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], approximately 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.

Arginine transport through system y(+)L in cultured human fibroblasts: normal phenotype of cells from LPI subjects.

In lysinuric protein intolerance (LPI), impaired transport of cationic amino acids in kidney and intestine is due to mutations of the SLC7A7 gene. To assess the functional consequences of the LPI defect in nonepithelial cells, we have characterized cationic amino acid (CAA) transport in human fibroblasts obtained from LPI patients and a normal subject. In both cell types the bidirectional fluxes of arginine are due to the additive contributions of two Na(+)-independent, transstimulated transport systems. One of these mechanisms, inhibited by N-ethylmaleimide (NEM) and sensitive to the membrane potential, is identifiable with system y(+). The NEM- and potential-insensitive component, suppressed by L-leucine only in the presence of Na(+), is mostly due to the activity of system y(+)L. The inward and outward activities of the two systems are comparable in control and LPI fibroblasts. Both cell types express SLC7A1 (CAT1) and SLC7A2 (CAT2B and CAT2A) as well as SLC7A6 (y+LAT2) and SLC7A7 (y+LAT1). We conclude that LPI fibroblasts exhibit normal CAA transport through system y(+)L, probably referable to the activity of SLC7A6/y+LAT2.

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.

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.

Smith-Lemli-Opitz syndrome: evidence of T93M as a common mutation of delta7-sterol reductase in Italy and report of three novel mutations.

The Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder of cholesterol biosynthesis characterised by facial dysmorphisms, mental retardation and multiple congenital anomalies. SLOS is caused by mutations of the human Delta7-sterol reductase (DHCR7) gene and, so far, 19 different mutations have been described. Among these, mutations impairing the activity of the C-terminus appear to be the most severe. Here we report the mutational analysis of the DHCR7 gene in nine Italian SLOS patients. The T93M mutation, previously reported in one patient, results the most frequent one (7/18 alleles) in our survey. Furthermore, we identified three novel mutations, two missense mutations (N407Y and E448K), and a 33 bp deletion spanning part of exon 5 and the donor splice site of intron 5.

SLC7A8, a gene mapping within the lysinuric protein intolerance critical region, encodes a new member of the glycoprotein-associated amino acid transporter family.

Using a bioinformatic approach, we have identified a new transcript, SLC7A8, mapping to 14q11.2, within the lysinuric protein intolerance (LPI) critical region. This gene is highly expressed in skeletal muscle, intestine, kidney, and placenta and encodes a predicted protein of 535 amino acids, homologous to the amino acid permease CD98 light chain and cationic amino acid transporters. RNA in situ hybridization data on mouse embryos confirm the expression in kidney and intestine and, interestingly, reveal that SLC7A8 is also highly expressed in eye, in retinal pigmented epithelium, and in tooth buds at day 16.5 of gestation. Mutational analysis excluded any direct involvement of the SLC7A8 gene product in LPI disease. The homology data and the expression pattern are in agreement with the hypothesis that SLC7A8 represents a novel light chain interacting with the 4F2 heavy chain in the multimeric complex mediating neutral and/or cationic amino acid transport and cystine/glutamate exchange.

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.

Mosaicism for the full mutation and a microdeletion involving the CGG repeat and flanking sequences in the FMR1 gene in eight fragile X patients.

The molecular mechanism of the fragile X syndrome is based on the expansion of an unstable CGG repeat in the 5' untranslated region of the FMR1 gene in most patients. This expansion is associated with an abnormal DNA methylation leading to the absence of production of FMR1 protein (FMRP). Such expansion apparently predisposes the repeat and flanking regions to further instability that may lead to mosaic conditions with a full mutation and a premutation or, rarely, with normal or reduced alleles that can sometimes be transcriptionally active. In this study we describe eight unrelated fragile X patients who are mosaic for both a full mutation and an allele of normal (four cases) or reduced size (four cases). Sequencing analysis of the deletion breakpoints in 6 patients demonstrated an internal deletion confined to the CGG repeat in four of them, which represents the most likely explanation for the regression of the full mutation to a normal sized allele. In two patients with a reduced allele, the deletion encompassed the entire CGG repeat and part of the flanking regions. Analysis of FMRP by Western blot was performed in one of the mosaics with a normal sized allele and in three of those with a reduced allele. In the first patient's lymphocytes FMRP was detected, whereas in the three other patients the deletion is likely to impair transcription as no FMRP was present in their lymphocytes.

Cystathionine beta-synthase mutations in homocystinuria.

The major cause of homocystinuria is mutation of the gene encoding the enzyme cystathionine beta-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.

SLC7A7, encoding a putative permease-related protein, is mutated in patients with lysinuric protein intolerance.

