Achromatopsia (ACHM) is an early-onset retinal dystrophy characterized by photophobia, nystagmus, color blindness and severely reduced visual acuity. Currently mutations in five genes CNGA3, CNGB3, GNAT2, PDE6C and PDE6H have been implicated in ACHM. We performed homozygosity mapping and linkage analysis in a consanguineous Pakistani ACHM family and mapped the locus to a 15.12-Mb region on chromosome 1q23.1-q24.3 with a maximum LOD score of 3.6. A DNA sample from an affected family member underwent exome sequencing. Within the ATF6 gene, a single-base insertion variant c.355_356dupG (p.Glu119Glyfs*8) was identified, which completely segregates with the ACHM phenotype within the family. The frameshift variant was absent in public variant databases, in 130 exomes from unrelated Pakistani individuals, and in 235 ethnically matched controls. The variant is predicted to result in a truncated protein that lacks the DNA binding and transmembrane domains and therefore affects the function of ATF6 as a transcription factor that initiates the unfolded protein response during endoplasmic reticulum (ER) stress. Immunolabeling with anti-ATF6 antibodies showed localization throughout the mouse neuronal retina, including retinal pigment epithelium, photoreceptor cells, inner nuclear layer, inner and outer plexiform layers, with a more prominent signal in retinal ganglion cells. In contrast to cytoplasmic expression of wild-type protein, in heterologous cells ATF6 protein with the p.Glu119Glyfs*8 variant is mainly confined to the nucleus. Our results imply that response to ER stress as mediated by the ATF6 pathway is essential for color vision in humans.

Dendritic spines represent the major site of neuronal activity in the brain; they serve as the receiving point for neurotransmitters and undergo rapid activity-dependent morphological changes that correlate with learning and memory. Using a combination of homozygosity mapping and next-generation sequencing in two consanguineous families affected by nonsyndromic autosomal-recessive intellectual disability, we identified truncating mutations in formin 2 (FMN2), encoding a protein that belongs to the formin family of actin cytoskeleton nucleation factors and is highly expressed in the maturing brain. We found that FMN2 localizes to punctae along dendrites and that germline inactivation of mouse Fmn2 resulted in animals with decreased spine density; such mice were previously demonstrated to have a conditioned fear-learning defect. Furthermore, patient neural cells derived from induced pluripotent stem cells showed correlated decreased synaptic density. Thus, FMN2 mutations link intellectual disability either directly or indirectly to the regulation of actin-mediated synaptic spine density.

Inherited retinal dystrophies are phenotypically and genetically heterogeneous. This extensive heterogeneity poses a challenge when performing molecular diagnosis of patients, especially in developing countries. In this study, we applied homozygosity mapping as a tool to reduce the complexity given by genetic heterogeneity and identify disease-causing variants in consanguineous Pakistani pedigrees. DNA samples from eight families with autosomal recessive retinal dystrophies were subjected to genome wide homozygosity mapping (seven by SNP arrays and one by STR markers) and genes comprised within the detected homozygous regions were analyzed by Sanger sequencing. All families displayed consistent autozygous genomic regions. Sequence analysis of candidate genes identified four previously-reported mutations in CNGB3, CNGA3, RHO, and PDE6A, as well as three novel mutations: c.2656C > T (p.L886F) in RPGRIP1, c.991G > C (p.G331R) in CNGA3, and c.413-1G > A (IVS6-1G > A) in CNGB1. This latter mutation impacted pre-mRNA splicing of CNGB1 by creating a -1 frameshift leading to a premature termination codon. In addition to better delineating the genetic landscape of inherited retinal dystrophies in Pakistan, our data confirm that combining homozygosity mapping and candidate gene sequencing is a powerful approach for mutation identification in populations where consanguineous unions are common.

