El Ghouzzi

Di Cunto

Mutations in Citron-Kinase a cause of autosomal recessive micro-lissencephaly with multinucleated neurons

Keywords: Cytokinesis, Neurogenesis, Primary Microcephaly, Lissencephaly, Autosomal Recessive, Citron Kinase

Abstract:

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Primary microcephaly is a neurodevelopmental disorder caused by a reduction in brain size 27" attributed to defects in early neurogenesis. Mutations in numerous genes encoding proteins that 28" localize to the mitotic spindle and centrosome are implicated in the pathogenicity of primary

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microcephaly. In contrast, involvement of the contractile ring and midbody required for cleavage

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furrow constriction and abscission have not previously been implicated in primary microcephaly.

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Citron Kinase (CIT), a component of the mitotic contractile ring, is a multi-domain protein that 32" localizes to the cleavage furrow and midbody of mitotic cells, where it is required for the 33" completion of cytokinesis. Rodent models of Cit deficiency highlighted the role of this gene in

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neurogenesis and microcephaly over a decade ago. Here we identify recessively inherited

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pathogenic variants in CIT as the genetic basis of severe microcephaly and neonatal death. We

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present postmortem data showing that CIT is critical to build a normally sized human brain.

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Consistent with cytokinesis defects attributed to CIT, multinucleated neurons are observed

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throughout the cerebral cortex and cerebellum of an affected proband, expanding our

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understanding of mechanisms attributed to primary microcephaly.

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Main:

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Primary microcephaly is a genetically heterogeneous neurodevelopmental disorder

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characterized by a severe reduction in brain growth [START_REF] Gilmore | Genetic causes of microcephaly and lessons for neuronal 290" development[END_REF] . This decreased brain volume often stems

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from a primary defect in neurogenesis. The cerebral cortex is composed of neurons born from

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neural progenitor cells (NPCs) that reside adjacent to the lateral ventricle during early 45" neurodevelopment [START_REF] Paridaen | Neurogenesis during development of the vertebrate 292" central nervous system[END_REF] . The mitotic machinery of NPCs is critical not only for rapid expansion of

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the progenitor pool required for normal brain growth, but also for maintaining the balance

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between proliferation and differentiation. Human genetics has identified many primary 48" microcephaly genes which impact the fidelity of mitotic spindle placement and centrosome

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stability required to build a human brain of normal size [START_REF] Bond | ASPM is a major determinant of cerebral cortical size[END_REF][START_REF] Bond | A centrosomal mechanism involving CDK5RAP2 and CENPJ controls 296" brain size[END_REF][START_REF] Kumar | Mutations in STIL, encoding a 298" pericentriolar and centrosomal protein, cause primary microcephaly[END_REF][START_REF] Bilgüvar | Whole-exome sequencing identifies recessive WDR62 mutations in 301" severe brain malformations[END_REF][START_REF] Guernsey | Mutations in centrosomal protein CEP152 in primary microcephaly 303" families linked to MCPH4[END_REF][START_REF] Nicholas | WDR62 is associated with the spindle pole and is mutated in human 305" microcephaly[END_REF] . Surprisingly, other steps in mitosis 50"

have not been implicated by neurogenetics in the pathophysiology of microcephaly, despite a

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series of rodent models showing that cleavage furrow placement, constriction of the contractile

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ring and abscission by the midbody are critical for neurogenesis [START_REF] Gauthier-Fisher | Lfc and Tctex-1 regulate the genesis of neurons from cortical 307" precursor cells[END_REF][START_REF] Sarkisian | Citron-kinase, a 309" protein essential to cytokinesis in neuronal progenitors, is deleted in the flathead mutant 310[END_REF][START_REF] Di Cunto | Defective neurogenesis in citron kinase knockout mice by altered 312" cytokinesis and massive apoptosis[END_REF] .

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CIT is a multi-domain protein that localizes to the cleavage furrow and midbody where it

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functions in cytokinesis and abscission, the final steps of mitosis (Fig. 1a) [START_REF] El Amine | Opposing actions of septins and 314" Sticky on Anillin promote the transition from contractile to midbody ring[END_REF][START_REF] Green | Cytokinesis in animal cells[END_REF] . CIT facilitates

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protein-protein interactions between contractile ring components (with anillin, actin, myosin and

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RhoA) and has a N-terminal kinase domain, both of which are critical for abscission [START_REF] El Amine | Opposing actions of septins and 314" Sticky on Anillin promote the transition from contractile to midbody ring[END_REF][START_REF] Gai | Citron kinase controls abscission through RhoA and anillin[END_REF][START_REF] Bassi | Sticky/Citron kinase maintains proper RhoA localization at the cleavage 321" site during cytokinesis[END_REF] .

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Studies in drosophila and mammals demonstrate the evolutionarily conserved function of CIT in

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cytokinesis, with mutations that disrupt abscission resulting in binucleate cells. The impact of

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cytokinesis defects on brain development and size are evident in the Cit knockout mouse and

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the Flathead rat with a spontaneous nonsense mutation in Cit exon 1. In rodents Cit is critical

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for proliferation of NPCs and male germ cell precursors. Cit null animals are characterized by

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testicular hypoplasia, microcephaly, ataxia, growth deficiencies and lethal epilepsy [START_REF] Sarkisian | Citron-kinase, a 309" protein essential to cytokinesis in neuronal progenitors, is deleted in the flathead mutant 310[END_REF][START_REF] Di Cunto | Defective neurogenesis in citron kinase knockout mice by altered 312" cytokinesis and massive apoptosis[END_REF] . These

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phenotypes have been linked to cytokinesis defects and the presence of multinucleated cells

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throughout the cortex and cerebellum. Premature differentiation of early NPCs and widespread

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apoptosis are compounded across development, resulting in a cerebral cortex and cerebellum

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less than 50% the size of normal brain. Cortical hypoplasia and layer disorganization are

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predicted to account for the recurrent spontaneous seizures and premature death in the mice [START_REF] Ackman | Citron kinase is required for 323" postnatal neurogenesis in the hippocampus[END_REF] .

69"

Roles for CIT in human brain development and primary microcephaly have been predicted but 70" not yet reported.

71"

72"

Here we describe three independent families, each with multiple affected children who

73"

presented with severe micro-lissencephaly associated with neonatal mortality (Fig. 1b). The

74"

clinical features of the probands are provided in Table 1. Egyptian Family A is consanguineous

75"

with two microcephalic male siblings. The first affected child died in the neonatal period (Fig.

76"

1b). Proband A was delivered at term and weighed 2600 grams (-2SD) consistent with 5th

77"

percentile for growth. Head circumference, not measured until 3.5 months of age, was 27cm (-

78"

11 to -12 SD below the age/sex mean). His length at 3.5 months was 53 cm (-4 to -5 SD), and

79"

he continued to experience failure to thrive thereafter. Dysmorphisms were noted including

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hypotelorism, sloping forehead and large ears (Fig. 2a). He exhibited axial hypotonia, upper and

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lower extremity hypertonia, increased deep tendon reflexes and lack of head support.

