Quantitative Fluorescent Multiplex PCR Research Articles

ADAR expression and copy number variation in patients with advanced gastric cancer

BackgroundGastric cancer (GC) is a world health problem and it is the third leading cause of cancer deaths worldwide. The current practice for prognosis assessment in GC is based on radiological and pathological criteria and they may not result in an accurate prognosis. The aim of this study is to evaluate expression and copy number variation of the ADAR gene in advanced GC and clarify its correlation with survival and histopathological characteristics.MethodsForty two patients with stage III and IV GC were included in this study. ADAR gene expression and copy number variation were measured by real-time PCR and Quantitative multiplex fluorescent-PCR, respectively. Survival analysis performed based on the Kaplan–Meier method and Mantel–Cox test.ResultsADAR mRNA was significantly overexpressed in the tumor tissues when compared to the adjacent normal tissues (p < 0.01). Also, ADAR expression level in stage IV was higher than stage III. 40% of patients showed amplification in ADAR gene and there was a positive correlation between ADAR copy number and expression. Increased ADAR expression was clearly correlated with poorer survival outcomes and Mantel–Cox test showed statistically significant differences between low and high expression groups (p < 0.0001). ADAR overexpression and amplification were significantly associated with metastasis, size and stage of tumor.ConclusionsTogether, our data indicate that amplification leads to over expression of ADAR and it could be used as a prognostic biomarker for disease progression, especially for the metastatic process in GC.

ERCC1 and telomere status in breast tumours treated with neoadjuvant chemotherapy and their association with patient prognosis.

Dysfunctional telomeres and DNA damage repair (DDR) play important roles in cancer progression. Studies have reported correlations between these factors and tumour aggressiveness and clinical outcome in breast cancer. We studied the characteristics of telomeres and expression of ERCC1, a protein involved in a number of DNA repair pathways and in telomere homeostasis, to assess their prognostic value, alone or in combination, in 90 residual breast tumours after treatment with neoadjuvant chemotherapy (NCT). ERCC1 status was investigated at different molecular levels (protein and gene expression and gene copy‐number variations) by immunohistochemistry, qRT‐PCR and quantitative multiplex fluorescent‐PCR (QMF‐PCR). A comprehensive analysis of telomere characteristics was performed using qPCR for telomere length and qRT‐PCR for telomerase (hTERT), tankyrase 1 (TNKS) and shelterin complex (TRF1, TRF2, POT1, TPP1, RAP1 and TIN2) gene expression. Short telomeres, high hTERT and TNKS expression and low ERCC1 protein expression were independently associated with worse survival outcome. Interestingly, ERCC1 gains and losses correlated with worse disease‐free (p = 0.026) and overall (p = 0.043) survival as compared to survival of patients with normal gene copy‐numbers. Unsupervised hierarchical clustering of all ERCC1 and telomere parameters identified four subgroups with distinct prognosis. In particular, a cluster combining low ERCC1, ERCC1 gene alterations, dysfunctional telomeres and high hTERT and a cluster with high TNKS and shelterin expression correlated with poor disease‐free (HR= 5.41, p= 0.0044) and overall survival (HR= 6.01, p= 0.0023) irrespective of tumour stage and grade. This comprehensive study demonstrates that telomere dysfunction and DDR can contribute synergistically to tumour progression and chemoresistance. These parameters are predictors of clinical outcome in breast cancer patients treated with NCT and could be useful clinically as prognostic biomarkers to tailor adjuvant chemotherapy post‐NCT.

Pitfalls in the detection of gross gene rearrangements using MLPA in Fabry disease

MLPA (Multiplex Ligation-dependent Probe Amplification) is a semiquantitative molecular technique developed to uncover gross gene rearrangements in several monogenic diseases, including the X-linked Fabry disease (FD) caused by mutations in the GLA gene. Heterozygosity of the X chromosome in females makes it important to combine routine sequencing analysis with at least one allelic dosage assay (i.e. MLPA).We identified two new gross GLA gene rearrangements, which were not properly detected by MLPA in male patients with FD. In Patient 1, routine sequencing did not amplify GLA exon 7. MLPA failed to confirm such deletion. An alternative allele dosage, based on the Quantitative Fluorescent Multiplex-PCR (QFM-PCR), confirmed the mutation in the proband and excluded it in the mother, revealing that the mutation was de novo in the proband. Patient 2 harboured a gross GLA gene deletion encompassing almost the entire exon 5, the entire intron 5 and part of exon 6. MLPA confirmed the deletion of exon 5 but missed the partial deletion of exon 6. We characterised the breakpoint (c.652_886del452) and carried out screening for possible heterozygosity among at risk female family members.MLPA can miss some gross GLA gene rearrangements making the combination with other allele dosage assays mandatory to confirm or exclude FD at the molecular level.

