Dysfunction of peripheral somatic and autonomic nervous system in patients with severe forms of Crohn's disease on biological therapy with TNFα inhibitors-A single center study.

OBJECTIVE: Crohn's disease (CD) can be associated with a wide range of extraintestinal manifestations (EIMs), including neurological ones. Published studies differ in their conclusions about the epidemiology and etiopathogenesis of neurological EIMs. The aims of this study were to demonstrate the presence and find risk factors of peripheral (somatic and autonomic) neuropathy patients with severe CD on anti-TNFα biological therapy. MATERIAL AND METHODS: A clinical examination focusing on detection of peripheral sensor-motor nervous dysfunction (including Sudoscan) and examination of autonomic nervous system dysfunction (using Ewing´s battery tests and spectral analysis) together with laboratory tests and collection of demographic data followed by administration of questionnaires were performed on a total of 30 neurologically asymptomatic outpatients with severe CD on anti-TNFα biological therapy. RESULTS: Peripheral sensor-motor nervous function via clinical neurological examination was pathological in 36.7% and Sudoscan in 33.3% of cases. Statistically significant associations between vibration perception test and age, CD and biological therapy duration, body mass index and Crohn's Disease Activity Index were proved while statistically significant associations between temperature perception test and age and BMI were proved as well. Additionally, a decrease of total protein in a patient´s serum below the physiological cut-off in the 6 months prior to measurement was associated with a pathological result of a Sudoscan. Cardiovascular autonomic neuropathy based on Ewing´s battery tests was present in 56.7% of patients, no statistically significant risk factors were found. Our peripheral neuropathy questionnaire correlated with the results of the Sudoscan test and some tests of the clinical examination of peripheral sensor-motor nervous function (discriminatory contact perception test, temperature perception test). CONCLUSIONS: This study demonstrated a relatively high prevalence of peripheral (especially autonomic) neuropathy and verified some risk factors for the development of peripheral somatic neuropathy in asymptomatic patients with severe form of CD on anti-TNFα biological therapy.

Exome sequencing reveals IFT172 variants in patients with non-syndromic cholestatic liver disease.

BACKGROUND AND AIM: Gene defects contribute to the aetiology of intrahepatic cholestasis. We aimed to explore the outcome of whole-exome sequencing (WES) in a cohort of 51 patients with this diagnosis. PATIENTS AND METHODS: Both paediatric (n = 33) and adult (n = 18) patients with cholestatic liver disease of unknown aetiology were eligible. WES was used for reassessment of 34 patients (23 children) without diagnostic genotypes in ABCB11, ATP8B1, ABCB4 or JAG1 demonstrable by previous Sanger sequencing, and for primary assessment of additional 17 patients (10 children). Nasopharyngeal swab mRNA was analysed to address variant pathogenicity in two families. RESULTS: WES revealed biallelic variation in 3 ciliopathy genes (PKHD1, TMEM67 and IFT172) in 4 clinically unrelated index subjects (3 children and 1 adult), heterozygosity for a known variant in PPOX in one adult index subject, and homozygosity for an unreported splice-site variation in F11R in one child. Whereas phenotypes of the index patients with mutated PKHD1, TMEM67, and PPOX corresponded with those elsewhere reported, how F11R variation underlies liver disease remains unclear. Two unrelated patients harboured different novel biallelic variants in IFT172, a gene implicated in short-rib thoracic dysplasia 10 and Bardet-Biedl syndrome 20. One patient, a homozygote for IFT172 rs780205001 c.167A>C p.(Lys56Thr) born to first cousins, had liver disease, interpreted on biopsy aged 4y as glycogen storage disease, followed by adult-onset nephronophthisis at 25y. The other, a compound heterozygote for novel frameshift variant IFT172 NM_015662.3 c.2070del p.(Met690Ilefs*11) and 2 syntenic missense variants IFT172 rs776310391 c.157T>A p.(Phe53Ile) and rs746462745 c.164C>G p.(Thr55Ser), had a severe 8mo cholestatic episode in early infancy, with persisting hyperbilirubinemia and fibrosis on imaging studies at 17y. No patient had skeletal malformations. CONCLUSION: Our findings suggest association of IFT172 variants with non-syndromic cholestatic liver disease.

