Clinical utility of genomic investigations in a Middle Eastern pediatric gastroenterology disease cohort

Abstract

BACKGROUND

The landscape and clinical utility of comprehensive genomic investigations for a wide range of pediatric gastrointestinal (GI) disorders have not been fully characterized in the Middle East.

AIM

To characterize the diagnostic yield and clinical utility of genomic investigations in a Middle Eastern pediatric cohort of GI disorders, and to dissect the pathogenic landscape of those disorders in this region.

METHODS

Sixty-nine pediatric patients of diverse Arab and Asian origins, were clinically and genetically assessed for a spectrum of GI diseases, including liver disease, inflammatory bowel disease, chronic diarrhea, and pancreatitis. Clinical genomic investigations included mainly (87%) next generation sequencing-based gene panels and whole exome or genome sequencing. Clinical information, including demographics, symptoms, management and clinical outcomes, was extracted from medical records.

RESULTS

The overall positive yield was 55%, whereas multiple molecular diagnoses were made in 3 patients (4%) including 2 with triple genetic findings, highlighting the utility of genetic investigations in delineating the phenotypic complexity in this cohort. A secondary medically actionable finding (MYBPC3-associated cardiomyopathy) was identified in one out of 12 patients (8%) who received exome or genome sequencing. Among all disease groups, the diagnostic yield was highest in patients with chronic diarrhea (73.3%) followed by those with cholestasis (62.5%). Copy number variants contributed substantially (18%) to the pathogenic variation spectrum. Consistent with consanguinity rates in this region, autosomal recessive conditions accounted for 66% of all diagnosed patients. Importantly, genetic findings guided clinical management plans and interventions in most cases (97%). Finally, we highlight a putative candidate gene, NR1I3, possibly associated with cholestasis identified in an undiagnosed Yemeni family with episodic transient disease.

CONCLUSION

Our study provides new insights into the pathogenic variation landscape in pediatric GI disorders in the Middle East and emphasizes the clinical utility of genomic investigations in managing those patients.

Key Words: Gastroenterology disease; Genomics; Diagnostic yield; Clinical utility; Middle East; Asia; Pediatrics

Core Tip: Here we characterize the diagnostic and clinical utility of genomic investigations for a wide range of pediatric gastrointestinal disorders in a pediatric cohort from the Middle East. We show that the cumulative diagnostic yield was 55%, including 6% having multiple molecular diagnoses, mostly attributed to autosomal recessive disorders (66%). Yield was highest for patients with congenital diarrhea (73.3%) and cholestasis (62.5%). Genomic findings guided management plans in 97% of diagnosed patients. We propose a novel gene-disease association most likely due to biallelic loss-of-function variants in the NR1I3 gene.

INTRODUCTION

The rapid advances in the genomics field have democratized clinical genetic testing which became widely accessible for diagnosing a variety of challenging diseases. The ability to sequence and analyze comprehensive gene lists, up to the whole exome, in a short period of time has shifted clinicians’ practice from ‘confirmatory’, often phenotype-driven testing, into a genotype-driven diagnostic paradigm especially for diseases with marked phenotypic and genetic heterogeneity. As of June 16, 2025, there are 7000 phenotypes for which the genetic and molecular basis are known[1], and there are ongoing studies to identify additional novel gene-disease associations. This is particularly important in the Arab population of the Middle East considering the high rate of consanguinity which can reach up to 20%-50%[2-6].

Several studies investigated the utility of genomics in gastroenterology with diagnostic rates varying based on clinical presentations. Jeyaraj et al[7] described a pediatric cholestatic cohort for which genetic testing yielded positive results in 8.5%, and emphasized the potential genotype-phenotype correlation. In pediatric Inflammatory bowel disease (IBD), the chance of getting a positive genetic result is higher with younger patients’ ages at which the diagnosis was established. The pathogenic variation spectrum in this cohort mostly impacts the immunological pathway rendering patients with unpredictable response to conventional treatment[8]. Mayerle et al[9] described the genetic basis of recurrent and chronic pancreatitis, and showed that targeting these receptors, receptors like inositol-tri-phosphate-receptor (types 2 and 3) where pathogenic variants have been identified, may help ameliorate pancreatitis. Another study showed genetic testing for congenital diarrhea or enteropathy was able to identify a molecular diagnosis in 64.2 % of the cohort (n = 137). The study showed that age and clinical presentation affected diagnostic yield, which was higher in neonates (75.4% vs 57.6%) and in cases of fatty (71.4%) or bloody diarrhea (68.0%) compared to watery diarrhea (48.1%)[10].

Besides diagnostic utility, genetic testing can assist in the management of various conditions and predict progression. For example, mutation class underlying cystic fibrosis disease can assist in anticipating and predicting the risk of developing pancreatitis[11]. In patients with microvillous inclusion disease (MVID), identifying pathogenic variants in MYO5B helps determine whether the disease will present as isolated intestinal involvement, a mixed intestinal-hepatic phenotype, or mimic other conditions such as progressive familial intrahepatic cholestasis (PFIC). This influences decisions around initiating parenteral nutrition, monitoring for cholestasis, and considering early intestinal or liver transplantation[12].

Currently, there are limited studies investigating the landscape and clinical utility of genomics in gastrointestinal (GI) diseases in pediatric populations across different ancestries, specifically those of Middle Eastern origin.