Application of quantitative fluorescence PCR for the prenatal diagnosis of common fetal chromosomal aneuploidies

OBJECTIVE To assess the value of quantitative fluorescence PCR (QF-PCR) for the prenatal diagnosis of common fetal chromosomal aneuploidies. METHODS A total of 2436 amniotic fluid samples were collected at 18 to 22 gestational weeks. Multiplex QF-PCR was performed with fluorescence-labeled primers specific for 32 polymorphic short tandem repeat (STR) sites on chromosomes 21, 18, 13, X and Y. The PCR products were assayed by capillary electrophoresis. All samples were also assayed by karyotyping. RESULTS Seventy-six (3.12%) samples were diagnosed as chromosomal aneuploidies by QF-PCR, among which 51 were trisomy 21, 12 were trisomy 18, 2 were trisomy 13, and 1 was triploidy. The results were all consistent with those of karyotyping. Ten samples were suspected as sex chromosomal aneuploidies, among which 9 were confirmed, except for 1 case with X structural abnormality. In addition, karyotyping has diagnosed 24 (0.99%) cases of structural abnormalities, only one of which was suspected by QF-PCR with partial abnormal STR results. Two (0.08%) samples were found to be mosaic by karyotyping, one of which was suggested by QF-PCR with cut-off ratios of STR markers. CONCLUSION QF-PCR is reliable for the diagnosis of numerical abnormalities of chromosomes 21, 18, 13, X and Y. The method can serve as an effective technique for rapid prenatal screening of common chromosome aneuploidies in fetus.

Establishment of a 10-Plex Quantitative Fluorescent-PCR Assay for rapid diagnosis of sex chromosome aneuploidies.

Sex chromosome aneuploidies occur commonly in the general population, with an incidence of 1 in 400 newborns. However, no tests specifically targeting sex chromosomes have been carried out in prenatal diagnosis or newborn screening, resulting in late recognition of these diseases. In this study, a rapid diagnostic method for sex chromosome aneuploidies was established using Quantitative Fluorescent-PCR (QF-PCR). Ten markers were included in one multiplex QF-PCR assay, including two sex determination genes (AMXY and SRY), five X-linked short tandem repeats (STRs; DXS1053, DXS981, DXS6809, DXS1187, and DXS8377), one X/Y-common STR (X22), and two autosomal STRs (D13S305 and D21S11). Retrospective tests of 70 cases with known cytogenetic results indicated that the 10-plex QF-PCR assay could well determine sex chromosome copy numbers by both allelic peak numbers and a sex chromosome dosage calculation with the autosomal STRs as internal controls. Prospective comparison with cytogenetic karyotyping on 534 cases confirmed that the 10-plex QF-PCR assay could be well employed for sex chromosome aneuploidy diagnosis in at least the Chinese Han population. This is the first QF-PCR test for the diagnosis of sex chromosome aneuploidies in the Chinese population. This test is superior to previous designs by including up to 8 sex-linked markers covering different parts of sex chromosomes as well as employing internal controls for copy number dosage calculation in a single PCR reaction. Due to simple technique and data analysis, as well as easy implementation within routine clinical services, this method is of great clinical application value and could be widely applied.

Identification of rare and novel deletions that cause (δβ)0‐thalassaemia and hereditary persistence of foetal haemoglobin in Indian population

Hereditary persistence of foetal haemoglobin (HPFH) and (δβ)(0) -thalassaemia are conditions caused by large deletions that involve δ- and β-globin genes in the β-globin cluster, and they are characterized by increased haemoglobin (HbF) levels in adults. Significant phenotypic diversity is observed between the different mutations that cause these conditions. Molecular characterization of these deletions is important for accurate molecular diagnosis, and they will also provide the information on the cis-acting genetic regulatory elements present in the β-globin cluster. We performed gap-PCR, multiplex ligation-dependent probe amplification (MLPA), quantitative fluorescent multiplex PCR (QF-MPCR) and DNA sequencing to detect and characterize the deletions in the β-globin cluster. We characterized six different deletions resulting in (δβ)(0) -thalassaemia or HPFH in 51 unrelated families. With the help of multiple genetic tools, we performed comprehensive genetic analysis of HPFH and (δβ)(0) -thalassaemia in Indian population and could define the molecular basis of these conditions in this population. We also identified two novel HPFH mutations, 49.98kb (HPFH-9) and 86.7kb (HPFH-10) deletions, in this population.

