Long-read genome sequencing resolves a complex 13q structural variant associated with syndromic anophthalmia.

Microphthalmia, anophthalmia, and coloboma (MAC) are a heterogeneous spectrum of anomalous eye development and degeneration with genetic and environmental etiologies. Structural and copy number variants of chromosome 13 have been implicated in MAC; however, the specific loci involved in disease pathogenesis have not been well-defined. Herein we report a newborn with syndromic degenerative anophthalmia and a complex de novo rearrangement of chromosome 13q. Long-read genome sequencing improved the resolution and clinical interpretation of a duplication-triplication/inversion-duplication (DUP-TRP/INV-DUP) and terminal deletion. Sequence features at the breakpoint junctions suggested microhomology-mediated break-induced replication (MMBIR) of the maternal chromosome as the origin. Comparing this rearrangement to previously reported copy number alterations in 13q, we refine a putative dosage-sensitive critical region for MAC that might provide new insights into its molecular etiology.

An approach to rapid characterization of DMD copy number variants for prenatal risk assessment.

Prenatal detection of structural variants of uncertain significance, including copy number variants (CNV), challenges genetic counseling, and creates ambiguity for expectant parents. In Duchenne muscular dystrophy, variant classification and phenotypic severity of CNVs are currently assessed by familial segregation, prediction of the effect on the reading frame, and precedent data. Delineation of pathogenicity by familial segregation is limited by time and suitable family members, whereas analytical tools can rapidly delineate potential consequences of variants. We identified a duplication of uncertain significance encompassing a portion of the dystrophin gene (DMD) in an unaffected mother and her male fetus. Using long-read whole genome sequencing and alignment of short reads, we rapidly defined the precise breakpoints of this variant in DMD and could provide timely counseling. The benign nature of the variant was substantiated, more slowly, by familial segregation to a healthy maternal uncle. We find long-read whole genome sequencing of clinical utility in a prenatal setting for accurate and rapid characterization of structural variants, specifically a duplication involving DMD.

Pathogenetics of alveolar capillary dysplasia with misalignment of pulmonary veins.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal lung developmental disorder caused by heterozygous point mutations or genomic deletion copy-number variants (CNVs) of FOXF1 or its upstream enhancer involving fetal lung-expressed long noncoding RNA genes LINC01081 and LINC01082. Using custom-designed array comparative genomic hybridization, Sanger sequencing, whole exome sequencing (WES), and bioinformatic analyses, we studied 22 new unrelated families (20 postnatal and two prenatal) with clinically diagnosed ACDMPV. We describe novel deletion CNVs at the FOXF1 locus in 13 unrelated ACDMPV patients. Together with the previously reported cases, all 31 genomic deletions in 16q24.1, pathogenic for ACDMPV, for which parental origin was determined, arose de novo with 30 of them occurring on the maternally inherited chromosome 16, strongly implicating genomic imprinting of the FOXF1 locus in human lungs. Surprisingly, we have also identified four ACDMPV families with the pathogenic variants in the FOXF1 locus that arose on paternal chromosome 16. Interestingly, a combination of the severe cardiac defects, including hypoplastic left heart, and single umbilical artery were observed only in children with deletion CNVs involving FOXF1 and its upstream enhancer. Our data demonstrate that genomic imprinting at 16q24.1 plays an important role in variable ACDMPV manifestation likely through long-range regulation of FOXF1 expression, and may be also responsible for key phenotypic features of maternal uniparental disomy 16. Moreover, in one family, WES revealed a de novo missense variant in ESRP1, potentially implicating FGF signaling in the etiology of ACDMPV.

Brain MRI abnormalities and spectrum of neurological and clinical findings in three patients with proximal 16p11.2 microduplication.

