Exome sequencing identifies mutations in KIF14 as a novel cause of an autosomal recessive lethal fetal ciliopathy phenotype.

Gene discovery using massively parallel sequencing has focused on phenotypes diagnosed postnatally such as well-characterized syndromes or intellectual disability, but is rarely reported for fetal disorders. We used family-based whole-exome sequencing in order to identify causal variants for a recurrent pattern of an undescribed lethal fetal congenital anomaly syndrome. The clinical signs included intrauterine growth restriction (IUGR), severe microcephaly, renal cystic dysplasia/agenesis and complex brain and genitourinary malformations. The phenotype was compatible with a ciliopathy, but not diagnostic of any known condition. We hypothesized biallelic disruption of a gene leading to a defect related to the primary cilium. We identified novel autosomal recessive truncating mutations in KIF14 that segregated with the phenotype. Mice with autosomal recessive mutations in the same gene have recently been shown to have a strikingly similar phenotype. Genotype-phenotype correlations indicate that the function of KIF14 in cell division and cytokinesis can be linked to a role in primary cilia, supported by previous cellular and model organism studies of proteins that interact with KIF14. We describe the first human phenotype, a novel lethal ciliary disorder, associated with biallelic inactivating mutations in KIF14. KIF14 may also be considered a candidate gene for allelic viable ciliary and/or microcephaly phenotypes.

Studies of the cells in the afferent and efferent lymph of lymph nodes draining the site of skin homografts.

Intermediate lymph (efferent from the prefemoral lymph node) was collected for 600 hr from both flanks of each of four sheep that had an autograft of skin on the left flank and a homograft of skin on the right flank. 8 days after the grafts had been applied considerable numbers of large basophilic cells, apparently identical with those that appear during immune responses to conventional antigens, appeared in the lymph draining from the homografts. No such cells appeared in the lymph draining from the autografts. At this time the homografts were already showing signs of rejection and were apparently dead well before the cellular response in the lymph reached a peak, about 350 hr (14-15 days) after the homografts had been applied. During the peak of the response up to 40% of the cells in the lymph were basophilic cells and in one experiment such cells were leaving the lymph node at a rate of 200 million per hr. Peripheral lymph (afferent to the popliteal lymph node) draining from the sites of homografts of skin was collected from five sheep. This lymph contained few white cells (<1000 per mm(3)) and showed only an insignificant lymphoid cell reaction. Although the percentage of macrophage-like cells was increased significantly there were few signs of a lymphoid cell reaction; the lymph also contained much amorphous debris. Experiments in which the basophilic cells from the efferent lymph were labeled in vitro with thymidine-(3)H and returned to the sheep by intravenous injections were carried out in six sheep. The presence of the labeled cells in the grafts, blood, and other tissue was detected by liquid scintillation counting of nucleic acid extracts of biopsy and postmortem material and by radioautography. 2-3 labeled cells out of every 1000 injected entered the homografts but hardly any entered the autografts. However, labeled basophilic cells that had originated in response to bacterial antigens entered the homografts with equal facility. It is thus hard to believe that the immunological specificity of a lymphoid cell endows it with a specific "homing" capability. Furthermore, in all the experiments the specific radioactivities of the nucleic acids extracted from the blood mononuclear cells were approximately of the same order as those of the nucleic acids extracted from the homografts. It was concluded that most of the mononuclear cells that infiltrate homografts represent a random selection from the mononuclear cell population of the blood.

The ultrastructure and function of the cells in lymph following antigenic stimulation.

