Genome Mapping Nomenclature.

Background Genome Mapping Technologies (optical and electronic) uses ultra high-molecular weight DNA to detect structural variation and has an application in constitutional genetic disorders, haematological neoplasms and solid tumours. Genome mapping can detect balanced and unbalanced structural variation, copy number changes and haplotypes. The technique is analogous to chromosomal microarray analysis although genome mapping has the added benefit of being able to detect and ascertain the nature of more abnormalities than array, karyotyping or FISH. Key Messages This paper describes a specific nomenclature for genome mapping that can be used by diagnostic and research centres to accurately report their findings. An international nomenclature is essential for patient results to be understood by different healthcare providers as well as clear communication in publications and consistency in databases. Summary Genome mapping can detect aneuploidy, balanced and unbalanced structural variation as well as copy number changes. The Standing Committee for the International System for Human Cytogenomic Nomenclature (ISCN), recognised there was a need for a specific nomenclature for genome mapping that encompasses the range of abnormalities detected by this technique. This paper explains the general principles of the nomenclature as well as giving specific ISCN examples for the different types of numerical and structural rearrangements.

Constitutional chromothripsis of the APC locus as a cause of genetic predisposition to colon cancer.

PURPOSE: Approximately 20% of patients with clinical familial adenomatous polyposis (FAP) remain unsolved after molecular genetic analysis of the APC and other polyposis genes, suggesting additional pathomechanisms. METHODS: We applied multidimensional genomic analysis employing chromosomal microarray profiling, optical mapping, long-read genome and RNA sequencing combined with FISH and standard PCR of genomic and complementary DNA to decode a patient with an attenuated FAP that had remained unsolved by Sanger sequencing and multigene panel next-generation sequencing for years. RESULTS: We identified a complex 3.9 Mb rearrangement involving 14 fragments from chromosome 5q22.1q22.3 of which three were lost, 1 reinserted into chromosome 5 and 10 inserted into chromosome 10q21.3 in a seemingly random order and orientation thus fulfilling the major criteria of chromothripsis. The rearrangement separates APC promoter 1B from the coding ORF (open reading frame) thus leading to allele-specific downregulation of APC mRNA. The rearrangement also involves three additional genes implicated in the APC-Axin-GSK3B-ß-catenin signalling pathway. CONCLUSIONS: Based on comprehensive genomic analysis, we propose that constitutional chromothripsis dampening APC expression, possibly modified by additional APC-Axin-GSK3B-ß-catenin pathway disruptions, underlies the patient's clinical phenotype. The combinatorial approach we deployed provides a powerful tool set for deciphering unsolved familial polyposis and potentially other tumour syndromes and monogenic diseases.

Genetic and phenotypic dissection of 1q43q44 microdeletion syndrome and neurodevelopmental phenotypes associated with mutations in ZBTB18 and HNRNPU.

Subtelomeric 1q43q44 microdeletions cause a syndrome associating intellectual disability, microcephaly, seizures and anomalies of the corpus callosum. Despite several previous studies assessing genotype-phenotype correlations, the contribution of genes located in this region to the specific features of this syndrome remains uncertain. Among those, three genes, AKT3, HNRNPU and ZBTB18 are highly expressed in the brain and point mutations in these genes have been recently identified in children with neurodevelopmental phenotypes. In this study, we report the clinical and molecular data from 17 patients with 1q43q44 microdeletions, four with ZBTB18 mutations and seven with HNRNPU mutations, and review additional data from 37 previously published patients with 1q43q44 microdeletions. We compare clinical data of patients with 1q43q44 microdeletions with those of patients with point mutations in HNRNPU and ZBTB18 to assess the contribution of each gene as well as the possibility of epistasis between genes. Our study demonstrates that AKT3 haploinsufficiency is the main driver for microcephaly, whereas HNRNPU alteration mostly drives epilepsy and determines the degree of intellectual disability. ZBTB18 deletions or mutations are associated with variable corpus callosum anomalies with an incomplete penetrance. ZBTB18 may also contribute to microcephaly and HNRNPU to thin corpus callosum, but with a lower penetrance. Co-deletion of contiguous genes has additive effects. Our results confirm and refine the complex genotype-phenotype correlations existing in the 1qter microdeletion syndrome and define more precisely the neurodevelopmental phenotypes associated with genetic alterations of AKT3, ZBTB18 and HNRNPU in humans.

