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Mitochondrial disorders are challenging to diagnose. Exome sequencing has greatly enhanced the diagnostic precision of these disorders although interpreting variants of uncertain significance (VUS) remains a formidable obstacle. Whether specific mitochondrial morphological changes can aid in the classification of these variants is unknown. Here, we describe two families (four patients), each with a VUS in a gene known to affect the morphology of mitochondria through a specific role in the fission-fusion balance. In the first, the missense variant in MFF, encoding a fission factor, was associated with impaired fission giving rise to a characteristically over-tubular appearance of mitochondria. In the second, the missense variant in DNAJA3, which has no listed OMIM phenotype, was associated with fragmented appearance of mitochondria consistent with its published deficiency states. In both instances, the highly specific phenotypes allowed us to upgrade the classification of the variants. Our results suggest that, in select cases, mitochondrial "dysmorphology" can be helpful in interpreting variants to reach a molecular diagnosis., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Erythrokeratoderma (EK) is heterogeneous clinical entity characterized by excessive scaling with resulting erythrokeratotic plaques. Several genes have been linked to EK and they encode a number of proteins that are important for the integrity of the keratinocyte layer of the epidermis. PERP is a transcription factor that is activated by both p53 and p63. However, its deficiency in a mouse model appears to only recapitulate p63-mediated role in skin development and organization. We report an extended multiplex consanguineous family in which an EK phenotype with a striking similarity to that observed in Perp -/- mice, is mapped to an autozygous region on chromosome 6 that spans PERP. Whole-exome sequencing revealed a novel variant in PERP that fully segregated with the phenotype. Functional analysis of patient- and control-derived keratinocytes revealed a deleterious effect of the identified variant on the intracellular localization of PERP. A previous report showed that PERP mutation causes a dominant form of keratoderma but a single patient in that report with a homozygous variant in PERP suggests that recessive inheritance is also possible. Our results, therefore, support the establishment of an autosomal recessive PERP-related EK phenotype in humans., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Acyl-CoA binding domain-containing 5 (ACBD5) is a peroxisomal protein that carries an acyl-CoA binding domain (ACBD) at its N-terminal region. The recent identification of a mutation in the ACBD5 gene in patients with a syndromic form of retinal dystrophy highlights the physiological importance of ACBD5 in humans. However, the underlying pathogenic mechanisms and the precise function of ACBD5 remain unclear. We herein report that ACBD5 is a peroxisomal tail-anchored membrane protein exposing its ACBD to the cytosol. Using patient-derived fibroblasts and ACBD5 knock-out HeLa cells generated via genome editing, we demonstrate that ACBD5 deficiency causes a moderate but significant defect in peroxisomal β-oxidation of very-long-chain fatty acids (VLCFAs) and elevates the level of cellular phospholipids containing VLCFAs without affecting peroxisome biogenesis, including the import of membrane and matrix proteins. Both the N-terminal ACBD and peroxisomal localization of ACBD5 are prerequisite for efficient VLCFA β-oxidation in peroxisomes. Furthermore, ACBD5 preferentially binds very-long-chain fatty acyl-CoAs (VLC-CoAs). Together, these results suggest a direct role of ACBD5 in peroxisomal VLCFA β-oxidation. Based on our findings, we propose that ACBD5 captures VLC-CoAs on the cytosolic side of the peroxisomal membrane so that the transport of VLC-CoAs into peroxisomes and subsequent β-oxidation thereof can proceed efficiently. Our study reclassifies ACBD5-related phenotype as a novel peroxisomal disorder., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

