Maurizia Baldi

Clinical impact of MEFV mutations in children with periodic fever in a prevalent western European Caucasian population

Objective To evaluate the actual impact of MEFV mutations on clinical manifestations associated with fever attacks in Caucasian children with periodic fever. Methods 113 children carrying MEFV mutations (44 with mutations in two alleles, 69 heterozygous) and 205 children negative for mutations in genes associated with periodic fevers were analysed. The following groups of patients were considered: patients carrying two high penetrance mutations (M694V, M694I, M680I); one high, one low penetrance mutation; two low penetrance mutations; one high penetrance mutation; one low penetrance mutation; genetically negative patients. Results Patients with two MEFV mutations displayed a shorter duration of fever attacks and higher prevalence of a positive family history than patients carrying one MEFV mutation and genetically negative patients. Severe abdominal pain, chest pain and pleurisy were also more frequent in patients with two MEFV mutations compared with children with one MEFV mutation and genetically negative patients. Conversely, a higher frequency of exudative and erythematous pharyngitis, enlargement of cervical lymph nodes, aphthous stomatitis and non-specific skin rash was observed in genetically negative patients and, to a lesser extent, in patients with one MEFV mutation. The frequency of ‘familial Mediterranean fever (FMF)-like symptoms’ decreases from patients carrying two high penetrance mutations towards patients with a single low penetrance mutation with an opposite trend for ‘periodic fever, aphthous stomatitis, pharyngitis, adenitis-like symptoms’. Conclusions This clinical observation supports recent findings contrasting the notion of FMF being a pure autosomal recessive disorder associated with recurrence of mutations leading to loss of protein function. A dosage effect could be invoked, giving rise to symptom onset even in the presence of one wild-type allele.

Sonographic and molecular diagnosis of thanatophoric dysplasia type I at 18 weeks of gestation

Thanatophoric dysplasia is the most common type of lethal skeletal dysplasia. It can usually be diagnosed with ultrasound, but differential diagnosis with other osteochondrodysplasias is not always possible. Mutations in the fibroblast growth factor receptor 3 (FGFR3) gene have been demonstrated to cause two distinct subtypes of the disorder. We describe a case of thanatophoric dysplasia type I diagnosed at 18 weeks of gestation by ultrasonography. Genomic DNA obtained by chorionic villus sampling showed a C to G substitution at position 746 in the FGFR3 gene, resulting in a Ser249Cys substitution already known to be associated with type I disease. Implications for perinatal management are discussed. Copyright

Germline mosaicism in achondroplasia detected in sperm DNA of the father of three affected sibs

We describe a sib recurrence for achondroplasia with parents of average stature. The three sibs shared the paternal allele and all carried the same causal mutation in the fibroblast growth factor receptor 3 gene (FGFR3): G > A nt1138 (Gly380Arg). We were able to identify this mutation on sperm DNA confirming paternal germinal mosaicism. Our family shows that a more precise definition of the recurrence risk is feasible using this approach, based on a single DNA test, which could be offered in selected cases.

Next-generation sequencing and its initial applications for molecular diagnosis of systemic auto-inflammatory diseases

Objectives Systemic auto-inflammatory disorders (SAIDs) are a heterogeneous group of monogenic diseases sharing a primary dysfunction of the innate immune system. More than 50% of patients with SAID does not show any mutation at gene(s) tested because of lack of precise clinical classification criteria and/or incomplete gene screening. To improve the molecular diagnosis and genotype interpretation of SAIDs, we undertook the development of a next-generation sequencing (NGS)-based protocol designed to simultaneous screening of 10 genes. Methods Fifty patients with SAID, already genotyped for the respective causative gene(s), were massively sequenced for the coding portions of MEFV, MVK, TNFRSF1A, NLRP3, NLRP12, NOD2, PSTPIP1, IL1RN, LPIN2 and PSMB8. Three different bioinformatic pipelines (Ion Reporter, CLC Bio Genomics Workbench, GATK-based in-house workflow) were compared. Results Once resulting variants were compared with the expected mutation list, no workflow turned out to be able to detect all the 79 variants known in the 50 DNAs. Additional variants were also detected, validated by Sanger sequencing and compared to assess true and false positive detection rates of the three workflows. Finally, the overall clinical picture of 34 patients was re-evaluated in the light of the new mutations found. Conclusions The present gene panel has resulted suitable for molecular diagnosis of SAIDs. Moreover, genotype–phenotype correlation has confirmed that the interpretation of NGS data in patients with an undefined inflammatory phenotype is remarkably difficult, thus supporting the need of evidence-based and validated clinical criteria to be used concurrently with the genetic analysis for the final diagnosis and classification of patients with SAIDs.

