Genetic and physical mapping of the locus for autosomal dominant renal Fanconi syndrome, on chromosome 15q15.3.

Autosomal dominant renal Fanconi syndrome is a genetic model for the study of proximal renal tubular transport pathology. We were able to map the locus for this disease to human chromosome 15q15.3 by genotyping a central Wisconsin pedigree with 10 affected individuals. After a whole-genome scan with highly polymorphic simple sequence repeat markers, a maximum LOD score of 3.01 was calculated for marker D15S659 on chromosome 15q15.3. Linkage and haplotype analysis for an additional 24 markers flanking D15S659 narrowed the interval to approximately 3 cM, with the two highest single-point LOD scores observed being 4.44 and 4.68 (for D15S182 and D15S537, respectively). Subsequently, a complete bacterial artificial chromosome contig was constructed, from the High Throughput Genomic Sequence Database, for the region bounded by D15S182 and D15S143. The identification of the gene and gene product altered in autosomal dominant renal Fanconi syndrome will allow the study of the physiology of proximal renal tubular transport.

The correlation of genotype and phenotype in Portuguese hyperphenylalaninemic patients.

To understand the basis for the clinical heterogeneity of phenylalanine hydroxylase deficiency among Portuguese hyperphenylalaninemic patients, genotype-phenotype correlations were established. A group of 61 patients was completely genotyped, leading to the identification of 20 different mutant alleles in 36 different genotypic combinations, including a mutant allele not reported previously. The severity of those mutations found within this hyperphenylalaninemic population, which have not been previously expressed in vitro, were assessed. The results obtained by the present study exhibit a strong correlation between the predicted residual enzyme activity, as deduced from the genotype of the patients, and the biochemical phenotype represented by the diagnostic parameters (phenylalanine levels before the beginning of treatment and the dietary phenylalanine tolerance). It was observed that only a judicious follow-up and compliance with the appropriate diet permits the correct assessment of the clinical phenotype of the patients. Additionally, based upon the correlation observed between genotypes and diagnostic parameters, it was possible to predict the potential residual enzyme activity of those mutations (identified in our patients) which have not yet been studied in vitro.

Human phenylalanine hydroxylase gene expression in kidney and other nonhepatic tissues.

Phenylalanine hydroxylase (PAH) is the key enzyme in phenylalanine metabolism. PAH deficiency results in hyperphenylalaninemia, leading to severe mental retardation in the classical form of the disease, phenylketonuria (PKU). Previously the expression of PAH could only unambiguously be demonstrated in human liver, whereas in rodents PAH expression has been established in kidney and liver. Reports concerning PAH activity in other human or rodent tissues were severely questioned by subsequent investigations such that they did not gain general recognition. Conducting Northern blot analyses, we detected the PAH transcript in RNA isolated from human liver, kidney, pancreas, and brain. PAH gene expression in human kidney was subsequently investigated by RNase protection assay analyses, RNA in situ hybridization, immunohistochemistry, enzyme assay, and cDNA isolation. These experiments allowed the conclusive verification of a functional PAH enzyme in human kidney. The primary structure of the kidney transcript corresponded to the structure of the liver transcript. Human kidney PAH may play a significant role in phenylalanine homeostasis of the organism, as impaired phenylalanine hydroxylation has been observed in renal failure and differences in the regulation of the kidney versus the liver enzyme have been indicated. These results provide new aspects to research into the basis for the heterogeneity of hyperphenylalaninemia phenotypes and establish that the expression of the human PAH gene is not limited to the liver.

Expression patterns of murine lysosome-associated membrane protein 2 (Lamp-2) transcripts during morphogenesis.

We report the isolation and characterization of the murine homologues to human and chicken lysosome-associated membrane protein (Lamp)-2 transcripts and their prevalent expression patterns during development. Lamp-2 transcripts code for proteins predominant in and specific for the lysosomal membrane. The function of these proteins is still under investigation. Other than in the lysosomal membrane, Lamp-2 proteins have been detected at the plasma membrane of cells in a differentiation dependent and activation dependent manner. They were also observed at the plasma membrane of cells, which secrete lysosomal hydrolases. Involvement of Lamp-2 in cell adhesion during such events has been proposed. A study of the developmental expression patterns of m-Lamp-2 transcripts was undertaken to help elucidate possible functions of their respective proteins. The m-Lamp-2b transcript was prevalent in neural crest derived ganglia. The m-Lamp-2a and -2c transcripts were similarly expressed in structures containing neural crest derived tissue with the strongest signals detected in thymus. However, m-Lamp-2a and -2c transcript expression differed in mesoderm or endoderm derived mesenchymal and epithelial tissues. M-Lamp-2c expression was pronounced in mesenchyme early in development, in limb connective tissue, and in lung parenchyma, whereas m-Lamp-2a was prevalent in the liver, the pancreas, and in differentiating kidney epithelium, and became increasingly prominent in the epithelial lining of the digestive and the respiratory tract during development. These results correlated with the detection of m-Lamp-2 protein in these tissues. In conclusion, all m-Lamp-2 transcripts were detected in tissues undergoing apoptosis during development requiring phagolysosome involvement. In addition, m-Lamp-2a and m-Lamp-2c transcripts were observed in epithelium and mesenchyme during the time of epithelial-mesenchymal interaction, mesenchymal-epithelial transformation, and branching. Their expression pattern became more tissue and cell type specific as differentiation progressed. These patterns indicate a possible involvement of m-Lamp-2 proteins in cell/cell or cell/extracellular matrix interaction, and appear to reflect tissue and cell type specific roles of lysosomes during morphogenesis.

