Light chains of rabbit immunoglobulin: assignment to the kappa class.

Normal rabbit gamma globulin was reduced under conditions presumed to break only interchain disulfide bridges, and the reduiced product was then blocked with C(14)-iodoacetamide. The light chains were separated from the heavy chains and subjected to peptic digestion. Two radioactive peptides were recovered from the digest. The peptides are apparently overlapping and represent the carboxyterminuis. Comparison of this region in the rabbit light chains with the corresponding amino acid sequences in various mouse and human light chains indicates that the rabbit light chains are of the (K)-class.

Lesch-Nyhan syndrome: preventive control by prenatal diagnosis.

The Lesch-Nyhan syndrome was detected in a fetus at a time sufficiently early to allow termination of the pregnancy. The feasibility of a preventive program for control of a severe sex-linked neurological disease through prenatal diagnosis is thus demonstrated.

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.

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 enzyme's 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.

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.

Acute intermittent porphyria: novel missense mutations in the human hydroxymethylbilane synthase gene.

PURPOSE: To identify mutations in families with acute intermittent porphyria, an autosomal dominant inborn error of metabolism that results from the half-normal activity of the third enzyme in the heme biosynthetic pathway, hydroxymethylbilane synthase. METHODS: Mutations were identified by direct solid phase sequencing. RESULTS: Two novel missense mutations E80G and T78P and three previously reported mutations, R173W, G111R, and the splice site lesion, IVS1+1, were detected, each in an unrelated proband. The causality of the novel missense mutations was demonstrated by expression studies. CONCLUSION: These findings provide for the precise diagnosis of carriers in these families and further expand the molecular heterogeneity of AIP.

Acute intermittent porphyria: a single-base deletion and a nonsense mutation in the human hydroxymethylbilane synthase gene, predicting truncations of the enzyme polypeptide.

Acute intermittent porphyria (AIP) is an autosomal-dominant inborn error of metabolism that results from the half-normal activity of the third enzyme in the heme biosynthetic pathway, hydroxymethylbilane synthase (HMB-synthase). AIP is an ecogenetic condition, since the life-threatening acute attacks are precipitated by various factors, including drugs, alcohol, fasting, and certain hormones. Biochemical diagnosis is problematic, and the identification of mutations in the HMB-synthase gene provides accurate detection of presymptomatic heterozygotes, permitting avoidance of the acute precipitating factors. By direct solid-phase sequencing, two mutations causing AIP were identified, an adenine deletion at position 629 in exon 11(629delA), which alters the reading frame and predicts premature truncation of the enzyme protein after amino acid 255, and a nonsense mutation in exon 12 (R225X). These mutations were confirmed by either restriction enzyme analysis or family studies of symptomatic patients, permitting accurate presymptomatic diagnosis of affected relatives.

Cholesteryl ester storage disease: pathologic changes in an affected fetus.

The prenatal diagnosis of cholesteryl ester storage disease, a rare autosomal recessive disorder, was made by demonstration of deficient lysosomal acid lipase activity in cultured amniocytes from an at-risk fetus. The histochemical and ultrastructural changes in the affected fetus (at 17 gestational weeks) are described and compared to findings in liver and duodenal biopsy specimens from a 9-year-old homozygous female. Massive lysosomal cholesterol and lipid accumulation was demonstrated in fetal hepatocytes, adrenal cells and syncytiotrophoblasts. Of particular note was the observation of extensive necrosis in the fetal adrenal glands. Necrosis of the adrenals may precede the calcification observed in some patients with cholesteryl ester storage disease and in most patients with Wolman disease, an allelic variant due to lysosomal acid lipase deficiency. Fibrosis of the liver and lipid accumulation in macrophages in liver and duodenum, which were present in the 9-year-old homozygote, were not observed in the affected fetus, and therefore, may represent later manifestations of the disease.

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

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.

delta-Aminolevulinic acid dehydratase isozymes and lead toxicity.

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.

Fabry disease: twenty-two novel mutations in the alpha-galactosidase A gene and genotype/phenotype correlations in severely and mildly affected hemizygotes and heterozygotes.

