Region 16p13 Research Articles

RPS19 and JAK2 Are Not Silenced by DNA Methylation in Diamond-Blackfan Anemia.

Diamond Blackfan Anemia (DBA) is a congenital disorder characterized by decreased red blood cell production accompanied by developmental abnormalities in 30% patients. Twenty-five percent of DBA patients display heterozygous mutations of ribosomal protein S19 (RPS19) on chromosome 19q13.2. No mutations were found in genes for other ribosomal proteins of the translation initiation complex. Although a second DBA locus has been proposed on in the region 8p23.3–8p22, the precise molecular defect is not known in 75% of DBA patients. The exact mechanism of how RPS19 mutations affect erythropoiesis remains unclear. Haploinsufficency of RPS19 may hamper translation machinery important for rapid erythroid differentiation. Reduced gene expression of a cluster of ribosomal proteins including RPS19 in DBA patients was recently reported. No causal therapy for DBA is available, with the exception of bone marrow transplantation. Some DBA patients benefit therapeutically from corticosteroids, cyclosporine A, or metoclopramide. Recently, a long-lasting remission was described in a DBA patient treated with valproic acid, a histone deacetylase inhibitor, suggesting epigenetic suppression of genes critical for erythropoiesis may be involved in the pathogenesis of DBA. DNA methylation of promoter-associated CpG islands is an epigenetic modification resulting in transcriptional silencing functionally equivalent to a loss-of-function mutation. Constitutive activation of JAK2 tyrosine kinase by a somatic V617F mutation leads to excessive erythropoiesis in polycythemia vera, an antonym of DBA. We hypothesized that silencing by DNA methylation of promoter-associated CpG island of the RPS19 or JAK2 genes may play a role in DBA. To test the hypothesis, we analyzed DNA methylation of RPS19 and JAK2 genes in 14 patients from the Czech DBA Registry. Genomic DNA isolated from blood cells of 3 DBA patients carrying heterozygous RPS19 mutations, 11 DBA patients without RPS19 mutation and 4 control samples was treated with bisulfite to convert all unmethylated cytosines to uracils while methylated cytosines were spared from the conversion. A region spanning 13 CpG sites positioned from 1–160 bases downstream from transcription start site (TSS) of RPS19 gene was PCR amplified and cloned in a sequencing vector. Individual bacterial clones were isolated and PCR inserts were sequenced in 8–12 clones per sample. Bisulfite cloning and sequencing revealed that more than 99% of CpG sites were converted to TpG and thus not methylated either in DBA samples (only 4/1466 CpG sites were methylated, methylation frequency was 0.3%) or control samples (2/555 CpG sites methylated, methylation frequency 0.4%). To explore a possibility of epigenetic suppression of erythropoietin signaling in DBA we analyzed DNA methylation of the CpG island of JAK2 tyrosine kinase gene in the same set of samples. Bisulfite-treated DNA was PCR amplified and T/C polymorphisms corresponding to unmethylated or methylated CpG sites were quantified by pyrosequencing. All DBA and control samples showed the absence of DNA methylation at four CpG sites located 12 to 25 bases downstream of TSS. We conclude that epigenetic silencing by DNA methylation is not involved in the expression of ribosomal structural protein RPS19; neither it affects the expression of a transducer of erythropoietin signaling JAK2 tyrosine kinase.

Comparative Genomic Hybridization Array Analysis and Real-Time PCR Reveals Genomic Copy Number Alteration for Lung Adenocarcinomas

Genomic alterations in lung cancer tissues have been observed in various studies. To analyze the aberrations in the genome of lung cancer patients, we used array comparative genomic hybridization (array CGH) in 15 lung adenocarcinoma (AdC) tissues. Copy number gains and losses in chromosomal regions were detected and corresponding genes were confirmed by real-time polymerase chain reaction (PCR). As for the results, several frequently altered loci, including gain of 16p (46% of samples), were found, and the most common losses were found in 14q32.33 (26% of samples). High-level DNA amplifications (> 0.8 log(2) ratio) were detected at 1p, 5p, 7p, 9p, 11p, 11q, 12q, 14q, 16p, 17q, 19q, 20p, 21q, and 22q. A subset of genes, gained or lost, was checked for over- or underrepresentation by means of real-time PCR. The degree of fold change was highest in ECGF1 (22q13.33), HOXA9 (7p15.2), MAFG (17q25.3), TSC2 (16p13.3), and ICAM1 (19p13.2) genes and the 16p chromosome terminal region (16p13.3pter). Taken together, these results show that array CGH could be used as a powerful tool for identification of genomic alteration for lung cancer, and the above-mentioned genes may represent potential candidate genes in the study of lung cancer pathogenesis and diagnosis.

