User Guide for Blood Samples Postnatal Section (Blood samples) Regional Cytogenetics Department St James’s Hospital Leeds LS9 7TF Head of section: Steve Morris Telephone: 0113 206 6566 Email: email@example.com Referral categories The main referral categories include abnormal newborns, patients with unexplained learning difficulties, dysmorphic children, investigation of infertility and recurrent miscarriage, parents of children with a known chromosome abnormality, followup from prenatal diagnosis, family followup and screening of patients and donors for IVF. Referral for Prader Willi and Angelman’s Syndrome: In addition to routine cytogenetic analysis all PraderWilli and Angelman syndrome cases are passed on to the Regional DNA laboratory to determine if there is an abnormal DNA pattern present and to check for uniparental disomy. Chromosome Breakage Syndrome: Routine and specialised cultures are prepared for these cases and more blood will be required (at least 5 ml). Conditions amenable to cytogenetic analysis include ataxia telangiectasia, Bloom’s syndrome, Fanconi’s anaemia and Nijmegen Breakage Syndrome. Please indicate on the request card which test is required. Microdeletion testing: A variety of genetic syndromes associated with specific chromosome microdeletions have been identified. Testing using an MLPA (Multiplex LigationDependent Probe Amplification) reaction for some of these syndromes (see list below) is now performed. Subtelomere testing: This is performed on patients following consultation with the Clinical Genetic’s Department, Chapel Allerton. Further clinical advice is available from the Department of Clinical Genetics, Chapel Allerton Hospital, Leeds TEST REQUIREMENTS. Chromosome analysis. Requirements: 12 mls (minimum) peripheral blood in a lithium heparin tube (green or orange top) & referral card completed with Name, NHS no., d.o.b. address and comprehensive clinical details. 1. Breakage Syndrome studies. Routine and specialised cultures are prepared for these cases and more blood will be required (at least 1.5 ml). Conditions amenable to cytogenetic analysis include ataxia telangectasia, Bloom’s syndrome, Fanconi’s anaemia and Nijmegen Breakage Syndrome. Please indicate on the request card which test is required. Requirements: 1.5mls (minimum) of peripheral blood in a lithium heparin tube (green or orange top). Sample Tubes Blood should be collected in a lithium heparin tube (orange or green top), and mixed well to prevent clotting. It is important to ensure that the sample tube contains lithium heparin irrespective of the colour of the tube top. Preferred volumes of blood Adults 5ml Children 25ml Infants 12ml More blood may be required if chromosome breakage studies are required. Blood in EDTA will also be accepted, however many bloods sent in EDTA do not grow and those that do often require a repeat sample. Blood in other containers is not suitable for culture. Clotted blood is unsuitable for cytogenetic analysis. Sample Transport • Samples should be addressed to: Cytogenetics Unit, St. James’s Hospital Beckett Street, Leeds LS9 7TF • Cytogenetic analysis requires living cells. Please ensure that the sample reaches us as quickly as possible (within 48 hours). First class post is satisfactory for nonurgent samples. • Urgent samples should be sent by courier or taxi. • Samples should not be frozen, exposed to excess heat or stored in formalin. • If there is a delay in transit please store the sample at 4°C (in a refrigerator). • Please try to avoid sending samples at weekends or bank holidays as post is not delivered on these days. ALL PACKAGES MUST CONFORM TO POST OFFICE REGULATIONS (Copies are available from the Post Office) Urgent Samples Cases treated as urgent are all new borns, cordocentesis specimens, pregnant couples including follow up of prenatal diagnosis, children awaiting surgery and others cases as discussed with the laboratory. Reporting of Results • Urgent cases. Usually within 57 days which is within the national guidelines. An aneuploidy screen can often be done overnight as a Fluorescent in situ Hybridisation (FISH) test (please contact the laboratory). • Other cases. We attempt to report within the national