We report an individual who presented with severe neurodevelopmental delay and an intractable infantile-onset seizure disorder. Exome sequencing identified a homozygous single nucleotide change that abolishes a splice donor site in the ARV1 gene (c.294 + 1G > A homozygous). This variant completely prevented splicing in minigene assays, and resulted in exon skipping and an in-frame deletion of 40 amino acids in primary human fibroblasts (NP_073623.1: p.(Lys59_Asn98del). The p.(Lys59_Asn98del) and previously reported p.(Gly189Arg) ARV1 variants were evaluated for protein expression and function. The p.(Gly189Arg) variant partially rescued the temperature-dependent growth defect in arv1Δ yeast, while p.(Lys59-Asn98del) completely failed to rescue at restrictive temperature. In contrast to wild type human ARV1, neither variant expressed detectable levels of protein in mammalian cells. Mice with a neuronal deletion of Arv1 recapitulated the human phenotype, exhibiting seizures and a severe survival defect in adulthood. Our data support ARV1 deficiency as a cause of autosomal recessive epileptic encephalopathy., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Limb reduction malformations are highly heterogeneous in their clinical presentation and so, predicting the underlying mutation on a clinical basis can be challenging. Molecular karyotyping is a powerful genomic tool that has quickly become the mainstay for the study of children with malformation syndromes. We describe three patients with major limb reduction anomalies in whom pathogenic copy number variants were identified on molecular karyotyping. These include a patient with hypoplastic phalanges and absent hallux bilaterally with de novo deletion of 11.9 Mb on 7p21.1-22.1 spanning 63 genes including RAC1, another patient with severe Holt-Oram syndrome and a large de novo deletion 2.2 Mb on 12q24.13-24.21 spanning 20 genes including TBX3 and TBX5, and a third patient with acheiropodia who had a nullizygous deletion of 102 kb on 7q36.3 spanning LMBR1. We discuss the potential of these novel genomic rearrangements to improve our understanding of limb development in humans., (© 2016 Wiley Periodicals, Inc.)

Golgi apparatus (GA) is a membrane-bound organelle that serves a multitude of critical cellular functions including protein secretion and sorting, and cellular polarity. Many Mendelian diseases are caused by mutations in genes encoding various components of GA. GOLGA2 encodes GM130, a necessary component for the assembly of GA as a single complex, and its deficiency has been found to result in severe cellular phenotypes. We describe the first human patient with a homozygous apparently loss of function mutation in GOLGA2. The phenotype is a neuromuscular disorder characterized by developmental delay, seizures, progressive microcephaly, and muscular dystrophy. Knockdown of golga2 in zebrafish resulted in severe skeletal muscle disorganization and microcephaly recapitulating loss of function human phenotype. Our data suggest an important developmental role of GM130 in humans and zebrafish.