The 9.6 kb long RNA genome of Hepatitis C virus (HCV) is under the control of RNA dependent RNA polymerase, an error-prone enzyme, for its transcription and replication. A high rate of mutation has been found to be associated with RNA viruses like HCV. Based on genetic variability, HCV has been classified into 6 different major genotypes and 11 different subtypes. However this classification system does not provide significant information about the origin of the virus, primarily due to high mutation rate at nucleotide level. HCV genome codes for a single polyprotein of about 3011 amino acids which is processed into structural and non-structural proteins inside host cell by viral and cellular proteases.

Causes of autosomal-recessive intellectual disability (ID) have, until very recently, been under researched because of the high degree of genetic heterogeneity. However, now that genome-wide approaches can be applied to single multiplex consanguineous families, the identification of genes harboring disease-causing mutations by autozygosity mapping is expanding rapidly. Here, we have mapped a disease locus in a consanguineous Pakistani family affected by ID and distal myopathy. We genotyped family members on genome-wide SNP microarrays and used the data to determine a single 2.5 Mb homozygosity-by-descent (HBD) locus in region 5p15.32-p15.31; we identified the missense change c.2035G>A (p.Gly679Arg) at a conserved residue within NSUN2. This gene encodes a methyltransferase that catalyzes formation of 5-methylcytosine at C34 of tRNA-leu(CAA) and plays a role in spindle assembly during mitosis as well as chromosome segregation. In mouse brains, we show that NSUN2 localizes to the nucleolus of Purkinje cells in the cerebellum. The effects of the mutation were confirmed by the transfection of wild-type and mutant constructs into cells and subsequent immunohistochemistry. We show that mutation to arginine at this residue causes NSUN2 to fail to localize within the nucleolus. The ID combined with a unique profile of comorbid features presented here makes this an important genetic discovery, and the involvement of NSUN2 highlights the role of RNA methyltransferase in human neurocognitive development.

A novel locus for autosomal recessive nonsyndromic hearing impairment (ARNSHI), DFNB96, was mapped to the 1p36.31-p36.13 region. A whole-genome linkage scan was performed using DNA samples from a consanguineous family from Pakistan with ARNSHI. A maximum two-point logarithm of odds (LOD) score of 3.2 was obtained at marker rs8627 (chromosome 1: 8.34 Mb) at θ=0 and a significant maximum multipoint LOD score of 3.8 was achieved at 15 contiguous markers from rs630075 (9.3 Mb) to rs10927583 (15.13 Mb). The 3-unit support interval and the region of homozygosity were both delimited by markers rs3817914 (6.42 Mb) and rs477558 (18.09 Mb) and contained 11.67 Mb. Of the 125 genes within the DFNB96 interval, the previously identified ARNSHI gene for DFNB36, ESPN, and two genes that cause Bartter syndrome, CLCNKA and CLCNKB, were sequenced, but no potentially causal variants were identified.

In a large consanguineous family of Turkish origin, genome-wide homozygosity mapping revealed a locus for recessive nonsyndromic hearing impairment on chromosome 14q24.3-q34.12. Fine mapping with microsatellite markers defined the critical linkage interval to a 18.7 cM region flanked by markers D14S53 and D14S1015. This region partially overlapped with the DFNB35 locus. Mutation analysis of ESRRB, a candidate gene in the overlapping region, revealed a homozygous 7 bp duplication in exon 8 in all affected individuals. This duplication results in a frame shift and premature stop codon. Sequence analysis of the ESRRB gene in the affected individuals of the original DFNB35 family and in three other DFNB35-linked consanguineous families from Pakistan revealed four missense mutations. ESRRB encodes the estrogen-related receptor beta protein, and one of the substitutions (p.A110V) is located in the DNA-binding domain of ESRRB, whereas the other three are substitutions (p.L320P, p.V342L, and p.L347P) located within the ligand-binding domain. Molecular modeling of this nuclear receptor showed that the missense mutations are likely to affect the structure and stability of these domains. RNA in situ hybridization in mice revealed that Esrrb is expressed during inner-ear development, whereas immunohistochemical analysis showed that ESRRB is present postnatally in the cochlea. Our data indicate that ESRRB is essential for inner-ear development and function. To our knowledge, this is the first report of pathogenic mutations of an estrogen-related receptor gene.

