Generation of human induced pluripotent stem cell line (NIDCRi001-A) from a Muenke syndrome patient with an FGFR3 p.Pro250Arg mutation.

Muenke syndrome is the leading genetic cause of craniosynostosis and results in a variety of disabling clinical phenotypes. To model the disease and study the pathogenic mechanisms, a human induced pluripotent stem cell (hiPSC) line was generated from a patient diagnosed with Muenke syndrome. Successful reprogramming was validated by morphological features, karyotyping, loss of reprogramming factors, expression of pluripotency markers, mutation analysis and teratoma formation.

Biallelic variants in KYNU cause a multisystemic syndrome with hand hyperphalangism.

Catel-Manzke syndrome is characterized by the combination of Pierre Robin sequence and radial deviation, shortening as well as clinodactyly of the index fingers, due to an accessory ossification center. Mutations in TGDS have been identified as one cause of Catel-Manzke syndrome, but cannot be found as causative in every patient with the clinical diagnosis. We performed a chromosome microarray and/or exome sequencing in three patients with hand hyperphalangism, heart defect, short stature, and mild to severe developmental delay, all of whom were initially given a clinical diagnosis of Catel-Manzke syndrome. In one patient, we detected a large deletion of exons 1-8 and the missense variant c.1282C > T (p.Arg428Trp) in KYNU (NM_003937.2), whereas homozygous missense variants in KYNU were found in the other two patients (c.989G > A (p.Arg330Gln) and c.326G > C (p.Trp109Ser)). Plasma and urine metabolomic analysis of two patients indicated a block along the tryptophan catabolic pathway and urine organic acid analysis showed excretion of xanthurenic acid. Biallelic loss-of-function mutations in KYNU were recently described as a cause of NAD deficiency with vertebral, cardiac, renal and limb defects; however, no hand hyperphalangism was described in those patients, and Catel-Manzke syndrome was not discussed as a differential diagnosis. In conclusion, we present unrelated patients identified with biallelic variants in KYNU leading to kynureninase deficiency and xanthurenic aciduria as a very likely cause of their hyperphalangism, heart defect, short stature, and developmental delay. We suggest performance of urine organic acid analysis in patients with suspected Catel-Manzke syndrome, particularly in those with cardiac or vertebral defects or without mutations in TGDS.

Identification of a novel PCNT founder pathogenic variant in the Israeli Druze population.

Majewski Osteodysplastic Primordial Dwarfism type II (MOPDII) is a form of dwarfism associated with severe microcephaly, characteristic skeletal findings, distinct dysmorphic features and increased risk for cerebral infarctions. The condition is caused by bi-allelic loss-of-function variants in the gene PCNT. Here we describe the identification of a novel founder pathogenic variant c.3465-1G > A observed in carriers from multiple Druze villages in Northern Israel. RNA studies show that the variant results in activation of a cryptic splice site causing a coding frameshift. The study was triggered by the diagnosis of a single child with MOPDII and emphasizes the advantages of applying next generation sequencing technologies in community genetics and the importance of establishing population-specific sequencing databases.

The CHD4-related syndrome: a comprehensive investigation of the clinical spectrum, genotype-phenotype correlations, and molecular basis.

PURPOSE: Sifrim-Hitz-Weiss syndrome (SIHIWES) is a recently described multisystemic neurodevelopmental disorder caused by de novo variants inCHD4. In this study, we investigated the clinical spectrum of the disorder, genotype-phenotype correlations, and the effect of different missense variants on CHD4 function. METHODS: We collected clinical and molecular data from 32 individuals with mostly de novo variants in CHD4, identified through next-generation sequencing. We performed adenosine triphosphate (ATP) hydrolysis and nucleosome remodeling assays on variants from five different CHD4 domains. RESULTS: The majority of participants had global developmental delay, mild to moderate intellectual disability, brain anomalies, congenital heart defects, and dysmorphic features. Macrocephaly was a frequent but not universal finding. Additional common abnormalities included hypogonadism in males, skeletal and limb anomalies, hearing impairment, and ophthalmic abnormalities. The majority of variants were nontruncating and affected the SNF2-like region of the protein. We did not identify genotype-phenotype correlations based on the type or location of variants. Alterations in ATP hydrolysis and chromatin remodeling activities were observed in variants from different domains. CONCLUSION: The CHD4-related syndrome is a multisystemic neurodevelopmental disorder. Missense substitutions in different protein domains alter CHD4 function in a variant-specific manner, but result in a similar phenotype in humans.

