Robust and easy-to-use STR-based method for 15q11-q13 rearrangements identification

doi:10.21203/rs.3.rs-1598143/v1

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Robust and easy-to-use STR-based method for 15q11-q13 rearrangements identification

https://doi.org/10.21203/rs.3.rs-1598143/v1

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The rearrangements of the 15q11-q13 region such as deletions, duplications, uniparental disomies (UPDs), and translocations caused different neurodevelopment phenotypes including Prader-Willy and Angelman syndromes, 5q11-q13 duplication syndrome, autism spectrum disorders.

The aim of our study is to elaborate on the most universal, convenient, and informative molecular test for rapid detection of 15q11-q13 alterations in patients with suspected 15q associated disorders. Eight short tandem repeats (STR) markers within the 15q11.1-q13 locus and 1 STR from 15q21.1 in a combination with SNRPN gene qPCR allowed us to detect both copy number variations (deletions and duplications) and different types of UPDs, determining the origin and boundaries of the reorganization.

The proposed strategy significantly improves medical genetic counseling for families - it allows the prediction of the disease severity, the estimation of genetic risks for families, and even applying the personalized approaches to therapy.

5q11-q13 region

STR-marker

deletion

duplication

uniparental disomy

Several neurodevelopmental disorders are caused by 15q11–q13 genetic and epigenetic abnormalities: i) so-called «sister imprinting diseases» Prader-Willy (OMIM #176270) and Angelman (#105830) syndromes; ii) 15q11-q13 duplication syndrome (# 608636); iii) 15q11.2 microdeletion syndrome (#615656) and iv) rare small deletions or duplications within 15q13.1-q14 associated with autism, schizophrenia and epilepsy. The 15q11-q13 region is a hotspot for recombination and includes five breakpoints (BP) regions of segmental duplications or low copy repeats, which result in increased susceptibility to genomic rearrangements, such as interstitial deletions, duplications, and multiplications. The deletions primarily occur in the frame of three breakpoints - BP1, BP2 and BP3, whereas in duplications and triplications two additional distal breakpoints (BP4 and BP5) may be also involved (Kalsner 2015). Besides, this chromosome region is an integral part of structural rearrangements such as reciprocal and Robertsonian translocation, ring derivation, isochromosome formation, or inversion, involving the chromosomes 15 and other acrocentric chromosomes.

The most common type of 15q alterations (60–70%) in PWS and AS is a heterozygous deletion. Class I deletions (apr. 30% of del 15q cases) located between BP1 and BP3. Class II deletions (70%) have the same as class I distal breakpoint and the proximal border is upstream MKRN3 gene (BP2) (Amos-Landgraf 1999; Chai 2003). It has been reported that individuals with larger class I deletions could have more language impairment or autistic traits than those with class II deletions (Sahoo 2006).

The second frequent cause of PWS/AS is uniparental disomy (UPD): 25–35% of PWS and 2–3% of AS patients have UPDs. Four general mechanisms can cause UPD: 1) gamete complementation – a zygote has formed by two abnormal gametes: one with an extra chromosome and another one that was missing the same chromosome; 2) trisomic rescue - loss of a supernumerary chromosome from a trisomic conceptus; 3) compensatory UPD - an abnormal chromosome is replaced with the normal homolog; 4) somatic recombination, in which segments of the chromosome may be homozygous (Robinson 2000). The types of UPDs (iso-, hetero or complex) are depending on the molecular mechanisms used for their formation.

Duplications that extend BP1, BP2 and BP3 lead to 15q11-q13 duplication syndrome. On chromosomal level 15q11-q13 duplication syndrome in most cases occurs due to formation of maternal supernumerary isodicentric chromosome 15q (idic(15)) resulting in tetrasomy for 15q11.2-q13.1. The distal breakpoint of truly idic(15) is typically BP3 on both sides but BP4 and BP5 are the most frequent sites in a case of asymmetric isochromosome (Kalsner 2015). Less common maternal interstitial duplication (15q11.2-q13.1 trisomy) causes dup15 syndrome (Lusk 2016). Conversely, the inheritance of paternal 15q duplications is generally regarded as an asymptomatic or reduced penetrant variant (Kalsner 2015; Marini 2013).