Intellectual disability (ID) is a highly heterogeneous disorder with hundreds of associated genes. Despite progress in the identification of the genetic causes of ID following the introduction of high-throughput sequencing, about half of affected individuals still remain without a molecular diagnosis. Consanguineous families with affected individuals provide a unique opportunity to identify novel recessive causative genes. In this report, we describe a novel autosomal recessive neurodevelopmental disorder. We identified two consanguineous families with homozygous variants predicted to alter the splicing of ATP9A which encodes a transmembrane lipid flippase of the class II P4-ATPases. The three individuals homozygous for these putatively truncating variants presented with severe ID, motor and speech impairment, and behavioral anomalies. Consistent with a causative role of ATP9A in these patients, a previously described Atp9a-/- mouse model showed behavioral changes.

Jalili syndrome (JS) is a rare cone-rod dystrophy (CRD) associated with amelogenesis imperfecta (AI). The first clinical presentation of JS patients was published in 1988 by Jalili and Smith. Pathogenic mutations in the Cyclin and CBS Domain Divalent Metal Cation Transport Mediator 4 (CNNM4) magnesium transporter protein have been reported as the leading cause of this anomaly.

Retinitis pigmentosa (RP) is a genetically heterogeneous trait with autosomal-recessive (ar) inheritance underlying 50% of genetic disease cases. Sixty-one arRP genes have been identified, and recently, DHX38 has been reported as a potential candidate gene for arRP with only a single family reported with a variant of unknown significance. We identified a missense variant in DHX38 that co-segregates with the arRP phenotype in two Pakistani families confirming the involvement of DHX38 in the etiology of early-onset RP.

Infantile and childhood-onset cataracts form a heterogeneous group of disorders; among the many genetic causes, numerous pathogenic variants in additional genes associated with autosomal-recessive infantile cataracts remain to be discovered. We identified three consanguineous families affected by bilateral infantile cataracts. Using exome sequencing, we found homozygous loss-of-function variants in DNMBP: nonsense variant c.811C>T (p.Arg271∗) in large family F385 (nine affected individuals; LOD score = 5.18 at θ = 0), frameshift deletion c.2947_2948del (p.Asp983∗) in family F372 (two affected individuals), and frameshift variant c.2852_2855del (p.Thr951Metfs∗41) in family F3 (one affected individual). The phenotypes of all affected individuals include infantile-onset cataracts. RNAi-mediated knockdown of the Drosophila ortholog still life (sif), enriched in lens-secreting cells, affects the development of these cells as well as the localization of E-cadherin, alters the distribution of septate junctions in adjacent cone cells, and leads to a ∼50% reduction in electroretinography amplitudes in young flies. DNMBP regulates the shape of tight junctions, which correspond to the septate junctions in invertebrates, as well as the assembly pattern of E-cadherin in human epithelial cells. E-cadherin has an important role in lens vesicle separation and lens epithelial cell survival in humans. We therefore conclude that DNMBP loss-of-function variants cause infantile-onset cataracts in humans.

Exploring genes and pathways underlying intellectual disability (ID) provides insight into brain development and function, clarifying the complex puzzle of how cognition develops. As part of ongoing systematic studies to identify candidate ID genes, linkage analysis and next-generation sequencing revealed Zinc Finger and BTB Domain Containing 11 (ZBTB11) as a novel candidate ID gene. ZBTB11 encodes a little-studied transcription regulator, and the two identified missense variants in this study are predicted to disrupt canonical Zn2+-binding residues of its C2H2 zinc finger domain, leading to possible altered DNA binding. Using HEK293T cells transfected with wild-type and mutant GFP-ZBTB11 constructs, we found the ZBTB11 mutants being excluded from the nucleolus, where the wild-type recombinant protein is predominantly localized. Pathway analysis applied to ChIP-seq data deposited in the ENCODE database supports the localization of ZBTB11 in nucleoli, highlighting associated pathways such as ribosomal RNA synthesis, ribosomal assembly, RNA modification and stress sensing, and provides a direct link between subcellular ZBTB11 location and its function. Furthermore, given the report of prominent brain and spinal cord degeneration in a zebrafish Zbtb11 mutant, we investigated ZBTB11-ortholog knockdown in Drosophila melanogaster brain by targeting RNAi using the UAS/Gal4 system. The observed approximate reduction to a third of the mushroom body size-possibly through neuronal reduction or degeneration-may affect neuronal circuits in the brain that are required for adaptive behavior, specifying the role of this gene in the nervous system. In conclusion, we report two ID families segregating ZBTB11 biallelic mutations disrupting Zn2+-binding motifs and provide functional evidence linking ZBTB11 dysfunction to this phenotype.

