Safe, efficient, and reproducible gene therapy of the brain in the dog models of Sanfilippo and Hurler syndromes.

Recent trials in patients with neurodegenerative diseases documented the safety of gene therapy based on adeno-associated virus (AAV) vectors deposited into the brain. Inborn errors of the metabolism are the most frequent causes of neurodegeneration in pre-adulthood. In Sanfilippo syndrome, a lysosomal storage disease in which heparan sulfate oligosaccharides accumulate, the onset of clinical manifestation is before 5 years. Studies in the mouse model showed that gene therapy providing the missing enzyme α-N-acetyl-glucosaminidase to brain cells prevents neurodegeneration and improves behavior. We now document safety and efficacy in affected dogs. Animals received eight deposits of a serotype 5 AAV vector, including vector prepared in insect Sf9 cells. As shown previously in dogs with the closely related Hurler syndrome, immunosuppression was necessary to prevent neuroinflammation and elimination of transduced cells. In immunosuppressed dogs, vector was efficiently delivered throughout the brain, induced α-N-acetyl-glucosaminidase production, cleared stored compounds and storage lesions. The suitability of the procedure for clinical application was further assessed in Hurler dogs, providing information on reproducibility, tolerance, appropriate vector type and dosage, and optimal age for treatment in a total number of 25 treated dogs. Results strongly support projects of human trials aimed at assessing this treatment in Sanfilippo syndrome.

[Lysosomal proteolysis; overview of lysosomal diseases]. / Le système lysosomial dans la protéolyse: panorama des maladies lysosomiales.

The lysosome plays a key role in recycling other cell organelles and long-lived cellular proteins, which undergo continuous turnover. Lying at the hub of vesicular pathways, the functions of which are tightly inter-regulated, the proteolytic machinery of the lysosome is essential for cellular homeostasis. Lysosomal diseases are rare genetic disorders due to deficiencies in one or several lysosomal protein(s). Their clinical manifestations are extremely diverse and depend on the total or partial nature of the protein deficiency and the specific functions of this organelle in different cell types.

Incidence and natural history of mucopolysaccharidosis type III in France and comparison with United Kingdom and Greece.

Sanfilippo syndrome, or mucopolysaccharidosis type III (MPSIII) is a lysosomal storage disease with predominant neurological manifestations in affected children. It is considered heterogeneous with respect to prevalence, clinical presentation, biochemistry (four biochemical forms of the disease referred to as MPSIIIA, B, C, and D are known), and causative mutations. The perspective of therapeutic options emphasizes the need for better knowledge of MPSIII incidence and natural history. We performed parallel retrospective epidemiological studies of patients diagnosed with MSPIII in France (n = 128), UK (n = 126), and Greece (n = 20) from 1990 to 2006. Incidences ranged from 0.68 per 100,000 live-births in France to 1.21 per 100,000 live-births in UK. MPSIIIA, which predominates in France and UK, was absent in Greece, where most patients have MPSIIIB. The study confirmed the large allelic heterogeneity of MPSIIIA and MPSIIIB and detected several yet undescribed mutations. Analysis of clinical manifestations at diagnosis and over a 6-7 years follow-up indicated that almost all patients, whatever the disease subtype, expressed neurological manifestations before the age of 5 years, including language acquisition delay, cognitive delay, and/or abnormal behavior. In contrast to relatively homogeneous early onset manifestations, disease progression showed significant variation depending on subtype and age at diagnosis. Different severities of disease progressions and different allele distribution between France and UK suggested that mutations are not equally deleterious, although genotype-phenotype correlation could not be established. Notwithstanding the rapidity of further clinical deterioration, all MPSIII patients suffer early onset devastating neurological manifestations that deserve early treatment when available.

[Metachromatic leucodystrophy. Clinical, biological, and therapeutic aspects]. / La leucodystrophie métachromatique Aspects clinique, biologique et thérapeutique.

