Novel biallelic multi-exon duplication in the PIGN gene associated with multiple congenital anomalies–hypotonia–seizures syndrome 1

Abstract

Background

Multiple congenital anomalies–hypotonia–seizures syndrome type 1 (MCAHS1) is a rare autosomal recessive disorder caused by PIGN variants affecting glycosylphosphatidylinositol (GPI) anchor biosynthesis. Exon-level duplications are exceptionally uncommon.

Case presentation

We describe a male neonate with multiple congenital anomalies, including auricular malformation, submucous cleft palate, accessory nipple, cryptorchidism, microphallus, clubfoot, and digital hypoplasia, accompanied by hypotonia and poor sucking. Whole-exome trio sequencing identified a biallelic duplication spanning exons 4–22 of PIGN, with an apparent four-copy state in the proband.

Conclusion

This report expands the mutational spectrum of PIGN-related disorders and emphasizes the diagnostic value of trio-based exome sequencing in neonates with multiple congenital anomalies and hypotonia.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12887-026-06923-2.

Introduction

Multiple congenital anomalies–hypotonia–seizures syndrome 1 (MCAHS1) is a rare autosomal recessive disorder characterized by neonatal hypotonia, global developmental delay, and variable congenital malformations, often accompanied by intractable seizures and poor survival [1, 2]. MCAHS1 belongs to the broader group of glycosylphosphatidylinositol biosynthesis defects (GPIBDs), a spectrum of inherited disorders caused by pathogenic variants in genes involved in GPI-anchor formation [3–5]. These GPI anchors are essential glycolipids that tether more than 150 cell-surface proteins to the plasma membrane, playing vital roles in neural development, cell signaling, and embryonic morphogenesis [6].

Among the 20 enzymes participating in GPI-anchor assembly, PIGN encodes GPI ethanolamine phosphate transferase 1, which transfers phosphoethanolamine to the first mannose residue during early GPI-anchor synthesis [7, 8]. Pathogenic PIGN variants lead to loss of this enzymatic step, resulting in defective cell-surface expression of GPI-anchored proteins. To date, over 30 distinct PIGN mutations—including missense, nonsense, and splice-site variants—have been reported [4], producing a continuum of clinical severity ranging from neonatal lethality to mild developmental delay. Because of this variability, PIGN-related disease is considered a core representative of the MCAHS spectrum.

Most previously reported PIGN mutations are single-nucleotide substitutions or small indels predicted to cause loss of function. Structural variants involving PIGN remain rare, and exon-level duplications have seldom been documented [9]. The identification of such duplications may provide insights into the molecular consequences of intragenic structural variants, which can disrupt transcript architecture (e.g., aberrant splicing or frameshift) rather than simple gene overexpression.

Here, we report a male infant with multiple congenital anomalies, generalized hypotonia, and developmental delay, in whom exome sequencing identified a biallelic duplication encompassing exons 4–22 of PIGN. Segregation analysis revealed that both parents carried the same heterozygous duplication, and copy-number analysis indicated an apparent four-copy state in the proband.

This represents, to our knowledge, the first reported multi-exon duplication of PIGN with an apparent biallelic copy-number pattern. Clinically, the patient exhibited a milder phenotype than typical MCAHS1 cases—surviving infancy without overt seizures—which may reflect partial preservation of GPI-anchor biosynthetic function. This case expands the mutational and phenotypic spectrum of PIGN-related disorders and emphasizes the importance of copy-number variant analysis in exome-based diagnostic pipelines for infants with congenital anomaly syndromes.

Case description

The proband was a male infant, the first child of non-consanguineous Chinese parents, born at term after an uneventful pregnancy and spontaneous vaginal delivery. His birth weight was 3.15 kg (25th–50th percentile), length 50 cm (25th–50th percentile), and head circumference 34 cm (25th–50th percentile). Apgar scores were 9 and 10 at 1 and 5 min, respectively. Dysmorphic features were noted immediately after birth, including a low-set right ear with malformed helix, submucous cleft palate, an accessory nipple on the left chest, bilateral cryptorchidism, micropenis, bilateral talipes equinovarus, and digital hypoplasia of the fifth fingers. Distal digital hypoplasia was noted, involving the fifth fingers and the toes. He exhibited profound generalized hypotonia, weak spontaneous movement, and poor sucking that required nasogastric feeding within the first week of life.