Here, we characterize the diagnostic yield, molecular findings, and clinical outcomes of genomic investigations in 69 pediatric patients, of primarily Middle Eastern origin, who presented with a range of GI associated symptoms.

MATERIALS AND METHODS

Study cohort

This study includes gastroenterology patients referred for clinical genomic testing from April 2019 to December 2023. Review of electronic medical records was performed by gastroenterologists to retrieve all clinical and demographics data and to identify interventions guided by genetic results.

Clinical indications included six groups: (1) Liver disease: Defined in patients with direct hyperbilirubinemia, persistent or recurrent episodes of indirect hyperbilirubinemia, elevated liver enzymes, hepatomegaly or liver failure; (2) IBD: Diagnosed by endoscopy and confirmed by histopathology. All patients were younger than 6 years old, which by definition is labelled as very early-onset IBD (VEOIBD); (3) Chronic diarrhoea: For more than 4 weeks; (4) Failure to thrive: Characterized by weight below 3^rd percentile or failing to gain weight over time; (5) Recurrent pancreatitis: With more than one episode; and (6) Polyposis: With multiple colonic polyps in the colon.

Ethical approval

This study was reviewed and approved by the Dubai Scientific Research Ethics Committee, Dubai Health Authority (Approval No. DSREC-08/2025_03). All patients were consented for clinical genetic testing under an approved de-identified research protocol, which permits the publication of de-identified analyses.

Exome sequencing

Genomic testing was performed in our College of American Pathologists-accredited genomics facility. Exome sequencing was performed as previously described[6]. Briefly, following DNA fragmentation by ultrasonication (Covaris, United States), the coding regions of the genome, also known as the exome, were captured using the Agilent Clinical Research Exome V2 capture probes (Agilent, United States). Libraries were prepared using the SureSelect^XT protocol (Agilent, Agilent) and then sequenced (2 × 150 bp) using the NovaSeq⁶⁰⁰⁰ system (Illumina, Agilent) to a minimum average depth of 100 ×.

Bioinformatic analysis

Sequencing data were processed using an in-house custom-made bioinformatics pipeline to retain high-quality sequencing reads across all coding regions. High-quality variants were annotated for allele frequency [using mainly the Genome Aggregation Database (gnomAD) and the Greater Middle East variome (MEV) database], predicted protein effects, and presence or absence in disease databases, such as ClinVar and the Human Gene Mutation Database (HGMD). Only rare variants (< 0.5% minor allele frequency if novel, and < 1% if present in disease databases) were retained for downstream filtration and analysis[13]. During the final interpretation of reportable variants, the analysts retrieved the variants’ allele frequencies from the MEV database, which includes whole exome and genome data from 2116 individuals from the Middle East[14].

For indication-based analysis, only rare, known pathogenic, or novel variants in the relevant genes associated with the patient’s indication, including cholestasis, pancreatitis, congenital diarrhea, and VEOIBD (Supplementary Table 1) were retained for interpretation.

For whole exome sequencing (WES), all known pathogenic variants in ClinVar/HGMD and novel loss-of-function variants in disease genes were retained. In addition, segregation analysis was performed for trio WES to identify dominant, de novo, homozygous, X-linked, and compound heterozygous variants. Copy number variants, mainly hemizygous or homozygous events, were called using normalized next-generation sequencing (NGS) read depth data as previously described[15-17]. Copy number changes detected by NGS were confirmed by microarrays, specific multiplex ligation-dependent probe amplification, PCR and gel electrophoresis, or customized droplet digital PCR assays[15].

Chromosomal microarray analysis

Chromosomal microarray analysis (CMA) was preformed using the Affymetrix CytoScan^HD.

Variant interpretation and reporting

All retained sequence and copy number variants were classified following the American College of Medical Genetics and Genomics/Association for Molecular Pathology or the American College of Medical Genetics and Genomics/Clinical genome Resource variant interpretation guidelines, respectively[18-20]. Pathogenic and likely pathogenic variants in genes relevant to the patients’ primary indications were reported and were considered diagnostic if the patient’s phenotype (based on physician’s notes and feedback), disease mechanism, and inheritance were all consistent. Clinically significant heterozygous variants in genes with recessive inheritance and all variants of uncertain significance relevant to patients’ primary indications were also reported, though were not considered diagnostic, leading to inconclusive reports.

RESULTS

Study cohort

Sixty-nine patients (average age: 2.8 years, range 2 weeks-18 years; 62.3% males; Figure 1) underwent genetic testing for a range of GI indications. Most patients (71%) were Arabs with majority being Emiratis (52.1%) and Yemenis (5.7%); 29% were mostly non-Arabs Asians (Figure 1).

Figure 1 Patient demographics. A: Distribution by age; B: Distribution by nationality and ancestry (Arab vs non-Arab). UAE: The United Arab Emirates.

Genomic findings and diagnostic yield

Patients mainly presented with liver disease (34.8%), chronic diarrhoea (21.8%), IBD (23.2%), failure to thrive (11.6%), recurrent pancreatitis (5.8%), and polyposis (2.9%; Figure 2A). Most patients were tested by genomic sequencing (86.9%), while CMA was done in 7 patients (10.1%; Supplementary Table 1).

Figure 2 Genomic findings and diagnostic yield. A: Genetic testing outcomes stratified by clinical category (disease groups); B: Breakdown of inheritance patterns in genes where pathogenic variants were identified. AR: Autosomal recessive; AD: Autosomal dominant.

