Breakpoints Identification of a Balanced Complex Chromosome Rearrangement Case: 46,XX, t(6;15;10;9)(q13;q15;p11.2;q34.3) ins(9;8)(q22.33;q21.1q21.3).

BACKGROUND: Balanced complex chromosome rearrangement (CCR) carriers are phenotypically normal but at high risk of reproductive failure, recurrent miscarriages, and affected offspring, so that cytogenetic characterizations of CCR carriers are crucial. METHODS: We report a case of CCR: 46,XX, t(6;15;10;9)(q13;q15;p11.2;q34.3) ins(9;8)(q22.33;q21.1q21.3). The peripheral blood was collected for karyotyping, single nucleotide polymorphism array (SNP-array) analysis, and whole genome mate-pair sequencing. RESULTS: The patient's karyotype is detected and identified as 46,XX, t(6;15;10;9)(q13;q15;p11.2;q34.3) ins(9;8) (q22.33;q21.1q21.3), with no significant duplication and deletion found by SNP-array analysis. There are 16 break-points among chromosomes 6, 8, 9, 10, and 15 identified by whole genome sequencing. CONCLUSIONS: With a variety of detection techniques, we can deeply study the genetic characteristics of CCRs, thus providing a basis for genetic counseling and choice of fertility.

Compound heterozygous variants in MYH11 underlie autosomal recessive megacystis-microcolon-intestinal hypoperistalsis syndrome in a Chinese family.

Megacystis-microcolon-intestinal-hypoperistalsis syndrome (MMIHS) is a rare and severe disorder characterized by functional obstruction in the urinary and gastrointestinal tract. The molecular basis of this condition has been defined recently. Heterozygous variants in ACTG2, homozygous mutations in LMOD1, MYLK, and MYH9 were related to the pathogenesis of the syndrome, which encodes proteins involved in the process of smooth muscle contraction, supporting a myopathic basis for the disease. Recent studies have identified homozygous or compound heterozygous variants in MYH11 as a candidate gene of MMIHS. In this report, we described a nonconsanguineous Chinese family with three male fetuses affected with megacystis. Trio-targeted exome sequencing identified compound heterozygous variants, c.2051 G > A (p.R684H) and c.3540_3541delinsTT (p.(E1180D, Q1181Ter)), in MYH11 (NM_001040114). The variants were inherited from the parents, respectively. Western blotting showed a marked decrease in MYH11 protein in the proband's umbilical cord tissue compared with the control sample. The study's results confirmed that MYH11 is a candidate gene for MMIHS with autosomal recessive (AR) inheritance and expanded the mutation spectrum for this clinical condition. Combining clinical phenotype with molecular diagnosis may enable the identification of candidate genes for potential monogenic diseases and facilitate accurate genetic counseling, informed decision-making, and prenatal diagnosis.

A novel SLC6A8 mutation associated with intellectual disabilities in a Chinese family exhibiting creatine transporter deficiency: case report.

BACKGROUND: X-linked creatine transporter deficiency (OMIM#300036,CRTR-D) is characterized by cerebral creatine deficiency, intellectual disabilities, severe speech impairment, seizures and behavioral problems. Mutations in the creatine transporter gene SLC6A8, a member of the solute-carrier family 6 mapped to Xq28, have been reported to cause the creatine transporter deficiency. CASE PRESENTATION: The proband presented at 5 yrs. 1 month of age with delays in intellectual and development, seizures and behavioral problems. A novel missense mutation, c.1181C > A (p.Thr394Lys), in the SLC6A8 gene (NM_005629.3) was detected via targeted exome sequencing, and then validated by Sanger sequencing. Multiple in silico variant effect analysis methods, including SIFT, PolyPhen2, PROVEAN, and Mutation Taster predicted that this variant was likely damaging or diseasing-causing. This hemizygous variation was also identified in the affected brother with the same clinical condition and inherited from the heterozygous carrier mother. The diagnosis was suggested by increased urinary creatine/creatinine (Cr:Crn) ratio and markedly reduced creatine content peak by brain proton magnetic resonance spectroscopy (MRS). The proband's mother became pregnant with a 3rd sibling, in whom the Sanger sequencing result of c.1181C > A was negative. CONCLUSION: The novel mutation c.1181C > A in the SLC6A8 gene reported in a Chinese family has expanded the mutation spectrum of CRTR-D. The combination of powerful new technologies such as targeted exome sequencing with thorough systematic clinical evaluation of patients will improve the diagnostic yield, and assist in genetic counselling and prenatal diagnosis for suspected genetic disorders.

