35-Year Follow-Up of a Case of Ring Chromosome 2: Array-CGH Analysis and Literature Review of the Ring Syndrome.

Côté et al. [1981] suggested that ring chromosomes with or without deletions share a common pattern of phenotypic anomalies, regardless of which chromosome is involved. The phenotype of this 'general ring syndrome' consists of growth failure without malformations, few or no minor anomalies, and mild to moderate mental retardation. We reconsidered the ring chromosome 2 case previously published by Côté et al. [1981], and we characterized it by array CGH, polymorphic markers as well as subtelomere MLPA and FISH analysis. A terminal deletion (q37.3qter) of maternal origin of the long arm of the ring chromosome 2 was detected and confirmed by all the above-mentioned methods. Ring chromosome 2 cases are exceedingly rare. Only 18 cases, including the present one, have been published so far, and our patient is the longest reported survivor, with a 35-year follow-up, and the third case characterized by array-CGH analysis.

A truncating mutation in SERPINB6 is associated with autosomal-recessive nonsyndromic sensorineural hearing loss.

More than 270 million people worldwide have hearing loss that affects normal communication. Although astonishing progress has been made in the identification of more than 50 genes for deafness during the past decade, the majority of deafness genes are yet to be identified. In this study, we mapped a previously unknown autosomal-recessive nonsyndromic sensorineural hearing loss locus (DFNB91) to chromosome 6p25 in a consanguineous Turkish family. The degree of hearing loss was moderate to severe in affected individuals. We subsequently identified a nonsense mutation (p.E245X) in SERPINB6, which is located within the linkage interval for DFNB91 and encodes for an intracellular protease inhibitor. The p.E245X mutation cosegregated in the family as a completely penetrant autosomal-recessive trait and was absent in 300 Turkish controls. The mRNA expression of SERPINB6 was reduced and production of protein was absent in the peripheral leukocytes of homozygotes, suggesting that the hearing loss is due to loss of function of SERPINB6. We also demonstrated that SERPINB6 was expressed primarily in the inner ear hair cells. We propose that SERPINB6 plays an important role in the inner ear in the protection against leakage of lysosomal content during stress and that loss of this protection results in cell death and sensorineural hearing loss.

Development of novel LOXL1 genotyping method and evaluation of LOXL1, APOE and MTHFR polymorphisms in exfoliation syndrome/glaucoma in a Greek population.

PURPOSE: In the Greek population of Epirus, exfoliation syndrome (XFS) and exfoliation glaucoma (XFG) occur at a high prevalence. In this study, we validate a novel lysyl oxidase-like 1 (LOXL1) genotyping method, investigate the previously reported association of LOXL1 with XFS/XFG, and evaluate apolipoprotein E (APOE) and methylenetetrahydrofolate reductase (MTHFR) polymorphisms as genetic risk factors for both conditions in our population. METHODS: Blood samples were collected from 82 patients with XFG, 69 patients with XFS, 52 patients with primary open-angle glaucoma (POAG), and 107 controls. APOE and MTHFR 677C>T genotyping was performed from extracted genomic DNA with established methods. A novel methodology of real-time PCR and melting curve analysis was developed and validated to accurately genotype the LOXL1 G153D and R141L polymorphisms by using two different fluorescent channels of the LightCycler instrument (Roche) examining each SNP separately. RESULTS: No significant differences were observed for the APOE and MTHFR polymorphisms between the patients with XFS, the patients with XFG, and the control subjects. The APOE ε2 allele appears to be associated with elevated risk of POAG in our population. Our novel LOXL1 genotyping method was easy to perform, fast, and accurate. A statistically significant association was found for the LOXL1 gene with XFS/XFG in this Greek population. The association of XFS and XFG with G153D appeared to be less powerful in this population (XFS: odds ratio [OR]=2.162, p=0.039, XFG: OR=2.794, p=0.002) compared to other populations, and for R141L, the association was proven only with XFG (OR=3.592, p<0.001). Neither of the two LOXL1 SNPs was significantly associated with POAG. CONCLUSIONS: We confirmed the association between LOXL1 and XFS/XFG, but the APOE and MTHFR polymorphisms are not significant risk factors for the development of XFS/XFG in our population of patients from Epirus (Greece).

Biomarkers in primary open angle glaucoma.

