Immunomodulatory, liver depot gene therapy for Pompe disease.

Pompe disease is caused by mutations in acid alpha glucosidase (GAA) that causes accumulation of lysosomal glycogen affecting the heart and skeletal muscles, and can be fatal. Enzyme replacement therapy (ERT) with recombinant human GAA (rhGAA) improves muscle function by reducing glycogen accumulation. Limitations of ERT include a short half-life and the formation of antibodies that result in reduced efficacy. By harnessing the immune tolerance induction properties of the liver, liver-targeted gene delivery (with an adeno-associated virus vector containing a liver specific promoter), suppresses immunity against the GAA introduced by gene therapy. This induces immune tolerance to rhGAA by activating regulatory T cells and simultaneously, corrects GAA deficiency. Potentially, liver-targeted gene therapy can be performed once with lasting effects, by administering a relatively low dose of an adeno-associated virus type 8 vector to replace and induce immune tolerance to GAA.

Rescue administration of a helper-dependent adenovirus vector with long-term efficacy in dogs with glycogen storage disease type Ia.

Glycogen storage disease type Ia (GSD-Ia) stems from glucose-6-phosphatase (G6Pase) deficiency and causes hypoglycemia, hepatomegaly, hypercholesterolemia and lactic acidemia. Three dogs with GSD-Ia were initially treated with a helper-dependent adenovirus encoding a human G6Pase transgene (HDAd-cG6Pase serotype 5) on postnatal day 3. Unlike untreated dogs with GSD-Ia, all three dogs initially maintained normal blood glucose levels. After 6-22 months, vector-treated dogs developed hypoglycemia, anorexia and lethargy, suggesting that the HDAd-cG6Pase serotype 5 vector had lost efficacy. Liver biopsies collected at this time revealed significantly elevated hepatic G6Pase activity and reduced glycogen content, when compared with affected dogs treated only by frequent feeding. Subsequently, the HDAd-cG6Pase serotype 2 vector was administered to two dogs, and hypoglycemia was reversed; however, renal dysfunction and recurrent hypoglycemia complicated their management. Administration of a serotype 2 HDAd vector prolonged survival in one GSD-Ia dog to 12 months of age and 36 months of age in the other, but the persistence of long-term complications limited HDAd vectors in the canine model for GSD-Ia.

Hydrostatic isolated limb perfusion with adeno-associated virus vectors enhances correction of skeletal muscle in Pompe disease.

Glycogen storage disease type II (Pompe disease; MIM 232300) stems from the inherited deficiency of acid-α-glucosidase (GAA; acid maltase; EC 3.2.1.20), which primarily involves cardiac and skeletal muscles. We hypothesized that hydrostatic isolated limb perfusion (ILP) administration of an adeno-associated virus (AAV) vector containing a muscle-specific promoter could achieve relatively higher transgene expression in the hindlimb muscles of GAA-knockout (GAA-KO) mice, in comparison with intravenous (IV) administration. ILP administration of AAV2/8 vectors encoding alkaline phosphatase or human GAA-transduced skeletal muscles of the hindlimb widely, despite the relatively low number of vector particles administered (1 × 10¹¹), and IV administration of an equivalent vector dose failed to transduce skeletal muscle detectably. Similarly, ILP administration of fewer vector particles of the AAV2/9 vector encoding human GAA (3 × 10¹°) transduced skeletal muscles of the hindlimb widely and significantly reduced glycogen content to, in comparison with IV administration. The only advantage for IV administration was moderately high-level transduction of cardiac muscle, which demonstrated compellingly that ILP administration sequestered vector particles within the perfused limb. Reduction of glycogen storage in the extensor digitorum longus demonstrated the potential advantage of ILP-mediated delivery of AAV vectors in Pompe disease, because type II myofibers are resistant to enzyme replacement therapy. Thus, ILP will enhance AAV transduction of multiple skeletal muscles while reducing the required dosages in terms of vector particle numbers.

