MLPA SAMPLES USER GUIDE

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1 MLPA SAMPLES USER GUIDE MLPA microdeletion studies Requirements: 1-2 mls (minimum) of peripheral blood in a lithium heparin tube (green or orange top) AND 1-2 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. MLPA sub-telomere studies Referrals for this test must be arranged through the Clinical Genetics Department, Chapel Allerton Hospital, Leeds. Requirements: 1-2 mls (minimum) of peripheral blood in a lithium heparin tube (green or orange top) AND 1-2 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. 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. Summary of Microdeletion syndromes tested for by the P245 MLPA microdeletion kit Loci Tested Associated Microdeletion/duplication Syndrome 1p p36 Deletion syndrome 2p16.1 2p16 Deletion syndrome 3q29 3q29 Deletion syndrome 4p16.3 Wolf-Hirschhorn syndrome 5p15.33 Cri-du-Chat syndrome 5q35.3 SOTOS syndrome 7q11.2 Williams syndrome/7q11.23 duplication 8p23.1 8p23 Deletion syndrome 8q24 Langer-Gideon syndrome 9q q22 Deletion syndrome 10p14 DiGeorge syndrome region 2 11p13 WAGR syndrome 15q11.2 Prader-Willi/Angelmans syndrome 15q q24 Deletion syndrome 16p13.3 Rubinstein-Taybi syndrome 17p11.2 Smith-Magenis syndrome/17p11.2 duplication 17p13.3 Miller-Dieker syndrome 17q11.2 NF1 Deletion 17q q21 Deletion syndrome 22q11.2 DiGeorge syndrome/22q11.2 duplication 22q13.3 Phelan-McDermid syndrome Xq28 MECP2 duplication syndrome

2 Technical details of P245 kit (Base positions based on Human GRCh37 Assembly hg19) General Reference: DiGeorge (22q11.2) 3 probes for 22q11.2: CLDN5 at 19.51Mb GP1BB at 19.71Mb 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: , DiGeorge Region 2 (10p14) 2 probes for 10p14: GATA3 at 8.10Mb Sensitivity: DKFZp566L0824 at Mb 2 Critical regions: 1. DGCR2 with cardiac defect and T cell deficiency is defined as between D10S547 ( Mb) 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, , J Med Genet. 37:33-37, J Mol Med. 80: , p36 Deletion Syndrome (1p36.33) 3 probes: TNFRSF4 at 1.15Mb GNB1 at 1.76Mb GABRD at 1.96Mb Sensitivity: These probes will detect all deletions that fall within the 1p36 microdeletion region as defined by Decipher; however some patients have been described that have deletions of 1p36 that fall outside this region. References: Am. J. Hum. Genet. 72: , p16.1 Deletion Syndrome (2p16.1) 2 probes: FANCL at 58.45Mb REL at 61.15Mb. Common region spans RP11-426N8 (57.68Mb) and RP11-470L12 (61.58Mb). Sensitivity: Will detect all occurrences of 2p16.1 deletion described in the literature. Reference: J. Med. Genet. 45: , q29 Deletion Syndrome (3q29) 2 probes: DGL1 at Mb DGL1 at Mb Common deletion and duplication spans Mb to Mb. 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: , 2005 Cytogenet Genome Res. 123:65 78, 2008

3 WHS 4p probes: LETM1 at 1.84Mb WHSC1 at 1.93Mb. Sensitivity: The majority of Wolf-Hirschhorn 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: , 2003 J Med Genet. 45:71-80, 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: , 2001 Am. J. Hum. Genet. 76: , 2005 Sotos Syndrome (5q35.3) 2 probes: NSD1 Exon17 at Mb NSD1 Exon 22 at Mb Sensitivity: 10-15% 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: , 2005 European Journal of Human Genetics. 15, , 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 G-banding. References: Human Molecular Genetics. Vol. 12, Review Issue 2, Am. J. Hum. Genet. 59: , Langer-Giedion Syndrome (8q24) 2 probes: TRPS1 at Mb EIF3S3 at Mb 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: , q22.3 Deletion Syndrome (9q22.3) 2 probes: TGFBR1 at Mb TGFBR1 at Mb 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, , 2006 WAGR Syndrome (11p13) 1 probe: PAX6 at 31.82Mb.

4 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: , 2002 Prader-Willi/Angelman Syndrome (15q11.2) 4 probes: NDN at 23.93Mb SNRPN at 25.10Mb SNRPN at 25.21Mb UBE3A at 25.62Mb. Sensitivity: 70-75% Prader-Willi 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 Prader-Willi/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 ~ Mb therefore these probes will detect all described occurrences of this deletion. Reference: Human Molecular Genetics. 16(5): , Rubinstein-Taybi 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: , Miller-Dieker Syndrome (17p13.3) 2 probes: PAFAH1B1 (LIS1) at 2.568Mb PAFAH1B1 (LIS1) at 2.570Mb Sensitivity: All cases of Miller-Dieker show deletions which encompass PAFAH1B1 along with other genes. These MLPA probes will therefore detect all cases of Miller-Dieker syndrome. All known duplications of this regions also encompass PAFAH1B1 and will be detected with this assay. References: Am. J. Hum. Genet. 72: , 2003 J Med Genet. 46: , Smith-Magenis 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: , NF1 (17q11.2) 2 probes: NF1 at 29.53Mb NF1 at 29.55Mb

5 Sensitivity: 5-20% NF1 patients carry a micro-deletion, 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: , q21.31 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):710-20, Phelan-McDermid 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 : , Nat Genet. 39(1): 25 27, MECP2 Duplication Syndrome (Xq28) 3 probes: MECP2 at Mb MECP2 at Mb MECP2 at Mb Sensitivity: Minimum duplicated region encompasses entire MECP2 gene and will therefore be detected by this assay. Reference: Am. J. Hum. Genet. 77: , p probes: TNKS at 9.44Mb GATA4 at 11.57Mb Sensitivity: Commonly deleted region spans ~ Mb, therefore these probes will detect the common deletion. Mutations of GATA4 have also been implicated with heart defects. Reference: Am. J. Hum. Genet. 64: , Distal 22q 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): , 2008.