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1.  Somatic Mutations Are Not Observed by Exome Sequencing of Lymphocyte DNA from Monozygotic Twins Discordant for Congenital Hypothyroidism due to Thyroid Dysgenesis 
Background/Aims
Congenital primary hypothyroidism (CH) is a rare pediatric disorder estimated to occur in about 1: 2,500 live births. Approximately half of these cases entail ectopic thyroid tissue, which is believed to result from a migration defect during embryogenesis. Approximately 3% of CH cases are explained by mutation(s) in known genes, most of which are transcription factors implicated in the embryology of the thyroid gland. Surprisingly, monozygotic (MZ) twins are usually discordant for CH due to thyroid dysgenesis, suggesting that most cases are not caused by transmitted genetic variation. One possible explanation is somatic mutation in genes involved in thyroid migration occurring after zygotic twinning. Such mutations should be observed only in the affected twin.
Methods
To test the hypothesis of somatic mutation, we performed whole exome sequencing of DNA from three pairs of MZ twins discordant for CH with ectopic glands.
Results
We found no somatic mutations exclusive to any of the three affected twins or in any of the unaffected twins.
Conclusion
Either somatic mutations are not significant for the etiology of CH or else such mutations lie outside regions of the genome accessible by exome sequencing technology.
doi:10.1159/000365393
PMCID: PMC5050031  PMID: 25277881 CAMSID: cams5404
Congenital hypothyroidism; Monozygotic twins; Exome sequencing
2.  Family Based Whole Exome Sequencing Reveals the Multifaceted Role of Notch Signaling in Congenital Heart Disease 
PLoS Genetics  2016;12(10):e1006335.
Left-ventricular outflow tract obstructions (LVOTO) encompass a wide spectrum of phenotypically heterogeneous heart malformations which frequently cluster in families. We performed family based whole-exome and targeted re-sequencing on 182 individuals from 51 families with multiple affected members. Central to our approach is the family unit which serves as a reference to identify causal genotype-phenotype correlations. Screening a multitude of 10 overlapping phenotypes revealed disease associated and co-segregating variants in 12 families. These rare or novel protein altering mutations cluster predominantly in genes (NOTCH1, ARHGAP31, MAML1, SMARCA4, JARID2, JAG1) along the Notch signaling cascade. This is in line with a significant enrichment (Wilcoxon, p< 0.05) of variants with a higher pathogenicity in the Notch signaling pathway in patients compared to controls. The significant enrichment of novel protein truncating and missense mutations in NOTCH1 highlights the allelic and phenotypic heterogeneity in our pediatric cohort. We identified novel co-segregating pathogenic mutations in NOTCH1 associated with left and right-sided cardiac malformations in three independent families with a total of 15 affected individuals. In summary, our results suggest that a small but highly pathogenic fraction of family specific mutations along the Notch cascade are a common cause of LVOTO.
Author Summary
Left-ventricular outflow tract obstructions comprise a group of developmental heart disorders that are genetically and phenotypically heterogeneous, with no single gene accounting for the majority of cases. In order to identify mutations contributing to disease, we selected patients from 51 families with a history of congenital cardiac malformations. We interrogated the entire coding sequences of 106 patients and identified a small but highly pathogenic fraction of mutations that are likely to contribute to disease in 12 families (23.5%). Furthermore, we present a strategy for identifying candidate mutations based on familial segregation in a genetically heterogeneous disorder.
doi:10.1371/journal.pgen.1006335
PMCID: PMC5070860  PMID: 27760138
3.  A homozygous loss-of-function variant in MYH11 in a case with megacystis-microcolon-intestinal hypoperistalsis syndrome 
European Journal of Human Genetics  2014;23(9):1266-1268.
Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS) is characterized by marked dilatation of the bladder and microcolon and decreased intestinal peristalsis. Recent studies indicate that heterozygous variants in ACTG2, which codes for a smooth muscle actin, cause MMIHS. However, such variants do not explain MMIHS cases that show an autosomal recessive mode of inheritance. We performed exome sequencing in a newborn with MMIHS and prune belly phenotype whose parents are consanguineous and identified a homozygous variant (c.3598A>T: p.Lys1200Ter) in MYH11, which codes for the smooth muscle myosin heavy chain. Previous studies showed that loss of Myh11 function in mice causes a bladder and intestinal phenotype that is highly reminiscent of MMIHS. All together, these observations strongly suggest that loss-of-function variants in MYH11 cause MMIHS. The documentation of variants in ACTG2 and MYH11 thus points to the involvement of the contractile apparatus of the smooth muscle in MMIHS. Interestingly, dominant-negative variants in MYH11 have previously been shown to cause thoracic aortic aneurism and dilatation. Different mechanisms of MYH11 disruption may thus lead to distinct patterns of smooth muscle dysfunction.
doi:10.1038/ejhg.2014.256
PMCID: PMC4538215  PMID: 25407000
4.  Genetic Mosaics and the Germ Line Lineage 
Genes  2015;6(2):216-237.
