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Genetic testing
April 17, 2009
Nirmal Paudel, Nirmal Acharya, Sajana Khadgi, Prakash Raut
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Index
- Introduction
Reproductive Issues.
Clinical Issues.
Uncertainties.
Health and Environmental Issues - Commercialization of Products
- Genetic Testing Policy for CLIA Regulation
- Cost of genetic testing
- Available Genetic Tests:
Illness caused by abnormalities in chromosome.Most of the disorders are quiet rare.It affects one person in several thousands or millions.Environmental factors may have affect on genetic disorders.Some type of recessive gene disorder has an advantage in the heterozygous state in certain environment.These disorders are diagnosed and treated by geneticist.Study of genetic disease is a scientific discipline and it is based on population genetics and molecular genetics.
- Single-gene
- Multifactorial
- Chromosomal
- Mitochondrial
Gina
Psychological Impact and Stigmatization
People who have lived in same geographic region for several generations may have some common alleles but not alleles are common within a population .Department of energy sponsored a Nature Genetics supplement called,Genetics for the human Race.The National Human Genome Center at Howard University held a workshop in Washington DC.It was financially supported by US DOE.Workshop focused on several important questions, such as "What does the current body of scientific information say about the connections among race, ethnicity, genetics and health? What remains unknown? Does the actual DNA sequences we each carry, clarify and inform our history and relationships?Each of us carries a set of genes that affects the color of his or her skin. But they represent only a small fraction of the estimated 30,000 total genes in our genomesWe are genetically far more nuanced and variable than is reflected in just skin coloration.
Reproductive Issues:Including adequate informed consent for complex and potentially controversial procedures, use of genetic information in reproductive decision making, and reproductive rights.Gene testing is done for :
Including the education of doctors and other health service providers, patients, and the general public in genetic capabilities, scientific limitations, and social risks; and implementation of standards and quality-control measures in testing procedures. Uncertainties:>Associated with gene tests for susceptibilities and complex conditions (e.g., heart disease) linked to multiple genes and gene-environment interactions. name="ref g">Health and Environmental Issues :Concerning genetically modified foods (GM) and microbes.
Molecular genetics isa branch of biochemical genetics and analyses are performed on any nucleic acids to detect heritable or acquired disease-related genotypes, including chromosome rearrangements and mutations involving as little as a single nucleotide. The fundamental tools of molecular genetics includes:
- TaqMan PCR
- LATE- PCR endpoint assays
- Quantitative Real time PCR
- Probe based qPCR
- SYBR real-time PCR
- PCR arrays: gene expression profiling technology utilizing real time PCR
- Quantitative multiplex methylation-specific PCR or QM-MSP
- Methylation-specific PCR (MSP)
- Differential Display- PCR
- Robust dosage-PCR
Carrier screening.-Patient has the abnormal gene but the patient doesnot show any symptoms or disease condition.
Newborn screening.-Done in the newborn to identify the serious condition before it begins.It is done before the baby leaves the hospital.
Prenatal screening.-It helps to identify health problems that affect both mother and baby.
Genetic Testing Policy for CLIA regulation:
It is the goal of CLIA to promote integration of validated genetic tests into clinical and public health practice. this is done under the following guidelines:
- Developing regulatory requirements for genetic testing Under CLIA :-CLIA spervises the laboratories that perform genetic testing.
- The Role of CLIA in the Oversight of Genetic Testing:-Goals of oversight:-Achieve balanced,timely,long lasting rules.
- Recommendations of the CLIAC:-For modifying the current CLIA regulations to establish specific requirements for genetic testing during 1997 – 1999.
- Publication of a Notice of Intent:-CLIA operates certain programs that monitors the quality assurance of genetic testing.
