Search

Item does not contain fulltext p63 mutations have been associated with EEC syndrome (ectrodactyly, ectodermal dysplasia, and cleft lip/palate), as well as with nonsyndromic split hand-split foot malformation (SHFM). We performed p63 mutation analysis in a sample of 43 individuals and families affected with EEC syndrome, in 35 individuals affected with SHFM, and in three families with the EEC-like condition limb-mammary syndrome (LMS), which is characterized by ectrodactyly, cleft palate, and mammary-gland abnormalities. The results differed for these three conditions. p63 gene mutations were detected in almost all (40/43) individuals affected with EEC syndrome. Apart from a frameshift mutation in exon 13, all other EEC mutations were missense, predominantly involving codons 204, 227, 279, 280, and 304. In contrast, p63 mutations were detected in only a small proportion (4/35) of patients with isolated SHFM. p63 mutations in SHFM included three novel mutations: a missense mutation (K193E), a nonsense mutation (Q634X), and a mutation in the 3' splice site for exon 5. The fourth SHFM mutation (R280H) in this series was also found in a patient with classical EEC syndrome, suggesting partial overlap between the EEC and SHFM mutational spectra. The original family with LMS (van Bokhoven et al. 1999) had no detectable p63 mutation, although it clearly localizes to the p63 locus in 3q27. In two other small kindreds affected with LMS, frameshift mutations were detected in exons 13 and 14, respectively. The combined data show that p63 is the major gene for EEC syndrome, and that it makes a modest contribution to SHFM. There appears to be a genotype-phenotype correlation, in that there is a specific pattern of missense mutations in EEC syndrome that are not generally found in SHFM or LMS.

Congenital contracture syndromes affect 1 out of every 1000 live births, and of those syndromes, distal arthrogryposis (DA), characterized by contractures of the hands and feet, is the most predominant. In one subtype of DA, Freeman Sheldon Syndrome (FSS), 97% of the cases are caused by mutations in the embryonic myosin heavy chain gene, MYH3. To assess the effects of this mutation on adult muscle contractility, skeletal muscle was obtained from a needle biopsy of the gastrocnemius muscle in an FSS individual (MYH3 R672C) and a control subject were performed and skinned single muscle fibers were dissected for measurements of contractile performance as the [Ca2+] of physiological solutions was varied. The magnitude of passive stiffness was 2x greater for patient fibers. There was no difference in maximal Ca2+ activated force found in the affected adult muscle fibers (0.204uN ± 0.044) compared to normal adult muscle fibers (0.259uN ± 0.028). However specific force was 69% less; this was attributable to hypertrophy of the patient fibers (159um ± 8 as compared to normal control myofibers of 87um ± 3). Little to no change was observed in Ca2+ sensitivity (pCa50) or in cooperativity of the force-pCa relationship. Relaxation was dramatically slower in patient fibers, taking 4x longer to reach 50% relaxation and 10x longer to reach 90% relaxation. Control experiments suggested this is not due to the larger patient fiber size. Preliminary analysis, using a 12.5% agarose gel, and Western Blots, indicated that these differences were not fiber type dependent. Interestingly, we have identified that embryonic myosin (MYH3) is present in single adult muscle cells. This work was supported by HL65497 (Regnier) and HD48895 (Bamshad).

If CC chemokine receptor 5 (CCR5)-dependent mechanisms at the time of initial virus exposure are important determinants of virus entry and disease outcome, then the polymorphisms in CCR5 that influence risk of transmission and disease progression should be similar; this hypothesis was tested in a cohort of 649 Argentinean children exposed perinatally to human immunodeficiency virus type 1 (HIV-1). Two lines of evidence support this hypothesis. First, CCR5 haplotype pairs associated with enhanced risk of transmission were the chief predictors of a faster disease course. Second, some of the haplotype pairs associated with altered rates of transmission and disease progression in children were similar to those that we previously found influenced outcome in European American adults. This concordance suggests that CCR5 haplotypes may serve as genetic rheostats that influence events occurring shortly after initial virus exposure, dictating not only virus entry but, by extension, also the extent of early viral replication.

EEC syndrome is an autosomal dominant disorder characterized by ectrodactyly, ectodermal dysplasia, and facial clefts. We have mapped the genetic defect in several EEC syndrome families to a region of chromosome 3q27 previously implicated in the EEC-like disorder, limb mammary syndrome (LMS). Analysis of the p63 gene, a homolog of p53 located in the critical LMS/EEC interval, revealed heterozygous mutations in nine unrelated EEC families. Eight mutations result in amino acid substitutions that are predicted to abolish the DNA binding capacity of p63. The ninth is a frameshift mutation that affects the p63 alpha, but not p63 beta and p63 gamma isotypes. Transactivation studies with these mutant p63 isotypes provide a molecular explanation for the dominant character of p63 mutations in EEC syndrome.

