Identifying Genetic Etiology in Patients with Intellectual Disability: An Experience in Public Health Services in Northeastern Brazil

Abstract

Intellectual disability (ID) is considered a common neuropsychiatric disorder that affects up to 3% of the population. The etiologic origin of ID may be genetic, environmental, and multifactorial. Chromosomopathies are relatively common among the genetic causes of ID, especially in the most severe cases and those associated with dysmorphic features. Currently, the application of new molecular cytogenetics technologies has increasingly allowed the identification of microdeletions, microduplications, and unbalanced translocations as causes of ID. The objective of this study was to investigate the etiology of ID in patients admitted to a public hospital in Northeastern Brazil. In total, 119 patients with ID who had normal karyotypes and fragile X exams participated in this study. The patients were initially physically examined for microdeletion syndromes and then tested using fluorescence in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA), methylation-sensitive polymerase chain reaction (MS-PCR), and chromosome microarray analysis (CMA), according to clinical suspicion. Patients with no diagnoses after FISH, MLPA, and/or MS-PCR evaluations were subsequently tested by CMA. The rate of etiologic diagnoses of ID in the current study was 28%. FISH diagnosed 25 out of 79 tested (31%), MLPA diagnosed 26 out of 79 tested (32%), MS-PCR diagnosed 7 out of 20 tested (35%), and the single nucleotide polymorphism array diagnosed 6 out of 27 tested (22%). Although the CMA is the most complete and recommended tool for the diagnosis of microdeletions, microduplications, and unbalance translocations in patients with ID, FISH, MLPA, and MS-PCR testing can be used as the first tests for specific syndromes, as long as the patients are first physically screened clinically, especially in the public health networks system in Brazil, where resources are scarce.

Introduction

Intellectual disability (ID) affects approximately 3% of the world population. This disability is classified by specific limitations in intellectual functioning, such as in adaptive behavior, characterized by delayed neuropsychomotor development, and starts before the age of 18 years for both acquired and hereditary causes. ^{1 2}

The etiology of ID is heterogeneous with genetic, environmental, and multifactorial being common causes. ³ Within the genetic etiologies, there are notably both Mendelian and copy number variations (CNVs) conditions causing ID. ^{4 5} CNVs are small deletions, duplications, and rearrangements of chromosomal material.

More than half of ID cases are still considered idiopathic. ⁶ However, the use of cytogenomic tools shows that 10 to 25% of ID cases involve small subtelomeric or interstitial rearrangements resulting in genomic diseases from CNV. ^{7 8}

For decades, G-banding chromosomal studies, or karyotyping, were the initial examination in the investigation of patients with idiopathic ID. ⁹ However, to detect submicroscopic alterations (less than 5 Mb) and to overcome the limitations of the G-band karyotype, the use of more advanced techniques, such as fluorescence in situ hybridization (FISH), the multiplex ligation-dependent probe amplification (MLPA), and the chromosome microarray analysis (CMA) have been developed. The use of these tools has increased the detection of CNV alterations from 5% to approximately 17% in patients with ID. ^{6 10 11 12 13}

Since 2010, the International Cytogenomic Array Consortium (ISCA) has recommended the analysis of CMA as the first test for the diagnosis of patients with ID and developmental disabilities, and congenital anomalies (CA). ⁸ The CMA is the principal method used to detect CNVs, which have been shown to be the pathogenesis of many of the above disorders. ^{14 15}

Here, we describe the results of a multicentric study, “Implementation of a Genetic Research Network for Mental Disability in the Northeast Regions within the Scope of the SUS/Brazil (Health Unic System),” approved by the Ethics Committee, Protocol 13/11, and financed by CNPq 402025/2010-5.

This study describes an experience in the etiologic investigation of ID in patients from public health services in Northeastern Brazil, using cytogenomic tools: FISH, MLPA, single nucleotide polymorphism (SNP)-array, methylation-sensitive polymerase chain reaction (MS-PCR), and CMA.

Methods

In the current study, 119 patients were evaluated between 2011 and 2014, from the Medical Genetics Clinic of SGM/HUPES/UFBA (Bahia) and collaborating institutions from the Northeast region: Albert Sabin Children's Hospital (Ceará); State Secretariat of Public Health of Rio Grande do Norte (RN); and Potiguar University—(UNP/Rio Grande do Norte); Association of Parents and Friends of Exceptional People—APAE, São Luiz (Maranhão). Each collaborating institution forwarded their respective samples, informed consent, and clinical data of patients to UFBA.

