Targeted long-read sequencing identifies missing disease-causing variation.

Despite widespread clinical genetic testing, many individuals with suspected genetic conditions lack a precise diagnosis, limiting their opportunity to take advantage of state-of-the-art treatments. In some cases, testing reveals difficult-to-evaluate structural differences, candidate variants that do not fully explain the phenotype, single pathogenic variants in recessive disorders, or no variants in genes of interest. Thus, there is a need for better tools to identify a precise genetic diagnosis in individuals when conventional testing approaches have been exhausted. We performed targeted long-read sequencing (T-LRS) using adaptive sampling on the Oxford Nanopore platform on 40 individuals, 10 of whom lacked a complete molecular diagnosis. We computationally targeted up to 151 Mbp of sequence per individual and searched for pathogenic substitutions, structural variants, and methylation differences using a single data source. We detected all genomic aberrations-including single-nucleotide variants, copy number changes, repeat expansions, and methylation differences-identified by prior clinical testing. In 8/8 individuals with complex structural rearrangements, T-LRS enabled more precise resolution of the mutation, leading to changes in clinical management in one case. In ten individuals with suspected Mendelian conditions lacking a precise genetic diagnosis, T-LRS identified pathogenic or likely pathogenic variants in six and variants of uncertain significance in two others. T-LRS accurately identifies pathogenic structural variants, resolves complex rearrangements, and identifies Mendelian variants not detected by other technologies. T-LRS represents an efficient and cost-effective strategy to evaluate high-priority genes and regions or complex clinical testing results.

Damaging variants in FOXI3 cause microtia and craniofacial microsomia.

PURPOSE: Craniofacial microsomia (CFM) represents a spectrum of craniofacial malformations, ranging from isolated microtia with or without aural atresia to underdevelopment of the mandible, maxilla, orbit, facial soft tissue, and/or facial nerve. The genetic causes of CFM remain largely unknown. METHODS: We performed genome sequencing and linkage analysis in patients and families with microtia and CFM of unknown genetic etiology. The functional consequences of damaging missense variants were evaluated through expression of wild-type and mutant proteins in vitro. RESULTS: We studied a 5-generation kindred with microtia, identifying a missense variant in FOXI3 (p.Arg236Trp) as the cause of disease (logarithm of the odds = 3.33). We subsequently identified 6 individuals from 3 additional kindreds with microtia-CFM spectrum phenotypes harboring damaging variants in FOXI3, a regulator of ectodermal and neural crest development. Missense variants in the nuclear localization sequence were identified in cases with isolated microtia with aural atresia and found to affect subcellular localization of FOXI3. Loss of function variants were found in patients with microtia and mandibular hypoplasia (CFM), suggesting dosage sensitivity of FOXI3. CONCLUSION: Damaging variants in FOXI3 are the second most frequent genetic cause of CFM, causing 1% of all cases, including 13% of familial cases in our cohort.

Isolated frontosphenoidal craniosynostosis: An argument for genetic testing.

Isolated frontosphenoidal craniosynostosis (IFSC) is a rare congenital defect defined as premature fusion of the frontosphenoidal suture in the absence of other suture fusion. Until now, IFSC was regarded as a phenomenon with an unclear genetic etiology. We have identified three cases with IFSC with underlying syndromic diagnoses that were attributable to pathogenic mutations involving FGFR3 and MN1, as well as 22q11.2 deletion syndrome. These findings suggest a genetic predisposition to IFSC may exist, thereby justifying the recommendation for genetic evaluation and testing in this population. Furthermore, due to improved imaging resolution, cases of IFSC are now readily identified. With the identification of IFSC with underlying genetic diagnoses, in combination with significant improvements in imaging resolution, we recommend genetic evaluation in children with IFSC.

A mutational hotspot in AMOTL1 defines a new syndrome of orofacial clefting, cardiac anomalies, and tall stature.

