Goldberg-Shprintzen syndrome is determined by the absence, or reduced expression levels, of KIFBP.

Goldberg-Shprintzen syndrome (GOSHS) is caused by loss of function variants in the kinesin binding protein gene (KIFBP). However, the phenotypic range of this syndrome is wide, indicating that other factors may play a role. To date, 37 patients with GOSHS have been reported. Here, we document nine new patients with variants in KIFBP: seven with nonsense variants and two with missense variants. To our knowledge, this is the first time that missense variants have been reported in GOSHS. We functionally investigated the effect of the variants identified, in an attempt to find a genotype-phenotype correlation. We also determined whether common Hirschsprung disease (HSCR)-associated single nucleotide polymorphisms (SNPs), could explain the presence of HSCR in GOSHS. Our results showed that the missense variants led to reduced expression of KIFBP, while the truncating variants resulted in lack of protein. However, no correlation was found between the severity of GOSHS and the location of the variants. We were also unable to find a correlation between common HSCR-associated SNPs, and HSCR development in GOSHS. In conclusion, we show that reduced, as well as lack of KIFBP expression can lead to GOSHS, and our results suggest that a threshold expression of KIFBP may modulate phenotypic variability of the disease.

Development and Function of Immune Cells in an Adolescent Patient With a Deficiency in the Interleukin-10 Receptor.

OBJECTIVE: Monogenic defects in the interleukin-10 (IL-10) pathway are extremely rare and cause infantile-onset inflammatory bowel disease (IBD)-like pathology. Understanding how immune responses are dysregulated in monogenic IBD-like diseases can provide valuable insight in "classical" IBD pathogenesis. Here, we studied long-term immune cell development and function in an adolescent IL-10 receptor (IL10RA)-deficient patient who presented in infancy with severe colitis and fistulizing perianal disease and is currently treated with immune suppressants. METHODS: Biomaterial was collected from the IL10RA-deficient patient, pediatric patients with IBD, and healthy controls. The frequency and phenotype of immune cells were determined in peripheral blood and intestinal biopsies by flow cytometry and immunohistochemistry. Functional changes in monocyte-derived dendritic cells and T cells were assessed by in vitro activation assays. RESULTS: The IL10RA-deficient immune system developed normally with respect to numbers and phenotype of circulating immune cells. Despite normal co-stimulatory molecule expression, bacterial lipopolysaccharide-stimulated monocyte-derived dendritic cells from the IL10RA-deficient patient released increased amounts of tumor necrosis factor α compared to healthy controls. Upon T-cell receptor ligation, IL10RA-deficient peripheral blood mononuclear cells released increased amounts of T-cell cytokines interferon γ and IL-17 agreeing with high numbers of T-bet and IL-17 cells in intestinal biopsies taken at disease onset. In vitro, the immunosuppressive drug thalidomide used to treat the patient's decreased peripheral blood mononuclear cell-derived tumor necrosis factor production. CONCLUSIONS: With time and during immunosuppressive treatment the IL10RA-deficient immune system develops relatively normally. Upon activation, IL-10 is crucial for controlling excessive inflammatory cytokine release by dendritic cells and preventing interferon γ and IL-17-mediated T-cell responses.

Identification and characterization of aberrant GAA pre-mRNA splicing in pompe disease using a generic approach.

Identification of pathogenic variants in monogenic diseases is an important aspect of diagnosis, genetic counseling, and prediction of disease severity. Pathogenic mechanisms involved include changes in gene expression, RNA processing, and protein translation. Variants affecting pre-mRNA splicing are difficult to predict due to the complex mechanism of splicing regulation. A generic approach to systematically detect and characterize effects of sequence variants on splicing would improve current diagnostic practice. Here, it is shown that such approach is feasible by combining flanking exon RT-PCR, sequence analysis of PCR products, and exon-internal quantitative RT-PCR for all coding exons. Application of this approach to one novel and six previously published variants in the acid-alpha glucosidase (GAA) gene causing Pompe disease enabled detection of a total of 11 novel splicing events. Aberrant splicing included cryptic splice-site usage, intron retention, and exon skipping. Importantly, the extent of leaky wild-type splicing correlated with disease onset and severity. These results indicate that this approach enables sensitive detection and in-depth characterization of variants affecting splicing, many of which are still unrecognized or poorly understood. The approach is generic and should be adaptable for application to other monogenic diseases to aid in improved diagnostics.

The expanding phenotype of COL4A1 and COL4A2 mutations: clinical data on 13 newly identified families and a review of the literature.

