Gain-of-function mutation of microRNA-140 in human skeletal dysplasia.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression. Heterozygous loss-of-function point mutations of miRNA genes are associated with several human congenital disorders1-5, but neomorphic (gain-of-new-function) mutations in miRNAs due to nucleotide substitutions have not been reported. Here we describe a neomorphic seed region mutation in the chondrocyte-specific, super-enhancer-associated MIR140 gene encoding microRNA-140 (miR-140) in a novel autosomal dominant human skeletal dysplasia. Mice with the corresponding single nucleotide substitution show skeletal abnormalities similar to those of the patients but distinct from those of miR-140-null mice6. This mutant miRNA gene yields abundant mutant miR-140-5p expression without miRNA-processing defects. In chondrocytes, the mutation causes widespread derepression of wild-type miR-140-5p targets and repression of mutant miR-140-5p targets, indicating that the mutation produces both loss-of-function and gain-of-function effects. Furthermore, the mutant miR-140-5p seed competes with the conserved RNA-binding protein Ybx1 for overlapping binding sites. This finding may explain the potent target repression and robust in vivo effect by this mutant miRNA even in the absence of evolutionary selection of miRNA-target RNA interactions, which contributes to the strong regulatory effects of conserved miRNAs7,8. Our study presents the first case of a pathogenic gain-of-function miRNA mutation and provides molecular insight into neomorphic actions of emerging and/or mutant miRNAs.

Patients' Attitudes Towards Disclosure of Genetic Test Results to Family Members: The Impact of Patients' Sociodemographic Background and Counseling Experience.

Many factors predict the intention to disclose genetic information to relatives. The article examines the impact of patients' socio-demographic factors on their intention to disclose genetic testing results to their relatives. Data were collected in eight genetic clinics in Israel. Patients were requested to fill in a questionnaire after counseling. A convenience sample of 564 participants who visited these clinics was collected for a response rate of 85 %. Of them, 282 participants came for susceptibility testing for hereditary cancers (cancer group), and 282 for genetic screening tests (prenatal group). In the cancer group, being secular and having more years of education correlated positively with the intention to disclose test results to relatives. In the prenatal group, being married and female correlated positively with the intention to disclose. In the cancer group, being religious and with less years of education correlated positively with the view that the clinician should deliver the results to the family. In the prenatal group, being male and unmarried correlated positively with this belief. In both groups, being of young age correlated with the perception that genetic information is private. Varied sociodemographic factors affect the intention to inform family members. Thus, knowing the social background of patients will shed light on people's attitudes to genetic information and will help clinicians provide effective counseling in discussions with patients about the implications of test results for relatives.

Novel SMARCAL1 bi-allelic mutations associated with a chromosomal breakage phenotype in a severe SIOD patient.

PURPOSE: Chromosomal instability syndromes include a group of rare diseases characterized by defective DNA-damage-response and increased risk of chromosomal breakage. Patients display defects in the recognition and/or repair of DNA damage, with a subsequent high rate of malignancies and abnormal gene rearrangements. Other clinical manifestations, such as immunodeficiency, neurodevelopmental delay and skeletal abnormalities, are present in some of these syndromes. We studied a patient with profound T-lymphocyte defect, neurodevelopmental delay, facial dysmorphism, nephrotic syndrome and spondyloepiphyseal bone dysplasia typical of SIOD. METHODS: Karyotype and chromosome fragility assays on patients' peripheral blood mononuclear cells showed an abnormal rate of spontaneous breaks. Cell cycle analysis of patient's fibroblasts following replication stress induced by hydroxyhurea revealed a delay in their release from S-phase to G2. When using higher concentrations of hydroxyhurea no patient fibroblast colonies could survive, compared with control fibroblasts. Whole-exome sequencing revealed novel compound heterozygote mutations in SMARCAL1 gene, resulting in putative frame shifts of encoded SMARCAL1 from each allele and no detected protein in patient's cells. The patient's youngest brother was found to have similar manifestations of SIOD but of less severity, including short stature, facial dysmorphism and typical osseous dysplasia, but no clinical findings suggestive of immunodeficiency and no chromosomal fragility. Similar to his sister, the brother carries both bi-allelic mutations in SMARCAL1 gene. CONCLUSIONS: We present here the first evidence of intrinsic chromosomal instability in a severe SMARCAL1-deficient patient with a clinical picture of SIOD. Our results are consistent with the recently outlined role of SMARCAL1 protein in DNA damage response.

BRCA1/2 mutations and FMR1 alleles are randomly distributed: a case control study.

