Caroline Michot

Cardio-facio-cutaneous and Noonan syndromes due to mutations in the RAS/MAPK signalling pathway: genotype–phenotype relationships and overlap with Costello syndrome

Cardio-facio-cutaneous (CFC) syndrome, Noonan syndrome (NS), and Costello syndrome (CS) are clinically related developmental disorders that have been recently linked to mutations in the RAS/MEK/ERK signalling pathway. This study was a mutation analysis of the KRAS, BRAF, MEK1 and MEK2 genes in a total of 130 patients (40 patients with a clinical diagnosis of CFC, 20 patients without HRAS mutations from the French Costello family support group, and 70 patients with NS without PTPN11 or SOS1 mutations). BRAF mutations were found in 14/40 (35%) patients with CFC and 8/20 (40%) HRAS-negative patients with CS. KRAS mutations were found in 1/40 (2.5%) patients with CFC, 2/20 (10%) HRAS-negative patients with CS and 4/70 patients with NS (5.7%). MEK1 mutations were found in 4/40 patients with CFC (10%), 4/20 (20%) HRAS-negative patients with CS and 3/70 (4.3%) patients with NS, and MEK2 mutations in 4/40 (10%) patients with CFC. Analysis of the major phenotypic features suggests significant clinical overlap between CS and CFC. The phenotype associated with MEK mutations seems less severe, and is compatible with normal mental development. Features considered distinctive for CS were also found to be associated with BRAF or MEK mutations. Because of its particular cancer risk, the term “Costello syndrome” should only be used for patients with proven HRAS mutation. These results confirm that KRAS is a minor contributor to NS and show that MEK is involved in some cases of NS, demonstrating a phenotypic continuum between the clinical entities. Although some associated features appear to be characteristic of a specific gene, no simple rule exists to distinguish NS from CFC easily.

Exome Sequencing Identifies PDE4D Mutations as Another Cause of Acrodysostosis

Acrodysostosis is a rare autosomal-dominant condition characterized by facial dysostosis, severe brachydactyly with cone-shaped epiphyses, and short stature. Moderate intellectual disability and resistance to multiple hormones might also be present. Recently, a recurrent mutation (c.1102C>T [p.Arg368*]) in PRKAR1A has been identified in three individuals with acrodysostosis and resistance to multiple hormones. After studying ten unrelated acrodysostosis cases, we report here de novo PRKAR1A mutations in five out of the ten individuals (we found c.1102C>T [p.Arg368(∗)] in four of the ten and c.1117T>C [p.Tyr373His] in one of the ten). We performed exome sequencing in two of the five remaining individuals and selected phosphodiesterase 4D (PDE4D) as a candidate gene. PDE4D encodes a class IV cyclic AMP (cAMP)-specific phosphodiesterase that regulates cAMP concentration. Exome analysis detected heterozygous PDE4D mutations (c.673C>A [p.Pro225Thr] and c.677T>C [p.Phe226Ser]) in these two individuals. Screening of PDE4D identified heterozygous mutations (c.568T>G [p.Ser190Ala] and c.1759A>C [p.Thr587Pro]) in two additional acrodysostosis cases. These mutations occurred de novo in all four cases. The four individuals with PDE4D mutations shared common clinical features, namely characteristic midface and nasal hypoplasia and moderate intellectual disability. Metabolic screening was normal in three of these four individuals. However, resistance to parathyroid hormone and thyrotropin was consistently observed in the five cases with PRKAR1A mutations. Finally, our study further supports the key role of the cAMP signaling pathway in skeletogenesis.

Extensive investigation of the IGF2/H19 Imprinting Control Region reveals novel OCT4/SOX2 binding site defects associated with specific methylation patterns in Beckwith-Wiedemann Syndrome

