Addressing ethical and laboratory challenges for initiation of a rapid whole genome sequencing program.

Rapid whole genome sequencing (rapid WGS) is a powerful diagnostic tool that is becoming increasingly practical for widespread clinical use. However, protocols for its use are challenging to implement. A significant obstacle to clinical adoption is that laboratory certification requires an initial research development phase, which is constrained by regulations from returning results. Regulations preventing return of results have ethical implications in cases which might impact patient outcomes. Here, we describe our experience with the development of a rapid WGS research protocol, that balanced the requirements for laboratory-validated test development with the ethical needs of clinically relevant return of results.

Effective variant filtering and expected candidate variant yield in studies of rare human disease.

In studies of families with rare disease, it is common to screen for de novo mutations, as well as recessive or dominant variants that explain the phenotype. However, the filtering strategies and software used to prioritize high-confidence variants vary from study to study. In an effort to establish recommendations for rare disease research, we explore effective guidelines for variant (SNP and INDEL) filtering and report the expected number of candidates for de novo dominant, recessive, and autosomal dominant modes of inheritance. We derived these guidelines using two large family-based cohorts that underwent whole-genome sequencing, as well as two family cohorts with whole-exome sequencing. The filters are applied to common attributes, including genotype-quality, sequencing depth, allele balance, and population allele frequency. The resulting guidelines yield ~10 candidate SNP and INDEL variants per exome, and 18 per genome for recessive and de novo dominant modes of inheritance, with substantially more candidates for autosomal dominant inheritance. For family-based, whole-genome sequencing studies, this number includes an average of three de novo, ten compound heterozygous, one autosomal recessive, four X-linked variants, and roughly 100 candidate variants following autosomal dominant inheritance. The slivar software we developed to establish and rapidly apply these filters to VCF files is available at https://github.com/brentp/slivar under an MIT license, and includes documentation and recommendations for best practices for rare disease analysis.

Genotype-Phenotype Correlations of Glucose-6-Phosphate-Deficient Variants Throughout an Activity Distribution.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked disorder that may manifest as neonatal jaundice or acute hemolytic anemia. Quantitative assessment of G6PD activity in erythrocytes is required to definitively diagnose a deficiency. Most males and homozygous females have low enzyme activities, whereas heterozygous females may have a range of activities. We sought to examine G6PD genotype-phenotype associations to identify an activity cutoff above which G6PD deficiency is unlikely. METHODS: Ninety-five residual samples were randomly selected to represent the various regions of a G6PD activity distribution. DNA was isolated from the leukocyte fraction and sequenced using the Sanger method. ROC curves were used to establish cutoffs. RESULTS: Thirteen variant alleles were identified, including 1 not previously reported. In the very deficient activity range, we found males and homozygous females of both class II and III variants. In the deficient category, we found predominantly class III males and heterozygous females. The presumed deficient category contained class III and IV variants and nonvariants. An activity cutoff of <7.85 U/g hemoglobin (Hb) was 100% sensitive and 94% specific for identifying a G6PD-deficient male, and a cutoff of <8.95 U/g Hb was 90% sensitive and 82% specific for a deficient female. CONCLUSIONS: The observed activity groupings were not because of a particular variant class. Cutoffs to identify the presence of a deficiency variant for males and females may be useful when trying to decide whether to recommend genetic analysis.

Pulmonary veno-occlusive disease: Two children with gradual disease progression.

Pulmonary veno-occlusive disease and pulmonary capillary hemangiomatosis are rare forms of pulmonary vascular disease. We report two cases of affected children who had evidence of pulmonary hypertension 3-5 years before developing radiographic findings of pulmonary veno-occlusive disease or pulmonary capillary hemangiomatosis. Both patients experienced a moderate decrease in pulmonary arterial pressure during acute vasodilator testing. Both patients experienced an improvement in six-minute walk performance without an increase in pulmonary edema when treated with targeted therapy for pulmonary hypertension. In some patients, pulmonary veno-occlusive disease and pulmonary capillary hemangiomatosis may progress slowly over a period of months to years. A favorable acute vasodilator response may identify patients who will tolerate, and demonstrate transient clinical improvement with, medical therapy.

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.

Infantile pulmonary capillary haemangiomatosis: a lethal form of pulmonary hypertension.

We describe the cases of two children who both presented in infancy with recurrent severe pulmonary hypertensive crises. Exhaustive clinical work-up failed to identify an underlying aetiology. The patients had no clinical response to steroids, immunoglobulins, or pulmonary vasodilators. Post-mortem examination revealed extensive invasive pulmonary capillary haemangiomatosis. There was no evidence of pulmonary venous occlusive disease. Given the lethal nature of this condition, early consideration of referral to a lung transplant centre should be considered in selected patients.

