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Congratulations to Professor Mark McCarthy who has been appointed as a National Institute for Health Research Senior Investigator.
Happle-Tinschert, Curry-Jones and segmental basal cell naevus syndromes, overlapping disorders caused by somatic mutations in hedgehog signalling genes: the mosaic hedgehog spectrum.
Happle-Tinschert syndrome (HTS) and Curry-Jones syndrome (CJS; OMIM 601707) are rare, sporadic, multisystem disorders characterized by hypo- and hyperpigmented skin patches following Blaschko's lines, plus acral skeletal and other abnormalities. The blaschkoid pattern implies mosaicism, and indeed CJS was found in 2016 to be caused by a recurrent postzygotic mutation in a gene of the hedgehog signalling pathway, namely SMO, c.1234C>T, p.Leu412Phe. More recently the original case of HTS was found to carry the same somatic mutation. Despite this genetic and phenotypic overlap, two significant differences remained between the two syndromes. The histological hallmark of HTS, basaloid follicular hamartomas, is not a feature of CJS. Meanwhile, the severe gastrointestinal manifestations regularly reported in CJS had not been described in HTS. We report a patient whose phenotype was entirely consistent with HTS apart from intractable constipation, and a second patient with classic features of CJS plus early-onset medulloblastoma, a feature of basal cell naevus syndrome (BCNS). Both had the same recurrent SMO mutation. This prompted a literature review that revealed a case with the same somatic mutation, with basaloid follicular hamartomas and other features of both CJS and BCNS. Segmental BCNS can also be caused by a somatic mutation in PTCH1. We thus demonstrate for the first time phenotypic and genetic overlap between HTS, CJS and segmental BCNS. All of these conditions are caused by somatic mutations in genes of the hedgehog signalling pathway and we therefore propose the unifying term 'mosaic hedgehog spectrum'. What's already known about this topic? Happle-Tinschert syndrome (HTS) and Curry-Jones syndrome (CJS) are rare mosaic multisystem disorders with linear skin lesions. CJS is characterized by severe constipation, which has not previously been reported in HTS. HTS is characterized by basaloid follicular hamartomas, which are not a recognized feature of CJS. The recurrent mosaic SMO mutation found in CJS was recently reported in a patient with HTS. What does this study add? We describe a patient with HTS and intractable constipation, and a case of CJS with medulloblastoma. Both patients had the same recurrent somatic SMO mutation also found in a case reported as segmental basal cell naevus syndrome. SMO functions in the hedgehog pathway, explaining phenotypic overlap between HTS, CJS and mosaic basal cell naevus syndrome. We propose the term 'mosaic hedgehog spectrum' for these overlapping conditions.
Correction: SMAD6 variants in craniosynostosis: genotype and phenotype evaluation.
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Factors influencing success of clinical genome sequencing across a broad spectrum of disorders.
To assess factors influencing the success of whole-genome sequencing for mainstream clinical diagnosis, we sequenced 217 individuals from 156 independent cases or families across a broad spectrum of disorders in whom previous screening had identified no pathogenic variants. We quantified the number of candidate variants identified using different strategies for variant calling, filtering, annotation and prioritization. We found that jointly calling variants across samples, filtering against both local and external databases, deploying multiple annotation tools and using familial transmission above biological plausibility contributed to accuracy. Overall, we identified disease-causing variants in 21% of cases, with the proportion increasing to 34% (23/68) for mendelian disorders and 57% (8/14) in family trios. We also discovered 32 potentially clinically actionable variants in 18 genes unrelated to the referral disorder, although only 4 were ultimately considered reportable. Our results demonstrate the value of genome sequencing for routine clinical diagnosis but also highlight many outstanding challenges.
Gastrointestinal disorders in Curry-Jones syndrome: Clinical and molecular insights from an affected newborn.
