Clinical descriptions of the SHFM family

The proband (I:2) exhibits the characteristic presentation of median division malformed clefts in both hands and feet, resulting in a diagnosis of split hand and feet malformation based on the patient’s clinical manifestations. Notably, the proband’s husband (I:1) demonstrates no clinical abnormalities. As depicted in the pedigree chart (Fig. 1a), the family manifests a hereditary genetic trait indicative of autosomal dominant inheritance, which suggests the likelihood of occurrence in each successive generation. Approximately 3 years ago, the couple experienced a pregnancy resulting in a child with hand-foot deformity (II:1). Following medical advice, they opted for induced labor. Subsequently, the couple welcomed the birth of a healthy son (II:2).

Fig. 1

Family pedigree and clinical characteration. Pedigree of the family. I2 is the proband. a, b Photos of the hands and the feet of the fetus

Approximately 4 months ago, the patient became pregnant once again. Ultrasound assessment indicated an intrauterine pregnancy with a single live fetus at a gestational age of 12 weeks and 4 days. Fetal NT measurements fell within the normal range. Further ultrasound observations revealed specific parameters, including a head and hip diameter (CRL) of 60 mm, a double parapedal diameter (BPD) of 21 mm, a heart rate of 151 beats per minute, and a normal cranial appearance with no evidence of anencephaly, open brain deformity, or severe sternoabdominal wall defects. The placenta was situated on the posterior wall with a thickness of 12 mm, and the maximum depth of amniotic fluid reached 35 mm. Notably, the inner cervix was closed. However, the development of both hands and feet in the fetus (II:3) exhibited anomalies characterized by a median laceration. In light of the family’s medical history, the couple made the decision to terminate the pregnancy at 13 weeks (Fig. 1b).

Chromosome copy number variations screen of the family

Subsequently, the family underwent chromosome copy number variation (CNV) screening. Traditionally, a combination of next-generation sequencing technology and SNP assay was employed for CNV detection, with a focus on chromosome aneuploidy and the presence of fragments exceeding 100 Kb in size. In this specific pedigree, standard DNA samples were subjected to CNV analysis using next-generation sequencing. The results indicated that the husband and their son exhibited no chromosome aneuploidy or CNV at loci related to SHFM. Conversely, both the proband (I:2) and the fetus displayed a microduplication involving chromosome 10 (Fig. 2a). Further validation through SNP array analysis yielded consistent findings (Fig. 2b). In-depth analysis via next-generation sequencing unveiled approximately 0.62 Mb duplications in the q24.31q24.32 region of chromosome 10 for the proband (seq [GRCh37]dup(10)(q24.31q24.32) chr10:g.102860001_103480000dup). Chromosome analysis of the aborted fetal tissue (II:3) demonstrated a similar 0.64 Mb copy number repeat in the q24.31q24.32 region of chromosome 10 (seq [GRCh37]dup(10)(q24.31q24.32) chr10:g.102820001_103460000dup) (Fig. 2c). Importantly, a comprehensive query revealed the presence of several consecutive gene repeats within this genomic region, including LBX1, BTRC, POLL, and DPCD. These genes significantly overlap with the core pathogenic region associated with congenital hand and foot cleft malformation, as documented in the OMIM database and relevant literature [5]. The evaluation of pathogenicity was conducted in accordance with the guidelines set forth by the American Society of Medical Genetics and Genomics (ACMG) [31], thereby suggesting that the observed variation was likely pathogenetic (LP) (Table 1). Subsequent scrutiny of the DECIPHER database revealed potential clinical manifestations among individuals with duplications in close proximity to this locus, notably hand-foot cleft deformity and the presence of multiple fingers anterior to the axis (DECIPHER ID: 360984, 368251, 368247). No CNV report was discerned within the general population database known as DGV. In summary, through extensive examination of cells utilizing NGS and SNP assays, it was ascertained that the overlap of pathogenic sites between the proband and the fetus included a span of 0.62 Mb, thus indicating a likelihood of pathogeny. Furthermore, no mutations were detected within the healthy son and the husband of the proband.

Fig. 2

Copy number variation detection in the SHFM3 family using both SNP array and NGS. a The result of copy number repeats of low coverage NGS. The proband (left) and the fetus (right). b The result of copy number repeats of SNP assay. The proband (left) and the fetus (right). All suggest that the proband and her fetus have the 0.6 Mb microduplication at chromosome 10. c The display of CNVs at chr10

Table 1 Pathogenicity of the family is assessed according to ACMG guidelinesIdentification of cnvs at the single-cell level and validation using SNP based platforms

The identification of CNVs at the single-cell level and their subsequent validation using SNP-based platforms pose formidable challenges in clinical practice due to the limitations in cell acquisition arising from technical constraints. The resolution of microduplication variants at the single-cell level constitutes a pressing issue warranting attention, with the potential to enhance the realm of clinical genetic diagnosis. Consequently, to address the endeavor of identifying microduplications at the single-cell level, DNA samples were subjected to further dilution, reducing them to approximately 10 pg, corresponding to the single-cell level. This was followed by comprehensive genome amplification via MDA and MALBAC techniques, respectively. These processes facilitated the detection of copy number variations within chromosomes and mutations within genes using linkage analysis.

