NF1 gene mutations represent the major molecular event underlying neurofibromatosis-Noonan syndrome.

Neurofibromatosis type 1 (NF1) demonstrates phenotypic overlap with Noonan syndrome (NS) in some patients, which results in the so-called neurofibromatosis-Noonan syndrome (NFNS). From a genetic point of view, NFNS is a poorly understood condition, and controversy remains as to whether it represents a variable manifestation of either NF1 or NS or is a distinct clinical entity. To answer this question, we screened a cohort with clinically well-characterized NFNS for mutations in the entire coding sequence of the NF1 and PTPN11 genes. Heterozygous NF1 defects were identified in 16 of the 17 unrelated subjects included in the study, which provides evidence that mutations in NF1 represent the major molecular event underlying this condition. Lesions included nonsense mutations, out-of-frame deletions, missense changes, small inframe deletions, and one large multiexon deletion. Remarkably, a high prevalence of inframe defects affecting exons 24 and 25, which encode a portion of the GAP-related domain of the protein, was observed. On the other hand, no defect in PTPN11 was observed, and no lesion affecting exons 11-27 of the NF1 gene was identified in 100 PTPN11 mutation-negative subjects with NS, which provides further evidence that NFNS and NS are genetically distinct disorders. These results support the view that NFNS represents a variant of NF1 and is caused by mutations of the NF1 gene, some of which have been demonstrated to cause classic NF1 in other individuals.

Familial recurrence of nonsyndromic congenital heart defects in first degree relatives of patients with deletion 22q11.2.

The majority of nonsyndromic congenital heart defects (CHDs) are considered to follow a multifactorial model of inheritance. Multiple family members affected by CHD can occasionally be detected, and the involvement of several genetic loci interacting with environmental factors is suspected to be implicated. The DiGeorge/velo-cardio-facial syndrome related to microdeletion 22q11.2 (del22) is a genetic condition associated with CHD in most of the cases. We report here on five pedigrees of patients with del22, showing occurrence of nonsyndromic CHD in a first-degree relative of the proband case. Familial aggregation of syndromic and nonsyndromic CHD as observed in our series is to be considered as an unusual pattern of recurrence. The interaction between several different genes and environmental factors, a familial susceptibility predisposing to a specific cardiac malformation, or chance association can all be hypothesized searching an explanation for these particular observations.

ZFPM2/FOG2 and HEY2 genes analysis in nonsyndromic tricuspid atresia.

Tricuspid atresia (TriAt), the third most common cyanotic congenital heart defect (CHD), consists of complete lack of tricuspid valve formation, with no connection between the right atrium and the right ventricle. To date, the genetic mechanism responsible of TriAt is still obscure. However, animal models have suggested a role of cardiogenic Zfpm2/Fog2 and Hey2 genes in the pathogenesis of TriAt. Therefore, we screened 40 individuals affected by nonsyndromic TriAt for ZFPM2/FOG2 and HEY2 gene mutations. No pathogenetic mutation has been identified, thus failing to demonstrate a major role of ZFPM2/FOG2 and HEY2 genes in the pathogenesis of human TriAt.

Mutations of the Nogo-66 receptor (RTN4R) gene in schizophrenia.

Schizophrenia (SCZD) or schizoaffective disorders are quite common features in patients with DiGeorge/velo-cardio-facial syndrome (DGS/VCFS) as a result of chromosome 22q11.2 aploinsufficiency. We evaluated the Nogo-66 receptor gene (RTN4R), which maps within the DGS/VCFS critical region, as a potential candidate for schizophrenia susceptibility. RTN4R encodes for a functional cell surface receptor, a glycosylphosphatidylinositol (GPI)-linked protein, with multiple leucine-rich repeats (LRR), which is implicated in axonal growth inhibition. One hundred and twenty unrelated Italian schizophrenic patients were screened for mutations in the RTN4R gene using denaturing high performance liquid chromatography (DHPLC). Three mutant alleles were detected, including two missense changes (c.355C>T; R119W and c.587G>A; R196H), and one synonymous codon variant (c.54G>A; L18L). The two schizophrenic patients with the missense changes were strongly resistant to the neuroleptic treatment at any dosage. Both missense changes were absent in 300 control subjects. Molecular modeling revealed that both changes lead to putative structural alterations of the native protein.

A novel PTPN11 gene mutation bridges Noonan syndrome, multiple lentigines/LEOPARD syndrome and Noonan-like/multiple giant cell lesion syndrome.

