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Objective: To evaluate the clinical value of positive copy number variations (CNVs) results by non‐invasive prenatal testing (NIPT) without fetal ultrasonography‐identified structural anomalies, especially with several known CNVs results. Methods: A total of 135,981 results of NIPT performed between April 1, 2017, and March 31, 2020, enrolled in the free NIPT service program implemented by the local government were retrospectively analyzed. Of these, 87 cases with positive NIPT screens for CNVs and no fetal ultrasonography‐identified anomalies were recalled and provided genetic counseling. After obtaining full informed consent, these cases were provided invasive prenatal diagnosis by karyotyping and chromosomal microarray analysis (CMA)/copy number variation sequencing (CNV‐seq) with follow‐up. One case was lost, while 86 cases were successfully followed up. Results: A total of 44 (50.6%) cases underwent invasive prenatal diagnosis, of which six cases were detected with abnormal karyotype. CMA/CNV‐Seq revealed 11 fetuses with positive results for CNVs, among whom eight were consistent with NIPT results, two were partially consistent, one was inconsistent, and positive predictive value (PPV) was 22.7% (10/44). For known CNVs, PPVs were 20% (15q11.2‐q13 microdeletion) and 33.3% (5p end deletions). Among 11 pregnant women with positive prenatal diagnosis, seven were confirmed to have pathogenic CNVs in their fetuses; four had CNVs of unknown clinical significance. Conclusions: Even in pregnancies without ultrasonography‐identified anomalies, a positive NIPT screen for CNVs must be interpreted with caution and validated by additional diagnostic study. [ABSTRACT FROM AUTHOR]

Abstract Background Congenital microtia is a common craniofacial malformation resulting from both environmental and genetic factors. Recurrent chromosomal imbalances were observed in patients with microtia. The 22q11.2 deletion is one of the most common microdeletions in human beings. The cell division cycle 45 gene (CDC45) embedded in the proximal 22q11.2 deleted region is involved in craniofacial development. However, only a few studies have focused on the 22q11.2 deletion as genetic etiology in microtia patients and studied its associated external ear deformity characteristics in detail. Methods In this research, a total of 65 patients from north China with sporadic microtia were studied. Copy number variations of CDC45 were screened using AccuCopy assay. The 22q11.2 deletion harboring CDC45 was identified by whole‐genome sequencing and targeted next‐generation sequencing. A parental test was carried out to determine the origin of the deletion. Results CDC45 copy number loss was identified in two patients with microtia. A set of qPCR assays demonstrated two patients carried a typical proximal 22q11.2 deletion between the low‐copy repeats on chromosome 22q11.2 (LCR22A and LCR22D), encompassing CDC45. The 22q11.2 deletions were de novo in each patient. In‐depth auricular phenotype assessment showed these two patients have a distinct concha‐type ear malformation while other microtia patients have lobule‐type microtia among the 65 microtia patient cohort in this study. Conclusion Here we present two additional Chinese microtia patients with de novo 22q11.2 proximal deletion harboring CDC45 and further report these patients’ distinct ear malformation.

Background: Congenital microtia is a common craniofacial malformation resulting from both environmental and genetic factors. Recurrent chromosomal imbalances were observed in patients with microtia. The 22q11.2 deletion is one of the most common microdeletions in human beings. The cell division cycle 45 gene (CDC45) embedded in the proximal 22q11.2 deleted region is involved in craniofacial development. However, only a few studies have focused on the 22q11.2 deletion as genetic etiology in microtia patients and studied its associated external ear deformity characteristics in detail. Methods: In this research, a total of 65 patients from north China with sporadic microtia were studied. Copy number variations of CDC45 were screened using AccuCopy assay. The 22q11.2 deletion harboring CDC45 was identified by whole‐genome sequencing and targeted next‐generation sequencing. A parental test was carried out to determine the origin of the deletion. Results: CDC45 copy number loss was identified in two patients with microtia. A set of qPCR assays demonstrated two patients carried a typical proximal 22q11.2 deletion between the low‐copy repeats on chromosome 22q11.2 (LCR22A and LCR22D), encompassing CDC45. The 22q11.2 deletions were de novo in each patient. In‐depth auricular phenotype assessment showed these two patients have a distinct concha‐type ear malformation while other microtia patients have lobule‐type microtia among the 65 microtia patient cohort in this study. Conclusion: Here we present two additional Chinese microtia patients with de novo 22q11.2 proximal deletion harboring CDC45 and further report these patients' distinct ear malformation. [ABSTRACT FROM AUTHOR]

