Ординатура / Офтальмология / Английские материалы / Mechanisms of the Glaucomas_Shields, Tombran-Tink, Barnstable_2008

Fig. 1. Distribution of OPTN gene mutations (bottom) and regional locations for eight of its known interacting proteins (top). The reported mutations are depicted below each of their respective coding exons. Note that OPTN gene has a total of 16 exons, of which the first three are untranslated.

Screening of the optineurin-coding regions in 11 Finnish subjects, eight with POAG and three with pseudo-exfoliation syndrome (PEX), identified OPTN-T34T polymorphism in four POAG and two PEX and OPTN-E163E in one POAG subject (90). Tang et al. (91) studied the presence of OPTN mutation in 148 NTG, 165 POAG, and 196 controls of Japanese population. They found non-significant higher frequency for variants of T34T, M98K, and R545Q in NTG as compared to POAG and controls. The OPTN T49T and L389L were found exclusively in POAG patients but not in NTG or controls. In another study, presence of similar OPTN mutations in 86 POAG and 80 control subjects was examined by sequencing of exons 4 and 5 and denaturing high performance liquid chromatography (DHPLC) screening of other exons (92). This study reported a similar frequency of the M98K variant between POAG and control subjects. Leung et al. (85) studied the association of OPTN sequence alterations with glaucoma in 72 POAG and 47 NTG and 126 control subjects in a Chinese population. In addition to the previously reported alterations of T34T, M98K, and R545Q, this study identified novel OPTN-coding variants of T49T, E103D, and V148V exclusively in the NTG patients. Another study screened OPTN sequence variants in the four portions of the genes that had been previously associated with glaucoma in 1048 glaucoma and 251 control cases by single-strand conformation polymorphism (SSCP) and sequencing (93). This study found the E50K mutation in 3.5% of studied familial NTG and a significantly higher frequency for the M98K variant in the Japanese NTG patients. The R545Q variant was found with higher but not significant frequency in Japanese NTG subjects than the ethnically matched controls. Sequence variants of T49T, Q142P, A134A, T145T, H571H, and S297S were observed exclusively in glaucoma subjects but it was considered as non-significant association by authors (as only one of each was observed). In evaluation of the OPTN-M98K variant by Melki et al. (94) in a group of glaucoma subjects, 237 from France and 56 from Morocco, no significant frequency was found for this variant. However, in the French patients, M98K variant was associated with higher initial IOP. Fuse et al. (87) studied OPTN mutations in a cohort of 89 POAG and 65 NTG Japanese subjects and found OPTN variants of H26D and R545Q exclusively in glaucoma subjects. The M98K frequency

was found in 16.9% and 15.4% of POAG and NTG cases, respectively, showing significantly higher frequencies than in controls (5%). A Canadian group screened 66 JOAG subjects and reported OPTN-H486R mutation exclusively in JOAG but not in POAG or controls (95). In addition to the H486R, the previously reported variants of T34T, L41L, T49T, and M98K were also observed in the JOAG cases. A significantly higher frequency was observed for the M98K in 49 POAG patients but not in the JOAG subjects. Umeda et al. (96) studied clinical relevance of OPTN mutations in 55 POAG and 28 NTG subjects in Japan. The M98K variant showed a significant higher frequency in POAG and NTG (14.5% and 14.2%, respectively) than in controls (1.7%). However, the R545Q showed no significant differences between glaucoma and controls individuals. Baird et al. (97) studied the presence of OPTN mutations in 18 POAG and 9 NTG subjects from the Blue Mountains Eye Study population. A higher, but not significant, frequency of M98K was found in glaucoma subjects than controls. Also the P556P variant was found in one POAG subject. Funayama et al. (86) studied OPTN gene and TNF-alpha polymorphisms in a Japanese cohort of 194 POAG, 217 NTG, and 218 controls. They concluded that visual field loss was more severe in patients with both OPTN-T34T and TNF-alpha/-857T than in those without an OPTN polymorphism. TNF-alpha/-863A was found with a significantly higher frequency in POAG and NTG than in controls, and visual field loss was more severe in patients with TNF-alpha/-863A and OPTN-M98K than without the OPTN polymorphism.