Axenfeld-Rieger syndrome: more than meets the eye.

BACKGROUND: Axenfeld-Rieger syndrome (ARS) is characterised by typical anterior segment anomalies, with or without systemic features. The discovery of causative genes identified ARS subtypes with distinct phenotypes, but our understanding is incomplete, complicated by the rarity of the condition. METHODS: Genetic and phenotypic characterisation of the largest reported ARS cohort through comprehensive genetic and clinical data analyses. RESULTS: 128 individuals with causative variants in PITX2 or FOXC1, including 81 new cases, were investigated. Ocular anomalies showed significant overlap but with broader variability and earlier onset of glaucoma for FOXC1-related ARS. Systemic anomalies were seen in all individuals with PITX2-related ARS and the majority of those with FOXC1-related ARS. PITX2-related ARS demonstrated typical umbilical anomalies and dental microdontia/hypodontia/oligodontia, along with a novel high rate of Meckel diverticulum. FOXC1-related ARS exhibited characteristic hearing loss and congenital heart defects as well as previously unrecognised phenotypes of dental enamel hypoplasia and/or crowding, a range of skeletal and joint anomalies, hypotonia/early delay and feeding disorders with structural oesophageal anomalies in some. Brain imaging revealed highly penetrant white matter hyperintensities, colpocephaly/ventriculomegaly and frequent arachnoid cysts. The expanded phenotype of FOXC1-related ARS identified here was found to fully overlap features of De Hauwere syndrome. The results were used to generate gene-specific management plans for the two types of ARS. CONCLUSION: Since clinical features of ARS vary significantly based on the affected gene, it is critical that families are provided with a gene-specific diagnosis, PITX2-related ARS or FOXC1-related ARS. De Hauwere syndrome is proposed to be a FOXC1opathy.

Rare disease gene association discovery from burden analysis of the 100,000 Genomes Project data.

To discover rare disease-gene associations, we developed a gene burden analytical framework and applied it to rare, protein-coding variants from whole genome sequencing of 35,008 cases with rare diseases and their family members recruited to the 100,000 Genomes Project (100KGP). Following in silico triaging of the results, 88 novel associations were identified including 38 with existing experimental evidence. We have published the confirmation of one of these associations, hereditary ataxia with UCHL1 , and independent confirmatory evidence has recently been published for four more. We highlight a further seven compelling associations: hypertrophic cardiomyopathy with DYSF and SLC4A3 where both genes show high/specific heart expression and existing associations to skeletal dystrophies or short QT syndrome respectively; monogenic diabetes with UNC13A with a known role in the regulation of ß cells and a mouse model with impaired glucose tolerance; epilepsy with KCNQ1 where a mouse model shows seizures and the existing long QT syndrome association may be linked; early onset Parkinson's disease with RYR1 with existing links to tremor pathophysiology and a mouse model with neurological phenotypes; anterior segment ocular abnormalities associated with POMK showing expression in corneal cells and with a zebrafish model with developmental ocular abnormalities; and cystic kidney disease with COL4A3 showing high renal expression and prior evidence for a digenic or modifying role in renal disease. Confirmation of all 88 associations would lead to potential diagnoses in 456 molecularly undiagnosed cases within the 100KGP, as well as other rare disease patients worldwide, highlighting the clinical impact of a large-scale statistical approach to rare disease gene discovery.

Posterior corneal vesicles are not associated with the genetic variants that cause posterior polymorphous corneal dystrophy.

