Intellectual disability inherited in an autosomal-recessive fashion represents an important fraction of severe cognitive-dysfunction disorders. Yet, the extreme heterogeneity of these conditions markedly hampers gene identification. Here, we report on eight affected individuals who were from three consanguineous families and presented with severe intellectual disability, absent speech, shy character, stereotypic laughter, muscular hypotonia that progressed to spastic paraplegia, microcephaly, foot deformity, decreased muscle mass of the lower limbs, inability to walk, and growth retardation. Using a combination of autozygosity mapping and either Sanger sequencing of candidate genes or next-generation exome sequencing, we identified one mutation in each of three genes encoding adaptor protein complex 4 (AP4) subunits: a nonsense mutation in AP4S1 (NM_007077.3: c.124C>T, p.Arg42(∗)), a frameshift mutation in AP4B1 (NM_006594.2: c.487_488insTAT, p.Glu163_Ser739delinsVal), and a splice mutation in AP4E1 (NM_007347.3: c.542+1_542+4delGTAA, r.421_542del, p.Glu181Glyfs(∗)20). Adaptor protein complexes (AP1-4) are ubiquitously expressed, evolutionarily conserved heterotetrameric complexes that mediate different types of vesicle formation and the selection of cargo molecules for inclusion into these vesicles. Interestingly, two mutations affecting AP4M1 and AP4E1 have recently been found to cause cerebral palsy associated with severe intellectual disability. Combined with previous observations, these results support the hypothesis that AP4-complex-mediated trafficking plays a crucial role in brain development and functioning and demonstrate the existence of a clinically recognizable syndrome due to deficiency of the AP4 complex.

Because of the high diagnostic yield of 36.8% and the possibility of identifying treatable diseases or the coexistence of several disease-causing variants, using exome sequencing as a first-line diagnostic approach in consanguineous families with neurodevelopmental disorders is recommended. Furthermore, the literature is enriched with 52 convincing candidate genes that are awaiting confirmation in independent families.

AMPA-type glutamate receptors (AMPARs), key elements in excitatory neurotransmission in the brain, are macromolecular complexes whose properties and cellular functions are determined by the co-assembled constituents of their proteome. Here we identify AMPAR complexes that transiently form in the endoplasmic reticulum (ER) and lack the core-subunits typical for AMPARs in the plasma membrane. Central components of these ER AMPARs are the proteome constituents FRRS1l (C9orf4) and CPT1c that specifically and cooperatively bind to the pore-forming GluA1-4 proteins of AMPARs. Bi-allelic mutations in the human FRRS1L gene are shown to cause severe intellectual disability with cognitive impairment, speech delay and epileptic activity. Virus-directed deletion or overexpression of FRRS1l strongly impact synaptic transmission in adult rat brain by decreasing or increasing the number of AMPARs in synapses and extra-synaptic sites. Our results provide insight into the early biogenesis of AMPARs and demonstrate its pronounced impact on synaptic transmission and brain function.

Rhizomelic chondrodysplasia punctata (RCDP) is a group of disorders with overlapping clinical features including rhizomelia, chondrodysplasia punctata, coronal clefts, cervical dysplasia, congenital cataracts, profound postnatal growth retardation, severe intellectual disability, and seizures. Mutations in PEX7, GNPAT, and AGPS, all involved in the plasmalogen-biosynthesis pathway, have been described in individuals with RCDP. Here, we report the identification of mutations in another gene in plasmalogen biosynthesis, fatty acyl-CoA reductase 1 (FAR1), in two families affected by severe intellectual disability, early-onset epilepsy, microcephaly, congenital cataracts, growth retardation, and spasticity. Exome analyses revealed a homozygous in-frame indel mutation (c.495_507delinsT [p.Glu165_Pro169delinsAsp]) in two siblings from a consanguineous family and compound-heterozygous mutations (c.[787C>T];[1094A>G], p.[Arg263(∗)];[Asp365Gly]) in a third unrelated individual. FAR1 reduces fatty acids to their respective fatty alcohols for the plasmalogen-biosynthesis pathway. To assess the pathogenicity of the identified mutations, we transfected human embryonic kidney 293 cells with plasmids encoding FAR1 with either wild-type or mutated constructs and extracted the lipids from the cells. We screened the lipids with gas chromatography and mass spectrometry and found that all three mutations abolished the reductase activity of FAR1, given that no fatty alcohols could be detected. We also observed reduced plasmalogens in red blood cells in one individual to a range similar to that seen in individuals with RCDP, further supporting abolished FAR1 activity. We thus expand the spectrum of clinical features associated with defects in plasmalogen biosynthesis to include FAR1 deficiency as a cause of syndromic severe intellectual disability with cataracts, epilepsy, and growth retardation but without rhizomelia.