The inclusion criteria for participating in the study were that individuals had varying degrees of IDs, with or without dysmorphic findings, and had normal karyotype and fragile X exams. Those meeting criteria then had FISH, MLPA, MS-PCR, and SNP-array testing.

Based on clinical criteria, the patients were physically screened for the following syndromes: Williams–Beuren syndrome (WBS) OMIM: #194050, Rubinstein–Taybi (RSTS1) OMIM: #180849, Miller–Dieker lissencephaly OMIM: #247200, Smith–Magenis syndrome (SMS) OMIM: #182290, Langer–Giedion syndrome (LGS) OMIM: #150230: #133700, Wolf–Hirschhorn syndrome (WHS) OMIM: #194190, cri-du-chat (CDChS) OMIM: #123450, Sotos (SOTOS1) OMIM: #117550, and Prader–Willi syndrome (PWS) OMIM: #176270/Angelman syndrome (AS) OMIM: #105830. All patients with these suspected disorders then underwent FISH testing.

In addition, patients with suspected PWS/AS also underwent MS-PCR testing and patients with a clinical suspicion of WBS also had MLPA. If the etiology of ID remained undetermined after these investigations, their medical records were reviewed, and the Vries protocol was applied. ¹⁶ Individuals who obtained scores of six or more were selected for analysis by CMA.

DNA Extraction

For the extraction of genomic DNA, 5 mL of peripheral blood was obtained from each patient. The DNA was extracted using the Wizard Genomic Purification Kit (Promega) according to a protocol provided by the manufacturer.

Hybridization In Situ Fluorescence

FISH analyses were performed on slides of chromosomal preparations according to the methods described by Pinkel et al. ¹⁷ The following Cytocell probes were used: ELN-7q11.23 (WBS), CREBBP-16p13.3 (MD), RAI1-SMCR 17q11.2 (SM), LIS1-MDS/ILS 17p13.3 (LG), tricho-rhino-phalangeal syndrome, type 1 (TRPS1) 8q23.3 and EXT1 8q24.11–8q24.12 (LG), WHSCR 4p16.3 (WH), CTNND2 5p15.2 (CDCh), NSD1 5q35 (SS), SNRPN/IC (PWS), and UBE3A (AS) 15q11–13. The FISH was performed following the guidelines of the probe's manufacturer. The analyses were performed using a Cytovision computerized image analysis system (Applied Biosystem).

Multiplex Ligation-Dependent Probe Amplification

The MLPA was performed with the SALSA MLPA probe mix P029-B1 WBS 1 kit following the manufacturer's guidelines. The data generated were analyzed using software and Coffalyser (MRC-Holland).

Methylation-Sensitive Polymerase Chain ReactionThe extracted genomic DNA was modified with sodium bisulfite, based on the method of Kosaki et al. ¹⁸ MS-PCR was performed at the promoter region of the SNRPN gene, with modified DNA. ¹⁹

Chromosome Microarray Analysis

The CMA was performed using SNP-array, the platform Scan SQsistem (Bead Array based sequencing Techologies-Illumina), and the chips Human CytoSNP-12 Bead Chip (Illumina Inc., San Diego, CA, United States). The procedures were performed following the manufacturer's instructions. The slides of the array were scanned on the iScan Reader, and the data analysis was performed using IlluminaGenomeStudio software, version 2010.1 and KaryoStudio version 1.4.3.0 Build 37 (CNV Plugin V3.0.7.0). CNVs identified in the SNP-array were evaluated using the USCS Genome Bioinformatics genomic variation databases ( http://genome.ucsc.edu ), DGV database ( http://projects.tcag.ca/variation/ ), and DECIPHER ( http://decipher.sanger.ac.uk/ ). The CNVs were classified according to the criteria of the American College of Medical Genetics and Genomics. ²⁰

Results

In the current study, it was possible to identify the etiologies of dysmorphic patients with ID in 34 of 119 patients ( Table 1 ), a diagnostic rate of 28%. FISH analyses were performed on 79 patients who were suspected to have microdeletion syndromes. This latter testing detected microdeletions in 25 (31%) of those patients investigated.

Table 1. Syndromes diagnosed in patients evaluated in this study.