Scholz's disease or metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by a deficiency in arylsulfatase A (ARSA: EC 3.1.6.8). This enzyme is responsible for the degradation of sulfatides commonly called cerebroside-3-sulfate or 3-O-sulfogalactosylcéramide in galactocérébroside and sulfate. The success of hydrolysis of these sphingolipids by ARSA necessarily depends on the presence of saposine B forms a complex with the substrate. The pathological accumulation of sulfatides in the nervous system (myelin, neurons and glial cells) results most often neurological, mental retardation, nervous disorders, blindness. The metachromatic granules accumulated in the central nervous system and peripheral compounds are highly toxic. These are at high levels in the urine of patients affected by the MLD. Arylsulfatase A activity is collapsed in these patients. Unfortunately, the value of enzyme activity is not a predictor of clinical severity of the neuropathology. In contrast, the study of the gene that codes for the ARSA is seen as a way to diagnose the simplest and most reliable of the disease to avoid misdiagnosis due to the presence of pseudodeficit. The conventional therapeutic approaches are essentially symptomatic. They were made in order to restore the enzyme activity of arylsulfatase A and prevent the progression of the pathological accumulation of sulfatides and consequently reduce morbidity associated with MLD.

Abnormal expression of truncated CRMP-1 protein in the brain cortex of MPSIIIB mice.

Mucopolysaccharidosis IIIB is a lysosomal disease characterized by a severe neurological deterioration, the pathophysiological mechanisms of which are poorly understood. Recently FGF pathway was shown to be altered leading us to explore a downstream target involved in brain development: the collapsin response mediator protein-1 (CRMP-1). CRMP-1 transcript level was normal but a cleavage of CRMP-1 was observed with an abnormal expression of the truncated form until adult age. This truncated CRMP-1 protein could play a role in post-natal cortex maturation and be involved in neuronal alterations occurring in lysosomal diseases.

Mucopolysaccharidosis type II: an update on mutation spectrum.

UNLABELLED: Mucopolysaccharidosis type II (MPS II; Hunter disease) is caused by deficiency of the enzyme iduronate-2-sulphatase (IDS) and patients present with a wide range of clinical signs and symptoms. The level of activity of IDS, however, does not allow prediction of phenotype. In our study of unrelated individuals with MPS II, alterations in the IDS gene could be identified in all 155 patients. Investigations in families in which the occurrence of MPS II was sporadic revealed mosaicism in the mothers of a small number of patients and a high frequency of de novo mutations occurring preferentially during male meiosis. Mutations identified in our patients include 27 large alterations and 128 small gene alterations (96 different alterations). These data further confirm the extreme heterogeneity of IDS gene alterations, as more than 330 have been reported to date. This genetic heterogeneity may explain the high degree of clinical heterogeneity in MPS II. Therefore, attempts have been made to establish genotype-phenotype correlations in order to provide an indication of the likely prognosis and a basis on which to evaluate treatment. To date, some progress has been made in predicting the clinical phenotype from the genotype although it remains difficult in a few individual cases. However, as the crystallographic 3D structure of IDS is yet to be determined, evaluation of the impact of mutations on IDS activity is often time consuming. Furthermore, if a given mutation is recurrent, some patients carrying the same change may present with different phenotypes, suggesting that factors other than the IDS gene (e.g. other genes, environmental factors) can modulate the clinical phenotype. CONCLUSION: Although genotype-phenotype correlations may be difficult to establish, they will be of increasing importance for choosing the most appropriate therapy for an individual patient, as new therapeutic strategies may be targeted according to phenotype.

[Lysosomal storage diseases: functional classification and treatment principles]. / Maladies de surcharge lysosomale: classification fonctionnelle et principes thérapeutiques.

Until recently, lysosomes were only considered as the place of macromolecules degradation. To date, these organelles are considered as playing a key role in cellular homeostasis. The knowledge of their complex role allows a better understanding of the physiopathology of the diseases resulting from their dysfunction. Biosynthesis of lysosomal enzymes and routes for macromolecules influx within lysosomes are briefly described and a functional classification of lysosomal diseases, which allows a comprehensive approach of treatment rationale, is proposed. Except cystinosis which is a lysosomal membrane transport disorder the majority of current treatments aim at treating enzymatic defects. Normal enzyme can be brought by hoematopoietic cell transplant (or other cell therapies) or by infusions of human recombinant enzyme. The normal enzyme can be also produced in situ by bringing the normal gene: it is the objective of gene therapy The other therapeutic approaches aim at decreasing the quantity of non degraded substrate reaching the lysosomes by limiting its biosynthesis or thanks to small pharmacologic molecules, at increasing the residual enzyme activity by stabilizing the protein (chaperones) or by improving read through possibilities of some stop codons in special contexts. The better knowledge of secondary phenomena set by the cell in order to try to restore homeostasis which are sometimes more deleterious than the storage itself should allow to develop complementary treatments. The rationale of these different therapeutic approaches and their limits are described in this paper

Differences in methylation patterns in the methylation boundary region of IDS gene in Hunter syndrome patients: implications for CpG hot spot mutations.

