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Mutation analysis of the cationic trypsinogen gene in patients with pancreatic cancer.

Recently, an Arg to His mutation at residue 117 of the cationic trypsinogen gene (Arg117His) has been shown to be associated with hereditary pancreatitis (hp). A serious complication of hp is development of pancreatic cancer. Patients suffering from hp have been reported to have a 53-fold increased risk to die from pancreatic cancer. However, the quantitative contribution of mutations in the cationic trypsinogen gene to all pancreatic cancer cases is unknown. A relevant contribution of the Arg117His-mutation to pathogenesis of pancreatic cancer might be possible, since also asymptomatic individuals have been reported to carry this mutation and individuals with only mild symptoms may be undiagnosed as hp. In the present study we analyzed genomic DNA obtained from pancreatic cancer tissue from 34 patients and corresponding normal tissue from 28 of these individuals. The third exon of the cationic trypsinogen gene was amplified by nested PCR and digested with AflIII, since the Arg117His mutation creates an AflIII-restriction site. None of the examined samples carried the Arg117His mutation, whereas the amplification product obtained from a patient with known hp was clearly positive. Sequencing of the complete third exon of the cationic trypsinogen gene in 10 of the pancreatic cancer patients resulted exclusively in the wild-type sequence. In addition DNA obtained from venous blood of 116 further patients with pancreatic cancer did not carry the Arg117His mutation. Our results show that the Arg117His mutation does not contribute to pathogenesis of a substantial fraction of all pancreatic adenocarcinomas. In contrast to most oncogenes or tumor suppressor genes the cationic trypsinogen gene (3rd exon) does not contain mutational hot spots.

Adenocarcinoma↗

Development of a point mutation assay for the detection of human cytomegalovirus UL97 mutations associated with ganciclovir resistance.

A point mutation assay was developed to detect the quantitative prevalence of mutations at codons 460 (M to I; M to V), 520 (H to Q), 594 (A to V) and 595 (L to F; L to S) within the UL97 gene of human cytomegalovirus which segregate with ganciclovir resistance. Synthetic mixtures of wild-type and mutant plasmids containing the UL97 gene were amplified by nested polymerase chain reaction and the 700 base pair amplicon subsequently subjected to the point mutation assay. In plasmid reconstruction experiments, there was a high correlation between experimentally derived percentage mutant with the theoretical values. The assay was then used to assess the changes in the genetic composition of the UL97 gene in three patients on prolonged ganciclovir therapy. All three patients developed genotypic resistance against ganciclovir involving mutation at codon L595S, L595F and double mutation at codons L595F and M460I. In one patient, alteration of therapy to foscarnet did not affect the composition of UL97 and virus remained genotypically resistant to ganciclovir. In contrast, in two patients whose therapy was altered to cidofovir (HPMPC), repopulation with cytomegalovirus strains carrying the wild-type (ganciclovir-sensitive) codon at positions 595 and 460 occurred. The potential use of this assay for the rapid detection of cytomegalovirus resistance in patients on long-term ganciclovir therapy is discussed.

Acquired Immunodeficiency Syndrome↗

Clonal populations of hematopoietic cells with paroxysmal nocturnal hemoglobinuria genotype and phenotype are present in normal individuals.

In paroxysmal nocturnal hemoglobinuria (PNH), acquired somatic mutations in the PIG-A gene give rise to clonal populations of red blood cells unable to express proteins linked to the membrane by a glycosylphosphatidylinositol anchor. These proteins include the complement inhibitors CD55 and CD59, and this explains the hypersensitivity to complement of red cells in PNH patients, manifested by intravascular hemolysis. The factors that determine to what extent mutant clones expand have not yet been pinpointed; it has been suggested that existing PNH clones may have a conditional growth advantage depending on some factor (e.g., autoimmune) present in the marrow environment of PNH patients. Using flow cytometric analysis of granulocytes, we now have identified cells that have the PNH phenotype, at an average frequency of 22 per million (range 10-51 per million) in nine normal individuals. These rare cells were collected by flow sorting, and exons 2 and 6 of the PIG-A gene were amplified by nested PCR. We found PIG-A mutations in six cases: four missense, one frameshift, and one nonsense mutation. PNH red blood cells also were identified at a frequency of eight per million. Thus, small clones with PIG-A mutations exist commonly in normal individuals, showing clearly that PIG-A gene mutations are not sufficient for the development of PNH. Because PIG-A encodes an enzyme essential for the expression of a host of surface proteins, the PIG-A gene provides a highly sensitive system for the study of somatic mutations in hematopoietic cells.