82"

Microcephaly was confirmed by MRI, which also revealed lissencephaly, enlarged ventricles,

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agenesis of the corpus callosum, cerebellar hypoplasia and brainstem hypoplasia (Fig. 2b). T2

84"

hyperintensity was also noted throughout the white matter consistent with spastic tetraplegia.

85"

86"

Family B has two affected children born to first cousin parents from United Arab Emirates. Male

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Proband B was noted by ultrasound at 30 gestational weeks (GWs) to have microcephaly,

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intrauterine growth retardation, and oligohydramnios. Echocardiogram indicated cardiomegaly,

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biventricular dilatation and tricuspid regurgitation. He was delivered by spontaneous vaginal

90"

delivery at 39 GWs with a head circumference of 24cm, 8 standard deviations below the

91"

age/sex mean, and a body length measurement of 27.2cm crown-rump (2.5 SD below the 92" mean) consistent with 36 GWs. Despite Apgar scores of 8 at one minute, and 9 at 5 minutes, he

93"

died the following day.

94"

95"

At birth, the brain weight of Proband B was 40g (10% the average newborn brain weight of 96" 390g). The two plate-like brain hemispheres were lissencephalic, separated by shallow Sylvian

97"

fissures. Corpus callosum agenesis, large ventricles and small well-delineated basal ganglia

98"

and thalami were noted (Fig. 2c-e). These structural defects were accompanied by brain stem

99"

and cerebellum hypoplasia with small cerebellar folia (Fig. 3a). Dysmorphic facial features were 100" noted including prominent occiput, absent fontanelle, large ears, wide nasal bridge, prominent 101" upper lip, highly arched palate, cloudy corneas, right-sided single palmar crease and

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hypoplastic nails. There was renal aplasia with few primitive glomerular and tubular structures

103"

embedded in connective tissue and absence of ureters and bladder. These features were 104" consistent with those observed during the pregnancy and birth of a previously affected female

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infant from the same pedigree who presented with microcephaly and renal agenesis, and 106" survived only 4 hours following birth (Fig. 1b).

107" 108"

Family C is French and had no reported consanguinity. Proband C was born at full term with 109" weight 2900g (-1 SD), length 46cm (-2 SD), and a head circumference of 30cm (-3.

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showed a simplified gyral pattern and hypoplastic cerebellum (Fig. 2f-g). A second child in this 117" family (II:2) was also confirmed to have microcephaly by fetal MRI at 29 GWs (Fig. 2h-i). The

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biparietal diameter measured from the fetal MRI at that time was consistent with 23 GWs.

119" 120"

Whole exome sequencing (WES) and candidate gene screening were employed to identify a 121" molecular diagnosis for these probands. Following WES for Family A (mother, father, proband

122"

and unaffected sibling), bioinformatics filtering removed common variants (minor allele 123" frequency (MAF) >0.005). Annotated rare variants analyzed according to a consanguineous

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recessive mode of inheritance identified a homozygous G>A transition (c.1111+1 G>A; g. chr12:

125" 120,260,623, hg19) disrupting the splice donor site of exon 9 in CIT (NM_007174) (Fig. 1c).

126"

Sanger sequencing confirmed the presence of this mutation and its appropriate segregation in

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the family, with both parents being heterozygous carriers. In silico analysis of the exonic and 128" intronic sequence flanking the c.1111+1 G>A nucleotide variant was evaluated with Human

129"

Splicing Finder (HSF) and identified cryptic splice donor sites 54 bp 5' of and 2 bp 3' of the

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cryptic splice site usage [START_REF] Gaildrat | Use of splicing reporter minigene assay to evaluate the effect on 325" splicing of unclassified genetic variants[END_REF] . Sequencing the RT-PCR products confirmed that both cryptic splice

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donors are alternatives, with the splice site 2 bp 3' of the WT site most frequently used (Supp.

135"

Fig 1b,c). This major splice product results in a premature stop mutation in exon 10 that is

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predicted to undergo nonsense mediated decay. The spliced CIT transcript generated from the 137" 54 bp 5' splice donor is a minor product and generates an 18 amino acid in-frame deletion in the 138" kinase domain of CIT (CIT -18aa ). The stability of mutant CIT -18aa has not been evaluated, but

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could disrupt kinase activity.

140" 141"

Resequencing of CIT coding exons and splice junctions in 35 probands with primary 142" microcephaly identified two additional patients, Probands B and C, with recessive pathogenic

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variants. A homozygous pathogenic mutation consistent with consanquinity was identified in

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Proband B. This 10bp deletion in exon 2 of CIT, creates a premature stop codon after 15 145" codons (NM_007174; c.29_38delATCCTTTGGA; g.chr12:120,313,935-120,313,944 hg19;

146"

p.Asn10Metfs*15) and is predicted to function as a null allele (Fig. 1a,d). Proband C and Subject

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II:2 of Family C were found to carry compound heterozygous CIT variants. Both variants are 148" located in the kinase domain; one generates a nonsense variant in exon 4 (c.412 C>T; g.

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chr.12: 120,295,329 hg19; p. Gln138*) and one generates a missense variant in exon 5 (c.473 150" C>G; g. chr12: 120,288,021 hg19; p. Pro158Arg) (Fig. 1a,f). The p. Pro158Arg may express a 151" partially functional protein that ameliorates the severity in the patient, compared with the 152" homozygous null phenotypes in Probands A and B.

153" 154"

The CIT mutations presented here were absent from online genomic databases, including the 155" 1000 Genomes Project, National Heart, Lung and Blood Institute (NHLBI) ESP6500, dbSNP 156" 141, and the Exome Aggregation Consortium (ExAC) [START_REF] Abecasis | A map of human genome variation from population-scale 327" sequencing[END_REF][START_REF] Psaty | Cohorts for Heart and Aging Research in Genomic Epidemiology 329" (CHARGE) Consortium: Design of prospective meta-analyses of genome-wide 330" association studies from 5 cohorts[END_REF][START_REF] Lek | Analysis of protein-coding genetic variation in 60,706 humans[END_REF] . Bioinformatic variant annotation

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using SeattleSeq Variation Annotation revealed that the point mutations in our probands affect

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highly conserved bases with high GERP and CADD scores that are predicted to be pathogenic

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(Table 1) [START_REF] Cooper | Distribution and intensity of constraint in mammalian genomic 334" sequence[END_REF][START_REF] Kircher | A general framework for estimating the relative pathogenicity of human 336" genetic variants[END_REF] . Further analysis of the genetic variation in CIT showed that loss-of-function 160" alleles are infrequent. Fifteen heterozygous and no homozygous nonsense or frameshift alleles

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were present in the ExAC browser, and each had a frequency of <0.001. This suggests high

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evolutionarily pressure against these alleles and supports the deleteriousness of the CIT

163"

variants. These findings correspond to a Residual Variation Intolerance Score (RVIS) of -2.35

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indicating CIT is in the top 1.14% of genes in the genome intolerant to common functional 165" genetic variation [START_REF] Petrovski | Genic intolerance 338" to functional variation and the interpretation of personal genomes[END_REF] .

