DISP1 deficiency: monoallelic and biallelic variants cause a spectrum of midline craniofacial malformations.

PURPOSE: DISP1 encodes a transmembrane protein that regulates the secretion of the morphogen, Sonic hedgehog (SHH), a deficiency of which is a major cause of holoprosencephaly (HPE). This disorder covers a spectrum of brain and midline craniofacial malformations. The objective of the present study was to better delineate the clinical phenotypes associated with DISP1 variants. METHODS: This study was based on the identification of at least one pathogenic variant of the DISP1 gene in individuals for whom detailed clinical data were available. RESULTS: A total of 23 DISP1 variants were identified in heterozygous, compound heterozygous or homozygous states in 25 individuals with midline craniofacial defects. Most cases were minor forms of HPE, with craniofacial features such as orofacial cleft, solitary median maxillary central incisor (SMMCI), and congenital nasal pyriform aperture stenosis (CNPAS). These individuals had either monoallelic loss-of-function variants or biallelic missense variants in DISP1. In individuals with severe HPE, the DISP1 variants were commonly found associated with a variant in another HPE-linked gene (i.e. oligogenic inheritance). CONCLUSION: The genetic findings we have acquired demonstrate a significant involvement of DISP1 variants in the phenotypic spectrum of midline defects. This underlines its importance as a crucial element in the efficient secretion of SHH. We also demonstrated that the very rare SMMCI-CNPAS combination is part of the DISP1-related phenotype. The present study highlights the clinical risks to be flagged up during genetic counseling after the discovery of a pathogenic DISP1 variant.

Primary Failure of Dental Eruption Due to Variants Parathyroid Hormone Receptor 1: Retrospective Study and Proposal of Guidelines Treatment.

OBJECTIVE: Primary failure of eruption is characterized by a nonsyndromic defect in tooth eruption in the absence of mechanical obstruction. It is correlated to rare heterozygous variants in the parathyroid hormone receptor 1 gene. The management of primary failure of eruption is complex because many therapies are ineffective. The present study aimed to compare the clinical outcomes of our patients with the findings reported in the literature, and to propose a treatment guideline based on the literature and our experience. METHODS: Retrospective study of patients affected by primary dental eruption failure in the department and analyse of the results and compare with those of the litterature. RESULTS: Twelve patients belonging to 5 families (9 males, 3 females; 13-52 y old) diagnosed and treated in the maxillofacial surgery and stomatology department of the Lille University Hospital were included. All patients showed posterior tooth involvement, and most patients showed bilateral defects. None of the affected teeth had coronal alveolar bone, whereas 6 patients showed root resorption in the affected teeth. Genetic analyses, performed on 11 patients, identified a parathyroid hormone receptor 1 disease-causing variant in 7 of them (63%). Multidisciplinary treatment was required to rehabilitate these patients. Orthodontic interventions, even at an early age, are difficult in affected teeth, which are often blocked or have internal resorption. Moreover, retention of these affected teeth during growth leads to dentoskeletal malocclusions, requiring difficult surgical management in the long term. Therefore, early extraction of these teeth is frequently recommended once the diagnosis has been confirmed. An implant-borne prosthetic rehabilitation can then be achieved at the end of growth after correction of the jaw discrepancy. In case of a late diagnosis, other surgical or noninvasive techniques may be used depending on the clinical situation. Distraction osteogenesis or segmental osteotomy could be discussed for patients with mild phenotypes. CONCLUSIONS: Early diagnosis of primary eruption defects is crucial to offer appropriate management as early as possible, and so to avoid late complicated treatments.

Parenting stress and needs for social support in mothers and fathers of deaf or hard of hearing children.

