Formation of human long intergenic non-coding RNA genes, pseudogenes, and protein genes: Ancestral sequences are key players

Pathways leading to formation of non-coding RNA and protein genes are varied and complex. We report finding a conserved repeat sequence present in human and chimpanzee genomes that appears to have originated from a common primate ancestor. This sequence is repeatedly copied in human chromosome 22 (chr22) low copy repeats (LCR22) or segmental duplications and forms twenty-one different genes, which include the human long intergenic non-coding RNA (lincRNA) family FAM230, a newly discovered lincRNA gene family termed conserved long intergenic non-coding RNAs (clincRNA), pseudogene families, as well as the gamma-glutamyltransferase (GGT) protein gene family and the RNA pseudogenes that originate from GGT sequences. Of particular interest are the GGT5 and USP18 protein genes that appear to have formed from an homologous repeat sequence that also forms the clincRNA gene family. The data point to ancestral DNA sequences, conserved through evolution and duplicated in humans by chromosomal repeat sequences that may serve as functional genomic elements in the development of diverse genes.


Introduction
Models presented for the pathways in formation of genes are diverse [1]. These include formation of long non-coding RNA (lncRNA) genes from protein genes [2][3][4][5], with one study based on similarities in open reading frames [5], and the reverse pathway of human protein gene formation from lncRNA genes that are found in rhesus macaque and chimpanzee [6]. Here we report new findings on an evolutionarily conserved repeat sequence that is present in multiple and diverse RNA and protein genes and propose that a similar sequence serves as a proto-gene forming unit, a nucleation site for formation of new genes, both non-coding RNA (ncRNA) and protein genes. The repeat sequence is highly prevalent in different segmental duplications or low copy repeats (LCR22) of human chromosome 22 (chr22), specifically in region 22q11.2. Chr22 has the largest number of segmental duplications per unit chromosomal length of any human chromosome [7]. These duplications are dynamic [8]. Several may have arisen after the separation of human and macaque lineages [9] and they are continuously evolving, as shown a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 by comparisons and differences found in current human populations [10]. Segmental duplications have been considered to be important for new gene formation and human evolution [11][12][13]. Additionally, the 22q11.2 region in itself is of special interest as it is prone to genetic deletions formed during fetal development that result in a high rate of genetic abnormalities [14]. Segmental duplications have been shown to participate in the deletion process via meiotic nonallelic homologous recombination [9,11].
In this paper we propose a model for the evolutionarily conserved human/chimpanzee repeat sequence and show that it serves as a starting point for formation of new lncRNA genes with subsequent base pair changes, sequence additions and/or deletions. The core sequence consists of the common sequence shared by the gamma-glutamyltransferase (GGT) protein gene family, where GGT is linked to three phylogenetically conserved and distinct sequences. In humans, these sequences form families of long intergenic non-coding RNA genes and pseudogenes that are linked to GGT sequences present in chromosomal segmental duplications. The presence of GGT in the long arm of human chr22 was determined several decades ago [15], and its duplication in segmental duplications has also been reported [11]. The GGT family is well characterized [16,17].
In addition to the GGT-linked gene segments, we describe another protein gene family in LCR22s, the ubiquitin specific peptidase (USP) family that is also found linked to lincRNA genes. The formation of two specific genes, GGT5 and USP18 protein genes are presented here. We demonstrate that these genes originated in a primate ancestor and with use of the homologous sequence that forms the clincRNA genes; this suggests that the clincRNA ancestral sequence may be a nucleation for development of both lincRNA and protein genes.
The significance of chromosomal segmental duplications to gene development described here has parallels to the importance of human genome expansion of repeat units in the evolution of regulatory elements [18].

