Abstract
Background/Objectives: Hardikar syndrome (HDKR) is an X-linked dominant disorder caused by pathogenic variants in the Mediator complex subunit 12 (MED12) gene, predominantly affecting females. It is characterized by multisystem congenital anomalies involving the foregut, biliary tract, craniofacial structures, eyes, skeleton, and genitourinary system, with generally preserved neurodevelopment. Only 34 cases have been reported to date, and most exhibit multiple congenital anomalies. We describe a female infant who presented with progressive cholestatic liver disease and complex hepatobiliary malformations, including an absent gallbladder and paucity of bile ducts, with unremarkable prenatal imaging. The clinical course was notable for hepatosplenomegaly, markedly elevated total bile acids, portal hypertension with esophageal varices, and eventual liver failure. Methods: Whole-exome sequencing (WES) was performed to investigate the underlying genetic etiology, followed by parental segregation analysis using Sanger sequencing to confirm and characterize the identified variant. Results: WES identified a novel de novo intronic heterozygous variant in MED12 (c.3868-5C>G). Unlike most previously reported cases, the predominant and early manifestation in our case was severe hepatobiliary disease with limited additional anomalies, suggesting possible phenotypic variability within the MED12-related Hardikar syndrome spectrum. The identified MED12 variant is classified as a variant of uncertain significance (VUS). Conclusions: This case underscores the diagnostic utility of WES in infants with unexplained cholestasis, highlights the importance of considering noncoding variants, and illustrates the value of reporting well-characterized patients carrying novel VUS, thereby contributing to the growing body of clinical and molecular evidence on MED12-related Hardikar syndrome.
Keywords: Hardikar syndrome, MED12, rare disease, cholestasis, genotype-phenotype correlation, segregation analysis, whole-exome sequencing
1. Introduction
Hardikar Syndrome (HDKR) (MIM 301068) is an ultra-rare female-specific genetic disorder characterized by multiple congenital anomalies. HDKR is an X-linked dominant disorder, caused by heterozygous pathogenic variants in the MED12 gene (MIM 300188). To the best of our knowledge, only 34 cases are documented in the medical literature to date, and the true prevalence of HDKR remains unknown [1].
The MED12 gene, located on the X chromosome (Xq13.1) [2], encodes a subunit of the Mediator complex that serves as an interface between transcription factors and RNA polymerase II, thereby regulating their respective functions and transcriptional activity [3]. The human Mediator complex is a ~1.4 MDa multiprotein assembly playing a central role in transcriptional regulation. It facilitates the interaction between DNA-binding transcription factors and RNA polymerase II [4]. Additionally, MED12 interacts with chromatin and transcription factors, thereby regulating transcriptional activity [5,6,7]. MED12 deficiency impairs ciliogenesis and reduces Hedgehog and YAP pathway activity, suggesting that disruption of these signaling mechanisms may contribute to the HDKR phenotype [8].
Germline pathogenic variants in MED12 are associated with a spectrum of overlapping X-linked disorders, including HDKR (MIM 301068), Lujan–Fryns syndrome (MIM 309520), X-linked Ohdo syndrome (MIM 300895), Opitz–Kaveggia syndrome/FG syndrome-1 (MIM 305450), and nonspecific intellectual disability (NSID). Missense variants are predominantly identified in males with FG syndrome-1, Lujan–Fryns syndrome, X-linked Ohdo syndrome, or nonspecific intellectual disability [9]. In contrast, truncating loss-of-function variants appear to be poorly tolerated in males and are presumed to result in embryonic lethality. HDKR is mainly associated with de novo truncating variants, most commonly nonsense or frameshift [10].
The clinical spectrum of HDKR includes foregut malformations, intestinal malrotation, hepatobiliary disease, genitourinary anomalies, cleft lip and/or palate, and pigmentary retinopathy. Some affected individuals also present with congenital cardiac defects or vascular abnormalities, such as aortic coarctation and carotid or intracranial aneurysms. Neurodevelopment and cognitive function are generally preserved despite multisystem involvement [1]. Recently, the phenotypic variability of HDKR has expanded, with some affected females reported to present with congenital diaphragmatic hernia [10] or pulmonary hypoplasia [11]. This phenotypic heterogeneity in females was suggested to be attributed to skewed X-chromosome inactivation (XCI) across different tissues [1].
The diagnosis of HDKR can be particularly challenging, given the broad phenotypic and molecular spectrum of MED12-related disorders [8], as well as the intra-syndromic variability observed between affected individuals [11]. In this context, the use of whole-exome sequencing (WES) represents a valuable tool to help overcome these clinical and phenotypic diagnostic challenges.
We hereby report a Lebanese infant with a phenotype highly suggestive of Hardikar syndrome in whom WES identified a novel de novo intronic MED12 variant currently classified as a VUS. This report expands the clinical and molecular spectrum of MED12-related Hardikar syndrome while contributing evidence that may assist future variant interpretation. Additionally, we provide a review of the literature summarizing all reported cases of HDKR.
2. Materials and Methods
2.1. Literature Review
A literature review was conducted using PubMed. The following search terms were used to identify eligible articles: “Hardikar syndrome”, “MED12 AND syndrome”, “Hardikar syndrome AND MED12”, and “MED12-related disorder”. The search was limited to English-language articles reporting clinical, genetic, and molecular findings related to Hardikar syndrome and the MED12-related Hardikar syndrome spectrum. Studies were screened based on their titles and abstracts, and full texts were reviewed when available and relevant.
Germline MED12 variants classified as pathogenic or likely pathogenic were compiled from published reports and curated variant databases, including the Human Gene Mutation Database (HGMD Professional), ClinVar, and DECIPHER. Database interrogation was last performed on 24 June 2026.
2.2. Patient
The proband is a 3-year-old Lebanese girl (Figure 1A, II.2), the second child from a non-consanguineous marriage. Due to her symptoms (Results section), the patient (Figure 1A, II.2) and her parents (Figure 1A, I.1 and I.2) were referred for genetic evaluation to the Centre Jacques Loiselet for Medical Genetics and Genomics (CGGM) at the Faculty of Medicine at Saint Joseph University of Beirut (USJ), Lebanon. There was no known family history of similar conditions. However, her family history was notable for the presence of Sandhoff disease (MIM #268800) due to compound heterozygous pathogenic variants in the HEXB gene (MIM #606873) (NM_000521.3: c.850C>T and c.1082+5G>A) previously documented in her brother, deceased at the age of 18 months (Figure 1, II.1). Her parents were molecularly confirmed to be heterozygote carriers of the identified HEXB variants. Genetic counseling was provided, and informed consent for genetic analyses and publication was obtained from both parents.
