Thyroid hormone and folinic acid in young children with Down syndrome: the phase 3 ACTHYF trial.

PURPOSE: To determine whether folinic acid (FA) and thyroxine, in combination or alone, benefit psychomotor development in young patients with Down syndrome (DS). METHODS: The Assessment of Systematic Treatment With Folinic Acid and Thyroid Hormone on Psychomotor Development of Down Syndrome Young Children (ACTHYF) was a single-center, randomized, double-blind, placebo-controlled phase 3 trial in DS infants aged 6-18 months. Patients were randomly assigned to one of four treatments: placebo, folinic acid (FA), L-thyroxine, or FA+L-thyroxine, administered for 12 months. Randomization was done by age and sex. The primary endpoint was adjusted change from baseline in Griffiths Mental Development Scale global development quotient (GDQ) after 12 months. RESULTS: Of 175 patients randomized, 143 completed the study. The modified intention-to-treat (mITT) population included all randomized patients who did not prematurely discontinue due to elevated baseline thyroid stimulating hormone (TSH). Baseline characteristics in the mITT were well balanced between groups, with reliable developmental assessment outcomes. Adjusted mean change in GDQ in the mITT showed similar decreases in all groups (placebo: -5.10 [95% confidence interval (CI) -7.84 to -2.37]; FA: -4.69 [95% CI -7.73 to -1.64]; L-thyroxine: -3.89 [95% CI -6.94 to -0.83]; FA+L-thyroxine: -3.86 [95% CI -6.67 to -1.06]), with no significant difference for any active treatment group versus placebo. CONCLUSION: This trial does not support the hypotheses that thyroxine and/or folinic acid improve development of young children with DS or are synergistic. This trial is registered with ClinicalTrials.gov number, NCT01576705.

The complex SNP and CNV genetic architecture of the increased risk of congenital heart defects in Down syndrome.

Congenital heart defect (CHD) occurs in 40% of Down syndrome (DS) cases. While carrying three copies of chromosome 21 increases the risk for CHD, trisomy 21 itself is not sufficient to cause CHD. Thus, additional genetic variation and/or environmental factors could contribute to the CHD risk. Here we report genomic variations that in concert with trisomy 21, determine the risk for CHD in DS. This case-control GWAS includes 187 DS with CHD (AVSD = 69, ASD = 53, VSD = 65) as cases, and 151 DS without CHD as controls. Chromosome 21-specific association studies revealed rs2832616 and rs1943950 as CHD risk alleles (adjusted genotypic P-values <0.05). These signals were confirmed in a replication cohort of 92 DS-CHD cases and 80 DS-without CHD (nominal P-value 0.0022). Furthermore, CNV analyses using a customized chromosome 21 aCGH of 135K probes in 55 DS-AVSD and 53 DS-without CHD revealed three CNV regions associated with AVSD risk (FDR ≤ 0.05). Two of these regions that are located within the previously identified CHD region on chromosome 21 were further confirmed in a replication study of 49 DS-AVSD and 45 DS- without CHD (FDR ≤ 0.05). One of these CNVs maps near the RIPK4 gene, and the second includes the ZBTB21 (previously ZNF295) gene, highlighting the potential role of these genes in the pathogenesis of CHD in DS. We propose that the genetic architecture of the CHD risk of DS is complex and includes trisomy 21, and SNP and CNV variations in chromosome 21. In addition, a yet-unidentified genetic variation in the rest of the genome may contribute to this complex genetic architecture.

Trisomy for synaptojanin1 in Down syndrome is functionally linked to the enlargement of early endosomes.

