A novel 196Leu to Pro substitution in the beta3 subunit of the alphaIIbbeta3 integrin in a patient with a variant form of Glanzmann thrombasthenia

Glanzmann thrombasthenia (GT) is an inherited disorder where an absence of platelet aggregation is associated with quantitative or qualitative abnormalities of the alphaIIbbeta3 integrin. In rare patients, amino acid substitutions have provided information on the functional significance of specific domains within alphaIIb or beta3. We now report an elderly male GT patient (R.M.) from the south west of France whose platelets possess a small residual expression of alphaIIbbeta3. Furthermore, the integrin failed to undergo the necessary conformational changes following platelet activation to permit the binding of fibrinogen or activation-dependent monoclonal antibodies despite the presence of an RGD-binding site. Screening of the alphaIIb and beta3 genes by PCR-SSCP revealed a heterozygous mutation at position 685 in exon 5 of the beta3 gene leading to a 196Leu to Pro substitution. 196Leu is a highly conserved amino acid of beta3. The other beta3 allele appeared to be silent. This mutation, inherited from his mother and present in other family members with intermediate levels of alphaIIbbeta3, was close to the MIDAS-like domain of beta3, a fact that appears to explain its effect on alphaIIbbeta3 activation and fibrinogen binding.     

Megakaryocyte-targeted synthesis of the integrin beta(3)-subunit results in the phenotypic correction of Glanzmann thrombasthenia

Glanzmann thrombasthenia is an inherited bleeding disorder characterized by qualitative or quantitative defects of the platelet-specific integrin, alphaIIbbeta(3). As a result, alphaIIbbeta(3) cannot be activated and cannot bind to fibrinogen, leading to a loss of platelet aggregation. Thrombasthenia is clinically characterized by mucocutaneous hemorrhage with episodes of intracranial and gastrointestinal bleeding. To develop methods for gene therapy of Glanzmann thrombasthenia, a murine leukemia virus (MuLV)-derived vector, -889Pl(A2)beta(3), was transduced into peripheral blood CD34(+) cells from 2 patients with thrombasthenia with defects in the beta(3) gene. The human alphaIIb promoter was used in this vector to drive megakaryocyte-targeted expression of the wild-type beta(3) subunit. Proviral DNA and alphaIIbbeta(3) biosynthesis were detected after in vitro differentiation of transduced thrombasthenic CD34(+) cells with megakaryocyte growth and development factor. Flow cytometric analysis of transduced patient samples indicated that 19% of megakaryocyte progeny expressed alphaIIbbeta(3) on the surface at 34% of normal receptor levels. Treatment of transduced megakaryocytes with a combination of agonists including epinephrine and the thrombin receptor-activating peptide induced the alphaIIbbeta(3) complex to form an activated conformation capable of binding fibrinogen as measured by PAC-1 antibody binding. Transduced cells retracted a fibrin clot in vitro similar to megakaryocytes derived from a normal nonthrombasthenic individual. These results demonstrate ex vivo phenotypic correction of Glanzmann thrombasthenia and support the potential use of hematopoietic CD34(+) cells as targets for alphaIIb promoter-driven MuLV vectors for gene therapy of platelet disorders. (Blood. 2000;95:3645-3651)     

Homozygous Cys542-->Arg substitution in GPIIIa in a Swiss patient with type I Glanzmann's thrombasthenia

Glanzmann's thrombasthenia (GT) arises from a qualitative or quantitative defect in the GPIIb-IIIa complex (integrin alphaIIbbeta3), the mediator of platelet aggregation. We describe a patient in whom clinical and laboratory findings typical of type I GT were found together with a second pathology involving neurological and other complications symptomatic of tuberous sclerosis. Analysis of platelet proteins by Western blotting revealed trace amounts of normally migrating GPIIb and equally small amounts of GPIIIa of slightly slower than normal migration. Flow cytometry confirmed a much decreased binding to platelets of monoclonal antibodies to GPIIb, GPIIIa or GPIIb-IIIa, and an antibody to the alphav subunit also showed decreased binding. Nonradioactive PCR single-strand conformation polymorphism analysis followed by direct sequencing of PCR-amplified DNA fragments showed a homozygous point mutation (T to C) at nucleotide 1722 of GPIIIa cDNA and which led to a Cys542-->Arg substitution in the GPIIIa protein. The mutation gave rise to a HinP1 I restriction site in exon 11 of the GPIIIa gene and allele-specific restriction enzyme analysis of family members confirmed that a single mutated allele was inherited from each parent. This amino acid substitution presumably changes the capacity for disulphide bond formation within the cysteine-rich core region of GPIIIa and its study will provide new information on GPIIb-IIIa and alphavbeta3 structure and biosynthesis.     

