Impact of deep phenotyping: high diagnostic yield in a diverse pediatric population of 172 patients through clinical whole-genome sequencing at a single center.

Background: Pediatric patients with undiagnosed conditions, particularly those suspected of having Mendelian genetic disorders, pose a significant challenge in healthcare. This study investigates the diagnostic yield of whole-genome sequencing (WGS) in a pediatric cohort with diverse phenotypes, particularly focusing on the role of clinical expertise in interpreting WGS results. Methods: A retrospective cohort study was conducted at Acibadem University's Maslak Hospital in Istanbul, Turkey, involving pediatric patients (0-18 years) who underwent diagnostic WGS testing. Clinical assessments, family histories, and previous laboratory and imaging studies were analyzed. Variants were classified and interpreted in conjunction with clinical findings. Results: The cohort comprised 172 pediatric patients, aged 0-5 years (62.8%). International patients (28.5%) were from 20 different countries. WGS was used as a first-tier approach in 61.6% of patients. The diagnostic yield of WGS reached 61.0%, enhanced by reclassification of variants of uncertain significance (VUS) through reverse phenotyping by an experienced clinical geneticist. Consanguinity was 18.6% of the overall cohort. Dual diagnoses were carried out for 8.5% of solved patients. Discussion: Our study particularly advocates for the selection of WGS as a first-tier testing approach in infants and children with rare diseases, who were under 5 years of age, thereby potentially shortening the duration of the diagnostic odyssey. The results also emphasize the critical role of a single clinical geneticist's expertise in deep phenotyping and reverse phenotyping, which contributed significantly to the high diagnostic yield.

A Retrospective Review of 18 Patients With Childhood-Onset Hereditary Spastic Paraplegia, Nine With Novel Variants.

BACKGROUND: Hereditary spastic paraplegias (HSPs) are a group of genetically heterogeneous neurodegenerative disorders. Our objective was to determine the clinical and molecular characteristics of patients with genetically confirmed childhood-onset HSPs and to expand the genetic spectrum for some rare subtypes of HSP. METHODS: We reviewed the charts of subjects with genetically confirmed childhood-onset HSP. The age at the disease onset was defined as the point at which the delayed motor milestones were observed. Delayed motor milestones were defined as being unable to hold the head up by four months, sitting unassisted by nine months, and walking independently by 17 months. If there were no delayed motor milestones, age at disease onset was determined by leg stiffness, frequent falls, or unsteady gait. Genetic testing was performed based on delayed motor milestones, progressive leg spasticity, and gait difficulty. The variant classification was determined based on the American College of Medical Genetics standard guidelines for variant interpretation. Variants of uncertain significance (VUS) were considered disease-associated when clinical findings were consistent with the previously described disease phenotypes for pathogenic variants. In addition, in the absence of another pathogenic, likely pathogenic, or VUS variant that could explain the phenotype of our cases, we concluded that the disease is associated with VUS in the HSP-causing gene. Segregation analysis was also performed on the parents of some patients to demonstrate the inheritance model. RESULTS: There were a total of 18 patients from 17 families. The median age of symptom onset was 18 months (2 to 84 months). The mean delay between symptom onset and genetic diagnosis was 5.8 years (5 months to 17 years). All patients had gait difficulty caused by progressive leg spasticity and weakness. Independent walking was not achieved at 17 months for 67% of patients (n = 12). In our cohort, there were two subjects each with SPG11, SPG46, and SPG 50 followed by single subject each with SPG3A, SPG4, SPG7, SPG8, SPG30, SPG35, SPG43, SPG44, SPG57, SPG62, infantile-onset ascending spastic paralysis (IAHSP), and spastic paraplegia and psychomotor retardation with or without seizures (SPPRS). Eight novel variants in nine patients were described. Two affected siblings had a novel variant in the GBA2 gene (SPG46), and one subject each had a novel variant in WASHC5 (SPG8), SPG11 (SPG11), KIF1A (SPG30), GJC2 (SPG44), ERLIN1 (SPG62), ALS2 (IAHSP), and HACE1 (SPPRS). Among the novel variants, the variant in the SPG11 was pathogenic and the variants in the KIF1A, GJC2, and HACE1 were likely pathogenic. The variants in the GBA2, ALS2, ERLIN1, and WASHC5 were classified as VUS. CONCLUSIONS: There was a significant delay between symptom onset and genetic diagnosis of HSP. An early diagnosis may be possible by examining patients with delayed motor milestones, progressive spasticity, gait difficulties, and neuromuscular weakness in the context of HSP. Eight novel variants in nine patients were described, clinically similar to the previously described disease phenotype associated with pathogenic variants. This study contributes to expanding the genetic spectrum of some rare subtypes of HSP.

