Irene M.J. Mathijssen

RAB23 Mutations in Carpenter Syndrome Imply an Unexpected Role for Hedgehog Signaling in Cranial-Suture Development and Obesity

Carpenter syndrome is a pleiotropic disorder with autosomal recessive inheritance, the cardinal features of which include craniosynostosis, polysyndactyly, obesity, and cardiac defects. Using homozygosity mapping, we found linkage to chromosome 6p12.1-q12 and, in 15 independent families, identified five different mutations (four truncating and one missense) in RAB23, which encodes a member of the RAB guanosine triphosphatase (GTPase) family of vesicle transport proteins and acts as a negative regulator of hedgehog (HH) signaling. In 10 patients, the disease was caused by homozygosity for the same nonsense mutation, L145X, that resides on a common haplotype, indicative of a founder effect in patients of northern European descent. Surprisingly, nonsense mutations of Rab23 in open brain mice cause recessive embryonic lethality with neural-tube defects, suggesting a species difference in the requirement for RAB23 during early development. The discovery of RAB23 mutations in patients with Carpenter syndrome implicates HH signaling in cranial-suture biogenesis--an unexpected finding, given that craniosynostosis is not usually associated with mutations of other HH-pathway components--and provides a new molecular target for studies of obesity.

Mutations in TCF12 , encoding a basic helix-loop-helix partner of TWIST1, are a frequent cause of coronal craniosynostosis

Craniosynostosis, the premature fusion of the cranial sutures, is a heterogeneous disorder with a prevalence of ∼1 in 2,200 (refs. 1,2). A specific genetic etiology can be identified in ∼21% of cases, including mutations of TWIST1, which encodes a class II basic helix-loop-helix (bHLH) transcription factor, and causes Saethre-Chotzen syndrome, typically associated with coronal synostosis. Using exome sequencing, we identified 38 heterozygous TCF12 mutations in 347 samples from unrelated individuals with craniosynostosis. The mutations predominantly occurred in individuals with coronal synostosis and accounted for 32% and 10% of subjects with bilateral and unilateral pathology, respectively. TCF12 encodes one of three class I E proteins that heterodimerize with class II bHLH proteins such as TWIST1. We show that TCF12 and TWIST1 act synergistically in a transactivation assay and that mice doubly heterozygous for loss-of-function mutations in Tcf12 and Twist1 have severe coronal synostosis. Hence, the dosage of TCF12-TWIST1 heterodimers is critical for normal coronal suture development.

Frontorhiny, a Distinctive Presentation of Frontonasal Dysplasia Caused by Recessive Mutations in the ALX3 Homeobox Gene

We describe a recessively inherited frontonasal malformation characterized by a distinctive facial appearance, with hypertelorism, wide nasal bridge, short nasal ridge, bifid nasal tip, broad columella, widely separated slit-like nares, long philtrum with prominent bilateral swellings, and midline notch in the upper lip and alveolus. Additional recurrent features present in a minority of individuals have been upper eyelid ptosis and midline dermoid cysts of craniofacial structures. Assuming recessive inheritance, we mapped the locus in three families to chromosome 1 and identified mutations in ALX3, which is located at band 1p13.3 and encodes the aristaless-related ALX homeobox 3 transcription factor. In total, we identified seven different homozygous pathogenic mutations in seven families. These mutations comprise missense substitutions at critical positions within the conserved homeodomain as well as nonsense, frameshift, and splice-site mutations, all predicting severe or complete loss of function. Our findings contrast with previous studies of the orthologous murine gene, which showed no phenotype in Alx3(-/-) homozygotes, apparently as a result of functional redundancy with the paralogous Alx4 gene. We conclude that ALX3 is essential for normal facial development in humans and that deficiency causes a clinically recognizable phenotype, which we term frontorhiny.