The phenotype of recurrent ∼600 kb microdeletion and microduplication on proximal 16p11.2 is characterized by a spectrum of neurodevelopmental impairments including developmental delay and intellectual disability, epilepsy, autism and psychiatric disorders which are all subject to incomplete penetrance and variable expressivity. A variety of brain MRI abnormalities were reported in patients with 16p11.2 rearrangements, but no systematic correlation has been studied among patients with similar brain anomalies, their neurodevelopmental and clinical phenotypes. We present three patients with the proximal 16p11.2 microduplication exhibiting significant developmental delay, anxiety disorder and other variable clinical features. Our patients have abnormal brain MRI findings of cerebral T2 hyperintense foci (3/3) and ventriculomegaly (2/3). The neuroradiological or neurological findings in two cases prompted an extensive diagnostic work-up. One patient has exhibited neurological regression and progressive vision impairment and was diagnosed with juvenile neuronal ceroid-lipofuscinosis. We compare the clinical course and phenotype of these patients in regard to the clinical significance of the cerebral lesions and the need for MRI surveillance. We conclude that in all three patients the lesions were not progressive, did not show any sign of malignant transformation and could not be correlated to specific clinical features. We discuss potential etiologic mechanisms that may include overexpression of genes within the duplicated region involved in control of cell proliferation and complex molecular mechanisms such as the MAPK/ERK pathway. Systematic studies in larger cohorts are needed to confirm our observation and to establish the prevalence and clinical significance of these neuroanatomical abnormalities in patients with 16p11.2 duplications.

Mutations in B4GALNT1 (GM2 synthase) underlie a new disorder of ganglioside biosynthesis.

Glycosphingolipids are ubiquitous constituents of eukaryotic plasma membranes, and their sialylated derivatives, gangliosides, are the major class of glycoconjugates expressed by neurons. Deficiencies in their catabolic pathways give rise to a large and well-studied group of inherited disorders, the lysosomal storage diseases. Although many glycosphingolipid catabolic defects have been defined, only one proven inherited disease arising from a defect in ganglioside biosynthesis is known. This disease, because of defects in the first step of ganglioside biosynthesis (GM3 synthase), results in a severe epileptic disorder found at high frequency amongst the Old Order Amish. Here we investigated an unusual neurodegenerative phenotype, most commonly classified as a complex form of hereditary spastic paraplegia, present in families from Kuwait, Italy and the Old Order Amish. Our genetic studies identified mutations in B4GALNT1 (GM2 synthase), encoding the enzyme that catalyzes the second step in complex ganglioside biosynthesis, as the cause of this neurodegenerative phenotype. Biochemical profiling of glycosphingolipid biosynthesis confirmed a lack of GM2 in affected subjects in association with a predictable increase in levels of its precursor, GM3, a finding that will greatly facilitate diagnosis of this condition. With the description of two neurological human diseases involving defects in two sequentially acting enzymes in ganglioside biosynthesis, there is the real possibility that a previously unidentified family of ganglioside deficiency diseases exist. The study of patients and animal models of these disorders will pave the way for a greater understanding of the role gangliosides play in neuronal structure and function and provide insights into the development of effective treatment therapies.

Uniparental disomy: can SNP array data be used for diagnosis?

Purpose:Single-nucleotide polymorphism microarray analysis identifies copy-number variants and blocks of homozygosity, suggestive of consanguinity or uniparental disomy. The purpose of this study was to validate chromosomal microarray analysis for the identification of uniparental disomy in a clinical laboratory.Methods:In phase I of this retrospective study, nine cases with uniparental disomy for chromosomes 7 (n = 1), 14 (n = 1), and 15 (n = 7), identified by conventional polymorphic microsatellite marker analysis were analyzed on the Affymetrix 6.0 single-nucleotide polymorphism array. In phase II, four cases of uniparental disomy 15 showing heterozygosity for all microsatellite markers were analyzed using the same array.Results:Chromosomal microarray analysis detected blocks of homozygosity in eight of the nine cases in phase I. Phase II analysis of molecularly defined heterodisomy failed to detect blocks of homozygosity in three of the four cases. The four cases in which microarray did not detect blocks of homozygosity all involved chromosome 15.Conclusion:A failure to recombine may predispose to nondisjunction and, therefore, to uniparental disomy. Four cases of heterodisomy 15 were not detected by array, suggesting a lack of recombination. Therefore, a normal chromosomal microarray result for chromosome 15 does not exclude the possibility of uniparental disomy. This observation may apply to other chromosomes; however, further study is needed.Genet Med advance online publication 26 April 2012.