- Analysis of public comments:-The CLIAC formed Genetics Workgroup to evaluate the NOI(Notice of Intent) comments and provide input to assist the CLIAC in making further recommendations on genetic test
- CLIAC recommendations were revised
- Notice of Proposed rule Making
| Costs for Selected Genetic Tests by Disorder and Type of Test | |||||
| LAB Test | Sequencing | Heteroduplex Analysis | DGGE | ASO | PTT |
| HNPCC | $500-3000 | $260 | $250-800 | -- | -- |
| FAP | $800-1000 | -- | -- | -- | $235 |
| BRCA1 | $1290 | -- | -- | $350-450 | -- |
| BRCA2 | $1290 | -- | -- | $350-450 | -- |
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BRCA-Ashkenazi mutations
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-- | -- | -- | $190-354 | -- |
| DGGE=Denaturing gradient gel electrophoresis ASO=Allele specific oligonucleotide PTT=Protein truncation test |
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| Range of Turn Around Time for Genetic Tests | |
| Type of Genetic Test | Range of Time for Test Result* |
| HNPCC | 4-20 weeks |
| FAP | 2-4 weeks |
| BRCA1 (sequencing) | 3-4 weeks |
| BRCA2 (sequencing) | 3-4 weeks |
| BRCA (Ashkenazi Mutations) | 1-4 weeks |
| *Based on a Genetic Health survey of 38 clinical laboratories conducting these tests.As | |
Tests
Pathogen Detection
- Field deployable detection of multiple pathogens and other organisms
- Homeland security and environmental testing
- Clinical testing at point-of-care
- At-home common pathogen diagnostics
Advantages
- Sensitive and specific for pathogens of interest
- <$20 per multi-pathogen test
- Handheld and simple to operate
- Disposable
Index
Genetic characterization of highly pathogenic H5N1 avian influenza viruses
[See also the CLS research page: Avian influenza]
The molecular diagnostic methods have also gained ground and traditional as well as real-time PCR methods targeting the conserved region of the matrix protein gene are widely used for the detection of influenza A viruses. In positive cases the subtype of the detected virus is determined with other PCRs targeting the haemagglutinin (HA) and neuraminidase (NA) genes. In case of H5 or H7 subtypes it is followed by the pathotyping, i.e. the characterization of the nucleotide composition of the region flanking the HA cleavage site. If the detected virus turns out to be an HPAI (that is subsequently confirmed by biological assays as well), strictly regulated measures must be applied as detailed in the respective National Contingency Plans. Although the above steps carried out in National Reference laboratories accomplish the compulsory laboratory investigations, thorough analysis of every isolate has become a routine in the more sophisticated laboratories in order to be better prepared for a possible outbreak.In the case of the highly pathogenic H5N1 avian influenza, full-length sequencing protocols are being used in the diagnostic laboratories. These protocols can quickly provide comprehensive data on a detected H5N1 avian influenza virus (AIV) to animal and public health authorities facilitating their actions. Part of the analysis is the handling of the obtained raw nucleotide sequence data. For this purpose, we use the CLC Combined Workbench software that provides fast, versatile, and flexible ways of handling and analyzing sequences. For instance, the accuracy of the assembly of several raw nucleotide sequences is supported by showing the translation of the particular sequence into amino acids, which aids in spotting conflicts among sequence reads.
Detection of pathogenic organisms in the routine monitoring of contaminants in food
The detection of pathogenic organisms plays an important role in the routine monitoring of contaminants in food.Based on a combination of microarray techniques, PCR and bioinformatics, twelve pathogens including: Staphylococcus, Vibrio cholerae, E. coli, Listeria Spp. Listeria, Monocytogenes, Salmonella, ß -Hemolytic streptococcus, Vibrio parahaemolyticus, Campylobacter jejuni, Campylobacter, Enterobacter sakazakii and Shigella can be rapidly and simultaneously detected with high accuracy and sensitivity.Genetic Tests for foodborne pathogens:
Rapid techniques were developed and applied to the determination of total viable bacteria and to the detection of food borne pathogens (Listeria monocytogenes, Salmonella, Campylobacter jejuni and E. coli O157:H7).The techniques developed for the detection of food borne pathogens are based on an initial short enrichment of the pathogens in the food sample, isolation of the food pathogen onto a polycarbonate membrane followed by detection using molecular techniques (polymerase chain reaction).
Molecular Diagnostic Tests: Cancer
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Types of cancer |
Genes Involved |
Diagnostic Test |
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|---|---|---|---|
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i) Adrenocortical carcinoma- most common type. Symptoms include increased hormone type if it is functioning type of tumor. ii) Pheochromocytoma- rare and seen in cells responsible for producing Epinephrine or Norepinephrine. iii) Neuroblastoma- Generally seen in adults in their 30s or 40s. Originates from undeveloped nerve cells and develops in adrenal medulla. |
Cancer presentation Wiedemann-Beckwith syndrome (WBS)- 11p15.5 Li-Fraumeni syndrome (LFS)- TP53 gene on 17p congenital adrenal hyperplasia (CAH)- CYP21B gene |
Fluorescent in situ hybridization (FISH) Comparative genomic hybridization (CGH) |
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Mainly due to chromosomal loses and mutation of KRAS oncogene. Genes involved in chromosomal loses are APC (5q), DCC/MADH2/MADH4 (18q), and TP53 (17p). |
Diagnostic method similar to the colorectal cancer. |
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Mutation of the tumor suppressor TP53 gene and PTEN gene.