Figure 1.
(A) Family pedigree. The proband (II.2, arrow) is affected with Hardikar syndrome (red). Her brother (II.1) was affected with Sandhoff disease (blue). Both parents are unaffected. (B) Sanger sequencing chromatograms showing the novel MED12 variant c.3868-5C>G (NM_005120.3) in the proband. The variant is absent in both parents, confirming its de novo occurrence. The position of the variant at the intron 27–exon 28 boundary is indicated.
2.3. Whole Exome Sequencing (WES)
WES was performed, guided by the following Human Phenotype Ontology (HPO) terms: Cholestasis (HP:0001396), cirrhosis (HP:0001394) and hepatic failure (HP:0001399). Genomic DNA was enzymatically fragmented, and target regions were enriched using DNA capture probes covering approximately 41 Mb of the human coding exome, targeting >98% of coding RefSeq regions (GRCh37/hg19), as well as the mitochondrial genome. Sequencing was performed on an Illumina platform, achieving a mean coverage depth sufficient to ensure ≥20× coverage for 99.52% of targeted nucleotides. An in-house bioinformatics pipeline was applied, including read alignment to GRCh37/hg19 genome assembly and the revised Cambridge Reference Sequence (rCRS) of the Human Mitochondrial DNA (NC_012920), followed by variant calling, annotation, and comprehensive variant filtering.
2.4. Sanger Sequencing
A subsequent familial segregation study was performed using standard PCR followed by Sanger sequencing. The following set of primers were used to amplify the region of interest in MED12 (NM_005120.3): Forward primer: 5′-TGATCACACCAGCTCCCTAC-3′ and Reverse primer: 5′-ACTGCCCCTCTCCTTTTCTC-3′. PCR products were purified and used for bidirectional Sanger sequencing on an ABI 3500 sequencer (Applied Biosystems, Foster City, CA, USA).
3. Results
3.1. Literature Review
From our literature search, 12 published reports comprising 33 individuals with clinical and/or molecular findings consistent with the MED12-related Hardikar syndrome spectrum were considered eligible. The reported molecular spectrum was predominantly composed of heterozygous MED12 loss-of-function variants, including nonsense, frameshift, splice-region, and copy-number variants. Most variants occurred de novo, although mosaicism and maternal transmission have also been reported.
In addition, one female individual registered in the DECIPHER database (ID 482687) carried a de novo heterozygous intragenic MED12 deletion (GRCh38: chrX: (71129456_71131855)x1). Her phenotype included intrahepatic biliary atresia, impaired liver function, cardiac and pulmonary abnormalities, urogenital anomalies, mild microcephaly, abducens palsy, and global developmental delay.
The two original cases described by Hardikar et al. were excluded because no molecular diagnosis was available. Including the present patient, a total of 35 individuals were analyzed and assigned case numbers 1–35. The clinical and molecular findings are summarized in Table 1 and Table 2.
Table 1.
Clinical overview of all reported cases of Hardikar syndrome (HDKR).
| CASES 1-35 | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Clinical Feature/Field | CASE 1 | CASE 2 | CASE 3 | CASE 4 | CASE 5 | CASE 6 | CASE 7 | CASE 8 | CASE 9 |
| Sex | F | F | F | F | F | F | F | F | F |
| Age at diagnosis | 7 years | 43 years | 8 years | 4 months | 5 months | 11 years | 1 year | 7 years | 5 years |
| Age at last evaluation | 7 years | 43 years | 8 years | 4 months | 5 months | 11 years | 1 year | 7 years | 5 years |
| Development—intellectual (HP:0001249) | Normal | Normal | Normal | Normal | NA | Normal | NA | Normal | Normal |
| Development—motor (HP:0001263) | Normal | Normal | Normal | Normal | NA | Normal | NA | Normal | Normal |
| Development—language (HP:0000750) | Normal | Normal | Normal | Normal | NA | Normal | NA | Normal | Normal |
| Cleft lip (HP:0410030) | No | Yes, not specified | No | Yes, unilateral, not specified | No | No | No | No | Yes, not specified |
| Cleft palate (HP:0000175) | No | Yes, not specified | No | Yes, unilateral, not specified | No | Yes, not specified | No | No | Yes, not specified |
| Hearing impairment (HP:0000365) | NA | Yes, unspecified | NA | NA | NA | NA | NA | NA | Yes, right sided, severe, type not specified |
| Pigmentary retinopathy (HP:0000580) | No | NA | Yes | No | NA | Yes | Yes | Yes | Yes |
| GI—Intestinal malrotation (HP:0002566) | Yes | No | No | No | No | Yes | No | Yes | Yes |
| GI—cholangitis (HP:0030151) | No | NA | NA | No | NA | NA | NA | NA | NA |
| GI—cholestasis (HP:0001396) | No | NA | NA | No | NA | NA | NA | NA | NA |
| GI—choledochal cyst (HP:0100890) | No | NA | NA | No | NA | NA | Yes | NA | Yes |
| GI—hepatomegaly (HP:0002240) | NA | NA | NA | NA | NA | NA | NA | NA | NA |
| GI—splenomegaly (HP:0001744) | NA | NA | NA | NA | NA | NA | NA | NA | NA |
| GI—absent gallbladder (HP:0011467) | No | No | No | No | No | No | No | No | No |
| GI—liver fibrosis (HP:0001395) | NA | NA | NA | NA | NA | NA | NA | NA | NA |
