The number of protein-coding genes within the human genome remains a subject of active investigation. A 2012 analysis of the human genome based on in vitro gene expression in multiple cell lines identified 20,687 protein-coding genes. Although the estimate of the number of protein genes has varied widely, ranging up to 2,000,000 in the late 1960s, several researchers pointed out in the early 1970s that the estimates of mutational load from deleterious mutations placed an upper limit of approximately 40,000 for the total number of functional loci (this includes protein-coding and functional non-coding genes).