Title 1 Pathogenic variants causing ABL 1 malformation syndrome cluster in a myristoyl-2 binding pocket and increase tyrosine kinase activity 3 4 Running title 5 ABL 1 pathogenic variants 6

33 ABL1 is a proto-oncogene encoding a nonreceptor tyrosine kinase, best known in the somatic 34 BCR-ABL fusion gene associated with chronic myeloid leukaemia. Recently, germline 35 missense variants in ABL1 have been found to cause an autosomal dominant developmental 36 syndrome with congenital heart disease, skeletal malformations and characteristic facies. 37 Here, we describe a series of six new unrelated individuals with heterozygous missense 38 variants in ABL1 (including four novel variants) identified via whole exome sequencing. All 39 the affected individuals in this series recapitulate the phenotype of the ABL1 developmental 40 syndrome and additionally we affirm that hearing impairment is a common feature of the 41 condition. Four of the variants cluster in the myristoyl-binding pocket of ABL1, a region 42 critical for auto-inhibitory regulation of the kinase domain. Bio-informatic analysis of 43 transcript-wide conservation and germline/somatic variation reveals that this pocket region is 44 subject to high missense constraint and evolutionary conservation. Functional work to 45 investigate ABL1 kinase activity in vitro by transient transfection of HEK293T cells with 46 variant ABL1 plasmid constructs revealed increased phosphorylation of ABL1-specific 47 substrates compared to wild-type. The increased tyrosine kinase activity was suppressed by 48 imatinib treatment. This case series of six new patients with germline heterozygous ABL1 49 missense variants further delineates the phenotypic spectrum of this condition and recognises 50 microcephaly as a common finding. Our analysis supports an ABL1 gain-of-function 51 mechanism due to loss of auto-inhibition, and demonstrates the potential for pharmacological 52 inhibition using imatinib. 53