An assessment of gene-by-environment interactions in developmental dyslexia-related phenotypes.

While the genetic and environmental contributions to developmental dyslexia (DD) have been studied extensively, the effects of identified genetic risk susceptibility and of specified environmental hazardous factors have usually been investigated separately. We assessed potential gene-by-environment (GxE) interactions on DD-related reading, spelling and memory phenotypes. The presence of GxE effects were investigated for the DYX1C1, DCDC2, KIAA0319 and ROBO1 genes, and for seven specified environmental moderators in 165 nuclear families in which at least one member had DD, by implementing a general test for GxE interaction in sib-pair-based association analysis of quantitative traits. Our results support a diathesis-stress model for both reading and memory composites: GxE effects were found between some specified environmental moderators (i.e. maternal smoke during pregnancy, birth weight and socio-economic status) and the DYX1C1-1259C/G marker. We have provided initial evidence that the joint analysis of identified genetic risk susceptibility and measured putative risk factors can be exploited in the study of the etiology of DD and reading-related neuropsychological phenotypes, and may assist in identifying/preventing the occurrence of DD.

A large analphoid invdup(3)(q22.3qter) marker chromosome characterized by array-CGH in a child with malformations, mental retardation, ambiguous genitalia and Blaschko's lines.

We report a new case of mosaic chromosome 3-derived marker chromosome, present in fibroblasts but not in lymphocytes, found in a child with malformations, mental retardation and ambiguous genitalia. Cytogenetic and molecular analysis showed that the supernumerary invdup(3)(q22.3qter) chromosome was negative at FISH with alpha satellite probe. The presence of a functional neocentromere was confirmed by immunofluorescence with antibodies to centromere proteins (CENPs). Definition of the marker breakpoints has been done through array-CGH. The skin of the patient presented dyschromic areas ordered along Blaschko's lines. The invdup(3q) marker chromosome was present only in fibroblasts from the dark skin biopsy, while lymphocytes and fibroblasts from the normal skin showed a normal male karyotype. Expression of the HPS3 gene (MIM: 606118) was more than two times higher in dark skin fibroblasts. Neocentromeres are most often observed on chromosomal arm fragments that have separated from an endogenous centromere, and therefore actually confer mitotic stability to what would have been acentric fragments. To our knowledge, this invdup(3q) analphoid marker is the largest among the several reported so far. Parental origin and possible mode of formation have been defined by DNA polymorphisms studies. The size of the duplicated marker chromosome and its frequency and tissue distribution may be relevant to the severity of the propositus' phenotype.

The dystrophin gene is alternatively spliced throughout its coding sequence.

We have analysed splicing patterns in the human dystrophin gene region encoding the rod and cysteine-rich domains in normal skeletal muscle, brain and heart tissues. Sixteen novel alternative transcripts were identified, the majority of them being present in all three tissues. Tissue-specific variants were also identified, suggesting a functional role of transcriptional diversity. Transcript analysis in dystrophinopathic autoptic and bioptic specimens revealed that pre-mRNAs secondary structure formation and relative strength of exon/exon association play little or no role in directing alternative splicing events. This analysis also showed that independent deletion events leading to the loss of the same exons may be associated with transcriptional variability.

Primary beta-sarcoglycanopathy manifesting as recurrent exercise-induced myoglobinuria.

We report an unusual presentation of a primary beta-sarcoglycanopathy (LGMD type 2E). A 12- year-old boy came to our attention after six episodes of exercise-induced myoglobinuria. Electromyogram showed mild myopathic features of the proximal lower limb muscles. Electrocardiogram was normal. Neurological examination revealed normal muscle strength and reduced deep tendon reflexes. A muscle biopsy showed rare regenerating fibers; the immunohistochemistry was normal for dystrophin, while all the sarcoglycans were diffusely decreased. Western blot analysis showed a relevant decrease of all sarcoglycan proteins and a mild dystrophin reduction. beta-Sarcoglycan gene analysis demonstrated a compound heterozygous status for these mutations: a novel A-T base pair substitution at nucleotide 85 in exon 2, changing the codon Arg to a stop codon; a C-T base pair substitution at nucleotide 272 in exon 3 changing a Arg to a Cys residue. We consider that exercise-induced myoglobinuria may be the presenting sign of primary beta-sarcoglycanopathy.