[Combined G-banded karyotyping and multiplex ligation-dependent probe amplification for the detection of chromosomal abnormalities in fetuses with congenital heart defects].

OBJECTIVE: To assess the value of G-banded karyotyping in combination with multiplex ligation-dependent probe amplification (MLPA) as a tool for the detection of chromosomal abnormalities in fetuses with congenital heart defects. METHODS: The combined method was used to analyze 104 fetuses with heart malformations identified by ultrasonography. Abnormal findings were confirmed with chromosomal microarray analysis (CMA). RESULTS: Nineteen (18%) fetuses were found to harbor chromosomal aberrations by G-banded karyotyping and MLPA. For 93 cases, CMA has detected abnormalities in 14 cases including 10 pathogenic copy number variations (CNVs) and 4 CNVs of uncertain significance (VOUS). MLPA was able to detect all of the pathogenic CNVs and 1 VOUS CNV. CONCLUSION: Combined use of G-banded karyotyping and MLPA is a rapid, low-cost and effective method to detect chromosomal abnormalities in fetuses with various heart malformations.

[Genetic and prenatal diagnosis for a Chinese family with primary carnitine deficiency].

OBJECTIVE: To identify potential mutation of SLC22A5 gene in a 5-month-old boy affected with primary carnitine deficiency and provide genetic counseling and prenatal diagnosis for the members of his family. METHODS: DNA was extracted from peripheral blood samples derived from the proband, his parents and elder sister, as well as amniotic fluid from his pregnant mother. All of the 10 exons of the SLC22A5 gene were amplified by PCR and subjected to Sanger sequencing. The amniotic fluid sample was also subjected to G-banded karyotyping and multiplex ligation-dependent probe amplification (MLPA). RESULTS: A homozygous mutation c.760C>T (p.R254X) of the SLC22A5 gene was detected in the proband. Heterozygous mutation c.760C>T (p.R254X) was also found in other family members including the fetus. The karyotyping and chromosomal microdeletion testing for the amniotic fluid sample were both normal. CONCLUSION: The newly identified homozygous nonsense c.760C>T (p.R254X) mutation of the SLC22A5 gene probably underlies the primary carnitine deficiency of the proband. Genetic counseling and prenatal diagnosis have been provided for this family.

[Genetic and prenatal diagnosis for four families with Williams-Beuren syndrome].

Williams-Beuren syndrome is a common chromosome microdeletion syndrome. Early diagnosis and treatment are very helpful for patients and their families. This study identified the chromosome karyotype in one fetus with ultrasonography abnormalities and three children with developmental disorders from four families. This provided guidance for subsequent pregnancy and prenatal diagnosis by using routine G-banding chromosome karyotyping analysis, multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (array-CGH). In one amniotic fluid sample from a pregnant woman with fetal abnormalities on an ultrasound screen and three peripheral blood samples from three children with developmental disorders, the decreased signal of ELN gene probes at 7q11.23 and heterozygous deletions at 7q11.23 were detected by MLPA and array-CGH analysis. The laboratory genetic tests of amniotic fluid samples were normal when the mothers from the four families became pregnant again. It was concluded that MLPA and array-CGH are rapid and accurate tools for the diagnosis of Williams-Beuren syndrome and can provide more information for clinical genetic counseling.

[Genetic analysis for a family with Cockayne syndrome].

OBJECTIVE: To identify potential mutations among three sisters from a Chinese family suspected with Cockayne syndrome for growth and psychomotor retardation, and to offer genetic counseling and prenatal diagnosis for the family. METHODS: G-banded karyotyping, microarray comparative genomic hybridization (CM-CGH), whole genome exon high-throughput sequencing and Sanger sequencing were employed to identify potential genetic variations for the three patients and their parents. RESULTS: Whole exome sequencing has identified two novel missense mutations, i.e., c.1595A>G (p.Asp532Gly) and c.1607T>G (p.Leu536Trp), in exon 7 of excision repair cross-complementing rodent repair deficiency, complementation group 6 (ERCC6) gene. Sanger sequencing confirmed that all of the three sisters have inherited one of the mutations (c.1607T>G) from their father and another (c.1595A>G) from their mother. CONCLUSION: Three sisters have all been identified as double heterozygote for mutations c.1607T>G and c.1595A>G and were diagnosed with Cockayne syndrome.

