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A previously undescribed mutation of hereditary gamma-glutamylcysteine synthetase (GCS) deficiency was found in a 5 year old boy of Moroccan origin. He presented with chronic haemolytic anaemia, delayed psychomotor development and progressive motor sensitive neuropathy of lower extremities. The parents were third degree relatives. The activity of glycolytic enzymes were found to be normal in the propositus, his parents and a sister, but and a complete lack of GSH was found in the propositus. Accordingly, the measurement of de novo GSH synthetic enzymes was undertaken, and severe GCS deficiency was found in the propositus. Both parents and his sister presented GCS activity ranging from 69% to 90% of normal. GCS gene sequencing showed that the propositus was homozygous for a 1241C>T mutation in exon 11 and both parents and his sister were heterozygous. This mutation predicts a Pro414Leu amino acid substitution. Even though the homology between GCS and crystallographically solved, functionally related proteins is not very high, a three-dimensional model of GCS was derived using Modeller Software. GCS deficiency is a very rare autosomal recessive disorder reported so far in only 8 unrelated probands with severe haemolytic anaemia. In only 3 of these was the anaemia associated with severe neurological dysfunction. We report here the fourth case of GCS deficiency presenting neuropathy, giving further support to the eventual relationship between this enzymopathy and neurological damage.

Background/Aims: To determine the molecular basis of a mild hemochromatosis phenotype in a man of Scottish-Irish descent. Methods: We sequenced genomic DNA to detect mutations of HFE, SLC40A1, TFR2, HAMP, and HFE2. RNA isolated from blood mononuclear cells was used to make cDNA. RT-PCR was performed to amplify ferroportin from cDNA, and amplified products were visualized by electrophoresis and sequenced. Results: The proband was heterozygous for the novel mutation c.1402G→A (predicted G468S) in exon 7 of the ferroportin gene (SLC40A1). Located in the last nucleotide before the splice junction, this mutation results in aberrant splicing to a cryptic upstream splice site located at nt 990 within the same exon. This causes truncation of ferroportin after glycine 330 and the addition of 4 irrelevant amino acids before terminating. The truncated ferroportin protein, missing its C-terminal 241 amino acids, would lack all structural motifs beyond transmembrane region 7. The patient was also heterozygous for the common HFE H63D polymorphism, but did not have coding region mutations in TFR2, HAMP, or HFE2. Conclusions: We conclude that this patient represents a unique example of hemochromatosis due to a single base-pair mutation of SLC40A1 that results in aberrant splicing and truncation of ferroportin.

Insertion of a P element in one copy of the Drosophila Indy gene results in a significant increase in Drosophila lifespan. In order to examine whether mutations or polymorphisms in the human Indy gene promoted human longevity, the Indy gene was sequenced in 13 subjects of age 90-99, 12 subjects between age 38-49 and 18 subjectsage 23. Three coding region polymorphisms resulting in an amino acid change, F62L, V80L and I550V, two silent coding region polymorphisms, A99A and I551I, and three intron polymorphisms, IVS2+10 C deletion, IVS9+40T/A and IVS10+38C/T were identified. The I550V polymorphism occurred in whites, Hispanics, Asians and African-Americans at allele frequencies of 0.3514, 0.5104, 0.4274 and 0.5375, respectively. The I550V polymorphism was genotyped in 2366 subjects and examined for its association with age and weight.

Summary. High frequencies of the C282Y and H63D mutations of the HFE gene occur in European populations, even though homozygous and compound heterozygous states are associated with hereditary haemochromatosis, which is a disease that decreases fitness. This suggests that heterozygotes may possess a selective advantage. HFE mutations increase iron absorption in patients with haemochromatosis, and the mean transferrin saturations and ferritin levels are mildly increased in heterozygotes, suggesting that HFE mutations may protect against iron depletion and iron deficiency anaemia. In this study of 23 681 Caucasian adults, mean transferrin saturation, serum ferritin and haemoglobin levels were significantly higher in subjects carrying HFE mutations compared with wild types. Analysed by ethnicity, mean haemoglobin and mean erythrocyte volume (MCV) were significantly lower in those with a southern versus northern European ancestry. C282Y mutation carriers had an increased mean haemoglobin level in both ethnic groups. Prevalence of non-anaemic iron deficiency was significantly lower among female carriers of the C282Y mutation compared with HFE wild types. However, prevalence of frank iron deficiency anaemia did not differ significantly among genotypes. Quantile:quantile plots showed a small but significant upward shift in the mid-range of the haemoglobin distribution among C282Y mutation carriers that was consistent with an increased mean haemoglobin level without significant changes in the anaemic range.

We undertook a three-year screening program for mutations of the HFE gene among 41,000 subjects attending the Kaiser Permanente Health Appraisal Center in San Diego, California. Our results show that the C282Y and H63D mutations of the HFE gene associated with hemochromatosis have measurable and consistent effects on iron indicators and are associated with liver disorders, but have no measurable effect on other iron overload-related symptoms and life-expectancy. The very low clinical penetrance of the HFE mutations must be taken into account in calculating cost/benefit and risk/benefit ratios in screening for hemochromatosis.

ABSTRACT A patient with chronic hemolytic anemia and G6PD deficiency was noted to be severely jaundiced and to have a high serum ferritin level. Analysis of his DNA revealed only heterozygosity for the c.187 C→G (H63D) mutation of HFE, but showed that he was homozygous for the UDP glucuronosyltransferase promoter mutation of Gilbert's disease and that he had a previously undescribed mutation of G6PD, c.832 T→C (Ser278Pro). The new variant was named G6PD La Jolla.