guidelines which are within 28 days. If results are required earlier please contact the lab. • Complex abnormalities, or abnormalities that are difficult to interpret, may require FISH to resolve the karyotype, this may delay some results. • Results are sent to the referring clinician. Complex abnormal results are usually telephoned prior to the written report being sent and the interpretation and implication discussed. Abnormal results from urgent cases will be telephoned to the referring clinician. • In response to telephone enquiries, only normal results or those which confirm a previous finding are given to a clinicians’ secretary or the clinic sister. All other results are only given to clinicians. * Please note that FISH testing might be performed if the sample received is suboptimal (sent without an EDTA sample or too small (<1.0mls)) or where it is considered by the lab to be the most cost effective approach. 1. MLPA microdeletion studies. Requirements: 12 mls (minimum) of peripheral blood in a lithium heparin tube (green or orange top) AND 12 mls of peripheral blood in a EDTA tube (purple top) & referral card completed with Name, NHS no., d.o.b. address and comprehensive list of clinical details. 2. MLPA subtelomere studies. Referrals for this test must be arranged through the Clinical Genetics Department, Chapel Allerton Hospital, Leeds. Requirements: 12 mls (minimum) of peripheral blood in a lithium heparin tube (green or orange top) AND 12 mls of peripheral blood in a EDTA tube (purple top) & referral card completed with Name, NHS no., d.o.b. address and comprehensive list of clinical details. Please note this test will only detect unbalanced rearrangements. Summary of Microdeletion syndromes tested for by the P245 MLPA microdeletion kit Loci Tested Associated Microdeletion/duplication Syndrome 1p36.33 1p36 Deletion syndrome 2p16.1 2p16 Deletion syndrome 3q29 3q29 Deletion syndrome 4p16.3 WolfHirschhorn syndrome 5p15.33 CriduChat syndrome 5q35.3 SOTOS syndrome 7q11.2 Williams syndrome/7q11.23 duplication 8p23.1 8p23 Deletion syndrome 8q24 LangerGideon syndrome 9q22.33 9q22 Deletion syndrome 10p14 DiGeorge syndrome region 2 11p13 WAGR syndrome 15q11.2 PraderWilli/Angelmans syndrome 15q24.1 15q24 Deletion syndrome 16p13.3 RubinsteinTaybi syndrome 17p11.2 SmithMagenis syndrome/17p11.2 duplication 17p13.3 MillerDieker syndrome 17q11.2 NF1 Deletion 17q21.31 17q21 Deletion syndrome 22q11.2 DiGeorge syndrome/22q11.2 duplication 22q13.3 PhelanMcDermid syndrome Xq28 MECP2 duplication syndrome Technical details of P245 kit (Base positions based on Human GRCh37 Assembly hg19) General Reference: https://decipher.sanger.ac.uk DiGeorge (22q11.2) 3 probes for 22q11.2: CLDN5 at 19.51Mb 1.5Mb deletion GP1BB at 19.71Mb 3 Mb deletion SNAP29 at 21.24Mb Sensitivity: ~96% patients carry either the 1.5Mb or 3Mb deletion and will be detected using this assay. Mutations of TBX1 have also been described. Reference: Lancet. 362:13661373, 2003. Digeorge Region 2 (10p14) 2 probes for 10p14: GATA3 at 8.10Mb DKFZp566L0824 at 10.548966Mb Sensitivity: 2 Critical region s :– 1. DGCR2 with cardiac defect and T cell deficiency is defined as between D10S547 (10.550410Mb) and D10S585 (11.25Mb). 2. HDR1 with hypoparathyroidism, deafness and renal dysplasia is defined as between D10S189 (6.72Mb) and D10S226 (8.92Mb). Whilst the DKFZp566L0824 probe lies outside the defined critical region it will detect the majority of cases as only one published case has a deletion not covered by this probe. The GATA3 probe lies within the HDR1 critical region and therefore will detect all deletions associated with HDR. References: European Journal of Human Genetics. 6, 213–225, 1998. J Med Genet. 37:3337, 2000. J Mol Med. 80:431–442, 2002. 1p36 Deletion Syndrome (1p36.33) 3 probes: TNFRSF4 at 1.15Mb GNB1 at 1.76Mb GABRD at 1.96Mb Sensitivity: Will detect up to 98% of 1p36 deletions Reference: Am. J. Hum. Genet. 72:1200–1212, 2003 2p16.1 Deletion Syndrome (2p16.1) 2 probes: FANCL at 58.45Mb REL at 61.15Mb. Common region spans RP11426N8 (57.68Mb) and RP11470L12 (61.58Mb). Sensitivity: Will detect all occurrences of 2p16.1 deletion described in the literature. Reference: J. Med. Genet. 