Repeat-directed isolation of a novel gene preferentially expressed from the maternal allele in human placenta

Using a repetitive sequence of tandemly arrayed pentanucleotides in the human H19 3′-flanking region, we isolated a phage clone (λ PEN11) which localized to chromosome 11p15.5. The λ PEN11 phage encodes a 2.3-kb cDNA consisting of seven exons at least. The gene was mainly expressed in brain and pancreas (and less abundantly in testis), and demonstrated differential allele usage, with maternal expression being predominant in placenta, which indicates the gene is an atypical imprinted gene. While the pentamer repeat might contribute to this effect, it is also possible that the differential allele usage might reflect the local chromosomal structure known as the imprinting domain. Received: July 24, 1998 / Accepted: August 31, 1998     

A novel Sac I RFLP in the 3′ untranslated region of the myotonin protein kinase gene

We found a novel Sac I polymorphism downstream of CTG repeats in the 3′ untranslated region of the myotonin protein kinase (MT-PK) gene. A C to G transition at nucleotide 13,590 in the gene was revealed by Southern blotting and confirmed by sequencing analyses. The allelic frequency of the C : G polymorphism in 63 unrelated Japanese individuals was estimated to be 0.98: 0.02. When Southern blotting is employed in the analysis of the CTG repeat length in the MT-PK gene, this Sac I polymorphism should be taken into consideration. Received: 15 October, 1998 / Accepted: 31 October, 1998     

Identification of three novel polymorphisms in the <Emphasis Type="Italic">MJD1</Emphasis> gene and study of their frequency in the Portuguese population

Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder of late onset, caused by the expansion of a (CAG)_n tract in the MJD1 gene. Using BLAST2 sequences between known cDNA variants transcribed by the MJD1 gene and a clone of human genomic DNA, six possible unknown intragenic single-nucleotide polymorphisms (SNPs), at variable positions in the MJD1 gene, were identified. To confirm this, we studied a Portuguese control population, using polymerase chain reaction amplification and single-strand conformation polymorphism analysis for each potential SNP. For four of the possible polymorphisms there was no variability in our population, but the existence of three novel polymorphisms was confirmed: GTT_⁵²⁷/GTC_⁵²⁷, C_¹¹⁷⁸/A_¹¹⁷⁸, and A_¹²⁹⁴/G_¹²⁹⁴. The polymorphism GTT_⁵²⁷/GTC_⁵²⁷ (Val/ Val) is located in the coding region, whereas C_¹¹⁷⁸/A_¹¹⁷⁸ and A_¹²⁹⁴/G_¹²⁹⁴ are located in the 3′noncoding region of cDNA variants of the MJD1 gene, MJD2-1 and MJD1-1, respectively. All these novel SNPs are in Hardy-Weinberg equilibrium. These intragenic polymorphisms can be useful for (1) the study of the origin of the MJD mutation(s), (2) the study of recombination events, (3) distinction of chromosomes with alleles of identical (CAG)_n size in genetic tests (homoallelism), (4) the study of genetic modifiers in the region flanking the MJD1 gene, and (5) association studies in other diseases. Received: December 19, 2001 / Accepted: January 28, 2002     

An NcoI polymorphism in the human complement component 7 (C7) gene

A novel polymorphic site has been found in the 3′ untranslated region (UTR) of the human complement component 7 (C7) gene. The polymorphic site at 14-bp downstream from the TAG stop codon was either C or A (Nco I-digested), with allele frequencies of 0.660 and 0.340. This NcoI polymorphism would be useful to perform a DNA marker haplotype study in patients with deficiencies of the complement genes, such as C6, C7, C9, which are located closely on chromosome 5p13. Received: March 25, 1999 / Accepted: April 3, 1999     

High-density single-nucleotide polymorphism (SNP) map in the 96-kb region containing the entire human DiGeorge syndrome critical region 2 (DGCR2) gene at 22q11.2

