Clinical and Molecular Findings in Early Infant Death Associated with Genetic Diseases: A Multicenter Cohort Study in China

Xia Wang Shanghai Jiaotong University School of Medicine Xinhua Hospital Tianwen Zhu Shanghai Jiaotong University School of Medicine Xinhua Hospital Xiaohui Gong Shanghai Children's Hospital: Children's Hospital of Shanghai Fei Bei Shanghai Childrens Medical Center A liated to Shanghai Jiaotong University School of Medicine Li Ma Shanghai Children's Hospital: Children's Hospital of Shanghai Yan Chen Shanghai Jiaotong University School of Medicine Xinhua Hospital Dongying Zhao Shanghai Jiaotong University School of Medicine Xinhua Hospital Jingjing Sun Shanghai Children's Hospital: Children's Hospital of Shanghai Jian Wang Shanghai Childrens Medical Center A liated to Shanghai Jiaotong University School of Medicine Gang Qiu Shanghai Children's Hospital: Children's Hospital of Shanghai Jianhua Sun Shanghai Childrens Medical Center A liated to Shanghai Jiaotong University School of Medicine Yu Sun Shanghai Jiaotong University School of Medicine Xinhua Hospital yongjun Zhang (  zhangyongjun@sjtu.edu.cn ) Shanghai Jiaotong University School of Medicine Xinhua Hospital https://orcid.org/0000-0001-93261012


Background
Genetic diseases are common among infants in neonatal intensive care units (NICUs), with an incidence of 16-20% [1][2][3]. Disease progression can be very rapid, and a signi cant proportion of the affected individuals succumb to their diseases early in life [4]. Early preventive measures implemented for decreasing genetic diseases mortality would reduce mortality rate under ve years of age signi cantly [5,6].
The clinical manifestations of more than 14,000 genetic diseases may be atypical and overlapped [4,7].
The prognosis and complications of each genetic disease are variable. Previous studies usually compared the mortality risk based on phenotypic system, chromosome disorder categories, recognized or unrecognized syndromes, and single or multiple defects [8,9]. Because the patients' symptoms are overlapped and may not be fully developed or recognized in young infants, the phenotypic category system would lead to an under-estimated [10]. Fortunately, the clinical application of next-generation sequencing empowers us to detect genetic etiology e ciently. Cause-speci c mortality classi cation of the underlying cause of death will be necessary for public health interventions to etiology-speci c populations, including limited diagnostic resources [11][12][13]. Currently, there are few published studies comparing the odds ratio of infantile mortality among categories of genetic disease according to phenotype related genes.
Here, we retrospectively examined the odds ratio for 180-day mortality in a total of 439 infants who underwent exome sequencing in our multicenter genetic cohort.
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 immunode ciencies (OR = 3.79, 95% CI: 1.25-11.53) had signi cantly 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 signi cantly lower survival rates than G0 infants (< 60% survival) (Fig. 1b).
Diagnostic ndings in the diagnosed cases involving inherited pathogenic variants prompted us to address prenatal diagnosis. 73 families were referred for reproductive counseling.