Molecular characterization of Turkish patients with pyrimidine 5' nucleotidase-I deficiency

Pyrimidine 5' nucleotidase-I (P5N-I) deficiency is a rare autosomal recessive disorder associated with hemolytic anemia, marked basophilic stippling, and accumulation of high concentrations of pyrimidine nucleotides within the erythrocyte. Recently, the structure and location of the P5N-I gene have been published. This paper presents the results of a study characterizing the molecular pathologies of P5N-I deficiency in a total of 6 Turkish patients from 4 unrelated families of consanguineous marriages. Mutation analysis in the P5N-I gene led to the identification of 3 novel mutations in these patients. In 4 patients from 2 families, a homozygous insertion of double G at position 743 was detected in exon 9 (743-744insGG), leading to premature termination of translation 23 bp downstream. In one family, a homozygous T to G transition at position 543 (543T>G) in exon 8 resulted in the replacement of tyrosine (Tyr) with a stop codon (Tyr181Stop). In another family, a homozygous insertion of a single A in exon 7 (384-385insA) created a stop signal at the codon nearby. In all families, the parents were heterozygous for the relevant mutations. None of these changes was detected in 200 chromosomes from a healthy Turkish population. These mutations were not correlated with any particular phenotype.     

The molecular genetic basis of Glanzmann's thrombasthenia in a gypsy population in France: identification of a new mutation on the alpha IIb gene

Glanzmann's thrombasthenia is a rare inherited bleeding disorder caused by a qualitative or quantitative defect of platelet alpha IIb beta 3. We describe here a new mutation that is the molecular genetic basis of Glanzmann's thrombasthenia in two gypsy families. Our investigation was focused on the alpha IIb gene as a result of biochemical and immunologic analysis of patients' platelets showing undetectable alpha IIb but residual beta 3 levels. The entire alpha IIb cDNA was polymerase chain reaction (PCR) amplified using patients platelet RNA. Sequence analysis showed an 8-bp deletion located at the 3' end of exon 15. This deletion causes a reading-frame shift leading to a premature stop codon and the synthesis of a severely truncated form of alpha IIb. Genomic DNA study showed a G-->A substitution, the Gypsy mutation, at the splice donor site of intron 15. This mutation results in an abnormal splicing occurring at an alternative donor site located 8 bp upstream from the mutation. Based on those results, an allele-specific PCR analysis was developed to allow a rapid identification of the mutation in patients and potential carriers of the gypsy community. This PCR analysis can also be used for genetic counseling and antenatal diagnosis.     

A naturally occurring mutation near the amino terminus of alphaIIb defines a new region involved in ligand binding to alphaIIbbeta3

Decreased expression of functional alphaIIbbeta3 complexes on the platelet surface produces Glanzmann thrombasthenia. We have identified mutations of alphaIIb(P145) in 3 ethnically distinct families affected by Glanzmann thrombasthenia. Affected Mennonite and Dutch patients were homozygous and doubly heterozygous, respectively, for a P(145)A substitution, whereas a Chinese patient was doubly heterozygous for a P(145)L substitution. The mutations affect expression levels of surface alphaIIbbeta3 receptors on their platelets, which was confirmed by co-transfection of alphaIIb(P145A) and beta3 cDNA constructs in COS-1 cells. Each mutation also impaired the ability of alphaIIbbeta3 on affected platelets to interact with ligands. Moreover, when alphaIIb(P145A) and beta3 were stably coexpressed in Chinese hamster ovary cells, alphaIIbbeta3 was readily detected on the cell surface, but the cells were unable to adhere to immobilized fibrinogen or to bind soluble fluorescein isothiocyanate-fibrinogen after alphaIIbbeta3 activation by the activating monoclonal antibody PT25-2. Nonetheless, incubating affected platelets with the peptide LSARLAF, which binds to alphaIIb, induced PF4 secretion, indicating that the mutant alphaIIbbeta3 retained the ability to mediate outside-in signaling. These studies indicate that mutations involving alphaIIb(P145 )impair surface expression of alphaIIbbeta3 and that the alphaIIb(P145A) mutation abrogates ligand binding to the activated integrin. A comparative analysis of other alphaIIb mutations with a similar phenotype suggests that these mutations may cluster into a single region on the surface of the alphaIIb and may define a domain influencing ligand binding. (Blood. 2000;95:180188)     

