PHIP-associated Chung-Jansen syndrome: Report of 23 new individuals.

In 2016 and 2018, Chung, Jansen and others described a new syndrome caused by haploinsufficiency of PHIP (pleckstrin homology domain interacting protein, OMIM *612,870) and mainly characterized by developmental delay (DD), learning difficulties/intellectual disability (ID), behavioral abnormalities, facial dysmorphism and obesity (CHUJANS, OMIM #617991). So far, PHIP alterations appear to be a rare cause of DD/ID. "Omics" technologies such as exome sequencing or array analyses have led to the identification of distinct types of alterations of PHIP, including, truncating variants, missense substitutions, splice variants and large deletions encompassing portions of the gene or the entire gene as well as adjacent genomic regions. We collected clinical and genetic data of 23 individuals with PHIP-associated Chung-Jansen syndrome (CHUJANS) from all over Europe. Follow-up investigations (e.g. Sanger sequencing, qPCR or Fluorescence-in-situ-Hybridization) and segregation analysis showed either de novo occurrence or inheritance from an also (mildly) affected parent. In accordance with previously described patients, almost all individuals reported here show developmental delay (22/23), learning disability or ID (22/23), behavioral abnormalities (20/23), weight problems (13/23) and characteristic craniofacial features (i.e. large ears/earlobes, prominent eyebrows, anteverted nares and long philtrum (23/23)). To further investigate the facial gestalt of individuals with CHUJANS, we performed facial analysis using the GestaltMatcher approach. By this, we could establish that PHIP patients are indistinguishable based on the type of PHIP alteration (e.g. missense, loss-of-function, splice site) but show a significant difference to the average face of healthy individuals as well as to individuals with Prader-Willi syndrome (PWS, OMIM #176270) or with a CUL4B-alteration (Intellectual developmental disorder, X-linked, syndromic, Cabezas type, OMIM #300354). Our findings expand the mutational and clinical spectrum of CHUJANS. We discuss the molecular and clinical features in comparison to the published individuals. The fact that some variants were inherited from a mildly affected parent further illustrates the variability of the associated phenotype and outlines the importance of a thorough clinical evaluation combined with genetic analyses for accurate diagnosis and counselling.

Next Generation Sequencing in Pediatric Epilepsy Using Customized Panels: Size Matters.

INTRODUCTION: Next generation sequencing (NGS) with customized gene panels is a helpful tool to identify monogenic epilepsy syndromes. The number of genes tested within a customized panel may vary greatly. The aim of the present study was to compare the diagnostic yield of small (<25 kb) and large (>25 kb) customized epilepsy panels. METHODS: This retrospective cohort study investigated data of 190 patients of 18 years or younger, with the diagnosis of an epilepsy of unknown etiology who underwent NGS using customized gene panels. Small (<25 kb) and large (>25 kb) panels were compared regarding the distribution of benign/likely benign and pathogenic/likely pathogenic variants and variants of unclear significance. In addition, differences of the diagnostic yield with respect to epilepsy severity, i.e., developmental and epileptic encephalopathy [DEE] vs. non-DEE, were analyzed. RESULTS: The diagnostic yield defined as pathogenic or likely pathogenic variants in large panels was significantly increased (29% [n = 14/48] vs. 13% [n = 18/142], p = 0.0198) compared with smaller panels. In non-DEE patients the increase of the diagnostic yield in large panels was significant(35% n = 6/17 vs. 13% n = 12/94, p = 0.0378), which was not true for DEE patients. DISCUSSION: This study indicates that large panels are superior for pediatric patients with epilepsy forms without encephalopathy (non-DEE). For patients suffering from DEE small panels of a maximum of 10 genes seem to be sufficient. The proportion of unclear findings increases with rising panel sizes. CONCLUSION: Customized epilepsy panels of >25 kb compared with smaller panels show a significant higher diagnostic yield in patients with epilepsy especially in non-DEE patients.

Novel variants in TECRL cause recessive inherited CPVT type 3 with severe and variable clinical symptoms.

