Clinical validation and application of targeted long-range PCR and long-read sequencing-based analysis for haemophilia: experience from a haemophilia treatment centre in China.

BACKGROUND: Targeted long-read sequencing (LRS) is expected to comprehensively analyse diverse complex variants in haemophilia A (HA) and B (HB), caused by the F8 and F9 genes, respectively. However, its clinical applicability still requires extensive validation. OBJECTIVES: To evaluate the clinical applicability of targeted LRS-based analysis, compared with routine PCR-based methods. METHODS: Gene variants of retrieved subjects were retrospectively and prospectively analysed. Whole-genome sequencing (WGS) was performed to further analyse undiagnosed cases. Breakpoints of novel genomic rearrangements were mapped and validated using long-distance-PCR and long-range-PCR combined with sequencing. RESULTS: Totally, 122 subjects were retrieved. In retrospective analysis of the 90 HA cases, HA-LRS assay showed consistent results in 84 cases compared with routine methods, and characterized six large deletions with their exact breakpoints confirmed by further validation in six cases (routine methods only presented failure in amplifying the involved exons). In prospective analysis of the 21 HA subjects, 20 variants of F8 were identified in 20 cases. For the remaining HA patient, no duplication/deletion or SNV/InDel was found, but a potential recombination involving exons 14 and 21 of F8 was observed by LRS. WGS analysis and further verification defined a 30,478bp tandem repeat involving exons 14-21 of F8. Among the 11 HB patients, HB-LRS analysis detected 11 SNVs/InDels in F9, consistent with routine methods. CONCLUSIONS: Targeted LRS-based analysis is efficient and comprehensive to identify SNVs/InDels and genomic rearrangements of haemophilia genes, especially we first expanding the panel including F9. However, further investigation for complex gross rearrangement is still essential.

The cryptic complex rearrangements involving the DMD gene: etiologic clues about phenotypical differences revealed by optical genome mapping.

BACKGROUND: Deletion or duplication in the DMD gene is one of the most common causes of Duchenne and Becker muscular dystrophy (DMD/BMD). However, the pathogenicity of complex rearrangements involving DMD, especially segmental duplications with unknown breakpoints, is not well understood. This study aimed to evaluate the structure, pattern, and potential impact of rearrangements involving DMD duplication. METHODS: Two families with DMD segmental duplications exhibiting phenotypical differences were recruited. Optical genome mapping (OGM) was used to explore the cryptic pattern of the rearrangements. Breakpoints were validated using long-range polymerase chain reaction combined with next-generation sequencing and Sanger sequencing. RESULTS: A multi-copy duplication involving exons 64-79 of DMD was identified in Family A without obvious clinical symptoms. Family B exhibited typical DMD neuromuscular manifestations and presented a duplication involving exons 10-13 of DMD. The rearrangement in Family A involved complex in-cis tandem repeats shown by OGM but retained a complete copy (reading frame) of DMD inferred from breakpoint validation. A reversed insertion with a segmental repeat was identified in Family B by OGM, which was predicted to disrupt the normal structure and reading frame of DMD after confirming the breakpoints. CONCLUSIONS: Validating breakpoint and rearrangement pattern is crucial for the functional annotation and pathogenic classification of genomic structural variations. OGM provides valuable insights into etiological analysis of DMD/BMD and enhances our understanding for cryptic effects of complex rearrangements.

Complex genotype-phenotype correlation of MYH11: new insights from monozygotic twins with highly variable expressivity and outcomes.