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.

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.

Genetics of pulmonary hypertension.

PURPOSE OF REVIEW: The identification of the genetic basis for heritable predisposition to pulmonary arterial hypertension (PAH) has altered the clinical and research landscape for PAH patients and their care providers. This review aims to describe the genetic discoveries and their impact on clinical medicine. RECENT FINDINGS: Since the landmark discovery that bone morphogenetic protein receptor type II (BMPR2) mutations cause the majority of cases of familial PAH, investigators have discovered mutations in genes that cause PAH in families without BMPR2 mutations, including the type I receptor ACVRL1 and the type III receptor ENG (both associated with hereditary hemorrhagic telangiectasia), caveolin-1 (CAV1), and a gene (KCNK3) encoding a two-pore potassium channel. Mutations in these genes cause an autosomal-dominant predisposition to PAH in which a fraction of mutation carriers develop PAH (incomplete penetrance). In 2014, scientists discovered mutations in eukaryotic initiation factor 2 alpha kinase 4 (EIF2AK4) that cause pulmonary capillary hemangiomatosis and pulmonary veno-occlusive disease, an autosomal recessively inherited disorder. SUMMARY: The discovery that some forms of pulmonary hypertension are heritable and can be genetically defined adds important opportunities for physicians to educate their patients and their families to understand the potential risks and benefits of genetic testing.

Noncontinuously binding loop-out primers for avoiding problematic DNA sequences in PCR and sanger sequencing.

We present a method in which noncontinuously binding (loop-out) primers are used to exclude regions of DNA that typically interfere with PCR amplification and/or analysis by Sanger sequencing. Several scenarios were tested using this design principle, including M13-tagged PCR primers, non-M13-tagged PCR primers, and sequencing primers. With this technique, a single oligonucleotide is designed in two segments that flank, but do not include, a short region of problematic DNA sequence. During PCR amplification or sequencing, the problematic region is looped-out from the primer binding site, where it does not interfere with the reaction. Using this method, we successfully excluded regions of up to 46 nucleotides. Loop-out primers were longer than traditional primers (27 to 40 nucleotides) and had higher melting temperatures. This method allows the use of a standardized PCR protocol throughout an assay, keeps the number of PCRs to a minimum, reduces the chance for laboratory error, and, above all, does not interrupt the clinical laboratory workflow.

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.

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

BACKGROUND: The 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. RESULTS: We 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. CONCLUSIONS: Based 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 patient's clinical response to disease-specific therapy is complex, involving the severity of the patient's 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.

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.

Exogenous expression of synaptotagmin XIII suppresses the neoplastic phenotype of a rat liver tumor cell line through molecular pathways related to mesenchymal to epithelial transition.

The molecular pathogenesis of hepatocellular carcinoma is well-studied but not completely understood. We utilized a microcell-hybrid model of tumor suppression in rat liver tumor cells to facilitate the identification of liver tumor suppressor genes located on human chromosome 11. These investigations confirmed a liver tumor suppressor locus at human 11p11.2, identified Wt1 as a potential effector of 11p11.2-mediated tumor suppression, and subsequently identified human SYT13 as a strong candidate for the 11p11.2 liver tumor suppressor gene. In the studies presented here, we introduced SYT13 into the GN6TF rat liver tumor cell line to characterize a functional role for SYT13 in this model system. Transfected clones expressing an SDS-resistant dimer form of the SYT13 protein displayed induction of Wt1 gene expression and a significant attenuation of the neoplastic phenotype exhibited by the parental tumor cell line. Saturation densities and anchorage-independent growth of SYT13 dimer-positive cell lines were reduced in vitro, and tumorigenicity was significantly decreased or ablated in syngeneic host rats in vivo. In addition, restoration of the contact-inhibited, epithelioid morphology observed in normal liver epithelial cells accompanied ectopic expression of the SYT13 protein dimer, suggesting that SYT13 may be mediating an epithelial differentiation coordinate with tumor suppression in these cells. Accordingly, the expression of E-cadherin (Cdh1) mRNA was increased >100-fold in SYT13-dimer-positive cell lines and the Cdh1 transcriptional repressor Snail was decreased >3-fold in these cells compared to the parental tumor cells. These studies combine to suggest that SYT13 is a liver tumor suppressor gene and that its function may be mediated through pathways implicated in mesenchymal to epithelial transition.

Liver regeneration by small hepatocyte-like progenitor cells after necrotic injury by carbon tetrachloride in retrorsine-exposed rats.