Enlarged early endosomes have been observed in neurons and fibroblasts in Down syndrome (DS). These endosome abnormalities have been implicated in the early development of Alzheimer's disease (AD) pathology in these subjects. Here, we show the presence of enlarged endosomes in blood mononuclear cells and lymphoblastoid cell lines (LCLs) from individuals with DS using immunofluorescence and confocal microscopy. Genotype-phenotype correlations in LCLs carrying partial trisomies 21 revealed that triplication of a 2.56 Mb locus in 21q22.11 is associated with the endosomal abnormalities. This locus contains the gene encoding the phosphoinositide phosphatase synaptojanin 1 (SYNJ1), a key regulator of the signalling phospholipid phosphatidylinositol-4,5-biphosphate that has been shown to regulate clathrin-mediated endocytosis. We found that SYNJ1 transcripts are increased in LCLs from individuals with DS and that overexpression of SYNJ1 in a neuroblastoma cell line as well as in transgenic mice leads to enlarged endosomes. Moreover, the proportion of enlarged endosomes in fibroblasts from an individual with DS was reduced after silencing SYNJ1 expression with RNA interference. In LCLs carrying amyloid precursor protein (APP) microduplications causing autosomal dominant early-onset AD, enlarged endosomes were absent, suggesting that APP overexpression alone is not involved in the modification of early endosomes in this cell type. These findings provide new insights into the contribution of SYNJ1 overexpression to the endosomal changes observed in DS and suggest an attractive new target for rescuing endocytic dysfunction and lipid metabolism in DS and in AD.

How many entities exist for the spectrum of disorders associated with brachydactyly, syndactyly, short stature, microcephaly, and intellectual disability?

We describe a French young man with digital anomalies consisting of brachydactyly, F1-5 bilateral camptodactyly, interdigital webbing, F5 bilateral radial clinodactyly, and partial syndactyly of some fingers and toes. He had psychomotor retardation, short stature, umbilical hernia, a secundum atrial septal defect, seizures, hearing impairment, and dysmorphic features consisting of microcephaly, a prominent metopic ridge, upslanting palpebral fissures, synophrys, enophthalmia, large ears, a bulbous nose, a high palate, a smooth and short philtrum, a low hanging columella, a thin upper vermillion, an everted lower lip, prognathism, pectum excavatum, and supernumerary nipples. Osteotendinous reflexes were brisk. Mild nystagmus, myopia, and astigmatia were also noted. Total body X-rays showed short terminal phalanges of the hands, short middle phalanges of the index and little fingers, clinodactyly of the little fingers, short and fused proximal 4th and 5th metacarpals of the right hand, a short 5th metacarpal of the left hand, a fused left lunate-triquetrum, fused capitate-hamates, a prominent mandibula, and partial sacral agenesis. A thin posterior corpus callosum was apparent by MRI. Differential diagnoses for mainly the Rubinstein-Taybi syndrome, the Tsukahara syndrome, the Filippi syndrome, the Feingold syndrome, and the Tonoki syndrome are discussed, and the possibility that we might be reporting a novel entity is raised. © 2011 Wiley-Liss, Inc.

Recessive marfanoid syndrome with herniation associated with a homozygous mutation in Fibulin-3.

We have previously reported on a consanguineous family where 2 siblings, a girl and a boy, presented with tall stature, long and triangular faces, prominent forehead, telecanthus, ptosis, everted lower eyelids, downslanting palpebral fissures, large ears, high arched palate, long arm span, arachnodactyly, advanced bone age, joint laxity, pectus excavatum, inguinal hernia, and myopia, suggestive of a new subtype of connective tissue disorder (Megarbane et al. AJMG, 2012; 158(A)5: 1185-1189). On clinical follow-up, both patients had multiple inguinal, crural, and abdominal herniae, intestinal occlusions, several huge diverticula throughout the gut and the bladder, and rectal prolapse. In addition, the girl had a mild hearing impairment, and the boy a left diaphragmatic hernia. Here we describe the molecular characterization of this disorder using Whole Exome Sequencing, revealing, in both siblings, a novel homozygous missense variant in the EFEMP1 gene, c.163T > C; p.(Cys55Arg) whose homozygous by descent, autosomal recessive transmission was confirmed through segregation analysis by Sanger sequencing. In addition, the girl exhibited a homozygous mutation in the MYO3A gene, c.1370_1371delGA; p.(Arg457Asnfs*25), associated with non-syndromic deafness. The siblings were also found to harbor a homozygous nonsense variant in the VCPKMT gene. We review the literature and discuss our updated clinical and molecular findings that suggest EFEMP1 to be the probable candidate gene implicated in this novel connective tissue disease.