Glaucoma, a leading cause of blindness worldwide, is currently defined as a disturbance of the structural or functional integrity of the optic nerve that causes characteristic atrophic changes in the optic nerve, which may lead to specific visual field defects over time. This disturbance usually can be arrested or diminished by adequate lowering of intraocular pressure (IOP). Glaucoma can be divided roughly into two main categories, ' open angle ' and ' closed angle ' glaucoma.Open angle, chronic glaucoma tends to progress at a slower rate and patients may not notice loss of vision until the disease has progressed significantly. Primary open angle glaucoma(POAG) is described distinctly as a multifactorial optic neuropathy that is chronic and progressive with a characteristic acquired loss of optic nerve fibers. Such loss develops in the presence of open anterior chamber angles, characteristic visual field abnormalities, and IOP that is too high for the healthy eye. It manifests by cupping and atrophy of the optic disc, in the absence of other known causes of glaucomatous disease. Several biological markers have been implicated with the disease. The purpose of this study was to summarize the current knowledge regarding the non-genetic molecular markers which have been predicted to have an association with POAG but have not yet been validated.

Erythrokeratodermia variabilis: report of two cases and a novel missense variant in GJB4 encoding connexin 30.3.

Erythrokeratodermia variabilis (EKV) is characterized by migrating red patches resembling a geographical map, and by localized or generalized hyperkeratosis with scaling of the skin. The onset is usually at birth or during infancy, and the disease persists throughout life. EKV is mainly inherited as an autosomal dominant disease, although recessive transmission has occasionally been reported. Mutations associated with EKV have been identified in the connexin (Cx) genes GJB3 (Cx31) and GJB4 (Cx30.3), however, several cases of EKV have been tested negative for mutations in these two Cx genes. Here, we report our findings of the clinical, histological, and molecular examinations performed in two unrelated sporadic cases of EKV. The molecular screening involved bidirectional sequencing of the coding regions of the GJB3 and GJB4 genes and revealed the existence of a novel c.295G>A missense variant in the GJB4 gene found in homozygosity in one case. The substitution was found to result in a p.E99K change of the Cx30.3 protein, an alteration predicted to have a benign rather than a damaging effect on the protein function.

A novel PIKFYVE mutation in fleck corneal dystrophy.

PURPOSE: To report the findings of the clinical and molecular evaluation in a Greek family with fleck corneal dystrophy (CFD). METHODS: A 58-year-old woman was seen on routine ophthalmic examination and diagnosed as having CFD. All available family members were examined to evaluate the clinical findings and inheritance of the disease. Twenty members of the family in five generations underwent slit-lamp examination. Eleven were females and nine males, aged from two years to 85 years old. Blood samples were available from four patients with CFD and seven unaffected relatives, and the DNAs were subjected to molecular screening of the phosphoinositide kinase, five finger-containing (PIKFYVE) gene by direct sequencing or denaturing high performance liquid chromatography (DHPLC). RESULTS: The clinical evaluation revealed six family members (five females and one male) with CFD. In two CFD patients early cataract formation was noticed. All patients affected with the corneal dystrophy were asymptomatic. The molecular analyses demonstrated the existence of a novel c.3060-3063delCCTT (p.P968Vfs23) mutation in PIKFYVE in all CFD patients tested but in none of the six unaffected family members. No molecular screening was performed in the seventh unaffected member as the causative mutation was clearly transmitted from his affected wife to his affected son. CONCLUSIONS: We report on the clinical and molecular findings of a five generation Greek family with CFD and we conclude that the novel c.3060-3063delCCTT (p.P968Vfs23) mutation in PIKFYVE, which segregated with the disease, was the causative mutation in this family.

Complex distal 10q rearrangement in a girl with mild intellectual disability: follow up of the patient and review of the literature of non-acrocentric satellited chromosomes.

