Novel Ameloblastin Variants, Contrasting Amelogenesis Imperfecta Phenotypes.

Amelogenesis imperfecta (AI) comprises a group of rare, inherited disorders with abnormal enamel formation. Ameloblastin (AMBN), the second most abundant enamel matrix protein (EMP), plays a critical role in amelogenesis. Pathogenic biallelic loss-of-function AMBN variants are known to cause recessive hypoplastic AI. A report of a family with dominant hypoplastic AI attributed to AMBN missense change p.Pro357Ser, together with data from animal models, suggests that the consequences of AMBN variants in human AI remain incompletely characterized. Here we describe 5 new pathogenic AMBN variants in 11 individuals with AI. These fall within 3 groups by phenotype. Group 1, consisting of 6 families biallelic for combinations of 4 different variants, have yellow hypoplastic AI with poor-quality enamel, consistent with previous reports. Group 2, with 2 families, appears monoallelic for a variant shared with group 1 and has hypomaturation AI of near-normal enamel volume with pitting. Group 3 includes 3 families, all monoallelic for a fifth variant, which are affected by white hypoplastic AI with a thin intact enamel layer. Three variants, c.209C>G; p.(Ser70*) (groups 1 and 2), c.295T>C; p.(Tyr99His) (group 1), and c.76G>A; p.(Ala26Thr) (group 3) were identified in multiple families. Long-read AMBN locus sequencing revealed these variants are on the same conserved haplotype, implying they originate from a common ancestor. Data presented therefore provide further support for possible dominant as well as recessive inheritance for AMBN-related AI and for multiple contrasting phenotypes. In conclusion, our findings suggest pathogenic AMBN variants have a more complex impact on human AI than previously reported.

Phenotype and Variant Spectrum in the LAMB3 Form of Amelogenesis Imperfecta.

Amelogenesis imperfecta (AI) is a heterogeneous group of inherited disorders characterized by abnormal formation of dental enamel, either in isolation or as part of a syndrome. Heterozygous variants in laminin subunit beta 3 ( LAMB3) cause AI with dominant inheritance in the absence of other cosegregating clinical features. In contrast, biallelic loss-of-function variants in LAMB3 cause recessive junctional epidermolysis bullosa, characterized by life-threatening skin fragility. We identified 2 families segregating autosomal dominant AI with variable degrees of a distinctive hypoplastic phenotype due to pathogenic variants in LAMB3. Whole exome sequencing revealed a nonsense variant (c.3340G>T, p.E1114*) within the final exon in family 1, while Sanger sequencing in family 2 revealed a variant (c.3383-1G>A) in the canonical splice acceptor site of the final exon. Analysis of cDNA from family 2 revealed retention of the final intron leading to a premature termination codon. Two unerupted third molar teeth from individual IV:5 in family 2 were subject to computerized tomography and scanning electron microscopy. LAMB3 molar teeth have a multitude of cusps versus matched controls. LAMB3 enamel was well mineralized but pitted. The architecture of the initially secreted enamel was abnormal, with cervical enamel appearing much less severely affected than coronal enamel. This study further defines the variations in phenotype-genotype correlation for AI due to variants in LAMB3, underlines the clustering of nonsense and frameshift variants causing AI in the absence of junctional epidermolysis bullosa, and highlights the shared AI phenotype arising from variants in genes coding for hemidesmosome proteins.

Hypomaturation amelogenesis imperfecta due to WDR72 mutations: a novel mutation and ultrastructural analyses of deciduous teeth.

