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Defects in hydroxymethylbilane synthase (HMBS) can cause Acute Intermittent Porphyria (AIP), an acute neurological disease. Although sequencing-based diagnosis can be definitive, ~⅓ of clinical HMBS variants are missense variants, and most clinically-reported HMBS missense variants are designated as “variants of uncertain significance” (VUS). Using saturation mutagenesis,en masseselection, and sequencing, we applied a multiplexed validated assay to both the erythroid-specific and ubiquitous isoforms of HMBS, obtaining confident functional impact scores for >84% of all possible amino-acid substitutions. The resulting variant effect maps generally agreed with biochemical expectation. However, the maps showed variants at the dimerization interface to be unexpectedly well tolerated, and suggested residue roles in active site dynamics that were supported by molecular dynamics simulations. Most importantly, these HMBS variant effect maps can help discriminate pathogenic from benign variants, proactively providing evidence even for yet-to-be-observed clinical missense variants.

The Mendelian inheritance pattern of acute intermittent porphyria, hereditary coproporphyria, and variegate porphyria is autosomal dominant, but the clinical phenotype is heterogeneous. Within the general population, penetrance is low, but among first-degree relatives of a symptomatic proband, penetrance is higher. These observations suggest that genetic factors, in addition to mutation of the specific enzyme of the biosynthetic pathway of heme, contribute to the clinical phenotype. Recent studies by others suggested that the genotype of the transporter protein ABCB6 contribute to the porphyria phenotype. Identifying the molecule(s) that are transported by ABCB6 has been problematic and has led to uncertainty with respect to how or if variants/mutants contribute to phenotypic heterogeneity. Knockout mouse models of Abcb6 have not provided a direction for investigation as homozygous knockout animals do not have a discrete phenotype. To address the proposed link between ABC6 genotype and porphyria phenotype, a large cohort of patients with acute hepatic porphyria and erythropoietic protoporphyria was analyzed. Our studies showed that ABCB6 genotype did not correlate with disease severity. Therefore, genotyping of ABCB6 in patients with acute hepatic porphyria and erythropoietic protoporphyria is not warranted.

Purpose: Patients with erythropoietic protoporphyria (EPP), a severe painful photodermatosis, experience prodromal sensations when exposed to sunlight, which are the “warning signals” to exit the sun, as prolonged exposure causes an excruciatingly painful phototoxic attack. The unique prodromal cutaneous sensations are reversible and differ from the severe burning pain attack lasting 2–7 days. Previously, afamelanotide treatment was studied using time to pain or time outside as primary outcome measures. Since patients have an ingrained fear of sunlight, these measures did not capture the full treatment effect. We retrospectively characterized and evaluated time to prodrome (TTP) as a safer, patient-reported outcome (PRO) measure in afamelanotide-treated patients. Methods: Structured interviews recorded TTP before and during afamelanotide treatment in retrospective US and Dutch cohort studies. Results: Thirty-one US and 58 Dutch EPP patients participated. Before afamelanotide treatment, 54.8% US and 39.7% Dutch patients reported TTP onset

BACKGROUND AND AIMS The risk for hepatocellular carcinoma (HCC) is increased in acute hepatic porphyrias (AHP). The aim of this study was to explore the clinicopathologic characteristics, outcomes, and frequency of HCC in patients with AHP in the United States. APPROACH AND RESULTS This cross-sectional analysis evaluated patients with HCC in a multicenter, longitudinal study of AHP. Among 327 patients with AHP, 5 (1.5%) were diagnosed with HCC. Of the 5 HCC cases, 4 had acute intermittent porphyria and 1 had variegate porphyria, confirmed by biochemical and/or genetic testing. All patients were white females, with a median age of 27 years (range 21-75) at diagnosis. The median age at HCC diagnosis was 69 years (range 61-74). AHP was asymptomatic in 2 patients; 2 reported sporadic attacks; and 1 reported recurrent attacks (>4 attacks/year). All patients had a single HCC lesion on liver imaging that was 1.8-6.5 centimeters in diameter. Serum alpha fetoprotein levels were below 10 ng/mL in all 4 patients with available results. Four patients underwent liver resection, and 1 was treated with radioembolization. No significant inflammation or fibrosis was found in adjacent liver tissues of 3 patients who underwent liver resection. Two patients developed recurrence of HCC at 22 and 26 months following liver resection. All patients are alive with survival times from HCC diagnosis ranging from 26-153 months. CONCLUSION In this U.S. study, 1.5% of patients with AHP had HCC. HCC in AHP occurred in the absence of cirrhosis, which contrasts with other chronic liver diseases. Patients with AHP, regardless of clinical attacks, should be screened for HCC, beginning at age 50. The pathogenesis of hepatocarcinogenesis in AHP is unknown and needs further investigation.

