..............................................................Myosin gene mutationcorrelates with anatomicalchanges in the human lineageHansell H. Stedman1,3, Benjamin W. Kozyak1, Anthony Nelson1,Danielle M. Thesier2, Leonard T. Su1, David W. Low1,5, Charles R. Bridges1,Joseph B. Shrager1,3, Nancy Minugh-Purvis2,4,5& Marilyn A. Mitchell11Department of Surgery and2Cell and Developmental Biology,3the Pennsylvania Muscle Institute, School of Medicine, and4Department ofAnatomy and Cell Biology, School of Dental Medicine, University of Pennsylvania,and5Division of Plastic Surgery, The Children’s Hospital of Philadelphia,Philadelphia, Pennsylvania 19104, USA.............................................................................................................................................................................Powerful masticatory muscles are found in most primates,including chimpanzees and gorillas, and were part of a promi-nent adaptation of Australopithecus and Paranthropus, extinctgenera of the family Hominidae1,2.Incontrast,masticatorymuscles are considerably smaller in both modern and fossilmembers of Homo. The evolving hominid masticatory appar-atus—traceable to a Late Miocene, chimpanzee-like mor-phology3—shifted towards a pattern of gracilization nearlysimultaneously with accelerated encephalization in earlyHomo4. Here, we show that the gene encoding the predominantmyosin heavy chain (MYH) expressed in these muscles wasinactivated by a frameshifting mutation after the lineages leadingto humans and chimpanzees diverged. Loss of this protein iso-form is associated with marked size reductions in individualmuscle fibres and entire masticatory muscles. Using the codingsequence for the myosin rod domains as a molecular clock, weestimate that this mutation appeared approximately 2.4 millionyears ago, predating the appearance of modern human body size5and emigration of Homo from Africa6. This represents the firstproteomic distinction between humans and chimpanzees thatcan be correlated with a traceable anatomic imprint in the fossilrecord.We obtained a DNA sequence by degenerate polymerase chainreaction (PCR) that suggested the existence of a hitherto unrecog-nized human sarcomeric myosin gene (MYH16, see SupplementaryInformation). An unannotated accession file posted during thefinalization of the human genome sequence (AC112711) containedthe only exact match to the query sequence on a BLAST search.Here, we use homology to other sarcomeric myosins to annotatethis locus and generate the complete sequence of a hypotheticalhuman sarcomeric myosin distantly related to the other knownmembers of this subfamily (Fig. 1; see also Supplementary Infor-mation). This reconstruction suggested that a frameshift deletion atcodon 660 of the messenger RNA of the deduced polypeptide wasFigure 1 Molecular evolution of MYH16. a, Distribution of 42 predicted coding exonsspanning 67,983 base pairs (bp) in the region of human chromosome 7q22 flanked 50bySMURF1 and 30by ARPC1A. b, Phylogenetic reconstruction for all human sarcomericmyosin genes (heavy chain), showing early divergence of MYH16 from others. Branchlengths shown are derived from a maximum likelihood analysis of the aligned cDNAs,beginning with the conserved proline at the head–rod junction. Non-sarcomeric class IImyosins (designated MYH9, -10 and -11; data not shown) are used to root the tree.c, Aligned DNA sequences for MYH16 exon 18 representing seven non-human primatespecies and six geographically dispersed human populations, revealing the effect offrameshift on reading frame and deduced amino acid sequence. Note stop codon atposition 72–74.letters to natureNATURE | VOL 428 | 25 MARCH 2004 | www.nature.com/nature 415© 2004Nature PublishingGroupeither a rare allele or a sequencing artefact. To resolve this uncer-tainty, we sequenced this region in DNA samples from members ofgeographically disparate human populations. As shown in Fig. 1c,the mutation is found in all modern humans sampled, includingnatives of Africa, South America, Western Europe, Iceland, Japanand Russia; thus, the inactivating mutation seems to be fixed inHomo sapiens. In contrast, all of the non-human primates for whichsequence was obtained have an ACC codon that encodes a highlyconserved threonine. The frameshift in the human coding sequencetruncates the predicted 224-kDa myosin heavy chain to a 76-kDafragment containing an unstable portion of the myosin headdomain (Fig. 1c).Animals homozygous for null mutations in sarcomeric myosinheavy chain genes sometimes possess severe functional defects inindividual muscles that ordinarily accumulate the highest levels ofspecific isomyosins7,8. In reconstructing the pattern of expression ofthe MYH16 gene in a recent common ancestor, scarcity of chim-panzee tissue led us to examine transcription in a wide range ofmuscles from macaque (Macaca fascicularis) and modern humans.Cross-species sequence similarity facilitated the design and use ofseveral isoform-specific RT–PCR primers, with the finding that theamplification products uniquely contain the sequences predicted byour human MYH16 gene annotation (Fig. 2a). Transcription isdetectable only in the muscles of the head, specifically those derivedfrom the embryonic first pharyngeal arch, including temporalis andtensor veli palatini (Fig. 2b).In gross anatomical comparisons between humans versus greatapes and monkeys, the relative size of individual masticatory musclehomologues contrasts remarkably, as is evident in Fig. 3a–k. Therelative sizes of the temporalis and masseter muscles are reflected inthe morphology of such craniofacial features as the temporal fossaand zygomatic arch (highlighted). Figure 3a–i illustrates the differ-ence in these sites of bony attachment in modern macaque, gorillaand human skulls. At the histological level, the difference betweenhuman and non-human primate temporalis muscle is highlightedby staining for type II (all fast twitch) sarcomeric myosin andinterstitial laminin (Fig. 3j–l). Although the unstained type I/slowfibres are nearly identical in cross-sectional area, as confirmed in areciprocal stain (data not shown), the type II fibres of H. sapiens areabout one-eighth the size of those of M. fascicularis. The relativehypotrophy of the type II fibres resembles that seen in limb musclein inclusion body myopathy-3 (IBM3)—a rare disease for which theonly proven molecular aetiology is a myosin gene mutation(MYH2)9.