PURPOSE: Posterior corneal vesicles (PCVs) have clinical features that are similar to posterior polymorphous corneal dystrophy (PPCD). To help determine whether there is a shared genetic basis, we screened 38 individuals with PCVs for changes in the three genes identified as causative for PPCD. METHODS: We prospectively recruited patients for this study. We examined all individuals clinically, with their first-degree relatives when available. We used a combination of Sanger and exome sequencing to screen regulatory regions of OVOL2 and GRHL2, and the entire ZEB1 coding sequence. RESULTS: The median age at examination was 37.5 years (range 4.7-84.0 years), 20 (53%) were male and in 19 (50%) the PCVs were unilateral. Most individuals were discharged to optometric review, but five had follow-up for a median of 12 years (range 5-13 years) with no evidence of progression. In cases with unilateral PCVs, there was statistically significant evidence that the change in the affected eye was associated with a lower endothelial cell density (p = 0.0003), greater central corneal thickness (p = 0.0277) and a steeper mean keratometry (p = 0.0034), but not with a higher keratometric astigmatism or a reduced LogMAR visual acuity. First-degree relatives of 13 individuals were available for examination, and in 3 (23%), PCVs were identified. No possibly pathogenic variants were identified in the PPCD-associated genes screened. CONCLUSION: We found no evidence that PCVs share the same genetic background as PPCD. In contrast to PPCD, we confirm that PCVs is a mild, non-progressive condition with no requirement for long-term review. However, subsequent cataract surgery can lead to corneal oedema.

Novel disease-causing variants and phenotypic features of X-linked megalocornea.

PURPOSE: The aim of the study was to describe the phenotype and molecular genetic causes of X-linked megalocornea (MGC1). We recruited four British, one New Zealand, one Vietnamese and four Czech families. METHODS: All probands and three female carriers underwent ocular examination and Sanger sequencing of the CHRDL1 gene. Two of the probands also had magnetic resonance imaging (MRI) of the brain. RESULTS: We identified nine pathogenic or likely pathogenic and one variant of uncertain significance in CHRDL1, of which eight are novel. Three probands had ocular findings that have not previously been associated with MGC1, namely pigmentary glaucoma, unilateral posterior corneal vesicles, unilateral keratoconus and unilateral Fuchs heterochromic iridocyclitis. The corneal diameters of the three heterozygous carriers were normal, but two had abnormally thin corneas, and one of these was also diagnosed with unilateral keratoconus. Brain MRI identified arachnoid cysts in both probands, one also had a neuroepithelial cyst, while the second had a midsagittal neurodevelopmental abnormality (cavum septum pellucidum et vergae). CONCLUSION: The study expands the spectrum of pathogenic variants and the ocular and brain abnormalities that have been identified in individuals with MGC1. Reduced corneal thickness may represent a mild phenotypic feature in some heterozygous female carriers of CHRDL1 pathogenic variants.

Clinical and Genetic Study of X-Linked Juvenile Retinoschisis in the Czech Population.

The aim of this study was to identify RS1 pathogenic variants in Czech patients with X-linked retinoschisis (XLRS) and to describe the associated phenotypes, including natural history, in some cases. Twenty-one affected males from 17 families were included. The coding region of RS1 was directly sequenced and segregation of the identified mutations was performed in available family members. In total, 12 disease-causing variants within RS1 were identified; of these c.20del, c.275G>A, c.[375_379del; 386A>T], c.539C>A and c.575_576insT were novel, all predicted to be null alleles. The c.539C>A mutation occurred de novo. Three patients (aged 8, 11 and 19 years) were misdiagnosed as having intermediate uveitis and treated with systemic steroids. Repeat spectral domain optical coherence tomography examinations in four eyes documented the transition from cystoid macular lesions to macular atrophy in the fourth decade of life. Four individuals were treated with topical dorzolamide and in two of them, complete resolution of the cystic macular lesions bilaterally was achieved, while one patient was noncompliant. Rebound phenomenon after discontinuation of dorzolamide for 7 days was documented in one case. Misdiagnosis of XLRS for uveitis is not uncommon; therefore, identification of disease-causing variants is of considerable benefit to the affected individuals.

Pigmentary retinopathy can indicate the presence of pathogenic LAMP2 variants even in somatic mosaic carriers with no additional signs of Danon disease.