Abstract Background Benign hereditary chorea (BHC) is an autosomal dominant disorder characterized by early‐onset non‐progressive involuntary movements. Although NKX2‐1 mutations or deletions are the cause of BHC, some BHC families do not have pathogenic alterations in the NKX2‐1 gene, indicating that mutations of non‐coding regulatory elements of NKX2‐1 may also play a role. Methods and Results By using whole‐genome microarray analysis, we identified a 117 Kb founder deletion in three apparently unrelated BHC families that were negative for NKX2‐1 sequence variants. Targeted next generation sequencing analysis confirmed the deletion and showed that it was part of a complex local genomic rearrangement. In addition, we also detected a 648 Kb de novo deletion in an isolated BHC case. Both deletions are located downstream from NKX2‐1 on chromosome 14q13.2‐q13.3 and share a 33 Kb smallest region of overlap with six previously reported cases. This region has no gene but contains multiple evolutionarily highly conserved non‐coding sequences. Conclusion We propose that the deletion of potential regulatory elements necessary for NKX2‐1 expression in this critical region is responsible for BHC phenotype in these patients, and this is a novel disease‐causing mechanism for BHC.

Background: Benign hereditary chorea (BHC) is an autosomal dominant disorder characterized by early‐onset non‐progressive involuntary movements. Although NKX2‐1 mutations or deletions are the cause of BHC, some BHC families do not have pathogenic alterations in the NKX2‐1 gene, indicating that mutations of non‐coding regulatory elements of NKX2‐1 may also play a role. Methods and Results: By using whole‐genome microarray analysis, we identified a 117 Kb founder deletion in three apparently unrelated BHC families that were negative for NKX2‐1 sequence variants. Targeted next generation sequencing analysis confirmed the deletion and showed that it was part of a complex local genomic rearrangement. In addition, we also detected a 648 Kb de novo deletion in an isolated BHC case. Both deletions are located downstream from NKX2‐1 on chromosome 14q13.2‐q13.3 and share a 33 Kb smallest region of overlap with six previously reported cases. This region has no gene but contains multiple evolutionarily highly conserved non‐coding sequences. Conclusion: We propose that the deletion of potential regulatory elements necessary for NKX2‐1 expression in this critical region is responsible for BHC phenotype in these patients, and this is a novel disease‐causing mechanism for BHC. [ABSTRACT FROM AUTHOR]

Abstract Background Genetic factors associated with intellectual disability (ID) include chromosomal aberrations, copy number variations (CNVs), and pathogenic variants. Identifying the genetic etiologies is beneficial for patient classification, therapy, management, and prognostic evaluation. Emerging genetic tests are helpful in identifying these genetic causes. Methods We enrolled two girl siblings with ID. Trio whole‐exome sequencing (WES) and Copy number variation sequencing (CNV‐Seq) were performed for genetic molecular analysis in these probands and their parents. The parents also accepted high‐resolution G‐banded karyotype studies. Results No significant homozygous or heterozygous variants were identified through WES. By CNV‐seq, we identified an abnormal 3p26.3p25.3 microdeletion and 14q32.13q32.33 microduplication in the two girl siblings but not in their parents. A balanced translocation 46, XX, t (3, 14) (p25; q32) was found in their mother. Conclusion The affected siblings have similar phenotype, including ID, short stature, and microcephaly. Their mother had a history of seven first‐trimester miscarriages and one elective termination because of multiple malformations. This abnormal karyotype was also thought to be responsible for the mother's recurrent miscarriage. WES in combination with CNV‐seq analysis is very helpful for identification of the genetic causes of ID without positive karyotype findings.

Background: Genetic factors associated with intellectual disability (ID) include chromosomal aberrations, copy number variations (CNVs), and pathogenic variants. Identifying the genetic etiologies is beneficial for patient classification, therapy, management, and prognostic evaluation. Emerging genetic tests are helpful in identifying these genetic causes. Methods: We enrolled two girl siblings with ID. Trio whole‐exome sequencing (WES) and Copy number variation sequencing (CNV‐Seq) were performed for genetic molecular analysis in these probands and their parents. The parents also accepted high‐resolution G‐banded karyotype studies. Results: No significant homozygous or heterozygous variants were identified through WES. By CNV‐seq, we identified an abnormal 3p26.3p25.3 microdeletion and 14q32.13q32.33 microduplication in the two girl siblings but not in their parents. A balanced translocation 46, XX, t (3, 14) (p25; q32) was found in their mother. Conclusion: The affected siblings have similar phenotype, including ID, short stature, and microcephaly. Their mother had a history of seven first‐trimester miscarriages and one elective termination because of multiple malformations. This abnormal karyotype was also thought to be responsible for the mother's recurrent miscarriage. WES in combination with CNV‐seq analysis is very helpful for identification of the genetic causes of ID without positive karyotype findings. [ABSTRACT FROM AUTHOR]

