH from heritable PVOD or PCH.

When to Offer Genetic Testing for Pulmonary Arterial Hypertension

Genetic testing is poised to play a greater role in the diagnosis and management of pulmonary arterial hypertension (PAH). Physicians who manage PAH should know the heritable PAH phenotypes, inheritance patterns, and responsible genes. They also should know indications, potential risks and benefits, and the issues surrounding genetic counselling and testing for patients with PAH.

Genomic Applications in Inherited Genetic Disorders

Next-generation sequencing (NGS) technology is revolutionizing clinical diagnostics for inherited disorders. The paradigm shift driven by NGS is rooted in its fundamental advances over Sanger sequencing, primarily its ultrahigh throughput and significantly lower cost per base of sequence. These enhanced capabilities have enabled clinical laboratories to go from testing single genes to simultaneously sequencing tens, hundreds, and thousands of genes, even the whole human genome. This dramatic leap in capabilities has come with dramatic success stories, but is also accompanied by numerous new challenges that will face the field for years to come. While clinical application of NGS is still a nascent field, it has been quickly adopted by academic and commercial laboratories which are expanding the applications of this technology at a rapid pace. Indeed, it is clear that NGS based genetic tests are applicable to inherited disorders in all stages of life, from preconception screening to diagnosis of fetal, infant, young child, and adult onset disorders. This chapter presents a brief description of NGS panels, exome and whole-genome tests for germ-line variations and discusses their current clinical applications.

Cystic fibrosis testing in a referral laboratory: results and lessons from a six-year period

BackgroundThe recent introduction of high throughput sequencing technologies into clinical genetics has made it practical to simultaneously sequence many genes. In contrast, previous technologies limited sequencing based tests to only a handful of genes. While the ability to more accurately diagnose inherited diseases is a great benefit it introduces specific challenges. Interpretation of missense mutations continues to be challenging and the number of variants of uncertain significance continues to grow.ResultsWe leveraged the data available at ARUP Laboratories, a major reference laboratory, for the CFTR gene to explore specific challenges related to variant interpretation, including a focus on understanding ethnic-specific variants and an evaluation of existing databases for clinical interpretation of variants. In this study we analyzed 555 patients representing eight different ethnic groups. We observed 184 different variants, most of which were ethnic group specific. Eighty-five percent of these variants were present in the Cystic Fibrosis Mutation Database, whereas the Human Mutation Database and dbSNP/1000 Genomes had far fewer of the observed variants. Finally, 21 of the variants were novel and we report these variants and their clinical classifications.ConclusionsBased on our analyses of data from six years of CFTR testing at ARUP Laboratories a more comprehensive, clinical grade database is needed for the accurate interpretation of observed variants. Furthermore, there is a particular need for more and better information regarding variants from individuals of non-Caucasian ethnicity.

Genetics and Pharmacogenomics in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is an uncommon disease in the general population, but a disease with significant morbidity and mortality. The prevalence of heritable PAH (HPAH) remains unknown. The reason for incomplete penetrance of HPAH is not well understood. A patients clinical response to disease-specific therapy is complex, involving the severity of the patients disease, other comorbidities, appropriateness of the prescribed therapy, and patient compliance. Warfarin is often used as an adjuvant therapy in patients with PAH.

Activation and regulation of reserve liver progenitor cells.

The mammalian liver possesses an extraordinary capacity for regeneration of tissue mass and cell numbers following loss of hepatocytes due to partial tissue loss (surgical resection) or hepatotoxic injury (necrosis). Restoration of liver mass can be obtained through the outgrowth and expansion of a number of different cell types depending upon the nature and extent of injury and/or tissue deficit. In an otherwise healthy liver, the replacement of hepatocytes (and tissue mass) is achieved through the proliferation of differentiated, normally quiescent hepatocytes contained in the residual (viable) tissue. However, in certain forms of liver injury, the capacity of differentiated hepatocytes to proliferate in response to liver tissue deficit is significantly impaired. When this occurs, reserve stem-like progenitor cells are activated to proliferate and replace lost hepatocytes. In this review, we will discuss the currently available information regarding the activation and outgrowth of each of these liver progenitor cell populations.