Genetic mosaics provide information about cellular lineages that is otherwise difficult to obtain, especially in humans. De novo mutations act as cell markers, allowing the tracing of developmental trajectories of all descendants of the cell in which the new mutation arises. De novo mutations may arise at any time during development but are relatively rare. They have usually been observed through medical ascertainment, when the mutation causes unusual clinical signs or symptoms. Mutational events can include aneuploidies, large chromosomal rearrangements, copy number variants, or point mutations. In this review we focus primarily on the analysis of point mutations and their utility in addressing questions of germ line versus somatic lineages. Genetic mosaics demonstrate that the germ line and soma diverge early in development, since there are many examples of combined somatic and germ line mosaicism for de novo mutations. The occurrence of simultaneous mosaicism in both the germ line and soma also shows that the germ line is not strictly clonal but arises from at least two, and possibly multiple, cells in the embryo with different ancestries. Whole genome or exome DNA sequencing technologies promise to expand the range of studies of genetic mosaics, as de novo mutations can now be identified through sequencing alone in the absence of a medical ascertainment. These technologies have been used to study mutation patterns in nuclear families and in monozygotic twins, and in animal model developmental studies, but not yet for extensive cell lineage studies in humans.
doi:10.3390/genes6020216
PMCID: PMC4488662  PMID: 25898403
mosaicism; germ line; lineage; de novo mutation
5.  Identification and Biochemical Characterization of a Novel Mutation in DDX11 Causing Warsaw Breakage Syndrome 
Human mutation  2012;34(1):103-107.
Mutations in the gene encoding the iron–sulfur-containing DNA helicase DDX11 (ChlR1) were recently identified as a cause of a new recessive cohesinopathy, Warsaw breakage syndrome (WABS), in a single patient with severe microcephaly, pre- and postnatal growth retardation, and abnormal skin pigmentation. Here, using homozygosity mapping in a Lebanese consanguineous family followed by exome sequencing, we identified a novel homozygous mutation (c.788G>A [p.R263Q]) in DDX11 in three affected siblings with severe intellectual disability and many of the congenital abnormalities reported in the WABS original case. Cultured lymphocytes from the patients showed increased mitomycin C-induced chromosomal breakage, as found in WABS. Biochemical studies of purified recombinant DDX11 indicated that the p.R263Q mutation impaired DDX11 helicase activity by perturbing its DNA binding and DNA-dependent ATP hydrolysis. Our findings thus confirm the involvement of DDX11 in WABS, describe its phenotypical spectrum, and provide novel insight into the structural requirement for DDX11 activity. Hum Mutat 34:103-107, 2013.
doi:10.1002/humu.22226
PMCID: PMC4599780  PMID: 23033317
DDX11; Warsaw breakage syndrome; WABS; helicase
6.  A homozygous loss-of-function variant in MYH11 in a case with megacystis-microcolon-intestinal hypoperistalsis syndrome 
European Journal of Human Genetics  2014;23(9):1266-1268.
Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS) is characterized by marked dilatation of the bladder and microcolon and decreased intestinal peristalsis. Recent studies indicate that heterozygous variants in ACTG2, which codes for a smooth muscle actin, cause MMIHS. However, such variants do not explain MMIHS cases that show an autosomal recessive mode of inheritance. We performed exome sequencing in a newborn with MMIHS and prune belly phenotype whose parents are consanguineous and identified a homozygous variant (c.3598A>T: p.Lys1200Ter) in MYH11, which codes for the smooth muscle myosin heavy chain. Previous studies showed that loss of Myh11 function in mice causes a bladder and intestinal phenotype that is highly reminiscent of MMIHS. All together, these observations strongly suggest that loss-of-function variants in MYH11 cause MMIHS. The documentation of variants in ACTG2 and MYH11 thus points to the involvement of the contractile apparatus of the smooth muscle in MMIHS. Interestingly, dominant-negative variants in MYH11 have previously been shown to cause thoracic aortic aneurism and dilatation. Different mechanisms of MYH11 disruption may thus lead to distinct patterns of smooth muscle dysfunction.
doi:10.1038/ejhg.2014.256
PMCID: PMC4538215  PMID: 25407000
7.  Mutations in NFKB2 and potential genetic heterogeneity in patients with DAVID syndrome, having variable endocrine and immune deficiencies 
BMC Medical Genetics  2014;15:139.
Background
DAVID syndrome is a rare condition combining anterior pituitary hormone deficiency with common variable immunodeficiency. NFKB2 mutations have recently been identified in patients with ACTH and variable immunodeficiency. A similar mutation was previously found in Nfkb2 in the immunodeficient Lym1 mouse strain, but the effect of the mutation on endocrine function was not evaluated.