Variation in human skin/hair pigmentation is due to varied amounts of eumelanin (brown/black melanins) and phaeomelanin (red/yellow melanins) produced by the melanocytes. The melanocortin 1 receptor (MC1R) is a regulator of eu- and phaeomelanin production in the melanocytes, and MC1R mutations causing coat color changes are known in many mammals. We have sequenced the MC1R gene in 121 individuals sampled from world populations with an emphasis on Asian populations. We found variation at five nonsynonymous sites (resulting in the variants Arg67Gln, Asp84Glu, Val92Met, Arg151Cys, and Arg163Gln), but at only one synonymous site (A942G). Interestingly, the human consensus protein sequence is observed in all 25 African individuals studied, but at lower frequencies in the other populations examined, especially in East and Southeast Asians. The Arg163Gln variant is absent in the Africans studied, almost absent in Europeans, and at a low frequency (7%) in Indians, but is at an exceptionally high frequency (70%) in East and Southeast Asians. The MC1R gene in common and pygmy chimpanzees, gorilla, orangutan, and baboon was sequenced to study the evolution of MC1R. The ancestral human MC1R sequence is identical to the human consensus protein sequence, while MC1R varies considerably among higher primates. A comparison of the rates of substitution in genes in the melanocortin receptor family indicates that MC1R has evolved the fastest. In addition, the nucleotide diversity at the MC1R locus is shown to be several times higher than the average nucleotide diversity in human populations, possibly due to diversifying selection.

A major goal of biology has been to understand the developmental mechanisms behind evolutionary trends. This has led to a growing interest in studying the molecular basis of the evolution of developmental programs such as those mediating the diversification of tetrapod limbs. Over the last 10 y, it has become clear that the genes and general developmental programs used to build a limb are strongly conserved among widely disparate species. This finding suggests that altered regulation of the timing and locations of developmental events may be responsible for the morphologic variation observed among some species. However, genetic analyses of the regulatory regions of genes controlling vertebrate developmental programs are very limited. Characterization of the genetic basis of human birth defects of the limb provides an opportunity to dissect the developmental programs used to modify the architecture of the hominoid limb. This may allow us to assess the relative contributions of altered gene regulation to morphologic variation among species and reconstruct the evolutionary history of the hominid limb. Such insight is also important because morphologic differences in the hominid upper limb have been correlated with the use of tools, and tool making is often regarded as the milestone that marked the emergence of the genus Homo.

Scientists have long been interested in understanding how social processes modulate evolutionary forces1. A good example of this is the intensively studied Hindu caste system, which governs the mating practices of nearly one-sixth of the world's population2,3. However, there is controversy concerning the effect of social stratification on the genetic structure of caste communities. Here we show that differences in social rank between castes correspond to mitochondrial DNA (mtDNA) distances between castes but not genetic distances estimated from Y-chromosome data.

To examine the signature of population expansion on genetic variability at microsatellite loci, we consider a population that evolves according to the time-continuous Moran model, with growing population size and mutations that follow a general asymmetric stepwise mutation model. We present calculations of expected allele-size variance and homozygosity at a locus in such a model for several variants of growth, including stepwise, exponential, and logistic growth. These calculations in particular prove that population bottleneck followed by growth in size causes an imbalance between allele size variance and heterozygosity, characterized by the variance being transiently higher than expected under equilibrium conditions. This effect is, in a sense, analogous to that demonstrated before for the infinite allele model, where the number of alleles transiently increases after a stepwise growth of population. We analyze a set of data on tetranucleotide repeats that reveals the imbalance expected under the assumption of bottleneck followed by population growth in two out of three major racial groups. The imbalance is strongest in Asians, intermediate in Europeans, and absent in Africans. This finding is consistent with previous findings by others concerning the population expansion of modern humans, with the bottleneck event being most ancient in Africans, most recent in Asians, and intermediate in Europeans. Nevertheless, the imbalance index alone cannot reliably estimate the time of initiation of population expansion.

SummaryCongenital limb malformations rank behind only congenital heart disease as the most common birth defects observed in infants. Finding genes that cause defects in human limb patterning should be straightforward but has been limited, in part, by the bewildering spectrum of phenotypes, which are difficult to separate into etiologically distinct disorders. One approach to the identification of relevant genes is to take advantage of unique extended kindreds in which a defect in limb patterning is segregating. Recently, a large Dutch family with ectrodactyly, ectodermal dysplasia, cleft palate, and urogenital defects (EEC) was described by Maas et al. We have studied this kindred and localized a gene causing EEC to a locus on chromosome 19, in a region defined by D19S894 and D19S416. A second extended kindred with EEC does not map to this locus, indicating that EEC is a genetically heterogeneous disorder. Growth and patterning of the limbs, teeth, hair, and genitourinary system are mediated in part by epithelial-mesenchyme inductive interactions. The identification of both the gene causing EEC and its mutation may further elucidate the general signals mediating inductive mechanisms.