Forensic evaluation of three closely linked STR markers in a 13 kb region at Xp11.23

Searching for markers located in the region p11 of the X-chromosome the clone AF196972 was checked for its content of microsatellites. The two tetranucleotide STRs DXS10076 and DXS10078 and the trinucleotide STR DXS10077 were characterised and evaluated for their forensic efficiency in a population sample of 352 unrelated individuals from North-West Germany. Whereas at locus DXS10076 10 alleles with PIC and HET values of 0.767 and 0.747 and at locus DXS10078 13 alleles with PIC and HET values of 0.811 and 0.861 could be observed locus DXS10077 only consists of 5 alleles leading to much lower HET and PIC values (0.492 and 0.507) and therefore to a decreased individualization capacity.

Posterior Polymorphous Corneal Dystrophy in Czech Families Maps to Chromosome 20 and Excludes theVSX1Gene

Posterior polymorphous corneal dystrophy (PPCD) is an autosomal dominant disorder, affecting both the corneal endothelium and Descemet's membrane. In the Czech Republic, PPCD is one of the most prevalent corneal dystrophies. The purpose of this study was to determine the chromosomal locus of PPCD in two large Czech families, by using linkage analysis. Linkage analysis was performed on 52 members of two Czech families with PPCD and polymorphic microsatellite markers and lod scores were calculated. The candidate gene VSX1 was also screened for mutations. Significant lod scores were obtained with microsatellite markers on chromosome 20. Linkage analysis delineated the Czech PPCD locus to a 2.7-cM locus on chromosome 20, region p11.2, between flanking markers D20S48 and D20S139, which excluded VSX1 as the disease-causing gene in both families. In addition, the exclusion of VSX1 was confirmed by sequence analysis. This study reports the localization of PPCD in patients of Czech origin to chromosome 20 at p11.2. Linkage data and sequence analysis exclude VSX1 as causative of PPCD in two Czech families. This refined locus for PPCD overlaps the congenital hereditary endothelial dystrophy (CHED1) disease interval, and it is possible that these corneal dystrophies are allelic.

Identification of the gene responsible for methylmalonic aciduria and homocystinuria, cblC type

Methylmalonic aciduria and homocystinuria, cblC type (OMIM 277400), is the most common inborn error of vitamin B(12) (cobalamin) metabolism, with about 250 known cases. Affected individuals have developmental, hematological, neurological, metabolic, ophthalmologic and dermatologic clinical findings. Although considered a disease of infancy or childhood, some individuals develop symptoms in adulthood. The cblC locus was mapped to chromosome region 1p by linkage analysis. We refined the chromosomal interval using homozygosity mapping and haplotype analyses and identified the MMACHC gene. In 204 individuals, 42 different mutations were identified, many consistent with a loss of function of the protein product. One mutation, 271dupA, accounted for 40% of all disease alleles. Transduction of wild-type MMACHC into immortalized cblC fibroblast cell lines corrected the cellular phenotype. Molecular modeling predicts that the C-terminal region of the gene product folds similarly to TonB, a bacterial protein involved in energy transduction for cobalamin uptake.

Complex Translocation (8;16;21); a New Variant of t(8;21), with t(13;22) in Acute Myeloid Leukemia.