The overall positive diagnostic yield was 55% (38/69), whereas multiple molecular diagnoses were made in 3 patients (4%) including 2 with triple genetic findings. In the liver (cholestasis) disease group, 15 patients (62.5%) had positive genetic results, while 4 patients with IBD (25%) were positive. A secondary medically actionable finding (MYBPC3-associated cardiomyopathy) was identified in one out of 12 patients (8%) who received exome or genome sequencing. Aside from the two patients with polyposis and positive findings, the highest diagnostic yield in this cohort was in the chronic diarrhea group where 11 patients (73.3%) had diagnostic results. The diagnostic yields in patients with pancreatitis and failure to thrive, were 50% each (Figure 2A). A list of all genetic findings is summarized in Table 1.

Table 1 Summary of positive genetic testing results and detected variants in the study cohort.

ID	Primary indication	Technology used	Test done	Variants	Inheritance	Zygosity	Classification
6	Very early onset IBD	NGS	WESTRIO	NM_000377.3(WAS):c.383T>C; p.(Phe128Ser)	X-linked recessive	Hemizygous	LP
14	Very early onset IBD	CMA	CMA	Xp21.1p11.4 (CYBB gene) deletion 1.83 Mb	X-linked recessive	Hemizygous	Pathogenic
15	Recurrent peri-anal abscess	CMA	CMA	Xp21.1p11.4 (CYBB gene) deletion 1.83 Mb	X-linked recessive	Hemizygous	Pathogenic
16	Very early onset IBD	NGS	WES	TTC7A: NM_020458.4, c.133_166del (p.Gly45SerfsTer23)	AR	Heterozygous	LP
17	Liver failure and direct hyperbilirubinemia	NGS	Custom gene panel	NM_002437.5(MPV17):c.280G>C; p.(Gly94Arg)	AR	Homozygous	LP
19	Direct hyperbilirubinemia, multiple congenital anomalies	CMA	CMA	arr[GRCh37] 18p11.32q23(136,227_78,014,123)x3	Chromosomal disorder	-	Pathogenic
20	Persistent indirect hyperbilirubinemia	NGS	UGT1A1 full gene sequencing	NM_000463.3(UGT1A1):c.625C>T; p.(Arg209Trp) NM_000463.3(UGT1A1): C.-41_-40dupTA; p.?	AR AR	Homozygous homozygous	Pathogenic LP
21	Persistent indirect hyperbilirubinemia	NGS	Cholestasis panel	NM_000463.3(UGT1A1): C.-41_-40dupTA; p.?	AR	Homozygous	Pathogenic
23	Persistent indirect hyperbilirubinemia	NGS	Cholestasis panel	NM_005603.4(ATP8B1):c.3040C>T; p.(Arg1014*)	AR	Homozygous	Pathogenic
24	Persistent indirect hyperbilirubinemia	NGS	Cholestasis panel	NM_000463.2(UGT1A1): C.1021C>T; p.R341*	AR	Homozygous	Pathogenic
25	Persistent elevated liver enzymes, congenital heart anomalies	NGS	Alagile syndrome	NM_000214.2(JAG1): C.1052delG; p.(Cys351 Leufs*61)	AD	Heterozgous	Pathogenic
26	Persistent elevated liver enzymes, congenital heart anomalies	NGS	Unknown	NM_000214.2(JAG1): C.1052delG; p.(Cys351 Leufs*61)	AD	Heterozgous	Pathogenic
27	Elevated liver enzymes and hepatosplenomegaly	NGS	WES	NM_000443.4(ABCB4):c.3634-4A>G; p.? NM_000443.4(ABCB4):c.1864G>T; p.(Gly622Trp)	AD/AR	Compound heterozygous	VUS
28	Elevated liver enzymes and hepatosplenomegaly	NGS	Custom gene panel	NM_000443.4(ABCB4):c.158A>T; p.(Asp53Val) NM_004004.6(GJB2):c.-23+1G>A; p.?NM_001042351.3(G6PD):c.563C>T; p.(Ser188Phe)	AR AR X-linked recessive	Homozygous homozygous hemizygous	VUS pathogenic pathogenic
34	Direct hyperbilirubinemia	Sanger	Targeted variant analysis	NM_025193.4(HSD3B7):c.45_46del; p.(Gly17 Leufs*26)	AR	Homozygous	Pathogenic
35	Persistent indirect hyperbilirubinemia	NGS	Gilbert syndrome genetic test	NM_000463.3(UGT1A1): C.-41_-40dupTA; p.?	AR	Homozygous	Pathogenic
37	Persistent indirect hyperbilirubinemia	NGS	Crigler-Najjar syndrome genetic test	NM_000463.3(UGT1A1): C.-41_-40dupTA; p.?	AR	Homozygous	Pathogenic
38	Persistent indirect hyperbilirubinemia	NGS	UGT1A1 full gene sequencing	NM_000463.3(UGT1A1): C.-41_-40dupTA; p.?	AR	Homozygous	Pathogenic
40	Direct hyperbilirubinemia	NGS	WGS	4.70 Mb deletion JAG1 gene 20p12	AD	Heterozygous	Pathogenic
43	Chronic pancreatitis	NGS	WES	NM_002769.4(PRSS1):c.365G>A; p.(Arg122His)	AD	Heterozygous	Pathogenic
44	Recurrent pancreatitis	NGS	Pancreatitis panel	NM_007272.3 (CTRC):c.738_761del; p.(Lys247_Arg254del)	AD	Heterozygous	Pathogenic
45	Chronic congenital diarrhea	NGS	Custom gene panel	NM_021102.4(SPINT2):c.442C>T; p.(Arg148Cys) NM_000277.3(PAH):c.157C>T; p.(Arg53Cys) NM_144687.4(NLRP12):c.1854C>G;p.(Tyr618*)	AR AR AD	Homozygous homozygous heterozygous	LP LP LP
48	Chronic congenital diarrhea	NGS	Unknown	NM_001080467.3(MYO5B):c.1966C>T; p.(Arg656Cys)	AR	Homozygous	LP
49	Chronic congenital diarrhea	NGS	Unknown	17 kb deletion in EPCAM gene 2p21	-	-	-
50	Chronic congenital diarrhea	NGS	Unknown	NM_001080467.3(MYO5B):c.1966C>T; p.(Arg656Cys)	AR	Homozygous	LP
51	Chronic congenital diarrhea, albinism, dysmorphism	NGS	Unknown	NM_014639.4(SKIC3):c.4070del; p.(Pro1357 Leufs*10)	AR	Homozygous	LP
52	Chronic congenital diarrhea	NGS	Chronic Congenital Diarrhea panel	NM_001080467.3(MYO5B):c.82del; p.(Thr28Profs*47)	AR	Homozygous	LP
53	FTT, persistent diarrhea, direct hyperbilirubinemia	NGS	Cholestasis panel	NM_020198.3(CCDC47):c.1234C>T; p.(Arg412*)	AR	Homozygous	LP
56	Non-mechanical intestinal obstruction, diarrhea	NGS	WES	NM_000111.2(SLC26A3):c.559G>T; p.(Gly187*)	AR	Homozygous	Pathogenic
57	FTT	NGS	Custom gene panel	NM_006408.4(AGR2):c.104del; p.(Asp35Alafs*38)	AR	Homozygous	LP
58	Chronic congenital diarrhea	NGS	WESTRIO	NM_001080467.2(MYO5B):c.2062C>T; p.(Arg688*) NM_000463.2(UGT1A1):c.1075G>A; p.(Asp359Asn) NM_000492.3(CFTR):c.1163C>T; p.(Thr388Met)	AR	Homozygous heterozygous heterozygous	Pathogenic VUS VUS
59	FTT, and chronic congenital diarrhea	NGS	WES	NM_012079.5(DGAT1):c.1374G>A; p.(Trp458*)	AR	Homozygous	LP
61	FTT	NGS	Cystic fibrosis panel	NM_000492.4(CFTR):c.1521_1523del; p.(Phe508del)	AR	Homozygous	Pathogenic
63	FTT	Sanger	Targeted variant analysis	NM_001012331.1(NTRK1):c.1624del; p.(Glu542Argfs*110)	AR	Homozygous	LP
64	FTT	NGS	Comprehensive lung panel	NM_001013838.3(CARMIL2):c.950dup; p.(Pro318Thrfs*44)	AR	Homozygous	LP
65	FTT	NGS	WESTRIO	NM_004333.6(BRAF):c.1574T>C; p.(Leu525Pro)	AD	Heterozygous	Pathogenic
68	Ileal mass and polyps	NGS	Unknown	NM_000455.5(STK11):c.300dup; p.(Gly622Trp)	AD	Heterozygous	LP
69	Multiple Polyp on multiple occasions	NGS	Unknown	STK11 gene deletion (unknown)	AD	Heterozygous	Pathogenic