Abstract Background Mucopolysaccharidosis type I (MPS I) is a rare, recessively inherited lysosomal storage disorder, characterized by progressive multi‐systemic disease. It is caused by a reduced or absent alpha‐l iduronidase (IDUA) enzyme activity secondary to biallelic loss‐of‐function variants in the IDUA. Over 200 causative variants in IDUA have been identified. Nevertheless, there is a fraction of MPS I patients with only a single mutated IDUA allele detectable. Methods As genetic testing of MPS I is usually based on sequencing methods, copy number variations (CNVs) in IDUA can be missed and therefore presumably remain underdiagnosed. The aim of this study was the detection of CNVs using an IDUA‐specific in house multiplex ligation‐dependent probe amplification (MLPA) assay. Results A total of five unrelated MPS I patient samples were re‐analyzed after only a single heterozygous IDUA mutation c.979G>C (p.A327P), c.1469T>C (p.L490P), c.1598C>G (p.P533R), c.1205G>A (p.W402X), c.973‐7C>G (p.?) could be identified. We detected a novel splice site variant c.973‐7C>G (p.?), as well as two novel CNVs, a large deletion of IDUA exon 14 and 3’UTR c.(1828 + 1_1829‐1)_(*1963_?)del, and a large duplication extending from IDUA exon 2 to intron 12 c.(157 + 1_158‐1)_(1727 + 1_1728‐1)dup. Conclusion Together with the CNVs we previously identified, a total of four pathogenic IDUA CNVs have now been reported.

Abstract Background There is a strong evidence for genetic factors as the main causes of Autism Spectrum Disorders (ASD). To date, hundreds of genes have been identified either by copy number variations (CNVs) and/or single nucleotide variations. However, despite all the findings, the genetics of these disorders have not been totally explored. Methods Thus, the aim of our work was to identify rare CNVs and genes present in these regions in ASD children, using a high‐resolution comparative genomic hybridization technique and quantitative PCR (qPCR) approach. Results Our results have shown 60–70 chromosomal aberrations per patient. We have initially selected 66 CNVs that have been further assessed using qPCR. Finally, we have validated 22 CNVs including 11 deletions and 11 duplications. Ten CNVs are de novo, 11 are inherited and one of unknown origin of transmission. Among the CNVs detected, novel ASD candidate genes PJA2, SYNPO, APCS, and TAC1 have been identified in our group of Lebanese patients. In addition, previously described CNVs have been identified containing genes such as SHANK3, MBP, CHL1, and others. Conclusion Our study broadens the population spectrum of studied ASD patients and adds new candidates at the list of genes contributing to these disorders.

Abstract Background The implementation of molecular karyotyping has resulted in an improved diagnostic yield in the genetic diagnostics of congenital anomalies, detected prenatally or after the termination of pregnancy. However, the systematic epidemiologic ascertainment of copy number variations in the etiology of congenital anomalies has not yet been sufficiently explored. Methods Consecutive fetuses, altogether 204, with major single or multiple congenital anomalies were ascertained by using the SLOCAT registry for the period from 2011 to 2015. After excluding aneuploidies by using conventional karyotyping or Quantitative Fluorescence‐Polymerase Chain Reaction, array comparative genomic hybridization was performed for the detection of copy number variations. Results We identified pathogenic or likely pathogenic copy number variations in 14 fetuses (6.8%); 2.9% in fetuses with isolated, and 3.9% in fetuses with multiple congenital anomalies. Additionally, aneuploidies and major structural chromosomal abnormalities were detected in 40.2%. Conclusion Our systematic approach of ascertaining congenital anomalies resulted in explaining the etiology of congenital anomalies in 47% of fetuses after the termination of pregnancy. By using array comparative genomic hybridization, we found that copy number variations represent an important part in the etiology of multiple, as well as isolated congenital anomalies, which indicates the importance of analyzing copy number variations in the diagnostic approach of fetuses with congenital anomalies after the termination of pregnancy.

