Generoso Andria

A cluster of sulfatase genes on Xp22.3: Mutations in chondrodysplasia punctata (CDPX) and implications for warfarin embryopathy

X-linked recessive chondrodysplasia punctata (CDPX) is a congenital defect of bone and cartilage development characterized by aberrant bone mineralization, severe underdevelopment of nasal cartilage, and distal phalangeal hypoplasia. A virtually identical phenotype is observed in the warfarin embryopathy, which is due to the teratogenic effects of coumarin derivatives during pregnancy. We have cloned the genomic region within Xp22.3 where the CDPX gene has been assigned and isolated three adjacent genes showing highly significant homology to the sulfatase gene family. Point mutations in one of these genes were identified in five patients with CDPX. Expression of this gene in COS cells resulted in a heat-labile arylsulfatase activity that is inhibited by warfarin. A deficiency of a heat-labile arylsulfatase activity was demonstrated in patients with deletions spanning the CDPX region. These data indicate that CDPX is caused by an inherited deficiency of a novel sulfatase and suggest that warfarin embryopathy might involve drug-induced inhibition of the same enzyme.

Cystathionine β‐synthase mutations in homocystinuria

The major cause of homocystinuria is mutation of the gene encoding the enzyme cystathionine β‐synthase (CBS). Deficiency of CBS activity results in elevated levels of homocysteine as well as methionine in plasma and urine and decreased levels of cystathionine and cysteine. Ninety‐two different disease‐associated mutations have been identified in the CBS gene in 310 examined homocystinuric alleles in more than a dozen laboratories around the world. Most of these mutations are missense, and the vast majority of these are private mutations. The two most frequently encountered of these mutations are the pyridoxine‐responsive I278T and the pyridoxine‐nonresponsive G307S. Mutations due to deaminations of methylcytosines represent 53% of all point substitutions in the coding region of the CBS gene. Hum Mutat 13:362–375, 1999.

Prevalence and clinical picture of celiac disease in italian down syndrome patients: a multicenter study.

Background A multicenter research study of Down syndrome patients was carried out to estimate the prevalence of celiac disease in patients with Down syndrome and to show clinical characteristics and laboratory data of Down syndrome patients. Methods The authors studied 1,202 Down syndrome patients. Fifty-five celiac disease patients (group 1) were compared with 55 immunoglobulin A antigliadin–positive antiendomysium antibodies–negative patients (group 2) and with 57 immunoglobulin A antigliadin–negative antiendomysium antibodies–negative patients (group 3). Results Celiac disease was diagnosed in 55 of 1,202 Down syndrome patients (4.6%). In group 1, weight and height percentiles were shifted to the left, whereas these parameters were normally distributed in groups 2 and 3. In celiac patients, diarrhea, vomiting, failure to thrive, anorexia, constipation, and abdominal distension were higher than in the other two groups. Low levels of hemoglobinemia, serum iron, and calcium were observed more frequently in group 1. The diagnosis of celiac disease was made after a mean period of 3.8 years from the initiation of symptoms. Sixty-nine percent of patients showed a classic presentation, 11% had atypical symptoms, and 20% had silent celiac disease. Autoimmune disorders were more frequent (30.9%) in group 1 than in the other two groups examined (15%;P < 0.05). Conclusions This study reconfirms a high prevalence of celiac disease in Down syndrome. However, the diagnostic delay, the detection of atypical symptoms or silent form in one third of the cases, and the increased incidence of autoimmune disorders suggest the need for the screening of celiac disease in all Down syndrome patients.

