Recessive Dystrophic Epidermolysis bullosa due to Hemizygous 40 kb Deletion of COL7A1 and the Proximate PFKFB4 Gene Focusing on the Mutation c.425A>G Mimicking Homozygous Status

Background: Dystrophic Epidermolysis bullosa (DEB) is a rare inherited mechanobullous disease characterised by the hyperfragility of the skin and mucous membranes. It is (typically) caused by (loss-of-function) mutations in the COL7A1 gene that impair the formation of collagen type VII, which represents the major constituent of anchoring fibrils within the basement membrane zone of epithelialised tissues. In a 4-year-old patient diagnosed with the clinical features of recessive DEB, genotyping via Next-Generation EB Panel Sequencing initially revealed the homozygosity of the maternal c.425A>G mutation, while the paternal heterozygosity in exon 3 was lacking. This genetic profile suggested incongruent gene transmission due to uniparental isodisomy (UPD) or the occurrence of a hemizygous deletion of unknown size. Methods: Thus, the EB panel sequencing of genomic DNA, followed by a paternity test and analysis of microsatellite markers, as well as multiplex ligation-dependent probe amplification (MLPA) copy number analysis using patient and parental DNA, were performed. Results: This approach revealed a paternally derived hemizygous deletion spanning from exon 3 to exon 118. Linear amplification-mediated PCR (LAM-PCR) determined the breaking points within intron 2 of the COL7A1 gene, comprising a 40kb segment within intron 1 of the adjacent PFKFB4 gene. Conclusion: This report highlights the relevance of advanced molecular profiling to determine new/exceptional/unusual genotypes and the accurate mode of genetic transmission in DEB.

A spoonful of L-fucose-an efficient therapy for GFUS-CDG, a new glycosylation disorder.

Congenital disorders of glycosylation are a genetically and phenotypically heterogeneous family of diseases affecting the co- and posttranslational modification of proteins. Using exome sequencing, we detected biallelic variants in GFUS (NM_003313.4) c.[632G>A];[659C>T] (p.[Gly211Glu];[Ser220Leu]) in a patient presenting with global developmental delay, mild coarse facial features and faltering growth. GFUS encodes GDP-L-fucose synthase, the terminal enzyme in de novo synthesis of GDP-L-fucose, required for fucosylation of N- and O-glycans. We found reduced GFUS protein and decreased GDP-L-fucose levels leading to a general hypofucosylation determined in patient's glycoproteins in serum, leukocytes, thrombocytes and fibroblasts. Complementation of patient fibroblasts with wild-type GFUS cDNA restored fucosylation. Making use of the GDP-L-fucose salvage pathway, oral fucose supplementation normalized fucosylation of proteins within 4 weeks as measured in serum and leukocytes. During the follow-up of 19 months, a moderate improvement of growth was seen, as well as a clear improvement of cognitive skills as measured by the Kaufmann ABC and the Nijmegen Pediatric CDG Rating Scale. In conclusion, GFUS-CDG is a new glycosylation disorder for which oral L-fucose supplementation is promising.

De novo variants in TCF7L2 are associated with a syndromic neurodevelopmental disorder.

TCF7L2 encodes transcription factor 7-like 2 (OMIM 602228), a key mediator of the evolutionary conserved canonical Wnt signaling pathway. Although several large-scale sequencing studies have implicated TCF7L2 in intellectual disability and autism, both the genetic mechanism and clinical phenotype have remained incompletely characterized. We present here a comprehensive genetic and phenotypic description of 11 individuals who have been identified to carry de novo variants in TCF7L2, both truncating and missense. Missense variation is clustered in or near a high mobility group box domain, involving this region in these variants' pathogenicity. All affected individuals present with developmental delays in childhood, but most ultimately achieved normal intelligence or had only mild intellectual disability. Myopia was present in approximately half of the individuals, and some individuals also possessed dysmorphic craniofacial features, orthopedic abnormalities, or neuropsychiatric comorbidities including autism and attention-deficit/hyperactivity disorder (ADHD). We thus present an initial clinical and genotypic spectrum associated with variation in TCF7L2, which will be important in informing both medical management and future research.

Delineation of epileptic and neurodevelopmental phenotypes associated with variants in STX1B.

