Molecular characterization, by digital PCR analysis of four HMBS gene mutations affecting the ubiquitous isoform of Porphobilinogen Deaminase (PBGD) in patients with Acute Intermittent Porphyria (AIP)

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Regular Article

Molecular characterization, by digital PCR analysis of four HMBS gene mutations affecting the ubiquitous isoform of Porphobilinogen Deaminase (PBGD) in patients with Acute Intermittent Porphyria (AIP) Francesca Granataa, Manuel Mendezb, Valentina Brancaleonia, Francisco J. Castelbonb, ⁎ Giovanna Graziadeia, Paolo Venturac, Elena Di Pierroa, a

Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, U.O.C. Medicina Generale, Milano, Italy Instituto de Investigación, Hospital 12 de Octubre, Madrid, Spain Division of Internal Medicine 2 - Centre for Porphyrias, Dept. of Medical and Surgical Sciences for Children and Adults, University of Modena and Reggio Emilia, Policlinico Hospital, Modena, Italy b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Porphyria HMBS Splicing isoform Promoter variants Digital PCR Gene expression

Genetic variants in promoters and alternative-splicing lesions require to be experimentally tested in order to validate them as causatives of a disease. The digital PCR (dPCR) approach, which is an alternative to the classical qPCR, is an innovative and a more sensitive method for the detection and quantification of nucleic acids. In the present study, we identified four HMBS gene mutations affecting the ubiquitous isoform of porphobilinogen deaminase (PBGD) and established a dPCR protocol which would be able to detect the different transcripts of this gene. With the application of this method, we were able to characterize the functional roles of these four genetic variants, demonstrating that all these mutations were causatives of the non-erythroid variant of the acute intermittent porphyria (AIP) disease.

1. Introduction Acute intermittent porphyria (AIP; OMIM#176000) is an autosomal dominant disorder caused as a result of mutations in the HMBS gene, which is located at the chromosomal region 11q24.1–24.2 (GRCh38.p11:119,084,003–119,094,417). The HMBS gene codes for the porphobilinogen deaminase (PBGD; EC 2.5.1.61; MIM#609806), an enzyme involved in the third step of the heme biosynthesis pathway [1]. In the peripheral blood, the porphobilinogen deaminase enzyme is present in two isoforms, namely, the ubiquitous and the erythroid isoforms, which arise from the two distinct mRNAs that are transcribed from the ubiquitous promoter and the erythroid-specific promoter, respectively [2]. The ubiquitous promoter is located in the 5′ flanking region upstream of the exon 1, and directs the synthesis of a transcript containing the exons 1 and 3–15. On the other hand, the erythroidspecific promoter is located in a region 3-kb downstream of the intron 1, and it directs the production of a transcript that contains the exons 2–15 [3–5] (Fig. 1). Most of the patients (95%–98%) exhibit the classical form of the AIP disease, which arises as a result of mutations in the

common region of the HMBS gene, from exon 3 to exon 15, and therefore, affects both the ubiquitous and the erythroid isoforms of PBGD. In the patients with the non-erythroid variant of the disease (5% of all the cases), the defects are reported within or close to the coding region of the exon 1, which is specific to the ubiquitous isoform of PBGD. So far, no mutations are known in the erythroid-specific promoter or in the exon 2 [6]. In both the forms of AIP, the symptomatology is a result of the acute systemic accumulation of the precursors of heme biosynthesis pathway as porphobilinogen (PBG) and δ-aminolevulinic acid (ALA) [7]. The increase in the levels of these metabolites is attributed to the exposition of several trigger factors, endogenous or exogenous, requiring greater amounts of heme [8]. The clinical manifestations of this disease include subtle neurological symptoms, such as weakness, dysesthesia, severe fatigue, and the inability to concentrate, which are followed by acute and progressively-worsening abdominal pain, nausea, vomiting, and psychiatric complications [9,10]. However, the acute attacks occur only in < 10% of the at-risk population, which reflects a key role of the environmental factors and possibly that of the genetic modifiers [11]. > 50% of the symptomatic patients develop the

