[Analysis of PAH gene variants and prenatal diagnosis for 43 Chinese pedigrees affected with Phenylketonuria].

OBJECTIVE: To explore the characteristics of phenylalanine hydroxylase (PAH) gene variants and prenatal diagnosis for 43 Chinese pedigrees affected with Phenylketonuria (PKU). METHODS: Forty three PKU pedigrees diagnosed at the First Affiliated Hospital of Zhengzhou University between 2019 and 2021 were selected as the study subjects. Variants of the PAH gene of the probands were screened by high-throughput sequencing, and candidate variants were verified by Sanger sequencing. Negative cases were further analyzed by multiplex ligation-dependent probe amplification (MLPA) to detect large fragment deletions and duplications of the PAH gene. For 43 women undergoing subsequent pregnancy, Sanger sequencing, MLPA, combined with short tandem repeats (STR) sequence-based linkage analysis, were carried out for prenatal diagnosis. RESULTS: Among the 86 alleles carried by the 43 probands, 78 nucleotide variants (90.70%) and 3 large deletions (3.49%) were found based on high-throughput sequencing and MLPA. The 81 mutant alleles had included 21 missense variants, 5 splice site variants, 4 nonsense variants, 2 microdeletions, 1 insertional variant and 2 large fragment deletions. Relatively common variants have included p.Arg243Gln (23.26%), p.Arg111Ter (8.14%), EX6-96A>G (6.98%), p.Val399Val (5.81%) and p.Arg413Pro (4.65%). Most of the variants were located in exons 7, 11, 3, 6 and 12. For the 43 families undergoing prenatal diagnosis, 9 fetuses (20.45%) were diagnosed with PKU, 20 (45.45%) were heterozygous carriers, and 15 (34.09%) did not carry the same pathogenic allele as the proband. All neonates were followed up till 6 months old, and the accuracy of prenatal diagnosis was 100%. CONCLUSION: The combination of high-throughput sequencing, Sanger sequencing, MLPA and linkage analysis can increase the diagnostic rate of PKU and attain accurate prenatal diagnosis.

[Analysis of a child with Microvillus inclusion disease due to variants of MYO5B gene and a literature review].

OBJECTIVE: To explore the clinical and genetic characteristics of a neonate with Microvillus inclusion disease (MVID). METHODS: A neonate with MVID admitted to the First Affiliated Hospital of Zhengzhou University in May 2019 was selected as the study subject. Clinical data were collected. Whole exome sequencing (WES) was carried out, and candidate variants were verified by Sanger sequencing and multiple ligation-dependent probe amplification (MLPA). A literature was also carried out to summarize the clinical and genetic characteristics of MVID. RESULTS: The prematurely born neonate had presented with unexplained refractory diarrhea and metabolic acidosis. Active symptomatic treatment was ineffective, and the child had died at 2 months old. WES revealed that he had harbored compound heterozygous variants of the MYO5B gene, namely c.1591C>T (p.R531W) and deletion of exon 9. Sanger sequencing showed that the R531W variant was inherited form his father, and MLPA confirmed that the exon 9 deletion was inherited from his mother. Seven children with MVID were reported in China, of which one was lost during follow-up and six had deceased. One hundred eighty eight patients were reported worldwide and only one was cured. The clinical features of MVID had included refractory diarrhea, metabolic acidosis and poor prognosis. CONCLUSION: The child was diagnosed with MVID due to the compound heterozygous variants of the MYO5B gene, which has provided a basis for genetic counseling and prenatal diagnosis.

[Genetic analysis of a rare case with Disorder of sex development due to structural rearrangement of Y chromosome].

OBJECTIVE: To explore the genetic basis for a rare case with Disorder of sex development. METHODS: Clinical data of the patient was collected. Chromosomal karyotyping, SRY gene testing, whole exome sequencing (WES), low-coverage massively parallel copy number variation sequencing (CNV-seq), fluorescence in situ hybridization (FISH), and whole genome sequencing (WGS) were carried out. RESULTS: The patient, a 14-year-old female, had manifested short stature and dysplasia of second sex characteristics. She was found to have a 46,XY karyotype and positive for the SRY gene. No pathogenic variant was found by WES, except a duplication at Yp11.32q12. The result of CNV-seq was 47,XYY. FISH has confirmed mosaicism for a dicentric Y chromosome. A 23.66 Mb duplication on Yp11.32q11.223 and a 5.16 Mb deletion on Yq11.223q11.23 were found by WGS. The breakpoint was mapped at chrY: 23656267. The patient's karyotype was ultimately determined as 46,X,psu idic(Y)(q11.223)/46,X,del(Y)(q11.223). CONCLUSION: The combination of multiple methods has facilitated clarification of the genetic etiology in this patient, which has provided a reference for the clinical diagnosis and treatment.