Bi-allelic truncating variants in CASP2 underlie a neurodevelopmental disorder with lissencephaly.

Lissencephaly (LIS) is a malformation of cortical development due to deficient neuronal migration and abnormal formation of cerebral convolutions or gyri. Thirty-one LIS-associated genes have been previously described. Recently, biallelic pathogenic variants in CRADD and PIDD1, have associated with LIS impacting the previously established role of the PIDDosome in activating caspase-2. In this report, we describe biallelic truncating variants in CASP2, another subunit of PIDDosome complex. Seven patients from five independent families presenting with a neurodevelopmental phenotype were identified through GeneMatcher-facilitated international collaborations. Exome sequencing analysis was carried out and revealed two distinct novel homozygous (NM_032982.4:c.1156delT (p.Tyr386ThrfsTer25), and c.1174 C > T (p.Gln392Ter)) and compound heterozygous variants (c.[130 C > T];[876 + 1 G > T] p.[Arg44Ter];[?]) in CASP2 segregating within the families in a manner compatible with an autosomal recessive pattern. RNA studies of the c.876 + 1 G > T variant indicated usage of two cryptic splice donor sites, each introducing a premature stop codon. All patients from whom brain MRIs were available had a typical fronto-temporal LIS and pachygyria, remarkably resembling the CRADD and PIDD1-related neuroimaging findings. Other findings included developmental delay, attention deficit hyperactivity disorder, hypotonia, seizure, poor social skills, and autistic traits. In summary, we present patients with CASP2-related ID, anterior-predominant LIS, and pachygyria similar to previously reported patients with CRADD and PIDD1-related disorders, expanding the genetic spectrum of LIS and lending support that each component of the PIDDosome complex is critical for normal development of the human cerebral cortex and brain function.

TRAPPC9-Related Intellectual Disability: Report of Two New Cases and Review of the Literature.

Introduction: Hereditary forms of intellectual disability (ID), an estimated prevalence ranging between 1% and 3% in the general population, are among the most important problems in health care. Especially, autosomal-recessive ID has a very heterogeneous molecular basis and a lack of specific phenotypic features. Methods: Here, we report on two unrelated patients with autosomal-recessive ID, microcephaly, and autistic features and review the patients with TRAPPC9-related ID. Whole-exome sequencing and array CGH were performed for molecular diagnosis of the patients. Results: The first case has a microdeletion on chromosome 8q24.23-q24.3 region which is 1.7 Mb in length and includes the last 5 exons of TRAPPC9, and c.3435delG [p.Thr1146Profs*8] deletion. The second case has a homozygous missense c.623A>C (p.His208Pro) variant in TRAPPC9 which is detected by means of whole-exome sequencing study of the proband. We also reviewed the clinical findings and mutation spectrum of all patients with TRAPPC9-related ID reported so far. Conclusions: Our study showed that the most consistent clinical findings for TRAPPC9-related ID are ID, microcephaly, and some structural brain MRI abnormalities. The mutations in the TRAPPC9 are scattered throughout all exons of TRAPPC9 indicating there is no hot spot mutation region in this gene.

Frequency of germline pathogenic variants in breast cancer predisposition genes among young Turkish breast cancer patients.