IBD: Inflammatory bowel disease; NGS: Next-generation sequencing; CMA: Chromosomal microarray analysis; WES: Whole exome sequencing; FTT: Failure to thrive; AD: Autosomal dominant; AR: Autosomal recessive, LP: Likely pathogenic; VUS: Variant of uncertain significance; WESTRIO: Whole exome sequencing trio.

Sequencing-based testing (exome, genome, targeted gene panels) yielded an overall diagnostic rate of 49% (26/53) while CMA resulted in a lower diagnostic yield of 42.9% (3/7). Notably, the VEOIBD panel was performed in 12 patients but did not identify a positive result in any case (Table 2).

Table 2 Genetic testing modalities and diagnostic yield.

Test/panel	Positive	Total	%
WES	5	8	62.5
WESTRIO	3	3	100.0
WGS	1	1	100.0
VEOIBD panel	0	12	0.0
Custom gene panel	4	4	100.0
Cholestasis panel	4	10	40.0
CMA	3	7	42.9
UGT1A1 full gene sequencing	2	2	100.0
Gilbert syndrome genetic test	1	2	50.0
Crigler-Najjar syndrome genetic test	1	1	100.0
Targeted variant analysis	2	2	100.0
Pancreatitis panel	1	2	50.0
Chronic congenital diarrhea panel	1	3	33.3
Cystic fibrosis panel	1	1	100.0
Comprehensive lung panel	1	1	100.0
Alagille syndrome	1	1	100.0
Congenital mono- and disaccharide disorders panel	0	2	0.0
Unknown	7	7	100.0

WGS: Whole genome sequencing; WES: Whole exome sequencing; CMA: Chromosomal microarray analysis; VEOIBD: Very early-onset inflammatory bowel disease; WESTRIO: Whole exome sequencing trio.