Syndromes	Number of patients	Number of patients diagnosed by test
Williams–Beuren syndrome (OMIM: # 194050)	16	FISH (16)/MLPA (16)
Chromosome 7q11.23 duplication syndrome (OMIM: # 609757	01	FISH (0)/MLPA (1)
Prader–Willi syndrome (OMIM: # 176270)	05	FISH (4) /MS-PCR (5)
15q26 deletion syndrome	01	FISH (1)
Angelman syndrome (OMIM: # 105830)	02	FISH (1) /MS-PCR (2)
Wolf–Hirschhorn syndrome (OMIM: #194190)	01	FISH (1)
Langer–Giedion syndrome (OMIM: 150230, #133700)	02	FISH (2)
Phelan–McDermid syndrome (OMIM: #606232)	01	SNP-array
DiGeorge syndrome (OMIM: #188400)	01	SNP-array
1q41.q42.11q42.12q42.13q42.2q42.3q43q44 duplication	01	SNP-array
5p15.33p15.32 duplication/18q22.2q22.3q23 deletion	01	SNP-array
2p25.3 deletion and 2q37.1q37.2q37.3 duplication	01	SNP-array
4q32.3q33q34.1 and 4q34.1q34.2q34.3q35.1q35.2 deletion /9p24.3p24.2p24.1p23 and 9p13.1p12 duplication	01	SNP-array
Total patients	34

Abbreviations: FISH, fluorescence in situ hybridization; MLPA, multiplex ligation-dependent probe amplification; MS-PCR, methylation-sensitive polymerase chain reaction.

Twenty-four patients were suspected to have WBS and were tested both by FISH and MLPA. By this testing, WBS syndrome was confirmed in 16 patients ( Table 1 ). Additionally, MLPA detected a patient with 7q11.23 duplication syndrome (OMIM #609757) not identified by FISH.

In total, 16 patients were tested for PWS using FISH and MS-PCR, which confirmed five PWS diagnoses. By MS-PCR, four patients possessed 15q11.2-q13 deletions with the paternal allele methylated, and one patient without a deletion who had an absence of the methylated allele and may have had uniparental disomy (UPD) or imprinting changes. Additionally, one patient suspected to have PWS syndrome was found to have an atypical distal 15q26 deletion with a normal methylation pattern. ²¹ Deletion in the SNPRN and UBE3A genes of maternal origin was found in one of the other two and all had total absence of the maternal methylated alleles, suggestive of UPD or an alteration in the imprinting mechanism.

Four patients were tested for LGS syndrome with two having deletions of both the EXT1 and TRPS1 genes. Among the others, four patients were tested for WHS, but only one patient had a deletion in the WHS critical region. This deletion was of approximately 165 kb in size.

Twenty-seven patients were screened for other syndromes by FISH: SOTOS1 (18 patients), SMS (six patients), RSTS1 (two patients), and CDChS (one patient); however, results were normal in all these cases. In total, 27 patients were evaluated by CMA and six patients had pathogenic CNV, a detection rate of 22% for this tool ( Table 2 ).

Table 2. Phenotype of patients with results of SNP-array showing pathogenic CNV.

Case	Sex	Age	SNP-array [hg19]	Size (Mb)	Phenotype
1	F	12	22q11.21q11.23 (20,737,903–23,971,028)X1	3.2	ADNPM; learning difficulties; short stature; microcephaly; narrow palpebral fissures; elongated, small and triangular face; micrognathia; facial skull disproportion; cleft palate; high nasal bridge; ocular hypertelorism; clinodactyly; and limb asymmetry
2	M	7	22q13.31q13.32q13.33(44,368,741–51,169,045)X1	6.8	ADNPM, epicanthal folds, enlarged face, anteverted nostrils, and autistic behavior
3	F	5	1q41.q42.11q42.12q42.13q42.2q42.3q43q44(215,394,162–249,202,755)X3,	33.8 a	ID, delayed speech, arched palate, micrognathia, delayed growth, and autistic behavior
4	M	13	5p15.33p15.32(38,139–4,599,400)X3 18q22.2q22.3q23(68,426,681–78,014,582)X1	4.5 9.5	ID, neuropsychotropic delay, craniosynostosis, low anterior hair implantation and other dysmorphic features
5	F	8	2p25.3 (72,184–3,149,987) X1 2q37.1q37.2q37.3(234,799,176–243,029,573)X 3	3.2 8.2	Global developmental delay, ID, central obesity, and small hands
6	M	11	4q32.3q33q34.1(168,768,607–174,366,240)X1 4q34.1q34.2q34.3q35.1q35.2(174,906,286–190,880,409)X1, 9p24.3p24.2p24.1p23(46,587–9,264,439)X3 9p13.1p12(40,294,324–42,374,011)X3	5.5 15.9 9.2 2.0	ID, protruding ears, ocular hypertelorism, low anterior hair line, flattened skull, micrognathia, short neck, and cryptorchidism