p15 and p16 genes |
PCR amplification to detect deletion of different genes associated with brain tumor. Read more… |
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AR, ATM, BARD1, BRIP1, CHEK2, DIRAS3, ERBB2, NBN, PALB2, RAD50, RAD51, RASSF1A, TWIST, Cyclin D2, HIN1, RARB |
Quantitative multiplex methylation-specific PCR Breast Cancer DNA Methylation PCR Array- Utilizes simple Restriction Enzyme digestion and Real time PCR to analyze 96 liver cancer genes for their methylation status |
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HPV is nowadays commonly associated with the Cervical cancers. |
Hybrid Capture II (HC II) assay- Enzyme-linked immunosorbent assay hybridization followed by a non-radioactive alkaline phosphatasein indicator in microplates. PCR |
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APC and MUTYH genes miRNA, short non-coding RNA’s has also been implied in colorectal cancer. They are regulatory RNA molecules responsible for repressing translation or cleaving RNA transcripts. |
Real Time PCR- the genes are expressed and normalized using selectable stable internal control gene. |
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Epidermolysis bullosa simplex (EBS) |
Mutation in COL7A1
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RT- PCR and direct sequencing- To detect the mutation and polymorphism in the COL7A1 gene. |
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RT- PCR- for Carcinoembryonic Antigen (CEA) transcript MethylPlex whole methylome Amplification DNA- Commercial kit available for the esophageal cancer detection |
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Seen frequently with overexpression and mutation of c-Kit oncogene. Generally distinguished from leiomyosarcomas (LMSs) by comparing relative expression of OBSCN and C9orf65 genes. |
Quantitative RT-PCR- Two-gene relative expression classifier which utilized RT- PCR showed 100% accuracy to distinguish between GIST and LMS and has the possibility to be rapidly implemented in a clinical settings. |
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Hodgkin's lymphoma |
The type varies with the age. |
ALK, NPM1, IL6, , CD44, DDX6, RSN, BCL2, PCNA, MIC2, MAGE-4A, CT45, CD99 |
DNA microarray Technology Methylation specific PCR (MSP) DNA sequencing |
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Leukemia |
CML- formation of fusion gene BCR/ABL that encodes for tyrosine kinase B- cell- Rearrangement of BCL2 and MYC
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Fluorescent PCR combined with capillary electrophoresis, restriction enzyme digestion and GeneScan analysis to detect the insertion mutation and internal tandem duplications.
RT-PCR- to amplify specific chromosomal break points which encode for fusion proteins affecting normal hematopoietic homeostasis and ultimately leukemia. |
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Genes associated- CHEK2 and TP53 genes |
DNA Sequence analysis- to detect the mutation in p53 gene which is further confirmed on second prep by independent amplification using PCR where sequencing is carried out in opposite direction. Robust Dosage-PCR- to detect large heterozygous deletion for exon 2-11 for p53 gene Read more about the assay |
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Comparative genomic hybridization Representational difference analysis |
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Primary Lung Cancer
Squamous cell carcinoma Adenocarcinoma Large cell carcinoma
Mesothelioma |
Germline mutation of EGFR & TP53, Somatic mutation of KRAS, BRAF, ERBB2, MET, STK11, PIK3CA Amplified genes like EGFR, ERBB2, MET, PIK3CA and NKX2-1 Fusion genes- ALK/EML4 Polymorphism seen in genes like ERCC6, LNCR2 COX-2 |
Quantitative real time PCR- able to detect free circulating DNA which is generally seen during early stages of the tumor development with sensitivity (90 %) and specificity (86 %) with positive predictive value of 90 % and negative predictive value of 90 %. Methylation specific PCR- to detect hypermethylation of the cytosines in CpG islands of the promoter regions of different genes including tumor suppressor genes leading to gene silencing. |
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CDKN2A (cyclin dependent kinase inhibitor 2A) gene, CDK4, MMP3, MMP1, MC1R, EFNB2, ARHC, S100A6, S100A2 |
cDNA microarray analysis- different gene expression level obtained is helpful for the staging as well as tracking the disease progression. |
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SDC1, LTA, TNF, IRF4, IL6, HGF, CCND1, B2M, CD19, DLC-1 |
Methylation specific- PCR Bisulphite genomic sequencing RT- PCR |
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Non-Hodgkin's Lymphoma |
LMO2, BCL6, FN1, CCND2, SCYA3,BCL2, CCND1, CDC37, glutathione-S-transferase pi (GST-pi) |
Light-up probe-based real-time PCR- detects B-lymphocyte monoclonality by comparing kappa and lambda immunoglobulin light chain expression. PCR- to detect hypermethylation of the genes associated. |