Two proα1(IV) chains, encoded by COL4A1, form trimers that contain, in addition, a proα2(IV) chain encoded by COL4A2 and are the major component of the basement membrane in many tissues. Since 2005, COL4A1 mutations have been known as an autosomal dominant cause of hereditary porencephaly. COL4A1 and COL4A2 mutations have been reported with a broader spectrum of cerebrovascular, renal, ophthalmological, cardiac, and muscular abnormalities, indicated as "COL4A1 mutation-related disorders." Genetic counseling is challenging because of broad phenotypic variation and reduced penetrance. At the Erasmus University Medical Center, diagnostic DNA analysis of both COL4A1 and COL4A2 in 183 index patients was performed between 2005 and 2013. In total, 21 COL4A1 and 3 COL4A2 mutations were identified, mostly in children with porencephaly or other patterns of parenchymal hemorrhage, with a high de novo mutation rate of 40% (10/24). The observations in 13 novel families harboring either COL4A1 or COL4A2 mutations prompted us to review the clinical spectrum. We observed recognizable phenotypic patterns and propose a screening protocol at diagnosis. Our data underscore the importance of COL4A1 and COL4A2 mutations in cerebrovascular disease, also in sporadic patients. Follow-up data on symptomatic and asymptomatic mutation carriers are needed for prognosis and appropriate surveillance.

Targeted Next Generation Sequencing reveals previously unidentified TSC1 and TSC2 mutations.

BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in TSC1 and TSC2. Conventional DNA diagnostic screens identify a TSC1 or TSC2 mutation in 75 - 90% of individuals categorised with definite TSC. The remaining individuals either have a mutation that is undetectable using conventional methods, or possibly a mutation in another as yet unidentified gene. METHODS: Here we apply a targeted Next Generation Sequencing (NGS) approach to screen the complete TSC1 and TSC2 genomic loci in 7 individuals fulfilling the clinical diagnostic criteria for definite TSC in whom no TSC1 or TSC2 mutations were identified using conventional screening methods. RESULTS: We identified and confirmed pathogenic mutations in 3 individuals. In the remaining individuals we identified variants of uncertain clinical significance. The identified variants included mosaic changes, changes located deep in intronic sequences and changes affecting promoter regions that would not have been identified using exon-only based analyses. CONCLUSIONS: Targeted NGS of the TSC1 and TSC2 loci is a suitable method to increase the yield of mutations identified in the TSC patient population.

Screening of TGFBR1, TGFBR2, and FLNA in familial mitral valve prolapse.

So far only mutations in the filamin A gene (FLNA) have been identified as causing familial mitral valve prolapse (MVP). Previous studies have linked dysregulation of the transforming growth factor beta (TGF-ß) cytokine family to MVP. We investigated whether mutations in the TGF-ß receptors genes type I (TGFBR1) and II (TGFBR2) underlie isolated familial MVP cases. Eight families with isolated familial MVP were evaluated clinically and genetically. Ventricular arrhythmias were present in five of the eight families and sudden cardiac death occurred in six patients. Tissue obtained during mitral valve surgery or autopsy was available for histological examination in six cases; all demonstrated myxomatous degeneration. A previously described FLNA missense mutation (p.G288R) was identified in one large family, but no mutations were discovered in TGFBR1 or TGFBR2. An FLNA missense mutation was identified in one family but we found no TGFBR1 or TGFBR2 mutations. Our results suggest that TGFBR1 and TGFBR2 mutations do not play a major role in isolated myxomatous valve dystrophy. Screening for FLNA mutations is recommended in familial myxomatous valvular dystrophy, particularly if X-linked inheritance is suspected.

Functional assessment of TSC2 variants identified in individuals with tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in the TSC1 or TSC2 genes. The TSC1 and TSC2 gene products, TSC1 and TSC2, form a complex that inhibits the mammalian target of rapamycin (mTOR) complex 1 (TORC1). Here, we investigate the effects of 78 TSC2 variants identified in individuals suspected of TSC, on the function of the TSC1-TSC2 complex. According to our functional assessment, 40 variants disrupted the TSC1-TSC2-dependent inhibition of TORC1. We classified 34 of these as pathogenic, three as probably pathogenic and three as possibly pathogenic. In one case, a likely effect on splicing as well as an effect on function was noted. In 15 cases, our functional assessment did not agree with the predictions of the SIFT amino acid substitution analysis software. Our data support the notion that different, nonterminating TSC2 mutations can have distinct effects on TSC1-TSC2 function, and therefore, on TSC pathology.

Novel no-stop FLNA mutation causes multi-organ involvement in males.

