Background

Birth defects are a major public health problem that lead to miscarriage, foetal death, premature birth and childhood disabilities [1]. In China, approximately 5.6% of newborns are affected by birth defects annually; of these, chromosome aberrations account for more than 80% of the genetic causes, including abnormalities in chromosome number (aneuploidy) or structure, large fragment deletion/duplication, and pathogenic copy number variations (CNVs) [2, 3]. With the implementation of the universal “two child” policy, the proportion of birth defects has increased. This increase might be partly due to the increase in maternal age at delivery, the proportion of mothers with complications, and the number of multiple pregnancies. However, the increase in the number of prenatal screening or prenatal diagnoses for pregnant women of advanced age in China might have alleviated this increasing trend in birth defects [4].

For decades, karyotype analysis has been widely used as the “gold standard” for chromosome aberrations, as it can identify aneuploidy, translocation and inversion of chromosomes. However, karyotyping cannot detect abnormalities in chromosome fragments smaller than 5–10 Mb. Notably, more than 300 types of microdeletion/microduplication syndromes that are caused by CNVs smaller than 5 Mb have been identified, and they account for half of the birth defects caused by chromosome aberrations. CNV sequencing (CNV-seq) technology has brought opportunities and challenges to the detection of chromosome aberrations smaller than 5 Mb. In 2019, genetic experts suggested that CNV-seq could be used as a first-line prenatal diagnosis test for pregnant women who may have foetal chromosome abnormalities in China [5, 6].

Large fragment deletions in chromosome Xq22 might cause neurodevelopmental disorders, including severe intellectual disability and behavioural abnormalities. In this study, we report a female foetus who carries a heterozygous 5.29 Mb deletion in chromosome Xq22.1–q22.3 (including 7 known morbid genes), which was inherited from her healthy mother who had a normal phenotype with normal intelligence.

Methods

Karyotype analysis

The pregnant women underwent amniocentesis for karyotype analysis to identify chromosome aberrations of the foetus. In addition, karyotype analysis of peripheral blood were performed in the nonconsanguineous parents to determine the possible causes of chromosome aberration. Using conventional G-banding analysis technology, twenty-five metaphases were analysed at the 550 chromosome band resolution.

CNV sequencing analysis

CNV sequencing procedures, including DNA extraction, library construction, next-generation sequencing (NGS), bioinformatics analysis, and quality control (QC), were performed in our NGS laboratory with the Ion Torrent platform (BioelectronSeq 4000 sequencing system: Life Technologies, USA) according to the manufacturer’s protocol (Product No. S30030).

Results

A healthy pregnant woman, who was 37 years old with G3P1A1, had a 12-year-old healthy daughter. The pregnant woman requested prenatal diagnosis due to advanced maternal age. 3D ultrasound examination showed no evidence of foetal anomalies. She underwent amniocentesis for karyotype analysis and CNV-seq at 23 + 5 weeks gestation at Taizhou Hospital of Zhejiang Province. The foetal karyotype analysis showed a normal female karyotype of 46,XX (Additional file 1). However, the results of CNV-seq analysis indicated a 5.29 Mb deletion in chromosome Xq22.1–q22.3 (GRCh37/hg19: chrX: 100,460,000–105,740,000), which may affect 98 genes from DRP2 to NAP1L4P2 according to the Ensembl genome browser (https://asia.ensembl.org/) (Fig. 1) and the ClinGen database (https://www.clinicalgenome.org/) (Additional file 2). According to the DECIPHER database (https://www.deciphergenomics.org), this CNV deletion encompassed 42 OMIM genes, including DRP2, TAF7L, TIMM8A, BTK, RPL36A, GLA, HNRNPH2, ARMCX1 ~ 6, ZMAT1, BEX1 ~ 5, NXF2 ~ 5, TMSB15A, GPRASP1 ~ 2, BHLHB9, RAB40AL, TCEAL7, RAB40A, TCEAL1, MORF4L2, PLP1, RAB9B, TMSB15B, H2BW1, SLC25A53, ESX1, IL1RAPL2, TEX13A, NRK, and SERPINA7 [7]. Among these OMIM genes, there are 7 known morbid genes, including TIMM8A, BTK, GLA, HNRNPH2, GPRASP2, PLP1, and SERPINA7. In addition, the nonconsanguineous parents have a normal phenotype and are of normal intelligence. Their intellectual levels have not been precisely tested, but they judged to be normal from their normal social activities.