| GI—jaundice (HP:0000952) | No | NA | NA | No | NA | NA | NA | NA | NA |
| GI—Biliary atresia (HP:0005912) | No | NA | NA | No | NA | Yes | NA | NA (biliary dysgenesis) | Yes |
| GI—obstructive liver disease (HP:0005230) | No | NA | NA | No | NA | NA | NA | NA | NA |
| GI—liver biopsy/histology notes | NA | NA | NA | NA | Dilated infrahepatic ducts | NA | NA | NA | NA |
| GI—congenital diaphragmatic hernia (CDH) (HP:0000776) | NA | NA | NA | NA | Yes, left sided | NA | Yes, left sided | NA | NA |
| GU—hydronephrosis (HP:0000126) | Yes, unspecified | NA | Yes, unspecified | No | NA | NA | NA | NA | NA |
| GU—hydroureters (HP:0000072) | NA | NA | NA | No | NA | NA | NA | NA | NA |
| GU—recurrent urinary tract infections (HP:0000010) | NA | NA | NA | NA | NA | NA | NA | NA | NA |
| GU—vesicoureteral reflux (HP:0000076) | NA | NA | Yes | No | NA | NA | NA | Yes | NA |
| GU—structural anomalies (HP:0000119) | Ectopic ureters | Ectopic ureter, side not specified | NA | No | Ectopic ureters; renal pelviectasis | Ectopic ureter | Right kidney dilatation | NA | Ectopic ureters; congenital urogenital sinus |
| GU—functional complications (HP:0000025) | NA | NA | NA | NA | NA | NA | NA | Chronic kidney disease, not specified | Chronic kidney disease, not specified |
| Cardiovascular—structural anomalies (HP:0030680) | Left ventricular noncompaction | Aortic coarctation; bicuspid aortic valve | Left ventricular noncompaction; ascending aortic dilatation; pulmonary artery stenosis | No | Aortic coarctation | Left ventricular noncompaction; aorta ascendens dilatation | Left ventricular noncompaction; aortic coarctation with postoperative restenosis; pulmonic vein stenosis with postoperative restenosis | Left ventricular noncompaction; atrial/ventricular septal defects; aortic coarctation; outpouching of the left cavernous internal carotid artery | Left ventricular noncompaction; aorta ascendens dilatation with aorta descendens narrowing; tricuspid regurgitation |
| Cardiovascular—functional complications (HP:0011025) | NA | Atrial and ventricular arrhythmia; ventricular fibrillation | NA | NA | NA | NA | Decreased left ventricular function | NA | Arterial hypertension |
| Skeletal (HP:0000924) | NA | Scoliosis | NA | NA | NA | NA | Scoliosis | Scoliosis | NA |
| Clinical feature/field | CASE 10 | CASE 11 | CASE 12 | CASE 13 | CASE 14 | CASE 15 | CASE 16 | CASE 17 | CASE 18 |
| Sex | F | F | F | F | F | F | F | F | F |
| Age at diagnosis | 14 years | 9 months | NA | 18 years | Prenatal | Prenatal | Postnatal | 14 months | 21 months |
| Age at last evaluation | 14 years | 9 months | 21 months | 20 years, deceased | 12 months | TOP | <1 year | 2 years + 8 months | 6 years |
| Development—intellectual (HP:0001249) | Normal | Mild delay | Delayed | Normal | Normal | NA | NA | Normal | Normal |
| Development—motor (HP:0001263) | Normal | Mild delay | Delayed | NA | Normal | NA | NA | Normal | Normal |
| Development—language (HP:0000750) | Normal | Mild delay | Delayed | Slightly below average | Normal | NA | NA | Normal | Normal |
| Cleft lip (HP:0410030) | Yes, right sided | No | Yes | Yes, not specified | Yes, not specified | Bilateral | Unilateral, side not specified | Unilateral, left | No |
| Cleft palate (HP:0000175) | Yes, soft palate | Yes, not specified | Yes | Yes, not specified | Yes, not specified | Bilateral | Unilateral, side not specified | No | Unilateral, side not specified |
| Hearing impairment (HP:0000365) | NA | Bilateral, type not specified | NA | NA | Yes, mixed | NA | NA | NA | NA |
| Pigmentary retinopathy (HP:0000580) | NA | NA | NA | Yes | Yes | NA | NA | No | Yes |
| GI—Intestinal malrotation (HP:0002566) | No | Yes | NA | Yes | No | Yes | NA | Yes | NA |
| GI—cholangitis (HP:0030151) | NA | NA | NA | NA | NA | NA | NA | NA | NA |
| GI—cholestasis (HP:0001396) | NA | NA | NA | NA | NA | NA | NA | NA | NA |
| GI—choledochal cyst (HP:0100890) | NA | NA | NA | Yes | Yes | NA | NA | Yes | Yes |
| GI—hepatomegaly (HP:0002240) | NA | NA | NA | NA | NA | NA | NA | NA | NA |
| GI—splenomegaly (HP:0001744) | Yes | NA | NA | NA | NA | NA | NA | NA | NA |
| GI—absent gallbladder (HP:0011467) | No | No | NA | No | No | NA | NA | NA | Yes |
| GI—liver fibrosis (HP:0001395) | NA | NA | NA | NA | NA | NA | NA | Yes | NA |
| GI—jaundice (HP:0000952) | NA | NA | NA | NA | NA | NA | NA | NA | NA |
| GI—Biliary atresia (HP:0005912) | Yes | NA | NA | No | NA | NA | Yes | NA | NA |
| GI—obstructive liver disease (HP:0005230) | NA | NA | NA | NA | NA | NA | NA | NA | NA |
| GI—liver biopsy/histology notes | NA | NA | NA | Fatal intrahepatic cholangiocarcinoma | NA | NA | NA | NA | NA |
| GI—congenital diaphragmatic hernia (CDH) (HP:0000776) | NA | Yes, left sided | NA | NA | NA | Yes | NA | Yes | NA |
| GU—hydronephrosis (HP:0000126) | NA | NA | NA | Yes, not specified, secondary to uretral stenosis | Bilateral | NA | Bilateral | Unilateral, left | NA |