Introduction: Hearing parents of deaf or hard of hearing (DHH) children may experience parenting stress and social support could be a buffer to this stress. Differences in levels of these two indicators may exist between mothers and fathers. This study focuses on the parenting stress and social support needs of mothers and fathers of DHH children. Methods: Twenty-seven French parental couples of DHH children completed the Parenting Stress Index and the Family Needs Survey, a questionnaire on social support needs. Results: Their overall stress scores showed no difference, but subdomain scores show that mothers and fathers are more stressed by the child's hyperactivity, and fathers by the child's adaptability, than parents of children with normal hearing. Mothers are more stressed than fathers by role restriction; they feel less free because of their parenting role. Fathers have a lower quality of attachment to their child than mothers. Parents have a high social support need, especially for obtaining information about their child's individual characteristics and health situation. The ranking of mothers and fathers in the top 10 needs reveals different needs profiles. Parenting stress profiles show that mothers and fathers with higher-than-normal stress levels have a greater overall need for social support than mothers and fathers with lower than normal stress levels. Discussion: This study highlights the value of assessing parenting stress and social support needs in parents of DHH children for a better understanding of their situation in research and its clinical implications, as well as the importance of differentiating outcomes for mothers and fathers.

Low risk of embryonic and other cancers in PIK3CA-related overgrowth spectrum: Impact on screening recommendations.

The PIK3CA-related overgrowth spectrum (PROS) encompasses various conditions caused by mosaic activating PIK3CA variants. PIK3CA somatic variants are also involved in various cancer types. Some generalized overgrowth syndromes are associated with an increased risk of Wilms tumor (WT). In PROS, abdominal ultrasound surveillance has been advocated to detect WT. We aimed to determine the risk of embryonic and other types of tumors in patients with PROS in order to evaluate surveillance relevance. We searched the clinical charts from 267 PROS patients for the diagnosis of cancer, and reviewed the medical literature for the risk of cancer. In our cohort, six patients developed a cancer (2.2%), and Kaplan Meier analyses estimated cumulative probabilities of cancer occurrence at 45 years of age was 5.6% (95% CI = 1.35%-21.8%). The presence of the PIK3CA variant was only confirmed in two out of four tumor samples. In the literature and our cohort, six cases of Wilms tumor/nephrogenic rests (0.12%) and four cases of other cancers have been reported out of 483 proven PIK3CA patients, in particular the p.(His1047Leu/Arg) variant. The risk of WT in PROS being lower than 5%, this is insufficient evidence to recommend routine abdominal imaging. Long-term follow-up studies are needed to evaluate the risk of other cancer types, as well as the relationship with the extent of tissue mosaicism and the presence or not of the variant in the tumor samples.

Pathogenic variants in the paired-related homeobox 1 gene (PRRX1) cause craniosynostosis with incomplete penetrance.

PURPOSE: Studies have previously implicated PRRX1 in craniofacial development, including demonstration of murine Prrx1 expression in the preosteogenic cells of the cranial sutures. We investigated the role of heterozygous missense and loss-of-function (LoF) variants in PRRX1 associated with craniosynostosis. METHODS: Trio-based genome, exome, or targeted sequencing were used to screen PRRX1 in patients with craniosynostosis; immunofluorescence analyses were used to assess nuclear localization of wild-type and mutant proteins. RESULTS: Genome sequencing identified 2 of 9 sporadically affected individuals with syndromic/multisuture craniosynostosis, who were heterozygous for rare/undescribed variants in PRRX1. Exome or targeted sequencing of PRRX1 revealed a further 9 of 1449 patients with craniosynostosis harboring deletions or rare heterozygous variants within the homeodomain. By collaboration, 7 additional individuals (4 families) were identified with putatively pathogenic PRRX1 variants. Immunofluorescence analyses showed that missense variants within the PRRX1 homeodomain cause abnormal nuclear localization. Of patients with variants considered likely pathogenic, bicoronal or other multisuture synostosis was present in 11 of 17 cases (65%). Pathogenic variants were inherited from unaffected relatives in many instances, yielding a 12.5% penetrance estimate for craniosynostosis. CONCLUSION: This work supports a key role for PRRX1 in cranial suture development and shows that haploinsufficiency of PRRX1 is a relatively frequent cause of craniosynostosis.

Exome sequencing efficacy and phenotypic expansions involving esophageal atresia/tracheoesophageal fistula plus.

