Heritable Deficiency Research Articles

Epidemiological Molecular Study of Combined Severe Immune Deficiency in Arabian Horses in Tunisia

Background: Severe Combined Immuno Deficiency (SCID) is a heritable deficiency transmitted through autosomal recessive gene, carried on by purebred Arabian and crossbred horses. A deletion of five base pairs at the gene encoding the catalytic subunit of the Protein Kinase DNA-dependent (DNA-PKcs) is responsible for this disease and SCID-affected animals always die in the first six months of life. Considering this problem, it is important to perform a molecular epidemiological study to estimate the frequency of the SCID allele in Arabian purebred horses in Tunisia. Methods: The DNA of the peripheral blood lymphocytes of 164 purebred Arabian horses belonging to the Sidi Thabet stud was extracted by the spin-column method, in order to verify the quality of the DNAs used, the samples were dosed by a spectrophotometer. The amplification of the DNA was carried out by two specific primers in a Polymerase Chain Reaction (PCR) to flank the deletion zone of the DNA sample by adding to each PCR sample a marker or a standard of size knowing that the fragment we wanted to amplify is composed of 163 base pairs. Then, the PCR products were sequenced using an automatic sequencer. Result: By analyzing the electropherograms results, we noted the absence of the SCID deletion in the studied group of Arab purebreds. Hence, it is essential to carry out molecular screening of the SCID deletion in other sites in order to determine its prevalence in the country’s purebred Arabian horse population.

Diagnostic challenge of the newborn patients with heritable protein C deficiency

AbstarctObjectiveThe diagnosis of neonatal-onset protein C (PC) deficiency is challenging. This study aimed to establish the neonatal screening of heritable PC deficiency in Japan.Study designWe determined the changes in plasma activity levels of PC and protein S (PS) in healthy neonates, and studied newborn patients with PROC mutation in the Japanese registry.ResultPhysiological PC and PS levels increased with wide range. The PC/PS-activity ratios converged after birth. The PC/PS-activity ratios of 19 patients with biallelic mutations, but not, 9 with monoallelic mutation, were lower than those of 13 without mutation. The logistic regression analyses established a formula including two significant variables of PC activity (cut-off < 10%, odds ratio = 30.0) and PC/PS-activity ratio (cut-off < 0.35, odds ratio = 22.7), with 93% sensitivity and 44% specificity for determining patients with mutation(s).ConclusionThe PC/PS-activity ratio is an effective parameter for the genetic screening of neonatal-onset PC-deficiency in Japanese population.

GSTM1 and GSTT1 Polymorphisms and Susceptibility to Prostate Cancer: A Case-Control Study of the Algerian Population

Objective:Prostate cancer (PCa) is a major public health problem worldwide, with high morbidity and mortality levels. Advanced age, androgen stimulation, and ethnicity have been reported to be possible risk factors. It has been suggested that particular genetic polymorphisms in glutathione S-transferases (GST), xenobiotic-metabolising enzymes, could predispose to prostate cancer through heritable deficiency in detoxification of environmental carcinogens. Conflicts in the published results and the absence of similar in depth studies in Algeria prompted us to perform the present case-control study of GSTM1 and GSTT1 polymorphisms and their possible association with PCa in an Algerian population.Methods:We determined GSTM1 and GSTT1 genotypes for 49 histologically verified prostate cancer patients and in 41 age-matched healthy controls by multiplex polymerase chain reaction (PCR) using peripheral blood DNA samples.Result:While an association between the GSTM1 null genotype and PCa risk (OR= 3.69, 95% CI= 1.30-10.44; P = 0.01) was evident, the GSTT1 null genotype (OR= 0.92, 95% IC= 0.32-2.62; P = 0.49) appeared without influence. Furthermore, no statistically significant differences between the double null genotype and PCa is detected, also no statistically significant differences between smoking status and PCa is detected.Conclusion:The GSTM1 null genotype may increase individual susceptibility to prostate cancer. On the other hand, the null-activity genotype of GSTT1 did not appear to contribute to the risk of prostate cancer in our population.

Human RECQ Helicase Pathogenic Variants, Population Variation and "Missing" Diseases.