Abbreviation: HMBS, Hydroxymethylbilane Synthase.; AIP, Acute Intermittent Porphyria.; PBGD, Porphobilinogen deaminase.; dPCR, Digital PCR.; PBG, Porphobilinogen.; ALA, δ-Aminolevulinic acid.; PAKD, Porphyria-associated kidney disease.; vAIP, Variant of the acute intermittent porphyria. ⁎ Corresponding author at: Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Via F. Sforza 35, 20122 Milano, Italy. E-mail addresses: [email protected], [email protected] (E. Di Pierro). https://doi.org/10.1016/j.ymgme.2018.09.002 Received 17 July 2018; Received in revised form 3 September 2018; Accepted 3 September 2018 1096-7192/ © 2018 Published by Elsevier Inc.

Please cite this article as: Granata, F., Molecular Genetics and Metabolism, https://doi.org/10.1016/j.ymgme.2018.09.002

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Fig. 1. Scheme of HMBS gene splicing: the ubiquitous promoter is located in the 5′ flanking region upstream of the exon 1, and directs the synthesis of a transcript containing the exons 1 and 3–15. On the other hand, the erythroid-specific promoter is located in a region 3-kb downstream of the intron 1, and it directs the production of a transcript that contains the exons 2–15. The ubiquitous and the erythroid isoforms arise from the two distinct mature mRNAs.

2.2. Mutation analysis

porphyria-associated kidney disease (PAKD) [8,12,13]. The laboratory diagnosis for AIP consists of various steps of evaluation. In the first step, biochemical screenings are performed, which include the following: urinary ALA, PBG, and porphyrin measurements; the assessment of the erythrocyte PBGD activity [14]; and the plasma fluorometric emission scanning (a peak could be detected at around 620 nm) [15]. As there are multiple variables of the biochemical parameters, these results are not reliable for being sure of the diagnosis. The second step, therefore, is the genetic evaluation of the entire HMBS gene in order to identify point mutations as well as large deletions [16,17]. Nevertheless the identification of genetic variants in the ubiquitous promoter or outside of the coding region makes necessary a subsequent, though not always conclusive, step of validation of the diagnosis by in vivo RNA quantification studies or in vitro reporter gene assays [18]. In this paper, we report the molecular characterization of four noncanonical HMBS gene mutations, identified in four unrelated patients affected by the non-erythroid variant of the acute intermittent porphyria (vAIP) disease. With the use of a novel and better-performing method for the in vivo RNA quantification– the digital PCR system [19,20], we demonstrated that these mutations affect the ubiquitous isoform of PBGD causing disease.

The study was conducted in accordance with the Declaration of Helsinki (DoH), and an informed consent was signed by all the subjects (patients) at Fondazione IRCCS Ca’ Granda–Ospedale Maggiore Policlinico, prior to their inclusion in the study. The genomic DNA was extracted from the peripheral blood using the Maxwell®16 Automated System (Promega Corp.) according to the manufacturer's instructions. The ubiquitous regions of the HMBS gene, including the promoter, exon 1, and the region upstream of the exon 3, were all PCR-amplified, using the primer pairs listed in Table 2 (NM_0000190.3 region from chr11:119,088,201–119,088,275). The genomic DNA (200 ng) was amplified in the presence of 1.5 mM Mg2+, 0.2 mM dNTPs, 1× buffer, 2.5 U of BioTaq DNA polymerase (Bioline, London, UK), and 10 pmol/ μL of each primer. The amplifications were performed using the following PCR program: a denaturation step at 94 °C for 5 min; followed by 30 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min; and a final elongation step at 72 °C for 5 min. The sequencing was performed using the BigDye Terminator v1.1 Cycle Sequencing Ready reaction kit for ABI Prism 310 Genetic Analyzer (Applied Biosystems by Thermo Fisher, San Francisco, USA). The frequency assessment for each variation was conducted using the Single Nucleotide Polymorphism Database (dbSNP) and the Exome Aggregation Consortium (ExAC) database. The large deletions were detected by using the multiplex ligation-dependent probe amplification (MLPA), as described in a previous study [16]. The sequencing of the breakpoints was performed using the longrange PCR and chromosome walking (Table 2).