Esophageal atresia/tracheoesophageal fistula (EA/TEF) is a life-threatening birth defect that often occurs with other major birth defects (EA/TEF+). Despite advances in genetic testing, a molecular diagnosis can only be made in a minority of EA/TEF+ cases. Here, we analyzed clinical exome sequencing data and data from the DECIPHER database to determine the efficacy of exome sequencing in cases of EA/TEF+ and to identify phenotypic expansions involving EA/TEF. Among 67 individuals with EA/TEF+ referred for clinical exome sequencing, a definitive or probable diagnosis was made in 11 cases for an efficacy rate of 16% (11/67). This efficacy rate is significantly lower than that reported for other major birth defects, suggesting that polygenic, multifactorial, epigenetic, and/or environmental factors may play a particularly important role in EA/TEF pathogenesis. Our cohort included individuals with pathogenic or likely pathogenic variants that affect TCF4 and its downstream target NRXN1, and FANCA, FANCB, and FANCC, which are associated with Fanconi anemia. These cases, previously published case reports, and comparisons to other EA/TEF genes made using a machine learning algorithm, provide evidence in support of a potential pathogenic role for these genes in the development of EA/TEF.

Monoallelic and biallelic variants in LEF1 are associated with a new syndrome combining ectodermal dysplasia and limb malformations caused by altered WNT signaling.

PURPOSE: LEF1 encodes a transcription factor acting downstream of the WNT-ß-catenin signaling pathway. It was recently suspected as a candidate for ectodermal dysplasia in 2 individuals carrying 4q35 microdeletions. We report on 12 individuals harboring LEF1 variants. METHODS: High-throughput sequencing was employed to delineate the genetic underpinnings of the disease. Cellular consequences were characterized by immunofluorescence, immunoblotting, pulldown assays, and/or RNA sequencing. RESULTS: Monoallelic variants in LEF1 were detected in 11 affected individuals from 4 unrelated families, and a biallelic variant was detected in an affected individual from a consanguineous family. The phenotypic spectrum includes various limb malformations, such as radial ray defects, polydactyly or split hand/foot, and ectodermal dysplasia. Depending on the type and location of LEF1 variants, the inheritance of this novel Mendelian condition can be either autosomal dominant or recessive. Our functional data indicate that 2 molecular mechanisms are at play: haploinsufficiency or loss of DNA binding are responsible for a mild to moderate phenotype, whereas loss of ß-catenin binding caused by biallelic variants is associated with a severe phenotype. Transcriptomic studies reveal an alteration of WNT signaling. CONCLUSION: Our findings establish mono- and biallelic variants in LEF1 as a cause for a novel syndrome comprising limb malformations and ectodermal dysplasia.

Toward clinical and molecular dissection of frontonasal dysplasia with facial skin polyps: From Pai syndrome to differential diagnosis through a series of 27 patients.

Unique or multiple congenital facial skin polyps are features of several rare syndromes, from the most well-known Pai syndrome (PS), to the less recognized oculoauriculofrontonasal syndrome (OAFNS), encephalocraniocutaneous lipomatosis (ECCL), or Sakoda complex (SC). We set up a research project aiming to identify the molecular bases of PS. We reviewed 27 individuals presenting with a syndromic frontonasal polyp and initially referred for PS. Based on strict clinical classification criteria, we could confirm only nine (33%) typical and two (7%) atypical PS individuals. The remaining ones were either OAFNS (11/27-41%) or presenting with an overlapping syndrome (5/27-19%). Because of the phenotypic overlap between these entities, OAFNS, ECCL, and SC can be either considered as differential diagnosis of PS or part of the same spectrum. Exome and/or genome sequencing from blood DNA in 12 patients and from affected tissue in one patient failed to identify any replication in candidate genes. Taken together, our data suggest that conventional approaches routinely utilized for the identification of molecular etiologies responsible for Mendelian disorders are inconclusive. Future studies on affected tissues and multiomics studies will thus be required in order to address either the contribution of mosaic or noncoding variation in these diseases.

First evidence of SOX2 mutations in Peters' anomaly: Lessons from molecular screening of 95 patients.