166" 167"

Given the well established role for CIT in cytokinesis, our three cases implicate a novel 168" pathogenic mechanism for microcephaly, a disorder previously linked primarily to gene products

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that localize to the centrosome or mitotic spindle [START_REF] Morris-Rosendahl | What next-generation sequencing (NGS) 341" technology has enabled us to learn about primary autosomal recessive microcephaly 342" (MCPH)[END_REF] . Severe microcephaly with widespread 170" multinucleated neurons is characteristic of the structural and cellular pathology observed in the

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Cit knockout mouse and the Flathead rat (Supplemental table 1) [START_REF] Sarkisian | Citron-kinase, a 309" protein essential to cytokinesis in neuronal progenitors, is deleted in the flathead mutant 310[END_REF][START_REF] Di Cunto | Defective neurogenesis in citron kinase knockout mice by altered 312" cytokinesis and massive apoptosis[END_REF] . In accordance with

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institutional research board and ethics committee approval, post-mortem analysis of Proband B

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allowed the human cell and molecular neuropathology associated with pathogenic variants in

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CIT to be analyzed. All non-neural tissue examined had normal cytology, but multinucleated 175" neurons were observed throughout the neuraxis of Proband B, a hallmark of cytokinesis defects 176" (Supplemental table 2). Microscopically, the profoundly microcephalic cerebral cortex showed

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both cytological and organizational abnormalities in many areas. The neocortex was excessively 178" thick, with the six cortical layers replaced by a molecular layer and two broad layers comprised

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of loosely and irregularly scattered neurons (Fig. 2j,k). The underlying white matter was 180" unmyelinated and contained scattered ectopic neurons. The overlying leptomeninges were

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greatly thickened with reticulin and collagen fibers, prominent vascularity, and heterotopic

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astrocytes and neurons, some of which were multinucleated (Fig. 2l). The hippocampi were

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dysplastic, small and under-rotated, with hypoplastic dentate gyri (Fig. 2m-o). Cortical

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hypoplasia and layer disorganization account for the recurrent spontaneous seizures associated

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with premature death in rodent models [START_REF] Ackman | Citron kinase is required for 323" postnatal neurogenesis in the hippocampus[END_REF] . Extrapolating from these models, the cytoarchitectural

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abnormalities observed in Proband B are consistent with cytokinesis defects that contribute to 187" reduced neuronal precursor proliferation, multinucleation of neurons and increased cell death.

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Despite the phenotypic similarities, pathogenic CIT variants have not been detected in epilepsy 189" cohorts, including the Epi4K exome sequence collection of 264 epileptic encephalopathy trios [START_REF] Consortium | Epi4K: gene discovery in 4,000 genomes[END_REF] .

190" 191"

The cerebellar cortex of Proband B was hypoplastic and dysplastic (Fig. 3). Laminar

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disorganization was more evident in the hemispheres than the vermis, where folia were fused

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and Purkinje cells were observed in multilayered islands interspersed by abnormal granule cell

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domains (Fig. 3b-h). At birth, the external granule cell layer (EGL) is usually compact, with 4-6

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rows of bipolar cells abutting the molecular layer (ML) (Fig. 3b,d,g). In Proband B the EGL was

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wider, the ML narrower, and were merged with the Purkinje cell layer (Fig. 3g-h). In this case

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the EGL was comprised of 2 or 3 compact rows of cells overlying a looser band of horizontally 198" oriented, thin, elongated cell bodies, some clearly binucleate (Fig. 3e,f,h). Purkinje cells had

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short simplified dendritic arborization compared to controls and many were multinucleated (Fig.

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3g-j). Likewise, the internal granular layer (IGL) was severely hypo-cellular (Fig. 3g-h).

201" 202"

In summary, we report the identification of pathogenic variants in CIT as a genetic basis for

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primary microcephaly and cerebellar hypoplasia, with phenotypic features remarkably similar to

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Cit -/-rodent models. These findings highlight the evolutionarily conserved function of CIT in

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neurodevelopment and the disproportionate sensitivity of the neuroaxis to pathogenic variants in

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this gene. Multinucleated neurons are rarely observed in the nervous system, apart from their

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occurrence in ganglion cell tumors. These genetic findings implicate novel mechanisms in the 208" pathogenesis of primary microcephaly and allow us to describe the human presentation of this

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very interesting multinucleated neuronal phenotype.

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Materials and Methods

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Growth Standards The Child Growth Standards published by World Health Organization were

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applied for weight, height and head circumference assessment. These standards can be found

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at http://www.who.int/childgrowth/standards/en/.

215" 216"

DNA Extraction DNA was extracted from peripheral blood lymphocytes for Family A and C

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using Qiagen Blood DNA extraction kit according to manufacturer's instructions. Proband B

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DNA was extracted from formalin-fixed, paraffin-embedded brain tissue using Qiagen QIAamp

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DNA FFPE Tissue Kit (Catalog Number 56404) per the manufacturer's instructions. Three

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independently extracted Proband B DNA samples were analyzed. Extracted DNA was

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quantified with the use of Life Technologies Qubit dsDNA BR Assay Kit (Catalog Number

222"

Q32850) and a Qubit 2.0 Fluorometer.

223" 224"

Whole exome sequencing Quad family WES was performed for family A, including Proband A,

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father, mother and unaffected sibling. The exome was captured with in-solution Agilent

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SureSelect All Exon XT2 50 Mb Kit and sequenced with an Illumina HiSeq 2000 instrument at

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Beijing Genomics Institute, resulting in 97X coverage of more than 99% of the targeted exons.

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SNPs and indels were mapped and called with the SOAPAligner/SOAP2-SOAPSnp-BWA-

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GATK BGI analysis pipeline [START_REF] Li | SNP detection for massively parallel whole-genome resequencing[END_REF][START_REF] Li | SOAP2: an improved ultrafast tool for short read alignment[END_REF] . Reads were mapped against hg19, with an average of 65457

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SNPs and 4455 Indels called per individual. Annotation, ranking, and stringent filtering of

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genetic variants was performed to exclude non-pathogenic variants with a MAF > 0.005 in

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dbSNP, 1000 Genomes, EVS or an in-house database. Nonsynonymous rare variants were

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filtered against reference databases and as potential candidates. Databases

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generated using http://genic-intolerance.org

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Mini-gene Construction and Splicing Assay

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Functional analysis of splicing abnormalities caused by c.1111+1 G>A was performed using an

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in vitro minigene splicing assay according to previously published guidelines [START_REF] Gaildrat | Use of splicing reporter minigene assay to evaluate the effect on 325" splicing of unclassified genetic variants[END_REF] . CIT exon 9 and

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approximately 150 bp of flanking intronic sequences were amplified from a control DNA sample

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and then cloned in the pCAS2 vector. The c.1111+1 G>A substitution was subsequently

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introduced using site-directed mutagenesis and analyzed for specific splicing abnormalities

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following transfection in HeLa cells. Forty-eight hours after transfection, transcriptional analysis

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was performed and the effect on splicing identified using RT-PCR and further confirmed using

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direct Sanger sequencing and agarose gel electrophoresis.

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273"

Immunohistochemistry Brain sections were examined with hematoxylin-eosin. Selected

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sections were stained with Kluver-Barrera, and immunostained for GFAP, 1:400 (Dako,

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Carpenteria, CA, #M0761), phosphorylated neurofilament NF-P, 1:10 (clone TA51, gift of Dr. J.