Summary Background There has been much interest in screening populations for disease-associated mutations. A favoured candidate has been the HFE gene, mutations of which are the most common cause of haemochromatosis in the European population. About five people in 1000 are homozygotes for the 845G→A mutation, but little is known of how many have mutation-caused clinical manifestations. Methods We screened 41 038 individuals attending a health appraisal clinic in the USA for the 845G→A and 187C→G HFE mutations, and analysed laboratory data and data on signs and symptoms of haemochromatosis as elicited by questionnaire. Findings The most common symptoms of haemo-chromatosis, including poor general health, diabetes, arthropathies, arrhythmias, impotence, and skin pigmentation were no more prevalent among the 152 identified homozygotes than among the controls. The age distribution of homozygotes and compound heterozygotes did not differ significantly from that of controls: there was no measurable loss of such individuals from the population during ageing. However, there was a significantly increased prevalence of a history of hepatitis or "liver trouble" among homozygotes and in the proportion of homozygotes with increased concentrations of serum aspartate amino-transferase and collagen IV; these changes were not related to iron burden or to age. Only one of the 152 homozygotes had signs and symptoms that would suggest a diagnosis of haemochromatosis. Interpretation The normal age distribution of people with the haemochromatosis genotype, and the lack of symptoms in patients of all ages, indicate that the penetrance of hereditary haemochromatosis is much lower than generally thought. The clinical penetrance of a disorder is an essential consideration in screening for genetic disease; disorders with low penetrance are more expensive candidates for screening than disorders with high penetrance. Our best estimate is that less than 1% of homozygotes develop frank clinical haemochromatosis.

Hereditary atransferrinemia is a rare but instructive disorder that has previously been reported in only 8 patients in 6 families. It is characterized by microcytic anemia and by iron loading, and can be treated effectively by plasma infusions. We now report the first case known in the United States. We determined the sequences flanking the exons of the human transferrin gene and sequenced all of the exons and some of the flanking regions of the patient's DNA and that of her parents. The patient's DNA revealed a 10-base pair (bp) deletion, followed by a 9-bp insertion of a duplicated sequence. There was also a G→C transversion at complementary DNA (cDNA) nt 1429, predicting that a proline was substituted for the alanine in amino acid position 477 (Ala 477 Pro). The latter mutation occurs at an evolutionarily highly conserved site; 704 control alleles were screened and this point mutation was not found. Each of the patient's transferrin genes contains one mutation, ie, the patient is a compound heterozygote for these mutations, because one was found in each of her parents. In addition to these mutations, which we regard to be causative in the patient's atransferrinemia, a silent polymorphism at cDNA 1572 G→C was found in exon 13 as well as 2 previously unreported polymorphisms at IVS8 + 62 c→t and IVS14-4 c→a. The mutation in nt 1572 and that in intron 8 were common in the general population; the intron 14 mutation is rare.

Pyruvate kinase (PK) deficiency is the most common cause of hereditary nonspherocytic hemolytic anemia. The prevalence of this deficiency is unknown, though some estimates have been made based on the frequency of low red cell PK activity in the population. An additional 20 patients with hereditary nonspherocytic hemolytic anemia caused by PK deficiency have been genotyped. One previously unreported mutation 1153C→T (R385W) was encountered. The relative frequency of PK mutations in patients with hemolytic anemia caused by PK deficiency was calculated from the 18 white patients reported here and from 102 patients previously reported in the literature. DNA samples from 3785 subjects from different ethnic groups have been screened for the 4 more frequently encountered mutations—c.1456 C→T(1456T), c.1468 C→T(1468T), c.1484 C→T(1484T), and c.1529 G6A (1529A)—by allele-specific oligonucleotide hybridization. Among white patients the frequency of the 1456T mutation was 3.50 × 10−3; that of the 1529A mutation was 2.03 × 10−3. Among African Americans the frequency of the 1456T mutation was 3.90 × 10−3 The only mutation found in the limited number of Asians tested was 1468T at a frequency of 7.94 × 10−3. Based on the gene frequency of the 1529A mutation in the white population and on its relative abundance in patients with hemolytic anemia caused by PK deficiency, the prevalence of PK deficiency is estimated at 51 cases per million white population. This number would be increased by inbreeding and decreased by failure of patients with PK deficiency to survive.

γ-Glutamylcysteine synthetase catalyzes the first step in glutathione synthesis. The enzyme consists of 2 subunits, heavy and light, with the heavy subunit serving as the catalytic subunit. A patient with hemolytic anemia and low red blood cell glutathione levels was found to have a deficiency of γ-glutamylcysteine synthetase activity. Examination of cDNA from the patient and her mother showed that she was homozygous and that her mother was heterozygous for a A→T transversion at nt1109 producing a deduced amino acid change of His370Leu. The partial genomic structure of the catalytic subunit of γ-glutamylcysteine synthetase (GLCLC) was determined, providing some intron/exon boundaries to make it possible to sequence an affected part of the coding region from genomic DNA. The 1109A→T mutation was not present in the DNA of 38 normal subjects. In the course of these studies we found a diallelic polymorphism in nt +206 of an intron and another polymorphism that consisted of a duplication of a CAGC at cDNA nt1972-1975 in the 3′ untranslated region. The 2 polymorphisms were found to be only in partial linkage disequilibrium.

