Maria Pia Sperandeo

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.

Lysinuric Protein Intolerance: Reviewing Concepts on a Multisystem Disease

Lysinuric protein intolerance (LPI) is an inherited aminoaciduria caused by defective cationic amino acid transport at the basolateral membrane of epithelial cells in intestine and kidney. LPI is caused by mutations in the SLC7A7 gene, which encodes the y+LAT‐1 protein, the catalytic light chain subunit of a complex belonging to the heterodimeric amino acid transporter family. LPI was initially described in Finland, but has worldwide distribution. Typically, symptoms begin after weaning with refusal of feeding, vomiting, and consequent failure to thrive. Hepatosplenomegaly, hematological anomalies, neurological involvement, including hyperammonemic coma are recurrent clinical features. Two major complications, pulmonary alveolar proteinosis and renal disease are increasingly observed in LPI patients. There is extreme variability in the clinical presentation even within individual families, frequently leading to misdiagnosis or delayed diagnosis. This condition is diagnosed by urine amino acids, showing markedly elevated excretion of lysine and other dibasic amino acids despite low plasma levels of lysine, ornithine, and arginine. The biochemical diagnosis can be uncertain, requiring confirmation by DNA testing. So far, approximately 50 different mutations have been identified in the SLC7A7 gene in a group of 142 patients from 110 independent families. No genotype–phenotype correlation could be established. Therapy requires a low protein diet, low‐dose citrulline supplementation, nitrogen‐scavenging compounds to prevent hyperammonemia, lysine, and carnitine supplements. Supportive therapy is available for most complications with bronchoalveolar lavage being necessary for alveolar proteinosis.

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.

Potential Celiac Patients: A Model of Celiac Disease Pathogenesis

Background and Aim Potential celiacs have the ‘celiac type’ HLA, positive anti-transglutaminase antibodies but no damage at small intestinal mucosa. Only a minority of them develops mucosal lesion. More than 40 genes were associated to Celiac Disease (CD) but we still do not know how those pathways transform a genetically predisposed individual into an affected person. The aim of the study is to explore the genetic features of Potential CD individuals. Methods 127 ‘potential’ CD patients entered the study because of positive anti-tissue transglutaminase and no mucosal lesions; about 30% of those followed for four years become frankly celiac. They were genotyped for 13 polymorphisms of ‘candidate genes’ and compared to controls and celiacs. Moreover, 60 biopsy specimens were used for expression studies. Results Potential CD bear a lighter HLA-related risk, compared to celiac (χ2 = 48.42; p value = 1×10−8). They share most of the polymorphisms of the celiacs, but the frequency of c-REL* G allele was suggestive for a difference compared to celiac (χ2 = 5.42; p value = 0.02). One marker of the KIAA1109/IL-2/IL-21 candidate region differentiated potentials from celiac (rs4374642: χ2 = 7.17, p value = 0.01). The expression of IL-21 was completely suppressed in potentials compared to celiacs (p value = 0.02) and to controls (p value = 0.02), in contrast IL-2, KIAA1109 and c-REL expression were over-expressed. Conclusions Potential CD show genetic features slightly different from celiacs. Genetic and expression markers help to differentiate this condition. Potential CD is a precious biological model of the pathways leading to the small intestinal mucosal damage in genetically predisposed individuals.

Relaxation of insulin-like growth factor 2 imprinting and discordant methylation at KvDMR1 in two first cousins affected by Beckwith-Wiedemann and Klippel-Trenaunay-Weber syndromes.

Beckwith-Wiedeman syndrome (BWS) and Klippel-Trenaunay-Weber syndrome (KTWS) are different human disorders characterized, among other features, by tissue overgrowth. Deregulation of one or more imprinted genes located at chromosome 11p15.5, of which insulin-like growth factor 2 (IGF2) is the most likely candidate, is believed to cause BWS, whereas the etiology of KTWS is completely obscure. We report a case of BWS and a case of KTWS in a single family. The probands, sons of two sisters, showed relaxation of the maternal IGF2 imprinting, although they inherited different 11p15.5 alleles from their mothers and did not show any chromosome rearrangement. The patient with BWS also displayed hypomethylation at KvDMR1, a maternally methylated CpG island within an intron of the KvLQT1 gene. The unaffected brother of the BWS proband shared the same maternal and paternal 11p15.5 haplotype with his brother, but the KvDMR1 locus was normally methylated. Methylation of the H19 gene was normal in both the BWS and KTWS probands. Linkage between the insulin-like growth factor 2 receptor (IGF2R) gene and the tissue overgrowth was also excluded. These results raise the possibility that a defective modifier or regulatory gene unlinked to 11p15.5 caused a spectrum of epigenetic alterations in the germ line or early development of both cousins, ranging from the relaxation of IGF2 imprinting in the KTWS proband to disruption of both the imprinted expression of IGF2 and the imprinted methylation of KvDMR1 in the BWS proband. Analysis of these data also indicates that loss of IGF2 imprinting is not necessarily linked to alteration of methylation at the KvDMR1 or H19 loci and supports the notion that IGF2 overexpression is involved in the etiology of the tissue hypertrophy observed in different overgrowth disorders, including KTWS.

