Michella Ghassibé

Four common glomulin mutations cause two thirds of glomuvenous malformations (“familial glomangiomas”): evidence for a founder effect

Background: Glomuvenous malformation (GVM) (“familial glomangioma”) is a localised cutaneous vascular lesion histologically characterised by abnormal smooth muscle-like “glomus cells” in the walls of distended endothelium lined channels. Inheritable GVM has been linked to chromosome 1p21-22 and is caused by truncating mutations in glomulin. A double hit mutation was identified in one lesion. This finding suggests that GVM results from complete localised loss of function and explains the paradominant mode of inheritance. Objective: To report on the identification of a mutation in glomulin in 23 additional families with GVM. Results: Three mutations are new; the others have been described previously. Among the 17 different inherited mutations in glomulin known up to now in 43 families, the 157delAAGAA mutation is the most common and was present in 21 families (48.8%). Mutation 108C→A was found in five families (11.8%), and the mutations 554delA+556delCCT and 1179delCAA were present together in two families (4.7% each). Polymorphic markers suggested a founder effect for all four mutations. Conclusions: Screening for these mutations should lead to a genetic diagnosis in about 70% of patients with inherited GVM. So far, a mutation in glomulin has been found in all GVM families tested, thus demonstrating locus homogeneity.

Interferon regulatory factor-6: a gene predisposing to isolated cleft lip with or without cleft palate in the Belgian population.

Cleft lip with or without cleft palate is the most frequent craniofacial malformation in humans (∼1/700). Its etiology is multifactorial; some are a result of a genetic mutation, while others may be due to environmental factors, with genetic predisposition playing an important role. The prevalence varies widely between populations and the mode of inheritance remains controversial. The interferon regulatory factor-6 (IRF6) gene has been shown to harbor mutations in patients with van der Woude syndrome, a dominant form of clefts associated with small pits of the lower lip. Moreover IRF6 has been associated with nonsyndromic cleft of the palate (CL/P) in two separate studies. We investigated the role of IRF6 in a set of 195 trios from Belgium. Cleft occurred as an isolated feature. We studied association of the IRF6 locus using two variants: one in the IRF6 gene and the other 100 kpb 3′ of the gene. Our independent study group confirms that the IRF6 locus is associated with nonsyndromic cleft lip with or without palate. This result, with previous studies performed in the United States and Italy, shows for the first time the implication of IRF6 in isolated CL/P in northern Europe. It is likely that association to this locus can be identified in various populations and that the IRF6 locus thus represents an important genetic modifier for this multifactorial malformation.

Prevalence and nonrandom distribution of exonic mutations in interferon regulatory factor 6 in 307 families with Van der Woude syndrome and 37 families with popliteal pterygium syndrome.

Purpose: Interferon regulatory factor 6 encodes a member of the IRF family of transcription factors. Mutations in interferon regulatory factor 6 cause Van der Woude and popliteal pterygium syndrome, two related orofacial clefting disorders. Here, we compared and contrasted the frequency and distribution of exonic mutations in interferon regulatory factor 6 between two large geographically distinct collections of families with Van der Woude and between one collection of families with popliteal pterygium syndrome.Methods: We performed direct sequence analysis of interferon regulatory factor 6 exons on samples from three collections, two with Van der Woude and one with popliteal pterygium syndrome.Results: We identified mutations in interferon regulatory factor 6 exons in 68% of families in both Van der Woude collections and in 97% of families with popliteal pterygium syndrome. In sum, 106 novel disease-causing variants were found. The distribution of mutations in the interferon regulatory factor 6 exons in each collection was not random; exons 3, 4, 7, and 9 accounted for 80%. In the Van der Woude collections, the mutations were evenly divided between protein truncation and missense, whereas most mutations identified in the popliteal pterygium syndrome collection were missense. Further, the missense mutations associated with popliteal pterygium syndrome were localized significantly to exon 4, at residues that are predicted to bind directly to DNA.Conclusion: The nonrandom distribution of mutations in the interferon regulatory factor 6 exons suggests a two-tier approach for efficient mutation screens for interferon regulatory factor 6. The type and distribution of mutations are consistent with the hypothesis that Van der Woude is caused by haploinsufficiency of interferon regulatory factor 6. On the other hand, the distribution of popliteal pterygium syndrome-associated mutations suggests a different, though not mutually exclusive, effect on interferon regulatory factor 6 function.