Mutations in the gene TBX5 cause Holt-Oram syndrome (HOS), an autosomal dominant disorder characterized by anterior (i.e., radial ray) upper limb malformations and congenital heart defects and/or cardiac conduction anomalies. The detection rate for TBX5 mutations in HOS patients has been given as 30–35% in most reports. However, a detection rate of 74% was reported when strict clinical inclusion criteria for HOS were applied prior to TBX5 analysis. Still, in a significant proportion of typical HOS cases no mutation can be found within the TBX5 coding region and flanking intronic sequences. One explanation could be that large but submicroscopic deletions of TBX5 could cause HOS, yet only one such TBX5 deletion has been reported to date. We developed a quantitative Real Time PCR strategy to detect large, submicroscopic deletions in TBX5. Using this assay, we screened a total of 102 TBX5 mutation negative patients and discovered two novel intragenic deletions. One deletion of 7756 bp removes exon 6 and a considerable part of the neighboring intronic sequences, and the other of 3695 bp removes exon 9 with the stop codon and the 3′UTR completely as well as a part of the preceding intron 8. We conclude that quantitative Real Time PCR is a reliable method to detect submicroscopic deletions within TBX5. However, such deletions explain only ∼2% of the TBX5 mutational spectrum in HOS cases. In addition, we also present eight novel TBX5 mutations (three nonsense, one splice mutation, four short deletions) as detected by direct sequencing in 21 families not previously analyzed for mutations. © 2006 Wiley-Liss, Inc.

The mechanisms underlying most forms of Distal Arthrogryposis (DA), a group of congenital contracture syndromes, are unknown. Our previous functional studies from adult individuals with DA caused by the heterozygous embryonic myosin heavy chain mutation, MYH3 R672C, showed that the time required by DA skinned skeletal myofibers to relax completely after calcium-induced contraction was several-fold longer than controls (Racca et al. (2010) Biophys J 98:542-3a). Here we measured force and kinetics of activation & relaxation, and compared chemomechanical analysis using myofibrils and myofibers sampled from gastrocnemius muscle from two affected individuals vs. three control (non-DA) individuals. The prolonged relaxation was reflected in isolated myofibrils from DA patients, as 50% relaxation time from maximal activation was 36% longer, and 90% relaxation time was prolonged by 58%. The kinetics of the slow phase of relaxation were significantly slower, in both the rate and duration (DA: kREL,SLOW=0.36±0.07s−1;tREL,SLOW=285±21ms vs. Control: 0.79±0.18s−1;199±23ms), implying slower cross-bridge release. Use of ADP prolonged relaxation of control samples to a greater extent than DA preparations, suggesting that slower ADP release from myosin in DA myofibrils may be the mechanism of slower relaxation and cross-bridge release. We also found that both mRNA and protein for this "embryonic" myosin (gene MYH3) were present in adult skeletal muscle, such that a small amount of this slower myosin may prolong relaxation. Although the mechanism that leads to the congenital contractures must begin prenatally, these results suggest that MYH3 R672H also affects ongoing function of adult skeletal muscle. Understanding the mechanism by which myosin mutations affect muscle cell contractility could provide a model for exploring the pathogenesis of more common contractures such as idiopathic clubfoot and facilitate the development of novel therapeutic approaches. Supported by F31AR06300(A.R.), 5K23HD057331(A.B.), HD048895(M.B.,M.R.).

We have examined differences in diversity at 60 microsatellite loci among human population samples from three major continental groups to evaluate the hypothesis of greater African diversity in this rapidly evolving class of loci. Application of a statistical test that assumes equal mutation rates at all loci fails to demonstrate differences in microsatellite diversity, while a randomization test that does not make this assumption finds that Africans have significantly greater microsatellite diversity ( P < 10 −8 ) than do Asians and Europeans. Greater African diversity is most apparent at loci with smaller overall variance in allele size, suggesting that the record of population history has been erased at repeat loci with higher mutation rates. A power analysis shows that only 35–40 microsatellites are needed to establish this difference statistically, demonstrating the considerable evolutionary information contained in these systems. On average, African populations have ≈20% greater microsatellite diversity than do Asian and European populations. A comparison of continental diversity differences in microsatellites and mtDNA sequences suggests earlier demographic expansion of the ancestors of Africans.