Lysinuric protein intolerance (LPI, MIM 222700) is an autosomal recessive multisystem disorder found mainly in Finland and Italy. On a normal diet, LPI patients present poor feeding, vomiting, diarrhoea, episodes of hyperammoniaemic coma and failure to thrive. Hepatosplenomegaly, osteoporosis and a life-threatening pulmonary involvement (alveolar proteinosis) are also seen. LPI is caused by defective cationic amino acid (CAA) transport at the basolateral membrane of epithelial cells in kidney and intestine. Metabolic derangement is characterized by increased renal excretion of CAA, reduced CAA absorption from intestine and orotic aciduria. The gene causing LPI was assigned using linkage analysis to chromosome 14q11.2 near the T-cell receptor alpha/delta chains locus, and a critical region has been defined. We have identified two new transcripts (SLC7A8 and SLC7A7) homologous to amino acid transporters, highly expressed in kidney and mapping in the LPI critical region. Mutational analysis of both transcripts revealed that SLC7A7 (for solute carrier family 7, member 7) is mutated in LPI. In five Italian patients, we found either an insertion or deletion in the coding sequence, which provides evidence of a causative role of SLC7A7 in LPI. Furthermore, we detected a splice acceptor change resulting in a frameshift and premature translation termination in four unrelated Finnish patients. This mutation may represent the founder LPI allele in Finland.

The C677T mutation of the 5,10-methylenetetrahydrofolate reductase gene is a moderate risk factor for spina bifida in Italy.

OBJECTIVE: To estimate the risk for spina bifida associated with the common mutation C677T of the MTHFR gene in a country with a relatively low prevalence of NTDs. DESIGN: Case-control study. CASES: 203 living patients affected with spina bifida (173 myelomeningocele and 30 lipomeningocele); controls: 583 subjects (306 young adults and 277 unselected newborns) from northern and central-southern Italy. CASES: three spina bifida centres; young adult controls: DNA banks; newborn controls: regional neonatal screening centres. MAIN OUTCOME MEASURES: Prevalence of the C677T genotypes in cases and controls by place of birth; odds ratios for spina bifida and estimated attributable fraction. RESULTS: The prevalence of T/T, T/C, and C/C genotype was 16.6%, 53.7%, and 29.7% in controls and 25.6%, 43.8%, and 30.6% in cases, respectively. We found no differences between type of defect or place of birth. The odds ratio for spina bifida associated with the T/T genotype v C/C plus T/C was 1.73 (95% CI 1.15, 2.59) and the corresponding attributable fraction was 10.8%. No increased risk was found for heterozygous patients (OR=0.79, 95% CI 0.53-1.18). CONCLUSION: This study, as well as the meta-analysis we updated, shows that homozygosity for the MTHFR C677T mutation is a moderate risk factor in Europe, and even in Italy where there is a relatively low prevalence of spina bifida. The estimated attributable fraction associated with this risk factor explains only a small proportion of cases preventable by periconceptional folic acid supplementation. Thus, other genes involved in folate-homocysteine metabolism, their interaction, and the interaction between genetic and environmental factors should be investigated further.

Mild phenotype associated with an interstitial deletion of the long arm of chromosome 1.

We report on a 21 month old child referred to us because of facial dysmorphism and psychomotor retardation. The patient's phenotype was characterised by a wide and receding forehead, broad nasal bridge, redundant retronuchal skin, low set and poorly shaped ears, micrognathia, and small hands and feet. High resolution R and G banding karyotype analysis of peripheral blood lymphocytes showed an interstitial deletion of the long arm of chromosome 1 spanning bands q22 to q24. The cytogenetic results were confirmed by molecular analysis. The phenotype observed in our patient was relatively milder than those reported in other patients with an interstitial deletion of chromosome 1q.

The gene encoding a cationic amino acid transporter (SLC7A4) maps to the region deleted in the velocardiofacial syndrome.

By screening an expressed sequence tag database, we identified a novel human gene, SLC7A4, encoding a solute carrier family 7 [cationic amino acid (CAA) CAT-4 transporter, y+ system] member 4. The SLC7A4 cDNA is 2325 nt long and includes 78, 1911, and 336 nt in the 5' noncoding, coding, and 3'-noncoding regions, respectively. SLC7A4 displays high homology with SLC7A1 and SLC7A2, two previously known CAA transporters. By chromosomal in situ hybridization and YAC identification, SLC7A4 was mapped to 22q11.2, the commonly deleted region of the velocardiofacial syndrome (VCFS, Shprintzen syndrome). In a patient affected by VCFS, deletion of SLC7A4 was demonstrated by chromosomal FISH. By Northern analysis, an abundant transcript was detected in brain, testis, and placenta. Microinjection of SLC7A4 mRNA into Xenopus laevis oocytes demonstrates a significant stimulation of CAA transport.

Clinical aspects of cystathionine beta-synthase deficiency: how wide is the spectrum? The Italian Collaborative Study Group on Homocystinuria.