OBJECTIVE: To further delineate the clinical and genetic spectrum of epileptic and neurodevelopmental conditions associated with variants in STX1B. METHODS: We screened our diagnostic in-house database (comprising >20,000 exome sequencing datasets) for pathogenic and likely pathogenic variants inSTX1B. The detected cases were phenotyped in detail, and the findings were compared to previously published case reports. RESULTS: We identified four unrelated individuals with pathogenic or likely pathogenic variants in STX1B (one missense and three loss-of-function variants). All patients displayed epileptic phenotypes, including epileptiform discharges on electroencephalography (without apparent seizures), developmental and epileptic encephalopathy and focal epilepsy. Three of the four patients had developmental delay. Febrile seizures occurred in two individuals. One patient with focal epilepsy underwent epilepsy surgery without lasting improvement. The neuropathological workup of brain tissue revealed a mild malformation of cortical development without alterations of cortical lamination or dysplastic neurons. CONCLUSIONS: Our findings confirm the wide clinical range ofSTX1B-related epileptic conditions and highlight the necessity of genetic testing prior to epilepsy surgery in cases with monogenic epilepsy. The identification of loss-of-function variants in very differently affected individuals suggests that no clear genotype-phenotype correlation can be established.

The clinical significance of A2ML1 variants in Noonan syndrome has to be reconsidered.

The RASopathies are a group of clinically and genetically heterogeneous developmental disorders caused by dysregulation of the RAS/MAPK signalling pathway. Variants in several components and regulators of this pathway have been identified as the pathogenetic cause. In 2015, missense variants in A2ML1 were reported in three unrelated families with clinical diagnosis of Noonan syndrome (NS) and a zebrafish model was presented showing heart and craniofacial defects similar to those caused by a NS-associated Shp2 variant. However, a causal role of A2ML1 variants in NS has not been confirmed since. Herein, we report on 15 individuals who underwent screening of RASopathy-associated genes and were found to carry rare variants in A2ML1, including variants previously proposed to be causative for NS. In cases where parental DNA was available, the respective A2ML1 variant was found to be inherited from an unaffected parent. Seven index patients carrying an A2ML1 variant presented with an alternate disease-causing genetic aberration. These findings underscore that current evidence is insufficient to support a causal relation between variants in A2ML1 and NS, questioning the inclusion of A2ML1 screening in diagnostic RASopathy testing.

FIG4 mutations leading to parkinsonism and a phenotypical continuum between CMT4J and Yunis Varón syndrome.

BACKGROUND: Charcot-Marie-Tooth disease type 4J (CMT4J) originates from mutations in the FIG4 gene and leads to distal muscle weakness. Two null alleles of FIG4 cause Yunis Varón syndrome with severe central nervous system involvement, cleidocranial dysmorphism, absent thumbs and halluces and early death. OBJECTIVES: To analyse the phenotypic spectrum of FIG4-related disease and explore effects of residual FIG4 protein. METHODS: Phenotyping of five new patients with FIG4-related disease. Western Blot analyses of FIG4 from patient fibroblasts. RESULTS: Next generation sequencing revealed compound heterozygous variants in FIG4 in five patients. All five patients presented with peripheral neuropathy, various degree of dysmorphism and a central nervous system involvement comprising Parkinsonism in 3/5 patients, cerebellar ataxia (1/5), spasticity of lower limbs (1/5), epilepsy (1/5) and/or cognitive deficits (2/5). Onset varied between the first and the seventh decade. There was no residual FIG4 protein detectable in fibroblasts of the four analysed patients. CONCLUSION: This study extends the phenotypic spectrum of FIG4-related disease to Parkinsonism as a feature and demonstrates new phenotypes on a continuum between CMT4J and Yunis Varón syndrome.

HIST1H1E heterozygous protein-truncating variants cause a recognizable syndrome with intellectual disability and distinctive facial gestalt: A study to clarify the HIST1H1E syndrome phenotype in 30 individuals.