We constructed a high-density single-nucleotide polymorphism (SNP) map in the 96-kb region containing the DiGeorge syndrome critical region 2 (DGCR2) gene at chromosome 22q11.2, a human counterpart of mouse seizure-related gene SEZ-12. A total of 102 SNPs were isolated from the region by systematic screening among 48 Japanese individuals: 9 SNPs in the 5′ flanking region, 3 in the 5′ untranslated region, 2 in the coding regions, 77 in introns, 7 in the 3′ untranslated region, and 4 in the 3′ flanking region. By a comparison of our data with SNPs deposited in the dbSNP database in the National Center for Biotechnology Information, 80 SNPs (78.4%) were considered to be novel. The ratio of transition to transver-sion was 3.08 : 1. In addition, eight other types of genetic variations (one GA dinucleotide polymorphism and seven insertion/deletion polymorphisms) were discovered. The high-resolution map that we constructed will be a useful resource for analyzing gene scans of complex diseases mapped to this local segment on chromosome 22. Received: July 4, 2001 / Accepted: July 11, 2001     

Isolation and characterization of a novel serine threonine kinase gene on chromosome 3p22-21.3

Through large-scale DNA sequencing of a genomic region on chromosome 3p22-p21.3, we isolated a novel gene encoding a 527-amino-acid protein. Its 18 exons spanned a genomic region of about 90 kilobases, and the 4536-nucleotide cDNA contained an open reading frame of 1581 base pairs. The gene was expressed in all 16 human tissues examined by Northern blotting. The amino acid sequence of the predicted protein was 39% identical to that of human SOK1 (Ste20/oxidant stress response kinase-1), a molecule that is activated by oxidative stress. In view of its significant similarity to SOK1, we suspect that the novel gene, which we named OSR1, is a member of the SOK family of kinases in terms of function. Received: October 26, 1998 / Accepted: November 30, 1998     

A highly polymorphic CA repeat marker at the human interleukin 6 receptor (IL6R) locus

A polymorphic dinucleotide (CA) sequence was isolated from a genomic clone containing the human interleukin 6 receptor (IL6R) gene. High heterozygosity (0.81) makes this polymorphism a useful marker in the genetic study of disorders affecting the inflammation process and bone resorption. Received: July 27, 1998 / Accepted: July 29, 1998     

Isolation and mapping of a polymorphic CA repeat sequence at the human VRK1 locus

VRK1 is a novel human putative serine/threonine kinase, and is located on chromosome 14 at band q32 where an autosomal recessive congenital microphthalmia (CMIC) is mapped. We isolated a polymorphic dinucleotide CA repeat marker from a genomic clone containing the human VRK1 gene. This polymorphism will be useful in genetic studies of disorders localized at the 14q32 region, such as CMIC. Received: October 8, 1998 / Accepted: October 16, 1998     

Novel germline mutations of the MEN1 gene in Japanese patients with multiple endocrine neoplasia type 1

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by tumors of the parathyroid glands, the pancreatic islet cells, and the anterior pituitary. Germline mutations of the MEN1 gene in three independent Japanese cases with MEN1 were analyzed. Case 1 has revealed a 2-bp (TA) insertion at nucleotide position 341 (341insTA) in exon 2, which shifts the reading frame such that the mutant protein has a completely different amino acid sequence from codon 78 to the premature stop codon at 119. In case 2, a nucleotide substitution, i.e., TAG in place of TGG, which encodes tryptophan at codon 198 was identified (nonsense mutation). These mutations were heterozygously present and have not been reported previously. Case 3 showed no mutations in the protein-coding exons and exon–intron junctions of the MEN1 gene by single-strand conformation polymorphism or direct sequencing of the polymerase chain reaction (PCR) fragments. We confirmed the finding that patients with MEN1 carry heterozygous germline mutations in the MEN1 gene, which is compatible with the idea that the MEN1 gene is a tumor suppressor gene. The reason why mutations in the coding region of the MEN1 gene could not be detected by PCR-based analysis in some of the MEN1 patients, e.g. case 3, needs to be clarified further. Received: July 30, 1998 / Accepted: August 31, 1998     

Identification of single-nucleotide polymorphisms (SNPs) of human N-acetyltransferase genes NAT1, NAT2, AANAT, ARD1, and L1CAM in the Japanese population