Severe congenital neutropenia (SCN) is a rare autosomal dominant hematopoietic disorder with an estimated incidence of 1 in 200,000 for individuals of European descent. Although several causal genes have been identified, the genetic basis for >30% of cases remains unknown. We report finding a novel single nucleotide variant (SNV) R736S identified in TCIRG1, utilizing SNP chip analysis, whole exome sequencing and confirmatory Sanger sequencing, in a multi-generation family (median blood neutrophil counts 0.486 x 109/L, range 0.074 to 1.235 x 109/L for 11 affected members and 3.808 x 109/L, range 1.548 to 6.488 x 109/L for 7 unaffected members). There is a perfect cosegregation of the variant with neutropenia in this family; all 11 affected, but none of the unaffected individuals share this novel variant. TCIRG1 is located at 11q13 and encodes the vacuolar H+-ATPase a3 subunit, a 116-kD component of V-ATPases called OC116, and is essential for bone tissue maintenance and homeostasis. Homozygous or compound heterozygous mutations in this gene cause autosomal recessive malignant osteopetrosis (arOP) characterized by bone resorption defect due to osteoclast malfunction. Interestingly, the same gene, through an alternative splicing and usage of an alternative initiation codon in exon 7, gives rise to another protein, i.e., TIRC7, which is a T cell-specific membrane protein demonstrated to play an essential role for T-lymphocyte activation and immune response. Recent publications suggest the existence of additional splice variants expressed in various tissues in normal volunteers. SCN due to mutations in TCIRG1 have not been previously described. The arginine (R) to serine (S) substitution is a critical change because arginine at position 736 is essential for proton translocation and is evolutionarily highly conserved with a Genomic Evolutionary Rate Profiling (GERP) score = 3.78. Although direct structural data on TCIRG1’s protein products are lacking, the effect of this mutation on the protein structure is predicted to be “probably damaging” by Polyphen (an automatic tool for prediction of possible impact of an amino acid substitution on the structure and function of the protein). Western blot analysis of protein products of this gene performed using peripheral blood mononuclear cells show reduced expression of TCIRG1 protein in the affected family members from this family utilizing a commercially available polyclonal antibody developed against the N-terminal cytoplasmic domain of human OC116. The ∼45kDa TCIRG1 product was down-regulated by 26%, 49%, and 35% in the 3 affected individuals, compared to 2 normal controls. Another antibody, raised against the TCIRG1 fragment 121aa-220aa, detects a ∼120 kDa protein band with similar expression levels in both affected individuals and healthy controls. These assays confirm the multi-product nature of TCIRG1 and suggest deregulatory involvement of this protein in the pathogenesis of the neutropenia in this family. Sanger sequencing of additional 20 unrelated SCN patients known to be negative for mutations in the known causal genes, revealed two different SNVs in TCIRG1 in two unrelated SCN patients, based on Minor Allele Frequency < 0.5% and high conservation among mammals (GERP score > 3). The first variant, g.16065 G>A, (IVS 14 -1G>A), is located in the acceptor splice site of the intron 14, and predicted to alter the splicing. The second variant, c.479 G>A (p.GLY160GLU), is located in exon 5 and predicted to be possibly damaging by Polyphen. No other SNV’s predicted to alter protein structure have been found. We conclude that mutations in TCIRG1 are a rare but important cause of autosomal dominant congenital neutropenia. Further studies are in progress to define the pathophysiological mechanisms for neutropenia and the importance of the vATPases in maintaining the integrity of neutrophil production and deployment. Disclosures: No relevant conflicts of interest to declare.

Beare-Stevenson cutis gyrata syndrome (MIM 123790) is an autosomal dominant condition characterized by the furrowed skin disorder of cutis gyrata, acanthosis nigricans, craniosynostosis, craniofacial dysmorphism, digital anomalies, umbilical and anogenital abnormalities and early death. Many of these features are characteristic of some of the autosomal dominant craniosynostotic syndromes. Mutations in Crouzon, Jackson-Weiss, Pfeiffer and Apert syndromes have been reported in the FGFR2 extracellular domain. In Crouzon syndrome patients with acanthosis nigricans, a recurrent mutation occurs in the transmembrane domain of FGFR3. We now describe the detection of FGFR2 mutations in the Beare-Stevenson cutis gyrata syndrome. In three sporatic cases, a novel missense mutation was found causing an amino acid to be replaced by a cysteine; two had the identical Ty375Cys mutation in the transmembrane domain and one had a Ser372Cys mutation in the carboxyl-terminal end of the linker region between the immunoglobulin III-like (Iglll) and transmembrane domains. In two patients, neither of these mutations were found suggesting further genetic heterogeneity.

Nature 493, 216–220 (2013); doi:10.1038/nature11690 In this Letter, Mark J. Rieder (Department of Genome Sciences, University of Washington, Seattle, Washington 98195, USA) was inadvertently omitted from the author list. He oversaw data generation and quality control and is a member of the Seattle Grand Opportunity group.