Noonan (NS) and multiple lentigines/LEOPARD syndromes (LS) have proved to be associated with distinct PTPN11 mutations. Noonan-like/multiple giant cell lesion syndrome (NLS) is a rare disease, characterised by short stature, facial dysmorphisms, congenital heart defect (CHD) and central giant cell lesions. PTPN11 gene mutations have been reported in a single NLS family and two sporadic patients. Here we report a patient with a complex phenotype progressing throughout the years from NS at birth towards LS and NLS. PTPN11 gene analysis disclosed a novel missense mutation (Ala461Thr) in exon 12, affecting the consensus sequence of the SHP2-active site. This observation joins together NS and LS to NLS into a unique genetic defect, broadening the clinical and molecular spectrum of PTPN11-related disorders.

LEOPARD syndrome: a new polyaneurysm association and an update on the molecular genetics of the disease.

LEOPARD syndrome, one of many cardiocutaneous syndromes, is an acronym for some of the obvious manifestations of the disease, such as lentigines or ocular hypertelorism. The synonymous name progressive cardiomyopathic lentiginosis better indicates the morbid cardiac features that patients with the syndrome have. A patient with LEOPARD syndrome is presented. He had recurrent upper extremity aneurysms requiring multiple operations and finally PTFE reinforced venous grafts to prevent further aneurysmal degeneration. He has multiple other peripheral aneurysms, thus far asymptomatic. His diagnosis of LEOPARD syndrome was confirmed on a genetic basis. Review of the literature reveals no previous reports of severe aneurysmal disease in these patients.

Familial aggregation of genetically heterogeneous hypertrophic cardiomyopathy: a boy with LEOPARD syndrome due to PTPN11 mutation and his nonsyndromic father lacking PTPN11 mutations.

BACKGROUND: Nonsyndromic hypertrophic cardiomyopathy (HCM) is a primary cardiac disease transmitted as an autosomal dominant trait. Multiple chromosomal loci have been found to be involved in the etiology of this defect. LEOPARD syndrome is a genetic condition characteristically associated with HCM. Additional features of the syndrome include multiple lentigines, facial anomalies, sensorineural deafness, and growth retardation. Mutations in PTPN11, a gene encoding the protein tyrosine phosphatase SHP-2 located at chromosome 12q24, have been identified in patients with LEOPARD syndrome. CASES: We report here on a patient with HCM presenting with classic clinical features of LEOPARD syndrome, whose father also has HCM, but lacks phenotypic anomalies of the syndrome. Molecular analysis searching for PTPN11 mutations was performed in this family. A missense mutation (836A-->G; Tyr279Cys) in exon 7 of PTPN11 gene was identified in the patient with LEOPARD syndrome, whereas no mutation in PTPN11 gene was detected in the father or in additional family members. CONCLUSIONS: Aggregation of syndromic and nonsyndromic HCM in the same family is an unusual pattern of recurrence. Although genetic heterogeneity of LEOPARD and nonsyndromic HCM is not disputed, the existence of peculiar interactions linking genes causing nonsyndromic HCM and HCM in LEOPARD syndrome can be hypothesized. Different genes can work together, and a more severe cardiac phenotype can be due to additive effects. The involvement of familial susceptibility to specific cardiac malformations based on the presence of common predisposing factors can also be considered. Further molecular studies may shed light on these observations.

Mutations of ZFPM2/FOG2 gene in sporadic cases of tetralogy of Fallot.

Two out of 47 patients with sporadic tetralogy of Fallot (TOF), the most common cyanotic conotruncal heart defect (CTD), showed heterozygous missense mutations of the ZFPM2/FOG2 gene. Knockout mice carrying mutations in the ZFPM2/FOG2 gene have similarly been found to exhibit TOF. While both mutant ZFPM2/FOG2 proteins, E30G (c.88A>G) and S657G (c.1968A>G), retain the ability to bind the partner protein GATA4 and repress GATA4 mediated gene activation, the S657G, but not the E30G, mutation is subtly impaired in this function. ZFPM2/FOG2 gene mutations may contribute to some sporadic cases of TOF.

Analysis of intracellular distribution and apoptosis involvement of the Ufd1l gene product by over-expression studies.

UFD1L is the human homologue of the yeast ubiquitin fusion degradation 1 (Ufd1) gene and maps on chromosome 22q11.2 in the typically deleted region (TDR) for DiGeorge/velocardiofacial syndromes (DGS/VCFS). In yeast, Ufd1 protein is involved in a degradation pathway for ubiquitin fused products (UFD pathway). Several studies have demonstrated that Ufd1 is a component of the Cdc48-Ufd1-Npl4 multiprotein complex which is active in the recognition of several polyubiquitin-tagged proteins and facilitates their presentation to the 26S proteasome for protein degradation or even more specific processing. The multiprotein complex Cdc48-Ufd-Npl4 is also active in mammalian cells. The biochemical role of UFD1L protein in human cells is unknown, even though the interaction between UFD1L and NPL4 proteins has been maintained. In order to clarify this issue, we examined the intracellular distribution of the protein in different mammalian cells and studied its involvement in the Fas and ceramide factors-mediated apoptotic pathways. We established that in mammalian cells, Ufd1l is localized around the nucleus and that it does not interfere with Fas-and ceramide-mediated apoptosis.