Heritable loss of function mutations in the human RECQ helicase genes BLM, WRN, and RECQL4 cause Bloom, Werner, and Rothmund-Thomson syndromes, cancer predispositions with additional developmental or progeroid features. In order to better understand RECQ pathogenic and population variation, we systematically analyzed genetic variation in all five human RECQ helicase genes. A total of 3,741 unique base pair-level variants were identified, across 17,605 potential mutation sites. Direct counting of BLM, RECQL4, and WRN pathogenic variants was used to determine aggregate and disease-specific carrier frequencies. The use of biochemical and model organism data, together with computational prediction, identified over 300 potentially pathogenic population variants in RECQL and RECQL5, the two RECQ helicases that are not yet linked to a heritable deficiency syndrome. Despite the presence of these predicted pathogenic variants in the human population, we identified no individuals homozygous for any biochemically verified or predicted pathogenic RECQL or RECQL5 variant. Nor did we find any individual heterozygous for known pathogenic variants in two or more of the disease-associated RECQ helicase genes BLM, RECQL4, or WRN. Several postulated RECQ helicase deficiency syndromes-RECQL or RECQL5 loss of function, or compound haploinsufficiency for the disease-associated RECQ helicases-may remain missing, as they likely incompatible with life.

Association of thiopurine methyltransferase status with azathioprine side effects in Chinese patients with systemic lupus erythematosus

Azathioprine (AZA) is indicated for the treatment of systemic lupus erythematosus (SLE). Thiopurine methyltransferase (TPMT) is the rate-limiting enzyme in the steps of AZA metabolization. Heritable deficiency of TPMT enzyme activity and polymorphisms may lead to leukopenia. This study aims to detect TPMT polymorphisms and TPMT enzyme activity in Chinese SLE patients and to describe the association between TPMT genotypes and adverse effects of AZA. One hundred and twenty-six SLE patients with present or previous thiopurine therapy were identified from a local database. Adverse effects were documented. No TPMT*2, TPMT*3A, or TPMT*3B mutant alleles were detected. TPMT*3C was detected in four patients (3.17%). The heterozygotes had significantly lower mean TPMT activity as compared to the homozygotes (2.38 ± 1.24 vs. 12.56 ± 7.02U/mL, P < 0.001). Twenty-seven cases (21.42%) exhibited adverse effects. All of the heterozygotes (4/4, 100%) developed severe leukopenia, and three cases (3/4, 75%) of whom exhibited alopecia simultaneously. The specificity of TPMT*3C for predicting leukopenia and alopecia was 100 and 99.17%, respectively, and the sensitivity was 28.57 and 60.00%, respectively. The mean value of TPMT activity with leukopenia (4.67 ± 3.01 vs. 13.2 ± 6.94U/mL RBC, P < 0.001) or alopecia (2.31 ± 1.16 vs. 12.65 ± 6.98U/mL RBC, P < 0.001) was significantly lower than those without. TPMT*3C was the most common mutant polymorphism found in the study group. TPMT activity is reduced in TPMT*3C mutant. AZA-induced leukopenia and alopecia were partly correlated to TPMT*3C heterozygotes and low TPMT activity. The results of this study suggest that the value of TPMT genotyping before AZA therapy was limited in Chinese SLE patients, considering the low sensitivity. Routine monitoring of TPMT activity before prescribing and continuous hematological monitoring dose were recommended.

The phenotypic and genetic assessment of antithrombin deficiency

Antithrombin (AT) deficiency is associated with an increased risk of deep vein thrombosis and pulmonary embolism which are major causes of morbidity and death. The incidence of deficiency in healthy populations has been reported to vary from 1/600 to 1/5000, with the variation being due to the different populations studied and detection methods used. When reduced activity levels are identified it is important to measure the AT antigen levels to differentiate type I from type II disorders, as type II defects have varying thrombotic risk. Functional AT assays detect the ability of AT to inactivate thrombin or factor Xa, and AT antigen assays detect the quantity of AT in plasma. In functional assays, reducing the incubation time of sample with enzyme/heparin reagent may increase sensitivity to type II defects. An excess of antigen over activity level suggests the presence of functionally defective AT, which can be characterized further by assaying AT in the absence of heparin, electrophoresis to investigate the ability of heparin to bind to AT, and gene sequencing. Many patients with AT deficiency have a type II defect and these defects may not be detected by all routine diagnostic assays. Assays using human thrombin may lack specificity and assays that use factor Xa may fail to detect the common variant, AT Cambridge II, which can be detected by assays using bovine thrombin, especially if activity is compared to antigen by ratio. Factor Xa based assays may be particularly sensitive to certain heparin binding defects, and sensitivity of assays to both heparin binding and reactive site defects can be improved by shortening the incubation time with enzyme. uAT activity assays are essential for the detection of AT deficiency because type II defects are relatively common in patients with heritable deficiency. No one functional assay can be assumed to detect all forms of AT deficiency, and assays can sometimes be improved by reducing reaction time of AT with thrombin or factor Xa.