BRCA mutation carriers were reported to display a skewed distribution of FMR1 genotypes, predominantly within the low normal range (CGG repeat number <26). This observation led to the interpretation that BRCA1/2 mutations are embryo-lethal, unless rescued by 'low FMR1 alleles'. We undertook to re-explore the distribution of FMR1 alleles subdivided into low, normal and high (<26, 26-34, and >34 CGG repeats, respectively) subgenotypes, on a cohort of 125 Ashkenazi women, carriers of a BRCA1/2 founder mutation. Ashkenazi healthy females (n=368), tested in the frame of the Israeli screening population program, served as controls. BRCA1/2 carriers and controls demonstrated a comparable and non-skewed FMR1 subgenotype distribution. Taken together, using a homogeneous ethnic group of Ashkenazi BRCA1/2 mutation carriers, we could not confirm the reported association between FMR1 low genotypes and BRCA1/2 mutations. The notion that BRCA1/2 mutations are embryo-lethal unless rescued by the low FMR1 subgenotypes is hereby refuted.

Morbid obesity resulting from inactivation of the ciliary protein CEP19 in humans and mice.

Obesity is a major public health concern, and complementary research strategies have been directed toward the identification of the underlying causative gene mutations that affect the normal pathways and networks that regulate energy balance. Here, we describe an autosomal-recessive morbid-obesity syndrome and identify the disease-causing gene defect. The average body mass index of affected family members was 48.7 (range = 36.7-61.0), and all had features of the metabolic syndrome. Homozygosity mapping localized the disease locus to a region in 3q29; we designated this region the morbid obesity 1 (MO1) locus. Sequence analysis identified a homozygous nonsense mutation in CEP19, the gene encoding the ciliary protein CEP19, in all affected family members. CEP19 is highly conserved in vertebrates and invertebrates, is expressed in multiple tissues, and localizes to the centrosome and primary cilia. Homozygous Cep19-knockout mice were morbidly obese, hyperphagic, glucose intolerant, and insulin resistant. Thus, loss of the ciliary protein CEP19 in humans and mice causes morbid obesity and defines a target for investigating the molecular pathogenesis of this disease and potential treatments for obesity and malnutrition.

Exome sequencing identifies INPPL1 mutations as a cause of opsismodysplasia.

Opsismodysplasia (OPS) is a severe autosomal-recessive chondrodysplasia characterized by pre- and postnatal micromelia with extremely short hands and feet. The main radiological features are severe platyspondyly, squared metacarpals, delayed skeletal ossification, and metaphyseal cupping. In order to identify mutations causing OPS, a total of 16 cases (7 terminated pregnancies and 9 postnatal cases) from 10 unrelated families were included in this study. We performed exome sequencing in three cases from three unrelated families and only one gene was found to harbor mutations in all three cases: inositol polyphosphate phosphatase-like 1 (INPPL1). Screening INPPL1 in the remaining cases identified a total of 12 distinct INPPL1 mutations in the 10 families, present at the homozygote state in 7 consanguinous families and at the compound heterozygote state in the 3 remaining families. Most mutations (6/12) resulted in premature stop codons, 2/12 were splice site, and 4/12 were missense mutations located in the catalytic domain, 5-phosphatase. INPPL1 belongs to the inositol-1,4,5-trisphosphate 5-phosphatase family, a family of signal-modulating enzymes that govern a plethora of cellular functions by regulating the levels of specific phosphoinositides. Our finding of INPPL1 mutations in OPS, a severe spondylodysplastic dysplasia with major growth plate disorganization, supports a key and specific role of this enzyme in endochondral ossification.

A system for exact and approximate genetic linkage analysis of SNP data in large pedigrees.

MOTIVATION: The use of dense single nucleotide polymorphism (SNP) data in genetic linkage analysis of large pedigrees is impeded by significant technical, methodological and computational challenges. Here we describe Superlink-Online SNP, a new powerful online system that streamlines the linkage analysis of SNP data. It features a fully integrated flexible processing workflow comprising both well-known and novel data analysis tools, including SNP clustering, erroneous data filtering, exact and approximate LOD calculations and maximum-likelihood haplotyping. The system draws its power from thousands of CPUs, performing data analysis tasks orders of magnitude faster than a single computer. By providing an intuitive interface to sophisticated state-of-the-art analysis tools coupled with high computing capacity, Superlink-Online SNP helps geneticists unleash the potential of SNP data for detecting disease genes. RESULTS: Computations performed by Superlink-Online SNP are automatically parallelized using novel paradigms, and executed on unlimited number of private or public CPUs. One novel service is large-scale approximate Markov Chain-Monte Carlo (MCMC) analysis. The accuracy of the results is reliably estimated by running the same computation on multiple CPUs and evaluating the Gelman-Rubin Score to set aside unreliable results. Another service within the workflow is a novel parallelized exact algorithm for inferring maximum-likelihood haplotyping. The reported system enables genetic analyses that were previously infeasible. We demonstrate the system capabilities through a study of a large complex pedigree affected with metabolic syndrome. AVAILABILITY: Superlink-Online SNP is freely available for researchers at http://cbl-hap.cs.technion.ac.il/superlink-snp. The system source code can also be downloaded from the system website. CONTACT: omerw@cs.technion.ac.il SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Short stature, onychodysplasia, facial dysmorphism, and hypotrichosis syndrome is caused by a POC1A mutation.

