Down-regulated and Commonly mutated ALPK1 in Lung and Colorectal Cancers.

The ALPK1 gene located in the 4q25 region encodes a newly explored protein kinase which could phosphorylate the amino acid of a domain full of α-helices. Recently, several studies have indicated that the expression of ALPK1 is related to inflammation and various diseases; therefore, the purpose of this investigation was to determine whether the expression of ALPK1 has an influence on tumorigenesis and to further scrutinize its gene polymorphism in order to better understand its clinical importance. In lung and colorectal cancer tissues, the ALPK1 RNA level of the normal part was higher than that of the tumor part using the RT-qPCR analysis. Moreover, differences in HRM melting curves could effectively separate the known mutation sites and be used to identify the two novel variants that might cause the bio-dysfunctions of ALPK1 found in silico predictions. Additionally, in both Lovo colorectal and A549 lung cancer cells with enhanced and depleted expression of ALPK1, the encoded ALPK1 could exert its activity on cell migration without interfering with cell viability. Taken together, these findings suggested that ALPK1 might play a vital role in cancer development and that the newly explored SNPs are found in a Taiwanese cohort.

Mutational analysis of CYP21A2 gene and CYP21A1P pseudogene: long-range PCR on genomic DNA.

CYP21A2, the gene that codes for P450c21 (Steroid 21-hydroxylase), has a duplicated pseudogene called CYP21A1P. The gene and the pseudogene share 98 % and 96 % sequence homology in exons and in noncoding sequences, respectively, and are located 30 kb apart within the HLA class III human histocompatibility complex locus on chromosome 6p21.3. CYP21A1P is inactive due to the presence of 11 deteriorated mutations in its coding region. These mutations can be transferred to the functional CYP21A2 through intergenic recombination during meiosis or mitosis and lead to the congenital adrenal hyperplasia (CAH) resulting from 21-hydroxylase deficiency. Conversely, portions of CYP21A2 sequence can be transferred to CYP21A1P, modifying the haplotype. Here, we describe a well-established protocol that can be used to unambiguously study the mutational profile of CYP21A2 gene and CYP21A1P pseudogene. The protocol is based on long-range PCR amplification with allele-specific primers, followed by DNA sequencing of smaller fragments.

Variants of the CYP21A2 and CYP21A1P genes in congenital adrenal hyperplasia.

More than 90% of congenital adrenal hyperplasia cases are caused by mutation of the CYP21A2 gene which converted from the CYP21A1P pseudogene. Sizes of the 3.7-kb TaqI-produced fragment that exists downstream of the TNXB gene, representing the CYP21A2, and the 3.2-kb TaqI-produced fragment that exists downstream of the XA gene, representing the CYP21A1P pseudogene, are used as size markers in the restriction fragment length polymorphism (RFLP) analysis. However, the size of and location for distinguishing these two genes might not be completely precise or reliable. Recent studies indicated that the 3.2-kb TaqI fragment may include multiple variants of chimeric CYP21A1P/CYP21A2 genes, a haplotype with dual mutations of IVS2-12A/C>G and 707-714del, and a functional CYP21A2 gene caused by small-scale conversions of the 5' end of the CYP21A1P sequence. In addition, a 3.7-kb TaqI fragment with more than 4 haplotypes of CYP21A2-like downstream of the TNXA gene and a 6.2-kb TaqI fragment of the CYP21A2 that results from a nucleotide mutation in the 3' end sequence were also identified. Accordingly, these structural variants reveal that traditional recognition of these two genes based on the TaqI fragment size analysis may lead to misinterpretation and increasingly interfere with the molecular diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

Analysis of CYP21A1P and the duplicated CYP21A2 genes.

The RCCX module on chromosome 6p21.3 has 3 possible forms: monomodular, bimodular, and trimodular. Chromosomes with 4 RCCX modules are very rare. In the monomodule, most of the CYP21A1P genes do not exist. However, haplotypes of the RCCX module with more than one CYP21A2 gene were observed. Obviously, the gene located downstream of the XA gene can possibly include the CYP21A2 as well as the CYP21A1P gene.

