Kb Deletion Of Exon Research Articles

Continuous Glucose Monitoring in the Management of Hypoglycemia in TANGO2

Abstract Introduction: The TANGO2 gene encodes a transport and Golgi organization protein of unclear function; mutations should be considered in patients presenting with acute metabolic crisis, hypoglycemic episodes, cardiac arrhythmias, and other endocrinopathies. We report the novel use of a continuous glucose monitor (CGM) to help predict and prevent significant hypoglycemic episodes in a patient with TANGO2 mutation. Clinical Case: A 14-month old previously healthy, developmentally normal female who presented with unresponsive hypoglycemia (glucose 26 mg/dL) was demonstrated by Next Generation Sequencing to have a pathogenic 31.8 kb deletion of exon 3 to 9 in the TANGO-2 gene and a suspected pathogenic hemizygous c.569_592dup, p.Ile190_Leu197dup in TANGO-2. Her hospital course was notable for MRI showing hypoxic ischemic encephalopathy and both physical and electrical cardiac dysfunction. Continuous intravenous dextrose corrected the hypoglycemia, and transient hyperglycemia followed after several days of a glucose infusion rate between 3.2 to 5.8 mg/kg/min. After transitioning to ad lib oral feeds without restrictions, she was discharged. A second admission for acute unresponsive hypoglycemia and metabolic acidosis (glucose 30 mg/dL) occurred at 17 months of age with no clear inciting cause. Continuous IV dextrose at 9.9 mg/kg/min corrected the hypoglycemia and again resulted in transient hyperglycemia up to 271 mg/dl. Levothyroxine was also started for a TSH of 27 mIU/mL and a T4 of 4.6 ug/dL. Immediately after discharge, a DexCom G6 CGM was placed. Data over 2 weeks shows an average glucose of 104 ng/dL with 99% of the BS in target range. Parents report that CGM predictive low alerts have allowed intervention to abort fasting-related metabolic crises. Conclusion: In TANGO-2 deficiency, the liver may not adequately store and/or release glycogen in response to glucagon due to abnormal endoplasmic reticulum, Golgi apparatus, and mitochondrial functioning in states of stress or illness. Recent reports are conflicting with some showing reduced mitochondrial respiration in TANGO-2 patients in steady state with others finding normal values, opening the possibility that a combination of factors in the setting of stress may precipitate a metabolic crisis. Our patient quickly returns to near-normal physiological functioning; consequently, we suggest that use of a CGM can help prevent fasting related metabolic crisis in TANGO2 patients and can help guide feeding schedule and food choices to limit hyper- and hypoglycemia. In addition, CGM data can help further investigate if any beta cell dysregulation exists in non-acute states.

RNA Splicing Defects in Cancer-Linked Genes Indicate Mutation or Focal Gene Deletion and Are Associated with TKI Resistance in CML

