Kenneth H. Astrin

Fabry disease: six gene rearrangements and an exonic point mutation in the alpha-galactosidase gene.

Fabry disease, an X-linked recessive disorder of glycosphingolipid catabolism, results from the deficient activity of the lysosomal hydrolase, alpha-galactosidase. Southern hybridization analysis of the alpha-galactosidase gene in affected hemizygous males from 130 unrelated families with Fabry disease revealed six with different gene rearrangements and one with an exonic point mutation resulting in the obliteration of an Msp I restriction site. Five partial gene deletions were detected ranging in size from 0.4 to greater than 5.5 kb. Four of these deletions had breakpoints in intron 2, a region in the gene containing multiple Alu repeat sequences. A sixth genomic rearrangement was identified in which a region of about 8 kb, containing exons 2 through 6, was duplicated by a homologous, but unequal crossover event. The Msp I site obliteration, which mapped to exon 7, was detected in an affected hemizygote who had residual enzyme activity. Genomic amplification by the polymerase chain reaction and sequencing revealed that the obliteration resulted from a C to T transition at nucleotide 1066 in the coding sequence. This point mutation, the first identified in Fabry disease, resulted in an arginine356 to tryptophan356 substitution which altered the enzymes kinetic and stability properties. The detection of these abnormalities provided for the precise identification of Fabry heterozygotes, thereby permitting molecular pedigree analysis in these families which revealed paternity exclusions and the first documented new mutations in this disease.

Human δ-aminolevulinate synthase: Assignment of the housekeeping gene to 3p21 and the erythroid-specific gene to the X chromosome

delta-Aminolevulinate synthase (ALAS) catalyzes the first committed step of heme biosynthesis. Previous studies suggested that there were erythroid and nonerythroid ALAS isozymes. We have isolated cDNAs encoding the ubiquitously expressed housekeeping ALAS isozyme and a related, but distinct, erythroid-specific isozyme. Using these different cDNAs, the human ALAS housekeeping gene (ALAS1) and the human erythroid-specific (ALAS2) gene have been localized to chromosomes 3p21 and X, respectively, by somatic cell hybrid and in situ hybridization techniques. The ALAS1 gene was concordant with chromosome 3 in all 26 human fibroblast/murine(RAG) somatic cell hybrid clones analyzed and was discordant with all other chromosomes in at least 6 of 26 clones. The regional localization of ALAS1 to 3p21 was accomplished by in situ hybridization using the 125I-labeled human ALAS1 cDNA. Of the 43 grains observed over chromosome 3, 63% were localized to the region 3p21. The gene encoding ALAS2 was assigned by examination of a DNA panel of 30 somatic cell hybrid lines hybridized with the ALAS2 cDNA. The ALAS2 gene segregated with the human X chromosome in all 30 hybrid cell lines analyzed and was discordant with all other chromosomes in at least 8 of the 30 hybrids. These results confirm the existence of two independent, but related, genes encoding human ALAS. Furthermore, the mapping of the ALAS2 gene to the X chromosome and the observed reduction in ALAS activity in X-linked sideroblastic anemia suggest that this disorder may be due to a mutation in the erythroid-specific gene.

δ‐Aminolevulinic Acid Dehydratase Isozymes and Lead Toxicitya

ALAD is a zinc metalloenzyme whose inhibition by lead is the first and most sensitive indicator of lead exposure and whose decreased activity has been implicated in the pathogenesis of lead poisoning. This heme biosynthetic enzyme is encoded by a gene located at chromosome 9q34, which has two codominant alleles, ALAD1 and ALAD2. The occurrence of two frequent alleles for ALAD stimulated an investigation into the possible pharmacogenetic role of the enzyme polymorphism in lead poisoning. In a New York City population at high risk for lead exposure, individuals heterozygous or homozygous for the less common allele, ALAD2, had blood lead levels greater than or equal to 30 micrograms/dl more frequently than expected. These findings suggest a potential genetic susceptibility to lead poisoning in individuals with the ALAD 1-2 and 2-2 phenotypes.