Transcriptomic study in women with trisomy 21 identifies a possible role of the GTPases of the immunity-associated proteins (GIMAP) in the protection of breast cancer.

BACKGROUND: People with trisomy 21 (T21) are predisposed to developing hematological tumors, but have significantly lower-than-expected age-adjusted incidence rates of having a solid tumor. MATERIAL AND METHODS: To identify novel genetic factors implicated in the lower breast cancer (BC) frequency observed in women with T21 than in the general population, we compared the transcriptome pattern of women with a homogeneous T21, aged more than 30 years, with or without BC, and tumoral BC tissue of control women with a normal karyotype from the study of Varley et al. (2014). RESULTS: Differential analysis of gene expression between the 15 women in the T21 without BC group and BC patients in the other groups (two women with T21 and fifteen control women, respectively) revealed 154 differentially expressed genes, of which 63 were found to have similar expression profile (up- or downregulated). Of those 63 genes, four were in the same family, namely GIMAP4, GIMAP6, GIMAP7 and GIMAP8, and were strongly upregulated in the T21 without BC group compared to the other groups. A significant decrease in mRNA levels of these genes in BC tissues compared to non-tumor breast tissues was also noted. CONCLUSION: We found that the expression of some GIMAPs is significantly higher in women with T21 without BC than in patients with sporadic BC. Our findings support the hypothesis that GIMAPs may play a tumor-suppressive role against BC, and open the possibility that they may also have the same role for other solid tumors in T21 patients. The search for new prognostic factors and hopefully new therapeutic or preventive strategies against BC are discussed.

A novel PDE6D mutation in a patient with Joubert syndrome type 22 (JBTS22).

Joubert syndrome (JS) is an autosomal or X-linked recessive syndrome principally characterized by hypotonia, ataxia, cognitive impairment, and a specific finding on brain imaging called a "molar tooth sign" (MTS), which can be isolated or in conjunction with variable organ involvement. The genetic basis of JS is heterogeneous, with over 35 ciliary genes being implicated in its pathogenesis. However, some of these genes (such as PDE6D) have been associated to JS only in single families, seeking confirmation. Here we report a boy, born to first cousin parents, presenting with developmental delay, hypotonia, microcephaly, post axial polydactyly, oculomotor apraxia, and MTS. Whole exome sequencing revealed the presence of a novel homozygous truncating variant in the PDE6D gene: NM_002601.3:c.367_368insG [p.(Leu123Cysfs*13)]. The variant was confirmed by Sanger sequencing and found at the heterozygous state in both parents. A review of the literature pertaining to the role of PDE6D in JS is discussed.

Report of a Second Lebanese Family with Basel-Vanagaite-Smirin-Yosef Syndrome: Possible Founder Mutation.

Basel-Vanagaite-Smirin-Yosef syndrome (OMIM 616449) is a rare autosomal recessive genetic disorder characterized by severe developmental delay and variable craniofacial, neurological, cardiac, and ocular anomalies in the presence of variants in the MED25 gene. So far, only a handful of patients have been reported with this condition globally. Here, we report an additional Lebanese family with 2 affected siblings presenting with severely delayed psychomotor and language development as well as craniofacial anomalies. By whole-exome sequencing (WES), a homozygous variant was found in the MED25 gene, c.518T>C, predicted to result in a p.Ile173Thr change in the MED25 protein. This change has recently been reported in another Lebanese family. Review of the literature, the importance of this mutation in the Lebanese population, and the possibility that this condition may be underdiagnosed and only effectively detected using molecular techniques such as WES are discussed.

COQ8A and MED25 Mutations in a Child with Intellectual Disability, Microcephaly, Seizures, and Spastic Ataxia: Synergistic Effect of Digenic Variants?

