Genome-wide linkage and sequence analysis challenge CCDC66 as a human retinal dystrophy candidate gene and support a distinct NMNAT1-related fundus phenotype.

To uncover the genotype underlying early-onset cone-rod dystrophy and central nummular macular atrophic lesion in 2 siblings from an endogamous Arab family, we performed targeted next-generation sequencing (NGS) of 44 retinal dystrophy genes, whole-exome sequencing (WES) and genome-wide linkage analysis. Targeted NGS and WES in the index patient highlighted 2 homozygous variants, a CCDC66 frameshift deletion and a novel missense NMNAT1 variant, c.500G>A (p.Asn167Ser). Linkage and segregation analysis excluded the CCDC66 variant and confirmed the NMNAT1 mutation. Biallelic NMNAT1 mutations cause Leber congenital amaurosis with a central nummular macular atrophic lesion (LCA9). The NMNAT1 mutation reported here underlied cone-rod dystrophy rather than LCA but the fundus lesion was compatible with that of LCA9 patients, highlighting that such a fundus appearance should raise suspicion for biallelic mutations in NMNAT1 when in the context of any retinal dystrophy. Although Ccdc66 mutations have been proposed to cause retinal disease in dogs, our results and public databases challenge CCDC66 as a candidate gene for human retinal dystrophy.

Positional cloning of the gene for X-linked retinitis pigmentosa 2.

X-linked retinitis pigmentosa (XLRP) results from mutations in at least two different loci, designated RP2 and RP3, located at Xp11.3 and Xp21.1, respectively. The RP3 gene was recently isolated by positional cloning, whereas the RP2 locus was mapped genetically to a 5-cM interval. We have screened this region for genomic rearrangements by the YAC representation hybridization (YRH) technique and detected a LINE1 (L1) insertion in one XLRP patient. The L1 retrotransposition occurred in an intron of a novel gene that consisted of five exons and encoded a polypeptide of 350 amino acids. Subsequently, nonsense, missense and frameshift mutations, as well as two small deletions, were identified in six additional patients. The predicted gene product shows homology with human cofactor C, a protein involved in the ultimate step of beta-tubulin folding. Our data provide evidence that mutations in this gene, designated RP2, are responsible for progressive retinal degeneration.

Mutations in PHF8 are associated with X linked mental retardation and cleft lip/cleft palate.

Truncating mutations were found in the PHF8 gene (encoding the PHD finger protein 8) in two unrelated families with X linked mental retardation (XLMR) associated with cleft lip/palate (MIM 300263). Expression studies showed that this gene is ubiquitously transcribed, with strong expression of the mouse orthologue Phf8 in embryonic and adult brain structures. The coded PHF8 protein harbours two functional domains, a PHD finger and a JmjC (Jumonji-like C terminus) domain, implicating it in transcriptional regulation and chromatin remodelling. The association of XLMR and cleft lip/palate in these patients with mutations in PHF8 suggests an important function of PHF8 in midline formation and in the development of cognitive abilities, and links this gene to XLMR associated with cleft lip/palate. Further studies will explore the specific mechanisms whereby PHF8 alterations lead to mental retardation and midline defects.

Focussing human B cell specificity by immunoselection via antigen-presenting cells in vitro.

We present a protocol for in vitro immunization of B cells using monocyte-derived accessory cells (MoAC). MoAC are developed from human peripheral blood monocytes in culture and represent functionally competent inducers of antigen-specific immune responses. Using MoAC, we attempted to immunoselect TT-specific lymphocytes by rosetting. Adherent human MoAC were pulsed with tetanus toxoid (TT) and allowed to form clusters with autologous lymphocytes, followed by removal of non-adherent cells. After one week of culture, a specific anti-TT antibody response emerged on a low background of unspecific Ig. In comparison, cultures which had not been selected for adherent cells produced a high polyclonal background. Our results demonstrate that from peripheral blood cells, previously not a favourable source for in vitro immunization, in a majority of tests antigen-specific B cells could efficiently be immunoselected via adherence to autologous antigen-presenting cells, leading to a high-titre in vitro immunization.

Mutations in the X-linked RP2 gene cause intracellular misrouting and loss of the protein.

Mutations in RP2 cause the second most frequent form of X-linked retinitis pigmentosa, a severe retinal degeneration that leads to loss of visual acuity and blindness. The RP2 gene encodes a protein with homology to cofactor C, a tubulin-folding chaperone. By searching protein sequence databases, we identified a whole set of similar molecules from diverse organisms. Protein sequence alignments show that RP2 and cofactor C represent members of two distinct orthologous groups. All previously identified missense mutations affect amino acid residues which are conserved in all RP2 orthologues or both orthologous groups. Intracellular localization of the wild-type protein and mutated variants was determined by fluorescence microscopy of cells expressing RP2 with a green fluorescent protein tag. A mutation in the N-terminus of RP2 abolishes localization to the plasma membrane in HeLa cells. C-terminal protein truncation mutations, which account for 2/3 of the pathogenic RP2 variants, lead to scattered fluorescent foci in the cytoplasm of COS-7 and HeLa cells. Analysis of protein extracts from the respective cells with anti-RP2 antibodies identified truncated proteins of expected size in a low-speed centrifugation pellet while the wild-type protein appeared in the supernatant. Moreover, no protein was detected in immortalized cell lines from patients with protein truncation mutations while mRNA was still present. Thus, loss of the protein and/or aberrant intracellular distribution might be the basis for the photoreceptor cell degeneration in most RP2 cases.