Inverted duplications are recurrent rearrangements always associated with a distal deletion: description of a new case involving 2q.

We studied the case of a subject with an inverted duplication of 40 cM of 2q33-q37 concurrent with a 10 cM deletion of the distal 2q, the latter not being detectable by cytogenetics. Microsatellite analysis demonstrated the absence of maternal alleles in the deleted region and a double dosage for one of the maternal alleles in the duplication region. We hypothesised that this type of rearrangement occurs at meiosis I, while the two homologues are synapsed for most of their length. The presence of inverted duplicons in the same chromosome arm would favour the partial refolding of one homologue into itself so leading to the intrachromatid synapsis and recombination of the inverted repeats. The arising recombinant chromosome is deleted for the region beyond the most distal repeat and with the chromatids joined together at the level of the region located between the two duplicons. At meiosis II, the two linked chromatids can join the opposite poles provided that a breakage between the two centromeres occurs leading to a duplicated/deleted chromosome and a simply deleted chromosome. This model can be extended to all the so-called inverted duplication cases and to part of the terminal deletions. In fact the finding that, in our invdup(2q), the entire 40 cM duplication region involves only one of the two maternal alleles, indeed indicates that the abnormal crossover occurs between sister chromatids. The phenotype associated with our 2q rearrangement led us to narrow the critical region for the Albright-like syndrome to 10 cM in the subterminal 2q region.

Constitutive expression and role of the TNF family ligands in apoptotic killing of tumor cells by human NK cells.

Natural killer cells mediate spontaneously secretory/necrotic killing against rare leukemia cell lines and a nonsecretory/apoptotic killing against a large variety of tumor cell lines. The molecules involved in nonsecretory/apoptotic killing are largely undefined. In the present study, freshly isolated, nonactivated, human NK cells were shown to express TNF, lymphotoxin (LT)-alpha, LT-beta, Fas ligand (L), CD27L, CD30L, OX40L, 4-1BBL, and TNF-related apoptosis-inducing ligand (TRAIL), but not CD40L or nerve growth factor. Complementary receptors were demonstrated to be expressed on the cell surface of solid tumor cell lines susceptible to apoptotic killing mediated by NK cells. Individually applied, antagonists of TNF, LT-alpha1beta2, or FasL fully inhibited NK cell-mediated apoptotic killing of tumor cells. On the other hand, recombinant TNF, LT-alpha1beta2, or FasL applied individually or as pairs were not cytotoxic. In contrast, a mixture of the three ligands mediated significant apoptosis in tumor cells. These findings demonstrate that human NK cells constitutively express several of the TNF family ligands and induce apoptosis in tumor cells by simultaneous engagement of at least three of these cytotoxic molecules.

The cytoplasmic domain of rat NKR-P1 receptor interacts with the N-terminal domain of p56(lck) via cysteine residues.

NKR-P1 is a type II transmembrane protein which acts as an activation receptor on natural killer (NK) cells. The cytoplasmic domains of the CD4, CD8 and 4-1BB receptors contain the sequence Cys-X-Cys-Pro which is directly involved in coupling to another pair of cysteines in the N-terminal domain of the src family tyrosine kinase p56(lck). The cytoplasmic domain of NKR-P1 in rodents also contains the Cys-X-Cys-Pro sequence, but the capacity of the receptor to bind p56(lck) is presently unknown. We tested for direct coupling between these proteins using both protein biochemistry and the yeast two-hybrid technique. Immunoprecipitation studies showed that p56(lck) can be co-immunoprecipitated with NKR-P1 from a rat NK tumor cell line. In addition, the cytoplasmic domain of NKR-P1 interacted with the N-terminal domain of p56(lck) in yeast as assessed by reporter gene activation. Integrity of the cysteine pairs in both proteins was critical in mediating the interaction. The experiments suggest that the association of p56(lck) with NKR-P1 is somewhat weaker than the p56(lck) association with CD8alpha, but of much lower avidity than between CD4 and p56(lck). This could reflect a higher activation threshold for the NKR-P1 and CD8 receptors, which are involved in cytolytic responses, compared to CD4 which is involved in T cell helper function.