Novel mutation in two brothers with Hermansky Pudlak syndrome type 3.

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder causing oculocutaneous albinism, bleeding disorder and ceroid lipofuscinosis. Platelets from HPS patients are characterized by impaired secretion of dense (δ)-bodies (CD63). Meanwhile, there are ten known human HPS genes, each leading to a particular clinical HPS subtype (HPS1-HPS10). We report on two Turkish brothers showing typical HPS phenotype comprising oculocutaneous albinism and bleeding symptoms. Pathological bleeding time as well as platelet aggregometry analyses revealed impaired platelet function. The brothers demonstrated absence of platelet δ-granule secretion as measured by flow cytometry. Using molecular genetic analyses, a novel homozygous 1bp-deletion in the HPS3 gene was identified in both brothers. In addition, the younger brother with HPS3 demonstrated psychomotoric retardation and cranial gliosis (magnetic resonance imaging, MRI). Array-CGH analysis revealed a de novo 0.482Mb deletion on chromosome 17 which is not present in his brother and parents. In this study, we identified a novel 1bp-deletion in the HPS3 gene causing HPS3 phenotype in two brothers. In patients with oculocutaneous albinism and increased bleeding symptoms platelet function should be analyzed. The identification of the molecular genetic defect allows the classification to a particular HPS subtype and is important for therapy and prognosis.

Optical Genome Mapping as a Potential Routine Clinical Diagnostic Method.

Chromosome analysis (CA) and chromosomal microarray analysis (CMA) have been successfully used to diagnose genetic disorders. However, many conditions remain undiagnosed due to limitations in resolution (CA) and detection of only unbalanced events (CMA). Optical genome mapping (OGM) has the potential to address these limitations by capturing both structural variants (SVs) resulting in copy number changes and balanced rearrangements with high resolution. In this study, we investigated OGM's concordance using 87 SVs previously identified by CA, CMA, or Southern blot. Overall, OGM was 98% concordant with only three discordant cases: (1) uncalled translocation with one breakpoint in a centromere; (2) uncalled duplication with breakpoints in the pseudoautosomal region 1; and (3) uncalled mosaic triplication originating from a marker chromosome. OGM provided diagnosis for three previously unsolved cases: (1) disruption of the SON gene due to a balanced reciprocal translocation; (2) disruption of the NBEA gene due to an inverted insertion; (3) disruption of the TSC2 gene due to a mosaic deletion. We show that OGM is a valid method for the detection of many types of SVs in a single assay and is highly concordant with legacy cytogenomic methods; however, it has limited SV detection capabilities in centromeric and pseudoautosomal regions.

CD133 induces tumour-initiating properties in HEK293 cells.

The pentaspan protein CD133 (Prominin-1) is part of the signature of tumour-initiating cells for various cancer entities. The aim of the present study was to investigate the impact of ectopic CD133 expression on tumourigenic properties of otherwise CD133-negative, non-tumourigenic cells in vitro and in vivo. CD133 was stably transfected into human embryonic kidney 293 (HEK293) which was then sorted for the expression of CD133. The effects of CD133 on cell proliferation were assessed upon standard cell counting of sorted cells at various time points. Severe combined immunodeficient (SCID) mice (n = 30) were injected with HEK293 CD133(high) and CD133(low) transfectants (5 × 10(3), 1 × 10(5), or 5 × 10(6) cells per injection). The expression of CD133, Ki67, CD44s, CD44v6, and EpCAM was analysed upon immunohistochemical staining of cryosections with specific antibodies. In vitro, ectopic expression of CD133 did influence neither cell proliferation nor cell cycle distribution of otherwise CD133-negative HEK293 cells. However, CD133(high) cells generated tumours in vivo in SCID mice with at least 1,000-fold increased frequency compared to CD133(low) cells. Tumour load was also significantly increased in CD133(high) cells as compared to those tumours formed by high numbers of CD133(low) cells. Immunohistochemistry stainings disclosed no changes in Ki67, CD44s, CD44v6, or EpCAM once tumours were formed by either cell type. CD133 induces tumour-initiating properties in HEK293 cells in vivo and is potentially involved in the regulation of tumourigenicity. Future research will aim at the elucidation of molecular mechanisms of CD133-induced tumourigenicity.

The phenotypic spectrum of duplication 5q35.2-q35.3 encompassing NSD1: is it really a reversed Sotos syndrome?