Disproportionate short stature refers to a heterogeneous group of hereditary disorders that are classified according to their mode of inheritance, clinical skeletal and nonskeletal manifestations, and radiological characteristics. In the present study, we report on an autosomal-recessive osteocutaneous disorder that we termed SOFT (short stature, onychodysplasia, facial dysmorphism, and hypotrichosis) syndrome. We employed homozygosity mapping to locate the disease-causing mutation to region 3p21.1-3p21.31. Using whole-exome-sequencing analysis complemented with Sanger direct sequencing of poorly covered regions, we identified a homozygous point mutation (c.512T>C [p.Leu171Pro]) in POC1A (centriolar protein homolog A). This mutation was found to cosegregate with the disease phenotype in two families. The p.Leu171Pro substitution affects a highly conserved amino acid residue and is predicted to interfere with protein function. Poc1, a POC1A ortholog, was previously found to have a role in centrosome stability in unicellular organisms. Accordingly, although centrosome structure was preserved, the number of centrosomes and their distribution were abnormal in affected cells. In addition, the Golgi apparatus presented a dispersed morphology, cholera-toxin trafficking from the plasma membrane to the Golgi was aberrant, and large vesicles accumulated in the cytosol. Collectively, our data underscore the importance of POC1A for proper bone, hair, and nail formation and highlight the importance of normal centrosomes in Golgi assembly and trafficking from the plasma membrane to the Golgi apparatus.

A distinctive autosomal recessive syndrome of severe disproportionate short stature with short long bones, brachydactyly, and hypotrichosis in two consanguineous Arab families.

Disproportionate short stature is a heterogeneous group of hereditary disorders, which are classified according to their mode of inheritance, their clinical skeletal and non-skeletal manifestations, and their radiological characteristics. Herein, we inform on eight individuals with severe disproportionate short stature from two unrelated consanguineous families of Arab-Muslim ancestry. The adult height of the affected individuals is between 112 cm and 127 cm, and is due to pre- and post-natal growth retardation, which probably manifests as early as the second trimester of pregnancy. At a young age, the phenotype is characterized by a short stature, a relatively large head, and a long triangular face, and this phenotype later evolves to one with in which the head is relatively small, the mandible is large and pointy. The affected individuals have normal cognitive abilities and lack any neurological deficits. Other typical features include a prominent nose, a voice with an unusual high-pitched sound, relatively small ears, clinodactyly, brachydactyly, small hands, hypoplastic fingernails, a waddling gait, and sparse hair post-pubertally. Typical skeletal changes include short long bones, especially the femurs and humeri, with mild metaphyseal changes and very short femoral necks. After due consideration of the other hereditary causes of disproportionate short stature and close examination of the pedigrees of the two families, we concluded that these eight individuals have the same hitherto unreported form of severe disproportionate short stature that is inherited in the autosomal recessive mode.

[60 years of medical genetics in Israel].

The principle deeds of genetics in Israel consist of a wide array of disciplines including agriculture, nutrients, biotechnology, pharmacology and pharmacogenetics, pertaining to criminal as well as medical aspects. In the scope of this state of the art historical review, the authors emphasize the medical issues. The initial stimulus for genetic studies and medical awareness among the various ethnic populations in Israel was the immigration, in the early 1950s, of over a million Jewish immigrants from more than 100 countries from all continents. It was soon recognized that frequencies of genetic diseases differed markedly among the various communities, serving as a trigger for studying and managing these populations. In this state of the art historical review, particular emphasize was given to the historical events concerning genetics in the land of Israel, as well as in the state of Israel. Highlights of genetic diversity of the various ethnic and sub-populations are added, along with the advances and major achievements of the human genetics discipline in the state of Israel.

Myotonia congenita in a large consanguineous Arab family: insight into the clinical spectrum of carriers and double heterozygotes of a novel mutation in the chloride channel CLCN1 gene.