Fig. 1
figure 1

Ensembl genome browser image showing the Xq22.1–q22.3 deletions (GRCh37: ChrX: 100,460,000–105,740,000). Red frame indicate the location of the deletion regions identified in the Chinese pedigree in this study

Full size image

When the pregnant woman had genetic counselling in our prenatal diagnosis centre, we learned that she had a term birth of a healthy girl in 2010 and suffered a termination of pregnancy due to the 46, XXX karyotype of the foetus in 2018. The family wanted to know whether the foetus would have genetic defects after birth. Therefore, we further investigated this pedigree to determine the possible causes of the Xq22.1–q22.3 deletion (Fig. 2). Is it due to parental inheritance or a novel foetal mutation? Further pedigree analysis indicated that the CNV deletion of this foetus was inherited from her healthy mother. Moreover, two more healthy female family members (the pregnant woman’s daughter and mother) were identified to carry the same Xq22.1–q22.3 deletion (Fig. 3). The pregnant woman has a normal clinical phenotype with regular menses and normal fertility. There were no problems during pregnancy or delivery. Her daughter is now 12 years old with normal physical and psychomotor development. Her mother is now 65 years old with normal physical and psychomotor development. Through a genotype–phenotype correlation analysis, although the 5.29 Mb deletion in chromosome Xq22.1–q22.3 was inherited from a normal phenotype parent, it is still considered to be a pathogenic CNV in this pedigree as it contains 7 known morbid genes (TIMM8A, BTK, GLA, HNRNPH2, GPRASP2, PLP1, and SERPINA7).

Fig. 2
figure 2

Three-generation pedigree of a Chinese family and carries a heterozygous 5.29 Mb deletion in chromosome Xq22.1–q22.3

Full size image
Fig. 3
figure 3

Chromsomal aberrations revealed by CNV-seq analysis are shown with Agilent Genomic Workbench (Agilent Technologies) in chromosome view. X- and Y-axes indicate chromosomal location and signal log2 ratio, respectively. A microdeletion is shown in Xq22.1–q22.3 region (5.29 Mb)

Full size image

After genetic counselling, the couple decided to continue with the pregnancy. On February 28, 2022, a female neonate weighing 4.4 kg and 49 cm in length was born at 39 plus 3 weeks of pregnancy by spontaneous labour. The foetus had a five-minute Apgar score of 10 points, and no abnormal clinical symptoms or signs have been observed to date.

Discussion

In this rare Chinese pedigree, no abnormality was found in the G-banding karyotype analysis of the foetus or her parents. As the “gold standard” for chromosome aberrations, conventional Giemsa-banding karyotype analysis cannot detect chromosome abnormalities at a resolution of smaller than 5–10 Mb. However, CNV-seq technology provides opportunities and challenges to detect chromosome aberrations smaller than 5 Mb. In this study, CNV-seq analysis of uncultured amniotic fluid cells showed a 5.29 Mb deletion (GRCh37: chrX: 100,460,000–105,740,000) in chromosome Xq22.1–q22.3. It appears that the 5.29 Mb deletion in Xq22.1–q22.3 is a rare chromosome aberration. The foetus inherited this CNV deletion from her healthy mother.