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Trojanowski, Hospital of University of Pennsylvania, PA), synaptophysin 1:100 (Dako,

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monoclonal #M7315) and calbindin 1:100 (monoclonal, Novocasta).

278" 279"

Funding

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This work was supported by the National Institutes of Health (R00HD069624 to S.L.B).

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282"

363"

amplified from Proband B that creates a stop after 15 codons (not shown). (e) Chromatographs

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and schematic of reference CIT exon 4 and exon 5 and the corresponding nonsense (c.412

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C>T; p. Gln138*) and missense (c.473 C>G; p. Pro158Arg) variants identified in Proband C.

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367"

395"

interspersed with granule cells (GC) within fused folia (c, e) as compared to control cerebellum

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(b, d). (f) Many GC appear binucleated (double blue arrowheads). (g-j) Cerebellum

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immunostained for calbindin shows abnormally thick EGL, reduced ML (blue line) and

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multilayered collections of small PCs (arrows) with stubby irregular dendritic processes in

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Proband B (h), as compared with control (g).

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(pink nucleotides) and cryptic splice sites (blue, orange and green nucleotides and asterisks)

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were delineated and scored with Human Splice Finder (HSF). Exonic sequence is boxed and 410" upper case. Intronic sequence is lower case. Proband A c.1111+1 G>A substitution (red

411"

arrowhead) breaks the WT splice donor site. (b) CIT exon 9 and approximately 150 bp of

412"

flanking intronic sequence was amplified from genomic DNA and cloned into the pCAS2 vector

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between pseudo-exon A and exon B. The c.1111+1 G>A substitution was introduced using

414"

site-directed mutagenesis. Forty-eight hours after transfection in HeLa cells, alternative spliced

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mRNA transcripts from empty (V), WT CIT exon 9 (WT), and c.1111+1 G>A CIT exon 9 (Mut)

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pCAS2 vectors were amplified by RT-PCR. (c) Differentially spliced products were detected by

417"

size separation on an agarose gel. Each product is labeled with a corresponding composition of

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the various splice products. Transcript composition was confirmed by Sanger sequencing. With

419"

loss of the WT splice donor, alternative splice transcripts were generated from nearby cryptic

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splice sites with the highest HSF scores. The major product included exon 9 along with an

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additional four base pairs of intron 9 (green asterisk and exon). The minor product includes only

422"

a portion of exon 9 with 54 base pairs excluded (blue asterisk and exon).

423"

Supplemental Table 1. Mammalian models of null mutations in CIT.

424" 425"

  site (Supp. Fig 1a). A minigene reporter assay was used to assess alternative 131" splicing. CIT WT and c.1111+1 G>A variant exon 9 were cloned into the pCAS2 reporter 132" plasmid between pseudoexons A and B. Plasmids were transfected into HeLa cells to test

Figure 1

 1 Figure 1 Autosomal recessive CIT variants in micro-lissencephaly. (a) Protein domain

Figure 2 .

 2 Figure 2. Structural and cellular neocortical phenotypes. (a) Proband A displays a

Figure 3

 3 Figure 3 Structural and cellular cerebellar CIT phenotypes. (a) Section though Proband B

Supplemental Figure 1 .

 1 (i-j) Dashed boxes are enlarged to show 400" binucleated PC (blue arrow heads) within a small soma in Proband B tissue (j) as compared to 401" controls (i). EGL=external granule layer, IGL=internal granule layer, ML=molecular layer Alternative splicing between CIT exon 9 and pseudo-exon B (exon B) 406" in minigene assay. Minigene splicing assay was performed according to previously published 407" guidelines 17 . (a) Schematic of CIT sequence across the exon-intron border of CIT exon 9. WT

Table 1 .

 1 Genetic and major clinical features

	352" 353"
		Proband A	Proband B	Proband C	Subject II:2 of Family C
		Chr12: 120,260,623	Chr12: 120,313,935 -120,313,944	Chr12: 120,295,329 and Chr12: 120,288,021	Chr12: 120,295,329 and Chr12: 120,288,021
	CIT variant (hg19, NM_007174)	c.1111+1 G>A	c.29_38delATCCTTTGGA	c.412 C>T and c.473 C>G	c.412 C>T and c.473 C>G
			p. Asn10Metfs*15	p. Gln138* and p. Pro158Arg	p. Gln138* and p. Pro158Arg
	GERP Score	5.900	N/A	4.660 and 5.280	4.660 and 5.280
	CADD Score	31.000	N/A	38.000 and 27.800	38.000 and 27.800
	Gender	Male	Male	Male	Male
	Gestational Length	Full Term	Full Term	Full Term	Pregnancy terminated at GW 29+2
	Birth Weight	2.6 kg (-2 SD)	1.730 kg (-4 SD)	2.92 kg (-1 SD)	N/A
	Birth Length	Unknown (home birth)	27.2 cm (crown-rump -2.5 SD)	46 cm (-2 SD)	N/A
	Birth Head
	Circumference	Unknown (home birth)	24 cm (-8 SD)	30 cm (-3.5 SD)	N/A
	(HC)
	HC at Most Recent Evaluation	27 cm (-11 to -12 SD) measured at 3.5 months	N/A	43 cm (-6.5 SD) measured at 10.5 years	N/A
			Autopsy revealed micro-
	Brain Abnormalities	MRI revealed micro-lissencephaly, agenesis of corpus callosum, cerebellar and brainstem hypoplasia, and T2 hyperintensity of the whole white matter	lissencephaly, absent corpus callosum, hindbrain and cerebellum hypotrophy, cerebral cortex hypotrophy, and comparatively large neuropathology included ventricles. Detailed	Microcephaly (MRI not performed)	Fetal MRI revealed a microcephaly, biparietal diameter equivalent to a fetus of 23 WG, and gyration equivalent to a fetus of 26 WG
			in the main text.
	Neurological Findings	Upper and lower extremity hypertonia, axial hypotonia, increased tetraplegia DTRs, and spastic	N/A	Upper and lower extremities extremity hypertonia, and brisk DTRs for the lower	N/A
	Seizures	No	N/A	No	N/A
	Intellectual Disability	Not assessed	N/A	Moderate to severe ID with mild autistic features	N/A
	Development	Delayed	N/A	Delayed	N/A
			Prominent occiput, absent
			fontanelle, large ears,
	Dysmorphisms	Hypotelorism, sloping forehead, and relatively large ears	wide nasal bridge, prominent upper lip, high corneas, a right-sided arched palate, cloudy	Sloping forehead, relatively large ears prominent nose, and	N/A
			sinlge palmar crease and
			small nails
	Status	Unknown	Deceased; one day after birth	Alive	Terminated pregnancy
	354"

  Coronal section of newborn control brain. Width is in centimeters (cm) and lateral ventricles

