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Background Hirschsprung’s disease (HSCR) is a serious congenital bowel disorder with a prevalence of 1/5000. Currently, there is a lack of systematically developed guidelines to assist clinical decision-making regarding diagnostics and management. Aims This guideline aims to cover the diagnostics and management of rectosigmoid HSCR up to adulthood. It aims to describe the preferred approach of ERNICA, the European Reference Network for rare inherited and congenital digestive disorders. Methods Recommendations within key topics covering the care pathway for rectosigmoid HSCR were developed by an international workgroup of experts from 8 European countries within ERNICA European Reference Network from the disciplines of surgery, medicine, histopathology, microbiology, genetics, and patient organization representatives. Recommendation statements were based on a comprehensive review of the available literature and expert consensus. AGREE II and GRADE approaches were used during development. Evidence levels and levels of agreement are noted. Results Thirty-three statements within 9 key areas were generated. Most recommendations were based on expert opinion. Conclusion In rare or low-prevalence diseases such as HSCR, there remains limited availability of high-quality clinical evidence. Consensus-based guidelines for care are presented.

Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital visceral myopathy characterized by severe dilation of the urinary bladder and defective intestinal motility. The genetic basis of MMIHS has been ascribed to spontaneous and autosomal dominant mutations in actin gamma 2 (ACTG2), a smooth muscle contractile gene. However, evidence suggesting a recessive origin of the disease also exists. Using combined homozygosity mapping and whole exome sequencing, a genetically isolated family was found to carry a premature termination codon in Leiomodin1 (LMOD1), a gene preferentially expressed in vascular and visceral smooth muscle cells. Parents heterozygous for the mutation exhibited no abnormalities, but a child homozygous for the premature termination codon displayed symptoms consistent with MMIHS. We used CRISPR-Cas9 (CRISPR-associated protein) genome editing of Lmod1 to generate a similar premature termination codon. Mice homozygous for the mutation showed loss of LMOD1 protein and pathology consistent with MMIHS, including late gestation expansion of the bladder, hydronephrosis, and rapid demise after parturition. Loss of LMOD1 resulted in a reduction of filamentous actin, elongated cytoskeletal dense bodies, and impaired intestinal smooth muscle contractility. These results define LMOD1 as a disease gene for MMIHS and suggest its role in establishing normal smooth muscle cytoskeletal–contractile coupling.

Hirschsprung disease (HSCR) is a complex genetic disease characterized by absence of ganglia in the intestine. HSCR etiology can be explained by a unique combination of genetic alterations: rare coding variants, predisposing haplotypes and Copy Number Variation (CNV). Approximately 18% of patients have additional anatomical malformations or neurological symptoms (HSCR-AAM). Pinpointing the responsible culprits within a CNV is challenging as often many genes are affected. Therefore, we selected candidate genes based on gene enrichment strategies using mouse enteric nervous system transcriptomes and constraint metrics. Next, we used a zebrafish model to investigate whether loss of these genes affects enteric neuron development in vivo . This study included three groups of patients, two groups without coding variants in disease associated genes: HSCR-AAM and HSCR patients without associated anomalies (HSCR-isolated). The third group consisted of all HSCR patients in which a confirmed pathogenic rare coding variant was identified. We compared these patient groups to unaffected controls. Predisposing haplotypes were determined, confirming that every HSCR subgroup had increased contributions of predisposing haplotypes, but their contribution was highest in isolated HSCR patients without RET coding variants. CNV profiling proved that specifically HSCR-AAM patients had larger Copy Number (CN) losses. Gene enrichment strategies using mouse enteric nervous system transcriptomes and constraint metrics were used to determine plausible candidate genes located within CN losses. Validation in zebrafish using CRISPR/Cas9 targeting confirmed the contribution of UFD1L , TBX2 , SLC8A1 , and MAPK8 to ENS development. In addition, we revealed epistasis between reduced Ret and Gnl1 expression and between reduced Ret and Tubb5 expression in vivo . Rare large CN losses—often de novo —contribute to HSCR in HSCR-AAM patients. We proved the involvement of six genes in enteric nervous system development and Hirschsprung disease.