The sphingosine-1-phosphate receptors (S1PRs) are a well-studied class of transmembrane G protein-coupled sphingolipid receptors that mediate multiple cellular processes. However, S1PRs have not been previously reported to be involved in the genetic etiology of human traits. S1PR2 lies within the autosomal-recessive nonsyndromic hearing impairment (ARNSHI) locus DFNB68 on 19p13.2. From exome sequence data we identified two pathogenic S1PR2 variants, c.323G>C (p.Arg108Pro) and c.419A>G (p.Tyr140Cys). Each of these variants co-segregates with congenital profound hearing impairment in consanguineous Pakistani families with maximum LOD scores of 6.4 for family DEM4154 and 3.3 for family PKDF1400. Neither S1PR2 missense variant was reported among ∼120,000 chromosomes in the Exome Aggregation Consortium database, in 76 unrelated Pakistani exomes, or in 720 Pakistani control chromosomes. Both DNA variants affect highly conserved residues of S1PR2 and are predicted to be damaging by multiple bioinformatics tools. Molecular modeling predicts that these variants affect binding of sphingosine-1-phosphate (p.Arg108Pro) and G protein docking (p.Tyr140Cys). In the previously reported S1pr2(-/-) mice, stria vascularis abnormalities, organ of Corti degeneration, and profound hearing loss were observed. Additionally, hair cell defects were seen in both knockout mice and morphant zebrafish. Family PKDF1400 presents with ARNSHI, which is consistent with the lack of gross malformations in S1pr2(-/-) mice, whereas family DEM4154 has lower limb malformations in addition to hearing loss. Our findings suggest the possibility of developing therapies against hair cell damage (e.g., from ototoxic drugs) through targeted stimulation of S1PR2.

Anophthalmia and microphthalmia (A/M) are a group of rare developmental disorders that affect the size of the ocular globe. A/M may present as the sole clinical feature, but are also frequently found in a variety of syndromes. A/M is genetically heterogeneous and can be caused by chromosomal aberrations, copy number variations and single gene mutations. To date, A/M has been caused by mutations in at least 20 genes that show different modes of inheritance. In this study, we enrolled eight consanguineous families with A/M, including seven from Pakistan and one from India. Sanger and exome sequencing of DNA samples from these families identified three novel mutations including two mutations in the Aldehyde Dehydrogenase 1 Family Member A3 (ALDH1A3) gene, [c.1310_1311delAT; p.(Tyr437Trpfs*44) and c.964G > A; p.(Val322Met)] and a single missense mutation in Forkhead Box E3 (FOXE3) gene, [c.289A > G p.(Ile97Val)]. Additionally two previously reported mutations were identified in FOXE3 and in Visual System Homeobox 2 (VSX2). This is the first comprehensive study on families with A/M from the Indian subcontinent which provides further evidence for the involvement of known genes with novel and recurrent mutations.

Hepatitis C virus (HCV), a member of the Flaviviridae family of viruses, is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Currently, the only treatment available consists of a combination of Pegylated interferon alpha (INF-α) and ribavirin, but only half of the patients treated show a sufficient antiviral response. Thus there is a great need for the development of new treatments for HCV infections. RNA interference (RNAi) represents a new promising approach to develop effective antiviral drugs and has been extremely effective against HCV infection.

