Craniofacial and dental development in cardio-facio-cutaneous syndrome: the importance of Ras signaling homeostasis.

Cardio-facio-cutaneous syndrome (CFC) is a RASopathy that is characterized by craniofacial, dermatologic, gastrointestinal, ocular, cardiac, and neurologic anomalies. CFC is caused by activating mutations in the Ras/mitogen-activated protein kinase (MAPK) signaling pathway that is downstream of receptor tyrosine kinase (RTK) signaling. RTK signaling is known to play a central role in craniofacial and dental development, but to date, no studies have systematically examined individuals with CFC to define key craniofacial and dental features. To fill this critical gap in our knowledge, we evaluated the craniofacial and dental phenotype of a large cohort (n = 32) of CFC individuals who attended the 2009 and 2011 CFC International Family Conferences. We quantified common craniofacial features in CFC which include macrocephaly, bitemporal narrowing, convex facial profile, and hypoplastic supraorbital ridges. In addition, there is a characteristic dental phenotype in CFC syndrome that includes malocclusion with open bite, posterior crossbite, and a high-arched palate. This thorough evaluation of the craniofacial and dental phenotype in CFC individuals provides a step forward in our understanding of the role of RTK/MAPK signaling in human craniofacial development and will aid clinicians who treat patients with CFC.

Prader-Willi syndrome resulting from an unbalanced translocation: characterization by array comparative genomic hybridization.

Prader-Willi syndrome (PWS) is caused by lack of expression of paternally inherited genes on chromosome 15q11-->15q13. Most cases result from microdeletions in proximal chromosome 15q. The remainder results from maternal uniparental disomy of chromosome 15, imprinting center defects, and rarely from balanced or unbalanced chromosome rearrangements involving chromosome 15. We report a patient with multiple congenital anomalies, including craniofacial dysmorphology, microcephaly, bilateral cryptorchidism, and developmental delay. Cytogenetic analysis showed a de novo 45,XY,der(5)t(5;15)(p15.2;q13), -15 karyotype. In effect, the proband had monosomies of 5p15.2-->pter and 15pter-->15q13. Methylation polymerase chain reaction analysis of the promoter region of the SNRPN gene showed only the maternal allele, consistent with the PWS phenotype. The proband's expanded phenotype was similar to other patients who have PWS as a result of unbalanced translocations and likely reflects the contribution of the associated monosomy. Array comparative genomic hybridization (array CGH) confirmed deletions of both distal 5p and proximal 15q and provided more accurate information as to the size of the deletions and the molecular breakpoints. This case illustrates the utility of array CGH in characterizing complex constitutional structural chromosome abnormalities at the molecular level.

Expression of fast myosin heavy chain transcripts in developing and dystrophic chicken skeletal muscle.

The expression of fast myosin heavy chain (MyHC) genes was examined in vivo during fast skeletal muscle development in the inbred White Leghorn chicken (line 03) and in adult muscles from the genetically related dystrophic White Leghorn chicken (line 433). RNA dotblot and northern hybridization was employed to monitor MyHC transcript levels utilizing specific oligonucleotide probes. The developmental pattern of MyHC gene expression in the pectoralis major (PM) and the gastrocnemius muscles was similar during embryonic development with three embryonic MyHC isoform genes, Cemb1, Cemb2, and Cemb3, sequentially expressed. Following hatching, MyHC expression patterns in each muscle differed. The expression of MyHC genes was also studied in muscle cell cultures derived from 12-day embryonic pectoralis muscles. In vitro, Cvent, Cemb1, and Cemb2 MyHC genes were expressed; however, little if any Cemb3 MyHC gene expression could be detected, even though Cemb3 was the predominant MyHC gene expressed during late embryonic development in vivo. In most adult muscles other than the PM and anterior latissimus dorsi (ALD), the Cemb3 MyHC gene was the major adult MyHC isoform. In addition, two general patterns of expression were identified in fast muscle. The fast muscles of the leg expressed neonatal (Cneo) and Cemb3 MyHC genes, while other fast muscles expressed adult (Cadult) and Cemb3 MyHC genes. MyHC gene expression in adult dystrophic muscles was found to reflect the expression patterns found in corresponding normal muscles during the neonatal or early post-hatch developmental period, providing additional evidence that avian muscular dystrophy inhibits muscle maturation.