The prevalence of OPTN mutations was examined in a cohort of 112 NTG and 100 controls of German subjects (88). This study identified two novel sequence alterations of A336G and A377T for OPTN that was exclusively found in NTG subjects. However, no significantly higher frequency was observed for the M98K variant in glaucoma subjects. A Swedish cohort study of 200 POAG, 200 PEX, and 200 matched control subjects revealed no disease-causing variants for OPTN (98). The M98K alteration was found with similar frequency in glaucoma and control subjects. Mukhopadhyay et al. (99) studied OPTN sequence alterations in 200 POAG and 200 controls of Indian population. The OPTN-R545Q was found in 3% of POAG but not in the controls. This study found a non-significant difference for variants T34T, M98K, R149R, and N303K between patients and controls. Another group studied exons 4 and 5 of OPTN gene in 170 glaucoma and 100 control subjects (100) and found a highly significant frequency for the OPTN-M98K variant.

The study conducted by Ariani et al. (101) did not identify any association between OPTN mutations and glaucoma in 53 studied subjects. Study of OPTN variants by Yao et al. (89) in 94 POAG, 48 NTG, and 77 control subjects identified I407T and L211L exclusively in the POAG cases. OPTN-T34T was found with a significantly higher frequency in POAG than NTG and controls. Frequency of OPTN IVS8 + 20G>A in POAG and NTG subjects was significantly higher than controls.

As it is evident from these studies, there is an extensive variation in the frequency and type of DNA alterations that have been identified by different investigators from various parts of the world. Although these studies confirm the role of OPTN mainly in the NTG patients, it is less likely that OPTN mutations play a major role in the etiology of other forms of glaucoma. In addition to the 15 amino acid mutations that have been exclusively observed in the glaucoma patients, a number of other synonymous

and non-synonymous as well as intronic variations have been reported in much higher frequencies in the glaucoma patients as compared to the normal control subjects. Although it is likely that such variations are true polymorphisms that are due to natural selection of allele frequencies in their respective populations, it is equally likely that these variations may play a silent role in the etiology of glaucoma. As a number of such variations may disrupt potential DNA–DNA or DNA–protein interaction of OPTN, one should consider that such variations might either have a direct effect or, at least, act as a modifying risk factor for these glaucomas. Therefore, the potential role of such distorted DNA alterations and their contribution to the glaucoma phenotype still require further investigation.

In summary, OPTN mutations are now generally considered to be involved in the etiology of familial forms of NTG, with little contribution to classical forms of highpressure POAG. However, recently obtained data suggest that OPTN cellular function and its biological contribution and known interaction with many other important proteins may provide a significant opportunity for better understanding of the role of this gene in the etiology of not only glaucoma but also in other neurodegenerative conditions, such as Huntington’s disease and sensorineural deafness.

OPTN Gene and Protein Structure, Distribution, and Expression

In addition to the above-mentioned mutation screening of the OPTN gene in a diverse group of glaucoma patients, other ongoing efforts are aiming to characterize and elucidate the functional significance of OPTN and the biological mechanisms through which the mutant forms of this protein lead to the pathogenesis of glaucoma. Our previous studies showed that the normal OPTN is an evolutionarily conserved secretory protein; localized to the Golgi apparatus (22); expressed in ocular tissues of the anterior segment, retina, and optic nerve of human, mouse, and monkey (102,103); and expressed during early stages of eye development in the mouse embryos (104,105). By immunoblotting, we showed that the endogenous OPTN is a 66-kDa protein that is present in aqueous humor and that it secretes into cell-culture media by various ocular and non-ocular cell lines that were tested. The endogenous protein is localized to the Golgi apparatus and vesicular structures near the cell membrane (22). Our studies further established expression of this protein in ocular cell lines of human TM, NPCE, and optic nerve head astrocytes, as well as in other tissues of the anterior segment, retina and optic nerve of the human eyes (102,106). The evolutionary significance of OPTN was evaluated by gene cloning and genomic characterization of the homologous sequences in both Mouse and Macaca (Rhesus Monkey). The mouse and monkey genes encode for 584 and 571 amino acids proteins, respectively, and a very high similarity was observed between gene structure, protein expression, and intercellular localization of these orthologs to their human counterparts (103,105). The intron–exon boundaries of these genes are evolutionarily conserved from mouse to human. Alignment of the OPTN protein showed 81% identity between mouse and monkey; 80% between mouse and human; and 97% between monkey and human (103,105). Studies of Optn in mouse showed mRNA expression of this gene in 7- and 11-day-old mouse embryos as well as in certain adult tissues. By in situ hybridization, we showed a very restrictive and specific Optn mRNA expression in the anterior chamber angle of the developing mouse

eye at 9.0 d.p.c. (days post-conception) to 13.5 d.p.c. intervals both in whole mount embryos as well as in various eye sections (104,105).