Craniosynostosis: prenatal diagnosis by means of ultrasound and SSSE-MRI. Family series with report of neurodevelopmental outcome and review of the literature

IntroductionCraniosynostosis is a condition characterized by a premature closure of one or more skull sutures and refers to a wide spectrum of cranial malformation with an estimated birth of 1:2,000–1:4,000 live births. Four receptors (FGFR 1, FGFR 2, FGFR 3, FGFR 4) involving mutation in the fibroblast growth factor have been identified.Materials and methodsTwo cases occurred in the same family and diagnosed prenatally by means of ultrasound, and antenatal and postnatal MR imaging are reported. Molecular biology regarding identification of craniosynostosis type has been analyzed. A revision of the medical literature is also provided.ConclusionThe premature closure of sagittal suture is characterized by a disproportionately large occipito-frontal and short biparietal diameter (scaphocephaly). The prenatal ultrasound diagnosis of craniosynostosis in utero may be difficult and be suspected when the cephalic index, the cranial shape or the fetal face shape are abnormal. Fetal karyotype is recommended and DNA testing plays a critical role in achieving an appropriate diagnosis, when possible. The prognosis of craniosynostosis is primarily dependent on the presence of associated anomalies as craniosynostosis are correlated with three to fivefold increased risk for cognitive disabilities.

Craniosynostosis, hydrocephalus, Chiari I malformation and radioulnar synostosis: probably a new syndrome.

We report on clinical and molecular findings of two brothers that both presented with sagittal craniosynostosis, hydrocephalus, Chiari I malformation, blepharophimosis, small low-set ears, hypoplastic philtrum, radioulnar synostosis, kidney malformation, and hypogenitalism. Their father presented mild brachydactyly. Conventional cytogenetic and array CGH screening did not show any chromosomal gains or losses. Furthermore, molecular genetic screening of genes involved in different craniosynostosis syndromes, namely FGFR1, FGFR2, FGFR3, TWIST, RECQL4, and POR genes failed to detect any mutations in genomic DNA. The unique range of clinical manifestations in these two patients and the negative findings of the molecular genetic screening suggest the hypothesis of a previously unrecognized syndrome.

Pfeiffer syndrome type 2 associated with a single amino acid deletion in the FGFR2 gene

To the Editor: Pfeiffer syndrome (PS) is an autosomal dominant disease characterised by coronal craniosynostosis, midface hypoplasia, broad thumbs and great toes, brachydactyly and a variable degree of syndactyly (1). PS type 2 patients have cloverleaf skull, severe proptosis, central nervous system involvement, broad and deviated thumbs and halluces. Prognosis is unfavourable with early death (2, 3). Mutations in FGFR1 and FGFR2 have been found in patients affected with PS. A single missense mutation in exon 5 of FGFR1 in the IgII–IgIII linker region causes familial PS (4). The mutations found in FGFR2 are missense in frame insertions or splice-site mutations (5–17). Two mutational hot spots have been identified in FGFR2 involving cysteine residues at codons 290 and 342, respectively (15). Different missense mutations at these codons have been found in patients affected with Crouzon syndrome, Jackson–Weiss syndrome and PS (15). One 18 bp in frame deletion has been found in a PS sporadic case (17). All the affected codons are located within the first segment of the Ig-like loop (IIIa) (17, 18) with the exception of the 342 codon. The patient reported here is affected with PS type 2 and carries an unknown mutation in FGFR2. He was admitted at 1 month of age to the Paediatric Neurosurgery Department because of craniofacial dysmorphism and neurological signs of intracranial hypertension. Severe acrocephalotrygonocephaly with cloverleaf skull, flat occipitus, downward displacement of the ears to a horizontal position with respect to the neck and severe ocular proptosis were noticed. Radial clinodactyly of the thumbs and valgus deviation of the halluces were also present. Computerised tomographic and magnetic resonance imaging investigations showed progressive triventricular hydrocephalus, callosal dysgenesis and Chiari I malformation. A cerebrospinal fluid ventriculo-peritoneal shunt was applied. Later, a cranioplastic was performed. Molecular analysis was performed on the FGFR2 DNA fragment encoding for the extracellular region containing the first half of the third Ig-like domain (IIIa) and a portion of the linker region between Ig-like domains I and III. Amplification of the relevant fragment by polymerase chain reaction (PCR) was performed using previously described primers (19), resulting in a 349-bp segment which contained FGFR2 exon 7 (IIIa). PCR products were sequenced directly in both directions by cycle sequencing method using a-33P radiolabelled dideoxynucleotides and the Thermo Sequenase Kit (Amersham Pharmacia Biotech, Milan, Italy). Sequencing analysis showed an ‘in frame’ 3-bp deletion (GAC), which removes the aspartic amino acid at position 273 (DD273). In order to confirm the result, ASO dot blot analysis by hybridisation was carried out (Fig. 1) according to standard procedures using the following g-32P end-labelled allele-specific oligonucleotides: 5%-CGG AGG AGA CGT AGA GTT T-Y as wild-type probe and 5%-CGG AGG AGT AGA GTT WT C-3% as mutant probe. Sequencing and ASO analysis were also performed on both parents, neither of whom carried the DD273. No ASO mutant cases were found in 60 control normal chromosomes. The present case is the first example of a minimal deletion affecting FGFR and associated with craniosynostosis. The deletion involves a negatively charged amino acid and is located in the IgII–IgIII linker region which consists of a sequence of 16 amino acids identical for all human fibroblast growth factor receptors highly conserved among different species (18, 20–22). The 273 aspartic acid is a highly conserved residue (18) and is very close to the first cysteine critical in the intramolecular bridge that defines the IgIIIl loop (18, 21, 23). The deletion of this single polar amino acid might affect the correct disulphide bridge formation, as previously hypothesised for the Phe276Val missense mutation (16) or, on the other hand, it might affect ligand binding (24). Single,