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.

Clinical and Demographic Evaluation of a Holoprosencephaly Cohort From the Kyoto Collection of Human Embryos.

Holoprosencephaly (HPE) is a genetically and phenotypically heterogeneous disorder involving developmental defects. HPE is a rare condition (1/10,000-20,000 newborns) but can be found as frequently as 1/250 among conceptions, suggesting that most HPE embryos are incompatible with postnatal life and result in spontaneous abortions during the first trimester of gestation. Beginning in 1961, the Kyoto University in Japan collected over 44,000 human conceptuses in collaboration with several hundred domestic obstetricians. Over 200 cases of HPE have been identified in the Kyoto collection, which represents the largest single cohort of HPE early stage embryo specimens. In this study, we present a comprehensive clinical and demographic evaluation of this HPE cohort prior to genomic analysis. The total percentage of the threatened abortion among HPE embryos in the Kyoto collection was 67%. Almost 20% of the women with embryos affected by HPE had experienced spontaneous miscarriage. In addition, there was a significant tendency that the mothers with HPE cases had fewer live births than the control. Moreover, in 70% of cases, the mother reported bleeding during pregnancy, a higher percentage than expected, indicating that most of the conceptions with HPE embryos tend to be terminated spontaneously. There were no differences in smoking between mothers with HPE affected and unaffected pregnancies; however, alcohol use was higher in women with pregnancies affected by HPE. In this study, we precisely characterize the phenotype and environmental influences of embryos affected by HPE allowing the future leveraging of genomic technologies to further understand the genetics of forebrain development. Anat Rec, 301:973-986, 2018. © 2018 Wiley Periodicals, Inc.

Human germline hedgehog pathway mutations predispose to fatty liver.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is the most common form of liver disease. Activation of hedgehog (Hh) signaling has been implicated in the progression of NAFLD and proposed as a therapeutic target; however, the effects of Hh signaling inhibition have not been studied in humans with germline mutations that affect this pathway. METHODS: Patients with holoprosencephaly (HPE), a disorder associated with germline mutations disrupting Sonic hedgehog (SHH) signaling, were clinically evaluated for NAFLD. A combined mouse model of Hh signaling attenuation (Gli2 heterozygous null: Gli2+/-) and diet-induced NAFLD was used to examine aspects of NAFLD and hepatic gene expression profiles, including molecular markers of hepatic fibrosis and inflammation. RESULTS: Patients with HPE had a higher prevalence of liver steatosis compared to the general population, independent of obesity. Exposure of Gli2+/- mice to fatty liver-inducing diets resulted in increased liver steatosis compared to wild-type mice. Similar to humans, this effect was independent of obesity in the mutant mice and was associated with decreased expression of pro-fibrotic and pro-inflammatory genes, and increased expression of PPARγ, a potent anti-fibrogenic and anti-inflammatory regulator. Interestingly, tumor suppressors p53 and p16INK4 were found to be downregulated in the Gli2+/- mice exposed to a high-fat diet. CONCLUSIONS: Our results indicate that germline mutations disrupting Hh signaling promotes liver steatosis, independent of obesity, with reduced fibrosis. While Hh signaling inhibition has been associated with a better NAFLD prognosis, further studies are required to evaluate the long-term effects of mutations affecting this pathway. Lay summary: Non-alcoholic fatty liver disease (NAFLD) is characterized by excess fat deposition in the liver predominantly due to high calorie intake and a sedentary lifestyle. NAFLD progression is usually accompanied by activation of the Sonic hedgehog (SHH) pathway leading to fibrous buildup (scar tissue) and inflammation of the liver tissue. For the first time patients with holoprosencephaly, a disease caused by SHH signaling mutations, are shown to have increased liver steatosis independent of obesity. This observation was recapitulated in a mouse model of attenuated SHH signaling that also showed increased liver steatosis but with decreased fibrosis and inflammation. While SHH inhibition is associated with a good NAFLD prognosis, this increase in liver fat accumulation in the context of SHH signaling inhibition must be studied prospectively to evaluate its long-term effects, especially in individuals with Western-type dietary habits.

An Ultraconserved Brain-Specific Enhancer Within ADGRL3 (LPHN3) Underpins Attention-Deficit/Hyperactivity Disorder Susceptibility.