Methods
We ascertained six unrelated DAVID syndrome families. We performed whole exome and traditional Sanger sequencing to search for causal genes. Lym1 mice were examined for endocrine developmental anomalies.
Results
Mutations in the NFKB2 gene were identified in three of our families through whole exome sequencing, and in a fourth by direct Sanger sequencing. De novo origin of the mutations could be demonstrated in three of the families. All mutations lie near the C-terminus of the protein-coding region, near signals required for processing of NFΚB2 protein by the alternative pathway. Two of the probands had anatomical pituitary anomalies, and one had growth and thyroid hormone as well as ACTH deficiency; these findings have not been previously reported. Two children of one of the probands carried the mutation and have to date exhibited only an immune phenotype. No mutations were found near the C-terminus of NFKB2 in the remaining two probands; whole exome sequencing has been performed for one of these. Lym1 mice, carrying a similar Nfkb2 C-terminal mutation, showed normal pituitary anatomy and expression of proopiomelanocortin (POMC).
Conclusions
We confirm previous findings that mutations near the C-terminus of NFKB2 cause combined endocrine and immunodeficiencies. De novo status of the mutations was confirmed in all cases for which both parents were available. The mutations are consistent with a dominant gain-of-function effect, generating an unprocessed NFKB2 super-repressor protein. We expand the potential phenotype of such NFKB2 mutations to include additional pituitary hormone deficiencies as well as anatomical pituitary anomalies. The lack of an observable endocrine phenotype in Lym1 mice suggests that the endocrine component of DAVID syndrome is either not due to a direct role of NFKB pathways on pituitary development, or else that human and mouse pituitary development differ in its requirements for NFKB pathway function.
Electronic supplementary material
The online version of this article (doi:10.1186/s12881-014-0139-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s12881-014-0139-9
PMCID: PMC4411703  PMID: 25524009
8.  Recessive TTN truncating mutations define novel forms of core myopathy with heart disease 
Human Molecular Genetics  2013;23(4):980-991.
Core myopathies (CM), the main non-dystrophic myopathies in childhood, remain genetically unexplained in many cases. Heart disease is not considered part of the typical CM spectrum. No congenital heart defect has been reported, and childhood-onset cardiomyopathy has been documented in only two CM families with homozygous mutations of the TTN gene. TTN encodes titin, a giant protein of striated muscles. Recently, heterozygous TTN truncating mutations have also been reported as a major cause of dominant dilated cardiomyopathy. However, relatively few TTN mutations and phenotypes are known, and titin pathophysiological role in cardiac and skeletal muscle conditions is incompletely understood. We analyzed a series of 23 families with congenital CM and primary heart disease using TTN M-line-targeted sequencing followed in selected patients by whole-exome sequencing and functional studies. We identified seven novel homozygous or compound heterozygous TTN mutations (five in the M-line, five truncating) in 17% patients. Heterozygous parents were healthy. Phenotype analysis identified four novel titinopathies, including cardiac septal defects, left ventricular non-compaction, Emery–Dreifuss muscular dystrophy or arthrogryposis. Additionally, in vitro studies documented the first-reported absence of a functional titin kinase domain in humans, leading to a severe antenatal phenotype. We establish that CM are associated with a large range of heart conditions of which TTN mutations are a major cause, thereby expanding the TTN mutational and phenotypic spectrum. Additionally, our results suggest titin kinase implication in cardiac morphogenesis and demonstrate that heterozygous TTN truncating mutations may not manifest unless associated with a second mutation, reassessing the paradigm of their dominant expression.
