A novel large intragenic DPYD deletion causing dihydropyrimidine dehydrogenase deficiency: a case report.

BACKGROUND: Dihydropyrimidine dehydrogenase (DPD), is the initial and rate-limiting enzyme in the catabolic pathway of pyrimidines. Deleterious variants in the DPYD gene cause DPD deficiency, a rare autosomal recessive disorder. The clinical spectrum of affected individuals is wide ranging from asymptomatic to severely affected patients presenting with intellectual disability, motor retardation, developmental delay and seizures. DPD is also important as the main enzyme in the catabolism of 5-fluorouracil (5-FU) which is extensively used as a chemotherapeutic agent. Even in the absence of clinical symptoms, individuals with either complete or partial DPD deficiency face a high risk of severe and even fatal fluoropyrimidine-associated toxicity. The identification of causative genetic variants in DPYD is therefore gaining increasing attention due to their potential use as predictive markers of fluoropyrimidine toxicity. METHODS: A male infant patient displaying biochemical features of DPD deficiency was investigated by clinical exome sequencing. Bioinformatics tools were used for data analysis and results were confirmed by MLPA and Sanger sequencing. RESULTS: A novel intragenic deletion of 71.2 kb in the DPYD gene was identified in homozygosity. The deletion, DPYD(NM_000110.4):c.850 + 23455_1128 + 8811del, eliminates exons 9 and 10 and may have resulted from a non-homologous end-joining event, as suggested by in silico analysis. CONCLUSIONS: The study expands the spectrum of DPYD variants associated with DPD deficiency. Furthermore, it raises the concern that patients at risk for fluoropyrimidine toxicity due to DPYD deletions could be missed during pre-treatment genetic testing for the currently recommended single nucleotide polymorphisms.

Sodium Channel Gene Variants in Fetuses with Abnormal Sonographic Findings: Expanding the Prenatal Phenotypic Spectrum of Sodium Channelopathies.

Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of action potentials in the brain and muscle. Pathogenic variants in genes encoding VGSCs have been associated with severe disorders including epileptic encephalopathies and congenital myopathies. In this study, we identified pathogenic variants in genes encoding the α subunit of VGSCs in the fetuses of two unrelated families with the use of trio-based whole exome sequencing, as part of a larger cohort study. Sanger sequencing was performed for variant confirmation as well as parental phasing. The fetus of the first family carried a known de novo heterozygous missense variant in the SCN2A gene (NM_001040143.2:c.751G>A p.(Val251Ile)) and presented intrauterine growth retardation, hand clenching and ventriculomegaly. Neonatally, the proband also exhibited refractory epilepsy, spasms and MRI abnormalities. The fetus of the second family was a compound heterozygote for two parentally inherited novel missense variants in the SCN4A gene (NM_000334.4:c.4340T>C, p.(Phe1447Ser), NM_000334.4:c.3798G>C, p.(Glu1266Asp)) and presented a severe prenatal phenotype including talipes, fetal hypokinesia, hypoplastic lungs, polyhydramnios, ear abnormalities and others. Both probands died soon after birth. In a subsequent pregnancy of the latter family, the fetus was also a compound heterozygote for the same parentally inherited variants. This pregnancy was terminated due to multiple ultrasound abnormalities similar to the first pregnancy. Our results suggest a potentially crucial role of the VGSC gene family in fetal development and early lethality.

CHD2 pathogenic nonsense variant in a three-generation family with variable phenotype and a paracentric inversion 16: Case report.