Isolated gain of methylation (GOM) at the IGF2/H19 imprinting control region 1 (ICR1) accounts for about 10% of patients with BWS. A subset of these patients have genetic defects within ICR1, but the frequency of these defects has not yet been established in a large cohort of BWS patients with isolated ICR1 GOM. Here, we carried out a genetic analysis in a large cohort of 57 BWS patients with isolated ICR1 GOM and analyzed the methylation status of the entire domain. We found a new point mutation in two unrelated families and a 21 bp deletion in another unrelated child, both of which were maternally inherited and affected the OCT4/SOX2 binding site in the A2 repeat of ICR1. Based on data from this and previous studies, we estimate that cis genetic defects account for about 20% of BWS patients with isolated ICR1 GOM. Methylation analysis at eight loci of the IGF2/H19 domain revealed that sites surrounding OCT4/SOX2 binding site mutations were fully methylated and methylation indexes declined as a function of distance from these sites. This was not the case in BWS patients without genetic defects identified. Thus, GOM does not spread uniformly across the IGF2/H19 domain, suggesting that OCT4/SOX2 protects against methylation at local sites. These findings add new insights to the mechanism of the regulation of the ICR1 domain. Our data show that mutations and deletions within ICR1 are relatively common. Systematic identification is therefore necessary to establish appropriate genetic counseling for BWS patients with isolated ICR1 GOM.

Fatal Rhabdomyolysis in 2 Children with LPIN1 Mutations

We report 2 cases of fatal rhabdomyolysis in children carrying an LPIN1 mutations preceded by similar electrocardiogram changes, including diffuse symmetrical high-amplitude T waves. Our report underlines the severity of this disease and the need for active management of episodes of rhabdomyolysis in a pediatric intensive care unit.

Myhre syndrome: Myhre syndrome

Myhre syndrome (MS) is a developmental disorder characterized by typical facial dysmorphism, thickened skin, joint limitation and muscular pseudohypertrophy. Other features include brachydactyly, short stature, intellectual deficiency with behavioral problems and deafness. We identified SMAD4 as the gene responsible for MS. The identification of SMAD4 mutations in Laryngotracheal stenosis, Arthropathy, Prognathism and Short stature (LAPS) cases supports that LAPS and MS are a unique entity. The long‐term follow up of patients shows that these conditions are progressive with life threatening complications. All mutations are de novo and changing in the majority of cases Ile500, located in the MH2 domain involved in transcriptional activation. We further showed an impairment of the transcriptional regulation via TGFβ target genes in patient fibroblasts. Finally, the absence of SMAD4 mutations in three MS cases may support genetic heterogeneity.

RPL10 mutation segregating in a family with X‐linked syndromic Intellectual Disability

Intellectual disability is a neurodevelopmental disorder of impaired adaptive skills and low intelligence quotient. The overall prevalence is estimated at 2–3% in the general population with extreme clinical and genetic heterogeneity, and it has been associated with possibly causative mutations in more than 700 identified genes. In a recent review, among over 100 X‐linked intellectual disability causative genes, eight were reported as “awaiting replication.” Exome sequencing in a large family identified a missense mutation in RPL10 highly suggestive of X‐linked intellectual disability. Herein, we report on the clinical description of four affected males. All patients presented apparent intellectual disability (4/4), psychomotor delay (4/4) with syndromic features including amniotic fluid excess (3/4), microcephaly (2/4), urogenital anomalies (3/4), cerebellar syndrome (2/4), and facial dysmorphism. In the literature, two mutations were reported in three families with affected males presenting with autism. This report confirms the implication of RPL10 mutations in neurodevelopmental disorders and extends the associated clinical spectrum from autism to syndromic intellectual disability.

Sleep-disordered breathing and its management in children with achondroplasia

Sleep‐disordered breathing is a common feature in children with achondroplasia. The aim of our study was to review the poly(somno)graphic (P(S)G) findings and consequent treatments in children with achondroplasia followed in the national reference center for skeletal dysplasia. A retrospective review of the clinical charts and P(S)G of 43 consecutive children (mean age 3.9 ± 3.5 years) with achondroplasia seen over a period of 2 years was performed. Twenty four (59%) children had obstructive sleep apnea (OSA). Thirteen children had an obstructive apnea‐hypopnea index (OAHI) < 5/hr, four had an OAHI between 5 and 10/hr, and seven had an OAHI ≥ 10/hr. Ten of the 15 children who had previous upper airway surgery still had an abnormal P(S)G. All the patients with an AHI ≥ 10/hr were under 7 years of age and none had a prior tonsillectomy. The children who underwent adeno‐tonsillectomy, coupled in most cases with turbinectomy, were significantly older (mean age 7.5 ± 3.5 vs. 3.5 ± 1.7 years old, P = 0.015) and had significantly better P(S)G results than those who underwent only adeno‐turbinectomy. No correlation was observed between the mean AHI value at the baseline P(S)G and the type of academic course (standard, supported or specialized). In conclusion, OSA is common in children with achondroplasia. The observation of a reduced prevalence of OSA after (adeno‐)tonsillectomy is in favor of this type of surgery when possible.