The translocation (8;21)(q22;q22) is a common recurrent chromosome aberration present in 10% to 15% of all acute myeloid leukemias. In approximately 3% of all cases with t(8;21), a variant of t(8;21) involving chromosomes 8,21 and other chromosomes is present. We report here a case of AML-M2 with a new complex translocation (8;16;21)(q22;p16.3;q22) associated with additional abnormality t(13;22)(q22;q13) not described before. Fluorescent in-situ hybridization analysis with probes for ETO and AML1 genes, and probes for the subtelomeric regions of chromosome 16 demonstrated an ETO-AML1 fusion signal on der(8). The 5′ region of ETO gene with chromosome arm distal to translocation breakpoint had moves to der(16) while region of AML1 probe proximal to translocation brealpoint stayed on der(21). The signal for the subtelomeric probe for 16p was present on der(21). Use of painting probe for chromosome 13 confirmed t(13;22). In a hematopoietic cell the expression of chimeric AML1- ETO protein resulting from t(8;21) plays a key role in leukemic transformation by targeting AML1-CBFB transcription factor complex, an essential regulator of genes required for normal hematopoietic cell development, by dominant negative effect on normal AML1 protein. However, the expression of AML1- ETO does not block normal differentiation of stem cells. Additional mutational events must occur in hematopoietic progenitor cell to block normal path of differentiation. Although clinical features of variant translocation is same as cases with conventional t(8;21), prognostic implications of variant (8;21) are yet to be established. Chromosome region 16p13 has been involved in clinically important translocations with poor prognosis such as t(8;16)(p11;p13) and t(11;16)(q23;p13) and may have similar implications. Translocation (8;21) is frequently accompanied by additional chromosome abnormalities i.e. loss of sex chromosome, trisomy 8 and structural abnormalities of 9q. Additional reports on secondary genetic alterations accompanying t(8;21) are needed to understand their cumulative effect. Chromosome region 16p13, 13q22 and 22q13 may harbor putative genes influencing normal path of differentiation.

Alpha-thalassémies et troubles bipolaires : liens génétiques possibles ?

After a previous paper discussing the possible association between beta-thalassemias and bipolar disorder, this article considers a possible association between alpha-thalassemia and the bipolar disorder. We report the case of a 36 year old woman with bipolar disorder and alpha-thalassemia. The patient, native of Reunion Island, has a family history of bipolar disorder (both parents, one brother, and a paternal uncle). The severity of the bipolar disorder type I in her family, is illustrated by the suicides of both parents, one brother and the paternal uncle, in intervals of only a few years. After a Medline review (1980-2004) we found only two studies suggesting a possible relationship between bipolar disorders and alpha-thalassemias, but without clinical case report information. Some genetic studies described the existence of possible genetic susceptibility for bipolar disorder on the short arm of chromosome 16, close to the gene involved in certain alpha-thalassemias, on the region 16p13.3. An interesting finding is that the sequencing of 258 kb of the chromosome region 16p13.3 not only allowed the identification of genes involved in the alpha-thalassemia and in the vulnerability to bipolar disorders, but also the identification of genes implicated in tuberous sclerosis, in polycystic kidney disease, in cataract with microophtalmia, and in vulnerability genetic factors for ATR-16 syndrome, asthma, epilepsy, certain forms of autism and mental retardation. Numerous clinical descriptions and some familial studies on linkage suggested a possible relationship between tuberous sclerosis, polycystic kidney disease, cataract with microophtalmia, ATR-16 syndrome, asthma, epilepsy, certain forms of autism, mental retardation and bipolar disorder, given the closeness of these vulnerability genes on the short arm of the chromosome 16. A vulnerability gene of alcohol dependence was also identified on this same chromosome region (16p13.3), by a study concerning 105 families. Taking into account the methodological difficulties due to the clinical and genetic heterogeneity of bipolar disorder, we suggest that linkage techniques should be used to confirm the presence of susceptibility genetic factor for bipolar disorders on chromosome 16. Thus a known genetic disease (alpha-thalassemia) could contribute to confirming the presence on the short arm of chromosome 16 of a susceptibility genetic factor for bipolar disorders. Linkage studies should be performed in families with a strong association for both diseases. Thanks to linkage techniques, one could hope for an improvement in understanding the physiopathology of bipolar disorder, with possible implications at a therapeutic level.