OPTN-Interacting Proteins

OPTN was initially discovered as an interacting protein for adenoviral protein of E3-14.7K (14.7K-interacting protein-2 and named FIP-2) and subsequently shown to protect the infected cells from TNF-a-induced cytolysis (107). Since then, seven other binding partners have been identified for the OPTN protein (see Fig. 1). These include: Huntingtin, the mutated protein in the neurodegenerative disorder of Huntington’s disease (108); RAB8, a small GTPase protein (109); GTF3A, the transcription factor IIIA (110); MYO6, myosin VI (111); FOS (112); RNF11, ring finger protein 11 (113); and mGluR1a, metabotrophic glutamate receptor 1-a (114). Different regions of the OPTN protein are involved in interaction with various domains of its binding partners.

Transcription Factor IIIA

Transcription factor IIIA (GTF3A) was the first transcription factor identified in eukaryotes. Subunit A of this protein together with the B and C components is required for RNA polymerase III-mediated transcription of 5S ribosomal RNA genes (115). In a study of GTF3A-interacting proteins using yeast two-hybrid library, the leucine-rich central domain of OPTN was identified as an interacting region for the transcription factor IIIA (110).

Huntingtin

Huntingtin (HTT) is a large protein with unidentified function that is linked to the Huntington’s disease, a late-onset neurodegenerative disorder that is caused by expansion of an unstable CAG repeat in the Huntingtin gene, which translates as a polyglutamine repeat at the N-terminus of the protein product. Intracellularly, HTT is localized to the Golgi complex and with speculated role in vesicle trafficking (116). Optineurin was identified as one of the several HTT-interacting partners to this multidomain protein (108).

Metabotrophic Glutamate Receptor 1-a

Metabotrophic glutamate receptor 1-a (mGluR1a) interacts with both OPTN and HTT proteins (114), and OPTN participates in inhibition of the mGluR1a signaling. Glutamate receptors are involved in RGC death and elevated IOP (117,118). One study showed that the H486R mutant form of OPTN impairs its binding to the mutant HTT protein (114). Direct inhibition of mGluR1a by OPTN and its expression in the RGC is a further evidence for involvement of OPTN in the RGC death.

Ring Finger Protein 11

Ring finger protein 11 is encoded by RNF11 gene and contains a specialized type of Zn-finger domain known as RING domain (amino acid 99–139) that mediates protein– protein interactions. RNF11 was identified as a binding partner to optineurin and

myosin VI (113) through mapping of the interacting proteins in the SMAD-signaling system that in turn is regulated by TGFsuperfamily.

Myosin VI

Myosin VI (MIM # 600970), a member of myosin motor protein family, uses the energy from ATP hydrolysis to generate force for movement along the actin filament tracks. Mutations in myosin VI are associated with sensorineural deafness. In the myosin VI knockout mouse, maturation of the stereocillia in the sensory hair cells of the inner ear is impaired and results in hearing loss (119). Myosin VI moves toward the minus end of the actin filaments and in the opposite direction of almost all other myosins (120). OPTN was identified as the first binding partner for myosin VI and probably targets this protein to the Golgi complex and vesicular structures. Studies conducted by Sahlender et al. (111) showed that absence of OPTN causes a noticeable decrease in the amount of myosin VI at the Golgi complex and leads to significant morphology changes and fragmentation of Golgi cisternae.

GTPase RAB8

GTPaseRAB8 is a member of the RAB superfamily and belongs to Ras-related small GTPases that regulates the delivery of basolateral proteins from the trans-Golgi network to the plasma membrane (121). RAB8 is associated with Rhodopsin-containing post-Golgi membranes of photoreceptors (122) and plays a role in docking of postGolgi membranes in rods (123). Hattula et al. (109) showed that OPTN binds to the GTPase Rab8 through its N-terminal domain and to HTT through its C-terminal domain, thus linking RAB8 to HTT. Based on participation of these three proteins in vesicle trafficking, a protein network model has been purposed in which HTT, OPTN and RAB8 form a complex to regulate membrane trafficking and cell morphogenesis.