| GU—hydroureters (HP:0000072) | NA | NA | NA | NA | Bilateral | NA | Unilateral, left | NA | NA |
| GU—recurrent urinary tract infections (HP:0000010) | NA | NA | NA | NA | NA | NA | NA | NA | NA |
| GU—vesicoureteral reflux (HP:0000076) | NA | NA | NA | NA | Bilateral | NA | NA | NA | NA |
| GU—structural anomalies (HP:0000119) | Ectopic ureters; urogenital sinus; right renal artery stenosis with post-procedure renal artery restenosis | Abnormal renal morphology | NA | Uretral stenosis; partial bladder agenesis with urethral atresia; bicornuate noncommunicating uterus with hematosalpinx and hematometra; vaginal atresia | Small right renal cyst; ectopic ureters | Uterus, vagina and ovaries not identifiable | NA | NA | Cloacal anomaly |
| GU—functional complications (HP:0000025) | NA | NA | NA | Chronic kidney disease; kidney transplant | No | NA | NA | NA | NA |
| Cardiovascular—structural anomalies (HP:0030680) | Aortic dilatation | Atrial septal defect; patent ductus arteriosus | Congenital heart disease: Ebstein anomaly; thickened tricuspid valve with regurgitation | Patent ductus arteriosus; aortic coarctation | No | VSD; right sided aortic arch; double outlet right ventricle | Aortic coarctation | Aortic coarctation | Aortic coarctation |
| Cardiovascular—functional complications (HP:0011025) | Arterial hypertension | NA | Tricuspid regurgitation | No | Atrial ectopy | NA | No | No | No |
| Skeletal (HP:0000924) | NA | NA | Digit anomalies | Kyphosis (11 years); incomplete fusion of S1 posterior elements; Sprengel deformity; C2/C3 retrolisthesis | NA | NA | NA | NA | NA |
| Clinical feature/field | CASE 19 | CASE 20 | CASE 21 | CASE 22 | CASE 23 | CASE 24 | CASE 25 | CASE 26 | CASE 27 |
| Sex | F | F | F | F | F | F | F | F | F |
| Age at diagnosis | 12 months | Postnatal | 4 years | 19 months | Prenatal | Prenatal | Prenatal | Prenatal | 9 months |
| Age at last evaluation | 14 years | 21 years, deceased | 4 years | 33 months | TOP | TOP | TOP | Fetal demise | 9 months |
| Development—intellectual (HP:0001249) | Normal | Normal | Normal | Normal | NA | NA | NA | NA | Mild delay |
| Development—motor (HP:0001263) | Normal | Normal (initially delayed at 1 year) | Normal | Normal | NA | NA | NA | NA | Mild delay |
| Development—language (HP:0000750) | Normal | Normal | Normal | Normal | NA | NA | NA | NA | Mild delay |
| Cleft lip (HP:0410030) | Bilateral | Bilateral | Yes, not specified | No | Yes | Bilateral | Yes, right sided | Yes, not specified | No |
| Cleft palate (HP:0000175) | Bilateral | Bilateral | Yes, not specified | Yes, not specified | Yes | Bilateral | Yes, right sided | No | Yes, not specified |
| Hearing impairment (HP:0000365) | NA | Yes, conductive, bilateral | NA | Yes, sensorineural | NA | NA | NA | NA | Bilateral, type not specified |
| Pigmentary retinopathy (HP:0000580) | Yes | Yes | Yes | NA | NA | NA | NA | NA | NA |
| GI—Intestinal malrotation (HP:0002566) | Yes | Yes | NA | NA | NA | No | NA (no autopsy) | No | Yes |
| GI—cholangitis (HP:0030151) | No | No | NA | NA | NA | NA | NA | NA | NA |
| GI—cholestasis (HP:0001396) | Yes | Yes | Yes | NA | NA | NA | NA | NA | NA |
| GI—choledochal cyst (HP:0100890) | Yes, type 1 | No | NA | NA | NA | NA | NA | NA | NA |
| GI—hepatomegaly (HP:0002240) | Yes | Yes | NA | NA | NA | NA | NA | NA | NA |
| GI—splenomegaly (HP:0001744) | Yes | Yes | NA | NA | NA | NA | NA | NA | NA |
| GI—absent gallbladder (HP:0011467) | NA | No | Yes | NA | NA | NA | NA | No | No |
| GI—liver fibrosis (HP:0001395) | Yes | Yes | NA | NA | NA | NA | NA | NA | NA |
| GI—jaundice (HP:0000952) | Yes | Yes | NA | NA | NA | NA | NA | NA | NA |
| GI—Biliary atresia (HP:0005912) | No | Yes | NA | NA | NA | NA | NA | NA | NA |
| GI—obstructive liver disease (HP:0005230) | NA | Yes | NA | NA | NA | NA | NA | NA | NA |
| GI—liver biopsy/histology notes | At 1 month: bile duct proliferation + fibrosis; at 7.5 months: cirrhosis with regenerating nodules + chronic portal inflammation with bile duct proliferation + lobular cholestasis | Fibrosis + porto-portal septa + marked ductular proliferation + polymorphonuclear infiltration | NA | NA | NA | NA | NA | NA | NA |
| GI—congenital diaphragmatic hernia (CDH) (HP:0000776) | NA | NA | NA | NA | Yes | Yes | Yes, left sided | Yes | Yes, left sided |
| GU—hydronephrosis (HP:0000126) | Bilateral | Bilateral | NA | NA | NA | Bilateral | NA | Yes, bilateral | NA |
| GU—hydroureters (HP:0000072) | Bilateral | Bilateral | NA | NA | NA | NA | NA | NA | NA |
| GU—recurrent urinary tract infections (HP:0000010) | Yes | Yes | NA | NA | NA | NA | NA | NA | NA |
| GU—vesicoureteral reflux (HP:0000076) | Yes, not specified | Bilateral | NA | NA | NA | NA | NA | NA | NA |
| GU—structural anomalies (HP:0000119) | Ectopic ureters; bladder exstrophy; vaginal atresia; urogenital sinus | Ectopic ureters; vaginal atresia | Ectopic ureters, bilateral | NA | NA | No | NA | Gingival atrophy | Abnormal renal morphology |