2. Methods 2.1. Patients and biochemical determinations Four unrelated patients with the non-erythroid form of the acute intermittent porphyria (vAIP) disease were included in the study. The diagnosis was made on the basis of the clinical history of at least one typical acute attack, as well as an increased excretion of porphyrins and precursors in the urine in relation to the normal value of the erythrocyte PBGD activity (Table 1). Urinary ALA, PBG, and porphyrins, plasma porphyrin peak, and the erythrocyte PBGD activity were determined regularly for the routine analysis [21].

2.3. Reverse transcription polymerase chain reaction (RT-PCR) The total RNA was isolated from the peripheral blood of all the patients (except one), using the LEV simplyRNA Blood Kit for Maxwell®16 (Promega Corporation, Madison, USA), according to the protocols already published in a previous study [22]. Only for one patient (Pt4), the total RNA was obtained from a pellet of nucleated

Table 1 Patients data. Variable

pt 1

pt 2

pt 3

pt 4

Sex HMBS genotype Predicted Protein Age at diagnosis (yrs) Fluorimetric Peak Enzymatic activity PBGD [72,8–179,6 pmol/h/mg] Total urine porphyrin [ < 150 μg/L] Urin δ-Aminolevulinic acid (ALA) [ < 5,0 mg/g creatinine] Urin Porphobilinogen (PBG) [ < 2,0 mg/g creatinine] Treatment

Female c.[1–2996_33 + 1591del4620bp];[=] p.?/p.? 19 Neg 82.01

Male c.[33 + 4A > G];[=] p.?/p.? 6 Neg 150.36

Female c.[34–21A > G];[=] p.?/p.? 41 Neg 140.56

Female c.[−150 T > A];[=] p.?/p.? 53 620 126.2

912 μg/L 25.8 mg/g creatinine

2773 μg/L 52.9 mg/g creatinine

1117 μg/L 30.6 mg/g creatinine

358 μg/24 h 25.5 mg/L [ < 5,5 mg/L]

27.0 mg/g creatinine

107 mg/g creatinine

184 mg/g creatinine

67.7 mg/L [ < 2 mg/L]

Glucose infusion

Glucose infusion Normosang ®

Glucose infusion Normosang ®

Glucose infusion Normosang ®

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(application version 3.1.2; algorithm version 4.4.10) [23].

Table 2 Primers used in experiments.

2.5. Statistical analysis

PCR Fragment

PCR Primers

Prom UBI

HMBS-UBI-F HMBS-UBI-R HMBS–3F HMBS–3R XLF Ivs1R2 VPSdel1F

5′-gaaattagtcccacgtggtgatg–3′ 5′-cacactttgggcttctggacgag–3′ 5′-gccagtgattctggttcttggac–3′ 5′-ccccgagaggagagaacacaaca–3′ 5′-tcactggagggcttgtttccttatcacct–3′ 5′-tgggtaaagagataaggccc–3′ 5′-ggtccttgctctcagagtctataaat–3′

RNA Primers 1Fb IVS10R 2F IVS10R 3BF IVS10R

5′-catgtctggtaacggcaatgcggc–3′ 5′-cttcggaagctggacgagcag–3′ 5′-ctactatcgcctccctctagtc–3′ 5′-cttcggaagctggacgagcag–3′ 5′-atgagagtgattcgcgtggg–3′ 5′-cttcggaagctggacgagcag–3′

