TRAPPC9-related autosomal recessive intellectual disability: report of a new mutation and clinical phenotype

Intellectual disability (ID) with autosomal recessive (AR) inheritance is believed to be common; however, very little is known about causative genes and genotype–phenotype correlations. The broad genetic heterogeneity of AR-ID, and its usually nonsyndromic nature make it difficult to pool multiple pedigrees with the same underlying genetic defect to achieve consistent nosology. Nearly all autosomal genes responsible for recessive cognitive disorders have been identified in large consanguineous families from the Middle East, and nonsense mutations in TRAPPC9 have been reported in a total of 5. Although several recurrent phenotypic abnormalities are described in some of these patients, the associated phenotype is usually referred to as nonsyndromic. By means of single-nucleotide polymorphism-array first and then by exome sequencing, we identified a new pathogenic mutation in TRAPPC9 in two Italian sisters born to healthy and apparently nonconsanguineous parents. It consists of a homozygous splice site mutation causing exon skipping with frameshift and premature termination, as confirmed by mRNA sequencing. By detailed phenotypic analysis of our patients, and by critical literature review, we found that homozygous TRAPPC9 loss-of-function mutations cause a distinctive phenotype, characterized by peculiar facial appearance, obesity, hypotonia (all signs resembling a Prader–Willi-like phenotype), moderate-to-severe ID, and consistent brain abnormalities.     

A patient with POLA1 splice variant expands the yet evolving phenotype of Van Esch O'Driscoll syndrome

Van Esch-O’Driscoll syndrome (VEODS) is a rare cause of syndromic X-linked intellectual disability characterised by short stature, microcephaly, variable degree of intellectual disability, and hypogonadotropic hypogonadism. To date, heterozygous hypomorphic variants in the gene encoding the DNA Polymerase α subunit, POLA1, have been observed in nine patients from five unrelated families with VEODS. We report a three-year-old child with VEODS having borderline intellectual disability due to a novel splice site variant causing exon 6 skipping and reduced POLA1 expression.     

Further insights into the spectrum phenotype of TRAPPC9 and CDK5RAP2 genes,segregating independently in a large Tunisian family with intellectual disability and microcephaly

Intellectual disability (ID) often co-occurs with other neurologic phenotypes making molecular diagnosis more challenging particularly in consanguineous populations with the co-segregation of more than one ID-related gene in some cases.In this study, we investigated the phenotype of three patients from a large Tunisian family with significant ID phenotypic variability and microcephaly and performed a clinical exome sequencing in two cases. We identified, within the first branch, a homozygous variant in the TRAPPC9 gene (p.Arg472Ter) in two cases presenting severe ID, absent speech, congenital/secondary microcephaly in addition to autistic features, supporting the implication of TRAPPC9 in the “secondary” autism spectrum disorders and congenital microcephaly. In the second branch, we identified a homozygous variant (p.Lys189ArgfsTer15) in the CDK5RAP2 gene associated with an heterozygous TRAPPC9 variant (p.Arg472Ter) in one case harbouring primary hereditary microcephaly (MCPH) associated with an inter-hypothalamic adhesion, mixed hearing loss, selective thinning in the retinal nerve fiber layer and parafoveal ganglion cell complex, and short stature. Our findings expand the spectrum of the recently reported neurosensorial abnormalities and revealed the variable phenotype expressivity of CDK5RAP2 defect. Our study highlights the complexity of the genetic background of microcephaly/ID and the efficiency of the exome sequencing to provide an accurate diagnosis and to improve the management and follow-up of such patients.     

TRAPPC9-CDG: A novel congenital disorder of glycosylation with dysmorphic features and intellectual disability