| GU—functional complications (HP:0000025) | Chronic renal insufficiency (KDIGO 2) | Chronic renal insufficiency (KDIGO 2) | NA | NA | NA | NA | NA | NA | NA |
| Cardiovascular—structural anomalies (HP:0030680) | Patent ductus arteriosus; aortic coarctation | Patent ductus arteriosus; patent foramen ovale; coarctatio aortae; partial anomalous pulmonary venous return; dilated ascending aorta (27 mm); bicuspid aortic valve; tricuspid valve regurgitation (3/4); mitral valve regurgitation (2/4) | Patent ductus arteriosus | Mild aortic dilatation | Normal | Atrioventricular septal defect; patent ductus arteriosus | NA | NA | Atrial septal defect; patent ductus arteriosus |
| Cardiovascular—functional complications (HP:0011025) | No | Systolic murmur | NA | NA | Normal | NA | NA | NA | NA |
| Skeletal (HP:0000924) | Osteoporosis | No | NA | NA | dolichocephaly and turricephaly | NA | NA | NA | NA |
| Clinical feature/field | CASE 28 | CASE 29 | CASE 30 | CASE 31 | CASE 32 | CASE 33 | CASE 34 | CASE 35 (current case) | |
| Sex | F | F | F | F | F | F | F | F | |
| Age at diagnosis | 34 years | 19 months | 2 years | Prenatal | NA | Prenatal | NA | 3 years | |
| Age at last evaluation | 36 years | 3 years | 7 years | TOP | 4 years | TOP | 7 years | 3 years, deceased | |
| Development—intellectual (HP:0001249) | Normal | Normal | Normal | NA | Delayed at 3 years | NA | Delayed | Normal | |
| Development—motor (HP:0001263) | Normal | Normal | Normal | NA | Delayed at 3 years | NA | Delayed | Normal | |
| Development—language (HP:0000750) | Normal | Normal | Normal | NA | Delayed at 3 years | NA | Delayed | Normal | |
| Cleft lip (HP:0410030) | No | No | No | Bilateral | Unilateral, left | Yes, left sided | No | No | |
| Cleft palate (HP:0000175) | No | No | No | Bilateral | No | Yes, left sided | No | No | |
| Hearing impairment (HP:0000365) | NA | NA | Yes, sensorineural, right sided | NA | Yes, unspecified | NA | NA | No | |
| Pigmentary retinopathy (HP:0000580) | NA | NA | NA | NA | NA | NA | NA | No | |
| GI—Intestinal malrotation (HP:0002566) | No | No | Yes | NA | Yes | NA | NA | No | |
| GI—cholangitis (HP:0030151) | No | No | No | NA | NA | NA | NA | No | |
| GI—cholestasis (HP:0001396) | No | No | Yes | NA | NA | NA | NA | Yes | |
| GI—choledochal cyst (HP:0100890) | Yes | Yes | No | Yes | NA | NA | NA | No | |
| GI—hepatomegaly (HP:0002240) | No | No | No | NA | NA | NA | NA | Yes | |
| GI—splenomegaly (HP:0001744) | No | No | No | NA | NA | NA | NA | Yes | |
| GI—absent gallbladder (HP:0011467) | No | No | No | No | NA | NA | NA | Yes | |
| GI—liver fibrosis (HP:0001395) | No | No | No | NA | NA | NA | NA | Yes | |
| GI—jaundice (HP:0000952) | Yes | Yes | No | NA | NA | NA | NA | Yes | |
| GI—Biliary atresia (HP:0005912) | Yes | No | No | NA | NA | NA | Yes | No | |
| GI—obstructive liver disease (HP:0005230) | No | No | No | NA | NA | NA | NA | No | |
| GI—liver biopsy/histology notes | NA | NA | Bilirubinostasis | NA | NA | NA | NA | Cholestasis and paucity of interlobular bile ducts, Portal fibrosis with a few septa and no evidence of cirrhosis, Mild intrahepatocytic and Kupffer cell hemosiderosis. | |
| GI—congenital diaphragmatic hernia (CDH) (HP:0000776) | NA | NA | NA | NA | NA | Yes | NA | No | |
| GU—hydronephrosis (HP:0000126) | Bilateral | Bilateral | Bilateral | Bilateral | Bilateral | Yes, left sided | NA | No | |
| GU—hydroureters (HP:0000072) | Bilateral | Bilateral | Bilateral | Bilateral | NA | Yes, left sided | NA | No | |
| GU—recurrent urinary tract infections (HP:0000010) | Yes | Yes (E. cloacae) | Yes | NA | NA | NA | NA | No | |
| GU—vesicoureteral reflux (HP:0000076) | Yes | Yes | Yes | NA | NA | NA | NA | No | |
| GU—structural anomalies (HP:0000119) | Ectopic ureters; bicornuate uterus; vaginal atresia | Ectopic ureters; uterus didelphys; distal vaginal atresia | Ectopic ureters; hypoplastic uterus; vaginal atresia | NA | Ectopic ureters | NA | Bicornuate uterus; urogenital sinus anomaly | No | |
| GU—functional complications (HP:0000025) | Chronic kidney disease (KDIGO 3a) | No | No | NA | NA | NA | NA | No | |
| Cardiovascular—structural anomalies (HP:0030680) | NA | No | Patent foramen ovale; biventricular hypertrophy; aortic coarctation; dilated ascending aorta (13 mm); bicuspid aortic valve; tricuspid valve regurgitation (1/4) | Dextrocardia | Patent foramen ovale | Large VSD | Abnormal heart morphology | No | |
| Cardiovascular—functional complications (HP:0011025) | NA | No | Supraventricular tachycardia | NA | NA | NA | NA | No | |
| Skeletal (HP:0000924) | No | No | Sacral dimple | NA | NA | NA | NA | sacrococcygeal agenesis | |
| Other | Abnormal pulmonary interstitial morphology, bronchomalacia, restrictive ventilatory defect, abducens palsy and global developmental delay. | Possible immunodeficiency, suggested by recurrent respiratory infections and sepsis. | |||||||
F: female, GI: gastrointestinal, GU: genitourinary, NA: not applicable, TOP: termination of pregnancy.
Table 2.
