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The fact that hereditary hearing loss is the most common sensory disorder in humans is reflected by, among other things, an extraordinary allelic and nonallelic genetic heterogeneity. X-chromosomal hearing impairment represents only a minor fraction of all cases. In a study of a Spanish family the locus for one of the X-chromosomal forms was assigned to Xp22 (DFNX4). We mapped the disease locus in the same chromosomal region in a large German pedigree with X-chromosomal nonsyndromic hearing impairment by using genome-wide linkage analysis. Males presented with postlingual hearing loss and onset at ages 3-7, whereas onset in female carriers was in the second to third decades. Targeted DNA capture with high-throughput sequencing detected a nonsense mutation in the small muscle protein, X-linked (SMPX) of affected individuals. We identified another nonsense mutation in SMPX in patients from the Spanish family who were previously analyzed to map DFNX4. SMPX encodes an 88 amino acid, cytoskeleton-associated protein that is responsive to mechanical stress. The presence of Smpx in hair cells and supporting cells of the murine cochlea indicates its role in the inner ear. The nonsense mutations detected in the two families suggest a loss-of-function mechanism underlying this form of hearing impairment. Results obtained after heterologous overexpression of SMPX proteins were compatible with this assumption. Because responsivity to physical force is a characteristic feature of the protein, we propose that long-term maintenance of mechanically stressed inner-ear cells critically depends on SMPX function., (Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of end-stage renal failure. Identification of single-gene causes of SRNS has generated some insights into its pathogenesis; however, additional genes and disease mechanisms remain obscure, and SRNS continues to be treatment refractory. Here we have identified 6 different mutations in coenzyme Q10 biosynthesis monooxygenase 6 (COQ6) in 13 individuals from 7 families by homozygosity mapping. Each mutation was linked to early-onset SRNS with sensorineural deafness. The deleterious effects of these human COQ6 mutations were validated by their lack of complementation in coq6-deficient yeast. Furthermore, knockdown of Coq6 in podocyte cell lines and coq6 in zebrafish embryos caused apoptosis that was partially reversed by coenzyme Q10 treatment. In rats, COQ6 was located within cell processes and the Golgi apparatus of renal glomerular podocytes and in stria vascularis cells of the inner ear, consistent with an oto-renal disease phenotype. These data suggest that coenzyme Q10-related forms of SRNS and hearing loss can be molecularly identified and potentially treated.

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., (Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Deafness is genetically very heterogeneous and forms part of several syndromes. So far, delayed rectifier potassium channels have been linked to human deafness associated with prolongation of the QT interval on electrocardiograms and ventricular arrhythmia in Jervell and Lange-Nielsen syndrome. Ca(v)1.3 voltage-gated L-type calcium channels (LTCCs) translate sound-induced depolarization into neurotransmitter release in auditory hair cells and control diastolic depolarization in the mouse sinoatrial node (SAN). Human deafness has not previously been linked to defects in LTCCs. We used positional cloning to identify a mutation in CACNA1D, which encodes the pore-forming α1 subunit of Ca(v)1.3 LTCCs, in two consanguineous families with deafness. All deaf subjects showed pronounced SAN dysfunction at rest. The insertion of a glycine residue in a highly conserved, alternatively spliced region near the channel pore resulted in nonconducting calcium channels that had abnormal voltage-dependent gating. We describe a human channelopathy (termed SANDD syndrome, sinoatrial node dysfunction and deafness) with a cardiac and auditory phenotype that closely resembles that of Cacna1d(-/-) mice.

Functional impairment of DNA damage response pathways leads to increased genomic instability. Here we describe the centrosomal protein CEP152 as a new regulator of genomic integrity and cellular response to DNA damage. Using homozygosity mapping and exome sequencing, we identified CEP152 mutations in Seckel syndrome and showed that impaired CEP152 function leads to accumulation of genomic defects resulting from replicative stress through enhanced activation of ATM signaling and increased H2AX phosphorylation.

Purpose: To determine the cause of Leber congenital amaurosis (LCA) and developmental cataracts in a consanguineous Pakistani family., Methods: The diagnosis was established in all affected individuals of a Pakistani LCA family by medical history, funduscopy, and standard ERG. We performed genome-wide linkage analysis for mapping the disease locus in this family., Results: Congenitally severely reduced visual acuity and nystagmus were reported for all patients who, in the later phase of the disease, also developed cataracts. LCA in the family cosegregated with homozygosity for a single nucleotide polymorphism (SNP) haplotype on chromosome 6p14.1. The respective candidate region contained Leber congenital amaurosis 5 (LCA5), a gene previously reported to underlie LCA. We subsequently identified a novel truncating mutation in exon 4 of LCA5, c.642delC, in homozygous state in all affected persons of the family., Conclusions: We report a novel LCA5 mutation causing LCA in a Pakistani family. Developmental cataracts were present in two of the four patients, raising the possibility that LCA5 mutations may predispose to this additional ocular pathology.

Altered Bone Morphogenetic Protein (BMP) signaling leads to multiple developmental defects, including brachydactyly and deafness. Here we identify chondroitin synthase 1 (CHSY1) as a potential mediator of BMP effects. We show that loss of human CHSY1 function causes autosomal-recessive Temtamy preaxial brachydactyly syndrome (TPBS), mainly characterized by limb malformations, short stature, and hearing loss. After mapping the TPBS locus to chromosome 15q26-qterm, we identified causative mutations in five consanguineous TPBS families. In zebrafish, antisense-mediated chsy1 knockdown causes defects in multiple developmental processes, some of which are likely to also be causative in the etiology of TPBS. In the inner ears of zebrafish larvae, chsy1 is expressed similarly to the BMP inhibitor dan and in a complementary fashion to bmp2b. Furthermore, unrestricted Bmp2b signaling or loss of Dan activity leads to reduced chsy1 expression and, during epithelial morphogenesis, defects similar to those that occur upon Chsy1 inactivation, indicating that Bmp signaling affects inner-ear development by repressing chsy1. In addition, we obtained strikingly similar zebrafish phenotypes after chsy1 overexpression, which might explain why, in humans, brachydactyly can be caused by mutations leading either to loss or to gain of BMP signaling., (Copyright © 2010 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The hypothalamic-pituitary system controls homeostasis during feed energy reduction. In order to examine which pituitary proteins and hormone variants are potentially associated with metabolic adaptation, pituitary glands from ad libitum and energy restrictively fed dairy cows were characterized using RIA and 2-DE followed by MALDI-TOF-MS. We found 64 different spots of regulatory hormones: growth hormone (44), preprolactin (16), luteinizing hormone (LH) (1), thyrotropin (1), proopiomelanocortin (1) and its cleavage product lipotropin (1), but none of these did significantly differ between feeding groups. Quantification of total pituitary LH and prolactin concentrations by RIA confirmed the results obtained by proteome analysis. Also, feed energy restriction provoked increasing non-esterified fatty acid, decreasing prolactin, but unaltered glucose, LH and growth hormone plasma concentrations. Energy restriction decreased the expression of glial fibrillary acidic protein, triosephosphate isomerase, purine-rich element-binding protein A and elongation factor Tu, whereas it increased expression of proline synthetase co-transcribed homolog, peroxiredoxin III, β-tubulin and annexin A5 which is involved in the hormone secretion process. Our results indicate that in response to feed energy restriction the pituitary reservoir of all posttranslationally modified hormone forms remains constant. Changing plasma hormone concentrations are likely attributed to a regulated releasing process from the gland into the blood., (Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aicardi-Goutières syndrome (AGS) is a rare inborn multisystemic disease, resembling intrauterine viral infection and resulting in psychomotor retardation, spasticity and chilblain-likeskin lesions. Diagnostic criteria include intracerebral calcifications and elevated interferon-alpha and pterin levels in cerebrospinal fluid (CSF). We report on four adult siblings with unknown neurodegenerative disease presenting with cerebrovascular stenoses, stroke and glaucoma in childhood, two of whom died at the age of 40 and 29 years. Genome-wide homozygosity mapping identified 170 candidate genes embedded in a common haplotype of 8Mb on chromosome 20q11-13. Next generation sequencing of the entire region identified the c.490C>T (p.Arg164X) mutationin SAMHD1, a gene most recently described in AGS, on both alleles in all affected siblings.Clinical diagnosis of AGS was then confirmed by demonstrating intracerebral calcifications on cranial computed tomography in all siblings and elevated pterin levels in CSF in three of them. Inpatient fibroblasts, lack of SAMHD1 protein expression was associated with increased basal expression of IL8, while stimulated expression of IFNB1 was reduced. We conclude that cerebrovascular stenoses and stroke associated with the Arg164X mutation in SAMHD1 extend the phenotypic spectrum of AGS. The observed vascular changes most likely reflect a vasculitis caused by dysregulated inflammatory stress response., (©2010 Wiley-Liss, Inc.)

