Maximum Two-point LOD Score Research Articles

A missense allele of PEX5 is responsible for the defective import of PTS2 cargo proteins into peroxisomes

Peroxisomes, single-membrane intracellular organelles, play an important role in various metabolic pathways. The translocation of proteins from the cytosol to peroxisomes depends on peroxisome import receptor proteins and defects in peroxisome transport result in a wide spectrum of peroxisomal disorders. Here, we report a large consanguineous family with autosomal recessive congenital cataracts and developmental defects. Genome-wide linkage analysis localized the critical interval to chromosome 12p with a maximum two-point LOD score of 4.2 (θ = 0). Next-generationexome sequencing identified a novel homozygous missense variant (c.653T > C; p.F218S) in peroxisomal biogenesis factor 5 (PEX5), a peroxisome import receptor protein. This missense mutation was confirmed by bidirectional Sanger sequencing. It segregated with the disease phenotype in the family and was absent in ethnically matched control chromosomes. The lens-specific knockout mice of Pex5 recapitulated the cataractous phenotype. In vitro import assays revealed a normal capacity of the mutant PEX5 to enter the peroxisomal Docking/Translocation Module (DTM) in the presence of peroxisome targeting signal 1 (PTS1) cargo protein, be monoubiquitinated and exported back into the cytosol. Importantly, the mutant PEX5 protein was unable to form a stable trimeric complex with peroxisomal biogenesis factor 7 (PEX7) and a peroxisome targeting signal 2 (PTS2) cargo protein and, therefore, failed to promote the import of PTS2 cargo proteins into peroxisomes. In conclusion, we report a novel missense mutation in PEX5 responsible for the defective import of PTS2 cargo proteins into peroxisomes resulting in congenital cataracts and developmental defects.

Mapping of Papillon-Lefevre syndrome to the chromosome 11q14 region.

Papillon-Lefevre syndrome (PLS) is an autosomal recessive disease which belongs to the palmo-plantar keratoderma (PPK) group. It is characterized by a premature loss of primary and permanent teeth and early onset periodontitis. High consanguinity has been observed in over one-third of PLS families. No candidate genes or gene localizations have been described to date for this disorder. A primary genome-wide search by homozygosity mapping using samples from a large consanguineous family in which 4 siblings were affected by the disease showed homozygosity and linkage in the region of 11q14. Linkage was confirmed in 4 additional families with diverse ethnic and geographic backgrounds, 2 of which were consanguineous. A maximum two-point lod score of 8.19 was obtained for the marker AFM063yg1 (D11S901= for theta = 0. Analysis of recombination events places the gene within a 7-cM interval between AFM063yg1 and AFM269yg9 (D11S4175). No shared haplotype was found for the 5 families analysed.

Linkage of DFNB1 to non-syndromic neurosensory autosomal-recessive deafness in Mediterranean families.

Recent studies show a susceptibility locus (DFNB1) responsible for non-syndromic neurosensory autosomal-recessive deafness (NSRD) mapping to the pericentromeric region of chromosome 13q. In order to better understand the frequency with which DFNB1 is the gene for deafness in our patient population and the role of DFNB1 in Caucasians, we performed a genetic linkage study with four microsatellite markers linked to DFNB1 in a total of 48 independent Mediterranean families, of which 30 and 18 were of Italian and Spanish descent, respectively. A maximum two-point lod score of 7.28 was found with marker D13S115 at a recombination frequency of theta 0.1. Significant lod scores were also obtained for D13S143, D13S292 and D13S175. Genetic heterogeneity was confirmed using the HOMOG program which indicated absence of linkage to DFNB1 in approximately 21% of the sample. This study clearly demonstrates that DFNB1 plays an important role in 79% of Mediterranean families with NSRD. Furthermore, results from multipoint analysis predict that the DFNB1 gene maps between markers D13S175 and D13S115 which are separated by approximately 14.2 cM.