A polymorphism in the promoter of the UDP-glucuronosyltransferase 1 ( UGT1A1 ) gene has been shown to cause Gilbert syndrome, a benign form of unconjugated bilirubinemia. Promoters containing seven thymine adenine (ta) repeats have been found to be less active than the wild-type six repeats, and the serum bilirubin levels of persons homozygous or even heterozygous for seven repeats have been found to be higher than those with the wild-type six repeats. We have now examined the genotypes in persons of Asian, African, and Caucasian ancestry. Although within the Caucasian ethnic group there is a strong correlation between promoter repeat number and bilirubin level, between ethnic groups we found that this relationship to be inverse. Among people of African ancestry there are, in addition to those with six and seven repeats, also persons who have five or eight repeats. Using a reporter gene we show that there is an inverse relationship between the number of ta repeats and the activity of the promoter through the range of 5–8 ta repeats. An incidental finding was a polymorphism at nucleotide −106, tightly linked to the (ta) 5 haplotype. Serum bilirubin levels are influenced by many factors, both genetic and environmental. We suggest that the unstable UGT1A1 polymorphism may serve to “fine-tune” the plasma bilirubin level within population groups, maintaining it at a high enough level to provide protection against oxidative damage, but at a level that is sufficiently low to prevent kernicterus in infants.

ABSTRACT: Nramp2 is a gene encoding a transmembrane protein that is important in metal transport, in particular iron. Mutations in nramp2 have been shown to be associated with microcytic anemia in mk/mk mice and defective iron transport in Belgrade rats. Nramp2 contains a classical iron responsive element in the 3′ untranslated region that confers iron dependent mRNA stabilization. In this report, we describe a splice variant form of human nramp2 that has the carboxyl terminal 18 amino acids substituted with 25 novel amino acids and has a new 3′ untranslated region lacking a classical iron-responsive element. This splice form of nramp2, nramp2 non-IRE, was found to be derived from splicing of an additional exon into the terminal coding exon. The nramp2 gene is comprised of 17 exons and spans more than 36 kb. It contains an additional 5′ exon and intron (exon and intron 1) and an additional 3′ exon (exon 17) and intron (intron 16) as compared to nramp1, a homologous gene. The additional exons and introns account for much of the difference in length between nramp2 (>36 kb) and nramp1 (12 kb). The exon-intron borders of nramp2 exons 3-15 are homologous to nramp1 exons 2-14. The nramp2 5′ regulatory region contains two CCAAT boxes but lacks a TATA box. The 5′ regulatory region of nramp2 also contains five potential metal response elements (MRE's) that are similar to the MRE's found in the metallothionein-IIAgene, three potential SP1 binding sites and a single γ-interferon regulatory element. Five single nucleotide mutations or polymorphisms were identified within the nramp2 gene. One of these, 1303C→A, occurs in the coding region of nramp2 and results in an amino acid change from leucine to isolecine. A polymorphism, 1254T/C, also occurs in the coding region of nramp2 but does not cause an amino acid change. The other 3 polymorphisms are within introns (IVS2+11A/G, IVS4+44C/A, and IVS6+538G/Gdel). In addition, a polymorphic microsatellite TATATCTATATATC (TA)6-7(CA)10-11CCCCCTATA (TATC)3(TCTG)5TCCG (TCTA)6was identified in intron 3. Analysis of cDNA derived by direct amplification of reversed transcribed RNA or cDNA clones isolated from a library provide evidence of skipping of exons 10 and 12 of nramp2. Deletion of either of these exons would result in a sequence that remains in frame yet would generate a protein that would lack transmembrane spanning region 7 or 8 respectively. The deletion of a single transmembrane domain would have severe topological consequences. The coding region of the nramp2 gene of hemochromatosis patients with or without mutations in the hemochromatosis gene,HFE, were examined and found to be normal. One hemochromatosis patient, with a normalHFEgenotype, was heterozygous for the 1303C→A mutation. Furthermore, in an examination of hemochromatosis patients with mutantHFEand normalHFEgenes, we did not observe a linkage disequilibrium of either group with a particular nramp2 haplotype. These data suggest that mutations in nramp2 are not commonly associated with hemochromatosis.

Hereditary hemochromatosis is a common disorder in people of European origin. The HLA-H gene has been found to have two mutations that apparently cause hemochromatosis. The principal mutation, 845G-->A (C282Y), is believed to have arisen relatively recently in the Celtic population. To determine the incidence of this mutation and the other hemochromatosis-associated mutation, 187C-->G (H63D), among Ashkenazi Jews, a people who are believed to have arrived in Europe in about the 8th Century A.D., we have examined the DNA from 381 unrelated Jewish subjects and 206 non-Jewish white controls. The gene frequency for the 845G-->A mutation among Jewish subjects was only 0.013 compared with a frequency of 0.070 among controls, a difference that is significant at the 0.00001 level. The phenotypically milder nt 187C-->G mutation had a frequency of 0.155 in the non-Jewish population and 0.097 in the Jewish population, a difference that was also statistically significant at the