Lack of association between glutathione S-transferase polymorphisms and primary glioma in a case-control study in Rio de Janeiro

The glutathione S-transferases (GSTs), a family of phase II isozymes, detoxify several carcinogens. Genetic variations in GSTs have been associated with increased risk for cancer due to a heritable deficiency in detoxification pathways for environmental carcinogens. Conflicting findings have been reported about the association between constitutive GST polymorphisms and gliomas in different populations. The present case-control study examined 78 patients with primary glioma and 347 controls from Rio de Janeiro. DNA was isolated from whole blood, and four genetic polymorphisms (GSTM1, GSTM3, GSTT1, and GSTP1) were determined by PCR-RFLP. The distributions of the genotypic frequencies of these polymorphisms did not differ significantly between cases and controls and were as expected by Hardy-Weinberg equilibrium (P > 0.05). Risk analysis did not show an association between GSTs and primary glioma, suggesting that these polymorphisms do not influence the risk of primary glioma, at least in this population in Rio de Janeiro, Brazil.

Abstract 5302: Glutathione Peroxidase-3 Deficiency Promotes Platelet-dependent Thrombosis and Thrombotic Stroke in vivo

Plasma glutathione peroxidase (GPx-3) is a selenocysteine-containing protein with antioxidant activity. GPx-3 plays an important role in plasma by scavenging reactive oxygen species. We have previously shown that a heritable deficiency of this enzyme is associated with platelet-dependent thrombotic stroke in patients. We recently developed a GPx-3 knockout mouse to assess platelet function and platelet-dependent thrombosis in models of vascular injury. We hypothesized that GPx-3 deficiency promotes platelet-dependent thrombus formation in vivo . GPx-3 (−/−) mice showed an attenuated bleeding time compared with wild-type mice (94.5±28.8 s versus 153.4±32.3, P&lt;0.05). We also noted an increase in the plasma levels of soluble P-selectin, a marker of platelet activation and prothrombotic activity, in GPx-3 (−/−) mice compared with wild-type mice (137.8±12.3 ng/ml plasma versus 101.5±8.8, P&lt;0.05). Using a no-flow ischemia-reperfusion stroke model, we found that GPx-3 (−/−) mice had significantly larger cerebral infarctions compared with wild-type mice (117.5±7.9 mm 3 versus 17.2±2.7, P&lt;0.001) as well as higher neurological deficit scores (3.33±0.33 versus 1.3±0.33, P=0.02). To investigate the effect of platelet inhibition on stroke size in GPx-3 deficiency, GPx-3 deficient mice were pre-treated with clopidogrel prior to producing the stroke. We found that clopidogrel treatment reduced stroke size significantly compared with vehicle-treated controls (4.42±3.0 mm 3 versus 105.1±15.2, P=0.003) as well as lowered neurological deficit scores (0.4±0.2 versus 2.83±0.44, P=0.018). These findings demonstrate that GPx-3 deficiency promotes platelet activation resulting in a prothrombotic state. These data illustrate the importance of this plasma antioxidant enzyme in regulating platelet activity, platelet-dependent thrombosis, and vascular thrombotic propensity.

Polymorphisms of glutathione-S-transferase M1, T1, P1 and the risk of prostate cancer: a case-control study

BackgroundIt has been suggested that polymorphisms in glutathione-S-transferases (GST) could predispose to prostate cancer through a heritable deficiency in detoxification pathways for environmental carcinogens. Yet, studies linking GST polymorphism and prostate cancer have so far failed to unambiguously establish this relation in patients. A retrospective study on healthy, unrelated subjects was conducted in order to estimate the population GST genotype frequencies in the Slovak population of men and compare our results with already published data (GSEC project-Genetic Susceptibility to Environmental Carcinogens). A further aim of the study was to evaluate polymorphisms in GST also in patients with prostate cancer in order to compare the evaluated proportions with those found in the control subjects.MethodsWe determined the GST genotypes in 228 healthy, unrelated subjects who attended regular prostate cancer screening between May 2005 and June 2007 and in 129 histologically verified prostate cancer patients. Analysis for the GST gene polymorphisms was performed by PCR and PCR-RFLP.ResultsWe found that the GST frequencies are not significantly different from those estimated in a European multicentre study or from the results published by another group in Slovakia. Our results suggest that Val/Val genotype of GSTP1 gene could modulate the risk of prostate cancer, even if this association did not reach statistical significance. We did not observe significantly different crude rates of the GSTM1 and GSTT1 null genotypes in the men diagnosed with prostate cancer and those in the control group.ConclusionUnderstanding the contribution of GST gene polymorphisms and their interactions with other relevant factors may improve screening diagnostic assays for prostate cancer. We therefore discuss issues of study feasibility, study design, and statistical power, which should be taken into account in planning further trials.