Peters' anomaly (PA) is a rare anterior segment dysgenesis characterized by central corneal opacity and irido-lenticulo-corneal adhesions. Several genes are involved in syndromic or isolated PA (B3GLCT, PAX6, PITX3, FOXE3, CYP1B1). Some copy number variations (CNVs) have also been occasionally reported. Despite this genetic heterogeneity, most of patients remain without genetic diagnosis. We retrieved a cohort of 95 individuals with PA and performed genotyping using a combination of comparative genomic hybridization, whole genome, exome and targeted sequencing of 119 genes associated with ocular development anomalies. Causative genetic defects involving 12 genes and CNVs were identified for 1/3 of patients. Unsurprisingly, B3GLCT and PAX6 were the most frequently implicated genes, respectively in syndromic and isolated PA. Unexpectedly, the third gene involved in our cohort was SOX2, the major gene of micro-anophthalmia. Four unrelated patients with PA (isolated or with microphthalmia) were carrying pathogenic variants in this gene that was never associated with PA before. Here we described the largest cohort of PA patients ever reported. The genetic bases of PA are still to be explored as genetic diagnosis was unavailable for 2/3 of patients. Nevertheless, we showed here for the first time the involvement of SOX2 in PA, offering new evidence for its role in corneal transparency and anterior segment development.

The natural history of a family with aortic dissection associated with a novel ACTA2 variant.

Disease-causing heterozygous variants in the ACTA2 gene cause an autosomal dominant heritable thoracic aortic disease (HTAD) with thoracic aortic aneurysm and dissection as main phenotype, and occasional extravascular abnormalities such as livedo reticularis. ACTA2-HTAD accounts for an important part of non-syndromic HTAD, with detection rates varying between 1.5-21% according to different studies. A consensus statement for the screening and management of patients with pathogenic ACTA2 variants has been recently published by the European reference network for rare vascular diseases (VASCERN). However, management of ACTA2 patients is often challenged by extremely variable inter- and intra-familial clinical courses of the disease. Here we report a family harboring a disease-causing ACTA2 variant. The proband and two siblings presented with acute type A aortic dissection and rupture involving nondilated aortic segments before the age of 30. Their mother died at 49 years-old from type B aortic dissection and rupture. Genetic testing revealed the heterozygous novel p.(Pro335Arg) variant in the ACTA2 gene in the proband and in the affected siblings. The clinical history of this family highlights the difficulty of adopting effective prevention strategies in ACTA2 patients.

Phenotypic spectrum of TGFB3 disease-causing variants in a Dutch-French cohort and first report of a homozygous patient.

Disease-causing variants in TGFB3 cause an autosomal dominant connective tissue disorder which is hard to phenotypically delineate because of the small number of identified cases. The purpose of this retrospective cross-sectional multicenter study is to elucidate the genotype and phenotype in an international cohort of TGFB3 patients. Eleven (eight novel) TGFB3 disease-causing variants were identified in 32 patients (17 families). Aortic root dilatation and mitral valve disease represented the most common cardiovascular findings, reported in 29% and 32% of patients, respectively. Dissection involving distal aortic segments occurred in two patients at age 50 and 52 years. A high frequency of systemic features (65% high-arched palate, 63% arachnodactyly, 57% pectus deformity, 52% joint hypermobility) was observed. In familial cases, incomplete penetrance and variable clinical expressivity were noted. Our cohort included the first described homozygous patient, who presented with a more severe phenotype compared to her heterozygous relatives. In conclusion, TGFB3 variants were associated with a high percentage of systemic features and aortic disease (dilatation/dissection) in 35% of patients. No deaths occurred from cardiovascular events or pregnancy-related complications. Nevertheless, homozygosity may be driving a more severe phenotype.

Multiplex targeted high-throughput sequencing in a series of 352 patients with congenital limb malformations.

Congenital limb malformations (CLM) comprise many conditions affecting limbs and more than 150 associated genes have been reported. Due to this large heterogeneity, a high proportion of patients remains without a molecular diagnosis. In the last two decades, advances in high throughput sequencing have allowed new methodological strategies in clinical practice. Herein, we report the screening of 52 genes/regulatory sequences by multiplex high-throughput targeted sequencing, in a series of 352 patients affected with various CLM, over a 3-year period of time. Patients underwent a clinical triage by expert geneticists in CLM. A definitive diagnosis was achieved in 35.2% of patients, the yield varying considerably, depending on the phenotype. We identified 112 single nucleotide variants and 26 copy-number variations, of which 52 are novel pathogenic or likely pathogenic variants. In 6% of patients, variants of uncertain significance have been found in good candidate genes. We showed that multiplex targeted high-throughput sequencing works as an efficient and cost-effective tool in clinical practice for molecular diagnosis of congenital limb malformations. Careful clinical evaluation of patients may maximize the yield of CLM panel testing.