45:122124, 2008 3q29 Deletion Syndrome (3q29) 2 probes: DGL1 at 196.79Mb DGL1 at 197.02Mb Common deletion and duplication spans 195.92Mb to 197.30Mb. Other cases of duplication described are bigger and cover this region. Sensitivity: Will detect all reported occurrences of 3q29 deletion/duplication. Reference: Am. J. Hum. Genet. 77:154–160, 2005 Cytogenet Genome Res. 123:65–78, 2008 WHS 4p16.3 2 probes: LETM1 at 1.84Mb WHSC1 at 1.93Mb. Sensitivity: The majority of WolfHirschhorn deletions are terminal and will be detected by this assay. A small number of interstitial deletions may not be detected by these probes. References: Am. J. Hum. Genet. 72:590 –597, 2003 J Med Genet. 45:7180, 2008. Cri du Chat Syndrome (5p15.3) 2 probes: TERT at 1.28Mb CRR9p at 1.34Mb Sensitivity: ~91.25% deletions are terminal and will be detected using these probes. ~8.75% patients have been described with an interstitial deletion, which will not be picked up by this assay. References: J Med Genet. 38:151–158, 2001 Am. J. Hum. Genet. 76:312–326, 2005 Sotos Syndrome (5q35.3) 2 probes: NSD1 Exon17 at 176.68Mb NSD1 Exon 22 at 176.72Mb Sensitivity: 1015% of NSD1 abnormalities in Europe and US are microdeletions (>50% in Japan), which will be detected with this assay. Intragenic mutations ac count for the remainder of patients. References: Am. J. Hum. Genet. 77:193–204, 2005 European Journal of Human Genetics. 15, 264–271, 2007 Williams Syndrome (7q11.23) 3 probes: ELN at 73.44Mb ELN at 73.47Mb LIMK1 at 73.51Mb Sensitivity: Up to 94% caused by deletion of ELN, which will be detected by this assay. Balanced rearrangements with breakpoints within ELN may account for other patients, which would be detected by Gbanding. References: Human Molecular Genetics. Vol. 12, Review Issue 2, 2003. Am. J. Hum. Genet. 59: 781792, 1996. LangerGiedion Syndrome (8q24) 2 probes: TRPS1 at 116.68Mb EIF3S3 at 117.66Mb Sensitivity: Phenotype in all cases is due to deletion of both TRPS1 and EXT1 genes, which will be detected by this assay. There is one report of a deletion that does not encompass TRPS1 but does cover EIF3S3. Reference: Am J Med Genet A 146A:1587–1592, 2008 9q22.3 Deletion Syndrome (9q22.3) 2 probes: TGFBR1 at 101.908Mb TGFBR1 at 101.910Mb Sensitivity: All deletions described encompass the TGFBR1 gene. Therefore all cases described in literature will be detected by this assay. References: European Journal of Human Genetics. 14, 759– 767, 2006 WAGR Syndrome (11p13) 1 probe: PAX6 at 31.82Mb. Sensitivity: ~66.6% patients with WAGR carry a deletion of PAX6 which will be detected with this probe. Due to the lack of a probe for WT1, FISH analysis is also recommended. Reference: Am. J. Hum. Genet. 71:1138–1149, 2002 PraderWilli/Angelman Syndrome (15q11.2) 4 probes: NDN at 23.93Mb SNRPN at 25.10Mb SNRPN at 25.21Mb UBE3A at 25.62Mb. Sensitivity: 7075% PraderWilli patients carry a deletion, which will be detected by this assay. The remaining cases are caused by maternal UPD15 or an imprinting defect. ~70% Angelman patients carry a deletion, which will be detected by this assay. The remaining cases are caused by paternal UPD15 or an imprinting effect. In all suspected cases of PraderWilli/Angelman syndrome molecular genetic testing is recommended. 15q24.1 Deletion Syndrome (15q24.1) 2 probes: SEMA7A at 74.71Mb CYP1A1 at 75.01Mb. Sensitivity: The minimal deleted region for this syndrome spans ~74.5076.00Mb therefore these probes will detect all described occurrences of this deletion. Reference: Human Molecular Genetics . 16(5):567–572, 2007. RubinsteinTaybi Syndrome (16p13.3) 1 probe: CREBBP at 3.93Mb (Exon 1) Sensitivity: Deletions of CREBBP account for ~10% RSTS cases. Partial deletions of CREBBP are common and this probe will only detect ~50% deletions. Intragenic mutations of CREBBP and mutations of EP300 accounting for the vast majority of cases. This assay cannot exclude a diagnosis of RSTS. Reference: J. Med. Genet. 37:168176, 2000 . MillerDieker Syndrome (17p13.3) 2 probes: PAFAH1B1 (LIS1) at 2.568Mb PAFAH1B1 (LIS1) at 2.570Mb Sensitivity: All cases of MillerDieker show deletions which encompass PAFAH1B1 along with other genes. These MLPA probes will therefore detect all cases of MillerDieker syndrome. All known duplications of this regions also encompass PAFAH1B1 and will be detected with this assay. References: Am. J. Hum. Genet. 