Comprehensive characterization of the genetic landscape of familial Hirschsprung’s disease

Background Hirschsprung’s disease (HSCR) is one of the most common congenital digestive tract malformations and can cause stubborn constipation or gastrointestinal obstruction after birth, causing great physical and mental pain to patients and their families. Studies have shown that more than 20 genes are involved in HSCR, and most cases of HSCR are sporadic. However, the overall rate of familial recurrence in 4331 cases of HSCR is about 7.6%. Furthermore, familial HSCR patients show incomplete dominance. We still do not know the penetrance and genetic characteristics of these known risk genes due to the rarity of HSCR families. Methods To find published references, we used the title/abstract terms “Hirschsprung” and “familial” in the PubMed database and the MeSH terms “Hirschsprung” and “familial” in Web of Science. Finally, we summarized 129 HSCR families over the last 40 years. Results The male-to-female ratio and the percentage of short segment-HSCR in familial HSCR are much lower than in sporadic HSCR. The primary gene factors in the syndromic families are ret proto-oncogene (RET) and endothelin B receptor gene (EDNRB). Most families show incomplete dominance and are relevant to RET, and the RET mutation has 56% penetrance in familial HSCR. When one of the parents is a RET mutation carrier in an HSCR family, the offspring’s recurrence risk is 28%, and the incidence of the offspring does not depend on whether the parent suffers from HSCR. Conclusion Our findings will help HSCR patients obtain better genetic counseling, calculate the risk of recurrence, and provide new insights for future pedigree studies. Supplementary Information The online version contains supplementary material available at 10.1007/s12519-023-00686-x.


Introduction
Hirschsprung's disease (HSCR) is one of the most common congenital digestive tract malformations, with an incidence of 1 in 5000 and a male-female ratio of 4:1. Due to the loss of enteric neurons in the distal colon, HSCR can cause stubborn constipation or gastrointestinal obstruction after birth due to loss of enteric neurons in the distal colon, bringing great physical and mental pain to patients and their families [1]. With the development of sequencing technology and bioinformatics analysis, more than 20 genes [ret proto-oncogene (RET), endothelin B receptor gene (EDNRB), pairedlike homeobox 2B gene (PHOX2B), etc.] have been linked to HSCR [2][3][4][5].
Although most cases of HSCR are sporadic, some families with two or more HSCR family members are classified as HSCR families. Three studies reported a series of HSCR families [6][7][8], implying that HSCR has a genetic predisposition. Mc Laughlin and Puri reported a 7.6% overall rate of familial recurrence in 4331 HSCR index cases [9]. Familial HSCR is also due to a list of pathogenic genes, such as RET [10], EDNRB [11], and PHOX2B [12]. However, familial HSCR does not follow Mendelian inheritance, and RET and PHOX2B show incomplete penetrance in members of familial HSCR [10,12]. Because HSCR families are uncommon, current gene studies are based on a summary of a few families. The penetrance and genetic characteristics of these known risk genes in familial HSCR, particularly the major pathogenic gene RET, which occurs in 50% of familial HSCR and 35% of sporadic HSCR, remain unknown [13].
In this study, we summarized 129 HSCR families reported in 53 references to analyze the penetrance, recurrence risk, and genetic characteristics of familial HSCR. Our study will elucidate the genetic characteristics of familial HSCR, provide preferable genetic counseling for HSCR patients, help in calculating the risk of recurrence, and provide new insights for future pedigree studies.

