Variants in myelin regulatory factor (MYRF) cause autosomal dominant and syndromic nanophthalmos in humans and retinal degeneration in mice.

Nanophthalmos is a rare, potentially devastating eye condition characterized by small eyes with relatively normal anatomy, a high hyperopic refractive error, and frequent association with angle closure glaucoma and vision loss. The condition constitutes the extreme of hyperopia or farsightedness, a common refractive error that is associated with strabismus and amblyopia in children. NNO1 was the first mapped nanophthalmos locus. We used combined pooled exome sequencing and strong linkage data in the large family used to map this locus to identify a canonical splice site alteration upstream of the last exon of the gene encoding myelin regulatory factor (MYRF c.3376-1G>A), a membrane bound transcription factor that undergoes autoproteolytic cleavage for nuclear localization. This variant produced a stable RNA transcript, leading to a frameshift mutation p.Gly1126Valfs*31 in the C-terminus of the protein. In addition, we identified an early truncating MYRF frameshift mutation, c.769dupC (p.S264QfsX74), in a patient with extreme axial hyperopia and syndromic features. Myrf conditional knockout mice (CKO) developed depigmentation of the retinal pigment epithelium (RPE) and retinal degeneration supporting a role of this gene in retinal and RPE development. Furthermore, we demonstrated the reduced expression of Tmem98, another known nanophthalmos gene, in Myrf CKO mice, and the physical interaction of MYRF with TMEM98. Our study establishes MYRF as a nanophthalmos gene and uncovers a new pathway for eye growth and development.

BetaB2-crystallin mutations associated with cataract and glaucoma leads to mitochondrial alterations in lens epithelial cells and retinal neurons.

Crystallin proteins are the most prominent protein of the lens and have been increasingly shown to play critical roles in other tissues, especially the retina. Members of all 3 sub-families of crystallins, alpha-, beta- and gamma-crystallins have been reported in the retina during diabetes, traumatic injury and other retinal diseases. While their specific role in the retina is still unclear and may vary, beta-crystallin proteins have been shown to play a critical role in ganglion cell survival following trauma. We recently reported the correlation between a gene conversion in the betaB2-crystallin gene and a phenotype of familial congenital cataract. Interestingly, in half of the patients, this phenotype was associated with glaucoma. Taken together, these data suggested that the mutations we recently reported could have an impact on the role of betaB2-crystallin in both lens epithelial cells and retinal neurons. Consistent with this hypothesis, we show in the current study that the gene conversion leading to an amino acid conversion lead to a loss of solubility and a change of subcellular localization of betaB2-crystallin in both cell types. While the overall observations were similar in both cell types, there were some important nuances between them, suggesting different roles and regulation of betaB2-crystallin in lens cells versus retinal neurons. The data reported in this study strongly support a significant role of betaB2-crystallin in both lenticular and retinal ocular tissues and warrant further analysis of its regulation and its impact not only in cataract formation but also in retinal neurodegenerative diseases.

Genome-wide association study and meta-analysis of intraocular pressure.

Elevated intraocular pressure (IOP) is a major risk factor for glaucoma and is influenced by genetic and environmental factors. Recent genome-wide association studies (GWAS) reported associations with IOP at TMCO1 and GAS7, and with primary open-angle glaucoma (POAG) at CDKN2B-AS1, CAV1/CAV2, and SIX1/SIX6. To identify novel genetic variants and replicate the published findings, we performed GWAS and meta-analysis of IOP in >6,000 subjects of European ancestry collected in three datasets: the NEI Glaucoma Human genetics collaBORation, GLAUcoma Genes and ENvironment study, and a subset of the Age-related Macular Degeneration-Michigan, Mayo, AREDS and Pennsylvania study. While no signal achieved genome-wide significance in individual datasets, a meta-analysis identified significant associations with IOP at TMCO1 (rs7518099-G, p = 8.0 × 10(-8)). Focused analyses of five loci previously reported for IOP and/or POAG, i.e., TMCO1, CDKN2B-AS1, GAS7, CAV1/CAV2, and SIX1/SIX6, revealed associations with IOP that were largely consistent across our three datasets, and replicated the previously reported associations in both effect size and direction. These results confirm the involvement of common variants in multiple genomic regions in regulating IOP and/or glaucoma risk.

Congenital cataracts: de novo gene conversion event in CRYBB2.