72:918–930, 2003 J Med Genet. 46:703710, 2009. SmithMagenis Syndrome (17p11.2) 3 probes: RAI1 at 17.58Mb LRRC48 at 17.89Mb LLGL1 at 18.14Mb Sensitivity: ~90.5% patients carry a deletion which encompasses RAI1. Duplication sizes are reciprocal. This assay will detect all deletions and duplications of this region. ~9.5% patients carry an intragenic mutation of RAI1, which will not be detected. Reference: Clin Genet. 71: 540–550, 2007. NF1 (17q11.2) 2 probes: NF1 at 29.53Mb NF1 at 29.55Mb Sensitivity: 520% NF1 patients carry a microdeletion, which encompasses NF1, these will be detected with this assay. The remainder of patients carry a point mutation of NF1. Reference: Am. J. Hum. Genet. 75:410–423, 2004 17q21.31 Deletion Syndrome (17q21.31) 3 probes: CRHR1 at 43.91Mb MAPT at 44.09Mb MAPT at 44.10Mb Sensitivity: Critical deletion region encompasses MAPT gene. This assay will detect all described occurrences of this deletion. Reference: J Med Genet. 45(11):71020, 2008. PhelanMcDermid Syndrome (22q13) 2 probes: SHANK3 at 51.14Mb SHANK3 at 51.16Mb Sensitivity: Critical deletion region encompasses 3’ end of SHANK3 gene. This assay will detect all known cases of this deletion. Some intragenic mutations of SHANK3 have been described associated with autistic spectrum disorder. References: Hum Genet. 110 :439–443, 2002. Nat Genet. 39(1): 25–27, 2007. MECP2 Duplication Syndrome (Xq28) 3 probes: MECP2 at 153.290Mb MECP2 at 153.296Mb MECP2 at 153.364Mb Sensitivity: Minimum duplicated region encompasses entire MECP2 gene and will therefore be detected by this assay. Reference: Am. J. Hum. Genet. 77:442–453, 2005. 8p23.1 2 probes: TNKS at 9.44Mb GATA4 at 11.57Mb Sensitivity: Commonly deleted region spans ~8.9211.48Mb, therefore these probes will detect the common deletion. Mutations of GATA4 have also been implicated with heart defects. Reference: Am. J. Hum. Genet. 64:1119–1126, 1999. Distal 22q11.2 2 probes: PPIL2 at 22.05Mb SMARCB1 at 24.14Mb Sensitivity: Abnormalities in this region have not been well characterised. Studies with MLPA have shown the majority of abnormalities in this region to encompass PPIL2 and sometimes SMARCB1. Reference: HUMAN MUTATION. 29(3):433440, 2008. Explanation of riders on reports. 1. The P245 microdeletion kit from MRCHolland (www.mpla.com) contains probes to detect deletions and duplications of the following regions or syndromes: 1p36; 2p16; 3q29; WolfHirschhorn; Criduchat; Sotos; Williams; LangerGideon; 9q22; 10p14; WAGR; PraderWilli/Angelman Syndrome; 15q24; RubinsteinTaybi; SmithMagenis; MillerDieker; NF1; 17q21; DiGeorge; 22qter; Xq28 (MECP2). The kit contains more than 40 separate probes and is designed to aid in the diagnosis of 24 recognised microdeletion syndromes. Hence, the kit contains several probes for each locus. 2. Modifications of the kit enable the detection of deletions and duplications of distal 22q11.2 and 8p23.1. At the request of our clinicians and in conjunction with MRC Holland, we have modified the P245 kit to include probes for 22q11.2 and 8p23.1. 3. This test is unlikely to detect small intragenic mutations, which account for a proportion of some of the above mentioned syndromes. The kit is designed to assist in the diagnosis of microdeletion syndromes, however, a normal result does not mean the patient does not have the queried syndrome e.g. 70% of PraderWilli syndrome have a deletion detectable by the P245 kit – the remaining 30% of patients would not be detected by this kit. This point should be reflected in the text of the report. 4. MLPA is unable to detect mosaicism at levels below 50%. The presence of mosaicism might go undetected by the test as the presence of normal cells can mask the presence of abnormal cells. 3. MLPA is unable to detect balanced rearrangements. The technique relies on comparing quantities of DNA from a patient against a control. A patient with a balanced rearrangement has a change in the order of their DNA but not a change in quantity therefore patients with balanced rearrangements are not detected by the test.
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