Methods
We used the title/abstract terms "Hirschsprung" and "familial" in the PubMed database and the MeSH terms "Hirschsprung" and "familial" in the Web of Science to search published references. At least two family members with HSCR were required for inclusion. Studies of multicenter investigated data were excluded to avoid including duplicate families in the analysis. Finally, we confirmed and analyzed 53 references containing 129 families (Supplemental Table 1) [6-8, 10-12, 14-60].
The group-divisible designs are as follows: (1) HSCR subtypes: short segment-HSCR (S-HSCR), long segment-HSCR (L-HSCR), total colonic aganglionosis (TCA), total bowel aganglionosis, not available (NA); (2) syndromic HSCR and non-syndromic HSCR/not mentioned, syndromic HSCR refers to HSCR patients suffering other syndromic symptoms, such as familial medullary thyroid cancer, multiple endocrine neoplasia type 2a (MEN2A) and Waardenburg syndrome; (3) male and female; (4) familial genetic characteristics: "parent to child, lineal 3 generation" means determined HSCR occurs in both the proband's parents (father, mother, or both) and grandparents (grandfather, grandmother, or both), "parent to child, lineal 2 generation" means determined HSCR occurs in the proband's parents (father, mother, or both), "siblings" means determined HSCR occurs in the proband's brothers or sisters, "collateral relatives" means determined HSCR occurs in the proband's cousins or relatives; (5) syndromic symptoms: familial medullary thyroid cancer (FMTC), MEN2A, Waardenburg syndrome, Bardet-Biedl syndrome, special physical characteristics, respiratory symptoms, external auditory canal agenesis, anisocoria, multiple sclerosis, congenital central hypoventilation syndrome, Currarino syndrome, neuroblastoma, congenital heart disease, trisomy 21, meningocele and intellectual disability; (6) genetic patterns or pathogenic mechanisms: dominant inheritance, recessive inheritance, incomplete dominance, compound heterozygous inheritance, epistasis; (7) transmission pattern: affected father to affected son, affected father to unaffected son, affected father to affected daughter, affected father to unaffected daughter, unaffected father to affected son, unaffected father to unaffected son, unaffected father to affected daughter, unaffected father to unaffected daughter, affected mother to affected son, affected mother to unaffected son, affected mother to affected daughter, affected mother to unaffected daughter, unaffected mother to affected son, unaffected mother to unaffected son, unaffected mother to affected daughter, unaffected mother to unaffected daughter (Supplementary Table 2).
We analyzed the penetrance and transmission patterns of the RET gene. Inclusion criteria are as follows: (1) families with equal to or more than two members diagnosed with HSCR; (2) the RET mutation was linked to families; (3) detailed RET mutation sites are available; and (4) there is only one variation of RET. The exclusion criteria were as follows: (1) consanguineous marriage; (2) unavailable RET mutation information or de novo variants; and (3) equal to or more than two variations of RET. Finally, we summarized 110 RET carriers from 21 references (Supplementary Table 3

Sex ratio of HSCR subtypes and syndromic HSCR in familial HSCR
We finally discovered 53 references (Fig. 1), including 129 families with 416 HSCR cases (Tables 1 and 2). The ratio of males to females in S-HSCR was 1.11 (39/35); the ratio of males to females in L-HSCR was 1.48 (43/29); the ratio of males to females in TCA/total bowel aganglionosis was 0.78 (7/9); and the ratio of males to females in all familial HSCR cases was 1.51 (250/166) ( Table 1), which is much lower than the sporadic HSCR ratio (4:1) [61]. The male-to-female ratio in syndromic HSCR was 1.11 (39/35), and the male-to-female ratio in non-syndromic HSCR/not mentioned was 1.61 (211/131) ( Table 2). The percentages of S-HSCR, L-HSCR, TCA and total bowel ananglionos in familial HSCR cases

The primary genetic factors of syndromic familial HSCR are RET and EDNRB
There were 74 HSCR patients with syndromic symptoms in 39 families (Table 4). FMTC, MEN2A, and Waardenburg syndrome families accounted for 41% (16/39) and 31% (12/39), respectively, of the total; FMTC/MEN2A    [61]. Table 3 Analysis between family characteristics, syndromic Hirschsprung's disease (HSCR) and non-syndromic HSCR When a large family exists multiple patterns, we choose the priority order refer to "parents to child > siblings > collateral relatives". a Determined HSCR occurs in both proband's parents (father, mother or both) and grandparents (grandfather, grandmother or both); b determined HSCR occurs in proband's parents (father, mother or both); c determined HSCR occurs in proband's brothers or sisters; d determined HSCR occurs in proband's cousins or relatives

Familial HSCR has complicated genetic patterns
There were 62 families with detailed gene information in this analysis (

The penetrance of the RET mutation and recurrence risk in familial HSCR
There are 110 RET mutation carriers in 21 familial HSCR references. The number of affected carriers (HSCR and the RET mutation) is 62, implying that the RET mutation is 56% (62/110) penetrant in familial HSCR ("carriers" means RET gene mutation members; "affected carriers" means HSCR along with RET mutation). As a result, when one of the parents is a RET mutation carrier in an HSCR family, the offspring's recurrence risk is 28% (1/2 of 56%). We counted the number of different transmission patterns (Fig. 2): unaffected RET-carrying parents transmitted the variation to children, and the percentage of affected children was 73% (16/22); affected RET-carrying parents transmitted the variation to children, and the percentage of affected children was 78% (7/9). They seem to have a similar HSCR rate, meaning that the incidence of HSCR in offspring does not depend on whether the parents suffer from it.

