Gianfranco Sebastio

Molecular subtypes and phenotypic expression of Beckwith-Wiedemann syndrome

Beckwith–Wiedemann Syndrome (BWS) results from mutations or epigenetic events involving imprinted genes at 11p15.5. Most BWS cases are sporadic and uniparental disomy (UPD) or putative imprinting errors predominate in this group. Sporadic cases with putative imprinting defects may be subdivided into (a) those with loss of imprinting (LOI) of IGF2 and H19 hypermethylation and silencing due to a defect in a distal 11p15.5 imprinting control element (IC1) and (b) those with loss of methylation at KvDMR1, LOI of KCNQ1OT1 (LIT1) and variable LOI of IGF2 in whom there is a defect at a more proximal imprinting control element (IC2). We investigated genotype/epigenotype–phenotype correlations in 200 cases with a confirmed molecular genetic diagnosis of BWS (16 with CDKN1C mutations, 116 with imprinting centre 2 defects, 14 with imprinting centre 1 defects and 54 with UPD). Hemihypertrophy was strongly associated with UPD (P<0.0001) and exomphalos was associated with an IC2 defect or CDKN1C mutation but not UPD or IC1 defect (P<0.0001). When comparing birth weight centile, IC1 defect cases were significantly heavier than the patients with CDKN1C mutations or IC2 defect (P=0.018). The risk of neoplasia was significantly higher in UPD and IC1 defect cases than in IC2 defect and CDKN1C mutation cases. Kaplan–Meier analysis revealed a risk of neoplasia for all patients of 9% at age 5 years, but 24% in the UPD subgroup. The risk of Wilms’ tumour in the IC2 defect subgroup appears to be minimal and intensive screening for Wilms’ tumour appears not to be indicated. In UPD patients, UPD extending to WT1 was associated with renal neoplasia (P=0.054). These findings demonstrate that BWS represents a spectrum of disorders. Identification of the molecular subtype allows more accurate prognostic predictions and enhances the management and surveillance of BWS children such that screening for Wilms’ tumour and hepatoblastoma can be focused on those at highest risk.

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.

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.

The molecular bases of cystinuria and lysinuric protein intolerance

Cystinuria and lysinuric protein intolerance are inherited aminoacidurias caused by defective amino-acid transport activities linked to a family of heteromeric amino-acid transporters (HATs). HATs comprise two subunits: co-expression of subunits 4F2hc and y(+)LAT-1 induces the efflux of dibasic amino acids from cells, whereas co-expression of subunits rBAT and b(o,+)AT induces the renal reabsorption and intestinal absorption of cystine and dibasic amino acids at the brush border of epithelial cells. Recently, the role of b(o,+)AT (SLC7A9) in cystinuria (non Type I) and the role of y(+)LAT-1 (SLC7A7) in lysinuric protein intolerance have been demonstrated.

Molecular analysis of aldolase B genes in hereditary fructose intolerance

The molecular basis of hereditary fructose intolerance (HFI) was studied in 50 subjects (41 pedigrees, 82 apparently independent mutant alleles of aldolase B) by direct analysis of aldolase B genes amplified by means of the polymerase chain reaction. The mutation A149P (ala 149----pro) was found in 67% of alleles but was significantly more common in patients from northern than from southern Europe. Two other point mutations of aldolase B were identified. A174D (C----A; ala 174----asp) was found in subjects from Italy, Switzerland, and Yugoslavia (overall frequency 16%) but not in those from the United Kingdom, France, or the United States. L288 delta C carried a single base-pair deletion causing frameshift at codon 288 and was restricted to Sicilian subjects. By testing for these mutations in amplified DNA with a limited panel of allele-specific oligonucleotides, more than 95% of HFI patients will be susceptible to genetic diagnosis.

Mowat–Wilson syndrome: Facial phenotype changing with age: Study of 19 Italian patients and review of the literature

Mowat–Wilson syndrome (MWS; OMIM #235730) is a genetic condition caused by heterozygous mutations or deletions of the ZEB2 gene, and characterized by typical face, moderate‐to‐severe mental retardation, epilepsy, Hirschsprung disease, and multiple congenital anomalies, including genital anomalies (particularly hypospadias in males), congenital heart defects, agenesis of the corpus callosum, and eye defects. Since the first delineation by Mowat et al. [Mowat et al. ( 1998 ); J Med Genet 35:617–623], ∼179 patients with ZEB2 mutations, deletions or cytogenetic abnormalities have been reported primarily from Europe, Australia and the United States. Genetic defects include chromosome 2q21–q23 microdeletions (or different chromosome rearrangements) in few patients, and ZEB2 mutations in most. We report on clinical and genetic data from 19 Italian patients, diagnosed within the last 5 years, including six previously published, and compare them with patients already reported. The main purpose of this review is to underline a highly consistent phenotype and to highlight the phenotypic evolution occurring with age, particularly of the facial characteristics. The prevalence of MWS is likely to be underestimated. Knowledge of the phenotypic spectrum of MWS and of its changing phenotype with age can improve the detection rate of this condition.

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.

The biosynthesis of intestinal sucrase-isomaltase in human embryo is most likely controlled at the level of transcription

Although sucrase-isomaltase appears in the small intestine at quite different stages of development in man as compared with most mammals, we find that in human embryo also the appearance of sucrase-isomaltase mRNA closely parallels that of sucrase and isomaltase activities, as we have previously found to be the case in baby rabbits. Also, in the proximal-distal gradient of human embryonic intestine (proximal small intestine greater than distal small intestine greater than colon) the levels of these enzyme activities and those of the corresponding mRNA correlate closely. Finally, glucocorticosteroid treatment of a human colon carcinoma cell line (Caco-2) in vitro or of baby rabbits in vivo leads to a parallel increase of both sucrase and isomaltase activities and of sucrase-isomaltase mRNA. We conclude that in man also, in spite of the different timing in development, the biosynthesis of sucrase-isomaltase is most likely to be controlled at the level of transcription or perhaps of the mRNA stability.

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.