The aims of this study were to (1) characterize the clinical phenotype, (2) define the causative mutation, and (3) correlate the clinical phenotype with genotype in a large consanguineous Arab family with myotonia congenita. Twenty-four family members from three generations were interviewed and examined. Genomic DNA was extracted from peripheral blood samples for sequencing the exons of the CLCN1 gene. Twelve individuals with myotonia congenita transmitted the condition in an autosomal dominant manner with incomplete penetrance. A novel missense mutation [568GG>TC (G190S)] was found in a dose-dependent clinical phenotype. Although heterozygous individuals were asymptomatic or mildly affected, the homozygous individuals were severely affected. The mutation is a glycine-to-serine residue substitution in a well-conserved motif in helix D of the CLC-1 chloride channel in the skeletal muscle plasmalemma. A novel mutation, 568GG>TC (G190S) in the CLCN1 gene, is responsible for autosomal dominant myotonia congenita with a variable phenotypic spectrum.

Mutations in the heparan-sulfate proteoglycan glypican 6 (GPC6) impair endochondral ossification and cause recessive omodysplasia.

Glypicans are a family of glycosylphosphatidylinositol (GPI)-anchored, membrane-bound heparan sulfate (HS) proteoglycans. Their biological roles are only partly understood, although it is assumed that they modulate the activity of HS-binding growth factors. The involvement of glypicans in developmental morphogenesis and growth regulation has been highlighted by Drosophila mutants and by a human overgrowth syndrome with multiple malformations caused by glypican 3 mutations (Simpson-Golabi-Behmel syndrome). We now report that autosomal-recessive omodysplasia, a genetic condition characterized by short-limbed short stature, craniofacial dysmorphism, and variable developmental delay, maps to chromosome 13 (13q31.1-q32.2) and is caused by point mutations or by larger genomic rearrangements in glypican 6 (GPC6). All mutations cause truncation of the GPC6 protein and abolish both the HS-binding site and the GPI-bearing membrane-associated domain, and thus loss of function is predicted. Expression studies in microdissected mouse growth plate revealed expression of Gpc6 in proliferative chondrocytes. Thus, GPC6 seems to have a previously unsuspected role in endochondral ossification and skeletal growth, and its functional abrogation results in a short-limb phenotype.

Dynamic modification strategy of the Israeli carrier screening protocol: inclusion of the Oriental Jewish Group to the cystic fibrosis panel.

PURPOSE: A retrospective population study was conducted to determine the carrier frequencies of recently identified mutations in Oriental Jewish cystic fibrosis patients. METHODS: Data were collected from 10 medical centers that screened the following mutations: two splice site mutations-3121-1G>A and 2751 + 1insT-and one nonsense mutation-the Y1092X in Iraqi Jews. One missense mutation, I1234V, was screened in Yemenite Jews. RESULTS: A total of 2499 Iraqi Jews were tested for one, two, or all three mutations. The 3121-1G>A, Y1092X, and 2751 + 1insT mutations had a carrier frequency of 1:68.5, 1:435, and 0, respectively. In 1435 Yemenite Jews screened, I1234V had a carrier frequency of 1:130. CONCLUSION: The 0.84% allele frequency of the three Iraqi founder mutations falls within the Israeli Society of Medical Geneticists' inclusion criteria for screening of 1:60 carrier frequency; hence, Iraqi Jews were added to the carrier screening policy with a panel including the three Iraqi founder mutations in addition to the five Ashkenazi mutations previously detected in Eastern Jews. 2751 + 1insT that was detected in patients only was included in the screening panel to increase the detection rate. I1234V does not meet the inclusion criteria but is now offered on a diagnostic basis and can be added to the screening panel for individuals whose mixed origin includes Yemenite, in addition to protocol-recommended origins. This study demonstrates the dynamic modifications of the Israeli carrier cystic fibrosis screening protocol based on newly detected founder mutations in a large cohort, taking into account mutation impact and intercommunal admixture.

Mutations in DDR2 gene cause SMED with short limbs and abnormal calcifications.

The spondylo-meta-epiphyseal dysplasia [SMED] short limb-hand type [SMED-SL] is a rare autosomal-recessive disease, first reported by Borochowitz et al. in 1993.(1) Since then, 14 affected patients have been reported.(2-5) We diagnosed 6 patients from 5 different consanguineous Arab Muslim families from the Jerusalem area with SMED-SL. Additionally, we studied two patients from Algerian and Pakistani ancestry and the parents of the first Jewish patients reported.(1) Using a homozygosity mapping strategy, we located a candidate region on chromosome 1q23 spanning 2.4 Mb. The position of the Discoidin Domain Receptor 2 (DDR2) gene within the candidate region and the similarity of the ddr2 knockout mouse to the SMED patients' phenotype prompted us to study this gene(6). We identified three missense mutations c.2254 C > T [R752C], c. 2177 T > G [I726R], c.2138C > T [T713I] and one splice site mutation [IVS17+1g > a] in the conserved sequence encoding the tyrosine kinase domain of the DDR2 gene. The results of this study will permit an accurate early prenatal diagnosis and carrier screening for families at risk.