Adult↗

[Rapid identification of bacteria by PCR and hybridization].

The amplification of bacteria DNA by PCR followed by rapid identification with hybridization are described. In the case of Mycobacteria, a 206 bases in dnaJ gene was amplified by nested PCR with conserved primers. The amplified DNAs were then hybridized with species-specific oligoprobes. Theses oligoprobes are capable of identifying the amplified DNA as M. tuberculosis, M. avium, M. intracellulare, M. kansasii or others. In the case of MRSA, clinical samples were examined to amplify mecA, femA and tsst-1 genes in the same tube, using mixed primers and the resultant 680 bp, 307 bp and 121 bp were identified by hybridization. For Helicobacter pylori, a 203 base were amplified and identified with [32p]-labeled oligoprobe as described (Valentine, J.L. et al., J. Clin. Microbiol., 29:689-695, 1991.). The sensitivity was 10(2) CFU/tube on agarose gel and 10 CFU/tube by hybridization.

Base Sequence↗

Most probable number method combined with nested polymerase chain reaction for detection and enumeration of enterotoxigenic Clostridium perfringens in intestinal contents of cattle, pig and chicken.

The most probable number (MPN) method combined with a nested polymerase chain reaction (nested PCR) for the detection and enumeration of enterotoxigenic Clostridium perfringens in the intestinal contents of cattle, pig and chicken was examined. Ten-fold serial dilutions of samples were added to three tubes of enrichment medium, which were incubated at 37 degrees C for 20-24 hr, and the C. perfringens enterotoxin gene was detected by nested PCR from the enrichment culture without isolating the organism. The results obtained by this method with artificially contaminated intestinal contents were significantly correlated with those obtained by a plate count method. When the method was applied to the detection and enumeration of indigenous enterotoxigenic C. perfringens, the organism was found in two, two and three samples of 10 intestinal contents of cattle, pig and chicken, respectively. Most of the positive samples contained fewer than 10 MPN/g of enterotoxigenic C. perfringens, except one sample of chicken, which contained 1.5 x 10(2) MPN/g. The MPN method combined with nested PCR is easy to perform and may be a useful tool for the detection and enumeration of enterotoxigenic C. perfringens in intestinal contents.

Animals↗

[DNA polymorphisms in strains of Staphylococcus aureus. I. Usefulness of coagulase gene as an epidemiological marker].

In this study, we report the genotyping of Staphylococcus aureus by restriction enzyme digestion of the nested PCR products of coagulase gene from clinical strains of 83 MRSA (methicillin-resistant S. aureus) and 23 MSSA (methicillin-sensitive S. aureus). Using Alu I restriction enzyme, a total of 106 strains could be classified into 19 different restriction patterns, arbitrarily designated the genotypes 1 to 19 for strains. The DNA patterns of the PCR coagulase gene products were visualized by ethidium bromide after 2% agarose gel electrophoresis and hybridized with alkaline-phosphatase labeled probe. Because of unique sequences of the coagulase gene containing 81-bp tandem repeats and Alu I recognition sites, most of clinical strains had bands in multiples of 81 bp which allowed easy discrimination among isolates. The deduced DNA fragment sizes ranged from approximately 80 to 900 bp, and the numbers of DNA fragments generated were 2 to 9. Eighteen of the 20 MRSA strains taken from the patients hospitalized in 1991 belonged to the genotypes 2 and 3, and 27 of the 29 MRSA strains taken from patients in 1992 belonged to the genotypes 1, 2 and 3, suggesting the possibility of nosocomial outbreaks caused by these strains. In conclusion, this coagulase gene restriction fragment length polymorphism (RFLP) technique will be useful for epidemiological evaluations of nosocomial MRSA infections.

Base Sequence↗

Two Hyposoter didmator ichnovirus genes expressed in the lepidopteran host encode secreted or membrane-associated serine and threonine rich proteins in segments that may be nested.