Exome genotyping and linkage analysis identifies two novel linked regions and replicates two others for myopia in Ashkenazi Jewish families

BackgroundMyopia is one of most common eye diseases in the world and affects 1 in 4 Americans. It is a complex disease caused by both environmental and genetics effects; the genetics effects are still not well understood. In this study, we performed genetic linkage analyses on Ashkenazi Jewish families with a strong familial history of myopia to elucidate any potential causal genes.MethodsSixty-four extended Ashkenazi Jewish families were previously collected from New Jersey. Genotypes from the Illumina ExomePlus array were merged with prior microsatellite linkage data from these families. Additional custom markers were added for candidate regions reported in literature for myopia or refractive error. Myopia was defined as mean spherical equivalent (MSE) of -1D or worse and parametric two-point linkage analyses (using TwoPointLods) and multi-point linkage analyses (using SimWalk2) were performed as well as collapsed haplotype pattern (CHP) analysis in SEQLinkage and association analyses performed with FBAT and rv-TDT.ResultsStrongest evidence of linkage was on 1p36(two-point LOD = 4.47) a region previously linked to refractive error (MYP14) but not myopia. Another genome-wide significant locus was found on 8q24.22 with a maximum two-point LOD score of 3.75. CHP analysis also detected the signal on 1p36, localized to the LINC00339 gene with a maximum HLOD of 3.47, as well as genome-wide significant signals on 7q36.1 and 11p15, which overlaps with the MYP7 locus.ConclusionsWe identified 2 novel linkage peaks for myopia on chromosomes 7 and 8 in these Ashkenazi Jewish families and replicated 2 more loci on chromosomes 1 and 11, one previously reported in refractive error but not myopia in these families and the other locus previously reported in the literature. Strong candidate genes have been identified within these linkage peaks in our families. Targeted sequencing in these regions will be necessary to definitively identify causal variants under these linkage peaks.

Detection of Large Structural Variants Causing Inherited Retinal Diseases.

Current application of next-generation sequencing (NGS) leads to detection of the underlying disease-causing gene and mutation or mutations in from 60% to 85% of patients with inherited retinal diseases (IRDs), depending on the methods used, disease type, and population tested. In a cohort of 320 families with autosomal dominant retinitis pigmentosa (adRP), we have detected the mutation in 82% of cases using a variety of methods, leaving more than 50 families with "elusive" disease genotypes. All of the remaining families have been screened for mutations in known IRD genes using retinal-targeted-capture NGS, and most have been tested by whole-exome NGS. Linkage mapping has been conducted in several large families. In one of these families, with DNA samples from ten affected family members and six unaffected, linking members, we observed substantial maximum two-point LOD scores for linkage to both chromosomes 2 and 4. Subsequent 10X Genomics Chromium™ sequencing, which facilitates linked-read, phase-known chromosomal analysis, revealed a balanced translocation of the q terminus arms of chromosomes 2 and 4 involving 35 Mb and 73 Mb of 2 and 4, respectively. The balanced translocation is present in all affected family members and absent from all unaffected individuals. Family histories suggest multiple miscarriages are associated with the translocation. The breakpoint on chromosome 4 is within or 5' to the LRAT gene, whereas the chromosome 2 break is in a gene-poor region. We conclude that the balanced translocation is the cause of adRP in this family, which may lead to dysregulation of the LRAT gene. Since multiple miscarriages are a hallmark of balanced translocations, this possibility should be considered in evaluating family histories. Further, large structural variants, which are not easily detected by conventional sequencing methods, may account for a significant fraction of the remaining unsolved families.

A recurrent mutation of CRYGD gene in a northern Chinese family with autosomal dominant congenital nuclear cataract

Background Congenital cataract is a major cause for blindness of childhood. Genetic gene mutation accounts for almost 1/3 of congenital cataract patients. The most common inheritance type is autosomal dominant congenital cataract (ADCC). Over 100 mutations in 26 genes have been found to be associated with ADCC. Objective This study was to identify the disease-causing gene mutation in a family with ADCC. Methods This study was approved by Ethic Committee of Beijing Tongren Hospital and followed Declaration of Helsinki. A northern Chinese family with autosomal dominant congenital nuclear cataract was entrolled in Beijing Tongren Hospital in January 2011. Ocular examinations were performed and periphery blood specimens were collected from each family member under the informed consent. Genomic DNA was extracted. Twenty-one microsatellite markers around 17 ADCC genes were selected for linkage analysis, and two-point LOD score was calculated. CRYGC gene and CRYGD gene were amplified and screened for mutations using direct sequencing. ProtScale software was used to analyze the changes of hydrophobicity of the mutated protein. Co-segregation of the observed change with the disease phenotype was further detected by restriction fragment length polymorphism (RFLP). Results This family included 20 members of 4 generations, and 9 patients were examined in serial 4 passages, which conformed to autosomal dominant inheritance pattern. Clinical examination revealed binocular congenital nuclear cataract in the 9 patients. Maximum two-point LOD score was 4. 68 at marker D2S325 (θ=0). A known T→C change at position 127 of cDNA sequence was found by mutations screening of CRYGD gene. ProtScale programs showed an obvious increase of the local hydrophobicity in the mutant protein. RFLP results indicated that this missense mutation co-segregated with affected members of the family, but was absent in unaffected members and 100 unrelated controls. Conclusions c. T127C mutation of CRYGD gene appears to be the molecular pathogenesis of this ADCC family. Aberrant structure of mutant CRYGD protein caused by hydrophobicity change may lead to opacification of lens. Key words: Cataract/congenital; Cataract/genetics; DNA mutational analysis; Lens nucleus, crystalline/pathology; Pedigree; gamma-Crystallin/genetics; Mutations; Chinese