Chromosomal inversions are usually balanced structural chromosomal rearrangements that do not have an impact on the clinical phenotype of a carrier. The main clinical consequence of inversions is the risk for unbalanced gametes and offspring with severe phenotypes. Rarely though, inversions are associated with a phenotype, mainly due to submicroscopic Copy Number Variants (CNVs) or disruption at the breakpoints of a functionally important gene and/or genomic elements. In this study, a paracentric inversion of chromosome 16 [inv(16)(q22.3q24.1)] was identified in a three-generation family with discordant phenotypes with/without epilepsy and/or intellectual impairment, as well as with an unaffected carrier. This finding was confirmed by fluorescence in situ hybridization (FISH). Genetic investigation, initially with chromosomal microarray (CMA), did not reveal any copy number variants. Finally, Clinical Exome Sequencing (CES), detected the presence of a pathogenic nonsense variant (rs797044912) in the Chromodomain Helicase DNA-binding protein 2 (CHD2) gene [NM_001271.4:c.5035C>T p.(Arg1679Ter)]. CHD2 pathogenic variants have been associated with Developmental and Epileptic Encephalopathy-94 (DEE-94), a rare yet severe condition, characterized by developmental delay, seizures with an early onset, intellectual impairment, autism spectrum disorder, and sometimes behavioral issues. Family testing showed that the variant segregated with phenotypic heterogeneity in the affected individuals and appears to be causative. To the best of our knowledge, this is the first CHD2 pathogenic variant segregating in a three-generation family and the fourth familial case reported. These results further support our previous findings that familial, balanced rearrangements with discordant phenotypes in the same family are, in the vast majority, coincidental.

GAA variants associated with reduced enzymatic activity but lack of Pompe-related symptoms, incidentally identified by exome sequencing.

Pompe disease is a rare metabolic myopathy caused by pathogenic variants affecting the activity of the lysosomal glycogen-degrading enzyme acid alpha-glucosidase (GAA). Impaired GAA function results in the accumulation of undegraded glycogen within lysosomes in multiple tissues but predominantly affects the skeletal, smooth and cardiac muscle. The degree of residual enzymatic activity appears to roughly correlate with the age of onset and the severity of the clinical symptoms. Here, we report four siblings in which the GAA variants NM_000152.5:c.2237G > C p.(Trp746Ser) and NM_000152.5:c.266G > A p.(Arg89His) were identified as an incidental finding of clinical exome sequencing. These variants are listed in the ClinVar and the Pompe disease GAA variant databases but are reported here for the first time in compound heterozygosity. All four siblings displayed normal urine tetrasaccharide levels and no clinical manifestations related to Pompe disease. Nevertheless, GAA enzymatic activity was within the range for late onset Pompe patients. Our report shows an association between a novel genotype and attenuated GAA enzymatic activity. The clinical significance can only be established by the regular monitoring of these individuals. The study highlights the major challenges for clinical care arising from incidental findings of next generation sequencing.

Hereditary multiple exostoses caused by a chromosomal inversion removing part of EXT1 gene.

BACKGROUND: Hereditary multiple exostoses (HME) is an autosomal dominant skeletal disorder characterized by the development of multiple, circumscript and usually symmetric bony protuberances called osteochondromas. Most HME are caused by EXT1 and EXT2 loss of function mutations. Most pathogenic mutations are nonsense followed by missense mutations and deletions. CASE PRESENTATION: Here we report on a patient with a rare and complex genotype resulting in a typical HME phenotype. Initial point mutation screening in EXT1 and EXT2 genes by Sanger sequencing did not reveal any pathogenic variants. The patient along with the healthy parents was subsequently referred for karyotype and array-Comparative Genomic Hybridization (CGH) analyses. Chromosomal analysis revealed two independent de novo apparently balanced rearrangements: a balanced translocation between the long arms of chromosomes 2 and 3 at breakpoints 2q22 and 3q13.2 and a pericentric inversion with breakpoints at 8p23.1q24.1. Both breakpoints were confirmed by Fluorescence In Situ Hybridization (FISH). Subsequently, array-CGH revealed a novel heterozygous deletion within the EXT1 gene at one of the inversion breakpoints, rendering the inversion unbalanced. The mode of inheritance, as well as the size of the deletion were further investigated by Quantitative Real-time PCR (qPCR), defining the deletion as de novo and of 3.1 kb in size, removing exon 10 of EXT1. The inversion in combination with the 8p23.1 deletion most likely abolishes the transcription of EXT1 downstream of exon 10 hence resulting in a truncated protein. CONCLUSIONS: The identification of a rare and novel genetic cause of HME, highlights the importance of additional comprehensive investigation of patients with typical clinical manifestations, even when EXT1 and EXT2 mutation analysis is negative.

