Loss-of-function mutations of ILDR1 cause autosomal-recessive hearing impairment DFNB42

By using homozygosity mapping in a consanguineous Pakistani family, we detected linkage of nonsyndromic hearing loss to a 7.6 Mb region on chromosome 3q13.31-q21.1 within the previously reported DFNB42 locus. Subsequent candidate gene sequencing identified a homozygous nonsense mutation (c.1135G>T [p.Glu379X]) in ILDR1 as the cause of hearing impairment. By analyzing additional consanguineous families with homozygosity at this locus, we detected ILDR1 mutations in the affected individuals of 10 more families from Pakistan and Iran. The identified ILDR1 variants include missense, nonsense, frameshift, and splice-site mutations as well as a start codon mutation in the family that originally defined the DFNB42 locus. ILDR1 encodes the evolutionarily conserved immunoglobulin-like domain containing receptor 1, a putative transmembrane receptor of unknown function. In situ hybridization detected expression of Ildr1, the murine ortholog, early in development in the vestibule and in hair cells and supporting cells of the cochlea. Expression in hair cell- and supporting cell-containing neurosensory organs is conserved in the zebrafish, in which the ildr1 ortholog is prominently expressed in the developing ear and neuromasts of the lateral line. These data identify loss-of-function mutations of ILDR1, a gene with a conserved expression pattern pointing to a conserved function in hearing in vertebrates, as underlying nonsyndromic prelingual sensorineural hearing impairment.     

Loss-of-function mutations in RAB18 cause Warburg micro syndrome

Warburg Micro syndrome and Martsolf syndrome are heterogenous autosomal-recessive developmental disorders characterized by brain, eye, and endocrine abnormalities. Previously, identification of mutations in RAB3GAP1 and RAB3GAP2 in both these syndromes implicated dysregulation of the RAB3 cycle (which controls calcium-mediated exocytosis of neurotransmitters and hormones) in disease pathogenesis. RAB3GAP1 and RAB3GAP2 encode the catalytic and noncatalytic subunits of the hetrodimeric enzyme RAB3GAP (RAB3GTPase-activating protein), a key regulator of the RAB3 cycle. We performed autozygosity mapping in five consanguineous families without RAB3GAP1/2 mutations and identified loss-of-function mutations in RAB18. A c.71T > A (p.Leu24Gln) founder mutation was identified in four Pakistani families, and a homozygous exon 2 deletion (predicted to result in a frameshift) was found in the fifth family. A single family whose members were compound heterozygotes for an anti-termination mutation of the stop codon c.619T > C (p.X207QextX20) and an inframe arginine deletion c.277_279 del (p.Arg93 del) were identified after direct gene sequencing and multiplex ligation-dependent probe amplification (MLPA) of a further 58 families. Nucleotide binding assays for RAB18(Leu24Gln) and RAB18(Arg93del) showed that these mutant proteins were functionally null in that they were unable to bind guanine. The clinical features of Warburg Micro syndrome patients with RAB3GAP1 or RAB3GAP2 mutations and RAB18 mutations are indistinguishable, although the role of RAB18 in trafficking is still emerging, and it has not been linked previously to the RAB3 pathway. Knockdown of rab18 in zebrafish suggests that it might have a conserved developmental role. Our findings imply that RAB18 has a critical role in human brain and eye development and neurodegeneration.     

Loss-of-function mutations in growth differentiation factor-1 (GDF1) are associated with congenital heart defects in humans

Congenital heart defects (CHDs) are among the most common birth defects in humans (incidence 8-10 per 1,000 live births). Although their etiology is often poorly understood, most are considered to arise from multifactorial influences, including environmental and genetic components, as well as from less common syndromic forms. We hypothesized that disturbances in left-right patterning could contribute to the pathogenesis of selected cardiac defects by interfering with the extrinsic cues leading to the proper looping and vessel remodeling of the normally asymmetrically developed heart and vessels. Here, we show that heterozygous loss-of-function mutations in the human GDF1 gene contribute to cardiac defects ranging from tetralogy of Fallot to transposition of the great arteries and that decreased TGF- beta signaling provides a framework for understanding their pathogenesis. These findings implicate perturbations of the TGF- beta signaling pathway in the causation of a major subclass of human CHDs.     