High-resolution melting curve (HRM) analysis to establish CYP21A2 mutations converted from the CYP21A1P in congenital adrenal hyperplasia.

BACKGROUND: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease of an inborn error of steroid metabolism in humans. More than 90% of CAH cases are caused by mutations of the steroid 21-hydroxylase (CYP21A2) gene, and approximately 75% of the defective CYP21A2 genes are generated through an intergenic recombination with the neighboring CYP21A1P pseudogene. METHODS: A high-resolution melting (HRM) curve analysis was designed to characterize 11 mutation sites of the CYP21A2 gene that commonly appeared in 21-hydroxylase deficiency. Among these 11 mutations, 9 were found in CAH patients, and 2 were mutations created from normal individuals. RESULTS: From the HRM analysis using 6 fragments of amplicons, we have successfully identified these 11 common disease-causing mutations of the CYP21A2 gene, among which 3 showed 3 distinguishable melting plots; the heteroduplexes showed an upcurved plot, a horizontal plot of homoduplexes of wild-type (WT), and a downcurved plot of homoduplexes of compound mutations. CONCLUSIONS: The HRM analysis is a 1-step of non-gel resolution technique which saves time and is a low-cost method to undertake such a program for screening CAH patients with the 21-hydroxylase deficiency caused by intergenic conversions from the neighboring CYP21A1P pseudogene.

Analysis of the CYP21A1P pseudogene: indication of mutational diversity and CYP21A2-like and duplicated CYP21A2 genes.

The CYP21A1P gene downstream of the XA gene, carrying 15 deteriorated mutations, is a nonfunctional pseudogene that shares 98% nucleotide sequence homology with CYP21A2 located on chromosome 6p21.3. However, these mutations in the CYP21A1P gene are not totally involved in each individual. From our analysis of 100 healthy ethnic Chinese (i.e., Taiwanese) (n=200 chromosomes) using the polymerase chain reaction (PCR) products combined with an amplification-created restriction site (ACRS) method and DNA sequencing, we found that approximately 10% of CYP21A1P alleles (n=195 chromosomes) presented the CYP21A2 sequence; frequencies of P30, V281, Q318, and R356 in that locus were approximately 24%, 21%, 11%, and 34%, respectively, and approximately 90% of the CYP21A1P alleles had 15 mutated loci. In addition, approximately 2.5% (n=5 chromosomes) showed four haplotypes of the 3.7-kb TaqI-produced fragment of the CYP21A2-like gene and one duplicated CYP21A2 gene. We conclude that the pseudogene of the CYP21A1P mutation presents diverse variants. Moreover, the existence of the CYP21A2-like gene is more abundant than that of the duplicated CYP21A2 gene downstream of the XA gene and could not be distinguished from the CYP21A2-TNXB gene; thus, it may be misdiagnosed by previously established methods for congenital adrenal hyperplasia caused by a 21-hydroxylase deficiency.

Analysis of the CYP21A2 gene with intergenic recombination and multiple gene deletions in the RCCX module.

The most frequent bimodular RCCX module of the RP1-C4A-CYP21A1P-TNXA-RP2-C4B-CYP21A2-TNXB gene sequence is located on chromosome 6p21.3. To determine RCCX alterations, we used the polymerase chain reaction (PCR) product containing the tenascin B (TNXB) and CYP21A2 genes with TaqI digestion and Southern blot analysis with AseI and NdeI endonuclease digestion of genomic DNA from congenital adrenal hyperplasia patients with common mutations resulting from an intergenic conversion of CYP21A1P, such as an I2 splice, I172N, V281L, F306-L307insT, Q318X, and R356W, and dual mutations of I236N/V237E in the CYP21A2 gene. The results showed that a 3.7-kb fragment of the CYP21A2 gene was detected in each case, and 21.6- and 11.3-kb DNA fragments were found in the RCCX region by a Southern blot analysis with these corresponding mutations. However, the IVS2-12A/C- > G (I2 splice) haplotype in combination with the 707-714delGAGACTAC (without the P30L mutation) mutation produced a 3.2-kb TaqI fragment in the PCR product analysis and a specific 9.3-kb fragment by the Southern blot method. Therefore, we concluded that the rearrangement in the RCCX region resulting from processing of either an intergenic recombination or multiple gene deletions can be identified by the PCR analysis and Southern blot method based on a fragment-distinguishing configuration without a family study.

