Rosanna Gatti

Genome Search in Celiac Disease

Celiac disease (CD), a malabsorption disorder of the small intestine, results from ingestion of gluten. The HLA risk factors involved in CD are well known but do not explain the entire genetic susceptibility. To determine the localization of other genetic risk factors, a systematic screening of the genome has been undertaken. The typing information of 281 markers on 110 affected sib pairs and their parents was used to test linkage. Systematic linkage analysis was first performed on 39 pairs in which both sibs had a symptomatic form of CD. Replication of the regions of interest was then carried out on 71 pairs in which one sib had a symptomatic form and the other a silent form of CD. In addition to the HLA loci, our study suggests that a risk factor in 5qter is involved in both forms of CD (symptomatic and silent). Furthermore, a factor on 11qter possibly differentiates the two forms. In contrast, none of the regions recently published was confirmed by the present screening.

Genotype/phenotype correlation in glycogen storage disease type 1b: a multicentre study and review of the literature

We studied the genotype/phenotype correlation in a cohort of glycogen storage disease type (GSD) 1b patients. A total of 25 GSD1b patients, 13 females and 12 males, age range: 4.3–28.4 years, mean:14.6±6.8 years; median: 15 years, representing the entire case load of Italian GSD1b patients, were enrolled in the study. Molecular analysis of the glucose 6-phosphate translocase (G6PT1) gene was performed in all patients. We analysed the presence of a correlation among both the clinical features associated with GSD1b (neutropenia, frequency of admission to the hospital for severe infections) and the presence of systemic complications (liver adenomas, nephropathy, bone mineral density defect, polycystic ovaries, short stature, inflammatory bowel disease) and the mutations detected in each patient. Nine patients were homozygous or compound heterozygous for mutations causing stop codons. In particular, three patients were homozygous for the same mutation (400X); of these patients, one showed chronic neutropenia with severe and frequent infections and severe inflammatory bowel disease, another patient cyclic neutropenia associated with rare bacterial infections and mild bowel involvement and the last one normal neutrophil count. Two patients were homozygous for the mutation 128X; one of these patients did not show neutropenia, whereas the other one had severe neutropenia needing frequent hospital admission and was under granulocyte-colony stimulating factor treatment. In three patients no mutations were detected. Conclusion:no correlation was found between individual mutations and the presence of neutropenia, bacterial infections and systemic complications. These results suggest that different genes and proteins modulate neutrophil differentiation, maturation and apoptosis and thus the severity and frequency of infections. The absence of detectable mutations in three patients could suggest that a second protein plays a role in microsomal phosphate transport.

Efficacy of ACE‐inhibitor therapy on renal disease in glycogen storage disease type 1: a multicentre retrospective study

Background  The efficacy of ACE‐inhibitors in decreasing microalbuminuria and proteinuria has been reported in a few patients with glycogen storage disease type 1 (GSD1); however, no case‐control study has ever been published.

An Asn > Lys substitution in saposin B involving a conserved amino acidic residue and leading to the loss of the single N-glycosylation site in a patient with metachromatic leukodystrophy and normal arylsulphatase A activity.

Sphingolipid activator proteins are small glycoproteins required for the degradation of sphingolipids by specific lysosomal hydrolases. Four of them, called saposins, are encoded by the prosaposin gene, the product of which is proteolytically cleaved into the four mature saposin proteins (saposins A, B, C, D). One of these, saposin B, is necessary in the hydrolysis of sulphatide by arylsulphatase A where it presents the solubilised substrate to the enzyme. As an alternative to arylsulphatase A deficiency, deficiency of saposin B causes metachromatic leukodystrophy. We identified a previously undescribed mutation (N215K) in the prosaposin gene of a patient with metachromatic leukodystrophy but with normal arylsulphatase A activity and elevated sulphatide in urine. The mutation involves a highly conserved amino acidic residue and abolishes the only N-glycosylation site of saposin B.

Jagged-1 mutation analysis in Italian Alagille syndrome patients

Alagille syndrome (AGS) is an autosomal dominant disorder with developmental abnormalities affecting the liver, heart, eyes, vertebrae, and craniofacial region. The Jagged‐1 (JAG1) gene, which encodes a ligand of Notch, has recently been found mutated in AGS. In this study, mutation analysis of the JAG1 gene performed on 20 Italian AGS patients led to the identification of 15 different JAG1 mutations, including a large deletion of the 20p12 region, six frameshift, three nonsense, three splice‐site, and two missense mutations. The two novel missense mutations were clustered in the 5′ region, while the remaining mutations were scattered throughout the gene. The spectrum of mutations in Italian patients was similar to that previously reported. We also studied in detail a complex splice site mutation, 3332dupl8bp, which was shown to lead to an abnormal JAG1 mRNA, resulting in a premature stop codon. With the exception of the missense mutations, the majority of the JAG1 mutations are therefore likely to produce truncated proteins. Since the phenotype of the patient with a complete deletion of the JAG1 gene is indistinguishable from that of patients with intragenic mutations, our study further supports the hypothesis that haploinsufficiency is the most common mechanism involved in AGS pathogenesis. Furthermore, our data confirmed the absence of a correlation between the genotype of the JAG1 gene and the AGS phenotype. Hum Mutat 14:394–400, 1999.