Liver regeneration after partial hepatectomy (PH) in rats exposed to the pyrrolizidine alkaloid retrorsine is accomplished through the proliferation and differentiation of a population of small hepatocyte-like progenitor cells (SHPCs). The activation, emergence, and outgrowth of SHPCs in response to the liver deficit generated through surgical PH have been well characterized. However, the participation of these cells in the restoration of hepatocyte numbers and regeneration of liver tissue mass following necrotic injury has not been investigated. To investigate the capacity of SHPCs to respond to necrotizing liver injury, we combined retrorsine treatment with the centrilobular-specific toxin carbon tetrachloride (CCl(4)). Male Fischer 344 rats were treated with retrorsine (30 mg/kg ip) at 6 and 8 weeks of age, followed by CCl(4) treatment (1500 mg/kg ip) 5 weeks later. Liver tissues were harvested at 3, 7, 14, 21, and 30-days post-injection. The dose of CCl(4) employed resulted in the necrotic destruction of 59±2% of liver mass and elicited a regenerative response equivalent to that of surgical PH. Livers from retrorsine-exposed CCl(4)-treated rats exhibit SHPC proliferation similar to retrorsine-exposed rats subjected to PH (RP). SHPCs appear at 3-days post-injection, continue to expand at 7-days and 14-days post-injection, and completely regenerate/restore the liver mass and structure in these animals by 30-days post-injection. The magnitude of SHPC response observed in the undamaged periportal zone of the liver in these animals is unaffected (versus RP rats) by the loss of the centrilobular region. The results of this study show that SHPCs are capable of regenerating liver after exposure to necrotizing agents and suggest that the progenitor cell of origin of the SHPCs is not restricted to the centrilobular zone of the liver parenchyma.

Cytokine-dependent activation of small hepatocyte-like progenitor cells in retrorsine-induced rat liver injury.

Complete liver regeneration after partial hepatectomy (PH) in rats exposed to the pyrrolizidine alkaloid retrorsine is accomplished through the activation, expansion, and differentiation of a population of small hepatocyte-like progenitor cells (SHPCs). The mechanism(s) governing activation of SHPCs after PH in retrorsine-injured rats has not been investigated. We examined the possibility that SHPCs require cytokine priming prior to becoming growth factor responsive in this model of liver injury and regeneration. Male Fischer 344 rats were treated with retrorsine (30 mg/kg ip) at 6 and 8 weeks of age. Retrorsine-exposed and age-matched control rats were randomized into dexamethasone-treated and no DEX groups. DEX-treated animals were either given a single dose of DEX (2 mg/kg ip) at the time of PH or multiple DEX treatments (2 mg/kg ip each) at 24 and 1 h before PH and 1, 2, and 3 days post-PH. A subset of rats received 10 microg of recombinant IL6 protein, administered intravenously 30 min after PH. Liver tissues were harvested at 7, 14, 21, and 30 days post-PH. Treatment of retrorsine-exposed rats with the cytokine inhibitor dexamethasone (DEX) effectively blocked the emergence of SHPCs resulting in an inhibition of liver regeneration and producing significant short-term mortality. The livers of DEX-treated retrorsine-exposed rats displayed decreased numbers and smaller SHPC clusters compared to retrorsine-exposed rats in the absence of DEX treatment. Administration of recombinant IL6 to DEX-treated retrorsine-exposed rats restored the emergence of SHPCs and SHPC-mediated regenerative response. The livers of DEX-treated retrorsine-exposed rats that received IL6 displayed numbers of expanding SHPC clusters comparable to that of retrorsine-exposed rats in the absence of DEX treatment. These results combine to suggest that SHPC activation after PH in retrorsine-exposed rats is cytokine dependent and may specifically require IL6.

A B-cell lymphoma diagnosed in "floater" tissue: implications of the diagnosis and resolution of a laboratory error.

Contamination of a paraffin-embedded tissue block with another patient's tissue can lead to an incorrect diagnosis. We report the use of short tandem repeats for the analysis of DNA extracted from microdissected tissue from unstained slides prepared from a decalcified block from a 33-year-old woman who was previously diagnosed with a low-grade B-cell lymphoma. This diagnosis was based on a single fragment of tissue found among bone fragments taken during orthopedic surgery at a referring hospital. Our results confirm that the B-cell lymphoma tissue was not derived from our patient. Furthermore, we suggest that in cases for which the definitive identification of a tissue contaminant can resolve clinically, therapeutically, and prognostically significant questions, short tandem repeat analysis of DNA derived from microdissected surgical pathology samples should be considered to minimize errors and enhance the quality of care.