A recurrent 8 bp frameshifting indel in FOXF1 defines a novel mutation hotspot associated with alveolar capillary dysplasia with misalignment of pulmonary veins

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a rare lethal lung developmental disease. Affected infants manifest with severe respiratory distress and refractory pulmonary hypertension and uniformly die in the first month of life. Heterozygous point mutations or copy‐number variant deletions involving FOXF1 and/or its upstream lung‐specific enhancer on 16q24.1 have been identified in the vast majority of ACDMPV patients. We have previously described two unrelated families with a de novo pathogenic frameshift variant c.691_698del (p.Ala231Argfs*61) in the exon 1 of FOXF1. Here, we present a third unrelated ACDMPV family with the same de novo variant and propose that a direct tandem repeat of eight consecutive nucleotides GCGGCGGC within the ~4 kb CpG island in FOXF1 exon 1 is a novel mutation hotspot causative for ACDMPV.     

Alveolar capillary dysplasia with misalignment of the pulmonary veins and hypoplastic left heart sequence caused by an in frame deletion within FOXF1

Alveolar capillary dysplasia with misalignment of the pulmonary veins (ACDMPV) is a rare, autosomal dominant disorder of interstitial lung development, leading to pulmonary hypertension, and death in infancy. Associated features include malformations of the heart, gastrointestinal tract, and genitourinary system. ACDMPV is caused by heterozygous variants in the FOXF1 gene or microdeletions involving FOXF1. We present a male infant with ACDMPV, hypoplastic left heart sequence (HLHS), duodenal atresia, and imperforate anus due to a de novo, in frame deletion in FOXF1: c.209_214del (p.Thr70_Leu71del). Previous reports have suggested that microdeletions involving FOXF1 are associated with ACDMPV with congenital heart defects, including HLHS, gastrointestinal atresias, and other anomalies; whereas likely pathogenic variants within FOXF1 have not been reported with ACDMPV and HLHS. This is the first patient reported with ACDMPV, HLHS, imperforate anus, and duodenal atresia associated with a likely pathogenic variant in the FOXF1 gene.     

Maternal mutations of FOXF1 cause alveolar capillary dysplasia despite not being imprinted

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a rare cause of pulmonary hypertension in newborns. Maternally inherited point mutations in Forkhead Box F1 gene (FOXF1), deletions of the gene, or its long‐range enhancers on the maternal allele are responsible for this neonatal lethal disorder. Here, we describe monozygotic twins and one full‐term newborn with ACD and gastrointestinal malformations caused by de novo mutations of FOXF1 on the maternal‐inherited alleles. Since this parental transmission is consistent with genomic imprinting, the parent‐of‐origin specific monoallelic expression of genes, we have undertaken a detailed analysis of both allelic expression and DNA methylation. FOXF1 and its neighboring gene FENDRR were both biallelically expressed in a wide range of fetal tissues, including lung and intestine. Furthermore, detailed methylation screening within the 16q24.1 regions failed to identify regions of allelic methylation, suggesting that disrupted imprinting is not responsible for ACDMPV.     

Intronic deletion and duplication proximal of the EXT1 gene: A novel causative mechanism for multiple osteochondromas

Multiple osteochondromas (MO) is a syndrome in which benign cartilage‐capped neoplasms develop at the surface of the long bones. Most cases are caused by exonic changes in EXT1 or EXT2, but 15% are negative for these changes. Here we report for the first time a family of MO patients with germline genomic alterations at the EXT1 locus without detectable mutations or copy number alterations of EXT exonic sequences. Array‐CGH showed an 80.7 kb deletion of Intron 1 of EXT1 and a 68.9 kb duplication proximal of EXT1. We identified a breakpoint between the distal end of the duplicated region and a sequence distal of the deleted region in the first intron. This breakpoint was absent in non‐affected family members. The configuration of the breakpoint indicates a direct insertion of the duplicated region into the deletion. However, no other breakpoint was found, which suggests a more complex genomic rearrangement has occurred within the duplicated region. Our results reveal intronic deletion and duplication as a new causative mechanism for MO not detected by conventional diagnostic methods. © 2013 Wiley Periodicals, Inc.     

A novel de novo point mutation of the OCT‐binding site in the IGF2/H19‐imprinting control region in a Beckwith–Wiedemann syndrome patient

The IGF2/H19‐imprinting control region (ICR1) functions as an insulator to methylation‐sensitive binding of CTCF protein, and regulates imprinted expression of IGF2 and H19 in a parental origin‐specific manner. ICR1 methylation defects cause abnormal expression of imprinted genes, leading to Beckwith–Wiedemann syndrome (BWS) or Silver–Russell syndrome (SRS). Not only ICR1 microdeletions involving the CTCF‐binding site, but also point mutations and a small deletion of the OCT‐binding site have been shown to trigger methylation defects in BWS. Here, mutational analysis of ICR1 in 11 BWS and 12 SRS patients with ICR1 methylation defects revealed a novel de novo point mutation of the OCT‐binding site on the maternal allele in one BWS patient. In BWS, all reported mutations and the small deletion of the OCT‐binding site, including our case, have occurred within repeat A2. These findings indicate that the OCT‐binding site is important for maintaining an unmethylated status of maternal ICR1 in early embryogenesis.     