PurposeTRAPPC9 deficiency is an autosomal recessive disorder mainly associated with intellectual disability (ID), microcephaly, and obesity. Previously, TRAPPC9 deficiency has not been associated with biochemical abnormalities.MethodsExome sequencing was performed in 3 individuals with ID and dysmorphic features. N-Glycosylation analyses were performed in the patients’ blood samples to test for possible congenital disorder of glycosylation (CDG). TRAPPC9 gene, TRAPPC9 protein expression, and N-glycosylation markers were assessed in patient fibroblasts. Complementation with wild-type TRAPPC9 and immunofluorescence studies to assess TRAPPC9 expression and localization were performed. The metabolic consequences of TRAPPC9 deficiency were evaluated using tracer metabolomics.ResultsAll 3 patients carried biallelic missense variants in TRAPPC9 and presented with an N-glycosylation defect in blood, consistent with CDG type I. Extensive investigations in patient fibroblasts corroborated TRAPPC9 deficiency and an N-glycosylation defect. Tracer metabolomics revealed global metabolic changes with several affected glycosylation-related metabolites.ConclusionWe identified 3 TRAPPC9 deficient patients presenting with ID, dysmorphic features, and abnormal glycosylation. On the basis of our findings, we propose that TRAPPC9 deficiency could lead to a CDG (TRAPPC9-CDG). The finding of abnormal glycosylation in these patients is highly relevant for diagnosis, further elucidation of the pathophysiology, and management of the disease.     

Revealing the functions of novel mutations in RAB3GAP1 in Martsolf and Warburg micro syndromes

Purpose: Martsolf (MS) and Warburg micro syndromes (WARBM) are rare autosomal recessive inherited allelic disorders, which share similar clinical features including microcephaly, intellectual disability, brain malformations, ocular abnormalities, and spasticity. Here, we revealed the functions of novel mutations in RAB3GAP1 in a Turkish female patient with MS and two siblings with WARBM. We also present a review of MS patients as well as all reported RAB3GAP1 pathogenic mutations in the literature. Methods: We present a female with MS phenotype and two siblings with WARBM having more severe phenotypes. We utilized whole‐exome sequencing to identify the molecular basis of these syndromes and confirmed suspected variants by Sanger sequencing. Quantitative (q) RT‐PCR analysis was carried out to reveal the functions of novel splice site mutation detected in MS patient. Results: We found a novel homozygous c.2607‐1G>C splice site mutation in intron 22 of RAB3GAP1 in MS patient and a novel homozygous c.2187_2188delinsCT, p.(Met729_Lys730delinsIleTer) mutation in exon 19 of RAB3GAP1 in the WARBM patients. We showed exon skipping in MS patient by Sanger sequencing and gel electrophoresis. qRT‐PCR analysis demonstrated the reduced expression of RAB3GAP1 in the patient with the c.2607‐1G>C splice site mutation compared to a healthy control individual. Conclusion: Here, we have studied two novel RAB3GAP1 mutations in two different phenotypes; a MS associated novel splice site mutation, and a WARBM1 associated novel deletion–insertion mutation. Our findings suggest that this splice site mutation is responsible for milder phenotype and the deletion–insertion mutation presented here is associated with severe phenotype.     

Autosomal dominant SCA5 and autosomal recessive infantile SCA are allelic conditions resulting from SPTBN2 mutations

Although many genes have been identified for the autosomal recessive cerebellar ataxias (ARCAs), several patients are unlinked to the respective loci, suggesting further genetic heterogeneity. We combined homozygosity mapping and exome sequencing in a consanguineous Egyptian family with congenital ARCA, mental retardation and pyramidal signs. A homozygous 5-bp deletion in SPTBN2, the gene whose in-frame mutations cause autosomal dominant spinocerebellar ataxia type 5, was shown to segregate with ataxia in the family. Our findings are compatible with the concept of truncating SPTBN2 mutations acting recessively, which is supported by disease expression in homozygous, but not heterozygous, knockout mice, ataxia in Beagle dogs with a homozygous frameshift mutation and, very recently, a homozygous SPTBN2 nonsense mutation underlying infantile ataxia and psychomotor delay in a human family. As there was no evidence for mutations in 23 additional consanguineous families, SPTBN2-related ARCA is probably rare.     

