Genome-wide linkage and sequence analysis challenge CCDC66 as a human retinal dystrophy candidate gene and support a distinct NMNAT1-related fundus phenotype.

To uncover the genotype underlying early-onset cone-rod dystrophy and central nummular macular atrophic lesion in 2 siblings from an endogamous Arab family, we performed targeted next-generation sequencing (NGS) of 44 retinal dystrophy genes, whole-exome sequencing (WES) and genome-wide linkage analysis. Targeted NGS and WES in the index patient highlighted 2 homozygous variants, a CCDC66 frameshift deletion and a novel missense NMNAT1 variant, c.500G>A (p.Asn167Ser). Linkage and segregation analysis excluded the CCDC66 variant and confirmed the NMNAT1 mutation. Biallelic NMNAT1 mutations cause Leber congenital amaurosis with a central nummular macular atrophic lesion (LCA9). The NMNAT1 mutation reported here underlied cone-rod dystrophy rather than LCA but the fundus lesion was compatible with that of LCA9 patients, highlighting that such a fundus appearance should raise suspicion for biallelic mutations in NMNAT1 when in the context of any retinal dystrophy. Although Ccdc66 mutations have been proposed to cause retinal disease in dogs, our results and public databases challenge CCDC66 as a candidate gene for human retinal dystrophy.

Extension of the clinical and molecular phenotype of DIAPH1-associated autosomal dominant hearing loss (DFNA1).

In about 20% of non-syndromic hearing loss (NSHL) cases, inheritance is autosomal dominant (ADNSHL). DIAPH1 mutations define the ADNSHL locus DFNA1. We identified two new families with heterozygous truncating DIAPH1 mutations (p.Ala1210Serfs*31 and p.Arg1213*). In contrast to the extensively studied original DFNA1 family, hearing loss was not confined to low frequencies, but congenital manifestation and rapid progression were confirmed. In line with a recent unrelated study, we identified an association with thrombocytopenia, reclassifying DFNA1 as a syndrome. Consequently, we suggest to include the blood count into the initial clinical workup of patients with autosomal dominant hearing loss to guide the genetic diagnosis. We provide the first data on DIAPH1 expression in the organ of Corti, where it localizes to the inner pillar cells, at the base of the outer hair cells. Homozygous truncating DIAPH1 mutations located N-terminally to the DFNA1 mutations have recently been identified in autosomal recessive microcephaly. It is therefore noteworthy that we found DIAPH1 expression also in spiral ganglion neurons and in the barrier between the myelinating glia of the peripheral nervous system and oligodendrocytes that form the myelinating glia of the central nervous system (CNS).

[Next-Generation Sequencing: A Quantum Leap in Ophthalmology Research and Diagnostics]. / Next-Generation Sequencing: Quantensprung für Forschung und Diagnostik in der Ophthalmologie.

Many eye diseases have a genetic basis, and most can be caused by mutations in many different genes (extensive genetic heterogeneity). The retinal dystrophies are a good example: More than 200 genes have been identified for the isolated forms (Leber's congenital amaurosis, retinitis pigmentosa, cone-rod dystrophy, congenital stationary night blindness), and for syndromes that comprise additional dysfunctions or malformations of extraocular tissues and organs. Selecting genes for diagnostic testing has been difficult, and their analysis with the hitherto predominant DNA sequencing method (Sanger sequencing) has been extremely laborious: The phenotype rarely indicates the affected gene, and the contributions of the particular genes to the disease (e.g., to LCA) were largely unknown. Consequently, comprehensive genetic analyses were impossible in most cases. In the recent years, high-throughput sequencing technologies, summarized as next-generation sequencing (NGS), have revolutionized genetic research and, subsequently, genetic diagnostics. The latter has far-reaching implications for the individual management of patients with genetic eye diseases and their families.

[Segregation Analysis in Inherited Eye Disorders: An Academic Add-on or An Essential Effort?] / Die Segregationsanalyse bei erblichen Augenerkrankungen ­ akademisches Extra oder notwendiger Aufwand?