Background 5-Aminolevulinic acid dehydratase (ALAD) porphyria (ADP) is an ultrarare autosomal recessive disease, with only eight documented cases, all of whom were males. Although classified as an acute hepatic porphyria (AHP), induction of the rate limiting hepatic enzyme 5-aminolevulinic acid synthase-1 (ALAS1) has not been demonstrated, and the marrow may also contribute excess 5-aminolevulinic acid (ALA). Two patients have died and reported follow up for the others is limited, so the natural history of this disease is poorly understood and treatment experience limited. Methods We report new molecular findings and update the clinical course and treatment of the sixth reported ADP patient, now 31 years old and the only known case in the Americas, and review published data regarding genotype-phenotype correlation and treatment. Results Circulating hepatic 5-aminolevulinic acid synthase-1 (ALAS1) mRNA was elevated in this case, as in other AHPs. Gain of function mutation of erythroid specific ALAS2 – an X-linked modifying gene in some other porphyrias – was not found. Seven reported ADP cases had compound heterozygous ALAD mutations resulting in very low residual ALAD activity and symptoms early in life or adolescence. One adult with a germline ALAD mutant allele developed ADP in association with a clonal myeloproliferative disorder, polycythemia vera. Conclusions Elevation in circulating hepatic ALAS1 and response to treatment with hemin indicate that the liver is an important source of excess ALA in ADP, although the marrow may also contribute. Intravenous hemin was effective in most reported cases for treatment and prevention of acute attacks of neurological symptoms.

Background: Acute Intermittent Porphyria (AIP) is a rare inborn error of heme biosynthesis characterized clinically by life-threatening acute neurovisceral attacks. Few studies have assessed quality of life (QoL) tools in the AIP population, and none are validated for use in AIP. In this study, the Patient Reported Outcomes Measurement Information System 57 (PROMIS-57) scales were assessed in AIP patients to determine the factor structure of PROMIS items and to compare this to previously proposed factor structures in the general population.Methods: Baseline data from the Porphyrias Consortium’s Longitudinal Study of the Porphyrias was obtained for 259 AIP patients. This included detailed disease and medical history information as well as PROMIS-57 data. Exploratory and confirmatory factor analyses (EFA and CFA, respectively) were conducted on baseline PROMIS-57 data. CFA was used to test the hypothesized three-factor structure from the PROMIS literature. Results: Internal consistency was high for all the PROMIS scales assessed. The EFA revealed a five-factor model, each consisting of a separate domain: pain interference, anxiety, depression, sleep disturbance, and fatigue. Model fit was good overall. A CFA of the hypothesized three-factor model did not converge suggesting an inappropriate structural model. Conclusion: The EFA showed a five-factor model that fit the data well, however a CFA of the three-factor model observed in the general population did not fit the data well. These findings, together with previous studies assessing correlations with clinical features, indicate that the PROMIS scales may be valid and reliable measures for AIP patients. However, further analyses are needed to confirm this. Further studies should assess correlations with other tools, whether the PROMIS scales are responsive to treatment, if they capture QoL longitudinally, and a CFA in a second sample.

Background Porphyrias are a group of rare diseases leading to dysregulation in heme biosynthesis and the accumulation of heme precursors, including porphyrinogens, which in their oxidized states [porphyrins] are reddish or purple. Acute hepatic porphyrias (AHP) comprise four diseases that cause acute debilitating neurovisceral attacks. Despite diagnostic advances, AHP is often undiagnosed or misdiagnosed due to a lack of disease awareness, low clinical suspicion, variable presentation, and nonspecific symptoms that mimic more common diseases. Delays in diagnosis and treatment increase the risk of serious acute and chronic complications. Aim In order to assess whether symptoms alone or in combination might be utilized as important indicators or “purple flags” that, when present, should alert clinicians to suspect AHP and pursue specific diagnostic testing, we conducted a comprehensive review of the literature on AHP, including cohort studies and case reports over two epochs, from 1980 to 2006 and from 2012 to 2018. Results We found that severe abdominal pain, with or without acute central nervous system manifestations and peripheral neuropathy, continues to be recognized the most frequent symptom. Hyponatremia, change in urine color, and certain chronic symptoms were also identified as features that should raise suspicion of AHP. To improve diagnosis of AHP, clinicians need to take a broad perspective, including demographic data and medical history, into consideration. Conclusions The clinical features of AHP continue to be severe pain, especially pain in the abdomen. Other features that should raise suspicion are autonomic, peripheral, or central neuropathies, hyponatremia, and red-purple urine color.

Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the alpha-galactosidase A (GLA) gene, which encodes the exogalactosyl hydrolase, alpha-galactosidase A (α-Gal A). Deficient α-Gal A activity results in the progressive, systemic accumulation of its substrates, globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3), leading to renal, cardiac, and/or cerebrovascular disease and early demise. The current standard treatment for Fabry disease is enzyme replacement therapy, which necessitates lifelong biweekly infusions of recombinant enzyme. A more long-lasting treatment would benefit Fabry patients. Here, a gene therapy approach using an episomal adeno-associated viral 2/6 (AAV2/6) vector that encodes the human GLA cDNA driven by a liver-specific expression cassette was evaluated in a Fabry mouse model that lacks α-Gal A activity and progressively accumulates Gb3 and Lyso-Gb3 in plasma and tissues. A detailed 3-month pharmacology and toxicology study showed that administration of a clinical-scale-manufactured AAV2/6 vector resulted in markedly increased plasma and tissue α-Gal A activities, and essentially normalized Gb3 and Lyso-Gb3 at key sites of pathology. Further optimization of vector design identified the clinical lead vector, ST-920, which produced several-fold higher plasma and tissue α-Gal A activity levels with a good safety profile. Together, these studies provide the basis for the clinical development of ST-920., Graphical Abstract

Background Fabry disease is a rare, X-linked genetic disorder that, if untreated in patients with the Classic phenotype, often progresses to end-stage kidney disease. This meta-analysis determined the effect of agalsidase beta on loss of estimated glomerular filtration rate (eGFR) in the Classic phenotype using an expansive evidence base of individual patient-level data. Methods The evidence base included four Sanofi-Genzyme studies and six studies from a systematic literature review. These were restricted to Classic Fabry patients meeting the eligibility criteria from Phases III and IV agalsidase beta trials, including 315 patients (161 treated). Linear regression was first used to model annual change in eGFR for each patient and the resulting annualized eGFR slopes were modelled with treatment and covariates using quantile regression. These results were then used to estimate median annualized eGFR change in agalsidase beta treated versus untreated groups. Results Imbalances across treatment groups were found in baseline age, sex and proteinuria, but not in the use of renin–angiotensin system blockers. The adjusted model suggests that treated (agalsidase beta) patients experienced a slower median eGFR decrease [2.46 mL/min/1.73 m2/year slower; 95% confidence interval (CI) 0.63–4.29; P = 0.0087] than comparable untreated patients. The median eGFR decrease was 2.64 mL/min/1.73 m2/year slower (95% CI 0.53–4.78; P = 0.0141) in treated Classic males. Conclusions Using an expansive evidence base and robust modelling approach, these data indicate that agalsidase beta-treated patients with the Classic phenotype conserve their renal function better than untreated patients.

Accumulation of protoporphyrin IX (PPIX) and Zn-PPIX, are the clinical hallmarks of protoporphyria. Phenotypic expression of protoporphyria is due to decreased activity of ferrochelatase (FECH) or to increased activity of aminolevulinic acid synthase (ALAS) in red blood cells. Other genetic defects have been shown to contribute to disease severity including loss of function mutations in the mitochondrial AAA-ATPase, CLPX and mutations in the Iron-responsive element binding protein 2 (IRP2), in mice. It is clear that multiple paths lead to a common phenotype of excess plasma PPIX that causes a phototoxic reaction on sun exposed areas. In this study we examined the association between mitochondrial iron acquisition and utilization with activity of FECH. Our data show that there is a metabolic link between the activity FECH and levels of MFRN1 mRNA. We examined the correlation between FECH activity and MFRN1 mRNA in cell lines established from patients with the classical protoporphyria, porphyria due to defects in ALAS2 mutations. Our data confirm MFRN1 message levels positively correlated with FECH enzymatic activity in all cell types.

Acute Intermittent Porphyria (AIP), an autosomal dominant hepatic disorder, results from hydroxymethylbilane synthase (HMBS) mutations that decrease the encoded enzymatic activity, thereby predisposing patients to life-threatening acute neurovisceral attacks. The ~1% penetrance of AIP suggests that other genetic factors modulate the onset and severity of the acute attacks. Here, we characterized the hepatic transcriptomic response to phenobarbital (PB) administration in AIP mice, which mimics the biochemical attacks of AIP. At baseline, the mRNA profiles of 14,138 hepatic genes prior to treatment were remarkably similar between AIP and the congenic wild-type (WT) mice. After PB treatment (~120mg/kg × 3d), 1,347 and 1,120 genes in AIP mice and 422 and 404 genes in WT mice were uniquely up- and down-regulated, respectively, at a False Discovery Rate 1.9-fold) and Cyp21a1 was upregulated only in PB-induced WT mice (>9-fold). Notably, the genes differentially expressed in induced AIP mice were enriched in circadian rhythm, mitochondria biogenesis and electron transport, suggesting these pathways were involved in AIP mice responding to PB treatment. These results advance our understanding of the hepatic metabolic changes in PB-induced AIP mice and have implications in the pathogenesis of AIP acute attacks.