Association of achondroplasia with sagittal synostosis and scaphocephaly in two patients, an underestimated condition?

We report on two patients with an unusual combination of achondroplasia and surgically treated sagittal synostosis and scaphocephaly. The most common achondroplasia mutation, p.Gly380Arg in fibroblast growth factor receptor 3 (FGFR3), was detected in both patients. Molecular genetic testing of FGFR1, FGFR2, FGFR3 and TWIST1 genes failed to detect any additional mutations. There are several reports of achondroplasia with associated craniosynostosis, but no other cases of scaphocephaly in children with achondroplasia have been described. Recently it has been demonstrated that FGFR3 mutations affect not only endochondral ossification but also membranous ossification, providing new explanations for the craniofacial hallmarks in achondroplasia. Our report suggests that the association of isolated scaphocephaly and other craniosynostoses with achondroplasia may be under recognized.

A de novo balanced translocation t(7;12)(p21.2;p12.3) in a patient with Saethre–Chotzen-like phenotype downregulates TWIST and an osteoclastic protein-tyrosine phosphatase, PTP-oc

Saethre-Chotzen syndrome (SCS) is an autosomal dominant craniosynostosis syndrome with variable expression. Here we report on a female infant with a de novo balanced translocation 46, XX, t(7;12)(p21.2;p12.3) and presenting at birth brachycephaly, antimongolic palpebral fissures, ocular hypertelorism, broad nose with low nasal bridge and low-set ears. This phenotype is suggestive of a subtle form of SCS, given the absence of limbs anomalies. Cloning of both breakpoints revealed that the translocation does not interrupt the TWIST1 coding region, on 7p21, known to be causative for SCS, but downregulates TWIST1 expression due to a position effect. On chromosome 12, the breakpoint translocates a shorter transcript of PTPRO gene, the osteoclastic protein-tyrosine phosphatase, PTP-oc, near to regulatory region of 7p leading to down-regulation of PTP-oc in the probands fibroblasts. This is a confirmatory case report providing further evidence for TWIST1 haploinsufficiency in SCS, although a possible role of PTP-oc as genetic factor underlying or at least influencing the development of craniosynostosis could not be a priori excluded.

Dysmorphic choroid plexuses and hydrocephalus associated with increased nuchal translucency: early ultrasound markers of de novo thanatophoric dysplasia type II with cloverleaf skull (Kleeblattschaedel).

Prenatal diagnosis of thanatophoric dysplasia (TD) type II presenting in the first trimester with increased nuchal translucency (NT) and cloverleaf skull (Kleeblattschaedel) have been scantly reported in the medical record. Abnormal choroid plexus has been seen in association with fetal anomalies. Here we described a case of increased NT associated with indented choroid plexuses, early onset hydrocephalus and cloverleaf skull in a fetus subsequently diagnosed at early second trimester to carry a de novo mutation encoding for TD type II. The findings of dysmorphic choroid plexus, early onset hydrocephalus and cloverleaf skull at first trimester scan may be early, useful ultrasound markers of TD type II. Molecular analysis to control for possible overlapping syndromes were performed and resulted negative. Postmortem X‐ray and 3D‐CT scan confirmed the cloverleaf skull, narrow thorax, straight femur with rhizomelic shortening of the limbs and the presence of a communicating hydrocephalus.