Due to some overlap of the PWS, AS and dup/del 15q syndromes clinical features with other neurodevelopmental pathologies (Dykens 2017, Livak 2001, Robinson 2000, Smith 2017, Tan 2020, Varela 2004), a definitive clinical diagnosis alone is uncertain and requires DNA testing. While there are various molecular tests available for PWS/AS (fluorescence in-situ hybridization - FISH, methyl-specific PCR – MS-PCR, methyl-specific MLPA, comparative genomic hybridization – array-CGH, etc.), the problems arise due to the complexity of determining the molecular mechanism, origin and type of inheritance of 15q alterations. The FISH is a method of choice for a common 15q11–q13 deletion but in case of UPD 15, it turns false-negative result. MS-PCR testing can identify PWS/AS but not dup15 patients and have a limitation in distinguishing UPD and large deletions or imprinting mutations (Amos-Landgraf 1999, Kubota 1997). So, the correct DNA testing algorithm of 15q associated syndromes has to be upfront about not only the type of 15q abnormality such as deletion, duplication, triplication or UPD, but also about its inheritance (de novo/inherited), and origin (paternal/maternal). The most cost-effective and informative diagnostic method to achieve these objectives is important to establish, as it would improve the clinical prognosis, the recurrence risk and, possibly, would be helpful in the personalized therapy (Coupaye 2016, Dykens 2017, Smith 2017). In the recent past, short tandem repeats (STRs) markers had been widely used in the diagnosis of hereditary diseases. Now, modern genome-wide analysis technologies have supplanted pre-existing STR-based analysis in many diagnostic algorithms. However, the 15q syndromes diagnostics using STR-markers could provide answers to most of the above questions in the least costly way.

Therefore, the aim of our study is to elaborate on the most universal, convenient and informative molecular test for rapid detection of 15q11-q13 alterations in patients with suspected 15q associated neurodevelopment disorders.

Patients

The study group was formed of 29 families of Ukrainian patients with suspected 15q associated pathologies and their relatives: PWS – 12 patients; PWS-like congenital hypotonia – 6 patients; AS -4 patients; AS or FraX like intellectual disability – 4 patients (males); AS-like with epilepsy – 2 patients; 1 patient with AS or Rett Syndrome. Informed consent was obtained from all the individuals participating in our study. The main inclusion criteria were the exclusion of the common genetic pathologies and the availability patient’s parents for further DNA and clinical investigation. According to these criteria, the patients with congenital hypotonia caused by SMN1 deletions as well as intellectual disabled patients with FMR1 and MECP2 common mutations have been ruled out.

DNA extraction. Genomic DNA was extracted from peripheral blood samples using Zymo Quick-DNA Universal Kit (Zymo Research). DNA samples quantity and quality were determined with ND-1000 (Nano-Drop).

Table 1

Characteristics and location of the STR-markers

Marker	Ch.band	15q11.1-q13 BPs	STR	Size (bp)	Heterozygosity
D15S541	15q11.2	BP1-BP2	(CA)n	126–152	0.51
D15S817	15q11.2	BP2-SNRPN	(GATA)n	138–178	0.82
D15S1513	15q12	UBE3A-BP3	(ATCT)n	232–244	0.61
D15S822	15q12	UBE3A-BP3	(GATA)n	244–312	0.86
D15S1002	15q12	UBE3A-BP3	(CA)n	105–129	0.79
D15S1233	15q13.1	BP3-BP4	(TCTA)n	180–210	0.51
D15S1048	15q13.1	BP3-BP4	(CA)n	300–320	0.84
D15S165	15q13.3	BP4-BP5	(CA)n	220–240	0.77
D15S659	15q21.1	outside, telomeric	(AGAT)n	164–188	0.74

STR genotyping. Eight STR markers within 15q11.1-q13 locus and 1 STR from 15q21.1 were selected according to their high informative (Heterozygosity indexes) and location nearby 15q typical deletions BPs and PWS/AS adjacent regions (Kong 2002) (table 1).

Reverse primers for D15S817, D15S1513 and D15S659 locus amplification were Cy5-labeled. For D15S541, D15S822, D15S1002, D15S1233, D15S165, D15S1048 markers Cy5-labeling of PCR fragments were performed with three primers: a forward primer with M13 (-21) tail at its 5′ end, a reverse primer, and the universal Cy5-labeled M13 (-21) primer. The STR alleles PCR products were analyzed on an automated laser fluorometer «ALFexpress II» («Pharmacia Biotech») using Fragment Manager 2.0 software.