Curry-Jones syndrome (CJS) is a pattern of malformation that includes craniosynostosis, pre-axial polysyndactyly, agenesis of the corpus callosum, cutaneous and gastrointestinal abnormalities. A recurrent, mosaic mutation of SMO (c.1234 C>T; p.Leu412Phe) causes CJS. This report describes the gastrointestinal and surgical findings in a baby with CJS who presented with abdominal obstruction and reviews the spectrum of gastrointestinal malformations in this rare disorder. A 41-week, 4,165 g, female presented with craniosynostosis, pre-axial polysyndactyly, and cutaneous findings consistent with a clinical diagnosis of CJS. The infant developed abdominal distension beginning on the second day of life. Surgical exploration revealed an intestinal malrotation for which she underwent a Ladd procedure. Multiple small nodules were found on the surface of the small and large bowel in addition to an apparent intestinal duplication that seemed to originate posterior to the pancreas. Histopathology of serosal nodules revealed bundles of smooth muscle with associated ganglion cells. Molecular analysis demonstrated the SMO c.1234 C>T mutation in varying amounts in affected skin (up to 35%) and intestinal hamartoma (26%). Gastrointestinal features including structural malformations, motility disorders, and upper GI bleeding are major causes of morbidity in CJS. Smooth muscle hamartomas are a recognized feature of children with CJS typically presenting with abdominal obstruction requiring surgical intervention. A somatic mutation in SMO likely accounts for the structural malformations and predisposition to form bowel hamartomas and myofibromas. The mutation burden in the involved tissues likely accounts for the variable manifestations.
Localized TWIST1 and TWIST2 basic domain substitutions cause four distinct human diseases that can be modeled in Caenorhabditis elegans.
Twist transcription factors, members of the basic helix-loop-helix family, play crucial roles in mesoderm development in all animals. Humans have two paralogous genes, TWIST1 and TWIST2, and mutations in each gene have been identified in specific craniofacial disorders. Here, we describe a new clinical entity, Sweeney-Cox syndrome, associated with distinct de novo amino acid substitutions (p.Glu117Val and p.Glu117Gly) at a highly conserved glutamic acid residue located in the basic DNA binding domain of TWIST1, in two subjects with frontonasal dysplasia and additional malformations. Although about one hundred different TWIST1 mutations have been reported in patients with the dominant haploinsufficiency Saethre-Chotzen syndrome (typically associated with craniosynostosis), substitutions uniquely affecting the Glu117 codon were not observed previously. Recently, subjects with Barber-Say and Ablepharon-Macrostomia syndromes were found to harbor heterozygous missense substitutions in the paralogous glutamic acid residue in TWIST2 (p.Glu75Ala, p.Glu75Gln and p.Glu75Lys). To study systematically the effects of these substitutions in individual cells of the developing mesoderm, we engineered all five disease-associated alleles into the equivalent Glu29 residue encoded by hlh-8, the single Twist homolog present in Caenorhabditis elegans. This allelic series revealed that different substitutions exhibit graded severity, in terms of both gene expression and cellular phenotype, which we incorporate into a model explaining the various human disease phenotypes. The genetic analysis favors a predominantly dominant-negative mechanism for the action of amino acid substitutions at this highly conserved glutamic acid residue and illustrates the value of systematic mutagenesis of C. elegans for focused investigation of human disease processes.
Diagnostic value of exome and whole genome sequencing in craniosynostosis.
BACKGROUND: Craniosynostosis, the premature fusion of one or more cranial sutures, occurs in ∼1 in 2250 births, either in isolation or as part of a syndrome. Mutations in at least 57 genes have been associated with craniosynostosis, but only a minority of these are included in routine laboratory genetic testing. METHODS: We used exome or whole genome sequencing to seek a genetic cause in a cohort of 40 subjects with craniosynostosis, selected by clinical or molecular geneticists as being high-priority cases, and in whom prior clinically driven genetic testing had been negative. RESULTS: We identified likely associated mutations in 15 patients (37.5%), involving 14 different genes. All genes were mutated in single families, except for IL11RA (two families). We classified the other positive diagnoses as follows: commonly mutated craniosynostosis genes with atypical presentation (EFNB1, TWIST1); other core craniosynostosis genes (CDC45, MSX2, ZIC1); genes for which mutations are only rarely associated with craniosynostosis (FBN1, HUWE1, KRAS, STAT3); and known disease genes for which a causal relationship with craniosynostosis is currently unknown (AHDC1, NTRK2). In two further families, likely novel disease genes are currently undergoing functional validation. In 5 of the 15 positive cases, the (previously unanticipated) molecular diagnosis had immediate, actionable consequences for either genetic or medical management (mutations in EFNB1, FBN1, KRAS, NTRK2, STAT3). CONCLUSIONS: This substantial genetic heterogeneity, and the multiple actionable mutations identified, emphasises the benefits of exome/whole genome sequencing to identify causal mutations in craniosynostosis cases for which routine clinical testing has yielded negative results.
A Recurrent Mosaic Mutation in SMO, Encoding the Hedgehog Signal Transducer Smoothened, Is the Major Cause of Curry-Jones Syndrome.