The DNA of 147 patients of European origin clinically diagnosed with idiopathic hemochromatosis and 193 controls was examined for mutations of the HLA-H gene at nt 845 and nt 187. One hundred twenty-one (82.3%) of the hemochromatosis patients were homozygous and 10 (6.8%) heterozygous for the 845A (C282Y) mutation. All of the homozygous patients were also homozygous for nt 187C, and all 845A heterozygotes had at least one copy of 187C. Thus, the nt 845 and nt 187 mutations were in complete linkage disequilibrium; nt 187 was a C on all chromosomes with the 845A mutation. Eight of the 10 heterozygotes for 845A were heterozygous for 187G(H63D). The excess of heterozygotes at both nt 187 and nt 845 suggested either the presence of as yet undiscovered mutations existing in trans with 845A and in linkage disequilibrium with 187G, or that the 187G itself is a deleterious mutation, which in concert with the 845A can give rise to hemochromatosis. None of the 193 normal controls were homozygous for 845A and 29/193 (15%) were heterozygous for 845A. Although 47/193 (24.3%) of normal controls were heterozygous for the 187G mutation only two of these carried the 845A mutation. If the 187G mutation complemented the 845A mutation with high penetrance in causing hemochromatosis, then the population frequency of the two genes would require that a high proportion of patients with hemochromatosis be heterozygous for 845A and 187G. Instead, the frequency of homozygotes for the 845A mutation was much higher than that of the 845A/187G genotype. Based on our data, the penetrance of the 845A/187G genotype is only 1.5% and based on the data of Feder et al. only 0.5%. In contrast, the penetrance of the homozygous 845A/845A genotype seems to be very high. Thus, screening for this genotype should be very useful.

Selective hybridization of small intestine and liver cDNA libraries was carried out using yeast artificial chromosomes (YACs) surrounding D6S105, the microsatellite that appears to be close to the gene for hereditary hemochromatosis ( HFE ). Of 14 candidate probes hybridizing with these YACs, only one, designated LD5-1, detected abnormalities in Southern blots of patients with hemochromatosis. Two different abnormalities were detected in 3 of 55 patients with hemochromatosis with the LD5-1 probe, and one of these was detected in one of 44 normal subjects. The gene that hybridizes with this probe is located about 300-400 kb centromeric of D6S105. It is transcribed into mRNA that is about 8.5 kb in length in many tissues, including peripheral blood leukocytes. The available sequence indicates that it codes for a zinc finger protein. We propose that there is a reasonable probability that LD5-1 hybridizes with the gene for hereditary hemochromatosis.

DNA from 17 individuals with 20 unidentified alleles was subjected to single-stranded conformation polymorphism analysis and/or sequencing and 5 previously undescribed mutations have been identified: 245T, 259T, 635G, 914C del, and IVS10(+2). Two of these mutations, 914C del and IVS10(+2), are null, or "lethal" mutations. Because the other mutation each of these two patients carried was "mild", the phenotype was type I disease. In addition to the new mutations we describe, the second example of the rare 1448G mutation has been documented in one of the patients. This mutation is particularly interesting because in samples studied by restriction analysis with NciI it can readily be confused with the common 1448C mutation. Reexamination of 28 patients who had previously been diagnosed as carrying the 1448C mutation were confirmed to be 1448C.

A patient with clinically manifest hereditary hemochromatosis was found to be heterozygous for the c.845 A-->G (C282Y) mutation. As simple heterozygotes for this mutation do not develop the hemochromatosis phenotype, the coding region of the patient's HFE gene was sequenced and a previously undescribed nonsense mutation was identified at c.211 C-->T (R74X). The patient's brother who also had the hemochromatosis phenotype shared his HFE genotype. To determine how common such mutations might be, the coding and 5' region of the HFE genes of 11 subjects who had been found in a large population survey to be heterozygous for the C282Y mutation and had elevated ferritin levels were sequenced. No mutations were found. Sequencing of the HFE gene also revealed two polymorphisms that had not previously been noted, -467 C-->G and -970 T-->G. Neither of these mutations appear to cause an abnormality in iron metabolism.

Contains fulltext : 89715.pdf (Publisher’s version ) (Closed access) Male subjects with iron deficiency from the general population were examined for polymorphisms or sporadic mutations in TMPRSS6 to identify genetic risk factors for iron deficiency anemia. Three uncommon non-synonymous polymorphisms were identified, G228D, R446W, and V795I (allele frequencies 0.0074, 0.023 and 0.0074 respectively), of which the R446W polymorphism appeared to be overrepresented in the anemic population. In addition, three children with iron refractory iron deficiency anemia, and one sibling with iron responsive iron deficiency anemia were also examined for polymorphisms or sporadic mutations in TMPRSS6. Two children (family 1) were compound heterozygotes for a L674F mutation and a previously described splicing defect predicted to cause skipping of exon 13 (IVS13+1 G>A). One child from the second family was homozygous for a deletion (497T) causing a frameshift (L166X+36) and premature termination. The sibling and mother from the second family were compound heterozygotes for the L166X mutation and the uncommon R446W polymorphism. Although in vitro expression studies demonstrated that the R446W isoform was biologically similar to wildtype Tmprss6, clinical data indicate that the R446W produces a milder disease when carried in trans with severe mutation in Tmprss6. The four children carrying mutations in TMPRSS6 all exhibited inappropriately high urinary hepcidin levels for the degree of iron deficiency.