Hunter syndrome, an X-linked disorder, results from deficiency of iduronate-2-sulfatase (IDS). Around 40% of independent point mutations at IDS were found at CpG sites as transitional events. The 15 CpG sites in the coding sequences of exons 1 and 2, which are normally hypomethylated, account for very few of transitional mutations. By contrast, the CpG sites in the coding sequences of exon 3, though also normally hypomethylated, account for much higher fraction of transitional mutations. To better understand relationship between methylation status and CpG transitional mutations in this region, the methylation patterns of 11 Hunter patients with transitional mutations at CpG sites were investigated using bisulfite genomic sequencing. The patient cohort mutation spectrum is composed of one mutation in exon 1 (one patient) and three different mutations in exon 3 (10 patients). We confirmed that in normal males, cytosines at the CpG sites from the promoter region to a portion of intron 3 were hypomethylated. However, specific CpG sites in this area were more highly methylated in patients. The patients with p.R8X (exon 1), p.P86L (exon 3), and p.R88H (exon 3) mutations had a hypermethylated condition in exon 2 to intron 3 but retained hypomethylation in exon 1. The same trend was found in four patients with p.A85T (exon 3), although the degree of hypermethylation was less. These findings suggest methylation patterns in the beginning of IDS genomic region are polymorphic in humans and that hypermethylation in this region in some individuals predisposes them to CpG mutations resulting in Hunter syndrome.

Type 2 Gaucher disease: 15 new cases and review of the literature.

OBJECTIVES: To provide a description of type 2 Gaucher disease. To attempt to define type 2 Gaucher disease within the spectrum of early-onset neuronopathic Gaucher disease. BACKGROUND: Type 2 Gaucher disease is a rare disorder due to glucocerebrosidase deficiency that comprises a rapidly progressing neurological degeneration associated with visceral signs. Most data collected rely on the description of single cases or siblings. Cases of perinatal-lethal Gaucher disease are frequently considered as type 2 Gaucher patients, though the clinical presentation is different. METHODS: We retrospectively studied the clinical history of 15 original acute Gaucher disease patients and reviewed the available data of 104 published cases of early-onset neuronopathic Gaucher disease, including 61 patients with the acute type and 43 cases of the perinatal-lethal form. RESULTS: The neurological presentation of type 2 Gaucher disease is homogeneous and characterized by precocious, severe, and rapidly progressive brainstem degeneration in the foreground. The most frequent initial signs are hyperextension of the neck, swallowing impairment, and strabismus. Provoked asphyxic episodes generally appear in a second time. They are followed by prolonged spontaneous apneas that seem to be the main pejorative feature. Other neurological signs may be observed, but epilepsy, myoclonic epilepsy/myoclonus, trismus, stridor, and progressive microcephaly are less characteristic. Psychomotor regression may occur, but is not a typical feature of the disease onset. Chronic or subacute pulmonary disease predominates in the visceral involvement. Hepatosplenomegaly, failure to thrive, thrombocytopenia, and anemia are the other remarkable, albeit non-specific, features. The inflammatory component of Gaucher disease is underlined by the addition of unexplained fever to this systemic clinical picture. The natural history and particular signs of perinatal-lethal Gaucher disease do not belong to the type 2 Gaucher disease phenotype. CONCLUSION: Type 2 Gaucher disease is a clinically homogeneous entity. The specificity of the neurological involvement is sufficient to suspect the diagnosis at the onset of the disease. Type 2 and perinatal-lethal Gaucher diseases are easily distinguishable in most cases.

Improved behavior and neuropathology in the mouse model of Sanfilippo type IIIB disease after adeno-associated virus-mediated gene transfer in the striatum.