We report on a girl, born to first-cousin Lebanese parents, with severe intellectual disability, congenital hip luxation, cardiac malformation, short stature, facial dysmorphic features including microcephaly, sparse hair, bilateral epicanthal folds, ataxia, seizures, and elevated lactate and pyruvate levels in serum. Whole exome sequencing was carried out on the patient's DNA. Potentially causal homozygous variants in the MED25 (p.Ile173Thr) and COQ8A (p.Arg512Trp) genes were found. The potential pathogenicity of these variants, and the possibility that the 2 variants could synergistically act to produce the phenotype reported, is discussed.

Methylomic profiling in trisomy 21 identifies cognition- and Alzheimer's disease-related dysregulation.

BACKGROUND: Trisomy 21 (T21) is associated with intellectual disability that ranges from mild to profound with an average intellectual quotient of around 50. Furthermore, T21 patients have a high risk of developing Alzheimer's disease (AD) early in life, characterized by the presence of senile plaques of amyloid protein and neurofibrillary tangles, leading to neuronal loss and cognitive decline. We postulate that epigenetic factors contribute to the observed variability in intellectual disability, as well as at the level of neurodegeneration seen in T21 individuals. MATERIALS AND METHODS: A genome-wide DNA methylation study was performed using Illumina Infinium® MethylationEPIC BeadChips on whole blood DNA of 3 male T21 patients with low IQ, 8 T21 patients with high IQ (4 males and 4 females), and 21 age- and sex-matched control samples (12 males and 9 females) in order to determine whether DNA methylation alterations could help explain variation in cognitive impairment between individuals with T21. In view of the increased risk of developing AD in T21 individuals, we additionally investigated the T21-associated sites in published blood DNA methylation data from the AgeCoDe cohort (German study on Ageing, Cognition, and Dementia). AgeCoDe represents a prospective longitudinal study including non-demented individuals at baseline of which a part develops AD dementia at follow-up. RESULTS: Two thousand seven hundred sixteen differentially methylated sites and regions discriminating T21 and healthy individuals were identified. In the T21 high and low IQ comparison, a single CpG located in the promoter of PELI1 was differentially methylated after multiple testing adjustment. For the same contrast, 69 differentially methylated regions were identified. Performing a targeted association analysis for the significant T21-associated CpG sites in the AgeCoDe cohort, we found that 9 showed significant methylation differences related to AD dementia, including one in the ADAM10 gene. This gene has previously been shown to play a role in the prevention of amyloid plaque formation in the brain. CONCLUSION: The differentially methylated regions may help understand the interaction between methylation alterations and cognitive function. In addition, ADAM10 might be a valuable blood-based biomarker for at least the early detection of AD.

Contribution of next generation sequencing in pediatric practice in Lebanon. A Study on 213 cases.

BACKGROUND: According to the Catalogue of Transmission Genetics in Arabs, less than half of diseases reported in Lebanese patients are mapped. In the recent years, Next Generation Sequencing (NGS) techniques have significantly improved clinical diagnosis, compared to traditional sequencing methods. METHODS: A total of 213 analyses by NGS (167 by whole exome sequencing (WES) and 46 by multigene panels tests) were performed on pediatric patients across different regions of Lebanon over a period of two years (December 2015-December 2017). RESULTS: Neurological disorders were the most frequent referral demand for both WES and gene panels (122/213). Pathogenic, likely pathogenic, or variants of unknown significance were identified in 69.5% of the WES and panel patients combined. Over half of the patients with such variants had an autosomal recessive disorder. A definite molecular diagnosis (pathogenic or likely pathogenic variants) was achieved in 34.1% and 47.8% of the patients studied by WES and the multigene panels, respectively. Thirty-three novel variants were found in the cases that were molecularly solved; 26 of these being identified by WES and seven by the multigene panels. In three consanguineous families, autosomal recessive inheritance of genes previously reported as showing dominant inheritance patterns were found. Biallelism was found in six cases, digenism in four cases, and one case was trigenic. CONCLUSION: Our study thus suggests that NGS tools are valuable for an improved clinical diagnosis, and highlights that the increased adoption of such techniques will significantly further improve our understanding of the genetic basis of inherited diseases in Lebanon.