Non-radioisotopic typing of human leukocyte antigen class II genes on microplates.

We describe a new nonradioisotopic method for HLA class II molecular typing performed in 96-well plates of the same size as those used in enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA). Biotinylated sequence-specific oligonucleotide (SSO) probes are bound to avidin-coated plates. Digoxigenin-labeled PCR-amplified DNA samples are hybridized, washed and detected with a peroxidase conjugated antibody assay. The method was tested by performing a partial HLA DQA1 and DQB1 typing on 69 randomly selected blood samples. The results are completely concordant with a traditional SSO-PCR typing performed on the same samples. This procedure is simple, fast and could be adapted for performance in semi-automated or automated fashion using equipment already available for ELISA and RIA assays.

Rat class III Fc gamma receptor isoforms differ in IgG subclass-binding specificity and fail to associate productively with rat CD3 zeta.

Several new rat class III Fc gamma R isoforms are described here, extending the genetic complexity of this receptor family and further distinguishing rat CD16 from mouse CD16, represented by only one receptor isoform, and human CD16, represented by only two isoforms. RNase protection assays reveal that three rat tumor cell lines--RBL-1 basophilic leukemia cells, RM-SV1 macrophages, and CRNK-16 NK cells--all coordinately express multiple and probably identical rtFc gamma RIII-related transcripts in similar relative proportions but at significantly different levels. These results indicate that no single isoform predominates in these cell types but that the overall level of rtFc gamma RIII-related transcripts is differentially regulated. Two of the rtFc gamma RIII isoforms found to have extensive amino acid sequence differences in their second extracellular (EC2) domains are shown to bind rat and mouse IgG subclasses differently. This result suggests that the receptor isoform diversity in this species may function as a mechanism for extending the IgG-binding capacity of rat leukocytes. Cloned cDNA for the rat CD3 zeta protein was also isolated in this study and its ability to augment surface expression of class III Fc gamma R was tested by rosetting of cDNA-transfected COS cells. Like the structurally homologous mouse CD3 zeta, rat CD3 zeta fails to promote surface expression of Fc gamma RIII, sharply contrasting the efficient receptor expression produced by human CD3 zeta. Variations in the transmembrane amino acid sequences correlate with the divergent capacities of these CD3 zeta molecules to augment receptor expression. The high levels of CD3 zeta message expressed in rat NK cells may indicate that other unidentified hetero-subunits are required for assembly of rat CD3 zeta into functional CD16 receptors.

The T-cell receptor V beta gene usage in tumor-infiltrating lymphocytes and blood of patients with hepatocellular carcinoma.

To determine a possibly restricted T-cell receptor (TCR) repertoire in tumor-infiltrating lymphocytes (TIL) in response to tumor-associated antigens in patients with hepatocellular carcinoma (HCC), freshly isolated TIL (n = 5) and peripheral blood lymphocytes (PBL; n = 6; 3 paired with TIL) were studied for expression of TCR variable (V) beta regions. RNA purified from TIL or PBL was reverse-transcribed into complementary DNA. This complementary DNA was amplified by quantitative polymerase chain reaction with 22 primers specific for 20 TCR V beta gene families and a 3' constant (C) beta primer. As a reference for later quantitation, a fragment of TCR C alpha was coamplified with each V beta region. Using 32P-labeled 3' primers, the percentage of total V beta expression was calculated by measuring the cpm of each of the amplified products. In contrast to PBL of 6 control, healthy individuals, whose range of expression of each TCR V beta gene varied from 0 to 13%, the expression of some V beta genes in HCC TIL was as high as 33%, indicating a restricted TCR V beta usage in HCC TIL. When polymerase chain reaction-amplified complementary DNAs of the V beta 1 or V beta 3 genes obtained from two TIL preparations were cloned and sequenced, the same rearrangements were found in the majority of DNA clones. The particular V beta genes that were over- or underrepresented in TIL varied among the patients. In 3 of 6 PBL and 3 of 5 TIL, the V beta 3 gene was expressed with a relatively high frequency. The V beta 4 gene expression was consistently low in patients' TIL or PBL. In 3 paired PBL and TIL, V beta expression was similar. In 5 of 6 cases, HCC PBL had different TCR V beta frequencies from those seen in normal PBL. This analysis of TCR V beta usage in freshly isolated TIL and in PBL indicated that T-lymphocytes in patients with HCC might have restricted immunological reactivity and that V beta 3-restricted TIL might represent antitumor effector cells.