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MGMT, MMP14, CYP1A1, MMP2, MMP9, ECGFB, DIA1, BIK, PDGFB |
Differential Display- PCR- to assess the gene expression profile of oral tumor against the normal epithelial cells. |
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TGM4, ACPP, FGF2, FGF1, SPARC, MSMB, FGF8, MKI67, KLK3, KLK2, HPCX, TMPRSS2, PCA3 |
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PRCC, RCA1, VHL, IL6, MET, TSC1, TSC2, CA9, TFE3 |
Comparative genomic hybridization and array analysis- for single nucleotide polymorphisms High-Density Affymetrix 10K Single Nucleotide Polymorphism Mapping Array |
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Deletion of the chromosome 13 containing RB1 gene, MYCN |
RT- PCR-was able to detect metastatic retinoblastoma cell which was also effective for monitoring therapeutic effects of the treatment. |
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CDKN2A, HMGIC, TP53, SST, HLA-A, HLA-B, TNFRSF5 |
RT- PCR- performed using SuperScript™ III One-Step RT-PCR System with Platinum® Taq DNA Polymerase which was able to detect commonly found translocation in soft tissue tumor. Nested PCR method - for detection of Kaposi sarcoma associated herpes virus |
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MDM2, TOP1, TGCT1, TSPY, MGMT |
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NTRK1, TPO, TGFBR2, ELE1, RET |
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The following descriptions were taken fpund at the Baaylor College of Medicine, Genetics laboratories; and ARUP
| Disease | Target | Methodology | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Achondroplasia (ACH) | growth factor receptor 3 (FGFR3) gene on chromosome 4p16.3 | DNA amplification (PCR), primer extension, and allele resolution by Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) Mass Spectrometry | ||||||||
| Androgen insensitivity syndrome (AIS) | androgen receptor (AR; OMIM 313700) | PCR amplification of the exons contained in the AR gene coding region. Direct sequencing of amplification products in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. | ||||||||
| ANGELMAN SYNDROME (AS) | UBE3A the E6-AP ubiquitin ligase protein | Direct sequencing of amplification, in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. | ||||||||
| Argininosuccinic aciduria |
ASL gene that comprised of 16 coding exons and located on chromosome 7q11.2 | Direct sequencing of amplification, both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. | ||||||||
| ARX Related Disorders | ARX gene located on chromosome Xp21.1 |
Bi-directional sequence analylsis of the entire ARX gene coding region is performed using automated fluorescence sequencing in a 96-capillary format. |
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| ARYLSULFATASE A DEFICIENCY (ARSA) | (ARSA) gene | PCR amplification of the exons contained in the ARSA gene coding region. Direct sequencing of amplification, both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. | ||||||||
| Ashkenazic Genetic Disease Screen |
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Direct DNA analysis is performed on 14 different mutations in four different genes that incorporates DNA amplification (PCR), primer extension, and allele resolution by Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) Mass Spectrometry | ||||||||
| Aspirin-Like Platelet Disorder | entire RUNX1 gene |
PCR amplification of the exons contained in the RUNX1 gene coding region. Direct sequencing of amplification, both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. |
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| Autoimmune polyendocrinopathy syndrome type I (APS1) | AIRE gene associated with Autoimmune polyendocrinopathy syndrome type I | A PCR-based assay is used to amplify all 14 exons of the AIRE gene coding region. Direct sequencing of amplification, the forward and reverse directions using automated fluorescence dideoxy sequencing methods. | ||||||||
| Bloom's syndrome | BLM gene, which is located on chromosome 15 at 15q26.1 | PCR amplification of the exons contained in the BLM gene coding region. Direct sequencing of amplification, forward and reverse directions using automated fluorescence dideoxy sequencing methods | ||||||||
| CANAVAN DISEASE | Y231X E285A A305E |
Direct DNA analysis for the 3 mutations that incorporates DNA amplification (PCR), primer extension, and allele resolution by Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) Mass Spectrometry | ||||||||
| Cartilage Hair Hypoplasia (CHH) | RMRP gene (70A>G) | PCR amplification of one exon contained in the RMRP gene coding region. Direct sequencing of amplification, forward and reverse directions using automated fluorescence dideoxy sequencing methods | ||||||||
