Janus-faced EPHB4-associated disorders: novel pathogenic variants and unreported intrafamilial overlapping phenotypes.

PURPOSE: Several clinical phenotypes including fetal hydrops, central conducting lymphatic anomaly or capillary malformations with arteriovenous malformations 2 (CM-AVM2) have been associated with EPHB4 (Ephrin type B receptor 4) variants, demanding new approaches for deciphering pathogenesis of novel variants of uncertain significance (VUS) identified in EPHB4, and for the identification of differentiated disease mechanisms at the molecular level. METHODS: Ten index cases with various phenotypes, either fetal hydrops, CM-AVM2, or peripheral lower limb lymphedema, whose distinct clinical phenotypes are described in detail in this study, presented with a variant in EPHB4. In vitro functional studies were performed to confirm pathogenicity. RESULTS: Pathogenicity was demonstrated for six of the seven novel EPHB4 VUS investigated. A heterogeneity of molecular disease mechanisms was identified, from loss of protein production or aberrant subcellular localization to total reduction of the phosphorylation capability of the receptor. There was some phenotype-genotype correlation; however, previously unreported intrafamilial overlapping phenotypes such as lymphatic-related fetal hydrops (LRFH) and CM-AVM2 in the same family were observed. CONCLUSION: This study highlights the usefulness of protein expression and subcellular localization studies to predict EPHB4 variant pathogenesis. Our accurate clinical phenotyping expands our interpretation of the Janus-faced spectrum of EPHB4-related disorders, introducing the discovery of cases with overlapping phenotypes.

Bohring-Opitz syndrome caused by an ASXL1 mutation inherited from a germline mosaic mother.

Bohring-Opitz syndrome (BOS) is characterized clinically by severe developmental delays, microcephaly, failure to thrive, and characteristic facial features (prominent eyes, facial nevus simplex [flammeus], and others). Most patients meeting the clinical criteria for BOS (MIM: 605039) have a de novo nonsense or frameshift variant in ASXL1. We report a case of BOS caused by a pathogenic ASXL1 variant inherited from a germline mosaic mother. The ASXL1 mutation was detected via trio exome sequencing. The sequencing data demonstrated that the variant was inherited maternally but that the maternal variant was underrepresented in comparison to the normal allele. These results suggested maternal mosaicism for the variant. Additional testing on the mother was performed on buccal cell DNA, which was also consistent with mosaicism. The mother had been reported to be healthy and the family history is unremarkable. This is the first report of BOS caused by a mutation inherited from an unaffected, presumed germline mosaic parent. This phenomenon has been reported for other traditionally de novo dominant disorders like CHARGE syndrome and Cornelia de Lange syndrome. This case emphasizes the need for accurate low but non-negative recurrence risk counseling for families with children with BOS and it impacts exome interpretation strategy.

A Recurrent De Novo Variant in NACC1 Causes a Syndrome Characterized by Infantile Epilepsy, Cataracts, and Profound Developmental Delay.

Whole-exome sequencing (WES) has increasingly enabled new pathogenic gene variant identification for undiagnosed neurodevelopmental disorders and provided insights into both gene function and disease biology. Here, we describe seven children with a neurodevelopmental disorder characterized by microcephaly, profound developmental delays and/or intellectual disability, cataracts, severe epilepsy including infantile spasms, irritability, failure to thrive, and stereotypic hand movements. Brain imaging in these individuals reveals delay in myelination and cerebral atrophy. We observe an identical recurrent de novo heterozygous c.892C>T (p.Arg298Trp) variant in the nucleus accumbens associated 1 (NACC1) gene in seven affected individuals. One of the seven individuals is mosaic for this variant. NACC1 encodes a transcriptional repressor implicated in gene expression and has not previously been associated with germline disorders. The probability of finding the same missense NACC1 variant by chance in 7 out of 17,228 individuals who underwent WES for diagnoses of neurodevelopmental phenotypes is extremely small and achieves genome-wide significance (p = 1.25 × 10-14). Selective constraint against missense variants in NACC1 makes this excess of an identical missense variant in all seven individuals more remarkable. Our findings are consistent with a germline recurrent mutational hotspot associated with an allele-specific neurodevelopmental phenotype in NACC1.