Mutations in FLNA (Filamin A, OMIM 300017) cause X-linked periventricular nodular heterotopia (XL-PNH). XL-PNH-associated mutations are considered lethal in hemizygous males. However, a few males with unusual mutations (including distal truncating and hypomorphic missense mutations), and somatic mosaicism have been reported to survive past infancy. Two brothers had an atypical presentation with failure to thrive and distinct facial appearance including hypertelorism. Evaluations of these brothers and their affected cousin showed systemic involvement including severe intestinal malfunction, malrotation, congenital short bowel, PNH, pyloric stenosis, wandering spleen, patent ductus arteriosus, atrial septal defect, inguinal hernia, and vesicoureteral reflux. The unanticipated finding of PNH led to FLNA testing and subsequent identification of a novel no-stop FLNA mutation (c.7941_7942delCT, p.(*2648Serext*100)). Western blotting and qRT-PCR of patients' fibroblasts showed diminished levels of protein and mRNA. This FLNA mutation, the most distal reported so far, causes in females classical XL-PNH, but in males an unusual, multi-organ phenotype, providing a unique insight into the FLNA-associated phenotypes.

Update of the pompe disease mutation database with 60 novel GAA sequence variants and additional studies on the functional effect of 34 previously reported variants.

Pompe disease is an autosomal recessive lysosomal glycogen storage disorder, characterized by progressive muscle weakness. Deficiency of acid α-glucosidase (EC; 3.2.1.20/3) can be caused by numerous pathogenic variants in the GAA gene. The Pompe Disease Mutation Database at http://www.pompecenter.nl aims to list all variants and their effect. This update reports on 94 variants. We examined 35 novel and 34 known mutations by site-directed mutagenesis and transient expression in COS-7 cells or HEK293T cells. Each of these mutations was given a severity rating using a previously published system, based on the level of acid α-glucosidase activity in medium and transfected cells and on the quantity and quality of the different molecular mass species in the posttranslational modification and transport of acid α-glucosidase. This approach enabled to classify 55 missense mutations as pathogenic and 13 as likely nonpathogenic. Based on their nature and the use of in silico analysis (Alamut® software), 12 of the additional 25 novel mutations were predicted to be pathogenic including 4 splicing mutations, 6 mutations leading to frameshift, and 2 point mutations causing stop codons. Seven of the additional mutations were considered nonpathogenic (4 silent and 3 occurring in intron regions), and 6 are still under investigation.

Functional assessment of TSC1 missense variants identified in individuals with tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in the TSC1 or TSC2 genes. The TSC1 and TSC2 gene products, TSC1 and TSC2, form a complex that inhibits the mammalian target of rapamycin (mTOR) complex 1 (TORC1). Previously, we demonstrated that pathogenic amino acid substitutions in the N-terminal domain of TSC1 (amino acids 50-224) are destabilizing. Here we investigate an additional 21 unclassified TSC1 variants. Our functional assessment identified four substitutions (p.L61R, p.G132D, p.F158S, and p.R204P) between amino acids 50 and 224 that reduced TSC1 stability and prevented the TSC1-TSC2-dependent inhibition of TORC1. In four cases (20%), our functional assessment did not agree with the predictions of the SIFT amino acid substitution analysis software. Our new data confirm our previous finding that the N-terminal region of TSC1 is essential for TSC1 function.

The TSC1-TSC2 complex consists of multiple TSC1 and TSC2 subunits.

BACKGROUND: Mutations to the TSC1 and TSC2 genes cause the disease tuberous sclerosis complex. The TSC1 and TSC2 gene products form a protein complex that integrates multiple metabolic signals to regulate the activity of the target of rapamycin (TOR) complex 1 (TORC1) and thereby control cell growth. Here we investigate the quaternary structure of the TSC1-TSC2 complex by gel filtration and coimmunoprecipitation. RESULTS: TSC1 and TSC2 co-eluted in high molecular weight fractions by gel filtration. Coimmunoprecipitation of distinct tagged TSC1 and TSC2 isoforms demonstrated that TSC1-TSC2 complexes contain multiple TSC1 and TSC2 subunits. CONCLUSIONS: TSC1 and TSC2 interact to form large complexes containing multiple TSC1 and TSC2 subunits.

Asymmetric polymicrogyria and periventricular nodular heterotopia due to mutation in ARX.