Loss-of-function mutations in the keratin 5 gene lead to Dowling-Degos disease

Dowling-Degos disease (DDD) is an autosomal dominant genodermatosis characterized by progressive and disfiguring reticulate hyperpigmentation of the flexures. We performed a genomewide linkage analysis of two German families and mapped DDD to chromosome 12q, with a total LOD score of 4.42 ( theta =0.0) for marker D12S368. This region includes the keratin gene cluster, which we screened for mutations. We identified loss-of-function mutations in the keratin 5 gene (KRT5) in all affected family members and in six unrelated patients with DDD. These represent the first identified mutations that lead to haploinsufficiency in a keratin gene. The identification of loss-of-function mutations, along with the results from additional functional studies, suggest a crucial role for keratins in the organization of cell adhesion, melanosome uptake, organelle transport, and nuclear anchorage.     

Loss-of-function mutations in filaggrin gene associate with psoriasis vulgaris in Chinese population

Loss-of-function mutations in filaggrin gene (FLG; OMIM #135940) have been reported to cause the semi-dominant keratinizing disorders such as ichthyosis vulgaris (IV; OMIM #146700) and atopic dermatitis (AD; OMIM #605803). Recent linkage analysis and immunohistochemical studies suggest the possible contribution of FLG to psoriatic susceptibility. However, no susceptibility variant in FLG gene associated with psoriasis (OMIM #177900) has been identified. In this study, we identified a non-sense mutation of FLG (p.K4022X) in a Chinese psoriasis/IV coexisting family. The homozygous p.K4022X mutation was detected in a psoriasis patient, whereas the heterozygous p.K4022X mutation was identified in two IV patients and four apparently normal family members. We also genotyped p.K4022X variant in 441 sporadic Chinese psoriasis patients and found homozygous mutation in two patients, while no homozygous variant was found in 500 control individuals. After sequencing the entire coding region of FLG gene in 441 psoriasis patients, we identified another five mutations (p.R826X, p.W2583X, c.7945delA, c.3321delA and p.Q2417X). Although all six FLG mutations as a whole was not significantly associated with psoriasis (P = 0.105), mutation p.K4022X was significantly associated with psoriasis (P < 0.05). Our data thus indicates an association of FLG with psoriasis in Chinese population.     

Loss-of-function mutations in the ethylene receptor ETR1 cause enhanced sensitivity and exaggerated response to ethylene in Arabidopsis

Ethylene signaling in Arabidopsis begins at a family of five ethylene receptors that regulate activity of a downstream mitogen-activated protein kinase kinase kinase, CTR1. Triple and quadruple loss-of-function ethylene receptor mutants display a constitutive ethylene response phenotype, indicating they function as negative regulators in this pathway. No ethylene-related phenotype has been described for single loss-of-function receptor mutants, although it was reported that etr1 loss-of-function mutants display a growth defect limiting plant size. In actuality, this apparent growth defect results from enhanced responsiveness to ethylene; a phenotype manifested in all tissues tested. The phenotype displayed by etr1 loss-of-function mutants was rescued by treatment with an inhibitor of ethylene perception, indicating that it is ethylene dependent. Identification of an ethylene-dependent phenotype for a loss-of-function receptor mutant gave a unique opportunity for genetic and biochemical analysis of upstream events in ethylene signaling, including demonstration that the dominant ethylene-insensitive phenotype of etr2-1 is partially dependent on ETR1. This work demonstrates that mutational loss of the ethylene receptor ETR1 alters responsiveness to ethylene in Arabidopsis and that enhanced ethylene response in Arabidopsis not only results in increased sensitivity but exaggeration of response.     