BPES with atypical premature ovarian insufficiency, and evidence of mitotic recombination, in a woman with trisomy X and a translocation t(3;11)(q22.3;q14.1).

Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is a rare autosomal dominant disorder characterized by a complex dysgenesis of the eyelids and premature ovarian insufficiency. FOXL2 located at 3q22.3, encoding a forkhead transcription factor, is the only gene known to be responsible for BPES. We describe a patient diagnosed with BPES with atypical ovarian failure, characterized by normal levels of gonadotropins, who was found to have trisomy X as well as a translocation (3;11)(q22.3;q14.1). The translocation breakpoint at 3q22.3 is located upstream of the FOXL2 gene and most likely causes BPES by separating the FOXL2 transcription unit from its cis-regulatory sequences. By array analysis we detected mosaicism for the balanced and an unbalanced form of the translocation in blood cells. We propose mitotic recombination as the likely mechanism of the mosaicism formation. Mitotic recombination is a common phenomenon in human cells. Thus, we hypothesize that it may be one of the mechanisms responsible for cryptic imbalances and possible abnormal phenotypes in some carriers of balanced rearrangements.

A co-occurrence of osteogenesis imperfecta type VI and cystinosis.

Osteogenesis imperfecta type VI (OI type VI) is a rare autosomal recessive disorder caused by mutations in the SERPINF1 gene that encodes pigment epithelium-derived factor (PEDF). Cystinosis is an autosomal recessive lysosomal transport disorder caused by mutations in the CTNS gene. Both SERPINF1 and CTNS are located on chromosome 17p13.3. We describe an individual presenting with both OI type VI and cystinosis. The patient was diagnosed with cystinosis at the age of 11 months and OI type VI on bone biopsy at the age of 8 years. He has sustained over 30 fractures during his lifetime, and at the age of 19 years entered end-stage renal disease and subsequent renal transplant. An Affymetrix 6.0 array was used to look for areas of loss of heterozygosity on chromosome 17. Sequencing of the SERPINF1 and CTNS genes was performed, followed by quantitative PCR and Western blot of PEDF to characterize the identified mutation. A 6.58 Mb region of homozygosity was identified on the Affymetrix 6.0 array, encompassing both the SERPINF1 and CTNS genes. Sequencing of the genes identified homozygosity for a known pathogenic CTNS mutation and for a novel in-frame duplication in SERPINF1. Skin fibroblasts produced a markedly reduced amount of SERPINF1 transcript and PEDF protein. This patient has the concurrent phenotype of two rare recessive diseases, cystinosis and OI type VI. We identified for the first time an in-frame duplication in SERPINF1 that is responsible for the OI type VI phenotype in this patient.

Mosaic embryo transfer-first report of a live born with nonmosaic partial aneuploidy and uniparental disomy 15.