We present in this work two novel Hyposoter didymator ichnovirus genes expressed in parasitized Spodoptera larvae. These genes, named HdCorfS6 and HdGorfP30, are unrelated and present in two different genome segments, possibly nested, SH-C and SH-G respectively. HdCorfS6 encodes a predicted transmembrane protein, putatively glycosylated. HdCorfS6 transcripts appear to be abundant in lepidopteran host hemocytes compared to the other tissues analyzed. The second gene described, HdGorfP30, is well expressed in hemocytes, but also in other tissues, such as the fat body, nervous system and epidermis. This gene is peculiar since it presents 17 perfectly conserved repeated sequences arranged in tandem arrays. Each of these repeats contains 58% of serine and threonine residues and therefore several potential sites for glycosylation. This mucin-like protein, predicted as highly glycosylated, could be involved in host immune suppression.

Amino Acid Sequence↗

[Study of sequence variations of Epstein-Barr virus LMP1 gene in nasopharyngeal carcinoma].

OBJECTIVE: To detect the sequence variations frequently found within the N- and C-terminal regions of Epstein-Barr virus (EBV) LMP1 gene in nasopharyngeal carcinoma (NPC) and to study the underlying mechanisms. METHODS: Fresh tumor tissues were sampled from 63 patients with untreated NPC encountered in Affiliated Tumor Hospital of Sun Yat-sen University, Guangzhou. The N-terminal region of EBV LMP1 gene was amplified with nested polymerase chain reaction (PCR), followed by XhoI enzyme digestion. Nested PCR was also employed to detect the 30 base pairs deletion within the C-terminal region. Four-colored fluorescence terminator sequencing method was applied for bi-directional solid-phase sequencing of the 8 representative PCR products in 4 cases of NPC. The DNA sequence within the N- and C-terminal regions of LMP1 gene was then analyzed. RESULTS: There were 4 patterns of sequence variations, namely, wt-XhoI/wt-LMP1 (4 cases, 6.3%), wt-XhoI and XhoI-loss/del-LMP1 (4 cases, 6.3%), wt-XhoI/del-LMP1 (5 cases, 7.9%) and XhoI-loss/del-LMP1 (50 cases, 79.5%), detected in the 63 studied cases. Sequence analysis showed that the EBV LMP1 gene had underwent non-synonymous and synonymous substitutions, as compared with the prototype of B95-8 cells. The ratio of non-synonymous to synonymous substitutions was 2.25. CONCLUSIONS: XhoI-loss/del-LMP1 is the predominant sequence variation pattern of EBV LMP1 gene in NPC from Guangzhou. The XhoI-loss variation seems to develop on top of del-LMP1. When compared with the EBV LMP1 gene in peripheral blood B-lymphocytes of virus carriers and in preinvasive epithelial lesions (reported previously), it is likely that the sequence variation patterns of LMP1 gene may represent 4 different phases of intrahost evolution of EBV during nasopharyngeal carcinogenesis.

Adult↗

Structural and promoter regions of the murine pyruvate carboxylase gene.

We have cloned and sequenced the gene encoding mouse pyruvate carboxylase (mPC) [EC 6.4.1.1]. The coding region contains 19 exons, one 5'-untranslated region exon, and 19 introns in 22 kb of genomic DNA. This gene's exon/intron organization is highly conserved with respect to rat and human PC genes. The mPC gene promoter lacks canonical TATA and CCAAT boxes, in common with a number of housekeeping genes. Transient expressions in COS-1 of a luciferase reporter gene under the control of 5'-nested deletions of the 5'-flanking sequence of the mPC gene have identified the 166-bp minimal sequence required for basal transcription. Alternative splicing at the 5'-untranslated region exon of the mouse PC gene results in the production of two alternate transcripts bearing different 5'-noncoding regions. Both transcripts are highly expressed in kidney and liver and moderately expressed in heart and testis and expressed at a low level in spleen.

Animals↗

Developmental extinction of liver lipoprotein lipase mRNA expression might be regulated by an NF-1-like site.

