Prenatal diagnosis of Hartsfield syndrome with a novel genetic variant.

A G2P0, 24-year-old woman presented at 17 weeks 3 days gestation for a fetal anatomy scan. Ultrasound identified bilateral upper and lower extremity ectrodactyly, semilobar holoprosencephaly, midface hypoplasia, and cleft lip and palate. Amniocentesis for a chromosome microarray demonstrated no significant copy number changes. Whole exome sequencing was subsequently completed, which revealed a de novo, likely pathogenic variant in FGFR1, c.2044G>A (D682N), consistent with FGFR1-related Hartsfield syndrome. This case highlights the first presumed molecularly confirmed prenatal diagnosis of Hartsfield syndrome and identifies a new pathogenic variant.

Fetal Description of the Pancreatic Agenesis and Holoprosencephaly Syndrome Associated to a Specific CNOT1 Variant.

Holoprosencephaly (HPE) is a clinically and genetically heterogeneous disease, which can be associated with various prenatal comorbidities not always detectable on prenatal ultrasound. We report on the case of a foetus carrying a semi-lobar HPE diagnosed at ultrasound, for which a fetal autopsy and a whole exome sequencing were performed following a medical termination of pregnancy. Neuropathological examination confirmed the semi-lobar HPE and general autopsy disclosed a total pancreas agenesis. Whole exome sequencing found the CNOT1 missense c.1603C>T, p.(Arg535Cys), occurring de novo in the foetus. The same variant was previously reported in 5 unrelated children. All individuals had HPE, and 4 out of 5 presented endo- and exocrine pancreatic insufficiency or total pancreas agenesis. CNOT1 encodes a subunit of the CCRN4-NOT complex, expressed at the early stage of embryonic development. This report is the first fetal description of the phenotype associating HPE and pancreatic agenesis linked to the recurrent CNOT1 missense c.1603C>T, p.(Arg535Cys). This finding strengthens the hypothesis of a specific recurrent variant associated with a particular phenotype of HPE and pancreas agenesis. The fetal autopsy that revealed the pancreas agenesis was crucial in guiding the genetic diagnosis and enabling accurate genetic counselling.

Alobar holoprosencephaly: Exploring mothers' perspectives on prenatal decision-making and prognostication.

OBJECTIVES: Parents receiving a prenatal diagnosis of alobar holoprosencephaly (HPE) often experience uncertainty regarding the pregnancy prognosis. There is little known about how best to counsel and support families receiving the diagnosis. This study explored parental experiences and wishes after receiving a prenatal diagnosis of alobar HPE. METHODS: This was a retrospective qualitative study. Semi-structured interviews were conducted to determine factors impacting parents' decision-making process, experiences with healthcare providers, and expectations for their child's length and quality of life. Interviews were analyzed using qualitative content analysis. RESULTS: Eight mothers who received a prenatal diagnosis of alobar HPE between 2013 and 2019 participated in the study. Parental expectations were based on information conveyed during prenatal counseling. Religious and personal beliefs, perceived suffering, and provider prognostication contributed to parent decisions and goals of care. Participants reported pressure to terminate the pregnancy. Parents were not prepared for the possibility of survival beyond the perinatal period. Most parents reported no regret in their choices. CONCLUSION: Patients receiving a prenatal diagnosis of alobar HPE desire access to balanced prenatal counseling about prognosis, morbidity, and mortality. Providers should explore values and beliefs that contribute to parents' goals of care and offer appropriate information and referrals.

Compound heterozygous splicing CDON variants result in isolated ocular coloboma.

Ocular coloboma is caused by failure of optic fissure closure during development and recognized as part of the microphthalmia, anophthalmia, and coloboma (MAC) spectrum. While many genes are known to cause colobomatous microphthalmia, relatively few have been reported in coloboma with normal eye size. Genetic analysis including trio exome sequencing and Sanger sequencing was undertaken in a family with two siblings affected with bilateral coloboma of the iris, retina, and choroid. Pathogenic variants in MAC genes were excluded. Trio analysis identified compound heterozygous donor splice site variants in CDON, a cell-surface receptor known to function in the Sonic Hedgehog pathway, c.928 + 1G > A and c.2650 + 1G > T, in both affected individuals. Heterozygous missense and truncating CDON variants are associated with dominant holoprosencephaly (HPE) with incomplete penetrance and Cdon-/- mice display variable HPE and coloboma. A homozygous nonsense allele of uncertain significance was recently identified in a consanguineous patient with coloboma and a second molecular diagnosis. We report the first compound heterozygous variants in CDON as a cause of isolated coloboma. CDON is the first HPE gene identified to cause recessive coloboma. Given the phenotypic overlap, further examination of HPE genes in coloboma is indicated.