Clinical Versus Research Sequencing.

Historically, sequencing has been the key technology to assess variation in the genetic code, and has been widely accepted in clinical diagnostics of genetic disease. The advent of next-generation sequencing (NGS) methods increased the size of the analyzed target by several orders of magnitude, while at the same time drastically reducing the cost of sequencing. Current research allows sequencing of germline and tumor whole genomes. However, with the arrival of cutting-edge technology to the clinical diagnostic field, strict regulatory oversight is required to use the advances of the latest research when applied to routine clinical practice. We discuss the differences between sequencing in a research setting and sequencing in a clinical diagnostics setting, as applied to next-generation technology.

Whole exome sequencing reveals de novo pathogenic variants in KAT6A as a cause of a neurodevelopmental disorder.

Neurodevelopmental disorders (NDD) are common, with 1-3% of general population being affected, but the etiology is unknown in most individuals. Clinical whole-exome sequencing (WES) has proven to be a powerful tool for the identification of pathogenic variants leading to Mendelian disorders, among which NDD represent a significant percentage. Performing WES with a trio-approach has proven to be extremely effective in identifying de novo pathogenic variants as a common cause of NDD. Here we report six unrelated individuals with a common phenotype consisting of NDD with severe speech delay, hypotonia, and facial dysmorphism. These patients underwent WES with a trio approach and de novo heterozygous predicted pathogenic novel variants in the KAT6A gene were identified. The KAT6A gene encodes a histone acetyltransfrease protein and it has long been known for its structural involvement in acute myeloid leukemia; however, it has not previously been associated with any congenital disorder. In animal models the KAT6A ortholog is involved in transcriptional regulation during development. Given the similar findings in animal models and our patient's phenotypes, we hypothesize that KAT6A could play a role in development of the brain, face, and heart in humans. © 2016 Wiley Periodicals, Inc.

Pathogenic and likely pathogenic variant prevalence among the first 10,000 patients referred for next-generation cancer panel testing.

PURPOSE: Germ-line testing for panels of cancer genes using next-generation sequencing is becoming more common in clinical care. We report our experience as a clinical laboratory testing both well-established, high-risk cancer genes (e.g., BRCA1/2, MLH1, MSH2) as well as more recently identified cancer genes (e.g., PALB2, BRIP1), many of which have increased but less well-defined penetrance. METHODS: Clinical genetic testing was performed on over 10,000 consecutive cases referred for evaluation of germ-line cancer genes, and results were analyzed for frequency of pathogenic or likely pathogenic variants, and were stratified by testing panel, gene, and clinical history. RESULTS: Overall, a molecular diagnosis was made in 9.0% of patients tested, with the highest yield in the Lynch syndrome/colorectal cancer panel. In patients with breast, ovarian, or colon/stomach cancer, positive yields were 9.7, 13.4, and 14.8%, respectively. Approximately half of the pathogenic variants identified in patients with breast or ovarian cancer were in genes other than BRCA1/2. CONCLUSION: The high frequency of positive results in a wide range of cancer genes, including those of high penetrance and with clinical care guidelines, underscores both the genetic heterogeneity of hereditary cancer and the usefulness of multigene panels over genetic tests of one or two genes.Genet Med 18 8, 823-832.

Findings from the Peutz-Jeghers syndrome registry of uruguay.

BACKGROUND: Peutz-Jeghers syndrome (PJS) is characterized by intestinal polyposis, mucocutaneous pigmentation and an increased cancer risk, usually caused by mutations of the STK11 gene. This study collected epidemiological, clinical and genetic data from all Uruguayan PJS patients. METHODS: Clinical data were obtained from public and private medical centers and updated annually. Sequencing of the STK11 gene in one member of each family was performed. RESULTS AND DISCUSSION: 25 cases in 11 unrelated families were registered (15 males, 10 females). The average age of diagnosis and death was 18 and 41 years respectively. All patients had characteristic PJS pigmentation and gastrointestinal polyps. 72% required urgent surgery due to intestinal obstruction. 3 families had multiple cases of seizure disorder, representing 20% of cases. 28% developed cancer and two patients had more than one cancer. An STK11 mutation was found in 8 of the 9 families analyzed. A unique M136K missense mutation was noted in one family. Comparing annual live births and PJS birth records from 1970 to 2009 yielded an incidence of 1 in 155,000. CONCLUSION: The Uruguayan Registry for Peutz-Jeghers patients showed a high chance of emergent surgery, epilepsy, cancer and shortened life expectancy. The M136K missense mutation is a newly reported STK 11 mutation.