The knowledge of the genetic basis of many eye diseases is constantly increasing. Besides retinal degeneration, developmental defects of the anterior segment, cataracts, and the development of the basic structure are often associated with genetic defects. Moreover, a lot of genetic syndromes involve eye abnormalities. The impact of genetics has become more and more evident in ophthalmological practice. Although genetic counselling is usually carried out by human geneticists, the increasing availability of therapeutic options requires ophthalmologists to have some basic knowledge of the genetic causes and how to identify them. The first step in this regard is to recognise potential genetic eye disorders and to initiate an adequate genetic analysis to confirm the diagnosis. This review discusses possible and necessary investigations within the patient's family facing ophthalmologists after the genetic cause of an eye disease has been identified.

[Genotype-Phenotype Correlations in Patients with CRB1 Mutations]. / Genotyp-Phänotyp-Korrelation bei Patienten mit CRB1-Mutationen.

Background Mutations in the CRB1 gene were identified in patients with early-onset severe retinal dystrophy (EOSRD), childhood-onset and juvenile-onset rod-cone dystrophy. This study describes the phenotypic spectrum of disease-causing CRB1-mutations in the first two decades of life. Materials and Methods Eight patients, aged three months to 20 years, underwent a full comprehensive ophthalmological examination including best corrected visual acuity testing (BCVA), color vision testing, funduscopy, spectral-domain optical coherence tomography (SD-OCT), and fundus autofluorescence (FAF) recording. Automated and manual retinal layer segmentation of SD-OCT recordings was performed using DIOCTA software. A full-field electroretinography (ffERG, ISCEV Standard) and visual fields were performed in cooperative patients. Results Five patients carried mutations causing a loss of the corresponding gene product (splice-mutation, nonsense-mutation, frame-shifting mutation). These patients presented with generally reduced vision in the first months of life that never exceeded 0.04 during the observational period. The sixth patient carried a homozygous missense mutation and reached maximal BCVA 0.05 at the age of 6 years. Two further patients, carrying at least one hypomorphic missense-mutation, presented with better preserved visual function with up to 0.5 at the age of 20 years. The recorded ffERG was below threshold in the majority of patients. Visual fields were severely restricted. The photoreceptor layers were significantly reduced in SD-OCT whenever stratification of retinal layers was possible. The inner nuclear layer thickness increased with progressing retinal degeneration. A-Scan analysis revealed better preservation of the retinal stratification in patients with missense mutations. Conclusions Patients with CRB1-mutations presented with a severe phenotype with severely reduced visual acuity from birth. Missense mutations with predicted residual function of the gene product were associated with moderate expression of the disease. Severe and progressive restriction of visual fields occurred in the first decade of life. The reduced retinal stratification indicates a general loss of structural integrity of the retinal layers.

Mutation of CDH23, encoding a new member of the cadherin gene family, causes Usher syndrome type 1D.

Usher syndrome type I (USH1) is an autosomal recessive disorder characterized by congenital sensorineural hearing loss, vestibular dysfunction and visual impairment due to early onset retinitis pigmentosa (RP). So far, six loci (USH1A-USH1F) have been mapped, but only two USH1 genes have been identified: MYO7A for USH1B and the gene encoding harmonin for USH1C. We identified a Cuban pedigree linked to the locus for Usher syndrome type 1D (MIM 601067) within the q2 region of chromosome 10). Affected individuals present with congenital deafness and a highly variable degree of retinal degeneration. Using a positional candidate approach, we identified a new member of the cadherin gene superfamily, CDH23. It encodes a protein of 3,354 amino acids with a single transmembrane domain and 27 cadherin repeats. In the Cuban family, we detected two different mutations: a severe course of the retinal disease was observed in individuals homozygous for what is probably a truncating splice-site mutation (c.4488G-->C), whereas mild RP is present in individuals carrying the homozygous missense mutation R1746Q. A variable expression of the retinal phenotype was seen in patients with a combination of both mutations. In addition, we identified two mutations, Delta M1281 and IVS51+5G-->A, in a German USH1 patient. Our data show that different mutations in CDH23 result in USH1D with a variable retinal phenotype. In an accompanying paper, it is shown that mutations in the mouse ortholog cause disorganization of inner ear stereocilia and deafness in the waltzer mouse.

A novel DFNB1 deletion allele supports the existence of a distant cis-regulatory region that controls GJB2 and GJB6 expression.