Holoprosencephaly in Kabuki syndrome.

Kabuki syndrome is a rare, multi-systemic disorder of chromatin regulation due to mutations in either KMT2D or KDM6A that encode a H3K4 methyltransferase and an H3K27 demethylase, respectively. The associated clinical phenotype is a direct result of temporal and spatial changes in gene expression in various tissues including the brain. Although mild to moderate intellectual disability is frequently recognized in individuals with Kabuki syndrome, the identification of brain anomalies, mostly involving the hippocampus and related structures remains an exception. Recently, the first two cases with alobar holoprosencephaly and mutations in KMT2D have been reported in the medical literature. We identified a de novo, pathogenic KMT2D variant (c.6295C > T; p.R2099X) using trio whole-exome sequencing in a 2-year-old female with lobar holoprosencephaly, microcephaly and cranio-facial features of Kabuki syndrome. This report expands the spectrum of brain anomalies associated with Kabuki syndrome underscoring the important role of histone modification for early brain development.

Cohesin complex-associated holoprosencephaly.

Marked by incomplete division of the embryonic forebrain, holoprosencephaly is one of the most common human developmental disorders. Despite decades of phenotype-driven research, 80-90% of aneuploidy-negative holoprosencephaly individuals with a probable genetic aetiology do not have a genetic diagnosis. Here we report holoprosencephaly associated with variants in the two X-linked cohesin complex genes, STAG2 and SMC1A, with loss-of-function variants in 10 individuals and a missense variant in one. Additionally, we report four individuals with variants in the cohesin complex genes that are not X-linked, SMC3 and RAD21. Using whole mount in situ hybridization, we show that STAG2 and SMC1A are expressed in the prosencephalic neural folds during primary neurulation in the mouse, consistent with forebrain morphogenesis and holoprosencephaly pathogenesis. Finally, we found that shRNA knockdown of STAG2 and SMC1A causes aberrant expression of HPE-associated genes ZIC2, GLI2, SMAD3 and FGFR1 in human neural stem cells. These findings show the cohesin complex as an important regulator of median forebrain development and X-linked inheritance patterns in holoprosencephaly.

Novel heterozygous variants in KMT2D associated with holoprosencephaly.

Lysine methyltransferase 2D (KMT2D; OMIM 602113) encodes a histone methyltransferase involved in transcriptional regulation of the beta-globin and estrogen receptor as part of a large protein complex known as activating signal cointegrator-2-containing complex (ASCOM). Heterozygous germline mutations in the KMT2D gene are known to cause Kabuki syndrome (OMIM 147920), a developmental multisystem disorder. Neither holoprosencephaly nor other defects in human forebrain development have been previously associated with Kabuki syndrome. Here we report two patients diagnosed with alobar holoprosencephaly in their antenatal period with de novo monoallelic KMT2D variants identified by trio-based exome sequencing. The first patient was found to have a stop-gain variant c.12565G>T (p.Gly4189*), while the second patient had a missense variant c.5A>G (p.Asp2Gly). Phenotyping of each patient did not reveal any age-related feature of Kabuki syndrome. These two cases represent the first report on association between KMT2D and holoprosencephaly.

Novel synonymous and missense variants in FGFR1 causing Hartsfield syndrome.

Hartsfield syndrome is a rare clinical entity characterized by holoprosencephaly and ectrodactyly with the variable feature of cleft lip/palate. In addition to these symptoms patients with Hartsfield syndrome can show developmental delay of variable severity, isolated hypogonadotropic hypogonadism, central diabetes insipidus, vertebral anomalies, eye anomalies, and cardiac malformations. Pathogenic variants in FGFR1 have been described to cause phenotypically different FGFR1-related disorders such as Hartsfield syndrome, hypogonadotropic hypogonadism with or without anosmia, Jackson-Weiss syndrome, osteoglophonic dysplasia, Pfeiffer syndrome, and trigonocephaly Type 1. Here, we report three patients with Hartsfield syndrome from two unrelated families. Exome sequencing revealed two siblings harboring a novel de novo heterozygous synonymous variant c.1029G>A, p.Ala343Ala causing a cryptic splice donor site in exon 8 of FGFR1 likely due to gonadal mosaicism in one parent. The third case was a sporadic patient with a novel de novo heterozygous missense variant c.1868A>G, p.(Asp623Gly).

Holoprosencephaly, orofacial cleft, and frontonaso-orbital encephaloceles: Genetic evaluation of a possible new syndrome.