Molecular spectrum of MED12 variants in all reported patients with Hardikar syndrome in the literature, including the present case.
| Cases | HGVS NC_000023.11: |
cDNA NM_005120.3: |
Protein | ACMG Classification | ACMG Scoring | Variant Type | Inheritance | Sex | References |
|---|---|---|---|---|---|---|---|---|---|
| CASE 1 | g.71121146del | c.729delG | p.(Met243Ilefs*13) | Pathogenic | PVS1, PS2, PM2 | Frameshift | De Novo | F | Strong et al. [8] |
| CASE 2 | g.71123713del | c.1737del | p.(Met580Cysfs*2) | Pathogenic | PVS1, PS2, PM2 | Frameshift | De Novo | F | |
| CASE 3 | g.71124338_71124405dup | c.1924_1974+17dup | p.? | Likely Pathogenic | PS2, PM2 | Splice | De Novo | F | |
| CASE 4 | g.71126059C>T | c.2446C>T | p.(Arg816*) | Pathogenic | PVS1, PS2, PM2 | Nonsense | De Novo | F | |
| CASE 5 | g.71129860G>A | c.3867+5G>A | p.? | Likely Pathogenic | PS2, PM2 | Splice | De Novo | F | |
| CASE 6 | g.71131549G>A | c.4048-1G>A | p.? | Pathogenic | PVS1, PM2, PM6 | Splice | NA | F | |
| CASE 7 | g.71132072G>A | c.4120-1G>A | p.? | Pathogenic | PVS1, PS2, PM2 | Splice | De Novo | F | |
| CASE 8 | g.71136366G>A | c.5111G>A | p.(Trp1704*) | Pathogenic | PVS1, PS2, PM2 | Nonsense | De Novo | F | |
| CASE 9 | g.71136390_ 71136393dup |
c.5135_5138dup | p.(Val1714Alafs*37) | Pathogenic | PVS1, PS2, PM2 | Frameshift | De Novo | F | |
| CASE 10 | g.71136401C>T | c.5146C>T | p.(Arg1716*) | Pathogenic | PVS1, PS2, PM2 | Nonsense | De Novo, Mosaic | F | |
| CASE 11 | g.71140759C>T | c.6169C>T | p.(Gln2057*) | Pathogenic | PVS1, PS2, PM2 | Nonsense | De Novo | F | |
| CASE 12 | g.71137942dup | c.6043dup | p.(Arg2015Lysfs*36) | Pathogenic | PVS1, PM2, PM6 | Frameshift | NA | F | Wang et al. [12] |
| CASE 13 | g.71130185C>T | c.4018C>T | p.(Gln1340*) | Pathogenic | PVS1, PS2 | Nonsense | De Novo, Mosaic | F | Pillai et al. [13] |
| CASE 14 | g.71125144C>T | c.2224C>T | p.(Gln742*) | Pathogenic | PVS1, PS2, PM2 | Nonsense | De Novo | F | |
| CASE 15 | g.71140786C>T | c.6196C>T | p.(Gln2066*) | Pathogenic | PVS1, PS2, PM2 | Nonsense | De Novo | F | Faergerman et al. [14] |
| CASE 16 | g.71119803C>T | c.322C>T | p.(Arg108*) | Pathogenic | PVS1, PS2, PM2 | Nonsense | De Novo, Mosaic | F | Li et al. [1] |
| CASE 17 | g.71125127_ 71125130del |
c.2207_2210del | p.(Thr736Ilefs*43) | Pathogenic | PVS1, PS2, PM2 | Frameshift | De Novo | F | |
| CASE 18 | g.71126462dup | c.2663dup | p.(Leu889Profs*11) | Pathogenic | PVS1, PS2, PM2 | Frameshift | De Novo | F | |
| CASE 19 | g.71135131_ 71135134delinsCCAGCA |
c.4903_4906delinsCCAGCA | p.(Val1635Profs*61) | Pathogenic | PVS1, PM2, PM6 | Frameshift | De Novo | F | |
| CASE 20 | g.71136366G>A | c.5111G>A | p.(Trp1704*) | Pathogenic | PVS1, PS2, PM2 | Nonsense | De Novo | F | |
| CASE 21 | g.71137257C>A | c.5622C>A | p.(Tyr1874*) | Pathogenic | PVS1, PS2, PM2 | Nonsense | De Novo | F | |
| CASE 22 | g.71140759C>T | c.6169C>T | p.(Gln2057*) | Pathogenic | PVS1, PS2, PM2 | Nonsense | De Novo | F | |
| CASE 23 | g.71118855T>G | c.99+2T>G | p.? | Likely Pathogenic | PVS 1 PM2 PS2 |
Splice | De Novo | F | Ramachandran et al. [11] |
| CASE 24 | g.71136978_TACAGGCAdel | c.5500_5507del | p. (Tyr1834Argfs*58) | Pathogenic | PVS1 PS2 PM2 |
Frameshift | De Novo | F | Dai et al. [15] |
| CASE 25 | arr[GRCh38] Xq13.1(71,043,894–71,344,313)x1 | - | - | Likely Pathogenic | - | Deletion (large CNV) | De Novo | F | Wu et al. [16] |
| CASE 26 | g.71137260del | c.5625del | p.(Gly1876Glufs*7) | Pathogenic | PVS1, PM2, PP5 | Frameshift | De Novo | F | Kao et al. [10] |
| CASE 27 | g.71140759C>T | c.6169C>T | p.(Gln2057*) | Pathogenic | PVS1, PM2, PP5 | Nonsense | De Novo | F | |
| CASE 28 | g.71121435C>T | c.844C>T | p.(Arg282*) | Likely pathogenic | PVS1, PM2 | Nonsense | De Novo, Mosaic | F | Warmoeskerken et al. [17] |
| CASE 29 | g.71121435C>T | c.844C>T | p.(Arg282*) | Likely pathogenic | PVS1, PM2 | Nonsense | Maternal | F | Warmoeskerken et al. [17] |
| CASE 30 | g.71130042del | c.3875del | p.(Val1292Glufs*21) | Likely pathogenic | PVS1, PM2 | Frameshift | De Novo | F | Warmoeskerken et al. [17] |
| CASE 31 | g.71127962G>A | c.3051G>A | p.(Trp1017*) | Likely pathogenic | PVS1, PM2 | Nonsense | De Novo | F | Warmoeskerken et al. [17] |
| CASE 32 | g.71129189del | c.3551delA | p.(Gln1184fs) | Likely pathogenic | PVS1, PM2 | Frameshift | De Novo | F | Munabi et al. [18] |
| CASE 33 | g.71132844G>C | c.4416-1G>C | p. ? | Likely pathogenic | PVS1, PM2, PP5 | Splice | De Novo | F | Taniguchi et al. [19] |
| CASE 34 | g.71129456_71131855del | - | p.(Gly1967Valfs*12) | Likely pathogenic | - | Frameshift | De Novo | F | Decipher |
| CASE 35 | g.71130030C>G | c.3868-5C>G | p. ? | VUS | PP3, PM2, PS2 | Splice | De Novo | F | El Kahy et al. (Current Case) |
ACMG: American College of Medical Genetics and Genomics [20], CNV: Copy Number Variant; F: female, HGVS: Human Genome Variation Society [21] (https://hgvs-nomenclature.org/, accessed on 12 May 2026), NA: Not Applicable., VUS: Variant of Uncertain Significance.