BACKGROUND: Thoracic aortic aneurysm/dissection (TAAD) and patent ductus arteriosus (PDA) are serious autosomal-dominant diseases affecting the cardiovascular system. They are mainly caused by variants in the MYH11 gene, which encodes the heavy chain of myosin 11. The aim of this study was to evaluate the genotype-phenotype correlation of MYH11 from a distinctive perspective based on a pair of monozygotic twins. METHODS: The detailed phenotypic characteristics of the monozygotic twins from the early fetal stage to the infancy stage were traced and compared with each other and with those of previously documented cases. Whole-exome and Sanger sequencing techniques were used to identify and validate the candidate variants, facilitating the analysis of the genotype-phenotype correlation of MYH11. RESULTS: The monozygotic twins were premature and presented with PDA, pulmonary hypoplasia, and pulmonary hypertension. The proband developed heart and brain abnormalities during the fetal stage and died at 18 days after birth, whereas his sibling was discharged after being cured and developed normally post follow-up. A novel variant c.766 A > G p. (Ile256Val) in MYH11 (NM_002474.2) was identified in the monozygotic twins and classified as a likely pathogenic variant according to the American College of Medical Genetics/Association for Molecular Pathology guidelines. Reviewing the reported cases (n = 102) showed that the penetrance of MYH11 was 82.35%, and the most common feature was TAAD (41.18%), followed by PDA (22.55%), compound TAAD and PDA (9.80%), and other vascular abnormalities (8.82%). The constituent ratios of null variants among the cases with TAAD (8.60%), PDA (43.8%), or compound TAAD and PDA (28.6%) were significantly different (P = 0.01). Further pairwise comparison of the ratios among these groups showed that there were significant differences between the TAAD and PDA groups (P = 0.006). CONCLUSION: This study expands the mutational spectrum of MYH11 and provides new insights into the genotype-phenotype correlation of MYH11 based on the monozygotic twins with variable clinical features and outcomes, indicating that cryptic modifiers and complex mechanisms beside the genetic variants may be involved in the condition.

[Analysis of a child with DIGFAN syndrome due to variant of MORC2 gene].

OBJECTIVE: To explore the clinical features and genetic etiology for a child with developmental delay, impaired growth, facial dysmorphism, and axonal neuropathy (DIGFAN). METHODS: A child who was admitted to the Second Affiliated Hospital of Guangxi Medical University on March 22, 2021 was selected the study subject. Clinical data of the child was collected. Following extraction of genomic DNA, the child and his parents were subjected to whole exome sequencing (WES), and candidate variant was verified by Sanger sequencing and bioinformatic analysis. RESULTS: The child, a 10-year-and-9-month-old boy, had manifested with short stature, intellectual disability, delayed speech, motor and language development, and facial dysmorphism. WES and Sanger sequencing revealed that he has harbored a novel de novo c.800T>C (p.Leu267Pro) variant of the MORC2 gene. The Leucine at position 267, which is highly conserved among various species, is located in the S5 domain of ribosome protein in the ATPase binding region of MORC2. And the Leu267Pro may affect the function of MORC2 by altering the spatial conformation and activity of ATPase. Based on the guidelines from the American College of Medical Genetics and Genomics, the c.800T>C variant was classified as likely pathogenic (PS2+PM2_Supporting+PP2+PP3). CONCLUSION: The MORC2: c.800T>C (p.Leu267Pro) variant probably underlay the pathogenesis of DIGFAN syndrome in this child.

Novel variants and phenotypic heterogeneity in a cohort of 11 Chinese children with Wiedemann-Steiner syndrome.

Objective: Wiedemann-Steiner syndrome (WSS) is a rare autosomal dominant disorder caused by deleterious heterozygous variants of the KMT2A gene. This study aims to describe the phenotypic and genotypic features of Chinese WSS patients, and assess therapeutic effects of recombinant human growth hormone (rhGH). Methods: Eleven Chinese children with WSS were enrolled in our cohort. Their clinical, imaging, biochemical and molecular findings were analyzed retrospectively. Moreover, the phenotypic features of 41 previously reported Chinese WSS patients were reviewed and included in our analysis. Results: In our cohort, the 11 WSS patients presented with classic clinical manifestations, but with different frequencies. The most common clinical features were short stature (90.9%) and developmental delay (90.9%), followed by intellectual disability (72.7%). The most frequent imaging features were patent ductus arteriosus (57.1%) and patent foramen ovale (42.9%) in cardiovascular system, and abnormal corpus callosum (50.0%) in the brain. In the set comprising 52 Chinese WSS patients, the most common clinical and imaging manifestations were developmental delay (84.6%), intellectual disability (84.6%), short stature (80.8%) and delayed bone age (68.0%), respectively. Eleven different variants, including three known and eight novel variants, of the KMT2A gene were identified in our 11 WSS patients without a hotspot variant. Two patients were treated with rhGH and yielded satisfactory height gains, but one developed acceleration of bone age. Conclusion: Our study adds 11 new patients with WSS, reveals different clinical characteristics in Chinese WSS patients, and extends the mutational spectrum of the KMT2A gene. Our study also shares the therapeutic effects of rhGH in two WSS patients without GH deficiency.

