Disruptions of topological chromatin domains cause pathogenic rewiring of gene-enhancer interactions.

Mammalian genomes are organized into megabase-scale topologically associated domains (TADs). We demonstrate that disruption of TADs can rewire long-range regulatory architecture and result in pathogenic phenotypes. We show that distinct human limb malformations are caused by deletions, inversions, or duplications altering the structure of the TAD-spanning WNT6/IHH/EPHA4/PAX3 locus. Using CRISPR/Cas genome editing, we generated mice with corresponding rearrangements. Both in mouse limb tissue and patient-derived fibroblasts, disease-relevant structural changes cause ectopic interactions between promoters and non-coding DNA, and a cluster of limb enhancers normally associated with Epha4 is misplaced relative to TAD boundaries and drives ectopic limb expression of another gene in the locus. This rewiring occurred only if the variant disrupted a CTCF-associated boundary domain. Our results demonstrate the functional importance of TADs for orchestrating gene expression via genome architecture and indicate criteria for predicting the pathogenicity of human structural variants, particularly in non-coding regions of the human genome.

Personal journeys to and in human genetics and dysmorphology.

Genetics has become a critical component of medicine over the past five to six decades. Alongside genetics, a relatively new discipline, dysmorphology, has also begun to play an important role in providing critically important diagnoses to individuals and families. Both have become indispensable to unraveling rare diseases. Almost every medical specialty relies on individuals experienced in these specialties to provide diagnoses for patients who present themselves to other doctors. Additionally, both specialties have become reliant on molecular geneticists to identify genes associated with human disorders. Many of the medical geneticists, dysmorphologists, and molecular geneticists traveled a circuitous route before arriving at the position they occupied. The purpose of collecting the memoirs contained in this article was to convey to the reader that many of the individuals who contributed to the advancement of genetics and dysmorphology since the late 1960s/early 1970s traveled along a journey based on many chances taken, replying to the necessities they faced along the way before finding full enjoyment in the practice of medical and human genetics or dysmorphology. Additionally, and of equal importance, all exhibited an ability to evolve with their field of expertise as human genetics became human genomics with the development of novel technologies.

Evolution in the clinic: Maladaptive units and "minor anomalies".

It is here argued that the application of the term "minor anomalies" is often imprecise and likely outdated. In the past, the designation was used indiscriminately to refer to a great variety of unrelated morphogenetic phenomena. Also, the term does not discriminate between mild qualitative defects of development (mild malformations) and quantitative variants of normal structure. The human face was formed by natural and sexual selection. Morphological and morphogenetic analyses have shown that the human face with its skin, muscles, nerves, arteries, veins, glands, and lymphatics is a complex structure made up of progeny of ectoderm and mesoderm. Holoprosencephaly demonstrates graphically how these embryonic derivatives fit together sequentially. These derivatives are the adaptive units of the human organism, the result of stringent evolutionary forces uniting essential function to a minimum of structure. Before an "unusual" facial appearance is diagnosed as "abnormal", phenotype analysis is required to determine if there is a family resemblance or if it is a pleiotropic structure. The facial structures of chimps and humans are homologous by virtue of descent from a common ancestor (Darwin, 1859). Differences in the appearance of these species reflect adaptive divergence over some 6-7 million years of evolution while retaining over 98-99% genetic identity. Both species may develop Down syndrome, evidence of similarly retained developmental plasticity. It has occurred to us that Dobzhansky's axiom ("Nothing in biology makes sense except in the light of evolution") applies not only to genetics, but to all of medicine.

Identification and characterization of a missense mutation in the O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase gene that segregates with X-linked intellectual disability.