[A novel mutation of cartilage oligomeric matrix protein gene underlies multiple epiphyseal dysplasia].

OBJECTIVE: To perform mutation analysis for a female with multiple epiphyseal dysplasia (MED) and provide pre-symptomatic and prenatal diagnosis. METHODS: Mutation screening of cartilage oligomeric matrix protein (COMP) gene was carried out through targeted next-generation DNA sequencing and Sanger sequencing. RESULTS: A novel c.956 A>T resulting in substitution of Aspartic acid 319 for Valine (p.Asp319Val) has been identified in exon 9 of the COMP gene in the patient. As predicted by a SIFT software, above mutation can cause damage to the structure of COMP protein. CONCLUSION: A novel c.956 A>T substitution mutation has been identified in a patient featuring MED.

[Application of array-CGH and MLPA for detection of 4 cryptic unbalanced translocations].

OBJECTIVE: To use array comparative genomic hybridization (array-CGH) and multiplex ligation-dependent probe amplification (MLPA) to detect unbalanced rearrangements in 4 cases suspected to have chromosome disease but were undetected with conventional karyotype analysis, and to assess the applicability of array-CGH and MLPA for detection of unbalanced translocation. METHODS: Genomic DNA was extracted with standard procedures. All cases were analyzed by array-CGH and subtelomeric MLPA. RESULTS: All of the cases were identified to have unbalanced translocations by array-CGH analysis, among which 3 were consistent with subtelomeric MLPA analysis. For the remaining one, its chromosomal abnormality was not detected by MLPA as the imbalance has occurred outside of target regions. CONCLUSION: Both array-CGH and MLPA techniques can complement conventional karyotyping for detecting unbalanced translocations. The combination features both high resolution and efficiency for clinical use.

[Analysis of copy number variations in an infant with Cri du Chat syndrome by array-based comparative genomic hybridization].

OBJECTIVE: To analyze genomic copy number variations in an infant with Cri du Chat syndrome, and to explore the underlying genetic cause. METHODS: G-banding analysis was carried out on cultured peripheral blood sample from the patient. Copy number variation analysis was performed using microarray comparative genomic hybridization, and the result was verified with fluorescence in situ hybridization. RESULTS: The infant was found to have a 46, XY, der(5) (p?) karyotype. By microarray comparative genomic hybridization, a 23.263 Mb deletion was detected in 5p14.2-p15.3 region in addition to a 14.602 Mb duplication in 12p31 region. A derivative chromosome was formed by rejoining of 12p31 region with the 5p14.2 breakpoint. The patient therefore has a karyotype of arr cgh 5p15.3p14.2 (PLEKHG4B>CDH12)× 1 pat, 12p13.33p13.1 (IQSEC3>GUC Y2C)× 3 pat. Loss of distal 5p and gain of distal 12p were verified with fluorescence in situ hybridization. CONCLUSION: The Cri du Chat syndrome manifested by the patient was caused by deletion of distal 5p from an unbalanced translocation involving chromosome 5. Microarray comparative genomic hybridization is a powerful tool for revealing genomic copy number variations for its high-resolution, high-throughput and high accuracy.

[Cytogenetic and molecular analysis of idic(Yp) in 1 infertile man and 1 prenatal fetus].

OBJECTIVE: To evaluate idic(Yp) in genetic diagnosis by examining 1 infertile man and 1 prenatal fetus using cytogenetic and molecular techniques. METHODS: Following conventional chromosome preparation, we performed G- and C-banding karyo. typing and fluorescence in situ hybridization (FISH). Then we extracted genomic DNA using standard procedures and analyzed it by array-CGH and multiplex ligation-dependent probe amplification (MLPA). RESULTS: Both cases were diagnosed as 45, X/46, X, idic (Yp11.31) mosaicism. The man showed 2 intact copies of Yp11.31-q12 (chrY:2, 710, 250-57, 428, 567, SRY, ZFY, UTY and AZF), and the prenatal fetus exhibited similar findings except a paternal deletion in the AZFc region. CONCLUSION: idic(Y) (p11.31) causes short stature and male infertility. Array-CGH and MLPA can improve the accuracy of the diagnosis of 45, X/46, X, idic (Y) mosaicism, which may contribute to the studies of the phenotype-genotype correlation and clinical genetic counseling.