Characterization of a ceramide kinase-like protein.

Ceramide is a key player governing cell fate, and its conversion to ceramide-1-phosphate by ceramide kinase (CERK) is emerging as an important mean to regulate apoptosis and inflammatory processes. We identified a new ceramide kinase homolog, designated CERK-like protein (CERKL) and we compared it to the known CERK. Real time-PCR analysis of human tissues revealed a restricted pattern of expression for CERKL mRNA. Surprisingly, various ceramides, known substrates for CERK, were not phosphorylated by CERKL in vitro. Upon 32P(i)-pulse labeling of COS-1 cells transiently expressing CERKL, or incubation with NBD-C6-ceramide, ceramide-1-phosphate was not detected. After recombinant expression in COS-1 cells, CERKL was partially recovered in the soluble fraction, as a phosphorylated protein. Live cell imaging indicated localization of GFP-tagged CERKL to many cell compartments, including specific association with nucleoli. Two splice variants of CERKL did not localize to nucleoli nor did a CERKL variant with a point mutation in the putative ATP binding site. We also studied a naturally occurring CERKL mutant (R257X), recently linked to the pathology of retinitis pigmentosa. It accumulated in the nucleus but was not associated with nucleoli. Treatment with the calcium ionophore A23187 led to clearing of CERKL from nucleoli, but had no effect on the R257X CERKL mutant. Collectively, although kinase activity of CERKL remains to be proven, these findings suggest a functional link between CERKL and its nucleolar localization. Furthermore, we propose that the cause for retinitis pigmentosa in patients bearing the CERKL R257X mutation might be the accumulation of a truncated CERKL protein in the nucleus.

Neuroinflammation and Aß accumulation linked to systemic inflammation are decreased by genetic PKR down-regulation.

Alzheimer's disease (AD) is a neurodegenerative disorder, marked by senile plaques composed of amyloid-ß (Aß) peptide, neurofibrillary tangles, neuronal loss and neuroinflammation. Previous works have suggested that systemic inflammation could contribute to neuroinflammation and enhanced Aß cerebral concentrations. The molecular pathways leading to these events are not fully understood. PKR is a pro-apoptotic kinase that can trigger inflammation and accumulates in the brain and cerebrospinal fluid of AD patients. The goal of the present study was to assess if LPS-induced neuroinflammation and Aß production could be altered by genetic PKR down regulation. The results show that, in the hippocampus of LPS-injected wild type mice, neuroinflammation, cytokine release and Aß production are significantly increased and not in LPS-treated PKR knock-out mice. In addition BACE1 and activated STAT3 levels, a putative transcriptional regulator of BACE1, were not found increased in the brain of PKR knock-out mice as observed in wild type mice. Using PET imaging, the decrease of hippocampal metabolism induced by systemic LPS was not observed in LPS-treated PKR knock-out mice. Altogether, these findings demonstrate that PKR plays a major role in brain changes induced by LPS and could be a valid target to modulate neuroinflammation and Aß production.

The intellectual disability of trisomy 21: differences in gene expression in a case series of patients with lower and higher IQ.

Trisomy 21 (T21), or Down syndrome (DS), is the most frequent and recognizable cause of intellectual disabilities. The level of disability, as evaluated by the intelligence quotient (IQ) test, varies considerably between patients independent of other factors. To determine the genetic or molecular basis of this difference, a high throughput transcriptomic analysis was performed on twenty T21 patients with high and low IQ, and 10 healthy controls using Digital Gene Expression. More than 90 millions of tags were sequenced in the three libraries. A total of 80 genes of potential interest were selected for the qPCR experiment validation, and three housekeeping genes were used for normalizing purposes. HLA DQA1 and HLA DRB1 were significantly downregulated among the patients with a low IQ, the values found in the healthy controls being intermediate between those noted in the IQ+ and IQ- T21 patients. Interestingly, the intergenic region between these genes contains a binding sequence for the CCCTC-binding factor, or CTCF, and cohesin (a multisubunit complex), both of which are essential for expression of HLA DQA1 and HLA DRB1 and numerous other genes. Our results might lead to the discovery of genes, or genetic markers, that are directly involved in several phenotypes of DS and, eventually, to the identification of potential targets for therapeutic interventions.

