Acute Porphyria Research Articles

Orphan designation: Synthetic double-stranded siRNA oligonucleotide directed against delta-aminolevulinic acid synthase 1 mRNA, covalently linked to a ligand containing three N-acetylgalactosamine residues (givosiran), Treatment of acute hepatic porphyria

Orphan designation: Synthetic double-stranded siRNA oligonucleotide directed against delta-aminolevulinic acid synthase 1 mRNA, covalently linked to a ligand containing three N-acetylgalactosamine residues (givosiran), Treatment of acute hepatic porphyria

Hypertensive Crisis in Patients with Acute Intermittent Porphyria

Abstract Introduction Acute intermittent porphyria (AIP) is the most common and the most severe form of acute hepatic porphyria. Case report Patient, 39 years old, was admitted to the Emergency Department because of abdominal pain. Abdominal pain started 5 days before the admission. The diagnostic research in his hospital showed presence of a stone in the right kidney, and the patient was transported to the other Clinical Centre, where a common urine test showed: high values of delta - aminolevulinic acid and porphobilinogen. The patient was transported to our Clinical Centre. At the admission, abdominal pain decreased, but the patient had a hypertensive crisis with a headache, tearing eyes, swelling, anxiety. Common laboratory tests were in reference range, except creatinine, CRP, arterial blood gas analysis, urine test. The hypertensive crisis was treated by beta blockers and diuretics in maximal doses, but without a positive effect, so we decided to try with Glyceryl trinitrate intravenously. Control blood pressure was 170/100mmHg….130/80mmHg. Discussion Porphyria can be a diagnostic problem, because one of the manifestations can be abdominal pain. Conclusions Comorbidities can be critical in the therapy of life threating conditions.

Sick leave, disability, and mortality in acute hepatic porphyria: a nationwide cohort study

BackgroundAcute hepatic porphyria (AHP) consists of three rare metabolic disorders. We investigated the risk of long-term sick leave, disability pension, and premature death in individuals with AHP compared to the general population.MethodsIn a nationwide cohort study from 1992 to 2017, records of 333 persons (total person-years = 6728) with a confirmed AHP diagnosis were linked to several national compulsory registries (reference population = 5,819,937). We conducted survival analyses to assess additional risk.ResultsPersons with AHP had higher risks of accessing long-term sick leave (adjusted hazard ratio (aHR): 1.5, 95% confidence interval (CI): 1.3, 1.7) and disability pension (aHR: 1.9, CI: 1.5, 2.4). The risk was highest in persons who had been hospitalised for acute attacks, while no additional risk was observed in asymptomatic AHP gene mutation carriers. The median age when accessing disability pension was 45 years, 21 years younger than the general population. AHP was associated with increased risk of mortality due to hepatocellular carcinoma (adjusted mortality rate ratio (aMRR): 84.4, CI: 37.8, 188.2), but no overall increased risk of premature death was observed.ConclusionsPersons with symptomatic AHP were at increased risk of accessing long-term sick leave and disability pension but not of premature death.

Real-world annualized healthcare utilization and expenditures among insured US patients with acute intermittent porphyria (AIP) treated with hemin

