Autosomal recessive mental retardation: homozygosity mapping identifies 27 single linkage intervals, at least 14 novel loci and several mutation hotspots

Mental retardation (MR) has a worldwide prevalence of around 2% and is a frequent cause of severe disability. Significant excess of MR in the progeny of consanguineous matings as well as functional considerations suggest that autosomal recessive forms of MR (ARMR) must be relatively common. To shed more light on the causes of autosomal recessive MR (ARMR), we have set out in 2003 to perform systematic clinical studies and autozygosity mapping in large consanguineous Iranian families with non-syndromic ARMR (NS-ARMR). As previously reported (Najmabadi et al. in Hum Genet 121:43-48, 2007), this led us to the identification of 12 novel ARMR loci, 8 of which had a significant LOD score (OMIM: MRT5-12). In the meantime, we and others have found causative gene defects in two of these intervals. Moreover, as reported here, tripling the size of our cohort has enabled us to identify 27 additional unrelated families with NS-ARMR and single-linkage intervals; 14 of these define novel loci for non-syndromic ARMR. Altogether, 13 out of 39 single linkage intervals observed in our cohort were found to cluster at 6 different loci on chromosomes, i.e., 1p34, 4q27, 5p15, 9q34, 11p11-q13 and 19q13, respectively. Five of these clusters consist of two significantly overlapping linkage intervals, and on chr 1p34, three single linkage intervals coincide, including the previously described MRT12 locus. The probability for this distribution to be due to chance is only 1.14 × 10(-5), as shown by Monte Carlo simulation. Thus, in contrast to our previous conclusions, these novel data indicate that common molecular causes of NS-ARMR do exist, and in the Iranian population, the most frequent ones may well account for several percent of the patients. These findings will be instrumental in the identification of the underlying genes.     

The H3 receptor is involved in cholecystokinin inhibition of food intake in rats.

We investigated the peripheral effects of an H3-receptor agonist and an H3-receptor antagonist (R)alpha-methylhistamine (Ralpha-MeHA) and thioperamide, respectively, on basal feeding and the CCK8-induced inhibition of food intake in rat. Intraperitoneal injection of thioperamide reduced food intake in a dose-dependent manner with maximal inhibition (35%, P<0.01 vs saline) at 3 mg/kg. (R)alpha-MeHA (0.3-3 mg/kg i.p.), an H3-receptor agonist alone had no effect on feeding but reversed the thioperamide-induced inhibition of food intake in a dose-dependent manner. The maximal feeding inhibitory dose of thioperamide (3 mg.kg i.p) increased by 40% and 22 % (P<0.01 vs saline) brain and stomach histamine contents, respectively. Histamine (0.3 - 6 mg/kg i.p.) and CCK-8 (3 - 30 microg/kg i.p) also inhibited food intake in a dose-dependent manner. Inhibition was 20% to 40% for histamine and 40% to 80% (P<0.01 vs saline) for CCK8. CCK-8 inhibition of feeding was increased by thioperamide and prevented by (R)alpha-MeHA in a dose-dependent way. In addition, CCK-8 did not reduce food intake if rats were pretreated with pyrilamine or ranitidine postsynaptic H1- and H2-receptor antagonists respectively. Our data suggest that the H3-receptor is involved in basal feeding. They also suggest that CCK satiety depends upon the release of histamine which acts on the H2- and H1-receptors, the final mediators of this effect.     

Loss-of-function mutations of ILDR1 cause autosomal-recessive hearing impairment DFNB42

By using homozygosity mapping in a consanguineous Pakistani family, we detected linkage of nonsyndromic hearing loss to a 7.6 Mb region on chromosome 3q13.31-q21.1 within the previously reported DFNB42 locus. Subsequent candidate gene sequencing identified a homozygous nonsense mutation (c.1135G>T [p.Glu379X]) in ILDR1 as the cause of hearing impairment. By analyzing additional consanguineous families with homozygosity at this locus, we detected ILDR1 mutations in the affected individuals of 10 more families from Pakistan and Iran. The identified ILDR1 variants include missense, nonsense, frameshift, and splice-site mutations as well as a start codon mutation in the family that originally defined the DFNB42 locus. ILDR1 encodes the evolutionarily conserved immunoglobulin-like domain containing receptor 1, a putative transmembrane receptor of unknown function. In situ hybridization detected expression of Ildr1, the murine ortholog, early in development in the vestibule and in hair cells and supporting cells of the cochlea. Expression in hair cell- and supporting cell-containing neurosensory organs is conserved in the zebrafish, in which the ildr1 ortholog is prominently expressed in the developing ear and neuromasts of the lateral line. These data identify loss-of-function mutations of ILDR1, a gene with a conserved expression pattern pointing to a conserved function in hearing in vertebrates, as underlying nonsyndromic prelingual sensorineural hearing impairment.     