Molecular Basis Of (δβ)0 Thalassaemia and HPFH In Indian Population and The Role Of Non-Coding Transcripts In Increased Foetal Hemoglobin In Adults

Hereditary persistence of fetal haemoglobin (HPFH) and (δβ)0-thalassaemia are conditions caused by large deletions of δ- and β-globin genes and are characterized by high fetal haemoglobin (HbF) in heterozygotes of these mutations. Complete characterization of these deletions is important for understanding the transcriptional and epigenetic mechanisms involved in increase in HbF levels by these mutations. Previous studies demonstrated that these large deletions remove the regulatory sequences which suppress γ-globin gene expression and increase the interaction of enhancers with γ-globin promoter. However, the exact mechanism for elevated HbF in these conditions and differences in HbF levels and phenotypes between (δβ)0-thalassaemia and HPFH still remains unclear. We screened 90 individuals from 51 families by gap-PCR for previously reported common deletions and by MLPA and quantitative fluorescent multiplex -PCR (QFM-PCR) for new deletions in the β-globin cluster. After identification of the approximate locations of the deletions by MLPA and QFM-PCR, we developed PCR strategies to amplify across the breakpoints and the products were sequenced to characterize the breakpoints. We identified six different deletions in Indian population. The frequencies of the previously reported common mutations, Asian inversion deletion and HPFH-3, were 46.66% and 24.44%, respectively. The rare mutations which were not found earlier in Indian population include 49.3kb deletion (12.22%) and 32.6kb deletion (10%). We found 2 novel deletions, 49.98kb deletion and 86.7kb deletion, which accounted 5.55% and 1.11%, respectively. The breakpoints of the 49.98kb deletion were found to be very close to those of HPFH-3 and this mutation was found in compound heterozygous with β-thalassaemia in a 8 year old boy with thalassaemia intermedia (Hb=8.8g/dL). Four heterozygotes in the family had mean MCV = 79.7fL (range: 76-85.6), mean HbF%= 26.9% (range: 23.3-33.0) and pancellular distribution of HbF suggesting that this mutation causes HPFH. The 86.7kb region encompassing Aγ-, δ-and β- globin genes was found in heterozygous state in one individual and it also had haematological parameters of HPFH (MCV=80.8fL and HbF=32.2%). To develop an experimental model to study the molecular mechanisms for elevated HbF in these conditions we established ex-vivo erythropoiesis system with the peripheral blood mononuclear cells obtained from patients with Asian inversion deletion and HPFH-3. Quantitative real time PCR (qPCR) showed increased expression of γ-globin transcripts in the cultured erythroid cells and chromatin immunoprecipitation (ChIP) of RNA Polymerase II (RNAPII) also showed increased transcriptional activation of γ-globin gene in the erythroid cells obtained from the patients with these mutations. To understand the roles of non-coding transcripts at the 5' and 3' breakpoints of the deletions in the transcriptional activation of γ-globin, we performed qPCR with the cDNA obtained from cultured erythroid cells using the primers that bind upstream and downstream of these breakpoints. The results showed that the level of BGL3, the noncoding transcript present near the 5'breakpoint, is enhanced by these deletions. We found activation of a novel non-coding transcript at the 3' breakpoints and they were absent in the erythroid cells of normal individuals. ChIP also showed significant binding of RNAPII at the genomic regions from where these non-coding transcripts are produced. The role of non-coding transcripts has not been reported earlier as a mechanism for increased γ-globin transcription in HPFH and (δβ)0-thalassaemia. It is possible that these deletions remove the genetic elements that cause transcription repression of γ-globin gene in normal adults and they also cause activation of non-coding transcripts which change the epigenetic status of γ-globin gene resulting in its active transcription. To conclude, using QFM-PCRs we could perform a comprehensive study of (δβ)0-thalassaemia and HPFH in Indian population and detected rare and novel deletions that cause these conditions. Using ex-vivo erythropoiesis system which mimicked the pattern of globin gene expression in normal individuals and patients with globin gene mutations, we could identify the role of noncoding transcripts in increased HbF expression in the patients with HPFH and (δβ)0-thalassaemia. Disclosures:No relevant conflicts of interest to declare.