First known microdeletion within the Wolf‐Hirschhorn syndrome critical region refines genotype–phenotype correlation

Deletions within HSA band 4p16.3 cause Wolf‐Hirschhorn syndrome (WHS), which comprises mental retardation and developmental defects. A WHS critical region (WHSCR) of approximately 165 kb has been defined on the basis of 2 atypical interstitial deletions; however, genotype–phenotype correlation remains controversial, due to the large size of deletion usually involving several megabases. We report on the first known patient with a small de novo interstitial deletion restricted to the WHSCR who presented with a partial WHS phenotype consisting only of low body weight for height, speech delay, and minor facial anomalies; shortness of stature, microcephaly, seizures and mental retardation were absent. The deletion was initially demonstrated by FISH analysis, and breakpoints were narrowed with a “mini‐FISH” technique using 3–5 kb amplicons. A breakpoint‐spanning PCR assay defined the distal breakpoint as disrupting the WHSC1 gene within intron 5, exactly after an AluJb repeat. The proximal breakpoint was not found to be associated with a repeated sequence or a known gene. The deletion encompasses 191.5 kb and includes WHSC2, but not LETM1. Thus, manifestations attributable to this deletion are reduced weight for height, minor facial anomalies, ADHD and some learning and fine motor deficiencies, while seizures may be associated with deletions of LETM1. © 2001 Wiley‐Liss, Inc.     

SHOX far‐downstream copy‐number variations involving cis‐regulatory nucleotide variants in two sisters with Leri‐Weill dyschondrosteosis

SHOX haploinsufficiency leading to Leri‐Weill dyschondrosteosis (LWD) and idiopathic short stature typically results from intragenic mutations or copy‐number variations (CNVs) involving SHOX and/or its putative enhancer regions that are distributed in the genomic interval between 400 kb and 840 kb from Xpter/Ypter. Here, we report two sisters with LWD, who carried a deletion in the far‐downstream region of SHOX. The 0.62 Mb deletion contained 50 single nucleotide polymorphisms (SNPs) and short insertions and deletions (indels), whose genotypes were linked to SHOX expression levels in the Genotype‐Tissue Expression portal. Notably, most of these SNPs/indels accumulated within a ~20 kb interval that was positioned ~900 kb away from Xpter/Ypter. These SNPs/indels showed similar minor allele frequencies, indicating that they reside within a haplotype block. The ~20 kb interval was not evolutionarily conserved; however, it was associated with the previously determined peak of chromosome conformation capture profiling (4C)‐seq. Importantly, the deletion in the present cases partially overlapped with CNVs of three previous cases with skeletal deformity and/or short stature. The results indicate that far‐downstream CNVs constitute rare genetic causes of SHOX haploinsufficiency. These CNVs possibly impair SHOX expression through copy‐number changes of a human‐specific cis‐regulatory haplotype block. This notion awaits further validation.     

1q24 deletion syndrome. Two cases and new insights into genotype‐phenotype correlations

1q24q25 deletions cause a distinctive phenotype including proportionate short stature, microcephaly, brachydactyly, dysmorphic facial features and intellectual disability. We present a mother and son who have a 672 kb microdeletion at 1q24q25. They have the typical skeletal features previously described but do not have any associated intellectual disability. We compare the genes within our patients' deletion to those in the deletions of previously reported cases. This indicates two genes that may be implicated in the intellectual disability usually associated with this deletion syndrome; PIGC and C1orf105. In addition, our cases provide supporting evidence to recent published work suggesting that the skeletal features may be linked to the microRNAs miR199 and miR214, encoded within intron 14 of the Dynamin‐3 gene.     