Genome-wide scan of homogeneous subtypes of NIMH genetics initiative schizophrenia families

In the light of the potential etiological heterogeneity of schizophrenia, we reanalyzed the NIMH genetics initiative data for schizophrenia. We performed linkage analyses on schizophrenia families divided into more homogeneous subgroups. The African-American and European-American families were divided into groups that were successively more homogeneous. The first group included schizophrenia families that were highly familial, meaning that they contained a minimum specified number of affected individuals. We also excluded patients with environmental influences that may affect disease status. These influences included obstetric complications (OC) and viral infections during the neurodevelopmental stage (VIN). In the African-American sample, a linkage analysis of highly familial schizophrenia families without any environmental influences resulted in a single-point LOD (SLOD) score of 2.90, a multipoint LOD (MLOD) of 2.11, a single-point heterogeneity LOD (SHLOD) score of 3.04, and a multipoint heterogeneity LOD (MHLOD) score of 2.11 at marker D8S1819 (8p23.1) under a dominant parametric model. The highly familial European-American schizophrenia families resulted in an SLOD of 0.91 and an MLOD of 1.85, an SHLOD of 1.64 and an MHLOD of 1.97 at marker D22S1169 (22q13.32) using a recessive parametric model. Although this work should be interpreted cautiously and requires replication, these results suggest that schizophrenia may be linked to chromosomal regions 8p23.1 and 22q12.3–q13.32.

Overexpression through amplification of genes in chromosome region 17p11.2∼p12 in high-grade osteosarcoma

Osteosarcomas are malignant tumors of the bone that are characterized by complex genetic changes, including loss and amplification of chromosome regions. Region 17p11.2∼p12 is frequently found to be amplified in this tumor, suggesting the presence of an oncogene (or oncogenes) important in osteosarcoma tumorigenesis. We had previously determined amplification profiles for this region. Reasoning that amplification of a causative oncogene in a tumor should result in increased expression of that gene, we have now determined the expression status of genes and expressed sequence tags (ESTs) in 17p11.2∼p12. We constructed a 17p11.2∼p12-specific macroarray containing 40 genes and 21 ESTs from this region, which was used for expression profiling of 11 osteosarcoma samples (9 tumors and 2 cell lines) and of normal human osteoblasts. Compared to normal osteoblasts, genes with at least threefold increased expression were considered to be overexpressed in the tumor. Genes PMP22 and COPS3, EST AA126939 (encoding part of the hypothetical protein FLJ20343), and two anonymous ESTs (AA918483 and R02360) were found to be most consistently overexpressed after amplification. By real-time reverse transcriptase polymerase chain reaction, we could confirm the overexpression status of PMP22 and COPS3 but not of FLJ20343. We conclude that PMP22 and COPS3, and possibly also the three ESTs, are candidate amplification targets in 17p11.2∼p12 in osteosarcoma.

Differential deletions in 3p are associated with the development of head and neck squamous cell carcinoma in Indian patients

In this study we performed detailed deletion mapping of two broad regions in the short arm (p) of chromosome 3 (i.e., 3p21.2∼p22 and 3p12∼p13), which were shown to have a high rate of deletions in head and neck lesions in our previous study. Using 18 highly polymorphic microsatellite markers, the deletion mapping was done in 35 dysplastic lesions and 46 primary head and neck squamous cell carcinoma (HNSCC) samples from Indian patients. Within the 21.6-megabase (Mb) region of 3p21.1∼p21.33, we have identified four areas (D1, 3p21.33; D2, 3p21.32; D3, 3p21.31; D4, 3p21.1) that showed a high frequency (46%–69%) of deletions in our samples. In the 3p12∼p13 region, we narrowed down the deletion within the 0.7-Mb region (D5, 3p12.1). Among these five regions (D1–D5), deletion in D3 is suggested to be necessary for the development of early dysplastic lesions, whereas the deletion in D2 may be necessary for dysplastic lesions and tumor progression. On the other hand, the deletion in D5 is significantly associated with progression of the lesions from mild/moderate to severe dysplasia. The deletions in D1 and D4, however, are required for tumor progression. As in our previous study, microsatellite size alterations (MA) were observed to be high in and around the highly deleted regions and gradually increased during the progression of the tumor. Loss of normal copy/interstitial alterations of chromosome 3 in the late stages of the tumor as well as rare biallelic alterations around the highly deleted regions also were seen in our samples. Human papilloma virus infection has been found to be associated with the deletion in the D5 region and MA in the D1 region, whereas nodal involvement of the tumor correlated only with the MA in D1 and D5. Thus, this study indicates that multiple tumor suppressor genes whose differential deletions are associated with the development of HNSCC may be present in 3p.