Six families with van der Woude and/or popliteal pterygium syndrome: all with a mutation in the IRF6 gene

. an der Woude syndrome (VWS, OMIM #119300) is a dominantly inherited developmental disorder characterised by pits and/or sinuses of the lower lip, cleft lip with or without cleft palate (CL/P), isolated cleft palate (CP), bifid uvula (BU), and hypodontia (H). 1–3 Cleft lip deformity is established during the first 6 weeks of life due to failure of fusion of maxillary and medial nasal processes or to incomplete mesodermal ingrowth into the processes. Palatal clefts result from failure of fusion of the palatal shelves that normally change from a vertical to horizontal position and fuse during the sixth to ninth weeks of gestation. The severity of VWS varies widely, even within families. About 25% of individuals with the syndrome have minimal findings, such as absent teeth or trivial indentations in the lower lips. Clefting of the lip or palate is present in ,50% of cases. Lip pits and/or sinuses are the cardinal features of this syndrome, present in 70–80% of VWS patients. 2 They are often associated with accessory salivary glands that empty into the pits, sometimes leading to embarrassing visible discharge. 45

IRF6 screening of syndromic and a priori non-syndromic cleft lip and palate patients : identification of a new type of minor VWS sign.

Van der Woude syndrome (VWS), caused by dominant IRF6 mutation, is the most common cleft syndrome. In 15% of the patients, lip pits are absent and the phenotype mimics isolated clefts. Therefore, we hypothesized that some of the families classified as having non-syndromic inherited cleft lip and palate could have an IRF6 mutation. We screened in total 170 patients with cleft lip with or without cleft palate (CL/P): 75 were syndromic and 95 were a priori part of multiplex non-syndromic families. A mutation was identified in 62.7 and 3.3% of the patients, respectively. In one of the 95 a priori non-syndromic families with an autosomal dominant inheritance (family B), new insights into the family history revealed the presence, at birth, of lower lip pits in two members and the diagnosis was revised as VWS. A novel lower lip sign was observed in one individual in this family. Interestingly, a similar lower lip sign was also observed in one individual from a 2nd family (family A). This consists of 2 nodules below the lower lip on the external side. In a 3rd multiplex family (family C), a de novo mutation was identified in an a priori non-syndromic CL/P patient. Re-examination after mutation screening revealed the presence of a tiny pit-looking lesion on the inner side of the lower lip leading to a revised diagnosis of VWS. On the basis of this data, we conclude that IRF6 should be screened when any doubt rises about the normality of the lower lip and also if a non-syndromic cleft lip patient (with or without cleft palate) has a family history suggestive of autosomal dominant inheritance.

Disruption of ST5 is associated with mental-retardation and multiple congenital anomalies

Background The authors observed a patient with a cryptic subtelomeric de novo balanced translocation 46,XY.ish t(11;20)(p15.4;q13.2) presenting with severe mental retardation, muscular hypotonia, seizures, bilateral sensorineural hearing loss, submucous cleft palate, persistent ductus Botalli, unilateral cystic kidney dysplasia and frequent infections. Methods and Results Fluorescence in situ hybridisation mapping and sequencing of the translocation breakpoints showed that no known genes are disrupted at 20q13.2 and that ST5 (suppression of tumorigenicity 5; MIM 140750) is disrupted on 11p15.4. By quantitative PCR from different human tissues, the authors found ST5 to be relatively evenly expressed in fetal tissues. ST5 expression was more pronounced in adult brain, kidney and muscle than in the corresponding fetal tissues, whereas expression in other tissues was generally lower than in the fetal tissue. Using RNA in situ hybridisation in mouse, the authors found that St5 is expressed in the frontal cortex during embryonic development. In adult mouse brain, expression of St5 was especially high in the hippocampal area and cerebellum. Conclusion Hence, the authors suppose that ST5 plays an important role in central nervous system development probably due to disturbance of DENN-domain-mediated vesicle formation and neurotransmitter trafficking. Thus, these findings implicate ST5 in the aetiology of mental retardation, seizures and multiple congenital anomalies.