A Novel Locus for Ectodermal Dysplasia of Hair, Nail and Skin Pigmentation Anomalies Maps to Chromosome 18p11.32-p11.31.

Ectodermal dysplasias (EDs) are a large heterogeneous group of inherited disorders exhibiting abnormalities in ectodermally derived appendages such as hair, nails, teeth and sweat glands. EDs associated with reticulated pigmentation phenotype are rare entities for which the genetic basis and pathophysiology are not well characterized. The present study describes a five generation consanguineous Pakistani family segregating an autosomal recessive form of a novel type of ectodermal dysplasia. The affected members present with sparse and woolly hair, severe nail dystrophy and reticulate skin pigmentation. After exclusion of known gene loci related with other skin disorders, genome-wide linkage analysis was performed using Illumina HumanOmniExpress beadchip SNP arrays. We linked this form of ED to human chromosome 18p11.32-p11.31 flanked by the SNPs rs9284390 (0.113Mb) and rs4797100 (3.14 Mb). A maximum two-point LOD score of 3.3 was obtained with several markers along the disease interval. The linkage interval of 3.03 Mb encompassed seventeen functional genes. However, sequence analysis of all these genes did not discover any potentially disease causing-variants. The identification of this novel locus provides additional information regarding the mapping of a rare form of ED. Further research, such as the use of whole-genome sequencing, would be expected to reveal any pathogenic mutation within the disease locus.

Gene mapping and analysis of candidate genes in a Chinese family with autosomal dominant congenital coralliform cataract

To map and to identify the causal gene of autosomal dominant congenital coralliform cataract (ADCC) in a Chinese family. Case series study. Clinical features of all affected members in this family were examined. Blood samples were collected from nine family members for genetic linkage analysis. Polymorphic microsatellite markers were selected from the regions which harbor all known loci linked with ADCC. Direct genomic sequencing was used to evaluate the candidate gene. The affected members in this family showed classic phenotype of ADCC. The maximum two-point LOD score of 1.51 was obtained for marker D2S325 (θ = 0). The LOD score of 1.20 was obtained for marker D11S925. The CRYGC/CRYGD gene located on 2q33-q35 and the CRYAB gene located on 11q22-q22.3. Therefore, the CRYGC/CRYGD and CRYAB gene may be the candidate gene in this family. Sequencing of the coding regions of the CRYGD gene showed the presence of a heterozygous C→A transversion at nucleotide 70 in exon 2 of CRYGD that is associated with cataracts in this family. This mutation resulted in a proline to threonine substitution at amino acid 23 of the protein in the first of the four Greek key motifs that characterized this protein. No mutation in all exons of CRYGC and CRYAB gene were found in the family. Direct DNA sequence analysis revealed a C-to-A transition at nucleotide 70 of the CRYGD gene in this ADCC family.

Identification of a locus (DSP2) for disseminated superficial porokeratosis at chromosome 12q21.2-24.21

Porokeratosis is a rare disorder of epidermal keratinization that is characterized by the presence of a border called the cornoid lamella. Disseminated superficial porokeratosis (DSP) is a subtype of porokeratosis, which is inherited as an autosomal trait. The first locus for DSP was localized to chromosome 18p11.3, but no causative gene has yet been identified. In this study, we recruited and analysed a large six-generation Chinese family with autosomal dominant DSP. The genome-wide screening identified a maximum two-point LOD score of 3.06 at θ = 0.00 with the microsatellite marker D12S78. Fine mapping and haplotype analysis defined a critical region of 38 Mb between D12S326 and D12S79 on chromosome 12q21.2-24.21, which is a probable second locus identified for DSP (DSP2). We sequenced 50 candidate genes in this region, but no causative mutation was found. This study provides a map location for isolation of a gene causing DSP.