[Application of low-depth whole genome sequencing for copy number variation analysis in children with disorders of sex development].

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.

Molecular study and genotype-phenotype in Chinese female patients with 46, XY disorders of sex development.

OBJECTIVE: The rare condition 46, XY disorders of sex development (DSDs) is characterized by the female phenotype and male karyotype. We aimed to describe the genetic basis of 46, XY DSDs in nine patients and the genotype-phenotype relationships of the genes involved. METHODS: Targeted next-generation sequencing (NGS) was used to analyze the underlying hereditary etiology in nine female patients with 46, XY DSDs. In silico analyses were used to predict the effects of novel variants on the protein function of the identified genes. RESULTS: Primary amenorrhea with the absence of puberty, inguinal hernia, and clitoridauxe were common complaints. All enrolled patients had a differential etiology by genetic testing, and five novel genetic variants involved in four genes (SRY, AR, NR5A1, and LHCGR) were identified. A novel nonsense variant of SRY c.51C > G was found in XY patients without testicles. Two novel heterozygous variants, i.e. c.265A > T (Ile89Leu) and c.422T > C (Val141Ala), of the LHCGR gene were found in male pseudo-hermaphroditism. CONCLUSIONS: We expanded the genetic mutation spectrum and described in detail the genotype-phenotype relationships of 46, XY DSDs. DNA sequencing for SRY should be a priority in female patients with 46, XY DSDs. NGS is useful for clarifying genetic pathogenesis and could provide a basis for clinical diagnosis and treatments of patients with 46, XY DSDs.

Two females presenting primary amenorrhea diagnosed with Kallmann syndrome caused by novel FGFR1 variants.

Primary amenorrhea as the common symptom has a complicated etiology, and genetic disorders are non-negligible. Kallmann syndrome (KS) is a rare inherited disease characterized by hypogonadotropic hypogonadism and anosmia. KS is uncommon in women and is an unusual cause of primary amenorrhea. Herein, we described the clinical features in two female patients presenting primary amenorrhea without puberty. Magnetic resonance imaging showed dysplastic or absent olfactory bulbs and tracts. Eventually, they were diagnosed with KS caused by FGFR1 novel variants, c.315_317delCCCinsTT and c.1081G>A, using whole-exome sequencing (WES). We emphasize that KS should be considered in females presenting primary amenorrhea and anosmia, and recommend that WES should be a priority in the patients presenting primary amenorrhea without secondary sex characteristics.

Novel mutations of the CYP17A1 gene in four Chinese 46,XX cases with partial 17a-hydroxylase/17,20-lyase deficiency.

The condition 17a-Hydroxylase/17,20-lyase deficiency (17-OHD) is a rare kind of congenital adrenal hyperplasia (CAH) characterized by failure to synthetize cortisol, adrenal androgens and gonadal steroids. Partial deficiency is much rarer, presenting with subtler symptoms. In this study, we summarized the clinical characteristics and identified the underlying gene mutation in four Chinese 46,XX patients with partial 17-OHD. Mutational analysis of the CYP17A1 gene was performed by polymerase chain reaction (PCR) and Sanger sequencing. Clinical and hormonal findings in these patients were consistent with typical manifestations of partial 17-OHD. All patients were found to have a compound heterozygous mutation of the CYP17A1 gene, with five mutations identified. Among them, c.887 T > C(p. I296T), c.1019G > A(p. R340H) and c.1346G > A(p. R449H) were novel missense mutations. In conclusion, we identified three novel missense mutations of the CYP17A1 gene from four patients with partial 17-OHD deficiency. Genotype-phenotype correlation analysis revealed that these novel mutations can lead to partial 17-OHD. Our findings thus provide novel insight into the clinical evaluations and molecular basis of 17-OHD.