TurboPrep II: an inexpensive, high-throughput plasmid template preparation protocol.

In an effort to reduce plasmid template preparation costs for large-scale genomic sequencing projects, a boiling minipreparation protocol has been developed that enables a single individual to easily prepare 768 sequence-quality templates in 8 h, without automation. The maximum throughput for one individual using one centrifuge in the manual configuration is 1920 templates in about 8 h. The most time-consuming manual steps of this method involve pipetting, which can be automated, resulting in a significant increase in throughput and about a 60% increase in yield. This method in the fully manual configuration yields sufficient double-stranded template for two sets of cycle sequencing reactions, membrane spotting for hybridization analysis and host cell transformation for the recovery of the original recombinant. The current materials cost per template using this method is less than twenty cents. The quality of the sequence generated has been evaluated by manual 35S radioactive cycle sequencing. Initial results have shown templates prepared by this method to yield greater than 300 bp of readable sequence when the radioactively labeled products were resolved on 6% modified denaturing polyacrylamide gels.

Phenylalanine hydroxylase genotypes, predicted residual enzyme activity and phenotypic parameters of diagnosis and treatment of phenylketonuria.

The interdependence of the predicted in vitro residual enzyme activity (PRA), as deduced from the complete genotypes of 64 hyperphenylalaninaemic patients, and parameters for diagnosis of hyperphenylalaninaemic disorders, the fluctuation of the phyenlylalanine (Phe) values during treatment, long-term dietary control during treatment, and a parameter for the outcome of therapy (IQ) was investigated by correlation analysis. A highly significant correlation was found between the PRA and diagnostic parameters, as well as the fluctuation of the Phe values during treatment. Significant correlations were also observed between the parameter describing the fluctuation of the Phe values and the IQ, as well as between the quality of dietary control and IQ. The PRA is a valuable tool for the differential diagnosis of hyperphenylalaninaemic disorders and for the prediction of one aspect of the course of the disease which is related to the intellectual outcome of therapy. The quality of dietary control was independent of the genotype, indicating that the outcome of therapy can be successfully manipulated in spite of the genetic make-up.

Point mutation analysis of the FMR-1 gene in autism.

We have analysed all 17 exons of the human FMR-1 gene for mutations in autistic individuals using single-stranded conformational polymorphism (SSCP) analysis. We have identified three new polymorphisms. SSCP DNA fragment shifts were found for exons, 5, 10 and 11 in autistic individuals and in normal controls. Sequence analysis showed the exon 10 and 11 polymorphisms to result from base substitutions within introns, 14 and 73 bp downstream from the splice site respectively. In exon 5, a G to A base substitution at codon 138 has no effect on amino acid sequence. The intronic polymorphism adjacent to exon 10 was analysed amongst two groups of unrelated autistic individuals-one from the UK and one from Germany- and amongst a control population. Comparison of allele frequencies between Caucasian autism cases and Caucasian controls show a significant increase in the presence of the polymorphic intronic sequence 3' to exon 10 (Fisher's exact test, P = 0.01). The base change is at a position where it is unlikely to affect splicing of the FMR-1 transcript and is most likely a neutral variant that has only a spurious false positive association with autism. However further linkage disequilibrium analyses are justifiable. The positive association with autism should be explored in further samples to determine whether it has any validity as a genetic marker for autism.

An alternatively spliced form of the human lysosome-associated membrane protein-2 gene is expressed in a tissue-specific manner.

We report the isolation of an alternatively spliced human lysosome-associated membrane protein-2 (h-lamp-2) transcript which is overexpressed in human muscle. The cloning of this transcript is an indication for the tissue-specific expression of lysosomal membrane proteins and implicates the possibility of multiple functions for the protein products of the h-lamp-2 gene, as well as other lysosome-associated membrane proteins. The new transcript, designated h-lamp-2b, results from the alternative splicing of the last exon, exon 9, the alternative form of which is approximately 2800 bp in length. The resulting protein is identical in length to the previously reported h-lamp-2 protein, 410 amino acids including the leader peptide. This final exon, which encodes the last eleven amino acids of the luminal domain, the 24 amino acid transmembrane spanning region, and an eleven amino acid cytoplasmic tail, shows complete conservation of the Gly.Tyr.X.X lysosomal targeting signal with regard to its position relative to the transmembrane spanning region and the carboxy terminus of the protein. Immune electron microscopy studies verified localization of this alternative gene product to the lysosomal membrane.

Complete cDNA sequence of human lysosome-associated membrane protein-2.

