Acute intermittent porphyria (AIP) is an autosomal dominant disorder caused by a partial deficiency of hydroxymethylbilane synthase (HMBS), the third enzyme of the heme biosynthetic pathway. The clinical features of AIP are intermittent attacks of neurological dysfunction, including abdominal pain and neuropsychiatric symptoms. Most patients remain asymptomatic throughout their life but 10–20% have severe acute attacks. AIP is normally diagnosed on the basis of clinical symptoms and urinary overproduction of porphyrin precursors, d-aminolevulinic acid (ALA) and porphobilinogen (PBG). The measurement of erythrocyte HMBS activity can confirm the diagnosis and it is also used to detect asymptomatic relatives. So far, more than 290 different mutations in the HMBS gene have been identified among AIP patients. Most of the reported mutations have been detected only in single families. In an Italian family with typical clinical and biochemical signs of AIP, no mutation was found by direct sequencing of 5′ UTR, 3′ UTR, erythroid promoter and the entire coding region of HMBS gene. All sympomatic patients showed apparent homozigosity for eleven common polymorphisms along the HMBS gene. Further family studies established the absence of Mendelian segregation. Excluding interference of polymorphisms in the primer sites, we assumed the presence of a complete deletion of the HMBS gene. In order to verify this hypothesis we performed a semiquantitative RT-PCR analysis on fresh RNA and different long PCR semiquantitative analysis on DNA. All analyses revealed the presence of a normal length fragment which was less abundant compared to controls. In order to identify the size of the deletion, SNPs analysis was extended to flanking genes, H2A Histone Family member X (H2AX) and Dolichyl-Phosphate N-Acetylglucosamine Phosphotransferase 1 (DPAGT1), downstream and Vacuolar protein sorting 11 (VPS11), upstream. We found heterozygotic polymorphisms in VPS11 and DPAGT1 genes. We performed XL-PCR of 19317bp with primers situated in regions outside the heterozygotic polymorphisms that showed a shorter band of 5434bp. Sequence analysis of the abnormal fragment revealed a loss of 77 intergenic base pairs upstream of the HMBS and H2AX genes and 2500bp of DPAGT1 gene. Simultaneously, an inverted complementary sequence of 223bp of intron 9 of HMBS gene was incorporated. Use of ElDorado-Genomatix software revealed the presence of different Alu sequences near breakpoints, that could cause this deletion. The H2AX gene is a member of a classical gene family and its loss has no adverse effects on phenotype. Defects in DPAGT1 are the cause of a congenital disorder of glycosylation type Ij, characterized by severe mental and psychomotor retardation. Our patients had no other symptoms than AIP, supporting the suggestion that DPAGT1 inheritance is probably recessive.

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