Loss-of-function mutations of NSD1 and 5q35 microdeletions encompassing NSD1 are a major cause of Sotos syndrome (Sos), which is characterized by overgrowth, macrocephaly, characteristic facies, and variable intellectual disability (ID). Microduplications of 5q35.2-q35.3 including NSD1 have been reported in only five patients so far and described clinically as a reversed Sos resulting from a hypothetical gene dosage effect of NSD1. Here, we report on nine patients from five families with interstitial duplication 5q35 including NSD1 detected by molecular karyotyping. The clinical features of all 14 individuals are reviewed. Patients with microduplications including NSD1 appear to have a consistent phenotype consisting of short stature, microcephaly, learning disability or mild to moderate ID, and distinctive facial features comprising periorbital fullness, short palpebral fissures, a long nose with broad or long nasal tip, a smooth philtrum and a thin upper lip vermilion. Behavioral problems, ocular and minor hand anomalies may be associated. Based on our findings, we discuss the possible etiology and conclude that it is possible, but so far unproven, that a gene dosage effect of NSD1 may be the major cause.

Generalized epilepsy in two patients with 5p duplication.

BACKGROUND: There are only few reports on patients with duplications of the short arm of chromosome 5, with little information about concomitant epilepsy. PATIENTS: We report on two patients with generalized epilepsies since age 2.5 years, in whom array comparative genomic hybridization revealed microduplications of different sizes in the short arm of chromosome 5. Both patients showed developmental delay, and magnetic resonance images were normal. The larger duplication in patient 1, who additionally exhibited dysmorphic features and photosensivity on electroencephalogram, occurred de novo, whereas the smaller duplication in patient 2 was paternally inherited. DISCUSSION: The overlapping duplicated region in band 5p13.1 comprises four genes (RICTOR, FYB, C9, and DAB2). Further investigation on patients with duplication 5p and epilepsy are needed to possibly identify a potential susceptibility locus for epilepsy at chromosome 5p13.1.

Cardiac malformation of partial trisomy 7p/monosomy 18p and partial trisomy 18p/monosomy 7p in siblings as a result of reciprocal unbalanced malsegregation--and review of the literature.

We report two unbalanced translocations involving the short arms of chromosomes 7 and 18 due to a balanced translocation 7;18 in the mother. Karyotyping and fluorescence in situ hybridization analysis of the female fetus revealed an unbalanced subtelomeric translocation(karyotype 46,XX,der(18)t(7;18)(p22.3;p11.32)mat resulting in a partial trisomy 7p and a partial monosomy 18p.Array comparative genomic hybridization (CGH) detected a4.44-Mb heterozygous duplication at 7p22.3 to 7p22.1 and a0.178-Mb heterozygous deletion at 18p11.32. Clinical characteristics comprised a mildly stenotic bicuspid aortic valve and a small aortic arch without coarctation. The patient's older brother displayed a reciprocal version of her chromosomal aberration (46,XY,der(7)t(7;18)(p22;p11.32) resulting in a partial monosomy 7p and a partial trisomy 18p. Array CGH revealed a 4.75-Mb heterozygous deletion at 7p22.3p22.1 and a 0.579-Mb duplication at 18p11.32. He presented with tetralogy of Fallot, cleft palate, microcephalus without craniosynostosis, growth retardation, ptosis of the right eyelid, right-sided renal agenesis, unilateral cryptorchism,and mental retardation. In this report, we present the clinical phenotype in patients with aberrations of chromosomes 7p and 18p and reviewed the literature to summarize cardiovascular malformations in these patients.

Biallelic MLH1 SNP cDNA expression or constitutional promoter methylation can hide genomic rearrangements causing Lynch syndrome.

BACKGROUND: A positive family history, germline mutations in DNA mismatch repair genes, tumours with high microsatellite instability, and loss of mismatch repair protein expression are the hallmarks of hereditary non-polyposis colorectal cancer (Lynch syndrome). However, in ~10-15% of cases of suspected Lynch syndrome, no disease-causing mechanism can be detected. METHODS: Oligo array analysis was performed to search for genomic imbalances in patients with suspected mutation-negative Lynch syndrome with MLH1 deficiency in their colorectal tumours. RESULTS AND CONCLUSION: A deletion in the LRRFIP2 (leucine-rich repeat flightless-interacting protein 2) gene flanking the MLH1 gene was detected, which turned out to be a paracentric inversion on chromosome 3p22.2 creating two new stable fusion transcripts between MLH1 and LRRFIP2. A single-nucleotide polymorphism in MLH1 exon 8 was expressed from both alleles, initially pointing to appropriate MLH1 function at least in peripheral cells. In a second case, an inherited duplication of the MLH1 gene region resulted in constitutional MLH1 promoter methylation. Constitutional MLH1 promoter methylation may therefore in rare cases be a heritable disease mechanism and should not be overlooked in seemingly sporadic patients.