DNA from 100 unrelated patients, 97 of whom were Jewish and three half- Jewish, was analyzed for 22 mutations known to cause Gaucher disease. All but seven of the alleles were identified as having previously described mutations. Five of the unidentified mutations proved to be a previously undescribed nucleotide substitution in a splice junction (IVS2+1) that causes skipping of exon 2. Thus, only 2 of 197 alleles remained unidentified. Homozygotes for the most common mutation, that a nucleotide (nt) 1226, manifested, on average, the mildest disease and the latest age of onset. The mutation at nt 84 and the newly described IVS2+1 mutation, which do not produce any enzyme, were associated with earlier onset and more severe disease. Five of the mutations were considered to be “public,” in the sense that they were found in more than one unrelated individual. Screening for these five mutations permitted detection of 97.5% of all Gaucher alleles in this patient population. Because the mutation at nt 1226 is underrepresented in the patient population and because not all homozygotes come to medical attention, screening the Ashkenazi population using DNA analysis should detect approximately 99% of all heterozygotes.

Mice with targeted disruptions in the iron-responsive binding protein 2 (IRP2) gene accumulate iron in distinct regions of the brain and develop neurodegenerative characteristics resembling Parkinson's disease after 6 months of age. To determine whether polymorphisms in IRP2 predispose humans to Parkinson's disease (PD), we sequenced the IRP2 gene of subjects with sporadic PD and normal controls. Three polymorphisms which result in an amino acid change were identified: L159V, F272L, and T560I. The L159V and T560I polymorphisms, identified in an African-American PD subject, were found in the African-American population at an allele frequency of 0.102 (n = 1,236) and 0.111 (n = 1,228), respectively, and were not associated with an increased prevalence of PD. The F272L polymorphism was found in a normal 58-year-old, Caucasian subject whose father had PD, but it was not observed in 38 additional patients with sporadic PD. The F272L polymorphism occurred at an allele frequency of 0.0014 (n = 1,384) in the normal Caucasian population. Additional F272L heterozygous subjects identified in the normal population did not have a family or personal history of PD. We conclude that these IRP2 polymorphisms do not play an important role in the development of sporadic cases of PD. It remains to be determined whether other polymorphisms in IRP2 play a role in familial PD. © 2002 Movement Disorder Society

We established the molecular basis for pyruvate kinase (PK) deficiency in a white male patient with severe nonspherocytic hemolytic anemia. The paternal allele exhibited the commonPKLR cDNA sequence (c.) 1529G>A mutation, known to be associated with PK deficiency. On the maternal allele, 3 in cis mutations were identified in the erythroid-specific promoter region of the gene: one deletion of thymine −248 and 2 single nucleotide substitutions, nucleotide (nt) −324T>A and nt −83G>C. Analysis of the patient's RNA demonstrated the presence of only the 1529A allele, indicating severely reduced transcription from the allele linked to the mutated promoter region. Transfection of promoter constructs into erythroleukemic K562 cells showed that the most upstream −324T>A and −248delT mutations were nonfunctional polymorphisms. In contrast, the −83G>C mutation strongly reduced promoter activity. Site-directed mutagenesis of the promoter region revealed the presence of a putative regulatory element (PKR-RE1) whose core binding motif, CTCTG, is located between nt −87 and nt −83. Electrophoretic mobility shift assay using K562 nuclear extracts indicated binding of an as-yet-unidentified trans-acting factor. This novel element mediates the effects of factors necessary for regulation of pyruvate kinase gene expression during red cell differentiation and maturation.

The four most common mutations account for 97% of the Gaucher disease-producing alleles in Jewish patients and 75% of the alleles in non-Jewish patients. Although at least 15 other mutations and some examples of gene conversion and/or fusion genes have been described, a number of mutations remain unidentified. We have now identified six new mutations, a deletion of a C at the 72 position of the cDNA, a 481C → T mutation (122Pro → Ser), a 751T → C (212Tyr → His), a 1549G → A (478Gly → Ser), a 1604G → A (496Arg → His), and a 55-bp deletion. All but one of these were found in single families. The 1604A mutation, however, was observed in four unrelated individuals.

Large-scale detection of mutations at the DNA level requires the development of cost-effective methods for the screening of thousands of samples. Hemochromatosis is an appropriate testing ground for the development of such technologies, and we report the methods that we have developed to screen large numbers of samples using equipment available in most laboratories. We are able to examine DNA samples for two mutations in the HFE gene at a cost of only slightly over $8 per sample, a cost that includes overhead and the approximately 40 hr per week of technician time required to perform the studies. The technologies involved in mutation analysis are evolving rapidly and ultimately more highly automated, lower-cost technologies may become available. At present, however, we find the methodology described to be very suitable for large-scale, low-cost mutation screening.

Amplification of the region of the HFE gene that contains the c.845G-->A (C282Y) mutation is usually performed using one amplimer that binds to a sequence in intron 3 and another that binds to a sequence in intron 4. Previously, a mutation that interferes with efficient binding to the intron 4 site has been described. We now find that another common mutation in intron 3, IVS3 -48c-->g, prevents binding of the amplimer to that site. This polymorphism occurs at a gene frequency of 0.128 in the African-American population and at a frequency of only 0.006 in the European population. DNA samples heterozygous for the IVS3 -48c-->g polymorphism and C282Y were undistinguishable from samples homozygous for the C282Y mutation when they were examined by allele-specific oligonucleotide hybridization (ASOH) and showed only a weak normal band when examined by electrophoresis following restriction endonuclease digestion. Although the polymorphism occurs in a DNA sequence almost identical to the intron 3 splice donor site, we found no evidence of alternative splice forms. Moreover, serum iron, transferrin saturation, and serum ferritin levels were normal in subjects heterozygous for the polymorphism. It appears, therefore, that the main importance of this polymorphism is that it may lead to misdiagnosis of heterozygotes for the C282Y mutation as homozygotes. We therefore recommend exonic amplimers that avoid sites that contain polymorphisms and that can be multiplexed for detection of the c.187C-->G (H63D) and c.845G-->A (C282Y) mutations.