Consistent with the higher consanguinity rates in the Middle East[4], conditions with autosomal recessive (AR) inheritance pattern were the most common, accounting for 64.4% (n = 29) of the 45 genetic variants identified in the study. Autosomal dominant (AD) and X-linked recessive conditions accounted for 20% (n = 9) and 8.8% (n = 4), respectively. Less frequent patterns included AR/AD inheritance and chromosomal anomalies, each observed in 2.2% (n = 1) of positive cases (Figure 2B). Notably, in two patients, a triple genetic diagnosis was identified (patients No. 28 and No. 45), with three distinct pathogenic variants detected, each associated with a different genetic disorder. Among the 45 identified pathogenic or likely pathogenic variants, 37 (82%) were single nucleotide polymorphisms (SNPs) and 8 (18%) were copy number variations.

Clinical utility

When considering the clinical context, diagnostic genetic results altered the clinical approach and management in 97% (37/38) of the positive cases and 53.6% of the whole cohort. Clinical management varied by indication and the genetic findings. Figure 3 illustrates the proportion of cases with altered management within each disease group, as well as among the total cohort and total genetically positive cases. In addition, all families with positive findings received genetic counselling to assess recurrence risks and to guide families in considering preventive strategies for future pregnancies. Table 3 summarizes the clinical diagnoses and corresponding management changes applied to each patient.

Figure 3  Rates of management changes per clinical category and across total and genetically positive cohort.

Table 3 Patient-specific clinical diagnoses and impact on medical management.

ID	Clinical diagnosis	Clinical implication
6	Wiskott-Aldrich syndrome or related phenotypes	Nil
14	X-linked chronic granulomatous disease	Requires BMT; no response to conventional therapy
15	X-linked chronic granulomatous disease	Requires BMT; no response to conventional therapy
16	Combined immunodeficiency with multiple intestinal atresias	Initiated leflunomide, counseled on immunodeficiency risk
17	Mitochondrial DNA depletion syndrome	Liver transplant referral; neurology referral; prenatal screening recommended
19	Trisomy 18	Liver transplant not pursued
20	Crigler-Najjar (and Gilbert syndrome)	Intensive phototherapy; phenobarbital; transplant counseling; potential gene therapy
21	Gilbert syndrome	Avoidance of further testing
23	PFIC 1	UDCA, IBAT inhibitor; hearing test; anticipate cirrhosis and possible liver transplant
24	Crigler-Najjar	Intensive phototherapy; phenobarbital; transplant counseling; potential gene therapy
25	Alagille syndrome	Screening for associated disorders: Cardiac, ocular, vascular, and renal
26	Alagille syndrome	Screening for associated disorders: Cardiac, ocular, vascular, and renal
27	PFIC 3	UDCA, IBAT inhibitor; slower disease progression
28	PFIC 3/autosomal recessive nonsyndromic hearing loss/glucose-6-phosphate dehydrogenase deficiency	UDCA, IBAT inhibitor; slower disease progression; counseling on post–liver transplant recurrence; G6PD precautions; hearing test; ENT referral
34	Congenital bile acid synthesis defect	Bile acid replacement therapy; liver transplant considered
35	Gilbert syndrome	Avoidance of further testing
37	Gilbert syndrome	Avoidance of further testing
38	Gilbert syndrome	Avoidance of further testing
40	Alagille syndrome	IBAT inhibitor, screening for associated disorders: Cardiac, ocular, vascular, and renal
43	Hereditary pancreatitis	Annual pancreatic cancer screening; regular monitoring of exocrine/endocrine function
44	Chronic pancreatitis	Regular monitoring of exocrine/endocrine function
45	TE, hyperphenylalaninemia, familial cold autoinflammatory syndrome	Family counseling for future pregnancies; MVT not favored as symptoms may improve
48	MVID	Family counseling for future pregnancies; renal screening; MVT likely as symptoms unlikely to improve; lifelong TPN anticipated
49	TE	Family counseling for future pregnancies; MVT not favored as symptoms may improve
50	MVID	Family counseling for future pregnancies; renal screening; MVT likely as symptoms unlikely to improve; lifelong TPN anticipated
51	Trichohepatoenteric syndrome	Screening for hypogammaglobulinemia, screening for liver disease
52	MVID	Family counseling for future pregnancies; renal screening; MVT likely as symptoms unlikely to improve; lifelong TPN anticipated
53	Trichohepatoneurodevelopmental syndrome	Screening for developmental delay, screening for liver disease
56	Congenital chloride diarrhea	Chloride supplementation
57	Recurrent respiratory infections and failure to thrive with or without diarrhea	Family counseling for future pregnancies
58	MVID	Family counseling for future pregnancies; renal screening; MVT likely as symptoms unlikely to improve; lifelong TPN anticipated
59	Congenital diarrheal disorder due to DGAT1 deficiency	Family counseling for future pregnancies; considering MVT
61	Cystic fibrosis	Annual screening for pancreatic insufficiency, started Trikafta
63	Autosomal recessive TRK 1 positive congenital insensitivity to pain with anhydrosis	Antipyretics and cooling measures; trauma precautions due to absent pain; screen for immunoglobulin deficiency
64	Immunodeficiency	Screening for immunodeficiency
65	BRAF gene related disorders (cardiofaciocutaneous syndrome, Noonan syndrome and LEOPARD syndrome)	Family counseling regarding high risk of developing tumors, referral to cardiology, started Growth hormone
68	Peutz-Jeghers syndrome	Regular endoscopic screening; genetic testing for first-degree relatives
69	Peutz-Jeghers syndrome	Regular endoscopic screening; genetic testing for first-degree relatives

