A. Jeannette M. Hoogeboom

Mutations in genes encoding subunits of RNA polymerases I and III cause Treacher Collins syndrome.

We identified a deletion of a gene encoding a subunit of RNA polymerases I and III, POLR1D, in an individual with Treacher Collins syndrome (TCS). Subsequently, we detected 20 additional heterozygous mutations of POLR1D in 252 individuals with TCS. Furthermore, we discovered mutations in both alleles of POLR1C in three individuals with TCS. These findings identify two additional genes involved in TCS, confirm the genetic heterogeneity of TCS and support the hypothesis that TCS is a ribosomopathy.

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 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.

Phenotype and genotype in 103 patients with tricho-rhino-phalangeal syndrome.

Tricho-rhino-phalangeal syndrome (TRPS) is characterized by craniofacial and skeletal abnormalities, and subdivided in TRPS I, caused by mutations in TRPS1, and TRPS II, caused by a contiguous gene deletion affecting (amongst others) TRPS1 and EXT1. We performed a collaborative international study to delineate phenotype, natural history, variability, and genotype-phenotype correlations in more detail. We gathered information on 103 cytogenetically or molecularly confirmed affected individuals. TRPS I was present in 85 individuals (22 missense mutations, 62 other mutations), TRPS II in 14, and in 5 it remained uncertain whether TRPS1 was partially or completely deleted. Main features defining the facial phenotype include fine and sparse hair, thick and broad eyebrows, especially the medial portion, a broad nasal ridge and tip, underdeveloped nasal alae, and a broad columella. The facial manifestations in patients with TRPS I and TRPS II do not show a significant difference. In the limbs the main findings are short hands and feet, hypermobility, and a tendency for isolated metacarpals and metatarsals to be shortened. Nails of fingers and toes are typically thin and dystrophic. The radiological hallmark are the cone-shaped epiphyses and in TRPS II multiple exostoses. Osteopenia is common in both, as is reduced linear growth, both prenatally and postnatally. Variability for all findings, also within a single family, can be marked. Morbidity mostly concerns joint problems, manifesting in increased or decreased mobility, pain and in a minority an increased fracture rate. The hips can be markedly affected at a (very) young age. Intellectual disability is uncommon in TRPS I and, if present, usually mild. In TRPS II intellectual disability is present in most but not all, and again typically mild to moderate in severity. Missense mutations are located exclusively in exon 6 and 7 of TRPS1. Other mutations are located anywhere in exons 4-7. Whole gene deletions are common but have variable breakpoints. Most of the phenotype in patients with TRPS II is explained by the deletion of TRPS1 and EXT1, but haploinsufficiency of RAD21 is also likely to contribute. Genotype-phenotype studies showed that mutations located in exon 6 may have somewhat more pronounced facial characteristics and more marked shortening of hands and feet compared to mutations located elsewhere in TRPS1, but numbers are too small to allow firm conclusions.

Expanding the genetic and phenotypic spectrum of popliteal pterygium disorders

The popliteal pterygia syndromes are a distinct subset of the hundreds of Mendelian orofacial clefting syndromes. Popliteal pterygia syndromes have considerable variability in severity and in the associated phenotypic features but are all characterized by cutaneous webbing across one or more major joints, cleft lip and/or palate, syndactyly, and genital malformations. Heterozygous mutations in IRF6 cause popliteal pterygium syndrome (PPS) while homozygous mutations in RIPK4 or CHUK (IKKA) cause the more severe Bartsocas–Papas syndrome (BPS) and Cocoon syndrome, respectively. In this study, we report mutations in six pedigrees with children affected with PPS or BPS. Using a combination of Sanger and exome sequencing, we report the first case of an autosomal recessive popliteal pterygium syndrome caused by homozygous mutation of IRF6 and the first case of uniparental disomy of chromosome 21 leading to a recessive disorder. We also demonstrate that mutations in RIPK4 can cause features with a range of severity along the PPS‐BPS spectrum and that mutations in IKKA can cause a range of features along the BPS‐Cocoon spectrum. Our findings have clinical implications for genetic counseling of families with pterygia syndromes and further implicate IRF6, RIPK4, and CHUK (IKKA) in potentially interconnected pathways governing epidermal and craniofacial development.

Gain-of-Function Mutations in ZIC1 Are Associated with Coronal Craniosynostosis and Learning Disability.