Nephronophthisis-related ciliopathies (NPHP-RC) are recessive disorders that feature dysplasia or degeneration occurring preferentially in the kidney, retina and cerebellum. Here we combined homozygosity mapping with candidate gene analysis by performing 'ciliopathy candidate exome capture' followed by massively parallel sequencing. We identified 12 different truncating mutations of SDCCAG8 (serologically defined colon cancer antigen 8, also known as CCCAP) in 10 families affected by NPHP-RC. We show that SDCCAG8 is localized at both centrioles and interacts directly with OFD1 (oral-facial-digital syndrome 1), which is associated with NPHP-RC. Depletion of sdccag8 causes kidney cysts and a body axis defect in zebrafish and induces cell polarity defects in three-dimensional renal cell cultures. This work identifies loss of SDCCAG8 function as a cause of a retinal-renal ciliopathy and validates exome capture analysis for broadly heterogeneous single-gene disorders.

We have previously shown that mutations in the genes encoding DNA Ligase IV (LIGIV) and RAD50, involved in DNA repair by nonhomologous-end joining (NHEJ) and homologous recombination, respectively, lead to clinical and cellular features similar to those of Nijmegen Breakage Syndrome (NBS). Very recently, a new member of the NHEJ repair pathway, NHEJ1, was discovered, and mutations in patients with features resembling NBS were described. Here we report on five patients from four families of different ethnic origin with the NBS-like phenotype. Sequence analysis of the NHEJ1 gene in a patient of Spanish and in a patient of Turkish origin identified homozygous, previously reported mutations, c.168C>G (p.Arg57Gly) and c.532C>T (p.Arg178Ter), respectively. Two novel, paternally inherited truncating mutations, c.495dupA (p.Asp166ArgfsTer20) and c.526C>T (p.Arg176Ter) and two novel, maternal genomic deletions of 1.9 and 6.9 kb of the NHEJ1 gene, were found in a compound heterozygous state in two siblings of German origin and in one Malaysian patient, respectively. Our findings confirm that patients with NBS-like phenotypes may have mutations in the NHEJ1 gene including multiexon deletions, and show that considerable clinical variability could be observed even within the same family., (Copyright 2010 Wiley-Liss, Inc.)

Generalized peeling skin disease is an autosomal-recessive ichthyosiform erythroderma characterized by lifelong patchy peeling of the skin. After genome-wide linkage analysis, we have identified a homozygous nonsense mutation in CDSN in a large consanguineous family with generalized peeling skin, pruritus, and food allergies, which leads to a complete loss of corneodesmosin. In contrast to hypotrichosis simplex, which can be associated with specific dominant CDSN mutations, peeling skin disease is characterized by a complete loss of CDSN expression. The skin phenotype is consistent with a recent murine Cdsn knockout model. Using three-dimensional human skin models, we demonstrate that lack of corneodesmosin causes an epidermal barrier defect supposed to account for the predisposition to atopic diseases, and we confirm the role of corneodesmosin as a decisive epidermal adhesion molecule. Therefore, peeling skin disease will represent a new model disorder for atopic diseases, similarly to Netherton syndrome and ichthyosis vulgaris in the recent past.

Mutations in the visual system homeobox 2 gene (VSX2, also known as CHX10), which encodes a retinal transcription factor from the paired homeobox family, have been implicated in recessive isolated microphthalmia. In this study, we use genome-wide single nucleotide polymorphism homozygosity mapping in unrelated small consanguineous pedigrees and a candidate gene approach to identify three further causative VSX2 mutations (two novel and one previously reported). All affected individuals with homozygous mutations had bilateral anophthalmia or severe microphthalmia with absent vision. In addition, we identified a novel inner retinal dystrophy in two carrier parents suggesting a semidominant effect for this particular VSX2 mutation. A further study of individuals with retinal degenerative conditions may reveal a causative role for heterozygous mutations in VSX2.

Werner syndrome (WS) is an autosomal recessive segmental progeroid syndrome caused by null mutations at the WRN locus, which codes for a member of the RecQ family of DNA helicases. Since 1988, the International Registry of Werner syndrome had enrolled 130 molecularly confirmed WS cases from among 110 worldwide pedigrees. We now report 18 new mutations, including two genomic rearrangements, a deep intronic mutation resulting in a novel exon, a splice consensus mutation leading to utilization of the nearby splice site, and two rare missense mutations. We also review evidence for founder mutations among various ethnic/geographic groups. Founder WRN mutations had been previously reported in Japan and Northern Sardinia. Our Registry now suggests characteristic mutations originated in Morocco, Turkey, The Netherlands and elsewhere.