Targeted therapy in patients with PIK3CA-related overgrowth syndrome

CLOVES syndrome (congenital lipomatous overgrowth, vascular malformations, epidermal naevi, scoliosis/skeletal and spinal syndrome) is a genetic disorder that results from somatic, mosaic gain-of-function mutations of the PIK3CA gene, and belongs to the spectrum of PIK3CA-related overgrowth syndromes (PROS). This rare condition has no specific treatment and a poor survival rate. Here, we describe a postnatal mouse model of PROS/CLOVES that partially recapitulates the human disease, and demonstrate the efficacy of BYL719, an inhibitor of PIK3CA, in preventing and improving organ dysfunction. On the basis of these results, we used BYL719 to treat nineteen patients with PROS. The drug improved the disease symptoms in all patients. Previously intractable vascular tumours became smaller, congestive heart failure was improved, hemihypertrophy was reduced, and scoliosis was attenuated. The treatment was not associated with any substantial side effects. In conclusion, this study provides the first direct evidence supporting PIK3CA inhibition as a promising therapeutic strategy in patients with PROS.A PI3KCA inhibitor reverses symptoms in a mouse model of PROS/CLOVES syndrome, which results from gain-of-function mutations in PI3KCA, and produces improvements in patients with PROS/CLOVES syndrome.

Monozygotic twins discordant for 18q21.2qter deletion detected by array CGH in amniotic fluid

Discordant chromosomal anomalies in monozygotic twins may be caused by various timing issues of erroneous mitosis and twinning events. Here, we report a prenatal diagnosis of heterokaryotypic monozygotic twins discordant for phenotype. In a 28-year-old woman, ultrasound examination performed at 26 weeks of gestation, detected intrauterine growth restriction and unilateral cleft lip and palate in twin B, whereas twin A had normal fluid, growth and anatomy. Molecular karyotyping in twin B identified a 18q21.2qter deletion, further confirmed by FISH analysis on amniocytes. Interestingly, in twin A, cytogenetic studies (FISH analysis and karyotype) on amniocytes were normal. Genotyping with microsatellite markers confirmed the monozygosity of the twins. At 32 weeks of gestation, selective termination of twin B was performed by umbilical cord coagulation and fetal blood samples were taken from the umbilical cord in both twins. FISH analyses detected mosaicism in both twins with 75% of cells being normal and 25% harboring the 18qter deletion. After genetic counseling, the parents elected to terminate the second twin at 36 weeks of gestation. In postmortem studies, FISH analyses revealed mosaicism on several tissues in both twins. Taking into account this observation, we discuss the difficulties of genetic counseling and management concerning heterokaryotypic monozygotic twins.

Chromosomal rearrangements in the 11p15 imprinted region: 17 new 11p15.5 duplications with associated phenotypes and putative functional consequences

Background The 11p15 region contains two clusters of imprinted genes. Opposite genetic and epigenetic anomalies of this region result in two distinct growth disturbance syndromes: Beckwith-Wiedemann (BWS) and Silver-Russell syndromes (SRS). Cytogenetic rearrangements within this region represent less than 3% of SRS and BWS cases. Among these, 11p15 duplications were infrequently reported and interpretation of their pathogenic effects is complex. Objectives To report cytogenetic and methylation analyses in a cohort of patients with SRS/BWS carrying 11p15 duplications and establish genotype/phenotype correlations. Methods From a cohort of patients with SRS/BWS with an abnormal methylation profile (using ASMM-RTQ-PCR), we used SNP-arrays to identify and map the 11p15 duplications. We report 19 new patients with SRS (n=9) and BWS (n=10) carrying de novo or familial 11p15 duplications, which completely or partially span either both telomeric and centromeric domains or only one domain. Results Large duplications involving one complete domain or both domains are associated with either SRS or BWS, depending on the parental origin of the duplication. Genotype-phenotype correlation studies of partial duplications within the telomeric domain demonstrate the prominent role of IGF2, rather than H19, in the control of growth. Furthermore, it highlights the role of CDKN1C within the centromeric domain and suggests that the expected overexpression of KCNQ1OT1 from the paternal allele (in partial paternal duplications, excluding CDKN1C) does not affect the expression of CDKN1C. Conclusions The phenotype associated with 11p15 duplications depends on the size, genetic content, parental inheritance and imprinting status. Identification of these rare duplications is crucial for genetic counselling.