UNLABELLED: Homocystinuria due to cystathionine beta-synthase (CBS) deficiency is an autosomal recessive disease of sulphur amino acid metabolism. Major clinical manifestations include disorders of the eye, the skeleton, the central nervous system and the vascular system. A wide clinical spectrum of the disease has been reported. We discuss the role of genetic factors (e.g. different mutations of the CBS gene and a variable genetic background) and the importance of environmental factors (e.g. diet, vitamins, perinatal factors and drugs) in explaining the phenotypic variability observed in homocystinuria. CONCLUSION: Homocystinuria represents a good model to explain the clinical differences frequently observed among patients affected by monogenic diseases.

Genetic homogeneity of lysinuric protein intolerance.

Lysinuric protein intolerance (LPI) is an autosomal recessive disorder in which transport of the cationic amino acids lysine, arginine and ornithine is defective at the basolateral membrane of the epithelial cells in the intestine and renal tubules. LPI is unusually common in Finland, but patients have been described on all continents. Linkage analysis in Finnish LPI families recently assigned the LPI gene locus to a 10 cM interval between markers D14S72 and MYH7 on the long arm of chromosome 14. In the present study linkage analysis of LPI families from six different non-Finnish populations strongly suggests genetic homogeneity in LPI. Peak lod scores were obtained at the chromosomal area between D14S72 and MYH7 with the same markers as in the Finnish families. The non-Finnish families showed no linkage disequilibrium except in an Italian family cluster, whereas strong allelic association in the Finnish families implies that LPI in Finland is caused by a founder mutation.

Combined immunodeficiency phenotype associated with inappropriate spontaneous and activation-induced apoptosis.

Programmed death of T cells has been proposed as one of the mechanisms by which HIV induces a decline in the number and functions of T cells in advanced AIDS. In this study we report on a patient affected by a congenital form of combined immunodeficiency presenting as a profound T cell activation deficiency. Subsequently, a gradual loss of T cells occurred, eventually resulting in a classical form of severe combined immunodeficiency (SCID). In this patient a sizeable fraction of apoptotic cells was documented in the first phase of the disease by either propidium iodide staining or DNA fragmentation analysis. The presence of anergic T cells of maternal origin and engrafted in the child was excluded by analysis of DNA polymorphic regions. At 4 years of age the patient died of disseminated interstitial pneumopathy, while still awaiting an HLA-matched bone marrow transplantation. On the occasion of a new pregnancy in the mother, the prenatal immunological evaluation of the female fetus revealed a T B+ SCID phenotype. This is the first observation of a primary immunodeficiency associated with inappropriate apoptosis.

Fragile X founder chromosomes in Italy: a few initial events and possible explanation for their heterogeneity.

A total of 137 fragile X and 235 control chromosomes from various regions of Italy were haplotyped by analyzing two neighbouring marker microsatellites, FRAXAC1 and DXS548. The number of CGG repeats at the 5' end of the FMR1 gene was also assessed in 141 control chromosomes and correlated with their haplotypes. Significant linkage disequilibrium between some "major" haplotypes and fragile X was observed, while other "minor" haplotypes may have originated by subsequent mutation at the marker microsatellite loci and/or recombination between them. Recent evidence suggests that the initial mechanism leading to CGG instability might consist of rare (10 (-6/-7)) CGG repeat slippage events and/or loss of a stabilizing AGG via A-to-C transversion. Also, the apparently high variety of fragile X chromosomes may be partly due to the relatively high mutation rate (10 (-4/-5)) of the microsatellite markers used in haplotyping. Our fragile X sample also showed a higher than expected heterozygosity when compared to the control sample and we suggest that this might be explained by the chance occurrence of the few founding events on different chromosomes, irrespective of their actual frequency in the population. Alternatively, a local mechanism could enhance the microsatellite mutation rate only on fragile X chromosomes, or fragile X mutations might occur more frequently on certain background haplotypes.

Homocysteine response to methionine challenge in four obligate heterozygotes for homocystinuria and relationship with cystathionine beta-synthase mutations.

Fasting and post-methionine load plasma total homocysteine concentrations were investigated in the parents of two homocystinuric patients. Three genetic mutations in the cystathionine beta-synthase gene were found. In the patient of family 1, a frequent Caucasian mutation. T833C, was found on one allele, while the mutation on the other allele has not yet been defined. In the patient of family 2, a mutation C569T, recently described by Sperandeo and colleagues, was found on one allele, while a novel mutation, G346A, was characterized on the other allele. The frequent gene mutation T833C was detected in a heterozygous mother who, surprisingly, exhibited strictly normal fasting and post-methionine load homocysteinaemia. In contrast, in the other family, we found a novel mutation (G346A) in the mother located near Lys 119, the putative binding site of phosphopyridoxal phosphate. This mother exhibited increased fasting and post-methionine load homocysteinaemia. These observations could explain the conflicting results reported for vascular pathologies in parents of homocystinuric patients and direct the search for genetic mutations in these vascular pathologies.