Background on GGT-linked gene repeat sequences
The DNA repeat sequence was detected in human chr22 segmental duplications LCR22A and LCR22D while analyzing the FAM230 lincRNA family genes [19]. The repeat represents three gene families, whose sequences are linked ( Fig 1A): the FAM230 lincRNA gene family (highlighted in yellow), a newly found conserved long intergenic non-coding RNA (clincRNA) gene family (highlighted in green) and the sequence of the GGT protein family as well as GGTrelated pseudogenes (highlighted in red). An uncharacterized spacer sequence that resides between the clincRNA and GGT sequences (highlighted in gray) is also highly conserved in LCR22A and LCR22D. We refer to GGT as the sequence shared by GGT1 and GGT2 that com-prises~20,000 bp. Fig 1B is a representation of the linked gene segment FAM230B-LOC105372935-GGT2, which we use as a guide and model for sequence comparisons. Listed are bp numbers that show the ends of genes present in the linked gene segment, which comprises a total of 116,120 bp. The drawings are representational and not to scale. Table 1 lists the linked gene segments, which represent copies of the conserved sequence, and their location in LCR22s. The clincRNA genes are those starting with the prefix LOC or AC and are linked to FAM230 genes in LCR22A and LCR22D. Also grouped together in Table 1 are linked genes in segmental duplications LCR22E, LCR22G and LCR22H; these carry the repeat sequence but do not have the FAM230 sequence, and some also differ with respect to the uncharacterized spacer sequence, which may be partially or totally missing. In segmental duplications LCR22E and LCR22H, pseudogenes POM121 transmembrane nucleoporin like 1 pseudogene POM121L1P and the BCR activator of RhoGEF family pseudogene BCRP3 are found linked to GGT; these pseudogenes stem from the clincRNA sequence. Thus the homologous sequence that forms the clincRNA gene family in LCR22A and D is found to generate pseudogenes in chromosomal segmental duplications LCR22E, and H. The FAM230C gene and linked genes reside in chr13 and not in chr22 or an LCR22 (Table 1).  A diagrammatic representation of the eight LCR22s in human chr22 shows the location of the GGT-linked gene segments in LCR22s (Fig 2). The four linked-gene units that contain the FAM230 gene family (Fig 1) are present only in LCR22A and LCR22D.
The human DNA repeat sequence is also found present in the chimpanzee genome with high identity. A nt sequence alignment of four human GGT-linked gene sequences together with two homologous sequences from chr22 of the chimpanzee genome reveals the high similarity between most of the human and chimpanzee sequences (S1 Fig). Fig 3 shows a small segment of the sequences, which is taken from the complete nt sequence alignment of six GGTlinked gene segments. It visually displays the near perfect similarity in shared sequences at the FAM230B gene/LOC105372935 (clincRNA) gene junction site (yellow/green highlighted junction). The divergence between the six sequences can be seen in (S1 Fig). As a model for the conserved repeat sequence, the sequence of FAM230B-LOC105372935-GGT2 is used here for all comparisons as it contains 94% of the length of the GGT2-linked gene counterpart in chimpanzee, LOC112206744-LOC107973052-GGT2, and displays a high nt sequence identity with the chimpanzee sequence (97%-98%). A phylogram representing a phylogenetic analysis shows a close similarity between the GGT2-linked gene sequence of chimpanzee and the human FAM230B-LOC105372935-GGT2 (S2 Fig). A complete nt sequence alignment between the chimpanzee and human sequences is in S3 Fig. In this manuscript the term FAM230-clincRNA -GGT is used to signify the conserved repeat sequence ( Fig  1A) and to represent the putative ancestral conserved sequence.

Analyses of GGT-linked genes in segmental duplications LCR22A and LCR22D
NCBI displays maps of GGT genes and surrounding genes (www.ncbi.nlm.nih.gov/gene).  Table 2 shows the percent nt sequence identity obtained from sequence alignments of the GGT-linked gene segments with the sequence of FAM230B-LOC105372935-GGT2. Fig 1B serves as a guide for the association of the percent identity relative to each gene family as it shows the positional ends of genes. Table 2 shows the conservation of sequence, which reveals a 98%-99% identity throughout most of the lengths of the four segments. Lower identities largely correspond to changes between FAM230 lincRNA genes.
A comparison of FAM230E-LOC105377182-GGT3P and FAM230B-LOC105372935-GGT2 sequences indicates that the major sequence changes are between lincRNA FAM230B and FAM230E genes. Fig 5 shows significant mutational changes in one region involving a large sequence deletion and several point mutations between the two FAM230 sequences. This region is followed by over 8 kbp that show no major additions/deletions/point mutation. These differences may show the development of the FAM230 genes into distinct structures and possibly different functions. For example, lincRNA transcripts from FAM230B and FAM230E differ in nt sequence, length and exon sequences [20,21]. Although the expression of RNA in normal somatic tissues from these genes is found only in testes [22,19], the expression of circular RNAs (circRNA)s during fetal development shows differences between certain tissues [23] (S4 Fig). FAM230E circRNA is expressed in fetal heart tissue at 10 weeks and 17 weeks development, whereas FAM230B circRNA is not expressed in this tissue. This may be of significance in terms of possible FAM230E RNA gene function in the 22q11.2 chromosomal region during fetal development, as a 22q11.2 deletion results in abnormal heart development [14]. It has been shown there are genetic factors that may influence expression of circRNAs, resulting in differences in circRNA expression and the onset of various diseases [24].
The clincRNA genes LOC105372935 and LOC105377182 (Fig 4, right panel, top two drawings) are nearly identical, both in sequence and length. The RNA transcripts from these genes show small a difference in length but they are nearly the identical in sequence. The expression of RNA from these two genes in somatic tissues, as well as the expression of circular RNA