Importance of comprehensive genetic testing for patients with suspected vascular Ehlers-Danlos syndrome: a family case report and literature review.

Vascular Ehlers-Danlos syndrome (vEDS), the most severe type of Ehlers-Danlos syndrome, is caused by an autosomal-dominant defect in the COL3A1 gene. In this report, we describe the clinical history, specific phenotype, and genetic diagnosis of a man who died of vEDS. The precise diagnosis of this case using whole-exome sequencing provided solid evidence for the cause of death, demonstrating the practical value of genetic counseling and analysis. Early diagnosis for the proband's son, who was also affected by vEDS, revealed initial complications of vEDS in early childhood, which have rarely been reported. We also reviewed the literature on COL3A1 missense mutations and related phenotypes. We identified an association between digestion tract events and non-glycine missense variants, which disproves a previous hypothesis regarding the genotype-phenotype correlation of vEDS. Our results demonstrate the necessity of offering comprehensive genetic testing for every patient suspected of having vEDS.

Case report: A novel heterozygous synonymous variant in deep exon region of NIPBL gene generating a non-canonical splice donor in a patient with cornelia de lange syndrome.

Cornelia de Lange syndrome (CdLS) is an autosomal dominant or X-linked genetic disease with significant genetic heterogeneity. Variants of the NIPBL gene are responsible for CdLS in 60% of patients. Herein, we report the case of a patient with CdLS showing distinctive facial features, microcephaly, developmental delay, and growth retardation. Whole exome sequencing was performed for the patient, and a novel de novo heterozygous synonymous variant was identified in the deep region of exon 40 in the NIPBL gene (NM_133433.4: c. 6819G > T, p. Gly2273 = ). The clinical significance of the variant was uncertain according to the ACMG/AMP guidelines; however, based on in silico analysis, it was predicted to alter mRNA splicing. To validate the prediction, a reverse transcriptase-polymerase chain reaction was conducted. The variant activated a cryptic splice donor, generating a short transcript of NIPBL. A loss of 137 bp at the 3' end of NIPBL exon 40 was detected, which potentially altered the open reading frame by inserting multiple premature termination codons. Quantitative real-time PCR analysis showed that the ratio of the transcription level of the full-length transcript to that of the altered short transcript in the patient was 5:1, instead of 1:1. These findings may explain the relatively mild phenotype of the patient, regardless of the loss of function of the truncated protein due to a frameshift in the mRNA. To the best of our knowledge, this study is the first to report a synonymous variant in the deep exon regions of the NIPBL gene responsible for CdLS. The identified variant expands the mutational spectrum of the NIPBL gene. Furthermore, synonymous variations may be pathogenic, which should not be ignored in the clinical and genetic diagnosis of the disease.

Phenotypic variability of syndromic craniosynostosis caused by c.833G > T in FGFR2: Clinical and genetic evaluation of eight patients from a five-generation family.

OBJECTIVE: Craniosynostosis is the result of the early fusion of cranial sutures. Syndromic craniosynostosis includes but not limited by Crouzon syndrome and Pfeiffer syndrome. Considerable phenotypic overlap exists among these syndromes and mutations in FGFR2 may cause different syndromes. This study aims to investigate the explanation of the phenotypic variability via clinical and genetic evaluation for eight patients in a large pedigree. METHODS: For each patient, comprehensive physical examination, cranial plain CT scan with three-dimensional CT reconstruction (3D-CT), and eye examinations were conducted. Whole exome sequencing was applied for genetic diagnosis of the proband. Variants were analyzed and interpreted following the ACMG/AMP guidelines. Sanger sequencing was performed to reveal genotypes of all the family members. RESULTS: A pathogenic variant in the FGFR2 gene, c.833G > T (p.C278F), was identified and proved to be co-segregate with the disease. Some symptoms of head, hearing, vision, mouth, teeth expressed differently by affected individuals. Nonetheless, all the eight patients manifested core symptoms of Crouzon syndrome without abnormality in the limbs, which could exclude diagnosis of Pfeiffer syndrome. CONCLUSION: We have established clinical and genetic diagnosis of Crouzon syndrome for eight patients in a five-generation Chinese family. Variability of clinical features among these familial patients was slighter than that in previously reported sporadic cases.