Usher syndrome is a genetically heterogeneous recessive disease characterized by hearing loss and retinitis pigmentosa (RP). It frequently presents with unexplained, often intrafamilial, variability of the visual phenotype. Although 9 genes have been linked with Usher syndrome, many patients do not have mutations in any of these genes, suggesting that there are still unidentified genes involved in the syndrome. Here, we have determined that mutations in PDZ domain-containing 7 (PDZD7), which encodes a homolog of proteins mutated in Usher syndrome subtype 1C (USH1C) and USH2D, contribute to Usher syndrome. Mutations in PDZD7 were identified only in patients with mutations in other known Usher genes. In a set of sisters, each with a homozygous mutation in USH2A, a frame-shift mutation in PDZD7 was present in the sister with more severe RP and earlier disease onset. Further, heterozygous PDZD7 mutations were present in patients with truncating mutations in USH2A, G protein-coupled receptor 98 (GPR98; also known as USH2C), and an unidentified locus. We validated the human genotypes using zebrafish, and our findings were consistent with digenic inheritance of PDZD7 and GPR98, and with PDZD7 as a retinal disease modifier in patients with USH2A. Pdzd7 knockdown produced an Usher-like phenotype in zebrafish, exacerbated retinal cell death in combination with ush2a or gpr98, and reduced Gpr98 localization in the region of the photoreceptor connecting cilium. Our data challenge the view of Usher syndrome as a traditional Mendelian disorder and support the reclassification of Usher syndrome as an oligogenic disease.

Cenani-Lenz syndrome (CLS) is an autosomal-recessive congenital disorder affecting distal limb development. It is characterized mainly by syndactyly and/or oligodactyly and is now shown to be commonly associated with kidney anomalies. We used a homozygosity-mapping approach to map the CLS1 locus to chromosome 11p11.2-q13.1. By sequencing candidate genes, we identified recessive LRP4 mutations in 12 families with CLS. LRP4 belongs to the low-density lipoprotein (LDL) receptor-related proteins (LRPs), which are essential for various developmental processes. LRP4 is known to antagonize LRP6-mediated activation of canonical Wnt signaling, a function that is lost by the identified mutations. Our findings increase the spectrum of congenital anomalies associated with abnormal lipoprotein receptor-dependent signaling., (Copyright (c) 2010 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

We performed genome-wide homozygosity mapping in a large consanguineous family from Morocco and mapped the autosomal-recessive nonsyndromic hearing loss (ARNSHL) in this family to the DFNB79 locus on chromosome 9q34. By sequencing of 62 positional candidate genes of the critical region, we identified a causative homozygous 11 bp deletion, c.42_52del, in the TPRN gene in all seven affected individuals. The deletion is located in exon 1 and results in a frameshift and premature protein truncation (p.Gly15AlafsX150). Interestingly, the deleted sequence is part of a repetitive and CG-rich motive predicted to be prone to structural aberrations during crossover formation. We identified another family with progressive ARNSHL linked to this locus, whose affected members were shown to carry a causative 1 bp deletion (c.1347delG) in exon 1 of TPRN. The function of the encoded protein, taperin, is unknown; yet, partial homology to the actin-caping protein phostensin suggests a role in actin dynamics., (Copyright 2010 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Detection of discriminating patterns in gene expression data can be accomplished by using various methods of statistical learning. It has been proposed that sample pooling in this context would have negative effects; however, pooling cannot always be avoided. We propose a simulation framework to explicitly investigate the parameters of patterns, experimental design, noise, and choice of method in order to find out which effects on classification performance are to be expected. We use a two-group classification task and simulated gene expression data with independent differentially expressed genes as well as bivariate linear patterns and the combination of both. Our results show a clear increase of prediction error with pool size. For pooled training sets powered partial least squares discriminant analysis outperforms discriminance analysis, random forests, and support vector machines with linear or radial kernel for two of three simulated scenarios. The proposed simulation approach can be implemented to systematically investigate a number of additional scenarios of practical interest.

Genetic defects affecting motility of cilia and flagella cause chronic destructive airway disease, randomization of left-right body asymmetry, and, frequently, male infertility in primary ciliary dyskinesia (PCD). The most frequent defects involve outer and inner dynein arms (ODAs and IDAs) that are large multiprotein complexes responsible for cilia-beat generation and regulation, respectively. Here, we demonstrate that large genomic deletions, as well as point mutations involving LRRC50, are responsible for a distinct PCD variant that is characterized by a combined defect involving assembly of the ODAs and IDAs. Functional analyses showed that LRRC50 deficiency disrupts assembly of distally and proximally DNAH5- and DNAI2-containing ODA complexes, as well as DNALI1-containing IDA complexes, resulting in immotile cilia. On the basis of these findings, we assume that LRRC50 plays a role in assembly of distinct dynein-arm complexes.

Hereditary sensory and autonomic neuropathy type II (HSAN II) leads to severe mutilations because of impaired nociception and autonomic dysfunction. Here we show that loss-of-function mutations in FAM134B, encoding a newly identified cis-Golgi protein, cause HSAN II. Fam134b knockdown results in structural alterations of the cis-Golgi compartment and induces apoptosis in some primary dorsal root ganglion neurons. This implicates FAM134B as critical in long-term survival of nociceptive and autonomic ganglion neurons.

Context: Familial glucocorticoid deficiency (FGD) is an autosomal recessive disorder resulting from resistance to the action of ACTH on the adrenal cortex. Affected individuals are deficient in cortisol and, if untreated, are likely to succumb to hypoglycemia and/or overwhelming infection. Mutations of the ACTH receptor (MC2R) and the melanocortin 2 receptor accessory protein (MRAP), FGD types 1 and 2 respectively, account for approximately 45% of cases., Objective: A locus on chromosome 8 has previously been linked to the disease in three families, but no underlying gene defect has to date been identified., Design: The study design comprised single-nucleotide polymorphism genotyping and mutation detection., Setting: The study was conducted at secondary and tertiary referral centers., Patients: Eighty probands from families referred for investigation of the genetic cause of FGD participated in the study., Interventions: There were no interventions., Results: Analysis by single-nucleotide polymorphism array of the genotype of one individual with FGD previously linked to chromosome 8 revealed a large region of homozygosity encompassing the steroidogenic acute regulatory protein gene, STAR. We identified homozygous STAR mutations in this patient and his affected siblings. Screening of our total FGD patient cohort revealed homozygous STAR mutations in a further nine individuals from four other families., Conclusions: Mutations in STAR usually cause lipoid congenital adrenal hyperplasia, a disorder characterized by both gonadal and adrenal steroid deficiency. Our results demonstrate that certain mutations in STAR (R192C and the previously reported R188C) can present with a phenotype indistinguishable from that seen in FGD.

Rapid intracellular transport and secretion of cytotoxic granules through the immunological synapse requires a balanced interaction of several proteins. Disturbance of this highly regulated process underlies familial hemophagocytic lymphohistiocytosis (FHL), a genetically heterogeneous autosomal-recessive disorder characterized by a severe hyperinflammatory phenotype. Here, we have assigned FHL-5 to a 1 Mb region on chromosome 19p by using high-resolution SNP genotyping in eight unrelated FHL patients from consanguineous families. Subsequently, we found nine different mutations, either truncating or missense, in STXBP2 in twelve patients from Turkey, Saudi Arabia, and Central Europe. STXBP2 encodes syntaxin binding protein 2 (Munc18-2), involved in the regulation of vesicle transport to the plasma membrane. We have identified syntaxin 11, a SNARE protein mutated in FHL-4, as an interaction partner of STXBP2. This interaction is eliminated by the missense mutations found in our FHL-5 patients, which leads to a decreased stability of both proteins, as shown in patient lymphocytes. Activity of natural killer and cytotoxic T cells was markedly reduced or absent, as determined by CD107 degranulation. Our findings thus identify a key role for STXBP2 in lytic granule exocytosis.