Understanding the Genomic Structure of Copy‐Number Variation of the Low‐Affinity Fcγ Receptor Region Allows Confirmation of the Association of FCGR3B Deletion with Rheumatoid Arthritis

Fcγ receptors are a family of cell–surface receptors that are expressed by a host of different innate and adaptive immune cells, and mediate inflammatory responses by binding the Fc portion of immunoglobulin G. In humans, five low‐affinity receptors are encoded by the genes FCGR2A , FCGR2B , FCGR2C , FCGR3A , and FCGR3B , which are located in an 82.5‐kb segmental tandem duplication on chromosome 1q23.3, which shows extensive copy‐number variation (CNV). Deletions of FCGR3B have been suggested to increase the risk of inflammatory diseases such as systemic lupus erythematosus and rheumatoid arthritis (RA). In this study, we identify the deletion breakpoints of FCGR3B deletion alleles in the UK population and endogamous native American population, and show that some but not all alleles are likely to be identical‐by‐descent. We also localize a duplication breakpoint, confirming that the mechanism of CNV generation is nonallelic homologous recombination, and identify several alleles with gene conversion events using fosmid sequencing data. We use information on the structure of the deletion alleles to distinguish FCGR3B deletions from FCGR3A deletions in whole‐genome array comparative genomic hybridization (aCGH) data. Reanalysis of published aCGH data using this approach supports association of FCGR3B deletion with increased risk of RA in a large cohort of 1,982 cases and 3,271 controls (odds ratio 1.61, P = 2.9×10^?3).     

Germline Chromothripsis Driven by L1‐Mediated Retrotransposition and Alu/Alu Homologous Recombination

Chromothripsis (CTH) is a phenomenon where multiple localized double‐stranded DNA breaks result in complex genomic rearrangements. Although the DNA‐repair mechanisms involved in CTH have been described, the mechanisms driving the localized “shattering” process remain unclear. High‐throughput sequence analysis of a familial germline CTH revealed an inserted SVA_E retrotransposon associated with a 110‐kb deletion displaying hallmarks of L1‐mediated retrotransposition. Our analysis suggests that the SVA_E insertion did not occur prior to or after, but concurrent with the CTH event. We also observed L1‐endonuclease potential target sites in other breakpoints. In addition, we found four Alu elements flanking the 110‐kb deletion and associated with an inversion. We suggest that chromatin looping mediated by homologous Alu elements may have brought distal DNA regions into close proximity facilitating DNA cleavage by catalytically active L1‐endonuclease. Our data provide the first evidence that active and inactive human retrotransposons can serve as endogenous mutagens driving CTH in the germline.     

Pure intronic rearrangements leading to aberrant pseudoexon inclusion in dystrophinopathy: a new class of mutations?

We report on two unprecedented cases of pseudoexon (PE) activation in the DMD gene resulting from pure intronic double‐deletion events that possibly involve microhomology‐mediated mechanisms. Array comparative genomic hybridization analysis and direct genomic sequencing allowed us to elucidate the causes of the pathological PE inclusion detected in the RNA of the patients. In the first case (Duchenne phenotype), we showed that the inserted 387‐bp PE was originated from an inverted ～57 kb genomic region of intron 44 flanked by two deleted ～52 kb and ～1 kb segments. In the second case (Becker phenotype), we identified in intron 56 two small deletions of 592 bp (del 1) and 29 bp (del 2) directly flanking a 166‐bp PE located in very close proximity (134 bp) to exon 57. The key role of del 1 in PE activation was established by using splicing reporter minigenes. However, the analysis of mutant constructs failed to identify cis elements that regulate the inclusion of the PE and suggested that other splicing regulatory factors may be involved such as RNA structure. Our study introduces a new class of mutations in the DMD gene and emphasizes the potential role of underdetected intronic rearrangements in human diseases. Hum Mutat 32:1–9, 2011. © 2011 Wiley‐Liss, Inc.     

Copy‐number variations (CNVs) of the human sex steroid metabolizing genes UGT2B17 and UGT2B28 and their associations with a UGT2B15 functional polymorphism

UGT2B17 and UGT2B28 are among the most commonly deleted genes in humans and encode members of the uridine diphosphate (UDP)‐glucuronosyltransferase 2B (UGT2B) subfamily. They are involved, along with UGT2B15, in the catabolism of sex‐steroid hormones. Despite the recent biomedical interest in UGT2B17 and UGT2B28 copy‐number variations (CNVs) within human populations, the impact of their gene dosage has been hampered by the lack of precise molecular identification of the common deletion breakpoints within high homology sequence regions on chromosome 4. We have characterized these common deletions and report their coexistence in Caucasians, along with the p.D85Y (rs1902023:G>T) functional polymorphism of UGT2B15. Segmental duplications of 4.9 kb for UGT2B17 and 6.8 kb for UGT2B28 comprise purine‐rich recombination sites located 117 kb and 108 kb apart on both ends of the deletions. CNVs of UGT2B17 and UGT2B28 occur in Caucasians at 27% and 13.5%, respectively. While only 43% have two copies of both genes, 57% harbor at least one deletion. Their co‐occurrence on 5% of chromosomes creates a 225‐kb genomic gap. CNVs of both UGT2B17 and UGT2B28, with the co‐occurrence of UGT2B15:p.D85Y, generate seven distinct haplotypes. Restricting the analyses to CNV of the UGT2B17 gene without evaluating UGT2B28 CNV, along with the genotype of UGT2B15, may over‐ or underestimate the impact of each gene under physiological conditions or disease states. Hum Mutat 30:1–10, 2009. © 2009 Wiley‐Liss, Inc.     

