Comprehensive Genetic Study of a Monozygotic Triplet Discordant for Autism Spectrum Disorder.

There are a few comprehensive genetic studies on autism spectrum disorders (ASD) in India. Children of multiple births are valuable for genomics studies of complex disorders such as ASD. We report whole-exome sequencing (WES) in a triplet family in which only one among the triplet has ASD. The objective of this study was to identify potential candidate genes for ASD. Exome DNA was enriched using a twist human customized core exome kit, and paired-end sequencing was performed. Proband-specific de novo variants included 150 single nucleotide polymorphisms (SNPs) and 74 indels. Thirteen SNPs were in exonic regions, 7 of them being missense variations. Seventeen variants were previously reported in ASD. Genes harboring variants have functions in the development and maintenance of the central nervous system and are enriched in biological processes involving cell adhesion. This is the first comprehensive genetic study of a monozygotic triplet in ASD.

A rare case of monozygotic triplets with Duchenne muscular dystrophy.

Twins with Duchenne muscular dystrophy (DMD) have been widely studied. We report the first rare case of monozygotic triplets with DMD who shared consistent phenotypes, including delayed motor and language milestones, muscle wasting and weakness, joint contracture, and lumbar lordosis. Muscle magnetic resonance imaging and biopsy revealed the similar muscle injury characteristics and dystrophin absence. Short tandem repeat analysis confirmed monozygosity. A de novo mutation (exon 49-52 deletion) was found in the triplets but not in their mother. Treatment included prednisone, idebenone, and rehabilitation management. At the 2-year follow-up, motor function had deteriorated, and muscle fatty infiltration was more extensive and severe. Our case offers a unique opportunity for genetic and therapeutic research. Furthermore, it highlights the critical role of genetic factors in DMD phenotypes and provides a potential choice for treatment observations.

A novel homozygous TPM1 mutation in familial pediatric hypertrophic cardiomyopathy and in silico screening of potential targeting drugs.

OBJECTIVE: Familial hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disease. While sarcomeric gene mutations explain many HCM cases, the genetic basis of about half of HCM cases remains elusive. Here we aimed to identify the gene causing HCM in a non-consanguineous Saudi Arabian family with affected family members and a history of sudden death. The impact of the identified mutation on protein structure and potential drug targets were evaluated in silico. MATERIALS AND METHODS: Triplets (two HCM subjects and one patent ductus arteriosus (PDA) case) and unaffected parents were screened by targeted next-generation sequencing (NGS) for 181 candidate cardiomyopathy genes. In silico structural and functional analyses, including protein modeling, structure prediction, drug screening, drug binding, and dynamic simulations were performed to explore the potential pathogenicity of the variant and to identify candidate drugs. RESULTS: A homozygous missense mutation in exon 1 of TMP1 (assembly GRCh37-chr15: 63340781; G>A) was identified in the triplets [two HCM and one patent ductus arteriosus (PDA)] that substituted glycine for arginine at codon 3 (p.Gly3Arg). The parents were heterozygous for the variant. The mutation was predicted to cause a significant and deleterious change in the TPM1 protein structure that slightly affected drug binding, stability, and conformation. In addition, we identified several putative TPM1-targeting drugs through structure-based in silico screening. CONCLUSIONS: TPM1 mutations are a common cause of HCM and other congenital heart defects. To date, TPM1 has not been associated with isolated PDA; to our knowledge, this is the first report of the homozygous missense variation p.Gly3Arg in TPM1 associated with familial autosomal recessive pediatric HCM and PDA. The identified candidate TPM1 inhibitors warrant further prospective investigation.

Novel BRCA2 pathogenic genotype and breast cancer phenotype discordance in monozygotic triplets.