SNRPN quantification. Copy number analysis of SNRPN gene was performed by dye-based qPCR. Intron 2 of SNRPN (target gene) and exon 12 ALB (reference gene) were amplified using locus-specific primers. qPCR was carried out on CFX96 Touch Real-Time PCR Detection System (Bio-Rad) in 15 ml reaction volume containing 1x HOT FIREPol® EvaGreen® qPCR Supermix (Solis Biodyne), 10 mM SNRPN or ALB primers and 25 ng of genomic DNA (5 µl). The cycling conditions were: preincubation stage of 12 min at 95°C to activate hot-start polymerase followed by 35 cycles of two-stage reaction − 20 s at 95°C and 30 s at 60°C. Each DNA sample was run in duplicate. The Real-Time data were analyzed by the CFX Maestro Software (Bio-Rad). The relative gene copy number of unknown sample was estimated using a 2^−ΔΔCq Livak method (Livak 2001). The efficiency of qPCR for both genes was estimated by the calibration curve of the control DNA samples. DNA samples of PWS patients with and without SRNPN deletion identified by FISH were used as controls − 1 and 2 SRNPN copies, correspondingly (data not shown).

We identified 15q structural alterations in 17 families out of 29.

15q11-q13 deletions

In 12 patient de novo deletions were identified by STR-segregation analysis; the affected probands lost paternal or maternal alleles of several STR-markers. Since all the probands were hemizygous or homozygous for D15S817 alleles close to SNRPN gene additional investigation were needed to differentiate deletion and UPD15 isodisomy. qPCR analysis detected 1 copy of SNRPN gene in all these patient, therefore the deletion was confirmed.

Ten our patients had PWS deletion (fig.1). It has been proven, that the size and chromosomal location of paternal 15q11-q13 deletions are associated with rather distinct PWS phenotypes (Hassan 2016, Kim 2012, Tan 2014, Varela 2004), therefore, determining the exact boundaries of deletions is one of the tasks of PWS diagnosing. In two cases exact borders of the deletions were not defined; seven deletions were typical type I (BP1-BP3) or type II (BP2-BP3); and in one 3 month old infant female patient atypical large BP1-BP4 deletion was detected. This patient with large deletion has atypical PWS including lower-than-typical birth weight, the much more severe developmental delay, microcephaly, atypical facial features, increased hypotonia and the prolonged failure-to-thrive. Among the patients with typical or unclassified PWS deletions three were infants under 6 months of age with severe hypotonia. One of the infants has congenital polyneuropathy due to preterm birth and incomplete myelination, in this case Dejerine-Sottas disease had been previously suspected. In other 2 probands spinal muscular atrophy was suspected and excluded. Other patients expressed classical PWS phenotypes.

In two patients maternal origin deletions associated with classical AS-phenotype were identified (fig. 1).

15q uniparental disomies

STR-segregation analysis revealed 5 patients with maternal UPD15 heterodisomy (hetUPD) associated with PWS (fig.1). These patients had both maternal alleles and lost paternal ones in several or all STR markers. In two patients, who had hemizygous/homozygous genotype by all STR markers (only maternal alleles were present), SNRPN qPCR analysis detect 2 copies of 15q11.2 region, which confirmed UPD15 isodisomy (isoUPD15) (fig.1).

And one 1,5-years-old male patient (case #147) showed a rare variant of segmental iso-heterosomic UPD, for D15S541 (BP1-BP2), D15S817 (BP2-SNRPN) and D15S1513 (UBE3A-BP3) loci the patient was homozygous meanwhile, mother was heterozygous, and for at least D15822, D151002 loci patient had the same heterozygous genotype as his mother (fig. 2). It was confirmed that the centromeric region of the maternal origin segmental UPD is isodisomic (D15S1513 marker inclusive). So we can assume that a possible mechanism of such segmental UPD is recombination before meiosis 1 during oocyte formation, which was followed by nondisjunction in meiosis 2, successive fertilization by a normal sperm resulting in trisomic zygote and final trisomic rescue (Niida 2018).

One hetUPD proband was 2 years old boy with congenital polyneuropathy due to preterm birth and incomplete myelination, in this case Dejerine-Sottas disease had been suspected in the age of 6 months (EGR2 and PMP22 mutations were excluded), and PWS like phenotype was detected in the second clinical examination.