A y(+)LAT-1 mutant protein interferes with y(+)LAT-2 activity: implications for the molecular pathogenesis of lysinuric protein intolerance.

Lysinuric protein intolerance (LPI) is an inherited aminoaciduria caused by defective cationic amino acid (CAA) transport at the basolateral membrane of epithelial cells in the intestine and kidney. The SLC7A7 gene, mutated in LPI, encodes the y+LAT-1 protein, which is the light subunit of the heterodimeric CAA transporter in which 4F2hc is the heavy chain subunit. Co-expression of 4F2hc and y+LAT-1 induces the y+L activity. This activity is also exerted by another complex composed of 4F2hc and y+LAT-2, the latter encoded by the SLC7A6 gene and more ubiquitously expressed than SLC7A7. On the basis of both the pattern of expression and the transport activity, y+LAT-2 might compensate for CAA transport when y+LAT-1 is defective. By expression in Xenopus laevis oocytes and mammalian cells, we functionally analysed two SLC7A7 mutants, E36del and F152L, respectively, the former displaying a partial dominant-negative effect. The results of the present study provide further insight into the molecular pathogenesis of LPI: a putative multiheteromeric structure of both [4F2hc/y+LAT-1] and [4F2hc/y+LAT-2], and the interference between y+LAT-1 and y+LAT-2 proteins. This interference can explain why the compensatory mechanism, that is, an increased expression of SLC7A6 as seen in lymphoblasts from LPI patients, may not be sufficient to restore the y+L system activity.

Improving the estimation of celiac disease sibling risk by non-HLA genes.

Celiac Disease (CD) is a polygenic trait, and HLA genes explain less than half of the genetic variation. Through large GWAs more than 40 associated non-HLA genes were identified, but they give a small contribution to the heritability of the disease. The aim of this study is to improve the estimate of the CD risk in siblings, by adding to HLA a small set of non-HLA genes. One-hundred fifty-seven Italian families with a confirmed CD case and at least one other sib and both parents were recruited. Among 249 sibs, 29 developed CD in a 6 year follow-up period. All individuals were typed for HLA and 10 SNPs in non-HLA genes: CCR1/CCR3 (rs6441961), IL12A/SCHIP1 and IL12A (rs17810546 and rs9811792), TAGAP (rs1738074), RGS1 (rs2816316), LPP (rs1464510), OLIG3 (rs2327832), REL (rs842647), IL2/IL21 (rs6822844), SH2B3 (rs3184504). Three associated SNPs (in LPP, REL, and RGS1 genes) were identified through the Transmission Disequilibrium Test and a Bayesian approach was used to assign a score (BS) to each detected HLA+SNPs genotype combination. We then classified CD sibs as at low or at high risk if their BS was respectively < or ≥ median BS value within each HLA risk group. A larger number (72%) of CD sibs showed a BS ≥ the median value and had a more than two fold higher OR than CD sibs with a BS value < the median (O.R = 2.53, p = 0.047). Our HLA+SNPs genotype classification, showed both a higher predictive negative value (95% vs 91%) and diagnostic sensitivity (79% vs 45%) than the HLA only. In conclusion, the estimate of the CD risk by HLA+SNPs approach, even if not applicable to prevention, could be a precious tool to improve the prediction of the disease in a cohort of first degree relatives, particularly in the low HLA risk groups.

Gene expression profile of peripheral blood monocytes: a step towards the molecular diagnosis of celiac disease?

