Carlo Marcelis

Identity-by-descent filtering of exome sequence data identifies PIGV mutations in hyperphosphatasia mental retardation syndrome

Hyperphosphatasia mental retardation (HPMR) syndrome is an autosomal recessive form of mental retardation with distinct facial features and elevated serum alkaline phosphatase. We performed whole-exome sequencing in three siblings of a nonconsanguineous union with HPMR and performed computational inference of regions identical by descent in all siblings to establish PIGV, encoding a member of the GPI-anchor biosynthesis pathway, as the gene mutated in HPMR. We identified homozygous or compound heterozygous mutations in PIGV in three additional families.

A Post-Hoc Comparison of the Utility of Sanger Sequencing and Exome Sequencing for the Diagnosis of Heterogeneous Diseases

The advent of massive parallel sequencing is rapidly changing the strategies employed for the genetic diagnosis and research of rare diseases that involve a large number of genes. So far it is not clear whether these approaches perform significantly better than conventional single gene testing as requested by clinicians. The current yield of this traditional diagnostic approach depends on a complex of factors that include gene‐specific phenotype traits, and the relative frequency of the involvement of specific genes. To gauge the impact of the paradigm shift that is occurring in molecular diagnostics, we assessed traditional Sanger‐based sequencing (in 2011) and exome sequencing followed by targeted bioinformatics analysis (in 2012) for five different conditions that are highly heterogeneous, and for which our center provides molecular diagnosis. We find that exome sequencing has a much higher diagnostic yield than Sanger sequencing for deafness, blindness, mitochondrial disease, and movement disorders. For microsatellite‐stable colorectal cancer, this was low under both strategies. Even if all genes that could have been ordered by physicians had been tested, the larger number of genes captured by the exome would still have led to a clearly superior diagnostic yield at a fraction of the cost.

A SWI/SNF-related autism syndrome caused by de novo mutations in ADNP

Despite the high heritability of autism spectrum disorders (ASD), characterized by persistent deficits in social communication and interaction and restricted, repetitive patterns of behavior, interests or activities, a genetic diagnosis can be established in only a minority of patients. Known genetic causes include chromosomal aberrations, such as the duplication of the 15q11-13 region, and monogenic causes, as in Rett and fragile-X syndromes. The genetic heterogeneity within ASD is striking, with even the most frequent causes responsible for only 1% of cases at the most. Even with the recent developments in next-generation sequencing, for the large majority of cases no molecular diagnosis can be established. Here, we report ten patients with ASD and other shared clinical characteristics, including intellectual disability and facial dysmorphisms caused by a mutation in ADNP, a transcription factor involved in the SWI/SNF remodeling complex. We estimate this gene to be mutated in at least 0.17% of ASD cases, making it one of the most frequent ASD-associated genes known to date.

Repetitive disruptions of the nuclear envelope invoke temporary loss of cellular compartmentalization in laminopathies.

The nuclear lamina provides structural support to the nucleus and has a central role in nuclear organization and gene regulation. Defects in its constituents, the lamins, lead to a class of genetic diseases collectively referred to as laminopathies. Using live cell imaging, we observed the occurrence of intermittent, non-lethal ruptures of the nuclear envelope in dermal fibroblast cultures of patients with different mutations of lamin A/C. These ruptures, which were absent in normal fibroblasts, could be mimicked by selective knockdown as well as knockout of LMNA and were accompanied by the loss of cellular compartmentalization. This was demonstrated by the influx of cytoplasmic transcription factor RelA and regulatory protein Cyclin B1 into the nucleus, and efflux of nuclear transcription factor OCT1 and nuclear structures containing the promyelocytic leukemia (PML) tumour suppressor protein to the cytoplasm. While recovery of enhanced yellow fluorescent protein-tagged nuclear localization signal in the nucleus demonstrated restoration of nuclear membrane integrity, part of the mobile PML structures became permanently translocated to the cytoplasm. These satellite PML structures were devoid of the typical PML body components, such as DAXX, SP100 or SUMO1. Our data suggest that nuclear rupture and loss of compartmentalization may add to cellular dysfunction and disease development in various laminopathies.

PLS3 Mutations in X-Linked Osteoporosis with Fractures

Plastin 3 (PLS3), a protein involved in the formation of filamentous actin (F-actin) bundles, appears to be important in human bone health, on the basis of pathogenic variants in PLS3 in five families with X-linked osteoporosis and osteoporotic fractures that we report here. The bone-regulatory properties of PLS3 were supported by in vivo analyses in zebrafish. Furthermore, in an additional five families (described in less detail) referred for diagnosis or ruling out of osteogenesis imperfecta type I, a rare variant (rs140121121) in PLS3 was found. This variant was also associated with a risk of fracture among elderly heterozygous women that was two times as high as that among noncarriers, which indicates that genetic variation in PLS3 is a novel etiologic factor involved in common, multi-factorial osteoporosis.