Objective: To inform clinicians of the first known case of a live born diagnosed with syndromic partial trisomy 15 and maternal uniparental disomy 15 resulting from a mosaic embryo transfer (MET). We believe that this case will highlight the need for standardized practice guidelines to address the potential risk of MET and the importance of prenatal follow-up after a pregnancy is achieved from a MET. Design: Case report. Setting: In vitro fertilization with preimplantation genetic testing for aneuploidy (PGT-A) and MET was completed at a fertility clinic in Canada. Postnatal testing and diagnosis were performed at the Medical Genetics Department of a hospital in Canada. Patients: A newborn male with a diagnosis of partial trisomy 15 and uniparental disomy (UPD) 15. Interventions: Mosaic embryo transfer after PGT-A was performed. Diagnostic testing performed after birth included a karyotype, fluorescence in situ hybridization analysis, chromosomal microarray, and microsatellite UPD testing. Main Outcome Measures: Confirmed nonmosaic partial aneuploidy of trisomy 15 and UPD15 in a symptomatic newborn conceived from MET. Results: Singleton pregnancy was achieved after a double embryo transfer involving 1 embryo diagnosed by PGT-A with high-level mosaic trisomy 15 and high-level mosaic deletion on chromosome 20 (mos(del(20)(q11.23-qter)). Routine prenatal screening and detailed fetal ultrasound did not identify any concerns. Postnatal genetic investigations, triggered by feeding difficulties in the newborn period, diagnosed the proband with maternal UPD15 and a supernumerary marker chromosome composed of 2 noncontiguous regions of chromosome 15. This karyotype is likely resulting from incomplete trisomy rescue occurring on the paternal chromosome 15. Conclusions: This case highlights the need for better guidelines and management of pregnancies achieved after MET.

Clinical and molecular characterization of an almost complete ring chromosome 4 in two sisters, with recurrence due to gonadal mosaicism.

Autosomal ring chromosomes are rare cytogenetic findings that arise from breakage and fusion of the chromosome ends. Rings are mitotically unstable, usually sporadic and associated with a 'ring syndrome', characterized by a variable phenotype: growth retardation, no significant dysmorphisms and normal to moderately disabled intelligence. We describe the clinical features and molecular characterization of two sisters with ring chromosome 4. Karyotype analysis was performed on both sisters and parents. Chromosome microarray was performed on both sisters to delineate the breakpoint imbalance. Both sisters had a large ring 4 chromosome in the majority of cells analyzed on karyotype. Microarray results were identical in the sisters, showing a 55.8 kb duplication on the terminal 4p arm and a 1.5 Mb deletion on the terminal 4q arm. No genes of interest were identified in these regions. Parental karyotypes on lymphocytes and fibroblasts were normal, with no finding of mosaicism for the ring 4 chromosome. Polymorphic marker analysis revealed the maternal origin of the ring. To our knowledge, this is the first reported instance of a ring 4 chromosome recurring in siblings after extensive parental testing, which suggests this was due to maternal gonadal mosaicism.

A child with terminal 14q deletion syndrome: consideration of genotype-phenotype correlations.

Patients with terminal deletions of chromosome 14 usually share a number of clinical features. The syndrome is thought not to be associated with multiple congenital anomalies. We report on a patient having a terminal deletion of about 3.2 Mb, with the breakpoint in 14q32.32. Multiple health problems led to his early death. By molecular techniques (array comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH)), we identified two previously reported patients with deletions in the terminal part of chromosome 14 of almost exactly the same size and compare the phenotypes of all three children. The phenotype of the current patient is much more severe than the phenotypes of the two patients reported previously. The patients also present different sets of dysmorphic features described previously as characteristic for 14q deletion syndrome. Molecular cytogenetic mapping showed that the breakpoints in all three patients were clustered within a 240 kb interval. The possibility of recurrent breakpoint location in terminal 14q deletion syndrome, as well as detailed characterization of the spectrum of phenotypes associated with the syndrome, will require the investigation of multiple patients with similar deletions in 14q.

A child with deletion (14)(q24.3q32.13) and auditory neuropathy.

An interstitial deletion in the middle and distal part of chromosome 14 is a rare chromosomal abnormality characterized by a wide spectrum of phenotypic manifestations. We present a patient with a nearly 20 Mb interstitial deletion of chromosome 14q24.3q32.13 determined by FISH, that is associated with minor dysmorphic features, developmental delay, absent speech and auditory neuropathy. The deleted region contains 130 known genes, among them 48 with reported function or association with human disease. The patient's phenotype is compared with interstitial deletions of the distal part of chromosome 14 reported previously. We hypothesize, that there is (are) a gene (genes) in the 14q32.11-q32.13 that is (are) important for the hearing process and for which haploinsufficiency can cause auditory neuropathy. Several genes in the region, among them calmodulin, chromogranin A, the goosecoid and FOXN3, can contribute to the observed phenotype. Detailed mapping in additional patients with 14q32 deletions and hearing loss could further define the candidate region.