Emerging therapies for glycogen storage disease type I.

Glycogen storage disease type I (GSD I) is caused by deficiency of the glucose-6-phosphatase catalytic subunit in type Ia or of glucose-6-phosphate transporter in type Ib. The cellular bases for disruptions of homeostasis have been increasingly understood in GSD I, including those for anemia, renal failure and neutropenia. Advances in the understanding of cellular abnormalities in GSD I have provided rationales for new therapy, and recent developments in gene therapy have led to potential curative treatments for GSD I. These advances will benefit patients with GSD I in the future, improving both quality of life and survival, as well as illuminating the molecular effects of altered metabolism upon multiple organ systems.

Three successful pregnancies through dietary management of fructose-1,6-bisphosphatase deficiency.

Fructose-1,6-bisphosphatase (FBPase) deficiency (OMIM 229700) has been characterized as the cause of life-threatening hypoglycaemia and lactic acidaemia following prolonged fasting. The patient, an adult African-American woman, presented during the second trimester of her first pregnancy with recurrent episodes of lactic acidaemia and hypoglycaemia. She had recently been admitted to a nearby intensive care unit after presentation with profound hypoglycaemia and lactic acidosis, and was found to be pregnant. The history was remarkable for approximately 30 hospitalizations for hypoglycaemia and acidosis. She had previously undergone liver biopsy at another centre and was diagnosed with a 'glycogen storage disease', although no enzyme testing had been done for confirmation. Based on clinical symptoms, a diagnosis of FBPase deficiency was accomplished through gene sequencing, which revealed homozygosity for a panethnic, common mutation, 960/961insG in exon 7. The availability of mutation testing facilitated the confirmation of FBPase deficiency in this patient, obviating liver biopsy for enzyme activity confirmation. The patient underwent three successful pregnancies by strict compliance with dietary management, including nocturnal uncooked cornstarch to manage hypoglycaemia. The pregnancies were complicated by mild gestational diabetes, increased cornstarch requirements, and hypoglycaemia at the time of discharge from the hospital. The three infants had normal birth weights and experienced no complications during the neonatal period. The patient subsequently developed sensorineural hearing loss and early-onset cognitive impairment, despite compliance with the monitoring and treatment of hypoglycaemia. The experience with multiple pregnancies in this FBPase-deficient patient provides insight into the management of hypoglycaemia in inherited disorders of gluconeogenesis.

Glycogen storage disease types I and II: treatment updates.

Prior to 2006 therapy for glycogen storage diseases consisted primarily of dietary interventions, which in the case of glycogen storage disease (GSD) type II (GSD II; Pompe disease) remained essentially palliative. Despite improved survival and growth, long-term complications of GSD type I (GSD I) have not responded to dietary therapy with uncooked cornstarch or continuous gastric feeding. The recognized significant risk of renal disease and liver malignancy in GSD I has prompted efforts towards curative therapy, including organ transplantation, in those deemed at risk. Results of clinical trials in infantile Pompe disease with alglucosidase alfa (Myozyme) showed prolonged survival reversal of cardiomyopathy, and motor gains. This resulted in broad label approval of Myozyme for Pompe disease in 2006. Furthermore, the development of experimental therapies, such as adeno-associated virus (AAV) vector-mediated gene therapy, holds promise for the availability of curative therapy in GSD I and GSD II/Pompe disease in the future.

Early, sustained efficacy of adeno-associated virus vector-mediated gene therapy in glycogen storage disease type Ia.