TE: Tufting enteropathy; BMT: Bone marrow transplant; UDCA: Ursodeoxycholic acid; IBAT: Ileal bile acid transporter; MVT: Multi-visceral transplant; TPN: Total parenteral nutrition; PFIC: Progressive familial intrahepatic cholestasis; ENT: Ear, nose, throat; MVID: Microvillous inclusion disease.

DISCUSSION

Liver disease

This was largest group of patients in our cohort where 24 presented mainly with elevated liver enzymes, hyperbilirubinemia (both direct and indirect) and liver failure. Majority of these cases were encountered during infancy, and the genetic testing was considered at some point the only modality left to establish the diagnosis. Several pathologies were identified such as: Mitochondrial DNA depletion syndrome, Trisomy 18, Crigler-Najjar syndrome, Gilbert syndrome, PFIC, Alagille syndrome, and congenital bile acid synthesis defect. As shown in Figure 2A, the yield of the genetic testing in this group was high (62.5%) and it helped to identify the diagnosis where other modalities have failed. The management or intervention of these patients has been altered in all cases, either directly or indirectly, based on the genetic results.

Patients with unexplained indirect hyperbilirubinemia often undergo frequent testing, and possibly invasive procedures like liver biopsy. The uncertain diagnosis is also stressful for parents. Using genetic testing to establish the diagnosis of Gilbert syndrome (n = 4) can help set expectations for parents, allowing them to understand the benign nature of the disease, avoid frequent blood tests, and also prevent negative triggers such as dehydration, fasting and stress[21]. On the other hand, Crigler-Najjar syndrome does not have the same benign nature as in Gilbert syndrome; patients may develop bilirubin-induced neurological dysfunction and even face the risk of death. These risks were discussed with the families, and patients (n = 2) were offered intense phototherapy and initiation of phenobarbital as an enzyme inducing agent. Additionally, parents were made aware of the choice of liver transplant which can be offered later in life to cure the disease[22]. Recently, gene therapy for this condition has been studied in a phase 1 and 2 trial, with promising results though this therapy require more time before it becomes an option for those patients[23].

Cholestasis or itching can manifest as presenting symptoms in variety of cases. In PFIC (n = 2), molecular findings can confirm the diagnosis and aids in identifying subtypes, which significantly influence prognosis. This information helped to counsel the parents and to decide management. One patient had ATP8B1 homozygous pathogenic variants (PFIC 1) and needed referral for hearing assessment since this disease is associated with hearing loss[24]. Family was informed that the prognosis remains poor despite liver transplantation patient No. 27 with ABCB4 compound heterozygous variant diagnosed with PFIC 3 disease had a milder disease and responded well to ursodeoxycholic acid. Both families were informed that ileal bile acid transporter (IBAT) inhibitors may be considered in cases of inadequate treatment response or disease progression. On the other hand, patient No. 34 was diagnosed with bile acid synthesis defects, and was started on bile acid replacement therapy.

Genetic testing has proven invaluable in guiding the management of complex and multisystem presentations. In confirmed Alagille syndrome cases (n = 3), identifying associated defects of the heart, eyes, vascular system (including the risk of intracranial bleeding), and kidneys are crucial for comprehensive care planning[25]. Accordingly, those patients were referred for several speciality clinics to screen for the associated defects. Also, patient No. 40 was started on IBAT inhibitors. In the case of mitochondrial DNA depletion syndrome (n = 1), the patient presented with acute liver failure and she was referred for liver transplant at another center[25]. Patient No. 19 was diagnosed with trisomy 18 and was ineligible for liver transplant due to poor outcomes associated with the syndrome and we did not offer the family this therapy option[26]. These examples highlight the importance of genetic testing in taking vital and critical decisions which can be quite significant to patients and their families.

A novel gene potentially underlying a transient form of cholestasis

Patient No. 29 presenting with transient episodic itching and cholestasis had negative exome sequencing, though further analysis identified a homozygous 4 bp deletion in exon 4 (NM_005122.5) of a novel gene, NR1I3 (Figure 4). This 4 bp deletion (c.318_321del), which is extremely rare (allele frequency 0.0022%) in the general population, namely in the gnomAD (causes a frameshift which is predicted to alter the protein’s amino acid sequence beginning at position 107 and lead to a premature termination codon 6 amino acids downstream (p.Ser107Argfs*6). This alteration is then predicted to lead to a truncated receptor lacking the ligand-binding domain or, most likely, an absent protein given the expected nonsense mediated decay.