Holt-Oram syndrome: clinical and molecular description of 78 patients with TBX5 variants.

Holt-Oram syndrome (HOS) is an autosomal dominant condition characterised by the association of congenital heart defect (CHD), with or without rhythm disturbances and radial defects, due to TBX5 variants. The diagnosis is challenged by the variability of expression and the large phenotypic overlap with other conditions, like Okihiro syndrome, TAR syndrome or Fanconi disease. We retrospectively reviewed 212 patients referred for suspicion of HOS between 2002 and 2014, who underwent TBX5 screening. A TBX5 variant has been identified in 78 patients, representing the largest molecular series ever described. In the cohort, 61 met the previously described diagnostic criteria and 17 have been considered with an uncertain HOS diagnosis. A CHD was present in 91% of the patients with a TBX5 variant, atrial septal defects being the most common (61.5%). The genotype-phenotype study highlights the importance of some critical features in HOS: the septal characteristic of the CHD, the bilateral and asymmetric characteristics of the radial defect and the presence of shoulder or elbow mobility defect. Besides, 21 patients presented with an overlapping condition. Among them, 13 had a typical HOS presentation. We discuss the strategies that could be adopted to improve the molecular delineation of the remaining typical patients.

Delineating the psychiatric and behavioral phenotype of recurrent 2q13 deletions and duplications.

Recurrent deletions and duplications at the 2q13 locus have been associated with developmental delay (DD) and dysmorphisms. We aimed to undertake detailed clinical characterization of individuals with 2q13 copy number variations (CNVs), with a focus on behavioral and psychiatric phenotypes. Participants were recruited via the Unique chromosomal disorder support group, U.K. National Health Service Regional Genetics Centres, and the DatabasE of genomiC varIation and Phenotype in Humans using Ensembl Resources (DECIPHER) database. A review of published 2q13 patient case reports was undertaken to enable combined phenotypic analysis. We present a new case series of 2q13 CNV carriers (21 deletion, 4 duplication) and the largest ever combined analysis with data from published studies, making a total of 54 deletion and 23 duplication carriers. DD/intellectual disabilities was identified in the majority of carriers (79% deletion, 70% duplication), although in the new cases 52% had an IQ in the borderline or normal range. Despite the median age of the new cases being only 9 years, 64% had a clinical psychiatric diagnosis. Combined analysis found attention deficit hyperactivity disorder (ADHD) to be the most frequent diagnosis (48% deletion, 60% duplication), followed by autism spectrum disorders (33% deletion, 17% duplication). Aggressive (33%) and self-injurious behaviors (33%) were also identified in the new cases. CNVs at 2q13 are typically associated with DD with mildly impaired intelligence, and a high rate of childhood psychiatric diagnoses-particularly ADHD. We have further characterized the clinical phenotype related to imbalances of the 2q13 region and identified it as a region of interest for the neurobiological investigation of ADHD.

19p13 microduplications encompassing NFIX are responsible for intellectual disability, short stature and small head circumference.

Syndromes caused by copy number variations are described as reciprocal when they result from deletions or duplications of the same chromosomal region. When comparing the phenotypes of these syndromes, various clinical features could be described as reversed, probably due to the opposite effect of these imbalances on the expression of genes located at this locus. The NFIX gene codes for a transcription factor implicated in neurogenesis and chondrocyte differentiation. Microdeletions and loss of function variants of NFIX are responsible for Sotos syndrome-2 (also described as Malan syndrome), a syndromic form of intellectual disability associated with overgrowth and macrocephaly. Here, we report a cohort of nine patients harboring microduplications encompassing NFIX. These patients exhibit variable intellectual disability, short stature and small head circumference, which can be described as a reversed Sotos syndrome-2 phenotype. Strikingly, such a reversed phenotype has already been described in patients harboring microduplications encompassing NSD1, the gene whose deletions and loss-of-function variants are responsible for classical Sotos syndrome. Even though the type/contre-type concept has been criticized, this model seems to give a plausible explanation for the pathogenicity of 19p13 microduplications, and the common phenotype observed in our cohort.

Cerebellar hypoplasia with endosteal sclerosis is a POLR3-related disorder.

