Effect of Genetic Testing on Diagnosing Gastrointestinal Pediatric Patients with Previously Undiagnosed Diseases

Figures & data

Figure 1 Pedigrees of the four participating families. F1 refers to family #1; F2, family #2; F3, family #3, and F4 refers to family #4. Squares and circles indicate male and female members, respectively. Black filled symbols represent affected individuals of relevance in this study, red filled symbols represent affected siblings with different disease, empty symbols for unaffected individuals, crossed symbols for deceased individuals, double lines represent consanguinity, and finally, arrows refer to probands in each case.

Table 1 Clinical Data for Participated Probands in Each Family

Family	Member	Gender	Age Onset	Initial Diagnosis	Symptoms	Outcome
F1	IV-3	Female	AB	Renal tubular acidosis, thalassemic, growth deficiency	Growth delay, splenomegaly, vomiting, diarrhea, painful distended abdomen, anemia, liver steatohepatitis and cirrhosis	Dead (15 Y)
	IV-1	Male	1.5 M	Suspected mitochondrial/ metabolic disorder	Vomiting, hypotonia, failure to thrive, anemia, hypothyroidism, distended abdomen, gastroesophageal reflux, skin peeling, chronic diarrhea	Stabilized through respiratory support (3 Y)
F2	II-1	Female	AB	Hirschsprung's disease	Distended abdomen, fecaloma, food intolerance, enterocolitis, vomiting, failure to thrive	Dead (1.5 Y)
F3	II-1	Female	2 M	Severe cholestatic hepatitis; metabolic disease	Prolonged neonatal jaundice	Clinically stable (11 M)
F4	II-2	Female	AB	Hypertrichosis related syndrome	Developmental delay, breathing difficulties including apnea, hypertrichosis, elevated liver enzyme, feeding difficulties with dysmorphic features, hypotonic and severe anasarca	Dead (2.5 M)

Abbreviations: AB, at birth; M, month; Y, year.

Table 2 Identified Variants in Each Participating Family

F#	Gene Transcript_ ID	MI	Variant coordinate (hg19)^a	Exon	Genotype	HGVS cDNA		HGVS aa	Consequence		GNOMAD Max MAF	dbSNP ClinVar HGMD IDs	Classification
1	SLC7A7 ENST00000555702.1	AR	chr14: 23243620–23243623	9	Homozygous	c.1185_1188del		p.Ser396fs	Frameshift deletion		0.0084%	rs386833800 56352 CD000276	Pathogenic
2	ACTG2 ENST00000345517.3	AD	chr2: 74141962	7	Heterozygous	c.769C>T		p.Arg257 Cys	Missense		N/A	rs587777387 132803 CM143757	pathogenic
3	HSD3B7 ENST00000297679.5	AR	chr16: 30997024–30997025	2	Homozygous	c.45_46del		p.Gly17fs	Frameshift deletion		0.0128%	rs786200876 2885 CD031506	Pathogenic
4	DPAGT1 ENST00000409993.2	AR	chr11: 118968581	8	Homozygous	c.901C>T		p.Arg301 Cys	Missense		0.0028%	rs776632995─ CM126444	VUS

Abbreviations: F, family; MI, mode inheritance; AR, autosomal recessive; AD, autosomal dominant; HGVS, Human genome variation society; cDNA, coding DNA sequence; aa, amino acid level; MAF, minor allele frequency; N/A, not available; VUS, virulence of uncertain significance; a Variant coordinate according to the Genome Reference Consortium Human Build 37 (hg19).

Table 3 Laboratory Tests for Both Patients in Family F1

Proband	Plasma Amino Acid Levels (µmol/L)			Urine Amino Acid Levels (nmol/mg)			Ammonia (µmol/L)	Ferritin (ng/mL)	LDH^a (U/L)
	Lysine (120–290)	Arginine (44–120)	Ornithine (44–90)	Lysine (19–1988)^b (10–240)^c	Arginine (10–560)^b <153 ^c	Ornithine <265^b <18^c	(11–32)	(12–64)^b(15−77)^c	(180–435)^b(110–283)^c
IV-1	72	0	0	5957	473	0	111.2	1814	2698
IV-3	216	46	83	2614	257	0	128.5	1917	4231

Notes: ^aLDH: lactate dehydrogenase. Reference ranges designated with ^b refer to group < 24 months old. Reference ranges designated with ^c refer to group between the ages 9 and 17 years.