Brain channelopathies represent a growing class of brain disorders that usually result in paroxysmal disorders, although their role in other neurological phenotypes, including the recently described NALCN-related infantile encephalopathy, is increasingly recognized. In three Saudi Arabian families and one Egyptian family all affected by a remarkably similar phenotype (infantile encephalopathy and largely normal brain MRI) to that of NALCN-related infantile encephalopathy, we identified a locus on 2q34 in which whole-exome sequencing revealed three, including two apparently loss-of-function, recessive mutations in UNC80. UNC80 encodes a large protein that is necessary for the stability and function of NALCN and for bridging NALCN to UNC79 to form a functional complex. Our results expand the clinical relevance of the UNC79-UNC80-NALCN channel complex., (Copyright © 2016 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Meckel-Gruber syndrome (MKS) is a perinatally lethal disorder characterized by the triad of occipital encephalocele, polydactyly and polycystic kidneys. Typical of other disorders related to defective primary cilium (ciliopathies), MKS is genetically heterogeneous with mutations in a dozen genes to date known to cause the disease. In an ongoing effort to characterize MKS clinically and genetically, we implemented a gene panel and next-generation sequencing approach to identify the causal mutation in 25 MKS families. Of the three families that did not harbor an identifiable causal mutation by this approach, two mapped to a novel disease locus in which whole-exome sequencing revealed the likely causal mutation as a homozygous splicing variant in TMEM107, which we confirm leads to aberrant splicing and nonsense-mediated decay. TMEM107 had been independently identified in two mouse models as a cilia-related protein and mutant mice display typical ciliopathy phenotypes. Our analysis of patient fibroblasts shows marked ciliogenesis defect with an accompanying perturbation of sonic hedgehog signaling, highly concordant with the cellular phenotype in Tmem107 mutants. This study shows that known MKS loci account for the overwhelming majority of MKS cases but additional loci exist including MKS13 caused by TMEM107 mutation., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Purpose: To describe three siblings with childhood cone-rod dystrophy and macular cystic degeneration in a family with apparently variable phenotypes of CRB1-related recessive retinal dystrophy., Methods: Ophthalmologic examination (including electroretinography (ERG), ocular coherence tomography (OCT), and intravenous fluorescein angiography when possible) and homozygosity analysis guided candidate gene testing., Results: When the proband was evaluated at 7 years old for progressive visual loss, fundus exam was unremarkable (including no macular thickening clinically or by OCT) but ERG revealed cone-rod dysfunction with an electronegative waveform. Four years later repeat examination was significant for bilateral macular cystic degeneration and immediate family members were evaluated. Both the older sister (15 years old) and the younger brother (7 years old) had cone-rod dystrophy with macular cystic degeneration. Both the father (45 years old) and mother (35 years old) had had early adult-onset nyctalopia with later eventual loss of central vision; examination revealed dystrophic retinas with mostly peripheral clumped and/or nummular pigment and macular atrophy. ERG for both the older sister and younger brother confirmed cone-rod dysfunction (without an electronegative waveform) and was non-recordable for both the parents. Homozygosity analysis guided candidate gene analysis and confirmatory Sanger sequencing for the family uncovered a homozygous CRB1 mutation (c.80G > T [p.Cys27Phe]) in affected family members., Conclusions: The phenotypic spectrum of recessive CRB1 mutation includes childhood cone-rod dystrophy with macular cystic degeneration and the associated ERG can be electronegative.

We have previously described a syndrome characterized by facial dysmorphism, lens dislocation, anterior-segment abnormalities, and spontaneous filtering blebs (FDLAB, or Traboulsi syndrome). In view of the consanguineous nature of the affected families and the likely autosomal-recessive inheritance pattern of this syndrome, we undertook autozygosity mapping and whole-exome sequencing to identify ASPH as the disease locus, in which we identified two homozygous mutations. ASPH encodes aspartyl/asparaginyl β-hydroxylase (ASPH), which has been found to hydroxylate aspartic acid and asparagine residues on epidermal growth factor (EGF)-domain-containing proteins. The truncating and missense mutations we identified are predicted to severely impair the enzymatic function of ASPH, which suggests a possible link to other forms of ectopia lentis given that many of the genes implicated in this phenotype encode proteins that harbor EGF domains. Developmental analysis of Asph revealed an expression pattern consistent with the proposed link to the human syndrome. Indeed, Asph-knockout mice had a foreshortened snout, which corresponds to the facial abnormalities in individuals with Traboulsi syndrome. These data support a genetic basis for a syndromic form of ectopia lentis and the role of aspartyl hydroxylation in human development., (Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: Epileptic encephalopathy is a broad clinical category that is highly heterogeneous genetically., Objective: To describe a multiplex extended consanguineous family that defines a molecularly novel subtype of early infantile epileptic encephalopathy., Methods: Autozygosity mapping and exome sequencing for the identification of the causal mutation. This was followed by expression analysis of the candidate gene., Results: In an extended multigenerational family with six affected individuals, a single novel disease locus was identified on chromosome 12p13.31-p13.2. Within that locus, the only deleterious novel exomic variant was a homozygous truncating mutation in NECAP1, encoding a clathrin-accessory protein. The mutation was confirmed to trigger nonsense-mediated decay. Consistent with previous reports, we show that NECAP1 is highly enriched in the central nervous system., Conclusions: NECAP1 is known to regulate clathrin-mediated endocytosis in synapses. The mutation we report here links for the first time this trafficking pathway in early infantile epileptic encephalopathy.