Exploring the Genetic Causality of Discordant Phenotypes in Familial Apparently Balanced Translocation Cases Using Whole Exome Sequencing.

Familial apparently balanced translocations (ABTs) are usually not associated with a phenotype; however, rarely, ABTs segregate with discordant phenotypes in family members carrying identical rearrangements. The current study was a follow-up investigation of four familial ABTs, where whole exome sequencing (WES) was implemented as a diagnostic tool to identify the underlying genetic aetiology of the patients' phenotypes. Data were analysed using an in-house bioinformatics pipeline alongside VarSome Clinical. WES findings were validated with Sanger sequencing, while the impact of splicing and missense variants was assessed by reverse-transcription PCR and in silico tools, respectively. Novel candidate variants were identified in three families. In family 1, it was shown that the de novo pathogenic STXBP1 variant (NM_003165.6:c.1110+2T>G) affected splicing and segregated with the patient's phenotype. In family 2, a likely pathogenic TUBA1A variant (NM_006009.4:c.875C>T, NP_006000.2:p.(Thr292Ile)) could explain the patient's symptoms. In family 3, an SCN1A variant of uncertain significance (NM_006920.6:c.5060A>G, NP_008851.3:p.(Glu1687Gly)) required additional evidence to sufficiently support causality. This first report of WES application in familial ABT carriers with discordant phenotypes supported our previous findings describing such rearrangements as coincidental. Thus, WES can be recommended as a complementary test to find the monogenic cause of aberrant phenotypes in familial ABT carriers.

Unravelling the genetic causes of multiple malformation syndromes: A whole exome sequencing study of the Cypriot population.

Multiple malformation syndromes (MMS) belong to a group of genetic disorders characterised by neurodevelopmental anomalies and congenital malformations. Here we explore for the first time the genetic aetiology of MMS using whole-exome sequencing (WES) in undiagnosed patients from the Greek-Cypriot population after prior extensive diagnostics workup including karyotype and array-CGH. A total of 100 individuals (37 affected), from 32 families were recruited and family-based WES was applied to detect causative single-nucleotide variants (SNVs) and indels. A genetic diagnosis was reported for 16 MMS patients (43.2%), with 10/17 (58.8%) of the findings being novel. All autosomal dominant findings occurred de novo. Functional studies were also performed to elucidate the molecular mechanism relevant to the abnormal phenotypes, in cases where the clinical significance of the findings was unclear. The 17 variants identified in our cohort were located in 14 genes (PCNT, UBE3A, KAT6A, SPR, POMGNT1, PIEZO2, PXDN, KDM6A, PHIP, HECW2, TFAP2A, CNOT3, AGTPBP1 and GAMT). This study has highlighted the efficacy of WES through the high detection rate (43.2%) achieved for a challenging category of undiagnosed patients with MMS compared to other conventional diagnostic testing methods (10-20% for array-CGH and ~3% for G-banding karyotype analysis). As a result, family-based WES could potentially be considered as a first-tier cost effective diagnostic test for patients with MMS that facilitates better patient management, prognosis and offer accurate recurrence risks to the families.

An unusual combination of an atypical maternally inherited novel 0.3 Mb deletion in Williams-Beuren region and a de novo 22q11.21 microduplication in an infant with supravalvular aortic stenosis.