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Formation of human long intergenic non-coding RNA genes, pseudogenes and protein genes during fetal development, are also nearly identical [21,23]. Formation of the clincRNA genes may be recent, as they have not significantly diverged in sequence or in tissue-specific transcript expression.
GGT3P (Fig 4, right panel, second drawing from top) is an unprocessed pseudogene and comprises 18,273 bp. Its entire sequence is homologous to the 3' end nt sequence of GGT2 and GGT1 protein genes. RNA transcript expression from GGT2 and GGT3P in normal somatic

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Formation of human long intergenic non-coding RNA genes, pseudogenes and protein genes tissues between the two genes is similar [21]. The expression of circular RNAs during fetal development also shows similar patterns [23]. GGT2 (Fig 4, right panel, top drawing) is a complex gene that encodes thirteen different transcripts. Most transcripts differ in size due to the presence of multiple exons in the GGT2 5' UTR (see: www.ncbi.nlm.nih.gov/gene/728441) (20). The transcript that is used here as a model for gene size is the longest (NCBI GenBank ACCESSION NM_001351304 XM_016999937). GGT2 produces a protein product [www.uniprot.org/uniprot/P36268], however the protein is inactive in glutathione hydrolase activity and its enzymatic activity has not been fully characterized [17].

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Formation of human long intergenic non-coding RNA genes, pseudogenes and protein genes Two other GGT-linked gene segments found in LCR22A, FAM230A-AC023490.3-GGTLC3 and FAM230J-LOC105372942-GGTLC5P also show a high identity with FAM230B-LOC105372935-GGT2 (Table 2), but here there are regions of major sequence changes within FAM230 and differences in GGT-related genes in sequence lengths. In these segments, the GGT sequence forms the protein gene GGTLC3, the gamma-glutamyltransferase light chain family member 3, and the unprocessed pseudogene GGTLC5P is the gamma-glutamyltransferase light chain 5 pseudogene. The GGTLC3 sequence consists only of the 3' end sequences of GGT1/GGT2 and displays an identity of 97% with GGT1, but closer identity with GGT2, 99%. An alignment of the three gene sequences reveals thirty-nine point mutations and three deletion/insertion mutations that are unique to GGT1 relative to the other two sequences, and only one point mutation that is unique to GGT2 and there are no deletions/insertions. This highly biased mutational pattern suggests that GGTLC3 originated from a sequence similar to that of GGT2.

GGT-linked genes in segmental duplications LCR22E, H and G
Linked gene sequences in segmental duplications LCR22E, H and G (Table 1) differ from those in LCR22A and D. They do not carry the FAM230 sequence, and in one case, sections of the clincRNA sequence and the spacer region are missing. Fig 6 shows a schematic drawing depicting the differences relative the FAM230B-LOC105373935-GGT2 model.