Phenotypic spectrum associated with CASK loss-of-function mutations.

BACKGROUND: Heterozygous mutations in the CASK gene in Xp11.4 have been shown to be associated with a distinct brain malformation phenotype in females, including disproportionate pontine and cerebellar hypoplasia. METHODS: The study characterised the CASK alteration in 20 new female patients by molecular karyotyping, fluorescence in situ hybridisation, sequencing, reverse transcriptase (RT) and/or quantitative real-time PCR. Clinical and brain imaging data of a total of 25 patients were reviewed. RESULTS: 11 submicroscopic copy number alterations, including nine deletions of ~11 kb to 4.5 Mb and two duplications, all covering (part of) CASK, four splice, four nonsense, and one 1 bp deletion are reported. These heterozygous CASK mutations most likely lead to a null allele. Brain imaging consistently showed diffuse brainstem and cerebellar hypoplasia with a dilated fourth ventricle, but of remarkably varying degrees. Analysis of 20 patients in this study, and five previously reported patients, revealed a core clinical phenotype comprising severe developmental delay/intellectual disability, severe postnatal microcephaly, often associated with growth retardation, (axial) hypotonia with or without hypertonia of extremities, optic nerve hypoplasia, and/or other eye abnormalities. A recognisable facial phenotype emerged, including prominent and broad nasal bridge and tip, small or short nose, long philtrum, small chin, and/or large ears. CONCLUSIONS: These findings define the phenotypic spectrum associated with CASK loss-of-function mutations. The combination of developmental and brain imaging features together with mild facial dysmorphism is highly suggestive of this disorder and should prompt subsequent testing of the CASK gene.

Splicing analyses for variants in MMR genes: best practice recommendations from the European Mismatch Repair Working Group.

Over 20% of the DNA mismatch repair (MMR) germline variants in suspected Lynch syndrome patients are classified as variants of uncertain significance (VUS). Well-established functional assays are pivotal for assessing the biological impact of these variants and provide relevant evidence for clinical classification. In our collaborative European Mismatch Repair Working Group (EMMR-WG) we compared three different experimental approaches for evaluating the effect of seven variants on mRNA splicing in MMR genes: (i) RT-PCR of full-length transcripts (FLT), (ii) RT-PCR of targeted transcript sections (TTS), both from patient biological samples and (iii) minigene splicing assays. An overall good concordance was observed between splicing patterns in TTS, FLT and minigene analyses for all variants. The FLT analysis depicted a higher number of different isoforms and mitigated PCR-bias towards shorter isoforms. TTS analyses may miss aberrant isoforms and minigene assays may under/overestimate the severity of certain splicing defects. The interpretation of the experimental findings must be cautious to adequately discriminate abnormal events from physiological complex alternative splicing patterns. A consensus strategy for investigating the impact of MMR variants on splicing was defined. First, RNA should be obtained from patient's cell cultures (such as fresh lymphocyte cultures) incubated with/without a nonsense-mediated decay inhibitor. Second, FLT RT-PCR analysis is recommended to oversee all generated isoforms. Third, TTS analysis and minigene assays are useful independent approaches for verifying and clarifying FLT results. The use of several methodologies is likely to increase the strength of the experimental evidence which contributes to improve variant interpretation.

In vitro supplementation with dAMP/dGMP leads to partial restoration of mtDNA levels in mitochondrial depletion syndromes.