AlphaIIbG236E causes Glanzmann thrombasthenia by impairing association with beta3

Glanzmann thrombasthenia (GT) is a recessively inherited bleeding disorder caused by the quantitative or qualitative deficiency of the platelet fibrinogen receptor, integrin alphaIIbbeta3. The N-terminal domain of the alphaIIb subunit is folded in a beta-propeller that plays the role of binding fibrinogen and associating with the ligand-binding region of beta3. Analysing the mutations of Italian GT patients we found that a patient had a alphaIIb G236E missense substitution that substitutes a glycine from the highly conserved PhiPhiGPhi motif of blade 4 of the beta-propeller. To verify experimentally the effect of the substitution of glycine 236 human embryonic kidney (HEK) cells were transfected with normal or mutated alphaIIb in conjunction with normal beta3. Using flow cytometry analysis we found the percentage of HEK cells transfected with alphaIIbG236Ebeta3 that reacted with anti alphaIIbbeta3 was very low. In HEK cells transfected with either alphaIIbbeta3 or alphaIIbG236Ebeta3 and lysed, when immunoblotting was done in non-reducing conditions a band reacting with an antibody against alphaIIb was present in both lysates, although less intense in cells transfected with alphaIIbG236Ebeta3. In reducing condition alphaIIb from cells transfected with alphaIIbbeta3 was nearly all mature, while in cells transfected with alphaIIbG236Ebeta3 the ratio pro-alphaIIb: alphaIIb was 1 : 1, with signs of degradation of the mutated protein. Cell lysates were then immunoprecipitated with antibodies against alphaIIb and immunoblotted with an antibody reacting with beta3. While in immunoblots from cells transfected with alphaIIbbeta3 a band corresponding to beta3 was strongly detectable, in immunoblots originating from cells transfected with alphaIIbG236Ebeta3 no band at the same level of normal beta3 was detected. Immunofluorescence studies showed accumulation of alphaIIbG236Ebeta3 in the endoplasmic reticulum and minimal transport to the Golgi. In conclusion we demonstrated that the alphaIIbG236E mutation causes GT by impairing the association with beta3 during biogenesis of the receptor.     

A Leu55 to Pro substitution in the integrin alphaIIb is responsible for a case of Glanzmann's thrombasthenia

Glanzmann's thrombasthenia (GT) is a hereditary bleeding disorder caused by a quantitative or qualitative defect in the integrin alphaIIbbeta3. A new mutation, a T to C substitution at base 258 in the alphaIIb gene, leading to the replacement of Leu55 with Pro, was found by sequence analysis of a patient's alphaIIb cDNA. In transfection experiments using COS7 cells, the cells co-transfected with the mutated alphaIIb cDNA containing C258 and wild-type beta3 cDNA scarcely expressed the alphaIIbbeta3 complex. The Leu55 to Pro substitution in the alphaIIb gene was found to be responsible for this case of Glanzmann's thrombasthenia.     