POM121L1P-GTTLC2
The POM121L1P-GTTLC2 linked gene segment (in LCR22E) resides in a complex chromosomal region, the immunoglobulin lambda gene locus IGL. There are six genes packed into a space of~3.2 kbp that also contains the POM121L1P-GTTLC2 linked gene segment [20]. There is evidence that the conserved repeat sequence was duplicated in this chromosomal region but it is significantly different; there is a partial clincRNA gene sequence present in POM121L1P-GGTLC2 and FAM230 and spacer sequences are missing (Fig 6A) (the symbol | XXX| refers to clincRNA and spacer sequences missing).
POM121L1P is termed a POM121 transmembrane nucleoporin like 1 unprocessed pseudogene [22]. 2279 bp of the POM121L1P pseudogene sequence has an identity of 96% with aligned sequences of the clincRNA gene LOC105372935 of FAM230B-LOC105372935-GGT2. Thus, part of the conserved sequence that forms clincRNA genes in LCR22A and LCR22D (green highlight, Fig 6) forms part of this pseudogene in LCR22E.
GGTLC2 encodes a gamma-glutamyltransferase light chain 2 protein and displays glutathione hydrolase activity [https://www.ebi.ac.uk/interpro/protein/Q14390]. It shares most of its sequence with GGT2 and GGT1 and displays an identity of 96% with GGT2 but 98% with GGT1, however a mutational analysis to determine the closeness of GGTLC2 with GGT1 relative to GGT2 is inconclusive.

POM121L10P-BCRP3-GGT1
POM121L10P-BCRP3-GGT1 resides in LCR22H. Based on a sequence alignment with FAM230B-LOC105372935-GGT2, the clincRNA sequence and the entire uncharacterized spacer sequence are present and are linked to GGT1 (Fig 6B). Sequences of the POM121L10P and BCRP3 genes both stem from the clincRNA sequence and are highly similar to sections of the clincRNA LOC105372935 sequence: 2264 bp of the POM121L10P sequence are homologous to LOC105372935 (clincRNA) and an adjacent 6498 bp encoding BCRP3 are also homologous to the clincRNA sequence with 96% identity. BCRP3 is one of the eight BCRP family of pseudogenes that contain sequences from the breakpoint cluster region (BCR) gene. The BCRP pseudogenes are complex. Part of the BCR gene is in LCR22F. Of the eight BCRP family genes only one, BCRP8 resides within the BCR gene sequence and thus stems from the BCR gene locus. Functions of BCRP3 are needed to understand the relationship of this pseudogene to BCR.
GGT1 is a well-characterized enzyme. Over two decades ago it was pointed out that there are several human genes for GGT that produce different mRNAs but that GGT1 produces an active gamma-glutamyltransferase enzyme [25]. This was confirmed by Heisterkamp et al. [17].

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Formation of human long intergenic non-coding RNA genes, pseudogenes and protein genes

POM121L9P(BCRP1)-GGTLC4P-GGT5
The POM121L9P(BCRP1)-GTLC4P-GGT5 segment resides in LCR22G ( Table 1). The BCRP1 gene is situated entirely within the POM121L9P sequence and is an antisense gene. However, GGT5 is an anomaly. Although gene positions relative to each other in chr22 are POM121L9P (BCRP1)-GTLC4P-GGT5, there is no evidence that GGT5 originates from a GGT locus, however data point to the origin from a clincRNA sequence.
An alignment of the POM121L9P (BCRP1)-GGTLC4P-GGT5 sequence with that of FAM230B-LOC105372935-GGT2 shows that POM121L9P (BCRP1)-GGTLC4P-GGT5 contains spacer and GGT sequences, and most of the clincRNA sequence. POM121L9P (BCRP1) carries sequences 5949 to 8219 of the clincRNA LOC105372935 sequence. Of significance, a sequence alignment of the GGT5 sequence with that of the clincRNA LOC105372935 shows that GGT5 contains part of the clincRNA sequence (positions 1-5958) but carries no GGT sequences (Table 3 and S5 Fig). GGT5 is 25489 bp in length and carries 5958 bp that are homologous to the LOC105372935 clincRNA sequence. Thus~23% of GGT5 contains clincRNA sequences with an identity of 90-92% (Table 3). It has been pointed out before that there is little nt sequence homology between the GGT5 and GGT1 genes [17]. Table 3 shows the close similarity of pseudogene GGTLC4P with GGT2 sequences, where the entire sequence of GGTLC4P consists of GGT2 sequences. GGTLC4P also displays a high identity with GGT1 (not shown).
Although there is no significant nt sequence homology between GGT5 and GGT2, amino acid sequences of the protein products have similarities where approximately half of the amino acid residues are identical [17]. In addition, the GGT5 protein displays gamma-glutamyltransferase activity [17].
The chimpanzee GGT5 nt sequence is also present in the chimpanzee genome and it is found to be highly similar to the human sequence (with 98% identity over 90% of the human GGT5 sequence). In addition, the chimpanzee gene also contains the clincRNA signature sequence. A phylogenetic analysis was performed with aligned GGT nt sequences that generated a phylogram tree. Included also are GGT sequences from the gorilla and Rhesus monkey, as these sequences are available from the NCBI (Fig 7). The tree shows that all GGT5 genes and the human clincRNA LOC105372935 gene form a branch that is separate from the branch grouping of GGT1, GGT2, and GGTLC (Fig 7). The close association of the various primate GGT5 genes with the human clincRNA gene LOC105372935 sequence is consistent with and adds to the data of Table 3. Because of the close similarities between the human and chimpanzee GGT5 gene sequences as well as with the other two primate GGT5 genes, GGT5 appears to have originated in a common primate ancestor.