PURPOSE: One of the most important risk factors for hereditary breast and ovarian cancer is young age. We aim to report the frequency of pathogenic/likely pathogenic variants in breast cancer predisposing genes in young (≤ 40 years old) breast cancer patients who undergone 26-gene inherited cancer panel at our Breast Health Center. METHODS: Medical records of breast cancer patients who were referred to genetic counseling based on NCCN criteria and were ≤ 40 years of age are reviewed. The frequency of germline pathogenic/likely pathogenic variants who undergone 26-gene inherited cancer panel was analyzed. RESULTS: Among 414 breast cancer patients who were ≤ 40 years of age, 308 undergone 26-gene inherited cancer panel and 108 had next generation sequencing (NGS)-based BRCA 1 and 2 genetic testing. Median age was 35 (22-40), Family history in first degree relatives was present in 14% of patients. Forty-five percent of patients met one of the NCCN criteria for genetic testing, 41% of them met two criteria, and 14% of patients fulfilled ≥ 3 criteria. Seventy pathogenic/likely pathogenic variants (PV/LPV) were found in 65 (21%) patients. PV/LPs in BRCA genes and non-BRCA genes represented 53% and 44% of all PV/LPVs, accounting for 12% and 10% of patients in the study cohort respectively. Two PVs were present in 5 patients and eleven PVs were novel. The most common PVs were in BRCA 1 (n:18), BRCA 2 (n:19), ATM (n:7), CHEK2 (n:7) and TP53 (n:5) genes. Thirty-one percent of the patients with triple-negative tumors and 25% of the patients with hormone receptor-positive tumors had PV/LPVs with panel testing. Family history in first degree relatives (p = 0.029), the number of met NCCN criteria (p = 0.036) and axillary nodal involvement (p = 0.000) were more common in patients with PVs. When combined with patient group (n:106) who had only BRCA1 and 2 gene testing, 16% of Turkish breast cancer patients ≤ 40 years of age had PVs in BRCA genes. CONCLUSION: One fifth of Turkish breast cancer patients ≤ 40 years of age had at least one PV/LPV in breast cancer predisposing genes with 26-gene inherited cancer panel. The frequency of PV/LPVs was higher in triple-negative young-onset patients compared to hormone receptor and Her-2 positive subtypes. Our findings regarding to frequency PV/LPVs in BRCA 1/2 and non-BRCA genes in young-onset breast cancer patients are in line with the literature.

Evaluation of AlphaFold structure-based protein stability prediction on missense variations in cancer.

Identifying pathogenic missense variants in hereditary cancer is critical to the efforts of patient surveillance and risk-reduction strategies. For this purpose, many different gene panels consisting of different number and/or set of genes are available and we are particularly interested in a panel of 26 genes with a varying degree of hereditary cancer risk consisting of ABRAXAS1, ATM, BARD1, BLM, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, MEN1, MLH1, MRE11, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53, and XRCC2. In this study, we have compiled a collection of the missense variations reported in any of these 26 genes. More than a thousand missense variants were collected from ClinVar and the targeted screen of a breast cancer cohort of 355 patients which contributed to this set with 160 novel missense variations. We analyzed the impact of the missense variations on protein stability by five different predictors including both sequence- (SAAF2EC and MUpro) and structure-based (Maestro, mCSM, CUPSAT) predictors. For the structure-based tools, we have utilized the AlphaFold (AF2) protein structures which comprise the first structural analysis of this hereditary cancer proteins. Our results agreed with the recent benchmarks that computed the power of stability predictors in discriminating the pathogenic variants. Overall, we reported a low-to-medium-level performance for the stability predictors in discriminating pathogenic variants, except MUpro which had an AUROC of 0.534 (95% CI [0.499-0.570]). The AUROC values ranged between 0.614-0.719 for the total set and 0.596-0.682 for the set with high AF2 confidence regions. Furthermore, our findings revealed that the confidence score for a given variant in the AF2 structure could alone predict pathogenicity more robustly than any of the tested stability predictors with an AUROC of 0.852. Altogether, this study represents the first structural analysis of the 26 hereditary cancer genes underscoring 1) the thermodynamic stability predicted from AF2 structures as a moderate and 2) the confidence score of AF2 as a strong descriptor for variant pathogenicity.

KCNB1 frameshift variant caused inherited intellectual disability, developmental delay, and seizure.