The deficiency of glucose-6-phosphatase (G6Pase) underlies life-threatening hypoglycemia and growth retardation in glycogen storage disease type Ia (GSD-Ia). An adeno-associated virus (AAV) vector encoding G6Pase was pseudotyped as AAV8 and administered to 2-week-old GSD-Ia mice (n = 9). Median survival was prolonged to 7 months following vector administration, in contrast to untreated GSD-Ia mice that survived for only 2 weeks. Although GSD-Ia mice were initially growth-retarded, treated mice increased fourfold in weight to normal size. Blood glucose was partially corrected by 2 weeks following treatment, whereas blood cholesterol normalized. Glucose-6-phosphatase activity was partially corrected to 25% of the normal level at 7 months of age in treated mice, and blood glucose during fasting remained lower in treated, affected mice than in normal mice. Glycogen storage was partially corrected in the liver by 2 weeks following treatment, but reaccumulated to pre-treatment levels by 7 months old (m.o.). Vector genome DNA decreased between 3 days and 3 weeks in the liver following vector administration, mainly through the loss of single-stranded genomes; however, double-stranded vector genomes were more stable. Although CD8+ lymphocytic infiltrates were present in the liver, partial biochemical correction was sustained at 7 m.o. The development of efficacious AAV vector-mediated gene therapy could significantly reduce the impact of long-term complications in GSD-Ia, including hypoglycemia, hyperlipidemia and growth failure.

The tandem mass spectrometry newborn screening experience in North Carolina: 1997-2005.

North Carolina (NC) was the first US state to initiate universal tandem mass spectrometry (MS/MS) newborn screening. This began as a statewide pilot project in 1997 to determine the incidence and feasibility of screening for fatty acid oxidation, organic acid and selected amino acid disorders. The MS/MS analyses were done by a commercial laboratory and all follow-up and confirmatory testing was performed through the NC Newborn Screening (NBS) Program. In April 1999, the NC NBS Laboratory began the MS/MS analyses in-house. Between 28 July 1997 and 28 July 2005, 944,078 infants were screened and 219 diagnoses were confirmed on newborns with elevated screening results, for an overall incidence of 1:4,300. Ninety-nine infants were identified with fatty acid oxidation disorders, 58 with organic acidaemias and 62 with aminoacidopathies. Medium-chain acyl-CoA dehydrogenase deficiency, 3-methylcrotonyl-CoA carboxylase deficiency and disorders of phenylalanine metabolism were the most common disorders detected. Identification of affected infants has allowed retrospective testing of other family members, resulting in an additional 16 diagnoses. Seven neonates died from complications of their metabolic disorders/prematurity despite timely MS/MS screening. In addition, there were six infants who were not identified by elevated NBS results but who presented with symptoms later in infancy. The NC MS/MS NBS Program uses a two-tier system, categorizing results as either 'borderline' or 'diagnostic' elevated, for both the cutoffs and follow-up protocol. Infants with an initial borderline result had only a repeat screen. Infants with a diagnostic or two borderline results were referred for confirmatory testing. The positive predictive value of the NC MS/MS NBS for those infants requiring confirmatory testing was 53% for 2003 and 2004. The success of the NC MS/MS NBS Program in identifying infants with metabolic disorders was dependent on a comprehensive follow-up protocol integrating the public health laboratory and the academic metabolic centres.

Complete correction of hyperphenylalaninemia following liver-directed, recombinant AAV2/8 vector-mediated gene therapy in murine phenylketonuria.

Novel recombinant adeno-associated virus vectors pseudotyped with serotype 8 capsid (rAAV2/8) have recently shown exciting promise as effective liver-directed gene transfer reagents. We have produced a novel liver-specific rAAV2/8 vector expressing the mouse phenylalanine hydroxylase (Pah) cDNA and have administered this vector to hyperphenylalaninemic PAH-deficient Pah(enu2) mice, a model of human phenylketonuria (PKU). Our hypothesis was that this vector would produce sufficient hepatocyte transduction frequency and PAH activity to correct blood phenylalanine levels in murine PKU. Portal vein injection of recombinant AAV2/8 vector into five adult Pah(enu2) mice yielded complete and stable (up to 17 weeks) correction of serum phenylalanine levels. Liver PAH activity was corrected to 11.5+/-2.4% of wild type liver activity and was associated with a significant increase in phenylalanine clearance following parenteral phenylalanine challenge. Although questions of long-term safety and stability of expression remain, recombinant AAV2/8-mediated, liver-directed gene therapy is a promising novel treatment approach for PKU and allied inborn errors of metabolism.