Congenital contractures such as clubfoot are present in ∼1/250 live births. Several congenital contractures syndromes are caused by mutations in genes that code for skeletal myofilament proteins, including MYH3 and MYH8. Because embryonic (MYH3) and perinatal (MYH8) myosin heavy chains are unique to the prenatal development of muscle, it is important to understand the contractile properties of human embryonic (HE) myosin and human fetal (HF) muscle to determine how mutations affect performance and development. However, information on HF skeletal muscle function is lacking. We previously reported HE myosin crossbridge cycling (measured by in vitro motility) was much slower that rabbit psoas (RP) myosin (Biophys. J. (2010), 98:542a). Here we characterized the contraction and relaxation properties of HF muscle using myofibril mechanics techniques. HF skeletal muscle and myofibrils were isolated from a 15.4 week gestation fetus. During maximal calcium activation (15°C) HF myofibrils produced much lower force (FMAX=5.9±1.2mN/mm2) as compared to myofibrils from human adult (HA) skeletal muscle (84±34mN/mm2) or RP muscle (220±40mN/mm2). Unlike control HA and RP fibrils, no striation pattern was apparent for HF myofibrils, suggesting that immature sarcomeres could explain the lower force production. HF myofibrils had slower kinetics of force development (kACT=0.66±0.1s−1) vs. HA (7.5±6.3s−1) and RP (5.7±0.5s−1) myofibrils. The initial (slow) phase of relaxation upon return to low calcium solution was slower and prolonged (kREL,SLOW=0.59±0.22s−1; tREL,SLOW=174±13ms) vs. HA (2.9±1.7s−1; 71±18ms) or RP (2.1±0.4s−1; 73±14ms) myofibrils. The larger, faster phase of relaxation was also slower (HF=1.5±0.2s−1 vs. HA=12±5s−1 and RP=21±4s−1). Our previous in vitro motility experiments indicated a similar inhibition of filament speed by increasing [ADP] for HE and RP myosin, but this was under low load, thus ongoing experiments will determine if ADP release is responsible for the slower kinetics of HF muscle. Funded by F31AR06300(A.R.), 5K23HD057331(A.B.), HD048895(M.B., M.R.).

Ulnar-mammary syndrome (UMS) is an autosomal dominant disorder characterized by posterior limb deficiencies or duplications, apocrine/mammary gland hypoplasia and/or dysfunction, abnormal dentition, delayed puberty and genital anomalies. We report the mapping of a gene causing UMS to chromosome 12q23-24.1. Linkage analysis generated a positive lod score of 6.21 at theta = 0.00 with the marker D12S79, and recombinants bracket the UMS gene to a 21 cM region. This region contains a locus for Holt-Oram syndrome (HOS) suggesting that the genes for UMS and HOS may be allelic or closely linked. The identification of the gene causing UMS will be an important step toward understanding the molecular mechanisms that control limb and apocrine gland development.

Trinucleotide repeats at five disease loci (DM, DRPLA, HD, SBMA and SCA1) were surveyed in phenotypically normal individuals from three continental populations. This is the first analysis to examine the population dynamics of these five disease-related trinucleotide repeats in the same individuals from worldwide populations. Roughly half of all alleles observed at each locus are shared between all continental groups. For three loci, disease prevalence in each population corresponds with the number of alleles in the upper tail of the allele-size distribution. The allele-size distributions of African, Asian and Caucasian groups show a high degree of variation, and gene diversity estimates for trinucleotide repeat loci exceed estimates derived from dinucleotide or tetranucleotide repeats. Analyses that compared infinite alleles and stepwise mutation models suggest that normal variation at trinucleotide loci is not generated by stepwise mutation alone. Trees constructed for subpopulations using trinucleotide repeat loci show accurate continental clustering. Interpopulation genetic distance estimates show remarkable similarity to distance estimates produced from tetranucleotide repeats or nuclear restriction site polymorphisms. This finding is especially noteworthy in light of the fact that trinucleotide repeat polymorphisms at these loci can cause disease, while restriction site and tetranucleotide polymorphisms appear to be selectively neutral. In contrast, genetic distance estimates from trinucleotide loci are poorly correlated with genetic distance estimates from mitochondrial sequence data.

INTRODUCTIONGenetic association and gene mapping studies must be carefully designed if they are to provide meaningful data. An important consideration in designing such studies is the type of population to be analyzed. This article discusses founder populations, homozygosity mapping, and admixed populations, and considers the advantages of each in mapping human disease genes.

Exome sequencing is poised to yield substantial insights into human genetic variation and evolutionary history, but there are significant challenges to overcome before this becomes a reality.

This analysis examines the association between genetic heterozygosity and individual morphologic variation in a captive population of Papio hamadryas hamadryas consisting of 403 juveniles and adults. The population structure of the colony was artificially generated and maintained and is thus rigorously defined. Subpopulations delimited by age, sex, and degree of inbreeding are also explored. Heterozygosity, as enumerated from six simple Mendelian biochemical loci, is compared with the residual morphologic variation of each individual for each of 20 quantitative traits. Use of a sequential Bonferroni technique nullifies all significant correlations. Principal-components analysis reduces the morphometrics to a single or few significant axes in each population. The first axis of the total population contains 86.07% of the variation in the sample and the absolute values of the factor scores exhibit a significant positive correlation with heterozygosity at P < 0.05. Correcting for age- and sex-related variation in the total population with a linear model subsequently demonstrates that no significant correlation between heterozygosity and morphologic variation exists. No significant relationship is found in the inbred animals or subpopulations when age and sex are controlled. Previous studies have indicated that individuals proximal to the population mean for a specific polygenic trait exhibit a higher biochemical heterozygosity than individuals distant from the mean. The results presented here, which are based on more loci than many studies and a well-defined population, do not support this relationship. Substructuring of a population by age and sex can lead to spurious correlations with univariate or multivariate techniques. Comprehensive indices of genetic variation and rigorous statistical techniques should be used in future analyses. Studies that fail to recognize these design elements should be interpreted with caution.