Potassium voltage-gated channel subfamily B member 1 (KCNB1) encodes Kv2.1 potassium channel. KCNB1 mutations are known to cause global developmental delay, behavioral disorders, and various epilepsies. Most variants occur de novo and are rarely inherited. Here, we report a 14-year-old male patient who was admitted to our clinic with seizures, developmental delay history, and intellectual disability. Brain magnetic resonance image (MRI) was normal and electroencephalogram (EEG) showed spike and sharp-wave complexes emerging in the left hemisphere parietooccipital areas, which were paroxysmally generalized. We performed whole exome sequence analysis (WES) and identified a heterozygous frameshift mutation c.522delA in exon 1 of KCNB1 (NM_004975.4) predicting a premature stop codon p.Lys174Asnfs*20 in the proband. Sanger sequencing confirmed the heterozygous c.522delA mutation in the proband and his mother who also had epilepsy and learning difficulties. His 45 year old mother had used antiepileptic drugs for 9 years after a seizure episode at 12 years old. Also, his mother's uncle's son is nonverbal and has developmental delay and epilepsy. Our study shows that frameshift mutation cytoplasmic domain of KCNB1 gene can cause intrafamilial phenotypic variability and relatively mild clinical findings in these patients.

Homozygous SQSTM1 nonsense variant identified in a patient with brainstem involvement.

In recent years, with advances in molecular genetics, many new mutations with various ataxic syndromes have been identified. Recently, homozygous sequestosome 1 (SQSTM1) gene variant with a progressive childhood-onset cerebellar ataxia, dystonia and gaze palsy was described. Here we describe a patient with progressive cerebellar ataxia and gaze palsy, as well as myoclonus, cognitive impairment and growth retardation with a homozygous SQSTM1 variant NM_003900.5:c.55G > T (p.Glu19*). Our case had brainstem lesions on brain magnetic resonance imaging that have not been previously reported. This novel finding expands the SQSTM1 gene-associated neuroradiologic spectrum. Homozygous SQSTM1 variant should be considered in the differential diagnosis in patients presenting with cerebellar findings, gaze palsy, and cognitive impairment to facilitate early diagnosis and genetic counseling.

Association of vitamin D receptor gene FokI and TaqI polymorphisms and risk of RDS.

Background: Vitamin D and its receptor (VDR) have important roles in perinatal lung development. The objective of this study was to investigate the possible association between VDR FokI and TaqI polymorphism and development of respiratory distress syndrome (RDS) in preterm infants.Method: A total of 173 premature infants <34 weeks: 82 with RDS and 91 without RDS were enrolled. Genotyping of VDR polymorphisms was assayed by real-time PCR. Serum 25-hydroxyvitamin D (25-OHD) levels were measured by ELISA in blood samples that were obtained at the time of admission to the neonatal intensive care unit.Results: Gestational age (GA) was significantly lower in the RDS group compared with the controls. In univariate analysis, VDR TaqI CT and CC genotypes were associated with the increased risk of RDS (OR = 3.264, p = .001, 95% CI = 1.597-6.672 and OR = 5.222, p < .001, 95% CI = 2.165-12.597, respectively); while VDR FokI showed no association with RDS. In multivariate logistic regression analysis, variant TaqI genotype increased risk of RDS (p = 0.001, OR = 3.464, 95% CI = 1.655-7.251) independent of gestational age, birth weight and gender. 25-OHD levels in the RDS group were significantly lower compared with those in without the RDS group (p = .002). Serum 25-OHD levels were not significantly different among the different FokI and TaqI genotypes in RDS group.Conclusions: This is the first report of association of VDR polymorphism with RDS development in preterm neonates. Current study suggests that VDR TaqI polymorphism may be involved in predisposition to RDS in premature neonates. Further studies are needed to assess the contribution of vitamin D and VDR signaling to the pathogenesis RDS.

Efficacy of stem cell therapy in ambulatory and nonambulatory children with Duchenne muscular dystrophy - Phase I-II.