Whole Exome Sequencing Uncovered the Genetic Architecture of Growth Hormone Deficiency Patients.

Purpose: Congenital growth hormone deficiency (GHD) is a rare and etiologically heterogeneous disease. We aim to screen disease-causing mutations of GHD in a relatively sizable cohort and discover underlying mechanisms via a candidate gene-based mutational burden analysis. Methods: We retrospectively analyzed 109 short stature patients associated with hormone deficiency. All patients were classified into two groups: Group I (n=45) with definitive GHD and Group II (n=64) with possible GHD. We analyzed correlation consistency between clinical criteria and molecular findings by whole exome sequencing (WES) in two groups. The patients without a molecular diagnosis (n=90) were compared with 942 in-house controls for the mutational burden of rare mutations in 259 genes biologically related with the GH axis. Results: In 19 patients with molecular diagnosis, we found 5 possible GHD patients received known molecular diagnosis associated with GHD (NF1 [c.2329T>A, c.7131C>G], GHRHR [c.731G>A], STAT5B [c.1102delC], HRAS [c.187_207dup]). By mutational burden analysis of predicted deleterious variants in 90 patients without molecular diagnosis, we found that POLR3A (p = 0.005), SUFU (p = 0.006), LHX3 (p = 0.021) and CREB3L4 (p = 0.040) represented top genes enriched in GHD patients. Conclusion: Our study revealed the discrepancies between the laboratory testing and molecular diagnosis of GHD. These differences should be considered when for an accurate diagnosis of GHD. We also identified four candidate genes that might be associated with GHD.

Heterozygous Recurrent Mutations Inducing Dysfunction of ROR2 Gene in Patients With Short Stature.

PURPOSE: ROR2, a member of the ROR family, is essential for skeletal development as a receptor of Wnt5a. The present study aims to investigate the mutational spectrum of ROR2 in children with short stature and to identify the underlying molecular mechanisms. METHODS: We retrospectively analyzed clinical phenotype and whole-exome sequencing (WES) data of 426 patients with short stature through mutation screening of ROR2. We subsequently examined the changes in protein expression and subcellular location in ROR2 caused by the mutations. The mRNA expression of downstream signaling molecules of the Wnt5a-ROR2 pathway was also examined. RESULTS: We identified 12 mutations in ROR2 in 21 patients, including 10 missense, one nonsense, and one frameshift. Among all missense variants, four recurrent missense variants [c.1675G > A(p.Gly559Ser), c.2212C > T(p.Arg738Cys), c.1930G > A(p.Asp644Asn), c.2117G > A(p.Arg706Gln)] were analyzed by experiments in vitro. The c.1675G > A mutation significantly altered the expression and the cellular localization of the ROR2 protein. The c.1675G > A mutation also caused a significantly decreased expression of c-Jun. In contrast, other missense variants did not confer any disruptive effect on the biological functions of ROR2. CONCLUSION: We expanded the mutational spectrum of ROR2 in patients with short stature. Functional experiments potentially revealed a novel molecular mechanism that the c.1675G > A mutation in ROR2 might affect the expression of downstream Wnt5a-ROR2 pathway gene by disturbing the subcellular localization and expression of the protein.

CNV profiles of Chinese pediatric patients with developmental disorders.

PURPOSE: To examine the overall genomic copy-number variant (CNV) landscape of Chinese pediatric patients with developmental disorders. METHODS: De-identified chromosomal microarray (CMA) data from 10,026 pediatric patients with developmental disorders were collected for re-evaluating the pathogenic CNV (pCNV) yields of different medical conditions and for comparing the frequency and phenotypic variability of genomic disorders between the Chinese and Western patient populations. RESULTS: The overall yield of pCNVs in the Chinese pediatric patient cohort was 21.37%, with variable yields for different disorders. Yields of pCNVs were positively associated with phenotypic complexity and intellectual disability/developmental delay (ID/DD) comorbidity for most disorders. The genomic burden and pCNV yield in neurodevelopmental disorders supported a female protective effect. However, the stratification analysis revealed that it was seen only in nonsyndromic ID/DD, not in nonsyndromic autism spectrum disorders or seizure. Furthermore, 15 known genomic disorders showed significantly different frequencies in Chinese and Western patient cohorts, and profiles of referred clinical features for 15 known genomic disorders were also significantly different in the two cohorts. CONCLUSION: We defined the pCNV yields and profiles of the Chinese pediatric patients with different medical conditions and uncovered differences in the frequency and phenotypic diversity of genomic disorders between Chinese and Western patients.