Williams-Beuren syndrome (WBS) is a rare neurodevelopmental disorder characterized by supravalvular aortic stenosis (SVAS), intellectual disability, overfriendliness and dysmorphic features. It is typically caused by 1.5-1.8 Mb deletions on 7q11.23. The 22q11.21 microduplication syndrome has a variable phenotype and is frequently associated with congenital heart disease. Here we present a unique patient, carrying aberrations within both of the above syndrome regions, referred for possible diagnosis of WBS because of SVAS. The patient was a boy who died suddenly 47 days after birth, possibly due to cardiac complications. Genetic testing was carried out, including array Comparative Genomic Hybridization (aCGH), Fluorescence In situ Hybridization (FISH) and Multiplex Ligation-Dependent Probe Amplification (MLPA) showing that the proband was heterozygous for a novel and atypical 0.3 Mb deletion in WBS region (7q11.23) encompassing the ELN gene. In addition, he was found heterozygous for a 22q11.21 microduplication. Parental studies revealed that the 7q11.23 deletion was inherited from the mother who also exhibited a cardiovascular phenotype, however very mild. The same maternally inherited deletion was detected in one of the proband's siblings, born two years later with a less severe SVAS. The 22q11.2 microduplication was de novo in origin. Detection and investigation of atypical deletions within known syndrome regions are crucial for better genotype-phenotype correlations and more accurate characterization of critical regions. The combined effect of two different genetic defects - one in a known syndrome region and one with variable clinical significance, is valuable for revealing gene interactions and enabling more accurate predictions, especially in prenatal diagnosis.

Novel pericentric inversion inv(9)(p23q22.3) in unrelated individuals with fertility problems in the Southeast European population.

Pericentric inversions are among the known polymorphisms detected in the general population at a frequency of 1-2%. Despite their generally benign nature, pericentric inversions affect the reproductive potential of carriers by increasing the risk for unbalanced live-born offspring, miscarriages, or other fertility problems. Here we present a novel large pericentric inversion of chromosome 9, inv(9)(p23q22.3), detected in 30 heterozygote carriers, 24 from seven apparently unrelated families and 6 isolated patients, where the probands were mainly referred for fertility and prenatal problems. The inversion carries a significant risk for recombinant abnormal chromosomes, as in two families one supernumerary rec(9)dup(9p) and one rec(9)dup(9q) were identified, leading to neonatal death and miscarriage, respectively. The inversion carriers were identified by three different laboratories in Greece, Cyprus and Germany respectively, however all carriers have Southeast European origin. The inversion appears to be more frequent in the Greek population, as the majority of the carriers were identified in Greece. We were able to determine that the inversion is identical in all individuals included in the study by applying a combination of several methodologies, such as karyotype, fluorescence in situ hybridization (FISH), chromosomal microarrays (CMA) and haplotype analysis. In addition, haplotype analysis supports that the present inversion is identical by descent (IBD) inherited from a single common ancestor. Our results are, therefore, highly indicative of a founder effect of this inversion, presumably reflecting an event that was present in a small number of individuals that migrated to the current Southeast Europe/Northern Greece from a larger population.

De novo mosaic MECP2 mutation in a female with Rett syndrome.

We describe a female with Rett syndrome carrying a rare de novo mosaic nonsense mutation on MECP2 gene, with random X-chromosome inactivation. Rett syndrome severity in females depends on mosaicism level and tissue specificity, X-chromosome inactivation, epigenetics and environment. Rett syndrome should be considered in both males and females.

Position effect, cryptic complexity, and direct gene disruption as disease mechanisms in de novo apparently balanced translocation cases.

The majority of apparently balanced translocation (ABT) carriers are phenotypically normal. However, several mechanisms were proposed to underlie phenotypes in affected ABT cases. In the current study, whole-genome mate-pair sequencing (WG-MPS) followed by Sanger sequencing was applied to further characterize de novo ABTs in three affected individuals. WG-MPS precisely mapped all ABT breakpoints and revealed three possible underlying molecular mechanisms. Firstly, in a t(X;1) carrier with hearing loss, a highly skewed X-inactivation pattern was observed and the der(X) breakpoint mapped ~87kb upstream an X-linked deafness gene namely POU3F4, thus suggesting an underlying long-range position effect mechanism. Secondly, cryptic complexity and a chromothripsis rearrangement was identified in a t(6;7;8;12) carrier with intellectual disability. Two translocations and a heterozygous deletion disrupted SOX5; a dominant nervous system development gene previously reported in similar patients. Finally, a direct gene disruption mechanism was proposed in a t(4;9) carrier with dysmorphic facial features and speech delay. In this case, the der(9) breakpoint directly disrupted NFIB, a gene involved in lung maturation and development of the pons with important functions in main speech processes. To conclude, in contrast to familial ABT cases with identical rearrangements and discordant phenotypes, where translocations are considered coincidental, translocations seem to be associated with phenotype presentation in affected de novo ABT cases. In addition, this study highlights the importance of investigating both coding and non-coding regions to decipher the underlying pathogenic mechanisms in these patients, and supports the potential introduction of low coverage WG-MPS in the clinical investigation of de novo ABTs.