BACKGROUND: Fabry disease, an inborn error of glycosphingolipid catabolism, results from mutations in the X-chromosomal gene encoding the lysosomal exoglycosidase, alpha-galactosidase A (alpha-Gal A; EC 3.2.1.22). The nature of the molecular lesions in the alpha-Gal A gene in 36 unrelated families was determined in order to provide precise heterozygote detection, prenatal diagnosis, and to define genotype/phenotype correlations. METHODS: Genomic DNA was isolated from affected males and/or carrier females from 36 unrelated families with Fabry disease. The entire alpha-Gal A coding region and flanking intronic sequences were analyzed by PCR amplification and solid-phase or cycle sequencing. Markers closely linked to the alpha-Gal A gene were analyzed to determine if probands with the same mutations were related. RESULTS: Twenty-two novel mutations were identified including 10 missense (P40L, W95S, S148N, C172R, M187V, N224S, W226R, A230T, D266H, N320Y), three nonsense (Y134X, C142X, W204X in two families), three splice-site defects (IVS2(+1), IVS3(+1), IVS4(+1)) and six small deletions or insertions (26delA in two families, 672ins37, 774delAC, 833insA, 1139delC, 1188insT). Of the remaining 12 families (33.3%), each had a previously identified mutation, eight of which occurred at CpG dinucleotides including R112C (two families), R112H, R227Q, R227X (three families), and R301Q. Haplotype analysis of the mutant alleles that occurred in two or three presumably unrelated families revealed that the families with the rare novel alleles (W204X and 26delA) were probably related, whereas those with mutations involving CpG dinucleotides (R112C and R227X) were not, the latter being consistent with their origins as independent mutational events. Genotype/phenotype correlations revealed that certain mutations previously found in mild variant patients also were found in classic patients. In addition, the genotypes and spectrum of phenotypic severity were determined in five heterozygotes with no family history. CONCLUSIONS: These results illustrate the molecular heterogeneity of the lesions causing Fabry disease and emphasize the fact that CpG dinucleotides constitute important hot spots for mutation in the alpha-Gal A gene. These studies also permit precise heterozygote detection and prenatal diagnosis in these families, and delineate phenotype-genotype correlations in this disease.

Molecular basis of acute intermittent porphyria: mutations and polymorphisms in the human hydroxymethylbilane synthase gene.

Acute intermittent porphyria (AIP) is an autosomal dominant inborn error of metabolism that results from the half-normal activity of the third enzyme in the heme biosynthetic pathway, hydroxymethylbilane synthase (HMB-synthase). AIP is an ecogenetic condition, with life-threatening acute attacks precipitated by various factors including drugs, alcohol, fasting, and certain hormones. Biochemical diagnosis is problematic and the identification of mutations in the HMB-synthase gene provides accurate detection of presymptomatic heterozygotes, permitting avoidance of the acute precipitating factors. Two HMB-synthase isozymes are encoded by the HMB-synthase gene: one unique to erythroid cells and the other a housekeeping isozyme present in all cells. These two isozymes arise from a single gene by alternative splicing. The recent isolation of the cDNAs and entire genomic sequence encoding the HMB-synthase isozymes has facilitated the detection of diagnostically useful intragenic polymorphisms and disease-causing mutations. Of the 36 mutations identified to date, most caused the classic form of AIP. These mutations included small deletions and insertions, point mutations and RNA splice junction alterations and resulted in the half-normal activity of both the erythroid-specific and housekeeping isozymes. Most AIP mutations were private; however, certain mutations were frequently found in Dutch (R116W) and Swedish (W198X) AIP families. A variant form of AIP, in which patients have normal erythroid activity, but half-normal activity of the housekeeping isozyme, resulted from two mutations at the exon 1/intron 1 boundary, each altering splicing of the hepatic-specific transcript. In addition, 10 polymorphisms in the HMB-synthase gene have been identified that are useful for the diagnosis of presymptomatic AIP heterozygotes in families whose specific mutations have not been determined.

Comparison of enzyme and DNA analysis in a Tay-Sachs disease carrier screening program.