Five genes encoding zinc finger proteins of the Cys 2 His 2 (or Kruppel) family were identified by direct cDNA hybridization to YACs 753H12 and 638D7, which encompass a region of human chromosome 6p21.3 extending from just centromeric of the microsatellite marker D6S306 to telomeric of D6S1260. The genes span a distance of approximately 1750 kb. The complete cDNA sequence, genomic structure, and tissue distribution of three of the zinc finger proteins, LD65/ZNF165, ZNF192 (previously called LD5-1), and ZNF193, are described. The three zinc finger proteins do not contain either Kruppel-associated box (KRAB) A or KRAB B domain, present in about one-third of all Kruppel-type zinc finger proteins (E. J. Bellefroid et al., 1991, Proc. Natl. Acad. Sci. USA 88: 3608–3612). The three zinc finger proteins do contain the conserved SCAN box domain (A. J. Williams et al., 1995, J. Biol. Chem. 270: 22143–22152). SCAN boxes are found in eight other genes in the GenBank database, five of which are also in the Kruppel family of zinc finger proteins lacking KRAB A and B domains and thereby define a new subclass of zinc finger proteins. In addition, three polymorphisms were identified in ZNF192, one of the zinc finger proteins. One of the three polymorphisms, Pro163Leu, is the second proline in a proline cluster (PEPP) in a region separating the SCAN box from the zinc finger motifs.

BACKGROUND: The 845A(C282Y) mutation in the HLA-H gene accounts for most cases of hereditary hemochromatosis in patients who are of European origin. Some lack this mutation, however, and it is not present in Asian patients. Thus, other mutations either in HLA-H or associated proteins may be present in such patients. HLA-H associates with beta-2-microglobulin. Calreticulin associates with class 1 HLA proteins and appears to be identical with mobilferrin, a putative iron transport protein. These two proteins are therefore candidates for mutations in patients with hemochromatosis. MATERIALS AND METHODS: We have sequenced the coding region and parts of introns of the HLA-H gene, the beta-2-microglobulin gene, and the calreticulin (mobilferrin) gene of 10, 7, and 5 hemochromatosis patients, respectively, selecting those who were not homozygous for the 845A(C282Y) mutation. The number of chromosomes at risk studied were 18 for HLA-H, 14 for beta-2-microglobulin and 10 for calreticulin. RESULTS: We detected 3 new intronic polymorphisms in the HLA-H gene, each a point mutation. Some differences from published sequences of beta-2-microglobulin and calreticulin were documented, but these were uniformly present in all samples. CONCLUSIONS: The lack of additional mutations in the HLA-H gene is remarkable, and we speculate that the C282Y mutation may be a gain-of-function change.

Gaucher disease is the most common glycolipid storage disorder, characterized by storage of the glycolipid, glucocerebroside in the liver, spleen, and marrow. The most prevalent form of Gaucher disease is designated type I (MIM 230800). Patients with type I disease may have hepatomegaly, splenomegaly, bone lesions, and less commonly, lung disease, but are free of neurological involvement. Types II (MIM 230900) and III (MIM 2310000), the acute infantile and juvenile forms, respectively, of Gaucher disease, are characterized by the fact that the central nervous system is affected. © 1996 Wiley-Liss, Inc.

BACKGROUND: Thirty-six mutations that cause Gaucher disease, the most common glycolipid storage disorder, are known. Although both alleles of most patients with the disease contain one of these mutations, in a few patients one or both disease-producing alleles have remained unidentified. Identification of mutations in these patients is useful for genetic counseling. MATERIALS AND METHODS: The DNA from 23 Gaucher disease patients in whom at least one glucocerebrosidase allele did not contain any of the 36 previously described mutations has been examined by single strand conformation polymorphism (SSCP) analysis, followed by sequencing of regions in which abnormalities were detected. RESULTS: Eight previously undescribed mutations were detected. In exon 3, a deletion of a cytosine at cDNA nt 203 was found. In exon 6, three missense mutations were identified: a C-->A transversion at cDNA nt 644 (Ala176-->Asp), a C-->A transversion at cDNA nt 661 that resulted in a (Pro182-->Thr), and a G-->A transition at cDNA nt 721 (Gly202-->Arg). Two missense mutations were found in exon 7: a G-->A transition at cDNA nt 887 (Arg257-->Gln) and a C-->T at cDNA nt 970 (Arg285-->Cys). Two missense mutations were found in exon 9: a T-->G at cDNA nt 1249 (Trp378-->Gly) and a G-->A at cDNA nt 1255 (Asp380-->Asn). In addition to these disease-producing mutations, a silent C-->G transversion at cDNA nt 1431, occurring in a gene that already contained the 1226G mutation, was found in one family. CONCLUSIONS: The mutations described here and previously known can be classified as mild, severe, or lethal, on the basis of their effect on enzyme production and on clinical phenotype, and as polymorphic or sporadic, on the basis of the haplotype in which they are found. Rare mutations such as the new ones described here are sporadic in nature.