doi:10.1093/hmg/ddt494
PMCID: PMC3954110  PMID: 24105469
9.  An international effort towards developing standards for best practices in analysis, interpretation and reporting of clinical genome sequencing results in the CLARITY Challenge 
Brownstein, Catherine A | Beggs, Alan H | Homer, Nils | Merriman, Barry | Yu, Timothy W | Flannery, Katherine C | DeChene, Elizabeth T | Towne, Meghan C | Savage, Sarah K | Price, Emily N | Holm, Ingrid A | Luquette, Lovelace J | Lyon, Elaine | Majzoub, Joseph | Neupert, Peter | McCallie Jr, David | Szolovits, Peter | Willard, Huntington F | Mendelsohn, Nancy J | Temme, Renee | Finkel, Richard S | Yum, Sabrina W | Medne, Livija | Sunyaev, Shamil R | Adzhubey, Ivan | Cassa, Christopher A | de Bakker, Paul IW | Duzkale, Hatice | Dworzyński, Piotr | Fairbrother, William | Francioli, Laurent | Funke, Birgit H | Giovanni, Monica A | Handsaker, Robert E | Lage, Kasper | Lebo, Matthew S | Lek, Monkol | Leshchiner, Ignaty | MacArthur, Daniel G | McLaughlin, Heather M | Murray, Michael F | Pers, Tune H | Polak, Paz P | Raychaudhuri, Soumya | Rehm, Heidi L | Soemedi, Rachel | Stitziel, Nathan O | Vestecka, Sara | Supper, Jochen | Gugenmus, Claudia | Klocke, Bernward | Hahn, Alexander | Schubach, Max | Menzel, Mortiz | Biskup, Saskia | Freisinger, Peter | Deng, Mario | Braun, Martin | Perner, Sven | Smith, Richard JH | Andorf, Janeen L | Huang, Jian | Ryckman, Kelli | Sheffield, Val C | Stone, Edwin M | Bair, Thomas | Black-Ziegelbein, E Ann | Braun, Terry A | Darbro, Benjamin | DeLuca, Adam P | Kolbe, Diana L | Scheetz, Todd E | Shearer, Aiden E | Sompallae, Rama | Wang, Kai | Bassuk, Alexander G | Edens, Erik | Mathews, Katherine | Moore, Steven A | Shchelochkov, Oleg A | Trapane, Pamela | Bossler, Aaron | Campbell, Colleen A | Heusel, Jonathan W | Kwitek, Anne | Maga, Tara | Panzer, Karin | Wassink, Thomas | Van Daele, Douglas | Azaiez, Hela | Booth, Kevin | Meyer, Nic | Segal, Michael M | Williams, Marc S | Tromp, Gerard | White, Peter | Corsmeier, Donald | Fitzgerald-Butt, Sara | Herman, Gail | Lamb-Thrush, Devon | McBride, Kim L | Newsom, David | Pierson, Christopher R | Rakowsky, Alexander T | Maver, Aleš | Lovrečić, Luca | Palandačić, Anja | Peterlin, Borut | Torkamani, Ali | Wedell, Anna | Huss, Mikael | Alexeyenko, Andrey | Lindvall, Jessica M | Magnusson, Måns | Nilsson, Daniel | Stranneheim, Henrik | Taylan, Fulya | Gilissen, Christian | Hoischen, Alexander | van Bon, Bregje | Yntema, Helger | Nelen, Marcel | Zhang, Weidong | Sager, Jason | Zhang, Lu | Blair, Kathryn | Kural, Deniz | Cariaso, Michael | Lennon, Greg G | Javed, Asif | Agrawal, Saloni | Ng, Pauline C | Sandhu, Komal S | Krishna, Shuba | Veeramachaneni, Vamsi | Isakov, Ofer | Halperin, Eran | Friedman, Eitan | Shomron, Noam | Glusman, Gustavo | Roach, Jared C | Caballero, Juan | Cox, Hannah C | Mauldin, Denise | Ament, Seth A | Rowen, Lee | Richards, Daniel R | Lucas, F Anthony San | Gonzalez-Garay, Manuel L | Caskey, C Thomas | Bai, Yu | Huang, Ying | Fang, Fang | Zhang, Yan | Wang, Zhengyuan | Barrera, Jorge | Garcia-Lobo, Juan M | González-Lamuño, Domingo | Llorca, Javier | Rodriguez, Maria C | Varela, Ignacio | Reese, Martin G | De La Vega, Francisco M | Kiruluta, Edward | Cargill, Michele | Hart, Reece K | Sorenson, Jon M | Lyon, Gholson J | Stevenson, David A | Bray, Bruce E | Moore, Barry M | Eilbeck, Karen | Yandell, Mark | Zhao, Hongyu | Hou, Lin | Chen, Xiaowei | Yan, Xiting | Chen, Mengjie | Li, Cong | Yang, Can | Gunel, Murat | Li, Peining | Kong, Yong | Alexander, Austin C | Albertyn, Zayed I | Boycott, Kym M | Bulman, Dennis E | Gordon, Paul MK | Innes, A Micheil | Knoppers, Bartha M | Majewski, Jacek | Marshall, Christian R | Parboosingh, Jillian S | Sawyer, Sarah L | Samuels, Mark E | Schwartzentruber, Jeremy | Kohane, Isaac S | Margulies, David M
Genome Biology  2014;15(3):R53.
Background
There is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance.
Results
A total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization.
Conclusions
The CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups.
doi:10.1186/gb-2014-15-3-r53
PMCID: PMC4073084  PMID: 24667040
10.  Germline Mutations in MAP3K6 Are Associated with Familial Gastric Cancer 
PLoS Genetics  2014;10(10):e1004669.