A polymorphism in thrombospondin-1 associated with familial premature coronary artery disease alters Ca2+ binding

A single nucleotide polymorphism that results in substitution at residue 700 of a serine (Ser-700) for an asparagine (Asn-700) in thrombospondin-1 is associated with familial premature coronary artery disease. The polymorphism is located in the first of 13 Ca2+ -binding motifs, within a consensus sequence in which Asn-700 likely coordinates Ca2+. Equilibrium dialysis of constructs comprised of the adjoining epidermal growth factor-like module and the Ca2+ -binding region (E3Ca) demonstrated that E3Ca Ser-700 binds significantly less Ca2+ than E3Ca Asn-700 at low [Ca2+]. The hypothesis that this difference is due to loss of a binding site in Ser-700 protein was tested with truncations of E3Ca containing four (Tr4), three (Tr3), two (Tr2), or one (Tr1) N-terminal Ca2+ -binding motifs. The Ser-700 truncation constructs bound 1 fewer Ca2+ than matching Asn-700 constructs and exhibited decreased binding affinities. Intrinsic fluorescence of a tryptophan at residue 698 (Trp-698) in the most N-terminal motif was cooperatively quenched by the addition of Ca2+ to Asn-700 Tr2, Tr3, and Tr4 constructs. In Ser-700 constructs, quenching of Trp-698 was incomplete in the Tr2 and Tr3 constructs and complete only in the Tr4 construct. Ca2+ -induced quenching of Ser-700 constructs required higher [Ca2+] and was slower as shown in stopped-flow experiments than quenching of Asn-700 constructs. Such differences were not found with Tb3+, which quenched the fluorescence of Asn-700 and Ser-700 constructs equivalently. Thus, the Ser-700 polymorphism alters a rapidly filled, high affinity Ca2+ -binding site in the first Ca2+ -binding motif. Slower Ca2+ binding to adjoining motifs partly compensates for the change.     

Loss-of-function mutations in euchromatin histone methyl transferase 1 (EHMT1) cause the 9q34 subtelomeric deletion syndrome

A clinically recognizable 9q subtelomeric deletion syndrome has recently been established. Common features seen in these patients are severe mental retardation, hypotonia, brachycephaly, flat face with hypertelorism, synophrys, anteverted nares, cupid bow or tented upper lip, everted lower lip, prognathism, macroglossia, conotruncal heart defects, and behavioral problems. The minimal critical region responsible for this 9q subtelomeric deletion (9q-) syndrome has been estimated to be <1 Mb and comprises the euchromatin histone methyl transferase 1 gene (EHMT1). Previous studies suggested that haploinsufficiency for EHMT1 is causative for 9q subtelomeric deletion syndrome. We have performed a comprehensive mutation analysis of the EHMT1 gene in 23 patients with clinical presentations reminiscent of 9q subtelomeric deletion syndrome. This analysis revealed three additional microdeletions that comprise the EHMT1 gene, including one interstitial deletion that reduces the critical region for this syndrome. Most importantly, we identified two de novo mutations--a nonsense mutation and a frameshift mutation--in the EHMT1 gene in patients with a typical 9q- phenotype. These results establish that haploinsufficiency of EHMT1 is causative for 9q subtelomeric deletion syndrome.     

Translation initiator EIF4G1 mutations in familial Parkinson disease

Genome-wide analysis of a multi-incident family with autosomal-dominant parkinsonism has implicated a locus on chromosomal region 3q26-q28. Linkage and disease segregation is explained by a missense mutation c.3614G>A (p.Arg1205His) in eukaryotic translation initiation factor 4-gamma (EIF4G1). Subsequent sequence and genotype analysis identified EIF4G1 c.1505C>T (p.Ala502Val), c.2056G>T (p.Gly686Cys), c.3490A>C (p.Ser1164Arg), c.3589C>T (p.Arg1197Trp) and c.3614G>A (p.Arg1205His) substitutions in affected subjects with familial parkinsonism and idiopathic Lewy body disease but not in control subjects. Despite different countries of origin, persons with EIF4G1 c.1505C>T (p.Ala502Val) or c.3614G>A (p.Arg1205His) mutations appear to share haplotypes consistent with ancestral founders. eIF4G1 p.Ala502Val and p.Arg1205His disrupt eIF4E or eIF3e binding, although the wild-type protein does not, and render mutant cells more vulnerable to reactive oxidative species. EIF4G1 mutations implicate mRNA translation initiation in familial parkinsonism and highlight a convergent pathway for monogenic, toxin and perhaps virally-induced Parkinson disease.     