A prospective study of brachytelephalangic chondrodysplasia punctata: identification of arylsulfatase E mutations, functional analysis of novel missense alleles, and determination of potential phenocopies.

PURPOSE: The only known genetic cause of brachytelephalangic chondrodysplasia punctata is X-linked chondrodysplasia punctata 1 (CDPX1), which results from a deficiency of arylsulfatase E (ARSE). Historically, ARSE mutations have been identified in only 50% of male patients, and it was proposed that the remainder might represent phenocopies due to maternal-fetal vitamin K deficiency and maternal autoimmune diseases. METHODS: To further evaluate causes of brachytelephalangic chondrodysplasia punctata, we established a Collaboration Education and Test Translation program for CDPX1 from 2008 to 2010. Of the 29 male probands identified, 17 had ARSE mutations that included 10 novel missense alleles and one single-codon deletion. To determine pathogenicity of these and additional missense alleles, we transiently expressed them in COS cells and measured arylsulfatase E activity using the artificial substrate, 4-methylumbelliferyl sulfate. In addition, clinical data were collected to investigate maternal effects and genotype-phenotype correlations. RESULTS: In this study, 58% of males had ARSE mutations. All mutant alleles had negligible arylsulfatase E activity. There were no obvious genotype-phenotype correlations. Maternal etiologies were not reported in most patients. CONCLUSION: CDPX1 is caused by loss of arylsulfatase E activity. Around 40% of male patients with brachytelephalangic chondrodysplasia punctata do not have detectable ARSE mutations or known maternal etiological factors. Improved understanding of arylsulfatase E function is predicted to illuminate other etiologies for brachytelephalangic chondrodysplasia punctata.

Clinical and radiological features in young individuals with nevoid basal cell carcinoma syndrome.

PURPOSE: Nevoid basal cell carcinoma syndrome is an autosomal dominant disorder characterized by multiple basal cell carcinomas, jaw cysts, palmar/plantar pits, spine and rib anomalies, and falx cerebri calcification. Current diagnostic criteria are suboptimal when applied to pediatric populations, as most common symptoms often do not begin to appear until teenage years. METHODS: We studied minor and major clinical features in 30 children/teenagers and compared the findings with 75 adults from 26 families with nevoid basal cell carcinoma syndrome. RESULTS: Fifty percent of children/teenagers and 82% of adults had at least one basal cell carcinoma. Jaw cysts occurred in 60% of children/teenagers and 81% of adults. Palmar/plantar pits were the most frequent feature seen in affected individuals at all ages. Macrocephaly was seen in 50% of affected and 8% of unaffected children/teenagers. Frontal bossing, hypertelorism, Sprengel deformity, pectus deformity, and cleft lip/palate were seen among affected children/teenagers but not among their unaffected siblings. Falx calcification, the most frequent radiological feature, was present in 37% of individuals <20 and 79% of those >20 years. CONCLUSION: We report clinical and radiological manifestations of nevoid basal cell carcinoma syndrome in children/teenagers, many of whom lacked major features such as basal cell carcinomas, jaw cysts, and falx calcification. Evaluations for palmar/plantar pits, craniofacial features, and radiological manifestations permit early diagnosis and optimum surveillance.

Comparative analysis of IRF6 variants in families with Van der Woude syndrome and popliteal pterygium syndrome using public whole-exome databases.