Curry-Jones syndrome (CJS) is a multisystem disorder characterized by patchy skin lesions, polysyndactyly, diverse cerebral malformations, unicoronal craniosynostosis, iris colobomas, microphthalmia, and intestinal malrotation with myofibromas or hamartomas. Cerebellar medulloblastoma has been described in a single affected individual; in another, biopsy of skin lesions showed features of trichoblastoma. The combination of asymmetric clinical features, patchy skin manifestations, and neoplastic association previously led to the suggestion that this could be a mosaic condition, possibly involving hedgehog (Hh) signaling. Here, we show that CJS is caused by recurrent somatic mosaicism for a nonsynonymous variant in SMO (c.1234C>T [p.Leu412Phe]), encoding smoothened (SMO), a G-protein-coupled receptor that transduces Hh signaling. We identified eight mutation-positive individuals (two of whom had not been reported previously) with highly similar phenotypes and demonstrated varying amounts of the mutant allele in different tissues. We present detailed findings from brain MRI in three mutation-positive individuals. Somatic SMO mutations that result in constitutive activation have been described in several tumors, including medulloblastoma, ameloblastoma, and basal cell carcinoma. Strikingly, the most common of these mutations is the identical nonsynonymous variant encoding p.Leu412Phe. Furthermore, this substitution has been shown to activate SMO in the absence of Hh signaling, providing an explanation for tumor development in CJS. This raises therapeutic possibilities for using recently generated Hh-pathway inhibitors. In summary, our work uncovers the major genetic cause of CJS and illustrates strategies for gene discovery in the context of low-level tissue-specific somatic mosaicism.
EFNB1 and EFNA4 in craniofrontonasal syndrome and craniosynostosis.
However, we are on the brink of a revolution in this area, and Inborn Errors is at the forefront. It is the first book to connect the disease-causing gene to its biochemical pathway and to the structural/functional disorder.
De novo and rare inherited mutations implicate the transcriptional coregulator TCF20/SPBP in autism spectrum disorder.
BACKGROUND: Autism spectrum disorders (ASDs) are common and have a strong genetic basis, yet the cause of ∼70-80% ASDs remains unknown. By clinical cytogenetic testing, we identified a family in which two brothers had ASD, mild intellectual disability and a chromosome 22 pericentric inversion, not detected in either parent, indicating de novo mutation with parental germinal mosaicism. We hypothesised that the rearrangement was causative of their ASD and localised the chromosome 22 breakpoints. METHODS: The rearrangement was characterised using fluorescence in situ hybridisation, Southern blotting, inverse PCR and dideoxy-sequencing. Open reading frames and intron/exon boundaries of the two physically disrupted genes identified, TCF20 and TNRC6B, were sequenced in 342 families (260 multiplex and 82 simplex) ascertained by the International Molecular Genetic Study of Autism Consortium (IMGSAC). RESULTS: IMGSAC family screening identified a de novo missense mutation of TCF20 in a single case and significant association of a different missense mutation of TCF20 with ASD in three further families. Through exome sequencing in another project, we independently identified a de novo frameshifting mutation of TCF20 in a woman with ASD and moderate intellectual disability. We did not identify a significant association of TNRC6B mutations with ASD. CONCLUSIONS: TCF20 encodes a transcriptional coregulator (also termed SPBP) that is structurally and functionally related to RAI1, the critical dosage-sensitive protein implicated in the behavioural phenotypes of the Smith-Magenis and Potocki-Lupski 17p11.2 deletion/duplication syndromes, in which ASD is frequently diagnosed. This study provides the first evidence that mutations in TCF20 are also associated with ASD.
A new locus for split hand/foot malformation with long bone deficiency (SHFLD) at 2q14.2 identified from a chromosome translocation.
Split hand/foot malformation (SHFM) with long bone deficiency (SHFLD) is a distinct entity in the spectrum of ectrodactylous limb malformations characterised by associated tibial a/hypoplasia. Pedigrees with multiple individuals affected by SHFLD often include non-penetrant intermediate relatives, making genetic mapping difficult. Here we report a sporadic patient with SHFLD who carries a de novo chromosomal translocation t(2;18)(q14.2;p11.2). Characterisation of the breakpoints revealed that neither disrupts any known gene; however, the chromosome 2 breakpoint lies between GLI2 and INHBB, two genes known to be involved in limb development. To investigate whether mutation of a gene in proximity to the chromosome 2 breakpoint underlies the SHFLD, we sought independent evidence of mutations in GLI2, INHBB and two other genes (RALB and FLJ14816) in 44 unrelated patients with SHFM, SHFLD or isolated long bone deficiency. No convincing pathogenic mutations were found, raising the possibility that a long-range cis acting regulatory element may be disrupted by this translocation. The previous description of a translocation with a 2q14.2 breakpoint associated with ectrodactyly, and the mapping of the ectrodactylous Dominant hemimelia mouse mutation to a region of homologous synteny, suggests that 2q14.2 represents a novel locus for SHFLD.