Gastric cancer is among the leading causes of cancer-related deaths worldwide. While heritable forms of gastric cancer are relatively rare, identifying the genes responsible for such cases can inform diagnosis and treatment for both hereditary and sporadic cases of gastric cancer. Mutations in the E-cadherin gene, CDH1, account for 40% of the most common form of familial gastric cancer (FGC), hereditary diffuse gastric cancer (HDGC). The genes responsible for the remaining forms of FGC are currently unknown. Here we examined a large family from Maritime Canada with FGC without CDH1 mutations, and identified a germline coding variant (p.P946L) in mitogen-activated protein kinase kinase kinase 6 (MAP3K6). Based on conservation, predicted pathogenicity and a known role of the gene in cancer predisposition, MAP3K6 was considered a strong candidate and was investigated further. Screening of an additional 115 unrelated individuals with non-CDH1 FGC identified the p.P946L MAP3K6 variant, as well as four additional coding variants in MAP3K6 (p.F849Sfs*142, p.P958T, p.D200Y and p.V207G). A somatic second-hit variant (p.H506Y) was present in DNA obtained from one of the tumor specimens, and evidence of DNA hypermethylation within the MAP3K6 gene was observed in DNA from the tumor of another affected individual. These findings, together with previous evidence from mouse models that MAP3K6 acts as a tumor suppressor, and studies showing the presence of somatic mutations in MAP3K6 in non-hereditary gastric cancers and gastric cancer cell lines, point towards MAP3K6 variants as a predisposing factor for FGC.
Author Summary
The underlying genetic mutations involved in 60% of inherited gastric cancer cases remain unknown. Here we present a large, extended pedigree with familial gastric cancer and an association in part of the family with a mutation in MAP3K6. The conservation, predicted pathogenicity of the variant, tissue distribution, and known function of MAP3K6 made this a strong candidate that warranted further investigation. Examination of an additional 115 unrelated probands identified additional mutations in MAP3K6, including a truncating mutation.
doi:10.1371/journal.pgen.1004669
PMCID: PMC4207611  PMID: 25340522
11.  Deficiency of asparagine synthetase causes congenital microcephaly and a progressive form of encephalopathy 
Neuron  2013;80(2):10.1016/j.neuron.2013.08.013.
SUMMARY
We analyzed four families that presented with a similar condition characterized by congenital microcephaly, intellectual disability, progressive cerebral atrophy and intractable seizures. We show that recessive mutations in the ASNS gene are responsible for this syndrome. Two of the identified missense mutations dramatically reduce ASNS protein abundance, suggesting that the mutations cause loss of function. Hypomorphic Asns mutant mice have structural brain abnormalities, including enlarged ventricles and reduced cortical thickness, and show deficits in learning and memory mimicking aspects of the patient phenotype. ASNS encodes asparagine synthetase, which catalyzes the synthesis of asparagine from glutamine and aspartate. The neurological impairment resulting from ASNS deficiency may be explained by asparagine depletion in the brain, or by accumulation of aspartate/glutamate leading to enhanced excitability and neuronal damage. Our study thus indicates that asparagine synthesis is essential for the development and function of the brain but not for that of other organs.
doi:10.1016/j.neuron.2013.08.013
PMCID: PMC3820368  PMID: 24139043
12.  Mitochondrial damage and cholesterol storage in human hepatocellular carcinoma cells with silencing of UBIAD1 gene expression 
Heterozygous mutations in the UBIAD1 gene cause Schnyder corneal dystrophy characterized by abnormal cholesterol and phospholipid deposits in the cornea. Ubiad1 protein was recently identified as Golgi prenyltransferase responsible for biosynthesis of vitamin K2 and CoQ10, a key protein in the mitochondrial electron transport chain. Our study shows that silencing UBIAD1 in cultured human hepatocellular carcinoma cells causes dramatic morphological changes and cholesterol storage in the mitochondria, emphasizing an important role of UBIAD1 in mitochondrial function.
doi:10.1016/j.ymgmr.2014.09.001
PMCID: PMC5121353  PMID: 27896114
Mitochondrion; Reactive oxygen species; Cholesterol; CoQ10; Schnyder corneal dystrophy
13.  A generalizable pre-clinical research approach for orphan disease therapy 
With the advent of next-generation DNA sequencing, the pace of inherited orphan disease gene identification has increased dramatically, a situation that will continue for at least the next several years. At present, the numbers of such identified disease genes significantly outstrips the number of laboratories available to investigate a given disorder, an asymmetry that will only increase over time. The hope for any genetic disorder is, where possible and in addition to accurate diagnostic test formulation, the development of therapeutic approaches. To this end, we propose here the development of a strategic toolbox and preclinical research pathway for inherited orphan disease. Taking much of what has been learned from rare genetic disease research over the past two decades, we propose generalizable methods utilizing transcriptomic, system-wide chemical biology datasets combined with chemical informatics and, where possible, repurposing of FDA approved drugs for pre-clinical orphan disease therapies. It is hoped that this approach may be of utility for the broader orphan disease research community and provide funding organizations and patient advocacy groups with suggestions for the optimal path forward. In addition to enabling academic pre-clinical research, strategies such as this may also aid in seeding startup companies, as well as further engaging the pharmaceutical industry in the treatment of rare genetic disease.