Novel compound heterozygous variant of BSCL2 identified by whole exome sequencing and multiplex ligation­dependent probe amplification in an infant with congenital generalized lipodystrophy.

Congenital generalized lipodystrophy (CGL) is a clinically and genetically heterogeneous condition with autosomal recessive inheritance. CGL is classified into four subtypes on the basis of causative genes. This study reported on a 2­month­old male infant diagnosed with CGL with generalized lipoatrophy and skin hyperpigmentation. Whole exome sequencing (WES) identified a heterozygous small insertion (c.545_546insCCG) in Berardinelli­Seip congenital lipodystrophy 2 (BSCL2) that was inherited from the infant's mother. Copy number variation analysis using exome data suggested a heterozygous deletion involving exon 3 that was inherited from the infant's father. This finding was confirmed by multiplex ligation­dependent probe amplification test. Gap­PCR revealed breakpoints and confirmed a 1274 bp heterozygous deletion encompassing exon 3 of BSCL2 (c.213­1081_c.294+111). This deletion is different from the founder 3.3 kb deletion involving exon 3 of BSCL2 in the Peruvian population. An 11­bp microhomology at the breakpoints may mediate the deletion, and its presence indicates the independent origins of the exon 3 deletion between Chinese and Peruvian populations. The present results expanded the mutational spectrum of the BSCL2 gene in the Chinese population and suggested that introns 2 and 3 of BSCL2 are prone to recombination. Thus, exon 3 deletion should be considered for patients with CGL2 when only one BSCL2 variant is detected through WES.

[Clinical practice guidelines for Prader-Willi syndrome].

Prader-Willi syndrome (PWS) is the first multi-systemic genetic disorder known to be caused by imprinting defect. The clinical manifestations of PWS vary with age. At the prenatal stage, decreased fetal movements are frequent. The major clinical manifestations during neonatal period include hypotonia, weak cry, poor suck and feeding difficulties. Growth retardation and delayed language and motor development are observed during infancy. Short stature, small hands and feet, cognitive deficiency are noticed in the childhood. At adolescence, prominent growth retardation, obesity, gonadal dysplasia, abnormal behavior and learning difficulties are the major issues. Morbid obesity caused by insatiable appetite is the major factor for prognosis. Early diagnosis and intervention play a significance role in improving the quality of life, preventing serious complications and prolonging survival. This guideline covers the clinical manifestations, developmental process, pathogenesis, molecular diagnosis and genetic counseling of PWS, with an aim to provide reference for clinicians for early identification, proper intervention and genetic counseling for this disease.

Novel compound heterozygous pathogenic variants in ASCC1 in a Chinese patient with spinal muscular atrophy with congenital bone fractures 2 : Evidence supporting a "Definitive" gene-disease relationship.

BACKGROUND: A very limited spectrum of ASCC1 pathogenic variants had been reported in six (mostly consanguineous) families with spinal muscular atrophy with congenital bone fractures 2 [OMIM #616867] since 2016. METHODS: A proband from a non-consanguineous Chinese family presented with neonatal severe hypotonia, respiratory distress, muscle weakness, and atrophy, as well as congenital bone fractures was performed by exome sequencing. RESULTS: A compound heterozygosity of a nonsense (c.932C>G,p.Ser311Ter) and an exon 5 deletion in ASCC1 segregating with phenotypes was detected, both variants are novel and pathogenic. Since ASCC1 is a relatively new disease gene, we performed the gene curation by ClinGen SOP. The existing evidence is sufficient to support a "Definitive" level of disease-gene relationship. CONCLUSION: This case report expended the mutation spectrum of ASCC1 and support the notion that this novel disease also occurs in outbreed populations and this is a rare disease but may still be underdiagnosed due to its perinatal lethal outcomes.