Background and aims: Patients with acute intermittent porphyria (AIP) may suffer from acute non-specific attacks that often result in hospitalizations or emergency room (ER) visits. Prior to the recent approval of givosiran (November 2019), hemin was the only FDA-approved therapy for AIP attacks in the US. Our aim was to estimate the annual healthcare utilization and expenditures for AIP patients treated with hemin using real-world data.Methods: Patients with ≥1 hemin claim and confirmed AIP diagnosis – 1 inpatient claim or 2 outpatient claims ≥30 d apart for AIP (2015–2017) or acute porphyria (prior to 2015) – were identified in MarketScan administrative claims dataset between 2007 and 2017. Continuous enrolment for ≥6 months from confirmed diagnosis was required. A secondary analysis (“active disease population”) limited the sample to adult patients with ≥3 attacks or 10 months of prophylactic use of hemin within a 12-month pre-index period. AIP-related care was defined by hemin use during an attack (daily glucose and/or hemin use) or prophylaxis (non-attack hemin use). Outcomes were annualized and expenditures were inflated to 2017.Results: Across 10 years, patients with a confirmed AIP diagnosis (N = 8,877) and ≥1 hemin claim (N = 164) were restricted by ≥6 months continuous follow-up (N = 139). AIP patients were mostly female (N = 112; 81%), had median age of 40 and 3 years average follow-up. Annualized average total expenditures for AIP-related care were $113,477. Annualized average all-cause (any diagnosis) hospitalizations were statistically significantly lower for patients treated with hemin prophylaxis vs. acute treatment (1.0 vs. 2.1; p < .001). In the secondary analysis (N = 27), annualized average total expenditures for AIP-related care were higher ($187,480).Conclusions: For AIP patients treated with hemin, patients treated for acute attacks may use a greater number of resources compared to patients treated prophylactically.

Penetrance and predictive value of genetic screening in acute porphyria

ObjectivePenetrance, predictive value and female patients' perspectives on genetic testing were evaluated among Finnish patients with acute porphyria. We conducted a retrospective study to evaluate prognosis among at-risk female family members depending on the primary method of diagnosis. MethodsThe penetrance was calculated among 23 genetically heterogeneous families selected from the Finnish porphyria registry (n = 515, AIP 333; VP 182). We included kindreds with ≥9 patients in a family (range 9–23 patients, total 216 AIP; 129 VP). In 2015, the registry included 164 living female subjects between 14 and 85 years of age. A questionnaire was sent to 143 women, of whom 107 (75%, AIP 67; VP 40) replied. Female at-risk relatives (AIP 54; VP 30) were divided into two groups based on the primary method of diagnosis: mutation analysis (Group A, n = 40) or biochemical analysis (Group B, n = 44). ResultsMean penetrance for all acute symptoms was 35% among AIP and 40% among VP families. In both study groups, the penetrance was higher among female (AIP 50%; VP 44%) than male patients (AIP 17%; VP 33%). Penetrance for hospitalized attacks was 30% among AIP families (range 10–80%, for women 41%) and 25% in VP (range 0–50%, for women 27%) demonstrating wide variations among families even with the similar genotype. Acute porphyria was diagnosed at the median age of 26 years (range 0–76 years) among female patients, commonly after the onset of acute symptoms. Diagnostic delay was an average of 7.4 years (range 1–30 years). Acute symptoms occurred at the median age of 24 years (range 10–57 years) and the first hospitalization at the median age of 26.5 years (range 15–57 years). At the onset of symptoms, 38% of the women were ≤ 20 years of age. According to the life table analysis, acute attacks occurred mainly during the following five years after the diagnosis and the attack risk diminished after 35 years of age. The annual risk for hospitalization due to an acute attack during fertile years was lower in Group A than Group B (0.002 vs. 0.010, p = .018), but the risk of all subsequent acute symptoms did not diminish (Group A 0.017 vs. Group B 0.019, p = .640). The cumulative risk of acute symptoms among asymptomatic patients at the time of diagnosis was 26.7% for Group A and 58.3% for Group B. The cumulative risk of the first subsequent attack requiring hospitalization after the diagnosis among all at-risk relatives was similarly less frequent in Group A than in Group B (OR 0.180; 95% CI 0.041–0.789, p = .041). If attacks were followed among symptomatic patients only, attack-free years were more frequent in Group A than in Group B. Patients preferred genetic screening before puberty to minimize the risk of acute symptoms and genetic discrimination was rare. 44% of the patients reported social, psychological or physical impairment due to acute hepatic porphyria, emphasizing the importance of supporting patients' emotional and resilience capacity. ConclusionsAmong female at-risk relatives the annual risk for hospitalization due to an acute attack is <1% and for acute symptoms <2% during the fertile years. Genetic testing of relatives diminishes the risk of acute attacks. Diagnosis before symptom onset is key for subjects to remain asymptomatic during follow-up, and genetic screening should be done earlier than currently.

