Dysregulation of the NRG1/ERBB pathway causes a developmental disorder with gastrointestinal dysmotility in humans.

Hirschsprung disease (HSCR) is the most frequent developmental anomaly of the enteric nervous system, with an incidence of 1 in 5000 live births. Chronic intestinal pseudo-obstruction (CIPO) is less frequent and classified as neurogenic or myogenic. Isolated HSCR has an oligogenic inheritance with RET as the major disease-causing gene, while CIPO is genetically heterogeneous, caused by mutations in smooth muscle-specific genes. Here, we describe a series of patients with developmental disorders including gastrointestinal dysmotility, and investigate the underlying molecular bases. Trio-exome sequencing led to the identification of biallelic variants in ERBB3 and ERBB2 in 8 individuals variably associating HSCR, CIPO, peripheral neuropathy, and arthrogryposis. Thorough gut histology revealed aganglionosis, hypoganglionosis, and intestinal smooth muscle abnormalities. The cell type-specific ErbB3 and ErbB2 function was further analyzed in mouse single-cell RNA sequencing data and in a conditional ErbB3-deficient mouse model, revealing a primary role for ERBB3 in enteric progenitors. The consequences of the identified variants were evaluated using quantitative real-time PCR (RT-qPCR) on patient-derived fibroblasts or immunoblot assays on Neuro-2a cells overexpressing WT or mutant proteins, revealing either decreased expression or altered phosphorylation of the mutant receptors. Our results demonstrate that dysregulation of ERBB3 or ERBB2 leads to a broad spectrum of developmental anomalies, including intestinal dysmotility.

Bi-allelic Variations of SMO in Humans Cause a Broad Spectrum of Developmental Anomalies Due to Abnormal Hedgehog Signaling.

The evolutionarily conserved hedgehog (Hh) pathway is essential for organogenesis and plays critical roles in postnatal tissue maintenance and renewal. A unique feature of the vertebrate Hh pathway is that signal transduction requires the primary cilium (PC) where major pathway components are dynamically enriched. These factors include smoothened (SMO) and patched, which constitute the core reception system for sonic hedgehog (SHH) as well as GLI transcription factors, the key mediators of the pathway. Here, we report bi-allelic loss-of-function variations in SMO in seven individuals from five independent families; these variations cause a wide phenotypic spectrum of developmental anomalies affecting the brain (hypothalamic hamartoma and microcephaly), heart (atrioventricular septal defect), skeleton (postaxial polydactyly, narrow chest, and shortening of long bones), and enteric nervous system (aganglionosis). Cells derived from affected individuals showed normal ciliogenesis but severely altered Hh-signal transduction as a result of either altered PC trafficking or abnormal activation of the pathway downstream of SMO. In addition, Hh-independent GLI2 accumulation at the PC tip in cells from the affected individuals suggests a potential function of SMO in regulating basal ciliary trafficking of GLI2 when the pathway is off. Thus, loss of SMO function results in abnormal PC dynamics of key components of the Hh signaling pathway and leads to a large continuum of malformations in humans.

Growth charts in Kabuki syndrome 1.

Kabuki syndrome (KS, KS1: OMIM 147920 and KS2: OMIM 300867) is caused by pathogenic variations in KMT2D or KDM6A. KS is characterized by multiple congenital anomalies and neurodevelopmental disorders. Growth restriction is frequently reported. Here we aimed to create specific growth charts for individuals with KS1, identify parameters used for size prognosis and investigate the impact of growth hormone therapy on adult height. Growth parameters and parental size were obtained for 95 KS1 individuals (41 females). Growth charts for height, weight, body mass index (BMI) and occipitofrontal circumference were generated in standard deviation values for the first time in KS1. Statural growth of KS1 individuals was compared to parental target size. According to the charts, height, weight, BMI, and occipitofrontal circumference were lower for KS1 individuals than the normative French population. For males and females, the mean growth of KS1 individuals was -2 and -1.8 SD of their parental target size, respectively. Growth hormone therapy did not increase size beyond the predicted size. This study, from the largest cohort available, proposes growth charts for widespread use in the management of KS1, especially for size prognosis and screening of other diseases responsible for growth impairment beyond a calculated specific target size.

PAICS deficiency, a new defect of de novo purine synthesis resulting in multiple congenital anomalies and fatal outcome.

