Demographics of the study population
A total of 439 NICU infants were enrolled. There were 266 males (60.6%) and 173 females (39.4%), with a median (quartile) age of 7.0 (1.0, 26.0) days at the time of hospital admission. There were 49 (11.2%) preterm newborns (gestational week ≤ 32), with a median gestational age of 38.1 (35.9, 39.5) weeks. A total of 147 (33.5%) newborns were hospitalized on the first day of life. The median (days) (quartile) age at onset was 1.0 (1.0, 11.0), age at admission was 3.5 (1.0, 26.0), and NICU stay length was 16.0 (8.0, 30.0). 99 infants died before 180 days of life, with a median age of 55.0 (19.0, 109.0) days at death. The main symptoms leading to genetic testing included multiple malformation (162, 36.9%), seizures and hypotonia (158, 35.9%), metabolic crisis and endocrine disturbance (105, 23.9%), recurrent and severe infection (67, 15.3%), cardiac anomalies (24, 5.5%), and others (95, 21.6%). The clinical characteristics of the genetic cohort are shown in Supplement Table 1.
Table 1
Summary of exome sequencing and medical management in the enrolled infants.
Characteristic | N (%) Median (quartiles) | 2016-2017a | 2018 | 2019 | P value b c |
N | 439 | 125 | 154 | 160 | NA |
Exome sequencing | | | | | 0.23 |
TES | 83 | 21 | 25 | 37 | |
WES | 356 | 104 | 129 | 123 | |
Sequencing type | | | | | 0.33 |
Singleton | 84 | 29 | 25 | 30 | |
Trios | 355 | 96 | 129 | 130 | |
Age at genetic testing (days) | 19.0 (8.0, 41.0) | 23.0 (10.0, 42.0) | 18.0 (9.0, 33.0) | 15.0 (6.0, 45.0) | 0.36 |
Time to genetic diagnosis (days) | | | | | |
Admission to attainment of parents’ consent | 6.0 (3.0, 12.0) | 6.0 (2.0, 11.0) | 7.0 (3.0,16.0) | 6.0 (3.0, 9.5) | 0.15 |
Sample receipt to report | 46.0 (33.0, 66.0) | 64.0 (45.0, 66.0) | 48.0 (35.0, 66.0) | 28.0(24.0, 36.0) | < 0.001 |
Molecular positivity rate | | | | | 0.82 |
Diagnosed | 203 (46.2) | 58 (46.4) | 68 (44.2) | 77 (48.1) | |
Undiagnosed | 236 (53.8) | 67 (53.6) | 86 (55.8) | 83 (51.9) | |
Medical management | | | | | NA |
Redirection of care | 12 | 3 | 5 | 4 | |
Initiation of new subspecialist care | 35 | 7 | 13 | 15 | |
Changes in medication or diet | 18 | 3 | 6 | 9 | |
Major procedures | 5 | 0 | 2 | 3 | |
Total proportion in diagnosed cases (%) | 70 (34.5) | 13 (22.4) | 26 (38.2) | 31 (40.3) | 0.06 |
180-day mortality in diagnosed infants | | | | | 0.31 |
Deceased | 60 (29.5) | 21 (35.9) | 21 (30.9) | 18 (29.6) | |
Survived | 143 (70.5) | 38 (64.41) | 47 (69.1) | 58 (70.4) | |
P trend of 180-day mortality across years | | | | | 0.08 |
TES: Targeted exome sequencing; WES: Whole exome sequencing. NA, not applicable. |
Categorical variables are presented as numbers (%). |
Continuous variables are presented as medians (quartiles). |
a Merged data from 2016 and 2017 due to a moderate sample size. |
b 2-Tailed t test or Fisher exact test, when applicable. |
c Comparation among three periods, 2016–2017, 2018 and 2019. |
Diagnostic And Turnaround Time Of Exome Sequencing
The number of enrolled infants suspected of genetic diseases was 30, 95, 154, and 160 in 2016, 2017, 2018, and 2019, respectively. Due to the moderate sample size, we merged the 2016 and 2017 data. The genetic testing methods included singleton (n = 84, 19.1%), trios (n = 355, 80.9%), TES (n = 83, 18.9%), and WES (n = 356, 81.1%), depending on the availability of parental samples and the suggestion of our multidisciplinary team. The molecular diagnosis rate was 59 (47.2%), 68 (44.2%), and 76 (47.5%) over three periods, without a significant difference. The time from patient admission to the attainment of the parents’ consent remained unchanged. However, the median days (quartiles) from sample receipt to report (positive and/or negative) was respectively 64.0 days (45.0, 66.0), 48.0 days (35.0, 66.0), and 28.0 days (24.0, 36.0) across 2016–2017, 2018 and 2019 with significant difference (p < 0.001) (Table 1).