Improving pharmacovigilance in Europe: TPMT genotyping and phenotyping in the UK and Spain

Thiopurine S-methyltransferase (TPMT) is the rate-limiting step in the conversion of thiopurine drugs including azathioprine (AZA) to inactive metabolites. Heritable deficiency of TPMT activity increases risk for adverse events, most notably, myelosuppression leading to leukopenia and neutropenic sepsis. The reported European Commission study was undertaken to identify current evidence for the clinical utility of testing for TPMT status and extent of uptake, by either genotyping or phenotyping, in the clinical setting. Data presented here for the UK and Spain indicate that there has been a considerable increase in the uptake of TPMT testing in recent years. There are some data that support routine TPMT testing before AZA prescribing for reducing AZA-related adverse events. Key data include evidence in favor of TPMT testing in addition to the current practice of routine monitoring for reducing the number of AZA-related episodes of myelosuppression, averting deaths from neutropenic sepsis and improving health-related quality of life. Further data are needed for determining the cost-effectiveness of routine TPMT testing.

Oxidative Enzymopathies and Vascular Disease

In the vasculature, reactive oxygen species (ROS) generated by both mitochondrial respiration and enzymatic sources serve as integral components of cellular signaling and homeostatic mechanisms. Because ROS are highly reactive biomolecules, the cellular redox milieu is carefully maintained by small-molecule antioxidants and antioxidant enzymes to prevent the deleterious consequences of ROS excess. When this redox balance is perturbed, because of either increased ROS production or decreased antioxidant capacity, oxidant stress is increased in the vessel wall and, if not offset, vascular dysfunction ensues. A number of heritable polymorphisms of pro-oxidant enzymes, including 5-lipoxygenase, cyclooxygenase-2, nitric oxide synthase-3, and NAD(P)H oxidase, have been identified and found to modulate ROS production and, thereby, the risk of atherothrombotic cardiovascular disease in individuals with these genetic polymorphisms. Similarly, heritable deficiency of the antioxidant enzymes catalase, glutathione peroxidases, glutathione-S-transferases, heme oxygenase, and glucose-6-phosphate dehydrogenase favors ROS accumulation, and has been associated with an increased risk of vascular disease. Individually, each of these polymorphisms imposes a state of uncompensated oxidant stress on the vasculature and collectively comprise the oxidative enzymopathies.

Deletion of macrophage-inflammatory protein 1 alpha retards neurodegeneration in Sandhoff disease mice.

Sandhoff disease is a prototypical lysosomal storage disorder in which a heritable deficiency of a lysosomal enzyme, beta-hexosaminidase, results in the storage of the enzyme's substrates in lysosomes. As with many of the other lysosomal storage diseases, neurodegeneration is a prominent feature. Although the cellular and molecular pathways that underlie the neurodegenerative process are not yet fully understood, macrophage/microglial-mediated inflammation has been suggested as one possible mechanism. We now show that the expanded macrophage/microglial population in the CNS of Sandhoff disease mice is compounded by the infiltration of cells from the periphery. Coincident with the cellular infiltration was an increased expression of macrophage-inflammatory protein 1alpha (MIP-1alpha), a leukocyte chemokine, in astrocytes. Deletion of MIP-1alpha expression resulted in a substantial decrease in infiltration and macrophage/microglial-associated pathology together with neuronal apoptosis in Sandhoff disease mice. These mice without MIP-1alpha showed improved neurologic status and a longer lifespan. The results indicate that the pathogenesis of Sandhoff disease involves an increase in MIP-1alpha that induces monocytes to infiltrate the CNS, expand the activated macrophage/microglial population, and trigger apoptosis of neurons, resulting in a rapid neurodegenerative course.