Five other patients, including the probands with isoUPD15 and iso-hetUPD15, have typical PWS phenotypes.

15q11-q13 duplications

Among the patients with atypical AS-like phenotypes two patients were diagnosed with 15q11.2-q13 duplication syndrome.

Case 1, 9.8 y.o., female

The patient is the second child of unconsanguineous parents, born from uncomplicated full term pregnancy with birth weight 2800 g (15th centile), and height 52 cm (85th centile). Age of the parents at the time of the child’s conception was 26.5 y.o. (mother) and 27.3 (father). The patient has a healthy elder brother. In early perinatal period till the age of 12 months the parents had noticed a gastroesophageal reflux disease and delay in motor development (delayed sitting without support). The first psychiatric examination had been conducted at the age of 4 years, at that age she just become able to walk independently and had moderate learning and communication difficulties, reproducing an autistic behavior. At that age she was diagnosed with F 07.9 and F 84.8, since that time she has been treated with memantine hydrochloride 10-15 mg per day. The treatment interruption, according to her parents, had resulted in behavioral and learning regress. According to the last psychiatric evaluation at the age of 6.8 years old the patient was diagnosed with psycho-organic syndrome with cognitive and visiomotor coordination deficiency, mild intellectual disability (ID), severe dyspraxic impairment, A.D.D., expressive-receptive speech and language disorder, moderate dysarthria associated with autism spectrum disorder, abnormal bioelectric activity of the brain in EEG. ADOS scale: autistic feathers– 17 points, stereotyped and restricted patterns of behavior – 4 points. At the age of 6.8 years her psychomotor developmental age as assessed by the PEP-R scale was 3y 1m. At the age of 8 years the patient had developed spastic seizures. On physical examination at the age of 9.8 she had a height of 141 cm (75th centile), and a weight of 40 kg (85th centile). She had no evident dysmorphic features.

As the proband demonstrated atypical possibly X-linked phenotype with ID and spastic seizures, Rett syndrome was suspected, but MECP2 common mutation screening didn’t confirm it. Therefore NGS Epilepsy panel (146 genes, Invitae) was analyzed, which revealed 2 genes from 15q11-q13 region UBE3A and GABRB3 to be triplicated (4 copies). In 2 genes outside 15q chromosomal region (MECP2 and SYNGAP1) heterozygous missense mutations were found, both are inherited (probably not pathogenic): paternal MECP2 and maternal SYNGAP1 (data not shown).

The segregation analysis of the STR markers confirmed maternal origin de novo triplication (fig.3). The most probable mechanism of the 15q triplication is isodicentric chromosome formation.

Case 2, 10.1 y.o., male

The patient is the only child of unconsanguineous parents, born from full term pregnancy by caesarean birth delivery for medical reasons (maternal urolithiasis). Patient’s birth weight was 2095 g (<1^st centile), and normal height - 50 cm, 50^th centile. Age of the parents at the time of the child’s conception was 26.4 y.o. (mother) and 36.6 y.o. (father). The family history mentioned learning disability in the mother. But, at the time of medical genetic counseling no evident autistic features or decreased intelligence ware observed in probands mother.

From early childhood, the boy has demonstrated a developmental delay and poor motor coordination. The patient motor development was mildly delayed, with independently walking at the age of 1,5 years old. Speech development and communication skills were more severely delayed. At the age of 3 he was diagnosed with mild motor delay, severe growth impairment and speech apraxia. The EEG revealed focal epileptiform activity with negative dynamics on follow-up. The antiepileptic treatment by Dekapin (sodium valproate) was prescribed.

On physical examination at the age of 10 his height was 126 cm (3^rd centile), and the weight was 26 kg (15th centile). The boy demonstrated an epilepsy, autistic behavior, speech dysarthria and developmental delay.

Therefore NGS Epilepsy panel (146 genes, Invitae) was analyzed, that revealed 2 genes from 15q11-q13 region UBE3A and GABRB3 were duplicated (3 copies), in RELN gene outside 15q chromosomal region heterozygous missense mutation with uncertain significance was found (data not shown).

The segregation analysis of the STR markers confirmed interstitial 15q11-q13 duplication in proband. The duplication was inherited from probands mother. She only has mild learning disability due to incomplete penetrance (fig. 4).