INTRODUCTION: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome characterized by adrenergically stimulated ventricular tachycardia. The most common form of CPVT is due to autosomal dominant variants in the cardiac ryanodine-receptor gene (RYR2). However, trans-2,3-enoyl-CoA reductase-like (TECRL) was recently suggested to be a novel candidate gene for life-threatening inherited arrhythmias. Patients previously reported with pathogenic changes in TECRL showed a special mixed phenotype of CPVT and long-QT-syndrome (LQTS) termed CPVT type 3 (CPVT3), an autosomal recessive disorder. METHODS AND RESULTS: We implemented TECRL into our NGS panel diagnostics for CPVT and LQTS in April 2017. By December 2018, 631 index patients with suspected CPVT or LQTS had been referred to our laboratory for genetic testing. Molecular analysis identified four Caucasian families carrying novel variants in TECRL. One patient was homozygous for Gln139* resulting in a premature stop codon and loss-of-function of the TECRL protein. Another patient was homozygous for Pro290His, probably leading to an altered folding of the 3-oxo-5-alpha steroid 4-dehydrogenase domain of the TECRL protein. The LOF-variant Ser309* and the missense-variant Val298Ala have been shown to be compound heterozygous in another individual. NGS-based copy number variation analysis and quantitative PCR revealed a quadruplication of TECRL in the last individual, which is likely to be a homozygous duplication. CONCLUSION: The data from our patient collective indicate that CPVT3 occurs much more frequently than previously expected. Variants in TECRL may be causative in up to 5% of all CPVT cases. According to these findings, the default analysis of this gene is recommended if CPVT is suspected.

Impact on Clinical Decision Making of Next-Generation Sequencing in Pediatric Epilepsy in a Tertiary Epilepsy Referral Center.

Background. Next-generation sequencing (NGS) describes new powerful techniques of nucleic acid analysis, which allow not only disease gene identification diagnostics but also applications for transcriptome/methylation analysis and meta-genomics. NGS helps identify many monogenic epilepsy syndromes. Pediatric epilepsy patients can be tested using NGS epilepsy panels to diagnose them, thereby influencing treatment choices. The primary objective of this study was to evaluate the impact of genetic testing on clinical decision making in pediatric epilepsy patients. Methods. We completed a single-center retrospective cohort study of 91 patients (43 male) aged 19 years or less undergoing NGS with epilepsy panels differing in size ranging from 5 to 434 genes from October 2013 to September 2017. Results. During a mean time of 3.6 years between symptom onset and genetic testing, subjects most frequently showed epileptic encephalopathy (40%), focal epilepsy (33%), and generalized epilepsy (18%). In 16 patients (18% of the study population), "pathogenic" or "likely pathogenic" results according to ACMG criteria were found. Ten of the 16 patients (63%) experienced changes in clinical management regarding their medication and avoidance of further diagnostic evaluation, that is, presurgical evaluation. Conclusion. NGS epilepsy panels contribute to the diagnosis of pediatric epilepsy patients and may change their clinical management with regard to both preventing unnecessary and potentially harmful diagnostic procedures and management. Thus, the present data support the early implementation in order to adopt clinical management in selected cases and prevent further invasive investigations. Given the relatively small sample size and heterogeneous panels a larger prospective study with more homogeneous panels would be helpful to further determine the impact of NGS on clinical decision making.

Biallelic COL3A1 mutations result in a clinical spectrum of specific structural brain anomalies and connective tissue abnormalities.

Vascular Ehlers-Danlos syndrome (type IV) is an autosomal dominant disorder caused by heterozygous variants of COL3A1. We identified biallelic COL3A1 variants in two unrelated families. In a 3-year-old female with developmental delay the nonsense variant c.1282C>T, p.(Arg428*) was detected in combination the c.2057delC, p.(Pro686Leufs*105) frame shift variant. Both compound heterozygous variants were novel. This patient was born with bilateral clubfoot, joint laxity, and dysmorphic facial features. At the age of 2 years she developed an aneurysmal brain hemorrhage. Cerebral MRI showed a peculiar pattern of profound cerebral abnormalities including bilateral frontoparietal polymicrogyria of the cobblestone variant. In the second family, the two affected siblings were homozygous for the missense variant c.145C

Phenotypic spectrum of GABRA1: From generalized epilepsies to severe epileptic encephalopathies.