Overall Health, Daily Functioning, and Quality of Life in Acute Hepatic Porphyria Patients: ENVISION, a Phase 3 Global, Multicenter, Randomized, Double-Blind, Placebo-Controlled Trial

Objectives: Acute Hepatic Porphyria (AHP) is a family of rare genetic diseases leading to an enzyme deficiency in the heme biosynthesis pathway, causing accumulation of neurotoxic heme intermediates, resulting in neurovisceral attacks and chronic manifestations. Givosiran, an investigational RNAi therapeutic, is being evaluated for its ability to reduce the levels of neurotoxic intermediates thus decreasing attacks and disease manifestations.

Givosiran: First Approval.

Givosiran (Givlaari™) is an aminolevulinate synthase 1 (ALAS1)-directed small interfering RNA (siRNA) covalently linked to a ligand to enable specific delivery of the siRNA to hepatocytes. This results in downregulation of ALAS1 mRNA and prevents accumulation of neurotoxic δ-aminolevulinic acid and porphobilinogen levels that are associated with acute porphyria attacks. Givosiran is being developed by Alnylam Pharmaceuticals for the treatment of acute hepatic porphyria (AHP). In November 2019, givosiran was approved in the USA for the treatment of adults with AHP based on the positive results from the multinational, phase III ENVISION trial. In the EU, givosiran received a positive opinion in January 2020 for the treatment of AHP in adults and adolescents aged 12 years and older. This article summarizes the milestones in the development of givosiran leading to this first approval for the treatment of adults with AHP.

Porfirias agudas: manifestaciones inespecíficas y manejo terapéutico específico

Revisión clínica, científica, teórica y documental teniendo como objetivo principal realizar una revisión sobre la porfiria aguda, su enfoque, diagnóstico y tratamiento. Se utilizó una metodología descriptiva, cualitativa y teórica que permitió la revisión bibliográfica de los estudios relacionados con la porfiria aguda, trastorno congénito poco frecuente que causa alteraciones en las enzimas de la biosíntesis del grupo hem. La importancia de esta patología radica en que se confunde con otras enfermedades debido al cuadro clínico complejo difícil de identificar, lo que ocasiona un diagnóstico tardío que puede comprometer la vida del paciente y por ello se conoce como enfermedad silenciosa. Como conclusión se recomienda mayor atención a los factores precipitantes de los ataques agudos por la elevada morbimortalidad, así como hacer un diagnóstico rápido y oportuno por medios cualitativos y cuantitativos solicitando PBG (porfobilinógeno) y ALA (ácido delta aminolevulínico).

Accelerated Porphyria Hepatopathy after Liver Resection in Eryhtropoeitic Proptoporphyria

Background: Direct toxic effects on hepatocytes and bile duct occlusion by protoporphyrin aggregates result in liver damage in Eryhthropoetic Protoporphyria(EPP).Fulminanat hepatic failure in EPP has been described but no definite trigger has been found. Effects of liver resection in EPP are also not known.We describe the first case of liver resection in a patient of EPP which led to acute fatal porphyria hepatopathy of previously normal liver.

Трудности диагноза острой порфирии в терапевтическом стационаре: описание клинических случаев

Two cases of acute porphyria are presented. Acute porphyria is a rare disease, a pathology associated with hereditary or acquired abnormalities of the biosynthesis of heme. The main features distinguishing these nosological forms are: the prevalence of precursors of porphyrins among laboratory-defined metabolites, rapid clinical progression of the disease; the absence of a correct diagnosis or late diagnosis resulting in life threatening neurological disorders. The main diagnostic difficulties are related to a non-specific character of clinical signs of acute intermittent porphyria, the clinical signs mimic a large range of somatic conditions, including emergency conditions. Certain difficulties for doctors are caused by the orphan nature of porphyria, clinical polymorphism, the difficulty to obtain pathogenetic drugs in medical institution for treatment of the disease in the Russian Federation.