Loss-of-function presenilin mutations in Alzheimer disease. Talking Point on the role of presenilin mutations in Alzheimer disease

Presenilin mutations are the main cause of familial Alzheimer disease. From a genetic point of view, these mutations seem to result in a gain of toxic function; however, biochemically, they result in a partial loss of function in the gamma-secretase complex, which affects several downstream signalling pathways. Consequently, the current genetic terminology is misleading. In fact, the available data indicate that several clinical presenilin mutations also lead to a decrease in amyloid precursor protein-derived amyloid beta-peptide generation, further implying that presenilin mutations are indeed loss-of-function mutations. The loss of function of presenilin causes incomplete digestion of the amyloid beta-peptide and might contribute to an increased vulnerability of the brain, thereby explaining the early onset of the inherited form of Alzheimer disease. In this review, I evaluate the implications of this model for the amyloid-cascade hypothesis and for the efficacy of presenilin/gamma-secretase as a drug target.     

Loss-of-function mutations in a glutathione S-transferase suppress the prune-Killer of prune lethal interaction

The prune gene of Drosophila melanogaster is predicted to encode a phosphodiesterase. Null alleles of prune are viable but cause an eye-color phenotype. The abnormal wing discs gene encodes a nucleoside diphosphate kinase. Killer of prune is a missense mutation in the abnormal wing discs gene. Although it has no phenotype by itself even when homozygous, Killer of prune when heterozygous causes lethality in the absence of prune gene function. A screen for suppressors of transgenic Killer of prune led to the recovery of three mutations, all of which are in the same gene. As heterozygotes these mutations are dominant suppressors of the prune-Killer of prune lethal interaction; as homozygotes these mutations cause early larval lethality and the absence of imaginal discs. These alleles are loss-of-function mutations in CG10065, a gene that is predicted to encode a protein with several zinc finger domains and glutathione S-transferase activity.     

Loss-of-function and gain-of-function mutations in FAB1A/B impair endomembrane homeostasis, conferring pleiotropic developmental abnormalities in Arabidopsis

In eukaryotic cells, PtdIns 3,5-kinase, Fab1/PIKfyve produces PtdIns (3,5) P(2) from PtdIns 3-P, and functions in vacuole/lysosome homeostasis. Herein, we show that expression of Arabidopsis (Arabidopsis thaliana) FAB1A/B in fission yeast (Schizosaccharomyces pombe) fab1 knockout cells fully complements the vacuole morphology phenotype. Subcellular localizations of FAB1A and FAB1B fused with green fluorescent protein revealed that FAB1A/B-green fluorescent proteins localize to the endosomes in root epidermal cells of Arabidopsis. Furthermore, reduction in the expression levels of FAB1A/B by RNA interference impairs vacuolar acidification and endocytosis. These results indicate that Arabidopsis FAB1A/B functions as PtdIns 3,5-kinase in plants and in fission yeast. Conditional knockdown mutant shows various phenotypes including root growth inhibition, hyposensitivity to exogenous auxin, and disturbance of root gravitropism. These phenotypes are observed also in the overproducing mutants of FAB1A and FAB1B. The overproducing mutants reveal additional morphological phenotypes including dwarfism, male-gametophyte sterility, and abnormal floral organs. Taken together, this evidence indicates that imbalanced expression of FAB1A/B impairs endomembrane homeostasis including endocytosis, vacuole formation, and vacuolar acidification, which causes pleiotropic developmental phenotypes mostly related to the auxin signaling in Arabidopsis.     

Loss-of-function mutations at the rim of the funnel of mechanosensitive channel MscL

MscL is a bacterial mechanosensitive channel that is activated directly by membrane stretch. Although the gene has been cloned and the crystal structure of the closed channel has been defined, how membrane tension causes conformational changes in MscL remains largely unknown. To identify the site where MscL senses membrane tension, we examined the function of the mutants generated by random and scanning mutagenesis. In vitro (patch-clamp) and in vivo (hypoosmotic-shock) experiments showed that when a hydrophilic amino acid replaces one of the hydrophobic residues that are thought to make contact with the membrane lipid near the periplasmic end of the M1 or M2 transmembrane domain, MscL loses the ability to open in response to membrane tension. Hydrophilic (asparagine) substitution of the other residues in the lipid-protein interface did not impair the channel's mechanosensitivity. These observations suggest that the disturbance of the hydrophobic interaction between the membrane lipid and the periplasmic rim of the channel's funnel impairs the function of MscL.     