Discussion
In our study, a molecular diagnosis achieved in 203 of 439 (46.2%) sequenced infants, in agreement with published rates of diagnosis of genetic diseases by exome sequencing [3,14,15]. Our study found that infants diagnosed with metabolic and endocrine disorders, early-onset muscle disease, and primary immunode ciencies had a high risk of 180-day mortality. Furthermore, the 180-day mortality rate decreased over four years, most likely due to the decreased turnaround time and the improvement of medical management.
With extensive use of next-generation sequencing technology, the contribution of genetic diseases to early infant mortality has been reported to progressively increase. Wojcik et al [16] reported that the proportion of genetic diseases con rmed by genetic testing among 1-year infant deaths reached 22%, higher than previous reports. Yang et al [17] showed that infants genetic diagnosed as multiple congenital malformations was one-third of neonatal mortality. The molecular etiology of pediatric mortality is becoming clearer [4,18,19].
Our study found that the 180-day mortality rate were related to genetic diseases of metabolic-endocrine disorders, primary immunode ciencies, and early-onset muscle disease, which were potentially curable and preventive [20][21][22]. Developing a rapid diagnostic method with a short turnaround time is an urgent need and has an important impact on clinical decision and management. Interestingly, our study found that multiple congenital malformations was not the leading cause of the 180-day mortality rate, which contrasted to previous studies [27,28]. Several explanations for this difference are presented below. First, the de nition of congenital malformation in our study was genetic diagnosis with a pathogenic molecular or cytogenetic aberration, which was different from the clinical diagnoses which was often based on abnormal facial features, limbs and organs. However, the structural anomalies may or may not be genetic [29,30]. Second, the decreased mortality rate can be explained by the fact that termination of pregnancy is legal in China in the case of fetal congenital anomalies based on the interpretation of neonatal and infant mortality statistics [31,32]. Finally, improved prenatal detection and early treatment would almost certainly result in longer survival for the affected patients.
Additionally, our study found that catalytic activity and transporter activity are likely to be life-threatening.
First, the catalytic activity category relates to enzyme proteins, which are present in multiple biological processes and are pivotal to health and disease [33]. The high-risk categories of metabolic and endocrine disorders and primary immunode ciency are usually associated with catalytic activity de ciency. Second, it was reported that transporter activity is another molecular function that is essential for life. Transporter gene mutations in neonates often show an early onset and result in metabolic crises that can be lifethreatening if not treated in a timely manner [34]. For example, twin brothers developed cardiac arrhythmia due to severe hyponatremia, hyperkalemia, and metabolic acidosis at 9 days of life. One sibling died of cardiac arrest shortly after NICU admission, while the other died within 5 days after discharge. The homozygous mutation c.1439 + 1G > C in the SCNN1A gene was con rmed by exome sequencing.
Although the molecular function of DNA/RNA metabolism is also important for life, our results show that diagnosis with signal pathway-associated activity de ciency and DNA/RNA metabolism de ciency was not related to the 180-day mortality rate. Infants diagnosed with congenital malformations were mostly distributed into these two categories. The at odds ratios of congenital malformation may be the reason for the lowered risk. The molecular function analysis in our study showed that monogenic diseases caused by a mutation in a single gene with a clear molecular pathogenesis are usually lethal, and these results provided new targets and clues for further gene therapy research.
A strength of our study is that our application enabled us to study a large patient population over more than four years, ensure a follow-up time of at least 180 days after birth to measure outcomes, and compare groups of patients according to phenotype-related genes. Importantly, we found that infants genetically diagnosed with metabolic-endocrine disorders, primary immunode ciencies and early-onset muscle disease showed signi cant risk for 180-day mortality. This study has some limitations. It is the retrospective design, yet we draw conclusion based on completed medical history and follow-up information. Another limitation is that we performed exome sequencing in the diagnostic pathway for those clinically suspected of genetic diseases, including newborns and infants with structural changes and those who were di cult to diagnose with other modalities, which may fail to identify all of the pathogenic variants. However, exome sequencing has shown the highest testing e ciency [19]. Our genetics team also reanalyzed sequencing data, which were negatives for the undiagnosed cases.
Additionally, to minimize differences in medical diagnosis and treatment level, all the infants were recruited from three specialized children's hospitals a liated to Jiao Tong University Medicine School. To draw more general conclusions, a prospective study with a larger cohort will be required.

Conclusions
Knowledge of genetic disease categories on infantile mortality is of signi cance for integrating preventive strategies to effectively care for patients and their families. More prospective studies focused on etiologyspeci c causes of mortality in genetic diseases are warranted.