Congenital erythropoietic porphyria: advances in pathogenesis and treatment

The inherited porphyrias are a diverse group of inborn errors of haem biosynthesis, each resulting from the deficient activity of a specific enzyme in the pathway (Fig 1). Porphyrias are classified as either hepatic or erythropoietic, according to whether the excess production of porphyrin precursors and porphyrins occurs primarily in the liver or in the erythron (Anderson et al, 2001). Patients with erythropoietic porphyrias have elevated bone marrow and erythrocyte porphyrins, and their clinical manifestations usually include anaemia and ⁄ or cutaneous photosensitivity causing mild to severe dermatological involvement. The erythropoietic porphyrias include congenital erythropoietic porphyria (CEP) and erythropoietic protoporphyria (autosomal dominant ferrochelatase deficiency). The inheritance patterns, enzymatic defects and accumulated porphyrins in the erythropoietic porphyrias are shown in Table I. CEP results from the markedly deficient, but not absent, activity of the fourth enzyme of the haem biosynthetic pathway, uroporphyrinogen III synthase (URO-synthase; EC 4Æ2.1Æ75) (Romeo & Levin, 1969; Anderson et al, 2001). In patients with this autosomal recessive disease, the clinical manifestations are markedly heterogeneous, ranging from non-immune hydrops fetalis to milder, later onset forms which have only cutaneous lesions in adult life (Desnick et al, 1998). Severely affected patients are transfusiondependent throughout life, have secondary hypersplenism and marked cutaneous involvement. Here, we present the clinical features, molecular genetics and pathology, and the successful treatment by bone marrow transplantation (BMT) of patients with this erythropoietic porphyria.

Uroporphyrinogen III synthase erythroid promoter mutations in adjacent GATA1 and CP2 elements cause congenital erythropoietic porphyria.

Congenital erythropoietic porphyria, an autosomal recessive inborn error of heme biosynthesis, results from the markedly deficient activity of uroporphyrinogen III synthase. Extensive mutation analyses of 40 unrelated patients only identified approximately 90% of mutant alleles. Sequencing the recently discovered erythroid-specific promoter in six patients with a single undefined allele identified four novel mutations clustered in a 20-bp region: (a) a -70T to C transition in a putative GATA-1 consensus binding element, (b) a -76G to A transition, (c) a -86C to A transversion in three unrelated patients, and (d) a -90C to A transversion in a putative CP2 binding motif. Also, a -224T to C polymorphism was present in approximately 4% of 200 unrelated Caucasian alleles. We inserted these mutant sequences into luciferase reporter constructs. When transfected into K562 erythroid cells, these constructs yielded 3 +/- 1, 54 +/- 3, 43 +/- 6, and 8 +/- 1%, respectively, of the reporter activity conferred by the wild-type promoter. Electrophoretic mobility shift assays indicated that the -70C mutation altered GATA1 binding, whereas the adjacent -76A mutation did not. Similarly, the -90C mutation altered CP2 binding, whereas the -86A mutation did not. Thus, these four pathogenic erythroid promoter mutations impaired erythroid-specific transcription, caused CEP, and identified functionally important GATA1 and CP2 transcriptional binding elements for erythroid-specific heme biosynthesis.

Twenty novel mutations in the alpha-galactosidase A gene causing Fabry disease.

BackgroundFabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from the deficient activity of the lysosomal exoglycohydrolase α-galactosidase A (EC 3.2.1.22; α-Gal A). The nature of the molecular lesions in the α-Gal A gene in 30 unrelated families was determined to provide precise heterozygote detection, prenatal diagnosis, and define genotype-phenotype correlations.Materials and MethodsGenomic DNA was isolated from affected males and/or carrier females from 30 unrelated families with Fabry disease. The entire α-Gal A coding region and flanking intronic sequences were analyzed by PCR amplification and automated sequencing.ResultsTwenty new mutations were identified, each in a single family: C142R, G183D, S235C, W236L, D244H, P259L, M267I, I289F, Q321E, C378Y, C52X, W277X, IVS4+4, IVS6+2, IVS6−1, 35del13, 256del1, 892ins1, 1176del4, and 1188del1. In the remaining 10 unrelated Fabry families, 9 previously reported mutations were detected: M42V, R112C, S148R, D165V, N215S (in 2 families), Q99X, C142X, R227X, and 1072del3. Haplotype analysis using markers closely flanking the α-Gal A gene indicated that the two patients with the N215S lesion were unrelated. The IVS4+4 mutation was a rare intronic splice site mutation that causes Fabry disease.ConclusionsThese studies further define the heterogeneity of mutations in the α-Gal A gene causing Fabry disease, permit precise heterozygote detection and prenatal diagnosis, and help delineate phenotype-genotype correlations in this disease.

Familial Porphyria Cutanea Tarda: Characterization of Seven Novel Uroporphyrinogen Decarboxylase Mutations and Frequency of Common Hemochromatosis Alleles

Familial porphyria cutanea tarda (f-PCT) results from the half-normal activity of uroporphyrinogen decarboxylase (URO-D). Heterozygotes for this autosomal dominant trait are predisposed to photosensitive cutaneous lesions by various ecogenic factors, including iron overload and alcohol abuse. The 3.6-kb URO-D gene was completely sequenced, and a long-range PCR method was developed to amplify the entire gene for mutation analysis. Four missense mutations (M165R, L195F, N304K, and R332H), a microinsertion (g10insA), a deletion (g645Delta1053), and a novel exonic splicing defect (E314E) were identified. Expression of the L195F, N304K, and R332H polypeptides revealed significant residual activity, whereas reverse transcription-PCR and sequencing demonstrated that the E314E lesion caused abnormal splicing and exon 9 skipping. Haplotyping indicated that three of the four families with the g10insA mutation were unrelated, indicating that these microinsertions resulted from independent mutational events. Screening of nine f-PCT probands revealed that 44% were heterozygous or homozygous for the common hemochromatosis mutations, which suggests that iron overload may predispose to clinical expression. However, there was no clear correlation between f-PCT disease severity and the URO-D and/or hemochromatosis genotypes. These studies doubled the number of known f-PCT mutations, demonstrated that marked genetic heterogeneity underlies f-PCT, and permitted presymptomatic molecular diagnosis and counseling in these families to enable family members to avoid disease-precipitating factors.

