Maria Teresa Bassi

Non-type I cystinuria caused by mutations in SLC7A9, encoding a subunit (b(o,+)AT) of rBAT

Cystinuria (MIM 220100) is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids. Mutations in SLC3A1, encoding rBAT, cause cystinuria type I (ref. 1), but not other types of cystinuria (ref. 2). A gene whose mutation causes non-type I cystinuria has been mapped by linkage analysis to 19q12–13.1 (refs 3,4). We have identified a new transcript, encoding a protein (bo,+AT, for bo,+ amino acid transporter) belonging to a family of light subunits of amino acid transporters, expressed in kidney, liver, small intestine and placenta, and localized its gene (SLC7A9) to the non-type I cystinuria 19q locus. Co-transfection of bo,+AT and rBAT brings the latter to the plasma membrane, and results in the uptake of L-arginine in COS cells. We have found SLC7A9 mutations in Libyan-Jews, North American, Italian and Spanish non-type I cystinuria patients. The Libyan Jewish patients are homozygous for a founder missense mutation (V170M) that abolishes b o,+AT amino-acid uptake activity when co-transfected with rBAT in COS cells. We identified four missense mutations (G105R, A182T, G195R and G295R) and two frameshift (520insT and 596delTG) mutations in other patients. Our data establish that mutations in SLC7A9 cause non-type I cystinuria, and suggest that bo,+AT is the light subunit of rBAT.

De novo mutations in ATP1A3 cause alternating hemiplegia of childhood

Alternating hemiplegia of childhood (AHC) is a rare, severe neurodevelopmental syndrome characterized by recurrent hemiplegic episodes and distinct neurological manifestations. AHC is usually a sporadic disorder and has unknown etiology. We used exome sequencing of seven patients with AHC and their unaffected parents to identify de novo nonsynonymous mutations in ATP1A3 in all seven individuals. In a subsequent sequence analysis of ATP1A3 in 98 other patients with AHC, we found that ATP1A3 mutations were likely to be responsible for at least 74% of the cases; we also identified one inherited mutation in a case of familial AHC. Notably, most AHC cases are caused by one of seven recurrent ATP1A3 mutations, one of which was observed in 36 patients. Unlike ATP1A3 mutations that cause rapid-onset dystonia-parkinsonism, AHC-causing mutations in this gene caused consistent reductions in ATPase activity without affecting the level of protein expression. This work identifies de novo ATP1A3 mutations as the primary cause of AHC and offers insight into disease pathophysiology by expanding the spectrum of phenotypes associated with mutations in ATP1A3.

Identification and expression of NEU3, a novel human sialidase associated to the plasma membrane

Several mammalian sialidases have been described so far, suggesting the existence of numerous polypeptides with different tissue distributions, subcellular localizations and substrate specificities. Among these enzymes, plasma-membrane-associated sialidase(s) have a pivotal role in modulating the ganglioside content of the lipid bilayer, suggesting their involvement in the complex mechanisms governing cell-surface biological functions. Here we describe the identification and expression of a human plasma-membrane-associated sialidase, NEU3, isolated starting from an expressed sequence tag (EST) clone. The cDNA for this sialidase encodes a 428-residue protein containing a putative transmembrane helix, a YRIP (single-letter amino acid codes) motif and three Asp boxes characteristic of sialidases. The polypeptide shows high sequence identity (78%) with the membrane-associated sialidase recently purified and cloned from Bos taurus. Northern blot analysis showed a wide pattern of expression of the gene, in both adult and fetal human tissues. Transient expression in COS7 cells permitted the detection of a sialidase activity with high activity towards ganglioside substrates at a pH optimum of 3.8. Immunofluorescence staining of the transfected COS7 cells demonstrated the proteins localization in the plasma membrane.

Identification and characterisation of human xCT that co-expresses, with 4F2 heavy chain, the amino acid transport activity system xc-.