OBJECTIVE: To delineate phenotypic heterogeneity, we describe the clinical features of a cohort of patients with GABRA1 gene mutations. METHODS: Patients with GABRA1 mutations were ascertained through an international collaboration. Clinical, EEG, and genetic data were collected. Functional analysis of 4 selected mutations was performed using the Xenopus laevis oocyte expression system. RESULTS: The study included 16 novel probands and 3 additional family members with a disease-causing mutation in the GABRA1 gene. The phenotypic spectrum varied from unspecified epilepsy (1), juvenile myoclonic epilepsy (2), photosensitive idiopathic generalized epilepsy (1), and generalized epilepsy with febrile seizures plus (1) to severe epileptic encephalopathies (11). In the epileptic encephalopathy group, the patients had seizures beginning between the first day of life and 15 months, with a mean of 7 months. Predominant seizure types in all patients were tonic-clonic in 9 participants (56%) and myoclonic seizures in 5 (31%). EEG showed a generalized photoparoxysmal response in 6 patients (37%). Four selected mutations studied functionally revealed a loss of function, without a clear genotype-phenotype correlation. CONCLUSIONS: GABRA1 mutations make a significant contribution to the genetic etiology of both benign and severe epilepsy syndromes. Myoclonic and tonic-clonic seizures with pathologic response to photic stimulation are common and shared features in both mild and severe phenotypes.

[Current methods in genetic analysis : an approach for genetics-based preventive medicine]. / Moderne genetische Analysemethoden : Grundlagen für eine genetisch basierte Prävention.

Modern genetic analysis methods such as DNA arrays (gene chips) or high-throughput DNA sequencing of the next generation (Next Generation Sequencing, NGS) have once again accelerated the pace of innovation that has been powered by genome research over the past 10 years of the "post-genomic era". The present paper introduces array and NGS methods as two important innovation driving methods and provides examples for their application in large-scale scientific projects. However, a broad application of these very powerful technologies for genetic screening for the purpose of disease prevention is currently not yet in sight. The complexity of the interaction of genes, gene products and the environment has so far exceeded all expectations, suggesting that reliable statements about the medical relevance of common genetic variants can presently only be made in a few areas such as pharmacogenetics and oncology. We also discuss ethical issues raised by genetic population screening. The aim of this paper is to provide a brief outline of the development of methods in molecular genetics to the now dominant modern technologies and present their applications in research, in the diagnosis of rare diseases, and in terms of screening approaches.

A boy with mild mental retardation, mild sensorineural hearing loss and mild facial dysmorphism caused by a 19p13.2 deletion: a case report and review of the literature.

The investigation of patients with congenital anomalies and/or intellectual disability with modern genetic methods allows the recognition of an increasing number of cases with these chromosomal rearrangements. Here, we present a mildly mentally retarded boy with mild facial dysmorphism, language development delay, mild sensorineural hearing loss due to a deletion of 1,14 Mb on chromosome 19p 13.2. The deletion was de novo and familial history negative for this disorder. To our knowledge this is the first description of a patient with symptoms mentioned above associated with a 19p13.2-p13.2 deletion.

Combined deletion 18q22.2 and duplication/triplication 18q22.1 causes microcephaly, mental retardation and leukencephalopathy.

Chromosome 18 abnormalities rank among the most common autosomal anomalies with 18q being the most frequently affected. A deletion of 18q has been attributed to microcephaly, mental retardation, short stature, facial dysmorphism, myelination disorders, limb and genitourinary malformations and congenital aural atresia. On the other hand, duplications of 18q have been associated with the phenotype of Edwards syndrome. Critical chromosomal regions for both phenotypes are contentious. In this report, we describe the first case of an 11-year old male with a combined interstitial duplication 18q22.1, triplication 18q22.1q22.2 and terminal deletion 18q22.2q23 with phenotypic features of isolated 18q deletion syndrome and absence of phenotypic features characteristic of Edwards syndrome despite duplication of the suggested critical region. This report allows for reevaluation of proposed critical intervals for the phenotypes in deletion 18q syndrome and Edwards syndrome.

Expanding the mutation spectrum for Fraser syndrome: identification of a novel heterozygous deletion in FRAS1.

Fraser syndrome (FS) is a rare autosomal recessive inherited disorder characterized by cryptophthalmos, laryngeal defects and oral clefting, mental retardation, syndactyly, and urogenital defects. To date, 250 patients have been described in the literature. Mutations in the FRAS1 gene on chromosome 4 have been identified in patients with Fraser syndrome. So far, 26 mutations have been identified, most of them are truncating mutations. The mutational spectrum includes nucleotide substitutions, splicing defects, a large insertion, and small deletions/insertions. Moreover, single heterozygous missense mutations in FRAS1 seem to be responsible for non-syndromic unilateral renal agenesis. Here we report the first case of a family with two patients affected by Fraser syndrome due to a deletion of 64 kb (deletion 4q21.21) and an additional novel frameshift mutation in exon 66 of the FRAS1 gene. To date, large deletions of the FRAS1 gene have not yet been described. Large deletions seem to be a rare cause for Fraser syndrome, but should be considered in patients with a single heterozygous mutation.

