Mutation in vascular endothelial growth factor-C, a ligand for vascular endothelial growth factor receptor-3, is associated with autosomal dominant milroy-like primary lymphedema.

RATIONALE: Mutations in vascular endothelial growth factor (VEGF) receptor-3 (VEGFR3 or FLT4) cause Milroy disease, an autosomal dominant condition that presents with congenital lymphedema. Mutations in VEGFR3 are identified in only 70% of patients with classic Milroy disease, suggesting genetic heterogeneity. OBJECTIVE: To investigate the underlying cause in patients with clinical signs resembling Milroy disease in whom sequencing of the coding region of VEGFR3 did not reveal any pathogenic variation. METHODS AND RESULTS: Exome sequencing of 5 such patients was performed, and a novel frameshift variant, c.571_572insTT in VEGFC, a ligand for VEGFR3, was identified in 1 proband. The variant cosegregated with the affected status in the family. An assay to assess the biological function of VEGFC activity in vivo, by expressing human VEGFC in the zebrafish floorplate was established. Forced expression of wild-type human VEGFC in the floorplate of zebrafish embryos leads to excessive sprouting in neighboring vessels. However, when overexpressing the human c.571_572insTT variant in the floorplate, no sprouting of vessels was observed, indicating that the base changes have a marked effect on the activity of VEGFC. CONCLUSIONS: We propose that the mutation in VEGFC is causative for the Milroy disease-like phenotype seen in this family. This is the first time a mutation in one of the ligands of VEGFR3 has been reported to cause primary lymphedema.

Rapid identification of mutations in GJC2 in primary lymphoedema using whole exome sequencing combined with linkage analysis with delineation of the phenotype.

BACKGROUND: Primary lymphoedema describes a chronic, frequently progressive, failure of lymphatic drainage. This disorder is frequently genetic in origin, and a multigenerational family in which eight individuals developed postnatal lymphoedema of all four limbs was ascertained from the joint Lymphoedema/Genetic clinic at St George's Hospital. METHODS: Linkage analysis was used to determine a locus, and exome sequencing was employed to look for causative variants. RESULTS: Linkage analysis revealed cosegregation of a 16.1 Mb haplotype on chromosome 1q42 that contained 173 known or predicted genes. Whole exome sequencing in a single affected individual was undertaken, and the search for the causative variant was focused to within the linkage interval. This approach revealed two novel non-synonymous single nucleotide substitutions within the chromosome 1 locus, in NVL and GJC2. NVL and GJC2 were sequenced in an additional cohort of individuals with a similar phenotype and non-synonymous variants were found in GJC2 in four additional families. CONCLUSION: This report demonstrates the power of exome sequencing efficiently applied to a traditional positional cloning pipeline in disease gene discovery, and suggests that the phenotype produced by GJC2 mutations is predominantly one of 4 limb lymphoedema.

Linkage and sequence analysis indicate that CCBE1 is mutated in recessively inherited generalised lymphatic dysplasia.

Generalised lymphatic dysplasia (GLD) is characterised by extensive peripheral lymphoedema with visceral involvement. In some cases, it presents in utero with hydrops fetalis. Autosomal dominant and recessive inheritance has been reported. A large, non-consanguineous family with three affected siblings with generalised lymphatic dysplasia is presented. One child died aged 5 months, one spontaneously miscarried at 17 weeks gestation, and the third has survived with extensive lymphoedema. All three presented with hydrops fetalis. There are seven other siblings who are clinically unaffected. Linkage analysis produced two loci on chromosome 18, covering 22 Mb and containing 150 genes, one of which is CCBE1. A homozygous cysteine to serine change in CCBE1 has been identified in the proband, in a residue that is conserved across species. High density SNP analysis revealed homozygosity (a region of 900 kb) around the locus for CCBE1 in all three affected cases. This indicates a likely ancestral mutation that is common to both parents; an example of a homozygous mutation representing Identity by Descent (IBD) in this pedigree. Recent studies in zebrafish have shown this gene to be required for lymphangiogenesis and venous sprouting and are therefore supportive of our findings. In view of the conserved nature of the cysteine, the nature of the amino acid change, the occurrence of a homozygous region around the locus, the segregation within the family, and the evidence from zebrafish, we propose that this mutation is causative for the generalised lymphatic dysplasia in this family, and may be of relevance in cases of non-immune hydrops fetalis.

Emberger syndrome-primary lymphedema with myelodysplasia: report of seven new cases.