Clinical and Genetic Characteristics of 17 α-Hydroxylase/17, 20-Lyase Deficiency: c.985_987delTACinsAA Mutation of CYP17A1 Prevalent in the Chinese Han Population.

OBJECTIVE: 17 α-hydroxylase/17, 20-lyase deficiency (17-OHD) is a rare recessive hereditary disease that can be attributed to cytochrome P450 17 α-hydroxylase deficiency caused by CYP17A1 gene mutations. METHODS: A large cohort of 10 Chinese Han patients with 17-OHD from 2012 to 2020 were enrolled. The clinical and biochemical features were investigated, and genetic mutations of CYP17A1 were analyzed by polymerase chain reaction-Sanger sequencing. Karyotype identification and the SRY gene test were also carried out. In silico analysis was used to predict the effects of genetic mutations on the protein function. RESULTS: All patients were female. Common complaints were hypertension, hypokalemia, and primary amenorrhea. The karyotype was 46, XY, and the SRY gene was detected in 7 patients; the karyotype was XX in the remaining 3 patients. A total of 7 mutations including Y329N, Y329X, Y329Lfs∗, R96W, A82D, S380N, and A487_P489del have been identified in the CYP17A1 gene. The Y329Lfs∗ mutation was found in 9/10 (90%) of patients with a high allele frequency of 70%. In silico prediction showed that a novel variant of c.1139G>A (S380N) occurs at a conserved residue and can cause disease. CONCLUSION: We presented a detailed description of the clinical and genetic characteristics in Chinese patients with 17-OHD and concluded that Y329Lfs∗ mutation of CYP17A1 is prevalent in the Chinese Han population. Therefore, hotspot screening by polymerase chain reaction-Sanger sequencing for exon 6 of CYP17A1 could contribute to the rapid diagnosis of 17-OHD in China. Genetic counseling based on the genetic diagnosis for at-risk relatives is advised.

[Genetic analysis of a case with ectodermal dysplasia using whole exome sequencing].

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.

[Genetic diagnosis for a patient with Leydig cell hypoplasia caused by two novel variants of LHCGR gene].

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.

[Phenotypic and genetic characteristics of a child with 7p15 deletion syndrome].

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.

Mix-breeding with HEV-infected swine induced inapparent HEV infection in SPF rabbits.

Studies have shown that swine HEV (sHEV) and rabbit HEV (rHEV) can experimentally infect rabbits and swine, respectively. However, no published data have documented isolating sHEV strains from rabbits in natural environment so far. To clarify the possibility of natural cross-species transmission of sHEV to rabbits, the pigs with HEV infection were farmed along with SPF rabbits in the same enclosed space. Five of 10 rabbits had seroconversion for anti-HEV antibody from the third week after mix-breeding. However, HEV RNA remained undetectable in feces, serum, liver and bile of the ten rabbits; and no obvious elevation of ALT was observed. The results possibly suggested that sHEV might lead to an inapparent infection of SPF rabbits by fecal-oral route.

Swine and rabbits are the main reservoirs of hepatitis E virus in China: detection of HEV RNA in feces of farmed and wild animals.

Hepatitis E virus (HEV) infection is recognized as a zoonosis. The prevalence of HEV RNA and anti-HEV antibodies in many animal species has been reported, but the host range of HEV is unclear. The aims of this study were to investigate HEV infection in various animal species and to determine the reservoirs of HEV. Eight hundred twenty-two fecal samples from 17 mammal species and 67 fecal samples from 24 avian species were collected in China and tested for HEV RNA by RT-nPCR. The products of PCR were sequenced and analyzed phylogenetically. The positive rates of HEV RNA isolated from pigs in Beijing, Shandong, and Henan were 33%, 30%, and 92%, respectively, and that from rabbits in Beijing was 5%. HEV RNA was not detectable in farmed foxes, sheep or sika deer, or in wild animals in zoos, including wild boars, yaks, camels, Asiatic black bears, African lions, red pandas, civets, wolves, jackals and primates. Sequence analysis revealed that swine isolates had 97.8%-98.4% nucleotide sequence identity to genotype 4d isolates from patients in Shandong and Jiangsu of China. Phylogenetic analysis showed that swine HEV isolates belong to genotype 4, including subgenotype 4h in Henan and 4d in Beijing and Shandong. The rabbit HEV strains shared 93%-99% nucleotide sequence identity with rabbit strains isolated from Inner Mongolia. In conclusion, swine and rabbits have been confirmed to be the main reservoirs of HEV in China.