We report on an intellectually disabled girl with a de novo satellited chromosome 10 (10qs) and performed a review of the literature of the non-acrocentric satellited chromosomes (NASC). Satellites and stalks normally occur on the short arms of acrocentric chromosomes; however, the literature cites several reports of satellited non-acrocentric chromosomes, which presumably result from a translocation with an acrocentric chromosome. This is, to our knowledge, the third report of a 10qs chromosome. The phenotype observed in the proband prompted a search for a structural rearrangement of chromosome 10q. By microsatellite analysis we observed a 4 Mb deletion on the long arm of chromosome 10, approximately 145 kb from the telomere. FISH and array CGH analyses revealed a complex rearrangement involving in range from the centromere to the telomere: A 9.64 Mb 10q26.11-q26.2 duplication, a 1.3 Mb region with no copy number change, followed by a 5.62 Mb 10q26.2-q26.3 deletion and a translocation of satellite material. The homology between the repeat sequences at 10q subtelomere region and the sequences on the acrocentric short arms may explain the origin of the rearrangement and it is likely that the submicroscopic microdeletion and microduplication are responsible for the abnormal phenotype in our patient. The patient presented here, with a 15-year follow-up, manifests a distinct phenotype different from the 10q26 pure distal monosomy and trisomy syndromes.

Age-related macular degeneration: genetic and clinical findings.

Age-related macular degeneration (AMD) is a sight threatening eye disease that affects millions of humans over the age of 65 years. It is considered to be the major cause of irreversible blindness in the elderly population in the developed world. The disease is prevalent in Europe and the United States, which has a large number of individuals of European descent. AMD is characterized by a progressive loss of central vision attributable to degenerative and neovascular changes that occur in the interface between the neural retina and the underlying choroid. This location contains the retinal photoreceptors, the retinal pigmented epithelium, a basement membrane complex known as Bruch's membrane and a network of choroidal capillaries. AMD is increasingly recognized as a complex genetic disorder where one or more genes contribute to an individual's susceptibility to development of the condition, while the prevailing view is that the disease stems from the interaction of multiple genetic and environmental factors. Although it has been proposed that a threshold event occurs during normal aging, the sequelae of biochemical, cellular, and molecular events leading to AMD are not fully understood. Here, we review the clinical aspects of AMD and summarize the genes which have been reported to have a positive association with the disease.

Multiple enhancers located in a 1-Mb region upstream of POU3F4 promote expression during inner ear development and may be required for hearing.

POU3F4 encodes a POU-domain transcription factor required for inner ear development. Defects in POU3F4 function are associated with X-linked deafness type 3 (DFN3). Multiple deletions affecting up to ~900-kb upstream of POU3F4 are found in DFN3 patients, suggesting the presence of essential POU3F4 enhancers in this region. Recently, an inner ear enhancer was reported that is absent in most DFN3 patients with upstream deletions. However, two indications suggest that additional enhancers in the POU3F4 upstream region are required for POU3F4 function during inner ear development. First, there is at least one DFN3 deletion that does not eliminate the reported enhancer. Second, the expression pattern driven by this enhancer does not fully recapitulate Pou3f4 expression in the inner ear. Here, we screened a 1-Mb region upstream of the POU3F4 gene for additional cis-regulatory elements and searched for novel DFN3 mutations in the identified POU3F4 enhancers. We found several novel enhancers for otic vesicle expression. Some of these also drive expression in kidney, pancreas and brain, tissues that are known to express Pou3f4. In addition, we report a new and smallest deletion identified so far in a DFN3 family which eliminates 3.9 kb, comprising almost exclusively the previous reported inner ear enhancer. We suggest that multiple enhancers control the expression of Pou3f4 in the inner ear and these may contribute to the phenotype observed in DFN3 patients. In addition, the novel deletion demonstrates that the previous reported enhancer, although not sufficient, is essential for POU3F4 function during inner ear development.

Are GJB2 mutations an aggravating factor in the phenotypic expression of mitochondrial non-syndromic deafness?

Hearing impairment is a frequent condition, and genes have an important role in its etiology. The majority of hearing loss occurs in non-syndromic form, with deafness being the only clinically recognizable feature. More than 60 nuclear genes or loci have been shown to be involved in non-syndromic hearing loss, but mutations in mitochondrial DNA also cause hearing impairment. Mitochondrial DNA mutations usually lead to progressive hearing loss with an age of onset varying from childhood to early adulthood. It is interesting to note that there is a great variability among phenotypes between individuals harboring the same mitochondrial mutation, even within the same family, and the phenotype may range from profound deafness to completely normal hearing. In the past years, the debate on mitochondrial mutations has been about the penetrance, the tissue specificity and the mechanisms of modifier genes that can modulate the severity of the phenotypic expression of the deafness-associated mitochondrial DNA mutations. Here we summarize evidence regarding modifying genes, and we discuss the effect of the coexistence of mitochondrial and GJB2 mutations in families reported to date.