Abbreviations: ADNPM, neuropsychomotor developmental delay; CNV, copy number variation; ID, intellectual disability; SNP, single nucleotide polymorphism.

^aPatient not availed by karyotype.

Discussion

Genomic imbalances are the most common genetic causes of ID. ^{4 5} The vast majority of ID cases remains without diagnosis after karyotyping due to methodologic limitations, primarily due to the inability to detect genomic variations smaller than 3 to 5 Mb. Currently, molecular biology techniques such as FISH, MLPA, and CMA, have made it possible to detect such microalterations. ^{6 10 13} The etiologic diagnostic rate for ID using molecular techniques depends on both the screening process of patients enrolled and the testing techniques of chosen, for example, MLPA kits, FISH probes, and array platforms. In general, the detection rate in patients with ID is estimated at 5% by G-banded karyotyping, 5.5% by FISH ²² , 9 to 30% by MLPA (depending on the combination of kits and previous screening ²³ ), and 20% by CMAs. ²⁴ The limitation of both MLPA and FISH is that those two techniques evaluate fewer target regions than CMA.

In the present study, FISH detected microdeletions in 25 of the 79 patients (31%) investigated, a rate that is considered high. Halder et al, using FISH to evaluate 301 patients with clinical suspicions of microdeletion syndromes, confirmed the diagnosis in only 7.6% of cases. ²⁵ Of the 219 patients with ID investigated by Baroncini et al., 12 (5.5%) were found to have subtelomeric rearrangements. ²⁶ The frequency of diagnosis for microdeletion syndromes using FISH depends on clinical suspicion and on the classification of syndromes investigated.

For WBS research in this study, both FISH and MLPA proved to be excellent diagnostic tests. MLPA was able to identify duplication of the WBS region in one of the patients, which was not identified by FISH.

Deletion of the critical region in WBS (the ELN gene) is found in 96% of cases using FISH. ^{27 28 29} Indeed, FISH is widely used and considered the gold standard for the molecular diagnosis of WBS. ^{30 31} By MLPA, all patients in this study with WBS demonstrated the same deletion involving the genes FKBP6 , FZD9 , TBL2 , STX1A , ELN , LIMK1 , RFC2 , and CLIP2 , within the WBSCR, while the POR and HSPB1 genes, located in more telomeric duplicons to the WBSCR, were not deleted. This enables us to state that these patients have deletions typical of WBS. All the patients with WBS had well-defined clinical characteristics: developmental and/or IDs (100%), an engaging personality (100%), hyperacusis (92.8%), delayed speech initiation (93.3%), and congenital heart disease (80%). Among the facial aspects, the following stood out: thick and prominent lips (93.7%), short nose or anteversion of the nostrils (92.8%), periorbital fullness (92.3%), bulbous nasal tip (92.3%), full cheeks (92.3%), and large mouth (86.6%).

In our study, a case of familial WBS was identified in a mother–daughter transmission of a typical WBS deletion. In the vast majority of cases, the deletion of WBS occurs sporadically, indicating a de novo deletion. ^{32 33 34 35} However, in 1993, Morris et al also reported parents and children were affected by WBS. ²⁷ Also, concordant monozygotic twins with WBS have been reported.