PURPOSE: Duchenne muscular dystrophy (DMD) is an X-linked recessive pediatric disorder that ultimately leads to progressive muscle degeneration. It has been known that cell-based therapies were used to promote muscle regeneration. The main purpose of this study was to investigate the effects of allogeneic Wharton jelly-derived mesenchymal stem cells therapy in Duchenne muscular dystrophy. PATIENTS AND METHODS: Four ambulatory and five nonambulatory male patients were assessed as having acceptance criteria. Gene expression and immunohistochemical analysis were performed for dystrophin gene expression. The fluorescent in situ hybridization method was used for detection of chimerism and donor-recipient compatibility. Complement dependent lymphocytotoxic crossmatch test and detection of panel reactive antigen were performed. All patients were treated with 2 × 106 cells/kg dose of allogeneic Wharton jelly-derived mesenchymal stem cells via intra-arterial and intramuscular administration. Stability was maintained in patient follow-up tests, which are respiratory capacity tests, cardiac measurements, and muscle strength tests. RESULTS: The vastus intermedius muscle was observed in one patient with MRI. Chimerism was detected by fluorescent in situ hybridization and mean gene expression was increased to 3.3-fold. An increase in muscle strength measurements and pulmonary function tests was detected. Additionally, we observed two of nine patients with positive panel reactive antigen result. CONCLUSION: All our procedures are well tolerated, and we have not seen any application-related complications so far. Our main purpose of this study was to investigate the effects of allogeneic mesenchymal stem cell therapy and determine its suitability and safety as a form of treatment in this untreatable disorder.

Maturity Onset Diabetes of the Young due to Glucokinase, HNF1-A, HNF1-B, and HNF4-A Mutations in a Cohort of Turkish Children Diagnosed as Type 1 Diabetes Mellitus.

BACKGROUND/AIMS: Maturity onset diabetes of the young (MODY) is a rare condition often misdiagnosed as type 1 diabetes (T1D). The purposes of this study were: to identify any patients followed in a large Turkish cohort as T1D, with an atypical natural history, who may in fact have MODY, and to define the criteria which would indicate patients with likely MODY as early as possible after presentation to allow prompt genetic testing. METHODS: Urinary C-peptide/creatinine ratio (UCPCR) was studied in 152 patients having a diagnosis of T1D for at least 3 years. Those with a UCPCR ≥0.2 nmol/mmol were selected for genetic analysis of the Glucokinase (GCK), Hepatocyte nuclear factor 1a (HNF1A), Hepatocyte nuclear factor 4a (HNF4A), and Hepatocyte nuclear factor 1b (HNF1B) genes. This UCPCR cut-off was used because of the reported high sensitivity and specificity. Cases were also evaluated using a MODY probability calculator. RESULTS: Twenty-three patients from 152 participants (15.1%) had a UCPCR indicating persistent insulin reserve. The mean age ± SD of the patients was 13.6 ± 3.6 years (range 8.30-21.6). Of these 23, two (8.7%) were found to have a mutation, one with HNF4A and one with HNF1B mutation. No mutations were detected in the GCK or HNF1A genes. CONCLUSION: In Turkish children with a diagnosis of T1D but who have persistent insulin reserve 3 years after diagnosis, up to 9% may have a genetic mutation indicating a diagnosis of MODY.

Vitamin D status, serum lipid concentrations, and vitamin D receptor (VDR) gene polymorphisms in Familial Mediterranean fever.

Vitamin D (VitD) is critical for the regulation of inflammatory processes, and VitD deficiency has been linked to several chronic inflammatory disorders. We aimed to investigate the concentrations of serum 25(OH)D3, lipid parameters, and three known VDR polymorphisms (BsmI, FokI, and TaqI) in patients with Familial Mediterranean fever (FMF), an autosomal recessive autoinflammatory disease. The study included 123 FMF patients and 105 controls. Seventy patients had no attack (group 1), 30 had 1-2 attacks (group 2), and 23 had 3 or more attacks (group 3) within last three months. Serum 25(OH)D3 concentrations were determined using liquid chromatography-tandem mass spectrometry. BsmI, FokI, and TaqI polymorphisms were analyzed by a competitive allele specific polymerase chain reaction assay (KASPar). Serum lipid parameters were measured with enzymatic colorimetric methods. 25(OH)D3 concentrations were lower in FMF patients compared to controls (p < 0.001). No difference was observed in 25(OH)D3 concentration between groups 1, 2, and 3. The distributions of FokI and TaqI genotypes were not significantly different between FMF patients and controls. There was a significant difference in the distribution of AA BsmI genotype between male FMF patients and male controls. Increased concentrations of triglycerides (p = 0.012) and decreased concentrations of high-density lipoprotein cholesterol [HDL-C] (p = 0.006) were found in FMF patients compared to controls. Although lower 25(OH)D3 concentrations were observed in FMF patients versus controls, no association was determined between FMF attack frequency and 25(OH)D3 concentrations. We showed that the AA genotype of BsmI polymorphism is associated with FMF in males but not in females. The effects of decreased HDL-C and increased triglyceride concentrations on cardiovascular events in FMF patients should be further investigated.