BACKGROUND: Genetic factors predispose individuals to attention-deficit/hyperactivity disorder (ADHD). Previous studies have reported linkage and association to ADHD of gene variants within ADGRL3. In this study, we functionally analyzed noncoding variants in this gene as likely pathological contributors. METHODS: In silico, in vitro, and in vivo approaches were used to identify and characterize evolutionary conserved elements within the ADGRL3 linkage region (~207 Kb). Family-based genetic analyses of 838 individuals (372 affected and 466 unaffected patients) identified ADHD-associated single nucleotide polymorphisms harbored in some of these conserved elements. Luciferase assays and zebrafish green fluorescent protein transgenesis tested conserved elements for transcriptional enhancer activity. Electromobility shift assays were used to verify transcription factor-binding disruption by ADHD risk alleles. RESULTS: An ultraconserved element was discovered (evolutionary conserved region 47) that functions as a transcriptional enhancer. A three-variant ADHD risk haplotype in evolutionary conserved region 47, formed by rs17226398, rs56038622, and rs2271338, reduced enhancer activity by 40% in neuroblastoma and astrocytoma cells (pBonferroni < .0001). This enhancer also drove green fluorescent protein expression in the zebrafish brain in a tissue-specific manner, sharing aspects of endogenous ADGRL3 expression. The rs2271338 risk allele disrupts binding of YY1 transcription factor, an important factor in the development and function of the central nervous system. Expression quantitative trait loci analysis of postmortem human brain tissues revealed an association between rs2271338 and reduced ADGRL3 expression in the thalamus. CONCLUSIONS: These results uncover the first functional evidence of common noncoding variants with potential implications for the pathology of ADHD.

De Novo Mutations in CHD4, an ATP-Dependent Chromatin Remodeler Gene, Cause an Intellectual Disability Syndrome with Distinctive Dysmorphisms.

Chromodomain helicase DNA-binding protein 4 (CHD4) is an ATP-dependent chromatin remodeler involved in epigenetic regulation of gene transcription, DNA repair, and cell cycle progression. Also known as Mi2ß, CHD4 is an integral subunit of a well-characterized histone deacetylase complex. Here we report five individuals with de novo missense substitutions in CHD4 identified through whole-exome sequencing and web-based gene matching. These individuals have overlapping phenotypes including developmental delay, intellectual disability, hearing loss, macrocephaly, distinct facial dysmorphisms, palatal abnormalities, ventriculomegaly, and hypogonadism as well as additional findings such as bone fusions. The variants, c.3380G>A (p.Arg1127Gln), c.3443G>T (p.Trp1148Leu), c.3518G>T (p.Arg1173Leu), and c.3008G>A, (p.Gly1003Asp) (GenBank: NM_001273.3), affect evolutionarily highly conserved residues and are predicted to be deleterious. Previous studies in yeast showed the equivalent Arg1127 and Trp1148 residues to be crucial for SNF2 function. Furthermore, mutations in the same positions were reported in malignant tumors, and a de novo missense substitution in an equivalent arginine residue in the C-terminal helicase domain of SMARCA4 is associated with Coffin Siris syndrome. Cell-based studies of the p.Arg1127Gln and p.Arg1173Leu mutants demonstrate normal localization to the nucleus and HDAC1 interaction. Based on these findings, the mutations potentially alter the complex activity but not its formation. This report provides evidence for the role of CHD4 in human development and expands an increasingly recognized group of Mendelian disorders involving chromatin remodeling and modification.

Craniosynostosis and Noonan syndrome with KRAS mutations: Expanding the phenotype with a case report and review of the literature.

Noonan syndrome (NS) is a multiple congenital anomaly syndrome caused by germline mutations in genes coding for components of the Ras-mitogen-activated protein kinase (RAS-MAPK) pathway. Features include short stature, characteristic facies, congenital heart anomalies, and developmental delay. While there is considerable clinical heterogeneity in NS, craniosynostosis is not a common feature of the condition. Here, we report on a 2 month-old girl with Noonan syndrome associated with a de novo mutation in KRAS (p.P34Q) and premature closure of the sagittal suture. We provide a review of the literature of germline KRAS mutations and find that approximately 10% of published cases have craniosynostosis. Our findings expand on the NS phenotype and suggest that germline mutations in the KRAS gene are causally involved in craniosynostosis, supporting the role of the RAS-MAPK pathway as a mediator of aberrant bone growth in cranial sutures. The inclusion of craniosynostosis as a possible phenotype in KRAS-associated Noonan Syndrome has implications in the differential diagnosis and surgical management of individuals with craniosynostosis.