Background Mutated cancer genes in patients (pts) with TKI failure and blast crisis (BC) CML have recently been described. RUNX1 mutations, namely single nucleotide variants (SNVs) and indels, were the most frequently detected besides BCR-ABL1 [reviewed in Branford, Kim Leuk 2019]. They were found in ~18% of pts, although splice variants were rarely described. RNA splicing events were associated with focal deletion of IKZF1 and RUNX1 in TKI resistant pts that were identified by copy number analysis and RNAseq [Branford Blood 2018]. Novel splicing associated with mutation of cancer genes is an unexplored area of study in resistance. RNA sequencing can assess the functional effect of splice site variants, which lead to splicing errors due to the use of alternative or cryptic splice sites and cause alterations to protein function. Aim We determined whether novel splicing can identify cancer genes with potential altered function. Methods RNAseq analysis was performed for 48 pts at diagnosis and 33 at BC using a protocol that preserved intron-retaining precursor RNA. Coverage of intron-exon borders was sufficient to detect intronic splice region variants. The STAR aligner was used to bioinformatically collate unannotated RNA splice junctions. 54 cancer genes were assessed and aberrant splice events were filtered based on the number of samples in which a splice junction occurred. Manual inspection of the splice junctions was performed using the Integrative Genomics Viewer. This approach identified previously verified aberrant splicing associated with IKZF1 and RUNX1 deletions. Results Ten previously undetected novel splice junctions were revealed in 9/33 pts (27%) in BC within key tumor suppressor genes CDKN2A/B (5), RB1 (1), ATM (1), and RUNX1 (3). The aberrant splicing pattern of CDKN2A and RB1 (Fig A/B) in 6 pts suggested large deletions, as previously described in our cohort with IKZF1 and RUNX1 deletions. Breakpoints associated with deletions ranging from 53 to 181 Kb were detected in the 5 pts with CDKN2A aberrant splicing. Similarly, a 90 Kb deletion of exons 18-27 of the RB1 gene led to the aberrant splicing. The pts transformed to lymphoid BC (median 5 months). 4 of these 6 pts were tested at diagnosis and the deletions were not detected, indicating they were gained at resistance. The aberrant splicing patterns of ATM and RUNX1 did not predict large deletions. These were related to somatic SNVs at canonical splice sites in ATM and in 2 of the pts with RUNX1 aberrant splicing. A splice acceptor site SNV in ATM resulted in skipping of exon 61 (Fig C) and protein truncation. This novel SNV has not been reported in any population or somatic variant database. Two pts in myeloid BC at 28 and 48 months after diagnosis had an identical somatic RUNX1 mutation at the canonical splice donor site of exon 5. The SNV was not detectable prior to imatinib treatment in both pts. The splice site SNV led to activation of a cryptic splice site within exon 5 in both pts (Fig D), which predicted premature termination. While this mutation is novel, an adjacent intronic SNV occurs in familial platelet disorder, leading to activation of the same cryptic splice site. The atypical RUNX1 splicing of the 3rd patient was associated with retention of 55 bp of intron 6 as a cryptic exon (Fig E), leading to protein truncation. A deep intronic SNV identified at lymphoid BC at 6 months of imatinib was detected near the cryptic exon by RNAseq and verified as somatic by DNA Sanger sequencing. This was predicted to activate cryptic RNA splicing elements and lead to intron sequence retention in a RUNX1 transcript. We sequenced the diagnosis sample using an RNA-based gene panel method under development that provides enhanced sensitivity of variant detection. The same pattern of atypical splicing was observed and the intronic SNV was present at low level. The RUNX1 mutation at diagnosis may have contributed to early BC. To our knowledge this is the first report of a RUNX1 truncating variant in CML involving a cryptic exon. Conclusion Enhanced bioinformatic analysis of RNAseq data has revealed a high proportion of pts with truncating mutations in cancer genes indicated by novel RNA splicing (27% pts in BC). Using RNA-based sequencing allows an evaluation of the potential functional effect of variants that are not apparent by DNA-based mutation analysis. We suggest that future studies include RNA sequencing to detect a broader spectrum of mutations associated with TKI resistance. Disclosures Shanmuganathan: Gilead: Other: Travel Support; Janssen: Other: Travel Support; Amgen: Other: Travel Support; Bristol-Myers Squibb: Honoraria, Other: Travel Support; Novartis: Honoraria, Other: Travel Support. Yeung:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria; Amgen: Honoraria. Scott:Celgene: Honoraria. Hughes:Novartis, Bristol-Myers Squibb, Celgene: Research Funding; Novartis, Bristol-Myers Squibb: Consultancy, Other: Travel. Branford:Cepheid: Consultancy, Honoraria; Qiagen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau.