Retinal and Choroidal Thickness in Adult Patients with Familial Mediterranean Fever.

PURPOSE: We aimed to evaluate changes in retinal, choroidal, ganglion cell complex (GCC) and retinal nerve fiber layer (RNFL) thicknesses in genetically diagnosed adult patients with familial Mediterranean fever (FMF). METHODS: A total of 50 eyes of 50 genetically diagnosed patients with FMF and 50 eyes of controls were analyzed. Patients were recruited from the Genetic Diagnostic Center of Diskapi Yildirim Beyazit Research and Training Hospital, Turkey. Retinal and choroidal thicknesses were obtained using spectral-domain optical coherence tomography from choroid, retina, GCC, and RNFL. RESULTS: Average baseline choroidal thickness was statistically significantly thinner in patients with FMF than controls at Ccenter (325.85 ± 30.8 µm and 338.97 ± 23.9 µm, respectively, p = 0.038), Cnasal500 (328.77 ± 31.6 µm and 349.00 ± 23.3 µm, respectively, p = 0.002), Cnasal1000 (324.97 ± 33.6 µm and 351.23 ± 23.8 µm respectively, p = 0.0001) and Cnasal1500 (324.75 ± 37.1 µm and 344.61 ± 27.3 µm, respectively, p = 0.008). However, there was no significant difference in temporal choroidal thickness (Ctemporal500, Ctemporal1000 and Ctemporal1500) in patients with FMF compared to controls (p > 0.05). There were no significant differences in retinal, GCC and RNFL thicknesses between the groups (p > 0.05). CONCLUSION: We hypothesize that the chronic inflammation seen in FMF could be the reason for the reduction seen in choroidal thickness in adult patients with FMF. Retinal, GCC and RNFL thicknesses did not differ from controls.

The effect of phototherapy on sister chromatid exchange with different light density in newborn hyperbilirubinemia.

AIM: Concerns of possible genotoxic effects of hyperbilirubinemia and phototherapy were raised from experimental and observational studies in neonates. The purpose of this study was to assess the effect of hyperbilirubinemia and phototherapy with three different methods on DNA damage by investigating sister chromatid exchange frequency. MATERIAL AND METHODS: Patients whose gestational ages were >37 weeks and bilirubin levels above phototherapy limits were enrolled into three groups and each group was planned to receive 25 babies. Group 1 received enhanced light-emitting diode phototherapy, group 2 had light-emitting diode phototherapy, and group 3 received conventional phototherapy. Infants with hyperbilirubinemia but did not require phototherapy comprised the control group, which was subdivided into two groups regarding bilirubin levels (10mg/dL). Blood samples were collected before and after phototherapy for sister chromatid exchange frequency and samples were examined by a biologist who was blinded to the study groups. RESULTS: The mean pre-treatment sister chromatid exchange frequency was 1.41±0.34/cell, post-treatment 2.65±0.68/cell, and 1.61±0.61/cell for the control group (p<0.05). A statistically significant increase in sister chromatid exchange frequency after phototherapy was observed in all three intervention groups (p values: 0.01, 0.01, and 0.008, respectively). When the treatment groups were compared with each other in terms of irradiance, no significant difference was found (p=0.08). CONCLUSIONS: Phototherapy causes an increase in the frequency of sister chromatid exchange regardless of the irradiance. Phototherapy could have some genotoxic adverse effects on chromosomes; however, further investigations are warranted to enlighten as to whether these effects are permanent or clinically important.

Analysis of CYP1B1 in pediatric and adult glaucoma and other ocular phenotypes.