The association of cyclin A and cyclin kinase inhibitor p21 in response to γ-irradiation requires the CDK2 binding region, but not the Cy motif

The cyclin kinase inhibitor p21 associates with and inhibits cyclin–CDKs to retard the progress of the cell cycle in response to DNA damage. The recognition sites for cyclin binding on the various cell cycle-related molecules have been identified as RXL motifs. In the case of p21, the dependence of the Cy1 (18CRRL) or Cy2 (154KRRL) motifs on cyclin E, but not on cyclin A has been demonstrated by in vitro experiments. In this study, to clarify the mechanism of p21 association with cyclin A, we constructed a p21 expression system in mammalian cells. After transfection with an expression vector containing cDNA of various p21-mutants, cells were irradiated with 10 Gy of γ-rays to introduce DNA damage, followed by quantification of the p21-cyclin A association. The p21-mutant constructs were single or multiple deletions in Cy1, Cy2, and the CDK2 binding region, and a nonphosphorylatable alanine mutant of the C-terminal phosphorylation site. We demonstrated that the association of p21 and cyclin A in response to γ-irradiation requires the CDK binding region, 49–71 aa, but not the Cy motifs. We believe the mechanism by which p21 inhibits cyclin–CDKs is distinct in each phase of the cell cycle. Furthermore, the increase in the association of p21 and cyclin A was not correlated with the levels of p21. This suggests that DNA damage triggers a signal to the p21 region between 21 and 96 aa to allow cyclin A association.

Identification of two new members of the CSMD gene family☆

CSMD1 is a putative suppressor of squamous cell carcinomas mapping to human chromosomal region 8p23. We have cloned two new members of this gene family, CSMD2 and CSMD3. The three CSMD proteins have very similar structures, each consisting of 14 CUB domains separated from one another by a sushi domain, an additional uninterrupted array of sushi domains, a single transmembrane domain, and a short cytoplasmic tail. CUB and sushi domains are thought to be sites of protein–protein or protein–ligand interactions, suggesting that CSMD proteins are either transmembrane receptors or adhesion proteins. The cytoplasmic tail sequences are highly conserved within the vertebrate lineage. CSMD2 maps to a chromosomal region that may contain a suppressor of oligodendrogliomas, yet its expression is elevated in some head and neck cancer cell lines. Functional overlap between the CSMD1 and the CSMD2 proteins may modify the phenotype resulting from the loss of either protein in tumors.

Abstracts of the 8th Meeting of the Italian Peripheral Nerve Study Group: 64

Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant, demyelinating peripheral neuropathy. HNPP is a genetically homogeneous disease with a single disease locus at chromosome 17p11.2‐p12. The major cause of HNPP is a 1.5 Mb deletion containing the PMP22 gene, which causes a dosage effect of peripheral myelin protein 22. The deletion is found in 85% of patients with HNPP. A few HNPP patients show smaller deletions, still containing the PMP22 gene. Point mutations in the PMP22 gene are rare cause of HNPP. To date, nine point mutations were reported and all these mutations most likely cause a loss of function of PMP22. We describe a HNPP patient who at the age of 20 years developed muscle weakness and paresthesias. The neurophysiological examination showed a sensory motor demyelinating polyneuropathy at four limbs. The sural nerve biopsy showed signs of a demyelinating neuropathy with abundance of classical tomacula and mild inflammatory reaction in endo‐ and epineurial tissues. The CMT1A duplication and HNPP deletion of the region 17p11.2 was excluded by Southern blot analysis and pulse field gel electrophoresis. The PMP22 gene was screened for mutations by single strand conformation polymorphism (SSCP) analysis. An altered SSCP fragment was observed in the exon 2. Direct sequencing demonstrated a nucleotide substitution in the exon 2‐intron 2 junction (c.179 + 1G?C). To define the molecular consequences of this splice site mutation, RT‐PCR experiments on the sural nerve biopsy are in progress. The pathophysiological role of this mutation will be discussed in the light of the clinical, neurophysiological and pathological features.Partially supported by grants MURST 2000 to F.A. and Ministero della Sanità to P.M.