A novel locus for restless legs syndrome maps to chromosome 19p in an Irish pedigree

Restless legs syndrome (RLS) is a common, sleep-related movement disorder. The symptoms follow a circadian pattern, worsening in the evening or night, leading to sleep disruption and daytime somnolence. Familial forms of RLS have been described and usually display an autosomal dominant pattern of inheritance. To date, linkage analysis has identified nine RLS loci, but no specific causative gene has been reported. Association mapping has highlighted a further four genomic areas of interest. We have conducted a genome-wide linkage analysis in an Irish autosomal dominant RLS pedigree with 11 affected members. Significant linkage was found on chromosome 19p for a series of microsatellite markers, with a maximum two-point LOD score of 3.59 at θ = 0.0 for marker D19S878. Recombination events, identified by haplotype analysis, define a genetic region of 6.57 cM on chromosome 19p13.3, corresponding to an interval of 2.5 Mb. This study provides evidence of a novel RLS locus and provides further evidence that RLS is a genetically heterogenous disorder.

GNAT1Associated with Autosomal Recessive Congenital Stationary Night Blindness

Congenital stationary night blindness is a nonprogressive retinal disorder manifesting as impaired night vision and is generally associated with other ocular symptoms, such as nystagmus, myopia, and strabismus. This study was conducted to further investigate the genetic basis of CSNB in a consanguineous Pakistani family. A consanguineous family with multiple individuals manifesting cardinal symptoms of congenital stationary night blindness was ascertained. All family members underwent detailed ophthalmic examination, including fundus photographic examination and electroretinography. Blood samples were collected and genomic DNA was extracted. Exclusion and genome-wide linkage analyses were completed and two-point LOD scores were calculated. Bidirectional sequencing of GNAT1 was completed, and quantitative expression of Gnat1 transcript levels were investigated in ocular tissues at different postnatal intervals. The results of ophthalmic examinations were suggestive of early-onset stationary night blindness with no extraocular anomalies. The genome-wide scan localized the critical interval to chromosome 3, region p22.1-p14.3, with maximum two-point LOD scores of 3.09 at θ = 0, flanked by markers D3S3522 and D3S1289. Subsequently, a missense mutation in GNAT1, p.D129G, was identified, which segregated within the family, consistent with an autosomal recessive mode of inheritance, and was not present in 192 ethnically matched control chromosomes. Expression analysis suggested that Gnat1 is expressed at approximately postnatal day (P)7 and is predominantly expressed in the retina. These data suggest that a homozygous missense mutation in GNAT1 is associated with autosomal recessive stationary night blindness.

DFNB86, a novel autosomal recessive non-syndromic deafness locus on chromosome 16p13.3

DFNB86, a novel autosomal recessive non-syndromic deafness locus on chromosome 16p13.3

Preliminary mapping of an autosomal dominant retinitis pigmentosa gene by linkage analysis

To map the gene responsible for a three generations family with autosomal dominant retinitis pigmentosa (ADRP) and screen the mutation in candidate genes. For a 14-member family with 7 suspected patients, systematic and ophthalmic examinations were performed including visual acuity, ophthalmoscopy, perimetry and electrophysiologic test. After the genomic DNA was extracted, genome scanning was performed, and candidate genes were screened for potential mutations. The maximal two-point LOD score was obtained at D1S252 with a value of 2.02 (θ=0.00), and multi-point LOD score reached its maximum 2.28 at D1S252. However, no mutations were detected in two candidate genes in this region, namely PRPF3 and SEMA4A by direct sequencing. Significant evidence for linkage was found at the chromosome region of 1p21.1-q23.3. However, neither PRPF3 nor SEMA4A has harbored a mutation. There may exist an additional gene which is responsible for this disease.

Autosomal Recessive Nonsyndromic Hearing Impairment due to a Novel Deletion in the RDX Gene

The RDX gene anchors cytoskeletal actin of stereocilia to hair cell transmembrane and is responsible for autosomal recessive nonsyndromic hearing impairment (ARNSHI) due to DFNB24. A genome scan was performed using DNA samples from a consanguineous Pakistani family with ARNSHI. A significant maximum two-point LOD score of 4.5 (θ = 0) and multipoint LOD score of 5.8 were achieved at marker D11S1998 (chr11 : 117.20 Mb). The region of homozygosity is bounded by markers D11S2000 (105.06 Mb) and D11S4464 (123.13 Mb) and contains the NSHI genes TECTA and RDX. Although no potentially causal variants were identified in the TECTA gene, within the RDX gene a novel deletion c.1076_1079delTTAA (p.Ile359Lysfs∗6) was identified. The RDX deletion segregates with ARNSHI within the family and was not observed in 500 control chromosomes. It is predicted to cause premature truncation of radixin at the α-helical domain and to result in nonfunctional transcripts within the cochlea. RDX isoforms which encode the coiled-coil region of the α-helical domain are deemed necessary for proper function of hair cell stereocilia.