Clinical and molecular characteristics of 1qter microdeletion syndrome: delineating a critical region for corpus callosum agenesis/hypogenesis

Background: Patients with a microscopically visible deletion of the distal part of the long arm of chromosome 1 have a recognisable phenotype, including mental retardation, microcephaly, growth retardation, a distinct facial appearance and various midline defects including corpus callosum abnormalities, cardiac, gastro-oesophageal and urogenital defects, as well as various central nervous system anomalies. Patients with a submicroscopic, subtelomeric 1qter deletion have a similar phenotype, suggesting that the main phenotype of these patients is caused by haploinsufficiency of genes in this region. Objective: To describe the clinical presentation of 13 new patients with a submicroscopic deletion of 1q43q44, of which nine were interstitial, and to report on the molecular characterisation of the deletion size. Results and conclusions: The clinical presentation of these patients has clear similarities with previously reported cases with a terminal 1q deletion. Corpus callosum abnormalities were present in 10 of our patients. The AKT3 gene has been reported as an important candidate gene causing this abnormality. However, through detailed molecular analysis of the deletion sizes in our patient cohort, we were able to delineate the critical region for corpus callosum abnormalities to a 360 kb genomic segment which contains four possible candidate genes, but excluding the AKT3 gene.

Whole-exome resequencing reveals recessive mutations in TRAP1 in individuals with CAKUT and VACTERL association

Congenital abnormalities of the kidney and urinary tract (CAKUT) account for approximately half of children with chronic kidney disease and they are the most frequent cause of end-stage renal disease in children in the US. However, its genetic etiology remains mostly elusive. VACTERL association is a rare disorder that involves congenital abnormalities in multiple organs including the kidney and urinary tract in up to 60% of the cases. By homozygosity mapping and whole exome resequencing combined with high-throughput mutation analysis by array-based multiplex PCR and next-generation sequencing, we identified recessive mutations in the gene TNF receptor-associated protein 1 (TRAP1) in two families with isolated CAKUT and three families with VACTERL association. TRAP1 is a heat shock protein 90-related mitochondrial chaperone possibly involved in antiapoptotic and endoplasmic reticulum-stress signaling. Trap1 is expressed in renal epithelia of developing mouse kidney E13.5 and in the kidney of adult rats, most prominently in proximal tubules and in thick medullary ascending limbs of Henle’s loop. Thus, we identified mutations in TRAP1 as highly likely causing CAKUT or CAKUT in VACTERL association.

Intragenic deletion in DYRK1A leads to mental retardation and primary microcephaly

To the Editor : For years, human genotype–phenotype studies have suggested DYRK1A (MIM 600855) as a candidate gene for microcephaly and mental retardation (MR) (1–3). These findings have been supported by various animal models such as Drosophila and mice (4, 5). DYRK1A is a member of the dual-specificity tyrosine phosphorylationregulated kinase (DYRK) family and participates in various cellular processes. It is a highly conserved gene located in the Down Syndrome Critical Region, a part on chromosome 21 that is responsible for the majority of phenotypic features in Down syndrome (6, 7). Convincing evidence for a brain developmental function of DYRK1A in humans was recently provided by a report on two mentally retarded individuals with a balanced translocation truncating this gene (8). However, so far, no pure DYRK1A deletions in humans have been reported. In this study, we investigated whether smaller copy number variations (CNVs), below the routine diagnostic threshold set at 150 kb, may be present at the DYRK1A locus. Therefore, the intensity log2 ratios of all single nucleotide polymorphism (SNP) probes located within and surrounding the DYRK1A gene (37,647,772–37,813,457 bp) were studied in 3009 mentally retarded individuals, in whom no pathogenic CNVs (>150 kb) were detected during routine diagnostic 250K SNP array analysis (Affymetrix Inc., Santa Clara, CA) (9). Patients in which at least four consecutive SNP probes showed an abnormal log2 ratio (<−0.2 or >0.25) were considered to be candidates for having a DYRK1A deletion or duplication. Of the 16 candidate aberrations fulfilling this criterion one deletion could be confirmed by quantitative polymerase chain reaction (qPCR, Supporting information, Table S1) and was shown to be de novo (Fig. 1b). A high-resolution NimbleGen hg18 chromosome 21 specific 385K array subsequently revealed that the deletion had a size of 52 kb (37,796,500–37,849,000 bp) and deleted the last three exons of DYRK1A (Fig. 1c). The female with the DYRK1A deletion was born with a length of 48 cm (−2 SD), weight of 3195 g (−1 SD) and a head circumference of 33 cm (<−2 SD). From birth onward, she had feeding problems with failure to thrive and non-fluent motoric movements and hypoactivity were noted. During infancy she regularly had febrile seizures. At 24 years of age, a cerebral magnetic resonance imaging (MRI) showed a mild atrophic brain and medulla without structural congenital anomalies. At 33 years of age, she had severe MR without speech and showed anxious autistic behavior. She walked with a stiff motoric gait and jerky movements. She had a slender posture with a length of 161.5 cm (−1.5 SD) and an occipitofrontal circumference (OFC) of 50.5 cm (−3 SD). Facial dysmorphisms included bitemporal narrowing, deep-set eyes, large simple ears and a pointed nasal tip. In addition, she had eczema, hypoplasia of breasts, hallux valgus of feet and an irregular implant of toes. Several features in this patient, such as severe developmental delay, growth retardation, nonfluent motoric movements, microcephaly in the absence of structural brain anomalies, hypoactivity and anxious behavior, show a striking similarity with features observed in different animal models with mutated DYRK1A. The Drosophila ortholog of DYRK1A is the minibrain (mnb) gene (5). The mnb gene plays an important role in regulating the number of distinct neuronal cell types. These mutants are slightly smaller in size and show behavior problems, characterized by poor visual pattern fixation, reduced walking activity and speed, and poor odor discrimination (5). Dyrk1A+/− mice show preand postnatal growth retardation, developmental delay, specific motor and learning deficits, visual impairment, behavioral defects and increased anxiety (4, 10–13). Brains of mutant mnb flies and Dyrk1A+/− mice are greatly reduced in size, although the neuronal architecture remains unchanged. On the basis of human genotype–phenotype studies of partial monosomies 21, DYRK1A has