We report for the first time an autosomal recessive inborn error of de novo purine synthesis (DNPS)-PAICS deficiency. We investigated two siblings from the Faroe Islands born with multiple malformations resulting in early neonatal death. Genetic analysis of affected individuals revealed a homozygous missense mutation in PAICS (c.158A>G; p.Lys53Arg) that affects the structure of the catalytic site of the bifunctional enzyme phosphoribosylaminoimidazole carboxylase (AIRC, EC 4.1.1.21)/phosphoribosylaminoimidazole succinocarboxamide synthetase (SAICARS, EC 6.3.2.6) (PAICS). The mutation reduced the catalytic activity of PAICS in heterozygous carrier and patient skin fibroblasts to approximately 50 and 10% of control levels, respectively. The catalytic activity of the corresponding recombinant enzyme protein carrying the mutation p.Lys53Arg expressed and purified from E. coli was reduced to approximately 25% of the wild-type enzyme. Similar to other two known DNPS defects-adenylosuccinate lyase deficiency and AICA-ribosiduria-the PAICS mutation prevented purinosome formation in the patient's skin fibroblasts, and this phenotype was corrected by transfection with the wild-type but not the mutated PAICS. Although aminoimidazole ribotide (AIR) and aminoimidazole riboside (AIr), the enzyme substrates that are predicted to accumulate in PAICS deficiency, were not detected in patient's fibroblasts, the cytotoxic effect of AIr on various cell lines was demonstrated. PAICS deficiency is a newly described disease that enhances our understanding of the DNPS pathway and should be considered in the diagnosis of families with recurrent spontaneous abortion or early neonatal death.

Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes.

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.

Trans-ethnic meta-analysis of genome-wide association studies for Hirschsprung disease.

Hirschsprung disease (HSCR) is the most common cause of neonatal intestinal obstruction. It is characterized by the absence of ganglia in the nerve plexuses of the lower gastrointestinal tract. So far, three common disease-susceptibility variants at the RET, SEMA3 and NRG1 loci have been detected through genome-wide association studies (GWAS) in Europeans and Asians to understand its genetic etiologies. Here we present a trans-ethnic meta-analysis of 507 HSCR cases and 1191 controls, combining all published GWAS results on HSCR to fine-map these loci and narrow down the putatively causal variants to 99% credible sets. We also demonstrate that the effects of RET and NRG1 are universal across European and Asian ancestries. In contrast, we detected a European-specific association of a low-frequency variant, rs80227144, in SEMA3 [odds ratio (OR) = 5.2, P = 4.7 × 10-10]. Conditional analyses on the lead SNPs revealed a secondary association signal, corresponding to an Asian-specific, low-frequency missense variant encoding RET p.Asp489Asn (rs9282834, conditional OR = 20.3, conditional P = 4.1 × 10-14). When in trans with the RET intron 1 enhancer risk allele, rs9282834 increases the risk of HSCR from 1.1 to 26.7. Overall, our study provides further insights into the genetic architecture of HSCR and has profound implications for future study designs.

Blepharophimosis, short humeri, developmental delay and hirschsprung disease: expanding the phenotypic spectrum of MED12 mutations.

We report on two male sibs, a fetus and a newborn, with short humeri and dysmorphic facial features including blepharophimosis. The newborn also had Hirschsprung disease. Goldberg-Shprintzen syndrome and the Say-Barber-Biesecker-Young-Simpson type of Ohdo syndrome were suspected but direct sequencing of KBP and KAT6B failed to identify a mutation. Finally, direct sequencing of MED12, the gene mutated in Opitz-Kaveggia syndrome, Lujan-Fryns syndrome and X-linked Ohdo syndrome identified in the two sibs the missense mutation c.3443G>A (p.Arg1148His) inherited from the mother. This report further expands the phenotypic spectrum of MED12 mutations.

Finger creases lend a hand in Kabuki syndrome.

Kabuki syndrome (KS) is a rare syndrome associating malformations with intellectual deficiency and numerous visceral, orthopedic, endocrinological, immune and autoimmune complications. The early establishment of a diagnostic of KS leads to better care of the patients and therefore prevents complications such as perception deafness, severe complications of auto-immune diseases or obesity. However, the diagnosis of KS remains difficult because based on the appreciation of facial features combined with other highly variable features. We describe a novel sign, namely the attenuation and/or congenital absence of the IPD crease of the third and fourth fingers associated with limitation of flexion of the corresponding joints, which seems to be specific of KS and could help the clinician to diagnose KS.