The increase of metopic synostosis: A pan-European observation

Metopic synostosis is thought to have an incidence of about 1 in 15,000 births. Traditionally, this makes it the third most frequent single-suture craniosynostosis, after scaphocephaly (1 in 4200-8500) and plagiocephaly (1 in 11,000). Our units have, independently from each other, noted a marked increase in the number of metopic synostosis over the recent years. This is a pan-European, retrospective epidemiological study on the number of cases with metopic synostosis born between January 1, 1997, and January 1, 2006. This number was compared to the prevalence of scaphocephaly, the most frequently seen craniosynostosis. In the 7 units, a total of 3240 craniosynostosis were seen from 1997 until 2006. Forty-one percent (n = 1344) of those were sagittal synostosis, and 23% (n = 756) were metopic synostosis. There was a significant increase of the absolute number as well as of the percentage of metopic synostosis over these years (regression analysis, P = 0.017, R2 = 0.578) as opposed to a nonsignificant increase in the percentage of sagittal synostosis (P > 0.05, R2 = 0.368). The most remarkable increase occurred around 2000-2001, with the average of metopics being 20.1% from 1997 to 2000 and 25.5% from 2001 to 2005 (independent t-test, P = 0.002). The sagittal synostosis showed a smaller and nonsignificant increase in the same years: from 39.9% in 1997-2000 leading up to 42.5% in 2001-2005 (independent t-test, P > 0.05). The number of metopic synostosis has significantly increased over the reviewed period in all of our units, both in absolute numbers as in comparison to the total number of craniosynostosis.

Inactivation of IL11 Signaling Causes Craniosynostosis, Delayed Tooth Eruption, and Supernumerary Teeth

Craniosynostosis and supernumerary teeth most often occur as isolated developmental anomalies, but they are also separately manifested in several malformation syndromes. Here, we describe a human syndrome featuring craniosynostosis, maxillary hypoplasia, delayed tooth eruption, and supernumerary teeth. We performed homozygosity mapping in three unrelated consanguineous Pakistani families and localized the syndrome to a region in chromosome 9. Mutational analysis of candidate genes in the region revealed that all affected children harbored homozygous missense mutations (c.662C>G [p.Pro221Arg], c.734C>G [p.Ser245Cys], or c.886C>T [p.Arg296Trp]) in IL11RA (encoding interleukin 11 receptor, alpha) on chromosome 9p13.3. In addition, a homozygous nonsense mutation, c.475C>T (p.Gln159X), and a homozygous duplication, c.916_924dup (p.Thr306_Ser308dup), were observed in two north European families. In cell-transfection experiments, the p.Arg296Trp mutation rendered the receptor unable to mediate the IL11 signal, indicating that the mutation causes loss of IL11RA function. We also observed disturbed cranial growth and suture activity in the Il11ra null mutant mice, in which reduced size and remodeling of limb bones has been previously described. We conclude that IL11 signaling is essential for the normal development of craniofacial bones and teeth and that its function is to restrict suture fusion and tooth number. The results open up the possibility of modulation of IL11 signaling for the treatment of craniosynostosis.

Long-term functional outcome in 167 patients with syndromic craniosynostosis; defining a syndrome-specific risk profile

OBJECTIVE Little is known about the long-term prevalence of elevated intracranial pressure (ICP), obstructive sleep apnoea (OSA), level of education, language and motor skills, impaired sight and hearing in craniosynostosis syndromes. The objective of this study was to define the prevalence per syndrome of elevated ICP, OSA, impaired sight and impaired hearing. METHODS A retrospective study was undertaken on 167 consecutive patients diagnosed with Apert, Crouzon, Pfeiffer, Muenke or Saethre-Chotzen syndrome, aged 1-25 years and treated between 1983 and 2008. The mean age at time of referral and review was 1 years and 2 months and 10 years and 3 months, respectively. RESULTS Patients with Apert and Crouzon/Pfeiffer syndromes had the highest prevalence of elevated ICP (33% and 53%, respectively) and OSA (31% and 27%, respectively), while Saethre-Chotzen syndrome was also associated with a fair risk for elevated ICP (21%). The prevalence of impaired sight (61%) and hearing (56%) was high in all syndromes. CONCLUSION Based on these data, a syndrome-specific risk profile with suggestions for screening and treatment is presented.

The origin of EFNB1 mutations in craniofrontonasal syndrome: Frequent somatic mosaicism and explanation of the paucity of carrier males