Molecular analysis of Cypriot families with aniridia reveals a novel PAX6 mutation.

The present study investigated the clinical and mutational spectrum of aniridia in a cohort of 17 affected individuals from six families from Cyprus. Each proband was initially evaluated for copy number variants at the PAX6 locus and subsequently underwent PAX6 mutation screening. Sequence analysis of FOXC1 and PITX2 was performed in patients who did not carry a PAX6 mutation. The most common clinical features in the group of aniridia patients associated with aniridia were nystagmus, cataracts and glaucoma. PAX6 pathogenic mutations were identified in five out of six families (a diagnostic yield of 84%). Previously reported pathogenic mutations in PAX6 were identified in four families, which comprise p.R203*, p.R240* and p.R317*. In addition, a novel pathogenic variant (p.E220Gfs*23) was identified in a single family. No pathogenic mutations were detected in PAX6, FOXC1 or PITX2 in the only patient with a sporadic form of aniridia­like phenotype, confirming the genetic heterogeneity associated with this disease. To the best of our knowledge this is the first report on the mutational spectrum of PAX6 in aniridia patients of Cypriot ancestry. Mutational screening of PAX6 serves a crucial role in distinguishing isolated from syndromic forms of aniridia, and it may therefore eliminate the need for renal ultrasound scan surveillance, delineate the phenotype and improve genetic counseling.

Aniridia due to a novel microdeletion affecting PAX6 regulatory enhancers: case report and review of the literature.

Aniridia is a rare congenital ocular malformation that follows an autosomal dominant mode of inheritance. Most patients carry pathogenic point mutations in the paired box 6 gene (PAX6), but some carry deletions involving the 11p13 region, encompassing partly or completely PAX6 or the region downstream. We identified a novel deletion, ~564 kb in size located about 46.5 kb downstream of PAX6 in a family with bilateral aniridia and foveal hypoplasia using array-CGH and multiplex ligation-dependent probe amplification. We also reviewall of the reported deletions downstream of PAX6 in patients with aniridia and/or other congenital malformations and define the overlapping region that leads to aniridia when deleted.

Identification of a novel 15.5 kb SHOX deletion associated with marked intrafamilial phenotypic variability and analysis of its molecular origin.

Haploinsufficiency of the short stature homeobox contaning SHOX gene has been shown to result in a spectrum of phenotypes ranging from Leri-Weill dyschondrosteosis (LWD) at the more severe end to SHOX-related short stature at the milder end of the spectrum. Most alterations are whole gene deletions, point mutations within the coding region, or microdeletions in its flanking sequences. Here, we present the clinical and molecular data as well as the potential molecular mechanism underlying a novel microdeletion, causing a variable SHOX-related haploinsufficiency disorder in a three-generation family. The phenotype resembles that of LWD in females, in males, however, the phenotypic expression is milder. The 15523-bp SHOX intragenic deletion, encompassing exons 3-6, was initially detected by array-CGH, followed by MLPA analysis. Sequencing of the breakpoints indicated an Alu recombination-mediated deletion (ARMD) as the potential causative mechanism.

Deletion of SNURF/SNRPN U1B and U1B* upstream exons in a child with developmental delay and excessive weight.