C-FOS

C-FOS is the human oncogene homologous to the Finkel–Biskis–Jinkins (FBJ) murine osteosarcoma virus oncogene. Strong implication of c-FOS expression in activation of apoptosis in various neuronal cells (124,125), its upregulation in RGCs by retinal or ischemic injuries (126,127), and its role in mechanism of neurite regeneration in damaged RGCs (128) have been documented. C-FOS was also identified as a responsive gene to hydrostatic pressure (HP) in human optic nerve head astrocytes (129) and showed an increased nuclear and decreased cytoplasmic localization in the cells exposed to HP. A significant upregulation and nuclear localization of c-FOS was seen in monkeys with experimental elevated IOP and glaucomatous damage (130). Recently, Miyamoto-Sato et al. (112) identified OPTN as a binding partner to Fos protein.

OPTN is localized to the Golgi complex, but its particular cellular function at this organelle is not known. The characteristics of OPTN-binding partners of RAB8, HTT, and MYO6, all localized to the Golgi, and function in membrane trafficking events may link OPTN to membrane trafficking pathways.

OPTN and Glaucoma

Significantly higher frequency of OPTN sequence alterations, as observed between glaucoma and controls subjects, further supports the contribution of this gene to the development of glaucoma. The synonymous and intronic OPTN variations may affect different biological functions such as mRNA stability and protein function (131). But their association with disease phenotype and their segregation in the families, sequence analysis of relevant cDNA regions, and evaluation of mRNA expression are all prerequisites before they can be concluded to be glaucoma-causing mutations. Additional OPTN mutation analysis by other investigators may provide complementary evidence for the involvement of this gene and its relationship to the glaucoma phenotype. Similar to our initial observation, other studies also found that the E50K mutation is exclusively associated with the familial forms of NTG glaucoma (93,132,133). Study of clinical features of NTG patients revealed that NTG phenotype is more severe in subjects with the OPTN-E50K mutation than in a control group of subjects with NTG but without this mutation (134). Another study also reported a more severe glaucomatous phenotype in a patient with OPTN-E50K mutation (133) that further supported this observation.

Another study reported that optineurin-E50K mutation leads to significant cupping of the optic disc in the transgenic mice while the IOP remains within the normal range (135). Also it has been shown that the OPTN-binding activity with the RAB8 protein is impaired by both the E50K and the M98K sequence alterations (136).

Association of the OPTN-H486R mutation is reported for both NTG (85) and JOAG (95) phenotypes. This observation supports the possibility that OPTN functions through an IOP-independent pathway and that its mutations are not limited to NTG. The Histidine 486 is an evolutionarily conserved residue located at the C-terminus and within an OPTN domain that interacts with five other proteins of adenovirus E314.7K, HTT, mGluR1a, MYO6, and RNF11. The downstream effect of this mutation on binding ability of OPTN to these interacting proteins still remains to be investigated. More recently, it was demonstrated that OPTN-H486R mutant protein looses its binding activity to the mutant HTT protein (114).

Inconsistent association of the OPTN-M98K sequence variation with glaucoma phenotype has been discussed in studies involving different populations. Evaluation of OPTN-M98K sequence variation as a potential risk factor for the French POAG subjects concluded that pre-treatment of IOP in patients with this variant is lower than patients without this sequence alteration (94). Two other studies reported a significantly higher frequency of the M98K variant in Japanese POAG and NTG patients (87,96). Another study confirmed the association of M98K variant in Japanese but not in Caucasians NTG patients (93). Study of 315 British sporadic glaucoma patients estimated the M98K frequency in 10.6% of NTG, 4.4% of high-tension POAG, and 3.2% of controls (132). Similarly, mutation analysis of OPTN in the Indian glaucoma patients identified the M98K sequence alteration in 6% of NTG and 4.1% of POAG patients but not in controls (137). This study also found significant association between IVS7+24G>A and the HTG phenotype, and by using computer programs for evaluation of putative transcription factor binding sites, they further suggested that IVS7+24G>A polymorphism creates a new binding site for the transcription factors NF-1 and CPE.