FAM230C-LOC101060145-GGT4P
The FAM230C-LOC101060145-GGT4P linked gene locus is in chr13, which distinguishes it from the other FAM230 family genes that are all in chr 22. What stands out in the FAM230-C-LOC101060145-GGT4P linked gene segment is that no genes stem from the clincRNA

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Formation of human long intergenic non-coding RNA genes, pseudogenes and protein genes sequence and that two pseudogenes, LOC101060145 and GGT4P originate from the GGT sequence (S6 Fig). LOC101060145 is annotated as a glutathione hydrolase light chain 1-like pseudogene by NCBI and GGT4P is a gamma-glutamyltransferase 4 pseudogene annotated by Ensembl. S6 Fig also shows that the FAM230C-LOC101060145-GGT4P linked gene sequence has a high identity with the FAM230B-LOC105372935-GGT2 sequence. This shows the presence of the FAM230-clincRNA-spacer-GGT repeat sequence outside of chr22.

FAM230D-USP18 linked gene sequences
In addition to the GGT-linked genes, there is another example of the human FAM230-lincRNA sequence that forms genes, sequences that are linked to the USP protein genes; USP is an ubiquitin specific peptidase. The USP18-linked FAM230D sequence does not carry the uncharacterized spacer; the FAM230D gene is directly linked to USP18. Of major significance, a nt sequence alignment of the USP18 gene sequence with the clincRNA DNA sequence shows that part of the clincRNA sequence forms part of the USP18 gene (3174 bp of the clincRNA sequence is present in the USP18 gene with 97% identity); the presence of the clincRNA sequence is also found in the USP18 genes of other primates. Fig 8 shows the segregation of the clincRNA sequence with the primate USP18 genes. The phylogram is similar to the GGT5 gene findings, where the USP18 protein genes cluster with the clincRNA sequence. But unlike the GGT5 gene that does not contain GGT sequences, USP18 and the related USP41 share some sequences.
With respect to the USP18 mRNA, the entire exon 11 consists of the clincRNA sequence. Exon 11 is the last and largest exon (626 nt) in the USP18 mRNA and it has an identity of 99% compared with the clincRNA sequence. A section of a translated aa sequence of clincRNA shows an oligo-peptide sequence of QETAYLLVYMKMEC; this is the identical sequence of the last 14 aa of the USP18 peptide chain. Thus the carboxy terminal amino acid sequence of the protein appears to have been formed from the clincRNA sequence. This suggests that information carried in the clincRNA nt sequence is used to partly form the USP18 protease. This further supports the concept that a sequence homologous to human clincRNA LOC105372935 may participate in forming protein genes at chromosomal loci where the sequence is duplicated.
USP41 is termed ubiquitin specific peptidase 41, and the USP41 gene is found linked to FAM230G in chr22. The FAM230G-USP41 segment resides is in LCR22B. There are major changes in the USP41-linked sequence compared to the model FAM230-clincRNA-spacer segment of the conserved repeat depicted in Fig 1. In humans, FAM230G is linked directly to the USP41 sequence without clincRNA or spacer sequences and the FAM230G gene lacks the 3' half sequence of FAM230B.
USP41 does not carry the clincRNA sequence. USP18 and USP41 share a 14170 bp sequence, but this is outside of the USP18 sequence that contains the clincRNA sequence and there is no overlap.