BRCA1/2 genes with high-penetrance are tumor suppressor and tumor susceptibility genes that play important roles in the homologous recombination mechanism in DNA repair and increase breast cancer risk. Variants in BRCA1 or BRCA2 are the main causes of familial and early-onset breast cancer. This study investigated pathogenic variant belonging to the BRCA2 gene splice region in monozygotic triplets. A 44-year-old woman was diagnosed with breast cancer when she was 32 years old. Her monozygotic sister had a history of breast cancer. No malignancy was detected in the third one of the monozygotic triplets. Sanger sequencing was used to evaluate the BRCA1/2 gene status of the patient and family members. It was figured out that they had the same genetic variant, a heterozygous germ-line splice region variant (c.7008-1G > C) in the BRCA2 gene. This novel splice region variant may be a new pathogenic variant of the BRCA2 gene. Its association with breast cancers needs to be further verified in more patient cases.

Genetic and epigenetic study of an Alzheimer's disease family with monozygotic triplets.

Age at onset of Alzheimer's disease is highly variable, and its modifiers (genetic or environmental) could act through epigenetic changes, such as DNA methylation at CpG sites. DNA methylation is also linked to ageing-the strongest Alzheimer's disease risk factor. DNA methylation age can be calculated using age-related CpGs and might reflect biological ageing. We conducted a clinical, genetic and epigenetic investigation of a unique Ashkenazi Jewish family with monozygotic triplets, two of whom developed Alzheimer's disease at ages 73 and 76, while the third at age 85 has no cognitive complaints or deficits in daily activities. One of their offspring developed Alzheimer's disease at age 50. Targeted sequencing of 80 genes associated with neurodegeneration revealed that the triplets and the affected offspring are heterozygous carriers of the risk APOE ε4 allele, as well as rare substitutions in APP (p.S198P), NOTCH3 (p.H1235L) and SORL1 (p.W1563C). In addition, we catalogued 52 possibly damaging rare variants detected by NeuroX array in affected individuals. Analysis of family members on a genome-wide DNA methylation chip revealed that the DNA methylation age of the triplets was 6-10 years younger than chronological age, while it was 9 years older in the offspring with early-onset Alzheimer's disease, suggesting accelerated ageing.

The Southern Illinois Twins/Triplets and Siblings Study (SITSS): A Longitudinal Study of Early Child Development.

This article reviews the Southern Illinois Twins/Triplets and Siblings Study (SITSS) and describes some of the findings related to recent projects that were completed using this sample. At this time, the SITSS has enrolled 375 twin pairs, 12 triplet families, 1 family of quadruplets, 98 nontwin sibling pairs and 287 singletons. Testing begins for twins and triplets as young as age 1 and then occurs yearly on their birthdays until 5 years of age. Through age 20, various follow-up studies have been conducted on the SITSS sample to examine their social, emotional, and cognitive development across childhood and adolescence from a behavioral genetic perspective. A variety of methodologies have been used to investigate gene-environment correlations (rGE) and gene-environment interactions (GxE). Advanced statistical procedures (e.g., genetic likelihood indices and multilevel modeling) have been utilized to further investigate genetic underpinnings of behavior. Recent results have indicated genetic influences on the aggressiveness of preschoolers' media preferences, increased problem behaviors related to young children's overestimation of self-competence, and the influence of early life temperament and internalizing problems on adolescent health behaviors. Additionally, the SITSS has provided evidence for evocative rGE for various behaviors (aggression, prosocial and play), as well as findings supporting interactions between the dopamine receptor D4 gene (DRD4) and the environment (peer victimization, prenatal birth complications and parental sensitivity). Together, by use of multitrait and multimethodological investigations, this behavior genetic data set assists in furthering our understanding of biological and environmental influences on children's development.

Breast Milk for Preterm Multiples: More Proteins, Less Lactose.