Prenatal and postnatal diagnosis of Phelan-McDermid syndrome: A report of 21 cases from a medical center and review of the literature.

Background: Phelan-McDermid syndrome (PMS), caused by deletions at 22q13.3 and pathogenic variants in the SHANK3 gene, is a rare developmental disorder characterized by hypotonia, developmental delay (DD), intellectual disability (ID), autism spectrum disorder (ASD), dysmorphic features, absence of or delayed language, and other features. Methods: Conventional karyotyping, chromosomal microarray analysis (CMA), and whole exome sequencing (WES) have been used to detect genetic defects causing PMS. We summarized the genetic and clinical findings from prenatal to postnatal stages of detected cases of PMS and mapped potential candidate haploinsufficient genes for deletions of 22q13. This study aimed to summarize the laboratory findings, genetic defects, and genotype-phenotype correlations for Chinese patients with PMS. Results: Seven prenatal cases and fourteen postnatal cases were diagnosed with PMS in our center. Thirteen cases had a deletion ranging in size from 69 to 9.06 Mb at 22q13.2-q13.33, and five cases had a pathogenic variant or an intragenic deletion in the SHANK3 gene. Three familial cases with a parental carrier of a balanced translocation were noted. A review of the literature noted another case series of 29 cases and a report of five cases of PMS in China. Genotype-phenotype correlations confirmed haploinsufficiency of the SHANK3 gene for PMS and suggested other candidate haploinsufficient genes TNFRSFI3C and NFAM1 genes for immunological features and TCF20, SULT4A1, PARVB, SCO2, and UPK3A genes for intellectual impairment and behavioral abnormality, neurological features, macrocephaly/hypotonia, oculopathy, and renal adysplasia, respectively. Conclusion: Indications for prenatal diagnosis of PMS are not specific, and approximately 85% prenatally diagnosed PMS elected termination of pregnancies after genetic counseling. For postnatal cases, 62.5% were caused by a deletion at 22q13 and 37.5% were caused by a pathogenic variant or an intragenic deletion in the SHANK3 gene. Approximately 6.7% of cases with a deletion were familial, and almost all pathogenic variants were de novo. Combined karyotype, CMA, and WES should be performed to increase the diagnostic yield. The identification of other candidate haploinsufficient genes in deletions of 22q13.2-q13.33 could relate to more severe dysmorphic features, neurologic defects, and immune deficiency. These results provided evidence for diagnostic interpretation, genetic counseling, and clinical management for the Chinese cases of PMS.

[A novel mutation Glu441stop (GAA to TAA) of androgen receptor gene resulting in complete androgen insensitivity syndrome].

OBJECTIVE: To identify the mutation of human androgen receptor gene (AR) in a patient with complete androgen insensitivity syndrome (CAIS). METHODS: DNA sequences of 8 exons and their exon/intron boundaries of the AR gene in the patient were amplified by PCR and directly sequenced. RESULTS: DNA sequencing revealed a nonsense mutation in exon 1, resulting in a change of codon 441 GAA (glutamic acid) to a stop codon (TAA). CONCLUSION: A novel mutation Glu441stop (GAA to TAA) of the androgen receptor gene leading to complete androgen insensitivity syndrome was identified in this study in a Chinese patient. It may help us further understanding the pathogenesis of CAIS.

[Analysis of fetal chromosomal karyotype and etiology in 252 cases of early spontaneous abortion].