O-GlcNAc is a regulatory post-translational modification of nucleocytoplasmic proteins that has been implicated in multiple biological processes, including transcription. In humans, single genes encode enzymes for its attachment (O-GlcNAc transferase (OGT)) and removal (O-GlcNAcase (OGA)). An X-chromosome exome screen identified a missense mutation, which encodes an amino acid in the tetratricopeptide repeat, in OGT (759G>T (p.L254F)) that segregates with X-linked intellectual disability (XLID) in an affected family. A decrease in steady-state OGT protein levels was observed in isolated lymphoblastoid cell lines from affected individuals, consistent with molecular modeling experiments. Recombinant expression of L254F-OGT demonstrated that the enzyme is active as both a glycosyltransferase and an HCF-1 protease. Despite the reduction in OGT levels seen in the L254F-OGT individual cells, we observed that steady-state global O-GlcNAc levels remained grossly unaltered. Surprisingly, lymphoblastoids from affected individuals displayed a marked decrease in steady-state OGA protein and mRNA levels. We observed an enrichment of the OGT-containing transcriptional repressor complex mSin3A-HDAC1 at the proximal promoter region of OGA and correspondingly decreased OGA promoter activity in affected cells. Global transcriptome analysis of L254F-OGT lymphoblastoids compared with controls revealed a small subset of genes that are differentially expressed. Thus, we have begun to unravel the molecular consequences of the 759G>T (p.L254F) mutation in OGT that uncovered a compensation mechanism, albeit imperfect, given the phenotype of affected individuals, to maintain steady-state O-GlcNAc levels. Thus, a single amino acid substitution in the regulatory domain (the tetratricopeptide repeat domain) of OGT, which catalyzes the O-GlcNAc post-translational modification of nuclear and cytosolic proteins, appears causal for XLID.

Santos syndrome is caused by mutation in the WNT7A gene.

We have recently described a family with a condition (Santos syndrome (SS; MIM 613005)) characterized by fibular agenesis/hypoplasia, hypoplastic femora and grossly malformed/deformed clubfeet with severe oligodactyly, ungual hypoplasia/anonychia, sometimes associated with mild brachydactyly and occasional pre-axial polydactyly. Autosomal dominant inheritance with incomplete penetrance was suggested, but autosomal recessive inheritance could not be ruled out, due to the high frequency of consanguineous matings in the region where the family lived. This report deals with linkage studies and exome sequencing, disclosing a novel variant in WNT7A, c.934G>A (p.Gly312Ser), as the cause of this syndrome. This variant was present in homozygous state in five individuals typically affected by the SS syndrome, and in heterozygous state in the son of one affected homozygous individual. The heterozygous boy presented only unilateral complex polysyndactyly and we hypothesize that he either presents a distinct defect or that his phenotype results from a rare, mild clinical manifestation of the variant in heterozygous state. Variants in WNT7A are known to cause at least two other limb defect disorders, the syndromes of Fuhrmann and Al-Awadi/Raas-Rothschild. Despite their variable degree of expressivity and overlap, the three related conditions can be differentiated phenotypically in most instances.

Sudden infant death "syndrome"-Insights and future directions from a Utah population database analysis.

"Sudden Infant Death syndrome" (SIDS) represents the commonest category of infant death after the first month of life. As genome scale sequencing greatly facilitates the identification of new candidate disease variants, the challenges of ascribing causation to these variants persists. In order to determine the extent to which SIDS occurs in related individuals and their pedigree structure we undertook an analysis of SIDS using the Utah Population Database, recording, for example, evidence of enrichment for genetic causation following the back-to-sleep recommendations of 1992 and 1994. Our evaluation of the pre- and post back-to-sleep incidence of SIDS in Utah showed a decrease in SIDS incidence on the order of eightfold following back-to-sleep. An odds ratio of 4.2 for SIDS recurrence among sibs was identified from 1968 to 2013 which was similar to the odds ratio of 4.84 for death due to other or unknown cause among sibs of SIDS cases for the same time period. Combining first through thid degree relatives yielded an odds ratio of SIDS recurrence of 9.29 in the post-back-to-sleep (1995-2013) subset of SIDS cases where similar calculations of first-third degree relatives for the entire time period of 1968-2013 showed an odds ratio of 2.95. Expanded multigenertional pedigrees showing enrichment for SIDS were also identified. Based on these findings we hypothesize that post back-to-sleep SIDS, especially recurrences within a family, are potentially enriched for genetic causes due to the impact of safe sleeping guidelines in mitigating environmental risk factors. © 2016 Wiley Periodicals, Inc.

Annals of morphology fields and prepatterns. Editorial Festschrift for John C. Carey, MD, MPH.