AMOTL1 encodes angiomotin-like protein 1, an actin-binding protein that regulates cell polarity, adhesion, and migration. The role of AMOTL1 in human disease is equivocal. We report a large cohort of individuals harboring heterozygous AMOTL1 variants and define a core phenotype of orofacial clefting, congenital heart disease, tall stature, auricular anomalies, and gastrointestinal manifestations in individuals with variants in AMOTL1 affecting amino acids 157-161, a functionally undefined but highly conserved region. Three individuals with AMOTL1 variants outside this region are also described who had variable presentations with orofacial clefting and multi-organ disease. Our case cohort suggests that heterozygous missense variants in AMOTL1, most commonly affecting amino acid residues 157-161, define a new orofacial clefting syndrome, and indicates an important functional role for this undefined region.

Mutations in the Epithelial Cadherin-p120-Catenin Complex Cause Mendelian Non-Syndromic Cleft Lip with or without Cleft Palate.

Non-syndromic cleft lip with or without cleft palate (NS-CL/P) is one of the most common human birth defects and is generally considered a complex trait. Despite numerous loci identified by genome-wide association studies, the effect sizes of common variants are relatively small, with much of the presumed genetic contribution remaining elusive. We report exome-sequencing results in 209 people from 72 multi-affected families with pedigree structures consistent with autosomal-dominant inheritance and variable penetrance. Herein, pathogenic variants are described in four genes encoding components of the p120-catenin complex (CTNND1, PLEKHA7, PLEKHA5) and an epithelial splicing regulator (ESRP2), in addition to the known CL/P-associated gene, CDH1, which encodes E-cadherin. The findings were also validated in a second cohort of 497 people with NS-CL/P, comprising small families and singletons with pathogenic variants in these genes identified in 14% of multi-affected families and 2% of the replication cohort of smaller families. Enriched expression of each gene/protein in human and mouse embryonic oro-palatal epithelia, demonstration of functional impact of CTNND1 and ESRP2 variants, and recapitulation of the CL/P spectrum in Ctnnd1 knockout mice support a causative role in CL/P pathogenesis. These data show that primary defects in regulators of epithelial cell adhesion are the most significant contributors to NS-CL/P identified to date and that inherited and de novo single gene variants explain a substantial proportion of NS-CL/P.

De novo loss-of-function variants in X-linked MED12 are associated with Hardikar syndrome in females.

PURPOSE: Hardikar syndrome (MIM 612726) is a rare multiple congenital anomaly syndrome characterized by facial clefting, pigmentary retinopathy, biliary anomalies, and intestinal malrotation, but with preserved cognition. Only four patients have been reported previously, and none had a molecular diagnosis. Our objective was to identify the genetic basis of Hardikar syndrome (HS) and expand the phenotypic spectrum of this disorder. METHODS: We performed exome sequencing on two previously reported and five unpublished female patients with a clinical diagnosis of HS. X-chromosome inactivation (XCI) studies were also performed. RESULTS: We report clinical features of HS with previously undescribed phenotypes, including a fatal unprovoked intracranial hemorrhage at age 21. We additionally report the discovery of de novo pathogenic nonsense and frameshift variants in MED12 in these seven individuals and evidence of extremely skewed XCI in all patients with informative testing. CONCLUSION: Pathogenic missense variants in the X-chromosome gene MED12 have previously been associated with Opitz-Kaveggia syndrome, Lujan syndrome, Ohdo syndrome, and nonsyndromic intellectual disability, primarily in males. We propose a fifth, female-specific phenotype for MED12, and suggest that nonsense and frameshift loss-of-function MED12 variants in females cause HS. This expands the MED12-associated phenotype in females beyond intellectual disability.

Cleft palate morphology, genetic etiology, and risk of mortality in infants with Robin sequence.

Robin sequence (RS) has many genetic and nongenetic causes, including isolated Robin sequence (iRS), Stickler syndrome (SS), and other syndromes (SyndRS). The purpose of this study was to determine if the presence and type of cleft palate varies between etiologic groups. A secondary endpoint was to determine the relationship of etiologic group, cleft type, and mortality. Retrospective chart review of patients with RS at two high-volume craniofacial centers. 295 patients with RS identified. CP was identified in 97% with iRS, 95% with SS, and 70% of those with SyndRS (p < .0001). U-shaped CP was seen in 86% of iRS, 82% with SS, but only 27% with SyndRS (p < .0001). At one institution, 12 children (6%) with RS died, all from the SyndRS group (p < .0001). All died due to medical comorbidities related to their syndrome. Only 25% of children who died had a U-shaped CP. The most common palatal morphology among those who died was an intact palate. U-shaped CP was most strongly associated with iRS and SS, and with a lower risk of mortality. RS with submucous CP, cleft lip and palate or intact palate was strongly suggestive of an underlying genetic syndrome and higher risk of mortality.