The M98K polymorphism is also reported to be significantly associated with POAG and with increased cup–disc ratio in Chinese glaucoma patients (85).

Elevated IOP and OPTN Expression in Trabecular Meshwork

The OPTN function and its potential role in mechanisms that regulate aqueous humor outflow are poorly understood. OPTN gene expression is significantly upregulated in response to sustained elevated IOP and prolonged exposure to TNF-a and dexamethasone treatment (138). Another study clearly showed that no significant alteration in OPTN gene expression occurs after short period of raised IOP (139).

Optineurin Summary

Our recent cloning and identification of OPTN as a new glaucoma-causing protein has now provided an opportunity to study the complex biochemical pathways that are anticipated to be involved in the etiologies of the glaucomas. A number of investigators have now followed up on our original report and have provided clear associations between OPTN mutations and the POAG phenotype, as well as evidence for involvement of mutant forms of this protein in glaucomatous optic neuropathy. Certain mutations in this gene cause significant damage to the optic nerve and development of a similar phenotype in mice. Association of OPTN protein and its interaction with a number of other proteins suggests that this protein may have a significant biological function in the normal eye development, protein–protein interaction, and possibly other intercellular protein trafficking and secretion. However, much information on the basic characteristics of OPTN protein, as well as the molecular processes in which OPTN mutations contribute to the glaucomatous optic neuropathies, await further investigation.

WD REPEAT DOMAIN 36 (WDR36)

Historical Review

In early 2003, genome scans of two large families with classical form of adultonset POAG (20 affected and 9 glaucoma suspects) provided provisional evidence for a new POAG locus on chromosome 5q (140,141). Screening of additional POAG families for possible linkage to the same region identified a total of seven families that showed consistent evidence of linkage to 5q. Subsequent saturation mapping in these seven POAG families (194 individuals and 31 living affected subjects) confined this linkage to a region of approximately 35 mega base (Mb) of DNA on 5q21.3-q31.3. Furthermore, one ocular hypertension (OHT) subject showed a recombination for one of the DNA markers (D5S404) on 5q23.1 thus indicating that the region of interest might be limited to an interval of approximately 7 Mb on 5q21.3-q23.1. In 2004, another group (142,143) used a single adult-onset POAG family and confirmed our original linkage. Their family mapped to a region of 6.6-Mb on 5q21.3-q22.2 that shared a common interval of 2-Mb within the same region that was identified in our families (see Fig. 2). The Human Genome Organization (HUGO) Gene Nomenclature Committee (HGNC) had designated this newly identified locus on 5q22.1 as GLC1G. Identification of this new POAG locus provided an opportunity for rapid mutation screening and identification of a defective glaucoma gene at this locus.

Fig. 2. Partial genetic map of the long arm of chromosome 5. On the top, map boundaries of the GLC1G locus and its defective gene, WDR36, are shown in the box. The GLC1G overlapping interval identified by the Oregon family (147) is also indicated. The relative position of the GLC1M locus (152) that maps immediately below the GLC1G locus is illustrated. Below, the West African IOP/Type 2 diabetes (NIDDM) locus (153) overlapping with the GLC1G locus is depicted.

The critical region of the GLC1G locus as defined by the two above-mentioned studies was approximately 2 Mb and contained seven known genes (MAN2A1,

AK125070, BC017169, TSLP, WDR36, CAMK4, and STARD4) as well as a number of other uncharacterized cDNA clones (23). Taken altogether, the transcript length of these seven genes was 18,397 bp that divided into 80 coding exons and collectively predicted to encode for 3654 amino acids. Originally, we screened these seven genes in all of our GLC1G-linked families by SSCP but did not find any DNA alterations in them. However, as the evidence for linkage was very strong for this region of chromosome 5, we changed our strategy and decided to directly sequence every one of the 80 coding exons and the adjacent flanking sequences of these 7 genes in our largest GLC1G-linked family. After generating over 34,000-bp of DNA sequences from these seven genes, two amino acid variations were identified, one in CAMK4 and another in WDR36 gene. However, subsequent investigation of the CAMK4 variation (A413V in Exon 11) showed that the observed DNA alteration does not segregate with the affected phenotype of our GLC1G-linked family and was, therefore, considered as a polymorphism. Conversely, the amino acid change observed in the WDR36 gene was perfectly segregated in all the seven affected subjects of our original linked family. It was further absent in all public DNA variation databases as well as in a total of 238 normal-matched control subjects that subsequently tested in our laboratory. Therefore, WDR36 was considered as a novel causative gene at the GLC1G locus (23), and it was selected for further screening in a group of familial and sporadic cases.