Meckel-Gruber syndrome (MKS, OMIM #249000) is a multiple congenital malformation syndrome that represents the severe end of the ciliopathy phenotypic spectrum. Despite the relatively common occurrence of this syndrome among Arabs, little is known about its genetic architecture in this population. This is a series of 18 Arab families with MKS, who were evaluated clinically and studied using autozygome-guided mutation analysis and exome sequencing. We show that autozygome-guided candidate gene analysis identified the underlying mutation in the majority (n=12, 71%). Exome sequencing revealed a likely pathogenic mutation in three novel candidate MKS disease genes. These include C5orf42, Ellis-van-Creveld disease gene EVC2 and SEC8 (also known as EXOC4), which encodes an exocyst protein with an established role in ciliogenesis. This is the largest and most comprehensive genomic study on MKS in Arabs and the results, in addition to revealing genetic and allelic heterogeneity, suggest that previously reported disease genes and the novel candidates uncovered by this study account for the overwhelming majority of MKS patients in our population.

Background: Meckel-Gruber syndrome (MKS) is a genetically heterogeneous severe ciliopathy characterised by early lethality, occipital encephalocele, polydactyly, and polycystic kidney disease., Purpose: To report genetic analysis results in two families in which all known MKS diseases genes have been excluded., Methods: In two consanguineous families with classical MKS in which autozygome-guided sequencing of previously reported MKS genes was negative, we performed exome sequencing followed by autozygome filtration., Results: We identified one novel splicing mutation in TMEM231, which led to complete degradation of the mutant transcript in one family, and a novel missense mutation in the other, both in the homozygous state., Conclusions: TMEM231 represents a novel MKS locus. The very recent identification of TMEM231 mutations in Joubert syndrome supports the growing appreciation of the overlap in the molecular pathogenesis between these two ciliopathies.

Purpose: To biometrically and molecularly characterize clinically diagnosed posterior microphthalmos., Design: Prospective case series., Methods: Twenty-five affected patients from 13 families diagnosed by ophthalmologists experienced with the condition at the King Khaled Eye Specialist Hospital were studied. All participants underwent axial length measurement, keratometry, corneal pachymetry, and candidate gene analysis (MFRP, PRSS56). Main outcome measures were the results of ocular biometry and gene analysis., Results: All patients (2-47 years of age) had high hyperopia, normal-appearing anterior segments, posterior chamber foreshortening, and characteristic papillomacular folds/wrinkles. For the right eye, mean cycloplegic refraction was +15.09 diopters (D) (range 9.88-18.75). Axial length (mean 16.25 mm [range 14.88-19.88]) had strong inverse correlation (Pearson coefficient -0.88, P < .0001) with corneal power (mean 48.89 D [range 41.91-52.25]) and a positive correlation with corneal diameter (Pearson 0.64, P = .001). Corneal thickness and anterior chamber dimensions were within normal ranges. Left eye data were similar. Nineteen Saudi patients (8/13 families) harbored 4 different homozygous PRSS56 mutations, 1 Indian and 1 Saudi patient harbored 2 different homozygous MFRP mutations, and 4 Saudi patients (3/13 families) had no detectable mutation in either gene. Patients with MFRP mutations were not clinically different from patients with PRSS56 mutations or no identified mutation. Truncating PRSS56 mutations were associated with shorter axial lengths (mean 15.72 mm) than missense PRSS56 mutations (mean 16.37 mm) or no identified mutation (mean 17.57 mm)., Conclusions: These data define posterior microphthalmos biometrically and reveal that corneal steepening proportional to the degree of axial foreshortening is part of the phenotype. Corneal diameter decreases with decreasing axial length, suggesting posterior microphthalmos and nanophthalmos represent a spectrum of high hyperopia rather than distinct phenotypes. In the Saudi population PRSS56 mutations are the major cause, and in our cohort truncating mutations were associated with a more severe phenotype., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Purpose: Microphthalmia is a condition in which eyes are small in size, often associated with coloboma, as a result of aberrant eye development. Isolated microphthalmia is a model disease for studying early development of the human eye, and mutations in several key genes related to eye development have been linked to this phenotype., Methods: In our search for novel genes that cause autosomal recessive microphthalmia when mutated, we enrolled a family that consists of third-cousin parents and two children with isolated colobomatous microphthalmia., Results: Exome and autozygome analysis identified a null mutation in ODZ3, one of four vertebrate orthologs of odz in Drosophila., Conclusion: Our data highlight a role for ODZ3 in the early development of the human eye.