The patient with the 7q11.23 duplication syndrome (OMIM #609757) who was clinically suspected to have WBS showed an atypical duplication in 7q11.23, as the duplication covered a region larger than the one normally deleted in WBS and involved the genes POR and HSPB1 in telomeric duplicons. The phenotype associated with this duplication is highly variable, but language impairment, behavioral problems, and ID are frequent characteristics in these patients. ^{36 37 38 39 40 41 42 43} The complete phenotype of 7q11.23 duplication is unclear, making clinical screening unreliable. ^{44 45} Of the characteristics described to date for 7q11.23 duplication syndrome, our patient had impaired language, ID, behavioral problems (phobia/schizophrenia), seizures, hypotonia, joint laxity, short palpebral fissures, slightly elongated columella, and short philtrum. These characteristics are not specific enough to clinically recognize this duplication. The literature on atypical duplication in 7q11.23, covering telomeric duplicons, shows no additional or more severe characteristics in these cases when compared with the typical duplication. ³⁹ On the contrary, our patient's characteristics were suggestive of WBS including macrostomia, micrognathia, malocclusion, long neck, friendly personality, musical affinity, and hyperacusis.

For PWS and AS diagnoses, MS-PCR obtained a better result compared with FISH, as it was able to identify AS cases with maternal imprinting defects. Different genetic mechanisms cause PWS/AS, including deletions, UPD, and imprinting center mutations. ⁴⁶ FISH and CMA are only able to identify cases with deletions. Of the five patients with PWS, four had deletions. The clinical findings were compatible with criteria described in the literature such as mild ID, neonatal hypotonia; obesity or weight gain after 1 year; insatiable appetite (hyperphagia); characteristic facies, mainly almond-shaped eyes and ocular abnormalities; and small hands and feet and hypogenitalism. ⁴⁷ However, for AS, one of our patients possessed a deletion of the SNPRN and UBE3A genes of maternal origin, while the other patient had the absence of the methylated allele of maternal origin, suggestive of UPD or alteration in the imprinting mechanism. The UBE3A gene is located at the distal part of the 15q11-q13 region and is expressed from the maternal allele alone. In approximately 70% of AS patients, there is a 4 Mb deletion at 15q11-q13 of the maternally derived chromosome 15 resulting in multiple genes being deleted including UBE3A. ⁴⁸

The patients diagnosed with AS typically have dysmorphic facial features, delayed psychomotor development, a happy disposition, absence of speech, ataxia, skin hypopigmentation, prognathism, sleep disturbance, and hyperactivity. ⁴⁹

Among the patients suspected to have PWS, one had 15q26-qter deletion. ²¹ The clinical evaluation of this patient revealed short stature (1.38 m; <3rd percentile and the average height of a 10 years old) and central obesity (weight 65 kg; above the 97th percentile when corrected for height). Physical findings included micrognathia; head circumference of 52.5 cm (10th percentile); short neck; posteriorly rotated ears; sharp, widely-spaced teeth with malocclusion; sparse eyebrows, almond-shaped eyes; divergent strabismus; and small hands and feet (total hand length of 15 cm, <3rd percentile).

FISH identified two patients with LGS, both with deletions of the EXT1 and TRPS1 genes. The patients presented clinically with multiple exostoses due to the deletion of EXT1 gene and typical facies from the deletion of the TRPS1 gene. Facial features included sparse, slowly growing scalp hair, a bulbous-shaped nose, eyebrows that were thick in the middle and sparse laterally, long flat philtra, thin upper lips, and protruding ears. LGS is an autosomal dominant, contiguous gene deletion syndrome, resulting from a microdeletion that includes the EXT1 and TRPS1 genes at 8q24. ⁵⁰ In 1969, Giedion noted that LGS had features of both TRPS1 and multiple cartilaginous exostoses (MCE), which eventually lead to the recognition that LGS was a contiguous gene deletion syndrome. ⁵¹ Only one of our patients had WHS. This patient presented with growth delay, microcephaly, seizures, and the typical facial features for this syndrome, usually referred to as “Greek helmet facies.” Different mechanisms result in WHS, however, in approximately 75% of cases, the syndrome is due to de novo chromosomal 4p-terminal deletions and can be diagnosed using FISH. ⁵²

By using CMA, our study diagnosed six patients who had both ID and pathogenic CNV ( Table 2 ). The percentage of patients diagnosed in this study by CMA was higher than that described in the literature. This finding may have been due to the criteria used in the selection of our patients. Other studies ^{14 53} have also demonstrated that CMA has revolutionized the clinical diagnosis of patients with undiagnosed ID and CA and has made it possible to detect CNV that cannot be seen by the conventional cytogenetics method. Using CMA for ID patients has a diagnostic yield of 15–20%. ^{54 55}

The six chromosomal abnormalities detected by CMA include the following cases.