Two polymorphisms, one in the liver-type pyruvate kinase gene (PKLR) and one in the glucocerebrosidase gene (GBA), both of which are on band q21 of chromosome 1, were found to be tightly linked. Each of three Gaucher disease mutations in 112 chromosomes studied was associated with a unique haplotype. With a conservative assumption about the length of time that the Gaucher disease mutation has been present in the Jewish population, we deduce that the genetic distance between these two loci is probably under 0.2 centimorgans. Four haplotypes are produced by these polymorphic loci, but two of these are relatively uncommon because the polymorphic sites are in linkage disequilibrium. Nonetheless these markers are potentially useful in the prenatal diagnosis of pyruvate kinase deficiency in families who have at least one affected child and may also be helpful in heterozygote detection in families with Gaucher disease where a specific mutation producing the disease in unknown.

In screening a rat mucosa cDNA subtraction library, a clone that exhibited a remarkable degree of homology with a previously described cDNA from the green garden pea, designated the 26g pea turgor protein, was found. A partial cDNA sequence from rat and a complete cDNA sequence from human were obtained. The deduced human protein had a molecular weight of 55,285 and was designated antiquitin because of its remarkable level of conservation through evolution. Human antiquitin was 60% homologous to the green pea 26g with only a single amino acid gap in each sequence. The 66 amino acids at the carboxyl ends of the human antiquitin and pea 26g proteins were 86% identical, and one segment of 52 amino acids was 92% identical. A similar partial sequence encoding 164 amino acids has been detected in Caenorhabditis elegans. Yeast DNA was found to have sequences that hybridize with a human antiquitin probe on Southern blotting. Analysis of the amount of mRNA in various rat and human tissues indicated that the largest amounts were found in rat kidney and liver and in cultured human hepatoma cells. Only minimal amounts were detected in human peripheral blood leukocytes, rat lung, or cultured human fibroblasts. Attempts to induce the mRNA by heat-shock, dehydration, ionizing irradiation, or treatment with iron, t-butylhydroperoxide, or glucocorticoids were unsuccessful. The function of the protein remains unknown.

Recently, a mutation at nucleotide 1193 of the glucocerebrosidase gene was described in a patient with type 1 Gaucher disease. This mutation destroys a TaqI site in a polymerase chain reaction (PCR)-amplified fragment. We used digestion with this enzyme to screen DNA samples from Gaucher disease patients representing 23 previously unidentified alleles and discovered that this site had been destroyed in three samples. However, the mutation that caused this change proved to be a CT substitution at cDNA nucleotide 1192 (Genomic 5408; 359Arg-->End). Fortuitously, another TaqI site was destroyed by a different mutation, a GA mutation at nt 1312 (Genomic 5927; 399AspAsn). Both of these mutations were functionally severe in that they were associated with type 2 (acute neuronopathic) Gaucher disease.

Two sisters with moderately severe Gaucher disease were diagnosed as having the usualy relatively benign 1226G/1226G genotype by examination of DNA amplified from exon 9, where this mutation is located. Because of the discrepancy between the apparent genotype and the phenotype, we suspected that one of the alleles had not amplified. Therefore, the DNA of both parents was examined. The father was heterozygous for the 1226G mutation but the mother did not have this abnormality. It was shown that the mother and both daughters had a deletion of the glucocerebrosidase gene: only about one-half of the polymerase chain reaction (PCR) amplification product of the glucocerebrosidase gene in this region was found, compared to internal controls consisting of the glucocerebrosidase pseudogene and of the adjacent liver pyruvate kinase (PKLR) gene. The appearance of Southern blots developed with full length glucocerebrosidase cDNA probes showed that the band unique to the functional glucocerebrosidase gene had reduced intensity, and no abnormal bands were present after digestion with any restriction endonuclease, indicating that the entire coding region was deleted. © 1994 Wiley-Liss, Inc.

Summary The DNA from 64 non-Jewish patients with Gaucher disease was examined for 29 different glucocerebrosidase mutations that are known to cause the disease. The most common was mutation 1448C and the second most common 1226G. Twenty of the mutations remained unidentified. These results were compared to those obtained from 122 Jewish patients, in whom the most common mutations were 1226G and 84GG. Among the non-Jewish patients 15.6% of the mutations remained unidentified. while among the Jewish patients only 1.2% remained unidentified, and even screening for the five most common mutations identified all but 2.9% of the Jewish mutations. The greater diversity of mutations among the non-Jewish patients has important implications with respect to DNA-based screening. diagnosis and counselling.

The two glucocerebrosidase genes from a patient with Gaucher disease were cloned and 8850 bp of each sequenced. Each clone had a single nucleotide change accounting for the clinical glucocerebrosidase deficiency, an A to G transition at cDNA nucleotide 1226 in one clone, and an insertion of a G at cDNA nucleotide 84 in the other clone. Sequence analysis revealed that there were 11 additional differences between the two clones. The clone with the nt 1226 mutation was, as is always the case, Pv1.1- (polymorphic PvuII site present). The 84GG clone was Pv1.1+. Examination of 35 normal subjects and 51 Gaucher disease patients was consistent with the existence of only two major haplotypes. Two additional minor haplotypes were found, one in Africans and one in the white population. These represented additional mutations superimposed on the basic two haplotypes. Two unrelated patients with Gaucher disease seemed to be exceptions in the 5' end of the gene was heterozygous for the + and - haplotypes but the most 3' marker was homozygous. These patients are believed to have a gene deletion on one allele. In addition to these studies, we correct 28 minor errors in the originally published sequence.