Chromosome 21 scan in Down syndrome reveals DSCAM as a predisposing locus in Hirschsprung disease.

Hirschsprung disease (HSCR) genetics is a paradigm for the study and understanding of multigenic disorders. Association between Down syndrome and HSCR suggests that genetic factors that predispose to HSCR map to chromosome 21. To identify these additional factors, we performed a dose-dependent association study on chromosome 21 in Down syndrome patients with HSCR. Assessing 10,895 SNPs in 26 Caucasian cases and their parents led to identify two associated SNPs (rs2837770 and rs8134673) at chromosome-wide level. Those SNPs, which were located in intron 3 of the DSCAM gene within a 19 kb-linkage disequilibrium block region were in complete association and are consistent with DSCAM expression during enteric nervous system development. We replicated the association of HSCR with this region in an independent sample of 220 non-syndromic HSCR Caucasian patients and their parents. At last, we provide the functional rationale to the involvement of DSCAM by network analysis and assessment of SOX10 regulation. Our results reveal the involvement of DSCAM as a HSCR susceptibility locus, both in Down syndrome and HSCR isolated cases. This study further ascertains the chromosome-scan dose-dependent methodology used herein as a mean to map the genetic bases of other sub-phenotypes both in Down syndrome and other aneuploidies.

Clinical and molecular spectrum of renal malformations in Kabuki syndrome.

OBJECTIVE: To determine the frequency and types of renal malformations, and to evaluate renal function in a cohort of patients with Kabuki syndrome (KS). STUDY DESIGN: Renal ultrasound scans and plasma creatinine measurements were collected from a French cohort of 94 patients with genotyped KS. Renal function was evaluated based on the estimated glomerular filtration rate. A genotype-phenotype study was conducted for renal and urinary tract malformations. RESULTS: Renal malformations were present in 22% of cases, and urinary tract anomalies were present in 15%. Renal malformations were observed in 28% of the MLL2 mutation-positive group and in 0% of the MLL2 mutation-negative group (P = .015). No correlation was found between the presence or absence of renal or urinary tract malformations and the location or type of MLL2 mutation. Renal function was normal except for 1 patient with a MLL2 mutation diagnosed in the first days of life and severe renal disease due to unilateral renal agenesia and controlateral severe hypoplasia that progressed to the terminal stage at age 2 years. CONCLUSION: Our study emphasizes the need for ultrasound and renal function screening in children diagnosed with KS.

Fine mapping of whole RB1 gene deletions in retinoblastoma patients confirms PCDH8 as a candidate gene for psychomotor delay.

Retinoblastoma (Rb) results from inactivation of both alleles of the RB1 gene located in 13q14.2. Whole-germline monoallelic deletions of the RB1 gene (6% of RB1 mutational spectrum) sometimes cause a variable degree of psychomotor delay and several dysmorphic abnormalities. Breakpoints in 12 Rb patients with or without psychomotor delay were mapped to specifically define the role of chromosomal regions adjacent to RB1 in psychomotor delay. A high-resolution CGH array focusing on RB1 and its flanking region was designed to precisely map the deletion. Comparative analysis detected a 4-Mb critical interval, including a candidate gene protocadherin 8 (PCDH8). PCDH8 is thought to function in signalling pathways and cell adhesion in a central nervous system-specific manner, making loss of PCDH8 one of the probable causes of psychomotor delay in RB1-deleted patients. Consequently, we propose to systematically use high-resolution CGH in cases of partial or complete RB1 deletion encompassing the telomeric flanking region to characterize the putative loss of PCDH8 and to better define genotype/phenotype correlations, eventually leading to optimized genetic counselling and psychomotor follow-up.

Male and female differential reproductive rate could explain parental transmission asymmetry of mutation origin in Hirschsprung disease.

Hirschsprung disease (HSCR, aganglionic megacolon) is a complex and heterogeneous disease with an incidence of 1 in 5000 live births. Despite the multifactorial determination of HSCR in the vast majority of cases, there is a monogenic subgroup for which private rare RET coding sequence mutations with high penetrance are found (45% of HSCR familial cases). An asymmetrical parental origin is observed for RET coding sequence mutations with a higher maternal inheritance. A parent-of-origin effect is usually assumed. Here we show that a differential reproductive rate for males and females also leads to an asymmetrical parental origin, which was never considered as a possible explanation till now. In the case of HSCR, we show a positive association between penetrance of the mutation and parental transmission asymmetry: no parental transmission asymmetry is observed in sporadic RET CDS mutation carrier cases for which penetrance of the mutation is low, whereas a parental transmission asymmetry is observed in affected sib-pairs for which penetrance of the mutation is higher. This allows us to conclude that the explanation for this parental asymmetry is that more severe mutations have resulted in a differential reproductive rate between male and female carriers.