Polymorphism in nucleotide excision repair gene XPC correlates with bleomycin-induced chromosomal aberrations.

Chromosomal aberrations (CAs) are important genetic alterations in the development and progression of the majority of human cancers. The frequency with which such alterations occur depends to a large extent on polymorphisms of DNA-repair genes and in genes coding for xenobiotic metabolizing enzymes, which are involved in the processes of activation and inactivation of xenobiotics. The frequency of bleomycin (BLM)-induced CAs is an indirect measure of the effectiveness of DNA repair mechanisms, and a predictor of environment-related risk of cancer. Our study was conducted on the human peripheral blood lymphocytes of 82 healthy volunteers. The aim of the study was to elucidate whether the frequency of BLM-induced CAs is correlated with polymorphisms of selected genes involved in different mechanisms of DNA repair such as: XRCC1 [base excision repair]; XPA, XPC, XPG, XPD, XPF, ERCC1 [nucleotide excision repair], NBS1, RAD51, XRCC2, XRCC3, RAD51, and BRCA1 [homologous recombination], as well as in genes encoding xenobiotic metabolizing enzymes, such as CYP1A, CYP2E1, NAT2, GSTT1, and EPHX (mEH). Our study indicated that, of the polymorphisms studied, only XPC (exon 15 and intron 11) is associated with BLM-induced CAs, suggesting a role of the NER pathway in the repair of BLM-induced chromosomal aberrations.

Significant involvement of chromosome 13q deletions in progression of larynx cancer, detected by comparative genomic hybridization.

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumours with various clinical characteristics. These tumours generally exhibit complex karyotypes. Few studies of genomic imbalances have been performed exclusively in subgroups of larynx cancer samples at different stages of the disease. In the present study, chromosomal gains and losses were investigated in 52 larynx tumours, by using comparative genomic hybridization (CGH). The mean number of observed alterations was 37.7 per tumour. The most common sites of losses were 1p, 13q, Xp, and the most common gains were located in 1p, 9q, 16q. The overall number of gains was negatively associated with cancer grading. G1 tumours were also characterized by a higher frequency of deletions in 13q32 and amplifications in 1q23, than tumours in other grades (p < 0.05). The frequency of losses of 13q22 also positively associated with tumour size. There was no association between the frequency of losses in 13q and the presence of lymph node metastases at the time of diagnosis. Another statistically significant association was observed for gains at 1q22-23 and tumour size (p < 0.01). No statistically significant difference in the frequency of most common imbalances was detected between primary tumours with lymph node metastases and those without metastases. In conclusion, we discovered a significant involvement of 13q deletions in the progression of larynx cancer. All the other significant changes observed in the present study were reported previously as being important for HNSCC progression. It seems that multiple genes are disrupted in the process of neoplastic transformation in the larynx, and the networks of events remain to be elucidated.

Alternating hypoglycemia and hyperglycemia in a toddler with a homozygous p.R1419H ABCC8 mutation: an unusual clinical picture.

BACKGROUND: Inheritance of two pathogenic ABCC8 alleles typically causes severe congenital hyperinsulinism. We describe a girl and her father, both homozygous for the same ABCC8 mutation, who presented with unusual phenotypes. METHODS: Single nucleotide polymorphism microarray and Sanger sequencing were performed. Western blot, rubidium efflux, and patch clamp recordings interrogated the expression and activity of the mutant protein. RESULTS: A 16-month-old girl of consanguineous descent manifested hypoglycemia. She had dysregulation of insulin secretion, with postprandial hyperglycemia followed by hypoglycemia. Microarray revealed homozygosity for the regions encompassing KCNJ11 and ABCC8. Her father had developed diabetes at 28 years of age. Sequencing of ABCC8 identified a homozygous missense mutation, p.R1419H, in both individuals. Functional studies showed absence of working KATP channels. CONCLUSION: This is the first description of a homozygous p.R1419H mutation. Our findings highlight that homozygous loss-of-function mutations of ABCC8 do not necessarily translate into early-onset severe hyperinsulinemia.