Sanfilippo syndrome is a mucopolysaccharidosis (MPS) caused by a lysosomal enzyme defect interrupting the degradation pathway of heparan sulfates. Affected children develop hyperactivity, aggressiveness, delayed development, and severe neuropathology. We observed relevant behaviors in the mouse model of Sanfilippo syndrome type B (MPSIIIB), in which the gene coding for alpha-N-acetylglucosaminidase (NaGlu) is invalidated. We addressed the feasibility of gene therapy in these animals. Vectors derived from adeno-associated virus serotype 2 (AAV2) or 5 (AAV5) coding for NaGlu were injected at a single site in the putamen of 45 6-week-old MPSIIIB mice. Normal behavior was observed in treated mice. High NaGlu activity, far above physiological levels, was measured in the brain and persisted at 38 weeks of age. NaGlu immunoreactivity was detected in neuron intracellular organelles, including lysosomes. Enzyme activity spread beyond vector diffusion areas. Delivery to the entire brain was reproducibly obtained with both vector types. NaGlu activity was higher and distribution was broader with AAV5-NaGlu than with AAV2-NaGlu vectors. The compensatory increase in the activity of various lysosomal enzymes was improved. The accumulation of gangliosides GM2 and GM3 present before treatment and possibly participating in neuropathology was reversed. Characteristic vacuolations in microglia, perivascular cells, and neurons, which were prominent before the age of treatment, disappeared in areas in which NaGlu was present. However, improvement was only partial in some animals, in contrast to high NaGlu activity. These results indicate that NaGlu delivery from intracerebral sources has the capacity to alleviate most disease manifestations in the MPSIIIB mouse model.

Molecular pathology of NEU1 gene in sialidosis.

Lysosomal sialidase (EC 3.2.1.18) has a dual physiological function; it participates in intralysosomal catabolism of sialylated glycoconjugates and is involved in cellular immune response. Mutations in the sialidase gene NEU1, located on chromosome 6p21.3, result in autosomal recessive disorder, sialidosis, which is characterized by the progressive lysosomal storage of sialylated glycopeptides and oligosaccharides. Sialidosis type I is a milder, late-onset, normosomatic form of the disorder. Type I patients develop visual defects, myoclonus syndrome, cherry-red macular spots, ataxia, hyperreflexia, and seizures. The severe early-onset form, sialidosis type II, is also associated with dysostosis multiplex, Hurler-like phenotype, mental retardation, and hepatosplenomegaly. We summarize information on the 34 unique mutations determined so far in the sialidase gene, including four novel missense and one novel nonsense mutations found in two Czech and two French sialidosis patients. The analysis of sialidase mutations in sialidosis revealed considerable molecular heterogeneity, reflecting the diversity of clinical phenotypes that make molecular diagnosis difficult. The majority of sialidosis patients have had missense mutations, many of which have been expressed; their effects on activity, stability, intracellular localization, and supramolecular organization of sialidase were studied. A structural model of sialidase allowed us to localize mutations in the sialidase molecule and to predict their impact on the tertiary structure and biochemical properties of the enzyme.

Fabry disease: characterization of alpha-galactosidase A double mutations and the D313Y plasma enzyme pseudodeficiency allele.

Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from mutations in the gene encoding the lysosomal exoglycohydrolase, alpha-galactosidase A (alpha-Gal A; GLA). In two unrelated classically affected males, two alpha-Gal A missense mutations were identified: R112C + D313Y (c.334C>T + c.937G>T) and C172G + D313Y (c.514T>G + c.937G>T). The D313Y lesion was previously identified in classically affected males as the single mutation [Eng et al., 1993] or in cis with another missense mutation, D313Y + G411D (c.937G>T + c.1232G>A) [Guffon et al., 1998]. To determine whether the D313Y mutation was a deleterious mutation or a coding region sequence variant, the frequency of D313Y in normal X-chromosomes, as well as its enzymatic activity and subcellular localization in COS-7 cells was determined. D313Y occurred in 0.45% of 883 normal X-chromosomes, while the R112C, C172G, and G411D missense mutations were not detected in over 500 normal X-chromosomes. Expression of D313Y in COS-7 cells resulted in approximately 60% of wild-type enzymatic activity and showed lysosomal localization, while R112C, C172G, G411D, and the double-mutated constructs had markedly reduced or no detectable activity and were all retained in the endoplasmic reticulum. The expressed D313Y enzyme was stable at lysosomal pH (pH 4.6), while at neutral pH (pH 7.4), it had decreased activity. A molecular homology model of human alpha-Gal A, based on the X-ray crystal structure of chicken alpha-galactosidase B (alpha-Gal B; alpha-N-acetylgalactosaminidase) was generated [Garman et al., 2002], which provided evidence that D313Y did not markedly disrupt the alpha-Gal A enzyme structure. Thus, D313Y is a rare exonic variant with about 60% of wild-type activity in vitro and reduced activity at neutral pH, resulting in low plasma alpha-Gal A activity.