Genetic Disease Spectrum Of The Diagnosed Infants
Of the 439 infants, 203 (46.2%) were molecularly diagnosed with 143 disorders. Autosomal dominant, autosomal recessive, and X-linked disorders were observed in 93 (45.8%), 83 (40.9%), and 28 (13.8%) infants, respectively. Of the genetically diagnosed cases, 178 (87.7%) infants had monogenic disorders. Although 23 genes were hit multiple times, we found that 120 (59.1%) genes were hit only once, reflecting genetic heterogeneity in NICU infants. Among the monogenic disorders, familial hyperinsulinemia hypoglycemia (OMIM 256450), Kabuki syndrome (OMIM 147920), methylmalonic aciduria and homocystinuria (OMIM 277400), ornithine trans-carboxylase deficiency (OMIM 311250), inflammatory bowel disease 28 (OMIM 613148), and spinal muscular atrophy (OMIM 253300), caused by variants in ABCC8, KMT2D, MMACHC, OTC, IL-10RA, and SMN1, respectively, were the most frequent monogenetic diseases and were observed in 24 infants in the cohort. 25 (25/203, 12.3%) infants had CNVs, including three aneuploidy variants and 22 microdeletion/duplication variants. The recurrent causative aberrations were Prader-Willi Syndrome (28.0%), 16p11.2p12.2 deletion (12.0%), Jacobsen syndrome (8.0%), and Xp11.23-p11.22 duplication (8.0%) (Supplement Table 2).
Table 2
Comparison of 180-day mortality rate across eight categories based on phenotype-related genes.
Category | Total number | 180-day mortality (%) | OR (95% CI) | Phenotype-related genes |
G0 | 236 | 39 (16.5) | Ref. | / |
G1 | 55 | 20 (36.4) | 2.89 (1.51,5.52) ** | ABCC8; ACADVL; ALG12; ALPL; BSCL2; CASR; CPS1; CYP21A2; DUOX2; FOXP3; G6PD; GNAS; GSS; KCNJ11; MARS; MMACHC; MUT; NPC1; OTC; PAX8; PCSK1; PEX1; PEX26; PKLR; PRKAR1A; SCNN1A; SCNN1G; SLC6A19; STAR; TBX19; TRPM6; UGT1A1; |
G2 | 36 | 9 (25.0) | 1.68 (0.74, 3.86) | 11q24.1-25 del; 12p13.33-11.21 dup; 15q11.2-13.1 del; 16p11.2-p12.2 del; 16q Trisomy; 18 Trisomy; 19p13.12 del; 21q22.12-22.13 del; 7q11.23 del; ATP6V1B2; BRAF; CREBBP; DDX58; EFTUD2; HRAS; KMT2D; NIPBL; NOTCH1; PTPN11; SHOC2; |
G3 | 29 | 6 (20.7) | 1.32 (0.50, 3.45) | 20q13.33 del; 2q37.3 del; 9q33.3 del; AP4E1; AP4S1; ATN1; CHRNA2; FGFR2; FOXP1; GLDC;KAT6B; KCNQ2; MECP2; NPRL2; PACS2; POLE; PPP2R5D; PUM1; SCN2A; SCN3A; SCN8A; SMO; STAG2; TSC2; UBA5; Xp11.23-p11.22 dup; ZC4H2 |
G4 | 16 | 9 (56.3) | 6.49 (2.28, 18.48) ** | CHRNA1; DMD; GTPBP3; IGHMBP2; MTM1; MYBPC3; MYH7; SMN1 |
G5 | 15 | 1 (6.7) | 0.36 (0.05, 2.82) | ALOX12B;COL17A1;COL7A1; EDA; IKBKG; KPT9; KRT14; TGM1; |
G6 | 14 | 6 (42.9) | 3.79 (1.25, 11.53) ** | 22q11.2 del; CXCR4; CYBB; IKBKG; IL10RA; IL2RG; ITGB2;JAK3; PRF1; SPINK5;WASP |
G7 | 38 | 9 (23.7) | 1.56 (0.68, 3.57) | Hematological: F13A1; FANCC; FLI1; GP1BA; HBG2; RPL5; RPS19; TUBB1; UNC13D; Cardiac: CHD7; FBN2; RAF1; TGFBR2; Gastrointestinal: EPCAM; JAG1; MYO5B; Skeletal: AMER1; CLCN7; COL2A1; PLOD1; Nephrological: ATP6V0A4; AQP2; AVPR2; NPHS1; Respiratory: ABCA3; DNAH11; DNAH5; Vascular: FGFR3; RASA1 ; Lymphatic: EPHB4; KIF11; Eyes: EYA1; Development Disorders: EZH2. |
G0: undiagnosed; G1: metabolic and endocrine disorders; G2: multiple congenital malformation; G3: neurological disorder; G4: early-onset muscle disease; G5: dermatological disorder; G6: primary immunodeficiencies; G7: other, including cases involving the following systems: hematological, cardiac; gastrointestinal, skeletal, nephrological, respiratory, vascular, lymphatic, eyes and development disorders. |
**: P < 0.01. |
Comparison of 180-day mortality rates across eight categories according to phenotype-related genes
Among the 203 diagnosed infants, 60 infants died before 180 days. 25 infants died prior to the genetic diagnosis. The 180-day mortality rate in the diagnosed infants was 18/59 (30.5%) in 2016–2017, 21/68 (30.9%), and 21/76 (27.6%), respectively. This represents a moderate decreasing trend (p trend = 0.08) (Table 1).