Lipoprotein metabolism in subjects with hepatic lipase deficiency

A heritable deficiency of hepatic lipase (HL) provides insights into the physiologic function of HL in vivo. The metabolism of apolipoprotein B (apoB)-100 in very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and low-density lipoprotein (LDL) and of apoA-I and apoA-II in high-density lipoprotein (HDL) particles lipoprotein (Lp)(AI) and Lp(AI:AII) was assessed in 2 heterozygous males for compound mutations L334F/T383M or L334F/R186H, with 18% and 22% of HL activity, respectively, compared with 6 control males. Subjects were provided with a standard Western diet for a minimum of 3 weeks. At the end of the diet period, apo kinetics was assessed using a primed-constant infusion of [5,5,5- 2H 3] leucine. Mean plasma triglyceride (TG) and HDL cholesterol levels were 55% and 12% higher and LDL cholesterol levels 19% lower in the HL patients than control subjects. A higher proportion of apoB-100 was in the VLDL than IDL and LDL fractions of HL patients than control subjects due to a lower VLDL apoB-100 fractional catabolic rate (FCR) (4.63 v 9.38 pools/d, respectively) and higher hepatic production rate (PR) (33.24 v 10.87 mg/kg/d). Delayed FCR of IDL (2.78 and 6.31 pools/d) and LDL (0.128 and 0.205 pools/d) and lower PR of IDL (3.67 and 6.68 mg/kd/d) and LDL 4.57 and 13.07 mg/kg/d) was observed in HL patients relative to control subjects, respectively. ApoA-I FCR (0.09 and 0.13 pools/d) and PR (4.01 and 6.50 mg/kg/d) were slower in Lp(AI:AII) particles of HL patients relative to control subjects, respectively, accounting for the somewhat higher HDL cholesterol levels. HL deficiency may result in a lipoprotein pattern associated with low heart disease risk.

Crystallographic Evidence for Substrate-assisted Catalysis in a Bacterial β-Hexosaminidase

beta-Hexosaminidase, a family 20 glycosyl hydrolase, catalyzes the removal of beta-1,4-linked N-acetylhexosamine residues from oligosaccharides and their conjugates. Heritable deficiency of this enzyme results in various forms of GalNAc-beta(1,4)-[N-acetylneuraminic acid (2,3)]-Gal-beta(1,4)-Glc-ceramide gangliosidosis, including Tay-Sachs disease. We have determined the x-ray crystal structure of a beta-hexosaminidase from Streptomyces plicatus to 2.2 A resolution (Protein Data Bank code ). beta-Hexosaminidases are believed to use a substrate-assisted catalytic mechanism that generates a cyclic oxazolinium ion intermediate. We have solved and refined a complex between the cyclic intermediate analogue N-acetylglucosamine-thiazoline and beta-hexosaminidase from S. plicatus to 2.1 A resolution (Protein Data Bank code ). Difference Fourier analysis revealed the pyranose ring of N-acetylglucosamine-thiazoline bound in the enzyme active site with a conformation close to that of a (4)C(1) chair. A tryptophan-lined hydrophobic pocket envelopes the thiazoline ring, protecting it from solvolysis at the iminium ion carbon. Within this pocket, Tyr(393) and Asp(313) appear important for positioning the 2-acetamido group of the substrate for nucleophilic attack at the anomeric center and for dispersing the positive charge distributed into the oxazolinium ring upon cyclization. This complex provides decisive structural evidence for substrate-assisted catalysis and the formation of a covalent, cyclic intermediate in family 20 beta-hexosaminidases.

Protoporphyria.

Human protoporphyria results from mutations in the ferrochelatase gene. Heritable deficiency of ferrochelatase causes overproduction of protoporphyrin IX, principally in the erythron. Photosensitivity is a universal feature of protoporphyria but hepatic clearance of the hydrophobic protoporphyrin molecule with excretion in bile may lead to precipitation within biliary pathways. Thus cholestatic injury and protoporphyrin gallstones occur. Minor hepatic abnormalities are frequent, but at least 30 patients have been reported with a progressive liver disease that requires transplantation. Fulminant hepatic disease appears to be recessively inherited in some pedigrees. Hazards of liver transplantation include tissue photolysis, hemolysis, and an unexplained neurological syndrome, but most of the 15 patients reported after transplantation have survived for several months to > 6 years. Aspects of protoporphyria, its pathogenesis and contemporary therapeutic strategies are considered, with emphasis on hepatic sequelae.

Heritable deficiency of glutathione S-transferases T1 and M1 and UV-light sensitivity of the skin.

Heritable deficiency of glutathione S-transferases T1 and M1 and UV-light sensitivity of the skin.

Identification of a heritable deficiency of the folate-dependent enzyme 10-formyltetrahydrofolate dehydrogenase in mice.