A novel interstitial deletion of 10q24.2q24.32 in a patient with renal coloboma syndrome.

Renal coloboma syndrome (RCS) is considered to be a rare autosomal dominant inherited disorder characterized by renal malformations and optic disc coloboma. Ocular anomalies range from asymptomatic abnormalities in retinal blood vessel patterning to large excavations of the optic nerve associated with reduced visual acuity. Commonly observed manifestations of the kidney are renal hypoplasia and vesicoureteric reflux leading to end-stage renal disease. Mutations in the PAX2 gene on chromosome 10 have been identified in patients with RCS. Up to date, nucleotide substitutions, insertions, small deletions, one de novo translocation, and one 240 kb deletion of the coding region of the PAX2 gene have been described to be responsible for RCS. We report here a new case of a patient with RCS due to a deletion of 3.8 Mb on chromosome 10q. Deletions on the long arm of chromosome 10 harboring the PAX2 gene seem to be a rare cause for RCS. Nevertheless, array-CGH testing should represent an important and valuable addition to PAX2 gene sequencing in diagnostic of RCS.

Deep sequencing reveals 50 novel genes for recessive cognitive disorders.

Common diseases are often complex because they are genetically heterogeneous, with many different genetic defects giving rise to clinically indistinguishable phenotypes. This has been amply documented for early-onset cognitive impairment, or intellectual disability, one of the most complex disorders known and a very important health care problem worldwide. More than 90 different gene defects have been identified for X-chromosome-linked intellectual disability alone, but research into the more frequent autosomal forms of intellectual disability is still in its infancy. To expedite the molecular elucidation of autosomal-recessive intellectual disability, we have now performed homozygosity mapping, exon enrichment and next-generation sequencing in 136 consanguineous families with autosomal-recessive intellectual disability from Iran and elsewhere. This study, the largest published so far, has revealed additional mutations in 23 genes previously implicated in intellectual disability or related neurological disorders, as well as single, probably disease-causing variants in 50 novel candidate genes. Proteins encoded by several of these genes interact directly with products of known intellectual disability genes, and many are involved in fundamental cellular processes such as transcription and translation, cell-cycle control, energy metabolism and fatty-acid synthesis, which seem to be pivotal for normal brain development and function.

Parental origin and functional relevance of a de novo UBE3A variant.

Sequence analysis of the imprinted UBE3A gene in a 3-year-old girl suspected of having Angelman syndrome had revealed a de novo 3bp in frame deletion predicted to encode a protein lacking the amino acid G538 (based on sequence NM_130838). In order to assess the clinical relevance of this unknown variant, we determined the parental origin and the functional consequences of the deletion. We separated the two chromosomes 15 by microdissection of metaphase spreads and used cytogenetic and molecular markers to demonstrate that the deletion is on the maternal chromosome. For determining the functional consequences of the deletion, we modelled the structure of the deletion mutant based on the wildtype X-ray structure and simulated the molecular dynamics of the wildtype and mutant protein in complex with UcbH7. Our simulations showed that deletion of G538 destroys a network of salt bridges between highly conserved residues in the catalytic cleft of UBE3A. In conclusion, our results strongly suggest that the 3bp deletion is a loss-of-function mutation of the maternal UBE3A allele that has caused Angelman syndrome in our patient. Our study may serve as a paradigm to determine the parental origin of a de novo mutation.

Phenotypic features of carbohydrate sulfotransferase 3 (CHST3) deficiency in 24 patients: congenital dislocations and vertebral changes as principal diagnostic features.