doi:10.1186/1750-1172-7-39
PMCID: PMC3458970  PMID: 22704758
Orphan disease therapy; Preclinical drug development; Generalizable screening methods; Translational toolbox
14.  Meier–Gorlin syndrome genotype–phenotype studies: 35 individuals with pre-replication complex gene mutations and 10 without molecular diagnosis 
Meier–Gorlin syndrome (MGS) is an autosomal recessive disorder characterized by microtia, patellar aplasia/hypoplasia, and short stature. Recently, mutations in five genes from the pre-replication complex (ORC1, ORC4, ORC6, CDT1, and CDC6), crucial in cell-cycle progression and growth, were identified in individuals with MGS. Here, we report on genotype–phenotype studies in 45 individuals with MGS (27 females, 18 males; age 3 months–47 years). Thirty-five individuals had biallelic mutations in one of the five causative pre-replication genes. No homozygous or compound heterozygous null mutations were detected. In 10 individuals, no definitive molecular diagnosis was made. The triad of microtia, absent/hypoplastic patellae, and short stature was observed in 82% of individuals with MGS. Additional frequent clinical features were mammary hypoplasia (100%) and abnormal genitalia (42% predominantly cryptorchidism and hypoplastic labia minora/majora). One individual with ORC1 mutations only had short stature, emphasizing the highly variable clinical spectrum of MGS. Individuals with ORC1 mutations had significantly shorter stature and smaller head circumferences than individuals from other gene categories. Furthermore, compared with homozygous missense mutations, compound heterozygous mutations appeared to have a more severe effect on phenotype, causing more severe growth retardation in ORC4 and more frequently pulmonary emphysema in CDT1. A lethal phenotype was seen in four individuals with compound heterozygous ORC1 and CDT1 mutations. No other clear genotype–phenotype association was observed. Growth hormone and estrogen treatment may be of some benefit, respectively, to growth retardation and breast hypoplasia, though further studies in this patient group are needed.
doi:10.1038/ejhg.2011.269
PMCID: PMC3355263  PMID: 22333897
Meier–Gorlin syndrome; ear-patella-short stature syndrome; origin recognition complex; pre-replication complex; genotype–phenotype
15.  Loss of the E3 ubiquitin ligase LRSAM1 sensitizes peripheral axons to degeneration in a mouse model of Charcot-Marie-Tooth disease 
Disease Models & Mechanisms  2013;6(3):780-792.
SUMMARY
Charcot-Marie-Tooth disease (CMT) is a clinically and genetically heterogeneous condition characterized by peripheral axon degeneration with subsequent motor and sensory deficits. Several CMT gene products function in endosomal sorting and trafficking to the lysosome, suggesting that defects in this cellular pathway might present a common pathogenic mechanism for these conditions. LRSAM1 is an E3 ubiquitin ligase that is implicated in this process, and mutations in LRSAM1 have recently been shown to cause CMT. We have generated mouse mutations in Lrsam1 to create an animal model of this form of CMT (CMT2P). Mouse Lrsam1 is abundantly expressed in the motor and sensory neurons of the peripheral nervous system. Both homozygous and heterozygous mice have largely normal neuromuscular performance and only a very mild neuropathy phenotype with age. However, Lrsam1 mutant mice are more sensitive to challenge with acrylamide, a neurotoxic agent that causes axon degeneration, indicating that the axons in the mutant mice are indeed compromised. In transfected cells, LRSAM1 primarily localizes in a perinuclear compartment immediately beyond the Golgi and shows little colocalization with components of the endosome to lysosome trafficking pathway, suggesting that other cellular mechanisms also merit consideration.
doi:10.1242/dmm.010942
PMCID: PMC3634660  PMID: 23519028
16.  Saturation of the Human Phenome 
Current Genomics  2010;11(7):482-499.
The phenome is the complete set of phenotypes resulting from genetic variation in populations of an organism. Saturation of a phenome implies the identification and phenotypic description of mutations in all genes in an organism, potentially constrained to those encoding proteins. The human genome is believed to contain 20-25,000 protein coding genes, but only a small fraction of these have documented mutant phenotypes, thus the human phenome is far from complete. In model organisms, genetic saturation entails the identification of multiple mutant alleles of a gene or locus, allowing a consistent description of mutational phenotypes for that gene. Saturation of several model organisms has been attempted, usually by targeting annotated coding genes with insertional transposons (Drosophila melanogaster, Mus musculus) or by sequence directed deletion (Saccharomyces cerevisiae) or using libraries of antisense oligonucleotide probes injected directly into animals (Caenorhabditis elegans, Danio rerio). This paper reviews the general state of the human phenome, and discusses theoretical and practical considerations toward a saturation analysis in humans. Throughout, emphasis is placed on high penetrance genetic variation, of the kind typically asociated with monogenic versus complex traits.
doi:10.2174/138920210793175886
PMCID: PMC3048311  PMID: 21532833
Human genome; phenome; genetics; saturation mutagenesis.