Exome sequencing identifies a recurrent de novo ZSWIM6 mutation associated with acromelic frontonasal dysostosis.

Acromelic frontonasal dysostosis (AFND) is a rare disorder characterized by distinct craniofacial, brain, and limb malformations, including frontonasal dysplasia, interhemispheric lipoma, agenesis of the corpus callosum, tibial hemimelia, preaxial polydactyly of the feet, and intellectual disability. Exome sequencing of one trio and two unrelated probands revealed the same heterozygous variant (c.3487C>T [p. Arg1163Trp]) in a highly conserved protein domain of ZSWIM6; this variant has not been seen in the 1000 Genomes data, dbSNP, or the Exome Sequencing Project. Sanger validation of the three trios confirmed that the variant was de novo and was also present in a fourth isolated proband. In situ hybridization of early zebrafish embryos at 24 hr postfertilization (hpf) demonstrated telencephalic expression of zswim6 and onset of midbrain, hindbrain, and retinal expression at 48 hpf. Immunohistochemistry of later-stage mouse embryos demonstrated tissue-specific expression in the derivatives of all three germ layers. qRT-PCR expression analysis of osteoblast and fibroblast cell lines available from two probands was suggestive of Hedgehog pathway activation, indicating that the ZSWIM6 mutation associated with AFND may lead to the craniofacial, brain and limb malformations through the disruption of Hedgehog signaling.

Compound heterozygosity of low-frequency promoter deletions and rare loss-of-function mutations in TXNL4A causes Burn-McKeown syndrome.

Mutations in components of the major spliceosome have been described in disorders with craniofacial anomalies, e.g., Nager syndrome and mandibulofacial dysostosis type Guion-Almeida. The U5 spliceosomal complex of eight highly conserved proteins is critical for pre-mRNA splicing. We identified biallelic mutations in TXNL4A, a member of this complex, in individuals with Burn-McKeown syndrome (BMKS). This rare condition is characterized by bilateral choanal atresia, hearing loss, cleft lip and/or palate, and other craniofacial dysmorphisms. Mutations were found in 9 of 11 affected families. In 8 families, affected individuals carried a rare loss-of-function mutation (nonsense, frameshift, or microdeletion) on one allele and a low-frequency 34 bp deletion (allele frequency 0.76%) in the core promoter region on the other allele. In a single highly consanguineous family, formerly diagnosed as oculo-oto-facial dysplasia, the four affected individuals were homozygous for a 34 bp promoter deletion, which differed from the promoter deletion in the other families. Reporter gene and in vivo assays showed that the promoter deletions led to reduced expression of TXNL4A. Depletion of TXNL4A (Dib1) in yeast demonstrated reduced assembly of the tri-snRNP complex. Our results indicate that BMKS is an autosomal-recessive condition, which is frequently caused by compound heterozygosity of low-frequency promoter deletions in combination with very rare loss-of-function mutations.

Tracheal cartilaginous sleeves in children with syndromic craniosynostosis.

PURPOSE: Because a tracheal cartilaginous sleeve (TCS) confers a significant mortality risk that can be mitigated with appropriate intervention, we sought to describe the prevalence and associated genotypes in a large cohort of children with syndromic craniosynostosis. METHODS: Chart review of patients with syndromic craniosynostosis across two institutions. RESULTS: In a cohort of 86 patients with syndromic craniosynostosis, 31 required airway evaluation under anesthesia. TCS was found in 19, for an overall prevalence of 22%. FGFR2, TWIST1, and FGFR3 mutations were identified in children with TCS. All five children with a W290C mutation in FGFR2 had TCS, and most previously reported children with W290C had identification of TCS or early death. In contrast, TCS was not associated with other mutations at residue 290. CONCLUSION: There is an association between TCS and syndromic craniosynostosis, and it appears to be particularly high in individuals with the W290C mutation in FGFR2. Referral to a pediatric otolaryngologist and consideration of operative airway evaluation (i.e., bronchoscopy or rigid endoscopy) in all patients with syndromic craniosynostosis should be considered to evaluate for TCS. Results from genetic testing may help providers weigh the risks and benefits of early airway evaluation and intervention in children with higher-risk genotypes.Genet Med 19 1, 62-68.