Congenital Insensitivity to Pain with Anhidrosis: Novel Mutations in the TRKA (NTRK1) Gene Encoding A High-Affinity Receptor for Nerve Growth Factor

Congenital insensitivity to pain with anhidrosis (CIPA) is characterized by recurrent episodes of unexplained fever, anhidrosis (inability to sweat), absence of reaction to noxious stimuli, self-mutilating behavior, and mental retardation. Human TRKA encodes a high-affinity tyrosine kinase receptor for nerve growth factor (NGF), a member of the neurotrophin family that induces neurite outgrowth and promotes survival of embryonic sensory and sympathetic neurons. We have recently demonstrated that TRKA is responsible for CIPA by identifying three mutations in a region encoding the intracellular tyrosine kinase domain of TRKA in one Ecuadorian and three Japanese families. We have developed a comprehensive strategy to screen for TRKA mutations, on the basis of the genes structure and organization. Here we report 11 novel mutations, in seven affected families. These are six missense mutations, two frameshift mutations, one nonsense mutation, and two splice-site mutations. Mendelian inheritance of the mutations is confirmed in six families for which parent samples are available. Two mutations are linked, on the same chromosome, to Arg85Ser and to His598Tyr;Gly607Val, hence, they probably represent double and triple mutations. The mutations are distributed in an extracellular domain, involved in NGF binding, as well as the intracellular signal-transduction domain. These data suggest that TRKA defects cause CIPA in various ethnic groups.

Lysosomal Storage Diseases: From Pathophysiology to Therapy

Lysosomal storage diseases are a group of rare, inborn, metabolic errors characterized by deficiencies in normal lysosomal function and by intralysosomal accumulation of undegraded substrates. The past 25 years have been characterized by remarkable progress in the treatment of these diseases and by the development of multiple therapeutic approaches. These approaches include strategies aimed at increasing the residual activity of a missing enzyme (enzyme replacement therapy, hematopoietic stem cell transplantation, pharmacological chaperone therapy and gene therapy) and approaches based on reducing the flux of substrates to lysosomes. As knowledge has improved about the pathophysiology of lysosomal storage diseases, novel targets for therapy have been identified, and innovative treatment approaches are being developed.

Analysis of seven maternal polymorphisms of genes involved in homocysteine/folate metabolism and risk of Down syndrome offspring

PURPOSE: We present a case-control study of seven polymorphisms of six genes involved in homocysteine/folate pathway as risk factors for Down syndrome. Gene-gene/allele-allele interactions, haplotype analysis and the association with age at conception were also evaluated.METHODS: We investigated 94 Down syndrome-mothers and 264 control-women from Campania, Italy.RESULTS: Increased risk of Down syndrome was associated with the methylenetetrahydrofolate reductase (MTHFR) 1298C allele (OR 1.46; 95% CI 1.02–2.10), the MTHFR 1298CC genotype (OR 2.29; 95% CI 1.06–4.96), the reduced-folate-carrier1 (RFC1) 80G allele (1.48; 95% CI 1.05–2.10) and the RFC1 80 GG genotype (OR 2.05; 95% CI 1.03–4.07). Significant associations were found between maternal age at conception ≥34 years and either the MTHFR 1298C or the RFC 180G alleles. Positive interactions were found for the following genotype-pairs: MTHFR 677TT and 1298CC/CA, 1298CC/CA and RFC1 80 GG/GA, RFC1 80 GG and methylenetetrahydrofolate-dehydrogenase 1958 AA. The 677–1298 T-C haplotype at the MTHFR locus was also a risk factor for Down syndrome (P = 0.0022). The methionine-synthase-reductase A66G, the methionine-synthase A2756G and the cystathionine-beta-synthase 844ins68 polymorphisms were not associated with increased risk of Down syndrome.CONCLUSION: These results point to a role of maternal polymorphisms of homocysteine/folate pathway as risk factors for Down syndrome.

Mitochondrial DNA methylation as a next-generation biomarker and diagnostic tool

Recent expansion of our knowledge on epigenetic changes strongly suggests that not only nuclear DNA (nDNA), but also mitochondrial DNA (mtDNA) may be subjected to epigenetic modifications related to disease development, environmental exposure, drug treatment and aging. Thus, mtDNA methylation is attracting increasing attention as a potential biomarker for the detection and diagnosis of diseases and the understanding of cellular behavior in particular conditions. In this paper we review the current advances in mtDNA methylation studies with particular attention to the evidences of mtDNA methylation changes in diseases and physiological conditions so far investigated. Technological advances for the analysis of epigenetic variations are promising tools to provide insights into methylation of mtDNA with similar resolution levels as those reached for nDNA. However, many aspects related to mtDNA methylation are still unclear. More studies are needed to understand whether and how changes in mtDNA methylation patterns, global and gene specific, are associated to diseases or risk factors.

