Assisted reproduction mediated resurrection of a feline model for Chediak-Higashi syndrome caused by a large duplication in LYST

Abstract

chediak-Higashi Syndrome (cHS) is a well-characterized, autosomal recessively inherited lysosomal disease caused by mutations in lysosomal trafficking regulator (LYST). the feline model for cHS was originally maintained for ~20 years. However, the colonies were disbanded and the CHS cat model was lost to the research community before the causative mutation was identified. To resurrect the cat model, semen was collected and cryopreserved from a lone, fertile, cHS carrier male. Using cryopreserved semen, laparoscopic oviductal artificial insemination was performed on three queens, two queens produced 11 viable kittens. To identify the causative mutation, a fibroblast cell line, derived from an affected cat from the original colony, was whole genome sequenced. Visual inspection of the sequence data identified a candidate causal variant as a ~20 kb tandem duplication within LYST, spanning exons 30 through to 38 (NM_001290242.1:c.8347-2422_9548 + 1749dup). PCR genotyping of the produced offspring demonstrated three individuals inherited the mutant allele from the CHS carrier male. this study demonstrated the successful use of cryopreservation and assisted reproduction to maintain and resurrect biomedical models and has defined the variant causing Chediak-Higashi syndrome in the domestic cat. Chediak-Higashi syndrome (CHS) (OMIM Accession: 214500) is a rare autosomal recessive disorder characterized in humans by severe immune deficiency, oculocutaneous albinism, bleeding tendencies, recurrent pyogenic infections, progressive neurologic defects and a lymphoproliferative syndrome. The most common cause of death from CHS is from recurrent infections or the development of an accelerated phase with hemophagocytic lymphohistiocytosis. Approximately 90% of deaths occur in the first decade of life, and those who survive into adulthood develop progressive neurological symptoms 1. The disease was first described in the 1940s to early 1950s 2-6 and has been characterized in a host of diverse species, including cow 7-10 , mink 10-14 , killer whale 15,16 , fox 17,18 and domestic cat 19-23 (OMIA: 000185-9913, 9733, 494514, 452646, 9685). Continuing studies of CHS models have demonstrated their value in deciphering delta storage pool deficiencies 24 , heritable platelet disorders 25,26 and cellular cytotoxicity 27. Bone marrow transplantation has been a viable option for management of human CHS for over 30 years 28-31. The genetic cause for CHS was first defined in rodents 32-36 , with the locus historically known as beige due to the associated hypopigmentation phenotype 33. The human homolog of the mouse beige locus revealed the first causative mutations for CHS in humans 37,38 and was defined as lysosomal trafficking regulator (LYST) 37. In humans, LYST encodes a 3,801 amino acid protein (11.4 kb transcript), which regulates intracellular protein trafficking to and from the lysosome (GCID:GC01M235824). In many species with CHS, mutations have been

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