When a lymph node receives an antigenic stimulus the cell population in the efferent lymph changes and large basophilic cells appear. During a secondary immune response cells of this type may account for over 50% of the cells present in lymph. When examined by electron microscopy, many of these cells were found to be primitive undifferentiated blast cells with many free ribosomes in their cytoplasm and only an occasional piece of endoplasmic reticulum. Their nuclear chromatin was sparse and the nuclei contained several nucleoli. Many other cells which were judged to be more differentiated had large numbers of ribosomes arranged in clusters which took the form of rosettes or spirals. These cells also had more ergastoplasm but this occurred usually in the form of short pieces of disorganized endoplasmic reticulum. No cells with the ultrastructure of classical plasma cells were found in efferent lymph although these cells were abundant in the stimulated lymph nodes. It was shown that when the lymph which contained these cells was collected quantitatively no systemic immunity developed even though a vigorous immune response took place in the lymph node with the formation of many plasma cells. Failure of the systemic immune response to develop could not be explained merely in terms of the loss of antibody. It was concluded that these basophilic cells rather than antigen are responsible for propagating the immune response throughout the body and that they depend on an intact lymphatic pathway for their immediate transport. This view was supported by experiments which showed that these cells are capable of initiating immune responses in other lymph nodes of the same animal and of transferring active immunity between chimeric twins. The most likely explanation of these results is that the basophilic lymphoid cells carry out their messenger function by developing into plasma cells at sites remote from the site at which antigen is localized. However this has yet to be proven and the possibility remains that these mobile, highly motile, RNA-rich cells may express their messenger function by transferring information to other effector cells.

Preservation of erniettomorph fossils in clay-rich siliciclastic deposits from the Ediacaran Wood Canyon Formation, Nevada.

Three-dimensionally preserved Ediacaran fossils occur globally within sandstone beds. Sandy siliciclastic deposits of the Ediacaran Wood Canyon Formation (WCF) in the Montgomery Mountains, Nevada, contain two fossil morphologies with similar shapes and sizes: one exhibits mm-scale ridges and a distinct lower boundary and the other is devoid of these diagnostic features. We interpret these as taphomorphs of erniettomorphs, soft-bodied organisms with uncertain taxonomic affinities. We explore the cast-and-mould preservation of both taphomorphs by petrography, Raman spectroscopy, X-ray fluorescence microprobe and X-ray diffraction. All fossils and the surrounding sedimentary matrix contain quartz grains, iron-rich chlorite and muscovite. The ridged fossils contain about 70% larger quartz grains compared to the ridgeless taphomorph, indicating a lower abundance of clay minerals in the ridged fossil. Chlorite and muscovite likely originated from smectite and kaolinite precursors that underwent lower greenschist facies metamorphism. Kaolinite and smectite are inferred to have been abundant in sediments around the ridged fossil, which enabled the preservation of a continuous, distinct, clay- and kerogen-rich bottom boundary. The prevalence of quartz in the ridged fossils of the WCF and in erniettomorphs from other localities also suggests a role for this mineral in three-dimensional preservation of erniettomorphs in sandstone and siltstone deposits.

Association between potassium concentration and serological type of sheep red blood cells.

When red blood cells from 115 sheep were classified for the presence or absence of antigenic factor M and for high (as opposed to low) potassium concentration levels, the cells of the 22 M-negative sheep were low in potassium.

Genomic imprinting.

In the last few years, the importance of genomic imprinting as a basic biological phenomenon has become clear. Work from different areas has demonstrated that parent-of-origin differences in phenotype occur on a regular basis. This article summarizes some of the recent advances made in this field.

Twinning: mechanisms and genetic implications.

In the past, twins have been studied to determine the genetic contribution to various disease processes. Recent work, however, suggests that monozygotic (MZ) twins are not truly identical. Many genetic forms of discordance have been described within MZ twin pairs and may even play a role in causing MZ twinning. Intra-uterine environmental differences in the allocation of the number of cells and in the placental vascular supply to each twin, as well as stochastic development events, may lead to major discordance at birth between the twins of a MZ pair.

Clinical and molecular cytogenetic characterisation of a newly recognised microdeletion syndrome involving 2p15-16.1.