The first confirmed case with C3 deficiency caused by compound heterozygous mutations in the C3 gene; a new aspect of pathogenesis for C3 deficiency

The complement system is an ancient cascade system that has a major role in innate and adaptive immunity. Component C3 is central to the three complement pathways. Hereditary compliment 3 (C3) deficiency characterized by severe recurrent infections and immune complex disorders is extremely rare disease. Since 1972, inherited C3 deficiency has been described in many families representing a variety of national origins; however, only 8 families of these cases have been identified their genetic defects. Interestingly, all except one (incomplete analysis) were shown to harbor homozygous C3 gene mutations. Previously we proposed a hypothesis, based on the unique process of C3 synthesis; C3 deficiency is not inherited as a simple autosomal recessive trait. Here, we report the first confirmed case with C3 deficiency caused by compound heterozygous mutations. They were a novel one base insertion (3176insT) in exon 24 which is predicted to result in a frameshift and a premature downstream stop codon (K1105X) in exon 26, and a nonsense mutation of C3303G (Y1081X) in exon 26 which was previously reported as homozygous mutations. This confirmed case suggests that our proposed hypothesis has prospects of a new aspect of pathogenesis for C3 deficiency.     

Novel mutations responsible for autosomal recessive multisystem pseudohypoaldosteronism and sequence variants in epithelial sodium channel alpha-, beta-, and gamma-subunit genes

Multisystem pseudohypoaldosteronism (PHA), is a syndrome of unresponsiveness to aldosterone with autosomal recessive inheritance. Previously we showed that mutations in the epithelial sodium channel (ENaC) alpha-, beta-, and gamma-subunits are responsible for PHA. In this study we examined four independent probands with multisystem PHA, three of whom were born to consanguineous parents. In our search for mutations we also determined the complete coding sequences of each of the three genes encoding alpha-, beta-, and gamma-subunits in individuals representing different ethnic groups. Our analyses revealed the following homozygous mutations in three probands: 1) insertion of a T in exon 8 of the alpha ENaC gene that causes a frameshift error at Tyr(447) and leads to a premature stop codon at K459 in a Pakistani patient; 2) R508stop mutation in exon 11 of the alpha ENaC gene in an Indian patient; and 3) a splice site mutation in intron 12 of the beta ENaC gene (1669 + 1 g-->a) in a Scottish patient. The parents were heterozygous for the latter two mutations. The second mutation was previously observed in an Iranian Jewish patient. Our sequencing of the alpha-, beta-, and gamma-coding sequences revealed some sequence variants, some of which may represent single nucleotide polymorphisms. The gamma-subunit protein sequence was completely conserved in the six subjects examined. The homozygous mutations identified in the alpha and beta ENaC genes should result in reduced or abolished ENaC activity in PHA patients, explaining the disease symptoms.     

Homozygous factor V splice site mutation associated with severe factor V deficiency

We investigated a family whose proband has a severe bleeding disorder and factor V antigenic and functional levels of 8% and less than 1% of control values, respectively. Molecular analysis of the factor V gene revealed a novel homozygous mutation in the last nucleotide of exon 10. 1701G>T causes activation of a cryptic exonic splice site in exon 10, which encodes part of the factor V heavy chain (A2 domain). This leads to the deletion of 35 nucleotides and results in a frameshift with a premature stop codon at amino acid position 498. The G1701 and corresponding Gln509 are conserved in murine, bovine, and porcine factor V and in human factor VIII. Few factor V deficiency mutations have been identified as yet. Several are present in the heterozygous form in combination with factor V Leiden (Arg506Gln). This is the first reported homozygous splice site mutation in a patient with factor V deficiency.     

Peutz-Jeghers syndrome: molecular analysis of a three-generation kindred with a novel defect in the serine threonine kinase gene STK11

The Peutz-Jeghers syndrome, phenotypically characterized by mucocutaneous pigmentation and hamartomatous polyposis, is an autosomal dominant disease with variable expression and incomplete penetrance. Moreover, affected patients are at increased risk for gastrointestinal and other malignancies. Recently, a mutated gene encoding abnormal forms of the novel serine threonine kinase STK11 has been identified as a genetic cause of Peutz-Jeghers syndrome. Here, we report the molecular analysis of the STK11 gene in a patient with Peutz-Jeghers syndrome, which in exon 1 revealed a guanine (G) insertion in the 5 G repeat of codons 51–53. The insertion leads to a frameshift with a premature TGA stop codon 324 bp downstream in codon 162, predicting the expression of a truncated protein without kinase activity. This heterozygous germline mutation was also found in the affected father and in one affected sister of the index patient, but not in any phenotypically unaffected family member or in unrelated control subjects. In DNA isolated from microdissected hamartomatous polyps of the index patient, exon 1 of the STK11 gene could not be amplified suggesting that both alleles of STK11 exon 1 were lost in the hamartomatous polyps. Identification of a STK11 gene mutation in an index patient offers the possibility of a predictive diagnosis, and initiation of specific screening programs in the genetically affected kindred.     