Here we report on a Brazilian child who presented semilobar holoprosencephaly, frontonasal encephaloceles and bilateral cleft lip and palate. Malformations also included agenesis of the corpus callosum, abnormal cortical gyres, dilation of the aqueduct, bilateral endolymphatic sac, bilateral cystic cocci-vestibular malformation, and a cribriform defect. The 3D TC craniofacial images showed abnormal frontonasal transition region, with a bone bifurcation, and partial agenesis of nasal bone. The trunk and upper and lower limbs were normal. To our knowledge, this rare association of holoprocensephaly with frontonaso-orbital encephaloceles without limb anomalies has never been reported before. Karyotype was normal. SNP-array showed no copy-number alterations but revealed 25% of regions of homozygosity (ROH) with normal copy number, indicating a high coefficient of inbreeding, which significantly increases the risk for an autosomal recessive disorder. Whole exome sequencing analysis did not reveal any pathogenic or likely pathogenic variants. We discuss the possible influence of two variants of uncertain significance found within the patient's ROHs. First, a missense p.(Gly394Ser) in PCSK9, a gene involved in the regulation of plasma low-density lipoprotein cholesterol. Second, an inframe duplication p.(Ala75_Ala81dup) in SP8, a zinc-finger transcription factor that regulates signaling centers during craniofacial development. Further studies and/or the identification of other patients with a similar phenotype will help elucidate the genetic etiology of this complex case.

The solitary median maxillary central incisor (SMMCI) syndrome: Associations, prenatal diagnosis, and outcomes.

Solitary median maxillary central incisor (SMMCI) syndrome is a complex disorder consisting of multiple, developmental defects involving midline structures of the head, which includes the cranial bones, the maxilla, and its container dentition (specifically the central incisor tooth germ), together with other midline structures of the body. SMMCI may appear as an isolated trait or in association with other midline developmental anomalies. We describe the case of a patient with SMMCI. He presented with a solitary median maxillary incisor, short stature, corpus callosum anomalies and a microform of holoprosencephaly (HPE), diabetes insipidus, and neurodevelopmental delay. The diagnosis was performed postnatally based on clinical features, radiological imaging, and a comprehensive genetic study. SMMCI can be diagnosed during the prenatal or neonatal periods or during infancy. Evaluation of the superior maxillary bone is important for prenatal diagnosis. Direct evaluation through bidimensional ultrasound or the use of multiplanar ultrasound or tridimensional reconstruction should be performed in cases of brain or face malformations. Early diagnosis can contribute to improved prenatal assessment and postnatal management.

Common genetic causes of holoprosencephaly are limited to a small set of evolutionarily conserved driver genes of midline development coordinated by TGF-ß, hedgehog, and FGF signaling.

Here, we applied targeted capture to examine 153 genes representative of all the major vertebrate developmental pathways among 333 probands to rank their relative significance as causes for holoprosencephaly (HPE). We now show that comparisons of variant transmission versus nontransmission among 136 HPE Trios indicates some reported genes now lack confirmation, while novel genes are implicated. Furthermore, we demonstrate that variation of modest intrinsic effect can synergize with these driver mutations as gene modifiers.

Syndromes associated with holoprosencephaly.

Holoprosencephaly (HPE) is partial or complete failure of the forebrain to divide into hemispheres and can be an isolated finding or associated with a syndrome. Most cases of HPE are associated with a syndrome and roughly 40%-60% of fetuses with HPE have trisomy 13 which is the most common etiology of HPE. Other syndromes associated with HPE include additional aneuploidies like trisomy 18 and single gene disorders such as Smith-Lemli-Opitz syndrome. There are a number of syndromes such as pseudotrisomy 13 which do not have a known molecular etiology; therefore, this review has two parts: syndromes with a molecular diagnosis and syndromes where the etiology is yet to be found. As most HPE is syndromic, this review provides a comprehensive list and description of syndromes associated with HPE that may be used as a differential diagnosis and starting point for evaluating individuals with HPE.

Molecular testing in holoprosencephaly.

Holoprosencephaly (HPE) is a structural brain anomaly characterized by failure of the forebrain to separate during early embryogenesis. Both genetic and environmental etiologies of HPE have been discovered over the last three decades. Traditionally, the genetic workup for HPE has been a karyotype, chromosomal microarray, and/or Sanger sequencing of select genes. The recent increased availability of next-generation sequencing has changed the molecular diagnostic landscape for HPE, associating new genes with this disorder such as FGFR1. We conducted a systematic review of the medical literature for the molecular testing of HPE for studies published in the last 20 years. We also queried known commercial diagnostic laboratories and used information on their websites to construct a list of available commercial testing. Our group released its first recommendations in 2010 and this update incorporates the technology shifts and gene discoveries over the last decade. These recommendations provide a guide for genetic diagnosis of HPE, which is paramount for patients and their families for prognosis, treatment, and genetic counseling.