Discussion
Previous studies only looked at a few HSCR families due to their rarity. They usually focus on one of these pathogenic genes and study its biological function. However, there is a lack of systematic research on the genetic characteristics and gene penetrance of familial HSCR. We summarized all 129 families reported in this study over the last 40 years, concluded that the genetic feature of familial HSCR exists, provided preferable genetic counseling for HSCR patients, assisted in calculating the risk of recurrence, and provided new insights for future pedigree studies. We determined that 65% of HSCR families were associated with RET in our study. RET shows complex genetic patterns, including dominant inheritance (30%) and incomplete dominance (58%). We determined the penetrance of the RET mutation to be 56% in familial HSCR. That is, when a member of a RET-associated family is diagnosed with HSCR, other carriers with the RET mutation have a 56% chance of developing HSCR. When one of the parents is a RET mutation carrier in an HSCR family, the offspring's recurrence risk is 28%.
A few risk genes are highly associated with familial HSCR and show multiple genetic characteristics. The function of RET in HSCR is complicated and varied. Previous research has revealed the dominant inheritance and incomplete dominance of RET. However, some scholars have also proposed dosage-dependent penetrance and epistasis to explain the phenomenon of incomplete dominance and different subtypes of HSCR [30,33]. For other risk genes, EDNRB shows dominant inheritance, recessive inheritance and incomplete dominance, and PHOX2B shows dominant inheritance and incomplete dominance. Thirty percent of families are syndromic, and 56% (22/39) of syndromic HSCR families are sibling groups. Syndromic symptoms (FMTC/MEN2A and Waardenburg syndrome) are mainly caused by RET and EDNRB. Syndrome symptoms are common and varied, so it is important to focus on other complications in HSCR patients and pay attention to risk genes, such as RET, EDNRB and PHOX2B.
Twelve percent (15/129) of the families had consanguineous marriages. Consanguineous marriage appears to be a risk factor for HSCR, and it is recommended that consanguineous marriage be avoided. We also observed 11 pairs of twins (six pairs of monozygotic twins and five pairs of dizygotic twins). In six pairs of monozygotic twins, four pairs (six males and two females) were diagnosed with HSCR, while there was only one case diagnosed with HSCR in the remaining two pairs. Two pairs of dizygotic twins were diagnosed with HSCR, while the remaining pairs had only one HSCR patient. Thus, it seems that the risk of HSCR was independent in both monozygotic and dizygotic twins due to the incomplete dominance of HSCR.
The mutated genes or loci, especially the gene RET, reported in the families in the references we included conform to the law of genetic coseparation and are predicted or proven to be highly pathogenic. Based on this, all reported mutated genes or loci were included and analyzed. We performed a large familial HSCR study and determined a series of ratios and percentages. However, there may be some statistical bias, as the reported HSCR families are typical and characteristic.
In conclusion, the male-to-female ratio in familial HSCR is close to one. Most families show incomplete dominance and are relevant to RET, and the RET mutation has 56% penetrance in familial HSCR. The incidence of HSCR in the offspring does not depend on whether the parent suffers from HSCR. Overall, our findings will enhance the comprehensive characterization of the genetic landscape for familial HSCR and help HSCR patients obtain better genetic counseling.
Author contributions FJX and MXY contributed to conceptualization, data curation, formal analysis, funding acquisition, and writing of the original draft. XJ contributed to conceptualization, data curation, project administration, writing of the original draft, and reviewing and editing. HLW, WJ and YXS contributed to investigation, methodology, project administration, and resources. YJY, LZJ and MHD contributed to resources, software, supervision, validation, and visualization. All authors approved the final manuscript as submitted and agreed to be accountable for all aspects of the work. The first author is XJ. FJX and MXY contributed equally to the work and are co-corresponding authors. Data availability All data generated or analyzed during this study are included in this published article (and its supplementary information files).