Mitochondrial DNA depletion syndrome, a frequent cause of childhood (hepato)encephalomyopathies, is defined as a reduction of mitochondrial DNA copy number related to nuclear DNA. It was previously shown that mtDNA depletion can be prevented by dAMP/dGMP supplementation in deoxyguanosine kinase-deficient fibroblasts. We investigated myotubes of patients diagnosed with mtDNA depletion carrying pathogenic mutations in DGUOK, POLG1 (Alpers syndrome) and TYMP. Differentiating myotubes of all patients and controls were supplemented with different doses of dAMP/dGMP or dAMP/dGMP/dCMP in TYMP deficiency, and analysed for mtDNA/nDNA ratio and for cytochrome c oxidase (COX) activity. Serum deprivation and myotube formation triggered a decrease in mtDNA copy number in DGUOK or POLG1 deficient myotubes, but not in TYMP deficiency and healthy controls. Supplementation with dAMP/dGMP leads to a significant and reproducible rescue of mtDNA depletion in DGUOK deficiency. POLG1 deficient myotubes also showed a mild, not significant increase in mtDNA copy number. MtDNA depletion did not result in deficient COX staining in DGUOK and POLG1-deficient myotubes. Treatment with ethidium bromide resulted in very severe depletion and absence of COX staining in all cell types, and no recovery was observed after supplementation with dAMP/dGMP. We show that supplementation with dAMP/dGMP increases mtDNA copy number significantly in DGUOK deficient myotubes and, leads to a mild, non-significant improvement of mtDNA depletion in POLG1 deficiency. No adverse effect on mtDNA copy number was observed on high-dose supplementation in vitro. Further studies are needed to determine possible therapeutic implications of dAMP/dGMP supplementation for DGUOK deficiency in vivo.

A Novel Likely Pathogenic Variant in the BLOC1S5 Gene Associated with Hermansky-Pudlak Syndrome Type 11 and an Overview of Human BLOC-1 Deficiencies.

Hermansky-Pudlak syndrome (HPS) is a heterogeneous disorder combining oculocutaneous albinism (OCA) and a platelet function disorder of varying severity as its most prominent features. The genes associated with HPS encode for different BLOC- (biogenesis of lysosome-related organelles complex) complexes and for the AP-3 (adaptor protein-3) complex, respectively. These proteins are involved in maturation, trafficking, and the function of lysosome-related organelles (LROs) such as melanosomes and platelet δ-granules. Some patients with different types of HPS can develop additional complications and symptoms like pulmonary fibrosis, granulomatous colitis, and immunodeficiency. A new type of HPS has recently been identified associated with genetic alterations in the BLOC1S5 gene, which encodes the subunit Muted of the BLOC-1 complex. Our aim was to unravel the genetic defect in two siblings with a suspected HPS diagnosis (because of OCA and bleeding symptoms) using next generation sequencing (NGS). Platelet functional analysis revealed reduced platelet aggregation after stimulation with ADP and a severe secretion defect in platelet δ-granules. NGS identified a novel homozygous essential splice site variant in the BLOC1S5 gene present in both affected siblings who are descendants of a consanguine marriage. The patients exhibited no additional symptoms. Our study confirms that pathogenic variants of BLOC1S5 cause the recently described HPS type 11.

A novel 1p31.3p32.2 deletion involving the NFIA gene detected by array CGH in a patient with macrocephaly and hypoplasia of the corpus callosum.

Interstitial deletions or apparently balanced translocations involving bands 1p31 and 1p32 in the short arm of chromosome 1 are rarely described chromosomal imbalances. To our knowledge, there have been six cases documented to date. Five of these cases, where the NFIA gene is involved, show complex central nervous system malformations and in some cases urinary tract defects. We report another case of a microdeletion with involvement of the NFIA gene in the short arm of chromosome 1 (del(1)(p31.3p32.2)) with, amongst other features, hypoplasia of the corpus callosum, ventriculomegaly, and dysmorphic features. A microdeletion 1p31.3p32.2 which includes the NFIA gene is associated with hypoplasia of the corpus callosum, ventriculomegaly, and dysmorphic features.

Single particle adsorbing transfer system.

Here we present a novel approach for horizontal transfer of single particles after laser microdissection. The developed technique is a single particle adsorbing system for highly selective and gentle horizontal transfer of microdissected fixed and living material. As mediated via low-pressure technology, the transfer process can be precisely controlled, thus facilitating horizontal particle transfer of any isolated material, e.g. tissue material, single cells or chromosomes, in addition to precise positioning for sample release. This collection method allows one to predefine target positions and enables material transfer without contamination to any planar microchip device. This contamination free transfer is indispensable for novel lab-on-a-chip systems performing nanoscale polymerase chain reaction analyses. Using virtual reaction chamber microdevices, small amounts of microdissected material--as little as one single cell--can be directly transmitted and immediately used for single cell analysis.