Routine laboratory testing revealed normal hematologic and biochemical parameters, including normal serum alkaline phosphatase levels. Metabolic screening, tandem mass spectrometry, and urinary organic acid analysis showed no abnormalities. Cranial magnetic resonance imaging (MRI) revealed no major structural abnormalities. Echocardiography and abdominal ultrasound were unremarkable. Electroencephalography (EEG) demonstrated multifocal irritative discharges but no overt seizures.

At 12 months of age, the patient remained alive but showed severe global developmental delay. He could not roll over or laugh responsively and exhibited persistent generalized hypotonia with minimal antigravity movement. Feeding difficulty persisted, requiring nasogastric tube support due to weak suck and frequent choking. Despite these challenges, growth parameters—including head circumference (42 cm), weight (7.2 kg), and length (68 cm)—were within normal ranges for age. He was easily irritable but had no clinical seizures, and follow-up MRI remained unremarkable. Supportive therapies including physiotherapy and early developmental intervention were continued. Representative clinical features are shown in Fig. 1.

Fig. 1.

Clinical features of the patient with PIGN-related MCAHS1. a Both feet showing bilateral talipes equinovarus with overlapping toes and hypoplasia of the fifth digits. b Frontal view of the trunk demonstrating accessory nipple on the left chest and mild pectoral asymmetry. c Right lateral view of the face revealing a low-set and malformed right auricle with small helix. d External genitalia showing micropenis and bilateral undescended testes. e Oral examination displaying a submucous cleft palate with bifid uvula. f Renal ultrasound image showing mild left renal pelvic dilatation without cortical malformation

Trio-based whole-exome sequencing (WES) was subsequently performed to identify the molecular basis of the disorder.

Genetic analysis

Trio-based WES was performed on genomic DNA extracted from peripheral blood samples of the proband and his parents. Exome capture was conducted using the NEXome XP Panel v1.0 (Nanodigmbio, China), and sequencing was carried out on the NovaSeq 6000 platform (Illumina, San Diego, CA, USA). All sequencing and primary bioinformatic analyses were performed at Guangzhou JiaJian Medical Laboratory, China. Sequence alignment and variant calling were performed with reference to the human genome build hg19 (GRCh37).

Copy number variant (CNV) analysis was performed using AmCareCNV, a read-depth–based algorithm optimized for exon-level CNV detection from WES data. CNV calling was conducted using a batch-based multi-sample analysis strategy, in which 17 samples from the same sequencing batch were jointly analyzed to normalize capture efficiency and sequencing depth. To improve reliability, CNV findings were independently cross-validated using the ClinCNV approach. This analysis identified a biallelic duplication encompassing exons 4–22 of PIGN in the proband, while both parents were heterozygous carriers.

Based on WES data, the minimal duplicated region was defined as chr18:59768307–59,828,586 (hg19), and the maximal duplicated interval spanned chr18:59763184–59,854,738 (hg19). According to the MANE Select transcript (NM_176787.5), PIGN comprises 31 exons, of which exons 1–3 are non-coding. According to the capture design information provided by the diagnostic laboratory, probe coverage was limited within the coding regions, and no probes were designed in the upstream region beyond chr18:59828586. Therefore, the non-coding exons 1–3 of PIGN were not captured by the WES panel, and extension of the duplication toward the 5′ region could not be assessed. Due to the intrinsic limitations of WES-based CNV analysis, the precise structure, orientation (e.g., tandem or inverted), insertion site, and potential extension of the duplication beyond the captured regions could not be determined. This multi-exon duplication has not been reported in public databases, including the Database of Genomic Variants (DGV), ClinVar, or gnomAD, and was classified as a variant of uncertain significance (VUS) according to ACMG guidelines.

Quantitative PCR (qPCR) was performed to validate the CNV findings using two primer pairs targeting PIGN coding regions (CDS4 and CDS18) within the duplicated interval. qPCR validation using two independent primer sets consistently demonstrated an apparent four-copy state in the proband and three-copy states in both parents, in agreement with the WES-based CNV analysis. Primer sequences and genomic coordinates are provided in Supplementary Table S1.

Taken together, these results support the PIGN duplication as the most plausible molecular explanation for MCAHS1 and highlight the diagnostic value of integrating exon-level CNV detection into WES workflows for rare congenital anomaly syndromes. The genetic findings are illustrated in Fig. 2.

Fig. 2.