BACKGROUND: Mutations in WDR72 have been identified in autosomal recessive hypomaturation amelogenesis imperfecta (AI). OBJECTIVE: to describe a novel WDR72 mutation and report the ultrastructural enamel phenotype associated with a different WDR72 mutation. METHODS: A family segregating autosomal recessive hypomaturation AI was recruited, genomic DNA obtained and WDR72 sequenced. Four deciduous teeth from one individual with a previously published WDR72 mutation, extracted as part of clinical care, were subjected to scanning electron microscopy, energy-dispersive X-ray analysis and transverse microradiography. RESULTS: A novel homozygous nonsense mutation, R897X, was identified in WDR72 in a family originating from Pakistan. Ultrastructural analysis of enamel from the deciduous teeth of an AI patient with the WDR72 mutation S783X revealed energy-dispersive X-ray analysis spectra with normal carbon and nitrogen peaks, excluding retention of enamel matrix protein. However, transverse microradiography values were significantly lower for affected teeth when compared to normal teeth, consistent with reduced mineralisation. On scanning electron microscopy the enamel rod form observed was normal, yet with inter-rod enamel more prominent than in controls. This appearance was unaltered following incubation with either α-chymotrypsin or lipase. CONCLUSIONS: The novel WDR72 mutation described brings the total reported WDR72 mutations to four. Analyses of deciduous tooth enamel in an individual with a homozygous WDR72 mutation identified changes consistent with a late failure of enamel maturation without retention of matrix proteins. The mechanisms by which intracellular WDR72 influences enamel maturation remain unknown.

Ultrastructural analyses of deciduous teeth affected by hypocalcified amelogenesis imperfecta from a family with a novel Y458X FAM83H nonsense mutation.

BACKGROUND: Nonsense mutations in FAM83H are a recently described underlying cause of autosomal dominant (AD) hypocalcified amelogenesis imperfecta (AI). OBJECTIVE: This study aims to report a novel c.1374C>A p.Y458X nonsense mutation and describe the associated ultrastructural phenotype of deciduous teeth. METHODS: A family of European origin from the Iberian Peninsula with AD-inherited AI was ascertained. Family members were assessed through clinical examination and supporting investigations. Naturally exfoliated deciduous teeth from 2 siblings were investigated by scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX) and transverse microradiography (TMR). RESULTS: On clinical and radiographic investigation the appearances of the affected deciduous and permanent teeth were consistent with hypocalcified AI with small focal areas of more normal looking enamel. DNA sequencing identified a novel c.1374C>A p.Y458X FAM83H nonsense mutation in affected, but not in either unaffected family members or unrelated controls. Exfoliated teeth were characterised by substantial post-eruptive enamel loss on gross examination. Irregular, poor quality enamel prisms were observed on SEM. These were coated in amorphous material. TMR and EDX confirmed reduced mineral and increased organic content in enamel, respectively. CONCLUSIONS: FAM83H nonsense mutations have recently been recognised as a cause of AD hypocalcified AI. We report a novel nonsense FAM83H mutation and describe the associated preliminary ultrastructural phenotype in deciduous teeth. This is characterised by poorly formed enamel rods with inappropriate retention of amorphous material, which is likely to represent retained organic matrix that contributes to the overall hypomineralised phenotype.

The natural history of OPA1-related autosomal dominant optic atrophy.

BACKGROUND/AIMS: Autosomal dominant optic atrophy (ADOA) is a genetically heterogenous disease. However, a large proportion of this disease is accounted for by mutations in OPA1. The aim of this longitudinal study was to investigate disease progression in Australian ADOA patients with confirmed OPA1 mutations. METHODS: Probands with characteristic clinical findings of ADOA were screened for OPA1 mutations, and relatives of identified mutation carriers were invited to participate. Disease progression was determined by sequential examination or using historical records over a mean of 9.6 (range 1-42) years. RESULTS: OPA1 mutation carriers (n = 158) were identified in 11 ADOA pedigrees. Sixty-nine mutation carriers were available for longitudinal follow-up. Using the right eye as the default, best-corrected visual acuity (BCVAR) remained unchanged (defined as visual acuity at or within one line of original measurement) in 43 patients (62%). BCVAR worsened by 2 lines in 13 patients (19%). BCVAR deteriorated by more than 2 lines in six patients (9%). Ten per cent of patients had an improvement in visual acuity. Mean time to follow-up was 9.6 years with the mean visual acuity being 6/18 for both the initial and subsequent measurements. There was no statistical significance in the rate of BCVAR loss across different OPA1 mutations (p = 0.55). CONCLUSION: OPA1-related ADOA generally progresses slowly and functional visual acuity is usually maintained. Longitudinal disease studies are important to enable appropriate counselling of patients. This study enables a better understanding of the natural history of ADOA.

Reduced bone mineral density and hyaloid vasculature remnants in a consanguineous recessive FEVR family with a mutation in LRP5.