PURPOSE: To identify the cause of congenital cataracts in a consanguineous family of Ashkenazi Jewish ancestry. METHODS: We performed genome-wide linkage analysis and whole-exome sequencing for the initial discovery of variants, and we confirmed the variants using gene-specific primers and Sanger sequencing. RESULTS: We found significant evidence of linkage to chromosome 22, under an autosomal dominant inheritance model, with a maximum logarithm of the odds (LOD) score of 3.91 (16.918 to 25.641 Mb). Exome sequencing identified three nonsynonymous changes in the CRYBB2 exon 5 coding sequence that are consistent with the sequence of the corresponding region of the pseudogene CRYBB2P1. The identification of these changes was complicated by possible mismapping of some mutated CRYBB2 sequences to CRYBB2P1. Sequencing with gene-specific primers confirmed that the changes--rs2330991, c.433 C>T (p.R145W); rs2330992, c.440A>G (p.Q147R); and rs4049504, c.449C>T (p.T150M)--present in all ten affected family members are located in CRYBB2 and are not artifacts of cross-reaction with CRYBB2P1. We did not find these changes in six unaffected family members, including the unaffected grandfather who contributed the affected haplotype, nor did we find them in the 100 Ashkenazi Jewish controls. CONCLUSIONS: Our data are consistent with a de novo gene conversion event, transferring 270 base pairs at most from CRYBB2P1 to exon 5 of CRYBB2. This study highlights how linkage mapping can be complicated by de novo mutation events, as well as how sequence-analysis pipeline mapping of short reads from next-generation sequencing can be complicated by the existence of pseudogenes or other highly homologous sequences.

Psychometric analysis of Marathi version of an updated European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Head and Neck Module (EORTC QLQ-H&N43).

BACKGROUND: The aim of this study was to perform a psychometric analysis of the Marathi version of an updated European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Head and Neck Module (EORTC QLQ-H&N43) in patients of head and neck squamous cell carcinoma (HNSCC). MATERIAL AND METHODS: After an institutional ethics committee approval and linguistic validation, the Marathi version of EORTC QLQ-H&N43 was served to consecutive eligible, Marathi-speaking HNSCC patients of oral cavity, oropharynx, hypopharynx and larynx, registered from August 2019 to March 2021 and who consented to the study. Performance status scale was used for perceptive assessment of organ function. Psychometric analysis was performed using SYSTAT v. 12 (Cranes software, Bengaluru). RESULTS: A total of 129 patients participated in the study with a median age of 67 years and the man-to-woman ratio of 100:29. The commonest sub-site reported was the oral cavity (61.24%) and the majority were in stage IV disease (69.76%). Ninety patients completed the questionnaire before being started on active oncology treatment at our center. 39 patients completed the questionnaire at pre-treatment and first follow-up. The questionnaire was filled on their own (module) by 32.55% of patients while 67.44% of patients required some kind of assistance (schedule). Internal consistency was moderate to high. The criterion and construct validities were acceptable. The questionnaire was sensitive to change with stage and time. No significant difference was observed in module versus schedule subgroup except for 2 out of 19 item-scales. CONCLUSION: Marathi translation of EORTC QLQ-H&N43 is a sensitive, reliable and valid tool when applied to patients of HNSCC. It can be considered as a schedule.

Association between depressive symptoms and socioeconomic status in head and neck cancer patients attending rural tertiary health care center.

AIM: To study the association between depressive symptoms and socio-economic status (SES) in patients with head and neck cancer (HNC). MATERIALS AND METHODS: Histopathology-proven non-metastatic and non-recurrent HNC patients attending radiation oncology services in February and June 2021 who consented to the study were evaluated with an interview. Hamilton Depression Rating Scale (HDRS) and modified BG Prasad classification were used to assess depressive symptoms and SES, respectively. Statistical analysis was done using SYSTAT version 12 (by Crane's software, Bangalore). RESULTS: The study cohort comprised 100 patients. The median age was 55 years (minimum: 28, maximum: 86) with a male-to-female ratio of 2.57:1. The majority of patients were educated beyond middle-school level (44%), of middle SES (47%), oral cavity subsite (68%), stage IVA (36%) disease, and were on treatment (79%). HDRS scores showed absent, moderate, and severe depressive symptoms in 21, 63, and 16 patients, respectively, that is, the prevalence of depressive symptoms was 79%. Lower socioeconomic strata showed a significantly higher prevalence of severe depressive symptoms (P-value = 0.01). HDRS score was significantly higher in females (P-value = 0.024) and in patients who were illiterate or were educated less than middle-school level (P-value = 0.010). No statistically significant difference was found between HDRS score and age of patient, stage and site of disease, and on treatment or follow-up status. CONCLUSION: Depressive symptoms are common in patients with head and neck cancer. Low socioeconomic status (SES), female gender, and education less than middle school are associated with severe depressive symptoms. Psycho-oncological and psychotherapeutic interventions are the need of the hour.