Congenital contracture syndromes associated with myofilament proteins are present in 1 out of every 1000 live births. Because the myosin heavy chain protein family has several isoforms unique to the pre-natal development of muscle, it is important to know the contractile properties of this tissue to determine how mutations affect performance and development. However, information on human fetal muscle contractile properties is lacking. We are beginning to characterize the contractile properties of this tissue using in vitro motility assays, where we can deconstruct which proteins of developing muscle can be attributed to differences seen in patients with congenital contracture syndromes. Embryonic myosin was isolated from human fetal muscle samples at 15.4 weeks gestation. An in vitro motility assay was performed at 30 degrees C with an ionic strength of 0.17 to determine the predominant myosin's enzymatic properties. The max speed of filaments on human fetal skeletal myosin (1.9 um/s) was significantly lower than filaments on adult rat skeletal myosin (4.8 um/s) under the same conditions. The Km values were similar between the human fetal (0.02 mM) and adult rat (0.03mM) myosins. Ongoing work includes studying the effects of increased ADP, substitution of ATP with dATP, studying Ca2+ regulation of in vitro motility assays using actin and thin filament regulatory proteins purified from fetal muscle of approximately the same gestational period, and skinned muscle cell experiments. By utilizing these assays, we can develop a more mature understanding of muscle contraction during development and form better hypotheses about the mechanism of how specific mutations lead to these contracture syndromes. Establishing these contractile properties will lead to better hypotheses regarding the development of contractures in utero and how it is affected by mutations associated with congenital contracture syndromes. Supported by HL65497 (Regnier) and HD48895 (Bamshad).

BackgroundMajor population movements, social structure, and caste endogamy have influenced the genetic structure of Indian populations. An understanding of these influences is increasingly important as gene mapping and case-control studies are initiated in South Indian populations.ResultsWe report new data on 155 individuals from four Tamil caste populations of South India and perform comparative analyses with caste populations from the neighboring state of Andhra Pradesh. Genetic differentiation among Tamil castes is low (RST= 0.96% for 45 autosomal short tandem repeat (STR) markers), reflecting a largely common origin. Nonetheless, caste- and continent-specific patterns are evident. For 32 lineage-defining Y-chromosome SNPs, Tamil castes show higher affinity to Europeans than to eastern Asians, and genetic distance estimates to the Europeans are ordered by caste rank. For 32 lineage-defining mitochondrial SNPs and hypervariable sequence (HVS) 1, Tamil castes have higher affinity to eastern Asians than to Europeans. For 45 autosomal STRs, upper and middle rank castes show higher affinity to Europeans than do lower rank castes from either Tamil Nadu or Andhra Pradesh. Local between-caste variation (Tamil Nadu RST= 0.96%, Andhra Pradesh RST= 0.77%) exceeds the estimate of variation between these geographically separated groups (RST= 0.12%). Low, but statistically significant, correlations between caste rank distance and genetic distance are demonstrated for Tamil castes using Y-chromosome, mtDNA, and autosomal data.ConclusionGenetic data from Y-chromosome, mtDNA, and autosomal STRs are in accord with historical accounts of northwest to southeast population movements in India. The influence of ancient and historical population movements and caste social structure can be detected and replicated in South Indian caste populations from two different geographic regions.

Okihiro syndrome results from truncating mutations in the SALL4 locus on the chromosome 20q13.13-q13.2. Deletions of the whole SALL4 coding region as well as single exon deletions are also a common cause of Okihiro syndrome and indicate haploinsufficiency as the disease causing mechanism. The phenotypes caused by SALL4 deletions are not different from those caused by point mutations. No multigene deletion including SALL4 has been documented to date. Here we report the detection and molecular characterization of four novel, overlapping microdeletions, all spanning SALL4 and flanking genes, in four unrelated cases with features of Okihiro syndrome and variable degrees of psychomotor delay. All deletions were first identified and mapped by quantitative Real Time PCR. Subsequently, three of four deletions were mapped in further detail by high-resolution array CGH (244k oligo-arrays). All cases had larger deletions of varying size (1.76-1.78 Mb, 2.01-2.05 Mb, 2.16-2.17 Mb, and 1.3-2.8 Mb, respectively), which included SALL4 plus 3 to 7 additional functional genes. While three cases with largely overlapping deletions are mildly developmentally delayed, the only patient with a more centromeric deletion is clearly mentally retarded. In this patient, four genes (MOCS3, DPM1, ADNP, BCAS4) are deleted, which were not affected in the other three cases, suggesting that the deletion of one or more of these genes contributes to the mental retardation. Since two of the four cases presented with choanal atresia, large deletions including SALL4 should be considered in the differential diagnosis of children with suspected CHARGE syndrome but without detectable CHD7 mutations.