Craniofrontonasal syndrome (CFNS) is an X-linked disorder that exhibits a paradoxical sex reversal in phenotypic severity: females characteristically have frontonasal dysplasia, craniosynostosis, and additional minor malformations, but males are usually mildly affected with hypertelorism only. Despite this, males appear underrepresented in CFNS pedigrees, with carrier males encountered infrequently compared with affected females. To investigate these unusual genetic features of CFNS, we exploited the recent discovery of causative mutations in the EFNB1 gene, which encodes ephrin-B1, to survey the molecular alterations in 59 families (39 newly investigated and 20 published elsewhere). We identified the first complete deletions of EFNB1, catalogued 27 novel intragenic mutations, and used Pyrosequencing and analysis of nearby polymorphic alleles to quantify mosaic cases and to determine the parental origin of verified germline mutations. Somatic mosaicism was demonstrated in 6 of 53 informative families, and, of 17 germline mutations in individuals for whom the parental origin of mutation could be demonstrated, 15 arose from the father. We conclude that the major factor accounting for the relative scarcity of carrier males is the bias toward mutations in the paternal germline (which present as affected female offspring) combined with reduced reproductive fitness in affected females. Postzygotic mutations also contribute to the female preponderance, whereas true nonpenetrance in males who are hemizygous for an EFNB1 mutation appears unusual. These results highlight the importance of considering possible origins of mutation in the counseling of families with CFNS and provide a generally applicable approach to the combined analysis of mosaic and germline mutations.

Advancement of the midface, from conventional Le Fort III osteotomy to Le Fort III distraction: review of the literature

Since its introduction in about 1950, the Le Fort III (LF III) procedure has become a widely accepted treatment for correction of midface hypoplasia and related functional and esthetic problems. As long-term surgical experience grows and improvements are made in technique, equipment and peri-operative care, the number of LF III procedures performed worldwide is increasing. A number of fundamental questions concerning the technique remain unclear, and large, conclusive studies are lacking owing to the relative rarity of severe midface hypoplasia. This literature review aims to address problems, such as the indication field, timing of surgery, rate of relapse and the use of distraction osteogenesis. An overview of the history and technique of LF III osteotomy and distraction is provided, together with a comprehensive review of the available clinical data.

Obstructive sleep apnea in children with syndromic craniosynostosis: long-term respiratory outcome of midface advancement

Almost 50% of patients with Apert, Crouzon or Pfeiffer syndrome develop obstructive sleep apnea (OSA), mainly due to midface hypoplasia. Midface advancement is often the treatment of choice, but the few papers on long-term outcome report mixed results. This paper aimed to assess the long-term respiratory outcome of midface advancement in syndromic craniosynostosis with OSA and to determine factors contributing to its efficacy. A retrospective study was performed on 11 patients with moderate or severe OSA, requiring oxygen, continuous positive airway pressure (CPAP), or tracheostomy. Clinical symptoms, results of polysomnography, endoscopy and digital volume measurement of the upper airways on CT scan before and after midface advancement were reviewed. Midface advancement had a good respiratory outcome in the short term in 6 patients and was ineffective in 5. In all patients without respiratory effect or with relapse, endoscopy showed obstruction of the rhino- or hypopharynx. The volume measurements supported the clinical and endoscopic outcome. Despite midface advancement, long-term dependence on, or indication for, CPAP or tracheostomy was maintained in 5 of 11 patients. Pharyngeal collapse appeared to play a role in OSA. Endoscopy before midface advancement is recommended to identify airway obstruction that may interfere with respiratory improvement after midface advancement.

Percutaneous aponeurotomy and lipofilling: a regenerative alternative to flap reconstruction?

Background: The application of a new approach is presented, percutaneous aponeurotomy and lipofilling, which is a minimally invasive, incisionless alternative to traditional flap reconstructions. Methods: The restrictive subdermal cicatrix and/or endogenous aponeurosis is punctured, producing staggered nicks. Expansion of the restriction reconstructs the defect and creates a vascularized scaffold with micro-openings that are seeded with lipografts. Wide subcutaneous cuts that lead to macrocavities and subsequent graft failure are avoided. Postoperatively, a splint to hold open the neomatrix/graft construct in its expansive state is applied until the grafts mature. Thirty-one patients underwent one to three operations (average, two) for defects that normally require flap tissue transfer: wounds where primary closure was not possible (n = 9), contour defects of the trunk and breast requiring large-volume fat grafts (n = 8), burn contractures (n = 5), radiation scars (n = 6), and congenital constriction bands (n = 3). Results: The regenerated tissue was similar in texture and consistency to the surrounding tissues. Wider meshed areas had greater tissue gain (range, 20 to 30 percent). There were no significant wound-healing issues, scars, or donor-site morbidities. Advancement tension was relieved without flap undermining or decreased perfusion. Conclusions: Realizing that, whether scar or endogenous fascia, the subdermal aponeurosis limits tissue stretch and/or its three-dimensional expansion, a minimally invasive procedure that expands this cicatrix into a matrix ideally suited for fat micrografts was developed. Grafting this scaffold applies tissue-engineering principles to generate the needed tissue and represents a regenerative alternative to reconstructive flap surgery. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.