Over 400 supposedly biochemically and genetically distinct variants of glucose-6-phosphate dehydrogenase (G6PD) have been described in the past. In order to investigate these variants at the DNA sequence level we have now determined the relevant sequences of introns of G6PD and describe a method which allows us to rapidly determine the sequence of the entire coding region of G6PD. This technique was applied to six variants that cause G6PD deficiency to be functionally so severe as to result in nonspherocytic hemolytic anemia. Although the patients were all unrelated, G6PD Marion, Gastonia, and Minnesota each had identical mutations, a G----T at nucleotide (nt) 637 in exon 6 leading to a Val----Leu substitution at amino acid 213. The mutations of Nashville and Anaheim were identical to each other, viz. G----A at nt 1178 in exon 10 producing a Arg----His substitution at amino acid 393. G6PD Loma Linda had a C----A substitution at nt 1089 in exon 10, producing a Asn----Lys change at amino acid 363. The results confirm our earlier results suggesting that the NADP-binding site is in a small region of exon 10 and suggest the possibility that this area is also concerned with the binding of glucose-6-P.

The most common Gaucher disease-producing mutation among Ashkenazi Jews is an A → G substitution at cDNA nt 1226 (genomic nt 5841). We describe a simple method for detecting this mutation both in genomic DNA and in cDNA by performing polymerase chain reaction (PCR) using a 5'-primer mismatched at one nucleotide so as to create an Xho I restriction site. When the mutation is present, the 105 bp fragment formed is cleaved to 89 and 16 nt fragments. The 89 bp fragment is easily visualized on a gel making it possible to distinguish individuals who do not have the mutation from heterozygotes and homozygotes.

We report the sequence of the entire human gene encoding beta-glucocerebrosidase and that of the associated pseudogene. The gene contains 11 exons extending from base pair 355 to base pair 7232 in the overall sequence. The gene promoter contains TATA- and CAT-like boxes upstream of the major 5' end of the glucocerebrosidase RNA. The two TATA boxes lie between nucleotides (-23)-(-27) and (-33)-(-39) and the two possible CAT boxes reside between nucleotides (-90)-(-94) and (-96)-(-99) in relation to the major 5' end of the mRNA. The functionality of the promoter region was monitored by coupling it to the bacterial gene coding for chloramphenicol acetyltransferase (CAT) and assaying the expression of the enzyme in cells transfected with this vector. The glucocerebrosidase promoter not only directs synthesis of the bacterial enzyme but also exhibits the same pattern of tissue-specific expression as that of the endogenous gene. An apparently tightly linked pseudogene is approximately 96% homologous to the functional gene. However, introns 2, 4, 6, and 7 have large "deletions" consisting of Alu sequences 313, 626, 320, and 277 bp in length, respectively. It is entirely possible that the ancestral gene lacks these sequences and that they have been inserted into the introns of the functioning gene. There is also a 55-bp deletion from a part of exon 9 flanked by a short inverted repeat. The sequence data should facilitate development of methods for diagnosis of Gaucher disease at the molecular level.

A cloned fragment of human glucocerebrosidase cDNA has been used as a probe to study restriction polymorphisms in the region of the gene for Gaucher disease. Variability in the size of fragments produced by digestion with the restriction endonucleases Pvu II and Kpn I was discovered. The Pvu II polymorphism was found to be a very prevalent one with a gene frequency of 0.65 for the Pv1.1- allele and 0.35 for the Pv1.1+ allele. Similar frequencies were encountered among diverse ethnic groups. Five of eight Jewish patients with Gaucher disease were found to be heterozygous for the Pvu II restriction polymorphism. One non-Jewish patient with type I Gaucher disease was heterozygous for the Kpn I variant. The existence of Gaucher disease genes in association with either allele of the ancient Pvu II polymorphism clearly indicates that, even within the Jewish population, the Gaucher disease mutation has occurred independently more than once. Presumably, different mutations have also occurred in the non-Jewish population.

A new G-6-PD variant, G-6-PD Walter Reed, causing hereditary nonspherocytic hemolytic anemia is characterized. This variant is unusual in that its stability requires the presence of high concentrations of NADP, while its Km for NADP is normal. This finding is consistent with the suggestion that G-6-PD has two separate binding sites, a high affinity "structural" site and a lower affinity catalytic site. The mutation in G-6-PD Walter Reed, like that of the previously described variant, G-6-PD Torrance, may be due to a mutation of the "structural" site for NADP.

Noting the scarcity of CpG dinucleotide in total genomic DNA derived from higher organisms and the scarcity of TpA dinucleotide in total genomic DNA derived from most life forms, we examined the distribution of these dinucleotides in sequences derived from functionally distinct types of human DNA, including mitochondrial DNA, intergenic DNA, intron DNA, and DNA destined to be represented in the cytoplasm as mRNA, tRNA, or rRNA. While CpG frequency has fallen to its lowest levels in DNA that is transcriptionally silent, TpA is most stringently excluded in DNA destined to be expressed as mRNA in the cytosol. This observation suggests that the selective pressures leading to the removal of CpG and TpA operate at different levels. With respect to TpA, dinucleotide scarcity may reflect a requirement for mRNA stability and may indicate the action of UpA-selective ribonucleases. We propose that, by reason of its instability, UpA must have been very rare in primordial RNA. Therefore, tRNA with the anticodon for this dinucleotide may have failed to evolve, making UpA the primordial doublet "stop" codon. The modern triplet code has faithfully conserved this arrangement in the two universal stop codons, UAA and UAG.