PURPOSE: Mutations in the transcription factor IRF6 cause allelic autosomal dominant clefting syndromes, Van der Woude syndrome, and popliteal pterygium syndrome. We compared the distribution of IRF6 coding and splice-site mutations from 549 families with Van der Woude syndrome or popliteal pterygium syndrome with that of variants from the 1000 Genomes and National Heart, Lung, and Blood Institute Exome Sequencing Projects. METHODS: We compiled all published pathogenic IRF6 mutations and performed direct sequencing of IRF6 in families with Van der Woude syndrome or popliteal pterygium syndrome. RESULTS: Although mutations causing Van der Woude syndrome or popliteal pterygium syndrome were nonrandomly distributed with significantly increased frequencies in the DNA-binding domain (P = 0.0001), variants found in controls were rare and evenly distributed in IRF6. Of 194 different missense or nonsense variants described as potentially pathogenic, we identified only two in more than 6,000 controls. PolyPhen and SIFT (sorting intolerant from tolerant) reported 5.9% of missense mutations in patients as benign, suggesting that use of current in silico prediction models to determine function can have significant false negatives. CONCLUSION: Mutation of IRF6 occurs infrequently in controls, suggesting that for IRF6 there is a high probability that disruption of the coding sequence, particularly the DNA-binding domain, will result in syndromic features. Prior associations of coding sequence variants in IRF6 with clefting syndromes have had few false positives.

Development and characterization of reference materials for MTHFR, SERPINA1, RET, BRCA1, and BRCA2 genetic testing.

Well-characterized reference materials (RMs) are integral in maintaining clinical laboratory quality assurance for genetic testing. These RMs can be used for quality control, monitoring of test performance, test validation, and proficiency testing of DNA-based genetic tests. To address the need for such materials, the Centers for Disease Control and Prevention established the Genetic Testing Reference Material Coordination Program (GeT-RM), which works with the genetics community to improve public availability of characterized RMs for genetic testing. To date, the GeT-RM program has coordinated the characterization of publicly available genomic DNA RMs for a number of disorders, including cystic fibrosis, Huntington disease, fragile X, and several genetic conditions with relatively high prevalence in the Ashkenazi Jewish population. Genotypic information about a number of other cell lines has been collected and is also available. The present study includes the development and commutability/genotype characterization of 10 DNA samples for clinically relevant mutations or sequence variants in the following genes: MTHFR; SERPINA1; RET; BRCA1; and BRCA2. DNA samples were analyzed by 19 clinical genetic laboratories using a variety of assays and technology platforms. Concordance was 100% for all samples, with no differences observed between laboratories using different methods. All DNA samples are available from Coriell Cell Repositories and characterization information can be found on the GeT-RM website.

Genetic testing in ectodermal dysplasia: availability, clinical utility, and the nuts and bolts of ordering a genetic test.

"Ectodermal Dysplasia syndromes" comprise a diverse group of heritable conditions characterized by congenital anomalies of one or more ectodermal structures and their appendages: hair, teeth, nails, and sweat glands. Genetic testing is available for many types of ectodermal dysplasia (ED) through clinical and/or research laboratories. We address the distinctions between genetic testing as performed on a clinical versus research basis, and summarize the clinical aspects, testing methodology, and sensitivity for those ED syndromes for which testing is available in a clinical laboratory. Lastly, we leave the laboratory for the clinical setting to discuss the utility of genetic testing for patients and their families, and summarize the practical issues involved in ordering a genetic test.

Oligonucleotide array CGH studies in myeloproliferative neoplasms: comparison with JAK2V617F mutational status and conventional chromosome analysis.

Comparative genomic hybridization (CGH), using oligo arrays with either 44,000 or 105,000 oligonucleotides, was performed on granulocyte-derived DNA from 71 patients with BCR-ABL-negative classic myeloproliferative neoplasms (MPNs): 32 primary myelofibrosis (PMF), 26 polycythemia vera (PV) and 13 essential thrombocythemia (ET). Copy number changes (CNCs) were detected in 44%, 35%, and 15% of the cases with PMF, PV and ET, respectively. In ET and PMF, CNCs were more frequently detected in the presence of JAK2V617F (50% vs. 19%; p=0.05). Conventional chromosome analysis was obtained in 57 patients either at diagnosis or within 1 year of the array CGH study; all 21 patients with PV and 11 with ET displayed normal cytogenetic findings despite the presence of CNCs in 29% and 18%, respectively. In PMF, the respective rates of CNCs and abnormal karyotype were 48% and 36%; karyotypic abnormalities, including unbalanced translocations, were often detected by array CGH as chromosomal gains or losses. This preliminary report suggests a potential value for array CGH in terms of both clinical diagnostics and genomic research in MPNs.