Prenatal diagnosis of cri-du-chat syndrome by SNP array: report of twelve cases and review of the literature.

BACKGROUND: Cri-du-chat syndrome (CdCS; OMIM#123450) is a classic contiguous gene syndrome caused by chromosome 5p terminal deletion (5p-), which characterized by a high-pitched cat-like cry, developmental delay, severe psychomotor, mental retardation, and dysmorphic features in infancy. Prenatal diagnosis of CdCS is difficult due to the non-specific ultrasound features. And reports using array analysis are rare. This study presented the first retrospective analysis of prenatal series of CdCS fetuses diagnosed by single nucleotide polymorphism (SNP) array in China. CASE PRESENTATION: A total of 35,233 pregnant women were enrolled from Jan 2014 to April 2019 in our center, there are twelve 5p- cases with abnormal sonographic signs revealed by SNP array, giving an incidence of 0.034% (12/35,233). Clinical information and molecular basis included: maternal demographics, indications for invasive testing, sonographic findings and SNP array results. Among all the 5p- cases revealed, nine cases were diagnosed by both karyotyping and SNP array, three cases were detected only by SNP array. Half of our cases (6/12) had an isolated 5p terminal deletion, which sizes ranged from 9.0 Mb to 30 Mb. The other half of cases (6/12) characterized by unbalanced translocation, with sex ratio 7:5 (female: male), when combine the clinical features observed from this study and available literature, the most frequent anomaly observed in prenatal ultrasound examination of CdCS was cerebral abnormalities, accounted for 44.4% (16/36) of the existing cases. Features that are less consistent included: choroid plexus cyst (13.8%, 5/36), single umbilical artery (13.3%, 4/30), ventricular septal defect (11.1%, 4/36), hydrops fetalis (8.3%, 3/36), ascites (8.3%, 3/36), increased NT/NF (8.3%, 3/36), absent/severely hypoplastic nasal bone (5.5%, 2/36), in order. CONCLUSION: Prenatal findings such as cerebral abnormalities, absent/hypoplastic nasal bone, hydrops fetalis, ascites or encephalocele may act as suggestive signs of CdCS or other microdeletion/duplication syndromes. Combining typical karyotyping with chromosomal microarray analysis (CMA) is a definitive method for a precise diagnosis of CdCS and provides more accurate results in order to offer genetic counseling to families which need to deal with cryptic aberrations.

Etiology and Outcome of non-immune Hydrops Fetalis in Southern China: report of 1004 cases.

Non-immune hydrops fetalis (NIHF) is a complex condition with a high mortality and morbidity rate. Here we report the etiology and outcome of 1004 fetuses with NIHF, in a large single Maternal and Children's hospital of Southern China, since the year of 2009 to 2016. Among these 1004 fetuses with NIHF, the etiology was identified prenatally in 722 of them (72%). The most common ones were hematologic diseases and chromosomal abnormalities. There were eight spontaneous abortions, 18 intrauterine fetal demise, 672 pregnancy terminations and 87 were lost to follow-up. 219 of the 1004 fetuses were live-born and the overall survival rate was 21.8% at this point. After birth 16 perinatal or early neonatal deaths were encountered and five lost to follow-up. Of the remaining 198 newborns, 153 thrived without apparent morbidity. The most significant factors associated with mortality were prematurity and low birthweight. In conclusion, we described the largest report of underlying causes and outcome of NIHF in Southern China. Etiologies were identified for 72% of 1004 fetuses with NIHF. And two poor prognostic factors for survival are preterm birth at less than 36.5 weeks and birthweight lower than 2575 g respectively.

Genetic analyses of oculocutaneous albinism types 1 and 2 with four novel mutations.