Identification of Intragenic Exon Deletions and Duplication of TCF12 by Whole Genome or Targeted Sequencing as a Cause of TCF12-Related Craniosynostosis.
TCF12-related craniosynostosis can be caused by small heterozygous loss-of-function mutations in TCF12. Large intragenic rearrangements, however, have not been described yet. Here, we present the identification of four large rearrangements in TCF12 causing TCF12-related craniosynostosis. Whole-genome sequencing was applied on the DNA of 18 index cases with coronal synostosis and their family members (43 samples in total). The data were analyzed using an autosomal-dominant disease model. Structural variant analysis reported intragenic exon deletions (of sizes 84.9, 8.6, and 5.4 kb) in TCF12 in three different families. The results were confirmed by deletion-specific PCR and dideoxy-sequence analysis. Separately, targeted sequencing of the TCF12 genomic region in a patient with coronal synostosis identified a tandem duplication of 11.3 kb. The pathogenic effect of this duplication was confirmed by cDNA analysis. These findings indicate the importance of screening for larger rearrangements in patients suspected to have TCF12-related craniosynostosis.
Integrating mapping-, assembly- and haplotype-based approaches for calling variants in clinical sequencing applications.
High-throughput DNA sequencing technology has transformed genetic research and is starting to make an impact on clinical practice. However, analyzing high-throughput sequencing data remains challenging, particularly in clinical settings where accuracy and turnaround times are critical. We present a new approach to this problem, implemented in a software package called Platypus. Platypus achieves high sensitivity and specificity for SNPs, indels and complex polymorphisms by using local de novo assembly to generate candidate variants, followed by local realignment and probabilistic haplotype estimation. It is an order of magnitude faster than existing tools and generates calls from raw aligned read data without preprocessing. We demonstrate the performance of Platypus in clinically relevant experimental designs by comparing with SAMtools and GATK on whole-genome and exome-capture data, by identifying de novo variation in 15 parent-offspring trios with high sensitivity and specificity, and by estimating human leukocyte antigen genotypes directly from variant calls.
Phenotypes of craniofrontonasal syndrome in patients with a pathogenic mutation in EFNB1.
Craniofrontonasal syndrome (CFNS) is an X-linked developmental malformation, caused by mutations in the EFNB1 gene, which have only been described since 2004. A genotype-phenotype correlation seems not to be present. As it is of major importance to adequately counsel patients with EFNB1 mutations and their parents, and to improve diagnosis of new patients, more information about the phenotypic features is needed. This study included 23 patients (2 male, 21 female) with confirmed EFNB1 mutations. All patients underwent a thorough physical examination and photographs were taken. If available, radiological images were also consulted. Hypertelorism, longitudinal ridging and/or splitting of nails, a (mild) webbed neck and a clinodactyly of one or more toes were the only consistent features observed in all patients. Frequently observed phenotypic features were bifid tip of the nose (91%), columellar indentation (91%) and low implantation of breasts (90%). In comparison with anthropometric data of facial proportions, patients with CFNS had a significantly different face in multiple respects. An overview of all phenotypic features is shown. Patients with EFNB1 mutations have a clear phenotype. This study will facilitate genetic counseling of parents and patients, and contribute to the diagnostic and screening process of patients with suspected CFNS.
Duplication of the EFNB1 gene in familial hypertelorism: imbalance in ephrin-B1 expression and abnormal phenotypes in humans and mice.
Familial hypertelorism, characterized by widely spaced eyes, classically shows autosomal dominant inheritance (Teebi type), but some pedigrees are compatible with X-linkage. No mechanism has been described previously, but clinical similarity has been noted to craniofrontonasal syndrome (CFNS), which is caused by mutations in the X-linked EFNB1 gene. Here we report a family in which females in three generations presented with hypertelorism, but lacked either craniosynostosis or a grooved nasal tip, excluding CFNS. DNA sequencing of EFNB1 was normal, but further analysis revealed a duplication of 937 kb including EFNB1 and two flanking genes: PJA1 and STARD8. We found that the X chromosome bearing the duplication produces ∼1.6-fold more EFNB1 transcript than the normal X chromosome and propose that, in the context of X-inactivation, this difference in expression level of EFNB1 results in abnormal cell sorting leading to hypertelorism. To support this hypothesis, we provide evidence from a mouse model carrying a targeted human EFNB1 cDNA, that abnormal cell sorting occurs in the cranial region. Hence, we propose that X-linked cases resembling Teebi hypertelorism may have a similar mechanism to CFNS, and that cellular mosaicism for different levels of ephrin-B1 (as well as simple presence/absence) leads to craniofacial abnormalities.