By direct sequencing of regions of the human genome containing five genes belonging to the acetyltransferase family, arylamine N-acetyltransferase (NAT1), arylamine N-acetyltransferase (NAT2), arylalkylamine N-acetyltransferase (AANAT), L1 cell adhesion molecule (L1CAM), and the human homolog of Saccharomyces cerevisiae N-acetyltransferase ARD1, we identified 53 single-nucleotide polymorphisms (SNPs) and two insertion/deletion polymorphisms in 48 healthy Japanese volunteers. NAT1 and NAT2 are so-called drug-metabolizing enzymes. In the NAT1 gene we found two SNPs and a 3-bp insertion/deletion polymorphism that corresponded to the NAT1^*3, ^*10, and ^*18A/^*18B alleles reported in other populations. The frequencies of NAT1^* alleles in our Japanese subjects were 52.6% for NAT1^*4, 1.0% for NAT1^*3, 40.6% for NAT1^*10, 2.6% for NAT1^*18A and 3.1% for NAT1^*18B. In the NAT2 gene we found 32 SNPs and a 1-bp insertion/deletion polymorphism; 6 SNPs within the coding region were reported previously and belonged to the slow acetylator group (NAT2^*5, NAT2^*6 and NAT2^*7), and 2 of the 8 SNPs in the 5′ flanking region were reported in the dbSNP of GenBank, but the remaining 24 SNPs and the insertion/deletion polymorphism were novel. The frequencies of NAT2^* alleles in Japanese (51.3% for NAT2^*4, 1.6% for ^*5B, 26.1% for ^*6A, 2.2% for ^*6B, 1.2% for ^*7A, 10.1% for ^*7B, 7.4% for ^*12A, and 1.1% for ^*13) were significantly different from those reported in Caucasian populations. In the AANAT gene we found 4 novel SNPs: 2 in the 5′ flanking region, 1 in exon 4, and 1 in intron 3. In the two genes belonging to the N-terminal N-acetyltransferase family, we identified 9 SNPs, 7 of them novel, for ARD1, and six novel SNPs for L1CAM. Variations at these loci may contribute to an understanding of the way in which different genotypes may affect the activities of human N-acetyltransferases, especially as regards the therapeutic efficacy of certain drugs and antibiotics. Received: February 9, 2001 / Accepted: February 19, 2001     

Analysis of cyclin-dependent kinase inhibitor genes (CDKN2A,CDKN2B,and CDKN2C) in childhood rhabdomyosarcoma

p16^INK4A, p15^INK4B, and p18 proteins are highly specific inhibitors of cyclin-dependent serine/threonine kinase (CDK) activities required for G1-S transition in the eukaryotic cell division cycle. Mutations, mainly homozygous deletions, of the CDKN2A (p16^INK4A/MTSI) gene have been recently found in tumor cell lines and in many primary tumors. We looked for homozygous deletions of CDKN2A, CDKN2B (p15^INK4B), and CDKN2C (p18) in 12 primary rhabdomyosarcoma (RMS) specimens and in five cell lines established from this cancer type. By means of polymerase chain reaction (PCR) and PCR-single strand conformation polymorphism (PCR-SSCP), we analyzed the presence of biallelic gene deletion or point mutation causing gene function loss. All the examined tumor cell lines (100%) and three of 12 (25%) primary tumors showed homozygous deletion of CDKN2A. Furthermore, no aberrant bands in primary tumors were detected via SSCP, suggesting the absence of mutations in the coding region. In all cases the deleted area at 9p21 also involved the CDKN2B gene. Conversely, no homozygous deletion or point mutations were detected when CDKN2C was analyzed. Our results strongly indicate that the p16^INK4A (and/or p15^INK4B) protein plays a key role in the development and/or progression of childhood rhabdomyosarcoma and suggest that this CDK-inhibitor protein might control proliferation and/or differentiation of human muscle cells. Moreover, alteration of CDKN2C does not appear to be involved in the genesis of rhabdomyosarcoma. Genes Chromosom Cancer 15:217–222 (1996). © 1996 Wiley-Liss, Inc.     