CHES (cerebellar hypoplasia with endosteal sclerosis) syndrome (OMIM#213002) associates hypomyelination, cerebellar atrophy, hypogonadism and hypodontia. So far, only five patients have been described. The condition is of neonatal onset. Patients have severe psychomotor delay and moderate to severe intellectual disability. Inheritance is assumed to be autosomal recessive due to recurrence in sibs, consanguinity of parents and absence of vertical transmission. CHES syndrome is reminiscent of 4H-leukodystrophy, a recessive-inherited affection due to variations in genes encoding subunits of the RNA polymerase III (POLR3A-POLR3B-POLR1C). POLR3B variants have been identified in one CHES patient. Here we report on a novel CHES patient, carrying compound heterozygous variations in POLR3B. This report confirms affiliation of CHES to POLR3-related disorders and suggests that CHES syndrome represents a severe form of 4H-leukodystrophy.

Blepharocheilodontic syndrome is a CDH1 pathway-related disorder due to mutations in CDH1 and CTNND1.

PURPOSE: Blepharocheilodontic (BCD) syndrome is a rare autosomal dominant condition characterized by eyelid malformations, cleft lip/palate, and ectodermal dysplasia. The molecular basis of BCD syndrome remains unknown. METHODS: We recruited 11 patients from 8 families and performed exome sequencing for 5 families with de novo BCD syndrome cases and targeted Sanger sequencing in the 3 remaining families. RESULTS: We identified five CDH1 heterozygous missense mutations and three CTNND1 heterozygous truncating mutations leading to loss-of-function or haploinsufficiency. Establishment of detailed genotype-phenotype correlations was not possible because of the size of the cohort; however, the phenotype seems to appear more severe in case of CDH1 mutations. Functional analysis of CDH1 mutations confirmed their deleterious impact and suggested accelerated E-cadherin degradation. CONCLUSION: Mutations in CDH1 encoding the E-cadherin were previously reported in hereditary diffuse gastric cancer as well as in nonsyndromic cleft lip/palate. Mutations in CTNND1 have never been reported before. The encoded protein, p120ctn, prevents E-cadherin endocytosis and stabilizes its localization at the cell surface. Conditional deletion of Cdh1 and Ctnnd1 in various animal models induces features reminiscent of BCD syndrome and underlines critical role of the E-cadherin-p120ctn interaction in eyelid, craniofacial, and tooth development. Our data assert BCD syndrome as a CDH1 pathway-related disorder due to mutations in CDH1 and CTNND1 and widen the phenotypic spectrum of E-cadherin anomalies.Genet Med advance online publication 09 March 2017.

15q11.2 microdeletion (BP1-BP2) and developmental delay, behaviour issues, epilepsy and congenital heart disease: a series of 52 patients.

Proximal region of chromosome 15 long arm is rich in duplicons that, define five breakpoints (BP) for 15q rearrangements. 15q11.2 microdeletion between BP1 and BP2 has been previously associated with developmental delay and atypical psychological patterns. This region contains four highly-conserved and non-imprinted genes: NIPA1, NIPA2, CYFIP1, TUBGCP5. Our goal was to investigate the phenotypes associated with this microdeletion in a cohort of 52 patients. This copy number variation (CNV) was prevalent in 0.8% patients presenting with developmental delay, psychological pattern issues and/or multiple congenital malformations. This was studied by array-CGH at six different French Genetic laboratories. We collected data from 52 unrelated patients (including 3 foetuses) after excluding patients with an associated genetic alteration (known CNV, aneuploidy or known monogenic disease). Out of 52 patients, mild or moderate developmental delay was observed in 68.3%, 85.4% had speech impairment and 63.4% had psychological issues such as Attention Deficit and Hyperactivity Disorder, Autistic Spectrum Disorder or Obsessive-Compulsive Disorder. Seizures were noted in 18.7% patients and associated congenital heart disease in 17.3%. Parents were analysed for abnormalities in the region in 65.4% families. Amongst these families, 'de novo' microdeletions were observed in 18.8% and 81.2% were inherited from one of the parents. Incomplete penetrance and variable expressivity were observed amongst the patients. Our results support the hypothesis that 15q11.2 (BP1-BP2) microdeletion is associated with developmental delay, abnormal behaviour, generalized epilepsy and congenital heart disease. The later feature has been rarely described. Incomplete penetrance and variability of expression demands further assessment and studies.