• (1) The patient had 22q11.21-q11.23 deletion consistent with DiGeorge syndrome (OMIM: #188400)/velocardiofacial syndrome (OMIM: #192430) or 22q11.3 deletion syndrome. In this patient, the deletion did not include the TBX1 gene (OMIN: #602054), which is responsible for most of the physical malformations of the syndrome. ⁵⁶ The deletion in this patient was outside the LCRs normally described in typical 22q11.3 deletion syndrome and involved the LCRs from C to F. The deletion in this patient involves 32 genes, with genes SCARF2 , LZTR1BCR, IGLL1, and SNAP29 totted to be disease causing. The last of these genes may be related to the patient's phenotype, as when both allelic genes are mutated, it leads to cerebral dysgenesis, microcephaly, psychomotor retardation, growth deficit, and facial dysmorphism. ⁵⁶ The FISH was normal in this patient as the deletion was outside the region of the probe used.

• (2) The patient had 22q13 deletion resulting in Phelan–McDermid syndrome (OMIM: #606232) and had ID, neonatal hypotonia, global developmental delay, severe speech delay, autistic behavior, and minor dysmorphic characteristics. ^{57 58} According to the OMIM Genes/Genome Browser (Feb. 2009 GRCh37/hg19), in this region, there are 30 genes, with the ARSA, MAPK8IP2, CHKB, SHANK3, ATXN10/SCA10 , PPAR, TMRU, ALG12 , MLC1, and CPT1B being associated with this syndrome. The gene, SHANK3 (OMIM: #606230), has been identified as the critical gene for the neurologic and behavioral aspects in patients with 22q13 deletions. Mutations in this gene can also cause autism spectrum disorder ⁵⁸ and the behavioral problems presented by our patient, who had deficiencies in social interaction and communication, and other behavioral problems.

• (3) The patient had trisomy 1q41q44. There are approximately 115 genes in this region, 30 of which have been associated with various disorders. Among these 30 genes, only ZBTB18 is described as associated with autosomal dominant ID. Partial trisomy 1q is a relatively rare chromosomal alteration and frequently described in the literature as being associated with other chromosomal abnormalities. ⁵⁹ For instance, there are six cases described in the literature with 1q41-qter duplication. ⁶⁰

• (4) The patient had trisomy 5p15.33 and monosomy 18q22. According to the OMIM Genes Genome Browser (Feb. 2009 GRCh37/hg19) in the region of 5p15.33p15.32, 20 genes are found, and of these genes, SDHA, SLC6A19, TERT, NDUFS6 , and SLC6A are associated with diseases, whereas in the 18q22.2q22.3q23 region, 30 genes are described and only the TSHZ1 , CYB5A , and CTDP1 are associated with disorders. Trisomies of the distal segment of 5p13.3 may result in minor malformations, developmental delay, and learning difficulties. ^{61 62} Distal deletions of 18q are relatively frequent and appear to cause a variable phenotypic spectrum, including growth deficit, microcephaly, micrognathia, cleft palate with or without cleft lip, congenital aural atresia, genitourinary system malformations, myelination disorders, hypotonia, and ID. ⁶³

• (5) The patient has monosomy 2p25.3 and trisomy 2q37.1-q37.3. According to the OMIM Genes in region 2p25.3, the TPO and PXDN genes are associated with diseases. In 2014, Bonaglia et al detected a 1.9 Mb deletion in the 2p25.3 region, which included the genes ACP1 , TMEM18 , SNTG2 , TPO, and PXDN in a patient who had ID, hyperactivity, and obesity. ⁶⁴ Obesity was attributed to haploinsufficiency of the ACP1 gene and deletion of the MYT1L gene was associated with ID, obesity, and hyperactivity, ⁶⁴ a clinical picture like that of our patient. In the region involving duplication in 2q37.1-q37.3, genes that are found there and are associated with diseases are COL6A3, MLPH, PER2, CAPN10, TWIST2, NDUFA10, AGXT, D2HGDH, PDCD1, HDAC4, and KIF1A, with the last two being associated with the ID.