A novel mutation in the MSX2 homeobox gene of a family with foramina parietalia permagna, headache and vascular anomaly.

Enlarged parietal foramina (PFM, MIM 168500) are secondary to an ossification default of the parietal fontanelles. They are bilateral, situated on both sides of the sagittal suture. They can be more than 2 cm in diameter and are occasionally seen on a routine skull radiograph. They are of no pathological significance, but are clinically associated with headache, scalp defects and structural or vascular malformation of the brain [4, 5]. A surgical intervention is sometimes required. PFM is caused by haploinsufficiency of the ALX4 gene, being part of the DEFECT 11 syndrome (MIM 601224) [2] or due to mutations in MSX2 on 5q34-q35. MSX2 is an MSX homeobox transcription factor. MSX2 has been shown to be involved in craniofacial development. Msx2-deficient mice show defects of skull ossification and persistent calvarial foramen. Altogether, nine MSX2 loss-of-function mutations have been reported [1, 3, 6, 7]. We studied a family with eight affected members showing cranial foramina associated with flash headaches and venous malformation (Fig. 1). Clinical examination and skull X-rays detected the presence of two symmetrical parietal foramen on each side of the sagittal suture in individuals VP-1, 3, 30, 31, 32 and 310. Moreover, VP-310 had a dilated vein on the right parietal part of the skull, arising from a right swelling of the vertex. Mild flash headaches from the right hole, often heat-dependent, were noted in individuals VP-1 and 3. VP-31 presented occasional painless flashes. Clinical examination of VP11, 32, 311, 312, 313 and 314 did not show a foramen. MSX2 screening revealed a 17-nucleotide duplication (CCTGGAGCGCAAGTTCC) in exon 2 for all affected individuals (Fig. 1). The c.468-485dup mutation occurs in helix I at the 21st amino acid of the DNA-binding domain of MSX2. It changes the open reading frame by abolishing 105 amino acids. In addition, it creates 23 new amino acids, followed by a premature stop codon. Co-segregation of the mutation was verified for all family members. It was not observed in 100 control alleles. This duplication results in protein-truncation and is consistent with MSX2 haploinsufficiency. Two protein-truncating duplications were previously identified. In one family, PFM was associated with cleidocranial dysplasia (PFMCCD, MIM168550) [1]. In the other, one individual had ventricular septal defect [3]. Interestingly, this is the first time that a mutation in the MSX2 gene is responsible for PFM associated with headache and venous anomaly, although the latter has been reported twice in clinical PFM cases [4, 5]. The presence of a venous anomaly underlying a skull defect raises the question whether MSX2 is directly linked to craniovascular development or whether the loss of parietal bone permits abnormal vascular morphogenesis. We need to identify the downstream targets of MSX2 and to characterise the associated cellular processes, including proliferation, apoptosis and cell adhesion, before we can understand howMSX2 dosage effect alters cranial bony and vascular development. Studying additional families is also important to evaluate the phenotype/genotype correlation. M. Ghassibe . L. M. Boon . M. Vikkula (*) Laboratory of Human Molecular Genetics, Christian de Duve Institute of Cellular Pathology and Universite catholique de Louvain, Avenue Hippocrate 74 (+5), 1200 Brussels, Belgium e-mail: vikkula@bchm.ucl.ac.be Tel.: +32-2-7647496 Fax: +32-2-7647460 e-mail: Michella.Ghassibe@bchm.ucl.ac.be