FOXE3 is a lens-specific transcription factor with a highly conserved forkhead domain previously implicated in congenital primary aphakia and anterior segment dysgenesis. Here, we identify new recessive FOXE3 mutations causative for microphthalmia, sclerocornea, primary aphakia, and glaucoma in two extended consanguineous families by SNP array genotyping followed by a candidate gene approach. Following an additional screen of 236 subjects with developmental eye anomalies, we report two further novel heterozygous mutations segregating in a dominant fashion in two different families. Although the dominant mutations were penetrant, they gave rise to highly variable phenotypes including iris and chorioretinal colobomas, Peters' anomaly, and isolated cataract (cerulean type and early onset adult nuclear and cortical cataract). Using in situ hybridization in human embryos, we demonstrate expression of FOXE3 restricted to lens tissue, predominantly in the anterior epithelium, suggesting that the extralenticular phenotypes caused by FOXE3 mutations are most likely to be secondary to abnormal lens formation. Our findings suggest that mutations in FOXE3 can give rise to a broad spectrum of eye anomalies, largely, but not exclusively related to lens development, and that both dominant and recessive inheritance patterns can be represented. We suggest including FOXE3 in the diagnostic genetic screening for these anomalies.

Autosomal recessive cutis laxa (ARCL) describes a group of syndromal disorders that are often associated with a progeroid appearance, lax and wrinkled skin, osteopenia and mental retardation. Homozygosity mapping in several kindreds with ARCL identified a candidate region on chromosome 17q25. By high-throughput sequencing of the entire candidate region, we detected disease-causing mutations in the gene PYCR1. We found that the gene product, an enzyme involved in proline metabolism, localizes to mitochondria. Altered mitochondrial morphology, membrane potential and increased apoptosis rate upon oxidative stress were evident in fibroblasts from affected individuals. Knockdown of the orthologous genes in Xenopus and zebrafish led to epidermal hypoplasia and blistering that was accompanied by a massive increase of apoptosis. Our findings link mutations in PYCR1 to altered mitochondrial function and progeroid changes in connective tissues.

We have earlier described a syndrome characterized by microcephaly, cutis verticis gyrata, retinitis pigmentosa, cataracts, hearing loss and mental retardation (Mendelian inheritance in man (MIM) no: 605685) in two brothers from a non-consanguineous Lebanese family. In view of the rarity of the disorder and the high rate of inbreeding in the Lebanese population, we assumed an autosomal recessive trait inherited from a common ancestor. A genomewide scan was performed. The single locus on the long arm of chromosome 8 that showed homozygosity by descent comprised the gene responsible for Cohen syndrome (CS), VPS13B. We then sequenced VPS13B in the patients and found a homozygous splice site mutation. Several possible explanations for the overlap between CS and the clinical features observed in our patients are discussed. Our data highlight the potential of high-resolution homozygosity mapping in small populations with a high rate of inbreeding.

We examined the repertoire and extent of inflammation dependent gene regulation in a bovine mammary epithelial cell (MEC) model, to better understand the contribution of the MEC in the immune defence of the udder. We challenged primary cultures of MEC from cows with heat inactivated Escherichia coli pathogens and used Affymetrix DNA-microarrays to profile challenge related alterations in their transcriptome. Compared to acute mastitis, the most prominently activated genes comprise those encoding chemokines, interleukins, beta-defensins, serum amyloid A and haptoglobin. Hence, the MEC exert sentinel as well as effector functions of innate immune defence. E. coli stimulated a larger fraction of genes (30%) in the MEC belonging to the functional category Inflammatory Response than we recorded with the same microarrays during acute mastitis in the udder (17%). This observation underscores the exquisite immune capacity of MEC. To more closely examine the adequacy of immunological regulation in MEC, we compared the inflammation dependent regulation of factors contributing to the complement system between the udder versus the MEC. In the MEC we observed only up regulation of several complement factor-encoding genes. Mastitis, in contrast, in the udder strongly down regulates such genes encoding factors contributing to both, the classical pathway of complement activation and the Membrane Attack Complex, while the expression of factors contributing to the alternative pathway may be enhanced. This functionally polarized regulation of the complex complement pathway is not reflected in the MEC models.

Purpose: Identification of the causal mutations in 28 unrelated families and individuals with hereditary congenital cataract identified from a national Danish register of hereditary eye diseases. Seven families have been published previously, and the data of the remaining 21 families are presented together with an overview of the results in all families., Methods: A combined screening approach of linkage analysis and sequencing of 17 cataract genes were applied to mutation analyses of total 28 families., Results: The study revealed a disease locus in seven of eight families that were amenable to linkage analysis. All loci represented known genes, and subsequent sequencing identified the mutations. Mutations were found in eight genes, among them crystallins (36%), connexins (22%), and the transcription factors HSF4 and MAF (15%). One family carried a complex CRYBB2 allele of three DNA variants, and a gene conversion is the most likely mutational event causing this variant. Ten families had microcornea cataract, and a mutation was identified in eight of those. Most families displayed mixed phenotypes with nuclear, lamellar, and polar opacities and no apparent genotype-phenotype correlation emerged., Conclusions: In total, 28 families were analyzed, and mutations were identified in 20 (71%) of them. Despite considerable locus heterogeneity, a high mutation identification rate was achieved by sequencing a limited number of major cataract genes. Provided these results are representative of Western European populations, the applied sequencing strategy seems to be suitable for the exploration of the large group of isolated cataracts with unknown etiology.

Hypoxia, a growing worldwide problem, has been intermittently present in the modern Baltic Sea since its formation ca. 8000 cal. yr BP. However, both the spatial extent and intensity of hypoxia have increased with anthropogenic eutrophication due to nutrient inputs. Physical processes, which control stratification and the renewal of oxygen in bottom waters, are important constraints on the formation and maintenance of hypoxia. Climate controlled inflows of saline water from the North Sea through the Danish Straits is a critical controlling factor governing the spatial extent and duration of hypoxia. Hypoxia regulates the biogeochemical cycles of both phosphorus (P) and nitrogen (N) in the water column and sediments. Significant amounts of P are currently released from sediments, an order of magnitude larger than anthropogenic inputs. The Baltic Sea is unique for coastal marine ecosystems experiencing N losses in hypoxic waters below the halocline. Although benthic communities in the Baltic Sea are naturally constrained by salinity gradients, hypoxia has resulted in habitat loss over vast areas and the elimination of benthic fauna, and has severely disrupted benthic food webs. Nutrient load reductions are needed to reduce the extent, severity, and effects of hypoxia.