PURPOSE: Danon disease (DD) is a rare X-linked disorder caused by pathogenic variants in LAMP2. DD primarily manifests as a severe cardiomyopathy. An early diagnosis is crucial for patient survival. The aim of the study was to determine the usefulness of ocular examination for identification of DD. METHODS: Detailed ocular examination in 10 patients with DD (3 males, 7 females) and a 45-year-old asymptomatic female somatic mosaic carrier of a LAMP2 disease-causing variant. RESULTS: All patients with manifest cardiomyopathy had pigmentary retinopathy with altered autofluorescence and diffuse visual field loss. Best corrected visual acuity (BCVA) was decreased (<0.63) in 8 (40%) out of 20 eyes. The severity of retinal pathology increased with age, resulting in marked cone-rod involvement overtime. Spectral-domain optical coherence tomography in younger patients revealed focal loss of photoreceptors, disruption and deposition at the retinal pigment epithelium/Bruch's membrane layer (corresponding to areas of marked increased autofluorescence), and hyperreflective foci in the outer nuclear layer. Cystoid macular oedema was seen in one eye. In the asymptomatic female with somatic mosaicism, the BCVA was 1.0 bilaterally. An abnormal autofluorescence pattern in the left eye was present; while full-field electroretinography was normal. CONCLUSIONS: Detailed ocular examination may represent a sensitive and quick screening tool for the identification of carriers of LAMP2 pathogenic variants, even in somatic mosaicism. Hence, further investigation should be undertaken in all patients with pigmentary retinal dystrophy as it may be a sign of a life-threatening disease.

The Phenotypic Spectrum of 47 Czech Patients with Single, Large-Scale Mitochondrial DNA Deletions.

BACKGROUND: In this retrospective study, we analysed clinical, biochemical and molecular genetic data of 47 Czech patients with Single, Large-Scale Mitochondrial DNA Deletions (SLSMD). METHODS: The diagnosis was based on the long-range PCR (LX-PCR) screening of mtDNA isolated from muscle biopsy in 15 patients, and from the buccal swab, urinary epithelial cells and blood in 32 patients. RESULTS: A total of 57% patients manifested before the age of 16. We did not find any significant difference between paediatric and adult manifestation in either the proportion of patients that would develop extraocular symptoms, or the timespan of its progression. The survival rate in patients with Pearson Syndrome reached 60%. Altogether, five patients manifested with atypical phenotype not fulfilling the latest criteria for SLSMD. No correlation was found between the disease severity and all heteroplasmy levels, lengths of the deletion and respiratory chain activities in muscle. CONCLUSIONS: Paediatric manifestation of Progressive External Ophthalmoplegia (PEO) is not associated with a higher risk of multisystemic involvement. Contrary to PEO and Kearns-Sayre Syndrome Spectrum, Pearson Syndrome still contributes to a significant childhood mortality. SLSMD should be considered even in cases with atypical presentation. To successfully identify carriers of SLSMD, a repeated combined analysis of buccal swab and urinary epithelial cells is needed.

Brittle cornea syndrome: Disease-causing mutations in ZNF469 and two novel variants identified in a patient followed for 26 years.

AIMS: Brittle cornea syndrome (BCS) is a rare autosomal recessive disorder. The aim of this study was to review ZNF469 mutations associated with BCS type 1 to date and to describe an additional case of Czech/Polish background. METHODS: Whole genome sequencing was undertaken to identify the molecular genetic cause of disease in the proband. Sequence variants in ZNF469 previously reported as BCS type 1-causing were searched in the literature, manually curated and aligned to the reference sequence NM_001127464.2. RESULTS: The proband has been reviewed since childhood with progressive myopia and hearing loss. Aged 13 years had been diagnosed with Stickler syndrome. Aged 16.5 years, he developed acute hydrops in the left eye managed by corneal transplantation. At the age of 26, he experienced right corneal rupture after blunt trauma, also managed by grafting. He had a number of secondary complications and despite regular follow-up and timely management, the right eye became totally blind and the left eye had light perception at the last follow-up visit, aged 42. He was found to be a compound heterozygote for two novel mutations c.1705C>T; p.(Gln569*) and c.1402_1411del; p.(Pro468Alafs*31) in ZNF469. In total 22 disease-causing variants in ZNF469 have been identified, mainly in consanguineous families or endogamous populations. Only four probands, including the case described in the current study, harboured compound heterozygous mutations. CONCLUSION: BCS occurs very rarely in outbred populations which may cause diagnostic errors due to poor awareness of the disease. Investigation into the underlying molecular genetic cause in patients with connective tissue disorders may lead to a re-evaluation of their clinical diagnosis.

