Turnner syndrome is a common sex chromosome disorder. We reported a rare case with Turnner syndrome caused by abnormal number and structure of sex chromosomes. Hereby fluorescence in situ hybridization (FISH) and copy number variation by whole genome low depth sequencing (CNV-seq) were used to clarify the abnormal chromosome. This study provides a diagnostic strategy for clinicians and genetic researchers.

Objective:To analyze the applicability and feasibility of a cell-free DNA barcode-enabled single-molecule test (cfBEST) in non-invasive prenatal diagnosis of phenylketonuria.Methods:This study recruited four pregnant women who were prenatally diagnosed as heterozygous carriers of hot spot mutations in the PAH gene from pedigrees with phenylketonuria at the First Affiliated Hospital of Zhengzhou University from July to September 2019. The frequency of mutations in maternal plasma cell-free DNA and the fetuses' genotypes were analyzed by cfBEST. Nested polymerase chain reaction primers were designed to amplify the mutation sites in each pedigree. The results of cfBEST were compared with those of invasive prenatal diagnosis. Descriptive analysis was used for data analysis. Results:In pedigree 1, the frequency of c.603T>G and c.842+2T>A mutations in maternal plasma cell-free DNA were 48.40% (291/601) and 9.70% (61/628), which was detected by cfBEST. The fetus was diagnosed with phenylketonuria with two heterozygous mutations. In pedigree 2, the frequency of c.1238G>C and c.842+2T>A mutations in maternal plasma cell-free DNA was 43.70% (786/1 798) and 0% (0/1 550), respectively. Both mutations were wild-type, and the fetus was neither phenylketonuria nor a carrier. In pedigree 3, the frequency of c.1045T>G and c.728G>A mutations in maternal plasma cell-free DNA was 44.00% (930/2 112) and 0% (0/705), respectively, suggesting that both mutations in the fetus were wild-type, and the fetus was neither phenylketonuria nor a carrier. In pedigree 4, the frequency of c.755G>A and c.728G>A mutations were 45.40% (743/1 637) and 4.50% (28/849), respectively, which indicated that the former was wild-type, and the latter was heterozygous; namely the fetus was a carrier of phenylketonuria. The results of cfBEST were consistent with those of invasive prenatal diagnosis. Three pedigrees (Pedigree 2, 3 and 4) continued the pregnancy to full-term, and the phenylalanine levels in the neonates were all below 120 μmol/L. No abnormalities were reported in those three infants during follow-ups at one, three, and six months after birth.Conclusions:The cfBEST could be used for non-invasive prenatal diagnosis of phenylketonuria caused by PAH gene mutation, but further studies with a larger sample size are needed.

OBJECTIVE: To assess the value of copy number variation sequencing (CNV-seq) for the diagnosis of children with disorders of sex development (DSD). METHODS: Five children with DSD who presented at the First Affiliated Hospital of Zhengzhou University from October 2019 to October 2020 were enrolled. In addition to chromosomal karyotyping, whole exome sequencing (WES), SRY gene testing, and CNV-seq were also carried out. RESULTS: Child 1 and 2 had a social gender of female, whilst their karyotypes were both 46,XY. No pathogenic variant was identified by WES. The results of CNV-seq were 46,XY,+Y (1.4) and 46,XY,-Y (0.75), respectively. The remaining three children have all carried an abnormal chromosome Y. Based on the results of CNV-seq, their karyotypes were respectively verified as 45,X[60]/46,X,del(Y)(q11.221)[40], 45,X,16qh+[76]/46,X,del(Y)(q11.222),16qh+[24], and 45,X[75]/46,XY[25]. CONCLUSION CNV-seq may be used to verify the CNVs on the Y chromosome among children with DSD and identify the abnormal chromosome in those with 45,X/46,XY. Above results have provided a basis for the clinical diagnosis and treatment of such children.
Humans ; Child ; Female ; DNA Copy Number Variations ; Chromosome Aberrations ; Karyotyping ; Exome Sequencing ; Disorders of Sex Development/genetics*

