Progress in instrument used for diagnosis of obstructive jaundice. 4. MRCP

OBJECTIVE: The objective of the study was to determine the prevalence and associations of abnormal alpha1-antitrypsin phenotypes in Caucasian adults with end stage liver disease with particular emphasis on heterozygous phenotypes and disease from hepatitis C virus. METHODS: All patients (788) with end stage liver disease considered for liver transplantation from July 1990 to June 1996 in a referral-based university hospital transplant center (University of Nebraska Medical Center, Omaha, NE) comprised the study population. Data for the study population was determined by retrospective review of the transplantation database at the transplant center. Hepatitis C virus infection was determined by a second generation ELISA method, and alpha1-antitrypsin phenotyping was performed on agarose gel with serum quantitation using a Behring Nephelometer. RESULTS: Among 683 Caucasian patients with severe liver disease, the prevalences of Pi ZZ, Pi MZ, and Pi MS were 0.4, 7.3, and 8.2%, respectively, compared with 0, 2.8, and 4.2% in the control population. The odds of having a heterozygous Z phenotype were significantly increased in Caucasian patients with hepatitis C virus (odds ratio (OR) = 4.3, 95% confidence interval (CI) = 2.1-9.0), alcoholic liver disease (OR = 5.0, 95% CI = 2.6-9.6), primary hepatic malignancy (OR = 7.4, 95% CI = 2.9-19.0), and cryptogenic cirrhosis (OR = 2.6, 95% CI = 1.1-6.3) compared with the control population. Caucasian patients with hepatitis C or B virus were 3.6 times more likely to have a heterozygous Z phenotype than a normal phenotype compared with patients with diseases of autoimmune etiology. CONCLUSION: This study provides evidence of an association of heterozygous Z alpha1-antitrypsin phenotype with end stage liver disease of several etiologies, not hepatitis C virus alone.     

Host response to initial and challenge infections, following treatment, of Gyrodactylus bullatarudis and G. turnbulli (Monogenea) on the guppy (Poecilia reticulata)

Patients homozygous for the Z allele of alpha-1-antitrypsin (alpha 1AT) have very low serum levels and are predisposed to emphysema. There have also been reports of emphysema being associated with the heterozygous phenotype FZ. To investigate whether F alpha 1AT was dysfunctional, the inhibitory activity of F alpha 1AT against human neutrophil elastase (HNE) was compared with that of common alpha 1AT phenotypes. Time-dependent inhibition of HNE by alpha 1AT was used to calculate the association rate constant (k assoc) for M, MZ, FM, FZ, F (partially purified from FZ or FS), Z and S alpha 1AT phenotypes in human sera. The results for k assoc at 25 degrees C were 9.1 (SD 0.9), 9.7 (SD 0.9), 8.0 (SD 0.8), 4.0 (SD 0.4), 4.2 (SD 0.8), 5.1 (SD 0.6) and 8.6 (SD 0.6) x 10(6) M-1s-1 respectively. F was found to have reduced activity much like that of Z, the alpha 1AT most commonly associated with emphysema. MZ (low risk for disease) and FZ heterozygotes had similar intermediate alpha 1AT levels. However the in vivo inhibition time for FZ was almost three times longer than for MZ, indicating greater exposure to proteolytic damage from free elastase for FZ than MZ individuals. In conclusion, F alpha 1AT is expressed in serum at low normal levels but is dysfunctional in its ability to inhibit HNE. Individuals who coinherit the F and a deficiency allele such as Z or Null, are likely to have a high risk for the development of emphysema. The disease risk for F homozygotes remains to be determined.     

Carcinoid tumours of the thymus

Analysis by isoelectric focusing of serum alpha 1-antitrypsin phenotype of 167 consecutive cases from an occupational health outpatient clinic, from university hospital departments and from private practitioners showed an excessive presence of rare gene alleles S, Z, I and anodal variants compared to their frequencies in blood donors from Lausanne or in the general Swiss population. It seems that analysis has been well grounded in most cases and helps to establish diagnosis in many respiratory diseases, in unexplained liver cirrhosis and even in aortic rupture.     