Givosiran. ALAS1 expression inhibitor, Treatment of acute porphyria

Givosiran. ALAS1 expression inhibitor, Treatment of acute porphyria

EXPLORE: A Prospective, Multinational, Natural History Study of Patients with Acute Hepatic Porphyria with Recurrent Attacks.

Background and AimsAcute hepatic porphyria comprises a group of rare genetic diseases caused by mutations in genes involved in heme biosynthesis. Patients can experience acute neurovisceral attacks, debilitating chronic symptoms, and long‐term complications. There is a lack of multinational, prospective data characterizing the disease and current treatment practices in severely affected patients.Approach and ResultsEXPLORE is a prospective, multinational, natural history study characterizing disease activity and clinical management in patients with acute hepatic porphyria who experience recurrent attacks. Eligible patients had a confirmed acute hepatic porphyria diagnosis and had experienced ≥3 attacks in the prior 12 months or were receiving prophylactic treatment. A total of 112 patients were enrolled and followed for at least 6 months. In the 12 months before the study, patients reported a median (range) of 6 (0‐52) acute attacks, with 52 (46%) patients receiving hemin prophylaxis. Chronic symptoms were reported by 73 (65%) patients, with 52 (46%) patients experiencing these daily. During the study, 98 (88%) patients experienced a total of 483 attacks, 77% of which required treatment at a health care facility and/or hemin administration (median [range] annualized attack rate 2.0 [0.0‐37.0]). Elevated levels of hepatic δ‐aminolevulinic acid synthase 1 messenger ribonucleic acid levels, δ‐aminolevulinic acid, and porphobilinogen compared with the upper limit of normal in healthy individuals were observed at baseline and increased further during attacks. Patients had impaired quality of life and increased health care utilization.ConclusionsPatients experienced attacks often requiring treatment in a health care facility and/or with hemin, as well as chronic symptoms that adversely influenced day‐to‐day functioning. In this patient group, the high disease burden and diminished quality of life highlight the need for novel therapies.

International Porphyria Molecular Diagnostic Collaborative: an evidence-based database of verified pathogenic and benign variants for the porphyrias

With the advent of precision and genomic medicine, a critical issue is whether a disease gene variant is pathogenic or benign. Such is the case for the three autosomal dominant acute hepatic porphyrias (AHPs), including acute intermittent porphyria, hereditary coproporphyria, and variegate porphyria, each resulting from the half-normal enzymatic activities of hydroxymethylbilane synthase, coproporphyrinogen oxidase, and protoporphyrinogen oxidase, respectively. To date, there is no public database that documents the likely pathogenicity of variants causing the porphyrias, and more specifically, the AHPs with biochemically and clinically verified information. Therefore, an international collaborative with the European Porphyria Network and the National Institutes of Health/National Center for Advancing Translational Sciences/National Institute of Diabetes and Digestive and Kidney Diseases (NIH/NCATS/NIDDK)-sponsored Porphyrias Consortium of porphyria diagnostic experts is establishing an online database that will collate biochemical and clinical evidence verifying the pathogenicity of the published and newly identified variants in the AHP-causing genes. The overall goal of the International Porphyria Molecular Diagnostic Collaborative is to determine the pathogenic and benign variants for all eight porphyrias. Here we describe the overall objectives and the initial efforts to validate pathogenic and benign variants in the respective heme biosynthetic genes causing the AHPs.