The Pharmacological Chaperone N-butyldeoxynojirimycin Enhances Enzyme Replacement Therapy in Pompe Disease Fibroblasts

In spite of the progress in the treatment of lysosomal storage diseases (LSDs), in some of these disorders the available therapies show limited efficacy and a need exists to identify novel therapeutic strategies. We studied the combination of enzyme replacement and enzyme enhancement by pharmacological chaperones in Pompe disease (PD), a metabolic myopathy caused by the deficiency of the lysosomal acid α-glucosidase. We showed that coincubation of Pompe fibroblasts with recombinant human α-glucosidase and the chaperone N-butyldeoxynojirimycin (NB-DNJ) resulted in more efficient correction of enzyme activity. The chaperone improved α-glucosidase delivery to lysosomes, enhanced enzyme maturation, and increased enzyme stability. Improved enzyme correction was also found in vivo in a mouse model of PD treated with coadministration of single infusions of recombinant human α-glucosidase and oral NB-DNJ. The enhancing effect of chaperones on recombinant enzymes was also observed in fibroblasts from another lysosomal disease, Fabry disease, treated with recombinant α-galactosidase A and the specific chaperone 1-deoxygalactonojirimycin (DGJ). These results have important clinical implications, as they demonstrate synergy between pharmacological chaperones and enzyme replacement. A synergistic effect of these treatments may result particularly useful in patients responding poorly to therapy and in tissues in which sufficient enzyme levels are difficult to obtain.

Pharmacological enhancement of mutated alpha-glucosidase activity in fibroblasts from patients with Pompe disease.

We investigated the use of pharmacological chaperones for the therapy of Pompe disease, a metabolic myopathy due to mutations of the gene encoding the lysosomal hydrolase α-glucosidase (GAA) and characterized by generalized glycogen storage in cardiac and skeletal muscle. We studied the effects of two imino sugars, deoxynojirimycin (DNJ) and N-butyldeoxynojirimycin (NB-DNJ), on residual GAA activity in fibroblasts from eight patients with different forms of Pompe disease (two classic infantile, two non-classic infantile onset, four late-onset forms), and with different mutations of the GAA gene. We demonstrated a significant increase of GAA activity (1.3-7.5-fold) after imino sugar treatment in fibroblasts from patients carrying the mutations L552P (three patients) and G549R (one patient). GAA enhancement was confirmed in HEK293T cells where the same mutations were overexpressed. No increase of GAA activity was observed for the other mutations. Western blot analysis showed that imino sugars increase the amount of mature GAA molecular forms. Immunofluorescence studies in HEK293T cells overexpressing the L552P mutation showed an improved trafficking of the mutant enzyme to lysosomes after imino sugar treatment. These results provide a rationale for an alternative treatment, other than enzyme replacement, to Pompe disease.

Lathosterolosis, a Novel Multiple-Malformation/Mental Retardation Syndrome Due to Deficiency of 3β-Hydroxysteroid-Δ5-Desaturase

We report the clinical, biochemical, and molecular characterization of a patient with a novel defect of cholesterol biosynthesis. This patient presented with a complex phenotype, including multiple congenital anomalies, mental retardation, and liver disease. In the patient’s plasma and cells, we found increased levels of lathosterol. The biosynthesis of cholesterol in the patient’s fibroblasts was defective, showing a block in the conversion of lathosterol into 7-dehydrocholesterol. The activity of 3β-hydroxysteroid-Δ5-desaturase (SC5D), the enzyme involved in this reaction, was deficient in the patient’s fibroblasts. Sequence analysis of the SC5D gene in the patient’s DNA, showing the presence of two missense mutations (R29Q and G211D), confirmed that the patient is affected by a novel defect of cholesterol biosynthesis.