Eleven affected members of a large German-American family segregating recessively inherited, congenital, non-syndromic sensorineural hearing loss (SNHL) were found to be homozygous for the common 35delG mutation of GJB2, the gene encoding the gap junction protein Connexin 26. Surprisingly, four additional family members with bilateral profound SNHL carried only a single 35delG mutation. Previously, we demonstrated reduced expression of both GJB2 and GJB6 mRNA from the allele carried in trans with that bearing the 35delG mutation in these four persons. Using array comparative genome hybridization (array CGH), we have now identified on this allele a deletion of 131.4 kb whose proximal breakpoint lies more than 100 kb upstream of the transcriptional start sites of GJB2 and GJB6. This deletion, del(chr13:19,837,344-19,968,698), segregates as a completely penetrant DFNB1 allele in this family. It is not present in 528 persons with SNHL and monoallelic mutation of GJB2 or GJB6, and we have not identified any other candidate pathogenic copy number variation by arrayCGH in a subset of 10 such persons. Characterization of distant GJB2/GJB6 cis-regulatory regions evidenced by this allele may be required to find the 'missing' DFNB1 mutations that are believed to exist.

GPR98 mutations cause Usher syndrome type 2 in males.

Mutations in the large GPR98 gene underlie Usher syndrome type 2C (USH2C), and all patients described to date have been female. It was speculated that GPR98 mutations cause a more severe, and eventually lethal, phenotype in males. We describe for the first time two male patients with USH2 with novel GPR98 mutations. Clinical characterization of a male patient and his affected sister revealed a typical USH2 phenotype in both. GPR98 may have been excluded from systematic investigation in previous studies, and the proportion of patients with USH2C probably underestimated. GPR98 should be considered in patients with USH2 of both sexes.

Slow progression of a small Wilms' tumor.

We report the uncommon clinical course of a female with right-sided hemi-hyperplasia. At the age of 2 years and 2 months, a small spherical lesion of the right kidney was detected by ultrasound and magnetic resonance tomography. When the patient was 4 years and 7 months, the very slowly growing tumor was removed completely and diagnosed as intermediate risk stage I nephroblastoma. The case demonstrates that even small renal lesions require diagnostic work-up and adequate treatment.

[Neuro-ophthalmological and ophthalmological findings in Joubert syndrome]. / Neuroophthalmologische und ophthalmologische Befunde beim Joubert-Syndrom.

BACKGROUND: Joubert syndrome (JS) belongs to the ciliopathies and is a mostly autosomal recessively inherited disease (in the case of OFD1 mutations, JS is an X-linked trait). It is characterised by midbrain-hindbrain malformations with developmental delay, hypotonia and ataxia and a broad spectrum of other facultative findings. The aim of our study was to examine the ophthalmological and neuro-ophthalmological features of JS in our patients and to compare our findings to those of other studies. METHODS: In a retrospective study we evaluated the ophthalmological and neuro-ophthalmological findings of 9 consecutive patients who met the diagnostic criteria of JS. RESULTS: All patients had abnormalities of ocular motility, 4/9 used head thrusts to shift gaze (oculomotor apraxia OMA). In 6/8 patients, the optokinetic reflex (OKN) was absent. Furthermore, 8/9 children showed nystagmus, mostly see-saw nystagmus. Manifest strabismus was found in 8/9 while 3/9 had a retinopathy with either abnormal ERG and/or fundus appearance with or without visual impairment. Chorioretinal colobomata were present in 5/9 cases. Two patients showed a unilateral congenital ptosis, one a facial nerve paresis. CONCLUSIONS: The early neuro-ophthalmological findings in JS are not pathognonomic, but may lead to the diagnosis of JS. The syndrome should be suspected in patients with nystagmus, especially see-saw nystagmus, and abnormal OKN and/or OMA, and/or colobomata of the fundus, and further paediatric examinations should be initiated.

[Genetic diagnostics of retinal dystrophies : Breakthrough with new methods of DNA sequencing]. / Genetische Diagnostik von Netzhautdystrophien : Revolutionierung durch neue Methoden der DNA-Sequenzierung.