A single-nucleotide polymorphism in the human bone morphogenetic protein-4 (BMP 4) gene

A polymorphic site has been found in the human bone morphogenetic protein-4 (BMP4) gene. To our knowledge, it is the first defined polymorphism in this gene, being present with similar frequencies in Caucasian, Hispanic, and African populations. Received: July 21, 1998 / Accepted: August 25, 1998     

Linkage disequilibrium and haplotype analysis among eight novel single-nucleotide polymorphisms in the human tissue-type plasminogen activator (t-PA) gene

Tissue-type plasminogen activator (t-PA), a serine protease, activates the conversion of plasminogen to the fibrinolytic protein, plasmin. The t-PA gene, mapped to chromosome 8p12-p11.2, contains 14 exons. An Alu insertion/deletion (I/D) polymorphism in this gene has been associated with an increased risk for myocardial infarction. In the work reported here we sequenced 11 kilobases (kb) of genomic DNA from 50 normal Japanese volunteers (100 alleles), to include all 14 exons of the t-PA gene, flanking intronic sequences, and 6 kb of the 5′ sequence. These experiments identified eight novel single-nucleotide polymorphisms (SNPs), in addition to the known Alu I/D polymorphism, from which genotypic data we constructed 12 haplotypes in the tested population. Two-way comparisons of SNPs and the Alu polymorphism revealed strong linkage disequilibrium between the Alu site and SNPs at positions 20,209 (χ² = 92.263) and 27,555 (χ² = 47.53), and between SNPs at positions 27,849 and 28,902 (χ² = 66.331). A phylogenic tree was constructed to infer a process of genome construction that would reflect the sequence variations we observed. Our results help to explain the lack of agreement among results of various disease-association studies in which a contribution of the human t-PA gene has been suspected but not always confirmed. Received: January 22, 2001 / Accepted: March 26, 2001     

Alteration of methylation status in archival DNA samples: A qualitative assessment by methylation specific polymerase chain reaction

DNA methylation is an epigenetic mechanism that regulates gene expression, which also facilitates genomic imprinting. Genomic imprinting is responsible for differential expression of genes based on parent of origin. Altered methylation of parental alleles results in imprinting disorders diagnosed by methylation specific polymerase chain reaction (MS-PCR) technique. With increasing evidence of genes under epigenetic influence, methylation studies are extensively performed on archival samples. To evaluate effect of storage and storage conditions on DNA methylation, a systematic MS-PCR based analysis was planned on an imprinted gene, SNRPN, located on chromosome 15q11.2. It was assessed by MS-PCR on fresh, 4 −20, and −80°C stored DNA samples for different time periods for systematic evaluation of methylation status. Technical factors like type of sample processing, method of DNA isolation, primer region polymorphism, sample heterogeneity were also evaluated. DNA methylation was observed to be altered for SNRPN gene after storage at −80°C from 2 months onwards. Long-term storage of DNA at −80°C results in altered DNA methylation status. This may lead to false MS-PCR diagnosis of imprinting disorders. Our proof of concept study should be followed with quantitative validation since the findings have critical implications in the present era of biobanking.     

Genetic Polymorphism (rs329498) in the Pellino-1 Gene as Possible Predisposal Factor for Systemic Lupus Erythematosus in a Chinese Population

Objective: The purpose of this study was to analyze the association of two single nucleotide polymorphisms (SNPs) in Peli-1 gene with systemic lupus erythematosus (SLE) in a Chinese population.

Methods: We conducted a case–control study and a total of 738 SLE patients and 827 healthy controls were finally recruited. Peli-1 rs329498 and rs10496105 polymorphisms were specified from genomic DNA using TaqMan genotyping assay on Fluidigm 192.24 system.

Results: Allele contrast showed the minor allele C was associated with decreased risk for SLE when compared with the A allele (OR = 0.851, 95% CI = 0.737–0.983, p = 0.028). Significant difference was observed in genotype distribution of rs329498 polymorphism between lupus nephritis (LN) patients and non-LN patients (χ² = 8.18, p = 0.017). Furthermore, we also found a decreased frequency of the minor allele C in LN patients (29.2%) than in non-LN patients (37.7%) (χ² = 8.67, p = 0.003). Moreover, a significant difference was also detected under a dominant model with regard to the distribution of genotype frequencies between LN patients and non-LN patients (CC + AC vs. AA: OR = 0.632, 95% CI = 0.451–0.884, p = 0.007). Clinical features analysis showed a significant difference in the distribution of genotypic frequencies between patients with malar rash and patients without this feature (χ² = 6.63, p = 0.036). Unfortunately, we failed to find any significant results between Peli-1 gene rs10496105 and SLE susceptibility.

Conclusions: Our observations suggested that Peli-1 gene polymorphism rs329498 might contribute to SLE susceptibility in Chinese Han Population. Likewise, the rs329498 SNP was also associated with the clinical features LN and malar rash in SLE patients.     