Histone Gene Cluster 1 Member E, HIST1H1E, encodes Histone H1.4, is one of a family of epigenetic regulator genes, acts as a linker histone protein, and is responsible for higher order chromatin structure. HIST1H1E syndrome (also known as Rahman syndrome, OMIM #617537) is a recently described intellectual disability (ID) syndrome. Since the initial description of five unrelated individuals with three different heterozygous protein-truncating variants (PTVs) in the HIST1H1E gene in 2017, we have recruited 30 patients, all with HIST1H1E PTVs that result in the same shift in frame and that cluster to a 94-base pair region in the HIST1H1E carboxy terminal domain. The identification of 30 patients with HIST1H1E variants has allowed the clarification of the HIST1H1E syndrome phenotype. Major findings include an ID and a recognizable facial appearance. ID was reported in all patients and is most frequently of moderate severity. The facial gestalt consists of a high frontal hairline and full lower cheeks in early childhood and, in later childhood and adulthood, affected individuals have a strikingly high frontal hairline, frontal bossing, and deep-set eyes. Other associated clinical features include hypothyroidism, abnormal dentition, behavioral issues, cryptorchidism, skeletal anomalies, and cardiac anomalies. Brain magnetic resonance imaging (MRI) is frequently abnormal with a slender corpus callosum a frequent finding.

A further case of familial ring chromosome 20 mosaicism - molecular characterization of the ring and review of the literature.

Ring chromosome 20 syndrome is a rare chromosomal disorder characterized by childhood-onset drug-resistant epilepsy, behavioral problems and variable cognitive impairment. While most cases occur sporadically, parent-to-child transmission of ring 20 mosaicism has only been reported in a few exceptional families. We identified a further family with mother-to-child transmission of ring 20 mosaicism. Detailed characterization of the ring chromosome showed a complete ring with preserved telomere repetitive sequences. SNP genotyping excluded mosaic uniparental disomy and indicated that the chromosome was transmitted without recombination from mother to child. These results corroborate the findings of a previous study and support the hypothesis that inherited mosaicism is due to transmission of an unstable chromosome either prone to ring opening or to ring re-formation.

Loss-of-Function Mutations in YY1AP1 Lead to Grange Syndrome and a Fibromuscular Dysplasia-Like Vascular Disease.

Fibromuscular dysplasia (FMD) is a heterogeneous group of non-atherosclerotic and non-inflammatory arterial diseases that primarily involves the renal and cerebrovascular arteries. Grange syndrome is an autosomal-recessive condition characterized by severe and early-onset vascular disease similar to FMD and variable penetrance of brachydactyly, syndactyly, bone fragility, and learning disabilities. Exome-sequencing analysis of DNA from three affected siblings with Grange syndrome identified compound heterozygous nonsense variants in YY1AP1, and homozygous nonsense or frameshift YY1AP1 variants were subsequently identified in additional unrelated probands with Grange syndrome. YY1AP1 encodes yin yang 1 (YY1)-associated protein 1 and is an activator of the YY1 transcription factor. We determined that YY1AP1 localizes to the nucleus and is a component of the INO80 chromatin remodeling complex, which is responsible for transcriptional regulation, DNA repair, and replication. Molecular studies revealed that loss of YY1AP1 in vascular smooth muscle cells leads to cell cycle arrest with decreased proliferation and increased levels of the cell cycle regulator p21/WAF/CDKN1A and disrupts TGF-ß-driven differentiation of smooth muscle cells. Identification of YY1AP1 mutations as a cause of FMD indicates that this condition can result from underlying genetic variants that significantly alter the phenotype of vascular smooth muscle cells.