PURPOSE: The CYP1B1 gene encodes an enzyme that is a member of the cytochrome P450 superfamily. Mutations in CYP1B1 have been mainly reported in recessive pediatric ocular phenotypes, such as primary congenital glaucoma (PCG) and congenital glaucoma with anterior segment dysgenesis (CG with ASD), with some likely pathogenic variants also identified in families affected with adult-onset primary open angle glaucoma (POAG). METHODS: We examined CYP1B1 in 158 pediatric patients affected with PCG (eight), CG with ASD (22), CG with other developmental ocular disorders (11), juvenile glaucoma with or without additional ocular anomalies (26), and ASD or other developmental ocular conditions without glaucoma (91); in addition, a large cohort of adult patients with POAG (193) and POAG-negative controls (288) was examined. RESULTS: Recessive pathogenic variants in CYP1B1 were identified in two PCG pedigrees, three cases with CG and ASD, and two families with CG and other ocular defects, such as sclerocornea in one patient and microphthalmia in another individual; neither sclerocornea nor microphthalmia has been previously associated with CYP1B1. Most of the identified causative mutations are new occurrences of previously reported pathogenic alleles with two novel variants identified: a c.1325delC, p.(Pro442Glnfs*15) frameshift allele in a family with PCG and a c.157G>A, p.(Gly53Ser) variant identified in a proband with CG, Peters anomaly, and microphthalmia. Analysis of the family history in the CYP1B1-positive families revealed POAG in confirmed or presumed heterozygous relatives in one family with PCG and two families with ASD/CG; POAG was associated with the c.1064_1076del, p.(Arg355Hisfs*69) allele in two of these pedigrees. Screening of an unrelated POAG cohort identified the same c.1064_1076del heterozygous allele in one individual with sporadic POAG but not in age- and ethnicity-matched POAG-negative individuals. Overall, there was no significant enrichment for mutant alleles in CYP1B1 within the POAG cases compared to the controls. CONCLUSIONS: In summary, these data expand the mutational and phenotypic spectra of CYP1B1 to include two novel alleles and additional developmental ocular phenotypes. The contribution of CYP1B1 to POAG is less clear, but loss-of-function variants in CYP1B1, especially c.1064_1076del, p.(Arg355Hisfs*69), may be associated with an increased risk for POAG.

The effects of commonly used intravitreal steroids on proliferation index of ciliary body-derived mesenchymal stem cells: an in vitro study.

AIM: To investigate the effects of commonly used intravitreal steroids on survival and proliferation (namely, proliferation index) of ciliary body-derived mesenchymal stem cells (CB-MSC). METHODS: CB-MSCs were isolated from newborn rats' eye, and they were expanded in the medium. Commonly used intravitreal steroids such as dexamethasone (Dex) and triamcinolone acetonide (TA) were added into the medium at commonly used concentration in clinical practice (0.1 mg/mL) and at lower concentration (0.01 mg/mL). Proliferation indexes of CB-MSCs were analyzed with the xCELLigence system at nine consecutive times (at 3rd, 6th, 21th, 30th, 45th, 60th, 75th, 90th and 100th h). RESULTS: Both TA and Dex at both 0.01 mg/mL and 0.1 mg/mL concentrations had negative effect on proliferation indexes of CB-MSC. Although negative effect of TA on proliferation index of CB-MSC at both concentrations was not statistically significant, statistically significant negative effect of Dex at 0.01 mg/mL concentration started 60th h (p = 0.017) and 0.1 mg/mL concentration started 30th h (p = 0.014). DISCUSSION: Even therapeutic doses of intravitreal corticosteroid agents might have negative effects on limited numbers of stem cells. Especially, Dex caused statistically significant toxic effects on CB-MSCs even at lower concentrations of those used clinically. These novel findings deserve further in vivo investigations.

Association Between Keratoconus and Familial Mediterranean Fever in Turkey.