Author(s): Naik, Hetanshi; Overbey, Jessica R; Desnick, Robert J; Anderson, Karl E; Bissell, D Montgomery; Bloomer, Joseph; Bonkovsky, Herbert L; Phillips, John D; Wang, Bruce; Singal, Ashwani; Balwani, Manisha | Abstract: BackgroundErythropoietic protoporphyria (EPP) and X-linked Protoporphyria (XLP) are rare photodermatoses presenting with severe phototoxicity. Although anecdotally, providers who treat EPP patients acknowledge their life-altering effects, tools that fully capture their impact on quality of life (QoL) are lacking.MethodsAdult patients with EPP/XLP were given four validated QoL tools: the Patient Reported Outcomes Measurement Information System 57 (PROMIS-57), the Hospital Anxiety and Depression Scale (HADS), the Illness Perception Questionnaire Revised (IPQR), and an EPP-Specific tool. All patients received the PROMIS-57 while the HADS, IPQR, and EPP-Specific tools were introduced at a later date. Associations between responses and clinical phenotypes were explored.ResultsTwo hundred and two patients were included; 193 completed PROMIS-57, 104 completed IPQR, 103 completed HADS, and 107 completed the EPP-Specific tool. The IPQR showed that patients strongly believed EPP/XLP had a negative impact on their lives. Mean scores in anxiety and depression domains of both HADS and PROMIS-57 were normal; however, anxiety scores from HADS were borderline/abnormal in 20% of patients. The EPP-Specific tool revealed a decreased QoL in most patients. The PROMIS-57 showed that 21.8% of patients have clinically significant pain interference. Several tool domains correlated with measures of disease severity, most being from the PROMIS-57.ConclusionsImpaired QoL is an important consequence of EPP/XLP. PROMIS-57 was most sensitive in evaluating impaired QoL in EPP/XLP. Further research is needed to compare the effectiveness of it for assessing response to treatment.

BACKGROUND AND AIMS: Acute intermittent porphyria (AIP) results from a partial deficiency of porphobilinogen deaminase (PBGD). Symptomatic AIP patients, most of whom are women, experience acute attacks characterized by severe abdominal pain and abrupt increases in blood pressure. Here, we characterized the reactivity of mesenteric arteries from male and female AIP mice with ~30% of normal PBGD activity and wild type C57BL/6 mice. METHODS: An acute porphyric attack was induced in AIP mice by treatment with phenobarbital. Vascular responses to K(+), phenylephrine (PE), acetylcholine (ACh), and hemin were determined (Wire Multi Myograph). RESULTS: Maximal contraction to PE was increased in arteries from male and female AIP mice (p

Erythropoietic protoporphyria (EPP), the most common porphyria of childhood and the third most common porphyria of adulthood, is characterized clinically by painful, non-blistering cutaneous photosensitivity. Two distinct inheritance patterns involving mutations affecting genes that encode enzymes of the heme biosynthetic pathway underlie the clinical phenotype. Aminolevulinic acid synthase 2 (ALAS2), the rate limiting enzyme of the heme pathway in the erythron, is a therapeutic target in EPP because inhibiting enzyme function would reduce downstream production of protoporphyrin IX (PPIX), preventing accumulation of the toxic molecule and thereby ameliorating symptoms. Isoniazid (INH) is widely used for treatment of latent and active M. tuberculosis (TB). Sideroblastic anemia is observed in some patients taking INH, and studies have shown that this process is a consequence of inhibition of ALAS2 by INH. Based on these observations, we postulated that INH might have therapeutic activity in patients with EPP. We challenged this hypothesis in a murine model of EPP and showed that, after 4 weeks of treatment with INH, both plasma PPIX and hepatic PPIX were significantly reduced. Next, we tested the effect of INH on patients with EPP. After eight weeks, no significant difference in plasma or red cell PPIX was observed among the 15 patients enrolled in the study. These results demonstrate that while INH can lower PPIX in an animal model of EPP, the standard dose used to treat TB is insufficient to affect levels in humans.