Alu-mediated Xq24 deletion encompassing CUL4B, LAMP2, ATP1B4, TMEM255A, and ZBTB33 genes causes Danon disease in a female patient.

Cullin 4B (CUL4B), lysosomal-associated membrane protein Type 2 (LAMP2), ATP1B4, TMEM255A, and ZBTB33 are neighboring genes on Xq24. Mutations in CUL4B result in Cabezas syndrome (CS). Male CS patients present with dysmorphic, neuropsychiatric, genitourinary, and endocrine abnormalities. Heterozygous CS females are clinically asymptomatic. LAMP2 mutations cause Danon disease (DD). Cardiomyopathy is a dominant feature of DD present in both males and heterozygous females. No monogenic phenotypes have been associated with mutations in ATP1B4, TMEM255A, and ZBTB33 genes. To facilitate diagnostics and counseling in CS and DD families, we present a female DD patient with a de novo Alu-mediated Xq24 rearrangement causing a deletion encompassing CUL4B, LAMP2, and also the other three neighboring genes. Typical to females heterozygous for CUL4B mutations, the patient was CS asymptomatic, however, presented with extremely skewed X-chromosome inactivation (XCI) ratios in peripheral white blood cells. As a result of the likely selection against CUL4B deficient clones, only minimal populations (~3%) of LAMP2 deficient leukocytes were identified by flow cytometry. On the contrary, myocardial LAMP2 protein expression suggested random XCI. We demonstrate that contiguous CUL4B and LAMP2 loss-of-function copy number variations occur and speculate that male patients carrying similar defects could present with features of both CS and DD.

Genetic Variants Associated With Corneal Biomechanical Properties and Potentially Conferring Susceptibility to Keratoconus in a Genome-Wide Association Study.

IMPORTANCE: Keratoconus is an important cause of visual loss in young adults, but little is known about its genetic causes. Understanding the genetic determinants of corneal biomechanical factors may in turn teach us about keratoconus etiology. OBJECTIVES: To identify genetic associations with corneal biomechanical properties and to examine whether these genetic variants are associated with keratoconus. DESIGN, SETTING, AND PARTICIPANTS: A stage 1 discovery and replication genome-wide association study (GWAS) of corneal biomechanical properties was performed in 2 cross-sectional populations (6645 participants from the European Prospective Investigation into Cancer and Nutrition [EPIC]-Norfolk Eye Study and 2384 participants from the TwinsUK study). In stage 2, the association of genetic determinants identified in stage 1 with keratoconus was examined in a case-control study. A total of 752 patients with keratoconus were compared with 974 TwinsUK participants (undergoing direct sequencing) or 13 828 EPIC-Norfolk participants (undergoing genotyping and imputation) who were not part of the stage 1 analysis. Data were collected from March 1, 1993, through March 13, 2017, and analyzed from November 1, 2015, through February 1, 2018. EXPOSURES: In stage 1, allele dosage at genome-wide single-nucleotide polymorphisms (SNPs); in stage 2, allele dosage at SNPs with genome-wide significance (P < 5 × 10-8) in stage 1 and not previously reported as associated with corneal disease. MAIN OUTCOMES AND MEASURES: In stage 1, corneal hysteresis (CH) and corneal resistance factor (CRF), measured with the Ocular Response Analyzer (ORA); in stage 2, association with keratoconus compared with controls. RESULTS: Among 6645 participants in the discovery cohort (3635 women (54.7%); mean age, 69 years [range, 48-92 years]), 7 genome-wide significant loci associated with CH or CRF were identified that were independently replicated. Two further suggestive loci were identified after meta-analysis. To date, 5 of the identified loci, at ANAPC1, ADAMTS8, ADAMTS17, ABCA6, and COL6A1, have not previously been reported as associated with corneal disease. The ABCA6 locus (rs77542162) was associated with keratoconus using the TwinsUK (odds ratio [OR], 0.50; 95% CI, 0.27-0.92; P = .03) and EPIC-Norfolk controls (OR, 0.39; 95% CI, 0.22-0.70; P = .002). The other loci were associated with keratoconus using TwinsUK (OR per effect allele for ADAMTS8, 0.51 [95% CI, 0.37-0.71; P = 7.9 × 10-5]; for COL6A1, 1.65 [95% CI, 1.05-2.59; P = .03]) or EPIC-Norfolk (OR per effect allele for ANAPC1, 0.78 [95% CI, 0.68-0.89; P = 3.7 × 10-4]; for ADAMTS17, 0.82 [95% CI, 0.68-0.99; P = .04]) controls. CONCLUSIONS AND RELEVANCE: Five loci that are associated with corneal biomechanical properties and that have suggestive associations with keratoconus were reported. These findings suggest the role of type VI collagen, extracellular matrix, and connective-tissue development for corneal biomechanics and keratoconus and the role of CH and CRF as biomarkers for keratoconus.