Exclusive breastfeeding is currently recommended until at least 6 months of postnatal age, due to maternal breast milk (BM) unique composition and beneficial properties. In fact, BM modifies itself according to gestational age (GA) at birth, adapting its composition to neonatal requests during lactation. Multiple births represent about 3% of the whole pregnancies; such neonates result more vulnerable than full-term newborns, due to lower GA and birth weight (BW) and the higher incidence of perinatal complications. Although an adequate nutrition is fundamental for twins and other multiples, studies on this topic are lacking. We collected and analyzed BM from mothers of 19 twins and 5 triplets showing GA < 33 weeks and BW < 1500 g, comparing it to a control group of 28 preterm singletons. As a result, at GA ≤ 28 weeks, we observed that protein content is higher in BM for multiples (1.53 vs. 1.29 g per 100 ml), lactose concentration is greater in BM for singletons (6.72 vs. 6.34 g per 100 ml) and GA results the most relevant factor influencing BM protein composition. BM for multiples results higher in proteins and lower in lactose, if compared with singleton's samples; this could promote and sustain growth and organ development in this vulnerable category. BM from multiples shows a trophic and immunologic role, since these neonates often show lower GA and BW instead of singletons. These findings could help in optimizing nutritional strategies and improving BM individualized fortification.

Monozygotic twins and triplets discordant for amyotrophic lateral sclerosis display differential methylation and gene expression.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the loss of upper and lower motor neurons. ALS exhibits high phenotypic variability including age and site of onset, and disease duration. To uncover epigenetic and transcriptomic factors that may modify an ALS phenotype, we used a cohort of Australian monozygotic twins (n = 3 pairs) and triplets (n = 1 set) that are discordant for ALS and represent sporadic ALS and the two most common types of familial ALS, linked to C9orf72 and SOD1. Illumina Infinium HumanMethylation450K BeadChip, EpiTYPER and RNA-Seq analyses in these ALS-discordant twins/triplets and control twins (n = 2 pairs), implicated genes with consistent longitudinal differential DNA methylation and/or gene expression. Two identified genes, RAD9B and C8orf46, showed significant differential methylation in an extended cohort of >1000 ALS cases and controls. Combined longitudinal methylation-transcription analysis within a single twin set implicated CCNF, DPP6, RAMP3, and CCS, which have been previously associated with ALS. Longitudinal transcriptome data showed an 8-fold enrichment of immune function genes and under-representation of transcription and protein modification genes in ALS. Examination of these changes in a large Australian sporadic ALS cohort suggest a broader role in ALS. Furthermore, we observe that increased methylation age is a signature of ALS in older patients.

The first case of congenital blood chimerism in two of the triplets in Korea.

BACKGROUND: Chimeras are composed of two or more different populations that originated from different zygotes. Blood chimerism arising from twins have been reported in the literature. Herein, we report the first blood group chimerism in triplets. METHODS: ABO blood grouping was carried out by manual tile methods (Merck Millipore, UK) and micro-column agglutination method (Bio-Rad, Cressier sur Morat, Switzerland). Flow cytometric analysis was performed with Anti-A-PE conjugated monoclonal antibodies (BD Biosciences, San Jose, CA, USA) and FACS Canto II (BD Biosciences). Molecular analysis was performed with allele-specific polymerase chain reaction (AS-PCR) and direct sequencing of the exons 6 and 7. RESULTS: Mixed-field agglutination and weak agglutination against anti-A were revealed by ABO blood grouping. Flow cytometric analysis revealed the presence of both A cells and O cells. AS-PCR and sequencing showed two neonates with chimerism, with each neonate`s genotype being A102/O01/O02. CONCLUSION: This is the first recorded case of blood chimera from a triplet in Korea. We recommend full investigation of blood group chimerism in neonates with ABO discrepancy, as blood chimerism is subject to certain caution in the clinical environment.

[Formula Derivation and Validation of Probability of Exclusion in the Cases of Standard Triplet Parentage Testing].