| CDKL5 Related Atypical Rett Syndrome | CDKL5 gene on Xp22 | PCR amplification of 21 exons contained in the CDKL5 gene coding region. Direct sequencing of amplification, forward and reverse directions using automated fluorescence dideoxy sequencing methods. | ||||||||
| chromodomain helicase DNA-binding protein 7gene (CHD7) | PCR amplification of 38 exons contained in the CHD7 gene coding region. Direct sequencing of amplification, forward and reverse directions using automated fluorescence dideoxy sequencing methods. | |||||||||
| Citrullinemia I | The ASS gene chromosome 9q34.1 and 16 exon | A PCR-based assay to amplify all 16 exons of the ASS gene coding region. Direct sequencing of amplification, forward and reverse directions using automated fluorescence dideoxy sequencing methods. | ||||||||
| Cleidocranial Dysplasia | 8 exons of the RUNX2 gene | A PCR-based assay is used to amplify all 8 exons of the RUNX2 gene coding region. Direct sequencing of amplification, forward and reverse directions using automated fluorescence dideoxy sequencing methods. | ||||||||
| CONNEXIN 26/GJB2 RELATED HEREDITARY HEARING LOSS | GJB2 gene, which encodes the connexin 26 protein |
Amplification of the GJB2 gene coding region iPCR on patient genomic DNA. Bi-directional DNA sequence analysis on PCR products using automated, fluorescent dideoxy sequencing methods. |
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| Costello Syndrome | HRAS gene | PCR amplification of the exons contained in the HRAS gene coding region. Direct sequencing of amplification products is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. | ||||||||
| Cystic Fibrosis (CF) | 27 exons contained in the Cystic Fibrosis Transmembrane Regulator gene (CFTR)gene coding region | PCR amplification of 27 exons contained in the CFTR gene coding region. Direct sequencing of amplification, forward and reverse directions using automated fluorescence dideoxy sequencing methods | ||||||||
| Dentatorubral Pallidoluysian Atrophy (DPRLA) | trinucleotide repeat sizes in the DRPLA gene | PCR analysis across the CAG repeat region to determine allele size(s). Southern analysis is used to identify extremely large-sized CAG expansions | ||||||||
| Diamond-Blackfan Anemia (DBA) | 6 exons of the RPS19 gene | A PCR-based assay is used to amplify all 6 exons of the RPS19 gene. Direct sequencing of amplification, the forward and reverse directions using automated fluorescence dideoxy sequencing methods. | ||||||||
| Fabry disease | GLA gene localized to Xq22.1 |
PCR amplification of seven exons contained in the GLA gene coding region is performed. Direct sequencing of amplification products in both forward and reverse directions using automated fluorescence dideoxy sequencing methods using capillary electrophoresis. |
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| FACTOR V LEIDEN | Factor V Leiden R506Q | DNA amplification (PCR) and an automated mini-sequencing methodology, Pyrosequencing, to test for the R506Q mutation in the Factor V gene. | ||||||||
| Familial adenomatous polyposis (FAP) | APC gene that encodes a protein with 2,843 amino acids | A PCR-based assay, amplify all 15 exons of the APCAPC coding region is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. gene. Direct sequence analysis of PCR products corresponding to the entire | ||||||||
| Familial Dysautonomia (FD) | IKBKAP, a gene encoding IKB kinase complex-associated protein | DNA analysis for the 2507 +6 T to C and R696P mutations by PCR and allele-specific oligonucleotide (ASO) hybridization | ||||||||
| Familial hypercholesterolemia (FH) | LDLR gene on chromosome 19 |
PCR amplification of the promoter region and exons contained in the LDLR gene coding region. Direct sequencing of amplification products is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. |
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| Familial Platelet Disorder with associated Myeloid Malignancy | Exons contained in the RUNX1 gene | PCR amplification of the exons contained in the RUNX1 gene coding region. Direct sequencing of amplification products is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. | ||||||||
| Focal dermal hypoplasia (FDH) | 10 exons contained in the PORCN gene | PCR amplification of 10 exons contained in the PORCN gene coding region. Direct sequencing of amplification products is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. | ||||||||
| Fragile X syndrome | FMR-1 gene |
PCR analysis across the CGG region to determine allele size(s). This assay is particularly sensitive for identifying premutation alleles. Southern analysis with the pE5.1 probe identifies large expansions of the CGG repeat region, and determines methylation status using Eco RI and BssH II. Southern analysis with the px6 probe and PstI digestion can be used to detect smaller expansions. |
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| Friedreich ataxia (FRDA) | frataxin gene (FXN) which maps to chromosome 9q13 | PCR amplification of the 5 exons of the FXN coding region of both isoforms. Direct sequencing of amplification products is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. | ||||||||