Exon 3 Long PCR Seq RNA Fragments 1/10 2/10 3/10

Internal RNA sequencing primers 3BF IVS4R 5B

The data analysis was performed using GraphPad Prism software version 7. The normality of the data from each measurement was assessed by using D'Agostino–Pearson's normality test, Shapiro–Wilk normality test, and KS normality test. Statistical analyses were performed using parametric tests, such as one-way ANOVA or t-test. In order to identify the outliers, ROUT test was performed for each experiment. The nominal statistical significance threshold was set at P < 0.05. 3. Results 3.1. Identification of mutation Four non-canonical mutations were identified in four unrelated patients who exhibited the typical biochemical profile of the non-erythroid variant of the acute intermittent porphyria (vAIP) disease. A large deletion of 4620 bp (c.1–2996_33 + 1591del), in a region including a portion of the upstream intergenic region, the entire ubiquitous promoter, exon 1, as well as a portion of intron 1 of the HMBS gene, was identified in patient 1 (pt 1) and in two other asymptomatic family members. Fig. 2 depicts the MLPA Plot, the long-range PCR results, and the sequence of the breakpoints of mutation that were already reported in the HGMD database. Two splicing variants (c.33 + 4A > G and c.34–21A > G), located outside the canonical donor and acceptor splicing sites, were identified in pt. 2 and pt. 3, respectively, and also in two other asymptomatic family members of each of these patients. Both of these mutations are already known to be associated with this form of the disease; however, the molecular characterization had not been performed so far [24,25]. Finally, a novel point mutation (c.–150 T > A) in the ubiquitous promoter region was identified in pt. 4, as summarized in Table 1. Only the c.33 + 4A > G variant had been previously reported in NCBI's SNP database (rs782312612). However, the provided MAF level (0.000008/1) was calculated on ExAC database on the basis of the exome analysis of the disease-specific carriers as well as the common population.

5′-atgagagtgattcgcgtggg–3′ 5′-tgaaagcctcgtaccctggc–3′ 5′-acaagattcttgatactgcac–3′

cells using the TRIZOL extraction protocol. Of the total RNA, 200 ng was reverse transcribed using the VILO Master Mix (Applied Biosystems by Thermo Fisher, San Francisco, USA). The cDNA synthesis was performed using the following program: the first step at 25 °C for 10 min; the next step at 42 °C for 60 min; and the last step at 85 °C for 5 min. The different sets of primer pairs were used to amplify the alternativelyspliced mRNAs corresponding to the ubiquitous isoform, the erythroidspecific isoform, or both the isoforms (Table 2). Of cDNA, 50 ng was amplified using 10 pmol/μL of each primer, in the presence of 1× buffer, 1.5 mM Mg2+, 0.2 mM dNTPs, and 1.25 U of Taq polymerase, under the following conditions: denaturation at 94 °C for 1 min; 35 cycles of amplification at 60 °C for 30 s and 72 °C for 1 min; and a final extension at 72 °C for 5 min. The PCR products were then submitted for direct sequencing. 2.4. Digital polymerase chain reaction (dPCR) A dPCR was performed for absolute quantification, on the Quantstudio®3D Digital PCR System (Applied Biosystems by Thermo Fisher, San Francisco, USA). In order to distinguish the two different isoforms of the HMBS gene, specific and differentially-labeled TaqMan® probe mixes were used in the dPCR. The probe HMBS 1 (Hs00609297_m1) was known to be located in the region between exon 1 and exon 3, and was used for the quantification of the ubiquitous mRNA with a FAM dye label. The HMBS 2 probe was designed to be located in the region between exon 2 and exon 3, and it was used for the detection of the erythroid mRNA using the custom TaqMan®MGB Probe mix with a VIC dye label. GUSB_VIC (Hs00939627_m1) and GUSB _FAM (Hs99999909_m) were used as the housekeeping genes in this analysis (Table 2). Of the total RNA, 200 ng was reverse transcribed using the VILO Master Mix (Applied Biosystems by Thermo Fisher, San Francisco, USA). Of cDNA, 10 ng, in a reaction mixture consisting of 2× Quantstudio®3D digital PCR Master mix and 1× of each of the TaqMan® Probes, was loaded onto the Digital PCR Chip using the QuantStudio®3D Chip Loader. The chips were then sealed and loaded onto the ProFlex™ 2 × flat PCR System (Applied Biosystems® by Thermo Fisher), and cycled under the following conditions: 96 °C for 10 min, followed by 45 cycles at 56 °C for 2 min and 98 °C for 30 s, and a final extension at 60 °C for 2 min, for HMBS 1; and 96 °C for 10 min, followed by 50 cycles of 60 °C for 2 min and 98 °C for 30 s, and a final extension at 60 °C for 2 min, for HMBS 2. Following the PCR cycling, the end-point fluorescence signal in each partition on the chips was measured by transferring the chips to QuantStudio®3D; the secondary analysis was performed using the QuantStudio®3D AnalysisSuite Cloud software