PILOT RESEARCH OF A GENETIC PREDISPOSITION FOR CLINICAL MANIFESTATIONS OF ACUTE INTERMITTENT PORPHYRIA

Introduction.Acute intermittent porphyria (AIP) is the most common and severe form of acute hepatic porphyria. AIP is caused by a deficiency in the third enzyme of the heme biosynthesis system — hydroxymethylbilanine synthase (HMBS) — and has a dominant inheritance type. However, the probability of the clinical manifestation of this condition in carriers of the mutation in the HMBS gene constitutes only 10–20 %. Thi s suggests that the presence of such a mutation can be a necessary but not a sufficient condition for the development of the disease.Aim.To search for additional genetic factors, which determine the clinical penetrance of AIP using Whole-Exome Sequencing.Materials and methods.Sequencing of the whole exome was performed using a TruSeqExomeLibraryPrepkit (Illumina) kit by an Illumina HiSeq4000 instrument for 6 women with API with known mutations in the HMBS gene. All the patients suffered from a severe form of the disease. As a reference, a version of the hg19 human genome was used.Results.No common mutations were found in the examined patients. However, in each patient, functional variations were found in the genes related to detoxification systems, regulation of the heme biosynthesis cascade and expression of delta-aminolevulinic acid synthase (ALAS1) and in genes of proteins regulating nervous system. These variations require further study involving an extended number of patients with AIP manifestations and their relatives, who are asymptomatic carriers of disorders in the gene HMBS.Conclusions.The results obtained have allowed us to formulate a hypothesis about a possible role of genetic defects in the penetrance of AIP, which determine the development of other neurological pathologies. This is evidenced by the presence of gene pathogenic variations in 5 out of 6 examined patients, defects in which are associated with hereditary myasthenia and muscle atrophy.

1025 Frequency of Diagnosed Acute Intermittent Porphyria in a National Health Care Database

INTRODUCTION: Acute hepatic porphyria (AHP) refers to a family of rare, genetic diseases caused by deficient activity of enzymes involved in the heme biosynthesis pathway in the liver, with acute intermittent porphyria (AIP) being the most common subtype. Accumulation of neurotoxic heme intermediates may result in potentially life-threatening acute attacks and chronic symptoms that negatively impact daily functioning and quality of life. Although AIP-related genetic mutations are relatively common (1:1,700), clinical penetrance of the disease is estimated around 1%. European data suggest that the diagnosed prevalence of AHP is approximately 1 per 100,000 individuals. Epidemiologic data specific to AIP are limited. The study aimed to identify AIP patients diagnosed in a nationally representative health care database to estimate prevalence, commonly reported clinical presentations, and health care resource utilization of AIP in the United States. METHODS: This retrospective analysis utilized the IBM® MarketScan® Commercial Claims and Medicare Supplemental Databases. Patients with at least one claim for AIP (ICD-10 diagnosis code E80.21) between October 1, 2015 – June 30, 2018 were selected for analyses. The subset of AIP patients with continuous enrollment for one year preceding and following their first observed AIP diagnosis (index diagnosis) were identified for the measurement of baseline characteristics and outcomes post-index, including healthcare resource utilization (HCRU), medications (including hemin), symptoms, and the frequency of comorbid diagnoses. RESULTS: Approximately 37 million patients within the MarketScan® databases had a medical or pharmacy claim between October 1, 2015 and June 30, 2018; a total of 361 of these patients were identified with a diagnosis of AIP. Among these 361 AIP patients, a partial prevalence estimate of AIP on June 30, 2018 was calculated to be 0.98 per 100,000 (214 AIP patients per 21,807,137 people enrolled on June 30, 2018). Sensitivity analyses around claims-based definitions for identifying AIP revealed 140 patients with ≥2 claims for AIP and 31 AIP patients receiving hemin (Table 1). CONCLUSION: Results from this national representative healthcare claims database demonstrated the frequency of diagnosed AIP in the United States to be similar to estimations previously published in global and European-specific AIP epidemiology studies.