Liver circadian clock, a pharmacologic target of cyclin-dependent kinase inhibitor seliciclib.

Circadian disruption accelerates malignant growth and shortens survival, both in experimental tumor models and cancer patients. In previous experiments, tumor circadian disruption was rescued with seliciclib, an inhibitor of cyclin-dependent kinases (CDKs). This effect occurred at a selective dosing time and was associated with improved antitumor activity. In the current study, seliciclib altered robust circadian mRNA expression of the clock genes Rev-erb alpha, Per2, and Bmal1 in mouse liver following dosing at zeitgeber time (ZT) 3 (i.e., 3 h after the onset of the 12 h light span), when mice start to rest, but not at ZT19, near the middle of the 12 h dark span, when mice are most active. However, liver exposure to seliciclib, as estimated by the liver area under the concentration x time curve (AUC), was approximately 80% higher at ZT19 than at ZT3 (p = 0.049). Circadian clock disruption was associated with increased serum liver enzymes and modified glycogen distribution in hepatocytes, as revealed by biochemical determinations and optic and electronic microscopy. The extent of increase in liver enzymes was most pronounced following dosing at ZT3, as compared to ZT19 (p < 0.04). Seliciclib further up-regulated the transcriptional activity of c-Myc, a cell cycle gene that promotes cell cycle entry and G1-S transition (p < 0.001), and down-regulated that of Wee1, which gates cell cycle transition from G2 to M (p < 0.001). These effects did not depend upon drug dosing time. Overall, the results suggest the circadian time of seliciclib delivery is more critical than the amount of drug exposure in determining its effects on the circadian clock. Seliciclib-induced disruption of the liver molecular clock could account for liver toxicity through the resulting disruption of clock-controlled detoxification pathways. Modifications of cell cycle gene expression in the liver likely involve other mechanisms. Circadian clocks represent relevant targets to consider for optimization of therapeutic schedules of CDK inhibitors.

Early neurodegeneration progresses independently of microglial activation by heparan sulfate in the brain of mucopolysaccharidosis IIIB mice.

BACKGROUND: In mucopolysaccharidosis type IIIB, a lysosomal storage disease causing early onset mental retardation in children, the production of abnormal oligosaccharidic fragments of heparan sulfate is associated with severe neuropathology and chronic brain inflammation. We addressed causative links between the biochemical, pathological and inflammatory disorders in a mouse model of this disease. METHODOLOGY/PRINCIPAL FINDINGS: In cell culture, heparan sulfate oligosaccharides activated microglial cells by signaling through the Toll-like receptor 4 and the adaptor protein MyD88. CD11b positive microglial cells and three-fold increased expression of mRNAs coding for the chemokine MIP1alpha were observed at 10 days in the brain cortex of MPSIIIB mice, but not in MPSIIIB mice deleted for the expression of Toll-like receptor 4 or the adaptor protein MyD88, indicating early priming of microglial cells by heparan sulfate oligosaccharides in the MPSIIIB mouse brain. Whereas the onset of brain inflammation was delayed for several months in doubly mutant versus MPSIIIB mice, the onset of disease markers expression was unchanged, indicating similar progression of the neurodegenerative process in the absence of microglial cell priming by heparan sulfate oligosaccharides. In contrast to younger mice, inflammation in aged MPSIIIB mice was not affected by TLR4/MyD88 deficiency. CONCLUSIONS/SIGNIFICANCE: These results indicate priming of microglia by HS oligosaccharides through the TLR4/MyD88 pathway. Although intrinsic to the disease, this phenomenon is not a major determinant of the neurodegenerative process. Inflammation may still contribute to neurodegeneration in late stages of the disease, albeit independent of TLR4/MyD88. The results support the view that neurodegeneration is primarily cell autonomous in this pediatric disease.

SIMPLE mutation analysis in dominant demyelinating Charcot-Marie-Tooth disease: three novel mutations.

Charcot-Marie-Tooth disease type 1C (CMT1C) is caused by mutations in the small integral membrane protein of the lysosome/late endosome (SIMPLE). We analyzed the coding sequence of SIMPLE in DNA of 53 unrelated cases of dominant demyelinating CMT disease with no mutations in PMP22, GJB1, MPZ, EGR2, and NEFL genes. Four different missense mutations were observed in six families. The mutation Gly112Ser was found in two families confirming its frequent occurrence in SIMPLE mutations. Three novel mutations were also identified: Ala111Gly (two families), Pro135Ser, and Pro135Thr. Familial studies revealed that all carriers of mutations (n = 38), aged from 1 to 78 years, were symptomatic, notably children under 10 years (n = 8). Motor conduction velocities in the median nerve ranked from 16.4 to 32.8 m/s (n = 20). In our series of 968 unrelated dominant demyelinating CMT cases (1992-2005), the percentage of SIMPLE mutations was 0.6 (6/968).