27 - Rare Focal Deletions Involving SNRPN and UBE3A Genes

Prader-Willi (PWS) and Angelman (AS) syndromes are genetic disorders with multiple well-known mechanisms. Approximately 70% of individuals with PWS or AS have a ~4 Mb deletion of 15q11-q13, while the remainder have UPD15, an imprinting defect or, in the case of UBE3A, a sequence variant. Rarely have focal deletions been reported. We report here on three such unusual cases from our clinical microarray experience. Case 1 was a 2-1/2 year old boy with global developmental delay, short stature, microcephaly, failure to thrive and possible hearing loss. This boy, his healthy mother and maternal grandfather had a 36 kb deletion involving only noncoding exons 1 and 2 of UBE3A (NM_130839). Although previously unreported, this deletion is considered likely pathogenic based upon the AS phenotype in the proband and the inheritance pattern in this family. Case 2 was a 2-1/2 year old girl with epilepsy referred for testing on our childhood epilepsy panel which includes sequencing and copy number analysis of 58 relevant genes. Exon-level array showed an intragenic deletion of exons 2–4 of UBE3A. Methylation -specific MLPA (MS-MLPA) showed normal imprinting. Parental testing may be informative in assisting diagnosis since only maternal inheritance would be consistent with AS. Case 3 was the phenotypically normal father of a boy with PWS and a known SNRPN deletion. Array showed that the father had a 109 kb deletion of exons 4–12 of SNRPN (NM_022808) as well as the upstream reading frame (SNURF). MS-MLPA showed that this deletion was on the maternally derived chromosome. Although rare, intragenic or gene deletion of either UBE3A or SNRPN rather than the common 4 Mb deletion can be observed with PWS and AS. An assay which provides exon level coverage of these genes is important when interrogating this region.

Next generation sequencing with copy number variant detection expands the phenotypic spectrum of HSD17B4-deficiency

BackgroundD-bifunctional protein deficiency, caused by recessive mutations in HSD17B4, is a severe, infantile-onset disorder of peroxisomal fatty acid oxidation. Few affected patients survive past two years of age. Compound heterozygous mutations in HSD17B4 have also been reported in two sisters diagnosed with Perrault syndrome (MIM # 233400), who presented in adolescence with ovarian dysgenesis, hearing loss, and ataxia.Case presentationAn adult male presented with cerebellar ataxia, peripheral neuropathy, hearing loss, and azoospermia. The clinical presentation, in combination with biochemical findings in serum, urine, and muscle biopsy, suggested a mitochondrial disorder. Commercial genetic testing of 18 ataxia and mitochondrial disease genes was negative. Targeted exome sequencing followed by analysis of single nucleotide variants and small insertions/deletions failed to reveal a genetic basis of disease. Application of a computational algorithm to infer copy number variants (CNVs) from exome data revealed a heterozygous 12 kb deletion of exons 10–13 of HSD17B4 that was compounded with a rare missense variant (p.A196V) at a highly conserved residue. Retrospective review of patient records revealed mildly elevated ratios of pristanic:phytanic acid and arachidonic:docosahexaenoic acid, consistent with dysfunctional peroxisomal fatty acid oxidation.ConclusionOur case expands the phenotypic spectrum of HSD17B4-deficiency, representing the first male case reported with infertility. Furthermore, it points to crosstalk between mitochondria and peroxisomes in HSD17B4-deficiency and Perrault syndrome.

Mitochondrial Carbonic Anhydrase VA Deficiency Resulting from CA5A Alterations Presents with Hyperammonemia in Early Childhood