The 180-day mortality rates across the eight categories were as follows: G0 (39/236, 16.5%); G1 (20/55, 36.4%); G2 (9/36, 25.0%); G3 (6/29, 20.7%); G4 (9/16, 56.3%); G5 (1/15, 6.7%); G6 (6/14, 42.9%); and G7, other (9/38, 23.7%), involving systems including hematological (1/9, 14.3%), cardiac (0/6), gastrointestinal (2/6, 33.3%), skeletal (1/4, 205.0%), nephrological (1/4, 25.0%), respiratory (3/3, 100.0%), vascular (0/2), lymphatic (0/2), eyes (0/1) and development disorders (0/1).
When observing the odds ratios for obtaining the odds ratios of 180-day mortality, we set the undiagnosed G0 infants as the reference group. The G1 infants with metabolic and endocrine disorders (OR = 2.89, 95% CI: 1.51–5.52), G4 infants with early-onset muscle disease (OR = 6.49, 95% CI: 2.28–18.48), and G6 infants with primary immunodeficiencies (OR = 3.79, 95% CI: 1.25–11.53) had significantly high odds ratios of 180-day mortality rate. However, infants diagnosed with the category of G2, G3, G5 or G7 were not related to 180-day mortality (Table 2). Furthermore, Kaplan-Meier curves showed that the survival proportion of patients with G3, G5, and G7 categories shown the same trend as that of G0 infants (Fig. 1a). In contrast, the mortality rate of patients with G1, G4 and G6 presented significantly lower survival rates than G0 infants (< 60% survival) (Fig. 1b).
Comparison of 180-day mortality rates across six molecular function categories
Among the diagnosed infants with monogenic disorder, the 180-day mortality rates across six molecular function categories were as follows: M0, undiagnosed (38/235, 16.17%); M1, catalytic activity deficiency (16/43, 37.2%); M2: signaling pathway associated activity deficiency (12/45, 26.7%); M3 transporter activity deficiency (12/27, 44.4%); M4, DNA/RNA metabolism activity deficiency (10/42, 23.8%); and M5, other (6/22, 27.3%). Compared with M0, the M1 (OR = 3.07, 95% CI: 1.51–6.25) and M3 (OR = 4.15, 95% CI: 1.80–9.56) categories were significantly related to the 180-day mortality rate. However, M2 (OR = 2.11, 95% CI: 0.99–4.38), M4 (OR = 1.41, 95% CI: 0.63–3.19) and M5 (OR = 1.95, 95% CI: 0.72–5.29) had low odds ratios for the 180-day mortality rate (Table 3).
Table 3
Comparison of 180-day mortality rate across five categories of molecular function.