Until the mid-2000s, knowledge about the genetic causes of retinal dystrophies was not adequately translated into molecular diagnostics and genetic counselling offered to the patients. Although many genes whose mutations underlie retinal degeneration, e.g., retinitis pigmentosa, Leber congenital amaurosis and cone-rod dystrophies were known, they could not be analyzed on a routine diagnostic basis because DNA sequencing was too expensive and time-consuming. New methods summarized under the term next-generation sequencing (NGS) procedures for high-throughput sequencing have changed this completely. In its initial application in research NGS greatly accelerated the pace of novel disease gene identification: the mutations of most patients with retinal dystrophies can today be found in genes which are known to be associated with the condition. Since approximately 2010, NGS has expanded into routine diagnostics. In most patients, this now enables a genetic diagnosis and therefore specific genetic counselling and medical treatment. Constantly improving bioinformatics and comprehensive databases facilitate the evaluation of the complex NGS data. Nevertheless, profound scientific knowledge regarding the genetics of retinal dystrophies is indispensable to avoid erroneous data interpretation. This is also true for the close interaction between ophthalmologists and medical geneticists.

MKS1 mutations cause Joubert syndrome with agenesis of the corpus callosum.

Joubert syndrome (JS) is a clinically and genetically heterogeneous ciliopathy characterized by episodic hyperpnea and apnea, hypotonia, ataxia, cognitive impairment and ocular motor apraxia. The "molar tooth sign" is pathognomonic of this condition. Mutations in the MKS1 gene are a major cause of Meckel-Gruber syndrome (MKS), the most common form of syndromic neural tube defects, frequently resulting in perinatal lethality. We present the phenotype and genotype of a child with severe JS and agenesis of the corpus callosum (ACC). In our patient, a next generation sequencing (NGS) approach revealed the following two variants of the MKS1 gene: first, a novel missense variant [ c.240G > T (p.Trp80Cys)], which affects a residue that is evolutionarily highly conserved in mammals and ciliates; second, a 29 bp deletion in intron 15 [c.1408-35_1408-7del29], a founder mutation, which in a homozygous state constitutes the major cause of MKS in Finland. We review the MKS1-variants in all of the eleven JS patients reported to date and compare these patients to our case. To our knowledge, this is the first patient with Joubert syndrome and agenesis of the corpus callosum where a potentially causal genotype is provided.

Characterization of ADAMTS14, a novel member of the ADAMTS metalloproteinase family.

ADAMTS (a disintegrin-like and metalloproteinase domain with thrombospondin type 1 modules) proteins constitute a family of zinc metalloproteinases which target and process extracellular matrix proteins. We cloned and characterized a novel human ADAMTS gene, ADAMTS14, which is located on human chromosome 10q2. ADAMTS14 exhibits the characteristic multidomain structure of ADAMTS proteins including four thrombospondin modules and shows highest similarity to ADAMTS3 and ADAMTS2. By RT-PCR analysis we demonstrated that ADAMTS14 is expressed in human retina and also at low levels in adult brain, lung and placenta.

[Genetics of retinal dystrophies--an overview]. / Die Genetik der retinalen Dystrophien -- ein Uberblick.

Vision requires complex retinal functions, involving multiple genes with different functions. Retinal degeneration results from disturbance of retina-specific processes such as the visual transduction cascade, but also from defects in basic functions such as pre-mRNA splicing and nucleotide synthesis. As a consequence, the retinal dystrophies are genetically extremely heterogeneous (as shown in the table). Thanks to the Human Genome Project, the identification of retinal disease genes and additional loci has skyrocketed. Today, a typical search for the causative gene in a disease-linked genomic interval starts at the computer. Genes from a particular region can be displayed, and multiple gene-specific data such as expression patterns are immediately accessible. Candidate genes can then be investigated in DNA from affected individuals.

Phenotypic variability in rippling muscle disease.

OBJECTIVE: To characterize the phenotype of hereditary rippling muscle disease (RMD) and to report the results of genetic linkage studies. BACKGROUND: RMD is a rare autosomal-dominant inherited muscle disorder. Individuals complain of muscle stiffness, exercise-induced muscle pain, and cramp-like sensations. The characteristic feature of RMD is increased mechanical muscle irritability, which is electrically silent in electromyographic examinations. METHODS: Forty-six individuals from two unrelated German kindreds with RMD were examined. Linkage analysis to the RMD locus on chromosome 1q41-q43 was performed. RESULTS: In kindred A, 15 individuals from four generations, and in kindred B, four individuals from three generations had clinical features of RMD. The most consistent clinical findings were percussion-induced rapid muscle contractions (PIRCs) and muscle mounding, which were present in all 19 affected individuals. Only 12 individuals exhibited muscle rippling, indicating that rippling is not always present in RMD. Twelve of 19 individuals had muscle-related complaints, primarily exertional cramps and stiffness. The mean age at the onset of complaints was 22 years (range, 5 to 54 years). Seven of 19 individuals showed only mechanical-induced muscle irritability but did not have muscular symptoms. Genetic analysis excluded linkage to the RMD locus on chromosome 1q4 in both kindreds. CONCLUSIONS: The phenotype of RMD is variable but generalized PIRCs are the most obvious and reliable clinical feature of RMD. Diagnostic criteria of RMD should include generalized PIRCs in addition to muscle mounding, rippling, and creatine kinase elevation.