Four children in three unrelated families (one consanguineous) presented with lethargy, hyperlactatemia, and hyperammonemia of unexplained origin during the neonatal period and early childhood. We identified and validated three different CA5A alterations, including a homozygous missense mutation (c.697T>C) in two siblings, a homozygous splice site mutation (c.555G>A) leading to skipping of exon 4, and a homozygous 4 kb deletion of exon 6. The deleterious nature of the homozygous mutation c.697T>C (p.Ser233Pro) was demonstrated by reduced enzymatic activity and increased temperature sensitivity. Carbonic anhydrase VA (CA-VA) was absent in liver in the child with the homozygous exon 6 deletion. The metabolite profiles in the affected individuals fit CA-VA deficiency, showing evidence of impaired provision of bicarbonate to the four enzymes that participate in key pathways in intermediary metabolism: carbamoylphosphate synthetase 1 (urea cycle), pyruvate carboxylase (anaplerosis, gluconeogenesis), propionyl-CoA carboxylase, and 3-methylcrotonyl-CoA carboxylase (branched chain amino acids catabolism). In the three children who were administered carglumic acid, hyperammonemia resolved. CA-VA deficiency should therefore be added to urea cycle defects, organic acidurias, and pyruvate carboxylase deficiency as a treatable condition in the differential diagnosis of hyperammonemia in the neonate and young child.

Comprehensive genetic analyses of primary adrenal failure without enzymatic defects

Our objective is to estimate frequencies of mutations in STAR, CYP11A1, NR0B1, NR5A1, MC2R, and MRAP in a cohort of Japanese patients with primary adrenal failure without enzymatic defects. Twenty-one patients were included, who were diagnosed as having primary adrenal failure without enzymatic defect, namely 21-hydroxylase deficiency, 3βHSD deficiency, 11β-hydroxylase deficiency, and P450 oxidoreductase deficiency. Sixteen patients presented with primary adrenal failure before the age of 2 years. Fourteen patients had apparent mineralocorticoid deficiency. Fourteen patients were 46, XY and 7 patients 46, XX. Three had 46, XY disorders of sex development. Mutation analyses of STAR, NR0B1, NR5A1, MC2R, and MRAP were done by PCR-based sequencing and next generation sequencing. In case of no amplification of NR0B1 by PCR, we performed oligonucleotide array CGH. We descried clinical findings in each patients and determined possible genotype-phenotype correlation. Five patients were diagnosed as having DAX-1 deficiency. NR0B1 mutations were found hemizygously in 3 patients (c.116delG, c.846_865del, and p.Q283X). NR0B1 deletions were found in 2 patients (400kb deletion including NR0B1 and 2.4kb deletion of exon 1). Four patients presented with primary adrenal failure in newborn, and the other patient presented at the age of 6 years. STAR mutations were found in 3 patients. One patient was 46, XY, and 2 patients were 46, XX. One patient, who presented with primary adrenal failure in newborn, had c.712delA/p.Q258X. Two patients, who presented at preschool age, had p.Q258X/p.R272C and p.Q258X/p.R188H. No mutations were found in CYP11A1, NR5A1, MC2R, and MRAP. In conclusion, NR0B1 mutations and deletions are relatively common in 46, XY normal male phenotype patients (5/11). STAR mutations might be found in cases, being older than 2 years of age. 3. CYP11A1, NR5A1, MC2R, and MRAP mutations are rare.

Targeted array comparative genomic hybridization – A new diagnostic tool for the detection of large copy number variations in nemaline myopathy-causing genes

Nemaline myopathy (NM) constitutes a heterogeneous group of congenital myopathies. Mutations in the nebulin gene (NEB) are the main cause of recessively inherited NM. NEB is one of the most largest genes in human. To date, 68 NEB mutations, mainly small deletions or point mutations have been published. The only large mutation characterized is the 2.5 kb deletion of exon 55 in the Ashkenazi Jewish population. To investigate any copy number variations in this enormous gene, we designed a novel custom comparative genomic hybridization microarray, NM-CGH, targeted towards the seven known genes causative for NM. During the validation of the NM-CGH array we identified two novel deletions in two different families. The first is the largest deletion characterized in NEB to date, (∼53 kb) encompassing 24 exons. The second deletion (1 kb) covers two exons. In both families, the copy number change was the second mutation to be characterized and shown to have been inherited from one of the healthy carrier parents. In addition to these novel mutations, copy number variation was identified in four samples in three families in the triplicate region of NEB. We conclude that this method appears promising for the detection of copy number variations in NEB.