Tay-Sachs disease (GM2 gangliosidosis, type 1; TSD) is an autosomal recessive GM2 gangliosidosis resulting from the deficient activity of the lysosomal hydrolase beta-hexosaminidase A (Hex A). With a carrier frequency estimated at 1 in 25, it is a common lysosomal disorder in the Ashkenazi Jewish population. Tay-Sachs disease has provided the prototype for the prevention of severe recessive genetic diseases. Molecular analysis of the Hex A gene (HEXA) of Ashkenazi Jewish individuals affected with Tay-Sachs disease revealed that three common mutations cause the infantile and adult onset forms of the disease; a four base insertion in exon 11, a splice junction mutation in intron 12 and a point mutation in exon 7 (G269S). A study was undertaken to determine whether mutation analysis would be useful in TSD screening programs in identifying carriers and clarifying the status of individuals whose enzyme assays are inconclusive. Ashkenazi Jewish individuals who had been diagnosed as carriers, inconclusives by enzyme assay and non-carriers with low normal enzyme levels in the Mount Sinai Tay-Sachs Disease Prevention Program were examined for the presence of the three mutations using polymerase chain reaction (PCR) and allele specific oligonucleotide (ASO) hybridization. The insertion mutation was present in 29 of 34 carriers and 2 of 36 inconclusive individuals, the splice junction mutation was found in 4 of 34 carriers and the G269S mutation was found in 1 of 34 carriers. Of the 313 non-carrier individuals with normal enzyme activity in the lower normal range, one was positive for the splice junction mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

Fabry disease: molecular genetics of the inherited nephropathy.

Originally described as a dermatologic curiosity by Fabry in 1898 and independently by Anderson in the same year, Fabry disease is now recognized as an inborn error of glycosphingolipid metabolism resulting from the defective activity of the lysosomal enzyme, alpha-galactosidase A (see Desnick and Sweeley for a comprehensive review). The enzymatic defect, transmitted by an X-linked recessive gene, leads to the accumulation of neutral glycosphingolipids with terminal alpha-galactosyl residues in the plasma and in the lysosomes of endothelial, perithelial, and smooth muscle cells of the cardiovascular-renal system and, to a lesser extent, in reticuloendothelial, myocardial, and connective tissue cells. Epithelial cells in the kidney, cornea, and other tissues contain the lysosomal depositions, as do the ganglia and perineural cells of the autonomic nervous system. The major accumulated substrate is globotriaosylceramide [galactosyl-(alpha 1----4)-galactosyl-(beta 1----4)-glucosyl-(beta 1----1')-ceramide]; another substrate, galabiosylceramide [galactosyl-(alpha 1----4)-galactosyl-(beta 1----1')-ceramide] is deposited primarily in renal lysosomes. The clinical manifestations of Fabry disease are the sequelae of the anatomical and physiologic alterations produced by progressive glycosphingolipid deposition. Clinical onset of the disease in hemizygous males usually occurs during childhood or adolescence, with periodic crises of severe pain in the extremities (acroparesthesias), the appearance of the vascular cutaneous lesions (angiokeratoma), hypohidrosis, and the characteristic corneal dystrophy. With increasing age, the major morbid symptoms of the disease result from the progressive infiltration of glycosphingolipid in the cardiovascular-renal system. Death usually occurs from renal, cardiac, or cerebral complications of the vascular disease. Prior to the availability of treatment by renal transplantation or dialysis, the average age at death for affected males was about 40 years. Heterozygous females, who may exhibit the disease in an attenuated form, are most likely to have only corneal opacities. Previously, the diagnosis of affected hemizygous males and heterozygous females was based on clinical findings and the levels of alpha-galactosidase A activity in easily obtained sources, e.g., plasma and isolated lymphocytes or granulocytes. Because the gene encoding alpha-galactosidase A undergoes random X-inactivation, the expressed level of enzymatic activity in females heterozygous for the disease gene may vary significantly, thereby making accurate carrier detection difficult.(ABSTRACT TRUNCATED AT 400 WORDS)

Amplification of human polymorphic sites in the X-chromosomal region q21.33 to q24: DXS17, DXS87, DXS287, and alpha-galactosidase A.

Methods for the PCR amplification of five polymorphic sites in the region Xq21.33 to Xq24 were developed and used to predict heterozygosity for Fabry disease in informative families. Clones containing polymorphic sites associated with DNA segments DXS17, DXS87, and DXS287, and the alpha-galactosidase A gene were isolated from genomic libraries. Surrounding nucleotide sequences and optimal conditions for amplification of each polymorphic site were determined. These amplifiable polymorphisms provided predictions of heterozygosity for Fabry disease and should be useful for diagnostic linkage analyses in Alport syndrome, X-linked cleft palate and ankyloglossia, Pelizaeus-Merzbacher disease, and X-linked agammaglobulinemia as well as sequence-tagged sites for gene mapping.