Comparing the Southern blot method and polymerase chain reaction product analysis for chimeric RCCX detection in CYP21A2 deficiency.

The 3.2-kb TaqI-produced fragment of the CYP21A1P pseudogene and the 3.7-kb TaqI-produced fragment of the functional CYP21A2 gene exist on chromosome 6p21.3. We used the polymerase chain reaction (PCR) product and Southern blot method with TaqI endonuclease digestion to identify a chimeric RCCX module in two unrelated patients with congenital adrenal hyperplasia (CAH). After TaqI cleavage, the PCR product analysis revealed that patient 1 with the chimeric CYP21A1P/CYP21A2 gene in one allele and IVS2-12A/C>G in combination with the 707-714del mutation in the other allele produced a configuration of 3.2- and 2.4-kb fragments. Patient 2, who carried IVS2-12A/C>G in combination with the 707-714del mutation in one allele and the chimeric TNXA/TNXB gene in the other allele, presented with 3.2- and 2.3-kb fragments. However, Southern blot analysis showed that patients 1 and 2 produced 3.2-, 2.4-, and 2.5-kb fragments. We conclude that the chimeric CYP21A1P/CYP21A2 gene, IVS2-12A/C>G in combination with the 707-714del mutation, and the chimeric TNXA/TNXB gene cannot be distinguished by the Southern blot method. Conversely, the chimeric TNXA/TNXB gene was identified in the PCR product analysis due to the appearance of the 2.37-kb fragment, which indicates the occurrence of the chimeric TNXA/TNXB formation extending to the boundary of TNXA in the RCCX region.

Application of the DHPLC method for mutational detection of the CYP21A2 gene in congenital adrenal hyperplasia.

BACKGROUND: More than 90% of cases of congenital adrenal hyperplasia (CAH) are caused by a steroid 21-hydroxylase deficiency. Approximately 75% of the defective CYP21A2 genes are generated through an intergenic recombination with the neighboring CYP21A1P pseudogene. These 2 duplicated genes share a 98% nucleotide sequence homology. Therefore, precisely identifying the CYP21A2 gene in CAH patients is absolutely necessary. METHODS: We describe an established PCR-based amplification method, a denaturing high-performance liquid chromatography (DHPLC) analysis, to directly identify 11 different mutations commonly appearing in the CYP21A1P gene. Among these 11 mutations, 9 are found in CAH patients and 2 created mutations were from normal individuals. RESULTS: From the DHPLC analysis using 6 fragments of amplicons, the elution profiles of the 11 mutation sites were successfully used to distinguish these common disease-causing mutations of the CYP21A2 gene. Based on this resolution, we were able to rapidly search existing sequences of mutations in the CYP21A1P gene for this malady. CONCLUSION: DHPLC is an efficient and specific means to undertake such a program for screening patients with CAH caused by defects of the CYP21A2 gene resulting from the neighboring CYP21A1P pseudogene.

The gene founder effect of two spontaneous mutations in ethnic Chinese (Taiwanese) CAH patients with 21-hydroxylase deficiency.