Evaluation of 3-methylcrotonyl-CoA carboxylase deficiency detected by tandem mass spectrometry newborn screening.

Since the addition of tandem mass spectrometry (MS/MS) to the North Carolina Newborn Screening Program, 20 infants with two consecutive elevated 3-hydroxyisovalerylcarnitine (C5OH) levels have been evaluated for evidence of inborn errors of metabolism associated with this metabolite. Ten of these 20 infants had significant concentrations of both 3-hydroxyisovaleric acid and 3-methylcrotonylglycine in their urine, suggestive of 3-methylcrotonyl-CoA carboxylase (3-MCC) deficiency. Four of these 10 were infants whose abnormal metabolites were found to be of maternal origin. Of 8 patients with probable 3-MCC deficiency, 7 have been tested and found to have the enzyme deficiency confirmed in lymphoblasts or cultured fibroblasts; one of these 7 infants had only marginally decreased 3-MCC activity in lymphocytes but deficient 3-MCC in fibroblasts. We estimate the incidence of 3-MCC deficiency at 1:64000 live births in North Carolina. We conclude that MS/MS newborn screening will detect additional inborn errors of metabolism, such as 3-MCC deficiency, not traditionally associated with newborn screening. The evaluation of newborns with two abnormally elevated C5OH levels on MS/MS newborn screening should include, at least, urine organic acid analysis by capillary GC-MS and a plasma acylcarnitine profile by MS/MS. Long-term follow-up is needed to determine the outcome of presymptomatically diagnosed patients with 3-MCC deficiency by MS/MS newborn screening.

Delivery of glucose-6-phosphatase in a canine model for glycogen storage disease, type Ia, with adeno-associated virus (AAV) vectors.

Therapy in glycogen storage disease type Ia (GSD Ia), an inherited disorder of carbohydrate metabolism, relies on nutritional support that postpones but fails to prevent long-term complications of GSD Ia. In the canine model for GSD Ia, we evaluated the potential of intravenously delivered adeno-associated virus (AAV) vectors for gene therapy. In three affected canines, liver glycogen was reduced following hepatic expression of canine glucose-6-phosphatase (G6Pase). Two months after AAV vector administration, one affected dog had normalization of fasting glucose, cholesterol, triglycerides, and lactic acid. Concatamerized AAV vector DNA was confirmed by Southern blot analysis of liver DNA isolated from treated dogs, as head-to-tail, head-to-head, and tail-to-tail concatamers. Six weeks after vector administration, the level of vector DNA signal in each dog varied from one to five copies per cell, consistent with variation in the efficiency of transduction within the liver. AAV vector administration in the canine model for GSD Ia resulted in sustained G6Pase expression and improvement in liver histology and in biochemical parameters.

Glutaric acidemia, type I, missed by newborn screening in an infant with dystonia following promethazine administration.

We report a child initially diagnosed with promethazine-induced dystonia despite a lack of response to diphenhydramine therapy. On further evaluation, the child was diagnosed with glutaric acidemia, type I (GA-I), an autosomal recessive inborn error of metabolism caused by the deficiency of glutaryl-CoA dehydrogenase. The characteristic clinical feature of GA-I is an acute encephalopathic and neurologic crisis typically occurring during a catabolic state. Despite slow improvement, many patients do not fully recover from a neurologic crisis, and residual neurologic morbidity can be significant. Although newborn screening using tandem mass spectrometry is expected to enable presymptomatic diagnosis of GA-I, this patient was not detected by newborn screening with tandem mass spectrometry. Therefore, a high suspicion of GA-I must be maintained in the evaluation of childhood dystonia, even when newborn screening results are reportedly normal.