| GALC Sequencing (KRABBE DEFICIENCY) | GALC gene | PCR amplification of the exons contained in the GALC gene coding region and the junction fragment from the common 30 kb deletion will be performed on patient genomic DNA. Direct sequencing of amplification products is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. | ||||||||
| GALNS Sequencing (Mucopolysaccharidosis IVA) | GALNS gene | PCR amplification of the exons contained in the GALNS gene coding region. Direct sequencing of amplification products is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. | ||||||||
| Gaucher Disease (GD) | the acid ß-glucosidase (GBA) gene on chromosome 1q21 |
Direct DNA analysis is performed on four mutations in the GBA gene using DNA amplification (PCR) and an allele-specific oligonucleotide hybridization methodology. The mutations tested are: N370S, IVS2+1A, 84GG, L444P. |
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| GJB6-RELATED DFNB1 HEREDITARY HEARING LOSS |
232 kb and 309 kb GJB6 gene | PCR primers were designed to amplify across the 232 kb and 309 kb GJB6 deletion regions; amplification occurs only when a deletion is present. The assay is performed as one multiplex reaction with co-amplification of GJB6 exon 1 as an internal control. | ||||||||
| Hereditary hemochromatosis (HH) | C282Y and the H63D and S65C alleles in the HFE gene. | DNA amplification (PCR) and an automated mini-sequencing methodology, Pyrosequencing, to test for the C282Y mutation, H63D and S65C alterations in the HFE gene. | ||||||||
| Hereditary nonpolyposis colorectal cancer (HNPCC) | exons of the hMLH1 and/or hMSH2 genes |
A quantitative real time PCR-based assay is used to amplify all exons of the hMLH1 and/or hMSH2 genes. The amplified products are monitored for dosage differences by real time quantitative PCR. Gene regions showing dosage variations are subsequently confirmed with additional methods which can include MLPA, long range PCR and SNP marker analysis when applicable. |
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| Huntington disease (HD) | A trinucleotide CAG repeat expansion in the huntingtin gene | PCR analysis across the CAG region to determine allele sizes. Additional PCR assays are available for unusual or complex cases. All normal size alleles and the vast majority of expanded alleles will be detected in this analysis. Southern analysis is used to identify extremely large CAG expansions (>100 repeats). | ||||||||
| Incontinentia Pigmenti | the NEMO gene (NF-kB Essential MOdulator) that maps to chromosome Xq28. | Southern analysis for the common NEMO gene deletion is performed with an exon 2 probe and a genomic HindIII restriction digest. The assay detects an 8kb deletion junction fragment and a normal 12kb band. | ||||||||
| Kennedy Disease | androgen receptor gene at Xq11-q12 | PCR analysis across the CAG region of the androgen receptor to determine allele size(s). All normal and expanded alleles will be detected by this assay. | ||||||||
| Lesch-Nyhan syndrome (LNS) | 9 exons of the HPRT gene | A PCR-based assay is used to amplify all 9 exons of the HPRT gene. Direct sequencing of amplification products is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. | ||||||||
| Li–Fraumeni syndrome (LFS) | 11 exons contained in the TP53 gene coding region |
PCR amplification of 11 exons contained in the TP53 gene coding region. Direct sequencing of amplification products is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. |
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| LOWE SYNDROME, OCRL Sequencing | OCRL1 gene at chromosome Xq25-q26 | Genomic DNA from this individual was used for PCR amplification of all exons contained in the coding region of the OCRL1 gene. Primers for these exons flank the respective portions of the OCRL1 coding region and the appropriate intron/exon junctions. Direct sequence analysis of PCR products corresponding to the entire OCRL1 gene coding region is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. | ||||||||
| MTHFR VARIANT ANALYSIS | methylenetetrahydrofolate reductase (MTHFR) gene 677 C-T variant (leading to an alanine to valine substitution) | DNA amplification (PCR), primer extension, and allele resolution by Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) Mass Spectrometry to test for the 677 C-T variant in the MTHFR gene. | ||||||||
| Mucopolysaccharidosis I (MPS1) - IDUA Sequencing | IDUA gene coding region | PCR amplification of the exons contained in the IDUA gene coding region. Direct sequencing of amplification products is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. | ||||||||
| i duronate sulfatase(IDS) gene | PCR amplification of the exons contained in the IDS gene coding region. Direct sequencing of amplification products is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. | |||||||||