Mouse models of the human porphyrias have proven useful for investigations of disease pathogenesis and to facilitate the development of new therapeutic approaches. To date, mouse models have been generated for the major porphyrias, with the exception of X-linked protoporphyria (XLP) and the ultra rare 5-aminolevulinic acid dehydratase deficiency porphyria (ADP). Mouse models have been generated for all three autosomal dominant acute hepatic porphyrias, acute intermittent porphyria (AIP), hereditary coproporphyria (HCP), and variegate porphyria (VP). The AIP mice, in particular, provide a useful investigative model as they have been shown to have acute biochemical attacks when induced with the prototypic porphyrinogenic drug, phenobarbital. In addition to providing important insights into the disease pathogenesis of the neurological impairment in AIP, these mice have been valuable for preclinical evaluation of liver-targeted gene therapy and RNAi-mediated approaches. Mice with severe HMBS deficiency, which clinically and biochemically mimic the early-onset homozygous dominant AIP (HD-AIP) patients, have been generated and were used to elucidate the striking phenotypic differences between AIP and HD-AIP. Mice modeling the hepatocutaneous porphyria, porphyria cutanea tarda (PCT), made possible the identification of the iron-dependent inhibitory mechanism of uroporphyrinogen III decarboxylase (UROD) that leads to symptomatic PCT. Mouse models for the two autosomal recessive erythropoietic porphyrias, congenital erythropoietic porphyria (CEP) and erythropoeitic protoporphyria (EPP), recapitulate many of the clinical and biochemical features of the severe human diseases and have been particularly useful for evaluation of bone marrow transplantation and hematopoietic stem cell (HSC)-based gene therapy approaches. The EPP mice have also provided valuable insights into the underlying pathogenesis of EPP-induced liver damage and anemia.

We conducted a consented pilot newborn screening (NBS) for Pompe, Gaucher, Niemann–Pick A/B, Fabry, and MPS 1 to assess the suitability of these lysosomal storage disorders (LSDs) for public health mandated screening. At five participating high–birth rate, ethnically diverse New York City hospitals, recruiters discussed the study with postpartum parents and documented verbal consent. Screening on consented samples was performed using multiplexed tandem mass spectrometry. Screen-positive infants underwent confirmatory enzymology, DNA testing, and biomarker quantitation when available. Affected infants are being followed for clinical management and long-term outcome. Over 4 years, 65,605 infants participated, representing an overall consent rate of 73%. Sixty-nine infants were screen-positive. Twenty-three were confirmed true positives, all of whom were predicted to have late-onset phenotypes. Six of the 69 currently have undetermined disease status. Our results suggest that NBS for LSDs is much more likely to detect individuals at risk for late-onset disease, similar to results from other NBS programs. This work has demonstrated the feasibility of using a novel consented pilot NBS study design that can be modified to include other disorders under consideration for public health implementation as a means to gather critical evidence for evidence-based NBS practices.

Acute intermittent porphyria (AIP) is an inborn error of heme biosynthesis due to the deficiency of hydroxymethylbilane synthase (HMBS) activity. Human AIP heterozygotes have episodic acute neurovisceral attacks that typically start after puberty, whereas patients with homozygous dominant AIP (HD-AIP) have early-onset chronic neurological impairment, including ataxia and psychomotor retardation. To investigate the dramatically different manifestations, knock-in mice with human HD-AIP missense mutations c.500G>A (p.Arg167Glu) or c.518_519GC>AG (p.Arg173Glu), designated R167Q or R173Q mice, respectively, were generated and compared with the previously established T1/T2 mice with ~30% residual HMBS activity and the heterozygous AIP phenotype. Homozygous R173Q mice were embryonic lethal, while R167Q homozygous mice (R167Q(+/+)) had ~5% of normal HMBS activity, constitutively elevated plasma and urinary 5-aminolevulinic acid (ALA) and porphobilinogen (PBG), profound early-onset ataxia, delayed motor development and markedly impaired rotarod performance. Central nervous system (CNS) histology was grossly intact, but CNS myelination was delayed and overall myelin volume was decreased. Heme concentrations in liver and brain were similar to those of T1/T2 mice. Notably, ALA and PBG concentrations in the cerebral spinal fluid and CNS regions were markedly elevated in R167Q(+/+) mice compared with T1/T2 mice. When the T1/T2 mice were administered phenobarbital, ALA and PBG markedly accumulated in their liver and plasma, but not in the CNS, indicating that ALA and PBG do not readily cross the blood–brain barrier. Taken together, these studies suggest that the severe HD-AIP neurological phenotype results from decreased myelination and the accumulation of locally produced neurotoxic porphyrin precursors within the CNS.