Figure 4 A novel gene potentially underlying a transient form of cholestasis. Karyogram view of chromosome 1 vertical red bar marks the cytogenetic location (1q23.1) of the NR1I3 gene. Blue lines represent a zoomed-in view into the 4 bp deletion identified in the patient No. 29 with episodic cholestasis. The 4 bp deletion is shown in Integrated Genome Viewer where each horizonal grey bar represents a sequencing read while vertical grey bars representing depth of coverage at each genomic position. The 4 bp deletion is found on every read suggesting that it is homozygous in the patient as also shown by the lack of coverage across those positions. An Alamut software view on the right shows that this deletion is in exon 4 of NR1I3 transcript NM_005122.5. Bottom left, expression pattern from the Genotype-Tissue Expression project showing exclusive expression of the NR1I3 gene in the liver consistent with a role in bile acid metabolism. Bottom middle, family pedigree where squares represent males where circles representing females. Dashed symbols represent affected individuals while the “+” and “-“ signs represent presence or absence of the NR1I3 deletion, respectively, on either allele. Bottom right, a schematic showing the molecular interaction of the NR1I3 protein with other partners involved in bile acid clearance in the hepatocyte.

The NR1I3 gene encodes a nuclear receptor which is strictly expressed in the liver, based on human gene expression data, and is thought to regulate the transcription of genes involved in drug metabolism and bilirubin clearance (Figure 4). A previous study suggested a link between NR1I3 variants and hyperbilirubinemia, with preliminary evidence indicating a possible role in bilirubin metabolism[27].

Familial testing showed that the similarly affected two siblings were also homozygous for the NR1I3 frameshift variant. However, their father, who does not recall a history of liver disease, was also homozygous while an affected cousin was heterozygous carrier for this variant (Figure 4). While this information may suggest non-segregation and may not support a causal role for NRI13 in cholestasis, functional analyses are needed to characterize the penetrance expressivity, and inheritance of this potentially novel gene in light of the above evidence and given the progressive and episodic nature of this disease. Notably, the patient’s older sibling’s symptoms resolved over time, raising the possibility that the father may have experienced a similar self-limited disease course during childhood.

IBD

A total of 16 patients presented with IBD in this cohort. Patient No. 16 presented with diarrhoea and early onset of bowel inflammation, ultimately leading to bowel atresia and short gut syndrome necessitating lifelong parenteral nutrition. Exome sequencing (targeted gene panel sequencing) identified a homozygous pathogenic variant in the TTC7A gene pathogenic variant was identified. TTC7A pathogenic variants typically coincide with immunodeficiency in up to 90% of cases and necessitate screening, our patient did not display overt immunodeficiency[28]. This genetic finding was helpful for anticipating prognosis, highlighting the possible associated immunodeficiency problems reassuring the family that GI symptoms might slightly improve with time[28]. Furthermore, the patient was treated with leflunomide without clinical benefit and was later offered a MVT, which could potentially provide long-term survival and independence from parenteral nutrition.

A significant outcome of genetic testing in VEOIBD patients lies in identifying underlying immunodeficiency as a primary condition. We documented the case of two male siblings (patients No. 14 and No. 15) harbouring an X-linked recessive CYBB pathogenic deletion, confirming the diagnosis of chronic granulomatous disease. This finding was important for management as it indicated that existing therapy for IBD may not be effective and the response to different IBD medicine classes is unpredictable[29]. Once the diagnosis was made, hematopoietic stem cells transplantation (HSCT) became the optimal treatment modality. There are ongoing clinical trials to study gene therapy for this condition[30]. Interestingly, the siblings exhibited different clinical presentations, with one experiencing bowel inflammation while the other suffered from recurrent perianal abscess formation. Both siblings were referred for assessment and are currently awaiting HSCT.

Pancreatitis

Genetic testing was performed for four cases with pancreatitis, revealing positive results in two individuals. Specifically, pathogenic variants were identified in the PRSS1 and CTRC genes. PRSS1 pathogenic variants are the most common cause of pancreatitis. This gene plays a role in regulating trypsin function, which can lead to immature and excessive activation leading to inflammation and pancreatitis[31]. On the other hand, the CTRC gene is responsible for facilitating trypsin lysis. Loss of function variants in this gene result in the loss of its protective role against trypsin activation[31]. Both cases were scheduled for regular follow up to screen for pancreatic exocrine and endocrine function, in addition to ultrasound screening for pancreatic cancer in the patient with the PRSS1 variant which has increased risk of malignancy.

In patient No. 41, although a definitive diagnosis explaining pancreatitis was not identified, a homozygous variant of uncertain clinical significance was identified in the TULP3 gene which has been linked to liver and kidney disease, as well as cardiomyopathy[32]. Interestingly, the same variant has been described in the literature in two patients with chronic pancreatitis[32]. This patient also had a secondary pathogenic variant in MYBPC3, which is associated with risk of dilated cardiomyopathy and was therefore referred for additional surveillance and familial testing[33].

Chronic congenital diarrhoea

A total of 15 patients were included in this cohort. They presented with chronic diarrhea and were genetically confirmed to have the following diagnoses: MVID, tufting enteropathy (TE), tricho-hepato-neurodevelopmental syndrome (THNS), tricho-hepato-enteric syndrome (THES), glucose galactose malabsorption, congenital sucrase-isomaltase deficiency and others. These patients typically present with diarrhea, which can vary from mild to severe. However, it is not possible to differentiate the underlying diagnosis based solely on the clinical presentation.