| MYH Associated Polyposis | Y165C and G382D in the MYH gene | DNA amplification (PCR) and an automated mini-sequencing methodology, Pyrosequencing, to test for the Y165C and G382D mutations in the MYH gene | ||||||||
| Myotonic dystrophy | unstable polymorphic trinucleotide CTG repeat. | PCR analysis across the CTG repeat region to determine allele size(s). This assay is particularly sensitive for identifying alleles between 30-80 repeats. Southern analysis: The MDY1 probe from the myotonin protein kinase gene is used to identify large expansions of the CTG repeat region with Nco I or BamHI digested DNA. | ||||||||
| Nail-Patella syndrome (NPS) | 8 exons of the LMX1B gene | A PCR-based assay is used to amplify all 8 exons of the LMX1B gene. Direct sequencing of amplification products is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. | ||||||||
| Niemann-Pick disease (NPD) type A | The gene encoding ASM, mapped to the chromosomal location 11p15.1-15.4 | Direct DNA analysis is performed on 3 different mutations in the ASM gene using DNA amplification (PCR) and an allele-specific oligonucleotide hybridization methodology. The mutations tested are: R496L, L302P, fsP330 | ||||||||
| Niemann-Pick Disease Type C | gene encoding for NPC1 or NPC2. | PCR amplification of the exons contained in the NPC1 and NPC2 gene coding region will be performed on patient genomic DNA. Direct sequencing of amplification products is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. | ||||||||
| NOONAN SYNDROME PTPN11 Sequencing |
PTPN11 gene on chromosome 12q24.1 and encodes the non-receptor protein tyrosine phosphatase SHP-2. |
The PTPN11 gene coding region which contains fifteen exons is amplified by PCR, and direct sequencing is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods in a capillary electrophoresis format. |
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| NOONAN SYNDROME KRAS Sequencing |
KRAS gene | PCR amplification of the exons contained in the KRAS gene coding region. Direct sequencing of amplification products is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. | ||||||||
| NOONAN SYNDROME SOS1 Sequencing |
SOS1 gene coding region | PCR amplification of the exons contained in the SOS1 gene coding region. Direct sequencing of amplification products is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. | ||||||||
| NOONAN SYNDROME RAF1 Sequencing |
RAF1 gene coding region | PCR amplification of the exons contained in the RAF1 gene coding region will be performed on patient genomic DNA. Direct sequencing of amplification products is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. | ||||||||
| Osteogenesis imperfecta (CRTAP Sequencing) | CRTAP gene on chromosome 3 | PCR amplification of seven exons contained in the CRTAP gene coding region. Direct sequencing of amplification,forward and reverse directions using automated fluorescence dideoxy sequencing methods using capillary electrophoresis. | ||||||||
| OSTEOGENESIS IMPERFECTA, (LEPRE1 (P3H1) Sequencing) | The prolyl 3- hydroxylase 1 (P3H1) encoded by LEPRE1 gene on chromosome 1p34. | PCR amplification of fourteen exons contained in the LEPRE1 gene coding region. Direct sequencing of amplification products is performed in both forward and reverse directions using automated fluorescence dideoxy sequencing methods using capillary electrophoresis | ||||||||
| PELIZAEUS-MERZBACHER DISEASE (PMD) | PLP1 gene, which encodes proteolipid protein 1 | PCR amplification of seven exons contained in the PLP1 gene coding region. Direct sequencing of amplification products is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. | ||||||||
| Prader-Willi syndrome (PWS) | 15q11-q13 chromosome | Direct detection involves Southern blot analysis to identify the origin of mutation by detecting differences in methylation within the PWS critical region using the methylation sensitive SNRPN probe | ||||||||
| PROTHROMBIN (FACTOR II) | 20210 G-A mutation in the prothrombin (Factor II) gene | DNA amplification (PCR), primer extension, and allele resolution by Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) Mass Spectrometry to test for the 20210 G-A mutation in the Prothrombin (Factor II) gene. | ||||||||
PTEN-RELATED DISORDERS |
PTEN gene coding region |
PCR amplification of the core promoter region (approximately from c.-700 to c.-1300) and exons contained in the PTEN gene coding region. Direct sequencing of amplification products is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. |
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RETT SYNDROME |
MECP2 exons 1 |
DNA Sequencing of MECP2 exons 1 through 4 in both directions using a 96-capillary sequencer. |
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RhD GENOTYPING |
RhD locus on chromosome 1p36 encode the RhD antigen (the RhD gene) |
PCR amplification of the RhD gene specific sequence with co-amplification of the RhCE sequence as an internal control to determine the presence or absence of RhD. |