We describe a recessively inherited frontonasal malformation characterized by a distinctive facial appearance, with hypertelorism, wide nasal bridge, short nasal ridge, bifid nasal tip, broad columella, widely separated slit-like nares, long philtrum with prominent bilateral swellings, and midline notch in the upper lip and alveolus. Additional recurrent features present in a minority of individuals have been upper eyelid ptosis and midline dermoid cysts of craniofacial structures. Assuming recessive inheritance, we mapped the locus in three families to chromosome 1 and identified mutations in ALX3, which is located at band 1p13.3 and encodes the aristaless-related ALX homeobox 3 transcription factor. In total, we identified seven different homozygous pathogenic mutations in seven families. These mutations comprise missense substitutions at critical positions within the conserved homeodomain as well as nonsense, frameshift, and splice-site mutations, all predicting severe or complete loss of function. Our findings contrast with previous studies of the orthologous murine gene, which showed no phenotype in Alx3(-/-) homozygotes, apparently as a result of functional redundancy with the paralogous Alx4 gene. We conclude that ALX3 is essential for normal facial development in humans and that deficiency causes a clinically recognizable phenotype, which we term frontorhiny.

Effects of dietary fat supplementation prepartum on liver lipids and metabolism in dairy cows are contradictory. Thus, we examined in 18 German Holstein cows (half-sib; first lactation 305-d milk yield >9,000 kg) whether dietary fat:carbohydrate ratio during the last trimester of gestation affects lipid metabolism and milk yield. The diets were formulated to be isoenergetic and isonitrogenous but differed in rumen-protected fat (FD; 28 and 46.5 g/kg of dry matter during far-off and close-up dry period; mainly C16:0 and C18:1) and starch concentration [carbohydrate diet (CD); 2.3 times as much starch as FD]. Diets were given ad libitum starting 12 wk before expected parturition. After parturition all cows were fed a single lactation diet ad libitum for 14 wk. With the FD treatment, dry matter intake was depressed prepartum, milk yield during first 4 wk of lactation was lower (36.9 vs. 41.0 kg/d), and postpartum energy balance during this period was more negative. During the first 4 wk, cows in the FD group had lower lactose percentage and yield but higher milk fat, whereas milk protein and fat yield as well as energy-corrected milk did not differ. Between wk 5 and 14, milk fat and milk protein percentage was lower in CD than in FD. Milk fat C14:0 was lower and C16:1 was higher in the FD group. For FD cows, plasma triacylglycerol, nonesterified fatty acids, and cholesterol concentrations were higher prepartum, whereas plasma beta-hydroxybutyrate and glucose concentrations were lower. During the first 10 d after parturition, plasma triacylglycerol concentration was higher in FD, and prepartum plasma glucose and cholesterol differences persisted during the first 14 wk of lactation. Irrespective of prepartum nutrient composition, concentrations of plasma leptin and subcutaneous fat leptin mRNA decreased between -10 d to +10 d relative to parturition, and liver lipids and glycogen reached maximum and minimal values, respectively, 10 d after parturition. Acetyl-coenzyme A carboxylase alpha mRNA abundance in subcutaneous fat decreased between -10 d to +1 d relative to parturition by 97%, whereas it was generally much lower in the liver and remained at a low level until wk 14 of lactation. In conclusion, feeding a diet containing rumen-protected fat during late lactation and dry period until calving negatively affected dry matter intake, energy balance, and milk yield during subsequent lactation, did not change acetyl-coenzyme A carboxylase alpha mRNA abundance in subcutaneous fat, and was not beneficial for liver lipid accumulation.

We extend the spectrum of phenotypes caused by mutations in the Wnt/Norrin coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) by identifying two novel types of mutation in related individuals whose presenting features were profound muscle hypotonia, mild mental retardation, blindness, and growth retardation. One mutation removes 6 out of 9 consecutive leucine residues in the LRP5 signal peptide (c.43_60del or p.Leu15_Leu20del), which impairs polypeptide entry into the endoplasmic reticulum (ER), trafficking to the cell membrane, and signal transduction. The second mutation resulted from nonhomologous recombination between Alu repeat sequences, which deleted exons 14-16 and would produce a nonfunctional, truncated, and frameshifted polypeptide, if expressed [chr11:g.(13871447_1387511)_(13879636_13879700)del (NW_925106.1) or p.Pro1010GlnfsX38]. We confirmed that the length of the LRP5 signal peptide poly-leucine repeat is polymorphic in the general population, and, importantly, we were able to demonstrate in independent in vitro assays that different allele sizes affect receptor processing and signal transduction. Consequently, this polymorphism may have physiologic effects in vivo. This latter finding is relevant since through a genomewide search we identified nearly 400 human proteins that contain poly-leucine repeats within their signal peptide. We chose 18 of these proteins and genotyped the underlying trinucleotide repeat in healthy Caucasian individuals. More than one length allele was observed in one-half of the proteins. We therefore propose that natural variation in poly-leucine-stretches within signal peptides constitutes a currently unrecognized source of variability in protein translation and expression., ((c) 2009 Wiley-Liss, Inc.)

This study was conducted to investigate whether the isoflavones genistein and daidzein, which are components of soy-based diets, and the estrogen 17beta-estradiol affect differentiation and protein metabolism of porcine skeletal muscle cells in vitro. Serum-free porcine myotube cultures expressing the estrogen receptors ERalpha and ERbeta were treated with various concentrations of genistein, daidzein, or 17beta-estradiol for 26 h. The degree of differentiation by creatine phosphokinase activity was not altered by treatment. At 100 micromol/L both genistein and daidzein caused decreases in protein amount due to cell loss. In addition, 100 micromol/L genistein reduced protein synthesis rate of the surviving cells (P < 0.05) measured as [3H]-phenylalanine incorporation. Interestingly, genistein (0.1 micromol/L), daidzein (10, 100 micromol/L), and 17beta-estradiol (0.1, 1 nmol/L) slightly reduced protein degradation (P < 0.05). The results suggest that both genistein and daidzein affect protein metabolism in a dose-dependent manner and that estrogenic actions may play a role in decreasing protein degradation in porcine skeletal muscle.

Vitamin B(12) (cobalamin) is essential in animals for metabolism of branched chain amino acids and odd chain fatty acids, and for remethylation of homocysteine to methionine. In the cblF inborn error of vitamin B(12) metabolism, free vitamin accumulates in lysosomes, thus hindering its conversion to cofactors. Using homozygosity mapping in 12 unrelated cblF individuals and microcell-mediated chromosome transfer, we identified a candidate gene on chromosome 6q13, LMBRD1, encoding LMBD1, a lysosomal membrane protein with homology to lipocalin membrane receptor LIMR. We identified five different frameshift mutations in LMBRD1 resulting in loss of LMBD1 function, with 18 of the 24 disease chromosomes carrying the same mutation embedded in a common 1.34-Mb haplotype. Transfection of fibroblasts of individuals with cblF with wild-type LMBD1 rescued cobalamin coenzyme synthesis and function. This work identifies LMBRD1 as the gene underlying the cblF defect of cobalamin metabolism and suggests that LMBD1 is a lysosomal membrane exporter for cobalamin.