Serum 25-hydroxy vitamin-D levels in head and neck cancer chemoradiation therapy: Potential in cancer therapeutics.

Background: To evaluate the relation between serum-25-hydroxy Vitamin-D levels (S25OHVDL) and concurrent chemoradiation therapy (CTRT) toxicities in patients of head and neck squamous cell cancer (HNSCC). Methods: After an institutional ethics committee approval, consecutive HNSCC patients who received radical/adjuvant CTRT were prospectively evaluated. Patients were assessed for CTRT toxicities using Common Terminology Criteria for Adverse Events version 5.0 (CTCAE-v5.0) and the response was evaluated according to Response Evaluation Criteria In Solid Tumors version 1.1 (RECIST-1.1). S25OHVDL was assessed at the time of the first follow-up. Patients were divided into group A (Optimal) and group B (suboptimal) according to S25OHVDL. The treatment toxicities were correlated with S25OHVDL. Results: Twenty-eight patients were evaluated for the study. S25OHVDL was optimal in eight (28.57%) and suboptimal in 20 patients (71.42%). Mucositis and radiation dermatitis were significantly more in subgroup B (P-value 0.0011 and 0.0505, respectively). Relatively lower but nonsignificant hemoglobin and peripheral white blood cell counts were observed in subgroup B. Conclusion: Suboptimal S25OHVDL was associated with significantly more skin and mucosal toxicities in HNSCC patients treated with CTRT.

Subjective and perceptive assessment of speech/voice and swallowing function before and after radiation therapy in patients of head-and-neck squamous cell cancer.

Aim: To prospectively assess subjective and perceptive speech/voice and swallowing function before and after radiation therapy (RT) in patients of head-and-neck squamous cell cancer (HNSCC). Materials and Methods: The study cohort comprised eligible consecutive HNSCC patients planned for curative RT from April 2018 to July 2018 who consented for the study. Prospective evaluation of speech/voice and swallowing function was done before and after RT. For subjective and perceptive evaluation of speech/voice, speech handicap index (SHI) and Grade, Roughness, Asthenia, Breathiness, and Strain (GRABS) Scale was used, respectively. For subjective and perceptive evaluation of swallowing, M D Anderson Dysphagia Inventory (MDADI) and Performance Status Scale for head and neck (PSSHN) were used, respectively. All patients were taught speech/voice and swallowing exercises before RT. Statistical analysis was performed using SYSTAT version-12 (Cranes software, Bengaluru). Results: The study cohort comprised 30 patients of HNSCC with a median age of 57 years and male-to-female ratio of 4:1. The most common subsite was the oral cavity (43.33%) and a majority (76.66%) presented in the locally advanced stage. Post-RT there was significant improvement in speech/voice function (SHI P = 0.0006, GRABS score P = 0.003). Perceptive assessment of swallowing function by PSSHN showed significant improvement (P = 0.0032), but subjective assessment by MDADI showed no significant (P = 0.394) improvement until the first follow-up. Conclusion: Speech/voice function improved significantly after radiotherapy when combined with rehabilitation exercises. Swallowing function did not improve till the first follow-up. Future studies with the large number of patients and long-term follow-up are needed to document the changes in organ function.

Pretreatment hematological parameters as predictors of tumor granulocyte-colony-stimulating factor expression in patients of head-and-neck squamous cell carcinoma.