The investigation of mammalian malformations began to approach human needs in the 19th century with, for example, Meckel's dissection of sibs with the "Meckel" syndrome, his intimation of Heredität as cause of the condition, his conclusion as to the common causal origin of this specific combination of congenital anomalies, the clear enunciation of the concept of primary malformations, the recognition that many human malformations are normal developmental states in other animals, and that some were normal anatomical states in remote ancestors and now still normal in collateral descendants (atavisms, Darwin's "reversions"; for example, four wings in dipterans, normal in dragonflies and their common ancestor). Later in the century, Wilhelm His Sr. had proposed a schematic map of "organ-forming districts" for prospective chick development, a concept that did not sit well with early workers in developmental biology (e.g., Boveri) until methods became available for a direct experimental "attack" on the embryo. This approach was pioneered by Spemann and Mangold through interspecies transplantation of embryonic rudiments with the spectacular result that set the research stage in developmental biology for the next many years. But it was not until mid-century that the late, great geneticist, Curt Stern, made the His model of chick development more intellectually and experimentally approachable with his meticulous analysis of cuticular appendages of Drosophila, one bristle and one bristle group (field) at a time, in mosaics or gynandromorphs, leading to the ingenious concept of prepatterns. As a basic scientist, Stern did not broaden prepatterns into medicine or to human malformations where it has now found a most gratifying application. This contribution to the Carey Festschrift is to summarize, briefly, field and prepattern theory. © 2016 Wiley Periodicals, Inc.

Screening of CD96 and ASXL1 in 11 patients with Opitz C or Bohring-Opitz syndromes.

Opitz C trigonocephaly (or Opitz C syndrome, OTCS) and Bohring-Opitz syndrome (BOS or C-like syndrome) are two rare genetic disorders with phenotypic overlap. The genetic causes of these diseases are not understood. However, two genes have been associated with OTCS or BOS with dominantly inherited de novo mutations. Whereas CD96 has been related to OTCS (one case) and to BOS (one case), ASXL1 has been related to BOS only (several cases). In this study we analyze CD96 and ASXL1 in a group of 11 affected individuals, including 2 sibs, 10 of them were diagnosed with OTCS, and one had a BOS phenotype. Exome sequences were available on six patients with OTCS and three parent pairs. Thus, we could analyze the CD96 and ASXL1 sequences in these patients bioinformatically. Sanger sequencing of all exons of CD96 and ASXL1 was carried out in the remaining patients. Detailed scrutiny of the sequences and assessment of variants allowed us to exclude putative pathogenic and private mutations in all but one of the patients. In this patient (with BOS) we identified a de novo mutation in ASXL1 (c.2100dupT). By nature and location within the gene, this mutation resembles those previously described in other BOS patients and we conclude that it may be responsible for the condition. Our results indicate that in 10 of 11, the disease (OTCS or BOS) cannot be explained by small changes in CD96 or ASXL1. However, the cohort is too small to make generalizations about the genetic etiology of these diseases.

A recurrent mutation in MED12 leading to R961W causes Opitz-Kaveggia syndrome.

Opitz-Kaveggia syndrome (also known as FG syndrome) is an X-linked disorder characterized by mental retardation, relative macrocephaly, hypotonia and constipation. We report here that the original family for whom the condition is named and five other families have a recurrent mutation (2881C>T, leading to R961W) in MED12 (also called TRAP230 or HOPA), a gene located at Xq13 that functions as a thyroid receptor-associated protein in the Mediator complex.

ADAM "sequence" part II: hypothesis and speculation.

Noted for centuries in humans, a relatively hairless mammal [e.g., Hallero, 1766; Hohl, 1828 in Klunker, 2003], the so-called amniotic deformities, adhesions, mutilations (ADAM) sequence remains causally and pathogenetically incognito. In 1930 Streeter stated " apodictically" that no evidence has been found that intra-uterine amputation is due to amniotic bands or adhesions …" and that his 16 cases provided (histological) evidence for a "germinal origin." He concluded that an amniotic cord was "not an adhesion or inflammatory product but … an anomalous developmental structure and present from the outset." In survivors the "traces" of damaged limb-buds "reveal the scars of poor germ-plasm." In 1958, Willis, in dismissing the amniotic origin of the ADAM defects (or "Streeter" or "Simonart" bands) quoted Keith [1940] to the effect that "(a)mniotic adhesions … are always produced by … the fetus ­ as a result of dysplasia in foetal tissues. They are the result, not the cause, of foetal malformations." Streeter [1930] mentions a potential familial case (56-year-old man and his mother), not controlled by photographs or other records and concluded "that the (ADAM) deformity is not easily transmissible," but "due to the constitution of the germ-plasm." Torpin [1968] concluded, as apodictically as Streeter and Willis, that "… proof of amnion rupture without damage to the chorionic sac is no longer "in question." Considering Torpin's decades-long study of the ADAM phenomenon and review of 494 references (missing many) it is surprising that he does not discuss the relationship between the apparent ADAM defects and other, internal anomalies that maybe present in an affected fetus or infant not evidently caused by the amniotic disruptions, adhesions or mutilations, unless his mind was made up. Our review of these internal and other presumed primary malformations in ADAM is ongoing. However, on a preliminary basis, it seems likely to us that: (1) there is an increased prevalence of such primary anomalies in the ADAM condition confirming the view and experience of others, for example Czeizel et al. [1993]; (2) these malformations (e.g., heterotaxy) may arise as early as gastrulation; (3) that, given the ADAM phenomenon is exclusively ascertained as the ADAM phenotype in fetuses and infants, that is, that its cause and ascertainment are completely congruent, then the apparent amniotic defect must also be regarded as a malformation; (4) that in such a case the ADAM phenomenon with associated primary malformation(s) is a form of syndromal pleiotropy due to one cause yet to be elucidated. To that end we recommend archiving DNA from all affected fetuses coming to autopsy and their parents and placentas and surgical tissues of all viable affected infants for ultimate exome or genome sequencing perhaps with special attention to the syncytin genes.