Challenging issues arising in counseling families experiencing holoprosencephaly.

The provision of information and support to families experiencing holoprosencephaly (HPE) in a loved one is unequivocally challenging, even for the most experienced clinicians. It deserves the balance of pertinent information coupled with medical guidance that forms the basis for shared decision-making; all of which is ideally contained within a supportive environment. It requires a willingness to carefully listen to the specific concerns of the parents and family allowing them to revisit challenging issues as much as needed to encourage existing road blocks to be resolved. It necessitates that professionals see each and every family as unique, without preconceived notions about what is or is not important and being prepared to accept thoughts and decisions that may not fit with the professional's own beliefs. To some, this may sound impractical, inefficient, or even impossible within the time constrained models of modern day clinical services. However, in practice, this patient-focused approach is arguably the most essential step in providing "personalized medicine" to the populations we encounter. This manuscript is intended to provide a brief review of relevant literature and case discussions to highlight issues for families learning of the diagnosis of HPE during a pregnancy, at birth, during childhood or more rarely, in adolescence.

Cytogenetics and holoprosencephaly: A chromosomal microarray study of 222 individuals with holoprosencephaly.

Holoprosencephaly (HPE), a common developmental forebrain malformation, is characterized by failure of the cerebrum to completely divide into left and right hemispheres. The etiology of HPE is heterogeneous and a number of environmental and genetic factors have been identified. Cytogenetically visible alterations occur in 25% to 45% of HPE patients and cytogenetic techniques have long been used to study copy number variants (CNVs) in this disorder. The karyotype approach initially demonstrated several recurrent chromosomal anomalies, which led to the identification of HPE-specific loci and, eventually, several major HPE genes. More recently, higher-resolution cytogenetic techniques such as subtelomeric multiplex ligation-dependent probe amplification and chromosomal microarray have been used to analyze chromosomal anomalies. By using chromosomal microarray, we sought to identify submicroscopic chromosomal deletions and duplications in patients with HPE. In an analysis of 222 individuals with HPE, a deletion or duplication was detected in 107 individuals. Of these 107 individuals, 23 (21%) had variants that were classified as pathogenic or likely pathogenic by board-certified medical geneticists. We identified multiple patients with deletions in established HPE loci as well as three patients with deletions encompassed by 6q12-q14.3, a CNV previously reported by Bendavid et al. In addition, we identified a new locus, 16p13.2 that warrants further investigation for HPE association. Incidentally, we also found a case of Potocki-Lupski syndrome, a case of Phelan-McDermid syndrome, and multiple cases of 22q11.2 deletion syndrome within our cohort. These data confirm the genetically heterogeneous nature of HPE, and also demonstrate clinical utility of chromosomal microarray in diagnosing patients affected by HPE.

Extracephalic manifestations of nonchromosomal, nonsyndromic holoprosencephaly.

Nonchromosomal, nonsyndromic holoprosencephaly (NCNS-HPE) has traditionally been considered as a condition of brain and craniofacial maldevelopment. In this review, we present the results of a comprehensive literature search supporting a wide spectrum of extracephalic manifestations identified in patients with NCNS-HPE. These manifestations have been described in case reports and in large cohorts of patients with "single-gene" mutations, suggesting that the NCNS-HPE phenotype can be more complex than traditionally thought. Likely, a complex network of interacting genetic variants and environmental factors is responsible for these systemic abnormalities that deviate from the usual brain and craniofacial findings in NCNS-HPE. In addition to the systemic consequences of pituitary dysfunction (as a direct result of brain midline defects), here we describe a number of extracephalic findings of NCNS-HPE affecting various organ systems. It is our goal to provide a guide of extracephalic features for clinicians given the important clinical implications of these manifestations for the management and care of patients with HPE and their mutation-positive relatives. The health risks associated with some manifestations (e.g., fatty liver disease) may have historically been neglected in affected families.

Modeling the complex etiology of holoprosencephaly in mice.