Genomic structure and strain-specific expression of the natural killer cell receptor NKR-P1.

NK cells are able to lyse a variety of virally infected and neoplastic cells in an MHC-unrestricted manner. The cell-surface protein NKR-P1 is thought to play a key role in this process. NKR-P1, initially identified in rat IL-2 activated NK cells, is encoded in the mouse by at least three similar, but not identical, genes. We previously reported the isolation and characterization of three different NKR-P1 cDNA, termed cDNA 2, 34, and 40, from IL-2 activated mouse NK cells. This report describes the structure of the gene encoding NKR-P1 cDNA 2, the smallest of these three cDNA. Gene 2 is composed of six exons spanning approximately 14 kb of genomic DNA. The first exon encodes the N-terminal intracellular domain, and exons 4, 5, and 6 contain the sequences coding for the CRD. This organization is similar to that of other genes that encode C-type animal lectins. The expression of the NKR-P1 genes in A-LAK cells from 13 mouse strains was examined by Northern blot analysis. NKR-P1 expression appears to coincide with that of the NK1.1 Ag. This observation further supports the hypothesis that the NK1.1 Ag is encoded by one of the NKR-P1 genes. Nucleotide sequence analysis of the promoter region of the three NKR-P1 genes in BALB/c and C57BL/6 mice suggests that differences in the level of expression probably do not result from alterations in the upstream regions of these genes, but may be caused by the expression of strain-specific transacting factors.

Mouse NKR-P1. A family of genes selectively coexpressed in adherent lymphokine-activated killer cells.

NK cells are a subpopulation of large granular lymphocytes. They are able to recognize and lyse a wide variety of virally infected or neoplastic target cells without previous sensitization or MHC restriction. The molecules involved in target recognition and subsequent triggering of the killing process are still undefined. Recently, a 30-kDa protein highly expressed on rat NK cells and capable of mediating transmembrane signaling was identified and the gene coding for it cloned and sequenced. To better understand the role of this protein in NK cell-mediated cytotoxicity, we cloned its mouse homologue by cross-hybridization of the rat gene to a cDNA library generated from highly purified mouse lymphokine-activated NK cells. Three messages, differing in size and sequence and encoded by different genes, are specifically cotranscribed in mouse NK cells. The protein products of this gene family express the lectin-like motif characteristic of type II transmembrane molecules. Both the rat and mouse proteins have conserved tyrosine and serine residues in their cytoplasmatic portion that are potential phosphorylation sites. They also share a sequence that could be the binding site of the P56lck tyrosine kinase. These observations are consistent with the signaling function hypothesized for these proteins.

NKR-P1, a signal transduction molecule on natural killer cells.

Natural killer (NK) cells are a subpopulation of large granular lymphocytes characterized by densely staining azurophilic granules. NK cells are able to recognize and lyse various virally infected or neoplastic target cells without previous sensitization or major histocompatibility complex restriction. A 60-kD disulfide-linked dimer, highly expressed on NK cells, was found capable of mediating transmembrane signaling. The gene encoding this signal transduction molecule was cloned and its nucleotide sequence determined. The encoded protein showed significant homology with a number of lectin-related membrane proteins that share receptor characteristics. This protein may function as a receptor able to selectively trigger NK cell activity.