OBJECTIVE: To investigate the relationship between fetal chromosomal karyotype and early spontaneous abortion, and the effect of the environmental factors on spontaneous abortion. METHODS: Choronic villi from 252 cases of missed abortion were sampled as patient group and 50 normal pregnancies as control group. Chorionic villi were cultured and karyotype analysis was performed by G-banding. Clinical information was collected. RESULTS: The rate of chorion chromosome abnormality in the patient group was 58.09%, significantly higher than that in the control group (4.17%) (P<0.01). Among the 140 cases of karyotype abnormalities, 81 were trisomy, 29 were monosomy X and 17 were polyploidy, accounting for 57.86%, 20.71% and 12.14% of total abnormalities, respectively. Long time and low dose radiation exposure of the pregnant female seemed to be related with spontaneous abortion(P<0.01). CONCLUSION: Chorion chromosome abnormality is a major reason of early spontaneous abortion and karyotype analysis of chorionic villus is of clinical importance. For pregnant women, long-term exposure to computers and television seems a risk factor for missed abortion.

[Mutation analysis of the androgen receptor gene in a complete androgen insensitivity syndrome family].

OBJECTIVE: To study the mutation of the androgen receptor gene in a family with complete androgen insensitivity syndrome and to explore the pathogenicity of the mutation. METHODS: PCR and DNA sequencing were performed to study the AR gene mutation; Mbo I restriction endonuclease was used to detect existence of the mutation in normal controls; conservation of the mutation site was analyzed by comparison of the sequence of amino acid among different species. RESULTS: The DNA sequence of the three patients contained the same substitution of a single nucleotide on codon 681 GAG to GAT of exon 4, which located in the ligand binding domain of the AR receptor and led to substitution of glutamic acid to aspartic acid in the AR receptor. Their mother was heterozygote of E681D. E681D was not observed in the normal controls. The E681 site was extremely conservative in different species. CONCLUSION: The E681D mutation of the AR gene is a novel mutation of leading to complete androgen insensitivity syndrome.

[Karyotype analysis of chorionic villi from pregnant women with missed abortion using multiplex ligation-dependent probe amplification].

OBJECTIVE: To evaluate the clinical value of multiplex ligation-dependent probe amplification (MLPA) technique used in karyotype analysis of chorionic villi from missed abortion. METHODS: Feb 2008 to Oct 2008, 91 patients with missed abortion diagnosed by hormonal measurement, type B ultrasound and physical exam matched with 20 normal pregnant women undergoing artificial abortion were enrolled in this study. Chorionic villi was obtained by suction dilation and curettage in aseptic condition, then those villi was cultured and analyzed by traditional cytogenetic karyotyping method, in the mean time, the DNA extracted from villi was detected by MLPA. The results of chromosomal G-banding of chorionic villi were compared between two methods. RESULTS: The diagnostic concordance of MLPA and traditional karyotyping was observed in 92% (84/91) cases, there were 84 cases in the case group with diagnostic concordance by traditional karyotyping and MLPA except 7 cases of euploidy could not be detected by MLPA. The 84 cases included 40 normal karyotype, 29 trisomy of euchromosome, 1 double trisomy of euchromosome, 10 monosomy X, 1 monosomy X combined with trisomy of euchromosome, 2 chimaera of X chromosome, 1 structural abnormity of euchromosome. Among 7 cases with discordance diagnosis, 2 cases with trisomy and 5 cases with tetrasomy of euchromosome were identified in traditional karyotyping, however, they were all diagnosed with normal disomy by MLPA. Of 20 villi from normal pregnancy, two methods got the consistent results. CONCLUSION: The MLPA was rapid and efficacy method used for analyzing aneuploids in chorionic villi.

[Chromosome aberration in a full-term neonate with low birth weight using microarray comparative genomic hybridization].

OBJECTIVE: To analyze the chromosome aberration in a full-term male neonate with low birth weight, and to explore the possible causes for growth retardation in intrauterine development for the neonate. METHODS: Genomic DNA was extracted from peripheral leukocytes of the neonate. Detection of genomic DNA copy number gain and loss was performed using microarray comparative genomic hybridization. Chromosome karyotype was obtained from cultured lymphocytes for the neonate and his parents in order to identify the origin of chromosome aberration. RESULTS: Gain of 10q25.2-->qter (22 Mb) was observed in the full-term neonate with low birth weight. In addition, one chromosomal region, 15q26.2-->qter (5 Mb) was lost. The karyotype of the neonate was 46, XY, -15, +der(15), t(10;15)(q25;q26)pat. CONCLUSION: The full-term neonate with low birth weight had a partial trisomy of 10q25.2-->qter with a partial monosomy of 15q26.2-->qter, both of them may contribute to the growth retardation in intrauterine development for the neonate case.