Stickler syndrome caused by COL2A1 mutations: Genotype-phenotype correlation in a series of 100 patients

Stickler syndrome is an autosomal dominant connective tissue disorder caused by mutations in different collagen genes. The aim of our study was to define more precisely the phenotype and genotype of Stickler syndrome type 1 by investigating a large series of patients with a heterozygous mutation in COL2A1. In 188 probands with the clinical diagnosis of Stickler syndrome, the COL2A1 gene was analyzed by either a mutation scanning technique or bidirectional fluorescent DNA sequencing. The effect of splice site alterations was investigated by analyzing mRNA. Multiplex ligation-dependent amplification analysis was used for the detection of intragenic deletions. We identified 77 different COL2A1 mutations in 100 affected individuals. Analysis of the splice site mutations showed unusual RNA isoforms, most of which contained a premature stop codon. Vitreous anomalies and retinal detachments were found more frequently in patients with a COL2A1 mutation compared with the mutation-negative group (P<0.01). Overall, 20 of 23 sporadic patients with a COL2A1 mutation had either a cleft palate or retinal detachment with vitreous anomalies. The presence of vitreous anomalies, retinal tears or detachments, cleft palate and a positive family history were shown to be good indicators for a COL2A1 defect. In conclusion, we confirm that Stickler syndrome type 1 is predominantly caused by loss-of-function mutations in the COL2A1 gene as >90% of the mutations were predicted to result in nonsense-mediated decay. On the basis of binary regression analysis, we developed a scoring system that may be useful when evaluating patients with Stickler syndrome.

PORCN mutations in focal dermal hypoplasia: coping with lethality.

The X‐linked dominant trait focal dermal hypoplasia (FDH, Goltz syndrome) is a developmental defect with focal distribution of affected tissues due to a block of Wnt signal transmission from cells carrying a detrimental PORCN mutation on an active X‐chromosome. Molecular characterization of 24 unrelated patients from different ethnic backgrounds revealed 23 different mutations of the PORCN gene in Xp11.23. Three were microdeletions eliminating PORCN and encompassing neighboring genes such as EBP, the gene associated with Conradi‐Hünermann‐Happle syndrome (CDPX2). 12/24 patients carried nonsense mutations resulting in loss of function. In one case a canonical splice acceptor site was mutated, and 8 missense mutations exchanged highly conserved amino acids. FDH patients overcome the consequences of potentially lethal X‐chromosomal mutations by extreme skewing of X‐chromosome inactivation in females, enabling transmission of the trait in families, or by postzygotic mosaicism both in male and female individuals. Molecular characterization of the PORCN mutations in cases diagnosed as Goltz syndrome is particularly relevant for genetic counseling of patients and their families since no functional diagnostic test is available and carriers of the mutation might otherwise be overlooked due to considerable phenotypic variability associated with the mosaic status.