Serendipity or prepared mind? Recollections of the KOP translocation (1967) and of one form of Perrault syndrome.

The human X/autosome translocation, designated KOP, was discovered by Dr. Philip D. Pallister in Montana in 1967 in a young man with apparent Klinefelter syndrome. Collaboratively it was possible to elucidate the genetic nature of his unprecedented chromosome rearrangement and its developmental effects in mother and son. In retrospect, these clinical and genetic studies at the height of the somatic cell genetics era (Ruddle, Siniscalco, etc.) presented human genetics with a highly productive opportunity to begin gene mapping of autosomes and the X chromosome. The late Victor McKusick considered the discovery of the KOP translocation, as he determined personally in Montana, one of the major transforming events in human genetics. The Perrault syndrome evaluated in two families in Montana and one in Sicily for familial deafness, primary amenorrhea and neurologic impairment (progressive in some), turned out to be heterogeneous. In the "hands" of Dr. M.-C. King of Seattle four forms of Perrault syndrome have been identified. The autosomal recessive mutation present in the P family studied with Dr. Pallister in Helena, turned out to affect the mitochondrial histidyl tRNA synthetase gene present in prokaryotes, annelids, fungi and mammals, hence, must already have been present in LUCA some 3.8 billion years ago.

Using VAAST to identify an X-linked disorder resulting in lethality in male infants due to N-terminal acetyltransferase deficiency.

We have identified two families with a previously undescribed lethal X-linked disorder of infancy; the disorder comprises a distinct combination of an aged appearance, craniofacial anomalies, hypotonia, global developmental delays, cryptorchidism, and cardiac arrhythmias. Using X chromosome exon sequencing and a recently developed probabilistic algorithm aimed at discovering disease-causing variants, we identified in one family a c.109T>C (p.Ser37Pro) variant in NAA10, a gene encoding the catalytic subunit of the major human N-terminal acetyltransferase (NAT). A parallel effort on a second unrelated family converged on the same variant. The absence of this variant in controls, the amino acid conservation of this region of the protein, the predicted disruptive change, and the co-occurrence in two unrelated families with the same rare disorder suggest that this is the pathogenic mutation. We confirmed this by demonstrating a significantly impaired biochemical activity of the mutant hNaa10p, and from this we conclude that a reduction in acetylation by hNaa10p causes this disease. Here we provide evidence of a human genetic disorder resulting from direct impairment of N-terminal acetylation, one of the most common protein modifications in humans.

A novel germline PIGA mutation in Ferro-Cerebro-Cutaneous syndrome: a neurodegenerative X-linked epileptic encephalopathy with systemic iron-overload.