During the analysis of liver protein expression in the offspring of male mice irradiated with fission-spectrum neutrons, one offspring displayed a heritable 50% decrease in the abundance of two proteins. Homozygous mice lacking detectable quantities of these proteins were obtained through breeding. Characterization of this protein deficiency has identified these liver proteins as forms of the enzyme 10-formyltetrahydrofolate dehydrogenase (10-formyl-THF DH; 10-formyltetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.6). NH2-terminal sequence analysis demonstrated that both proteins share identical sequences in the first 25 residues, and this sequence matches (96% identity) that of rat and human 10-formyl-THF DH. In addition, these proteins showed cross-reactivity to polyclonal antiserum raised against purified rat 10-formyl-THF DH. Southern (DNA) blot analysis revealed a restriction fragment length polymorphism consistent with a deletion mutation in the 10-formyl-THF DH structural gene in homozygous mice. Results of Northern (RNA) blot analysis demonstrated the absence of 10-formyl-THF DH mRNA in mice lacking 10-formyl-THF DH protein. Furthermore, liver cytosolic 10-formyl-THF DH enzymatic activity was undetectable in homozygotes. Measurement of hepatic folate pools showed that in homozygotes the total folate pool is decreased and the level of tetrahydrofolate is markedly depleted.

Hurler Syndrome With Special Reference to Histologic Abnormalities of the Growth Plate

Hurler syndrome is a mucopolysaccharide disorder resulting from an heritable deficiency in alpha-L-iduronidase, an enzyme required in the catabolism of heparan sulfate and dermatan sulfate glycosaminoglycan (GAGs). The resultant intracellular accumulation of GAG leads to disruption of the intracellular and extracellular environment and dysfunction of multiple organ systems. Among the most noted manifestations of this disease is disproportionate short trunk dwarfism, which develops during the first years of life. Histochemical and electron-microscopic observations on a 30-month-old child with Hurler syndrome showed marked irregularities in chondrocyte orientation within the growth plate, along with disruption of the normal columnar architecture. Vacuolization with enlargement of the cellular border was the characteristic ultrastructural finding. An heritable abnormality in the enzymatic degradation of structural glycosaminoglycans leads to profound disruption of the normal mechanisms of growth and development.

The Role of the Type 3 Complement Receptor in the Induced Recruitment of Myelomonocytic Cells to Inflammatory Sites in the Mouse

The type 3 complement receptor (CR3), initially identified as the leukocyte cell surface receptor for iC3b, is now known to form part of the extended integrin family of cell adhesion molecules that mediate both cell-cell and cell-extracellular matrix interactions. The identification of a heritable deficiency of human leukocyte adhesion together with the advent of monoclonal antibodies has shed some light on the central role of CR3 in the transendothelial migration of macrophages and neutrophils to sites of inflammation. We review the general structural features of CR3 and then examine our understanding of its role in both nonspecific and T cell-dependent inflammatory processes based on our murine in vivo experiments. CR3-dependent inflammation seems to contribute to the pulmonary response to some stimuli (lipopolysaccharide) but not to others (bacillus Calmette-Guerin). These studies highlight the potential therapeutic benefits, as well as the significant risks of potentiating acute bacterial infections, of CR3 blockade in vivo.

Leukocyte Adhesion Deficiency: Aberrant splicing of a conserved integrin sequence causes a moderate deficiency phenotype

Leukocyte adhesion deficiency (LAD) is a heritable deficiency of the LFA-1, Mac-1, p150,95 family of leukocyte alpha beta heterodimers (the leukocyte integrins). We have studied the defect in patients who synthesize an aberrantly small form of the beta subunit common to all three proteins. S1 nuclease protection showed the presence of a 90-nucleotide mismatch in RNA from patients and relatives, correlating with inheritance of the disease. Use of the Taq polymerase chain reaction to amplify this region of RNA after first strand cDNA synthesis and sequencing showed an in-frame deletion of 90 nucleotides in the extracellular domain. Thus, this highly conserved region, 63% and 53% identical in amino acid sequence to two other beta subunits of the integrin family, is required for association of the beta subunit with alpha subunits. The 90-nucleotide region corresponds to a single exon present in both the normal and patient genome. The patient DNA has a single G to C substitution in the 5' splice site. This results in the direct joining of nonconsecutive exons in an unusual type of abnormal RNA splicing. A small amount of normally spliced message, detected by S1 nuclease protection and Taq polymerase chain reaction, encodes a normal sized beta subunit which is surface-expressed and accounts for the low levels of leukocyte integrin expression observed in these patients, and hence the moderate phenotype.