BACKGROUND: During whole genome microarray-based comparative genomic hybridisation (array CGH) screening of subjects with idiopathic intellectual disability, we identified two unrelated individuals with a similar de novo interstitial microdeletion at 2p15-2p16.1. Both individuals share a similar clinical phenotype including moderate to severe intellectual disability, autism/autistic features, microcephaly, structural brain anomalies including cortical dysplasia/pachygyria, renal anomalies (multicystic kidney, hydronephrosis), digital camptodactyly, visual impairment, strabismus, neuromotor deficits, communication and attention impairments, and a distinctive pattern of craniofacial features. Dysmorphic craniofacial features include progressive microcephaly, flat occiput, widened inner canthal distance, small palpebral fissures, ptosis, long and straight eyelashes, broad and high nasal root extending to a widened, prominent nasal tip with elongated, smooth philtrum, rounding of the upper vermillion border and everted lower lips. METHODS: Clinical assessments, and cytogenetic, array CGH and fluorescence in situ hybridisation (FISH) analyses were performed. RESULTS: The microdeletions discovered in each individual measured 4.5 Mb and 5.7 Mb, spanning the chromosome 2p region from 57.2 to 61.7 Mb and from 56 to 61.7 Mb, respectively. Each deleted clone in this range demonstrated a dosage reduction from two to one copy in each proband except for clone RP11-79K21, which was present in three copies in each proband and in four copies in their respective parents (two per each chromosome 2 homologue). DISCUSSION: The common constellation of features found in the two affected subjects indicates that they have a newly recognised microdeletion syndrome involving haploinsufficiency of one or more genes deleted within at least a 4.5-Mb segment of the 2p15-16.1 region.

A DNA computing readout operation based on structure-specific cleavage.

We describe a structure-specific cleavage-based READOUT strategy for surface-based DNA computing. The strategy was demonstrated in the solution of a 4-variable/3-satisfiability (SAT) problem. The READOUT step identifies the DNA molecules present at the end of the computational process. The specificity of the sequence detection used here derives from the sequence specificity of DNA hybridization coupled with the structure specificity of the enzymatic cleavage. The process is linear, yielding a higher uniformity of detection of the DNA computing products compared to that obtained with PCR amplification. The structure-specific cleavage-based readout is simple, accurate, and compatible with multiple-word DNA computing.

Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes.

Flap endonucleases (FENs) isolated from archaea are shown to recognize and cleave a structure formed when two overlapping oligonucleotides hybridize to a target DNA strand. The downstream oligonucleotide probe is cleaved, and the precise site of cleavage is dependent on the amount of overlap with the upstream oligonucleotide. We have demonstrated that use of thermostable archaeal FENs allows the reaction to be performed at temperatures that promote probe turnover without the need for temperature cycling. The resulting amplification of the cleavage signal enables the detection of specific DNA targets at sub-attomole levels within complex mixtures. Moreover, we provide evidence that this cleavage is sufficiently specific to enable discrimination of single-base differences and can differentiate homozygotes from heterozygotes in single-copy genes in genomic DNA.

An invasive cleavage assay for direct quantitation of specific RNAs.

RNA quantitation is becoming increasingly important in basic, pharmaceutical, and clinical research. For example, quantitation of viral RNAs can predict disease progression and therapeutic efficacy. Likewise, gene expression analysis of diseased versus normal, or untreated versus treated, tissue can identify relevant biological responses or assess the effects of pharmacological agents. As the focus of the Human Genome Project moves toward gene expression analysis, the field will require a flexible RNA analysis technology that can quantitatively monitor multiple forms of alternatively transcribed and/or processed RNAs (refs 3,4). We have applied the principles of invasive cleavage and engineered an improved 5'-nuclease to develop an isothermal, fluorescence resonance energy transfer (FRET)-based signal amplification method for detecting RNA in both total RNA and cell lysate samples. This detection format, termed the RNA invasive cleavage assay, obviates the need for target amplification or additional enzymatic signal enhancement. In this report, we describe the assay and present data demonstrating its capabilities for sensitive (<100 copies per reaction), specific (discrimination of 95% homologous sequences, 1 in > or =20,000), and quantitative (1.2-fold changes in RNA levels) detection of unamplified RNA in both single- and biplex-reaction formats.

Analysis of single nucleotide polymorphisms with solid phase invasive cleavage reactions.