17.  Mutation in the Gene Encoding Ubiquitin Ligase LRSAM1 in Patients with Charcot-Marie-Tooth Disease 
PLoS Genetics  2010;6(8):e1001081.
Charcot-Marie-Tooth disease (CMT) represents a family of related sensorimotor neuropathies. We studied a large family from a rural eastern Canadian community, with multiple individuals suffering from a condition clinically most similar to autosomal recessive axonal CMT, or AR-CMT2. Homozygosity mapping with high-density SNP genotyping of six affected individuals from the family excluded 23 known genes for various subtypes of CMT and instead identified a single homozygous region on chromosome 9, at 122,423,730–129,841,977 Mbp, shared identical by state in all six affected individuals. A homozygous pathogenic variant was identified in the gene encoding leucine rich repeat and sterile alpha motif 1 (LRSAM1) by direct DNA sequencing of genes within the region in affected DNA samples. The single nucleotide change mutates an intronic consensus acceptor splicing site from AG to AA. Direct analysis of RNA from patient blood demonstrated aberrant splicing of the affected exon, causing an obligatory frameshift and premature truncation of the protein. Western blotting of immortalized cells from a homozygous patient showed complete absence of detectable protein, consistent with the splice site defect. LRSAM1 plays a role in membrane vesicle fusion during viral maturation and for proper adhesion of neuronal cells in culture. Other ubiquitin ligases play documented roles in neurodegenerative diseases. LRSAM1 is a strong candidate for the causal gene for the genetic disorder in our kindred.
Author Summary
Sensory motor neuropathies are diseases of the peripheral nervous system, involving primarily the nerves which control our muscles. These can result from either genetic or non-genetic causes, with genetic causes usually referred to as Charcot-Marie-Tooth (CMT) disease after the three clinicians who first described the key diagnostic markers. CMT patients lose muscle function, mainly in their arms and legs, with increasing severity during their lives. There are almost two dozen known genes that can mutate to cause CMT, and these fall into a wide variety of biochemical cellular pathways. We identified a group of patients with CMT from a small rural community, with good reason to suspect a genetic basis for their disease. Using high-throughput mapping and DNA sequencing technologies developed as part of the Human Genome Project, we were able to find the likely mutated gene, which was not any of the previously known CMT genes. Based on its sequence, the gene, called LRSAM1, probably plays a role in the correct metabolism of other proteins in the cell. Among the known CMT genes, some are also involved in protein metabolism, suggesting that this is a generally important pathway in the neurons that control muscle activity.
doi:10.1371/journal.pgen.1001081
PMCID: PMC2928813  PMID: 20865121
18.  Compound heterozygosity in sodium channel Nav1.7 in a family with hereditary erythermalgia 
Molecular Pain  2008;4:21.
Hereditary erythermalgia is a painful and debilitating genetic disorder associated with mutations in voltage-gated sodium channel Nav1.7. We have previously reported a Canadian family segregating erythermalgia consistently with a dominant genetic etiology. Molecular analysis of the proband from the family detected two different missense mutations in Nav1.7. In the present study we have performed a long-term follow-up clinical study of disease progression in three affected family members. A more extensive molecular study has also been completed, analyzing the segregation of the two missense variants in the family. The two variants (P610T, L858F) segregate independently with respect to clinical presentation. Detailed genotype/phenotype correlation suggests that one of the two variants (L858F) is causal for erythermalgia. The second variant (P610T) may modify the phenotype in the proband. This is the second reported study of potential compound heterozygosity for coding polymorphisms in Nav1.7, the first being in a patient with paroxysmal extreme pain disorder.
doi:10.1186/1744-8069-4-21
PMCID: PMC2430949  PMID: 18518989
19.  Mutations in a novel serine protease PRSS56 in families with nanophthalmos 
Molecular Vision  2011;17:1850-1861.
Purpose
Nanophthalmos is a rare genetic ocular disorder in which the eyes of affected individuals are abnormally small. Patients suffer from severe hyperopia as a result of their markedly reduced axial lengths, but otherwise are capable of seeing well unlike other more general forms of microphthalmia. To date one gene for nanophthalmos has been identified, encoding the membrane-type frizzled related protein MFRP. Identification of additional genes for nanophthalmos will improve our understanding of normal developmental regulation of eye growth.
Methods
We ascertained a cohort of families from eastern Canada and Mexico with familial nanophthalmos. We performed high density microsatellite and high density single nucleotide polymorphism (SNP) genotyping to identify potential chromosomal regions of linkage. We sequenced coding regions of genes in the linked interval by traditional PCR-based Sanger capillary electrophoresis methods. We cloned and sequenced a novel cDNA from a putative causal gene to verify gene structure.