Genetic findings in the proband. a Pedigree of the family showing segregation of the PIGN exon 4–22 duplication. Squares indicate males and circles indicate females. The proband (II-1, arrow) showed an apparent four-copy state (CN = 4) by copy-number analysis, while both parents (I-1 and I-2) showed three-copy states (CN = 3). b CNV plot for chromosome 18 showing a duplication region at 18q21 (red dashed box). c Zoom-in CNV plot of the PIGN locus (chr18: 59,768,307–59,828,586) showing a multi-exon duplication (exons 4–22) involving PIGN. d qPCR validation of the PIGN duplication. Relative quantification values were used to estimate copy number, with the control set as 1.0, corresponding to two copies. The proband showed an RQ value of approximately 2.3, consistent with an apparent four-copy state, while both parents showed RQ values of approximately 1.5, consistent with three-copy states

Runs of homozygosity (ROH) analysis was not reliably performed because the duplicated region was relatively small (approximately 60 kb) and the SNP density within the WES capture intervals was insufficient for robust ROH inference.

Discussion

We report a male infant with an exon 4–22 duplication of PIGN, with an apparent four-copy state in the proband and three-copy states in both parents. Segregation analysis demonstrated that both parents carried the same heterozygous duplication, and copy-number analysis indicated an apparent four-copy state in the proband. To our knowledge, this represents the first documented biallelic multi-exon duplication in PIGN, thereby expanding the known mutational spectrum beyond previously described single-nucleotide or small indel variants. Clinically, the patient survived infancy, exhibited persistent feeding difficulties requiring nasogastric support, and showed developmental delay without overt seizures or major structural brain abnormalities. These features suggest that exon-level duplications may result in partial rather than complete loss of enzymatic function, contributing to a milder phenotype within the continuum of PIGN-related MCAHS1. Although no consanguinity was reported in the family, the apparent four-copy state in the proband is consistent with inheritance of the same rare duplication allele from both parents, each of whom showed a three-copy state by CNV analysis and qPCR. Unrecognized distant relatedness or shared ancestry cannot be completely excluded. Because ROH analysis was not reliable in this case owing to the small duplicated interval and limited SNP density within the WES capture regions, we interpreted the finding conservatively as a biallelic duplication rather than claiming proven homozygosity.

Pathogenic variants in PIGN disrupt the biosynthesis of GPI anchors, impairing the attachment of GPI-anchored proteins to the cell membrane [10]. More than 30 PIGN variants have been reported, most being missense, nonsense, or splice-site mutations that typically cause severe neonatal presentations with intractable seizures, profound hypotonia, and multiple organ malformations [11–13]. A comparison of the clinical and molecular findings between our case and previously reported patients is summarized in Table 1 [1, 3, 14, 15]. Most reported patients had a severe course with early death, although a small number of survivors have been described. In contrast, our patient showed preserved growth parameters, absence of clinical seizures, and relatively unremarkable brain MRI findings, indicating a comparatively attenuated phenotype within the PIGN-related spectrum.

Table 1.

Comparison of clinical and molecular features in the present case and previously reported PIGN-related MCAHS1 patients

Feature	Present case	Khayat et al., 2016 (Am J Med Genet A)	Alessandri et al., 2018 (EJHG)	Bukowska-Olech et al., 2023 (Eur J Med Genet)	Loong et al., 2023 (Genet Med)
Origin / Ethnicity	Chinese	Israeli Arab	La Réunion Island (founder population)	Polish	Multi-ethnic (international cohort)
Sex / Age at report	Male, 12 mo	Male, 2 mo	Multiple (neonatal–infantile)	Female, 10 mo	61 pts (1 mo – 10 y)
Genotype	PIGN exon 4–22 dup (homozygous four-copy state)	c.755A>T (p.Asp252Val) homozygous	Biallelic LoF variants, including intragenic deletion	c.1247_1251delAAGTG + c.1434+5G>A compound het	Various (frameshift, missense, nonsense, CNV)
Variant type	Multi-exon duplication	Missense	Truncating / Splice / Intragenic deletion	Frameshift + Splice	Truncating / Missense / Duplication
Onset	Neonatal	Neonatal	Neonatal	Neonatal	Mostly neonatal
Hypotonia	+++ (severe, persistent)	+++	+++	+++	+++
Seizures	− (no clinical seizures, irritable EEG)	+++ (intractable)	++ to +++	++ (controlled)	+ to +++ (variable)
Dysmorphic / congenital anomalies	Low-set ear, submucous cleft palate, accessory nipple, cryptorchidism, micropenis, clubfoot	Facial dysmorphism, diaphragmatic hernia	Fryns-like features (diaphragmatic hernia, facial and limb anomalies)	Facial dysmorphism, microcephaly, heart defect	Variable (organ malformations, limb defects)
Neuroimaging	Mildly increased white-matter signal, no structural abnormalities	Cortical atrophy, ventriculomegaly	Cortical atrophy / white-matter loss	Delayed myelination	Variable (atrophy to normal)
Serum ALP	Normal	Elevated	Normal or mildly increased	Normal	Mostly normal or slightly ↑
Outcome / follow-up	Alive at 12 mo, feeding tube, no seizures	Died at 3 mo	Deceased (neonatal–infantile)	Alive at 10 mo with delay	25% survived > 1 y with variable severity
Phenotypic severity	Mild (viable infant)	Severe (lethal)	Severe	Moderate	Spectrum (mild to severe)