The molecular mechanism underlying the extinction of lipoprotein lipase (LPL) expression in rat liver during development was investigated. A mouse (BWTG3) and a rat (7777) hepatoma, both of which exhibit characteristics of fetal hepatocytes, were found to contain LPL mRNA, whereas the more differentiated human (Hep G2 and Hep 3B) or rat (Fa32) hepatoma cell lines did not. Somatic cell hybrids between LPL-producing hepatoma cells and non-LPL-producing cells, such as adult rat hepatocytes or fibroblasts, exhibited extinction of LPL gene expression. Assay of expression of nested deletions in the 5' regulatory sequences of the LPL gene in the Hep G2 cell line and in BWTG3 cells localized sequences involved in the suppression of LPL production to a region between -591 and -288 relative to the transcription initiation site. A site with sequence homology to a glucocorticoid responsive element (GRE) was shown not to play an important role in the extinction process. A novel transcription factor, termed RF-1-LPL, was shown to bind to an NF-1-like site in this region. In contrast to neonatal animals, in adult animals an additional protein complex (RF-2-LPL), was formed on the NF-1-like site, suggesting that this sequence might recruit a trans-acting factor involved in the extinction of LPL gene expression in adult rat liver.

Animals↗

Association between telomere length and BCL2 gene rearrangements in low- and high-grade non-Hodgkin lymphomas.

Telomere length based on terminal restriction fragment (TRF) assay was evaluated in cells of bone marrow, lymph node, or both from 53 non-Hodgkin lymphoma (NHL) patients: 44 with follicular lymphoma (FL) and 9 with secondary diffuse large B-cell lymphoma (S-DLBCL) to FL. The TRF data were correlated with BCL2 gene rearrangement evaluated by nested and long-distance polymerase chain reaction approaches. Peripheral blood cells from 12 healthy donors were studied as controls. Both groups of NHL patients revealed significant telomere shortening compared with controls (8.50 +/- 0.50 kb; P < 0.001), with significantly shorter TRFs in S-DLBCL (3.49 +/- 0.26 kb) than in FL cases (4.09 +/- 0.12 kb; P = 0.047). Patients carrying BCL2 gene rearrangements showed longer telomere length (4.19 +/- 0.14 kb) than those without (3.51 +/- 0.14 kb; P = 0.05). Among patients with FL, telomere length was shortest in patients without t(14;18), intermediate when breakpoints occurred in the minor breakpoint cluster region (3.97 +/- 0.33 kb), and greater when breakpoints occurred in the major breakpoint region (MBR) (4.24 +/- 0.15 kb), with significant differences between MBR and BCL2-negative cases (P = 0.033). Our findings support the existence of alternative genetic pathways involved in the origin of these FL subsets. Even though the number of S-DLBCL cases was small, they showed the shortest telomere length, suggesting that this parameter reflects another genetic alteration involved in the progression from FL to a higher-grade lymphoma.

Adult↗

Sensitive detection of Ehrlichia chaffeensis in cell culture, blood, and tick specimens by reverse transcription-PCR.

Ehrlichia chaffeensis is an obligatory intracellular bacterium of monocytes and macrophages and the etiologic agent of human monocytic ehrlichiosis, an emerging zoonosis. The Lone Star tick (Amblyomma americanum) has been implicated as the primary vector of E. chaffeensis. The present study examined the sensitivity of the nested reverse transcription (RT)-PCR based on the 16S rRNA gene relative to that of the nested PCR for detection of E. chaffeensis in infected DH82 cells, experimentally infected dog peripheral blood mononuclear cells, or experimentally infected A. americanum tick samples. The RT-PCR was found to be approximately 100 times more sensitive than the PCR for detection of E. chaffeensis regardless of the nature of the specimens. Thus, this RT-PCR is useful for detection of E. chaffeensis when a high sensitivity is required. Positive results by RT-PCR also imply the presence of viable pathogens. This is the first demonstration of RNA of E. chaffeensis in infected blood and acquisition-fed male, nymphal, and larval A. americanum ticks.

Animals↗

Detection of babesia species from infected dog blood by polymerase chain reaction.

Polymerase chain reaction (PCR) was first applied to diagnosis of canine babesiosis in Japan. Blood samples from 13 dogs suffering from canine babesiosis were used for examination of specificity and sensitivity of the PCR diagnosis. Of the 13 dogs, three were experimentally infected, and ten were naturally infected with Babesia species in west part of Japan. We designed a nested PCR to amplify the babesial small subunit ribosomal RNA gene and found that only the nested PCR produced a visual band, which were not apparent by the first-round PCR to the positive samples. Specificity of the nested PCR was confirmed by amplification after the second-round PCR. Sensitivity of the nested PCR was examined by diluting the blood samples from infected and uninfected dogs. The nested PCR was found to show positive results on the most diluted blood at 0.0001% parasitemia. These results indicate that the nested PCR is highly sensitive and useful for diagnosis of canine babesiosis.