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Rickets-Alopecia Syndrome |
VDR gene is located on chromosome 12q13.11 |
PCR amplification of the exons contained in the VDR gene coding region will be performed on patient genomic DNA. Direct sequencing of amplification products is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. |
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Rothmund–Thomson syndrome (RTS) |
the RECQL4 gene on chromosome 8q24.3 |
PCR amplification of 21 exons and introns contained in the RECQL4 gene will performed on patient genomic DNA. Direct sequencing of amplification products is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. |
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Schmid Metaphyseal Chondrodysplasia (SMCD) |
the COL10A1 gene for collagen X |
A PCR-based assay is used to amplify all 3 exons of the COL1OA1 gene. Direct sequencing of amplification products is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. | ||||||||
| SICKLE CELL DISEASE |
the b-globin gene on chromosome 11p |
Direct DNA analysis is performed on two mutations in the b-globin gene using DNA amplification (PCR) and Matrix Assisted Laser Desorption Ionization-Time Of Flight (MALDI-TOF) Mass Spectrometry. The mutations tested are: E6V and E6K |
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| SPINAL MUSCULAR ATROPHY | SMN (survival motor neuron) gene on chromosome 5q |
PCR amplification is performed on exon 7 and exon 8 sequences from the SMN gene and the homologous copy gene, followed by restriction digestion of the exon 7 and 8 PCR products of the copy gene with Dral and Ddel. The undigested PCR products of the SMN gene are separated from the digested products of the copy gene by agarose gel electrophoresis. |
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| Spinocerebellar ataxia type 1 (SCA1) | the ataxin-1 gene which maps to 6p22-p23 | PCR analysis across the CAG region of the SCA1 gene to determine allele sizes. All normal sized alleles and the vast majority of expanded alleles will be detected in this analysis. Southern analysis is used to identify extremely large CAG repeat expansions. . | ||||||||
| Spinocerebellar ataxia type 10 (SCA10) | the SCA10 (also known as E46) gene that maps to chromosome 22q13-qter. | PCR analysis across the ATTCT region of the SCA10 gene to determine allele sizes. All normal sized alleles and the vast majority of expanded alleles will be detected in this analysis. Southern analysis is used to identify extremely large ATTCT repeat expansions. | ||||||||
| SRY GENE |
SRY gene | PCR amplification of SRY gene specific sequence from the Y chromosome. Sequences from the ZP3 gene (a human sperm receptor gene located on chromosome 7) is co-amplified and used as an internal control. PCR products from ZP3 are generated from DNA of both normal males and females. SRY-specific PCR products are found exclusively in normal males and abnormal females carrying an intact or derivative Y chromosome or a part of it | ||||||||
| TAY-SACHS DISEASE | 1277insTATC 1421+1G>C G269S |
Direct DNA analysis is performed for the 3 mutations using an automated high-throughput system that incorporates DNA amplification (PCR), primer extension, and allele resolution by Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) Mass Spectrometry | ||||||||
| WOLMAN DISEASE | LIPA gene coding region |
PCR amplification of the exons contained in the LIPA gene coding region will be performed on patient genomic DNA. Direct sequencing of amplification products is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. |
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| X-linked Angelman-Like Syndrome | the SLC9A6 gene, which is localized to Xq26.3 |
PCR amplification of the exons contained in the SLC9A6 gene coding region will be performed on patient genomic DNA. Direct sequencing of amplification products is performed in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. |
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| X-LINKED OCULAR ALBINISM (XLOA) |
the amplifications of all the exons individually and screening them for point mutations and deletions by full sequencing of the coding region of the XLOA gene. |
Patient DNA samples will undergo amplification of all 9 exons of the OA1 gene individually and in a multiplex format. The multiplexed PCR products are electrophoresed to detect exon deletions/duplication. In the absence of a male proband, carrier testing on high-risk females for exon deletions/ duplications has a lower estimated detection rate. The individual amplified products of samples negative for deletions/ duplications are scanned for point mutations and deletions by direct sequence analysis of PCR products which corresponds to the entire XLOA coding region in both the forward and reverse directions using automated fluorescence dideoxy sequencing methods. |