3.2. Clinical Presentation
Prenatal ultrasounds were unremarkable, with no reported intrauterine anomalies in the proband. At birth, laboratory evaluation revealed elevated liver enzymes: AST 331U/L, ALT 96U/L, GGT 24U/L, and alkaline phosphatase 237U/L. On day 4 of life, conjugated hyperbilirubinemia was documented (12.92 mg/dL). Congenital coccygeal agenesis was identified at 22 months of age.
Abdominal ultrasound performed at 22 days of life demonstrated hepatomegaly with a liver measuring 7.2 cm in maximal height, an absence of the gallbladder, and non-visualization of the intrahepatic and extrahepatic bile ducts, including the common bile duct. No triangular cord sign was identified. The spleen was normal in size (5.5 cm).
After excluding biliary atresia, based on the overall clinical, imaging, and histopathological findings, microscopic examination of liver biopsy performed at 2 months of age revealed cholestasis with paucity of intralobular bile ducts, portal fibrosis with a few septa in the absence of cirrhosis, mild intrahepatocytic and Kupffer cell hemosiderosis, and absence of interlobular bile ducts in more than half of the portal tracts. Persistent worsening of hepatomegaly was noted on follow-up by serial imaging, reaching 12 cm at 19 months of age, and 15.2 cm at 3 years and 2 months.
At 3 years of age, the patient exhibited short stature (height: 88 cm, <3rd percentile) with relatively preserved weight (16 kg, 75th percentile). Clinical examination revealed symptoms of malnutrition and advanced liver disease, including severe jaundice, abdominal collateral venous circulation, marked ascites, increased abdominal circumference (68 cm), and mild edema of the lower extremities. The patient’s relatively preserved weight was likely influenced by ascites related to advanced liver disease. Signs of portal hypertension were suggested by the detection of esophageal varices on upper gastrointestinal endoscopy. The parents reported recurrent upper respiratory tract infections associated with wheezing. No formal immunological evaluation was performed; however, WES did not identify any pathogenic or likely pathogenic variants in genes associated with primary immunodeficiency. The list of analyzed genes was based on the recommendations of the International Union of Immunological Societies (IUIS) for the genetic diagnosis of inborn errors of immunity.
Progressive clinical deterioration, including the development of splenomegaly (16.2 cm), ultimately led to consideration of liver transplantation. However, the patient developed unresolved septicemia caused by Pasteurella multocida and died at the age of 3 years and 8 months before liver transplantation could be performed and before the genetic test results became available.
3.3. Molecular Results
Given the family history of autosomal recessive Sandhoff disease, targeted sequencing of the previously identified HEXB pathogenic variants was initially performed. Carriership of the familial variants was not detected in the proband with subsequent exclusion of Sandhoff disease as an explanation for the clinical findings. Consequently, broader genetic testing was considered, and WES was performed.
A novel heterozygous intronic variant was identified in the MED12 gene (NM_005120.3:c.3868-5C>G) and subsequently confirmed by Sanger sequencing. This variant affects a nucleotide upstream of the canonical splice-site and is predicted to affect exon splicing. It is absent from population databases, including gnomAD, the Exome Sequencing Project (ESP), and the 1000 Genomes Project. To the best of our knowledge, this variant has not been previously reported in the literature. In silico prediction tools support a deleterious effect on protein function. SpliceAI indicates a significant probability of splice acceptor loss (Δ score = 0.60), suggesting disruption of the native acceptor site and potential alteration of normal MED12 pre-mRNA splicing. The database splicing consensus single nucleotide variant (dbscSNV) classifies this variant as Pathogenic Moderate, with a score of 0.9995 (version v1.1). According to the ACMG/AMP/ClinGen SVI guidelines, the c.3868-5C>G variant is currently classified as a variant of uncertain significance (VUS), with moderate evidence supporting pathogenicity (PP3, PM2). No additional clinically relevant sequence variants, copy number variants, or structural variants related to the patient’s phenotype were identified by WES.
Segregation analysis by Sanger sequencing indicated the absence of this variant in both parents (Figure 1B), consistent with a de novo occurrence (PS2). In view of the rapid disease progression and death, subsequent RNA analysis could not be performed, preventing assessment of the variant’s effect on the transcript.
Altogether, the clinical and molecular findings are compatible with a MED12-related disorder within the Hardikar syndrome spectrum.
4. Discussion
HDKR is an ultra-rare X-linked dominant multisystem disorder caused by germline pathogenic variants in the MED12 gene [1]. To date, the majority of reported HDKR cases result from de novo nonsense or frameshift variants leading to loss of function [10,12]. In contrast, intronic variants remain less frequently observed in the literature, and their contribution to the molecular landscape of HDKR is not yet well defined. The identification of a novel de novo intronic MED12 variant in our patient expands the molecular and clinical spectrum of this syndrome.
MED12 encodes a critical component of the Mediator kinase module, which regulates transcriptional activity through interaction with RNA polymerase II and multiple signaling pathways [3]. Disruption of MED12 function has been linked to impaired ciliogenesis and altered Hedgehog and YAP signaling, mechanisms that plausibly contribute to the complex developmental phenotype observed in HDKR [8]. While most previously reported variants directly truncate the protein (Figure 2); intronic variants, such as the one identified in our patient, may exert pathogenic effects through aberrant RNA splicing, potentially resulting in exon skipping, intron retention, or cryptic splice site activation. Variants located at the −5 position relative to the splice acceptor site may have variable effects on splicing efficiency, potentially allowing partial preservation of normal transcripts. This residual splicing activity could contribute to phenotypic variability and may partly explain the relatively restricted clinical presentation observed in our patient. Additionally, the presence of multiple MED12 transcript isoforms may modulate the functional impact of such splice variants, further contributing to variability in clinical expression.
Figure 2.