SERKAL syndrome: an autosomal-recessive disorder caused by a loss-of-function mutation in WNT4.

The WNT-signaling pathway plays a major role during mammalian embryogenesis. We report a novel autosomal-recessive syndrome that consists of female to male sex reversal and renal, adrenal, and lung dysgenesis and is associated with additional developmental defects. Using a candidate-gene approach, we identified a disease-causing homozygous missense mutation in the human WNT4 gene. The mutation was found to result in markedly reduced WNT4 mRNA levels in vivo and in vitro and to downregulate WNT4-dependent inhibition of beta-catenin degradation. Taken together with previous observations in animal models, the present data attribute a pivotal role to WNT4 signaling during organogenesis in humans.

Language in boys with fragile X syndrome.

The current paper reports of language production in 15 Hebrew-speaking boys, aged 9;0-13;0, with fully methylated, non-mosaic fragile X syndrome and no concomitant diagnosis of autism. Contrary to expectations, seven children were non-verbal. Language production in the verbal children was studied in free conversations and in context-bound speech. Despite extra caution in calculating MLU, participants' language level was not predicted by mean utterance length. Context bound speech resulted in grammatically more advanced performance than free conversation, and performance in both contexts differed in important ways from performance of typically developing MLU-matched controls. The relevance of MLU as a predictor of productive grammar in disordered populations is briefly discussed.

Molecular epidemiology of hereditary epidermolysis bullosa in a Middle Eastern population.

Epidermolysis bullosa (EB) encompasses a large group of inherited blistering skin disorders caused by mutations in at least 10 genes. Numerous studies, mainly performed in European and US families with EB, have revealed a number of characteristic epidemiological and genetic features, which form the basis for current diagnostic and counseling strategies. However, little is currently known about the molecular epidemiology of EB in Middle East populations. In the present study, we assessed 55 EB families for pathogenic sequence alterations in the 10 genes known to be associated with EB. Our results show unique EB subtype distribution and patterns of inheritance in our cohort. We also failed to detect recurrent mutations frequently encountered in Europe and the US, and did not consistently observe genotype-phenotype correlations formerly established in Western populations. Thus, the molecular epidemiology of EB in the Middle East is significantly different from that previously delineated in Europe and the US. Our data raise the possibility that similar differences may also be found in other genetically heterogeneous groups of disorders, and indicate the need for population-specific diagnostic and management approaches.

Autosomal recessive Alport syndrome: an in-depth clinical and molecular analysis of five families.

BACKGROUND: Alport syndrome (ATS) is a progressive inherited nephropathy characterized by irregular thinning, thickening and splitting of the glomerular basement membrane (GBM) often associated with hearing loss and ocular symptoms. ATS has been shown to be caused by COL4A5 mutations in its X-linked form and by COL4A3 and COL4A4 mutations in its autosomal forms. METHODS: Five families with a suspicion of ATS were investigated both from a clinical and molecular point of view. COL4A3 and COL4A4 genes were analysed by DHPLC. Automated sequencing was performed to identify the underlying mutation. RESULTS: Molecular analysis indicated that in all 5 cases the correct diagnosis was autosomal recessive ATS. In three families in which parental consanguinity clearly pinpointed to autosomal recessive ATS, we found COL4A4 homozygous mutations in two of them and COL4A3 homozygous mutation in the other one. In the remaining two families a differential diagnosis including X-linked ATS, autosomal recessive ATS and thin basement membrane nephropathy was considered. The molecular analysis demonstrated that the probands were genetic compounds for two different mutations in the COL4A4 gene pinpointing to the correct diagnosis of autosomal recessive ATS. CONCLUSIONS: A clinical evaluation of probands and their relatives of the five families carrying mutations in either the COL4A3 or the COL4A4 gene was carried out to underline the natural history of the autosomal recessive ATS. In addition, this paper stresses the complexity of the clinics and genetics of ATS and how a correct diagnosis is based on a combination of: (i) an in-depth clinical investigation; (ii) a detailed formal genetic analysis; (iii) a correct technical choice of the gene to be investigated; (iv) a correct technical choice of the family member to be included in the mutational screening. A correct diagnosis is the basis for an appropriate genetic counselling dealing with both the correct prognosis and the accurate recurrence risk for the patients and family members.