Aim: To comprehensively interrogate CYP2D6 by integrating genotyping, copy number analysis and novel strategies to identify CYP2D6*36 and characterize CYP2D6 duplications. Methods: Genotyping of 16 CYP2D6 alleles, multiplex ligation-dependent probe amplification (MLPA) and CYP2D6*36 and duplication allele-specific genotyping were performed on 427 African–American, Asian, Caucasian, Hispanic, and Ashkenazi Jewish individuals. Results: A novel PCR strategy determined that almost half of all CYP2D6*10 (100C>T) alleles are actually *36 (isolated or in tandem with *10) and all identified duplication alleles were characterized. Integrated results from all testing platforms enabled the refinement of genotype frequencies across all studied populations. Conclusion: The polymorphic CYP2D6 gene requires comprehensive interrogation to characterize allelic variation across ethnicities, which was enabled in this study by integrating multiplexed genotyping, MLPA copy number analysis, novel PCR strategies and duplication allele-specific genotyping.

Acute intermittent porphyria (AIP), an autosomal dominant disorder due to the half-normal activity of hydroxymethylbilane synthase (HMBS), is characterized by acute neurovisceral attacks that are precipitated by factors that induce heme biosynthesis. Molecular diagnosis is the most sensitive and specific diagnostic test for AIP, and importantly, it permits the identification of asymptomatic family members for genetic counseling and avoidance of precipitating factors. Here, we report the identification of 40 novel HMBS mutations, including 11 missense, four nonsense, 16 small insertions or deletions, eight consensus splice site mutations, and a complex insertion-deletion mutation in unrelated individuals with AIP. Prokaryotic expression of the missense mutations demonstrated that all mutants had ≤5% of expressed wildtype activity, except for c.1039G>C (p.A347P), which had 51% residual HMBS activity but was markedly thermolabile. Of note, the mutation c.612G>T (p.Q204H) altered the last nucleotide of exon 10, which resulted in an alternative HMBS transcript with an in-frame nine base-pair deletion at the 3'-terminus of exon 10 (encoding protein Q204HΔ3). When expressed, Q204HΔ3 and an in-frame three base-pair deletion (c.639_641delTGC) had no detectable HMBS activity. Western blot analyses and mapping of the missense mutations on the human HMBS crystal structure revealed that mutations near the active site or at the dimerization interface resulted in stably expressed proteins, while most that altered surface residues resulted in unstable proteins, presumably due to improper protein folding. These studies identified novel pathogenic HMBS mutations and expanded the molecular heterogeneity of AIP.

The SLC6A4 gene has been implicated in psychiatric disorder susceptibility and antidepressant response variability. The SLC6A4 promoter is defined by a variable number of homologous 20&ndash, 24 bp repeats (5-HTTLPR), and long (L) and short (S) alleles are associated with higher and lower expression, respectively. However, this insertion/deletion variant is most informative when considered as a haplotype with the rs25531 and rs25532 variants. Therefore, we developed a long-read single molecule real-time (SMRT) sequencing method to interrogate the SLC6A4 promoter region. A total of 120 samples were subjected to SLC6A4 long-read SMRT sequencing, primarily selected based on available short-read sequencing data. Short-read genome sequencing from the 1000 Genomes (1KG) Project (~5X) and the Genetic Testing Reference Material Coordination Program (~45X), as well as high-depth short-read capture-based sequencing (~330X), could not identify the 5-HTTLPR short (S) allele, nor could short-read sequencing phase any identified variants. In contrast, long-read SMRT sequencing unambiguously identified the 5-HTTLPR short (S) allele (frequency of 0.467) and phased SLC6A4 promoter haplotypes. Additionally, discordant rs25531 genotypes were reviewed and determined to be short-read errors. Taken together, long-read SMRT sequencing is an innovative and robust method for phased resolution of the SLC6A4 promoter, which could enable more accurate pharmacogenetic testing for both research and clinical applications.

To review the peripheral neurological complications of the acute hepatic porphyrias, as well as the latest advances in their pathophysiology and management. The diagnosis of porphyric neuropathy remains challenging as varying neuropathic patterns are encountered depending on disease stage, including a non-length-dependent distribution pattern. The major pathophysiologic mechanism is δ-aminolevulinic acid (ALA)–induced neurotoxicity. The less restrictive blood-nerve barrier in the autonomic ganglia and myenteric plexus may explain the frequency of dysautonomic manifestations. Recently, a prophylactic small interfering RNA (siRNA)-based therapy that reduces hepatic ALA Synthase-1 mRNA was approved for patients with recurrent neuro-visceral attacks. Neurologists should appreciate the varying patterns of porphyric neuropathy. As with most toxin-induced axonopathies, long-term outcomes depend on early diagnosis and treatment. While the short-term clinical and biochemical benefits of siRNA-based therapy are known, its long-term effects on motor recovery, chronic pain, and dysautonomic manifestations are yet to be determined.