A novel and de novo spontaneous point mutation (Glu271STOP) of the antithrombin gene results in a type I deficiency and thrombophilia

We describe a novel, de novo point mutation in one antithrombin (AT) allele resulting in type I AT deficiency and thrombophilia. Low plasma AT activity as well as low plasma AT antigen were documented in the propositus, but not in the parents, or in a male sibling. AT gene analysis by sequencing polymerase chain reaction-amplified genomic DNA from exon 5 of the propositus revealed a novel point mutation, GAG-->TAG at codon 271, resulting in a stop codon (Glu271STOP). This mutation was not demonstrable in the other members of his immediate family. DNA marker polymorphism analysis indicated the expected parentage. Based on allele frequency data for Caucasians in the United States the cumulative paternity index, or CPI, for the propositus and his father is 219,077. This corresponds to a probability of paternity of 99.9995% based on a prior probability of 50%. Included in this analysis is a linkage analysis of a trinucleotide repeat in intron 5 of the AT gene of the various family members, which also confirmed maternity and paternity. These studies provide documentation of the first spontaneous mutation of an AT gene in a thrombophilic individual, resulting in a type I AT deficiency.     

Reactive arthritis with a severe lesion of the cervical spine

The porphyrias are disorders that result from the inherited or acquired dysregulation of one of the eight enzymes in the heme biosynthetic pathway. Variegate porphyria (VP) is characterized by deficiencies in protoporphyrinogen oxidase (PPO) and has recently been genetically linked (Z = 6.62) to the PPO gene on chromosome 1q21. In this study, we have identified two sequence variants in the PPO gene in a family with VP. The first is a neutral polymorphism at the -47 position of intron 2; this polymorphism is present in the general population and is unlikely to underlie the VP phenotype. The second is a mutation in the PPO gene in a patient with VP; the mutation consists of an apparently de novo 2-bp insertion in exon 3 of PPO and results in a frameshift and downstream premature termination codon. These data establish that a frameshift mutation in PPO is the underlying mutation in this patient with VP and explain the sporadic occurrence of the phenotype in this family.     

Color stability of heat-activated and chemically activated fluid resin acrylics

The incidence of complete thyroxine-binding globulin (TBG) deficiency (TBG-CD) was determined for a Japanese population from a comprehensive health examination, in which a T3 resin uptake test of the upper 5% (78 subjects from among 1,589 men) was the screening line for TBG-CD. Further analysis of the known mutation in TBG-CD gene of the Japanese population (reported as TBG-CDJ with codon 352 deletion) was performed on 72 subjects, and three were found to have TBG-CDJ, two of whom were siblings. Only those three subjects had a serum TBG concentration of less than 5 mg/l. The six subjects for whom the DNA analysis was not performed, did not have a serum TBG level of less than 5 mg/l. From these findings, the gene frequency of TBG-CDJ was calculated to be 0.13%. The incidence of TBG-CDJ in the total Japanese population is suggested to be 0.09%.     

Laboratory diagnosis of immune inner ear disease

Inherited antithrombin deficiency is associated with an increased risk of thrombosis, primarily venous rather than arterial. Most affected individuals have inherited only a single copy of an abnormal antithrombin (AT) gene. Homozygously affected individuals, although rare, have a severe thrombotic history of early onset and often affecting the arteries. We report two new cases of type II HBS (heparin binding site) deficiency in which the propositi are homozygous for the previously reported mutation 99 Leu to Phe, and who have a severe thrombotic history. These cases are considered alongside existing homozygote and compound heterozygote cases.     

Genetics of vitamin D 1alpha-hydroxylase deficiency in 17 families

Vitamin D-dependent rickets type I (VDDR-I), also known as pseudo-vitamin D-deficiency rickets, appears to result from deficiency of renal vitamin D 1alpha-hydroxylase activity. Prior work has shown that the affected gene lies on 12q13.3. We recently cloned the cDNA and gene for this enzyme, mitochondrial P450c1alpha, and we and others have found mutations in its gene in a few patients. To determine whether all patients with VDDR-I have mutations in P450c1alpha, we have analyzed the P450c1alpha gene in 19 individuals from 17 families representing various ethnic groups. The whole gene was PCR amplified and subjected to direct sequencing; candidate mutations were confirmed by repeat PCR of the relevant exon from genomic DNA from the patients and their parents. Microsatellite haplotyping with the markers D12S90, D12S305, and D12S104 was also done in all families. All patients had P450c1alpha mutations on both alleles. In the French Canadian population, among whom VDDR-I is common, 9 of 10 alleles bore the haplotype 4-7-1 and carried the mutation 958DeltaG. This haplotype and mutation were also seen in two other families and are easily identified because the mutation ablates a TaiI/MaeII site. Six families of widely divergent ethnic backgrounds carried a 7-bp duplication in association with four different microsatellite haplotypes, indicating a mutational hot spot. We found 14 different mutations, including 7 amino acid replacement mutations. When these missense mutations were analyzed by expressing the mutant enzyme in mouse Leydig MA-10 cells and assaying 1alpha-hydroxylase activity, none retained detectable 1alpha-hydroxylase activity. These studies show that most if not all patients with VDDR-I have severe mutations in P450c1alpha, and hence the disease should be referred to as "1alpha-hydroxylase deficiency."     