Four reports have been published on an association between acute myeloid leukaemia (AML) and primary lymphedema, with or without congenital deafness. We report seven new cases, including one extended family, confirming this entity as a genetic syndrome. The lymphedema typically presents in one or both lower limbs, before the hematological abnormalities, with onset between infancy and puberty and frequently affecting the genitalia. The AML is often preceded by pancytopenia or myelodysplasia with a high incidence of monosomy 7 in the bone marrow (five propositi and two relatives). Associated anomalies included hypotelorism, epicanthic folds, long tapering fingers and/or neck webbing (four patients), recurrent cellulitis in the affected limb (four patients), generalized warts (two patients), and congenital, high frequency sensorineural deafness (one patient). Children with lower limb and genital lymphedema should be screened for hematological abnormalities and immunodeficiency.

CD14 C(-260)T promoter polymorphism and prevalence of acute coronary syndromes.

BACKGROUND: Inflammation and infection have been implicated in atherosclerosis and its complications. The CD14 receptor mediates monocyte activation by lipopolysaccharide (LPS) of Gram-negative bacteria. The aim of this study was to assess whether the C(-260)T polymorphism in the promoter of the CD14 receptor gene is associated with a higher prevalence of acute coronary syndromes (ACS) and severity of coronary atherosclerosis. METHODS: We studied 428 patients (mean age: 63+/-10 years, 67% men) consisting of 334 patients with coronary artery disease (CAD) and 94 patients with normal coronary arteriogram. Patients with CAD were subdivided in two groups: (1) no previous history of ACS (n=140; 64+/-9 years; 79% men) and (2) patients with a history of ACS (n=194; 64+/-10 years; 80% men). CD14 genotypes were determined by a Polymerase Chain Reaction (PCR)-Restriction Fragment Length Polymorphism Analysis (RFLP) technique. RESULTS: Patients with a prior ACS had a significantly higher frequency of the T/T genotype than CAD patients without prior ACS (33% vs. 20%; P=0.009), even after multivariate analysis (odd ratio [OR] 1.8 [1.1-3.1]; confidence intervals [CI] 95%; P=0.023). T/T genotype was not significantly different in CAD patients without prior ACS compared to controls (20% vs. 22.3%; P=0.67), and there was no significant association between genotypes, or allele frequencies, and severity of CAD. CONCLUSIONS: The CD14 C(-260)T polymorphism is associated with a history of ACS and it may represent a genetically determined risk factor for the development of ACS and atheromatous plaque vulnerability in angina patients.

Association of the -159C/T polymorphism of the endotoxin receptor (CD14) with carotid artery disease and cardiovascular mortality in dialysis patients.

BACKGROUND: Atherosclerosis is a major problem in end-stage renal disease (ESRD) patients treated by hemodialysis and the prevalence of carotid artery disease is much higher in this group than in the general population. Repeated exposure to cytokine-inducing material, derived from dialysate, may induce a chronic inflammatory state, that could contribute to the atherosclerotic process. Endotoxin is mainly cleared from plasma by the sCD14, the soluble form of the endotoxin receptor CD14. The levels of sCD14 are associated with a polymorphism, -159 C/T, of the CD14 gene. METHODS AND RESULTS: We determined the genotype for the -159 C/T polymorphism in 158 haemodialysis patients and 168 healthy controls. In patients we investigated the association between the CD14 polymorphism and carotid artery disease. With a prospective follow-up study we assessed whether the CD14 polymorphism shows any relationship with cardiovascular mortality. The polymorphic frequency was comparable between patients and controls. In patients, we found a significant difference in the prevalence of carotid artery disease between groups divided by genotype: CC 87.0%, CT 71.7%, TT 48.9% (p = 0.0093). In dialysis patients with hypertension the CC polymorphism was associated with an increased cardiovascular mortality. CONCLUSIONS: These results demonstrate an association between the -159 C/T polymorphism of the CD14 gene and carotid artery disease in dialysis patients. We hypothesize that the low plasma clearance of endotoxin associated with the CC genotype facilitates the atherogenic action of endotoxin-derived cytokines in haemodialysis patients.

Genetic heterogeneity in LEOPARD syndrome: two families with no mutations in PTPN11.

LEOPARD syndrome (lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonary stenosis, abnormal genitalia, retardation of growth, and sensorineural deafness) is an autosomal dominant condition. The main clinical features include multiple lentigines, cardiovascular defects, and facial anomalies, some of which are shared with Noonan syndrome (NS). Recent reports have shown that LEOPARD syndrome can be caused by mutations in PTPN11, the gene in which mutations can produce NS. Here we report the findings of mutation screening and linkage analysis of PTPN11 in three families with LEOPARD syndrome. We identified a novel mutation in one family. The mutation (1529A>C) substitutes proline for glutamine at amino acid 510 (Gln510Pro). No variations in sequence were observed in the other two families, and negative LOD scores excluded linkage to the PTPN11 locus, showing that LEOPARD syndrome is genetically heterogeneous.

