Table1_Case report: identification of one frameshift variant and two in cis non-canonical splice variants of NEB gene in prenatal arthrogryposis.XLSX

NEB mutation is associated with congenital nemaline myopathies. Here, we report a family with recurrent prenatal arthrogryposis. Trio whole exome sequencing (WES) disclosed three novel NEB (NM_001271208.2) variants including one paternal frameshift c.19049_19050delCA (p.Thr6350Argfs*14) and two double maternal variants in cis c. [24871G>T;24871-10C>G] (p. [Val8291Phe;?]). They are evaluated as “likely pathogenic (LP)”, “variant of uncertain of significance (VUS)”, and “VUS”, respectively. After further prediction, the c.24871G>T, c.24871-10C>G, and c.[24871G>T;24871-10C>G] were respectively genetically engineered into the three plasmids. Compared with their wild-type counterparts, the three plasmids all produced truncated transcripts, and also a significant proportion of the full-length transcripts, which allowed us to reclassify NEB c.24871G>T and c.24871-10C>G variants as LP. As far as we know, this is the first case carrying NEB allele-specific function of partial loss. This result helped the couple make informed reproductive choices and opt for assisted reproduction for future pregnancies. This study also increased awareness to the phenotype of prenatal nemaline myopathy and expanded the variant spectrum of NEB.</p

Phylogenetic tree and classification of 214 plant CYP93 proteins.

The neighbor joining (NJ) tree includes 214 CYP93 proteins from 60 eukaryotes. Proteins are clustered into eight subfamilies (e.g., CYP93A). The colored lines and names symbolize the species to which the proteins in each clade belong. The black dots represent the major clades in the phylogenetic tree, and the corresponding bootstrap support values from 1000 replications are shown beside the black dots. Bootstrap values 50% are shown as black dots in the phylogenetic tree. The numbers in brackets indicate the dN/dS value for each subfamily or branch. The information of species abbreviations used for the tree of Fig 2 is listed in S2 Table. The scale bar represents amino acid substitutions per site.</p

Genome-Wide Analysis, Classification, Evolution, and Expression Analysis of the Cytochrome P450 93 Family in Land Plants

<div><p>Cytochrome P450 93 family (CYP93) belonging to the cytochrome P450 superfamily plays important roles in diverse plant processes. However, no previous studies have investigated the evolution and expression of the members of this family. In this study, we performed comprehensive genome-wide analysis to identify CYP93 genes in 60 green plants. In all, 214 CYP93 proteins were identified; they were specifically found in flowering plants and could be classified into ten subfamilies—CYP93A–K, with the last two being identified first. CYP93A is the ancestor that was derived in flowering plants, and the remaining showed lineage-specific distribution—CYP93B and CYP93C are present in dicots; CYP93F is distributed only in Poaceae; CYP93G and CYP93J are monocot-specific; CYP93E is unique to legumes; CYP93H and CYP93K are only found in <i>Aquilegia coerulea</i>, and CYP93D is Brassicaceae-specific. Each subfamily generally has conserved gene numbers, structures, and characteristics, indicating functional conservation during evolution. Synonymous nucleotide substitution (<i>d</i>_N/<i>d</i>_S) analysis showed that CYP93 genes are under strong negative selection. Comparative expression analyses of CYP93 genes in dicots and monocots revealed that they are preferentially expressed in the roots and tend to be induced by biotic and/or abiotic stresses, in accordance with their well-known functions in plant secondary biosynthesis.</p></div

Summary of functionally characterized CYP93 genes in plants.

Summary of functionally characterized CYP93 genes in plants.</p

qRT-PCR analyses of the expression profiles of CYP93 homologous genes in <i>Arabidopsis</i>, soybean, rice, and maize.

(A) Expression profiles of CYP93 genes in soybean. (B) Expression profiles of the AtCYP93D1 gene in Arabidopsis. (C) Expression profiles of CYP93 genes in maize. (D) Expression profiles of CYP93 genes in rice.</p

Weblogo of SRSs based on the amino acid alignments across the eight subgroups of the plant CYP93 family.

The bit score indicates the information content for each position in the sequence. A−K indicate subfamilies CYP93A−CYP93K.</p

Sequence logos of the multiple alignments of the 214 CYP93 proteins in plants.

<p>The sequence logos of plant CYP93 proteins based on amino acid alignment using MAFFT are shown. The logos were generated using Weblogo. The bit score indicates the information content for each position in the sequence. The height of the letter designating the amino acid residue at each position represents the degree of conservation. The key conserved motifs are underlined; the red lines indicate the less conserved regions; the black ones, the P450 motifs; and the blue ones, the substrate recognition sites (SRSs). The white triangles indicate the conserved intron insertion location of plant CYP93 genes; the numbers within the triangles indicate the splicing phase of the intron (0 refers to phase 0). The red and black dots indicate the conserved amino acid insertion or deletion sites, respectively, in a given subfamily and/or clade; the number below each dot indicates the corresponding subfamily, i.e., B indicates the CYP93B subfamily.</p

Expression profiles of <i>GmCYP93</i> genes in response to biotic stresses.

<p>(A) Expression profiles of GmCYP93 genes after infection with root-knot nematode (GSE33410). (B) Expression profiles of GmCYP93 genes after infection with <i>Phytophthora sojae</i> (GSE9687). (C) Expression profiles of GmCYP93 genes after aphid infestation (GSE35427). Color bar at the base represents log2 expression values.</p

Expression profiles of plant CYP93 genes in response to abiotic stresses.

<p>(A) Expression profiles of <i>AtCYP93</i> and representative P450 genes in response to abiotic stresses. (B) Expression profiles of eight probe sets representing eight soybean CYP93 genes based on four microarray datasets of abiotic stresses. (C) Expression profiles of rice CYP93 genes based on four microarray datasets of abiotic stresses. Color bar at the base represents log2 expression values.</p

Phylogenetic relationships of the 60 species investigated in the present study.

<p>Phylogenetic relationships (branch lengths are arbitrary) among these species have been described previously (<a href="http://www.phytozome.net/" target="_blank">http://www.phytozome.net/</a>). The total number of cytochrome P450 93 (CYP93) proteins identified in each genome is indicated on the right.</p