Race is frequently used by clinicians and biomedical researchers to make inferences about an individual's ancestry and to predict whether an individual carries specific genetic risk factors that influence health. The extent to which race is useful for making such predictions depends on how well race corresponds with genetic inferences of ancestry, how frequently common diseases in different racial groups are influenced by the same vs different gene variants, and whether such variants have the same effects in different racial groups. New studies of human genetic variation show that while genetic ancestry is highly correlated with geographic ancestry, its correlation with race is modest. Therefore, while data on the correspondence of race, ancestry, and health-related traits are still limited, particularly in minority populations, geographic ancestry and explicit genetic information are alternatives to race that appear to be more accurate predictors of genetic risk factors that influence health. Making accurate ancestry inferences is crucial because common diseases and drug responses are sometimes influenced by gene variants that vary in frequency or differ altogether among racial groups. Thus, operationalizing alternatives to race for clinicians will be an important step toward providing more personalized health care.

To test hypotheses about the origin of modern humans, we analyzed mtDNA sequences, 30 nuclear restriction-site polymorphisms (RSPs), and 30 tetranucleotide short tandem repeat (STR) polymorphisms in 243 Africans, Asians, and Europeans. An evolutionary tree based on mtDNA displays deep African branches, indicating greater genetic diversity for African populations. This finding, which is consistent with previous mtDNA analyses, has been interpreted as evidence for an African origin of modern humans. Both sets of nuclear polymorphisms, as well as a third set of trinucleotide polymorphisms, are highly consistent with one another but fail to show deep branches for African populations. These results, which represent the first direct comparison of mtDNA and nuclear genetic data in major continental populations, undermine the genetic evidence for an African origin of modern humans.

SummaryUlnar-mammary syndrome (UMS) is a pleiotropic disorder affecting limb, apocrine-gland, tooth, hair, and genital development. Mutations that disrupt the DNA-binding domain of the T-box gene, TBX3, have been demonstrated to cause UMS. However, the 3′ terminus of the open reading frame (ORF) of TBX3 was not identified, and mutations were detected in only two families with UMS. Furthermore, no substantial homology outside the T-box was found among TBX3 and its orthologues. The subsequent cloning of new TBX3 cDNAs allowed us to complete the characterization of TBX3 and to identify alternatively transcribed TBX3 transcripts, including one that interrupts the T-box. The complete ORF of TBX3 is predicted to encode a 723-residue protein, of which 255 amino acids are encoded by newly identified exons. Comparison of other T-box genes to TBX3 indicates regions of substantial homology outside the DNA-binding domain. Novel mutations have been found in all of eight newly reported families with UMS, including five mutations downstream of the region encoding the T-box. This suggests that a domain(s) outside the T-box is highly conserved and important for the function of TBX3. We found no obvious phenotypic differences between those who have missense mutations and those who have deletions or frameshifts.

Achromatopsia (ACHM) is an early-onset retinal dystrophy characterized by photophobia, nystagmus, color blindness and severely reduced visual acuity. Currently mutations in five genes CNGA3, CNGB3, GNAT2, PDE6C and PDE6H have been implicated in ACHM. We performed homozygosity mapping and linkage analysis in a consanguineous Pakistani ACHM family and mapped the locus to a 15.12-Mb region on chromosome 1q23.1–q24.3 with a maximum LOD score of 3.6. A DNA sample from an affected family member underwent exome sequencing. Within the ATF6 gene, a single-base insertion variant c.355_356dupG (p.Glu119Glyfs*8) was identified, which completely segregates with the ACHM phenotype within the family. The frameshift variant was absent in public variant databases, in 130 exomes from unrelated Pakistani individuals, and in 235 ethnically matched controls. The variant is predicted to result in a truncated protein that lacks the DNA binding and transmembrane domains and therefore affects the function of ATF6 as a transcription factor that initiates the unfolded protein response during endoplasmic reticulum (ER) stress. Immunolabeling with anti-ATF6 antibodies showed localization throughout the mouse neuronal retina, including retinal pigment epithelium, photoreceptor cells, inner nuclear layer, inner and outer plexiform layers, with a more prominent signal in retinal ganglion cells. In contrast to cytoplasmic expression of wild-type protein, in heterologous cells ATF6 protein with the p.Glu119Glyfs*8 variant is mainly confined to the nucleus. Our results imply that response to ER stress as mediated by the ATF6 pathway is essential for color vision in humans. Electronic supplementary material The online version of this article (doi:10.1007/s00439-015-1571-4) contains supplementary material, which is available to authorized users.