Background: Tumor secreting granulocyte-colony-stimulating factor (G-CSF) and/or G-CSF therapy has been documented as a poor prognostic factor. Tumor G-CSF study is a relatively costly and sparsely available investigation. Therefore, this study was undertaken to predict tumor G-CSF score from pretreatment hematological parameters (PTHP) in patients of head-and-neck squamous cell carcinoma (HNSCC). Materials and Methods: This pilot study was performed after institutional ethics committee approval. Consecutive nonmetastatic HNSCC patients of oral cavity, oropharynx, hypopharynx, and larynx registered from February to December 2019 were analyzed. Patients whose PTHP and formalin-fixed-paraffin-embedded tissue were available, were included. PTHP (absolute neutrophil count [ANC], absolute monocyte count [AMC], absolute lymphocyte count [ALC], neutrophil-to-lymphocyte ratio [NLR], and platelet-to-lymphocyte ratio [PLR]) done before any active oncology treatment, were noted. A semiquantitative tumor G-CSF score was calculated. Tumor G-CSF score and PTHP were correlated with clinicopathological factors. Statistical analysis was performed using SYSTAT version 12. Results: Data of 47 eligible patients were analyzed. The median age at presentation was 60 years. The male-to-female ratio was 43:4. The most common head-and-neck subsite was oropharynx (31.92%), and majority of patients presented with Stage IVA disease (51.1%). Higher tumor G-CSF score was significantly associated with a higher T-stage (P = 0.013). Tumor G-CSF score was directly proportional to ANC, AMC, and ALC while it was inversely proportional to NLR and PLR. Regression equations to predict the tumor G-CSF score when PTHP are known, were determined. Conclusions: PTHP can predict the tumor G-CSF score which may guide G-CSF-directed therapy. Future studies with large number of patients are needed to elucidate its clinical use.

Oncology treatment outcome in human immunodeficiency virus-positive cancer cervix patients: Where we are and what we achieved?

Objective: To find out the epidemiological factors and oncology treatment outcome in human immunodeficiency virus-positive cancer cervix patients (HPCCP). Materials and Methods: After institutional ethics committee approval, hospital case records of HPCCP registered at the radiation oncology department from January 2011 to December 2018 were retrospectively studied. Results: The case records of 22 eligible HPCCP were studied. Median age at presentation was 42.5 years. 90.90% of the patients were below 55 years of age. The duration of symptom was <3 months in 63.64% of patients. 68.18% of the patients were FIGO Stage III. Only 11 patients completed the planned treatment. Total target equivalent dose of 2 Gy per fraction delivered was 66 Gy. Seven patients had complete response. Four patients had local recurrence. Median disease-free and overall survival was 27 (14-38) and 18 months (2-48), respectively. Conclusion: HPCCP present at relatively early age and advanced stage despite short symptom duration. Poor patient compliance and treatment alteration have led to suboptimal outcome.

Marathi Translation and Linguistic Validation of an Updated European Organization for Research and Treatment of Cancer Quality of Life Module for Head and Neck.

Aim This study was aimed to translate an updated European Organization for Research and Treatment of Cancer (EORTC) quality of life module for head and neck (EORTC QLQ-H&N43) in grammatically and conceptually acceptable Marathi language and its linguistic validation. Materials and Methods Approval was obtained from the Institutional Ethics Committee. The permission for translation was obtained from the EORTC translation unit (TU). The EORTC guidelines for the translation were followed to form a translation for pilot testing which was administered to 10 Marathi speaking head and neck squamous cell cancer (HNSCC) patients who gave informed written consent for the participation in the study. Patients were interviewed personally. The final Marathi translation was prepared and sent to EORTC TU for approval. Statistical analysis was performed using SYSTAT version 12 by Cranes software, Bengaluru, Karnataka, India. Results After getting permission, the translation files were received from EORTC TU, including Marathi EORTC QLQ-H&N35 for reference. Two forward translations, reconciled translation, back translations, first interim translation, translation for proof editing, and second interim translation (SIT) were prepared. This SIT was pilot tested in 10 Marathi-speaking HNSCC patients. Each patient was interviewed regarding difficulty in answering, confusing or offensive word, and reframing sentence. The questionnaire was well understood by patients reflecting its linguistic validity. After incorporating the changes as per the patient's interview, updated translation was prepared and sent to EORTC TU which was accepted and approved by EORTC. The psychometric analysis of pilot testing showed that the questionnaire is acceptable. Conclusion Marathi translation of EORTC QLQ-H&N43 is well accepted and understandable. It can be used for future studies.

Novel TMEM98, MFRP, PRSS56 variants in a large United States high hyperopia and nanophthalmos cohort.