CYP21A2 mutations resulting from microconversions of the CYP21A1P sequence in congenital adrenal hyperplasia (CAH) commonly appear in all populations. However, it has not often been described as being due to the gene founder effect. Herein, we investigated two spontaneous mutations of IVS2+1G>A and R316X in ethnic Chinese (Taiwanese) CAH patients to determine whether they share the same haplotype of ancient origin by the analysis of sequence-specific oligonucleotide (SSO) for HLA class I B and sequence-based typing (SBT) for HLA class II DRB1 gene-typing methods. From over 200 CAH families, eight unrelated CAH patients were found and examined: five had the IVS2+1G>A mutation and three had the R316X mutation. Based on HLA typing data, five alleles in five patients with the IVS2+1G>A mutation were consistent with a shared haplotype of the B *3909-DRB1 *160201 allele, and the three alleles in the three patients with the R316X mutation were all the B *460101-DRB1 *080302 allele. The evidence indicates that the haplotype of single-base substitutions of either the IVS2+1G>A or R316X mutation came from the same allele rather than a mutational hot spot, suggesting that the gene founder effect has occurred in the Taiwanese population. This is the first report of the gene founder effect of the CYP21A2 mutation occurring in ethnic Chinese (Taiwanese) CAH patients with 21-hydroxylase deficiency.

An investigation of the C4 gene arrangement in ethnic Chinese (Taiwanese).

C4 complement components are encoded by two genes, C4A and C4B , located on chromosome 6p21.3 of the major histocompatibility complex class III region. The isotypic residues at position 1101-1106 of the C4A gene contain the Pro-Cys-Pro-Val-Leu-Asp sequence which has a higher affinity for binding amino group-containing antigens, while C4B contains the Leu-Ser-Pro-Val-Ileu-His sequence which has a higher affinity for hydroxyl group-containing antigens. These two genes show different reaction rates which infer solubilization of antibody-antigen aggregates and propagation of the activation pathway to form the membrane attack complex. Using a polymerase chain reaction-based amplification method to identify and differentiate the locations of the C4A and C4B genes adjacent to the respective CYP21A2P and CYP21A2 genes, the isotypic residues at position 1101-1106 for the C4 isotype were categorized into five haplotypes of C4 gene arrangements. Among them, we found that 65% of the gene proportions between C4A and C4B were balanced, while 35% of them were unbalanced in this ethnic Chinese (i.e. Taiwanese) cohort. We consider that the unbalanced arrangements of the C4 locus in the individuals might have influenced the clearance of apoptotic debris and immune complexes which may injure tissue by initiating autoimmune diseases and immunity responses associated with susceptibility to viral and bacterial infections.

Low frequency of the CYP21A2 deletion in ethnic Chinese (Taiwanese) patients with 21-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder which causes more than 90% of CAH cases due to defects in the steroid 21-hydroxylase gene (CYP21A2). The frequency of large mutations was determined in 200 ethnic Chinese (i.e., Taiwanese) CAH patients belonging to 200 families with different clinical forms of CYP21A2 deficiency over 10 years of molecular diagnoses. For a large-gene deletion (or conversion) and the CYP21A2 deletion identification, a PCR product covering the TNXB gene and the 5'-end of the CYP21A2 gene with TaqI endonuclease digestion was analyzed by electrophoresis on agarose gels. For CYP21A2 mutational analysis, secondary PCR amplification of the amplification-created restriction site method was applied. From the results of the analysis, we found that large-gene deletions (or conversions) occurred in 7.5% of the alleles including three different types of the chimeric CYP21A1P/CYP21A2 genes and the haplotype of IVS2-12A/C>G in combination with the 707-714del mutation (without the P30L mutation). The CYP21A2 deletion occurred in 2.0% of the alleles which contained three types of the chimeric TNXA/TNXB genes with two novel ones. We concluded that the CYP21A2 deletion in the ethnic Chinese (Taiwanese) patients exhibits a low occurrence, with the haplotype of the IVS2-12A/C>G in combination with the 707-714del mutation (without the P30L mutation) being prevalent among large gene deletions or conversions.

Use of a PCR-based amplification analysis as a substitute for the Southern blot method to determine the C4A and C4B genes.