Differential contributions of rare and common, coding and noncoding Ret mutations to multifactorial Hirschsprung disease liability.

The major gene for Hirschsprung disease (HSCR) encodes the receptor tyrosine kinase RET. In a study of 690 European- and 192 Chinese-descent probands and their parents or controls, we demonstrate the ubiquity of a >4-fold susceptibility from a C-->T allele (rs2435357: p = 3.9 x 10(-43) in European ancestry; p = 1.1 x 10(-21) in Chinese samples) that probably arose once within the intronic RET enhancer MCS+9.7. With in vitro assays, we now show that the T variant disrupts a SOX10 binding site within MCS+9.7 that compromises RET transactivation. The T allele, with a control frequency of 20%-30%/47% and case frequency of 54%-62%/88% in European/Chinese-ancestry individuals, is involved in all forms of HSCR. It is marginally associated with proband gender (p = 0.13) and significantly so with length of aganglionosis (p = 7.6 x 10(-5)) and familiality (p = 6.2 x 10(-4)). The enhancer variant is more frequent in the common forms of male, short-segment, and simplex families whereas multiple, rare, coding mutations are the norm in the less common and more severe forms of female, long-segment, and multiplex families. The T variant also increases penetrance in patients with rare RET coding mutations. Thus, both rare and common mutations, individually and together, make contributions to the risk of HSCR. The distribution of RET variants in diverse HSCR patients suggests a "cellular-recessive" genetic model where both RET alleles' function is compromised. The RET allelic series, and its genotype-phenotype correlations, shows that success in variant identification in complex disorders may strongly depend on which patients are studied.

Epistasis between RET and BBS mutations modulates enteric innervation and causes syndromic Hirschsprung disease.

Hirschsprung disease (HSCR) is a common, multigenic neurocristopathy characterized by incomplete innervation along a variable length of the gut. The pivotal gene in isolated HSCR cases, either sporadic or familial, is RET. HSCR also presents in various syndromes, including Shah-Waardenburg syndrome (WS), Down (DS), and Bardet-Biedl (BBS). Here, we report 3 families with BBS and HSCR with concomitant mutations in BBS genes and regulatory RET elements, whose functionality is tested in physiologically relevant assays. Our data suggest that BBS mutations can potentiate HSCR predisposing RET alleles, which by themselves are insufficient to cause disease. We also demonstrate that these genes interact genetically in vivo to modulate gut innervation, and that this interaction likely occurs through complementary, yet independent, pathways that converge on the same biological process.

Interaction between a chromosome 10 RET enhancer and chromosome 21 in the Down syndrome-Hirschsprung disease association.

Individuals with Down syndrome (DS) display a 40-fold greater risk of Hirschsprung disease (HSCR) than the general population of newborns implicating chromosome 21 in HSCR etiology. Here we demonstrate that the RET enhancer polymorphism RET+9.7 (rs2435357:C>T) at chromosome 10q11.2 is associated with HSCR in DS individuals both by transmission disequilibrium (P=0.0015) and case-control (P=0.0115) analysis of matched cases. Interestingly, the RET+9.7 T allele frequency is significantly different between individuals with DS alone (0.26+/-0.04), HSCR alone (0.61+/-0.04), and those with HSCR and DS (0.41+/-0.04), demonstrating an association and interaction between RET and chromosome 21 gene dosage. This is the first report of a genetic interaction between a common functional variant (rs2435357) and a not infrequent copy number error (chromosome 21 dosage) in two human developmental disorders.

Highly conserved non-coding elements on either side of SOX9 associated with Pierre Robin sequence.

Pierre Robin sequence (PRS) is an important subgroup of cleft palate. We report several lines of evidence for the existence of a 17q24 locus underlying PRS, including linkage analysis results, a clustering of translocation breakpoints 1.06-1.23 Mb upstream of SOX9, and microdeletions both approximately 1.5 Mb centromeric and approximately 1.5 Mb telomeric of SOX9. We have also identified a heterozygous point mutation in an evolutionarily conserved region of DNA with in vitro and in vivo features of a developmental enhancer. This enhancer is centromeric to the breakpoint cluster and maps within one of the microdeletion regions. The mutation abrogates the in vitro enhancer function and alters binding of the transcription factor MSX1 as compared to the wild-type sequence. In the developing mouse mandible, the 3-Mb region bounded by the microdeletions shows a regionally specific chromatin decompaction in cells expressing Sox9. Some cases of PRS may thus result from developmental misexpression of SOX9 due to disruption of very-long-range cis-regulatory elements.