Persistent, therapeutically relevant levels of human granulocyte colony-stimulating factor in mice after systemic delivery of adeno-associated virus vectors.

Adeno-associated virus (AAV) vectors have been shown to preferentially transduce hepatocytes after systemic administration in adult mice and to provide long-term expression of introduced genes. One application of this technology would be for the production of granulocyte colony-stimulating factor (G-CSF), which increases mature neutrophil numbers in humans and in animals, and has therapeutic effects in disorders featuring chronic neutropenia, including cyclic, severe congenital, and idiopathic neutropenia, and glycogen storage disease type Ib. We have treated mice by tail vein injection of AAV vectors encoding human G-CSF, and have detected high G-CSF levels and marked elevation of neutrophil counts for at least 5 months. A therapeutically relevant amount of G-CSF production was obtained when the liver-specific mouse albumin promoter-enhancer was used to drive G-CSF expression. In mice receiving higher amounts of vector, plasma levels of human G-CSF gradually increased over 3 weeks to high concentrations, whereas for lower amounts human G-CSF remained at initial, low levels. The previously observed effect of gamma irradiation, to increase AAV transduction rates, was diminished when large amounts of vector were used. Absolute neutrophil counts increased 10- to 50-fold for the period of observation to levels that would be therapeutic in the treatment of cyclic neutropenia. In conclusion, gene therapy with AAV vectors synthesizing G-CSF shows promise for the treatment of disorders featuring neutropenia.

Persistent expression of human clotting factor IX from mouse liver after intravenous injection of adeno-associated virus vectors.

We previously found that gene transduction by adeno-associated virus (AAV) vectors in cell culture can be stimulated over 100-fold by treatment of the target cells with agents that affect DNA metabolism, such as irradiation or topoisomerase inhibitors. Here we show that previous gamma-irradiation increased the transduction rate in mouse liver by up to 900-fold, and the topoisomerase inhibitor etoposide increased transduction by about 20-fold. Similar rates of hepatic transduction were obtained by direct injection of the liver or by systemic delivery via tail vein injection. Hepatocytes were much more efficiently transduced than other cells after systemic delivery, and up to 3% of all hepatocytes could be transduced after one vector injection. The presence of wild-type AAV, which contaminates many AAV vector preparations, was required to observe a full response to gamma-irradiation. Injection of mice with AAV vectors encoding human clotting factor IX after gamma-irradiation resulted in synthesis of low levels of human clotting factor IX for the 5-month period of observation. These studies show the potential of targeted gene transduction of the liver by AAV vectors for treatment of various hematological or metabolic diseases.

Prenatal diagnosis of 45,X/46,XX mosaicism and 45,X: implications for postnatal outcome.

The prognosis for 45,X/46,XX mosaicism diagnosed prenatally has yet to be established. We report our experience with 12 patients in whom prenatal diagnosis of 45,X/46,XX mosaicism was detected by amniocentesis for advanced maternal age or decreased maternal serum alpha-feto protein and compared them with 41 45,X/46,XX patients diagnosed postnatally. The girls in the prenatal group range in age from 3 mo to 10 years. All have had normal linear growth. Four had structural anomalies including: ASD (n = 1); ptosis and esotropia (n = 1); labial fusion (n = 1); and urogenital sinus, dysplastic kidneys, and hydrometrocolpos (n = 1). Gonadotropins were measured in seven; one had elevated luteinizing hormone/FSH at 3 mo of age. One has developmental delay and seizures as well as ophthalmologic abnormalities. None would have warranted karyotyping for clinical suspicion of Turner syndrome. The prevalence of 45,X/46,XX mosaicism is 10-fold higher among amniocenteses than in series of postnatally diagnosed individuals with Turner syndrome, which suggests that most individuals with this karyotype escape detection and that an ascertainment bias exists toward those with clinically evident abnormalities. The phenomenon of a milder phenotype for the prenatal group is similar to that observed for 45,X/46,XY diagnosed prenatally. Prenatal counseling for 45,X/46,XX in the absence of such ultrasound abnormalities as hydrops fetalis should take into account the expectation of a milder phenotype (except, possibly, with respect to developmental delay) than that of patients ascertained postnatally. The same does not hold true for 45,x diagnosed prenatally.