Novel biallelic SZT2 mutations in 3 cases of early‐onset epileptic encephalopathy

The seizure threshold 2 (SZT2) gene encodes a large, highly conserved protein that is associated with epileptogenesis. In mice, Szt2 is abundantly expressed in the central nervous system. Recently, biallelic SZT2 mutations were found in 7 patients (from 5 families) presenting with epileptic encephalopathy with dysmorphic features and/or non‐syndromic intellectual disabilities. In this study, we identified by whole‐exome sequencing compound heterozygous SZT2 mutations in 3 patients with early‐onset epileptic encephalopathies. Six novel SZT2 mutations were found, including 3 truncating, 1 splice site and 2 missense mutations. The splice‐site mutation resulted in skipping of exon 20 and was associated with a premature stop codon. All individuals presented with seizures, severe developmental delay and intellectual disabilities with high variability. Brain MRIs revealed a characteristic thick and short corpus callosum or a persistent cavum septum pellucidum in each of the 2 cases. Interestingly, in the third case, born to consanguineous parents, had unexpected compound heterozygous missense mutations. She showed microcephaly despite the other case and previous ones presenting with macrocephaly, suggesting that SZT2 mutations might affect head size.     

Biallelic variants in LINGO1 are associated with autosomal recessive intellectual disability,microcephaly, speech and motor delay

PurposeTo elucidate the novel molecular cause in two unrelated consanguineous families with autosomal recessive intellectual disability.MethodsA combination of homozygosity mapping and exome sequencing was used to locate the plausible genetic defect in family F162, while only exome sequencing was followed in the family PKMR65. The protein 3D structure was visualized with the University of California–San Francisco Chimera software.ResultsAll five patients from both families presented with severe intellectual disability, aggressive behavior, and speech and motor delay. Four of the five patients had microcephaly. We identified homozygous missense variants in LINGO1, p.(Arg290His) in family F162 and p.(Tyr288Cys) in family PKMR65. Both variants were predicted to be pathogenic, and segregated with the phenotype in the respective families. Molecular modeling of LINGO1 suggests that both variants interfere with the glycosylation of the protein.ConclusionLINGO1 is a transmembrane receptor, predominantly found in the central nervous system. Published loss-of-function studies in mouse and zebrafish have established a crucial role of LINGO1 in normal neuronal development and central nervous system myelination by negatively regulating oligodendrocyte differentiation and neuronal survival. Taken together, our results indicate that biallelic LINGO1 missense variants cause autosomal recessive intellectual disability in humans.     

Mutations in the mitochondrial gene C12ORF65 lead to syndromic autosomal recessive intellectual disability and show genotype phenotype correlation

Homozygosity mapping and exome sequencing in two affected siblings of a consanguineous family with mild intellectual disability, spastic paraplegia, and strabismus revealed a homozygous premature stop mutation at codon 139 of C12ORF65. Two previous studies reported truncating mutations at positions 84 and 132 of the protein. However, symptoms of the referred patients were only partially overlapping. Considering our findings, we now conclude that truncating mutations in C12ORF65 lead to a variable phenotype with intellectual disability, spastic paraplegia, and ophthalmoplegia as common symptoms. Further, we confirm a genotype–phenotype correlation between increasing length of the truncated protein and decreasing severity of symptoms.     

Delineation of C12orf65-related phenotypes: a genotype–phenotype relationship

C12orf65 participates in the process of mitochondrial translation and has been shown to be associated with a spectrum of phenotypes, including early onset optic atrophy, progressive encephalomyopathy, peripheral neuropathy, and spastic paraparesis.We used whole-genome homozygosity mapping as well as exome sequencing and targeted gene sequencing to identify novel C12orf65 disease-causing mutations in seven affected individuals originating from two consanguineous families. In four family members affected with childhood-onset optic atrophy accompanied by slowly progressive peripheral neuropathy and spastic paraparesis, we identified a homozygous frame shift mutation c.413_417 delAACAA, which predicts a truncated protein lacking the C-terminal portion. In the second family, we studied three affected individuals who presented with early onset optic atrophy, peripheral neuropathy, and spastic gait in addition to moderate intellectual disability. Muscle biopsy in two of the patients revealed decreased activities of the mitochondrial respiratory chain complexes I and IV. In these patients, we identified a homozygous splice mutation, g.21043 T>A (c.282+2 T>A) which leads to skipping of exon 2. Our study broadens the phenotypic spectrum of C12orf65 defects and highlights the triad of optic atrophy, axonal neuropathy and spastic paraparesis as its key clinical features. In addition, a clear genotype–phenotype correlation is anticipated in which deleterious mutations which disrupt the GGQ-containing domain in the first coding exon are expected to result in a more severe phenotype, whereas down-stream C-terminal mutations may result in a more favorable phenotype, typically lacking cognitive impairment.     