The selected panel of markers showed high rates of heterozygosity, which ranged from 51% (markers D15S822 and D15S1233) to 86% (marker D15S541). The highest diagnostic information content was achieved for loci D15S817, D15S1513, D15S822 and D15S10002, amounting approx. 90%. The least informative were pericentromeric markers. However, this panel is highly informative for identifying all possible genomic reorganizations: deletions, duplications and uniparental disomies (iso-, hetero- and complex), as well as for determining their origin and inheritance (de novo/inherited, maternal or paternal). Therefore, the panel of markers could be used for Prader-Willi / Angelman syndromes and related diseases diagnostics, allowing identification both typical and atypical 15q deletions and duplications, different types of 15q UPDs (even complex ones), as well as assess the genetic risk of re-inheritance of 15q11–q13 reorganizations in each specific family. Since all of the above can be determined using the same markers, with additional analysis using routine qPCR, this method is inexpensive and fast enough in comparison with other strategies (Smith 2017).

The main limitation of this approach is the segregation analysis, which needs to examine at least 2 family members (the proband and at least one of the parents). However, in most cases, one or both parents are available for analysis.

It should be noted that different types of genomic reorganizations of 15q11-q13 are a consequence of different molecular genetic mechanisms of their occurrence, which, in turn, causes different family risks of inheritance of the defined mutation.

1. Deletions of different size are associated with non-allelic recombination events in different BPs. These events occur nearly always de novo, not related to the age of the parents. Therefore the genetic risk of the recurrent event of having the next child with the same reorganization does not increase and is equal to the population risk. On the other hand, atypical deletions (longer or shorter than typical ones) may be due to balanced translocation in proband’s parents which significantly increases the risk of the mutation re-inheritance.
2. In case of UPD different mechanisms lead to iso- and heterodisomies, which means different recurrent risks, depending on the mother's age, and can be the result of a balanced translocation (then there is a risk of a recurrent case). It could be illustrated by PWS het-UPD, which in most cases is a result of "trisomic rescue" - the loss of a single copy of paternal chromosome 15 when the zygote becomes trisomic due to the inheritance of a nondisjunction pair of maternal chromosomes and the single paternal chromosome. In this case, the risk of chromosome nondisjunction is much higher in oogenesis and it increases significantly with the age of the mother. Our study recorded a significant predominance of children with Prader-Willi syndrome as a result of maternal isodisomy among women who conceived at the age of over 40 compared to families of younger mothers in whom children with PWS had a paternal deletion (data not shown).
3. Complex iso-heterodisomies also have different mechanisms of formation. In our case (Fig. 4), we assume that this reorganization in a proband with PWS was inherited as a result of the following sequence of events: recombination before M1 in oogenesis - nondisjunction of chromosomes in M1 - trisomic rescue (Niida 2018).
4. Duplication or multiplication is the most common genetic cause of ASD (Miles 2011). In the described 2 cases duplication and multiplication differ in the mechanism of occurrence - one is de novo, the other one is inherited, which corresponds to the different risks of re-inheritance in these two families.

The proposed STR-based strategy for 15q11-q13 reorganizations identification allows to determine the type, origin, and boundaries of the reorganization, significantly improves medical genetic counseling for families - it allows prediction of the disease severity, the estimation of genetic risks for families, and even applies the personalized approaches to therapy.

Funding

All funding for this work was received from The National Academy of Sciences of Ukraine, grant title: “Genetic, epigenetic factors and cross genome interactions in neurological and oncological pathogenesis in human”, grant number 0121U109972.

Author Contributions

All authors contributed to the study design. Chernushyn S.Yu.: planning, conduct of experiments, data analysis, manuscript preparation; Kravchenko S.A.: conduct of experiments, data analysis; Patskun E.I.: clinical data collection and analysis; Hryshchenko N.V.: conceptualization, conduct of experiments, data analysis, and manuscript preparation. The first draft of the manuscript was written by Chernushyn S.Yu, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data availability statement

The authors declare data sharing and adopt Experts Data Policy.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This study was performed in line with the principles of the Declaration of Helsinki. Ethics approval was granted by the Ethics Committee of Institute of molecular biology and genetics NASU (Date 19.04.2022/No. 31).”

Consent to participate

Informed consent was obtained from all individuals included in the study. The probands and his parents agreed to participate in this study.

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Robust and easy-to-use STR-based method for 15q11-q13 rearrangements identification

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