Myhre syndrome is characterized by short stature, brachydactyly, facial features, pseudomuscular hypertrophy, joint limitation and hearing loss. We identified SMAD4 mutations as the cause of Myhre syndrome. SMAD4 mutations have also been identified in laryngotracheal stenosis, arthropathy, prognathism and short stature syndrome (LAPS). This study aimed to review the features of Myhre and LAPS patients to define the clinical spectrum of SMAD4 mutations. We included 17 females and 15 males ranging in age from 8 to 48 years. Thirty were diagnosed with Myhre syndrome and two with LAPS. SMAD4 coding sequence was analyzed by Sanger sequencing. Clinical and radiological features were collected from a questionnaire completed by the referring physicians. All patients displayed a typical facial gestalt, thickened skin, joint limitation and muscular pseudohypertrophy. Growth retardation was common (68.7%) and was variable in severity (from -5.5 to -2 SD), as was mild-to-moderate intellectual deficiency (87.5%) with additional behavioral problems in 56.2% of the patients. Significant health concerns like obesity, arterial hypertension, bronchopulmonary insufficiency, laryngotracheal stenosis, pericarditis and early death occurred in four. Twenty-nine patients had a de novo heterozygous SMAD4 mutation, including both patients with LAPS. In 27 cases mutation affected Ile500 and in two cases Arg496. The three patients without SMAD4 mutations had typical findings of Myhre syndrome. Myhre-LAPS syndrome is a clinically homogenous condition with life threatening complications in the course of the disease. Our identification of SMAD4 mutations in 29/32 cases confirms that SMAD4 is the major gene responsible for Myhre syndrome.

Silver-Russell syndrome (SRS) is a heterogeneous disorder characterized by intrauterine and postnatal growth retardation. HMGA2 variants are a rare cause of SRS and its functional role in human linear growth is unclear. Patients with suspected SRS negative for 11p15LOM/mUPD7 underwent whole-exome and/or targeted-genome sequencing. Mutant HMGA2 protein expression and nuclear localization were assessed. Two Hmga2-knockin mouse models were generated. Five clinical SRS patients harbored HMGA2 variants with differing functional impacts: 2 stop-gain nonsense variants (c.49G>T, c.52C>T), c.166A>G missense variant, and 2 frameshift variants (c.144delC, c.145delA) leading to an identical, extended-length protein. Phenotypic features were highly variable. Nuclear localization was reduced/absent for all variants except c.166A>G. Homozygous knockin mice recapitulating the c.166A>G variant (Hmga2K56E) exhibited a growth-restricted phenotype. An Hmga2Ter76-knockin mouse model lacked detectable full-length Hmga2 protein, similarly to patient 3 and 5 variants. These mice were infertile, with a pygmy phenotype. We report a heterogeneous group of individuals with SRS harboring variants in HMGA2 and describe the first Hmga2 missense knockin mouse model (Hmga2K56E) to our knowledge causing a growth-restricted phenotype. In patients with clinical features of SRS but negative genetic screening, HMGA2 should be included in next-generation sequencing testing approaches.

Background: Pediatric Intestinal Pseudo-obstruction (PIPO) is a congenital enteric disorder characterized by severe gastrointestinal (GI) dysmotility, without mechanical obstruction. Although several genes have been described to cause this disease, most patients do not receive a genetic diagnosis. Here, we aim to identify the genetic cause of PIPO in a patient diagnosed with severe intestinal dysmotility shortly after birth. Methods: Whole exome sequencing (WES) was performed in the patient and unaffected parents, in a diagnostic setting. After identification of the potential disease-causing variant, its functional consequences were determined in vitro and in vivo . For this, expression constructs with and without the causing variant, were overexpressed in HEK293 cells. To investigate the role of the candidate gene in GI development and function, a zebrafish model was generated where its expression was disrupted using CRISPR/Cas9 editing. Results: WES analysis identified a de novo heterozygous deletion in TFAP2B (NM_003221.4:c.602-5_606delTCTAGTTCCA), classified as a variant of unknown significance. In vitro studies showed that this deletion affects RNA splicing and results in loss of exon 4, leading to the appearance of a premature stop codon and absence of TFAP2B protein. Disruption of tfap2b in zebrafish led to decreased enteric neuronal numbers and delayed transit time. However, no defects in neuronal differentiation were detected. tfap2b crispants also showed decreased levels of ednrbb mRNA, a downstream target of tfap2b . Conclusion: We showed that TFAP2B haploinsufficiency leads to reduced neuronal numbers and GI dysmotility, suggesting for the first time, that this gene is involved in PIPO pathogenesis.

Background Epilepsy belongs to a group of chronic and highly heterogeneous brain disorders. Many types of epilepsy and epileptic syndromes are caused by genetic factors. The neural amino acid y‐aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the mammalian central nervous system. It regulates activity of channel pores by binding to transmembrane GABA‐receptors (GABRs). The GABRs are heteropentamers assembled from different receptor subunits (α1‐6, β1‐3, γ1‐3, δ, ε, θ, π, and ρ1‐3). Several epileptic disorders are caused by mutations in genes encoding single GABRs. Methods We applied trio‐ and single‐whole exome sequencing to search for genetic sequence variants associated with a wide range of epileptic phenotypes accompanied by intellectual disability and/or global developmental delay in the investigated patients. Results We identified four hemizygous sequence variants in the GABAA receptor subunit ε gene (GABRE), including one nonsense (NM_004961.3: c.399C>A, p.Tyr133*), two missense variants (NM_004961.3: c.664G>A, p.Glu222Lys; NM_004961.3: c.1045G>A, p.Val349Ile), and one variant affecting the translation initiation codon (NM_004961.3: c.1A>G, p.Met1?) in four unrelated families. Conclusion Our clinical and molecular genetic findings suggest that GABRE is a likely candidate gene for epilepsy. Nevertheless, functional studies are necessary to better understand pathogenicity of the GABRE‐mutations and their associations with epileptic phenotypes. The neural amino acid y‐aminobutyric acid (GABA) regulates activity of channel pores by binding to transmembrane GABA‐receptors (GABRs). We identified four hemizygous sequence variants in the GABAA receptor subunit ε gene (GABRE) in four epileptic patients. Our clinical and molecular genetic findings suggest that GABRE is a likely candidate gene for epilepsy.