JNK-interacting leucine zipper protein is a novel scaffolding protein in the Galpha13 signaling pathway

Scaffolding proteins play a critical role in conferring specificity and fidelity to signaling pathways. The JNK-interacting leucine zipper protein (JLP) has been identified as a scaffolding protein involved in linking components of the JNK signaling module. Galpha(12) and Galpha(13), the alpha-subunits of heterotrimeric G proteins G12 and G13, respectively, stimulate the JNK module in diverse cell types. Here, we report that Galpha(13) physically interacts with JLP, and this interaction enhances Galpha(13)-mediated JNK activation. We also demonstrate endogenous interaction between JLP and Galpha(13) in MCF-7 cells. JLP interaction is specific to the G12 family of alpha-subunits via its C-terminal domain (termed GID-JLP), spanning amino acids 1165-1307, and this interaction is more pronounced with the mutationally or functionally activated form of Galpha(13) compared to that of wild-type Galpha(13). The presence of a ternary complex consisting of Galpha(13), JLP, and JNK suggests a role for JLP in tethering Galpha(13) to the signaling components involved in JNK activation. Coexpression of GID-JLP disrupts ternary complex formation in addition to attenuating Galpha(13)-stimulated JNK activity. These findings identify JLP as a novel scaffolding protein in the Galpha(13)-mediated JNK signaling pathway.     

A study of hyperelastic models for predicting the mechanical behavior of extensor apparatus

Biomechanics and Modeling in Mechanobiology - In this research, the nonlinear elastic behavior of human extensor apparatus was investigated. To this goal, firstly the best material parameters of...     

Effects of Hexaflumuron and Pyriproxyfen on the purified Phenoloxidase of Chilo suppressalis Walker (Lepidoptera: Crambidae)

Effects of two insect growth regulators (IGRs), hexaflumuron and pyriproxyfen, were studied on the purified phenoloxidase (PO) of Chilo suppressalis. Purification procedure revealed two isozymes of PO, namely POI and POII. IC₅₀ concentrations of hexaflumuron and pyriproxyfen on POI were 0.36, 0.23?μg/ml and on POII were 0.105, 0.42?μg/ml, respectively. Determination of optimal pH and temperature revealed pH 5 and temperature 40?°C as the optimal values for the enzymatic activity. Treating POs with IC₅₀ concentrations of two IGRs was pH and temperature dependent. Effects of these IGRs on POI caused significant increase of K_m value versus control suggesting competitive inhibition. Hexaflumuron and pyriproxyfen cause reduction in V_max value of POII versus control suggesting non-competitive inhibition. The current study shows direct effects of two IGRs on purified PO of C. suppressalis for the first time. These findings could be helpful to develop safe compounds with inhibitory mechanism on PO to neutralise insect immune responses against entomopathogenic agents.     

Molecular characterization of WFS1 in an Iranian family with Wolfram syndrome reveals a novel frameshift mutation associated with early symptoms

Wolfram syndrome (WS) is a rare autosomal recessive neurodegenerative disorder that represents a likely source of childhood diabetes especially among countries in the consanguinity belt. The main responsible gene is WFS1 for which over one hundred mutations have been reported from different ethnic groups. The aim of this study was to identify the molecular etiology of WS and to perform a possible genotype–phenotype correlation in Iranian kindred.     

Homozygous SLC6A17 Mutations Cause Autosomal-Recessive Intellectual Disability with Progressive Tremor,Speech Impairment,and Behavioral Problems

We report on Dutch and Iranian families with affected individuals who present with moderate to severe intellectual disability and additional phenotypes including progressive tremor, speech impairment, and behavioral problems in certain individuals. A combination of exome sequencing and homozygosity mapping revealed homozygous mutations c.484G>A (p.Gly162Arg) and c.1898C>G (p.Pro633Arg) in SLC6A17. SLC6A17 is predominantly expressed in the brain, encodes a synaptic vesicular transporter of neutral amino acids and glutamate, and plays an important role in the regulation of glutamatergic synapses. Prediction programs and 3D modeling suggest that the identified mutations are deleterious to protein function. To directly test the functional consequences, we investigated the neuronal subcellular localization of overexpressed wild-type and mutant variants in mouse primary hippocampal neuronal cells. Wild-type protein was present in soma, axons, dendrites, and dendritic spines. p.Pro633Arg altered SLC6A17 was found in soma and proximal dendrites but did not reach spines. p.Gly162Arg altered SLC6A17 showed a normal subcellular distribution but was associated with an abnormal neuronal morphology mainly characterized by the loss of dendritic spines. In summary, our genetic findings implicate homozygous SLC6A17 mutations in autosomal-recessive intellectual disability, and their pathogenic role is strengthened by genetic evidence and in silico and in vitro functional analyses.     

Optimization of continuous purification of recombinant patchoulol synthase from Escherichia coli with membrane adsorbers

The natural production of patchouli oil in developing countries cannot meet the increasing demand any more. This leads to socioecological consequences, such as the use of arable land, which is actually intended for food. Hence, the world market price increased up to $150/kg. An alternative is the biotechnological production of patchouli oil using a multiproduct sesquiterpene synthase, the patchoulol synthase (PTS). Here, we report the optimization of recombinant PTS purification from Escherichia coli lysate using continuous immobilized metal affinity chromatography. First, the purification conditions of the batch process were optimized in regard to optimal buffer composition and optimized chromatographic conditions. The best purification result was achieved with Co²⁺-immobilized metal affinity chromatography (Sartobind® IDA 75) with a triethanolamine buffer at pH 7, 0.5 M NaCl, 10% [vol/vol] glycerol, 5 mM MgCl₂ and 250 mM imidazole for product elution. This optimized method was then transferred to a continuous chromatography system using three membrane adsorber units (surface of 75 cm² each). Within 1.5 hr in total, 4.55 mg PTS with a final purity of 98% and recovery of 68% could be gained. The purified enzyme was used to produce 126 mg/L (-)-patchoulol from farnesyl pyrophosphate. Here, for the first time bioactive PTS was successfully purified using membrane adsorbers in a continuous downstream process.