Radiological features in 82 patients with nevoid basal cell carcinoma (NBCC or Gorlin) syndrome.

PURPOSE: Nevoid Basal Cell Carcinoma Syndrome (NBCCS) is an autosomal-dominant disorder characterized by multiple basal cell carcinomas, jaw cysts, palmar/plantar pits, calcification of the falx cerebri, and spine and rib anomalies. NBCCS is due to mutations in PTCH1, the human homologue of the Drosophila segment polarity gene patched. Mutations are detected in approximately 60% to 85% of individuals tested by sequencing of PTCH1; therefore, clinical examination and x-rays remain important in diagnosis of NBCCS. METHODS: We studied 82 NBCCS patients and 38 of their unaffected siblings at the NIH between 1985 and 1994. Chest, rib, spine, skull, hand and foot x-rays, brain MRI or CT, and pelvic ultrasound (in females) were obtained in the affected individuals and compared to their unaffected relatives. RESULTS: The following features were significantly more frequent in those with NBCCS: calcification of the falx cerebri, the most frequent radiological feature, was present in 79% of patients > 20 years and in 37% <20 years, calcification of the tentorium cerebellum was present in 20%, bridging of the sella in 68%, and abnormal frontal sinus aeration in 18% of affected individuals. Bifid ribs most often involving the third, fourth, and fifth ribs were seen in 26%; splayed, fused, and misshapen ribs in a further 16%, and widened ends of clavicles in 12%. Spine X-rays revealed calcification of the nuchal ligament in 18%, fusion of vertebrae in 10%, and hemivertebrae in 15%. Flame-shaped lucencies of the metacarpals and/or phalanges were present in 30%, modeling deformities of the phalanges in 14%, and polydactyly of the feet in 4%. The frequency of scoliosis, cervical ribs, absent or rudimentary ribs, spina bifida occulta, or short 4th metacarpal was not higher in the affected individuals as compared to their unaffected relatives. Except for falx calcification, the frequency of radiological manifestations was similar in different age groups. Cranial CT or MRI in 42 affected individuals revealed asymmetric or dilated ventricles in 24%, cerebral atrophy in 10%, cavum septum pellucidum in 19%, dysgenesis or agenesis of the corpus callosum in 10%, and meningioma in 5%. Ovarian fibromas were detected in 17% of females. CONCLUSIONS: This study reports the varied radiological manifestations of NBCCS. In the absence of major features such as basal cell carcinomas, jaw cysts, or falx calcification, which is often not evident until the teen years, other radiological manifestations of the disorder can permit early diagnosis of NBCCS in childhood. This will allow optimum surveillance for medulloblastoma and other neoplasms (cardiac fibromas and basal cell carcinomas) associated with NBCCS.

Refined mapping of Naegeli-Franceschetti- Jadassohn syndrome to a 6 cM interval on chromosome 17q11.2-q21 and investigation of candidate genes.