An AciI polymorphism in the 3′ untranslated region of the human phosphomannomutase 2 (PMM2) gene

We found an AciI polymorphism in the 3′ untranslated region of the phosphomannomutase 2 (PMM2) gene located at 16p13. A G-to-C transition at nucleotide position 96 bp downstream from the PMM2 stop codon was detected in polymerase chain reaction (PCR) products after AciI digestion. The heterozygosity of the polymorphic alleles was 0.375 in a Japanese population. This polymorphism is useful for genetic analysis in patients with carbohydrate-deficient glycoprotein syndromes, of which there are four subtypes. Received: March 17, 1999 / Accepted: April 3, 1999     

Thymidylate synthase enhancer region: Novel allele in Indians

Background: Thymidylate synthase (TS) is the major target for fluoropyrimidine drugs like 5-Fluorouracil (5-FU). There are polymorphic tandem repeats in the TYMS gene enhancer region (TSER). The number of tandem repeats varies in different populations. The aim of this study was to determine the frequencies of the TSER tandem repeats (rs34743033) and compare the observed frequencies with those of other populations.

Methods: This study genotyped 350 healthy individuals by Polymerase Chain Reaction (PCR).

Results: A novel allele *1 (only a single repeat) was observed in four individuals, the individuals were heterozygous (TSER*1/*2) for TYMS. Another variant rs2853542 affecting the expression of Thymidylate synthase was also analysed. The observed genotype frequencies were compared with frequencies observed in other populations for understanding differences between various population groups. There was a statistically significant difference between Indians and Chinese, Kenyans, Ghanians, African-Americans, Americans of European Ancestry, British, Hungarians, Turkish, Australians and Brazilians.

Conclusion: This study identified a novel single repeat in the TYMS gene which might have an impact on the expression of this gene, which needs to be confirmed by functional studies.     

Identification of novel polymorphisms in the<Emphasis Type="Italic"> Adam33</Emphasis> gene

Adam33 is a member of a family of genes that encode membrane-anchored proteins with a disintegrin and a metalloprotease domain and that are primarily expressed in lung fibroblasts and bronchial smooth muscle. The human Adam33 gene is located on chromosome 20p13, a region that has been linked to asthma and bronchial hyperresponsiveness. Recently, the polymorphisms in Adam33 have been found to be associated with asthma. In this study, we performed polymorphism scanning of the entire genomic region, including the promoter region of Adam33, by direct sequencing. We identified 16 novel polymorphisms in the Adam33 gene. Among these novel polymorphisms, three polymorphisms (−2154G→A, −753T→A, and −330C→T) were found to be in the promoter region and one polymorphism (13491 G→A) was located in 3' untranslated region of the Adam33 gene. Electronic Publication     

Functional evaluation of paraplegin mutations by a yeast complementation assay

An autosomal recessive form of hereditary spastic paraplegia (AR‐HSP) is primarily caused by mutations in the SPG7 gene, which codes for paraplegin, a subunit of the hetero‐oligomeric m‐AAA protease in mitochondria. In the current study, sequencing of the SPG7 gene in the genomic DNA of 25 unrelated HSP individuals/families led to the identification of two HSP patients with compound heterozygous mutations (p.G349S/p.W583C and p.A510V/p.N739KfsX741) in the coding sequence of the SPG7 gene. We used a yeast complementation assay to evaluate the functional consequence of novel SPG7 sequence variants detected in the HSP patients. We assessed the proteolytic activity of hetero‐oligomeric m‐AAA proteases composed of paraplegin variant(s) and proteolytically inactive forms of AFG3L2 (AFG3L2^E575Q or AFG3L2^K354A) upon expression in m‐AAA protease‐deficient yeast cells. We demonstrate that the newly identified paraplegin variants perturb the proteolytic function of hetero‐oligomeric m‐AAA protease. Moreover, commonly occurring silent polymorphisms such as p.T503A and p.R688Q could be distinguished from mutations (p.G349S, p.W583C, p.A510V, and p.N739KfsX741) in our HSP cohort. The yeast complementation assay thus can serve as a reliable system to distinguish a pathogenic mutation from a silent polymorphism for any novel SPG7 sequence variant, which will facilitate the interpretation of genetic data for SPG7. Hum Mutat 31:1–5, 2010. © 2010 Wiley‐Liss, Inc.