Very-long-chain fatty acids (VLCFAs) play important roles in membrane structure and cellular signaling, and their contribution to human health is increasingly recognized. Fatty acid elongases catalyze the first and rate-limiting step in VLCFA synthesis. Heterozygous mutations in ELOVL4, the gene encoding one of the elongases, are known to cause macular degeneration in humans and retinal abnormalities in mice. However, biallelic ELOVL4 mutations have not been observed in humans, and murine models with homozygous mutations die within hours of birth as a result of a defective epidermal water barrier. Here, we report on two human individuals with recessive ELOVL4 mutations revealed by a combination of autozygome analysis and exome sequencing. These individuals exhibit clinical features of ichthyosis, seizures, mental retardation, and spasticity-a constellation that resembles Sjögren-Larsson syndrome (SLS) but presents a more severe neurologic phenotype. Our findings identify recessive mutations in ELOVL4 as the cause of a neuro-ichthyotic disease and emphasize the importance of VLCFA synthesis in brain and cutaneous development., (Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Hereditary Spastic Paraplegia (HSP) is a clinically and genetically heterogeneous group of neurological disorders that are characterized by progressive spasticity of the lower extremities. We describe an extended consanguineous Saudi family in which HSP is linked to SPG18, a previously reported autosomal recessive locus, and show that it is associated with a nullimorphic deletion of ERLIN2, a component of endoplasmic reticulum associated degradation. This finding adds to the growing diversity of cellular functions that are now known to be involved in the maintenance of the corticospinal tract neurons.

Meckel Gruber syndrome (MKS) is an autosomal recessive multisystem disorder that represents a severe form of ciliopathy in humans and is characterized by significant genetic heterogeneity. In this article, we describe the identification of a novel MKS locus MKS8 that we map to TCTN2, in a multiplex consanguineous family. TCTN2 is a paralog of the recently identified Tectonic 1, which has been shown to modulate sonic hedgehog signaling. Expression analysis at different developmental stages of the murine ortholog revealed a spatial and temporal pattern consistent with the MKS phenotype observed in our patient. The exclusion of this and the other seven MKS genes in our collection of consanguineous Arab MKS families confirms the existence of two additional MKS loci., (© 2011 Wiley-Liss, Inc.)

Peroxisomal biogenesis disorders represent a group of genetically heterogeneous conditions that have in common failure of proper peroxisomal assembly. Clinically, they are characterized by a spectrum of dysmorphia, neurological, liver, and other organ involvement. To date, mutations in 13 PEX genes encoding peroxins have been identified in patients with peroxisomal biogenesis disorders. Mutations in PEX13, which encodes peroxisomal membrane protein PEX13, are among the least common causes of peroxisomal biogenesis disorders with only three mutations reported so far. Here, we report on two infants whose clinical and biochemical profile was consistent with classical Zellweger syndrome and whose complementation analysis assigned them both to group H of peroxisomal biogenesis disorders. We show that they harbor two novel mutations in PEX13. One patient had a genomic rearrangement resulting in a 147 kb deletion that spans the whole of PEX13, while the other had an out-of-frame deletion of 14 bp. This represents the first report of a PEX13 deletion and suggests that further work is needed to examine the frequency of PEX13 mutations among Arab patients with peroxisomal biogenesis disorders., ((c) 2009 Wiley-Liss, Inc.)