3.2. Qualitative RNA analysis In order to examine the effect of the c.33 + 4A > G and c.34–21A > G mutations on splicing, different sets of primer pairs were used to amplify the alternatively-spliced mRNAs of the HMBS gene (Table 2). The ubiquitous isoform of the gene was selected by using the 1Fb and IVS10R primers, located on exon 1 and exon 10, respectively; while the erythroid-specific isoform of the gene was selected by using the two primers located on exon 2 and exon 10. The PCR performed for the ubiquitous isoform of the gene identified the presence of a shorter band only for the c.34–21A > G variant; whereas in the PCR performed for the erythroid-specific isoform of the gene, no possible insertions or deletions were identified for both the variants. The sequence analysis confirmed that the c.34–21A > G mutation caused the skipping of exon 3 only in the ubiquitous mRNA, while the splicing between the exon 2 and exon 3 remained unaffected (Fig. 3). Surprisingly, the sequences of the mRNAs associated with the c.33 + 4A > G mutation exhibited variation at a known polymorphic level in exon 10 (rs1131488). This variation resulted in homozygous genotype in the ubiquitous isoform, and heterozygous genotype in the erythroid-specific isoform (Fig. 4). Even when the PCR was performed with primer F which was located in the region common to the two isoforms of the mRNA – exon 3, the polymorphism resulted in the heterozygous state. These results suggested that in this case as well, only the ubiquitous isoform was affected. However, the actual effect of the mutations on the splicing remains to be elucidated. 3

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Fig. 2. c.1–2996_33 + 1591 deletion in patient 1: the upper right panel shows the electrophoresis run of DNA analysis for patient 1 (pt1), the selected deleted band (del, 1940 bp) and the wt band in the control subject (C, 6560 bp); the lane M is the molecular weight marker (λ/Hind III digest). The sequence in the left indicates the deletion breakpoint; the panel in the bottom reports the multiplex ligation-dependent probe amplification plot (MLPA)

loaded chip, precision values in the desired range, and a homogeneous fluorescence. The quantity of 10 ng of the cDNA provided the best results in both the assays: a correct chip-loading with a homogenous fluorescence and sample distribution, and an optimal cluster division with precision levels below the recommended threshold of 10% (Table 3). In order to obtain a precise quantification, we set a threshold of 14,000 data points for the quality control of the chip. In these experiments, all the four enrolled patients were included. Three independent experiments were performed for each patient and compared with the respective three healthy controls. The relative

3.3. Absolute RNA quantification In order to assess the effects of c.1–2996_33 + 1591del and c.–150 T > A mutations in the transcriptional promoter activity, we established a novel method using the digital PCR strategy. We used two differentially - labeled TaqMan assays (HMBS1 and HMBS2) for the detection of the two HMBS mRNA species, each designed to work in a duplex with the housekeeping gene, GUSB. Different amounts of cDNA were used (10 ng, 5 ng, 2.5 ng, and 1.25 ng) in the assays, in order to establish the most suitable conditions that would ensure a correctly-

Fig. 3. c.34–21A > G mutation in patient 3: the upper right panel shows the electrophoresis run of RNA analysis for patient 3 (pt3) and the control subject (C); the lane M is the molecular weight marker (X 174/Hae III digest) and the lane blank is the negative control of PCR. The sequence in the left indicates the deletion breakpoint in the mature mRNA; the bottom panel reports the sequence of both normal and abnormal RNAs.

