Identification and characterization of hydroxymethylbilane synthase mutations causing acute intermittent porphyria: Evidence for an ancestral founder of the common G111R mutation

Abstract

Acute intermittent porphyria (AIP), the most common hepatic porphyria, results from the half-normal activity of hydroxymethylbilane synthase (HMB-synthase; EC 4.3.1.8), the third enzyme in the heme biosynthetic pathway. Because life-threatening acute neurologic attacks of this autosomal dominant disease are triggered by various ecogenic factors (e.g., certain drugs, hormones, alcohol, and starvation), efforts have been directed to identify and counsel presymptomatic heterozygotes in affected families to avoid the precipitating factors. Thus, to determine the nature of the mutations causing AIP in 26 unrelated enzyme-confirmed patients from Argentina, a long-range polymerase chain reaction method was developed to amplify the entire 10-kb gene in two fragments for efficient cycle sequencing and mutation detection. Eight new mutations were identified including two missense mutations (Q34P and G335S), four small deletions (728delCT, 815delAGGA, 948delA, and 985del12), a single base insertion (666insA), and a splice site mutation (IVS12(+1)). In addition, five previously reported mutations (G111R, R173W, Q204X, R201W, and 913insC) were detected. Notably, G111R was identified in 12 of the 26 (46%) presumably unrelated propositi; however, haplotype analysis with intragenic and flanking markers indicated an ancestral founder. Expression of the two new missense mutations (Q34P and G335S) in f1 E. coli resulted in 2.5% or less of the normal expressed enzyme, confirming their defective function. Thus, eight new and five previously reported HMB-synthase mutations, including a common lesion, were detected, permitting accurate identification and counseling of presymptomatic carriers in these 26 unrelated Argentinean AIP families with this dominant porphyria.

Two deletion mutations in the hydroxymethylbilane synthase gene in two unrelated Japanese patients with acute intermittent porphyria

“…Recently, this mutation has been documented as the first mutation in AIP associated with a founder effect in North America with the identified founding couple (Greene-Davis et al 1997). Such a founder effect has been well documented in variegate porphyria, the other form of acute porphyria (Dean 1971;Meissner et al 1996;Warnich et al 1996;Groenewald et al 1998), and has also been observed in other AIP patients (De Siervi et al 1999). The other mutation in Japan, reported by Morita et al (1995), is a single base substitution C-to-T in exon 8 of the gene, which was the same as a common mutation in the Netherlands (Gu et al 1994).…”

Hydroxymethylbilane Synthase Gene Mutations and Polymorphisms in Brazilian Families with Acute Intermittent Porphyria

“…It was also reported in Slavic (Rosipal et al, 1997), French , British (Whatley et al, 1999), Swedish (Andersson et al, 1995;and Floderus et al, 2002) and Polish patients . In Argentina, the G111R mutation was detected in 12 families (De Siervi et al, 1999) which seem Normal individuals showed one band whereas a heteroduplex was observed in asymptomatic gene carriers. (B).…”

“…This finding suggests that HMBS is very prone to mutations which are usually family specific, since in only a few cases has the same mutation been found in nonrelated AIP patients. For example, some populations may present the same mutation, as with the missense mutation R173W which occurs in nonrelated Scottish patients (Greene-Davis et al, 1997) and G111R detected in 12 Argentine families (De Siervi et al, 1999). This molecular variability requires gene investigation of the 15 exons by single-stranded conformational polymorphism (Kauppinen et al, 1995), denaturing gradient gel electrophoresis (Gu et al, 1994;Puy et al, 1997), chemical cleavage mismatch (Ong et al, 1998), heteroduplex (Schreiber et al, 1995) or high-resolution melting analysis (Ulbrichova-Douderova & Martasek, 2009).…”

Diagnosis of latent acute intermittent porphyria by genetic analysis

“…In this PCR protocol two sets of primers were used to amplify the entire PBG-D gene (10 kb) in two fragments, one including the promoter region to intron 3 (4.5 kb) and the other from exon 2 to exon 15 (5.5 kb). 5 The products obtained were assayed on 1% agarose gels stained with ethidium bromide and puri®ed employing the QIAquick PCR puri®cation kit from QIAGEN, USA.…”

“…5 The cycle sequencing reaction (30 cycles) was performed with a denaturation step of 30 s at 958C, annealing 30 s at 608C and extension 60 s at 728C. The reaction was stopped by adding 4.5 mL of stop solution (95% formamide, 20 mmol/L EDTA, 0.05% bromophenol blue and 0.02% xylene cyanole) and, after a denaturation step of 3 min at 948C, samples were loaded in a 4% acrylamide solution (19:1) containing 7 mol/L urea gel preheated at about 508C, and was run at 80 W for different periods depending on both the gene region to be read and the primer used.…”