Duplication of exon 2 of the GPC3 gene in a case of Simpson‐Golabi‐Behmel syndrome

In the May issue of the Journal, Mateos et al. [2013] reported a case of Simpson-Golabi-Behmel syndrome (SGBS), with a novel mutation of theGPC3 gene consisting of a duplication of exons 2–4 which causes a frameshift leading to a protein truncated of its last 183 amino-acids including the C-terminus. This report pinpointed that duplication of the GPC3 gene had not been reported before. We would like to emphasize that duplication screening should be part of the molecular analysis in SGBS by describing another duplication of GPC3. In a recent paper [Cottereau et al., 2013] we reported a large series of patientswith SGBS including one casewith aduplication of exon 2. This duplication was detected bymultiplex ligation-dependent probe amplification (MLPA) analysis in a series of 48 patients referred for SGBS, in whom no GPC3 deletion or point mutation had been identified (Fig. 1). This result was confirmed by quantitativemultiplex fluorescent PCR (PCR of exons 2, 3, and 8 ofGPC3 compared with exon 22 of the CFTR gene as internal control) (results not shown). RT-PCR analysis performed on mRNA extracted from a lymphoblastoid cell line of the patient, with primers designed in exons 1 and 3, respectively, showed a fragment with a size increase of around 160 pb compared to a normal control (Fig. 2). Sequencing of this fragment showed that it was a tandem duplication of exon 2. Unlike the previously reported case, this duplication conserves the reading frame and leads to an insertion of 54 residues from Gly59 to Gln112, in the GPC3 protein. This insertion includes, in the fourteen cysteine motif, three additional cysteine residues (p.Cys65, p.Cys72 and p.Cys73) which probably disrupt the conserved glypican three-dimensional structure of the protein thus altering its function [Filmus and Capurro, 2008]. This duplication was observed in a sporadic case (DNA of his mother was unavailable) who had a clinical picture typical of SGBS. He had been examined at the age of 20 years for tall stature. The diagnosis of SGBS had been suggested on themedical history of the patient because of macrosomia at birth followed by advanced statural growth (height at 214 cm) but normal OFC (þ0.8 SD) and BMI (20.1), diaphragmatic hernia operated on in the neonatal period, cryptorchidism, and on examination pectus excavatum, one pair of extra nipples, inguinal hernia, large hands, and suggestive dysmorphic features (macrognathia, median groove of the

Characterization of two deletions of the CTRC locus

Novel variants associated with chronic pancreatitis are being increasingly reported. However, most studies have so far only analyzed point mutations and small insertions or deletions. Here we report the characterization of two distinct deletions of the CTRC locus. Variants in four chronic pancreatitis genes, PRSS1, SPINK1, CTRC and CFTR, were systematically analyzed in the studied cases. Copy number change of the CTRC gene was analyzed by quantitative fluorescent multiplex PCR (QFM-PCR). Walking QFM-PCR followed by long-range PCR and direct sequencing were employed to identify the deletion breakpoints at the nucleotide level. A heterozygous CTRC-deleting complex rearrangement, which was co-inherited with different trans variants in SPINK1, CFTR or PRSS1, is associated with variable phenotypes (chronic pancreatitis; pancreatic cancer and chronic pancreatitis; and type 1 diabetes). Moreover, a different homozygous deletion of the CTRC locus was found in an unrelated patient with asymptomatic chronic pancreatitis. Our findings revealed a hitherto unrecognized level of complexity of genotype–phenotype correlation in chronic pancreatitis. The CTRC-deleting complex rearrangement probably resulted from LINE-1-mediated Alu insertion, which represents a novel mutational mechanism causing chronic pancreatitis.