Mutations in the ARX gene, at Xp22.3, cause several disorders, including infantile spasms, X-linked lissencephaly with abnormal genitalia (XLAG), callosal agenesis and isolated intellectual disability. Genotype/phenotype studies suggested that polyalanine tract expansion is associated with non-malformative phenotypes, while missense and nonsense mutations cause cerebral malformations, however, patients with structural normal brain and missense mutations have been reported. We report on a male patient born with cleft lip and palate who presented with infantile spasms and hemiplegia. MRI showed agenesis of corpus callosum (ACC), an interhemispheric cyst, periventricular nodular heterotopia (PVNH), and extensive left frontal polymicrogyria (PMG). Sequencing of the ARX gene in the patient identified a six basepair insertion (c.335ins6, exon 2). The insertion leads to a two-residue expansion of the first polyalanine tract and was described previously in a family with non-syndromic X-linked mental retardation. To our knowledge, ARX mutation causing PMG and PVNH is unique, but the spasms and ACC are common in ARX mutations. Clinicians should be aware of the broad clinical range of ARX mutations, and further studies are necessary to investigate the association with PMG and PVNH and to identify possible modifying factors.

Functional assessment of variants in the TSC1 and TSC2 genes identified in individuals with Tuberous Sclerosis Complex.

The effects of missense changes and small in-frame deletions and insertions on protein function are not easy to predict, and the identification of such variants in individuals at risk of a genetic disease can complicate genetic counselling. One option is to perform functional tests to assess whether the variants affect protein function. We have used this strategy to characterize variants identified in the TSC1 and TSC2 genes in individuals with, or suspected of having, Tuberous Sclerosis Complex (TSC). Here we present an overview of our functional studies on 45 TSC1 and 107 TSC2 variants. Using a standardized protocol we classified 16 TSC1 variants and 70 TSC2 variants as pathogenic. In addition we identified eight putative splice site mutations (five TSC1 and three TSC2). The remaining 24 TSC1 and 34 TSC2 variants were classified as probably neutral.

Analysis of TSC1 truncations defines regions involved in TSC1 stability, aggregation and interaction.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterised by the development of hamartomas in a variety of organs and tissues. The disease is caused by mutations in either the TSC1 gene on chromosome 9q34, or the TSC2 gene on chromosome 16p13.3. The TSC1 and TSC2 gene products, TSC1 and TSC2, interact to form a protein complex that inhibits signal transduction to the downstream effectors of the target of rapamycin complex 1 (TORC1). Here we investigate TSC1 structure and function by analysing a series of truncated TSC1 proteins. We identify specific regions of the protein that are important for TSC1 stability, localisation, interactions and function.

Mucopolysaccharidosis type IIIA: clinical spectrum and genotype-phenotype correlations.

OBJECTIVE: Mucopolysaccharidosis (MPS) IIIA (Sanfilippo syndrome type A) is a lysosomal storage disorder caused by deficiency of the enzyme sulfamidase. Information on the natural course of MPS IIIA is scarce, but is much needed in view of emerging therapies. METHODS: Clinical history and molecular defects of all 110 MPS IIIA patients identified by enzymatic studies in the Netherlands were collected and included in this study. RESULTS: First clinical signs, mainly consisting of delayed speech development and behavioral problems, were noted between the ages of 1 and 6 years. Other symptoms included sleeping and hearing problems, recurrent upper airway infections, diarrhea, and epilepsy. The clinical course varied remarkably and could be correlated with the molecular defects. The frequent pathogenic mutations p.R245H, p.Q380R, p.S66W, and c.1080delC were associated with the classical severe phenotype. Patients compound heterozygous for the p.S298P mutation in combination with 1 of the mutations associated with the classical severe phenotype had a significantly longer preservation of psychomotor functions and a longer survival. Two patients homozygous for the p.S298P mutation, and 4 patients from 3 families heterozygous for 3 missense variants not reported previously (p.T421R, p.P180L, and p.L12Q), showed a remarkably attenuated phenotype. INTERPRETATION: We report the natural history and mutational analysis in a large unbiased cohort of MPS IIIA patients. We demonstrate that the clinical spectrum of MPS IIIA is much broader than previously reported. A significant genotype-phenotype correlation was established in this cohort.

Long-term follow-up of type 1 lissencephaly: survival is related to neuroimaging abnormalities.

AIM: To evaluate survival, clinical, and genetic characteristics of all patients with classic or type 1 lissencephaly born between 1972 and 1990 in the Netherlands, who at the time were enrolled in an observational study. METHOD: We re-evaluated 24 patients (11 males, 13 females) for long-term follow-up and survival information. RESULTS: Mean length of follow-up was 14 years (SD 9 y 8 mo). Eleven patients were alive at follow-up. All patients showed severe intellectual disability, intractable epilepsy, and complete dependency on care. Life expectancy was related to the severity of the lissencephaly on neuroimaging. Molecular analysis of the LIS1 gene was not possible at the time of the original study and was now requested by eight parents. This revealed a pathogenic nonsense mutation or deletion in seven patients. INTERPRETATION: Our study provides information about the long-term course of lissencephaly and the relationship between lissencephaly severity and prognosis. It also shows that renewed attention to genetic counselling remains valued by families of patients with a severe congenital neurological disease.