The human C4 complement components of the C4 gene are encoded by two genes, C4A and C4B, located on chromosome 6p21.3 of the major histocompatibility complex (MHC) of the human leukocyte antigen (HLA) class III region. Genetic determination of these two genes was by the Southern blot method: the 276- and 191-bp NlaIV fragments represent the C4A gene with the sequence, PCPVLP, at residues 1101-1106; the 467-bp NlaIV fragment represents the C4B gene with the sequence, LSPVIH, at residues 1101-1106. Here, we describe a PCR-based approach for differential amplification of the C4 genes adjacent to the respective CYP21A1P and CYP21A2, followed by NlaIV restriction digestion in a secondary PCR product and direct analysis by electrophoresis on an agarose gel to determine the C4A and C4B genes. From the results of this study, we concluded that 87% and 85% of the C4 genes adjacent to the CYP21A1P and CYP21A2 genes carried the C4A and C4B genes, respectively. The frequencies of the C4A and C4B genes comprising the C4 locus were 51.5 and 49%, respectively in this ethnic Chinese (Taiwanese) cohort. Since no radiolabelling application is involved, the protocol is reliable as a substitute for the Southern blot method for C4A and C4B determination.

Identification of the size and antigenic determinants of the human C4 gene by a polymerase chain-reaction-based amplification method.

The human C4 complement components of the C4 locus are encoded by two genes, C4A and C4B, located on chromosome 6p21.3 of the major histocompatibility complex of the human leukocyte antigen class III region. The size difference between the two genes is due to the presence of HERV-K (C4), an endogenous retroviral sequence (6.7 kb long), in intron 9 of the long C4 gene. Whether the C4 is the long (L) or short (S) gene was determined by the Southern blot method, and the antigenic determinants in residues 1,054-1,106 of Rodgers and Chido were generally identified by immunoblot analysis. Herein, we explore a polymerase chain reaction (PCR) amplification method for directly determining the size of C4 loci adjacent to the respective RP1 and RP2 genes and antigenic determinants by DNA sequencing. From the results of this study, we concluded that all of the C4 genes adjacent to the RP1 gene presented the long gene. In addition, 47% of the C4 genes adjacent to the RP2 gene were the short gene and 53% were the long gene. This result was consistent with that of the Southern blot analysis. The PCR method is practical for identifying the C4 genotype and can be used to detect other polymorphisms among variants of C4 genes.

Diversity of the CYP21A2 gene: a 6.2-kb TaqI fragment and a 3.2-kb TaqI fragment mistaken as CYP21A1P.

The 3.7- and 3.2-kb fragments produced by TaqI digestion are respective crucial markers of the CYP21A2 and CYP21A1P genes for the analysis of the RCCX module in chromosome 6p21.3. Herein, we report two distinct CYP21A2 haplotypes. One occurred in a CAH patient with a 6.2-kb TaqI fragment caused by a mutation at the TaqI site (TCGA) located downstream of the CYP21A2 gene, and the other was a parent in a suspected CAH family with a 3.2-kb TaqI fragment resulting from a 156-bp fragment conversion of the CYP21P promoter sequence which led to the production of a TaqI site at nt -209 and two additional CYP21A1P nucleotides at nt -198 (C>T) and -188/-189 (T insertion). From further sequencing analysis, the promoter region of the 3.2-kb allele showed four normal transcriptional sequences positioned at nt -126C, -113G, -110T, and -103A. However, other nucleotides such as at nt -294T, -293A, and -282A were unchanged. We concluded that the 6.2-kb TaqI fragments of the CYP21A2 haplotype may lead to an incorrect result in the analysis between CYP21A2 and CYP21A1P. The formation of the 3.2-kb TaqI fragment allele which can be mistaken for the CYP21A1P gene may be caused by small-scale conversions of the CYP21A1P gene located between nt -126C and -282A. Therefore, the CYP21A2 haplotype not only presents a 3.7-kb TaqI fragment but also may possibly exist in multiple forms including both 6.2- and 3.2-kb fragments.

Novel missense mutations, GCC [Ala306]- > GTC [Val] and ACG [Thr318]- > CCG [Pro], in the CYP11B1 gene cause steroid 11beta-hydroxylase deficiency in the Chinese.