In this cohort, 4 patients with MVID exhibited homozygous pathogenic variants in the MYO5B gene. Clinical presentation commonly includes acidosis, hypotonic dehydration with high stool sodium excretion, and absence of inflammatory markers. Additional investigations can raise suspicion, while genetic testing was crucial for confirming the diagnosis. Diagnosing MVID is challenging, as relying solely on electron or light microscopy can lead to false negatives. Accurate interpretation of histological and immuno-staining findings necessitates the expertise of a skilled pathologist. Fortunately, genetic testing provided us with a reliable means of confirmation[34]. Once diagnosed, parents were informed about the need of lifelong parenteral nutrition. MVID can adversely affect kidney function, necessitating regular monitoring. The only curative option available is a multi-visceral transplant (MVT) which was offered to the parents. Ongoing clinical trials are exploring medications like Crofelemer to alleviate symptoms and reduce the dependence on parenteral nutrition in MVID patients, which was also offered to our patients[35].

TE can be considered a favourable diagnosis since its prognosis is good with long-term survival reaching up to 90%, and enteral autonomy can range between 50%-75% at age of 25 years[36]. In patient No. 45, two homozygous likely pathogenic variants were identified in SPINT2 and PAH, explaining the chronic diarrhoea and suggesting phenylketonuria, respectively. However, biochemical testing showed phenylalanine levels within the non-treatment range, consistent with benign hyperphenylalaninemia, which does not require dietary management. Given the diagnosis of TE, MVT was not recommended, as over 50% of affected patients eventually achieve enteral autonomy[36]. Additionally, a likely pathogenic NLRP12 variant was detected, correlating with recurrent fevers and elevated serum amyloid A. Despite trials of colchicine and Anakinra, there was no therapeutic response; symptoms resolved spontaneously by age two. Genetic testing clarified the diagnosis, avoided unnecessary interventions, and guided targeted (though ultimately ineffective) therapy.

In patient No. 49, who presented with diarrhoea shortly after birth, a pathogenic deletion in the EPCAM gene confirmed the diagnosis of MVID. The genetic diagnosis influenced the decision not to advocate for MVT, given the known potential for clinical improvement over time[36]. In two other patients (No. 51 and No. 53), we were able to diagnose rarer diseases such as THES and THNS caused by homozygous pathogenic variants in the SKIC3 and CCDC47 genes, respectively. Both patients had abnormalities of the hair color, and liver involvement. These genetic findings promoted screening for hypogammaglobulinemia in THES and for neurodevelopmental delay in THNS. Additionally, regular surveillance for hepatic dysfunction was carried out to prevent unnoticed progression.

Failure to thrive

The main reason for performing genetic testing in this group, which included 8 patients, was failure to thrive, yet all the patients had other symptoms such as diarrhea, vomiting, developmental delay, and dysmorphism and therefore genetic findings, which were positive in 4 out of the 8 patients (50%) in this group, helped characterize the underlying disease. For example, the identification of a mutation in the NTRK1 gene confirmed the diagnosis of AR congenital insensitivity to pain with anhidrosis. This condition requires special care of the limbs and appropriate temperature control. A BRAF gene mutation was found in another patient which is associated with Noonan syndrome characterized with failure to thrive, short stature, dysmorphic features, and congenital heart defects[37]. Therefore, patient was referred to cardiology for further evaluation and was started on growth hormone therapy. A pathogenic variant in the CARMIL2 gene was found in patient No. 64 who presented with recurrent chest infection and failure to thrive. Loss of function of this gene causes primary immunodeficiency along with failure to thrive, and recently was linked to the development of VEOIBD[38]. This patient was therefore referred for immunological screening. Finally, a homozygous pathogenic variant in the CFTR gene was identified in patient No. 61. Subsequently, the patient was diagnosed with cystic fibrosis and referred to a specialized clinic for regular follow-up and surveillance.

Polyposis

This small group comprised only two patients, both presenting with multiple polyps, and one experiencing intussusception. Both patients exhibited heterozygous pathogenic variants in the STK11 gene, which causes an AD Peutz-Jeghers syndrome. These individuals displayed distinctive polyp characteristics on histopathological analysis, though the diagnosis was confirmed by genetic testing. Following diagnosis, patients required routine endoscopic surveillance and regular screening for extraintestinal cancers due to the increased risk. Moreover, genetic test screening was offered to first-degree relatives of affected individuals with known genetic mutations[39].

Limitations

This study has several limitations. First, the sample size is relatively small, which may limit the generalizability of our findings across the broader pediatric gastroenterology population. Second, we were unable to obtain long-term follow-up for many patients. A significant proportion of families relocated to other countries or transferred care to other institutions, which restricted our ability to monitor the sustained clinical impact of the genetic results over time. Consequently, while the short-term clinical utility of genetic testing was clearly demonstrated, the long-term outcomes and potential evolution of management decisions could not be fully assessed. Future studies with larger cohorts and more consistent longitudinal follow-up are needed to confirm and expand upon these findings.

CONCLUSION

In conclusion, genomic investigations serve as a critical tool in pediatric GI diseases, aiding in diagnosis, etiological understanding, and treatment guidance. Despite ongoing gaps in our knowledge of many genes, the expanding genomic databases offer promising prospects for further insights. Our findings underscore the significant impact of genetic testing on altering management strategies and providing valuable prognostic information. Furthermore, we identified a novel gene potentially associated with a new form cholestasis. Continued research and technological advancements in genetic testing hold the promise of improving diagnostic and therapeutic approaches, ultimately enhancing patient outcomes and quality of life.