	372"
	373"	marked by red asterisks. (d) Lateral view of lissencephalic newborn brain from Proband B (scale
	374"	in cm). (e) Coronal section through mid-thalamus of Proband B brain showing enlarged lateral
	375"	ventricles (red asterisks). (f) Coronal brain MRI of Proband C at 10 years of age showing
	376"	microcephaly, simplified gyral pattern and enlarged ventricles (red asterisks). (g) Mid-sagittal
	377"	Proband C MRI showing sloping forehead, hypoplastic brainstem and cerebellum (red arrow)
	378"	(h) Coronal and (i) axial fetal brain MRI of affected subject II:2 of Family C at 29 GWs. Reduced
	379"	brain volume, gyrification and enlarged ventricles (red asterisks) were noted. (j) Control and (k)
	380"	Proband B cerebral cortex stained with hematoxylin and eosin (H&E). Compared to control (j)
	381"	leptomeninges (LM; blue arrow) are excessively thick and contiguous with molecular layer (ML;
	382"	pink arrow) in proband (k). Cortex (CX; dark purple arrow) is thickened and cortical layering
	383"	obscure when compared with control (j). (l) High magnification of Proband B cortex stained with
	384"	Kluver-Barrera. Disorganized parenchyma includes many multinucleated neurons: inserts show
	385"	detail of multinucleated (red double arrowheads) cells labeled 1,2,3. (m-n) Kluver-Barrera
	386"	stained sections of control (m) and Proband B (n) temporal lobe. The proband hippocampus (n)
	387"	is very small with hypoplastic pyramidal layer (red asterisk) and greatly reduced dentate gyrus
	388"	(arrowheads). Image of normal control hippocampus (m) imaged at half the magnification to
	389" 391"	allow for comparison of overall structure of the hippocampus. (o) Detail of Proband B dentate
	390"	gyrus (red box in H), showing binucleated granule cells (double arrowheads).

  Proband B Cit -/-Knockout Mouse (Di Cunto et al. 2000) Flathead fh/fh Mutant Rat (Sarkisian et al. 2002) (Ackman et al. 2007)

		Citron Kinase Mutation	10 bp deletion in exon 2	Conditional excision exon 2 by Cre mediated homologous recombination	Spontaneous G/C bp deletion in exon 1
		Predicted Mutational Impact	Frameshift causing a premature stop codon 25 amino acids from start site	Premature stop codon	Frameshift causing a premature stop 27 amino acids from start site
		CIT Transcript	Predicted NMD	NMD	NMD
		Brain Size and Weight	Microcephalic and ~1/10 of the brain weight of an average newborn	Microcephalic 50% brain weight reduction	50% brain size reduction
		Cerebral Cortex Abnormalities	Cortex shows low cellularity and disorganized three-layer arrangement. Presence of multinucleated neurons.	40% reduction in cerebral cortex thickness. Disorganized lamination of 6-layer cortex. Presence of binucleated cells.	Neocortex displays a reduced number of neurons but normal layering. Presence of binucleated neurons in neocortex.
		Cerebellar Abnormalities	Global hypotrophy and disrupted laminar architecture. Crowded layer of Purkinje cells with simple dendrite projections. and granule cells. Binucleated Purkinje cells	70% cerebellum size reduction. Crowded layer of Purkinje cells with simple dendrite projections. cells. Presence of binucleated	Cerebellum displays a reduced number of neurons. Presence of binucleated neurons.
			Small and disorganized	Normal Ammon's horn cell	Dentate gyrus displays a
		Hippocampal	Ammon's horns. Only a small	density and lamination.	reduced number of neurons.
		Abnormalities	remnant of dentate fascia.	Essentially absent dentate	Presence of binucleated
			Binucleated granule cells.	gyrus.	neurons.
		Additional Locations of Multinucleated Cells	Thalamus, striatum, brain stem and spinal cord	Thalamus	Striatum, thalamus, midbrain, hindbrain, and spinal cord
		Other Phenotypoic Abnormalities	Kidney and heart defects, facial dysmorphisms, and rotated lower limbs	Ataxia, seizures, and failure to thrive	Seizures and disrupted development of the retina
	426" 427"	NMD=Nonsense mediated decay

Acknowledgements

283"

We thank the families for their participation and contribution to this research. We appreciate

284"

Ritesh KC and Brian McGrath for stimulating discussion during manuscript preparation. Dr.

285"

Joseph Loturco provided feedback regarding CIT.

286"

287"

433"


Elisabeth Lajeunie

Solange Heuertz

Vincent El Ghouzzi

Jelena Martinovic

Dominique Renier

Martine Le Merrer

Jacky Bonaventure
email: bonavent@necker.fr

Mutation

Keywords: FGFR1, FGFR2, FGFR3, Pfeiffer syndrome, Crouzon syndrome, Jackson-Weiss syndrome, mutations

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Crouzon (MIM# 123500), Jackson-Weiss (MIM# 123150) and Pfeiffer syndromes (MIM# 101600) are three clinically related but distinct craniosynostoses with common clinical features including craniosynostosis, ocular hypertelorism with proptosis and midface hypoplasia. They differ by the absence in CS and presence in PS and JWS of limb abnormalities. Clinical distinction between PS and JWS is based on the presence in JWS of broad great toes with medial deviation and tarsal-metatarsal coalescence in the absence of hand anomalies, but clinical overlap between the two entities does exist, making the diagnosis difficult [START_REF] Cohen | Jackson-Weiss syndrome[END_REF]]. In 1993, Pfeiffer syndrome has been subdivided into 3 types [START_REF] Cohen | Pfeiffer syndrome update, clinical subtype and guidelines for differential diagnosis[END_REF]]. Type I, the most frequent form is usually associated with a benign course and satisfactory prognosis, whereas type II and III represent severe forms of the disease with a poor outcome and early demise in some cases. Subdivision was based on the presence (type II) or absence (type III) of cloverleaf skull.

In 1994, CS has been ascribed to de novo mutations in the FGFR2 gene [START_REF] Reardon | Mutations in the fibroblast growth factor receptor 2 gene cause Crouzon syndrome[END_REF]. FGFR2 belongs to a family of four receptors comprising an extracellular ligand-binding domain, a transmembrane domain and an intracellular domain carrying the tyrosine kinase activity. Further studies have shown that FGFR2 mutations also accounted for JWS and PS [START_REF] Jabs | Jackson-Weiss and Crouzon syndromes are allelic with mutations In fibroblast growth factor receptor 2[END_REF][START_REF] Lajeunie | FGFR2 mutations in Pfeiffer syndrome[END_REF][START_REF] Rutland | Identical mutations in the FGFR2 gene cause both Pfeiffer and Crouzon syndrome phenotypes[END_REF], but genetic heterogeneity of this latter syndrome was demonstrated by the identification in several families of a recurrent FGFR1 mutation causing mild forms of the disease [START_REF] Muenke | A common mutation in the fibroblast growth factor receptor 1 gene in Pfeiffer syndrome[END_REF][START_REF] Schell | Mutations in FGFR1 and FGFR2 cause familial and sporadic Pfeiffer syndrome[END_REF]. Sporadic cases of CS and PS have been associated with advanced paternal age and the origin of FGFR2 mutations in these two conditions, like in Apert syndrome, was demonstrated to be exclusively paternal [START_REF] Moloney | Exclusive paternal origin of new mutations in Apert syndrome[END_REF][START_REF] Glaser | Paternal origin of FGFR2 mutations in sporadic cases of Crouzon Syndrome and Pfeiffer syndrome[END_REF].