Our initial screening of the WDR36 gene in a group of 130 unrelated POAG subjects revealed a total of 24 DNA variants of which 12 were amino acid coding and the remaining involved intronic alterations (23). Four of the 12 coding variations (N355S, A449T, R529Q, and D658G) were observed in a total of 17 glaucoma subjects (5.02%) of whom 11 (65%) showed typical high-tension POAG and the remaining 6 (35%) presented with NTG (23). In silico analysis showed that the observed mutations are evolutionarily conserved between the WDR36 orthologs in human, chimp, rat, mouse, and dog. Three other amino acid changes (L25P, A163V, and H212P) were

also observed significantly more in the POAG subjects (11.55%) as compared to the normal-matched control subjects (2.21%). Therefore, these alterations were considered as potential disease-susceptibility mutations, and their presence in these individuals probably serves as another contributing risk factor for POAG (23). In addition to these amino acid alterations, a number of other silent and intronic variations were exclusively observed in the POAG subjects that were not present in any of the normal control subjects. Although the exact role of such DNA alterations in the etiology of POAG is currently unclear, these variations may one day prove to be the silent partner of this complicated clinical phenotype.

WDR36 Screening in Different Populations

Further to our publication of WDR36 as a new gene for POAG (23), other investigators screened this gene in their respective POAG population. In addition to 130 British and American POAG subjects that we originally screened, another 311 FrenchCanadian (144), 309 German (145), and 118 North-American (146) glaucoma subjects have also been screened for this gene. As of this writing (see Table 6), a total of 868 unrelated POAG subjects have been screened for WDR36, and altogether, a total of 67 variants have been identified. Of these, 19 amino acid alterations are predicted as disease-causing mutations (11.23% in patients and 0% in controls), 4 considered as synonymous variations (2.36% in patients and 0% in controls), and an additional 5 amino acid changes were classified as potential disease-susceptibility mutations (10.32% in patients and 5.7% in controls) (23,144–146).

A number of WDR36 mutations and DNA variations have been observed in different populations. The common disease-causing mutation of A449T has been identified in all the four populations studied while N355S and R529Q mutations were reported only in two and three studies, respectively (see Table 6). So far, the two mutations of I604V and A449T with 2.54% and 1.3% frequency in glaucoma patients, respectively, are the most recurrent disease-causing mutations. Among potential disease-susceptibility mutations, L25P (2.9% of patients vs. 1.2% controls) and H212P (2.9% of patients vs. 1.35% controls) are the two most frequently observed variants.

So far, meta-analysis from four different studies suggests that mutations in WDR36 may be responsible for between 10% and 17% of glaucoma subjects (23,144–146). These studies not only confirmed the role of WDR36 as a glaucoma-causing gene but also validated its importance in augmenting glaucoma phenotypes.

We sequenced a number of affected and normal subjects from a large Oregon adultonset POAG family that is linked to the GLC1G locus. However, no disease-causing mutations were identified in the coding exons and the immediate adjacent flanking sequences of the WDR36 gene in this family (147). There are four possibilities for this observation: First, a mutation within the non-coding regions, promoter, splicing sites, or un-translated regions or probably presence of large and undetected DNA rearrangements (i.e., insertion, deletion, or duplication) within the WDR36 gene may still be responsible for the POAG phenotype in this pedigree. Second, this family may not be linked to the GLC1G locus as one of the affected members in this pedigree (FIV:14) showed recombination for the entire of the GLC1G region. Third, occurrence

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Table 6

Distribution of 28 Coding Mutant Variants Identified in the WDR36 Gene

of excessive homozygousity in a group of highly polymorphic DNA markers and presence of multiple and distanced branches as well as the large size of this family may all suggest that more than one gene is involved in etiology of the affected subjects of this family. Fourth, a second POAG gene may lie in the close proximity of WDR36 and mutations in this gene could be responsible for the glaucoma phenotype in this family (see Fig. 2). Further investigation into molecular genetics of this family is still needed to resolve these issues.