De novo paternal origin duplication of chromosome 11p15.5: report of two Chinese cases with Beckwith-Wiedemann syndrome.

BACKGROUND: The molecular etiology of Beckwith-Wiedemann syndrome (BWS) is complex and heterogeneous. Several subtypes of epigenetic-genetic alterations including aberrant methylation patterns, segmental uniparental disomy, single gene mutations, and copy number changes have been described. An integrated molecular approach to analyze the epigenetic-genetic alterations is required for accurate diagnosis of BWS. CASE PRESENTATION: We reported two Chinese cases with BWS detected by genome-wide copy number analysis and locus-specific methylation profiling. Prenatal analysis on cord blood of patient 1 showed a de novo paternal origin duplication spanning 896Kb at 11p15.5. Patient 2 was referred at 2-month old and the genetic analysis showed a de novo 228.8Kb deletion at 11p15.5 telomeric end and a de novo duplication of 2.5 Mb at 11p15.5-15.4. Both the duplications are of paternal origin with gain of methylation at the imprinting center 1 and thus belong to the subgroup of a low tumor risk. CONCLUSION: Results from these two cases and other reported cases from literature indicated that paternally derived duplications at 11p15.5 region cause BWS. Combined chromosome microarray analysis and methylation profiling provided reliable diagnosis for this subtype of BWS. Characterization of genetic defects in BWS patients could lead to better understanding the genetic mechanisms of this clinically and genetically heterogeneous disorder.

Novel homozygous FANCL mutation and somatic heterozygous SETBP1 mutation in a Chinese girl with Fanconi Anemia.

Fanconi Anemia (FA) is a rare genetically heterogeneous disorder with 17 known complement groups caused by mutations in different genes. FA complementation group L (FA-L, OMIM #608111) occurred in 0.2% of all FA and only eight mutant variants in the FANCL gene were documented. Phenotype and genotype correlation in FANCL associated FA is still obscure. Here we describe a Chinese girl with FA-L caused by a novel homozygous mutation c.822_823insCTTTCAGG (p.Asp275LeufsX13) in the FANCL gene. The patient's clinical course was typical for FA with progression to bone marrow failure, and death from acute myelomonocytic leukemia (AML-M4) at 9 years of age. Mutation analysis also detected a likely somatic c.2608G > A (p.Gly870Ser) in the SETBP1 gene. Consistent copy number losses of 7q and 18p and gains of 3q and 21q and accumulated non-clonal single cell chromosomal abnormalities were detected in blood leukocytes as her FA progressed. This is the first Chinese FA-L case caused by a novel FANCL mutation. The somatic gene mutation and copy number aberrations could be used to monitor disease progression and the clinical findings provide further information for genotype-phenotype correlation for FA-L.

Copy number changes and methylation patterns in an isodicentric and a ring chromosome of 15q11-q13: report of two cases and review of literature.

BACKGROUND: The low copy repeats (LCRs) in chromosome 15q11-q13 have been recognized as breakpoints (BP) for not only intrachromosomal deletions and duplications but also small supernumerary marker chromosomes 15, sSMC(15)s, in the forms of isodicentric chromosome or small ring chromosome. Further characterization of copy number changes and methylation patterns in these sSMC(15)s could lead to better understanding of their phenotypic consequences. METHODS: Routine G-band karyotyping, fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH) analysis and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay were performed on two Chinese patients with a sSMC(15). RESULTS: Patient 1 showed an isodicentric 15, idic(15)(q13), containing symmetrically two copies of a 7.7 Mb segment of the 15q11-q13 region by a BP3::BP3 fusion. Patient 2 showed a ring chromosome 15, r(15)(q13), with alternative one-copy and two-copy segments spanning a 12.3 Mb region. The defined methylation pattern indicated that the idic(15)(q13) and the r(15)(q13) were maternally derived. CONCLUSIONS: Results from these two cases and other reported cases from literature indicated that combined karyotyping, aCGH and MS-MLPA analyses are effective to define the copy number changes and methylation patterns for sSMC(15)s in a clinical setting. The characterized genomic structure and epigenetic pattern of sSMC(15)s could lead to further gene expression profiling for better phenotype correlation.