Paraproteinemic keratopathy associated with monoclonal gammopathy of undetermined significance (MGUS): clinical findings in twelve patients including recurrence after keratoplasty.

PURPOSE: To describe the ocular findings of 12 subjects with paraproteinemic keratopathy associated with monoclonal gammopathy of undetermined significance (MGUS). METHODS: Ocular examination included corneal spectral domain optical coherence tomography. In three individuals with an initial diagnosis of a lattice or Thiel-Behnke corneal dystrophy, the TGFBI gene was screened by conventional Sanger sequencing. RESULTS: We confirmed a diagnosis of MGUS by systemic examination and serum protein electrophoresis in 12 individuals (9 males and 3 females), with a mean age at presentation of 52.2 years (range 24-63 years) and mean follow-up 6.4 years (range 0-17 years). The best-corrected visual acuity (BCVA) at presentation ranged from 1.25 to 0.32. In all individuals, the corneal opacities were bilateral. The appearances were diverse and included superficial reticular opacities and nummular lesions, diffuse posterior stromal opacity, stromal lattice lines, superficial and stromal crystalline deposits, superficial haze and a superficial ring of hypertrophic tissue. In one individual, with opacities first recorded at 24 years of age, we documented the progression of corneal disease over the subsequent 17 years. In another individual, despite systemic treatment for MGUS, recurrence of deposits was noted following bilateral penetrating keratoplasties. The three individuals initially diagnosed with inherited corneal dystrophy were negative for TGFBI mutations by direct sequencing. CONCLUSION: A diagnosis of MGUS should be considered in patients with bilateral corneal opacities. The appearance can mimic corneal dystrophies or cystinosis. In our experience, systemic treatment of MGUS did not prevent recurrence of paraproteinemic keratopathy following keratoplasty.

SD-OCT imaging as a valuable tool to support molecular genetic diagnostics of Usher syndrome type 1.

A girl with profound congenital deafness and balance problems was found at 3.5 years of age to be a carrier of two novel compound heterozygous mutations in MYO7A that were predicted to be disease-causing. She also carried one known pathogenic mutation and one rare variant in USH2A. Fundus examination performed at 4.75 years revealed one small peripheral pigment deposit in the right eye, indicating probable retinal degeneration. Spectral domain optical coherence tomography (SD-OCT) showed a loss of photoreceptors throughout the macular area, except for the foveolar region, clearly confirming a diagnosis of Usher syndrome type 1. This case demonstrates that SD-OCT may be easily used in young children to confirm retinal disease, quantify the extent of retinal damage, and monitor disease progression.

Ex vivo 3D human corneal stroma model for Schnyder corneal dystrophy - role of autophagy in its pathogenesis and resolution.