Category | Total number | 180-day mortality (%) | OR (95% CI) | Mutated gene products |
M0 | 197 | 38 (16.2) | Ref. | / |
M1 | 43 | 16 (37.2) | 3.07 (1.51, 6.25) * | ACADVL; ALG12; ALOX12B; ALPL; BSCL2; CASR; CPS1; CYP21A2; DUOX2; G6PD; GLDC; GNAS; GSS; MARS; MMACHC; MUT; NPC1; OTC; PAX8; PCSK1; PEX1; PEX26; PKLR; STAR; TBX19; TRPM6; UGT1A1; |
M2 | 45 | 12 (26.7) | 2.11 (0.99,4.38) | AMER1; BRAF; CXCR4; CYBB; EDA; EPHB4; F13A1; FGFR2; FGFR3; GTPBP3; HRAS; IGHMBP2; IKBKG; IL10RA; IL2RG; ITGB2; JAG1; JAK3; MTM1; NPRL2; PLOD1; PPP2R5D; PRF1; PRKAR1A; PTPN11; RAF1; RASA1; SHOC2; SMO; SPINK5; TGFBR2; TGM1; TSC2; UBA5; |
M3 | 27 | 12 (44.4) | 4.15 (1.80, 9.56) * | ABCA3; ABCC8; AQP2; ATP6V0A4; ATP6V1B2; AVPR2; CHRNA1; CHRNA2; KCNJ11; KCNQ2; MYO5B; NPHS1; SCN2A; SCN3A; SCN8A; SCNN1A; SCNN1G; SLC6A19; |
M4 | 42 | 10 (23.8) | 1.41 (0.63, 3.19) | ATN1; CHD7; CREBBP; DDX58; DMD; DNAH11; DNAH5; EFTUD2; EYA1; EZH2; FANCC; FLI1; FOXP1; FOXP3; KAT6B; KMT2D; KRT14; MECP2; NIPBL; NOTCH1; POLE; PUM1; RPL5; RPS19; SMN1; STAG2; TUBB1; WAS; ZC4H2; |
M5 | 22 | 6 (27.3) | 1.95 (0.72, 5.29) | AP4E1; AP4S1; CLCN7; COL17A1; COL2A1; COL7A1; EPCAM; FBN2; GP1BA; HBG2; KIF11; KRT9; MTM1; MYBPC3; MYH7; PACS2; UNC13D; |
M0: undiagnosed; M1: catalytic activity deficiency, defined as catalysis of a biochemical reaction at physiological temperatures. M2: signaling pathway-associated activity deficiency, defined as a process beginning with an active signal and ending when a cellular response has been triggered. M3: transporter activity deficiency, defined as the process in which a solute is transported across a lipid bilayer from one side of a membrane to the other. M4: DNA/RNA metabolism activity deficiency, defined as interacting selectively and non-covalently with any nucleic acid. M5: other. |
*: P < 0.05 |
A: redirection of care; B: initiation of new subspecialist care; |
C: changes in medication or diet; D: major procedures; |
The red boxes indicate a change to palliative therapy, the orange boxes indicate the initiation of new subspecialist care, the yellow boxes indicate medication or diet modifications, and the green boxes indicate major procedures, such as transplants. |
The Influence Of Medical Management On Genetic Findings
Among 203 genetic diagnosed infants, 70 of 203 (34.5%) patients were under specific management based on molecular diagnosis. The exceptions included the following: 25 (12.3%) infants died prior to genetic diagnosis. 41 (20.2%) infants with specific phenotypes have implemented surgical or medical treatments under the clinical diagnosis. The proportion of medical management corresponding to genetic diagnosis was 13 (22.0%), 26 (38.2%), and 31 (40.3%) in 2016–2017, 2018 and 2019. Moderate improvement was achieved over a 4-year period (p = 0.06).
Of these 4 categories, we observed that 12 (5.9%) patients with critical and serious conditions underwent redirection of care, including palliative care and withdrawal of life support, such as patient 401, who presented with mechanical ventilator-dependent respiratory failure, was found to have pathogenetic variants in MTM1, relating to myotubular myopathy (OMIM: 310400). These patients underwent a palliative trajectory and family genetic counsel. Second, 35 (17.2%) patients benefitted from the initiation of additional subspecialist evaluation, such as by neurology, endocrinology, cardiology and surgery specialty teams. For example, patient 434 diagnosed with homozygous loss of the SMN1 exon 7 causes spinal muscular atrophy (OMIM: 253300) at 15 days of life were referred to participate in ongoing gene therapy (BP 39056). Patient 315 was a 5-day-old girl with severe hypercalcemia and hyperparathyroidism who was transferred for partial parathyroidectomy on 25 days of life, earlier than the standard surgical day, as sequence-data revealed two inherited pathogenic variants in CASR, diagnostic of CASR-related-hyperparathyroidism (OMIM: 239200). Third, 18 (8.9%) patients had a change in their medical status and/or diet. For example, patient 355, diagnosed with NPC1-related Niemann-Pick disease type C1 (OMIM: 257220), was treated with Migalastat beginning at 75 days of life, avoiding severe progressive hepatic dysfunction, psychomotor delay and multiple organ damage. Finally, major procedures, such as transplant, were implemented for 5 patients (2.5%) who are currently alive. Hematopoietic stem cell transplant was undertaken in 4 affected infants, 2 patients (patient 457 and 464) with an IL-10RA mutation that caused early-onset Inflammatory Bowel Disease 28 (OMIM: 613148), a patient 38 with ITGB2 homozygous mutations causing leukocyte adhesion deficiency (OMIM: 116920), and a patient 475 with WAS mutations that contributed to Wiskott-aldrich syndrome (OMIM: 301000). Another infant (patient 374) had successfully undergone partial-liver transplantation due to complicated heterozygous mutations in the UGT1A1 gene (OMIM: 606785) (Fig. 2).
Diagnostic findings in the diagnosed cases involving inherited pathogenic variants prompted us to address prenatal diagnosis. 73 families were referred for reproductive counseling.