BACKGROUND/AIMS: Familial exudative vitreoretinopathy (FEVR) is an inherited blinding condition characterised by abnormal development of the retinal vasculature. FEVR has multiple modes of inheritance, and homozygous mutations in LRP5 have recently been reported as underlying the recessive form of this disease. The aim of this study was to examine LRP5 in a consanguineous recessive FEVR family and to clarify the eye and bone phenotype associated with recessive FEVR. METHODS: All family members were examined by slit lamp biomicroscopy and indirect ophthalmoscopy. Linkage to LRP5 was determined by genotyping microsatellite markers, constructing haplotypes and calculating lod scores. Mutation screening of LRP5 was performed by polymerase chain reaction amplification of genomic DNA followed by direct sequencing. Bone mineral density (BMD) was evaluated in all family members using dual energy x ray absorptiometry (DEXA). RESULTS: The clinical features observed in this family were consistent with a diagnosis of recessive FEVR. A homozygous LRP5 missense mutation, G550R, was identified in all affected individuals and all unaffected family members screened were heterozygous carriers of this mutation. Reduced BMD, hyaloid vasculature remnants, and nystagmus were features of the phenotype. CONCLUSION: Recessive mutations in LRP5 can cause FEVR with reduced BMD and hyaloid vasculature remnants. Assessment of a patient with a provisional diagnosis of FEVR should therefore include investigation of BMD, with reduced levels suggestive of an underlying LRP5 mutation.

Further evidence of genetic heterogeneity in familial exudative vitreoretinopathy; exclusion of EVR1, EVR3, and EVR4 in a large autosomal dominant pedigree.

BACKGROUND/AIMS: Familial exudative vitreoretinopathy (FEVR) is an inherited blinding condition characterised by abnormal development of the retinal vasculature. The aim of this study was to perform linkage analysis in a large family affected with FEVR to determine whether the mutation involved was in one of the three known autosomal dominant FEVR loci or in another as yet unidentified gene. METHODS: Genomic DNA samples from family members were polymerase chain reaction (PCR) amplified with fluorescently tagged microsatellite markers spanning the EVR1/EVR4 locus (11q13-14) and the EVR3 locus (11p12-13). The resulting PCR products were resolved using an automated DNA sequencer and the alleles sized. These data were used to construct haplotypes across each locus and linkage analysis was performed to prove or exclude linkage. RESULTS: The clinical evaluation in this family suggested features typical of FEVR, with deficient peripheral retinal vascularisation being the common phenotype in all affected individuals. However, linkage analysis proved that this family has a form of FEVR genetically distinct from the EVR1, EVR3 and EVR4 loci. CONCLUSION: The exclusion of linkage in this family to any of the known FEVR loci proves the existence of a fourth locus for autosomal dominant FEVR and shows that this rare disorder is far more heterogeneous than previously thought.

Retinitis pigmentosa: genes, proteins and prospects.

The name retinitis pigmentosa (RP) describes a heterogeneous group of inherited progressive retinal dystrophies, primarily affecting the peripheral retina. Patients experience night blindness and visual field loss, often leading to complete blindness. RP can be inherited in autosomal dominant, autosomal recessive, X-linked, mitochondrial and genetically more complex modes. To date, 39 loci have been implicated in non-syndromic RP, for which 30 of the genes are known. Many of these can be grouped by function, giving insights into the disease process. These include components of the phototransduction cascade, proteins involved in retinol metabolism and cell-cell interaction, photoreceptor structural proteins and transcription factors, intracellular transport proteins and splicing factors. Current knowledge of each grouping is reviewed briefly herein and consistent patterns of inheritance, which may have functional significance, are noted. The complexity of these diseases has in the past made it difficult to counsel patients or to envisage widely applicable therapies. As a more complete picture is emerging however, possibilities exist for streamlining screening services and a number of avenues for possible therapy are being investigated.

Progression of phenotype in Leber's congenital amaurosis with a mutation at the LCA5 locus.