A new locus in chromosome 2q37-qter is associated with posterior polar cataract

To study the genetic basis of autosomal dominant posterior polar cataracts in two Chinese pedigrees. Peripheral blood samples were collected and genomic DNA was isolated. A genome-wide scan, using microsatellite markers at approximately 10-cm intervals and additional microsatellite markers for the positive region, was performed. Haplotype data were processed using Cyrillic software (version 2.1) to define the region of the disease gene. Mutation analysis was carried out for candidate genes. Sequencing data were analyzed with the software Sequence Scanner v1.0. A maximum two-point LOD score (Z (max)) of 2.53 and 2.03 was obtained at marker D2S125 with recombination θ = 0.00 in the two families. The possible disease genes were located at approximately 8.44-cM between the marker D2S125 and the terminal of chromosome 2q, namely, 2q37-qter. Candidate genes, such as Gamma-crystallins (CRYGA-D), septin 2 (SEPT2), aquaporin 12B (AQP12B), and chemokine orphan receptor 7 (CXCR7), were sequenced but no causative mutations were found. Our results suggest that an unidentified gene in chromosome 2q37-qter is associated with posterior polar cataract, which may have an implication in understanding the genetic and molecular mechanisms of cataracts.

A dominant mutation in RPE65 identified by whole-exome sequencing causes retinitis pigmentosa with choroidal involvement

Linkage testing using Affymetrix 6.0 SNP Arrays mapped the disease locus in TCD-G, an Irish family with autosomal dominant retinitis pigmentosa (adRP), to an 8.8 Mb region on 1p31. Of 50 known genes in the region, 11 candidates, including RPE65 and PDE4B, were sequenced using di-deoxy capillary electrophoresis. Simultaneously, a subset of family members was analyzed using Agilent SureSelect All Exome capture, followed by sequencing on an Illumina GAIIx platform. Candidate gene and exome sequencing resulted in the identification of an Asp477Gly mutation in exon 13 of the RPE65 gene tracking with the disease in TCD-G. All coding exons of genes not sequenced to sufficient depth by next generation sequencing were sequenced by di-deoxy sequencing. No other potential disease-causing variants were found to segregate with disease in TCD-G. The Asp477Gly mutation was not present in Irish controls, but was found in a second Irish family provisionally diagnosed with choroideremia, bringing the combined maximum two-point LOD score to 5.3. Mutations in RPE65 are a known cause of recessive Leber congenital amaurosis (LCA) and recessive RP, but no dominant mutations have been reported. Protein modeling suggests that the Asp477Gly mutation may destabilize protein folding, and mutant RPE65 protein migrates marginally faster on SDS-PAGE, compared with wild type. Gene therapy for LCA patients with RPE65 mutations has shown great promise, raising the possibility of related therapies for dominant-acting mutations in this gene.

Linkage analysis of one Chinese family with autosomal dominant congenital cataract

Background About one third of congenital cataract is associated with inheriting factor.The inherited heterogeneity has been found in congenital cataract.To seek the pathogenic gene is essential for the gene therapy. Objective Present study was to map and identify the causal gene for autosomal dominant congenital cataract (ADCC) in a Chinese family. Methods The clinical features of all affected members in this family were examined.Blood samples were collected from eleven family members for genetic linkage analysis.Polymorphic microsatellite markers were selected from the regions which harbor all known loci linked with ADCC.Universal fluorescent-labeled M13 primer was used in linkage analysis.Direct genomic sequencing was used to evaluate the candidate gene for example CRYBB2 gene.This study followed Helsinki Declaration and was proved by Tianjin City Ethic Committee.Written informed consent was obtained from each SUbject before any medical procees. Results The maximum two-point LOD score of 1.20 was obtained for marker D22S315 (θ=0).The LOD score of 0.6 was obtained for marker D16S3068.No mutation in all exons of CRYBB2 gene was found in the family. Conclusion CRYBB2 gene associated with ADCC was excluded from the family.A genome-wide linkage screening should be conducted.Genotyping with microsatellite markers using Universal fluorescent-labeled M13 primer can decrease the cost and obtain the same result. Key words: congenital cataract; Linkage analysis; fluorescent labeling; universal M13