Acute intermittent porphyria: identification and expression of exonic mutations in the hydroxymethylbilane synthase gene. An initiation codon missense mutation in the housekeeping transcript causes "variant acute intermittent porphyria" with normal expression of the erythroid-specific enzyme.

Acute intermittent porphyria (AIP), an autosomal dominant inborn error, results from the half-normal activity of the heme biosynthetic enzyme, hydroxymethylbilane synthase (EC 4.3.1.8). Diagnosis of AIP heterozygotes is essential to prevent acute, life-threatening neurologic attacks by avoiding various precipitating factors. Since biochemical diagnosis is problematic, the identification of hydroxymethylbilane synthase mutations has facilitated the detection of AIP heterozygotes. Molecular analyses of unrelated AIP patients revealed six exonic mutations: an initiating methionine to isoleucine substitution (M1I) in a patient with variant AIP, which precluded translation of the housekeeping, but not the erythroid-specific isozyme; four missense mutations in classical AIP patients, V93F, R116W, R201W, C247F; and a nonsense mutation W283X in a classical AIP patient, which truncated the housekeeping and erythroid-specific isozymes. Each mutation was confirmed in genomic DNA from family members. The W283X lesion was found in another unrelated AIP family. Expression of each mutation in Escherichia coli revealed that R201W, C247F, and W283X had residual activity. In vitro transcription/translation studies indicated that the M1I allele produced only the erythroid-specific enzyme, while the other mutant alleles encoded both isozymes. These mutations provide insight into the molecular pathology of classic and variant AIP and facilitate molecular diagnosis in AIP families.

Regional assignment of the human uroporphyrinogen III synthase (UROS) gene to chromosome 10q25.2→q26.3

SummaryUroporphyrinogen III synthase [UROS; hydroxymethylbilane hydro-lyase (cyclizing), EC 4.2.1.75] is the fourth enzyme in the human heme biosynthetic pathway. The recent isolation of the cDNA encoding human UROS facilitated its chromosomal localization. Human UROS sequences were specifically amplified by the polymerase chain reaction (PCR) from genomic DNA of two independent panels of human-rodent somatic cell hybrids. There was 100% concordance for the presence of the human UROS PCR product and human chromosome 10. For each of the other chromosomes, there was 19%–53% discordance with human UROS. The chromosomal assignment was confirmed by Southern hybridization analysis of DNA from somatic cell hybrids with the full-length UROS cDNA. Using human-rodent hybrids containing different portions of human chromosome 10, we assigned the UROS gene to the region 10q25.2→ q26.3.

Uroporphyrinogen III Synthase AN ALTERNATIVE PROMOTER CONTROLS ERYTHROID-SPECIFIC EXPRESSION IN THE MURINE GENE

Uroporphyrinogen III synthase (URO-synthase, EC4.2.1.75) is the fourth enzyme of the heme biosynthetic pathway and is the defective enzyme in congenital erythropoietic porphyria. To investigate the erythroid-specific expression of murine URO-synthase, the cDNA and ∼24-kilobase genomic sequences were isolated and characterized. Three alternative transcripts were identified containing different 5′-untranslated regions (5′-UTRs), but identical coding exons 2B through 10. Transcripts with 5′-UTR exon 1A alone or fused to exon 1B were ubiquitously expressed (housekeeping), whereas transcripts with 5′-UTR exon 2A were only present in erythroid cells (erythroid-specific). Analysis of the TATA-less housekeeping promoter upstream of exon 1A revealed binding sites for ubiquitously expressed transcription factors Sp1, NF1, AP1, Oct1, and NRF2. The TATA-less erythroid-specific promoter upstream of exon 2A had nine putative GATA1 erythroid enhancer binding sites. Luciferase promoter/reporter constructs transfected into NIH 3T3 and mouse erythroleukemia cells indicated that the housekeeping promoter was active in both cell lines, while the erythroid promoter was active only in erythroid cells. Site-specific mutagenesis of the first GATA1 binding site markedly reduced luciferase activity in K562 cells (<5% of wild type). Thus, housekeeping and erythroid-specific transcripts are expressed from alternative promoters of a single mouse URO-synthase gene.