482 Prevalence and Predictors of Symptom Complexes Commonly Seen in Acute Hepatic Porphyria in the General Population

INTRODUCTION: Acute hepatic porphyria (AHP), a family of rare genetic disorders, can present with life-threatening attacks with a predominant symptom of abdominal pain. Literature states that patients with AHP have been misdiagnosed with other conditions such as irritable bowel syndrome, functional abdominal pain, and endometriosis, among others. We aimed to determine the prevalence and predictors of symptom complexes commonly seen in AHP among community-dwelling adults in the US. METHODS: We partnered with Cint, an online survey research firm, to recruit a representative sample of US adults aged ≥18 y with abdominal pain. We asked if they had recurrent episodes of excruciating belly pain lasting ≥1 day with any of these co-occurring symptoms: urine color change to dark red/brown; skin blisters on sun-exposed areas; peripheral (e.g., muscle weakness, extremity pain), central (e.g., depression, seizures), or autonomic (e.g., tachycardia, hypertension) nervous system manifestations. Multivariable logistic regression models were used to adjust for confounding. RESULTS: 24,932 individuals accessed the survey, with 10,300 (41.3%) reporting abdominal pain (Table 1). Of those with abdominal pain, 23.4% had symptoms consistent with AHP (≥2 episodes of excruciating abdominal pain and ≥2 concomitant symptoms). Those with AHP-like symptoms experienced the following: autonomic (97.7%), central (96.5%), and peripheral (58.8%) nervous system manifestations; urine color change (21.9%); and skin blisters (6.6%). Most (90.9%) sought medical care for their symptoms: emergency room (57.6%), doctor's office (52.1%); urgent care facility (29.4%); clinic/health center (19.1%); hospital admission (18.4%). Table 2 shows results of the regression analysis on presence of AHP-like symptoms. Younger individuals, Latinos, those who identified as other race, and those with a usual source of care, comorbidities, lower locus of control, more severe pain, and worse physical health were more likely to have AHP-like symptoms. CONCLUSION: In this population-based survey, 1 of 4 individuals with prior abdominal pain had symptoms consistent with AHP manifestations. Although AHP prevalence is very low, clinicians should ask those experiencing multiple episodes of severe abdominal pain about concomitant neurologic, psychiatric, skin, and urologic symptoms. This practice may prompt earlier testing for AHP, decreasing the delay to diagnosis and improving outcomes.

996 Disease Characteristics of Acute Hepatic Porphyria Patients: ENVISION, a Phase 3 Global, Multicenter, Randomized, Double-Blind, Placebo-Controlled Trial

INTRODUCTION: Acute Hepatic Porphyria (AHP) is a family of rare genetic diseases due to enzyme deficiencies in heme biosynthesis in the liver that can lead to neurovisceral attacks and chronic symptoms. (1, 2) AHP is also associated with multiple long-term complications, including neuropathy (3,4), hypertension (5,6), chronic kidney disease (7), and hepatocellular carcinoma (8,9). METHODS: ENVISION (NCT03338816) was a Phase 3 global, multicenter, randomized, double-blind, placebo-controlled trial with an open label extension to evaluate the efficacy and safety of subcutaneous givosiran in AHP. Eligible patients had an AHP diagnosis and ≥2 attacks within the prior 6 months. At baseline detailed medical history and laboratory assessments were obtained. RESULTS: Ninety-four AHP patients enrolled, median (range) age 37.5 (19-65) years, 89% female, and median (range) of 6.5 (0.1–43) years since diagnosis. Patients had a median of 4 composite attacks in the preceding 6 months and 40% were on hemin prophylaxis prior to study. Half the patients experienced daily chronic symptoms and 29% used opioids daily/most days between attacks. Comorbidities reported on medical history included neuropathy (38%), central venous access complications (71%), iron overload (33%), liver disease (28%), and chronic kidney disease (18%). At baseline 17% of patients had liver transaminases &gt; ULN and 34% had an eGFR &lt; 60 mL/min/1.73m2. CONCLUSION: The baseline disease characteristics of the Phase 3 study population are consistent with findings reported in the AHP literature, underscoring the high rate of liver disease and central venous access complications in these patients, and highlighting the multi-systemic nature of AHP and the medical challenge it presents for diagnosis and treatment.