D.P.13 Targeted array CGH analysis of the nebulin gene: Identification of large deletions causing nemaline myopathy

Nemaline myopathy (NM) constitutes a heterogeneous group of disorders among the congenital myopathies. Mutations in the nebulin gene (NEB) are a main cause of recessively inherited NM. Sixty-eight different mutations causing NM have been published in NEB to date. Almost all are point mutations or small deletions. The only large mutation described in NEB thus far is the 2.5kb deletion of exon 55 in the Ashkenazi Jewish population. To investigate any copy number variations in NEB we designed a novel custom NM-CGH microarray targeted towards the seven known NM genes. During the validation and optimization of the NM-CGH array we identified three novel disease-causing aberrations in three different families. Two of these aberrations are the largest deletions characterized in NEB to date (53kb and 88kb) encompassing 24 and 68 exons, respectively. The third mutation is a 1kb deletion, covering two exons. In two of the families, the copy number change was the second mutation to be characterized. The novel aberrations were shown to have been inherited from one of the healthy carrier parents. However, the largest aberration was detected in a family with three affected family members in three different generations. Further studies are ongoing to refute or corroborate the potentially dominant effect of this mutation. In addition to these aberrations, copy number variation, both deletions and duplications, in the triplicate region of NEB, have been identified in 11 samples from six different families. Further study is ongoing to elucidate the potential pathogenicity of these variants. We conclude that the NM-CGH-array appears promising for the detection of copy number variations in NEB, and already, it has given a lot of new information about the vast nebulin gene.

Analysis of severely affected patients with dihydropyrimidine dehydrogenase deficiency reveals large intragenic rearrangements of DPYD and a de novo interstitial deletion del(1)(p13.3p21.3)

Dihydropyrimidine dehydrogenase (DPD) deficiency is an infrequently described autosomal recessive disorder of the pyrimidine degradation pathway and can lead to mental and motor retardation and convulsions. DPD deficiency is also known to cause a potentially lethal toxicity following administration of the antineoplastic agent 5-fluorouracil. In an ongoing study of 72 DPD deficient patients, we analysed the molecular background of 5 patients in more detail in whom initial sequence analysis did not reveal pathogenic mutations. In three patients, a 13.8 kb deletion of exon 12 was found and in one patient a 122 kb deletion of exon 14-16 of DPYD. In the fifth patient, a c.299_302delTCAT mutation in exon 4 was found and also loss of heterozygosity of the entire DPD gene. Further analysis demonstrated a de novo deletion of approximately 14 Mb of chromosome 1p13.3-1p21.3, which includes DPYD. Haploinsufficiency of NTNG1, LPPR4, GPSM2, COL11A1 and VAV3 might have contributed to the severe psychomotor retardation and unusual craniofacial features in this patient. Our study showed for the first time the presence of genomic deletions affecting DPYD in 7% (5/72) of all DPD deficient patients. Therefore, screening of DPD deficient patients for genomic deletions should be considered.

The Delta>15 Kb deletion French Canadian founder mutation in familial hypercholesterolemia: rapid polymerase chain reaction-based diagnostic assay and prevalence in Quebec.

Approximately one in 500 individuals in Western population has autosomal dominant familial hypercholesterolemia due to mutations in the low-density lipoprotein receptor (LDLR) gene. Screening for these mutations is hampered by their large number, except in founder populations. We identified the breakpoint of the >15 kb deletion involving the LDLR gene promoter and exon 1, responsible for more than 60% of French Canadian hypercholesterolemia cases, as well as the breakpoint of the 5 kb deletion of exons 2 and 3 that accounts for an additional 5% of cases. Both deletions appear to be because of homologous recombination by unequal crossing-over between the left arms of Alu repeats. Using RepeatMasker, we determined that 55% of the LDLR gene is composed of Alu elements; thus, it is not surprising that most LDLR rearrangements involve at least one Alu. Furthermore, we developed a rapid polymerase chain reaction-based assay for the French Canadian-1 (>15 kb) and French Canadian-5 (5 kb) hypercholesterolemia alleles. Screening a representative population sample of 943 French Canadian youths whose LDL cholesterol levels were above the 50th percentile allowed us to estimate the prevalence of the >15 kb allele as 0.11% (95% confidence interval, 0.03-0.38).