The A1555G mitochondrial DNA mutation in Greek patients with non-syndromic, sensorineural hearing loss.

Mitochondrial DNA mutations are undoubtedly a factor that contributes to sensorineural, non-syndromic deafness. One specific mutation, the A1555G, is associated with both aminoglycoside-induced and non-syndromic hearing impairment. The mutation is considered to be the most common of all mitochondrial DNA deafness-causing mutations but its frequency varies between different populations. Here we report on the first large screening of the A1555G mitochondrial DNA mutation in the Greek population. The aim of this study was to determine the frequency of the A1555G mutation in Greek sensorineural, non-syndromic deafness patients, with childhood onset. We screened 478 unrelated Greek patients with hearing loss of any degree and found two individuals harboring the A1555G mutation (0.42%). Both cases had been subjected to aminoglycosides. They were prelingual, familial and homoplasmic for the A1555G mutation. One of the cases was also found heterozygous for the frequent GJB2 35delG mutation, while the other case was negative. The A1555G mutation seems to be less common than in other European populations.

Strong linkage disequilibrium for the frequent GJB2 35delG mutation in the Greek population.

Approximately one in 1,000 children is affected by severe or profound hearing loss at birth or during early childhood (prelingual deafness). Up to 40% of congenital, autosomal recessive, severe to profound hearing impairment cases result from mutations in a single gene, GJB2, that encodes the connexin 26 protein. One specific mutation in this gene, 35delG, accounts for the majority of GJB2 mutations detected in Caucasian populations. Some previous studies have assumed that the high frequency of the 35delG mutation reflects the presence of a mutational hot spot, while other studies support the theory of a common founder. Greece is among the countries with the highest carrier frequency of the 35delG mutation (3.5%), and a recent study raised the hypothesis of the origin of this mutation in ancient Greece. We genotyped 60 Greek deafness patients homozygous for the 35delG mutation for six single nucleotide polymorphisms (SNPs) and two microsatellite markers inside or flanking the GJB2 gene. The allele distribution in the patients was compared to 60 Greek normal hearing controls. A strong linkage disequilibrium was found between the 35delG mutation and markers inside or flanking the GJB2 gene. Furthermore, we found a common haplotype with a previous study, suggesting a common founder for the 35delG mutation.

Cohen syndrome resulting from a novel large intragenic COH1 deletion segregating in an isolated Greek island population.

Cohen syndrome, caused by mutations in the COH1 gene, is an autosomal recessive disorder consisting of mental retardation, microcephaly, growth delay, severe myopia, progressive chorioretinal dystrophy, facial anomalies, slender limbs with narrow hands and feet, tapered fingers, short stature, kyphosis and/or scoliosis, pectus carinatum, joint hypermobility, pes calcaneovalgus, and, variably, truncal obesity. Here, we describe the clinical and molecular findings in 14 patients from an isolated Greek island population. The clinical phenotype was fairly homogeneous, although microcephaly was not constant, and some patients had severe visual disability. All patients were homozygous for a novel intragenic COH1 deletion spanning exon 6 to exon 16, suggesting a founder effect. The discovery of this mutation has made carrier detection and prenatal diagnosis possible in this population.

Sudden hearing loss in a family with GJB2 related progressive deafness.

Mutations of GJB2, the gene encoding connexin 26, have been associated with prelingual, sensorineural hearing loss of mild to profound severity. One specific mutation, the 35delG, has accounted for the majority of mutations detected in the GJB2 gene in Caucasian populations. Recent studies have described progression of hearing loss in a proportion of cases with GJB2 deafness. We report an unusual family with four 35delG homozygous members, in which the parents were deaf-mute whilst both children had a postlingual progressive hearing loss. Furthermore, the son suffered from sudden hearing loss.

SLITRK6 mutations cause myopia and deafness in humans and mice.

Myopia is by far the most common human eye disorder that is known to have a clear, albeit poorly defined, heritable component. In this study, we describe an autosomal-recessive syndrome characterized by high myopia and sensorineural deafness. Our molecular investigation in 3 families led to the identification of 3 homozygous nonsense mutations (p.R181X, p.S297X, and p.Q414X) in SLIT and NTRK-like family, member 6 (SLITRK6), a leucine-rich repeat domain transmembrane protein. All 3 mutant SLITRK6 proteins displayed defective cell surface localization. High-resolution MRI of WT and Slitrk6-deficient mouse eyes revealed axial length increase in the mutant (the endophenotype of myopia). Additionally, mutant mice exhibited auditory function deficits that mirrored the human phenotype. Histological investigation of WT and Slitrk6-deficient mouse retinas in postnatal development indicated a delay in synaptogenesis in Slitrk6-deficient animals. Taken together, our results showed that SLITRK6 plays a crucial role in the development of normal hearing as well as vision in humans and in mice and that its disruption leads to a syndrome characterized by severe myopia and deafness.