Novel deletion in 11p15.5 imprinting center region 1 in a patient with Beckwith-Wiedemann syndrome provides insight into distal enhancer regulation and tumorigenesis.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is an early-onset overgrowth disorder with a high risk for embryonal tumors. It is mainly caused by dysregulation of imprinted genes on chromosome 11p15.5; however, the driving forces in the development of tumors are not fully understood. PROCEDURE: We report on a female patient presenting with macrosomia, macroglossia, organomegaly and extensive bilateral nephroblastomatosis. Adjuvant chemotherapy was initiated; however, the patient developed hepatoblastoma and Wilms tumor at 5 and 12 months of age, respectively. Subsequent radiofrequency ablation of the liver tumor and partial nephrectomy followed by consolidation therapy achieved complete remission. RESULTS: Molecular genetic analysis revealed a maternally derived large deletion of the complete H19-differentially methylated region (H19-DMR; imprinting control region-1 [ICR1]), the whole H19 gene itself as well as large parts of the distal enhancer region within the imprinting cluster-1 (IC1). Extended analysis showed highly elevated insulin-like growth factor 2 (IGF2) expression, possibly explaining at least in part the distinct BWS features and tumor manifestations. CONCLUSIONS: This study of a large maternal deletion encompassing the H19 gene and complete ICR1 is the first to demonstrate transcriptional consequences on IGF2 in addition to methylation effects resulting in severe overgrowth and occurrence of multiple tumors in a BWS patient. Studying this deletion helps to clarify the complex molecular processes involved in BWS and provides further insight into tumorigenesis.

Bisphosphonates in multicentric osteolysis, nodulosis and arthropathy (MONA) spectrum disorder - an alternative therapeutic approach.

Multicentric osteolysis, nodulosis and arthropathy (MONA) spectrum disorder is a rare inherited progressive skeletal disorder caused by mutations in the matrix metalloproteinase 2 (MMP2) gene. Treatment options are limited. Herein we present successful bisphosphonate therapy in three affected patients. Patients were treated with bisphosphonates (either pamidronate or zoledronate) for different time periods. The following outcome variables were assessed: skeletal pain, range of motion, bone densitometry, internal medical problems as well as neurocognitive function. Skeletal pain was dramatically reduced in all patients soon after initiation of therapy and bone mineral density increased. Range of motion did not significantly improve. One patient is still able to walk with aids at the age of 14 years. Neurocognitive development was normal in all patients. Bisphosphonate therapy was effective especially in controlling skeletal pain in MONA spectrum disorder. Early initiation of treatment seems to be particularly important in order to achieve the best possible outcome.

Clinical, cytogenetic, and molecular findings in a patient with a 46,XX,del(18)(q22)/46,XX,idic(18)(q22) karyotype.

Pseudoisodicentric or asymmetrical dicentric chromosomes 18 are rare findings in clinical cytogenetics. So far, only 8 patients with breakpoints in 18q have been reported and in none of them breakpoints were narrowed down to the molecular level. Here, we describe a 17 months old girl with a perimembranous ventricular septal defect, cleft palate, and minor dysmorphism including hypertelorism, flat nose, frontal bossing and low set ears as well as mosaicism for a cell line with a pseudoisodicentric chromosome 18q and a second cell line with a terminal deletion of 11 Mb in 18q22.2→qter. SNP-array investigation revealed a symmetric breakpoint in 18q22.2 and most likely postzygotic formation from the maternal chromosome 18. Clinical findings in all patients reported so far as well as in the patient presented here were in part overlapping with the clinical phenotypes of trisomy 18 and partial monosomy 18q.

Iron Supplementation Associated With Loss of Phenotype in Autosomal Dominant Hypophosphatemic Rickets.

CONTEXT: Autosomal dominant hypophosphatemic rickets (ADHR) is the only hereditary disorder of renal phosphate wasting in which patients may regain the ability to conserve phosphate. Low iron status plays a role in the pathophysiology of ADHR. OBJECTIVE: This study reports of a girl with ADHR, iron deficiency, and a paternal history of hypophosphatemic rickets that resolved without treatment. The girl's biochemical phenotype resolved with iron supplementation. SUBJECTS: A 26-month-old girl presented with typical features of hypophosphatemic rickets, short stature (79 cm; -2.82 SDS), and iron deficiency. Treatment with elemental phosphorus and calcitriol improved her biochemical profile and resolved the rickets. The girl's father had presented with rickets at age 11 months but never received medication. His final height was reduced (154.3 cm; -3.51 SDS), he had undergone corrective leg surgery and had an adult normal phosphate, fibroblast growth factor 23, and iron status. Father and daughter were found to have a heterozygous mutation in exon 3 of the FGF23 gene (c.536G>A, p.Arg179Gln), confirming ADHR. INTERVENTION: Withdrawal of rickets medication was attempted off and on iron supplementation. RESULTS: Withdrawal of rickets medication in the girl was unsuccessful in the presence of low-normal serum iron levels at age 5.6 years but was later successful in the presence of high-normal serum iron levels following high-dose iron supplementation. CONCLUSIONS: We report an association between iron supplementation and a complete loss of biochemical ADHR phenotype, allowing withdrawal of rickets medication. Experience from this case suggests that reduction and withdrawal of rickets medication should be attempted only after iron status has been optimized.