Surprisingly, the same FGFR2 mutation can give rise to Crouzon, Jackson-Weiss or Pfeiffer syndrome [START_REF] Rutland | Identical mutations in the FGFR2 gene cause both Pfeiffer and Crouzon syndrome phenotypes[END_REF][START_REF] Tartaglia | Trp290cyst mutation in exon IIIa of the fibroblast growth factor receptor 2 (FGFR2) gene is associated with Pfeiffer syndrome[END_REF]. Until 2002, FGFR2 mutations had been identified exclusively in the extracellular domain of the receptor mainly in exons 8 (IIIa) and 10 (IIIc) encoding the third immunoglobulin-like (Ig III) loop and appeared to account for only 50% of CS and PS cases [START_REF] Passos-Bueno | Clinical spectrum of Fibroblast Growth Factor Receptor mutations[END_REF]. In 2002, novel mutations in other regions of the receptor including the IgII loop and the tyrosine kinase sub-domains TK1 and TK2 have been reported [START_REF] Kan | Genomic screening of fibroblast growth factor receptor 2 reveals a wide spectrum of mutations in patients with syndromic craniosynostosis[END_REF]. Likewise, this study demonstrated that in a clinically homogeneous group of CS and PS patients, FGFR2 mutations were detectable in more than 90% of all cases, thus making the existence of an additional locus [START_REF] Passos-Bueno | Clinical spectrum of Fibroblast Growth Factor Receptor mutations[END_REF] very unlikely. A recurrent mutation, A391E, in a third gene, FGFR3, accounts for a peculiar form of CS associating craniosynostosis with acanthosis nigricans [Meyers et al.,95].

Molecular screening in a large cohort of CS, JWS and PS patients diagnosed in our hospital was performed. The diagnosis was based on the clinical presentation of the proband and family members. Mutations in one of the three FGFR genes (FGFR 1, 2 or 3) were found in 94% of our cases. Five novel FGFR2 mutations were identified. FGFR2 mutations, although clustered mainly in exons 8 and 10, were also present with a lower frequency in exons 3, 14 and 16. The recurrent FGFR3 mutation A391E in exon 10 caused CS with acanthosis nigricans whereas the P252R FGFR1 mutation produced PS with slight facial anomalies and absence of neurological defects. Altogether our results confirm that CS and JWS are genetically homogeneous at the FGFR2 locus whereas PS is heterogeneous, being caused by FGFR1 or FGFR2 mutations. Clinical and radiological examination of PS patients further revealed that FGFR2 mutations creating cysteine residues at positions 290 and 340 are associated with the most severe phenotypes.

PATIENTS AND METHODS

Patients and samples

During the past 10 years, clinical diagnosis and surgical correction of the skull deformation in patients with syndromic craniosynostoses was achieved by the same team of physicians and surgeons at the Craniofacial Surgery Department of the Hôpital Necker-Enfants Malades. Each patient showed characteristic clinical features including synostosis of one or several cranial sutures, ocular proptosis, maxillary hypoplasia and midface retrusion. Crouzon patients were distinguishable from PS and JWS by the absence of hand and foot anomalies. Among 2594 children with proven craniosynostosis, 116 children from 100 families were diagnosed as Crouzon patients and 35 children from 33 families as Pfeiffer patients. Diagnosis of JWS was established in two patients. In our series, the birth prevalence of CS was 1/50,000 and this entity appeared to account for 4.5 % of all craniosynostoses. PS was less frequent with an estimated birth prevalence of 1/150,000 and accounted for 1.3 % of all craniosynostoses. DNA samples were obtained from eighty four patients with Crouzon, Jackson-Weiss or Pfeiffer syndromes. Informed consents for molecular studies and photographs were obtained from all patients or their parents.

Mutation analysis

Molecular analysis was performed on a cohort of patients comprising 62 unrelated CS (24 familial cases and 38 sporadic cases) including two patients with acanthosis nigricans (one sporadic and one familial case), two familial cases of JWS and 20 unrelated PS (5 familial cases and 15 sporadic cases).

PCR amplification of genomic DNA was performed using previously described primers and conditions [START_REF] Lajeunie | FGFR2 mutations in Pfeiffer syndrome[END_REF][START_REF] Kan | Genomic screening of fibroblast growth factor receptor 2 reveals a wide spectrum of mutations in patients with syndromic craniosynostosis[END_REF]. PCR fragments were directly sequenced on an ABI 3100 capillary sequencer (Applied Biosystems) with BigDye terminator mix. Since FGFR2 mutations preferentially occur in exons 8 and 10, these two exons were tested first.

Novel mutations were validated by sequencing exons 8 and 10 in 65 control genomic DNAs. When no mutation was found, screening of other exons was undertaken. Exon numbering for FGFR2 was based on a recently proposed nomenclature [Ingersoll et al 2002]. GenBank accession numbers for FGFR1: BC015035; FGFR2: AF410480; FGFR3: AY768549

RT-PCR amplifications

For RT-PCR studies, total RNA was extracted from cultured skin fibroblasts using the RNeasy Mini Kit (Qiagen). Complementary DNA was synthesized by priming with either random hexamers or oligo-dT in the presence of MuLV reverse transcriptase using the manufacturer's protocol (GeneAmp RNA PCR Core Kit, Roche). Twenty five to 40 PCR cycles were then performed to amplify fragments specific for either the IIIb isoform (exon 9) or the IIIc isoform (exon 10) of FGFR2 by using the following primers: 5'-AAGCACTCGGGGATAAATAG-3' (F) and 5'-GTTTTGGCAGGACAGTGAGC-3' (R) for exon 9 ; 5'-CACAGTGGTCGGAGGAGA-3' (F) and 5'-AGTTACATTCCGAATATAGAG-3' (R) for exon 10. Sense and antisense primers used for GAPDH amplification were as follows: 5'-CATGTGGGCCATGAGGTCCACCAC-3' and 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3'. Samples were analysed on 1% agarose gels.

Specificity of all RT-PCR products was tested by direct sequencing

RESULTS

Mutation screening was performed by direct sequencing of FGFR1, 2 and 3 genes. In FGFR1 and FGFR3 genes, sequence analyses were restricted to exons 7 and 10 respectively, whereas 16 exons of the FGFR2 gene were studied. FGFR mutations were detected in 79/84 (94%) unrelated patients (Table 1). The recurrent FGFR1 mutation P252R was recorded in 3 Pfeiffer cases (one familial and two sporadic). In two Crouzon patients with acanthosis nigricans, the recurrent A391E FGFR3 mutation was identified. All other mutations causing CS, JWS and PS were found in the FGFR2 gene.

As expected, mutations in exons 8 and 10 of the FGFR2 gene were largely predominant in our cohort, representing 92% (68/74) of the overall FGFR2 mutations. Among these 68 mutations, 36 (53%) either created or eliminated cysteine residues, thus generating unpaired cysteine able to induce disulfide bond formation between two mutant receptors. Mutations outside exons 8 and 10 (6/74) were located in either exon 3 encoding the first Ig-like loop (Y105C) or exons 14 and 16 encoding the tyrosine kinase sub-domains. Mutations N549H in the TK1 and K659N in the TK2 domains are homologous, respectively, to the N540K and K650N FGFR3 mutations causing hypochondroplasia. In 5 patients (4 CS and 1 PS) no FGFR2 mutation was found.