Gordon syndrome (GS), or distal arthrogyposis type 3, is a rare, autosomal-dominant disorder characterized by cleft palate and congenital contractures of the hands and feet. Exome sequencing of five GS-affected families identified mutations in piezo-type mechano-sensitive ion channel component 2 (PIEZO2) in each family. Sanger sequencing revealed PIEZO2 mutations in five of seven additional families studied (for a total of 10/12 [83%] individuals), and nine families had an identical c.8057G>A (p.Arg2686His) mutation. The phenotype of GS overlaps with distal arthrogryposis type 5 (DA5) and Marden-Walker syndrome (MWS). Using molecular inversion probes for targeted sequencing to screen PIEZO2, we found mutations in 24/29 (82%) DA5-affected families and one of two MWS-affected families. The presence of cleft palate was significantly associated with c.8057G>A (Fisher's exact test, adjusted p value

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal-recessive disorder, characterized by oto-sino-pulmonary disease and situs abnormalities. PCD-causing mutations have been identified in 14 genes, but they collectively account for only ∼60% of all PCD. To identify mutations that cause PCD, we performed exome sequencing on six unrelated probands with ciliary outer dynein arm (ODA) defects. Mutations in CCDC114, an ortholog of the Chlamydomonas reinhardtii motility gene DCC2, were identified in a family with two affected siblings. Sanger sequencing of 67 additional individuals with PCD with ODA defects from 58 families revealed CCDC114 mutations in 4 individuals in 3 families. All 6 individuals with CCDC114 mutations had characteristic oto-sino-pulmonary disease, but none had situs abnormalities. In the remaining 5 individuals with PCD who underwent exome sequencing, we identified mutations in two genes (DNAI2, DNAH5) known to cause PCD, including an Ashkenazi Jewish founder mutation in DNAI2. These results revealed that mutations in CCDC114 are a cause of ciliary dysmotility and PCD and further demonstrate the utility of exome sequencing to identify genetic causes in heterogeneous recessive disorders.

Professional recommendations for the return of results from exome and whole-genome sequencing (ES/WGS) have been controversial. The lack of clear guidance about whether and, if so, how to return ES/WGS incidental results limits the extent to which individuals and families might benefit from ES/WGS. The perspectives of genetics professionals, particularly those at the forefront of using ES/WGS in clinics, are largely unknown. Data on stakeholder perspectives could help clarify how to weigh expert positions and recommendations. We conducted an online survey of 9,857 genetics professionals to learn their attitudes on the return of incidental results from ES/WGS and the recent American College of Medical Genetic and Genomics Recommendations for Reporting of Incidental Findings in Clinical Exome and Genome Sequencing. Of the 847 respondents, 760 completed the survey. The overwhelming majority of respondents thought that incidental ES/WGS results should be offered to adult patients (85%), healthy adults (75%), and the parents of a child with a medical condition (74%). The majority thought that incidental results about adult-onset conditions (62%) and carrier status (62%) should be offered to the parents of a child with a medical condition. About half thought that offered results should not be limited to those deemed clinically actionable. The vast majority (81%) thought that individual preferences should guide return. Genetics professionals' perspectives on the return of ES/WGS results differed substantially from current recommendations, underscoring the need to establish clear purpose for recommendations on the return of incidental ES/WGS results as professional societies grapple with developing and updating recommendations.

Background: Nonphotosensitive trichothiodystrophy (TTDN) is a rare autosomal recessive disorder of neuroectodermal origin. The condition is marked by hair abnormalities, intellectual impairment, nail dystrophies and susceptibility to infections but with no UV sensitivity. Methods: We identified three consanguineous Pakistani families with varied TTDN features and used homozygosity mapping, linkage analysis, and Sanger and exome sequencing in order to identify pathogenic variants. Haplotype analysis was performed and haplotype age estimated. A splicing assay was used to validate the effect of the MPLKIP splice variant on expression. Results: Affected individuals from all families exhibit several TTDN features along with a heart-specific feature, i.e. mitral regurgitation. Exome sequencing in the probands from families ED168 and ED241 identified a homozygous splice mutation c.339 + 1G > A within MPLKIP. The same splice variant co-segregates with TTDN in a third family ED210. The MPLKIP splice variant was not found in public databases, e.g. the Exome Aggregation Consortium, and in unrelated Pakistani controls. Functional analysis of the splice variant confirmed intron retention, which leads to protein truncation and loss of a phosphorylation site. Haplotype analysis identified a 585.1-kb haplotype which includes the MPLKIP variant, supporting the existence of a founder haplotype that is estimated to be 25,900 years old. Conclusion: This study extends the allelic and phenotypic spectra of MPLKIP-related TTDN, to include a splice variant that causes cardiomyopathy as part of the TTDN phenotype.