Homozygous STIL Mutation Causes Holoprosencephaly and Microcephaly in Two Siblings

February Homozygous STIL Mutation Causes Holoprosencephaly and Microcephaly in Two Siblings Charlotte Mouden 0 1 Marie de Tayrac 0 1 Christle Dubourg 0 1 Sophie Rose 0 1 Wilfrid Carr 0 1 Houda Hamdi-Roz 0 1 Marie-Claude Babron 0 1 Linda Akloul 0 1 Bndicte Hron-Longe 0 1 Sylvie Odent 0 1 Valrie Dup 0 1 Rgis Giet 0 1 Vronique David 0 1 0 1 Institut de Genetique et Developpement de Rennes, Equipe Genetique des Pathologies Liees au Developpement, Faculte de Medecine , Universite de Rennes 1, 35043 Rennes , France , 2 Institut de Genetique et Developpement de Rennes, Equipe Cytosquelette et Proliferation Cellulaire, Faculte de Medecine , Universite de Rennes 1, 35043 Rennes , France , 3 Laboratoire de Genetique Moleculaire et Genomique , CHU Pontchaillou, 35033 Rennes , France , 4 Inserm U946, Variabilite Genetique et Maladies Humaines, Universite Paris-Diderot , 75010 Paris , France , 5 Service de Genetique Clinique , Hopital Sud, 35200 Rennes , France , 6 Service de Pediatrie, Hopital Jean Verdier , APHP, 93140 Bondy, France, 7 Plateforme Genomique Sante, Biosit, Universite Rennes 1, 35033 Rennes , France 1 Academic Editor: Coro Paisan-Ruiz, Icahn School of Medicine at Mount Sinai, UNITED STATES Holoprosencephaly (HPE) is a frequent congenital malformation of the brain characterized by impaired forebrain cleavage and midline facial anomalies. Heterozygous mutations in 14 genes have been identified in HPE patients that account for only 30% of HPE cases, suggesting the existence of other HPE genes. Data from homozygosity mapping and whole-exome sequencing in a consanguineous Turkish family were combined to identify a homozygous missense mutation (c.2150G>A; p.Gly717Glu) in STIL, common to the two affected children. STIL has a role in centriole formation and has previously been described in rare cases of microcephaly. Rescue experiments in U2OS cells showed that the STIL p.Gly717Glu mutation was not able to fully restore the centriole duplication failure following depletion of endogenous STIL protein indicating the deleterious role of the mutation. In situ hybridization experiments using chick embryos demonstrated that expression of Stil was in accordance with a function during early patterning of the forebrain. It is only the second time that a STIL homozygous mutation causing a recessive form of HPE was reported. This result also supports the genetic heterogeneity of HPE and increases the panel of genes to be tested for HPE diagnosis. - Competing Interests: The authors have declared that no competing interests exist. Holoprosencephaly (HPE) (#236100) is the most frequent congenital malformation of the brain (1 in 10,000 live births; 1 in 250 conceptuses). HPE is characterized by impaired forebrain cleavage, midline facial anomalies and wide phenotypic spectrum. The clinical spectrum ranges from alobar HPE to semilobar and lobar HPE generally associated with facial anomalies [1]. HPE is a severe pathology with mental retardation and developmental delay in all affected live newborns, with poor or symptomatic treatment. In addition, the midline malformation affects the development of the hypothalamus and the pituitary gland, leading to frequent endocrine disorders like temperature, heart rate and respiration instabilities, hypogonadism, thyroid hypoplasia or diabetes insipidus. The oromotor dysfunction is also affected, with feeding and swallowing difficulties [2]. Only 20% of children with alobar HPE survive after the first year of life, and 50% of infants with semi-lobar HPE are alive after 12 months [3]. Isolated HPE presents a high genetic heterogeneity and to date heterozygous mutations in 14 genes have been identified in HPE patients, 4 major genes (Sonic hedgehog or SHH, ZIC2, SIX3, TGIF1) and 10 genes considered as minor genes (PTCH1, TDGF1, FAST1, GLI2, DISP1, FGF8, GAS1, CDON, NODAL and DLL1) [47]. These genes encode proteins playing a role in early development, belonging mostly to signaling pathways like Sonic Hedgehog (SHH) or Nodal [8]. However, many patients remain without a molecularly confirmed diagnosis. In 70% of isolated cases, mutations in SHH, SIX3 and TGIF1 are inherited from a parent unaffected or harboring a microform of HPE [1], suggesting that other events are necessary to develop the disease. Thus, the mode of inheritance described as autosomal dominant with an incomplete penetrance and a variable expression has evolved to the assumption of a multi-hit pathology requiring two or more events involving several genes from the same or different signaling pathways. The existence of rare consanguineous families suggests the possibility that autosomal recessive inheritance may account for a substantial part of this disorder. In the case of a rare recessively inherited disorder and known consanguinity, the initial assumptions are that the disease is caused by a homozygous variant inherited from both parents and that this variant resides within a large homozygous region. To test this hypothesis and to improve the genetic basis of HPE, we have employed homozygosity mapping in two affected siblings and their mother issued from a Turkish consanguineous family (parents were first cousins), coupled with a next-generation sequencing approach on DNA from the mother and only one of the affected siblings (Fig. 1A). DNA of the father was not available. Material and Methods All patient samples in this study were obtained with informed consent according to the protocols approved by the local ethics committee (Rennes hospital). Genome-wide genotyping was undertaken using Illumina 300K single nucleotide polymorphism (SNP) mapping array beadchip (Illumina), on DNA of individuals I2 (mother), II3 (siblinggirl) and II5 (siblingboy). The BlueFuse Multi software v3.3 (BlueGnome) was used to identify homozygous regions in all the family members. Only the regions longer than 1Mb and carrying at least 100 consecutive homozygous SNPs were selected. In parallel, homozygous regions and inbreeding coefficients were estimated/analyzed using FSuite pipeline [9]. Whole exome sequencing and variants filtering Exome sequencing was performed on DNA of the boy II5 and of the mother I2 by Integragen using SureSelect V5 capture kit (Agilent) on HiSeq system (Illumina). The mean coverage was 80x, 95% of sequences had at least 10-times coverage and 91% of sequenced bases had a quality score greater than or equal to Q30. Bioinformatics analysis has been conducted with the Illumina pipeline analysis CASAVA v1.8. The sequenced reads were aligned to the hg19 human Fig 1. Pedigree of the consanguineous family, brain MRI of the affected siblings, Sanger validation of the c.2150G>A (p.Gly717Glu) STIL mutation, and schematic report of all STIL mutations reported so far. (A) Pedigree of the inbred family. Closed symbols indicate individuals affected with holoprosencephaly. Family members marked with an asterisk were analyzed by whole exome sequencing. (B) Coronal (on the left) and axial (on (...truncated)