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expression compared to healthy controls (ANOVA test *p = 0.02); however the same level of HMBS 2 expression was registered for a control of well-purified buffy coat of nucleated cells (Fig. 5C). Moreover, a considerable difference was identified in the expression levels of total HMBS 1 and HMBS 2 (****p < 0.0001), through a paired t-test performed by excluding the pt. 4 following the ROUT test (Fig. 5D). 4. Discussion In the present study, we identified and characterized, at the molecular level, one novel and three previously-reported HMBS gene mutations. The molecular epidemiology of the classical AIP disease has been studied extensively so far. To date, over 400 mutations causing the AIP disease have been reported in the Human Gene Mutation Database (HGMD) [6]; however, only 20 mutations have been identified to cause the non-erythroid form of this disease. All the mutations reported in this study affect the ubiquitous isoform of PBGD, causing the non-erythroid variant of the AIP disease. The large deletion of 4620 bp (c.1–2996_33 + 1591del) begins in the intergenic region close to the VPS11 upstream gene, and ends in the intron 1 of the HMBS gene. This mutation deletes the entire ubiquitous promoter, completely preventing the transcription of the ubiquitous mRNA (NM_000190). As demonstrated by the digital PCR (dPCR) for pt. 1(c.1–2996_33 + 1591del), the ubiquitous mRNA was expressed at a level 50% of that in the normal control. At the same time, the erythroid-specific HMBS mRNA (NM_001024382) was neither impaired nor increased, as expected. The two splicing variants c.33 + 4A > G and c.34–21A > G were located close to the donor and acceptor splice sites of exon 1 and exon 3, respectively. The majority of the mutations that cause the non-erythroid form of AIP are splicing defects in exon 1; also, seven different substitutions from +1 to +5 positions in the canonical splicing donor site have been reported previously. All these mutations resulted in the activation of a cryptic splicing site located 67 bp downstream of the intron 1, leading to a frameshift and the formation of a premature stop codon in the exon 4 [26,27]. It is possible that the c.33 + 4 A > G mutation described in the present study could have had a similar effect on the HMBS mRNA, even though the abnormal isoform was not identified during the qualitative mRNA analysis. The differences observed between the sequencing of the ubiquitous and that of the erythroid-specific HMBS mRNAs suggested that the abnormal mRNA that was produced as a result of the mutation was lost during the analysis. At the same time, the highly-sensitive digital RNA quantification revealed that pt. 2 (c.33 + 4A > G) contained only 50% of the ubiquitous mRNA compared to the normal control. Considering that we used different primers located on exon 1, for both RNA analysis, we cannot completely exclude an implication of exon 1. Therefore, we strongly suggest that the mutation produced an aberrant mRNA, which degraded rapidly. However, we cannot distinguish whether the skipping of exon 1, the alternative 5′ splicing, or the retention of the entire intron 1 occurred. On the contrary, the qualitative RNA analysis confirmed that the c.34–21A > G mutation was involved in the suppression of splicing between the exon 1 and the exon 3 of the ubiquitous transcript variant, leading to the deletion of exon 3 in the mature transcript. This in-frame deletion could explain the reason behind the abnormal transcript not being exposed to the nonsense-mediated mRNA decay mechanism. Furthermore, the splicing between the exon 2 and exon 3 remained unaffected. These data were supported by the results from the dPCR experiments, where the use of a primer located on the exon 3 resulted in reduced levels of the ubiquitous transcript only. It is noteworthy that for both the splicing variants, the expression levels of the erythroidspecific transcript were higher than normal, and the difference in the expression levels was statistically more significant for pt. 3 (c.34–21A > G). This data suggested that the alterations in the canonical splicing sites of the ubiquitous transcript could switch the splicing toward the use of the alternative exon 2, forming more of the erythroidspecific transcript. Finally, in the present study, we demonstrated that

Fig. 4. Sequence analysis of mRNA in patient 2 carrying the c.33 + 4A > G mutation: the upper panel shows the electropherogram relative to ubiquitous mRNA sequencing; in the bottom panel the sequence of the erythroid-specific mRNA is shown. Table 3 Digital PCR condition setting. mRNA concentration