Prevalence and complications of single-gene and chromosomal disorders in craniosynostosis.
OBJECTIVES: We describe the first cohort-based analysis of the impact of genetic disorders in craniosynostosis. We aimed to refine the understanding of prognoses and pathogenesis and to provide rational criteria for clinical genetic testing. METHODS: We undertook targeted molecular genetic and cytogenetic testing for 326 children who required surgery because of craniosynostosis, were born in 1993-2002, presented to a single craniofacial unit, and were monitored until the end of 2007. RESULTS: Eighty-four children (and 64 relatives) had pathologic genetic alterations (86% single-gene mutations and 14% chromosomal abnormalities). The FGFR3 P250R mutation was the single largest contributor (24%) to the genetic group. Genetic diagnoses accounted for 21% of all craniosynostosis cases and were associated with increased rates of many complications. Children with an initial clinical diagnosis of nonsyndromic craniosynostosis were more likely to have a causative mutation if the synostoses were unicoronal or bicoronal (10 of 48 cases) than if they were sagittal or metopic (0 of 55 cases; P = .0003). Repeat craniofacial surgery was required for 58% of children with single-gene mutations but only 17% of those with chromosomal abnormalities (P = .01). CONCLUSIONS: Clinical genetic assessment is critical for the treatment of children with craniosynostosis. Genetic testing of nonsyndromic cases (at least for FGFR3 P250R and FGFR2 exons IIIa/c) should be targeted to patients with coronal or multisuture synostoses. Single-gene disorders that disrupt physiologic signaling in the cranial sutures often require reoperation, whereas chromosomal abnormalities follow a more-indolent course, which suggests a different, secondary origin of the associated craniosynostosis.
Expanding the phenotype of craniofrontonasal syndrome: two unrelated boys with EFNB1 mutations and congenital diaphragmatic hernia.
Craniofrontonasal syndrome (CFNS, MIM 304110) is an X-linked craniofacial disorder that shows paradoxically greater severity in heterozygous females than in hemizygous males. Mutations have been identified in the EFNB1 gene that encodes a member of the ephrin-B family of transmembrane ligands for Eph receptor tyrosine kinases. Here, we describe two unrelated families, in both of which a mother and her son have proven mutations in EFNB1. The mothers have classical features of CFNS; although the sons have no major craniofacial features other than telecanthus, both had a congenital diaphragmatic hernia (CDH). Our cases represent the first in which CDH has been confirmed in males with mutations in EFNB1, highlighting an important role for signalling by ephrin-B1 in the development of the diaphragm.
The origin of EFNB1 mutations in craniofrontonasal syndrome: frequent somatic mosaicism and explanation of the paucity of carrier males.
Craniofrontonasal syndrome (CFNS) is an X-linked disorder that exhibits a paradoxical sex reversal in phenotypic severity: females characteristically have frontonasal dysplasia, craniosynostosis, and additional minor malformations, but males are usually mildly affected with hypertelorism only. Despite this, males appear underrepresented in CFNS pedigrees, with carrier males encountered infrequently compared with affected females. To investigate these unusual genetic features of CFNS, we exploited the recent discovery of causative mutations in the EFNB1 gene, which encodes ephrin-B1, to survey the molecular alterations in 59 families (39 newly investigated and 20 published elsewhere). We identified the first complete deletions of EFNB1, catalogued 27 novel intragenic mutations, and used Pyrosequencing and analysis of nearby polymorphic alleles to quantify mosaic cases and to determine the parental origin of verified germline mutations. Somatic mosaicism was demonstrated in 6 of 53 informative families, and, of 17 germline mutations in individuals for whom the parental origin of mutation could be demonstrated, 15 arose from the father. We conclude that the major factor accounting for the relative scarcity of carrier males is the bias toward mutations in the paternal germline (which present as affected female offspring) combined with reduced reproductive fitness in affected females. Postzygotic mutations also contribute to the female preponderance, whereas true nonpenetrance in males who are hemizygous for an EFNB1 mutation appears unusual. These results highlight the importance of considering possible origins of mutation in the counseling of families with CFNS and provide a generally applicable approach to the combined analysis of mosaic and germline mutations.