Isolation and characterization of a cDNA that encodes the core protein of the cytolytic granule proteoglycan in rat natural killer cells.

In this report, we describe the transcription of messenger ribonucleic acid (mRNA) specific for the core protein of chondroitin sulfate proteoglycan (CSPG), the entire sequence of the message for CSPG core protein and the presence of CSPG in the granules of a highly purified population of recombinant interleukin-2 (rIL-2)-stimulated rat natural killer (NK), i.e. adherent lymphokine-activated Killer (A-LAK), cells. The presence of CSPG in A-LAK cell granules was demonstrated by a variety of biochemical and immunologic methods. Further, we have demonstrated the presence of a 1.1-kilobase (kb) transcript for the core protein of CSPG by Northern blot analysis using a specific probe (pPG6) derived from a rat yolk sac tumor cell line (L-2). Three cell types that contained CSPG in granules produced a transcript of 1.1 kb, whereas L-2 cells, which localize CSPG on the cell surface, produced a transcript of 1.3 kb. A complementary DNA (cDNA) library was prepared from rat A-LAK cells and the gene for the CSPG core protein was cloned. From approximately 1.2 X 10(5) recombinant phages, 5 positive clones were obtained. The longest clone, PG-NK-5, was sequenced in its entirety and it was found to encode the entire sequence of the CSPG core protein. The other 4 clones were partially sequenced and were identical to PG-NK-5. Comparison of the sequence of PG-NK-5 with pPG6 indicated that they were nearly identical. However, all NK-cell-derived cDNAs contained a poly(A) tail that started 20 basepairs upstream from other published sequences for CSPG core proteins. These data represent the first description of the sequence of the core protein of CSPG contained in NK cells.

Molecular organization of developmentally regulated Dictyostelium discoideum ubiquitin cDNAs.

Dictyostelium discoideum ubiquitin mRNAs are regulated in a complex fashion during spore germination and multicellular development. Species of mRNA of 1900, 1400, 1100, 840, 580, and 500 nucleotides (nt) are found which are expressed differentially during different stages of development. DNA blot analysis indicates that ubiquitin genes constitute a multigene family of at least six genes. cDNAs representing all the ubiquitin mRNA transcripts were isolated and sequenced. The Dictyostelium mRNAs are organized as tandem repeats of the 76 amino acid ubiquitin unit (228 nt). We isolated one cDNA containing seven of these tandem repeats, and two different five- and three-repeat cDNAs. In addition, 2 cDNAs containing a single ubiquitin repeat fused at its 3' end to an unrelated 52 and 78 amino acid extension were identified. There is a remarkable similarity in the sequences of the non-ubiquitin extensions among yeast and mammalian counterparts. The extensions are very basic, containing approximately 30% lysine/arginine. Another common feature of these proteins is the presence of a common structural motif containing cysteine residues at conserved positions, suggesting a metal binding domain that matches a consensus sequence of Xenopus transcription factor TFIIIA and other nucleic acid binding proteins. The characterization of ubiquitin cDNAs and genomic sequences in D. discoideum now makes the understanding of its developmental regulation feasible.

Structure of two developmentally regulated Dictyostelium discoideum ubiquitin genes.

A previously isolated cDNA clone, pLK229, that is specific for mRNA developmentally expressed during Dictyostelium discoideum spore germination and multicellular development, was used to screen two genomic libraries. Two genomic sequences homologous to pLK229 were isolated and sequenced. Genomic clone p229 is identical to the cDNA clone pLK229 and codes for a polypeptide of 381 amino acids. This polypeptide is composed of five tandem repeats of the same 76-amino-acid sequence. Clone lambda 229 codes for a protein of 229 amino acids, containing three tandem repeats of the identical 76-amino-acid sequence. A computer search for homology to known proteins revealed that the 76-amino-acid repeat was identical to human and bovine ubiquitin except for two amino acid differences.