The identification of recessive disease-causing genes by homozygosity mapping is often restricted by lack of suitable consanguineous families. To overcome these limitations, we apply homozygosity mapping to single affected individuals from outbred populations. In 72 individuals of 54 kindred ascertained worldwide with known homozygous mutations in 13 different recessive disease genes, we performed total genome homozygosity mapping using 250,000 SNP arrays. Likelihood ratio Z-scores (ZLR) were plotted across the genome to detect ZLR peaks that reflect segments of homozygosity by descent, which may harbor the mutated gene. In 93% of cases, the causative gene was positioned within a consistent ZLR peak of homozygosity. The number of peaks reflected the degree of inbreeding. We demonstrate that disease-causing homozygous mutations can be detected in single cases from outbred populations within a single ZLR peak of homozygosity as short as 2 Mb, containing an average of only 16 candidate genes. As many specialty clinics have access to cohorts of individuals from outbred populations, and as our approach will result in smaller genetic candidate regions, the new strategy of homozygosity mapping in single outbred individuals will strongly accelerate the discovery of novel recessive disease genes., Competing Interests: The authors have declared that no competing interests exist.

The congenital splay leg syndrome in piglets is characterized by a temporarily impaired functionality of the hind leg muscles immediately after birth. Etiology and pathogenetic mechanisms for the disease are still not well understood. We compared genome wide gene expression of three hind leg muscles (M. adductores, M. gracilis and M. sartorius) between affected piglets and their healthy littermates with the GeneChip Porcine Genome Array (Affymetrix) in order to identify candidate genes for the disease. Data analysis with standard algorithms revealed no significant differences between both groups. By application of an alternative approach, we identified 63 transcripts with differences in two muscles and 5 genes differing between the groups in three muscles. The expression of six selected genes (SQSTM1, SSRP1, DDIT4, ENAH, MAF, and PDK4) was investigated with SYBRGreen RT-Real time PCR. The differences obtained with the microarray analysis could be confirmed and demonstrate the validity of the alternative approach to microarray data analysis. Four genes with different expression levels in at least two muscles (SQSTM1, SSRP1, DDIT4, and MAF) are assigned to transcriptional cascades related to cell death and may thus indicate pathways for further investigations on congenital splay leg in piglets.

Familial thrombosis (FT) has been described as a rare autosomal-dominant disorder, mostly caused by activating mutations of the thrombopoietin gene (THPO). Other cases of FT have been linked to one of two different germline mutations in the myeloproliferative leukaemia virus oncogene gene (MPL), which codes for the thrombopoietin receptor MPL. We studied an Arab family with two siblings with severe thrombocytosis by linkage analysis and obtained evidence for linkage to MPL. Sequencing revealed homozygosity for the novel MPL germline mutation p.Pro106Leu (c.317C > T) in the two siblings. Subsequently, homozygosity for p.Pro106Leu was identified in six further FT patients from three other Arab families. Of 18 heterozygous carriers, 14 had normal platelet counts, while four had mild thrombocytosis. Strong support for association of the novel MPL mutation p.Pro106Leu with development of familial thrombocytosis has been obtained. Overall, p.Pro106Leu was absent on 386 alleles of 193 healthy German controls and present on 14 of 426 alleles (3.3%) of 213 unrelated Arabs, which was statistically significantly different (P < 0.001, Fisher's exact test). We assume that p.Pro106Leu is a frequent MPL mutation in the Arab population, leading to severe thrombocytosis in homozygotes and occasionally to mild thrombocytosis in heterozygotes. In the families described the mode of inheritance could be regarded as autosomal-recessive with possible mild heterozygote manifestation rather than autosomal-dominant with high penetrance as usually seen in FT.

The incidence of hyper-contracted giant fibres in pig postmortem skeletal muscle is closely related to poor meat quality in terms of pale, soft, and exudative pork. Detection of a predisposition to develop giant fibres in live pigs could help to predict pork quality and to exclude affected pigs from genetic selection. The abundance and proportion of giant fibres in longissimus muscle were highest in Piétrain followed by Landrace, Large White, and Leicoma pigs of market weight. The postmortem development of giant fibres could be successfully simulated in vitro incubating biopsy samples from longissimus muscle at 37°C for 60min. For repeated measurements on three samples the intraclass correlation coefficient for the number of giant fibres/cm(2) was ϑˆ(3)=0.69 for biopsy and ϑˆ(3)=0.87 for carcass samples. "Simulated" giant fibres exhibited ultrastructural changes in plasma membrane, myofibrils, mitochondria, and sarcoplasmatic reticulum as shown previously for giant fibres in carcass samples.

Soy-derived isoflavones have been reported to be specific inhibitors of protein tyrosine kinases like the type 1 insulin-like growth factor receptor (IGF-1R) and the epidermal growth factor receptor (EGFR). This study was conducted to investigate, whether IGF-I and EGF stimulate porcine myoblast growth and whether the responses are influenced by isoflavones. Satellite cell-born myoblasts derived from the semimembranosus muscle of newborn piglets were treated for 26 h with IGF-I or EGF alone and in combination with genistein or daidzein. The DNA amount was measured and DNA synthesis was recorded as 6 h-[(3)H]thymidine incorporation during exponential growth in serum-free basal medium. IGF-I and EGF synergistically stimulated DNA synthesis of porcine myoblast with EGF causing a greater response. Genistein (100 micromol/l) effectively reduced the growth factor-mediated DNA synthesis, which was associated with an inhibition of growth factor receptor protein expression. In response to daidzein no reduction in growth factor-mediated DNA synthesis was found. Daidzein (1; 10 micromol/l) combined with IGF-I caused even a slight increase in DNA amount compared with the untreated control. The expression of the IGF-1R precursor protein was reduced with 10 and 100 micromol/l daidzein, whereas the EGFR expression remained unchanged with daidzein. The results suggest that dietary isoflavones may interact with growth factor-induced stimulation of pig skeletal muscle growth.

Pontocerebellar hypoplasias (PCH) represent a group of neurodegenerative autosomal recessive disorders with prenatal onset, atrophy or hypoplasia of the cerebellum, hypoplasia of the ventral pons, microcephaly, variable neocortical atrophy and severe mental and motor impairments. In two subtypes, PCH2 and PCH4, we identified mutations in three of the four different subunits of the tRNA-splicing endonuclease complex. Our findings point to RNA processing as a new basic cellular impairment in neurological disorders.