Nanophthalmos is a rare condition defined by a small, structurally normal eye with resultant high hyperopia. While six genes have been implicated in this hereditary condition (MFRP, PRSS56, MYRF, TMEM98, CRB1,VMD2/BEST1), the relative contribution of these to nanophthalmos or to less severe high hyperopia (≥ + 5.50 spherical equivalent) has not been fully elucidated. We collected probands and families (n = 56) with high hyperopia or nanophthalmos (≤ 21.0 mm axial length). Of 53 families that passed quality control, plausible genetic diagnoses were identified in 10/53 (18.8%) by high-throughput panel or pooled exome sequencing. These include 1 TMEM98 family (1.9%), 5 MFRP families (9.4%), and 4 PRSS56 families (7.5%), with 4 additional families having single allelic hits in MFRP or PRSS56 (7.5%). A novel deleterious TMEM98 variant (NM_015544.3, c.602G>C, p.(Arg201Pro)) segregated with disease in 4 affected members of a family. Multiple novel missense and frameshift variants in MFRP and PRSS56 were identified. PRSS56 families were more likely to have choroidal folds than other solved families, while MFRP families were more likely to have retinal degeneration. Together, this study defines the prevalence of nanophthalmos gene variants in high hyperopia and nanophthalmos and indicates that a large fraction of cases remain outside of single gene coding sequences.

Effects of timolol on MYOC, OPTN, and WDR36 RNA levels.

OBJECTIVES: To evaluate if timolol affects expression of 3 open-angle glaucoma genes and to study its ability to modulate dexamethasone-induced up-regulation of MYOC. METHODS: We used quantitative polymerase chain reaction assay of glaucoma gene transcript levels from human trabecular meshwork (HTM) cultures exposed to 3 different doses of timolol. Three HTM cell cultures were grown with or without 1 of 3 timolol doses in the presence or absence of dexamethasone. RESULTS: All 3 concentrations of timolol reduced MYOC RNA levels in 1 HTM culture compared with an untreated control and showed negligible effects in the other 2 cultures. Timolol had no effect on dexamethasone-induced MYOC transcript levels in any of the 3 cultures. Timolol, dexamethasone, and dexamethasone plus timolol had a negligible effect on OPTN and WDR36 RNA levels. CONCLUSIONS: Timolol can reduce MYOC RNA levels in HTM cultures from some individuals. Timolol does not alter OPTN or WDR36 levels or ameliorate MYOC induction by dexamethasone in vitro. CLINICAL RELEVANCE: It remains to be determined whether timolol could reduce production of misfolded myocilin protein by HTM cells in individuals with MYOC missense mutations. A broader survey of interindividual variation in response to timolol as well as mechanistic studies are still needed before potential therapeutic implications can be explored.

Recurrent Myocilin Asn480Lys glaucoma causative mutation arises de novo in a family of Andean descent.

PURPOSE: To search for MYOC mutations in Peruvian primary open angle glaucoma (POAG) families. PATIENTS AND METHODS: Two patients from each of the 11 POAG Peruvian families were screened for sequence variants in MYOC coding exons by conformational sensitive gel electrophoresis and sequencing was performed on the samples indicating probable sequence changes. RESULTS: We detected 2 families bearing distortions of conformational sensitive gel electrophoresis indicating mutations. Sequencing of these samples revealed coding sequence changes. A native Andean descent family presented with a MYOC mutation, Asn480Lys (C-->G at nucleotide 1440). This is different from the previously reported C-->A change at nucleotide 1440 that causes Asn480Lys in 2 unrelated French and Dutch families with glaucoma of variable expressivity, and indicates a third independent event. A second family of admixed origin showed the presence of the known Arg76Lys polymorphism. CONCLUSIONS: In the study of MYOC variants in 11 POAG Peruvian families, we have found a family of ethnically admixed origin with polymorphism Arg76Lys and a family of Andean descent bearing a third event of the Asn480Lys, the MYOC mutation that has been reported in the highest number of POAG patients (>80 cases). Analysis of this family could contribute with information about disease manifestation, progression, and treatment response in the context of a distinct genetic background and also climatic, altitude, and socioeconomical conditions.

Variation in optineurin (OPTN) allele frequencies between and within populations.