Human ZIC1 (zinc finger protein of cerebellum 1), one of five homologs of the Drosophila pair-rule gene odd-paired, encodes a transcription factor previously implicated in vertebrate brain development. Heterozygous deletions of ZIC1 and its nearby paralog ZIC4 on chromosome 3q25.1 are associated with Dandy-Walker malformation of the cerebellum, and loss of the orthologous Zic1 gene in the mouse causes cerebellar hypoplasia and vertebral defects. We describe individuals from five families with heterozygous mutations located in the final (third) exon of ZIC1 (encoding four nonsense and one missense change) who have a distinct phenotype in which severe craniosynostosis, specifically involving the coronal sutures, and variable learning disability are the most characteristic features. The location of the nonsense mutations predicts escape of mutant ZIC1 transcripts from nonsense-mediated decay, which was confirmed in a cell line from an affected individual. Both nonsense and missense mutations are associated with altered and/or enhanced expression of a target gene, engrailed-2, in a Xenopus embryo assay. Analysis of mouse embryos revealed a localized domain of Zic1 expression at embryonic days 11.5–12.5 in a region overlapping the supraorbital regulatory center, which patterns the coronal suture. We conclude that the human mutations uncover a previously unsuspected role for Zic1 in early cranial suture development, potentially by regulating engrailed 1, which was previously shown to be critical for positioning of the murine coronal suture. The diagnosis of a ZIC1 mutation has significant implications for prognosis and we recommend genetic testing when common causes of coronal synostosis have been excluded.

A de novo GLI3 mutation in a patient with acrocallosal syndrome

Acrocallosal syndrome is characterized by postaxial polydactyly, macrocephaly, agenesis of the corpus callosum, and severe developmental delay. In a few patients with this disorder, a mutation in the KIF7 gene has been reported, which was associated with impaired GLI3 processing and dysregulaton of GLI3 transcription factors. A single patient with acrocallosal syndrome and a de novo p.Ala934Pro mutation in GLI3 has been reported, whereas diverse and numerous GLI3 mutations have also been described in syndromes with overlapping clinical manifestations, including Greig cephalopolysyndactyly syndrome, Pallister–Hall syndrome, trigonocephaly with craniosynostosis and polydactyly, oral–facial‐digital syndrome, and non‐syndromic polydactyly. Here, we describe a second patient with acrocallosal syndrome, who has a de novo, novel c.2786T > C mutation in GLI3, which predicts p.Leu929Pro. This mutation is in the same domain as the mutation in the previously reported patient. These data confirm that mutations in GLI3 are a cause of the acrocallosal phenotype.

Boston type craniosynostosis: Report of a second mutation in MSX2

We describe a family that segregated an autosomal dominant form of craniosynostosis characterized by variable expression and limited extra‐cranial features. Linkage analysis and genome sequencing were performed to identify the underlying genetic mutation. A c.443C>T missense mutation in MSX2, which predicts p.Pro148Leu was identified and segregated with the disease in all affected family members. One other family with autosomal dominant craniosynostosis (Boston type) has been reported to have a missense mutation in MSX2. These data confirm that missense mutations altering the proline at codon 148 of MSX2 cause dominantly inherited craniosynostosis.

Additional phenotypic features of Muenke syndrome in 2 Dutch families.

In about 30% of the patients with syndromal craniosynostosis, a genetic mutation can be traced. For the purpose of adequate genetic counseling and treatment of these patients, the full spectrum of clinical findings for each specific mutation needs to be appreciated. The Pro250Arg mutation in the FGFR3 gene is found in patients with Muenke syndrome and is one of the most frequently encountered mutations in craniosynostosis syndromes. A number of studies on the relationship between genotype and phenotype concerning this specific mutation have been published. Two Dutch families with Muenke syndrome were screened for the reported characteristics of this syndrome and for additional features. New phenotypical findings were hypoplasia of the frontal sinus, ptosis of the upper eyelids, dysplastic elbow joints with restricted elbow motion, and mild cutaneous syndactyly. Incidentally, polydactyly, severe ankylosis of the elbow, fusion of cervical vertebrae, and epilepsy were found. Upper eyelid ptosis is thought to be pathognomonic for Saethre-Chotzen syndrome but was also observed in our series of patients with Muenke syndrome. Because Muenke and Saethre-Chotzen syndrome can have similar phenotypes, DNA analysis is needed to distinguish between these syndromes, even when a syndrome diagnosis is already made in a family member.