BACKGROUND: Leber's congenital amaurosis (LCA) accounts for 5% of inherited retinal disease and is usually inherited as an autosomal recessive trait. Genetic and clinical heterogeneity exist. Mutations have been described in the RPE65, CRB1, RPGRIP1, AIPL1, GUCY2D, and CRX genes and other pedigrees show linkage to the LCA3 and LCA5 loci. The latter is a new locus which maps to 6q11-q16. The ocular findings and the evolution of the macula staphyloma are described in five members of a Pakistani family with consanguinity and a mutation in the LCA5 gene. METHODS: 13 family members including five affected individuals consented to DNA analysis and ocular examination including fundal photography. RESULTS: Ocular abnormalities are described. The most striking feature was the progression of macula abnormalities in three brothers resulting in a colobomatous appearance in the eldest compared to only mild atrophy in the youngest. The phenotypic pattern of this mutation in this Pakistani family contrasts with the "Old Order River Brethren" who were of Swiss descent, in whom the mutation was first described. CONCLUSION: The evolution of a new phenotypic picture is presented to a mutation in LCA5.

CORD9 a new locus for arCRD: mapping to 8p11, estimation of frequency, evaluation of a candidate gene.

PURPOSE: To determine the locus of the mutant gene causing autosomal recessive cone-rod dystrophy (arCRD) in a consanguineous pedigree, to evaluate a candidate gene expressed in retina that maps to this locus, and to estimate the percentage of arCRD cases caused by mutations in this gene. METHODS: DNAs from family members were genotyped for markers covering the entire genome at an average spacing of approximately 9 centimorgans (cM). The data were input into a pedigree computer program to produce output files used to calculate lod scores. Significant linkage was revealed at 8cen, prompting the genotyping of a number of additional markers. Exons of a candidate gene were sequenced directly by standard fluorescent dideoxy methods. Haplotype analysis was performed with markers in this locus in 13 multiplex and 2 simplex CRD families in which neither parent had disease. RESULTS: Four-point linkage analysis gave a maximum lod score of approximately 7.6 at both D8S1769 and GATA101H09 in the large consanguineous family. Recombination events defined an interval of 8.7 cM between D8S1820 and D8S532 within which the gene must lie. This 8p11 locus (CORD9) is immediately distal to but distinct from the RP1 autosomal dominant RP (adRP) locus. Two islands of homozygosity were found in this locus: The alleles of 6 of 10 markers in one of the islands and 2 of 4 in the other were homozygous. The UniGene cluster Hs.8719 (UniGene System, provided by the National Center for Biotechnology Information and available at http://www.ncbi.nlm.nih.gov/UniGene), which tags a gene with significant homology to Dual Specificity Phosphatase 3, maps within the CORD9 interval and is highly expressed in the retina. To evaluate this gene as a potential disease candidate, intron-exon structure was determined, and exons were screened in the consanguineous family. No variants were found that could be related to disease. Haplotype analysis of 15 other families with CRD, using markers at CORD9, excluded this locus in 9 of 15. CONCLUSIONS: A new arCRD locus (CORD9) has been identified corresponding to a yet unidentified gene in the 8.7-cM interval D8S1820-D8S532. No mutations were found in one candidate gene in affected members of the primary study family. Haplotype analysis of a cohort of 13 multiplex and 2 simplex families with CRD ruled out the CORD9 gene in 9 of 15 of the families. To date, a total of 126 loci carrying gene mutations causing various forms of retinal degeneration have been mapped, and the mutant gene has been identified in 64 of them. However, only 2 loci for arCRD have been documented. This is the report of a third.

Mutations in the pre-mRNA splicing factor gene PRPC8 in autosomal dominant retinitis pigmentosa (RP13).

Retinitis pigmentosa (RP) is a genetically heterogeneous disorder characterized by progressive degeneration of the peripheral retina leading to night blindness and loss of visual fields. With an incidence of approximately 1 in 4000, RP can be inherited in X-linked, autosomal dominant or autosomal recessive modes. The RP13 locus for autosomal dominant RP (adRP) was placed on chromosome 17p13.3 by linkage mapping in a large South African adRP family. Using a positional cloning and candidate gene strategy, we have identified seven different missense mutations in the splicing factor gene PRPC8 in adRP families. Three of the mutations cosegregate within three RP13 linked families including the original large South African pedigree, and four additional mutations have been identified in other unrelated adRP families. The seven mutations are clustered within a 14 codon stretch within the last exon of this large 7 kb transcript. The altered amino acid residues at the C-terminus exhibit a high degree of conservation across species as diverse as humans, Arabidopsis and trypanosome, suggesting that some functional significance is associated with this part of the protein. These mutations in this ubiquitous and highly conserved splicing factor offer compelling evidence for a novel pathway to retinal degeneration.