Novel findings of left ventricular non-compaction cardiomyopathy, microform cleft lip and poor vision in patient with SMC1A-associated Cornelia de Lange syndrome.

Relatively few patients with Cornelia de Lange syndrome (CdLS) due to SMC1A mutation have been reported, limiting understanding of the full extent of the phenotype. Compared to children with classic NIPBL-associated CdLS, patients with SMC1A-associated CdLS have a milder physical phenotype with prominent intellectual disability, high rate of cleft palate and absence of limb reductions. We present a patient with SMC1A-associated CdLS who had typical features including developmental delay, seizure disorder, feeding difficulties, hirsutism, and cleft palate. She also was found to have three novel features: (i) left ventricular non-compaction (LVNC) cardiomyopathy; (ii) microform cleft lip; and (iii) severe hyperopia and astigmatism. These features have implications regarding potential insight into the pathogenesis of the disorder, screening, and medical management. Hypertrophic cardiomyopathy has previously been reported in SMC1A-associated CdLS, but to our knowledge this is the first reported child with LVNC. Previous reports have included children with isolated clefts of the palate without involvement of the lip. When cleft palate alone is associated with a disorder, the underlying pathophysiology for clefting is sometimes secondary due to mechanical blocking of the fusion of the palatal shelves with the developing tongue. The presence of microform cleft lip in this patient suggests that the pathophysiology of clefting in SMC1A is primary rather than secondary. Few studies report ophthalmologic findings specific to SMC1A. Based on these findings, LVNC cardiomyopathy and cleft lip should be considered features of SMC1A-associated CdLS. All patients should receive echocardiogram and undergo thorough ophthalmologic evaluation as part of routine CdLS care. © 2016 Wiley Periodicals, Inc.

Characterizing facial features in individuals with craniofacial microsomia: A systematic approach for clinical research.

BACKGROUND: Craniofacial microsomia (CFM) is a congenital condition with wide phenotypic variability, including hypoplasia of the mandible and external ear. We assembled a cohort of children with facial features within the CFM spectrum and children without known craniofacial anomalies. We sought to develop a standardized approach to assess and describe the facial characteristics of the study cohort, using multiple sources of information gathered over the course of this longitudinal study and to create case subgroups with shared phenotypic features. METHODS: Participants were enrolled between 1996 and 2002. We classified the facial phenotype from photographs, ratings using a modified version of the Orbital, Ear, Mandible, Nerve, Soft tissue (OMENS) pictorial system, data from medical record abstraction, and health history questionnaires. RESULTS: The participant sample included 142 cases and 290 controls. The average age was 13.5 years (standard deviation, 1.3 years; range, 11.1-17.1 years). Sixty-one percent of cases were male, 74% were white non-Hispanic. Among cases, the most common features were microtia (66%) and mandibular hypoplasia (50%). Case subgroups with meaningful group definitions included: (1) microtia without other CFM-related features (n = 24), (2) microtia with mandibular hypoplasia (n = 46), (3) other combinations of CFM- related facial features (n = 51), and (4) atypical features (n = 21). CONCLUSION: We developed a standardized approach for integrating multiple data sources to phenotype individuals with CFM, and created subgroups based on clinically-meaningful, shared characteristics. We hope that this system can be used to explore associations between phenotype and clinical outcomes of children with CFM and to identify the etiology of CFM. Birth Defects Research (Part A) 106:915-926, 2016.© 2016 Wiley Periodicals, Inc.

A human homeotic transformation resulting from mutations in PLCB4 and GNAI3 causes auriculocondylar syndrome.