Gene therapy of the brain in the dog model of Hurler's syndrome.

OBJECTIVE: A defect of the lysosomal enzyme alpha-L-iduronidase (IDUA) interrupts the degradation of glycosaminoglycans in mucopolysaccharidosis type I, causing severe neurological manifestations in children with Hurler's syndrome. Delivery of the missing enzyme through stereotactic injection of adeno-associated virus vectors coding for IDUA prevents neuropathology in affected mice. We examined the efficacy and the safety of this approach in enzyme-deficient dogs. METHODS: Because deficient dogs raise antibodies against IDUA in response to infusion, intracerebral vector injections were combined with an immunosuppressive regimen. RESULTS: Treatment was tolerated well. We observed broad dispersion of vector genomes in the brain of efficiently immunosuppressed dogs. The delivery of IDUA to large areas, which could encompass the entire brain, prevented glycosaminoglycan and secondary ganglioside accumulations. This condition was associated with drastic reduction of neuropathology throughout the encephalon. In contrast, vector injection combined with partial immunosuppression was associated with subacute encephalitis, production of antibodies against IDUA in brain tissues, and elimination of genetically modified cells. INTERPRETATION: Gene therapy directed to the entire brain is feasible and may be beneficial to children with Hurler's syndrome. The possibility of subacute encephalitis emphasizes the importance of preventing immune response against IDUA, a problem that needs to be considered in similar therapies for other genetic defects.

Mutations in TMEM76* cause mucopolysaccharidosis IIIC (Sanfilippo C syndrome).

Mucopolysaccharidosis IIIC (MPS IIIC, or Sanfilippo C syndrome) is a lysosomal storage disorder caused by the inherited deficiency of the lysosomal membrane enzyme acetyl-coenzyme A: alpha -glucosaminide N-acetyltransferase (N-acetyltransferase), which leads to impaired degradation of heparan sulfate. We report the narrowing of the candidate region to a 2.6-cM interval between D8S1051 and D8S1831 and the identification of the transmembrane protein 76 gene (TMEM76), which encodes a 73-kDa protein with predicted multiple transmembrane domains and glycosylation sites, as the gene that causes MPS IIIC when it is mutated. Four nonsense mutations, 3 frameshift mutations due to deletions or a duplication, 6 splice-site mutations, and 14 missense mutations were identified among 30 probands with MPS IIIC. Functional expression of human TMEM76 and the mouse ortholog demonstrates that it is the gene that encodes the lysosomal N-acetyltransferase and suggests that this enzyme belongs to a new structural class of proteins that transport the activated acetyl residues across the cell membrane.

Fabry disease: D313Y is an alpha-galactosidase A sequence variant that causes pseudodeficient activity in plasma.

Fabry disease, an X-linked recessive lysosomal storage disease, results from the deficient activity of the exogalactosidase, alpha-galactosidase A (alpha-Gal A). To date, over 270 disease-causing mutations have been identified; however, no coding sequence variants have been reported. In the course of enzyme diagnostic testing, a normal female control had low plasma and leukocyte alpha-Gal A activities. Sequencing her alpha-Gal A gene revealed the D313Y substitution (GAT to TAT at cDNA nucleotide 937). alpha-Gal A mutation and enzyme analyses of family members revealed X-linked transmission and leukocyte alpha-Gal A enzymatic activities in females, consistent with Lyonization. Since D313Y was reported in a classically affected male who had the double mutation, D313Y and G411D, efforts were undertaken to characterize these lesions. Expression of D313Y, G411D, and the doubly mutated construct, D313Y/G411D, resulted in alpha-Gal A levels of 76, 2.9, and 1.7% of mean expressed wild-type activity, respectively. Biosynthetic studies revealed essentially normal processing of the D313Y subunit, but the absence of the mature subunit encoded by the G411D and D313Y/G411D constructs. Thus, G411D is the disease-causing mutation, while D313Y is the first coding sequence variant identified in the human alpha-Gal A gene.