PURPOSE: To evaluate the extent to which mutations in the optineurin (OPTN) glaucoma gene play a role in glaucoma in different populations. METHODS: Case-controlled study of OPTN sequence variants in individuals with or without glaucoma in populations of different ancestral origins and evaluate previous OPTN reports. We analyzed 314 subjects with African, Asian, Caucasian and Hispanic ancestries included 229 cases of primary open-angle glaucoma, 51 cases of juvenile-onset open-angle glaucoma, 33 cases of normal tension glaucoma, and 371 controls. Polymerase chain reaction-amplified OPTN coding exons were resequenced and case frequencies were compared to frequencies in controls matched for ancestry. RESULTS: The E50K sequence variant was identified in one individual from Chile with normal tension glaucoma, and the 691_692insAG variant was found in one Ashkenazi Jewish individual from Russia. The R545Q variant was found in two Asian individuals with primary open-angle glaucoma; one of Filipino ancestry and one of Korean ancestry. In addition to presenting OPTN allele frequencies for Caucasian and Asian populations that have been the subject of previous reports, we also present information for populations of Hispanic and black African ancestries. CONCLUSIONS: Our study contributes additional evidence to support the previously reported association of the OPTN E50K mutation with glaucoma. After finding an additional 691_692insAG OPTN variant, we can still only conclude that this variant is rare. Combined analysis of our data with data from more than a dozen other studies indicates no association of R545Q with glaucoma in most populations. Those same studies disagree in their conclusions regarding the role of M98K in glaucoma. Our analysis of the combined data provides statistically significant evidence of association of M98K with normal tension glaucoma in Asian populations, but not in Caucasian populations; however, the validity of this conclusion is questionable because of large differences in allele frequencies between and within populations. It is currently not possible to tell how much of the underlying cause of the allele frequency difference is attributable to demographic, technical, or ascertainment differences among the studies.

Differential expression profile prioritization of positional candidate glaucoma genes: the GLC1C locus.

OBJECTIVES: To develop and apply a model for prioritization of candidate glaucoma genes. METHODS: This Affymetrix GeneChip (Affymetrix, Santa Clara, Calif) study of gene expression in primary culture human trabecular meshwork cells uses a positional differential expression profile model for prioritization of candidate genes within the GLC1C genetic inclusion interval. RESULTS: Sixteen genes were expressed under all conditions within the GLC1C interval. TMEM22 was the only gene within the interval with differential expression in the same direction under both conditions tested. Two genes, ATP1B3 and COPB2, are of interest in the context of a protein-misfolding model for candidate selection. SLC25A36, PCCB, and FNDC6 are of lesser interest because of moderate expression and changes in expression. Transcription factor ZBTB38 emerges as an interesting candidate gene because of the overall expression level, differential expression, and function. CONCLUSIONS: Only 1 gene in the GLC1C interval fits our model for differential expression under multiple glaucoma risk conditions. The use of multiple prioritization models resulted in filtering 7 candidate genes of higher interest out of the 41 known genes in the region. CLINICAL RELEVANCE: This study identified a small subset of genes that are most likely to harbor mutations that cause glaucoma linked to GLC1C.

Gene expression profile of human trabecular meshwork cells in response to long-term dexamethasone exposure.

PURPOSE: Topical use of dexamethasone has long been associated with steroid induced-glaucoma, although the mechanism is unknown. We applied a strict filtering of comparative microarray data to more than 18,000 genes to evaluate global gene expression of cultured human trabecular meshwork cells in response to treatment with dexamethasone. METHODS: Three human trabecular meshwork cell primary cultures from nonglaucomatous donors were incubated with and without dexamethasone for 21 days. Relative gene expression was evaluated by analysis of U133A GeneChip and the results validated using quantitative polymerase chain reaction (PCR). RESULTS: Application of strict filtering to include only genes with statistically significant differences in gene expression across all three trabecular meshwork cell cultures produced a list of 1,260 genes. Significant changes in signal level were observed, including 23 upregulated and 18 downregulated genes that changed greater than three fold in each of three cell cultures. Using quantitative PCR we found changes greater than a thousand fold for two genes (SLP1 and SAA2) and changes greater than a hundred fold for another five genes (ANGPTL7, MYOC, SAA1, SERPINA3, and ZBTB16). CONCLUSIONS: Expression changes in trabecular meshwork cells in response to dexamethasone treatment indicate that a group of actins and actin-associated proteins are involved in the development of cross-linked actin networks that form in response to dexamethasone. A trend was identified toward decreased expression of protease genes accompanied by an increased expression of protease inhibitors. Such a trend in nonproteasomal proteolysis conceivably affects gene product levels above the level of transcription. Only two genes, MYOC and IGFBP2, showed significantly elevated expression after dexamethasone treatment in our study and the other three previously published reports of primary culture trabecular meshwork cell gene expression.