The Asp84Glu variant of the melanocortin 1 receptor (MC1R) is associated with melanoma

Melanocyte stimulating hormone (MSH) plays an important role in determining the cutaneous response to ultraviolet radiation and may also influence melanoma progression. We have previously shown that variants of the melanocortin receptor present on melanocytes, MC1R, are associated with sun sensitivity and red hair in a UK population and therefore now consider the gene as a candidate for melanoma susceptibility. We have compared the frequency of known MC1R variants in the second and seventh transmembrane domains in 43 melanoma cases and 44 controls. MC1R variants were more common in cases than controls (chi 2 = 6.75, 1 d.f.; P = 0.0094) with a relative risk to carriers of variant alleles compared with normal homozygotes of 3.91 (95% c.l.: 1.48-10.35), and a population risk attributable to carriers of 34.6% (95% c.i. 10.7-52.1%). The Asp84Glu variant was only present in melanoma cases and appears to be of particular significance. The contribution of variant MC1R alleles was largely independent of skin type. Variants of the MC1R gene are likely to be causally associated with the development of melanoma.     

A model to describe how a point mutation of the estrogen receptor alters the structure-function relationship of antiestrogens

The antiestrogen tamoxifen [(Z)-1(p-beta-dimethylamino-ethoxyphenyl)-1,2- diphenylbut-1-ene] is an effective anticancer agent for the treatment of hormone responsive breast cancer. Previous studies have demonstrated that a point mutation in the estrogen receptor (ER) resulted in an alteration of the pharmacology of 4-hydroxytamoxifen, the active metabolite of tamoxifen (Jiang et al, Mol Endocrinol 6:2167-2174, 1992). We have extended our studies to evaluate the effect of a point mutation, a Val substitution for Gly at amino acid 400 in the ligand binding domain of ER, on the pharmacology of other antiestrogens in ER stable transfectants derived from the ER-negative breast cancer cell line MDA-MB-231 CL10A. The compounds were tested with or without estradiol-17 beta (E2) for their effects on cell growth in cells expressing the wild type ER (S30) or the mutant ER (ML alpha 2H) or in control antisense ER transfectant AS23 which does not express ER protein. MCF-7 cells, which express the wild type ER, were also used as a control. The growth of AS23 cells was not affected by any of the compounds at a concentration of 1 microM. E2 stimulated the growth of MCF-7 cells but inhibited the growth of ER transfectants S30 and ML alpha 2H. The ML alpha 2H cells were about 10 to 100-fold less sensitive to E2 and antiestrogens than S30 and MCF-7 cells. Keoxifene, an antiestrogen with a high affinity for the ER, maintained antiestrogenic activities in both ER transfectants and MCF-7 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Targeted inactivation of Npt2 in mice leads to severe renal phosphate wasting, hypercalciuria, and skeletal abnormalities

Npt2 encodes a renal-specific, brush-border membrane Na+-phosphate (Pi) cotransporter that is expressed in the proximal tubule where the bulk of filtered Pi is reabsorbed. Mice deficient in the Npt2 gene were generated by targeted mutagenesis to define the role of Npt2 in the overall maintenance of Pi homeostasis, determine its impact on skeletal development, and clarify its relationship to autosomal disorders of renal Pi reabsorption in humans. Homozygous mutants (Npt2(-/-)) exhibit increased urinary Pi excretion, hypophosphatemia, an appropriate elevation in the serum concentration of 1,25-dihydroxyvitamin D with attendant hypercalcemia, hypercalciuria and decreased serum parathyroid hormone levels, and increased serum alkaline phosphatase activity. These biochemical features are typical of patients with hereditary hypophosphatemic rickets with hypercalciuria (HHRH), a Mendelian disorder of renal Pi reabsorption. However, unlike HHRH patients, Npt2(-/-) mice do not have rickets or osteomalacia. At weaning, Npt2(-/-) mice have poorly developed trabecular bone and retarded secondary ossification, but, with increasing age, there is a dramatic reversal and eventual overcompensation of the skeletal phenotype. Our findings demonstrate that Npt2 is a major regulator of Pi homeostasis and necessary for normal skeletal development.     