OBJECTIVES: To derive and experiment validate the probability of exclusion （PE） formulas in the cases of standard triplet parentage testing. METHODS: The formulas were derived voluntarily based on the PE definition: [Formula: see text]. This formula was compared with the 5 formulas （1）-（5） reported previously, and the PE values of 19 autosomal STR loci in AGCU EX20 system were calculated. Based on 1 000 samples of single-parentage cases and 1 000 unrelated individuals, the real experiment was designed and the real experiment results of PE were calculated. Ten million pairs of simulated biological mother and son and 10 million random individuals were gained by random simulation method, and the simulated experiment was designed and the simulated values of PE were calculated. In 19 STR loci, the sum of all allele frequency （S） was calculated, and the formula values of PE were compared with the values of real and simulated experiments. RESULTS: If S=1, the calculation values of formula （1）, （2）, （5） and （6） were quite the same, which accord with the double verification of real and simulated experiments. If S≠1, there was a minor error in the calculation results of formula （1）, （2）, （5） and （6）, while which had a large error in formula （3） and （4）. CONCLUSIONS: The formula （6） derived in present study and the classical formulas （1）, （2） and （5） can be applied to the standard triplet parentage testing. The S value has a certain influence on PE calculation.

Seasonality in Multiple Maternities.

In the 19th century, a series of international statistical congresses introduced common rules for the national demographic registers. This activity contributed to the genesis of statistical research. During the history of twin research, Hellin's law has played a central role because it is an approximately correct association between the rates of multiple maternities. However, it has been mathematically proven that Hellin's law cannot hold exactly. The majority of all studies of Hellin's law are based on empirical rates of multiple maternities. Such studies can never confirm the law, but only identify errors too large to be characterized as random. It is of particular interest to examine why the rates of higher multiple maternities are sometimes too high or too low when Hellin's law is used as a benchmark. However, divergences from the law are often difficult to explain and/or eliminate. Different improvements to the law have been proposed. In this article, we study the seasonality of multiple maternities. We apply Hellin's law to compare the seasonality of twin and triplet rates.

Mixed pineal mature teratoma and germinoma in two brothers of the fraternal triplets.

INTRODUCTION: Intracranial teratomas are rare germ cell neoplasms that contain tissues derived from all three germ cell layers and most commonly occurring during childhood. This is the first report of pineal region mixed mature teratoma and germinoma in two fraternal brothers of fraternal triplets. CASE PRESENTATION: We report the case of a mixed mature teratoma and germinoma of the pineal region in two brothers of fraternal triplets. Older brother was initially diagnosed at the age of 11 years with the pure teratoma of the pineal region but the review of the pathology 3 years after initial surgery revealed the mixed mature teratoma with 5% germinomatous component. The younger brother was diagnosed at the age of 13 years with the mixed mature teratoma with 10% germinomatous component tumor of the pineal region. Younger brother has been treated with adjuvant chemo-radiotherapy and older brother was treated without adjuvant therapy. Both brothers had no recurrence. CONCLUSION: Pineal mature teratomas have a good prognosis, in contrast to their immature or mixed counterparts. A rigorous histological examination of the tumor samples is mandatory, in order to not omit a mixed contingent within the tumor.

The gut microbiota composition in dichorionic triplet sets suggests a role for host genetic factors.

Monozygotic and dizygotic twin studies investigating the relative roles of host genetics and environmental factors in shaping gut microbiota composition have produced conflicting results. In this study, we investigated the gut microbiota composition of a healthy dichorionic triplet set. The dichorionic triplet set contained a pair of monozygotic twins and a fraternal sibling, with similar pre- and post-natal environmental conditions including feeding regime. V4 16S rRNA and rpoB amplicon pyrosequencing was employed to investigate microbiota composition, and the species and strain diversity of the culturable bifidobacterial population was also examined. At month 1, the monozygotic pair shared a similar microbiota distinct to the fraternal sibling. By month 12 however, the profile was more uniform between the three infants. Principal coordinate analysis (PCoA) of the microbiota composition revealed strong clustering of the monozygotic pair at month 1 and a separation of the fraternal infant. At months 2 and 3 the phylogenetic distance between the monozygotic pair and the fraternal sibling has greatly reduced and by month 12 the monozygotic pair no longer clustered separately from the fraternal infant. Pulse field gel electrophoresis (PFGE) analysis of the bifidobacterial population revealed a lack of strain diversity, with identical strains identified in all three infants at month 1 and 12. The microbiota of two antibiotic-treated dichorionic triplet sets was also investigated. Not surprisingly, in both triplet sets early life antibiotic administration appeared to be a major determinant of microbiota composition at month 1, irrespective of zygosity. By month 12, early antibiotic administration appeared to no longer exert such a strong influence on gut microbiota composition. We hypothesize that initially host genetics play a significant role in the composition of an individual's gut microbiota, unless an antibiotic intervention is given, but by month 12 environmental factors are the major determinant.