Animals↗

Developmental regulation of B lymphocyte immune tolerance compartmentalizes clonal selection from receptor selection.

B lymphocyte development is a highly ordered process that involves immunoglobulin gene rearrangements, antigen receptor expression, and a learning process that minimizes the development of cells with reactivity to self tissue. Two distinct mechanisms for immune tolerance have been defined that operate during early bone marrow stages of B cell development: apoptosis, which eliminates clones of cells, and receptor editing, which spares the cells but genetically reprograms their autoreactive antigen receptors through nested immunoglobulin L chain gene rearrangements. We show here that sensitivity to antigen-induced apoptosis arises relatively late in B cell development and is preceded by a functionally distinct developmental stage capable of receptor editing. This regulation compartmentalizes clonal selection from receptor selection.

Amino Acid Sequence↗

Treatment of low grade gastric mucosa-associated lymphoid tissue lymphoma in stage I with Helicobacter pylori eradication. Long-term results after sequential histologic and molecular follow-up.

BACKGROUND AND OBJECTIVES: Most cases of gastric low-grade mucosa-associated lymphoid tissue (MALT) lymphoma are associated with H. pylori. In localized disease (stage I), eradication of H. pylori can result in histologic regression of the lymphoma in 50% to 100% of the patients. Moreover, in half of the apparently cured patients a monoclonal rearrangement of the IgH gene can be demonstrated. However, data on the long-term outcome of the patients are scarce. We report the evolution of a series of patients followed-up since 1994 in order to evaluate the long-term outcome of the apparently cured lymphoma. DESIGN AND METHODS: From January 1994 to July 2000, 19 consecutive patients with stage I gastric low grade MALT lymphoma were sequentially studied in our hospital. They had all been diagnosed by endoscopy and had had a complete staging (including CT-scan, contrast X-ray of the small bowel, bone marrow biopsies, immunophenotyping of bone marrow and peripheral blood and, in the later years, endoscopic ultrasonography). Diagnosis required established histologic criteria for low grade MALT lymphoma in the samples obtained by endoscopy. The investigation of H. pylori status included histologic search, serology and breath test urea-(13)C. Only patients in stage I disease associated with H. pylori were included in the study. Patients received standard triple therapy for eradication of H.pylori and after treatment were sequentially followed-up with endoscopies performed every 2-3 months in the first year, every 6 months in the second year and then yearly. Post-treatment biopsies were obtained by endoscopy for histologic studies, H. pylori cultures and molecular studies. The criteria of Wotherspoon et al. were used for the histological evaluation. Molecular studies were performed with a polymerase chain reaction analysis of the IgH gene using semi-nested procedures with consensus primers for the V(H) (Fr3A/Fr2A) and J(H) (LJH and VLJH) regions. RESULTS: After the eradication treatment, 18 of the 19 patients (94.7%) achieved histologic regression of the MALT lymphoma that occurred after a mean of 4.6 months (range 2-19). In 11 of the 18 histologically cured patients (61%) a monoclonal rearrangement of the IgH gene was demonstrated. In 2 patients the monoclonality disappeared completely, but 9 of the 11 patients (82%) had either persistent (3 patients) or intermittently persistent (5 patients) monoclonality for as long as 64 months. None of the patients who achieved a histologic remission (either with or without monoclonality) relapsed after a mean follow-up of 37 months (range 2-78). Two patients were lost to follow-up and another patient died of a gastric carcinoma; the remaining 15 patients are still in histologic remission after a mean period of 43 months (range 5-78). Ten patients studied between 1994 and the end of 1996 are in remission after a mean of 59 months (range 33-78). INTERPRETATIONS AND CONCLUSIONS: In most cases of gastric low-grade MALT lymphoma in stage I eradication of H. pylori can produce histologic regression of the lymphoma and this regression can be maintained for years. However, IgH gene monoclonality can be detected and persists in most cases. Although this persistent monoclonality seems to indicate the presence of a latent lymphoma population, over a period of 6 years it has not so far influenced the outcome. These findings indicate that in cases of localized gastric low-grade MALT lymphoma associated with H. pylori, the first step of treatment should be eradication of the H. pylori; however, a close and long follow-up is essential to determine the ultimate outcome of these patients and the possible significance of the persistent monoclonality.