Nephronophthisis (NPHP), Joubert (JBTS), and Meckel-Gruber (MKS) syndromes are autosomal-recessive ciliopathies presenting with cystic kidneys, retinal degeneration, and cerebellar/neural tube malformation. Whether defects in kidney, retinal, or neural disease primarily involve ciliary, Hedgehog, or cell polarity pathways remains unclear. Using high-confidence proteomics, we identified 850 interactors copurifying with nine NPHP/JBTS/MKS proteins and discovered three connected modules: "NPHP1-4-8" functioning at the apical surface, "NPHP5-6" at centrosomes, and "MKS" linked to Hedgehog signaling. Assays for ciliogenesis and epithelial morphogenesis in 3D renal cultures link renal cystic disease to apical organization defects, whereas ciliary and Hedgehog pathway defects lead to retinal or neural deficits. Using 38 interactors as candidates, linkage and sequencing analysis of 250 patients identified ATXN10 and TCTN2 as new NPHP-JBTS genes, and our Tctn2 mouse knockout shows neural tube and Hedgehog signaling defects. Our study further illustrates the power of linking proteomic networks and human genetics to uncover critical disease pathways.

Hearing loss or hearing impairment is a clinically and genetically heterogeneous disorder. More than 117 genes were discovered to date in hereditary, nonsyndromic hearing loss (NSHL). Identifying novel gene variants and their frequency in specific populations is valuable for public health and potentially for genetic screening of NSHL.

Wolfram syndrome (WFS) is characterized by deafness, diabetes mellitus, and diabetes insipidus along with optic atrophy. WFS has an autosomal recessive mode of inheritance and is due to variants in WFS1 and CISD2.

Biallelic mutations in G-Protein coupled receptor kinase 1 (GRK1) cause Oguchi disease, a rare subtype of congenital stationary night blindness (CSNB). The purpose of this study was to identify disease causing GRK1 variants and use in-depth bioinformatic analyses to evaluate how their impact on protein structure could lead to pathogenicity. Patients' genomic DNA was sequenced by whole genome, whole exome or focused exome sequencing. Disease associated variants, published and novel, were compared to nondisease associated missense variants. The impact of GRK1 missense variants at the protein level were then predicted using a series of computational tools. We identified twelve previously unpublished cases with biallelic disease associated GRK1 variants, including eight novel variants, and reviewed all GRK1 disease associated variants. Further structure-based scoring revealed a hotspot for missense variants in the kinase domain. In addition, to aid future clinical interpretation, we identified the bioinformatics tools best able to differentiate disease associated from nondisease associated variants. We identified GRK1 variants in Oguchi disease patients and investigated how disease-causing variants may impede protein function in-silico.

Craniofacial microsomia (CFM; also known as Goldenhar syndrome), is a craniofacial developmental disorder of variable expressivity and severity with a recognizable set of abnormalities. These birth defects are associated with structures derived from the first and second pharyngeal arches, can occur unilaterally and include ear dysplasia, microtia, preauricular tags and pits, facial asymmetry and other malformations. The inheritance pattern is controversial, and the molecular etiology of this syndrome is largely unknown. A total of 670 patients belonging to unrelated pedigrees with European and Chinese ancestry with CFM, are investigated. We identify 18 likely pathogenic variants in 21 probands (3.1%) in FOXI3. Biochemical experiments on transcriptional activity and subcellular localization of the likely pathogenic FOXI3 variants, and knock-in mouse studies strongly support the involvement of FOXI3 in CFM. Our findings indicate autosomal dominant inheritance with reduced penetrance, and/or autosomal recessive inheritance. The phenotypic expression of the FOXI3 variants is variable. The penetrance of the likely pathogenic variants in the seemingly dominant form is reduced, since a considerable number of such variants in affected individuals were inherited from non-affected parents. Here we provide suggestive evidence that common variation in the FOXI3 allele in trans with the pathogenic variant could modify the phenotypic severity and accounts for the incomplete penetrance.