Crigler-Najjar syndrome in The Netherlands: identification of four novel UGT1A1 alleles, genotype-phenotype correlation, and functional analysis of 10 missense mutants.

Crigler-Najjar syndrome (CN), caused by deficiency of UGT isoform 1A1 (UGT1A1), is characterized by severe unconjugated hyperbilirubinemia. In this study we have analyzed 19 CN patients diagnosed in The Netherlands (18) and in Belgium (1), and have identified 14 different UGT1A1 mutations, four of which are novel. Two mutations were present in several unrelated patients, suggesting the presence of two founder effects in The Netherlands. In addition, we show linkage of the UGT1A1 *28 promoter polymorphism (rs5719145insTA) to three structural mutations. Functional studies of partial active UGT1A1 mutants are limited. Therefore, we performed in vitro studies to determine the functional activity of seven missense mutants identified in this study and of three reported previously. In addition to bilirubin, we also determined their activity toward eight other UGT1A1 substrates. We demonstrate that five mutants have residual activity that, depending on the substrate, varies from not detectable to 94% of wild-type UGT1A1 activity. The identification of four novel pathogenic mutations and the analysis of residual activity of 10 UGT1A1 missense mutants are useful for clinical diagnosis, and provides new insights in enzyme activity, whereas the identification of two founder mutations will speed up genetic counseling for newly identified CN patients in The Netherlands.

Mucopolysaccharidosis type IIID: 12 new patients and 15 novel mutations.

Mucopolysaccharidosis III D (Sanfilippo disease type D, MPS IIID) is a rare autosomal recessive lysosomal storage disorder previously described in only 20 patients. MPS IIID is caused by a deficiency of N-acetylglucosamine-6-sulphate sulphatase (GNS), one of the enzymes required for the degradation of heparan sulphate. So far only seven mutations in the GNS gene have been reported. The clinical phenotype of 12 new MPS IIID patients from 10 families was studied. Mutation analysis of GNS was performed in 16 patients (14 index cases). Clinical signs and symptoms of the MPS IIID patients appeared to be similar to previously described patients with MPS III. Early development was normal with onset of behavioral problems around the age of 4 years, followed by developmental stagnation, deterioration of verbal communication and subsequent deterioration of motor functions. Sequence analysis of the coding regions of the gene encoding GNS (GNS) resulted in the identification of 15 novel mutations: 3 missense mutations, 1 nonsense mutation, 4 splice site mutations, 3 frame shift mutations, 3 large deletions and 1 in-frame small deletion. They include the first missense mutations and a relatively high proportion of large rearrangements, which warrants the inclusion of quantitative techniques in routine mutation screening of the GNS gene.

Mucopolysaccharidosis type IIIB may predominantly present with an attenuated clinical phenotype.

Mucopolysaccharidosis type IIIB (MPS IIIB, Sanfilippo syndrome type B) is a lysosomal storage disorder caused by deficiency of the enzyme N-acetyl-α-D-glucosaminidase (NAGLU). Information on the natural course of MPS IIIB is scarce but much needed in view of emerging therapies. To improve knowledge on the natural course, data on all 52 MPS IIIB patients ever identified by enzymatic studies in the Netherlands were gathered. Clinical data on 44 patients could be retrieved. Only a small number (n = 9; 21%) presented with a classical MPS III phenotype; all other patients showed a much more attenuated course of the disease characterized by a significantly slower regression of intellectual and motor abilities. The majority of patients lived well into adulthood. First signs of the disease, usually mild developmental delay, were observed at a median age of 4 years. Subsequently, patients showed a slowing and eventually a stagnation of development. Patients with the attenuated phenotype had a stable intellectual disability for many years. Molecular analysis was performed in 24 index patients. The missense changes p.R643C, p.S612G, p.E634K, and p.L497V were exclusively found in patients with the attenuated phenotype. MPS IIIB comprises a remarkably wide spectrum of disease severity, and an unselected cohort including all Dutch patients showed a large proportion (79%) with an attenuated phenotype. MPS IIIB must be considered in patients with a developmental delay, even in the absence of a progressive decline in intellectual abilities. A key feature, necessitating metabolic studies, is the coexistence of behavioral problems.