Expanding the phenotype of IQSEC2 mutations: truncating mutations in severe intellectual disability

Intellectual disability (ID) is frequent in the general population, with 1 in 50 individuals directly affected worldwide. The multiple etiologies include X-linked ID (XLID). Among syndromic XLID, few syndromes present severe ID associated with postnatal microcephaly and midline stereotypic hand movements. We report on three male patients with ID, midline stereotypic hand movements, hypotonia, hyperkinesia, strabismus, as well as seizures (2/3), and non-inherited and postnatal onset microcephaly (2/3). Using array CGH and exome sequencing we characterised two truncating mutations in IQSEC2, namely two de novo intragenic duplication mapped to the Xp11.22 region and a nonsense mutation in exon 7. We propose that truncating mutations in IQSEC2 are responsible for syndromic severe ID in male patients and should be screened in patients without mutations in MECP2, FOXG1, CDKL5 and MEF2C.     

Autosomal recessive retinitis pigmentosa and cone-rod dystrophy caused by splice site mutations in the Stargardt's disease gene ABCR

Ophthalmological and molecular genetic studies were performed in a consanguineous family with individuals showing either retinitis pigmentosa (RP) or cone-rod dystrophy (CRD). Assuming pseudodominant (recessive) inheritance of allelic defects, linkage analysis positioned the causal gene at 1p21-p13 (lod score 4.22), a genomic segment known to harbor the ABCR gene involved in Stargardt's disease (STGD) and age- related macular degeneration (AMD). We completed the exon-intron structure of the ABCR gene and detected a severe homozygous 5[prime] splice site mutation, IVS30+1G->T, in the four RP patients. The five CRD patients in this family are compound heterozygotes for the IVS30+1G- >T mutation and a 5[prime] splice site mutation in intron 40 (IVS40+5G- >A). Both splice site mutations were found heterozygously in two unrelated STGD patients, but not in 100 control individuals. In these patients the second mutation was either a missense mutation or unknown. Since thus far no STGD patients have been reported to carry two ABCR null alleles and taking into account that the RP phenotype is more severe than the STGD phenotype, we hypothesize that the intron 30 splice site mutation represents a true null allele. Since the intron 30 mutation is found heterozygously in the CRD patients, the IVS40+5G->A mutation probably renders the exon 40 5[prime] splice site partially functional. These results show that mutations in the ABCR gene not only result in STGD and AMD, but can also cause autosomal recessive RP and CRD. Since the heterozygote frequency for ABCR mutations is estimated at 0.02, mutations in ABCR might be an important cause of autosomal recessive and sporadic forms of RP and CRD.      

Large deletions encompassing the TCOF1 and CAMK2A genes are responsible for Treacher Collins syndrome with intellectual disability

Mandibulofacial dysostosis is part of a clinically and genetically heterogeneous group of disorders of craniofacial development, which lead to malar and mandibular hypoplasia. Treacher Collins syndrome is the major cause of mandibulofacial dysostosis and is due to mutations in the TCOF1 gene. Usually patients with Treacher Collins syndrome do not present with intellectual disability. Recently, the EFTUD2 gene was identified in patients with mandibulofacial dysostosis associated with microcephaly, intellectual disability and esophageal atresia. We report on two patients presenting with mandibulofacial dysostosis characteristic of Treacher Collins syndrome, but associated with unexpected intellectual disability, due to a large deletion encompassing several genes including the TCOF1 gene. We discuss the involvement of the other deleted genes such as CAMK2A or SLC6A7 in the cognitive development delay of the patients reported, and we propose the systematic investigation for 5q32 deletion when intellectual disability is associated with Treacher Collins syndrome.     