Single cell analysis of mutations in the APC gene.

INTRODUCTION: Mutation analysis of inherited monogenic diseases is an important aspect of preimplantation genetic diagnosis. Familial adenomatous polyposis of the colon is an autosomal dominant inherited disorder caused by mutations in the tumor suppressor gene adenomatous polyposis coli (APC). A characteristic of this severe disease is the development of thousands of polyps in the colon which untreated evolve into malignant colon carcinomas. Here we present a novel approach for the establishment of mutation detection in the APC gene in single cells applicable for preimplantation genetic diagnosis. METHODS: Single fixed lymphocytes were isolated via laser microdissection and transferred to reaction centers of planar chemically structured glass slides via a recently developed horizontal low-pressure single particle adsorbing transfer system (SPATS). A multiplex nested polymerase chain reaction protocol in a 1-microl reaction volume, followed by sequencing and fragment length analysis was applied in order to detect mutations in the APC gene. RESULTS: Reliable isolation and transfer of single lymphocytes was demonstrated. High amplification efficiency and low allelic drop out (ADO) rates for polymorphic microsatellite markers and mutation specific amplification products of various mutations in the APC gene were obtained from fixed single cells. CONCLUSIONS: This novel nanotechnological downscaling approach enables a reliable validation of genetic testing using diploid single lymphocytes, and will open a wide range of single cell diagnostics.

Hereditary neuralgic amyotrophy in childhood caused by duplication within the SEPT9 gene: A family study.

Hereditary neuralgic amyotrophy (HNA) is an autosomal dominant disorder associated with episodic, recurrent, and painful neuropathies affecting the nerves of the brachial plexus. In this study, we report on a family of Lebanese descent with HNA onset in early childhood. The affected family members presented with platelet dysfunction. Platelet aggregation was reduced after stimulation with the agonists ADP and epinephrine in all affected family members. Flow cytometric analyses revealed impaired platelet δ-secretion. The index patient and one brother suffered from kidney cysts. Molecular genetic analysis revealed a heterozygous duplication of exon 2 within the septin 9 (SEPT9) gene in all the affected family members. Such a young child with HNA (aged 2 years) caused by SEPT9 duplication has not been described so far.

Full-length transcript amplification and sequencing as universal method to test mRNA integrity and biallelic expression in mismatch repair genes.

In pathogenicity assessment, RNA-based analyses are important for the correct classification of variants, and require gene-specific cut-offs for allelic representation and alternative/aberrant splicing. Beside this, the diagnostic yield of RNA-based techniques capable to detect aberrant splicing or allelic loss due to intronic/regulatory variants has to be elaborated. We established a cDNA analysis for full-length transcripts (FLT) of the four DNA mismatch repair (MMR) genes to investigate the splicing pattern and transcript integrity with active/inhibited nonsense-mediated mRNA-decay (NMD). Validation was based on results from normal controls, samples with premature termination codons (PTC), samples with splice-site defects (SSD), and samples with pathogenic putative missense variants. The method was applied to patients with variants of uncertain significance (VUS) or unexplained immunohistochemical MMR deficiency. We categorized the allelic representation into biallelic (50 ± 10%) or allelic loss (≤10%), and >10% and <40% as unclear. We defined isoforms up to 10% and exon-specific exceptions as alternative splicing, set the cut-off for SSD in cDNA + P to 30-50%, and regard >10% and <30% as unclear. FLT cDNA analyses designated 16% of all putative missense variants and 12% of VUS as SSD, detected MMR-defects in 19% of the unsolved patients, and re-classified >30% of VUS. Our method allows a standardized, systematic cDNA analysis of the MMR FLTs to assess the pathogenicity mechanism of VUS on RNA level, which will gain relevance for precision medicine and gene therapy. Diagnostic accuracy will be enhanced by detecting MMR defects in hitherto unsolved patients. The data generated will help to calibrate a high-throughput NGS-based mRNA-analysis and optimize prediction programs.

Extending the critical regions for mutations in the non-coding gene RNU4ATAC in another patient with Roifman Syndrome.

Compound heterozygosity of a previously described pathogenic variant and a second novel nucleotide substitution (NR_023343.1:n.116A>C) affecting a highly conserved nucleotide in the noncoding RNU4ATAC gene could be identified in a patient with overlapping features of Roifman Syndrome. These data extend the spectrum of pathogenic variants in RNU4ATAC.