INTRODUCTION: Multilamellar bodies (MLBs) are concentric cytoplasmic membranes which form through an autophagy-dependent mechanism. In the cornea, the presence of MLBs is associated with Schnyder corneal dystrophy (SCD). Ex vivo 3D modelling of the corneal stroma and SCD can help study pathogenesis and resolution of the disorder. METHODS: Corneal stroma explants were isolated from cadavers and cultivated long-term for more than 3 months to achieve spontaneous 3D outgrowth of corneal stroma-derived mesenchymal stem-like cells (CSMSCs). The 3D tissues were then examined by transmission electron microscopy (TEM) for presence of MLBs, and by immunofluorescent labelling against markers for autophagy (p62, LC3). Autophagy was induced by classical serum starvation or rapamycin (RAP) treatment (50 nM), and inhibited by the autophagy inhibitor 3-methyladenine (3-MA, 10 mM) for 24 hours. RESULTS: CSMSCs can form spontaneously 3D outgrowths over a 3-4 weeks period, depositing their own extracellular matrix containing collagen I. TEM confirmed the presence of MLBs in the long-term (>3 months) 3D cultures, which became more abundant under starvation and RAP treatment, and decreased in number under autophagy inhibition with 3-MA. The presence of autophagy and its disappearance could be confirmed by an inversely related increase and decrease in the expression of LC3 and p62, respectively. CONCLUSIONS: MLB formation in long-standing CSMSC cultures could serve as a potential ex vivo model for studying corneal stroma diseases, including SCD. Inhibition of autophagy can decrease the formation of MLBs, which may lead to a novel treatment of the disease in the future.

Segregation of a novel p.(Ser270Tyr) MAF mutation and p.(Tyr56∗) CRYGD variant in a family with dominantly inherited congenital cataracts.

A bilaterally blind woman, with a three generation family history of autosomal dominant congenital cataracts, variably associated with iris colobomata and microcornea, sought preconception genetic consultation. Whole-exome sequencing was performed in three affected family members, one unaffected first degree relative, and one spouse. The sequence variant c.168C>G; p.(Tyr56∗) in CRYGD, previously reported as pathogenic, and a novel mutation c.809C>A; p.(Ser270Tyr) in MAF, were identified in two affected family members; the grandmother, and half-brother of the proband. The proband inherited only the MAF mutation, whereas her clinically unaffected sister had the CRYGD change. In silico analysis supported a pathogenic role of p.(Ser270Tyr) in MAF, which was absent from publicly available whole-exome datasets, and 1161 Czech individuals. The frequency of CRYGD p.(Tyr56∗) in the ExAC dataset was higher than the estimated incidence of congenital cataract in the general population. Our study highlights that patients with genetically heterogeneous conditions may exhibit rare variants in more than one disease-associated gene, warranting caution with data interpretation, and supporting parallel screening of all genes known to harbour pathogenic mutations for a given phenotype. The pathogenicity of sequence variants previously reported as cataract-causing may require re-assessment in light of recently released datasets of human genomic variation.

Early detection of bilateral cataracts in utero may represent a manifestation of severe congenital disease.

We observed bilateral cataracts on second trimester ultrasound, in two consecutive pregnancies, with no other structural defects detected. The parents were unrelated and had no family history for the disease. The first pregnancy was terminated in week 22. Copy number variation analysis revealed, in both the aborted fetus and the mother, a 495 kb duplication at 22q11.23 encompassing CRYBB3 and CRYBB2, and not present in variation databases. In the second pregnancy, lens hyperechogenicity was detected by ultrasound at week 13 and 4 days. The identical duplication at 22q11.23 was found in the fetus and considered as possibly pathogenic. At weeks 22 and 30, smaller orbit measurements were elucidated on ultrasound, raising concerns as to the underlying molecular genetic cause, necessitating further investigation. Whole-exome sequencing, using DNA of the first fetus, was performed shortly after the birth of a male child, and two truncating RAB3GAP1 mutations were detected: c.538G>T; p. (Glu180*) and c.943C>T; p. (Arg315*). Neither mutation has been previously reported to be disease-causing; however, evaluation in the context of previously published literature indicated their deleterious nature, implying a clinical diagnosis of Warburg micro syndrome or Martsolf syndrome. Sanger sequencing confirmed segregation of the two mutations within the family, consistent with autosomal recessive inheritance. The child born from the second pregnancy showed features typical of Warburg micro syndrome, with the exception of microcephaly, at age 31 months. © 2016 Wiley Periodicals, Inc.

Phenotypic features of CRB1-associated early-onset severe retinal dystrophy and the different molecular approaches to identifying the disease-causing variants.