Family history of von Hippel–Lindau disease was uncommon in Chinese patients: suggesting the higher frequency of de novo mutations in VHL gene in these patients

Von Hippel-Lindau (VHL) disease is an autosomal dominant familial cancer syndrome caused by germline mutations in VHL tumor suppressor gene. It is characterized by hemangioblastoma in central nervous system and retina, renal cell carcinoma or cyst, pheochromocytoma, pancreatic cyst and tumor, endolymphatic-sac tumor, and papillary cystadenoma in epididymis and broad ligament. Here, we used PCR-direct sequencing and universal primer quantitative fluorescent multiplex PCR (UPQFM-PCR) to detect VHL mutations in 16 patients clinically diagnosed with VHL disease. PCR-direct sequencing detected 12 germline mutations (75%, 12/16), in which a novel mutation of c.451A>T/p.Ile151Phe found in one proband had not been reported previously. UPQFM-PCR found two large deletions (12.5%, 2/16). The two remaining patients carried non-typical disease-causing mutations, including one silent mutation (c.481C>A/p.Arg161Arg) and one mutation in 3'-UTR (c.642+70C>A). Remarkably, 56.3% (9/16) probands did not have family history of VHL disease, suggesting the higher frequency of de novo mutations in Chinese patients. We also summarized Chinese VHL disease patients with VHL mutation findings published in the literature to provide information about the spectrum of VHL mutations in Chinese VHL disease patients.

Detection of cell-free fetal DNA in plasma of pregnant women using multiplex quantitative fluorescent-PCR

Detection of cell-free fetal DNA in plasma of pregnant women using multiplex quantitative fluorescent-PCR

Clinical utility gene card for: Gorlin syndrome

1.6 Analytical methods Sequencing of all PTCH1 exons and their intron–exon boundaries,1 MLPA, QMP (Quantitative Multiplex fluorescent PCR) or array-CHG for deletions spanning the whole gene.2,3 Based on the type of mutations most frequently detected,1 start with sequencing (point mutations) followed by MLPA, QMP or array-CHG (large mutations). In patients who test negative for mutations in PTCH1, promoter analysis, testing of SUFU4 and PTCH25 may be considered. The detection specificity of sequencing is almost 100% for point mutations and small deletions and insertions. MLPA, QMP or arrayCHG are applicable only for exon-spanning mutations (o5% of all mutations), the detection specificity is 95–100%. Pathogenicity of missense PTCH1 alterations is verified by testing a set of 100 controls (200 chromosomes) and by in silico prediction methods.

High resolution mapping of OCA2 intragenic rearrangements and identification of a founder effect associated with a deletion in Polish albino patients

Oculocutaneous albinism type 2 (OCA2) represents about 30% of OCA worldwide. Using quantitative multiplex fluorescent PCR and very high-resolution array-CGH focussed on the OCA2 gene and surrounding regions in 15q12, we identified new rearrangements. Deletion 1, encompassing exons 3-20, was present in three patients (including one in the homozygous state), and Deletion 2 (exons 1-20) was found in one patient (heterozygous state). The duplication (exons 3-20) was found in one patient in the homozygous state. Using 14 microsatellite markers we determined haplotypes associated with these rearrangements. Deletion 1 was associated with the same haplotype in three patients who were all of Polish origin, which is strongly in favour of a founder effect. Deletion 2 was associated with a distinct haplotype. The homozygous duplication was inherited from the two unrelated parents of the patients on two different haplotypes. Analysis of the sequences around the breakpoints of these rearrangements showed that all occurred within complex arrays of repetitive sequences. The combined use of very high-resolution array-CGH and of microsatellites (including new intragenic ones described here) constitutes a powerful approach for the precise characterization of OCA2 rearrangements, which have been found in more than 20% of OCA2 patients.

Identification of copy number variants in miscarriages from couples with idiopathic recurrent pregnancy loss

Recurrent pregnancy loss (RPL), defined as two or more miscarriages, affects 3-5% of couples trying to establish a family. Despite extensive evaluation, no factor is identified in ∼40% of cases. In this study, we investigated the possibility that submicroscopic chromosomal changes, not detectable by conventional cytogenetic analysis, exist in miscarriages with normal karyotypes (46,XY or 46,XX) from couples with idiopathic RPL. Array comparative genomic hybridization (array-CGH) was used to assess for DNA copy number variants (CNVs) in 26 miscarriages with normal karyotypes. Parental array-CGH analysis was performed to determine if miscarriage CNVs were de novo or inherited. There were 11 unique (previously not described) CNVs, all inherited, identified in 13 miscarriages from 8 couples. The maternal origin of two CNVs was of interest as they involved the imprinted genes TIMP2 and CTNNA3, which are only normally expressed from the maternal copy in the placenta. Two additional cohorts, consisting of 282 women with recurrent miscarriage (RM) and 61 fertile women, were screened for these two CNVs using a Quantitative Multiplex Fluorescent PCR of Short Fragments assay. One woman with RM, but none of the fertile women, carried the CTNNA3-associated CNV. This preliminary study shows that array-CGH is useful for detecting CNVs in cases of RPL. Further investigations of CNVs, particularly those involving genes that are imprinted in placenta, in women with RPL could be worthwhile.