To review the peripheral neurological complications of the acute hepatic porphyrias, as well as the latest advances in their pathophysiology and management.The diagnosis of porphyric neuropathy remains challenging as varying neuropathic patterns are encountered depending on disease stage, including a non-length-dependent distribution pattern. The major pathophysiologic mechanism is δ-aminolevulinic acid (ALA)-induced neurotoxicity. The less restrictive blood-nerve barrier in the autonomic ganglia and myenteric plexus may explain the frequency of dysautonomic manifestations. Recently, a prophylactic small interfering RNA (siRNA)-based therapy that reduces hepatic ALA Synthase-1 mRNA was approved for patients with recurrent neuro-visceral attacks. Neurologists should appreciate the varying patterns of porphyric neuropathy. As with most toxin-induced axonopathies, long-term outcomes depend on early diagnosis and treatment. While the short-term clinical and biochemical benefits of siRNA-based therapy are known, its long-term effects on motor recovery, chronic pain, and dysautonomic manifestations are yet to be determined.

Fabry disease, a lysosomal storage disorder resulting from the deficient activity of α-galactosidase A (α-Gal A), is characterized by cardiac, renal, and/or cerebrovascular disease due to progressive accumulation of the enzyme's substrates, globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3). We report here the preclinical evaluation of liver-targeted in vivo genome editing using zinc-finger nuclease (ZFN) technology to insert the human α-galactosidase A (hGLA) cDNA into the albumin "safe harbor" locus of Fabry mice, thereby generating an albumin-α-Gal A fusion protein. The mature α-Gal A protein is secreted into the circulation for subsequent mannose-6-phosphate receptor-mediated tissue uptake. Donor vector optimization studies showed that replacing the hGLA cDNA signal peptide sequence with that of human iduronate 2-sulfatase (IDS) achieved higher transgene expression. Intravenous adeno-associated virus (AAV) 2/8-mediated co-delivery of the IDS-hGLA donor and ZFNs targeting the albumin locus resulted in continuous, supraphysiological plasma and tissue α-Gal A activities, which essentially normalized Gb3 and Lyso-Gb3 levels in key tissues of pathology. Notably, this was achieved with10% of hepatocytes being edited to express hGLA, occurring mostly via non-homologous end joining (NHEJ) rather than homology-directed repair (HDR). These studies indicate that ZFN-mediated in vivo genome editing has the potential to be an effective one-time therapy for Fabry disease.

5′-aminolevulinate synthase (ALAS) catalyzes the first step in heme biosynthesis, generating 5′-aminolevulinate from glycine and succinyl-CoA. Inherited frameshift indel mutations of human erythroid-specific isozyme ALAS2, within a C-terminal (Ct) extension of its catalytic core that is only present in higher eukaryotes, lead to gain-of-function X-linked protoporphyria (XLP). Here, we report the human ALAS2 crystal structure, revealing that its Ct-extension folds onto the catalytic core, sits atop the active site, and precludes binding of substrate succinyl-CoA. The Ct-extension is therefore an autoinhibitory element that must re-orient during catalysis, as supported by molecular dynamics simulations. Our data explain how Ct deletions in XLP alleviate autoinhibition and increase enzyme activity. Crystallography-based fragment screening reveals a binding hotspot around the Ct-extension, where fragments interfere with the Ct conformational dynamics and inhibit ALAS2 activity. These fragments represent a starting point to develop ALAS2 inhibitors as substrate reduction therapy for porphyria disorders that accumulate toxic heme intermediates., Mutation of the C-terminal extension of 5′-aminolevulinate synthase 2 (ALAS2) is the molecular cause for X-linked protoporphyria, but the underlying mechanism is unclear. Based on crystal structures and MD simulations of ALAS2, the authors here show how the C-terminal extension regulates ALAS2 activity.

EVIDENCE, an automated variant prioritization system, has been developed to facilitate whole exome sequencing analyses. This study investigated the diagnostic yield of EVIDENCE in patients with suspected genetic disorders. DNA from 330 probands (age range, 0‐68 years) with suspected genetic disorders were subjected to whole exome sequencing. Candidate variants were identified by EVIDENCE and confirmed by testing family members and/or clinical reassessments. EVIDENCE reported a total 228 variants in 200 (60.6%) of the 330 probands. The average number of organs involved per patient was 4.5 ± 5.0. After clinical reassessment and/or family member testing, 167 variants were identified in 141 probands (42.7%), including 105 novel variants. These variants were confirmed as being responsible for 121 genetic disorders. A total of 103 (61.7%) of the 167 variants in 95 patients were classified as pathogenic or probably to be pathogenic before, and 161 (96.4%) variants in 137 patients (41.5%) after, clinical assessment and/or family member testing. Factor associated with a variant being regarded as causative includes similar symptom scores of a gene variant to the phenotype of the patient. This new, automated variant interpretation system facilitated the diagnosis of various genetic diseases with a 42.7% diagnostic yield., Schematic diagram showing the number of patients with and without variant identification and family member testing and the proportion of variant classification.