MPZ mutation in an early-onset Charcot-Marie-Tooth disease type 1B family by genome-wide linkage analysis

Charcot-Marie-Tooth disease (CMT) is a clinically and genetically heterogeneous peripheral neuropathy. The objective of this study was to find the causative mutation(s) in a demyelinating autosomal dominant CMT family. A high density SNP-based genome-wide linkage scan was performed, and causative mutations were determined by sequencing of candidate genes in the linkage disequilibrium region. Linkage analysis mapped the underlying gene to a region on chromosome 1q22-q23 with a maximum two-point LOD score of 2.036. Sequencing analysis revealed a novel c.243C>G (His81Gln) mutation in the MPZ gene, which encodes the major integral membrane protein of the peripheral nerve system. MPZ is well known as a CMT-causative gene with wide phenotypic spectrum. The clinical symptoms were more similar to those of patients with the His81Arg than patients with the His81Tyr mutation. The novel mutation completely co-segregated with affected members, and was not found in controls. Therefore, we suggest that the identified mutation in MPZ is the underlying cause of CMT in the family. In addition, this study demonstrated that the clinical phenotypes may be variable with different mutations at the same site in the MPZ gene.

Mutation in PVRL4 gene encoding nectin-4 underlies ectodermal-dysplasia-syndactyly syndrome (EDSS1)

Ectodermal-dysplasia-syndactyly syndrome (EDSS1) is a rare form of ectodermal dysplasia (ED), affecting skin and its appendages mainly hair, teeth and nails. In the present study, we have investigated a large consanguineous Pakistani family with 10 individuals showing features of EDSS1. Human genome was screened using highly polymorphic microsatellite markers to identify the gene causing EDSS1. The disease locus for EDSS1 was assigned to chromosome 1q23.1-q23.3. This region corresponds to 5.63 Mb according to the sequenced based physical map (Build 36.2) of the human genome and flanked by markers D1S1653 and D1S1677. A maximum two-point LOD score of 5.05 was obtained with the marker D1S484. Sequence analysis revealed a homozygous missense mutation (c.635C>G; p.Pro212Arg) in the recently reported PVRL4 gene causing EDSS1. The involvement of mutant nectin-4 in causing EDSS1 may open up interesting prospectives into the role of cell adhesion molecules in causing syndromic forms of EDs.

A clinically complex form of dominant optic atrophy (OPA8) maps on chromosome 16

Dominant optic atrophy (DOA) is genetically heterogeneous and pathogenic mutations have been identified in the OPA1 and OPA3 genes, both encoding for mitochondrial proteins. We characterized clinical and laboratory features in a large OPA1-negative family with complicated DOA. Search for mitochondrial dysfunction was performed by studying muscle biopsies, fibroblasts, platelets and magnetic resonance (MR) spectroscopy. Genetic investigations included mitochondrial DNA (mtDNA) analysis, linkage analysis, copy number variation (CNV) analysis and candidate gene screening. Optic neuropathy was undistinguishable from that in OPA1-DOA and frequently associated with late-onset sensorineural hearing loss, increases of central conduction times at somato-sensory evoked potentials and various cardiac abnormalities. Serum lactic acid after exercise, platelet respiratory complex activities, adenosine triphosphate (ATP) content in fibroblasts and muscle phosphorus MR spectroscopy all failed to reveal a mitochondrial dysfunction. However, muscle biopsies and their mtDNA analysis showed increased mitochondrial biogenesis. Furthermore, patient's fibroblasts grown in the galactose medium were unable to increase ATP content compared with controls, and exhibited abnormally high rate of fusion activity. Genome-wide linkage revealed a locus on chromosome 16q21-q22 with a maximum two-point LOD score of 8.84 for the marker D16S752 and a non-recombinant interval of ∼ 6.96 cM. Genomic screening of 45 genes in this interval including several likely candidate genes (CALB2, CYB5B, TK2, DHODH, PLEKHG4) revealed no mutation. Moreover, we excluded the presence of CNVs using array-based comparative genome hybridization. The identification of a new OPA locus (OPA8) in this pedigree demonstrates further genetic heterogeneity in DOA, and our results indicate that the pathogenesis may still involve mitochondria.