Improved criterion‐referenced assessment in indirect tracking of haemophilia A using a 0.23 cM‐resolution dense polymorphic marker set

In haemophilia A, linkage analysis with coagulation factor VIII (F8) intragenic and/or neighbouring extragenic short tandem repeats (STRs) enables indirect tracking F8 pathogenic allelic variant-carriers. Even where DNA sequencing is available, linkage analysis still has a role if no causative or candidate mutation is unveiled. The cumulative heterozygosity rate of the available multiplexed STRs haplotyping assays rarely reaches 100%. This means that in a proportion of women these loci are uninformative. The norm-referenced assessment is based on at least one informative marker criterion. We reasoned that by typing a dense market set, spanning a small fraction of recombination, we should be able to improve assessment. The aim of this study was to improve criterion-referenced assessment in polymorphism segregation analyses using a low-recombination fraction and dense informative STRs set. The multiplex quantitative fluorescence PCR assay comprises four novel tetranucleotide and pentanucleotide STRs distant < or = 0.15 cM from the F8 gene, and three F8 intragenic dinucleotide STRs, mapped to a 0.23 cM interval spanning the F8 on human chromosome band Xq28. We determined heterozygosity rates and allele frequencies from 100 unrelated healthy females. To investigate about segregation stability, we typed 50 true trios (mother, daughter and father) and 50 true mother-and-son duos from the general population. The heterozygosity rates for the extragenic markers ranged 0.49-0.76. The 0.23 cM-resolution heptaplex rendered a cumulative heterozygosity of 0.89 for a minimum of two informative markers, with at least one F8 intragenic. The heptaplex assay enabled improving the criterion-referenced assessment in indirect carrier-detection.

A new large deletion in the DFNB1 locus causes nonsyndromic hearing loss

Mutations in the GJB2 gene encoding the gap junction protein connexin 26 are responsible for up to 30% of all cases of autosomal recessive nonsyndromic hearing impairment (HI) with prelingual onset in most populations. The corresponding locus DFNB1, located on chromosome 13q11–q12, is also affected by three distinct deletions. These deletions extended distally to GJB2, which remains intact. We report a novel large deletion in DFNB1 observed in a patient presenting profound prelingual HI. This deletion was observed in trans to a GJB2 mutated allele carrying the p.Val84Met (V84M) mutation and was shown to be associated with hearing loss. The deletion caused a false homozygosity of V84M in the proband. Quantification of alleles by quantitative fluorescent multiplex PCR (QFM-PCR) enabled us to study the breakpoints of the deletion. The deleted segment extended through at least 920 kb and removed the three connexin genes GJA3, GJB2 and GJB6. The distal breakpoint inside intron 2 of CRYL1 gene differed from the breakpoints of the known DFNB1 deletions. This case highlights the importance of screening for large deletions in molecular studies of GJB2.

Application of multiplex quantitative fluorescent PCR with non-polymorphic loci in prenatal diagnosis