Discussion
The proposed ancestral proto-gene forming element is based on the findings that GGT, USP, and the three distinct linked DNA sequences, FAM230, clincRNA and spacer are conserved between humans, chimpanzee and other primates, and that different genes have formed from these sequences. The clincRNA sequence appears to have been the focal point in the development of human and non-human primate GGT5 and USP18 protein genes and formation of these genes occurred in a common primate ancestor. The informational content of the clin-cRNA DNA element appears to be such that it can serve as a foundation for development of either lncRNA or protein genes. The FAM230 DNA sequence is not as clear. Although FAM230 sequences do carry large open reading frames, for example, the nonsense mediated decay transcript from FAM230A (21) that translates to a 454 aa sequence, no human protein has yet been found to contain an FAM230 open reading frame, although several uncharacterized proteins such as unnamed protein product, GenBank: BAG63817.1 do carry the FAM230A nt sequence and the FAM230A open reading frame.
In humans, the FAM230-clincRNA-spacer-GGT sequence has been repeatedly duplicated in the genome by chromosomal expansion through segmental duplications where the FAM230-clincRNA sequences are found to form lincRNA gene and pseudogene families. The concept of segmental duplications as vehicles for the proliferation of GGT-and USP-related

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Formation of human long intergenic non-coding RNA genes, pseudogenes and protein genes repeat sequences concerned with the development of new genes parallels the findings of the effects of human chromosomal expansion, which consists primarily of repeat sequences, on the evolution and development of gene regulation [18]. In addition, there is a parallel of the proposed ancestral proto-gene forming element described here with that of enhancer regulatory elements that have developed from ancestral sequences or proto-enhancers [26]. These studies point to the role of ancestral sequences in the evolution of regulatory elements, and in the work here, that of gene development.
The study here adds another aspect to the work of others that suggests a number of lncRNA genes originated from protein genes [2][3][4][5]. For example, Talyan et al. [5] showed that RNA and protein genes share partial open reading frames and that a number of RNA genes may have originated from protein genes. In the work presented here, some of the GGT-related RNA pseudogenes stem from protein GGT sequences, however the BCRP and POM121 family pseudogenes and lincRNA genes originate from the evolutionarily conserved clincRNA or FAM230 RNA sequences, and not from existing protein genes. On another scale, other lncRNAs have been shown to come from enhancer sequences [27]. Thus various studies show that lncRNAs can have very different origins. With respect to protein gene formation, open reading frames in lincRNA sequences may have the potential to provide a foundation for protein gene development, however lincRNA genes lack protein coding capacity, as was previously pointed out [28].
The clincRNA contributes to the structure of the USP18 protein. Part of the clincRNA sequence provides the entire exon 11, the last exon in the ubiquitin specific peptidase 1 (USP18) mRNA and it thus provides the carboxy terminal thirteen amino acid sequence of the protein. On the other hand, the entire putative ancestral clincRNA sequence is used to form the clincRNA family of genes. Thus the informational content of the ancestral DNA sequence is such that it can lead to the development of either lincRNA or protein genes.
There are sixteen lincRNA genes and pseudogenes found to arise from the FAM230-clin-cRNA sequence in humans, and in addition, the protein genes, GGT5 and USP18. GGT5 belongs to the GGT family of protein genes. It is a well-characterized gene whose protein product displays gamma-glutamyltransferase activity but the gene nt sequence displays no significant DNA sequence homology with other members of the GGT family, as shown by Heisterkamp et al [17] and the work presented here. Thus GGT5 is an anomaly in that its DNA sequence does not stem from a GGT locus. Although its gene position in chr22 is: POM121L9P(BCRP1)-GTLC4P-GGT5, there is no evidence that GGT5 contains GGT DNA sequences but data point to the origin from the clincRNA locus.
Why is GGT5 formed from an unrelated DNA sequence and not from the GGT sequence itself? The primate cell may have performed its own "genetic and molecular engineering" to form a protein similar in aa sequence and function to the GGT family proteins but from a different genomic sequence. This does not address why the GGT nt sequence is not used to form the GGT5 gene as it is for other GGT-related genes. Genes that are descended from an ancestral gene, share nucleotide sequences, have similar translated protein aa sequences and share similar functions are generally classified as a gene family. The GGT5 gene offers an interesting variation to this definition.
The clincRNA gene family found in LCR22 A and D may have formed recently, as there is little or no difference in sequence or in RNA transcript expression in different tissues. Whether some of these genes will develop important functions or eventually disappear is not known. The FAM230 genes show more development with differences in DNA nt sequence, specific RNA transcript structures and a differential expression of circRNAs in fetal tissues [21].
The uncharacterized spacer sequence is not found to form a part of genes. This sequence is highly conserved in homologous sequences of the linked gene loci found in LCR22A and D, and it is also present in chimpanzee counterparts and conserved to 97%. But it has totally dissipated in linked genes POM121L1P-GGTLC2, found in LCR22E and in FAM230D-USP18 in LCR22A and FAM230G-USP41 in LCR22B. Its evolutionary conservation points to a function, but perhaps a non-essential one as it has disappeared in some segmental duplications.
The formation of the FAM230 lincRNA family, which consists of a total of eight lincRNA genes in chr22, is only partially understood. This gene family is formed from the 3' half sequence of FAM230C with a remnant of the 5' half FAM230C sequence found in the upstream region of seven of the genes [19,29]. FAM230C resides in chr13. Six FAM230 genes are part of the conserved repeat units described here, either with GGT or USP. The remaining three FAM230 genes have not been found to be part of a repeat element and may have formed by a separate process. The origin of FAM230C, which is found in chr13 is also uncertain.
With the FAM230 sequences found linked to GGT and/or USP genes in chimpanzee and other primates, such as Rhesus monkey, gorilla or orangutan, gene annotations have not progressed enough to be able to compare genes that may stem from non-human primate FAM230 sequences with those of the human genes. Any major differences would be of interest.