NaF potentiates a K(+)-selective ion channel in G292 osteoblastic cells

In a previous study, isoelectrofocusing of serum from liver-diseased and healthy dogs revealed three different types of the acute phase protein, alpha-1-antitrypsin: Pi (protease inhibitor) F, Pi I and Pi S. Moreover, accumulated alpha-1-antitrypsin was found immunohistochemically in liver sections from dogs with chronic liver disease, predominantly in association with Pi I in serum. The present study was made to further the relationship between alpha-1-antitrypsin and the pathogenesis of chronic liver disease in dogs. Aliquots of samples of purified canine Pi F, Pi I and Pi S were examined for elastase inhibitory capacity, the main function of alpha-l-antitrypsin, and for polymerization tendency, a possible cause of accumulation of alpha-1-antitrypsin in the liver. These parameters were studied after incubation of the proteins at different temperatures (4, 37 and 42 degrees C) and pH values (6.8, 7.8 and 8.5) and for different periods (< or = 24 h and 5 days). In contrast to findings with Pi Z, the human alpha-1-antitrypsin variant associated with liver disease, polymers of canine Pi F, Pi I or Pi S could not be detected under any of the conditions tested. However, Pi I was sensitive to pH, as was demonstrated by reduced elastase inhibitory capacity after incubation at pH 6.8 for < or = 24 h or 5 days, or at pH 8.5 for 5 days. However, after incubation at pH 7.8 for < or = 24 h or 5 days at 4, 37 or 42 degrees C, Pi I was completely stable. Pi F retained its elastase inhibitory capacity, even after prolonged incubation, at all pH values and temperatures tested. Due to low yield, Pi S was tested only after incubation for < or = 24 h at pH 6.8 and at 4 degrees C; under these conditions its elastase inhibitory capacity was equal to that of Pi F. Taken together, these findings indicate molecular and functional differences between Pi I and Pi F and further support a role for alpha-1-antitrypsin in the pathogenesis of canine liver disease.     

The significance of haemochromatosis gene mutations in the general population: implications for screening

BACKGROUND: Haemochromatosis is associated with mutations in the HFE gene but the significance of these mutations in the general population is unknown. AIMS: To determine the frequency of HFE gene mutations in the general population, their effect on serum iron indexes, and their role in screening for haemochromatosis. METHODS: Deoxyribonucleic acid (DNA) from 1064 randomly selected subjects was analysed for the C282Y and H63D mutations in the HFE gene. Serum iron, transferrin saturation, and ferritin were measured and individuals with increased iron indexes were investigated to confirm or exclude a clinical diagnosis of haemochromatosis. RESULTS: Mutations were identified in 409 individuals (38.4%) with heterozygote (carrier) frequencies of 13.2% and 24.3% for the C282Y and H63D mutations respectively. Heterozygosity for either mutation significantly increased serum iron and transferrin saturation but despite a similar trend for ferritin, this was only significant for C282Y homozygotes. Five individuals (0.47%) were homozygous for the C282Y mutation, three of whom had haemochromatosis confirmed by liver biopsy (0.28%). The other two C282Y homozygotes would not have been detected by phenotypic screening alone. CONCLUSIONS: HFE mutations are present in 38.4% of the population, affect serum iron indexes, and are important determinants of iron status. The population frequency of genetically defined haemochromatosis (C282Y homozygosity) is approximately one in 200 and is higher than the prevalence of clinically apparent haemochromatosis.     

Association of systemic lupus erythematosus with promoter polymorphisms of the mannose-binding lectin gene

OBJECTIVE: To investigate the distribution of promoter variants of the mannose-binding lectin (MBL) gene and correlations between the promoter variants and serum MBL concentrations in Chinese patients with systemic lupus erythematosus (SLE) and in healthy Chinese controls. METHODS: We studied the serum MBL levels and codon 54 mutation in 112 Chinese patients with SLE and 110 healthy controls. Genotyping of promoter variants of the MBL gene were done by polymerase chain reaction and allele-specific oligonucleotide hybridization. RESULTS: We found significant differences in the distribution of the 2 pairs of promoter polymorphisms, H/L and Y/X, between SLE patients and controls (P=0.018 and P=0.019, respectively). Analysis of the correlation between promoter haplotypes and serum MBL levels revealed HY as the highest-producing, LY as the intermediate-producing, and LX as the lowest-producing haplotypes. The LX haplotype was present at a frequency of 0.259 in SLE patients and 0.154 in controls and was significantly associated with SLE (P=0.019, odds ratio 1.79, 95% confidence interval 1.12-2.85). CONCLUSION: The low-producing promoter polymorphism of the MBL gene is associated with SLE, and a low serum MBL level is a risk factor for SLE. Even allowing for promoter polymorphisms and structural mutations of the MBL gene, serum MBL levels in SLE patients are still lower than those in controls, suggesting a trans-factor in regulating serum MBL levels.     