Using microparticles as the capture surface and fluorescence resonance energy transfer as the detection technology, we have demonstrated the feasibility of performing the invasive cleavage reaction on a solid phase. An effective tool for many genomic applications, the solution phase invasive cleavage assay is a signal amplification method capable of distinguishing nucleic acids that differ by only a single base mutation. The method positions two overlapping oligonucleotides, the probe and upstream oligonucleotides, on the target nucleic acid to create a complex recognized and cleaved by a structure-specific 5'-nuclease. For microarray and other multiplex applications, however, the method must be adapted to a solid phase platform. Effective cleavage of the probe oligonucleotide occurred when either of the two required overlapping oligonucleotides was configured as the particle-bound reagent and also when both oligonucleotides were attached to the solid phase. Positioning probe oligonucleotides away from the particle surface via long tethers improved both the signal and the reaction rates. The particle-based invasive cleavage reaction was capable of distinguishing the ApoE Cys158 and Arg158 alleles at target concentrations as low as 100 amol/assay (0.5 pM).

The early history of Pallister-Hall syndrome-Buried treasure of a sort.

Pallister-Hall syndrome was initially recognized under fairly unique circumstances involving exhumation of the very first case. The first two cases had dramatic and unusual features including a hypothalamic hamartoblastoma, imperforate anus, an unusual type of polydactyly with the extra digit being central, hypopituitarism with secondary hypoadrenalism, and lethality after birth (probably due to hypoadrenalism). Within a short time frame, four additional cases were identified. As the full spectrum and variability of anomalies was recognized, it became clear that it was not such a rare disorder. Shortly after familial cases were recognized, the responsible gene was identified at GLI3. However, since other different conditions also involved GLI3, elaborating the domains of the gene and the types of mutations needed to be defined in order to have a clear correlation of the genotype-phenotype relations.

The relationship between GSH, GSSG and non-GSH thiol in GSH-deficient erythrocytes from Finnish landrace and Tasmanian merino sheep.

1. Two automated colorimetric methods have been developed for assaying the GSH and total thiol in protein-free extracts of erythrocytes. They employ as chromogens 5,5'-dithiobis-(2-nitrobenzoate) (DTNB) and alloxan. 2. The concentrations of GSH, GSSG and total non-protein thiol have been estimated in high and low GSH erythrocytes from Finnish Landrace and Tasmanian Merino sheep. 3. In both breeds of sheep low GSH cells were found to have low concentrations of total non-protein thiol and GSSG as well as of GSH. 4. Nevertheless high and low GSH cells have similar values for the oxidation-reduction potential of the GSH : GSSG couple.

Functional alcohol dehydrogenase mutants of Saccharomyces cerevisiae conferring temperature-conditional allyl alcohol resistance.

Selection for allyl alcohol resistance in respiratory incompetent yeast is a highly specific method for isolating functional mutations at ADH1, the gene coding for the cytoplasmic alcohol dehydrogenase, ADHI. Because of the nature of this selection scheme, the ADHI activity of such mutants is retained, but the kinetic characteristics of the enzymes are altered. The high specificity for targeting functional mutations at this locus suggested that selection for enzyme variants with more subtle phenotypic effects might be possible. Here, we describe functional ADHI mutants that are temperature-conditional in their allyl alcohol resistance. Haploid cells of one of these mutants grow well on plates at 10 mM allyl alcohol at 19 degrees, but not at 37 degrees, the restrictive temperature. A second mutant grows well at 10 mM at 37 degrees, but its growth is restricted at 19 degrees. What distinguishes these mutants from other temperature-sensitive mutants is that the temperature-conditional growth phenotypes described here must be due to interactions between allyl alcohol levels and ADHI functional properties and cannot be due to lability of the enzyme at the restrictive temperature. This system shows promise for the investigation of functional enzyme variants that differ by only one or two amino acid residues but have significant temperature- and substrate-conditional effects on growth phenotypes in both the haploids and the diploids.

Conditional overdominance at an alcohol dehydrogenase locus in yeast.

Documented examples of heterosis attributable to overdominance at specific protein-encoding gene loci have rarely been reported, the association of sickle cell hemoglobin with malarial resistance being the best documented example of this phenomenon. Here we report an example of overdominance that is temperature- and allyl alcohol-dependent and due to heterozygosity at the ADH1 locus, involving two ADHI functional mutants. Overdominance appears to be due in part to an intermediate level of ADHI activity in the heterozygote. Unlike previous work with this this system using haploid strains, the NAD+/NADH ratios show no negative correlation with allyl alcohol resistance. This system is formally equivalent to that of sickle cell hemoglobin and shows promise as a tool for investigating the physiological basis for overdominance.