Analysis of renal anomalies in VACTERL association.

BACKGROUND: VACTERL association refers to a combination of congenital anomalies that can include: vertebral anomalies, anal atresia, cardiac malformations, tracheo-esophageal fistula with esophageal atresia, renal anomalies (typically structural renal anomalies), and limb anomalies. METHODS: We conducted a description of a case series to characterize renal findings in a cohort of patients with VACTERL association. Out of the overall cohort, 48 patients (with at least three component features of VACTERL and who had abdominal ultrasound performed) met criteria for analysis. Four other patients were additionally analyzed separately, with the hypothesis that subtle renal system anomalies may occur in patients who would not otherwise meet criteria for VACTERL association. RESULTS: Thirty-three (69%) of the 48 patients had a clinical manifestation affecting the renal system. The most common renal manifestation (RM) was vesicoureteral reflux (VUR) in addition to a structural defect (present in 27%), followed by unilateral renal agenesis (24%), and then dysplastic/multicystic kidneys or duplicated collected system (18% for each). Twenty-two (88%) of the 25 patients with a structural RM had an associated anorectal malformation. Individuals with either isolated lower anatomic anomalies, or both upper and lower anatomic anomalies were not statistically more likely to have a structural renal defect than those with isolated upper anatomic anomalies (p = 0.22, p = 0.284, respectively). CONCLUSION: Given the high prevalence of isolated VUR in our cohort, we recommend a screening VCUG or other imaging modality be obtained to evaluate for VUR if initial renal ultrasound shows evidence of obstruction or renal scarring, as well as ongoing evaluation of renal health.

GWAS reveals new recessive loci associated with non-syndromic facial clefting.

We have applied a GWAS to 40 consanguineous families segregating cases of non-syndromic cleft lip with or without cleft palate (NS CL/P) (a total of 160 affected and unaffected individuals) in order to trace potential recessive loci that confer susceptibility to this common facial malformation. Pedigree-based association test (PBAT) analyses reported nominal evidence of association and linkage over SNP markers located at 11q25 (rs4937877, P = 2.7 × 10(-6)), 19p12 (rs4324267, P = 1.6 × 10(-5)), 5q14.1 (rs4588572, P-value = 3.36 × 10(-5)), and 15q21.1 (rs4774497, P = 1.08 × 10(-4)). Using the Versatile Gene-Based Association Study to complement the PBAT results, we found clusters of markers located at chromosomes 19p12, 11q25, and 8p23.2 overcome the threshold for GWAS significance (P < 1 × 10(-7)). From this study, new recessive loci implicated in NS CL/P include: B3GAT1, GLB1L2, ZNF431, ZNF714, and CSMD1, even though the functional association with the genesis of NS CL/P remains to be elucidated. These results emphasize the importance of using homogeneous populations, phenotypes, and family structures for GWAS combined with gene-based association analyses, and should encourage. other researchers to evaluate these genes on independent patient samples affected by NS CL/P.

Identification of NFAT binding sites that mediate stimulation of cathepsin K promoter activity by RANK ligand.