Hepatitis E vaccine immunization for rabbits to prevent animal HEV infection and zoonotic transmission.

Hepatitis E virus (HEV) infection has become a significant global public health concern as increasing cases of acute and chronic hepatitis E are reported. HEV of animal origin was proved to be a possible source of human infection and a previous study showed that the recent licensed HEV 239 vaccine can serve as a candidate vaccine to manage animal sources of HEV infection. However, previous immunization strategy for rabbits was the same as that for human, which is too costly to conduct large-scale animal vaccination. In an effort to reduce the costs, three vaccination schemes were assessed in the present study. Forty specific pathogen-free (SPF) rabbits were divided randomly into five groups with eight animals for each and inoculated intramuscularly with different doses of HEV 239 and placebo, respectively. All animals were challenged intravenously with swine HEV-4 and rabbit HEV of different titers 7 weeks after the initial immunization and then fecal virus excretion was monitored for 10 weeks. The results indicated that immunizing rabbits with two 10µg doses of the vaccine is superior to vaccination with two 20µg doses or a single 30µg dose, which can protect rabbits against homologous and heterologous HEV infection. These findings could enable implementation of large-scale animal vaccination to prevent rabbit HEV infection and zoonotic transmission.

Hepatitis E virus infection in farmed rabbits and swine in the Eastern Chinese city Lianyungang: showing no potential interspecies transmission.

Rabbit HEV isolated recently from farmed rabbits in China has been shown experimentally to be able to infect both cynomolgus macaques and pigs. The purpose of the present study was to investigate the extent to which cross-species transmission of rabbit HEV in farm settings is a significant factor in the spread of this zoonotic infection. Rabbit and swine feces were collected from the same area in Eastern China (Lianyungang City) and analyzed by RT-PCR. Partial genome sequencing of a 365 bp region of ORF2 from the HEV positive rabbit samples revealed that they had 92-99% sequence identity with rabbit strains (rbIM163-c1 and rbIM004) isolated from Inner Mongolia. Similarly, sequencing of a 765 bp region of ORF2 of HEV positive swine samples showed 96-98% sequence identity with genotype 4d isolates collected from patients in the Yantai and Nanjing regions of China. By contrast, the sequence identity between the rabbit and swine isolates was only 73-75%, with no molecular biological evidence of interspecies transmission having occurred. It is concluded that whilst interspecies infection with rabbit HEV can be achieved experimentally, in the field it is not a significant factor in zoonotic disease transmission at least in the area of China where this study was undertaken.

SPF rabbits infected with rabbit hepatitis E virus isolate experimentally showing the chronicity of hepatitis.

This study focused on investigating the pathogenesis seen in specific-pathogen-free (SPF) rabbits following infection with a homologous rabbit HEV isolate (CHN-BJ-rb14) and comparing it to that seen following infection with a heterologous swine genotype 4 HEV isolate (CHN-XJ-SW13). Three of the four animals inoculated with the homologous rabbit HEV became infected, exhibiting an intermittent viremia, obvious fluctuations of liver function biomarkers alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and persistent fecal virus shedding throughout the nine month study. In addition, liver histopathology showed both chronic inflammation and some degree of fibrosis. Both positive and negative-stranded HEV RNA and HEV antigen expression were detected in liver, brain, stomach, duodenum and kidney from the necropsied rabbits. Inflammation of extrahepatic tissue (duodenum and kidney) was also observed. Three of the four rabbits inoculated with the heterologous genotype 4 swine HEV also became infected, showing similar levels of anti-HEV antibody to that generated following infection with the homologous virus isolate. The duration of both viremia and fecal shedding of virus was however shorter following infection with the heterologous virus and there was no significant elevation of liver function biomarkers. These results suggest that rabbit HEV infection may cause more severe hepatitis and prolong the course of the disease, with a possible chronic trend of hepatitis in SPF rabbits.