Therapeutic expression of the platelet-specific integrin, alphaIIbbeta3, in a murine model for Glanzmann thrombasthenia

Integrins mediate the adhesion of cells to each other and to the extracellular matrix during development, immunity, metastasis, thrombosis, and wound healing. Molecular defects in either the alpha- or beta-subunit can disrupt integrin synthesis, assembly, and/or binding to adhesive ligands. This is exemplified by the bleeding disorder, Glanzmann thrombasthenia (GT), where abnormalities of the platelet-specific integrin, alphaIIbbeta3, prevent platelet aggregation following vascular injury. We previously used a retrovirus vector containing a cDNA cassette encoding human integrin beta3 to restore integrin alphaIIbbeta3 on the surface of megakaryocytes derived from peripheral blood stem cells of GT patients. In the present study, bone marrow from beta3-deficient (beta3-/-) mice was transduced with the ITGbeta3-cassette to investigate whether the platelet progeny could establish hemostasis in vivo. A lentivirus transfer vector equipped with the human ITGA2B gene promoter confined transgene expression to the platelet lineage. Human beta3 formed a stable complex with murine alphaIIb, effectively restoring platelet function. Mice expressing significant levels of alphaIIbbeta3 on circulating platelets exhibited improved bleeding times. Intravenous immunoglobulin effectively diminished platelet clearance in animals that developed an antibody response to alphaIIbbeta3. These results indicate the feasibility of targeting platelets with genetic therapies for better management of patients with inherited bleeding disorders.     

A novel beta-Thalassemic allele due to a two nucleotide deletion: beta76 (-GC)

We have identified and characterized a novel beta-thalassemic mutation in a North African adult. The molecular defect consists of a two nucleotide (nt) deletion in the beta-globin gene at codon 76 [beta76 (-GC), c.229-230delGC]. This frameshift mutation generates a TGA stop codon at position 89. The carrier presented with mild microcytic anemia (Hb 12.8 g/dL, MCV 60 fL), no detectable Hb F, an elevated Hb A2 level (5.5%) with no other mutation in the beta-globin gene and none of the more common known deletions in the alpha-globin cluster. No abnormal hemoglobin (Hb) was present in routine electrophoresis or in high performance liquid chromatography (HPLC) analyses. Pathologic inclusions were absent in both mature red cells and in reticulocytes. This observation reinforces the hypothesis that nonsense and frameshift mutations that result in a premature stop codon in exon 1 or exon 2 inherited in the heterozygous state do not generate dominant beta-thalassemia (thal). This is the first example of a premature stop codon at position 89.     

Variable phenotypic expression of chylomicron retention disease in a kindred carrying a mutation of the Sara2 gene

Chylomicron retention disease is a recessive inherited disorder characterized by fat malabsorption and steatorrhea and is associated with failure to thrive in infancy. We describe a kindred carrying a mutation of Sara2 gene causing a chylomicron retention phenotype. The proband was a 5-month-old baby, born of consanguineous, apparently healthy parents from Morocco, with failure to thrive. There was a large quantity of fats in feces and malabsorption of fat-soluble vitamins. Intestinal biopsies showed a diffused enterocyte vacuolization with large cytosolic lipid droplets. Chylomicron retention disease or Anderson disease was hypothesized, and the Sara2 gene was analyzed by direct sequencing. Analysis of the Sara2 gene in the proband identified a 2-nucleotide homozygous deletion in exon 3 leading to a premature stop codon (c.75-76 del TG-L28fsX34). The father was heterozygous for the same mutation, whereas the proband's mother was homozygous, suggesting a variable phenotypic expression of the molecular defect. More studies are needed to understand the reasons of the phenotypic variability of the same molecular defect in the same family.     