The isolation and sequencing of 15 independent human lysosome-associated membrane protein-2 (h-lamp-2) recombinants from a primary human liver cDNA library has resulted in the determination of a transcript sequence significantly longer than previously reported and reveals the utilization of each of the four potential polyadenylation signals (AATAAA) present in the 3' untranslated region. The most 5' extending cDNA clone initiates upstream of the proposed transcription initiation site. A number of differences with published sequences for the h-lamp-2 transcript were observed, some of which result in amino acid changes in the predicted primary structure of the h-lamp-2 protein, and two of which give rise to restriction fragment length polymorphisms. The knowledge of these sequence alterations and polymorphisms is an important consideration for the further analysis of the h-lamp-2 locus with regard to the delineation of function and association with human inherited disorders.

Evolutionary conservation and genomic organization of XAP-4, an Xq28 located gene coding for a human rab GDP-dissociation inhibitor (GDI).

After the development of efficient methods for the construction of transcription maps of defined genomic regions, the rate-limiting step in the analysis of the coding potentials of these regions is the elucidation of function of the novel genes and the examination of their possible involvement in hereditary diseases localized to the region. This can be greatly facilitated by the detection of sequence homology to a gene of known function. XAP-4 is one of the genes identified in the G6PD region of the human Xq28 by direct cDNA selection. The rapid assembly of this gene and the determination of its function was possible because of its sequence homology with the bovine smg p25A/rab3A GDP dissociation inhibitor (GDI). Sequence comparison with other GDIs in the databases has revealed that XAP-4 belongs to one of at least two distinct classes of mammalian rab GDIs. The rab GDIs, which play an important role in the regulation of cellular transport, are highly evolutionarily conserved, as are several other genes identified in the neighborhood of XAP-4. This genomic region is very gene dense, and all the cDNA clones from the approximately 2.5-kb-long transcript of XAP-4 map to a single 7.5-kb genomic EcoRI fragment. The genomic organization of XAP-4 has been examined to determine the distribution of the exonic sequences within this short segment of genomic DNA. It was found that, similar to several other genes from the region, XAP-4 is split into exons of average size, which are interrupted by very short introns.

DNA sequence polymorphisms in exonic and intronic regions of the human phenylalanine hydroxylase gene aid in the identification of alleles.

Five sequence polymorphisms at the phenylalanine hydroxylase (PAH) gene locus were observed to be in tight association with specific alleles of this locus. Since these polymorphisms can be detected using polymerase chain reaction (PCR) methodology, application of a combination of these polymorphisms reduces the effort involved in PAH DNA haplotype analysis, which is needed for population genetic analysis or diagnosis of the disease status. In addition our results indicate the evolution of haplotype 3, 4 and 7 PAH alleles from a common ancestor, whereas PAH haplotypes 5, 6, and 11 arose from another common ancestor allele. These data reveal that two of the polymorphisms investigated originated before the separation of races.

Construction of a transcription map of a 300 kb region around the human G6PD locus by direct cDNA selection.

A transcription map covering a 300 kb region around the G6PD gene in the human Xq28 region was constructed by the direct cDNA selection method and the analysis of the resulting region-specific enriched cDNA sublibrary. Seven new genes and two loci of endogenous retrovirus HERV-K were identified. The distribution of the genes across the region is strongly non-uniform and follows the non-uniform distribution of GpG islands in the area. While one of the novel genes was found to be highly homologous to bovine smg p25A GDP-dissociation inhibitor, the remaining genes did not detect any homology to known genes. The analysis of region-specific cDNA sublibraries represents a simple, rapid and efficient tool for the generation of a regional transcription map.

Direct selection of DNA sequences conserved between species.

An essential requirement in the analysis of genomes is the identification of functionally important sequence elements, which are often evolutionarily conserved. We describe here the development of a novel procedure for the selective isolation of conserved sequences which is based on hybridization of PCR-amplifiable DNA fragments from the whole genome of one species to biotinylated DNA from a genomic region of another species. The interspecies DNA hybrids are immobilized and the PCR-amplifiable DNA fragments are eluted, amplified and after further hybridization-amplification rounds cloned. This method was used for the generation of sublibraries of conserved sequences from mouse and pig DNA from regions corresponding to cosmids from the human Xq28 region. Mouse and pig homologs of sequences containing exons of known human genes, as well as exons from novel genes have been identified.

Alternative splicing in the fragile X gene FMR1.

The FMR1 gene, associated with fragile X syndrome, has recently been cloned and the sequence of partial cDNA clones is known. We have determined additional cDNA sequences both at the 5' and 3' end. We have characterized the expressed gene by means of RT-PCR in various tissues and have found that alternative splicing takes place in the FMR1 gene, which does not seem to be tissue specific. When the different alternative splicing events are combined, 12 distinct mRNA products could result from FMR1 expression in each tested tissue. In all these transcripts the open reading frame is maintained until the same stop codon. At the 3' end alternative use of polyadenylation signals is found. The alternative splicing allows functional diversity of the FMR-1 gene. Whether all the possible proteins will be synthesized and whether they will be functionally active has to be determined.