Drug competition for thyroxine binding to transthyretin (prealbumin): comparison with effects on thyroxine-binding globulin.

We examined the effect of 26 drugs on T4 binding to transthyretin (TTR; prealbumin) and T4-binding globulin (TBG) by determining their ability to inhibit [125I]T4 binding to TTR isolated from normal human plasma and to serum diluted 1:10,000, respectively. The hierarchies for drug inhibition of T4 binding differed greatly for these two proteins. Relative to T4, the drugs were much more potent inhibitors of [125I]T4 binding to TTR than to TBG. Compounds of the anthranilic acid class, such as flufenamic, meclofenamic, and mefenamic acids, interacted particularly strongly with TTR. Flufenamic acid was more potent than T4 itself in inhibiting [125I]T4 binding [175 +/- 17% (+/- SD); cf. T4; n = 3; P less than 0.001], while mefenamic acid, diflunisal, and meclofenamic acid were 20-26% as potent as T4 in their interaction with TTR. The reactivity of diclofenac, fenclofenac, indomethacin, sulindac, and the diuretic ethacrynic acid was 0.8-2.1% relative to that of T4. In contrast, furosemide, the drug most highly reactive with TBG, was only 0.11 +/- 0.03% (n = 7) as potent as T4, followed by meclofenamic acid greater than mefenamic acid greater than fenclofenac greater than flufenamic acid greater than diflunisal greater than milrinone. Aspirin and sodium salicylate were, respectively, 0.05% and 0.20% as active as unlabeled T4 as inhibitors of [125I]T4 binding to TTR, but these compounds had only 3-4 x 10(-6)% of the activity of T4 for TBG binding. Diphenylhydantoin had no detectable effect on T4 binding to TTR and was 2.9 x 10(-4)% as reactive as T4 with TBG. Amiodarone did not interact with either binding site. Drug interactions with TTR may be important when this protein becomes a major circulating T4-binding protein, as in patients with complete or partial TBG deficiency, or when serum T4 is markedly elevated. Such interactions may also be important where TTR is the dominant tissue T4-binding protein, as in the choroid plexus. In addition, the drug competitors described here may be useful as probes to further define the structural basis for specific ligand interactions with different classes of T4-binding sites.

Thrombocytopenia with absent radius (TAR).

Forty examples (27 from the literature and 13 new cases) of a syndrome of hypomegakaryocytic thrombocytopenia with bilateral absence of the radius have been analyzed. This syndrome is designated in this paper as "thrombocytopenia with absent radius (TAR)". The onset of hematologic complications usually occurs at birth or during early infancy. Thrombocytopenia may be episodic and sometimes is accompanied by leukemoid reactions and eosinophilia. Bone marrow examination reveals decreased and/or abnormal megakaryocytes, with normal myeloid and erythroid precursors. Congenital skeletal deformities include bilateral absence of radius, shortening and deformity of the ulnae, and occasionally absence of all the long bones in the arm. The fingers and thumbs are always present. Other skeletal anomalies are frequent. Cardiac anomalies, particularly the tetralogy of Fallot and atrial septal defects, may be present. Other non-skeletal congenital abnormalities are rare. The prognosis is good if the patient survives to one year of age. The syndrome has been compared to Fanconi's anemia, thalidomide embryopathy, limb-cardiovascular syndrome, and a syndrome of multiple congenital malformations, from which it can be distinguished.

Joubert's syndrome associated with congenital ocular fibrosis and histidinemia.

We describe a 16-month-old girl with Joubert's syndrome (JS), congenital ocular fibrosis, and histidinemia. Abnormal respiration, ptosis, and minimal eye movements were observed in the neonatal period. Intraoperative examination of the eyes later demonstrated severely restricted eye movements and abnormal insertions and fibrosis of the extraocular muscles. Computed tomography of the head revealed absence of the corpus callosum and brain stem. Histidine levels were elevated in the blood, urine, and cerebrospinal fluid. The patient was ataxic and developmentally delayed. To our knowledge, the association of JS with congenital ocular fibrosis has not previously been described. This report indicates that jerky eye movements are not an invariable finding in JS.