Analysis of exon 7 of FGFR1, exons 7 and 10 of FGFR3 and exon 1 of TWIST also failed to reveal an abnormal sequence in those cases.

CROUZON PATIENTS

Missense substitutions were detected in 57/60 unrelated cases including 21 familial forms and 36 sporadic cases. In familial forms, presence of the single base change was confirmed by DNA sequencing of at least one additional affected member.

The Y105C mutation in exon 3 was found in one familial case. Three other family members carried the same substitution. Sixteen missense mutations corresponding to 10 distinct heterozygous amino-acid changes were identified in exon 8. Two of these mutations (I288N and Y308C) are novel (Table 2). They were found neither in the DNA of unaffected parents, nor in 65 control DNA samples. Thirty three mutations corresponding to 12 distinct amino-acid changes occurred in exon 10. The most frequent mutation C342Y was detected in 12 unrelated cases (22%). The L357S mutation is reported for the first time.

Clinical re-examination of Crouzon patients carrying FGFR2 mutations showed that proptosis although variable was systematically present in all patients. Coronal sutures were the most frequently affected as 86% of patients presented bicoronal fusions (brachycephaly), 18% exhibited both coronal and sagittal fusions (oxycephaly) and 5% showed pansynostosis (fusion of all sutures). In the remaining 9%, fusion was restricted to the sagittal suture (scaphocephaly) or the sagittal and lambdoids.

A close examination of patients carrying novel FGFR2 mutations revealed that the Y308C mutation occurred in a girl with a typical form of CS. The L357S mutation was detected in a familial case and segregated with the disease. The proband was a 12 year-old girl with pansynostosis and chronic tonsillar herniation. Her older brother who had sagittal and bicoronal synostosis had never required surgery because facial anomalies were mild.

JACKSON-WEISS PATIENTS

An heterozygous dinucleotide deletion (AC) at position 958-959 was detected in exon 10 of FGFR2 in a familial form of JWS with typical tarsal/metatarsal coalescence in the absence of hand anomalies (Fig. 1a-d). This frameshift mutation that occurred in a mother and her son, was predicted to induce translation of four illegitimate aminoacids, immediately followed by a premature termination codon (TGA) at position 324 of the receptor (Fig. 2b). RT-PCR analysis of mRNA extracted from fibroblasts of the affected mother, revealed an absence of the mutant transcripts, indicating that the 2bp deletion induced RNA instability of the IIIc transcripts (Fig. 2a). Interestingly, ectopic expression of the FGFR2 IIIb transcripts was observed in the mother's fibroblasts (Fig. 2c) suggesting that illegitimate expression of the IIIb isoform in cells of mesenchymal origin might account for the phenotype.

PFEIFFER PATIENTS

FGFR mutations were detected in 19/20 (95%) Pfeiffer samples. Three patients carried the recurrent P252R FGFR1 substitution that gave rise to a relatively mild phenotype (Table 3). Sixteen patients including 4 familial forms and 12 sporadic cases harbored a FGFR2 mutation. Although variable, the phenotype was more severe (Fig. 1j) than in patients carrying FGFR1 mutations as attested by marked facial deformities, common hydrocephaly (in 10/16 patients), mental retardation and premature death (Table 3). In exon 8, the W290C FGFR2 mutation was identified in three cases, two of them died prematurely and the third was prenatally diagnosed by ultrasound examination at 29 weeks of gestation. Radiological and clinical examination after termination of pregnancy confirmed the diagnosis of Pfeiffer type II with very severe proptosis, cloverleaf skull and humero-radio-ulnar synostosis (Fig. 1k). In exon 10, we found 11 mutations of 6 different types including the common splice mutation (940-2A→G) that creates a cryptic donor site. Both patients harboring the Y340C mutation had a severe phenotype consistent with the diagnosis of Pfeiffer type II. One case died in the early childhood of respiratory distress while the second case was prenatally diagnosed and pregnancy was terminated at 25 weeks of gestation. Radiological examination showed cloverleaf skull, elbow ankylosis with bilateral humero-radio-ulnar synostosis, broad and deviated big toes and vertebral anomalies including sacrococcygeal eversion (Fig. 1e-g)

Mutations in exons 14 and 16 encoding the tyrosine kinase domains TK1 and TK2 were identified in two sporadic cases. Mutation in the TK2 domain was associated with a more severe phenotype (Fig. 1h,i) than in the TK1.

DISCUSSION

We have carried out a molecular study of three FGFR genes in a large series of 84 cases with CS, JWS and PS. Based on previous studies [START_REF] Kan | Genomic screening of fibroblast growth factor receptor 2 reveals a wide spectrum of mutations in patients with syndromic craniosynostosis[END_REF][START_REF] Cornejo-Roldan | Analysis of the mutational spectrum of the FGFR2 gene in Pfeiffer syndrome[END_REF] and our own experience, the mutation detection rate appears to depend largely on the accuracy of the original diagnosis and the sensitivity of the detection method. In our study, all patients were examined by the same physicians so that the clinical criteria for phenotypic classification were highly homogeneous allowing recognition of the Crouzonoid facies.

Based on our clinical diagnosis and using direct sequencing, we identified FGFR2 mutations in 95% (57/60) of our Crouzon patients. This situation is similar to two previous studies [START_REF] Kress | Clustering of FGFR2 gene mutations in patients with Pfeiffer and Crouzon syndromes (FGFR2associated craniosynostoses)[END_REF][START_REF] Kan | Genomic screening of fibroblast growth factor receptor 2 reveals a wide spectrum of mutations in patients with syndromic craniosynostosis[END_REF] in which mutations were found in 25/28 and 18/20 Crouzon patients respectively. In PS, we found 95% of mutations in our cohort of 20 patients. A similar percentage was obtained in the Oxford series [START_REF] Kan | Genomic screening of fibroblast growth factor receptor 2 reveals a wide spectrum of mutations in patients with syndromic craniosynostosis[END_REF]. In contrast, Cornejo-Roldan et al. [1999] studying a total of 78 unrelated Pfeiffer patients identified FGFR mutations in only 40 cases (51%). The difference between the three studies could rely on the stringency of clinical diagnosis. In the Cornejo-Roldan's group, the criteria for inclusion in the Pfeiffer group might be too broad. Hence, the presence of mild radiological findings like shortened middle phalanges could be insufficient to make a diagnosis of PS.

Although previous studies have failed to disclose genotype-phenotype relationships in craniosynostoses caused by FGFR2 mutations, analysis of amino-acid substitutions eliminating cysteine 342 in our cohort of patients suggest that the phenotype relies on the nature of the newly created residue. Tyrosine would preferentially cause CS (12/12) while serine, arginine or tryptophane would mainly account for PS (5/6). A similar situation exists in FGFR3-related skeletal dysplasias.

