Enzo Ranieri

Submicroscopic Duplications of the Hydroxysteroid Dehydrogenase HSD17B10 and the E3 Ubiquitin Ligase HUWE1 Are Associated with Mental Retardation

Submicroscopic copy-number imbalances contribute significantly to the genetic etiology of human disease. Here, we report a novel microduplication hot spot at Xp11.22 identified in six unrelated families with predominantly nonsyndromic XLMR. All duplications segregate with the disease, including the large families MRX17 and MRX31. The minimal, commonly duplicated region contains three genes: RIBC1, HSD17B10, and HUWE1. RIBC1 could be excluded on the basis of its absence of expression in the brain and because it escapes X inactivation in females. For the other genes, expression array and quantitative PCR analysis in patient cell lines compared to controls showed a significant upregulation of HSD17B10 and HUWE1 as well as several important genes in their molecular pathways. Loss-of-function mutations of HSD17B10 have previously been associated with progressive neurological disease and XLMR. The E3 ubiquitin ligase HUWE1 has been implicated in TP53-associated regulation of the neuronal cell cycle. Here, we also report segregating sequence changes of highly conserved residues in HUWE1 in three XLMR families; these changes are possibly associated with the phenotype. Our findings demonstrate that an increased gene dosage of HSD17B10, HUWE1, or both contribute to the etiology of XLMR and suggest that point mutations in HUWE1 are associated with this disease too.

Expanded newborn screening: Outcome in screened and unscreened patients at age 6 years

OBJECTIVE: Tandem mass spectrometry is widely applied to routine newborn screening but there are no long-term studies of outcome. We studied the clinical outcome at six years of age in Australia. METHODS: In a cohort study, we analyzed the outcome at 6 years for patients detected by screening or by clinical diagnosis among >2 million infants born from 1994 to 1998 (1 017 800, all unscreened) and 1998 to 2002 (461 500 screened, 533 400 unscreened) recording intellectual and physical condition, school placement, other medical problems, growth, treatment, diet, and hospital admissions. Results were analyzed separately for medium-chain acyl-CoA dehydrogenase deficiency (MCADD) and other disorders, and grouped patients as those who presented clinically or died in the first 5 days of life; patients presented later or diagnosed by screening, and those with substantially benign disorders. RESULTS: Inborn errors, excluding phenylketonuria, were diagnosed in 116 of 1 551 200 unscreened infants (7.5/100 000 births) and 70 of 461 500 screened infants (15.2/100 000 births). Excluding MCADD, 21 unscreened patients with metabolic disorders diagnosed after 5 days of life died or had a significant intellectual or physical handicap (1.35/100 000 population) compared with 2 of the screened cohort (0.43/100 000; odds ratio: 3.1 [95% CI: 0.73–13.32]). Considering the likely morbidity or mortality among the expected number of never-diagnosed unscreened patients, there would be a significant difference. Growth distribution was normal in all cohorts. CONCLUSION: Screening by tandem mass spectrometry provides a better outcome for patients at 6 years of age, with fewer deaths and fewer clinically significant disabilities.

Newborn Screening for Lysosomal Storage Disorders: Clinical Evaluation of a Two-Tier Strategy

Objective. To evaluate the use of protein markers using immune-quantification assays and of metabolite markers using tandem mass spectrometry for the identification, at birth, of individuals who have a lysosomal storage disorder. Methods. A retrospective analysis was conducted of Guthrie cards that were collected from newborns in Denmark during the period 1982–1997. Patients whose lysosomal storage disorder (LSD; 47 representing 12 disorders) was diagnosed in Denmark during the period 1982–1997 were selected, and their Guthrie cards were retrieved from storage. Control cards (227) were retrieved from the same period. Additional control cards (273) were collected from the South Australian Screening Centre (Australia). Results. From 2 protein and 94 metabolite markers, 15 were selected and evaluated for their use in the identification of LSDs. Glycosphingolipid and oligosaccharide markers showed 100% sensitivity and specificity for the identification of Fabry disease, α-mannosidosis, mucopolysaccharidosis (MPS) IVA, MPS IIIA, Tay-Sachs disease, and I-cell disease. Lower sensitivities were observed for Gaucher disease and sialidosis. No useful markers were identified for Krabbe disease, MPS II, Pompe disease, and Sandhoff disease. The protein markers LAMP-1 and saposin C were not able to differentiate individuals who had an LSD from the control population. Conclusions. Newborn screening for selected LSDs is possible with current technology. However, additional development is required to provide a broad coverage of disorders in a single, viable program.

Neonatal screening for cystic fibrosis using immunoreactive trypsinogen and direct gene analysis: four years' experience.