Genotype-phenotype correlations in Down syndrome identified by array CGH in 30 cases of partial trisomy and partial monosomy chromosome 21.

Down syndrome (DS) is one of the most frequent congenital birth defects, and the most common genetic cause of mental retardation. In most cases, DS results from the presence of an extra copy of chromosome 21. DS has a complex phenotype, and a major goal of DS research is to identify genotype-phenotype correlations. Cases of partial trisomy 21 and other HSA21 rearrangements associated with DS features could identify genomic regions associated with specific phenotypes. We have developed a BAC array spanning HSA21q and used array comparative genome hybridization (aCGH) to enable high-resolution mapping of pathogenic partial aneuploidies and unbalanced translocations involving HSA21. We report the identification and mapping of 30 pathogenic chromosomal aberrations of HSA21 consisting of 19 partial trisomies and 11 partial monosomies for different segments of HSA21. The breakpoints have been mapped to within approximately 85 kb. The majority of the breakpoints (26 of 30) for the partial aneuploidies map within a 10-Mb region. Our data argue against a single DS critical region. We identify susceptibility regions for 25 phenotypes for DS and 27 regions for monosomy 21. However, most of these regions are still broad, and more cases are needed to narrow down the phenotypic maps to a reasonable number of candidate genomic elements per phenotype.

Allele dosage-dependent penetrance of RET proto-oncogene in an Israeli-Arab inbred family segregating Hirschsprung disease.

Hirschsprung disease (HSCR) is characterised by intestinal obstruction resulting from an absence of ganglion cells in the intestinal tract. The mutations in the major gene, RET, associated with isolated HSCR, are dominant loss-of-function mutations with incomplete penetrance and variable expressivity. We have ascertained a large inbred Israeli-Arab family segregating HSCR. Sequencing of the RET gene showed a splicing mutation, IVS6+5G- >A, in the homozygous state in all the females with severe forms of HSCR and in the heterozygous state in the male patient with short-segment HSCR. The recently described hypomorphic-RET predisposing allele, rs2435357, was transmitted in the heterozygous state to the male patient, but was not transmitted to the three affected females. Although the heterozygous IVS6+5G- >A is of low-penetrance for short-segment HSCR disease, the homozygous state is fully penetrant for total aganglionosis or long-segment HSCR. As in other inbred populations segregating a weakly penetrant RET allele (Mennonite), our findings support the hypothesis that the penetrance of RET gene mutations for the HSCR phenotype depends on: (i) the nature of the mutation, (ii) the allele dosage and (iii) modifier-loci.

Allgrove syndrome with features of familial dysautonomia: a novel mutation in the AAAS gene.

UNLABELLED: Allgrove syndrome (or triple-A syndrome) is a rare autosomal recessive disorder characterized by alacrima, achalasia, adrenal insufficiency (glucocorticoid in the majority of cases) and autonomic/neurological abnormalities. This disease is now known to be caused by mutation in the AAAS gene located on chromosome 12q13. Diagnosis should be readily available when the full-blown features are there, but it becomes less apparent when presentation is atypical or in the evolving process. We present a brother and sister (12 and 19 y old, respectively) born to consanguineous parents of Palestinian origin with Allgrove syndrome. The index patient was erroneously diagnosed to be a case of familial dysautonomia before the diagnosis of adrenal insufficiency was made at the age of 7.5 y, while his elder sister had only alacrima from birth and developed achalasia at the age of 15 y. She started to develop early evidence of adrenal disease at the age of 19 y. Both of them had neuroautonomic dysfunction. The diagnosis of Allgrove syndrome was confirmed in these two patients by studying the gene mutation in the family. The sequencing of the AAAS gene in the two patients identified a novel homozygous mutation within intron 5 (IVS5+1G-->A). Both parents as well as all three other children were heterozygous for the same mutation. CONCLUSION: These two cases illustrate the heterogenous nature and the intrafamilial phenotypic variability of Allgrove syndrome.