Klinische und molekulargenetische Befunde bei Familien mit Cherubismus über 3 Generationen

Cherubism is a rare fibro-osseous disorder that almost exclusively affects the maxilla and mandible. We report on three affected males in three generations in family A, and ten affected patients in family B. The youngest affected relative in family A also had craniosynostosis. His father and grandfather had cherubism and clubbed fingers. Cherubism was mapped to region 4p16.3. Because of the associated craniosynostosis, we excluded the FGFR3 gene as a candidate gene for cherubism. The inheritance pattern is autosomal dominant with variable expression. The penetrance is 100% in males and 50-70% in females. We found incomplete penetrance in males, which does not conform with all publications.

Proteolytic Processing of the Hepatitis B Virus e Antigen Precursor: CLEAVAGE AT TWO FURIN CONSENSUS SEQUENCES

The Hepatitis B virus P22 protein is a nonstructural protein that is the precursor of the 17-kDa secreted e antigen (HBeAg). The mature HBeAg is obtained after the removal of the C-terminal region of P22, a process which involves a proprotein convertase. Our studies show first that the protease could cleave P22 at the C-terminal side of Arg(167) or Arg(154) and second, that the maturation process can be either done in one step or in two steps with the generation of a processing intermediate (P20). Our data also demonstrate that the removal of the P22 C terminus, which occurs mainly in the trans-Golgi network, can also be achieved after exocytosis. Keeping in mind this characteristic and the amino acid sequence of the cleavage sites, we concluded that furin is involved in the maturation of the HBeAg. In addition, we show that in our experimental system, the HBeAg is a 164-amino acid protein and not a 159-amino acid protein as previously reported.

Amplification of 17p11.2∼p12, including PMP22, TOP3A, and MAPK7, in high-grade osteosarcoma

Amplification of region 17p11.2∼p12 has been found in 13%–29% of high-grade osteosarcomas, suggesting the presence of an oncogene or oncogenes that may contribute to their development. To determine the location of these putative oncogenes, we established 17p11.2∼p12 amplification profiles by semiquantitative PCR, using 15 microsatellite markers and seven candidate genes in 19 high-grade osteosarcomas. Most of the tumors displayed complex amplification profiles, with frequent involvement of marker D17S2041 in 17p12 and TOP3A in 17p11.2 and, in some cases, very high-level amplification of PMP22 and MAPK7 in 17p11.2. Our findings suggest that multiple amplification targets, including PMP22, TOP3A, and MAPK7 or genes close to these candidate oncogenes, may be present in 17p11.2∼p12 and thus contribute to osteosarcoma tumorigenesis.

Comparative genomic hybridization analysis identifies gains of 1p35∼p36 and chromosome 19 in osteosarcoma

Osteosarcomas (OS) are aggressive tumors of the bone and often have a poor prognosis. Conventional cytogenetic analyses of OS have revealed highly complex karyotypes, with numerous abnormalities. In this study, we analyzed 18 untreated OS tumors from 17 patients of the younger incidence age group by comparative genomic hybridization (CGH), 4 tumors by spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH). Comparative genomic hybridization identified frequent copy number changes of the chromosomal region 1p (10/17) and gain of part or all of chromosome 19(8/17). In addition gains were observed at 5p(3/17), 8q(3/17), 16p(3/17), and 17p(5/17); and losses at chromosomes 2q(3/17), 10(4/17) and 13(3/17). High level gains were detected in the 8q23∼q24 region in two tumors as well as at 17p in one primary and a metastatic tumor. Minimal regions of gain were present at 1p35∼p36.3 (8/17); 5p14∼p15.2 (3/17), and 8q22∼q24.3 (3/17). SKY analysis demonstrated that OS has a complex pattern of clonal and non-clonal rearrangements and helped confirm the structural basis for the imbalances detected by CGH. Spectral karyotyping confirmed an overall pattern of chromosomal gain affecting 1p in all four tumors. Fluorescence in situ hybridization analysis from these tumors confirmed the gain of the 1p36 region in 2 tumors as determined by CGH analysis as well as the amplification of 8q.