BACKGROUND: Oculocutaneous albinism (OCA) is a human autosomal-recessive hypopigmentation disorder with hypopigmentation in the skin, hair, and eyes. OCA1 and OCA2 are caused by mutations of the TYR and OCA2 genes, respectively, which are responsible for most oculocutaneous albinism. However, the incidence of oculocutaneous albinism patients in Guangxi remains unclear. METHODS: To evaluate the molecular basis of oculocutaneous albinism in thirty-six patients in Guangxi, China. Peripheral venous blood samples were collected from these unrelated patients. The TYR and OCA2 genes of all individuals were analyzed by direct DNA sequencing and the sequences compared with are reference database and bioinformatics analysis. RESULTS: Among the 36 OCA patients, 8(22.2%) were found mutations on TYR gene, 28 (77.8%) on OCA2. And we identified Twenty-seven different TYR and OCA2 mutations in these patients, including one novel TYR framshift mutation c.561_562insTTATTATGTGTCAAATTATCCCCCA, three novel OCA2 mutations: one nonsense mutation c.2195C > G(p.S732X), one deletation mutation(c.1139-1141delTGG), one missense mutations c.2495A > C(p.H832P). The population screening and the bioinformatic analysis to determined the effects of the mutations, which revealed these four novel mutations were pathogenic. CONCLUSIONS: This study expands the mutation spectrum of oculocutaneous albinism. Four novel mutational alleles c.1139-1141delTGG, c.1832 T > C and c.2195C > G and of the OCA2 gene and c.561_562insTTATTATGTGTCAAATTATCCCCCA of TYR were associated with OCA. The genotype-phenotype correlations suggest that molecular diagnosis is more accurate and important in OCA.

Two novel pathogenic variants of L1CAM gene in two fetuses with isolated X­linked hydrocephaly: A case report.

Hydrocephalus due to aqueductal stenosis (HSAS; Online Mendelian Inheritance in Man #307000) is a rare X­linked, recessively­inherited disease characterized by severe hydrocephaly and occasionally adducted thumbs, in addition to intellectual disability and spasticity in surviving individuals. The present study described two fetuses with severely enlarged ventricles of the brain. The clinical diagnosis of HSAS was made on the basis of family history and sonographic findings. Molecular testing of the L1 cell adhesion molecule (L1CAM) gene revealed two novel hemizygous L1CAM variants, c.998C>T(p.Pro333Leu) and c.2362G>T(p.Val788Phe). The variants affect the highly conserved amino acids which are located in the key domains of the protein (the fourth Ig domain and second FnIII domain, respectively). The two variants were predicted to be 'disease causing' by a number of prediction tools, and have been classified as likely pathogenic following the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines. The present study highlights the importance of combining family history, prenatal ultrasonography and molecular testing in the diagnosis of HSAS. The novel variants expand the mutational spectrum of L1CAM gene in the Chinese population, and could be used in genetic counseling, carrier testing of female relatives, and prenatal, as well as preimplantation genetic diagnosis.

Genetic Testing and Pregnancy Outcome Analysis of 362 Fetuses with Congenital Heart Disease Identified by Prenatal Ultrasound / Análise de Teste Genético e de Desfecho da Gestação de 362 Fetos com Cardiopatia Congênita Identificados por Ultrassom Pré-Natal

Abstract Background: Congenital heart defects (CHD), as the most common congenital anomaly, have been reported to be associated with chromosomal abnormalities. Currently, patients with CHD are routinely offered karyotyping and chromosomal microarray (CMA) testing, but the genotype-phenotype relationship has not yet been fully established. Objective: To determine the type and frequency of chromosomal abnormalities in fetuses with CHD and to analyze pregnancy outcomes of fetuses with heart abnormalities caused by different genetic factors. Methods: A total of 362 cases of CHD were enrolled from 2009 to 2016. Detailed ultrasound and laboratory examinations, including karyotyping and CMA, were performed. Outcome was obtained from discharge summaries. Results: Of the 362 fetuses, 220 were found with an isolated CHD, and 142 had CHD with extracardiac anomaly. Among these 362 fetuses, 140 were identified with a genetic cause, including 111 cases with aneuploidy, 10 cases with abnormality of chromosomal structure by karyotyping and 19 cases with pathogenic or likely pathogenic copy-number variations (CNVs) by CMA. The detection rate is close to 38.7%. Only one (identified as trisomy 18 syndrome) in 140 positive cases resulted in perinatal death, with the others being induced. The remaining 222 cases had negative results for both genetic testing and of these cases, 56 resulted in induced labor, and 77 had natural childbirth or caesarean births. The pregnancy outcome of the remaining 89 cases was uncertain. Conclusions: Karyotyping and CMA are effective and accurate prenatal genetic techniques for identifying fetal chromosomal abnormalities associated with cardiac defects, and this can assist clinical doctors to perform appropriate genetic counselling with regard to the etiology and outcome of CHD.

