BackgroundSequence analysis of the regulators of complement activation (RCA) cluster of genes at chromosome position 1q32 shows evidence of several large genomic duplications. These duplications have resulted in a high degree of sequence identity between the gene for factor H (CFH) and the genes for the five factor H-related proteins (CFHL1–5; aliases CFHR1–5). CFH mutations have been described in association with atypical haemolytic uraemic syndrome (aHUS). The majority of the mutations are missense changes that cluster in the C-terminal region and impair the ability of factor H to regulate surface-bound C3b. Some have arisen as a result of gene conversion between CFH and CFHL1. In this study we tested the hypothesis that nonallelic homologous recombination between low-copy repeats in the RCA cluster could result in the formation of a hybrid CFH/CFHL1 gene that predisposes to the development of aHUS.Methods and FindingsIn a family with many cases of aHUS that segregate with the RCA cluster we used cDNA analysis, gene sequencing, and Southern blotting to show that affected individuals carry a heterozygous CFH/CFHL1 hybrid gene in which exons 1–21 are derived from CFH and exons 22/23 from CFHL1. This hybrid encodes a protein product identical to a functionally significant CFH mutant (c.3572C>T, S1191L and c.3590T>C, V1197A) that has been previously described in association with aHUS.Conclusions CFH mutation screening is recommended in all aHUS patients prior to renal transplantation because of the high risk of disease recurrence post-transplant in those known to have a CFH mutation. Because of our finding it will be necessary to implement additional screening strategies that will detect a hybrid CFH/CFHL1 gene.

Glycogen storage disease type Ia (GSD-Ia) patients deficient in glucose-6-phosphatase-alpha (G6Pase-alpha or G6PC) manifest disturbed glucose homeostasis. We examined the efficacy of liver G6Pase-alpha delivery mediated by AAV-GPE, an adeno-associated virus (AAV) serotype 8 vector expressing human G6Pase-alpha directed by the human G6PC promoter/enhancer (GPE), and compared it to AAV-CBA, that directed murine G6Pase-alpha expression using a hybrid chicken beta-actin (CBA) promoter/cytomegalovirus (CMV) enhancer. The AAV-GPE directed hepatic G6Pase-alpha expression in the infused G6pc(-/-) mice declined 12-fold from age 2 to 6 weeks but stabilized at wild-type levels from age 6 to 24 weeks. In contrast, the expression directed by AAV-CBA declined 95-fold over 24 weeks, demonstrating that the GPE is more effective in directing persistent in vivo hepatic transgene expression. We further show that the rapid decline in transgene expression directed by AAV-CBA results from an inflammatory immune response elicited by the AAV-CBA vector. The AAV-GPE-treated G6pc(-/-) mice exhibit normal levels of blood glucose, blood metabolites, hepatic glycogen, and hepatic fat. Moreover, the mice maintained normal blood glucose levels even after 6 hours of fasting. The complete normalization of hepatic G6Pase-alpha deficiency by the G6PC promoter/enhancer holds promise for the future of gene therapy in human GSD-Ia patients.

Glycogen storage disease type Ib (GSDIb) is caused by a deficiency in the glucose-6-phosphate (G6P) transporter (G6PT) that works with a liver/kidney/ intestine-restricted glucose-6-phosphatase-␣ (G6Pase-␣) to maintain glucose homeostasis between meals. Clinically, GSD-Ib patients manifest disturbed glucose homeostasis and neutrophil dysfunctions but the cause of the latter is unclear. Neutrophils express the ubiquitously expressed G6PT and G6Pase-␤ that together transport G6P into the endoplasmic reticulum (ER) lumen and hydrolyze it to glucose. Because we expected G6PT-deficient neutrophils to be unable to produce endogenous glucose, we hypothesized this would lead to ER stress and increased apoptosis. Using GSD-Ib mice, we showed that GSD-Ib neutrophils exhibited increased production of ER chaperones and oxidative stress, consistent with ER stress, and increased annexin V binding and caspase-3 activation, consistent with an increased rate of apoptosis. Bax activation, mitochondrial release of proapoptotic effectors, and caspase-9 activation demonstrated the involvement of the intrinsic mitochondrial pathway in these processes. The results demonstrate that G6P translocation and hydrolysis are required for normal neutrophil functions and support the hypothesis that neutrophil dysfunction in GSD-Ib is due, at least in part, to ER stress and increased apoptosis. (Blood. 2008;111: 5704-5711) IntroductionGlycogen storage disease type I (GSD-I) is a group of autosomal recessive disorders caused by a deficiency in the glucose-6-phosphatase-␣ (G6Pase-␣) complex that consists of a glucose-6-phosphate transporter (G6PT, also known as SLC37A4) and the hydrolytic enzyme G6Pase-␣ (also known as G6PC). 1,2 Between meals, blood glucose homeostasis is maintained by glucose produced in the terminal steps of gluconeogenesis and glycogenolysis, 1,2 via G6Pase-␣-mediated hydrolysis of glucose-6-phosphate (G6P). Both G6Pase-␣ 3 and G6PT 4 are anchored in the endoplasmic reticulum (ER) by multiple transmembrane domains with G6PT transporting cytoplasmic G6P across the ER membrane to the G6Pase-␣ active site situated inside the ER lumen. 5 A deficiency of G6PT causes GSD type Ib (GSD-Ib, MIM232220 6 ) and a deficiency in G6Pase-␣ causes GSD type Ia (GSD-Ia, MIM232200 6 ). Because G6PT and G6Pase-␣ must be functionally coupled to transport and hydrolyze G6P to glucose, 7,8 a detrimental mutation in either protein prevents the other from functioning efficiently and leads to a common metabolic phenotype of disturbed glucose homeostasis manifested initially by changes in the glucose and lipid profiles of blood, and in the longer term with liver and kidney disease. 1,2 This profile is consistent with the expression pattern of G6Pase-␣, which is restricted primarily to the liver, and kidney, with a low level of expression in the small intestine 9 and G6PT, which is expressed ubiquitously. 10 Although G6Pase-␣ is not expressed in myeloid cells, GSD-Ib patients do manifest symptoms of neutropenia and neutrophil dysfunctions. 1,2,11,12 Their...