Molecular defects in the α-N-acetylglucosaminidase gene in Italian Sanfilippo type B patients

Sanfilippo syndrome type B (mucopolysaccharidosis IIIB) is a rare autosomal recessive disorder characterized by the inability to degrade heparan sulfate because of a deficiency of the lysosomal enzyme α-N-acetylglucosaminidase (NAGLU). We performed mutation screening in a group of 20 patients, identyifing 28 mutations, 14 of which were novel (L35F, 204delC, 221insGCGCG, G82D, W156C, 507delC, IVS3+1G→A, E336X, V501G, R520W, S534Y, W649C, 1953insGCCA, 2185delAGA). Four of these mutations were found in homozygosity and only one was seen in two different patients, showing the remarkable molecular heterogeneity of the disease. Mutation IVS3+1G→A produces aberrant RNA splicing: it represents a base substitution from G to A of the invariant GT dinucleotides at the splicing donor site of intron 3 resulting in the skipping of exon 3 and both exons 2 and 3. Transient transfection of COS cells, by DNA mutagenized with NAGLU mutations, produced enzymatic molecules without activity, demonstrating the deleterious nature of the defects. Metabolic labeling of transfected mutants suggested a normal synthesis of the involved polypeptide for missense alterations, whereas increased protein or mRNA instability was shown for nonsense and most of the frameshift mutations.

Multiple congenital anomalies, brain hypomyelination, and ocular albinism in a female with dup(X)(pter→q24::q21.32→qter) and random X inactivation

We report on an 18-month-old girl with multiple congenital anomalies (prominence of the metopic suture, fine hair, club foot, absence of the 12th rib, brachydactyly) and severe mental retardation. The funduscopic examination showed diffuse retinal hypopigmentation. Brain magnetic resonance image (MRI) showed signs of diffuse hypomyelination. On cytogenetic and molecular evidence, the karyotype was 46,X,dirdup(X) (pter-->q24::q21.32-->qter). The duplication of the PLP gene, involved in Pelizaeus-Merzbacher disease, was confirmed by fluorescent in situ hybridization (FISH). Both cytogenetic and molecular studies on the X chromosome inactivation status indicated a random pattern in lymphocytes and fibroblasts. This patient appears to be the first case of a female bearing a large duplication of Xq with a random X inactivation. The phenotype of this patient is compared to that of previously reported cases with Xq duplication.

Minor facial anomalies in combined methylmalonic aciduria and homocystinuria due to a defect in cobalamin metabolism.

Methylmalonic acidaemia and homocystinuria (MMA/HC) is a very rare inborn error of cellular metabolism of cobalamin (Cbl), that results in functional defects of both methylmalonyl-CoA mutase and methionine synthase because of deficient synthesis of 5-deoxyadenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl). AdoCbl is the cofactor of methylmalonyl-CoA mutase, the enzyme catalysing the intramitochondrial isomerization of methylmalonyl-CoA to succinyl-CoA, while MeCbl is the cofactor of the cytosolic remethylation from homocysteine to methionine with recycling of the folate derivative tetrahydrofolate (THF). Seven different defects of the intracellular metabolism of Cbl have been described; of these seven, three forms involve the synthesis of both AdoCbl and MeCbl and are distinct genetically and biochemically, namely Cbl-C (the most frequent variant), Cbl-D and Cbl-F. Age of onset and clinical findings vary widely among the three complementation groups and among known patients with Cbl-C disease: the affected patients generally show feeding difficulties, failure to thrive, neurological dysfunction (developmental delay, hypotonia, seizures, extrapyramidal signs in childhood, dementia, psychosis), haematological changes, ocular abnormalities and renal involvement. The acute neonatal onset is severe and can lead to death (Fenton and Rosenblatt 1995; Rosenblatt et al 1997)). To date, minor facial anomalies have been reported only in two cases of the Cbl-F form, but not in the other variants of combined MMA and HC, whereas peculiar facial dysmorphism has been described in several different metabolic diseases such as peroxisomal disorders, carbohydrate glycoprotein deficiency syndrome, Smith-Lemli-Opitz syndrome, mevalonic aciduria, pyruvate dehydrogenase deficiency and respiratory chain deficiencies. We report here our personal observation of these clinical findings in seven patients with MMA and HC.

A homozygous missense arginine to histidine substitution at position 482 of the β-galactosidase in an Italian infantile GM1-gangliosidosis patient

We have studied, by the polymerase chain reaction, the β-galactosidase cDNA from several Italian patients with infantile GM1-gangliosidosis. One homozygote for a previously undiscovered G > A mutation at position 1479, causing an arginine to histidine change, was detected. The same mutation, in heterozygosis, was identified in 6 unrelated patients, but not in 100 normal chromosomes.

An Italian severe Salla disease variant associated with a SLC17A5 mutation earlier described in infantile sialic acid storage disease

The present study reports two Italian brothers affected by severe Salla disease (sialic acid storage disease), a slowly progressive autosomal recessive neurodegenerative disorder prevalent in the Finnish population. Mutations of the SLC17A5 gene, which encodes a protein called sialin, are the primary cause of both Salla disease and infantile sialic acid storage disease (ISSD), a clinically distinct severe disorder. All Finnish patients with Salla disease show a R39C mutation. Both patients showed moderate intellectual disability, spastic ataxic syndrome, hypomyelination and cerebellar atrophy on magnetic resonance imaging (MRI), and lysosomal storage, all typical of Salla disease. Mutation analysis of the SLC17A5 gene in the younger brother revealed no R39C mutation, but a 15‐bp deletion in exon 6 on one of the alleles. This mutation is the same described in French‐Canadian patients with ISSD. Salla disease must be suspected in patients with unexplained psychomotor retardation associated with ataxia and/or pyramidal symptoms, and MRI findings consistent with cerebral hypomyelination, irrespective of the patients ethnic origin. A mutation screening based on R39C change does not exclude Salla disease outside Finland. Conversely, mutations found in ISSD can be expected, even in patients showing the Salla phenotype (e.g. symptoms at the milder end of the spectrum).