Substitutions of lysine 650 by methionine or glutamic residues give rise to thanatophoric dysplasia or SADDAN whereas replacement of lysine by glutamine, asparagine or threonine generates hypochondroplasia, a much milder condition [START_REF] Bellus | Identical mutations in three different fibroblast growth factor receptor genes in autosomal dominant craniosynostosis syndromes[END_REF][START_REF] Bellus | Distinct missense mutations of the FGFR3 Lys 650 codon modulate receptor kinase activation and the severity of the skeletal dysplasia phenotype[END_REF]. Differences in the receptor activation levels due to conformational changes of the receptor three-dimensional structure induced by amino-acids of variable charge and size could explain this result.

Mutations affecting tryptophan 290 were also of particular interest. Three different amino-acid substitutions have been recorded W290C, W290G and W290R. The four patients carrying W290G and W290R substitutions had Crouzon syndrome. Similarly, four previously reported patients with the same mutations have been diagnosed as Crouzon [START_REF] Passos-Bueno | Clinical spectrum of Fibroblast Growth Factor Receptor mutations[END_REF]. By contrast, the three patients carrying the W290C mutation in our series had a severe sub-lethal form of Pfeiffer syndrome. The same mutation has been described in at least seven additional patients. Six of them had a severe form of PS with cloverleaf skull and severe proptosis and two of them died prematurely [START_REF] Tartaglia | Trp290cyst mutation in exon IIIa of the fibroblast growth factor receptor 2 (FGFR2) gene is associated with Pfeiffer syndrome[END_REF][START_REF] Schaefer | Novel mutation in the FGFR2 gene at the same codon as the Crouzon syndrome mutations in a severe Pfeiffer syndrome type 2 case[END_REF][START_REF] Kan | Genomic screening of fibroblast growth factor receptor 2 reveals a wide spectrum of mutations in patients with syndromic craniosynostosis[END_REF][START_REF] Zackai | Craniosynostosis with tracheal sleeve: a patient with Pfeiffer syndrome, tracheal sleeve and additional malformations in whom an FGFR2 mutation was found[END_REF][START_REF] Nazarro | Prenatal ultrasound diagnosis of a case of Pfeiffer syndrome without cloverleaf skull and review of the literature[END_REF]. The seventh patient presented a severe non classifiable craniosynostosis syndrome with limb and joint anomalies and mental retardation [START_REF] Shotelersuk | Distinct craniofacial-skeletal-dermatological dysplasia in a patient with W290C mutation in FGFR2[END_REF]. Taken together, these observations strongly suggest that conversion of tryptophane 290 into cysteine is likely to induce formation of disulfide bonded dimer receptors resulting in a severe phenotype, mostly Pfeiffer syndrome type II [START_REF] Cohen | Pfeiffer syndrome update, clinical subtype and guidelines for differential diagnosis[END_REF], characterized by marked disabling and early death.

Of note was the relatively uncommon Y340C substitution. This mutation has been previously described in three patients with PS [START_REF] Cornejo-Roldan | Analysis of the mutational spectrum of the FGFR2 gene in Pfeiffer syndrome[END_REF][START_REF] Kan | Genomic screening of fibroblast growth factor receptor 2 reveals a wide spectrum of mutations in patients with syndromic craniosynostosis[END_REF][START_REF] Blaumeiser | Prenatal diagnosis of Pfeiffer syndrome type II[END_REF] and in our series, it was identified in two PS cases.

Both exhibited a very severe form of the disease with cloverleaf skull, severe ocular proptosis, hydrocephalus, abnormal great toes and thumbs consistent with the diagnosis of PS type II. Premature death occurred in one case. It is tempting to conclude that this mutation, like the W290C substitution, is preferentially associated with severe forms of PS with a poor prognosis regarding neurological functions and survival.

Interestingly, another amino-acid substitution creating an unpaired cysteine residue, namely S351C, also results in severe forms of Pfeiffer syndrome (type III) in most reported cases [START_REF] Gripp | Phenotype of the fibroblast growth factor receptor 2 Ser 351Cys mutation: Pfeiffer syndrome type III[END_REF][START_REF] Sweeney | Sacral appendage associated with a mutation in FGFR2[END_REF]. Although confusion might exist between PS type III and the so-called 'Antley-Bixler-like' phenotype, we consider that the heterozygous S351C FGFR2 mutation is a hallmark of severe forms of PS, as typical forms of Antley-Bixler syndrome are caused by recessive mutations in the P450 oxido-reductase (POR) gene [START_REF] Fluck | Mutant P450 oxidoreductase causes disordered steroidogenesis with and without Antley-Bixler syndrome[END_REF].

Unlike FGFR3, mutations in the tyrosine kinase subdomains of FGFR2 have been described only recently [START_REF] Kan | Genomic screening of fibroblast growth factor receptor 2 reveals a wide spectrum of mutations in patients with syndromic craniosynostosis[END_REF] and since, only one additional case has been reported [START_REF] Zankl | Novel mutation in the tyrosine kinase domain of FGFR2 in a patient with Pfeiffer syndrome[END_REF]. In our series, five additional cases were identified including three CS and two PS. In keeping with previous data, the K641R mutation caused PS. The N549H mutation caused both CS and a mild form of PS suggesting that mutations in the TK domains are phenotypically less deleterious than mutations creating cysteine residues in the extracellular domain.

Although no definitive genotype-phenotype relationships can be drawn from the literature on craniosynostoses and FGFR genes, our results suggest that some specific FGFR2 residues namely tryptophan 290 and tyrosine 340 account for the most severe forms of Pfeiffer syndrome when converted into cysteine residues.

Legends

Figure 1 .

 1 Figure 1. Phenotype of a mother and her son with Jackson-Weiss syndrome. Both patients have brachycephaly, midface hypoplasia, facial dysmorphism and proptosis (a, b). Note partial tarsal fusion and extra delta-shaped small bone between the first and the second phalanx of the left hallux in the boy (at 15 years) and complete tarsal fusion in the mother

Figure 2 .

 2 Figure 2. Direct sequencing of genomic DNA from the JWS mother showing a heterozygous frameshift deletion (delAC) in exon 10 of FGFR2 (b). The normal sequence of the RT-PCR product derived from the patient fibroblasts indicates that the mutant allele is not detectable at the cDNA level (a). RT-PCR amplification of the IIIc isoform of FGFR2 encoded by exon 10 gives a signal both in the patient and control cells whereas amplification of the IIIb isoform encoded by exon 9 gives a signal in the patient's fibroblasts only (c). M : affected mother; C : control

TABLE 1 .

 1 Patients analyzed for FGFR mutations

	Clinical	Number of	Patients with	Patients with	Patients with	Patients with
	diagnosis	patients	FGFR2	FGFR1	FGFR3	no FGFR
			mutation	mutation	mutation	mutation
	Crouzon	60	57 (95%)	0	0	3 (5%)
	syndrome
	Pfeiffer	20	16 (80%)	3 (15%)	0	1 (5%)
	syndrome
	Crouzon	2	0	0	2	0
	syndrome
	with A.N. *
	Jackson-	2	1	0	0	1
	Weiss
	syndrome
	Total	84	74 (88%)	3 (3.5%)	2 (2.5%)	5 (6%)

* AN = acanthosis nigricans

ACKNOWLEDGMENTS

We thank patients and their families for their participation in this study.