Abstract. We have identified a new human complementary deoxyribonucleic acid (cDNA), for the xc– amino acid transporter (HGMW-approved name SLC7A11; also known as human xCT), that, when co-expressed with the heavy chain of surface antigen 4F2 (4F2hc, also termed CD98), but not with rBAT, (related to the bo,+ amino acid transporter), induces system xc– transport activity in Xenopus oocytes. Human xCT is the seventh human member of the family of amino acid transporters that are subunits of 4F2hc or rBAT and, inview of its amino acid sequence identity (89%) with mouse xCT, is most probably the human orthologue thereof. The amino acid transport activity induced by the co-expression of human 4F2hc and xCT in Xenopus oocytes was sodium independent and specific for L-cystine, L-glutamate and L-aspartate. This activity also functioned in an exchange mode (e.g. cystine/glutamate) with a substrate stoichiometry of 1:1. Expression of human xCT alone in oocytes did not induce amino acid transport activity and the expressed xCT protein was localised intracellularly. When human xCT was co-expressed with 4F2hc, the former localised to the oocyte plasma membrane. Tissue-expression studies showed that human SLC7A11 mRNA is expressed mainly in the brain, but also in pancreas and in cultured cell lines. The transport characteristics of human xCT and the distribution of its tissue expression strongly suggest that it corresponds to the human amino acid transporter system xc–.

A novel mutation in the ATP1A2 gene causes alternating hemiplegia of childhood

Alternating hemiplegia of childhood (AHC, MIM 104290) is a rare syndrome, characterised by early onset of episodic hemi- or quadriplegia lasting minutes to days. This disorder, first reported by Verret and Steel in 1971,1 has historically been thought to represent a migraine equivalent1 or an unusual form of epilepsy or a movement disorder,2 as it typically presents with complex and variable clinical features. In most patients, the earliest manifestations clearly related to AHC are tonic–dystonic attacks and paroxysmal nystagmus associated with autonomic changes and paroxysmal dyspnoea and usually appear between 3 and 6 months of age. The hemiplegic episodes develop before 18 months of age lasting anywhere from minutes to days at a time and involving either side of the body or shifting from one side to the other during the same episode with a period of bilateral weakness when the second side becomes involved.3 Some attacks are characterised by a bilateral involvement, which is apparent from the beginning, and do not follow an hemiplegic episode,3 which determines extreme hypotonia of the whole body with inability to move and a level of consciousness markedly depressed. A characteristic feature of AHC is the disappearance of all abnormalities when the child falls asleep.3 With increasing age, hemiplegic episodes follow a general pattern of initially increasing frequency and duration, followed by a plateau, and finally by a decrease in the number and duration of attacks. Analogously, the paroxysmal manifestations associated with hemiplegia tend to decrease in frequency and intensity with time and usually disappear after 5–7 years. Development before the onset of hemiplegic episodes may be normal or delayed. Epileptic seizures are reported in a variable percentage of sporadic patients.3–5 An association with migraine has also been noticed since the earliest description.1 Indeed, for …

Structure of the SLC7A7 gene and mutational analysis of patients affected by lysinuric protein intolerance.

Lysinuric protein intolerance (LPI) is a rare autosomal recessive defect of cationic amino acid transport caused by mutations in the SLC7A7 gene. We report the genomic structure of the gene and the results of the mutational analysis in Italian, Tunisian, and Japanese patients. The SLC7A7 gene consists of 10 exons; sequences of all of the exon-intron boundaries are reported here. All of the mutant alleles were characterized and eight novel mutations were detected, including two missense mutations, 242A-->C (M1L) and 1399C-->A (S386R); a nonsense mutation 967G-->A (W242X); two splice mutations IVS3 +1G-->A and IVS6 +1G-->T; a single-base insertion, 786insT; and two 4-bp deletions, 455delCTCT and 1425delTTCT. In addition, a previously reported mutation, 1625insATCA, was found in one patient. It is noteworthy that 242A-->C causes the change of Met1 to Leu, a rare mutational event previously found in a few inherited conditions. We failed to establish a genotype/phenotype correlation. In fact, both intrafamilial and interfamilial phenotypic variability were observed in homozygotes for the same mutation. The DNA-based tests are now easily accessible for molecular diagnosis, genetic counseling, and prenatal diagnosis of LPI.