The Koolen-de Vries syndrome (KdVS; OMIM #610443), also known as the 17q21.31 microdeletion syndrome, is a clinically heterogeneous disorder characterised by (neonatal) hypotonia, developmental delay, moderate intellectual disability, and characteristic facial dysmorphism. Expressive language development is particularly impaired compared with receptive language or motor skills. Other frequently reported features include social and friendly behaviour, epilepsy, musculoskeletal anomalies, congenital heart defects, urogenital malformations, and ectodermal anomalies. The syndrome is caused by a truncating variant in the KAT8 regulatory NSL complex unit 1 (KANSL1) gene or by a 17q21.31 microdeletion encompassing KANSL1. Herein we describe a novel cohort of 45 individuals with KdVS of whom 33 have a 17q21.31 microdeletion and 12 a single-nucleotide variant (SNV) in KANSL1 (19 males, 26 females; age range 7 months to 50 years). We provide guidance about the potential pitfalls in the laboratory testing and emphasise the challenges of KANSL1 variant calling and DNA copy number analysis in the complex 17q21.31 region. Moreover, we present detailed phenotypic information, including neuropsychological features, that contribute to the broad phenotypic spectrum of the syndrome. Comparison of the phenotype of both the microdeletion and SNV patients does not show differences of clinical importance, stressing that haploinsufficiency of KANSL1 is sufficient to cause the full KdVS phenotype.

Witteveen-Kolk syndrome (OMIM 613406) is a recently defined neurodevelopmental syndrome caused by heterozygous loss-of-function variants in SIN3A. We define the clinical and neurodevelopmental phenotypes related to SIN3A-haploinsufficiency in 28 unreported patients. Patients with SIN3A variants adversely affecting protein function have mild intellectual disability, growth and feeding difficulties. Involvement of a multidisciplinary team including a geneticist, paediatrician and neurologist should be considered in managing these patients. Patients described here were identified through a combination of clinical evaluation and gene matching strategies (GeneMatcher and Decipher). All patients consented to participate in this study. Mean age of this cohort was 8.2 years (17 males, 11 females). Out of 16 patients ≥ 8 years old assessed, eight (50%) had mild intellectual disability (ID), four had moderate ID (22%), and one had severe ID (6%). Four (25%) did not have any cognitive impairment. Other neurological symptoms such as seizures (4/28) and hypotonia (12/28) were common. Behaviour problems were reported in a minority. In patients ≥2 years, three were diagnosed with Autism Spectrum Disorder (ASD) and four with Attention Deficit Hyperactivity Disorder (ADHD). We report 27 novel variants and one previously reported variant. 24 were truncating variants; three were missense variants and one large in-frame gain including exons 10–12.

Background Hirschsprung disease (HSCR), which is congenital obstruction of the bowel, results from a failure of enteric nervous system (ENS) progenitors to migrate, proliferate, differentiate, or survive within the distal intestine. Previous studies that have searched for genes underlying HSCR have focused on ENS-related pathways and genes not fitting the current knowledge have thus often been ignored. We identify and validate novel HSCR genes using whole exome sequencing (WES), burden tests, in silico prediction, unbiased in vivo analyses of the mutated genes in zebrafish, and expression analyses in zebrafish, mouse, and human. Results We performed de novo mutation (DNM) screening on 24 HSCR trios. We identify 28 DNMs in 21 different genes. Eight of the DNMs we identified occur in RET, the main HSCR gene, and the remaining 20 DNMs reside in genes not reported in the ENS. Knockdown of all 12 genes with missense or loss-of-function DNMs showed that the orthologs of four genes (DENND3, NCLN, NUP98, and TBATA) are indispensable for ENS development in zebrafish, and these results were confirmed by CRISPR knockout. These genes are also expressed in human and mouse gut and/or ENS progenitors. Importantly, the encoded proteins are linked to neuronal processes shared by the central nervous system and the ENS. Conclusions Our data open new fields of investigation into HSCR pathology and provide novel insights into the development of the ENS. Moreover, the study demonstrates that functional analyses of genes carrying DNMs are warranted to delineate the full genetic architecture of rare complex diseases.