Through the utilization of karyomapping technology, the process of karyotype localization was executed on couples, sons, and malformed fetuses, alongside the amplification products of individual cells. Subsequently, an elucidation of the DNA arrangement rule governing genetic material ensued. The identification of the haplotype DNA harboring pathogenic genes within this familial context was achieved by means of analysis conducted with BlueFuse Multi Software in accordance with the provided manual. As delineated in the illustrative model diagram, it was ascertained that the malformed fetus had inherited the haplotype of the mother (Fig. 3a). The presentation of the B allele Frequency plots and Log R Ratio plots adhered to stringent criteria to determine diploid presence. The yellow region denoted the locus of the BTRC (Fig. 3b-f). Notably, the LogR diagram exhibited an upward shift in the mother and the fetus within this familial cohort, indicative of SNP sites within this domain. Further scrutiny unveiled the presence of 230 Informative SNPs (effective SNPs) located in the 5′ terminus of the malformed fetus within the BTRC gene region, of which 53 originated from the maternal lineage and 20 from the paternal lineage. In the intragenic region, 33 active SNPs were identified, with 5 emanating from the maternal lineage and 1 from the paternal lineage. In the 3′ terminus, 287 active SNPs were observed, with 22 attributed to the maternal lineage and 45 to the paternal lineage. Subsequent analysis elucidated that the malformed fetus harbored a 442 kb copy number repeat in the 10q24.31 to 10q24.32 region. The pathogenicity of this SNP was conclusively assessed as pathogenic, including the BTRC gene and other genes associated with hand-foot deformity. Thus, corroborated by Karyomapping, the fetal chromosome and the genotype of the genetic disease remained entirely congruent with the SNP linkage analysis results.

Fig. 3

The pathogenic CNV detection on single cell level using Karyomapping. a Schematic representation of the linkage identified for the disease-carrying allele. Yellow indicates the allele carrying the heterozygous SNPs, which is inherited from the mother. b–f. From top to bottom represent the results of father (b), mother (c), fetus (d), normal son (e), and single cell expansion product (f). The selected area in the yellow box indicates the area where BTRC is located

Identification of cnvs at the single-cell level and validation using next generation sequencing and single molecular sequencing methods

Subsequently, further confirmation was undertaken through NGS analysis employing trace genomic DNA (gDNA) and the MDA products from aborted fetal tissue. The findings consistently revealed a duplication spanning 0.64 Mb. Notably, the identified gene was situated in the 10q24.31q24.32 region, and the findings disclosed the absence of the maternal mutation in the healthy son (II:2), while the aborted fetus (II:3) harbored the maternal heterozygous duplicationchr10:g.102860001_103480000dup (Fig. 4a).

Fig. 4

The pathogenic CNV detection on single cell level using sequencing. a The pathogenic CNV of the fetus at chromosome 10 using NGS. b The linkage analys of the family. c The pathogenic CNV of the fetus at chromosome 10 using single molecular sequencing method at gDNA and WGA products

To substantiate the feasibility of successfully identifying subtle variations at the single-cell level, SNP linkage analysis was conducted within this familial context. Genotypic information pertaining to SNP alleles at sites located within 2 M of upstream and downstream of the microduplication region was analyzed in the genomic DNA of the family members, as well as in the WGA products derived from single cells. As depicted in our results, the linkage analysis concurred with the findings of mutation site detection. Specifically, there were 13 informative SNPs within 1 M in healthy boy at the single-cell level (II:2-single cell), whereas the aborted fetus (II:3-single cell) exhibited 12 effective SNPs within 1 M (Fig. 4b). Based on the genotype of the SNP locus in the family member carrying the 620 kb region mutation, the haplotype carrying the 620 kb region mutation was inferred (Table 2). For instance, if rs11190644 is C/C in father, T/C in mother, and C/T in the malformed fetus, we can infer that the T bases are inherited from females, and so on for the other SNPs (we usually utilized at least five informative SNPs), then we can easily constructed the pathogenic and non-pathogenic haplotype; thus, utilizing the haplotype to deduce the inheritance of the variation. The results of SNP site analysis in single cell amplification products were congruent with those of conventional mutation detection; thus, signifying the successful recognition of 620 kb microduplications at the single-cell level in this study.

Table 2 Informative SNPs flanking the microduplication at chr10 of SHFM

Concurrently, the data underwent validation utilizing the Single-Molecule Gene Sequencing approach, employing both extensive and limited DNA WGA products, which also demonstrated a 0.64 Mb duplication positioned within the 10q24.31q24.32 locus (Fig. 4c). This conveys the congruence of outcomes obtained via distinct methodologies. It is noteworthy that the MDA product’s results exhibited discretization, rendering the detection of variations challenging.

The new strategy for identification of micro-variation

In summary, a novel methodology for discerning microrepeat variants at the single-cell level was devised and implemented (Fig. 5). Initially, both whole blood and tissue specimens were collected from members of the SHFM3-afflicted family. Subsequently, we conducted copy number variation and single nucleotide polymorphic site detection using NGS, SNP, and single molecular sequencing at the standard DNA level. These analyses revealed a 620 kb microduplication variation in both the proband and the affected fetus. we adopted single-cell-level DNA amplification techniques employing MDA and MALBAC. Furthermore, we used single-cell level DNA for whole gene amplification by MDA and MALBAC. Additionally, Karyomapping was executed following the amplification step. Karyomapping technology employs Illumina’s SNP chip to acquire SNP site information from the target sample, which is subsequently processed using BlueFuse Multi software for comprehensive SNP analysis. Consequently, haploblock information for each embryo chromosome is obtained and visualized within the software interface. Based on the haplotype segment data, as well as the distinctions between “key SNP” and “non-key SNP” for each segment, the pathogenic gene-carrying status of each embryo chromosome was assessed. Last but not least, next generation sequencing and single molecular sequencing methods will further help us to identify microduplicate variants at the single-cell level. Our analytical findings corroborate the detection of the 620 kb micro-repeat mutation after MDA amplification at the single-cell level, with linkage analysis attributing the mutation to the maternal lineage.

Fig. 5

The new strategy for identification of microduplication and microdeletion at single cell level