Diagnostic whole genome sequencing and split‐read mapping for nucleotide resolution breakpoint identification in CNTNAP2 deficiency syndrome

Whole genome sequencing (WGS) has the potential to report on all types of genetic abnormality, thus converging diagnostic testing on a single methodology. Although WGS at sufficient depth for robust detection of point mutations is still some way from being affordable for diagnostic purposes, low‐coverage WGS is already an excellent method for detecting copy number variants (“CNVseq”). We report on a family in which individuals presented with a presumed autosomal recessive syndrome of severe intellectual disability and epilepsy. Array comparative genomic hybridization (CGH) analysis had revealed a homozygous deletion apparently lying within intron 3 of CNTNAP2. Since this was too small for confirmation by FISH, CNVseq was used, refining the extent of this mutation to approximately 76.8 kb, encompassing CNTNAP2 exon 3 (an out‐of‐frame deletion). To characterize the precise breakpoints and provide a rapid molecular diagnostic test, we resequenced the CNVseq library at medium coverage and performed split read mapping. This yielded information for a multiplex polymerase chain reaction (PCR) assay, used for cascade screening and/or prenatal diagnosis in this family. This example demonstrates a rapid, low‐cost approach to converting molecular cytogenetic findings into robust PCR‐based tests. © 2014 The Authors. American Journal of Medical Genetics Part A Published by Wiley Periodicals, Inc.     

Extended runs of homozygosity at 17q11.2: an association with type‐2 NF1 deletions?

Large deletions in the NF1 gene region at 17q11.2 are caused by nonallelic homologous recombination (NAHR). The recurrent type‐2 NF1 deletions span 1.2 Mb, with breakpoints in the SUZ12 gene and SUZ12P. Type‐2 NF1 deletions occur preferentially during mitosis and are associated with somatic mosaicism. A panel of 16 type‐2 NF1 deletions was used as a model system in which to investigate whether extended homozygosity across 17q11.2 might be associated with somatic deletion. Using SNP arrays, a 3.2 Mb interval encompassing the NF1 deletion region was found to harbor runs of homozygosity (ROHs) in different human populations. However, ROHs ≥500 kb directly flanking the NF1 deletion region on both sides were not found to occur disproportionately in NF1 patients harboring type‐2 deletions compared to controls. Although low allelic diversity in 17q11.2 is unlikely to be a key factor in promoting NAHR‐mediated somatic type‐2 deletions, a specific ROH of 588 kb (roh1), located some 525 kb proximal to the deletion interval, was found to occur more frequently (P=0.012) in the type‐2 deletion patients compared with controls. We postulate that roh1 may act remotely, via an as yet unknown mechanism, to increase the frequency of somatic recombination between the distally duplicated SUZ12 sequences. Hum Mutat 30:1–10, 2010. © 2010 Wiley‐Liss, Inc.     

Novel intragenic deletion in OPHN1 in a family causing XLMR with cerebellar hypoplasia and distinctive facial appearance

Al‐Owain M, Kaya N, Al‐Zaidan H, Al‐Hashmi N, Al‐Bakheet A, Al‐Muhaizea M, Chedrawi A, Basran RK, Milunsky A. Novel intragenic deletion in OPHN1 in a family causing XLMR with cerebellar hypoplasia and distinctive facial appearance. X‐linked mental retardation (XLMR) is notably a heterogeneous condition and often poses a diagnostic challenge. The oligophrenin 1 gene (OPHN1) is a protein with a Rho‐GTPase‐activating domain required in the regulation of the G‐protein cycle. Mutations in the OPHN1 cause XLMR with cerebellar hypoplasia and distinctive facial appearance. We report a large Saudi family of four boys and one girl affected with XLMR. The boys had moderate MR, seizure disorder, facial dysmorphism, and cerebellar vermis hypoplasia. The girl had mild MR, seizures, and mild cerebellar hypoplasia. A novel deletion of at least exons 7–15 was identified by polymerase chain reaction analysis and multiple ligation probe amplification of the OPHN1 gene. The array comparative genomic hybridization further delineated approximately 68 kb deletion of the 7–15 exons and nearly half of intron 15. In addition, the X‐inactivation confirmed random pattern in the girl. Although the affected boys have remarkably similar phenotype, there was some variability in the severity of the seizure disorder and the cerebellar hypoplasia. The report confirms the previous findings that carrier females may be symptomatic.