Genomic structure and comparative analysis of nine Fugu genes: conservation of synteny with human chromosome Xp22.2-p22.1.

The pufferfish Fugu rubripes has a compact 400-Mb genome that is approximately 7.5 times smaller than the human genome but contains a similar number of genes. Focusing on the distal short arm of the human X chromosome, we have studied the evolutionary conservation of gene orders in Fugu and man. Sequencing of 68 kb of Fugu genomic DNA identified nine genes in the following order: (SCML2)-STK9, XLRS1, PPEF-1, KELCH2, KELCH1, PHKA2, AP19, and U2AF1-RS2. Apart from an evolutionary inversion separating AP19 and U2AF1-RS2 from PHKA2, gene orders are identical in Fugu and man, and all nine human homologs map to the Xp22 band. All Fugu genes were found to be smaller than their human counterparts, but gene structures were mostly identical. These data suggest that genomic sequencing in Fugu is a powerful and economical strategy to predict gene orders in the human genome and to elucidate the structure of human genes.

RPGR transcription studies in mouse and human tissues reveal a retina-specific isoform that is disrupted in a patient with X-linked retinitis pigmentosa.

X-linked retinitis pigmentosa (XLRP) is a genetically heterogeneous group of progressive retinal degenerations. The disease process is initiated by premature apoptosis of rod photoreceptor cells in the retina, which leads to reduced visual acuity and, eventually, complete blindness. Mutations in the retinitis pigmentosa GTPase regulator ( RPGR ), a ubiquitously expressed gene at the RP3 locus in Xp21.1, account for approximately 20% of all X-linked cases. We have analysed the expression of this gene by northern blot hybridization, cDNA library screening and RT-PCR in various organs from mouse and man. These studies revealed at least 12 alternatively spliced isoforms. Some of the transcripts are tissue specific and contain novel exons, which elongate or truncate the previously reported open reading frame of the mouse and human RPGR gene. One of the newly identified exons is expressed exclusively in the human retina and mouse eye and contains a premature stop codon. The deduced polypeptide lacks 169 amino acids from the C-terminus of the ubiquitously expressed variant, including an isoprenylation site. Moreover, this exon was found to be deleted in a family with XLRP. Our results indicate tissue-dependent regulation of alternative splicing of RPGR in mouse and man. The discovery of a retina-specific transcript may explain why phenotypic abberations in RP3 are confined to the eye.

Preparation of human and murine monoclonal antibodies: antigens combined with or conjugated to lipopeptides constitute potent immunogens for in vitro and in vivo immunizations.

Lipopeptide analogues of bacterial lipoprotein constitute polyclonal B lymphocyte activators. Combined with or covalently coupled to antigens, they act as potent adjuvants. We could show that antigens (BSA-DNP, TNP-SRBC, saxitoxin, HIV-1 gp160(BH10303-329, EGFR516-523) combined with or coupled to the synthetic lipodipeptide N-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-(R)-cysteinyl-s erine (P3CS) constitute active immunogens in vivo in mice. They were also able to induce an in vitro humoral immune response in the murine and human systems, and B lymphocytes thus activated were suitable for fusion. Thus, the antigens chaperonin/phytochrome, BSA-saxitoxin, histamine, HIV-1 gp160 (BH10(303-329)), HIV-1 gp160 (RF316-341)), and HIV-2 p17 (ROD111-121) combined with or conjugated to P3CS could be used for in vitro immunization followed by the preparation of murine and human monoclonal antibodies. Our novel immunization procedure offers reproducibility, high antibody titers often after one immunization, lack of toxicity of the adjuvants, easy chemical preparation of the conjugates in mg amounts, and the applicability of the conjugates for screening for the antibodies obtained.

Inhibition of human immunodeficiency virus-1 proliferation by liposome-encapsulated sense DNA to the 5' tat splice acceptor site.

A liposome formulation containing a distearoylphosphatidylethanolamine analog was developed that was endocytosed by both lymphocytes and monocytes. This formulation was used to encapsulate sense and antisense 20-mer oligodeoxynucleotides to the 5' tat splice acceptor site of human immunodeficiency virus type 1. At a DNA concentration of 140 nM, the liposome-encapsulated sense DNA inhibited p24 production by as much as 84% in human peripheral blood leukocytes infected with "wild-type" virus. This treatment also reduced the number of peripheral blood leukocytes producing intracellular viral antigen by 71%. Of interest, no reduction in either parameter was observed for the antisense-containing liposomes. The results demonstrate the promise of a new liposomal delivery vehicle to inhibit human immunodeficiency virus replication by an entrapped oligodeoxynucleotide.