To study the genotype-phenotype and genetic characteristics of Kallmann syndrome. Five patients with Kallmann syndrome were enrolled. Clinical data collection, chromosome karyotyping, whole exome sequencing (WES), and multiplex ligation-dependent probe amplification (MLPA) were used. All the five patients were males, aging from 2 months to 45 years old. Three of the five patients complained cryptorchidism, one complained gonadal dysgenesis, and one complained fasting hyperglycemia. The clinical feature was hypogonadotropic hypogonadism with anosmia, and all karyotype was 46 XY. Magnetic resonance imaging (MRI) showed undeveloped olfactory bulbs and tracts. Kallmann syndrome related gene novel variants were found in all the 5 patients. The hypoplasia of right kidney was found in a patient with c. 1795_1799del (p.Asn599Profs*66) of anosmin 1 (ANOS1) variant. Clinical heterogeneity and incomplete penetrance were seen in a patient with c. 2824A>G (p.Thr942Ala) of chromodomain helicase DNA binding protein 7 (CHD7). Besides, WES indicated a 109 bp-deletion on Xp22.31 (chrX: 8507699-8507804), which was the deletion of exon 10 on ANOS1 gene verified by MLPA. The deletion variant was inherited form his mother, and conformed to X-linked recessive inheritance. Kallmann syndrome is genetic and clinical heterogeneous. WES is helpful for early diagnosis. MLPA and genome copy number variation analysis (CNV) are also recommend if necessary.

We report a case of non-invasive prenatal diagnosis of fetal ectodermal dysplasia caused by EDA gene mutations. The pregnant woman underwent prenatal diagnosis at 11 gestational weeks because of a childbearing history of ectodermal dysplasia. Cell-free DNA barcode-enabled single-molecule test (cfBEST) was used to detect the ectodermal dysplasia gene mutation, and chorionic villus sampling was also performed. The cfBEST results showed that the genotype of maternal EDA gene c.340C> T(p.Gln114*) was heterozygous, while the genotype of fetal EDA was normal wild-type (C/C), which were consistent with the results of villus sampling, suggesting that cfBEST can be used for non-invasive prenatal diagnosis of ectodermal dysplasia caused by EDA gene mutation.

OBJECTIVE: To explore the genetic cause of a patient suspected for congenital ectodermal dysplasia with repeated hyperthermia and to assess the reproductive risk for his family. METHODS: Medical whole-exome sequencing (WES) were used to detect single-nucleotide variations and low-coverage massively parallel copy number variation sequencing (CNV-seq) were employed to verify suspected CNVs. PCR and real-time quantitative PCR were applied to confirm the deletion of EDA gene. RESULTS: The results of WES suggested that the patient carried a hemizygous deletion for chrX:69 243 016-69 395 730. CNV-seq indicated that the patient carried a deletion of approximately 0.12 Mb on Xq13.1, which encompassed the EDA gene. The PCR results confirmed that there was a hemizygous deletion of exons 3 to 8 of the EDA gene. The same deletion was not found in his mother. CONCLUSION The congenital ectodermal dysplasia of the patient may be attributed to deletion of exons 3 to 8 of the EDA gene, which could be de novo or derive from germline mosaicism of his mother. The WES and CNV-seq are of great value for the diagnosis of rare diseases.
DNA Copy Number Variations ; Ectodermal Dysplasia/genetics* ; Ectodysplasins/genetics* ; Exons ; Genetic Testing ; High-Throughput Nucleotide Sequencing ; Humans ; Mosaicism ; Sequence Deletion ; Whole Exome Sequencing

Objective:To explore the genotype-phenotype relationship of Wilson's disease (WD) and further study the mutation spectrum in the ATP7B gene.Methods:The clinical data and genetic test results of 115 cases with WD diagnosed in the First Affiliated Hospital of Zhengzhou University from 2015 to 2022 were retrospectively analyzed. The rank sum test was used for quantitative data comparison, and χ2 test was used for count data comparison. Multivariate logistic regression was used to analyze the relationship between patients' genotype and phenotype. Results:The onset of liver manifestations (hepatic type) accounted for 60.9%, neurological symptoms (cerebral type) for 13.0%, and mixed hepato-cerebral symptoms for 26.1%. Presymptomatic individuals (hepatic types) accounted for 62.9%. Next-generation sequencing- diagnosed WD cases accounted for 87.8%. Combined multiplex ligation-dependent probe amplification assay-diagnosed WD cases accounted for 89.6%. A single case with a detected pathogenic locus accounted for 10.4%. The diagnostic rate of WD by genetic testing combined with clinical data was 100%. A total of 76 ATP7B mutations were detected, and the top three mutation frequencies were c.2333G>T (p.Arg778Leu) (30.7%), c.2975C>T (p.Pro992Leu) (7.3%), and c.2621C>T (p.Ala874Val) (6.4%). The mutations were mainly distributed in exons 8, 11-13, and 15-18, accounting for more than 90% of the total mutations. Eight new mutations were found, including c.3724G>A (p.Glu1242Lys), c.3703G>C (p.Gly1235Arg), c.3593T>C (p.Val1198Ala), c.2494A>C (p.Lys832Gln), c.1517T>A (p.Ile506Lys), c.484G>T (p.Glu162Ter), c.1870-49A>G, and the missing of exons 10-21. Liver histopathology showed cellular edema, degeneration, inflammation, and necrosis, as well as a 42.8% copper staining positive rate. Genotype-phenotype analysis showed that the p.Arg778Leu mutation had higher alanine aminotransferase (ALT) levels than those carrying other mutations ( P=0.024), while the homozygous mutation of p.Arg778Leu was associated with cerebral-type patients ( P=0.027). Conclusion:Genetic testing plays an important role in the diagnosis of WD. p.Arg778Leu is the first high-frequency mutation in the Chinese population, and patients carrying it have higher ALT levels. The p.Arg778Leu homozygous mutation is prone to causing cerebral-type WD. This study expands the ATP7B gene mutation spectrum.