• (6) The patient had loss of a copy in the regions 4q32.3q33q34.1 and 4q34.1q34.2q34.3q35.1q35.2 and the gain of a copy number in the regions 9p24.3p24.2p24.1p23, and 9p13.1p12. Subsequently, FISH was performed on the parents, which showed a balanced translocation in the father involving chromosomes 4 and 9. In 4q deletion syndromes, both terminal or interstitial ones are relatively rare and have been described in patients with a variable phenotypic spectrum including congenital heart disease, moderate ID, and minor dysmorphic features. ⁶⁵ Partial duplications of 9p24 are relatively frequent, with the clinical findings including craniofacial anomalies typical of trisomy 9p chromosome, developmental delay, ID, and brain anomalies. ⁶⁶

In the study presented here, we were able to determine the etiology of 28% of both individuals with ID and CA. For those cases submitted for CMA, the diagnostic rate was 22%. Chong et al, in a study of patients with ID, developmental delay, and/or multiple CA, detected pathogenic CNV in 19% of these patients. ⁶⁷ A higher rate was described by Hochstenbach et al in a retrospective review of over 36,000 cases from 29 studies of patients with ID and CA. ⁶⁸ We believe that this higher rate is due to the expertise of the geneticists who performed the assessment. In addition, that higher rate may also be attributed to the screening process of patients enrolled in the study, given that only patients who were already at higher risk for chromosomal disorders (as well as dysmorphic features associated with ID) were eligible to participate. The ISCA has recommended CMA as the first cytogenetic diagnostic test for patients with ID, neuropsychomotor developmental delay (ADNPM), autism, and multiple congenital malformations. ⁶⁷ Our study demonstrated that the evaluation by CMA enabled the identification of genomic alterations not detected by classical cytogenetics, which provides a much more powerful tool for diagnosing individuals with ID and dysmorphic defects. However, the use of CMA as the first test for all ID patients in the public health network in Brazil is not yet feasible, mainly for financial reasons. For this reason, other diagnostic tools such as FISH and MLPA can still be used and we have shown them here to be quite efficient for confirming the diagnoses in patients suspected to have specific microdeletion syndromes.

In 2014, Ordinance No. 199, January 30, 2014, was approved by the Ministry of Health and published in the Official Gazette/DOU. Then, the institutes of National Policy for Comprehensive Care for People with Rare Diseases approved the Guidelines for Comprehensive Care for People with Rare Diseases within the scope of the Unified Health System (SUS) and the institute's financial funding incentives. ID and CA are classified as the structural axes in the Rare Disease Policy. Currently, in Brazil, the specific diagnosis of these diseases frequently is difficult and time-consuming. The result may be that it takes considerably longer for patients to be diagnosed and to receive adequate counseling and treatments.

In November 2020, the Ministry of Health (Brazil) then published the Clinical Protocol and Therapeutic Guidelines for ID, Ordinance No. 21, which recommended CMA for the investigation of ID.

The CMA, as well as the other molecular techniques used in the current study (FISH, MLPA), are included among the laboratory procedures necessary for the diagnosis of ID and CA. During this work, CMA analysis had not yet been incorporated by the SUS. Nowadays, it has changed and due to the implementation of those diagnostic tools in public health services, it has been possible to perform CMA analyses more frequently. Currently, the biggest challenge to overcome is that of budgetary restrictions for public health in Brazil, at the current demand, far exceeds available funding, especially in the north-northeast region of the country. This study has made it possible to provide laboratory support for the diagnosis and treatment of tested individuals, and when specific diagnoses are established, the risk of recurrence can be provided and directing those patients to personalized therapy. Diagnoses also provided epidemiologic information, diagnostic tools, and resources to the SUS. Those actions are compliant with what had been proposed by the National Policy for Comprehensive Care for People with Rare Diseases, which is a public health policy with the aims to needs of the population of this study.

Conclusion

Although CMA is the most complete and recommended tool for the diagnosis of microdeletions, microduplications, and unbalanced translocations in patients with ID, the FISH, MLPA, and MS-PCR tests can be used as the first test for specific syndromes, as long as these patients are well screened clinically, especially in the public health networks in Brazil, where resources are scarce. In the current study, we were able to observe that all the tools used contributed successfully to the diagnosis of patients with various chromosomal anomalies. The establishment of the etiology of ID in the cases evaluated here was extremely important for the prognosis, clinical management, and genetic counseling of these patients and their families. We highly recommend expanding this service by making available all the diagnostic tools available to all patients with ID.

Funding Statement

Funding This study was supported by funding from CNPq (National Council of Technological and Scientific Development).