Resumo Fundamento: As cardiopatias congênitas (CCs) são as anomalias congênitas mais comuns, e têm sido associadas a anormalidades cromossômicas. Atualmente, a cariotipagem e a análise cromossômica por microarray (CMA) são oferecidas rotineiramente aos pacientes, mas a relação genótipo-fenótipo ainda não foi totalmente estabelecida. Objetivo: Determinar o tipo e a frequência das anomalias cromossômicas em fetos com CC e analisar os desfechos da gestação de fetos com anormalidades cardíacas causadas por diferentes fatores genéticos. Métodos: No total, foram admitidos 362 casos de CC entre 2009 e 2016. Ultrassonografia e exames laboratoriais detalhados foram realizados, incluindo cariotipagem e CMA. O resultado foi obtido a partir das folhas de epicrise. Resultados: Dos 362 fetos, 220 apresentaram doença coronariana isolada e 142 apresentaram doença coronariana com anomalia extracardíaca. Entre esses 362 fetos, foram identificados 140 com causa genética, incluindo 111 casos com aneuploidia, 10 casos com anormalidade da estrutura cromossômica por cariotipagem e 19 casos com variações no número de cópias (CNVs) patogênicas ou provavelmente patogênicas por CMA. A taxa de detecção é de aproximadamente 38,7%. Apenas um (identificado como síndrome da trissomia do cromossomo 18) em 140 casos positivos resultou em morte perinatal, com as demais sendo induzidas. Os 222 casos restantes tiveram resultados negativos para ambos os testes genéticos e, destes, 56 resultaram em trabalho de parto induzido e 77 tiveram partos naturais ou cesarianas. O desfecho da gravidez dos 89 casos restantes foi incerto. Conclusões: A cariotipagem e a CMA são técnicas genéticas pré-natais eficazes e precisas para a identificação de anomalias cromossômicas fetais associadas a defeitos cardíacos, e isso pode ajudar os médicos a realizar aconselhamento genético adequado com relação à etiologia e ao desfecho das cardiopatias congênitas.

Genetic Testing and Pregnancy Outcome Analysis of 362 Fetuses with Congenital Heart Disease Identified by Prenatal Ultrasound.

BACKGROUND: Congenital heart defects (CHD), as the most common congenital anomaly, have been reported to be associated with chromosomal abnormalities. Currently, patients with CHD are routinely offered karyotyping and chromosomal microarray (CMA) testing, but the genotype-phenotype relationship has not yet been fully established. OBJECTIVE: To determine the type and frequency of chromosomal abnormalities in fetuses with CHD and to analyze pregnancy outcomes of fetuses with heart abnormalities caused by different genetic factors. METHODS: A total of 362 cases of CHD were enrolled from 2009 to 2016. Detailed ultrasound and laboratory examinations, including karyotyping and CMA, were performed. Outcome was obtained from discharge summaries. RESULTS: Of the 362 fetuses, 220 were found with an isolated CHD, and 142 had CHD with extracardiac anomaly. Among these 362 fetuses, 140 were identified with a genetic cause, including 111 cases with aneuploidy, 10 cases with abnormality of chromosomal structure by karyotyping and 19 cases with pathogenic or likely pathogenic copy-number variations (CNVs) by CMA. The detection rate is close to 38.7%. Only one (identified as trisomy 18 syndrome) in 140 positive cases resulted in perinatal death, with the others being induced. The remaining 222 cases had negative results for both genetic testing and of these cases, 56 resulted in induced labor, and 77 had natural childbirth or caesarean births. The pregnancy outcome of the remaining 89 cases was uncertain. CONCLUSIONS: Karyotyping and CMA are effective and accurate prenatal genetic techniques for identifying fetal chromosomal abnormalities associated with cardiac defects, and this can assist clinical doctors to perform appropriate genetic counselling with regard to the etiology and outcome of CHD.