[Genetics of congenital aniridia]. / Genetik der kongenitalen Aniridie.

BACKGROUND: Mutations in the PAX6 gene mostly cause non-syndromic aniridia with autosomal dominant inheritance and familial occurrence. The underlying point mutations and deletions in the PAX6 locus cause loss-of-function of one gene copy (haploinsufficiency). Mutations with residual PAX6 function often result in milder disease expression but may also cause distinct and more severe ocular phenotypes. Combined deletion of PAX6 and the adjacent WT1 tumor suppressor gene causes Wilms tumor, aniridia, genitourinary anomalies and mental retardation (WAGR) syndrome with a high risk for Wilms tumors in infancy. PURPOSE: Genetic diagnostics are important for confirming the clinical diagnosis, for the assessment of the risk of recurrence and early recognition of children with associated tumor risk. RESULTS AND DISCUSSION: Sequencing of the PAX6 gene and quantitative analysis of the PAX6 locus allow for efficient molecular genetic evaluation of the clinical diagnosis of both isolated and syndromic aniridia. In cases of clinical overlap with other entities, high-throughput sequencing of multiple additional genes can simultaneously cover genes for differential diagnoses (e.g. microphthalmia syndromes). Optimal care of aniridia patients requires close cooperation of ophthalmologists and medical geneticists.

[Genetics of Usher syndrome]. / Genetik des Usher-Syndroms.

Since the first gene (MYO7A) for Usher syndrome was identified 14 years ago, there has been substantial progress in the elucidation of the genetic basis of this disorder, revealing extensive genetic heterogeneity (with nine genes known to date). Most Usher genes have similar functions, localize to similar regions in inner ear hair cells and retinal photoreceptors, and interact with each other. Approximately 80% of the patients carry mutations in one of the known Usher genes. One major challenge for the scientific community is to identify the remaining causative genes. Moreover, it is still largely unclear which genetic factors are responsible for the clinical variability that can be observed even between affected siblings. The establishment of high-throughput techniques shall soon provide comprehensive genetic testing covering all genes, which would be desirable: Early confirmation (or exclusion) of the diagnosis would be important for the individual patient, as it could help predict whether retinal degeneration can be expected in addition to the congenital hearing impairment.

Characterisation of severe rod-cone dystrophy in a consanguineous family with a splice site mutation in the MERTK gene.

AIM: To characterise the ocular phenotype of a family segregating the splice site mutation c.2189+1G>T in the tyrosine kinase receptor gene MERTK. METHODS: Five affected children of a consanguineous Moroccan family were investigated by ophthalmic examinations, including fundus photography, autofluorescence (FAF) imaging, optical coherence tomography (OCT), psychophysical and electrophysiological methods. RESULTS: Affected children were between 5 and 19 years of age, allowing an estimation of disease progression. Electroretinography demonstrated loss of scotopic and photopic function in the first decade of life. Younger siblings showed drusen-like deposits with focal relatively increased FAF in the macular area. With increasing age, a yellowish lesion with relatively increased FAF and subsequent macular atrophy developed. Visual acuity deteriorated with age and ranged between 20/50 in the best eye of the youngest affected and 20/400 in the worst eye of the oldest affected sibling. Spectral-domain OCT revealed debris-like material in the subneurosensory space. CONCLUSION: The splice site mutation c.2189+1G>T in MERTK causes rod-cone dystrophy with a distinct macular phenotype. The debris in the subneurosensory space resembles that in the Royal College of Surgeons (RCS) rat being the mertk animal model. Patients might therefore benefit from advances in gene therapy that were previously achieved in the RCS rat.