Acute 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. EXPLORE 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 prior to 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 healthcare 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 healthcare utilization. Conclusions: Patients experienced attacks often requiring treatment in a healthcare facility and/or with hemin, as well as chronic symptoms that adversely influence day-to-day functioning. In this patient group, the high disease burden and diminished quality of life highlight the need for novel therapies. This article is protected by copyright. All rights reserved.

Acute intermittent porphyria (AIP) is an autosomal dominant inborn error of heme biosynthesis due to a pathogenic mutation in the Hmbs gene, resulting in half-normal activity of hydroxymethylbilane synthase. Factors that induce hepatic heme biosynthesis induce episodic attacks in heterozygous patients. The clinical presentation of acute attacks involves the signature neurovisceral pain and may include psychiatric symptoms. Here we used a knock-in mouse line that is biallelic for the Hmbs c.500G > A (p.R167Q) mutation with ~ 5% of normal hydroxymethylbilane synthase activity to unravel the consequences of severe HMBS deficiency on affective behavior and brain physiology. Hmbs knock-in mice (KI mice) model the rare homozygous dominant form of AIP and were used as tool to elucidate the hitherto unknown pathophysiology of the behavioral manifestations of the disease and its neural underpinnings. Extensive behavioral analyses revealed a selective depression-like phenotype in Hmbs KI mice; transcriptomic and immunohistochemical analyses demonstrated aberrant myelination. The uncovered compromised mitochondrial function in the hippocampus of knock-in mice and its ensuing neurogenic and neuroplastic deficits lead us to propose a mechanistic role for disrupted mitochondrial energy production in the pathogenesis of the behavioral consequences of severe HMBS deficiency and its neuropathological sequelae in the brain.

PurposeAcute intermittent porphyria (AIP) is a rare inborn error of heme biosynthesis characterized by life-threatening acute attacks. Few studies have assessed quality of life (QoL) in AIP and those that have had small sample sizes and used tools that may not have captured important domains.MethodsBaseline data from the Porphyrias Consortium's Longitudinal Study were obtained for 259 patients, including detailed disease and medical history data, and the following Patient-Reported Outcomes Measurement Information System (PROMIS) scales: anxiety, depression, pain interference, fatigue, sleep disturbance, physical function, and satisfaction with social roles. Relationships between PROMIS scores and clinical and biochemical AIP features were explored.ResultsPROMIS scores were significantly worse than the general population across all domains, except depression. Each domain discriminated well between asymptomatic and symptomatic patients with symptomatic patients having worse scores. Many important clinical variables like symptom frequency were significantly associated with domain scores in univariate analyses, showing responsiveness of the scales, specifically pain interference and fatigue. However, most regression models only explained ~20% of the variability observed in domain scores.ConclusionPain interference and fatigue were the most responsive scales in measuring QoL in this AIP cohort. Future studies should assess whether these scales capture longitudinal disease progression and treatment response.

Two distinct metabolic derangements are included under the eponym “Niemann–Pick disease (NPD)”. The first is due to the deficient activity of the lysosomal enzyme acid sphingomyelinase (ASM). Patients with this disorder [also referred to as ASM-deficient (ASMD) NPD] are broadly classified as having types A, A/B, and B NPD depending on their degree of neurologic involvement. Type A NPD patients, also known as the infantile neurovisceral form, exhibit hepatosplenomegaly in infancy and profound central nervous system (CNS) involvement. They rarely survive beyond 2–3 years of age. Type B patients, also known as the chronic visceral form, have hepatosplenomegaly and pathologic alterations of their lungs, but there are usually no CNS signs. The age of onset and rate of disease progression varies greatly among type B patients, and many may survive into adulthood. Patients with phenotypes intermediate between types A and B NPD (type A/B), also known as the chronic neurovisceral form, also have been identified. These individuals represent the expected continuum caused by inheriting different mutations in the ASM gene (SMPD1). Patients in the second NPD category are designated as having types C and D NPD. These individuals may have mild hepatosplenomegaly, but the CNS is profoundly affected. Impaired intracellular trafficking of cholesterol causes types C and D NPD, and two distinct gene defects have been found. All type D patients originate from a common Nova Scotian ancestry.