Holoprosencephaly (HPE) is a common developmental defect caused by failure to define the midline of the forebrain and/or midface. HPE is associated with heterozygous mutations in Nodal and Sonic hedgehog (SHH) pathway components, but clinical presentation is highly variable, and many mutation carriers are unaffected. It is therefore thought that such mutations interact with more common modifiers, genetic and/or environmental, to produce severe patterning defects. Modifiers are difficult to identify, as their effects are context-dependent and occur within the complex genetic and environmental landscapes that characterize human populations. This has made a full understanding of HPE etiology challenging. We discuss here the use of mice, a genetically tractable model sensitive to teratogens, as a system to address this challenge. Mice carrying mutations in human HPE genes often display wide variations in phenotypic penetrance and expressivity when placed on different genetic backgrounds, demonstrating the existence of silent HPE modifier genes. Studies with mouse lines carrying SHH pathway mutations on appropriate genetic backgrounds have led to identification of both genetic and environmental modifiers that synergize with the mutations to produce a spectrum of HPE phenotypes. These models favor a scenario in which multiple modifying influences-both genetic and environmental, sensitizing and protective-interact with bona fide HPE mutations to grade phenotypic outcomes. Despite the complex interplay of HPE risk factors, mouse models have helped establish some clear concepts in HPE etiology. A combination of mouse and human cohort studies should improve our understanding of this fascinating and medically important issue.

Further evidence for complex inheritance of holoprosencephaly: Lessons learned from pre- and postnatal diagnostic testing in Germany.

Holoprosencephaly (HPE) has been defined as a distinct clinical entity with characteristic facial gestalt, which may-or may not-be associated with the true brain malformation observed postmortem in autopsy or in pre- or postnatal imaging. Affected families mainly show autosomal dominant inheritance with markedly reduced penetrance and extremely broad clinical variability even between mutation carriers within the same families. We here present advances in prenatal imaging over the last years, increasing the proportion of individuals with HPE identified prenatally including milder HPE forms and more frequently allowing to detect more severe forms already in early gestation. We report the results of diagnostic genetic testing of 344 unrelated patients for HPE at our lab in Germany since the year 2000, which currently with the application of next generation sequencing (NGS) panel sequencing identifies causal mutations for about 31% (12/38) of unrelated individuals with normal chromosomes when compared to about 15% (46/306) using conventional Sanger sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA). More comprehensive genetic testing by our in house NGS panel sequencing of 10 HPE associated genes (MiSeq™ and NextSeq™500, Illumina, Inc., San Diego, CA) not only allowed to include genes with smaller contribution to the phenotype, but may also unravel additional low frequency or more common genetic variants potentially contributing to the observed large intrafamiliar variability and may ultimately guide our understanding of the individual clinical manifestation of this complex developmental disorder.

In-depth investigations of adolescents and adults with holoprosencephaly identify unique characteristics.

PurposeWith improved medical care, some individuals with holoprosencephaly (HPE) are surviving into adulthood. We investigated the clinical manifestations of adolescents and adults with HPE and explored the underlying molecular causes.MethodsParticipants included 20 subjects 15 years of age and older. Clinical assessments included dysmorphology exams, cognitive testing, swallowing studies, ophthalmic examination, and brain magnetic resonance imaging. Genetic testing included chromosomal microarray, Sanger sequencing for SHH, ZIC2, SIX3, and TGIF, and whole-exome sequencing (WES) of 10 trios.ResultsSemilobar HPE was the most common subtype of HPE, seen in 50% of the participants. Neurodevelopmental disabilities were found to correlate with HPE subtype. Factors associated with long-term survival included HPE subtype not alobar, female gender, and nontypical facial features. Four participants had de novo pathogenic variants in ZIC2. WES analysis of 11 participants did not reveal plausible candidate genes, suggesting complex inheritance in these cases. Indeed, in two probands there was a history of uncontrolled maternal type 1 diabetes.ConclusionIndividuals with various HPE subtypes can survive into adulthood and the neurodevelopmental outcomes are variable. Based on the facial characteristics and molecular evaluations, we suggest that classic genetic causes of HPE may play a smaller role in this cohort.

Management Strategies for Hydrocephalus in Alobar Holoprosencephaly: A Case Report and Discussion.

Holoprosencephaly is a rare congenital malformation resulting from an impaired midline division of the prosencephalon into distinct cerebral hemispheres. Hydrocephalus is a frequent problem among the few survivors with alobar holoprosencephaly (aHPE), its most severe form. The literature about neurosurgical management of hydrocephalus in this condition is limited and dispersed, and there are still some points that need to be resolved. We report the case of a newborn with aHPE, hydrocephalus, and central diabetes insipidus. We delineate the complexity of the management of these patients and emphasize the benefits of using an initial programmable shunt valve. Further discussion about management strategies includes reviewing previous reports and the benefits of shunting for hypothalamic osmoreceptor function.