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Thrombotic Microangiopathy Sequencing Panels

The fifteen genes evaluated in this panel include: ADAMTS13, C3, CD46, CFB, CFH, CFHR1, CFHR2, CFHR3, CFHR4, CFHR5, CFI, DGKE, MMACHC, PLG and THBD

Background:

The term Thrombotic Microangiopathy (TMA) describes the pathologic changes of endothelial injury, and subsequent thrombosis and microvascular occlusion. Additional laboratory findings include low haptoglobin levels, elevated LDH and bilirubinemia. There are multiple acquired and inherited causes for TMA. The latter include mutations in alternative complement pathway components or regulators that clinically manifest as atypical hemolytic uremic syndrome (aHUS) or thrombotic thrombocytopenic purpura (TTP). These genetic alterations render the patient at risk for unregulated complement activation due to environmental triggers.1

aHUS is a relatively rare subtype of complement mediated TMA which clinically manifests as acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia (schistocytes on peripheral blood smear).  It can occur in both children and adults, and extra-renal involvement is common; including neurologic, cardiac, and gastrointestinal organ systems.2  Gene mutations have been identified in approximately 60% of aHUS patients.3 These mutations may involve genes encoding complement cascade proteins and/or other non-complement proteins, and increase the risk of over-active and/or prolonged activation of the alternative complement pathway due to impaired homeostasis (ability to maintain or return to normal low level activity from fully amplified state).  In a recent review variants in CFH gene were identified in 25-27% of sporadic and 40% of familial cases; CFH family member genes CFHR5 <1 – 4% and CHFR1CHFR4 3-10%; MCP (CD46) 5-9%; CFI 4-8%; C3 2-8%; CFB <1 – 4%; THBD 5%; Mutations in non-complement genes include DGKE <1%, and PLG (plasminogen), were also identified. However, in up to 30% of aHUS patients, no causative genetic abnormality was identified.4  Secondary aHUS may occur in the setting of other acquired TMAs due to an underlying genetic predisposition in the patient for overactive or persistent activation of the alternative complement pathway.  Thus, secondary aHUS may be seen in the context of primary TMA due to thrombotic thrombocytopenia (TTP), malignancy, autoimmune disease, infection, drugs, pregnancy (eclampsia, pre-eclampsia, and HELP syndrome), malignant hypertension, and transplantation.

Inherited non-autoantibody associated thrombotic thrombocytopenic purpura (TTP) is caused by homozygous or compound heterozygous mutations in ADAMTS13 gene.  Its clinical spectrum ranges from asymptomatic to life threatening in severity, and from neonatal to adult disease in regards to age of onset. The ADAMTS13 protein is responsible for cleavage of von Willebrand factor (VWF). Deficiency of ADAMTS13 leads to recurrent episodes of hemolytic anemia and widespread platelet-rich microvascular thrombosis within capillaries and arterioles of multiple organs, including brain, heart, kidney, pancreas and mesentery.5

Indications for testing:

Renal biopsy with morphologic and/or clinical features of TMA, including:

Atypical hemolytic uremic syndrome (aHUS), primary and secondary forms

Thrombotic thrombocytopenic purpura (TTP)

Transplant associated microangiopathy (TAM) following allogeneic stem cell transplantation

Glomeruli with fibrin thrombi and mesangiolysis

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Test Details:

Genes in this panel: C3, C8A, CD46 (MCP), CFB, CFH, CFHR1, CFHR2, CFHR3, CFHR4, CFHR5, and CFI

Methods:

Next Generation Sequencing (NGS)

Direct/Sanger Sequencing (Big Dye Terminator)

Performance:

Coverage: >99.8% of targeted areas (exons +/- 10bp to include splice junctions)

Depth: 98.7% at >20x (average 210)

Frequency:  Can be performed bi-weekly (depending on receipt of samples to be tested)

Turn around time: 3 months

Cost of test:  $2500.00

Specimen Requirements:

Peripheral blood:  At least 2ml in lavender top (EDTA) or DNA PaxGene tube

Extracted DNA (quantity): 100ng total extracted

Shipping Instructions:

Peripheral blood: Ship in a protective container at room temperature.

DNA: Ship in a protective container at room temperature.

Use Arkana Laboratories FedEx number to pay for the shipping charges (FedEx #229571460).

Attention: Arkana Laboratories Molecular Division

10810 Executive Center Drive, Suite 100

Little Rock, AR 72211

Phone: (501) 604-2695

References:

  1. Barbour T, et al. Thrombotic microangiopathy and associated renal disorders. Nephrol Dial Transplant

2012;27:2673-2685

  1. Laurence J. Atypical hemolytic uremic syndrome (aHUS): making the diagnosis. Clin Adv

Hematol Oncol 2012;10 Suppl 17:1-12

  1. Noris M, et al. Genetic Atypical Hemolytic-Uremic Syndrome. In: Pagon RA, Adam MP, Ardinger HH,

Wallace SE, Amemiya A, Bean LJH, Bird TD, Fong CT, Mefford HC, Smith RJH, Stephens K, editors.

GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2016.2007 Nov 16

[updated 2016 Jun 9].

  1. Nester CM, et al. Atypical aHUS: State of the art. Molecular Immunology 2015;67:31-42
  2. Lotta L, et al. ADAMTS13 Mutations and Polymorphisms in Congenital Thrombotic

Thrombocytopenic Purpura. Human Mutation 20010;31:11-19

Contact information:

Arkana Laboratories Molecular Division

10810 Executive Center Drive, Suite 100

Little Rock, AR 72211

Email: support@arkanalabs.com

Phone: (501) 604-2695

Fax: (501) 604-2699

 

Co-Director: Chris Larsen, MD

Co-Director: Jon D. Wilson, MD

Lab Manager: Marjorie Beggs, PhD