The receptor activator of NFkappaB ligand (RANKL) is a critical mediator of osteoclastogenesis and regulates cathepsin K (CTSK) expression, which is essential for normal bone resorption. RANKL acts, in part, via the Ca(2+)/calmodulin/calcineurin signaling pathway, which in turn, activates NFATc1 (nuclear factor of activated T-cells) and downstream gene expression. We investigated the signals and promoter elements that regulate CTSK gene expression in RAW 264.7 cells, which can be differentiated to osteoclasts by RANKL. Disrupting Ca(2+) signaling, by blocking Ca(2+) channels, thus inhibiting calcineurin or chelation of intracellular Ca(2+), prevented the stimulation of CTSK expression by RANKL. Both RANKL treatment and overexpression of NFATc1 dramatically enhanced CTSK promoter activity, but not in an identical manner. NFATc1 regulates CTSK promoter activity, but the motifs have not been explicitly identified. We found that as few as 238 bp of the CTSK promoter were sufficient to elicit a marked response to both RANKL and NFATc1, truncations of the CTSK promoter illustrated differences in regional responsiveness. Transfection analysis of CTSK promoter-luciferase plasmids revealed that NFATc1 binding sites at 85, 289 and 345 bp upstream of the transcriptional start site mediated responses to RANKL and NFATc1. Deletion of a 4-bp core element from the site at -85 bp dramatically reduced the response of the CTSK promoter to both RANKL and NFATc1, whereas a similar deletion at -345 bp decreased NFATc1- but not RANKL-mediated responses. Mutation of the site at -289 bp did not affect NFAT-mediated stimulation of CTSK on its own, but did decrease responsiveness in combination with either or both of the other two deletions. Electrophoretic mobility shift assays demonstrated NFATc1 binding to oligonucleotides containing the -85-bp and -345-bp sites, while chromatin immunoprecipitation assays demonstrated enhanced in situ binding by NFATc1 to two analogous sites in the mouse CTSK promoter in response to RANKL treatment. Therefore, proximal NFAT binding sites play a significant role in the NFATc1-mediated stimulation of CTSK gene expression by RANKL.

AP-1 stimulates the cathepsin K promoter in RAW 264.7 cells.

Cathepsin K (CTSK) is a secreted protease that plays an essential role in osteoclastic bone resorption, and CTSK levels increase with osteoclast differentiation and activation, a process that is controlled by a complex physiological network of hormones and cytokines. A critical regulator of this process is receptor activator of NF-kappaB ligand (RANKL), a member of the tumor necrosis factor (TNF) superfamily of cytokines that can act via the TNF receptor activating factor (TRAF6)/AP-1 signaling pathway. However, the mechanism whereby RANKL modulates CTSK expression is not fully understood. Therefore, we investigated the regulation of CTSK expression and promoter activity in RAW 264.7 osteoclast precursor cells, which can be readily differentiated to osteoclasts upon RANKL stimulation. Western blot analysis, real-time RT-PCR and luciferase reporter gene assays revealed that RANKL stimulated CTSK expression and promoter activity in a dose- and time-dependent manner and that this activation was inhibited by either dominant negative (DN) TRAF6 or DN-c-fos. TRAF6 stimulated the basal activity of a truncated CTSK promoter, and DN-c-fos blocked this stimulation. JunB alone also stimulated basal CTSK promoter activity, whereas c-jun, JunD or c-fos alone did not. However, co-transfection of any of these jun-family members with c-fos (AP-1) significantly increased CTSK promoter expression. siRNA targeted against c-jun or junB suppressed RANKL-mediated CTSK expression. Therefore, both TRAF6 and AP-1 help regulate the basal and RANKL-mediated stimulation of CTSK gene expression in RAW 264.7 cells.

RANK ligand and interferon gamma differentially regulate cathepsin gene expression in pre-osteoclastic cells.

Receptor activator of NF-kappaB ligand (RANKL) and interferon gamma (IFN-gamma) are critical and opposing mediators of osteoclastogenesis, exerting stimulatory and inhibitory effects, respectively. Cathepsin K (CTSK) is a secreted protease that plays an essential role in osteoclastic bone resorption. We have examined the role of IFN-gamma in the regulation of CTSK expression in the murine monocytic RAW 264.7 cell line, which can be readily differentiated to bone-resorbing osteoclasts upon RANKL treatment. Real-time RT-PCR reveals that RANKL stimulates CTSK mRNA expression in a dose- and time-dependent fashion, but that RANKL does not alter the expression of cathepsin L (CTSL) and cathepsin S (CTSS) mRNA. IFN-gamma stimulates both CTSL and CTSS expression after 3 days, but fails to significantly alter CTSK expression. IFN-gamma markedly inhibits the stimulation of CTSK mRNA and protein by RANKL, whereas RANKL suppresses the stimulation of CTSL and CTSS mRNA by IFN-gamma. IFN-gamma also ablates the RANKL induced osteoclastic differentiation of RAW cells. In RAW cells stably transfected with a CTSK promoter-luciferase plasmid containing the 1618 bp upstream of the transcription initiation site, IFN-gamma inhibits CTSK promoter activity and ablates its induction by RANKL. In conclusion, IFN-gamma and RANKL differentially regulate cathepsin K, S, and L gene expression in pre-osteoclastic cells, and there appears to be significant cross talk between the signal transduction pathways mediating the responses to RANKL and IFN-gamma.