Results
We identified a linked locus on chromosome 2q37 with a peak logarithm (base 10) of odds (LOD) score of 4.7. Sequencing of coding exons of all genes in the region identified multiple segregating variants in one gene, recently annotated as serine protease gene (PRSS56), coding for a predicted trypsin serine protease-like protein. One of our families was homozygous for a predicted pathogenic missense mutation, one family was compound heterozygous for two predicted pathogenic missense mutations, and one family was compound heterozygous for a predicted pathogenic missense mutation plus a frameshift leading to obligatory truncation of the predicted protein. The PRSS56 gene structure in public databases is based on a virtual transcript assembled from overlapping incomplete cDNA clones; we have now validated the structure of a full-length transcript from embryonic mouse brain RNA.
Conclusions
PRSS56 is a good candidate for the causal gene for nanophthalmos in our families.
PMCID: PMC3137557  PMID: 21850159
20.  Application of Homozygosity Haplotype Analysis to Genetic Mapping with High-Density SNP Genotype Data 
PLoS ONE  2009;4(4):e5280.
Background
In families segregating a monogenic genetic disorder with a single disease gene introduction, patients share a mutation-carrying chromosomal interval with identity-by-descent (IBD). Such a shared chromosomal interval or haplotype, surrounding the actual pathogenic mutation, is typically detected and defined by multipoint linkage and phased haplotype analysis using microsatellite or SNP genotype data. High-density SNP genotype data presents a computational challenge for conventional genetic analyses. A novel non-parametric method termed Homozygosity Haplotype (HH) was recently proposed for the genome-wide search of the autosomal segments shared among patients using high density SNP genotype data.
Methodology/Principal Findings
The applicability and the effectiveness of HH in identifying the potential linkage of disease causative gene with high-density SNP genotype data were studied with a series of monogenic disorders ascertained in eastern Canadian populations. The HH approach was validated using the genotypes of patients from a family affected with a rare autosomal dominant disease Schnyder crystalline corneal dystrophy. HH accurately detected the ∼1 Mb genomic interval encompassing the causative gene UBIAD1 using the genotypes of only four affected subjects. The successful application of HH to identify the potential linkage for a family with pericentral retinal disorder indicates that HH can be applied to perform family-based association analysis by treating affected and unaffected family members as cases and controls respectively. A new strategy for the genome-wide screening of known causative genes or loci with HH was proposed, as shown the applications to a myoclonus dystonia and a renal failure cohort.
Conclusions/Significance
Our study of the HH approach demonstrates that HH is very efficient and effective in identifying potential disease linked region. HH has the potential to be used as an efficient alternative approach to sequencing or microsatellite-based fine mapping for screening the known causative genes in genetic disease study.
doi:10.1371/journal.pone.0005280
PMCID: PMC2670504  PMID: 19399176
21.  Exome sequencing identifies mutations in the gene TTC7A in French-Canadian cases with hereditary multiple intestinal atresia 
Journal of Medical Genetics  2013;50(5):324-329.
Background
Congenital multiple intestinal atresia (MIA) is a severe, fatal neonatal disorder, involving the occurrence of obstructions in the small and large intestines ultimately leading to organ failure. Surgical interventions are palliative but do not provide long-term survival. Severe immunodeficiency may be associated with the phenotype. A genetic basis for MIA is likely. We had previously ascertained a cohort of patients of French-Canadian origin, most of whom were deceased as infants or in utero. The goal of the study was to identify the molecular basis for the disease in the patients of this cohort.
Methods
We performed whole exome sequencing on samples from five patients of four families. Validation of mutations and familial segregation was performed using standard Sanger sequencing in these and three additional families with deceased cases. Exon skipping was assessed by reverse transcription-PCR and Sanger sequencing.
Results
Five patients from four different families were each homozygous for a four base intronic deletion in the gene TTC7A, immediately adjacent to a consensus GT splice donor site. The deletion was demonstrated to have deleterious effects on splicing causing the skipping of the attendant upstream coding exon, thereby leading to a predicted severe protein truncation. Parents were heterozygous carriers of the deletion in these families and in two additional families segregating affected cases. In a seventh family, an affected case was compound heterozygous for the same 4bp deletion and a second missense mutation p.L823P, also predicted as pathogenic. No other sequenced genes possessed deleterious variants explanatory for all patients in the cohort. Neither mutation was seen in a large set of control chromosomes.
Conclusions
Based on our genetic results, TTC7A is the likely causal gene for MIA.
doi:10.1136/jmedgenet-2012-101483
PMCID: PMC3625823  PMID: 23423984
Gastroenterology; Genetics; Developmental; Molecular genetics

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