Human β‐defensin 4: a novel inducible peptide with a specific salt‐sensitive spectrum of antimicrobial activity

SPECIFIC AIMSThe aim of this study was to identify and characterize a novel human member of the β-defensin family by screening genomic sequences, analyze its genomic structure, tissue distribution, and regulation, and evaluate its antimicrobial and chemoattractant activities.PRINCIPAL FINDINGS1. Analysis of the genomic and cDNA sequences of the novel β-defensinTo identify genomic sequences around human β-defensin 2 at the chromosomal region 8p23, the peptide sequence of this β-defensin was used to perform a ‘basic local alignment search tool’ (BLAST) search in the High Throughput Genomic (HTG) division of the GenBank. Accession numbers AF202031, AF252831, AF189745, and AC074340 were found and subsequently screened for the presence of the β-defensin consensus pattern. Analysis of the clone AF202031 revealed a genomic sequence coding for the carboxy-terminal region of a putative novel β-defensin, which was found in several HTG clones available at GenBank and subsequently termed hBD-4. The full-length cDNA f...

Scandinavian CDG-Ia patients: genotype/phenotype correlation and geographic origin of founder mutations.

Congenital disorders of glycosylation type Ia, (previous name carbohydrate-deficient glycoprotein syndrome type Ia; CDG-Ia) is an inherited disorder of the glycosylation of certain glycoproteins. The defect is caused by mutations in the phosphomannomutase 2 (PMM2) gene located in chromosome region 16p13. The purpose of this study was twofold: (1) to investigate the possible correlation between certain genotypes and the phenotype of the patients and their PMM activity, and (2) to study further the founder origin of the Scandinavian mutations. Sixty-four CDG-Ia patients were studied. Regardless of mutation combination, the patients showed the basic neurological symptoms associated with CDG-Ia. However, patients carrying the mutation 548T-->C had less severe disease, e.g., no pericardial effusions, malnutrition, or clinical coagulation disturbances. Liver dysfunction and peripheral neuropathy were milder. In contrast, patients carrying mutation 691G-->A showed a high incidence of severe malnutrition and hepatopathy, and they had the highest mortality including affected siblings. Heterozygotes for the two most common mutations (422G-->A and 357C-->A) displayed a phenotype of variable severity sometimes leading to early death. PMM activity showed no correlation with either genotype or phenotype but was reduced in most patients. There was a pronounced geographic clustering for some of the Scandinavian mutations. For example, 548T-->C was almost exclusively found in patients stemming from southeastern parts of Sweden, whereas 26G-->A was found to cluster in a region in the most southern parts of Sweden, suggesting that these mutations originated in these two regions separately as founder mutations. The most frequent mutation (422G-->A) did not show a specific geographic focus. The widespread 422G-->A mutation is probably an older mutation, although haplotype data from intragenic polymorphisms indicate that this mutation also arose only once. The detailed information of the origin of mutations and their respective associated phenotypic pattern should enable improvements to be made regarding tools for genetic counseling and for prenatal diagnoses in CDG-Ia families.

Fine mapping of a tumour suppressor candidate gene region in 1p36.2-3, commonly deleted in neuroblastomas and germ cell tumours

A common genetic feature of neuroblastomas, which is also an important prognostic factor, is deletion of chromosome region 1p. The deletion of 1p often involves a deletion of varying size, with a consensus region within the most distal bands 1p36.2-3. The neuroblastoma SRO (shortest region of overlap of (deletions) presented earlier by our group was defined distally by the cluster of loci D1S80/ D1Z2/CDC2L1 and proximally by loci D1S244, i.e., approximately 25 cM. The 1p deletions are, however, not restricted to neuroblastoma tumours. In fact, a large spectrum of tumour types display deletions to varying degrees of 1p. We have exploited the possibility of using deletions of other tumour types, preferentially that of germ cell tumours, and combining the deletions with that of the neuroblastoma SRO. Also in germ cell tumours, distal 1p-deletions have been shown to have prognostic significance. We found in our germ cell tumours a SRO ranging from D1S508 to D1S200. Interestingly, this region only partially overlapped (approximately 5 cm) with our neuroblastoma SRO in region D1S508 to D1S244. We have thus focused on analysing this smaller region in the search for genes involved in the genesis of different cancers. We have performed radiation hybrid mapping of a large number of markers, STSs, ESTs, and others known to reside in 1p. We have also initiated the development of a BAC contig of the region. FISH, and fibre-FISH mapping of BACs were also performed. The data presented here constitute an ongoing work with the aim of identifying and cloning gene(s) important for development of germ cell tumours, neuroblastomas, and possibly other tumours.