Mosaicism and uniparental disomy in prenatal diagnosis.

Chromosomal mosaicism is the presence of numerous cell lines with different chromosomal complements in the same individual. Uniparental disomy (UPD) is the inheritance of two homologous chromosomes from the same parent. These genetic anomalies arise from errors in meiosis and/or mitosis and can occur independently or in combination. Due to the formation mechanisms of UPD, low-level or undetected mosaicisms are assumed for a significant number of UPD cases. The pre- and postnatal clinical consequences of mosaicism for chromosomal aberrations and/or UPD depend on the gene content of the involved chromosome. In prenatal evaluation of chromosomal mosaicism and UPD, genetic counseling should be offered before any laboratory testing.

Elevated heart rate triggers action potential alternans and sudden death. translational study of a homozygous KCNH2 mutation.

BACKGROUND: Long QT syndrome (LQTS) leads to arrhythmic events and increased risk for sudden cardiac death (SCD). Homozygous KCNH2 mutations underlying LQTS-2 have previously been termed "human HERG knockout" and typically express severe phenotypes. We studied genotype-phenotype correlations of an LQTS type 2 mutation identified in the homozygous index patient from a consanguineous Turkish family after his brother died suddenly during febrile illness. METHODS AND RESULTS: Clinical work-up, DNA sequencing, mutagenesis, cell culture, patch-clamp, in silico mathematical modelling, protein biochemistry, confocal microscopy were performed. Genetic analysis revealed a homozygous C-terminal KCNH2 mutation (p.R835Q) in the index patient (QTc ∼506 ms with notched T waves). Parents were I° cousins - both heterozygous for the mutation and clinically unremarkable (QTc ∼447 ms, father and ∼396 ms, mother). Heterologous expression of KCNH2-R835Q showed mildly reduced current amplitudes. Biophysical properties of ionic currents were also only nominally changed with slight acceleration of deactivation and more negative V50 in R835Q-currents. Protein biochemistry and confocal microscopy revealed similar expression patterns and trafficking of WT and R835Q, even at elevated temperature. In silico analysis demonstrated mildly prolonged ventricular action potential duration (APD) compared to WT at a cycle length of 1000 ms. At a cycle length of 350 ms M-cell APD remained stable in WT, but displayed APD alternans in R835Q. CONCLUSION: Kv11.1 channels affected by the C-terminal R835Q mutation display mildly modified biophysical properties, but leads to M-cell APD alternans with elevated heart rate and could precipitate SCD under specific clinical circumstances associated with high heart rates.

Don't miss patients with atypical FMR1 mutations: dysmorphism and clinical features in a boy with a partially methylated FMR1 full mutation.

UNLABELLED: Fragile X syndrome characterized by intellectual disability (ID), facial dysmorphism, and postpubertal macroorchidism is the most common monogenic cause of ID. It is typically induced by an expansion of a CGG repeat in the fragile X mental retardation 1 (FMR1) gene on Xq27 to more than 200 repeats. Only rarely patients have atypical mutations in the FMR1 gene such as point mutations, deletions, or unmethylated/partially methylated full mutations. Most of these patients show a minor phenotype or even appear clinically healthy. Here, we report the dysmorphism and clinical features of a 17-year-old boy with a partially methylated full mutation of approximately 250 repeats. Diagnosis was made subsequently to the evaluation of a FMR1 premutation as the cause for maternal premature ovarian failure. Dysmorphic evaluation revealed no strikingly long face, no prominent forehead/frontal bossing, no prominent mandible, no macroorchidism, and a head circumference in the lower normal range. Acquisition of a driving license for mopeds and unaccompanied rides by public transport in his home province indicate rather mild ID (IQ = 58). CONCLUSION: This adolescent demonstrates that apart from only minor ID, patients with a partially methylated FMR1 full mutation present less to absent pathognomonic facial dysmorphism, thus emphasizing the impact of family history for a straightforward clinical diagnosis.