PGAP2 encodes a protein involved in remodeling the glycosylphosphatidylinositol (GPI) anchor in the Golgi apparatus. After synthesis in the endoplasmic reticulum (ER), GPI anchors are transferred to the proteins and are remodeled while transported through the Golgi to the cell membrane. Germline mutations in six genes (PIGA, PIGL, PIGM, PIGV, PIGN, and PIGO) in the ER-located part of the GPI-anchor-biosynthesis pathway have been reported, and all are associated with phenotypes extending from malformation and lethality to severe intellectual disability, epilepsy, minor dysmorphisms, and elevated alkaline phosphatase (ALP). We performed autozygosity mapping and ultra-deep sequencing followed by stringent filtering and identified two homozygous PGAP2 alterations, p.Tyr99Cys and p.Arg177Pro, in seven offspring with nonspecific autosomal-recessive intellectual disability from two consanguineous families. Rescue experiments with the altered proteins in PGAP2-deficient Chinese hamster ovary cell lines showed less expression of cell-surface GPI-anchored proteins DAF and CD59 than of the wild-type protein, substantiating the pathogenicity of the identified alterations. Furthermore, we observed a full rescue when we used strong promoters before the mutant cDNAs, suggesting a hypomorphic effect of the mutations. We report on alterations in the Golgi-located part of the GPI-anchor-biosynthesis pathway and extend the phenotypic spectrum of the GPI-anchor deficiencies to isolated intellectual disability with elevated ALP. GPI-anchor deficiencies can be interpreted within the concept of a disease family, and we propose that the severity of the phenotype is dependent on the location of the altered protein in the biosynthesis chain.

Many eukaryotic cell-surface proteins are anchored to the membrane via glycosylphosphatidylinositol (GPI). There are at least 26 genes involved in biosynthesis and remodeling of GPI anchors. Hypomorphic coding mutations in seven of these genes have been reported to cause decreased expression of GPI anchored proteins (GPI-APs) on the cell surface and to cause autosomal-recessive forms of intellectual disability (ARID). We performed homozygosity mapping and exome sequencing in a family with encephalopathy and non-specific ARID and identified a homozygous 3 bp deletion (p.Leu197del) in the GPI remodeling gene PGAP1. PGAP1 was not described in association with a human phenotype before. PGAP1 is a deacylase that removes an acyl-chain from the inositol of GPI anchors in the endoplasmic reticulum immediately after attachment of GPI to proteins. In silico prediction and molecular modeling strongly suggested a pathogenic effect of the identified deletion. The expression levels of GPI-APs on B lymphoblastoid cells derived from an affected person were normal. However, when those cells were incubated with phosphatidylinositol-specific phospholipase C (PI-PLC), GPI-APs were cleaved and released from B lymphoblastoid cells from healthy individuals whereas GPI-APs on the cells from the affected person were totally resistant. Transfection with wild type PGAP1 cDNA restored the PI-PLC sensitivity. These results indicate that GPI-APs were expressed with abnormal GPI structure due to a null mutation in the remodeling gene PGAP1. Our results add PGAP1 to the growing list of GPI abnormalities and indicate that not only the cell surface expression levels of GPI-APs but also the fine structure of GPI-anchors is important for the normal neurological development.

There are two known mRNA degradation pathways, 3' to 5' and 5' to 3'. We identified likely pathogenic variants in two genes involved in these two pathways in individuals with intellectual disability. In a large family with multiple branches, we identified biallelic variants in DCPS in three affected individuals; a splice site variant (c.636+1G>A) that results in an in-frame insertion of 45 nucleotides and a missense variant (c.947C>T; p.Thr316Met). DCPS decaps the cap structure generated by 3' to 5' exonucleolytic degradation of mRNA. In vitro decapping assays showed an ablation of decapping function for both variants in DCPS. In another family, we identified a homozygous mutation (c.161T>C; p.Phe54Ser) in EDC3 in two affected children. EDC3 stimulates DCP2, which decaps mRNAs at the beginning of the 5' to 3' degradation pathway. In vitro decapping assays showed that altered EDC3 is unable to enhance DCP2 decapping at low concentrations and even inhibits DCP2 decapping at high concentration. We show that individuals with biallelic mutations in these genes of seemingly central functions are viable and that these possibly lead to impairment of neurological functions linking mRNA decapping to normal cognition. Our results further affirm an emerging theme linking aberrant mRNA metabolism to neurological defects.

Next-generation sequencing combined with international data sharing has enormously facilitated identification of new disease-associated genes and mutations. This is particularly true for genetically extremely heterogeneous entities such as neurodevelopmental disorders (NDDs). Through exome sequencing and world-wide collaborations, we identified and assembled 20 individuals with de novo variants in FBXO11. They present with mild to severe developmental delay associated with a range of features including short (4/20) or tall (2/20) stature, obesity (5/20), microcephaly (4/19) or macrocephaly (2/19), behavioral problems (17/20), seizures (5/20), cleft lip or palate or bifid uvula (3/20), and minor skeletal anomalies. FBXO11 encodes a member of the F-Box protein family, constituting a subunit of an E3-ubiquitin ligase complex. This complex is involved in ubiquitination and proteasomal degradation and thus in controlling critical biological processes by regulating protein turnover. The identified de novo aberrations comprise two large deletions, ten likely gene disrupting variants, and eight missense variants distributed throughout FBXO11. Structural modeling for missense variants located in the CASH or the Zinc-finger UBR domains suggests destabilization of the protein. This, in combination with the observed spectrum and localization of identified variants and the lack of apparent genotype-phenotype correlations, is compatible with loss of function or haploinsufficiency as an underlying mechanism. We implicate de novo missense and likely gene disrupting variants in FBXO11 in a neurodevelopmental disorder with variable intellectual disability and various other features.