Auriculocondylar syndrome (ACS) is a rare, autosomal-dominant craniofacial malformation syndrome characterized by variable micrognathia, temporomandibular joint ankylosis, cleft palate, and a characteristic "question-mark" ear malformation. Careful phenotypic characterization of severely affected probands in our cohort suggested the presence of a mandibular patterning defect resulting in a maxillary phenotype (i.e., homeotic transformation). We used exome sequencing of five probands and identified two novel (exclusive to the patient and/or family studied) missense mutations in PLCB4 and a shared mutation in GNAI3 in two unrelated probands. In confirmatory studies, three additional novel PLCB4 mutations were found in multigenerational ACS pedigrees. All mutations were confirmed by Sanger sequencing, were not present in more than 10,000 control chromosomes, and resulted in amino-acid substitutions located in highly conserved protein domains. Additionally, protein-structure modeling demonstrated that all ACS substitutions disrupt the catalytic sites of PLCB4 and GNAI3. We suggest that PLCB4 and GNAI3 are core signaling molecules of the endothelin-1-distal-less homeobox 5 and 6 (EDN1-DLX5/DLX6) pathway. Functional studies demonstrated a significant reduction in downstream DLX5 and DLX6 expression in ACS cases in assays using cultured osteoblasts from probands and controls. These results support the role of the previously implicated EDN1-DLX5/6 pathway in regulating mandibular specification in other species, which, when disrupted, results in a maxillary phenotype. This work defines the molecular basis of ACS as a homeotic transformation (mandible to maxilla) in humans.

"Mandibulofacial dysostosis with microcephaly" caused by EFTUD2 mutations: expanding the phenotype.

Heterozygous mutations in the EFTUD2 were identified in 12 individuals with a rare sporadic craniofacial condition termed Mandibulofacial dysostosis with microcephaly (MIM 610536). We present clinical and radiographic features of three additional patients with de novo heterozygous mutations in EFTUD2. Although clinical features overlap with findings of the original report (choanal atresia, cleft palate, maxillary and mandibular hypoplasia, and microtia), microcephaly was present in two of three patients and cognitive impairment was milder in those with head circumference proportional to height. Our cases expand the phenotypic spectrum to include epibulbar dermoids and zygomatic arch clefting. We suggest that craniofacial computed tomography studies to assess cleft of zygomatic arch may assist in making this diagnosis. We recommend consideration of EFTUD2 testing in individuals with features of oculo-auriculo-vertebral spectrum and bilateral microtia, or individuals with atypical CHARGE syndrome who do not have a CHD7 mutation, particularly those with a zygomatic arch cleft. The absence of microcephaly in one patient indicates that it is a highly variable phenotypic feature.

Oculoauriculofrontonasal syndrome: case series revealing new bony nasal anomalies in an old syndrome.

Frontonasal Dysplasia (FND) and Oculo-auriculo-vertebral spectrum (OAVS) are two well-recognized clinical entities. With features of both FND and OAVS, the term oculoauriculofrontonasal syndrome (OAFNS) was coined in 1981. The OAFNS phenotype combines elements of abnormal morphogenesis of the frontonasal and maxillary process (derived from forebrain neural crest) with abnormal development of the first and second branchial arches (derived from hindbrain neural crest). We present a case series of 33 children with OAFNS ascertained from a comprehensive review of the literature and report an additional retrospective series of eight patients displaying features consistent with OAFNS. Notably, in a subset of our cases, we have observed abnormalities in nasal ossification and bony structures of the maxilla that have not previously described in OAFNS and are not seen in either FND or OAVS. We present the phenotype and novel naso-maxillary findings and explore potential etiologic and developmental pathways for OAFNS. We highlight the differences in phenotypic characteristics of OAFNS compared to OAVS and FND. These observations support the classification of OAFNS as a discrete syndrome. Further phenotypic refinements of OAFNS are needed to understand pathogenesis of this syndrome and the newly described nasal malformation may help identify the etiology.

Making the diagnosis: metopic ridge versus metopic craniosynostosis.