Abstract Objective : To assess the performance and impact of a two tier neonatal screening programme for cystic fibrosis based on an initial estimation of immunoreactive trypsinogen followed by direct gene analysis. >Design : Four year prospective study of two tier screening strategy. First tier: immunoreactive trypsinogen measured in dried blood spot samples from neonates aged 3-5 days. Second tier: direct gene analysis of cystic fibrosis mutations (δF508, δI506, G551D, G542X, and R553X) in samples with immunoreactive trypsinogen concentrations in highest 1% and in all neonates with meconium ileus20or family history of cystic fibrosis. Setting : South Australian Neonatal Screening Programme, Adelaide. Subjects : All 88 752 neonates born in South Australia between December 1989 and December 1993. Interventions : Neonates with two identifiable mutations were referred directly for clinical assessment and confirmatory sweat test; infants with only one identifiable mutation were recalled for sweat test at age 3-4 weeks. Parents of neonates identified as carriers of cystic fibrosis mutation were counselled and offered genetic testing. Main outcome measures - Identification of20all children with cystic fibrosis in the screened population. Results : Of 1004 (1.13%) neonates with immunoreactive trypsinogen >=99th centile, 912 (90.8%) had no identifiable mutation. 23 neonates were homozygotes or compound heterozygotes; 69 carried one identifiable mutation, of whom six had positive sweat tests. Median age at clinical assessment for the 29 neonates with cystic fibrosis was 3 weeks; six had meconium ileus and two had affected siblings. 63 neonates were identified as carriers of a cystic fibrosis mutation. Extra laboratory costs for measuring immunoreactive trypsinogen and direct gene analysis were

The sensitivity of ultrasound and serum alpha-fetoprotein in population-based antenatal screening for neural tube defects, South Australia 1986–1991

A1.50 per neonate screened. Conclusion : This strategy results in early and accurate diagnosis of cystic fibrosis and performs better than screening strategies based on immunoreactive trypsinogen measurement alone.

Endogenous plasma carnitine pool composition and response to erythropoietin treatment in chronic haemodialysis patients

Objective To determine the sensitivity of antenatal screening methods for neural tube defects in population‐based screening in South Australia in 1986–1991, and whether ultrasound can replace serum alpha‐fetoprotein screening in terms of achieving an equivalent level of sensitivity.

Lentiviral-mediated gene therapy for murine mucopolysaccharidosis type IIIA.

BACKGROUND Anaemia is a common complication associated with haemodialysis and is usually managed by treatment with recombinant human erythropoietin (rHuEPO). However, many patients remain hyporesponsive to rHuEPO treatment despite adequate iron therapy. The effect of L-carnitine administration on rHuEPO dose and/or haematocrit in haemodialysis patients has been previously reported with equivocal results. This study examined the relationship between endogenous carnitine pool composition and rHuEPO requirements in long-term haemodialysis patients. METHODS Pre-dialysis blood samples were collected from 87 patients and analysed for plasma L-carnitine and individual acylcarnitine levels by LCMS/MS. As an indication of rHuEPO responsiveness, erythropoietin resistance index (ERI) was calculated as rHuEPO dose/kg/week normalized for haemoglobin levels. RESULTS A significant negative correlation between L-carnitine levels and ERI was found (P = 0.0421). All patients categorized as high ERI (>0.02 microg/kg/week/gHb) exhibited subnormal L-carnitine levels (<30 microM); conversely, patients with normal L-carnitine levels (>30 microM) displayed low ERI values (<0.02 microg/kg/week/gHb). More importantly, the ratio of non-acetyl acylcarnitines/total carnitine was significantly positively correlated with ERI (P = 0.0062). CONCLUSIONS These data illustrate the relationship between carnitine levels and response to rHuEPO treatment in haemodialysis patients, in particular, the importance of the proportion of long-chain acylcarnitines within the plasma carnitine pool. This proportion may be more indicative of the response to L-carnitine supplementation than absolute L-carnitine levels alone.