Expanded Access Protocol of Givosiran for Patients With Acute Hepatic Porphyria

Expanded Access Protocol of Givosiran for Patients With Acute Hepatic Porphyria

Hypothesis: Metabolic targeting of 5-aminolevulinate synthase by tryptophan and inhibitors of heme utilisation by tryptophan 2,3-dioxygenase as potential therapies of acute hepatic porphyrias

Metabolic targeting of liver 5-aminolevulinate synthase (5-ALAS) by inhibition of heme utilisation by tryptophan (Trp) 2,3-dioxygenase (TDO) or the use of tryptophan is proposed as a therapy of acute hepatic porphyrias. 5-ALAS, the rate-limiting enzyme of heme biosynthesis, is under negative feedback control by a small regulatory heme pool in the hepatic cytosol. Acute porphyric attacks, precipitated by fasting, certain hormones and some drugs, involve induction of 5-ALAS secondarily to depletion of the above pool, and the resultant elevation of 5-ALA levels initiates the abdominal and neurological symptoms of attacks. By utilising the regulatory heme, cytosolic TDO undermines the feedback control, thus allowing 5-ALAS induction to occur, e.g. upon glucocorticoid induction of TDO during fasting (starvation) and exogenous glucocorticoid administration. Currently, glucose therapy is the preferred strategy for reversing moderate attacks induced by fasting (calorie restriction), with more severe attacks being treated by intravenous heme preparations. Reversal of fasting-induced attacks by glucose is explained by the previously demonstrated reversal of increased heme utilisation by TDO. Inhibitors of this utilisation are therefore potential therapeutic targets in acute attacks and also for maintenance of a symptomless state. Existing TDO inhibitors other than glucose include allopurinol, nicotinamide and recently developed potent inhibitors such as LM10 used in cancer therapy. Based on studies in rats, the hypothesis predicts that the safety or otherwise of drugs in the hepatic porphyrias is determined by their ability to inhibit TDO utilisation of heme under basal conditions or after glucocorticoid induction or heme activation of TDO, in parallel with reciprocal changes in 5-ALAS induction. Tryptophan is also proposed as a potential therapy of acute attacks either alone or as an adjunct to the recently proposed 5-ALAS1 gene silencing. Trp increases heme biosynthesis by enhancing 5-ALA dehydratase activity and, based on a Trp-5-ALA model presented herein, Trp offers several advantages over heme therapy, namely rapid conversion of 5-ALA into heme, a greatly enhanced heme availability, a near complete inhibition of 5-ALAS induction, assumed rapid clearance of 5-ALA and hence accelerated resolution of symptoms of attacks, and finally provision of the neuroprotective metabolite kynurenic acid to neutralise the neurological symptoms. The hypothesis also addresses heme regulation in species lacking the TDO free apoenzyme and its glucocorticoid induction mechanism and proposes detailed assessment of heme biosynthesis in these species. Detailed proposals for testing the hypothesis are presented.

A next-generation-sequencing panel for mutational analysis of dominant acute hepatic porphyrias

Molecular diagnosis of autosomal dominant acute hepatic porphyrias (AHPs) plays an important role in the management of these disorders. To introduce next generation sequencing (NGS) to the porphyria diagnosis, we designed a panel that contained four genes, ALAS1, HMBS, CPOX and PPOX for mutational analysis of acute intermittent porphyria (AIP), hereditary coproporphyria (HCP) and variegate porphyria (VP). To validate the AHP panel, 30 samples with known pathogenic variants as determined by Sanger sequencing, were analyzed using the Ion PGM™. Among them, nine have so far not been reported. The pathogenic variants were identified and annotated manually in IGV by three individuals who were blinded to the Sanger results. The AHP panel consists of 95 amplicons that covers 92% of the coding region of the four genes. Of the 95 amplicons, 93 had an average read-depth of >500 reads. In 29 of the 30 tested samples, pathogenic variants were correctly identified and annotated. The number of reads from the mutated alleles were approximately 50% of the total. The annotation of a 22-bp duplication with NGS differed from that of Sanger by one nucleotide. NGS showed an advantage in allelic discrimination over Sanger sequencing and was also able to detect a known somatic variant in the HMBS gene. The AHP panel will be applied in the initial diagnosis of new patients. Any sequence variations with a frequency of ≥10% will be confirmed by Sanger sequencing. The cost-effectiveness of a NGS approach for AHP in a diagnostic laboratory needs to be further assessed.