PURPOSE: The aim of this study was to determine the molecular genetic basis of an early-onset severe retinal dystrophy in three unrelated consecutive patients of Czech origin and to describe their ocular phenotype. METHODS: DNA samples from two probands were analyzed using a genotyping microarray (Asper) followed by either target analysis of 43 genes implicated in retinal disorders by next generation sequencing or whole-exome sequencing, respectively. The third proband underwent conventional Sanger sequencing of CRB1 based on her ocular findings. RESULTS: All three probands harboured a known disease-causing mutation c.2843G>A; p.(Cys948Tyr) in the CRB1 gene. One individual was homozygous for this mutation, while in the other two probands c.2308G>A; p.(Gly770Ser) and c.3121A>G; p.(Met1041Val) were also identified in the heterozygous state, respectively. Both variants were novel and evaluated by in silico analysis as pathogenic. A false-negative result was observed in one of the two samples examined by the genotyping microarray. Disease onset in all patients was before the age of 7 years. Hypermetropic refractive error, bilateral nummular retinal pigmentation, retinal thickening and cystoid spaces in the macula were observed in two probands, aged 6 and 7 years. The third proband, aged 28 years, had bone spicule-like pigmentary changes associated with increased retinal nerve fiber layer. CONCLUSIONS: The first study reporting on the molecular genetic cause of non-syndromic early-onset severe retinal dystrophy in Czech patients identified one homozygous and two compound heterozygote probands with CRB1 mutations. Retina nerve fibre layer measurements should be considered an integral part of the clinical evaluation of retinal dystrophies. Detailed clinical examination and imaging can both direct molecular screening and help to confirm or refute disease causation of identified variants.

Transmembrane segments of complement receptor 3 do not participate in cytotoxic activities but determine receptor structure required for action of Bordetella adenylate cyclase toxin.

Adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) of the whooping cough agent Bordetella pertussis penetrates phagocytes expressing the integrin complement receptor 3 (CR3, CD11b/CD18, α(M)ß(2) or Mac-1). CyaA translocates its adenylate cyclase (AC) enzyme domain into cell cytosol and catalyzes unregulated conversion of ATP to cAMP, thereby subverting cellular signaling. In parallel, CyaA forms small cation-selective membrane pores that permeabilize cells for potassium efflux, contributing to cytotoxicity of CyaA and eventually provoking colloid-osmotic cell lysis. To investigate whether the single-pass α-helical transmembrane segments of CR3 subunits CD11b and CD18 do directly participate in AC domain translocation and/or pore formation by the toxin, we expressed in CHO cells variants of CR3 that contained artificial transmembrane segments, or lacked the transmembrane segment(s) at all. The results demonstrate that the transmembrane segments of CR3 are not directly involved in the cytotoxic activities of CyaA but serve for maintaining CR3 in a conformation that is required for efficient toxin binding and action.

Identification of six novel mutations in ZEB1 and description of the associated phenotypes in patients with posterior polymorphous corneal dystrophy 3.

Posterior polymorphous corneal dystrophy 3 (PPCD3) is a rare autosomal dominant disorder caused by mutations in ZEB1. To date all identified disease-causing variants were unique to the studied families, except for c.1576dup. We have detected six novel ZEB1 mutations; c.1749_1750del; p.(Pro584*) and c.1717_1718del; p.(Val573Phefs*12) in two Czech families, c.1176dup; p.(Ala393Serfs*19), c.1100C>A; p.(Ser367*), c.627del; p.(Phe209Leufs*11) in three British families and a splice site mutation, c.685-2A>G, in a patient of Sri Lankan origin. An additional British proband had the c.1576dup; p.(Val526Glyfs*3) mutation previously reported in other populations. Clinical findings were variable and included bilateral congenital corneal opacity in one proband, development of opacity before the age of 2 years in another individual and bilateral iris flocculi in yet another subject. The majority of eyes examined by corneal topography (10 out of 16) had an abnormally steep cornea (flat keratometry 46.5-52.7 diopters, steep keratometry 48.1-54.0 diopters). One proband underwent surgery for cryptorchidism. Our study further demonstrates that PPCD3 can present as corneal edema in early childhood, and that an abnormally steep keratometry is a common feature of this condition. As cryptorchidism has been previously observed in two other PPCD3 cases, its association with the disease warrants further investigation.