Background: In a recent study it was demonstrated that a largely increased ovulation number is responsible for high prolificacy in two mouse lines selected for fertility performance. The objective of the present study was to identify genes that are involved in increasing the ovulation number in one of these lines, FL1. For differential expression profiling, ovaries of FL1 and of a non-selected control line, DUKsi, both lines derived from the same genetic pool, were analyzed with microarray analysis and quantitative polymerase chain reaction (qPCR). Ovaries from 30 animals of each line were collected at the metestrous stage, combined to 6 pools each, and processed for microarray analysis., Results: The actual number of ova shed in FL1 exceeded that of the DUKsi control line more than twofold (26.6 vs. 12.9). 148 differentially expressed ovarian transcripts could be identified, 74 of them up- and 74 down-regulated. Of these, 47 significantly mapped to specific Gene Ontology (GO) terms representing different biological processes as steroid metabolism, folliculogenesis, immune response, intracellular signal transduction (particularly of the G protein signaling cascade), regulation of transcription and translation, cell cycle and others. qPCR was used to re-evaluate selected transcripts and to estimate inter-individual variation of expression levels. These data significantly correlated with microarray data in 12 out of 15 selected transcripts but revealed partly large variations of expression levels between individuals., Conclusion: (1) The abundance of numerous ovarian transcripts was significantly different in FL1 compared to the non-selected control line DUKsi thus suggesting that at least some of the respective genes and corresponding biological processes are involved in improving reproductive traits, particularly by increasing the number of ovulation. (2) Selective qPCR re-evaluation largely confirmed the microarray data and in addition demonstrated that sample pooling can be beneficial to find out group-specific expression profiles despite of large inter-individual variation. (3) The present data will substantially help ongoing genetic association studies to identify candidate genes and causative mutations responsible for increased fertility performance in mice.

Purpose: The goal of this study was to identify mutations in X-chromosomal genes associated with retinitis pigmentosa (RP) in patients from Germany, The Netherlands, Denmark, and Switzerland., Methods: In addition to all coding exons of RP2, exons 1 through 15, 9a, ORF15, 15a and 15b of RPGR were screened for mutations. PCR products were amplified from genomic DNA extracted from blood samples and analyzed by direct sequencing. In one family with apparently dominant inheritance of RP, linkage analysis identified an interval on the X chromosome containing RPGR, and mutation screening revealed a pathogenic variant in this gene. Patients of this family were examined clinically and by X-inactivation studies., Results: This study included 141 RP families with possible X-chromosomal inheritance. In total, we identified 46 families with pathogenic sequence alterations in RPGR and RP2, of which 17 mutations have not been described previously. Two of the novel mutations represent the most 3'-terminal pathogenic sequence variants in RPGR and RP2 reported to date. In exon ORF15 of RPGR, we found eight novel and 14 known mutations. All lead to a disruption of open reading frame. Of the families with suggested X-chromosomal inheritance, 35% showed mutations in ORF15. In addition, we found five novel mutations in other exons of RPGR and four in RP2. Deletions in ORF15 of RPGR were identified in three families in which female carriers showed variable manifestation of the phenotype. Furthermore, an ORF15 mutation was found in an RP patient who additionally carries a 6.4 kbp deletion downstream of the coding region of exon ORF15. We did not identify mutations in 39 sporadic male cases from Switzerland., Conclusions: RPGR mutations were confirmed to be the most frequent cause of RP in families with an X-chromosomal inheritance pattern. We propose a screening strategy to provide molecular diagnostics in these families.

Dyschromatosis universalis hereditaria (DUH) and dyschromatosis symmetrica hereditaria (DSH) are pigmentary dermatoses most commonly seen in Japan. Both disorders usually show autosomal dominant inheritance, although in some cases autosomal recessive inheritance was reported. DSH was mapped to chromosome 1q21.3, and mutations in the gene ADAR (DSRAD) were identified in Japanese, Chinese and Taiwanese families with autosomal dominant DSH. A second locus for dyschromatosis was mapped on chromosome 6q24.2-q25.2 in two Chinese families initially reported to be affected with DSH, but later suggested to have autosomal dominant DUH. The aim of this study was to investigate whether one of these two loci is involved in the development of DUH in a consanguineous Bedouin family from Saudi Arabia with four affected and three unaffected sibs, clearly pointing to autosomal recessive inheritance. After excluding mutations in ADAR and linkage to the candidate regions on chromosomes 1 and 6, we performed an single nucleotide polymorphism-based genome-wide scan for linkage with other loci. Under the assumption of autosomal recessive inheritance, we have identified a new locus for dyschromatosis on chromosome 12q21-q23 in this Arab family with a maximum logarithm of the odds (LOD) score of 3.4, spanning a distance of 18.9 cM. Our study revealed the first locus for autosomal recessive DUH and supports recent evidence that DSH and DUH are genetically distinct disorders.

Autosomal dominant inheritance is described in about 20% of all nonsyndromic hearing loss with currently 54 distinct loci (DFNA1-54), and >20 different genes identified. Seven different unconventional myosin genes are involved in ten different types of syndromic and nonsyndromic hearing loss with different patterns of inheritance: MYO7A in DFNA11/DFNB2/USH1B, MYH9 in DFNA17, MYH14 in DFNA4, MYO6 in DFNA22/DFNB37, MYO3A in DFNB30, MYO1A in DFNA48, and MYO15A in DFNB3. Two missense mutations in MYO6 (p.C442Y and p.H246R) have been characterized in families of Italian and American Caucasian extraction with autosomal dominant hearing loss, respectively, and the latter was associated with cardiomyopathy in some patients. Three Pakistani families had homozygosity for three MYO6 mutations (c.36insT, p.R1166X, and p.E216V, respectively), and was in one instance associated with retinal degeneration. In the present study, we linked autosomal dominant hearing loss in a large Danish family to a 38.9 Mb interval overlapping with the DFNA22/DFNB37 locus on chromosome 6q13. A novel nonsense mutation in MYO6 exon 25 (c.2545C > T; p.R849X) was identified in the family. The mutation co-segregated with the disease and the mutant allele is predicted to encode a truncated protein lacking the coiled-coil and globular tail domains. These domains are hypothesized to be essential for targeting myosin VI to its cellular compartments. No other system was involved indicating nonsyndromic loss. In conclusion, a novel nonsense MYO6 mutation causes post-lingual, slowly progressive autosomal dominant nonsyndromic moderate to severe hearing loss in a Danish family., (Copyright 2008 Wiley-Liss, Inc.)