DiGeorge subtypes of nonsyndromic conotruncal defects: evidence against a major role of TBX1 gene.

The role of the 22q11 region genes, and among them TBX1, in nonsyndromic conotruncal defects (CTDs) is still unclear. Mice hemizygous at the Tbx1 locus show a remarkable incidence of heart outflow tract anomalies, of the same type commonly found in DiGeorge/Velo-cardio-facial syndrome (DGS/VCFS). Mutation analysis of the TBX1 gene in isolated, nonsyndromic CTDs has not demonstrated any functional pathogenetic variation so far. We screened the TBX1 gene in 41 patients affected by nonsyndromic CTDs of the DGS/VCFS subtype, principally "atypical" tetralogy of Fallot. Besides a few polymorphisms, we did not find any pathogenetic variation. These results do not support a major role of the TBX1 gene as responsible for human nonsyndromic CTDs.

Association study between CAG trinucleotide repeats in the PCQAP gene (PC2 glutamine/Q-rich-associated protein) and schizophrenia.

Schizophrenia or schizoaffective disorders are quite common features in patients with DiGeorge/velocardiofacial syndrome (DGS/VCFS) as a result of hemizygosity of chromosome 22q11.2. We evaluated the PCQAP gene, which maps within the DGS/VCFS interval, as a potential candidate for schizophrenia susceptibility. PCQAP encodes for a subunit of the large multiprotein complex PC2, which exhibits a coactivator function in RNA polymerase II mediated transcription. Using a case-control study, we searched association between schizophrenia and the intragenic coding trinucleotide polymorphism. The distribution of the CAG repeat alleles was significantly different between patients and controls with the Mann-Whitney test (z = -2.5694, P = 0.0051; schizophrenics: n = 378, W = 161,002.5, Mean rank = 425.9325; controls: n = 444, W = 177,250.5, Mean rank = 399.2128). This result may indicate a possible involvement of the multiprotein complex PC2 in schizophrenia susceptibility.

Grouping of multiple-lentigines/LEOPARD and Noonan syndromes on the PTPN11 gene.

Multiple-lentigines (ML)/LEOPARD (multiple lentigines, electrocardiographic-conduction abnormalities, ocular hypertelorism, pulmonary stenosis, abnormal genitalia, retardation of growth, and sensorineural deafness) syndrome is an autosomal dominant condition--characterized by lentigines and café au lait spots, facial anomalies, cardiac defects--that shares several clinical features with Noonan syndrome (NS). We screened nine patients with ML/LEOPARD syndrome (including a mother-daughter pair) and two children with NS who had multiple café au lait spots, for mutations in the NS gene, PTPN11, and found, in 10 of 11 patients, one of two new missense mutations, in exon 7 or exon 12. Both mutations affect the PTPN11 phosphotyrosine phosphatase domain, which is involved in <30% of the NS PTPN11 mutations. The study demonstrates that ML/LEOPARD syndrome and NS are allelic disorders. The detected mutations suggest that distinct molecular and pathogenetic mechanisms cause the peculiar cutaneous manifestations of the ML/LEOPARD-syndrome subtype of NS.

Functional characterization of the 5' flanking region of human ubiquitin fusion degradation 1 like gene (UFD1L).

UFD1L (Ubiquitin Fusion Degradation 1 Like) gene encodes for a component of a multi-complex involved in the degradation of ubiquitin fusion proteins. The gene maps on chromosome 22q11, in a region commonly deleted in severe congenital disorders such as DiGeorge (DGS) and velo-cardio-facial (VCFS) syndromes. UFD1L is a single copy gene ubiquitously expressed in high levels in the pharyngeal pouches and fourth branchial arch artery during development. To understand the regulation of UFD1L expression we performed a functional analysis of its 5' regulatory region. 5'-RACE and primer extension analyses revealed the presence of different transcription start sites in adult and fetal tissues. UFD1L 5' flanking region contains a TATA-box motif and is also very GC-rich with a CpG island encompassing exon 1. Transcriptional activity of this region was examined by transfection experiments of promoter-GFP reporter gene constructs in a human epithelial cell line. These experiments revealed the importance of the region between -17 and -463 nt which contains the TATA-box. EMSA assay resulted in the detection of five functional consensus sequences respectively for the transcription complex TFIID and for the transcription factors AP-1 (one site), AP-2 (one) and Sp1 (two).