Cytochrome oxidase subunit III from Arbacia lixula: detection of functional constraints by comparison with homologous sequences

The role of autoantibodies in the pathogenesis of autoimmune thyroiditis (AT) still remains poorly understood. Serum transfer experiments in experimental AT (EAT) and spontaneous AT (SAT) animal models frequently did not succeed or only resulted in minor thyroid lesions. The inconsistent results may have arisen because of technical problems inherent in serum transfer, the major of which is to obtain high enough concentrations of autoantibodies over long enough periods at the potential site of tissue injury. An attempt was made to circumvent this hurdle by repeated in situ perfusions of the rabbit thyroid via the superior thyroid artery. In situ perfusion of rabbit thyroids with high-titered homologous sera reactive with saline thyroid extract indeed led to thyroiditis characterized by granular deposits of rabbit IgG and C3 in the thyroid follicular basal laminae, cellular infiltrates consisting of mononuclear cells and granulocytes, destruction of the thyroid follicular architecture, and focal fibrosis. Perfusion with control sera lacking thyroid-reactive antibodies did not lead to thyroid lesions. These results demonstrate that humoral antibodies can induce severe AT comparable to actively induced thyroiditis.     

ThioTEPA pharmacokinetics during intravesical chemotherapy: the influence of dose and volume of instillate on systemic uptake and dose rate to the tumour

Hereditary tyrosinaemia type I (HTI), an autosomal recessive inborn error of metabolism, is caused by a deficiency of the enzyme fumarylacetoacetate hydrolase. The highest incidence of HTI is observed in the Saguenay-Lac-St-Jean region (SLSJ) (Québec, Canada), where 1 out of 22 individuals is thought to be a carrier. A splice mutation (IVS12 + 5G-->A) has recently been identified in this particular region. Here, we have determined the frequency of this mutation in a population of obligate carriers from the SLSJ region by allele-specific oligonucleotide hybridization and a method using a restriction enzyme digestion. Over 95 per cent of the HTI carriers were found to have the IVS12 + 5G-->A splice mutation. Screening for this mutation based on the two methods reported here is thus a reliable and rapid way of detecting carriers of hereditary tyrosinaemia type I in that region at high risk.     

Different molecular events result in low protein levels of mannan-binding lectin in populations from southeast Africa and South America

Previous studies have shown that three point mutations in exon 1 and a particular promoter haplotype of the mannan-binding lectin (MBL) gene lead to a dramatic decrease in the serum concentration of MBL. In this study, MBL genotypes and serum concentrations were determined in unrelated individuals in a population from Mozambique (n = 154) and in two native Indian tribes from Argentina (i.e., the Chiriguanos (n = 43) and the Mapuches (n = 25)). In both populations, the MBL concentrations were low compared with those found in Eskimo, Asian, and European populations. In Africans, the low serum concentrations were due to a high allele frequency (0.24) of the codon 57 (C) variant, which resulted in a high frequency of individuals with MBL deficiency (0.06), and were also due to the effect of a relatively high frequency (0.13) of low-producing promoter haplotypes. The low concentrations in the South American populations were primarily due to an extremely high allele frequency of the codon 54 (B) variant in both the Chiriguanos (0.42) and the Mapuches (0.46), resulting in high frequencies of individuals with MBL deficiency (0.14 and 0.16, respectively). In the search for additional genetic variants, we found five new promoter mutations that might help to elucidate the evolution of the MBL gene. Taken together, the results of this study show that different molecular mechanisms are the basis for low MBL levels on the two continents.     