Novel splice site mutation in CNNM4 gene in a family with Jalili syndrome

Jalili syndrome is a rare autosomal recessive genetic disease characterized by the association of amelogenesis imperfecta and cone-rod retinal dystrophy. This syndrome is caused by mutations in the CNNM4 gene. Different types of CNNM4 mutations have been reported; missense, nonsense, large deletions, single base insertion, and duplication.We used Sanger sequencing to analyze a large consanguineous family with three siblings affected with Jalili syndrome, suspected clinically after dental and ophthalmological examination. These patients are carrying a novel homozygous mutation in the splice site acceptor of intron 3 (c.1682-1G > C) in the CNNM4 gene.We compare the findings of the present family to those from literature, in order to further delineate Jalili syndrome.     

TRAPPC9-related neurodevelopmental disorder: Report of a homozygous deletion in TRAPPC9 due to paternal uniparental isodisomy

TRAPPC9 loss-of-function biallelic variants are associated with an autosomal recessive intellectual disability syndrome (Online Mendelian Inheritance of Man no. 613192), also characterized by microcephaly, hypertelorism, obesity, growth delay, and behavioral differences. Here, we describe an 8-year-old Hispanic female with neurodevelopmental disorder, partial epilepsy, microcephaly, bilateral cleft lip and alveolus, growth delay, and dysmorphic features. She had abnormal myelination, mega cisterna magna, and colpocephaly on brain magnetic resonance imaging (MRI). Microarray showed a single ~146 Mb region of homozygosity (ROH) encompassing all of Chromosome 8, consistent with uniparental isodisomy (UPD). Exome sequencing performed in-house did not identify single nucleotide variants to explain her phenotype. Algorithms developed in-house and further evaluation of BAM files revealed a homozygous deletion overlapping Exon 2 in TRAPPC9 within the ROH. Subsequent del/dup analyses with exon-level oligo array confirmed a likely pathogenic deletion in TRAPPC9 (NM_031466.5): arr[GRCh37] 8q24.3(141460661_141461780)x0. Our case highlights the implications of downstream analyses from UPD/ROH given the increased risk for AR conditions, the strengths of combining orthologous molecular methods to establish a diagnosis and further delineates the TRAPPC9-related phenotype in an individual of Hispanic ancestry.     

Spectrum of splicing errors caused by CHRNE mutations affecting introns and intron/exon boundaries

Background: Mutations in CHRNE, the gene encoding the muscle nicotinic acetylcholine receptor ε subunit, cause congenital myasthenic syndromes. Only three of the eight intronic splice site mutations of CHRNE reported to date have had their splicing consequences characterised. Methods: We analysed four previously reported and five novel splicing mutations in CHRNE by introducing the entire normal and mutant genomic CHRNEs into COS cells. Results and conclusions: We found that short introns (82–109 nucleotides) favour intron retention, whereas medium to long introns (306–1210 nucleotides) flanking either or both sides of an exon favour exon skipping. Two mutations are of particular interest. Firstly, a G→T substitution at the 3'' end of exon 8 predicts an R286M missense mutation, but instead results in skipping of exon 8. In human genes, a mismatch of the last exonic nucleotide to U1 snRNP is frequently compensated by a matching nucleotide at intron position +6. CHRNE intron 8 has a mismatch at position +6, and accordingly fails to compensate for the exonic mutation at position –1. Secondly, a 16 bp duplication, giving rise to two 3'' splice sites (g.IVS10-9_c.1167dup16), results in silencing of the downstream 3'' splice site. This conforms to the scanning model of recognition of the 3'' splice site, which predicts that the first "ag" occurring after the branch point is selected for splicing.     

Recurrent RTTN mutation leading to severe microcephaly,polymicrogyria and growth restriction

Autosomal recessive missense Rotatin (RTTN) mutations are responsible for syndromic forms of malformation of cortical development, ranging from isolated polymicrogyria to microcephaly associated with primordial dwarfism and other major malformations.We identified, by trio based whole exome sequencing, a homozygous missense mutation in the RTTN gene (c.2953A > G; p.(Arg985Gly)) in one Moroccan patient from a consanguineous family. The patient showed early onset primary microcephaly, detected in the fetal period, postnatal growth restriction, encephalopathy with hyperkinetic movement disorders and self-injurious behavior with sleep disturbance. Brain MRI showed an extensive dysgyria associated with nodular heterotopia, large interhemispheric arachnoid cyst and corpus callosum hypoplasia.