Quantification of Glutamine in Dried Blood Spots and Plasma by Tandem Mass Spectrometry for the Biochemical Diagnosis and Monitoring of Ornithine Transcarbamylase Deficiency

Mucopolysaccharidosis type IIIA (MPS IIIA) is a heritable glycosaminoglycan (GAG) storage disorder which is characterised by lysosomal accumulation of heparan sulphate, secondary to a deficiency of sulphamidase (heparan-N-sulphatase, N-sulphoglucosamine sulphohydrolase, EC No. 3.10.1.1.). There is currently no treatment for affected individuals who experience progressive CNS deterioration prior to an early death. As a first step towards developing gene therapy as a treatment for MPS IIIA, an MPS IIIA mouse model was used to examine the efficacy of intravenous lentiviral-mediated gene therapy. Five-week-old mice were injected with virus expressing murine sulphamidase and analysed 6 months after treatment. Transduction by the lentiviral vector was highest in the liver and spleen of treated animals, and sulphamidase activity in these tissues averaged 68% and 186% of normal, respectively. Storage was assessed using histochemical, chemical and mass spectrometric analyses. Storage in most somatic tissues was largely normalised, although chondrocytes were an obvious exception. Histologically, improvement of lysosomal storage within the brain was variable. However, beta-hexosaminidase activity, which is abnormally elevated in MPS IIIA, was significantly reduced in every treated tissue, including the brain. Total uronic acid was also significantly reduced in the brains of treated mice. The level of a disaccharide marker (hexosamine-N-sulphate[alpha-1,4]hexuronic acid; HNS-UA) of heparan sulphate storage was also decreased in the brains of treated mice, albeit non-significantly. These results suggest that lentiviral-mediated somatic gene transfer may affect not only the somatic, but possibly also the CNS pathology, found in MPS IIIA.

Lentiviral-mediated gene correction of mucopolysaccharidosis type IIIA

A notable deficiency in the use of tandem mass spectrometry (MS/MS) for newborn screening is the inability to screen for urea cycle defects. The most common of these, with an incidence of 1 in 14 000 births (1), is the inherited X-linked disorder ornithine transcarbamylase deficiency (OTCD). A majority (60%) of hemizygous males risk death from hyperammonemic coma during the first week of life. The remainder, including 10% of heterozygous females, exhibit lethargy, vomiting episodes, and behavioral problems during childhood. The severity of the disorder and the potential for correction of OTCD by liver transplantation and gene therapy (2) provide adequate justification for newborn screening. OTCD patients have low blood citrulline because of reduced conversion from carbamoyl phosphate. Citrulline is one of the amino acids routinely measured in MS/MS newborn-screening programs. Unfortunately, many protein-restricted newborns also have low blood citrulline (3). A more selective amino acid metabolite for OTCD is glutamine. The derivatization procedure used in many MS/MS screening programs (4), which uses butanol–hydrogen chloride, destroys glutamine. Approximately one-half of the glutamine is converted to glutamic acid dibutyl ester and is indistinguishable from that formed from endogenous glutamic acid in the blood. The surviving glutamine butyl ester is deaminated in acidic solution to a protonated form of pyroglutamic acid butyl ester in the electrospray source of the MS/MS. Again it is not possible to distinguish this pyroglutamic acid from what is already present in the blood. As a secondary consequence, the measurements of glutamic and pyroglutamic (and by analogy, aspartic) acids in blood spots after derivatization are grossly inaccurate. MS/MS newborn-screening programs that do not derivatize amino acids avoid solvolysis of glutamine and of pyroglutamic acid to glutamic acid. During electrospray ionization-MS/MS analysis, however, glutamine is again indistinguishable from pyroglutamic acid. Resolution is possible by separation with time-consuming liquid chromatography …

Genetic susceptibility to viral exposure may increase the risk of cerebral palsy.

BackgroundMucopolysaccharidosis type IIIA (MPS IIIA) is the most common of the mucopolysaccharidoses. The disease is caused by a deficiency of the lysosomal enzyme sulphamidase and results in the storage of the glycosaminoglycan (GAG), heparan sulphate. MPS IIIA is characterised by widespread storage and urinary excretion of heparan sulphate, and a progressive and eventually profound neurological course. Gene therapy is one of the few avenues of treatment that hold promise of a sustainable treatment for this disorder.MethodsThe murine sulphamidase gene cDNA was cloned into a lentiviral vector and high-titre virus produced. Human MPS IIIA fibroblast cultures were transduced with the sulphamidase vector and analysed using molecular, enzymatic and metabolic assays. High-titre virus was intravenously injected into six 5-week old MPS IIIA mice. Three of these mice were pre-treated with hyperosmotic mannitol. The weight of animals was monitored and GAG content in urine samples was analysed by polyacrylamide gel electrophoresis.ResultsTransduction of cultured MPS IIIA fibroblasts with the sulphamidase gene corrected both the enzymatic and metabolic defects. Sulphamidase secreted by gene-corrected cells was able to cross correct untransduced MPS IIIA cells. Urinary GAG was found to be greatly reduced in samples from mice receiving the vector compared to untreated MPS IIIA controls. In addition, the weight of treated mice became progressively normalised over the 6-months post-treatment.ConclusionLentiviral vectors appear promising vehicles for the development of gene therapy for MPS IIIA.