HMBS 1 (FAM) Precision

GUSB (VIC) Precision

CI copies/ μl FAM

CI copies/ μl VIC

# qualified by QT

no of filled

10 ng 5 ng 2.5 ng 1.25 ng

8.98% 13.37% 19.87% 25.59%

3.35% 4.44% 6.58% 8.88%

49.676 21.412 9.891 5.755

387.46 191.12 82.29 41.866

14,131 15,223 15,727 17,070

15,151 17,035 16,499 17,884

mRNA concentration

HMBS 2(VIC) Precision

GUSB (FAM) Precision

CI copies/ μl VIC

CI copies/ μl FAM

# qualified by QT

no of filled

10 ng 5 ng 2.5 ng

8.57% 16.71% 609.93%

2.88% 4.72% 6.76%

460.03 156.52 71.06

16,488 16,261 17,196

17,568 17,624 18,629

1.25 ng

609.93%

10.48%

46.443 13.187 7.70E2 8.16E2

31.98

16,223

17,054

quantification of RNA (%) was obtained by comparing the ratio between the absolute copy number of the target gene and the housekeeping gene in the patients and the ratio obtained for the controls. Fig. 5A and B present the mean results of these three independent experiments in terms of Log%. All the patients demonstrated a considerable decrease in the expression levels of HMBS1 compared to control (p ≤ 0.0001). On the contrary, different expression levels of HMBS2 were identified between the patients. The ANOVA test excluded a decrease in the expression levels of HMBS2 compared to control, for pt. 1 (c.1–2996_33 + 1591del) and pt. 2 (c.33 + 4A > G); whereas, a considerable increase was observed for pt. 3 (c.34–21A > G, p = 0.003). On the contrary, the mean value obtained for the result of the three experiments for pt. 4 (c.–150 T > A) was 0.03, similar to the null 5

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Fig. 5. Digital experiments results: (A) the log% of HMBS1 gene expression (ubiquitous transcript); (B) the log% of HMBS2 gene expression (erythroid-specific); (C) the log% of HMBS2 gene expression (erythroid-specific) for pt4 compared to a control with the same origin; (D) the differences in the expression of HMBS1 and HMBS2. pt. 1 (c.1–2996_33 + 1591del); pt. 2 (c.33 + 4A > G); pt. 3 c.34–21A > G; pt. 4 (c.–150 T > A).

expression is limited, assuming that a 50% variation in the gene expression is translated into the variation of a single Ct. In this paper, in a pioneer attempt, we designed and applied an easy, fast, reliable, and highly-sensitive method based on digital PCR, for the in vivo detection of small variations in the gene expression. This method could prove to be useful in clinical practice, as a functional rapid test for variants with an unclear or not-well-described pathogenetic role.

the c.–150 T > A substitution affected the transcriptional promoter activity of the ubiquitous promoter. Similar to the other patients, the amount of the ubiquitous transcript variant in pt. 4 (c.–150 T > A) was only about 50% of that in the control subjects. In contrast, very low expression levels of the erythroid-specific transcript variant were obtained. This difference could be explained by the fact that the RNA sample of this patient originated from a well-purified buffy coat of nucleated cells of the peripheral blood, instead of buffy coat of whole blood containing the red blood cells as well. The present study, in addition to large-scale genome sequencing, supports the clinical implications of the mutations in the promoter region, suggesting the importance of the examination of such mutations in the molecular diagnosis. However, while the non-sense, indels and the consensus splice-site mutations in the coding region of the genes are clearly pathogenic, the genetic variants of the promoters or the alternative-splicing lesions require experimental testing in order to receive validation as causatives of a disease. The in vitro reporter-gene assays are used widely to establish the functional consequences of such alterations on the residual promoter activity. However, these experiments are often expensive and time-consuming. Qualitative RNA analysis is a widely used method for the detection of the effect of splicing variants on the mature RNAs. However, as in the experiments of the present study, it is possible to lose the abnormal mRNAs due to their rapid degradation. Moreover, the results of the application of classical qPCR to analyze the in vivo residual-gene expression are not always conclusive, due to the technical limitations of this analysis. The sensitivity of this analysis for the detection of small variations in the gene

Funding This research was supported in part by grants from the Italian Ministry of Health (GR–2011–02347129 to Dr. Di Pierro) and from Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico (RC2018).

Authors’ contribution FG designed the study, planned and performed digital PCR experiments, analyzed data and wrote the paper; MM, FJC, GG, and PV recruited patients; VB collected samples and provided technical support for dPCR; EDP performed the qualitative RNA experiments, supervised research and wrote the paper. All authors read and agreed to the final version of the manuscript, revised it critically and they have no relevant conflict of interest to disclose.

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Acknowledgements

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