BDNF and DYRK1A are variable and inversely correlated in lymphoblastoid cell lines from Down syndrome patients.

Down syndrome or trisomy 21 is the most common genetic disorder leading to mental retardation. One feature is impaired short- and long-term spatial memory, which has been linked to altered brain-derived neurotrophic factor (BDNF) levels. Mouse models of Down syndrome have been used to assess neurotrophin levels, and reduced BDNF has been demonstrated in brains of adult transgenic mice overexpressing Dyrk1a, a candidate gene for Down syndrome phenotypes. Given the link between DYRK1A overexpression and BDNF reduction in mice, we sought to assess a similar association in humans with Down syndrome. To determine the effect of DYRK1A overexpression on BDNF in the genomic context of both complete trisomy 21 and partial trisomy 21, we used lymphoblastoid cell lines from patients with complete aneuploidy of human chromosome 21 (three copies of DYRK1A) and from patients with partial aneuploidy having either two or three copies of DYRK1A. Decreased BDNF levels were found in lymphoblastoid cell lines from individuals with complete aneuploidy as well as those with partial aneuploidies conferring three DYRK1A alleles. In contrast, lymphoblastoid cell lines from individuals with partial trisomy 21 having only two DYRK1A copies displayed increased BDNF levels. A negative correlation was also detected between BDNF and DYRK1A levels in lymphoblastoid cell lines with complete aneuploidy of human chromosome 21. This finding indicates an upward regulatory role of DYRK1A expression on BDNF levels in lymphoblastoid cell lines and emphasizes the role of genetic variants associated with psychiatric disorders.

A 56-year-old female patient with facio-oculo-acoustico-renal syndrome (FOAR) syndrome. Report on the natural history and of a novel mutation.

The facio-oculo-acoustico-renal syndrome (FOAR) is a rare autosomal recessive syndrome characterized by the presence of dysmorphic facial features, ocular anomalies, sensorineural hearing loss, and proteinuria. Diaphragmatic hernia, exomphalos, absent or abnormal corpus callosum, and myopia, can also be part of the syndrome. The disorder is caused by mutations of the LRP2 gene located on chromosome 2q23.3-q31.1. We hereby report the case of a 56-year-old female patient with typical FOAR features. Molecular study of the LRP2 gene revealed the presence of a novel splice-site mutation. In addition to what was reported in FOAR syndrome, this patient had a megadolichocolon complicated by a volvulus and a late-onset renal failure which necessitated hemodyalisis and renal transplantation. Reporting aging patients with genetic syndromes will provide information about their special needs and lead to improvements in their follow-up.

Ceramide kinase targeting and activity determined by its N-terminal pleckstrin homology domain.

The N-terminus of ceramide kinase (CERK) is thought to be myristoylated and to contain a pleckstrin homology (PH) domain. We found that deletion of this region (DeltaPH-CERK) ablates activity. This is not due to prevention of N-terminal myristoylation since a G2A CERK mutant, which cannot be myristoylated, was active. CERK was able to bind liposomes, as well as the isolated unmyristoylated PH domain; DeltaPH-CERK was not. Upon analysis of EGFP-tagged proteins, CERK was found associated with the Golgi complex. Osmotic cell swelling induced translocation of CERK to the plasma membrane and formation of large vesicles displaying bound CERK. None of these features occurred with DeltaPH-CERK, which remained disseminated throughout the cytoplasm. These findings show that the PH domain of CERK is essential for localization, translocation, and activity of this lipid kinase.