Myopia, or short-sightedness, is the most common form of vision disorder worldwide. Higher levels of myopia, usually defined as an axial eye length of >26 mm or a refractive error of < -5.00 diopters are often designated as 'pathologic' myopia, because of the predisposition to develop further eye disorders such as retinal detachment, macular degeneration, cataract, or glaucoma. Many distinct forms of autosomal dominant non-syndromic high-grade myopia are described in humans. While the underlying chromosomal locations and critical disease intervals have been identified and located to physical map positions, the gene defects and causative mutations responsible for autosomal dominant myopia remain elusive to date. Examination of a German six-generation kindred by 10K whole genome chips led to the identification of a 19-cM map segment as being the most likely familial myopia candidate region spanning from chromosomal band 12q14.3 to 12q21.31 (MYP3). In our family, a maximum multi-point LOD score of 3.9 was obtained between rs1373877 and rs717996. The recombination breakpoints in this family and the interval of the originally reported German/Italian family defining the MYP3 locus on chromosome 12 (OMIM 603221, two-point LOD score 3.85 for markers D12S1706 and D12S327 at 12q21-23) allowed us to significantly refine a minimum consensus region. This new composite region is located between microsatellite marker D12S1684 at 75.8 K and SNP&#95;A-1509586 (alias rs717996) at position 82,636,288 bp, and narrows the original 30.1 cM of the MYP3 interval to 6.8 cM. The refined MYP3 interval allowed us to restrict the list of database-indexed genes to 25, several of which are promising MYP3 candidates based on similarities with genes and proteins involved in vision physiology and eye disease. While autosomal dominant high-grade myopia is recognized to be genetically heterogeneous, our results suggest genetic homogeneity of the MYP3-based condition in families that share the same ethnic and geographical background. The future identification of this MYP3 gene may provide insights into the pathophysiology of myopia and eye development.

Many genetic diseases have been linked to the dysfunction of primary cilia, which occur nearly ubiquitously in the body and act as solitary cellular mechanosensory organelles. The list of clinical manifestations and affected tissues in cilia-related disorders (ciliopathies) such as nephronophthisis is broad and has been attributed to the wide expression pattern of ciliary proteins. However, little is known about the molecular mechanisms leading to this dramatic diversity of phenotypes. We recently reported hypomorphic NPHP3 mutations in children and young adults with isolated nephronophthisis and associated hepatic fibrosis or tapetoretinal degeneration. Here, we chose a combinatorial approach in mice and humans to define the phenotypic spectrum of NPHP3/Nphp3 mutations and the role of the nephrocystin-3 protein. We demonstrate that the pcy mutation generates a hypomorphic Nphp3 allele that is responsible for the cystic kidney disease phenotype, whereas complete loss of Nphp3 function results in situs inversus, congenital heart defects, and embryonic lethality in mice. In humans, we show that NPHP3 mutations can cause a broad clinical spectrum of early embryonic patterning defects comprising situs inversus, polydactyly, central nervous system malformations, structural heart defects, preauricular fistulas, and a wide range of congenital anomalies of the kidney and urinary tract (CAKUT). On the functional level, we show that nephrocystin-3 directly interacts with inversin and can inhibit like inversin canonical Wnt signaling, whereas nephrocystin-3 deficiency leads in Xenopus laevis to typical planar cell polarity defects, suggesting a role in the control of canonical and noncanonical (planar cell polarity) Wnt signaling.

Several years ago, autosomal dominant myofibrillar myopathy (MFM) in combination with arrhythmogenic right ventricular cardiomyopathy (ARVC7) was tentatively mapped to a 10.6-Mbp (million base pairs) region on chromosome 10q22.3 between D10S605 (78.9 Mbp) and D10S215 (89.5 Mbp) in a Swedish family assuming that ARVC7 was allelic with cardiomyopathy, dilated 1C (CMD1C). To date, neither the genetic defect in ARVC7 nor CMD1C has been reported. In a comprehensive follow-up study we re-examined and confirmed the previous linkage data for ARVC7 using a high-density single nucleotide polymorphism marker panel from Affymetrix (Human Mapping 10K Array). No other regions with significant evidence for linkage were discovered. The critical interval was narrowed down to 4.27 Mbp between D10S1645 and D10S1786. This reduced the total number of candidate genes to 18 of which 17 (RAI17, PPIF, C10ORF56, SFTPA1, SFTPA2, SFTPA1B, SFTPA2B, SFTPD, C10ORF57, PLAC9, ANXA11, MAT1A, DYDC1, DYDC2, C10ORF58, TSPAN14 and SH2D4B) are shared with the CMD1C region. No disease-causing mutation was found in their coding regions. Moreover, metavinculin (VCL) and ZASP/cypher (LDB3) proximal and distal to this linked region were excluded by sequence analysis. To search for submicroscopic and intragenic deletions by PCR, we generated hybrid cell lines carrying only the affected or normal chromosome 10 homolog. All sequence tagged sites and exons were present on both homologs. We speculate that regulatory mutations in 1 of the 18 genes from 10q22.3 are responsible for a heterogenous spectrum of clinically distinct myodegenerative disorders, affecting both skeletal and cardiac muscles to variable degrees.

Unobstructed vision requires a particular refractive index of the lens, a measure based on the organization of the structural proteins within the differentiated lens cells. To ensure an intact lens structure, homeostasis within the lens cells is indispensable. Alterations of the lens structure result in opacity and lead to cataract. Renal glucosuria is defined by elevated glucose level in the urine without hyperglycemia and without evidence of morphological renal anomalies. In a Swiss family with autosomal dominant juvenile cataract, microcornea, and renal glucosuria, we have identified a nonsense mutation in a member of the carboxylic acid transporter family SLC16. The underlying gene defect in SLC16A12 resides within a 3 cM region on chromosome 10q23.13 defined by linkage mapping of this phenotype. We found tissue-specific variability of SLC16A12 transcript levels in control samples, with high expression in the eye and kidney, the two organs affected by this syndrome. This report demonstrates biological relevance of this solute carrier. We hypothesize that SLC16A12 is important for lens and kidney homeostasis and discuss its potential role in age-related cataract.

The outcome of an udder infection is influenced by the pathogen species. We established a strictly defined infection model to better analyze the unknown molecular causes for these pathogen-specific effects, using Escherichia coli and Staphylococcus aureus strains previously asseverated from field cases of mastitis. Inoculation of quarters with 500 CFU of E. coli (n = 4) was performed 6 h, 12 h, and 24 h before culling. All animals showed signs of acute clinical mastitis 12 h after challenge: increased somatic cell count (SCC), decreased milk yield, leukopenia, fever, and udder swelling. Animals inoculated with 10 000 CFU of S. aureus for 24 h (n = 4) showed no or only modest clinical signs of mastitis. However, S. aureus caused clinical signs in animals, inoculated for 72 h-84 h. Real-time PCR proved that E. coli inoculation strongly and significantly upregulated the expression of beta-defensins, TLR2 and TLR4 in the pathogen inoculated udder quarters as well as in mammary lymph nodes. TLR3 and TLR6 were not significantly regulated by the infections. Immuno-histochemistry identified mammary epithelial cells as sites for the upregulated TLR2 and beta-defensin expression. S. aureus, in contrast, did not significantly regulate the expression of any of these genes during the first 24 h after pathogen inoculation. Only 84 h after inoculation, the expression of beta-defensins, but not of TLRs was significantly (> 20 fold) upregulated in five out of six pathogen inoculated quarters. Using the established mastitis model, the data clearly demonstrate a pathogen-dependent difference in the time kinetics of induced pathogen receptors and defense molecules.