Figure 2 Histopathological findings for patient IV-3 in family F1. (A and B) Hematoxylin and eosin staining shows steatosis, hepatocytes ballooning, and portal lymphocytic infiltration. (C and D) Masson’s trichrome and Van Gieson’s stain, respectfully, showing bridging fibrosis partially forming half nodules. Magnification: 40× (A and C) and 100× (B and D).

Figure 3 Sanger sequence chromatography for family F1 (left panel), family F2 (middle panel), and family F3 (right panel). In F1: the two probands (IV-1 and IV-3) have a pathogenic homozygous variant in SLC7A7 gene. Heterozygous carriers in the family are shown in middle part, while homozygous for the wild type allele is shown at the top. In F2 (middle panel): proband II-1 carries a de novo heterozygous variant in ACTG2 gene; whilst her parents carry the wild type homozygous allele. In the right panel, family F3, the two probands II-1 and II-2 have a homozygous deletion in HSD3B7 gene. While the parents along their children have the heterozygous allele.

Figure 4 Imaging tests for proband II-1 in family F2. (A and B) Abdominal X-ray and barium study showing severe dilated bowel loops. (C and D) Calretinin staining 2 cm away from the anal verge, 4 cm from the anal verge (E), and from the ileostomy site (F). Ganglion cells are present (magnification: 40×, 100×, 200×, and 40× for C, D, E, and F, respectively).

Table 4 Liver Function Tests and Blood Coagulation Tests Conforming Liver Damage in Proband II-1 from Family F3

Liver and Coagulation Tests	Proband II-1	Reference Range
Aspartate aminotransferase (AST)	68.5 U/L	8–50 U/L
Alanine aminotransferase (ALT)	89 U/L	7–45 U/L
Alkaline phosphatase (ALP)	691 U/L	122–469 μmol/L
Total bilirubin	50.9 μmol/L	2.0 to 21 μmol/L
Direct bilirubin	45.9 μmol/L	<8 μmol/L
Partial thromboplastin time (PPT)	41.8 seconds	30–45 seconds
Prothrombin time (PT)	17 seconds	12–13 seconds

Table 5 FAB-MS Test Results for Both Probands in Family F3 Indicate the Presence of Peaks Characteristic of 3-β-HSD Deficiency

Test	Proband II-1	Proband II-2	Reference Range
Creatinine	1.5 mmol/L	21.9 mmol/L	0.5–2.2 mmol/24 h
Cholic acid	1.08 μmol/L	0.47 μmol/L	Negative
Chenodeoxycholic acid	0.12 μmol/L	0.57 μmol/L	Negative
Deoxycholic acid	0.00 μmol/L	0.25 μmol/L	Negative
Lithocholic acid	0.00 μmol/L	0.0 0μmol/L	Negative
Ursodeoxycholic acid	0.00 μmol/L	136 μmol/L	Negative
Hyocholic acid	0.00 μmol/L	0.7 μmol/L	Negative
Hyodeoxycholic acid	0.00 μmol/L	0.00 μmol/L	Negative
3β,7α-Dihydroxy-5-cholestenoate	0.00 μmol/L	51.54 μmol/L	Negative
DiOH oxycholeoic acid	0.00 μmol/L	1.00 μmol/L	Negative

Figure 5 Histopathological findings for patient II-1 in family F3. Hematoxylin and eosin staining showing hepatocytes ballooning and feathery changes with marked intrahepatic cholestasis. Mild periportal inflammation and foci of spotty necrosis are also present (magnification: 100× and 200× left to right, respectively).

Figure 6 MRI and clinical features of proband II-2 in family F4. (A) Brain MRI; myelination is appropriate for proband’s age. Both thalami, both basal ganglia, corpus callosum, brainstem, cerebellum, and ventricular system appear normal. Central midline structures with no mass effect, no hemorrhage or collection seen. (B) Patient’s characteristic features; soft long ears, U-shaped vermilion of the upper lip, thick skin, hypertrichosis, moderate multiple contractures, hypotonia, and severe hypokinesia.