Mohammad Shboul

Mutations in PYCR1 cause cutis laxa with progeroid features.

Autosomal recessive cutis laxa (ARCL) describes a group of syndromal disorders that are often associated with a progeroid appearance, lax and wrinkled skin, osteopenia and mental retardation. Homozygosity mapping in several kindreds with ARCL identified a candidate region on chromosome 17q25. By high-throughput sequencing of the entire candidate region, we detected disease-causing mutations in the gene PYCR1. We found that the gene product, an enzyme involved in proline metabolism, localizes to mitochondria. Altered mitochondrial morphology, membrane potential and increased apoptosis rate upon oxidative stress were evident in fibroblasts from affected individuals. Knockdown of the orthologous genes in Xenopus and zebrafish led to epidermal hypoplasia and blistering that was accompanied by a massive increase of apoptosis. Our findings link mutations in PYCR1 to altered mitochondrial function and progeroid changes in connective tissues.

C5orf42 is the major gene responsible for OFD syndrome type VI

Oral-facial-digital syndrome type VI (OFD VI) is a recessive ciliopathy defined by two diagnostic criteria: molar tooth sign (MTS) and one or more of the following: (1) tongue hamartoma (s) and/or additional frenula and/or upper lip notch; (2) mesoaxial polydactyly of one or more hands or feet; (3) hypothalamic hamartoma. Because of the MTS, OFD VI belongs to the “Joubert syndrome related disorders”. Its genetic aetiology remains largely unknown although mutations in the TMEM216 gene, responsible for Joubert (JBS2) and Meckel-Gruber (MKS2) syndromes, have been reported in two OFD VI patients. To explore the molecular cause(s) of OFD VI syndrome, we used an exome sequencing strategy in six unrelated families followed by Sanger sequencing. We identified a total of 14 novel mutations in the C5orf42 gene in 9/11 families with positive OFD VI diagnostic criteria including a severe fetal case with microphthalmia, cerebellar hypoplasia, corpus callosum agenesis, polydactyly and skeletal dysplasia. C5orf42 mutations have already been reported in Joubert syndrome confirming that OFD VI and JBS are allelic disorders, thus enhancing our knowledge of the complex, highly heterogeneous nature of ciliopathies.

Haploinsufficiency for AAGAB causes clinically heterogeneous forms of punctate palmoplantar keratoderma

Palmoplantar keratodermas (PPKs) are a group of disorders that are diagnostically and therapeutically problematic in dermatogenetics. Punctate PPKs are characterized by circumscribed hyperkeratotic lesions on the palms and soles with considerable heterogeneity. In 18 families with autosomal dominant punctate PPK, we report heterozygous loss-of-function mutations in AAGAB, encoding α- and γ-adaptin–binding protein p34, located at a previously linked locus at 15q22. α- and γ-adaptin–binding protein p34, a cytosolic protein with a Rab-like GTPase domain, was shown to bind both clathrin adaptor protein complexes, indicating a role in membrane trafficking. Ultrastructurally, lesional epidermis showed abnormalities in intracellular vesicle biology. Immunohistochemistry showed hyperproliferation within the punctate lesions. Knockdown of AAGAB in keratinocytes led to increased cell division, which was linked to greatly elevated epidermal growth factor receptor (EGFR) protein expression and tyrosine phosphorylation. We hypothesize that p34 deficiency may impair endocytic recycling of growth factor receptors such as EGFR, leading to increased signaling and cellular proliferation.

Mutations in IRX5 impair craniofacial development and germ cell migration via SDF1

Using homozygosity mapping and locus resequencing, we found that alterations in the homeodomain of the IRX5 transcription factor cause a recessive congenital disorder affecting face, brain, blood, heart, bone and gonad development. We found through in vivo modeling in Xenopus laevis embryos that Irx5 modulates the migration of progenitor cell populations in branchial arches and gonads by repressing Sdf1. We further found that transcriptional control by Irx5 is modulated by direct protein-protein interaction with two GATA zinc-finger proteins, GATA3 and TRPS1; disruptions of these proteins also cause craniofacial dysmorphisms. Our findings suggest that IRX proteins integrate combinatorial transcriptional inputs to regulate key signaling molecules involved in the ontogeny of multiple organs during embryogenesis and homeostasis.

Gmnc Is a Master Regulator of the Multiciliated Cell Differentiation Program

Multiciliated cells (MCCs) differentiate hundreds of motile cilia that generate mechanical force required to drive fluid movement over epithelia [1, 2]. For example, metachronal beating of MCC cilia in the mammalian airways clears mucus that traps inhaled pathogens and pollutants. Consequently, abnormalities in MCC differentiation or ciliary motility have been linked to an expanding spectrum of human airway diseases [3–6]. The current view posits that MCC precursors are singled out by the inhibition of Notch signaling. MCC precursors then support an explosive production of basal bodies, which migrate to the apical surface, dock with the plasma membrane, and seed the growth of multiple motile cilia. At the center of this elaborate differentiation program resides the coiled-coil-containing protein Multicilin, which transcriptionally activates genes for basal body production and the gene for FoxJ1, the master regulator for basal body docking, cilia formation, and motility [7, 8]. Here, using genetic analysis in the zebrafish embryo, we discovered that Gmnc is a novel determinant of the MCC fate. Like Multicilin, Gmnc is a coiled-coil-containing protein of the Geminin family. We show that Gmnc functions downstream of Notch signaling, but upstream of Multicilin in the developmental pathway controlling MCC specification. Moreover, we find that loss of Gmnc in Xenopus embryos also causes loss of MCC differentiation and that overexpression of the protein is sufficient to induce supernumerary MCCs. Together, our data identify Gmnc as an evolutionarily conserved master regulator functioning at the top of the hierarchy of transcription factors involved in MCC differentiation.