Sequences within the coding regions of clotting factor VIII and CFTR block transcriptional elongation.

The clotting factor VIII (FVIII) and cystic fibrosis transmembrane conductance regulator (CFTR) cDNAs have dramatically reduced levels of expression compared to clotting factor IX (FIX) and other cDNAs (100 and 1,000-fold lower, respectively), when produced in cells by using an expression vector. Part of the inhibitory signal in the FVIII cDNA has been localized to a 1.2-kb inhibitory sequence (FVIII INS), which decreased steady-state RNA levels from a retroviral vector by 30- to 100-fold. An analysis of RNA degradation indicated that the FVIII INS vector RNA is relatively stable. Nuclear run-on experiments with the FVIII INS vector demonstrated a low signal for FVIII, in contrast to the high signal for a FIX vector. The low signal for FVIII INS was not due to a decrease in transcriptional initiation. Thus, FVIII expression is reduced through a block to transcriptional elongation, as has been found in c-myc and other genes. We show that the inhibitory effect of FVIII INS is orientation dependent with regard to the promoter. In addition, the inhibitory effect is position dependent, because expression of FVIII INS sequence increased when it was moved 1 kb further from the promoter in a retroviral vector. Similar results were observed by using a retroviral vector for expression of the CFTR cDNA. The CFTR retroviral vector produced 1,000-fold decreased steady-state RNA levels, compared to the parent vector. Nuclear run-on analysis with the CFTR vector revealed a block to transcriptional elongation within the CFTR cDNA. The presence of blocks to transcriptional elongation within the FVIII and CFTR cDNAs complicates efforts to produce high levels of these proteins for therapeutic purposes and to develop high-titer retroviral expression vectors for human gene therapy.

T296----M, a common mutation causing mild hemophilia B in the Amish and others: founder effect, variability in factor IX activity assays, and rapid carrier detection.

By direct genomic sequencing, we have delineated the causative mutation in 64 families of European decent with hemophilia B. Six (9%) had a C----T transition at base 31008, which substitutes methionine for threonine 296 (T296----M) in the catalytic domain of factor IX. Five of the patients had the same haplotype (frequency of 16% in the northern European population). These individuals are of Amish/German descent and they are likely to share a common ancestor. The sixth patient had a different haplotype, which indicates that his mutation had an independent origin. The data highlight the importance of clinical criteria for the classification of hemophilia B. All six patients had clinically mild disease and their factor IX coagulant activities were in the range of 3%-6% when tested simultaneously in one laboratory, yet the factor IX activities provided with patient records varied 40-fold. Due to the high frequency of this mutation, we have utilized the technique of polymerase chain reaction amplification of specific alleles (PASA) to perform rapid and inexpensive carrier diagnoses in the families with this mutation. This is of particular importance for the Amish since the mutation should account for much of, if not all, the mild hemophilia B that is commonly found in this population.

Mutations causing hemophilia B: direct estimate of the underlying rates of spontaneous germ-line transitions, transversions, and deletions in a human gene.