OBJECTIVE: Steroid 11beta-hydroxylase (CYP11B1) deficiency, an autosomal recessive inherited disease, accounts for 5-8% of congenital adrenal hyperplasia (CAH). It is mainly caused by mutations of nucleotide substitutions in the coding region. PATIENTS AND METHODS: The study reports on a 9-year-old Chinese boy who presented with a bone age of 16 years, an enlarged penis, an accelerated growth rate since early childhood and hypertension (160-170/100-110 mmHg) for 3 years. Because it shares 95% sequence homology with aldosterone synthetase (CYP11B2), we developed gene-specific primers for differential PCR amplification of the CYP11B1 gene. The secondary PCR products of nine exons of the CYP11B1 gene were then subjected to single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. The serum hormone levels were also determined. RESULTS: We found that the boy diagnosed with CAH due to 11beta-hydroxylase deficiency carried mutations of A306V (GCC- > GTC) and T318P (ACG- > CCG) in two respective chromosomes. The hormone assay showed that the 11-deoxycortisol level was higher (667 nmol/l) than normal and was further increased after ACTH stimulation (1206 nmol/l). CONCLUSIONS: These two mutations have not previously been described in the CYP11B1 gene. The discovery of these two novel mutations increases our knowledge of CAH caused by 11beta-hydroxylase deficiency.

Diversity of the CYP21P-like gene in CYP21 deficiency.

More than 90% of cases of congenital adrenal hyperplasia (CAH) are caused by mutations of the CYP21 gene. The occurrence of defective CYP21 genes, including 15 mutations, has been attributed to intergenic recombination of DNA sequences from CYP21P, and shows no influence on the RP1-C4A-CYP21P-XA-RP2-C4BCYP21- TNXB gene locus on chromosome 6p21.3. However, multiple gene deletions in this region produce at least three categories of gene arrangements: (a) C4A-CYP21P/CYP21-TNXB, in which there is a CYP21P/CYP21 fusion gene; (b) C4A-XCYP21-TNXB, where XCYP21 indicates that the CYP21 gene contains mutations of IVS2 (-12A/C>G and 707-714delGAGACTAC); and (c) C4A-CYP21P-TNXA/TNXB, in which the TNX A and B genes are fused. Among them, seven different structures of the CYP21 haplotype were found at these three loci. Formation of the C4A-CYP21P/CYP21-TNXB locus produced four distinct CYP21P/CYP21 chimeras. The C4A-XCYP21-TNXB locus contained the IVS2 mutation -12A/C>G and 707-714delGAGACTAC from the XCYP21 gene; and two kinds of TNXA/TNXB hybrids were found in the C4A-CYP21P-TNXA/TNXB locus. The seven different CYP21 alleles produced 3.2 kb Taq I fragments caused by deletion of the RP2-XA-C4B locus. Therefore, production of a 3.2-kb CYP21 allele shows diversity, but is not a unique feature of the CYP21P gene. Most of these gene arrangements probably exist in the C4A-XCYP21-TNXB and C4A-CYP21P/CYP21-TNXB gene loci. The existence of the C4A-CYP21P-TNXA/TNXB locus might not be common in CAH patients with 21-hydroxylase deficiency.

Chimeric CYP21P/CYP21 and TNXA/TNXB genes in the RCCX module.

Two types of chimeric RCCX modules found in chromosome 6p21.3 are the chimeras CYP21P/CYP21 and TNXA/TNXB. The CYP21P-specific sequence of chimera CYP21P/CYP21 has the 5'-end in common, but differs in the 3'-end of CYP21-specific genes. The sequence organization of the gene array is C4A-CYP21P/CYP21-TNXB, whereas chimera TNXA/TNXB is caused by a CYP21 deletion, and a partial TNXB replaced by the TNXA gene shows the C4A-CYP21P-TNXA/TNXB sequence. Therefore, chimeras CYP21P/CYP21 and TNXA/TNXB are two distinct hybrid genes produced in the RCCX module in HLA class III. In addition, the haplotype of CYP21 with chimera CYP21P/CYP21 causes 21-hydroxylase deficiency in congenital adrenal hyperplasia (CAH), while chimera TNXA/TNXB is associated with Ehlers-Danols syndrome as well as CAH.