A total of 98 genes were mapped to this 5.29 Mb deletion CNV. This fragment encompasses 7 known morbid genes, translocase of inner mitochondrial membrane 8A (TIMM8A), Bruton tyrosine kinase (BTK), galactosidase alpha (GLA), heterogeneous nuclear ribonucleoprotein H2 (HNRNPH2), G protein-coupled receptor associated sorting protein 2 (GPRASP2), proteolipid protein 1 (PLP1), and serpin family A member 7 (SERPINA7). According to the OMIM database (http://omim.org/), defects in the TIMM8A gene are the cause of Mohr–Tranebjaerg syndrome (MTS) [MIM #304700], defects in BTK are the cause of X-linked agammaglobulinemia (XLA) [MIM #300755], defects in GLA are the cause of Fabry disease (FD) [MIM #301500], defects in HNRNPH2 are the cause of the bain type of X-linked syndromic intellectual developmental disorder (MRXSB)[MIM #300986], defects in GPRASP2 are the cause of X-linked deafness-7 (DFNX7) [MIM #301018], defects in PLP1 are the cause of Pelizaeus–Merzbacher disease (PMD) [MIM #312080] or Spastic paraplegia 2 (SPG2) [MIM #312920], and defects in SERPINA7 are the cause of Thyroxine-binding globulin quantitative trait locus (TBGQTL) [MIM #300932].

A literature review identified that more than 43 families and 56 cases involving the affected region of Xq22.1–q22.3 deletion or a deletion that partially overlaps have been previously reported [8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31]. None of these previously reported cases had the same CNV deletion as the Chinese pedigree we reported. As shown in Table 1, we analysed the genotype–phenotype correlations of these patients with CNV deletions in chromosome Xq22.1–q22.3. Among them, the phenotype of female cases mainly include severe mental or physical limitations [9, 12,13,14,15,16,17, 27]. But so far, only one 4-year-old female of Xq22.1 → qter deletion had a normal phenotype [32]. Fortunately, in this Chinese pedigree, all three females with the same Xq22.1–q22.3 deletion have a normal phenotype, most likely due to complete inactivation of the abnormal X chromosomes in females [14, 17]. Notably, no abnormal clinical symptoms or signs have been observed in the fourth female neonate in this Chinese pedigree to date. However, further follow-up will still be necessary to evaluate the phenotype.

Table 1 Summary of the genotype–phenotype correlation of chromosome Xq22.1–q22.3 deletions
Full size table

In addition, we focus on the genetic patterns of these morbid genes. Mohr–Tranebjaerg syndrome is caused by mutations in the TIMM8A gene, which is a rare X-linked recessive disorder resulting in early-onset hearing impairment, progressive visual deterioration, and gradual dystonia. Some female carriers showed signs of minor neuropathy and mild hearing impairment [33, 34]. Fabry disease is a rare X-linked lipid storage disorder caused by a deficiency or absence of lysosomal alphagalactosidase A, which encoded by GLA gene. It is worth noting that heterozygous women should not be called carriers because they often been reported with a wide range of clinical symptoms. The early clinical manifestations mainly include acroparesthesias, angiokeratomas, pain crisis, and cornea verticillata, among other abnormalities. It therefore appears that Fabry disease affects both hemizygotes and heterozyotes, and should be considered an X-linked dominant disorder [35, 36]. Pelizaeus–Merzbacher disease is an X-linked recessive central nervous system disorder, which belongs to the group of hypomyelinating leukodystrophy (HLD1). PMD principally affect males and occasionally observed in carrier females, which is characterized clinically by nystagmus, spastic quadriplegia, ataxia, and developmental delay [9, 17, 37]. In addition, there was no report of male patients with large fragment Xq22 deletions. This is probably because larger Xq22 deletions may lead to embryonic lethality in males, since male patients with smaller nullisomy in the vicinity show more severe developmental delay [8, 10,11,12, 17].

Conclusions

X chromosomal deletions are infrequent findings in prenatal diagnosis and present a difficult counselling challenge when they occur. Genotype–phenotype correlation analysis can provide reliable clinical genetic counselling for chromosome abnormality reports. In addition, the X-inactivation pattern could provide an opportunity for more informative genetic counselling when a de novo CNV deletion in the X chromosome is detected.