Molecular basis of congenital erythropoietic porphyria: mutations in the human uroporphyrinogen III synthase gene.

Congenital erythropoietic porphyria (CEP) is an autosomal recessive inborn error of metabolism that results from the markedly deficient activity of the fourth enzyme in the heme biosynthetic pathway, uroporphyrinogen III synthase (URO-synthase). To date, 17 mutations have been described including 11 missense, one nonsense, two mRNA splicing defects, one deletion and two coding region insertions. Most mutations have been identified in one or a few unrelated families with the exception of C73R and L4F which occurred in 29.6% and 9.3% of the 54 mutant alleles studied, respectively. Interestingly, analysis of the mutant alleles identified only 83% of the causative mutations, suggesting that about 20% of the mutations causing CEP lie elsewhere in the gene. Of note, mutation V82F, resulting from a G to T transversion of the last nucleotide of exon 4, caused both a missense mutation and an aberrantly spliced RNA transcript. Prokaryotic expression of the mutant URO-synthase alleles identified those with significant residual activity, thereby permitting genotype/phenotype predictions for this clinically heterogeneous disease.

Human delta-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.

Fabry disease: molecular diagnosis of hemizygotes and heterozygotes.

Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from the deficient activity of the lysosomal hydrolase, alpha-galactosidase A. Previously, the diagnosis of affected hemizygous males and heterozygous females was based on clinical findings and the levels of alpha-galactosidase A activity in easily obtained sources such as plasma and isolated lymphocytes or granulocytes. Since the gene encoding alpha-galactosidase A undergoes random X-inactivation, the expressed level of enzymatic activity in females heterozygous for the disease gene may vary significantly, thereby making accurate carrier detection difficult. The recent cloning and characterization of the full-length cDNA encoding human alpha-galactosidase A now permits the accurate diagnosis of affected hemizygotes and heterozygous females. In families with gene rearrangements or an altered restriction endonuclease cleavage site, precise diagnosis can be accomplished by Southern hybridization analysis using the alpha-galactosidase A cDNA as probe. In families with normal restriction patterns, two restriction fragment length polymorphisms have been identified in and adjacent to the alpha-galactosidase A gene which also allow precise hemizygote and heterozygote diagnosis. In addition, the recent identification of polymorphic, random DNA sequences (DXS17 and DXS87) located near the alpha-galactosidase A locus permits molecular diagnosis in informative families. Further evaluation of DXS17, DXS87 and other closely linked random DNA probes is required in order to determine their informativeness, proximity to the alpha-galactosidase A locus and, hence, accuracy for molecular diagnosis.

Uroporphyrinogen III synthase. An alternative promoter controls erythroid-specific expression in the murine gene.

Uroporphyrinogen III synthase (URO-synthase, EC 4.2.1.75) 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 approximately 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.

Molecular genetics of congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP), an autosomal recessive inborn error of heme biosynthesis, results from the markedly deficient activity of the cytosolic enzyme, uroporphyrinogen III synthase (URO-synthase). The accumulation of the nonphysiological and pathogenic porphyrin isomers, uroporphyrin I and coproporphyrin I, leads to the clinical manifestations of CEP. Disease severity in unrelated patients is markedly heterogeneous, ranging from fetal demise or severe transfusion dependency throughout life to milder adult cases with only cutaneous photosensitivity. To date, 18 mutations causing CEP have been described in the URO-synthase gene, including single base substitutions, insertions and deletions, and splicing defects. Most mutations have been identified in one or a few unrelated families with the exception of C73R, L4F, and T228M which occurred in about 33%, 8%, and 7% of the mutant alleles studied, respectively. Prokaryotic expression of the mutant URO-synthase alleles identified those with significant residual activity, thereby permitting genotype/phenotype predictions for severe to milder phenotypes of this clinically heterogeneous disease. As successful bone marrow transplantation in severely affected patients has proven curative, current efforts are underway to develop hematopoietic stem cell gene therapy for CEP.