Compound heterozygosity of the novel c.292C>T (p.R98W) and the c.35delG GJB2 mutations in postlingual, non-syndromic, sensorineural deafness.

OBJECTIVES: Connexins (Cxs) are membrane-spanning proteins that co-assemble into intercellular gap junction channels. Gap junction channels mediate electrical and biochemical communication between adjacent cells and play vital roles as mediators of intercellular molecular signaling. Cx-linked deafness highlights the key role of gap junctions in the physiological processes of hearing. Co-localization of Cxs with the gap junction system in the inner ear suggests a role in cochlear electrolyte homeostasis. During auditory transduction, they are proposed to maintain membrane potentials by regulating the flow of potassium ions between the sensory epithelia of the inner ear. METHODS: Clinical and molecular genetic methods were employed in a Greek proband presenting with bilateral, postlingual, non-syndromic, sensorineural deafness. RESULTS: We detected a novel c.292C>T (p.R98W) mutation in compound heterozygosity with the c.35delG mutation in the GJB2 gene. CONCLUSION: Although mutations in the GJB2 gene usually cause prelingual, severe to profound deafness, compound heterozygosity of the novel c.292C>T (p.R98W) and the c.35delG GJB2 mutations appears to be the cause of postlingual, moderate, sensorineural deafness in our proband.

The novel c.247_249delTTC (p.F83del) GJB2 mutation in a family with prelingual sensorineural deafness.

Non-syndromic hearing loss is one of the most common hereditary determined diseases in human, and the disease is a genetically heterogeneous disorder. Mutations in the GJB2 gene, encoding connexin 26 (Cx26), are a major cause of non-syndromic recessive hearing impairment in many countries and are largely dependent on ethnic groups. Due to the high frequency of the c.35delG GJB2 mutation in the Greek population, we have previously suggested that Greek patients with sensorineural, non-syndromic deafness should be tested for the c.35delG mutation and the coding region of the GJB2 gene should be sequenced in c.35delG heterozygotes. Here we present on the clinical and molecular genetic evaluation of a family suffering from prelingual, sensorineural, non-syndromic deafness. A novel c.247_249delTTC (p.F83del) GJB2 mutation was detected in compound heterozygosity with the c.35delG GJB2 mutation in the proband and was later confirmed in the father, while the mother was homozygous for the c.35delG GJB2 mutation. We conclude that compound heterozygosity of the novel c.247_249delTTC (p.F83del) and the c.35delG mutations in the GJB2 gene was the cause of deafness in the proband and his father.

Detection of deafness-causing mutations in the Greek mitochondrial genome.

Mitochondrion harbors its own DNA, known as mtDNA, encoding certain essential components of the mitochondrial respiratory chain and protein synthesis apparatus. mtDNA mutations have an impact on cellular ATP production and many of them are undoubtedly a factor that contributes to sensorineural deafness, including both syndromic and non-syndromic forms. Hot spot regions for deafness mutations are the MTRNR1 gene, encoding the 12S rRNA, the MTTS1 gene, encoding the tRNA for Ser^{(UCN)}, and the MTTL1 gene, encoding the tRNA for Leu^{(UUR)}. We investigated the impact of mtDNA mutations in the Greek hearing impaired population, by testing a cohort of 513 patients suffering from childhood onset prelingual or postlingual, bilateral, sensorineural, syndromic or non-syndromic hearing loss of any degree for six mitochondrial variants previously associated with deafness. Screening involved the MTRNR1 961delT/insC and A1555G mutations, the MTTL1 A3243G mutation, and the MTTS1 A7445G, 7472insC and T7510C mutations. Although two patients were tested positive for the A1555G mutation, we failed to identify any subject carrying the 961delT/insC, A3243G, A7445G, 7472insC, or T7510C mutations. Our findings strongly support our previously raised conclusion that mtDNA mutations are not a major risk factor for sensorineural deafness in the Greek population.