To explore the feasibility of application of multiplex quantitative fluorescent PCR with non-polymorphic loci in prenatal diagnosis of aneuploidies. From Mar 2006 to Nov 2007, a total of 63 samples were collected from the Department of Obstetrics and Gynecology, Affiliated Drum Tower Hospital of Medical College, Nanjing University, including 54 villous samples obtained for karyotyping because of spontaneous abortion, six amniotic fluid samples of second trimester and three umbilical cord blood samples of third trimester. Blood samples of 60 healthy adults were obtained at the same time as a control group, including 30 males and 30 females. Non-polymorphic QF-PCR was performed on both testing group and control group for the detection of aneuploidies. The Amelogenin gene (AMXY) was selected as an internal control, and dosage quotiety (DQ) of each locus was calculated according to the known formula. If DQ was between 0.7 and 1.3, the sample was considered as normal. If the figure turned out to be > 1.3 or < 0.7, a potential duplication or deletion of the corresponding gene or chromosome was indicated. If the results implied numerical abnormalities in more than one euchromosome, sex chromosome aneuploidies should be considered. Cell culture and karyotyping were carried out for every sample simultaneously. The results of non-polymorphic QF-PCR were checked with karyotypes. (1) In the control group, all female samples presented only an AMX peak for sex chromosome while all males showed AMX and AMY amplified peaks. The AMY/AMX ratios were between 0.7 - 1.3, and SD was between 0.05 - 0.12. (2) Among 19 QF-PCR abnormal cases, 13 cases were proved by karyotyping. Of the six cases which turned out to be conflicting, one case of trisomy 18 shown by karyotyping was not completely detected by QF-PCR, a locus on chromosome 18 implied trisomy, while another turned out to be normal (DQ = 1.28). Four cases were detected by non-polymorphic QF-PCR as trisomies but showed normal female karyotype because of maternal contamination during cell culture. A karyotypingly '46, XY' case did not present an AMY peak. Thirty-six out of 44 (82%) normal results implied by non-polymorphic QF-PCR were in accordance with cytogenetic analysis. Of the other eight cases, one case which failed cytogenetic analysis was detected by QF-PCR as normal. Four cases showed multiploidy by karyotyping but normal in QF-PCR analysis, including three cases of 69, XXX, one case of 92, XXXX and one case of 45, XX, rob (13;21). The other two cases that showed normal male results turned out to be normal female karyotypes. Prenatal aneuploidy detection by non-polymorphic QF-PCR is feasible in a clinical diagnostic setting. With the advantages of high throughput, rapidness and low cost, this method shows a good prospect in clinical application.

Molecular diagnosis of oculocutaneous albinism: new mutations in the OCA1–4 genes and practical aspects

Molecular diagnosis of oculocutaneous albinism: new mutations in the OCA1–4 genes and practical aspects

Hereditary pancreatitis caused by a double gain-of-function trypsinogen mutation

Hereditary pancreatitis, an autosomal dominant disease with approximately 80% penetrance, can be caused by both 'gain-of-function' missense and copy number mutations in the cationic trypsinogen gene (PRSS1). Here we demonstrate a heterozygous hybrid PRSS2 (encoding anionic trypsinogen)/PRSS1 gene in a French white family with hereditary pancreatitis, by means of quantitative fluorescent multiplex PCR and RT-PCR analyses. The hybrid gene, in which exons 1 and 2 are derived from PRSS2 and exons 3-5 from PRSS1, apparently resulted from a non-allelic homologous recombination (NAHR) event between the chromosome 7 homologs or sister chromatids during meiosis. Interestingly, this hybrid gene causes the disease through a combination of its inherent 'double gain-of-function' effect, acting simultaneously as a 'quantitative' copy number mutation and a 'qualitative' missense mutation (i.e. the known disease-causing p.N29I mutation). Our finding reveals a previously unknown mechanism causing human inherited disease, enriches the lexicon of human genetic variation and goes beyond the known interaction between copy number variations (CNVs) and single nucleotide substitutions in health and disease. Our finding should also stimulate more interest in analyzing both types of genetic variation whenever one tries to determine the contribution of a specific locus to a given disease phenotype.

Development of a multiplex quantitative fluorescent PCR assay for identification of rearrangements in the AZFb and AZFc regions

The azoospermia factor b (AZFb) and azoospermia factor c (AZFc) regions in the human Y chromosome consist of five palindromes constructed from six distinct families of amplicons and are prone to rearrangement. Partial deletion and duplication in the region can cause azoospermia or oligozoospermia and male infertility. The aim of the study was to establish a quantitative fluorescent PCR (QF-PCR) assay to classify AZFb and AZFc rearrangements. A single pair of fluorescent primers was designed to amplify simultaneously the amplicon in AZFc and the length-variant homologous sequences outside of the region as control. Since the copy number of the control sequences is fixed in the human genome, dosage of the target could be easily obtained through comparing the height of the fluorescent peaks between the target and the control after amplification with limited PCR cycles. Most types of rearrangements in AZFb and AZFc regions could be classified with QF-PCR containing four such primer pairs. Eleven types of rearrangement in AZFb and AZFc regions were well discriminated with QF-PCR. In conclusion, QF-PCR is a simple and reliable method to detect rearrangements in AZFb and AZFc.