Identification of a novel mutation in the arginine vasopressin-neurophysin II gene in familial central diabetes insipidus.

Familial central diabetes insipidus is an inherited disease of predominant autosomal dominant trait characterized by a deficiency of arginine vasopressin. The arginine vasopressin-neurophysin II ( AVP-NPII) gene consists of three exons and is located on chromosome 20p13 encoding for the precursor protein of AVP. We investigated two Caucasian families with a typical autosomal dominant trait of familial central diabetes insipidus, defined by deficiency of arginine vasopressin. After PCR amplification of exon 1 and exon 2/3, fragments were pooled and purified. Nucleotide sequencing was performed with the Taq DyeDeoxy-terminator cycle sequencing method using nested primers. Two mutations in the coding region of NPII were identified. In family C we found a heterozygous G ==> C missense mutation (AA61) in exon 2 leading to the substitution of cystein with serine. In family D a novel heterozygous nonsense mutation in exon 3 (AA 83, GAG ==> TAG) was indentified, leading to a stop codon instead of glutamine. Both mutations were confirmed by restriction analysis and were found in all affected but not in healthy family members or control subjects. We therefore have identified a missense mutation of the AVP-NPII gene and a novel mutation predicting a truncated protein.     

A novel mutation that leads to a congenital factor XI deficiency in a Japanese family

We have identified a novel mutation leading to a congenital deficiency of the coagulation factor XI (FXI) in a Japanese family. A propositus was a 42-year-old female patient without bleeding tendency. Coagulant activity and the antigen level of FXI in her plasma were below the detectable range. The nucleotide sequences of the FXI gene of this patient were determined by a direct sequence method established in this study. A novel nonsense mutation (CAA; Gly263 --> TAA; stop) was identified in exon 8 of the FXI gene. Her parents are first cousins, and a polymerase chain reaction-restriction-fragment length polymorphism analysis revealed that her parents were heterozygous at this nucleotide position. This patient inherited mutant alleles from her parents and is homozygous at this nucleotide position. The nonsense mutation in the FXI gene is responsible for her deficiency of FXI.     

Family screening for a Glanzmann's thrombasthenia mutation using PCR-SSCP

Genetic counselling is often requested in Glanzmann's thrombasthenia, but measurements of GPIIb-IIIa density on platelets are often too inconclusive to allow a precise assessment of whether prospective parents are obligate heterozygotes for this disease by this measure alone. The recent application of PCR technology to Glanzmann's thrombasthenia has resulted in the identification of a large number of mutations, i.e. insertions/ deletions, splicing defects, in the genes for both GPIIb and GPIIIa. Among the reported abnormalities is an intronic G-->A substitution at the splice donor site of intron 15 in the GPIIb gene of a European gypsy tribe. This gives rise to an abnormal splicing, of an 8-bp deletion located at the 3' end of exon 15, a reading-frame shift and a premature stop codon in the mRNA for GPIIb. In applying PCR-SSCP to the elucidation of the genetic defects of a series of Glanzmann's patients, we have found the above-cited abnormality in three more gypsy families in France. The presence of the mutation was initially established by sequencing the amplified fragment, and its presence in family members was confirmed by both PCR-SSCP and HphI restriction analysis. Evaluation of the intronic G-->A mutation enabled genetic counselling to prospective parents within these families.     

A novel nonsense mutation in the Cyp11B1 gene from a subject with the steroid 11beta-hydroxylase form of congenital adrenal hyperplasia

11beta-Hydroxylase deficiency (11beta-OHD) inherited in an autosomal recessive manner accounts for about 5-8% of congenital adrenal hyperplasia (CAH). In order to clarify the underlying mechanism causing 11beta-OHD, we have done the molecular genetic analysis on the CYP11B1 gene in a patient diagnosed as 11beta-OHD. The nucleotide sequence of the patient's CYP11B1 revealed a novel nonsense mutation that converts codon 265 CAG (glutamine) to TAG (stop) of exon 4. Restriction fragment length polymorphism (RFLP) data showed that the patient was homozygous for the mutation. The above results confirm that the patient suffers from complete loss of the final step in cortisol biosynthesis pathway because of the nonsense mutation.