Genetic disease cohort
We performed a retrospective mortality analysis for the underlying genetic cause of death from three tertiary children's hospitals, including Xinhua Hospital, Shanghai Children's Hospital and Shanghai Children's Medical Center, a liated to Shanghai Jiao Tong University School of Medicine. These three hospitals served as training centers for medical students, pediatric and nurses, and family practice residents.
The patient enrollment inclusion criteria were as follows as detailed previously [35]: (1) Affected infants with suspected genetic conditions were referred for exome sequencing by a multidisciplinary medical team consisting of the treating neonatologists, medical geneticists and laboratory specialists from January 2016 to December 2019.
(2) Infants were admitted before 100 days of life.
(3) Clinical assessments of 180-day mortality (yes/no) were completed. The exclusion criteria were as follows: (1) one or more biological parents refused to participate; and (2) death of infants due to an acquired condition not clearly related to their underlying genetic etiology (Fig. 1).
Exome sequencing including whole exome sequencing (WES) and target exome sequencing (TES), were performed after genetic consultation by the multi-disciplinary medical team and attainment of consent from the parents who had children with the following conditions:1) multiple congenital malformation; 2) seizures and/or hypotonia; 3) metabolic crisis or endocrine disturbance; 4) recurrent and severe infection; 5) cardiac anomaly; and 6) other combined conditions suspected to be associated with genetic diseases in the absence of a clinical diagnosis (Fig. 3

Classi cation System
The enrolled infants were classi ed into eight categories according to the phenotype-related genes as follows: G0: undiagnosed, G1: metabolic and endocrine disorders, G2: multiple congenital malformation, G3: neurological disorder, G4: early-onset muscle disease, G5: dermatologic disorder, G6: primary immunode ciency, and G7: other, meaning those who did not fall into the above categories, including those with defects in the systems of hematological, cardiac, gastrointestinal, skeletal, nephrological, respiratory, vascular, lymphatic, eyes and development disorders.
Second, infants diagnosed with a monogenic disorder were classi ed into six categories on the basis of the molecular function of the gene (http://geneontology.org) as follows[38]: M0: undiagnosed; M1: catalytic activity de ciency, de ned as catalysis of a biochemical reaction at physiological temperatures; M2: signaling pathway associated activity de ciency, de ned as a process beginning with an active signal and ending when a cellular response has been triggered; M3: transporter activity de ciency, de ned 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 de ciency, de ned as interacting selectively and non-covalently with any nucleic acid; and M5: other, including those who did not fall into the above categories.
Genetic diagnosis provides medical treatment in amenable cases, and comfort care to reduce patients' suffering in incurable cases. To evaluate the medical management of genetics ndings, we classi ed medical managements into four categories (1) redirection of care, (2) initiation of new subspecialist care, (3) changes in medicine or diet, and (4) major procedures, such as organ transplant. However, there are three categories of exceptions: (A) infants who were genetically diagnosed postmortem, (B) infants with congenital malformation who underwent surgical procedures prior to the return of genetics results, and (C): infants whose biochemical phenotypes suggested a speci c metabolic disease and were administrated a speci c diet and medicine with clinical diagnoses.

Statistical analysis
Continuous data are presented as medians and quartiles. Categorical data are presented as numbers and constituent ratios. Data were compared using the chi-square test, Fisher's exact test, and ANOVA. A p value of 0.05 was used as a signi cance threshold and was calculated using SAS 9.2. This study involving human participants was reviewed and approved by the Ethics Committee of Xin Hua Hospital A liated to Shanghai Jiao Tong University School of Medicine (Approval number: XHEC-D-2020-095).

Consent for publication
All presentations of case reports have consent for publication.
Availability of data and material The datasets used during and/or analyzed during the current study are available from the corresponding author on request.

Competing interests
None of the authors has any con ict of interest to disclose.

Funding
National Natural Science Foundation of China (81671501) and Shanghai Municipal Commission of Health and Family Planning (2016ZB0103).

Contributors
YZ conceptualized and designed the study. XW, TZ, XG and FB prepared an analytical plan, analyzed the data, and drafted the initial manuscript. LM, JS, GQ and JS collected the original data. YC and DZ provided advice on statistical analysis of data. JW and YS provided essential suggestions on genetic data analysis. YZ critically reviewed the manuscript for important intellectual content and interpreted the data and results.