Expression studies of delta-globin gene alleles associated with reduced hemoglobin A2 levels in Greek Cypriots

We previously identified five delta-globin gene alleles associated with reduced hemoglobin (Hb) A2 (Trifillis, P., Ioannou, P., Schwartz, E., and Surrey, S. (1991) Blood 78, 3298-3305). We have now evaluated functional consequences of the changes after expression in COS-1 cells to monitor effects on RNA splicing. In addition, variant Hb A2 tetramers were expressed in yeast to assess effects of amino acid changes on oxygen binding and stability to heat and mechanical agitation. The G --> T change at codon 27 and the A --> G change in IVS-2 both affect RNA splicing, whereas the C --> T change at codon 97 and the AT deletion in IVS-2 have no effect. Oxygen equilibrium curves of the Hb A2 variants expressed in yeast were similar to that of wild type Hb A2. None of the three variant Hb A2 tetramers (Thr --> Ile at codon 4 (Hb deltaT4I), Ala --> Ser at codon 27 (Hb deltaA27S), and Arg --> Cys at codon 116 (Hb deltaR116C)) showed decreased heat stability compared with Hb A2, whereas the Hb deltaT4I variant showed highest instability to mechanical agitation. Co-expression in yeast of alpha-globin chain and the delta-chain variant containing a Leu --> Pro change at codon 141 yielded no identifiable tetramers, suggesting lack of assembly or severe tetramer instability. These studies show the probable cause for decreased Hb A2 for two alleles is due to defective splicing, whereas decreased protein stability, increased tetramer association with red cell membranes, increased interdisulfide bond formation of delta-chains, which inhibits assembly with alpha-chains, and/or reduced assembly is suggested for the other three alleles.     

Erratum to Mutation spectrum of 2-chloroethyl methanesulfonate in Drosophila melanogaster premeiotic germ cells' [Mutation Res. 331 (1995) 213-224]

The 2-chloroethyl methanesulfonate (2CIEMS)-induced alcohol dehydrogenase (Adh) null germline mutation frequency in treated Drosophila melanogaster second instar larval gonia was two orders of magnitude greater than the spontaneous mutation frequency. DNA sequence analysis of 83 Adh null mutations showed that 40 mutations of independent origin were at 23 sites in the Adh gene. The mutation spectrum contained only GC --> AT transitions with 35 mutations (87.5%) at the middle or 3' guanine. In addition, characteristics of glutathione (GSH)-mediated bioactivation were determined for 2CIEMS in vitro. Rates of GSH-mediated conjugation, catalyzed by purified rat liver glutathione-S-transferase (GST), and binding of [35S]GSH-mediated conjugation products to calf thymus DNA were determined for 2CIEMS, 1,2-dichloroethane (EDC) and 1,2-dibromoethane (EDB). The relative rates of GSH-mediated conjugation were the following: 5 mM EDB > 40 mM 2CIEMS > 40 mM EDC. A similar trend was observed for DNA binding of the [35S]GSH-mediated conjugation products when differences in mutagen concentration were considered: EDB > 2CIEMS > EDC. The ratios of DNA binding to GSH conjugation calculated for EDB, EDC and 2CIEMS were 6.8 x 10(-5), 9.3 x 10(-5) and 19.1 x 10(-5), respectively. A narrow range, less than a 3-fold difference, in the ratios of DNA binding to GSH conjugation indicates that the bioactivation of 2CIEMS is mediated by the same mechanism as EDB and EDC. Consequently, 2CIEMS, EDC and EDB may induce a specific mutation in premeiotic germ cells.     

Impaired cotranslational processing as a mechanism for type I antithrombin deficiency

Most secretory proteins, including antithrombin (AT), are synthesized with a signal peptide, which is cleaved before the mature protein is exported from the cell. The signal peptide is important in the process whereby nascent protein is recognized as requiring subsequent modification within the endoplasmic reticulum (ER). We have identified a novel mutation, 2436T-->C L(-10)P, which affects the central hydrophobic domain of the AT signal peptide, in a proband presenting with venous thrombotic disease and type I AT deficiency. We investigated the basis of the phenotype by examining expression in mammalian cells of a range of variant AT cDNAs with mutations affecting the -10 residue. Glycosylated AT was secreted from COS-7 cells transfected with wild-type AT, -10L deletion, -10V or -10M variants, but not variants with P, T, R, or G at -10. Cell-free expression of wild-type and variant AT cDNAs was then performed in the presence of canine pancreatic microsomes, as a substitute for ER. Variant AT proteins with P, T, R, or G at residue -10 did not undergo posttranslational glycosylation, and their susceptibility to trypsin digestion suggested they had not been translocated into microsomes. Our results suggest that the ability of AT signal peptide to direct the protein to ER for cotranslational processing events appears to be critically dependent on maintaining the hydrophobic nature of the region including residue -10. The investigations have defined impaired cotranslational processing as a hitherto unrecognized cause of hereditary AT deficiency.