Integrated schematic representation of the MED12 gene (NM_005120.3) and its encoded protein (NP_005111.2), showing the location of previously reported MED12 variants associated with Hardikar syndrome. The 45 exons (purple boxes) and intervening introns (purple lines) are represented schematically. Exonic variants, including substitutions and small insertions/deletions, are indicated above the gene structure (blue lines), whereas intronic splice-region variants are shown below (green lines). The novel splice-region variant identified in the present study, c.3868-5C>G, is highlighted with a red star. The principal functional domains of the MED12 protein are shown above the gene structure: MED (Mediator complex subunit 12 domain), LCEWAV (Leu-Cys-Glu-Trp-Ala-Val conserved motif), PQL (proline-, glutamine-, and leucine-rich domain), and OPA (opa/polyglutamine-rich domain).
To date, few studies have reported data on the molecular and clinical aspects of HDKR. Only 34 previous cases have been documented, highlighting the rarity of the syndrome and the need for more research on HDKR. Table 2 summarizes the molecular results of MED12 variants in all HDKR cases reported in the literature including our patient (Table 2).
Functional analyses would have been valuable to assess the biological impact of the identified variant. However, RNA or protein studies could not be performed because the patient had passed away, representing an important limitation of the present study. Functional validation could, in principle, be pursued using minigene splicing assays or RNA-based approaches to assess the impact of the variant on MED12 splicing. Although the variant occurred de novo (PS2), was absent from population databases (PM2), and showed computational evidence supporting a potential effect on splicing (PP3), computational predictions alone cannot establish pathogenicity, particularly for non-canonical splice-region variants. Consequently, the variant is conservatively classified as a VUS. Nevertheless, documenting such rare non-canonical splice variants remains important to facilitate future genotype–phenotype correlations and potential reinterpretation as additional functional or clinical evidence becomes available.
Although the identified MED12 variant currently remains classified as a VUS according to ACMG criteria, reporting carefully phenotyped patients carrying novel VUS is an important component of the iterative process of clinical variant interpretation. Such reports contribute to evidence accumulation by documenting genotype–phenotype associations and may ultimately support variant reclassification when additional unrelated patients or functional studies become available. Therefore, we consider the present case valuable as a contribution to the expanding molecular and phenotypic spectrum of MED12-related Hardikar syndrome while acknowledging that definitive pathogenicity cannot yet be established.
In contrast to most published cases of HDKR, which typically display a broad constellation of multisystem anomalies including orofacial clefting, pigmentary retinopathy, cardiovascular and genitourinary malformations along with biliary anomalies [8], our patient’s phenotype was most notable for severe hepatobiliary disease including biliary tract malformation, cholestasis, and absent gallbladder. Additional findings included coccygeal agenesis, possible immunodeficiency, suggested by recurrent respiratory infections and sepsis. Importantly, regular prenatal screening was unremarkable, emphasizing the challenge of early detection in atypical presentations. The described absence of classical extra-hepatic features in our case and the predominantly hepatobiliary involvement with limited extra-hepatic features suggest a milder or variable expressivity of HDKR, which may be attributed to tissue-specific skewed XCI or differential nonsense-mediated decay of MED12 transcripts, phenomena increasingly recognized to contribute to the phenotypic heterogeneity of MED12-related disorders [1,22]. Finally, the early mortality in our patient, driven by progression of liver disease and inability to undergo timely transplant, underscores the severity of hepatobiliary progression in isolated presentations and reinforces the importance of genetic diagnosis to inform prognosis and management decisions.
Interestingly, some MED12 variants, such as c.6169C>T, c.5111G>A and c.844C>T (Table 2), have been reported in multiple independent cases across different studies, suggesting possible recurrent variation within the MED12-related Hardikar syndrome spectrum.
Although a recent review published in March 2026 provided a comprehensive overview of the clinical and molecular spectrum of Hardikar syndrome [17], two subsequently identified MED12-related cases with features compatible with the Hardikar spectrum were not included [11,12]. Together with the present case, these additional reports warranted an updated literature review, providing a more comprehensive and up-to-date synthesis of the clinical manifestations and molecular findings associated with this rare disorder.
In cases of early-onset cholestasis with hepatobiliary anomalies, several differential diagnoses should be considered. These include biliary atresia, Alagille syndrome, progressive familial intrahepatic cholestasis (PFIC), and mitochondrial hepatopathies. The absence of characteristic features of these conditions, combined with the genetic findings, was suggestive of a MED12-related disorder in our patient. This highlights the importance of comprehensive genomic evaluation in complex and atypical presentations.
The expanding application of molecular diagnostics increasingly reveals rare noncoding variants that would previously have remained undetected. Our findings underscore the importance of carefully evaluating intronic regions while sequencing the MED12 gene, particularly in females presenting with features suggestive of typical or atypical HDKR. Continued analysis of such variants is essential to refine genotype–phenotype correlations and to improve variant classification frameworks for MED12-related disorders.
In conclusion, this study highlights the value of WES in diagnosing rare disorders with overlapping clinical features. Although the identified de novo intronic MED12 variant remains classified as a VUS, the present report expands the clinical and molecular spectrum of MED12-related Hardikar syndrome and emphasizes the importance of documenting well-characterized rare cases to facilitate future evidence accumulation and variant interpretation. Additional unrelated patients and/or functional studies will be required to confirm pathogenicity and potentially support future variant reclassification.
Acknowledgments
We thank the parents for their participation and their full cooperation throughout this study.
Author Contributions
N.E.K. wrote the manuscript. N.E.K. and A.C. prepared the figures. A.M. and N.A. performed the clinical investigation of the patient and conducted the follow-up with the parents. N.E.K., R.C., R.M. and N.S. performed the molecular analyses. A.C. supervised the study. All authors have read and agreed to the published version of the manuscript.
Institutional Review Board Statement
This study was approved by the Ethics Committee of Saint Joseph University of Beirut (USJ) and Hôtel-Dieu de France hospital (HDF) in Beirut (CEHDF-2784. on 4 December 2025).
Informed Consent Statement
Written informed consent for participation and publication was obtained from the patient’s parents.
Data Availability Statement
The data that supports the findings of this study are available from the corresponding author upon reasonable request.
Conflicts of Interest
The authors declare no conflicts of interest.
Funding Statement
Molecular study was funded by a grant from the research council at Saint Joseph University of Beirut (Grant no. FM451).
Footnotes
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data that supports the findings of this study are available from the corresponding author upon reasonable request.