Adult↗

[The identification of recombinant HIV-1 strains in IDUs in southwest and northwest China].

OBJECTIVE: To search for possible recombination of HIV-1 strains in China. METHODS: The blood samples were collected from the HIV infected individuals in areas where epidemic of more than two subtypes of HIV-1 were found. The HIV env and tat genes were amplified by nested PCR from PBMCs and directly sequenced and analyzed. RESULTS: The sequence analysis were performed on 14 HIV-1 strains collected in the subtype B' and C epidemic areas of China. No evidence of recombination after sequencing of env was found. The recombnant HIV-1 strains of subtype B' and C were found in 10 of the 14 samples tested when the first exon of the tat gene was sequenced. In addition, three subtype B' and one subtype C non-recombinant HIV-1 strains were found among IDUs in Sichuan Province. CONCLUSIONS: The recombinant HIV-1 strains of subtype B' and C were firstly identified in Sichuan Province of southwest China and Xinjiang Autonomous Region of western China. Their identical sequences and recombinant patterns indicated the same origin of the recombinant strains, it strongly indicates a close correlation between these two HIV epidemic regions. Since no other subtype of HIV-1 except the recombinant was found in Xinjiang, whereas both subtype B and C as well as B'/C recombinant were found in Sichuan, the recombination was most probably happened in Sichuan Province rather than in Xinjiang.

Amino Acid Sequence↗

Structure of the mouse myelin P2 protein gene.

Myelin P2 protein is a small (14,800 Da) protein found in peripheral and central nervous system myelin. To investigate the regulation of expression of this myelin protein, a mouse genomic library was screened with a rabbit P2 cDNA (pSN2.2-2), and a single positive phage clone containing a 20-kb insert was obtained. This insert contained a single internal SalI restriction site and several EcoRI sites. The EcoRI fragments from this insert were subcloned into Bluescript. The rabbit P2 cDNA (pSN2.2-2) hybridized with a 4-kb EcoRI fragment, and this 4-kb fragment was then sequenced after the creation of nested deletions. The mouse gene contained four exons: exon 1 coded for amino acids 1-24, exon 2 for amino acids 24-81, exon 3 for amino acids 82-115, and exon 4 for amino acids 116-131. The three introns were 1.2 kb, 150 bp, and 1.3 kb in length. Primer extension analysis revealed two transcription start sites at +1 and +47, consistent with the presence of two P2 mRNAs, with the larger transcript appearing more abundant. The amino acid sequences predicted from the mouse DNA indicate that the mouse protein differs from the rabbit protein at 16 different positions, with most of the differences located in exon 3. When the gene structure of fatty acid binding protein (FABP) genes were compared, the P2 gene had the same overall structure as others in the FABP family, suggesting a common ancestral gene for members of this family. The 5'-flanking region contains candidate TATA and CAAT sequences, as well as two AP-1 binding sites that may be important in modulation of the expression of this gene.

Animals↗

[Acute myeloid leukemia M2and t (8; 19) (q22; q13)].

OBJECTIVE: Report for the first time of two cases of acute myeloid leukemia (AML) M(2) with t (8; 19) (q22; q13). METHODS: Chromosome specimens were prepared by short-term culture of bone marrow cells and karyotype analyses were carried out using R-and G-banding techniques. Immunophenotyping of the blast cells was analyzed by flow cytometry with a panel of monoclonal antibodies. AML1/ETO fusion gene was tested by "nested" reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Karyotype analyses showed 46, XX, t (8; 19) (q22; q13) [28]/46, XX [2] in case 1 and t (8; 19) (q22; q13), del (9) (q12q22) [23]/46, XY [2] in case 2. In case 2, the blast cells expressed CD(13) (38.8%), CD(33) (31.8%), CD(34) (80.9%), and CD(19) (63.9%) and RT-PCR assay revealed no AML1/ETO fusion gene transcript. CONCLUSION: t (8; 19) (q22; q13) is a variant form of t (8; 21) (q22; q22). Its molecular entity remains to be elucidate.

Adolescent↗