Thymic hypoplasia induced by copy number variations contributed to explaining sudden infant death based on forensic autopsies

Highlights

• Thymic hypoplasia of infants could contribute to the infant sudden death.

• The reduction of thymus size and mature T-cells are obvious in thymic hypoplasia.

• IHC is necessary to the degree of T-cell maturation in death without malformations.

• MLPA is a necessary auxiliary examination in diagnosing thymic hypoplasia.

• Both of the genetics and molecular pathology are important in explaining SUDI.

Abstract

Thymic hypoplasia is a primary cellular immunodeficiency that causes susceptibility to serious infections leading to sudden death in infants. Some genetic disorders in humans could result in the evident permanent hypoplasia or occasional aplasia of the thymus at birth. However, determining the genetic etiology of thymic hypoplasia is challenging for the sudden infant death due to primary cellular immunodeficiency. In this study, in order to find the fundamental reasons for sudden death of infants with thymic hypoplasia, 5 infants with suspected thymic hypoplasia and 10 control infants were assessed, and the immunohistochemistry and DNA analysis were used to investigate whether the infants with thymic hypoplasia had DiGeorge syndrome (DGS) with copy number variations (CNVs) in 22q11.2 and other chromosomes. The results showed that the weight of the thymus was significantly lower than the normal except the case 4, and that all the infants had hypocalcemia and a significant decrease or even absence of the markers CD1a, CD2, CD3, CD4 and CD8, which are related to T-cell maturation. In addition, multiplex ligation-dependent probe amplification (MLPA) analysis showed that these infants carried CNVs in 22q11.2 and other associated chromosomes with deletion and duplication of 25 genes. The results of thymus weight, histopathology, molecular pathology and MLPA analysis suggested that DGS predominantly with thymic hypoplasia induced by CNVs caused the sudden death of these infants under various infections or other unexplained reasons, which may provide new insights into the diagnosis of sudden infant death and could help the parents of deceased infants to attach more importance of genetic screening and thymus ultrasound to reduce the postnatal mortality of the infant, and demonstrates the value of genetic diagnosis in the forensic pathology.

Introduction

The thymus is responsible for the maturation of lymphoid precursors that differentiate into T cells and is necessary for the establishment of the T-cell pool during prenatal and early postnatal life in humans. Previous studies have shown that the thymic microenvironment is related to sudden infant death [1]. A strong correlation between sudden neonatal death during the perinatal period and hypoplasia of the thymus has been reported [2]. Thymic hypoplasia is a disease in which T cell function is defective due to congenital agenesis or dysplasia of the thymus, cell-mediated immune deficiency or suppression, infants with thymic hypoplasia are susceptible to immune deficiency and, consequently, are vulnerable to various infections, which is a contributing factor in sudden unexpected death in infancy (SUDI) [3], [4]. However, the routine pathological anatomy excluding histopathology was easy to ignore the underlying cause of death due to abnormal thymus quality, resulting in not fully reflect the clinical value of forensic pathological diagnosis. Therefore, it was necessary to perform the histopathology, further experimental test and genetic assessment to diagnose the thymic hypoplasia.

DGS is characterized by congenital thymus hypoplasia accompanied by diverse clinical phenotypes with varying severity of symptoms, including distinct facial features, hypoplasia or aplasia of the parathyroid glands, and conotruncal cardiac defects. Cardiac cone malformation, thymic hypoplasia, and hypocalcemia caused by parathyroid hypoplasia comprise the DGS triad. Studies have demonstrated that approximately 35%− 90% of patients with a clinical diagnosis of DGS1 have a hemizygous 3-Mb deletion in chromosome 22q at band 11.2 [5], and those with other CNVs at chromosomes 4, 8, 10, etc., are diagnosed with DGS2 [6], [7], which could be considered as the genetic etiology of thymic hypoplasia. To date, at least 30 genes have been mapped to that deleted region in 22q11.2, including TBX1, which encodes a pivotal transcription factor regulating thymus development [8], [9]. Subsequently, studies related to understanding the genetic etiology of DGS extended the definition of this syndrome to 22q11.2 deletion syndrome (22q11.2 DS). 22q11.2 DS is the most common microdeletion syndrome in humans, occurring in approximately 1:4000 births, and is the major cause of infant mortality and morbidity associated with birth defects nearly 5% of all live birth; 22q11.2 DS is also related to cardiac abnormalities, T-cell deficits, cleft palate facial anomalies, and hypocalcemia [8], [10], [11]. The overall mortality of pediatric patients with 22q11.2 DS has been reported to be 4% in one study and 14% in another, with median ages of death of 5 months and 3.4 months, respectively; notably, these cases most commonly involved immune dysfunction or allergies, hypocalcemia, and serious heart defects [10], [12], [13], [14]. However, some children with DGS caused by chromosome 22q11.2 microdeletion have persistent low thymic activity and do not die of heart disease [15]. One report has suggested that the expected survival rate for patients with complete DGS without treatment is approximately 27% at 1 year [16].

During the process of forensic autopsy, some cases of unknown cause of death, especially cases involving infants without obvious congenital malformations, confused forensic doctors. Because it is difficult to identify the causative pathogen in infants with a small thymus who died of serious infections, most scholars diagnosed thymic dysplasia according to pathological histology and thymus weight. However, few studies have focused on genetic screening and molecular detection to further clarify the thymic hypoplasia. Therefore, the objective of the study was to provide more evidences to diagnose the thymic hypoplasia and clear the genetic etiology, 5 infants with a abnormal development thymus by gross anatomy and HE staining, which was diagnosed initially with thymic dysplasia were selected, and 10 control infants were collected to serve as a normal reference for molecular pathology and genetic etiology detection of thymic dysplasia. And the molecular analysis related to T-cell development and DNA analysis were used to further clarify the thymic dysplasia, which could provide more evidence as the forensic reference to definite the diagnosis of thymic hypoplasia, and prompt the parents of deceased infants to make a genetic secerning timely, and remind the clinician to perform the clinical intervention to reduce the infant sudden death.