Association of interleukin-6 -174G/C promoter polymorphism with hypertension and left ventricular hypertrophy in dialysis patients.

BACKGROUND: Gene polymorphisms of proinflammatory cytokines, such as interleukin-6 (IL-6) and the chemokine receptor CX3CR1, have been found in association with cardiovascular disease in the general population. In dialysis patients, in whom the prevalence of cardiovascular comorbidity is strikingly high, these polymorphisms have not been investigated. METHODS: The -174G/C polymorphism of the IL-6 gene and the chemokine receptor CX3CR1 polymorphisms 249V/I and 280T/M were examined for their association with cardiovascular abnormalities in a cohort of 161 patients with end-stage renal disease (ESRD) treated by hemodialysis. Arterial blood pressure, electrocardiogram (ECG) ischemic changes, and left ventricular mass index (LVMI) were the parameters examined for the association study. The control group was made up of 169 healthy subjects. RESULTS: We found that for both IL-6 and chemokine receptor, genotype frequency and allelic distribution in both ESRD patients and controls were comparable. The genetic association study showed that in the whole group of dialysis patients, individuals with GC + CC genotype for the -174G/C polymorphism had a higher diastolic blood pressure (P = 0.008) and LVMI (P = 0.026) than GG homozygotes. The prevalence of left ventricular hypertrophy (LVH) in the former group was 58.6% vs. 39.2% in the latter (P = 0.02). The same analysis limited to diabetic patients in dialysis, showed that the prevalence of LVH in those with CG + CC genotype was 87.5% vs. 36.3% in those with GG genotype (P = 0.02). In diabetic patients, lower levels of serum albumin was found in the GC + CC genotypic group than in GG subjects; 34.63 +/- 5.18 g/L vs. 41.75 +/- 4.79 g/L (P = 0.003). CONCLUSION: These data demonstrate an association between the IL-6 promoter polymorphism -174G/C and high blood pressure and LVH in hemodialysis patients, especially those with diabetes. The results strengthen the hypothesis that chronic inflammation is a mechanism of cardiovascular damage in dialysis patients and the role played by the IL-6 system in this mechanism.

Polymorphism of renin-angiotensin system genes in dialysis patients--association with cerebrovascular disease.

BACKGROUND: Polymorphisms of genes of the renin-angiotensin system (RAS) have been found in association with cerebrovascular and cardiovascular diseases in the general population. In dialysis patients, RAS gene polymorphisms have been studied in combination and separately and have yielded conflicting results. METHODS: In this study we have analysed, in 160 dialysis patients, the distribution of the following genetic polymorphisms: M235T and T174M of the angiotensinogen gene, A1166C of the angiotensin II type 1 receptor gene and the insertion/deletion (I/D) of the ACE gene. The association of these polymorphisms with cerebrovascular and cardiovascular diseases was also tested. Healthy blood donors and hospital staff (169) were the control group for the distribution of the polymorphisms. RESULTS: The distribution of the polymorphisms in dialysis patients as a whole did not differ significantly from that of healthy controls. However, for patients with severe cerebrovascular disease, 70% carried the D allele compared with 52% of patients without cerebrovascular disease (P=0.035). We also found that the degree of carotid artery stenosis was significantly correlated with the presence of the ACE 'D' allele in subjects on dialysis (P=0.0348). CONCLUSIONS: The distribution of RAS genes in dialysis patients is similar to that of the normal population. The presence of the D allele of ACE gene is associated with cerebrovascular disease and the degree of carotid artery stenosis. We postulate that the ACE gene polymorphism is a risk factor for cerebrovascular disease in dialytic patients.

Different mutations in the NF1 gene are associated with Neurofibromatosis-Noonan syndrome (NFNS).

The association of the Noonan phenotype with neurofibromatosis type 1 (NF1) was first noted by Allanson et al. [Am J Med Genet 1985;21:457-462.] and 30 further cases have subsequently been reported. It has been suggested that this phenotype is more common than previously appreciated, as Colley et al. [Clin Genet 1996;49:59-64.] examined 94 sequentially identified patients with NF1 from their genetic register and found Noonan features in 12. A 3-bp deletion of exon 17 of the NF1 neurofibromin gene was described in one family by Carey et al. [Proc Greenwood Genet Center 1997;17:52-53]. However, it remains unclear whether Neurofibromatosis-Noonan syndrome (NFNS) represents a form of NF1 (with mutations in the NF1 neurofibromin gene) or a separate syndrome. We have used a new, rapid sequence analysis technique-comparative sequence analysis (CSA)-to examine the NF1 gene in six patients with NFNS. None of the six patients had the previously identified mutation, nor did we observe other mutations within this exon. However, two other mutations were found: in exon 25, a 3-bp deletion 4312 del GAA, and in exon 23-2, a 2-bp insertion 4095 ins TG. The PTPN11 gene, now known to cause over 50% of Noonan syndrome was also examined in four cases of NFNS, and no mutations were found. These results show that NFNS can in some cases result from different mutations in the NF1 gene and therefore represents a variant form of NF1.