Reference genomes for primate species
Homo sapiens chromosome 22, GRCh38.p12 Primary Assembly NCBI was the source of sequences and properties of RNA and protein genes. Pan troglodytes, isolate Yerkes chimp pedigree #C0471 (Clint) chromosome 22, Clint_PTRv2, NCBI was the source of chimpanzee sequences and protein genes. In addition, a cloned sequence from Pan troglodytes, clone rp43-41g5, complete sequence GenBank: AC099533.36 provided an additional copy of the conserved repeat sequence. Gorilla gorilla (western gorilla) chromosome 22, gorGor4, NCBI Reference Sequence: NC_018446.2, locus: NC_018446 [20] (was used to search for the presence of GGT genes and the conserved repeat sequence. The Rheus monkey GGT5 sequence (annotated as LOC720345) was from the NCBI sequence of Macaca mulatta isolate AG07107 chromosome 10, Mmul_10, whole genome shotgun sequence, ACCESSION: NC_041763, REGION: complement 28391649..28419652).

Genomic coordinates
The NCBI and Ensembl gene coordinates differ for a number of genes, especially at the 5' ends. For uniformity, all coordinates used here were according to NCBI with the expectation of AC023490.3 that has been annotated only by Ensembl. The description of linked gene segments in Table 1 is in the order of FAM230-clincRNA-GGT or pseudogenene-GGT. However, in the genome, the gene order for several of the repeat segments is in the reverse orientation. For consistency, all linked genes are shown in the same orientation as in Table 1.

Nucleotide and amino acid sequence alignments and identity determinations
The EMBL-EBI Clustal Omega Multiple Sequence Alignment program,website: http://www. ebi.ac.uk/Tools/msa/clustalo/ was used for alignment of two or more nucleotide or amino acid sequences. This program was also used to determine phylogenetic relationships via generation of a phylogram.
The identity between two sequences was determined by the NCBI Basic Local Alignment Search Tools, blastn and blastp, align two or more sequences with the Program Selection: Optimize for Highly similar sequences (megablast) [35]. The identities represent only aligned sequences and do not including gaps sequences.
RNA expression. The expression of RNA from normal tissues were obtained from website: www.ncbi.nlm.nih.gov/gene/, human tissue-specific expression (HPA) RNA-seq normal tissues [22]. The expression of circular RNAs: Tissue-specific circular RNA induction during human fetal development was obtained from website: www.ncbi. nlm.nih.gov/gene/, that presents data by Szabo et al [23], where RNA-seq was performed on 27 different human tissues with samples from 95 individuals.

Protein properties
UniProtKB (uniprot.org/uniprot/) was the source of human protein amino acid sequences. For chimpanzee proteins, amino acid sequences and regions of the protein sequence that have predicted functional domains were from: www.ncbi.nlm.nih.gov/protein [36].
Availability of data on websites.