We recently reported on the deficiency of carbohydrate sulfotransferase 3 (CHST3; chondroitin-6-sulfotransferase) in six subjects diagnosed with recessive Larsen syndrome or humero-spinal dysostosis [Hermanns et al. (2008); Am J Hum Genet 82:1368-1374]. Since then, we have identified 17 additional families with CHST3 mutations and we report here on a series of 24 patients in 23 families. The diagnostic hypothesis prior to molecular analysis had been: Larsen syndrome (15 families), humero-spinal dysostosis (four cases), chondrodysplasia with multiple dislocations (CDMD "Megarbane type"; two cases), Desbuquois syndrome (one case), and spondylo-epiphyseal dysplasia (one case). In spite of the different diagnostic labels, the clinical features in these patients were similar and included dislocation of the knees and/or hips at birth, clubfoot, elbow joint dysplasia with subluxation and limited extension, short stature, and progressive kyphosis developing in late childhood. The most useful radiographic clues were the changes of the lumbar vertebrae. Twenty-four different CHST3 mutations were identified; 16 patients had homozygous mutations. We conclude that CHST3 deficiency presents at birth with congenital dislocations of knees, hips, and elbows, and is often diagnosed initially as Larsen syndrome, humero-spinal dysostosis, or chondrodysplasia with dislocations. The incidence of CHST3 deficiency seems to be higher than assumed so far. The clinical and radiographic pattern (joint dislocations, vertebral changes, normal carpal age, lack of facial flattening, and recessive inheritance) is characteristic and distinguishes CHST3 deficiency from other disorders with congenital dislocations such as filamin B-associated dominant Larsen syndrome and Desbuquois syndrome.

Mapping translocation breakpoints by next-generation sequencing.

Balanced chromosome rearrangements (BCRs) can cause genetic diseases by disrupting or inactivating specific genes, and the characterization of breakpoints in disease-associated BCRs has been instrumental in the molecular elucidation of a wide variety of genetic disorders. However, mapping chromosome breakpoints using traditional methods, such as in situ hybridization with fluorescent dye-labeled bacterial artificial chromosome clones (BAC-FISH), is rather laborious and time-consuming. In addition, the resolution of BAC-FISH is often insufficient to unequivocally identify the disrupted gene. To overcome these limitations, we have performed shotgun sequencing of flow-sorted derivative chromosomes using "next-generation" (Illumina/Solexa) multiplex sequencing-by-synthesis technology. As shown here for three different disease-associated BCRs, the coverage attained by this platform is sufficient to bridge the breakpoints by PCR amplification, and this procedure allows the determination of their exact nucleotide positions within a few weeks. Its implementation will greatly facilitate large-scale breakpoint mapping and gene finding in patients with disease-associated balanced translocations.

Endochondral gigantism: a newly recognized skeletal dysplasia with pre- and postnatal overgrowth and endocrine abnormalities.

We report on a 3-year-old male, born at 34 weeks of gestation, with marked pre- and postnatal overgrowth, birth weight of 6,600 g, length of 61 cm, and head circumference of 38.5 cm. A striking phenotype was recorded at birth, which became more evident during the follow-up period. He had macrobrachycephaly, facial abnormalities, small thoracic cage, long trunk, deformed spine, rhizomelia, large hands and feets, absent subcutaneous fat, small umbilical hernia, inguinal hernias, and large joints with mild contractures. Hypoglycemic episodes and obstructive apnea complicated the neonatal period. During follow-up, overgrowth continued with a height of 146 cm (+11.65 SDS) and a weight of 39 kg (BMI 18.3 kg/m(2)) at 3.5 years. Endocrinological work-up disclosed extremely low levels of growth hormone, insulin-like growth factors, and insulin. What makes our patient unique is the association of marked prenatal overgrowth; unusual phenotype; skeletal dysplasia caused by accelerated endochondral ossification resulting in cartilage hyperplasia of the skull base and spine, and postnatal gigantism; and complete absence of subcutaneous fat. Other well-known overgrowth syndromes were excluded. We hypothesize that autocrine/paracrine growth factors could be the cause of excessive endochondral ossification. Alternately, activating mutations in transcription factors involved in both growth and endocrine/metabolic homeostasis could be responsible for this unusual phenotype.

MRX42: two linkage intervals, one in the pericentromeric region and one in Xq26, and the impact for carrier risk estimation.

A nonspecific X-linked mental retardation (MRX) family is reported with four mild to moderately affected males and no intellectual impairment in their obligate carrier mothers. Linkage analysis obtained the same multipoint lod score of 2.08 for two intervals on the X chromosome already reported to be linked to other MRX and syndromic X-linked mental retardation (XLMR) families: one pericentromeric and the other at Xq26. Since the responsible gene is not yet characterized, haplotyping is presently the only means available for carrier and prenatal testing for this form of MRX. Carrier risk estimation using pedigree and haplotype data for five females at risk is presented, and the difficulties of prenatal diagnosis given linkage to two different regions is discussed.