INTRODUCTION: The metopic suture is the only calvarial suture which normally closes during infancy. Upon closure, a palpable and visible ridge often forms which can be confused with metopic craniosynostosis. Metopic ridging (MR) is treated nonsurgically while metopic craniosynostosis (MCS) is treated surgically. Differentiating between the two is paramount; however, consensus is lacking about where a clear diagnostic threshold lies. The goal of this study is to describe the physical examination and CT scan characteristics which may help to differentiate between physiological closure of the metopic suture with ridging (MR) and MCS. METHODS: A retrospective chart review of all patients seen at Seattle Children's Hospital between 2004 and 2009 with the diagnosis of either MCS or MR (n = 282) was performed. Physical examination characteristics described by diagnosing practitioners were analyzed. Clinical photos were assessed by 3 expert raters to determine the importance of these characteristics. CT scan findings were abstracted and compared between the two diagnoses. RESULTS: The "classic" triad of narrow forehead, biparietal widening, and hypotelorism was present in only 14% of patients with MCS. Ninety-eight percent of patients in both groups had a palpable metopic ridge. The photographic finding of narrow forehead and pterional constriction was present in all patients with MCS, but only in 11.2% and 2.8% of patients with MR. On CT scan, the presence of 3 or more MCS findings was diagnostic of MCS in 96% of patients. Patients with MCS were more likely to present before 6 months of age (66% vs. 32%). CONCLUSIONS: Patients with MCS tend to present earlier than those with MR. Upon physical examination, the relationship between the lateral frontal bone and the lateral orbit is important in distinguishing between the two diagnoses. A CT scan can be helpful in making the diagnosis not to confirm a closed suture but to identify 3 or more MCS characteristics.

Bathrocephaly: a head shape associated with a persistent mendosal suture.

Bathrocephaly, a deformity of the posterior skull with bulging of the midportion of the occipital bone, is often associated with a benign variant of the mendosal suture ( Mulliken and Le, 2008 ). The endochondral and membranous portions of the occipital bone converge at the mendosal suture, which normally closes during fetal life or early infancy. When it persists, it is associated with a characteristic head shape that requires no intervention. We review the clinical findings associated with postnatal persistence of the mendosal suture and discuss other factors that may be associated with bathrocephaly.

Dominant-negative variant in SLC1A4 causes an autosomal dominant epilepsy syndrome.

SLC1A4 is a trimeric neutral amino acid transporter essential for shuttling L-serine from astrocytes into neurons. Individuals with biallelic variants in SLC1A4 are known to have spastic tetraplegia, thin corpus callosum, and progressive microcephaly (SPATCCM) syndrome, but individuals with heterozygous variants are not thought to have disease. We identify an 8-year-old patient with global developmental delay, spasticity, epilepsy, and microcephaly who has a de novo heterozygous three amino acid duplication in SLC1A4 (L86_M88dup). We demonstrate that L86_M88dup causes a dominant-negative N-glycosylation defect of SLC1A4, which in turn reduces the plasma membrane localization of SLC1A4 and the transport rate of SLC1A4 for L-serine.

Mechanism of KMT5B haploinsufficiency in neurodevelopment in humans and mice.

Pathogenic variants in KMT5B, a lysine methyltransferase, are associated with global developmental delay, macrocephaly, autism, and congenital anomalies (OMIM# 617788). Given the relatively recent discovery of this disorder, it has not been fully characterized. Deep phenotyping of the largest (n = 43) patient cohort to date identified that hypotonia and congenital heart defects are prominent features that were previously not associated with this syndrome. Both missense variants and putative loss-of-function variants resulted in slow growth in patient-derived cell lines. KMT5B homozygous knockout mice were smaller in size than their wild-type littermates but did not have significantly smaller brains, suggesting relative macrocephaly, also noted as a prominent clinical feature. RNA sequencing of patient lymphoblasts and Kmt5b haploinsufficient mouse brains identified differentially expressed pathways associated with nervous system development and function including axon guidance signaling. Overall, we identified additional pathogenic variants and clinical features in KMT5B-related neurodevelopmental disorder and provide insights into the molecular mechanisms of the disorder using multiple model systems.