Chromosomal rearrangements in patients with clinical features of Silver-Russell syndrome.

Silver-Russell syndrome (SRS) is characterized by pre- and postnatal growth retardation, relative macrocephaly, asymmetry, and a triangular facial gestalt. In 5-10% of the patients the phenotype is caused by maternal UPD 7, and 38-64% of the patients present with hypomethylation at the imprinting center region 1 (ICR1) on 11p15.5. The etiology of the remaining cases is so far not known and various (sub-)microscopic chromosome aberrations with a phenotype resembling SRS have been published, especially duplication 11p15 (n = 15), deletion 12q14 (n = 19), ring chromosome 15, deletion 15qter, and various other mostly unique chromosomal aberrations (n = 30). In this study the phenotypes of these chromosomal aberrations were revisited and compared with the phenotypes of maternal UPD 7 and hypomethylation at ICR1 on 11p15.5. In some patients with a unique chromosomal aberration even the hallmarks of SRS were missing. Patients with duplication 11p15 show a more variable occipitofrontal head circumference at birth, a higher frequency of intellectual disability, and additional anomalies not reported in SRS. Deletion 12q14 is characterized by less severe pre- and postnatal growth retardation and less impressive relative macrocephaly. Patients with ring chromosome 15 and deletion 15qter have no relative macrocephaly (mostly even microcephaly) and more severe intellectual disability. Finally, deletion 15qter lacks the triangular facial gestalt. In summary, as SRS seems not an adequate diagnosis in many of these patients, diagnosis should focus on the chromosomal aberration than on SRS.

Identification and functional characterization of three NoLS (nucleolar localisation signals) mutations of the CDC73 gene.

Hyperparathyroidism Jaw-Tumour Syndrome (HPT-JT) is characterized by primary hyperparathyroidism (PHPT), maxillary/mandible ossifying fibromas and by parathyroid carcinoma in 15% of cases. Inactivating mutations of the tumour suppressor CDC73/HRPT2 gene have been found in HPT-JT patients and also as genetic determinants of sporadic parathyroid carcinoma/atypical adenomas and, rarely, typical adenomas, in familial PHPT. Here we report the genetic and molecular analysis of the CDC73/HRPT2 gene in three patients affected by PHPT due to atypical and typical parathyroid adenomas, in one case belonging to familial PHPT. Flag-tagged WT and mutant CDC73/HRPT2 proteins were transiently transfected in HEK293 cells and functional assays were performed in order to investigate the effect of the variants on the whole protein expression, nuclear localization and cell overgrowth induction. We identified four CDC73/HRPT2 gene mutations, three germline (c.679_680delAG, p.Val85_Val86del and p.Glu81_Pro84del), one somatic (p.Arg77Pro). In three cases the mutation was located within the Nucleolar Localisation Signals (NoLS). The three NoLS variants led to instability either of the corresponding mutated protein or mRNA or both. When transfected in HEK293 cells, NoLS mutated proteins mislocalized with a predeliction for cytoplasmic or nucleo-cytoplasmic localization and, finally, they resulted in overgrowth, consistent with a dominant negative interfering effect in the presence of the endogenous protein.

Medullary nephrocalcinosis in an adult patient with idiopathic infantile hypercalcaemia and a novel CYP24A1 mutation.

Idiopathic infantile hypercalcaemia (IIH) is an autosomal recessively inherited disease, presented in the first year of life with hypercalcaemia, precipitated by normal amounts of vitamin D supplementation. Recently loss-of-function mutations in the CYP24A1 gene, which encodes the vitamin D-metabolizing enzyme 24-hydroxylase, have been found in these patients. We describe a young man homozygous for a novel missense mutation (c.628T>C) of the CYP24A1 gene. He had suffered from severe hypercalcaemia in early childhood. At age 29 he presented with medullary nephrocalcinosis, chronic kidney disease (CKD) stage 2, microalbuminuria, mild hypertension and nephrogenic diabetes insipidus. He had mild hypercalcaemia and moderate hypercalciuria. As a novel finding, fibroblast growth factor 23 (FGF23) was elevated.