Spectrum, frequency and penetrance of OPA1 mutations in dominant optic atrophy.

Dominant optic atrophy (DOA) is the commonest form of inherited optic neuropathy. Although heterogeneous, a major locus has been mapped to chromosome 3q28 and the gene responsible, OPA1, was recently identified. We therefore screened a panel of 35 DOA patients for mutations in OPA1. This revealed 14 novel mutations and a further three known mutations, which together accounted for 20 of the 35 families (57%) included in this study. This more than doubles the number of OPA1 mutations reported in the literature, bringing the total to 25. These are predominantly null mutations generating truncated proteins, strongly suggesting that the mechanism underlying DOA is haploinsufficiency. The mutations are largely family-specific, although a common 4 bp deletion in exon 27 (eight different families) and missense mutations in exons 8 (two families) and 9 (two families) have been identified. Haplotype analysis of individuals with the exon 27 2708del(TTAG) mutation suggests that this is a mutation hotspot and not an ancient mutation, thus excluding a major founder effect at the OPA1 locus. The mutation screening in this study also identified a number of asymptomatic individuals with OPA1 mutations. A re-calculation of the penetrance of this disorder within two of our families indicates figures as low as 43 and 62% associated with the 2708del(TTAG) mutation. If haploinsufficiency is the mechanism underlying DOA it is unlikely that this figure will be mutation-specific, indicating that the penetrance in DOA is much lower than the 98% reported previously. To investigate whether Leber's hereditary optic neuropathy (LHON) could be caused by mutations in OPA1 we also screened a panel of 28 LHON patients who tested negatively for the three major LHON mutations. No mutations were identified in any LHON patients, indicating that DOA and LHON are genetically distinct.

A high-density transcript map of the human dominant optic atrophy OPA1 gene locus and re-evaluation of evidence for a founder haplotype.

Dominant optic atrophy (DOA, gene OPA1) is the commonest form of inherited optic atrophy. Linkage studies have shown that a locus for this disease lies in a 1.4-cM region at chromosome 3q28-->q29 and have suggested a founder haplotype for as many as 95% of the linked families. To aid the identification of candidate genes for this disease, we have constructed a Bacterial Artificial Chromosome (BAC) contig covering approximately 3.3 Mb and encompassing the OPA1 critical region (flanking markers D3S3669 and D3S3562). This physical map corrects errors in the marker order reported in the literature, allowing the OPA1 critical region to be precisely defined. A reassessment of the founder effect in the light of the revised marker order suggests that it may not be as significant as had previously been suggested. A high-density transcript map was created by precisely mapping genes and expressed sequence tags (ESTs) from GeneMap'99, that have been loosely assigned to the region by radiation hybrid mapping. One known gene (KIAA0567 protein) and 15 ESTs were found to lie within the minimal disease region. Analysis of the sequence data already available from within the OPA1 critical region allowed the identification and mapping of a further 31 ESTs. The work presented in this study provides the basis for the characterisation of candidate genes and the ultimate identification of the gene mutated in DOA.

A new locus for autosomal dominant familial exudative vitreoretinopathy maps to chromosome 11p12-13.

We report a new locus for familial exudative vitreoretinopathy (FEVR), on chromosome 11p12-13 in a large autosomal dominant pedigree. Statistically significant linkage was achieved across a 14-cM interval flanked by markers GATA34E08 and D11S4102, with a maximum multipoint LOD score of 6.6 at D11S2010. FEVR is a disease characterized by the failure of development of peripheral retinal blood vessels, and it is difficult to diagnose clinically because of the wide spectrum of fundus abnormalities associated with it. The identification of a new locus is important for genetic counseling and potentiates further studies aimed toward the identification of a gene with an important role in angiogenesis within neuroepithelial tissues. Such a gene may also have a role in the genetic predisposition to retinopathy of prematurity, a sporadic disorder with many clinical similarities to FEVR.