PTPN11 mutations in Noonan syndrome: molecular spectrum, genotype-phenotype correlation, and phenotypic heterogeneity.

Noonan syndrome (NS) is a developmental disorder characterized by facial dysmorphia, short stature, cardiac defects, and skeletal malformations. We recently demonstrated that mutations in PTPN11, the gene encoding the non-receptor-type protein tyrosine phosphatase SHP-2 (src homology region 2-domain phosphatase-2), cause NS, accounting for approximately 50% of cases of this genetically heterogeneous disorder in a small cohort. All mutations were missense changes and clustered at the interacting portions of the amino-terminal src-homology 2 (N-SH2) and protein tyrosine phosphatase (PTP) domains. A gain of function was postulated as a mechanism for the disease. Here, we report the spectrum and distribution of PTPN11 mutations in a large, well-characterized cohort with NS. Mutations were found in 54 of 119 (45%) unrelated individuals with sporadic or familial NS. There was a significantly higher prevalence of mutations among familial cases than among sporadic ones. All defects were missense, and several were recurrent. The vast majority of mutations altered amino acid residues located in or around the interacting surfaces of the N-SH2 and PTP domains, but defects also affected residues in the C-SH2 domain, as well as in the peptide linking the N-SH2 and C-SH2 domains. Genotype-phenotype analysis revealed that pulmonic stenosis was more prevalent among the group of subjects with NS who had PTPN11 mutations than it was in the group without them (70.6% vs. 46.2%; P<.01), whereas hypertrophic cardiomyopathy was less prevalent among those with PTPN11 mutations (5.9% vs. 26.2%; P<.005). The prevalence of other congenital heart malformations, short stature, pectus deformity, cryptorchidism, and developmental delay did not differ between the two groups. A PTPN11 mutation was identified in a family inheriting Noonan-like/multiple giant-cell lesion syndrome, extending the phenotypic range of disease associated with this gene.

Paternal germline origin and sex-ratio distortion in transmission of PTPN11 mutations in Noonan syndrome.

Germline mutations in PTPN11--the gene encoding the nonreceptor protein tyrosine phosphatase SHP-2--represent a major cause of Noonan syndrome (NS), a developmental disorder characterized by short stature and facial dysmorphism, as well as skeletal, hematologic, and congenital heart defects. Like many autosomal dominant disorders, a significant percentage of NS cases appear to arise from de novo mutations. Here, we investigated the parental origin of de novo PTPN11 lesions and explored the effect of paternal age in NS. By analyzing intronic portions that flank the exonic PTPN11 lesions in 49 sporadic NS cases, we traced the parental origin of mutations in 14 families. Our results showed that all mutations were inherited from the father, despite the fact that no substitution affected a CpG dinucleotide. We also report that advanced paternal age was observed among cohorts of sporadic NS cases with and without PTPN11 mutations and that a significant sex-ratio bias favoring transmission to males was present in subjects with sporadic NS caused by PTPN11 mutations, as well as in families inheriting the disorder.

Absence of PTPN11 mutations in 28 cases of cardiofaciocutaneous (CFC) syndrome.

CFC (cardiofaciocutaneous) syndrome (MIM 115150) has been considered by several authors to be a more severe expression of Noonan syndrome. Affected patients present with congenital heart defects, cutaneous abnormalities, Noonan-like facial features and severe psychomotor developmental delay. We have recently demonstrated that Noonan syndrome can be caused by missense mutations in PTPN11(MIM 176876), a gene that encodes the non-receptor protein tyrosine phosphatase SHP-2. In this report, we have evaluated the possible involvement of mutations in PTPN11 in CFC syndrome. A cohort of 28 CFC subjects rigorously assessed as having CFC based on OMIM diagnostic criteria was examined for mutations in the PTPN11 coding sequence by using DHPLC analysis. The results showed no abnormalities in the coding region of the PTPN11 gene in any CFC patient, nor any evidence of major deletions within the gene suggesting that mutations in other gene(s) are responsible for this syndrome.