Naegeli-Franceschetti-Jadassohn syndrome and dermatopathia pigmentosa reticularis are autosomal dominant ectodermal dysplasias characterized by the absence of dermatoglyphics, reticulate hyper pigmentation of the skin, hypohidrosis, and heat intolerance. Palmoplantar keratoderma, nail dystrophy, and enamel defects are common in Naegeli-Franceschetti-Jadassohn syndrome, whereas diffuse alopecia is only seen in dermatopathia pigmentosa reticularis. We studied a large Swiss family with Naegeli-Franceschetti-Jadassohn syndrome originally described by Naegeli in 1927 and assessed linkage to chromosome 17q, which was proposed to harbor the Naegeli-Franceschetti-Jadassohn syndrome gene. Our results considerably narrow the Naegeli-Franceschetti-Jadassohn syndrome gene region from 27 cM to 6 cM flanked by D17S933 and D17S934 with a maximum multipoint LOD score of 2.7 at marker locus D17S800. In addition, we studied a small family with dermatopathia pigmentosa reticularis, and our linkage data suggest that dermatopathia pigmentosa reticularis may map to the same chromosomal region. The Naegeli-Franceschetti-Jadassohn syndrome critical interval spans approximately 5.4 Mb and contains a minimum of 45 distinct genes. We scrutinized 13 new prime candidates in addition to five genes previously examined, established the genomic organization of 10 of these genes, and excluded all of them by mutation analysis. Moreover, we identified a cDNA (KRT24) encoding a new keratin protein that bears high similarity to the type I keratins and displays a unique expression profile. No pathogenic mutations were identified in this novel gene either, however. In summary, our results substantially refine the Naegeli-Franceschetti-Jadassohn syndrome region and will aid in identifying a gene that is critical for ontogenesis of multiple ectodermal tissues.

Auditory manifestations of Keratitis-Ichthyosis-Deafness (KID) syndrome.

OBJECTIVE: Evaluation of the auditory manifestations of Keratitis-Ichthyosis-Deafness (KID) syndrome, a rare genodermatosis characterized by follicular hyperkeratosis, vascularizing keratitis, and congenital hearing loss. STUDY DESIGN: Five individuals with sporadic KID syndrome were evaluated in the outpatient audiology clinic at the Warren Grant Magnuson Clinical Center of the National Institutes of Health. METHODS: Audiologic examinations included pure-tone audiometry, speech audiometry, and middle ear immittance testing. Auditory brainstem responses and otoacoustic emissions were analyzed in 2 subjects. RESULTS: Four subjects had prelingual, bilateral, profound sensorineural hearing loss, whereas the fifth subject had significant residual hearing that exhibited no progression on serial audiograms. All 5 subjects had a history of non-erosive keratosis obturans and cutaneous cysts in the external ear canals that prevented continuous use of ear molds. CONCLUSIONS: The sensorineural hearing loss in KID syndrome is generally prelingual and profound. This combination of auditory and cutaneous phenotypes is similar to those previously reported for KID syndrome. KID syndrome presents a difficult challenge for communication rehabilitation because keratitis may impair the perception of sign and spoken language, and the cutaneous manifestations routinely curtail use of external amplification devices.

Missense mutations in GJB2 encoding connexin-26 cause the ectodermal dysplasia keratitis-ichthyosis-deafness syndrome.

Keratitis-ichthyosis-deafness syndrome (KID) is a rare ectodermal dysplasia characterized by vascularizing keratitis, profound sensorineural hearing loss (SNHL), and progressive erythrokeratoderma, a clinical triad that indicates a failure in development and differentiation of multiple stratifying epithelia. Here, we provide compelling evidence that KID is caused by heterozygous missense mutations in the connexin-26 gene, GJB2. In each of 10 patients with KID, we identified a point mutation leading to substitution of conserved residues in the cytoplasmic amino terminus or first extracellular domain of Cx26. One of these mutations was detected in six unrelated sporadic case subjects and also segregated in one family with vertical transmission of KID. These results indicate the presence of a common, recurrent mutation and establish its autosomal dominant nature. Cx26 and the closely related Cx30 showed differential expression in epidermal, adnexal, and corneal epithelia but were not significantly altered in lesional skin. However, mutant Cx26 was incapable of inducing intercellular coupling in vitro, which indicates its functional impairment. Our data reveal striking genotype-phenotype correlations and demonstrate that dominant GJB2 mutations can disturb the gap junction system of one or several ectodermal epithelia, thereby producing multiple phenotypes: nonsyndromic SNHL, syndromic SNHL with palmoplantar keratoderma, and KID. Decreased host defense and increased carcinogenic potential in KID illustrate that gap junction communication plays not only a crucial role in epithelial homeostasis and differentiation but also in immune response and epidermal carcinogenesis.