PURPOSE: To evaluate the association between familial Mediterranean fever (FMF) and keratoconus (KC). METHODS: This retrospective case-control study was performed to compare the prevalence of KC in patients with FMF with the corresponding prevalence in control patients without FMF referred to Genetic Diagnostic Center at Diskapi Yildirim Beyazit Training and Research Hospital from June 2012 to June 2015. We included all 100 patients with FMF. Each FMF-affected patient was matched to 3 controls. RESULTS: None of the patients in the control group (0%, 0/300) had KC, whereas 4 of 100 patients with FMF (4%) had KC (P < 0.004). Three of 33 patients with a homozygous mutation (9.1%) (M694V/M694V in 2 cases and M680I/M680I in 1 case) and 1 of the 46 patients with a compound heterozygous mutation (2.2%) (M694V/M680I) had KC, whereas none of the 21 patients with a heterozygous mutation (0%) had KC. All patients with KC were women, and mean age was 40.8 years (range, 30-51). Although 1 of the 4 patients with KC had hypertension and type 2 diabetes mellitus, the other 3 patients did not have any systemic illness except FMF. When we compared the prevalence of KC in patients with FMF (4%) with the highest prevalence of KC reported in the literature (0.2%), FMF was a predisposing factor to develop KC [odds ratio: 18.1 (95% CI: 11.9-27.5)] especially in patients with a homozygous mutation [odds ratio: 43.4 (95% CI: 28.6-65.7)]. CONCLUSIONS: Mediterranean fever (MEFV) gene mutations, particularly in homozygous mutations of the MEFV gene, may be a predisposing factor in the development of KC.

Nonclassical Congenital Adrenal Hyperplasia and Pregnancy.

Objective. The most common form of congenital adrenal hyperplasia (CAH) is 21-hydroxylase (21-OH) deficiency due to mutation of the CYP21A2 gene. Patients with nonclassical CAH (NC-CAH) are usually asymptomatic at birth and typically present in late childhood, adolescence, or adulthood with symptoms of excessive androgen secretion. Subfertility is relative in NC-CAH, but the incidence of spontaneous miscarriage is higher. Here, we report a previously undiagnosed female who gave birth to a normal male child and is planning to become pregnant again. Case Report. A 32-year-old female was referred to our clinic for obesity. Her medical history revealed that she had had three pregnancies. She was planning to become pregnant again. Her laboratory results revealed that she had NC-CAH. Since her husband is the son of her aunt and she had miscarriages and intrauterin exitus in her history, their genetic analyses were performed. Conclusion. Since most patients with NC-CAH have a severe mutation, these patients may give birth to a child with the classical CAH (C-CAH) if their partner is also carrying a severe mutation. Females with NC-CAH who desire pregnancy must be aware of the risk of having an infant with C-CAH.

Thyroid hormone resistance in two patients with papillary thyroid microcarcinoma and their BRAFV600E mutation status.

Resistance to thyroid hormone (RTH) is a rare autosomal dominant hereditary disorder. Here in, we report two patients with RTH in whom differentiated thyroid cancer was diagnosed. Two patients were admitted to our clinic and their laboratory results were elevated thyroid hormone levels with unsuppressed TSH. We considered this situation thyroid hormone resistance in the light of laboratory and clinical datas. Thyroid nodule was palpated on physical examination. Thyroid ultrasonography showed multiple nodules in both lobes. Total thyroidectomy was performed. The pathological findings were consistent with papillary thyroid microcarcinoma. BRAFV600E mutation analysis results were negative. RTH is very rare and might be overlooked. There is no consensus on how to overcome the persistently high TSH in patients with RTH and differentiated thyroid cancer (DTC). Further studies are needed to explain the relationship between RTH and DTC which might be helpful for the treatment of these patients.

Thyroid hormone resistance in two patients with papillary thyroid microcarcinoma and their BRAFV600E mutation status

Resistance to thyroid hormone (RTH) is a rare autosomal dominant hereditary disorder. Here in, we report two patients with RTH in whom differentiated thyroid cancer was diagnosed. Two patients were admitted to our clinic and their laboratory results were elevated thyroid hormone levels with unsuppressed TSH. We considered this situation thyroid hormone resistance in the light of laboratory and clinical datas. Thyroid nodule was palpated on physical examination. Thyroid ultrasonography showed multiple nodules in both lobes. Total thyroidectomy was performed. The pathological findings were consistent with papillary thyroid microcarcinoma. BRAFV600E mutation analysis results were negative. RTH is very rare and might be overlooked. There is no consensus on how to overcome the persistently high TSH in patients with RTH and differentiated thyroid cancer (DTC). Further studies are needed to explain the relationship between RTH and DTC which might be helpful for the treatment of these patients.