A prospective cross-sectional study of fetal middle cerebral artery peak systolic velocity in a normal obstetric population attending an Indian Medical College.

PURPOSE: Fetal middle cerebral artery peak systolic velocity (MCA-PSV) is now an established method of noninvasive diagnosis of moderate to severe fetal anemia of different origins. This being a population-based parameter, it may have different values in individuals from different locations. A standard local reference range of normal individuals specific to each geographic locality is therefore needed. MATERIALS AND METHODS: Fetal MCA-PSV was measured in 1,015 healthy pregnant women attending the antenatal ultrasound clinic at least once between 12 and 40 weeks of gestation (35 patients per week of gestation). Statistical analysis was done using the SPSS 12 software. RESULTS: A positive correlation between MCA-PSV and gestational age was found. MCA-PSV was seen to increase with advancing gestational age. The Tukey-Kramer multiple comparisons test showed a normal distribution that was highly significant (p < 0.01). The multiples of median values of fetal MCA-PSV to label a fetus as severely anemic were consistently and significantly lower than internationally accepted values. CONCLUSIONS: A normal reference range of fetal MCA-PSV for local use was thus scientifically and successfully constructed. Significant differences between local values and internationally accepted value were found.

Developmental validation of the PrepFiler Forensic DNA Extraction Kit for extraction of genomic DNA from biological samples.

The PrepFiler Forensic DNA Extraction Kit enables isolation of genomic DNA from a variety of biological samples. The kit facilitates reversible binding of DNA with magnetic particles resulting in high DNA recovery from samples with very low and high quantities of biological materials: 0.1 and 40 microL of human blood (donor 2) provided 14 and 2883 ng of DNA, respectively. Following the revised SWGDAM guidelines, performance of the developed method was investigated using different sample types including saliva on swabs, semen stains on cotton fabric, samples exposed to environment, samples with polymerase chain reaction (PCR) inhibitors, blood stains (on denim, cotton cloth, and FTA paper), and touch evidence-type samples. DNA yields for all samples tested were equal or better than those obtained by both phenol-chloroform extraction and commercial kits tested. DNA obtained from these samples was free of detectable PCR inhibitors. Short tandem repeat profiles were complete, conclusive, and devoid of PCR artifacts.

Mutations in TCF8 cause posterior polymorphous corneal dystrophy and ectopic expression of COL4A3 by corneal endothelial cells.

Posterior polymorphous corneal dystrophy (PPCD, also known as PPMD) is a rare disease involving metaplasia and overgrowth of corneal endothelial cells. In patients with PPCD, these cells manifest in an epithelial morphology and gene expression pattern, produce an aberrant basement membrane, and, sometimes, spread over the iris and nearby structures in a way that increases the risk for glaucoma. We previously mapped PPCD to a region (PPCD3) on chromosome 10 containing the gene that encodes the two-handed zinc-finger homeodomain transcription factor TCF8. Here, we report a heterozygous frameshift mutation in TCF8 that segregates with PPCD in the family used to map PPCD3 and four different heterozygous nonsense and frameshift mutations in TCF8 in four other PPCD probands. Family reports of inguinal hernia, hydrocele, and possible bone anomalies in affected individuals suggest that individuals with TCF8 mutations should be examined for nonocular anomalies. We detect transcripts of all three identified PPCD genes (VSX1, COL8A2, and TCF8) in the cornea. We show presence of a complex (core plus secondary) binding site for TCF8 in the promoter of Alport syndrome gene COL4A3, which encodes collagen type IV alpha 3, and we present immunohistochemical evidence of ectopic expression of COL4A3 in corneal endothelium of the proband of the original PPCD3 family. Identification of TCF8 as the PPCD3 gene provides a valuable tool for the study of critical gene regulation events in PPCD pathology and suggests a possible role for TCF8 mutations in altered structure and function of cells lining body cavities other than the anterior chamber of the eye. Thus, this study has identified TCF8 as the gene responsible for approximately half of the cases of PPCD, has implicated TCF8 mutations in developmental abnormalities outside the eye, and has presented the TCF8 regulatory target, COL4A3, as a key, shared molecular component of two different diseases, PPCD and Alport syndrome.