Phospholipase A2 group v in benign familial fleck retina in a set of triplets.

PURPOSE: To evaluate the association of phospholipase A2, Group V (PLA2G5), with benign familial fleck retina in a consanguineous family with triplets. METHODS: Clinical eye examination, including fundus examination and spectral domain optical coherence tomography, was performed for all the family members. After blood sample collection and DNA extraction, polymerase chain reaction was performed to amplify regions spanning Exons 2, 3, 4, and 5 of PLA2G5. The amplified products were sequenced to observe the presence of any mutations. RESULTS: Fundus examination in two of the triplets revealed discrete yellow-white flecks and both had good vision and absence of night blindness, consistent with benign familial fleck retina. The flecks were hyperautofluorescent. Furthermore, spectral domain optical coherence tomography showed focal thickening of the retinal pigment epithelium because of the presence of these flecks. Molecular investigations showed that PLA2G5 Exons 2, 4, and 5 harbored no misalignments among all family members. However, PLA2G5 Exon 3 showed a p.Gly45Cys mutation for the father and the third triplet who was affected. CONCLUSION: The clinical findings in this family suggest a diagnosis of benign familial fleck retina with excellent prognosis, in which the PLA2G5 gene may play a role.

Repeated multiple maternities in triplet families.

In earlier studies, scientists have attempted to identify genetic and environmental factors affecting the rate of multiple maternities among humans. We contribute to these studies by analysing the frequencies of multiple maternities in sibships containing triplets. Use of the Hellin transformation is included in evaluation of the triplet rate. Our results indicate greater frequencies of repeated multiple maternities in the sibships than expected, based on population frequencies. The excesses obtained are more marked in triplet maternities than in twin maternities. The transformed triplet rate shows results similar to the twinning rate. The findings also indicate that in families, the influence of maternal factors on the frequencies of multiple maternities is stronger than the influence of paternal factors.

Extracranial carotid artery aneurysms in two of three monozygotic triplets with tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder with multisystem clinical manifestations. Dysplastic proliferations of small blood vessels including hemangiomas are common; however, anomalies of medium- and large-size vessels are rare. The only extracranial carotid artery aneurysm in a patient with TSC was reported in 1998. Since then, 19 cases have documented intracranial aneurysms in patients with TSC. The case of two of three identical triplet sisters with TSC who were treated for extracranial carotid artery aneurysms is presented. To the authors' knowledge, this is the first reported case of monozygotic siblings who both manifested this rare expression of TSC.

Monozygotic triplet pregnancies after single blastocyst transfer: two cases and literature review.

Following IVF, single blastocyst transfer has been thought to reduce the risks of high-order multiple pregnancies. This is a report of two cases of monozygotic triplet pregnancies after single blastocyst transfer and a review of the current concepts of the pathogenesis of multiple monozygotic pregnancies as well as the options for managing these high-risk pregnancies. Both cases were reduced to a twin pregnancy by selective cord coagulation at 15-16 weeks. Whereas one patient had uneventful pregnancy until labour was induced for growth arrest and cord Doppler abnormalities in one twin, the other developed a severe twin-to-twin transfusion syndrome which required fetoscopic laser surgery at 21 weeks. In both cases, healthy twins were delivered by Caesarean section at 34.5 and 34 weeks, respectively. As the predictors of their occurrence are not fully understood, patients should be informed of the risks of monozygotic pregnancies after single blastocyst transfer.