Fanconi anemia (FA) is a rare bone marrow failure disorder characterized by clinical and genetic heterogeneity with at least 17 genes involved, which make molecular diagnosis complex and time-consuming. Since next-generation sequencing technologies could greatly improve the genetic testing in FA, we sequenced DNA samples with known and unknown mutant alleles using the Ion PGMTM system (IPGM). The molecular target of 74.2 kb in size covered 96% of the FA-coding exons and their flanking regions. Quality control testing revealed high coverage. Comparing the IPGM and Sanger sequencing output of FANCA, FANCC, and FANCG we found no false-positive and a few false-negative variants, which led to high sensitivity (95.58%) and specificity (100%) at least for these two most frequently mutated genes. The analysis also identified novel mutant alleles, including those in rare complementation groups FANCF and FANCL. Moreover, quantitative evaluation allowed us to characterize large intragenic deletions of FANCA and FANCD2, suggesting that IPGM is suitable for identification of not only point mutations but also copy number variations. [ABSTRACT FROM AUTHOR]

Background: Mucopolysaccharidosis type I (MPS I) is a rare, recessively inherited lysosomal storage disorder, characterized by progressive multi‐systemic disease. It is caused by a reduced or absent alpha‐l iduronidase (IDUA) enzyme activity secondary to biallelic loss‐of‐function variants in the IDUA. Over 200 causative variants in IDUA have been identified. Nevertheless, there is a fraction of MPS I patients with only a single mutated IDUA allele detectable. Methods: As genetic testing of MPS I is usually based on sequencing methods, copy number variations (CNVs) in IDUA can be missed and therefore presumably remain underdiagnosed. The aim of this study was the detection of CNVs using an IDUA‐specific in house multiplex ligation‐dependent probe amplification (MLPA) assay. Results: A total of five unrelated MPS I patient samples were re‐analyzed after only a single heterozygous IDUA mutation c.979G>C (p.A327P), c.1469T>C (p.L490P), c.1598C>G (p.P533R), c.1205G>A (p.W402X), c.973‐7C>G (p.?) could be identified. We detected a novel splice site variant c.973‐7C>G (p.?), as well as two novel CNVs, a large deletion of IDUA exon 14 and 3'UTR c.(1828 + 1_1829‐1)_(*1963_?)del, and a large duplication extending from IDUA exon 2 to intron 12 c.(157 + 1_158‐1)_(1727 + 1_1728‐1)dup. Conclusion: Together with the CNVs we previously identified, a total of four pathogenic IDUACNVs have now been reported. [ABSTRACT FROM AUTHOR]

Background: There is a strong evidence for genetic factors as the main causes of Autism Spectrum Disorders (ASD). To date, hundreds of genes have been identified either by copy number variations (CNVs) and/or single nucleotide variations. However, despite all the findings, the genetics of these disorders have not been totally explored. Methods: Thus, the aim of our work was to identify rare CNVs and genes present in these regions in ASD children, using a high‐resolution comparative genomic hybridization technique and quantitative PCR (qPCR) approach. Results: Our results have shown 60–70 chromosomal aberrations per patient. We have initially selected 66 CNVs that have been further assessed using qPCR. Finally, we have validated 22 CNVs including 11 deletions and 11 duplications. Ten CNVs are de novo, 11 are inherited and one of unknown origin of transmission. Among the CNVs detected, novel ASD candidate genes PJA2, SYNPO, APCS, and TAC1 have been identified in our group of Lebanese patients. In addition, previously described CNVs have been identified containing genes such as SHANK3, MBP, CHL1, and others. Conclusion: Our study broadens the population spectrum of studied ASD patients and adds new candidates at the list of genes contributing to these disorders. [ABSTRACT FROM AUTHOR]

Background: The implementation of molecular karyotyping has resulted in an improved diagnostic yield in the genetic diagnostics of congenital anomalies, detected prenatally or after the termination of pregnancy. However, the systematic epidemiologic ascertainment of copy number variations in the etiology of congenital anomalies has not yet been sufficiently explored. Methods: Consecutive fetuses, altogether 204, with major single or multiple congenital anomalies were ascertained by using the SLOCAT registry for the period from 2011 to 2015. After excluding aneuploidies by using conventional karyotyping or Quantitative Fluorescence‐Polymerase Chain Reaction, array comparative genomic hybridization was performed for the detection of copy number variations. Results: We identified pathogenic or likely pathogenic copy number variations in 14 fetuses (6.8%); 2.9% in fetuses with isolated, and 3.9% in fetuses with multiple congenital anomalies. Additionally, aneuploidies and major structural chromosomal abnormalities were detected in 40.2%. Conclusion: Our systematic approach of ascertaining congenital anomalies resulted in explaining the etiology of congenital anomalies in 47% of fetuses after the termination of pregnancy. By using array comparative genomic hybridization, we found that copy number variations represent an important part in the etiology of multiple, as well as isolated congenital anomalies, which indicates the importance of analyzing copy number variations in the diagnostic approach of fetuses with congenital anomalies after the termination of pregnancy. [ABSTRACT FROM AUTHOR]