Abbreviations: ALP alkaline phosphatase, LoF loss of function, CNV copy-number variant, het heterozygous, dup duplication

“+” indicates presence and approximate severity of feature

However, due to the limitations of WES-based CNV analysis, the precise structure, orientation, and insertion site of the duplication could not be determined. Although the proband showed an apparent four-copy state by copy number analysis and qPCR validation, this does not necessarily imply functional overexpression of PIGN. Multi-exon duplications may lead to aberrant splicing, disrupted transcript architecture, or partial loss of function rather than increased gene dosage. Several structural scenarios are plausible. In a tandem duplication, the reading frame may be disrupted depending on the breakpoint junctions, and even an in-frame insertion affecting more than half of the coding region would be likely to impair protein function. In contrast, an inverted duplication would be even more likely to disrupt normal transcript architecture. Because the breakpoint junctions were not resolved, these possibilities remain speculative, but they support a loss-of-function or hypomorphic mechanism rather than simple overexpression. Functional assays such as flow-cytometric analysis of GPI-anchored proteins would be valuable to clarify the biological consequence of this duplication.

From a clinical perspective, this case highlights the importance of incorporating CNV detection into exome analysis pipelines [16]. Conventional variant calling focusing solely on single-nucleotide variants would have overlooked this clinically relevant duplication [17]. As demonstrated here, exon-level CNV analysis can substantially increase the diagnostic yield in rare congenital anomaly syndromes. Moreover, the relatively mild course observed in our patient indicates that PIGN-related disorders should be considered even in the absence of seizures or major neuroimaging abnormalities.

The primary limitation of this study is the lack of functional validation, particularly at the transcript level. We were unable to assess whether the duplication resulted in reduced PIGN mRNA abundance through nonsense-mediated decay or other RNA-level mechanisms. In addition, we lacked independent parental CNV confirmation beyond WES-based segregation data. Because the 5′non-coding exons were not covered by the capture design, the reported exon 4–22 duplication represents the WES-detectable interval rather than the definitive structural boundary. Due to the limited size of the duplicated region and the resolution of WES-based data, we were also unable to determine whether the duplication resided within a region of homozygosity. Long-term follow-up is also needed to assess developmental outcomes. Nevertheless, our findings broaden the genotypic and phenotypic spectrum of PIGN-related disorders and suggest that biallelic exon-level duplications, as indicated by copy number analysis, may be compatible with a viable and attenuated form of GPI biosynthesis defect.

Authors’ contributions

Xiaohua Li and Xia Wang contributed equally to this work and share first authorship. Xiaohua Li performed the clinical assessment, data collection, and initial manuscript drafting. Xia Wang conducted the genetic analysis and interpretation of sequencing data. Jimo Zhu participated in data analysis and literature review. Yiting Du assisted with patient follow-up and clinical data interpretation. Maoxiao Li supervised the genetic validation and contributed to critical manuscript revision. Hongping Li designed and supervised the overall study, interpreted clinical and molecular findings, and finalized the manuscript. All authors read and approved the final version of the manuscript.

Funding

This work was supported by the Shenzhen Science and Technology Program (No. RCJC20231211085923029), Sanming Project of Medicine in Shenzhen (No. SZSM202311027), Guangdong High-level Hospital Construction Fund and Clinical key specialty construction project of Guangdong Province (ynkt2021-2216).

Data availability

The datasets generated and analyzed during the current study are not publicly available due to privacy and ethical restrictions concerning patient anonymity. Reasonable requests for access to de-identified data should be directed to the corresponding author.

Declarations

Ethics approval and consent to participate

The study involving human participants was reviewed and approved by the Ethics Committee of Shenzhen Children's Hospital (Approval number: 2023[050]). Written informed consent to participate in this study and for the publication of any potentially identifiable images or data was obtained from the participant’s legal guardians in accordance with institutional and national regulations.

Competing interests

The authors declare no competing interests.