Objective:To investigate the clinical and genetic characteristics of children with 45, X/46, XY mosaicism.Methods:The retrospective study included 20 children diagnosed with 45, X/46, XY and 45, X/46, X,+mar mosaicism in the First Affiliated Hospital of Zhengzhou University from 2018 to 2022. The clinical features, gonadal pathology, treatment and follow-up were summarized. Genetic tests were performed by SRY gene test, azoospermia factor region (AZF) deletion test, copy number variation-sequencing (CNV-seq). Age at first diagnosis was compared between boys and girls using independent sample t-test. Results:The 20 patients included 3 boys and 17 girls, and the age at first diagnosis were (7.6±5.5) years, it is (2.1±1.9) years in boys, (8.7±5.4) years in girls, significantly younger for boys ( t=-3.86, P=0.004). The chief complaint was external genitalia malformation for boys, and short stature (13 cases) and dysplastic external genital for girls (4 cases). Five girls presented with features of Turner syndrome. The gonadal phenotypes included mixed gonadal dysplasia (MGD, 6 cases), complete gonadal dysplasia (CGD, 10 cases), unilateral ovotestis (2 cases), possible ovaries (1 case) and undetermined gonad (1 case). One female with dysplastic genital was reassigned to male, and the gender of the remaining cases remained unchanged. Seven females were treated with recombinant human growth hormone. The height increased by (17±7) cm during the (2.9±1.2) years follow-up. No gonadal malignancy was observed. The karyotype was 45, X/46, XY in 16 cases, and 45, X/46, X,+mar in 4 cases. All of the 4 marker chromosomes were derived from Y chromosome confirmed by CNV-seq. SRY gene was detected in all 20 patients genome, and AZF deletion was found in 7 girls. Conclusions:45, X/46, XY mosaicism presented with dysplastic external genital or female with remarkable short stature. Gonadal phenotypes included MGD, CGD and ovotestis. AZF microdeletions were found in the majority of female cases.

OBJECTIVE: To explore the genetic basis for a child with multiple malformation and growth retardation. METHODS: The child was subjected to low-coverage massively parallel copy number variation sequencing (CNV-seq) based on next generation sequencing (NGS) technique. RESULTS: G-banding karyotyping analysis has found no abnormality in the boy and his parents. CNV-seq analysis discovered that the child has carried a heterozygous 4.36 Mb deletion (24 020 000-28 380 000) at 7p15.3p15.1. The same deletion was not found in either parent. The deletion has encompassed 28 OMIM genes including HOXA13, CYCS, DFNA5, HOXA11 and HOXA2. Among these, HOXA13 has been associated with distal limb deformity, hypospadias and cryptorchidism. HOXA1, HOXA3 and HOXA4 are involved in the formation of cardiac primordia and primordial tube, and HOXA2 is involved in the development of auditory system. The clinical phenotype of the child was consistent with that of 7p15 deletion syndrome. CONCLUSION Haploinsufficiency of HOXA1, HOXA2, HOXA3, HOXA4 and HOXA13 genes may underlie the clinical phenotype of the child, which is comparable to 7p15 deletion syndrome.

OBJECTIVE: To explore the genetic basis for a patient with Leydig cell hypoplasia. METHODS: Whole exome sequencing was used to detect genetic variants in the patient. Suspect variants were verified by PCR and Sanger sequencing of the family members. RESULTS: The patient was found to carry two novel variants, namely c.265A>T (p.Ile189Leu) and c.422T>C (p.Val141Ala), of the luteinizing hormone receptor gene (LHCGR), where were respectively inherited from her father and mother. Upon prenatal diagnosis, the fetus was found to be a heterozygous carrier of the c.265A>T (p.Ile189Leu) variant. CONCLUSION The compound heterozygous variants of c.265A>T (p.Ile189Leu) and c.422T>C (p.Val141Ala) of the LHCGR gene probably underlie the Leydig cell hypoplasia in the patient.