Mitochondrial respiratory dysfunction and mutations in mitochondrial DNA in PINK1 familial Parkinsonism

A summary is presented of the cellular function and topology of the protein products of genes whose mutations are associated with familial forms of Parkinsonism, with particular emphasis on mitochondrial involvement. Observations are reviewed which show mitochondrial respiratory depression in the fibroblasts of a patient affected by familial Parkinsomism associated with homozygous PINK1 mutation. The respiratory depression, which was due to loss of mitochondrial cytochrome c, was associated with decreased capacity of respiratory chain oxidative phosphorylation and enhanced cellular level of ROS. Sequence analysis of the overall mtDNA revealed coexistence with the PINK1 mutation of homoplasmic point mutations in the ND5 and ND6 genes of complex I. The presence of these mutations appears to have an impact on the development of the Parkinsonism, which can also occur in the heterozygous PINK1 mutation state.     

Loss-of-function and residual channel activity of connexin26 mutations associated with non-syndromic deafness

Connexins are the protein subunits of gap junction channels that allow a direct signaling pathway between networks of cells. The specific role of connexin channels in the homeostasis of different organs has been validated by the association of mutations in several human connexins with a variety of genetic diseases. Several connexins are present in the mammalian cochlea and at least four of them have been proposed as genes causing sensorineural hearing loss. We have started our functional analysis by selecting nine mutations in Cx26 that are associated with non-syndromic recessive deafness (DFNB1). We have observed that both human Cx26 wild-type (HCx26wt) and the F83L polymorphism, found in unaffected controls, generated electrical conductance between paired Xenopus oocytes, which was several orders of magnitude greater than that measured in water-injected controls. In contrast, most recessive Cx26 mutations (identified in DFNB1 patients) resulted in a simple loss of channel activity. In addition, the V37I mutation, originally identified as a polymorphism in heterozygous unaffected individuals, was devoid of function and thus may be pathologically significant. Unexpectedly, we have found that the recessive mutation V84L retained functional activity in both paired Xenopus oocytes and transfected HeLa cells. Furthermore, both the magnitude of macroscopic junctional conductance and its voltage-gating properties were indistinguishable from those of HCx26wt. The identification of functional differences of disease causing mutations may lead to define which permeation or gating properties of Cx26 are necessary for normal auditory function in humans and will be instrumental in identifying the molecular steps leading to DFNB1.     

Mosaic SCN1A mutations in familial partial epilepsy with antecedent febrile seizures

SCN1A is the most relevant epilepsy gene. Mutations of SCN1A generate phenotypes ranging from the extremely severe form of Dravet syndrome (DS) to a mild form of generalized epilepsy with febrile seizures plus (GEFS+). Mosaic SCN1A mutations have been identified in rare familial DS. It is suspected that mosaic mutations of SCN1A may cause other types of familial epilepsies with febrile seizures (FS), which are more common clinically. Thus, we screened SCN1A mutations in 13 families with partial epilepsy with antecedent febrile seizures (PEFS+) using denaturing high-performance liquid chromatography and sequencing. The level of mosaicism was further quantified by pyrosequencing. Two missense SCN1A mutations with mosaic origin were identified in two unrelated families, accounting for 15.4% (2/13) of the PEFS+ families tested. One of the mosaic carriers with ~25.0% mutation of c.5768A>G/p.Q1923R had experienced simple FS; another with ~12.5% mutation of c.4847T>C/p.I1616T was asymptomatic. Their heterozygous children had PEFS+. Recurrent transmission occurred in both families, as noted in most of the families with germline mosaicism reported previously. The two mosaic mutations identified in this study are less destructive missense, compared with the more destructive truncating and splice-site mutations identified in the majority of previous studies. This is the first report of mosaic SCN1A mutations in families with probands that do not exhibit DS, but manifest only a milder phenotype. Therefore, such families with mild cases should be approached with caution in genetic counseling and the possibility of mosaicism origin associated with high recurrence risk should be excluded.