Aim Celiac disease (CD) is a multifactorial autoimmune disease induced by ingestion of gluten in genetically predisposed individuals. Despite technological progress, the diagnosis of CD is still based on duodenal biopsy as it was 50 years ago. In this study we analysed the expression of CD-associated genes in small bowel biopsies of patients and controls in order to explore the multivariate pathway of the expression profile of CD patients. Then, using multivariant discriminant analysis, we evaluated whether the expression profiles of these genes in peripheral blood monocytes (PBMs) differed between patients and controls. Participants Thirty-seven patients with active and 11 with treated CD, 40 healthy controls and 9 disease controls (Crohn’s disease patients) were enrolled. Results Several genes were differentially expressed in CD patients versus controls, but the analysis of each single gene did not provided a comprehensive picture. A multivariate discriminant analysis showed that the expression of 5 genes in intestinal mucosa accounted for 93% of the difference between CD patients and controls. We then applied the same approach to PBMs, on a training set of 20 samples. The discriminant equation obtained was validated on a testing cohort of 10 additional cases and controls, and we obtained a correct classification of all CD cases and of 91% of the control samples. We applied this equation to treated CD patients and to disease controls and obtained a discrimination of 100%. Conclusions The combined expression of 4 genes allows one to discriminate between CD patients and controls, and between CD patients on a gluten-free diet and disease controls. Our results contribute to the understanding of the complex interactions among CD-associated genes, and they may represent a starting point for the development of a molecular diagnosis of celiac disease.

Feasibility of prenatal diagnosis of lysinuric protein intolerance by linkage analysis: a case report

Lysinuric protein intolerance (LPI) is a rare autosomal recessive defect of cationic amino acid transport (CAA), relatively common in Finland and Italy. After weaning, LPI patients present poor feeding, vomiting and failure to thrive. A severe pulmonary complication and episodes of metabolic imbalance may lead to death. Prenatal diagnosis has not been available due to lack of either biochemical or molecular markers to be used in the fetal period. The LPI locus has recently been assigned to chromosome 14q12, very close to the T‐cell receptor alpha‐chain (TCRA) locus. We carried out a prenatal diagnosis for LPI by linkage analysis in one LPI Italian family after CVS. For the haplotype analysis 11 DNA markers from the LPI critical region were used (D14S742, D14S50, D14S283, five TCRA intragenic polymorphic sites, D14S990, MYH7 and D14S80). It was concluded that the haplotype analysis indicated that the fetus was healthy as he had inherited the two wild alleles of the LPI locus. After birth, the clearances of CAA were measured and found to be in the normal range, thus confirming the result of the prenatal diagnosis. The prenatal diagnosis of LPI can now be offered to families affected by LPI. Copyright

How many loci for the His475Tyr polymorphism of the glutamate carboxypeptidase II gene

Recently, a study on His475Tyr (C1561T) polymorphism of the glutamate carboxypeptidase II (GCPII) gene, encoding for the folylpoly-g-glutamate carboxypeptidase (FGCP) enzyme, has appeared in this Journal [Vieira et al., 2002]. By using primers previously described [Devlin et al., 2000], the authors reported the frequencies of the His475Tyr variant and of a (TAT)2/3 repeat sequence, located 77 bp downstream of it. A complete linkage disequilibrium was also observed between the 475Tyr substitution and the (TAT)3 repeat. In a previous report, a different couple of primers (Table I) was designed to investigate the His475Tyr polymorphism as a determinant of plasma folate or total homocysteine levels in the Framingham Offspring Study [Vargas-Martinez et al., 2002]. The results of this study exclude the proposed association among His475Tyr polymorphism, increased plasma homocysteine concentrations, and lower plasma folate levels. The GCPII gene (GenBank accession no. AF007544) was assigned to chromosome 11p11.2 [Maraj et al., 1998; O’Keefe et al., 1998]. We have noticed that another sequence, located on chromosome 2, is present in the H. sapiens genomic contig sequences database when the GCPII cDNA sequence (FGCP, GenBank accession no. AF176574) is used as a query. Similarly, when the AF007544 sequence is used as a query for a BLAST search in the same database, two nearly identical contigs are found on chromosome 11 (GenBank accession no. AP002369) and chromosome 2 (GenBank accession no. AC074003), respectively (Table II). Both sequences include exon 13 with the His475Tyr polymorphism and a potential (TAT)2/3 repeat located 77 bp downstream of the site of the His475Tyr variant. Interestingly, either couples of primers, used for the His475Tyr genotyping, may amplify a DNA fragment with the same size and oligonucleotidic sequence by annealing to both sequences. The amplified DNA fragment also contains the (TAT) tandem sequence but the reverse primer used by Vargas-Martinez et al. [2002] does not allow the allele discrimination of this repeat. Taking into account the evidence of two copies of a GCPII-like gene, we are concerned about the reliability of genetic studies carried out so far. By pooling all the epidemiological data produced so far on the His475Tyr variant (2,329 individuals), we found that the observed frequencies of the three genotypes are in agreement with the Hardy–Weinberg law (CHISQUARE1⁄4 1.414, P1⁄40.493, df1⁄42). In addition, the