Prader-Willi syndrome is a rare syndrome characterized by hypotonia, developmental delay and excessive appetite. This syndrome is caused by the loss of function of paternally-expressed genes located in an imprinting centre in 15q11-q13. Here, we report the case of a patient who was referred to us with Prader-Willi syndrome-like symptoms including obesity and developmental delay. Examination of this patient revealed that he was a carrier of a paternally inherited deletion that affected the U1B and U1B* upstream exons of the SNURF-SNRNP gene within the 15q11-q13 imprinted region. Mutations localized within this genomic region have not been previously reported in Prader-Willi syndrome patients. It is possible that disruption of upstream exons of SNURF-SNRNP could contribute to Prader-Willi phenotype by disrupting brain-specific alternative transcripts, although, case reports from further patients with a comparable phenotype are required.

Novel GLA Deletion in a Cypriot Female Presenting with Cornea Verticillata.

Fabry disease is an X-linked lysosomal storage disorder resulting from a deficiency of the hydrolytic enzyme α-galactosidase A (α-Gal-A). It is characterized by progressive lysosomal accumulation of globotriaosylceramide (Gb3) and multisystem pathology, affecting the skin, nervous and cerebrovascular systems, kidneys, and heart. Heterozygous females typically exhibit milder symptoms and a later age of onset than males. Rarely, they may be relatively asymptomatic throughout a normal life span or may have symptoms as severe as those observed in males with the classic phenotype. We report on a 17-year-old female in whom cornea verticillata was found during a routine ophthalmological examination but with no other clinical symptoms. Leucocyte α-galactosidase activity was within the overlap range between Fabry heterozygotes and normal controls. Sanger sequencing of the GLA gene failed to reveal any pathogenic variants. Multiplex Ligation-dependent Probe Amplification (MLPA) analysis revealed a deletion of exon 7. Using a long-range PCR walking approach, we managed to identify the deletion breakpoints. The deletion spans 1182 bp, with its 5' end located within exon 6 of the GLA gene and its 3' end located 612 bp downstream of exon 7. This finding represents a novel deletion identified in the first reported Cypriot female carrier of Fabry disease.

A novel HCFC1 variant in male siblings with intellectual disability and microcephaly in the absence of cobalamin disorder.

Approximately 10-15% of intellectual disability (ID) cases are caused by genetic aberrations affecting chromosome X, a condition termed X-linked ID (XLID). Examination by whole-exome sequencing of two male siblings with microcephaly and suspected XLID with an unknown genetic basis revealed that they were both hemizygous for a predicted pathogenic variant (p.Ala897Val) causing a non-synonymous substitution of an evolutionary conserved amino acid within the host cell factor C1 (HCFC1) gene. Subsequent analysis determined that this was a rare variant not identified in 100 control individuals or in online databases of control individuals. Recent studies have reported mutations affecting HCFC1 in patients with ID and dysmorphic features that are associated with defective cobalamin metabolism. Biochemical investigations did not find evidence of an association between the variant identified in the present study and cobalamin metabolic disorder. This study offers further support for mutations of HCFC1 being implicated in XLID and microcephaly, but that these are not necessarily associated with cobalamin disorder.

Identification of an AVP-NPII mutation within the AVP moiety in a family with neurohypophyseal diabetes insipidus: review of the literature.

Familial neurohypophyseal diabetes insipidus (FNDI) is a disorder characterized by excess excretion of diluted urine (polyuria) and increased uptake of fluids (polydipsia). The disorder is caused by mutations affecting the AVP-NPII gene, resulting in absent or deficient secretion of the antidiuretic hormone arginine vasopressin (AVP) by the neurohypophysis. In this study we examined a three-generation Cypriot kindred suspected to have FNDI. Direct sequencing analysis of AVP-NPII identified a missense mutation (NM_000490.4:c.61T>C; p.Tyr21His; rs121964893) within the AVP moiety on exon 1 of the gene in all affected family members. So far, only three studies have reported mutations within the AVP moiety of AVP-NPIIas being associated with FNDI, with the vast majority of identified FNDI mutations being located within the signalling peptide or the neurophysis II (NPII) moiety of the gene. The mutation within the AVP moiety identified here had been reported previously in a Turkish kindred with FNDI. Consequently, the findings of this study confirm the causal role of mutations within the AVP moiety in FNDI. Herein we review reported mutations within the AVP moiety of AVP-NPII and their contribution to FNDI.