Independent post-zygotic breaks of a dicentric chromosome result in mosaicism for an inverted duplication deletion 9p and terminal deletion 9p.

Mosaicism with two cell lines having different rearrangements of the same chromosome is rare. Only a few cases of mosaicism have been described in association with chromosomal inverted duplication deletion (inv dup del) rearrangements. A well-established mechanism of formation of inv dup del rearrangements involves a dicentric intermediate, which undergoes breakage during cell division, generating cells with either an inv dup del or a simple deletion. A patient with developmental delay and dysmorphic features was found to carry two cell lines with rearrangements of 9p: an inv dup del 9p and a terminal deletion 9p. Microarray and FISH analysis showed that these cell lines do not constitute the reciprocal products of a single dicentric breakage event. We propose that independent post-zygotic breaks of a dicentric chromosome as a likely mechanism leading to the generation of the observed cell lines. The post-zygotic origin of the inv dup del rearrangements and the associated mosaicism can be a more frequent phenomenon than currently appreciated. Therefore, genotype-phenotype correlations in the inv dup del rearrangements need to take into account the possible presence of other abnormal cell lines during early development.

A minigene approach for analysis of ATP7B splice variants in patients with Wilson disease.

Wilson disease (WND) is an autosomal recessive condition that results in accumulation of copper in the liver and brain when a membrane bound copper transporter, ATP7B, is defective. ATP7B is expressed in hepatic, brain and kidney cells, and a defect can lead to liver, neurological and renal damage in WND patients. Presentation is variable with a broad range of age of onset and symptoms, and not all biochemical signs used in diagnosis are found in every patient. Therefore, diagnosis by mutation analysis is particularly important. To date, there are approximately 380 probable disease-causing variants in ATP7B, 33 of which are splice site variants that are predicted to affect splicing, based on their location. Few of these splice site variants have been analyzed in vivo. Some exonic variations also have the potential to affect splicing. The aim of this project was to use minigenes for transcript analysis. We have chosen exon 8 as our focus and have cloned a wild-type three-exon minigene into a mammalian expression vector. After transfection, extracted RNA was analyzed by reverse transcription PCR and accurate splicing was detected. This minigene will facilitate the analysis of the numerous potential splice variants identified in exon 8 of ATP7B, with the advantage that patient cell lines are not required for each variant.

FISH-mapping of telomeric 14q32 deletions: search for the cause of seizures.

Ring chromosome 14 is a rare cytogenetic disorder. Individuals with r(14) generally have developmental delay and seizures. Other features include hypotonia, microcephaly, mild facial dysmorphism, and retinal pigmentation. Most of these features are also found in patients with linear terminal deletions of chromosome 14, except for seizures and retinal abnormalities. The objective of the study was to determine if deletion of a specific chromosome region is a possible explanation for the occurrence of seizures in patients with ring chromosome 14. Patients diagnosed either with r(14) (six patients) or a deletion of distal 14q (three patients) were analyzed by FISH (fluorescence in situ hybridization) with BAC probes. We observed differences in the size of deletions in the studied group. In two r(14) patients, we did not detect any deletion; the four other patients had deletions of various sizes, ranging from 0.8 Mb to 5 Mb. Two linear deletions were 3.2 Mb and 5.3 Mb in length, respectively; the third case had an interstitial deletion that did not overlap with the others. The deleted regions in ring chromosomes showed overlap with those in the two linear terminal deletions. We conclude that there is unlikely to be a specific deleted locus in 14q32.3 that predisposes r(14) patients to seizures or retinal pigmentation. The cause is probably related to the formation of the ring itself and the effect this may have on local chromatin structure.