Spontaneous mutation provides the substrate for evolution on one hand and for genetic susceptibility to disease on the other hand. X-linked diseases such as hemophilia B offer an opportunity to examine recent germ-line mutations in humans. By utilizing the direct sequencing method of genomic amplification with transcript sequencing, eight regions (2.46 kb) of likely functional significance in the factor IX gene have been sequenced in a total of 60 consecutive, unrelated hemophiliacs. The high frequency of patient ascertainment from three regions in the midwestern United States and Canada suggests that the sample is representative of hemophiliacs of northern European descent. Twenty-six of the delineated mutations are reported herein, and the group of 60 is analyzed as a whole. From the pattern of mutations causing disease and from a knowledge of evolutionarily conserved amino acids, it is possible to reconstruct the underlying pattern of mutation and to calculate the mutation rates per base pair per generation for transitions (27 x 10(-10)), transversions (4.1 x 10(-10), and deletions (0.9 x 10(-10)) for a total mutation rate of 32 x 10(-10). The proportion of transitions at non-CpG nucleotides is elevated sevenfold over that expected if one base substitution were as likely as another. At the dinucleotide CpG, transitions are elevated 24-fold relative to transitions at other sites. The pattern of spontaneous mutations in factor IX resembles that observed in Escherichia coli when the data are corrected for ascertainment bias. The aggregate data hint that most mutations may be due to endogenous processes. The following additional conclusions emerge from the data: (1) Although in recent decades reproductive fitness in individuals with mild and moderate hemophilia has been approximately normal, the large number of different mutations found strongly suggest that these levels of disease substantially compromised reproduction in previous centuries. (2) Mutations which putatively affect splicing account for at least 13% of independent mutations, indicating that the division of the gene into eight exons presents a significant genetic cost for the organism. In one individual a "silent" mutation at lysine 5 is likely to cause hemophilia by generating a perfect splice donor consensus sequence in exon b. (3) All the missense mutations occurred at evolutionarily conserved amino acids. As additional data are generated on the pattern of mutations caused by specific mutagens, it will be possible to utilize the pattern of spontaneous mutation to estimate the maximal contribution of that mutagen during the past century.

A past mutation at isoleucine 397 is now a common cause of moderate/mild haemophilia B.

Of the factor IX sequence changes that we have identified in 65 consecutive males with haemophilia B, 11 (17%) are the same mutation. This mutation is a T----C transition at base 31311 which substitutes threonine for isoleucine397 (ile397) in the factor IX molecule. The 11 patients are of Western European descent and have the same haplotype: Hinf1 (-), Xmn1 (-), Taq1 (-), BamH1 (+), Malmö allele = thr148. The frequency of this haplotype was estimated and the probability of the same mutation occurring independently 11 times in this haplotype was miniscule. We conclude that these patients have a common ancestor despite the lack of overlapping pedigrees. The clinical symptoms of the disease were consistently moderate/mild in these 11 patients, whereas factor IX coagulation values obtained from the medical records varied more than sixfold between individuals. However, when plasma from five individuals was assayed by the same laboratory concurrently, the values varied less than twofold. Thus, in routine practice, clinical severity may correlate better with the presence of a given mutation than the factor IX coagulant activity. The high frequency of the mutation at ile397 indicates that carrier testing in families of Northern European descent with moderate/mild haemophilia B can be expedited by first determining whether this particular mutation is present. We demonstrate here that the technique of polymerase chain reaction (PCR) amplification of specific alleles (PASA) can be used to rapidly perform carrier testing in families with the ile397 mutation.

Recurrent nonsense mutations at arginine residues cause severe hemophilia B in unrelated hemophiliacs.

Direct sequencing of the regions of the factor IX gene of likely functional significance was performed in four patients with severe hemophilia B. In two of the individuals, a transition at the dinucleotide CpG caused a nonsense mutation at arginine 333. In the other two individuals, a transition at CpG caused a nonsense mutation at arginine 29. Since these patients are all unrelated, as shown by differing alleles of the TaqI polymorphism in intron four or extensive nonoverlapping pedigrees, the mutations arose independently. In addition, the origin of one arginine 333 mutation in one family has been traced to the germline of the maternal grandfather. The frequent occurrence of mutations at arginine codons that contain the sequence CGN can be explained by the dramatic elevation of transitions at CpG. As a result, approximately one in four individuals with hemophilia B is expected to have a mutation at arginine and nonsense mutations at one of six arginine residues should be common causes of severe hemophilia.