Human Asunder promotes dynein recruitment and centrosomal tethering to the nucleus at mitotic entry

Proper coupling of centrosomes to the nuclear surface at prophase is essential for fidelity of mitotic events. A pool of dynein motors anchored to the nuclear surface mediates this step. The protein Asunder is required in human cultured cells for dynein localization and tethering of centrosomes to the nucleus at mitotic entry.

Loss of the scavenger mRNA decapping enzyme DCPS causes syndromic intellectual disability with neuromuscular defects

mRNA decay is an essential and active process that allows cells to continuously adapt gene expression to internal and environmental cues. There are two mRNA degradation pathways: 3′ to 5′ and 5′ to 3′. The DCPS protein is the scavenger mRNA decapping enzyme which functions in the last step of the 3′ end mRNA decay pathway. We have identified a DCPS pathogenic mutation in a large family with three affected individuals presenting with a novel recessive syndrome consisting of craniofacial anomalies, intellectual disability and neuromuscular defects. Using patients primary cells, we show that this homozygous splice mutation results in a DCPS loss-of-function allele. Diagnostic biochemical analyses using various m7G cap derivatives as substrates reveal no DCPS enzymatic activity in patients cells. Our results implicate DCPS and more generally RNA catabolism, as a critical cellular process for neurological development, normal cognition and organismal homeostasis in humans.

Nuclear-localized Asunder regulates cytoplasmic dynein localization via its role in the Integrator complex

A pool of dynein anchored to the nuclear surface mediates many processes at G2/M, although its spatial and temporal regulation is poorly understood. Asunder, a critical regulator of dynein recruitment to the nuclear envelope, works in the nucleus as part of Integrator, an snRNA-processing complex, to mediate this event.

ThalassoChip, an array mutation and single nucleotide polymorphism detection tool for the diagnosis of β-thalassaemia.

Abstract Background: The detection and diagnosis of β-thalassaemia for populations with molecular heterogeneity, or diverse ethnic groups, has increased the need for the development of an array high-throughput diagnostic tool that can deliver large scale genetic detection. We report on the update and validation of the ThalassoChip, a β-thalassaemia genetic diagnostic tool which is based on arrayed primer extension (APEX) technology. Methods: ThalassoChip slides with new and redesigned probes were prepared for testing the microarray. Six hundred and sixty DNA samples collected from eight Mediterranean countries were used for standardisation, optimisation and validation of the ThalassoChip. The β-globin gene region was amplified by PCR, the products were hybridised to the probes after fragmentation and the APEX reaction followed. Results: The ThalassoChip was updated with new probes and now has the ability to detect 57 β-globin gene mutations and three single nucleotide polymorphisms (SNPs) in a single test. The ThalassoChip as well as the PCR and APEX reactions were standardised and optimised using 500 DNA samples that were previously genotyped using conventional diagnostic techniques. Some probes were redesigned in order to improve the specificity and sensitivity of the test. Validation of the ThalassoChip performed using 160 samples analysed in blinded fashion showed no error. Conclusions: The updated version of the ThalassoChip is versatile, robust, cost-effective and easily adaptable, but most notably can provide comprehensive genetic diagnosis for β-thalassaemia and other haemoglobinopathies. Clin Chem Lab Med 2010;48:1713–8.

Long-Term Culture of Self-renewing Pancreatic Progenitors Derived from Human Pluripotent Stem Cells

Summary Pluripotent stem cells have been proposed as an unlimited source of pancreatic β cells for studying and treating diabetes. However, the long, multi-step differentiation protocols used to generate functional β cells inevitably exhibit considerable variability, particularly when applied to pluripotent cells from diverse genetic backgrounds. We have developed culture conditions that support long-term self-renewal of human multipotent pancreatic progenitors, which are developmentally more proximal to the specialized cells of the adult pancreas. These cultured pancreatic progenitor (cPP) cells express key pancreatic transcription factors, including PDX1 and SOX9, and exhibit transcriptomes closely related to their in vivo counterparts. Upon exposure to differentiation cues, cPP cells give rise to pancreatic endocrine, acinar, and ductal lineages, indicating multilineage potency. Furthermore, cPP cells generate insulin+ β-like cells in vitro and in vivo, suggesting that they offer a convenient alternative to pluripotent cells as a source of adult cell types for modeling pancreatic development and diabetes.