Significant contribution of large BRCA1 gene rearrangements in 120 French breast and ovarian cancer families.

Genetic linkage data have shown that alterations of the BRCA1 gene are responsible for the majority of hereditary breast-ovarian cancers. However, BRCA1 germline mutations are found much less frequently than expected, especially as standard PCR-based mutation detection approaches focus on point and small gene alterations. In order to estimate the contribution of large gene rearrangements to the BRCA1 mutation spectrum, we have extensively analysed a series of 120 French breast-ovarian cancer cases. Thirty-eight were previously found carrier of a BRCA1 point mutation, 14 of a BRCA2 point mutation and one case has previously been reported as carrier of a large BRCA1 deletion. The remaining 67 cases were studied using the BRCA1 bar code approach on combed DNA which allows a panoramic view of the BRCA1 region. Three additional rearrangements were detected: a recurrent 23.8 kb deletion of exons 8-13, a 17.2 kb duplication of exons 3-8 and a 8.6 kb duplication of exons 18-20. Thus, in our series, BRCA1 large rearrangements accounted for 3.3% (4/120) of breast-ovarian cancer cases and 9.5% (4/42) of the BRCA1 gene mutation spectrum, suggesting that their screening is an important step that should be now systematically included in genetic testing surveys.

Characterization of Partial Gene Deletions in Type III von Willebrand Disease with Alloantibody Inhibitors

von Willebrand factor gene deletions were characterized in four patients with severe type III von Wilebrand disease and alloantibodies to von Willebrand factor. A PCR-based strategy was used to characterize the boundaries of the deletions. Identical 30 kb von Willebrand factor gene deletions which include exons 33 through 38 were identified in two siblings of one family by this method. A small 5 base pair insertion (CCTGG) was sequenced at the deletion breakpoint. PCR analysis was used to detect the deletion in three generations of the family, including two family members who are heterozygous for the deletion. In a second family, two type III vWD patients, who are distant cousins, share an approximately 56 kb deletion of exons 22 through 43. The identification and characterization of large vWF gene deletions in these type III vWD patients provides further support for the association between large deletions in both von Willebrand factor alleles and the development of inhibitory alloantibodies.

Rearrangements in the LDL receptor gene in Dutch familial hypercholesterolemic patients and the presence of a common 4 kb deletion

DNA samples from 53 unrelated Dutch patients with familial hypercholesterolemia (FH) were screened for rearrangements in the gene for the LDL receptor (LDLR) by Southern analysis. Four different mutations have been detected by hybridisation of BglII digested genomic DNA with an exon 10–14 containing cDNA probe. The mutations are defined by a 7 kb insertion near exon 11, a partial gene duplication encompassing exons 9–12, a 4 kb deletion of exons 7 and 8 and an 0.4 kb deletion comprising the 5′-part of exon 16. These four different rearrangements in the LDLR gene account for 17% of the mutations in the Dutch FH population sample. Interestingly, the 4 kb deletion was detected in 5 unrelated FH patients (9.5%) and appeared to be identical to the deletion previously described (Russell, D.W. et al., Arteriosclerosis, 9 (Suppl. 1) (1989) 1–8; Russell, D.W. et al., Cold Spring Harbor Symp. Quant. Biol., 51 (1987) 401). in an FH patient of Dutch origin. This suggests that the 4 kb deletion is a common mutation in the Dutch FH population.