Three related males presented with a newly recognized x-linked syndrome associated with neurodegeneration, cutaneous abnormalities, and systemic iron overload. Linkage studies demonstrated that they shared a haplotype on Xp21.3-Xp22.2 and exome sequencing was used to identify candidate variants. Of the segregating variants, only a PIGA mutation segregated with disease in the family. The c.328_330delCCT PIGA variant predicts, p.Leu110del (or c.1030_1032delCTT, p.Leu344del depending on the reference sequence). The unaffected great-grandfather shared his X allele with the proband but he did not have the PIGA mutation, indicating that the mutation arose de novo in his daughter. A single family with a germline PIGA mutation has been reported; affected males had a phenotype characterized by multiple congenital anomalies and severe neurologic impairment resulting in infantile lethality. In contrast, affected boys in the family described here were born without anomalies and were neurologically normal prior to onset of seizures after 6 months of age, with two surviving to the second decade. PIGA encodes an enzyme in the GPI anchor biosynthesis pathway. An affected individual in the family studied here was deficient in GPI anchor proteins on granulocytes but not erythrocytes. In conclusion, the PIGA mutation in this family likely causes a reduction in GPI anchor protein cell surface expression in various cell types, resulting in the observed pleiotropic phenotype involving central nervous system, skin, and iron metabolism.

Mutations in mitochondrial histidyl tRNA synthetase HARS2 cause ovarian dysgenesis and sensorineural hearing loss of Perrault syndrome.

Perrault syndrome is a genetically heterogeneous recessive disorder characterized by ovarian dysgenesis and sensorineural hearing loss. In a nonconsanguineous family with five affected siblings, linkage analysis and genomic sequencing revealed the genetic basis of Perrault syndrome to be compound heterozygosity for mutations in the mitochondrial histidyl tRNA synthetase HARS2 at two highly conserved amino acids, L200V and V368L. The nucleotide substitution creating HARS2 p.L200V also created an alternate splice leading to deletion of 12 codons from the HARS2 message. Affected family members thus carried three mutant HARS2 transcripts. Aminoacylation activity of HARS2 p.V368L and HARS2 p.L200V was reduced and the deletion mutant was not stably expressed in mammalian mitochondria. In yeast, lethality of deletion of the single essential histydyl tRNA synthetase HTS1 was fully rescued by wild-type HTS1 and by HTS1 p.L198V (orthologous to HARS2 p.L200V), partially rescued by HTS1 p.V381L (orthologous to HARS2 p.V368L), and not rescued by the deletion mutant. In Caenorhabditis elegans, reduced expression by RNAi of the single essential histydyl tRNA synthetase hars-1 severely compromised fertility. Together, these data suggest that Perrault syndrome in this family was caused by reduction of HARS2 activity. These results implicate aberrations of mitochondrial translation in mammalian gonadal dysgenesis. More generally, the relationship between HARS2 and Perrault syndrome illustrates how causality may be demonstrated for extremely rare inherited mutations in essential, highly conserved genes.

Mutations in the DBP-deficiency protein HSD17B4 cause ovarian dysgenesis, hearing loss, and ataxia of Perrault Syndrome.

Perrault syndrome is a recessive disorder characterized by ovarian dysgenesis in females, sensorineural deafness in both males and females, and in some patients, neurological manifestations. No genes for Perrault syndrome have heretofore been identified. A small family of mixed European ancestry includes two sisters with well-characterized Perrault syndrome. Whole-exome sequencing of genomic DNA from one of these sisters revealed exactly one gene with two rare functional variants: HSD17B4, which encodes 17beta-hydroxysteroid dehydrogenase type 4 (HSD17B4), also known as D-bifunctional protein (DBP). HSD17B4/DBP is a multifunctional peroxisomal enzyme involved in fatty acid beta-oxidation and steroid metabolism. Both sisters are compound heterozygotes for HSD17B4 c.650A>G (p.Y217C) (maternal allele) and HSB17B4 c.1704T>A (p.Y568X) (paternal allele). The missense mutation is predicted by structural analysis to destabilize the HSD17B4 dehydrogenase domain. The nonsense mutation leads to very low levels of HSD17B4 transcript. Expression of mutant HSD17B4 protein in a compound heterozygote was severely reduced. Mutations in HSD17B4 are known to cause DBP deficiency, an autosomal-recessive disorder of peroxisomal fatty acid beta-oxidation that is generally fatal within the first two years of life. No females with DBP deficiency surviving past puberty have been reported, and ovarian dysgenesis has not previously been associated with this illness. Six other families with Perrault syndrome have wild-type sequences of HSD17B4. These results indicate that Perrault syndrome and DBP deficiency overlap clinically; that Perrault syndrome is genetically heterogeneous; that DBP deficiency may be underdiagnosed; and that whole-exome sequencing can reveal critical genes in small, nonconsanguineous families.