The evolution of the chicken sarcomeric myosin heavy chain multigene family.

This manuscript describes the chicken sarcomeric myosin heavy chain (MyHC) multigene family and how it differs from the sarcomeric MyHC multigene families of other vertebrates. Data is discussed that suggests the chicken fast MyHC multigene family has undergone recent expansion subsequent to the divergence of avians and mammals, and has been subjected to multiple gene conversion-like events. Similar to human and rodent MyHC multigene families, the chicken multigene family contains sarcomeric MyHC genes that are differentially regulated in developing embryonic, fetal, and neonatal muscles. However, unlike mammalian genes, chicken fast MyHC genes expressed in developing muscles are also expressed in mature muscle fibers as well. The potential significance of conserved and divergent sequences with the MyHC rod domain of five fast chicken isoforms that have been cloned and sequenced is also discussed.

The evolutionary relationship of avian and mammalian myosin heavy-chain genes.

Sequence comparisons of avian and mammalian skeletal and cardiac myosin heavy-chain isoforms are used to examine the evolutionary relationships of sarcomeric myosin multigene families. Mammalian fast-myosin heavy-chain isoforms from different species, with comparable developmental expression, are more similar to each other than they are to other fast isoforms within the same genome. In contrast, the developmentally regulated chicken fast isoforms are more similar to each other than they are to myosin heavy-chain isoforms in other species. Extensive regions of nucleotide identity among the chicken fast myosin heavy chains and in the mouse and rat alpha- and beta-cardiac myosin heavy-chain sequences suggest that gene-conversion-like mechanisms have played a major role in the concerted evolution of these gene families. We also conclude that the chicken fast myosin heavy-chain multigene family has undergone recent expansion subsequent to the divergence of birds and mammals and that both the developmental regulation and the specialization of myosin isoforms have likely developed independently in birds and mammals.

Analysis of the chicken fast myosin heavy chain family. Localization of isoform-specific antibody epitopes and regions of divergence.

cDNAs encoding the rod region of four different fast myosin heavy chains (MYCHs) in the chicken were identified, using anti-MYCH monoclonal antibodies, in two expression libraries prepared from 19-day embryonic and adult chicken muscle. These clones were used to determine the amino acid sequences that encompass the epitopes of five anti-MYHC monoclonal antibodies. Additionally, the amino acid sequences were compared to each other and to a full length embryonic MYHC. Although there is extensive homology in the chicken fast myosin rods, sequences within the hinge, within the central portion of the light meromyosin fragment, and at the carboxy terminus exhibit the largest number of amino acid substitutions. We propose that divergence within these subdomains may contribute to isoform-specific properties associated with skeletal myosin rods.

Gene conversions within the skeletal myosin multigene family.

Comparisons of the nucleotide sequences of the light meromyosin (LMM) region of developmentally regulated fast chicken myosin heavy chain (MHC) isoforms indicates that chicken MHC isoforms are more similar to each other than to MHC isoforms in other species. The sequence data provide evidence that gene conversion events have occurred recently among the isoforms. An embryonic (Cemb1) isoform and neonatal isoform have the most extensive regions of sequence identity. Similar gene conversion events are present in the rat alpha- and beta-cardiac MHCs, but were not obvious in the LMM of developmentally regulated fast human MHC isoforms. The data suggest that gene conversion events can play a significant role in the evolution of the MHC multigene families and that concerted evolution of the chicken multigene family occurred after the divergence of mammals and avians.